Biomedical subjects

F Peter Guengerich

Publications and source records attributed to F Peter Guengerich.

At least 19 recordsLinked to original sources

PubMed2004

Selection of human cytochrome P450 1A2 mutants with enhanced catalytic activity for heterocyclic amine N-hydroxylation.

Cytochrome P450 (P450) 1A2 is the major enzyme involved in the metabolism of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) and other heterocyclic arylamines and their bioactivation to mutagens. Random mutant libraries of human P450 1A2, in which mutations were made throughout the entire open reading frame, were screened with Escherichia coli DJ3109pNM12, a strain designed to bioactivate MeIQ and detect mutagenicity of the products. Mutant clones with enhanced activity were confirmed using quantitative measurement of MeIQ N-hydroxylation. Three consecutive rounds of random mutagenesis and screening were performed and yielded a highly improved P450 1A2 mutant, SF513 (E225N/Q258H/G437D), with >10-fold increased MeIQ activation based on the E. coli genotoxicity assay and 12-fold enhanced catalytic efficiency (k(cat)/K(m)) in steady-state N-hydroxylation assays done with isolated membrane fractions. SF513 displayed selectively enhanced activity for MeIQ compared to other heterocyclic arylamines. The enhanced catalytic activity was not attributed to changes in any of several individual steps examined, including substrate binding, total NADPH oxidation, or H(2)O(2) formation. Homology modeling based on an X-ray structure of rabbit P450 2C5 suggested that the E225N and Q258H mutations are located in the F-helix and G-helix, respectively, and that the G437D mutation is in the "meander" region, apparently rather distant from the substrate. In summary, the approach generated a mutant enzyme with selectively elevated activity for a single substrate, even to the extent of a difference of a single methyl group, and several mutations had interacting roles in the development of the selected mutant protein.

Amino Acid Sequence↗

PubMed2004

Functional characterization of four allelic variants of human cytochrome P450 1A2.

Human cytochrome P450 1A2 catalyzes important reactions in xenobiotic metabolism, including the N-hydroxylation of carcinogenic aromatic amines. In 2001, Chevalier et al. reported four new P450 1A2 sequence variants in the human population. We have now expressed these variants in Escherichia coli and measured protein expression (optical spectroscopy of holoenzyme and immunoblotting) and bioactivation of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and MeIQ (2-amino-2,4-dimethylimidazo[4,5-f]quinoline) in the lacZ reversion mutagenicity test. Enzyme kinetic analyses were performed for N-hydroxylation of five heterocyclic amine substrates and for O-deethylation of phenacetin. The most drastic effect was that of the R431W substitution: no holoenzyme was detectable. This residue is located in the "meander" peptide region and earlier site-directed mutagenesis studies demonstrated that it is critical for maintenance of protein tertiary structure. The other three variants had subtly different catalytic activities compared to the wild-type enzyme.

Alleles↗

PubMed2004

S-(2-chloroethyl)glutathione-generated p53 mutation spectra are influenced by differential repair rates more than sites of initial dna damage.

Several steps occur between the reaction of a chemical with DNA and a mutation, and each may influence the resulting mutation spectrum, i.e. nucleotides at which the mutations occur. The half-mustard S-(2-bro-moethyl)glutathione is the reactive conjugate implicated in ethylene dibromide-induced mutagenesis attributed to the glutathione-dependent pathway. A human p53-driven Ade reporter system in yeast was used to study the factors involved in producing mutations. The synthetic analog S-(2-chloroethyl)glutathione was used to produce DNA damage; the damage to the p53 exons was analyzed using a new fluorescence-based modification of ligation-mediated polymerase chain reaction and an automated sequencer. The mutation spectrum was strongly dominated by the G to A transition mutations seen in other organisms with S-(2-chloroethyl)glutathione or ethylene dibromide. The mutation spectrum clearly differed from the spontaneous spectrum or that derived from N-ethyl,N-nitrosourea. Distinct differences were seen between patterns of modification of p53 DNA exposed to the mutagen in vitro versus in vivo. In the four p53 exons in which mutants were analyzed, the major sites of mutation matched the sites with long half-lives of repair much better than the sites of initial damage. However, not all slowly repaired sites yielded mutations in part because of the lack of effect of mutations on phenotype. We conclude that the rate of DNA repair at individual nucleotides is a major factor in influencing the mutation spectra in this system. The results are consistent with a role of N(7)-guanyl adducts in mutagenesis.

