* Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and
Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599
Received March 12, 2002; accepted July 8, 2002
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ABSTRACT |
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Key Words: Eker rat; renal; drinking water; disinfection by-products; mixtures; kidney.
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INTRODUCTION |
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A rodent model of hereditary renal cancer was utilized to evaluate the carcinogenicity of a mixture of DBPs and to determine whether the mixture enhanced the carcinogenic effects of individual DBPs. The Eker rat model is characterized by a spontaneous germ-line mutation in the tuberous sclerosis complex (Tsc2) tumor-suppressor gene, which predisposes the animals to develop multiple spontaneous renal tumors as early as four months of age (Kobayashi et al., 1995; Yeung et al., 1994
). As a result, Eker rats are highly susceptible to the effects of renal carcinogens (Walker et al., 1992
). The utility of this model is that proliferative lesions can be identified in the kidney and counted microscopically. Because these lesions progress to tumors, they can be quantitated to predict tumor outcome. Potassium bromate (KBrO3), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform (CHCl3), and bromodichloromethane (BDCM) were selected for analysis because they are renal carcinogens and/or nephrotoxicants with previously defined modes of action or characterized mechanisms of toxicity (Table 1
).
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MX is one of the most potent mutagenic by-products identified in drinking water (Kronberg and Vartiainen, 1988; Meier et al., 1987
) and accounts for up to 70% of the total in vitro mutagenicity of chlorinated drinking water (Richardson, 1998
). MX is not a renal carcinogen when administered to male and female Wistar rats in the drinking water for 104 weeks (Komulainen et al., 1997
). However, MX is a nephrotoxicant in male Wistar rats, causing renal tubular damage (Komulainen et al., 1994
). MX is a direct-acting mutagen in several Salmonella typhimurium strains in the Ames test and is genotoxic in cultured mammalian cells (Meier et al., 1987
).
CHCl3 and BDCM are two of the most prevalent trihalomethanes in drinking water disinfected either by chlorination or by ozonation processes that involve posttreatment with chlorine or chloramines. CHCl3 is a nephrotoxicant and renal carcinogen in male Osborne-Mendel rats when given in the drinking water (Jorgenson et al., 1985). The proposed carcinogenic mode of action requires the metabolism of CHCl3 by cytochrome P450 (CYP2E1) in renal epithelial cells forming cytotoxic oxidative metabolites, resulting in sustained cytotoxicity and regenerative cell proliferation with subsequent tumor development (Reitz et al., 1982
). CHCl3 is not considered to be mutagenic or genotoxic (Larson et al., 1994
; Rosenthal, 1987
). BDCM is one of the most potent rodent carcinogens among the trihalomethanes. BDCM increased the incidences of tubular cell adenomas and adenocarcinomas in the kidneys of male and female F344 rats and male B6C3F1 mice when administered by corn oil gavage for 102 weeks (Dunnick et al., 1987
). BDCM did not induce kidney tumors in male F344 rats or B6C3F1 mice when administered in the drinking water for 104 weeks at concentrations up to 0.7 g/l in rats and 0.5 g/l in mice. However, a significant increase in renal tubular cell hyperplasia was observed in male rats at 0.7 g/L BDCM (George and DeAngelo, 1999
). There are conflicting reports of the mutagenicity and genotoxicity of BDCM in the literature (Dunnick et al., 1987
; Morimoto and Koizumi, 1983
; Pegram et al., 1997
).
The present study was designed to examine the potential carcinogenicity in the Eker rat of a mixture of DBPs that act on the same target organ and to characterize the nature of the interaction of these compounds.
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MATERIALS AND METHODS |
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The actual chemical concentrations and stability of the representative drinking water solutions were monitored monthly throughout the study. Drinking water samples were collected monthly for chemical analysis from the carboys holding the freshly prepared solutions and from water bottles removed from the animal cages. The solutions were pipetted into 7-ml liquid scintillation minivials that were capped tightly without any headspace and were stored at 5°C. The drinking water solutions were analyzed to determine KBrO3 concentrations according to EPA Method 300.0, part B (U.S. EPA, 1996) using a Waters high-pressure liquid chromatography system equipped with a Dionex CD-20 conductivity detector with a DS-3 conductivity cell and an ASRS-II anion self-regenerating suppressor module. MX concentrations were determined according to published methods (Coleman and Munch, 1991; Ogawa et al., 1993
) and CHCl3 and BDCM concentrations were determined using Standard Method 6232B (American Public Health Association, 1998
). Drinking water samples were analyzed for MX, CHCl3, and BDCM concentrations using a 5890 series II Hewlett Packard gas chromatography system equipped with an electron capture detector and a DB-5 column. The target chemical concentrations are shown in Table 2
, and the calculated chemical consumption for each group is shown in Table 3
.
