Polycyclic aromatic hydrocarbon-inducible DNA adducts: evidence by 32P-postlabeling and use of knockout mice for Ah receptor-independent mechanisms of metabolic activation in vivo

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens in laboratory animals and are suspected human carcinogens. The PAHs are bioactivated by cytochrome P450 (CYP)1A1/1B1 enzymes to reactive intermediates that bind to DNA, a critical step in the initiation of carcinogenesis. The Ah receptor (AHR) plays a critical role in the induction of CYP1 enzymes (i.e., CYP1A1, 1A2 and 1B1) by PAHs such as benzo[a]pyrene (BP) and 3-methylcholanthrene (MC). In our investigation, we tested the hypothesis that AHR-null animals are less susceptible to PAH-induced DNA adduct formation than wild-type animals. Wild-type [AHR (+/+)] mice or mice lacking the gene for the AHR were treated with a single dose (100 micromol/kg) of BP or MC, and hepatic DNA adducts were analyzed by (32)P-postlabeling. BP induced multiple hepatic DNA adducts in wild-type as well as AHR-null animals, suggesting the existence of AHR-independent mechanisms for BP metabolic activation. On the other hand, DNA adduct formation was markedly suppressed in AHR-null animals exposed to MC, although the major MC-DNA adduct was produced in these animals. Hepatic activities and apoprotein contents of 7-ethoxyresorufin O-deethylase (EROD) (CYP1A1) and 7-methoxyresorufin O-demethylase (MROD) (CYP1A2) activities were markedly induced by BP and MC in the wild-type, but not, in AHR-null animals. CYP1B1 expression was also induced, albeit to a lesser extent by the PAH MC, but not BP, in the wild-type animals. In conclusion, these results demonstrate the existence of AHR- and CYP1A1-independent mechanisms of PAH metabolic activation in mouse liver, a phenomenon that may have important implications for PAH-mediated carcinogenesis.     

In situ freezing of the rat urinary bladder: DNA adduct formation in the bladder epithelium demonstrated by 32P-postlabeling assay

In situ freezing of the urinary bladder has been demonstrated to exert tumor-initiating potential in two-stage urinary bladder carcinogenesis in the rat. In the present experiment, DNA modification was examined after in situ freezing of the whole urinary bladder performed by pinching with frozen forceps at -15 degrees C or -30 degrees C for 2 s. The 32P-postlabeling analysis revealed at least 2 DNA adducts in the epithelial cells of the urinary bladder collected 3 days after freezing. Single-strand breaks of DNA were also found by means of the alkaline elution assay in the bladder epithelium collected 10 min after freezing. Thus, the previously demonstrated tumor-initiating activity of in situ freezing in urinary bladder carcinogenesis was revealed to be associated with substantial DNA damage and adduct formation.     

Differences in metabolic activation of dibenzo[a,e]fluoranthene characterized by 32P-postlabeling in two mouse fibroblast models.

The formation of DNA adducts was investigated in mouse fibroblasts from two different tissues--embryos and adult lung--after incubation with dibenzo[a,e]fluoranthene (DBF) or its major proximate metabolites. The nuclease P1 modification of the 32P-postlabeling method was adapted for detection of DBF-DNA adducts. Quantitative and qualitative differences were observed in the metabolic activation mediated by the two cell types. DBF-DNA adducts generated three major spots reproducibly, and more than ten spots of medium or weak importance. The highest level of DNA binding occurred via the DBF-bay region vicinal dihydrodiol epoxide but with significant differences in the quantitative distribution of adducts. Striking qualitative differences were observed when lung fibroblasts were incubated with the DBF-pseudo bay region dihydrodiol (DBF-12,13-DHD). The spots representing adducts induced in embryo fibroblasts by DBF-3OH-12,13-DHD, a further metabolite of DBF-12,13-DHD, were totally absent from chromatograms of lung cells. These results show that both embryo and lung fibroblasts can activate DBF but that different cytochrome P-450 forms and substrate affinities are involved. The finding that different activation systems may be present in subcategories of the same tissue, may provide a partial explanation for the wide variations in sensitivity to carcinogens among species, organs and tissues.     

Microsome-mediated bioactivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling

Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60- fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.      

