Methanol (CH(3)OH), a common industrial solvent, is metabolized to toxic compounds by several enzymatic as well as free radical pathways. Identifying which process best enhances or prevents CH(3)OH-induced cytotoxicity could provide insight into the molecular basis for acute CH(3)OH-induced hepatoxicity. Metabolic pathways studied include those found in 1) an isolated hepatocyte system and 2) cell-free systems. Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques demonstrated that CH(3)OH had little toxicity towards rat hepatocytes in 95% O(2), even at 2M concentration, whereas 50 mM was the estimated LC(50) (2h) in 1% O(2), estimated to be the physiological concentration in the centrilobular region of the liver and also the target region for ethanol toxicity. Cytotoxicity was attributed to increased NADH levels caused by CH(3)OH metabolism, catalyzed by ADH1, resulting in reductive stress, which reduced and released ferrous iron from Ferritin causing oxygen activation. A similar cytotoxic mechanism at 1% O(2) was previous found for ethanol. With 95% O(2), the addition of Fe(II)/H(2)O(2), at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1M CH(3)OH, with a 3-fold increase in cytotoxicity and ROS formation. Iron chelators, desferoxamine, and NADH oxidizers and ATP generators, e.g. fructose, also protected hepatocytes and decreased ROS formation and cytotoxicity. Hepatocyte protein carbonylation induced by formaldehyde (HCHO) formation was also increased about 4-fold, when CH(3)OH was oxidized by the Fenton-like system, Fe(II)/H(2)O(2), and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system, Fe(II)/H(2)O(2) also increased CH(3)OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H(2)O(2) generating system increased HCHO-induced cytotoxicity and hepatocyte protein carbonylation. In addition, HCHO cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or GSH-depleted hepatocytes. Increased HCHO concentration levels correlated with increased HCHO-induced protein carbonylation in hepatocytes. These results suggest that CH(3)OH at 1% O(2) involves activation of the Fenton system to form HCHO. However, at higher O(2) levels, radicals generated through Fe(II)/H(2)O(2) can oxidize CH(3)OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death.