HPLC separation of 32P-postlabelled benzo[b]fluoranthene-DNA adducts.

Analysis using 32P-postlabelling and a recently developed HPLC method resolved the adduct formed by reaction of the benzo[b]fluoranthene (BbF) anti-bay-region diol-epoxide with DNA from the more polar major adduct produced by the hydrocarbon in three different biological systems. In each case, the adduct formed from the anti-bay-region diol-epoxide constituted only a minor proportion of the total DNA modification. Comparisons of the DNA adducts formed from the hydrocarbon with those formed in microsomal incubations from the putative metabolites BbF-9,10-diol, anti-BbF-9,10-diol-11,12-oxide and the 5,9,10- and 6,9,10-BbF-triols indicate that the predominant pathway for BbF activation in skin probably involves a bay-region triol-epoxide possessing a phenolic OH-group on the peninsula ring.     

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.     

DNA adduct formation in mice following treatment with used engine oil and identification of some of the major adducts by 32P-postlabelling.

Used engine oil from a petrol-powered vehicle was fractionated by column chromatography into seven parts for which the major polycyclic aromatic hydrocarbon (PAH) components were determined by GC. Topical treatment of mice with the fractions and 32P-postlabelling of the skin DNA resulted in the detection of multiple adduct spots on TLC for some, but not all, of the fractions. The majority of the DNA binding capacity of the used engine oil was possessed by the first three fractions, (equivalent to 25, 15 and 14.5%, respectively) of the adduct forming ability of the unfractionated oil. The chromatographic mobilities of the adduct spots induced by these fractions were compared to those produced by unfractionated used engine oil. In addition, mice were also treated topically with reference PAHs, either singly or as mixtures, dissolved in unused oil at the concentrations at which they were present in the used oil. Comparisons were made between the chromatographic mobilities of the adducts formed in mouse skin DNA by synthetic mixtures with those formed by the used oil. From these data, some of the major adducts produced by treatment with used engine oil are suggested to be formed by reactive metabolites of benzo[b]naphtho[1,2-d]thiophene, benzo[c]phenanthrene, benzo[g,h,i]fluoranthene, chrysene, benzo[a]pyrene and benzo[g,h,i]perylene.     

Identification of tumorigenic metabolites of benzo[j]fluoranthene formed in vivo in mouse skin

The metabolism of benzo[j]fluoranthene (BjF) in vivo in mouse skin was investigated. trans-4,5-Dihydro-4,5-dihydroxybenzo[j]fluoranthene (BjF-4,5-diol) and trans-9,10-dihydro-9,10-dihydroxybenzo[j]fluoranthene (BjF-9,10-diol) have been identified as major metabolites. In addition, 4- and 10-hydroxybenzo[j]fluoranthene and benzo[j]fluoranthen-4,5-dione have been tentatively identified among the metabolites formed in vivo in mouse skin. The enantiomeric purity of the metabolic dihydrodiols of BjF as formed in vivo in mouse skin was determined. The major enantiomer of BjF-4,5-diol was present in 57-62% enantiomeric excess while that of BjF-9,10-diol was present in 66-71% enantiomeric excess. In each case the later-eluting enantiomer on chiral stationary-phase high performance liquid chromatography predominated. The tumor-initiating activity of trans-2,3-dihydro-2,3-dihydroxybenzo[j]fluoranthene (BjF-2,3-diol), BjF-4,5-diol, BjF-9,10-diol, and BjF was evaluated on the skin of female CD-1 mice. As a total initiation dose of 3 mumol/mouse BjF-4,5-diol resulted in a 100% incidence of tumor-bearing mice with 5.0 tumors/mouse. In comparison, BjF-9,10-diol elicited a 60% incidence of tumor-bearing mice with 1.7 tumors/mouse, while BjF-2,3-diol was inactive. At the same dose, BjF gave rise to a 90% incidence of tumor-bearing mice with 7.8 tumors/mouse. At a 1-mumol dose, BjF-4,5-diol induced a 78% incidence of tumor-bearing mice with 4.3 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse. These studies indicate that while BjF-9,10-diol could contribute to the overall tumorigenic activity of BjF in mouse skin, BjF-4,5-diol is a more potent tumor initiator in the target tissue.     

Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis

Identification of DNA adducts in peripheral lymphocytes could serve as a means of monitoring human exposure to potential genotoxic agents. In this study, DNA from peripheral lymphocytes of smokers and nonsmokers was examined for adducts by the P1 nuclease 32P-postlabeling technique. Thin layer chromatography (TLC) maps from both groups revealed multiple DNA adducts which ranged from no adducts for one individual to six adducts for a different individual. The total DNA adduct concentrations were approximately one adduct in 10(8)-10(10) normal nucleotides. Comparison of the adduct TLC profiles revealed individual variation in both pattern and level of DNA adducts. The type and amount of adduct was not influenced by smoking history and remained unchanged in four out of six subjects who were resampled after a 1 month interval. The capacity of lymphocytes to form BaP-derived DNA adducts after a 72 h incubation with 10(-6) M [3H]BaP was measured by both high-performance liquid chromatography (HPLC) and 32P-postlabeling analysis. The in vitro adduct values detected by [3H]nucleoside concentrations on HPLC ranged from 1 to 7 fmol adduct per micrograms DNA (3.3-23.3 adducts per 10(7) nucleotides). The [3H]nucleoside values were consistent with values obtained by 32P-postlabeling of the same sample (correlation coefficient of 0.88). No relationship was apparent between the capacity of lymphocytes to form a [3H]BaP-derived adduct in vitro and the concentration of any adduct, or total adducts present in untreated lymphocytes. These results suggest that multiple DNA adducts are present in lymphocytes from nonsmokers as well as smokers, although the profile and extent of these adducts can vary among individuals. The relationship of the lymphocyte DNA adducts detected in this study to human cancer susceptibility remains to be determined.     

Analysis of the polycyclic aromatic hydrocarbon content of petrol and diesel engine lubricating oils and determination of DNA adducts in topically treated mice by 32P-postlabelling

Engine lubricating oils are known to accumulate carcinogenic polycyclic aromatic hydrocarbons (PAHs) during engine running. Oils from nine petrol-powered and 11 diesel-powered vehicles, in addition to samples of unused oil, were analysed for PAH content and ability to form DNA adducts when applied topically to mouse skin. The levels of 19 PAHs, determined by GC, were in total, approximately 22 times higher in used oils from petrol engines than in oils from diesel engines. Male Parkes mice were treated with 50 microliters of oil daily for 4 days before they were killed and DNA isolated from skin and lung tissue. DNA samples were analysed by nuclease P1-enhanced 32P-postlabelling. Used oils from both diesel and petrol engines showed several adduct spots on PEI-cellulose plates at total adduct levels of up to 0.57 fmol/microgram DNA [approximately 60 times greater than in experiments with samples of unused oil in which adduct levels (0.01-0.02 fmol adducts/microgram DNA) were close to the limit of detection]. Higher adduct levels were generally formed by petrol engine oils than by diesel engine oils. Lung DNA contained similar total adduct levels to those in skin although the adduct maps were less complex. Total adduct levels correlated with extent of oil use in the engine, the total PAH concentration in oils and with the concentrations of certain individual PAHs present in the oils. An adduct spot that co-eluted with that of the major benzo[a]pyrene-DNA adduct accounted for 9-26% of the total adducts in skin DNA, and approximately 8% of the adducts in lung DNA, of mice treated with petrol engine oils. A major, and as yet unidentified, adduct spot comprised up to 30% of the total adducts in skin DNA, and up to 89% of the total adducts in lung DNA, of these animals.     

