Rodent Leydig Cell Tumorigenesis: A Review of the ...

Critical Reviews in Toxicology, 29(2):169–261 (1999)

Critical Reviews in Toxicology Downloaded from informahealthcare.com by US EPA Environmental Protection Agency on 04/30/15 For personal use only.

Rodent Leydig Cell Tumorigenesis: A Review of the Physiology, Pathology, Mechanisms, and Relevance to Humans Jon C. Cook,1,* Gary R. Klinefelter,2 Jerry F. Hardisty,3 Richard M. Sharpe,4 and Paul M. D. Foster 5 1DuPont Haskell Laboratory, Newark, DE; 2USEPA-NHEERL, Research Triangle Park, NC; 3Experimental Pathology Laboratories, Research Triangle Park, NC; 4MRC Reproductive Biology Unit, Edinburgh, United Kingdom; 5Chemical Industry Institute of Toxicology, Research Triangle Park, NC

*

Address correspondence to: Dr. Jon C. Cook, Pfizer Inc., Central Research, Eastern Point Road, Groton, CT 06340

TABLE OF CONTENTS I. Introduction ............................................................................................................................. 172 II. Cytology and Ontogeny of the LC ........................................................................................ 172 A. Cytology .............................................................................................................................. 172 B. Ontogeny and Fate of the LC ........................................................................................... 174 1. The Fetal LC ................................................................................................................. 174 2. The Postnatal LC .......................................................................................................... 175 3. The Pubertal LC ........................................................................................................... 175 4. The Adult LC ................................................................................................................ 176 5. The Senescent LC ......................................................................................................... 179 III. Endocrine and Paracrine Regulation of the LC .................................................................. 179 A. Endocrine Regulation of LC Function ............................................................................ 179 B. Paracrine Regulation of LC Function ............................................................................. 188

Abbreviations: 11b-HSD, 11β-hydroxsteroid dehydrogenase; AIS, androgen insensitivity syndrome; bFGF, basic fibroblast factor; C8, ammonium perfluorooctanoate; CD, Crl:CD®BR; DBI, diazepam binding inhibitor; DBCP, dibromochloropropane; DES, diethylstilbestrol; DHT, dihydrotestosterone; E2, 17β-estradiol; EDS, ethane dimethane sulphonate; EE, ethinylestradiol; ETU, ethylenethiourea; FMPP, familial male precocious puberty; FSH, follicle stimulating hormone; GnRH, gonadotropin releasing hormone; hCG, human chorionic gonadotropin; HMG-CoA, 3-hydroxy-3-methylgutaryl coenzyme A; hpg, hypogonadal; HPT, hypothalamo-pituitary-testis; IARC, International Agency for Research on Cancer; IF, interstitial fluid; IGF-1, insulin-like growth factor-1; IL-1, interleukin-1; JP-4, Jet Petroleum-4; LCs, Leydig cells; LCTs, Leydig cell tumors; LH, luteinizing hormone; MIS, Mullerian Inhibiting Substance; MTBE, methyl tertiary butyl ether; NTP, National Toxicology Program; o,p′-DDD, 1,1-dichloro-2,2-bis (p-chlorophenyl)ethane; o,p′-DDT, 1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane; p,p′-DDE, 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene; P450, cytochrome P450; PBR, peripheral-type benzodiazapine receptors; PCE, perchloroethylene; PDR, Physician’s Desk Reference; PKA, protein kinase A; PNCB, p-nitrochlorobenzene; PTU, 6-n-propyl-2-thiouracil; SCP-2, sterol carrier protein; SER, smooth endoplasmic reticulum; SHBG, sex hormone-binding globulin; StAR, Steroidogenic Acute Regulatory Protein; TACE, tri-p-anisyl-chloroethylene; TCE, trichloroethylene; TDA, 2,4-toluenediamine; TFA, trifluoroacetic acid; TGFa, transforming growth factor-α; TGFb, transforming growth factor-β; TNG, trinitroglycerin; WY, Wyeth-14,643

169


IV. V. VI. VII.

VIII.

170

1. LC Changes After Disruption of Spermatogenesis .................................................. 188 2. Potential Paracrine Regulators of LC Number ........................................................ 189 a. Insulin-Like Growth Factor-1 ............................................................................... 189 b. Transforming Growth Factor-a ........................................................................... 189 c. Transforming Growth Factor-b ........................................................................... 190 d. Basic Fibroblast Growth Factor ............................................................................ 190 e. Interleukin-1 ............................................................................................................ 190 f. Inhibin and Activin ................................................................................................. 191 C. Potential Paracrine Regulators of LC Testosterone Production .................................. 191 Role of LH in LC Tumorigenesis .......................................................................................... 192 Role of Paracrine Factors in LC Tumorigenesis ................................................................. 192 Pathology of LCTs................................................................................................................... 194 Compounds That Induce LC Hyperplasia or Tumors ....................................................... 200 A. Nongenotoxic Compounds ................................................................................................. 210 1. Classified by Mode of Action ...................................................................................... 210 a. Androgen Receptor Antagonists ........................................................................... 210 b. 5α-Reductase Inhibitors ......................................................................................... 213 c. Testosterone Biosynthesis Inhibitors .................................................................... 213 d. Aromatase Inhibitors .............................................................................................. 217 e. Dopamine Agonists/Dopamine Enhancers ........................................................... 217 f. GnRH Agonists ........................................................................................................ 219 g. Estrogen Agonists/Antagonists .............................................................................. 219 2. Classified by Chemical Activity .................................................................................. 221 a. Antihypertensives .................................................................................................... 221 b. Calcium Channel Blockers ..................................................................................... 221 c. Fungicides ................................................................................................................ 222 d. Goitrogens ................................................................................................................ 222 e. Peroxisome Proliferators ........................................................................................ 222 3. Classified by Chemical Class ...................................................................................... 226 a. Fluorochemicals ....................................................................................................... 226 b. Nitroaromatics and Related Compounds ............................................................. 227 c. Organochlorines ...................................................................................................... 228 d. Sugars ....................................................................................................................... 228 4. Unclassified .................................................................................................................... 228 B. Genotoxic Compounds....................................................................................................... 231 1. Cadmium ....................................................................................................................... 231 2. Dibromochloropropane ................................................................................................ 232 3. Radiation ....................................................................................................................... 232 Human Relevance .................................................................................................................... 233 A. Human Incidence of LCTs ................................................................................................ 233 B. Comparative Biology ......................................................................................................... 234 C. Endocrine Disease States in Humans .............................................................................. 236 D. Epidemiology ....................................................................................................................... 237 1. 1,3-Butadiene ................................................................................................................. 237 2. Cadmium ....................................................................................................................... 237 3. Ethanol ........................................................................................................................... 238 4. Lactose ........................................................................................................................... 238 5. Lead Acetate.................................................................................................................. 238


6. Nicotine .......................................................................................................................... 238 7. Trichloroethylene .......................................................................................................... 239 IX. Conclusions............................................................................................................................... 240 ABSTRACT: Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has increased in recent years because of its common occurrence in rodent bioassays and the importance in assessing the relevance of this tumor type to humans. To date, there have been no comprehensive reviews to identify all the compounds that have been shown to induce LCTs in rodents or has any review systematically evaluated the epidemiology data to determine whether humans were at increased risk for developing LCTs from exposure to these agents. This review attempts to fill these deficiences in the literature by comparing the cytology and ontogeny of the LC, as well as the endocrine and paracrine regulation of both normal and tumorigenic LCs. In addition, the pathology of LCTs in rodents and humans is compared, compounds that induce LC hyperplasia or tumors are enumerated, and the human relevance of chemical-induced LCTs is discussed. There are plausible mechanisms for the chemical induction of LCTs, as typified by agonists of estrogen, gonadotropin releasing hormone (GnRH), and dopamine receptors, androgen receptor antagonists, and inhibitors of 5α-reductase, testosterone biosynthesis, and aromatase. Most of these ultimately involve elevation in serum luteinizing hormone (LH) and/or LC responsiveness to LH as proximate mediators. It is expected that further work will uncover additional mechanisms by which LCTs may arise, especially the role of growth factors in modulating LC tumorigenesis. Regarding human relevance, the pathways for regulation of the hypothalamo-pituitary-testis (HPT) axis of rats and humans are similar, such that compounds that either decrease testosterone or estradiol levels or their recognition will increase LH levels. Hence, compounds that induce LCTs in rats by disruption of the HPT axis pose a risk to human health, except for possibly two classes of compounds (GnRH and dopamine agonists). Because GnRH and prolactin receptors are either not expressed or are expressed at very low levels in the testes in humans, the induction of LCTs in rats by GnRH and dopamine agonists would appear not to be relevant to humans; however, the potential relevance to humans of the remaining five pathways of LCT induction cannot be ruled out. Therefore, the central issue becomes what is the relative sensitivity between rat and human LCs in their response to increased LH levels; specifically, is the proliferative stimulus initiated by increased levels of LH attenuated, similar, or enhanced in human vs. rat LCs? There are several lines of evidence that suggest that human LCs are quantitatively less sensitive than rats in their proliferative response to LH, and hence in their sensitivity to chemically induced LCTs. This evidence includes the following: (1) the human incidence of LCTs is much lower than in rodents even when corrected for detection bias; (2) several comparative differences exist between rat and human LCs that may contribute, at least in part, to the greater susceptibility of the rat to both spontaneous and xenobiotic-induced LCTs; (3) endocrine disease states in man (such as androgen-insensitivity syndrome and familial male precocious puberty) underscore the marked comparative differences that exist between rats and man in the responsiveness of their LC’s to proliferative stimuli; and (4) several human epidemiology studies are available on a number of compounds that induce LCTs in rats (1,3-butadiene, cadmium, ethanol, lactose, lead, nicotine) that demonstrate no association between human exposure to these compounds and induction of LC hyperplasia or adenomas. After considering the human incidence of LCTs, the comparative differences between rats and humans, human endocrine disease states, and epidemiology, the weight of evidence suggests that human LCs are quantitatively less sensitive than rat LCs in their proliferative response to LH, and hence in their sensitivity to chemically induced LCTs. It can be concluded that no observable effect levels for the induction of LCTs in rodent bioassays provide an adequate margin of safety for protection of human health and that the data support a nonlinear mode of action (i.e., threshold response). In conclusion, the data suggest that nongenotoxic compounds that induce LCTs in rats most likely have low relevance to humans under most exposure conditions because humans are quantitatively less sensitive than rats. In a recent international multidisciplinary workshop on LC tumorigenesis, seven research needs were identified. In this review, we have begun to address two of these needs (comparative sensitivity differences and epidemiology), and as the other areas of research are further investigated, these data will help to critically test the conclusions in this review. KEY WORDS: interstitial cell, Leydig cell, Leydig cell tumors, interstitial cell tumors, testis, review, chemically induced Leydig cell tumors, human relevance of rodent Leydig cell tumors.

171


I. INTRODUCTION Several book chapters and reviews have been written about compounds that induce Leydig cell tumors (LCTs) in rodents (Lacassagne, 1971; Vermeulen, 1982; Neumann, 1991; Bar, 1992; Ewing, 1992; Alison et al., 1994; Bosland et al., 1994; Prentice and Meikle, 1995; Huseby, 1996; Morris, 1996; Cook et al., 1997). An entire book has also been devoted to the physiology and pathophysiology of Leydig cells (LCs) (Payne et al., 1996). The number of reviews on LCTs has dramatically increased in recent years because of its common occurrence when compounds are evaluated in rodent cancer bioassays and the importance in assessing the relevance of this tumor type to humans. Recently, an international multidisciplinary workshop on LC tumorigenesis was held to evaluate the human relevance of compounds that induce LCTs in rodent bioassays (summarized in Clegg et al., 1997). With the wealth of information known about LCs and agents known to induce LCTs, it was surprising that no comprehensive review has been undertaken to identify all the compounds that have been shown to induce LCTs in rodents or has any review to date systematically evaluated the epidemiology data to determine whether humans were at increased risk for developing LCTs from exposure to these agents. Prentice and Meikle (1995) have reviewed pharmaceutical agents for their potential to induce LCTs. The goal of the present review is to build on the work by these authors and the above workshop on LC tumorigenesis by reviewing the chemicals identified by Clegg and co-workers (1997). The current review has searched the published literature exhaustively through May 1998. This review provides an overview of the cytology and ontogeny of the LC (Section II), a description of endocrine and paracrine regulation of the LC (Section III), a discussion of the role of luteinizing hormone (LH) (Section IV) and paracrine factors (Section V) in LC tumorigenesis, a summary of the pathology of LCTs in rodents and humans (Section VI), an enumeration of the compounds that induce LC hyperplasia or adenomas (Section VII), a discussion of the human relevance (Section VIII), and, lastly, conclusions regarding the knowledge gained from this effort (Section IX). This review builds on the 172

work of several other authors and is clearly the most comprehensive overview of agents known to induce LCTs to date.

II. CYTOLOGY AND ONTOGENY OF THE LC A. Cytology In mammalian testes, LCs reside within the interstitial tissue bordered by adjacent seminiferous tubules. The interstitial tissue that contains arterioles, venules, capillaries, lymphatic vessels, fibroblasts, macrophages, occasional nerve fibers, and clusters of LCs (Figure 1). Collectively, these components are embedded within a loose extracellular ground substance supported by collagen and elastic fibers. The peritubular tissue which is comprised of extracellular matrix, fibroblasts, and myoid cells actually forms the border of the interstitial space. LCs are often found between the peritubular tissue of two adjacent seminiferous tubules (Christensen, 1975). Peritubular myoid cells, by virtue of gap junctions between adjacent cells, form the proximate component of the bloodtestis barrier, a cellular barrier between capillaries and the luminal compartment of the seminiferous tubules that effectively restricts permeability. In both the rat and human, LCs appear predominantly in clusters. In the human testis, however, single LCs can often be found interspersed within loose interstitial connective tissue. Typically, the plasma membranes of adjacent LCs are spaced about 150 Å apart and gap junctions, which reduce the intermembrane distance to 20 Å, are commonly observed. In young adult human males, the interstitial tissue comprises 34% of the testicular parenchyma, with LCs occupying 12% of the testicular volume (Schinz and Slotopolsky, 1927; Neaves et al., 1984; Johnson et al., 1986). In rats, interstitial tissue and LCs constitute only 6 and 2% of the testis volume, respectively (Roosen-Runge, 1955). In the human testis, interstitial spaces contain distinct lymph vessels, and LCs are prevented from direct contact with the lymph by the wall of the lymph vessel. By contrast, the LCs in the rat testis are in continuous contact with the testicular lymph because lymphatic sinusoids, which occupy the majority of


FIGURE 1. (A) Cytology of the interstitium of the human testis. Note that lymphatic vessels are bounded by a continuous endothelial cell layer. Clusters of LCs are dispersed within a matrix of loose connective tissue. (B) The interstitium of the rat testis. Distinct lymphatic vessels are not present and discontinuity in the visceral layer of endothelium allows lymph to bathe the LC clusters directly. Abbreviations: LC, Leydig cell; LV, lymph vessel; L, lymph; M, macrophage; F, fibroblast; C, capillary; VE, visceral endothelium; PE, parietal endothelium; PM, peritubular myoid cells; SC, Sertoli cell. (Modified from Fawcett, 1973.)

173


the interstitial tissue, are bounded by discontinuous layers of endothelium (Fawcett et al., 1973; Holstein et al., 1979). LCs are epitheliod-type cells that contain mitochondria with tubular cristae, abundant smooth endoplasmic reticulum (SER), variable amounts of lipid, and a network of peroxisomes. Both rat and human LCs contain comparable amounts of SER, the organelle that confers the capacity of the LC to produce testosterone (Ewing and Zirkin, 1983). While lipid droplets in both adult human and mouse LCs are numerous, adult rat LCs contain relatively few lipid droplets (Christensen, 1975). A unique morphological feature of both the human LC and the Australian wild bush rat is the presence of Reinke crystals within the cytoplasm. These crystalline inclusions represent an hexagonal assembly of 50 Å-thick protein filaments. Reinke crystals may be associated with decreased steroidogenic capacity of the LC because Reinke crystals have been shown to increase with advancing age in the human testis (Paniagua et al., 1986). Moreover, these cytoplasmic inclusions also increase in LCs of the Australian wild bush rat when testosterone production is compromised (Irby et al., 1984).

B. Ontogeny and Fate of the LC

1. The Fetal LC Testosterone concentration in peripheral blood changes dramatically throughout the life cycle in both male rats and humans (Payne et al., 1996). These fluctuating concentrations of testosterone in the blood are directly related to LC development. In humans, an initial rise in fetal testosterone occurs around the fourth month of gestation. The testosterone produced at this time is responsible for androgen-induced differentiation of the epididymis, vas deferens, and seminal vesicles, and its 5α-reduced metabolite, dihydrotestosterone (DHT), masculinizes the genitalia and differentiates the prostate. LC precursors arise from undifferentiated interstitial mesenchymal cells, which in turn are derived from the intermediate mesoderm. These cells can be observed by week 8 of gestation as newly formed seminiferous cords containing Sertoli cells and germ cells and are seen bounded by a basement membrane 174

(Pelliniemi and Niemi, 1969). Subsequent cytodifferentiation of the LCs involves an increase in cytoplasmic volume in the size and number of mitochondria and in the number of lipid droplets. Crystals of Reinke are not evident in human fetal LCs. Functional differentiation seems to be coincident with cytodifferentiation. The factor that initiates fetal LC differentiation is unknown, but LH probably is not involved because testosterone production precedes detection of immunoreactive LH (Reyes et al., 1989). By week 15 of gestation, when circulating testosterone levels peak, LC clusters occupy more than half of the testis volume, and circulating levels of human chorionic gonadotropin (hCG) are increased significantly (Reyes et al., 1989). From week 16 of gestation until birth, there is a decline in hCG levels, a 60% reduction in the number of LCs, a significant decrease in the size of the remaining LCs, and a concomitant decline in testosterone production. These LCs undergo further involution during the first year of postnatal life (Codesal et al., 1990). In the rat, LCs appear in the fetal gonad by day 16 of gestation, shortly after a basement membrane forms around seminiferous cords containing Sertoli cell precursors and germ cells (Magre and Jost, 1980). It seems that these fetal LCs arise exclusively by differentiation of mesenchymal cells because no mitotic activity has been observed (Orth, 1982). They contain large mitochondria and numerous lipid droplets and are capable of binding LH and producing testosterone at this time (Gangnerau et al., 1982; Habert and Picon, 1984). The onset of fetal LC differentiation in the rat appears to be unrelated to genetic sex or gonadotropins. It is possible that differentiation is initiated via paracrine signalling from the fetal Sertoli cell because recombinant folliclestimulating hormone (FSH) has been shown to stimulate testosterone production in fetal testis explants (Lecerf et al., 1993) and Anti-Mullerian Hormone (also referred to as Mullerian Inhibiting Hormone, Mullerian Inhibiting Substance, or MIS), a fetal Sertoli cell product, has been implicated along with testosterone in gonadal morphogenesis and testicular descent (Lee and Donahoe, 1993). More recently, MIS-deficient mice (Behringer et al., 1994) were shown to have fully descended testes and functional sperm but testes with LC hyperplasia. In one instance, LC neopla-


sia was observed. This suggests that MIS is a potent, negative regulator of LC proliferation. The number of fetal rat LCs increases to a maximum of 100,000 per testis by gestation day 20 (Kerr and Knell, 1988). By this time, the number of LH receptors within the testis has also reached a maximum (Warren et al., 1984). Although LC number remains constant between gestation day 20 and postnatal week 2, fetal LC volume decreases because of a decrease in the size of individual LCs (Habert and Picon, 1982). It is unclear whether the fetal LC degenerates (Kuopio et al., 1989) or persists throughout adult life (Kerr and Knell, 1988). Even if fetal LCs do persist in the adult, this population would be relatively insignificant from both quantitative and qualitative perspectives. The adult rat testis typically contains 25 × 106 LCs so the fetal population would represent only 0.4% of the total number of LCs in the adult testis. Based on in vivo and in vitro data, testosterone production increases between gestation day 16 and 18, and then decreases until birth (Tapanainen et al., 1984; Weisz and Ward, 1980; Habert and Brignaschi, 1991). Because the steroidogenic capacity per fetal LC is markedly diminished by the end of fetal life, these LCs would contribute little to the total testosterone pool within the pubertal/adult testis. The cause for functional regression is unknown, but it is hypothesized that paracrine factors may be involved. Finally, unlike adult rat LCs, fetal LCs fail to become desensitized by LH or hCG stimulation (Warren et al., 1982). This developmental distinction also has been documented for human LCs (Leinonen and Jaffe, 1985).

2. The Postnatal LC In human males, a second peak in circulating testosterone occurs due to an increase in LH and results in a second generation of LCs. This change occurs around the second month of postnatal life (Forest et al., 1973). Presumably, this peak is involved in the imprinting of androgen-dependent tissues and masculinized sexual behavior (Forest et al., 1973). Subsequently, these LCs degenerate so that very few LCs exist in the testis at 1 year of age (Codesal et al., 1990). In the rat, a postnatal rise in testosterone also occurs; but this is an abrupt elevation and is observed within

only a few hours after birth (Gogan et al., 1981). This surge is associated with both an increased concentration of testosterone within the testis and decreased clearance of testosterone (Baum et al., 1988). As the number of fetal rat LCs remains constant between gestation day 20 and postnatal week 2, this postnatal surge probably reflects an increase in the steroidogenic capacity of preexisting fetal LCs resulting from an increase in secretion of LH.

3. The Pubertal LC The third and final peak in circulating testosterone occurs at the time of adolescence in humans and is associated with pubertal development: masculinization, the onset of spermatogenesis within the testis, and sperm maturation within the epididymis. During childhood, mesenchymal or fibroblastic cells representing undifferentiated precursor cells divide and differentiate into adulttype LCs (Mancini et al., 1963; Chemes et al., 1985). The differentiation of the precursor cells appears to be regulated by LH (Chemes et al., 1992). Once the population of adult LCs is formed, there is negligible cell division. Thus, the population is relatively stable until degeneration of LCs ensues later in life. In the rat, the pubertal rise in testosterone also is achieved with a new generation of LCs that become recruited between 2 and 4 weeks of age. These LCs appear to result from differentiation of mesenchymal-like cells (Hardy et al., 1989). After week 4, division of morphologically distinguishable LCs determines the ultimate number of adult LCs in the testis. In vivo and in vitro studies have demonstrated that both LH and androgen appear to be involved in the morphological and functional differentiation of LC precursors (Hardy et al., 1990; Shan et al., 1995). However, the relative contributions of LH and androgen in precursor differentiation remains unknown. Whether there is any similarity in role of LH in the differentiation of mesenchymal-like cells into LCs and the putative role of LH in the induction of LC hyperplasia in adult animals remains unclear. While LH appears to be pivotal in the differentiation of LC precursors, other hormones may play a role. There is extensive literature showing that administration of FSH to hypophysectomized 175


immature rats (e.g., Odell and Swerdloff, 1976; Chen et al., 1977; Kerr and Sharpe, 1985a; Teerds et al., 1989b; Vihko et al., 1991; reviewed in Sharpe, 1994), to the hypogonadal (hpg) mouse (O’Shaughnessy et al., 1992) or to hypophysectomized or photoperiod-inhibited hamsters (Niklowitz et al., 1989; Chandrashekar et al., 1994) results in increased size and number of the LCs (Kerr and Sharpe, 1985a; Teerds et al., 1989b). Notably, FSH treatment increases the volume of SER (Kerr and Sharpe, 1985b) and the level of steroidogenic enzymes (O’Shaughnessy et al., 1992; Murono and Payne, 1979), and, as a consequence, greatly increases the capacity of the testis or isolated LCs to secrete testosterone and other steroids when stimulated with LH (see references cited above). Many of these stimulatory effects of FSH can be reproduced in vitro (Benahmed et al., 1984; Tabone et al., 1984; Ojeifo et al., 1990). As puberty in both rats and human is associated with relatively high FSH levels (Odell and Swerdloff, 1976), it is reasonable to conclude that this hormone, acting via the Sertoli cells, regulates development of the adult LC population via the production of unknown paracrine factors (Sharpe, 1994). Moreover, as damage to the seminiferous tubules in adult rats and humans frequently leads to elevation of the blood levels of FSH, the LC hypertrophy, and hyperresponsiveness found in such situations (discussed below) may be related to the increase in FSH levels. The pubertal rise in testosterone in the rat is preceded by increased production of 5α-reduced androgens, a steroidogenic feature of immature LCs. The levels of 5α-reduced androgens reach a maximum around day 30. As final LC maturation is achieved, the capacity for 5α reduction diminishes (Rommerts and van der Molen, 1989). Both LH and prolactin appear to be involved in supporting 5α reductase activity in the immature testis (Chase and Payne, 1985), but the subsequent decline in activity as adult LCs are formed is not understood. Presumably, the declining 5α reductase activity also may result from paracrine influences within the testis (see below).

4. The Adult LC In the rat, a small number of progenitor cells persist throughout adult life. Recruitment of new 176

LCs also can occur with sufficient LH stimulation (Christensen and Peacock, 1980). Interestingly, recruitment of new LCs occurs after cytotoxic destruction of adult LCs. In this instance, both the rate and location of regenerating LCs after their destruction by administration of ethane dimethane sulfonate (EDS) are subject to paracrine influence, probably via the Sertoli cell. Induction of unilateral or bilateral cryptorchidism at the time of EDS treatment results in more rapid LC regeneration in the cryptorchid testes, with new LCs arising mainly in the vicinity of severely damaged seminiferous tubules (i.e., lacking germ cells: Kerr and Donachie, 1986; O’Leary et al., 1986; Sharpe et al., 1990). Indeed, the smaller the seminiferous tubule (i.e., more damaged), the greater the volume of adjacent regenerating LCs (Savage and Kerr, 1995). This suggests that the Sertoli cells produce one or more growth factors that regulate the pathway of LC development following toxic insult and that, in turn, germ cells modulate this function of Sertoli cells (Table 1). Other data indicate that testicular macrophages also play an important role in regulating LC regeneration in EDS-treated rats (Gaytan et al., 1994a,b). While the initial recruitment and proliferation of LC precursors occurs in the absence of LH and is presumed to be paracrine mediated, it appears that the final differentiation of these precursors into adult LCs is an LH-dependent process (Teerds et al., 1988, 1989a, 1990a). Perhaps the most important question, unanswered in the above studies, is what eventually halts LC regeneration after EDS treatment? The pattern of change in blood LH levels is probably important (Teerds et al., 1994), but LH is typically considered a “stimulus” for LC development. One candidate inhibitor of LC development is 17β-estradiol (E2). In EDS treated rats supplemented with hCG, co-administration of E2 from days 5 to 30 after EDS treatment largely prevents LC regeneration. By contrast, administration of E2 on days 0 to 5 or 16 to 30 after EDS treatment fails to prevent LC regeneration ( and Myers, 1991). While it was not determined if exogeneous E2 decreased circulating LH levels via negative feedback (see below), the data suggest that the initial proliferation of LC precursors after EDS treatment is unaffected by E2, but the final differentiation into mature LCs may be inhibited by E2. It is tempting to speculate that the acquisition

177

Testicular site(s) of productiona,b

SC, LC, GC (Smith et al., 1987; Cailleau et al., 1990; Naville et al., 1990; Saez, 1994)

SC, LC, PT (Skinner et al., 1989; Teerds et al., 1990b)

LC, GC (Teerds and Dorrington, 1993)

SC, LC, GC, PT (Ueno et al., 1987; Story et al., 1988; Smith et al., 1989; Murono et al., 1992; Han et al., 1993)

Growth factor

IGF-1

TGF-a

TGF-b

bFGF

Yes (immature LC) (Sordoillet et al., 1988; Murono et al., 1992; Han et al., 1993; Le MagueresseBettistoni et al., 1994)

?

Yes (Saez, 1994)

Yes (Bernier et al., 1986; Lin et al., 1986, 1988; Vanelli et al., 1988; Nagpal et al., 1991)

Presence of receptors on LCs

Inhibitory (immature LC) (Murono and Washburn, 1990a,b; Saez, 1994)

Inhibitory adult LC (Avallet et al., 1987; Morera et al., 1988; Van Bebaker et al., 1990) Immature LC (Lin et al., 1987b; Morera et al., 1988)

Variable effects (Saez, 1994)

Stimulatory (Bernier et al., 1986; Lin et al., 1986, 1987a; Benahmed et al., 1987; Saez et al., 1988; Gelber et al., 1992; Saez, 1994)

Probable effect on LC steroidogenesis

?

Inhibitory — immature LC Teerds et al., 1994)

Stimulatory — immature LC (Khan et al., 1992a; Teerds et al., 1994) Inhibitory (EGF) — adult LC (Ojeifo et al., 1990)

Stimulatory (with other factors) (Bernier et al., 1986; Khan et al., 1992a; Teerds et al., 1994)

Effect on LC numbers

TABLE 1 Summary of the Studies Indicating the Effects of the Major Growth Factors on LC Numbers and/or Function


178

b

a

SC(a), LC (b,?a), GC(a) MC(b) (Gerard et al., 1991; Wang et al., 1991; Lin et al., 1993; Haugen et al., 1994; Syed et al., 1995)

Testicular site(s) of productiona,b ?

Presence of receptors on LCs Inhibitory (Calkins et al., 1988, 1990; Verhoeven et al., 1988; Lin et al., 1991; Moore and Moger, 1991; Hales et al., 1992; Mauduit et al., 1992) Stimulatory (Verhoeven et al., 1988; Sharpe, 1993; Saez, 1994)

Probable effect on LC steroidogenesis

SC, Sertoli cells; LC, Leydig cells; GC, germ cells; PT, peritubular myoid cells; Mc, macrophages. Evidence is based on immunostaining, mRNA expression and/or direct measurement.

IL-1 a b

Growth factor Stimulatory (b) — immature LC (Khan et al., 1992b; Gaytan et al., 1994a,b)

Effect on LC numbers

TABLE 1 (continued) Summary of the Studies Indicating the Effects of the Major Growth Factors on LC Numbers and/or Function



of aromatizing ability by LCs during puberty (Payne et al., 1976; and Melner, 1979) and the dramatic increase in intratesticular E2 levels that occurs in the LH- or hCG-stimulated adult testis of both rats and humans (Heller and Leach, 1971; de Jong et al., 1974; Cigorraga et al., 1980; Wang et al., 1980) provide evidence that E2 secreted by differentiated LCs negatively regulates differentiation of new LCs via some feedback loop: autocrine, paracrine, or endocrine. Indeed, the presence of estrogen receptors in LC nuclei (Mulder et al., 1973; Fisher et al., 1997) is consistent with estrogen-mediated negative regulation of LC differentiation.

5. The Senescent LC In humans, senescence is accompanied by a decrease in circulating testosterone from 7 mg/d in a young adult (Horton and Tait, 1967) to 4 mg/ d in an aged man (Vermeulen et al., 1972). This decrease in androgens occurs despite maintenance or an increase in circulating LH levels (Vermeulen, 1978) and clearly is the result of a decline in LC numbers via degeneration and dissolution (Neaves et al., 1984; 1985). In the testis of aging beagle dogs, it appears that both LC atrophy and hyperplasia occur and are associated with an elevation in circulating LH levels (Ewing et al., 1987). By contrast, the decline in circulating testosterone that occurs during senescence in rats occurs while LH levels are declining (Pirke, 1979) and appears to be related to a reduction in the capacity of the LC to respond to LH rather than to a decline in LC number (Chen et al., 1994). In a study comparing young (2 months) and aged (24 months) Wistar rats (Ichihara et al., 1993), morphometric analysis revealed a significant increase in LC numbers in aged rats, together with a significant decrease in LC volume. The decrease in LC volume was associated with a reduction in the volume of LC SER per aged LC. In the study by Chen and co-workers (1994) comparing 6- and 24-month Brown Norway rats, an increase in LC numbers was observed, but this increase was not statistically significant. This study did, however, establish that declining LC steroidogenic responsiveness was not associated with

a decrease in circulating LH. Moreover, these investigators demonstrated that testosterone biosynthesis was compromised at some point beyond the adenylate cyclase-cAMP transduction step. Consistent with these results is the observation that cholesterol esters increase and the activities of cholesterol ester hydrolase and HMG-CoA reductase decline as rats age (Liao et al., 1993). Taken together, these results suggest that as rat LCs age they may increase in number without an increase in LH stimulation, and their ability to mobilize free cholesterol into the mitochondria declines, limiting the production of testosterone.

III. ENDOCRINE AND PARACRINE REGULATION OF THE LC A. Endocrine Regulation of LC Function The most proximate sites in the regulation of testosterone production in rats and humans are the hypothalamus and pituitary (Figure 2) (De Groot et al., 1995b). GnRH is synthesized and secreted by cell bodies in the preoptic and medial basal areas of the hypothalamus, which in turn stimulates the synthesis and release of LH by gonadotropes in the anterior pituitary. To prevent down-regulation (internalization and/or degradation) of the LH receptor on LCs, the levels of circulating LH are pulsatile. Both the upper and lower limits of these LH pulses are determined by the frequency and amplitude of GnRH pulses discharged into the pituitary portal vessels (Everett, 1988). In turn, the frequency and amplitude of the GnRH pulses generated in the medial-basal hypothalamus are regulated by the gonadal steroids testosterone and estrogen. Both testosterone and estrogen receptor-containing neurons have been demonstrated in the preoptic area of the hypothalamus (Page, 1988). The receptors for estrogen appear to be localized in interneurons within the preoptic area rather than co-localization with GnRH neurons (Silverman, 1988). In both rats and humans, testosterone production by LCs is initiated acutely once LH binds to its receptor and activates the Gs protein, which in turn activates adenylate cyclase, resulting in the conversion of ATP to cAMP (Figure 3). Next,

179


FIGURE 2. Diagram illustrating the common pathways of endocrine regulation along the HPT axis. The neurotransmitter-induced synthesis and release of GnRH from the preoptic-medial basal hypothalamus prompts the release of FSH and LH from the anterior pituitary. These gonadotropins exert positive influences on LCs and Sertoli cells, respectively. Increased levels of testosterone produced by the LC and/or estrogen produced via the action of aromatase in either Leydig or Sertoli cells (depending on age), feedback on the hypothalamus, and/or pituitary gland to suppress the release of GnRH, as well as locally to inhibit testosterone biosynthesis within the LC.

180


protein kinase A (PKA) is activated by cAMP, and this results in the phosphorylation of specific proteins that are pivotal to steroidogenesis (Wang and Ascoli, 1990; Moger, 1991). One protein activated by this phosphorylation is cholesterol ester hydrolase (Hall, 1994). Newly synthesized proteins also are requisite for cAMP-induced transcription of both the C17(-hydroxylase/- C17,20-lyase gene and the 3β-hydroxysteroid dehydrogenase gene. In contrast to immature LCs in which testosterone and DHT promote functional differentiation, these androgens appear to exert generally negative effects on the steroidogenic potential of adult LCs. In vitro perfusion of the adult rat testis with testosterone or DHT leads to reduced de novo secretion of testosterone (Darney and Ewing, 1981). In this context, testosterone has been shown to act via the androgen receptor to repress the expression of both the C17a-hydroxylase/C17,20lyase gene and the 3β-hydroxysteroid dehydrogenase gene (Payne et al., 1992). In addition to this mechanism of product inhibition, it has been demonstrated recently that testosterone also inhibits its own biosynthesis via specific, competitive binding to C17a-hydroxylase (Darney et al., 1995). Testosterone production by the LC can be inhibited by mechanisms that compromise the signal transduction pathway as well as those that reduce the activity of steroidogenic enzymes directly. For example, activation of the Gi protein inhibits the production of the second messenger cAMP, which in turn decreases the steroidogenic response (Platts et al., 1988). A number of potential paracrine factors have been shown to modulate both the transduction pathway as well as the activity of the various steroidogenic enzymes, and these are discussed below and are summarized in Table 1. The obligatory role of cAMP as a second messenger has been questioned because of the failure to measure an increase in cAMP. However, this inability appears related to compartmentalization of PKA. Maximal LH-stimulated testosterone production occurs with less than 1% saturation of LH receptors and with no detectable increase in cAMP levels (Cooke, 1990). Minor LH receptor occupancy can be explained by spare receptors required to maintain steroidogenesis during the continuous, pulsatile LH stimulation to

which LCs are exposed under normal physiological conditions (Catt and Dufau, 1973). Failure to elicit an increase in cAMP is best explained by compartmentalization of cAMP-bound PKA (Dufau et al., 1977). Two isoenzymes of adenylate cyclase are present in LCs and binding to each results in a synergistic increase in testosterone production. Moreover, PKA type I has preferential access to cAMP under LH stimulation (Moger, 1991). Although it is clear that cAMP plays a vital role in steroidogenesis, other second messengers have been proposed. Intracellular free calcium, stimulated by GnRH, increases testosterone production without a detectable rise in cAMP (Cooke, 1990). Calcium-calmodulin complexes stimulate steroidogenesis by increasing transport of cholesterol to the inner mitochondrial membrane (Hall et al., 1981). Indeed, it has been demonstrated using Xenopus oocytes that the LH receptor is coupled to different transduction systems, causing both adenylate cyclase activation and increased cAMP, as well as phospholipase C-stimulation of phosphotidyl inositol breakdown and increased calcium release from intracellular stores (Gudermann et al., 1992). It has been demonstrated using rat LCs that significant increases in intracellular calcium and testosterone can occur with less LH stimulation than that required to elicit a significant increase in cAMP (Sullivan and Cooke, 1986). Thus, it appears that the two signal transduction pathways may work synergistically to potentiate LH-stimulated testosterone production. However, although a role for the phosphotidyl inositol pathway is indicated, the phospholipase C-stimulated formation of inositol triphosphate and diacylglyerol has not been clearly established in rat LCs. Moreover, cAMP itself can increase cytosolic calcium levels (Sullivan and Cooke, 1986). Although protein kinase C is present in LCs, a role for its activation in the physiological regulation of LC steroidogenesis remains questionable. Studies do suggest, however, that continuous release of arachidonic acid within the LC stimulates protein kinase C, which in turn inhibits testosterone biosynthesis (Lopez-Ruiz et al., 1992). Thus, if protein kinase C does have a physiological role, it most likely functions in a tonic fashion to suppress testosterone production.

181

182


183

FIGURE 3. Diagram of the predominant mechanisms of testosterone biosynthesis within the LC. LH receptor binding results in activation of adenylate cyclase, and the cAMP-induced activation of PKA. Once activated, PKA modifies or activates proteins involved in the mobilization of cholesterol to the inner mitochondrial membrane, as well as induces transcription of steroidogenic enzymes. The transcription of some of these enzymes is inhibited when excess testosterones bind to the androgen receptor. Binding of LH to its receptor also stimulates the action of membrane-bound phospholipase C on phosphotidyl inositol, forming diacylglyceral and inositol triphosphate. Inositol triphosphate interacts with calcium channels on the outer membrane of the SER to facilitate the release of calcium from this organelle. The activity of adenylate cyclase also can be inhibited if Gi protein is activated. Abbreviations: LH, luteinizing hormone; I-PKA/A-PKA, inactivated or activated protein kinase A; PI, phosphotidyl inositol; DAG, diacylglycerol; ITP, inositol triphosphate; IL-1, interleukin 1; CEH, cholesterol ester hydrolase; StAR, Steroidogenic acute regulatory protein; DBI, diazepam binding inhibitor; PDR = peripheral-type benzodiazepine receptor; SAP, sterol activating protein; SCC, side chain cleavage enzyme; C17 H/C17,20 L, C17a-hydroxlase/C17,20-lyase; 3b-HSD/17b-HSD, 3β or 17β-hydroxysteroid dehydrogenase; AR, androgen receptor; PREG, pregnenolone; OH-PROG, 17-hydroxyprogesterone; ADIONE, androstenedione; T, testosterone.


184


185

FIGURE 4. Diagram of the predominant testosterone biosynthetic pathways in humans and rats. The predominant pathways in humans is represented by heavy dashed lines. Pregnenolone undergoes a series of ∆5 steroid conversions until dehydroepiandrosterone is formed and coverted to the ∆4 steroid androstenedione. The predominant pathway in rats is represented by solid lines. Pregnenolone is converted to the ∆4 steroid progesterone, which then undergoes a series of ∆4 steroid conversions. Minor conversions are represented by dotted lines.



Esterified cholesterol is sequestered within LCs in the form of lipid droplets. While lipid droplets are sparse in the adult rat, they are common in adult human and mouse LCs. This may have a bearing on the fact that less than 5% of the cholesterol in the rat testis is esterified (Van der Molen et al., 1972). LCs can also synthesize cholesterol de novo from acetate or mobilize cholesterol from blood-borne lipoprotein (Hall, 1994). Lipoprotein derived from the circulation, presumably becomes incorporated into the plasma membrane that then serves as the cellular source of cholesterol (Freeman, 1989). The internalization of lipoprotein also has been demonstrated (Benahmed et al., 1983). The type of lipoprotein that is utilized appears to depend on the species. Mouse MA-10 tumor LCs utilize low-density lipoprotein (Freeman and Ascoli, 1983), while rat LCs prefer high- rather than low-density lipoprotein to potentiate LH-stimulated testosterone production (Klinefelter and Ewing, 1988). Cholesterol ester hydrolase, phosphorylated by LH-stimulated PKA, deesterifies cholesterol esters to provide free cholesterol for transport to the mitochondria. This transport of cholesterol involves both microfilaments and intermediate filaments. Sterol carrier protein (SCP-2) transports cholesterol to the inner mitochondrial membrane to bring it in close proximity to the cytochrome P-450 (P450), C27-side chain cleavage enzyme (Vahouny et al., 1983). This protein has been localized within peroxisomes of the LC, and both the peroxisome volume and the peroxisome SCP-2 content are regulated acutely by LH (Chamindrani Mendis-Handagama et al., 1990). Endozepine (Besman et al., 1989) or diazepam binding inhibitor (DBI) binds to peripheraltype benzodiazapine receptors (PBR) to further assist in the rate-limiting movement of cholesterol from the outer to the inner mitochondrial membrane (Papadopoulos et al., 1990). It appears that LH regulates posttranslational activation of DBI to stimulate steroidogenesis (Papadopoulos et al., 1991; Cavallaro et al., 1992). It also has been speculated that this peptide accelerates C27-side chain cleavage enzyme activity directly by loading the enzyme with substrate (Hall, 1994). Although this benzodiazapine-mediated event appears to be important, different benzodiazapines have either stimulatory or inhibitory effects on 186

steroidogenesis (Garnier et al., 1993). Thus, the relative importance of the PBR-DBI receptorligand complex in regulating LC steroidogenesis under physiological conditions is unclear. There is no question that LH plays a role in the ratelimiting mobilization of cholesterol from the outer mitochodrial membrane to the inner mitochondrial membrane, and the mechanism by which it mediates this response is currently being elucidated (Stocco, 1997). Recently, the cDNA for a novel protein termed Steroidogenic Acute Regulatory Protein (StAR) has been identified in MA-10 tumor LCs (Clark et al., 1994) and human testicular tissue (Lin et al., 1995). The expression of this protein is LH dependent, and once synthesized, it appears that StAR intercalates between the outer and inner mitochondrial membranes to shuttle free cholesterol to the C27-side chain cleavage enzyme. Although this protein is induced by LH, expression of StAR in the absence of LH stimulation maintains steroidogenesis. It now is believed that the newly synthesized 37-kDa StAR protein is chaperoned to the outer mitochondrial membrane where it is processed to a 32-kDa molecule as contact occurs between the outer and inner mitochondrial membranes. Presumably, it is during this membrane fusion that cholesterol is transferred to the inner membrane (Stocco, 1997). Regardless of the mechanism(s), once cholesterol is mobilized to the inner mitochondrial membrane and positioned next to the cholesterol C27-side chain cleavage enzyme, an LH-dependent protein termed sterol-activating protein is thought to facilitate the binding of cholesterol to the P450 C27-side chain cleavage enzyme (Pederson, 1984). Once formed, the C21 steroid pregnenolone enters the SER. Here pregnenolone is converted via the action of a series of microsomal enzymes to testosterone. Within the SER either one of two steroidogenic pathways predominate, depending on the species. In the rat, the ∆4 pathway predominates (Ewing et al., 1980) and in man the ∆5 pathway predominates (Weuesten et al., 1987). In the rat, pregnenolone is converted to progesterone by the action of 3β-hydroxysteroid dehydrogenase ∆5∆4 isomerase. Next, the P450 enzyme, C17a-hydroxylase/C17,20-lyase, catalyzes two reactions: conversion of progesterone to hydroxyprogesterone followed by cleavage at the C17–20 bond


to form the C19 steroid, androstenedione. Finally, 17β-hydroxysteroid dehydrogenase catalyzes the conversion of androstenedione to testosterone. In the human testis, the activity of 3β-hydroxysteroid dehydrogenase ∆5∆4 isomerase is less, favoring the formation of the ∆ 5 precursors: 17αhydroxypregnenolone, dehydroepiandrosterone, and androstenediol. In addition, two other enzymes play key roles in LC function. The P450 aromatase catalyzes the conversion of testosterone to estrogen under the influence of LH in both adult rat and human LCs (Valladares and Payne, 1979a,b; Payne et al., 1976). Aromatase activity also is found in adipose tissue, liver, and skeletal muscle (Lephart and Simpson, 1991; Longcope et al., 1978). In males, the majority of circulating estradiol is probably synthesized in adipose tissue, although additional sites of synthesis such as bone are being elucidated. Estrogen exerts rapid negative effects on the activity of C17α-hydroxylase in adult rat LCs, an effect that can be triggered by overstimulation with LH or hCG and that appears to be receptor mediated (reviewed in Sharpe, 1982). As a consequence, the steroidogenic capacity of the LC is blunted. As discussed below, paracrine factors originating from the seminiferous tubule may be responsible for modulating aromatase activity within the LC. A discussion of testosterone regulation would be remiss without mention of the ability of glucocorticoids, elevated after stress, to suppress testosterone production in rats by directly inhibiting the activity of the key P450 steroidogenic enzymes: C27-side chain cleavage, C17α-hydroxylase, and C17,20-lyase (Hales and Payne, 1989; Klinefelter et al., 1996). The LC contains 11β-hydroxysteroid dehydrogenase (11β-HSD) that, depending on whether it is type I or type II, interconverts active glucocorticoid to inactive (or vice versa), which could be important in limiting inhibition of testosterone biosynthesis by glucocorticoids (Monder et al., 1994). In addition, glucocorticoids can also induce 11β-HSD, which can attenuate the glucocorticoid inhibition of testosterone production (Klinefelter et al., 1996). Because the appearance of 11β-HSD occurs during puberty, it is possible that glucocorticoids might be involved in the pubertal expression of steroidogenic enzymes and the subsequent rise in testosterone levels during puberty (Engel and Frowein, 1974). It is clear that

trophic LH stimulation regulates the amount of SER within the LC and that this is tightly correlated with LH-induced changes in LC volume and testosterone production (Zirkin et al., 1982; Keeney et al., 1988). Moreover, LH regulates both the activity of the P450 C17-hydroxylase/C17,20lyase located within the SER (Wing et al., 1984) and the amount of this microsomal enzyme (Klinefelter et al., 1987). Presumably, LH plays a role in the synthesis and/or degradation of P450 C17-hydroxylase/C17,20-lyase. Because LH does not appear to influence the activities of other enzymes in the smooth endoplasmic reticulum, P450 C17-hydroxylase/C17,20-lyase is rate limiting in the conversion of pregnenolone to testosterone (Wing et al., 1984). Interestingly, although long-term LH deprivation fails to maintain LC size and function, LC number is not reduced. Thus, LH is not required to maintain the number of adult LCs in the rat (Keeney et al., 1990). Although physiological concentrations of LH are required to maintain LH receptor number, increased LH or hCG reduces the number of available receptors and testosterone production becomes refractory (Catt et al., 1980). The time frame for this down-regulation is minutes in MA10 tumor LCs and hours in rat LCs. The fact that LH promotes cAMP-induced proteolytic cleavage may account for the initial phase of LH receptor down-regulation. In addition, LH/hCG stimulation results in a decrease in LH receptor mRNA. This decrease appears to be due to decreased transcription in MA-10 cells and increased degradation of the mRNA in rat LCs. The longer time required for down-regulation in the rat may be explained by recycling of the receptor back to the surface after initial internalization (Saez, 1994). Aside from LH-induced down-regulation of the LH receptor, LH-induced desensitization also occurs. With desensitization, the LH receptor-G protein complex becomes uncoupled, perhaps due to PKA regulated phosphorylation of the LH receptor itself (Saez et al., 1978). However, it must be noted that data regarding both down-regulation and desensitization should be viewed as pharmacological responses because LH stimulation of the LC in vivo is pulsatile not tonic, and the normal stimulating concentration of LH in vivo is physiological, not supraphysiological. To date, no in vitro system has successfully mimicked the 187

physiological conditions of LC stimulation by LH.


