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

The linoleic acid metabolite, 13-oxooctadecadienoic acid (13-OXO), is react ive with cellular thiols. In the present report, incubations of HT-29 or Ca Co-2 homogenates with 13-OXO and GSH indicate that HT-29 cell homogenates p roduce 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 consisten t with the relative ability of the two cell lines to conjugate GSH to 13-OX O. Incubation of intact HT-29 cells with either 13-OXO, or the metabolic pr ecursor 13-hydroxyoctadecadienoic acid (13-HODE), showed detectable 13-OXO- GSH conjugate in the media, but none in the cells. The stereochemistry of t he extracellular conjugate suggested an enzymatic origin. In additional exp eriments, the labeling of cellular protein by 13-HODE was much more specifi c 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 cellul ar thiols and 13-OXO. These results demonstrate that in HT-29 cells, 13-HOD E 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. (C) 1999 Published by Elsevier Science B.V. All rights reserved.