In vitro examination of the cytotoxic and cytoprotective mechanisms of linoleic acid, cis-epoxyoctadecenoic acids, and dihydroxyoctadecenoic acids in cellular and mitochondrial models

The toxicological significance of linoleic acid metabolism to the corresponding linoleic acid monoepoxides (EOAs) and diols (DHOAs) has been the subject of considerable research for approximately 15 years. However, the use of different fatty acid derivatives, as well as, different in vivo and in vitro models has made it difficult to make unifying conclusions about the overall importance of this metabolic pathway. The research presented in this dissertation is specifically designed to clarify discrepancies in current literature and to test the hypothesis: “Metabolism of linoleic acid to EOAs and DHOAs via extrahepatic cytochrome P450s and soluble epoxide hydrolase, respectively, represents an activation pathway which can explain many of pathological and cytoprotective properties of linoleic acid.” We tested the cytotoxic properties of these fatty acid metabolites in two different mammalian models, rabbit renal proximal tubules (RPT) and rabbit renal cortical mitochondria (RCM), and compared these results to an insect in vitro model, Sf-21 cells. The mechanism of toxicity for linoleic acid and the EOAs in rabbit RPT and RCM is mediated through the uncoupling of oxidative phoshphorylation. Of the tested metabolites, the DHOAs are the least toxic in rabbit RPT and RCM. These data suggest that the formation of EOAs and DHOAs is a detoxification pathway, and we have characterized human P450s (CYP2J2 and CYP2E1) which could be involved in this detoxification pathway. In contrast to the mammalian systems, the DHOAs appear to be the most toxic in Sf-21 cells and specifically inhibit the electron transport chain. Linoleic acid and the EOAs also uncouple oxidative phosphorylation in Sf-21 cells; however, these effects are transient in this model. In addition to studying the cytotoxic properties of these fatty acid metabolites, we examined the possibility that these compounds are produced in vivo as a protective response. Linoleic acid is a cytoprotective agent in rabbit RPT exposed to mitochondrial toxicants, and its cytoprotective mechanism occurs during the late phase of necrotic cell death and prevents the influx of extracellular chloride. Consistent with our toxicity data, metabolism of linoleic acid to the EOAs or DHOAs produces compounds which are not as cytoprotective as linoleic acid.