Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells
Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells
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DOI：
10.1124/mol.53.4.638
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发表时间：
1998-04-01
影响因子：
3.6
通讯作者：
Cederbaum, AI
中科院分区：
文献类型：
--
作者：
Chen, Q;Cederbaum, AI
用转染和有限稀释筛选技术建立了两个过表达CYP2E1的Hep G2亚克隆。Hep G2-C12E1 - 43和-47（E47）细胞（过表达CYP2E1的转导Hep G2亚克隆）的生长速率低于不表达CYP2E1的亲本Hep G2细胞或对照亚克隆，但仍完全存活。当GSH合成被丁硫克百威亚砜处理抑制时，GSH水平在E47细胞中迅速下降，而不是对照细胞，这很可能是CYP2E1催化活性氧形成的反映。在GSH耗竭的这些条件下，仅发现E47细胞的细胞毒性和凋亡。低水平的脂质过氧化作用，发现在E47细胞，这变得更加明显后，GSH耗尽。抗氧化剂维生素E，维生素C，或trolox防止脂质过氧化以及细胞毒性和细胞凋亡，转染含有反义CYP2E1或过表达Bcl-2的质粒。E47细胞中ATP水平较低，因为线粒体复合物I受损。当GSH耗尽，氧摄取量显着降低与所有基板在E47提取物。维生素E完全防止了氧摄取的减少。在CYP2E1过表达的条件下，可以在Hep G2细胞中观察到两种CYP2E1依赖性毒性模式：当细胞GSH水平维持时生长速率较慢，当细胞GSH水平耗尽时细胞活力丧失。脂质过氧化作用的升高在CYP2E1依赖性毒性和细胞凋亡中起重要作用。过表达CYP2E1的这种直接毒性可能反映了这种酶即使在不添加代谢底物的情况下也能产生活性氧的能力。
Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-C12E1-43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.