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Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha]. We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPAR[alpha] activator in a COS-7 cell expression system. Incubation with 10 [micro]M 14,15-DHET produced a 12-fold increase in PPAR[micro]-mediated luciferase activity, an increase similar to that produced by the PPAR[alpha] agonist Wy-14643 (20 [micro]M). Although 10 [micro]M 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5(Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPAR[alpha]. However, PPAR[alpha] was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[[sup.3]H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[[sup.3]H]EET. Binding studies indicated that 14,15-[[sup.3]H]DHET binds to the ligand binding domain of PPAR[alpha] with a [K.sub.d] of 1.4 [micro]M. Furthermore, 14,15-DHET increased the expression of camitine palmitoyltransferase lA, a PPAR[alpha]-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPAR[alpha]. cytochrome P-450; soluble epoxide hydrolase; 14,15-epoxyeicosatriconic acid analogs

Cytochrome P-450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels when [adenosine.sub.2A] receptors ([A.sub.2A]R) are stimulated. As high salt (HS) intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in HS-fed rats. Male Sprague-Dawley rats were fed either HS (4.0% NaCl) or normal salt (NS; 0.4% NaCl) diet. On day 8, isolated kidneys were perfused with Krebs' buffer containing indomethacin (10 [micro]M) and L-NAME (200 [micro]M) and preconstricted to ~150 mmHg with infusion of phenylephrine ([10.sup.7] M). Renal effluents were extracted for analysis of eicosanoids by gas chromatography-mass spectrometry. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1-10 [micro]g) resulted in dose-dependent dilation; at 10 [micro]g, perfusion pressure (PP) was lowered to a greater extent in the kidneys of HS rats compared with NS rats (-60 [+ or -] 4 vs. -31 [+ or -] 8 mmHg; P < 0.05) and the area of response was increased (27 [+ or -] 6 vs. 9 [+ or -] 4 [mm.sup.2]; P < 0.05), as was EET release (132 [+ or -] 23 vs. 38 [+ or -] 18 ng; P < 0.05). HS treatment increased [A.sub.2A]R and CYP2C23 protein expression. A selective epoxygenase inhibitor, MS-PPOH (12 [micro]M), significantly reduced the response to 2-CA in HS rats; PP, area of response, and EET release decreased by 40, 70, and 81%, respectively, whereas lesser changes were evident in NS kidneys. Thus the greater vasodilator response to 2-CA seen in kidneys obtained from HS-fed rats was mediated by increased EET release. As EETs are renal vasodilator and natriuretic eicosanoids, interactions between adenosine and EETs may contribute to the adaptive response to HS intake. adenosine receptors; cytochrome P-450

Elevated LPS and elevated cytochrome P-450 2E1 (CYP2E1) in liver are two major independent risk factors in alcoholic liver disease. We investigated possible synergistic effects of the two risk factors in causing oxidative stress and liver injury. Sprague-Dawley rats were injected intraperitoneally with pyrazole (inducer of CYP2E1) for 2 days, and then LPS was injected via tail vein. Other rats were treated with pyrazole alone or LPS alone or saline. Eight hours later, blood was collected and livers were excised. Pathological evaluation showed severe inflammatory responses and necroses only in liver sections from rats in the pyrazole plus LPS group; blood transaminase levels were significantly elevated only in the combination group. Activities of caspase-3 and -9 and positive terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were highest in the LPS alone and the LPS plus pyrazole group, with no significant difference between the two groups. Lipid peroxidation and protein carbonyls in liver homogenate as well as in situ superoxide production were maximally elevated in the LPS plus pyrazole group. Levels of nitrite plus nitrate and inducible nitric oxide (NO) synthase (iNOS) content were comparably elevated in LPS alone and the LPS plus pyrazole group; however, 3-nitrotyrosine adducts were elevated in the combined group but not the LPS group. It is likely that LPS induction of iNOS, which produces NO, coupled to pyrazole induction of CYP2E1 which produces superoxide, sets up conditions for maximal peroxynitrite formation and production of 3-nitrotyrosine adducts. CYP2E1 activity and content were elevated in the pyrazole and the LPS plus pyrazole groups. Immunohistochemical staining indicated that distribution of CYP2E1 was in agreement with that of necrosis and production of superoxide. These results show that pyrazole treatment enhanced LPS-induced necrosis, not apoptosis. The enhanced liver necrosis appears to involve an increase in oxidative and nitrosative stress generated by the combination of LPS plus elevated CYP2E1 levels. pyrazole; cytochrome P-450 2E1; oxidative stress; alcoholic liver disease

