Gong Pengfei, Cederbaum Arthur I, Nieto Natalia
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029.
Mol Pharmacol. 2004 Jan;65(1):130-8. doi: 10.1124/mol.65.1.130.
HepG2 cells expressing CYP2E1 (E47 cells) are more susceptible to toxicity by arachidonic acid (AA) or after glutathione depletion with an inhibitor of glutamate-cysteine ligase, l-buthionine-(S,R)-sulfoximine (BSO), compared with control HepG2 cells (C34 cells). The ability of nitric oxide (NO) to protect against CYP2E1-dependent toxicity has not been evaluated. We therefore studied the ability of O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective NO donor, to protect against CYP2E1-dependent toxicity and compared this with protection by chemical NO donors. E47 cells incubated with V-PYRRO/NO produced NO, whereas C34 cells did not. Incubation of E47 cells with 50 microM AA or 100 microM BSO for 2 days resulted in a 50% loss of cell viability. VPYRRO/NO (1 mM) blocked this toxicity of AA and BSO by a mechanism involving NO release via CYP2E1 metabolism of VPYRRO/NO. NO scavengers hemoglobin and 2-(4-carboxophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide blocked the protective effects of V-PYRRO/NO. V-PYRRO/NO inhibited CYP2E1 activity and production of reactive oxygen species, whereas hemoglobin prevented these events. AA and BSO induced lipid peroxidation and decreased mitochondrial membrane potential; both of these effects were blocked by V-PYRRO/NO. Unlike V-PYRRO/NO, the chemical donors spermine/NO and (S)-nitroso-N-acetylpenicillamine release NO directly when added to the medium; however, they could partially protect against the CYP2E1-dependent toxicity. These results suggest that VPYRRO/NO protects HepG2 cells against CYP2E1-dependent toxicity through inhibition of CYP2E1-derived reactive oxygen species production and lipid peroxidation by the generated NO and that this compound may be valuable in protecting against CYP2E1-dependent toxicity via liver P450-specific generation of NO.
与对照HepG2细胞（C34细胞）相比，表达CYP2E1的HepG2细胞（E47细胞）对花生四烯酸（AA）的毒性或在用谷氨酸 - 半胱氨酸连接酶抑制剂L-丁硫氨酸 - （S，R）-亚磺酰亚胺（BSO）耗尽谷胱甘肽后更敏感。一氧化氮（NO）预防CYP2E1依赖性毒性的能力尚未得到评估。因此，我们研究了肝脏选择性NO供体O2-乙烯基-1-（吡咯烷-1-基）重氮-1,2-二醇盐（V-PYRRO/NO）预防CYP2E1依赖性毒性的能力，并将其与化学NO供体的保护作用进行比较。用V-PYRRO/NO孵育的E47细胞产生NO，而C34细胞则不产生。用50 microM AA或100 microM BSO孵育E47细胞2天导致细胞活力丧失50%。VPYRRO/NO（1 mM）通过涉及V-PYRRO/NO经CYP2E1代谢释放NO的机制阻断了AA和BSO的这种毒性。NO清除剂血红蛋白和2-（4-羧基苯基）-4,4,5,5-四甲基咪唑啉-1-氧基-3-氧化物阻断了V-PYRRO/NO的保护作用。V-PYRRO/NO抑制CYP2E1活性和活性氧的产生，而血红蛋白阻止了这些事件。AA和BSO诱导脂质过氧化并降低线粒体膜电位；这两种作用均被V-PYRRO/NO阻断。与V-PYRRO/NO不同，化学供体精胺/NO和（S）-亚硝基-N-乙酰青霉胺添加到培养基中时直接释放NO；然而，它们可以部分预防CYP2E1依赖性毒性。这些结果表明，V-PYRRO/NO通过抑制CYP2E1衍生的活性氧产生和所生成的NO引起的脂质过氧化来保护HepG2细胞免受CYP2E1依赖性毒性，并且该化合物可能在通过肝脏P450特异性产生NO来预防CYP2E1依赖性毒性方面具有价值。
