Olfactory Epithelial Metaplasia and Hyperplasia in Female Harlan Sprague–Dawley Rats Following Chronic Treatment with Polychlorinated Biphenyls

Abstract

The National Toxicology Program recently completed a series of studies to evaluate the relative potency for toxicity and carcinogenicity of several polyhalogenated aromatic hydrocarbons including dioxin-like compounds (DLCs) and polychlorinated biphenyls. Female Sprague–Dawley rats were administered by gavage for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3′,4,4′,5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3′,4,4′,5-pentachlorobiphenyl (PCB118); control animals received corn oil-acetone vehicle (99:1) alone. Nasal epithelial changes were observed only in animals exposed for 2 years to the higher doses of the binary mixtures of PCB126 + PCB153 (1000 ng/kg and 1000 ug/kg) and PCB126 + PCB118 (216 and 360 ng TCDD equivalents/kg). In both studies, the changes were of the same nonneoplastic nature, localized to nasal sections II and III located, respectively, at the level of the incisive papilla anterior to the first palatial ridge (section II) and through the middle of the second molar teeth (section III). The changes consisted of hyperplasia of the respiratory epithelium (level II) and metaplasia of olfactory epithelium to respiratory epithelium with further hyperplasia of the metaplastic respiratory epithelium (levels II and III). Variable amounts of acute inflammatory exudate appeared within the lumen of the nasal cavity, overlying the affected epithelium. Occasionally, the inflammation eroded through the skull and into the adjacent olfactory bulbs.

Introduction

The nasal cavity is a site recognized for the development of chemically induced tumors in man and laboratory animals (Haseman and Hailey, 1997; Saracci et al. 1991). In a recent review of the carcinogenic outcome of nearly 500 chemicals tested by the National Toxicology Program (NTP), 12 produced nasal tumors. All of these 12 chemicals were genotoxic, usually affecting both sexes and always producing nasal tumors in the rat; while 5 proved carcinogenic to the mouse nasal cavity as well. Since inflammation, epithelial hyperplasia, and squamous metaplasia can often occur without nasal-cavity carcinogenesis in inhalation studies, investigators concluded that nasal carcinogenesis is generally not associated with the magnitude of chronic toxicity observed at this site. Increased risk for cancers of the nose and nasal cavity was reported in workers exposed to chlorophenoxy herbicides and/or chlorophenols, including those contaminated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), although a causal relationship with TCDD was not confirmed (Saracci et al., 1991).

Even chemicals not administered by inhalation may target the nasal cavity, inducing the development of a range of pathological changes. Significant metabolic enzymatic activity of the lining nasal epithelium imparts the ability to bioactivate or detoxify compounds (Reed, 1993; Nyska and Ghanayem, 2003). Rats exposed by gavage for 2 years to methacrylonitrile, a nongenotoxic and noncarcinogenic compound, developed olfactory epithelial atrophy and metaplasia of the nose (Nyska and Ghanayem, 2003). The location of these lesions was likely related to the species-specific disposition of this compound in the olfactory epithelium, due to the presence of a high content of the metabolizing enzyme, cytochrome P4502E1 (Wang et al., 2002).

Polyhalogenated aromatic hydrocarbons (PHAHs) comprise a large class of compounds, including polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs). Certain PCDDs, PCDFs, and PCBs have the ability to bind to the aryl hydrocarbon receptor (AhR) and exhibit biological actions similar to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); thus, they are commonly referred to as “dioxin-like compounds (DLCs).” The National Toxicology Program recently conducted a series of 2-year bioassays in rats to evaluate the chronic toxicity and carcinogenicity of DLCs, structurally-related PCBs, and mixtures of these compounds. Compounds used for these studies were TCDD; 3,3′,4,4′,5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3′,4,4′,5-pentachlorobiphenyl (PCB118). TCDD has been considered the most potent DLC and the reference compound to which all DLCs are compared in the TEF methodology.

