title: Cooperativity in CYP2E1 metabolism of acetaminophen and styrene mixtures.
abstract: Risk assessment for exposure to mixtures of drugs and pollutants relies heavily on in vitro characterization of their bioactivation and/or metabolism individually and extrapolation to mixtures assuming no interaction. Herein, we demonstrated that in vitro CYP2E1 metabolic activation of acetaminophen and styrene mixtures could not be explained through the Michaelis-Menten mechanism or any models relying on that premise. As a baseline for mixture studies with styrene, steady-state analysis of acetaminophen oxidation revealed a biphasic kinetic profile that was best described by negative cooperativity (Hill coefficient=0.72). The best-fit mechanism for this relationship involved two binding sites with differing affinities (Ks=830μM and Kss=32mM). Introduction of styrene inhibited that reaction less than predicted by simple competition and thus provided evidence for a cooperative mechanism within the mixture. Likewise, acetaminophen acted through a mixed-type inhibition mechanism to impact styrene epoxidation. In this case, acetaminophen competed with styrene for CYP2E1 (Ki=830μM and Ksi=180μM for catalytic and effector sites, respectively) and resulted in cooperative impacts on binding and catalysis. Based on modeling of in vivo clearance, cooperative interactions between acetaminophen and styrene resulted in profoundly increased styrene activation at low styrene exposure levels and therapeutic acetaminophen levels. Current Michaelis-Menten based toxicological models for mixtures such as styrene and acetaminophen would fail to detect this concentration-dependent relationship. Hence, future studies must assess the role of alternate CYP2E1 mechanisms in bioactivation of compounds to improve the accuracy of interpretations and predictions of toxicity.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Hartman
authors.affiliation: Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
authors.forename: Lynda G
authors.initials: LG
authors.lastname: Letzig
authors.affiliation: Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
authors.forename: Dean W
authors.initials: DW
authors.lastname: Roberts
authors.affiliation: Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
authors.forename: Laura P
authors.initials: LP
authors.lastname: James
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
authors.forename: E Kim
authors.initials: EK
authors.lastname: Fifer
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States. Electronic address: MillerGroverP@uams.edu.
authors.forename: Grover P
authors.initials: GP
authors.lastname: Miller
mesh_terms: D000082:Acetaminophen / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D001665:Binding Sites; D001667:Binding, Competitive; D001711:Biotransformation; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D065691:Cytochrome P-450 CYP2E1 Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D004785:Environmental Pollutants / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008954:Models, Biological; D008956:Models, Chemical; D010084:Oxidation-Reduction; D020058:Styrene / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D013379:Substrate Specificity
chemical_list: D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004785:Environmental Pollutants; D000082:Acetaminophen; D020058:Styrene; D019392:Cytochrome P-450 CYP2E1
keywords: Acetaminophen; CYP2E1; Cooperativity; Mixture; Styrene