Effects of the co-carcinogen catechol on benzo[a]pyrene metabolism and DNA adduct formation in mouse skin

We have studied the effects of the co-carcinogen catechol (1,2-dihydroxybenzene)on the metabolic activation of [³H] benzo[a]pyrene (BaP) inmouse skin, in vivo and on the binding of BaP metabolites toDNA and protein at intervals from 0.5–24 h. Upon topicalapplication of 0.015 mg [³H]BaP and 0.25 or 0.5 mg catecholper mouse, catechol had little effect on the total amount of[³H]BaP metabolized in mouse skin, but it affected the relativeproportions of [³H]BaP metabolites. Catechol (0.5 mg/mouse)decreased the proportion of watersoluble [³H]BaP metabolites,ethyl acetate-soluble polar metabolites and quinones, but doubledthe levels of unconjugated 3-hydroxy-BaP at all measured intervalsafter treatment. Catechol also caused a small increase in thelevels of trans-7,8-dihydroxy-7,8-dihydroBaP and trans-9,10-dihydroxy-9,10-dihydroBaP0.5 h after treatment. Two hours after treatment, the levelsof these metabolites subsided to those of the controls. Catecholdid not affect the levels of glutathione conjugates of BaP.However, it caused a decrease in glucuronide and sulphate conjugateformation from BaP. Catechol caused an 2-fold increase in theformation of anti-7, 8-dihydroxy-9, 10-epoxy-7, 8, 9, 10-tetrahydroBaP(BPDE) DNA adducts and elevated the ratio of anti-syn-BPDE-DNAadducts 1.6 to 2.9-fold. Catechol treatment increased the radioactivityassociated with epidermal proteins after [³H]BaP application.Because catechol increased levels of 3-hydroxyBaP, we consideredthe possibility that 3-hy-droxyBaP might enhance the tumor initiatingactivities of BaP or BPDE in mouse skin; a bioassay demonstratedthat this was not the case. The results of this study indicatethat one important effect of catechol related to its co-carcinogenicityis its ability to enhance formation of anti-BPDE-DNA adductsin mouse skin.     

Validation of a new fluorometric assay for benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells: comparisons with 32P-postlabeling and ELISA

A new fluorometric assay was validated for quantification ofbenzo[a]pyrene diolepoxide (BPDE)-DNA adducts in white bloodcells (WBC) from humans exposed to polycyclic aromatic hydrocarbons(PAH). This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetra-hydrobenzo[a]pyrenederived from add hydrolysis of BPDE-DNA, and can measure 1 BPDEadduct per 108 unmodified nucleotides. The quantity of WBC DNArequired depends on the modification level and varies between5 and 500 µg. The assay was applied to seven WBC DNA samplesfrom lung cancer patients, six of whom were heavy smokers, andto three WBC DNA samples from healthy subjects employed in analuminum production plant. High levels of BPDE-DNA adducts,ranging from 62 to 533 adducts/10⁸ nucleotides were found insix out of seven DNA samples from the lung cancer patients.In WBC DNA from healthy persons BPDE-DNA adducts were detectedonly in two non-smokers, but at a much lower level than in lungcancer patients (4-10 adducts/10⁸ nucleotides). Using codedWBC DNA samples, BPDE-DNA adduct levels measured by fluorometryof the B[a]P-tetrols, were compared with the results obtainedby 32P-postlabeling (nuclease P1 enrichment) and ELISA measurements.A good correlation and proportionality was found between thelevels of BPDE-DNA adducts measured by fluorometry and ³²P-postlabeling(r = 0.95, P < 0.001, n = 8). The correlation between fluorometryand ELISA was much lower and not significant (r = 0.61, P =0.1, n = 6). Moreover, the ELISA grossly overestimated BPDE-DNAadduct levels measured by the other two methods. The resultsdemonstrate that the highly sensitive and specific fluorometricassay is suitable for measuring BPDE-DNA adducts in WBC fromhumans exposed to benzo[a]pyrene.     

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.     

Myeloperoxidase--463A variant reduces benzo[a]pyrene diol epoxide DNA adducts in skin of coal tar treated patients.

