Relation between benzo[a]pyrene-DNA adducts, cell proliferation and p53 expression in tracheal epithelium of hamsters fed a high {beta}-carotene diet

Vitamin A and ß-carotene protect against respiratorytract cancer by inhibiting the formation of DNA damage and controllingcellular proliferation and differentiation. Recently, it hasbeen shown that the p53 tumor-suppressor gene plays a crucialrole in the etiology of respiratory tract cancer. In the presentstudy, we investigated the relationship between benzo[a]pyrene(B[a]P)-DNA adducts, cell proliferation and p53 expression andthe possible effect of ß-carotene on such a relationshipin tracheal epithelium of hamsters given intratracheal instillationsof B[a]P-Fe₂O₃ particles suspended in saline. DNA-adducts werequantified by the ³²P-postlabeling assay, cell proliferationwas quantified by immunocytochemical detection of incorporatedBrdU during S-phase, and p53 protein was detected by immuno-histochemistrywith an antibody that recognized both the wild-type and themutated protein (BioGenex, Clone BP53–12–1). A clearrelationship appeared to exist between the extent of B[a]P-DNAadduct formation, the induction of cell proliferation and theexpression of p53 protein in hamster tracheal epithelium. Theseresults suggest that B[a]P induces cell proliferation in hamstertracheal epithelial cells most likely by the induction of mutationsin the p53 gene. Furthermore, ß-carotene was not foundto influence the formation of B[a]P-DNA adducts, which is probablydue to the high B[a]P dose. Moreover, ß-carotene didnot statistically significantly affect cell proliferation andp53-protein expression in hamster tracheal epithelial cells.     

Comparison of DNA adduct formation in mice fed coal tar or benzo[a]pyrene

Coal tar is a complex mixture containing hundreds of compounds,including the carcinogenic poly cyclic aromatic hydrocarbon,benzo[a]pyrene. In order to compare the metabolic activationof a single carcinogen versus a complex mixture containing thecarcinogen, we determined the DNA adduct profiles in B6C3F₁mice fed doses of coal tar or benzo[a]pyrene at concentrationscorresponding to the amount of benzo[a]pyrene found in the respectivecoal tar treatments. DNA adduct formation was quantified inliver, lungs and forestomach by ³²P-postlabeling and was foundto increase as a function of dose in each tissue with both coaltar and benzo[a]pyrene. In mice fed benzo[a]pyrene, a majoradduct was detected with the same elution characteristics byTLC and HPLC as the major adduct, 10ß-(deoxyguanosin-N²-yl)-7ß,8     

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.     

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.     

DNA adduct formation and repair in hamster and rat tracheas exposed to benzo[a]pyrene in organ culture

Syrian golden hamsters are much more susceptible than Wistarrats to the induction of tracheal tumors by benzo[a]pyrene (B[a]P).To investigate whether this difference is reflected in the patternof DNA adduct induction and removal, tracheas from either specieswere isolated and exposed to B [a]P (5 µg/ml) in organculture. At various time- points B[a]P-DNA adducts were quantifiedby ³²P-postlabeling; unscheduled DNA synthesis (UDS) and cellproliferation were determined by [³H]thymidine incorporationduring the 18 h before sampling. In an induction-repair experimenttracheas were exposed to B[a]P for 2 days, and cultured foranother 4 days without B[a]P After 2 days of exposure totalB[a]P-DNA adduct levels were 10 times higher in hamster comparedto rat tracheas. In hamster tracheas one major adduct was formed(95%), namely the adduct between (+)-anti-benzo[a]pyrene diolepoxideand deoxy guanosine (BPDE-N²dG). In rat tracheas BPDE-N²dG comprised60% of the total B[a]P-DNA adduct level. The other major adductfound in rat tracheas is probably derived from interaction ofsyn-BPDE and deoxyadenosine. During exposure to B[a]P in hamstersthe adduct level increased to 36 ± 19 adducts/10⁶ nucleotides(add/10⁶n) on day 2. Two days after removal of B[a]P-the B[a]P-DNAadduct level had decreased to 60% of that on day 2; there wasno further decrease in the B[a]P-DNA adduct level, despite considerablecell proliferation at the end of the 6 day culture period. UDSincreased during exposure to B[a]P and decreased after removalof B[a]P. In rats removal of B[a]P did not lead to a decreasein the B[a]P adduct level, which agreed with the observed absenceof UDS. In a second experiment tracheas were exposed to B[a]Pcontinuously for 15 days. In hamster tracheas the total B[a]P-DNAadduct level increased from 11 ± 0.7 add/10⁶n after 1day of exposure to 105 ± 2 add/10 after 15 days; alsoUDS increased with increasing exposure until day 11. Cell proliferationwas low at the end of the culture period. In rat tracheas noprogressive increase in the B[a]P-DNA adduct level was seen,UDS was not increased and cell proliferation had increased significantlyat the end of the exposure period. The extent of adduct inductionin the trachea of the two species corresponded with the differentsusceptibilities to B[a]P-induced tumor formation.     

