Cob(I)alamin for Trapping Butadiene Epoxides in Metabolism with Rat S9 and for Determining Associated Kinetic Parameters

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• 作者：Hitesh V. Motwani ; Charlotta Fred ; Johanna Haglund ; Bernard T. Golding ; Margareta Trnqvist
• 刊名：Chemical Research in Toxicology
• 出版年：2009
• 出版时间：September 21, 2009
• 年：2009
• 卷：22
• 期：9
• 页码：1509-1516
• 全文大小：221K
• 年卷期：v.22,no.9(September 21, 2009)
• ISSN：1520-5010

文摘

The reduced state of vitamin B₁₂, cob(I)alamin, acts as a supernucleophile that reacts ca. 10⁵ times faster than standard nucleophiles, for example, thiols. Methods have been developed for trapping electrophilically reactive compounds by exploiting this property of cob(I)alamin. 1,3-Butadiene (BD) has recently been classified as a group 1 human carcinogen by the International Agency for Research on Cancer (IARC). The carcinogenicity of BD is considered to be dependent on the activation or deactivation of the reactive metabolites of BD, that is, the epoxides (oxiranes) 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EBdiol). Cytochrome P450 (P450) isozymes are involved in oxidation of BD to EB and further activation to DEB. EB and DEB are hydrolyzed by epoxide hydrolases (EH) to 3,4-dihydroxy-1-butene (BDdiol) and EBdiol, respectively. EBdiol can also be formed by oxidation of BDdiol. In the present study, cob(I)alamin was used for instant trapping of the BD epoxide metabolites generated in in vitro metabolism to study enzyme kinetics. The substrates EB, DEB, and BDdiol were incubated with rat S9 liver fraction, and apparent K_m and apparent V_max, were determined. The ratio of conversion of EB to DEB (by P450) to the rate of deactivation of DEB by EH was 1.09. Formation of EBdiol from hydrolysis of DEB was ca. 10 times faster than that from oxidation of BDdiol. It was also found that the oxidation of EB to DEB was much faster than that of BDdiol to EBdiol. The study offers comparative enzyme kinetic data of different BD metabolic steps, which is useful for quantitative interspecies comparison. Furthermore, a new application of cob(I)alamin was demonstrated for the measurement of enzyme kinetics of compounds that form electophilically reactive metabolites.