Stereoselective covalent binding of enantiomers of anti-benzo[a]pyrene diol epoxide to DNA as probed by optical detection of magnetic resonance

Phosphorescence and optical detection of magnetic resonance measurements applied to the covalent adducts of (+)- and (-)-anti-benzo[a]pyrene with DNA show a marked red shift of the pyrenyl phosphorescence and a lowering of the zero field splitting parameters of the (-) adduct, relative to the (+) adduct and the (solvent-exposed) benzo[a]pyrene tetraol. These results are consistent with a predominance of quasi-intercalative sites in the (-) adduct and external, solvent-exposed sites in the (+) adduct.     

Conformations of adducts and kinetics of binding to DNA of the optically pure enantiomers of anti-benzo(a)pyrene diol epoxide

Kinetic flow dichroism studies indicate that the (+) enantiomer of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene physically bound at intercalative-type sites in double-stranded DNA undergoes covalent binding reactions to form adducts at external binding sites. The conformation of the non-covalent complex derived from the (-) stereoisomer is also intercalative in nature, but the conformations of the covalent adducts are heterogeneous and are characterized by both intercalative-type and external conformations. It is suggested that the distinctly higher biological activity of the (+) enantiomer relative to the activity of the (-) enantiomer may be related to the preponderance of 7,8,9-triol benzo(a)pyrene residues covalently linked to deoxyguanine and located at external binding sites in the DNA adducts.     

The importance of intercalation in the covalent binding of benzo(a)pyrene diol epoxide to DNA

The primary mode of non-covalent interaction of the strong carcinogen, benzo(a)pyrene diol epoxide, with DNA is through intercalation. It has variously been suggested that intercalative complexes may be prerequisite for either covalent binding or DNA-catalysed hydrolysis of the epoxide or both. Geacintov [Geacintov, N. E. (1986). Carcinogenesis 7, 589.] has recently argued that intercalation is important in covalent binding and presented theoretical constructs consistent with this proposal. A more general theoretical model is presented here which includes the possibilities that either catalysis of hydrolysis or covalent binding of benzo(a)pyrene diol epoxide DNA can occur (a) in an intercalation complex, or (b) without formation of a detectable, physically bound complex. It is shown that a variety of possible mechanisms formulated under this general theory lead to equations for overall reaction rates and covalent binding fractions which are all of the same form with respect to DNA concentration dependence. A consequence of this is that experimental studies of the dependence of hydrolysis rates and covalent binding fractions on DNA concentration do not distinguish between the various possible mechanisms. These findings are discussed in relation to the interactions of benzo(a)pyrene diol epoxide with chromatin in cells.     

Conformations and selective photodissociation of heterogeneous benzo(a)pyrene diol epoxide enantiomer-DNA adducts

The covalent binding of the tumorigenic (+) enantiomer and the nontumorigenic (-) enantiomer of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,19-tetrahydrobenzo(a)pyrene (BPDE) to double-stranded native DNA gives rise to heterogeneous adducts, especially in the case of (-)-BPDE. The covalent (+)-BPDE-DNA adducts are predominantly of the external site II type, while the (-)-BPDE-DNA adducts are predominantly of the quasi-intercalative, site I type (65%), with 35% of site II adducts. The site I adducts can be selectively photodissociated with near-ultraviolet light (quantum yields in the range 0.0003-0.005); the external site II adducts (photodissociation quantum yield 3 X 10(-5) are 10-100-times more stable. The photolability of covalent (-)-BPDE-DNA adducts accounts for the discrepancies in the linear dichroism properties of these complexes reported previously. Fluorescence quenching data, previously utilized to assess the degree of solvent exposure of the pyrenyl residues in covalent adducts, were in some cases significantly influenced by the presence of highly fluorescent tetraol dissociation products. After correcting for this effect, it is shown that the fluorescence of the external site II (+)-BPDE-DNA adducts is sensitive to acrylamide, while the fluorescence of the dominant site I (-)-BPDE-DNA adducts is not affected by this fluorescence quencher, as expected for adducts with considerable carcinogen-base stacking interactions.     

Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo[a]pyrene diol epoxide.

The unwinding of supercoiled phi X174 RFI DNA induced by the tumorigenic (+) and non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) has been investigated by agarose slab-gel and ethidium titration tube gel electrophoresis. The differences in adduct conformations were verified by flow linear dichroism techniques. Both enantiomers cause a reversible unwinding by the formation of noncovalent intercalative complexes. The effects of covalently bound BPDE residues on the electrophoretic mobilities of the RF I DNA form in agarose gels were investigated in detail in the range of binding ratios rb approximately 0.0-0.06 (covalently bound BPDE residues/nucleotide). In this range of rb values, there is a striking difference in the mobilities of (+)-BPDE- and (-)-BPDE-adducted phi X174 DNA in agarose slab-gels, the covalently bound (+)-BPDE residues causing a significantly greater retardation than (-)-BPDE residues. Increasing the level of covalent adducts beyond rb approximately 0.06 in the case of the (+)-BPDE enantiomer, leads to further unwinding and a minimum in the mobilities (corresponding to comigration of the nicked form and the covalently closed relaxed modified form) at rb 0.10 +/- 0.01; at still higher rb values, rewinding of the modified DNA in the opposite sense is observed. From the minimum in the mobility, a mean unwinding angle (per BPDE residue) of theta = 12 +/- 1.5 degrees is determined, which is in good agreement the value of theta = 11 +/- 1.8 degrees obtained by the tube gel titration method. Using this latter method, values of theta = 6.8 +/- 1.7 degrees for (-)-BPDE-phi X174 adducts are observed. It is concluded that agarose slab gel techniques are not suitable for determining unwinding angles for (-)-BPDE-modified phi X174 DNA because the alterations in the tertiary structures for rb < 0.06 are too small to cause sufficiently large changes in the electrophoretic mobilities. The major trans (+)-BPDE-N2-guanosine covalent adduct is situated at external binding sites and the mechanisms of unwinding are therefore different from those relevant to noncovalent intercalative BPDE-DNA complexes or to classical intercalating drug molecules; a flexible hinge joint and a widening of the minor groove at the site of the lesion may account for the observed unwinding effects. The more heterogeneous (-)-BPDE-nucleoside adducts (involving cis and trans N2-guanosine, and adenosine adducts) are less effective in causing unwinding of supercoiled DNA for reasons which remain to be elucidated.     

High ratio of alkali-sensitive lesions to total DNA modification induced by benzo(a)pyrene diol epoxide

The ratio of alkali-labile lesions to total DNA adducts for DNA modified by an active metabolite of benzo(a)pyrene was investigated using DNA sequencing methodology. About 40% of the adducts formed result in alkali-labile sites. About 25% of the lesions were alkali-labile at positions of guanine, 10% at adenine, and 5% at cytosine. This study highlights the potential role of adducts other than the N2-substituted guanine in mutagenic and carcinogenic effects of benzo(a)pyrene.     

Excision of benzo[a]pyrene diol epoxide I adducts from nucleosomal DNA of confluent normal human fibroblasts

