Studies Of The Metabolism Of 1-Chloro-3-buten-2-ol With Rat Liver Microsomes

1,3-Butadiene?BD?is an important petrochemical manufactured in large amounts and is used primarily to produce synthetic rubber and plastics.It is a carcinogenic air pollutant and is ubiquitously present in the atmosphere with the major environmental source being the exhaust from automobile and incomplete combustion of biomass.The BD carcinogenicity stems from its metabolites,which are formed via two metabolic pathways.In one pathway,BD is metabolized to epoxidesbycytochromeP450 s,andintheotherpathwayto1-chloro-2-hydroxy-3-butene?CHB?by myeloperoxidase?MPO?in the presence of H₂O₂ and Cl^-.We have demonstrated that CHB can be bioactivated to1-chloro-3-buten-2-one?CBO?by alcohol dehydrogenase?ADH?,which is cytotoxic and genotoxic,and readily reacts with nucleosides and DNA to yield multiple adducts.However,the metabolism of CHB by cytochrome P450 s has not been reported yet.In the present study,biotransformation of CHB by rat liver microsomes was investigated.TheresultsshowedthatCHBwasmetabolizedto1-chloro-3,4-epoxy-2-butanol?CEB?,a novel BD metabolite,and also CBO.4-Methylpyrazole,a cytochrome P450 2E1-specific inhibitor,inhibited the formation of both CEB and CBO,while 1-benzylimidazole,a generic cytochrome P450 inhibitor,completely abolished the formation of CEB and CBO,suggesting that CHB metabolism was mediated by cytochrome P450 s.Because of the presence of two chiral centers in its molecule,CEB was detected as two stereoisomers,which were designated D-CEB and M-CEB.The two stereoisomers were synthesized and their structures were characterized as 2S,3R-/2R,3S-CEB and 2R,3R-/2S,3S-CEB,respectively.The amounts of M-CEB were more than those of D-CEB by 50-80%.The formation of CEB and CBO was time-dependent;their amounts increased over incubation time from 30 to 50 min and changed little from 50 to 60 min.The enzymatic reactions of CHB biotransformation to CEB and CBO followed Michaelis-Menten kinetics.The K_m and V_maxvalues were determined to be 6.4±0.7mM and 0.1±0.01 nmol/min/mg protein for D-CEB,4.2±0.5 mM and 0.16±0.01nmol/min/mg protein for M-CEB,and 4.0±0.5 mM and 4.6±0.5 nmol/min/mg protein for CBO,respectively.Therefore,CBO was the predominant product of CHB biotransformation.In addition,CEB was found to be genotoxic as examined by the comet assay.At 50?M and higher concentrations,both D-CEB and M-CEB caused statistically significant rates of DNA migration compared to the negative controls,indicating that they induced DNA damage.In summary,CHB can be biotransformed into CEB and CBO by cytochrome P450 s with CBO being the predominant metabolite.Therefore,the formation of CEB and CBO can be used as evidence of CHB production,and these products may also play a role in toxicity of CHB.