TCDD has a TEF value of 1.0, and PCB126, PeCDF, and PCB118 have values of 0.1, 0.5, and 0.0001, respectively. PCB 126, a non-ortho-substituted PCB, has been deemed the most potent dioxin-like PCB congener present in the environment, accounting for 40–90% of the total toxic potency of PCBs exhibiting “dioxin-like” activity. PeCDF, a dioxin-like PHAH, represents the most potent PCDF in human tissues. PCB118 is a mono-ortho-substituted PCB with partial dioxin-like activity and is currently included in the TEF scheme. In contrast, PCB153, which is a di-ortho-substituted nonplanar PCB, exhibits no dioxin-like activity. Since DLCs act through the AhR, one of the hypotheses tested in these studies was that both individual compounds and mixtures would elicit a similar spectrum of neoplastic and nonneoplastic responses following chronic exposure.

The objective of the present work was to describe the incidences and morphologic aspects of the nonneoplastic changes observed in the nasal cavity in 2 of these carcinogenicity studies conducted by the NTP. These 2 studies involved rats exposed for 2 years to the higher doses of the binary mixtures of PCB126 + PCB 153 and PCB 126 + PCB 118. This report constitutes the first morphologic documentation of hyperplastic and metaplastic changes within the nasal mucosa induced by these PCBs.

Materials and Methods

Study Design

Female Harlan Sprague–Dawley rats were used, since this strain has proven sensitive to the effects of TCDD (Kociba et al. 1978). A range of 50 to 66 animals per group was treated for 2 years with several doses of TCDD; PCB126; PeCDF; PCB153; the TEF tertiary mixture of TCDD, PCB126, and PeCDF; the binary mixture of PCB126 and 153; or the binary mixture of PCB126 and 118 (Table 1). Additional groups that received the highest doses of TCDD, PCB126, PeCDF, PCB153, and the mixture of PCB126 and 118 for 30 weeks, followed by vehicle treatment through the termination of the 2-year study, were designated stop groups. Animals were dosed once daily for 5 days per week by oral gavage. The doses of all compounds were based on the TEF values selected by the World Health Organization (WHO) (Van den Berg et al., 1998) (Table 1).

Chemicals

Dose formulations of TCDD (The IIT Research Institute, Chicago, IL), PCB 126 (AccuStandard, Inc., New Haven, CT), PeCDF (Cambridge Isotope Laboratories, Cambridge, MA), PCB153 (Radian International LLC, Austin, TX), and PCB118 (Radian International LLC, Austin, TX) were prepared for administration by gavage by formulating the test chemical in a corn-oil vehicle containing 1% USP-grade acetone. Purities of TCDD, PCB126, and PeCDF, and PCB153, were determined to be approximately 98%, 99.51%, 97%, and 99.8% respectively, with no change in purity observed over the duration of the studies. The PCB 126/PCB118 “mixture” was bulk synthesized PCB118 that was determined to be greater than 98.5% pure, but contained 0.622% PCB126 as a synthesis contaminant.

Dose formulations were prepared monthly for gavage administration by mixing the test chemical in a corn oil vehicle containing 1% USP-grade acetone. The corn oil was analyzed by potentiometric titration and the acetone by infrared spectroscopy. Homogeneity and stability studies of dose formulations indicated that chemicals could maintain an acceptable homogeneity for dosing and stability for 35 days when stored at room temperature. Dose formulations were analyzed at least every 3 months and were generally within 10% of the target concentrations. For the TEF mixture, the dose formulations were prepared by mixing volumes of the TCDD, PeCDF, and PCB 126 formulations. For the PCB 126/153 mixture the dose formulations were prepared by mixing volumes of the PCB126 and PCB153 formulations.

