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-C-14]-13-OXO or [1-C-14]-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-ethylmaleimide reduced the binding of [1-C-14]-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.