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.     

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.     

Anti-benzo[a]pyrene diolepoxide--DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking

The level of (±)-r-7, t-8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39coke oven workers exposed to polycyclic aromatic hydrocarbons(PAH) and 39 non-exposed persons (controls) were investigated,each of the groups consisting of smokers and non-smokers. Theadduct level was measured by an improved HPLC/fluorescence method(Rojas, M., Alexandrov, K., van Schooten, F.J., Hillebrand,M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557–560,1994) through the release of the corresponding benzo[a]pyrene(B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51%of coke oven workers exposed to PAH and in 18% of the non-exposed(control) subjects. The mean level of anti-BPDE-DNA adducts/10⁸nucleotides in coke oven workers (15.7 ± 37.8) was     

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.     

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.     

In vivo formation and persistence of DNA adducts in mouse and rat skin exposed to ({+/-})-trans-7, 8-dihydroxy-7, 8-dihydrobenzo[a]-pyrene and ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7, 8, 9, 10-tetrahydro-benzo(a)pyrene

The in vivo DNA adduct formation of (±)-trans-7, 8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BPD) and (±)-7ß, 8-dihydroxy-9,10-epoxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene (anti-BPDE) werecompared and the persistence and disappearance of the adductsin both mouse and rat epidermis determined. BPD (100 nmol/mousein 150 µl acetone and 200 nmol/rat in 300µl acetone)and anti-BPDE (77 nmol/mouse in 150 µJ tetrahydrofuran)and 154 nmol/rat in 300 µ tetra-hydrofuran) were topicallyapplied to 50-day-old male Swiss mice and 35-day-old Wistarrats. To improve the identification of the DNA adducts formed,an acid hydrolysis technique was used to convert the BPD- andanti-BPDE- de-oxyribonucleoside adducts formed in mouse andrat skin to BP tetrols. The modified deoxyribonucleosides andBP tetrols obtained by hydrolysis of adducts were isolated byreverse-phase h.p.l.c. At approximately similar doses per unitarea of treated skin, the initial total binding of these compoundsto epidermal DNA and the level of modified deox-yribonucleosideswas 6-fold lower in rat skin epidermis than in mouse skin epidermis.Similar ratios of (±)-anti-BPDE-deoxyguanosine (dGuo)to (±)-syn-BPDE-dGuo adducts (5.7 and 6.1, determinedby h.p.l.c. analysis of BP tetrols obtained by hydrolysis ofmodified dGuo) were found in both mouse and rat epidermis ashort time (6 h)after topical application of (±)-trans-BPD.Three hours after topical application of (±)-anti-BPDE,the ratios of BP-7, 10/8, 9-tetrol to 7/8, 9, 10-tetrol were9: 1 in mouse epidermal DNA and 6: 1 in rat epidermal DNA. Oneand three weeks after application of these two compounds, only(+)-anti-BPDE-dGuo was detected in mouse epidermis; 2 and 0.2%of the initial (+)-anti-BPDE-dGuo level was found to persistin the epidermal DNA from BPD- and anti-BPDE-treated mice respectively.No DNA adducts were detected in rat epidermis 3 weeks afterBPD and anti-BPDE treatment. Thus, 3 weeks after topical applicationof BPD and anti-BPDE to mouse and rat skin, the DNA adductscompletely disappeared form rat epidermis while they persistedin mouse epidermis. The results suggest that: (i) the persistenceof (+)-anti-BPDE-dGuo may be related to carcinogenesis in mouseepidermis by BPD and anti-BPDE; (ii) the complete disappearanceof the anti-BPDE-dGuo adduct may also account in part for therelative resistance of tissue from this species to the carcinogenicaction of benzo(a) pyrene.     

