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.
Dai
Jieyu
J
Center for Developmental Therapeutics, Seattle Children's Research Institute, Division of Gastroenterology, Department of Pediatrics, University of Washington, Seattle, WA 98101-1309, USA.
Zhang
Fan
F
Zheng
Jiang
J
D000111:Acetylcysteine / Q000737:chemistry; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007274:Injections, Intraperitoneal; D008745:Methylation; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D004852:Epoxy Compounds; D012710:Serum Albumin, Bovine; C013690:styrene oxide; D003545:Cysteine; D000111:Acetylcysteine
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