Animals

All experiments, for the duration of these studies, were conducted in the AAALAC-accredited facility of Battelle-Columbus Laboratories (Columbus, OH). Animal handling and husbandry met all NIH guidelines (Institute of Laboratory Animal Resources, 1996). Female Harlan Sprague–Dawley rats were approximately 8 weeks of age at the start of the study. Animals were randomly assigned to control or treated groups and housed 5 to a cage in solid-bottom polycarbonate cages (Lab Products, Inc., Maywood, NJ). The animal rooms were maintained at 69–75°F with 35–65% relative humidity and 12 hours each of light and darkness. Irradiated NTP-2000 pelleted feed (Zeigler Bros., Inc., Gardner, PA) and water were available ad libitum.

Pathology

Moribund and all scheduled-to-be-sacrificed animals were euthanized by carbon dioxide. Complete necropsies were performed on all animals using standardized methodology. At necropsy, all tissues, including masses and macroscopic abnormalities, were removed and fixed in 10% neutral buffered formalin. The maxillae, including the nose, were decalcified in a 5% Nitric Acid Decal Solution (Poly Scientific, Inc., Bay Shore, NY) for 3 days. Three nasal sections that included oral tissues were examined. The maxilla was trimmed immediately posterior to the upper incisor teeth (section I), through the level of the incisive papilla anterior to the first palatial ridge (section II), and through the middle of the second molar teeth (section III) (Boorman et al., 1990). After fixation and/or decalcification, all of the tissues were trimmed, dehydrated, cleared, embedded in paraffin, sectioned into 5-μm-thick sections, stained with hematoxylin and eosin (H&E), and examined microscopically. The severity of lesions was graded on a 4-point scale of 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked. The pathology results underwent comprehensive NTP peer review by Pathology Working Group pathologists (Boorman et al., 2002).

Statistical Analysis

The probability of survival was estimated by the product-limit procedure of Kaplan-Meier (Kaplan and Meier, 1958). Associations between dose and incidences of lesions were evaluated statistically by the poly-3 test (Bailer and Portier, 1988; Portier and Bailer, 1989), which adjusts for survival differences among groups. For animals in the 2-year studies, the incidences of all lesions, including findings from animals that survived until study termination and early-death animals, were included in the analysis.

Results

The individual animal data for all endpoints, including the nasal pathology reported in this paper are available on the NTP web site 〈http://ntp-apps.niehs.nih.gov/ntptox/index.cfm〉, and are summarized in individual NTP technical reports on the evaluation of the carcinogenicity of each agent tested (NTP, 2004a; NTP, 2004c). Treatment-related lesions in the nasal cavity were noted only in the animals exposed for 2 years with the PCB binary mixtures of PCB126 + PCB153, and PCB126 + PCB118. No significant effect on nasal pathology was observed in the studies of TCDD, PCB126 alone, PeCDF, PCB153, or the mixture of TCDD, PCB126, and PeCDF-The incidences of the lesions noted in this organ are presented in Tables 2 and 3. In both studies, the changes were of the same nonneoplastic nature and localized to sections II and III (Boorman et al., 1990). The changes consisted of inflammation, respiratory epithelial hyperplasia, and metaplasia of the olfactory epithelium into the respiratory epithelium (Figures 1–4). Within these 2 studies increased incidences of these lesions were seen only in animals treated with 1000 ng/kg PCB126 + 1000 μg/kg PCB153, and 216 ng TEQ/kg of PCB126 + PCB118. The relatively lower incidence in the group administered 360 ng TEQ/kg PCB126 + PCB118 compared to the one given 216 ng TEQ/kg is considered to be related to the low survival and early deaths observed in this dose group.

Respiratory epithelial hyperplasia was seen in the epithelium lining the nasal septum, nasoturbinates at level II, and ethmoid turbinates at level III. It was characterized by increased numbers of columnar cells and cells containing an abundance of mucin, formation of multifocal short papillary projections and a series of infoldings and/or “crypt-like” invaginations (Figures 1 and 2). The mucosa and underlying submucosa were infiltrated by mixed population of polymorphonuclear and mononuclear inflammatory cells. In both sections II and III, the respiratory epithelium hyperplasia was accompanied by prominent dilated submucosal glands, some lined by hyperplastic epithelium within which prominent, dilated openings of glandular ducts could be seen.