The formation and removal of covalent adducts of racemic 7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) was studied in nucleosomal DNA of confluent cultures of normal human fibroblasts (NF). For this purpose NF were prelabeled in their DNA with [14C]-thymidine and treated with [3H]BPDE I. The adducts were composed of 77% (7R)-N2-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl)deoxyguanosine, 12% of the corresponding 7S-enantiomer and of minor amounts of adducts to cytosine and adenine. The adduct composition did not change significantly in 24-h post treatment incubation. Bulk mononucleosomes were prepared from micrococcal nuclease digested nuclei and their DNA analyzed by gel electrophoresis. The adduct concentrations were determined in 145 base pair (b.p.) nucleosomal core-DNA, 165 b.p. chromatosomal DNA and in total nuclear DNA. From these data the concentration in nucleosomal linker-DNA was calculated. The initial adduct distribution was non-random and 6.3 times higher in 47 b.p. linker-DNA relative to 145 b.p. core-DNA and 9.2 times higher in 27 b.p. linker-DNA relative to 165 b.p. chromatosomal DNA. Adduct removal was very rapid during the first 8 h and more efficient from linker-DNA than from core-DNA. After this early phase the adducts located in 145 b.p. core-DNA became refractory to further excision and represent a major fraction of the adducts persisting in DNA of NF over a prolonged period. In contrast, further adduct removal was observed from nucleosomal linker-DNA.     

Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes

Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection. The rate of recovery of replicative capacity is inversely related to the initial adduct number. Holding the infected cells temporarily under conditions that prevent viral DNA replication results subsequently in increased recovery, proportional to the holding time. The mechanism of recovery appears to be constitutive and prereplicative. In addition, there is a second mode of recovery which is induced by pretreatment of the host cells with BPDE before infection. The effect of pretreatment is similar to that of extending the holding time before replication: the first molecules begin to replicate earlier but the subsequent rate of recovery is unchanged. The induced mechanism may be either a limited stoichiometric repair process or a slow replicative bypass.     

Sequence preferences of covalent DNA binding by anti-(+)- and anti-(-)-benzo[a]pyrene diol epoxides

The sequence preferences of formation of piperidine-labile adducts of guanine by individual (+)- and (-)-isomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [anti-(+)- and anti-(-)-BPDE] were examined by techniques analogous to chemical DNA sequencing. Data were obtained on over 1200 bases with anti-(-)-BPDE and 1000 bases with anti-(+)-BPDE. Guanines on average yielded more labile adducts than other bases, and the reactivities of guanines with both anti-(+)- and anti-(-)-BPDE isomers were found to be distinctly nonrandom with respect to DNA sequence. The most and least reactive guanines, defined in terms of the upper and lower 10 percentiles of reactivity, differed on average by a factor of 17. This range of guanine reactivities was correlated with distinct sequence preferences, which differed in part for the two isomers. The strongest determinant for preferred reaction of anti-(-)-BPDE to form a labile adduct at a guanine was the presence of a 3'-flanking guanine, but a thymine 5'-flanking a guanine also generally enhanced reactivity. The triplets containing central guanines most preferred by anti-(-)-BPDE were AGG, CGG, and TG(G greater than T greater than C,A). anti-(+)-BPDE also formed labile adducts preferentially at AGG and CGG triplets, but not at TGN triplets. Significant effects of next-nearest-neighbor bases on guanine reactivities were also noted.     

Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]G.C adduct structure and comparison with the (+)-trans-anti-[BP]G.C enantiomer.

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fate of SV40 DNA in infected cells after treatment with benzo(a)pyrene diol epoxide

Treatment of SV40-infected monkey kidney cells with $\text{anti}$-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide results in the alkylation of viral DNA. The specific infectivity of viral DNA isolated from diol epoxide-treated cells is markedly lower than that of viral DNA from untreated cells. Within 5–6 hours following treatment, approximately half of the alkylated viral DNA disappears; during this same period, single-stranded regions in SV40 DNA are detected. These data indicate that, in infected cells, a significant amount of the alkylated viral DNA is removed from the cellular DNA pool while the remainder stays intact. Whether BP-modified SV40 DNA undergoes repair is still uncertain.     

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.     

