Omega-hydroxylase Activity Research Articles

Postmenopausal hypertension: role of 20-HETE

Blood pressure (BP) increases after menopause. However, the mechanisms responsible have not been elucidated. In this study we tested the hypothesis that 20-hydroxyeicosatetraenoic acids (20-HETE), produced by cytochrome P-450 (CYP450) ω-hydroxylase, contributes to the hypertension in a model of postmenopausal hypertension, aged female spontaneously hypertensive rats (PMR). 1-Aminobenzotriazole, a nonselective inhibitor of arachidonic acid metabolism, for 7 days, reduced BP in PMR but had no effect in young females. Acute intravenous infusion of HET-0016, a specific inhibitor of 20-HETE, over 3 h, also reduced BP in PMR. CYP4A isoform mRNA expression showed no difference in renal CYP4A1 or CYP4A3 but increases in CYP4A2 and decreases in CYP4A8. CYP4A protein expression was decreased in kidney of PMR compared with young females. Endogenous 20-HETE was significantly higher in cerebral vessels of PMR than young females (YF) but was significantly lower in renal vessels of PMR. Omega-hydroxylase activity in cerebral vessels was also higher in PMR but was similar in kidney vessels in both groups. In renal microsomal preparations, endogenous 20-HETE was not different in PMR and young females, but ω-hydroxylase activity was significantly lower in PMR than YF. The data with blockers suggest that 20-HETE contributes to postmenopausal hypertension in SHR. The data also suggest that cerebral production of 20-HETE may be increased and renal tubular production may be decreased in PMR, thus both contributing to their elevated BP.

Isoform‐specific modulation of CYP450 omega hydroxylases by cyclosporine in glomerular mesangial cells

Cyclosprine A (CSA), an immunosuppressive agent used in the treatment of many proteinuric conditions, protects the glomerular filtration barrier. We have shown that 20‐hydroxyeicosatetraenoic acid (20‐HETE), an eicosanoid generated by the CYP4a and 4f omega hydroxylase activities, maintains and protects the glomerular filtration barrier. We hypothesize that CSA induces expression of CYP4a and/or 4f in mesangial cells to protect the filtration barrier and mitigate proteinuria. We incubated murine mesangial cells in CSA (10 nM, 100 nM, 1 μM, 10 μM) or vehicle for 1, 8 or 24 hrs. We then isolated total RNA and performed real time RT‐PCR using specific primers to measure expression of CYP4f13, CYP4a12a and CYP4a12b. CYP4f13 expression showed the greatest response to CSA treatment. At 8 hr CSA 1 μM and 10 μM increased CYP4f13 by 91% and 74%, respectively. At 24 hr CSA 100 nM, 1 μM and 10 μM increased CYP4f13 by 36%, 268% and 200%, respectively. At 24 hr, CSA 10nM and 100 nM increased expression of CYP4a12a by 75% and 88%, respectively, while CSA 10 μM appeared to suppress expression to 48% of control at 24 hr. CSA 10 μM increased expression of CYP4a12b by 85% at 8 hr; this concentration appeared to suppress expression at 24 hr. Thus, mesangial cells express CYP4a and 4f, and CSA modulates the expression of these CYP isoforms in this cell type in a time and dose‐dependent manner.Supported by NIH/NIDDK R01 DK064969.

Biochemical and structural characterization of CYP124: a methyl-branched lipid omega-hydroxylase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) produces a variety of methyl-branched lipids that serve important functions, including modulating the immune response during pathogenesis and contributing to a robust cell wall that is impermeable to many chemical agents. Here, we report characterization of Mtb CYP124 (Rv2266) that includes demonstration of preferential oxidation of methyl-branched lipids. Spectrophotometric titrations and analysis of reaction products indicate that CYP124 tightly binds and hydroxylates these substrates at the chemically disfavored omega-position. We also report X-ray crystal structures of the ligand-free and phytanic acid-bound protein at a resolution of 1.5 A and 2.1 A, respectively, which provide structural insights into a cytochrome P450 with predominant omega-hydroxylase activity. The structures of ligand-free and substrate-bound CYP124 reveal several differences induced by substrate binding, including reorganization of the I helix and closure of the active site by elements of the F, G, and D helices that bind the substrate and exclude solvent from the hydrophobic active site cavity. The observed regiospecific catalytic activity suggests roles of CYP124 in the physiological oxidation of relevant Mtb methyl-branched lipids. The enzymatic specificity and structures reported here provide a scaffold for the design and testing of specific inhibitors of CYP124.

