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编号:11154580
Gingival Carcinogenicity in Female Harlan Sprague-Dawley Rats following Two-Year Oral Treatment with 2,3,7,8-Tetrachlorodibenzo-p-dioxin and
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     ABSTRACT

    We evaluated gingival toxicities induced by chronic exposure of female Harlan Sprague-Dawley rats to dioxin and dioxin-like compounds (DLCs) and compared them to similarly induced oral lesions reported in the literature. This investigation represents part of an ongoing initiative of the National Toxicology Program to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. For two years, animals were administered by gavage 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. A full complement of tissues, including the palate with teeth, was examined microscopically. In the groups treated with TCDD and the mixtures of TCDD, PCB126, and PeCDF; PCB126 and 153; and PCB126 and 118, the incidences of gingival squamous hyperplasia increased significantly. Moreover, in the groups treated with TCDD, PCB126, and the mixture of PCB126 and 153, squamous cell carcinoma (SCC) in the oral cavity increased significantly. This investigation constitutes the first report documenting that chronic administration of dioxin-like PCBs can induce gingival SCC in rats. These results indicate that dioxin and DLCs target the gingiva of the oral cavity, in particular the junctional epithelium of molars.

    Key Words: gingival squamous hyperplasia; squamous cell carcinoma; rat; dioxin; dioxin-like compounds.

    INTRODUCTION

    Polyhalogenated aromatic hydrocarbons (PHAHs) comprise a large class of compounds including polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), and polybrominated diphenyl ethers (PBDEs). Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), commonly termed dioxin, receives the most attention. Certain PCDDs, PCDFs, and coplanar PCBs have the ability to bind to the aryl hydrocarbon receptor (AhR) and exhibit biologic actions similar to those of TCDD; they have been commonly designated dioxin-like compounds (DLCs). Dioxin and DLCs enter the environment primarily through pyrolysis activities, at sites of municipal, hospital, and hazardous-waste incineration and metal smelting and refining, and as unintentional trace contamination formed during the manufacture, use, and disposal of chlorinated organics (Bosetti et al., 2003; Brambilla et al., 2004; Brouwer et al., 1998). They may induce developmental, endocrine, and immunological toxicity and multi-organ carcinogenicity in animals and/or humans (ATSDR, 1998, 2000; Brouwer et al., 1998; Huff et al., 1994; McGregor et al., 1998; Steenland et al., 2001; United States Environmental Protection Agency, 1996).

    The incidences of cancer have been evaluated in several human populations that received elevated exposures to TCDD and DLCs (ATSDR, 1998, 2000). A study in Seveso, Italy, indicated that exposure apparently induced an increase in all cancers combined and several specific cancers, such as lung cancer, Hodgkin's disease, non-Hodgkin's lymphoma, and myeloid leukemia (Bertazzi et al., 2001). More than 2000 people each in Japan (1968) and Taiwan (1979) were reported to ingest accidentally PCBs and PCDFs in rice bran oil (Asahi, 1993; Guo et al., 1999; Miller, 1985; Wang et al., 2003); follow-up studies indicated increased mortality chiefly from liver cancer and other liver disease (McGregor et al., 1998). Several reports discuss whether exposure to PCBs causes an increased incidence of human cancer (Kimbrough, 1985; McGregor et al., 1998), and experimental studies provide meaningful evidence that they exert probable carcinogenic effects in humans (ATSDR, 2000; United States Environmental Protection Agency, 1996). Although much evidence exists of induction of cancer in humans by DLCs, a conclusive link between these compounds and increased incidences of oral tumors has not been established.

