Alcohol Dehydrogenase- and Rat Liver Cytosol-Dependent Bioactivation of 1-Chloro-2-hydroxy-3-butene to 1-Chloro-3-buten-2-one, a Bifunctional Alkylating Agent

Abstract

1,3-Butadiene (BD) is an air pollutant whose toxicity and carcinogenicity have been considered primarily mediated by its reactive metabolites, 3,4-epoxy-1-butene and 1,2,3,4-diepoxybutane formed in liver and extrahepatic tissues by cytochromes P450. A possible alternative metabolic pathway in bone marrow and immune cells is the conversion of BD to the chlorinated allylic alcohol 1-chloro-2-hydroxy-3-butene (CHB) by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. In the present study, we investigated the in vitro bioactivation of CHB by alcohol dehydrogenases (ADH) under in vitro physiological conditions (pH 7.4, 37 °C). The results provide clear evidence for CHB being converted to 1-chloro-3-buten-2-one (CBO) by purified horse liver ADH and rat liver cytosol. CBO readily reacted with glutathione (GSH) under assay conditions to form three products: two CBO-mono-GSH conjugates [1-chloro-4-(S-glutathionyl)butan-2-one (3) and 1-(S-glutathionyl)-3-buten-2-one (4)], and one CBO-di-GSH conjugate [1,4-bis(S-glutathionyl)butan-2-one (5)]. CHB bioactivation and the ratios of the three GSH conjugates formed were dependent upon incubation time, GSH and CHB concentrations, and the presence of ADH or rat liver cytosol. The ADH enzymatic reaction followed Michaelis-Menten kinetics with a Km at 3.5 mM and a kcat at 0.033 s−1. After CBO was incubated with freshly isolated mouse erythrocytes, globin dimers were detected using SDS-PAGE and silver staining, providing evidence that CBO can act as a protein cross-linking agent. Collectively, the results provide clear evidence for CHB bioactivation by ADH and rat liver cytosol to yield CBO. The bifunctional alkylating ability of CBO suggests that it may play a role in BD toxicity and/or carcinogenicity.

Cytotoxicity, genotoxicity, and mutagenicity of 1-chloro-2-hydroxy-3-butene and 1-chloro-3-buten-2-one, two alternative metabolites of 1,3-butadiene

“…We have demonstrated in the preceding paper that CBO could cause the formation of cross-links of globin (Elfarra and Zhang, 2012). Thus, it was logical to investigate if CBO could induce DNA cross-linking.…”

“…CHB was synthesized from BD and calcium hypochlorite as described in the literature (Kadesch, 1946). Its purity was ≥ 98% as determined by GC-MS. CBO was synthesized as reported in our preceding paper (Elfarra and Zhang, 2012). The CBO used in the experiments was purified on a silica gel column and the purity was at least 98% as determined by GC-MS.…”

“…Recently, we demonstrated that CHB could be oxidized to 1-chloro-3-buten-2-one (CBO) by alcohol dehydrogenase and rat liver cytosol (Elfarra and Zhang, 2012). CHB may also be converted to CBO by other enzymes, such as cytochrome P450 2E1 and 3A4, since 3-butene-1,2-diol, a structural analog of CHB, can be oxidized to hydroxymethyl vinyl ketone by cytochrome P450s (Krause et al, 2001).…”

“…CHB may also be converted to CBO by other enzymes, such as cytochrome P450 2E1 and 3A4, since 3-butene-1,2-diol, a structural analog of CHB, can be oxidized to hydroxymethyl vinyl ketone by cytochrome P450s (Krause et al, 2001). CBO is a Michael acceptor with bifunctional alkylating ability (Elfarra and Zhang, 2012). In reactions with glutathione (GSH) it readily formed a CBO-di-GSH conjugate.…”

“…In reactions with glutathione (GSH) it readily formed a CBO-di-GSH conjugate. Globin-chain cross-links were also detected upon in vitro incubation of CBO with freshly-isolated erythrocytes (Elfarra and Zhang, 2012). …”