Does 20-Hydroxyeicosatetraenoic Acid Contribute to Sex Differences in Cardiovascular Risk by Increasing Oxidative Stress?

Previous studies have indicated that there are large sex differences in the incidence of hypertension, and the role of androgens in enhancing the risk of cardiovascular disease is well recognized.1,2 However, the mechanisms by which androgens increase blood pressure are poorly understood. Recent studies have demonstrated that androgen-sensitive forms of hypertension are associated with increased production of 20-hydroxyeicosatetranoic acid (20-HETE) in the kidney and the vasculature.3 However, the role of 20-HETE in this form of hypertension has not been established, because this pathway has both prohypertensive and antihypertensive actions.4 At the level of the renal tubule, upregulation of the formation of 20-HETE inhibits sodium transport and opposes the development of hypertension, whereas in the vasculature, 20-HETE is a potent vasoconstrictor that promotes hypertension. The study of Singh et al5 in the current issue explores the role of 20-HETE in the development of endothelial dysfunction and hypertension in rats chronically treated with dihydrotestosterone. The results indicate that that the expression cytochrome P450 4A protein and the production of 20 HETE increase in renal arteries of rats treated with dihydrotestosterone. The increase in vascular 20-HETE production is because of upregulation of the expression of the cytochrome P450 4A8 isoform. The development of hypertension in these studies was associated with the development of endothelial dysfunction, as reflected by an impaired vasodilator response to acetylcholine, increased expression of the gp-91 …

Evolutionary relationship and substrate specificity of Arabidopsis thaliana fatty acid omega-hydroxylase

On the basis of a phylogenic tree constructed using the available Arabidopsis thaliana genomic sequence, new fatty acid (FA) omega-hydroxylases were cloned and expressed in yeast. Several uncharacterized cytochrome P450s (CYP, P450), comprising a phylogenic sub-cluster (CYP86A, 94B, 94C, 96A, 704A, 97B and 711A) demonstrated FA omega-hydroxylase activities towards saturated FAs with medium chain length. While CYP96A4 showed the highest catalytic activity among the enzymes characterized in this study, other CYP96A subfamily members did not display any potent activity. In addition, CYP704A2 and CYP711A1, which are phylogenetically distant to both CYP86 and CYP94 with FA omega-hydroxylase activities, did not show any metabolic conversion of FAs. As we have studied omega-hydroxylation of FAs catalyzed by a series of CYP94 isoforms, active site models were produced to compare inactive CYP704A2 and CYP711A1 to active CYP94C1. The modeled structures revealed that the hydrophobicity in the heme binding site is very different between active and inactive isoforms. CYP94C1 contains highly hydrophobic residues while CYP704A2 and CYP711A1 do not. Our study provides evidence that substrate specificity is conserved in phylogenetically related isoforms and suggests specific residues in the active site pocket that play a key role in determining substrate specificity.

Discovery, Characterization, and Significance of the Cytochrome P450 ω‐Hydroxylase Pathway of Vitamin E Catabolism