B. Paracrine Regulation of LC Function Thus far, we have focused on LH regulation of LC function. During recent years extensive work has been done to elucidate the role of other regulators of LC function. Two excellent reviews on the role of gonadal peptides in mediating the development and function of the testis summarize much of this work (Saez, 1994; Gnessi et al., 1997). While it is clear that normal functioning of the LCs is absolutely dependent on an appropriate endocrine environment within the testis, primarily that provided by LH stimulation, the paracrine environment also plays an important role. After disruption of the testis, and thus the paracrine environment, specific alterations in LC number, morphology, or testosterone production must occur to maintain intratesticular and circulating testosterone levels. For example, if the seminiferous tubules shrink in size due to experimentally induced loss of germ cells (Setchell and Galil, 1983), and there is a concomitant reduction in the transport of LH into the testis, testosterone production by the LCs decreases. These changes trigger either compensatory changes in LH levels and/or changes in LC number or size in order to restore steady-state testosterone levels. The ultimate, triggered response may differ depending on the age, species, and strain of the animal. The purpose of the following section is to review such differences and identify those that could be relevant to the ontogeny of LC hyperplasia and tumors.

1. LC Changes After Disruption of Spermatogenesis The first reports that induction of seminiferous tubule damage or cryptorchidism could lead to hyperplasia of LCs were noted from around the turn of the century (Maximov, 1899; Moore, 1924). These and later findings showing that cadmiuminduced testicular atrophy in rats and mice resulted in a high incidence of LCTs (Roe, 1964) fostered the belief that any form of seminiferous tubule damage resulted in an increase in LC num188

ber. Studies involving induction of focal damage to the seminiferous tubules of rats (Aoki and Fawcett, 1978) reinforced this belief. However, when morphometric measurements were applied, they mostly revealed that the LCs in testes from rats with seminiferous tubule damage had undergone hypertrophy and not hyperplasia (reviewed in Sharpe, 1993). In particular, the number and size of mitochondria and the volume of SER were increased, with corresponding increases in the capacity of these LCs to secrete testosterone in vitro when stimulated with LH (Kerr et al., 1979; Rich et al., 1979; Risbridger et al., 1981a,b,c). These changes were not simply a consequence of the raised LH (or FSH) levels that often accompany seminiferous tubule damage, as the changes in LC morphology were restricted to the “damaged” testis when this was induced unilaterally (Risbridger et al., 1981a,c) and preceded any elevation of gonadotropin levels. Thus, in the adult rat testis, the primary response to seminiferous tubule damage (i.e., germ cell loss) is hypertrophy of the steroidogenic organelles in mature LCs adjacent to the damaged tubules (Sharpe, 1993). There is no obvious change in LC number (Kerr and Sharpe, 1989b). Evidence from the bull (Schanbacher, 1979) and ram (Schanbacher, 1980; Byers, 1984; Byers and Glover, 1984) suggest that similar LC changes may occur in these species after induction of seminiferous tubule damage, but in the adult mouse there is a dramatically different response (MendisHandagama et al., 1990a,b; 1991). Instead of hypertrophy, the LCs shrink by more than half in size and in their cytoplasmic organelle content (including mitochondria and SER). To compensate for these changes, the number of LCs approximately doubles. These responses to seminiferous tubule damage are the opposite of those in the rat and suggest that different species may utilize different paracrine pathways to ensure steady-state levels of testosterone. However, there is an additional but important qualification to this general conclusion. All of the data discussed derive from studies in which seminiferous tubule damage has been induced experimentally in normal adult animals. If newborn rats are rendered unilaterally cryptorchid and the testis is examined in adulthood, the abdominal testis will have similar LC numbers to the contralateral scrotal testis,


but these cells are reduced significantly in size and have reduced amounts of SER and impaired steroidogenic potential (Bergh and Damber, 1978, Bergh et al., 1984). These changes are almost the opposite of those resulting from induction of cryptorchidism in the adult rat, implying that age or the stage of testicular development are also important in determining the LC response to seminiferous tubule damage. Similar to the rat, hyperplasia of the LCs have been reported in humans in various situations of seminiferous tubule damage (Dubin and Hotchkiss, 1969; Kothari and Gupta, 1974, Chemes et al., 1977; Guay et al., 1977; Honore, 1979, Agger and Johnsen, 1978) or following prolonged hCG treatment (Maddock and Nelson, 1952). However, when objective cell-counting methods have been used, the conclusion has been that hyperplasia does not occur in any of these situations but rather hypertrophy has occurred (Ahmad et al., 1971; Heller and Leach, 1971; Weiss et al., 1979; Neaves et al., 1984, 1985).

2. Potential Paracine Regulators of LC Number a. Insulin-Like Growth Factor-l (IGF-1) During puberty, the blood levels of growth hormone and IGF-1 increase substantially at around the same time as the increase in FSH and LH levels (see Daughaday and Rotwein, 1989) and the period of LC proliferation. In addition to IGF-1 derived from the blood, IGF-1 is produced in the testis by Sertoli and LCs and probably other testicular cells, including germ cells (Table 1). Secretion of IGF-1 by Sertoli and LCs can be stimulated in vitro by FSH and LH, respectively (Cailleau et al., 1990, Naville et al., 1990), and the level of mRNA for IGF-1 can be similarly enhanced in vivo (Closset et al., 1989; Lin et al., 1990; Spiteri-Grech and Nieschlag, 1993). Receptors for IGF-1 have been demonstrated in LCs from several species, including man, and the number of these receptors can also be enhanced by treatment with LH or hCG (Table 1). Exposure of LCs to IGF-1 can increase the levels of several of the key steroidogenic enzymes (notably 3β-HSD) and, accordingly, can enhance LH-stimulated tes-

tosterone production (Table 1). Of perhaps most significance is the observation that IGF-1 has a greater stimulatory effect on LH-stimulated androgen secretion by pubertal (i.e., immature) LCs than by adult LCs (Hardy et al., 1991; Gelber et al., 1992). Finally, IGF-1 can exert small mitogenic effects on LCs, although probably only when acting in concert with other hormones/growth factors (Table 1). Viewed as a whole, the data would suggest that IGF-1 is involved in promoting functional differentiation of adult type LCs (Saez, 1994; Teerds et al., 1994), and IGF-1 derived from both peripheral and intratesticular sources may play this role during puberty.

b. Transforming Growth Factor-α (TGFα) Pubertal expansion of the adult LC population in the rat is associated with increased immunoexpression of TGFα in the LCs (Teerds et al., 1990b) and addition of TGFα to immature rat LCs in vitro can stimulate [3H]thymidine incorporation to a small but significant extent, both in the absence and presence of LH (Table 1). However, this effect of TGFα is enhanced greatly when both LH and IGF-1 are present. The fact that Sertoli cells and peritubular cells from immature rats also produce TGFα and that receptors for TGFα are present on LCs from a number of species (Table 1) raises the possibility that TGFα from the seminiferous tubules could also participate in the potential effects of this growth factor on developing LCs. Taken together, the data suggest that the pubertal coincidence of raised IGF-1 and TGFα levels could be key interactive factors in the development of the adult LC population. The situation may be very different in the adult rat as addition of EGF (closely related to TGFα to isolated adult LCs results in a major reduction in DNA synthesis (Table 1). If IGF-1 and TGFα can be considered as stimulators of LC development, and possibly function, which of the paracrine factors provide the inevitable counterbalancing force? Reasonably strong cases can be made for three growth factors, namely, transforming growth factor-β (TGFβ), basic fibroblast growth factor (bFGF), and interleukin-1 (IL-1). In considering the potential effects of these factors it should be kept in mind, 189

however, that there is already evidence for multiple forms of each of these factors and their corresponding receptors (Saez, 1994), and this complexity is likely to increase. Therefore, it may prove that the discussion below is an oversimplification.


c. Transforming Growth Factor-β (TGF β) In the same way that IGF-1 and, to a lesser extent TGFα, are functional stimulators of adult LC differentiation, TGFβ appears to act in the reverse manner. Expression of TGFβ1 as detected immunohistochemically in LCs declines progressively during puberty (Teerds and Dorrington, 1993), being the mirror image of TGFα (Teerds et al., 1990b). Moreover, addition of TGFβ to immature rat LCs in vitro is able to antagonize the combined stimulatory effects of IGF-1 and TGFα on [3H]thymidine incorporation (Table 1). Virtually all of the available evidence suggests that TGFβ inhibits testosterone secretion by LCs in a number of species, as well as by immature LCs (Table 1). TGFβ also reduces the number of LC LH receptors in a dose-dependent manner (Avallet et al., 1987) and may affect the activity of certain steroidogenic enzymes (Saez, 1994).

d. Basic Fibroblast Growth Factor (bFGF) bFGF is a member of a growing family of growth factors that appear to play pervasive roles in stimulating replication and differentiation of cells from mesodermal tissues (Gospodarowicz, 1990). bFGF appears to be produced by Sertoli, germ, peritubular, and/or LCs in a number of species (Table 1), and, importantly, its production by immature rat Sertoli cells is stimulated by FSH (Mullaney and Skinner, 1992). Immature LCs possess receptors for bFGF, and addition of bFGF to these cells in vitro inhibits LH-stimulated testosterone production, probably by decreasing the activity of 3β-HSD and 17α-hydroxylase (Table 1), although enhanced activity of aromatase (Raeside et al., 1988) and reduction in the number of LH receptors could also be involved. A further observation of interest is that bFGF inhibits 5αreductase activity in immature rat LCs (Murono 190

and Washburn, 1990b), and, as production of bFGF by Sertoli cells is stimulated by FSH (Mullaney and Skinner, 1992), it raises the possibility of its involvement in the pubertal decrease in activity of this enzyme, as described earlier.

e. Interleukin-1 (IL-1) Both IL-1α and IL-1β are produced within the adult testis of several species, including man. The general consensus is that they exert inhibitory effects on LH-stimulated LC steroidogenesis, irrespective of whether the LCs are derived from immature or adult rats, immature pigs, or adult mice (Table 1). Moreover, there is convincing evidence for a positive effect of IL-1β on LC numbers. Thus, addition of IL-1β to LCs from immature rats (aged 10 or 20 days) caused dosedependent stimulation of [3H]thymidine incorporation, an effect that was exacerbated in the presence of other stimulators of [3H]thymidine uptake (e.g., hCG or TGFα + IGF-1); however, IL-1β had no effect on LCs from adult rats (Khan et al., 1992b). As IL-1α was far less effective in inducing immature LC DNA synthesis, the authors concluded that IL-1β from testicular macrophages perhaps played a role in regulating the pubertal proliferation of LCs. Two other pieces of evidence lend this suggestion strong support. First, the number of testicular macrophages increases more or less in parallel with LC number during puberty in the rat (Hardy et al., 1989). Second, regeneration of LCs in the EDS-treated rat is prevented by depletion of testicular macrophages (Gaytan et al., 1994ab). With these observations in mind, perhaps some of the contradictory or equivocal observations reported for the effects of IL-1α and IL-1β on LC steroidogenesis in vitro (see above; Sharpe, 1993; Saez, 1994) may be related to the age of the donor animals and/or the degree of macrophage contamination of the LC preparations. The intratesticular source of IL-1α and IL-1β is also more complex than was originally supposed. Initial findings demonstrated that large amounts of IL-1α were present in the rat and human testis (Khan et al., 1987, 1988) and that this IL-1α was secreted in increasing amounts by isolated seminiferous tubules during puberty in


the rat (Syed et al., 1988), and, in adulthood, secretion was stage-dependent (Soder et al., 1991). The secreted IL-1α is detectable in testicular interstitial fluid (Gustafsson et al., 1988), and it is clear that Sertoli cells secrete IL-1α although the mRNA for IL-1α is also present in LCs (Table 1). As the levels of testicular IL-1 (α and β) are reduced by hypophysectomy and can be restored by treatment with hCG but not by FSH or testosterone (Soder et al., 1988; von Euler et al., 1989), it is likely that LCs are the origin of this IL-1. However, these studies did not distinguish between IL-1α and IL-1β, and a number of pieces of information suggest that it was the latter that may have predominated. LCs express IL-1β mRNA at higher levels than IL-1α and expression is stimulated by hCG (Table 1). Testicular macrophages are also an important source of IL-1α, and stimulation of the mRNA for this factor following phagocytosis-induced activation in mice in vivo resulted in a marked but reversible decrease in the mRNA for C17a-hydroxylase in LCs (Hales et al., 1992). The same author subsequently demonstrated that IL-1α is able to suppress C17a-hydroxylase and testosterone production by mouse LCs in vitro, although this study and others using LCs from other species also show that cholesterol transport and/or C27-side chain cleavage can be inhibited by IL-1 (Table 1). To complicate the picture further, germ cells have now also been identified as another source of IL-1α in the rat (Table 1).

adult population of LCs is developing. Although effects of inhibin on LCs are equivocal (Mather and Krummem, 1992), selected deletion of the α-inhibin gene in mice results in the development of Sertoli cell and granulosa cell tumors in all of the male and female offspring, respectively (Matzuk et al., 1992), implying a function for inhibin, or at least the α-subunit, in controlling growth and differentiation. It is possible that these effects could extend to the LCs in certain situations, particularly as it is established that experimental induction of seminiferous tubule damage (i.e., germ cell loss) in rats results in increased secretion of inhibin into the interstitium (Sharpe, 1988). Moreover, normal adult rat LCs and two different LCT-derived cell lines from rats (Mather and Krummen, 1992), all express the inhibin α-subunit, as do LCs and LCTs in man (Bergh and Cajander, 1990). Although the general consensus is that adult LCs probably do not secrete dimeric inhibin, this needs to be confirmed using the latest specific assays. Activin, but not inhibin, is secreted by LCs from immature rats and pigs, but this ability is lost early during puberty (Lee et al., 1989; Mather and Krummen, 1992). Activin can affect LC steroidogenesis (Lin et al., 1989), although there is rather conflicting evidence as to the nature of this effect (see Mather and Krummen, 1992; Saez, 1994). As both activin (Mather et al., 1990) and inhibin (van Dissel Emiliani et al., 1989) appear able to affect spermatogonial proliferation, growth effects on the LCs also remains a possibility.

f. Inhibin and Activin This family of peptides, which have homology to the transforming growth factors, are produced within the testis and may exert a number of paracrine effects as well as the FSH-suppressing function associated more traditionally with inhibin. This area is presently in a state of flux because of uncertainties about what activity is being measured in inhibin assays, and so the following comments should be considered with this reservation in mind. Dimeric inhibin is produced mainly by Sertoli cells in the testis under FSH stimulatory control (Ying, 1988), and secretion of inhibin into the interstitium declines exponentially during puberty (Maddocks and Sharpe, 1990) when the

C. Potential Paracrine Regulators of LC Testosterone Production One of the most perplexing problems relating to the physiological roles in the testis of the growth factors described above is that they appear to originate from more than one cell type in the testis (often including the LCs themselves) and/or are present in the systemic circulation. Indeed, with the exception of TGFα (Skinner et al., 1989), all of the growth factors discussed are also produced by one or more meiotic/post-meiotic germ cell types (Table 1). This could be coincidence, but it might also be significant. For example, it is possible that by being responsive to growth factors 191


that may originate from multiple intratesticular and extratesticular origins, the LC is merely condensing the information from many sources to a single final message that will govern its response. In this way, the LC could be said to be responding to the broadest spectrum of changes within the body, a feature that is perhaps important for a cell type, the products of which have both vital endocrine and paracrine functions. This might also explain why such an extraordinary range of chemicals and drugs are able to induce LC changes in the rat (Section VII).

IV. ROLE OF LH IN LC TUMORIGENESIS Although there is compelling evidence that local damage to the seminiferous tubule in the rat results in juxtaposed areas of LC hyperplasia (see below), there also is an increased volume of cytoplasmic organelles that is similar to that observed in LCs exposed to elevated LH for an extended period (Zirkin et al., 1980). Therefore, an endocrine imbalance also may be the underlying cause of LC hyperplasia in conditions of seminiferous tubule damage. Interestingly, as stated above, LCs in the testes of aging beagles have been shown to undergo both atrophy and hyperplasia, and these alterations were associated with increased circulating levels of LH (Ewing et al., 1987). In the rat, conditions that increase levels of LH/hCG or GnRH have the potential to cause LC hyperplasia (Walsh, 1977). LC hyperplasia results when testicular parenchyma is implanted into castrate animals with an intact hypothalamic-pituitary axis, due to the hypersecretion of LH (Biskind and Biskind, 1945; Twombly et al., 1949). Exposure to antiandrogens such as flutamide, which blocks testosterone feedback at the level of the hypothalamo-pituitary axis, also causes hypersecretion of LH and LC hyperplasia and tumors (Viguier-Martinez et al., 1983a,b; PDR, 1995d). A diverse range of compounds such as the antimicrobial agent oxolinic acid (Yamada et al., 1994), the herbicide linuron (Cook et al., 1993), the calcium channel blocker SDZ-110 (Roberts et al., 1989), and the antacid lansoprazole (Fort et al., 1995) also have been shown to induce LCTs in the rat. In each of these cases, an increased secretion of LH has been found (Section VII). 192

While the percentage of LCs that undergo mitosis in an intact animal is extremely low (less than 0.1%), exogenous LH, but not FSH, appears capable of stimulating LC division (Keeney et al., 1990). Interestingly, LH or hCG has been shown to result in a transient elevation in expression of protooncogenes in vitro (Lin et al., 1988; Czerwiec et al., 1989). This might provide insight into the early mechanism of LH-induced mitogenesis in a relatively static LC population. Such a mechanism might be anticipated to lack species specificity. Indeed, chronically elevated LH levels may be pivotal in the induction of human LC hyperplasia (Jockenhovel et al., 1993). Interestingly, however, while hyperplastic rat LCs retain the morphological features of adult rat LCs, hyperplastic human LCs resemble fetal LCs (Pelliniemi et al., 1980). Finally, recent studies have shown that experimental manipulation of the thyroid during neonatal development can manifest significant changes in both the Sertoli cell and LC populations. Indeed, induction of neonatal hypothyroidism in rats produces a 40 to 70% increase in the Sertoli cell population, as well as a parallel increase in the LC population in adulthood (Hardy et al., 1993). Surprisingly, the increased number of LCs occurs in the presence of subnormal levels of LH and FSH (Kirby et al., 1992), and the testosterone produced in vitro by these adult LCs under conditions of maximum LH stimulation is roughly half that produced by normal adult LCs. Similarly, it is interesting that there is an increased incidence of LCTs in the aged rat, when circulating LH levels are reduced. Because compromised LH responsiveness occurs with age in humans (Chen et al., 1994), one wonders what role a sustained reduction in LH responsivity might play in LC tumorigenesis. These data suggest that nongonadotropin components of the endocrine milieu during reproductive development are critical in determining the Leydig and Sertoli cell populations.

V. ROLE OF PARACRINE FACTORS IN LC TUMORIGENESIS Although there is considerable evidence supporting the notion that LH promotes LC hyper-


plasia and adenomas, there are other data suggesting that hyperplasia may be also influenced by factors produced by the seminiferous tubules. Much of this evidence, relating to the induction of seminiferous tubule damage, has already been summarized above, but there is other data demonstrating the production of LC mitogenic factors by isolated seminiferous tubules and Sertoli cells. The study by Ojeifo et al. (1990) showed that Sertoli cells isolated from 19-day-old immature rats produce a protein of 30 kDa, under FSH and testosterone control, which exerts a major effect on DNA synthesis by isolated, purified LCs. This effect was much greater on LCs from immature rats (16 to 26 days of age) than those from adult rats and was dose dependent. Ojeifo et al. (1990) also found that basal DNA synthesis in LCs from rats aged 16 to 26 days was 5- to 12-fold higher than when LCs were isolated from rats aged 36 to 90 days. These findings fit well with LC replication in vivo during puberty, which is maximal at 24 to 28 days (Hardy et al., 1989; Russell et al., 1995), and the increase in LC numbers, which is highest at 21 to 28 days and lowest in animals aged 40 days or older (Hardy et al., 1989; Russell et al., 1995). These findings have been confirmed and extended by a recent study that demonstrated that although Sertoli cells from both immature and adult rats secreted a factor thtat stimulated DNA synthesis and proliferation of immature LCs, adult LCs were refractory to this factor (Wu and Murono, 1994). It is also reasonable to presume that the numerous studies of immature, hypophysectomized rats in which FSH administration has been shown to increase LC number and responsiveness to LH (see above) involves similar pathways. Interstitial fluid (IF) collected from adult rats clearly contains mitogenic activity (Drummond et al., 1988; Risbridger et al., 1987; Ojeifo et al., 1990), but this activity is unchanged in the first 2 weeks after EDS treatment (when proliferation of LC precursors is occurring [see above]) or after experimental cryptorchidism (when LC hypertrophy is occurring, see above). Presumably, the mitogen present in adult testicular IF is different from that which exerts the effects just described during puberty, although no studies of mitogenic activity in IF collected from immature rats have been reported.

It seems likely that paracrine factors are involved in the etiology of LCTs in rats, either as primary factors or secondary to changes in gonadotropin levels, blood flow, or spermatogenesis. From the data discussed above, there is clearly no shortage of potential pathways that could lead to tumor formation if production of one or more of the normal stimulators or inhibitors of LC development should change abnormally. Unfortunately, there is no direct information that points to where this failure might be, although it is conceivable that proliferation of LC precursors or of fully formed LCs could be involved. It appears reasonably certain that proliferation of these two points of the LC lineage are differentially regulated (Russell et al., 1995). Proliferation of LC precursors is LH independent, whereas that of immature and adult LCs is LH dependent. However, both pathways clearly involve paracrine input from the seminiferous tubules, presumably from the Sertoli cells, but again it seems likely that separate growth factors are involved. LCTs in rats are not usually induced rapidly, but appear only over a long period of time, whether occurring naturally in old age or as the result of chemical/drug treatment (Section VII). This is very different from the pattern of LC proliferation and development that occurs during fetal life, puberty, or after EDS treatment, where the changes are confined to a matter of weeks and occur in the presence of fairly dramatic endocrine hormone changes. This may indicate that the etiological paracrine changes associated with the early stages of LC hyperplasia and tumor formation are rather subtle and therefore difficult to detect. Alternatively, if a sudden change in a key parameter should occur (e.g., reduced testosterone production by LCs), it may be compensated for within a relatively short time span (e.g., by moderate LC hypertrophy and hyperresponsiveness, increased LH secretion, or LH pulse frequency) such that no indication of the original disturbance remains. Continuous resetting of the homeostatic mechanisms in this way during chronic exposure to a chemical could eventually lead to tumor formation, perhaps in association with the normal agerelated failure or decline of other paracrine pathways regulating LC function. Changes in testicular paracrine pathways are certain to occur in the laboratory rat during aging 193


because of the marked decline in LH levels, the increase in incidence of atrophic seminiferous tubules, and the tendency toward inhibited steroidogenesis. As a consequence, the normal process of LC renewal in an aged rat takes place in a radically different environment from that occurring at any other time of life. In view of the multiple paracrine pathways that are used to regulate LC number and function, adjustments in one or more of these pathways seems plausible. Whatever these are, they clearly predispose the testis toward uncontrolled proliferation of LCs. In this respect, other pieces of information are potentially significant. First, cadmium-induced atrophy of the testis results in an organ virtually devoid of Sertoli and germ cells, and these testes frequently go on to develop LCTs (reviewed in Waalkes et al., 1992) (Section VII), implying that absence of the Sertoli cells and/or germ cells is conducive to LC hyperplasia. Second, in the normal adult rat testis it is remarkable that the Sertoli cell:LC ratio is kept constant, even when the number of Sertoli cells is increased substantially (Kirby et al., 1992) by experimental manipulation during neonatal life. Finally, if the LCs are destroyed with EDS, they regenerate until the normal number is restored (Sharpe et al., 1990). In each of these situations testosterone levels are held steady or are returned to normal levels within a short period (i.e., EDS), by altering LH levels, LC numbers, LC size, LC responsiveness to LH, or a combination of the parameters. This must mean that the Sertoli cells and/or germ cells are regulating the LC supply by the balanced secretion of stimulators and inhibitors of cell division and differentiation; several candidates for these roles have been described above. Presumably, imbalance between the positive and negative regulators occurs with advancing age and, at least in the case of rats treated with cadmium, it must be the absence of inhibitory regulators that are important. It also seems likely that alteration in the hormonal milieu within the testis of rats with advancing age plays a role in LCT initiation (i.e., hyperplasia). Increased secretion of progesterone occurs with most LCTs from Fischer 344 rats (Amador et al., 1985; Bartke et al., 1985; Teerds et al., 1991), and during aging increased secretion of progesterone and decreased secretion of testoster194

one are characteristic (Kaler and Neaves, 1981; Gruenewald et al., 1992). This indicates a selective deficiency of P450 C17a-hydroxylase/C17,20lyase, an enzyme complex that is clearly regulated by paracrine factors from the Sertoli cells as well as by androgens and estrogens (see above). This deficiency would lead to mild impairment of spermatogenesis because of the decreased testosterone secretion (Sharpe, 1994), reduced secretion of LH (and probably FSH because of the negative feedback effects of the raised progesterone (e.g., Gruenewald et al., 1992), and alterations in the steroid-signaling pathways to developing LCs. For example, because lower levels of androgens are produced in aged rats, less substrate is available for aromatization and thus less estrogen may be produced to restrain development of new LCs. One or more such changes could lay the foundations for LCT development. The potential importance of this apparent agerelated deficiency in converting progesterone to testosterone is illustrated further by studies of the Brown Norway rat. This species, in contrast to the common strains of rat used in toxicology studies, does not develop LCTs in old age, yet still shows a decline in testosterone levels due to decreased LC responsiveness to LH (Zirkin et al., 1993; Chen et al., 1994), but shows no selective increase in progesterone levels (Gruenewald et al., 1994).

VI. PATHOLOGY OF LCTS The most frequently encountered neoplasm of the testis of the rat and mouse is the LC adenoma. The incidence rate varies according to strain, ranging from 1 to 5% in Sprague-Dawley rats to nearly 100% in the F344 rats (Table 2). Early neoplasms are commonly encountered in 1-year-old F344 rats and become increasingly more frequent with age (Boorman et al., 1990). Testicular neoplasia is less frequently observed in all strains of mice. In the mouse the reported incidence of LC adenoma ranges from less than 1 to 2.5% (Table 3). In the beagle dog, the spontaneous incidences of LC hyperplasia and LCTs are 8.3 and 6.3%, respectively, at 7.75 years of age (James and Heywood, 1979). Tumors of the testes comprise 1% of all human male neoplasms


TABLE 2 Incidence of LCTs of the Testes in Male Rates for Studies Terminated at 24 Months Strain

Diagnosis

Incidence

(%)

Ref.

Wistar Crl:CD®BR Sprague-Dawley F344/DuCrj Crl:CD®BR Sprague-Dawley

Adenoma Adenoma Adenoma Adenoma Adenoma Adenoma Carcinoma Adenoma Adenoma Adenoma Carcinoma Adenoma Adenoma

87/1249 37/721 16/349 537/569 38/585 11/1340 1/1340 27/685 39,253/51,230 59/1260 1/1260 741/946 29/496

7.0 5.1 4.6 94.4 6.5 0.82 0.07 3.94 76.6 4.68 0.08 78.3 5.8

Bomhard and Rinke, 1994 Hansen, 1993 Lewis, 1993 Iwata et al., 1991 McMartin et al., 1992 Chandra et al., 1992

Wistar F344 Crl:CD®BR CDF(F344)/CrlBR Crl:CD®SD

Walsh and Poteracki, 1994 Mitsumori and Elwell, 1988 Lang, 1992 Lang, 1990 IRI, 1995b

TABLE 3 Incidence of LCTs of the Testes in Male Mice for Studies Terminated at 24 Months Strain

Diagnosis

Incidence

(%)

Swiss CD-1® Crl:CD-1® BR IRC Crj:CD-1® CD-1®

Adenoma Adenoma Adenoma Adenoma Carcinoma Adenoma Adenoma

— 13/524 8/891 14/725 1/725 169/46,752 8/400

≤1.0 2.48 0.9 1.9 0.14 0.4 2.0

B6C3F1 Crl:CD-1® Swiss

(Prentice and Meikle, 1995). Most human testis tumors are either germ or Sertoli cell in origin. In contrast to the rat, LCTs in man are rare. Tumors of LC origin constitute about 2 to 3% of human testicular tumors (i.e., 0.02 to 0.03% of all human male neoplasms) (Mostofi, 1990). LCTs in rodents generally occur in older animals. In humans they arise at any age, but the majority of the reported cases have been noted between 20 and 60 years of age (Cotran et al., 1994; Rao and Reddy, 1987). Earlier reports found no differences between racial or cultural groups (Mostofi and Davis, 1985), but recently it appears that LCTs are more prevalent in white males (Dieckmann and Loy, 1993; Bosland, 1994). The estimated incidence of LCTs in man is 0.1 to 3 per million (Bar, 1992).

Ref. Engelhardt et al., 1993 Lang, 1995 Maita et al., 1988 Chandra and Frith, 1992 Mitsumori and Elwell, 1988 IRI, 1995a

The normal mammalian testis consists of a compound tubular gland covered by a dense fibrous capsule whose outer surface is lined by mesothelial cells. At the hilus, fibrovascular septae extend into the testicular parenchyma, dividing it into irregular lobules. Each lobule contains 1 to 3 convoluted seminiferous tubules. The surrounding fibrovascular stroma contains connective tissue, blood vessels, nerves, and the interstitial cells of Leydig. LCs are congregated in the angular spaces between the seminiferous tubules and form compact groups without any specific relationship to blood vessels (Figure 5). Normal LCs present a wide range of morphology, from elongated fibrous connective tissue-like cells to small rounded cells to polyhedral cells with eosinophilic or vacuolated cytoplasm (Mostofi et al., 1990). 195


FIGURE 5. Normal LCs (arrow) in an F344 rat. Note the LCs are located between seminiferous tubules (S). (H&E, magnification ×190.)

Regardless of the strain of rat or mouse, the morphologic appearance of the proliferative changes involving the LCs of the testis is similar. Proliferative lesions of the LCs of the testis are observed as a continuous spectrum starting with small nodular foci of hyperplasia leading to large LC adenomas that eventually replace the entire testis. The distinction between hyperplasia and adenoma is not always clear. Uniformity of diagnoses within and between studies is achieved only when morphologic criteria are applied consistently. The size of the proliferative lesion is often a consideration in diagnosis, because cytologic features are similar between small and large lesions. There is some debate concerning the exact criteria to be used for the distinction between focal hyperplastic changes and early neoplastic changes. While the LC adenomas increase in size, the diagnosis of neoplasia is not difficult. Because focal hyperplasia and early adenoma are similar in morphologic appearance, the size of the lesion has been used as the critical diagnostic feature to separate hyperplastic and neoplastic changes. There has been much discussion concerning the exact size that should be used to distinguish 196

between hyperplasia and adenoma, and two arbitrary criteria have been developed for rodents. Following the criteria established by the National Toxicology Program (NTP) (Boorman et al., 1987), an aggregate of LCs smaller than the diameter of a seminiferous tubule is classified as being focal hyperplasia. A mass of LCs greater than that of a seminiferous tubule is classified as an adenoma. Masses of this size generally produce some compression of adjacent tubules. Guidelines recommended by The Society of Toxicologic Pathologists for standardization of diagnoses proposes that three tubular diameters be set as the arbitrary separation of focal LC hyperplasia from LC neoplasia (McConnell et al., 1992). In addition, the proliferative focus must also have morphological features consistent with LC neoplasms. These features may include evidence of autonomous growth by symmetrical peripheral compression of adjacent seminiferous tubules, evidence of cellular pleomorphism or reduction in the nuclear/cytoplasmic ratio, and development of a typical endocrine sinusoidal vascular network. To date, the debate continues, and there is no widely accepted size criteria (Boorman et al., 1990;


Greaves, 1990; Maekawa and Hayashi, 1992; McConnell et al., 1992). Unfortunately, the references reported in Tables 2 and 3 do not describe which criteria were used and point to the need for this information to be included in the methods section of manuscripts. In a limited number of instances, the incidence of xenobiotic-induced LCTs were compared using both size criteria and fortunately the incidence is not dramatically different (S. R. Frame, DuPont unpublished data). In contrast to rodents, no size criteria is used to distinguish between hyperplasia and adenomas in humans. A distinct mass of LCs must be evident before it is considered a tumor in humans (Mostofi and Davis, 1990). Rodent LC hyperplasia is observed as collections of LCs between seminiferous tubules. The hyperplasia may be diffuse but is more frequently focal or multifocal (Figure 6). The cells characteristically have abundant eosinophilic cytoplasm with a central nucleus. Some LCs have a highly vacuolated cytoplasm (Figure 7). The cell boundaries are usually prominent. Occasional cells may have pyknotic nuclei and some areas of hyperpla-

FIGURE 6. × 76.)

sia have small spindle cells with dark basophilic nuclei, and scant cytoplasm. LC adenomas vary greatly in size. When small, they are usually detected as single or multiple, white, or yellow nodules. Small adenomas are usually composed of fairly uniform cells that have eosinophilic vacuolated cytoplasm (Figure 8). The nucleus is centrally located with marginally dispersed chromatin and a single distinct nucleolus. As these neoplasms progressively enlarge, they produce compression of the surrounding seminiferous tubules. The neoplasms may be focal or multifocal and unilateral or bilateral. Large tumors in rodents are well circumscribed and cut surfaces of testes show multiple, yellow, or yellow-brown lobules, demarcated clearly from the surrounding normal and/or atrophic tissue. In large tumors, cysts filled with clear or hemorrhagic fluid are frequently observed. Larger neoplasms may completely efface the normal testicular parenchyma (Figure 9). Large adenomas often develop a blood sinus network and commonly have cavities containing blood or brownish fluid. Foci of hemorrhage, necrosis, mineralization,

LC hyperplasia (H) in an F344 rat. Note the presence of multiple nodular areas. (H&E, magnification

197


FIGURE 7. LC hyperplasia (H) in an F344 rat. Note the presence of hyperplastic cells with eosinophilic cytoplasm and centrally located nuclei. (H&E, magnification × 238.)

FIGURE 8. Small LC adenoma (A) in an F344 rat. Note the presence of uniform cells arranged in a sheet. (H&E, magnification × 63.)

198


FIGURE 9. Large LC adenoma (A) in an F344 rat. Note how this large LC adenoma has effaced the normal testicular parenchymea. (H&E, magnification × 12.)

nsinflammation, and cystic degeneration are frequently present in the larger adenomas. Many large tumors may contain poorly differentiated small dark cells with scanty cytoplasm at the margin of the nodules composed of eosinophilic cells. Spontaneous LCTs in the rat are frequently endo-crinologically functional and may produce testosterone, although in the Fischer rat there is evidence that tumorous testes produce mainly progesterone (Prentice and Meikle, 1995). Almost all LC neoplasms of the testis in rodents are benign. By all accounts, malignant LCTs are rare in all strains of rat and mouse. The criteria for malignancy are cellular anaplasia, increased and abnormal mitotic figures, invasion of the testicular tunica and epididymis, and rarely distant metastases (Chandra and Riley, 1994). LC neoplasms should be classified as benign LC adenomas unless distinct evidence of malignancy is found. In humans, LCTs may elaborate androgens or androgens and estrogens, and some have also elaborated corticosteroids (Cotran et al., 1994). In children, they are invariably associated with mac-

rogenitosomia. In adult men, 40% of those with LCTs have gynecomastia and other feminizing features (Mostofi and Davis, 1985). As with other testicular tumors, the most common presenting feature is testicular swelling, but in some patients gynecomastia may be the first symptom. Grossly, human tumors are uniformly mahogany brown, lobulated, and more or less encapsulated. Histologically, the tumors are made up of hexagonal cells with ground glass eosinophilic or vacuolated cytoplasm and have a vesicular nucleus with a delicate nucleolus. Histologically, human LCTs resemble those of the rat, except for the presence of Reinke’s crystals, intracytoplasmic cigar-shaped inclusions of various size, which are observed in about 40% of the human tumors (Mostofi and Davis, 1985; Mostofi and Price, 1973). About 10% of LCTs are malignant in humans (Mostofi and Davis, 1990). The criterion for malignancy is metastasis, where the LCTs exhibit many of the following morphologic features: hemorrhage and necrosis, anaplasia, increased mitotic activity, vascular invasion, and/or local extension (Mostofi and Davis, 1990). Metastases, however, 199


are usually delayed, occurring 3 to 4 years postorchiectomy (Mostofi and Davis, 1985). Endocrine manifestations are common in benign tumors but uncommon with malignant tumors (Prentice and Meikle, 1995). The greatest difference between testicular tumors in man and in the rat is the high incidence of germ cell tumors in man (over 90%) and their occurrence predominantly in younger men. In contrast, germ cell tumors are rare in rats (Mostofi et al., 1990). As mentioned previously, tumors of LCs constitute about 2 to 3% of human testicular tumors with an estimated incidence of 0.1 to 3 per million, and they occur at various ages. In the rat, the incidence of LCTs is much higher, up to 100% in some strains, and occur most frequently in older animals. In rodents, LCTs are invariably benign, while in humans approximately 10% of LCTs are considered to be malignant.

VII. COMPOUNDS THAT INDUCE LC HYPERPLASIA OR TUMORS Figure 10 illustrates five mechanisms by which chemicals can disrupt the HPT axis and produce LCTs. The common theme in all these mechanisms is that these compounds disrupt the HPT axis and increase LH levels. Increased LH levels can produce LC hyperplasia and LCTs if the disruption is sustained (Bar, 1992; Ewing, 1992; Neumann, 1991; Prentice and Meikle, 1995). To date, there are no reported examples of compounds that can disrupt the HPT axis by enhancing the clearance of testosterone or estradiol, although felbamate (PDR, 1995e) and oxazepam (PDR, 1995n) are possible candidates because LCTs are seen at dosages that also produce liver tumors. An examination of the plethora of compounds that have been reported as producing rodent LCTs indicates that a wide variety of structures and potential modes of action are capable of eliciting LC hyperplasia and in longer-term studies the induction of benign LC adenomas. A number of factors emerge in attempting to draw inferences on potential mechanisms for induction of these tumors. For example, the large majority of agents, tested in conventional rodent bioassays in two species, have been reported to induce this tumor type in the rat, but not in the mouse. There are, however, notable exceptions that would include 200

those agents that have estrogenic activity (see below). That compounds possessing such hormonal activity should show this species specificity is readily addressed by reference to the major differences in hormonal control mechanisms between the rat and mouse (Sections III and VII.A.1.g; Huseby, 1976). In toxicology safety assessment studies, the animal models used include the F344, SpragueDawley (Crl:CD®BR), and Wistar strains of rats, the B6C3F1 and CD-1 strains of mice, and the beagle dog. The focus of this review is primarily on LC tumorigenesis in rodent strains typically used in safety assessment studies. Because the focus of this review is LCTs seen in safety assessment studies, other animal models such as the Wistar substrain U (Teerds et al., 1991) will not be discussed. Some compounds have been included in this review based on their induction of LCT in F344 rats. However, as previously noted (Section VI), the F344 strain of rat has a high spontaneous incidence of LCTs, which confounds detection of chemically induced LCTs. In these instances, the response was judged to be equivocal, and this is noted in the text as well as in Tables 4 and 5. For some references such as those from the PDR, tumor incidence was not reported. In addition, almost none of the references stated the pathological criteria used in distinguishing between LC hyperplasia and adenomas (Section VI). Hence, the reviewers could not apply a consistent criteria for judging whether a compound was positive for induction of LCTs and relied on professional judgement using a weight-of-evidence approach. Theoretically, one would predict that using a three-tubule criterion could result in an underreporting of LC tumor incidence. To investigate this point, three compounds that induced LC tumors in 2-year feeding studies were evaluated using both criteria. There was no marked difference in overall tumor incidence, suggesting that most LC tumors grow to a size greater than three tubules by the end of 2 years exposure (S. R. Frame, DuPont, unpublished data). Many of the studies reported data from dietary administration of the compound (i.e., ppm values). Readers can estimate the mg/kg daily intake of the compound by dividing the ppm value by 20. Overall, it is quite reasonable to conclude that agents inducing LCTs do so by a disturbance of normal hormonal homeostasis (be this endocrine,


FIGURE 10. Regulation of the HPT axis and control points for potential disruption. Symbols: (+) feedback stimulation; (–) feedback inhibition; ⊕ receptor stimulation; ⊗ enzyme or receptor inhibition.

paracrine, or autocrine), leading, at a critical stage, to a signal that permits LC growth or potentially inhibit LC death. It is clear that LH is the primary driver of LCTs in rats because testosterone administration by silastic tubules decreases serum LH levels and blocks both spontaneous and xenobiotic-induced LCTs (Chantani et al., 1990; Waalkes et al., 1997). In this section of the review, compounds were divided into two broad

groups, namely, genotoxic or nongenotoxic in nature. For the nongenotoxic compounds, they were further subdivided by their mode of action, chemical activity, chemical class, or as other (Table 4). For the genotoxic compounds, they were grouped into the following categories: alkylating agents, ability to induce base substitutions, metals, radiation, or as other (Table 5). An exhaustive literature search was conducted to iden201

202 Species (strain)a

Rat (CD) Mouse (CD-1)

3. Testosterone biosythesis inhibitors Calcium Channel Rat Blockers (see Section B) Cimetidine Rat (51481-61-9) (Wistar)

2. 5α -Reductase inhibitors Finasteride (98319-26-7)

1. Androgen receptor antagonists Bicalutamide Rat (90357-06-5) (Wistar) Cimetidine Rat (51481-61-9) (Wistar) Fenvalerate Rat (51630-58-1) (SD) Flutamide Rat (13311-84-7) Linuron Rat (330-55-2) (CD) Procymidone Rat (32809-16-8) (OsborneMendel) Vinclozolin Rat (50471-44-8) Zanoterone Dog (107000-34-0) (Beagle)

A. Classified by Mode of Action

Compound (CAS number)

None None

None None

Adenoma Adenoma

Adenoma Hyperplasia

None

None

Adenoma

Adenoma (equivocal)

None

None

Adenoma

None

Adenoma (equivocal) Adenoma

None

None

Adenoma

Hyperplasia

Thyroid

Other tumor sites

Adenoma

LC response

Leslie et al., 1981; Brimblecombe and Leslie, 1984; Morita et al., 1990

PDR, 1995c,i,l; Roberts et al., 1989

PDR, 1995m; Prahalada et al., 1994, George et al., 1989 PDR, 1995m; Prahalada et al., 1994

Juniewicz et al., 1990

Gray et al., 1994; Wong et al., 1995

Hosokawa et al., 1993a,b; Murakami et al., 1995

PDR, 1995d; Viguier-Martinez et al., 1983a,b; Cook et al., 1993 Cook et al., 1993

Leslie et al., 1981; Sivelle et al., 1982, Brimblecombe and Leslie, 1984 Parker et al., 1984; Eil and Nisula, 1990

Iswaran et al., 1998

Ref.