The skin of atopic dermatitis patients provides an excellent model to study the role of inflammation in benzo[a]pyrene (BaP) activation, since these individuals are often topically treated with ointments containing high concentrations of BaP. In this study we have determined, by HPLC with fluorescence detection, the BaP diol epoxide (BPDE)-DNA adduct levels in human skin after topical treatment with coal tar and their modulation by the -463G-->A myeloperoxidase (MPO) polymorphism, which reduces MPO mRNA expression. BPDE-DNA adduct levels were 2.2 and 14.2 adducts/10(8) nt for MPO-463AA/AG and -463GG, respectively. The predominant BaP tetrol observed was tetrol I-1, which is derived after hydrolysis of the anti-BPDE-DNA adduct. The tetrol I-1/II-2 ratio, corresponding to the anti/syn ratio, was 6.7. The (32)P-post-labeling assay was also performed and thin layer chromatograms showed a major spot with a chromatographic location corresponding to BPDE-DNA. The mean values of the BPDE-DNA adduct spots were 3.8 +/- 2.4 per 10(8) nt for MPO-463AA/AG (n = 3) and 18.4 +/- 11.0 per 10(8) nt for MPO-463GG (n = 7), respectively (P = 0.03). One individual with the homozygous mutant genotype (-463AA) even had a 13-fold lower adduct level (1.4 per 10(8) nt) as compared to MPO-463GG subjects. In conclusion, these data show for the first time: (i) the in vivo formation of BPDE-DNA adducts in human skin treated with coal tar; (ii) that the MPO-463AA/AG genotype reduced BPDE-DNA adduct levels in human skin.     

An applied synchronous fluorescence spectrophotometric assay to study benzo[a]pyrene-diolepoxide-DNA adducts

Synchronous scanning fluorescence with a fixed wavelength difference() of 34 nm between excitation and emission was used to quantitatebenzo[a]pyrene-diol epoxide (BPDE)-DNA adducts. Fluorescenceemission maxima occurred at 382 nm for BPDE-DNA and at 379 nmfor benzo[a]pyrene-tetrols and -triol, which are hydrolysisproducts of BPDE. Similarly, the peak for pyrene was at 372nm and for 1-nitropyrene at 386 nm. The minimum detectable amountof BPDE-moieties in in vitro modified BPDE-DNA, after hydrolysisin HCl, was 20 fmol in 100 µg of DNA, which is equivalentto 1 adduct per 1.4 x 10⁷ nucleotides. The correlation of fluorescenceintensity and the amount of BPDE-moieties was linear between20 fmol and 1 pmol. DNA isolated from human lymphoblasts incubatedwith BDPE had the same fluorescence peak and the correlationbetween the fluorescence intensity and the amount of BPDE inthe incubation mixture was linear. Among the DNA-samples fromperipheral blood lymphocytes of 30 aluminum plant workers, onlyone sample was found to contain a peak similar to BPDE-DNA.None of the DNA-samples from 10 persons not occupationally exposedwere positive. Measurement of BPDE-DNA adducts by synchronousfluorescence spectrophotometry should be useful in monitoringhuman exposure to benzo[a]pyrene.     

The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo[a]pyrene diol epoxide is dependent on the level of modification. Implications for quantitation of benzo[a]pyrene-DNA adducts in vivo

A number of polyclonal antibodies specific for DNA modifiedwith (±)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) were obtained from the sera of New Zealand white rabbitsimmunized with BPDE-DNA, complexed with methylated bovine serumalbumin (mBSA). Monoclonal antibodies were developed by fusionof mouse myeloma cells with spleen cells isolated from BALB/cmice immunized with the same complex of BPDE-DNA and mBSA. Theseantibodies have been characterized for specificity in a highlysensitive, enzyme-linked immunosorbent assay (ELISA). All antibodiesshowed a very high affinity for single-stranded BPDE-DNA, buthad lower affinity towards native BPDE-DNA. The affinity forthe free mononucleoside BPDE-dG was at least 100-fold lowerthan that for BPDE-DNA, and no affinity was detected for BPtetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene.A high cross reactivity was observed with DNA modified with(±)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene.Using five different antibodies, monoclonal or polyclonal, weobserved that the antibody affinity for BPDE-DNA was dependenton the level of modification; in the competitive ELISA as littleas 4 fmol BPDE-DNA (50 pmol/µg) was sufficient for 50%inhibition with our best antisera, but 17 fmol of the adductwas required when [³H]BPDE-DNA of low modification (1–10fmol/µg) was used as inhibitor. When samples of [³H]BP-DNAisolated from the livers of mice, treated i.p. with differentdoses of [³H]BP were examined by competitive ELISA and calibratedwith [³H]BPDE-DNA of low modification (1–10 fmol/µg),binding values calculated from the immunoassay were in goodagreement with those obtained from radioactivity measurements.In contrast, when this DNA was quantitated in competitive ELISAusing highly modified BPDE-DNA as standards, values by ELISAwere 20–40% of those obtained by radioactivity. Theseresults indicate that the use of serially diluted BPDE-DNA ofhigh modification as standard competitor in the ELISA will leadto erroneous results in the measurement of adducts in DNAs modifiedto a low extent (biological samples). The property of antiseraspecific for BP-DNA, recognizing highly modified DNA more efficientlythan DNA modified to a low extent, may be common to all antiseraelicited against highly modified DNA immunogens. Therefore weconclude that antibody affinity must be tested also with DNAsamples of low modification, obtained either in vitro or invivo.     

