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PubMed1997

Mechanism of differential efficacy of garlic organosulfides in preventing benzo(a)pyrene-induced cancer in mice.

The mechanism of differential efficacies of diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS) in preventing benzo(a)pyrene (BP)-induced cancer in mice has been investigated by determining their effects on the enzymes of BP activation/inactivation pathways. With the exception of DATS, treatment of mice with other organosulfides (OSCs) caused a small but significant increase (37-44%) in hepatic ethoxyresorufin O-deethylase (EROD) activity. However, the forestomach EROD activity did not differ significantly between control and treated groups. Only DAS treatment caused a modest but statistically significant reduction (about 25%) in pulmonary EROD activity. These results suggest that while reduction of EROD activity may, at least in part, contribute to the DAS-mediated inhibition of BP-induced lung cancer, anticarcinogenic effects of OSCs against BP-induced forestomach carcinogenesis seems to be independent of this mechanism. Treatment of mice with DAS, DADS and DATS resulted in a significant increase, as compared with control, in both hepatic (3.0-, 3.2- and 4.4-fold, respectively) and forestomach (1.5-, 2.7- and 2.7-fold, respectively) glutathione transferase (GST) activity toward anti-7beta,8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE), which is the ultimate carcinogen of BP. The pulmonary GST activity was not increased by any of the OSCs. Even though epoxide hydrolase (EH) activity was differentially altered by these OSCs, a correlation between chemopreventive efficacy of OSCs and their effects on EH activity was not apparent. The results of the present study suggest that differences in the ability of OSCs to modulate GST activity toward anti-BPDE may, at least in part, account for their differential chemopreventive efficacy against BP-induced cancer in mice.

Allyl Compounds↗
PubMed1998

Gender-related differences in susceptibility of A/J mouse to benzo[a]pyrene-induced pulmonary and forestomach tumorigenesis.

Benzo[a]pyrene (BP) is a suspected human carcinogen and is known to produce tumors in the lung and forestomach of mice. Glutathione (GSH) S-transferases (GST) play a major role in the detoxification of the ultimate carcinogen of BP, (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE). Previous studies have shown gender-related differences in the expression of GST isoenzymes in mice. The present study was designed to test the hypothesis whether gender-related differences in the expression of GST isoenzymes can affect the susceptibility of mice to BP-induced lung and forestomach tumorigenesis. The expression of pi class isoenzyme mGSTP1-1, which is highly efficient in the detoxification of (+)-anti-BPDE, was approximately 3.0- and 1.5-fold higher in the liver and forestomach of male A/J mouse, respectively, as compared with the female. The levels of other major GST isoenzymes, mGSTA3-3 (alpha class), mGSTM1-1 (mu class) and mGSTA4-4 (alpha class), were also significantly higher in the liver of the male mouse as compared with the female. While pulmonary mGSTP1-1 expression did not differ significantly between male and female A/J mice, the expression of mGSTA3-3, mGSTM1-1 and mGSTA4-4 was significantly higher (1.4-4.0-fold) in the lung of the male A/J mouse as compared with the female. At lower concentrations of BP (0.5 mg BP/mouse), the tumor incidence/multiplicity was significantly higher in the lung as well as in the forestomach of female mice as compared with male mice. For example, while 30% of the female mice developed pulmonary tumors 26 weeks after the first 0.5 mg BP administration, none of the male mice had tumors in their lungs. At higher doses of BP (1.5 mg BP/mouse), however, this differential was either abolished or relatively less pronounced. Our results suggest that up to a certain threshold of BP exposure the levels of GST isoenzymes may be an important determinant of susceptibility to BP-induced tumorigenesis in mice.

Animals↗
PubMed2000

Interactions between benzo[a]pyrene and UVA light affecting ATP levels, cytoskeletal organization, and resistance to trypsinization.