TABLE 4 Nongenotoxic Compounds that Produce LC Hyperplasia or Adenomas in Rats, Mice, or Dogs


203

Dog (Beagle) Dog (Beagle)

Rat (CD) Rat (CD) Rat (Wistar) Rat (Wistar) Rat

6. GnRH agonists Buserelin (57982-77-1) Histrelin (76712-82-8) Leuprolide (74381-53-6) Nafarelin (76932-56-4)

Pituitary

Adenoma Adenoma Adenoma

Rat Rat

Pituitary

Pituitary

None

Hyperplasia

Rat (Wistar) Rat

PDR, 1995p

PDR, 1995h

PDR, 1995o

Donaubauer et al., 1987

Yamada et al., 1994a,b; Yamada et al., 1995a,b

None None

Junker-Walker and Nogues, 1994

Prentice et al., 1992; Dirami et al., 1996 Roberts et al., 1993

None

Hyperplasia

Junker-Walker and Nogues, 1994

None

None

None

Adenoma

Hyperplasia

Rustia and Shubik, 1979,

Pituitary

Ronis et al., 1994; Gray et al., 1994; Wong et al., 1995

Zawirska and Medras, 1968

Cheever et al., 1990; Widenius et al., 1989 Fort et al., 1995; Meikle et al., 1994

None

None

None



5. Dopamine agonists/enhancement of dopamine levels Mesulergine Rat Adenoma (64795-35-3) (Wistar) Norprolac Rat Adenoma (87056-78-8) (CD) Oxolinic acid Rat Adenoma (14698-29-4) (Wistar)

4. Aromatase inhibitors Formestane (566-48-3) Letrozole (112809-51-5)

Ethanol (64-17-5) Lansoprazole (103577-45-3) Lead acetate (301-04-2/15347-57-6) Metronidazole (443-48-1) Vinclozolin (50471-44-8)



7. Estrogen agonists/antagonists Diethylstilbestrol Mouse (56-53-1) (BALB/c) Estradiol Mouse (50-28-2) (BALB/c) Ethinylestradiol Mouse (57-63-6) (ICR) Methoxychlor Mouse (72-43-5) (BALB/c) Sigetin Rat (60252-39-3) (Strong A) Mouse Tri-p-anisylchloroethylene (TACE) (C57x (569-57-3) CBA) Tamoxifen Mouse (10540-29-1) (Alderley Park) Triphenylethylene Mouse (58-72-0) (Strong A)

A. Classified by Mode of Action (continued)


None None None None None Liver

None

None

Adenoma Hyperplasia Adenoma Adenoma Adenoma

Adenoma

Adenoma

Other tumor sites

Adenoma

LC response

Bonser, 1942

Tucker et al., 1984

Gardner and Boddaert, 1950

Ird, 1983

Yasuda et al., 1986; Yasuda et al., 1988 Reuber, 1979

Huseby, 1980; Nishizawa et al., 1988

Baroni et al., 1966; Huseby, 1976

Ref.

TABLE 4 (continued) Nongenotoxic Compounds that Produce LC Hyperplasia or Adenomas in Rats, Mice, or Dogs


205

4. Goitrogens Ethylenethiourea (ETU) (96-45-7) 6-n-Propyl-2-thiouracil (PTU) (51-52-5)

Vinclozolin (50471-44-8) Folpet (133-07-3)

3. Fungicides Procymidone (32809-16-8)

Rat (SD)

Rat

Rat (CD)

Rat (OsborneMendel) Rat

None

Adenoma

Adenoma (equivocal) Hyperplasia

Adenoma

Adenoma

None

None

Thyroid

None

None

None

Adenoma

Adenoma

None

Adenoma

Liver

None

Adenoma

Rat

None

Adenoma

Adenoma

Rat

2. Calcium channel blockers Felodipine Rat (72509-76-3) Isradipine Rat (75695-93-1) (CD) Lacidipine Rat (103890-78-4) (CD) Nimodipine Rat (66085-59-4) (Wistar)

1. Antihypertensives Guanadrel (22195-34-2) Hydralazine (86-54-4)

B. Grouped by Chemical Activity

Mendis-Handagama and Sharma, 1994

Gak et al., 1976

Wong et al., 1995, Ronis et al., 1994 Quest et al., 1993

Hosokawa et al., 1993a,b; Murakami et al., 1995

PDR, 1995i

Hamada and Futamura, 1998

Roberts et al., 1989; PDR, 1995c

PDR, 1995l

PDR, 1995a

PDR, 1995f



Trichloroethylene (TCE) (79-01-6) Wyeth-14,643 (50892-23-4)

Rat (SD) Rat (CD)

5. Peroxisome proliferators Ammonium Rat Perfluorooctanoate (C8) (CD) (3825-26-1) Clofibrate Rat (637-07-0) (Alderley Park) Diethylhexylphthalate Rat (DEHP) (SD) (117-81-7) Gemfibrozil Rat (25812-30-0) (CD) HCFC-123 Rat (306-83-2) (CD) Methylclofenapate Rat (21340-68-1) (Alderley Park) Perchloroethylene (PCE) Rat (127-18-4) (F344)

B. Grouped by Chemical Activity (continued)


None

Liver pancreas Liver pancreas Liver pancreas

Adenoma

Adenoma

Adenoma

Adenoma

Adenoma (equivocal)

Adenoma

Leukemia kidney skin Leukemia kidney Liver pancreas

Liver pancreas

Adenoma

Adenoma

Liver pancreas

Other tumor sites

Adenoma

Leydig cell response

Cook et al., 1994

Maltoni et al., 1988

Clarke and Ragan, 1986

Tucker and Orton, 1995

Malley et al., 1995

Fitzgerald et al., 1981

Berger, 1995

PDR, 1995b

Sibinski, 1987; Cook et al., 1992; Cook et al., 1994

Ref.



207

Species (strain)a

Rat (CD) Rat (Wistar) Rat (Wistar) Rat (CD)

4. Sugars Lactose (63-42-3) Lactitol (585-86-4) Tara gum

o,p′-DDT (789-02-6)

3. Organochlorines o,p′-DDD

Rat (Wistar) Rat (Wistar) Rat (F344)

Rat (OsborneMendel)

Rat

2. Nitroaromatics and related compounds p-Nitrochlorobenzene Rat (100-00-5) (SD) Nitroglycerine Rat (55-63-0) (CD) 2,4-Toluenediamine Rat (95-80-7) (F344)

1. Fluorochemicals HCFC-123 (306-83-2) HCFC-133a (75-88-7) HFC-134a (811-97-2) HCFC-141b (1717-00-6)

C. Grouped by Chemical Class


None None

Adenoma Adenoma

Liver pancreas

Adenoma

None None None

Adenoma Adenoma Adenoma (equivocal)

Liver

None

Liver

Adenoma

Adenoma (equivocal) Adenoma (equivocal)

None

Adenoma

Adenoma

Liver pancreas None

Other tumor sites

Adenoma

LC response

Melnick et al., 1983

Sinkeldam et al., 1992

Sinkeldam et al., 1992

Fitzhugh and Nelson, 1947

Lacassagne and Hurst, 1965

Cardy, 1979

Ellis et al., 1984; PDR, 1995j

Schroeder and Daly, 1984

Turnbull et al., 1994

Collins et al., 1995

Longstaff et al., 1984

Malley et al., 1995

Ref.


208 a

Rat (Wistar) Rat (SD) Rat (F344) Rat (F344) Rat (F344) Rat (CD, F344) Rat (F344) Rat

Mouse (B6) Rat (SD) Rat

Species (strain)a

Strain is included when specified in citation.

d-Limonene (5989-27-5) MTBE (1634-04-4) Nicotine (54-11-5) Oxazepam (604-75-1)

Boric Acid (10043-35-3) Carbamazepine (298-46-4) Felbamate (25451-15-4) Flecainide (54143-55-4) Indomethacin (53-86-1) Isopropanol (67-63-0) JP-4

D. Unclassified


Thompson et al., 1973 PDR, 1995n

None Liver thyroid prostate

Hyperplasia (equivocal) Adenoma

Belpoggi et al., 1995

None

Jameson, 1990

Bruner et al., 1993

Adenoma

Kidney

Adenoma

Burleigh-Flayer et al., 1997

Kidney

None

Adenoma

Goerttler et al., 1992

Case et al., 1984

PDR, 1995e

PDR, 1995r

Dieter, 1994

Ref.

Adenoma

None

Adenoma

Liver

Adenoma None

None

Adenoma

Adenoma

None

Other tumor sites

Hyperplasia

LC response



TABLE 5 Genotoxic Compounds that Produce LC Hyperplasia or Adenomas Compound (CAS number)


1.

Alkylating agents N-Nitrosobis(2-oxopropyl)amine (BOP) (60599-38-4) 1,3-Butadiene (106-99-0) BrdU (59-14-3) BrdU + X-rays (59-14-3) 3-Chloro-2methylpropene (563-47-3) Cycasin (14901-08-7) Dibromochloropropane (DBCP) (96-12-8) Diethylnitrosoamine (DEN) (55-18-5) Dimethylnitrosoamine (DMN) (62-75-9) Isoprene (78-79-5) 8-Methoxypsoralen (298-81-7) Methy-CCNU (33073-59-5) Nitrosoethylene (NEU) (759-73-9) Streptozotocin (18883-66-4)

2.

3.

4.

5.

a

Base substitutions 5-Azacytidine (320-67-2) Vidarabine (5536-17-4) Metals Cadmium (7440-43-9)

Radiation X-Irradiation

Others Ethylene dichloride + Disulfiram (107-06-2/97-77-8)

Species (Strain)a

LC response

Rat (Wistar)

Adenoma

Rat (CD) Rat (LIO) Rat (LIO) Rat (F344)

Other tumor sites

Ref. Pour, 1986

Adenoma

Prostate, vas deferens, coagulating glands, liver Pancreas

Adenoma

Kidney, intestine

Anisimov, 1995

Adenoma

Anisimov and Osipova, 1993

Adenoma

Prostate, kidney, adrenal cortex, hematopoietic system, thyroid Forestomach, kidney

Rat (ACI) Humans

Adenoma

Intestine, liver, kidney

Fukunishi et al., 1971

Hyperplasia

None

Cortes-Gallegos et al., 1980

Mice (RF)

Adenoma

Lung, liver, forestomach

Clapp, 1973

Rat (Wistar)

Adenoma

Liver, hematopoietic system

Arai et al., 1979; Terao et al., 1978

Rat (F344) Rat (F344) Mice (SJL/J) Rat (Outbred) Rat (SD)

Adenoma

None

Melnick et al., 1994

Adenoma

Kidney, Zymbal’s, lung

Dunnick, 1989

Hyperplasia

None

Yegana et al., 1988

Adenoma

Nervous system

Ird and Smirnova, 1983

Adenoma

Pancreas, kidney, liver

Okawa and Doi, 1983

Adenoma

Hematopoietic system, skin, lung, kidney Liver, kidney, intestine, thyroid

Carr et al., 1988; Carr et al., 1984 Griffith, 1988

Rat (F344) Rat

Adenoma

Owen et al., 1987

NTP, 1986

Rat (Wistar/ F344)

Adenoma

Lung, prostate, hematopoietic system

Bomhard et al., 1987; Waalkes and Rehm, 1992; Waalkes et al., 1997

Rat (LongEvans)

Adenoma

None

Lindsay et al., 1969

Rat (SD)

Adenoma

Liver, skin

Cheever et al., 1990

Strain is included when specified in citation.

209


tify compounds that induce LC hyperplasia and/ or tumors as well as mechanistic information when available. The differentiation between hyperplasia and tumors is arbitary (Section VI) because the diagnosis is dependent on the size of the nodule of LCs in cross section. This, in turn, will be dependent on the number of testicular sections examined (frequently this is only one per testis) and where the cut has been made (i.e., is it in the middle or on the edge of the nodule). In a number of instances, particularly for pharmaceutical agents, the only published information available is the Physician’s Desk Reference (PDR). This does not consitute a peer-reviewed journal or publication. Even though the detail available from the PDR is often limited, it is useful in the overall discussion of the ability of agents to induce LCTs. Invariably, the reported tumors in the PDR occur at levels in excess of the normal therapeutic dose. Wherever possible, reference to peer-reviewed published material is used.

A. Nongenotoxic Compounds

1. Classified by Mode of Action a. Androgen Receptor Antagonists Androgen receptor antagonists such as flutamide compete with testosterone and DHT for binding to the androgen receptor (Simard et al., 1986). This competition reduces the net androgenic signal to the hypothalamus and adenohypophysis, resulting in an increase in LH secretion with a concomitant elevation of testosterone secretion (Cook et al., 1993; Viguier-Martinez et al., 1983a,b). Flutamide has been shown to produce LCTs in rats after 1 year of continuous exposure (PDR, 1995d). Other examples of androgen receptor antagonists include cimetidine (Brimblecombe and Leslie, 1984; Leslie et al., 1981; Sivelle et al., 1982), linuron (Cook et al., 1993), procymidone (Hosokawa et al., 1993a,b; Murakami et al., 1995), and vinclozolin (Wong et al., 1995), all of which can induce LCTs. Bicalutamide. Iswaran and co-workers (1998) have summarized the toxicological profile of bicalutamide (ICI 176,334). Bicalutamide is a nonsteroidal androgen receptor antagonist developed for treatment of prostate cancer. Based on a 210

battery of in vitro and in vivo studies of genotoxicity, bicalutamide was found not to be a genotoxic agent. In a 2 year rat oncogenicity study, Wistar rats were dietarily administered 0, 5, 15, or 75 mg/kg bicalutamide; both LC hyperplasia and LCTs were observed at all dose levels. The incidences of LCTs were 2.9, 37.3. 62.7, and 94.1% at the 0, 5, 15, and 75 mg/kg bicalutamide dose groups, respectively. In addition to the LCTs, bicalutamide also induced thyroid follicular cell adenomas in male and female rats, a finding secondary to hepatic enzyme induction, and uterine adenocarcinoma in female rats, a finding secondary to the pharmacological effects of bicalutamide interferring with the pituitary gonadal axis resulting in an unopposed estrogen action on the uterus. Using the same dose levels as the rat oncogenicity study, bicalutamide did not produce LCTs, thyroid follicular cell adenomas, or uterine adenomas in a 2-year mouse (C57BL/10JfCD-1) oncogenicity study. An increased incidence of hepatocellular carcinoma was observed in male mice treated with 75 mg/kg bicalutamide, a finding attributed to hepatic enzyme induction. In the clinic, metastatic prostate cancer patients treated with 50 mg/ kg/d bicalutamide had no evidence of LC hyperplasia (Jones et al., 1994). In developmental and reproduction studies, bicalutamide produced the expected alterations of an androgen receptor antagonist: reduction in anogenital distance in male fetuses, hyposadias, and nipple retention in male offspring. The authors concluded that nongenotoxic mechanisms were responsible for the tumors observed with bicalutamide: LCTs (rat) were induced via the elevated LH levels, and the thyroid follicular cell adenomas (rat) and hepatocellular carcinomas (mouse) were mediated via liver enzyme induction (Imai et al., 1998). Cimetidine. In a rodent bioassay (Leslie et al., 1981), Wistar rats were administered cimetidine by gavage daily for 2 years at levels of 0 (two control groups: distilled water; no vehicle treatment), 150, 378, or 950 mg/kg/d. At termination, there were decreased testis, prostate, and seminal vesicle weights at the highest dose level. The prostate and seminal vesicle weights were also significantly lower at the 150 and 378 mg/k/d dosage levels. There was an increase in the incidence of LCTs in cimetidine-treated groups, although the dose response was flat. The percentage incidences were 15.5 (0 mg/kg/d), 23.1


(150 mg/kg/d), 20.6 (378 mg/kg/d), and 23.5 (950 mg/kg/d). While this increase in LCTs appears to be equivocal, the large sample size for each group (n = 100) and the greater survival in the control groups resulted in the incidence being statistically increased in the cimetidine-treated rats. Leslie and co-workers concluded that the increased incidence of LCTs was compound related (Leslie et al., 1981). No data were provided on the incidence of LC hyperplasia, but it was stated not to be increased in the cimetidine-treated rats. Cimetidine is acknowledged to be a weak antiandrogen based on functional endpoints such as antagonizing the effects of testosterone in maintaining prostate and seminal vesicle weight in young castrated rats. It has also been shown to inhibit the androgenic action of DHT. Reductions in prostate and seminal vesicle weight have been noted in repeat dose studies with Wistar rats and Beagle dogs (Leslie and Walker, 1977; Brimblecomb and Leslie, 1984). Gynecomastia has been shown to be produced in a small percentage of patients administered cimetidine. The antiandogenic activity of cimetidine has been shown to be related to its ability to compete for binding to the androgen receptor based on data from classic receptor competition studies using radiolabeled DHT (Winters et al., 1979). In addition, cimetidine may also inhibit testosterone biosynthesis (discussed below). Hence, cimetidine may act as both an androgen receptor antagonist and a testosterone biosynthesis inhibitor, which illustrates an important point that compounds can have more than one type of endocrine activity. Fenvalerate. In a combined chronic-oncogenicity study with male and female SpragueDawley (Crl:CD®BR, CD) rats, fenvalerate was administered in the diet at 0, 1, 5, 25, 250, and 1000 ppm. At 1000 ppm fenvalerate, LCT incidence was numerically increased when compared with the controls (5/50 vs. 1/50); however, this difference was not statistically different (Parker et al., 1984). The authors noted that LCT incidence was within the range of historical controls, although these data were not included. No other compound-related changes in tumor incidence were reported except for a slight increase in mammary tumors in female rats that was within the historical control range. More recently, several pyrethroids, including fenvalerate, have been shown to compete for binding to the androgen

receptor (Eil and Nisula, 1990). In these studies, the ability of fenvalerate to compete with radiolabeled methyltrienolone (R1881) in an in vitro receptor competition assay was evaluated and an IC50 value of 1.8 × 10-4 for fenvalerate was determined. Clearly, the increase in LCTs as well as the in vitro receptor competition data with fenvalerate are equivocal. If fenvalerate does induce LCTs, which requires confirmation, it may be doing so because it is a weak androgen receptor antagonist. Flutamide. In two chronic studies (PDR, 1995), flutamide has been shown to induce LCTs in Sprague-Dawley rats, although the incidence was not reported. In the first study, flutamide was administered at levels of 0, 30, 90, or 180 mg/ kg/d for 1 year and produced increases in LCTs at all doses. In a 24-month carcinogenicity study, the dose levels of flutamide used were 0, 10, 30, or 50 mg/kg/d. This treatment again produced an increased number of LCTs at all dose levels tested. Flutamide is a classic nonsteroidal androgen receptor antagonist (Simard et al., 1986) producing elevations in both LH and testosterone, while decreasing prostate and seminal vesicle weight (Viguier-Martinex et al., 1983a,b). In immature male Wistar rats, 2-week gavage studies (5 mg/ rat/d) resulted in LC hypertrophy but not hyperplasia even though LH and testosterone were markedly increased (Viguier-Martinex et al., 1983a). In mature male Wistar rats (ViguierMartinex et al., 1983b), marked hormonal changes were again apparent, but without the LC hypertrophy. Cook and co-workers (1993) confirmed these findings in the CD rat when flutamide was administered (10 mg/kg/d) for a similar time period. These authors again noted the difference in sensitivity between the mature and immature rats, with immature animals having a greater sensitivity to the reductions in accessory sex organ weights, but mature animals having greater changes with respect to the increase in LH and testosterone levels. Diffuse hyperplasia was seen in the flutamide-treated CD rats after treatment for 2 weeks with 10 mg/kg/d (J. C. Cook, DuPont, personal communication). Linuron. In a 2-year feeding study with linuron (USEPA, 1988), there was a significant dose-dependent elevation in the incidence of LCTs in CD rats: 5.9% (0 ppm), 16.1% (50 ppm), 29.7% (125 ppm), and 56.1% (625 ppm). Linuron has 211


been shown to be negative in a battery of shortterm tests for genotoxicity. Cook and co-workers (1993) have compared the profile of linuron to the structurally similar compound flutamide (see above). These studies have essentially confirmed that linuron at dose levels of 200 mg/kg/d is an androgen receptor antagonist with a similar, if not identical, mode of action to flutamide in producing decreases in accessory sex organ weights and increased levels of serum LH and testosterone after subacute dosing, with distinct differences between sexually mature and immature male rats (see flutamide above). Androgen receptor competition studies would suggest that linuron is approximately 3.5 times less potent than flutamide. The mechanism for the production of LCTs is believed to be via the sustained elevation of LH. Procymidone. Procymidone is a fungicide that also shares structural similarity with flutamide (Hosokawa et al., 1993a,b). In two chronic rat studies, procymidone was administered at dietary levels of 0, 100, 300, 1000, or 2000 ppm in either Sprague-Dawley or Osborne-Mendel rats (Murakami et al., 1995). In the former study, LCT incidence at the highest dose level was 17%. The control incidence of LCTs was not stated by the authors, but the spontaneous incidence of LCTs is approximately 5% for Sprague-Dawley rats (Section VI). In the second study, the incidence of tumors was 2% (0 ppm), 2% (100 ppm), 0% (300 ppm), 20% (1000 ppm), and 49% (2000 ppm). The incidence of pituitary tumors was increased in female but not male rats. No LCTs were noted in a chronic mouse study (B6C3F1 strain). In Cynomolgus monkeys, no effects were noted on the reproductive organs or serum LH levels given dose levels up to 300 mg/kg/d for 13 weeks. However, testosterone was increased in the procymidone-treated monkeys, but not in a dose-dependent manner (2-fold increase) compared with control. Procymidone was shown to be negative in a battery of short-term tests for genotoxicity. A number of studies (discussed below) have been conducted that confirm that procymidone induces LCTs via an androgen receptor antagonist mechanism. In a 90-day mechanistic study using SpragueDawley rats (Murakami et al., 1995), dietary levels of 0, 700, 2000, or 6000 ppm procymidone were used. Testostrone levels in the testis were 212

elevated at 4 weeks at the highest dose level (>2-fold increase) as was serum LH (2-fold increase). Pituitary LH was increased at 700 ppm and above and maximally was 180% of control. At 13 weeks, testicular testosterone was significantly elevated at the highest level (circa 160% of control) with no increase in serum LH. Pituitary LH levels were raised to their highest levels (circa 30% above control). Serum testosterone was elevated at the highest dose level at both time points (not statistically significant): 155% at 4 weeks and 136% at 13 weeks (i.e., marginal for biological significance). These studies illustrate an important point that clear demonstration of an elevation in LH levels was seen in mechanistic studies conducted at higher levels than in the bioassay. This illustrates the difficulty in detecting changes in LH under bioassay conditions. In vitro studies were conducted with whole testes from procymidone-treated rats incubated in the presence or absence of hCG. These in vitro studies demonstrated that testes from procymidone-treated rats had elevated testosterone production, which was consistent with the in vivo findings. Even though the authors demonstrated only a transient increase in LH (at 4 but not 13 weeks), the pattern of hormonal chnages is consistent with an androgen receptor antagonism mechanism. The authors proposed that a threshold for the induction of LCTs probably exists. Vinclozolin. The dicarboximide fungicide, vincozolin, has been reported to induce LC hyperplasia and atrophy of the prostate and seminal vesicles when administered to adult male rats (van Ravenzwaay, 1992). In a developmental study, pregnant Long-Evans rats were gavaged with either 100 or 200 mg/kg vinclozolin beginning on gestation day 14 through postnatal day 3. The male offspring of vinclozolin-treated dams had reduced anogenital distance, cleft phallus, hypospadias, ectopic testes, and small accessory sex glands (Gray et al., 1994). While some of the malformations seen in male offspring from vinclozolin-treated dams could also be produced by exposure to estrogens, the vinclozolin-treated female offspring did not display any estrogen-like alterations in developmental landmarks. Because of these observations, the authors concluded that vinclozolin was acting as an antiandrogen. Subsequently, inclozolin and two of its metabolites have been shown to compete for binding


to the androgen receptor and to inhibit transcriptional activity using an androgen receptor reporter gene system (Wong et al., 1995). From the work by Wong and co-workers, vinclozolin has been clearly shown to be an androgen receptor antagonist. Vinclozolin has also been shown to inhibit several P450 isozymes from Sprague-Dawley rats, and this inhibition is related to its ability to form a Type II binding spectrum (Ronis et al., 1994). Because the testosterone biosynthetic pathway contains three P450 isozymes, it is possible that vinclozolin could also inhibit testosterone biosythesis. If this is shown, then vinclozolin would act as an antiandrogen through two mechanisms: androgen receptor antagonism and testosterone biosynthesis inhibition, both of which would facilitate the development of LC hyperplasia in adult male rats. Zanoterone. Zanoterone is a steroidal androgen receptor antagonist that was designed to treat benign prostatic hyperplasia in aging human males. Adult male beagle dogs were treated with either 0, 0.625, 2.5, 10, or 40 mg/kg/d zanoterone for 16 weeks (Juniewicz et al., 1990). A mild LC hyperplasia was seen in 2/4 dogs in the 40 mg/kg/d dosage group, but there were no effects on testis weight, daily sperm production, or spermatogenesis. Serum testosterone levels were increased at the 10 and 40 mg/kg/d dosage groups, a finding consistent with an androgen receptor antagonist. Serum LH levels were not measured.

b. 5α-Reductase Inhibitors 5α-Reductase inhibitors such as finasteride (PDR, 1995m; Prahalada et al., 1994; Rittmaster et al., 1992) block the conversion of testosterone to DHT. The latter amplifies the androgenic signal through several mechanisms: (1) unlike testosterone, DHT cannot be aromatized to estrogen and thus its effects are purely androgenic, and (2) DHT binds to the androgen receptor with greater affinity and stability than does testosterone (De Groot et al., 1995b). Hence, 5α-reductase inhibitors decrease DHT levels, which reduces the net androgenic signal received by the hypothalamus and pituitary and thereby causes a compensatory increase in LH levels. Unlike the other mechanisms shown in Figure 10, 5α-reductase inhibitors induce LCTs in mice and LC hyperplasia in

rats, while the other mechanisms exclusively induce LCTs in rats. Finasteride. Finasteride did not induce LCTs in a CD rat chronic study using dose levels of up to 160 mg/kg/d; however, LC hyperplasia was seen at dose levels ≥ 40 mg/kg/d. In CD-1 mice there was a statistically significant increase in LCTs at 250 mg/kg/d (PDR, 1997). It is possible that finasteride also may induce LCTs in rats if tested at higher levels as in the mouse bioassay. Increased LC hyperplasia was noted at 25 mg/kg/d with a positive correlation with serum LH (2- to 3-fold higher than control) in both species. No changes in LCs were reported at 1 year in rodents, or were effects seen in canine LCs at dose levels up to 45 mg/kg/d. Adverse effects were noted in the reproduction study and included a dose-dependent increase in hypospadias (up to 100% of male offspring at 100 mg/kg/d). Pregnant rats produced males with decreased prostate and seminal vesicle weights, delayed preputial separation, and decreased anogenital distance. In the developmental study, the critical period was determined as days 16 to 17 of gestation. Prahalada and co-workers (1994) summarized the mouse oncogenicity study referenced above. In this study, dose levels selected were 0, 2.5, 25, 250 mg/kg/d for 83 weeks. At study termination, there were increases in LC hyperplasia (52 vs. 24% in controls) at 25 mg/kg/d and adenomas (32 vs. 0.5% in control) at 250 mg/kg/d. No effects were reported on the seminiferous tubules, but it is unclear whether the authors looked for changes in androgen-dependent stages of the seminiferous epithilial cycle. The presence of LC hyperplasia was first noted at 1 month at 250 mg/ kg/d. Serum testosterone levels were unaffected in the study up to 14 weeks after dosing commenced, when elevations in LH and LC hyperplasia were noted. Surprisingly, DHT levels were not measured, especially as the agent is a 5α-reductase inhibitor. Studies in castrated mice with and without finasteride suggested that DHT may be decreased, leading to increased secretion of LH.

c. Testosterone Biosynthesis Inhibitors Testosterone biosynthesis inhibitors such as lansoprazole (Fort et al., 1995; Meikle et al., 1994) decrease testosterone levels and increase LH lev213


els, resulting in the development of LCTs. Ketoconazole is the most well-studied and widely known testosterone biosynthesis inhibitor, but was not included in Figure 10 because it has not been reported to induce LCTs (Feldman, 1986; PDR, 1997). In all likelihood, ketoconazole would induce LCTs if tested in a bioassay at levels which meet the USEPA criteria for a maximum tolerated dose. The ketoconazole bioassay was only 18 months in duration, and LCTs generally do not occur until very late in life (i.e., after 18 months). In addition, it appears that minimal toxicity was observed at the high dosage level. Other examples of testosterone biosynthesis inhibitors include the calcium channel blockers, felodipine (PDR, 1995l) and isradipine (Roberts et al., 1989), and the H2 receptor antagonist cimetidine (Brimblecombe and Leslie, 1984; Leslie et al., 1981; Sivelle et al., 1982, Morita et al., 1990). To date, most known testosterone biosynthesis inhibitors contain one of the following structural moieties: imidazole, benzimidazole, dicarboximide, or dimethylpyridine. At least for the imidazole and benzimidazole classes, it has been clearly shown that these compounds inhibit P450 by binding to its protoporphyrin iron, producing a Type II binding spectrum (430 nm peak, 393 trough) (Vanden Bossche, 1985; Feldman, 1986). These compounds inhibit testosterone biosynthesis because the testosterone pathway contains three P450 isozymes: C27-side chain cleavage, C17a-hydroxylase, and C17, 20-lyase enzymes. For instance, ketoconazole has been shown to differentially inhibit these three isozymes where sensitivity from most sensitive to least sensitive appears to be C17, 20-lyase, C17a-hydroxylase, and C27-side chain cleavage (De Coster et al., 1989; Morita et al., 1990; Kan et al., 1985; Sikka et al., 1985). Using isolated rat LCs, the inhibition of testosterone biosynthesis by ketoconazole has been shown to be reversible in in vitro (Kan et al., 1985) and ex vivo (Pont et al., 1982) studies. Calcium Channel Blockers. Felodipine. In a conventional chronic rat bioassay with felodipine, the dose levels were 0, 7.7, 23.1, or 69.3 mg/kg/d (PDR, 1995l). This study demonstrated a doserelated increase in the incidence of LCTs in male rats. No tumors were seen in studies with mice. The mode of action for the induction of tumors was believed to be by a lowering of intratesticular 214

and serum testosterone concentrations, giving rise to an increase in LH secretion. The agent was nongenotoxic in conventional studies. No effects were noted in a reproduction study at dose levels up to 29.6 mg/kg/d. Other potential hormonal effects were characterized as difficulties in dams during parturition and embryo-fetal loss at a dose level of 9.6 mg/kg/d. Isradipine. In a 2-year CD rat bioassay, isradipine (SDZ-200-110) has been shown to produce a 2.5-fold increase in LCT incidence at 62.5 mg/kg/d (Roberts et al., 1989). The compound was reported as being nongenotoxic in a range of conventional studies. The study reported some evidence for a decrease in LC testosterone production in vitro, and in vivo treatment with 62.5 mg/kg isradipine caused a 90% drop in circulating testosterone 4 h after dosing. In a long-term dietary study, there was a significant elevation of testicular testosterone levels. The authors stated that an increase in serum LH was seen during the course of this chronic study. In fact, a fairer assessment would be that there was a lesser rate of decline in serum LH levels than was observed in the control animals. Testicular LH receptor numbers were also found to be decreased. The establishment of a hormonal mechanism for the production of LCTs seems sound. However, the question of what causes the massive short-term reduction in testosterone production was not fully addressed and in all likelihood is due to inhibition of testosterone biosynthesis. It seems reasonable to surmise that this short-term change may have had an impact on the changes in serum LH. No male fertility effects were reported in studies using dose levels up to 60 mg/kg/d. Lacidipine. In a 2-year oncogenicity study with rats, lacidipine increased the incidence of LCTs in male Crj:CD(SD) rats in the 20 (weeks 1 to 18)/10 (weeks 19 to 104) mg/kg/d group, but not in the 0.2 or 2.0 mg/kg/d groups (summarized in Hamada and Futamura, 1998). There was no increase in the incidence of other tumors in the female Crj:CD(SD) rats or in an oncogenicity study with CD-1 mice. Lacidipine was also found not to be genotoxic. In a follow-up mechanisitic study, Crj:CD(SD) rats were administered by gavage either 0 or 15 mg/kg lacidipine/d (except on Sundays) for up to 78 weeks (Hamada and Futamura, 1998). At terminal sacrifice, the inci-


dences of LCH and LCTs in the lacidipine-treated rats were 16% (8/50) and 14% (7/50), respectively, and were statistically increased when compared with the concurrent control. In the control rats, the incidences of LCH and LCTs were 5% (1/20) and 0%, respectively. The authors also measured a battery of plasma hormone levels (testosterone, progesterone, prolactin, LH, and FSH) on weeks 51, 59, 67, and 75. Plasma LH levels were statistically elevated and plasma testosterone levels were statistically decreased at all sampling timepoints, as would be expected for a testosterone biosynthesis inhibitor. The effects of lacidipine and another calcium channel antagonist nicardipine on isolated LCs from three species (Crj:CD(SD) rats, Slc:ICR mice, and rhesus monkeys) were investigated. Both lacidipine and nicardipine decreased basal, and LH- and LHRHstimulated testosterone biosynthesis in all three species. Interestingly, BrdU incorporation was increased in LH-stimulated LCs from rats treated with both calcium channel antagonists, but not in LCs from mice or monkeys. These responses are consistent with the tumor responses in the oncogenicity studies with rats and mice, suggesting that the increase in LH in rats is responsible for the development of LCTs. In addition, the absence of an increase in BrdU incorporation in mice and monkeys would suggest that humans would be at low risk for LCT induction from usage of lacidipine. This absence of proliferative response was related to the low levels of LHRH receptors on mice and monkey LCs, suggesting that humans may also be at low risk for development of LCTs due to their low expression of LHRH receptors on LCs. Nimodipine. When administered at a dietary level of 1800 ppm (equivalent to 91 to 121 mg/ kg/d) for 2 years, nimodipine produced an increase in the incidence of LCTs in male and adenocarcinoma of the uterus in female Wistar rats (PDR, 1995i). This later finding was judged not to be compound related because the incidence was within historical control levels for the laboratory. LCTs were not found in the chronic mouse study. No male fertility effects were noted in studies at dose levels up to 30 mg/kg/d. Embryotoxicity was noted in rats at 30 mg/kg/d. Cimetidine. Cimetidine has clearly been shown to be an androgen receptor antagonist (Sec-

tion VII.A.1.a). In addition, cimetidine may also inhibit testosterone biosynthesis through formation of a Type II binding spectrum with P450. This conclusion is based on cimetidine having an imidazole structure (Feldman, 1986) and data that cimetidine inhibits testosterone biosynthesis in vitro (Morita et al., 1990). In addition, cimetidine has been reported to interfere with other drugs that are metabolized via P450 pathways (PDR, 1995q; Rowley-Jones and Flind, 1981), suggesting that at therapeutic doses significant inhibition of P450 does occur. Hence, cimetidine appears to induce LCTs via two modes of action: androgen receptor antagonism and testosterone biosynthesis inhibition. This example illustrates the important point that compounds can have more than one type of endocrine activity. Ethanol. In an oncogenicity study, male and female Spague-Dawley (CD) rats were exposed to 5% ethanol in their drinking water for 2 years as part of a larger study examining the modifying effects of ethanol and disulfiram on ethylene dichloride oncogenicity (Cheever et al., 1990). LCT incidence was numerically increased in the ethanol-treated rats when compared with the control group (7/50 vs. 2/50 for the control); however, this difference was not statistically different (Cheever et al., 1990). No other compound-related changes in tumor incidence were reported in male or female rats administered 5% ethanol in the drinking water. Clearly, the increase in LCTs with ethanol is equivocal, but appears to be compound related because the incidence was greater than both concurrent controls and was outside the reported historical control range for this strain of rat (Table 2). If ethanol does induce LCTs, its mode of action is in all likelihood via inhibition of testosterone biosynthesis. Several studies have shown that ethanol decreases serum testosterone levels via decreasing LH levels as well as by inhibition of testosterone biosynthesis (Widenius et al., 1989; Van Thiel et al., 1983; Johnston et al., 1981). The inhibition of serum testosterone levels has clearly been shown to be due to a direct effect on LCs, based on in vivo and in vitro experiements (Van Thiel et al., 1983). In time-course experiments using adult male Long-Evan rats treated with 1.5 g/kg ethanol, Widenius and co-workers (1989) observed that ethanol inhibited testosterone levels before 215


serum LH levels decreased. These data demonstrate that the decrease in serum testosterone levels were initially due to a direct effect on LC production of testosterone. This direct effect may be mediated by increased glucocorticoids that have been shown to suppress LC steroidogenesis by decreasing gonadotropin stimulation of cAMP production and by inhibition of C17a-hydroxylase, one of the enzymes of the testosterone biosynthetic pathway. Lansoprazole. Lansoprazole is a substituted benzimidazole and has been shown to induce LCTs in a Sprague-Dawley rat bioassay, but not in a CD-1 mouse bioassay (up to 600 mg/kg/d) study (Fort et al., 1995). In a 3-month mouse study, lansoprazole produced decreases in sperm count and seminiferous tubular atrophy at high doses (1200 and 2400 mg/kg/d). Similar findings were found in rats dosed at levels of 300 mg/kg/d or higher for 3 months. In a 1-year rat study, there was an increase in LC hyperplasia at 50 mg/kg/d with a single tumor at this dose level. In a 2-year rat study, there was an increased incidence of seminiferous tubular degeneration at 50 mg/kg/d or higher, and increased LC hyperplasia or LCTs at ≥15 mg/kg/d. Decreased plasma testosterone levels were noted in monkeys administered 150 mg/kg/d, but not 50 mg/kg/d for 1 month. In mechanistic studies with Sprague-Dawley rats, there was a decrease in responsiveness to hCG at 2 or 4 weeks after 50 or 150 mg/kg/d. After 4 weeks there was a decrease in testosterone levels (maximal change of 50%), with an increase in LH levels (68% above control). The authors conducted several in vitro studies using isolated LCs from rats, mice, and monkeys to identify where in the testosterone biosynthetic pathway lansoprazole and its metabolites were acting. These studies found that two metabolites were approximately 10 times more potent than the parent compound in inhibiting testosterone biosynthesis and that the most sensitive point for inhibiton was the C27-side chain cleavage enzyme. However, the authors also noted that cholesterol transport could also be affected, a finding seen with omeprazole (Dowie et al., 1988). This is another example of an agent where there are early changes in serum hormones that returned to normal (by approximately 3 months), but subsequently was shown to induce LCTs at 216

2 years. The authors proposed a LC “compensation-decompensation hypothesis” to explain these hormonal findings. Initial exposure to lansoprazol results in inhibition of testosterone biosynthesis followed by a return to homeostasis, which is termed “compensation”. This compensation can occur either by alterations in LH receptor signaling or steroidogenic enzyme induction to restore testosterone to normal levels even though there is constant inhibition by lansoprazole. It is known that homeostatic mechanisms deteriorate in aging animals (Lin et al., 1980). This deterioration would lead to the loss of the ability to overcome the inhibition of testosterone biosynthesis without an increase in LC mass, which the authors coin “decompensation”. The resulting decompensation would result in LC hyperplasia and eventually progression to LCTs. This pattern of hormal changes (inhibition-compensation-decompensation) has been seen with two other inhibitors of testosterone biosynthesis, isradipine (Roberts et al., 1989) and a triazole herbicide (Foster, 1992). Lead acetate. Lead acetate has been reported to induce LC hyperplasia (57%) and LCTs (24%) in Wistar rats when administered at approximately 8 mg/kg/d (Zawirska and Medras, 1968). The authors reported that there were no LCTs seen in the controls, but the control incidence of LC hyperplasia was not stated. These data were judged equivocal because this study had small sample sizes (approximately 20/group) when compared with guideline oncogencitiy study designs that use sample sizes of ≥50/group. Nonetheless, the incidence of LCTs is clearly greater than the spontaneous incidence for the Wistar strain of rat (Table 2), suggesting that this response is compound related. It has subsequently been shown that treatment of Sprague-Dawley rats with 8 mg/ kg/d lead acetate by intraperitoneal injection for 5 days per week decreased plasma and testicular testosterone levels to 20% of control, yet plasma LH levels were decreased to only 68% of control, suggesting an impairment of LC function (Thoreux-Manlay et al., 1995a). Using isolated LCs from Sprague-Dawley rats (in vitro, ex vivo assessments), lead acetate was shown to directly inhibit testosterone production (Thoreux-Manlay et al., 1995b). This decrease in testosterone biosynthesis was also shown by immunohistochemistry analysis to be related to lower expression of


cytochromes P 450scc (CYP11A1) and P 450c17 (CYP17) and 3β-hydroxysteroid dehydrogenase (3β-HSD) (Thoreux-Manlay et al., 1995b). Consistent with these findings, lead acetate has been shown to decrease StAR protein expression in a mouse Leydig cell tumor line, MA-10 (Huang et al., 1997). These data suggest that lead acetate may induce LCTs via downregulation of the enzymes of the testosterone biosynthesis pathway, a mechanism that is distinct from the other testosterone biosynthesis inhibitors described in this section that produce direct inhibition of the cytochrome P450 isoenzymes. Metronidazole. Rustia and Shubik (1979) evaluated metronidazole in a chronic Wistar rat study at dietary levels of 0, 600, 3000, or 6000 ppm. Animals were maintained on test for up to 150 weeks. Metronidazole improved the survival of treated animals compared with controls. LCT incidence at the different dietary levels of metronidazole was 18% (0 ppm), 30% (600 ppm), 20% (3000 ppm), and 46.7% (6000 ppm). Vinclozolin. Vinclozolin has also been shown to inhibit several P450 isozymes from SpragueDawley rats and this inhibition is related to its ability to form a Type II binding spectra (Ronis et al., 1994). If vinclozolin also inhibits testosterone biosynthesis, then vinclozolin would act as an antiandrogen through two mechanisms: androgen receptor antagonism and testosterone biosynthesis inhibition (Section VII.A.1.a), both of which would explain the development of LC hyperplasia in adult male rats.

