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A W Bull

Publications and source records attributed to A W Bull.

At least 19 recordsLinked to original sources
PubMed2001

Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles.

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,gamma-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K(m) of 325.5 microM and a V(max) of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K(m) of 169 microM and a V(max) of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.

Adenosine Triphosphate↗
PubMed2001

Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers.

Conjugated linoleic acid (CLA) has been shown to inhibit tumorigenesis in animal models and is cytostatic to numerous cell lines in vitro. However, the mechanism of action is unknown. In the current study, we determined the effects of CLA and specific isomers of CLA on the rate of oxygenation of arachidonic acid by prostaglandin H synthase (PGHS) in ram seminal vesicle microsomes. The enzyme was incubated with 0.1 to 100 microM CLA or specific isomers of CLA for 2 min prior to the addition of 44 to 176 microM arachidonate. The isomers tested were 9(E),11(E) CLA; 9(Z),11(E) CLA; 9(Z),11(Z) CLA, and 10(E),12(2) CLA. For a positive inhibitor control, flurbiprofen was used at 0.75 to 2.50 microM. Enzyme activity was assessed by measuring the rate of oxygen consumption. Inclusion of CLA or specific isomers of CLA in the incubation mixtures inhibits PGHS. The efficacy differs for each isomer, with the 9(Z),11 (E) CLA isomer being the most effective and the 9(Z),11 (Z) CLA isomer being the least effective inhibitor among the four CLA isomers tested. The Ki values obtained by Dixon replots range from 18.7 microM for the most effective isomer, 9(Z),11 (E) CLA, to 105.3 microM for the least effective isomer, 9(2),11(2) CLA. The Ki value for flurbiprofen with ram seminal vesicle microsomes was 0.33 microM. As the concentration of arachidonate was increased, the CLA-dependent inhibition of PGHS decreased, suggesting competitive inhibition. The results of this study demonstrate the potential of CLA and specific isomers of CLA to modulate prostaglandin biosynthesis.

Animals↗
PubMed2001

13-hydroxy- and 13-oxooctadecadienoic acids: novel substrates for human UDP-glucuronosyltransferases.

Although there are numerous studies of glucuronidation of endogenous compounds, information on the glucuronidation of fatty acids is lacking. In the present studies, both linoleic acid (LA) and its biologically active oxidized derivatives, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-oxooctadecadienoic acid (13-OXO), have been shown to be effective substrates for human liver UDP-glucuronosyltransferases (UGT) and recombinant UGT2B7. LA (carboxyl glucuronide) and 13-OXO (carboxyl glucuronide, unproven) were actively glucuronidated by human liver microsomes (HLM) and human recombinant UGT2B7 with similar activities, in the range of 2 nmol/mg. min. The hydroxyl derivative of LA, 13-HODE, was glucuronidated at both the hydroxyl and carboxyl functions with carboxyl glucuronidation predominating (ratio of COOH/OH, 2:1). For all substrates, the K(m) for formation of the carboxyl-linked glucuronide was in the range of 100 to 200 microM while that for the hydroxyl-linked glucuronide was somewhat lower (>100 microM). This is the first demonstration of glucuronidation of LA and its oxidized derivatives, 13-HODE and 13-OXO, by HLM and recombinant UGT2B7.

Autoradiography↗
PubMed1999

Specific protein targets of 13-oxooctadecadienoic acid (13-OXO) and export of the 13-OXO-glutathione conjugate in HT-29 cells.

The linoleic acid metabolite, 13-oxooctadecadienoic acid (13-OXO), is reactive with cellular thiols. In the present report, incubations of HT-29 or CaCo-2 homogenates with 13-OXO and GSH indicate that HT-29 cell homogenates produce a 13-OXO-GSH conjugate. The conjugate formed was likely of enzymatic origin as chiral-phase HPLC showed the major product consisted of only one of two possible diastereomers. The glutathione transferase activity (GST), using chlorodinitrobenzene, was found to be 126 nmol/mg/min in HT-29 cells and 21 nmol/mg/min in CaCo-2 cells. These levels of activity are consistent with the relative ability of the two cell lines to conjugate GSH to 13-OXO. Incubation of intact HT-29 cells with either 13-OXO, or the metabolic precursor 13-hydroxyoctadecadienoic acid (13-HODE), showed detectable 13-OXO-GSH conjugate in the media, but none in the cells. The stereochemistry of the extracellular conjugate suggested an enzymatic origin. In additional experiments, the labeling of cellular protein by 13-HODE was much more specific than the labeling of protein by 13-OXO suggesting that in situ generation of 13-OXO from 13-HODE confers selectivity on the reactions between cellular thiols and 13-OXO. These results demonstrate that in HT-29 cells, 13-HODE is converted to 13-OXO which then either reacts with cellular protein or is conjugated to GSH by GST. The 13-OXO-GSH conjugate is then exported from the cell.

