Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+).
Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), 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 Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-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 Cu(2+) redox cycling.
Pourahmad
J
J
Faculty of Pharmacy, University of Toronto, Toronto ON, Canada.
Ross
S
S
O'Brien
P J
PJ
D000165:Acridine Orange; D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D006051:Aurothioglucose / Q000494:pharmacology; D002104:Cadmium / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects*; D002738:Chloroquine / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005839:Gentamicins / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects; D008099:Liver / Q000187:drug effects* / Q000648:ultrastructure*; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008744:Methylamines / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D010084:Oxidation-Reduction; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism
D005839:Gentamicins; D007976:Leupeptins; D008744:Methylamines; D010436:Pepstatins; D011480:Protease Inhibitors; D017382:Reactive Oxygen Species; D002104:Cadmium; C014195:Streptomyces pepsin inhibitor; D006051:Aurothioglucose; C025946:3-methyladenine; D003300:Copper; D002738:Chloroquine; D008985:Monensin; C027451:methylamine; D010450:Endopeptidases; D000165:Acridine Orange; C032854:leupeptin; D000225:Adenine; C031375:pepstatin