title: Specific protein targets of 13-oxooctadecadienoic acid (13-OXO) and export of the 13-OXO-glutathione conjugate in HT-29 cells.
abstract: 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.
authors.affiliation: Department of Chemistry, Oakland University, Rochester, MI 48309-4477, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Blackburn
authors.forename: I
authors.initials: I
authors.lastname: Podgorski
authors.forename: A W
authors.initials: AW
authors.lastname: Bull
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D018938:Caco-2 Cells; D002250:Carbon Radioisotopes; D002458:Cell Fractionation; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D019073:HT29 Cells / Q000378:metabolism*; D006801:Humans; D008041:Linoleic Acids / Q000378:metabolism*; D008042:Linolenic Acids / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D008041:Linoleic Acids; D008042:Linolenic Acids; D011506:Proteins; C064441:13-oxo-9,11-octadecadienoic acid; C024348:13-hydroxy-9,11-octadecadienoic acid; D000429:Alcohol Oxidoreductases; C067701:13-hydroxyoctadecadienoic acid dehydrogenase; D005982:Glutathione Transferase; D005978:Glutathione