Morphology of Nasal Lesions in F344/N Rats ... - SAGE Journals

Toxicologic Pathology, 31:655–664, 2003 C by the Society of Toxicologic Pathology Copyright ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1080/01926230390242016

Morphology of Nasal Lesions in F344/N Rats Following Chronic Inhalation Exposure to Naphthalene Vapors PHILIP H. LONG,1 RONALD A. HERBERT,2 JOHN C. PECKHAM,3 SONDRA L. GRUMBEIN,4 CYNTHIA C. SHACKELFORD,3 AND KAMAL ABDO2 2

1 Pathology Associates, A Division of Charles River Laboratories, West Chester, Ohio 45069, USA National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA 3 Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709, USA, and 4 Battelle Northwest Laboratories, Richland, Washington 99352, USA

ABSTRACT Naphthalene (CAS No. 91-20-3) administered by inhalation at concentrations of 10, 30, or 60 ppm for 6 hours per day, 5 days per week for 105 weeks caused nonneoplastic and neoplastic effects in nasal respiratory and olfactory regions of male and female F344/N rats. Non-neoplastic nasal effects were characterized by an increase in the incidence and severity of a complex group of lesions, including atypical hyperplasia, atrophy, chronic inflammation, and hyaline degeneration of olfactory epithelium; hyperplasia, squamous metaplasia, hyaline degeneration, and goblet cell hyperplasia of the respiratory epithelium; and hyperplasia and squamous metaplasia of mucosal glands. Neoplastic effects were characterized by the induction of two types of rare primary nasal tumors, olfactory neuroblastomas and respiratory epithelial adenomas. The incidences of olfactory neuroblastomas in males at 0 ppm, 10 ppm, 30 ppm, and 60 ppm were, respectively, 0%, 0%, 8%, and 6%, whereas in females they were 0%, 4%, 6%, and 24%. The incidences of respiratory epithelial adenomas in males at 0 ppm, 10 ppm, 30 ppm, and 60 ppm were, respectively, 0%, 12%, 17%, and 31% and in females 0%, 0%, 8%, and 4%. The olfactory neuroblastomas and respiratory epithelial adenomas were considered carcinogenic effects related to naphthalene exposure based on their relatively high incidence in exposed rats, their absence in concurrent control rats and NTP historical controls, and their rare spontaneous occurrence in rats of any strain. Keywords. Naphthalene; rat; olfactory; inhalation; toxicity; carcinogenicity; neuroblastoma; adenoma.

INTRODUCTION Naphthalene, a white crystalline powder, is a commercially important aromatic hydrocarbon. The main use for naphthalene worldwide is in the production of phthalic anhydride, which is used as an intermediate in the production of polyvinyl chloride plasticizers. Naphthalene is used extensively as a chemical intermediate in the synthesis of anthranilic acids, naphthols, naphthyl-amines, naphthalene sulfonates, synthetic resins, celluloid, hydronaphthalenes, and dyes, and as an ingredient in some moth repellants (10, 15). Forest fires contribute to the presence of naphthalene in the environment, as the chemical is a natural combustion product of wood. Other sources of naphthalene in the environment are from coal tar and petroleum (15). Potential chronic exposure can also occur through cigarette smoke (3 µg naphthalene/cigarette) (25). In rats, the nasal cavity is a complex structure with regions lined by four distinct types of epithelium—squamous, transitional, respiratory, and olfactory (4). The region of squamous epithelium includes the nares, vestibule, anterior ventral meatus, and incisive duct. The region of transitional epithelium includes the anterior lateral wall and the anterior tips and lateral surfaces of the maxillary and nasal turbinates. The pseudostratified ciliated respiratory epithelial region includes the nasal septum and an area between the transitional epithelium

and olfactory region. Olfactory epithelium covers approximately 50% of the nasal cavity surface area and lines the posterior dorsal septum, the middle and posterior dorsal medial meatus, and the ethmoid turbinates (11). Olfactory epithelium is comprised of olfactory sensory neurons, sustentacular (supporting) cells, and basal (reserve) cells (4). The olfactory sensory cells are spindle-shaped and have a spherical nucleus. The supporting cells are tall and cylindrical and may contain lipofuscin. The basal cells are small and conical, lying with their base on the basement membrane. Beneath the olfactory mucosa is the lamina propria, which contains the Bowman’s glands. In 2-year toxicity/carcinogenicity studies in F-344/N rats recently reported by the NTP, inhalation exposure to naphthalene resulted in marked nasal-mucosa toxicity and the induction of rare nasal respiratory and olfactory epithelial neoplasms (1, 21). This paper describes the microscopic features of the neoplastic and non-neoplastic lesions that were observed in the nasal mucosa of male and female rats. Complete details on the conduct of the study were published as an NTP technical report (21). MATERIALS AND METHODS Chemical Naphthalene was obtained from Aldrich Chemical Co. (Milwaukee, WI) in one lot. The identification of the chemical, a white crystalline solid, was confirmed by infrared and proton nuclear magnetic resonance spectroscopy and gas chromatography/mass spectroscopy. The overall purity of the naphthalene was determined to be greater than 99%.

