Biomedical subjects

F Peter Guengerich

Publications and source records attributed to F Peter Guengerich.

At least 73 records · Page 4Linked to original sources

PubMed2004

Expression of cytochromes P450 1A1 and 1B1 in human lung from smokers, non-smokers, and ex-smokers.

Cytochromes P450 1A1 and 1B1 are known to bioactivate procarcinogens such as polycyclic aromatic hydrocarbons (PAHs) found in cigarette smoke and are inducible via an Ah receptor-mediated mechanism. The aim of this study was to examine the levels of expression of CYP1A1 and CYP1B1 in samples of lung from smokers (n = 18), non-smokers (n = 7), and ex-smokers (n = 7). Using immunoglobulin preparations of highly specific polyclonal antibodies and immunoblot analysis of microsomes from lung tissues, we determined the specific content for CYP1A1 and CYP1B1. For CYP1A1, we found median expression levels of 15.5 pmol/mg microsomal protein in smokers, 6.0 pmol/mg microsomal protein in non-smokers, and 19.0 pmol/mg microsomal protein in ex-smokers. The difference in median expression levels of smokers and ex-smokers compared to non-smokers was statistically significant. For CYP1B1, we found median expression levels of 1.8 pmol/mg microsomal protein in smokers, 1.0 pmol/mg microsomal protein in non-smokers, and 4.4 pmol/mg microsomal protein in ex-smokers. The difference in median expression levels between ex-smokers and non-smokers was statistically significant. These results suggest that levels of expression of CYP1A1 and CYP1B1 protein in lung tissues from smokers and ex-smokers are quantitatively greater than in non-smokers. By immunohistochemical analysis, we demonstrated the expression of CYP1A1 and CYP1B1 in normal human alveolar type I and II cells, ciliated columnar epithelial cells lining bronchoalveolar airways, and alveolar macrophages. These results confirm that CYP1A1 is expressed in normal human lung, appears to be induced in smokers, and show interindividual variation; the similar characteristics of CYP1B1 are demonstrated.

Adolescent↗

PubMed2004

Rate-limiting steps in oxidations catalyzed by rabbit cytochrome P450 1A2.

Several issues regarding the rate-limiting nature of individual reaction steps in catalysis by rabbit liver cytochrome P450 (P450) 1A2 were addressed using anisoles and other substrates. Substrate binding is very fast (k > 10(6) M(-1) s(-1)). Product release is not rate-limiting, as shown by the absence of bursts, placing rate-limiting steps at or before product formation. We had previously shown that the first 1-electron reduction step is fast (k > 700 min(-1)), even in the absence of ligand [Guengerich, F. P., and Johnson, W. W. (1997) Biochemistry 36, 14741-147500]. O(2) binding to ferrous P450 is fast (k >/= 10(6) M(-1) s(-1)). The decay of the P450 Fe(2+)-substrate-O(2) complex was slow in the absence of NADPH-P450 reductase, with a first-order rate constant of 14 min(-1) at 25 degrees C. During the decay, product was formed (from the substrate methacetin) in 61% theoretical yield, although this reaction requires electron transfer among P450 molecules and may not be related to normal turnover. Steady-state spectra suggest that one or more iron-oxygen complexes accumulate, representing entities between the Fe(2+)-O(2) complex and putative FeO(3+) entity. Kinetic isotope effect experiments were done with several substrates, mainly anisoles. Apparent intrinsic deuterium isotope effects as high as 15 were measured. In all cases, the C-H bond-breaking step is at least partially rate-limiting. The isotope effects were not strongly attenuated in noncompetitive or competitive experiments, consistent with relatively rapid P450-substrate exchange, except with the active enzyme Fe-O complex. Kinetic simulations with the available data (i) are consistent with the view that C-H bond breaking is a major rate-limiting step, (ii) demonstrate that increasing the rate of this step will affect k(cat), K(m), and kinetic hydrogen isotope effects but will only increase catalytic efficiency to a certain degree, (iii) indicate that increasing ground-state binding can increase catalytic efficiency but not k(cat), and (iv) suggest that nonproductive binding modes and abortive reduction of O(2) are factors that attenuate catalytic efficiency.

