Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes.

Cylindrospermopsin (CY) is a naturally occurring alkaloid produced by the cyanobacterium Cylindrospermopsis raciborskii, which has been linked to an outbreak of hepatoenteritis in humans. We previously showed that CY is cytotoxic to primary cultures of rat hepatocytes and that CY lowers cell reduced glutathione (GSH) at nontoxic doses. Lower cell GSH also potentiates CY-induced cytotoxicity (Runnegar et al., Biochem Biophys Res Commun 201: 235-241, 1994). Our current work examined the mechanism of the fall in cell GSH induced by CY. We excluded several possible explanations for the loss in GSH, namely increased formation of oxidized glutathione (GSSG), increased GSH efflux, hidden forms of GSH, decreased GSH precursor availability, or decreased cellular ATP level. To address whether the fall in GSH was due to decreased GSH synthesis or increased GSH consumption, we examined the rate of fall in total GSH after 5 mM buthionine sulfoximine (BSO, an irreversible inhibitor of GSH synthesis) treatment. The rates of fall in total GSH (nmol/10(6) cells/hr) were 8.2 +/- 2.5, 6.0 +/- 1.7 and 5.9 +/- 1.3 for control, 2.5 microM and 5 microM CY-pretreated cells, respectively. This suggests that the fall in GSH induced by CY was due to the inhibition of GSH synthesis rather than increased consumption, because in the latter case the rate of fall in GSH would have been accelerated by CY pretreatment. Furthermore, excess GSH precursor (20 mM N-acetylcysteine), which supported GSH synthesis in control cells, did not prevent the fall in GSH or toxicity induced by CY. Treatment of cells with the cytochrome P450 inhibitor alpha-naphthoflavone protected partially from CY-mediated toxicity and from the fall in cell GSH. Thus, it is likely that cytochrome P450 is involved in the metabolism of CY, and the metabolite(s) that is generated may be more toxic and/or potent in inhibiting GSH synthesis. Inhibition of GSH synthesis is most likely an important factor in the cytotoxicity of CY.