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Systemic and Histopathologic Changes in Beagle Dogs After Chronic Daily Oral Administration of Synthetic (Ethinyl Estradiol) or Natural (Estradiol) Estrogens, with Special Reference to the Kidney and Thyroid Ibrahim Zayed, Eric Van Esch and Robert F. Mcconnell Toxicol Pathol 1998 26: 730 DOI: 10.1177/019262339802600603 The online version of this article can be found at: http://tpx.sagepub.com/content/26/6/730

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Systemic and Histopathologic Changes in Beagle Dogs After Chronic Daily Oral Administration of Synthetic (Ethinyl Estradiol) or Natural (Estradiol) Estrogens, with Special Reference to the Kidney and Thyroid* IBRAHIM

1,4 ZAYED,

Department of Toxicology 2

ERIC

VAN

2AND ROBERT ESCH,

F. MCCONNELL 3

1 N . V. Organon, Oss, The Netherlands, and Drug Disposition, N. V. Organon, Oss, The Netherlands, and Flemington, New Jersey 08822, USA 3

Four groups of 3 male and 3 female sexually mature Beagle dogs were treated daily po with either ethinyl estradiol (EE) or estradiol A fifth group of 4 males and 4 females acted as a control group. Three groups of dogs were treated with EE: One group was treated at dose levels of 2.0, 1.5, and 1.0 mg/kg for 6 mo; the other 2 groups received either 0.5 mg/kg or 1.0 mg/kg for 1 yr. The fourth group was treated with 5.0 mg/kg E 2 for 1 yr. Results obtained for the clinical, hematological, and biochemical parameters and the histopathologic findings of most organs and tissues in EE- and E -treated dogs were essentially comparable to those documented 2 in the literature for dogs treated with synthetic or natural estrogens. Chronic treatment with EE or E 2 induced similar effects, with the exception of mesothelial proliferation of the genital serosa, which was observed in EE-treated dogs only. Additional new estrogenrelated findings were observed in the kidneys and thyroid glands of EE- and E 2 treated dogs. Increased interstitial fibrous tissue occurred at the corticomedullary junction and in the outer cortex of the kidney. It appeared to originate primarily from the perivascular fibrous tissue of branches of the renal arteries and veins. Extension of this lesion into the renal parenchyma resulted in secondary atrophic changes of tubules and glomeruli. The treatment relationship and specific characteristics of this renal alteration differentiated it from other chronic renal interstitial and vascular diseases. Squamous metaplasia of urogenital tract epithelia, including renal cortical tubule epithelium, occurred as expected in both EE- and E 2 treated dogs. Unexpectedly, squamous metaplasia of thyroid follicular 2 treated dogs. The renal and thyroid changes did not epithelium also occurred. It was present in scattered follicles of both EE- and -E alter clinicopathological function tests for either of these organs. These 2 new findings extend the list of estrogen-related effects in the dog.

). 2 (E

Keywords. dog

Estrogens; estrogen-induced

renal

disease; renal interstitium; squamous metaplasia; kidney; thyroid gland; histopathol-

ogy;

INTRODUCTION

used in humans as therapeutic agents or as components of oral contraceptives. The synthetic estrogens ethinyl estradiol (EE) and mestranol have been studied in rats, mice, dogs, and monkeys as part of regulatory preclinical safety requirements. Other estrogenic compounds, such as diethylstilbestrol, have been studied extensively. Very few of the preclinical safety studies were published because of the proprietary nature of the contraceptive compounds (9, 31, 32). Studies utilizing Beagle, mongrel, or other dog strains have been used to study the toxicologic effects of injectable and orally administered synthetic or natural estrogens. Several nonregulatory investigative studies have been published that described systemic and histopathologic effects of these estrogens (1, 3, 24, 26); other studies focused on specific parameters or histopathologic alterations in one or more organs or organ systems (2, 7, 10,

Estrogens

*

Address

are

widely

correspondence and reprint requests to: Dr. E. van Esch, of Toxicology and Drug Disposition, N. V. Organon, PO. Box 20, 5340 BH, Oss, The Netherlands. 4 Present address: Twaalf Apostelenweg 25, 6523 LV Nijmegen, The Netherlands. Department

11, 13-15, 18, 21, 22, 28, 29). Among those studies describing estrogens administered via the oral route, high-dose levels were administered for only short periods because of toxicity-related problems, while longterm (7-yr) studies were performed using relatively lowdose levels. The reason for this dose/time limitation is related to the extreme sensitivity of dogs to estrogens and to the development of estrogen-dependent thrombocytopenia. Despite these limiting factors, an extensive list of estrogen-related systemic and pathological changes in dogs receiving natural or synthetic estrogens for varying time periods has been established during the past decades. A direct comparison of EE with estradiol (E2) had not been previously reported. It was the purpose of this study to determine and compare the long-term (1-yr) effects of high-doses of EE to E~. Results obtained for the clinical, hematological, and biochemical parameters and the histopathologic findings in most organs and tissues were essentially comparable to those documented in the literature for dogs treated with synthetic or natural estrogens. The estrogen-induced renal and thyroid changes found in dogs in this study have not been previously reported, and these 730


731 TABLE Treatment groups.

groups)

on

the first

day

of treatment and then

weekly

thereafter. T&dquo;’B......~......

findings extend in this species.

the list of

MATERIALS

estrogen-related AND

effects found

METHODS

The test substances we used included 1-mg tablets of EE and 1-mg tablets of E,, both of which were administered orally (in the form of gelatin capsules). Test substances were supplied by N. V. Organon, Oss, The Netherlands. The dose we administered was determined weekly for each animal according to changes in body weight and was adjusted to the nearest milligram of the test substance.

