Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals

Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in the metabolism of all chemicals (general chemicals, natural, and physiological compounds, and drugs) are rather consistent in the findings that >90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs

Contributions of Human Enzymes in Carcinogen Metabolism

Considerable support exists for the roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are pro-carcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on the metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s1A1, 1A2, 1B1, 2A6, 2E1, and 3A4accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, interindividual variations, and risk assessment

Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals

Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in the metabolism of all chemicals (general chemicals, natural, and physiological compounds, and drugs) are rather consistent in the findings that >90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs

Bioactivation of Fluorinated 2-Aryl-benzothiazole Antitumor Molecules by Human Cytochrome P450s 1A1 and 2W1 and Deactivation by Cytochrome P450 2S1

Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and 5-fluoro-2-(3,4-dimethoxyphenyl)-benzothiazole (GW 610) contain the benzothiazole pharmacophore and possess potent and selective in vitro antitumor properties. Prior studies suggested the involvement of cytochrome P450 (P450) 1A1 and 2W1-mediated bioactivation in the antitumor activities and P450 2S1-mediated deactivation of 5F 203 and GW 610. In the present study, the biotransformation pathways of 5F 203 and GW 610 by P450s 1A1, 2W1, and 2S1 were investigated, and the catalytic parameters of P450 1A1- and 2W1-catalyzed oxidation were determined in steady-state kinetic studies. The oxidations of 5F 203 catalyzed by P450s 1A1 and 2W1 yielded different products, and the formation of a hydroxylamine was observed for the first time in the latter process. Liquid chromatography–mass spectrometry (LC-MS) analysis with the synthetic hydroxylamine and also a P450 2W1/5F 203 incubation mixture indicated the formation of dGuo adduct via a putative nitrenium intermediate. P450 2W1-catalyzed oxidation of GW 610 was 5-fold more efficient than the P450 1A1-catalyzed reaction. GW 610 underwent a two-step oxidation process catalyzed by P450 1A1 or 2W1: a regiospecific <i>O</i>-demethylation and a further hydroxylation. Glutathione (GSH) conjugates of 5F 203 and GW 610, presumably through a quninoneimine and a 1,2-quinone intermediate, respectively, were detected. These results demonstrate that human P450s 1A1 and 2W1 mediate 5F 203 and GW 610 bioactivation to reactive intermediates and lead to GSH conjugates and a dGuo adduct, which may account for the antitumor activities of 5F 203 and GW 610 and also be involved in cell toxicity. P450 2S1 can catalyze the reduction of the hydroxylamine to the amine 5F 203 under anaerobic conditions and, to a lesser extent, under aerobic conditions, thus attenuating the anticancer activity

Replication past the Butadiene Diepoxide-Derived DNA Adduct <i>S</i>‑[4‑(<i>N</i>⁶‑Deoxyadenosinyl)-2,3-dihydroxybutyl]glutathione by DNA Polymerases

1,2,3,4-Diepoxybutane (DEB), a metabolite of the carcinogen butadiene, has been shown to cause glutathione (GSH)-dependent base substitution mutations, especially A:T to G:C mutations in <i>Salmonella typhimurium</i> TA1535 [Cho, S. H., et al. (2010) <i>Chem. Res. Toxicol. 23</i>, 1544] and <i>Escherichia coli</i> TRG8 cells [Cho, S. H., and Guengerich, F. P. (2012) <i>Chem. Res. Toxicol. 25</i>, 1522]. We previously identified <i>S</i>-[4-(<i>N</i>⁶-deoxyadenosinyl)-2,3-dihydroxybutyl]­GSH [<i>N</i>⁶dA-(OH)₂butyl-GSH] as a major adduct in the reaction of <i>S</i>-(2-hydroxy-3,4-epoxybutyl)­glutathione (DEB-GSH conjugate) with nucleosides and calf thymus DNA and <i>in vivo</i> in livers of mice and rats treated with DEB [Cho, S. H., and Guengerich, F. P. (2012) <i>Chem. Res. Toxicol. 25</i>, 706]. For investigation of the miscoding potential of the major DEB-GSH conjugate-derived DNA adduct [<i>N</i>⁶dA-(OH)₂butyl-GSH] and the effect of GSH conjugation on replication of DEB, extension studies were performed in duplex DNA substrates containing the site-specifically incorporated <i>N</i>⁶dA-(OH)₂butyl-GSH adduct, <i>N</i>⁶-(2,3,4-trihydroxybutyl)­deoxyadenosine adduct (<i>N</i>⁶dA-butanetriol), or unmodified deoxyadenosine (dA) by human DNA polymerases (Pol) η, ι, and κ, bacteriophage polymerase T7, and <i>Sulfolobus solfataricus</i> polymerase Dpo4. Although dTTP incorporation was the most preferred addition opposite the <i>N</i>⁶dA-(OH)₂butyl-GSH adduct, <i>N</i>⁶dA-butanetriol adduct, or unmodified dA for all polymerases, the dCTP misincorporation frequency opposite <i>N</i>⁶dA-(OH)₂butyl-GSH was significantly higher than that opposite the <i>N</i>⁶dA-butanetriol adduct or unmodified dA with Pol κ or Pol T7. LC–MS/MS analysis of full-length primer extension products confirmed that Pol κ or Pol T7 incorporated the incorrect base C opposite the <i>N</i>⁶dA-(OH)₂butyl-GSH lesion. These results indicate the relevance of GSH-containing adducts for the A:T to G:C mutations produced by DEB

