Effect of inducer and inhibitor probes on DNA adduction of benzo[a]pyrene and 2-acetylaminofluorene and their roles in defining bioactivation mechanism(s)

In this study, we used DNA adducts as the endpoint to reflect on the type and amount of electrophilic metabolites formed in a cell-free system, which were 'trapped' with DNA and the resultant adducts analyzed by a highly sensitive (32)p- postlabeling assay. Incubation of benzo[a]pyrene (BP) (10 mu M) with liver microsomes and S-9 fractions from uninduced and beta-naphthoflavone (beta-NF)-induced rats and NADPH-generating system resulted in two major adducts, one derived from the interaction of benzo[a]pyrene diolepoxide with deoxyguanosine (BPDE-dG) and the other from further activation of 9-OH-BP. beta-NF treatment increased the microsomal DNA adduction capability: both BPDE-dG and 9-OH-BP adducts were enhanced to 19,600 and 26,600 adducts/10(9) nucleotides compared to 2,800 and 1,700 adducts/10(9) nucleotides with uninduced microsomes, respectively. An even greater enhancement of both adducts was observed when S-9 was substituted for microsomes. These results suggest the involvement of CYP1A1 in BP activation because of the known role of beta-NF in the induction of this enzyme. Further, evidence of the involvement of CYP1A1 was obtained by using alpha-naphthoflavone (alpha-NF), a known inhibitor of CYP1A family. Addition of alpha-NF (50 mu M) to the activation system almost completely (>95%) abolished both the adducts. These results are consistent with selective inhibition of CYP1A1, the isozyme involved in the conversion of BP to BP-7,8-diol. Further, cyclohexene oxide (Chox) (100 mu M), a known inhibitor of epoxide hydrolase reduced BPDE-dG adduct by 55% over that in the absence of the inhibitor, suggesting its epoxide origin; 9-OH-BP adduct was, however slightly increased. Enhanced DNA adduction of another class of carcinogen, 2-acetylaminofluorene (2-AAF) (20 mu M) with induced S-9 and microsomes, and inhibition in the presence of alpha-NF was also observed which is consistent with the involvement of CYP1A2 in the initial activation of aromatic amines. Results from these experiments suggest that the approach of using a battery of inducer/inhibitor probes and their influence on DNA adduction capability of subcellular fractions coupled with P-32-postlabeling can be fully exploited as important tools in defining metabolic pathway(s) that may be involved in the bioactivation of unknown compounds.     

Transport of DNA-adducting metabolites in mouse serum following benzo [a]pyrene administration

Metabolic activation of benzo[]pyrene (BaP) by cellular enzymesis required for DNA adduct formation. In vivo DNA adducts mightalso arise from BaP metabolites supplied via the systemic circulation,rather than from in situ activation. We determined whether electrophilicmetabolites could be detected in mouse serum 4 h after BaP dosing(i.p.) by trapping metabolites with salmon sperm DNA (ssDNA),followed by ³²P-postlabeling analysis for DNA adducts. In vitrostudies demonstrated that mouse serum sequesters BaP-7,8-diol-9,10-epoxide(BPDE) and protects it from hydrolysis. BPDE was rapidly transferredfrom serum to ssDNA or splenocytes, with adduct levels in ssDNA4- to 7-fold greater than in splenocytes. After BaP administration,mouse serum produced two adduct spots when incubated with ssDNA.The major adduct (spot 3) co-chromatographed with a BPDE adductstandard, while the minor adduct (spot 2) was unrelated to BPDE.A BPDE standard curve in control serum was developed to quantitateBPDE levels in dosed serum. These levels ranged from 13.1 to19.1 nM. Tissue DNA contained three adduct spots: spots 2 and3 appeared identical to the respective adducts arising fromdosed serum. BPDE-DNA adducts in tisues were highest in liver,lung and spleen, with kidney and stomach levels significantlylower. Levels of adduct 2 did not correlate with levels of adduct3, especially in spleen where the adduct 2/adduct 3 ratio wasvery low. In vitro studies in which splenocytes were presentedwith both adducting metabolites suggested that splenocytes preferentiallyform adduct 3. These results indicate that two of the threeBaP electrophilic metabolites responsible for cellular DNA damageare present in mouse serum. The levels of BPDE in serum maybe sufficient to account for a substantial portion of the tissueload of BPDE-DNA adducts.     

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.     

