Hartley D P, Kroll D J, Petersen D R
Department of Pharmaceutical Sciences, Hepatobiliary Research Center, University of Colorado Health Sciences Center, Denver 80262, USA.
Chem Res Toxicol. 1997 Aug;10(8):895-905. doi: 10.1021/tx960181b.
Toxicity associated with prooxidant-mediated hepatic lipid peroxidation is postulated to originate from the interaction of the aldehydic end products of lipid peroxidation with cellular constituents. The principal alpha,beta-unsaturated aldehydic products of lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), are known to modify proteins through covalent alkylation of lysine, histidine, and cysteine amino acid residues. To detect and characterize the formation of 4-HNE- and MDA-adducted proteins during prooxidant-initiated lipid peroxidation, rabbit polyclonal antibodies were raised to 4-HNE-sulfhydryl, dinitrophenylhydrazine (DNPH)-4-HNE-sulfhydryl, and MDA-amine conjugates of keyhole limpet hemocyanin (KLH). Each antiserum displayed high antibody titers to either 4-HNE-metallothionein, DNPH-albumin, or MDA-albumin adducts when measured by ELISA. To study the formation of 4-HNE- and MDA-protein adducts during prooxidant-initiated cellular injury, isolated hepatocytes were exposed to either carbon tetrachloride or iron/ascorbate for 2 h. Indices of hepatocellular oxidative stress (i.e., cell viability and glutathione status) and lipid peroxidation (i.e., formation of 4-HNE, protein carbonyls, and MDA) were monitored continuously. Hepatocellular viability was affected moderately by carbon tetrachloride, while cellular reduced glutathione status was moderately affected by both iron/ascorbate and carbon tetrachloride. Levels of MDA and protein carbonyls increased dramatically with both prooxidants, whereas 4-HNE levels did not change significantly over the time course studied. In addition, hepatocellular proteins were immunoprecipitated with each antiserum, and aldehyde-modified immunopositive proteins were detected by immunoblotting. Prooxidant-induced increases in MDA corresponded with increases in intensity and number of MDA-adducted proteins over the time course studied. A total of 13 MDA-modified proteins (20, 25, 28, 30, 33, 38, 41, 45, 80, 82, 85, 130, and 150 kDa) were detected with the MDA-amine antiserum. Additionally, both iron/ascorbate- and carbon tetrachloride-induced formation of DNPH-derivatizable protein carbonyls corresponded quantitatively with the ability to detect specific proteins (80, 100, 130, and 150 kDa) with the DNPH-4-HNE-cysteine antiserum. Neither CCl4 nor iron/ascorbate elicited changes in 4-HNE or induced the formation of 4-HNE-modified proteins when assessed by immunoprecipitation-immunoblot analysis with the 4-HNE-sulfhydryl antiserum. In all instances detection of aldehyde-modified proteins was not associated with cell death and may be related to the function of these proteins as aldehyde-binding proteins which sequester electrophilic molecules during oxidative liver injury.
与促氧化剂介导的肝脏脂质过氧化相关的毒性被推测源于脂质过氧化的醛类终产物与细胞成分的相互作用。脂质过氧化的主要α,β-不饱和醛类产物，4-羟基-2-壬烯醛（4-HNE）和丙二醛（MDA），已知可通过赖氨酸、组氨酸和半胱氨酸氨基酸残基的共价烷基化修饰蛋白质。为了检测和表征促氧化剂引发的脂质过氧化过程中4-HNE和MDA加合物蛋白的形成，制备了针对4-HNE-巯基、二硝基苯肼（DNPH）-4-HNE-巯基以及钥孔血蓝蛋白（KLH）的MDA-胺缀合物的兔多克隆抗体。通过酶联免疫吸附测定（ELISA）检测时，每种抗血清对4-HNE-金属硫蛋白、DNPH-白蛋白或MDA-白蛋白加合物均显示出高抗体滴度。为了研究促氧化剂引发的细胞损伤过程中4-HNE和MDA-蛋白加合物的形成，将分离的肝细胞暴露于四氯化碳或铁/抗坏血酸中2小时。持续监测肝细胞氧化应激指标（即细胞活力和谷胱甘肽状态）以及脂质过氧化指标（即4-HNE、蛋白质羰基和MDA的形成）。四氯化碳对肝细胞活力有中度影响，而铁/抗坏血酸和四氯化碳均对细胞内还原型谷胱甘肽状态有中度影响。两种促氧化剂均使MDA和蛋白质羰基水平显著升高，而在研究的时间进程中4-HNE水平未发生明显变化。此外，用每种抗血清对肝细胞蛋白进行免疫沉淀，通过免疫印迹检测醛修饰的免疫阳性蛋白。在研究的时间进程中，促氧化剂诱导的MDA增加与MDA加合物蛋白的强度和数量增加相对应。用MDA-胺抗血清共检测到13种MDA修饰的蛋白（20、25、28、30、33、38、41、45、80、82、85、130和150 kDa）。此外，铁/抗坏血酸和四氯化碳诱导的DNPH可衍生化蛋白质羰基的形成与用DNPH-4-HNE-半胱氨酸抗血清检测特定蛋白（80、100、130和150 kDa）的能力在数量上相对应。当用4-HNE-巯基抗血清通过免疫沉淀-免疫印迹分析评估时，四氯化碳和铁/抗坏血酸均未引起4-HNE的变化或诱导4-HNE修饰蛋白的形成。在所有情况下，醛修饰蛋白的检测与细胞死亡无关，可能与这些蛋白作为醛结合蛋白的功能有关，即在氧化肝损伤过程中隔离亲电分子。