32P-Post-labelling analysis of DNA adducts in mouse embryo fibroblasts treated with dibenzo[a,e]fluoranthene and its major metabolites

The formation of DNA adducts was investigated in mouse fibroblaststreated with dibenzo [a, e] fluoranthene (DBF), using the nucleaseP₁ modification of the ³²P-post-labeling method. In order toseparate the poorly soluble, bulky DNA adducts of this potentsarcomogenic, six-ring poly cyclic aromatic hydrocarbon, severalmodifications of the method were introduced. Chromatographicspots were identified by incubating fibroblasts with the fourmajor proximate metabolites of DBF and observing the co-migrationof adducts with those of DBF. DNA-DBF adducts chromatographedvery reproducibly in three major spots and in > 10 spotsof medium or low importance. The most prominent spots, 2 and3, were present characteristically after incubation of cellswith the DBF-bay region dihydrodial (±-trans-3, 4-dihydro-3,4-dihydroxy DBF; DBF-3, 4-DHD). Incubation with the DBF pseudo-bayregion dihydrodiol (±-trans-12, 13-dihydro 12, 13-dihydroxyDBF; DBF-12, 13-DHD) gave rise to a more complex pattern ofnine spots, two of which, spots 4 and 5, were prominent. Directin vitro reaction between DNA and the synthetic anti-isomerof the DBF-bay region DHD epoxide yielded adducts in spots 2and 3, while the DBF-anti-pseudobay region DHD epoxide yieldedadducts in spots 4 and 5. Peripheral, fast-migrating spots presentin the DBF chromatogram were identified as adducts of DBF-7OH-3,4-DHD and DBF-3OH-12, 13-DHD. Major spot 1 was present in allDBF chromatograms but not after incubation with the DBF bayand pseudo-bay region proximate metabolites. Its probable originas a non-bay region epoxide reaction is discussed. In previousexperiments, the physicochemically very similar DBF-bay regionand pseudo-bay region tritium-labeled adducts co-eluted in HPLCas a single peak. ³²P-Post-labeling analysis allowed reproducibleseparation of DBF-DNA adducts and showed in addition the existenceof several new adducts models of DBF. Quantification of DBFadducts made it possible to identify the DBF-bay region DHDepoxide and the metabolites responsible for spot 1 adducts asthe major ultimate DBF metabolites in fibroblasts.     

SHORT COMMUNICATION: TPA decreases the p53 response to benzo[a]pyrene-DNA adducts in vivo in mouse skin

TPA, a well-known tumor promoter, decreased the response ofnuclear P⁵³ immunoreactivity to benzo[a]pyrene-7, 8-diol-9,10-epoxide (BPDE)-DNA adducts in C57BL/6 mouse skin in vivo.A dose-dependent increase in both the level of BPDE-DNA adductsand nuclear p⁵³ immunoreactivity was found in mice treated topicallywith 50–750 µg benzo[a]pyrene. Such a positive correlationbetween the adducts and p53 positivity was suggested by an earlierstudy. Since p⁵³ probably functions in DNA damage control, interferenceby TPA with the p53 response could be a mechanism in TPA- inducedtumor promotion. Whether such a mechanism is more general intumor promotion deserves further study.     