Address correspondence to: Dr. Philip H. Long, Pathology Associates, A Division of Charles River Laboratories Inc, 6217 Centre Park Drive, West Chester, Ohio 45069, USA; e-mail: [email protected]

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Its stability during the duration of the study was monitored using gas chromatography. No degradation of the bulk chemical was detected. Vapor Generation The vapor generator consisted of a 2-L glass reaction flask surrounded by a heated mantle. Heated nitrogen metered into the flask carried the vaporized naphthalene out of the generator. A heated teflon line transported the vapor to the exposure room where it was diluted with heated HEPA- and charcoalfiltered air before entering a distribution manifold. Before the study began, a small particle detector (Type CN, Gardner Associates, Schenectady, NY) was used with and without animals in the exposure chambers to ensure that naphthalene vapor, not aerosol, was produced. Naphthalene concentrations in the exposure chambers were monitored frequently throughout the exposures by an online gas chromatograph. The average chamber concentrations were maintained within 1% of the target concentrations. Study Design Groups of 49 male and 49 female rats were exposed to naphthalene at concentrations of 0, 10, 30, or 60 ppm for 6 hours plus T90 (the time required to reach 90% of the target concentration; 12 minutes) per day, 5 days per week for 105 weeks, excluding holidays. The exposure concentrations were selected based on the results of a 2-year study in mice in which animals were exposed to 0, 10, or 30 ppm (21). The 10 ppm concentration was selected because it represented the threshold limit value (TLV) for naphthalene (2). The highest exposure concentration (60 ppm) was selected to allow for variations in the maximum achievable concentration that could result from changes in temperature or operating conditions within the exposure system, without aerosolization or condensation of naphthalene in the chambers. Animals Male and female F344/N rats were obtained from Taconic Laboratory Animals and Services (Germantown, NY). The animals were quarantined for 14 days before the beginning of the study and were approximately 6 weeks old at the beginning of the study. During the study the health of the animals was monitored according to the protocols of the NTP Sentinel Animal Program. Male and female rats were housed individually. Feed (NTP-2000 Diet, Zeigler Brothers, Inc, Gardners, PA) was available ad libitum, except during the exposure period. Tap water was available ad libitum. Cages and chambers were changed weekly. All animals were observed twice daily. Body weights were recorded on study day 1, every 4 weeks beginning at week 4, and every 2 weeks beginning at week 92. Clinical findings were recorded every 4 weeks beginning at week 4 and every 2 weeks beginning at week 92. Animal care and use procedures were in accordance with the policy of the U.S. Public Health Service on humane care and use of animals and the Guide for the Care and Use of Laboratory Animals (14). Pathology Complete necropsies and microscopic examinations were performed on all animals. At necropsy, all organs and tissues were examined for grossly visible lesions. A comprehen-

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sive list of protocol organs and tissues, including all grossly observed lesions, were fixed and preserved in 10% neutral buffered formalin (NBF). Tissues were trimmed and processed, embedded in paraffin, sectioned to a thickness of 4 to 6 microns, and stained with hematoxylin and eosin for microscopic examination. Nasal cavities were fixed by retrograde flushing with NBF through the nasopharynx. Following decalcification of the skull, 3 transverse sections through the nose were trimmed at specific anatomical landmarks for all animals: Level I, immediately posterior to the upper incisor teeth; Level II, through the level of the incisive papilla anterior to the first palatial ridge; and Level III, through the middle of the second molar teeth. Levels I and II contain the nasoand maxillo-turbinates that, along with the nasal passages (meatuses) and septum, are lined by ciliated respiratory-type epithelium. Level III encompasses the olfactory region of the nose with ethmoid turbinates and meatuses lined entirely by specialized olfactory neuroepithelium. Final diagnoses for all tumors and target organs represent a consensus among the laboratory study pathologist, an independent reviewing pathologist, and an NTP Pathology Working Group. RESULTS The survival of all naphthalene-exposed groups was similar to that of the chamber controls. The olfactory and respiratory regions of the nasal cavity were the only sites of naphthalene toxicity and carcinogenicity in male and female rats. With the exception of a few spontaneous age-related changes, the nasal cavities of control rats were grossly and histologically normal. At necropsy, nasal masses observed in several naphthalene-treated male and female rats partially occluded the nasal passages or obliterated the normal architecture of the nasal turbinates and, in some animals, invaded the brain. Microscopically, exposure to naphthalene resulted in the induction of 2 rare types of nasal tumors, olfactory neuroblastomas and respiratory epithelial adenomas (Table 1). These tumors occurred amid a spectrum of non-neoplastic proliferative, degenerative, metaplastic, and inflammatory lesions in the olfactory and respiratory epithelia and lamina propria of the olfactory region. Non-neoplastic olfactory epithelial lesions included atypical hyperplasia, atrophy, hyaline degeneration, and chronic inflammation (Table 2). Proprial olfactory epithelial lesions included hyperplasia and squamous metaplasia of the Bowman’s gland epithelium. Non-neoplastic respiratory epithelial lesions included epithelial and goblet hyperplasia, squamous metaplasia, and hyaline degeneration. Regardless of the concentration, nearly all animals exposed to naphthalene had non-neoplastic lesions involving the olfactory epithelium and Bowman’s glands. In general, these lesions increased in severity with increasing exposure concentrations (Table 2). Although the incidences of non-neoplastic lesions of the respiratory epithelium increased in all exposure groups compared to controls, they were lower than those of the olfactory epithelium and did not increase with increasing exposure concentrations (Table 2). In addition, the incidences of hyaline degeneration of the olfactory and respiratory epithelia and of goblet cell hyperplasia did not increase with increasing exposure concentrations. Multiple lesions were observed in the same animal. Olfactory-epithelium atypical hyperplasia, atrophy, chronic inflammation, and hyaline

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TABLE 1.—Incidence of neoplastic nasal lesions in rats exposed to naphthalene vapor for 2 years.

