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PubMedDate not supplied

Reaction of a chemotherapeutic agent, 6-mercaptopurine, with a direct-acting, electrophilic carcinogen, benzo[a]pyrene-7,8-diol 9,10-epoxide.

The chemotherapeutic agent 6-mercaptopurine (6-MP) has been shown to react covalently with the ultimate carcinogenic metabolite of benzo[a]pyrene, 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), in aqueous solution, forming a single adduct. NMR studies of the HPLC-purified product were consistent with its identification as 10(S)-(6'-mercaptopurinyl)-7,8,9-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene. Reaction kinetics were analyzed by using both HPLC separation of the products formed and a spectrophotometric assay for adduct formation. A simple model in which direct reaction between 6-MP and BPDE takes place without formation of a physical complex was found to adequately predict the dependence of product ratios on 6-MP concentration. Variations in the observed rate constant for this reaction with changes in temperature, pH, and buffer concentration were determined and compared to the effects of these variables on the observed rate constant for BPDE hydrolysis. In each case, the processes were affected quite differently, suggesting that different rate-determining steps are involved. The data suggest that the reaction mechanism involves SN2 attack of the anion of 6-MP, formed by ionization of the sulfhydryl group, on carbon 10 of BPDE, resulting in a trans-9,10 reaction product.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2005

High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts.

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

Animals↗
PubMed2006

Synthesis and characterization of enantiomeric anti-2-fluorobenzo[a]pyrene-7,8-dihydrodiol-9,10-epoxides and their 2'-deoxyguanosine and oligodeoxynucleotide adducts.

Benzo[a]pyrene diol epoxides (BPDEs) are the ultimate carcinogenic species of benzo[a]pyrene, a prototype polycyclic aromatic hydrocarbon (PAH). BPDE-modified DNA duplexes can adopt multiple conformations depending on the nature of the modified bases, the stereochemistry at the location of the covalent linkage, and the sequence context surrounding the lesion site. In this paper, we describe the preparation of enantiomeric 2-fluoro-BPDEs, trans-(7R,8S)-dihydroxy-(9S,10R)- and trans-(7S,8R)-(9R,10S)-epoxy-7,8,9,10-tetrtahydro-2-fluorobenzo[a]pyrene (22 and 23, respectively), as models for probing the BPDE-induced conformational heterogeneity. The multistep synthesis of the target diol epoxides described herein entails regiospecific succinoylation of 2-fluoropyrene, followed by a ring closure, regio- and stereospecific construction of the 7,8-dihydrodiol functionality, and a subsequent meta-chloroperbenzoic acid-mediated epoxidation. Stereoselectivity was achieved by using Jacobsen chiral catalysts, which produced greater than approximately 90% enantiomeric excess. Absolute configurations at the C(7,8) carbons of the FBP derivatives were determined by comparison of the circular dichroism (CD) spectra with those reported for the BP analogues. Analysis of the 3J(7,8) vicinal coupling constants, CD shape, and charge density calculations all indicated that the prepared anti-FBPDEs preferentially adopt the pseudo-diequatorial C(7,8) diol conformation. Hydrolysis of anti-FBPDEs produced a 9:1 ratio of trans- to cis-opened tetraols. Reactions of each of the anti-FBPDEs with deoxyguanosine 5'-monophosphate produced predominantly trans-anti-N2-dG as the major adducts. Analogous reactions with two 11-mer oligodeoxynucleotides (5'-CCATXGCTACC-3' where X = dT, dC) gave FBP-modified oligodeoxynucleotides with structures that were characterized by enzyme digest/HPLC and electrospray ionization time-of-flight mass spectrometry data. The oligonucleotide adducts were annealed with the appropriate sequences to form fully complementary duplexes [(5'-CCATXGCTACC-3')(5'-GGTAGCYATGG-3'), G = FBP-N2-dG adduct, X = dT, Y = dA in duplex I; X = dC, Y = dG in duplex II] for CD and UV melting studies. The results of the present study were consistent with those reported previously for BPDE-modified duplexes in the same sequence contexts and support the utility of FBPDEs as useful structural probes.

DNA Adducts↗
PubMed2006

Detection and quantitation of benzo[a]pyrene-derived DNA adducts in mouse liver by liquid chromatography-tandem mass spectrometry: comparison with 32P-postlabeling.

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.

Animals↗
PubMedDate not supplied

Benzo[a]pyrene-DNA adducts inhibit the DNA helicase activity of the bacteriophage T7 gene 4 protein.

