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 limited 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 interrelationship between CsA metabolism and production of ROS. Using cultures of rat aortic smooth muscle cells (RASMC), CsA (1 microM) produced a rapid (within 10 min) increase in reactive oxygen species, detected by oxidation of the fluorescent probes 2,7-dichlorofluorescin and dihydrorhodamine-123. DNA synthesis was increased in the presence of CsA as assessed by [3H]thymidine incorporation. The superoxide dismutase inhibitor diethyldithiocarbamate (1 mM) and the iron chelator desferal (5 microM), as well as ketoconazole (1 microM) and troleandomycin (10 microM), inhibitors of the cytochrome P-450 3A, were able to block both effects. High-performance liquid chromatography analysis revealed that RASMC were capable to metabolize CsA to its primary metabolites (AM1, AM9 and AM4N), and that their formation was inhibited by ketoconazole and troleandomycin. Furthermore, mRNAs encoding cytochrome P-450 3A1 and 3A2 were detected in RASMC by reverse transcriptase-polymerase chain reaction. Our data suggest that CsA is metabolized by cytochrome P-450 3A in RASMC producing reactive oxygen species, most likely superoxide and the hydroxyl radical, known to damage lipids and DNA.
Nguyen
N S
NS
Pharmacology Group, School of Pharmacy, University of Lausanne, Switzerland.
Cottet-Maire
F
F
Buetler
T M
TM
Lo Russo
A
A
Krauskopf
A S
AS
Armstrong
J M
JM
Vickers
A E
AE
Macé
K
K
Rüegg
U T
UT
D000818:Animals; D001011:Aorta; D001189:Aryl Hydrocarbon Hydroxylases; D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D016572:Cyclosporine / Q000378:metabolism* / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004247:DNA / Q000096:biosynthesis*; D003676:Deferoxamine / Q000494:pharmacology; D004050:Ditiocarb / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species / Q000378:metabolism*
D002614:Chelating Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016572:Cyclosporine; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D004050:Ditiocarb; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D011493:Protein Kinase C; D003676:Deferoxamine