Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: Carbonyl-metabolizing enzymes versus oxidative stress

Methanol (CH3OH), 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 CH3OH-induced cytotoxicity could provide insight into the molecular basis for acute CH3OH-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 CH3OH had little toxicity towards rat hepatocytes in 95% O-2, even at 2 M concentration, whereas 50 mM was the estimated LC50 (2 h) 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 CH3OH 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% 02 was previous found for ethanol. With 95% 02, the addition of Fe(II)/H2O2, at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1 M CH3OH, 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 CH3OH was oxidized by the Fenton-like system, Fe(II)/H2O2, and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system. Fe(II)/H2O2 also increased CH3OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H2O2 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 CH3OH at 1% O-2 involves activation of the Fenton system to form HCHO. However, at higher 02 levels, radicals generated through Fe(II)/H2O2 can oxidize CH3OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death. (C) 2011 Elsevier Ireland Ltd. All rights reserved.