Biphasic lindane-induced oxidation of glutathione and inhibition of gap junctions in myometrial cells

The insecticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells by a mechanism involving oxidative stress. We hypothesized that oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG) and subsequent S-glutathionylation provide a mechanistic link between lindane-induced oxidative stress and lindane's inhibition of myometrial gap junction communication. Gap junction communication between cultured rat myometrial myocytes was assessed by Lucifer yellow dye transfer after microinjection. A biphasic pattern was confirmed, with dye transfer nearly abolished after 1 h of exposure to 100 mu M lindane followed initially by recovery after lindane removal, and then the development 4 h after termination of lindane exposure of a delayed-onset, sustained inhibition that continued for 96 h. As measured by HPLC, cellular GSH varied over a 24-h period in a biphasic fashion that paralleled lindane-induced inhibition of dye transfer, whereas GSSG levels increased in a manner inversely related to GSH. In accordance, GSH/GSSG ratios were depressed at times when GSH and dye transfer were low. Lindane substantially increased S-glutathionylation in a concentration-dependent manner, measured biochemically by GSSG reductase-stimulated release of GSH from precipitated proteins. Furthermore, treatments that promoted accumulation of GSSG (50 mu M diamide and 25 mu M 1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU]) inhibited Lucifer yellow dye transfer between myometrial cells. Findings that lindane induced GSH oxidation to GSSG with increased S-glutathionylation, together with the diamide and BCNU results, suggest that oxidation of GSH to GSSG is a component of the mechanism by which lindane inhibits myometrial gap junctions.