Studies on the effect of intratesticular administration of opioid peptides, naloxone or N-acetyl beta-endorphin antiserum on some testicular parameters in rats

Substrate specificity and other properties of a fatty acid monooxygenase system in kidney microsomes of the Japanese house musk shrew (Suncus murinus) were examined. The suncus kidney microsomes catalyzed the hydroxylation of various saturated and unsaturated fatty acids to the omega- and (omega-1)-hydroxy derivatives. Laurate was most effectively hydroxylated among saturated and unsaturated fatty acids. The specific activity (53.79 +/- 5.59 [mean +/- SD, n = 6] nmol/nmol cytochrome P450/min) of laurate in suncus kidney microsomes was very high compared with that in liver and kidney microsomes of other species. C18 unsaturated fatty acids were converted to epoxides by a cytochrome P450-dependent fatty acid monooxygenase system in suncus kidney microsomes, in addition to omega- and (omega-1)-hydroxylation products. The monooxygenase system metabolized arachidonic acid only to omega- and (omega-1)-hydroxylation products, not to epoxidation products.     

Fatty acid omega- and (omega-1)-hydroxylation in experimental alcoholic liver disease: relationship to different dietary fatty acids

Arachidonic acid concentrations in liver are decreased in response to ethanol administration. In addition, the oxygenated products of arachidonic acid metabolites could affect the severity of alcoholic liver injury. Selective utilization of arachidonic acid by the cytochrome P-450 system could, in part, account for the decrease in arachidonic acid. To evaluate this pathway further, male Wistar rats were fed different dietary fats: medium chain triglycerides, palm oil, and corn oil or fish oil with either ethanol or isocaloric amounts of dextrose. Histopathology, cytochrome P-4502E1 (CYP2E1) and cytochrome P-4504A (CYP4A), and omega- and (omega-1)-hydroxylation products of lauric and arachidonic acids were evaluated. Ethanol induction of CYP2E1 was related to the concentration of polyunsaturated fatty acids in the diet; induction of CYP4A by ethanol was seen in all groups. The highest levels of 11-hydroxy-lauric acid and 19-hydroxyarachidonic acid (omega-1) were seen in rats fed ethanol with palm oil and corn oil. Highly significant correlations were seen between the (omega-1)-hydroxylation products and CYP2E1 activity. No correlation was seen between the omega-hydroxylation products and CYP2E1 activity. In contrast, the levels of omega-hydroxylation products correlated with CYP4A. The overall results showed a significant increase in (omega-1)-hydroxylation products in rats fed diets containing significant amounts of linoleic acid (i.e., palm oil and corn oil).     

Structural and metabolic requirements for activators of the peroxisome proliferator-activated receptor

Fatty acids have recently been demonstrated to activate peroxisome proliferator-activated receptors (PPARs) but specific structural requirements of fatty acids to produce this response have not yet been determined. Importantly, it has hitherto not been possible to show specific binding of these compounds to PPAR. To test whether a common PPAR binding metabolite might be formed, we tested the effects of long-chain omega-3 polyunsaturated fatty acids, differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. We determined the activation of a reporter gene by a chimaeric receptor encompassing the DNA binding domain of the glucocorticoid receptor and the ligand binding domain of PPAR. The omega-3 unsaturated fatty acids were slightly more potent PPAR activators in vitro than saturated fatty acids. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY 14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester. These data should aid understanding of signal transduction via PPAR and the identification of a receptor ligand.     

Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes

The effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems with respect to three cytochrome P-450 isozymes in rat liver microsomes were investigated. FK-506 non-competitively inhibited the aniline p-hydroxylase, p-nitroanisole O-demethylase and lidocaine N-deethylase activities of cytochrome P-450-linked monooxygenase systems, these activities being mainly catalyzed by cytochromes P-450 CYP2E1, CYP2C11 and CYP3A4, respectively, and the Ki values of the activities for FK-506 were determined to be 605, 491 and 97 microM, respectively. The inhibition of cytochrome P-450-linked monooxygenase systems by FK-506 seemed to involve the direct inhibition of cytochromes P-450 because the NADPH-cytochrome c reductase and NADPH-ferricyanide reductase activities of NADPH-cytochrome P-450 reductase were not affected by the presence of 1 mM FK-506 at all. A spectrophotometric study showed that a reverse type I spectral change was induced on the addition of FK-506 to rat liver microsomes, and the Ks value was apparently 125 microM. On the other hand, the EPR spectra of cytochromes P-450 in rat liver microsomes were not affected by 1 mM FK-506. These results suggest direct interaction between FK-506 and cytochrome P-450 apoproteins, except for the heme iron regions of cytochromes P-450, resulting in inhibition of the drug-metabolism activities catalyzed by cytochromes P-450.     

