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