Ethylbenzene induces microsomal oxygen free radical generation: antibody-directed characterization of the responsible cytochrome P450 enzymes.
Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to its fluorescent product 2',7'-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H(2)O(2)) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to influence free radical production and that the increase in free radical production in EB treated rats is consistent with the EB-mediated elevation of CYP2B, CYP 2E1, and CYP3A2. Such alterations in free radical generation in response to hydrocarbon treatment may contribute to the toxicity of these compounds.
Serron
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Department of Pharmacology and Experimental Therapeutics, The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, Louisiana, 70112, USA.
Dwivedi
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D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D001555:Benzene Derivatives / Q000494:pharmacology*; D015153:Blotting, Western; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004785:Environmental Pollutants / Q000494:pharmacology; D005609:Free Radicals; D008297:Male; D008565:Membrane Proteins; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D017382:Reactive Oxygen Species / Q000378:metabolism*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism
D000906:Antibodies; D001555:Benzene Derivatives; D004785:Environmental Pollutants; D005609:Free Radicals; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; C465260:Cyp3a2 protein, rat; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; C025619:steroid 16-beta-hydroxylase; D010089:Oxidoreductases, N-Demethylating; C004912:ethylbenzene