Alcohol Oxidoreductases↗
PubMed1997

Substrate specificity and characterization of partially purified rat liver 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase.

Oxidation products of linoleic acid, such as 13-hydroxyoctadecadienoic acid (13-HODE), exhibit biological activity in a number of systems. One major metabolic fate of 13-HODE is oxidation to the 2,4-dienone, 13-oxooctadecadienoic acid by an NAD(+)-dependent dehydrogenase (13-HODE dehydrogenase). The present work describes the partial purification and characterization of 13-HODE dehydrogenase from rat liver cytosol. The enzyme was purified using a combination of ammonium sulfate precipitation, as well as hydroxylapatite, gel permeation, and hydrophobic interaction chromatography. Analysis of the most purified preparation by SDS-polyacrylamide gel electrophoresis indicates two subunits of approximately 55 kDa, suggesting the possibility of a heterodimeric enzyme. However, due to aggregation in the purified preparation, an accurate molecular mass for the native enzyme has not yet been obtained. Using 13-HODE as a substrate, the purified enzyme has a Km of 6.3 microM and a Vmax of 5.7 nmol/min/mg. More importantly, the enzyme has a narrow substrate specificity with 13-HODE being the preferred substrate. From a series of 17 potential substrates, only 9-HODE (53% the activity of 13-HODE) and 15-hydroxyeicosatetraenoic acid (64% the activity of 13-HODE) showed significant activity as substrates. A number of other unsaturated hydroxy fatty acids, including several eicosanoids, are not substrates. The narrow substrate specificity displayed by the enzyme suggests that it could play a key role in modulating the effects of oxidized derivatives of linoleic acid.

Alcohol Oxidoreductases↗
PubMed1997

Production of 13-hydroxyoctadecadienoic acid (13-HODE) by prostate tumors and cell lines.

The major lipoxygenation product derived from linoleic acid, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), has been shown to be involved in cell proliferation and differentiation in a number of systems. Rapid detection of picogram amounts of this bioactive lipid in biological samples, however, has been hindered due to lack of immunological reagents. In the current report, we have used a polyclonal antibody specific for 13-(S)-HODE to detect this bioactive lipid for the first time in human prostate adenocarcinoma specimens (PCa) and the prostate cancer cell lines LNCaP and PC-3 by enzyme immunoassay. In addition, we have verified-the quantitation of 13-HODE by chiral-phase HPLC and examined the levels of lipoxygenase expression by Western, Northern, and RT-PCR analysis. Immunohistochemically detectable 13-HODE was observed in human PCa, whereas adjacent normal tissue showed no immunoreactivity. The presence of 15-lipoxygenase was evident by Western and RT-PCR analysis in both LNCaP and PC-3 cells, while Northern blot analysis showed the presence of 15-lipoxygenase message in LNCaP cells but failed to detect any 15-lipoxygenase message in PC-3 cells. In contrast, quantitation of 13-HODE by enzyme immunoassay and chiral-phase HPLC showed significant levels of the compound in PC-3 cells but minimal enzymatically produced 13-HODE in LNCaP cells. These data provide a link between linoleic acid metabolism and the development or progression of prostate cancer.

Arachidonate 15-Lipoxygenase↗
PubMed1997

Separation and quantitation of linoleic acid oxidation products in mammary gland tissue from mice fed low- and high-fat diets.

