Michael H. George

Carcinogenicity of Potassium Bromate Administered in the Drinking Water to Male B6C3F1 Mice and F344/N Rats

Ozone has been proposed for water disinfection because it is more efficient than chlorine for killing microbes and results in much lower levels of carcinogenic trihalomethanes than does chlorination. Ozone leads to formation of hypobromous acid in surface waters with high bromine content and forms brominated organic by-products and bromate. The carcinogenicity and chronic toxicity of potassium bromate (KBrO3) was studied in male B6C3F1 mice and F344/N rats to confirm and extend the results of previous work. Mice were treated with 0, 0.08, 0.4, or 0.8 g/L KBrO3 in the drinking water for up to 100 wk, and rats were provided with 0, 0.02, 0.1, 0.2, or 0.4 g/L KBr03. Animals were euthanatized, necropsied, and subjected to a complete macroscopic examination. Selected tissues and gross lesions were processed by routine methods for light microscopic examination. The present study showed that KBr03is carcinogenic in the rat kidney, thyroid, and mesothelium and is a renal carcinogen in the male mouse. KBr03 was carcinogenic in rodents at water concentrations as low as 0.02 g/L (20 ppm; 1.5 mg/kg/day). These data can be used to estimate the human health risk that would be associated with changing from chlorination to ozonation for disinfection of drinking water.

Dimethylarsinic acid treatment alters six different rat biochemical parameters: relevance to arsenic carcinogenesis.

In a previous study, we found that sodium arsenite increased hepatic ornithine decarboxylase (ODC) activity and hepatic heme oxygenase (HO) activity, but did not cause any DNA damage in adult female rat liver or lung, suggesting that arsenite may be a promoter of carcinogenesis. In this study sodium arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) were administered orally in equitoxic doses to adult female rats at 21 and 4 h prior to sacrifice. DNA damage (DD), cytochrome P450 content (P450), glutathione content (GSH), ODC, serum alanine aminotransferase (ALT) and HO were measured in liver and/or lung tissue. At 60 mg/kg in rat liver, sodium arsenate increased hepatic HO fivefold. MMA decreased ALT at 226 mg/kg, decreased ALT and GSH at 679 mg/kg and also increased P450 at 679 mg/kg in rat liver. DMA decreased ALT and hepatic GSH and increased hepatic HO at 387 mg/kg. In the lung, DMA decreased ODC at both 129 and 387 mg/kg. DD in lung tissue was significantly higher at 387 mg/kg DMA, demonstrating organ specific DNA damage. The biochemical effects and the inferred oncologic potential of the four major forms of arsenic (arsenate, arsenite, MMA and DMA) differ dramatically. The inorganic forms (arsenate and arsenite) are similar to each other (both good HO inducers); the methylated organic forms of arsenic (MMA and DMA) also share a similar pattern of biochemical effects (decreased GSH and ALT, increased P450). All six of the biochemical parameters studied were altered by DMA in either rat liver or lung.

Hepatocarcinogenicity in the male B6C3F1 mouse following a lifetime exposure to dichloroacetic acid in the drinking water: dose-response determination and modes of action.