Alkylating Agents↗

PubMed2004

Formation and mass spectrometric analysis of DNA and nucleoside adducts by S-(1-acetoxymethyl)glutathione and by glutathione S-transferase-mediated activation of dihalomethanes.

The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A >> C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.

Animals↗

PubMed2003

Characterization of a mutagenic DNA adduct formed from 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase.

It has been proposed that the DNA repair protein O6-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s).

Animals↗

PubMed2003

Hydroperoxy-10,12-octadecadienoic acid stimulates cytochrome P450 3A protein aggregation by a mechanism that is inhibited by substrate.

We recently demonstrated that microsomes from nicardipine-treated rats will form cytochrome P450 3A (CYP3A) aggregates when incubated at 37 degrees C. CYP3A substrates inhibited the protein aggregation and subsequent degradation, suggesting that this process is important in substrate-mediated stabilization of CYP3A. In this paper, we demonstrate that oxidative stress is a key factor in the formation of CYP3A aggregates in incubated microsomes and in a reconstituted system with purified enzymes. Our data further suggest that the effects of oxidative stress are mediated by lipid hydroperoxides, which are efficiently metabolized by CYP3A. In the presence of substrate, the CYP3A-mediated lipid hydroperoxide metabolism is inhibited along with the associated protein aggregation. Therefore, these studies provide a mechanistic model of why CYP3A has a relatively short half-life and how substrates stabilize CYP3A.

Animals↗

PubMed2003

O6-alkylguanine-DNA alkyltransferase: low pKa and high reactivity of cysteine 145.

The active site cysteine of human O(6)-alkylguanine-DNA alkyltransferase (hAGT), Cys145, was shown to be highly reactive with model electrophiles unrelated to substrates, including 1-chloro-2,4-dinitrobenzene. The high reactivity suggested that the Cys145 thiolate anion might be stable at neutral pH. The pK(a) was estimated from plots of UV spectra (A(239)) and reactivity toward 4,4'-dithiopyridine vs pH. The estimated pK(a) for hAGT was 4-5, depending upon the method used, and near that of the extensively characterized papain Cys25. Rates of reaction with 4,4'-dithiopyridine were similar for the thiolate forms of hAGT, papain, glutathione, and the bacterial hAGT homologue Ogt (the pK(a) of the latter was 5.4). Bound Zn(2+) has previously been shown to be required for the catalytic activity of hAGT (Rasimas, J. J. et al. (2003) Biochemistry 42, 980-990). Zn(2+) was shown to be required for the low pK(a) of hAGT. The high reactivity of hAGT Cys145 is postulated to be important in normal catalytic function, in cross-linking reactions involving bis-electrophiles, and in inhibition of the DNA repair function of hAGT by electrophiles.

Anions↗

PubMed2003

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis.

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.

Amines↗

PubMed2003

Roles of human liver cytochrome P450 3A4 and 1A2 enzymes in the oxidation of myristicin.

The aim of this work was to identify the form(s) of human liver cytochrome P450 (CYP) involved in the hepatic transformation of myristicin to its major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene. When microsomes prepared from different human liver samples were compared, the activity of 5-allyl-1-methoxy-2,3-dihydroxybenzene formation was well correlated (r(2)=0.87) with nifedipine oxidation (a marker of CYP3A4). With a microsomal sample having high CYP3A4 activity, microsomal oxidation of myristicin to the major metabolite (5-allyl-1-methoxy-2,3-dihydroxybenzene) was markedly inhibited by gestodene and ketoconazole, selective inhibitors of CYP3A enzymes, but not by any of several other P450 inhibitors. Antibodies raised against CYPs 3A4 and 1A2 could also inhibit the oxidation of myristicin, but antibodies recognizing other CYPs had no effect. The oxidation of myristicin to 5-allyl-1-methoxy-2,3-dihydroxybenzene was catalyzed by purified bacterial recombinant CYPs 3A4 and 1A2. These results provide evidence that CYP3A4 (and possibly other CYP3A enzymes) and CYP1A2 play roles in the formation of the major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene.