Acetamides↗

PubMed2004

Generation of new protein kinase inhibitors utilizing cytochrome p450 mutant enzymes for indigoid synthesis.

Indigoids, a class of bis-indoles, represent a promising protein kinase inhibitor scaffold. Oxidation of indole by cytochrome P450 (P450) has been shown to generate species (indoxyl, isatin) that couple to yield indigo and indirubin. Escherichia coli-expressed human P450 2A6 mutants isolated from a randomized library were incubated with 27 substituted indole derivatives. Extracts of the cultures were screened for inhibition of human cyclin-dependent kinases (CDK)-1 and -5 and glycogen synthase kinase-3 (GSK3). The extracts from cultures incubated with 5-methoxyindole were the most inhibitory. High-performance liquid chromatography (HPLC) separation yielded a mixture of seven colored indigoids. These indigoids included indigo, indirubin, the di(5-methoxy) derivatives of indigo and indirubin, and both of the possible mono 5-methoxy derivatives of indirubin, which were all identified by visible, mass, and NMR spectra. Cultures with 5-methylindole added to the media also yielded inhibitory material, and 5- and 5'-methylindirubin were characterized. The most inhibitory of these indigoids were the monosubstituted indirubins and 5,5'-dimethoxyindirubin, which was > or =10x more active than indirubin. Thus, the overall approach involves the use of a library of randomized enzyme mutants to activate component moieties of a desired set of larger molecules, thus yielding a library of drug candidates that can be screened and characterized. The general strategy may have additional applications.

Aryl Hydrocarbon Hydroxylases↗

PubMed2004

The human CYP1A1 gene is regulated in a developmental and tissue-specific fashion in transgenic mice.

Regulation and expression of human CYP1A1 is demonstrated in transgenic mice. We have developed two transgenic mouse lines. One mouse strain (CYPLucR) carries a functional human CYP1A1 promoter (-1612 to +293)-luciferase reporter gene, and the other strain (CYP1A1N) expresses CYP1A1 under control of the full-length human CYP1A1 gene and 9 kb of flanking regulatory DNA. With CYPLucR(+/-) mice, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and several other aryl hydrocarbon receptor ligands induced hepatocyte-specific luciferase activity. When other tissues were examined, TCDD induced luciferase activity in brain with limited induction in lung and no detectable luciferase activity in kidney. Treatment of CYP1A1N(+/-) mice with TCDD resulted in induction of human CYP1A1 in liver and lung, while mouse Cyp1a1 was induced in liver, lung, and kidney. Although induced CYP1A1/Cyp1a1 could not be detected by Western blot analysis in brains from CYP1A1N(+/-) mice, induction in brain was verified by detection of CYP1A1/Cyp1a1 RNA. The administration of TCDD to nursing mothers to examine the effect of lactational exposure via milk demonstrated prominent induction of luciferase activity in livers of CYPLucR(+/-) newborn pups with limited induction in brain. However, TCDD treatment of adult CYPLucR(+/-) mice led to a 7-10-fold induction of brain luciferase activity. Combined these results indicate that tissue-specific and developmental factors are controlling aryl hydrocarbon receptor-mediated induction of human CYP1A1.

Animals↗

PubMed2004

Aryl hydrocarbon receptor response to indigoids in vitro and in vivo.

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.

Animals↗

PubMed2004

Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase.

The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease(-) (T7(-)). RT and T7(-) effectively bypassed N(2)-methyl(Me)G and readily extended primers but were strongly blocked by N(2)-ethyl(Et)G, N(2)-isobutylG, N(2)-benzylG, and N(2)-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7(-) showed a decrease of 10(3) in k(cat)/K(m) for dCTP incorporation opposite N(2)-MeG and a further large decrease opposite N(2)-EtG. Misincorporation frequency was increased 10(2)-10(3)-fold by a Me group and another approximately 10(3)-fold by an Et group. dATP was preferentially incorporated opposite bulky N(2)-alkylG molecules. N(2)-MeG attenuated the pre-steady-state kinetic bursts with RT and T7(-), and N(2)-EtG eliminated the bursts. Large elemental effects with thio-dCTP(alphaS) were observed with N(2)-EtG (6- and 72-fold decreases) but were much less with N(2)-MeG, indicating that the N(2)-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of K(d(dCTP)) and K(d(DNA)) and k(off) rates of DNA substrates from RT and T7(-) indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.