Sexually mature Beagle dogs (Marshall Farms, North Rose, New York, USA), ranging in age from 10 to 14 mo and having body weights of 12.2 ± 0.4 kg (males) and 10.0 ± 0.6 kg (females), were used in this study. The animals were housed individually under standard laboratory conditions, had free access to tap water, and received 300 g of commercially available pelleted diet daily (U.A.R., 91360 Villemoisson-sur-Orge, France). Twenty-four animals were allocated by sex into 4 treatment groups that consisted of 3 males and 3 females each (Table I). Group 1, which served as a maximum-tolerated-dose finding group, received oral administration of daily doses of 2.0 mg/kg of EE from days 1 through 30, of 1.5 mg/kg of EE from days 31 to 49, and of 1.0 mg/ kg of EE from days 50 to 188 (6 mo)(hereafter this group of animals will be referred to as the 2.0/1.5/1.0-mg/kg group). The 2 remaining EE groups were treated with daily doses of 0.5 or 1.0 mg/kg of EE; group 4 (E2) dogs were treated with daily doses of 5.0 mg/kg of E2 for the 1-yr duration of the study. Results obtained were compared with those of the control group (group 5), which consisted of 4 males and 4 females, and with historical laboratory data. The animals were observed daily for morbidity or mortality and for any abnormal physical or clinical signs. A veterinary examination was performed on each animal once before the beginning of the treatment period and monthly thereafter. During these examinations, the prostate gland was monitored by rectal palpation. Food consumption was determined daily for 1 wk before the treatment period as well as throughout the study. Body weight was recorded for each animal once (before animals were separated into

Ophthalmologic examinations were performed on all animals before the beginning of the treatment period and then again during the week of terminal sacrifice for the survivors in all groups. These examinations included evaluation of visual reflexes (pupillary light and blink reflexes) and examination of the appendages, eyelids, conjunctiva, lens, cornea, anterior chamber, vitreous humor, and fundus (retina) by indirect ophthalmoscopy. Systolic and diastolic blood pressures were recorded using an oscillometric electrosphygmomanometer (Dynamap, Critikon, Trappes, France) and a pneumatic cuff placed around the foreleg (humeral artery). These measurements were accomplished once before the beginning of the treatment period and again during weeks 6 and 13. Clinicopathological evaluations were performed on all animals before the treatment period and again on all survivors in groups 2, 3, and 4 on weeks 4, 12, 20, 27, 36, 44, and 52 (evaluations were performed in group I animals only up to week 27). These evaluations were conducted on group 5 dogs prior to the initiation of the study and then again during weeks 25 and 51. These investigations included evaluation of the following: hematology (measures of erythrocyte count and morphology, hemoglobin, mean cell volume, packed cell volmean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, thrombocyte count, total leukocyte count, and differential cell count [with cell morphology, including neutrophils, eosinophils, basophils, lymphocytes, monocytes, and reticulocytes]), blood clotting (measures of prothrombin time, activated partial thromboplastin time, and fibrinogen), blood biochemistry (measures of sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, cholesterol, triglycerides, alkaline phospha-

ume,

tase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, ~y-glutamyl transferase, total proteins, and protein electrophoresis (including albumin, cox-1 globulins, a-2 globulins, (3-globulins, -y-globulins, albumin/globulin ratio]), urinalysis (measures of urine

volume, pH, specific gravity, sodium, potassium, creatinine, creatinine clearance, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, and cytology (measures of leukocytes, erythrocytes, other cells, casts,

magnesium ammonium phosphate crystals, calcium phosphate crystals, and calcium oxalate crystals). After completion of the treatment period (27 wk for group I and 1 yr for the remaining groups), all survivors were sacrificed. A thorough macroscopic examination was performed on all animals, including any that were sacrificed or that died during the study. Body weights were recorded prior to necropsy. The wet weights of adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, pituitary gland, prostate, spleen, testes, thymus, thyroid (with parathyroids), and uterus were recorded. These organs, together with the aorta, cecum, colon, duodenum, epididymides, eyes (with optic nerves), femoral bone (with articulation), gallbladder, ileum, jejunum,


732 TABLE II.-Factors

Abbreviations: EE

=

ethinyl estradiol; E=

=

contributing

thoracic, lumbar), sternum (with bone marrow), stomach, tongue, trachea, ureters (in the few animals that displayed gross abnormalities), urinary bladder, and vagina (includcervix and all macroscopic lesions), were preserved in 10% neutral-buffered formalin, except for the eyes and the pituitary glands, which were fixed in formol sublimate. All previously mentioned organs and tissues were processed, routinely embedded in paraffin, sectioned at 4 ~,m, and stained with hematoxylin and eosin. Paramedian whole organ sections of kidneys from all animals were prepared and stained with hematoxylin and eosin, van Gieson’s stain, and toluidine blue.

ing

RESULTS

Mortality and Morbidity Mortality and morbidity occurred among all groups treated with either EE or E,, regardless of the dose level given (Table II). The major contributory factors included hemorrhage following severe thrombocytopenia (sometimes accompanied by anemia), endometritis in a few females, and obstructive prostatic hypertrophy, leading to uremia in males.

