Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+

Previously we showed that the redox active Cu2+ was much more effective than Cd2+ at inducing reactive oxygen species (“ROS”) formation in hepatocytes and furthermore “ROS” scavengers prevented Cu2+-induced hepatocyte cytotoxicity (Pourahmad and O’Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu2+, but not Cd2+, was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, “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 Cu2+, but not Cd2+, as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu2+-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu2+-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu2+. Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu2+-induced hepatocyte cytotoxicity. It is proposed that Cu2+-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by “ROS” generated by lysosomal Cu2+ redox cycling.