We have developed an assay for the isolation and quantitation by gas chromatography-mass spectrometry (GC-MS) of free 9- and 13-hydroxyoctadecadienoic acid (9-HODE, 13-HODE) in the mammary glands of female mice. Internal standards consisting of 18O2-labeled analogs of 9- and 13-HODE are added to pulverized frozen tissue prior to extraction with ethanol. Nonlipid materials are removed in a chloroform/methanol/water step. The remaining lipid material is methylated with ethereal diazomethane, and much of the nonoxygenated fatty acid methyl esters are removed via silica solid-phase extraction. Samples are either further derivatized with bis(trimethylsilyl)trifluoroacetamide to form the trimethylsilyl ethers for quantitative analysis by GC-MS or are analyzed as the methyl esters by chiral high-performance liquid chromatography to determine the enantiomeric distribution of the 9- and 13-HODE. The extraction and quantitation protocol was applied to the analysis of mammary glands for free 9- and 13-HODE from mice fed isocaloric diets containing 20% corn oil, 5% corn oil, or 20% beef tallow. Chiral analysis of the products showed higher production of 13(S)-HODE relative to 13(R)-HODE; the enantiomeric excess is most likely due to enzymatic production of 13-HODE superimposed on a background of autoxidative production of 13(R)- plus 9(S)- and 9(R)-HODE. In addition, the effect of sample handling and storage conditions on the formation of 9- and 13-HODE in the samples was assessed by exposing aliquots of a common pool of rat mammary gland tissue to specified conditions prior to analysis. This methodology will be important during investigations of the contribution of linoleate oxidation products to the enhancement of mammary tumorigenesis by dietary fat.

Analysis of Variance↗
PubMed1997

Occurrence of 13(S)-hydroxyoctadecadienoic acid in biological samples.

The oxygenated metabolite of linoleic acid, 13(S)-hydroxyoctadecadienoic acid has recently been shown to play a role in cellular regulation. To detect this molecule in biological systems, we recently developed a specific polyclonal antibody. Using this antibody, we report the presence of 13(S)-hydroxyoctadecadienoic acid in human urine, cell culture media, and untreated goat serum for the first time by a specific, sensitive, and rapid enzyme immunoassay. Furthermore, the enzyme linked immunosorbent assay data are verified by gas chromatography/mass spectrometry analysis of the same samples.

Animals↗
PubMed1997

Characterization of the enzymatic and nonenzymatic reaction of 13-oxooctadecadienoic acid with glutathione.

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE by the NAD(+)-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). These oxidized derivatives of linoleic acid have been shown to be involved in several cellular regulatory processes. In the present study, we have examined the enzymatic and nonenzymatic reaction of 13-OXO with glutathione (GSH) and N-acetylcysteine (N-AcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optimum of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HPLC-purified products were examined by FAB MS as well as one- and two-dimensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addition of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transferase 8-8. In the case of the enzymatic reaction there is stereoselective product formation. Furthermore, submicromolar concentrations of the 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provide insight into the potential metabolic disposition of linoleate oxygenation products.

Acetylcysteine↗
PubMed1997

Fatty acids and colon cancer in experimental models.

Experimental models have several advantages in the study of colon cancer. They can be used to tightly control diet, examine putative intermediate markers, test hypotheses about mechanisms of carcinogenesis, and quantify development of tumors in a short time. Dietary issues that have been studied in animal models but are unresolved include the concept of the effects of total fat compared with energy intake, composition of the basal diet, linoleic acid requirements, and interactions of fat with other nutrients. Intermediate markers that have been probed in animal or in vitro studies include cytokinetics, aberrant crypt foci, eicosanoids and hydroxyoctadecadienoic acids, ornithine decarboxylase, tyrosine kinase, protein kinase C, and gene expression. Colon cancer is studied in animals primarily with use of chemicals that are relatively specific inducers of these tumors, but transplantable models and transgenic animals are also used. Total dietary fat is generally thought to affect colon tumorigenesis, but there does not appear to be any specific fatty acid that promotes the development of colon cancer. Several studies indicate that n-3 fatty acids from marine sources alter a variety of biological intermediates and inhibit colonic tumorigenesis; this is probably mediated via the eicosanoid pathway. Although there are undoubtedly multiple cellular changes elicited by certain fatty acids, our current knowledge of this area suggests that specific fatty acid metabolites or their targets are the likely mediators in this sequence.

Animals↗
PubMed1996

Formation of adducts between 13-oxooctadecadienoic acid (13-OXO) and protein-derived thiols, in vivo and in vitro.