The gene 4 protein of bacteriophage T7 provides the essential helicase and primase activities for the replication of the T7 genome. In addition, it also displays a DNA-dependent deoxyribonucleoside triphosphatase activity, the preferred substrate of which is dTTP. Previous investigations have demonstrated that the translocation of the gene 4 protein along single-stranded DNA is blocked by the presence of benzo[a]pyrene-DNA adducts and that the gene 4 protein is likely to be sequestered at the sites of these adducts. In the present study, we directly show that the helicase activity of the gene 4 protein is also profoundly inhibited by the benzo[a]pyrene-DNA adducts. The inhibitory effects of these adducts are strand-specific in that they block the DNA helicase activity of the gene 4 protein only when they are located in the DNA strand where the gene 4 protein translocates when it unwinds double-stranded DNA. Consistent with the hypothesis that the gene 4 protein is sequestered at the adduct site, we also show that the complexes formed by the gene 4 protein and benzo[a]pyrene-modified DNA are far more stable than those formed by the gene 4 protein and unmodified DNA.

Bacteriophage T7↗
PubMed1997

The effect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils.

The effects of several bioremediation stimulants, including potential metabolism pathway inducers, inorganic/organic nutrients, and surfactants on the metabolism of phenanthrene and pyrene, as well as the population dynamics of PAH degrading microorganisms was examined in five soils with differing background PAH concentrations, exposure histories and physical properties. Most of the supplements either had no significant effect or decreased the mineralization of [14C]-phenanthrene and [14C]-pyrene in soil slurry microcosms. The effect of a particular supplement, however, was often not uniform within or across soils. Decreased mineralization of [14C]-phenanthrene and [14C]-pyrene was usually due to either preferential use of the supplement as carbon source and/or stimulation of non-PAH degrading microorganisms. Many of the supplements increased populations of heterotrophic microorganisms, as measured by plate counts, but did not increase populations of phenanthrene degrading microorganisms, as measured by the [14C]-PAH mineralization MPN analysis or cellular incorporation of [14C]-PAH. These results suggest that the PAH degrading community at each site may be unique in their response to materials added in an attempt to stimulate PAH degradation. The characteristics of the site, including exposure history, soil type, and temporal variation may all influence their response.

Biodegradation, Environmental↗
PubMed1999

The p53 tumor suppressor protein reduces point mutation frequency of a shuttle vector modified by the chemical mutagens (+/-)7, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, aflatoxin B1 and meta-chloroperoxybenzoic acid.

p53 has been postulated to be the guardian of the genome. However, results supporting the prediction that point mutation frequencies are elevated in p53-deficient cells either have not been forthcoming or have been equivocal. To analyse the effect of p53 on point mutation frequency, we used the supF gene of the pYZ289 shuttle vector as a mutagenic target. pYZ289 was treated in vitro by ultraviolet irradiation, aflatoxin B1, (+/-)7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and meta-chloroperoxybenzoic acid and then transfected into p53-deficient cells with or without a p53 expression vector. p53 reduced the mutant frequency up to fivefold when pYZ289 was treated with aflatoxin B1, (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene or meta-chloroperoxybenzoic acid but not when it was ultraviolet-irradiated. The p53-dependent mutation frequency reduction was higher at a higher level of premutational lesions for aflatoxin B1 and (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and at a lower level of lesions for meta-chloroperoxybenzoic acid. This suggests that the chemical mutagens produce, in a dose-dependent fashion, two kinds of DNA damage, one subject to p53-dependent mutation frequency reduction and the other not. These results indicate that p53 can reduce the point mutation frequency in a shuttle vector treated by chemical mutagens and suggest that p53 can act as guardian of the genome for at least some kinds of point mutations.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2005

Determination of benzo[a]pyrene tetrols by column-switching capillary liquid chromatography with fluorescence and micro-electrospray ionization mass spectrometric detection.