    Recently, the NTP conducted two-year bioassays in female rats to evaluate the chronic pathology and carcinogenicity induced by dioxin and DLCs, structurally-related PCBs, and mixtures of these compounds, such as TCDD; 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachloro-dibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); the Toxic Equivalency Factor (TEF) tertiary mixture of TCDD, PCB126, and PeCDF; and the binary mixtures of PCB126 and 153 and PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118) (National Toxicology Program, 2004a,b,c,d,e,f,g). In these studies, in several tissues, a significant increase occurred in the incidence of neoplastic and nonneoplastic effects, such as cholangiocarcinoma and/or hepatocellular adenoma of the liver and cystic keratinizing epithelioma of the lung (Brix et al., 2004; Jokinen et al., 2003; National Toxicology Program, 2004a,b,c,d,e,f,g; Nyska et al., 2004; Tani et al., 2004; Walker et al., submitted manuscript). Additionally, the incidences of gingival squamous hyperplasia (GSH) and/or squamous cell carcinoma (SCC) were increased in all studies except PCB 153. Ten chemicals that induce oral tumors in rats have been reported by the National Toxicology Program (NTP) (Table 1, NTP Web Site: http://ntp-server.niehs.nih.gov/htdocs/pub.html).

    Study design. The original studies comprised part of a series of analyses undertaken by the NTP to determine the suitability of the TEF methodology for predicting chronic toxicity and carcinogenicity of TCDD and DLCs. Female Harlan Sprague-Dawley rats were used, since the Sprague-Dawley rat has proven sensitive to the effects of TCDD (Kociba et al., 1978). A range of 50 to 66 animals per group was treated for two years with several doses of TCDD; PCB126; PeCDF; PCB153; a TEF tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 118 (Tables 2 and 3). 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 two-year study, were designated stop-exposure groups. Animals were dosed once daily for five days per week by oral gavage. The doses of all compounds were based on the TEF values selected by the World Health Organization (Van den Berg et al., 1998) (Table 2). 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 present in human tissues. PCB118 is a mono-ortho-substituted PCB with partial dioxin-like activity; controversy exists, however, over whether mono-ortho-substituted PCBs should be included in the TEF methodology. In contrast, PCB153, which is a di-ortho-substituted nonplanar PCB, exhibits no dioxin-like activity and, therefore, merits no inclusion in the TEF methodology (Van den Berg et al., 1998).

    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 mixing the test chemical in a corn-oil vehicle containing 1% USP-grade acetone.

    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 (Grossblatt, 1996). Female Harlan Sprague-Dawley rats were approximately eight weeks of age at the start of the study. Animals were randomly assigned to control or treated groups and housed five 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 h 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 three days. Three nasal sections that included oral tissues were examined. The maxilla was trimmed just posterior to the upper incisors (Section I), midway between the incisors and first molar at the anterior surface of the incisive papilla (Section II), and at the middle of the first molar (Section III). 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 four-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). The tongue and mandible were not routinely examined histopathologically, and no gross abnormality was observed in these organs.

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

    RESULTS

    Several reports have dealt with oral-cavity lesions related to PCB exposure in animals or humans (Guo et al., 1999; Hashiguchi et al., 1987, 1995, 1997; Miller, 1985; Shimizu et al., 1992; Wang et al., 2003; Yamashita and Hayashi, 1985). In the adult human exposed to PCBs and PeCDFs, elongation of the depth of periodontal pockets, gingival pigmentation (Hashiguchi et al., 1995), and the sensation of "elevated" teeth (Yoshimura and Hayabuchi, 1985) occurred. Recently, the concentration of PCBs in saliva appeared to be one of the important risk factors for periodontal disease (Ogawa et al., 2003). PCB126 induced oral lesions in animals includes GSH in mink (Render et al., 2000a, 2001) and GSH, hyperkeratosis, dyskeratosis, keratocystic formation, squamous metaplasia of the ameloblast surrounding unerupted teeth, and/or hyperpigmentation in the monkey (Hashiguchi et al., 1983, 1987; McNulty, 1985; Tryphonas et al., 1986; Yoshihara et al., 1979). These lesions, however, were not observed in longer-exposure studies of PCBs in rats and monkeys (Arnold et al., 1999; Chu et al., 1994, 1995; Hori et al., 1982; Kimbrough, 1985; Schaeffer et al., 1984). Although several reports have indicated that exposure to PCBs causes an increased incidence of cancer in animals and possibly humans, no reports have documented the increased incidence of oral tumors as a result of such exposure (ATSDR, 2000; Kimbrough, 1985; United States Environmental Protection Agency, 1996).