The cytochrome P-450 catalyzed hydroxylation of a hypersensitive radical probe, (trans, trans-2-tert-butoxy-3-phenylcyclopropyl)methane, leads to unrearranged and rearranged products. The probe differentiates between the radical and cationic intermediates. The short-lived apparent radical intermediate is actually a reacting ensemble. Rearrangement of a cationic intermediate produces the major rearranged products. The lack of synchrony between the insertion reaction and the C-H bond scission is responsible for the small concentration of radically rearranged products.

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer [beta]-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.

Electrolytic systems containing a platinum electrode and a redox mediator with purified recombinant proteins containing P450A1 are applicable to various P450s for the synthesis of stereo and regio-specific hydroxylated products. Experimental results show that NADPH can be replaced by these systems for the electrocatalytically mediated omega-hydroxylation of lauric acid.

Isotope effect profiles show a hydrogen atom abstraction mechanism for the cytochrome P-450 oxidative dealkylation of substituted N,N-dimethylanilines. For several P-450 oxidants the reaction mechanisms appear to be common, as detected from similar isotope effect profiles. The mechanisms of other amine oxidations by P-450 and oxidations by other hemoproteins may also be investigated by the profiles.

Purified cytochrome p450(CAM) is used to attenuate dehalogenation. Various systems such as putidaredoxin, putidaredoxin reductase, and NADH or optional donor processes yield electrons for the attenuation. The binding of halogenated substrates is observed at the site where physiological substrate camphor is noticed. Cytochrome P450(CAM) is employed to create taut ES complexes. The environment is likely to be influenced by the speed at which dehalogenation decreases in the presence of pentachloroethane.

Microsomal cytochrome P-450 was employed to understand the isotopic influence for the N,N-bis(dideuteriomethyl)anilines 2a-d and the substituted N-methyl-N-(trideuteriomethyl)anilines 1a-d which underwent oxidative demethylation. Masking was evident in most of the intramolecular isotope effects, and was observed to be the same in the pairs of p-nitro- and p-cyano-N,N-dimethylanilines. An isotope effect of 2.5 was observed for the PhN(CH3)2 abstractions as against the PhN(CD3)2.

C--H hydroxylation is a fundamental process that is catalyzed in Nature by the enzyme cytochrome P450. Density functional theoretical calculations are used in a study to show that C--H hydroxylation of the Newcomb probe, trans-2-phenyl-methyl cyclopropane, and proceeds via a two-state rebound mechanism.

Research was conducted to examine the decomposition of reactive intermediates by active site water in the metabolism of 8-alkyl xanthines by P450 1A2 enzymes. It is proposed that the addition of water within the confines of the enzyme active site will substantially decompose the reactive intermediate. Results support the observation that reactive intermediates could not be trapped with alternative nucleophiles in the medium.

The rat liver microsome-catalyzed oxidation of n-butyraldoxime yields two volatile metabolites, nitrobutane and n-butyronitrile. The latter's formation via a Beckmann-type dehydration is unprecedented. Evidence suggests that the process is catalyzed by cytochrome P-450 through the interaction of its ferrous iron with the oxime's oxygen atom. The conversion is also unique in that the nitrile retains the oxime's original carboskeleton, in contrast to other Beckmann dehydration reactions. While n-butyraldoxime is known to inhibit hepatic aldehyde dehydrogenase, neither n-butyronitrile nor nitrobutane does so, suggesting other means of bioactivation for the oxime.