Tocopherols are known to undergo metabolism to phytyl chain-shortened metabolites excreted in urine. We sought to characterize the pathway, including associated enzymes, involved in this biotransformation. We previously found that human hepatoblastoma (HepG2) cultures metabolized tocopherols to their corresponding short-chain carboxychromanols. Putative metabolites of gamma-tocopherol that contained intact chromanol moieties were structurally identified using HepG2 cultures and electron impact gas chromatography-mass spectrometry. A microsomal assay for synthesis of the initial omega-oxidation metabolites was developed and used to screen several recombinant human liver cytochrome P450 isozymes for omega-hydroxylase activity. Seven metabolites of gamma-tocopherol were identified in HepG2 cultures, including 13'-hydroxy-gamma-TOH and all six carboxychromanols predicted by sequential omega-oxidation truncation. Rat and human liver microsomes catalyzed synthesis of 13'-OH- and 13'-COOH-gamma-TOH, but not other metabolites, in the presence of NADPH. Inclusion of NAD favored synthesis of the 13'-COOH metabolite. Recombinant CYP4F2, but not other major human liver CYP isoforms (including CYP3A4 and 3A7), exhibited tocopherol-omega-hydroxylase activity. Liver microsomes and recombinant CYP4F2 both exhibited substrate preference for gamma-TOH over alpha-TOH, and recent studies show that tocotrienols are catabolized more extensively than the corresponding tocopherols. Comparative rates of omega-oxidation of tocochromanols in hepatocytes are inversely related to biopotency and directly related to cytotoxicity of these substances in macrophages. The liver contains a cytochrome P450-mediated pathway that preferentially catabolizes "non-alpha" tocochromanols to excretable metabolites. This metabolic pathway appears central to the optimization of tissue tocochromanol status.

Catalytic activity and isoform-specific inhibition of rat cytochrome p450 4F enzymes.

Arachidonic acid is omega-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome p450 (p450) 4A isoforms are generally considered the major arachidonic acid omega-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B(4) (LTB(4)) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K(s) of 25 to 59 microM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of LTB(4) with a K(m) of 24 and 31 microM, respectively, and CYP4F5 had minor activity in LTB(4) metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of arachidonic acid with an apparent k(cat) of 9 and 11 min(-1), respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid omega-hydroxylase activity. The p450 omega-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed omega-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify p450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

Toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic cytochrome P450 in F344 rats.

The subacute toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic microsomal cytochrome P450 (P450) were investigated in male and female F344 rats. Rats were given 0.25, 0.5, and 1.0% terbutol for 28 days. Liver weights of male and female rats increased at all dose levels. The compound did not affect activity or amount of serum biochemical markers related to hepatic damage. The concentrations of terbutol in rat serum were less than 0.1 microM, and its major metabolites in serum were 2,6-di-tert-butyl-4-carboxyphenyl N-methyl-carbamate and 2,6-di-tert-butyl-4-carboxyphenol. In male rats, P450 and cytochrome b5 (b5) contents, and NADPH cytochrome c reductase (fp2) activity in liver microsomes were increased about 2-fold by 1% terbutol administration for 7 to 28 days. Among the P450-dependent monooxygenase activities in liver microsomes, 7-benzyloxyresorufin-O-debenzylase (BROD) activity was greatly increased by 100-fold, and 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), and aminopyrine-N-demethylase (APND) activities were elevated 2- to 3-fold. 7-Methoxyresorufin-O-deethylase (MROD), erythromycin-N-demethylase (EMND), estradiol 2-hydroxylase (ED2H), chlorzoxazone 6-hydroxylase (CZ6H), and lauric acid omega-hydroxylase (LAOH) activities were unchanged. For the activities of testosterone hydroxylation, testosterone 16beta-hydroxylase (T16BH) activity was markedly increased by 30-fold, and testosterone 6beta-hydroxylase (T6BH) and testosterone 7alpha-hydroxylase (T7AH) activities were slightly elevated. Testosterone 2alpha-hydroxylase (T2AH) activity was not affected. Terbutol 4-methylhydroxylase (T4MH) activity was increased 9-fold by 1% terbutol. In an immunoinhibition study, T4MH activity in liver microsomes from 1% terbutol-treated rats was decreased about 50% by polyclonal anti-rat CYP2B1, whereas polyclonal anti-rat CYP2A1 and CYP2C11 did not affected the activity. These results indicate that terbutol increased CYP2B subfamily in rat liver microsomes, and that the compound did not cause serious hepatic damage.

