Differences in metabolic activation of dibenzo[a,e]fluoranthene characterized by 32P-postlabeling in two mouse fibroblast models.

The formation of DNA adducts was investigated in mouse fibroblasts from two different tissues--embryos and adult lung--after incubation with dibenzo[a,e]fluoranthene (DBF) or its major proximate metabolites. The nuclease P1 modification of the 32P-postlabeling method was adapted for detection of DBF-DNA adducts. Quantitative and qualitative differences were observed in the metabolic activation mediated by the two cell types. DBF-DNA adducts generated three major spots reproducibly, and more than ten spots of medium or weak importance. The highest level of DNA binding occurred via the DBF-bay region vicinal dihydrodiol epoxide but with significant differences in the quantitative distribution of adducts. Striking qualitative differences were observed when lung fibroblasts were incubated with the DBF-pseudo bay region dihydrodiol (DBF-12,13-DHD). The spots representing adducts induced in embryo fibroblasts by DBF-3OH-12,13-DHD, a further metabolite of DBF-12,13-DHD, were totally absent from chromatograms of lung cells. These results show that both embryo and lung fibroblasts can activate DBF but that different cytochrome P-450 forms and substrate affinities are involved. The finding that different activation systems may be present in subcategories of the same tissue, may provide a partial explanation for the wide variations in sensitivity to carcinogens among species, organs and tissues.     

Marked differences between mutagenicity in Salmonella and tumour-initiating activities of dibenzo[a,e] fluoranthene proximate metabolites; initiation inhibiting activity of norharman

Dibenzofluoranthene-12,13-dihydrodiol (DBF-12,13-DHD) is sixtimes more mutagenic in Salmonella TA100 than diben-zofluoranthene-3,4-dihydrodiol(DBF-3,4-DHD). However, these two major dibenzo[a,e]fluoranthene(DBE) proximate metabolites, which are immediate precursorsof the corre-sponding diolepoxides, showed on an equiinolarbasis nearly identical initiation activities on mouse skin;they induced three times more papillomas than the parent hydrocarbon.On the other hand the epithelioma initiation capacities, i.e.the num-ber of papillomas progressing to malignant tumours,of DBF or the two dibenzofluoranthene dihydrodiols were equivalent.Norharman, a putative vicinal diolepoxidation inhibitor in DBFmetabolism when administered topically together with the initiationdose (100 nmol), strongly inhibited the induc-tion of tumoursby DBF-3,4-DHD and DBF. The relationship between Dibenzofluoranthene-12,13-dihydrodiol(DBF-12,13-DHD) is six times more mutagenic in Salmonella TA100than diben zofluoranthene-3,4-dihydrodiol (DBF-3,4-DHD). However,these two major dibenzo (DBE) proximate metabolites, which areimmediate precursors of the corre sponding diolepoxides, showedon an equiinolar basis nearly identical initiation activitieson mouse skin; they induced three times more papillomas thanthe parent hydrocarbon. On the other hand the epithelioma initiationcapacities, i.e. the num ber of papillomas progressing to malignanttumours, of DBF or the two dibenzofluoranthene dihydrodiolswere equivalent. Norharman, a putative vicinal diolepoxidationinhibitor in DBF metabolism when administered topically togetherwith the initiation dose (100 ninol), strongly inhibited theinduc tion of tumours by DBF-3,4-DHD and DBF. The relationshipbetween in vitro mutagemc activity in Salmonella and the carcinogenicityof DBF metabolites in mice appears to be qualitative ratherthan quantitative.in vitro mutagemc activity in Salmonella andthe carcinogenicity of DBF metabolites in mice appears to bequalitative rather than quantitative.     

Formation and removal of dibenzo[a,e]fluoranthene-DNA adducts in mouse embryo fibroblasts