d. Aromatase Inhibitors Aromatase inhibitors such as formestane and letrozole (Junker-Walker and Nogues, 1994) block the conversion of testosterone to estradiol resulting in a decrease in estradiol and an increase in LH levels (Figure 10). Aminoglutethemide is the most well-studied and widely known aromatase inhibitor, but was not included in Figure 10 because it has not been reported to induce LC hyperplasia or LCTs (Salhanick, 1982; Shaw et al., 1988). Formestane/Letrozole. In subchronic studies, formestane and letrozole have been shown to induce LC hyperplasia in Beagle dogs but not in

rodents. Junker-Walker and Nogues (1994) discuss how estradiol plays a more significant role in the feedback regulation of LH in dogs, primates, and humans than in rodents. Such a difference would account for dogs being more sensitive than rodents for the development of LC hyperplasia in response to chronic aromatase inhibition. However, this generality may not always be true because the authors have experience with two proprietary aromatase inhibitors that produce LCTs in rats after 2 years of treatment but not LC hyperplasia in dogs after 1 year of treatment (J. C. Cook, DuPont, unpublished data).

e. Dopamine Agonists/Dopamine Enhancers Dopamine agonists such as muselergine decrease serum prolactin levels. It has been proposed that this decrease in prolactin levels causes downregulation of LH receptors on LCs (Prentice et al., 1992). This receptor downregulation decreases testosterone production and results in a compensatory increase in serum LH to maintain testosterone at physiologic levels. The sustained compensatory increase in LH has been proposed to result in LC hyperplasia and LCTs (Prentice and Meikle, 1995). Based on the work with oxolinic acid, an alternative mechanism has been proposed, namely, that dopamine agonists/enhancers increase GnRH levels that subsequently increases LH levels (Yamada et al., 1995). The relative contribution of these two mechanisms toward the development of LCTs remains to be determined. Mesulergine. Prentice and co-workers (1992) reported the effects of acute dosages of 0.16, 1.6, and 16 mg mesulergine/kg/d when given to 8-week-old animals. Serum LH and testosterone levels were reduced in a dose-dependent manner. No effect was noted on serum estradiol. In a 2-week study with the same dose levels, no consistent changes in hormonal levels were reported apart from the decreased prolactin levels consistent with the pharmacological action of the drug. In a 4-week study, with the same dosage levels, there was a decrease in testosterone (67% of control at highest dose level) and an increase in LH (approximately 2-fold). After 10 weeks, there was 217


an increase in LH (at 16 mg/kg/d only), but no significant effect on circulating testosterone. After 13 weeks, there were significant increases in LH (50% increase) and an increase in testosterone (56% increase) at 0.16, 1.6, and 16 mg/kg/d. LH receptor levels on the LCs were reduced. No histopathological changes in the testes were noted. In a chronic study using Wistar rats (1.6 mg/ kg/d only), mesulergine increased serum LH levels for the first 79 weeks with less obvious changes thereafter. The combined LCT and hyperplasia incidences were 77% compared with 8% in controls (n = 12). In a 39-week study in aged rats (67 weeks old at start), there was a statistically significant increase in LH after 13 and 26 weeks (233 and 142%, respectively), with no changes in serum testosterone. The authors suggested that old age is a prerequisite for the induction of LCTs in that early changes in LH are noted, but there is no shortening in the onset for the induction of the tumors. In a subsequent study, mesulergine (2 mg/ kg/d) was administered via the diet to SpragueDawley rats for either 5 or 57 weeks, and LC hypertrophy was assessed using [3H]methionine incorporation (Dirami et al., 1996). LC hypertrophy was observed within 5 weeks of treatment followed by hyperplasia and nodules at 57 weeks of treatment. These data are significant because it correlates the progression of LC changes from hypertrophy/hyperplasia/adenomas as would be expected if LH were elevated and driving this response. In fact, increased circulating levels of LH have been demonstrated to occur after 4 weeks of treatment with mesulergine but not earlier (Prentice and Meikle, 1995), which is consistent with LH being the causative agent. Norprolac. Roberts and co-workers (1993) reported on the effects of norprolac (SDZ-205502) when administered at dosage levels of 0, 0.01, 0.06, or 0.2 mg/kg/d for 104 weeks to CD rats. No effects were noted on body weight or food consumption. After 1 year, 2 mid-dose animals (0.06 mg/kg/d) had LC hyperplasia and 1 high-dose animal (0.2 mg/kg/d) had a LCT. At 2 years, the incidence of LCTs was 1, 11, 38, and 45% at 0, 0.01, 0.06, or 0.2 mg/kg/d norprolac, respectively. LC hyperplasia was also increased with incidences of 1, 1, 13, and 11%, respectively, with ascending dosage level. Serum LH levels were increased 1.5- to 3-fold through week 218

88 for the mid- and high-dose groups, while serum testosterone and progesterone levels were unchanged. Decreased serum prolactin levels were seen throughout the study, as was expected due to norprolac being a dopamine agonist. The authors concluded that the mode of action for the induction of LCTs was similar to mesulergine. Oxolinic acid. Yamada and co-workers have published four papers investigating the potential mode of action of oxolinic acid (an antimicrobial containing a methylenedioxyphenyl moiety) in the induction of LCTs. In the first paper (Yamada et al., 1994a), oxolinic acid was fed to Wistar rats (0, 30, 100, 300, or 1000 ppm) or ICR mice (0, 50, 150, or 500 ppm). Decreases in body weight gain were noted in mice, but with no treament-related pathological changes. In the rat bioassay, there was an increase in testicular atrophy, LCTs, and LC hyperplasia at 1000 ppm. The incidence of LCTs was 22% in the 1000 ppm group compared with a 4% incidence in the control group. The authors reported a 6% historical control incidence for LCTs, which is consistent with other laboratories (Table 2). Oxolinic acid was reported as being nongenotoxic in conventional studies to assess genotoxicity. In their second study (Yamada et al., 1994b), the authors described the results from a 104-week study in Wistar rats which were fed diets containing 0, 100, 1000, and 3000 ppm oxolinic acid (0, 4, 43, 145 mg/kg/d, respectively). Statistically significant increases in serum LH were observed in rats at the mid- and high-dose levels when compared with controls (less than 2-fold increases); however, these increases in LH appear to be due more to the absence of the normal rate of decline of LH that is seen in aging rats. Serum testosterone levels in the high-dose group tended to be higher and achieved statistical significance on occasion, but the magnitude of the effect was small. When 3000 ppm oxolinic acid was administered to aged rats for 1 month, serum LH levels were increased statistically, but the magnitude of change was small (156% of control). No compound-related effects were noted on LH clearance when exogenous LH was administered to aged rats that had been treated for 1 month with 3000 ppm oxolinic acid. When haloperidol (D2 receptor antagonist that increases prolactin) was given in conjunction with 3000 ppm oxolinic acid for 1 month, serum LH levels were decreased (to


50% of control levels) and serum prolactin levels were increased (as expected), suggesting that the increase in LH levels by oxolinic acid is mediated by the dopamine pathway. Serum LH elevations after 1 month on 3000 ppm in aged rats returned to normal by 2 weeks after treatment cessation. The proposed mode of action is that oxolinic acid possesses dopamine agonist activity that increases the amount of GnRH released from the median eminence. Increased GnRH levels enhance LH release from the pituitary, which will then induce hyperplasia and tumors. Unfortunately, GnRH and prolactin levels were not measured to unequivocally support their hypothesis. In their third paper (Yamada et al., 1995a), the authors have attempted to further elucidate the mode of action by substituing oxolinic acid with high doses of L-DOPA. L-DOPA will increase dopamine levels, which leads to decreased serum prolactin and an increase in serum LH levels. In this study, serum LH was almost doubled 4 h following a single oral dose of 1 g/kg L-DOPA. Administration for 7 or 14 days at this dose level decreased relative prostate weight, but not testes or seminal vesicle weights. After 14 days of treatment with 500 or 1000 mg L-DOPA/kg, increased serum LH (approximately 2-fold) and decreased PRL (30% of control) levels were seen 24 h after the last dose. Serum testosterone also tended to be higher (up to 2-fold). The results suggested that prolonged treatment with L-DOPA in male rats induced the release of LH from the pituitary resulting in sustained higher levels of LH in the peripheral circulation. The implication of this paper is that oxolinic acid would mimic the effects of L-DOPA. In their fourth paper, microdialysis studies demonstrated that oxolinic acid increases dopamine levels in the preoptic area of the hypothalamus (Yamada et al., 1995b). These data suggest that oxolinic acid can increase dopamine levels that may be enhancing GnRH levels, although they were not measured.

of a non-LH-type mechanism that can induce either LC hyperplasia or LCTs. GnRH agonists induce LCTs in rats by binding to GnRH receptors on LCs. Because LCs from mice and humans do not contain GnRH receptors, these species are believed not to be susceptible to tumor induction by this class of compounds (Donaubauer et al., 1987; Hunter et al., 1982). Buserelin. In a 2-year carcinogenicity study, buserelin did not increase the incidence of LCTs when administered to Wistar rats via the subcutaneous route at dose levels of 0, 0.2, 0.6, or 1.8 µg/ kg/d buserelin (Donaubauer et al., 1987). There was a dose-related increase in the incidence of diffuse LC hyperplasia and an increase in lipid storage within LCs that were hyperplastic. This study also reported an increase in serum testosterone levels in animals and reduced testicular weight with associated effects on the seminiferous epithelium. Histrelin. In a rat chronic study, histrelin was administered at dose levels of 0, 5, 25, or 150 µg/ kg/d by injection (PDR, 1995o). Increases were noted in pancreatic islet cell tumors and LCTs (not dose related). These tumor types were not noted in the chronic study with mice. Fertility effects were reported in male rats and monkeys, which were fully reversible after 6 months of treatment at dose levels up to 180 µg/kg/d. Leuprolide. A chronic study in rats at dose levels of 600 to 4000 µg/kg/d leuprolide by the subcutaneous route resulted in increases in pancreatic islet cell and LCTs (not dose related) (PDR, 1995h). Such tumors were not noted in similar studies in mice. Some changes were reported following dosing with leuprolide in the testes of immature rats that were not reversible. Nafarelin. In a conventional rat study at dose levels of up to 100 µg/kg/d for 24 months, there was an increase in pancreatic islet cell tumors and LCTs in males and ovarian tumors in females (PDR, 1995p). There were no LCTs in the 18-month mouse study at dose levels up to 500 µg/kg/d intramuscularly.

f. GnRH Agonists g. Estrogen Agonists/Antagonists GnRH agonists such as buserelin (Donaubauer et al., 1987; Hunter et al., 1982), histrelin (PDR, 1995o), leuprolide (PDR, 1995h), and nafarelin (PDR, 1995p) are the only documented examples

In contrast to the other modes of action, estrogen agonists/antagonists induce LCTs almost exclusively in the mouse rather than the rat. This 219


difference in response reflects differences in the local effects of estrogens on LCs and the hormonal feedback pathways at the level of the hypothalamus and pituitary between the rat and mouse (Section III; Huseby, 1976; Huseby, 1996). In knockout mice that were deficient in Mullerian-inhibiting substance (MIS) and inhibin, LC hyperplasia and LCTs were observed in these male mice (Matzuk et al., 1995). The authors concluded that inhibins and MIS synergize to influence LCT development. The MIS-deficient male mice are pseudohermaphrodites with oviducts and uteri. The authors also demonstrated that the inhibin/MIS-deficient mice had elevated FSH levels and a subsequent increase in serum estradiol levels. Hence, based on data in this section that estradiol drives LCTs in mice, the development of LCTs in inhibin/MIS-deficient mice is in all likelihood due to the increase in serum estradiol levels secondary to the increase in FSH from the absence of MIS and inhibin. This conclusion is supported by the observation that pure estrogen receptor antagonist ICI-182,780 blocked the uterine fluid retention in these inhibin/MISdeficient male mice. Diethylstilbestrol (DES). DES has been used as a model for the induction of LCTs in mice where the contribution of pituitary hormones has been studied extensively (Huseby, 1976). For instance, DES, when administered by subcutaneous pellet to BALB/c mice, induces LC hyperplasia after 60 days of treatment and LCTs after 120 days of treatment (Baroni et al., 1966). Indeed, it has been shown that the response to estrogens is entirely different in the rat compared with the mouse to such an extent that the induction of hyperprolactinemia by giving a DES implant subcutaneously will reduce the incidence of LCTs in the aged F344 rat (Bartke et al., 1985). DES will also induce LCTs in the European and Syrian hamster (Reznik-Schuller, 1979). In these experiments DES was given as a subcutaneous implant (replaced every 5 months) containing 25 mg DES until the animals died. Incidences of LCTs for the European hamster were 0% (control) and 54% (DES) and for the Syrian hamster were 0% (control) and 10% (DES). All of the LCTs were found in animals possessing pituitary tumors, but pituitary tumors were also noted in animals without LCTs. 220

Estradiol. Estradiol has been shown to have a direct carcinogenic effect on the LCs of the mouse (Huseby, 1980). In these experiments, castrated BALB/c mice were grafted with the testis from a 1-day-old isologous animal (into the spleen) where a pellet containing 5 mg of 20% 17βestradiol: 80% cholesterol (w/w) was deposited near the graft. After 1 year of treatment, no tumors were seen in the controls, but in the estradiol pellet-treated mice LCTs or LC hyperplasia were observed in more than 80% of the animals. Placing the estradiol pellets at a distant site failed to produce LCTs. Ethinylestradiol (EE). Yasuda and co-workers (1986) have shown that EE will induce LC hyperplasia in fetal BALB/c mice treated transplacentally. Pregnant mice were given EE on days 11 to 17 of gestation. Dams were treated with 0, 0.02, 0.2, or 2 mg/kg/d EE by gavage. On day 18 the dams were sacrificed and the male offspring examined. Three out of 12 males from dams at the highest dose level showed cryptorchid testes with uterine tubes. Microscopically, LC hyperplasia was evident in both cryptorchid and normally descended testes with a dose-dependent increase in LC number. However, the number of LCs in the cryptorchid testes were less when compared with those from normally descended testes. Using the same protocol described above, these authors also showed that a dose of 0.02 mg/ kg resulted in an apparent delay in delivery (dystocia) as well as cannabilism of the pups by the dams (Yasuda et al., 1988). Of the four litters surviving, offspring were kept to 20 to 22 months of age. In the males from EE-treated dams, atrophy of the seminiferous tubules (5/6 mice) and LC hyperplasia (4/6 mice) were seen; none of these lesions were observed in the control BALB/c mice. These experiments demonstrated that the exposure to 10 times the therapeutic dose of EE induces developmental defects in the male reproductive organs that leads to sterility and to precancerous lesions when exposure occurs during the critical time for testicular differentiation. Methoxychlor. Methoxychlor is a structural analog of DDT. BALB/cJ and C3H (C3 HeB/FeJ) mice were fed diets containing 750 ppm methoxychlor for 2 years (Reuber, 1979). Methoxychlor induced LCTs in BALB/cJ mice but not in C3H mice. The incidence of LCTs in the BALB/cJ mice ingesting 750 ppm methoxychlor was 53%


(27/51) vs. 11% (8/71) in the control mice. Signetin. Signetin is a synthetic analog of DES used in the treatment of toxicosis in Russia. Outbred pregnant C57XCBA rats were treated subcutaneously with 0, 0.2, 2, and 20 mg signetin on the last 3 days of embryogenesis. Offspring were followed from birth to natural death (up to 32 months) (Ird, 1983). The incidence of LCTs was 2.2% (2/89), 30.4% (7/23), 12.5% (2/16), and 0% (0/16) in the 0, 0.2, 2, 20 mg signetin groups, respectively. The first LCTs in the signetin-treated rats was detected 17 months after treatment. The incidence of LCTs was statistically increased in the 0.2-mg signetin group when compared with the control. The reason for the inverse dose-response was not stated but may be due to the suppression of LH levels that results in decreased testosterone levels and testicular atrophy, both of which are commonly seen at high doses of estrogens (Biegel et al., 1995). Tamoxifen. In a mouse oncogenicity study, tamoxifen was administered by gavage at 0, 5, or 50 mg/kg/d for 3 months and then administered in the diet for an additional 12 months at levels that achieved similar mean daily intake levels (Tucker et al., 1984). LCT incidence was increased in the tamoxifen-treated Alderley Park mice: 0% (0 mg/ kg/d), 8% (5 mg/kg/d), and 84% (50 mg/kg/d). A chronic study in the Wistar rat did not report an increase in LCTs, but did report testicular atrophy (Greaves et al., 1993). Tri-ρ-anisyl-chloroethylene (TACE). TACE is a synthetic estrogen that was administered to C57 X CBA hybrid male mice weekly by subcutaneous injection at either 50 or 100 µg for up to 850 days (Gardner and Boddaert, 1950). The incidence of LCTs in the TACE-treated mice was 50% (46/92). There was no concurrent control included in this study; however, the authors reported that only three cases of LCTs had been seen in the colony of mice from this strain. The sample size and age of histologic examination for this historical data were not mentioned. Triphenylethylene. Beginning at 4 months, 10 Strong A male mice received weekly subcutaneously injections of 3 mg triphenylethylene (Bonser, 1942). Rats were treated until a natural death occurred (up to 75 weeks). Of the mice surviving greater than 50 weeks, 8/8 had bilateral LCTs. There was no concurrent control included

in this study or did the author report on the spontaneous incidence of LCTs for this mouse strain.

2. Classified by Chemical Activity a. Antihypertensives Guanadrel. Guanadrel is an orally active antihypertensive that enters the neuron via the norepinephrine pump. Once inside the neuron, guanadrel inhibits norepinephrine uptake and release. This inhibition results in depletion of norepinephrine from the nerve ending causing the desired pharmacologic activity (PDR, 1995f). In a 2-year chronic study in rats, guanadrel produced a dose-dependent increase in the incidence of LCTs at dose levels of 100 or 400 mg/kg/d (PDR, 1995f), although the incidence was not stated. In a reproduction study, guanadrel reduced libido at 30 mg/kg/d, and both fertility and libido were reduced at 100 mg/kg/d. There were no indications of LCTs or any other tumors in the 2-year mouse oncogenicity study. The mechanism by which guanadrel induces LCTs has not been investigated. If LC tumor induction by guanadrel was shown to be related to its pharmacologic activity, it would represent another mechanism for LCT induction. Hydralazine. Hydralazine is an antihypertensive, whose mechanism of action is not fully understood, but has been shown to lower blood pressure by exerting a peripheral vasodilating effect (PDR, 1995a). This vasodilating effect appears to be mediated by a direct relaxation of vascular smooth muscle. In a 2-year chronic study, rats were administered hydralazine by gavage at 0, 15, 30, and 60 mg/kg/d. LC and liver tumors were statistically increased in the male 60 mg/ kg/d dosage group. In a lifetime study in Swiss albino mice, hydralazine did not induce LCTs at dosages up to 250 mg/kg/d, but did increase the incidence of lung tumors at this dosage. No fertility studies have been conducted with hydralazine. The weight of evidence suggests that hydralazine is not genotoxic, although it was positive in some in vitro assays.

b. Calcium Channel Blockers Felodipine, Isradipine, Lacidipine, and Nimodipine. This class of compounds has been 221

discussed in the preceding section (Section VII.A.1.c). They appear to induce LCTs via inhibition of testosterone biosynthesis.


c. Fungicides Procymidone. Procymidone has been discussed in the preceding section (Section VII.A.1.a). Procymidone appears to induce LCTs via an androgen receptor antagonist mechanism. Vinclozolin. Vinclozolin has been discussed in the preceding sections (Sections VII.A.1.a and l.c). Vinclozolin appears to induce LCTs via a combination of two mechanisms: inhibition of testosterone biosynthesis and androgen receptor antagonism. The potency of vinclozolin may be related to its ability to act at two different pathways to disrupt the HPT axis. Folpet. Folpet is a chloroalkylthiodicarboximide fungicide that produced LCTs in Sprague-Dawley (CD) rats but not in two strains of mice (CD-1, B6C3F1), even though the dietary intake was greater in the mouse oncogenicity studies (Quest et al., 1993). Duodenal adenomas/carcinomas were seen in the two strains of mice fed diets containing folpet. The incidence of LCTs in the 2 year CD rat study was 2% (1/50), 10% (5/50), 8% (4/50), and 16% (8/50) in the 0, 200, 800, and 3200 ppm folpet groups, respectively. The LCT incidence was statistically increased only in the 3200 ppm folpet group. Folpet was not reported to induce LCTs in F344 rats. This negative finding is probably due to the F344 rat having a high spontaneous incidence of LCTs that confounds detection of LC tumorigens. This conclusion is based on the experience from datasets with methyl tertiary butyl ether (MTBE) and trichloroethylene (TCE), both of which were tested in strains of rats exhibiting a low- or high-spontaneous incidence of LCTs (see below).

d. Goitrogens The studies with ethylenethiourea (ETU) and 6-n-propyl-2-thiouracil (PTU) suggest that goitrogens may affect LC proliferation; however, the data for LCT induction are equivocal for ETU. 222

Clearly, more work is necessary to determine whether goitrogens as a class induce LCTs or whether it is compound specific. Interestingly, oxazepam induces liver, LC, and thyroid tumors, presumably via liver enzyme induction. It may be that goitrogens that produce enzyme induction rather than agents that inhibit thyroid hormone synthesis are more commonly associated with inducing LCTs. Ethylenethiourea (ETU). In a 2-year feeding study, ETU was reported to induce LCTs in rats at levels that induced thyroid tumors (Gak et al., 1976). However, it is difficult to judge this finding because tumor incidence was not reported. 6-n-Propyl-2-thiouracil (PTU). It is interesting that PTU, when administered neonatally, induces LCH in Sprague-Dawley rats (MendisHandagama and Sharma, 1994; Chapin et al., 1996). The neonatally PTU-treated rats have normal androgen status based on measurement of serum hormone levels, and studies with isolated LCs from such animals show that they have compensated by reducing steroid secretion and responsiveness to LH (Hardy et al., 1993). This hypotrophy of steroid production appears to be due to lower LH levels (Hardy et al., 1993) (see also Section IV). Hardy and co-workers (1996) have subsequently shown that proliferation of LCs rather than the proliferation of their mesenchymal precursors is responsible for the increase in LC number following neonatal hypothyroidism. The LC hyperplasia can be related to two potential mechanisms either hypothyroidism and/or the increased Sertoli cell population (Hardy et al., 1996).

e. Peroxisome Proliferators A large number of structurally and chemically diverse compounds have been shown to cause peroxisome proliferation, the induction of peroxisomal enzymes, and hepatocellular carcinoma (Ashby et al., 1994; Bentley et al., 1993; Gibson and Lake, 1993; Stott, 1988). In addition to the hepatic effects, several peroxisome proliferators have been shown to induce LCTs in rats: ammonium perfluorooctanoate (C8) (Cook et al., 1994; Sibinski, 1987), clofibrate (Tucker and Orton, 1995), gemfibrozil (Fitzgerald et al., 1981), HCFC-


123 (Malley et al., 1995), methylclofenapate (Tucker and Orton, 1995), perchloroethylene (Mennear, 1986), trichloroethylene (Maltoni, 1988; Mennear, 1988), and Wyeth-14,643 (WY) (Cook et al., 1994). Peroxisome proliferators do not induce peroxisomes in LCs, and, hence, may induce LCs tumors via a different mechanism than in the liver where peroxisome proliferation is seen (Biegel et al., 1992; Cook et al., 1994). Ammonium perfluorooctanoate (C8). C8 has been shown to produce LCTs in SpragueDawley (CD) rats in two oncogenicity studies. In a 2-year feeding study, C8 produced a dose-dependent increase in LCTs where the incidence was 0, 6, and 14% at 0, 30, and 300 ppm C8, respectively (Sibinski, 1987). In a mechanistic bioassay (discussed below) C8 was again shown to induce LCTs: 0% (0/80), 2.6% (2/78), and 10.5% (8/76) in the ad libitum control, pair-fed control, and 300 ppm C8 groups, respectively (Cook et al., 1994). In the second oncogenicity study, C8 was also shown to increase the incidence of hepatocellular adenoma/carcinoma (3, 4, and 13% for the ad libitum control, pair-fed control, and C8 groups, respectively) and pancreatic acinar cell adenomas (0, 1, and 9% for the ad libitum control, pair-fed control, and C8 groups, respectively) (Cook et al., 1994). Administration of C8 to adult male rats by gavage for 14 days was shown to decrease testosterone levels and increase serum estradiol levels (Cook et al., 1992). These endocrine changes were hypothesized to play a role in the C8-induction of LCTs. Subsequently, C8 has been shown to directly inhibit testosterone production when incubated with isolated LCs, while ex vivo studies demonstrated that this inhibition was reversible (Biegel et al., 1995). Several other peroxisome proliferators have been shown to inhibit testosterone production using isolated LCs (Liu et al., 1996a). This inhibition of testosterone biosynthesis may contribute to the development of LC tumorigenesis through disruption of the HPT axis. However, in a mechanistic bioassay with C8 and WY, serum testosterone and LH levels were not significantly altered at the levels of C8 and WY that were tested (Cook et al., 1994). C8 and WY have also been shown to produce a sustained increase in serum estradiol levels over 2 years, and these increases correlate with their

potency to induce LCTs in rats (Cook et al., 1994). A non-LH-type mechanism has been proposed for the induction of LCTs by peroxisome proliferators, where estradiol modulates growth factor expression in the testis to produce LC hyperplasia and neoplasia (Biegel et al., 1995; Cook et al., 1992). Several components of the estradiol hypothesis have been tested and are summarized as follows: (1) peroxisome proliferators induce hepatic aromatase activity (Biegel et al., 1995; Liu et al., 1996b), (2) hepatic aromatase induction increases serum estradiol levels (Biegel et al., 1995; Cook et al., 1992; Cook et al., 1994; Liu et al., 1996b), which increases testis estradiol levels (Biegel et al., 1995), (3) increased interstitial fluid estradiol levels modulate growth factors, specifically TGFα, within the testis (Biegel et al., 1995), and (4) altered growth factors stimulate LC proliferation. The last point in the hypothesis remains to be tested. However, in the mammary gland, estradiol has been shown to stimulate TGFα secretion and overexpression of TGF( has been associated with the proliferation of mammary epithelial cells and subsequent neoplasia (Liu et al., 1987). TGFα has also been shown to stimulate thymidine incorporation into LC precursors and appears to be a LC stimulant (Khan et al., 1992a) (Section III). Conflicting evidence exists for the role of estrogens in the development of LCTs in rats (Section VII.1.g). Estrogenic compounds do not induce LCTs in rats when given at doses that produce testicular atrophy, which can confound detection of LC hyperplasia (Gibson et al., 1967; Marselos and Tomatis, 1993; Schardein et al., 1970; Schardein, 1980). These earlier studies were also limited by small sample size and reduced survival. Interestingly, GnRH agonists induce LCTs at low doses but do not induce LCTs at higher doses where LH levels are suppressed and testicular atrophy occurs (PDR, 1995o; PDR, 1995h; PDR, 1995p; Donaubauer et al., 1987; Hunter et al., 1982) (Section VII.1.f). Hence, these negative bioassays with estrogenic compounds may be due to suppression of LH, which to date is the primary demonstrated “driver” of LCTs. In support of this conclusion, the DES analog, signetin, induces LCTs at low doses, but this response returns to control levels at the highest dosage (Section VII.A.1.g). Estradiol also appears 223


to play a role in enhancement of LC tumorigenesis based on data from aging studies. In F344 rats, which have a high spontaneous incidence of LCTs, there is an age-related increase in serum estradiol that correlates with the development of LC hyperplasia and tumor formation (Turek and Desjardins, 1979). However, in the CD rat, which has a low spontaneous incidence of LCTs, serum estradiol decreases with age (Cook et al., 1994). In a 2-year rat mechanistic bioassay, C8 and WY produced a sustained increase in serum estradiol levels that correlated with the potency of the two compounds to induce LCTs (Cook et al., 1994). These studies suggest that estradiol may play a role in enhancement of LCTs in the rat, and that peroxisome proliferators may induce LCTs via a non-LH type mechanism. Whether estradiol plays a role in the induction of LCTs by peroxisome proliferators can only be determined from an estradiol bioassay conducted at levels that do not induce testicular atrophy or reduce LH levels. Clofibrate. Clofibrate is believed to act as a hypolidemic agent via a number of mechanisms, namely, inhibition of hepatic release of lipoproteins (particularly VLDL), potentiation of lipoprotein lipase activity, and enhancement of fecal excretion of neutral sterols (PDR, 1995b). In a rat oncogenicity study (PDR, 1995b), clofibrate has been shown to induce LCTs in Alderley Park (Wistar-derived) rats when given at a dosage of 400 mg/kg. Arrest of spermatogenesis has been seen in both dogs and monkeys treated with clofibrate at dosages ranging from 160 to 240 mg/kg. In other oncogenicity studies conducted at a single dosage, clofibrate has been shown to induce liver tumors in mice (320 mg/kg) and rats (200 mg/kg) (PDR, 1995b). In studies using the Alderly Park (Wistar-derived) rat and two strains of mice (outbred albino Swiss and C57Bl/10J), clofibrate did not induce hepatic or LCTs tumors at dosages that approached those reported above (Tucker and Orton, 1995). No mechanistic data have been performed to understand why clofibrate induces LCTs. Diethylhexylphthalate (DEHP). In a lifetime feeding study, DEHP was administered to male Sprague-Dawley rats at dietary concentrations that achieved a mean daily intake of 30, 95, or 300 mg/kg/d of DEHP (Berger, 1997). DEHP induced LCTs but did not produce liver tumors. 224

Unfortunately, this report is based on an abstract and few experimental details were given. Gemfibrozil. In a 2 year oncogenicity study with Sprague-Dawley (CD) rats, gemfibrozil produced a dose-dependent increase in LCTs where the incidence was 2% (1/50), 16% (8/50), and 34% (17/50) at dosages of 0, 30, or 300 mg/kg/d gemfibrozil, respectively (Fitzgerald et al., 1981). Gemfibrozil also increased the incidence of hepatocellular carcinoma in male rats at both dosage levels. In an 18-month oncogenicity study with CD-1 mice, gemfibrozil (0, 30, 300 mg/kg/d) did not increase the incidence or decrease the latency of onset of tumors. HCFC-123. In a 90-day subchronic inhalation study with Sprague-Dawley (CD) rats, the alternative fluorocarbon, HCFC-123, was shown to be a peroxisome proliferator at levels ≥300 ppm based on measurement of hepatic β-oxidation activity (Rusch et al., 1994). The potential chronic toxicity and oncogenicity of HCFC-123 was also evaluated by exposing male and female CD rats to 0, 300, 1000, or 5000 ppm HCFC-123 for 6 h/d, 5 d/week, for 2 years (Malley et al., 1995). The incidence of LCTs was statistically increased in all the HCFC-123-treatment groups: 6% (4/67), 18% (12/66), 14% (9/66), and 21% (14/66) at 0, 300, 1000, and 5000 ppm HCFC123, respectively. Pancreatic acinar cell adenomas were increased in all the male test groups and acinar cell hyperplasia was increased in the 1000 and 5000 ppm male and female groups. Hepatocellular adenomas were increased in the 5000 ppm males and in all test groups of females. The ability of HCFC-123 to induce extrahepatic tumors in the testis and pancreas has been seen with other peroxisome proliferators, including C8, methylclofenapate, and WY. HCFC-123 did not produce developmental or reproductive toxicity, but did produce a decrease in offspring weight gain during lactation (Malinverno et al., 1996). This lactation effect subsequently has been shown to be related to peroxisome proliferation within the pups from HCFC-123 and trifluoroacetic acid (TFA) exposure in the milk. Methylclofenapate. In a 2-year oncogenicity study with Alderley Park (Wistar-derived) rats, methylclofenapate was administered in the diet at 0, 10, 50, or 250 ppm (Tucker and Orton, 1995). At the end of 2 years, methylclofenapate pro-


duced dose-dependent increases in LCTs where the incidence was 4.2% (1/24), 12.5% (3/24), 40% (10/25), and 39% (9/23) at 0, 10, 50, and 250 ppm, respectively. Methylclofenapate also increased the incidence of hepatocellular carcinoma and pancreatic acinar cell adenomas in the male 50- and 250-ppm methlyclofenapate groups. In contrast to the males, the methlyclofenapatetreated female rats only had an increased incidence of hepatocellar carcinoma in the 250-ppm group. A reversibility experiment was also conducted in male rats, where it was found that for all three tumor types more than 12 months of treatment with methylclofenapate was necessary for the induction of tumors (Tucker and Orton, 1995). In an 18-month oncogenicity study with Alderley Park mice (0, 10, 50, and 100 ppm methylclofenapate), methylclofenapate increased the incidence of hepatocellar carcinoma in all the treated male groups and only the 50- and 100-ppm female groups; no LCTs or pancreatic acinar cell tumors were seen (Tucker and Orton, 1995). Perchloroethylene (PCE). PCE is used as a dry cleaning agent and industrial solvent. The potential oncogenicity of PCE was evaluated by exposing male and female F344 rats to 0, 200, and 400 ppm PCE for 6 h/d, 5 d/week, for 2 years (NTP, 1986). The overall incidence of LCTs was 70% (35/50), 80% (39/49), and 82% (41/50) in the 0, 200, and 400 ppm PCE groups, respectively. The terminal rates (animals that survived to 2 years) of LCTs were 87% (20/23), 95% (19/20), and 100% (12/12) in the 0, 200, and 400 ppm PCE groups, respectively. Neither the overall nor terminal rates of LCTs in the PCEtreated rats were statistically different from the control. The NTP report concluded that this marginal increase in LCTs was not compound related. However, because of the reduced survival in 400 ppm PCE group and the difficulty in detecting compound-related LCTs in F344 rats, the LCT findings were judged by the reviewers to be equivocal but potentially compound related based on the following. As was demonstrated with folpet (Quest et al., 1993), TCE (Maltoni et al., 1988), and MTBE (Belpoggi et al., 1995), all of which were judged by the NTP to not induce LCTs in F344 rats, these compounds were subsequently shown to induce LCTs when tested in strains of rats with a low spontaneous incidence of LCTs.

Hence, PCE may also induce LCTs if tested in a rat with a low spontaneous incidence of LCTs. In a 2 year inhalation oncogenicity study with B6C3F1 mice (0, 100, and 200 ppm PCE for 6 h/d, 5 d/week), PCE did not induce LCTs but did increase the incidence of hepatocellular carcinoma in the 100- and 200-ppm male and female groups. Trichloroethylene (TCE). In a 2-year gavage study with four strains of rats, TCE was administered at 0, 500, and 1000 mg/kg/d for 5 d/ week to male and female ACI, August, Marshall, and Osborne-Mendel rats (Mennear, 1988). TCE produced a dose-related increase in LCTs only in the Marshall rats that was statisically elevated at the high dose. The incidence of LCTs in the Marshall rats was 34.8% (16/46), 34.8% (17/46), 43.7% (21/48), and 66.6% (32/48) in the untreated control, vehicle-treated control, 500, and 1000 mg/kg TCE groups, respectively. There were no other tumors seen in the four strains of rats. A 2-year gavage study was also conducted in F-344 rats using the same dosages as described above, but LCTs were not seen (Wilmer et al., 1994). In inhalation studies, the potential oncogenicity of TCE was evaluated by exposing male and female Sprague-Dawley rats for 2 years, and Swiss and B6C3F1 mice for 18 months to 0, 100, 300, or 600 ppm TCE for 7 h/d, 5 d/week (Maltoni et al., 1988). In the Sprague-Dawley rats, TCE produced a dose-dependent increase in LCTs where the incidence was 4.4%, 12.3%, 23.1%, and 23.8% at concentrations of 0, 100, 300, and 600 ppm TCE, respectively. A low incidence (3.1%) of renal tubuli adenocarcinoma was seen in the male 600ppm TCE group. TCE did not increase the incidence of LCTs in either strain of mice. In both the Swiss and B6C3F1 mice, small numerical increases in hepatocellular adenomas were observed. Wyeth-14,643 (WY). In a mechanistic bioassay study using Sprague-Dawley (CD) rats, WY was administered in the diet at 50 ppm through test day 300, but was reduced to 25 ppm for the remainder of the study due to mortality (Cook et al., 1994). At the end of 2 years, WY produced a statistically significant increase in LCTs where the incidence was 0% (0/80) and 24% (16/67) at 0 and 50 (25) ppm, respectively. Similar to C8, HCFC-123, and methylclofenapate, WY also increased the incidence of hepatocellular adenoma/ carcinoma (25% vs. 3% for the control) and pan225


creatic acinar cell adenomas (37% vs. 0% for the control). The potential mechanism(s) for peroxisome proliferator-induced LCTs have been discussed above with C8. WY also produced a sustained increase in serum estradiol levels. This increase in serum estradiol levels may be due to increased aromatase expression and/or decreased expression of enzymes that inactivate estradiol, CYP2C11 (Corton et al., 1997).

3. Classified by Chemical Class a. Fluorochemicals Alternative fluorochemicals such as hydrochlorofluorocarbons (HCFCs) and hydrofluorocarbons (HFCs) are being developed to replace the ozone-depleting chloroflurocarbons (CFCs). The alternative fluorochemicals, HCFC-123 (Malley et al., 1995), HCFC-133a (Longstaff et al., 1984), HCFC-141b (Turnbull et al., 1994), and HFC-134a (Collins et al., 1995), have been shown to induce LCTs in either gavage or inhalation bioassay studies with rats. HCFC-123 has been shown to be a peroxisome proliferator (Rusch et al., 1994) and may induce LCTs by a mechanism(s) similar to other peroxisome proliferators (Section VII.A.2.e), although mechanistic data for such a linkage have not been published with HCFC-123. HCFC-133a produces testicular atrophy possibly by formation of trifluoroacetaldehyde (Ellis et al., 1995). It is possible that testicular atrophy may induce LCTs via alteration of paracrine factors; however, this is purely conjecture, because the study of the role of paracrine factors in LCTs is in its infancy. HFC-134a and HCFC-141b are not peroxisome proliferators or do they produce testicular atrophy, so other potential mechanisms need to be explored. Based on these data to date, there does not appear to be a unifying mechanism for the induction of LCTs by these alternative fluorochemicals. HCFC-123. HCFC-123 has been discussed in the preceding section (Section VII.A.2.e). The induction of LCTs by HCFC-123 appears to be related to its ability to be a peroxisome proliferator. HCFC-133a. In a modified bioassay study design, Wistar rats were administered 300 mg/kg HCFC-133a by corn oil gavage for 5 days per 226

week for 1 year and then sacrificed at 2 years (Longstaff et al., 1984). There was a significant increase in the incidence of LCTs in the 300 mg/ kg/d HCFC-133a-treated rats (29/36, 80.6%) when compared with the controls (16/104, 15.4%). A high degree of seminiferous tubule atrophy was also observed in the HCFC-133a-treated male rats. The incidence of uterine adenocarcinoma in HCFC-133a-treated female rats was increased when compared with the current control (1% control vs. 42% HCFC-133a group). HCFC-133a was negative in a wide range of genotoxicity tests. HCFC-133a is metabolized to trifluroacetaldehyde (Ellis et al., 1995), a known testicular toxicant (Lloyd et al., 1988). Ellis and co-workers (1995) postulated that the testicular toxicity and formation of LCTs by HCFC-133a occurs following the formation of trifluroacetaldehyde. HFC-134a. In a 2 year inhalation study, male and female Wistar rats were exposed to atmospheres containing 0, 2,500, 10,000, or 50,000 ppm HFC-134a for 6 h/d, 5 d/week (Collins et al., 1995). At the highest concentration, there were no effects on body weight, food consumption, or clinical signs. However, there was a significant increase in the incidence of LC hyperplasia and tumors at 50,000 ppm HCFC-134a. The incidences (includes 1 year interim sacrifice rats) for LC hyperplasia were 31% (0 ppm), 29% (2500 ppm), 36% (10,000 ppm), and 47% (50,000 ppm). The comparable incidences for LCTs were 10%, 8%, 14%, and 27% in ascending order of exposure concentration. HFC-134a was judged to be nongenotoxic based on negative results from a battery of in vitro and in vivo genotoxicity tests. HCFC-141b. In a 2-year inhalation study, male and female Sprague-Dawley (CD) rats were exposed to atmospheres containing 0, 1500, 5000, or 20,000 ppm HCFC-141b for 6 h/d, 5 d/week (Turnbull et al., 1994; Millischer et al., 1995). There was a significant increase in the incidence of LCTs in the 5000 and 20,000 ppm HCFC-141b groups. The incidences for LCTs were 4.3% (3/70), 5.7% (4/70), 20% (14/70), and 17.1% (12/70) in the 0, 1500, 5000, and 20,000 ppm HCFC-141b groups, respectively. HCFC-141b was judged to be nongenotoxic based on negative results from a battery of in vitro and in vivo genotoxicity tests (Hodson-Walker et al., 1993; Millischer et al., 1995).


b. Nitroaromatics and Related Compounds p-Nitrochlorobenzene (NTIS, 1980), nitroglycerin (also known as trinitroglycerin) (PDR, 1995j; Ellis et al., 1984), and 2,4-toluenediamine (Cardy, 1979) have been shown to induce LCTs in bioassays with rats. Nitroglycerin has also been tested in mice and did not induce LCTs. To date, no mechanistic data have been performed with these compounds. Interestingly, p-nitrochlorobenzene and nitroglycerin induce methemoglobinemia, which suggests a capability to interact with heme iron. Linuron, which has been shown to be an androgen receptor antagonist, also induces methemoglobinemia (Section VII.A.1.a). Because testosterone biosynthesis is catalyzed partly by P450 isozymes that contain heme, it is possible that these compounds may inhibit steroidogenesis. Besides inducing LCTs, 2,4toluenediamine (Cardy, 1979) also induces liver and pancreatic acinar cell tumors, a tumor pattern that is seen many times with peroxisome proliferators (Cook et al., 1994). Hence, 2,4toluenediamine may be inducing LCTs via a peroxisome proliferator-like mechanism although this has not been investigated. r-Nitrochlorobenzene (PNCB). PNCB was tested in a combined chronic toxicity/oncogenicity study in Sprague-Dawley rats (NTIS, 1980). Rats were administered by gavage 0, 0.1, 0.7, or 5.0 mg/kg/d PNCB for 24 months. Control rats received a corn oil vehicle at a dose volume of 5 ml/kg. At the end of 2 years, PNCB produced statistically significant increases in LCTs at the mid- and high-dosage groups, where the incidence was 1.7% (1/60), 6.8% (4/59), 8.3% (5/60), and 10% (6/60) in the 0, 0.1, 0.7, and 5.0 mg/kg/d PNCB groups, respectively. The only other compound-related finding was a dose-related increase in methemoglobinemia in male and female rats treated with 0.7 or 5.0 mg/kg/d PNCB. Nitroglycerin/Trinitroglycerin (TNG). TNG is used in the treatment of angina via its ability to produce vasodilation, as an explosive in dynamite, and as a solid propellant for firearms and rockets (Ellis et al., 1984). Hydralazine (Section VII.A.2.a) has a similar pharmacology (i.e., vasodilation) to TNG and also induces LCTs. Sprague-Dawley (CD) rats and CD-1 mice were fed diets containing 0, 100, 1000, and 10,000 ppm

TNG for 2 years (Ellis et al., 1984). In these 2-year studies, the dietary administration of TNG at 10,000 ppm is equivalent to 363 and 434 mg/ kg/d in male and female rats, respectively, and 1022 and 1058 mg/kg/d in male and female mice, respectively. After 2 years, TNG produced doserelated increases in neoplastic nodules/hepatocelluar carcinoma in both sexes of rats that were statistically increased in the 10,000 ppm male (71.4% vs. 4.2% in the control) and female (64% vs. 0% in the control) groups. The incidence of LCTs was also statistically increased in the 10,000 ppm male group (52.4% [11/21] vs. 8.3% [2/24] in the control). A dose-related decrease in pituitary adenomas and mammary tumors were seen in the TNG-treated female rats. Although no mechanism was stated, the tumor pattern in the male and female rats suggest that TNG may be reducing serum estradiol levels or decreasing prolactin secretion similar to dopamine agonists (Section VII.A.1.e). In the 2-year oncogenicity study with CD-1 mice, TNG was not tumorigenic at dietary levels up to 10,000 ppm. In a 1-year Beagle dog study (0, 1, 5, or 25 mg/kg/d), the only evidence of chronic toxicity was a dose-related methemoglobin (Ellis et al., 1984). In a rat three-generation reproduction study (dosages up to 434 mg/kg/d), infertility was seen in the high-dose male F1- and F2-generation rats (PDR, 1995j). This infertility was judged to be due to aspermatogenesis secondary to the development of LCTs. No evidence of developmental effects were seen in this multigeneration study or in developmental studies in rats (up to 80 mg/ kg/d) and rabbits (up to 240 mg/kg/d) (PDR, 1995j). TNG also produces methemoglobinemia, as is seen with many other organic nitrates (Ellis et al., 1984). 2,4-Toluenediamine (TDA). TDA was fed to F344 rats at dietary levels of 50 and 100 ppm for 2 years (Cardy, 1979). TDA produced numerical increases in LCTs, where the incidence was 75% (15/20), 90% (45/50), and 88% (44/50) at 0, 10, 50, and 100 ppm, respectively. The poor survival in the high-dose group (no rats survived past 18 months) compares to the control (75% survival at 25 months) tenuous, suggesting that the higher incidence of LCTs in the TDA-treated rats is compound related. TDA also increased the incidence of hepatocellular carcinoma and pancre227


atic acinar cell adenomas in the male 50- and 100ppm groups. This tumor pattern is common to many peroxisome proliferators (Section VII.A.2.e) and suggests that TDA may be a peroxisome proliferator, although no evidence was presented. In contrast to the males, the TDA-treated female rats only had an increased incidence of hepatocellar carcinoma at the 100-ppm group and a dose-related increase in the incidence of mammary tumors.

c. Organochlorines 1,1-Dichloro-2,2-bis(r-chlorophenyl)ethane (o,p′-DDD). o,p′-DDD (Lacassagne and Hurst, 1965) has been reported to induce LCTs in rats; however, it is difficult to judge these findings because tumor incidence data were not reported. 1,1,1-Trichloro-2,2-bis(r-chlorophenyl) ethane (o,p′-DDT). o,p′-DDT (Fitzhugh and Nelson, 1947) has been reported to produce proliferative lesions in the testes of Osborne-Mendel rats, which suggests either an increased incidence of LC hyperplasia or LCTs. It is difficult to judge these findings because tumor incidence was not shown. o,p′-DDT did not produce LCTs in mice; however, the concentrations used were lower than in the rat bioassay (Reuber, 1979). 1,1-dichloro2,2-bis(ρ-chlorophenyl)ethylene (p,p′-DDE), which is a metabolite of o,p′-DDT, appears to possess androgen receptor antagonist activity (Kelce et al., 1995). Hence, o,p′-DDT may induce LCTs in rats through an androgen receptor antagonist mechanism through its metabolite, p,p′-DDE.

d. Sugars Lactose. Lactose has been shown to induce LCTs (Sinkeldam et al., 1992; Woutersen, 1987). Wistar rats chronically fed diets containing 20% lactose had significant increases in both the incidence (24 vs. 4% in controls) and multiplicity of LCTs (Sinkeldam et al., 1992). Lactitol. Lactitol has been shown to induce LCTs in Wistar rats (Sinkeldam et al., 1992; Woutersen, 1987). Wistar rats chronically fed diets containing 0, 2, 5, or 10% lactitol had a dose228

dependent increase in the incidence of LCTs: 4.2% (2/48), 4% (2/50), 8.3% (4/48), and 22.4% (11/49), respectively (Sinkeldam et al., 1992). Bar (1992) proposed two possible mechanisms for LCT induction by lactose and lactitol: (1) increased calcium absorption that would alter the calcium homeostatic hormones, calcitonin, and parathyroid hormone, potentially affecting prolactin secretion, or (2) blockage of steroid enterohepatic recycling presumably estradiol and/or testosterone. De Groot and co-workers (1995a) investigated whether the hypercalcemia and subsequent LCT induction are related to the acidifying effects of slowly digestible carbohydrates such as lactose. Their findings demonstrated that acidic end products or carbohydrate fermentation do not appear to influence LCT induction. Hence, alteration of enterohepatic recycling of steroids may be a more plausible mechanism. Tara gum. Tara gum has been shown to induce LCTs in F344 rats after 103 weeks of treatment (Melnick et al., 1983). The incidence of LCTs was 83% (40/48), 100% (46/46), and 100% (48/48) in the 0, 25,000 (2.5% of diet), and 50,000 (5% of diet) ppm tara gum groups, respectively. The incidence of LCTs was statistically increased in both treatment groups. The historical control incidence of LCTs was reported to be 87% (899/1032) in this strain of rat. LCTs were not seen in B6C3F1 mice fed the same dietary concentrations of tara gum. Even though the LCTs were statistically increased and outside the historical control range, Melnick and co-workers (1983) concluded that the increased incidence of LCTs were not compound related. The reviewers judgement is that this response should be considered equivocal evidence until tara gum is tested in a strain of rat with a low spontaneous incidence of LCTs based on the experience with folpet, MTBE, and TCE. To date, no mechanistic data have been generated for tara gum.

