Bioactivation of leukotoxins to their toxic diols by epoxide hydrolase.

Leukotoxin is a linoleic acic oxide produced by leukocytes and has been associated with the multiple organ failure and adult respiratory distress syndrome seen in some severe burn patients. Leukotoxin has been reported to be toxic when injected into animals intravenously. Herein, we report that this lipid is not directly cytotoxic in at least two in vitro systems. Using a baculovirus expression system we demonstrate that leukotoxin is only cytotoxic in the presence of epoxide hydrolases. In addition, it is the diol metabolite that proves toxic to pulmonary alveolar epithelial cells, suggesting a critical role for the diol in leukotoxin-associated respiratory disease. In vivo data also support the toxicity of leukotoxin diol. For the first time we demonstrate that soluble epoxide hydrolase can bioactivate epoxides to diols that are apparently cytotoxic. Thus leukotoxin should be regarded as a protoxin corresponding to the more toxic diol. This clearly has implications for designing new clinical interventions.

Development of an in situ toxicity assay system using recombinant baculoviruses.

A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern.

Effects of dietary pyrrolizidine (Senecio) alkaloids on vitamin A metabolism in rats.

In two experiments, the effect of feeding the pyrrolizidine alkaloid (PA)-containing plant tansy ragwort (Senecio jacobaea) on the metabolism of vitamin A in rats was examined. In Experiment 1, dietary levels of 0, 5 and 10% tansy ragwort and 0, 25,000 and 100,000 IU supplementary vitamin A/kg diet were used. In rats fed tansy ragwort, both plasma and liver concentrations of vitamin A were depressed (P less than 0.05). Plasma values were decreased to about 50% of control values. In Experiment 2, rats fed 5% tansy ragwort had depressed plasma and liver vitamin A concentrations 48 h after oral dosing with vitamin A. Fecal excretion of vitamin A was decreased in tansy ragwort-fed rats. In control rats, most fecal vitamin A was excreted in the first 24 h post-dosing, while in tansy ragwort-fed animals, the excretion was delayed, suggesting a possible effect of PA on gut motility. The results indicate that PA causes reductions in liver and plasma vitamin A concentrations. Postulated mechanisms include an effect of PA on hepatic retinol-binding protein synthesis, and impaired biliary excretion depressing vitamin A absorption.

An LC/MS/MS method for improved quantitation of the bound residues in the tissues of animals orally dosed with [(14)C]Benomyl.

Livers of goats orally dosed with [phenyl(U)-(14)C]benomyl contained radioactive residues which were not extractable using conventional, solvent-based extraction methods. We report a new residue method capable of enhanced extraction of benomyl-derived residues with selective and sensitive quantitation capability for methyl 4-hydroxybenzimidazol-2-ylcarbamate (4-HBC), methyl 5-hydroxybenzimidazol-2-ylcarbamate (5-HBC), and methyl benzimidazol-2-ylcarbamate (MBC). This method involves rigorous Raney-nickel reduction of hypothesized thioether bonds between benomyl residues and polar cellular components. Following acidic dehydration (desulfurization), the polar benomyl-derived residues are extracted into ethyl acetate and analyzed by LC/MS/MS. We have shown this method to be superior to alternative extraction approaches. When applied to goat liver tissue containing [phenyl(U)-(14)C]benomyl-bound residues, the extraction efficiency of total radioactive residues was approximately 30%, and the major benomyl-derived residue was 5-HBC (91-95% of extractable residue) with minor levels of carbendazim (MBC) (5-9%). HPLC/LSC data were consistent with the LC/MS/MS data. The overall method satisfies U.S. regulatory requirements in extraction efficiency, selectivity in detection, and limits of quantitation for benomyl-bound residues.

Discovery of 12-(S)-hydroxy-5,8,10,14-icosatetraenoic acid [12-(S)-HETE] in the tropical red alga Platysiphonia miniata.

The potent mammalian immunohormone, 12-(S)-hydroxy-5,8,10,14-icosatetraenoic acid (12-(S)-HETE), is a 12-lipoxygenase metabolite of arachidonic acid that is widely distributed in animal tissues. In humans, it is produced and secreted by platelet cells and elicits both chemotactic and degranulatory responses in target neutrophils. As widely as 12-lipoxygenase activity and one of its major products, 12-(S)-HETE, have been found in animal tissues, it has never been found in plants. Herein, we report the first isolation of the 12-lipoxygenase product, 12-(S)-HETE, from a plant, the tropical marine alga Platysiphonia miniata (C. Agardh) Børgesen.

Discovery of the mammalian insulin release modulator, hepoxilin B3, from the tropical red algae Platysiphonia miniata and Cottoniella filamentosa.

