Ernst H. Oliw

The selective cyclooxygenase-2 inhibitor rofecoxib reduces kainate-induced cell death in the rat hippocampus.

Treatment of male Sprague–Dawley rats with kainic acid (10 mg/kg, i.p.) triggered limbic seizures in 60% of the animals starting within 30 min and lasting for about 6 h. Cyclooxygenase‐2 (COX‐2) mRNA was strongly induced in the pyramidal cells of the hippocampus, in the amygdala and the piriform cortex after 8 h, as shown by in situ hybridization, and returned to control levels after 72 h. At this time marked cell loss occurred in the CA1–CA3 areas of the hippocampus. We hypothesize that rofecoxib, a selective COX‐2 inhibitor, might abbreviate the late neurotoxicity, possibly associated with COX‐2 induction. Animals which developed seizures were treated for 3 days with rofecoxib (10 mg/kg, i.p., n = 12) starting 6 or 8 h after kainic acid injection. Histological staining of viable cells confirmed that rofecoxib treatment selectively diminished cell loss in the hippocampus. The TdT‐mediated dUTP nick end labelling (TUNEL) technique was used to estimate delayed cell death. Abundant TUNEL‐positive cells were detected in seizure rats 72 h after kainic acid injection in pyramidal cells of the hippocampus (CA1–CA3), in cells of the thalamus, the amygdala and the piriform cortex. Treatment with rofecoxib selectively and significantly (P < 0.05) attenuated the number of TUNEL‐positive cells in the hippocampus, whereas the cells of the thalamus, amygdala and piriform cortex were not protected. Therefore we conclude that COX‐2 might contribute to cell death of pyramidal cells of the hippocampus as a consequence of limbic seizures.

Manganese Lipoxygenase PURIFICATION AND CHARACTERIZATION

A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gäumannomyces graminis, the take-all fungus, by hydrophobic interaction, cation exchange, lectin affinity, and size-exclusion chromatography. The purified dioxygenase lacked light absorption between 300 and 700 nm. Gel filtration indicated an apparent molecular mass of ∼135 kDa in 6 murea and ∼160 kDa in buffer. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was heterogeneous in size and consisted of diffuse protein bands of 100–140 kDa. Treatment with glycosidases for N- and O-linked oligosaccharides yielded a distinct protein of ∼73 kDa on SDS-PAGE. Atomic emission spectroscopy indicated 0.5–1.0 manganese atom/enzyme molecule. The isoelectric point was ∼9.7, and the enzyme was active between pH 5 and 11 with optimum activity at pH 7.0. For molecular oxygen,K m was 30 μm andV max 10 μmol mg−1min−1; for linoleic acid,K m was 4.4 μmol, V max 8.2 μmol mg−1min−1, and the turnover number 1100 min−1. The enzyme oxidized linolenic acid twice as fast as linoleic acid. The main products were identified by mass spectrometry as 13-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic and 13-hydroperoxy-(9Z,11E)-octadecadienoic acids, respectively. After reduction of the hydroperoxide, steric analysis of methyl 13-hydroxyoctadecadienoate by chiral high performance liquid chromatography yielded one enantiomer (>95%), which co-eluted with the R-stereoisomer of methyl (13R,13S)-hydroxyoctadecadienoate. Arachidonic and dihomogammalinolenic acids were not substrates, while oxygen consumption, UV analysis, and mass spectrometric analysis indicated that γ-linolenic acid was oxygenated both at C-11 and C-13. The enzyme was active at 60 °C and after treatment with 6 murea. It was strongly inhibited by 10–50 μmconcentrations of eicosatetraynoic acid and a lipoxygenase inhibitor (N-(3-phenoxycinnamyl)acetohydroxamic acid), but many other lipoxygenase inhibitors (100 μm) were without effect. We conclude that, after deglycosylation, the enzyme has the same size on SDS-PAGE as mammalian and marine lipoxygenases, but it differs from all previously described lipoxygenases in three ways. It is secreted, it forms (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid, and it contains manganese.