4. Unclassified Boric acid. In a 2-year B6 mouse oncogenicity study, boric acid was administered at 0, 2500, or 5000 ppm in the diet (Dieter, 1994). The authors reported that LC hyperplasia was seen only at the highest dietary concentration. This


was noted in combination with a high incidence of seminiferous tubule degeneration; however, no LCTs were observed. In the absence of LC morphometry, the report of LC hyperplasia should be judged cautiously because this diagnosis can be confounded by the presence of tubule atrophy. Carbamazepine. Carbamazepine is used as an anticonvulsant and a specific analgesic for trigeminal neuraglia; however, its mechanism of action remains unknown (PDR, 1995r). In a 2-year oncogenicity study with Sprague-Dawley rats (0, 25, 75, and 250 mg/kg/d carbamazepine), doserelated increases in hepatocellular tumors in females, and LCTs in males were observed (PDR, 1995r). Carbamazepine has been shown to be negative in bacterial and mammalian mutagenicity studies. In reproduction studies with rats, nursing offspring demonstrated a lack of body weight gain and an unkempt appearance at a maternal dosage level of 200 mg/kg/d. Felbamate. Felbamate (2-phenyl-1,3-propanediol dicarbamate) is an anticonvulsant, the mechanism of action of which is unknown. In a 2-year feeding study with rats (males— 0, 30, 100, 300; females— 0, 10, 30, 100 mg/kg/d), statistically significant increases in hepatocellar adenomas and LCTs were seen in the female 100 mg/kg/d and male 300 mg/kg/d groups, respectively (PDR, 1995e). In a 92-week feeding study with mice (0, 300, 600, 1200 mg/kg/d felbamate), a statistically significant increase in hepatocellular adenomas was seen in the male and female 1200 mg/kg/d groups; no LCTs were seen in the male mice. A dose-dependent increase in liver hypertrophy secondary to enzyme induction was also seen in felbamate-treated mice. Felbamate was found to be negative in four short-term tests for genotoxicity. Felbamate does not appear to be a reproductive or developmental toxin, but did produce an effect during lactation on pups. Flecainide. Flecainide [2,5-bis (2,2,2trifluorethoxyl)-N-(2-piperidylmethyl) benzamide acetate] is an antiarrhythmic compound. Flecainide was evaluated for oncogenicity in CD-1 mice (18-month duration), and Wistar rats (24-month duration) where the dietary levels were adjusted to achieve an intake of 0, 15, 30, and 60 mg/kg/d (Case et al., 1984). In the Wistar rats, a doserelated increase in the incidence of LCTs was observed that was statistically increased at the

high dosage (64 vs. 38% for the control). No compound-related increase in LCTs was observed in the CD-1 mice. The authors attributed the increased LCT incidence in the Wistar rats to increased survival in the flecainide-treated rats. However, it is difficult to interpret these conclusions, because survival curves were not presented. The differential survival suggests that the finding of LCTs is equivocal and requires confirmation. Indomethacin. Indomethacin is a prostaglandin synthetase inhibitor that is used as an antiinflammatory drug. A single appplication of indomethacin or its formulation Amuno® (2.5 mg indomethacin/100 g body weight) were applied to the shaved dorsal skin of adolescent (28 days of age) or adult (98 days of age) male SpragueDawley rats (Goerttler et al., 1992). A statistically significant increase in the incidence of LCTs was observed in both adolescent (12 vs. 1% for the control) or adult (6 vs. 0% for the control) male rats treated with indomethacin or Amuno®. The authors suggested that the indomethacin may induce LCTs via its ability to block the actions of prolactin (Rui et al., 1984). Such a mechanism would be analogous with the dopamine agonists producing LCTs via their ability to decrease prolactin (Section VII.A.1.e). Isopropanol. Isopropanol is used as a solvent in various consumer products and commercial sprays. Male and female CD-1 mice and F344 rats were exposed to isopropanol vapors (6 h/d, 5 d/week) at concentrations of 0, 500, 2500, or 5000 ppm for 78 and 104 weeks, respectively (Burleigh-Flayer et al., 1997). An interim sacrifice was performed during weeks 54 and 73 for the CD-1 mice and F344 rats, respectively. In the F344 rats, a concentration-related increase in LCTs was observed in at the interim sacrifice (statistically elevated at 5000 ppm) and among rats found dead or euthanized moribund (57.7, 72.2, 84.7, and 93.8% for the 0, 500, 2500, and 5000 ppm groups, respectively). The overall frequencies of LCTs for all male rats examined were 64.9, 77.3, 86.7, and 94.7% for the 0, 500, 2500, and 5000 ppm groups, respectively. There were no other neoplastic lesions observed in the F344 rats or in the CD-1 mice. The authors concluded that the increased incidence of LCTs was an artifact of the low spontaneous control incidence. This argument is tenuous based on several recent studies 229


with F344 rats discussed in this review that have similar control values as this study. In addition, the incidence of LCTs were increased at interim sacrifice coupled with the higher mortality in the 5000 ppm group, which would bias against detection of LCTs, suggest that this finding is not an artifact, and that isopropanol would in all likelihood induce LCTs if tested in a strain of rat with a low spontaneous incidence of LCTs. Jet Petroleum-4 (JP-4). JP-4 is used as a jet fuel by the military and is a complex mixture of aliphatic and aromatic hydrocarbon compounds with physiochemical properties similar to commercial gasoline. F344 rats and C57BL/6 mice were exposed to JP-4 vapors (6 /d, 5 d/week) at concentrations of 0, 1000, or 5000 mg/m3 for 12 months (Bruner et al., 1993). Animals were held for a 12-month postexposure tumorigenesis obervation period. In the male F344 rats, there was clear evidence that the 5000 mg/m3 male rat group had a treatment-related renal toxicity and neoplasia consistent with an α2µ-globulin nephropathy syndrome, which is unique to male rats. A statistically significant increase in LCTs also was observed at 5000 mg/m3 group (95 vs. 86% for the control). Clearly, the high spontaneous incidence of LCTs confounds the ability to conclude whether this observation is treatment related and must be considered equivocal until confirmed in a strain of rat that has a low spontaneous incidence of LCTs. Interestingly, d-limonene and MTBE (see below) also induce α2µ-globulin nephropathy syndrome and increase the incidence of LCTs in rats. In the male mice, the 5000 mg/m3 group had an increased incidence of LCH (18 vs. 0% in control); however, testicular atrophy was present. In the absence of LC morphometry, the report of LC hyperplasia should be judged cautiously because this diagnosis can be confounded by the presence of tubule atrophy. d-Limonene. d-Limonene is a natural component of a variety of foods and beverages and is found in many fruits, vegetables, meats, and spices (Jameson, 1990). It is found naturally in orange juice at an average concentration of 100 ppm. In 2-year studies, male (0, 75, or 150 mg/kg; 5 d/week) and female (0, 300, or 600 mg/kg; 5 d/week) F344 rats and male (0, 250, or 500 mg/kg; 5 d/week) and female (0, 500, or 1000 mg/kg; 5 d/week) B6C3F1 mice were ad230

ministered d-limonene by gavage. The LCT incidence was statistically increased at the 75 (96%) and 150 (96%) mg/kg/d dose groups when compared with controls (74%) (Jameson, 1990). The author concluded that the increased incidence of LCTs was probably not compound related because the control group had a lower survival rate; however, there are two reasons why this conclusion may be incorrect. The first is that this lesion is commonly dismissed in studies using F344 rats (e.g., folpet, MTBE, TCE), yet compounded-related increases in LCTs are replicated when these compounds were retested in strains of rats with a low spontaneous incidence of LCTs. Recently, it has been shown that the false-negative rate for LCTs in F344 is greater than expected by chance, suggesting that the tendency to dismiss this tumor type in F344 is occurring when, in fact, it is a compound-related finding (Haseman and Elwell, 1996). The second is that JP-4 (see above), MTBE (see below), and d-limonene all induce a compounded-related increase in kidney tumors that as attributed to the α2µ-globulin nephropathy syndrome, and each of these compounds also caused an increased incidence of LCTs. This circumstantial evidence may point to an association between α2µ-globulin nephropathy syndrome and LCTs in male rats. This hypothesis needs to be tested further by examining other 2-year rat bioassays with compounds that cause hyaline droplet nephropathy (e.g., trimethylpentane, dichlorobenzene, isophorone, decalin, and unleaded gas). d-Limonene did not induce tumors in female F344 rats or in the B6C3F1 mice. Methyl tertiary butyl ether (MTBE). MTBE is used as a gasoline additive, primarily to reduce carbon monoxide emissions and as an antiknock blending agent (Rudo, 1995). In a 2-year inhalation study, F344 rats were exposed to 0, 400, 3000, or 8000 ppm MTBE (6 h/d, 5 d/week) (Chun et al., 1992; Bird et al., 1997). MTBE induced a dose-related increase in LCTs that was statisically increased at the two highest concentrations: 64%, 70%, 82%, and 94% for the 0, 400, 3000, and 8000 ppm groups, respectively. This increase in LCTs was dismissed as being within the historical control range for F344 rats, even though there was reduced survival in the two highest concentrations. MTBE also induced a compound-related increase in kidney tumors (3000 and 8000 ppm


groups) that was attributed to the α2µ-globulin nephropathy syndrome. In a lifetime study, 0, 250, or 1000 mg/kg MTBE (4 d/week, 104 weeks) was administered by gavage to male and female Sprague-Dawley rats (Belpoggi et al., 1995). MTBE produced a statistically significant increase in the incidence of LCTs in the 1000 mg/kg group (18.3 vs. 3.3% for the control). MTBE did not induce any other tumors in the male rats. These data combined with the above study demonstrate that MTBE clearly induces LCTs in rats. No mechanistic data were presented; however, a reduced incidence of mammary tumors was reported in the SpragueDawley rats rats (Belpoggi et al., 1995). Such a tumor profile in male and female rats would suggest reduced serum estradiol levels or decreasing prolactin secretion similar to dopamine agonists (Section VII.A.1.e) as a possible mechanisms to be investigated. In addition, MTBE did not induce LCTs in male CD-1 mice, but did induce hepatocellular tumors at 8000 ppm MTBE (Burleigh-Flayer et al., 1992; Bird et al., 1997). Nicotine. Nicotine was administered subcutaneously to male F344 rats for either 2 or 22 months at 1000 µg/kg/d, a dosage that is equivalent to the absorption of nicotine from smoking approximately 15 to 20 cigarettes (Thompson et al., 1973). After 2 months, the incidence of LC hyperplasia in the nicotine group was 40% (4/10 rats) vs. 0% (0/6 rats). After 22 months of treatment, the incidence of LC hyperplasia in the nicotine group was 89% (34/38 rats) vs. 67% (4/6 rats). Clearly, the 40% incidence of LC hyperplasia at 2 months demonstrates that nicotine is capable of inducing LC proliferation. However, the absence of LCT at 22 months is highly unusual given the high spontaneous incidence of LCTs in F344 and suggests that the pathological criteria used were not consistent with current pathological nomenclature. Ideally, this type of experiment should be repeated in a strain of rat that has a low spontaneous incidence of LCTs, because there is a large epidemiology database on cigarette smokers that could be used to determine whether the testis is a target in humans. Oxazepam. Oxazepam (7-chloro-1,3-dihydro-3-hydroxy-5-phenyl-2H-1,4-benzodiazepin-2one) belongs to the 3-hydroxybenzodiazepinone class of compounds. Oxazepam is used to relieve

several different types of anxiety (PDR, 1995n). In a dietary oncogenicity study with rats, male rats receiving 30 times the human dose (estimated to be approximately 50 mg/kg) had statistically increased incidences of thyroid follicular cell tumors, LCTs, and prostatic adenomas. Mice fed dietary dosages of 35 or 100 times the human daily dose of oxazepam for 9 months developed a dose-related increase in hepatocellular adenomas/ carcinomas (Fox and Lahcen, 1974). No mechanistic, developmental, or reproduction studies were reported. Regarding potential mechanisms, the pattern of liver, thyroid, and LCTs suggest that enzyme induction with concomitant-enhanced hormone elimination may be responsible for these tumor types. If enhanced elimination of testosterone were demonstrated for oxazepam, it would be the first documented example.

B. Genotoxic Compounds Compounds that are clearly genotoxic and produce LC hyperplasia or LCTs are listed in Table 5. In contrast to nongenotoxic agents, the only genotoxic agents that have any hormonal or mechanistic data are cadmium (Bomhard et al., 1987; Laskey and Phelps, 1991; Waalkes and Rehm, 1992), dibromochloropropane (DBCP) (Cortes-Gallegos et al., 1980; ), and radiation (Lindsay et al., 1969; Pinon-Lataillade et al., 1991). These compounds are discussed below. These examples illustrate that even genotoxic compounds induce LCTs via similar mechanisms as nongenotoxic compounds. Clearly, additional work is necessary to determine whether genotoxic agents in general induce LCTs via disruption of the HPT axis.

1. Cadmium The ability of cadmium to induce LCTs has been attributed to testicular necrosis, which is typically induced by acute high doses of cadmium (Gunn and Gould, 1970; Bomhard et al., 1987). This conclusion was based on data that physical treatments that induce testicular necrosis, such as vascular ligation, also produce LCTs (Gunn et al., 1965). However, chronic dietary 231


administration of cadmium has been shown to induce LCTs in Wistar rats, but does not induce testicular necrosis based on the absence of fibrotic lesions (Waalkes and Rehm, 1992). These data suggest that cadmium can induce LCTs by mechanisms other than necrosis. Using isolated LCs from Sprague-Dawley rats, cadmium has also been shown to decrease testosterone biosynthesis (Laskey and Phelps, 1991). Hence, acute highlevel exposure to cadmium appears to induce LCTs by producing testicular necrosis, while chronic low-level exposure to cadmium may induce LCTs via inhibition of testosterone biosynthesis due to disruption of the HPT axis. In a recent study with F344 rats, testosterone silastic implants abolished both cadmium-induced and spontaneously occurring LCTs, but had no effect on cadmium-induced chronic testicular degeneration (Waalkes et al., 1997). Testosterone implantation acts to suppress LH levels and hence illustrates that LH is the primary mediator of LCTs in both spontaneous and cadmium-induced LCTs. Chatani and co-workers (1990) have also shown that testosterone silastic implants block spontaneous LCT development in F344 rats. Waalkes and co-workers (1997) have demonstrated that the cadmium-induced LCTs are due to inhibition of testosterone biosynthesis with the subsequent increase in LH rather than testicular degeneration. In addition, the authors have also demonstrated that even genotoxic agents such as cadmium can induce LCTs by a nongenotoxic mechanism, namely, elevated LH levels.

2. Dibromochloropropane (DBCP) DBCP is also a known human male reproductive toxicant. LC hyperplasia has been reported in men exposed to DBCP and confirmed histologically (Cortes-Gallegos et al., 1980). In men showing classic symptoms of male reproductive toxicity from DBCP exposure, investigators reported increased LH levels with normal testosterone levels. It is possible that the mechanism for LC hyperplasia seen in men is due to extensive tubular damage. DBCP has also been shown to be positive in a dominant lethal study in rats, suggesting that DBCP is a germ cell mutagen (Teramoto et al., 232

1980). In long-term animal studies, DBCP is carcinogenic, but there is no evidence for an increase in incidence of LCTs (Whorton and Foliart, 1983). It is interesting that DBCP does not produce testicular tumors in rats, even though it produces testicular atrophy.

3. Radiation LCTs were observed in Long-Evans rats 2 years after they had been subjected to localized testicular irradiation (Lindsay et al., 1969). The incidence of LCTs was 1/45 (2/2%), 6/62 (9.7%), and 40/74 (54%) in the control, 150R, and 500R groups, respectively. The ability of radiation to induce LCTs may be related to increased LH levels (Pinon-Lataillade et al., 1991), possibly secondary to tubule damage. Interestingly, other radiation studies with rats did not demonstrate an association with radiation and LCT formation (Hulse, 1977; Anisimov and Osipova, 1993). Comparison of the study designs between the positive and negative studies reveals that the negative studies were conducted on older rats (greater than 3 months of age), and the single positive study was conducted on younger rats (6 to 7 weeks of age). Whether LCT induction by radiation is age dependent requires further investigation; however, such a mechanism has been shown for the mammary gland (Welsch, 1985). Because of the paucity of mechanistic data for genotoxic compounds, one cannot determine whether the induction of LCTs is due to DNA adduct formation or to hormonally mediated mechanisms. However, all of the genotoxic compounds listed in Table 5 induced tumors (adenomas and/or carcinomas) at sites other than the testis, suggesting the former rather than the latter mechanism. The only exceptions to the above generalization were DBCP, isoprene, methyCCNU, and X-irradiation, and these exceptions appear to be related to study design. For instance, DBCP has been shown to produce LC hyperplasia in humans, presumably secondary to tubule damage. In rodents, DBCP only produces testicular atrophy (Cortes-Gallegos et al., 1980) and, consistent with DBCP being a genotoxic agent, induces multiple tumors at sites other than the testis in oncogenicity studies with mice and rats


(Whorton and Foliart, 1983). The isoprene and methyl-CCNU studies were 1 year and 70 days in duration, respectively; hence, their inability to induce tumors at other sites is in all likelihood a reflection of the short study duration (Melnick et al., 1994; Yegana et al., 1988). In the radiation studies, the X-irradiation was adminstered only to the testes; hence, the inability to observe tumors at other sites is attributed to localized exposure (Lindsay et al., 1969). Clearly, the data summarized in Table 5 demonstrate that the genotoxic agents that induce LCTs also induce tumors at other sites when tested in appropriately designed studies. In summary, two interpretations of the data are possible for genotoxic agents. One is that DNA-reactive agents initiate LCs and then LH promotes the development of the tumor. The other is that DNA-reactive agents act via an unidentified hormonal mechanism that may or may not be related to their mutagenic or clastogenic activity. Because such DNA-reactive agents cause tumors of other types as well, the LCTs would probably not be the endpoint of primary regulatory concern. Hence, genotoxic agents that produce LC hyperplasia or LCTs should be viewed as posing a risk to human health and traditional risk assessment methodology for genotoxic agents should be used (Clegg et al., 1997).

VIII. HUMAN RELEVANCE A. Human Incidence of LCTs Human LCTs were first described in 1895 by Dr. Franz Leydig and since that time a little more than 200 case reports have been documented in the scientific literature (Dilworth et al., 1991; Clegg et al., 1997). Approximately 1% of all cancers in men are of testicular origin and over 90% of these are exclusively of germ cell origin (Cotran et al., 1994; Gilliland and Key, 1995). Only about 1% of testicular tumors are LC adenomas (0.01% of all cancers in men), and these occur at an ageadjusted rate of 0.4 per million in the U.S. (Cotran et al., 1994; Gilliland and Key, 1995). Human LCTs are exclusively benign in children (Kaplan et al., 1986), but approximately 10 to 15% can be malignant in adults (Grem et al., 1986). The inci-

dence of LCTs appears to vary by ethnic background, where the highest incidence is seen in white males (Dieckmann et al., 1993; Bosland, 1994). In contrast to humans, spontaneous LCTs are commonly seen in laboratory animal species. Among the strains of rats commonly used in toxicology studies, the reported incidences of spontaneous LCTs are 76.8% for F344 (Iwata et al., 1991; Mitsumori and Elwell, 1988; Lang, 1990), 5.9% for Wistar (Bomhard and Rinke, 1994; Walsh and Poteracki, 1994), and 5.3% for CD (Hansen, 1993; McMartin et al., 1992; Lang, 1992; IRI, 1995) rats (Table 2). In mice, the reported spontaneous incidences of LCTs are 1.7% in CD-1® mice (Lang, 1995; Maita et al., 1988; Chandra and Frith, 1992; Inveresk Research International, 1995) and 0.4% in B6C3F1 mice (Mitsumori and Elwell, 1988) (Table 3). In the Beagle dog, the spontaneous incidence of LC hyperplasia and LCTs is 8.3 and 6.3%, respectively, at 7.75 years of age (James and Heywood, 1979). Thus, a high incidence of spontaneous LCTs is unique to the rat relative to the mouse and humans, with incidences in the latter much lower than in the two rodent species. The reported human incidence is 1,920,000- and 132,500-fold less than the spontaneous incidence seen in F344 and CD rats, respectively. In considering the above comparisons, it should be noted that the diagnosis of LCTs in laboratory animals is from histologic evaluation, while in humans it is commonly from palpation, often after presentation of gynecomastia or abnormal endocrine profile. Testicular ultrasonography in men with either gynecomastia or other predisposing indications found small, nonpalpable LC adenomas (Corrie et al., 1987; Horstman et al., 1994). Horstman and co-workers (1994) reported a 0.2% (4/1600 men) incidence of LCTs from men examined by scrotal ultrasonography for a variey of indications, but primarily gynecomastia. Using testicular ultrasonography, Buckspan and co-workers (1989) reported a 1% (4/400 men) incidence of impalable nodules that also were subsequently diagnosed as benign LCTs in oligospermic men. Therefore, small, benign LC adenomas that are not palpable may escape detection in humans. Hence, the true incidence of LCTs in humans may be higher than previously 233

thought, but it is nonetheless significantly lower than in rats (Clegg et al., 1997).


B. Comparative Biology As shown in Section VI, rats but not mice are commonly the target of xenobiotic-induced LCTs. Hence, the focus in the subsequent sections is exploring the differences in response between the rat and human. The apparent species differences in the incidence of LCTs between rats and humans may be due to several physiological or endocrine differences between the two species. For example, rats lack sex hormone binding globulin (SHBG), have enhanced sensitivity to the proliferative response to hCG, and greater LH receptor number per Leydig cell. In addition, rats exhibit a greater responsiveness to prolactin and have GnRH receptors on their LCs. All of these differences may contribute to the greater sensitivity for induction of hyperplasia when compared with human LCs. The absence of SHBG in rats may be an important contributing factor in the greater sensitivity of rats vs. humans to LCT induction. In man, SHBG is produced by the liver and the majority (~95%) of testosterone in peripheral blood is bound to this SHBG, which has the effect of retarding its metabolism and clearance (Moore and Bulbrook, 1988). Because bound and free (bioavailable) testosterone are kept in balance, it means that it is relatively difficult in man to perturb the peripheral levels of testosterone in any short-term way. In contrast to man, the rat has no peripheral SHBG and thus the blood levels of testosterone can potentially be altered more rapidly. This difference between the species is probably related to differences in LC mass to blood volume, which is greater in rats than in man (Simpson et al., 1987). Perhaps as a consequence of this difference, the rat testis is more susceptible to disruption of testosterone levels by xenobiotics, and hence the need for a battery of mechanisms to ensure maintenance of reasonably steady-state testosterone levels. A similar analogy has been described for the thyroid gland, where rats lack thyroid binding globulin and its this absense, which contributes to the species differences in response to long-term alterations in the thyroid axis (Capen, 1996). 234

Rat and human LCs have been shown to respond differently to hCG, a hormone equivalent to LH in its action on LCs. The contrast in responsiveness of the rat and human LC to the proliferative stimuli of hCG may explain the low incidence of LCTs in humans. In Sprague-Dawley rats administered hCG daily for up to 5 weeks, LC numbers were increased to three times that of controls (Christensen and Peacock, 1980). In contrast, humans administered hCG three times a week for 6 weeks or every second day for 16 weeks showed an increase in LC size but had no increase in LC number (Heller and Leach, 1971). Even though these two studies used different dosing schedules, the data suggest that rat LCs respond to hCG stimulation in a proliferative manner, while the human LC response is limited to hypertrophy. This comparative difference has been confirmed using isolated LCs where human LCs were 10- to 100-fold less sensitive than rat LCs in their testosterone secretory and mitogenic responses to hCG (Simpson et al., 1987). Another comparative difference is the number of LH receptors expressed per LC. Human LCs contain approximately 1500 LH receptors/ cell, whereas rodent LCs contain approximately 20,000 receptors/cell (Huhtaniemi, 1983), a 13-fold difference between rats and humans. Many hormones, including gonadotropins such as LH, produce maximal biological response(s) with only a fraction of the total cell receptors occupied (Dufau, 1988; DeGroot, 1995). This phenomenon has given rise to the term “spare receptors”. The effect of “spare receptors” is to amplify the signal response because there is a nonlinear coupling between occupancy and action. As receptor number/cell increases, there is a corresponding shift in the dose-response to the left (i.e., lower concentrations of hormone are required to produce a similar biologic response) (De Groot, 1995; Kahn et al., 1992). Hence, the greater number of LH receptors on rat vs. human LCs should, by “spare receptor theory”, make the rodent LCs more sensitive to the same level of circulating LH than human LCs and this appears to be the case, at least in vitro (Simpson et al., 1987). The presence of more LH receptors on rat LCs may explain why hCG stimulates rat LCs to proliferate, while human LCs only undergo hypertrophy (Christensen and Peacock, 1980; Heller and Leach, 1971).


In addition to LH receptors, rat LCs also contain a receptor for GnRH (Prentice and Meikle, 1995; Hunter et al., 1982; Cooke and Sullivan, 1985). GnRH receptors do not appear to be present on LCs from mice (Hunter et al., 1982; Wang et al., 1983), monkeys (Mann et al., 1989a,b), or humans (Clayton and Huhtaniemi, 1982). Low dosages of GnRH analogs in rats stimulate testosterone production at the level of the LC, while high concentrations and/or chronic administration of GnRH analogs suppress steroid production via suppression of LH release at the level of the hypothalamus and pituitary (Sharpe, 1988; Hunter et al., 1982). Thus by extension, low levels of GnRH may also stimulate LC proliferation in the rat both directly, through the GnRH receptor, and indirectly through stimulation of pituitary secretion of LH. Consistent with these effects, GnRH agonists produce LCTs when administered at low levels, but do not induce LCTs at high levels presumably due to suppression of LH centrally (Section VII.A.1.f). Because GnRH receptors are not present on human LCs, the GnRHinduced LCTs in rats would not appear to be relevant to humans (Clegg et al., 1997). The role of prolactin in modulating LH receptor expression on LCs is also different in rodents when compared with humans. Prolactin has been shown to be necessary for the maintenance of testicular LH receptors on rat LCs (Zipf et al., 1978). Dopamine agonists appear to induce LCTs in rats by their ability to decrease prolactin, which causes a decrease in LC LH receptor number and thus a decrease in testosterone production with a concomitant increase in LH to maintain testosterone levels (Prentice et al., 1992). Human LCs appear not to express measurable prolactin receptors on their surface (Wahlstrom et al., 1983). Because decreased prolactin levels do not decrease the number of LH receptors on human LCs, humans do not appear to be at risk for LCT induction by this mode of action (Clegg et al., 1997). However, a recent study in normal men has shown that bromocryptine administration decreases serum prolactin (25% of control) and testosterone (78% of control) levels, but has no effect on serum LH, FSH, or estradiol levels (Marin-Lopez et al., 1996). These data illustrate that significant decreases in serum prolactin levels can produce small decrements in testosterone

levels. Hence, humans are not totally refractory to the effects of dopamine agonists on androgen synthesis, but clearly are less sensitive than rodents. These examples provide possible mechanistic reasons for the apparent species differences in the incidence of LCTs observed between rats and humans, as well as the apparent enhanced sensitivity of rats to chemically induced LCTs in general. This sensitivity difference appears to have a molecular determinant; specifically, we postulate that human LCs appear to be less sensitive to the proliferative responses than the rat because human LCs do not have GnRH and prolactin receptors and have fewer LH receptors, all of which would attenuate the responsiveness to LH. Thus, the unique presence of GnRH receptors in rats and the greater number of LC LH receptors in rats compared with man may contribute, at least in part, to the greater susceptibility of the rat to both spontaneous and xenobiotic-induced LCTs. Additionally, the absence of SHBG in rats may also contribute to greater sensitivity of rats vs. humans to LCT induction by enhancing the potential of xenobiotics to disrupt steady-state testosterone levels. In addition to the biologic differences noted above, a lesser sensitivity of human LCs to trophic stimuli is suggested by comparative differences in LH levels. Testosterone levels decline with age in most strains of rats (Chan et al., 1977) as well as in humans (Horton and Tait, 1967; Vermeulen et al., 1972). In rats, this decrease is probably secondary to declining LH levels (Pirke, 1979; Roberts et al., 1989; Prentice et al., 1992; Chen et al., 1994), although this is somewhat paradoxical because one would expect LH to increase in order to maintain testosterone levels. This is in contrast to the situation in man where LH levels tend to increase with age, presumably because of decreasing testosterone levels (Rubens et al., 1974; Vermeulen, 1978). In addition, the half-life of circulating LH in humans is in excess of 100 min (Caron et al., 1994), while in the rat the half life is 5 to 10 min (De Groot et al., 1995b), demonstrating that human LCs are exposed to higher physiologic levels of LH over their lifetime. The apparent lower spontaneous LCT incidence in man compared with the rat, despite greater exposure to LH, further suggests the relative insen235

sitivity of human LCs to proliferative influences of LH.


C. Endocrine Disease States in Humans As noted above, LCTs apparently occur less frequently in man than in rodents, even though the human incidence cannot be directly compared due to different diagnostic criteria. There are, however, several endocrine disease states in man, the mechanisms of which underscore the marked comparative differences that exist between rat and man in the responsiveness of the LC to proliferative stimuli (summarized in Prentice and Meikle, 1995). Two such examples will be discussed in this review, namely, androgen insensitivity syndrome (AIS) and familial male precocious puberty (FMPP). AIS is a hormone-resistance disorder in which androgens are not recognized by tissues of the body due to defective androgen receptor function (reviewed in Quigley et al., 1995). The prevalence of this disease is estimated to be 1:20,400 male births (Bangsboll et al., 1992). Individuals who have AIS are genotypic males but phenotypically can range from male to female in appearance. AIS is divided into two clinical syndromes: (1) complete androgen insensitivity in which the androgen receptor is completely nonfunctional, causing the genotypic male to have a female phenotype, and (2) partial androgen insensitivity in which there is partial recognition of androgens by the androgen receptor such that the phenotype of the affected individual ranges from an infertile male to a female. Individuals with complete androgen insensitivity with the testes in situ (cryptorchid) have elevated estradiol and LH levels and normal-to-elevated FSH and testosterone levels when compared with normal men (Quigley et al., 1995). Testicular tumors of germ cell and non-germ cell origin (Sertoli, Leydig) occur with increased frequency in individuals with AIS, and with an incidence that is greater than that seen in simple cryptorchidism. The overall risk for gonadal tumors in AIS has been estimated to be 6 to 9% (Rutgers and Scully, 1991; Scully, 1981). LC adenomas are less common (Jockenhovel et al., 1993) and in one study appeared at a frequency of 2.3% in individuals with AIS (Rutgers and Scully, 1991). However, LC hyperplasia is frequently observed (Rutgers and Scully, 236

1991). For comparative purposes, the androgen receptor antagonist flutamide when given to rats induces almost a 100% incidence of LCTs. In a sense, the flutamide-treated rats can be considered a type of chemically induced AIS. If humans were as sensitive as rats, then all individuals with AIS would be expected to have LCTs because their androgen receptors were either partially or completely blocked from conception. Yet, as previously noted, the incidence of human LCTs is in the range of 2 to 3%. It must be noted, however, that LC hyperplasia was present in the majority of these cases (Rutgers and Scully, 1991). It should also be noted that this elevation in LC hyperplasia and adenomas is seen in males with cryptorchidism, a known risk factor for testicular neoplasia. In addition, the method of detection of the LC hyperplasia/LCTs is not stated clearly, but appeared to be in most instances from histopathological examination. This comparison illustrates that humans are clearly less sensitive than rats for the induction of LCTs. The second example, FMPP, is a gonadotropin-independent disease that is inherited in an autosomal dominant, male-limited pattern, but, unlike AIS, is not confounded by the presence of cryptorchidism (Shenker et al., 1993; Holland, 1991). Children with FMPP undergo puberty beginning at around 4 years of age when testosterone production and LC hyperplasia occur in the face of nondetectable levels of bioactive LH (Schedewie, 1981; Rosenthal et al., 1983; Holland, 1991). FMPP has been shown to be due to an activating mutation in the gene that encodes the LH receptor (Shenker et al., 1993; Kosugi et al., 1995). This mutation results in a state of constant LH receptor activation where the constitutive activation is estimated to be 42% of maximal hCG stimulation (Shenker et al., 1993), thus mimicking the primary mechanism of LCT formation in rats. While individuals with FMPP do develop focal LC hyperplasia, there are no reports of increased incidences of LCTs in these individuals despite the state of continued activation of the LH receptor throughout their life. Unlike AIS, these individuals have normal fertility. If this condition existed in rats, one could reasonably predict that all of them would develop LCTs, yet biopsies from men with FMPP report only LC hyperplasia (Schedewie, 1981; Rosenthal et al., 1983; Holland, 1991).


D. Epidemiology In rodents, testicular tumors are almost exclusively non-germ cell in origin (LC), while in humans testicular tumors are almost exclusively germ cell in origin (Section VI). Hence, there is a clear cell type difference between humans and rodents in regard to testicular tumors. In humans, the only risk factors for testicular cancer found consistently in epidemiological studies are cryptorchidism and higher socioeconomic status (reviewed in Bosland, 1994; Prentice and Meikle, 1995). Less consistent are the associations observed between testis cancer risk and low birth weight, premature birth, and prenatal exposure to hormones, such as DES. Human epidemiology studies are available on a number of compounds that induce LCTs in rats (Tables 4 to 5). These compounds include 1,3-butadiene (Himmelstein et al., 1996), cadmium (Waalkes et al., 1992), clofibrate (Oliver et al., 1978), ethanol (Longnecker, 1995), gemfibrozil (Frick et al., 1987), lactose (Ursin et al., 1990), lead (IARC, 1987), nicotine (IARC, 1986; Preston-Martin, 1991; Gupta et al., 1996), and TCE (Wilmer et al., 1994). The epidemiological studies of clofibrate and gemfibrozil are of limited usefulness for the evaluation of carcinogenic potential because of their short-exposure duration and/or limited follow-up period; clearly, reevaluation of individuals who have used these two drugs would be valuable. The remaining compounds are discussed in detail below. It must be noted that detection of LCTs in these epidemiology studies relied on relatively insensitive methods of detection such as palpable tumors and/or an abnormal endocrine profile rather than highresolution ultrasonography. In addition, the human exposure to these compounds are often less than those used in rodent bioassays. These limitations are common to most epidemiological studies. Nonetheless, the absence of any positive association is consistent with the findings from endocrine disease states in humans (Section VIII.C).

1. 1,3-Butadiene 1,3-Butadiene is used in the production of resins and plastics and is ranked in the top 20

synthetic organic chemicals produced in the U. S., with an annual production of over 3 billion lbs (Himmelstein et al., 1996). Because of its high use, 1,3-butadiene has been studied extensively in both human epidemiological studies and in animal bioassays. In a chronic inhalation study in Sprague-Dawley rats (Owen et al., 1987), exposure to 8000 ppm of 1,3-butadiene was associated with an increased incidence of LCTs (8 vs. 0% in controls). However, numerous human epidemiological studies involving large worker cohorts have shown no evidence of increased risk for LCTs in humans but do show an increased risk for leukemia (reviewed in Himmelstein et al., 1996). The most definitive study was by Divine and Hartman (1997) because of its large sample size, near lifetime follow-up for initial employees, and 1,3butadiene exposure assessment of workers. They found that 1,3-butadiene workers had elevated rates of non-Hodgkin’s lymphoma, but the testis was not identified as a target organ.

2. Cadmium The testes are a well-defined target site of cadmium carcinogenesis in the rat and mouse (reviewed in Waalkes et al., 1992). Cadmium appears to induce LCTs primarily via its ability to induce acute toxic effects such as severe hemorrhagic necrosis and loss of seminiferous tubules followed by fibrosis. More recent work has shown that cadmium can induce LCTs in the absence of obvious testicular degeneration, suggesting a possible direct effect on the LC. Systemic exposure of rats to cadmium has also been shown to induce adenomas of the prostate (Waalkes et al., 1988; Waalkes et al., 1989), while inhalation exposure produces lung carcinoma (Takenaka et al., 1983). Epidemiological studies have shown that humans occupationally exposed to cadmium are at increased risk for lung and prostate cancer (reviewed in Waalkes et al., 1992; Armstrong and Kazantzis, 1983; Elinder et al., 1985). The testis was not identified as a target organ in any of the epidemiological studies to date. These data demonstrate that even when humans have been exposed to cadmium at levels high enough to detect tumors identified in the rat (i.e., lung, prostate), there was no evidence of testicular tumors. 237


3. Ethanol Ethanol has been shown to produce a numerical increase in the incidence of LCTs in SpagueDawley rats (7/50 vs. 2/50 for the control) (Cheever et al., 1990). Although this increase was not statistically different and was judged to be equivocal, two pieces of information suggest that this effect may be compound related: (1) the reported incidence is outside the historical control range for this strain of rat (Table 2), and (2) it is mechanistically plausible because ethanol is known to inhibit testosterone biosynthesis (Widenius et al., 1989; Van Thiel et al., 1983; Johnston et al., 1981). Epidemiological studies have shown that alcoholic beverage consumption increases the risk for development of cancer of the mouth, pharynx, larynx, esophagus, and liver (reviewed in Longnecker, 1995). There are suggestions that alcoholic beverage consumption may increase the risk of cancer of the breast, large bowel, or pancreas. The testis is not mentioned as a target in humans for cancer, although alcohol is known to affect fertility and sperm count. If ethanol was shown definitively to induce LCTs in rats, then the human data would support the conclusion that humans are less suceptible to chemical induction of LCTs.

4. Lactose The response of rats to chronic administration of lactose allows comparative evaluation of a compound to which the general human population is routinely exposed. Lactose is a component of milk and all dairy products. Wistar rats chronically fed diets containing 20% lactose had significant increases in both the incidence (24 vs. 4% in controls) and multiplicity of LCTs (Sinkeldam et al., 1992). However, despite the commonplace exposure of humans to lactose-containing products, there is no evidence that lactose is associated with increased incidence of LCTs in man (Ursin et al., 1990; Roe, 1989). Thus, the Federation of American Societies for Experimental Biology concluded the following: “In view of the substantial per capita lactose consumption (about 25 g daily) in the United States as a component of milk and other dairy products, the low incidence rate of 238

Leydig-cell tumours indicates that lactose is not significantly tumorigenic in humans” (cited in Roe, 1989). In the rat carcinogenicity study, a 20% lactose diet would result in a mean daily intake of approximately 20 g/kg/d lactose. Hence, only infants and very young children would be likely to be exposed to levels approaching those seen in rats. Unfortunately, the lactose carcinogenicity study in rats (Sinkeldam et al., 1992) was conducted at a single dose so one cannot determine whether lower levels would also produce LCTs in rats. Hence, while the absence of doseresponse data lessens this study’s usefulness for risk assessment purposes, the similarity of doses in humans and rats and the differences in response suggest that there is a fundamental difference between the two species.

5. Lead Acetate Lead acetate has been reported to induce both LC hyperplasia (57%) and LCTs (24%) in Wistar rats when adminstered at approximately 8 mg/ kg/d (Zawirska and Medras, 1968). The authors reported that there were no LCTs seen in the controls, but the control incidence of LC hyperplasia was not stated. The epidemiology studies on workers exposed to lead and lead compounds (smelters, battery workers) were evaluated by the International Agency for Research on Cancer (IARC, 1987). The testis was not identified as a target organ, while excesses in cancers of the digestive and respiratory systems, and of the kidney, were reported, although the evidence for carcinogenicity was judged by IARC to be inadequate.

6. Nicotine Nicotine has been shown to produce LC hyperplasia after only 2 months of treatment in F344 rats when given at 1000 µg/kg/d, a dosage that is equivalent to the absorption of nicotine from smoking approximately 15 to 20 cigarettes (Thompson et al., 1973). Ideally, the oncogenicity study with nicotine should be repeated in a strain of rat that has a low spontaneous incidence of LCT to confirm that nicotine can induce LCTs


in rats. These data demonstrate that nicotine can cause a proliferative lesion in the testis of rats. Epidemiological data from individuals who smoke have demonstrated an increased risk for cancers of the lung, upper respiratory and digestive tract, bladder, kidney, and pancreas (IARC, 1986; Gupta et al., 1996). The testis was not identified as a target organ. Nicotine is known to enhance dopamine levels and as a result decrease prolactin levels (Fuxe et al., 1990). Hence, nicotine may be producing LC hyperplasia in rats via a mechanism that may be analogous to dopamine agonists/enhancers (Section VII.A.1.f), and therefore one would predict humans not to be at risk for LCTs.