Male Number examined microscopically Olfactory epithelium, neuroblastoma Overall rate Poly-3 test Respiratory epithelium, adenoma Overall ratea Poly-3 testb Female Number examined microscopically Olfactory epithelium, neuroblastoma Overall rate Poly-3 test Respiratory epithelium, adenoma Overall rate Poly-3 test

Chamber control

10 ppm

30 ppm

60 ppm

49

49

48

48

0/49 (0%) p = 0.027

0/49 (0%) c

4/48 (8%) p = 0.056

3/48 (6%) p = 0.109

0/49 (0%) p < 0.001

6/49 (12%) p = 0.013

8/48 (17%) p = 0.003

15/48 (31%) p < 0.001

49

49

49

49

0/49 (0%) p < 0.001

2/49 (4%) p = 0.214

3/49 (6%) p = 0.112

12/49 (24%) p < 0.001

0/49 (0%) p = 0.066

0/49 (0%) c

4/49 (8%) p = 0.053

2/49 (4%) p = 0.212

a

Number of animals with neoplasm per number of animlas with organ examined microscopically. Beneath the chamber control incidence are the p values associated with the trend test. Beneath the exposed group incidence are the p values corresponding to pairwise comparisons between the chamber controls and the exposed group. The Poly-3 test accounts for differential mortality in animlas that did not reach terminal sacrifice. c Value of statistic cannot be computed. b

degeneration, and Bowman’s gland hyperplasia were recorded in the same animal for 80% to 100% of animals, regardless of the exposure concentration. Less frequently, lesions in the respiratory epithelium accompanied the olfactory and Bowman’s gland lesions in the same animal. Nasal Olfactory Epithelium Neoplastic Lesions: Neuroblastomas of the olfactory epithelium occurred in male rats exposed to 30 or 60 ppm and in all groups of exposed females. The incidences of neuroblastoma occurred with positive trends in males and females and, in females exposed to 60 ppm, were significantly greater than those in the chamber controls (Table 1). Olfactory neuroblastomas were aggressively invasive masses of various sizes that developed in the ethmoid re-

gion (Level III) of the nasal cavity and extended rostrally to involve the respiratory epithelia in Levels II and I. Small lesions classified as early developing neuroblastomas were composed of tumor cells that thickened and replaced the olfactory epithelium and infiltrated the underlying lamina propria. Larger masses occluded the nasal passages and often distorted or completely obliterated the nasal architecture, invading nerves, nasal bones, the cribriform plate, and the olfactory lobes of the brain (Figures 1, 2). Tumor cells tended to exhibit a confluent endophytic growth pattern, infiltrating the lamina propria, along the length of the mucosa, as well as along submucosal nerve fibers, and gradually replaced the normal architecture of the turbinates and nasal septum. One male each in the 30 and 60 ppm groups had metastases in the lungs.

TABLE 2.—Incidence of nonneoplastic nasal lesions in rats exposed to naphthalene vapor for 2 years.

Male Number examined microscopically Olfactory epithelium, hyperplasia, atypical Olfactory epithelium, atrophy Olfactory epithelium, inflammation, chronic Olfactory epithelium, degeneration, hyaline Respiratory epithelium, hyperplasia Respiratory epithelium, metaplasia, squamous Respiratory epithelium, degeneration, hyaline Respiratory epithelium, goblet cell, hyperplasia Glands, hyperplasia Glands, metaplasia, squamous Female Number examined microscopically Olfactory epithelium, hyperplasia, atypical Olfactory epithelium, atrophy Olfactory epithelium, inflammation, chronic Olfactory epithelium, degeneration, hyaline Respiratory epithelium, hyperplasia Respiratory epithelium, metaplasia, squamous Respiratory epithelium, degeneration, hyaline Respiratory epithelium, goblet cell, hyperplasia Glands, hyperplasia Glands, metaplasia, squamous ∗∗ a b

Chamber control

10 ppm

30 ppm

60 ppm

49 0a 3 (1.3)b 0 3 (1.3) 3 (1.0) 0 0 0 1 (1.0) 0

49 48∗∗ (2.1) ∗∗ 49 (2.1) 49∗∗ (2.0) 46∗∗ (1.7) 21∗∗ (2.2) 15∗∗ (2.1) 20∗∗ (1.2) 25∗∗ (1.3) 49∗∗ (2.2) 3 (3.0)

48 45∗∗ (2.5) ∗∗ 48 (2.8) 48∗∗ (2.2) 40∗∗ (1.7) 29∗∗ (2.0) 23∗∗ (2.0) 19∗∗ (1.4) 29∗∗ (1.2) 48∗∗ (2.9) 14∗∗ (2.1)