In vivo binding of dibenzo[a, e]fluoranthene (DBF) to mouseembryo fibroblast DNA was compared with that observed previouslyin vitro on calf thymus DNA incubated with mouse liver microsomes.The h.p.l.c. elution patterns of the adducts formed by DBF metaboliteswith DNA and obtained in vivo at the optimal exposure time of42–48 h were qualitatively very similar to the patternsobtained in vitro, but their amplitude was quantitatively reduced.There are two striking differences between the in vivo and invitro results. Firstly, the most polar peak A, very abundantin vitro, was absent in vivo. Secondly, the reactivity of thetwo major proximate metabolites of DBF, the bay and pseudo-bayregion dihydrodiols, was very different in intact cells comparedwith the results in vitro. When incubated in vitro, pseudo-bayregion dihydrodiol DBF was twice as reactive as bay region dihydrodiolDBF. The opposite reactivities were observed in vivo. The majorDBF-DNA adducts formed in vivo were collected in the peaks E,B and C. The predominant peak E contained DNA adducts of bothbay and pseudo-bay region dihydrodiolepoxides which are themajor ultimate metabolites of DBF in vivo and in vitro. Theother two prominent peaks B and C contained DNA adducts of 3-hydroxyDBF pseudo-bay region dihydrodiolepoxide and the 7-hydroxy DBFbay region dihydrodiolepoxide, respectively. After adduct formation,post incubation of fibroblasts for a further 48 h, in the absenceof DBF, eliminated half the amount of adducts present. PeakB adducts were repaired more efficiently than those of peaksE, C D and F. The carcinogenic initiating activity of DBF appearsto be a complex process in which several DNA adducts play arole.     

7-Methylbenz[c]acridine: mutagenicity of some of its metabolites and derivatives, and the identification of trans-7-methylbenz[c]-acridine-3,4-dihydrodiol as a microsomal metabolite

The presence of the proposed proximate carcinogen, trans-3,4-dihydro-3,4-dihydroxy-7-methylbenz[c]acridine(7MBAC-3, 4-DHD) among the liver microsomal metabolites of 7-methylbenz[c]acridine(7MBAC) has been demonstrated using gas chromatography massspectrometry (GCMS) and by co-chromatography with syntheticstandards on reverse and normal phase h.p.l.c. 7MBAC-3, 4-DHDrepresented 2.2–3.4% of the total ethyl acetate-extractablemetabolites formed from 7MBAC by liver microsomes prepared fromuntreated and induced rats. About 2.3–2.7% of metabolitesformed by lung microsomes was identified as 7MBAC-3, 4-DHD.Mutagenicity studies with 7MBAC-3,4-DHD have been carried outin bacterial and mammalian systems using S9 fractions derivedfrom rats pre-treated with Aroclor and guinea pigs pre-treatedwith 3-methylcholanthrene. Comparative data with other 7MBACderivatives are also reported. The 7MBAC-3,4-DHD and the analogousdihydro derivative of 7MBAC were the most potent mutagens ofthose compounds requiring metabolic activation. The data implythat the 3,4-dihydrodiol is metabolised to a bay region diolepoxide as the ultimate carcinogen. In support of this, anti-1,2-epoxy-trans-3,4-dihydroxy-7-methyl-1,2,3,4-tetrahydrobenz[c]acridinewas a potent mutagen in the Ames and V79 cell systems withoutactivation. The syn-isomer was less active.     

Mutagenic potential of DNA adducts formed by diol-epoxides, triol-epoxides and the K-region epoxide of chrysene in mammalian cells

The syn- and anti-tsomers of chrysene-l,2-diol-3,4oxide (syn-diol-epoxideand anti-diol-epoxide) and of 9-hydroxychry-sene-l, 2-diol-3,4-oxide (syn-triol-epoxide and anti-triol-epoxide) and chrysene-5,6-oxide,the K-region epoxide, were tested for their ability to induce6-thioguanine-resistant mutants in V79 Chinese hamster cells.The levels of DNA adducts formed by each compound in the V79cells were determined by ³²P-post-labelling analysis. The mostpotent mutagen, in terms of the mutation frequency/nmol compoundadministered, was the anti-triol-epoxide, which was 1.7 timesas active as the anti-triol-epoxide. The anti-diol-epoxide was10 times more active than both the syn-triol-epoxide and thesyn-diol-epoxide, which in turn were several times more activethan the K-region epoxide. However, when the results were expressedas mutations/pmol total adducts formed, the anti-triol-epoxideand anti-diol-epoxide were shown to be of similar potency andapproximately twice as active as the other three compounds.Thus differences in the conformation of adducts formed withDNA by syn- and anti-isomers may be responsible for their differentmutagenic potentials; the presence of a phenolic OH-group atthe 9-position of a chrysene-l,2-diol-3,4-oxide appears to increaseits chemical reactivity.     

