Respiratory Tract Lesions in Noninhalation Studies

Nonneoplastic Nasal Lesions

Nonneoplastic lesions included hyperplasia of respiratory epithelium; degeneration, necrosis, and atrophy of the olfactory epithelium; and metaplasia of olfactory epithelium to respiratory type with further hyperplasia of the new respiratory type epithelium.

Respiratory epithelial hyperplasia was characterized by an increase in the number of columnar cells, by formation of multiple short papillary epithelial projections, and by the formation of multiple invaginations or “crypt-like” structures (Figure 1A). The mucosa and underlying submucosa were sometimes infiltrated with polymorphonuclear and mononuclear inflammatory cells. Dilated submucosal glands, some of which were lined by hyperplastic epithelium, often accompanied the respiratory epithelial hyperplasia. Respiratory epithelial hyperplasia was observed in the respiratory epithelium lining the nasal septum, the nasal turbinates and the ventral ethmoid turbinates. Respiratory epithelial hyperplasia was diagnosed in Sprague–Dawley rats administered a binary mixture of 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153), and a binary mixture of PCB 126 and 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118) by gavage (Nyska et al., 2005).

Metaplasia of olfactory epithelium to respiratory type epithelium was noted in the olfactory epithelium lining the nasal septum, nasoturbinates, and ethmoid turbinates. Morphologically the olfactory epithelium was replaced by respiratory epithelium (Figure 1B). Metaplasia to respiratory type epithelium was sometimes followed by hyperplasia of the respiratory epithelium with the formation of papillary projections similar to that observed with hyperplasia of respiratory epithelium. Inflammation was observed in association with the olfactory epithelial metaplasia in some instances. Atrophy of the olfactory nerve cell bundles often accompanied changes in the olfactory epithelium. In the vast majority of cases respiratory metaplasia of olfactory epithelium was a sequel to olfactory degeneration/regeneration. Modulation of normal patterns of cellular differentiation through direct actions on proliferating basal cells was suggested as a mechanism with some dioxin and dioxin-like compounds (Nyska et al., 2005). This finding was observed with a binary mixture of PCB 126 and PCB 153, and a binary mixture of PCB 126 and PCB 118, and in studies with methacrylonitrile (Nyska et al., 2003) and benzophenone, monochloracetic acid, and mercuric chloride (Table 1).

Degeneration and/or atrophy of the olfactory epithelium (Figure 1C and 1D) were characterized by a thinning and/or disorganization of the olfactory epithelial layer. Increased intercellular space and disruption of epithelium were early indicators of degeneration, which sometimes lead to vacuolization, apoptosis and necrosis. The sensory cells (bipolar neurons) were the most sensitive cells to injury in the olfactory epithelium and loss of these cells could lead to atrophy of the nerves; however, atrophy of the nerves did not occur with all chemicals that caused destruction of the olfactory epithelium (Gaskell, 1990). The damaged olfactory epithelium could repair or respiratory epithelial metaplasia could occur. Degeneration and/or atrophy was observed with methacrylonitrile (Nyska et al., 2003), benzyl acetate, dipropylene glycol, o-nitrotoluene, cyclohexanone oxime, butanal oxime, and methyl ethyl ketoxime (Table 1). Methacrylonitrile was metabolized by cytochrome P-450 2E1(CYP2E1) to acetone, which was eliminated along with the parent compound in the breath. CYP2E1 was increased in the lung and nasal tissues and it is possible that induction of CYP2E1 could play a role in the bioactivation of methacrylonitrile (Wang et al., 2002).

Pigmentation in the olfactory epithelium was observed with some chemicals (pentachloroanisole and trans-cinnamaldehyde) administered by the noninhalation route (Table 1). For pentachloroanisole the pigment appeared as coarse, golden brown to dark brown intracytoplasmic granules in the olfactory epithelium, which with special stains were negative for iron and bile, and did not stain with periodic acid-Schiff. In the trans-cinnamaldehyde study the pigment was located in the basal cytoplasm of the olfactory epithelium and was characterized as a finely granular golden brown pigment consistent with lipofuscin.

A homogenous hyaline globular material, which has been diagnosed as eosinophilic globules, hyaline accumulation, or accumulation of homogenous globular hyaline material, is often observed in the respiratory and olfactory epithelium. The material ultrastructurally appeared as membrane-bound ellipsoid bodies suggestive of dilated rough endoplasmic reticulum (Popp et al., 1986). These were nonspecific entities that tended to increase with age, although they were reported to increase with treatment in some studies.

Neoplastic Nasal Lesions

Nasal tumors induced by chemicals administered by noninhalation routes included adenoma, adenocarcinoma, squamous cell carcinoma, carcinoma (not otherwise specified), and neuroblastoma (Table 1). Sex differences were observed in the incidence of tumors with some chemicals (Brown et al., 1991).

