Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta (original) (raw)
Related papers
The AAPS Journal, 2013
Cytochrome P450 (P450)-derived arachidonic acid (AA) metabolites serve pivotal physiological roles. Therefore, it is important to determine the dominant P450 AA monooxygenases in different organs. We investigated the P450 AA monooxygenases protein expression as well as regioselectivity, immunoinhibition, and kinetic profile of AA epoxygenation and hydroxylation in rat heart, lung, kidney, and liver. Thereafter, the predominant P450 epoxygenases and P450 hydroxylases in these organs were characterized. Microsomes from heart, lung, kidney, and liver were incubated with AA. The protein expression of CYP2B1/2, CYP2C11, CYP2C23, CYP2J3, CYP4A1/2/3, and CYP4Fs in the heart, lung, kidney, and liver were determined by Western blot analysis. The levels of AA metabolites were determined by liquid chromatography-electrospray ionization mass spectroscopy. This was followed by determination of regioselectivity, immunoinhibition effect, and the kinetic profile of AA metabolism. AA was metabolized to epoxyeicosatrienoic acids and 19-and 20-hydroxyeicosatetraenoic acid in the heart, lung, kidney, and liver but with varying metabolic activities and regioselectivity. Anti-P450 antibodies were found to differentially inhibit AA epoxygenation and hydroxylation in these organs. Our data suggest that the predominant epoxygenases are CYP2C11, CYP2B1, CYP2C23, and CYP2C11/CYP2C23 for the heart, lung, kidney, and liver, respectively. On the other hand, CYP4A1 is the major ωhydroxylase in the heart and kidney; whereas CYP4A2 and/or CYP4F1/4 are probably the major hydroxlases in the lung and liver. These results provide important insights into the activities of P450 epoxygenases and P450 hydroxylases-mediated AA metabolism in different organs and their associated P450 protein levels.
Toxicology, 2014
Benzo[a]pyrene (BaP) is an environmental pollutant produced by combustive processes, such as cigarette smoke and coke ovens, and is implicated in the pathogenesis of atherosclerosis. Cytochrome P450 1A1 (CYP1A1) plays a role in both metabolic activation and detoxication of BaP in a context-dependent manner. The role of CYP1A1 in BaP-induced toxicity in aorta remains unknown. First, we fed Apoe(−/−) mice an atherogenic diet plus BaP and found that oral BaP-enhanced atherosclerosis is associated with increased reactive oxygen species (ROS) and inflammatory markers, such as plasma tumor necrosis factor levels and aortic mRNA expression of vascular endothelial growth factor A (Vegfa). We next examined the effect of an atherogenic diet plus BaP on ROS and inflammatory markers in Cyp1a1(−/−) mice. Although this treatment was not sufficient to induce atherosclerotic lesions in Cyp1a1(−/−) mice, plasma antioxidant levels were decreased in Cyp1a1(−/−) mice even in the absence of BaP treatment. The atherogenic diet plus BaP effectively elevated plasma ROS levels and expression of atherosclerosis-related genes, specifically Vegfa, in Cyp1a1(−/−) mice compared with wild-type mice. BaP treatment increased Vegfa mRNA levels in mouse embryonic fibroblasts from Cyp1a1(−/−) mice but not from wild-type mice. BaP-induced DNA adduct formation was increased in the aorta of Cyp1a1(−/−) mice, but not wild-type or Apoe(−/−) mice, and the atherogenic diet decreased BaP-induced DNA adducts in Cyp1a1(−/−) mice compared with mice on a control diet. These data suggest that ROS production contributes to BaP-exacerbated atherosclerosis and that CYP1A1 plays a protective role against oral BaP toxicity in aorta.
