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

Abstract

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 GC/MS. Three isomers, 2-, 3-, and 4-VP, were found in the exposed urine using authentic standards. Additionally, three novel minor urinary metabolites, arylmercapturic acids 2-, 3-, and 4-VPMA, were identified by LC-ESI-MS(2) 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 was 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 was always the main product, while 3-vinylphenol has never been detected. No mercapturic acid was found in the reaction of 3,4-STO with N-acetylcysteine 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, no mercapturic acid was found in the reaction of 3,4-STO with N-acetylcysteine 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 an aqueous environment. Moreover, the in vivo formation of 2- and 3-isomers of both VP and VPMA, neither of which was formed from 3,4-STO in vitro, strongly suggests that another arene oxide, styrene 2,3-oxide, might be a minor metabolic intermediate of styrene.

Biomarkers of toluene exposure in rats: mercapturic acids versus traditional indicators (urinary hippuric acid ando-cresol and blood toluene)

“…The ratios for the different TMA isomers (2/1/6: o/m/p) are similar to those found for the corresponding ring oxidation mercapturic acids of ethylbenzene in rat (1/2/6) (Cossec et al, 2010) and for styrene in mice (2/1/6) (Linhart et al, 2010). Thus, the rates for glutathione's reaction with the 2,3-oxide and 3,4-oxide appear to be the same for all monosubstituted aromatic compounds in rodents.…”

Evidence for cellular protein covalent binding derived from styrene metabolite

“…Not only does it indicate that the protein modification attributed to the conjugation of styrene oxide with cysteine residues, but also it tells us that the antibody binding resulted from immunochemical recognition and not from non-specific binding. Recently, Linhart and co-workers reported urinary aromatic mercapturic acids in mice given styrene, indicating the formation of arene oxides as metabolic intermediates [52]. We may not exclude possible involvement of the reported arene oxides in protein adduction.…”

Detection of Phenolic Metabolites of Styrene in Mouse Liver and Lung Microsomal Incubations

“…The ratio of the detected 2-VP, 3-VP, and 4-VP was 27.7:1.0:2.8 in mouse liver microsomes. In a recent study, Linhart et al (2010) reported the detection of urinary 3-VP, 4-VP, possible 2-VP, and mercapturic acids derived from styrene-3,4-oxide and styrene-2,3-oxide in mice given styrene. They found approxi- mately 3-fold more mercapturic acid derived from styrene-3,4-oxide than that derived from styrene-2,3-oxide in the urine, inferring that styrene-3,4-oxide dominated in the formation of styrene arene oxides.…”

“…2-Vinylphenol (2-VP) and 3-vinylphenol (3-VP) have been proposed as aromatic hydroxylation products of styrene (Sumner and Fennell, 1994), but they have never been identified either in experimental animals or in humans exposed to styrene. Most recently, Linhart et al (2010) reported urinary 3-VP, 4-VP, and possible 2-VP in mice exposed to airborne styrene. Cruzan et al (2009) stated that differences in styrene oxide blood concentrations did not account for the species selectivity and tumor response to styrene.…”