Mechanism of the Third Oxidative Step in the Conversion of Androgens to Estrogens by Cytochrome P450 19A1 Steroid Aromatase

Aromatase is the cytochrome P450 enzyme that cleaves the C10–C19 carbon–carbon bond of androgens to form estrogens, in a three-step process. Compound I (FeO³⁺) and ferric peroxide (FeO₂^–) have both been proposed in the literature as the active iron species in the third step, yielding an estrogen and formic acid. Incubation of purified aromatase with its 19-deutero-19-oxo androgen substrate was performed in the presence of ¹⁸O₂, and the products were derivatized using a novel diazo reagent. Analysis of the products by high-resolution mass spectrometry showed a lack of ¹⁸O incorporation in the product formic acid, supporting only the Compound I pathway. Furthermore, a new androgen 19-carboxylic acid product was identified. The rates of nonenzymatic hydration of the 19-oxo androgen and dehydration of the 19,19-<i>gem</i>-diol were shown to be catalytically competent. Thus, the evidence supports Compound I and not ferric peroxide as the active iron species in the third step of the steroid aromatase reaction

Conjugation of Butadiene Diepoxide with Glutathione Yields DNA Adducts in Vitro and in Vivo

1,2,3,4-Diepoxybutane (DEB) is reported to be the most potent mutagenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant. DEB is capable of inducing the formation of monoalkylated DNA adducts and DNA–DNA and DNA–protein cross-links. We previously reported that DEB forms a conjugate with glutathione (GSH) and that the conjugate is considerably more mutagenic than several other butadiene-derived epoxides, including DEB, in the base pair tester strain <i>Salmonella typhimurium</i> TA1535 [Cho (2010) Chem. Res. Toxicol. 23, 1544−1546]. In the present study, we determined steady-state kinetic parameters of the conjugation of the three DEB stereoisomers<i>R</i>,<i>R</i>, <i>S</i>,<i>S</i>, and <i>meso</i> (all formed by butadiene oxidation)with GSH by six GSH transferases. Only small differences (<3-fold) were found in the catalytic efficiency of conjugate formation (<i>k</i>_cat/<i>K</i>_m) with all three DEB stereoisomers and the six GSH transferases. The three stereochemical DEB–GSH conjugates had similar mutagenicity. Six DNA adducts (<i>N</i>³-adenyl, <i>N</i>⁶-adenyl, <i>N</i>⁷-guanyl, <i>N</i>¹-guanyl, <i>N</i>⁴-cytidyl, and <i>N</i>³-thymidyl) were identified in the reactions of DEB–GSH conjugate with nucleosides and calf thymus DNA using LC-MS and UV and NMR spectroscopy. <i>N</i>⁶-Adenyl and <i>N</i>⁷-guanyl GSH adducts were identified and quantitated in vivo in the livers of mice and rats treated with DEB ip. These results indicate that such DNA adducts are formed from the DEB–GSH conjugate, are mutagenic regardless of sterochemistry, and are therefore expected to contribute to the carcinogenicity of DEB

