No abstract

Several nonsymmetric polychlorinated biphenyl (PCB) congeners form atropisomers due to steric hindrance of free rotation around the phenyl-phenyl bond. It is evident from the literature that both chiral PCB congeners and their atropisomeric methylsulfonyl-PCB metabolites, formed in higher animals and in humans, are present in biota as nonracemic mixtures. Chiral methylsulfonyl-PCBs are strongly dominated by one of the atropisomers in mammalian tissues. The aim of the present study is to examine enantioselective metabolism, retention, and excretion of 2,2',3,3',4,6'-hexachlorobiphenyl (CB-132) in rat by administration of a CB-132 racemate and pure atropisomers. Chemical analysis of liver, lung, and adipose tissue from the rats showed a strong retention of one of the CB-132 atropisomers and a similar, but even more pronounced, accumulation of one of the atropisomers of the meta- and para-methylsulfonyl-substituted CB-132 metabolites in these tissues. Metabolites with R structures were predominately formed from one of the atropisomers of CB-132. The slower metabolism of the other atropisomer of CB-132 and its pronounced excretion in feces suggest an enantioselective metabolism. The results indicate enantio-selective formation of the methylsulfonyl-CB132 metabolites and confirm the critical role of stereochemistry of chemicals for their metabolism.

Belgium DNA-phosphate adducts are known to be formed by a variety of alkylating agents. Due to little or no repair of DNA-phosphate adducts, these adducts may offer increased possibilities of both identifying and quantifying DNA adducts. The formation of DNA-phosphate adducts leads to a complete esterification of the phosphate group giving rise to a phosphotriester configuration. This work consists of the characterization of ethyl phosphotriesters (Ethyl PTE) using miniaturized LC-ESI-MS/MS and column switching in enzymatic hydrolysate of DNA treated in vitro with the model compound N-ethyl-N-nitrosourea (ENU). In vitro ENU-treated DNA was enzymatically degraded using nuclease P1, phosphodiesterase, and alkaline phosphatase. The use of column switch allowed for large-volume injections, where unmodified nucleosides were discarded in the loading step. The analytes were forward flushed to the analytical column in the eluting step and separated using a linear gradient. Ten different ethyl PTEs (dGpEtdG, dApEtdA, dCpEtdC, TpEtT, dGpEtdA, dGpEtdC, dGpEtT, dApEtdC, dApEtT, and dCpEtT) were characterized by their masses and CAD product ion spectra. Measurements of accurate masses were carried out yielding experimental masses within 5 ppm of the calculated masses for 9 of the 10 ethyl PTEs. For comparison, the enzymatic hydrolysate of ENU-treated DNA was subjected to transalkylation of the DNA-phosphate adducts by cob(I)alamin. Formed ethyl-cobalamins were analyzed according to earlier developed methods. The limit of detection of an alkyl-cobalamin standard and an alkyl PTE standard was 2 fmol and 5 fmol, respectively. (J Am Soc Mass Spectrom 2004, 15, 593-606)

The reduced state of vitamin B(12), cob(I)alamin, acts as a supernucleophile that reacts ca. 10(5) 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.