Detection of deoxyadenosine-4-aminobiphenyl adduct in DNA of human uroepithelial cells treated with N-hydroxy-4-aminobiphenyl following nuclease P1 enrichment and 32P-postlabeling analysis

To characterize the DNA adducts in human uroepithelial cells(HUC) exposed to 4-aminobiphenyl and its proximate N-hydroxymetabolites, we used ³²P-analyses following butanol extractionof the DNA hydrolysates. Using this method, we identified N-(deoxyguanosin-3',5'-bisphospho-8-yl)-4-aminobiphenyl (pdGp-ABP) as a major adductand N-(deoxyguanosin-3', 5'-bisphospho-8-yl)-4- aminobiphenyl(pdAp-ABP) as a minor adduct in an immortalized non-tumorigeniccell line of HUC following exposure to N-hydroxy-4-aminobiphenyl(N-OH-ABP). Towards characterization of pdAp-ABP, we postlabeledthe synthetic N-(deoxyguanosin-3', 5'-bisphospho-8-yl)-4-aminobi-phenyl (dAp-ABP) adduct to generate pdAp-ABP and determinedits chromatographic (TLC and HPLC) proper ties and sensitivityto nuclease P1 digestion. In contrast to pdGp-ABP, which wascleaved to the corresponding 5' monophosphate by nuclease P1,the pdAp-ABP adduct was unaffected when incubated with nucleaseP1 under similar conditions. To test whether nuclease P1 digestioncould be adopted for enrichment of the dAp-ABP adduct in HUCsamples, postlabeling analyses were carried out after but anolextraction following nuclease P1 digestion of the DNA hydrolysate.Under these conditions, the pdAp-ABP adduct was detected inDNA from HUC E7 cells treated with N OH-ABP and in calf thymusDNA reacted with N-OH ABP under acidic (pH 5.0) conditions.These data indicate that pdGp-ABP and pdAp-ABP adducts are generatedin HUC E7 on treatment with N-OH-ABP and that nuclease P1 enrichmentmay provide a method for qualitative and quantitative analysesof the pdAp-ABP adduct in DNA.     

Strong intensification of mouse hepatic tamoxifen DNA adduct formation by pretreatment with the sulfotransferase inhibitor and ubiquitous environmental pollutant pentachlorophenol

Although negative in assays for mutagenicity, the clinicallyimportant antiestrogen tamoxifen induces hepakic DNA adductformation in mice, rats and hamsters, as indicated by ³²P-postlabeling,and is a potent hepatocardnogen in rats. Both phenolic and alcoholicmetabolites of tamoxifen have been reported. As these metabolitesare potential candidates for sulfate coqjugation, we examinedwhether the sulfe transferase inhibitor pentachlorophenol, aubiquitous environmental contaminant, modulates hepatic tamoxifenadduct formation in vivo. Female ICR mice were given tamoxifen(45 mg/kg) daily per os for up to 4 days, with and without i.p.pretreatment with pentachloropheno1 (20 mg/kg) 1 h before dosingwith tamoxifen. At days 1,2 and 4, liver DNA wm analyzed 5 hafter tamoxifen administration by a modified monophosphate versionof the ³²P-postlabeling assay. At day 4, patachrophenol pretreatmentled to a large increase (13- to 17-fold) of the levels of fourtamoxifen adduct fractions, while two adducts appeared unaffected,resulting in an     

DNA adduct formation in human and mouse skin by mixtures of polycyclic aromatic hydrocarbons

32P-Postlabelling analysis has been used to detect the formation in vivo of DNA adducts by components of complex mixtures of polycyclic aromatic hydrocarbons (PAHs) in coal-tar, creosote, bitumen, juniper tar, used engine oils and fuel exhaust condensates. The presence of DNA adducts derived from these agents has been investigated in mouse skin, in human skin explants maintained in short-term organ culture and in human skin in vivo, and the formation of many different PAH-DNA adducts was observed. Similar levels and patterns of adducts were found in DNA from human skin to those in mouse skin, which is known to be susceptible to the carcinogenic activity of PAH mixtures, thus demonstrating the potential hazard to man of epidermal contact with these materials.     

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.     

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.      

