Pérez María José, Cederbaum Arthur I
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA.
Free Radic Biol Med. 2003 Feb 15;34(4):443-55. doi: 10.1016/s0891-5849(02)01302-3.
Zinc has been shown to have antioxidant actions, which may be due, in part, to induction of metallothionein (MT). Such induction can protect tissues against various forms of oxidative injury because MT can function as an antioxidant. The objective of this study was to investigate if zinc or MT induction by zinc could afford protection against CYP2E1-dependent toxicity. HepG2 cells overexpressing CYP2E1 (E47cells) were treated with 60 microM arachidonic acid (AA), which is known to be toxic to these cells by a mechanism dependent on CYP2E1, oxidative stress, and lipid peroxidation. E47 cells were preincubated overnight in the absence or presence of metals such as zinc or cadmium that can induce MT. The culture medium containing the metals was removed, AA was added, and cell viability determined after 24 h incubation. Preincubation overnight with 150 microM zinc sulfate or 5 microM cadmium chloride induced a 20- to 30-fold increase of MT2A mRNA; high levels of MT2A mRNA were maintained during the subsequent challenge period with AA, even after the zinc was removed. MT protein levels were increased about 4- to 5-fold during the overnight preincubation with zinc and a 20- to 30-fold increase was observed 24 h after zinc removal during the AA challenge. The treatment with zinc was associated with significant protection against the loss of cell viability caused by AA in E47 cells. The zinc pretreatment protected about 50% against the DNA fragmentation, cell necrosis, the enhanced lipid peroxidation and increased generation of reactive oxygen species, and the loss of mitochondrial membrane potential induced by AA treatment in E47 cells. CYP2E1 catalytic activity and components of the cell antioxidant defense system such as glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxidase (GPX), catalase, Cu,Zn superoxide dismutase (SOD), and MnSOD were not altered under these conditions. Zinc preincubation also protected the E47 cells against BSO-dependent toxicity. When E47 cells were coincubated with zinc plus AA for 24 h (i.e., zinc was not removed, nor was there a preincubation period prior to challenge with AA), AA toxicity was increased. Thus, zinc had a direct pro-oxidant effect in this model and an indirect antioxidant effect, perhaps via induction of MT. MT may have potential clinical utility for the prevention or improvement of liver injury produced by agents known to be metabolized by CYP2E1 to reactive intermediates and to cause oxidative stress.
锌已被证明具有抗氧化作用，这可能部分归因于金属硫蛋白（MT）的诱导。这种诱导可以保护组织免受各种形式的氧化损伤，因为MT可以作为抗氧化剂发挥作用。本研究的目的是调查锌或锌诱导的MT是否能提供针对CYP2E1依赖性毒性的保护作用。用60微摩尔花生四烯酸（AA）处理过表达CYP2E1的HepG2细胞（E47细胞），已知AA通过依赖CYP2E1、氧化应激和脂质过氧化的机制对这些细胞有毒性。E47细胞在不存在或存在可诱导MT的金属（如锌或镉）的情况下预孵育过夜。去除含有金属的培养基，加入AA，并在孵育24小时后测定细胞活力。用150微摩尔硫酸锌或5微摩尔氯化镉预孵育过夜可使MT2A mRNA增加20至30倍；在随后用AA刺激期间，即使去除锌后，MT2A mRNA仍维持在高水平。在用锌预孵育过夜期间，MT蛋白水平增加约4至5倍，在AA刺激期间去除锌后24小时观察到增加20至30倍。锌处理与显著保护E47细胞免受AA引起的细胞活力丧失有关。锌预处理可保护约50%免受DNA片段化、细胞坏死、脂质过氧化增强、活性氧生成增加以及AA处理诱导的E47细胞线粒体膜电位丧失的影响。在这些条件下，CYP2E1催化活性以及细胞抗氧化防御系统的成分如谷胱甘肽（GSH）、谷胱甘肽-S-转移酶（GST）、谷胱甘肽过氧化物酶（GPX）、过氧化氢酶、铜锌超氧化物歧化酶（SOD）和锰超氧化物歧化酶（MnSOD）未发生改变。锌预孵育也保护E47细胞免受BSO依赖性毒性的影响。当E47细胞与锌加AA共孵育24小时（即未去除锌，在用AA刺激之前也没有预孵育期）时，AA毒性增加。因此，在该模型中锌具有直接的促氧化作用和间接的抗氧化作用，可能是通过诱导MT实现的。MT对于预防或改善由已知经CYP2E1代谢为反应性中间体并导致氧化应激的药物引起的肝损伤可能具有潜在的临床应用价值。
