Inorganic arsenic shows great promise in human cancer chemotherapy, although hepatotoxicity is a major limiting side-effect. O(2)-Vinyl 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) [Correction added after publication 19 December 2008: 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) was corrected to O(2)-Vinyl 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO)] is a nitric oxide (NO) donor prodrug that is metabolized by liver cytochromes P450 to release NO. Other NO-releasing agents have been shown to mitigate arsenic toxicity. Thus, the effects of V-PROLI/NO pretreatment on the toxicity of inorganic arsenic (as NaAsO(2)) were studied in vitro in a human liver (HepG2) cell line. HepG2 cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a dose- and time-dependent fashion to maximal levels of 57-fold above control levels. In cells pretreated with V-PROLI/NO (200 microM, 24 h) then exposed to arsenic for an additional 24 h, arsenic was much less toxic (LC(50) = 151.9 +/- 5.9 microM) than in control cells (LC(50) = 90.5 +/- 6.5 microM) and the reduced cytolethality was directly related to the level of NO produced. V-PROLI/NO also increased CYP2E1 transcriptional expression in a dose-dependent manner and CYP2E1 expression was directly related to the level of NO produced and the reduction in arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis as measured by DNA fragmentation. Pretreatment with V-PROLI/NO suppressed phosphorylation of JNK1/2 after arsenic exposure. Arsenic increased metallothionein, a metal-binding protein important in arsenic tolerance, and V-PROLI/NO pretreatment caused additional increases in metallothionein levels. Thus, the prodrug, V-PROLI/NO, protects against arsenic toxicity in cultured human liver cells, reducing cytolethality, apoptosis and dysregulation of mitogen-activated protein kinases, through generation of NO formed after metabolism by liver cell enzymes, possibly including CYP2E1.