Signs

Clinical signs of hemorrhage varied from the presence of blood on the prepuce or vulva to petechiae and ecchymoses on various mucous membranes and to paleness of the gingival mucosa, bloodstained urine, brownish vomit, and melena. These signs occurred in all treated groups, but the incidence and severity were lessened in the 0.5-mg/kg EE group. Other clinical signs, including alopecia, gray-gray brown-blackish spots on abdominal and thigh skin, swelling of the prepuce and vulva, and ptosis of the penis, were observed with similar incidence in all treatment groups.

Veterinary

premature sacrifice

or

death.

estradiol.

lymph nodes (mandibular and mesenteric), mammary glands, esophagus, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord (cervical,

Clinical

to

Examination

A time-related decrease in the size of the testis, progressive irregular enlargement of the prostate, nipple en-

in all males and females, slight gynecomastia in males, and enlargement of mammary glands in females of all treatment groups were noted during the study.

largement

Body Weight

and Food

Consumption

No effects on food consumption and body weight were noted in any survivors of EE- and E,-treated groups. A decrease in both parameters occurred only in moribund animals and was attributed to poor clinical condition preceding death or premature sacrifice.

Ophthalmologic Examination and Blood Pressure Ophthalmologic examinations and blood pressure recordings did not reveal any treatment-related changes. Hematology Red Blood Cell, Hemoglobin, and Packed Cell Volume Measures. No effect on red blood cell parameters was seen in males and females at 0.5 mg/kg EE, in males at 1.0 mg/kg EE, or in females treated with 5.0 mg/kg E,. Males and females in the 2.0/1.5/1.0-mg/kg EE group had slightly to moderately decreased values, except in the case of the 1 male that displayed normal values until the end of 6 mo. One female from this group, sacrificed in a moribund state early in the dosing period, developed anemia. Anemia also occurred in 2 females from the 1.0mg/kg EE group and in 2 males from the 5.0-mg/kg E2 group. These 4 dogs were sacrificed while moribund. White Blood Cell Count and Neutrophils. The changes in white blood cell count reflected the changes in absolute neutrophil count. Initially, at week 4, both values were markedly increased up to more than 4 times the normal level in all animals. Values returned to normal thereafter in nearly all animals of the 0.5-mg/kg EE group and in some animals from the other groups. However, a second increase of these values occurred later among a few animals in all treated groups. Except for animals treated with 0.5 mg/kg EE, marked leukocytopenia/neutropenia was observed among all animals that were sacrificed while moribund. Dogs affected included all females from the 1.0-mg/kg EE group, 1 female from the 2.0/1.5/1.0mg/kg EE group, and 1 male from the 5.0-mg/kg E2 group.


733

Eosinophils.

Absolute

eosinophil

counts

gradually

de-

and it occurred mainly among dogs that a moribund state or that died prematurely, despite treatment or dose level. In a male and a female of the 2.0/1.5/1.0-mg/kg EE-treated group, the changes resulted in aplastic bone marrow. In contrast, myeloid hyperplasia was observed among other EE- and E,-treated animals. This resulted in hypercellular bone marrow in a number of these animals. Moreover, in some cases a high number of mature elements dominated the picture (&dquo;right shift&dquo;). Mitotic figures among the myeloid elements were numerous. Megakaryocytes were markedly decreased or had disappeared completely from the bone marrow in one-half of the EE- and E,-treated animals. In a number of the remaining animals, active megakaryopoiesis, microscopically characterized by an increased number of megakaryoblasts, was present. Interestingly, this was always

row

creased from week 4 onward in both EE- and E,_-treated males and females. Other Blood Cell Types. Absolute lymphocyte, monocyte, and basophil counts remained normal during the

were

study. Thrombocytes. All animals treated with EE and the of females treated with E2 had a moderate (females, 0.5 mg/kg EE) to marked decrease in platelet

majority

count from week 4 onward. The values displayed a broad range of fluctuation. Females treated with 0.5 mg/kg EE regained normal levels after week 36. All males and 1 female treated with E2 had normal or increased platelet

counts, initially followed by moderate thrombocytopenia from week 20 onward. The exceptions included 1 male and 1 female from this group that had increased platelet counts at week 27. The increased value in the male was obtained prior to its moribund sacrifice. The platelet count in the female remained normal until the end of the 52-wk dosing period.

cellularity,

sacrificed in

found in relation to

an

increased number of

myeloid

el-

ements.

Male Genital

Coagulation Parameters. Fibrin, prothrombin time, and activated partial thromboplastin time were not affected by treatment.