7. Trichloroethylene (TCE) Since the begining of this century, widespread human exposure to TCE has occurred via its use as an industrial solvent, a metal-cleaning agent, and historically as an inhalation anaesthetic as well as an additive to drugs, food, and consumer products (Wilmer et al., 1994; IARC, 1979; ACGIH, 1992). As a result of its widespread use, TCE has been studied extensively in humans and rodents. In an inhalation study with SpragueDawley rats (Maltoni et al., 1988), TCE produced marked increases in LCTs with an incidence as high as 23.8% in rats exposed to 600 ppm (incidences were 4.4% in controls). In humans, five cohort studies with TCE are available that report on 18,183 workers with follow-up periods of 25 years (in four of the five studies) (reviewed in Wilmer et al., 1994). The most definitive studies were conducted by Axelson and co-workers (1994) and Spirtas and co-workers (1991). In the Spirtas et al. study (1991), the mortality was evaluated in 6929 workers with a follow-up period of up to 30 years and an extensive exposure assessment was conducted (maximum estimates of early exposure were 400 ppm) (Stewart et al., 1991). In the Axelson et al. study (1994), both mortality and morbidity (incidence) were evaluated in 1670 workers with a follow-up period of up to 37 years where exposure was estimated to be approximately 20 ppm, although this exposure may be underestimated (Wilmer et al., 1994). Deaths attributed to testicular cancer were reported in both studies; 2 were observed (vs. one expected) in the Axelson et al. study (1994), and 1 was observed (vs. two

expected) in the Spirtas et al. study (1994). Although not stated, the deaths from testicular cancer can be reasonably assumed to be germ cell in origin. In the Axelson et al. study, the standarized mortality ratio for the testis was not statistically elevated and the deaths from testicular cancer appear from the text to have occurred in the lowexposure groups. The authors from both studies concluded that there was no association between exposure to TCE and cancer in general or any specific cancer site. None of the five cohort studies demonstrated a link between TCE exposure and human cancer, including human LCTs (Wilmer et al., 1994). Further, notwithstanding the findings of TCE-induced LCTs in rats, the European Centre for Ecotoxicology and Toxicology of Chemicals concluded that “...exposure to TCE does not present a carcinogenic hazard to man” (Wilmer et al., 1994). Of the above compounds, the best datasets for both the rat and human exist for 1,3-butadiene, cadmium, lactose, and TCE. These epidemiology studies do not demonstrate an association between human exposure to these compounds and induction of LC adenomas. In addition, these studies do not demonstrate an association with testicular cancer in general, both germ and non-germ cell in origin. It must be noted that the epidemiological studies discussed in this section are based on cancer death registries (with the exception of TCE); therefore, nonpalable LCTs would not be recorded in these datasets. This absence of a response has been confirmed with a few compounds (1,3-butadiene, cadmium) that have produced human neoplasia at other target sites, demonstrating that humans were exposed to adequate levels to induce neoplasia. Regarding ethanol and nicotine, strengthening the rodent data by confirming that these compounds do induce LCTs would be useful because these two compounds represent widespread human exposure at levels producing adverse responses in humans. Overall, these epidemiological studies support the concept that species differences exist between rats and humans (discussed above), and no evidence exists to date that a compound induces LCTs in humans even when sufficient exposure has occurred to produce detectable tumors at other sites. In addition to these epidemiology studies, vast experience has been gained with many pharmaceutical agents that are sold in large volumes 239

throughout the world but that also induce LCTs in rats. To date, there is no evidence that such compounds induce LCTs in humans (reviewed in Prentice and Meikle, 1995).


IX. CONCLUSIONS There are a variety of plausible mechanisms for the chemical induction of LCTs, all of which have been discussed (Section VII), including agonists of the estrogen, GnRH, and dopamine receptors, androgen receptor antagonists, and inhibitors of 5α-reductase, testosterone biosynthesis, or aromatase activity. Most of these ultimately involve alterations in serum LH as the final proximate mediator. However, the interstitial fluid of the testis is awash in growth factors, and LCs and their precursors have been shown to respond to these at least in in vitro studies. Because culture conditions, cell age and source, cell purity, and co-culture of different cell types modulate the functional response to these growth factors (reviewed in Saez, 1994), it is tenuous to extend these in vitro observations to in vivo responses. Clearly, studies examining the effect of intratesticular administration of growth factors are needed to determine which growth factors are important regulators of LC proliferation in intact animals. It is expected that further work will uncover additional mechanisms by which LCTs may arise. Regarding human relevance, the pathways for the regulation of the HPT axis of rats and humans are similar, where compounds that either decrease testosterone or estradiol levels or their recognition will increase LH levels. Hence, compounds that induce LCTs in rats by disruption of the HPT axis pose a potential risk to human health, except for possibly two classes of compounds (GnRH and dopamine agonists). Because testicular GnRH and prolactin receptors are either not expressed or expressed at very low levels in humans, the induction of LCTs in rats by GnRH and dopamine agonists would appear to be not relevant to humans (Clegg et al., 1997); however, the relevance to humans of the remaining five mechanisms of action (Section VII) cannot be ruled out. Therefore, the central issue becomes the relative sensitivity between rat and human LCs in their response to increased LH levels; specifically, is the 240

proliferative stimulus initiated by increased levels of LH attenuated, similar, or enhanced in human vs. rat LCs? There are several lines of evidence that suggest that human LCs are quantitatively less sensitive than rats in their proliferative response to LH, and hence their sensitivity to chemically induced LCTs. This evidence includes the following: • Human incidence of LCTs. The incidence of human LCTs has been reported to be approximately 0.4 per million (0.00004%) (Cotran et al., 1994; Gilliland and Key, 1995), while in rats it ranges from 76.8% in F344 to 5.3% in Sprague-Dawley strains (Table 2). Based on these data, the human incidence is 1,920,000to 132,500-fold less than the spontaneous incidence seen in F344 and Sprague-Dawley rats, respectively. However, there is a detection bias toward rats as the diagnosis of LCTs in laboratory animals is from histologic evaluation, while in humans it is commonly from palpation, often after presentation with gynecomastia or from fertility problems. Therefore, small, benign LC adenomas that are not palpable may escape detection in humans. Hence, the true incidence of LCTs in humans may be higher than previously thought, but nonetheless is expected to be much lower than observed in rats even when one corrects for this detection bias (Clegg et al., 1997). • Comparative differences. Several physiological and endocrine differences exist between rat and human LCs: (1) the presence of more LH receptors per LC in rats than humans, (2) the presence and activity of GnRH and prolactin receptors on rat LCs and the apparent absence or low expression of these receptors in human LCs, (3) the modulation of LH receptor levels on LCs by prolactin in rats but not in humans, (4) the absence of SHBG in rats, and (5) the observation that exogenous hCG treatment produces LC hyperplasia in rats and hypertrophy in humans. These comparative differences may contribute, at least in part, to the greater susceptibility of the rat to both spontaneous and xenobiotic-induced LCTs. • Human Endocrine Diseases. Two endocrine disease states in man, AIS and FMPP, underscore the marked comparative differences that exist between rats and man in the responsive-


ness of their LCs to proliferative stimuli. AIS is a hormone-resistance disorder in which androgens are not recognized due to a defective androgen receptor (Quigley et al., 1995), and FMPP is a gonadotropin-independent disease in which a mutation in the LH receptor results in constitutive activation (42% of maximal hCG stimulation) (Shenker et al., 1993; Kosuga et al., 1995). In individuals with AIS, the incidence of LC adenomas has been estimated to be approximately 2.3% (Rutgers and Scully, 1991). For comparative purposes, the androgen receptor antagonist flutamide when given to rats induces almost a 100% incidence of LCTs and, in a sense, can be considered a chemically induced AIS. In individuals with FMPP, biopsies report only LC hyperplasia and there are no reports of LCTs (Schedewie, 1981; Rosenthal et al., 1983; Holland, 1991). If this condition existed in rats, one could reasonably predict that all of them would develop LCTs. One of the assumptions in these examples is that LH is the primary driver for the formation of LCTs, which is well supported by the available data; however, several growth factors have mitogenic activity (Sections III and V) and may also play a role in human LCTs • Epidemiology. Several human epidemiological studies are available on a number of compounds that induce LCTs in rats: 1,3-butadiene (Himmelstein et al., 1996); cadmium (Waalkes et al., 1992); ethanol (Longecker et al., 1995), lactose (component of diary products) (Ursin et al., 1990), lead (IARC, 1987), nicotine (IARC, 1986; Preston-Martin, 1991; Gupta et al., 1996), and TCE (Wilmer et al., 1994). These epidemiological studies do not demonstrate an association between human exposure to these compounds and induction of LC hyperplasia or adenomas. Of the above compounds, the best data sets for both the rat and human data are 1,3-butadiene, cadmium, lactose, and TCE. The absence of LCTs has been confirmed with a few compounds (1,3-butadiene, cadmium, ethanol, nicotine) that have produced human neoplasia at other target sites. Regarding ethanol and nicotine, strengthening the rodent data by confirming that these compounds do induce LCTs would be useful because these two compounds represent wide-

spread human exposure at levels producing adverse responses in humans. Overall, these epidemiological studies support the conclusion that species differences exist between rats and humans (Section VIII), and no evidence to date exists for chemical induction of LCTs in humans even when sufficient exposure has occurred to detect tumors at other sites (e.g., 1,3butadiene, cadmium, ethanol, nicotine). Again, it must be noted that the epidemiological studies discussed in this review are based on cancer registries (with the exception of TCE); therefore, nonpalable LCTs would not be recorded in these datasets. After considering the human incidence of LCTs, the physiological and endocrine differences between rats and humans, human endocrine disease states, and epidemiology, the weight of evidence suggest that human LCs are quantitatively less sensitive than rats in their proliferative response to LH, and hence their sensitivity to chemically induced LCTs. The role that growth factors may play in LC tumorigenesis remains to be determined, and hence this risk to humans cannot be evaluated at this time. Vast experience has also been gained with many pharmaceutical agents that are sold in large volumes throughout the world and that also induce LCTs in rats. Although the human dose is less than the tumorigenic dose in rats, to date there is no evidence that such compounds induce LCTs in humans (reviewed in Prentice and Meikle, 1995). Of the seven hormonal modes of induction of LCTs, two (GnRH and dopamine agonism) were considered not to be relevant to humans (Clegg et al., 1997). Androgen antagonism, estrogen receptor agonism, and inhibition of 5α-reductase, testosterone biosynthesis, or aromatase activity were considered potentially relevant. For all of these, clear quantitative differences exist between species, with rodents being more sensitive than humans (Clegg et al., 1997). Therefore, one can reasonably conclude that no-observable effect levels for the induction of LCTs in rodent bioassays provide an adequate margin of safety for protection of human health and that the data support a nonlinear mode of action (i.e., threshold response). A similar conclusion was reached by the USEPA Scientific Advisory Board with respect to nongenotoxic thyroid follicular cell tumorigens that also act via 241


disruption of the thyroid endocrine axis (Hill et al., 1996; Hill et al., 1989). In conclusion, the data suggest that nongenotoxic compounds that induce LCTs in rats most likely have low relevance to humans under most exposure conditions because humans are quantitatively less sensitive than rats. Other investigators have come to a similar conclusion (Bar, 1992; Alison et al., 1994; Prentice and Meikle, 1995; Bosland, 1996). In a recent international multidisciplinary workshop on LC tumorigenesis (Clegg et al., 1997), seven reseach needs were identified: (1) improve detection of LC hyperplasia and adenomas in men; (2) obtain accurate measures of the incidence of LCTs in human population; (3) examine age-dependent characteristics of LCT induction; (4) conduct more interspecies comparative research to examine mechanistic and sensitivity differences; (5) examine the role of paracrine factors in LCT induction; (6) conduct reversibility studies in rodents with LC tumorigens; and (7) critically evaluate the epidemiology data on agents known to induce LCTs in rats. In this review, we have begun to address items 4, 5, and 7, and as the other areas of research are further investigated, these data will help to critically test the conclusions in this review.

ACKNOWLEDGMENTS The authors would like to thank Dr. George M. Rusch, Chairperson, and the committee members of the Program for Alternative Fluorochemical Toxicity Testing for the partial funding of this review. Many thanks go to Richard C. Graham (DuPont), who performed the exhaustive database searching for chemicals that induce LCTs, and Janice Tartaglia (DuPont), who created and maintained the bibliographic database for this project.

DISCLAIMER This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication, but does not neces242

sarily reflect the views of the Agency and no official endorsement should be inferred.

REFERENCES Abney, T. and Melner, M., Characterization of estrogen binding in the developing rat testis; ontogeny of the testicular cytoplasmic estrogen receptor. Steroids 34, 413–427, 1979. Abney, T. O. and Myers, R. B., 17β-estradiol inhibition of Leydig cell regeneration in the ethane dimethylsulfonate-treated mature rat. J. Androl. 12, 295–304, 1991. ACGIH, Trichloroethylene. Notice of intended change. Appl. Occup. Environ. Hyg. 7, 786–791, 1992. Agger, P. and Johnsen, S., Quantitative evaluation of testicular biopsies in varicocele. Fertil Steril 29, 52– 63, 1978. Ahmad, K., Dykes, J., Ferguson-Smith, M., Lennox, B., and Mack, W., Leydig cell volume in chromatinpositive Klinefelter’s syndrome. J. Clin. Endocrinol. Metab. 33, 517–520, 1971. Alison, R. H., Capen, C. C. ,and Prentice, D. E., Neoplastic Lesions of Questionable Significance to Humans. Toxicologic Pathology 22(2), 179–186, 1994. Amador, A., Steiger, R. W., Bartke, A., Johns, A., SilerKhodr, T. M., Parker, C. R. J., and Shepherd, A. M., Testicular LH receptors during aging in Fischer 344 rats. J. Androl. 6, 61–64, 1985. Anisimov, V. N., Carcinogenesis induced by neonatal exposure to various doses of 5-bromo–2′-deoxyuridine in rats, Cancer Lett. 91, 63–71, 1995. Anisimov, V. N. and Osipova, G. Y., Carcinogenesis induced by combined neonatal exposure to 5-bromo–2′deoxyuridine and subsequent total-body X-ray irradiation in rats. Cancer Lett. 70, 81–90, 1993. Aoki, A. and Fawcett, D. W., Is there a local feedback from the seminiferous tubules affecting activity of the Leydig cell? Biol. Reprod. 19, 144–158, 1978. Arai, M., Aoki, Y., Nakanishi, K., Miyata, Y., Mori, T., and Ito, N., Long-term experiment of maximal noncarcinogenic dose of dimethylnitrosamine for carcinogenesis in rats. Gann. 70, 549–558, 1979. Armstrong, B. G. and Kazantzis, G., The mortality of cadmium workers. The Lancet 1(8339), 1425–1427, 1983. Ashby, J., Brady, A., Elcombe, C. R., Elliott, B. M., Ishmael, J., Odum, J., Tugwood, J. D., Kettle, S., and Purchase, I. F. H., Mechanistically based human hazard assessment of peroxisome proliferatorinduced hepatocarcinogenesis. Hum. Exp. Toxicol. 13(Suppl. 2), S1-S117, 1994. Avallet, O., Vigier, M., Perrard-Sapori, M. H., and Saez, J. M., Transforming growth factor-b1 inhibits Leydig cell function. Biochem. Biophys. Res. Commun. 146, 575–581, 1987.


Axelson, O., Selden, A., Andersson, K., and Hogstedt, C., Updated and expanded Swedish cohort study on trichloroethylene and cancer risk. J. Occup. Med. 36, 556–562, 1994. Bangsboll, S., Qvist, I., Lebech, P. E., and Lewinsky, M., Testicular feminization syndrome and associated gonadal tumors in Denmark. Acta Obstet Gynecol Scand. 71, 63–66, 1992. Bar, A., Significance of Leydig cell neoplasia in rats fed lactitol or lactose. J. Am. Coll. Toxicol. 11, 189–207, 1992. Baroni, C., Magrini, U., Martinazzi, M., and Bertoli, G., Testicular Leydig Cell tumourigenesis by diethylstilbestrol in the BALB/c mouse. Histologic and histochemical study. Eur. J. Cancer 7, 211–220, 1966. Bartke, A., Sweeney, C. A., Johnson, L., Castracane, V. D., and Doherty, P. C., Hyperprolactinemia inhibits development of Leydig cell tumors in aging Fischer rats. Exper. Aging Res. 11, 123–128, 1985. Baum, M. J., Brand, T., Ooms, M., Vreeburg, J. T. M., and Slob, A. K., Immediate postnatal rise in whole body androgen content in male rats: correlation with increased testicular content and reduced body clearance of testosterone. Biol. Reprod. 38, 980–986, 1988. Behringer, R. R., Finegold, M. J., and Cate, R. L., Mullerian-inhibiting substance function during mammalian sexual development. Cell 79, 415–425, 1994. Belpoggi, F., Soffritti, M., and Maltoni, C., Methyl-tertiary-butyl ether (MTBE) — a gasoline additive — causes testicular and lymphohaematopoietic cancers in rats. Toxicol. Industrial Health 11, 119–149, 1995. Benahmed, M., Reventos, J., and Saex, J. M., Steroidogenesis of cultured purified pig Leydig cells: effects of lipoproteins and human chorionic gonadotropin. Endocrinology 112, 1952–1957, 1983. Benahmed, M., Tabone, E., Revenkos, J., and Saez, J. M., Role of Sertoli cells in Leydig cell function. INSERM Colloquia 123, 363–385, 1984. Benahmed, M., Morera, A., and Chauvin, M., Somatomedin C/insulin-like growth factor I as a possible intratesticular regulator of Leydig cell activity. Mol. Cell Endocrinol. 50, 69–77, 1987. Bentley, P., Calder, I., Elcombe, C., Grasso, P., Stringer, D., and Wiegand, H-J., Hepatic peroxisome proliferation in rodents and its significance for humans. Food Chem. Toxicol. 31, 857–907, 1993. Berger, M. R., Combination effect of three non-genotoxic carcinogens in male SD rats. Cancer Res. 36, 788, 1995. Bergh, A. and Damber, J. E., Morphometric and functional investigation on the Leydig cells in experimental unilateral cryptorchidism in the rat. Int. J. Androl. 1, 549–560, 1978. Bergh, A., Asonbert, A., Damber, J. E., Hammer, M., and Selstam, G., Steroid biosynthesis and Leydig cell morphology in adult unilaterally cryptorchid rats. Acta. Endocrinol. 107, 556–564, 1984. Bergh, A. and Cajander, S., Immunohistochemical localization of inhibin-a in the testes of normal men and in

men with testicular disorders. Int. J. Androl. 13, 463– 469, 1990. Bernier, M., Chatelain, P., Mather, J., and Saez, J., Regulation of gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells. J. Cell Physiol. 129, 257–263, 1986. Besman, M. J., Yanagibashi, K., Lee, T. D., Kawamura, M., Hall, P. F., and Sively, J. E., Identification of des-(Gly-Ile)-endozepine as an effector of corticoprotein-dependent adrenal steriodogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazapine receptor. Proc. Natl. Acad. Sci. USA 86, 4897–4901, 1989. Biegel, L. B., Hurtt, M. E., Frame, S. R., Applegate, M., O’Connor, J. C., and Cook, J. C., Comparison of the effects of Wyeth-14,643 in Crl:CD BR and Fisher– 344 rats. Fundam. Appl. Toxicol. 19, 590–597, 1992. Biegel, L. B., Liu, R. C. M., Hurtt, M. E., and Cook, J. C., Effects of ammonium perfluorooctanoate on Leydig cell function: In vitro, in vivo, and ex vivo studies. Toxicol. Appl. Pharmacol. 134, 18–25, 1995. Bird, M. G., Burleigh-Flayer, H. D., Chun, J. S., Douglas, J. F., Kneiss, J. J., and Andrews, L. S., Oncogenicity studies of inhaled methyl tertiary-butyl ether (MTBE) in CD–1 mice and F-344 rats. J. Appl. Toxicol., Vol. 17(S1) S45–S55, 1997. Biskind, M. S. and Biskind, G. R., Tumor of rat testis produced by heterotransplantation of infantile testis to spleen of adult castrate. Proc. Soc. Exp. Biol. Med. 59, 4–8, 1945. Bomhard, E., Karbe, E., and Loeser, E., Spontaneous tumors of 2000 Wistar TNO/W.70 rats in two-year carcinogenicity studies. J. Environ. Pathol. Toxicol. Oncol. 7, 35–52, 1986. Bomhard, E. and Rinke, M., Frequency of spontaneous tumours in Wistar rats in 2-year studies. Exp. Toxic. Pathol. 46, 17–29, 1994. Bonser, G. M., Malignant tumours of the interstitial cells of the testis in Strong A mice treated with triphenylethylene. J. Pathol. Bacteriol. 54, 149–154, 1942. Boomhard, E., Vogel, O., and Loser, E., Chronic effects of single and multiple oral and subcutaneous cadmium administration on the testes of Wistar rats. Cancer Lett. 36, 307–315, 1987. Boorman, G. A., Hamlin, M. H., and Eustis, S. L., Focal interstitial cell hyperplasia, testes, rat, in Monographs on Pathology of Laboratory Animals Sponsored by the International Life Sciences Institute, Genital System. Jones, T. C., Mohr, U., and Hunt, R. D., Eds., SpringerVerlag, Berlin Heidelberg, 1987, 201–204. Boorman, G. A., Eustis, S. L., and Elwell, M. R., Neoplasms of the testes, in Atlas of Tumor Pathology of the Fischer Rat. Stinson, S. F., Schuller, H. M., and Resnik, G., Eds., CRC Press, 1990, 409–414. Boorman, G. A., Chapin, R. E., and Mitsumori, K., Testis and epididymis, in Pathology of the Fischer Rat. Boorman, G., Eustis, S., Montgomery, C., Elwell, M.,

243


and MacKenzie, W., Eds., Academic Press, San Diego, 1990, 405–418. Bosland, M. C., Male reproductive system, in Carcinogenesis. Waalkes, M. P. and Ward, J. M., Eds., Raven Press, New York, 1994, 339–402. Bosland, M. C., Hormonal factors in carcinogenesis of the prostate and testis in humans and in animal models, in Celluar and Molecular Mechanisms of Hormonal Carcinogenesis: Environmental Influences. Huff, J., Boyd, J., and Barrett, J. C., Eds., 1996, 309–352. Brimblecombe, R. W. and Leslie, G. B., Cimetidine, in Testing of New Drugs. Laurence, D. R., McLean, A. E. M. and Weatheral, M., Eds., Academic Press, London, 1984, 65–91. Bruner, R. H., Kinkead, E. R., O’Neill, T. P., Flemming, C. D., Mattie, D. R., Russell, C. A., and Wall, H. G., The toxicologic and oncogenic potential of JP–4 jet fuel vapors in rats and mice: 12-month intermittent inhalation exposures. Fundam. Appl. Toxicol. 20, 97–110, 1993. Buckspan, M. P., Klotz, P. G., Goldfinger, M., Stoll, S., and Fernandes, B., Intraoperative ultrasound in the conservative resection of testicular neoplasms. J. Urol. 141, 326–327, 1989. Burleigh-Flayer, H. D., Chun, J. S., and Kintish, W. J., Methylteriary butyl ether: vapor inhalation oncogenicity study in CD–1 mice, Export, Union Carbide, Bushy Run Research Center, 1992. Burleigh-Flayer, H., Garman, R., Neptun, D., Bevan, C., Gardiner, T., Kapp, R., Tyler, T., and Wright, G., Isopropanol vapor inhalation oncogenicity study in Fisher 344 Rats and CD–1 mice. Fundam. Appl. Toxicol. 36, 95–111, 1997. Byers, S. W., Effect of scrotal insulation on the ability of ram testes to produce testosterone in vivo. J. Reprod. Fertil. 71, 17–21, 1984. Byers, S. W. and Glover, T. D., Effect of scrotal insulation on the pituitary-testicular axis of the ram. J. Reprod. Fertil. 71, 23–31, 1984. Byyny, R. L., Orth, D. N., and Cohen, S., Radioimmunoassay of epidermal growth factor. Endocrinology 90, 1261–1266, 1972. Cailleau, J., Vermeire, S., and Verhoeven, G., Independent control of the production of insulin-like growth factor I and its binding protein by cultured testicular cells. Mol. Cell. Endocrinol. 69, 79–89, 1990. Calkins, J. H., Guo, H., Sigel, M. M., and Lin, T., Differential effects of recombinant interleukin–1a and b on Leydig cell function. Biochem. Biophy. Res. Commun. 167, 548–553, 1990. Calkins, J. H., Siegel, M. M., Nankin, H. R., and Lin, T., Interleukin–1 inhibits Leydig cell steroidogenesis in primary culture. Endocrinology 123, 1605–1610, 1998. Capen, C. C., Toxic responses of the endocrine system, in Casarett and Doull’s Toxicology, 5th ed., Klaassen, C. D., Ed., McGraw-Hill, New York, 1996, 607–640. Cardy, R. H., Carcinogenicity and chronic toxicity of 2,4toluenediamine in F344 rats. J. Natl. Cancer Inst. 62, 1107–1116, 1979.

244

Caron, P. J., Bennet, A. P., Plantavid, M. M., and Louvet, J., Luteinizing hormone secretory pattern before and after removal of Leydig cell tumor of the testis. Eur. J. Endocrinol. 131, . 156–159, 1994. Carr, B.I., Garrett-Reilly, J., Smith, S. S., Winberg, C., and Riggs, A., The tumorigenicity of 5-azacytidine in the male Fischer rat. Carcinogenesis 5, 1583–1590, 1984. Carr, B.I., Rahbar, S., Asmeron, Y., Riggs, A., and Winberg, C. D., Carcinogenicity and haemoglobin synthesis induction by cytidine analogues. Br. J. Cancer 57, 395–402, 1988. Case, M.T., Sibinski, L. J., and Ray Steffen, G., Chronic oral toxicity and oncogenicity studies of flecainide, an antiarrhythmic, in rats and mice. Toxicol. Appl. Pharmacol. 73, 232–242, 1984. Catt, K. J. and Dufau, M. L., Spare gondotropin receptors in the rat testis. Nature. New Biol. 244, 219–221, 1973. Catt, K. J., Clayton, R. N., Davies, T. F., Chan, V., Katikenini, M., Nozu, K., and Dufau, M. L., Regulation of peptide hormone receptors and gonadal steroidogenesis. Rec. Progr. Hormone Res. 36, 557–622, 1980. Cavallaro, S., Korneyev, A., Guidotti, A., and Costa, E., Diazepam-binding inhibitor (DBI) processing products, acting at the mitochondrial DBI receptor, mediate adrenocorticotropic hormone-induced steroidogenesis in rat adrenal gland. Proc. Natl. Acad. Sci. USA89, 10598–10602, 1992. Chamindrani Mendis-Handagama, S. M. L., Watkins, P. A., Gelber, S. J., Scallen, T. J., Zirkin, B. R., and Ewing, L. L., Luteinizing hormone causes rapid and transient changes in rat Leydig cell peroxisome volume and intraperoxisomal sterol carrier protein–2 content. Endocrinology 127, 2947–2954, 1990. Chan, S. W., Leathem, J. H., and Esashi, T., Testicular metabolism and serum testosterone in aging male rats. Endocrinology 101, 128–133, 1977. Chandra, M. and Frith, C. H., Spontaneous neoplasms in aged CD–1 mice. Toxicol. Lett. 61, 67–74, 1992. Chandra, M., Riley, M. G. I., and Johnson, D. E., Spontaneous neoplasms in aged Sprague-Dawley rats. Arch. Toxicol. 66, 496–502, 1992. Chandra, M. and Riley, M. G. I., Rarely occurring spontaneous metastasizing testicular tumors in rats, histopathologic and ultrastructural features. Exp. Toxic. Pathol. 46, 155–161, 1994. Chapin, R. E., Stevens, J. T., Hughes, C. L., Kelce, W. R., Hess, R. A., and Daston, G. P., Endocrine modulation of reproduction. Fundam. Appl. Toxicol. 29, 1–17, 1996. Chase, D. J. and Payne, A. H., Prolactin involvement in regulation of testicular 2-reductase activity in the immature rat. Biol. Reprod. 33, 637–643, 1985. Chatani, F., Nonoyama, T., Sudo, K., Miyajima, H., Takeyama, M., Takatsuka, D., Mori, H., and Matsumoto, K., Stimulatory effect of luteinizing hormone on the development and maintenance of 5α-reduced steroid-producing testicular interstital cell tumors in fischer 344 rats, Anticancer Res. 10, 337–342, 1990.


Cheever, K. L., Cholakis, J. M., Monaem El-Hawari, A., Kovatch, R. M., and Weisburger, E. K., Ethylene dichloride: The influence of disulfiram or ethanol on oncogenicity, metabolism, and DNA covalent binding in rats. Fundam. Appl. Toxicol. 14, 243–261, 1990. Chemes, H. E., Gottlieb, S. E., Pasqualini, T., Domenichini, E., Rivarola, M. A., and Bergada, C., Response to acute hCG stimulation and steroidogenic potential of Leydig cell fibroblastic precursors in humans. J. Androl. 6, 102–112, 1985. Chemes, H., Cigorraga, S., Bergada, C., Schteinhart, H., Rey, R., and Pellizzari, E., Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture. Biol. Reprod. 46, 793–801, 1992. Chemes, H. E., Dym, M., Fawcett, D. W., Javadpour, N., and Sherins, R. J., Pathophysiological observations of Sertoli cells in patients with germinal aplasia or severe germ cell depletion. Ultrastructural findings and hormone levels. Biol. Reprod. 17, 108–123, 1997. Chen, Y. D.I., Shaw, M. J., and Payne, A. H., Steroid and FSH action on LH receptors and LH-sensitive testicular responsiveness during sexual maturation of the rat. Mol. Cell. Endocrinol. 8, 291–299, 1977. Chen, H., Hardy, M. P., Huhtaniemi, I., and Zirkin, B. R., Age-related decreased Leydig cell testosterone production in the Brown Norway rat. J. Androl. 15, 551–557, 1994. Christensen, A. K., Leydig cells, in Handbook of Physiology, Male Reproductive System. Hamilton, D. W. and Greep, R. O., Eds., American Physiological Society, Washington, DC, 1975, 339–351. Christensen, A. K. and Peacock, K. C., Increase in Leydig cell number in testes of adult rats treated chronologically with an excess of hCG. Biol. Reprod. 22, 383– 391, 1980. Chun, J. S., Burleigh-Flayer, H. D., and Kintigh, W. J., Methyltertiary butyl ether: Vapor inhalation oncogenicity study in Fisher 344 rats, Export, Union Carbide, Bushy Run Research Center, 1992. Cigorraga, S., Sorrell, S., Bator, J., Catt, K., and Dufaua, M., Estrogen dependence of a gonadotropin-induced steroidogenic lesion in rat testicular Leydig cells. J. Clin. Invest. 65, 699–705, 1980. Clapp, N. K., Carcinogenicity of nitrosamines and methanesulphonate esters given intraperitoneally in RF mice. Int. J. Cancer 12, 728–733, 1973. Clark, B. J., Wells, J., King, S. R., and Stocco, D. M., The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA– 10 mouse Leydig tumor cells. J. Biol. Chem. 269, 28314–28322, 1994. Clarke, W. and Ragan, H., NTP technical report on the toxicology and carcinogenesis of tetrachlorethylene (perchloroethylene) in F344/N rats and B6C3F1 mice (inhalation studies), NTP publication no. 86–2567, 1986. Clayton, R. M. and Huhtaniemi, I. T., Absence of gonadotropin-releasing hormone receptors in human gonadal tissue. Nature 299, 56–59, 1982.

Clegg, E. D., Cook, J. C., Chapin, R. E., Foster, P. M. D., and Daston, G. P., Leydig cell hyperplasia and adenoma formation: mechanisms and relevance to humans. Reproductive Toxicol. 11, 107–121, 1997. Closset, J., Gothot, A., Sente, B., Scippo, M., Igout, A., Vandenbroeck, M., Donbrowicz, D., and Henne, G., Pituitary hormone dependent expression of insulin-like growth factors I and II in the immature hypophysectomized rat testis. Mol. Endocrinol. 89, 1125–1131, 1989. Codesal, J., Regadera, J., Mistal, M., Regadera, R. J., and Paniagua, R., Involution of human fetal Leydig cells. An immunohistochemical, ultrastructural and quantitative study. J. Anatomy 172, 103–114, 1990. Collins, M. A., Rusch, G. M., Sato, F., Hext, P. M., and Millischer, R-J., 1,1,1,2-Tetrafluoroethane: Repeat exposure inhalation toxicity in the rat, developmental toxicity in the rabbit, and genotoxicity in vitro and in vivo. Fundam. Appl. Toxicol. 25, 271–280, 1995. Cook, J. C., Murray, S. M., Frame, S. R., and Hurtt, M. E., Induction of Leydig cell adenomas by ammonium perfluorooctanoate: a possible endocrine-related mechanism. Toxicol. Appl. Pharmacol. 113, 209–217, 1992. Cook, J. C., Mullin, L. S., Frame, S. R., and Biegel, L. B., Investigation of a mechanism for Leydig cell tumorigenesis by linuron in rats. Toxicol. Appl. Pharmacol. 119, 195–204, 1993. Cook, J. C., Hurtt, M. E., Frame, S. R., and Biegel, L. B., Mechanisms of extrahepatic tumor induction by peroxisome proliferators in Crl:CD_BR (CD) rats. Toxicologist (Abstr.) 14, 1994. Cook, J. C., Frame, S. R., and Obourn, J. D., Leydig cell tumors, in Comprehensive Toxicology. Chapin, R. E. and Boekelheide, K., Eds., Pergamon, New York, 1997, 193–201. Cooke, B. A. and Sullivan, M., The mechanisms of LHRH agonist action in gonadal tissues. Mol. Cell Endocrinol. 41, 115–122, 1985. Cooke, B. A., Is cyclic AMP an obligatory second messenger for luteinizing hormone? Mol. Cell. Endocrinol. 69, 11–15, 1990. Corrie, D., Norbeck, J. C., and Thompson, I. M., Ultrasound detection of bilateral Leydig cell tumors in palpable normal testes. J. Urol. 137, 747–748, 1987. Cortes-Gallegos, V., Castaneda, G., Alonso, R., Arellano, H., Cervantes, C., and Parra, A., Effects of dibromochloropropane upon diurnal variations of gonadotropins and androgens. J. Androl. 1, 277–280, 1980. Corton, J. C., Bocos, C., Moreno, E. S., Merritt, A., Cattley, R. C., and Gustafsson, J-A., Peroxisome proliferators alter the expression of estrogen-metabolizing enzymes, Biochimie 79, 151–162, 1997. Cotran, R. S., Kumar, V., Robbins, S. L., and Schoen, F. J., Male genital system, in Robbins Pathologic Basis of Disease, 5th ed., Cotran, R. S., Kumar, V., and Robbins, S. L., Eds., W. B. Saunders, Philadelphia, 1994, 1015–1022. Czerwiec, F. S., Melner, M. H., and Puett, D., Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells

245


after addition of human chorionic gonadotropin. Mol. Endocrinol. 3, 105–109, 1989. Darney, K. J. and Ewing, L. L., Autoregulation of testosterone secretion in perfused rat testes. Endocrinology. 109, 993–995, 1981. Darney, K. J., Zirkin, B. R., and Ewing, L. L., Testosterone autoregulation of its biosynthesis in the rat testis: inhibition of 17-hydroxylase activity. J. Androl. 17, 137–142, 1996. Daughaday, W. and Rotwein, P., Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structure, serum and tissue concentration. Endocrin. Rev. 10, 68–91, 1989. De Coster, R., Coene, M. C., Van Camp, C., Van Camp, K., Beerens, D., and Cools, W., Comparative effects of ketoconazole on rat, dog, and human testicular steroidogenesis. J. Enzyme Inhibition 2, 261–268, 1989. De Groot, A. P., Lina, B. A. R., Hagenaars, A. J. M., Hollanders, V. M. H., Andringa, M., and Feron, V. J., Effects of a dietary load of acid or base on changes induced by lactose in rats. Food Chem. Toxicol. 33, 1– 14, 1995a. De Groot, L. J., Besser, M., Burger, H. G., Jameson, J. L., Loriaux, D. L., Marshall, J. C., Odell, W. D., Potts Jr., J. T., and Rubenstein, A. H., Endocrinology, 3rd ed., W. B. Saunders, Philadelphia, 1995b. de Jong, F. H., Hey, A. G., and Van Der Molen, H. J., Oestradiol–17b and testosterone in rat testis tissue; effect of gonodotrophins, localization and production in vitro. J. Endocrinol. 60, 409–419, 1974. Dieckmann, K. P. and Loy, V., Metachronous germ cell and Leydig cell tumors of the testis. Do testicular germ cell tumors and Leydig cell tumors share common etiologic factors? Cancer 72, 1305–1307, 1993. Dieter, M. P., Toxicity and carcinogenicity studies of boric acid in male and female B6C3F1 mice. Environ. Health Perspect. 102(Suppl. 7), 93–97, 1994. Dilworth, J. P., Farrow, G. M., and Oesterling, J. E., Nongerm cell tumors of testis. Urology 37, 399–417, 1991. Dirami, G., Teerds, K. J., and Cooke, B. A., Effect of a dopamine agonist on the development of Leydig cell hyperplasia in Sprague-Dawley rats, Toxicol. Appl. Pharmacol. 141, 169–177, 1996. Divine, B. J. and Hartman, C. M., Mortality update of butadiene production workers. Toxicology, 113, 169– 181, 1996. Donaubauer, H. H., Kramer, M., Kreig, K., Mayer, D., Von Rechenberg, W., Sandow, J., and Schutz, E., Investigations of the carcinogenicity of the LH-RH analog buserelin (HOE 766) in rats using the subcutaneous route of administration. Fundam. Appl. Toxicol. 9, 738–752, 1987. Dowie, L. J., Smith, J. E., MacGilchrist, A. J., Fraser, R., Honour, J. W., Reid, J. L., and Kenyon, C. J., In vivo and in vitro studies of the site of inhibitory action of omperazole on adrenocortical steroidogenesis. Eur. J. Clin. Pharmacol. 35, 625–629, 1988. Drummond, A. E., Risbriger, G. P., O’Leary, P. C. O., and De Kretser, D. M., Alteration in mitogenic and steroidogenic activities in rat testicular interstitial fluid

246

after administration of ethane dimethane sulphonate. J. Reprod. Fertil. 83, 141–147, 1988. Dubin, L. and Hotchkiss, R., Testis biopsy in subfertile men with varicocele. Fertil. Steril. 20, 50–57, 1969. Dufau, M. L., Tsuruhara, T., Horner, K. A., Podesta, E., and Catt, K. J., Intermediate role of adenosine 3',5'cyclic monophosphate and protein kinase during gonadotropin-induced steroidogenesis in testicular interstitial cells. Proc. Natl. Acad. Sci. USA 73, 3419–3423, 1977. Dufau, M. L., Endocrine regulation and communicating functions of the Leydig cell. Ann. Rev. Physiol. 50, 583–508, 1988. Dunnick, J. K., Toxicology and Carcinogenesis Studies of 8-Methoxypsoralen, NIH Publication No. 89–2814, Research Triangle Park, NC, 1989. Eil, C. and Nisula, B. C., The binding properties of pyrethroids to human skin fibroblast androgen receptors and to sex hormone binding globulin. J. Steroid Biochem. 35, 409–414, 1990. Elinder, C. G., Kjellstrom, T., Gogstedy, C., Andersson, K., and Spang, G., Cancer mortality of cadmium workers. Br. J. Indust. Med. 42, 651–655, 1985. Elliniemi, L. J. and Niemi, M., Fine structure of the human foetal testis. I. The interstitial tissue. Z. Zellforsch 99, 507–522, 1969. Ellis, M. K., Naylor, J. L., Green, T., and Collins, M. A., Identification and quantification of fluorine-containing metabolites of 1-chloro–2,2,2-trifluoroethane (HCFC133A) in the rat by 19F-NMR spectroscopy. Drug Metab. Dispos. 23, 102–106, 1995. Ellis III, H.V., Hong, C. B., Lee, C. C., Dacre, J. C., and Glennon, J. P., Subacute and chronic toxicity studies of trinitroglycerin in dogs, rats, and mice. Fundam. Appl. Toxicol. 4, 248–260, 1984. Elson, S. D., Browne, C. A., and Thorburn, G. D., Identification of epidermal growth factor-like activity in human male reproductive tissues and fluids. J. Clin. Endocrinol. Metab. 58, 589–594, 1984. Engel, W. and Frowein, J., Glucocorticoids and hCG sensitivity of rat testicular Leydig cell. Nature 251, 146– 148, 1974. Engelhardt, J. A., Gries, C. L., and Long, G. G., Incidence of spontaneous neoplastic and nonneoplastic lesions in Charles River CD–1 mice varies with breeding origin. Toxicol. Pathol. 21, 538–541, 1993. Everett, J. W., Pituitary and hypothalamus: perspectives and overviews, in The Physiology of Reproduction. Knobil, E. and Neill, J. D., Eds., Raven Press, New York, 1988, 1143–1160. Ewing, L. L., Cochran, C., Zirkin, B., and Chubb, C. E., Steroid synthesis and secretion by in vivo perfused rat, rabbit, dog, guinea pig, and hamster testes, in Testicular Development, Structure and Function. Steinberger, A. and Steinberger, E., Eds., Raven Press, New York, 1980, 117–127. Ewing, L. L. and Zirkin, B., Leydig cell structure and steroidogenic function. Recent Prog. Horm. Res. 39, 599–635, 1983. Ewing, L. L., Berry, S. J., Juniewicz, P. E., Zirkin, B. R., and Coffey, D. S., in Benign Prostatic Hypertrophy.


Rogers, C. H., Coffey, D. S., Cunha, G., Grayhack, J. T., Hinman, F., and Horton, R., Eds., 1987, 129–137. Ewing, L. L., The Leydig cell, in Reversibility in Testicular Toxicity Assessment. Scialli, A. R. and Clegg, E. D., Eds., CRC Press, Boca Raton, 1992, 89–126. Fauser, B. C. and Hsueh, A. J., Effect of transforming growth factor-β on human chronic gonadotropin induced testosterone production by cultured rat testicular cell. Life Sci. 43, 1363–1370, 1988. Fawcett, D. W., Neaves, W. B., and Flores, M. N., Observations on the organization of the interstitial tissue and on the occluding cell junctions in the seminiferous epithelium. Adv. Biosci. 10, 83–99, 1973. Fawcett, D. W., Ultrastructure and function of the Sertoli cell, in Handbook of Physiology. Hamilton, D. W. and Greep, R. O., Eds., 1973. Feldman, D., Ketoconazole and other imidazole derivatives as inhibitors of steroidogenesis. Endoc. Rev. 7, 409– 420, 1986. Fisher, J. S., Miller, M. R., Majdic, G., Saunders, P.T. K., Fraser, H. M., and Sharpe, R. M., Immunolocalisation of oestrogen receptor-α within the testis and excurrent ducts fo the rat and marmoset monkey from perinatal life to adulthood. J. Endocrinol. 153, 485–495, 1997. Fitzgerald, J. E., Sanyer, J. L., Schardein, J. L., Lake, R. S., McGuire, E. J., and de la Iglesia, F. A., Carcinogen bioassay and mutagenicity studies with the hypolipidemic agent gemfibrozil. JNCI 67, 1105–1116, 1981. Fitzhugh, O. G. and Nelson, A. A., The chronic oral toxicity of DDT (2,2-bis(p-chlorophenyl–1,1,1-trichloroethane). J. Pharmacol. Therap. 89, 18–30, 1947. Forest, M. G., Cathiard, A. M., and Bertrand, J. A., Evidence of testicular activity in early infancy. J. Clin. Endocrinol. Metab. 37, 148–151, 1973. Fort, F. L., Miyajima, H., Ando, T., Suzuki, T., Yamamoto, M., Hamashima, T., Sato, S., Kitazaki, T., Mahony, M. C., and Hodgen, G. D., Mechanism for speciesspecific induction of Leydig cell tumors in rats by lansoprazole. Fundam. Appl. Toxicol. 26, 191–202, 1995. Foster, P. M. D., Rat Leydig cell tumors induced by xenobiotics: etiology and possible mechanisms. Toxicologist 12, 3 (Abstr.), 1992. Fox, K. A. and Lahcen, R. B., Liver-cell adenomas and peliosis hepatitis in mice associated with oxazepam. Res. Commum. Chem. Pathol. Pharmacol. 8, 481–488, 1974. Freeman, D. A. and Ascoli, M., The LDL pathway of cultured Leydig tumor cells. Biochem. Biophys. Acta. 754, 72–83, 1983. Freeman, D. A., Plasma membrane cholesterol; removal and insertion into the membrane and utilization as substrate for steroidogenesis. Endocrinology 124, 2527– 2533, 1989. Frick, H., Elo. Haapa, K., and Heinonen, O. P., Helsinki heart study: primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. N. Engl. J. Med. 317, 1235–1247, 1987. Fukunishi, R., Watanabe, K., Terashi, S., and Kawaji, K., Shift of target organs in cycasin carcinogenesis.

