Gupta S, Rogers L K, Taylor S K, Smith C V
Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Toxicol Appl Pharmacol. 1997 Oct;146(2):317-27. doi: 10.1006/taap.1997.8228.
The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophen in vivo. In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS-PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question.
对乙酰氨基酚诱导肝坏死的主要机制被认为是通过共价结合和硫醚形成或通过诸如S-硫醇化等氧化反应来消耗蛋白质硫醇。然而，在先前的研究中，我们并未检测到在体内给予肝毒性剂量的对乙酰氨基酚后蛋白质硫醇含量有显著损失。在本研究中，我们采用硫醇特异性试剂单溴代联苯胺进行衍生化，并通过SDS-PAGE分离蛋白质，以研究在对乙酰氨基酚诱导的肝坏死过程中特定蛋白质硫醇的可能损失。禁食的成年雄性小鼠给予对乙酰氨基酚，随后在通过差速离心分离的亚细胞组分中检测蛋白质硫醇状态。除了荧光强度显著降低外，蛋白质硫醇含量没有降低，但考马斯亮蓝染色显示，在给予对乙酰氨基酚的小鼠线粒体组分中，一条约130 kDa的单带蛋白质含量并未降低。通过分离和N端序列分析，该蛋白质被鉴定为氨甲酰磷酸合成酶-I（CPS-I）（EC 6.3.4.16）。给予肝毒性剂量对乙酰氨基酚的小鼠肝脏CPS-I活性降低。此外，给予肝毒性剂量对乙酰氨基酚的小鼠肝脏谷氨酰胺合成酶活性较低，血浆氨水平升高。观察到的高氨血症可能导致对乙酰氨基酚毒性剂量的不良反应，并阐明导致高氨血症的具体机制可能在临床上有用。然而，内质网中产生的化学反应性代谢产物优先消耗线粒体蛋白质的蛋白质硫醇含量，这提出了一个具有挑战性且可能提供信息的机制问题。