    The location of the appearances of these oral lesions merits discussion. In our studies, we detected them lesions within the area of the molar-tooth ligament (tooth pocket, gingival sulcus). In the rat, the gingiva consists of a keratinized stratified squamous epithelium, connective tissue with fibroblasts, and the extracellular matrix composed chiefly of collagen fibers and ground substance containing sulfated glycosaminoglycans (Brunet et al., 1996; Haschek and Rousseaux, 1998; Tintari, 1983). The gingival epithelium in the molar-tooth area is classified as gingival oral epithelium, sulcular epithelium, and junctional epithelium, which appears nonkeratinized and forms the floor of the gingival sulcus. Gingival epithelium manifests a higher proliferative capacity and higher rate of absorption of drugs and chemicals than the skin (Haschek and Rousseaux, 1998; Shojaei, 1998). Mitotic activity appears greatest at the dento-gingival junction of molars, especially within the junctional epithelium (L?e et al., 1972; Shimono et al., 2003; Watanabe et al., 2004). Absorption of chemicals and the conversion by cytochrome P450 proteins to xenobiotic metabolites can occur in gingival epithelia (Vondracek et al., 2001). Our literature search revealed that, in animals, the molar teeth and their gingivae seem to be most sensitive to dioxin-induced toxicity. The junctional epithelium of molars, with high proliferative and metabolic activity, may change pathologically and constitute the earliest gingival change induced by TCDD and some kinds of DLCs.

    Reports implicate several underlying mechanisms of chemical induction of GH. Three classifications of drugs administered to humans–calcium channel blockers (e.g., nifedipine), immunosuppressants (e.g., cyclosporine), and anticonvulsants (e.g., phenytoin)—comprise the main causative agents of drug-induced gingival hyperplasia (GH) (Abdollahi and Radfar, 2003; Brunet et al., 1996; Butler et al., 1987; Guggenheimer, 2002). In association with lesions induced by phenytoin and cyclosporine, the occurrence of oral SCC has also been reported (McLoughlin et al., 1995; Varga and Tyldesley, 1991). Bacteria-associated inflammation manifested as gingivitis and the appearance of sulcular epithelium of the teeth have played essential roles in some cases of drug-induced GH (Brown et al., 1991). Although gingivitis appeared in all dosed groups in our rat studies, we observed no significant differences in its occurrence between control and dosed groups. Gingivitis with hair impaction has been occasionally observed in rat toxicity studies (Brown and Hardisty, 1990). A direct action upon the gingival epithelium, rather than the development of gingivitis, appears therefore to have played a key role in the induction of GSH in our rat studies. Indirect alterations in retinoid homeostasis in the liver may constitute another possible mechanism for the action of DLCs in the oral cavity. In rodents, mobilization of retinoid stores by TCDD and DLCs leads to a disruption in retinoid homeostasis, as well as vitamin A deficiency (Fattore et al., 2000; Fiorella et al., 1995; Schmidt et al., 2003; Van Birgelen et al., 1994, 1995). Abnormal epithelial differentiation creating a keratinized squamous phenotype characterizes retinoid deficiency (Lancillotti et al., 1992; Lotan, 1994). The action of DLCs may therefore involve a disruption of retinoid action leading to altered growth and differentiation of the oral gingival epithelium that result in development of GSH and, ultimately, neoplasia. Since the mechanisms of dioxin- and DLC-induced GSH and SCC remain to be elucidated, concentration on gene- and protein-related functions could enhance understanding of the pathogenesis of oral lesions. Additional research is needed to analyze the mechanism(s) of this induction and provide understanding of the potential extrapolations to humans of dioxin-induced oral lesions.

    ACKNOWLEDGMENTS

    The authors would like to thank all those involved in the design and conduct of these NTP studies, with special thanks to John Bucher, Rick Hailey, Angelique Braen (nee Van Birgelen), and Milton Hejtmancik. We gratefully acknowledge Dr. Rodney Miller, Dr. Adriana Doi, and Ms. JoAnne Johnson for critical review of the manuscript and Norris Flagler for expert preparation of the illustrations. The authors declare that they have no competing financial interests.

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