48 46∗∗ (3.0) ∗∗ 47 (3.5) 48∗∗ (3.0) 38∗∗ (1.5) 29∗∗ (2.2) 18∗∗ (1.8) 19∗∗ (1.2) 26∗∗ (1.2) 48∗∗ (3.5) 26∗∗ (2.5)

49 0 0 0 13 (1.1) 0 0 8 (1.0) 0 0 0

49 48∗∗ (2.0) ∗∗ 49 (1.9) 47∗∗ (1.9) 46∗∗ (1.8) 18∗∗ (1.6) 21∗∗ (1.6) 33∗∗ (1.2) 16∗∗ (1.0) 48∗∗ (1.9) 2∗∗ (2.0)

49 48∗∗ (2.4) ∗∗ 49 (2.7) 47∗∗ (2.6) 49∗∗ (2.1) 22∗∗ (1.9) 17∗∗ (1.5) 34∗∗ (1.4) 29∗∗ (1.2) 48∗∗ (3.1) 20∗∗ (2.5)

49 43∗∗ (2.9) ∗∗ 47 (3.2) 45∗∗ (3.4) 45∗∗ (2.1) 23∗∗ (1.7) 15∗∗ (1.8) 28∗∗ (1.2) 20∗∗ (1.0) 42∗∗ (3.3) 20∗∗ (2.8)

Significantly different ( p ≤ 0.01) from the chamber control group by the Poly-3 test. Number of animals with lesion. Average severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

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The neuroblastomas had 3 histological patterns, with more than one type occurring frequently within the same tumor. One type was composed of small islands and nests of round to polygonal cells with prominent round vesicular nuclei; single, centrally located prominent nucleoli; marginated chromatin; poorly-defined cytoplasmic borders; and a large nuclear-tocytoplasmic ratio (Figures 3, 4). Cell nuclei resembled those of neurons, suggesting sensory cell differentiation. Rare multinucleated cells were noted. Small nests and cords of tumor cells were often present in the lamina propria of the turbinates and nasal septum, and especially in and along olfactory nerve bundles located distant to the origin of the tumor (Figure 5). In some fields, small amounts of eosinophilic fibrillar material were interspersed with small islands of tumor cells. The degree of cytologic atypia varied from moderate to high. Cells within small tumors were relatively uniform in size and shape, whereas those in larger tumors appeared more pleomorphic. Tumor cells occasionally formed pseudorosettes (Homer-Wright rosettes) in which they were arranged in a circle around a small central lumen that was partly filled with tangled fibrillar material (Figure 6). Nuclei of cells forming the rosettes blended peripherally with the cells forming the rest of the tumor. True rosettes with a pseudoglandular appearance were noted in the larger tumors. Mitotic figures were abundant. A second morphologic type of neuroblastoma was composed of irregular lobules separated by thin bands of fibrovascular connective tissue (Figure 7). Cells within lobules were generally round to oval to polygonal and basophilic, with round to oval to elongate hyperchromatic nuclei, single nucleoli, poorly defined cytoplasmic borders, and a large nuclear-to-cytoplasmic ratio. There was a tendency for the cells within the center of the lobule (distant to the fibrovascular connective tissue) to be necrotic. Large lobules often contained cystic centers. Within each lobule, tumor cells adjacent to the fibrovascular connective tissue were cuboidal to columnar and oriented in pseudostratified rows, with their long axis perpendicular to the connective tissue base, suggestive of sustentacular cell differentiation (Figure 8). The degree of cytologic atypia varied from moderate to high. Tumor cells often formed pseudorosettes. Mitotic figures were abundant and often atypical. Also noted were pseudoglandular structures resembling true rosettes (Flexner-Wintersteiner rosettes) in which tumor cells surrounded an open central lumen bounded by distinct cell membranes, mimicking glandular structures (Figure 9). Foci of squamous epithelial differentiation were occasionally noted. A third, less common morphologic type of neuroblastoma was composed of interlacing cords of tumor cells with areas of spindle cell differentiation (Figure 10). Cells along the outer regions of the cords were undifferentiated basophilic cells,

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which appeared to have given rise to a population of spindle cells that produced eosinophilic, fibrillar, neural-like stroma (Figure 11). In some regions, the tumor cells appeared to be forming rudimentary nerve fibers (Figure 12). A few neoplasms of this type also had focal areas of squamous differentiation, sometimes with formation of keratin (Figure 13). Foci of necrosis were present but not prominent. The degree of cytologic atypia was moderate to high. A few pseudorosettes as well as true rosettes were noted, some of which mimicked glandular structures. Mitotic figures were abundant and often atypical. Nonneoplastic Lesions: Neuroblastomas occurred amid a complex spectrum of nonneoplastic lesions of the olfactory epithelium (Table 2). The principal nonneoplastic proliferative lesion was atypical hyperplasia, which occurred primarily along the nasal septum in the ethmoid region and consisted of proliferating nests of dysplastic olfactory epithelial cells within or beneath the epithelium and/or multifocal nodular proliferations of basal cells extending into the submucosa (Figure 14). The hyperplastic cells were deeply basophilic and, in many areas, continuous with the neoplasms. Such continuity was most clearly observed in association with small neuroblastomas. Atrophy of olfactory epithelium was characterized by a decrease in the height of the epithelium lining the dorsal meatuses of Level II and the ethmoid turbinates of Level III and was due to loss of epithelial cells. Mild atrophy consisted of sustentacular cell loss only. Moderate atrophy consisted of mostly sustentacular cell loss; however, there was also loss of olfactory neurons. The most severe lesions had complete loss of sustentacular cells and neurons, leaving only basal epithelial cells. Frequently, respiratory-like ciliated columnar cells had replaced atrophic olfactory epithelium. Although included here as a component of olfactory epithelial atrophy, this change is often given a separate diagnosis of epithelial metaplasia. Chronic inflammation of the olfactory region was characterized by infiltrates of primarily mononuclear inflammatory cells within the lamina propria accompanied by fibrosis. As a result of tissue repair involving previous inflammatory changes, synechia were often observed between adjacent turbinates. Nasal Respiratory Epithelium Neoplastic Lesions: Adenomas of the respiratory epithelium occurred with a positive trend in male rats and were significantly increased in all groups; the incidences in female rats exposed to 30 or 60 ppm were also increased, but not significantly (Table 1). Adenomas developed in Levels I and II of the nasal cavity along the medial or lateral aspects or tips of the

