The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA involves attack at the N7-position of guanine moieties

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE) with DNA prelabelled with [14C] and [3H]-purine precursors has indicated that in addition to the N2-position of guanine previously reported [10--12] reaction also involves the N7-position of guanine. The hydrocarbon-N7-guanine product was not detected earlier because it is lost from the DNA very readily at pH 7. The same N7-product was obtained by reaction of anti-BPDE with guanine in dimethylformamide.     

Antioxidative and antitumor promoting effects of [6]-paradol and its homologs

Benzo[a]pyrene diol epoxide, a metabolite of benzo[a]pyrene (BaP), and chlorohydrin, the reaction product of chloride and the epoxide, form in vitro the same trans- and cis-stereoisomeric DNA adducts, but in different proportions. In this study, we asked whether the DNA adduct concentration can be kept the same by applying the appropriate dose of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)and (+/-)-7r,8t,9t-trihydroxy-10c-chloro-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-BPDCH) to rodent skin and whether the DNA adducts formed differ only in their trans- and cis-stereoisomerism. Skin from C57Bl6 mice, spontaneous hypertension rats (SHR) and Sprague-Dawley (SD) rats was treated ex vivo immediately after the death of the animals with anti-BPDE and its corresponding bay region chlorohydrin trans-BPDCH and the epidermis was analyzed for DNA adducts 1h after the application. We found that adduct formation at the exocyclic amino groups of deoxyguanosine and deoxyadenosine in epidermal DNA followed a linear dose-response within 6--100 nmol/cm(2) with both chemicals. In order to achieve the same adduct concentration in mouse, spontaneous hypertension rat (SHR), and Sprague-Dawley (SD) rat skin, respectively, a 37-, 23- and 10-fold lower dose of anti-BPDE than of trans-BPDCH had to be applied. The order of 2'-deoxyguanosine (dGuo) adduct concentration with anti-BPDE was similar to what has been reported, but the order with trans-BPDCH was (+)-cis-BPDE-N(2)-dGuo adduct>(+)-trans-BPDE-N(2)-dGuo=(-)-trans-BPDE-N(2)-dGuo>(-)-cis-BPDE-N(2)-dGuo in mouse skin. Irrespective of species or strain, a significantly higher proportion of cis-adducts was obtained after treatment with trans-BPDCH than after treatment with anti-BPDE. Therefore, DNA adduct concentration can be kept the same by applying the appropriate dose of anti-BPDE and trans-BPDCH to rodent skin and the DNA adducts formed differ only in their trans- and cis-stereoisomerism.     

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.     

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.     

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.     

Identification and characterization of a novel benzo[a]pyrene-derived DNA adduct

The aim of this study was to generate and identify a novel benzo[a]pyrene (BP)-derived DNA adduct found both in vitro and in vivo. To date, the majority of studies have focused on N(2)-[10 beta(7 beta,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxyguanosine (anti-BPDE-dG), the major adduct generated following bioactivation of BP. However, a second adduct is also formed following bioactivation of BP which has been speculated to result from further metabolism of 9-OH-BP. In order to identify this second reaction pathway, the ultimate DNA binding species, and the DNA base involved, we have synthesized and characterized a dG-derived DNA adduct arising from further bioactivation of 9-OH-BP in the presence of rat liver microsomes. Analysis of the adducted nucleotides was conducted using both the (32)P-postlabeling assay and capillary electrophoresis-mass spectrometry (CE-MS).     

Sequence specificity in the reaction of benzopyrene diol epoxide with DNA

Benzopyrene diol epoxide (BPDE; (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene), the ultimate carcinogen derived from the polycyclic hydrocarbon benzo[a]pyrene, reacts principally with the guanine bases in DNA. Nineteen double stranded, self-complementary oligonucleotides, containing deoxyguanosine in various sequence contexts, were each treated with tritium labelled BPDE. The extent of reaction was determined by releasing the BPDE-guanine adduct with acid, isolating it by chromatography on a reverse-phase column, and estimating it by its radioactivity. Oligonucleotides containing an isolated guanine, such as AAGTACTT, were little affected by BPDE. Reactivity was increased where the guanine was flanked by another guanine on the same strand (e.g. TACCTAGGTA) or on the complementary strand (e.g. TATTCGAATA), and was highest in mixed G-C sequences such as ATCCGGAT. The results should help predict major sites of attack of BPDE on cellular proto-oncogenes.     

