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Rapid effects of pesticides on human granulosa-lutein cells

Department of Obstetrics and Gynecology, Reproductive Biology Division, McMaster University, Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5

Correspondence should be addressed to E Younglai; Email: younglai{at}mcmaster.ca

Following our previous demonstration that p,p'-DDE (dichlorodiphenylchloroethylene), at environmentally relevant concentrations, can rapidly increase intracellular calcium [Ca²⁺]_i concentrations in human granulosa-lutein cells, we examined whether other pesticides, such as Kepone, o,p-DDE and methoxychlor, have similar effects. Cultured human granulosa-lutein cells were loaded with Fura-2 AM, and changes in [Ca²⁺]_i concentrations within small areas of single cells were studied with a dynamic digital Ca²⁺ imaging system. Kepone, at concentrations of 0.2–2 nmol/ml, consistently increased [Ca²⁺]_i concentrations 2–6 times higher than baseline values within minutes of exposure. Methoxychlor at concentrations of 2.8–280 nmol/ml failed to alter [Ca²⁺]_i levels consistently in cells from 10 patients. However, at 0.28 and 1.4 nmol/ml, increases in [Ca²⁺]_i concentrations could be elicited by methoxychlor. The isomer o,p-DDE at 3 nmol/ml increased [Ca²⁺]_i in granulosa cells of 11/20 patients. Pertussis toxin treatment inhibited the [Ca²⁺]_i increases induced by estradiol, p,p'-DDE, o,p-DDE and methoxychlor, but not by Kepone or progesterone, indicating that Kepone and progesterone may act through an insensitive G protein-coupled receptor. The [Ca²⁺]_i increases induced by Kepone also occurred in Ca²⁺-free medium, suggesting that [Ca²⁺]_i mobilization occurred from the smooth endoplasmic reticulum. Thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump, also stimulated [Ca²⁺]_i increases but did not inhibit the Ca²⁺ response to all the pesticides. These results demonstrate that pesticides can have a rapid effect on human granulosa-lutein cells, and a nongenomic mechanism of action is suggested