FIGURE 1.—Neuroblastoma. Level III nasal cavity from a female rat exposed to 60 ppm naphthalene by inhalation for 2 years. The neoplasm has completely obliterated the nasal architecture. Bar = 1000 µm. 2.—Neuroblastoma. Level III nasal cavity from a female rat exposed to 60 ppm naphthalene by inhalation for 2 years. Tumor cells are invading the olfactory bulbs of the brain. Bar = 250 µm. 3.—Neuroblastoma. Level III nasal cavity from a female rat exposed to 60 ppm naphthalene by inhalation for 2 years. Tumor cells arranged in solid islands and small nests that have completely filled the nasal passages and invaded adjacent tissues. Bar = 250 µm. 4.—Higher magnification of Figure 3. The neoplastic cells are round to polygonal with prominent round vesicular nuclei; single, centrally located prominent nucleoli; marginated chromatin; and a large nuclear-to-cytoplasmic ratio. Multinucleated tumor cell also present. Bar = 30 µm. 5.—Higher magnification of Figure 3 showing tumor cells infiltrating along nerve fiber. Bar = 50 µm.

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Figures 6–11 FIGURE 6.—Tumor cells from same mass shown in Figure 3 showing pseudorosette formation (arrows). Bar = 50 µm. 7.—Neuroblastoma. Level III nasal cavity from a female rat exposed to 30 ppm naphthalene by inhalation for 2 years. The tumor cells are arranged in irregular lobules separated by thin bands of fibrovascular connective tissue. Large lobules often contained necrotic cystic centers. Bar = 250 µm. 8.—Higher magnification of Figure 7. Tumor cells adjacent to the fibrovascular connective tissue are cuboidal to columnar and oriented in rows with their long axis perpendicular to the connective tissue base, suggestive of sustentacular cell differentiation. Bar = 30 µm. 9.—Neuroblastoma with a pseudoglandular structure resembling a true rosette. Tumor cells surround an open central lumen bounded by distinct cell membranes. Bar = 50 µm. 10.—Neuroblastoma composed of interlacing cords of tumor cells with areas of spindle cell differentiation. Bar = 250 µm. 11.—Higher magnification of Figure 10. The basophilic cells appear to give rise to a population of spindle cells producing eosinophilic fibrillar neural-like stroma. Bar = 50 µm.

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Figures 12–17 FIGURE 12.—Higher magnification of Figure 10. In some regions the tumor cells appear to form rudimentary nerve fibers. Bar = 50 µm. 13.—Higher magnification of Figure 10 showing focal areas of squamous differentiation with keratin formation and cellular debris. Bar = 50 µm. 14.—Level III nasal cavity from a female rat exposed to 30 ppm naphthalene by inhalation for 2 years. Note atypical basal cell hyperplasia, atrophy of olfactory epithelium, and loss of Bowman’s glands. Bar = 50 µm. 15.—Large exophytic respiratory epithelial adenoma in Level I nasal cavity from female rat exposed to 30 ppm naphthalene by inhalation for 2 years. Bar = 1000 µm. 16.—Higher magnification of Figure 15. Note neoplastic cells are arranged in gland-like formations. Bar = 250 µm. 17.—Higher magnification of Figure 15. Note the cuboidal cells and cellular debris in lumen of some glandlike structures. Bar = 100 µm.