Alkylation↗

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↗

PubMed2004

Formation and reduction of aryl and heterocyclic nitroso compounds and significance in the flux of hydroxylamines.

Cytochrome p450 (p450) 1A2 and NADPH-P450 reductase (NPR) catalyzed the oxidation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), with consumption of NADPH. The oxidation rate of NADPH by p450 1A2/NPR increased with time in the presence of IQ until depletion of NADPH. This unusual autocatalytic pattern of NADPH oxidation could be rationalized by formation of a nitroso derivative (IQ-N=O) and the subsequent reduction of the hydroxylamine (IQ-NHOH) and IQ-N=O, which would consume more NADPH. The formation of IQ-NHOH and IQ-N=O from IQ was confirmed using HPLC/MS. Reduction of IQ-NHOH and IQ-N=O was NPR-dependent but did not require p450. Autocatalytic NADPH oxidation was also observed in the oxidation of other heterocyclic and arylamines. However, the N-hydroxyl and nitroso oxidation products of 2-aminofluorene and 4-aminobiphenyl were reduced nonenzymatically by NADPH, and NPR did not catalyze the reactions. We simulated the enzymatic kinetic model for possible pathways for IQ metabolism, which included the formation of IQ-N=O, using some kinetic parameters obtained from the experimental results. In the kinetic model, we could reproduce the similar curvature for NADPH oxidation and the formation of IQ-N=O, and the reduction of IQ-NHOH and IQ-N=O is required to explain the observed results for NADPH oxidation. Our results support a role for nitroso derivatives of HAAs in the unusual autocatalytic NADPH oxidation and may have relevance in terms of possible toxicities of the nitroso derivatives. Both IQ-NHOH and IQ-N=O were mutagenic in a bacterial tester system devoid of p450 and NPR; the mutagenicity of both was decreased by expression of NPR, consistent with the reduction of these compounds observed with purified NPR.

Carcinogens↗

PubMed2004

Human cytochromes P450 1A1 and 1B1 catalyze ring oxidation but not nitroreduction of environmental pollutant mononitropyrene isomers in primary cultures of human breast cells and cultured MCF-10A and MCF-7 cell lines.

The position of the nitro group determines the relative carcinogenic activities of mono-nitropyrene isomers (mono-NPs) in the rat mammary gland. To determine whether the results obtained in rodents treated with these environmental pollutants can be applicable to humans, we examined their metabolic activation in primary cultures of human breast cells derived from reduction mammoplasty, as well as in the cultured human breast cancer cell line MCF-7 and the immortalized human mammary epithelial cell line MCF-10A. Primary cultures as well as cell lines were competent in metabolizing all three isomers via both ring oxidation and nitro reduction pathways. Qualitatively similar metabolic patterns were observed but quantitative differences were evident. On the basis of cochromatography with synthetic standards in two HPLC systems, metabolites of 1-NP were identified as 1-OH-Py, 3-, 6-, and 8-OH-1-NP and 1-AP. In the case of 2-NP, 6-OH-2-NP and 2-AP were identified. 4-NP was metabolized to 9,10-DHD-4-NP, Py-4,5-Q, 9,10-Q-4-NP, 9/10-OH-4-NP, 6/ 8-OH-4-NP, and 4-AP. Varying degrees of sulfate and glucuronide conjugation of mono-NP metabolites were detected. In MCF-7 cells, we found that 1-, 2-, and 4-NP bind to DNA at levels of 68, 17, and 132 pmol/mg DNA, respectively. Following HPLC analysis of the DNA hydrolysates, we detected multiple DNA adducts including those derived from nitro reduction of 2- and 4-NP; however, none was detected in the case of 1-NP. To determine the P450 enzymes responsible for the metabolic activation of these carcinogens, we incubated [(3)H]mono-NPs with recombinant human P450 1A1 or 1B1. Metabolites identified were primarily derived from ring oxidation; both P450s 1A1 and 1B1 yielded similar metabolic profiles. This is the first report demonstrating that human breast (target organ) cells, immortalized human mammary epithelial cell line MCF-10A, and breast cancer cell line MCF-7 are capable of activating mono-NPs to metabolites that can damage DNA.

