Metabolism-dependent stimulation of reactive oxygen species and DNA synthesis by cyclosporin A in rat smooth muscle cells

The clinical use of the immunosuppressive drug cyclosporin A (CsA) is limit ed by its side effects, namely hypertension and nephrotoxicity. It has been proposed that reactive oxygen species (ROS) could be involved as mediators of the toxic effects of CsA. Here, we have studied the possible interrelat ionship between CsA metabolism and production of ROS. Using cultures of rat aortic smooth muscle cells (RASMC), CsA (1 mu M) produced a rapid (within 10 min) increase in reactive oxygen species, detected by oxidation of the f luorescent probes 2,7-dichlorofluorescin and dihydrorhodamine-123. DNA synt hesis was increased in the presence of CsA as assessed by [H-3]thymidine in corporation. The superoxide dismutase inhibitor diethyldithiocarbamate (1 m M) and the iron chelator desferal (5 mu M), as well as ketoconazole (1 mu M ) and troleandomycin (10 mu M), inhibitors of the cytochrome P-450 3A, were able to block both effects. High-performance liquid chromatography analysi s revealed that RASMC were capable to metabolize CsA to its primary metabol ites (AM1, AM9 and AM4N), and that their formation was inhibited by ketocon azole and troleandomycin. Furthermore, mRNAs encoding cytochrome P-450 3A1 and 3A2 were detected in RASMC by reverse transcriptase-polymerase chain re action. Our data suggest that CsA is metabolized by cytochrome P-450 3A in RASMC producing reactive oxygen species, most likely superoxide and the hyd roxyl radical, known to damage lipids and DNA. (C) 1999 Elsevier Science In c.