Mutagenicity and tumor initiating activity of methylated benzo[b]fluoranthenes

The mutagenicity toward S. typhimurium TA 100 and tumor initiatingactivity on mouse skin of benzo[b]fluoranthene (BbF), 1-methylbenzo[b]fluoranthene(1-MeBbF), 3-MeBbF, 7-MeBbF, 8-MeBbF, 9-MeBbF, 12-MeBbF, 5,6-dimethylbenzo[b]fluoranthene(5,6-diMeBbF) and 1,3-diMeBbF were assayed. Dose-dependent mutagenicactivity was observed for BbF, 3-MeBbF, and 1,3-diMeBbF; theother compounds were inactive at the doses tested. 3-MeBbF and1,3-diMeBbF were strong tumor initiators, with activity greaterthan that of BbF. All the other compounds were less tumorigenicthan BbF. The results suggest that the structural features favoringtumorigenicity of methylated non-alternant polynuclear aromatichydrocarbons such as BbF are different from those favoring tumorigenicityof methylated alternant polynuclear aromatic hydrocarbons suchas benzo[a]pyrene, chrysene and benz[a]anthracene.     

A pilot study of detection of DNA adducts in white blood cells of roofers by 32P-postlabelling

To assess the utility of DNA adducts as biomarkers of exposure to carcinogens in an industrial population, a pilot study of roofers occupationally exposed to a mixture of polycyclic aromatic hydrocarbons was conducted. DNA was isolated from peripheral white blood cells of roofers and non-occupationally exposed subjects matched for age, sex and smoking status. Occupational exposures to anthracene, fluoranthene, pyrene, benzanthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene and benzo[k]fluoranthene were assessed by personal breathing zone air sampling and skin wipes. Exposures to benzo[a]pyrene in air of exposed subjects ranged from 0.60 microgram/m3 to 1.39 micrograms/m3, and exposures to total polycyclic aromatic hydrocarbon (the sum of eight hydrocarbons) ranged from 6.0 micrograms/m3 to 13.8 micrograms/m3 on the day before blood collection. In the biomarker studies 10 of 12 roofers, but only 2 of 12 comparison subjects, had detectable levels of aromatic DNA adducts by 32P-postlabelling assay (p less than 0.01). The two non-roofers with detectable adducts had levels at or near the detection limit of 2 adducts per 10(9) nucleotides. In two roofer samples which were studied in a mixing experiment, the major adduct spots did not co-migrate with the guanosine N2 adduct of benzo[a]pyrene diol epoxide. These results suggest that the 32P-postlabelling assay may be useful for monitoring exposures to complex mixtures of aromatic hydrocarbons in industrial populations.     

Analysis of 7-methylbenz[a]anthracene-DNA adducts formed in SENCAR mouse epidermis by 32P-postlabeling

The present study has analysed the DNA adducts formed in SENCAR mouse epidermis following topical application of 7-methylbenz[a]anthracene (7- MBA). Mice were treated with 400 nmol of 7-MBA, which represents an initiating dose of this hydrocarbon for SENCAR mice. DNA adducts were analysed 24 h after topical application of the hydrocarbon by 32P- postlabeling coupled with either HPLC analysis or an improved TLC procedure giving better resolution of DNA adducts through the use of a D6 solvent [isopropanol:4N NH4OH (1:1)] following D5. Twenty-four hours after topical application of 400 nmol 7-MBA, the level of total covalent binding was 0.37 +/- 0.07 pmol/mg DNA as determined by 32P- postlabeling. This level of binding correlated well with the relative tumor initiating activity of this hydrocarbon compared to 7,12- dimethylbenz[a]anthracene (6.4 +/- 0.01 pmol/mg DNA) and dibenz[a,j]anthracene (0.03 +/- 0.01 pmol/mg DNA). Analysis of the 32P- labeled 3',5'-diphosphodeoxyribonucleosides by HPLC and TLC revealed the presence of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both the anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs). The major DNA adduct derived from 7-MBA in mouse epidermis was tentatively identified as (+) anti-7-MBADE-trans-N2- dGuo. In addition, a minor dGuo adduct derived from the bay-region syn- diol-epoxide of 7-MBA was detected as well as a minor dAdo adduct from this diol-epoxide. Another minor dAdo adduct was also detectably present which arose from either the anti- or syn-diol epoxide. Furthermore, several unidentified DNA adducts were present in both HPLC and TLC chromatograms of DNA samples from 7-MBA-treated mice. These results are discussed in terms of the role of specific 7-MBA-DNA adducts in tumor initiation by this hydrocarbon.      

