Role of oxidative stress and thiol/protein modifications of gap junctions in lindane's inhibition of uterine function

This dissertation examines mechanisms by which the pesticide lindane (γ-hexachlorocyclohexane) inhibits gap junction communication between cultured rat myometrial cells and contractility of pregnant rat uterine tissue in vitro. Lindane has been previously shown to inhibit gap junctions, measured by cell-cell transfer of microinjected Lucifer yellow dye, in a biphasic, time-dependent manner and to eliminate spontaneous oscillatory contractions of pregnant rat uterine strips hung in muscle baths. This dissertation hypothesized that lindane-induced inhibition of uterine contractility results from inhibition of gap junctional communication subsequent to oxidative stress-induced S-glutathionation of protein, including the gap junction protein connexin43.; To test this hypothesis, cultured myometrial cells and uterine tissue were examined in vitro. Exposure of myometrial cells to lindane caused an increase in formazan deposition and in thiobarbituric acid reactive substances, suggesting generalized oxidative stress and lipid peroxidation. Superoxide production was elevated and glutathione was depleted in cell cultures and uterine tissue, also indicative of oxidative stress. The antioxidants α-tocopherol, superoxide dismutase, diphenyl phenylene diamine and d-mannitol each reversed lindane's inhibition of Lucifer yellow dye transfer. Alpha-tocopherol also reversed the lindane-induced inhibition of uterine contractility. Augmentation of intracellular glutathione concentrations, however, failed to reverse lindane's inhibition of Lucifer yellow dye transfer or uterine contractility. Rather, depletion of reduced glutathione with buthionine sulfoximine pretreatment reversed lindane-induced Lucifer yellow dye transfer and contractility inhibitions.; Glutathione depletion in cell cultures and uterine tissue was concurrent with oxidation of glutathione to its disulfide form and with significant increases in protein-glutathione mixed disulfides. Diamide, used to directly oxidize glutathione to its disulfide form, inhibited Lucifer yellow dye transfer and uterine contractility similar to lindane. These experiments suggest that glutathione oxidation is an important element in lindane's inhibitory pathway. Lindane increased S-glutathionation of proteins, including the gap junction protein connexin43. This was demonstrated using isoelectric focusing followed by western blotting techniques to probe for connexin43 in lindane-exposed myometrial cells and uterine tissue. This study shows that oxidative stress may initiate lindane's inhibition of uterine contraction through S-glutathionation of connexin43, and is the first study known to provide evidence of a mechanism by which oxidative stress inhibits gap junction cell-cell communication.