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nasoturbinates, or the lateral wall originating from regions of the nasal mucosa lined by respiratory epithelium. They were irregular exophytic, polypoid, pedunculated, or broadbased sessile masses that varied in size. Exophytic adenomas partially occluded, compressed, and distorted the nasal passages (Figure 15). Neoplastic cells were welldifferentiated, cuboidal to columnar, and arranged primarily as variably-sized pseudoglandular structures surrounded by scant fibrovascular stroma with few focal solid areas of cells (Figure 16). The pseudoglandular structures appeared to form from outgrowths and folds of surface epithelium. Many pseudoglandular structures were variably distended with proteinaceous material, sloughed epithelial cells, leukocytes, and cell debris (Figure 17). Cystic pseudoglandular structures were also noted, especially in the larger adenomas. In some adenomas, the epithelium appeared pseudostratified, and in others squamous metaplasia was noted. A few adenomas were composed of less well-differentiated cells that were squamous; these cells were large, round to polygonal, and had scant to moderate amounts of eosinophilic cytoplasm and large round to oval nuclei that contained one or two prominent nucleoli. Nonneoplastic Lesions: Respiratory epithelial adenomas also occurred amid a spectrum of non-neoplastic lesions of the respiratory epithelium and the submucosal glandular epithelium (Table 2). Respiratory epithelial hyperplasia involved the lateral wall and medial surface of the naso- and maxillo-turbinates and was a focal to segmental lesion that sometimes involved most of the turbinate extending onto the lateral wall in Levels I and II of the nasal cavity. The affected epithelium was thickened by increased numbers of disorganized, often pseudostratified, epithelial cells. Individual cells were either nonciliated flattened, or ciliated cuboidal to columnar cells. In a few animals, focal proliferation of hyperplastic cuboidal respiratory epithelium resembled early adenoma formation. Frequently, the hyperplastic ciliated epithelium was folded in rugose fashion and sometimes extended into the submucosa forming pseudoglands. Respiratory epithelial squamous metaplasia involved the lateral surfaces of the nasoturbinates and the lateral wall in Level I of the nasal cavity. Metaplasia consisted of replacement of the normally ciliated respiratory epithelium by one to 6 layers of polygonal to flat squamous cells. Keratinization was seldom present. Nasal Glandular Epithelium Glandular hyperplasia primarily affected the Bowman’s glands of the nasal septum along the dorsal meatus in Level II and in the ethmoid turbinates in Level III of the nasal cavity. This hyperplasia was characterized by proliferation of the glandular epithelium resulting in enlarged glands that were often distended with cellular debris and proteinaceous material. Frequently, affected glands were lined by hyperplastic ciliated epithelium continuous with that of the mucosa. The hyperplastic cells were usually distended by intracytoplasmic protein or protein globules. Squamous metaplasia of glands often accompanied hyperplasia and was characterized by several layers of nonkeratinized squamous cells that filled the glandular lumen. Goblet cell hyperplasia was generally of minimal severity and primarily involved the respiratory epithelium of the nasal septum in Level I of the nasal cavity. Hyperplastic goblet cells were increased in size and number,


were swollen with mucus, and often formed in small glandlike clusters within the mucosal epithelium. Hyaline degeneration was a minimal to mild change that affected localized areas of both the respiratory and olfactory epithelium. Affected epithelial cells were swollen by intracytoplasmic homogenous, brightly eosinophilic material that often occurred as globules. DISCUSSION The olfactory and respiratory regions of the nasal cavity were the only sites of naphthalene toxicity and carcinogenicity in male and female rats. The resulting olfactory neuroblastomas and respiratory epithelial adenomas were considered to be carcinogenic effects related to naphthalene exposure based on their relatively high incidence in exposed rats, their absence in concurrent control rats and NTP historical controls (13, 21), and their rare spontaneous occurrence in rats of any strain. Though variable, similar nasal changes have been reported in laboratory animals after exposure to a variety of chemicals (8, 9, 22, 29, 30). The undifferentiated, olfactory basal cell has been proposed as the cell of origin of olfactory neuroblastomas. These cells are thought to be capable of differentiating into sustentacular (supporting), sensory (neuronal), and ductal epithelial cells of Bowman’s glands. The observations that the neuroblastomas in this study appeared to be arising from the olfactory epithelium and contained cells with morphologic features similar to these different cell types are consistent with their classification as olfactory neuroblastomas. The terminology and diagnostic criteria used in this publication were consistent with those proposed by the Society of Toxicologic Pathology, with some modification by others (4, 22, 26). In addition to the presence of nasal respiratory and olfactory neoplasms, a variety of proliferative and nonproliferative non-neoplastic nasal lesions were significantly increased in all groups as a result of naphthalene exposure. Except for atypical hyperplasia of the olfactory epithelium, these non-neoplastic lesions were of the type frequently observed in inhalation studies when animals are exposed to chemicals with irritant properties. They are generally considered nonspecific reparative, protective, or adaptive responses to chronic irritation. Although the incidence and severity of these non-neoplastic lesions frequently increase in an exposure-dependent manner, they commonly occur with no evidence of nasal carcinogenicity, indicating that factors other than the extent of tissue injury from chronic nasal toxicity contribute to nasal carcinogenesis (13, 16–20). Atypical hyperplasia of the olfactory basal cells occurred at very high frequencies in all male and female groups exposed to naphthalene. This was considered an unusual proliferative lesion, because it had not been reported in previous NTP inhalation studies. Morphologically, these cells were similar to, and frequently formed a continuum with, those of the neuroblastomas. This appearance suggests that the atypical hyperplasia may represent a precursor for nasal olfactory carcinogenesis. In addition, a few animals had localized proliferative changes of the respiratory epithelium that were morphologically similar to respiratory epithelial adenomas. These changes were not given a separate diagnosis but were included in the diagnosis “respiratory-epithelium hyperplasia.”