Fluorescence spectral evidence that benzo[a]pyrene is activated by metabolism in mouse skin to a diol-epoxide and a phenol-epoxide

Hydrolysates of DNA that had been isolated from mouse skin treated with 3H-labelled benzo[a]pyrene were subjected to chromatography on Sephadex LH20. Two major products were eluted in the region expected for deoxyribonucleoside-hydrocarbon adducts and these were purified further by h.p.l.c. The fluorescence emission and excitation spectra of one of the adducts were identical to that of the adduct obtained from DNA that was treated with BP-7,8-diol 9,10-oxide (r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a] pyrene). The fluorescence emission and excitation spectra of the other adducts were identical to the published spectra of 9-OHBP-4,5-diol (4,5-dihydro-4,5,9-trihydroxy-benzo[a]pyrene) and of the deoxyribonucleoside-hydrocarbon adduct obtained from DNA that had been incubated with 9-OHBP (9-hydroxybenzo[a] pyrene) in the presence of a rat-liver microsomal system. The metabolic activation of benzo[a]pyrene in mouse skin, a target tissue for carcinogenesis by this hydrocarbon, thus appears to involve the formation of adducts derived from both BP-7,8-diol 9,10-oxide and 9-OHBP 4,5-oxide (9-hydroxybenzo-[a]pyrene 4,5-oxide), although quantitatively, the adduct derived from 9-OHBP 4,5-oxide is a minor product.     

Comparison of the morphological transforming activities of dibenzo[a,l]pyrene and benzo[a]pyrene in C3H10T1/2CL8 cells and characterization of the dibenzo[a,l]pyrene-DNA adducts

C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts were used to study the in vitro carcinogenic activities of dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P). The morphological transforming activities of these rodent carcinogens were compared using replicate concentration- response studies. In concentration ranges where both polycyclic aromatic hydrocarbons (PAHs) were active, DB[a,l]P proved to be four to 12 times as potent as B[a]P based on concentration. At lower concentrations DB[a,l]P was active at 0.10 and 0.20 microM, concentrations where B[a]P was inactive. This makes DB[a,l]P the most potent non-methylated PAH evaluated to date in C3H10T1/2 cells. DNA adducts of DB[a,l]P in C3H10T1/2 cells were analyzed by both TLC and TLC/HPLC 32P-postlabeling methods using mononucleotide 3'-phosphate adduct standards derived from the reactions of anti-DB[a,l]P-11,12-diol- 13,14-epoxide (anti-DB[a,l]PDE) and syn-DB[a,l]P-11,12-diol-13,14- epoxide (syn-DB[a,l]PDE) with deoxyadenosine 3'-monophosphate and deoxyguanosine 3'-monophosphate. All of the DNA adducts observed in C3H10T1/2 cells treated with DB[a,l]P were identified as being derived from the metabolism of DB[a,l]P to its fjord region diol epoxides through DB[a,l]P-11,12-diol. The predominant adduct was identified as an anti-DB[a,l]PDE-deoxyadenosine adduct. Other major adducts were anti- DB[a,l]PDE-deoxyguanosine and syn-DB[a,l]PDE-deoxyadenosine adducts with minor amounts of syn-DB[a,l]PDE-deoxyguanosine adducts. These DNA adduct data are consistent with similar findings of DB[a,l]PDE- deoxyadenosine adducts in mouse skin studies and human mammary cells in culture.      

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.     