Polycyclic aromatic hydrocarbons affect cells in many ways, including covalent modifications of DNA, participation in redox cycling, and alterations in cellular signaling pathways. Similarly, exposure to ultraviolet (UV) light may modify DNA, generate reactive oxygen species, and alter signaling. Because environmental conditions may interact to affect cellular functions, we investigated the combined effects of benzo[a]pyrene (BaP) and UV light in a cell line in which BaP-induced alterations in Ca(2+) homeostasis have previously been shown. Exposure of MCF-10A cells to BaP (18 h) followed by a brief (5 min) exposure to UVA resulted in resistance to trypsinization of cells grown on type I collagen (Vitrogen). This effect was not seen following treatment with BaP or UVA alone nor with benzo(e)pyrene (BeP)+UVA. BaP+UVA light also caused actin filaments to reorganize from typical stress fibers to substrate-associated aggregates of actin and caused depletion of cellular adenosine triphosphate (ATP). The effects of BaP+UVA on adhesion and actin aggregate formation were partially prevented by treatment with reduced glutathione. Depletion of cellular ATP affected resistance to trypsinization and actin organization in a similar manner. Thus, these studies suggest a redox-sensitive interaction between BaP+UVA light to deplete cellular ATP levels, resulting in resistance to trypsinization and actin filament reorganization in MCF-10A cells.

Actins↗
PubMed1999

Priming effect of benzo[a]pyrene on monocyte oxidative metabolism: possible mechanisms.

Monocytes, separated from human peripheral blood, were preincubated with different polycyclic aromatic hydrocarbons (PAHs) for 24 h and the production of superoxide ions (O*2-) was then measured using as a stimulating agent phorbol 12-myristate 13-acetate. A significantly enhanced O*2- production is only observed when the cells are treated with benzo[a]pyrene (B[a]P); benzo[e]pyrene, benzo[a]anthracene and 3-methylcholanthrene induce a small but not significant increase of O*2-. Anthracene has no effect, while phenanthrene slightly inhibits. The priming activity of B[a]P is unrelated to variations in intracellular Ca2+ ([Ca2+]i), as demonstrated by the inability of B[a]P to increase [Ca2+]i concentration in both monocytes and the promonocytic cell line U937. Furthermore, in monocytes the sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin, which can increase [Ca2+]i evokes a differentiation-like event associated with a decrease in the production of superoxide ions. These results further support that the enhancing activity of B[a]P on monocytes superoxide production is not mediated by an increase of [Ca2+]i. In contrast, the role of the aryl hydrocarbon receptor (AhR) in B[a]P-induced superoxide ion enhancement is suggested by the inhibitory effect of the specific antagonist alpha-naphthoflavone (alphaNF), while the tumor necrosis factor (TNF-alpha) is not involved in the phenomenon. Thus, the interaction of B[a]P with its cytosolic receptor and either the metabolism of the compound into reactive intermediates or the over-expression of some unknown genes seem to be involved in an essential step in this process.

Benzo(a)pyrene↗
PubMed2003

Assessment of metabolites and AhR and CYP1A1 mRNA expression subsequent to prenatal exposure to inhaled benzo(a)pyrene.

Few studies have focused on environmental aerosol contaminant, mechanistically-based, dose-related neurotoxicity with respect to development of the central nervous system. To fill this important data gap and to highlight possible mechanistic pathways, a study was undertaken to determine metabolite concentrations associated with the transplacental disposition of inhaled benzo(a)pyrene (B(a)P) and the resulting effects on the status of aryl hydrocarbon receptor (AhR), and cytochrome P450 1A1 (CYP1A1) mRNA in preweaning F1 generation animals. In this study, laparotomy on GD 8 was performed on timed-pregnant rats followed by dosing via nose-only exposure for 4h a day for 10 days (GD 11-GD 20) to three concentrations of a B(a)P: carbon black aerosol (25, 75 and 100 microg/m(3)). A dose-dependent decrease in birth index was observed in the B(a)P exposed group as compared to the controls (P<0.05). Analysis of cerebrocortical extracts from F1 generation pups revealed a dose-dependent (P<0.05) increase in total B(a)P metabolites. Analysis of cerebrocortical and hippocampal mRNA developmental expression profiles for AhR and CYP1A1 using 18sRNA as the internal standard, revealed that inhaled B(a)P upregulates AhR during the first postnatal month. The present study suggest that prenatal exposure to inhaled B(a)P upregulates hepatic aryl hydrocarbon receptor dependent mechanisms in the F1 generation. Hepatic upregulation of the aryl hydrocarbon receptor may modulate the potential for benzo(a)pyrene toxicity via the activation of cytochrome P450 and the subsequent deposition of lipophillic metabolites to developing central nervous system structures such as cerebral cortex and hippocampus.

