Lesion selectivity in blockage of lambda exonuclease by DNA damage.

Various kinds of DNA damage block the 3' to 5' exonuclease action of both E. coli exonuclease III and T4 DNA polymerase. This study shows that a variety of DNA damage likewise inhibits DNA digestion by lambda exonuclease, a 5' to 3' exonuclease. The processive degradation of DNA by the enzyme is blocked if the substrate DNA is treated with ultraviolet irradiation, anthramycin, distamycin, or benzo[a]-pyrene diol epoxide. Furthermore, as with the 3' to 5' exonucleases, the enzyme stops at discrete sites which are different for different DNA damaging agents. On the other hand, digestion of treated DNA by lambda exonuclease is only transiently inhibited at guanine residues alkylated with the acridine mustard ICR-170. The enzyme does not bypass benzo[a]-pyrene diol epoxide or anthramycin lesions even after extensive incubation. While both benzo[a]-pyrene diol epoxide and ICR-170 alkylate the guanine N-7 position, only benzo[a]-pyrene diol epoxide also reacts with the guanine N-2 position in the minor groove of DNA. Anthramycin and distamycin bind exclusively to sites in the minor groove of DNA. Thus lambda exonuclease may be particularly sensitive to obstructions in the minor groove of DNA; alternatively, the enzyme may be blocked by some local helix distortion caused by these adducts, but not by alkylation at guanine N-7 sites.     

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.     

Formation and removal of benzo[a]pyrene metabolites--DNA adducts in cultured normal human fibroblasts using a microsome-activating system

The rate of removal of DNA adducts of several benzo[a]pyrene metabolites from nuclear DNA was compared by introducing a microsome-activating system in human fibroblast cells. Confluent human fibroblasts were exposed to benzo[a]pyrene in the presence of a microsomal activating system and DNA adducts were formed in the nuclear DNA. The adducts present in DNA were determined after 1 h of incubation and 48 h later. There was no difference in the rate of removal between 7S- and 7R -N2-[10-(7 beta, 8 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene)yl]deoxyguanosine, 7R -N2-[10(7beta, 8 alpha, 9 beta-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyguanosine and the covalent adduct of 9-hydroxybenzo[a]pyrene-4,5-epoxide to guanosine. This finding does not agree with the idea that metabolites forming 'persistent DNA adducts' are always responsible for the carcinogenicity of their parent compound.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

The role of hRev7, the accessory subunit of hPolζ, in translesion synthesis past DNA damage induced by benzo[a]pyrene diol epoxide (BPDE)

Background  

DNA polymerase zeta (Polζ) is a specialized DNA polymerase that, unlike classical replicative polymerases, is capable of replicating past DNA lesions, i.e. of performing translesion synthesis (TLS). The catalytic subunit of hPolζ, hRev3, has been shown to play a critical role in DNA damage-induced mutagenesis in human cells, but less is known about the role of hRev7, the accessory subunit of hPolζ, in such mutagenesis. To address this question, we recently generated human fibroblasts with very significantly reduced levels of hRev7 protein and demonstrated that hRev7 is required to protect cells from ultraviolet_{(254 nm)} (UV) radiation-induced cytotoxicity and mutagenesis (McNally et al., DNA Repair 7 (2008) 597-604). The goal of the present study was to determine whether hRev7 is similarly involved in the tolerance of DNA damage induced by benzo[a]pyrene diol epoxide (BPDE), the reactive form of the widespread environmental carcinogen benzo[a]pyrene.     

Induction of aryl hydrocarbon hydroxylase and DNA adduct formation in parental and carcinogen transformed C3H/10T1/2 clone 8 cells by benzo[a]pyrene

C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.     

The effect of caffeine on cytotoxicity, mutagenesis, and sister-chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges.     

Two-step error-prone bypass of the (+)- and (-)-trans-anti-BPDE-N2-dG adducts by human DNA polymerases eta and kappa

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase eta (Poleta) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Poleta predominantly inserted an A opposite a template (+)- and (-)-trans-anti-BPDE-N2-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Poleta. Error-prone nucleotide insertion by human Poleta was more efficient opposite the (+)-trans-anti-BPDE-N2-dG adduct than opposite the (-)-trans-anti-BPDE-N2-dG. However, translesion synthesis by human Poleta largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Poleta from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N2-dG adducts, the nucleotide opposite the lesion, and the sequence context 5' to the lesion. By combining the nucleotide insertion activity of human Poleta and the extension synthesis activity of human Polkappa, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts.     

Induction of alkaline-labile sites in DNA by benzo [a]pyrene and the repair of those lesions in cultured Chinese hamster cells

Formation of alkaline-labile sites in DNA by S9-activated benzo [a]pyrene (B [a]P) and the repair of those lesions were investigated using the technique of alkaline elution in cultured Chinese hamster V79 cells. When the cells were treated with B [a]P (1-5 micrograms/ml) there was negligible increase in DNA elution at pH 12.1 as compared to untreated controls. However, the elution of DNA increased at pH 12.6 with a concentration dependency, thereby indicating formation of alkaline-labile sites in DNA by B [a]P. After 4 h of repair incubation the elution of DNA at pH 12.6 of B [a]P (5 micrograms/ml) treated cells returned to the control levels. The half-life of alkaline-labile sites formed by B [a]P was approximately 1.5 h. Inhibitors of DNA-repair synthesis, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (ara-C) when added simultaneously with S9-activated B [a]P for 3 h showed an increase in elution of DNA at pH 12.1, indicating that a population of B [a]P-induced DNA lesions could be removed by a rapid DNA-repair process. These results indicate that at least two kinds of DNA lesions, repairable alkaline-labile sites and rapidly repairable DNA single-strand breaks, are detected after B [a]P treatment by the use of the alkaline elution procedure, by changing elution pH.     