Comparison of TLC-- and HPLC--32P-postlabelling assay for cisplatin--DNA adducts

Calf thymus DNA, treated with the antitumour agent cis-diamminediehloroplatinum(II)(cisplatin), was enzymatically digested with deoxyribonucleaseI, snake venom phosphodiesterase and prostatic acid phosphatase.As a result the adducts were released with an unmodified nucleotideat their 5’-side. The adducts were postlabelled with [-³²P]ATPand separated by thin-layer chromatography (TLC) and HPLC withon-line detection of ³²P. One of the standards, platinated d(TGG),could be analysed with a recovery of 31% with both the TLC andthe HPLC separation systems, when the amount of adduct was between0.5 and 100 fmol. In the case of platinated DNA the linear partof the concentration curve was from 100 fmol down to the lowestamount of adducts measured, 3.2 fmol, with the TLC system andbetween 8 and 40 fmol with the HPLC system. The recoveries ofthe adducts were 28% and 16% respectively.     

Molecular dosimetry of DNA adducts in C3H mice treated with glycidaldehyde.

The formation of DNA adducts in the skin of male C3H mice treated cutaneously with glycidaldehyde (2 or 10 mg/animal) in acetone has been investigated by HPLC coupled with fluorescence detection and 32P-postlabelling analysis. Following a 24 h exposure period, epidermal DNA was isolated from treated dorsal skin and enzymically digested to nucleoside-3'-monophosphates. HPLC-32P-postlabelling analysis of the DNA hydrolysate indicated that a single major cyclic adduct was formed from the reaction of glycidaldehyde with deoxyadenosine residues in mouse skin DNA. This adduct was identified as 3-beta-D-deoxyribofuranosyl-7-(hydroxymethyl)-3H- imidazo[2,1-i]purine-3'-monophosphate by comparison with a synthetic standard. This adduct was stable, strongly fluorescent and readily detected by HPLC with fluorescence detection. There was no evidence for the formation of deoxyguanosine adducts in epidermal DNA of treated animals. Glycidaldehyde also reacted with calf thymus DNA in vitro at pH 7.0 to give the same deoxyadenosine adduct observed in vivo. At pH 10, however, this was a relatively minor product and the major adduct was 5,9-dihydro-7-(hydroxymethyl)-9- oxo-3-beta-D-deoxyribofuranosyl-3H-imidazo[1,2-a]purine-3'- monophosphate formed by the initial reaction of glycidaldehyde with deoxyguanosine residues.     

Use of the 32P-postlabelling assay to study transplacental carcinogens and transplacental carcinogenesis

The formation of DNA adducts represents a key step in the postnatal initiation of the carcinogenic process. Little is known as yet about the role of prenatally induced adducts in transplacental carcinogenesis in offspring. Measurement of transplacental DNA damage in fetal organs of experimental animals has been difficult in the past because of the small amounts of DNA available and low adduct levels. In principle, these difficulties have been overcome by the recent development of a highly sensitive 32P-postlabelling assay which can be applied to a large number of DNA adducts of diverse structure and requires only microgram amounts of DNA for analysis. In this assay, tissue DNA is degraded to mononucleotides; these are enzymatically 32P-labelled via T4 polynucleotide kinase-catalysed [32P]phosphate transfer from [gamma--32P]ATP, to form 5'--32P-labelled 3',5'-bisphosphate derivatives; the labelled products are separated into normal and adducted [32P]nucleotides and quantified by thin-layer chromatography, autoradiography and scintillation (Cerenkov) counting. This technique allows the detection and quantitation of one adduct in 10(8)-10(10) DNA nucleotides (approximately 1-100 adducts/mammalian genome) using a 10-micrograms DNA sample and has been applied in studies of adduct formation from transplacental carcinogens in fetal and adult rodent tissues. In this paper, we review application of 32P-postlabelling to DNA adducts formed with transplacental or suspected transplacental carcinogens in fetal and maternal tissues. The carcinogens studied include diethylstilboestrol (DES), benzo[a]pyrene, safrole, 4-aminobiphenyl and 4-nitroquinoline-1-oxide, as well as cigarette smoke condensate. In DNA of DES-exposed hamsters, one major and several minor adduct spots were observed, which were absent from vehicle controls. A characteristic adduct, which resembled the major hamster adduct chromatographically, was detected in all exposed mouse tissue, except fetal kidney. Chronic administration of low doses of DES to male Syrian hamsters led to an entirely different pattern of adducts in kidney DNA, the target organ of carcinogenesis. These adducts did not contain covalently bound oestrogen moieties and appeared to be formed indirectly: oestrogen appeared to induce or enhance the synthesis of an endogenous electrophilic metabolite reacting with DNA. Thus, multiple mechanisms exist by which DES can damage DNA. Additional work using 32P-postlabelling has shown that non-hormonal genotoxicants (e.g., benzo[a]pyrene, safrole, 4-aminobiphenyl, 4-nitroquinoline-1-oxide) and cigarette smoke condensate given to pregnant mice can induce specific DNA adduct profiles in fetal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative 32P-analysis of cigarette smoke-induced DNA damage in human tissues and mouse skin

