Jágr Michal, Mráz Jaroslav, Linhart Igor, Stránský Vladimír, Pospísil Martin
Centre of Occupational Health, National Institute of Public Health, Prague, Czech Republic.
Chem Res Toxicol. 2007 Oct;20(10):1442-52. doi: 10.1021/tx700057t. Epub 2007 Sep 15.
Styrene 7,8-oxide (SO), a reactive metabolic intermediate of the industrial chemical styrene, binds covalently at nucleophilic amino acid residues of blood proteins in vivo and in vitro. In this study, SO adducts with cysteine, lysine, and histidine were synthesized, characterized, and then used as authentic standards to assign and quantitate the SO adducts in globin incubated with SO. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine were prepared by direct alkylation of cysteine with (R)-SO or (S)-SO. To prepare the SO adducts with lysine and histidine, Nalpha-Boc-protected amino acids were alkylated with (R)-SO or (S)-SO followed by deprotection of the Boc group to obtain Nepsilon-(2-hydroxy-1-phenylethyl)lysine and Nepsilon-(2-hydroxy-2-phenylethyl)lysine as well as Npi-(2-hydroxy-1-phenylethyl)histidine, Npi-(2-hydroxy-2-phenylethyl)histidine, Ntau-(2-hydroxy-1-phenylethyl)histidine, and Ntau-(2-hydroxy-2-phenylethyl)histidine. The individual regioisomers were isolated from their mixtures by semipreparative HPLC, and their structure was assigned using NMR techniques. The SO-modified globin, isolated from human hemoglobin incubated in vitro with racemic SO at a molar ratio SO/globin of 100:1 or 10:1, was digested with pronase and subjected to LC/MS and GC/MS analysis. All known regioisomers of the SO adducts were detected, with S-(2-hydroxy-1-phenylethyl)cysteine, Nepsilon-(2-hydroxy-1-phenylethyl)lysine, and Ntau-(2-hydroxy-2-phenylethyl)histidine being the most abundant in the modified globin. Deuterated analogues of the SO adducts were employed as internal standards. The SO-amino acid adducts described here appear to be suitable biomarkers for long-term exposures to styrene or SO.
苯乙烯7,8 - 氧化物（SO）是工业化学品苯乙烯的一种活性代谢中间体，在体内和体外均能与血液蛋白质的亲核氨基酸残基共价结合。在本研究中，合成、表征了SO与半胱氨酸、赖氨酸和组氨酸的加合物，然后将其用作真实标准品，以鉴定和定量与SO孵育的球蛋白中的SO加合物。通过用（R）-SO或（S）-SO直接烷基化半胱氨酸制备S -（2 - 羟基 - 1 - 苯乙基）半胱氨酸和S -（2 - 羟基 - 2 - 苯乙基）半胱氨酸。为了制备SO与赖氨酸和组氨酸的加合物，用（R）-SO或（S）-SO对Nα - Boc保护的氨基酸进行烷基化，然后脱除Boc基团，得到Nε -（2 - 羟基 - 1 - 苯乙基）赖氨酸和Nε -（2 - 羟基 - 2 - 苯乙基）赖氨酸以及Nπ -（2 - 羟基 - 1 - 苯乙基）组氨酸、Nπ -（2 - 羟基 - 2 - 苯乙基）组氨酸、Nτ -（2 - 羟基 - 1 - 苯乙基）组氨酸和Nτ -（2 - 羟基 - 2 - 苯乙基）组氨酸。通过半制备HPLC从混合物中分离出各个区域异构体，并使用NMR技术确定其结构。从体外以SO/球蛋白摩尔比100:1或10:1与外消旋SO孵育的人血红蛋白中分离出的SO修饰球蛋白，用链霉蛋白酶消化并进行LC/MS和GC/MS分析。检测到了SO加合物的所有已知区域异构体，其中S -（2 - 羟基 - 1 - 苯乙基）半胱氨酸、Nε -（2 - 羟基 - 1 - 苯乙基）赖氨酸和Nτ -（2 - 羟基 - 2 - 苯乙基）组氨酸在修饰球蛋白中含量最高。SO加合物的氘代类似物用作内标。本文所述的SO - 氨基酸加合物似乎是长期接触苯乙烯或SO的合适生物标志物。