Induction of alkaline-labile sites in DNA by benzo[a]pyrene and their repair of the lesions in cultured Chinese hamster cells

Formation of alkaline-labile sites in DNA by S9-activated benzo[a]pyrene (B[a]P) and the repair of those lesions were investigated using the technique of alkaline elution in cultured Chinese hamster V79 cells.When the cells were treated with B[a]P (1–5 μg/ml) there was negligible increase in DNA elution at pH 12.1 as compared to untreated controls. However, the elution of DNA increased at pH 12.6 with a concentration dependency, thereby indicating formation of alkaline-labile sites in DNA by B[a]P. After 4 h of repair incubation the elution of DNA at pH 12.6 of B[a]P (5 μg/ml) treated cells returned to the control lavels. The half-life of alkaline-labile sties formed by B[a]P was approximately 1.5 h. Inhibitors of DNA-repair synthesis, hydroxyrea (HU) and 1-β-arabinofuranosly cytosine (ara-C) when added simultaneously with S9-activated B[a]P for 3 h showed an increase in elution of DNA at pH 12.1 indicating that a population of B[a]P-induced DNA lesions could be removed by a rapid DNA-repair process.These results indicate that at least two kinds of DNA lesions, repairable alkaline-labile sites rapidly repairable DNA single-strand breaks, are detected after B[a]P treatment by the use of the alkaline elution procedure, by changing elution pH.     

DNA--benzo[a]pyrene adducts formed in a Salmonella typhimurium mutagenesis assay system.

The DNA adducts formed in Salmonella typhimurium when bacteria are incubated with radioactive benzo[a]pyrene and liver microsomal enzymes from several sources has been investigated. When enzyme preparations from Aroclor I254 or 3-methylcholanthrene induced C57BL/6N (B6) mice were used to mediate activation, the predominant product was an adduct between the 10 position of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the N-2 position of deoxyguanosine. Similar results were obtained with human liver and with Aroclor-induced rat-liver enzyme preparations. This adduct is also the major DNA product previously found when human tissues or certain rodent cells were incubated with benzo[a]pyrene. On the other hand, when activation of benzo[a]pyrene was mediated by a phenobarbital-induced B6 mouse-liver enzyme preparation, the extent of binding was quite low and the profile of DNA adducts in S. typhimurium DNA was quite different. Thus, under appropriate conditions, the activation and DNA binding of benzo[a]pyrene inthe microsome mediated S. typhimurium mutagenesis assay generally resembles that seen in intact mammalian cells. Caution must be exercised, however, in the choice of microsome-activation systems.     

Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis.

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.     

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.     

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5,6-dimethylchrysene, of benzo[g]chrysene and of 7-methylbenz[a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at AT or GC pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[a]pyrene or benzo[c]phenanthrene dihydrodiol epoxide–DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.     

Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system.

We have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. We show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. Polyacrylamide gels indicate that a shorter DNA product is produced on modified templates and this is confirmed by determining the average chain lengths from the ratio of chain initiations to chain elongation. Gene 4 protein primed synthesis reactions display a greater sensitivity to the presence of B[a]P adducts than do oligonucleotide-primed reactions. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, we present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies [Brown, W. C., & Romano, L. J. (1989) J. Biol. Chem. 264, 6748-6754] have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, we observe an increase in synthesis but to lesser extent than observed on oligonucleotide-primed synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres

Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.     

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites.

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N2-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.     

Benzo[a]yrenedione/benzo[a]pyrenediol oxidation-reduction couples and the generation of reactive reduced molecular oxygen.

The ability of the isomeric quinone metabolites of benzo[a]pyrene, benzo[a]pyrene-6,12-dione, benzo[a]pyrene-1,6-dione, and benzo[a]pyrene-3,6-dione to undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrenediols and intermediate semiquinone radicals has been characterized. Under anaerobic conditions, all three benzo[a]pyrenediones are easily reduced to benzo[a]pyrenediols, even by mild biological agents such as NAD(P)H, cysteamine, and glutathione. The benzo[a]pyrenediols, in turn, are very rapidly autoxidized to the benzo[a]pyrenediones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autoxidations, and other reactive reduced oxygen species, such as the superoxide and hydroxyl radicals, are probably formed transiently as well. The benzo[a]pyrenediol-benzo[a]pyrenedione interconversions proceed by one-electron steps; the corresponsing semiquinone radicals can be monitored by electron spin resonance spectroscopy as inter mediates during these reactions carried out at high pH. Benzo[a]pyrenediones induce DNA strand scission when incubated with bacteriophage T7 DNA. This damage is modified by conditions which indicate that reduced oxygen species propagate the free-radical reactions responsible for the strand scission. Benzo[a]pyrenediones are electron-acceptor substrates for NADH dehydrogenase from Clostridium kluyveri. Catalytic amounds of these benzo[a]pyrene metabolites, together with this respiratory enzyme function as cyclic oxidation-reduction couples which link NADH and molecular oxygen in the continuous production of hydrogen peroxide. These data, together with preliminary results with cells in culture, indicate that benzo[a]pyrenediones are potentially harmful metabolites of benzo[a]pyrene, acting by processes which lead to their regeneration rather than depletion; nucleic acid and call damage is probably produced by the reactive reduced oxygen species resulting from such regenerative oxidation-reduction cycles.     

Synthesis and DNA binding properties of a new benzo[f]imidazo[1,5b]-isoquinoline-butan-1,2-diol derivative

A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-pentafuranoside 2 in 22% yield. After deprotection, the resulting compound 5 was converted to a DMT protected phosphoramidite building block 7 for standard DNA synthesis. DNA/DNA, DNA/RNA duplexes with 5 inserted as bulges were destabilized, except when the new amidite was used for the synthesis of a zipping duplex.