Polypoid adenoma (Figure 1E) was the most commonly induced adenoma in the nasal cavity (Brown et al., 1991), arising in the anterior nasal cavity from the respiratory epithelium of the nasoturbinates, maxilloturbinates, or lateral wall of the nasal cavity. Polypoid adenomas were sessile or pedunculated tumors formed of nonciliated cuboidal to low columnar cells that formed solid sheets or microcysts (Kerns, 1985). The nose was identified as a site of metabolic transformation of chemicals/drugs when administered by a variety of routes (Bogdanffy, 1990). Adenomas were associated with the administration of 2,3-dibromo-1-propanol, and 2,6-xylidine (Table 1). Adenocarcinomas arose from the respiratory epithelium or glandular structures. Those that arose from respiratory epithelium were exophytic or grew by lateral extension (Figure 1F). Those that arose from glandular structures formed solid sheets of round or polyhedral cells or were formed of spindle-shaped cells.

Adenocarcinomas, transitional cell carcinomas, and squamous cell carcinomas were reported to occur in the nose of Sprague–Dawley rats administered phenacetin in the diet (Isaka et al., 1979). 1,4-dinitrosopiperazine given in the drinking water caused carcinomas (adenocarcinomas, adenosquamous carcinomas and undifferentiated carcinomas) in F344 rats (Takano et al., 1982). Adenocarcinomas, squamous cell carcinomas, and adenomas and/or papillomas were reported to be increased in the nose when p-cresidine was administered in the feed to F344 rats (Reznik et al., 1981).

Squamous cell carcinomas occurred at any location in the nasal cavity. They originated from the squamous epithelium in the nasal vestibule, the ventral meatus or the nasopalatine duct, or more often they arose in areas of respiratory, transitional, or olfactory epithelium that had undergone squamous metaplasia (Herbert et al., 1999). Squamous cell carcinomas were formed of nests or cords of squamous epithelial cells with variable degrees of anaplasia and keratin formation (Figure 1G). Squamous cell carcinomas were frequently aggressive and invaded surrounding structures. Squamous cell carcinomas were attributed to administration of 1,4-dioxane (Table 1).

Tumors arising from the olfactory epithelium were quite diverse and the cell of origin difficult to determine without special techniques. A variety of names were applied to these tumors (olfactory neuroblastoma, olfactory carcinoma, esthesioblastoma, esthesioneuroepithelioma, neuroepithelial carcinoma). Because of the diverse morphologic features that may occurred and difficulty in determining the cell of origin, the tumors were often lumped under the heading of neuroblastoma (Figure 1H) although neuroepithelial carcinoma was suggested as a term that would include tumors arising from the olfactory sensory, sustentacular cells, basal cells, or cells from Bowman’s glands (Herbert et al., 1999).

Several types of olfactory neuroblastomas were described (Brown et al., 1991). One type was composed of cells with features of sustentacular cells in solid masses, with gland-like structures or rosettes being formed. A second type had features of neuronal olfactory cells. The cells were basophilic with hyperchromatic nuclei and a large nuclear to cytoplasmic ratio, and formed serpentine bands, sheets of cells, gland-like structures, and/or rosettes. A third type was characterized by confluent growths of endophytically proliferating surface epithelium that formed sheets or lobules of cells that had large nuclei with marginated chromatin and single prominent nucleoli. Neuroblastoma was diagnosed in rats administered p-cresidine in the diet (Table 1), and with N′-nitrosonornicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone given by subcutaneous injection to rats (Hecht et al., 1980).

Rhabdomyosarcomas were observed in low incidence (2/sex for the high dose group) in feed studies with 2,6-xylidine (Table 1). Because of the rarity of this tumor at this location, it was potentially treatment related. A search of the larynx and trachea found little evidence of treatment-related lesions in noninhalation studies. The larynx, however, was rarely examined in noninhalation studies.

Nonneoplastic Lung Lesions

Alveolar histiocytosis (macrophage infiltration of the alveoli) consisted of the accumulation of macrophages in alveoli (Figure 2A). These cells had abundant foamy cytoplasm and were sometimes associated with inflammation, cholesterol cleft formation, an increase in type II cells, or were present in alveoli in response to increased production of surfactants by type II cells. Alveolar histiocytosis was found in control animals but was increased in severity and incidence with the administration of some compounds. Elmiron (Nyska et al., 2002), dimethoxybenzidine dihydrochloride, and titanocene dichloride induced alveolar histiocytosis (Table 2) as well as the drugs amiodarone used for cardiac arrhythmia, ipindole used as an antidepressant, and chlorophentermine used for anorexia (Boorman et al., 1990a).

