Abstract
In man the immunosuppressive drug Cyclosporine A (CsA) has been used successfully in organ transplantation and in the treatment of autoimmune disorders. The drug, however, causes side effects which occur mainly in the kidney but also in the liver. The mechanisms leading to the hepatic side effects are not yet fully understood. Because reactive oxygen production is a common mechanism of drug toxicity, the goal of this study was to evaluate whether CsA induces oxidative stress in rat liver cells. In primary rat hepatocyte 20-h cultures, CsA caused a concentration-dependent increase of free reactive oxygen species, thiobarbituric acid reactive substances, loss of protein thiols and decrease of molar ratios of glutathione and glutathione disulfide in the range of 0 to 50 μM CsA. The weakening or enforcement of the cellular glutathione state by the glutathione synthesis inhibitor buthionine sulfoximine or the glutathione disulfide -reducing agent dithiothreitol either increased or inhibited the CsA cytotoxicity, as determined by lactate dehydrogenase release. CsA also decreased the level of endogenous antioxidant ascorbic acid and increased its oxidation product dehydroascorbic acid. Supplementation of the cell cultures with ascorbic acid significantly reduced the CsA toxicity. The antioxidantdl-α-tocopherol-polyethylene-glycol-1000-succinate partly decreased CsA-mediated reactive oxygen species formation, totally decreased thiobarbituric acid reactive substances formation, prevented the loss of protein-bound sulfhydryl groups and in addition totally inhibited the CsA cytotoxicity. The present data provide good evidence that oxidative stress is part of the mechanism by which CsA causes toxicity in rat liver cells.
Footnotes
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Send reprint requests to: Armin Wolf, PhD, Novartis Pharma Ltd., Toxicology/ Pathology SMU-881.5.514, CH-4002 Basle, Switzerland.
- Abbreviations:
- AA
- ascorbic acid
- BSO
- butionine sulfoximine
- CsA
- cyclosporine A
- DCFdAc
- 2′,7′-dichlorofluorescin diacetate
- DHAA
- dehydroascorbic acid
- DTT
- dithiothreitol
- GSH
- glutathione
- GSSG
- glutathione disulfide
- LDH
- lactate dehydrogenase
- NADPH
- reduced nicotinamide adenine dinucleotide phosphate
- ROS
- reactive oxygen species
- TBA-RS
- thiobarbituric acid reactive substances
- TPGS
- dl-α-tocopherol-polyethylene-glycol-1000-succinate
- WME
- William’s medium E
- PBS
- phosphate-buffered saline
- Received August 5, 1996.
- Accepted November 29, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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