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High performance liquid chromatography/UV-absorption and 32 P-postlabeling were used to quantitate adducts generated by reaction of the four configurationally isomeric benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides with native or denatured DNA in vitro. For both the 4R,3S-dihydrodiol 2S,1R-epoxide and the 4S,3R-dihydrodiol 2S,1R-epoxide, the amount of product resulting from trans-opening of the epoxide ring by the exocyclic amino group of deoxyadenosine in denatured DNA was much less than the level found in native DNA, indicating that the native DNA structure probably intercalates the hydrocarbon residue in a fashion that promotes adenine reaction for 2S,1R-epoxides.

Introduction During the 30 years or so following the identification of the first pure chemical carcinogen (1), no common factors or pathways in the mechanism of action of carcinogens from different chemical classes were evident. For this reason perhaps, each class of carcinogen, e.g. the polycyclic aromatic hydrocarbons, the aromatic amines and the nitrosamines, was often reviewed and discussed separately. The discovery by the Millers and their colleagues of the role of metabolism in carcinogen activation (2) revealed a commonality amongst many carcinogens, i.e. a chemical reactivity towards cellular macromolecules, such as DNA (3,4). Today, DNA adduct formation is recognized as a common property of most potent carcinogens and the formation of such adducts is the basis of several current strategies in molecular epidemiology and biomonitoring. Despite this common aspect of mechanism for many chemical carcinogens, the complexities of metabolic activation and of the chemistry and stereochemistry of adduct formation have tended to keep some degree of compartmentalization in research and in literature reviews of DNA adduct formation by different classes of chemical compounds (5).

A 16mer oligonucleotide containing a single guanine residue at nucleotide 13 from the 3' end was treated with the (+)-enantiomer of the 7,8-dihydrodiol 9,10-epoxide of benzo[a]pyrene (B[a]P). Oligonucleotides containing either an adduct in which the epoxide ring was opened trans or cis by the amino group of the guanine residue were separated by chromatography and identified by 32P postlabeling and circular dichroism spectroscopy. In the presence of nucleotide triphosphates and DNA polymerase (either Sequenase, version 2.0 or human polymerase alpha), it was found that the B[a]P adducts inhibited extension of an 11mer primer opposite the nucleotide 3' to the adduct in the template. Under various conditions, this inhibition was greater for the cis adduct than for the trans adduct. After a 10 min incubation with Sequenase, primer extension was reduced to approximately 20% of that seen with unmodified oligonucleotide by the trans adduct and was almost completely inhibited by the cis adduct. When a 12mer primer was used to examine nucleotide incorporation directly across from the guanine or adducted guanine residues, it was clear that deoxycytidylic acid was preferentially incorporated in all cases but that the incorporation was severely inhibited by both the cis and trans adducts. These findings suggest that a cis adduct is a more effective block to replication than a trans adduct, and that these adducts may not be very efficient mutagenic lesions.

Purine deoxyribonucleoside 3'-phosphates were reacted with the (+)- and (-)-enantiomers of the anti dihydrodiol epoxide of benzo[a]pyrene. Products from cis and trans opening of the epoxide ring were separated by HPLC and they were identified by comparison of their CD spectra with those known for the corresponding nucleoside adducts. Thereafter, the eight known benzo[a]pyrene-purine deoxyribonucleoside-3'-phosphate adducts were postlabeled with [32P]ATP and T4 kinase and the positions of these individual bisphosphates were mapped by TLC. Though all eight adducts migrated to the same general region of the thin layer plates, the four possible adducts from each enantiomeric dihydrodiol epoxide were resolved.