Alveolar epithelial hyperplasia (AEH) was characterized by a focal increase in type II cells lining the interalveolar septa. The cells were cuboidal and some of the septa were slightly thickened, but normal architecture was maintained (Figure 2B). AEH was associated with chronic inflammation or was a primary lesion where it was considered a precursor to alveolar/bronchiolar adenomas and carcinomas. AEH was associated with administration of o-nitrotoulene, riddelliine, dibromoacetic acid, p-nitrotoulene, 4-methylimidazole, 3,3′-dimethylbenzidine dihydrochloride, and 2,2-bis (bromomethyl)-1,3-propanediol (Table 2).

Bronchiolar metaplasia (BM) of the alveolar epithelium was characterized by metaplasia of alveolar epithelium to respiratory type primarily at the junction of the terminal bronchioles and along alveolar ducts (Figure 2C). The metaplastic cells were cuboidal to columnar, with basally located nuclei, eosinophilic cytoplasm, and cilia. Alveolar duct space and alveolar lumens often contained an amorphous, lightly basophilic to amphophilic material. Periodic Acid-Schiff and Alcian Blue staining indicated that the material was mucuslike. Scattered cells with a smooth apical surface lacking cilia, which were consistent with Clara cells, were sometimes present. These cells were distinct from those seen with alveolar epithelial hyperplasia, which had features of type II alveolar epithelial cells. BM was not associated with progression to neoplasia. In rats, this finding was associated with administration of dioxin or dioxin-like compounds such as PCB 126 and with administration p-nitrotoluene (Table 2). The metaplastic cells were positive for glutathione S-transferase Pi (GSTPi) staining in the PCB 126 study, similar to the staining of normal bronchiolar epithelium, suggesting specific metabolic activity of cells in the metaplastic areas (Brix et al., 2004).

Squamous lesions in the lung associated with inhalation studies were reviewed and classified by a panel of international experts (Boorman et al., 1996). Some of that spectrum of lesions was observed in noninhalation studies. Squamous metaplasia was observed in gavage studies with dioxin and dioxin-like compounds (Table 2) and was characterized by small focal areas of squamous cells in the alveolar areas. This may have represented an early stage in the progression to keratin-filled squamous lesions. These keratin-filled lesions were considered to be a family of lesions with the hallmark being the formation of large keratin-containing cysts. Pulmonary squamous metaplasia (Figure 2D) was characterized by the transition of alveolar epithelial cells to squamous metaplastic cells with distortion but not obliteration of the normal architecture. Keratin formation was evident and inflammation sometimes accompanied the change. Pulmonary keratinizing cyst was a term used to describe a more expansive lesion with distortion of the normal architecture. These keratin-filled cysts were often several centimeters in diameter.

Diagnostic features of pulmonary keratinizing cyst consisted of a cystic structure with a thin uniform wall composed of mature squamous cells that contained various amounts of keratin. Such lesions could be quite large because of the amount of keratin accumulated.

Neoplastic Lung Lesions

Cystic keratinizing epithelioma (Figure 2E) was used as a diagnosis for a benign tumor in this family of squamous lung lesions. This lesion was differentiated from a pulmonary keratinizing cyst by the nature of the wall, which was more complex, thicker, and irregular. The lesion seemed to grow by expanding into contiguous alveoli giving it a roughened irregular surface. This was in contrast to pulmonary keratinizing cysts, which had a thin uniform inner and outer wall. Additionally, mitotic figures and a lack of orderly maturation were features of cystic keratinizing epithelioma, and most lesions had a large keratin-filled cavity. Cystic keratinizing epithelioma was observed in gavage studies with dioxin and dioxin-like compounds (Table 2). Pulmonary squamous cell carcinoma was used as a diagnosis for the malignant form of the lesion. Some arose from cystic keratinizing epitheliomas where focal areas of malignancy occurred in the wall. Other carcinomas lacked keratinization. Squamous cell carcinoma was characterized by invasion of the pulmonary tissue, cellular atypia, lack of normal maturation, increased mitotic activity, and rarely metastasis (Figure 2F).

Other primary pulmonary neoplasms were classified as alveolar/bronchiolar (A/B) adenoma or carcinoma. These tumors had a solid, papillary or mixed pattern and some had areas of squamous or mucinous differentiation. Adenomas were usually distinct masses and usually caused compression of surrounding parenchyma (Figure 2G). In contrast to hyperplasia, the normal alveolar architecture was lost. Neoplastic cells were cuboidal to round or polygonal with finely vacuolated cytoplasm. Carcinomas had a more irregular growth pattern and the neoplastic cells were more pleomorphic (Figure 2H). A/B adenomas and carcinomas were associated with the administration of a number of chemicals (Table 2) by noninhalation routes.