The fungicide mancozeb induces astrocyte atrophy and disrupts Ca²⁺ signaling via inhibition of Orai1/STIM1-mediated SOCE

Mancozeb, a widely used fungicide composed of manganese ethylene-bis-dithiocarbamate with zinc salts, has raised concerns due to its potential neurotoxic effects. In this study, we investigated how chronic oral administration of mancozeb affects astrocyte function and neurobehavior in mice, focusing on store-operated Ca²⁺ entry (SOCE), mediated by Orai1 and STIM1. Mancozeb treatment at 0.5 µg/kg/day for 4 weeks reduced glial fibrillary acidic protein (GFAP) expression in the hippocampus and corpus callosum of mice, indicating astrocyte atrophy. Further, administration at the human acceptable daily intake (30 µg/kg/day) for 1 week induced hippocampal astrocyte atrophy and hyperlocomotor activity in open field tests. In vitro experiments revealed that mancozeb specifically inhibited SOCE in astrocytes by targeting the Orai1/STIM1 complex, as its inhibitory effect was abolished by short hairpin RNA (shRNA)-mediated knockdown of Orai1 or STIM1, but not by knockdown of TRPA1 or scramble shRNA. This demonstrates that mancozeb-mediated SOCE inhibition critically depends on the presence of Orai1 and STIM1, highlighting the molecular specificity of its action. Furthermore, mancozeb diminished endoplasmic reticulum (ER) Ca²⁺ stores and P2Y1 receptor agonist-induced Ca²⁺ transients. Electrophysiological analyses revealed that mancozeb selectively decreased the inhibitory postsynaptic current frequency without affecting excitatory currents, suggesting reduced astrocyte-mediated GABA release. Collectively, these findings demonstrate that mancozeb disrupts astrocytic Ca²⁺ homeostasis through Orai1/STIM1-dependent SOCE inhibition, leading to astrocyte atrophy and altered inhibitory neurotransmission, which may underlie the observed behavioral changes. These results highlight the potential neurotoxic risk posed by mancozeb via the impairment of astrocyte function and intracellular Ca²⁺ regulation. Importantly, these neurotoxic effects occurred at concentrations below current regulatory safety limits (ADI), indicating that mancozeb-induced disruption of astrocytic Ca²⁺ signaling provides a mechanistic basis for re-evaluating established human safety exposure standards.