Excimer fluorescence of (+)-anti-benzo (a)pyrene diol epoxide covalently bound to poly(dG-dC): structural implications

Fluorescence of (+)-anti-benzo(a)pyrene diol epoxide [(+)-anti-BPDE] covalently bound to poly(dG-dC) has been studied with steady-state and time-resolved techniques. Extensive formation of excimers is found, even at small (0.008) BPDE/nucleotide ratios. This indicates favored covalent binding to bases close to already modified guanines. Both fluorescence excitation spectra and lifetime measurements reveal two populations of (+)-anti-BPDE adducts: one that can form excimers and one that cannot. Three excimer lifetimes (4.5, 29, and 83 ns) are observed. Differently shifted monomer and excimer excitation spectra are discussed in terms of pyrene-pyrene exciton interactions, consistent with a distance shorter than 7 A between the excimer-forming BPDE chromophores.     

Binding geometries of benzo[a]pyrene diol epoxide isomers covalently bound to DNA. Orientational distribution

Flow linear dichroism (LD) of different benzo[a]pyrene diol epoxide (BPDE) isomers covalently bound to calf thymus DNA or poly(dG-dC) provides information about binding geometry and DNA perturbation. With anti-BPDE the apparent angle between the long axis (z) of the pyrene chromophore and the DNA helix axis is approximately 30 degrees as evidenced from the LD of z-polarized absorption bands in the pyrenyl chromophore at 252 and 346 nm. The corresponding angle for the in-plane short axis (y) is determined to be approximately 70 degrees from a y-polarized band at 275 nm. The binding of (+)-anti-BPDE to DNA is found to cause a considerable reduction of the DNA orientation. This is ascribed to a decreased persistence length of DNA, owing either to increased flexibility ("flexible joints") or to permanent kinks at the points of binding. The reduced linear dichroism (LDr), i.e., the ratio between LD and isotropic absorbance, of the long-wavelength absorption band system of BPDE bound to DNA exhibits a wavelength dependence that indicates a relatively wide orientational distribution of the z axis of pyrene. Fluorescence data support the conclusion of a heterogeneous distribution, and a very low polarization anisotropy indicates a mobility between the different orientational states, which is rapid compared to the fluorescence lifetime (nanosecond time scale). Attempts are made to simulate the observed LDr features of the (+)-anti-BPDE-poly(dG-dC) complex using different distribution models on the assumption that the angular dependence of the spectral perturbation is due to dispersive interactions with DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of benzo(a)pyrene by isolated hepatocytes and factors affecting covalent binding of benzo(a)pyrene metabolites to DNA in hepatocyte and microsomal systems

Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrene-treated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxides) and of epoxide derivatives of BP-9-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:1 to 3:1 between 30 and 100 μm BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated.     

Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC).(dG-dC) studied by synchrotron excited fluorescence polarization anisotropy decay.

Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.     

Kinetics of DNA alkylation, depurination and hydrolysis of anti diol epoxide of benzo(a)pyrene and the effect of cadmium on DNA alkylation

Anti benzo[a]pyrene diol epoxide (BPDE) alkylates guanines of DNA at N7 in the major groove and at the exocyclic amino group in the minor groove. In this report we investigated the rates of BPDE hydrolysis, DNA alkylation and subsequent depurination of BPDE-adducted pBR322 DNA fragment using polyacrylamide gel electrophoresis. Preincubation studies showed that it hydrolyzed completely in triethanolamine buffer in <2 min. The depurination kinetics showed that a fraction of the N7 alkylated guanine depurinated rapidly; however a significant amount of N7 guanine alkylation remained stable to spontaneous depurination over a 4-h period. Similar results were obtained for the hydrolysis and alkylation rates of syn isomer but it required nearly 500 times more concentration to induce similar levels of N7 guanine alkylation. Cadmium ion strongly inhibited the N7 guanine alkylation of both isomers. But the minor groove alkylation was not affected as demonstrated by postlabeling assay which confirmed the presence of heat-and cadmium-stable minor groove adducts in BPDE-treated calf thymus DNA. Based on these and our earlier findings, we propose a mechanism for the synergistic effect of cadmium in chemically induced carcinogenesis.     

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Human DNA polymerase ι (polι) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of polι to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to polι-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 × 10^–3 and 6 × 10^–4. Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polκ, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that polι might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polκ.