Oxygen radical formation due to the effect of varying hydrogen ion concentrations on cytochrome P450-catalyzed cyclosporine metabolism in rat and human liver microsomes.

Abstract

Cyclosporin A (CyA) is a hydrophobic cyclic undecapeptide whose potent immunosuppressive effects have resulted in its widespread usage for suppressive allograft rejection. However, its metabolism by the cytochrome P450 mixed-function oxidase (MFO) system frequently results in hepatotoxicity as seen by jaundice and altered serum enzyme in hepatic transplant recipients, hyperbilirubinemia, hypoproteinemia, and albuminemia in rats (Whiting et al., 1983). In previous experiments, the sources of the hepatotoxicity associated with CyA metabolism was implicated as being oxygen radicals (Ahmed et al., 1993). Xenobiotics, like CyA or FK-506, lead to the production of free radicals by a redox cycle involving one-electron reductions by oxidoreductase that reduce molecular oxygen to superoxide. Not only can superoxide result in the observed toxicity, but it may, in the presence of transition metals such as iron, generate reactive hydroxy radicals by the Haber-Weiss or the Fenton reaction. Oxidoreductase is a vital element of the cytochrome P450 system which catalyzes the biotransformation of xenobiotics by either oxidation or reduction. Oxidoreductase usually transfers two reducing equivalents from NADPH to the cytochrome P450-substrate complex resulting in substrate oxidation and subsequent product release. This reductase-mediated transfer of electrons is characterized as an electron flux. Certain investigators have measured metabolite ratios of different substrates at varying pH (Grogan et al., 1993 and Hille et al., 1981) and have reported differences in the electron transfer from oxidoreductase to cytochrome P450. These results have prompted us to investigate the possible role of pH in either uncoupling the electron-flux at the level of oxidoreductase and P450 or that of P450 and CyA with the generation of hydroxy radicals as seen by lipid peroxidation.