Detection of protein adduction derived from styrene oxide to cysteine residues by alkaline permethylation.

Styrene oxide-cysteine adduction is predominantly involved in protein covalent modification after exposure in vivo to styrene or styrene oxide. In the present study, we developed an alkaline permethylation- and GC/MS-based approach to detect styrene oxide-derived protein adduction. Permethylation of the protein adducts produced two methylthiophenylethanols, namely 2-methylthio-2-phenyl-1-ethanol and 2-methylthio-1-phenyl-1-ethanol. To improve the permethylation efficiency, reaction conditions, including temperature, time, NaOH strength, and molar ratio of CH(3)I/NaOH, were explored. Under optimized conditions, the yields of the analyte formation resulting from permethylation of authentic standard alpha- and beta-mercapturic acids, representing alpha and beta isomers of cysteine adducts, were 35% and 28%, respectively. Permethylation of styrene oxide-modified bovine serum albumin released the two methylthiophenylethanols with an alpha-/beta-adduction ratio of 1.5. A concentration-dependent increase in both alpha- and beta-adduction was observed in mouse liver microsomes incubated with styrene at various concentrations. CD-1 mice were administered intraperitoneally with styrene at doses of 0, 50, and 400mg/kg daily for 5 days. The formation of protein adducts derived from styrene oxide in whole blood in 400mg/kg group was observed with an alpha/beta ratio of 4.8, suggesting that the reaction of styrene oxide with cysteine residues took place more likely at the alpha-carbon than the beta-carbon of styrene oxide. Copyright 2010 Elsevier Inc. All rights reserved.