The effect of a potent mammary carcinogen, anti benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide, on the progress of human mammary carcinoma MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G1 after treatment with actinomycin D (a positive control), treatment with the mammary carcinogen did not cause G1 arrest but instead delayed the cells in the DNA synthesis phase. In concert with the absence of a G1 arrest, it was found that though both chemical treatments led to increased levels of p53, only the p53 induced by actinomycin D was transcriptionally active and increased the levels of the cyclin dependent kinase inhibitor, p2l waf1/cip1 . Since treatment of the cells with the mammary carcinogen did not abrogate the G1 arrest induced by actinomycin D, the lack of p21 waf1/ cip1 and of G1 arrest, resulting from treatment with the mammary carcinogen alone, was not due to some general inhibition of transcription or translation. An analogous difference between these two chemicals was demonstrated also in other human cell systems. The stealth-like property of the mammary carcinogen that allows it to damage DNA without turning on the cells' 'guardian of the genome' defense mechanism presumably increases the likelihood of malignant change because DNA replication continues on a damaged template. It is suggested that this stealth characteristic may be a major contributor to the high carcinogenic potency of this mammary carcinogen and possibly to that of other highly potent carcinogens.

Mutation induction in the supF gene of the plasmid pS189 by 7-bromomethylbenz[a]anthracene and 7-bromomethyl-12-methylbenz[a]anthracene was examined. The former compound was substantially more mutagenic than the latter but a much greater proportion of the total mutations were located at mutation hotspots for the 12-methyl derivative. The overall correlation between sites of mutation and sites of polymerase arrest (an indicator of adduct formation) through the supF gene was poor. Although these bromocompounds should form only a single guanine adduct (unlike dihydrodiol epoxides that form both cis and trans adducts) more than one mutational change was found at a given site, although the predominant base substitution was G-->T for either compound.

Nitric oxide is an important bioregulatory agent that may also be an endogenous and exogenous human mutagen. In order to study mutations generated following exposure of a shuttle vector-borne target gene to nitric oxide, mutations were induced in the supF gene of the pSP189 shuttle vector by treatment with nitric oxide in aerobic buffered solution followed by replication of the plasmid in either human Ad293 or Escherichia coli MBM7070 cells. The induced mutation frequency, which increased with nitric oxide dose, was 44-fold greater than the spontaneous background in human cells and > 15-fold greater than background in the bacterial cells when a total of 100 mmol of nitric oxide was oxidatively absorbed/I of pH 7.4 buffer containing the plasmid. The majority of point mutations analysed (61 and 75% for human and E. coli cells respectively) were AT-->GC transitions with GC-->AT transitions (29 and 23%) being the next most prevalent. The overall frequencies of the various point mutations seen in the supF gene were similar in the two cell types, although the distribution of hotspots showed differences. The results are consistent with a mutational mechanism initiated by deamination of DNA bases.

The action of DNA polymerase (Sequenase Version 2.0) on an oligonucleotide template containing a 7-bromomethyl-benz[a]anthracene-deoxyadenosine adduct flanked by thymidine residues was investigated. The polymerase incorporated deoxyadenosine or deoxyguanosine residues opposite the thymidine 3' to the adduct with similar efficiencies. Whereas the normal A.T base pair led to arrest of polymerase progression along the template, formation of the G.T mismatch allowed incorporation of thymidine opposite the adduct and further primer extension. This mispair-mediated bypass was also seen with AMV reverse transcriptase and may represent a novel mechanism for overcoming the replication block of a bulky carcinogen--DNA adduct.

To examine the effect of DNA adducts on nucleotide incorporation by DNA polymerase at 3' neighboring bases, synthetic oligonucleotides (16mers) containing a purine at position 13 from the 3' end and any one of the four possible bases at position 12 were prepared and reacted with 7-bromomethylbenz[a]anthracene. Using HPLC, unmodified oligonucleotide was separated from oligonucleotide containing a single adduct, at either an adenine or a guanine residue. These products were annealed with a 32P 5'-end labeled primer (11mer) and incubated with modified T7 DNA polymerase (Sequence, version 2.0) in the presence of deoxyribonucleoside 5'-triphosphates. Analysis by gel electrophoresis showed that unmodified oligonucleotide template allowed the primer to be rapidly extended to the entire length of the template. However, the presence of an adduct caused primer extension to stop at the base 3' to the adduct. While correct base pairing occurred at this termination site with most adducted templates, there was a high frequency of misincorporation of guanine opposite a thymine located 3' to an adenine adduct. This result suggest that some bulky carcinogen--DNA adducts may lead to base mismatches at neighboring bases.

