A combinatorial approach for metal cytotoxic mechanisms

The molecular cytotoxic mechanisms of hepatocyte cell death induced by metal toxins, CuCl2, CdCl2, HgCl2, K 2Cr2O7 and also H2O2 (utilizing nitrofurantoin (NFT) to generate intracellular H2O2 by redox cycling) were compared using a combinatorial approach and accelerated cytotoxicity mechanism screening. The major differences found between the effect of Cu2+ and Cd2+ on hepatocyte were: (1) Cu2+ but not Cd2+ rapidly oxidized GSH which effluxed the hepatocyte likely as a Cu:GSSG complex whereas Cd2+ increased hepatocyte GSH efflux with much less GSH oxidation. (2) Na+ omission from the media or addition of the Na+/H+ exchange inhibitor 5-(N,N-dimethyl)-amiloride markedly increased Cu2+ cytotoxicity even though Cu2+ did not increase hepatocyte Na + when the media contained Na+. By contrast Cd 2+ induced cytotoxicity was prevented by Na+ omission from the media or by the addition of 5-(N,N-dimethyl)-amiloride. Intracellular Ca2+ levels however were markedly increased when the hepatocytes were incubated with Cu2+ but not Cd2+ in a Na + free media. Removing media Ca2+ with EGTA also prevented Cu2+ induced hepatocyte cytotoxicity. A hypotonic media also increased Cd2+ but not Cu2+ induced hepatocyte cytotoxicity. These results suggest that Cd2+ but not Cu 2+ cytotoxicity could be partly attributed to disruption of cell volume regulation mechanisms. (3) Hepatocyte cytotoxicity induced by Cu 2+ but not Cd2+ was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cu2+ but not Cd2+ induced cytotoxicity, reactive oxygen species ("ROS") formation and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu 2+ but not Cd2+ as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cr VI cytotoxicity was prevented by cytochrome P450 inhibitors, particularly CYP 2E1 inhibitors, but not inhibitors of DT diaphorase or glutathione reductase. This suggests that P450 reductase and/or reduced cytochrome P450 contributes to Cr VI reduction to Cr IV. Reduction of dichromate by glutathione or cysteine in vitro was also accompanied by oxygen uptake and was inhibited by Mn II (a Cr IV reductant). Cr VI induced cytotoxicity and "ROS" formation was also inhibited by Mn II which suggests that Cr IV and Cr IV.GSH mediate "ROS" formation in isolated hepatocytes. Nitrofurantoin (NFT) was also reductively activated by reduced cytochrome P450 and/or P450 reductase to form H2O2. Hepatocyte mitochondrial membrane potential (MPT) was only slightly decreased by NFT before cytotoxicity ensued. However NFT induced lysosomal damage and hepatocyte protease activation. It is concluded that H2O2 generated by the redox cycling of NFT reacts with lysosomal Fe2+ to form "ROS" which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death. Finally it was shown that hepatocyte lysis induced by CdCl2, HgCl2 or K2Cr2O7 was prevented by apoptosis inhibitors. This suggest that metal induced cytolysis may involve apoptotic death signals initiated by MPT and "ROS".