Administration, Inhalation↗
PubMed2001

Polycyclic aromatic compounds as anticancer agents: structure-activity relationships of chrysene and pyrene derivatives.

A large number of diamides and diamines were synthesized using 6-amino chrysene and 1-amino pyrene as starting materials. A structure activity study with cis-platinum as internal control against animal and human tumor lines was carried out in vitro. This study indicated that the in vitro cytotoxicity toward these lines depends on the functionality present in the molecules. The diamino compounds were found to be more potent than the diamides, and these were equally active irrespective of the end heterocyclic group, whereas the activity of the diamides was strongly dependent on the terminal unit. In general, the diamides containing chrysene as the chromophore were more active than those with a pyrene ring. The size of the end heterocyclic ring, along with the nature of the spacer connecting the polycyclic ring to the heterocyclic ring, seemed to affect the biological activity in certain cell lines. Hemolysis experiments on a lead compound established that it had activities similar to those described for membrane-stabilizing agents. This agent also demonstrated the capacity to produce differentiation in leukemia cell lines.

Animals↗
PubMed2001

Musk ketone enhances benzo(a)pyrene induced mutagenicity in human derived Hep G2 cells.

Musk ketone is a widely used artificial fragrance which has been identified in human fatty tissue and milk. The mutagenic and comutagenic effects of this compound were studied in micronucleus tests with a human derived hepatoma cell line (Hep G2). Exposure of the cells to MK alone in the range between 5 and 5000 ng/ml did not cause induction of MN. When the cells were treated simultaneously with MK (5-5000 ng/ml) and 0.2 microg/ml benzo(a)pyrene, no synergistic effects were detected; benzo(a)pyrene (B(a)P) itself caused an 1.5-fold increase of MN over the spontaneous background frequency (60 versus 39 MN/1000 binucleated cells). In a third experimental series, the cells were pretreated with MK for 28h and subsequently exposed to 0.2 microg/ml B(a)P. In this case, a pronounced comutagenic effect was observed: The LOAEL for MK was 0.05 microg/ml. With higher doses (0.5, 1.0 and 5.0 microg MK/ml), a significant increase of B(a)P induced MN frequencies was measured, the induction rates being 50, 66, and 88%, respectively. Additional measurements of 7-ethoxyresorufin deethylase indicated that MK induces cytochrome P450 isoenzymes (1A1) which play a key role in the activation of B(a)P. The results of the present study show that MK amplifies the genotoxic effects of B(a)P in human derived cells and indicate that exposure of humans to MK might increase their susceptibility to the health hazards of B(a)P and other polycyclic aromatic hydrocarbons.

Benzo(a)pyrene↗
PubMed2003

Inhibition of clastogenicity of benzo[a]pyrene and of its trans-7,8-dihydrodiol in mice in vivo by fruits, vegetables, and flavonoids.

