Oxygen radical formation during cytochrome P450-catalyzed cyclosporine metabolism in rat and human liver microsomes at varying hydrogen ion concentrations |
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| Authors: | S Sohail Ahmed Kimberly L Napoli Henry W Strobel |
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| Institution: | (1) Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, P.O. Box 20708, 77225 Houston, Texas, USA;(2) Division of Immunology/Organ Transplantation, The University of Texas Medical School at Houston, P.O. Box 20708, 77225 Houston, Texas, USA |
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| Abstract: | The role of pH in uncoupling the electron-flux between oxidoreductase and cytochrome P450 (P450) or P450 and cyclosporine (CyA) and resulting in the generation of oxygen radicals was investigatedin vitro in rat and human liver microsomal preparations. Since the electron-flux from NADPH to cytochrome c via oxidoreductase showed a fairly constant reduction activity from pH 7.0–9.5, the generation of oxygen radicals at the level of P450-Cyclosporine (instead of oxidoreductase-P450) was investigated. The effects of increasing pH on oxygen radical formation was measured by the thiobarbituric acid assay (TBA) and the adrenochrome reaction. The trends in oxygen radical production were correlated with benzphetamine metabolism (production of formaldehyde) and CyA metabolism (analyzed by high performance liquid chromatography). The TBA assay showed increased MDA-detected lipid peroxidation (unrelated to autooxidation) at pH<8.0 and pH>8.0 (rat and human, respectively) while the adrenochrome reaction showed decreased oxygen radical production. When these results were compared to benzphetamine (a substrate of P450 2B and 3A) metabolism and CyA (a substrate of P450 3A) metabolism, increased metabolism followed the pH-dependent trend of MDA-detected lipid peroxidation. Benzphetamine metabolism with formaldehyde production and depletion of parent compound during CyA metabolism were increased at pH<8.0 in the rat samples and at pH>8.0 in the human samples. This parallel relation suggests that the increased metabolism of CyA at lower pH in rats and higher pH in humans may be the result of favorable interactions of P450 with Cyclosporine that also result in increased oxygen radical-related lipid peroxidation.Abbreviations CCl4
carbon tetrachloride
- CyA
cyclosporin A
- EDTA
ethylenediaminetetraacetic acid
- HPLC
high performance liquid chromatography
- MDA
malondialdehyde
- MFO
mixed function oxidase
- MICROS
microsomes
- NADPH
nicotinamide adenine dinucleotide phosphate
- TBA
thiobarbituric acid
This work was supported by Grant No. CA-53191 from the National Cancer Institute DHHW |
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| Keywords: | cyclosporin A benzphetamine oxygen radicals hepatotoxicity hydrogen ion concentrations adrenochrome reaction chromatography thiobarbituric acid assay human liver microsomes rat liver microsomes cytochrome P-450 mixed-function oxidase system drug metabolism immunosuppression transplantation |
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