Co-exposure to Fe, Zn, and Cu induced neuronal ferroptosis with associated lipid metabolism disorder via the ERK/cPLA2/AA pathway

Excessive amounts of iron (Fe), zinc (Zn), and copper (Cu) can be toxic to neuronal cells, even though these are essential trace elements for animals and humans. However, the precise mechanisms underlying the neurotoxicity of exposure to mixtures of Fe, Zn, and Cu are still mostly unclear. The research aimed to investigate the influence of co-exposure to iron, zinc and copper and the related mechanisms in HT22 murine hippocampal neuronal cells. Intracellular metal content, markers of oxidative damage, and biomarkers of ferroptosis were respectively detected. Afterward, metabolomic analyses were performed to obtain a comprehensive understanding of the metal mixtures on metabolism, and the functions of key enzymes on metabolic pathways were validated. The results showed that metal co-exposure resulted in cellular iron overload and increased lipid peroxidation, accompanied by significant pathological damage and mitochondrial abnormalities in HT22 cells. Meanwhile, it was found that GSH depletion, decreased GPX4, and increased expression of the lipid metabolism gene ACSL4 play important roles in ferroptosis induced by metal mixture. Further, metabolomic analysis revealed metal co-exposure induced significant alterations in metabolite levels, especially in the glycerophospholipid metabolism pathway and the arachidonic acid metabolism pathway. The levels of cPLA2 and its metabolite, arachidonic acid, were significantly increased after metal co-exposure. Then, inhibition of cPLA2 decreased the level of arachidonic acid and attenuated ferroptosis in neuronal cells. Collectively, our findings unveiled ferroptosis induced by metal co-exposure associated with crucial molecular changes in neuronal cells, providing a novel perspective on the comprehensive toxicity risk assessment of metal mixtures.