Ellagic acid: a potent naturally occurring inhibitor of benzo[a]pyrene metabolism and its subsequent glucuronidation, sulfation and covalent binding to DNA in cultured BALB/C mouse keratinocytes

The metabolism of [³H]benzo[a]pyrene (BP) by cultured primarykeratinocytes prepared from BALB/C mouse epidermis was foundto be largely inhibited by the dietary plant phenol, ellagicacid. Varying concentrations of ellagic acid added to the keratinocytecultures resulted in a dosedependent inhibition of the cytochromeP-450-dependent monooxygenases aryl hydrocarbon hydroxylase(AHH) and 7-ethoxycoumarin-0-deethylase (ECD). The major organicsolvent-extractable metabolites found intracellularly in thecultured cells were trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene(BP-7,8-diol) and 3-hydroxybenzo[a]pyrene (3-OH-BP), althoughsmall amounts of 9-hydroxybenzo[a]pyrene, quinones and trans-9,10-dihydro-9,10-dihydroxybenzo[a]-pyrene(BP-9,10-diol) were also present. The major organic solvent-extractablemetabolites found in the extracellular culture medium were BP-7,8-dioland BP-9,10-diol, with smaller quantities of unconjugated phenolsand quinones. The major intracellular and extracellular water-solublemetabolites of BP were conjugated with glucuronide (primarily3-OH-BP and several BP-quinones), and to a lesser extent withsulfate (primarily BP-7,8-diol). Both intracellular and extracellularmetabolism of organic solvent-extractable and water-solubleconjugates was significantly inhibited by ellagic acid in adose-dependent manner. The intracellular enzyme-mediated bindingof BP to mouse keratinocyte DNA was also largely inhibited ina dose-dependent fashion by ellagic acid. Our results indicatethat cultured primary mouse keratinocytes offer a useful modelsystem for studying factors affecting the metabolic activationand detoxification of polycyclic aromatic hydrocarbon carcinogensin the epidermis, and that polyphenolic compounds such as ellagicacid may prove useful in modulating the risk of cutaneous cancerthat results from exposure to these environmental chemicals.     

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.     

Modification of aflatoxin B1 binding to DNA in vivo in rats fed phenolic antioxidants, ethoxyquin and a dithiothione

The effects of dietary administration of 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 2(3)-tert-butyl-4-hydroxyanisole (BHA),ethoxyquin (EQ) and 5-(2-pyrizinyl)-4-methyl-1,2- dithiol-3-thione(oltipraz) on aflatoxin B₁ (AFB₁) - DNA adduct formation invivo in livers and kidneys of rats were investigated. Male F344rats were treated with 1 mg/kg AFBI by i.p. administration andnucleic acids isolated 2 h post dosing. Animals were fed a semipurifleddiet supplemented with either 0.5% EQ, 0.45% BHT, 0.45% BHAor 0.1% oltipraz for 2 weeks prior to AFBI treatment. Analysisof nucleic acid bases by h.p.l.c. showed that several AFB metabolite-DNAadducts were formed in both tissues. The principal and relatedadducts of 8,9-dlhydro-8-(N² guanyl)-9-hydroxyaflatoxin represented80-90% of all adducts in both tissues and in all treatment groups.However, inclusion of the antioxidants in the diet resultedin substantial reductions in overall AFB modified DNA levels.EQ, BHT, BHA and oltipraz reduced the covalent binding of AFBto liver DNA by 91, 85, 65 and 76% and to kidney DNA by 80,35, 62 and 64%, respectively. Concordantly, the specific activitiesof hepatic enzymes of presumed importance to AFB₁ detoxification,epoxide hydrase, and glycuronyl and glutathione transferaseswere significantly elevated by all antioxidants. Reduced glutathionelevels were unchanged except by oltipraz, although activitiesof enzymes contributing to the maintenance of reduced gluta-thionepools, glutathione reductase and glucose-6- phosphate dehydrogenase,were elevated in most treatment groups. An excellent correlation(r = 0.95) was observed between the degree of inhibition ofDNA binding by AFB₁ and the induction of hepatic glutathioneS-transferase activities by the four antioxidants.     