32P-Post-labelling analysis of DNA adducts formed by food-derived heterocyclic amines: evidence for incomplete hydrolysis and a procedure for adduct pattern simplification

Food-derived aminoimidazoazarenes have been shown to be mutagenicand carcinogenic and to form covalent DNA adducts. ³²P-Post-labellinganalysis of DNA modified with these heterocyclic amines (HA),including 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimid-azo[4,5-b]pyridine(PhIP), 2-amino-3,4-dimethylimidazo [4,5-fquinoline (MeIQ),2-amino-3,4,8-trimethylimidazo [4,5-f1 quinoxaline (4,8-DiMeIQx),2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx)and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) hasresulted in considerable interlaboratory variation in the characteristicpatterns of DNA adduct spots, with up to six being detectedfor each compound. Similar complex patterns were observed whenazido-derivatives of HA were photoreacted with calf thymus DNA.When deoxyguanosine 3'-monophosphate was modified with the azidoderivatives and analysed using the ³²P-post-labelling procedure,one major spot was observed for IQ, 4,8-DilMeIQx, 7,8-DiMeIQxor PhIP and two major spots for MeIQ or MeIQx. In each case,these adducts were chromatographically indistinguishable fromthe major adducts formed with DNA. No major adduct spots wereobserved when 3'-phosphate derivatives of deoxyadenosine, deoxycytidineor thymidine were reacted with the azido-derivatives of HA.In an attempt to identify the additional spots, azido derivativesof PhIP or IQ were reacted with the synthetic homopolymer poly(dG)·poly(dC),the alternating copolymer poly(dC-dG) or a synthetic oligonucleotide(TTT-GTTTTTTCTTTCCCT): in each case a reduced number of adductspots were detected. The introduction of an additional nucleaseP1 hydrolysis step following the labelling reaction furtherreduced the number of adduct spots to only one or two majorspots. Reversed-phase HPLC analysis showed that the number ofpeaks of radioactivity was also reduced to one or two, presumablycorresponding to the [³²P]-5'-monophosphate deoxyguanosine adducts.We suggest that many of the additional spots commonly observedin conventional ³²P-post-labelling analysis of HA-modified DNAare adducted oligonucleotides that are partly resistant to hydrolysisby micrococcal nuclease and spleen phosphodiesterase but aresusceptible to hydrolysis by nuclease P1.     

Effect of arsenic on benzo[a]pyrene DNA adduct levels in mouse skin and lung

Concomitant exposures to arsenic and polycyclic aromatic hydrocarbons(PAHs) such as benzo[a]pyrene (BaP) are widespread. While BaPacts by binding to and inducing mutations in critical siteson DNA, the mechanism(s) of arsenic carcinogenesis remains unknown.Data from epidemiological studies of arsenic copper smelterworkers and arsenic ingestion in drinking water suggest a positiveinteraction for arsenic exposure and smoking and lung cancer.A previous in vitro study showed that arsenic potentiated theformation of DNA adducts at low doses of BaP and arsenic. Thepresent study was conducted to test the effect of arsenic onBaP–DNA adduct formation in vivo. We hypothesized thatarsenic co-treatment would significantly increase BaP adductlevels in C57BL/6 mouse target organs: skin and lung. Treatmentgroups were: five mice, -BaP/-arsenic; five mice, -BaP/+arsenic;15 mice, +BaP/-arsenic; 15 mice, +BaP/+arsenic. Mice in theappropriate groups were provided sodium arsenite in drinkingwater (2.1 mg/l), ad libitum, for 13 days (starting 9 days beforeBaP treatment), and 200 nmol BaP/25 ml acetone (or acetone alone)was applied topically, once per day for 4 days. DNA was extractedfrom skin and lung and assayed by ³²P-postlabeling. Statisticalcomparisons were made using independent t-tests (unequal variancesassumed). BaP–DNA adduct levels in the +BaP groups weresignificantly higher than -BaP controls. Arsenic co-treatmentincreased average BaP adduct levels in both lung and skin; theincrease was statistically significant in the lung (P = 0.038).BaP adduct levels in the skin of individual animals were positivelyrelated to skin arsenic concentrations. These results corroborateour in vitro findings and provide a tentative explanation forarsenic and PAH interactions in lung carcinogenesis.     