In the in vivo mouse bone marrow micronucleus assay, homogenates of spinach, artichoke, peaches, and blue grapes as well as commercial concentrates of these vegetables and fruits reduced induction of micronuclei by benzo[a]pyrene (BaP) by 43-50%. Concentrates of strawberries (31% reduction) and of cauliflower (20% reduction) were less potent. Inhibition of genotoxicity by spinach and peaches was not caused by any delay in maturation of micronucleated erythrocytes as shown by experiments with sampling times of 24, 48, and 72 h after dosing of BaP. Pre-treatment of the mice with spinach 48, 24, and 12h before application of BaP resulted in a 44% reduction of micronuclei while peaches generated only a marginal effect. A post-treatment procedure administering spinach or peaches 6h after dosing of BaP did not indicate any protective effects. When trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-OH) was applied for induction of micronuclei spinach and peaches reduced the number of micronuclei by 55 and 48%, respectively. Pre-treatment of mice with spinach 96, 72, and 60 h before sacrifice caused a decline of hepatic 7-ethoxyresorufin-O-dealkylase (EROD) and of 7-pentoxyresorufin-O-dealkylase (PROD) activities by factors of 2.2 and 1.4, respectively. However, statistical significance was not reached. On the other hand, peaches had no influence on hepatic EROD or PROD activities. The flavonoids quercetin and its glucoside isoquercitrin, administered orally in doses of 0.03 mmol/kg body weight simultaneously with intraperitoneally given BaP, reduced the number of micronuclei in polychromatic erythrocytes of the bone marrow of mice by 73 and 33%. Ten-fold higher concentrations, however, reversed the effects with a particular strong increase observed with isoquercitrin (+109%; quercetin: +16%).

Administration, Oral↗
PubMed1999

Cytochrome P450 1A1 in rat peripheral blood lymphocytes: inducibility in vivo and bioactivation of benzo[a]pyrene in the Salmonella typhimurium mutagenicity assay in vitro.

The presence and inducibility of CYP1A1 in freshly isolated peripheral blood lymphocytes was examined in untreated rats and in rats pretreated with agents known to induce the enzyme in other tissues, as well as dexamethasone [CAS #50-02-2], which is not commonly associated with CYP1A1 induction. CYP1A1 but not CYP1A2 was detected by Western blot analysis of lymphocytes from untreated rats and was induced in lymphocytes from rats treated with the known CYP1A inducers beta-naphthoflavone [CAS #6051-87-2] or 3-methylcholanthrene [CAS #56-49-5] (7.3-fold), cigarette smoke (2. 8-fold), and pyridine [CAS #108-86-1] (2.6-fold). CYP1A1 was also induced in lymphocytes from rats treated with the nonprototypic inducer dexamethasone (17.7-fold) or bromobenzene [CAS #108-86-1] (3. 9-fold). Lymphocyte homogenate from rats treated with the inducers also catalyzed NADPH-dependent bioactivation of benzo[a]pyrene [CAS #50-32-8] to mutagens. The benzo(a)pyrene mutagenicity was detected using Salmonella typhimurium TA100 in the Ames test, and correlated positively with lymphocyte CYP1A1 content. The data show that CYP1A1 is present in rat peripheral blood lymphocytes in vivo, and is inducible by prototypic, as well as nonprototypic, inducers of the enzyme.

Animals↗
PubMed2000

Highly selective fluorometric determination of polyamines based on intramolecular excimer-forming derivatization with a pyrene-labeling reagent.

We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipyrene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1 (Put), 1 (Cad), 5 (Spd), and 8 (Spm) fmol on the column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.

Chromatography, High Pressure Liquid↗
PubMed1993

KO2 chemical transformation/mass spectrometry detection of covalent damage to the DNA of cultured human lymphocytes exposed to benzo[a]pyrene.

