Detection Of Some Styrene Metabolites And Investigation Of Their Toxicity

Styrene(CAS:100-42-5)is an important material of plastic and rubber industry. Styrene can polymerize to polystyrene or form copolymers with other monomers.It was used to manufacture plastic resin and composite rubber.Plastic and rubber products containing styrene are widely used in daily life and industry,such as packaging materials,insulation,disposable food containers,and cups et al.The annual production of styrene is huge.The global annul production of styrene is 30 million tons,of which more than 1 million tons is produced in China.Styrene is also produced by certain plants,smoking and car exhaust.The main situation of toxic exposure is the workers exposed to styrene in the plastic production environment.The workers were mainly exposed through respiratory tract and skin contact.Short time exposure to styrene can cause irritation to skin and respiratory tract.Long time exposure cause lung injury and tend to have lung infection.Styrene is a potential carcinogen,but its carcinogenicity to human is still not confirmed.Styrene metabolism research was primarily performed in mice,rats,and human, exhibiting a significant species difference.Styrene was metabolized more fastly in mouse than in rat and human.Styrene is mainly metabolized to styrene oxide by cytochrome P450s.Styrene oxide is an oxidizing agent and can cause cell oxidizing injury.And styrene oxide is also an electrophilic compound and can react with nucleophilic biomolecules such as proteins and nucleotides,forming covalent binding and changing protein function,inducing mutation.The oxidation of phenyl ring is another minor metabolism pathway of styrene. This study is aimed to obtain further knowledge of the mechanism of styrene toxicity.We investigated styrene toxicity from the following three parts.1.Detection of diol metabolites of styrene and 1,3-butadiene by LC/ESI-MS methodStyrene and 1,3-butadiene are important intermediates extensively used in the plastics industry.They are mainly metabolized through cytochromes P450-mediated oxidation to the corresponding epoxides,which are subsequently converted to diols by epoxide hydrolase or through spontaneous hydration.The resulting styrene glycol and 3-butene-1,2-diol have been suggested as biomarkers of exposure to styrene and 1,3-butadiene, respectively.Unfortunately,poor ionization of the diols within electrospray mass spectrometers becomes the obstacle to the detection of the two diols by LC/ESI-MS. We developed an LC/ESI-MS approach to analyze styrene glycol and 3-butene-1,2 -diol by means of derivatization with 2-bromopyridine-5-boronic acid(BPBA), which not only dramatically increases the sensitivity of diol detection but also facilitates the identification of the diols.The analytical approach developed was simple,quick,and convincing without need of complicated chemical derivatization.In order to evaluate the feasibility of BPBA as a derivatizing reagent of diols,we investigated the impact of diol configuration on the affinity of a selection of diols to BPBA using the established LC/ESI-MS approach.We found that cis-diols showed higher affinity to BPBA than trans-diols and that the reactivity of vicinal diols to BPBA decreased with the increase in their molecular rigidity.In conclusion,BPBA may be used as a general derivatizing reagent for the detection of vicinal diols by LC/MS.2.Development of enantioselective polyclonal antibodies to detect styrene oxide protein adductsStyrene has been reported to be pneumotoxic and hepatotoxic in humans and animals.Styrene oxide,a major reactive metabolite of styrene,has been found to form covalent binding with proteins,such as albumin and hemoglobin.Styrene oxide has two optical isomers and it was reported that the(R)-enantiomer was more toxic than the (S)-enantiomer.The purpose of this study was to develop polyclonal antibodies that can stereoselectively recognize proteins modified by styrene oxide enantiomers at cysteine residues.Immunogens were prepared by alkylation of thiolated keyhole limpet hemocyanin(KLH)with styrene oxide enantiomers.Polyclonal antibodies were raised by immunization of rabbits with the chiral immunogens.Titration tests showed all six rabbits generated high titers of antisera that recognize(R)-or(S)-coating antigens accordingly.No cross-reaction was observed toward the carrier protein(BSA).All three rabbits immunized with(R)-immunogen produced antibodies that show enantioselectivity to the corresponding antigen,while only one among the three rabbits immunized with(S)-immunogen generated antibodies with enantioselectivity of the recognition.The enantioselectivity was also observed in competitive ELISA and immunoblot analysis.Additionally,competitive ELISA tests showed that the immuno-recognition required the hydroxyl group of the haptens.Affinity chromatography purification of antibody can improve the enatioselectivity but can not be removed totally. Immunoblot analysis demonstrated that the immuno-recognition depended on the amount of protein adducts blotted and hapten loading in protein adducts.3.The roles of vinylphenols in styrene toxicityOxidation on the phenyl ring is another reported metabolism pathway of styrene. 4-Vinylphenol(4-VP),which is a ring oxidation product,was found as a minor metabolite in the urine of the workers exposed to styrene.It was also observed in mice and rats after exposure to styrene.It was reported that approximately 6%of urinary styrene metabolites were ring oxidized productds in mouse,whereas in rat that accounted for only about 1%urinary metabolites and in human was less than 0.1%.4-VP is a minor metabolite of styrene,while its toxicity in vivo is much higher than styrene oxide.The importance of 4-VP in styrene-induced toxicity is unknown.We applied 4-fluorostyrene to probe the role of 4-VP in styrene-induced toxicity and found that 4-fluorostyrene showed similar toxicity potential as styrene both in cultured cells and mice.In mouse liver microsomal incubations,4-fluorostyene failed to be metabolized to 4-VP,while 4-fluorostyrene oxide was observed as the major metabolite of 4-fluorostyrene.After 10 min incubation in mouse liver microsome,4-VP/SG formation rate is only 0.07%.The observed toxicity of 4-fluorostyrene along with the lacking of the formation of 4-VP from 4-fluorostyrene implies that 4-VP has little contribution to the cytotoxicity of styrene.A comprehensive study was conducted to explore oxidative metabolites of styrene using mouse liver microsomes.Besides styrene 7,8-oxide and 4-VP,we identified 2-VP and 3-VP which have never been reported.In addition,2-VP,3-VP, and 4-VP were found to be metabolized to the corresponding catechol and dihydroxystyrene as well as their 7,8-oxides.