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.     

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.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P₁-enhanced ³²P-postlabeling to investigate DNA adductformation in the bone marrow of B6C3F1 mice treated intraperitoneallywith benzene (BZ). No adducts were detected in the bone marrowof controls or mice treated with various doses of BZ once aday. After twice-daily treatment with BZ, 440 mg/kg, for 1 to7 days, one major and two minor DNA adducts were detected. Therelative adduct levels ranged from 0.06–1.46x10^–7.In vitro treatment of bone marrow from B6C3F1 mice with variousdoses of hydroquinone (HQ) for 24 h also produced three DNAadducts. These adducts were the same as those formed after invivo treatment of bone marrow with BZ. Co-chromatography experimentsindicated that the principal DNA adduct detected in the bonemarrow of B6C3F1 mice was the same as that detected in HL-60cells treated with HQ. This finding suggests that HQ may bethe principal metabolite of BZ leading to DNA adduct formationin vivo. DNA adduct 2 corresponds to the DNA adduct formed inHL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remainsunidentified. After a 7-day treatment with BZ, 440 mg/kg twicea day, the number of cells per femur decreased from 1.6x10⁷to 0.85xlO⁷, indicating myelotoxicity. In contrast, administrationof BZ once a day produced only a small decrease in bone marrowcellularity. These studies demonstrate that metabolic activationof BZ leads to the formation of DNA adducts in the bone marrow.Further investigation is required to determine the role of DNAadducts and other forms of DNA damage in the myelotoxic effectsof exposure to BZ.     

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.     

Covalent binding of polycyclic aromatic hydrocarbon components of coal tar to DNA in mouse skin

Treatment of mouse skin with coal tar is known to initiate tumourformation, with the carcinogenic activity associated mainlywith polycyclic aromatic hydrocarbons (PAHs). A sample of pharmaceuticalcoal tar was analysed by gas chromatography and 19 major PAHswere identified. ³²P-postlabelling analysis was used to characterizethose PAHs that are responsible for the DNA binding of coaltar and, by inference, its biological activity. PAHs were groupedaccording to their reported carcinogenic activities and appliedas mixtures to mouse skin. Group A contained all of the 19 PAHs,group B seven PAHs for which there is sufficient evidence forcarcinogenicity and group C 12 PAHs with only limited or inadequateevidence of carcinogenicity in experimental animals. ³²P-LabelledDNA adducts formed by coal tar were resolved on TLC into a patternof three discrete spots (2, 4 and 6) and four areas of diffuseradioactivity (1, 3, 5 and 7). By comparison of the patternof adducts formed by coal tar with those formed by the syntheticmixtures it appeared that PAHs in group B formed coal tar–DNAadduct spots 4 and 6, and that adduct spot 2 was formed by PAHsin group C. Attempts to identify those PAHs responsible forthe formation of coal tar–DNA adducts 4 and 6 were madeby comparing the chromatographic mobilities of ³²P-labelledcoal tar-derived DNA adducts formed in mouse skin, using TLCand HPLC, with those formed by PAHs in group B. As benzo[ghi]perylene(B[ghi]P), a component of group C, has been demonstrated toexhibit significant DNA binding ability previously, the chromatographicmobility of coal tar–DNA adduct spot 2 was compared tothat of the major DNA adducts formed by B[ghi]P in vivo andin vitro. It appeared that coal tar adduct spot 2 was the majoradduct formed by B[ghi]P in vitro and that benzo[a]pyrene, benzo[b]fluoranthene,benzo [j]fluoranthene and benzo[k]fluoranthene contributed tothe formation of adduct spot 6. None of the PAHs examined appearedto be responsible for the formation of adduct spot 4.     