The present work displays capillary liquid chromatographic column switching methodology tailored for determination of benzo[a]pyrene tetrol isomers in biological matrices using on-line fluorescence and micro-electrospray ionization mass spectrometric detection. A well-established off-line crude solid phase extraction procedure was used in order to make the method compatible with several biological matrices. The solid phase extraction eluates were evaporated to dryness, redissolved in 1.0 ml methanol:water (10:90, v/v), loaded onto a 0.32 mm I.D. x 40 mm 5 microm Kromasil C(18) pre-column for analyte enrichment and back-flushed elution onto a 0.30 mm I.D. x 150 mm 3.5 microm Kromasil C(18) analytical column. The samples were loaded with a flow rate of 50 microl min(-1) and the tetrols were separated at a flow rate of 4 microl min(-1) with an acetonitrile:10 mM ammonium acetate gradient from 10 to 90%. A sample loading flow rate up to 50 microl min(-1) was allowed. The fluorescence excitation and emission were set to 342 and 385 nm, respectively, while mass spectrometric detection of the benzo[a]pyrene tetrols was obtained by monitoring their [M - H](-) molecular ions at m/z 319. The method was validated over the concentration range 0.1-50 ng ml(-1) benzo[a]pyrene tetrols in a cell culture medium with 100 microl injection volume, fluorescence detection and the first eluting tetrol isomer as model compound, resulting in a correlation coefficient of 0.993. The within-assay (n= 6) and between-assay (n= 6) precisions were determined to 2.6-8.6% and 3.8-9.6%, respectively, and the recoveries were determined to 97.9-102.4% within the investigated concentration range. The mass limit of detection (by fluorescence) was 3 pg for all the tetrol isomers, corresponding to a concentration limit of detection of 30 pg ml(-1) cell culture medium. The corresponding mass spectrometric mass limits of detection were 4-10 pg, corresponding to concentration limits of detection of 40-100 pg ml(-1) cell culture medium.

Carcinogens↗
PubMed1987

Glutathione S-transferases of mouse lung. Selective binding of benzo[a]pyrene metabolites by the subunits which are preferentially induced by t-butylated hydroxyanisole.

Six isoenzymes of glutathione S-transferase (GST) present in mouse lung have been purified and characterized. GST I (pI 9.8) is a dimer of Mr-26,500 subunits and GST II is a heterodimer of Mr-26,500 and -22,000 subunits, and GST III (pI 7.9) and IV (pI 6.4) are dimers of Mr-24,500 subunits. GST V (pI 5.7) is a heterodimer of Mr-24,500 and -23,000 subunits, whereas GST VI (pI 4.9) is a dimer of Mr-23,000 subunits. Immunological studies indicate that the Mr-24,500 subunits present in GST III (pI 7.9) are distinct from those present in GST IV (pI 6.4) and V (pI 5.7). Structural and immunological studies provide evidence that at least five distinct types of subunits in their different binary combinations give rise to various GST isoenzymes of mouse lung. These isoenzymes express varying degrees of catalytic activities towards a wide range of electrophilic substrates including benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 4,5-oxide. The dietary antioxidant t-butylated hydroxyanisole (BHA) preferentially induces GST II and III. Also, these two isoenzymes selectively bind benzo[a]pyrene (B[a]P) metabolites, indicating that they play an important physiological role in the detoxification of B[a]P metabolites. The preferential induction of the GST isoenzymes involved in the detoxification of activated B[a]P metabolites indicates that the anti-neoplastic activity of BHA against B[a]P-induced neoplasia in mouse lung [Wattenberg (1973) J. Natl. Cancer Inst. 50, 1541-1544] may be due to the enhanced detoxification of B[a]P metabolites.

Animals↗
PubMed1996

Degradation of pyrene-labelled phospholipids by lysosomal phospholipases in vitro. Dependence of degradation on the length and position of the labelled and unlabelled acyl chains.

The hydrolysis of pyrenylacyl phosphatidylcholines (PyrnPCs)(n indicates the number of aliphatic carbons in the pyrene-chain) by crude lysosomal phospholipases in vitro was investigated. PyrnPCs consist of several sets in which the length of the pyrene-labelled or the unlabelled acyl chain, linked to the sn-1 or sn-2 position, was systematically varied. Lysophosphatidylcholine and fatty acid were the only fluorescent breakdown products detected, thus indicating that PyrnPCs were degraded by A-type phospholipases and lysophospholipases. Of these, mainly A1-type phospholipases appear to be involved, as determined from the relative amounts of labelled fatty acid and lysolipid released from the positional isomers. Based on the effects of the length and position of the pyrene-labelled and unlabelled chains it is suggested that (1) the lysosomal A-type phospholipases acting on PyrnPCs recognize the carboxy-terminal part of the lipid acyl chains and (2) the relevant part of the binding site is relatively narrow. Thus phospholipids with added bulk in the corresponding region, such as those that are peroxidized and polymerized, may not be good substrates for the lysosomal phospholipases mentioned. The impaired hydrolysis of the most hydrophobic PyrnPCs indicates that lysosomal phospholipases may not be able to penetrate significantly into the substrate interphase, but upward movement of the lipid may be required for efficient hydrolysis. Finally, the rate of hydrolysis of many pyrenyl derivatives was found to be comparable to that of a natural phosphatidylcholine species, both in micelles and in lipoprotein particles, indicating that these derivatives can be used as faithful reporters of lysosomal degradation of natural lipids in vivo and in vitro.

