Oxidative Stress and an Alternative Glutathione-dependent Mechanism in the Developmental Toxicity of Methanol

The in vivo teratogenicity of methanol (MeOH) and its underlying molecular mechanisms were investigated, particularly the pathogenic contribution of reactive oxygen species (ROS) and oxidative stress. First, MeOH pharmacokinetics were compared among different strains of mice (CD-1, C3H and C57BL/6J) and species (New Zealand White rabbits and cynomolgus monkeys) to determine the potential contribution of metabolism to teratological risk. The mechanism was evaluated directly by manipulating important ROS-relevant pathways including: (1) altering a key antioxidative enzyme (genetically altered mice with high or low catalase activity), (2) inhibiting glutathione [GSH] synthesis using buthionine sulfoximine (BSO), and (3) blocking ROS effects with a free radical spin-trapping agent (phenylbutylnitrone, PBN). Rabbits and one mouse strain (C3H) were resistant to MeOH teratogenicity. Sensitive (C57BL/6J, CD-1) and resistant (C3H) mouse strains exhibited similar MeOH and formic acid (FA) pharmacokinetics, suggesting that developmental risk is not dependent upon MeOH or FA metabolism. MeOH did not increase embryonic ROS formation or DNA oxidation, nor was MeOH teratogenicity altered in C57BL/6J mice expressing elevated catalase levels, or by pretreatment with PBN, suggesting that ROS do not contribute to the in vivo mechanism of MeOH teratogenesis. This apparent ROS independence was in contrast to results from other studies implicating a ROS-dependent mechanism, based on different models of toxicity. These species- and strain-dependent results raise questions regarding the determinants of risk and the relevance of different animal models to the human situation. The spectrum of MeOH-initiated birth defects was expanded, and the incidences of multiple anomalies increased, by GSH depletion. GSH is both a ROS scavenger and a cofactor for formaldehyde dehydrogenase (ADH3), which detoxifies the MeOH metabolite formaldehyde. The results from this collection of studies suggest that formaldehyde is a proximate teratogen in vivo, and that GSH protects by serving as a cofactor for ADH3.Ph