The insulin release enhancer, 10-hydroxy-11,12-trans-epoxy-5(Z),8(Z),14(Z)-icosatrienoic acid (hepoxilin B3), is a 12-lipoxygenase metabolite of arachidonic acid that has been found in various mammalian tissues. Although lipoxygenase pathways are well documented in terrestrial plants, this lipoxygenase product has never been isolated from the plant kingdom. Herein, we report the first isolation of this lipoxygenase product, hepoxilin B3, from two plants, the tropical red marine algae Platysiphonia miniata (C. Agardh) Børgesen and Cottoniella filamentosa Børgesen. Furthermore, through application of two-dimensional NMR methodology to the structural description of this algal natural product, we demonstrate the tremendous power of this technique in this chemical class.

Novel cytotoxic peptides from the tropical marine cyanobacterium Hormothamnion enteromorphoides. 1. Discovery, isolation and initial chemical and biological characterization of the hormothamnins from wild and cultured material.

A Caribbean cyanobacterium, Hormothamnion enteromorphoides, was found to produce a complex mixture of ichthyotoxic peptides, perhaps explaining the apparent absence of predation upon these potentially palatable life forms. Bioassay-guided fractionation was used to isolate these toxic and antimicrobial natural products, and a variety of techniques including HR FAB mass spectrometry, 2D-NMR, traditional hydrolysis-amino acid analysis, and several chemical reactions were used to define the basic structural features of the major peptide, hormothamnin A. Hormothamnin A is a cyclic undecapeptide containing six common and five uncommon or new amino acid residues. HPLC analyses indicate that the relative proportions of these peptide natural products remain relatively constant between different collection locations and years, however, they do vary seasonally. Clonal isolates of this cyanobacterium in culture produce the full spectrum of toxic peptides.

Biotransformation, excretion kinetics, and tissue distribution of an N-pyrrolo[1,2-c]imidazolylphenyl sulfonamide herbicide in rats.

TY029, an N-pyrrolo[1,2-c]imidazolylphenyl sulfonamide herbicide, controls economically important weeds through inhibition of protoporphyrinogen oxygenase. Due to the potential for exposure to this compound in food and animal feed items, a rat metabolism study was required to define the biotransformation of this compound. Animals were exposed to single 50- and 2-mg/kg doses of TY029 [hydantoin-5-(14)C] by oral gavage. About 90% of the administered dose was excreted within 96 h after oral administration. Excretion plateaued after 48 h, and the cumulative sum of urinary or fecal excretion after 48 h was less than 5% of the orally administered dose. TY029 yielded seven major metabolites. While some metabolites were formed by epimerization around chiral centers, others were generated through hydrolytic bond cleavage and hydroxylations and subsequent oxidation of hydroxyl groups to carboxylic acids. One metabolite, about 6.1% of the dose, was observed only in the urine from low-dose female rats. This metabolite was characterized as a glutamate conjugate of an extensively oxidized analog of TY029. With the exception of the glutamate conjugate, the same metabolites were observed in the excreta of all dose groups. However, the relative ratios of the metabolites were different between various dose groups.

Properties of enzymes hydrating epoxides in human epidermis and liver.

1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different. 2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity chromatographic properties. 3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities. 4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes. 5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts. 6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

Unique metabolites of eicosapentaenoic acid interfere with corpus luteum function in the ewe.

Experiments were conducted to determine the in vivo and in vitro effects of metabolites of eicosapentaenoic acid on ovine luteal function. Injection of 750 micrograms methyl eicosapentaenoic acid (EPA) or methyl 12(R),13(S)-dihydroxyeicosapentaenoic acid (12,13-diHEPE) into the ovarian artery of ewes on day 10 of the estrous cycle caused a reduction in serum concentrations of progesterone by 48 h posttreatment compared with levels of this steroid in arachidic acid-treated controls (p < 0.005). Although mean serum concentrations of progesterone in methyl EPA-treated ewes during the remainder of the cycle did not differ from those in control ewes, levels in methyl 12,13-diHEPE-treated ewes remained significantly suppressed. Duration of the estrous cycle did not differ among treatment groups (p > 0.05), but more of the methyl 12,13-diHEPE-treated animals (3/5) had exhibited estrus within 3 days after injection than methyl EPA-treated (1/5) or control ewes (0/5). Slices of corpus luteum removed from ewes on day 10 of the estrous cycle were incubated with arachidic acid (controls), 12,13-diHEPE or docosatetraenoic acid (DTA). Regardless of fatty acid treatment, all tissues retained the ability to produce basal levels of progesterone during subsequent incubation. Luteal slices previously exposed to arachidic acid or DTA exhibited an increase in progesterone production in response to subsequent treatment with LH (p < 0.05). In contrast, luteal slices incubated with 12,13-diHEPE did not respond to LH with a significant increase in production of this steroid above that observed in controls. All tissues displayed a marked increase in progesterone synthesis upon treatment with 8-Br-cAMP relative to incubation of tissue alone (p < 0.001). Subcellular distribution of [14C]-12,13-diHEPE in luteal cells after incubation revealed that the majority of the fatty acid was associated with the plasma membrane. These data suggest that metabolites of eicosapentaenoic acid with hydroxyl groups on adjacent carbon atoms interfere with luteal function in the ewe, perhaps in part by altering luteal response to LH.