CYP1A2 protects against reactive oxygen production in mouse liver microsomes

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Abstract

H2O2 production was evaluated in liver microsomes prepared from Cyp1a1/1a2(+/+) wild-type and Cyp1a1(−/−) and Cyp1a2(−/−) knockout mice pretreated with 5 μg dioxin (TCDD)/kg body wt or vehicle alone. NADPH-dependent H2O2 production in TCDD-induced microsomes from wild-type mice was about one-third of that in noninduced microsomes. In Cyp1a2(−/−) mice, H2O2 production was the same for induced and noninduced microsomes, with levels significantly higher than those in wild-type mice. Cyp1a1(−/−) microsomes displayed markedly lower levels of H2O2 production in both induced and noninduced microsomes, compared with those in wild-type and Cyp1a2(−/−) microsomes. The CYP1A2 inhibitor furafylline in vitro exacerbated microsomal H2O2 production proportional to the degree of CYP1A2 inhibition, and the CYP2E1 inhibitor diethyldithiocarbamate decreased H2O2 production proportional to the degree of CYP2E1 inhibition. Microsomal H2O2 production was strongly correlated to NADPH-stimulated production of thiobarbituric acid-reactive substances, as well as to decreases in microsomal membrane polarization anisotropy, indicative of peroxidation of unsaturated membrane lipids. Our results suggest that possibly acting as an “electron sink,” CYP1A2 might decrease CYP2E1-and CYP1A1-mediated H2O2 production and oxidative stress. In this regard, CYP1A2 may be considered an antioxidant enzyme.

Introduction

Polynuclear polyhalogenated aromatic hydrocarbons (PHAHs) are environmental toxicants that include halogenated dibenzodioxins, dibenzofurans, and biphenyls. Many PHAHs, including 2,3,7,8-tetrahydrodibenzo-p-dioxin (TCDD, dioxin), are known or suspected carcinogens, toxicants, and teratogens in animals and humans [1], [2], [3], [4], [5], [6], [7], [8] TCDD was recently characterized by the U.S. Environmental Protection Agency as a probable human carcinogen [9] in a report challenged [10] and subsequently defended [11]. The controversy regarding the human carcinogenicity of TCDD and related compounds is based primarily on human levels of exposure to such compounds, rather than to their carcinogenic potential. Although TCDD is not directly genotoxic, exposure results in reactive oxygen production and an oxidative stress response in adult and fetal tissues of experimental animals [12], [13], [14], [15], [16], [17]. This reactive oxygen may in turn oxidize DNA bases, leading to strand breakage or clastogenic effects [14], [18]. Although the TCDD-mediated oxidative stress response has been characterized in many studies, the mechanism by which TCDD produces reactive oxygen is poorly understood.

A vast literature attributes most, if not all, of the effects of low concentrations of TCDD and other TCCD-like PHAHs to events following the activation of the aromatic hydrocarbon receptor (AHR). This cytosolic ligand-activated transcription factor has the potential to upregulate and downregulate the expression of hundreds of genes [19], including those of the Ah gene battery, such as CYP1A1 and CYP1A2 [6], [20]. Activation of the AHR is clearly associated with a cellular oxidative stress response [20], [21], mediated in part by the induction of cytochrome P450s (CYPs) that tend to exhibit relatively loose coupling between oxygen and NADPH utilization, on the one hand, and substrate oxidation, on the other. The mechanism of incomplete coupling involves the release of some of the heme-bound oxygen that is chemically reduced by one or two electrons. Specific CYPs that may be loosely coupled include CYP2E1 [22], [23] and members of the CYP2B and CYP3A [24], [25] and CYP1A [26], [27] subfamilies.