Linoleic acid is metabolized by numerous tissues to oxidized derivatives possessing biological activity. In the current experiments, we have investigated the reaction of 13-oxooctadecadienoic acid (13-OXO) and the metabolic precursor 13-hydroxyoctadecadienoic acid (13-HODE) with cellular macromolecules and model cellular nucleophiles. Colonic mucosal explants from Sprague-Dawley rats were incubated in the presence of [1-14C]-13-OXO or [1-14C]-13-HODE. The binding of radiolabel to the protein and nucleic acid fractions was analyzed by isopycnic centrifugation in Cs2SO4. Cellular homogenates incubated with either 13-OXO or 13-HODE resulted in the binding of radiolabel to cellular protein. No significant amounts of reaction with cellular RNA or DNA were observed. To assess possible modes of reaction with cellular constituents, the oxidized fatty acids were incubated in vitro with oxygen, sulfur, or nitrogen, nucleophiles including, serine, cysteine, glutathione, methionine, lysine, adenosine, and guanosine. Under physiologic conditions, in the absence of cellular homogenates, only 13-OXO was reactive. In addition, only the sulfur-containing compounds cysteine and glutathione showed significant rates of reaction. Furthermore, treatment of colonic homogenates with N-ethlymaleimide reduced the binding of [1-14C]-13-OXO to cellular protein. These data support the suggestion that 13-HODE requires metabolic activation, by dehydrogenation to 13-OXO, prior to binding to cellular protein and that protein-derived thiol groups are involved in the binding reactions.

Animals↗
PubMed1996

Decreased levels of 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase in neoplastic tissue of human colon biopsies.

Recent studies have identified a role for the oxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (13-HODE) in cell proliferation. The enzyme 13-HODE dehydrogenase catalyzes the conversion of 13-HODE to 13-oxooctadecadienoic acid. This enzyme has been shown to correlate with the degree of differentiation of intestinal cells in both in vitro and in vivo models. Higher enzyme levels are found in more differentiated cell types. The present study was done to determine if enzyme levels of 13-HODE dehydrogenase are predictive of the differentiation status of biopsies from human colonic mucosa. Twenty-eight patients who underwent diagnostic colonoscopy (10 patients with adenocarcinoma and 18 with adenomatous polyps) had biopsies taken from both normal rectal mucosa and neoplastic mucosa. The determination of 13-HODE dehydrogenase activity was conducted by high-performance liquid chromatography analysis of all biopsy samples. Sixteen of the 18 patients with polyps had lower 13-HODE dehydrogenase activity in the adenoma than in the uninvolved rectal mucosa (P = 0.001). The colon adenocarcinomas also had less 13-HODE dehydrogenase activity in the cancer biopsy tissue than in uninvolved rectal mucosa (P = 0.041) These data are consistent with a role for 13-HODE dehydrogenase in intestinal cell differentiation. Understanding the precise role of this enzymatic reaction could be important potentially in the therapy and biology of colon cancer. In addition, measurements of 13-HODE dehydrogenase may be a useful parameter by which to ascertain the differentiation status of intestinal cells in vitro.

Adenocarcinoma↗
PubMed1993

Effect of alkyl sulfides on diazomethane-induced methylation of DNA in vitro.

Organosulfur compounds found in Allium species such as garlic and onions inhibit carcinogenesis induced by alkylating agents. One potential mechanism for this activity involves scavenging of ultimate carcinogenic species by the nucleophilic sulfur atom. Carcinogenic methylating agents such as methyl-nitrosourea produce, among others, O6-methylguanine and N7-methylguanine in DNA both in vivo and in vitro. In the present study we have determined the effect of several alkyl sulfides on the formation of O6-methylguanine and N7-methylguanine in DNA reacted with the methylating agent diazomethane in vitro. Dipropyl sulfide and diallyl sulfide affect guanine methylation by increasing the O6/N7 ratio without drastic alterations in the total amount of adduct formed. Three similar compounds--diallyl disulfide, allyl methyl sulfide and diallyl ether--had no appreciable effect on the amount of alkylation at either position. These data suggest that scavenging of diazomethane-like ultimate carcinogens does not play a major role in the inhibition of carcinogenesis by organic sulfides.

Allyl Compounds↗
PubMed1993

Regulation of the induction of ornithine decarboxylase in short-term rat colon organ culture by dexamethasone and 13-hydroxyoctadecadienoic acid (13-HODE).

The activity of rat colon mucosal ornithine decarboxylase (ODC) was found to dramatically increase within three hours after placement of mucosal explants under organ culture conditions. Increases of 224-fold above the initial levels were observed 24 hours after establishment of cultures. During the next 72 hours, activity gradually declined although it never reached in vivo levels. Inclusion of either dexamethasone (DEX) or 13-hydroxyoctadecadienoic acid (13-HODE) in the media suppressed the early induction of ODC activity but did not abolish the increases. The effect of these compounds was reversible. Within 24 hours after removal of either dexamethasone or 13-HODE the ODC activity increased to the level found in untreated control cultures. These data suggest that glucocorticoids and 13-HODE may play a role in the regulation of colonic cellular proliferative activities in intact animals. The findings with 13-HODE add to the growing list of examples of regulation of biological activity by oxidized derivatives of linoleic acid.