Characterization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro

Bay region diolepoxide—DNA adducts of dibenz[a,h]anthracene(DBA) formed in vitro were identified and their absolute stereochemistrywas assigned. After activation of [5,12-¹⁴C]DBA with liver microsomesobtained from Aroclor 1254 treated male Sprague—Dawleyrats in the presence of calf thymus DNA for 1 h, the amountof DNA adducts was found to be 9.9 ± 2.4 pmol/mg DNA,calculated on the basis of the portion of radioactivity elutedfrom the HPLC reversed-phase column with a water/acetonitrilegradient. Bay region diolepoxide—DNA adducts represented27.5% of radioactivity associated with DNA adducts. The absoluteconfiguration of the various adducts was determined from thereaction of the (+)- and (–)-3,4-dihydrodiol after metabolicactivation and the reaction of the anti- andsyn-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracenewith DNA or with the individual deoxyribonucleotides. The mainbay region adduct was identified as a deoxyguanosine adductof (anti)-3S,4R-dihydroxy-1R,2S-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene, a metabolite of (–)-3,4-dihydroxy-3,4-dihydrodi-benz[a,h]anthracene.Anti bay region diolepoxide-deoxyguanosine adducts of DBA contributedto 17.7% and syn diolepoxide-derived deoxyguanosine adductsto 5.8% of adduct-associated radioactivity. The amount of bayregion deoxyadenosine adducts was calculated to be 4%. For sixof probably eight different deoxyadenosine adducts absolutestereochemistry could be assigned. ₃₂P-Postlabelling experimentsrevealed a binding of 23 ± 6 pmol/mg DNA for (–)-3,4-dihydrodioland of 1.5 ± 0.4 pmol/mg DNA for (+)-3,4-dihydrodiolof DBA.     

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.     

Inhibitory effect of vitamin C on the mutagenicity and covalent DNA binding of the electrophilic and carcinogenic metabolite, 6-sulfooxymethylbenzo[a]pyrene

6-Sulfooxymethylbenzo[a]pyrene has recently been shown to bea strong hepatocarcinogen in infant male B6C3F₁ mice (Y.-J.Surhet al., Biochem. Biophys. Res. Commun., 172, 85–91, 1990)and appears to be an ultimate carcinogenic metabolite of 6-hydroxymethylbenzo[a]pyreneand possibly of benzo[a]pyrene and 6-methylbenzo[a]pyrene Itproduced high levels of aralkyl DNA adducts in the livers ofB6C3F₁ mice and also exhibited strong direct mutagenicity towardSalmonella typhimurium TA98 without metabolic activation. Inthe present study we found that ascorbic acid significantlyreduced the bacterial mutagenicity and in vitro covalent DNAbinding of 6-sulfooxymethylbenzo[a]pyrene. Ascorbic acid formsa mutagenically inactive covalent adduct with 6-sulfooxymethylbenzo[a]pyrene,which appears to account for its novel protective mechanismagainst this reactive sulfuric acid ester. It seems likely thatthe formation of this adduct involves aralkylation of an ascorbicacid anion by a presumed carbo cation derived from the electrophilicsulfuric acid ester.     

Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in isolated rat hepatocytes and rabbit lung cells

Benz[j]aceanthrylene (B[j]A) and benz[l)aceanthrylene (B[l]A),two isomeric cyclopenta polycyclic aromatic hydrocarbons (CP-PAH)structurally related to 3-methylcholanthrene, were studied withrespect to their genotoxic effects in isolated liver and lungcells. Both compounds were found to cause DNA adducts measuredby the ³²P-postlabelling technique. The level of DNA-adductsin rat hepatocytes exposed to 30 µg/ml B[l]A and B[j]Afor 4 h were 46.5 ± 22.0 and 8.3 ± 5.1 fmol/µgDNA respectively. Using butanol extractions, the major and oneof the minor B[l] A adducts co-chromatographed with B[j]A-l,2-oxide adducts of 2'-deoxyadenosine and 2'-deoxyguanosine.Thus, oxidation at the cyclopenta-ring of B[j]A appears to bean important activation pathway. In hepatocytes, 3-30 µg/mlof B[j]A and B[j]A induced DNA damage and repair measured bothas increased alkaline elution of DNA and as increased incorporationof [³H]TdR in the DNA. B[l]A was somewhat more potent than B[j]Ain inducing DNA repair. Reactive CP-PAH intermediates formedin the hepatocytes caused mutations in Salmonella typhimuriumTA98 upon co-incubation. DNA adducts were also observed in isolatedrabbit lung cells exposed to 30 µg/ml B[l]A or B[j]A for2 h. A total of 14.5 ± 6.9, 2.9 ± 2.1 and 0.2± 0.6 fmol B[l] A adducts/µg DNA were observedin Clara cells, type II pneumocytes and alveolar macrophagesrespectively. The main B[l]A adduct observed in the liver cellswas not found in the lung cells. On the other hand, the levelsof B[j]A adducts in the lung cells were in the range 4-14% ofthat found in liver cells, and no major differences betweenthe various lung cells were observed. Neither B[l]A nor B[j]Ainduced DNA damage measured by alkaline elution in the lungcells, indicating that these adducts are not alkali labile.     

