Cyclosporin A‐induced free radical generation is not mediated by cytochrome P‐450

Abstract

1 Reactive oxygen species (ROS) have been proposed to play a role in the side e ects of the immunosuppressive drug cyclosporin A (CsA). 2 The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may in¯uence the CYP system to produce ROS. 3 We show that CsA (1 ± 10 mM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the¯uorescent probe 2,7-dichloro¯uorescin diacetate. 4 Using cytochrome c as substrate, we show that CsA (10 mM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. 5 CsA (10 mM) did not uncouple the electron¯ow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. 6 CsA (10 mM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. 7 Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. 8 These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes.

Molecular Mechanisms of TGFβ Receptor-Triggered Signaling Cascades Rapidly Induced by the Calcineurin Inhibitors Cyclosporin A and FK506

“…45) and in some cases may include a redox-sensitive mechanism (46,47). Previous studies have demonstrated that CsA at different concentrations promotes the generation of ROS by a mechanism that is independent of cytochrome P-450 oxidases (27) and NADPH oxidases (48), the major cellular superoxide-generating enzymes. In full agreement with these studies on smooth muscle cells, we found a similar increase in ROS production by both CNI that is blocked by the antioxidant NAC.…”

Vasopressin Type 1A Receptor Up-regulation by Cyclosporin A in Vascular Smooth Muscle Cells Is Mediated by Superoxide

“…These data suggest that shorter times of CsA exposure may be sufficient to induce V 1A receptor up-regulation. If ROS are the critical mediators of this response, it is questionable whether ROS are produced for the full 20 h. Our data have shown that CsAinduced ROS generation was increased within the first hour, peaking at 45 min, followed by a steady decline up to 2 h (20). Additional investigations have shown that after 20 h CsA does no longer generate any detectable levels of ROS when incubated with 2Ј,7Ј-DCFH for the last hour (data not shown).…”

“…Some studies have shown that CsA is able to produce ROS in vascular endothelial and mesangial cells (16,18,19). In addition, we have shown that CsA generates ROS in RASMC that were inhibited by antioxidants (20). ROS have been assigned a role of biological mediators in cellular signaling (21)(22)(23).…”

“…A, the medium was changed with medium containing CsA (1 M) but no PEG-SOD or PEG-CAT, and DCF-fluorescence was determined 1 h later. B, the medium was changed with medium containing CsA (1 M) but no PEG-SOD or PEG-CAT, and patients (44)) produced significant amounts of ROS in RASMC that could be blocked by antioxidants (20). Another study has also found that 1 M CsA generated ROS in cardiomyocytes (45).…”

Cyclosporin a Activates TGF-/Smad Signaling Pathway in Rat Liver

“…In this respect, oxidation of the latency-associated peptide can cause a conformational change that releases TGF-β (Barcellos-Hoff & Dix, 1996). Previously, it has been demonstrated that CsA generates ROS in different cell types (Krauskopf et al, 2002 andAkool et al, 2008). Furthermore, CsA has been shown to induce TGF- activation and Smads phosphorylation via generation of ROS in renal cells (Akool et al, 2008).…”