The effect of methamphetamine on the release of acetylcholine in the rat striatum

Comparative investigations were performed to study the effect of endogenous and exogenous N-nitrosodiethylamine on the dynamics of content variations of oxidized cytochrome P-450 and its isoforms in the monooxygenase system of rat liver. The variations of cytochrome P-450 contents in both cases were demonstrated to be of the same character correlating with hepatocarcinogenesis stages. Higher quantities of oxidized cytochrome P-450 and its isoforms with MM 52, 53, and 56 kDa in the rat liver when acted upon by NDEA precursors are seen as the precondition of enhancing the monooxygenase reaction of NDEA bioactivation and, as a result, of the carcinogenic effects. Ascorbic acid is assumed to block the synthesis of NDEA from its precursors giving use to a compound whose metabolism does not influence the activity of the monooxygenase system of liver cells.     

Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol

Previously we reported that eugenol (4-allyl-2-methoxyphenol) inhibits non-enzymatic peroxidation in liver mitochondria (E. Nagababu and N. Lakshmaiah, 1992, Biochemical Pharmacology. 43, 2393-2400). In the present study, we examined the effect of eugenol on microsomal mixed function oxidase mediated peroxidation using Fe+3-ADP-NADPH, carbon tetrachloride (CCL4)-NADPH and cumene hydroperoxide (CumOOH) systems. In the presence of eugenol the formation of thiobarbituric acid reactive substances (TBARS) was decreased in all the systems (IC50 values: 14 microM for Fe+3-ADP-NADPH, 4.0 microM for CCl4-NADPH and 15 microM for CumOOH). Oxygen uptake was also inhibited to a similar extent with Fe+3-ADP-NADPH and CumOOH systems. A comparative evaluation with other antioxidants showed that in Fe+3-ADP-NADPH and CumOOH systems, the antioxidant efficacy was in the order: butylated hydroxytoluene (BHT) > eugenol > alpha-tocopherol, while in CCl4-NADPH system the order was alpha-tocopherol > BHT > eugenol. Time course of inhibition by eugenol indicated interference in initiation as well as propagation of peroxidation. Eugenol did not inhibit cytochrome P-450 reductase activity but it inhibited P-450 - linked monooxygenase activities such as aminopyrine-N-demethylase, N-nitrosodimethylamine demethylase, benzo(a)pyrene hydroxylase and ethoxyresorufin-O-deethylase to different extents. However, CumOOH supported monooxygenases (aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase) required much higher concentrations of eugenol for inhibition. The concentration of eugenol required to inhibit monooxygenase activities was more than that required to inhibit peroxidation in all the systems. Eugenol elicited type 1 changes in the spectrum of microsomal cytochrome P-450. These results suggest that the inhibitory effect of eugenol on lipid peroxidation is predominantly due to its free radical quenching ability. Eugenol significantly protected against the degradation of cytochrome P-450 during lipid peroxidation with all the systems tested. These findings suggest that eugenol has the potential to be used as a therapeutic antioxidant. Further evaluation may throw more light on this aspect.     

Dietary omega-3 and omega-9 fatty acids uniquely enhance allograft survival in cyclosporine-treated and donor-specific transfusion-treated rats

BACKGROUND: Both laboratory and clinical studies have shown that dietary lipids may affect immunologic responses. This study was conducted to compare different classes of long-chain unsaturated fatty acids for their effect on allograft survival in animals receiving a donor-specific transfusion and a short course of low-dose cyclosporine (CsA). METHODS: Heterotopic ACI strain cardiac allografts were transplanted to Lewis strain rat recipients given diets with different lipid composition. In experiment 1, animals received CsA for 14 days and different diets were enriched with lipids with high concentrations of omega-3, omega-6, or omega-9 fatty acids. In experiment 2, animals received CsA for only 8 days and different diets were enriched with corn oil (omega-6), canola oil (omega-3 and omega-9), fish oil (omega-3) or a mixture of sunflower oil and fish oil (omega-3 and omega-9). RESULTS: In experiment 1, animals receiving the diet with 30% sunflower oil had the best allograft survival (200+/-42 days vs. 53+/-8 days for regular chow plus donor-specific transfusion and CsA, P<0.05). In experiment 2, diets containing canola oil (a mixture of omega-3 and omega-9 fatty acids) were associated with the best survival (P=0.0011 vs. regular chow). CONCLUSION: Dietary omega-3 and omega-9 fatty acids both enhanced cardiac allograft survival in a stringent rat strain combination. Canola oil is a convenient oil for administering both alpha-linoleic acid (omega-3) and oleic acid (omega-9) in a palatable form for human consumption. Further investigation of the potential usefulness of lipids in transplant therapy is warranted.     