The metabolic activation of dibenz[a,c]anthracene

In rat liver microsomal preparations, the 10, 11-dihydro-diolof dibenz[a, c]anthracene (DBA) is metabolized to r-10, t-11-dihydroxy-t-12,13-oxy-10, 11, 12, 13-tetrahydro-dibenz[a, c]anthracene (anti-DBA10, 11-diol 12, 13-oxide), the anti isomer of a non-bay-regiondiol-epoxide of DBA. When ³-labelled DBA or trans-10, 11-di-hydro-10,ll-dihydroxydibenz[a, c]anthracene were metabolized in thissystem in the presence of DNA or when ³H-labelled DBA was addedto primary cultures of hamster embryo cells, covalent reactionsof hydrocarbon metabolites with DNA occurred. The chromato-graphiccharacteristics of the radioactive hydrocarbon — deoxyribonucleosideadducts formed in these reactions were examined using SephadexLH20 column chromatography and high pressure liquid chromato-graphy.The results showed that whilst some of the radioactive hydrocarbon— deoxyribonucleoside adducts formed were indistinguishablefrom adducts that were formed when anti-DBA 10, 11-diol 12,13-oxide reacted with DNA, other, unidentified adducts, whichdid not apparently arise from reactions of this diol-epoxidewith DNA, were also present. Hydrocarbon — nucleosideadducts were not detected in hydrolysates of nucleic acids thatwere isolated from mouse skin that had been treated in vivowith DBA.     

32P-postlabeling analysis of inhibition by norharman of formation of dibenzo[a,e]fluoranthene--DNA adducts in mouse embryo fibroblasts.

Quantitative and qualitative changes in the inhibition of DNA adduct formation in the presence of increasing concentrations of norharman (NH) were investigated in vivo in mouse fibroblasts treated with dibenzo[a,e]fluoranthene (DBF), a potent carcinogen in mice. The nuclease P1 modification of the 32P-postlabeling technique was used to identify adducts. A dose-dependent reduction in DBF-DNA adduct formation was observed: an 80% reduction with 0.06 mM NH and 90% with 0.12 mM NH. At 0.12 mM NH, all of the spots coming from hydroxylated DBF vicinal dihydrodiol (DHD) epoxides were missing; the only clear spot was that of the major DBF adduct produced by the ultimate DBF metabolite, DBF-3,4-DHD-1,2 oxide. Spots representing other DBF-DHD epoxide adducts appeared only in trace amounts. These results can be interpreted as a dose-dependent competition or inhibition of some secondary metabolic step, most probably secondary epoxidation; however, a direct protective effect of NH during adduct formation cannot be excluded. NH is a strong inhibitor of DBF-DNA adduct formation in vivo.     

Further metabolism of diol-epoxides of chrysene and dibenz[a,c]anthracene to DNA binding species as evidenced by 32P-postlabelling analysis

Incubation of r-1t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydro-chrysene(anti-chrysene-1,2-diol 3,4-oxide), the bay-region diol-epoxideof chrysene, with rat liver microsomes in the presence of NADP⁺and DNA, followed by ³²P-postlabelling analysis of the DNA,revealed the presence of at least two adducts not detected whenanti-chrysene-1,2-diol 3,4-oxide was incubated with DNA alone.The formation of these adducts was not blocked by the epoxidehydrolase inhibitor 1,1,1-trichloropropane-2,3-oxide. One ofthe adducts cochromatographed with the adduct spot obtainedwhen authentic 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetra-hydrochrysene(anti-9-OH-chrysene-1,2-diol 3,4-oxide) was reacted with DNA.Evidence suggested that a second adduct could also be formedby further metabolism of anti-9-OH- chrysene-1,2-diol 3,4-oxide.In addition, evidence was obtained for the further metabolismof the syn-isomer of chrysene 1,2-diol 3,4-oxide and the anti-isomersof a non-bay-region diol epoxide of dibenz[a,c]anthracene toDNA binding species, but not for that of either the anti- orsyn- isomers of the bay-region diol-epoxide of benzo[a]pyrene,the anti-isomers of the bay-region or a non-bay-region diol-epoxideof benz[aanthracene, or the anti-isomer of the bay-region diol-epoxideof benzo[bfluoranthene.     