Mechanism of rat liver DNA methyltransferase interaction with anti-benzo[a]pyrenediol epoxide modified DNA templates

We investigated the methylation reaction catalyzed by 1500-fold purified rat liver DNA methyltransferase (DMase) on native Micrococcal luteus DNA (ML-DNA) and poly(dC-dG) templates containing covalently bound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the strongly carcinogenic, principal metabolite of benzo[a]pyrene. Since eukaryotic DNA methyltransferases recognize the dinucleotide 5'd[CG] in DNA as a substrate for methylation, the model polynucleotide poly(dC-dG) was used to study in more detail the mode of interaction and effect on incorporation. With either of these BPDE-modified templates, a progressive inhibition of methylation was correlated with increasing amount of BPDE substitution. The effect of BPDE-dG adducts did not alter the apparent km with respect to the concentration of d[CG] in either unmodified or BPDE-modified poly(dC-dG) (km = 10 microM) but lowered the relative apparent Vmax. In assays in which perturbation by salt of preformed enzyme-DNA complex is measured, no change in the relative stability to either unsubstituted or the carcinogen-modified template was noted, thus, excluding any change in the ionic component of this interaction. However, in competition-type experiments, BPDE-DNA is an inhibitor of the methylation reaction on native DNA. When BPDE-DNA is allowed to interact with the enzyme before the addition of native competitor DNA, the methylation rate is not stimulated, suggesting very tight hydrophobic binding of the enzyme to BPDE-DNA and an inhibition in the dissociation of DMase from the template following a methylation event.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of T7 DNA with a polycyclic aromatic hydrocarbon. Lack of structural perturbation

Bacteriophage T7 DNA reacts uniformly with trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene(anti-BPDE). The reaction product retains the native configuration so that only one site sensitive to S1 nuclease is produced for every 70 anti-BPDE adducts. DNA treated with anti-BPDE is retained on benzoylated naphthoylated DEAE-cellulose even after washing with 1.0 M salt solutions. About 100 adducts per T7 molecule are required for adherence which is not due to breaks or single-stranded regions since adherence is not affected by S1 nuclease treatment. The binding of anti-BPDE reacted DNA to benzoylated naphthoylated DEAE-cellulose is cooperative and requires many residues per bound fragment. Treatment of T7 DNA treated with anti-BPDE with restriction endonuclease yields smaller molecules, still containing adducts, which do not adhere. We interpret these results to mean that reaction with BPDE does not involve deformation of the DNA structure and that the adducts lie in a position which they are readily accessible for interaction with aromatic groups on the column resin.     

A comparison of cytotoxicity, ouabain-resistant mutation, sister-chromatid exchanges, and nascent DNA synthesis in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Chinese hamster V79 cells were treated with either (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide I) or (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide II) and the nascent DNA was labeled with [Me-3H]thymidine. The cells were harvested for determination of cytotoxicity, sister-chromatid exchanges (SCE), ouabain-resistant (Or) mutations and the size of newly synthesized daughter-strand DNA. Both isomers caused dose-dependent decreases in survival of cells and in the size of nascent DNA. Increases in the frequencies of SCE and of Or mutation were found in cells treated with either isomer. However, B[a]P-diol epoxide I caused 10--20-fold more Or mutations and 50-100% more SCE than did B[a]P-diol epoxide II at equal molar dose levels. In contrast to the marked difference in the frequencies of both SCE and Or mutations caused by both compounds, the isomers induced similar reductions in the size of the nascent DNA at equal dose levels. In comparing the molecular and biological effects of the two isomers the reduction in the size of nascent DNA was more closely related to cytotoxicity than to the induction of SCE or Or mutations.     