Cultured human lymphocytes were exposed to benzo[a]pyrene (B[a]P), and diol epoxide-type DNA adducts arising from this chemical were detected by a method consisting of the following sequence of steps: (1) isolate the DNA; (2) subject the DNA to mild acid hydrolysis to release the polyaromatic moiety as a tetrahydrotetrol; (3) add an internal standard; (4) oxidize the tetrahydrotetrol with potassium superoxide to pyrene-2,3-dicarboxylic acid; (5) derivatize the latter with pentafluorobenzyl bromide; (6) purify the diester product on a silica cartridge; and (7) detect this product by gas chromatography electron capture negative ion mass spectrometry. From the dose (1 microgram/mL) of B[a]P applied, five adducts in 10(7) normal nucleotides were found. Largely because steps 2-5 of the method take place sequentially in a single vial, the procedure is convenient and affords precise results. To demonstrate the potential of the method to detect KO2-susceptible polyaromatic hydrocarbon DNA adducts in general, including unknowns, it was also applied to picomole and femtomole amounts of a standard of chrysene-1,4-quinone using scanning and selected ion monitoring conditions, respectively, in the MS. Since standard products can be detected with selected ion monitoring at levels 10(4) below those encountered here (prior work), it should be possible in the future to extend the method to samples containing smaller amounts of such adducts.

Animals↗
PubMed1995

Oxygen sensors based on luminescence quenching: interactions of pyrene with the polymer supports.

Oxygen quenching of pyrene has been studied in a diverse series of polymers. Most measurements were made using homo- or copolymers containing a poly(dimethylsiloxane) region. Systematic variations in the polymer properties have been made in order to delineate the structural features important for satisfactory use as supports for oxygen sensors. In particular, quenching behavior was examined as a function of the type and amount of copolymer cross-linkers; these were added to produce domains of different polarity and rigidity that would segregate the sensor molecule. A domain model (Xu, W.; et al. Anal. Chem. 1994, 66, 4133-4141) was used to explain the variations in oxygen-quenching properties as a function of additives and cross-linkers. The relative affinity of the different domains for the pyrene and the efficacy of the domains for oxygen quenching controls the overall behavior of the sensing response.

Electrodes↗
PubMed2004

Probing the interaction of benzo[a]pyrene adducts and metabolites with monoclonal antibodies using fluorescence line-narrowing spectroscopy.

A new approach for studying antibody-antigen interactions of DNA adducts and metabolites of polycyclic aromatic hydrocarbons (PAHs) is demonstrated in which fluorescence line-narrowing spectroscopy (FLNS) is used. It is based on the fact that in an FLN spectrum the relative intensities of the line-narrowed bands (that correspond to the excited-state vibrations) are, in general, strongly dependent on the local environment of the fluorophore. Information on the nature of the interactions can be obtained by comparing the FLN spectra of the antigen-antibody complexes to the spectra of the antigen in different types of solvents (H-bonding, aprotic, and pi-electron-containing solvent molecules) recorded under the same conditions. The antigens used were the DNA adduct 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and the metabolite (+)-trans-anti-7,8,9,10-benzo[a]pyrenetetrol (BP-tetrol) of benzo[a]pyrene; two monoclonal antibodies (MAbs) have been developed to selectively bind these compounds. It is shown that, for BP-tetrol, H-bonding solvents have a pronounced effect on the FLN spectra. The presence of pi electrons in the solvent molecules results in relatively small but still significant changes in the spectra. When BP-tetrol is bound to its MAb, however, neither of these effects is observed; its spectrum is very similar to the one obtained with an aprotic solvent, methylcyclohexane. Therefore, we can conclude that this MAb has an internal binding site in which the interaction with BP-tetrol is of a hydrophobic character. For BP-6-N7Gua, however, there is a strong effect of the presence of pi electrons in the solvent molecules. The FLN spectrum of this antigen bound to its MAb is very similar to its spectrum in acetone, indicating that pi-pi interactions play an important role in the binding.

Antibodies, Monoclonal↗
PubMed1996

Detection and identification of benzo[a]pyrene diol epoxide adducts to DNA utilizing capillary electrophoresis-electrospray mass spectrometry.

