34 research outputs found
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 P450sî—¸1A1, 1A2, 1B1, 2A6, 2E1, and 3A4î—¸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><sup>6</sup>‑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><sup>6</sup>-deoxyadenosinyl)-2,3-dihydroxybutyl]ÂGSH [<i>N</i><sup>6</sup>dA-(OH)<sub>2</sub>butyl-GSH] as a major adduct
in the reaction of <i>S</i>-(2-hydroxy-3,4-epoxybutyl)Âglutathione
(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><sup>6</sup>dA-(OH)<sub>2</sub>butyl-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><sup>6</sup>dA-(OH)<sub>2</sub>butyl-GSH adduct, <i>N</i><sup>6</sup>-(2,3,4-trihydroxybutyl)Âdeoxyadenosine adduct (<i>N</i><sup>6</sup>dA-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><sup>6</sup>dA-(OH)<sub>2</sub>butyl-GSH adduct, <i>N</i><sup>6</sup>dA-butanetriol adduct,
or unmodified dA for all polymerases, the dCTP misincorporation frequency
opposite <i>N</i><sup>6</sup>dA-(OH)<sub>2</sub>butyl-GSH
was significantly higher than that opposite the <i>N</i><sup>6</sup>dA-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><sup>6</sup>dA-(OH)<sub>2</sub>butyl-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 (FeO<sup>3+</sup>) and ferric peroxide (FeO<sub>2</sub><sup>–</sup>) 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 <sup>18</sup>O<sub>2</sub>, and the products were derivatized using a novel diazo reagent.
Analysis of the products by high-resolution mass spectrometry showed
a lack of <sup>18</sup>O 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 stereoisomersî—¸<i>R</i>,<i>R</i>, <i>S</i>,<i>S</i>, and <i>meso</i> (all formed by butadiene oxidation)î—¸with GSH by six GSH transferases.
Only small differences (<3-fold) were found in the catalytic efficiency
of conjugate formation (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) 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><sup>3</sup>-adenyl, <i>N</i><sup>6</sup>-adenyl, <i>N</i><sup>7</sup>-guanyl, <i>N</i><sup>1</sup>-guanyl, <i>N</i><sup>4</sup>-cytidyl,
and <i>N</i><sup>3</sup>-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><sup>6</sup>-Adenyl and <i>N</i><sup>7</sup>-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><sub><i>d</i></sub> 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><sub><i>d</i></sub> 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (<i>K</i><sub><i>d</i></sub> 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><sub><i>d</i></sub> 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><sup>6</sup>‑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><sup>6</sup>-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><sup>6</sup>-deoxyadenosinyl)Âethyl]ÂGSH, 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><sup>6</sup>-deoxyadenosinyl)Âethyl]ÂGSH and
also showed the production of a −1 frameshift. These results
reveal the significance of <i>N</i><sup>6</sup>-dA GSH-DBE
adducts in blocking replication, as well as producing mutations, by
human translesion synthesis DNA polymerases
Effects of <i>N</i><sup>2</sup>‑Alkylguanine, <i>O</i><sup>6</sup>‑Alkylguanine, and Abasic Lesions on DNA Binding and Bypass Synthesis by the Euryarchaeal B‑Family DNA Polymerase Vent (exo<sup>–</sup>)
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<sup>–</sup>) from the euryarchaeon <i>Thermococcus litoralis</i> was
studied with three types of DNA lesionsî—¸<i>N</i><sup>2</sup>-alkylG, <i>O</i><sup>6</sup>-alkylG, and an abasic
(AP) siteî—¸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<sup>–</sup>) readily bypassed <i>N</i><sup>2</sup>-methylÂ(Me)ÂG and <i>O</i><sup>6</sup>-MeG,
but was strongly blocked at <i>O</i><sup>6</sup>-benzylÂ(Bz)ÂG
and <i>N</i><sup>2</sup>-BzG, whereas Dpo4 efficiently bypassed <i>N</i><sup>2</sup>-MeG and <i>N</i><sup>2</sup>-BzG
and partially bypassed <i>O</i><sup>6</sup>-MeG and <i>O</i><sup>6</sup>-BzG. Vent (exo<sup>–</sup>) bypassed
an AP site to an extent greater than Dpo4, corresponding with steady-state
kinetic data. Vent (exo<sup>–</sup>) showed ∼110-, 180-,
and 300-fold decreases in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) for nucleotide insertion
opposite an AP site, <i>N</i><sup>2</sup>-MeG, and <i>O</i><sup>6</sup>-MeG but ∼1800- and 5000-fold decreases
opposite <i>O</i><sup>6</sup>-BzG and <i>N</i><sup>2</sup>-BzG, respectively, as compared to G, whereas Dpo4 showed
little or only ∼13-fold decreases opposite <i>N</i><sup>2</sup>-MeG and <i>N</i><sup>2</sup>-BzG but ∼260–370-fold
decreases opposite <i>O</i><sup>6</sup>-MeG, <i>O</i><sup>6</sup>-BzG, and the AP site. Vent (exo<sup>–</sup>)
preferentially misinserted G opposite <i>N</i><sup>2</sup>-MeG, T opposite <i>O</i><sup>6</sup>-MeG, and A opposite
an AP site and <i>N</i><sup>2</sup>-BzG, while Dpo4 favored
correct C insertion opposite those lesions. Vent (exo<sup>–</sup>) and Dpo4 both bound modified DNAs with affinities similar to unmodified
DNA. Our results indicate that Vent (exo<sup>–</sup>) is as
or more efficient as Dpo4 in synthesis opposite <i>O</i><sup>6</sup>-MeG and AP lesions, whereas Dpo4 is much or more efficient
opposite (only) <i>N</i><sup>2</sup>-alkylGs than Vent (exo<sup>–</sup>), irrespective of DNA-binding affinity. Our data also
suggest that Vent (exo<sup>–</sup>) accepts nonbulky DNA lesions
(e.g., <i>N</i><sup>2</sup>- or <i>O</i><sup>6</sup>-MeG and an AP site) as manageable substrates despite causing error-prone
synthesis, whereas Dpo4 strongly favors minor-groove <i>N</i><sup>2</sup>-alkylG lesions over major-groove or noninstructive lesions