Inhibition of DNA synthesis, independent of DNA adduct formation, by benzo[a]pyrene diol epoxide in mammalian cells

Treatment of SV40-infected CV-1 cells with the ultimate carcinogenanti-benzo[a]pyrene diol epoxide (BPDE) at 1 x 10 ^–4 mg/mlor higher reduced the rate of viral DNA synthesis to an extentdependent on the BPDE concentration; similar reductions in cellularDNA synthesis were produced in infected and uninfected cells.Treatment of cells with BPDE, followed by removal of BPDE, atvarious times before infection with SV40 gave the same results.Recovery or partial recovery of DNA synthesis occurred whenthe BPDE concentration was below 6 x 10 ^–4 mg/ml; at higherconcentrations the cells were killed. Simultaneously replicatingviral DNA's from viruses infecting the same cells before andafter BPDE treatment of the cells exhibited the same reducedrate of synthesis. The evidence indicates that covalent adductsin viral DNA are not responsible for its reduced replicationrate; moreover, it is probable that an insignificant numberof adducts is produced in intracellular viral DNA at BPDE concentrationsthat do not kill CV-1 cells. Rather, it appears likely thatBPDE inhibits viral DNA synthesis by attacking cellular DNAor non-DNA targets. Caution is therefore required in relatingthe effects of BPDE and other carcinogens to DNA adduct formation.     

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.     

Comparison of hydrocarbon - DNA adducts formed in mouse skin in vivo and in organ culture in vitro following treatment with benzo[a]pyrene

Species differences in the metabolism of toxic chemicals canconfound the extrapolation of experimentally determined riskdata to man. However, it is often difficult to obtain reliableinformation on human metabolism, particularly of genotoxic agents.In this study, comparisons of chromatographic pro-flies of DNAadducts formed in vivo and in vitro have been applied to developand validate in vitro systems as models for the bioactivationof precursor genotoxic agents in vivo. Reversed phase h.p.l.c.analysis showed that the DNA adducts obtained from the skin(epidermal and dermal) of mice (CD₁, CF₁ and athymic nude mice)treated topically with [³H] or [¹⁴C]benzo[a]pyrene (BP) werequalitatively very similar to those formed in mouse (CD₁) skinexplant cultures. In each case the principal product was theN²-deoxyguanosine adduct, (+)-N²-(7R, 8S, 9R-trihydroxy-7, 8,9, 10-tetrahydrobenzo[a]-pyren-10S-yl)-2'-deoxyguanosine, derivedfrom (+)-7R, 8S,9R-trihydroxy-9R, 10R-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene.The use of [¹⁴C]BP has provided an accurate reference profileof BP - DNA adducts formed in mouse skin in vivo. These findingsshow that mouse skin explants maintained in organ culture effectivelymimic the bioactivation of BP and the binding of the productsto the DNA of mouse skin in vivo. Such culture techniques arereadily transferable to human skin thus permitting the indirectdetermination of bioactivation pathways in human skin in vivofor comparison with those of mouse skin and other models usedto determine the human hazard. In principle, this approach tovalidate in vitro bioactivation systems may be applied to allhuman tissues.     

Effect of butylated hydroxyanisole on the metabolism of benzo[a]-pyrene and the binding of metabolites to DNA, in vitro and in vivo, in the forestomach, lung, and liver of mice

The effects of butyldted hydroxyanisole (BHA) administrationon the amounts of benzo[a]pyrene (BP) metabolite-DNA adductsformed in vivo in the forestomach of A/HeJ mice were investigated48 h after oral administration of BP. BP was administered tomice in amounts known to result in BPInduced neoplasia in certaintissues. Analysis of deoxyribonudeosides by h.p.l.c. showedthat several BP metabolite-DNA adducts were formed in this tissue.The major identified adduct was the (±)-7ß,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDEI) deoxyguanosine adduct. Addition of BHA to the diet inhibitedBPDE I-DNA adduct formation in the forestomach. The inhibitionof BPDE I-DNA adduct formation by BHA occurred under the sameexperimental conditions as does inhibition of tumor formationby this compound. These results in forestomach and previousresults in lung and forestomach demonstrated that inhibitionof the formation of the BPDE I-DNA adduct in the target tissueis a possible mechanism by which BHA inhibits BP-induced neoplasia.BP metabolism and DNA binding were also studied under in vitroconditions using microsomes prepared from forestomach, lung,and liver of A/HeJ mice. The amount of BPDE-DNA adduct formedin vitro is either equal to or lower than the amount of BP phenol-oxide-DNAadduct formed. BPDE I-DNA adduct formation was not significantlydifferent in incubations containing microsomes prepared fromBHA-treated or untreated mice. These results suggest that alterationsof the microsomal monooxygenases induced by BHA feeding arenot sufficient to account for the observed decreases in BPDE-DNAadduct formation in vivo. The monooxygenases were apparentlyaltered by BHA feeding as indicated by the substantial changesin the metabolic profile of BP and the decrease in the formationof the BP phenol-oxide-DNA adducts in the forestomach. The exclusionof glutathione transferases from the in vitro incubations couldaccount for the lack of effect of BHA treatment on BPDE-DNAadduct formation. BHA enhancement of ghitathione transferaseactivity has been postulated to play a role in the anticarcinogenicaction of BHA.     

DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilicand a neurotoxic metabolite of o-tolyl phosphates. In a previouspaper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide is mutagenic in Salmonella typhimurium TA100 and formsDNA adducts in incubations with nucleotides, nucleosides andisolated DNA. In the present study we compare DNA adduct formationusing ³²P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide-treated bacteria (S.typhimurium TA100) and hepatomacells with DNA adducts formed in liver, kidney, lung and heartof tri-o-tolyl phosphate-exposed Fischer 344 male rats. In bothbacteria and hepatoma cells two DNA adducts could be detectedafter treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide. The minor adduct co-chromatographed with syntheticN³-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling.The major DNA adduct was a cytidine adduct, most likely N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate. Male Fischer 344 rats were treated orallyfor 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNAwas isolated from liver, kidney, lung, heart, brain and testes1,4,7 and 28 days after giving the last dose. Analysis by ³²P-postlabellingrevealed that two adducts were present in the DNA isolated fromliver, kidney, lung and heart on the first day after givingthe last dose; DNA adducts were not detected in the brain andtestes. The adduct pattern after in vivo treatment with tri-o-tolylphosphate was identical with that found in bacteria and hepatomacells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin2-oxide, the major adduct being N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate and the minor N³-(o-hydroxybenzyl)deoxyuridine3' monophosphate. Both DNA adducts persisted in the lungs forthe entire observation period, whereas in the kidney only thecytidine adduct could be detected 28 days after the last doseof tri-o-tolyl phosphate. In liver and heart the adducts weredetectable only on the first day after completion of the treatment.The results indicate that in addition to the well establishedneurotoxicity, some o-tolyl phosphates may have a carcinogenicpotential.     

Effect of ellagic acid and 3-O-decylellagic acid on the formation of benzo[a]pyrene-derived DNA adducts in vivo and on the tumorigenicity of 3-methylcholanthrene in mice

The effect of ellagic acid and its more lipophilic derivative,3-O-decylellagic acid, on the amount of DNA-bound adducts inthe epidermis or lung of CD-I mice treated with [³H]benzo-[a]pyrene([³H]B[a]P) was evaluated using several different treatmentprotocols. The i.v. administration of 50µmol/kg of ellagicacid or 3-O-decyIellagk acid either together with or 5 min beforea 0.2 µmol/kg i.v. dose of [³H]B[a]P did not inhibit theformation of pulmonary DNA-bound adducts. Feeding mice a dietthat contained 1% ellagic acid for 10 days or the i.p. administrationof 120 µmol/kg of ellagic acid 30 min before the i.v.administration of 0.2 µmol/kg of [³H)B(a)P did not inhibitthe formation of DNA-bound adducts in the lung. The applicationof 2500 nmol of ellagic acid or 3-O-decylellagic acid to mouseskin 5 min before the application of 2, 10 or 50 nmol of [³H]B[a]Phad little or no effect on the covalent binding of [³H]B[a]Pmetabolites to epidermal DNA. Feeding mice a diet containing1% ellagic acid for 10 days did not inhibit the formation ofepidermal DNA-bound adducts after a topical dose of 2 nmol of[³H]B[a]P. Similarly, the topical application of 2500 nmol ofellagic acid at 2 h, 1 h and 5 min before and at 10 min afterthe application of 2 nmol of [³H]B[a]P did not inhibit the formationof DNA-bound adducts, but the same dosing regimen of 3-O-decylellagicacid (total dose of 10 000 nmol) resulted in a modest inhibitionin the formation of DNA-bound adducts. The topical applicationof 1500 nmol of ellagic acid 1 h before the application of 1500nmol of 3-methylcholanthrene (3-MC) to CD-I or BALB/c mice twiceweekly did not inhibit the development of skin tumors. Our resultsindicate that ellagic acid and 3-O-decylellagic acid are noteffective in inhibiting [³H]B[a]P DNA adduct formation in mouseskin and lung and that ellagic acid does not inhibit 3-MC-inducedskin tumori-genesis in BALB/c or CD-I mice.     