British Journal of Pharmacology, 2009
There is a strong correlation between cytochrome P450 (P450)-dependent arachidonic acid metabolism and the pathogenesis of cardiac hypertrophy. Several aryl hydrocarbon receptor (AhR) ligands were found to alter P450-dependent arachidonic acid metabolism. Here, we have investigated the effect of 3-methylcholanthrene (3-MC) and benzo(a)pyrene (BaP), two AhR ligands, on the development of cardiac hypertrophy. Experimental approach: Male Sprague Dawley rats were injected (i.p.) daily with either 3-MC (10 mg·kg -1 ) or BaP (20 mg·kg -1 ) for 7 days. Then hearts were removed, and the heart to body weight ratio and the gene expression of the hypertrophic markers and P450 genes were determined. Levels of arachidonic acid metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry. Key results: Both 3-MC and BaP increased the heart to body weight ratio as well as the hypertrophic markers, atrial natriuretic peptide and brain natriuretic peptide. 3-MC and BaP treatment increased the gene expression of CYP1A1, CYP1B1, CYP2E1, CYP4F4, CYP4F5 and soluble epoxide hydrolase. Both 3-MC and BaP treatments increased the dihydroxyeicosatrienoic acids (DHETs) : epoxyeicosatrienoic acids (EETs) ratio and the 20-hydroxyeicosatetraenoic acid (20-HETE) : total EETs ratio. Treatment with benzo(e)pyrene, an isomer of BaP that is a poor ligand for the AhR, did not induce cardiac hypertrophy in rats, confirming the role of AhR in the development of cardiac hypertrophy. Treatment with the w-hydroxylase inhibitor, HET0016, significantly reversed BaP-induced cardiac hypertrophy. Conclusions and implications: 3-MC and BaP induce cardiac hypertrophy by increasing the ratio of DHETs : EETs and/or the ratio of 20-HETE : total EETs, through increasing soluble epoxide hydrolase activity.
Journal of Lipid Research, 2000
The demonstration of in vivo arachidonic acid epoxidation and-hydroxylation established the cytochrome P450 epoxygenase and /-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and /-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products.-Capdevila,
Cytochrome P450 expression and catalytic activity in coronary arteries and liver of cattle
Biochimica et Biophysica Acta (BBA) - General Subjects, 2005
There is increasing evidence that cytochrome P450 (CYP) enzymes are involved not only in the metabolism of xenobiotics, but also in vascular homeostasis. Among the CYP-derived vasoactive agents, special importance is assigned to endogenous products from arachidonic acid (AA). Specifically, the vasodilator epoxyeicosatrienoic acids (EETs), being linked to the CYP 2B, 2C, and 2J subfamilies, and the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) connected instead to the CYP 4A subfamily and, to a lesser degree, to isoforms of the CYP 1A and 2E subfamilies. Here, we have examined the occurrence of functional CYP isoforms in the coronary arteries of cattle by RT-PCR with sequence verification, Western immunoblotting, and analysis of distinct catalytic activities with fluorescent substrate probes. Liver tissue was examined comparatively. Coronary tissue expressed mRNA transcripts and immunoreactive proteins belonging to the CYP 1A, 2C, 2E, and 2J subfamilies. Appropriate catalytic activity was ascertained with all these CYP species except 2J. A broader spectrum of CYP enzymes (CYP 1A, 2B, 2C, 2D, 2E, 2J, 3A, 4A subfamilies) was found in liver tissue with catalytic activities exceeding many fold those of coronary tissue. We conclude that bovine coronary arteries are endowed with a full-fledged CYP system with potential for AAlinked vasoregulation through dilator rather than constrictor agents. The same tissue and, to a much larger degree, liver tissue possess the capability of metabolizing xenobiotics via the CYP pathway.
Molecular and Cellular Biochemistry, 2015
As exposure to polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) have been implicated in cardiovascular diseases, the ability of the aortic tissue to process these toxicants is important from the standpoint of abdominal aortic aneurysms and atherosclerosis. Benzo(a)pyrene (B(a)P), a representative PAH compound is released into the environment from automobile exhausts, industrial emissions, and considerable intake of B(a)P is also expected in people who are smokers and barbecued red meat eaters. Therefore, knowledge of B(a)P metabolism in the cardiovascular system will be of importance in the management of vascular disorders. Toward this end, subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) were isolated from the aortic tissues of Apo E mice that received a 5 mg/kg/week of B(a)P for 42 days and 0.71 mg/kg/day for 60 days. The fractions were incubated with 1 and 3 lM B(a)P. Post incubation, samples were extracted with ethyl acetate and analyzed by reverse-phase HPLC. Microsomal B(a)P metabolism was greater than the rest of the fractions. The B(a)P metabolite levels generated by all the subcellular fractions showed a B(a)P exposure concentrationdependent increase for both the weekly and daily B(a)P treatment categories. The preponderance of B(a)P metabolites such as 7,8-dihydrodiol, 3,6-, and 6,12-dione metabolites are interesting due to their reported involvement in B(a)P-induced toxicity through oxidative stress.