Organs

were reduced in size and weight in all treated males. The epithelium of the seminiferous tubules was atrophied, with only spermatogonia and Sertoli cells remaining. Leydig cells were also atrophied. In some animals, the testicular tunics were prominently thickened, and there were thickened fibrous septa. Epididymides. The epididymal ducts were lined with atrophied epithelium and were devoid of sperm in all treated males. There was a marked increase of the fibromuscular stroma, resulting in compression and atrophy of ducts. The adventitial layer of the epididymal arteries was thickened in 1 animal that had been treated with 1.0 mg/ kg EE. Focal mesothelial papillary hyperplasia occurred in 1 animal from the 2.0/1.5/1.0-mg/kg EE group. The lower epididymal weights obtained in most males of all treated groups were associated with aspermia and atrophy of the ductal epithelium. Prostate Gland. The prostate gland was markedly enlarged in all EE- and E2-treated dogs. The prostate weights ranged from 3 to 40 times the control values. This enlargement was caused primarily by extensive squamous metaplasia, often resulting in keratin-filled glandular cysts and increased fibromuscular stroma. These changes occurred in conjunction with urethral squamous metaplasia.

Testes. The testes

Clinical

Chemistry Urea-Nitrogen/Creatinine. Markedly increased

values for urea-nitrogen and creatinine were observed in 1 male from the 0.5-mg/kg EE group, 1 male from the 1.0-mg/ kg EE group, and 2 males from the 5.0-mg/kg E2 group. No significant changes were noted in any of the other measured clinical chemistry parameters. Urine Analysis. No treatment-related effects were noted in the urine parameters examined. Organ Weights. Values obtained for the weights of the brain, heart, liver, lungs, pituitary gland, thyroid, and parathyroid glands were comparable to those of the placebo group. For the remaining organs, weight changes are reported in correlation to morphological findings.

Pathology Lymphoid Organs: Thymus. Moderate to marked cortical thymic atrophy, occasionally with medullary epithelial cyst formation, was a consistent finding among all treated animals. In severe cases, the medullary thymocyte population was also diminished. The atrophic changes were reflected in the weights of the thymuses, which were decreased compared with those of control animals. Lymph Nodes. Although the changes in the lymph nodes were not that prominent, sinusoidal erythrophagocytosis and a decreased cellularity of the germinal centers were observed in a number of EE- and E~-treated dogs; male dogs were more often affected than females. Hematopoietic System Bone Marrow. The microscopic changes in the bone of both EE- and E2-treated animals varied dependent upon the time and condition of sacrifice or death. In most treated animals, erythroid elements were moderately to markedly decreased, while myeloid elements were either increased or decreased. The latter phenomenon was associated with an overall decrease in bone mar-

marrow

.

.

.

Female Genital Organs Ovaries. There was suppression of ovarian follicular development and an absence of corpora lutea in all treated groups. Moderate or marked hyperplasia of the germinal epithelium, referred to as mesothelial hyperplasia (Fig. 1), resulting in a granular or cauliflowerlike gross appearance, was noted in all females treated with EE but not in those treated with E,. Accordingly, E~-treated females had decreased ovarian weights compared with controls. In EE-treated females, the ovarian weights reflected the extent of mesothelial proliferation and varied from normal in the 0.5-mg/kg group to up to 10 times control values in high-dose groups. The progressive mesothelial proliferative growth seen


734

FIG. FIG. FIG.

1.-Ovary: marked serosal mesothelial proliferation. H&E. Original magnification (OM) X 12.5. OM X 25. 2.-Ovary: the papillary pattern of the mesothelial proliferation. H&E. 3.-Ovary: the pseudoglandular pattern of the mesothelial proliferation. Mitotic figures are sporadically

observed (arrowhead). H&E.

OM

X50. FIG. 4.-Uterus: uterine serosal mesothelial

OM X25. papillary proliferation. H&E. FIG. 5.-Liver: transplanted genital serosal mesothelial proliferative lesion on the Glisson’s capsule. H&E. OM X 50. FIG. 6.-Uterus: squamous metaplasia of the superficial and glandular epithelium resulting in a keratin-filled cyst. H&E.

among EE-treated females ranged from simple papillary structures with a supportive fibrovascular stroma (Fig. 2) to more densely populated solid complex growth with pseudoglandular structures (Fig. 3). Microcysts with intraluminal papillary projections occurred in some females. The cells within the more solid areas were slightly larger because they had more cytoplasm than those lining

OM X 12.5.

the

papillae. Microvilli were found to be present on a varying number of cells lining the papillae. Ciliated cells were not present. Mesothelial cell vacuolation and sporadic mitotic figures along with adventitial proliferation of hylar arteries were occasionally observed. Uterus. Slight cystic endometrial hyperplasia was present in 5 EE- and 2 E2-treated females. This was evidenced


735 )y a slight increase in the number of endometrial glands, ,vhich were dilated mainly at the basal part of the eniometrium. The epithelium lining these cystic glands ranged in shape from cubical to flattened, and some jlands were filled with inspissated secretory material. A variable degree of stromal hyalinization occurred among both EE- and E2-treated females. Five EE-treated females had endometritis, which led to loss of the endometrium in 2 of the animals, both of which were treated with 1.0 mg/kg EE. The luminal side of the uterine horns in these dogs consisted of inflammatory cells and markedly hypertrophied myometrial smooth muscle cells. A number of multinucleated giant cells were detected within these areas.

