P44 Humanized, Transgenic Worms to Study the Role of Mitochondrial CYP2E1 in Toxicant-Induced Mitochondrial Dysfunction and Neurodegeneration

Jessica Helene Hartman , Duke University, Durham, North Carolina
Kacy L Gordon , Duke University, Durham, NC
David R Sherwood , Duke University, Durham, NC
Joel N Meyer , Duke University, Durham, NC
Cytochrome P450 2E1 (CYP2E1) is a critical enzyme involved in the metabolism and bioactivation of many small hydrophobic pollutants, drugs, and other xenobiotic compounds. Although the vast majority of CYP2E1 research has focused on the endoplasmic reticulum-localized (microsomal) form of the enzyme, erCYP2E1, CYP2E1 also localizes to mitochondria. The mitochondrial form, mtCYP2E1, has been significantly less studied, but it appears that there is significant interindividual variation in organellar targeting. We hypothesize that mtCYP2E1 is driving mitochondrial dysfunction by generating reactive metabolites within mitochondria that damage mitochondrial DNA and/or proteins. Because mitochondrial dysfunction is a key hallmark of neurodegeneration, we further hypothesize that toxicant-induced neurodegeneration may be driven by mtCYP2E1 activity. To test this hypothesis, novel C. elegans nematode models have been generated that express wild-type CYP2E1, erCYP2E1, and mtCYP2E1. Transgenic humanized C. elegans were generated by gonadal microinjection of a codon-optimized human CYP2E1 gene driven by the ubiquitous C. elegans eft-3 (eef-1A) promoter. Purified microsomes and crude mitochondria from transgenic animals displayed robust CYP2E1 activity (44 and 53 pmol/min/mg protein 4-nitrophenol hydroxylase activity for microsomes and mitochondria, respectively) compared to wild-type N2 nematodes which do not have any detectable CYP2E1 activity (< 1 pmol/min/mg protein, limit of detection). Furthermore, a 48-hour exposure of adult CYP2E1-expressing nematodes to the classic CYP2E1-activated drug acetaminophen resulted in significantly more lethality (25% at 3 mM, 50% at 25 mM) compared to wild-type N2 nematodes which did not show any lethality up to 25mM acetaminophen. Ongoing experiments will include mtCYP2E1 and erCYP2E1 transgenic C. elegans strains and determine the relative sensitivity of CYP2E1-expressing nematodes to CYP2E1-activated mitochondrial toxicants acetaminophen and trichloroethylene, including lethality and sublethal endpoints such as neurodegeneration and mitochondrial dysfunction. Ultimately, the results of this study will provide critical insight into the role of mtCYP2E1 in driving toxicant-induced mitochondrial dysfunction and neurodegeneration and could identify mitochondrial CYP2E1 localization as a risk factor for these exposures.