Animals↗

PubMed2004

Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase.

O(6)-Alkylguanine DNA-alkyl transferase (AGT) has been shown to conjugate both 1,2-dibromoethane and dibromomethane, yielding AGT inactivation, DNA-AGT cross-linking, and enhanced mutagenicity. A variety of related chemicals were examined to determine if similar phenomena occur. Among the compounds examined in these systems (histidine operon reversion in Escherichia coli and Salmonella typhimurium tester strains), a strong halide order was generally observed, with increasing activities in the order I > Br >> Cl. At least one Br atom appeared to be required for human AGT-dependent mutations, and compounds with only Cl did not inhibit AGT and were not activated to genotoxins. Of a series of haloforms tested (CHX(3), X = Br or Cl), all were without effect. Among a series of alpha,omega-disubstituted dihaloalkanes (Br or I), the inactivation of AGT increased with methylene chain length (at least up to n = 5) but the most mutagenic activity (AGT-dependent) was seen with n = 1-3. The effects with n = 1 or 2 were expected from previous results; the mutagenic effect with n = 3 and the reduction with n > 3 may represent a balance between AGT reaction, stability, and reactivity, in the absence of anchimeric assistance. A strong AGT-dependent mutation was observed for 1,3-butadiene diepoxide. We conclude that numerous bis-electrophiles show AGT-dependent activation to mutagenic conjugates. Haloforms and dichloroalkanes are therefore not an issue, but bromohaloalkanes and 1,3-butadiene diepoxide are potential problems. These observations are of relevance in considering toxicity and risks of some chemicals used in industrial applications.

Biotransformation↗

PubMed2004

O6-alkylguanine-DNA alkyltransferase has opposing effects in modulating the genotoxicity of dibromomethane and bromomethyl acetate.

O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes O(6)-alkylguanine adducts. The interaction of dibromomethane (CH(2)Br(2)) and bromomethyl acetate (BrCH(2)OAc) with AGT was studied in vitro, and the effect of AGT on their toxicity and mutagenicity was investigated using Escherichia coli strain TRG8 (lacking endogenous AGT) that expressed human AGT or its inactive C145A mutant. Both CH(2)Br(2) and BrCH(2)OAc reacted with AGT at its cysteine acceptor site, abolishing its DNA repair activity with the latter agent being much more potent. The formation of AGT-Cys(145)S-CH(2)OAc by BrCH(2)OAc was confirmed by mass spectral analysis, but the presumed AGT-Cys(145)S-CH(2)Br adduct from CH(2)Br(2) was too unstable for such characterization. In the presence of CH(2)Br(2), AGT was covalently cross-linked to an oligodeoxyribonucleotide, 5'-d(AG)(8)-3', but no cross-link was formed by BrCH(2)OAc. Survival of cells exposed to CH(2)Br(2) was reduced, and the number of mutants was greatly increased when wild-type AGT was present. The cytotoxicity of CH(2)Br(2) was similar to that of BrCH(2)CH(2)Br(2), but the mutagenicity was about four times less. Virtually all of the AGT-mediated mutants induced by CH(2)Br(2) in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A. In contrast, BrCH(2)OAc was more than 10-fold less genotoxic than CH(2)Br(2) and the survival of cells exposed to BrCH(2)OAc was not affected by AGT. The number of mutations (almost all G:C to A:T transitions) induced by BrCH(2)OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant. These results with CH(2)Br(2) are consistent with a mechanism in which reaction at the active site Cys145 residue followed by attack of AGT-Cys(145)S-CH(2)Br at guanine in DNA forms a covalent adduct, which leads to cytotoxicity and to mutagenicity. The results with BrCH(2)OAc suggest that it reacts directly with DNA to form O(6)-(CH(2)OAc)guanine, which, if unrepaired, causes G:C to A:T transitions. Our experiments reveal two novel pathways (direct inactivation of AGT and formation of AGT-Cys(145)S-CH(2)-DNA adducts) by which CH(2)Br(2) may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs.