of the glandular as well as of the endometrial epithelium was present only in superficial females treated with 0.5 mg/kg EE or with 5.0 mg/kg E2. In the body of the uterus of 1 E2-treated female, large areas of the luminal endometrial lining were replaced by a keratinizing squamous epithelium, while some of the glands were transformed into keratin cysts (Fig. 6). All treated females had moderate to marked myometrial hypertrophy, accompanied in most animals by an increase in perivascular fibrous tissue. The myometrial hypertrophy was responsible for the slightly to markedly increased uterine weights in treated females. Mesothelial papillary hyperplasia (Fig. 4) occurred only in EE-treated females. The most severe mesothelial hyperplasia occurred at the parametrium along the length of the uterine horns, although it was less marked than that of the ovarian mesothelium. Vagina and Cervical Canal. The epithelium was markedly thickened and was usually heavily comified in almost all treated females. The hyperkeratotic squamous epithelium was comparable to, but more pronounced than, that seen in the estrus phase of the cycle. In 2 females from the 2.0/1.5/1.0-mg/kg EE group, the maturation of the squamous epithelium was disturbed. The superficial layers of the epithelium contained nucleated, nonkeratinized cells with large cytoplasmic vacuoles and occasional eosinophilic globules. Marked fibromuscular hypertrophy of the vaginal and cervical walls was seen in all treated females. Slight reactive serosal mesothelium, consisting of hypertrophied mesothelial cells, was present in EE-treated animals. Low-grade mesothelial micropapillary hyperplasia was present in 2 females from the 0.5- and the 2.0/1.5/1.0-mg/kg EE groups, respectively. Mesothelial changes were absent in E2-treated females.

Squamous metaplasia

Urinary System Kidney. Grossly, the kidneys were enlarged in some animals treated with EE or E,. The absolute and relative kidney weights were increased up to 2.5 times mean control values in several animals treated with EE and in animals treated with 5.0 mg/kg E,. These weight increases, especially those in the higher range, correlated with the morphologic changes observed. These were characterized microscopically by an increased amount of perivascular fibrous tissue surrounding interlobar vessels running within the pelvic recesses (Figs. 7-9) and around the ar-

and interlobular vessels. A well-demarcated inin fibrous tissue surrounding the arcuate and the ]lower branches of the interlobular vessels occurred at the < ~orticomedullary junction in some dogs (Fig. 10) and of1ten extended to vessels in the cortical parenchyma. Such < changes. ranged from well-demarcated foci to large areas, with more diffuse interstitial fibrous tissue replacing the inormal parenchymal structures (Figs. 11, 12, and 19). ,Some of these areas were located directly adjacent to the .renal capsule and were recognized grossly as grayishwhitish areas. The renal capsule was thickened because of the presence of increased fibrovascular tissue in a number of cases (Fig. 12). Affected kidneys did not appear to have any surface irregularities, even in those cases where areas of fibrous tissue extended to the outer cortical parenchyma. Most of these fibrous tissue areas contained multiple cysts of varying sizes. At higher magnification, it could be established that the cysts originated from cortical tubules and glomeruli, which had been entrapped by the expanding fibrous tissue. Occasionally, these cysts contained fine granular proteinaceous material. In a number of dogs, a third type of cyst was found in the peripelvic region beneath the urothelium. These cysts developed in some of the multiple urothelial pouches formed by downgrowths of hyperplastic urothelium (Figs. 8 and 9). Squamous metaplasia of entrapped urothelial and tubular structures was occasionally observed, which in some cases resulted in formation of keratin-filled cysts. Foci of tubular epithelial squamous metaplasia were sometimes found within fibrous areas in the outer cortex or at the corticomedullary junction (Fig. 13). Other tubular and glomerular structures entrapped within the expanding fibrous tissue finally collapsed and atrophied. The progressively expanding interstitial fibrous tissue contained active, young fibroblasts surrounded by thick collagen bundles (Fig. 14). The fibroblasts had large oval to round nuclei, which often contained nucleoli. Expansion of the fibrous lesion was evidenced by an obvious pericapillary increase of acellular, deep eosinophilic collagen between the otherwise morphologically normal tubules and glomeruli (Fig. 15). Tubular and glomerular basement membranes were normal; however, an increase in periglomerular fibrous tissue was observed as an early renal change. Apart from the perivascular changes, the arteries and veins both within and outside the affected areas were histologically normal. Dilated distal cortical tubules with flattened epithelium were present in only 2 males from the 1.0-mg/kg EE group. Nonkeratinizing squamous metaplasia of the hyperplastic urothelium lining the pelvis was occasionally seen. In a few dogs, the urothelial basement membrane was markedly thickened. Urinary Bladder. The urinary bladder was thickened in approximately one-half of the animals in all treatment groups. Microscopically, this gross change correlated with hypertrophy of the smooth muscle wall. Muscle fibers were plump with enlarged oval nuclei. Urothelial hyperplasia was a consistent finding in nearly all male and fe< curate

< crease

,

.

,


736

FIG.

(OM)

7.-Kidney:

interlobar blood vessels in

a

pelvic recess in a placebo

animal

(for comparison with Fig. 8). Toluidine blue. Original magnification

X 3.2.