Capillary zone electrophoresis (CZE) coupled with negative ion electrospray mass spectrometry (ES-MS) is used for the detection and identification of adducts formed from the reaction of DNA with (+/-)-anti-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE),an active metabolite of benzo[a]pyrene (BaP). Results presented in this paper demonstrate low nanogram detection limits ( < 10 ng or < 15 pmol) for normal scan spectra and collision-induced dissociation spectra of the main nucleotide adduct formed from this reaction. (BPDE reacts predominantly with the exocyclic amino group of guanine.) Exploitation of selective reaction monitoring (SRM) produces detection limits in the low picogram range ( < 85 pg or < 130 fmol). The application of sample stacking significantly increases the concentration detection limit (to approximately 10(-8) M). Nucleotide adducts are negatively charged at most pHs and are therefore ideally suited to the stacking process used in this research. These techniques have been applied to the analysis of the adducts formed from the in vitro reaction of BPDE with DNA. In addition it is shown that CZE-ES-MS, combined with solid-phase sample cleanup, can detect adducts at levels of four adducts in 10(7) unmodified bases or less.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMedDate not supplied

Reagent for introducing pyrene residues in oligonucleotides.

A novel pyrenyl-containing phosphoramidite reagent, N-[4-(1-pyrenyl)butyryl]-O1-(4,4'-dimethoxytrityl)-O2- [(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1 ,2-propanediol (5), has been synthesized from 4-(1-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction.

Base Sequence↗
PubMed1995

Transversal distribution of acyl-linked pyrene moieties in liquid-crystalline phosphatidylcholine bilayers. A fluorescence quenching study.

Quenching of the fluorescence of pyrene-labeled phospholipids by dibromolipids was used to determine the chain length dependence of the bilayer depths of the pyrenyl moieties. Six 1-palmitoyl-2-(pyrenyl-n-acyl)-phosphatidylcholines (PyrnPC) were examined, with end-labeled pyrenyl chains varying in length, n, from 4 to 14 carbons. These lipids were incorporated, at a concentration of 0.3 mol%, into bilayers composed of various mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and of one of three 1-palmitoyl-2-(x,y-dibromostearoyl)phosphatidylcholine quencher lipids (Brx,yPC; x,y = 6,7; 9,10; or 11,12). Parallel experiments were carried out with bilayers containing 50 mol % cholesterol. Quenching in these systems is dynamic, as demonstrated by the identical dependence of steady-state fluorescence intensities and excited state lifetimes of Pyr8PC on the mole fraction of Br6,7PC. Stern--Volmer analysis of the Brx,PC mole fraction dependence of PyrnPC fluorescence yielded apparent quenching constants, KSV, which show a systematic relation with both the length of the pyrenyl acyl chain and the position of the bromine atoms. The quenching data were further analyzed by plotting KSV as a function of n (defined above), or b (the average of the two bromine positions for each PyrnPC), or n--b (the separation between pyrenes and bromines). In all cases, the data were fit by Gaussian functions yielding estimates of the centers and the apparent 1/e half-widths of the transversal distributions of the pyrenyl moieties in methylene units (mu). Both in the absence and in the presence of cholesterol, the position of each PyrnPC Gaussian center is equal to the sum of n plus a constant d approximately 2.5 mu, corresponding to the distance from the effective center of the pyrenyl moiety to its point of attachment to the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization↗
PubMed1995

Structural alignments of (+)- and (-)-trans-anti-benzo[a]pyrene-dG adducts positioned at a DNA template-primer junction.

