Identification of glutathione and related cysteine conjugates derived from reactive metabolites of methyleugenol in rats

• Biliary GSH conjugates via metabolites of methyleugenol were identified in rats.
• The GSH conjugates were verified by microsomal incubations and chemical synthesis.
• Cysteine conjugates related with the GSH conjugates were detected in rat urine.

Methyleugenol (ME), an alkenylbenzene compound, is a constituent of many foods and is used as flavoring agent in foodstuffs and as fragrance in cosmetics. It has been reported that exposure to ME can cause carcinogenicity, cytotoxicity, and genotoxicity. Metabolic activation is suggested to play an important role in ME-induced toxicities. Electrophilic metabolites of ME have been reported to covalently bind to proteins and nucleic acids. The objective of this study was to identify GSH and related cysteine conjugates derived from these reactive metabolites in vivo. Five biliary GSH (M1-M5) and four urinary cysteine conjugates (M6-M9) were detected in rats given ME. M1 and M2 were GSH conjugates derived from the epoxide of ME. M3, M4, and M5 were GSH conjugates possibly generated from the corresponding α,β-unsaturated aldehyde, carbonium ion, and quinone methide, respectively. The structures of the GSH conjugates were verified by chemical synthesis. Cysteine conjugates M6, M7, M8, and M9 were found to correspond to the respective M1/M2, M3, M4, and M5. The data obtained from the present in vivo work facilitate the understanding of mechanism action of ME toxicities and may provide information suitable for use as biomarkers of exposure to ME.