Alkenes↗

PubMed2003

Activation of dihaloalkanes by thiol-dependent mechanisms.

Dihaloalkanes constitute an important group of chemicals because of their widespread use in industry and agriculture and their potential for causing toxicity and cancer. Chronic toxic effects are considered to depend upon bioactivation, either by oxidation or thiol conjugation. Considerable evidence links genotoxicity and cancer with glutathione conjugations reactions, and some aspects of the mechanisms have been clarified with 1,2-dihaloalkanes and dihalomethanes. Recently the DNA repair protein O6-alkylguanine transferase has been shown to produce cytotoxicity and genotoxicity by means of a thiol-dependent process with similarities to the glutathione reactions.

DNA Adducts↗

PubMed2003

Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions.

Much of the interest in the cytochrome P450 (P450) enzymes has been because of oxidation of chemicals to reactive products. The epoxides (oxiranes) have been a major topic of interest with olefins and aryl compounds. Epoxides vary considerably in their reactivity, with t(1/2) varying from 1s to several hours. The stability and reactivity influences not only the overall damage to biological systems but also the site of injury. Transformations of some xenobiotic chemicals may involve products other than epoxides. Chemicals considered here include olefins, aromatic hydrocarbons, heterocycles, vinyl halides, ethyl carbamate, vinyl nitrosamines, and aflatoxin B(1). These compounds either are unsaturated or are transformed to unsaturated products. The epoxides and other products provide a view of the landscape of P450-generated reactive products and the myriad of chemistry involved in the metabolism of drugs and protoxicants. Understanding the chemical nature of reactive products is necessary to develop rational strategies for intervention.

Aflatoxin B1↗

PubMed2003

Analysis of the kinetic mechanism of haloalkane conjugation by mammalian theta-class glutathione transferases.

Glutathione (GSH) transferases (GSTs) catalyze the conjugation of small haloalkanes with GSH. In the case of dihalomethanes and vic-1,2-dihaloalkanes, the reaction leads to the formation of genotoxic GSH conjugates. A generally established feature of the reaction of the mammalian theta-class GSTs, which preferentially catalyze these reactions, is the lack of saturability of the rate with regard to the substrate concentration. However, the bacterial GST DM11 catalyzes the same reactions with a relatively low K(m). Recently, DM11 has been shown to exhibit burst kinetics, with a rate-determining k(off) rate for product (Stourman et al. (2003) Biochemistry 42, 11048-11056). We examined rat GST 5-5 and human GST T1-1 and did not detect any burst kinetics in the conjugation of C(2)H(5)Cl, CH(2)Br(2), or CH(2)Cl(2), distinguishing these enzymes from GST DM11. The kinetic results were fit to a minimal mechanism in which the rate-limiting step is halide displacement. The differences in the steady state kinetics of conjugations catalyzed by bacterial GST DM11 and the mammalian GSTs 5-5 and T1-1 are concluded to be the result of differences in the rate-limiting steps and not to inherent enzyme affinity for the haloalkanes. The results may be interpreted in the context of a model in which the halide order affects the rate of carbon-halogen bond cleavage of all such reactions catalyzed by the GSTs. With GST DM11, the halide order is manifested in the K(m) parameter but not k(cat). With mammalian GSTs, the high K(m) is difficult to estimate. With all of the GSTs, the halide order is seen in the enzyme efficiency, k(cat)/K(m), with C-Br cleavage approximately 10-fold faster than C-Cl cleavage. The ratio k(cat)/K(m) is the most relevant parameter for issues of risk assessment.

Alkanes↗

PubMed2003

Diterpene quinone tanshinone IIA selectively inhibits mouse and human cytochrome p4501A2.