Separation and characterization of post-labeled DNA adducts of stereoisomers of benzo[a]pyrene-7,8-diol-9,10-epoxide by immobilized boronate chromatography and HPLC analysis

The carcinogenic polycydlic aromatic hydrocarbon (PAH) benzo[a]pyrene(BaP) is enzymatically activated in cells to an ultimate carcinogenicmetabolite, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BaPDE),which reacts with DNA to form covalent adducts involved in theinitiation of cancer. Previously, a post-labeling procedurethat uses adenosne-5'-O-(3'-[³⁵S]-thiotriphosphate) was developedto facilitate adduct analysis by HPLC. The much greater carcinogenicpotency of (+)-anti-BaPDE makes it essential to be able to separateand identify the adducts formed by all four BaPDE enantiomersin DNA of cells exposed to BaP. Reversed-phase HPLC (RPHPLC)resolved the major (+)-anti-BaPDE-N²-deoxyguanosine [(+)-anti-BaPDE-N²-dG]adduct from the (+)-syn-BaPDE-N²-dG adduct. However, anti-BaPDE-N²-dGadducts formed by (+)-and (–)-anti-BaPDE were not resolved.By using ion-pair RPHPLC (IP-RPHPLC) with tetrabutylammoniumphosphate, the [³⁵S]post-labeled (–)-anti-BaPDE-N²-dGadduct eluted 3 min prior to the [³⁵S]labeled (+)-anti-BaPDE-N²-dGadduct. In contrast, the major syn-BaPDE-N²-dG adducts wereresolved better by RPHPLC than by IP-RPHPLC. The differencein conditions required for optimal separation of anti- and syn-BaPDE-DNAadducts necessitated the development of an immobilized boronatechromatography technique for the separation of anti- from syn-BaPDE-DNAadducts prior to analytical HPLC analysis. At 4°C and withelution buffers containing high salt concentrations, the [³⁵S]post-labeledanti-BaPDE-DNA adducts were selectively retained by a boronatecolumn whereas the [³⁵S]labeled syn- BaPDE-DNA adducts werenot. Analysis of the multiple BaP-DNA adducts formed in BaP-treatedhamster embryo cells by these techniques gave results comparableto those obtained by other methods. The major BaP-DNA adductswere anti-BaPDE-N²-dG 14% from (–)- and 86% from (+)-anti-BaPDE.The ability of these techniques to detect low levels of PAH-DNAadducts because of the high specific radioactivity of ³⁵S andto separate the DNA adducts formed by stereolsomeric PAN diolepoxides adducts by boronate chromatography and HPLC will facilitatestudies of the role of individual PAH-DNA adducts in the inductionof biological effects such as toxicity and carcinogenesis.     

Co-chromatography of a tamoxifen epoxide-deoxyguanylic acid adduct with a major DNA adduct formed in the livers of tamoxifen-treated rats

Tamoxifen is a potent liver carcinogen in rats and has beenshown to form covalent DNA adducts in the livers of severalspecies of rodent. We have shown previously by ³²P-postlabelling(Carcinogenesis, 13, 2197–2203) that >85% of the totaladducts detected and resolved by multi-directional TLC migrateas a single spot. In the present study, this material was furtheranalysed by reverse-phase HPLC and resolved into two approximatelyequal components. Tamoxifen 1,2-epoxide, a postulated metaboliteof tamoxifen, was reacted with DNA and polydeoxyribonucleotidesand the products analysed. ³²P-Postlabelling revealed threemajor adduct spots on TLC which comigrated with the three majoradduct spots seen with DNA from livers of tamoxifen-treatedrats. Moreover, the major epoxide adduct, which contained guanineas the modified base, eluted on HPLC as a single major peakcoincident with one of the major peaks derived from the liverDNA of tamoxifen-treated rats. These results demonstrate that{small tilde}40% of the tamoxifen-DNA adducts formed in vivoare chromatographically indistinguishable with the major productof the reaction of tamoxifen epoxide with guanine residues inDNA and provide important clues to the mechanism of activationof tamoxifen to a genotoxic carcinogen.     

