Abstract
Cisplatin-induced nephrotoxicity was studied in porcine proximal tubular cells, focusing on the relationship between mitochondrial damage, reactive oxygen species (ROS) and cell death. Cisplatin specifically affected mitochondrial functions: complexes I to IV of the respiratory chain were inhibited 15 to 55% after 20 min of incubation with 50 to 500 μM, respectively. As a result, intracellular ATP was decreased to 70%. The mitochondrial glutathione (reduced form) (GSH)-regenerating enzyme GSH-reductase (GSH-Rd) activity was reduced by 20%, which contributed to a 70% reduction of GSH levels and ROS formation. The residual electron flow through the mitochondrial respiratory chain was the source of ROS because additional inhibition of the complexes I to IV reduced ROS formation. Because cisplatin affects both GSH-Rd and complexes I to IV, cells were incubated with N,N′-bis(2-chloroethyl)-N-nitrosourea (inhibitor of GSH-Rd) and inhibitors of the different complexes. Only N,N′-bis(2-chloroethyl)-N-nitrosourea with rotenone (complex I inhibitor) induced ROS formation, which indicates that inhibition of complex I and inhibition of the GSH-Rd is probably the cause of ROS formation. However, the resulting ROS is not the cause of cell death because diphenyl-p-phenylene-diamine and deferoxamine, which completely prevented ROS, could not prevent cell death. Similarly, the antioxidants did not completely prevent the decrease in activity of complexes I to IV, ATP or GSH levels. In conclusion, ROS formation does occur during cisplatin-induced toxicity, but it is not the direct cause of cell death.
Footnotes
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Send reprint requests to: Ms Marieke Kruidering, Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
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↵1 This project was financially supported by the Dutch Foundation Platform Alternatives to Animal Experiments.
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↵2 W.A. Jones Cell Science Center, Old Barn Road, P.O. Box 49, Lake Placid, NY 12946.
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↵3 Dept. of Pathology, Leiden University, P.O. Box 9603, 2300 RC Leiden, The Netherlands.
- Abbreviations:
- anti
- antimycin
- BCNU
- N,N′-bis(2-chloroethyl)-N-nitrosourea
- BSA
- bovine serum albumin
- CCCP
- cyanide m-chlorophenylhydrazone
- DES
- DesferalR(deferoxamine)
- Dih123
- dihydrorhodamine-123
- DPPD
- diphenyl-p-phenylene-diammine
- Δψ
- mitochondrial membrane potential
- EDTA
- ethylenediaminetetraacetate
- GSH
- glutathione (reduced form)
- GSH-Px
- GSH peroxidase
- GSH-Rd
- GSH reductase
- GSSG
- glutathione (oxidized form)
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- MRC
- mitochondrial respiratory chain
- Oxa
- oxaloacetic acid
- PPTC
- porcine proximal tubular cells
- Pt
- cis-diamine-dichloroplatinum (II)
- R123
- rhodamine-123
- ROS
- reactive oxygen species
- rot
- rotenone
- TBHP
- tertiary butyl hydroperoxide
- TCA
- trichloroacetic acid
- TTFA
- thenoyltrifluoroacetone
- Received July 25, 1996.
- Accepted October 21, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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