Synthesis and characterization of covalent adducts derived from the binding of benzo[a]pyrene diol epoxide to a -GGG- sequence in a deoxyoligonucleotide

Direct synthesis and purification procedures are described forthe preparation of adducts derived from the covalent bindingof 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene[(+)-ante-BPDE or (+)-BPDE 2] to each of the three guanine residues(trans-N² lesions) in the oligodeoxyribonucleotide d(CTATG¹G²G³TATC)The positions of the modified Gs are defined by Maxam-Gilbertsequencing techniques. Six different oligonucleotides with oneor two precisely positioned (+)-anti-BPDE residues are identified.The absorbance, circular dichroism and fluorescence characteristicsare changed upon formation of duplexes with the complementarystrands d(GATACCCATAG). In the doubly-modified oligonucleotides,a broad, excimer-like long wavelength fluorescence emissionband is observed with a maximum near 455 nm only if the two(+)-anti-BPDE-modified Gs are adjacent to one another. The covalentlyattached (+)-anti-BPDE residues decrease the thermodynamic stabilitiesof the duplexes; their melting points are markedly dependenton the position of the lesions, being highest with the (+)-anti-BPDEresidue at G¹ (T^m=40°C, only 2°C lower than in the caseof the unmodified oligonucleotide) and lowest when it is situatedat G³ (T^m=29°C). The implications of these and other physicalcharacteristics are discussed. The facile synthesis of theseor similar site-specific and stereochemically defined (+)-trans-anti-BPDE-N²-dGlesions in runs of contiguous guanines in oligodeoxyribonucleotidesof specified base sequence should be useful for the design ofsite-directed mutagenesis studies in vitro and in vivo.     

Formation and repair of benzo[a]pyrene--DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis

Hamster tracheal organ cultures were used to investigate therelationship between DNA adduct formation measured directlyby the ³²P-postlabeling assay, and the DNA damage measured indirectlyby the unscheduled DNA synthesis (UDS) assay. Hamster tracheaswere treated with three concentrations of benzo[a]pyrene (B[a]P)for 2 days. Postlabeling and UDS assays were also carried outa few days after removal of the B[a]P. Furthermore, the typesof B[a]P—DNA adducts formed in the in vitro organ culturewere qualitatively compared with those formed in vivo afterintratracheal intubation of B[a]P attached to Fe₂O₃ particles.In vivo only one adduct was detected by ³²P-postlabeling. Thisadduct co-chromatographed with the trans-addition produce ofdG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE). In vitro, a clear B[a]P-DNA adduct pattern was alsofound with the ³²P-postlabeling assay. Four different adductswere found. The main adduct spot migrated to the same positionon the thin-layer chromatogram as the in vivo adduct. B[a]P-DNAadduct formation was both time-and dose-dependent. During thefirst day after removal of B[a]P the adduct levels still increased,thereafter they decreased at all B[a]P concentrations. A time-and dose-dependent increase in UDS was observed in the trachealepithelial cells treated with B[a]P in vitro. After removalof the B[a]P, UDS decreased immediately, in contrast to theformation of DNA adducts. The results of the present study showthat B[a]P induces time- and dose-dependently both DNA adductsand UDS in hamster tracheal organ culture. Moreover, the mainDNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same asthat found in vivo.     

Spectroscopic studies on double-stranded poly d[(G-C)(G-C)] in B and Z form after covalent modification with the anti diastereomer of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo,[a] pyrene

Poly d[(G-C)(G-C)] in B or Z conformation has been incubatedwith the anti-diasterromer of trans-7,8-dU1ydroxy-9,10 epoxy-7,8,9,10-tetrahydrobenzo,[a]pyrrene(anti-BPDE). Enzymatic hydrolysis, circular dichroism (CD) andlight absorp tion were used to study the binding specificityof anti-BPDE to tbe right- and left-handed form of poly d[(G-C)(G-C)].The effect of anti-BPDE binding on the conformational transition,induced by MnCl₂ or NaCl, was also studied. The rsults obtaineddemonstrate that in Mn²⁺ -containing solutions, when the ionicconditions are identical, there is only a minor difference inthe extent of covalent binding of anti-BPDE to the polynucleotidein right- or left-handed form. A high degree of enanticlselectivityof poly d[(G-C(G-C)] towards (+)-anti-BPDE was observed withboth conformational states, although the B form seems to beslightly more selective. With either form, the major adductin the h.p.l.c. analysis is consistent with trans-N²-10[(7ß,8,9-trihydroxy-7,8,9,l0 tetrahydrobenzo[alpyrene)]-deoxyguanosine(+)-anti-BPDE-N-²-dG]adducts. A 3 nrn hathochromk shift of the light absorption ofthe pyrene chromophore was observed for tbe Mn²⁺-induced left-handedform of tbe polynucleotide. A similar shift was observed withthe NaCl-induced transition only at intermediate NaCl concentrations,where the CD spectra showed a mixture of right- and left-handedforms. The bathochromic shift may be a consequence of a reducedexposure of the chromophore to tbe aqueous solvent. In polyd[(G-C)(G-C)] modified with anti-BPDE, higher ionic strengthsare required to reach optimal B–Z transition comparedto unmodified samples. This indicates that there is a restrictionin the transition process at the sites of modification. Theresults have been discussed in terms of two distinct domainsin the mudifled polynucleotide: one ‘wd’ domain,where an initially rapid transition takes place, and another‘BPDE-domain’ involving the modification site anda few adjacent base pairs. It is likely that this domain isassociated with the inhibitory effect on the B–Z transition.     

