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.     

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.     

An analysis of DNA replication in synchronized CHO cells treated with benzo[a]pyrene diol epoxide

Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.     

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.     

Caffeine enhancement of the initiation of DNA replication in benzo[a]pyrene diol epoxide damaged cells

We have used a newly developed pH stepwise alkaline elution method to show that caffeine enhances DNA initiation (DNA replication in sub-replicon size nascent strands) in (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDEI) damaged mouse primary epidermal cells. Caffeine alone caused a dose-dependent increase in DNA initiation without an effect on DNA elongation (joining of replicon-sized nascent DNA). BPDEI alone inhibited DNA elongation as shown by a relative increase in sub-replicon size nascent DNA. When BPDEI treated cells were incubated with caffeine, there was a dose-dependent increase in sub-replicon size nascent DNA without a significant effect on the proportion of joined replicons. Therefore, caffeine can enhance DNA initiation in mammalian cells damaged with a reactive form of the carcinogen benzo[a]pyrene and this may account for the biological interaction between caffeine and the ultimate carcinogenic form of benzo[a]pyrene.     

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.     

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.     

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)     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

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.     

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.     

Stabilization of a reactive, electrophilic carcinogen, benzo[a]pyrene diol epoxide, by mammalian cells

We have studied several features of the interactions of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with a DNA repair-proficient derivative of Chinese hamster ovary cells (CHO), AT3-2, and with a UV-light sensitive mutant, UVL-10, derived from AT3-2. Methods were developed for quantitating the amount of unhydrolysed BPDE-I associated with cells and for purifying DNA from cells under conditions where artificial labeling during preparation is minimized. In both cell types, about 30% of the BPDE-I added to a cell culture is rapidly taken up by the cells and is maintained in a cellular compartment in which the half-life of BPDE-I is about 10-fold longer than in aqueous medium. The kinetics of covalent binding to DNA were measured in both cell types and found to be described well by a single exponential process with a half-life of about 60 min. This is virtually identical to the half-life for intracellular hydrolysis of BPDE-I (57 min), consistent with the suggestion that this intracellular, relatively stable BPDE-I is responsible for binding.     

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.     

Differential efficiency of mutagenesis at three genetic loci in CHO cells by a benzo[a]pyrene diol epoxide

The formation of DNA adducts by the ultimate carcinogen 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (BPDE-I) has been implicated in the process of carcinogenesis. In a line of Chinese hamster ovary (CHO) cells designated AT3-2 and in two derivative mutant lines, UVL-1 and UVL-10, originally selected for hypersensitivity to UV-irradiation, we have measured the formation of BPDE-I: DNA adducts and the production of biological damage. The quantity and quality of BPDE-I: DNA adducts formed initially in the 3 cell lines are identical over a wide range of BPDE-I doses. However, the UVL lines are unable to remove adducts from their DNA, while the AT3-2 cells remove about 50% of the BPDE-I: DNA adducts in a 24-h incubation. Correlated with this, the UVL lines are more sensitive to the lethal effects of BPDE-I than are the AT3-2 cells. Mutant frequencies were measured at the aprt, hprt and oua loci and were found to increase linearly with BPDE-I: DNA adduct formation at doses which gave greater than 50% survival. At the hprt and oua loci, the efficiency of mutation induction was similar for AT3-2 and UVL-10 cells. UVL-1 cells showed slightly higher (within a factor of 2-3) mutant frequencies in response to BPDE-I compared to AT3-2 at these two loci. However, at the aprt locus the repair-deficient cells were much more highly mutable (9-15-fold) than the repair-proficient AT3-2 cells. Based on the measured average level of adduct formation, it is calculated that 15% of the BPDE-I: DNA adducts in the aprt gene are converted into mutations. However, the possibility exists that the aprt locus is subject to higher levels of modification by BPDE-I than is the bulk DNA, which would lead to an artifactually high apparent conversion frequency.     

