In vitro metabolic activation of triphenyl phosphate leading to the formation of glutathione conjugates by rat liver microsomes

Abstract

The present study investigated the metabolism of the flame retardant and plasticizer chemical, triphenyl phosphate (TPHP), in a rat liver microsome-based in vitro assay with glutathione (GSH) in order to elucidate metabolic pathways leading to formation of conjugates. A highly sensitive and efficient method was developed for the detection and characterization of GSH reactive metabolites using LC-Q-TOF-MS/MS both in the negative and positive electrospray ionization modes. Seven GSH conjugates formed as a result of microsomal incubation, which were identified as S-conjugates based on MS/MS spectra, and confirmed by subsequent time-dependent incubation assays. With the exception of hydrolysis reactions leading to formation of a diester metabolite, diphenyl phosphate (DPHP), the results demonstrated that Phase I epoxidation on phenyl ring of TPHP leading to mono- and di-hydroxylated TPHP metabolites, which can further conjugate with GSH. Depending on hydroxylated TPHP formation, an o-hydroquinone intermediate formed in vitro via Phase I metabolism, and the o-benzoquinone form reacted with GSH and also formed GSH conjugates. The present study showed that via hydroxylated TPHP Phase I formation that GSH conjugates are important Phase II metabolites for TPHP metabolism in vitro. Some GSH conjugates may be valuable candidate biomarkers for monitoring TPHP exposure in biota.