Effects of cytochrome P450 inhibitors on peroxidase activity.
OBJECTIVE
Of several enzymes metabolizing xenobiotics, cytochrome P450 (CYP) and peroxidase enzymes seem to be most important. One of the major challenges in studies investigating metabolism of xenobiotics is to resolve which of these two groups of enzymes is predominant to metabolize individual xenobiotic compounds. Utilization of selective inhibitors of CYP and peroxidase enzymes might be a useful tool to identify the contribution of these enzymes to metabolism of xenobiotics in samples, where both types of enzymes are present. The aim of this study was to investigate specificities of several known CYP inhibitors to these enzymes; whether they inhibit only the CYP enzymes and do not inhibit peroxidases.


METHODS
Since the oxidation of o-anisidine catalyzed by a model peroxidase used, horseradish peroxidase (HRP), is a two-substrate reaction, the inhibition potential of tested chemicals was studied with respect to both peroxidase substrates, o-anisidine and hydrogen peroxide. Initial velocities of o-anisidine oxidation by HRP under various conditions were determined spectrophotometrically.


RESULTS
The CYP inhibitors metyrapone, troleandomycine, disulfiram, sulfaphenazole, quinidine and 1-aminobenzotriazole do not inhibit o-anisidine oxidation catalyzed by HRP. In contrast, ketoconazole, diethyldithiocarbamate, ellipticine, α-naphtoflavone, proadifen SKF525A, piperonylbutoxide, were found to inhibit not only the CYPs, but also the HRP-mediated oxidation of o-anisidine. Interestingly, α-naphtoflavone inhibits oxidation of o-anisidine by HRP with respect to H2O2, but not with respect to o-anisidine. Diethyldithiocarbamate is the most potent peroxidase inhibitor of o-anisidine oxidation with Ki with respect to o-anisidine of 10 μM and Ki with respect to H2O2 of 60 μM, being even the better peroxidase inhibitor than the classical "peroxidase inhibitor" - propyl gallate (Ki with respect to o-anisidine of 60 μM and Ki with respect to H2O2 of 750 μM).


CONCLUSIONS
The results of the present study demonstrate that 1-aminobenzotriazole, a potent inhibitor of various CYP enzymes, seems to be the best candidate suitable for utilization in studies evaluating participation of CYP enzymes in metabolism of xenobiotics in various complex biological materials containing both CYP and peroxidase enzymes. Moreover, precaution to prevent misinterpretation of results is necessary in cases when proadifen SKF525A, piperonylbutoxide, diethyldithiocarbamate, ketoconazole, α-naphtoflavone and ellipticine are used in similar studies (as CYP inhibitors in various complex biological materials containing both CYP and peroxidase enzymes), since these chemicals can except of CYP enzymes inhibit also peroxidase-mediated reactions.
Department of Biochemistry, Charles University, Prague, Czech Republic.
Marketa
M
Martinkova
Bozena
B
Kubickova
Marie
M
Stiborova
D001571:Benzoflavones / Q000737:chemistry / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D004221:Disulfiram / Q000737:chemistry / Q000494:pharmacology; D004050:Ditiocarb / Q000737:chemistry / Q000494:pharmacology; D004611:Ellipticines / Q000737:chemistry / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D007654:Ketoconazole / Q000737:chemistry / Q000494:pharmacology; D008797:Metyrapone / Q000737:chemistry / Q000494:pharmacology; D010882:Piperonyl Butoxide / Q000737:chemistry / Q000494:pharmacology; D011335:Proadifen / Q000737:chemistry / Q000494:pharmacology; D011802:Quinidine / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D013426:Sulfaphenazole / Q000737:chemistry / Q000494:pharmacology; D014230:Triazoles / Q000737:chemistry / Q000494:pharmacology*; D014217:Troleandomycin / Q000737:chemistry / Q000494:pharmacology
D001571:Benzoflavones; D065607:Cytochrome P-450 Enzyme Inhibitors; D004611:Ellipticines; D004791:Enzyme Inhibitors; D014230:Triazoles; D013426:Sulfaphenazole; C034192:ellipticine; C033020:1-aminobenzotriazole; C011512:alpha-naphthoflavone; D004050:Ditiocarb; D011335:Proadifen; D014217:Troleandomycin; D006735:Horseradish Peroxidase; D011802:Quinidine; D010882:Piperonyl Butoxide; D007654:Ketoconazole; D004221:Disulfiram; D008797:Metyrapone