Gann 62, 353–358, 1971. Fuxe, K., Andersson, K., Harfstrand, A., Eneroth, P., Perez de la Mora, M., and Agnati, L. F., Effects of nicotine on synaptic transmission in the brain, in Nicotine Psychopharmacology. Molecular, Cellular, and Behavioural Aspects. Wonnacott, S., Russell, M. A. H., and Stolerman, I. P., Eds., Oxford University Press, Oxford, 1990, 194–225. Gak, J.-C., Graillot, C., and Truhaut, R., Difference in sensitivity of the hamster and rat to long-term administration of ethylene thiourea. Eur. J. Toxicol. 9, 303– 312, 1976. Gangnerau, M. N., Funkenstein, B., and Picon, R., LH/ hCG receptors and stimulation of testosterone biosynthesis in the rat testis: changes during foetal development in vivo and in vitro. Mol. Cell. Endocrinol. 28, 499–512, 1982. Gardner, W. U. and Boddaert, J., Testicular interstitial cell tumors in hybrid mice given tri-p-anisyl chloroethylene. AMA Arch. Pathol. 50, 750–764, 1950. Garnier, M., Boujrad, N., Oke, B. O., Brown, A. S., Riond, J., Ferrara, P., Shoyab, M., Suarez-Quian, C. A., and Papadopoulos, V., Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms. Endocrinology 132, 444–458, 1993. Gaytan, F., Bellido, C., Morales, C., Reymundo, C., Aguilar, E., and van Rooijen, N., Selective depletion of testicular macrophages and prevention of leydig cell repopulation after treatment with ethylene dimethane sulphonate (EDS) in the rat. J. Reprod. Fertil. 101, 175–182, 1994a. Gaytan, F., Bellido, C., Moratle, C., Reymundo, C., Aguilar, E., and Van Rookjen, N., Effects of macrophage depletion at different times after treatment with ethylene dimethane sulfonate (EDS) on the regeneration of leydig cells in the adult rat. J. Androl. 15, 558– 564, 1994b. Gelber, S., Hardy, M., Mendis-Handagama, S., and Casella, S., Effect of insulin-like growth factor-I on androgen production by highly purified pubertal and adult Leydig cells. J. Androl. 13, 125–130, 1992. George, F. W., Johnson, L., and Wilson, J. D., The effect of a 5α-reductase inhibitor on androgen physiology in the immature male rat. Endocrinology 125, 2434–2438, 1989. Gerard, N., Syed, V., Bardin, C. W., Genetet, N., and Jegou, B., Sertoli cells are the site of interleukin–1α synthesis in the rat. Mol. Cell Endocrinol. 82, R13-R16, 1991. Gibson, J. P., Newberne, J. W., Kuhn, W. L., and Elsea, J. R., Comparative chronic toxicity of three oral estrogens in rats. Toxicol. Appl. Pharmacol. 11, 489–510, 1967. Gibson, G. and Lake, B., Peroxisomes: Biology and Importance in Toxicology and Medicine. Gibson, G. and Lake, B., Eds., Taylor and Francis, Washington, 1993. Gilliland, F. D. and Key, C. R., Male genital cancers. Cancer 75(Suppl. 1), 295–315, 1995.

247


Gnessi, L., Fabbri, A., and Spera, G., Gonadal peptides as mediators of development and functional control of the testis: an integrated system with hormones and local environment, Endocrine Rev. 18, 541–609, 1997. Goerttler, K., Edler, L., and Loehrke, H., Long-term tumorigenicity of a single application of indomethacin or amuno in adolescent and in adult Sprague Dawley rats. Exp. Toxic. Pathol. 44, 361–370, 1992. Gogan, F., Slama, A., Bizzini-Koutznetzova, B., Dray, F., and Kordon, C., Importance of perinatal testosterone in sexual differentiation in the male rat. J. Endocrinol. 91, 75–79, 1981. Gospodarowicz, D., Fibroblast growth factor and its involvement in developmental processes. Curr. Top. Dev. Biolo. 24, 57–93, 1990. Gray, L. E., Jr., Ostby, J. S., and Kelce, W. R., Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol. 129, 46–52, 1994. Greaves, M. B., Male genital tract - testis, in Histopathology of Preclinical Toxicity Studies, Elsevier, Amsterdam, 1990, 596–624. Greaves, P., Goonetilleke, R., Nunn, G., Topham, J., and Orton, T., Two-year carcinogenicity study of tamoxifen in Alderley Park Wistar-derived rats. Cancer Res. 53, 3919–3924, 1993. Grem, J. L., Ronins, H.I., Wilson, K. S., Gilchrist, K., and Trump, D. L., Metastatic Leydig cell tumor of the testis. Report of three cases and review of the literature. Cancer Res. 58, 2116–2119, 1986. Griffith, R. W., Carcinogenic potential of marketed drugs. J. Clin. Res. Drug Develop. 2, 141–144, 1988. Gruenewald, D. A., Hess, D. L., Wilkinson, C. W., and Matsumoto, A. M., Excessive testicular progesterone secretion in aged male fischer 344 rats: a potential cause of age-related gonadotropin suppression and confounding variable in aging studies. J. Gerontol. 47, B164-B170, 1992. Gruenewald, D. A., Naai, M. A., Hess, D. L., and Matsumoto, A. M., The Brown Norway Rat as a Model of Male Reproductive Aging: evidence for both primary and secondary testicular failure. J. Gerontol. 49, B42-B50, 1994. Guay, T., Tuthill, R., and Woolf, P., Germinal cell aplasia: response of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone to LH/FSHreleasing hormone with histopathologic correlation. Fertil. Steril. 28, 642–649, 1977. Gudermann, T., Nichols, C., Levy, F. O., Birnbaumer, M., and Birnbaumer, L., Ca2+ mobilization by the LH receptor expressed in Xenopus oocytes independent of 3′, 5′-cyclic adenosine monophosphate formation: evidence for parallel activation of two signaling pathways. Mol. Endocrinol. 6, 272–278, 1992. Gunn, S. A., Gould, T. C., and Anderson, W. A. D., Comparative study of interstitial cell tumors of rat testis induced by cadmium injection and vascular ligation. J. Natl. Cancer Inst. 35, 329–334, 1965.

248

Gunn, S. A. and Gould, T. C., Cadmium and other mineral elements, in The Testes, Volume 3. Influencing Factors. Johnson, A. D., Gomes, W. R., and Vandemark, N. L., Eds., Academic Press, New York, 1970, 377–481. Gupta, P. C., Murti, P. R., and Bhonsle, R. B., Epidemiology of cancer by tobacco products and the significance of TSNA. Crit. Rev. Toxicol. 26(2), 183–198, 1996. Gustafsson, K., Soder, O., Pollanen, P., and Ritzen, E. M., Isolation and partial characterization of an interleukinI-like factor from rat testis interstitial fluid. J. Reprod. Immunol. 14, 139–150, 1988. Habert, R. and Picon, R., Control of testicular steroidogenesis in foetal rat: effect of decapitation on testosterone and plasma luteinizing hormone-like activity. Acta Endocrinol. 99, 466–473, 1982. Habert, R. and Picon, R., Testosterone, dihydrotestosterone and estradiol 17-β levels in maternal and fetal testes in the rat. J. Steroid Biochem. 21, 193–198, 1984. Habert, R. and Brignaschi, P., Developmental changes in in vitro testosterone production by dispersed Leydig cells during fetal life in rats. Arch. Androl. 27, 65–71, 1991. Hales, D. B. and Payne, A. H., Glucocorticoid-mediated repression of P450scc mRNA and de novo synthesis in cultured Leydig cells. Endocrinology 124, 2099–2104, 1989. Hales, D. B., Xiong, Y., and Tur-Kaspa, I., The role of cytokines in the regulation of Leydig cell P450c17 gene expression. J. Steroid Biochem. Mol. Biol. 43, 907–914, 1992. Hall, P. F., Osawa, S., and Mroteck, J. J., Influence of calmodulin on steroid synthesis in Leydig cells from rat testis. Endocrinology 109, 1677–1684, 1981. Hall, P. F., Testicular steroid synthesis: organization and regulation, in The Physiology of Reproduction. Knobil, E. and Niell, J. D., Eds., Raven Press, New York, 1994, 1335–1362. Hamada, Y. and Futamura, Y., Induction of Leydig cell tumors by lacidipine via up-regulation of the LHRH receptor on Leydig cells in rats, J. Toxicol. Sci. 23, 35– 52, 1998. Han, I. S., Sylvester, S. R., Kim, K.U., Schelling, M. E., Venkateswaran, S., Blanckaert, N. D., McGuinness, M. P., and Griswold, M. D., Basic fibroblast growth factor is a testicular cell product which may regulate Sertoli cell function. Mol. Endocrinol. 7, 889–897, 1993. Hansen, J. F., Historical control data on testicular interstitial cell tumors in Crl:CD BR control rats (personal communication), Haskell Laboratory for Toxicology and Industrial Medicine Medicine, Newark, Delaware, 1993. Hardy, M. P., Zirkin, B. R., and Ewing, L. L., Kinetic studies on the development of the adult population of Leydig cells in testes of the pubertal rat. Endocrinology 124, 762–770, 1989. Hardy, M. P., Kelce, W. R., Klinefelter, G. R., and Ewing, L. L., Differentiation of Leydig cell precursors in vitro: a role for androgen. Endocrinology 127, 488–490, 1990.


Hardy, M. P., Gelber, S. J., Zhou, Z., Penning, T. M., Ricigliano, J. W., Ganjam, V. K., Nonneman, D. and Ewing, L. L., Hormonal control of Leydig cell differentiation. Ann. NY Acad. Sci. 637, 152–163, 1991. Hardy, M. P., Kirby, J. D., Hess, R. A., and Cooke, P. S., Leydig cells increase their numbers but decline in steroidogenic function after neonatal hypothyroidism. Endocrinology 132, 2417–2420, 1993. Hardy, M. P., Sharma, R. S., Arambepola, N. K., Sottas, C. M., Russell, L. D., Bunick, D., Hess, R. A., and Cooke, P. S., Increased proliferation of Leydig cells induced by neonatal hypothyroidism in the rat. J. Androl. 17, 231–238, 1996. Haseman, J. K. and Elwell, M. R., Evaluation of false positive and false negative outcomes in NTP long-term rodent carcinogenicity studies. Risk Analysis 16, 813– 820, 1996. Haugen, T. B., Landmark, B. F., Josefson, G. M., Hansson, V., and Hogset, A., The mature form of interleukin–1α is constitutively expresed in immature male germ cells from the rat. Mol. Cell Endocrinol. 105, R19-R23, 1994. Heller, C. and Leach, D., Quantification of Leydig cells and measurement of Leydig cell size following administration of human chorionic gonadotrophin to normal men. J. Reprod. Fertil. 25, 185–192, 1971. Hill, R. N., Erdeich, L. S., Paynter, O. E., Roberts, P. A., Rosenthal, S. L., and Wilkinson, C. F., Thyroid follicular cell carcinogenesis. Fundam. Appl. Toxicol. 12, 629–697, 1989. Hill, R. N., Hurley, P. M., Crisp, T. M., Rosenthal, S. L., and Singh, D.V., Assessment of thyroid follicular cell tumors. SAB Review Draft, 1996. Himmelstein, M. W., Acquavella, J. F., Recio, L., Medinsky, M. A., and Bond, J. A., Toxicology and epidemiology of 1,3-Butadiene. Crit. Rev. Toxicol. 27, 1–108, 1997. Hodson-Walker, G., Rusch, G. M., and Debets, F. M. H., Mutagenicity testing of a number of CFC replacements: Methodology and results. Environ. Mol. Mutagen. 21(Suppl. 22), 28(Abstr.), 1993. Holland, F. J., Gonadotropin-independent precocious puberty. Endocrinol. Metabolism Clin. N. Am. 20, 191– 210, 1991. Holstein, A. F., Orlandini, G. E., and Moller, R., Distribution and fine structure of the lymphatic system in the human testis. Cell Tissue Res. 200, 15–27, 1979. Honore, L., Testicular biopsy for infertility: a review of sixty-eight cases with a simplified histologic classification of lesions. Int. J. Fertil. 24, 49–52, 1979. Horstman, W. G., Haluszka, M. M., and Burkhard, T. K., Management of testicular masses incidentally discovered by ultrasound. J. Urol. 151, 1263–1265, 1994. Horton, R. and Tait, J. F., In vitro conversion of dehydroisoandrosterone to plasma androstenedione and testosterone in man. J. Clin. Endocrinol. Metab. 22, 79– 88, 1967. Hosokawa, S., Murakami, M., Ineyama, M., Yamada, T., Koyama, Y., Okuno, Y., Yoshitake, A., Yamada, H., and Miyamoto, J., Effects of procymidone on repro-

ductive organs and serum gonadotropins in male rats. J. Toxicol. Sci. 18, 111–124, 1993a. Hosokawa, S., Murakami, M., Ineyama, M., Yamada, T., Yoshitake, A., Yamada, H., and Miyamoto, J., The affinity of procymidone to androgen receptor in rats and mice. J. Toxicol. Sci. 18, 83–93, 1993b. Huang, B. M., Tsai, M. L., Li, P. H., Yang, H. Y., and Liu, M. Y., The effects of lead on StAR protein expression and steroidogenesis in MA–10 cells. Biol. Reprod. 56(Suppl. 1), 475, 1997. Huhtaniemi, I. T., Gonadotrophin receptors: correlates with normal and pathological functions of the human ovary and testis, in Clinical Endocrinology and Metabolism: Receptors in Health and Disease, Clayton, W.B. Saunders, Philadelphia, 1983, 117–132. Hulse, E. V., Can radiation induce interstitial-cell (Leydigcell) tumours of the testis? Int. J. Radiat. Biol. 32, 185– 190, 1977. Hunter, M. G., Sullivan, M. H. F., Dix, C. J., Aldred, L. F., and Cooke, B. A., Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells. Mol. Cell Endocrinol. 27, 31–34, 1982. Huseby, R. A., Estrogen-induced Leydig cell tumor in the mouse: A model system for the study of carcinogenesis and hormone dependency. J. Toxicol. Environ. Health Suppl. 1, 177–192, 1976. Huseby, R. A. and Samuels, L. T., Lack of influence of hypophysectomy on estrogen-induced DNA synthesis in Leydig cells of BALB/c Mice. J. Natl. Cancer Inst. 58, 1047–1049, 1977. Huseby, R. A., Demonstration of a direct carcinogenic effect of estradiol on Leydig cells of the mouse. Cancer Res. 40, 1006–1013, 1980. Huseby, R. A., Leydig cell neoplasia, in The Leydig Cell. Payne, A. H., Hardy, M. P., and Russell, L. D., Eds., Cache River Press, Vienna, IL, 1996, 555–572. IARC, Monographs on the evaluation of the carcinogenic risk of chemicals to humans 20, Some Halogenated Hydrocarbons, Lyon, 1979. IARC, Tobacco Smoking, Vol. 38, IARC, Lyon, 1986. IARC, Lead and lead compounds: Lead and inorganic lead compounds (Group 2B) Organolead compounds (Group 3), in IARC Monogr. Suppl. 7, 230–232, 1987. Ichihara, I., Kawamura, H., and Pelliniemi, L. J., Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats. Cell Tissue Res. 271, 241–255, 1993. Irby, D. C., Kerr, J. B., Risbridger, G. P., and de Kretser, D. M., Seasonally and experimentally induced changes in testicular function of the Australian bush rat Rattus fuscipes. J. Reprod. Fertil. 70, 657–666, 1984. Ird, E. A. and Smirnova, I. O., Carcinogenesis in rats induced during combined transplacental exposure to sex hormones and nitrosoethylurea. Vopr. Onkol. 29, 32– 36, 1983. Ird, E. A., Blastomogenesis in rats in transplacental administration of estrogens. Vopr. Onkol. 29, 61–66, 1983.

249


IRI, Background tumour incidences from carcinogenicity studies in Crl: CD–1 Swiss mice, Inveresk Research International, Tranent, 1995a. IRI, Background tumour incidences from carcinogenicity studies in Crl: CD Sprague-Dawley rats, Inveresk Research International, Tranent, 1995b. Iswaran, T. J., Imai, M., Betton, G. R., and Siddall, R. A., An overview of animal toxicology studies with bicalutamide (ICI 176, 334), J. Toxicol. Sci. 22, 75–88, 1998. Iwata, H., Hirouchi, Y., Koike, Y., Yamakawa, S., Kobayashi, K., Yamamoto, T., Inoue, H., and Enomoto, M., Historical control data of non-neoplastic and neoplastic lesions in F344/DuCrj rats. J. Toxicol. Pathol. 4, 1–24, 1991. James, R. W. and Heywood, R., Age-related variations in the testes and prostate of beagle dogs, Toxicology 12, 273–279, 1979. Jameson, C. W., Toxicology and carcinogenesis studies of d-limonene in F344/N rats and B6C3F1 mice, NIH Publication No. 90–2802, Research Triangle Park, 1990. Jockenhovel, J. K. L., Mason, J. S., Griffin, J. E., and Swerdloff, R. S., Leydig cell neoplasia in a patient with Reifenstein Syndrome. Exp. Clin. Endocrinol. 101, 365– 370, 1993. Johnson, L., George, F. W., Neaves, W. B., Rosenthal, I. M., Christensen, R. A., DeCristoforo, A., Schweikert, H.U., Sauer, M.V., Leshin, M., Griffin, J. E., and Wilson, J. D., Characterization of the testicular abnormality in 5-reductase deficiency. J. Clin. Endocrinol. Metab. 63, 1091–1099, 1986. Johnston, D. E., Chiao, Y.-B., Gavaler, J. S., and Van Thiel, D. H., Inhibition of testosterone synthesis by ethanol and acetaldehyde. Biochemical Pharmacol. 30, 1827–1831, 1981. Jones, H. B., Betton, G. R., Bowler, A. L., McFarquhar, R. L., Middleman, B. J., and Lunglmayr, G., Pathological and morphometric assessment of testicular parameters in patients with metastatic prostate cancer following treatment with either the anti-androgen Casodex (ZM176,334) or bilateral orchidectomy. Urol. Refs., 22, 191–195, 1994. Juniewicz, P. E., McCarthy, M., Lemp, B. M., Barbolt, T. A., Shaw, C., Hollenbaugh, D. M., Winneker, R. C., Reel, J. R., and Batzold, F. H., The effect of the steroidal androgen receptor antagonist, Win 49,596, on the prostate and testis of beagle dogs. Endocrinology 126, 2625–2634, 1990. Junker Walker, U. and Nogues, V., Changes induced by treatment with aromatase inhibitors in testicular Leydig cells of rats and dogs. Exp. Toxic. Pathol. 46, 211–213, 1994. Kahn, C. R., Smith, R. J., and Chin, W. W., Mechanism of action of hormones that act at the cell surface, in Textbook of Endocrinology, WB Saunders, Philadelphia, 1992, 91–134. Kaler, L. W. and Neaves, W. B., The androgen status of aging male rats. Endocrinology 108, 712–719, 1981.

250

Kan, P. B., Hirst, M. A., and Feldman, D., Inhibition of steroidogenic cytochrome P–450 enzymes in rat testis by ketoconazole and related imidazole anti-fungal drugs. J. Steroid Biochem. 23, 1023–1029, 1985. Kaplan, G. W., Cromie, W. J., Kelalis, P. P., Silber, I., and Tank, E. S., Gonadal stromal tumors: A report of the prepubertal testicular tumor registry. J. Urol. 136, 300–302, 1986. Keeney, D. S., Mendis-Handagama, S. M. L. C., Zirkin, B. R., and Ewing, L. L., Effect of long term deprivation of luteinizing hormone on Leydig cell volume, Leydig cell number, and steroidogenic capacity of the rat testis. Endocrinology 123, 2906–2915, 1988. Keeney, D. S., Sprando, R. L., Robaire, B., Zirkin, B. R., and Ewing, L. L., Reversal of long-term luteinizing hormone deprivation on testosterone secretion and Leydig cell volume, number, and proliferation in adult rats. J. Endocrinol. 127, 47–58, 1990. Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppainen, J. A., and Wilson, E. M., Persistent DDT metabolite p,p′-DDE is a potent androgen receptor antagonist. Nature 375, 581–585, 1995. Kerr, J. B., Rich, K. A., and de Kretser, D. M., Alterations of the fine structure and androgen secretion of the interstitial cells in the experimentally cryptorchid rat testis. Biol. Reprod. 20, 409–422, 1979. Kerr, J. B. and Sharpe, R. M., FSH induction of Leydig cell maturation. Endocrinology 116, 2592–2604, 1985a. Kerr, J. B. and Sharpe, R. M., Stimulatory effect of FSH on rat Leydig cells, A morphometric and ultrastructural study. Cell Tiss. Res. 239, 405–415, 1985b. Kerr, J. B. and Donachie, K., Regeneration of Leydig cells in unilaterally cryptorchid rats: evidence for stimulation by local testicular factors. Cell Tiss. Res. 245, 649– 655, 1986. Kerr, J. B. and Knell, C. M., The fate of fetal Leydig cells during the development of the fetal and postnatal rat testis. Development 103, 535–544, 1988. Kerr, J. B. and Sharpe, R. M., Sertoli-Leydig cell interactions in the unilaterally cryptorchid testis, in The Cryptorchid Testis. Abney, T. O. and Keel, B. A., Eds., CRC Press, Boca Raton, 1989, 133–159. Khan, S. A., Soder, O., Syed, V., Gustafsson, K., Lindh, M., and Ritzen, E. M., The rat testis produces large amounts of an interleukin-I-like factor. Int. J. Androl. 10, 495–503, 1987. Khan, S. A., Schmidt, K., Hallin, P., di Pauli, R., de Gyter, C., and Nieschlag, E., Human testis cytosol and ovarian follicular fluid contain high amounts of interleukinI-like factor(s). Mol. Cell Endocrinol. 58, 221–230, 1988. Khan, S., Teerds, K., and Dorrington, J., Growth factor requirements for DNA synthesis by Leydig cells from the immature rat. Biol. Reprod. 46, 335–341, 1992a. Khan, S. A., Khan, S. J., and Dorrington, J. H., InterleukinI stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro. Endocrinology 131, 1853–1857, 1992b.


Kirby, J. D., Jetton, A. E., Cooke, P. S., Hess, R. A., Brunick, D., Ackland, J. F., Turek, F. W., and Schwartz, N. B., Developmental hormonal profiles accompanying the neonatal hypothyroidism-induced increase in adult testicular size and sperm production in the rat. Endocrinology 131, 559–565, 1992. Klinefelter, G. R., Hall, P. F., and Ewing, L. L., Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol. Reprod. 36, 769–783, 1987. Klinefelter, G. R. and Ewing, L. L., Optimizing testosterone production by purified adult rat Leydig cells. In Vitro Cell. Dev. Biol. 24, 545–549, 1988. Klinefelter, G. R. and Kelce, W. R., A comparison of Leydig cell responsiveness to hormonal and nonhormonal factors In Vivo and In Vitro, in The Leydig Cell. Payne, A. H., Russell, L., and Hardy, M., Eds., Cache River Press, 1996, 535–553. Kosugi, S., Van Dop, C., Geffner, M. E., Rabl, W., Carel, J., Chaussain, J., Mori, T., Merendino, J. J., Jr., and Shenker, A., Characterization of heterogeneous mutations causing constitutive activation of the luteinizing hormone receptor in familial male precocious puberty. Hum. Mol. Genet. 4, 183–188, 1995. Kothari, L. and Gupta, A., Effect of aging on the volume, structure and total Leydig cell content of the human testis. Int. J. Fertil. 19, 140–146, 1974. Kuopio, T., Tapanainen, J., Pelliniemi, L. J., and Huhtaniemi, L., Developmental stages of fetal-type Leydig cells in prepubertal rats. Development 107, 213– 220, 1989. Kurtz, S. M., Fitzgerald, J. E., Fisken, R. A., Schardein, J. L., Reufner, T. H., and Lucas, J. A., Toxicology studies on gemfibrozil. Proc. Royal Soc. Med. 69(Suppl. 2), 15–23, 1976. Lacassagne, A. and Hurst, L., Experimental tumors of the rat interstitial gland: An oncogenic manifestation of o,p′-dichlorodiphenyldichloroethane in the testicle. Bull. Cancer 52, 89–104, 1965. Lacassagne, A., Critical review of experimental Leydig cell tumors, particularly in the rat. Bull. Cancer 58, 235– 275, 1971. Lang, P. L., Spontaneous Neoplastic Lesions in the B6C3F1/ CrlBR Mouse, Charles River Laboratory, Raleigh, 1989. Lang, P. L., Spontaneous Neoplastic Lesions in the CDF (F-344)/CrlBr Rat, Charles River Laboratory, Raleigh, 1990. Lang, P. L., Spontaneous Neoplastic Lesions and Selected Non-Neoplastic Lesions in the Crl:CD Br Rat, Charles River Laboratory, Raleigh, 1992. Lang, P. L., Spontaneous Neoplastic Lesions in the Crl:CD– 1´BR Mouse, Charles River Laboratory, Raleigh, 1995. Laroque, P. A., Prahalada, S., Gordon, L. R., Molon Noblot, S., Bagdon, W. J., Duprat, P., Peter, C. P., and Van Zwieten, M. J., Effects of chronic oral administration of a selective 5α-reductase inhibitor, finasteride, on the dog prostate. The Prostate 24, 93– 100, 1994.

Laskey, J. W. and Phelps, P. V., Effect of cadmium and other metal cations on in vitro Leydig cell testosterone production. Toxicol. Appl. Pharmacol. 108, 296–306, 1991. Le Magueresse-Battistoni, B. L., Wolf, J., Morera, A. M., and Benahmed, M., Fibroblast growth factor receptor type I expression during rat testicular development and its regulation in cultured sertoli cells. Endocrinology 135, 2404–2411, 1994. Lecerf, L., Rouiller-Fabre, V., Levacher, C., Gautier, C., Saez, J. M., and Habert, R., Stimulatory effects of follicle stimulating hormone on basal and LH-stimulated testosterone secretion by the fetal rat testis in vitro. Endocrinology 133, 2313–2218, 1993. Lee, W., Mason, A. J., Schwall, R., Szonyi, E., and Mather, J. P., Secretion of activin by interstitial cells in the testis. Science 243, 396–398, 1989. Lee, M. M. and Donahoe, P. K., Mullerian inhibiting substance: a gonadal hormone with multiple functions. Endocrin. Rev. 14, 152–164, 1993. Leinonen, P. and Jaffe, R. B., Leydig cell desensitization by human chorionic gonadotropin does not occur in the human fetal testis. J. Clin. Endocrinol. Metab. 61, 234– 238, 1985. Lephart, E. D. and Simpson, E. R., Assay of aromatase activity. Methods Enzymol. 206, 477–483, 1991. Leslie, G. B. and Walker, T. F., A toxicological profile of cimetidine, in Cimetidine: Proceedings of the Second International Symposium on Histamine Antagonists. Burland, W. L. and Simkins, M. A., Eds., Oxford Excepta Medica, Amsterdam, 1977, 24–33. Leslie, G. B., Noakes, D. N., Pollitt, F. D., Roe, F. J. C., and Walker, T. F., A two-year study with cimetidine in the rat: assessment for chronic toxicity and carcinogenicity. Toxicol. Appl. Pharmacol. 61, 119–137, 1981. Lewis, D., Incidence of benign interstitial cell tumours of the testis from seven recently completed chronic studies (personal communication), HRC, Huntingdon, England, 1993. Liao, C., Reaven, E., and Azhar, S., Age-related decline in the steroidogenic capacity of isolated rat Leydig cells: a defect in cholesterol mobilization and processing. J. Steroid Biochem. Mol. Biol. 46, 39–47, 1993. Lin, T., Murono, E., Osterman, J., Allen, D. O., and Nankin, H. R., The aging Leydig cell: Testosterone and adenosine 3′, 5′-monophosphate responses to gonadotropin stimulation in rats. Steroids 35, 653–663, 1980. Lin, T., Haskell, J., Vinson, N., and Terracio, L., Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary culture: a comparative study. Endocrinology 119, 1641–1647, 1986. Lin, T., Vinson, N., Haskett, J., and Murono, E., Induction of 3β-hydroxysteroid dehydrogenase activity by insulin-like growth factor-I in primary culture of purified Leydig cells. 219, 603–607, 1987a. Lin, T., Blaisdell, J., and Haskell, J. F., Transforming growth factor-β inhibits Leydig cell steroidogenesis in

251


primary culture. Biochem. Biophys. Res. Commun. 146, 387–394, 1987b. Lin, T., Blaisdell, J., Barbour, K. W., and Thompson, E. A., Transient activation of c-myc protooncogene expression in Leydig cells by human chorionic gonadotropin. Biochem. Biophys. Res. Comm. 157, 121– 126, 1988. Lin, T., Calkins, H., Morris, P. L., Vale, W. W., and Bardin, C. W., Regulation of Leydig cell function in primary culture by inhibin and activin. Endocrinology 125, 2134–2140, 1989. Lin, T. W. D., Calkins, J., Guo, H., Chi, R., and Housley, P., Regulation of insulin-like growth factor-I messenger ribonucleic acid expression in Leydig cells. Mol. Cell Endocrinol. 73, 147–152, 1990. Lin, T., Wang, D., Nagpal, M.T., Calkins, J. H., Chang, W. C., and Chi, R., Interleukin-I inhibits cholesterol side-chain cleavage cytochrome P450 expression in primary cultures of Leydig cells. Endocrinology 129, 1305–1311, 1991. Lin, T., Wang, D., and Nagpal, L., Human chorionic gonadotropin induces interleukin-I gene expression in rat Leydig cell in vitro. Mol. Cell Endocrinol. 95, 139–145, 1993. Lin, D., Sugawara, T., Strauss, J. F., Clark, B. J., Stocco, D. M., Saenger, P., Rogol, A., and Miller, W. L., Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267, 1828– 1831, 1995. Lindsay, S., Nichols, J., C. W., Sheline, G. E., and Chaikoff, I. L., Leydig cell tumors in rat testes subjected to low-dose X-irradiation. Radiat. Res. 40, 366– 378, 1969. Liu, S. C., Sanfilippo, B., Perroteau, I., Derynck, R., Salomon, D. S., and Kidwell, W. R., Expression of transforming growth factor a (TGFa) in differentiated rat mammary tumors: estrogen induction of TGFα production. Mol. Endocrinol. 1, 683–692, 1987. Liu, R. C. M., Hahn, C., and Hurtt, M. E., The direct effect of hepatic peroxisome proliferators on rat Leydig cell function in vitro. Fundam. Appl. Toxicol. 30, 102– 108, 1996a. Liu, R. C. M., Hurtt, M. E., Cook, J. C., and Biegel, L. B., Effect of the peroxisome proliferator, ammonium perfluorooctanoate (C8), on hepatic aromatase activity in adult male Crl:CDBR (CD) rats. Fundam. Appl. Toxicol. 30, 220–228, 1996b. Lloyd, S. C., Blackburn, D. M., and Foster, P. M. D., Trifluoroethanol and its oxidative metabolites: Comparison of in vivo and in vitro effects in rat testis. Toxicol. Appl. Pharmacol. 92, 390–401, 1988. Longcope, C., Pratt, J. H., Scheider, S. H., and Fineberg, S. E., Aromatization of androgens by muscle and adipose tissue in vivo. J. Clin. Endocrinol. Metab. 46, 146– 152, 1978. Longnecker, Alcohol consumption and risk of cancer in humans: an overview. Alcohol 12, 87–96, 1995. Longstaff, E., Robinson, M., Bradbrook, C., Styles, J. A., and Purchase, I. F. H., Genotoxicity and carcinogenicity of fluorocarbons: Assessment by short-term in

252

vitro tests and chronic exposure in rats. Toxicol. Appl. Pharmacol. 72, 15–31, 1984. Lopez-Ruiz, M. P., Choi, M. S. K., Rose, M. P., West, A. P., and Cooke, B. A., Direct effect of arachnidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells: evidence for tonic inhibitory control of steroidogenesis by protein kinase C. Endocrinology 130, 1122–1130, 1992. Maddock, W. O. and Nelson, W. O., The effects of chorionic gonadotropin in adult men: increased estrogen and 17-ketosteroid excretion, gynecomastia, Leydig cell stimulation and seminiferous tubule damage. J. Clin. Endocrinol. Metab. 12, 985–1007, 1952. Maddocks, S. and Sharpe, R. M., The effects of sexual maturation and altered steroid synthesis on the production and route of secretion of inhibin-α from the rat testis. Endocrinology 126, 1541–1550, 1990. Maekawa, A. and Hayashi, Y., Neoplastic lesions of the testis, in Pathobiology of the Aging Rat Sponsored by the International Life Sciences Institute, Blood and Lymphoid, Respiratory, Urinary, Cardiovascular, and Reproductive Systems. Mohr, U., Dungworth, D. L., and Capen, C. C., Eds., ILSI Press, Washington, D. C., 1992, 413–418. Magre, S. and Jost, A., The initial phases of testicular organogenesis in the rat. Arch. Anat. Mic. Morphol. Exp. 69, 297–318, 1980. Maita, K., Hirano, M., Hirada, T., Mitsumori, K., Yoshida, A., Takahashi, K., Nakashima, N., Kitazawa, T., Enomoto, A., Inuie, K., and Shirasus, Y., Mortality, major cause of morbundity and spontaneous tumors in CD–1 mice. Toxicol. Pathol. 16, 340–349, 1988. Malinverno, G., Rusch, G. M., Millischer, R. J., Hughes, E. W., Schroeder, R. E., and Coombs, D. W., Inhalation teratology and reproduction studies with1,1dichloro–2,2,2-trifluoroethane (HCFC–123), Fundam. Appl. Toxicol. 34, 276–287, 1996. Malley, L. A., Carakostas, M., Hansen, J. F., Rusch, G. M., Kelly, D. P., and Trochimowicz, H. J., Two-year inhalation toxicity study in rats with hydro-chlorofluorocarbon 123. Fundam. Appl. Toxicol. 25, 101–114, 1995. Maltoni, C., Lefemine, G., Cotti, G., and Perino, G., Long-term carcinogenicity bioassays on trichloroethylene administered by inhalation to Sprague-Dawley rats and Swiss and B6C3F1 mice. Ann. New York Acad. Sci. 534, 316–342, 1988. Mancini, R. E., Vilar, O., Laviere, J. C., Andrada, J. A., and Heinrich, J. J., Development of Leydig cells in the normal human testis. A cytological, cytochemical and quantitative study. Am. J. Anat. 112, 203–214, 1963. Mann, D. R., Adams, S. R., Gould, K. G., Orr, T. E., and Collins, D. C., Evaluation of the possible direct effects of gonadotrophin-releasing hormone analogues on the monkey. (Macaca mulatta) testis. J. Reprod. Fertil. 85, 89–95, 1989. Marselos, M. and Vainio, H., Carcinogenic properties of pharmaceutical agents evaluated in the IARC monographs programme. Carcinogenesis 12, 1751–1766, 1991.


Marselos, M. and Tomatis, L., Diethylstilbestrol. II. Pharmacology, toxicology and carcinogenicity in experimental animals. Eur. J. Cancer 29A, 149–155, 1993. Martin-Lopez, G., Vilchez-Martinez, J., HernandezYanez, L., Torres-Morales, A., and Bishop W., Leydig cells function in hyper or hipoprolactinaemic states in healthy men, Invest. Clin. 37, 153–156, 1996. Mather, J. P., Attie, K. M., Woodruff, T. K., Rice, G. C., and Phillips, D. M., Activin stimulates spermatogonial proliferation in germ-Sertoli cell cocultures from immature rat testis. Endocrinology 127, 3206–3214, 1990. Mather, J. P. and Krummen, L. A., Inhibin, activin and growth factors: Paracrine regulators of testicular function, in Spermatogenesis, Fertilization, Contraception. Nieschlag, E. and Habenicht, U.-F., Eds., SpringerVerlag, Berlin, 1992, 169–200. Matzuk, M. M., Finegold, M. J., Mishina, Y., Bradley, A., and Behringer, R. R., Synergistic effects of inhibins and mullerian-inhibiting substance on testicular tumorgenesis, Mol. Endocrinol. 9, 1337–1345, 1995. Matzuk, M. M., Finefold, M. J., Su, J. G. J., Hsueh, A. J. W., and Bradley, A., α-Inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 360, 313–319, 1992. Mauduit, C., Chauvin, M. A., Hartmann, D. J., Revol, A., Morera, A. M., and Benahmed, M., Interleukin–1α as a potent inhibitor of gonadotropin action in porcine Leydig cells: site(s) of action. Biol. Reprod. 46, 1119– 1126, 1992. Maximov, A., Die histologische vorgange bie der heilung von hodenverletzengen und die regenerations fahigheet des hodengewebes. Beitr Path. Anat. 26, 231–319, 1899. McConnell, R. F., Westen, H. H., Ulland, B. M., Bosland, M. C., and Ward, J. M., Proliferative lesions of the testes in rats with selected examples from mice, in Guides for Toxicologic Pathology, STP/ARP/AFIP, Washington, D. C., URG–3, 1992. McMartin, D. N., Sahota, P. S., Gunson, D. E., Hsu, H. H., and Spaet, R. H., Neoplasms and related proliferative lesions in control Sprague-Dawley rats from carcinogenicity studies. Historical data and diagnostic considerations. Toxicol. Pathol. 20, 212–225, 1992. Meikle, A. W., Sanders, S. W., Tolman, K. G., Jennings, D. E., Karol, M. D., and Ringham, G. L., Effect of lansoprazole on male hormone function. Drug Invest. 8, 191–202, 1994. Melnick, R. L., Huff, J., Haseman, J. K., Dieter, M. P., Grieshaber, C. K., Wyand, D. S., Russfield, A. B., Murthy, A. S. K., Fleischman, R. W., and Lilja, H. S., Chronic effects of agar, agar gum, gum arabic, locustbean gum, or tara gum in F344 rats and B6C3F1 mice. Food Chem. Toxicol. 21, 305–311, 1983. Melnick, R. L., Sills, R. C., Roycroft, J. H., Chou, B. J., Ragan, H. A., and Miller, R. A., Isoprene, an endogenous hydrocarbon and industrial chemical, induces multiple organ neoplasia in rodents after 26 weeks of inhalation exposure. Cancer Res. 54, 5333–5339, 1994. Mendis-Handagama, S. M. L. C., Kerr, J. B., and De Kretser, D. M., Experimental cryptorchidism in the

adult mouse. I. Qualitative and quantitative light microscopic morphology. J. Androl. 11, 539–547, 1990a. Mendis-Handagama, S. M. L. C., Kerr, J. B. and de Kretser, D. M., Experimental cryptorchidism in the adult mouse. II. A hormonal study. J. Androl. 11, 548– 547, 1990b. Mendis-Handagama, S. M. L. C., Kerr, J. B., and de Kretser, D. M., Experimental cryptorchidism in the adult mouse. III. Qualitative and quantitative electron microscopic morphology of Leydig cells. J. Androl. 12, 335–343, 1991. Mendis-Handagama, S. M. L. C., and Sharma, O. P., Effects of neonatal administration of the reversible goitrogen propylthiouracil on the testis interstitium in adult rats. J. Reprod. Fertil. 100, 85–92, 1994. Mennear, J. H., Toxicology and carcinogenesis studies of tetrachloroethylene (perchloroethylene) in F344/N rats and B6C3F1 mice, NTP Technical Report 311 (NIH Publication No. 86–2567), 1986. Mennear, J. H., Toxicology and carcinogenesis studies of trichloroethylene in four strains of rats, NTP Technical Report 273 (NIH Publication No. 88–2529), 1988. Millischer, R.-J., de Rooij, C. G., Rush, G. M., Farr, C. H., Ben-Dyke, R., Hardy, C. J., Lewis, D. J., and HodsonWalker, G., Evaluation of the genotoxicity potential and chronic inhalation toxicity of 1,1-dichloro–1fluoroethane (HCFC–141b). Fd. Chem. Toxicol. 33, 491–500, 1995. Mitsumori, K. and Elwell, M. R., Proliferative lesions in the male reproductive system of F344 rats and B6C3F1 mice: incidence and classification. Environ. Health Perspect. 77, 11–21, 1988. Moger, W., Evidence for compartimentalization of adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinases in rat Leydig cells using site-selective cAMP analogs. Endocrinology 128, 1414–1418, 1991. Monder, C., Miroff, Y., Marandici, A., and Hardy, M., 11b-Hydroxysteroid dehydrogenase alleviates glucocorticoid-mediated inhibition of steroidogenesis in rat Leydig cells. Endocrinology 134, 1199–1204, 1994. Monder, C., Hardy, M. P., Blanchard, R. J., and Blanchard, D. C., Comparative aspects of 11βhydroxysteroid dehydrogenase. Testicular 11βhydroxysteroid dehydrogenase: development of a model for the mediation of Leydig cell function by corticosteroids. Steroids 59, 69–73, 1994. Moore, C. R., Testicular reactions to experimental crypotrchidism. Am. J. Anat. 34, 269–276, 1924. Moore, J. W. and Bulbrook, R. D., The epidemiology and function of sex hormone binding globulin, in Oxford Reviews of Reproductive Biology, Vol. 10. Clark, J. R., Ed., Oxford University Press, Oxford, 1988, 180–236. Moore, C. and Moger, W. H., Interleukin–1α-induced changes in androgen and cyclic adenosine 3′, 5′-monophosphate release in adult rat Leydig cells in culture. J. Endocrinol. 129, 381–390, 1991. Moore, A. and Morris, I. D., The involvement of insulinlike growth factor-I in local control of steroidogenesis and DNA synthesis of Leydig and non-Leydig cells in

253


rat testicular interstitium. J. Endocrinol. 138, 107–114, 1993a. Moore, A. and Morris, I. D., Paracrine effects via the epidermal growth factor receptor in the rodent testis may be mediated by non-Leydig interstitial cells. J. Endocrinol. 136, 439–446, 1993b. Morera, A. M., Cochet, C., Keramidas, M., Chauvin, M. A., De Peretti, E., and Benahmed, M., Direct regulating effects of transforming growth factor-β on the Leydig cell steroidogenesis in primary culture. J. Steroid Biochem. 30, 443–447, 1988. Morita, K., Ono, T., and Shimakawa, H., Inhibition of testosterone biosynthesis in testicular microsomes by various imidazole drugs. Comparative study with ketoconazole. J. Pharmacobio-Dyn. 13, 336–343, 1990. Morris, I. D., Leydig Cell Toxicology, in The Leydig Cell. Payne, A. H., Hardy, M. P., and Russell, L. D., Eds., Cache River Press, Vienna, IL, 1996, 573–596. Mostofi, F. K. and Price, E. B., Jr., Tumours of the male genital system, in Atlas of Tumor Pathology, FASC 8. AFIP, Washington, 1973, 86–99. Mostofi, F. K. and Davis, J. D., Jr., Rare tumors of testes and adnexal tumors, in Testicular Cancer and Other Tumors of the Genitourinary Tract. Pavone-Macaluso, M., Smith, P. H., and Bagshaw, M. A., Eds., Plenum Press, New York, 1985, 29–30. Mostofi, F. K. and Davis, J. D., Jr., Male reproductive system and prostate, in Anderson’s Pathology, Kissane, J. M., Ed. C.V. Mosby Company, Philadelphia, 1990, 871–919. Mostofi, F. K., Sesterhenn, I. A., and Bresler, V. M., Tumors of the testis, in Pathology of Tumours in Laboratory Animals, 1, Tumors of the Rat. Turusov, V. and Mohr, U., Eds., IARC Scientific Publications, Lyon, 1990, 399–419. Mulder, E., Brinkman, A. D., Lamets-Stahlhoden, G. J. M., and vender Molen, H. J., Binding of estradiol by the nuclear fraction of rat testis interstitial tissues. FEBS Lett. 31, 131–136, 1973. Mullaney, B. P. and Skinner, M. K., Basic fibroblast growth factor (bFGF) gene expression and protein production during pubertal development of the seminiferous tubule: follicle-stimulating hormone-induced Sertoli cell bFGF expression. Endocrinology 131, 2928–2934, 1992. Murakami, M., Hosokawa, S., Yamada, T., Harakawa, M., Ito, M., Koyama, Y., Kimura, J., Yoshitake, A., and Yamada, H., Species-specific mechanism in rat Leydig cell tumorigenesis by procymidone. Toxicol. Appl. Pharmacol. 131, 244–252, 1995. Murono, E. P. and Payne, A. H., Testicular maturation in the rat. In vivo effect of gonadotropins on steroidogenic enzymes in the hypophysectomized immature rat. Biol. Reprod. 20, 911–918, 1979. Murono, E. P. and Washburn, A. L., Basic fibroblast growth factor inhibits ∆5–3β-hydroxysteroid dehydrogenase-isomerase activity in cultured immature Leydig cells. Biochem. Biophys. Res. Commun. 168, 248–253, 1990a.

