Original Contributions
Production of Reactive Oxygen Species by Microsomes Enriched in Specific Human Cytochrome P450 Enzymes

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Abstract

Few studies have evaluated the production of reactive oxygen intermediates by human microsomes, especially the influence of the specific form of cytochrome P450. Experiments were carried out to evaluate the ability of CYP1A1, 1A2, 2B6, and 3A4 to consume NADPH, reduce iron, and catalyze production of reactive oxygen species. Microsomes enriched in each of these CYPs were obtained from commercial ± lymphoblast cells that had been transfected with cDNA encoding the specific human CYP. On a per nanomole cytochrome P450 basis, CYP3A4 was the most active P450 evaluated in catalyzing NADPH oxidation, production of superoxide anion radical, NADPH-dependent chemiluminescence, oxidation of dichlorofluorescein diacetate, and reduction of either ferric-EDTA or ferric-citrate. CYP1A1 was the next most reactive CYP, whereas CYP1A2 and 2B6 displayed a comparable, lower activity. Nitric oxide, which reacts with and inactivates hemoproteins, inhibited superoxide production by all the CYPs to a similar extent. Because CYP3A4 is present in high amounts in human liver microsomes and is active in catalyzing the formation of reactive oxygen species, this CYP may make an important contribution in the overall ability of human liver microsomes to generate active oxygen species.

Introduction

Cytochrome P450s are a large superfamily of hemoproteins that catalyze the biotransformation of a wide variety of chemicals.1, 2, 3Their ubiquitous presence and similarities in primary and secondary structure as well as physiochemical characteristics suggest that all these enzymes have evolved from a common ancestral gene and have diversified through evolution. Multiple CYPs have been characterized in many mammalian species.1, 2, 3, 4Each CYP enzyme has its own profile of catalytic specificity, although overlapping substrate specificities among different CYP forms are also observed. Cytochromes P450 are able to convert xenobiotics into inert polar metabolites, or to activate them into becoming reactive species able to initiate DNA, protein, or lipid damage.

CYP1A1 is among the most active of the CYPs to catalyze the bioactivation of many promutagens and procarcinogens.[5]CYP1A1/1A2 are mainly induced in humans by cigarette smoking and eating charcoal-broiled food. CYP1A1 is involved in the activation of polycyclic aromatic hydrocarbons and the tobacco smoke nitrosamine NNK.[6]CYP1A2 catalytic activities include metabolism of 7-ethoxyresorufin, acetanilide, caffeine, estrogens, and phenacetin. CYP1A2 is present in most human livers but its levels of expression varies considerably.5, 6The CYP2B6 enzyme can activate aflatoxin B1[7]and is capable of activating the structurally related anticancer drugs cyclophosphamide and ifosphamide.[8]CYP3A4 is expressed in livers of most adult subjects[6]and can metabolize cortisol, estrogen, nifedipine, cyclosporin, testosterone, (R)-warfarin, gestodene, ketoconazole, miconazole, dapsone, FK-506, quinidine, lovastatin, erythromycin, and numerous other drugs.

The catalytic mechanism for CYPs involves reductive activation of molecular oxygen in a process in which several steps are one-electron transfers, that is, movement of either an electron or a hydrogen atom.4, 9The generation of radicals within the active site of CYPs is responsible for their unusual catalytic properties, and small amounts of O2•− and H2O2 are by-products of the normal P450 catalytic cycle.[10]To be catalytically active, microsomal CYPs require a supply of electrons, which are provided by NADPH-cytochrome P450 oxidoreductase (OR, EC 1.6.2.4).[11]The study of human cytochrome P450s has been greatly facilitated by their expression in heterologous systems, including mammalian cells,[7]insect cells,[12]yeast,[13]and Escherichia coli.[14]Few studies have evaluated the production of reactive oxygen intermediates by human microsomes,[15]especially the influence of the specific type of CYP present. CYP2E1 from rat liver was more active in producing H2O2 compared with several other CYPs16, 17; however, CYP2E1 from rabbit liver (P450 3a) was less reactive in producing H2O2 compared with three other rabbit CYPs (2, 3b, and 4).[18]Rat CYP1A1 was less reactive than rat CYP1A2 in catalyzing NADPH plus iron-dependent lipid peroxidation.[19]The goal of the current report was to compare the ability of several human CYPs to produce reactive oxygen species when microsomes enriched with a specific CYP were incubated with NADPH.

Section snippets

Materials and Methods

Microsomes enriched in CYP1A1, CYP1A2, CYP2B6, or CYP3A4 were obtained from Gentest Corp., Woburn, MA. These microsomes were prepared from metabolically competent derivatives of the human AHH-1 TK ± cell line (human β-lymphoblastoid cells) that have been engineered to stably express human CYP1A1,[20]CYP1A2,[21]CYP2B6,[22]or CYP3A4.[23]The parent AHH-1 TK ± cell line contains a low level of native human cytochrome P450 activity that is inducible by polycyclic aromatic hydrocarbons (PAH). The

Results

Table 1 summarizes the cytochrome P450 content and NADPH-cytochrome P450 reductase activity of microsomes obtained from the AHH-1 TK ± cell lines transfected with cDNA for human CYP1A1, 1A2, 2B6, and 3A4. The content of CYP was similar for the CYP1A1, 1A2, and 2B6 preparations, but was lower for the CYP3A4 microsomes. Reductase activity was also similar for the former three preparations and was equal to that of the vector control as this activity reflected that due to the endogenous content of

Discussion

The cytochrome P450 reaction cycle through unproductive pathways can give rise to side products such as superoxide, hydrogen peroxide, and water.4, 10, 18The one-electron-reduced oxy-P450 intermediate marks the first branch point at which autooxidation or second-electron reduction occurs. The two-electron reduced peroxy-P450 species undergoes either dioxygen cleavage or peroxide release at the second branch point. At the third fork, the putative iron-oxo intermediate is partitioned between

Acknowledgements

These studies were supported by USPHS Grants AA 09460 and AA 03312 from The National Institute on Alcohol Abuse and Alcoholism and by The Thalmann Fellowship from The University of Buenos Aires. We thank Ms. Pilar Visco Cenizal for typing the manuscript.

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