FIG. 8.-Kidney: increased fibrous tissue surrounding interlobar vessels within a pelvic recess and around the arcuate vessels at the corticomedullary junction (arrowhead). Toluidine blue. OM X3.2. FIG. 9.-Kidney: increased perivascular fibrous tissue and urothelial hyperplasia (arrowhead) resulting in cystic epithelial structures. Toluidine blue. OM X 12.5. OM X 12.5. FIG. 10.-Kidney: well-demarcated area of increased fibrous tissue at the corticomedullary junction with cystic epithelial structures. H&E. FIG. 11.-Kidney: diffuse interstitial fibrous tissue replacing normal cortical parenchymal structures. H&E. OM X 50. FIG. 12.-Kidney: a well-demarcated cortical area of increased fibrous tissue replacing normal parenchymal structures. (Note the absence of contraction of the lesion.) The renal capsule is thickened. H&E. OM X 12.5.

male treated

dogs. The proliferative epithelium was slightly to moderately higher in most animals. Minimal focal papillary hyperplasia occurred in only 1 female from the 0.5-mg/kg EE group. Severe erosion of the urothelium accompanied by cystitis was present among males from the

and 5.0-mg/kg E, groups. The submucosa was edematous and congested in a few animals and in one 1.0-mg/kg EE female, submucosal collagen and ground substance were increased. The serosal mesothelial surface was unaffected.

0.5-mg/kg EE, 1.0-mg/kg EE,


737

FIG. 13.-Kidney: tubular epithelial squamous metaplasia. H&E. Original magnification (OM) X25. FIG. 14.-Kidney: high-power magnification of active young fibroblasts within areas of fibroplasia. H&E. OM X 100. FIG. 15.-Kidney: pericapillary increase of acellular collagen between normal parenchymal structures expressing expansion of the fibrous lesion. H&E. OM X50. FIG. 16.-Adrenal gland: absence of normal arcade architecture of the zona glomerulosa. H&E. OM X 50. FIG. 17.-Thyroid gland: foci of nonkeratinizing follicular squamous metaplasia. H&E. OM X 25. OM FIG. 18.-Thyroid gland: high magnification of follicular squamous metaplasia with abrupt transition to normal follicular epithelium. H&E. X50. -

offo~,

EF

Ureter. Among animals from the 0.5-mg/kg EE, 1.0mg/kg EE, and 5.0-mg/kg E, groups, the ureters were thickened grossly. Smooth muscle hypertrophy of the wall and urothelial hyperplasia were evident and were sometimes accompanied by submucosal urothelial nests and foci of squamous metaplasia.

Integumentary System Skin. Areas of skin where alopecia and gray or graybrownish discoloration were present had rather uniform microscopic changes in both male and female treated dogs. The epithelial layer was thinned and atrophied with


738 a loss of dermal papillary ridges. Thickened collagen bundles increased the density of the dermis. The collagen fibers were often separated by spaces containing increased ground substance. Atrophy of pilosebaceous units and hair follicle epithelium was pronounced. The follicles were widely dilated and filled with concentric rings of keratinized debris (follicular keratosis). The affected follicles were generally devoid of hair shafts. There was no evidence of increased melanin or other pigment associated with the skin discoloration. Mammary Gland. Ductal epithelial hyperplasia, increased periductal connective tissue, and dilated to cystic ducts filled with scanty to prominent secretion occurred in males and females of all treatment groups. No significant acinar/lobular development was present. Nipple enlargement was usually associated with lactiferous ductal epithelial hyperplasia, hypertrophy of the smooth muscle bundles, and increased connective tissue stroma. Generally, mammary gland changes were more prominent in both females and males of the 2.0/1.5/1.0- and 1.0-mg/ kg EE treatment groups than in those receiving 0.5 mg/ kg EE or 5.0 mg/kg E2’

Endocrine

System

Grossly, adrenal glands of animals in 1.0-mg/kg EE, 2.0/1.5/1.0-mg/kg EE, and E2-treated groups were found to be enlarged and/or discolored. Adrenal gland weights were generally increased in all treated dogs, although they were not markedly increased in comparison with controls. This change was reflected microscopically by a widened cortex. In all animals except for 1 male (from the 2.0/1.5/1.0-mg/kg EE-treated Adrenal Gland.

the

group), the normal arcade architecture of the

zona

glom-

erulosa

partially or completely disappeared (Fig. 16). The cells of the zona fasciculata were slightly hypertrophied with foamy vacuolated cytoplasm, but they were less vacuolated than in controls, which indicated lipid depletion in treated animals. Focal nodular hyperplasia occurred in only 1 female (from the 2.0/1.5/1.0-mg/kg EE-treated group). Thyroid and Parathyroid Glands. Foci of nonkeratinsquamous metaplasia were found in thyroid glands of one-half the EE- and E2-treated dogs (Fig. 17). Both male and female animals were affected. A number of such foci were clearly found to be related to follicles and appeared as a dome of multilayered squamous epithelium within the follicular lumen, with an abrupt transition to normal follicular epithelium (Fig. 18). Follicles were sometimes completely filled by squamous epithelium. This lesion was first encountered in a male dog treated with 2.0/1.5/1.0 mg/kg EE; this dog was found dead on day 69 of the experiment. The most pronounced change was found in a 1.0-mg/kg EE male dog that was found dead on day 133. Multifocal nests of squamous epithelium were found throughout the thyroid gland in this dog. Foci of squamous metaplasia were also found within remnants of the thyroglossal duct and the ultimobranchial bodies, when present. Parathyroid glands were normal in all treated dogs.

izing

FIG. 19.-Distribution of renal fibroplasia. Left: schematic presentation of a paramedian section through a normal canine kidney; only the renal and interlobar arteries are depicted. Right: schematic presentation of a paramedian section through an affected kidney; (1) perivascular fibroplasia in the pelvic recesses, (2) well-demarcated area of fibroplasia surrounding the arcuate vessels at the level of the corticomedullary junction, (3) perivascular fibroplasia surrounding interlobular vessels, (4) well-demarcated cortical area of fibroplasia, and (5) diffuse cortical fibroplasia. The renal capsule and ureter are thickened. (C., cortex; M., medulla; R., recesses; R.A., renal artery; R.V. renal vein; U., ureter.)