Effect of sulfite on the covalent reaction of benzo(a)pyrene metabolites with DNA

Sulfur dioxide (SO₂) potentiates the carcinogenicity of polycyclicaromatic hydrocarbons. To investigate the mechanism of SO₂ cocarcinogenesis,the effect of sulfite, the hydrated form of SO₂, on the covalentreaction of benzo(a)pyrene (BaP) metabolites with DNA in vitrowas measured. (¹⁴C)BaP was incubated with rat lung or liverpost-mitochondrial supernatant (S9), an NADPH generating system,calf thymus DNA and sodium sulfite (0–20 mM). In the presenceof lung S9, covalent reaction increased linearly from 0.66 to1.20 pmol BaP metabolites per mg DNA with increasing sulfiteconcentrations. Addition of sulfite to rat liver S9 also increasedBaP-DNA adduct formation with BaP-DNA adducts increasing from80 to 120 pmol per mg DNA. Sulfite altered the amount and patternof BaP metabolites formed by either lung or liver enzyme preparations.BaP was metabolized more extensively and the amount of watersoluble BaP metabolites formed increased significantly withsulfite present. With lung S9, the amount of BaP-tetrols, diols,and phenols increased slightly. With liver S9, diol and phenolformation was significantly lower while tetrol formation wasunchanged. Incubation of rat lung S9 with sulfite resulted information of glutathione S-sulfonate (GSSO₃H), a known inhibitorof glutathione S-transferases mediating the conjugation of glutathione(GSH) and BaP epoxides. Our results suggest that sulfite may,by altering the overall metabolic activation and detoxicationof BaP, or by reacting directly with DNA, subsequently affectthe covalent reaction of BaP metabolites with DNA. These areoffered as possible mechanisms to explain the cocarcinogeniceffect of SO₂.     

Use of a microsome-mediated test system to assess efficacy and mechanisms of cancer chemopreventive agents

There is a growing need for short-term assays which can assessthe mechanisms and efficacy of cancer chemopreven-tive agents.In the present study we have employed a microsome-mediated testsystem concomitantly with DNA adduct detection to assess theefficacy of five chemopreven-tive agents, N-acetylcysteine,butylated hydroxytoluene (BHT), curcumin, oltipraz, and ellagicacid. ³²P-Postla-beling analysis of DNA incubated with benzo[a]pyrene(BP) in the presence of Aroclor 1254-induced microsomes producedtwo major adducts: one derived from the interaction of benzo[a]pyrene–7,8-diol–9, 10-epoxide (BPDE) with deoxyguanosine (dG) andthe other from further activation of 9-OH-BP (309 and 34 adducts/10⁷nucleotides, respectively). With the exception of N-acetylcysteine,all test agents significantly altered BP-DNA adduct levels:Intervention with ellagic acid and oltipraz substantially (64–94%)inhibited both BPDE-dG and 9-OH-BP adducts, while interventionwith curcumin and BHT inhibited the BPDE-dG adduct (57% and38%, respectively) and enhanced the 9-OH-BP adduct (230% and650%, respectively). Furthermore, ellagic acid was the onlytest agent observed to inhibit the anti BPDE-dG adduct in theabsence of Microsomal enzymes, which is consistent with theknown conjugation of ellagic acid with BPDE. These results suggestthat oltipraz may be acting as an inhibitor of P4501A1, theisozyme involved in activation of BP to BPDE, or by conjugationof the electrophilic species by a metabolite of oltipraz. Aplausible mechanism for inhibition of the BPDE-dG adduct andenhancement of the 9-OH-BP adduct by curcumin and BHT includesinhibition of epoxide hydrolase. Our results also indicate thatN-acetylcysteinedoes not act as an electrophilic trapping agent of BP metabolitesbut may exert its protective effect in vivo by various othermeans, including modulation of detoxification enzymes and alteringDNA repair processes. These data suggest that this cell-freesystem in conjunction with the sensitive ³²P-Postlabeling DNAadduct analysis may prove a viable test system for assessingthe mechanisms and efficacy of chemopreventive agents.     

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.     

Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32P-postlabelling analysis using different chromatographic procedures

We report the analysis of DNA adducts in the target organ (forestomach)of male Sprague–Dawley rats treated orally with two doses(10 mg/kg body wt) per week for 2 weeks of either aristolochicacid I (AAI), aristolochic acid II (AAII) or the plant extractaristolochic acid (AA). DNA adducts were detected and quantitatedusing the nuclease P1-enhanced version of the ³²P-postlabellingassay. For identification of adducts, reference compounds wereprepared by reaction of enzymatically activated AAI and AAIIwith 3'-purine phosphonucleosides and analysed by the ⁿ-butanolenrichment procedure. These reference compounds were assignedto the previously characterized DNA adducts of AAI [7-(deoxy-guanosin-N²-yl)-aristolactamI = dG-AAI, 7-(deoxyadenosin-N⁶-yl) I = dA-AAI] and AAII [7-(deoxyadenosin-N⁶-yl)-aristolactamII = dA-AAII]. Cross referencing of the carcinogen-modifiednucleoside bisphosphates obtained from forestomach DNA withthe synthetic standard compounds by ion-exchange chromatographyand reversed-phase HPLC demonstrated that the major DNA adductsformed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise,forestomach DNA isolated from AAII-treated rats showed two purinederived adduct spots, the major one being dA-AAII, the minorone being tentatively identified as 7-(deoxyguanosin-N²-yl)-aristolactamII. A minor adduct detected in forestomach DNA of rats treatedwith AAI was found to be chromatographically indistinguishablefrom the adduct identified as dA-AAII, indicating a possibledemethoxylation reaction of AAI. Quantitation of DNA adductsrevealed that in in vitro reactions with 3'-phosphonucleosidesthe adduct levels were approximately one order higher for bothAAI and AAII-derived adducts than in forestomach DNA modifiedwith AAI or AAII in vivo. In vitro as well as in vivo adductionby AAI was more efficient than adduction by AAII. The patternof adduct spots obtained from forestomach DNA of rats treatedwith the plant extract AA reflected the composition of the extractdetermined by HPLC analysis. Irrespective of the aristolochicacid used to induce DNA adducts, deoxyadenosine is the majortarget of modification, pointing to the general importance ofdeoxyadenosine adducts for chemical carcinogenesis of thesenaturally occurring products. This study shows that the combinationof two independent chromatographic systems considerably enhancesthe fidelity of identification of DNA adducts with the ³²P-posthbellingassay.     

SHORT COMMUNICATION: Cyproterone acetate is an integral part of hepatic DNA adducts induced by this steroidal drug

We have recently shown that cyproterone acetate (CPA), an activecomponent of some antiandrogenic drugs, induces the formationof DNA adducts detectable by the ³²P-DNA postlabelling techniquein rat liver. The postlabelling technique, however, does notprovide evidence for the chemical nature of the adducts observed.To ascertain whether the CPA-induced adducts do contain CPA,we have incubated tritiated CPA with cultured hepatocytes fromfemale rats, digested the DNA to 3'-monophospho-nucleosides,extracted the DNA adducts formed into but-anol and phosphorylatedthe adducts in the extract with unlabelled ATP. One major andone minor ³H-labelled adduct spot were detectable on the TLCchromatograms. The spots appeared at positions identical tothose observed in the ³²P-postlabelling experiments with unlabelledCPA. Furthermore, the ratio of ³H activity for the major versusthe minor adduct spot was 11.9±1.8, which agreed withthe corresponding ratio for the ³²P activities, which was 13.2±3.5.These findings indicate that the CPA-induced DNA adducts, whichwe have previously detected by ³²P-postlabelling do containCPA or CPA metabolites.     

Polycyclic aromatic hydrocarbon--DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung.

Smokers of cigarettes are exposed to a number of carcinogens, including polycyclic aromatic hydrocarbons (PAHs), and are at a high risk for lung cancer. PAHs exert their carcinogenic activity after metabolic activation to reactive intermediates that can damage DNA through adduct formation. Measuring DNA adducts in peripheral white blood cells (WBC) could serve as a means of monitoring human exposure to genotoxic agents and subsequently risk assessment. In this study, DNA from WBC obtained from 39 lung cancer patients was examined for PAH-DNA adducts both in an ELISA using a polyclonal antibody against benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE)-DNA and the 32P-post-labeling technique. The ELISA results showed BPDE-DNA antigenicity in WBC DNA from 12/38 (32%) patients and adduct levels ranged from 1.5 to greater than 150 adducts in 10(8) nucleotides. The autoradiographs of chromatograms of 32P-post-labeled digests of WBC DNA from the 38 patients showed a variety of adduct spots; relative adduct labeling (RAL) values ranged from 0.3 to 407 adducts in 10(8) nucleotides. In 18 of the 38 (47%) persons an adduct spot was detected that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG); RAL values ranged from 0.03 to 382 adducts in 10(8) nucleotides. Correlations were not significant between the adduct levels in WBC and smoking habits, age or sex. From 20 patients of the same group lung tissue was collected at surgery and examined for PAH-DNA adducts by ELISA and 32P-post-labeling assay. No significant correlation was found between DNA adduct levels in blood and lung. This finding stresses the limitations of the use of WBC as a surrogate for adduct levels in the target organ.     