Hydroperoxide-dependent oxygenation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen

Incubation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with ram seminal vesicle microsomes (RSVM) triggers the oxygenation of $\text{trans}$-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). The principal oxidation products are 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which are non-enzymatic hydrolysis products of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. At short incubation times, an additional product is isolated which is identified as r-7,t-8,t-9-trihydroxy-c-10-methoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product appears to arise by solvolysis of the extracted diolepoxide during high performance liquid chromatography using methanol-water solvent systems. The incubation of ¹⁸O-labeled 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with BP-7,8-diol and RSVM leads to very little incorporation of ¹⁸O into the stable solvolysis products (analyzed by gc-ms of their peracetates). Parallel incubations conducted with ¹⁶O-labeled hydroperoxide under an ¹⁸O₂ atmosphere indicate that the principle source of the epoxide oxygen is molecular oxygen.     

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.     

Conformation of dinucleoside monophosphates modified with benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide as measured by circular dichroism

The conformational properties of GpU modified with the reactive derivative of benzo[a]pyrene, (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, has been investigated utilizing circular dichroism spectroscopy. Binding of this carcinogen to the N2 of G residues in GpU resulted in the formation of four compounds (I to IV) representing two pairs of diastereoisomers. The molar ellipticity values of the modified dimers were approximately twofold higher than those of the modified guanosine monomers. These values were decreased appreciably when the spectra of the dimers were obtained at 80 degrees C or in methanol rather than at 25 degrees C in water, suggesting that under the latter conditions there is a stacking interaction between the carcinogen and the neighboring uridine residue. Based on these results, a conformation is proposed for modified GpU. It includes insertion of the benzo[a]pyrene moiety, by rotation of the modified guanine residue about its glycoside bond, coplanar to the neighboring uridine and perpendicular to the phosphodiester backbone.     

Studies on the further activation of benzo[a]pyrene diol epoxides by rat liver microsomes and nuclei

The syn- and anti-diastereoisomers of trans-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) were further metabolized by rat liver microsomes obtained from 3-methylcholanthrene(MC)-pretreated rats and NADPH to reactive intermediates, presumably 1,7,8- and 3,7,8-trihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (triol-epoxides), that bound to macromolecules or decomposed to products consistent with pentahydroxy derivatives of benzo[a]pyrene (BP-pentols). Three major metabolites of syn-BPDE and four major metabolites of anti-BPDE were isolated by high performance liquid chromatography and characterized by spectroscopic techniques. When fluorescence spectroscopy was employed all metabolites exhibited very similar spectral properties and showed substantial shifts in excitation and emission maxima to longer wavelengths when measured under alkaline conditions, consistent with the presence of a phenolic hydroxyl group. Furthermore, the spectral properties of the metabolites from syn- and anti-BPDE were similar to those of 1-hydroxypyrene. Previous data from this laboratory together with the data presented in this study thus strongly suggest that further metabolism of BPDE involves hydroxylation at the 1- and 3-positions to yield the corresponding triol-epoxides and various BP-pentols. The pentols could also be formed by incubating tetrols derived from syn- and anti-BPDE with microsomes and NADPH. However, the rate of formation of pentols from the BP-tetrols was much slower than the rate of further metabolism of BPDE. Accordingly, the major route of BP-pentol formation is likely to be via the intermediate formation of triol-epoxides. Isolated liver nuclei from MC-pretreated rats were also found to catalyze the activation of anti-BPDE in presence of NADPH to reactive intermediates. This resulted in a substantial increase in binding to histone and non-histone proteins, with a concomitant decrease in binding to DNA. No qualitative change in the distribution of DNA-bound products of anti-BPDE could be demonstrated as a result of the further metabolism of anti-BPDE.     

Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.     