In Vitro Cellular & Developmental Biology - Animal, 2005
Endothelial ceils are a structural barrier and an active regulator of many bodily processes. Cytochrome P4501A (CYP1A) activity is induced in the endothelimn of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney, and rete mirabile of the eel, Anguilla rostrata, to aryl hydrocarbon receptor (AhR) agonists. In heart endothelial cells, the maximum response (based on O-deethylation of 7-ethoxyresorufin to resorufin [EROD] activity) to 2,3,7,8-tetraehlorodibenzo-p-dioxin (TCDD), 113 pmol/mg/min, was at 1 nM TCDD and the peak response to ~-napthoflavone (~NF), 135 pmol/mg/min, was at 3 p~M ~NF. The maximum response to TCDD in the kidney endothelial cells is 12 pmolhng/min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and 13NF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg/min, respectively) in untreated or dimethylsulfoxide-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vaseulature.
In Vitro Cellular Developmental Biology Animal, 2005
Endothelial ceils are a structural barrier and an active regulator of many bodily processes. Cytochrome P4501A (CYP1A) activity is induced in the endothelimn of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney, and rete mirabile of the eel, Anguilla rostrata, to aryl hydrocarbon receptor (AhR) agonists. In heart endothelial cells, the maximum response (based on O-deethylation of 7-ethoxyresorufin to resorufin [EROD] activity) to 2,3,7,8-tetraehlorodibenzo-p-dioxin (TCDD), 113 pmol/mg/min, was at 1 nM TCDD and the peak response to ~-napthoflavone (~NF), 135 pmol/mg/min, was at 3 p~M ~NF. The maximum response to TCDD in the kidney endothelial cells is 12 pmolhng/min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and 13NF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg/min, respectively) in untreated or dimethylsulfoxide-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vaseulature.
Cytochrome P450, the arachidonic acid cascade, and hypertension: new vistas for an old enzyme system
The FASEB Journal, 1996
In the cytochromes P450 series, this review is the first of four articles addressing function and regulation of forms of cytochromes P450 involved in endogenous substrate metabolism. The arachidonic acid cascade leading to eicosinoids with strong vasoactive properties involves microsomal P450s. Capdevila and colleagues have pioneered study of the role of these P450 metabolites and in this review describe the biochemical characteristics of P450 arachidonic acid monooxygenases in rat kidney, which are found to be members of different CYP gene families. The functional significance of these enzymes and their products is described with particular attention to vasoactive properties and effects on ion transport. Study of renal P450 arachidonic acid monooxygenases indicates that their products significantly affect the physiological mechanisms that control fluid volume and composition. Studies in experimental models of hypertension indicate the importance of these enzymes in certain aspects of this complex disease.
Biochemical Pharmacology, 1982
Single intraperitoneal injections of octachlorostyrene (OCS) and hexachlorobenzene in genetically polycyclic aromatic hydrocarbon 'responsive' C57iBLi6 (B6) mice led to a time-and dosedependent increase in the levels of liver microsomal cytochromes P-450 and bs as well as in the activities of NADPH cytochrome P-450 (cytochrome c) reductase, ethylmorphine (EM) N-demethylase, 4nitroanisole (PNA) 0-demethylase and acetanilide 4-hydroxylase (AcA hydroxylase). No, or only a very moderate, increase in the activity of aryl hydrocarbon hydroxylase was seen after OCS and HCB, respectively. Pretreatments with phenobarbital (PB) or 3-methylcholanthrene (MC) both increased AcA hydroxylase activity to a similar degree, whereas pretreatment with polychlorinated biphenyls (Aroclor 1254) had an effect equal to the sum of PB and MC. Judged from sodium dodecylsulfate polyacrylamide gel electrophoresis studies, OCS and HCB predominantly increased a microsomal polypeptide of apparent mol. wt 52,000, similar to PB. A reduced response was seen after OCS or HCB treatment of aromatic hydrocarbon 'non-responsive' DBAR (D2) mice compared to B6 mice, both with respect to AcA hydroxylase as well as EM demethylase and PNA demethylase activities. OCS treatment of B6D2Fl mice resulted in a doubling of AcA hydroxylase activity, but in mice of the (B6D2)D2 backcross no distinct subgroupings of individual AcA hydroxylase activities were apparent. These results demonstrate that OCS is an inducer of the PB-type in mice and that induction of AcA hydroxylase by OCS is not regulated by the Ah locus.