Identification of an arene oxide metabolite of 2,2′,5,5′-tetrachlorobiphenyl by gas chromatography-mass spectroscopy

Abstract

Tritiated 2,2′,5,5′-tetrachlorobiphenyl ( 3H-TCB) was incubated with phenobarbital(PB)-induced rat liver microsomes in the presence of an epoxide hydrase inhibitor and a brominated analog (BrAO) of the expected metabolic intermediate, 2,2′,5,5′-tetrachlorobiphenyl-3,4-oxide (TCBAO). A putative arene oxide intermediate ( 3H-AO), which was radiolabeled, was separated from 3H-TCB and BrAO by column chromatography, high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC), and was analyzed for TCBAO by methods that were independent of radiometric techniques. The retention times (R t's) of TCBAO and 3H-AO on two gas chromatography (GC) columns were the same, both before and after acid catalyzed rearrangement. 3H-AO was further characterized by rearrangement to a mixture of 3- and 4-hydroxy-TCB that was identified by gas chromatography-mass spectroscopy (GC-MS). The rate of TCB metabolism and the production of 3H-AO by liver microsomes from a PB-induced, adult male rhesus monkey was less than that observed with rat microsomes. The 3H-AO from the monkey was also characterized as TCBAO by rearrangement to the characteristic TCB phenols that were analyzed by GC-MS using selective ion monitoring. This study is the first in which an arene oxide of a polychlorinated biphenyl (PCB) was actually isolated as a mammalian metabolite and subjected to direct chemical analysis. it is an obligatory intermediate in TCB oxidation. Direct hydroxylation, i.e. insertion of singlet oxygen (or the equivalent), as delineated by Tomaszewski et al. [29] may exist as an alternative to the addition of oxygen across the aromatic bond at the 3,4 position of TCB. Oxidative metabolism of biphenyl [30] and certain hexachlorobiphenyls [3, 4] appears to proceed both via arene oxide intermediates and by direct hydroxylation. Both mechanisms of aromatic hydroxylation, perhaps catalyzed by distinct P-450 cytochromes, may be involved in the production of the various TCB metabolites.