Determination of stereospecificity of benzo[a]pyrene diolepoxide-DNA antisera with site-specifically modified oligonucleotides

Antisera developed against benzo[a]pyrene diolepoxide (BPDE)—DNAadducts are sensitive tools for detection of DNA adducts inhuman samples. All antisera currently used for biomonitoringstudies were produced against DNA or guanosine modified withracemic anti-BPDE. Using a non-competitive enzyme-linked immunosorbentassay (ELISA), Venkatachalam and Wani (Carcinogenesis, 15, 565–572,1994) recently tested polyclonal and monoclonal (5D2) antiserafor cross-reactivity against oligonucleotides containing (+)-and (–)-trans-anti-BPDE-N²-guanine or N⁶-adenine adductsand showed different stereospecificity for the two antisera.Because of the importance of antiserum specificity in humanbiomonitoring studies, we have tested several monoclonal (Mab5D11 and 5D2) and polyclonal (Pab #29) antisera developed againstracemic anti-BPDE-DNA adducts, and Mab 8E11 developed againstanti-BPDE-guanosine adducts. Stereoisomeric anti-BPDE-modifiedoligonucleotide adducts in the sequence 5'-d(CCAT-CG^*CTACC)-3'where G^* = anti-BPDE-N²-dG with (+ )-trans, (–)-trans,(+ )-cis and (–)-cis adduct stereochemistry at the C10position of anti-BPDE were tested by competitive ELISA. Twostructurally related 5-methylchrysene diolepoxide adducts withG^* = (+)- and (–)-trans-anti-5-MeCDE-N²-dG in the sameoligonucleotide were also tested. While Mab5D2 had the highestaffinity for the (–)-trans-anti-BPDE-modified oligomer,Mab 5D11 and 8E11 and Pab #29 recognized the (+ )-trans-anti-BPDE-modifiedoligomer better than the (–)-trans-anti-BPDE modifiedoligomer. Mab 5D11 and Pab #29 recognized racemic anti-BPDE-modifiedDNA adducts better than trans-anti-BPDE-modified oligonucleotides;however, Mab 8E11 showed similar sensitivity to racemic anti-BPDE-DNAadducts and (+ )-and (–)-trans-anti-BPDE-modified oligomers.All antisera exhibited lower reactivities with both 5-MeCDEmodified oligomers. Because of their sensitive detection of(+)-trans-anti-BPDE-dG adducts, the primary adduct producedin vivo, Mab 8E11 and 5D11 and Pab #29 are appropriate for measurementof most adducts formed in humans.     

DNA repair in human cells: quantitative assessment of bulky anti-BPDE-DNA adducts by non-competitive immunoassays

Mutagenicity and carcinogenicity of the ubiquitous environmentalpollutant benzo[a]pyrene is mediated via its reactive diol epoxidemetabolite, anti-BPDE, with the predominant formation of N²-deoxyguanineadducts in genomic DNA. Polyclonal and monoclonal antibodiesspecific for ()-anti-BPDE DNA adducts were used for the quantitativedetection of genotoxic damage in DNA treated in vitro and invivo with ()-anti-BPDE. In non-competitive enzyme-linked immunosorbentassay the polyclonal antiserum (BP1) exhibited higher affinity,avidity and sensitivity than the monoclonal antibody (5D2).A linear antibody binding response was observed over a widecarcinogen dose range with a detection limit of <0.1 fmoladducts in immobilized DNA. Non-competitive immuno-slot blotassay could detect 0.2 adducts/10⁶ nucleotides induced by <1nM ()-anti-BPDE. The high sensitivity and mono-adduct specificityof non-competitive immunoassays allowed the detailed study of()-anti-BPDE-DNA adduct processing in human cells exposed tovery low levels of the genotoxin. Analysis of polyclonal antiserumbinding sites in DNA from repairproficient human fibroblastsrevealed adduct removal rates directly proportional to the initialgenotoxic insult. Despite efficient repair, substantial damagepersisted in repairproficient cells exposed to high doses ofthe carcinogen. At low levels of initial damage (0.882 and 3.44 0.17 adducts/ 10⁶ nucleotides)     

Mutagenic specificity of syn-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide in the dihydrofolate reductase gene of Chinese hamster ovary cells