Cytochrome P450 metabolites of arachidonic acid may be important mediators in angiotensin II-induced vasoconstriction in the rat mesentery in vivo

We have investigated the role of cytochrome P450 (CYP-450) metabolites of arachidonic acid in the modulation of vascular reactivity to angiotensin II in vivo using an in situ blood-perfused mesenteric preparation in anaesthetized spontaneously hypertensive rats (SHR). Miconazole, a non-selective inhibitor of CYP-450 that inhibits both hydroxylation and epoxidation, substantially suppressed mesenteric vasoconstrictor responses to angiotensin II in SHR, but had no effect on responses to noradrenaline or sympathetic nerve stimulation. In normotensive Wistar-Kyoto (WKY) rats, miconazole caused only a modest suppression of vasoconstrictor responses to angiotensin II. N-Methylsulphonyl-12, 12-dibromododec-11-enamide (DDMS), a new selective inhibitor of CYP-450 omega-hydroxylase activity, decreased mean intra-arterial blood pressure and significantly attenuated mesenteric angiotensin II-induced vasoconstrictor responses in SHR. Isolated mesenteric vessels were able to metabolize (14)C-labelled arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) in vitro, and this was substantially inhibited by DDMS. The results from the present studies combined with the existing evidence that angiotensin II stimulates the release of 20-HETE, a CYP-450 metabolite of arachidonic acid, suggest that CYP-450-derived HETEs may be important mediators in angiotensin II-induced vasoconstriction. However, the development of more sensitive assays for the detection in vivo of 20-HETE in mesenteric vessels would be required to confirm these findings.

The effect of immunosuppressive agents (FK-506, rapamycin) on renal P450 systems in rat models.

It is well known that cyclosporin, rapamycin and FK-506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes. Cyclosporin caused impairment of renal function with an increase in renal-specific P450 content, but FK-506 and rapamycin did not. Laurate omega- and (omega-1)-hydroxylase activity increased in rats treated with rapamycin but decreased in those treated with FK-506. Prostaglandin A1 (PGA1) omega-hydroxylase activity increased in rats treated with FK-506 but was reduced by treatment with cyclosporin. Aminopyrine N-demethylase activity increased in rats treated with cyclosporin or FK-506, but not in those treated with rapamycin. Western-blot analysis revealed significant induction of P450, (similar to CYP2B4 of the rabbit P450 isozyme) in kidneys from rats treated with cyclosporin but not in those from rats receiving FK-506 or rapamycin. Histochemical studies clearly demonstrated a form of P450 such as CYP4A2 in the proximal tubules of rats treated with cyclosporin, but not in those of rats treated with FK-506 or rapamycin. These results show that although cyclosporin has a strong effect on renal P450 systems and induces such a system in kidney cortex (microsomal P450), FK-506 and rapamycin have no substantial effect on the induction of renal P450. These findings might clarify the nephrotoxicity induced by these immunosuppressive drugs.

Peroxisome Proliferator-activated Receptor α Controls the Hepatic CYP4A Induction Adaptive Response to Starvation and Diabetes

The hepatic CYP4A enzymes are important fatty acid and prostaglandin omega-hydroxylases that are highly inducible by fibric acid hypolipidemic agents and other peroxisome proliferators. Induction of the CYP4A enzymes by peroxisome proliferators is mediated through the nuclear peroxisome proliferator-activated receptor alpha (PPARalpha). Fatty acids have recently been identified as endogenous ligands of PPARalpha, and this receptor has been implicated in the regulation of lipid homeostasis. In the present report we characterized the induction of the hepatic CYP4A genes in rats during the altered lipid metabolism associated with starvation and diabetes. The mRNA levels of CYP4A1, CYP4A2, and CYP4A3 were induced 7-17-fold in the livers of fasted animals and 3-8-fold in the livers of diabetic animals. This was accompanied by corresponding changes in CYP4A protein levels and arachidonic and lauric acid omega-hydroxylase activity. Interestingly, feeding animals after the fasting period caused as much as an 80% suppression of CYP4A mRNA levels, whereas CYP4A protein levels and functional activity returned to control values. A second PPARalpha-responsive gene, acyl-CoA oxidase, was also induced in rat liver by diabetes and fasting. By using PPARalpha-deficient mice, we unambiguously demonstrated that PPARalpha is strictly required for hepatic CYP4A induction by starvation and diabetes. Similarly, induction of hepatic thiolase and bifunctional enzyme also required expression of PPARalpha. This represents the first evidence for the pathophysiologically induced activation of a nuclear receptor.