32P-Postlabeling analysis of lipophilic DNA adducts resulting from interaction with (+/-)-3-hydroxy-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene.

Bay-region diol epoxides are considered the putative ultimate carcinogens of polynuclear aromatic hydrocarbons. However, the results of studies on tumorigenesis and DNA binding of benzo[a]pyrene (BP) and its bay-region diol epoxide, (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e [(+)-anti-BPDE] suggest that, in addition to anti-BPDE, other reactive metabolite(s) of BP may also be involved in BP-induced carcinogenesis. Recent studies have demonstrated that 3-hydroxy-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a ]pyrene (anti-BPTE) is another highly reactive metabolite of BP. In order to identify syn- and anti-BPTE-derived DNA adducts and their base selectivity, we synthesized both compounds by two different methods and reacted in vitro with calf thymus DNA and individual nucleotides. The resultant adducts were analyzed by nuclease P1-enhanced 32P-postlabeling. Anti-BPTE produced three major and several minor adducts with DNA; dAp and dGp were the preferred substrates, while dCp and dTp were the least reactive. In contrast, syn-BPTE produced two major adducts each with DNA and dGp; dAp generated only one adduct. Co-chromatography of anti-BPTE-derived DNA adducts with those of mononucleotide adducts revealed that the major adducts in DNA were guanine derived. Further, co-chromatographic results revealed that the anti-BPTE-DNA adducts were distinctly different from that of anti-BPDE-DNA adducts. These observations indicate that both syn- and anti-BPTE can react with DNA bases and these DNA adducts may also contribute to BP-induced carcinogenesis.     

Depurination of benzo[a]pyrene-diolepoxide treated DNA

Rat liver DNA was treated in vitro with benzo[a]pyrene-diolepoxide (BPDE), the ultimate carcinogenic metabolite derived from the polycyclic hydrocarbon benzo[a]pyrene. On incubation of the reacted DNA, apurinic sites developed which gave rise to strand breakage in alkaline solution. The reduction in molecular weight produced by these breaks was measured by analytical ultracentrifugation. In the case of anti-BPDE this depurination was shown to occur in two stages. The first was mainly due to attack at the 7-position of guanine, to yield an adduct which was lost from the DNA within a few hours. The second stage was due to much slower loss of the major N2-guanine adduct. The separated enantiomers, (+)- and (-)-anti-BPDE, and syn-BPDE all caused depurination to various extents. It is argued that although these processes are important in a study of the action of BPDE on DNA in vitro, their contribution to the biological activity of BPDE is probably negligible.     

Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

Interactions of Molecules with Nucleic Acids. X. Covalent Intercalat e Binding of the Carcinogenic BPDE I(+) to Kinked DNA

Abstract

A theoretical model is proposed for the covalent binding of (+) 7 β,8α-dihydroxy-9α, 10α- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene denoted by BPDE I(+), to N2 on guanine. The DNA must kink a minimum of 39° to allow proper hybrid configurations about the C10 and N2 atoms involved in bond formation and to allow stacking of the pyrene moiety with the non-bonded adjacent base pair. Conservative (same sugar puckers and glycosidic angles as in B-DNA) and non-conservative (alternating sugar puckers as in intercalation sites) conformations are found and they are proposed structures in pathways connecting B-DNA, an intercalation site, and a kink site in the formation of a covalently intercalative bound adduct of BPDE I(+) to N2 on guanine. Stereographic projections are presented for (3′) and (5′) binding in the DNA. Experimental data for bending of DNA by BPDE, orientation of BPDE in DNA and unwinding of superhelical DNA is explained. The structure of a covalent intercalative complex is predicted to result from the reaction. Also, an anti ? syn transition of guanine results in a structure which allows the DNA to resume its overall B-form. The only change is that guanine has been rotated by 200° about its glycosidic bond so that the BPDE I(+) is bound in the major groove. The latter step may allow the DNA to be stored with an adduct which may produce an error in the genetic code.     

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.