Male B6C3F, mice were exposed to dichloroacetic acid (DCA) in the drinking water in order to establish a dose response for the induction of hepatocellular cancer and to examine several modes of action for the carcinogenic process. Groups of animals were exposed to control, 0.05, 0.5, 1, 2, or 3.5 g/L DCA in the drinking water for 90-100 wk. Mean daily doses (MDD) of 8, 84, 168, 315, and 429 mg/kg/d of DCA were calculated. The prevalence (percent of animals) with hepatocellular carcinoma (HC) was significantly increased in the 1-g/L (71%), 2-g/L (95%), and 3.5-g/L (100%) treatment groups when compared to the control (26%). HC multiplicity (tumors/animal) was significantly increased by all DCA treatments-0.05 g/L (0.58), 0.5 g/L (0.68), 1 g/L (1.29), 2 g/L (2.47), and 3.5 g/L (2.90)-compared to the control group (0.28). Based upon HC multiplicity, a no-observed-effect level (NOEL) for hepatocarcinogenicity could not be determined. Hepatic peroxisome proliferation was significantly increased only for 3.5 g/L DCA treatment at 26 wk. and did not correlate with the liver tumor response. The severity of hepatotoxicity increased with DCA concentration. Below 1 g/L, hepatotoxicity was mild and transient as demonstrated by the severity indices and serum lactate dehydrogenase activity. An analysis of generalized hepatocyte proliferation reflected the mild hepatotoxicity and demonstrated no significant treatment effects on the labeling index of hepatocytes outside proliferative lesions. Consequently, the induction of liver cancer by DCA does not appear to be conditional upon peroxisome induction or chemically sustained cell proliferation. Hepatotoxicity, especially at the higher doses, may exert an important influence on the carcinogenic process.

Toxicity Profiles in Mice Treated with Hepatotumorigenic and Non-Hepatotumorigenic Triazole Conazole Fungicides: Propiconazole, Triadimefon, and Myclobutanil

Conazoles comprise a class of fungicides used in agriculture and as pharmaceutical products. The fungicidal properties of conazoles are due to their inhibition of ergosterol biosynthesis. Certain conazoles are tumorigenic in rodents; both propiconazole and triadimefon are hepatotoxic and hepatotumorigenic in mice, while myclobutanil is not a mouse liver tumorigen. As a component of a large-scale study aimed at determining the mode(s) of action for tumorigenic conazoles, we report the results from comparative evaluations of liver and body weights, liver histopathology, cell proliferation, cytochrome P450 (CYP) activity, and serum cholesterol, high-density lipoprotein and triglyceride levels after exposure to propiconazole, triadimefon, and myclobutanil. Male CD-1 mice were treated in the feed for 4, 30, or 90 days with triadimefon (0, 100, 500, or 1800 ppm), propiconazole (0, 100, 500, or 2500 ppm) or myclobutanil (0, 100, 500, or 2000 ppm). Alkoxyresorufin O-dealkylation (AROD) assays indicated that all 3 chemicals induced similar patterns of dose-related increases in metabolizing enzyme activity. PROD activities exceeded those of MROD, and EROD with propiconazole inducing the highest activities of PROD. Mice had similar patterns of dose-dependent increases in hepatocyte hypertrophy after exposure to the 3 conazoles. High-dose exposures to propiconazole and myclobutanil, but not triadimefon, were associated with early (4 days) increases in cell proliferation. All the chemicals at high doses reduced serum cholesterol and high-density lipoprotein (HDL) levels at 30 days of treatment, while only triadimefon had this effect at 4 days of treatment and only myclobutanil and propiconazole at 90 days of treatment. Overall, the tumorigenic and nontumorigenic conazoles induced similar effects on mouse liver CYP enzyme activities and pathology. There was no specific pattern of tissue responses that could consistently be used to differentiate the tumorigenic conazoles, propiconazole, and triadimefon, from the nontumorigenic myclobutanil. These findings serve to anchor other transcriptional profiling studies aimed at probing differences in key events and modes of action for tumorigenic and nontumorigenic conazoles.

Toxicity Profiles in Rats Treated with Tumorigenic and Nontumorigenic Triazole Conazole Fungicides: Propiconazole, Triadimefon, and Myclobutanil

Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. The particular mode of toxic and tumorigenic action for these compounds is not known, however it has been proposed that triadimefon-induced rat thyroid tumors arise through the specific mechanism of increased TSH. The present study was designed to identify commonalities of effects across the different conazoles and to determine unique features of the tissue responses that suggest a toxicity pathway and a mode of action for the observed thyroid response for triadimefon. Male Wistar/Han rats were treated with triadimefon (100, 500, 1800 ppm), propiconazole (100, 500, 2500 ppm), or myclobutanil (100, 500, 2000 ppm) in feed for 4, 30, or 90 days. The rats were evaluated for clinical signs, body and liver weight, histopathology of thyroid and liver, hepatic metabolizing enzyme activity, and serum T3, T4, TSH, and cholesterol levels. There was a dose-dependent increase in liver weight but not body weight for all treatments. The indication of cytochrome induction, pentoxyresorufin O-dealkylation (PROD) activity, had a dose-related increase at all time points for all conazoles. Uridine diphopho-glucuronosyl transferase (UDPGT), the T4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced to the same extent after 30 and 90 days for all three conazoles. Livers from all high dose treated rats had centrilobular hepatocyte hypertrophy after 4 days, while only triadimefon and propiconazole treated rats had hepatocyte hypertrophy after 30 days, and only triadimefon treated rats had hepatocyte hypertrophy after 90 days. Thyroid follicular cell hypertrophy, increased follicular cell proliferation, and colloid depletion were present only after 30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T4 was present after 4 days with all 3 compounds but only the high doses of propiconazole and triadimefon produced decreased T4 after 30 days. T3 was decreased after high-dose triadimefon after 4 days and in a dose-dependent manner for all compounds after 30 days. Thyroid hormone levels did not differ from control values after 90 days and TSH was not increased in any exposure group. A unique pattern of toxic responses was not identified for each conazole and the hypothesized mode of action for triadimefon-induced thyroid gland tumors was not supported by the data.

In vivo genotoxicity of dichloroacetic acid: evaluation with the mouse peripheral blood micronucleus assay and the single cell gel assay.

Chlorination is a widely used method for disinfection of drinking water supplies. Reaction of chlorine with naturally present organic compounds can result in toxic by‐products. One major disinfection by‐product from the chlorination of drinking water is dichloroacetic acid (DCA). This chemical has been shown to be carcinogenic in rodents, yet little genotoxicity data are available to assess the possible role of DNA and/or chromosomal damage in this process. We have used the peripheral blood erythrocyte micronucleus (MN) assay and the alkaline single cell gel electrophoresis (SCG) technique to investigate the in vivo genotoxicity of DCA in bone marrow and blood leukocytes, respectively. The MN assay detects chromosome breakage and/or malsegregation, while the SCG assay detects DNA damage (e.g., single strand breaks, alkali‐labile sites, crosslinking). Mice were exposed to this compound in drinking water, available ad libitum, for up to 31 weeks. Our results show a small but statistically significant dose‐related increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) after subchronic exposure to DCA for 9 days. In addition, at the highest dose of DCA tested (3.5 g/l), a small but significant increase in the frequency of micronucleated normochromatic erythrocytes (NCE) was detected following exposure for ≥ 10 weeks. Coadministration of the antioxidant vitamin E did not affect the ability of DCA to induce this damage, indicating that the small induction of MN by DCA was probably not due to oxidative damage. Based on the lack of any difference observed in the proportion of kinetochore‐positive micronuclei between the treated and control animals, we interpret the induced MN as arising from clastogenic events. The SCG technique suggested the presence of DNA crosslinking in blood leukocytes in mice exposed to 3.5 g/l DCA for 28 days. These data provide evidence that DCA may be an extremely weak inducer of chromosome damage when provided to mice in drinking water under conditions which lead to increased levels of tumors.

Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease P1 treatment

The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo[a]pyrene diol epoxide I (BPDE-I), nuclease P1 treatment resulted in a significant reduction in detection of major adducts from 1-amino-6-nitropyrene (1-amino-6-NP), 1-amino-8-nitropyrene (1-amino-8-NP), 2-aminofluorene (2-AF), 2-naphthylamine (2-NA) and 4-aminobiphenyl (4-ABP) modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene (2-AAF) modified DNA. These results suggest that, at least initially, both modifications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitroaromatics and aromatic amines.

