Role of oxidative stress and the mitochondrial permeability transition in methylmercury neurotoxicity

Methylmercury (MeHg) is a potent environmental neurotoxicant. Human poisonings and animal experiments demonstrate that the developing central nervous system (CNS) is more sensitive to MeHg than that of the adult, but the reasons for this difference are unknown. Data suggest that increased production of reactive oxygen species (ROS) by mitochondria is important in the pathogenesis of MeHg neurotoxicity. We hypothesized that differences in mitochondrial ROS production and mitochondrial permeability transition (MTP) are early events contributing to the enhanced sensitivity of the developing CNS.; MeHg-induced ROS production was examined in vivo in mitochondria and synaptosomal preparations from MeHg-treated mice using DCF fluorescence. Adult mice were treated with MeHg 5.0 mg/kg for 5 days or 10 mg/kg for 2 days, while pups received MeHg 4.0 mg/kg on postnatal days (PND) 6, 8 and 10. MeHg significantly decreased ROS production in adult cerebellar mitochondria but no significant changes in ROS were seen in samples from the PND 11 mice. Confocal microscopy studies were performed in live undifferentiated P19 cells and their day 5 neuronal derivatives exposed to MeHg 1.5 uM for 1 hr. The D5 neuron was more sensitive than undifferentiated cells to both MeHg-induced ROS production indexed by DCF and MeHg-induced loss of mitochondrial membrane potential (Psim), indicated by decreased TMRM fluorescence. Further, MeHg-treated neurons exhibited enhanced translocation of cytochrome c (cyt c) from the mitochondria to the cytosol. Pretreatment with the MPT pore blocking agent cyclosporin A (CsA) prevented the decline in (Psi m), and cyt c release in undifferentiated cells and impaired ROS generation in D5 neurons, but only delayed the MeHg-induced loss of (Psim), in D5 neurons. The antioxidant Trolox reduced MeHg-induced ROS production and virtually eliminated the MeHg-induced decline in Psim in D5 neurons, indicating that Trolox may directly protect at the mitochondrial membrane. Neuronal cultures derived from transgenic mice overexpressing the human intracellular isoform of glutathione peroxidase were largely refractory to MeHg-induced loss of mitochondrial membrane integrity. The protective effects of CsA implicate the MPT in MeHg cytotoxicity, and the protective effects of enhanced antioxidant defenses similarly suggest the mitochondrion is an important early target of MeHg and a critical mediator of downstream toxic responses in the adult brain. Overall, the data suggest that differences in mitochondrial energy metabolism, cellular antioxidant defenses, and the MPT pore contribute to the regional selectivity of MeHg intoxication in the mature CNS and to the differential susceptibility of neurons at various stages of development.