Abstract
We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of NAD(P)H: quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and enhances the ratio of BaP phenolic metabolites versus diol/diones in endothelial microsomes.
Highlights
Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound, has been shown to contribute to the development of atherosclerosis-related cardiovascular disease [1, 2]
We previously reported that overexpression of antioxidant enzyme catalase diminished BaP-induced atherosclerosis in hypercholesterolemic mice [4], reduced BaP intermediates [17], and enhanced BaP-induced expression of NAD(P)H: quinone oxidoreductase-1 (NQO1), glutathione S-transferases (GSTs)-pi (GSTP), CYP1A1 and 1B1 in mouse aortic endothelial cells (MAECs) [17, 18]
We previously reported that that endothelial cells obtained from human catalase transgene (hCatTg) mice had ~2.5 fold increase in their catalase activity, and no significant change in the activities of other antioxidant scavengers, including Cu/Zn-superoxide dismutase (SOD), Mn-SOD, extracellular-SOD, and glutathione peroxidase-1, when compared with the cells obtained from wild-type (WT) littermates [20]
Summary
Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound, has been shown to contribute to the development of atherosclerosis-related cardiovascular disease [1, 2]. Knockout of CYP1A1 augments BaP-DNA adducts and BaP-induced toxicity [13], while knockout of CYP1B1 results in protection against PAH-induced toxicity in mice [14]. A balance between expression of CYP1A1 and 1B1 enzymes governs BaP-induced toxicity Another explanation for the different effects of CYP1 proteins on BaP metabolism is that CYP1A1 is tightly coupled to phase II enzymes in the endoplasmic reticulum (ER). We previously reported that overexpression of antioxidant enzyme catalase diminished BaP-induced atherosclerosis in hypercholesterolemic mice [4], reduced BaP intermediates [17], and enhanced BaP-induced expression of NQO1, GST-pi (GSTP), CYP1A1 and 1B1 in mouse aortic endothelial cells (MAECs) [17, 18]. Overexpression of catalase reduced BaP intermediates, and upregulated BaP-induced expression of these XMEs in an order as follows: 1A1>NQO1>GSTP>1B1>EH1. Decrease in microsomal BaP intermediates in catalase-overexpressing cells unlikely result from an increased spatial association of phase I and phase II XMEs, but might result from an increased expression of XMEs in a way that shifts the balance between CYP1A1 and 1B1 and the balance between phase I and phase II XMEs in favor of reducing BaP intermediates
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