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 link ed to an outbreak of hepatoenteritis in humans. We previously showed t hat CY is cytotoxic to primary cultures of rat hepatocytes and that CY lowers cell reduced glutathione (GSH) at nontoxic doses. Lower cell G SH 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 p ossible explanations for the loss in GSH, namely increased formation o f oxidized glutathione (GSSG), increased GSH efflux, hidden forms of G SH, decreased GSH precursor availability, or decreased cellular ATP le vel. To address whether the fall in GSH was due to decreased GSH synth esis or increased GSH consumption, we examined the rate of fall in tot al GSH after 5 mM buthionine sulfoximine (BSO, an irreversible inhibit or of GSH synthesis) treatment. The rates of fall in total GSH (nmol/1 0(6) cells/hr) were 8.2 +/- 2.5, 6.0 +/- 1.7 and 5.9 +/- 1.3 for contr ol, 2.5 mu M and 5 mu M CY-pretreated cells, respectively. This sugges ts that the fall in GSH induced by CY was due to the inhibition of GSH synthesis rather than increased consumption, because in the latter ca se the rate of fall in GSH would have been accelerated by CY pretreatm ent. 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-media ted toxicity and from the fall in cell GSH. Thus, it is likely that cy tochrome 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 fa ctor in the cytotoxicity of CY.