Polycyclic aromatic hydrocarbons (PAHs) with sterically hinderedfjord region diol epoxides are interesting with respect to theirpotency as carcinogens, interactions with DNA and mutagenicspecificities. Unlike the bay region PAH derivative, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene(BPDE), reactive metabolites of two fjord region PAH, trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-Phenanthrene[(±)-anti-BcPHDE] and trans-11,12-dihydroxy-syn-13,14-epoxy-11,12,13,14-tetrabenzo[g]chrysene[(±)-syn-BgCDE], react with DNA to yield high levelsof adenine adducts. We previously found that forward mutationsinduced by (±)-anti-BcPHDE in the dihydrofolate reductase(dhfr) gene of Chinese hamster ovary (CHO) cells preferentiallytargeted mRNA splice acceptor sites. (±)-anti-BcPHDEand (±)-syn-BgCDE are structurally similar; they differonly by the presence of an additional benzene ring. Thus weused (±)-syn-BgCDE to learn if the mutational targetbias reflects aspects of the mutagen structure or its capacityto efficiently modify deoxyadenosine (dA) in vivo. dhfr^–mutants were induced after treatment of hemizygous UA21 cellswith a 0.75 µM dose of (±)-syn-BgCDE. Cell survivalafter carcinogen exposure was 40%. The induced mutation frequencywas 9x10^–6, nearly 10-fold higher than the spontaneousone, but     

5-Methylchrysene metabolism in mouse epidermis in vivo, diol epoxide--DNA adduct persistence, and diol epoxide reactivity with DNA as potential factors influencing the predominance of 5-methylchrysene-1,2-diol-3,4-epoxide--DNA adducts in mouse epidermis

5-Methylcbrysene (5-MeC) can form two bay region dihydrodiolepoxides: 1,2-dihydroxy-3-4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene(DE-I) which has the methyl group and the epoxide ring in thesame bay region, and 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene(DE-II). In a previous study, we observed that the ratio ofDE-I:DNA adduds to DE-II:DNA adducts in mouse epidermis, 24h after application of [³H]5-MeC metabolites was 2.7 to 1. Toinvestigate the basis for this observation we have now studied:(i) the formation of [³H]5-MeC in mouse epidermis in vivo atvarious time intervals from 0.33 to 24 h; (ii) the persistenceof DE-I:DNA adducts and DE-II:DNA adducts in mouse epidermisat 4–48 h after application of [³H]5-MeC and (iii) thereactions of DE-I and DE-II with calf thymus DNA in vitro. Incontrast to results obtained with mouse liver 9000 g supernatant,the dihydrodiol precursors of DE-I and DE-II were present inequivalent quantities in mouse epidermis in vivo at every timepoint studied. The ratio of DE-I:DNA adducts to DE-II:DNA adductsin mouse epidermis was constant throughout the time period studied.However, the extent of formation of DE-I:DNA adducts was greaterthan that of DE-II:DNA adducts upon reaction of DE-I or DE-IIwith calf thymus DNA in vitro. These results suggest that differencesin reactivity with DNA of DE-I and DE-II may be responsiblefor the higher levels in mouse epidermis of DE-I:DNA adductscompared with DE-II:DNA adducts and provide a possible basisfor the observed enhancing effect of a bay region methyl groupon the carcinogenicity of polynuclear aromatic hydrocarbons.     

Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: importance of adduct conformation

The formation and repair of benzo[a]pyrene diol epoxide-N²-deoxyguanosineadducts (BPDE-N²-dG) in DNA isolated from the skin of mice treatedtopically with benzo[a]pyrene (BP) was studied by ³²P-postlabelingand by low-temperature fluorescence spectroscopy under low resolutionand under high resolution fluorescence line narrowing (FLN)conditions. In agreement with earlier studies, total BP-DNAbinding reached a maximum at 24 h after treatment (dose: 1 µmol/mouse),then declined rapidly until 4 days after treatment and muchmore slowly thereafter. An HPLC method was developed which resolvedthe ³²P-postlabeled (–)-trans- from (–)-cis-anti-BPDE-N²-dG,and (+)-trans- from (+)-cis-anti-BPDE-N² High performance liquidchromatography analysis of the major TLC adduct spot (containing>80% of the total adducts) obtained by postlabeling BP-modifiedmouse skin DNA showed that it consisted of a major componentthat coeluted with (–)-cis-/(+)-trans-anti-BPDE-N²-dGand a minor component that coeluted with (–)-trans-/(+)-cis-anti-BPDE-N²-dGand that the minor component was repaired at a slower rate thanthe major component. Low-temperature fluorescence spectroscopyof the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N²-dGand the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N²In agreement with the ³²P-postlabeling results it was observedby fluorescence spectroscopy that the (+)-cis-adducts were repairedmore slowly than most other adducts. Moreover, the (+)-trans-adductsexhibited a broad distribution of base-stacked, partially base-stackedand helix-external conformations. Mouse skin DNA samples obtainedat early timepoints (2–8 h) after treatment with BP containedsubstantially more of the ‘external’ adducts, whilesamples at later timepoints (24–48 h) contained relativelymore adducts in the base-stacked conformation, indicating alsothat the latter adducts are repaired less readily than the former.The possible biological significance of these novel observationsof conformation-dependent rates of DNA adduct repair and theirpossible dependence on DNA sequence, are discussed.     