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 by N-acetylcysteine of carcinogen-DNA adducts in the tracheal epithelium of rats exposed to cigarette smoke

The ability of the aminothiol N-acetylcysteine (NAC) to preventthe formation of carcinogen-DNA adducts in tracheal epithelialcells was investigated in Sprague-Dawley rats exposed whole-bodyto mainstream cigarette smoke for either 40 or 100 consecutivedays. ³²P-Postlabelling analyses showed the occurrence of DNAadducts (12.49 adducts/10⁸ nucleotides) after 40 days of exposure,with a trend to formation of characteristic diagonal radioactivezones. Total adduct levels were not further enhanced after 100days of exposure to smoke, although significant changes occurredin the amounts of individual adducts. NAC, given by gavage inthe 40 day study and in drinking water in the 100 day study,significantly inhibited the formation of smoke-related carcinogen-DNAadducts in the tracheal epithelium, to such an extent that adductlevels were not significantly higher than those detected insham-exposed control rats. Together with a variety of othermolecular, clastogenicity, metabolic, cytological and histopathologicalend-points investigated in rodents and with the preliminaryevidence arising from a study in humans, these results documentthe considerable efficacy of oral NAC in inhibiting smoke-relatedcarcinogen-DNA adducts.     

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)     

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.     

The 32P-post-labeling method in quantitative DNA adduct dosimetry of 2-acetylaminofluorene-induced mutagenicity in Chinese hamster ovary cells and Salmonella typhimurium TA1538

The usefulness of the ³²P-post-labeling/t.l.c. method for quantitativeDNA adduct dosimetry was evaluated. 2-Acetylaminofluorene (2-AAF)-DNAadducts from three systems were characterized qualitativelyand quantitatively by the ³H-radiolabeled technique with subsequentanalysis by h.p.l.c. (pre-labeling method) and by the ³²-post-labelingmethod. Both methods showed N-acetoxyacetylaminofluorene (N-OAc-AAF)reaction products with calf thymus DNA were predominantly N-(deoxyguanosm-8-yl)-2-acetylaminofhiorene(dG-C8-AAF) with some N-(deoxyguanosin-8-yl)-2-amino-fluorene(dG-C8-AF) and N-(deoxyguanosin-N²-yl)-2-acetyl-aminofluorene(dG-N2-AAF). In contrast, Chinese hamster ovary (CHO) cellstreated with [³H]N-OAc-AAF gave 80 or 90% dG-C8-AF adducts and20 or 10% dG-C8-AAF adducts with the post- or pre-labeling method,respectively. Likewise in CHO cells treated with 2-AAF in thepresence of rat liver homogenate, {small tilde}90% dG-C8-AFand 10% dG-C8-AAF adducts were detected using the ³²P-post-labelingmethod. In Salmonella typhimurium strain TA1538 treated with2-AAF or [³H]2-AAF in the presence of a rat liver homogenate,one adduct, dG-C8-AF, was identified. Similar quantitative resultswere also obtained with the two methods. However, the ³²P-post-labelingmethod was more sensitive and also eliminated the use of radiolabeled-mutagentreatments. Quantitative DNA adduct dosimetry was applied toAAF-induced mutagenesis in the S. typhimurium and CHO/HPRT mutationassays. A linear and reproducible relationship existed betweendG-C8-AF levels and AAF-induced mutants in both systems.     

Interaction of ({+/-})-7r, 8t-dihydroxy-9t, 10t-oxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene with purified rat liver chromatin

Previous studies have shown that in addition to serving as atarget for covalent adduct formation, purified DNA catalyzesthe detoxification of (±)–7r, 8t-dihydroxy–9t,10t-oxy–7, 8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE). Tobegin to relate these in vitro findings with the processes importantin carcino-genesis in vivo, we have prepared native chromatinfrom rat liver nuclei and analyzed its interactions with BPDE.Using several different methods to follow the hydrolysis ofBPDE, we find the ability of chromatin to catalyse this detoxificationis severely reduced relative to purified DNA. The rate of formationof covalent adducts is also reduced, although the final levelof modification is almost the same in chromatin and purifiedDNA. The difference in rates could be an important in vivo protectionmechanism, especially in the presence of competing nucleophiles,e.g.-SH compounds. In addition, non-covalent, physical bindingto chromatin is altered, both quantitatively and qualitatively,compared with purified DNA. The specificity of covalent bindingof BPDE to histone proteins in chromatin is identical to thespecificity found in intact nuclei.     

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.