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.     

Flanking base effects on the structural conformation of the (+)-trans-anti-benzo[{alpha}]pyrene diolepoxide adduct to N2-dG in sequence-defined oligonucleotides

Conformations of the trans adduct of (+)-anti-benzo[a]8- pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) to N²-guanine, the majorstable DNA adduct of the environmental carcinogen benzo[a]pyrene,were studied as a function of flanking bases in single-strandedand in double-stranded oligonucleotides. Three llmer oligonucleotidesd(CTATG₁G₂G₃TATC) were synthesized containing the (+)-transanti-BPDEadduct at one specific guanine of the GGG sequence (a knownmutational hot spot). Polyacrylamide gel electrophoresis ofthe three single-stranded oligonucleotides showed that the adductbound to G₂ or G₃ (5’-flanking base guanine) caused significantlystronger retardation than the same adduct bound to Gt (5’-flankingbase thymine). The strength of the carcinogen-base interactionwas reflected in the spectroscopic properties of the pyrenylmoiety. Low temperature fluorescence measurements under line-narrowing(FLN) or non-line-narrowing (NLN) conditions showed that insingle-stranded form the adduct at G₂ or G₃ (5’-flankingbase guanine) adopts a conformation with strong interactionwith the bases. This was also observed for the same adduct atthe sequence AGA. In contrast, the (+)-trans-anti-BPDE adductwith a 5’ -flanking thymine exists in a primarily helix-externalconformation. Similar differences were observed in the double-strandedoligonucleotides: the adducts at G₂ and G₃ were found to existin similar conformational equilibria, again with significantcarcinogen-base interactions , while the adduct at G₁ showeda predominantly external conformation. The nature of the 3’-flankingbase appeared to have little influence on the conformationalequilibrium of the (+)-trans-anti-BTDE—guanine adductThe results could provide insight into the mutational specificityand flanking base effects observed for (+)-anti-BPDE.     

Fluoranthene-DNA adducts: identification and quantification by an HPLC-32P-postlabeling method

A ³²P-postlabeling procedure for identifying and quantifyingfhioranthene (FA) - DNA adducts has been developed through modificationsof the method of Randerath and collaborators. In this modifiedprocedure, labeled adducts are separated chromatographicallyby high-pressure liquid chromatography (HPLC) and quantifiedby liquid scintillation counting. FA-modified DNA is digestedto nucleotide 3'-monophosphates and nucleotide 3'-monophosphateadducts; unmodified nucleotides are then separated from adductsusing a disposable C₁₈ cartridge. Residual unmodified nucleotides,which reduce the efficiency of ³²P-postlabeling of FA adducts,are removed by brief digestion with nuclease P1. This treatmentselectively dephospborylates unmodified nucleotide 3-monophosphates,while FA adducts are minimally affected. FA adducts are then5-phosphorylated with polynucleotide kinase and [-³²ATP. Prolongedtreatment with nuclease P1 then is employed to remove the unlabeled3-phosphate from adducted diphosphate nucleotides, followingwhich adducts are separated by HPLC and quantified by liquidscintillation counting. Postlabeled microsomally-activated FA-modifiedDNA contained adducts derived from anti- and syn-2,3-dihydroxy-l,10b-epoxy-l,2,3-trihydrofluoranthene.The identity of the major adduct of DNA-bound microsomally-activatedFA was confirmed by this HPLC-P-postlabeling method as an a/tft-2,3-dihydroxy-l,10b-epoxy-fluoranthenenucleotide adduct. Each step in the procedure was optimizedwith respect to experimental conditions, and the recovery ofadducts was determined by analysis of DNA modified with [³H]FA.In repeated analyses of 2–50 µg DNA containing 1.8adducts per 10s nucleotides, 10–15% of total DNA-bound[³H]FA was recovered as the major adduct; recovery was greaterfrom DNA containing higher levels of adducts. The reproducibilityof multiple analyses of the same sample was {small tilde}5%,and multiple analyses at different times were reproducible withinexperimental error. The limit of detection of the method was{small tilde}0.1 finol adduct, representing a binding levelof {small tilde} 3 adducts per 10⁸ nucleotides in 1 µgDNA or {small tilde}1 adduct per 1010 nucleotides in 500 µgDNA. Because the method is not limited with respect to the amountof DNA that can be subjected to analysis, the inherent sensitivityfor adduct detection can be greatly enhanced by analysis oflarger quantities of DNA.     