7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching, suggesting binding at multiple protein sites

<p>Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (<i>α</i>-naphthoflavone, <i>α</i>NF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and <i>α</i>NF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern–Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with <i>α</i>NF; at the relatively higher [<i>α</i>NF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (<i>K</i>_<i>d</i> 1.8–2.5 and 6.5 μM). A range is given for the stronger interaction since <i>α</i>NF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (<i>K</i>_<i>d</i> 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (<i>K</i>_<i>d</i> 2.5 and 2.1 μM, respectively). In the reverse titration, <i>α</i>NF fluorescence was quenched by P450 3A4; at the lower [<i>α</i>NF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (<i>K</i>_<i>d</i> 0.048 and 1.0 μM). Thus, four binding interactions of <i>α</i>NF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.</p

Formation of <i>S</i>‑[2‑(<i>N</i>⁶‑Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases

1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA, notably to sites on 2′-deoxyadenosine and 2′-deoxyguanosine (Cmarik, J. L., et al. (1991) J. Biol. Chem. 267, 6672−6679). Adduction at the N6 position of 2′-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of <i>O</i>⁶-alkylguanine DNA alkyltransferase (Chowdhury, G., et al. (2013) Angew. Chem. Int. Ed. 52, 12879−12882). We identified and quantified a new adduct, <i>S</i>-[2-(<i>N</i>⁶-deoxyadenosinyl)­ethyl]­GSH, in calf thymus DNA using LC-MS/MS. Replication studies were performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) η, ι, and κ, as well as with <i>Sulfolobus solfataricus</i> Dpo4, <i>Escherichia coli</i> polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols η and ι, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol η and ι showed increased misincorporation opposite the adduct compared to that of unmodified 2′-deoxyadenosine. LC-MS/MS analysis of full-length primer extension products by hPol η confirmed the incorporation of dC opposite <i>S</i>-[2-(<i>N</i>⁶-deoxyadenosinyl)­ethyl]­GSH and also showed the production of a −1 frameshift. These results reveal the significance of <i>N</i>⁶-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases

Effects of <i>N</i>²‑Alkylguanine, <i>O</i>⁶‑Alkylguanine, and Abasic Lesions on DNA Binding and Bypass Synthesis by the Euryarchaeal B‑Family DNA Polymerase Vent (exo^–)

Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo^–) from the euryarchaeon <i>Thermococcus litoralis</i> was studied with three types of DNA lesions<i>N</i>²-alkylG, <i>O</i>⁶-alkylG, and an abasic (AP) sitein comparison with a model Y-family pol Dpo4 from <i>Sulfolobus solfataricus</i>, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo^–) readily bypassed <i>N</i>²-methyl­(Me)­G and <i>O</i>⁶-MeG, but was strongly blocked at <i>O</i>⁶-benzyl­(Bz)­G and <i>N</i>²-BzG, whereas Dpo4 efficiently bypassed <i>N</i>²-MeG and <i>N</i>²-BzG and partially bypassed <i>O</i>⁶-MeG and <i>O</i>⁶-BzG. Vent (exo^–) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo^–) showed ∼110-, 180-, and 300-fold decreases in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) for nucleotide insertion opposite an AP site, <i>N</i>²-MeG, and <i>O</i>⁶-MeG but ∼1800- and 5000-fold decreases opposite <i>O</i>⁶-BzG and <i>N</i>²-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ∼13-fold decreases opposite <i>N</i>²-MeG and <i>N</i>²-BzG but ∼260–370-fold decreases opposite <i>O</i>⁶-MeG, <i>O</i>⁶-BzG, and the AP site. Vent (exo^–) preferentially misinserted G opposite <i>N</i>²-MeG, T opposite <i>O</i>⁶-MeG, and A opposite an AP site and <i>N</i>²-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo^–) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo^–) is as or more efficient as Dpo4 in synthesis opposite <i>O</i>⁶-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) <i>N</i>²-alkylGs than Vent (exo^–), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo^–) accepts nonbulky DNA lesions (e.g., <i>N</i>²- or <i>O</i>⁶-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove <i>N</i>²-alkylG lesions over major-groove or noninstructive lesions