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Studies conducted in experimental animals and tissues using a variety of techniques provide some insight into the possible mechanism(s) for naphthalene toxicity and carcinogenicity. One potential mechanism of action for its carcinogenicity is demonstrated in assays that measure induction of clastogenic chromosomal effects. For example, naphthalene gave positive results in micronucleus assays (6, 24) and chromosomal aberration (21) and recombination tests (5), consistent with a clastogenic mechanism of action. For some endpoints, naphthalene-induced chromosomal effects require, or are enhanced by, cytochrome P450 enzymes. The reactive naphthalene intermediate responsible for the clastogenic, and presumably carcinogenic, effects is not clear; however, evidence for the reactivity of both naphthalene 1,2-epoxide and naphthoquinone exists. Naphthalene did not induce mutations in Salmonella or cultured human MCL-5 cells. Naphthalene also causes cellular injury and accelerated cell replication, suggesting a cytotoxic mode of action. For example, intraperitoneal administration of naphthalene produced injury (swelling, vacuolization, exfoliation, and necrosis) of tracheobronchial Clara cells in mice, but not rats (23). In the same study, naphthalene was cytotoxic to the olfactory epithelium of both rats and mice, but the effect was seen at much higher doses in mice, suggesting an increased sensitivity of rat olfactory epithelium. These site and species differences in toxicity correlate well with higher rates of metabolism by rat nasal tissue and mouse lung tissue. Experimentally, nasal olfactory neuroblastomas have been induced by oral, inhalation, or peritoneal exposure to several structurally unrelated chemicals (22). Some of these compounds undergo metabolic activation by the cytochrome P450 enzyme system before causing olfactory epithelial injury, chronic hyperplastic/regenerative lesions, and olfactory neoplasms. One such compound, the type IV phosphodiesterase inhibitor RP 73401, caused neuroblastomas and a spectrum of non-neoplastic lesions in the nasal olfactory epithelium of Sprague-Dawley rats that appeared to be similar to those observed in this study (22). RP 73401 toxicity was related to its metabolic activation in olfactory sustentacular cells, which contain high concentrations of metabolizing enzymes, especially those of the cytochrome P450 (CYP2F) system, and are important in xenobiotic metabolism (12, 28). Respiratory epithelial cells also contain xenobiotic-metabolizing enzymes, including those of the cytochrome P450 system (12). That naphthalene is also metabolized by cytochrome P450 enzymes suggests that the toxic nasal effects observed in this study may be related to its metabolism in respiratory and olfactory epithelium (7). The results of this study are consistent with the general observation that rats are more susceptible to epithelial tumors of the nasal cavity than mice (3, 4). In addition to differences in site-specific metabolism in rats and mice, the tendency for rats to develop nasal tumors, while mice develop lung tumors, following exposure to naphthalene may be partly related to anatomic differences in the nasal passages in these two species. Such distinctiveness could lead to differences in doses delivered at this site, especially when the dose is normalized to the surface area of the nasal passages (27). Rates of production and clearance of carcinogenic metabolites of naphthalene by the nasal epithelia and lungs could also account for species differences in sites of tumor development.

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ACKNOWLEDGMENTS This work was sponsored by the National Toxicology Program, National Institute of Environmental Health Sciences, US Department of Health and Human Services. The authors thank Drs. James Hailey and Robert Sills of the National Institute of Environmental Health Sciences for their critical review of this manuscript and Mr. Norris Flagler for his expert technical assistance in preparation of the illustrations. REFERENCES 1. Abdo KM, Grumbein S, Chou BJ, Herbert RA (2001). Toxicity and carcinogenicity study in F344 rats following 2 years of whole-body exposure to naphthalene. Inhal Toxicol 13: 931–950. 2. American Conference of Governmental Industrial Hygienists (ACGIH) (1999). 1999 Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices. Cincinnati, OH. 3. Brown HR (1990). Neoplastic and potentially preneoplastic changes in the upper respiratory tract of rats and mice. Environ Health Perspect 85: 291– 304. 4. Brown HR, Monticello TM, Maronpot RR, Randall HW, Hotchkiss JR, Morgan KT (1991). Proliferative and neoplastic lesions in the rodent nasal cavity. Toxicol Pathol 19: 358–372. 5. Delgado-Rodriguez A, Ortiz-Marttelo R, Graf U, Villalobos-Pietrini R, Gomez-Arroyo S (1995). Genotoxic activity of environmentally important polycyclic aromatic hydrocarbons and their nitro derivatives in the wing spot test of Drosophila melanogaster. Mutat Res 341: 235–247. 6. Djomo JE, Ferrier V, Gauthier L, Zoll-Moreux C, Marty J (1995). Amphibian micronucleus test in vivo: evaluation of the genotoxicity of some major polycyclic aromatic hydrocarbons found in a crude oil. Mutagenesis 10: 223–226. 7. Doherty MD, Makowski R, Gibson GG, Cohen GM (1985). Cytochrome P-450 dependent metabolic activation of 1-naphthol to naphthoquinones and covalent binding species. Biochem Pharmacol 34: 2261–2267. 8. Feron VJ, Kruysse A, Woutersen RA (1982). Respiratory tract tumors in hamsters exposed to acetaldehyde vapour alone or simultaneously to BP or DENA. Eur J Cancer Oncol 18: 13–31. 9. Feron VJ, Bruyntjes JP, Woutersen RA, Immel HR, Appleman LM (1988). Nasal tumors in rats after short-term exposure to a cytotoxic concentration of formaldehyde. Cancer Lett 39: 101–111. 10. Gosselin RE, Smith RP, Hodge HC (1984). Clinical Toxicology of Commercial Products, 5th ed. Williams and Wilkins, Baltimore, pp III–307. 11. Gross EA, Swenberg JA, Fields S, Popp JA (1982). Comparative morphometry of the nasal cavity in rats and mice. J Anat 135: 83–88. 12. Harkema JR, Morgan KT (1996). Proliferative and metaplastic lesions in nonolfactory nasal epithelia induced by inhaled chemicals. In: Respiratory System, 2nd ed, Jones TC, Dungworth DL, Mohr U (eds). Springer-Verlag, Berlin, pp 18–28. 13. Haseman JK, Hailey JR (1997). An update of the National Toxicology Program database on nasal carcinogens. Mutation Res 380: 3–11. 14. Institute of Laboratory Animal Resources (1978). Guide for the Care and Use of Laboratory Animals. NIH Publication No. 78-23, National Institutes of Health, Research Triangle Park, NC. 15. The Merck Index (1996). 12th ed., Budavari S (ed). Merck and Company, Inc, Whitehouse Station, New Jersey, p 1094. 16. Miller RR, Young JT, Kociba RJ, Keyes DG, Bonder KM, Calhoun LL, Ayres JA (1985). Chronic toxicity and oncogenicity bioassay of inhaled ethyl acrylate in Fischer 344 rats and B6C3F1 mice. Drug Chem Toxicol 8: 1–42. 17. National Toxicology Program (NTP) (1997). Toxicology and carcinogenesis studies of nitromethane (CAS No. 75-52-5) in F344/N rats and B6C3F1 mice (inhalation studies). Technical Report Series No. 461, NIH Publication No. 97-3377. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. 18. National Toxicology Program (NTP) (1998a). Toxicology and carcinogenesis studies of chloroprene (CAS No. 126-99-8) in F344/N rats and B6C3F1