The structural features of a chemically modified DNA template strand may promote error-prone DNA synthesis during replication. The resulting higher incidence of mutations, in turn, can eventually lead to tumor initiation. Structural insights into this process can be monitored by studying chemically modified base adducts of defined stereochemistry positioned site-specifically at a single strand--duplex template--primer junction. We have used a NMR-molecular mechanics approach to obtain the solution conformations of the covalent adducts derived from trans additions at the [BP]C10 position of the highly tumorigenic (+)-anti-benzo[a]pyrene diol epoxide [(+)-anti-BPDE] and nontumorigenic (-)-anti-benzo-[a]pyrene diol epoxide [(-)-anti-BPDE] to the N2 position of guanine [(+) and (-)-trans-anti-[BP]dG, respectively] in the d(A1-A2-C3-[BP]G4-C5-T6-A7-C8-C9-A10-T11-C12-C13).d (G14-G15-A16-T17-G18-G19-T20-A 21-G22) 13/9-mer DNA sequence. The modified 13-mer strand constitutes the template strand, while the complementary 9-mer strand constitutes a primer which has been synthesized from the 3'-end of the template toward the 5'-end up to the base preceding, but not including, the modified guanine. The modified guanine (denoted by [BP]dG4) is positioned at the junction site between the single-stranded and duplex segments. Structural features of the (+)-trans-anti-[BP]dG 13/9-mer have been determined by incorporating proton--proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The 3'-side duplex segment retains a minimally perturbed B-DNA conformation with all nine base pairs in Watson--Crick hydrogen-bonded alignments. Conformational heterogeneity is detected at the single-stranded d(A1-A2-C3) segment located 5' to the modified (+)-trans-anti-[BP]dG lesion which contrasts with an unperturbed alignment of these same residues in the unmodified control 13/9-mer. The modified guanine adopts a syn glycosidic torsion angle, is displaced into the major groove, and no longer stacks over the adjacent dC5.dG22 base pair. Such a base displacement is accompanied by stacking of one face of the pyrenyl ring with the dC5.dG22 base pair located on the duplex segment proximate to the modified guanine, while the other face of BP is exposed to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1993

Mutagenesis by the (+)-anti-diol epoxide of benzo[a]pyrene: what controls mutagenic specificity?

Mutagenesis by (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [(+)-anti-B[a]PDE], an important mutagenic/carcinogenic metabolite of benzo[a]pyrene (B[a]P), is being studied in order to understand the factors that influence mutagenesis both quantitatively and qualitatively. A new mutational system, which permits the selection of supF- mutations in an Escherichia coli plasmid, pUB3, was used. The work described herein is an extension of previous work, which involved plasmid adduction and then immediate transformation (Rodriguez & Loechler, 1993), and began with the observation that mutation frequency (MF) decreased approximately 2-fold when the (+)-anti-B[a]PDE-adducted plasmid pUB3 is either (1) frozen and then thawed prior to transformation or (2) heated at 80 degrees C for 10 min prior to transformation. Several results suggest that this decrease is not due to the loss of labile adducts. To begin to understand this phenomenon, the mutagenic spectra are compared for (+)-anti-B[a]PDE in supF for the unheated (187 mutants), the freeze/thawed (134 mutants), and the heated (254 mutants) samples. In general, freeze/thawing and heating cause a decrease in all classes of mutations. Considering substitution mutations at G.C base pairs, which predominate, the mutagenic specificity for the combined data sets is GC-->TA (57%), GC-->AT (23%), and GC-->CG (20%). This raises the question, how does (+)-anti-B[a]PDE generate this complex mutagenic specificity, which contrasts with the situation for, e.g., simple methylating agents? One factor is that mutagenic specificity at a particular guanine residue can be influenced by the base on its immediate 5'-side, most notably where mutations are virtually exclusively restricted to GC-->TA in 5'-TG-3' sequence contexts. One unexpected finding may provide additional insight. G115 in supF, which is the major hot spot for base-pairing mutagenesis, is the only site where the qualitative pattern of mutagenesis is significantly affected by heating the (+)-anti-B[a]PDE-adducted plasmid prior to transformation. Without heating, G115-->T mutations predominate, but following heating there is a statistically significant increase in the fraction of G115-->A and G115-->C mutations. The most likely model to explain this and other results is (1) a particular DNA adduct can adopt multiple conformations, (2) the conformation adopted by an adduct can be influenced by various factors, including DNA sequence context, as well as heating and freeze/thawing, and (3) each of these conformations can cause a different pattern of mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

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