Persistence of O6-guanine DNA adducts in styrene-exposed lamination workers determined by 32P-postlabelling

A modified ³²P-postlabelling method was used for the detectionof styrene-specific DNA adducts in lamination workers. The persistenceof O⁶-styrene DNA adducts was studied in DNA from lymphocytesand granulocytes of an exposed and a control group. We comparedO⁶-adduct levels obtained from a sampling prior to vacation,after 2 weeks of vacation and after an additional 1 month ofwork. In granulocytes, there was no significant difference inadduct levels between the control and the exposed groups inany individual samplings. In lymphocytes of laminators the detectedadduct levels were significantly higher (5.4 adducts/10⁸ nucleotides)than those in the controls (1.0 adduct/10⁸ nucleotides). The2 week interruption of exposure did not influence the totalO⁶-adduct level (4.9 adducts/10⁸ nucleotides in the first samplingversus 5.1 adducts/10⁸ nucleotides in the second), indicatingvery slow removal of the specific O⁶-styrene adducts from DNA.     

Catechol-induced alterations in metabolic activation and binding of enantiomeric and racemic 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes to DNA in mouse skin

Catechol (1,2-dihydroxybenzene) is a potent co-carcinogen withbenzo[a]pyrene (BaP) and with (?)-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BaP-7, 8-diol) in mouse skin. The effects of catechol on themetabolic activation of (+)- and (–)- [³H]BaP-7,8-diolsand on epidermal DNA adduct formation of racemic and enantiomeric[³H]BaP-7, 8-diols were examined by applying the tritlated diolsto mouse skin. The major metabolite of the (+)- [³H]BaP-7, 8-diolswas the hydrolysis product of (–)- [³H]-7,8ß-diolsepoxy-9ß,10ß-epoxy-7,8,9, 10-tetrahydroheiizo[a]pyrene (anti-BPDE).This suggests that a peroxyl radical-mediated pathway Is predominantlyresponsible for the epoxidation of this diol. Formation of (–)-anti[³H]BPDEfrom (+)-[³H]BaP-7,8-diol was greater than that of (+)-anti-BPDEfrom (–)-[³H]BaP-7,8-diol Co-application of catechol with[³H]BaP-7,8-diols inhibited epoxidation of the (+) enantiomerto a greater extent than that of the (–) enantiomer. Catecholdecreased the total DNA-binding and the formation of the majoradduct with (+)-[³H]BaP-7, 8-diols metabolites but catecholhad no significant effect on the binding and formation of (+)-anti-[³H]BPDE-deoxyguanosine the major DNA adduct derived from (–)-[³H]BaP-7,8-diolsCo-administration of catechol with (?)-[³H]BaP-7,8-diols increasedthe ratio of (–)- to (+)-[³H]BaP-7, 8-diols major DNAadducts in mouse skin suggesting that catechol selectively inhibitscertain pathways of metabolic activation of (? )-[³H]BaP-7,8-diols Thus, catechol modifies the tumorigenic activity of(?)- BaP-7 ,8.-diol either by alteration of the relative proportionof various hydrocarbon:DNA adducts or by a totally differentas yet unexplored mechanism.     

