UC Davis

Ubiquitous exposure to various exogenous factors imparts both acute and chronic adverse effects on human health. Common molecular modes of action include induction of inflammation and activation of the mitochondrial dysfunction-reactive oxygen species-endoplasmic reticulum (ER) stress axis. The kidney is especially susceptible to noxious impacts due to several structural and functional characteristics. Preservation of inflammation-mediated and organelle-linked health impacts is further hampered by a lack of safe and efficacious reno-protective agents and unreliable biomarkers.

The cytochrome P450 (CYP) branch of polyunsaturated fatty acid (PUFA) metabolism oxygenates PUFAs to a class of signaling molecules known as epoxy fatty acids (EpFAs). These are primarily inflammation resolving and mitochondria-ER stabilizing lipid mediators. Since numerous points within the P450 pathway can be capitalized upon and/or manipulated, CYP-mediated EpFA biosynthesis and metabolism offer novel therapeutic targets to ameliorate and monitor disease.

Thus, this dissertation describes new and complementing research methodologies that were explored to elucidate the pharmacological and diagnostic potential of this PUFA metabolizing route. More precisely, Chapter 1 reveals the prevalence and therapeutic relevance of an overlooked group of EpFAs. Chapter 2 furthers the quest for naturally occurring, dietarily-attained enzyme inhibitors that block the primary route of EpFA metabolism and exert desired medicinal effects. Chapter 3 illuminates a biochemically active functional group that can be employed as a lead for the design of new and improved EpFA-like therapies. Chapter 4 delivers a modern and clinically pertinent analytical method for quantitation of the downstream metabolites of EpFAs derived from a pervasive dietary fat to exploit their exceptional biomarker functions.

Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are the most widely pharmacologically utilized group of EpFAs. However, the presence and therapeutic activity of their 22 carbon- homologues, adrenic acid-derived epoxydocosatrienoic acids (EDTs), is unstudied, despite the abundance of its parent PUFA in the kidney and vasculature. EDTs were found to occur naturally in vivo and exert anti-ER stress effects in kidney cells and relief from inflammatory pain in an animal model, thus illuminating another medicinally relevant group of EpFAs. Inhibition of the soluble epoxide hydrolase (sEH) enzyme curbs the major route of EpFA degradation, facilitating their bioactivities. sEH inhibitors (sEHI) derived from natural sources, such as botanicals, offer unique applicatory benefits over synthetic drugs. Accordingly, macamides derived from maca (Lepidium meyenii) were found to be the most promising natural sEHI group to date for a combination of inhibitory potency towards sEH and abundance in plant root. The single most relevant macamide was orally available and alleviated inflammatory pain pre-clinically. Design of structural analogues of EpFAs provides biochemically robust therapeutics that mimic EpFA activity. Accordingly, replacement of the labile epoxide group with more stable alkyl ether groups yielded a new class of EpFA analogues. These alkoxy-based analogues of EETs attenuated nephrotoxicity of a widely used chemotherapeutic (i.e., cisplatin) in vitro through downregulation of mitochondrial oxidative stress and termination of downstream apoptotic signaling. Downstream sEH-generated dihydroxy fatty acids, most notably the predominant linoleic acid-derived dihydroxyoctadecenoic acids (DiHOMEs), have been closely implicated in numerous pro-inflammatory pathologies and hence provide a promising biomarker for assessment of health outcomes. Reliable analytical methods are necessary for their detection and enzyme-linked immunoassays (ELISA) possess distinct benefits as complements to classically used liquid chromatography-tandem mass spectrometry (LC-MS/MS). Therefore, utilizing polyHRP technology, a highly sensitive ELISA for DiHOMEs was developed, validated for use in human plasma, the most clinically relevant matrix, and authenticated with LC-MS/MS.

In conclusion, the multitude of strategies discussed here highlight the medicinal potential and malleability of the CYP route of PUFA metabolism and advance the pursuit of potential biochemical means to mitigate and track the deleterious health effects of environmental stressors.