Time- and Dose-Dependent Development of Potassium Bromate-Induced Tumors in Male Fischer 344 Rats

Potassium bromate (KBrO 3) is a rodent carcinogen and a nephro- and neurotoxicant in humans. KBrO3 is used in cosmetics and food products and is a by-product of water disinfection by ozonization. KBrO3 is carcinogenic in the rat kidney, thyroid, and mesothelium and is a renal carcinogen in the male mouse. The present study was designed to investigate the relationship of time and dose to bromate-induced tumors in male Fischer 344 (F344) rats and to provide some insight into the development of these tumors. KBrO 3 was dissolved in drinking water at nominal concentrations of 0, 0.02, 0.1, 0.2, and 0.4 g/L and administered to male F344 rats as the sole water source for 12, 26, 52, 78, or 100 wk. Renal cell tumors were present after 52 wk of treatment only in the high-dose group. Mesotheliomas developed after 52 wk of treatment on the tunica vaginalis. Mesotheliomas were present at sites other than the testicle after 78 wk of treatment, indicating that their origin was the testicular tunic. Thyroid follicular tumors were present as early as 26 wk in 1 rat each from the 0.1- and 0.2-g/L groups. The present study can be used as a basis for the determination of dose-time relationships of tumor development for a better understanding of KBrO3-induced cancer.

Characterization of Peroxisome Proliferator–Activated Receptor α—Independent Effects of PPARα Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate also Activates the Constitutive-Activated Receptor

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha. Recent studies indicate that the plasticizer di-(2-ethylhexyl) phthalate (DEHP) increased the incidence of liver tumors in PPARalpha-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPARalpha but dependent on other nuclear receptors, including the constitutive-activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate, and DEHP in rat liver and DEHP in mouse liver induced genes, including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and reverse transcription-PCR in the livers of DEHP-treated wild-type, PPARalpha-null, and CAR-null mice demonstrated that (1) most (approximately 94%) of the transcriptional changes induced by DEHP were PPARalpha-dependent, (2) many PPARalpha-independent genes overlapped with those regulated by PB, (3) induction of genes Cyp2b10, Cyp3a11, and metallothionine-1 by DEHP was CAR dependent but PPARalpha-independent, and (4) induction of a number of genes (Cyp8b1, Gstm4, and Gstm7) was independent of both CAR and PPARalpha. Our results indicate that exposure to PPARalpha activators including DEHP leads to activation of multiple nuclear receptors in the rodent liver.

Subchronic sodium chlorate exposure in drinking water results in a concentration-dependent increase in rat thyroid follicular cell hyperplasia

Chlorine dioxide (ClO 2) is an effective drinking water disinfectant, but sodium chlorate (NaClO3) has been identified as a potentially harmful disinfection by-product. Studies were performed to describe the development of thyroid lesions in animals exposed to NaClO3 in the drinking water. Male and female F344 rats and B6C3F1 mice were exposed to 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/L NaClO3 for 21 days. Additional male F344 rats were exposed to 0, 0.001, 0.01, 0.1, 1.0, or 2.0 g/L NaClO 3 for 90 days. Female F344 rats were exposed to 0, 0.5, 1.0, 2.0, 4.0, or 6.0 g/L of NaClO3 for 105 days. Thyroid tissues were processed by routine methods for light microscopic examination, and follicular cell hyperplasia was diagnosed using a novel method. Thyroid hormone levels were altered significantly after 4 and 21 days. NaClO3 treatment induced a concentration-dependen t increase in the incidence and severity of thyroid follicular cell hyperplasia. Male rats are more sensitive to the effects of NaClO3 treatment than females. Follicular cell hyperplasi a was not present in male or female B6C3F1 mice. These data can be used to estimate the human health risk that would be associated with using ClO 2, rather than chlorine, to disinfect drinking water.