Libin Xu - US grants

In this project, funded by the Chemical Structure, Dynamic & Mechanism B Program of the Chemistry Division, Professor Libin Xu of the Department of Medicinal Chemistry at the University of Washington aims to develop chemical tools to understand the reactivities and reaction mechanisms of oxidation of biologically important lipids. Understanding the fundamental chemistry underneath lipid peroxidation has broad impacts because lipid peroxidation plays significant roles in human pathologies, such as aging, atherosclerosis, and degenerative diseases. The project lies at the interface of physical organic chemistry and bioorganic chemistry and utilizes modern analytical chemistry techniques, and is well suited to the education of scientists with different training backgrounds. This research group also aims to integrate education with the research plan by disseminating the knowledge of lipid peroxidation through outreach to high school students and teachers and attracting undergraduates from diverse backgrounds to the research field.

The rate-determining step in lipid peroxidation is the propagation step of the peroxyl radical, where generally two types of reactions occur: a) hydrogen-atom transfer from a donor to the peroxyl radical; b) peroxyl-radical addition to a carbon-carbon double bond. The hydrogen-atom transfer mechanism is well studied, but much less is known about the reactivities and products of peroxyl-radical addition reactions. The goal of this project is to elucidate factors governing the reactivity of peroxyl-radical addition to a double bond and the contribution of this mechanism to peroxidation of a number of biologically important lipids, including cholesterol biosynthetic precursors, fat-soluble vitamins, and polyunsaturated fatty acids. There are three research objectives: 1) Establish a "radical clock" to measure the rate constants of both hydrogen-atom transfer and peroxyl-radical addition; 2) Evaluate factors controlling the reactivities of peroxyl-radical addition to a double bond; 3) Elucidate the products and reaction mechanisms of free radical peroxidation of biologically important lipids.

Project Summary Benzalkonium chlorides (BACs) are widely used antimicrobials in disinfecting products, medical products, consumer products, and food processing industries, suggesting humans may be exposed chronically and sys- temically to BACs through a variety of routes. Our preliminary study found that close to 50 of 100 random hu- man plasma samples contain detectable levels of BACs, suggesting BACs are indeed absorbed. The ongoing COVID-19 pandemic has led to greatly increased use of BAC-containing disinfectants, which is concerning given accumulating evidence in respiratory, developmental, reproductive, and neurological toxicities inflicted by BACs and BAC-induced disruption of cholesterol and lipid homeostasis in rodents. However, there is a lack of knowledge on the metabolism, transport, and biological consequences of BACs in humans. Our goal is to characterize the pathways of metabolism and transport of BACs and their impact on nephrotoxicity of BACs. The potential for nephrotoxicity is supported by previous studies in rats showing that BACs accumulate to the highest level in the kidney after oral intake and our preliminary studies showing that BACs exert potent cytotox- icity in a 3D ?kidney-on-a-chip? microphysiological system (MPS). Recently, we reported that BACs are metab- olized by human cytochrome P450 (CYP) isoforms CYP2D6 and CYP4s in vitro. Furthermore, we found that BACs are actively transported by human organic cation transporters (hOCTs). Because CYP2D6, CYP4s, and hOCTs are highly polymorphic with greatly varying protein activities, we hypothesize that toxicities of BACs in kidney are dependent on the activities of BAC-metabolizing and transporting proteins in both liver and kidney. In Aim 1, we will characterize pathways of metabolism and transport of BACs in vitro, including secondary me- tabolism by ?-oxidation and glucuronidation and transport by hOCTs, human multidrug and toxin extrusion pro- teins, and P-glycoprotein. In Aim 2, we will evaluate nephrotoxicity induced by BACs in human proximal tubule epithelial cells in 3D integrated liver-kidney ?organs-on-chips? MPS. An integrated sterolomics, lipidomics, and transcriptomics approach will be used to systemically assess the toxicity and biological activities of BACs. In Aim 3, we will assess BAC exposure levels and their correlation with lipid and kidney injury biomarkers in hu- mans, as well as the impact of genetic variations on BAC metabolism and disposition. The significance of this project lies in that it will, for the first time, address the knowledge gap in metabolism, transport, and toxicity of BACs in humans. Elucidation of the contribution of reduced activities in CYPs and/or transporters to BAC tox- icity would enable us to identify high-risk human population with genetic variations in these proteins. The gained knowledge could also inform federal agencies on setting more appropriate exposure limitations. The innovation of this project lies in a) a novel example of gene-environment interaction through xenobiotic- processing proteins, b) the use of an integrated liver-kidney MPS to assess the toxicological consequences of xenobiotics, and c) integrated omics for rigorous systems toxicology studies.