An SV40-based pS189 shuttle vector, which contained a supF target gene and was replicated in human cells (Ad293), was used to determine the mutational specificity of anti 5-methylchrysene 1,2-dihydrodiol 3,4-epoxide, the active metabolite of the environmentally prevalent carcinogen 5-methylchrysene. The frequency of supF mutants containing point mutations increased with dose to approximately 40 times the spontaneous frequency. The induced mutations were not randomly distributed but occurred preferentially at mutagenic hotspots, which were not all identical to those reported by others for benzo[a]pyrene dihydrodiol epoxide, a metabolite with similar chemistry.

In order to determine how individual hydrocarbon-DNA adducts give rise to specific mutations, a single-stranded oligonucleotide, 5'-T8GT10AT8C2T4CT3CT-3', was reacted with the carcinogen 7-bromomethylbenz[a]anthracene which generates both deoxyguanosine and deoxyadenosine adducts in DNA. The products were separated by HPLC to yield unmodified oligonucleotide and oligonucleotide modified either at the single guanine, or at the single adenine, residue. Incubation of these products with 32P-5'-end-labeled primer, 5'-AGA3GA4G2-3', modified T7 DNA polymerase (Sequenase) and deoxyribonucleoside-5'-triphosphates followed by gel electrophoretic analysis indicated that unmodified oligonucleotide template allowed the primer to be rapidly extended to give species of the same length as the template (40 nucleotides) and of 41 nucleotides in length. However, primer extension for the templates containing the guanine and adenine adducts was held up initially (1 min) at the nucleotide preceding the adduct. At longer times (up to 15 min) a nucleotide was added opposite the adduct and, to a lesser extent, another nucleotide was added beyond this. Some full-length oligonucleotide was also synthesized with these carcinogen-modified templates. When synthesis was allowed to proceed only to the nucleotide preceding the adduct, and this template-extended primer complex incubated with individual nucleotide triphosphates plus Sequenase, it was found that deoxyadenosine residues were most readily incorporated opposite the adduct irrespective of whether it was a deoxyguanosine or deoxyadenosine adduct. These results, which suggest that G.C----T.A and A.T----T.A transversions would be the mutagenic consequences of formation of bulky hydrocarbon adducts at guanines and adenines respectively, are consistent with the most frequent hydrocarbon-induced mutational changes reported thus far.

7,12-Dimethylbenz[a]anthracene (DMBA)--deoxyribonucleoside adducts formed in mouse skin DNA were quantified in order to determine whether these changed in any systematic fashion under conditions where the tumorigenic activity of DMBA is modified. Similar distributions of adducts were found in male NIH Swiss mice and C57BL mice which exhibit different sensitivities to initiation-promotion using DMBA as initiator, though in both these strains of mice the bay region syn dihydrodiol epoxide is responsible for a greater fraction of total binding at higher DMBA doses. Pretreatment with various chemicals known to inhibit the tumor initiating activity of DMBA in mouse skin did not lead to selective inhibition of the formation of any adduct in female NIH Swiss mice. However, the effects of these agents ranged from a clear inhibition of overall DNA binding (7,8-benzoflavone) to little or no effect on overall binding (butylated hydroxyanisole, butylated hydroxytoluene). The lack of any effect of the antioxidants on DMBA--DNA adduct formation suggests that they may affect some step in tumor initiation other than adduct formation.

The crystal structure of a novel nucleoside isolated from guanosine/p-methylbenzyl chloride reactions demonstrates linkage between the methylene carbon of the benzyl moiety and carbon-5 of guanosine, and loss of the carbonyl function at carbon-6 of guanosine, to yield 4-(p-methylbenzyl)-5-guanidino-1-beta-D-ribofurasylimidazole. These findings suggest that carbon-5 of guanine in DNA is a potential site of reaction for electrophilic ultimate carcinogens.

