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 = 830 µM and Kss = 32 mM). 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 = 830 µM and Ksi = 180 µM 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.

Development of an IgY Antibody-Based Immunoassay for the Screening of the CYP2E1 Inhibitor/Enhancer from Herbal Medicines

“…This enzyme is highly induced by food or beverage constituents (alcohol), drugs (acetaminophen) (Hartman et al, 2015), and pollutants (styrene). CYP2E1 may eliminate potentially toxic compounds and, paradoxically, bioactive compounds in toxins or carcinogens.…”

“…CYP2E1 may eliminate potentially toxic compounds and, paradoxically, bioactive compounds in toxins or carcinogens. For example, CYP2E1 is induced during alcohol or acetaminophen (APAP) overdose and results in the formation of ROS and the increased generation of hydroxyl radicals (Jin et al, 2013; Hartman et al, 2015). Increased oxidative stress from induction of CYP2E1 can damage cellular and mitochondrial components, including mitochondrial DNA and cytochrome c oxidase (Demeilliers et al, 2002).…”

Immature mice are more susceptible than adult mice to acetaminophen-induced acute liver injury

“…Although it is safe at therapeutic doses, APAP overdose induces acute liver injury 1 2 3 . APAP-induced acute liver injury is initiated by the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which is generated by several cytochrome P450 (CYP) isoenzymes, mainly CYP2E1 and CYP3A4 4 5 6 7 8 9 10 . Several studies demonstrate that the prolonged activation of hepatic c-Jun N-terminal kinase (JNK) is involved in APAP-induced hepatocyte death 11 12 .…”

Mechanisms of Herb-Drug Interactions Involving Cinnamon and CYP2A6: Focus on Time-Dependent Inhibition by Cinnamaldehyde and 2-Methoxycinnamaldehyde

“…CYP2A6 and CYP2E1 have more restricted active site volumes among human P450s (DeVore et al, 2008;Porubsky et al, 2008); yet there is evidence both enzymes accommodate multiple ligands simultaneously, and exhibit homotropic and heterotropic allosteric behavior (Harrelson et al, 2008;Hartman et al, 2012;Hartman et al, 2013;Hartman et al, 2015). Spectral binding analysis can provide evidence for multiple ligand binding; it may also overlook some multiple ligand binding scenarios as the binding of a second ligand may not always generate changes in the spin state of the heme iron (Davydov et al, 2002;Atkins, 2005).…”