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Fixed kidney tissues, gross lesions, and other tissues of interest were processed by routine methods to 5-mm paraffin sections and stained with hematoxylin and eosin for histological examination by light microscopy. A midsagittal section of each kidney was examined microscopically for tumors (adenomas and carcinomas). A renal adenoma is a solid or cystic focus of altered tubular epithelial cells the size of 3 average tubules and <10 mm in diameter, and may or may not breach the basement membrane. A renal carcinoma is a solid or cystic focus of altered tubular epithelial cells that is
10 mm in diameter. All slides were examined without knowledge of treatment group. The severity of chronic progressive nephropathy was graded semiquantitatively, based on the percentage of the renal cortex involved: 0 = no nephropathy, 1 = 110% of the renal cortex involved, 2 =
25%, 3 =
50%, 4 =
75%, 5
75% of the renal cortex involved with fibrosis and mineralization present.
Data analysis.
Data were analyzed using a one-way analysis of variance (ANOVA) to compare means against control followed by the least-square means test. In addition, the Dunnetts test was used to compare the data from the treatment groups against their respective controls and the Tukey-Kramer test was used for multiple comparisons among the treatment groups.
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RESULTS |
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Nonneoplastic pathology.
No significant changes were observed in the severity of chronic progressive nephropathy between any groups of either sex (data not shown). Transitional cell hyperplasia and karyomegaly were present in the urinary bladders from 7/7 females and in 4/8 (hyperplasia) and 5/8 (karyomegaly) males treated with the high dose of MX (Table 4). These lesions were less prevalent in females (4/14) and males (2/14) treated with the high-dose-mixture solution. Additional lesions in male Eker rats in the present study included mesothelial hypertrophy on the testicle of 1/8 males treated with the high dose of KBrO3. Centrilobular swelling was present in the livers of 5/8 males treated with the high dose of BDCM, 7/14 males treated with the low-dose mixture, and 1/14 males treated with the high-dose mixture. In addition to centrilobular hypertrophy, 3/8 high-dose BDCM, 3/14 low-dose mixture, and 4/14 high-dose mixture-treated male rats had clear cell foci of cellular atypia. One male (1/8) treated with the high dose of BDCM and one male (1/14) treated with the low dose mixture had basophilic foci. None of these lesions were present in control rats.
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Neoplastic pathology.
Each kidney was examined macroscopically and microscopically for the presence and number of renal adenomas and carcinomas. After four months of treatment, nearly all of the male rats had at least one renal tumor. For the CHCl3, BDCM, and mixture groups, there were more adenomas and total tumors in the high-dose than in the low-dose groups, but the differences were not statistically significant (Table 5). For KBrO3, the renal tumor response was similar between the low and high dose groups (Table 5
). For MX, more tumors were present in the low dose compared to the high dose group (Table 5
). No significant increases were observed in adenomas or carcinomas in any treatment group compared to control animals after four months of treatment (Table 5
).
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DISCUSSION |
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The potential of chemical mixtures to enhance cancer induction in exposed populations is an important consideration in human health risk assessment. Most studies of drinking water mixtures assess the toxicity of binary or tertiary mixtures of disinfection by-products. These studies have demonstrated increased toxicity and carcinogenicity (Pereira et al., 1997) above that expected for single chemicals and provided information about the potential interaction of DBPs in simple defined mixtures (Teuschler et al., 2000
). Other studies have assessed the toxicity or carcinogenicity of real drinking water samples obtained from public utility systems (Condie et al., 1994
) or whole mixtures produced by simulating disinfection scenarios. A carcinogenicity study was conducted in F344 rats that were exposed to concentrated drinking water supplied by the Denver Potable Water Department Reuse Plant. The plant provided 500-fold concentrates of drinking water that had been treated by reverse osmosis or ultrafiltration to remove microbial and chemical contaminants. Administration of drinking water concentrates for up to 104 weeks did not result in any overt toxicological or carcinogenic effects. While these types of studies assess the low dose effects of complex mixtures of DBPs, the chemical composition of the drinking water samples is often not well characterized.
The National Toxicology Program evaluated the toxicity of a chemically defined mixture of 25 common groundwater contaminants frequently found near toxic waste dumps. Male and female F344 rats and B6C3F1 mice were exposed for 26 weeks to a technically achievable stock solution of the 25-chemical mixture at various concentrations in drinking water. Rats developed inflammatory lesions in the liver, spleen, lymph nodes and adrenal gland (NTP, 1993). In addition, the mixture was shown to be immunosuppressive in female mice (Germolec et al., 1989
). A subsequent 14-day drinking water study with a 1:10 dilution of the 25-chemical stock solution demonstrated little or no toxicity or histopathology in the livers and kidneys of adult male F344 rats exposed to the chemical mixture (Simmons et al., 1994
).
The carcinogenic effects of a mixture of four DBPs were not additive for any endpoint examined. In fact, the renal tumor response to the high dose mixture was often less than that for the individual compounds. Male and female Eker rats responded similarly, indicating the absence of a sex difference in the tumor response to this chemical mixture. Although the high dose mixture produced the greatest number of renal cell tumors in female rats after four months of treatment, the response was not additive. Because the carcinogenicity of CHCl3 was not assessed in female Eker rats after four months of treatment, the potential contribution of CHCl3 to the tumorigenic response of the mixture is not known. The lack of additivity was unexpected given the present knowledge of the mode of action of these compounds.
