New metabolites of styrene, three isomeric vinylphenylmercapturic acids (2-, 3-, and 4-VPMA), were recently identified by LC-ESI-MS in the urine of mice. In this study, 4-VPMA together with traces of 2- and 3-VPMA were found also in the urine of hand-lamination workers, which were exposed to styrene vapours at concentrations ranging from 23 to 244mg/m(3). Concentrations of 4-VPMA in these end-of-shift samples were 4.59±3.64ng/mL (mean±S.D.; n=10), those found next morning after the work-shift were 2.14±2.07ng/mL (mean±S.D.; n=10). Strong correlation (R=0.959) was found in the next-morning samples between concentrations of 4-VPMA and phenylglyoxylic acid, whereas correlations found between 4-VPMA and mandelic acid in both end-of-shift and next-morning samples were much weaker. The excretion of 4-VPMA accounted for only about 3.5×10(-4)% of the absorbed dose of styrene. Despite very low metabolic yield, formation of VPMAs clearly indicates occurrence and extent of styrene ring oxidation considered to be a toxicologically relevant metabolic pathway.

ABSTRACTMetabolism of benzene, an important environmental and industrial carcinogen, produces three electrophilic intermediates, namely, benzene oxide and 1,2‐ and 1,4‐benzoquinone, capable of reacting with the DNA. Numerous DNA adducts formed by these metabolites in vitro have been reported in the literature, but only one of them was hitherto identified in vivo. In a search for urinary DNA adducts, specific LC‐ESI‐MS methods have been developed for the determination in urine of six nucleobase adducts, namely, 7‐phenylguanine, 3‐phenyladenine, 3‐hydroxy‐3,N4‐benzethenocytosine, N2‐(4‐hydroxyphenyl)guanine, 7‐(3,4‐dihydroxyphenyl)guanine and 3‐(3,4‐dihydroxyphenyl)‐adenine (DHPA), with detection limits of 200, 10, 260, 50, 400 and 200 pg ml−1, respectively. Mice were exposed to benzene vapors at concentrations of 900 and 1800 mg m−3, 6 h per day for 15 consecutive days. The only adduct detected in their urine was DHPA. It was found in eight out of 30 urine samples from the high‐exposure group at concentrations of 352 ± 146 pg ml−1 (mean ± SD; n = 8), whereas urines from the low‐exposure group were negative. Assuming the DHPA concentration in the negative samples to be half of the detection limit, conversion of benzene to DHPA was estimated to 2.2 × 10−6% of the absorbed dose. Thus, despite the known high mutagenic and carcinogenic potential of benzene, only traces of a single DNA adduct in urine were detected. In conclusion, DHPA is an easily depurinating adduct, thus allowing indication of only high recent exposure to benzene, but not long‐term damage to DNA in tissues. Copyright © 2012 John Wiley & Sons, Ltd.

A simple convenient method has been developed for the preparation of N-acetyl-S-arylcysteines based on the Chan-Lam-Evans arylation of N,N'-diacetylcystine dimethyl ester with arylboronic acids and used to synthesize a series of aryl-mercapturic acids. Unlike copper-mediated N-arylation, the S-arylation of neither cysteine nor cystine derivatives proceeded satisfactorily in the presence of air. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Urine samples from humans occupationally exposed to styrene, with mandelic acid levels ranging from 400 to 1145 mg/g creatinine and from 68 to 400mg/g creatinine for high and low exposure group, respectively, were analysed for N3 adenine DNA adducts, namely, 3-(2-hydroxy-1-phenylethyl)adenine (N3 alpha A) and 3-(2-hydroxy-2-phenylethyl)adenine (N3 beta A). A sensitive LC-ESI-MSMS method was developed with the limit of quantification of 1 pg/mL for both analytes. Peaks corresponding to N3 alpha A and/or N3 beta A were found in seven of nine end-of-shift samples of the high exposure group and in six of 19 end-of-shift samples of the low exposure group. Concentration of N3 alpha A+N3 beta A amounted to 2.8+/-1.6 pg/mL (mean+/-S.D.; n=9) and 1.8+/-1.3 pg/mL (mean+/-S.D.; n=19) in the high and low exposure group, respectively. Of other 10 samples taken the next morning after exposure, two contained low but quantifiable concentrations of N3 alpha A and none contained N3 beta A. However, interfering peaks were detected also in some control urine samples. Out of 22 controls, six and two samples contained peaks co-eluting with N3 alpha A and N3 beta A, respectively. Therefore, the method used was found insufficiently specific to be applicable for biological monitoring. Comparing the excretion of N3 alpha A+N3 beta A to that reported previously in mice it can be estimated that at the same absorbed dose, humans excreted not more than 1/30 of the amount of adenine adducts excreted by mice. As a consequence, the damage to DNA caused by styrene 7,8-oxide (SO), a reactive metabolite of styrene, appears to be much lower in humans than in mice.

A series of N3 and N9 aryladenines was prepared by arylation of 8bromoadenine under modified Chan-Lam-Evans coupling conditions followed by reductive debromination with hydrogen on palladium.Conditions of arylation were optimised to maximise the yield of N3-arylated products.The selectivity of the reaction varied with temperature the ligand used.The best results were obtained in DMF with phenanthroline as a ligand at 60°C.

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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.

New urinary adenine adducts, 3-(2-hydroxy-1-phenylethyl)adenine (N3αA), 3-(2-hydroxy-2-phenylethyl)adenine (N3βA), were found in the urine of mice exposed to styrene vapour. These styrene 7,8-oxide derived adenine adducts as well as previously identified guanine adducts, 7-(2-hydroxy-1-phenylethyl)guanine (N7αG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7βG) were quantified by HPLC–ESI–MS2 and the excretion profile during and after a repeated exposure to 600 mg/m3 or 1200 mg/m3 of styrene for 10 consecutive days (6 h/day) was determined. The excretion was dose dependent. Total N3 adenine adducts (N3αA + N3βA) excreted amounted to nearly 0.8 × 10−5% of the absorbed dose while urinary N7 guanine adducts (N7αG + N7βG) amounted to nearly 1.4 × 10−5% of the dose. No accumulation of the adducts was observed. Due to rapid depurination from the DNA, the excretion of both N3 adenine and N7 guanine adducts ceased shortly after finishing the exposure. Both N3 adenine and N7 guanine adducts may be used as non-invasive biomarkers of effective dose reflecting only a short time exposure to styrene.

3-(2-Hydroxy-2-phenylethyl)- and 3-(2-hydroxy-1-phenylethyl)adenine, DNA adducts derived from styrene, along with their 9-substituted analogues were prepared by alkylation of 8-bromoadenine with corresponding allyl-protected bromohydrins followed by a new deallylation procedure using tetrakis(triphenylphosphine)palladium catalyzed reductive cleavage by poly(methylhydrosiloxane) in the presence of p-toluenesulphonic acid. This novel procedure proved to be useful for purine derivatives, which were resistant to other deallylation protocols. Structure of positional isomers was assigned using 21) NMR experiments HMBC and HMQC.