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 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.
Linhart
Igor
I
Department of Organic Chemistry, Faculty of Chemical Technology, Institute of Chemical Technology, Prague, Technicka 1905, CZ-166 28 Prague, Czech Republic. linharti@vscht.cz
Mráz
Jaroslav
J
Scharff
Jan
J
Krouzelka
Jan
J
Dusková
Sárka
S
Nohová
Hana
H
Vodicková
L'udmila
L
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000652:urine*; D000818:Animals; D002396:Catechols / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D051379:Mice; D010636:Phenols / Q000652:urine*; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D020058:Styrene / Q000378:metabolism*
D002396:Catechols; D010636:Phenols; D020058:Styrene; C034221:catechol; C030626:4-vinylphenol; D000111:Acetylcysteine