Oxygenation of polyunsaturated fatty acids by cytochrome P450 monooxygenates

Polyunsaturated fatty acids can be oxygenated by P450 in different ways--by epoxidation, by hydroxylation of the omega-side chain, by allylic and bis-allylic hydroxylation and by hydroxylation with double bond migration. Major organs for these oxygenations are the liver and the kidney. P450 is an ubiquitous enzyme. It is therefore not surprising that some of these reactions have been found in other organs and tissues. Many observations indicate that P450 oxygenates arachidonic acid in vivo in man and in experimental animals. This is hardly surprising. omega-Oxidation was discovered in vivo 60 years ago. It was more unexpected that biological activities have been associated with many of the P450 metabolites of arachidonic acid, at least in pharmacological doses. Epoxygenase metabolites of arachidonic acid have attracted the largest interest. In their critical review on epoxygenase metabolism of arachidonic acid in 1989, Fitzpatrick and Murphy pointed out some major differences between the PGH synthase, the lipoxygenase and the P450 pathways of arachidonic acid metabolism. Their main points are still valid and have only to be modified slightly in the light of recent results. First, lipoxygenases show a marked regiospecificity and stereospecificity, while many P450 seem to lack this specificity. There are, however, P450 isozymes which catalyse stereospecific epoxidations or hydroxylations. Many hydroxylases and at least some epoxygenases also show regiospecificity, i.e. oxygenate only one double bond or one specific carbon of the fatty acid substrate. In addition, preference for arachidonic acid and eicosapentaenoic acid may occur in the sense that other fatty acids are oxygenated with less regiospecificity. A more important difference is that prostaglandins and leukotrienes affect specific and well characterised receptors in cell membranes, while receptors for epoxides of arachidonic acid or other P450 metabolites have not been characterised. Nevertheless, epoxides of arachidonic acid have been found to induce a large number of different pharmacological effects. In some systems, effects have been noted at pm concentrations which might conceivably be in the physiological concentration range of these epoxides, e.g. after release from phospholipids by phospholipase A2. An intriguing possibility is that the effects of [Ca]i on different ion channels might possibly explain their biological actions. In situations when pharmacological doses are used, metabolism to epoxyprostanoids or other interactions with PGH synthase could also be of importance. Finally, one report on a specific receptor for 14R,15S-EpETrE in mononuclear cell membranes has just been published.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of Dioxygenases Required for Aspergillus Development STUDIES OF PRODUCTS, STEREOCHEMISTRY, AND THE REACTION MECHANISM

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-2H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.

Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn

Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSn mass spectra of the main metabolites of oleic, linoleic, α-linolenic and γ-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS3 analysis of hydroperoxides and MS2 analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18∶2n−6, 18∶3n−3, and 18∶3n−6 in a ratio of ∼1∶1–3 at high substrate concentrations. 8-Hydroxy-9(10)expoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSn is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.

Cyclooxygenase-2, prostaglandin synthases, and prostaglandin H2 metabolism in traumatic brain injury in the rat.

Inflammatory mediators are important in traumatic brain injury (TBI). The objective of the present study was to investigate the expression of cyclooxygenase-2 (COX-2), prostaglandin E (PGE) and PGD synthases, and PGH2 metabolism in two rat models of TBI. Fluid percussion injury (FPI) resulted in bilateral induction of COX-2 mRNA in the dentate gyri and the cortex, whereas controlled cortical contusion injury (CCC) induced COX-2 mRNA in the ipsilateral dentate gyrus and intensely in the cortex as judged by in situ hybridization. The induction subsided within 24 h. COX-2 immunoreactivity was detectable in these areas and persisted in the ipsilateral cortex for at least 72 h after CCC. Regions with COX-2 induction co-localized with TUNEL staining, suggesting a link between COX-2 expression and cell damage. COX-2 forms PGH2, which can be isomerized to PGD2, PGE2, and PGF2alpha by enzymatic and non-enzymatic mechanisms. In situ hybridization showed that mRNA of PGD synthase and microsomal PGE synthase were present in the choroid plexus. The microsomal PGE synthase was induced bilaterally after FPI and unilaterally after CCC. Liquid chromatography-mass spectrometry showed that low speed supernatant of normal and traumatized cortex and hippocampus transformed PGH2 to PGD2 as main product. PGD2 was dehydrated in brain homogenates to biological active compounds, for example, 15-deoxy-delta12,14-PGJ2. Thus COX-2 increases in certain neurons following TBI without neuronal induction of PGD and microsomal PGE synthases, suggesting that PGH2 may decompose to PGD2 and its dehydration products by nonenzymatic mechanisms or to PGD2 by low constitutive levels of PGD synthase.