DNA Damage↗

PubMed2004

Cytochrome P450: what have we learned and what are the future issues?

The cytochrome P450 (P450) field came out of interest in the metabolism of drugs, carcinogens, and steroids, which remain major focal points. Over the years we have come to understand the P450 system components, the multiplicity of P450s, and many aspects of the regulation of the genes and also the catalytic mechanism. Many crystal structures are now becoming available. The significance of P450 in in vivo metabolism is appreciated, particularly in the context of pharmacogenetics. Current scientific issues involve posttranslational modification, gene regulation, component interactions, structures of P450 complexed with ligands, details of high-valent oxygen chemistry, the nature and influence of rate-limiting steps, greater details about some reaction steps, cooperativity, and the relevance of P450 variations to cancer risk. Some emerging research areas involve new methods of analysis of ligand interactions, roles of conformational changes linked to individual reaction steps, functions of orphan P450s, "molecular breeding" of new P450 functions and enhanced activity, and the utilization of P450s in chemical synthesis.

Animals↗

PubMed2004

Where is toxicology headed in the future?

Herbert Remmer was a pioneer in the study of the phenomenon of enzyme induction. He was also a leading figure, if not the foremost, in the development of the discipline of toxicology in Germany during his tenure as Professor of Toxicology at the University of Tübingen. Included here are some brief thoughts about where toxicology came from, where it is today, and what the future is. Toxicology is at a crossroads today, at an interface between basic science and applied projects. From its past as a descriptive discipline, it has incorporated a medley of concepts and technology from basic science. The usefulness of many approaches is now being evaluated. The hope is that toxicology will be able to be much more predictive in the future; a great need exists in the pharmaceutical industry. The shape of academic toxicology is also changing.

Animals↗

PubMed2004

Testosterone 1 beta-hydroxylation by human cytochrome P450 3A4.

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6 beta-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6 beta- and 2 beta-hydroxy products, was identified as 1 beta-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of approximately 6 microg of this product. Mass spectrometry established the formula as C(19)H(29)O(3) (MH(+) 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the beta-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1 alpha-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.

Animals↗

PubMed2004

Different in vitro metabolism of paclitaxel and docetaxel in humans, rats, pigs, and minipigs.

We investigated cytochrome P450 (P450)-catalyzed metabolism of the important cancer drugs paclitaxel and docetaxel in rat, pig, minipig, and human liver microsomes and cDNA-expressed P450 enzymes. In rat microsomes, paclitaxel was metabolized mainly to C3'-hydroxypaclitaxel (C3'-OHP) and to a lesser extent to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP), and another unknown monohydroxylated paclitaxel. In pig and minipig microsomes, this unknown hydroxypaclitaxel was the main metabolite, whereas C3'-OHP was a minor product. In minipigs, C2-OHP was the next minor product. In human liver microsomes, 6 alpha-hydroxypaclitaxel (6 alpha-OHP) was the main metabolite, followed by C3'-OHP and C2-OHP. Among different cDNA-expressed human P450 enzymes (CYP1A2, 1B1, 2A6, 2C9, 2E1, and 3A4), only CYP3A4 enzyme formed C3'-OHP and C2-OHP. Docetaxel was metabolized in pig, minipig, rat, and human liver microsomes mainly to hydroxydocetaxel (OHDTX), whereas CYP3A-induced rat microsomes produced primarily diastereomeric hydroxyoxazolidinones. Human liver microsomes from 10 different individuals formed OHDTX at different rates correlated with CYP3A4 content. Troleandomycin as a selective inhibitor of CYP3A inhibited the formation of C3'-OHP, C2-OHP, and di-OHP, as well as the unknown OHP produced in rat, minipig, and pig microsomes. In human liver microsomes, troleandomycin inhibited C3'-OHP and C2-OHP formation, and a suitable inhibitor of human CYP2C8, fisetin, strongly inhibited the formation of 6 alpha-OHP, known to be catalyzed by human CYP2C8. In conclusion, the metabolism of docetaxel is the same in all four species, but metabolism of paclitaxel is different, and 6 alpha-OHP remains a uniquely human metabolite. Pigs and minipigs compared with each other formed the same metabolites of paclitaxel.

Adolescent↗