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UNC-Chapel Hill ENVR 442 - Cytochrome P450 and Chemical Toxicology

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Cytochrome P450 and Chemical ToxicologyF. Peter Guengerich*Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine,638 Robinson Research Building, 23rd and Pierce AVenues, NashVille, Tennessee 37232-0146ReceiVed March 12, 2007The field of cytochrome P450 (P450) research has developed considerably over the past 20 years, andmany important papers on the roles of P450s in chemical toxicology have appeared in Chemical Researchin Toxicology. Today, our basic understanding of many of the human P450s is relatively well-established,in terms of the details of the individual genes, sequences, and basic catalytic mechanisms. Crystal structuresof several of the major human P450s are now in hand. The animal P450s are still important in the contextof metabolism and safety testing. Many well-defined examples exist for roles of P450s in decreasing theadverse effects of drugs through biotransformation, and an equally interesting field of investigation isthe bioactivation of chemicals, including drugs. Unresolved problems include the characterization of theminor “orphan” P450s, ligand cooperativity and kinetic complexity of several P450s, the prediction ofmetabolism, the overall contribution of bioactivation to drug idiosyncratic problems, the extrapolation ofanimal test results to humans in drug development, and the contribution of genetic variation in humanP450s to cancer incidence.Contents1. Introduction and Background701.1. Current Knowledge about P450s702. Roles of P450s in Reducing Toxicity723. Bioactivation by P450s733.1. Aflatoxin B1733.2. Ethyl Carbamate743.3. Coupling of Norharman and Aniline743.4. Troglitazone743.5. Other Bioactivation Reactions753.6. Mechanism-Based Activation753.7. P450s and Oxidative Damage764. Current and Future Issues774.1. Functions of “Orphan” P450s774.1.1. Analysis of Suspects774.1.2. Transgenic Animal Models774.1.3. Library Screening774.1.4. Untargeted Metabolomic Strategies inVitro774.1.5. Untargeted in Vitro Strategies withIsotope Editing774.2. Ligand Cooperativity774.3. Predictions of Metabolism774.4. Overlaps of Detoxication and Bioactivation784.5. Roles of P450s in Idiosyncratic DrugToxicity784.6. Predicting Human Toxicity784.7. Understanding P450 Gene Polymorphismsand Disease785. Conclusion791. Introduction and BackgroundCytochrome P450 (P450) research can be traced back to invitro studies on the metabolism of steroids, drugs, and carcino-gens in the 1940s (1). Some of the major developments werethe spectral observation of P450 (2), photochemical actionstudies implicating P450 as the oxidase in the electron transportsystem (3), the separation (4) and subsequent purification ofP450 (5, 6), and several studies implicating multiple P450enzymes (7, 8). Other early seminal studies include the extensivebiochemical and biophysical work with the bacterial P450101A1 (P450cam)(9) and the first complete nucleotide sequenceof a P450 (10). Studies on the chemistry of oxygen activationdeveloped, and one of the key studies underpinning our currentmodels was evidence for a stepwise process involving C–H bondbreaking (11).During the past 20 years, we have seen a major shift ofemphasis to human P450s, which had seemed almost impossiblein the early research. The knowledge about the human P450shas had important ramifications in understanding the metabolismof drugs. In comparison to the situation ∼25 years ago, far fewerdrugs fail in development due to pharmacokinetic problems inhumans, because of the reiterative approach of chemicalsynthesis, target screening, and in vitro metabolism studies inplace in pharmaceutical companies (Figure 1). However, lessprogress has been made in accurately predicting human toxicityproblems with drugs and the challenge remains considerable(13, 14). In retrospect, one of the driving forces for the studyof P450s has been the quest for information to better understandand predict the metabolism and toxicity of drugs and otherchemicals [e.g., thalidomide (15–17)].1.1. Current Knowledge about P450s. This section willfocus on important developments that have occurred over thepast 20 years, that is, since this journal began. One is certainlythe completion of the human genome project, which set thenumber of human P450 (“CYP”) genes at 57 (Table 1) (and thenumber of pseudogenes at 58) (http://drnelson.utmem.edu/* To whom correspondence should be addressed. Tel: 615-322-2261.Fax: 615-322-3141. E-mail: [email protected]. Res. Toxicol. 2008, 21, 70–837010.1021/tx700079z CCC: $40.75  2008 American Chemical SocietyPublished on Web 12/06/2007Downloaded by UNIV OF NORTH CAROLINA on August 31, 2009 | http://pubs.acs.org Publication Date (Web): December 6, 2007 | doi: 10.1021/tx700079zCytochromeP450.html), thus putting speculation about thisnumber to rest. However, some uncertainties exist about theexpression of some of the genes at the mRNA and particularlythe protein levels (e.g., P450 4A22).Twenty years ago, the biochemical purification of several ofthe major human liver P450s was achieved (20–22). Thedevelopment of recombinant DNA technology was well under-way, and heterologous expression was done in low-yieldsystems. Some breakthroughs in the early 1990s led to successfulhigh-level bacterial expression (23–25), which was critical forcrystallography work. In the past few years, the number ofcrystal structrures of human P450s has increased rapidly, andtoday, high-resolution structures are available for human P450s1A2 (26), 2A6 (27), 2C8 (28), 2C9 (29, 30), 2D6 (31), and3A4 (32–34), the major P450s involved in drug metabolism.These structures have replaced less accurate homology modelsbased mainly on bacterial P450s (P450s 101A1 and 102A1)and also serve as reasonable templates for other closely relatedsubfamily P450 members. One general observation with theseand other animal and bacterial P450 structures is that mostundergo major conformational changes upon ligand binding (35),and ligand-free structures are of limited use in understandingthe functions of these proteins. However, with some of the P450shaving large active sites, the positions of ligands in the crystalstructures still leave many questions open about the interactions(32, 34, 36).The generally accepted catalytic cycle for P450 reactions isshown in Figure 2. However, the point should be made thatthis is a simplified version and that the system is dynamic, andthe steps do not necessarily proceed in a linear


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UNC-Chapel Hill ENVR 442 - Cytochrome P450 and Chemical Toxicology

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