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver.

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2alpha-hydroxylase (T2AH) and testosterone 6beta-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C 11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19-73%). The Km values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The Vmax for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid omega-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7alpha-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA.

Glu-320 and Asp-323 are determinants of the CYP4A1 hydroxylation regiospecificity and resistance to inactivation by 1-aminobenzotriazole.

Little information is available on the active site structure of the CYP4A family of enzymes or the mechanism by which their omega-hydroxylation regiospecificity is enforced. We report here that the E320A, D323E, and E320/D323E mutations decrease the catalytic rate of CYP4A1 approximately 5-fold and cause up to a 10-fold shift from omega- to (omega-1)-hydroxylation. The decreased catalytic rate is due to an increase in the uncoupled reduction of molecular oxygen. Tighter binding of 1- and 4-substituted imidazoles to the double mutant than to the other proteins suggests that its active site is less constrained. The reaction of these proteins with phenyldiazene causes heme degradation without the detectable formation of a phenyl-iron complex. CYP4A1 and its E320A mutant are not inactivated by 1-aminobenzotriazole (1-ABT), but the D323E and E320A/D323E mutants are inactivated. The resistance of purified CYP4A1 to inactivation by 1-ABT is surprising in view of the fact that 1-ABT causes the loss of the omega-hydroxylase activity both in microsomal preparations and in vivo. Collectively, the results establish that Glu-320, and particularly Asp-323, help to define the active site dimensions, the degree of coupled versus uncoupled versus uncoupled turnover, the omega-versus (omega-1)-hydroxylation regiospecificity, and the susceptibility to inactivation by mechanism-based inhibitors. Furthermore, they provide experimental evidence for a structural analogy between the CYP4A1 and P450BM-3 active sites.

Developmentally regulated expression of the CYP4A genes in the spontaneously hypertensive rat kidney.

The CYP4A enzymes catalyze the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), which has potent effects on the renal vasculature and tubular ion transport. Based on an increased 20-HETE formation in renal microsomes from spontaneously hypertensive rats, it has been proposed that increased expression of the CYP4A genes is an early event in the development of hypertension in these animals. To test this hypothesis, we developed RNase protection assays for specific detection of the individual CYP4A genes in the kidneys of spontaneously hypertensive and Wistar-Kyoto rats. Distinct age-dependent patterns of expression were observed for the individual CYP4A genes, with only CYP4A3 mRNA measurable in the kidneys of 1-week-old rats. CYP4A1 and CYP4A8 mRNA were detectable by 3 weeks of age and CYP4A2 mRNA at 5 weeks of age. The expression of CYP4A1 and CYP4A3 varied 4-5-fold throughout development and was highest between 3 and 5 weeks of age, declining steadily thereafter to 20% of their maximal level by 9 weeks of age. CYP4A2 mRNA levels increased steadily between 5 and 9 weeks of age, whereas CYP4A8 mRNA levels were relatively constant throughout development. The CYP4A3 mRNA level was significantly increased 1. 6-2-fold in the cortex and outer medulla of 1-4-week-old spontaneously hypertensive rat kidneys relative to the corresponding level in the Wistar-Kyoto. A similar 1.4-1.7-fold increase in CYP4A8 mRNA was also found in 3- and 4-week-old spontaneously hypertensive kidneys. Accompanying the increased expression of CYP4A3 and CYP4A8 mRNA in the prehypertensive rats were corresponding changes in functional CYP4A measured as either arachidonic acid or lauric acid omega-hydroxylase activity (1.4-2.0-fold increases) and CYP4A protein levels. After 4 weeks of age, the level of CYP4A mRNA, enzyme activity, and protein were similar in the kidneys of Wistar-Kyoto and spontaneously hypertensive rats. The findings suggest that the expression of CYP4A3 and CYP4A8 may be critical to the early changes in eicosanoid formation and renal function in the young spontaneously hypertensive rat.