Retinoic acid metabolism in cultured retinal pigment epithelial cells

PURPOSE: To examine the stability of retinoic acid administered to cultured bovine retinal pigment epithelial (RPE) cells and to determine if RPE cells metabolize retinoic acid by a cytochrome P-450 mechanism. METHODS: Retinoic acid metabolism was examined in cultured RPE cells and subcellular fractions quantitatively by a thin-layer chromatography procedure and qualitatively by normal and reverse phase high-performance liquid chromatography. RESULTS: Cultured bovine RPE cells were found to have an activity that converts retinoic acid into more polar metabolites rapidly released from the cell. The highest specific activity for this process is found in the post-mitochondrial pellet (100,000g), is induced by retinoic acid, and is inhibited by ketoconazole. The major product of the RPE cell-mediated metabolism of retinoic acid is 4-oxo-retinoic acid, a known P-450 monooxygenase product of retinoic acid. The retinoic acid metabolizing activity is greatest in primary RPE cultures and decreases with aging in culture. CONCLUSIONS: These data suggest that a cytochrome P-450 monooxygenase is involved in the metabolism of retinoic acid in RPE cells, and this is similar to the findings of other investigators using other cells and tissues. The authors' findings suggest that the RPE may be important in the deactivation of this biologically potent retinoid in the retina.     

The catalytic site of cytochrome P4504A11 (CYP4A11) and its L131F mutant

CYP4A11, the principal known human fatty acid omega-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of cytochrome P450 reductase and NADPH.     

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon beta in mice

AIMS/METHODS: Interferon beta is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon beta on cytochrome P-450 in mice. RESULTS: Interferon beta (2 x 10(5) units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon beta reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In beta-naphthoflavone-treated mice, interferon beta lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). CONCLUSIONS: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon beta, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon beta.     

Changes in alveolar oxygen and carbon dioxide concentration and oxygen consumption during lung preservation. The maintenance of aerobic metabolism during lung preservation

Adult human liver microsomes supplemented with NADPH catalyzed the regioselective N-oxygenation of the aliphatic tertiary amine and S-oxidation of the phenothiazine sulfur atom of several 10-(N,N-dimethylaminoalkyl)phenothiazines. In addition, (+)- and (-)-4-bromophenyl-1,3-oxathiolane were converted to the corresponding S-oxides in the presence of NADPH and adult human liver microsomes. The (+) and (-) enantiomers of 4-bromophenyl-1,3-oxathiolane were converted to the S-oxides with low and high stereoselectivity, respectively. Studies on the biochemical mechanism for N-oxygenation of 10-(N,N-dimethylaminoalkyl)phenothiazines suggested that this reaction was catalyzed by the flavin-containing monooxygenase (form II), although cytochrome P-450 2D6 may also have contributed to N-oxide formation. S-Oxidation of chlorpromazine was catalyzed mainly by cytochrome P-450 3A. S-Oxidation of 10-(N,N-dimethylaminoalkyl)phenothiazines was catalyzed by a number of cytochromes P-450, including cytochromes P-450 2A6, 2C8, and 2D6. S-Oxygenation of (+)-4-bromophenyl-1,3-oxathiolane produced a mixture of the cis- and trans diastereomers in a process probably dependent on both hepatic monooxygenase systems. (-)-4-Bromophenyl-1,3-oxathiolane was converted almost exclusively to the trans-S-oxide in a process likely dependent on the adult human liver flavin-containing monooxygenase (form II). Development of regio- and stereochemical probes of adult human liver flavin-containing monooxygenase (form II) and cytochromes P-450 activity may be useful for eventual in vitro-in vivo correlations, but may require approaches quite distinct from that currently used for animal monooxygenases.     