Current models designed to evaluate the dose response for biological effects of TCDD and dioxin-like compounds are based primarily on their capacity to bind to and activate the AHR, leading to dose-dependent induction of CYP1A1 and CYP1A2 expression [28], [29]. CYP1A1 and CYP1A2 are the two best-characterized TCDD-inducible CYPs. Although the status of CYP1A1 and CYP1A2 in mediating chemical toxicity has been examined in many studies, a clear understanding has yet to emerge. CYP1A1 and CYP1A2 are involved primarily in the metabolism and biological clearance of a vast array of aromatic hydrocarbons and aryl amines, respectively [30]. In some instances metabolism may result in activation of substrates, leading to toxicity and carcinogenesis [31], [32]. The complicated relationship between the pharmacokinetic profile of toxicant and metabolite clearance, versus the pharmacokinetic profile of toxicant and metabolite interaction with critical cellular toxicologic target sites, may result in disparate findings, depending on the test system. These considerations are illustrated by recent findings that levels of benzo[a]pyrene–DNA adducts are dramatically increased in Cyp1a1(−/−) mice [33]. Furthermore, oxidative stress and DNA adducts due to 4-aminobiphenyl exposure are significantly enhanced in Cyp1a2(−/−) mice [34], [35]. Clearly, the pathways through which CYP1A1 and CYP1A2 mediate or ameliorate metabolic activation, chemical toxicity, and carcinogenesis require further consideration.

In previous studies, we reported an increase in succinate-dependent release of superoxide and H2O2 in mitochondria from TCDD-induced mice [36], [37]. Furthermore, the production of these reactive oxygen species was dependent on the presence of the AHR, but not dependent on either CYP1A1 or CYP1A2. We have also shown (accompanying article) that highly halogenated and coplanar aromatic hydrocarbons increase microsomal NADPH-dependent H2O2 production up to more than 7-fold in TCDD-induced mouse liver microsomes. For this study, we examined the basis for microsomal H2O2 production in noninduced and TCDD-induced mice. We had expected to find an increase in NADPH-dependent H2O2 production in liver microsomes from TCDD-treated animals. We found, however, that TCDD treatment significantly decreased microsomal H2O2 production and the associated oxidative stress response. This present study examines the mechanisms responsible for these findings.

Section snippets

Chemicals

TCDD was purchased from Accustandard (New Haven, CT, USA). All other chemicals and reagents were obtained from Sigma–Aldrich Chemical Company (St. Louis, MO, USA) as the highest available grades.

Animals and treatment

All experiments involving mice were conducted in accordance with the National Institutes of Health standards for care and use of experimental animals and the University of Cincinnati Institutional Animal Care and Use Committee (IACUC). Animals were group-housed, maintained on a 12-h light/dark cycle,

Results

We first determined H2O2 production using luminol luminescence in TCDD-induced and noninduced liver microsomes (Fig. 1). Without NADPH, no H2O2 was produced (data not shown). In Cyp1a1/1a2(+/+) wild-type microsomes, we found that NADPH-dependent H2O2 production was decreased in TCDD-induced microsomes (Fig. 1); the diminution of the integrated luminescence over time was 69% (legend to Fig. 1). In Cyp1a2(−/−) knockout mice, H2O2 production increased in both induced and noninduced microsomes,

Discussion

These studies demonstrate that the NADPH-dependent H2O2 production and the resulting microsomal oxidative stress response—including increases in lipid peroxidation and membrane fluidity—are decreased in liver microsomes from TCDD-induced mice, relative to those from vehicle-treated control mice. The decrease in oxidative stress parameters is mediated by CYP1A2, which appears to accept electrons from the pro-oxidant CYP2E1 and CYP1A1.

Our current finding, that in Cyp1a1(−/−) microsomes the CYP1A2

Conclusions

Our results suggest that, in hepatic noninduced microsomes, H2O2 is produced primarily by CYP2E1, whereas in TCDD-induced microsomes, CYP2E1 and likely CYP1A1 may be involved. CYP1A2 decreases the microsomal H2O2 production generated by CYP2E1 and CYP1A1, apparently by acting as an electron acceptor, or sink, to prevent the uncoupled electron transfer from NADPH to O2. These pathways for electron transfer appear to regulate the microsomal production of reactive oxygen and may have important

Acknowledgements

We thank our colleagues for a careful reading of the manuscript. The work was funded, in part, by NIH Grants R01 ES10133, RO1 ES06321, R01 ES08147, T32 ES07250, and P30 ES06096.

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