Previous studies using a highly sensitive 32P-postlabeling assay for the analysis of carcinogen/mutagen-induced DNA damage have shown the presence of tobacco smoking-related DNA adducts in human placenta (Everson, R.B., Randerath, E., Santella, R.M., Cefalo, R.C., Avitts, T. A., and Randerath, K., Science (Wash. DC), 231: 54-57, 1986). The occurrence of such adducts in smokers' bronchus and larynx is reported here. Since the chemical nature of these adducts could not be characterized by direct methods due to the extremely low levels of individual adducts (less than 0.03 fmol per microgram DNA), we have sought an experimental animal model for studying the formation of tobacco-related DNA adducts. Because cigarette smoke condensate is known to initiate tumors in mouse skin, ICR mice were treated topically with cigarette tar equivalent to 1.5, 3, 6, and 9 cigarettes for 0.4, 3, 5, and 7 days, respectively, and skin DNA was isolated 1 day after the last treatment. When DNA from exposed mice was analyzed by the 32P-postlabeling assay, 12 distinct 32P-labeled DNA adduct spots, as well as a diagonal radioactive zone, which presumably reflected the presence of incompletely resolved adducts, were noted on polyethyleneimine-cellulose TLC fingerprints. One derivative in particular (adduct 1) was seen to increase rapidly during the early treatment phase and also to persist to 8 days after treatment. The prominent adduct 1 was observed in the same location on the fingerprints of DNA samples from smokers. Cochromatography experiments suggested identity of human and mouse DNA adduct 1. Similarly, several other human and mouse adducts (adducts 3, 5, 6, and 9) appeared identical, and the diagonal radioactive zone was also present on DNA adduct maps from smokers. While absolute levels of individual human adducts were too low to be accurately quantitated, semiquantitative estimation of total tobacco-related aromatic DNA adducts in the human specimens gave values of 1 adduct in (1.7-2.9) X 10(7) nucleotides (0.10-0.18 fmol per micrograms DNA), with adduct 1 constituting 8.5-14% of the total. On the basis of these results, it appears now feasible to determine the chemical origin of smoking-induced DNA adducts in human tissues by preparation of authentic 32P-labeled reference adducts from animals treated with characterized subfractions of cigarette tar, 32P-postlabeling, and cochromatography of 32P-labeled human and animal adducts.     

Mutagenicity and tumor initiating activity of methylated benzo[k] fluoranthenes

The mutagenic activities toward Salmonella typhimurium TA100 and tumor initiating activities on mouse skin of the polynuclear aromatic hydrocarbons benzo[k]fluoranthene (BkF),2-methylBkF,8-methylBkF,9-methyl-BkF and 7,12-dimethylBkF were compared. BkF and 2-methylBkF were the most mutagenic of the compounds tested and had comparable activity; they were more active than 7,12-dimethylBkF. 8-MethylBkF and 9-methylBkF were not mutagenic. BkF and the methylated BkFs had similar tumor initiating activities on mouse skin. The results suggest that 8,9-dihydro-8,9-epoxy-BkF might be involved in the metabolic activation of BkF to a mutagen, but do not indicate which metabolite may be involved in BkF tumorigenesis.     