The domain architecture of cytochrome P450BM-3

Cytochromes P450 utilize redox partners to deliver electrons from NADPH/NADH to the P450 heme center. Microsomal P450s utilize an FAD/FMN reductase. The bacterial fatty acid hydroxylase, P450BM-3, is similar except the P450 heme and FAD/FMN proteins are linked together in a single polypeptide chain arranged as heme-FMN-FAD. Sequence comparisons indicate that the P450BM-3 FMN and FAD domains are similar to flavodoxin and ferredoxin reductase, respectively. Previous work has shown that the heme and FMN/FAD domains can be separately expressed and purified. In this study we have expressed, purified, and characterized the following additional domains: heme-FMN, FMN, and FAD. Each domain retains their prosthetic groups although the FMN domain is more labile. The FAD domain retains a high level of ferricyanide reductase activity but no cytochrome c reductase activity. In addition, we have deleted a 110-residue stretch in the FAD domain that is not present in ferredoxin reductase. This protein retains both FAD and heme but not FMN. We also have investigated the dimerization pattern of the individual domains that lead to the following conclusions. Holo-P450BM-3 appears to dimerize via interactions that do not involve disulfide bond formation, whereas the reductase and FAD domains form intermolecular disulfides. This indicates that the Cys residues not available for dimerization in holo-P450BM-3 are unmasked in the individual domains.     

Metabolism of nicotine by human liver microsomes: stereoselective formation of trans-nicotine N'-oxide

Liver microsomes from humans catalyze the NADPH-dependent oxidation of (S)-nicotine. The principal product is the 5'-carbon atom oxidation product, nicotine delta 1',5'-iminium ion, which is efficiently converted to the gamma-lactam derivative cotinine in the presence of aldehyde oxidase. Another major product is nicotine N'-oxide. In contrast to previous reports describing in vitro or in vivo studies, formation of only trans-nicotine N'-oxide was observed. Demethylation of nicotine was not observed. Studies on the biochemical mechanism of nicotine 5-carbon atom oxidation strongly implicate one major cytochrome P-450 isoenzyme (i.e., P-450 2A6) as largely responsible for delta 1',5'-iminium ion formation. Stereoselective formation of trans-nicotine N'-oxide may be catalyzed in large part by the flavin-containing monooxygenase (form II). These conclusions are based on the effects of alternate substrates for the flavin-containing monooxygenase, heat inactivation studies, immunoblot studies, and selective substrates for cytochromes P-450. The results suggest that (S)-nicotine trans N'-oxygenation and delta 1',5'-iminium ion formation may be selective probes of human liver flavin-containing monooxygenase form II and cytochrome P-450 2A6 activities, respectively, useful for in vivo phenotyping of humans.     

The changing sex ratio of the navajo tribe

Two approaches may be used to study the function of cytochrome P-450 in insects: (a) an evaluation of the spectral and catalytic properties of the hemoprotein while associated with microsomal membranes; (b) the solubilization, resolution and purification of the microsomal mixed-function oxidase system. The first approach has provided some understanding of the biochemical factors involved in the metabolism of a variety of compounds, including pesticides, drugs, hormones and many other xenobiotics. However, solubilization of the monooxygenase system allows the study of each of its components individually, providing a better insight on the sequence of events leading to the hydroxylation of a substrate, the type of intermediates formed, and the rate-limiting step(s). This report discusses studies carried out with the monooxygenase system associated with microsomal membranes, as well as procedures to solubilize and partially purify its components from housefly microsomes. The latter involves solubilization with either Triton X-100 or sodium cholate, followed by either ammonium sulfate fractionation, Sephadex G-200, DEAE-Sephadex A-50 column chromatography or by omega-amino-n-octyl-Sepharose 4B affinity chromatography. These procedures have shown that two cytochrome P-450 species (P-450 and P-450I) are present in microsomes isolated from a resistant housefly strain. Induction with either naphthalene or phenobarbital appears to increase cytochrome P-450I preferentially.     

Assessment of regional myocardial uptake and metabolism of omega-(p-123I-phenyl) pentadecanoic acid with serial single-photon emission tomography

The utility of myocardial imaging and assessment of regional myocardial metabolism of omega-(123I-paraphenyl-)pentadecanoic acid (I-PPA) by means of serial single-photon tomography is demonstrated in animal experiments. High quality cross sectional images of dog hearts with clear delineation of left ventricular walls are obtained. Myocardial infarcts are visualized as areas of deficient radioactivity uptake. I-PPA elimination from non-infarcted myocardial regions is significantly (p less than 0.001) prolonged when compared with unaffected controls. Hence, not only localized absence of uptake of free fatty acid by infarcted myocardium can be demonstrated with serial single-photon tomography but also general impairment of cardiac FFA-metabolism.     

Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450)-dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/kg markedly elevated benzo[a]pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/kg/d for 4 d resulted in significant increases of P-450 and cytochrome b5 contents and NADPH-cytochrome c reductase activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S-transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Expression of functionally active hyman cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and single-stage purification of it using metal-affinity chromatography

Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.     

