Cai H, Guengerich F P
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Chem Res Toxicol. 2001 Jan;14(1):54-61. doi: 10.1021/tx000185n.
Trichloroethylene (TCE) shows several types of toxicities, some of which may be the result of bioactivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed N(6)-acyllysine adducts formed from the reaction of TCE oxide with proteins [Cai, H., and Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327-335]; however, we had been unable to measure ester adducts under the prolonged conditions of proteolysis and derivatization. Protein amino acid adducts were directly observed by mass spectrometry during the reaction of TCE oxide with the model polypeptides insulin and adrenocorticotropic hormone (ACTH, residues 1-24). The majority (80%) of the protein adducts were unstable under physiological conditions and had a collective t(1/2) of approximately 1 h, suggesting that they are ester type adducts formed from reactions of Cys, Ser, Tyr, or Thr residues with intermediates formed in TCE oxide hydrolysis. Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr had half-lives of 1 h and 10 min at pH 8.0, respectively, similar to the stabilities of the protein adducts. The effects of TCE oxide adduct formation on catalytic activities were examined with five model enzymes. No recovery of catalytic activity was observed during the reaction of TCE oxide with two model enzymes for which the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate dehydrogenase. However, in the cases of papain (essential Cys residue in the active site), alpha-chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr residues), time-dependent recoveries of enzyme activity were observed following reaction with TCE oxide or either of two model nucleophiles (dichloroacetyl chloride and acetic formic anhydride), paralleling the kinetics of removal of adducts from insulin and ACTH. Formation of adducts ( approximately 2%) was detected in the direct reaction of TCE oxide with 2'-deoxyguanosine, but not with the other three nucleosides found in DNA. During the reaction of TCE oxide with a synthetic 8-mer oligonucleotide, formation of adducts was observed by mass spectrometry. However, the adducts had a t(1/2) of 30 min at pH 8.5. These results indicate the transient nature of the adducts formed from the reaction of TCE oxide with macromolecules and their biological effects.
三氯乙烯（TCE）具有多种毒性，其中一些可能是生物活化的结果。细胞色素P450s氧化可产生亲电试剂三氯乙烯氧化物。我们之前分析了三氯乙烯氧化物与蛋白质反应形成的N(6)-酰基赖氨酸加合物[Cai, H., and Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327 - 335]；然而，在长时间的蛋白水解和衍生化条件下，我们一直无法测量酯加合物。在三氯乙烯氧化物与模型多肽胰岛素和促肾上腺皮质激素（ACTH，1 - 24位残基）的反应过程中，通过质谱直接观察到了蛋白质氨基酸加合物。大多数（80%）蛋白质加合物在生理条件下不稳定，其集体半衰期约为1小时，这表明它们是由半胱氨酸、丝氨酸、酪氨酸或苏氨酸残基与三氯乙烯氧化物水解形成的中间体反应生成的酯类加合物。合成的O - 乙酰 - L - 丝氨酸和O - 乙酰 - L - 酪氨酸在pH 8.0时的半衰期分别为1小时和10分钟，与蛋白质加合物的稳定性相似。用五种模型酶研究了三氯乙烯氧化物加合物形成对催化活性的影响。在三氯乙烯氧化物与两种模型酶的反应过程中未观察到催化活性的恢复，文献表明这两种酶中赖氨酸起作用，即兔肌肉醛缩酶和葡萄糖 - 6 - 磷酸脱氢酶。然而，在木瓜蛋白酶（活性位点有必需半胱氨酸残基）、α - 胰凝乳蛋白酶（关键丝氨酸残基）和D - 氨基酸氧化酶（必需半胱氨酸和酪氨酸残基）的情况下，与三氯乙烯氧化物或两种模型亲核试剂（二氯乙酰氯和乙酸甲酸酐）反应后，观察到酶活性随时间的恢复，这与胰岛素和ACTH上加合物去除的动力学情况相似。在三氯乙烯氧化物与2'-脱氧鸟苷的直接反应中检测到了加合物（约2%）的形成，但在与DNA中发现的其他三种核苷的反应中未检测到。在三氯乙烯氧化物与合成的8聚体寡核苷酸的反应过程中，通过质谱观察到了加合物的形成。然而，这些加合物在pH 8.5时的半衰期为30分钟。这些结果表明三氯乙烯氧化物与大分子反应形成的加合物具有瞬时性及其生物学效应。