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.     

Fluorine probes for investigating the mechanism of activation of indeno[1,2,3-cd]pyrene to a tumorigenic agent

Indeno[1,2,3-cd]pyrene (IP) is a non-alternant polycyclic aromatic hydrocarbon that has tumor-initiating activity on mouse skin and is carcinogenic in newborn mice and in rat lungs. Previous studies have shown that 8- and 9-hydroxyIP and IP-1,2-diol are major metabolites formed in vivo in mouse skin. 8-HydroxyIP-1,2-diol and 9-hydroxyIP-1,2-diol are also observed as in vivo metabolites of IP. Although 8-hydroxyIP had marginal tumor-initiating activity on mouse skin, IP-1,2-diol and its epoxide precursor, IP-1,2-oxide, had similar tumorigenic activity as IP. In the present study fluorine probes have been employed to investigate the contribution of metabolic activation at the 1,2 and 7-10 positions of IP. At a total initiating dose of 4.0 mumol, 2-fluoroIP induced skin tumors in 76% of the treated animals with an average of 3.9 tumors/mouse. At the same dose, IP induced a 72% incidence of tumor-bearing mice with 2.1 tumors/mouse. In contrast, 8,9-difluoroIP elicited a tumorigenic response in 40% of the treated animals with 0.6 tumors/animal. Five mice from each experimental group were killed at the conclusion of the initiation phase of the bioassay and DNA was isolated from the treated areas of skin. 32P-Postlabeling analysis of the hydrolyzed DNA indicated that IP forms one major detectable DNA adduct that migrates close to the origin. This adduct is absent in mice treated with 8,9-difluoroIP. In contrast, 2-fluoroIP forms one major adduct spot with different retention behavior as compared with the adduct formed from IP. DNA from mice treated topically with IP-1,2-diol and IP-1,2-oxide was subjected to 32P-postlabeling analysis. IP-1,2-diol forms one major DNA adduct spot with mobility similar to that observed for the IP-DNA adduct. IP-1,2-oxide displayed an intense pattern of DNA adducts centered around the location of the IP-DNA adduct. No adducts were detected which had mobility similar to that formed from 2-fluoroIP. These results are consistent with IP undergoing metabolic activation at positions 7-10 either alone or in conjunction with dihydrodiol formation at the 1,2 position.     

Relative tumor initiating activity of benzo[a]fluoranthene, benzo[b]fluoranthene, naphtho[1,2-b]fluoranthene and naphtho[2,1-a]fluoranthene on mouse skin

The tumor-initiating activities of benzo[a]fluoranthene (BaF), benzo[b]fluoranthene (BbF), naphtho[1,2-b]fluoranthene (NbF) and naphtho[2,1-a]fluoranthene (NaF) were evaluated on the skin of female CD-1 mice. Each of these polycyclic aromatic hydrocarbons was assayed at total initiation doses of 1.0 and 4.0 mumol/mouse. These hydrocarbons were applied in 10 subdoses administered every other day. Promotion commenced 10 days after the last initiator dose and consisted of thrice weekly application of 2.5 micrograms of tetradecanoylphorbol acetate for 20 weeks. BbF was the most potent tumor initiator inducing a 100% incidence of tumor-bearing mice with an average of 8.5 tumors per mouse at a total initiator dose of 1.0 mumol. NaF was slightly more active as a tumor initiator than either NbF or BaF. NaF induced a 90% incidence of tumor-bearing mice with an average of 5.9 tumors per mouse at a total initiator dose of 1.0 mumol. BaF and NbF at a total initiator dose of 4.0 mumol exhibited similar tumor-initiating activity with both inducing a 90% incidence of tumor-bearing mice with an average of 4.3 and 6.6 tumors per mouse, respectively. However, at a total initiator dose of 1.0 mumol, BaF and NbF induced a 95% and 65% incidence of tumor-bearing mice with an average of 3.3 and 2.5 tumors per mouse, respectively.