Dietary fat and breast cancer metastasis by human tumor xenografts

Human breast cancer cell lines growing as xenografts in athymic nude mice have been used to examine the effects of dietary fat and fatty acids on tumor progression. The estrogen independent MDA-MB-435 cell line has the advantage that it metastasizes consistently to the lungs and forms quantifiable secondary nodules when injected into the mammary fat pads. With these breast cancer cells, the stimulating effects of polyunsaturated omega-6 fatty acids on both primary tumor growth and metastasis were demonstrated; in contrast, the long-chain omega-3 fatty acids were inhibitory. The model can also be adapted to examine dietary fatty acids, and inhibitors of their metabolism, as experimental adjuvant therapy after surgical excision of the primary tumors. Unfortunately, estrogen dependent human breast cancer cells do not metastasize, or do so rarely, in nude mice; in consequence, it is not possible to use the model to study estrogen-fatty acid interactions on the metastatic process. In addition to metastasis from a primary location, intravenous injection of MDA-MB-435 cells into the nude mouse host, particularly when combined with studies using Matrigel-based in vitro invasion assays, permits further dissection of the steps in the metastatic cascade which are influenced by dietary fatty acids. The results obtained by these several approaches have demonstrated distinct roles for the cyclooxygenase and lipoxygenase-mediated products of omega-6 fatty acid metabolism, and suggest new approaches to experimental breast cancer therapy.     

Xenobiotic-metabolizing enzymes in carp (Cyprinus carpio) liver, spleen, and head kidney following experimental Listeria monocytogenes infection

Infection of carp with Listeria monocytogenes 4b resulted in decreased liver, spleen, and head kidney enzyme activities, involved in the metabolism of xenobiotics. After infection, cytochrome P-450 levels and ethoxyresorufin O-deethylase (EROD) activity were decreased while conjugation enzymes remained unaffected. The maximum decrease for phase I enzymes occurred on d 3. This loss of monooxygenase levels and activity could not be directly correlated with an increase in the number of organisms, as consistently high bacterial counts were observed in all three organs during infection. The effect of L. monocytogenes infection was also measured in carp exposed to 3-methylcholanthrene (MCA). Cytochrome P-450 levels and EROD activity were significantly reduced, especially on d 3. A significant decreased activity of conjugation enzymes such as glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) was also observed for all days studied. Listeria infection inhibited MCA-induced increases in xenobiotic-metabolizing enzyme activities. These results indicate that infection may have deleterious effects on basal cytochrome P-450 monooxygenase levels. Furthermore, MCA treatment aggravates the insult to xenobiotic biotransformation enzymes by L. monocytogenes infection, by impairing a number of detoxification enzymes. These findings could result in significant changes in the susceptibility of fish to pollutants.     

Stereoselective epoxidation of arachidonic acid by cytochrome P-450s 2CAA and 2C2

In the present study, we determined the stereoselective epoxidation of arachidonic acid by cytochrome P-450 (P-450) 2CAA and P-450 2C2, two arachidonic acid epoxygenases found in rabbit renal cortex, by chiral normal-phase high-performance liquid chromatography (HPLC)-analysis. Purified P-450 2CAA reconstituted with P-450 oxidoreductase, lipid and cytochrome b5 or microsomes isolated from COS-1 cells expressing P-450 2C2 were incubated in the presence of [1-14C]arachidonic acid. The epoxide metabolites 14,15- and 11,12-epoxyeicosatrienoic acids (EETs) were purified by reverse-phase HPLC and derivatized to methyl (14,15-EET) and pentafluorobenzyl (11,12-EET) esters. Enantiomers of 14,15-EET-methyl ester and 11,12-EET-pentafluorobenzyl ester were resolved on Chiralcel OB and OD columns, respectively, with a mobile phase of 0.15% 2-propanol in n-hexane. P-450 2CAA and P-450 2C2 produce 11,12- and 14,15-EETs in distinct ratios but are equally stereoselective at the 11,12-position. P-450 2CAA produced 11(S), 12(R)-EET with 63% stereoselectivity, and P-450 2C2 produced the same enantiomer with 61% stereoselectivity. Both enzymes are also stereoselective at the 14,15- position, preferentially producing the 14(R), 15(S)-EET. P-450 2CAA produces this enantiomer with 72% selectivity, and P-450 2C2 produces it with 62% selectivity. The results of this study indicate that P-450 2CAA and P-450 2C2 are not only regioselective but also exhibit a high degree of stereoselectivity.