New Urinary Metabolites Formed from Ring-Oxidized Metabolic Intermediates of Styrene

The Urine from mice exposed to styrene vapors (600 and 1200 mg/m(3), 6 h) was analyzed for ring-oxidized metabolites of styrene. To facilitate the identification of metabolites in urine, the following potential metabolites were prepared: 2-, 3-, and 4-vinylphenol (2, 3-, and 4-VP), 4-vinylpyrocatechol, and 2, 3-, and 4-vinylphenylmercapturic acid (2-, 3-, and 4-VPMA). For the analysis of vinylphenols beta-glucuronidase-treated Urine was extracted and derivatized with acetanhydride/triethylamine before injection into GUMS. Three isomers, 2-, 3-, and 4-VP, were found in the exposed urine using authentic standards. Additionally, three novel minor urinary metabolites, arylmereapturic acids 2-. 3-, and 4-VPMA, were identified by LC-ESI-MS2 by comparison with authentic standards. Excretion of the most abundant isomer, 4-VPMA, amounted to 535 +/- 47 nmol/kg and 984 +/- 78 nmol/kg, representing approximately 0.047 and 0.043% of the absorbed dose for the exposure levels of 600 and 1200 mg/m(3), respectively. The ratio of 2-VPMA, 3-VPMA, and 4-VPMA wits approximately 2:1:6. In model reactions of styrene 3.4-oxide (3,4-STO) with N-acetylcysteine in aqueous solutions and of its methyl ester in methanol, 4-vinylphenol wits always the main product, while 3-vinylphenol has never been detected. No mercapturic acid Wits found in the reaction of 3,4-STO with N-acutylcysteine in aqueous Solution at pH 7.4 or 9.7, but a small amount of 4-VPMA methyl ester was detected by LC-ESI-MS after the reaction of 3,4-STO with N-acetylcysteine methyl ester. In contrast, 110 mercapturic acid was found in (lie reaction of 3,4-STO with N-acelylcysteine in aqueous solution at pH 7.4 or 9.7. These findings indicate a capability of 3,4-STO to react with cellular thiol groups despite its rapid isomerization to vinylphenol in all aqueous environment. Moreover, the in vivo formation of 2- and 3-isomers of both VP and VPMA, neither of which wits formed front 3,4-STO in vitro, strongly suggests that another arene oxide, styrene 2,3-oxide, might be a minor metabolic intermediate of styrene.