Effect of Methoxychlor on Ca²⁺ Movement and Viability in MDCK Renal Tubular Cells

The effect of the insecticide methoxychlor on the physiology of renal tubular cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells using the Ca²⁺-sensitive fluorescent dye fura-2. Methoxychlor at 5-20 μM increased [Ca²⁺]_i in a concentration-dependent manner. The signal was reduced by 80% by removing extracellular Ca²⁺. Methoxychlor-induced Ca²⁺ entry was not affected by nifedipine and SK&F96365 but was inhibited by econazole and protein kinase C modulators. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited methoxychlor-induced [Ca²⁺]_i rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 nearly abolished methoxychlor-induced [Ca²⁺]_i rise. At 5-15 μM, methoxychlor slightly increased cell viability, whereas at 20 μM, it decreased viability. 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 10 μM methoxychlor inhibited apoptosis, while 20 μM methoxychlor enhanced apoptosis. Methoxychlor (10 and 20 μM) increased the production of reactive oxygen species. Together, in renal tubular cells, methoxychlor induced [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from multiple stores and Ca²⁺ entry via protein kinase C- and econazole-sensitive channels. Methoxychlor slightly enhanced or inhibited cell viability in a concentration-dependent, Ca²⁺-independent manner. Methoxychlor induced cell death that may involve apoptosis via mitochondrial pathways.