Digestive System Liver. There

evidence of treatment-associated The livers of treated dogs were microhepatotoxicity. scopically comparable to those of controls. Focal mesothelial seeding, probably of ovarian origin, was found on the serosal surface of the liver near the gallbladder in I female dog treated with 1.0 mg/kg EE. Glisson’s capsule cells were unaffected and remained flattened, while the foreign cells were plump with a papillary and pseudoglandular pattern, typical of the growth covering the ovarian surface (Fig. 5). was no

DISCUSSION It is well known that estrogens induce a variety of changes and lesions in a broad spectrum of organs and tissues of the dog. The renal interstitial fibroplasia and thyroid squamous metaplasia that occurred in the dogs used in this study have not been previously reported. Although a similar renal interstitial lesion had been encountered in dogs treated with diethylstilbestrol (25, 28) and in 1 dog with a granulosa cell tumor of the ovary (33), the authors did not consider the change to have been causally related to the estrogenic activity of stilbestrol or to endogenous hyperestrogenism. Preclinical safety studies of oral contraceptive agents, usually combinations of a progestogen with either EE or mestranol, were conducted for 7 yr (9, 16). Renal interstitial fibroplasia was not produced; however, estrogen dosage levels were much lower than they were for dogs in this study. Renal interstitial fibroplasia occurred in dogs from all treatment groups of this study, which clearly linked the development and progression of the alteration to EE or E2 treatment. Initial development of the alteration began in the form of increased perivascular fibrous tissue surrounding interlobar vessels within the pelvic recesses. Arcuate and interlobular vessels were affected as the lesion


739

progressed, initially at the level of the corticomedullary junction, and finally extending into the cortex. The corchanges ranged from well-demarcated focal areas to large regions of diffuse fibrous tissue infiltration, which resulted in secondary glomerular and tubular cystic changes and atrophy. Expansion of the lesion within the cortex was evidenced by an obvious increase in pericapillary collagen between otherwise morphologically normal tubules and glomeruli. Induction of the lesion was tical

time related with great variation in individual animal

sus-

ceptibility. The estrogen-induced fibroplasia

in dogs included in this study was easily differentiated from other classical canine renal diseases characterized by interstitial fibrosis, such as chronic nephritis and renal infarction as well as induced hypertensive renal disease. The shape of the enlarged kidneys remained normal without surface irregularities despite the relatively large cortical and subcortical areas of fibrous tissue. Microscopically, there was no significant interstitial inflammatory change, and the fibroplasia did not result in contraction or surface irregularities, which occur in the case of chronic renal disease. The absence of renal contraction in dogs in this study was attributed to active progressive fibroplasia secondary to continuous estrogenic stimulation. Although some cortical areas of fibroplasia had a radial appearance, no vascular lesions were present. All renal vessels, including the renal arteries and veins, were normal, even though the early fibrous proliferation first occurred around vessels in the peripelvic region. There was no clinicopathological evidence of inflammatory renal disease. Increased plasma blood-urea nitrogen and creatinine values, which occurred in some males, were associated with obstructive uropathy secondary to prostatic and urethral epithelial and fibromuscular hypertrophy and

hyperplasia. Transitional cell hyperplasia occurred in the urinary bladder, ureter, and kidney pelvis. The urothelial hyperplasia of the renal pelvis was often accompanied by formation of multiple subepithelial microcysts, which were sometimes lined by metaplastic, nonkeratinized squamous epithelium. The muscular walls of both the urinary bladder and urethra were hypertrophied. The urinary and the genital systems are closely allied

during early embryonic development. It was not surprising to have stimulated fibrous tissue proliferation in the kidney similar to that occurring in the sex organs and their accessories following chronic estrogen treatment. The same applies for the squamous metaplasia encountered in the renal pelvis and tubules, as described in this study and by others (19, 28, 33). In apparent contrast, estrogen receptors have not been the canine kidney nor in the glandular portion of the prostate gland; however, estrogen receptors were identified in the prostatic urethral epithelium and the surrounding stroma (30). The authors theorized that the estrogenic changes in the urogenital system were either the result of the presence of masked or altered antigenic estrogen receptor determinants or were mediated by estrogen receptors in the underlying stroma. This may explain the EE- and E2-induced changes in the glandular and fi-

