Early cellular events couple covalent binding of reactive metabolites to cell killing by nephrotoxic cysteine conjugates.
Addition of the nephrotoxic cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), to the LLC-PK1 line of renal epithelial cells leads to covalent binding of reactive intermediates followed by thiol depletion, lipid peroxidation, and cell death (Chen et al., 1990, J. Biol. Chem., 265:21603-21611). The present study was designed to determine if increased intracellular free calcium might play a role in this pathway of DCVC-induced toxicity by comparing the temporal relationships among increased intracellular free calcium, lipid peroxidation, and cytotoxicity. Intracellular free calcium increased 1 hr after DCVC treatment, long before LDH release occurred. The elevation of intracellular free calcium and cytotoxicity was prevented by inhibiting DCVC metabolism with AOA. The cell-permeable chelators, Quin-2AM and EGTA-AM, prevented the toxicity. Pretreatment of cells with a nontoxic concentration of ionomycin increased intracellular free calcium and potentiated DCVC-induced LDH release. However, the antioxidant, DPPD, which blocks lipid peroxidation and toxicity, did not affect the increase in intracellular free calcium, whereas buffering intracellular calcium with Quin-2AM or EGTA-AM blocked both lipid peroxidation and toxicity without preventing the depletion of nonprotein sulfhydryls by DCVC. Ruthenium red, an inhibitor of mitochondrial calcium uptake, also blocked cell death. We hypothesize that covalent binding of the reactive fragment from DCVC metabolism leads to deregulation of intracellular calcium homeostasis and elevation of intracellular free calcium. Increased intracellular free calcium may in turn be coupled to mitochondrial damage and the accumulation of endogenous oxidants which cause lipid peroxidation and cell death.
Chen
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Q
W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
Jones
T W
TW
Stevens
J L
JL
D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D002614:Chelating Agents / Q000494:pharmacology; D003545:Cysteine / Q000031:analogs & derivatives* / Q000506:poisoning; D005110:Extracellular Space / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D018374:LLC-PK1 Cells; D008054:Lipid Peroxides / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D013552:Swine
D000975:Antioxidants; D002614:Chelating Agents; D008054:Lipid Peroxides; D012430:Ruthenium Red; D000625:Aminooxyacetic Acid; D015759:Ionomycin; C039961:S-(1,2-dichlorovinyl)cysteine; D003545:Cysteine; D002118:Calcium