Development of Polyclonal Antibodies for the Detection of Styrene Oxide Modified Proteins

Wei, Yuan; Chung, Jou-Ku; Gee, Shirley J.; Hammock, Bruce D.; Zheng, Jiang

doi:10.1021/tx600340c

Cited by 11 publications

(24 citation statements)

References 27 publications

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“…Protein adduction was monitored by immunoblot using the polyclonal antibodies we developed earlier [46]. The antibodies were raised to specifically recognize styrene oxide cysteinyl protein adducts 3 and 4 (Scheme 1).…”

Section: Resultsmentioning

confidence: 99%

“…Protein adduction was assessed by immunoblots, using the polyclonal antibodies we developed previously. The antibodies have been proved to selectively recognize styrene oxide cysteine adducts, and the recognition selectivity was probed by competitive ELISA and competitive immunoblots [46]. As shown in Figure 4, several chemiluminescent bands were detected in the lanes loaded with protein samples obtained from h2E1 cells after exposure to styrene, particularly at concentration of 1000 μM.…”

Section: Discussionmentioning

confidence: 99%

“…Styrene oxide-derived mercapturic acid I ( 5 , Scheme 1) (2-(acetylamino)-3-(2-hydroxy-1-phenylethylthio)propanoic acid) and styrene oxide-derived mercapturic acid II ( 6 , Scheme 1) (2-(acetylamino)-3-(2-hydroxy-2-phenylethylthio)propanoic acid) were synthesized as reported earlier by our laboratory [46] and their structures were confirmed by both mass spectrometry and NMR. Rabbit antiserum #1043 against styrene oxide cysteinyl protein adducts was developed in our laboratory [46].…”

Section: Methodsmentioning

confidence: 99%

“…Rabbit antiserum #1043 against styrene oxide cysteinyl protein adducts was developed in our laboratory [46]. …”

Section: Methodsmentioning

confidence: 99%

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Evidence for cellular protein covalent binding derived from styrene metabolite

Wei

Jin

Chung

et al. 2010

Chemico-Biological Interactions

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Styrene is one of the most important industrial intermediates consumed in the world. Human exposure to styrene occurs mainly in the reinforced plastics industry, particularly in developing countries. Styrene has been found to be hepatotoxic and pneumotoxic in humans and animals. The biochemical mechanisms of styrene-induced toxicities remain unknown. Albumin and hemoglobin adduction derived from styrene oxide, a major reactive metabolite of styrene, has been reported in blood samples obtained from styrene exposed workers. The objectives of the current study focused on cellular protein covalent binding of styrene metabolite and its correlation with cytotoxicity induced by styrene. We found that radioactivity was bound to cellular proteins obtained from mouse airway trees after incubation with 14C-styrene. Microsomal incubation studies showed that the observed protein covalent binding required the metabolic activation of styrene. The observed radioactivity binding in protein samples obtained from the cultured airways and microsomal incubations were significantly suppressed by co-incubation with disulfiram, a CYP2E1 inhibitor, although disulfiram apparently did not show a protective effect against the cytotoxicity of styrene. A 2-fold increase in radioactivity bound to cellular proteins was detected in cells stably transfected with CYP2E1 compared to the wild-type cells after 14C-styrene exposure. With the polyclonal antibody developed in our lab, we detected cellular protein adduction derived from styrene oxide at cysteinyl residues in cells treated with styrene. Competitive immunoblot studies confirmed the modification of cysteine residues by styrene oxide. Cell culture studies showed that the styrene-induced protein modification and cell death increased with the increasing concentration of styrene exposure. In conclusion, we detected cellular protein covalent modification by styrene oxide in microsomal incubations, cultured cells, and mouse airways after exposure to styrene and found a good correlation between styrene-induced cytotoxicity and styrene oxide-derived cellular protein adduction.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Rabbit antiserum #1043 against styrene oxide cysteinyl protein adducts was developed in our laboratory [46]. …”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Evidence for cellular protein covalent binding derived from styrene metabolite

Wei

Jin

Chung

et al. 2010

Chemico-Biological Interactions

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“…Polyclonal antibodies to detect protein adduction derived from styrene oxide were developed in our laboratory [16, 17]. The antibody approach shares the advantage of its application in immunoblot, immuno-histologic staining and immuno-affinity chromatography, but it lacks offering quantitative assessment of protein adduction.…”

Section: Introductionmentioning

confidence: 99%

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

Dai

Zhang

Zheng

2010

Analytical Biochemistry

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Styrene oxide-cysteine adduction predominantly involves in protein covalent modification undergoing in vivo after exposed to styrene or styrene oxide. In 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 CH3I/NaOH, were explored. Under the optimized condition, the yields of the analyte formation resulting from permethylation of authentic standard α- and β-mercapturic acids, representing α and β isomers of cysteine adducts, were 35% and 28%, respectively. Permethylation of styrene oxide-modified bovine serum albumin released the two methylthiophenylethanols with an α-/β-adduction ratio of 1.5. A concentration-dependent increase in both α- and β-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 400 mg/kg daily for 5 days. The formation of protein adducts derived from styrene oxide in whole blood in 400 mg/kg group was observed with an α/β ratio of 4.8, suggesting the reaction of styrene oxide with cysteine residues took place more likely at α-carbon than β-carbon of styrene oxide.

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Proteome changes in human bronchoalveolar cells following styrene exposure indicate involvement of oxidative stress in the molecular‐response mechanism

et al. 2009

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Styrene is a volatile organic compound (VOC) that is widely used as an intermediate in many industrial settings. There are known adverse health effects at environmentally significant concentrations, but little is known about the molecular effect of exposure to styrene at subacute toxic concentrations. We exposed human lung epithelial cells, at a wide range of concentrations (1 mg/m3-10 g/m3), to styrene and analyzed the effects on the proteome level by 2-DE, where 1,380 proteins spots were detected and 266 were identified unambiguously by mass spectrometry. A set of 16 protein spots was found to be significantly altered due to exposure to styrene at environmentally significant concentrations of 1-10 mg/m3 (0.2 – 2.3 ppm). Among these, superoxide dismutase [Cu-Zn] as well as biliverdin reductase A could be correlated with the molecular pathway of oxidative stress, while eukaryotic translation initiation factor 5A-1, ezrin, lamin B2 and voltage dependent anion channel 2 have been reported to be involved in apoptosis. Treatment with styrene also caused formation of styrene oxide-protein adducts, specifically for thioredoxin reductase 1. These results underline the relevance of oxidative stress as a primary molecular response mechanism of lung epithelial cells to styrene exposure at indoor-relevant concentrations.

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Development of Polyclonal Antibodies for the Detection of Styrene Oxide Modified Proteins

Cited by 11 publications

References 27 publications

Evidence for cellular protein covalent binding derived from styrene metabolite

Evidence for cellular protein covalent binding derived from styrene metabolite

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

Proteome changes in human bronchoalveolar cells following styrene exposure indicate involvement of oxidative stress in the molecular‐response mechanism

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