The nature of the metabolites of 7,12-dimethylbenz[a]anthracene (DMBA) binding to DNA in the presence of Aroclor-induced rat liver microsomes is not affected by the presence or absence of magnesium ions, but is dependent on the concentration of the hydrocarbon. At high concentrations of DMBA, the primary route of metabolic activation is through the K-region oxide while, at low concentrations of DMBA, activation is through other routes. A small proportion of these latter products elute from Sephadex LH-20 columns with mouse embryo cell [14C]DMBA-DNA adducts which are known to arise through reaction of the bay region diol-epoxide of DMBA with cellular DNA. In contrast to the dose-dependence of activation of DMBA by microsomes, the binding of this carcinogen to DNA in intact cultured mouse embryo cells is not qualitatively influenced by concentration over a forty-fold dose range. These findings suggest limitations in the use of microsomal systems as models for target tissue activation.

Compared with standard Sephadex LH-20 column chromatography, a newly developed high pressure liquid chromatographic separation of hydrocarbon deoxyribonucleoside adducts derived from the DNA of mouse embryo cell cultures exposed to 7,12-dimethylbenz[a]anthracene (DMBA) provides markedly superior resolution. Once resolved, the fluorescence spectroscopic properties of the three major DMBA--DNA adducts indicate that the fluorescence exhibited by adducts derived from a bay region syn dihydrodiol epoxide of DMBA differs subtly from that exhibited by adducts derived from the isomeric anti dihydrodiol epoxide.

[3H]7,12-Dimethylbenz[a]anthracene-modified DNA obtained from mouse cells in culture was enzymatically hydrolyzed to nucleoside 3'-phosphates, postlabeled with [32P]phosphate, and the carcinogen-modified nucleoside bisphosphates were separated by thin layer chromatography. Each adduct spot was eluted, dephosphorylated and the resulting [3H]nucleoside adducts were analyzed by high pressure liquid chromatography so that the structural information available for the liquid chromatographic peaks could be applied to the spots obtained from the postlabeling procedure. After this cross referencing, specific dihydrodiol epoxide-nucleotide adducts can now be monitored by the postlabeling technique.

Four 7,12-dimethylbenz[a]anthracene--deoxyribonucleoside adducts formed in mouse epidermis in vivo arise from the syn dihydrodiol epoxide metabolite of this carcinogen. With the synthetic syn dihydrodiol epoxide it was possible to identify three of these as deoxyadenosine adducts and to establish their structures. These three adducts account for the large majority of DNA adduct arising from this metabolite in vivo. The in vivo metabolite is unusual, therefore, in that it reacts almost exclusively with adenine residues in DNA while most carcinogen metabolites react preferentially with guanine residues.

Products of reaction of the racemic anti bay region 1,2-dihydrodiol-3,4-epoxide of 5-methylchrysene with DNA were identified by comparison with the products formed in reactions with individual nucleotides. The latter products, i.e. two deoxyguanosine adducts and four deoxyadenosine adducts, were characterized by various spectroscopic methods. In DNA, in addition to the major deoxyguanosine adduct already identified by Melikian et al. (Cancer Res., 44, 2524, 1984), we have now identified a second deoxyguanosine adduct arising from the trans opening of the epoxide ring by the amino group of deoxyguanosine. This differs from the adduct characterized by Melikian et al. only in that it arises from the opposite enantiomer of the dihydrodiol epoxide. Three deoxyadenosine adducts were also found in DNA. Two of these arose from the trans opening of the epoxide ring of each dihydrodiol epoxide enantiomer by the amino group of deoxyadenosine and the third from the cis opening of the epoxide ring of one enantiomer. Approximately 32% of the racemic dihydrodiol epoxide reacts with DNA rather than with water and this high extent of reaction with DNA is attributed to the out-of-plane deformations arising from the methyl substitution in the bay region.