reported in

bromuscular portions of the prostate gland as well as in the kidney of dogs of this study. It was concluded that the renal fibroplasia in dogs included in this study was attributable to the estrogenic activity of EE or E,. Thyroid follicular squamous metaplasia observed in EE- and E~-treated dogs in this study was also concluded to have been an estrogen-induced change. It was not associated with any other morphological change or clinicopathological alteration. The epithelial change was most often present in the form of squamous cell nests within follicles. There was usually an abrupt change from normal follicular epithelium into the multilayered squamous epithelium. Thyroid follicular squamous metaplasia has been described in man and has been associated with goiter or chronic thyroiditis (20). It has also been reported in cows with functional ovarian cysts or tumors (17). In line with our conclusion, this thyroidal change in cows might also have been the result of hyperestrogenism. The remaining EE- and E2-induced changes were not unlike those that have been previously reported. However, in our study, E2 administration did not cause a proliferative germinal epithelial response of the ovary or mesothelial proliferation of the serosal surface of the uterine horns or epididymides. This was apparently the result of selective receptor activity differences between EE and E,. The pronounced proliferative effect on the germinal epithelium of the ovary, which occurred among EE-treated female dogs, has been reported previously (13, 14, 27, 29). These changes were first observed in female dogs that were treated with diethylstilbestrol, and they were considered to have been malignant because of the appearance of distant metastases (13, 14). Such growths were thought to be derived from the ovarian germinal

epithelium (27). In none of the cases did we consider the serosal mesothelial proliferation in EE-treated dogs to be a true neoplasm ; we considered it to be a hyperplastic lesion. Even though a focus of this proliferative lesion was found on the liver surface, it was not considered to be a true metastasis. The close structural resemblance of the focus to that of the ovarian proliferative growth suggested that spread occurred via transplantation. Similar proliferative serosal mesothelial lesions were induced in dogs treated with trans-4,4’-dimethyl-a,a’-diethylstilbene (29). These proliferative lesions have been shown to regress after termination of treatment, which confirms their nonneoplastic origin (21, 29). Recently, estrogen receptors have been demonstrated in the canine ovarian germinal epithelium and subsurface epithelial ingrowths (4). Their presence potentially explains the estrogen responsiveness of the serosal mesothelial surfaces covering the genital tissues. The responsiveness of the genital serosa was not exclusively restricted to the female, since 1 male dog that had been treated with 2.0/1.5/1.0 mg/kg EE had a focal papillary mesothelial proliferation on the serosal surface of the epididymides. Males, however, are less susceptible. Although these proliferative growths are well known in dogs, a similar lesion has not been produced in rats, mice, rhesus


740

monkeys (12), or cynomolgus monkeys in a parallel study. The devastating effect of high-level estrogen administration on bone marrow activity in male and female dogs, as reported in the literature (5, 6, 8, 22), was again shown in this study. Mortality rates associated with thrombocytopenia and generalized bone marrow depression were high in all EE- and E2-treated groups. However, there

wide variation in individual animal suscepNevertheless, tibility. recovery occurred in some dogs despite continuation of treatment. Changes in the adrenal zona glomerulosa were comparable to those described in dogs treated with stilbestrol (24). There was no distinct zona glomerulosa, as the glomerulosa cells appeared to transform into fasciculatype cells. There was no apparent clinicopathological disturbance in mineralocorticoid function, as supported by the results obtained for electrolyte determination. Skin alterations were both grossly and microscopically similar to those that have been published (7, 15, 19, 24, 31). Patchy discoloration and hair loss, which was especially prominent on the abdomen and inner thighs of animals, was associated with hair follicle and pilosebaceous unit atrophy. The hair follicles were most often devoid of hair shafts and were widely distended, with a concentrically laminated mass of keratinized debris. The skin epithelium had thinned. The density of dermal collagen had increased and was often separated by increased ground substance. The skin discoloration in dogs included in this study was attributed to the thinning of the skin and to the deep keratinization of hair follicles and not associated with increased melanin (23). This dog study, like many others that have been published, points out the extreme sensitivity of the dog to estrogens (both synthetic and natural estrogens). The increase in renal interstitial fibrous tissue and the follicular squamous metaplasia in the thyroid gland that are described in the dogs of this study are attributable to the estrogenic activity of EE or E2 when it is administered chronically. Accordingly, this adds the kidney and thyroid glands to the extensive list of dog-specific target organs and tissues that are susceptible to chronic estrogen treatment. Furthermore, these renal changes appear to be species specific, since treatment of cynomolgus monkeys in a parallel study with comparable dose levels of EE and E2 did not induce such lesions. Long-term toxicity studies with estrogens in rats and mice have also not shown such was a

Capel-Edwards K, Hall DE, and Sansom AG (1971). Hematological changes observed in female Beagle dogs given ethinylestradiol. Toxicol. Appl. Pharmacol. 20: 319-326. 4. Cock de H, Ducatelle R, and Logghe JP (1997). Immunohistochem3.

5.

6.

7.

8.

9.

10.

11. 12.

13. 14.

15. 16.

17. 18. 19.

20. 21.

22.

changes. ACKNOWLEDGMENT

We thank Ms. Karin Vloet for her patience and expertise and for the many hours she devoted to organizing and preparing this manuscript.

23. 24.

25.

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ERRATUM In the article &dquo;Pathology of Minamata Disease&dquo; by Eto that appeared in the November-December 1997 issue of Toxicologic Pathology (Toxicol. Pathol. 25: 614623), there is an error in the introduction on page 614, second column, 4 lines down. The sentence &dquo;Of these patients, all 2,946 had already died&dquo; should be changed to &dquo;About 1,300 patients of Minamata disease are still alive.&dquo;