1. Tanshinone IIA is the main active diterpene quinone in the herbal medicine Salvia miltiorrhiza. In untreated mouse liver microsomes, tanshinone IIA selectively inhibited 7-ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, N-nitrosodimethylamine and nifedipine. Tanshinone IIA was a competitive inhibitor of MROD activity with a K(i) of 7.2 +/- 0.7 nM. 2. In 3-methylcholanthrene-treated mouse liver microsomes, tanshinone IIA and two minor tanshinones, tanshinone I and cryptotanshinone, inhibited liver microsomal MROD activity without affecting EROD and benzo(a)pyrene hydroxylation activities at the concentrations up to 1 microM. Tanshinone IIA induced a type I binding spectrum with a spectral dissociation constant K(s) of 2.3 +/-0.8 microM without cooperativity. 3. In human liver microsomes, tanshinone IIA decreased EROD and MROD activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, chlorzoxazone and nifedipine. 4. In Escherichia coli membranes expressing bicistronic human CYP1A enzymes, tanshinone IIA inhibited EROD activity of CYP1A1 with an IC(50) 48 times higher than that for CYP1A2. Tanshinone I and cryptotanshinone had the same IC(50) ratio (1A1/1A2) of 4. 5. The results indicate that tanshinone represents a new group of CYP1A inhibitors, and tanshinone IIA had the highest selectivity in inhibition of CYP1A2.

Abietanes↗

PubMed2003

Decreased coumarin 7-hydroxylase activities and CYP2A6 expression levels in humans caused by genetic polymorphism in CYP2A6 promoter region (CYP2A6*9).

One of seven poor metabolizers of coumarin found in Thai subjects was previously genotyped as heterozygote for the CYP2A6*4 (whole deletion) and CYP2A6*9. Thus, we aimed to investigate the relationship between the genetic polymorphism in the TATA box of the CYP2A6 gene (CYP2A6*9), expression levels of CYP2A6 mRNA and coumarin 7-hydroxylase activities in human livers. Levels of CYP2A6 mRNA were quantified by real-time quantitative reverse transcriptase-polymerase chain reaction. The mean expression levels of CYP2A6 mRNA in individuals with CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 58%, 71% and 21% of the individuals genotyped as CYP2A6*1/*1, respectively. The mean in-vitro coumarin 7-hydroxylase activities in subjects carrying CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 41%, 71% and 12%, respectively, compared to those of the subjects judged as wild-type. Vmax values for coumarin 7-hydroxylation in the liver microsomes from human subjects with genotypes of CYP2A6*1/*1, CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 0.58, 0.26, 0.44 and 0.13 nmol/min/nmol total P450, respectively. CYP2A6 protein levels in human liver microsomes with the CYP2A6*4 and the CYP2A6*9 alleles were markedly decreased. These results suggest that the genetic polymorphism in the promoter region of the CYP2A6 gene (CYP2A6*9) reduced the expression levels of CYP2A6 mRNA and protein in human livers, resulting in the decrease of coumarin 7-hydroxylase activities. Individuals judged as CYP2A6*4/*9 were expected to be poor metabolizers, having extremely low activity of CYP2A6.

Adult↗

PubMed2002

Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity.

Anticarcinogenic Agents↗

PubMed2002

Metabolism of heterocyclic aromatic amines by human hepatocytes and cytochrome P4501A2.

The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was investigated in primary human and rat hepatocytes. The genotoxic metabolites 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which are formed by cytochrome P4501A2 (CYP1A2), were detected as stable N(2)-glucuronide and N(2)- and N(3)-glucuronide conjugates, respectively. These products accounted for as much as 10% of the amount of MeIQx and 60% of PhIP added to human hepatocytes. Significantly lower amounts of these products were formed in rat hepatocytes. The phase II conjugates N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl-sulfamic acid (MeIQx-N(2)-SO(3)H) and N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx-N(2)-Gl), as well as the 7-oxo derivatives of MeIQx and N-desmethyl-MeIQx, 2-amino-3,8-dimethyl-6-hydro-7H-imidazo[4,5-f]quinoxalin-7-one (7-oxo-MeIQx), and 2-amino-6-hydro-8-methyl-7H-imidazo[4,5-f]quinoxalin-7-one (N-desmethyl-7-oxo-MeIQx) were also identified. A novel CYP1A2-derived metabolite was characterized as 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH) and was the predominant metabolite formed in human hepatocytes exposed to MeIQx at levels approaching human exposure. Unlike human hepatocytes, rat cell preparations, even following pretreatment with the potent CYP1A1/CYP1A2 inducer 3-methylcholanthrene (3-MC) did not produce IQx-8-COOH but did catalyze the formation of 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline (5-HO-MeIQx) as a major CYP-mediated detoxication product. In the case of PhIP, direct glucuronidation of the N(2) and N(3) positions also occurred in human and rat hepatocytes. Glucuronide and sulfate conjugates of 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-HO-PhIP) were detected as relatively minor metabolites in human hepatocytes but were the major products formed in rat hepatocytes, accounting for up to 50% of the metabolism. Rat CYP1A2, but not the human ortholog, significantly contributes to 4'-hydroxylation of PhIP. Important differences exist between human and rat liver enzymes in catalytic activity and regioselectivity of MeIQx and PhIP metabolism. Some human hepatocyte preparations are more active at transforming MeIQx and PhIP to a genotoxic species than rat hepatocytes pretreated with potent inducer 3-MC. These pronounced interspecies differences in metabolism of MeIQx and PhIP may affect the biological activity of these mutagens and must be considered when assessing human health risk.