Polycyclic aromatic hydrocarbon-DNA adducts in lung tissue from lung cancer patients

In an attempt to probe for polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human subjects resulting from smoking (or other chronic environmental exposure), lung tissue and lung tumours were obtained from patients hospitalized for lung cancer. DNA was isolated from the tissue samples and examined both in an ELISA using a polyclonal antibody against (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE)-DNA as well as by the nuclease P1-mediated modification of the 32P-post-labelling technique. The ELISA results showed BPDE-DNA antigenicity in lung DNA from 6 out of 21 patients, and adduct levels ranged from 2 to 134 adducts per 10(8) nucleotides. For all 21 patients, the autoradiographs of chromatograms of 32P-postlabelled digests of DNA from non-tumorous lung tissue showed a strong diagonal radioactive zone (DRZ). This DRZ was generally absent in tumorous tissue. DNA samples that were positive in the ELISA contained a dominant spot within the DRZ that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG). The quantities of the BPDE-dG spots ranged from 2.1 to 42 adducts in 10(9) nucleotides. These values were lower than the levels found in the ELISA but correlated well with the ELISA results (Kendall W = 0.97; P = 0.00). The levels of the DRZ adducts ranged from 1.9 to 34 adducts in 10(8) nucleotides. Correlations between smoking and DNA adduct levels were poor because of the small number of current smokers (n = 13). However, smokers of filter cigarettes had significantly lower DNA adduct levels compared with smokers of cigarettes without a filter (P = 0.02 by Fischer's exact test).     

2-Aminofluorene metabolism and DNA adduct formation by mononuclear leukocytes from rapid and slow acetylator mouse strains

Following exposure of mice to the arylamine carcinogen 2-aminofluorene,DNA-carcinogen adducts can be found in the target tissues liverand bladder, and also in circulating leukocytes. Evidence ispresented here that mouse mononuclear leukocytes (MNL) are capableof metabolizing 2-aminofluorene to DNA-binding metabolites whichgive rise to the adducts found in the MNL. Both lymphocytesand monocytes were able to acetylate arylamines during 18 hof culture. The degree of acetytation was determined by theN-acetyltransferase genotype of the mice as shown through useof acetylator congenic strains which differ only in the Nat-2gene. Cultured MNL from rapid acetylator mice (C57BL/6J andA.B6-Nat¹) produced about twice as much N-acetylaminofluorenefrom 2-aminofluorene and 6- to 8-fold as much N-acetyl-p-amino-benzoicacid from p-aminobenzoic acid as cells from slow acetylatormice (B6.A-Nat⁵ and A/J). Other differences in arylamine metabolismby MNL in culture were observed and shown to be due to geneticfactors, currently unidentified, other than N-acetyltransferase.DNA adduct formation following incubation of MNL with the arylaminecarcinogen 2-aminofluorene was related to both acetylation capacityand to other genetic metabolic factors in the mouse genome.MNL from rapid acetylator mice with the C57BL/6J background(B6) had 3-fold the DNA adduct levels of cells from the correspondingslow acetylator congenic (B6.A-Nat^$). Similarly, MNL from rapidacetylator mice with the A/J background (A.B6-Nat^r) had twicethe DNA adduct levels of those from their corresponding slowcongenic (A). Adduct levels in MNL from C57BL/6J were nearlythe same as those of MNL from A/J, again indicating the involvementof loci other than acetylation in DNA adduct formation. Thefinding of genetically dependent arylamine carcinogen metabolismand DNA adduct formation in cultured MNL suggests the possibilityof using cultured MNL for assessing individual susceptibilityto arylamine-induced DNA damage.     