Reactivity with DNA of three pyrenofuran analogues of benzo(a)pyrene and benzo(e)pyrene

Three pyrenofurans, the pyreno[1,2-b]furan (FP1), the pyreno[2,1-b] furan (FP2) and the pyreno[4,5-b]furan (FP3) have been synthesized as analogues of the mutagenic and carcinogenic benzo(a)pyrene (FP1 and FP2) and of its non-carcinogenic isomer benzo(e)pyrene (FP3). For each of the pyrenofurans, the reactivity with DNA has been tested in presence of liver microsomes of rats induced with 3-methylcholanthrene. Fluorescence spectroscopy showed that only FP2 and FP3 which possess a "bay region" react with DNA. In both cases, metabolites bound to DNA have a fluorescence emission comparable to that of the "bay region" dihydrodiols obtained after the "in vitro" metabolism of initial molecules. FP2 is shown to react similarly to benzo(a)pyrene whereas the reactivity of FP3 is different from that of benzo(e)pyrene, in spite of their structural similarities. This is probably due to reasons of three-dimensional space configuration. The peculiar reactivity of FP3 is predicted by calculations of the bond order values.     

Mutagenesis of benzo[a]pyrene diol epoxide in yeast: requirement for DNA polymerase zeta and involvement of DNA polymerase eta

Benzo[a]pyrene is a potent environmental carcinogen, which can be metabolized in cells to the DNA damaging agent anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE). We hypothesize that mutations induced by BPDE DNA adducts are mainly generated through an error-prone translesion synthesis that requires a specialized DNA polymerase (Pol). Using an in vivo mutagenesis assay in the yeast model system, we have examined the potential roles of Pol(zeta) and Pol(eta) in (+/-)-anti-BPDE-induced mutagenesis. In cells proficient in mutagenesis, (+/-)-anti-BPDE induced 85% base substitutions with predominant G --> C followed by G --> T transversions, 9% deletions of 1-3 nucleotides, and 6% insertions of 1-3 nucleotides. In rad30 mutant cells lacking Pol(eta), (+/-)-anti-BPDE-induced mutagenesis was reduced and accompanied by a moderate decrease in base substitutions and more significant decrease in deletions and insertions of 1-3 nucleotides. In rev3 mutant cells lacking Pol(zeta), (+/-)-anti-BPDE-induced mutagenesis was mostly abolished, leading to a great decrease in both base substitutions and deletions/insertions of 1-3 nucleotides. In contrast, large deletions/insertions were significantly increased in cells lacking Pol(zeta). Consistent with the in vivo results, purified yeast Pol(zeta) performed limited translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-N(2)-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (+/-)-anti-BPDE-induced mutagenesis in yeast requires Pol(zeta) and partially involves Pol(eta) and suggest that Pol(zeta) directly participates in nucleotide insertions opposite the lesion, while Pol(eta) significantly contributes to deletions and insertions of 1-3 nucleotides.     

Base-sequence dependence of noncovalent complex formation and reactivity of benzo[a]pyrene diol epoxide with polynucleotides

The base-sequence selectivity of the noncovalent binding of (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyr ene (BPDE) to a series of synthetic polynucleotides in aqueous solutions (5 mM sodium cacodylate buffer, 20 mM NaCl, pH 7.0, 22 degrees C) was investigated. The magnitude of a red-shifted absorbance at 353 nm, attributed to intercalative complex formation, was utilized to determine values of the association constant Kic. Intercalation in the alternating pyridine-purine polymers poly(dA-dT).(dA-dT) (Kic = 20,000 M-1), poly(dG-dC).(dG-dC) (4200 M-1), and poly(dA-dC).(dG-dT) (9600 M-1) is distinctly favored over intercalation in their nonalternating counterparts poly(dA).(dT) (780 M-1), poly(dG).(dC) (1800 M-1), and poly(dA-dG).(dT-dC) (5400 M-1). Methylation at the 5-position of cytosine gives rise to a significant enhancement of intercalative binding, and Kic is 22,000 M-1 in poly(dG-m5dG).(dG-m5dC). In a number of these polynucleotides, values of Kic for pyrene qualitatively follow those exhibited by BPDE, suggesting that the pyrenyl residue in BPDE is a primary factor in determining the extent of intercalation. Both BPDE and pyrene exhibit a distinct preference for intercalating within dA-dT and dG-m5dC sequences. The catalysis of the chemical reactions of BPDE (hydrolysis to tetrols and covalent adduct formation) is enhanced significantly in the presence of each of the polynucleotides studied, particularly in the dG-containing polymers. A model in which catalysis is mediated by physical complex formation accounts well for the experimentally observed enhancement in reaction rates of BPDE in the alternating polynucleotides; however, in the nonalternating polymers a different or more complex catalysis mechanism may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)