Metaplasia of the olfactory epithelium, which became altered to resemble respiratory epithelium was noted in the epithelium lining the nasal septum at levels II and III, and in the nasoturbinates (level II) and ethmoid turbinates (level III). It consisted of replacement of normal olfactory epithelium by respiratory-type epithelium with varying degrees of hyperplasia of the metaplastic respiratory epithelium. The hyper-plasia in section III was similar to but generally more severe than that seen in section II, with the hyperplastic epithelium often forming irregular, generally ramifying, papillary projections (Figures 3 and 4).

Variable amounts of acute inflammatory exudate were seen within the lumen of the nasal cavity, overlying the affected epithelium. Occasionally, the inflammation eroded through the skull and into the adjacent olfactory bulbs. The presence of inflammation within the nasal cavity lumen extending into the mucosa and sometimes to the brain was a prominent feature in these NTP studies. Acute inflammation of the nasal mucosa is a common sequelum of toxic insult, possibly attributable to opportunist infections following enhanced susceptibility due to metaplastic changes and/or atrophy of lymphoid tissue (Monticello et al., 1990).

Discussion

In this paper we compare the effects of chronic exposure to dioxin like compounds and binary mixtures containing PCBs on the nasal cavity in the female Harlan Sprague–Dawley rat. Our data indicate that the respiratory epithelium lining the nasoturbinates, and the olfactory epithelium lining the ethmoturbinates were target tissues of these PCB mixtures, inducing hyperplastic and metaplastic lesions only at high doses. Effects on the nasal epithelium were not seen in the studies of the dioxin-like compounds alone or in the study of PCB153 alone.

Testing of the toxicity of industrial and environmental chemicals has revealed that lesions are often confined to specific, distinct areas of the nasal cavity. Ozone induces lesion formation in the transitional epithelium, respiratory epithelium of the lateral wall, and olfactory epithelium, and lesions caused by methacrylonitrile are localized in the olfactory epithelium (Reed, 1993; Herbert et al., 1996; Nyska and Ghanayem, 2003). Regional selective damage within the nasal cavity may be related to inspiratory airflow patterns or the regional presence of elevated levels of cytochrome P450 (Voigt et al., 1993). An increase in cytochrome CYP1A1 and CYP1A2 characterizes a hallmark response to DLCs and is directly linked to binding and activation of the AhR (Whitlock, 1993). The liver is the principal target organ for toxicity of dioxin like-compounds, and this is likely due to the high retention of dioxin-like compound within the liver, relative to other organs. The hepatic sequestration of DLCs is due to binding to CYP1A2, which is itself induced by DLCs (Diliberto et al., 1997). Studies of the bio-distribution of radiolabeled [¹⁴C]TCDD in mice and rats have shown a selective localization of the radioactivity in not only the liver and nasal mucosa, without, however being covalently bound to the latter (Appelgren, 1983; Gillner et al., 1987). In addition region selective localization of CYP1A2 has been observed in the olfactory epithelium (Ling et al., 2004). It is believed that like the CYP1A2 mediated hepatic sequestration of DLCs, the localization in the nasal region is due to CYP1A2 binding. These data suggest that the toxic effects on the nasal epithelium may be the result of a direct action of DLCs in this tissue as a result of CYP1A2 mediated sequestration.

The observation that these effects were seen only in the PCB126/118 and the PCB126/153 mixture studies may be due to the fact that the PCB 126/118 studies had the highest administered dose levels (on a TEQ basis) of all the studies conducted as part of the NTPs dioxin TEF evaluation. For example the 216 ng TEQ/kg PCB126/118 group was over twice as high as the highest doses used (on a TEQ basis) in the studies of TCDD PeCDF, PCB126, and the mixture of TCDD, PeCDF, and PCB126. As such the observation of these lesions may simply reflect an increasing dose response.