Potent hypocholesterolemic activity of novel ureido phenoxyisobutyrates correlates with their intrinsic fibrate potency and not with their ACAT inhibitory activity

The hypocholesterolemic activity for novel ureido fibrate analogues was found to be over 100-fold greater than for any "second-generation" fibrate in cholesterol-fed rats. A comparison of 12 related analogues revealed that the optimal configuration for a urea-bridging region located between two aromatic rings consisted of a trisubstituted nitrogen, optimally substituted with a C7 alkyl chain and linked by dimethylene to a phenoxyisobutyrate moiety found in most fibrate analogues. The hypocholesterolemic potency of these compounds was found to correlate with their increased intrinsic fibrate activity as determined by the ability to induce omega-hydroxylase activity either in rat hepatocyte cultures or in vivo, and not with their 10-fold increased ACAT inhibitory potency when compared to other fibrates. The most active compound, 2-(4-(2-(N'-(2,4- difluorophenyl)-N-heptylureido)ethyl)phenoxy)-2-methylpropionic acid, referred to as (2), was found to induce omega-hydroxylase activity in hepatocytes at concentrations between 5 and 100 nM compared to 1-20 microM concentrations for bezafibrate, and lower serum VLDL+LDL cholesterol in rats at doses between 0.1 and 0.5 mg/kg per day compared to doses of 25-100 mg/kg per day for bezafibrate. Single-dose pharmacokinetic studies with 2 indicated that total drug exposure will be much lower at hypocholesterolemic doses due to the enhanced intrinsic activity, and may result in an improved safety profile for these novel trisubstituted ureido fibrate analogues in rats and humans compared to other fibrates.

Overexpression of the Heme Oxygenase Gene in Renal Cell Carcinoma

Heme oxygenase (HO) activity has been implicated in the regulation of renal function and cell growth in normal and disease states. Expression of HO genes has been shown to regulate important hemoprotein(s) such as cytochrome P450. In the present study, HO activity was measured in samples of human adenocarcinoma, juxtatumor, and normal renal tissues. The samples were histologically examined to verify the malignant and normal nature. HO activity was 4-fold higher in the adenocarcinoma than in either normal or juxtatumor tissues. We designed a reverse transcriptase-polymerase chain reaction (RT-PCR) method to assess the presence of HO-1 and HO-2 mRNA in biopsy samples of various human renal tissues. Total RNA from renal samples was reverse transcribed and amplified simultaneously by PCR using specific primers for HO-1 and HO-2. Results show that both HO-1 and HO-2 mRNAs were expressed in all renal tissues examined and that HO-1 appeared to be amplified more than HO-2. Northern blot analysis revealed that HO-1 mRNA was elevated by several-fold in adenocarcinoma compared with juxtatumor or normal tissues. In contrast, no differences in HO-2 mRNA levels were observed using either RT-PCR or Northern blot. Cytochrome P450 arachidonic acid epoxygenase and omega-hydroxylase activities were markedly reduced in the tumor tissues, whereas, in the juxtatumor tissue, cytochrome P450 omega-hydroxylase activity was significantly increased. Northern blot analysis using cytochrome P450 cDNA probe 4A2 cDNA for the omega-hydroxylase gene family revealed that mRNA levels for omega-hydroxylase transcripts were significantly decreased in the adenocarcinoma compared with juxtatumor. The decrease in cytochrome P450 4All mRNA levels correlated with a decrease in the arachidonic acid omega-hydroxylation metabolite, 20-HETE. The production of 20-HETE was significantly higher in juxtatumor in agreement with omega-hydroxylase mRNA. Higher levels of HO-1 may be a contributing factor for the undetectable levels of cytochrome P450 arachidonic acid metabolites, 20-HETE, in the adenocarcinoma. Our results suggest that increased generation of mitogenic activities by omega-hydroxylase and 20-HETE in the juxtatumor may be a contributing factor in the development and growth of neoplastic tissues, and the induction of HO in the tumor tissue may be an attempt to limit oxidative injury caused by the cytochrome P450 metabolites and other oxidative stress.