32P-Postlabeling and HPLC separation of DNA adducts formed by diesel exhaust extracts in vitro and in mouse skin and lung after topical treatment

Diesel exhaust extracts contain many carcinogenic compoundswhich have been shown to form polycyclic aromatic hydrocarbon(PAH)— and nitrated PAH—DNA adducts in rodent skinand lung. The aim of this study was to characterize by ³²P-postlabeling,TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formedin vitro and in vivo by diesel extracts. The diesel particleextracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes(B[b,j,k]F) and chrysene. DNA adducts were analyzed in calfthymus DNA incubated in vitro with PAHs activated by S9 mixand in skin and lung DNA from topically treated mice. The maindiesel-derived DNA adduct formed in vitro and in vivo did notco-migrate on HPLC and large TLC plates with ()-r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE)—,B[b]F—, B[j]F—, B[k]F— or chrysene—DNAadduct standards. By co-chromatography DNA adducts formed bychrysene from both in vitro and in vivo samples were identified.Nissan diesel extract containing higher PAH concentrations thanVolkswagen automobile extract formed skin DNA adducts that co-migratedwith chrysene— and anti BPDE—DNA-derived adducts.We conclude that the use of a highly sensitive ³²P-postlabelingmethod combined with HPLC improves the identification of PAHadducts formed by complex mixtures such as diesel exhaust extracts.     

32P-Postlabeling analysis of DNA adducts in human and rat mammary epithelial cells

The etiology of hwnan breast cancer is currently undefined.However, it has been hypothesized that exposure to chemicalcarcinogens may be an important factor. Extrapolation from rodentmodels for chemically-induced mammary cancer suggests the possibilitythat human mammary epithelial cells insitu might contain DNAadducts due to exposure to environmental chemicals. We havetherefore screened breast epithelial cells from 10 donors forthe existence of DNA adducts using the ³²P-postlabeling assay.In order to validate this analysis technique, we also examinedthe DNA adducts formed in human mammary cells exposed to benzo[a] Pyrene (B[a]P)in vitro, and adducts formed in rat mammaryepithelial cells exposed to B[a]P in vitro and in vivo. Confirmingprevious results using HPLC analysis of [³H]B[a]P-DNA adducts,the major B[a]P-adduct formed by human mammary epithelial cellsin vitro was (+)-anti-B[a]P-7, 8-dihydrodiol-9, 10-epoxide (BPDE):deoxyguanoslne.This adduct did not appear to be formed b rat mammary cellsexposed to B[a]P in vitro. However, ³²P-postlabeling analysisof mammary epithelial cell DNA from rats exposed to B[a]P invivo indicated that (+)-anti-BPDE-deoxyguanosine was a majorB[a]P-DNA adduct under these exposure conditions. When the mammaryepithelial cells from 10 human donors were screened for DNAadducts formed in situ, cells from three donors exhibited distinctadduct patterns. None of these adducts appeared to be (+)-anti-BPDE-deoxyguanosine.The existence of DNA adducts in human mammary epithelial cellsin situ, coupled with the data indicating that rat mammary cellsform different B[a]P adducts in vitro and in situ, suggeststhe need for further study of human breast cell adducts.     

SHORT COMMUNICATION: Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene

We have employed the P₁-enhanced ³²P-postlabeling procedureto detect the formation DNA of adducts in the white blood cells(WBC) of B6C3F1 mice treated by i.p. injection with benzene.Treatment twice a day with 440 mg/kg benzene for 1–7 daysresulted in the formation of one major (adduct 1) and one minor(adduct 2) DNA adduct in the WBCs of mice. The same DNA adductpattern was also found in the bone marrow (BM) of benzene treatedmice. The relative adduct levels were dependent upon both benzenedose from 100–440 mg/kg and treatment time from 1 to 7days. The relative adduct levels ranged between 0.11 and 1.33adducts in 10⁷ nucleotides for WBCs and 0.16–1.21 adductsin 107 nucleotides for BM. Following treatment with benzene,the levels of DNA adducts formed in WBCs were significantlycorrelated with the levels of DNA adducts formed in BM (r² =0.97, P <0.001). Our results suggest that measurement ofDNA adducts in WBCs may be an indicator of DNA adduct formationin BM following BZ exposure.     