KBrO3 and CHCl3 are both renal carcinogens in other rat strains when administered individually via the drinking water route, so the effect of the two chemicals given together could be additive. One plausible mechanism of chemical interaction could involve cytotoxicity or production of initiated cells by KBrO3 or MX with subsequent stimulation of cell proliferation by promoting agents such as CHCl3 or BDCM. MX, for example, is a direct acting mutagen, and mice receiving MX orally and tetradecanoyl phorbol acetate (TPA) topically had a statistically significant increase in both the percent incidence of tumors and the number of tumors per mouse, indicating the ability of MX to act as an initiator (Meier et al., 1990). KBrO3 and CHCl3 were not renal carcinogens in the Eker model. Although the high dose mixture solution containing these DBPs was carcinogenic in Eker rats after 10 months of treatment, the response to the mixture was not greater than the additive response of the individual chemicals or the chemical producing the greatest response. Since CHCl3 and BDCM are both metabolized by cytochrome P450 CYP2E1 (Guengerich et al., 1991
; Thornton-Manning et al., 1993
), it is possible that one compound inhibited the metabolism of another, contributing to the lack of additivity. Alternatively, an increased carcinogenic response may be observed with a longer period of chemical exposure. Two time points were utilized to accurately determine the renal carcinogenicity of the DBPs. Only female rats treated with the high dose mixture had a statistically significant tumor response after four months of treatment (6 months of age). In contrast, a statistically significant tumor response was observed in both male and female Eker rats after 10 months of treatment.
The results from this study suggest that the default assumption of additivity may overestimate the carcinogenic effect of chemical mixtures in drinking water. This observation is consistent with studies that demonstrate a lack of additivity with chemical carcinogens that act on the same target organ through similar or dissimilar modes of action. Elashoff et al. (1987) evaluated four hepatocarcinogens in a factorial experiment to determine if liver tumor production was synergistic. Of the 6 possible combinations, only one combination was found to interact synergistically to induce liver tumors. Likewise, combinations of five carcinogenic environmental polycyclic aromatic hydrocarbons (PAH) were evaluated in strain A/J mice to characterize the lung tumor response. The majority of the chemical interactions were less than additive for lung adenoma formation indicating that although interactions between PAHs do occur, they are limited in extent (Nesnow et al., 1998). Carpy et al. (2000) concluded that exposure to a combination of compounds does not cause effects stronger than the ones of their most active compound, provided components are present at low concentration levels. Similarly, DeMarini (1998) concluded that the mutation spectra of complex mixtures may reflect the dominance of one or a few chemical classes within the mixture.
The Eker rat is predisposed to develop multiple spontaneous renal cell carcinomas, splenic hemangiosarcomas, and uterine leiomyosarcomas due to an insertion mutation in the Tsc2 gene. Since Eker rats are genetically susceptible to renal tumor development, one of the objectives of this study was to determine if they were also more sensitive to the carcinogenic effects of DBPs compared to previous studies with F344 rats. Although MX is not classified as a renal carcinogen in other rat species, a significant renal tumor response was present in male and female Eker rats treated with 0.07 g/l MX for 10 months. These data indicate that MX has a greater carcinogenic effect in animals that are predisposed to tumor formation. On the other hand, 1.8 g/l CHCl3 was not carcinogenic in the Eker rat model, even though it was a renal carcinogen in the Osborne-Mendel rat when given in the drinking water. Low doses of the DBPs were not carcinogenic in Eker rats consistent with a lack of tumor formation in other rodent systems. Although thyroid tumors were not present in Eker rats treated with 0.4 g/l KBrO3 or 0.07 g/l MX, thyroid gland hypertrophy was present. In addition, mesothelial hypertrophy on the testicle was present in one male rat treated with 0.4 g/l KBrO3. Likewise, centrilobular hepatocellular swelling, and foci of cellular atypia were present in Eker rats treated with 0.7 g/l BDCM. These results suggest that a similar spectrum of proliferative lesions associated with these chemicals in other rodent models is also observed in the Eker rat model. Because the present study was only 10 months, it is not known if any of these lesions would have progressed to cancer.
Taken together, the results of this study demonstrate that the combined carcinogenic risk for exposure to mixtures of drinking water DBPs may be less than additive. Although the Eker rat model has proven to be a valuable tool for addressing the interaction of chemical toxicants and carcinogens, additional experimental approaches will be necessary to adequately characterize the potential human health risk from exposure to complex mixtures of drinking water disinfection by-products.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at U.S. EPA, MD-68, 86 T. W. Alexander Dr., Research Triangle Park, NC 27711. Fax: (919) 541-0694. E-mail: wolf.doug{at}epa.gov.
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