Acylation↗
PubMed2003

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.

Acholeplasma laidlawii↗
PubMed2000

Benzo[a]pyrene diol epoxide adducts in DNA are potent suppressors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages.

The catalytic intermediates of DNA topoisomerase I (top1) are cleavage complexes that can relax DNA supercoiling (intramolecular reaction) or mediate recombinations (intermolecular religation). We report here that DNA adducts formed from benzo[a]pyrene bay-region diol epoxides can markedly affect top1 activity. Four oligonucleotide 22-mers of the same sequence were synthesized, each of which contained a stereoisomerically unique benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the 2-amino group of a central 2'-deoxyguanosine residue. These four adducts correspond to either cis or trans opening at C-10 of the (+)-(7R, 8S, 9S, 10R)- or (-)-(7S, 8R, 9R, 10S)-7,8-diol 9,10-epoxides. Their solution conformations in duplex DNA (intercalated and minor-groove bound for the cis and trans opened adducts respectively) can be deduced from previous NMR studies. All four adducts completely suppress top1 cleavage activity at the alkylation site and induce the formation of new top1cleavage complexes on both strands of the DNA 3-6 bases away from the alkylation site. The trans opened adduct from the highly carcinogenic (+)-diol epoxide is the most active in inducing top1 cleavage independently of camptothecin, demonstrating that minor groove alkylation can efficiently poison top1. We also found that this isomer of the diol epoxide induces the formation of top1-DNA complexes in mammalian cells, which suggests a possible relationship between induction of top1 cleavage complexes and carcinogenic activity of benzo[a]pyrene diol epoxides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1978

Cyclopenta[c,d]pyrene: a highly mutagenic polycyclic aromatic hydrocarbon.

A polycyclic aromatic hydrocarbon recently isolated from carbon black and identified as cyclopenta[c,d]pyrene (CPP) is highly mutagenic. By the criteria of the Salmonella/mammalian-microsome mutagenicity test, the mutagenic potency of CPP is equalled by only two other naturally occurring polycyclic aromatic hydrocarbons--benzo[a]pyrene and dibenz[a,c]anthracene. The potent mutagenicity of CPP is noteworthy for two reasons: (i) CPP is a mutagenic polycyclic aromatic hydrocarbon without a "bay-region" and (ii) there is evidence that it is distributed widely in the environment. On the basis of experimental observations and perturbational molecular orbital calculations we propose that a mutagenic metabolite of CPP will be the 3,4-oxide. The carbonium ion derived from opening of CPP 3,4-oxide is identical to that derived from opening of benzo[a]pyrene 7,8-diol-9,10-oxide, the metabolite now thought to be an ultimate mutagenic and carcinogenic species.

Benzopyrenes↗
PubMed1984

Hydrolysis of benzo[a]pyrene diol epoxide and its covalent binding to DNA proceed through similar rate-determining steps.

The mutagenic and carcinogenic metabolite of benzo[a]pyrene, (7R,8S)-dihydroxy-(9R,10R)-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene, undergoes two major reactions in the presence of DNA: (i) hydrolysis and (ii) covalent binding. We report that hydrolysis and covalent binding are specific and general acid-catalyzed reactions with the same or similar rate-determining steps. To account for the similarity of rate-determining steps in covalent binding and hydrolysis we propose and test two models. In each model, the rate-determining step results in formation of a carbonium ion, which serves as a precursor for both tetrol and adduct. In model A the carbonium ion is partitioned between two domains (1 and 2), while in model B there is only one domain. Measurements of pseudo-first-order rate constants, product ratios, and rate ratios support model A, while kinetic results are inconsistent with model B. Domain 1 most likely represents activated benzo[a]pyrenes that are intercalated into DNA, while domain 2 hydrocarbons are physically bound to the outside of the DNA helix.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Turning On uracil-DNA glycosylase using a pyrene nucleotide switch.

Base flipping is a highly conserved process by which enzymes swivel an entire nucleotide from the DNA base stack into their active site pockets. Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the hydrolysis of the N-glycosidic bond of 2'-deoxyuridine (2'-dUrd) in DNA as the first step in uracil base excision repair. Flipping of 2'-dUrd by UDG has been proposed to follow a "pushing" mechanism in which a completely conserved leucine side chain (Leu-191) is inserted into the DNA minor groove to expel the uracil. Here we report a novel implementation of the "chemical rescue" approach to show that the weak binding affinity and low catalytic activity of L191A or L191G can be completely or partially restored by substitution of a pyrene (Y) nucleotide wedge on the DNA strand opposite to the uracil base (U/A to U/Y). These results indicate that pyrene acts both as a wedge to push the uracil from the base stack in the free DNA and as a "plug" to hinder its reinsertion after base flipping. Pyrene rescue should serve as a useful and novel tool to diagnose the functional roles of other amino acid side chains involved in base flipping.