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 DNA adducts in different target tissues of rats after multiple administration of benzo[a]pyrene

Exposure of humans to poly cyclic aromatic hydrocarbons is anongoing concern because of the carcinogenicity of these substances.DNA adducts are being increasingly used as indicators of carcinogenexposure. While considerable experimental evidence exists tosupport their use there are aspects that require further attention,especially after repeated exposure, which has led to this seriesof experiments. Male Sprague-Dawley rats were dosed with 10mg/kg benzo[a]pyrene (B[a]P) i.p., 3 times/week for 2 weeks.At 1, 3, 7, 14, 28 and 56 days after the last treatment liver,lung, spleen and peripheral blood mononuclear cells (PBMNs)were sampled. The DNA adduct levels, as measured by the ³²P-postlabellingtechnique, were significantlyincreased in all tissues, withlung having the highest levels. At day 14 total DNA adductsin lung, spleen and PBMNs were still >50% of the level atday 1. The removal of total DNA adducts was found to be fastestin liver > spleen < PBMNs > lung. A consistent correlationof total adducts between the lung and PBMNs was observed. Amajor adduct, designated adduct 1, was detected in all tissues,while adduct 4 was only found in liver and lung. Adduct 5 wasdetected only in lung, where it constituted     

32P-Post-labeling detection of DNA adducts in mononuclear cells of workers occupationally exposed to styrene

³²-Post-labeling was used to analyze for the presence of DNAadducts in 47 workers exposed to styrene in a boat manufacturingfacility. Individual airborne exposures measured several timesover the course of 1 year ranged from 1to 235 mg/m³ with a meanvalue of 65.6 mg/m³. Two adducts were detected in the DNA ofmononuclear cells of these workers. The following levels ofadducts were detected: adduct 1, range 0.6–102x10^–8(mean 15.8x10^–8); adduct 2, range 0.1–70.9x10^–8(mean 14.2x10^–8). Significant linear relationships werefound between styrene exposure and both DNA adducts (adduct2, r = 0.330, P = 0.012; adduct 1, r = 0.244, P = 0.049). Co-chromatographyexperiments identified DNA adduct 1 in the exposed samplesasN²-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3', 5'-bisphosphate.DNA adduct 2 remains unidentified. No significant linear relationshipswere observed between the level of DNA adducts and sister chromatidexchanges, possibly because of the poor precision of the ³²-post-labelingassay (the estimated coefficients of variation for adducts 1and 2 were 2.54 and 1.96, respectively). These results demonstratethat occupational exposure to styrene results in the formationof DNA adducts in human mononuclear cells.     

32P-Postlabelling analysis of the covalent binding of benzo[ghi]perylene to DNA in vivo and in vitro

Benzo[ghi]perylene (B[ghi]P) is a polycyclic aromatic hydrocarbon(PAH), present in complex combustion products, and evidencefor its carcinogenic activity in experimental animals is equivocal,and yet it has demonstrable mutagenic activity in vitro. Inorder to investigate the possible DNA binding properties ofB[ghi]P, groups of male Parkes mice were treated topically with1.0 µmol of B[ghi]P. Mice were killed up to 3 months aftertreatment, DNA was isolated from the treated areas of skin andanalysed for adducts by ³²P-postlabelling. Maximum levels ofbinding (0.57 fmol/µg DNA) were detected 2 days aftertreatment and adducts were found to persist for at least 12weeks after treatment, a property of many PAHs whh known tumor-initiatingactivity. B[ghi]P also became bound to DNA in vitro in the presenceof 3-methylcholanthrene-induced rat liver microsomal preparations.When chromatographed on PEI–cellulose, the major adductsformed by B[ghi]P in vivo and in vitro appeared to be identical.However, they were found to be different when compared by reversed-phaseHPLC. These differences might explain, in part, the differencesin the biological activity of B[ghi]P in vivo and in vitro,.The behaviour of B[ghi]P when present in a mixture was alsoexamined. B[ghi]P was applied topically to mouse skin with sixother PAHs at a dose level of 0.25 µmol/PAH/mouse. DNAisolated 24 h after treatment was analysed for adducts by ³²P-postlabelllng.Whilst the total level of binding was 30% lower than expectedfrom the sum of the binding levels that resulted when the hydrocarbonswere applied singly, the formation of B[ghi]P–DNA adductsdid not appear to be inhibited. The results have demonstratedthat B[ghi]P has significant DNA binding ability in vivo andin vitro and on the basis of its DNA binding ability in mouseskin it would be predicted to be at least a weak tumour initiator.The formation of DNA adducts by B[ghi]P when present in an artificialmixture of PAHs suggests that B[ghi]P may contribute to theDNA binding activity of more complex carcinogenic mixtures.     

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.