664

19.

20.

21.

22.

23.

LONG ET AL

mice (inhalation studies). Technical Report Series No. 467, NIH Publication No. 98-3957. US Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. National Toxicology Program (NTP) (1998b). Toxicology and carcinogenesis studies of cobalt sulfate heptahydrate (CAS No. 10026-24-1) in F344/N rats and B6C3F1 mice (inhalation studies). Technical Report Series No. 471, NIH Publication No. 98-3961. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. National Toxicology Program (NTP) (1999). Toxicology and carcinogenesis studies of glutaraldehyde (CAS No. 111-30-8) in F344/N rats and B6C3F1 mice (inhalation studies). Technical Report Series No. 490, NIH Publication No. 99-3980. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. National Toxicology Program (2000). Toxicology and carcinogenesis studies of naphthalene (CAS No. 91-20-3) in F344/N rats (inhalation studies). Technical Report Series No. 500, NIH Publication No. 01-4434. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Pino MV, Valerio MG, Miller GK, Larson JL, Rosolia DL, Jayyosi Z, Crouch CN, Trojanowski JQ, Geiger LE (1999). Toxicologic and carcinogenic effects of the Type IV phosphodiesterase inhibitor RP 73401 on the nasal olfactory tissue in rats. Toxicol Pathol 27: 383–394. Plopper CG, Suverkropp C, Morin D, Nishio S, Buckpitt A (1992). Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after

24.

25.

26.

27.

28.

29. 30.


parenteral administration of naphthalene. J Pharmacol Exp Ther 261: 353– 363. Sasaki JC, Arey J, Eastmond DA, Parks KK, Grosovsky AJ (1997). Genotoxicity induced in human lymphoblasts by atmospheric reaction products of naphthalene and phenanthrene. Mutat Res 393: 23–35. Schmeltz I, Tosk J, Hilfrich J, Horita N, Hoffman D, Wynder EL (1978). Bioassays of naphthalene and alkylnaphthalenes for co-carcinogenic activity: relation to tobacco carcinogenesis. In: Carcinogenesis—A Comprehensive Survey, Jones PW, Freudenthal RI (eds). Raven, New York, 3: 47–60. Schwartz LW, Hahn FF, Keenan KP, Brown HR, Mann PC (1994). Proliferative lesions in the rat respiratory tract. In: Guides for Toxicologic Pathology. Society of Toxicologic Pathologists/American Registry of Pathology/ Armed Forces Institute of Pathology, Washington, DC, pp 1–24. Swenberg JA, Heck HA, Morgan KT, Star TB (1985). A scientific approach to formaldehyde risk assessment. In: Risk Quantitation and Regulatory Policy: 19th Banbury Report, Hoel DG, Merrill RA, Perera FP (eds). Cold Spring Harbor, New York, pp 255–267. Thornton-Manning JR, Dahl AR (1997). Metabolic capacity of nasal tissue interspecies comparisons of xenobiotic-metabolizing enzymes. Mutat Res 380: 43–59. Woutersen RA, Appleman LM, Van Garderen-Hoetmer, Feron VJ (1986). Inhalation toxicity of acetaldehyde in rats. Toxicol 41: 213–231. Woutersen RA, Van Garderen-Hoetmer, Slootweg PJ, Feron VJ (1994). Upper respiratory tract carcinogenesis in experimental animals and in humans. In: Carcinogenesis, Waalkens MP, Ward JM (eds). Raven Press Ltd, New York.