Effect of Methoxychlor on Ca²⁺ Handling and Viability in OC2 Human Oral Cancer Cells

The effect of the insecticide methoxychlor on the physiology of oral cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in human oral cancer cells (OC2) by using the Ca²⁺-sensitive fluorescent dye fura-2. Methoxychlor at 5-20μM increased [Ca²⁺]_i in a concentration-dependent manner. The signal was reduced by 70% by removing extracellular Ca²⁺. Methoxychlor-induced Ca²⁺ entry was not affected by nifedipine, econazole, SK&F96365 and protein kinase C modulators but was inhibited by the phospholipase A2 inhibitor aristolochic acid. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished methoxychlor-induced [Ca²⁺]_i rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter methoxychlor-induced [Ca²⁺]_i rise. At 5-20μM, methoxychlor killed cells in a concentration-dependent manner. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that methoxychlor (10 and 20μM) evoked apoptosis in a concentration-dependent manner. Together, in human OC2, methoxychlor induced a [Ca²⁺]_i rise probably by inducing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A₂-sensitive channels. Methoxychlor induced cell death that may involve apoptosis.