Metabolic activation of 1,2-dibromo-3-chloropropane: evidence for the formation of reactive episulfonium ion intermediates

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Abstract
The nematocide and soil fumigant 1,2-dibromo-3-chloropropane (DBCP) is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidney. It was been proposed that both cytochrome P-450 mediated activation and glutathione (GSH) conjugation pathways are operative in DNA damage and organotropy induced by DBCP. To determine the chemical mechanisms involved in the bioactivation of DBCP and to assess a role for an episulfonium ion intermediate, the mechanism of formation of GSH conjugate metabolites of DBCP was investigated. Five biliary GSH conjugates of DBCP were isolated from rats and identified by fast atom bombardment tandem mass spectrometry: S-(2,3-dihydroxypropyl)glutathione (I), S-(2-hydroxypropyl)glutathione (IIA), S-(3-chloro-2-hydroxypropyl)glutathione (III), 1,3-di(S-glutathionyl)propan-2-ol (IV), and 1-(glycyl-S-cysteinyl)-3-(S-glutathionyl)propan-2-ol (V). The mechanisms of conjugate formation were addressed by assessing deuterium retention in conjugates derived from [1,1,2,3,3-2H5]DBCP (D5-DBCP). GSH conjugates, I, III, IV, and V displayed quantitative retention of deuterium, an observation consistent with the formation of an episulfonium ion intermediate. GSH conjugate IIA, however, retained three atoms of deuterium, thus invoking a P-450 mechanism in its genesis. The involvement of glutathione transferase (GST) and sequential episulfonium ion intermediates in the formation of metabolites I, III, and IV was demonstrated in vitro. Upon incubation of DBCP with GST, metabolites, I, III, and IV were identified by tandem mass spectrometry and were found to arise with quantitative retention of deuterium when D5-DBCP was employed as a substrate. An additional GSH conjugate, 1,2,3-tri(S-glutathionyl)propane (VI), was observed as the major metabolite in incubations of GST with DBCP. When the incubations of DBCP with GST were performed in H218O, metabolite I incorporated two atoms of 18O, and metabolites III and IV incorporated one atom of 18O. The ability of GST to catalyze the formation of the four GSH conjugates observed in vivo, with quantitative retention of deuterium and incorporation of 18O from H218O, may be rationalized by a mechanism invoking the initial formation of S-(2-bromo-3-chloropropyl)glutathione. Rearrangement of this unstable conjugate via several reactive episulfonium ions, with either hydrolysis by water or alkylation of GSH at various stages, would account for the pattern of metabolites and their status of isotopic enrichment observed under various incubation conditions. The compelling chemical evidence presented here for the formation of episulfonium ion intermediates is in accord with mechanistic studies conducted on DBCP-induced DNA damage and toxicity in extrahepatic tissues, in which indices of toxicity were found to be insensitive to deuterium substitution.