Inhibition of poly(ADP-ribose) polymerase increases ({+/-})-anti-benzo[a]pyrene diolepoxide-induced micronuclei formation and p53 accumulation in isolated human peripheral blood lymphocytes

In response to DNA damage, in particular DNA strand breaks,the proposed roles for normal tumour suppressor protein p53are to increase the period of time available for DNA repairprior to replication, or to direct damaged cells into programmedcell-death. Since treatment of mammalian cells with (±)-anti-benzo[a]pyrenediolepoxide [(±)-anti-BPDE]—a mixture of metabolitescomprising the most reactive (+ )-anti-enantiomer of the fullenvironmental carcinogen benzo[a]pyrene—has been shownto result in induction of DNA repair processes and consequentlyin DNA strand break formation, the aim of the present studywas to investigate whether p53 accumulation is induced in (±)-anti-BPDE-treatedphytohaemagglutinin-stimulated human peripheral blood lymphocytes(PBLs). Both immunocytochemical and immunoblot analysis indicatedthat treatment of PBLs with (±)-anti-BPDE results inp53 accumulation. Optimal accumulation was observed at 2.5 uM,while no increase of p53 levels was observed at concentrations<2.5 µM and >10 µM. Further, (±)-anti-BPDE-inducedp53 accumulation in PBLs was found to be time-dependent withaccumulation up to 24 h after the onset of treatment. Treatmentof PBLs with 2.5 µM of (±)-anti-BPDE and 1 mM of3-aminobenzamide, an inhibitor of the DNA strand break-dependentenzyme poly(ADP-ribose) polymerase, resulted in increased p53levels, in comparison to cells treated with (±)-anti-BPDEalone. This combination also potentiated the frequency of (±)-anti-BPDE-inducedmicronuclei. These findings suggest that (±)-anti-BPDE-inducedDNA strand break formation is responsible for the observed p53accumulation. It is unlikely that poly(ADP-ribose) polymer formationis a prerequisite in the process of p53 accumulation, as triggeredby DNA strand-break inducing agents like (±)-anti-BPDE.It is hypothesized that p53-dependent pathways may be activatedin phytohaemagglutinin-stimulated human peripheral blood lymphocytesexposed ex vivo to (±)-anti-BPDE.     

Benzo[a]pyrene-DNA adduct formation in target and activator cells in a Wistar rat embryo cell-mediated V79 cell mutation assay

To determine the relationship of the benzo[a]pyrene (BaP)-DNAadducts formed in the activator cells of a cell-mediated mutationassay to the adducts formed in the target cells and to mutationinduction, irradiated second passage Wistar rat embryo (WRE)cells and V79 Chinese hamster lung cells were exposed to [³H]BaPfor 5, 24 and 48 h under the conditions of a cell-mediated mutationassay. The V79 target cells were separated from the WRE activatorcells by an immunoseparation procedure; the resulting V79 cellpellet contained <7% WRE cells. The percentage of the BaP-DNAadducts containing cis vicinal hydroxyl groups and the h.p.l.cprofile of individual adducts in the V79 target cells were similarto those of the WRE activator cells for each time point. Thetransfer of reactive BaP metabolites from the activator cellsto the target cell DNA was detectable after only 5 h of exposureto BaP, however, exposure for this length of time did not resultin significant mutation induction. The (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(anti-BaPDE)-deoxyguanosine (dGuo) adduct was essentially absentafter 5 h of exposure, but the amount of this adduct increasedat longer times of exposure as did the mutation frequency. Thecomplex mixture of BaP-DNA adducts formed in the WRE cells wasalso present in the V79 cells after all three times of exposureto BaP, a result which demonstrates the value of this cell-mediatedmutation assay for investigating the role of species-specificdifferences in the activation of BaP. The correlation betweenthe increase in mutation induction and the relative amount ofthe (+)-anti-BaPDE-dGuo adduct present in V79 cell DNA suggeststhe importance of this adduct in mutation induction by BaP.     

Mutational specificity of the (+)-anti-diol epoxide of benzo[a]pyrene in a supF gene of an Escherichia coli plasmid: DNA sequence context influences hotspots, mutagenic specificity and the extent of SOS enhancement of mutagenesis

Mutagenesis by the suspected major mutagenic metabolite of activatedbenzo[a]pyrene, which is (+)-anti-BPDE, was analyzed with anew system, which permits the selection of supF^– mutationsin an Escherichia coli plasmid using lactose minimal plates.(+)-anti-BPDE enhances base pairing mutations—principallyat G:C base pairs, frameshift mutations and large deletions.Frameshift mutagenesis principally involves deletions and insertionsof a single G:C base pair in runs of G:C base pairs. Base pairingmutations are significantly enhanced by SOS induction, especiallyGC