DNA adducts of the carcinogen, 15, 16-dihydro-11-methylcyclo-penta(a)phenanthren-17-one, in vivo and in vitro: high pressure liquid chromatographic separation and partial characterization

The 11-methyl derivative (11-methyl ketone) is the most carcinogenicof the series of methylated derivatives based on 15, 16-dihydro-cyclopenta[a]phenanthren-17-one.The nudeoside adducts derived from [³H]-11-methyl ketone-modifiedmouse skin DNA have been separated by both Sephadex LH-20 chromatographyand reverse-phase h.p.l.c and compared to those derived fromDNA modified in vitro with the [¹⁴C]-11-methyl ketone usingrat liver microsomes. The in vivo modified DNA separated togive 6 adducts (designated I- VI) on h.p.l.c. The major in vivoadduct (80% total adducts) co-chromatographed with the majorin vitro adduct. The metabolites of the 11-methyl ketone (designateda-g) have been separated by h.p.l.c, and the adducts derivedfrom each of these individual metabolites determined by furthermetabolism in the presence of DNA. H.p.l.c. separation of theseadducts has allowed characterization of the in vivo adducts.The major adduct (V) and possibly one of the minor adducts (IV)were derived from the 3, 4-dihydro-3, 4-diol of the 11-methylketone (metabolite e). Adducts II and III were derived fromthe 16- and 15-monohydroxylated derivatives of the 11-methylketone and also from their corresponding 3, 4-diols and thereforeare likely to be the 16- and 15-hydroxy derivatives, respectively,of adduct V. Adduct VI, however, although derived from the 15-hydroxy-3,4-diol had a late retention time on h.p.l.c, suggesting eithera non-diol-epoxide adduct or a deoxyadenosine adduct. The useof [³H-G]DNA has established that the major adduct (V) and the16-hydroxy-derived adduct (II) contain deox-yguanosine. Reactionof the carcinogen with [³H-A]poly(dA-dT) gave adduct VI whichwas the only adduct peak shown to contain [³H]deoxyadenosine.     

Immunoaffinity chromatography of carcinogen DNA adducts with polyclonal antibodies directed against benzo[a]pyrene diol-epoxide-DNA

Polyclonal antibodies specific for (+/-)-trans-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(BPDE) CAS: 58917-67-2]-DNA adducts were obtained from the sera of New Zealand White rabbits immunized with BPDE-DNA. These antibodies did not recognize benzo[a]pyrene [(BP) CAS: 50-32-8] or DNA alone or other carcinogen adducts, such as aflatoxin (CAS: 1402-68-2)-DNA or aminopyrene (CAS: 58-15-1)-DNA, up to the concentrations used. In a competitive enzyme-linked immunosorbent assay, 0.18 microgram BPDE-DNA/ml (single stranded), equivalent to 7 pmol BPDE adduct, caused 50% inhibition with this antibody. (When referring to the DNA content of BPDE-DNA, the authors gave the concentration in microgram/ml; when referring to the BPDE content of BPDE-DNA, the authors gave the concentration as pmol/ml.) Chemical and enzymic modifications of the BPDE-DNA substrate suggested that the epitope for the antibody is greater than that represented by a BPDE-nucleoside adduct. The specific BPDE-DNA antibodies were covalently bound to cyanogen bromide-activated Sepharose 4B, and the extent of ligand binding to the immunoaffinity column was measured with the use of [3H]BPDE-DNA as substrate. Maximum binding to the immunoaffinity column was obtained after DNase 1 digestion of [3H]BPDE-DNA: The bound adducts could be readily eluted from the column with 50 mM NaOH. The binding of DNase 1-digested [3H]BPDE-DNA to the immunoaffinity column was dose related and not affected by the addition of unmodified DNA. The columns have proven to be reusable. Samples of [3H]BP-DNA isolated from the skin of mice treated topically with either 0.75 mumol [3H]BP/mouse or 1.5 mumol [3H]BP/mouse were examined by immunoaffinity chromatography. Binding values of 6.0 and 12.2 pmol BP/mg DNA were obtained; these values from immunoaffinity chromatography were slightly lower than those determined by high-pressure liquid chromatography analysis (9 and 17 pmol BP/mg DNA). With chemically reacted BPDE-DNA, around 70% of that applied was retained by immunoaffinity chromatography, whereas with [3H]BP-DNA isolated from the in vivo treatment of mouse skin, only 40% was retained--a possible reflection of the greater heterogeneity of the in vivo BP-DNA adducts. This immunoaffinity chromatography technique should prove useful in the selective examination of levels of BPDE-DNA adducts present in biological samples.