Suicide Inactivation of Cytochrome P450 by Midchain and Terminal Acetylenes: A MECHANISTIC STUDY OF INACTIVATION OF A PLANT LAURIC ACID ω-HYDROXLYASE

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega-hydroxylase (omega-LAH), with [1-(14)C]11-dodecynoic acid (11-DDYA) generates a major metabolite characterized as 1,12-dodecandioic acid. In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible binding of 11-DDYA (200 pmol of 11-DDYA bound/mg of microsomal protein) at a saturating concentration of 11-DDYA was observed. SDS-polyacrylamide gel electrophoresis analysis showed that 30% of the label was associated with several protein bands of about 53 kDa. The presence of beta-mercaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxidized 11-DDYA with the nucleophile. Although the alkylation of proteins was reduced, the lauric acid omega-hydroxylase activity was not restored, suggesting an active site-directed inactivation mechanism. Similar results were obtained when reconstituted mixtures of cytochrome P450 from family CYP4A from rabbit liver were incubated with 11-DDYA. In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cytochrome P450 2B4 protein and irreversible inhibition of activity. These results demonstrate that acetylenic analogues of substrate are efficient mechanism-based inhibitors and that a correlation between the position of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.

Identification of a putative microvascular oxygen sensor.

The vascular response to changes in oxygen levels in the blood and tissue is a highly adaptive physiological response that functions to match tissue oxygen supply to metabolic demand. Defining the cellular mechanisms that can sense physiologically relevant changes in PO2 and adjust vascular diameter are vital to our understanding of this process. A cytochrome P450 (P450) enzyme of the 4A family of omega-hydroxylases was localized in renal microvessels, renal cortex, and a striated muscle microvascular bed (cremaster) of the rat. In the presence of molecular oxygen, this P450 enzyme catalyzes formation of 20-HETE from arachidonic acid (AA). Prior studies have shown that 20-HETE potently contracts renal and cerebral arteries and arterioles. The present study demonstrates that 20-HETE constricts striated muscle arterioles as well. In both intact renal microvessels and enriched renal cortical microsomal enzyme preparations, the formation of 20-HETE was linearly dependent on PO2 between 20 and 140 mm Hg. Homogenates of cremaster tissue produced 20-oxygen HETE when incubated with AA. They also expressed message for P450 4A enzyme, as determined by Southern and Western blots. Administration of 17-octadecynoic acid (17-ODYA), which is a P450 4A inhibitor, attenuated the constriction of third-order cremasteric arterioles in response to elevation of superfusion solution PO2 from approximately equal to 3 to 5 mm Hg to approximately equal to 35 mm Hg. 17-ODYA had no effect on basal vascular tone or response of cremaster arterioles to vasoactive compounds. These results demonstrate the existence of P450 omega-hydroxylase activity and 20-HETE formation in the vasculature and parenchyma of at least two microvascular beds. Our data suggest that a P450 enzyme of the 4A family has the potential to function as an oxygen sensor in mammalian microcirculatory beds and to regulate arteriolar caliber by generating 20-HETE in an oxygen-dependent manner.

Altered leukotriene B4 metabolism in colonic mucosa with inflammatory bowel disease.

omega-Oxidation is regarded as the major pathway for leukotriene B4 (LTB4) metabolism. Very little is known about it in colonic mucosa with inflammatory bowel disease (IBD). We investigated the metabolic ratio of omega-oxidation to LTB4 biosynthesis in colonic mucosa from patients with IBD and control subjects. After incubation of colonic mucosa with 14C-arachidonic acid and ionophore A23187, we separated LTB4 and its omega-oxidative metabolites by high-performance liquid chromatography. The rate of LTB4 omega-oxidation was comparable to the rate of its biosynthesis. The metabolic ratio was significantly decreased in inflamed mucosa with ulcerative colitis. LTB4 omega-hydroxylase activity is an important factor in regulating LTB4 level in colonic mucosa, and the increased LTB4 level in inflamed mucosa with IBD, especially ulcerative colitis, is caused by decreased LTB4 omega-hydroxylase activity and increased 5-lipoxygenase activity.