Fluorescence HPLC methods for detecting benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide-deoxyadenosine adducts in enzyme-digests of modified DNA: improved sensitivity

The fluorescence of mononucleoside adducts derived from thebinding of anti-7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetra-hydrobenzo[a]pyrene(BPDE I) to N⁶-deoxyadenosine (BPDE-dA adducts) is 10–100times stronger (depending on the methanol/water solvent composition)than the fluorescence of adducts derived from the binding ofthis diol epoxide derivative to N²-deoxyguanosine. It is shownhere that these fluorescence characteristics can be used toquantitate the relatively low yields of BPDE-dA adducts by fluorescencedetection when BPDE–modified DNA is subjected to enzymaticdegradation to the mononucleoside levels, followed by HPLC analysisof the digests.     

Genotoxicity characteristics of reverse diol-epoxides of chrysene

Trans-3,4-dihydroxy-3,4-dihydrochrysene (chrysene-3,4-diol),a major metabolite of chrysene, is further metabolized by ratliver enzymes to products which effectively revert the his^–Salmonella typhimurium strain TA98 to histidine prototrophy,but are only weakly mutagenic in strain TA100 and in Chinesehamster V79 cells (acquisition of resistance to 6-thioguanine).The liver enzyme mediated mutagenicity of chrysene-3,4-diolis substantially enhanced in the presence of 1,1,1-trichloropropene2,3-oxide, an inhibitor of microsomal epoxide hydrolase. Thepredominant metabolites of chrysene-3,4-diol, namely the anti-and syn-isomers of its 1,2-oxide (termed reverse diol-epoxides),proved to be extraordinarily effective mutagens in S.typhimuriumstrain TA98, but were only moderately active in strains TA100and TA104, and in the SOS induction in Escherichia coli PQ37.These genotoxicity spectra in bacteria are completely differentfrom those observed with the bay-region diol-epoxides of chryseneand 3-hydroxychrysene. In V79 cells, the reverse diol-epoxidesformed low levels of DNA adducts and were very weak inducersof gene mutations. In M2 mouse prostate cells, however, highnumbers of transformed foci were induced by chrysene-3,4-dioland its diastereomeric 1,2-oxides. Chrysene-3,4-diol was somewhatmore potent than chrysene-1,2-diol. The potency of both reversediol-epoxides was similar to that of the syn-diastereomers ofthe bay-region diol-epoxides of chrysene and 3-hydroxychrysene,but lower than that of their anti-diastereomers. The reversediol-epoxides of chrysene, unlike the bay-region diol-epoxides,were inactivated by purified microsomal epoxide hydrolase. Noteworthyfindings were also made with regard to the chemical stabilityof the diol-epoxides in buffer, determined from the declinein mutagenicity after preincubation in the absence of the targetcells. Despite its lower     

Reactivity of benzo[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxyguanosine adducts