254

Murono, E. P. and Washburn, A. L., Fibroblast growth factor inhibits 5α-reductase activity in cultured immature Leydig cells. Mol. Cell Endocrinol. 68, R19-R23, 1990b. Murono, E. P., et al., Evidence for basic fibroblast growth factors in cultured immature Leydig cells. Endocrinology 88, 39–45, 1992. Nagpal, M. and et. al., Human chorionic gonadotropin upregulates insulin-like growth factor-I receptor gene expression of Leydig cells. Endocrinology 129, 2820–2826, 1991. Naville, D., Chatelain, P., Avallet, O., and Saez, J., Control of production of insulin-like growth factor-I by pig Leydig and Sertoli cells cultured alone or together. Cell-cell interactions. Mol. Cell Endocrinol. 70, 217– 224, 1990. Neaves, W. B., Johnson, L., Porter, J. C., Parker, C. R., and Petty, C. S., Leydig cell numbers, daily sperm production, and serum gonadotropin levels in aging men. J. Clin. Endocrinol. Metab. 59, 756–763, 1984. Neaves, W. B., Johnson, L., and Petty, C. S., Age-related change in numbers of other interstitial cells in the testes of adult men: evidence bearing on the fate of Leydig cells lost with increasing age. Biol. Reprod. 33, 259– 269, 1985. Neumann, F., Early indicators for carcinogenesis in sex hormone-sensitive organs. Mutat. Res. 248, 341–356, 1991. Niklowitz, P., Khan, S., Bergmann, M., Hoffmann, K., and Nieschalg, E., Differential effects of FSH and LH on Leydig cell function and restoration of spermatogenesis in hypophysectomized an photoinhibited Djungarian hamsters (Phodopus Sungorus). Biol. Reprod. 41, 871–880, 1989. Nishizawa, Y., Sato, B., Miyashita, Y., Tsukada, S., Hirose, T., Kishimoto, S., and Matsumoto, K., Autocrine regulation of cell proliferation by estradiol and hydroxytamoxifen of transformed mouse Leydig cells in serum-free culture. Endocrinology 122, 227–235, 1988. NTIS, A Chronic Oral Gavage Study in Rats with pNitrochlorobenzene, NTIS/OTS0526382, 1980. NTP, Toxicology and carcinogenesis studies of 3-chloro–2methylpropene in F344/N rats and B6C3F1 mice, National Toxicology Program NIH Publication No. 86–2556, Research Triangle Park, 1986. O’Leary, P., Jackson, A. E., Averill, S., and de Kretser, D. M., The effects of ethanedimethane sulphonate (EDS) on bilaterally cryptorchid rat testes. Mol. Cell. Endocrinol. 45, 183–190, 1986. O’Shaughnessy, P. J., Bennett, M. K., Scott, I. S., and Charlton, K. M., Effects of FSH on Leydig cell morphology and function in the hypogonadal mouse. J. Endocrinol. 135, 517–525, 1992. Odell, W. D. and Swerdloff, R. S., Etiologies of sexual maturation. Recent. Prog. Horm. Res. 32, 245–288, 1976. Ojeifo, J. O., Byers, S. W., Papadopoulos, V., and Dym, M., Sertoli cell-secreted protein(s) stimulates DNA synthesis in purified rat Leydig cells in-vitro. J. Reprod. Fertil. 90, 93–108, 1990.


Okawa, H. and Doi, K., Neoplastic lesions in streptozotocintreated rats. Exp. Anim. 32, 77–84, 1983. Oliver, M. F., Heady, J. A., Morris, J. N., and Cooper, J., A cooperative trial in the primary prevention of ischemic heart disease using clofibrate. (Report from the committee of principal investigators.). Brit. Heart J. 40, 1069–1118, 1978. Orth, J. M., Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study. Anat. Rec. 203, 458–492, 1982. Owen, P. E., Glaister, J. R., Gaunt, I. F., and Pullinger, D. H., Inhalation toxicity studies with 1,3-butadiene. 3 Two year toxicity/carcinogenicity study in rats. Am. Ind. Hyg. Assoc. J. 48, 407–413, 1987. Page, R. B., The anatomy of the hypothalmo-hypophyseal complex, in The Physiology of Reproduction. Knobil, E. and Neil, J. D., Eds., Raven Press, New York, 1988, 1161–1234. Paniagua, R., Amat, P., Nistal, M., and Martin, A., Ultrastructure of Leydig cells in human aging testes. J Anat. 146, 173–183, 1986. Papadopoulos, V., Mukhin, A. G., Cost, E., and Krueger, K. E., Peripheral type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis. J. Biol. Chem. 265, 3772–3779, 1990. Papadopoulos, V., Berkovich, A., and Krueger, K. E., The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: regulation of steroid biosynthesis. Neuropharmacology 30, 1417–1423, 1991. Parker, C. M., Patterson, D. R., Van Gelder, G. A., Gordon, E. B., Valerio, M. G., and Hall, W. C., Chronic toxicity and carcinogenicity evaluation of fenvalerate in rats. J. Toxicol. Environ. Health 13, 83– 97, 1984. Payne, A. H., Youngblood, G. L., Burgos-Trinidad, M., and Hammond, S. H., Hormonal regulation of steroidogenic enzyme gene expression in Leydig cells. J. Steroid Biochem. Mol. Biol. 43, 895–906, 1992. Payne, A. H., Hardy, M. P., and Russell, L. D., Payne, A. H., Hardy, M. P., and Russell, L. D., Eds., Cache River Press, Vienna, IL, 1996. PDR, Apresazide®(Hydralazine), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995a, 873–875. PDR, Atromid-S® (Clofibrate), in Physician’s Desk Reference, Oradell, Medical Economics Data, 1995b, 2647– 2649. PDR, Dynacirc® (Isradipine), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995c, 2155–2157. PDR, Eulexin® (Flutamide), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995d, 2253–2254. PDR, Felbatol® (Felbamate), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995e, 2605–2608. PDR, Hylorel® (Guanadrel), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995f, 1001–1002.

PDR, Lopid® (Gemfibrozil), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995g, 1851–1854. PDR, Lupron® (Leuprolide), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995h, 2503–2505. PDR, Nimotop® (Nimodipine), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995i, 1684–1686. PDR, Nitrolingual Spray® (Nitroglycerin), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995j, 1975–1976. PDR, Nolvadex® (Tamoxifen), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995k, 2757–2759. PDR, Pendil® (Felodipine), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995l, 544– 546. PDR, Proscar® (Finasteride), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995m, 1625–1628. PDR, Serax® (Oxazepam), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995n, 2739–2740. PDR, Supprelin® (Histrelin), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995o, 2000–2002. PDR, Synarel® (Nafarelin), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995p, 2486–2487. PDR, Tagamet® (Cimetidine), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995q, 2401–2404. PDR, Tegretol® (Carbamazepine), in Physician’s Desk Reference, 11th ed., Oradell, Medical Economics Data, 1995r, 603–605. PDR, Nizoral®, in Physician’s Desk Reference, 13th ed., Oradell, Medical Economics Data, 1997, 1344– 1355. Pederson, R. C., Polypeptide activators of cholesterol side-chain cleavage. Endocrin. Res. 10, 533–561, 1984. Pelliniemi, L. J. and Niemi, M., Fine structure of the human foetal testis. I. The interstitial tissue. Z. Zellforsch 99, 507–522, 1969. Pelliniemi, L. J., Dym, M., Crigler, J. F., Retik, A. B., and Fawcett, D. W., Development of Leydig cells in human fetuses and in patients with androgen insensitivity, in Testicular Development, Structure and Function. Steinberger, A. and Steinberger, E., Eds., Raven Press, New York, 1980, 49–54. Pinon-Lataillade, G., Viguier-Martinez, M. C., Touzalin, A. M., Maas, J., and Jegou, B., Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions. Reprod. Nutr. Dev. 31, 617–629, 1991. Pirke, K. M., Geiss, M., and Sintermann, R., A quantitative study on feedback control of LH by testosterone in young adult and old male rats. Acta Endocrinol. 89, 789–795, 1979.

255


Platts, E. A., Schulster, D., and Cooke, B. A., The inhibitory GTP-binding protein (Gi) occurs in rat Leydig cells and is differentially modified by lutropin and 12-Otetradecanoylphorbol 13-acetate. Biochem. J. 273, 895– 899, 1988. Pont, A., Williams, P. L., Azhar, S., Reitz, R. E., Bochra, C., Smith, E. R., and Stevens, D. A., Ketoconazole blocks testosterone synthesis. Arch. Intern. Med. 142, 2137–2140, 1982. Pour, P. M., Transplacental induction of gonadal tumors in rats by a nitrosamine. Cancer Res. 46, 4135–4138, 1986. Prahalada, S., Majka, J. A., Soper, K. A., Nett, T. M., Bagdon, W. J., Peter, C. P., Burek, J. D., MacDonald, J. S., and Van Zwieten, M. J., Leydig cell hyperplasia and adenomas in mice treated with finasteride, a 5 α-reductase inhibitor: a possible mechanism. Fundam. Appl. Toxicol. 22, 211–219, 1994. Prentice, D. E., Siegel, R. A., Donatsch, P., Qureshi, S., and Ettlin, R. A., Mesulergine induced Leydig cell tumours, a syndrome involving the pituitary-testicular axis of the rat. Arch. Toxicol. 15(Suppl.), 197–204, 1992. Prentice, D. E. and Meikle, A. W., A review of drug-induced Leydig cell hyperplasia and neoplasia in the rat and some comparisons with man. Hum. Exp. Toxicol. 14, 562–572, 1995. Preston-Martin, S. P., Evaluation of the evidence that tobacco-specific nitrosamines (TSNA) cause cancer in humans. Crit. Rev. Toxicol. 21, 295–298, 1991. Quest, J. A., Fenner-Crisp, P. A., Burnam, W., Copley, M., Dearfield, K. L., Hamernik, K. L., Saunders, D. S., Whiting, R. J., and Engler, R., Evaluation of the carcinogenic potential of pesticides. IV. Chloralkylthiodicarboximide compounds with fungicidal activity. Regul. Toxicol. Pharmacol. 17, 19–34, 1993. Quigley, C. A., De Bellis, A., Marschke, K. B., El-Awady, M. K., Wilson, E. M., and French, F. S., Androgen receptor defects: Historical, clinical, and molecular perspectives. Endocrine Reviews 16, 271–321, 1995. Raeside, J. I., Berthelon, M. C., Sanchez, P. and Saez, J. M., Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF). Biochem. Biophys. Res. Commun. 151, 151–159, 1988. Rao, M. S. and Reddy, J. K., Interstitial cell tumor, testis, rat, in Monographs on Pathology of Laboratory Animals Sponsored by the International Life Sciences Institute, Genital System. Jones, T. C., Mohr, U., and Hunt, R. D., Eds., Springer-Verlag, Berlin, 1987, 184–192. Reuber, M. D., Interstitial cell carcinomas of the testis in Balb/C male mice ingesting methoxychlor. J. Cancer Res. Clin. Oncol. 93, 173–179, 1979. Reyes, F.I., Winter, J. S. D., and Faiman, C., Endocrinology of the fetal testis, in The Testis. Burger, H. and de Kretser, D., Eds., Raven Press, New York, 1989, 119–142. Reznik-Schuller, H., Carcinogenic effects of diethylstilbestrol in male Syrian golden hamsters and European hamsters. J. Natl. Cancer Inst. 62, 1083–1088, 1979.

256

Rich, K. A., Kerr, J. B., and de Kretser, D. M., Evidence for Leydig cell dysfunction in rats with seminiferous tubule damage. Mol. Cell. Endocrinol. 13, 123–135, 1979. Risbridger, G. P., Kerr, J. B., and de Kretser, D. M., Evaluation of Leydig cell function and gonadotrophin binding in unilateral and bilateral cryptorchidism: evidence for local control of Leydig cell function by the seminiferous tubule. Biol Reprod. 24, 534–540, 1981a. Risbridger, G. P., Kerr, J. B., Peake, R., Rich, K. A., and de Kretser, D. M., The temporal changes in Leydig cell function after the induction of bilateral cryptorchidism. J. Reprod. 63, 415–423, 1981b. Risbridger, G. P., Kerr, J. B., Peake, R. A. and de Dretser, D. M., An assessment of Leydig cell function after bilateral or unilateral efferent duct ligation: further evidence for local control of Leydig cell function. Endocrinology 109, 1234–1241, 1981c. Risbridger, G. P., Kerr, J. B., and de Kretser, D. M., Effect of cryptorchidism on steroidogenesis and mitogenic activities in rat testicular interstitial fluid. J. Reprod. Fertil. 81, 617–624, 1987. Rittmaster, R. S., Lemay, A., Zwicker, H., Capizzi, T. P., Winch, S., Moore, E., and Gormley, G. J., Effect of finasteride, a 5 α-reductase inhibitor, on serum gonadotropins in normal men. J. Clin. Endocrinol. Metab. 75, 484–488, 1992. Roberts, S. A., Nett, T. M., Hartman, H. A., Adams, T. E., and Stoll, R. E., SDZ 200–100 induces Leydig cell tumors by increasing gonadotropins in rats. J. Am. Coll. Toxicol. 8, 487–505, 1989. Roberts, S. A., Robison, R. L., Van Ryzin, R. J., and Stoll, R. E., Increased incidence of Leydig cell tumors in male rats with the dopaminergic agonist, SDZ 205–502: correlation with increased serum LH levels. Toxicologist (Abstr.) 13, 1993. Roe, F. J. C., Cadmium neoplasia: testicular atrophy and Leydig cell hyperplasia and neoplasia in rats and mice following subcutaneous injection of cadmium salts. Br. J. Cancer 18, 674–681, 1964. Roe, F. J. C., Relevance for man of the effects of lactose, polyols and other carbohydrates on calcium metabolism seen in rats: A Review. Hum. Toxicol. 8, 87–98, 1989. Rommerts, F. R. G. and van der Molen, H. J., Testicular steroidogenesis, in The Testis. Burger, H. and deKretser, D. M., Eds., Raven Press, New York, 1989, 303– 328. Ronis, M. J. J., Ingelman-Sundberg, M., and Badger, T. M., Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and Bobwhite quail (colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs). Biochem. Pharmacol. 48, 1953–1965, 1994. Roosen-Runge, E. C., Quantitative studies on spermatogenesis in the albino rat. III. Volume changes in the cells of the seminiferous tubules. Anat. Rec. 123, 385–398, 1955. Rosenthal, S. M., Grumbach, M. M., and Kaplan, S. L., Gonadotropin-independent familial sexual precocity with


premature Leydig and germinal cell maturation (familial Testotoxicosis): Effect of a potent luteinizing hormone-releasing factor agonist and medroxyprogesterone acetate therapy in four cases. J. Clin. Endocrinol. Metab. 57, 571–579, 1983. Rowley-Jones, D. and Flind, A. C., Continuing evaluation of the safety of cimetidine, in Cimetidine in the 1980s. Baron, J. H., Ed., Churchill Livingstone, Edinburgh, 1981, 261–269. Rubens, R., Dhont, M., and Vermeulen, A., Further studies on Leydig cell function in old age. J. Clin Endocrinol. Metab. 39, 40–45, 1974. Rudo, K. M., Methyl tertiary butyl ether (MTBE) — Evaluation of MTBE carcinogenicity studies. Toxicol. Indust.Health 11, 167–173, 1995. Rui, H., Gordeladze, J. O., Gautvik, K. M., and Purvis, K., Prolactin desensitizes the prostaglandin E1-dependent adenylyl cyclase in the rat prostate gland. Mol. Cell. Endocrinol. 38, 53–60, 1984. Rusch, G. M., Trochimowicz, H. J., Malley, L. J., Kelly, D. P., Peckham, J., Hansen, J., and Charm, J. B., Subchronic inhalation toxicity studies with hydro-chlorofluorocarbon 123 (HCFC 123). Fundam. Appl. Toxicol. 23, 169–178, 1994. Rustia, M. and Shubik, P., Experimental induction of hepatomas, mammary tumors, and other tumors with metronidazole in noninbred Sas:MRC(WI)BR rats. JNCI 63, 863–868, 1979. Rutgers, J. L. and Scully, R. E., The androgen insensitivity syndrome (testicular feminization): a clinicopathological study of 43 cases. Int. J. Gynecol. Pathol. 10, 126– 144, 1991. Saez, J. M., Haour, F., and Cathiard, A. M., Human chorionic gonadotropin-induced Leydig cell refractoriness to gonadotropin stimulation. Mol. Pharmacol. 14, 1054– 1062, 1978. Saez, J. M., Leydig cells: endocrine, paracrine, and autocrine regulation. Endocrine Reviews 15, 574–626, 1994. Salhanick, H. A., Basic studies on aminoglutethimide. Cancer Res. 42, 3315s–3321s, 1982. Savage, G. N. and Kerr, J. B., Effect of seminiferous tubule size on hCG-induced regeneration of peritubular Leydig cells in hypophysectomized, EDS-treated rats. Int. J. Androl. 18, 35–44, 1995. Schanbacher, B. D., Testosterone secretion in cryptorchid and intact bulls injected with gonadotrophin-releasing hormone and luteinizing hormone. Endocrinology 104, 360–364, 1979. Schanbacher, B. D., Androgen response of cryptorchid and intact rams to ovine LH. J. Reprod. Fertil. 59, 151–154, 1980. Schardein, J. L., Kaump, D. H., Woosley, E. T., and Jellema, M. M., Long-term toxicologic and tumorigenesis studies on an oral contraceptive agent in albino rats. Toxicol. Appl. Pharmacol. 16, 10–23, 1970. Schardein, J. L., Studies of the components of an oral contraceptive agent in albino rats. I. Estrogenic component. J. Toxicol. Environ. Health 6, 885–894, 1980. Schedewie, H. K., Reiter, E. O., Beitins, I.Z., Seyed, S., Wooten, V. D., Jimenex, J. F., Aiman, E. J., DeVane,

G. W., Redman, J. F., and Elders, M. J., Testicular Leydig cell hyperplasia as a cause of familial sexual precocity. J. Clin. Endocrinol. Metab. 52, 271–278, 1981. Schinz, H. R. and Slotopolsky, B., Methodik experimenteller und histologischer Untersuchungen am Hoden, in Hanbuch de Biologischen Arbeitsmethoden. Abderhalden, E., Ed., Urgan and Schwarzenberg, Berlin, 1927, 238. Schottenfeld, D., Warshauser, M. E., Sherlock, S., Zauber, A. G., Leder, M., and Payne, R., The epidemiology of testicular cancer in young adults. Am. J. Epidemiol. 112, 232–246, 1980. Schroeder, R. E. and Daly, I. W., A chronic oral gavage study in rats with p-nitrochlorobenzene. NTIS OTS0526382, 1984. Scully, R. E., Neoplasia associated with anomalous sexual development and abnormal sex chromosomes, in The Intersex Child. Pediatric and Adolescent Endocrinology. Karger, Basel, 1981, 203–217. Setchell, B. P. and Galil, K. A. A., Limitations imposed by testicular blood flow on the function of Leydig cells in rats in vivo. Austral. J. Biol. Sci. 36, 285–294, 1983. Shan, L., Hardy, D., Catterall, J. F., and Hardy, M. P., Effects of luteinizing hormone (LH) and androgen on steady state levels of messenger ribonucleic acid for LH receptors, androgen receptors, and steroidogenic enzymes in rat Leydig cell progenitors in vivo. Endocrinology 136, 1686–1693, 1995. Sharpe, R. M., The hormonal regulation of the Leydig cell, in Oxford Reviews of Reproductive Biology, Vol. 4., Finn, C. A., Ed., Oxford University Press, Oxford, 1982, 241–317. Sharpe, R. M., Endocrinology and paracrinology of the testis, in Physiology and Toxicology of Male Reproduction. Lamb, J. C. and Foster, P. M. D., Eds., Academic Press, San Diego, 1988, 71–102. Sharpe, R. M., Maddocks, S., and Kerr, J. B., Cell-cell interactions in the control of spermatogenesis as studied using Leydig cell destruction and testosterone replacement. Am. J. Anat. 188, 3–20, 1990. Sharpe, R. M., Experimental evidence for Sertoli-germ cell and Sertoli-Leydig cell interactions, in The Sertoli Cell. Russell, L. D. and Griswold, M. D., Eds., Cache River Press, Clearwater, 1983, 391–413. Sharpe, R. M., Regulation of spermatogenesis, in The Physiology of Reproduction, 2nd ed., Knobil, E. and Neill, J. D., Eds., Raven Press, New York, 1994, 1363–1424. Shaw, M. A., Nicholls, P. J., and Smith, H. J., Aminoglutethimide and ketoconazole: historical perspectives and future prospects. J. Steroid Biochem. 31, 137–146, 1988. Shenker, A., Laue, L., Kosugi, S., Merendino Jr. , J. J., Minegishi, T., and Cutler, G. B., Jr., A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365, 652– 654, 1993. Sibinski, L. J., Two-year oral (diet) toxicity/carcinogenicity study of fluorochemical FC–143 in Rats, Riker Laboratories, Inc./3M Co., 1987.

257


Sikka, S. C., Swerdloff, R. S., and Raijfer, J., In vitro inhibition of testosterone biosynthesis by ketoconazole. Endocrinology 116, 1920–1925, 1985. Silverman, A. J., The gonadotropin-releasing hormone (GnRH) neuronal systems: immunocytochemistry, in The Physiology of Reproduction. Knobil, E. and Neill, J. D., Eds., Raven Press, New York, 1988, 1283–1304. Simard, J., Luthy, I., Guay, J., Belanger, A., and Labrie, F., Characteristics of interaction of the antiandrogen flutamide with the androgen receptor in various target tissues. Mol. Cell. Endocrinol. 44, 261–270, 1986. Simpson, B. J. B., Wu, F. C. W., and Sharpe, R. M., Isolation of human Leydig cells which are highly responsive to hCG. J. Clin. Endocrinol. Metab. 65, 415–422, 1987. Sinkeldam, E. J., Woutersen, R. A., Hollanders, V. M. H., Til, H. P., Van Garderen-Hoetmer, A., and Bar, A., Subchronic and chronic toxicity/carcinogenicity feeding studies with lactitol in rats. J. Am. Coll. Toxicol. 11, 165–188, 1992. Sivelle, P. C., Underwood, A. H., and Jelly, J. A., The effects of histamine H2 receptor antagonists on androgen action in vivo and dihydrotestosterone binding to the rat prostate androgen receptor in vitro. Biochem. Pharmacol. 31, 677–684, 1982. Skinner, M. K., Takacs, K., and Coffey, R. J., Transforming growth factor-a gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Endocrinology 124, 845–854, 1989. Skinner, M. K., Cell-cell interactions in the testis. Endocrin. Rev. 12, 45–67, 1991. Smith, E., Svoboda, M., Van Wyk, J., Kierzenbaum, A., and Tress, L., Partial characterization of a somatomedin-like peptide from the medium of cultured rat Sertoli cells. Endocrinology 120, 186–193, 1987. Smith, E. P., Hall, S. H., L., M., French, F. S., Wilson, E. M., and Conti, M., A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells. Mol. Endocrinol. 3, 954–961, 1989. Soder, O., Syed, V., Pollanen, P., Gustafsson, K., Granholm, K. K. S., Arver, S., Holst, M., von Euler, M., and Ritzen, E. M., Testicular interleukin–1-like factor, in The Molecular and Cellular Endocrinology of the Testis. Cooke, B. A. and Sharpe, R. M., Eds., Raven Press, New York, 1988, 325–332. Soder, O., Syed, V., and Callard, G.V., Production and secretion of an interleukin–1-like factor is stage-dependent and correlates with spermatogonial DNA synthesis in the rat seminiferous epithelium. Int. J. Androl. 14, 223–231, 1991. Sordoillet, C., Chauvin, M. A., Revol, A., Morera, A. M., and Benahmed, M., Fibroblast growth factor is a regulator of testosterone secretion in cultured immature Leydig cells. Mol. Cell. Endocrinol. 58, 283–286, 1988. Sordoillet, C., Chauvin, M. A., Hendrick, J. C., Franchimont, P., Morera, A. M., and Benahmed, M., Sites of interaction between epidermal growth fac-

258

tor and transforming growth factor-b1 in the control of steroidogenesis in cultured porcine Leydig cells. Endocrinology 130, 1352–1358, 1992. Spector, T. D., Ollier, W., Perry, L. A., Silman, A. J., Thompson, P. W., and Edwards, A., Free and serum testosterone levels in 276 males: a comparative study of rheumatoid arthritis, ankylosing spondylitis and health controls. Clin. Rheum. 8, 37–41, 1989. Spirtas, R., Stewart, P. and Lee, S., Retrospective cohort mortality study of workers at an aircraft maintenance facility I. Epidemiological results. Br. J. Ind. Med. 48, 515–530, 1991. Spiteri-Grech, J., Weinbauer, G., Bolze, P., Chandolia, R., and Bartlett, J., Effects of FSH and testosterone on intra-testicular IGF-I and specific germ cell populations in rats treated with a GnRH antagonist. J. Endocrinol. 137, 81–89, 1993. Stewart, P. A., Lee, J. G., Marano, D. E., Spirtas, R., Forbs, C. D., and Blair, A., Retrospective cohort mortality study of workers at an aircraft maintenance facility II. Exposures and their assessment. Br. J. Ind. Med. 48, 531–537, 1991. Stocco, D. M., A StAR Search: implications in controlling steroidogenesis. Bio. Reprod. 56, 328–336, 1997. Story, M.T., Sassee, J., Kakuska, D., Jacobs, S. C. and Lawson, K. L., A growth factor in bovine and human testes structurally related to basic fibroblast growth factor. J. Urol. 140, 422–427, 1988. Stott, W. T., Chemical induced proliferation of peroxisomes: Implications for risk assessment. Regul. Toxicol. Pharmacol. 8, 125–159, 1988. Sullivan, M. H. F. and Cooke, B. A., The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca+ by luteotropin (LH), luliberin (LHRH) agonist and cyclic AMP. Biochem. J. 236, 45–51, 1986. Syed, V., Soder, O., Arver, S., Lindh, M., Khan, S., and Ritzen, E. M., Ontogeny and cellular origin of an interleukin–1-like factor in the reproductive tract of the male rat. Int. J. Androl. 11, 437–447, 1988. Syed, V., Stephan, J. P., Gerard, N., Le Grand, A., Parvinen, M., Bardin, C. W., and Jegou, B., Residual bodies activate Sertoli cell IL–1α release which triggers IL–6 production by an autocrine mechanism through the lipoxygenase pathway. Endocrinology 136, 3070– 3078, 1995. Tabone, E., Benahmed, M., Reventos, J., and Saez, J. M., Interactions between immature porcine Leydig and Sertoli cells in vitro. An ultrastructural and biochemical study. Cell. Tiss. Res. 237, 357–362, 1984. Takenaka, S., Oldiges, H., Konig, H., Hochrainer, D. and Oberdorster, G., Carcinogenicity of cadmium chloride aerosols in Wistar rats. JNCI 70, 367–373, 1983. Tapanainen, J., Kuopio, T., Pelliniemi, L. J., and Huhtaniemi, I., Rat testicular endogenous steroids and number of Leydig cells between the fetal period and sexual maturity. Biol. Reprod. 31, 1027–1035, 1984. Teerds, K. J., De Rooij, D. G., Rommerts, F. F. G., and Wensing, C. J. G., The regulation of the proliferation


and differentiation of rat Leydig cell precursor cells after EDS administration or daily hCG treatment. J. Androl. 9, 343–351, 1988. Teerds, K. J., Closset, J., Rommerts, F. F. G., de Rooij, D. G., Stocco, D. M., Colenbrander, B., Wensing, C. J. G., and Hennen, G., Effects of pure FSH and LH preparations on the number and function of Leydig cells in immature hypophysectomized rats. J. Endocrinol. 120, 97–106, 1989a. Teerds, K. J., de Rooij, D. G., Rommerts, F. F. G., van den Hurk, R., and Wensing, C. J. G., Stimulation of the proliferation and differentiation of Leydig cell precursors after the destruction of existing Leydig cells with ethane dimethyl sulfonate (EDS) can take place in the absence of LH. J. Androl. 10, 472–477, 1989b. Teerds, K. J., de Rooij, D. G., Rommerts, F. F. G., and Wensing, C. J. G., Development of a new Leydig cell population after destruction of existing Leydig cells by ethane dimethane sulphonate in rats: an autoradiographic study. J. Endocrinol. 126, 229–236, 1990a. Teerds, K. J., Rommerts, F. F. G., and Dorrington, J. H., Immunohistochemical detection of transforming growth factor-a in Leydig cells during the development of the rat testis. Mol. Cell. Endocrinol. 69, R1-R6, 1990b. Teerds, K. J., de Rooij, D. G., de Jong, F. H. and Rommerts, F. F., Rapid development of Leydig cell tumors in Wistar rat substrain. J. Androl. 12, 171–179, 1991. Teerds, K. J. and Dorrington, J. H., Localization of transforming growth factor b1, and b2 during testicular development in the rat. Biol. Reprod. 48, 40–45, 1993. Teerds, K. J., Veldhuizen-Tsoerkan, M. B., Rommerts, F. F. G., de Rooij, D. G., and Dorrington, J. H., Proliferation and differentiation of testicular interstitial cells: aspects of Leydig cell development in the (pre) pubertal and adult testis. Journal 37–65, 1994. Teramoto, S., Saito, R., Aoyama, H. and Shirasu, Y., Dominant lethal mutation induced in male rats by 1,2dibromo–3-chloropropane (DBCP). Mutation Res. 77, 71–78, 1980. Terao, K., Aikawa, T. and Kera, K., A synergistic effect of nitrosodimethylamine on sterigmatocystin carcinogenesis in rats. Food Cosmet. Toxicol. 16, 591–596, 1978. Thompson, J. H., Irwin, R. D., Kanematsu, S., Seraydarian, K., and Suh, M., Effects of chronic nicotine administration and age in male Fischer–344 rats. Toxicol. Appl. Pharmacol. 26, 606–620, 1973. Tucker, M. J., Adam, H. K., and Patterson, J. S., Tamoxifen, in Safety Testing of New Drugs. Laboratory Predictions and Clinical Performance. Tucker, M. J. and Adams, H. K., Eds., Academic Press, London, 1984, 125–161. Tucker, M. J. and Orton, T. C., Comparative Toxicology of Hypolipidaemic Fibrates, Taylor & Francis Inc., Bristol, 1995. Turek, F. W. and Desjardins, C., Development of Leydig cell tumors and onset of changes in the reproductive and endocrine system of aging Fisher 344 rats. J. Natl. Cancer Inst. 63, 969–975, 1979.

Turnbull, D., Machado, R. J., and Boberg, R. E., Safety assessment of HCFC–141b: Use as a blowing agent for insulation in building construction and refrigeration. Regul. Toxicol. Pharmacol. 19, 282–296, 1994. Twombly, G. H., Meisel, D., and Stout, A. P., Leydig cell tumors induced experimentally in the rat. Cancer 2, 884–892, 1949. Ueno, N., Baird, A., Esch, F., Ling, N., and Guillemin, R., Isolation and partial characterization of basic fibroblast growth factor from bovine testis. Mol. Cell Endocrinol. 49, 189–194, 1987. Ursin, G., Bjelke, E., Heuch, I., and Vollset, S. E., Milk consumption and cancer incidence: a Norwegian prospective study. Br. J. Cancer 61, 454–459, 1990. USEPA, Environmental protection agency preliminary determination to conclude special review, continue registration of linuron, U. S. Environmental Protection Agency, Office of Pesticide and Toxic Substances, Washington, D.C., 1988. Vahouny, G.V., Chanderbahn, R., and Nolan, B. J., Sterol carrier protein 2. Identification of adrenal SCP and site of action for mitochondrial cholesterol utilization. J. Biol. Chem. 258, 11731–11737, 1983. Van Bebakar, N. M., Honour, J. W., Foster, D., Liu, Y. L., and Jacobs, H. S., Regulation of testicular function by insulin and transforming growth factor. Steroids 55, 266–272, 1990. Van der Molen, H. J., Bijleveld, M. J., Van der Vusse, G. J., and Cooke, B. A., Effect of gonadotropins on cholesterol and cholesterol ester as precursors of steroid production in the testis. J. Endocrinol. 57, 6–7, 1972. van Dissel-Emiliani, F. M., Grootenhuis, A. J., de Jong, F. H., and de Rooij, D. G., Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters. Endocrinology 125, 1899–1903, 1989. van Ravenzwaay, B., Discussion of prenatal and reproductive toxicity of Regul. No. 83–258 (Vinclozolin), BASF Co., 1992. Van Thiel, D. H., Gavaler, J. S., Cobb, C. F., Santucci, L., and Graham, T. O., Ethanol, a Leydig cell toxin: evidence obtained in vivo and in vitro. Pharmacol. Biochem. Behav. 18(Suppl.1), 317–323, 1983. Vanden Bossche, H., Biochemical targets for antifungal azole derivatives: hypothesis on the mode of action, in Current Topics in Medical Mycology. McGinnis, M. R., Ed., Springer-Verlag, New York, 1985, 313–351. Vanelli, B., Natali, A., Barni, T., Serio, M., Orlando, C., and Balboni, G., Insulin-like growth factor-I (IGF-1) and IGF-I receptor in human testis: an immunohistochemical study. Fertil. Steril. 49, 666–669, 1988. Verhoeven, G., Cailleau, J., van Damme, J., and Billiau, A., Interleukin–1 stimulates steroidogenesis in cultured rat Leydig cells. Mol. Cell Endocrinol. 57, 51–60, 1988. Vermeulen, A., Rubens, R., and Verdonck, L., Testosterone secretion and metabolism in male senescence. J. Clin. Endocrinol. Metab. 34, 730–735, 1972. Vermeulen, A., Effects of drugs on Leydig-cell function. Int. J. Androl. 5, 163–182, 1982.

259


Viguier-Martinez, M. C., Hochereau de Reviers, M.T., Barenton, B., and Perreau, C., Endocrinological and histological changes induced by flutamide treatment on the hypothalamo-hypophyseal testicular axis of the adult male rat and their incidences on fertility. Acta Endocrinologica 104, 246–252, 1983a. Viguier-Martinez, M. C., Hochereau de Reviers, M.T., Barenton, B., and Perreau, C., Effect of a non-steroidal antiandrogen, flutamide, on the hypothalamo-pituitary axis, genital tract and testis in growing rats: Endocrinological and histological data. Acta Endocrinologica 102, 299–306, 1983b. Vihko, K. K., Lapolt, P. S., Mishomor, K., and Hseuh, A. J. W., Stimulatory effects of recombinant FSH on Leydig cell function and spermatogenesis in immature hypophysectomized rats. Endocrinology 129, 1926– 1932, 1991. von Euler, M., Soder, O., Pollanen, P., Arver, S., and Ritzen, E. M., Human chorionic gonadotropin stimulates production and secretion of rat testicular interleukin–1 like activity, in Proceedings of the IVth International Congress of Andrology. Monduzzi, Ed., Milan, 1989, 289–293. Waalkes, M. P, Rehm, S., and Devor, D. E., The effects of continuous testosterone exposure on spontaneous and cadmium-induced tumors in the male Fisher (F344/ NCr) rat: Loss of testicular response. Toxicol. Appl. Pharmacol. 142, 40–46, 1997. Waalkes, M. P., Rehm, S., Riggs, C. W., Bare, R. M., Devor, D. E., Poirier, L. A., Wenk, M. L., Henneman, J. R., and Balaschak, M. S., Cadmium carcinogenesis in the male Wistar[Crl:(WI)BR] rats: dose-response analysis of tumor induction in the prostate and testes and at the injection site. Cancer Res. 48, 4656–4663, 1988. Waalkes, M. P., Rehm, S., Riggs, C. W., Bare, R. M., Devor, D. E., Poirier, L. A., Wenk, M. L., and Henneman, J. R., Cadmium carcinogenesis in male Wistar [Crl:(WI)BR] rats: dose-response analysis of effects of zinc on tumor induction in the prostate, in the testes, and at the injection site. Cancer Res. 49, 4282– 4288, 1989. Waalkes, M. P. and Rehm, S., Carcinogenicity of oral cadmium in the male Wistar (WF/NCr) rat: Effect of chronic dietary zinc deficiency. Fundam. Appl. Toxicol. 19, 512–520, 1992. Waalkes, M. P., Coogan, T. P., and Barter, R. A., Toxicological principles of metal carcinogenesis with special emphasis on cadmium. Crit. Rev. Toxicol. 22, 175–201, 1992. Wahlstrom, T., Huhtaniemi, I., Hovatta, O., and Seppala, M., Localization of luteinizing hormone, follicle-stimulating hormone, prolactin, and their receptors in human and rat testes using immunochemistry and radioreceptor assays. J. Clin. Endocrinol. Metab. 57, 826–830, 1983. Walsh, P. C., The endocrinology of testicular tumors. Recent results. Cancer Res. 60, 196–201, 1977. Walsh, K. M. and Poteracki, J., Spontaneous neoplasms in control Wistar rats. Fundam. Appl. Toxicol. 22, 65–72, 1994.

260

Wang, C., Rebar, R., Hopper, B., and Yen, S., Functional studies of the luteinizing hormone-Leydig cell-androgen axis. Exaggerated response in C–18 and C–21 testicular steroids to various modes of luteinizing hormone stimulation. J. Clin. Endocrinol. Metab. 51, 201–208, 1980. Wang, H. G., Sundaram, K., Pavou, S., Rivier, J., Vale, W., and Bardin, C. W., Mice are insensitive to the anti-testicular effects of luteinizing hormone-releasing hormone agonists. Endocrinology 112, 331–335, 1983. Wang, H. and Ascoli, M., Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA–10 cells with a mutant gene of the type I regulatory subunit of the cAMPdependent protein kinase. Mol. Endocrinol. 4, 80–90, 1990. Wang, D., L. M. L., N., Calkins, H., Chang, W., Sigel, M. M., and Lin, T., Interleukin–1β induces interleukin– 1a messenger ribonucleic acid expression in primary cultures of Leydig cells. Endocrinology 129, 2862– 2866, 1991. Warren, D. W., Dufau, M. L., and Catt, K. J., Hormonal regulation of gonadotropin receptors and steroidogenesis in cultured fetal rat testes. Science. 218, 375–377, 1982. Warren, D. W., Huhtaniemi, I.T., Tapanainen, J., Dufau, M. L., and Catt, K. J., Ontogeny of gonadotropin receptors in the fetal and neonatal rat testis. Endocrinology 114, 470–476, 1984. Warren, D. W., Wisner, J. R., Jr., and Ahmad, N., Effects of 1,2-dibromo–3-chloropropane on male reproductive function in the rat. Biol. Reprod. 31, 454–463, 1984. Weir, R. J. and Fisher, R. S., Toxicologic studies on borax and boric acid. Toxicol. Appl. Pharmacol. 23, 351–364, 1972. Weiss, D., Rodrigues-Rigau, L., Smith, K., Grotjan, E. J., and Steinberger, E., Leydig cell density and function and their relation to gonadotropins in infertile oligospermic men with varicocele. Isr. J. Med. Sci. 15, 556–563, 1979. Weisz, J. and Ward, I. L., Plasma testosterone and progesterone titres of pregnant rats, their male and female fetuses and neonatal offspring. Endocrinology 106, 306– 316, 1980. Welsch, C. W., Host factors affecting the growth of carcinogen-induced rat mammary carcinomas: a review and tribute to Charles Brenton Huggins. Cancer Res. 45, 3415–3443, 1985. Weuesten, J. J. A. M., Smals, A. G. H., Hofman, J. A., Kloppenborg, P. W. C., and Benraad, T. J., Early time sequence in pregnenolone metabolism to testosterone in homogenates of human and rat testis. Endocrinology 120, 1919–1923, 1987. Whorton, M. D. and Foliart, D. E., Mutagenicity, carcinogenicity and reproductive effects of dibromochloropropane (DBCP). Mutation Res. 123, 13–30, 1983. Widenius, T. V., Peter-Eriksson, C. J., Ylikahri, R. H., and Harkonen, M., Inhibition of testosterone synthesis by ethanol: Role of luteinizing hormone. Alcohol 6, 241–244, 1989.


Wilmer, J., Bloemen, L., Farrar, D., Garny, V., Green, T., Millischer, R. J., and Vrijhor, H., Trichloroethylene: Assessment of Human Carcinogenic Hazard, Vol. Technical Report No. 60, ECETOC, Brussels, 1994. Wing, T. Y., Ewing, L. L., and Zirkin, B. R., Effects of luteinizing hormone withdrawal on Leydig cell smooth endoplasmic reticulum and steroidogenic reactions which convert pregnenolone to testosterone. Endocrinology 115, 2290–2296, 1984. Winters, S. J., Banks, J. L., and Loriaux, D. L., Cimetidine is an anti-androgen in the rat. Gastroenterology 76, 504–512, 1979. Wong, C., Kelce, W. R., Sar, M., and Wilson, E. M., Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Biol. Chem. 270, 19998–20003, 1995. Woutersen, R. A., Chronic toxicity and carcinogenicity of lactitol in rats: comparison with lactose, in Low Digestibility Carbohydrates. Leegwater, D. C., Feron, V. J., and Hermus, R. J. J., Eds., Wageningen, Pudoc, 1987, 51–60. Wu, N. and Murono, E., A Sertoli cell-secreted paracrine factor(s) stimulates proliferation and inhibits steroidogenesis of rat Leydig cells. Mol. Cell. Endocrinol. 106, 99–109, 1994. Yamada, T., Maita, K., Nakamura, J., Murakami, M., Okuno, Y., Hosokawa, S., Matsuo, M., and Yamada, H., Carcinogenicity studies of oxolinic acid in rats and mice. Food Chem. Toxic. 32, 397–408, 1994a. Yamada, T., Nakamura, J., Murakama, M., Okuno, Y., Hosokawa, S., Matsuo, M., and Yamada, H., The correlation of serum luteinizing hormone levels with the induction of Leydig cell tumors in rats by oxolinic acid. Toxicolol. Appl. Pharmacol. 129, 146–154, 1994b. Yamada, T., Nakamura, J., Murakami, M., Okuno, Y., Hosokawa, S., Matsuo, M., and Yamada, H., Effect of chronic L-DOPA administration on serum luteinizing hormone levels in male rats. Toxicology 97, 173–182, 1995a. Yamada, T., Nakamura, J., Okuno, Y., Hosokawa, S., Matsuo, M., Yamada, H., and Ohta, M., A possible

mechanism for the increase of serum luteinizing hormone levels in rats by oxolinic acid. Toxicol. Appl. Pharmacol. 134, 35–42, 1995b. Yasuda, Y., Konishi, H., and Tanimura, T., Leydig cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol. Teratology 33, 281–288, 1986. Yasuda, Y., Ohara, I., Konishi, H., and Tanimura, T., Long-term effects on male reproductive organs of prenatal exposure to ethinyl estradiol. Am. J. Obstet. Gynecol. 159, 1246–1250, 1988. Yegana, Y., Zimber, A., and Perk, K., The effect of high doses of methyl-CCNU on male and female fertility in SJL/J mice. Anticancer Res. 8, 397–402, 1988. Ying, S. Y., Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocrinology 9, 267–293, 1988. Zawirska, B. and Medras, K., Tumors and disturbances of the porphyrin metabolism in rats with chronic experimental lead intoxication I. Morphological studies. Zbl. Allg. Path. 111, 1–12, 1968. Zipf, W. B., Payne, A. H., and Kelch, R. G., Prolactin, growth hormone and LH in the maintenance of testicular LH receptors. Endocrinology 103, 595–600, 1978. Zirkin, B. R., Ewing, L. L., Kromann, N., and Cochran, R. C., Testosterone secretion by rat, rabbit, guinea pig, dog, and hamster testes perfused in vitro: correlation with Leydig cell ultrastructure. Endocrinology 107, 1867–1874, 1980. Zirkin, B. R., Dykman, D. D., Kromann, N., Cochran, R. C., and Ewing, L. L., Inhibition and recovery of testosterone secretion in rats are tightly coupled to quantitative changes in Leydig cell smooth endoplasmic reticulum. Ann. New York Acad. Sci. 383, 17–27, 1982. Zirkin, B. R., Santulli, R., Strandbert, J. D., Wright, W. W., and Ewing, L. L., Testicular Steroidogenesis in the Aging Brown Norway rat. J. Andrology 14, 118– 123, 1993. Zirkin, B. R., Santulli, R., Strandberg, J. D., Wright, W. W., and Ewing, L. L., Testicular steroidogenesis in the aging Brown Norway rat. J. Androl. 14, 118–123, 1993.

261