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.

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.

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.

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.

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.

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.

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.

Fertility timing, wages, and human capital.

PIP: This theoretical model posits that women who delay child bearing will be more likely to invest in human capital (training that enhances productivity but is costly). This investment is conditioned by a greater discount rate than an economy-wide growth rate of wages for non-human capital investor women. The aim of the model is to present a more unified view of relationships between wages and fertility timing identified in earlier research. The empirical analyses, using ordinary least squares techniques, was based on data from the National Longitudinal Survey of Young Women, 1968-82 annually, for a sample of 1817 White working women aged 28-38 in 1982. Data were available for wages, education, work experience, age, number of children, and the percentage in occupations (manager, professional, administrative, service, and blue collar). First wages of women not in school and without a first birth were obtained for 991 women in the sample. Descriptive statistics revealed that the average early wage of late child bearers was 37% higher than the average early wage of early child bearers and 43% higher for 1982 wages. Childless women, compared to early child bearers, experienced a growth in wages from 31-38%. The assumptions in the theoretical model were 1) that all women were equally productive in the labor market in the beginning; 2) that women bore only one child; 3) that women worked continuously for a period of time, except for time out for child bearing; 4) that all women had the option of investing in one type of human capital, which cost the same for all women; 5) that the only source of income was the woman's own earnings; and 6) that a woman's lifetime utility was a function of the present value of her lifetime income and the intervening time period for child birth. Differences in education, experience, tenure, and wages were strongly associated with differences in fertility timing. The results revealed that wages were higher for delayed child bearers, primarily because of larger accumulations of human capital, assuming joint human capital and fertility timing decisions. The interpretation of regressions to test family influence was that unobserved heterogeneity partly explained the empirical relationship. Wages were affected by education, experience, and tenure, as proxies for human capital. Results were consistent with the hypothesis but did not confirm the theory.^ieng

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.