Toxicity of environmental chemicals often arises from metabolic activation. Cytochrome P450 2E1 (CYP2E1) metabolically activates a broad range of small hydrophobic pollutants, drugs, and dietary compounds. Assessment of risk associated with compounds activated by CYP2E1 relies on in vitro kinetics of CYP2E1 metabolism assuming a hyperbolic relationship between rate and the concentration of toxicant (Michaelis-Menten kinetics). CYP2E1 reactions were traditionally thought to conform to Michaelis-Menten kinetics; however, there are exceptions including 4-nitrophenol, which exhibits substrate inhibition during its metabolism, and 7-ethoxycoumarin, phenacetin, and m-xylene, which have sigmoidal kinetic profiles indicating positive cooperativity. Furthermore, in vivo pharmacokinetic modeling supports a non-Michaelis-Menten CYP2E1 model for dichloromethane-induced carcinogenesis in mice. Overall, this evidence suggests that CYP2E1 metabolism may involve complex kinetics, which impact the resulting toxicity of dichloromethane. Thus, my dissertation research utilized biochemical and biophysical tools to assess the mechanism of hepatic CYP2E1 metabolism of toxicants and to predict its biological consequences. Using recombinant CYP2E1 and pooled human liver microsomes, I showed that metabolism of the pollutant styrene to its genotoxic epoxide metabolite styrene oxide exhibited a sigmoidal kinetic profile, indicating positive cooperativity. Conversely, CYP2E1-mediated biochemical activation of the drug acetaminophen and the pollutant aniline both conformed to negative cooperativity. In all three cases, this cooperativity could be explained by a two-binding site mechanism involving substrate binding at catalytic and cooperative binding sites. Modeling of in vivo clearance (bioactivation) using the in vitro kinetic mechanisms and parameters revealed significant deviations from the traditional Michaelis-Menten model. Finally, I determined how cooperativity would impact CYP2E1 metabolism of styrene and acetaminophen mixtures. Based on the resulting kinetic data, clearance predictions indicated that acetaminophen may cooperatively increase styrene genotoxicity. Taken together, this thesis work challenges the paradigm of simple CYP2E1 Michaelis-Menten kinetics and demonstrates that cooperativity is more prevalent than initially assumed for biologically important CYP2E1-activated toxicants.
|Advisor:||Miller, Grover P.|
|Commitee:||Bhattacharyya, Sudeepa, Boysen, Gunnar, Eoff, Robert L., Roberts, Dean W., Ronis, Martin J.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 77/11(E), Dissertation Abstracts International|
|Subjects:||Toxicology, Surgery, Biochemistry|
|Keywords:||Acetaminophen, Aniline, CYP2E1, Cooperativity, Cytochrome P450, Styrene|
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