In vitro biotransformation and genotoxicity of the drinking water disinfection byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1

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Abstract

The drinking water disinfection byproduct bromodichloromethane (CHBrCl2) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl2. First, rodent hepatic cytosol incubations containing [14C]CHBrCl2, supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA. The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl2. Second, exposure of GSTT1-1-expressing S. typhimurium to [14C]CHBrCl2 resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity. Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis. A possible explanation for this observation was formation of a ‘transient’ adduct that was unstable in the DNA isolation and hydrolysis procedures employed. To circumvent problems of adduct instability, reactions of [14C]CHBrCl2 with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine. Hydroxyapatite chromatography of [14C]-labeled DNA or HPLC chromatography of [14C]-labeled deoxyguanosine derivatives demonstrated the covalent binding of [14C]CHBrCl2-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of [14C]CHBrCl2 biotransformed by GSTT1-1 resulted in DNA adducts). Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl2 in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl2 exposure were compared with rat liver (a nontarget tissue). Rat liver had a significant capacity to detoxify CHBrCl2 (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (Vmax/Km) were compared. In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl2 in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues. The significance of these findings is that conjugation of CHBrCl2 with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats).

Introduction

Halogenated water disinfection by-products are carcinogenic in rodents at high doses (IARC, 1991) and can cause adverse reproductive outcomes in laboratory animals (Nieuwenhuijsen et al., 2000). An association between chlorinated-drinking water consumption and increased incidences of human bladder and colorectal cancers has been suggested by epidemiology studies King and Marrett, 1996, Koivusalo et al., 1997, Morris, 1995. The trihalomethanes are the most abundant class of disinfection byproducts found in drinking water, and brominated trihalomethanes can predominate if source waters with high bromine content are chlorinated or ozonated Boorman et al., 1999, Krasner et al., 1989.

Brominated trihalomethanes may pose a greater human health risk than CHCl3 based on rodent studies demonstrating that these brominated compounds are more toxic than CHCl3. For instance, tumors were observed in the kidney and large intestine of male and female F-344 rats following chronic doses of bromodichloromethane (CHBrCl2) by corn oil gavage; a striking finding was the high incidence of large intestine adenocarcinomas observed in male rats Dunnick et al., 1987, National Toxicology Program, 1987. No evidence of liver tumors was seen in rats of either sex. In contrast, a gender-dependent induction of tumors was observed in B6C3F1 mice following gavage doses of CHBrCl2; liver tumors were induced only in female mice, and renal tumors formed only in males (National Toxicology Program, 1987). A separate study demonstrated that CHBrCl2 exposure via drinking water caused a greater incidence of preneoplastic aberrant crypt foci in the colons of rats compared to the number of lesions produced as a result of CHCl3 exposure (DeAngelo et al., 2002). In acute studies, dose-related pathological changes in the livers and kidneys of rats were observed following oral doses of CHBrCl2 that were greater and more persistent than lesions produced by equimolar doses of CHCl3 Keegan et al., 1998, Kroll et al., 1994, Lilly et al., 1997a. Results from Salmonella studies have indicated that brominated trihalomethanes can be bioactivated to intermediates that produce mutations by the enzyme glutathione transferase theta 1-1 (GSTT1-1) (Pegram et al., 1997). Greater than 90% of the mutations observed in this bacterial strain were a result of a single G→A transition at the selectable locus of the hisG46 gene (DeMarini et al., 1997). This result suggests the formation of a specific DNA adduct that is derived from a unique reactive intermediate produced by the initial conjugation of CHBrCl2 with glutathione (GSH). Similar bioactivations have previously been reported for several dihalomethanes and dihaloethanes in the same strain of bacteria (Thier et al., 1993). In contrast to CHBrCl2, CHCl3 did not produce mutations at equivalent doses in this bacterial strain (Pegram et al., 1997) and this likely reflects the enhanced ability of Br to act as a leaving group compared with Cl during the substitution reaction of the trihalomethanes with GSH. Thus, although CHCl3 may be a quantitatively more abundant disinfection by-product than CHBrCl2 in drinking water, it may not elicit as potent a toxic effect as CHBrCl2 following environmental exposure.

There are many examples of halogenated alkanes that yield intermediates that are more toxic than the parent compound following GSH conjugation (van Bladeren, 2000). Several of these compounds are substrates for GSTT1-1. This isoform is an evolutionarily conserved enzyme and has unique kinetic properties for a GST (Meyer, 1993). GSTT1-1 is expressed constitutively in several tissues of rodents and humans (reviewed by Landi, 2000) and is polymorphically expressed in the human population with some individuals having a null genotype (Pemble et al., 1994). Thus, GSTT1-1 expression in tissues may modulate disease susceptibility following human exposure to haloalkanes found in the environment, and it is hypothesized that bioactivation of brominated trihalomethanes catalyzed by GSTT1-1 may result in the transformation of cells that lead to cancers. At low environmental exposures to trihalomethanes, it is hypothesized that cytochrome P450 (CYP)-mediated metabolism acts as a detoxification pathway because the final product of this oxidative metabolism is CO2 (Mathews et al., 1990).

The purpose of the current study was to examine if reactive intermediates derived from the reaction of GSH with CHBrCl2 could produce DNA damage directly. Several in vitro approaches were used to examine this possibility. Comparisons are also made of the CYP- and GST-dependent biotransformation of CHBrCl2 in rat tissues that are target sites in cancer bioassays because CHBrCl2-dependent genotoxicity in vivo is likely a function of the balance in detoxification and activation pathways in these susceptible tissues.

Section snippets

Chemicals and reagents

[14C]CHBrCl2 was purchased from Dupont/NEN (Boston, MA) (radiolabeled purity 98%, specific activity, 5.2 mCi mmol−1 in all experiments), and 500 mM working stock solutions were prepared in methanol. [13C]CHBrCl2 (99 atom%) was purchased from Cambridge Isotopes Laboratories (Cambridge, MA). Non-radiolabeled CHBrCl2 (purity >98%) and trifluoroacetic acid (TFA) were from Aldrich (Milwaukee, WI). GSH, S-hexyl-GSH, 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrophenol, calf thymus DNA, DNaseI, nuclease

Correlation of GST theta 1-1 activity with the extent of DNA radiolabeling produced in rodent hepatic cytosol incubations

Male mouse and rat hepatic cytosols from naive animals were prepared in this study. The activity of mouse liver cytosol toward the standard GSTT1-1 substrate ENPP was significantly greater (8-fold more) than rat liver cytosol (Fig. 1A, open bars) and is consistent with literature values (Sherratt et al., 1998). Similarly, the efficiency (measured by the ratio Vmax/Km) of CHBrCl2 metabolism by mouse hepatic cytosol was greater than for rat (Fig. 1A, black bars, Ross and Pegram, 2003). The amount

DNA binding of reactive GSH conjugates

Conjugation of polyhalogenated methanes and vic-dihaloethanes has been shown to cause DNA damage (reviewed by van Bladeren, 2000). We have undertaken studies to measure the DNA binding efficiency of GSH conjugates of the water disinfection byproduct CHBrCl2. The average binding frequency of CHBrCl2-derived metabolites to DNA was estimated from in vitro total radioactive labeling studies to be approximately 2.5 mol DNA adducts per 104 mol CHBrCl2 metabolized by GSTT1-1, using rodent hepatic

Acknowledgements

M.K. Ross was supported by NIEHS Postdoctoral Fellowship F32 ES11111-01 and by UNC/EPA Cooperative Training Agreement CT827206. We thank Drs. Hugh Barton, Stephen Nesnow, and Linda Birnbaum for helpful comments regarding the manuscript. The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the

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