Animals↗

PubMed2002

Diversity in the oxidation of substrates by cytochrome P450 2D6: lack of an obligatory role of aspartate 301-substrate electrostatic bonding.

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions with substrates on the basis of homology modeling and site-directed mutagenesis experiments [Ellis, S. W., Hayhurst, G. P., Smith, G., Lightfoot, T., Wong, M. M. S., Simula, A. P., Ackland, M. J., Sternberg, M. J. E., Lennard, M. S., Tucker, G. T., and Wolf, C. R. (1995) J. Biol. Chem. 270, 29055-29058]. However, pharmacophore models based on the role of Asp301 in substrate binding are compromised by reports of catalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. We characterized a high-affinity ligand for P450 2D6, also devoid of a basic nitrogen atom, spirosulfonamide [4-[3-(4-fluorophenyl)-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl]benzenesulfonamide], with K(s) 1.6 microM. Spirosulfonamide is a substrate for P450 2D6 (k(cat) 6.5 min(-)(1) for the formation of a syn spiromethylene carbinol, K(m) 7 microM). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the binding of spirosulfonamide (K(s) 2.5-3.5 microM). However, the hydroxylation of spirosulfonamide was attenuated in these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effect of the D301N substitution was manifested on k(cat) but not K(m). Analogues of spirosulfonamide were also evaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to an amide, thioamide, methyl sulfone, or hydrogen were ligands with K(s) values of 1.7-32 microM. All were substrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue of the major spirosulfonamide product. The D301N mutation produced varying changes in the oxidation patterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone and testosterone had been reported to be substrates for P450 2D6, but the affinities (K(s)) were unknown; these were estimated to be 1.2, 1.5, and 15 microM, respectively (cf. 6 microM for the classic substrate bufuralol). The results are consistent with a role of Asp301 other than electrostatic interaction with a positively charged ligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our results show that it is not required for all substrates and explain why predictive models fail to recognize the proclivity for many substrates, especially those containing no basic nitrogen.

Amines↗

PubMed2002

Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase.

The presence of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) paradoxically increases the mutagenicity and cytotoxicity of 1,2-dibromoethane (DBE) in Escherichia coli. This enhancement of genotoxicity did not occur when the inactive C145A mutant of human AGT (hAGT) was used. Also, hAGT did not enhance the genotoxicity of S-(2-haloethyl)glutathiones that mimic the reactive product of the reaction of DBE with glutathione, which is catalyzed by glutathione S-transferase. These experiments support a mechanism by which hAGT activates DBE. Studies in vitro showed a direct reaction between purified recombinant hAGT and DBE resulting in a loss of AGT repair activity and a formation of an hAGT-DBE conjugate at Cys(145). A 2-hydroxyethyl adduct was found by mass spectrometry to be present in the Gly(136)-Arg(147) peptide from tryptic digests of AGT reacted with DBE. Incubation of AGT with DBE and oligodeoxyribonucleotides led to the formation of covalent AGT-oligonucleotide complexes. These results indicate that DBE reacts at the active site of AGT to generate an S-(2-bromoethyl) intermediate, which forms a highly reactive half-mustard at Cys(145). In the presence of DNA, the DNA-binding function of AGT facilitates formation of DNA adducts. In the absence of DNA, the intermediate undergoes hydrolytic decomposition to form AGT-Cys(145)-SCH(2)CH(2)OH.

Amino Acid Substitution↗