The Liver-Selective Nitric Oxide Donor O-Vinyl 1-(pyrrolidin-1- yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) Protects HepG2 Cells against Cytochrome P450 2E1-Dependent Toxicity

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 O-vinyl 1-(pyrrolidin1-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 M AA or 100 M 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. To study the biochemical and toxicological effects of CYP2E1, our laboratory established a HepG2 cell line that constitutively overexpresses CYP2E1 (E47 cells) (Chen and Cederbaum, 1998). Although no toxicity was found with control HepG2 cells that do not express CYP2E1 (C34 cells), the addition of ethanol, iron, or a polyunsaturated fatty acid such as arachidonic acid (AA) to E47 cells decreased cell viability and caused apoptosis (Chen et al., 1997; Cederbaum et al., 2001). These effects were enhanced when cellular glutathione (GSH) levels were lowered by treatment with L-buthionine-(S,R)-sulfoximine (BSO). Moreover, the treatment of E47 cells to deplete GSH with BSO, an inhibitor of the glutamate-cysteine ligase, resulted in apoptosis and necrosis (Wu and Cederbaum, 2001), whereas no toxicity was found with control C34 or HepG2 cells that expressed CYP3A4 instead of CYP2E1. The antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) partially prevented the apoptosis and necrosis after BSO treatment, whereas diallylsulfide, a CYP2E1 inhibitor, was fully protec-