P. J. Van Bladeren

The role of glutathione conjugation in the mutagenicity of 1,2-dibromoethane

Abstract Two mechanisms for the toxic actions of 1,2-dibromoethane have been postulated, both of which involve biotransformation. The first is oxidation to 2-bromoacetaldehyde, a highly reactive substance, the second a possible direct conjugation to glutathione, giving rise to a reactive half-mustard. It was the purpose of this investigation to determine to what extent these two reactive species are responsible for the mutagenicity of 1,2-dibromoethane. To assess quantitatively the importance of the conjugation to glutathione in vivo, rats were administered single doses of 1,2-dibromoethane; 30–55 per cent of the dose was excreted as mercapturic acid. The conjugation of 1,2-dibromoethane to glutathione was also studied in vitro. Specific activities of the metabolizing systems used in the mutagenicity experiments were determined. The mutagenicity of 1,2-dibromoethane towards Salmonella typhimurium TA100 was considerably enhanced by the addition of 100,000 g supernatant fraction, whereas the addition of microsomes had no effect, indicating that the primary glutathione adduct is responsible for the mutagenic effect. As a model for the mutagenic intermediate, S-2-bromoethyl-N-acetyl-cysteine methyl ester was synthesized. This proved to be a very reactive and highly mutagenic compound, which can be further metabolized and thereby detoxified by glutathione conjugation. A similar phenomenon is likely to occur in the mutagenicity test with 1,2-dibromoethane, where after an initial rise in the number of mutants with increasing amounts of glutathione, the number of mutations decreases again. These results clearly indicate that glutathione conjugation plays an important role in the mutagenicity of 1,2-dibromoethane.

Mutagenic activation of dibromomethane and diiodomethane by mammalian microsomes and glutathione S-transferases

The influence of mammalian metabolizing enzymes on the mutagenic activity of dibromomethane and diiodomethane was investigated by using Salmonella typhimurium strain TA100 as indicator. The 2 compounds are known to be metabolized via an oxidative pathway catalysed by microsomal enzymes as well as through direct enzymatic conjugation with glutathione; both pathways possibly give rise to reactive electrophilic intermediates. In mutagenicity plate assays with pre-incubation, dibromo- and diiodo-methane were directly mutagenic towards strain TA100; their mutagenic activity was enhanced upon incubation either with rat-liver microsomes or with the cytosol fraction of the same organ, containing the glutathione S-transferases. These data can be taken as an indication that both microsomal oxidation and conjugation to glutathione are indeed responsible for the mammalian mutagenic activation of dihalomethanes.

The influence of disulfiram and other inhibitors of oxidative metabolism on the formation of 2-hydroxyethyl-mercapturic acid from 1,2-dibromoethane by the rat

Abstract The mercapturic acid derivative, N- acetyl -S-2- hydroxyethyl - l - cysteine , is a major metabolite of 1,2-dibromoethane in vivo . This compound can be formed via two pathways, both involving a potentially dangerous reactive intermediate. One way involves the intermediacy of bromoacetaldehyde, formed by microsomal oxidation, followed by loss of hydrogen bromide. The second pathway, direct conjugation of 1,2-dibromoethane with glutathione, gives rise to S -2-bromoethyl glutathione. Using several inhibitors of microsomal mixed function oxidases, it was found that under these conditions about 10% of the mercapturic acid derivative formed via direct conjugation. Disulfiram, an inhibitor of aldehyde dehydrogenases, but also of microsomal oxidation, also markedly inhibits the excretion of the mercapturic acid, after administration of a single high dose (1 g/kg) or upon chronic treatment with a low dose (50 mg/kg). The inhibitory effect is maximal after 10 days of chronic treatment. Administration of large amounts of 1,2-dibromoethane (>0.20 nmole/rat) following a single lower dose of disulfiram (125 mg/kg) also leads to a lower excretion of mercapturic acid metabolite a phenomenon associated with a decrease in cytochrome P-450 levels. From these results it is concluded that the enhanced carcinogenic effect of the combination disulfiram (chronic)/1,2-dibromoethane is not caused by bromoacetaldehyde, since its formation is completely inhibited under these conditions, but by S -2-bromoethyl-glutathione, although a role for 1,2-dibromoethane itself cannot be excluded.

The Activating Role of Glutathione in the Mutagenicity of 1,2-Dibromoethane

1,2-Dibromoethane (DBE) is widely used as an insecticide, fungicide and gasoline additive.1 A number of adverse effects of this compound have been reported, notably its mutagenicity towards bacteria2 and carcinogenicity towards rats and mice.3 Apart from its direct alkylating ability, two mechanisms for these toxic actions can be envisaged, both of which involve biotransformation. The first one consists of oxidation, followed by loss of hydrogen bromide, leading to bromoacetaldehyde, a highly reactive substance which can bind covalently to macromolecules.4, 5 However, bromoacetaldehyde has been reported to be non-mutagenic.6 The second possible mechanism involves conjugation to glutathione (GSH). As has been shown for 1,2-dichloroethane7 and cis-1,2-dichlorocyclohexane,8 the 2-halogenothioether resulting from substitution of one of the halogen atoms by glutathione is responsible for enhanced mutagenic activity of these compounds in the presence of GSH and GSH-S-transferases (see Fig. 1).

Dietary chemoprevention in toxicological perspective

Nutrition is essential to support life, but at the same time it can paradoxically be considered the main cause of cancer. As concerns the latter, Doll and Peto (1981) estimated that in the USA the proportion of cancer deaths due to the diet was approximately 30%. Indeed, on the one hand, food contains a wide variety of mutagens and/or carcinogens, some of which occur naturally, and others that might be introduced during the preparation of food (Pariza et al., 1990; Wakabayashi et al., 1991), whereas, on the other hand, the human diet also contains a number of compounds that protect against cancer (Birt and Bresnick, 1991; Stich, 1991; Dragsted et al., 1993; Verhagen et al., 1993). This is in close agreement with epidemiological findings of negative associations between cancer and consumption of fibre-containing foods, fresh fruits, vegetables, vitamins and minerals (Archer, 1988; Birt and Bresnick, 1991; Steinmetz and Potter, 1991a,b). Many a compound of dietary origin has been claimed to have chemopreventive potential. Therefore chemoprevention of cancer is an area of great scientific, public and economic interest.