Gas Chromatographic Analysis with Chiral Cyclodextrin Phases Reveals the Enantioselective Formation of Hydroxylated Polychlorinated Biphenyls by Rat Liver Microsomes

Abstract

Chiral PCB congeners are major components of PCB mixtures and undergo enantioselective biotransformation to hydroxylated (OH-)PCBs by cytochrome P450 enzymes. While it is known that biotransformation results in an enantiomeric enrichment of the parent PCB, it is currently unknown if OH-PCBs are formed enantioselectively. The present study screened seven commercial capillary gas chromatography columns containing modified β- or γ-cyclodextrins for their potential to separate the atropisomers of methylated derivatives of OH-PCB. The atropisomers of 3-, 4- and 5-methoxy derivatives were at least partially separated on one or more columns. A subsequent biotransformation study was performed with rat liver microsomes to assess if hydroxylated metabolites are formed enantioselectively from PCBs 91, 95, 132 and 149. The OH-PCBs were extracted from the microsomal incubations, derivatized with diazomethane and analyzed as the respective methoxylated (MeO-)PCB derivatives using selected columns. The 5-hydroxylated metabolites of PCBs 91, 95, 132 and 149 were the major metabolites, which is consistent with PCB’s biotransformation by cytochrome P450 2B enzymes. All 5-hydroxylated metabolites displayed a clear, congener-specific enantiomeric enrichment. Overall, this study demonstrates for the first time that chiral PCBs, such as PCB 91, 95, 132 and 149, are enantioselectively metabolized to OH-PCBs by cytochrome P450 enzymes.

Chiral polychlorinated biphenyls: absorption, metabolism and excretion—a review

“…Incubations of individual, racemic PCB congeners or a mixture of several C-PCBs with rat CYP2B1 show preferential transformation of E 2 -PCB 45, (−)-PCB 84, E 1 -PCB 91, E 2 -PCB 95, (−)-PCB 132, (+)-PCB 136 and (−)-PCB 149. The same direction of atropisomeric enrichment is observed in rat liver microsomal incubations (Kania-Korwel et al 2011, Kania-Korwel and Lehmler 2013, Wu et al 2011) and, for PCB 136, in rat liver tissue slices (Wu et al 2013b). At the same time, the formation of HO-PCB metabolites by rat P450 enzymes is atropselective, thus resulting in an atropisomeric enrichment of the HO-PCBs.…”

“…A convenient nomenclature for PCB metabolites has been proposed by Maervoet et al and, as shown for PCB 136 in Fig 3, will be used throughout this manuscript (Maervoet et al 2004). The oxidation of C-PCBs, in particular those with a 2,3,6-trichloro substitution pattern in one phenyl ring, to HO-PCBs has been studied extensively using recombinant enzymes (Lu et al 2013, Lu and Wong 2011, Waller et al 1999, Warner et al 2009), hepatic microsomes (Kania-Korwel et al 2011, Kania-Korwel and Lehmler 2013, Schnellmann et al 1983, Wu et al 2014, Wu et al 2011), isolated hepatocytes (Vickers et al 1986) and, liver, hippocampus and skin slices (Garner et al 2006, Wu et al 2013a, Wu et al 2013b) obtained from mammalian species. To the best of our knowledge, the oxidation of C-PCBs in non-mammalian species, such as amphibians, fish or avian species, has not been investigated to date.…”

“…The 4,5-epoxide intermediate of C-PCBs with a 2,3,6-trichloro substitution pattern can rearrange to form both 4- or 5-hydroxylated PCB metabolites. In addition, a 1,2-shift (or NIH-shift) (Guroff et al 1967, Jerina and Daly 1974) of the 3-chloro substituent can lead to the formation of HO-PCBs with a 2,4,6-trichloro-3-hydroxy substitution pattern as minor metabolites (Kania-Korwel et al 2012, Kania-Korwel et al 2011, Wu et al 2013a, Wu et al 2014). Studies with recombinant P450 enzymes demonstrate that CYP2B isoforms, such as rat CYP2B1 (Lu et al 2013, Waller et al 1999, Warner et al 2009), human CYP2B6 (Warner et al.…”

“…Several studies using recombinant P450 enzymes (Lu et al 2013, Warner et al 2009), liver microsomes (Kania-Korwel et al 2011, Kania-Korwel and Lehmler 2013, Wu et al 2014, Wu et al 2011) or liver tissue slices (Wu et al 2013a, Wu et al 2013b) demonstrate that PCBs are atropselectively metabolized to HO-CPBs by P450 enzymes, thus resulting in an atropisomeric enrichment of PCBs and HO-PCBs in the microsomal incubation (Table 2). The atropselective biotransformation of C-PCBs to HO-PCB metabolites by recombinant rat and human cytochrome P450 enzymes has been studies in some detail (Lu et al 2013, Warner et al 2009).…”

“…The availability of small quantities of pure PCB atropisomers allowed the determination of the optical rotation (i.e., (+)- vs. (−)-enantiomer) (Haglund 1996b); the elution order on different enantioselective gas chromatography columns (Haglund and Wiberg 1996) and the absolute configuration (i.e., R - vs. S -enantiomer) of some, but not all C-PCBs (Toda et al 2012). Moreover, the metabolites of C-PCBs are also chiral due to the hindered rotation around the central C-C bond (Nezel et al 1997), and—as with the parent compounds—can be separated into their atropisomers using liquid chromatography (Pham-Tuan et al 2005, Zhai et al 2013) or gas chromatography (Kania-Korwel et al 2011, Kania-Korwel et al 2008c). The absolute configuration of several C-PCB metabolites has been determined using a combination of experimental and computational techniques (Pham-Tuan et al 2005).…”