Animals↗
PubMed1993

The correlation between 13-hydroxyoctadecadienoate dehydrogenase (13-HODE dehydrogenase) and intestinal cell differentiation.

The enzyme 13-hydroxyoctadecadienoate dehydrogenase (13-HODE dehydrogenase) catalyzes the NAD(+)-dependent oxidation of 13-hydroxyoctadecadienoic acid to 13-oxooctadecadienoic acid. In that the oxygenation of linoleic acid is increasingly being shown to be involved in the regulation of cellular function, this enzyme is poised to play a key role in the expression of the biological activity of these compounds. We have measured the activity of 13-HODE dehydrogenase in rat intestinal cells at various stages of differentiation. The specific activity of 13-HODE dehydrogenase shows a strong positive correlation with the degree of differentiation of intestinal mucosal cells from both the small and large intestines. In the small intestine the gradient of activity parallels that of alkaline phosphatase, while in the colon the incorporation of 3H-deoxythymidine and 13-HODE dehydrogenase are inversely related. Since the expression of 13-HODE dehydrogenase is most likely not associated with the nutritive function of the intestinal tract, these data raise the possibility the enzyme plays a role in the process of cellular differentiation.

Alcohol Oxidoreductases↗
PubMed1993

Increases in 13-hydroxyoctadecadienoic acid dehydrogenase activity during differentiation of cultured cells.

Recently, oxidation products of linoleic acid such as 13-hydroxyoctadecadienoic acid (HODE) have been implicated in the regulation of cellular physiology including the proliferative response to growth factor treatment. In addition, an NAD(+)-dependent 13-HODE dehydrogenase was recently described. To evaluate the contribution of this enzyme to cellular processes we have examined the behavior of the enzyme under different conditions. In the present report, changes in the activity of 13-hydroxyoctadecadienoic acid dehydrogenase during in vitro differentiation of two different cell lines were examined. The cell line HT-29 undergoes induced differentiation via manipulation of the medium while the Caco-2 line undergoes spontaneous differentiation upon attainment of confluence. In both cell lines, longer culture times were accompanied by increases in 13-HODE dehydrogenase activity. The increase in enzyme activity continued even after cell proliferation had ceased. Cellular differentiation was verified by the observation of increases in sucrase and alkaline phosphatase activities. In addition, the activity of 13-HODE dehydrogenase was measured in growing, early confluent and late confluent cultures of undifferentiating Swiss mouse 3T3 fibroblasts. In the fibroblast line, no significant changes in 13-HODE dehydrogenase activity were observed during the course of the experiment. The specific activity of 13-HODE dehydrogenase was also significantly different between the three cell lines, consistent with the extent of differentiation. Highest levels of activity were found in Caco-2 cells (200-400 pmol/min/mg) and barely detectable levels in the fibroblasts (0.6-2 pmol/min/mg). The correlation between 13-HODE dehydrogenase and cell differentiation suggests the enzyme may have a role to play in the partitioning of cells between proliferation and differentiation pathways.

3T3 Cells↗
PubMed1992

Failure of rectal ornithine decarboxylase to identify adenomatous polyp status.

Rectal mucosal ornithine decarboxylase (ODC) activity has been reported to distinguish between patients with and without adenomatous polyps (AP). In the present investigation, ODC activity has been measured in 28 patients with AP and 34 patients without AP. To assess the intraindividual variation in ODC activity, repeat biopsies were performed on 11 patients. In addition, the effect of postbiopsy sample handling was investigated by storage of samples on either dry or wet ice during transport to the laboratory. The mean rectal mucosal ODC activity in patients with AP was 196.0 +/- 195.5, whereas that in AP negative patients was 182.2 +/- 320.5. The rectal mucosal ODC activity in patients with colorectal cancer was 388.2 +/- 581. Repeat samples in individuals were generally within the same range as the original samples. The method of sample transport did not significantly affect the level of ODC measured in a particular biopsy. Because of high variability in rectal mucosal ODC activity within the population, there was wide overlap in ODC values between those patients with and without AP in an unselected general population. Thus, the measurement of flat rectal mucosal ODC activity is not a good predictor of the presence or absence of AP. Additional studies of the factors affecting mucosal ODC activity are necessary before the potential clinical utility of the method can be realized in the general population.

Aged↗