Polycyclic aromatic hydrocarbon (PAH) o-quinones are productsof the dihydrodiol dehydrogenase-catalyzed oxidation of trans-dihydrodiolswhich are proximate carcinogens. The PAH o-quinones are highlyreactive molecules and have the potential to alkylate DNA. Inthis study, the reactivity of [³H](+/–)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene([³H](+/–)-anti-BPDE), [³H]benzo[a]pyrene-7,8-dione ([³H]BPQ)and [³H](+/–)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ([³H](+/–)-B[a]P-diol) with DNA were compared.(+/–)-anti-BPDE reacted equally well with native, deproteinatedand deproteinated/sheared calf thymus DNA. In each case DNAadducts were formed which upon digestion to deoxyribonucleosidescomigrated on reverse-phase (RP)-HPLC with adducts synthesizedby reacting (+/–)-anti-BPDE with oligo-p(dG)₁₀. (+/–)-anti-BPDEalso reacted with plasmid (pGEM-3) DNA to yield multiple adductsone of which comigrated with the (+)-anti-BPDE-deoxyguanosineadduct. Under identical conditions [³H]BPQ reacted preferentiallywith native calf thymus DNA but displayed low reactivity withdeproteinated and deproteinated/sheared calf thymus DNA. RP-HPLCanalysis of deoxyribonucleoside—BPQ adducts indicatedthat the predominant adduct formed comigrated with a standardsynthesized by reacting BPQ with oligo-p(dG)₁₀. BPQ also reactedwith pGEM-3 DNA to yield multiple adducts one of which comigratedwith the BPQ—deoxyguanosine adduct. Reactions between[³H]BPQ and poly(dA), poly(dT), poly(dC) and oligo-p(dG)₁₀ indicatedthat BPQ preferentially formed deoxyguanosine adducts. In thisstudy, [³H]BPQ and [³H](+/–)-anti-BPDE covalently labelednative calf thymus DNA to an equal extent, however, less [³H]BPQwas recovered as deoxyguanosine adducts. By contrast, no covalentmodification of calf thymus DNA, pGEM-3 DNA or oligonucleotideswas observed with [³H](+/–)-B[a]P-diol. These studiesindicate that BPQ has the potential to be genotoxic in vitro;that reactivity is heightened in the presence of protein orcircular DNA and that the major adduct formed is a deoxyguanosineadduct.     

Metabolism and DNA adduct formation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in fish cell lines in culture

The metabolic activation of the carcinogens benzo[a]anthracene(BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was examinedin cell lines derived from bluegill fry (BF-2), rainbow trout(RTG-2) and brown bullhead (BB). All three cell lines metabolizedBP (0.5 µg/ml medium) almost completely to water-solublemetabolites within 120 h, but the maximum amount of BP boundto DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17in the BB cells and 44 in the RTG-2 cells. The major BP-DNAadduct in the BB and BF-2 cells was that formed by reactionof (+)-anti-BP-7,8-diol-9,10 epoxide [(+)anti-BPDE] with deoxyguanosine.This adduct was also present in the RTG-2 cell DNA, but therewere larger amounts of unidentified polar BP-DNA adducts. Exposureof the cells to [³H]BP-7,8-diol, a metabolic precursor of (+)anti-BPDE,resulted in binding of 1.5, 12 and 35 pmol BP per mg DNA inthe BF-2, BB and RTG-2 cells, respectively. More than 90% ofthe BP-7,8-diol added to the BF-2 cultures was recovered asa glucuronic acid conjugate, but the RTG-2 cells formed moreglutathione conjugates than glucuronide conjugates. The BB cellsformed both types of conjugates at a slower rate for more than75% of the 7,8-diol was recovered unchanged after 24 h. Thethree cell lines differed in the proportion of a 0.1 µg/mldose of DMBA metabolized in 48 h: the values ranged from 47%in the BF-2 cells to 78% in the BB cells and 97% in the RTG-2cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6pmol/mg DNA in the three cell lines: DMBA-3,4-diol-1,2-epoxide(DMBADE) adducts were present in the BB cell DNA, but no significantamounts of DMBADE-DNA adducts were detected in the RTG-2 orBF-2 cell DNA. These results demonstrate that fish cell culturescan activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE,but the level of binding of this metabolite to DNA is much lowerthan that which occurs in rodent embryo cell cultures. In BF-2cell cultures formation of BP-7,8-diol-glucuronide effectivelyprevents the activation of this diol to (+)anti-BPDE. A substantialproportion of the BP-7,8-diol is also metabolized to glucuromdeand glutathione conjugates in BB and RTG-2 cells. DMBA alsobinds to DNA at very low levels in these fish cell cultures.Thus effective conjugation of diols and their metabolites byfish cell lines appears to greatly reduce metabolic activationof hydrocarbons through the bay-region diol epoxide pathwaythat predominates in mammalian cell cultures.     

Characterization of 5-methylchrysene-1,2-dihydrodiol-3,4-epoxide--DNA adducts

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