Catalysis↗
PubMed2001

Specificity of 17beta-oestradiol and benzo[a]pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432.

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48 Ala119 Leu432, Arg48 Ala119 Val432 Gly48 Ala119 Leu432, Gly48 Ala119 Val432, Arg48 Ser119 Leu432, Arg48 Ser119 Val432, Gly48 Ser119 Leu432 and Gly48 Ser119 Va1432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17beta-oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+.CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg48 variants tended to have higher activities for 17beta-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17beta-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17beta-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms. 4. In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17beta-oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Alleles↗
PubMed1997

Effect of tannic acid on benzo[a]pyrene-DNA adduct formation in mouse epidermis: comparison with synthetic gallic acid esters.

Tannic acid, a naturally occurring plant phenol, was shown to inhibit the mutagenicity and/or tumorigenicity of several polycyclic aromatic hydrocarbons in mouse skin. In this study the effect of topical application of tannic acid on epidermal aryl hydrocarbon hydroxylase, glutathione S-transferase, and binding of benzo[a]pyrene (B[a]P) to epidermal DNA was compared with the activity of synthetic gallic acid esters. Single topical application of 8 mumol octyl and dodecyl gallate had no effect on the induction of aryl hydrocarbon hydroxylase, whereas propyl gallate and tannic acid increased the enzyme activity by nearly 200%. Application of the phenolics one hour before 0.2 mumol of B[a]P enhanced the enzyme activity, but the observed differences were not significant in comparison with a B[a]P-treated group of mice. Application of dodecyl and octyl gallates to mouse skin resulted in three- and twofold increases, respectively, in the activity of glutathione S-transferase. Combined treatment with dodecyl gallate and B[a]P also resulted in significant enhancement of this enzyme activity. Application of the same dose of tannic acid to mouse skin one hour before the application of 0.2 or 1 mumol of B[a]P afforded 60% inhibition of covalent benzo[a]pyrene-diol-epoxide binding to epidermal DNA. Gallic acid esters with the exception of dodecyl gallate were less effective inhibitors of benzo[a]pyrene-diol-epoxide binding, especially when the higher dose of B[a]P was used. These results indicate that the antitumorigenic activity of tannic acid involves the interaction of the ultimate carcinogen with DNA rather than an altered metabolism. The linkage between gallic acid and glucose in natural plant phenols is also more effective at inhibiting B[a]P binding to epidermal DNA than the linkage with the alkyl group in synthetic gallates.

Animals↗
PubMed1984

Stereoselective covalent binding of anti-benzo(a)pyrene diol epoxide to DNA conformation of enantiomer adducts.

The conformation of adducts derived from the reactions and covalent binding of the (+) and (-) enantiomers of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) with double-stranded calf thymus DNA in vitro were investigated utilizing the electric linear dichroism technique. The linear dichroism and absorption spectra of the covalent DNA complexes are interpreted in terms of a superposition of two types of binding sites. One of these conformations (site I) is a complex in which the plane of the pyrene residue is close to parallel (within 30 degrees) to the planes of the DNA bases (quasi-intercalation), while the other (site II) is an external binding site; this latter type of adduct is attributed to the covalent binding of anti-BaPDE to the exocyclic amino group of deoxyguanine (N2-dG), while site I adducts are attributed to the O6-deoxyguanine and N6-deoxyadenine adducts identified in the product analysis of P. Brookes and M.R. Osborne (Carcinogenesis (1982) 3, 1223-1226). Site II adducts are dominant (approximately 90% in the covalent complexes derived from the (+) enantiomer), but account for only 50 +/- 5% of the adducts in the case of the (-)-enantiomer. The orientation of site II complexes is different by 20 +/- 10 degrees in the adducts derived from the binding of the (+) and the (-) enantiomers to DNA, the long axis of the pyrene chromophore being oriented more parallel to the axis of the DNA helix in the case of the (+) enantiomer. These findings support the proposals by Brookes and Osborne that the difference in spatial orientation of the N2-dG adducts of (-)-anti-BaPDE together with their lower abundance may account for the lower biological activity of the (-) enantiomer. The external site II adducts, rather than site I adducts, appear to be correlated with the biological activity of these compounds.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