Metabolism of 18:2(n-6), 18:3(n-3), 20:4(n-6) and 20:5(n-3) by the fungus Gaeumannomyces graminis : identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.

Nimesulide Aggravates Kainic Acid‐Induced Seizures in the Rat

Treatment of rats with kainic acid (10 mg/kg, intraperitoneally) triggers limbic seizures. Cyclooxygenase-2 mRNA is expressed in the hippocampus and cortex after 8 hr and marked cell loss occurs after 72 hr in the CA1-CA3 areas of the hippocampus. We examined the effect of the cyclooxygenase-2 inhibitor, nimesulide (N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide), on kainate-induced seizures and delayed neurotoxicity. Nimesulide (10 mg/kg, intraperitoneally) was well tolerated given alone or 6-8 hr after kainate. However, pretreatment with nimesulide augmented seizures and increased the mortality rate from approximately 10% to 69%. We examined the effect of nimesulide on delayed cell loss after 72 hr in the surviving animals with histological staining. Cell loss did not seem to be reduced in animals treated with nimesulide 6-8 hr after kainate, but in the surviving animals pretreated with nimesulide less cell loss occurred. We conclude that nimesulide should be used with caution as an antiinflammatory drug in patients with convulsive disorders.

Bisallylic hydroxylation and epoxidation of polyunsaturated fatty acids by cytochrome P450.

Polyunsaturated fatty acids can be oxygenated by cytochrome P450 to hydroxy and epoxy fatty acids. Two major classes of hydroxy fatty acids are formed by hydroxylation of the ω-side chain and by hydroxylation of bisallylic methylene carbons. Bisallylic cytochrome P450-hydroxylases transform linoleic acid to 11-hydroxylinoleic acid, arachidonic acid to 13-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid, 10-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and 7-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and eicosapentaenoic acid to 16-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid, 13-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid and 10-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid as major metabolites. The bisallylic hydroxy fatty acids are chemically unstable and decompose rapidly tocis-trans conjugated hydroxy fatty acids during acidic extractive isolation. Bisallylic hydroxylase activity appears to be augmented in microsomes induced by the synthetic glucocorticoid dexamethasone and by some other agents, but the P450 gene families of these hydroxylases have yet to be determined. The fatty acid epoxides, which are formed by cytochrome P450, are chemically stable, but are hydrolyzed to diols by soluble epoxide hydrolases. Epoxidation of polyunsaturated fatty acids is a prominent pathway of metabolism int he liver and the renal cortex and epoxygenase activity appears to be under homeostatic control in the kidney. Many arachidonate epoxygenases have been identified belonging to the CYP2C gene subfamily. Epoxygenases have also been found in the central nervous system, endocrine organs, the heart and endothelial cells. Epoxides of arachidonic acid have been found to exert pharmacological effects on many cells.

Biosynthesis of 8R-hydroperoxylinoleic acid by the fungus Laetisaria arvalis

8-Hydroxylinoleic acid is known to be a fungicidal metabolite formed by the fungus Laetisaria arvalis (Bowers, W.S. et al. (1986) Science 232, 105-106). In the present report, the mechanism of formation of 8-hydroxylinoleic acid was investigated. L. arvalis metabolized [14C]linoleic acid to 8-hydroperoxylinoleic acid and 8-hydroxylinoleic acid as major metabolites. The identification is based on the reduction of the hydroperoxide to an alcohol with stannous chloride and gas chromatography-mass spectrometry. The absolute configuration of the hydroxyl was determined to be R by ozonolysis of the (-)-menthoxycarbonyl derivative of 8-hydroxylinoleic acid. Linoleic acid 8R-dioxygenase activity was present in the 100,000 x g supernatant of the cell lysate. In summary, the 8R-linoleic acid dioxygenase of L. arvalis shows many similarities with the 8R-dioxygenase recently described in the fungus Gaeumannomyces graminis.