For the PCB 126/153 study the same administered dose of PCB 126 was used as was used in the study of PCB 126 alone; a study in which no significant effects were seen in the nasal epithelium. However in the liver in the PCB 126/153 mixture study, there was a higher degree of hepatic toxicity and higher incidences of hepatic neoplasms (NTP, 2004b, NTP, 2004c) when compared to effects seen with PCB126 alone at the same administered dose. This was despite the fact that at high doses of PCB 153 there were lower levels of PCB 126 in the liver, and lower levels in the lung, fat and blood (when compared to the study of PCB 126 alone) (NTP, 2004c). Tissue levels in the nasal cavity were not measured in any of these studies. Assuming similar CYP1A2 mediated sequestration in the liver and nasal passage it is likely that the tissue levels in the nasal region were also lower than would be expected with PCB 126 alone at the same administered dose. This suggests that tissue concentration per se may not be the best dose metric related to pathological responses and that some subset of the total tissue dose, e.g., free, non-CYP1A2-bound PCB126 may the biologically effective dose of concern. Given the higher hepatotoxicity in the PCB126/153 study and the common role of CYP1A2 in the distribution of DLCs like PCB126 to the liver, the effects presented here in the PCB126/153 study may be due to a similar higher biologically effective dose of PCB126 in the nasal epithelium, i.e., the effects in both the PCB126/118 are due to the combined PCB126/118 TEQ driven biologically effective dose and the effects in the PCB126/153 study due to a higher PCB126 biologically effective dose. Alternatively it is known that mono-ortho and di-ortho PCB have non-AhR-mediated actions. It is possible that these non-AhR activities may to enhance the effectiveness of the PCB126-mediated effects, at the same biologically effective dose of PCB126 that one would have if PCB126 were administered alone.

The nasal mucosa, like the liver and kidney, possesses the capacity to promote development of chronic regeneration of damaged tissues following toxic insult causing degenerative and inflammatory changes, without the development of tumors. A list has been compiled Haseman and Hailey presenting chemicals tested in the NTP 2-year studies in which significant increases in acute inflammation, epithelial hyperplasia, and metaplasia were noted in the nasal mucosa, without development of cancer at this site (Ward et al., 1993; Haseman and Hailey, 1997). Nasal carcinogenesis was not associated with the magnitude of chronic toxicity and cellular proliferation at this site, but, rather, with positive genotoxicity. The lack of carcinogenesis resulting from chronic cellular proliferation may be related to the lack of involvement of stem cells, which are important targets for carcinogens, or the fact that some mutations are time-dependent instead of replication-related (Ward et al., 1993). With respect to this it should be noted that TCDD and related DLCs are not direct acting genotoxic agents. However they do have the ability to induce oxidative stress and hepatic oxidative DNA damage after prolonged exposure (Hassoun et al., 2000, 2001; Wyde et al., 2004).

Respiratory epithelial metaplasia of the olfactory epithelium encompasses atrophy and degeneration of the olfactory epithelium, with loss of sensory and sustentacular cells, and replacement by ciliated and nonciliated epithelium (Greaves, 2000). Disturbed differentiation related to TCDD exposure, leading to metaplasia of different types of cells, was shown in the epidermis, teeth, cervix, and lungs (Greenlee et al., 1985; Enan et al., 1998; Tritscher et al., 2000; Kiukkonen et al., 2002; Brix et al., 2004). That the mechanism of metaplasia is related to the presence of AhR specifically in the affected cells was suggested; TCDD may modulate normal patterns of cellular differentiation through direct actions on proliferating basal cells, altering the responsiveness of these cells to growth factors, such as epidermal growth factor (Greenlee et al., 1985).