DNA adduct formation after oral administration of 2-nitrofluorene and N-acetyl-2-aminofluorene, analyzed by 32P-TLC and 32P-HPLC

DNA adducts have been detected in laboratory animals after exposureto carcinogens as well as in human populations with known orsuspected risk of developing cancer. Examples are smokers, cokeand aluminium workers, urban citizens and roofers. The formationof DNA adducts is an early event in carcinogenesis which canbe used for measuring target dose and as a biomarker for genotoxkrisk. A method of analyzing ³²P-postlabeled DNA adducts on reverseHPLC with on-line detection of ³²P has been developed. The methodpermits direct injection of the ³²P-postlabeling mixture intothe analytical system without prior purification with backgroundradioactivity on a low level. The method can be used in parallelwith TLC analyses of ³²P-postlabeled DNA adducts to improvethe analytical capacity. The time for analysis of a typicalsingle sample by HPLC and TLC is 30–60 min and 6–24h respectively. A high (2 M) salt concentration in the HPLCeluent reduces the ³²P background considerably. Also the peaktailing was substantially diminished, giving an ability to separateDNA adducts equal to or better than the TLC method. The methodhas been applied to 2-nitrofluorene (NF), a carcinogenic airpollutant, and N-acetyl-2-aminofluorene (AAF), a model carcinogenwhich is also a metabolite of NF. A number of DNA adducts areformed in the livers of rats. After oral administration of AAFand NF, DNA adducts in the liver have been characterized asdG-C₈-AF and dG-C₈-AAF. The major DNA adduct found in both NF-and AAF-administered animals was dG-C₈-AF. The described HPLCmethod can, with minor adjustments, generally be used to analyze³²P-postlabeled DNA adducts.     

DNA-adduct formation in the forestomach of rats treated with 3-tert-butyl-4-hydroxyanisole and its metabolites as assessed by an enzymatic 32P-postlabeling method

Formation of DNA-adducts by 3-BHA or its metabolites, i.e., tert-butyl-1,4-benzoquinone (TBQ) and 5-methoxy-3-tert-butyl-1,2-benzoquinone (3-TBOQ), as well as DNA-adduct formation by 4-nitroquinoline-N-oxide (4NQO), in rat forestomach were examined by an enzymatic 32P-postlabeling assay. Four DNA-adducts were clearly detected in the forestomach after treatment of rats with 4NQO. The sensitivity was 1.9 certain adducts per 10(8) normal nucleotides. On the contrary, no DNA adducts were detected in the forestomach of rats given either a single or repeated oral administration (5 days) of 3-BHA, TBQ or 3-TBOQ. The analyses were carried out under conditions which could detect the DNA-adducts produced by reaction of TBQ with calf thymus DNA in vitro. The results suggest that formation of aromatic adducts in vivo by 3-BHA, TBQ or 3-TBOQ in the rat forestomach-DNA is not evident or at least below the detection limits of the current bioassay.     

Appearance of artefacts when using 32P-postlabelling to investigate DNA adduct formation by bile acids in vitro: lack of evidence for covalent binding

We reported (Scates et al. Carcinogenesis 1994, 15, 2945–2948)that incubating a range of bile acids with DNA in vitro, withor without exogenous metabolic activation, gave no evidenceof DNA adduct formation as judged by the nuclease P1 methodof ³²P-postlabelling. In contrast Hamada et al. (Carcinogenesis1994, 15, 1911–1915), also using postlabelling, claimedthat chenodeoxycholic acid, lithocholic acid, glycolithocholicacid and taurolithocholic acid bound covalently to DNA in vitro.To investigate this discordance we incubated solutions of salmonsperm DNA for 1 h at 37°C with 1 mg/ml of cholic acid, chenodeoxycholicacid, lithocholic acid, glycolithocholic acid or taurolithocholicacid. Each incubate was extracted extensively with diethyl etherafter which a sample of DNA was taken and ³²P-postlabelled usingthe nuclease P1 method. The DNA in the remaining incubate wasprecipitated from high salt solution with ethanol. Aliquotsof this DNA were postlabelled. The remainder of the DNA waspurified with proteinase-K, ribonuclease, phenol-chloroform,precipitated and postlabelled. Parallel incubates were madewith the same bile acids, under the same conditions but in theabsence of DNA and were then extracted, precipitated and postlabelledas described above. When DNA was present in the incubate butwas not precipitated, chenodeoxycholic acid, lithocholic acid,glycolithocholic acid and taurolithocholic acid, but not cholicacid, produced spots similar to those reported by Hamada etal. No such spots were seen when DNA was postlabelled afterprecipitation, or after precipitation and purification. Thesesame bile acids produced spots when postlabelled in the absenceof DNA, but spots were absent when these incubates were precipitatedand purified before postlabelling. We conclude that the spotsobtained when bile acids are incubated with DNA which is notprecipitated from high salt before it is postlabelled are technicalartefacts, and cannot be regarded as evidence that bile acidsbind covalently to DNA to form adducts. We also confirm reports(Vulimiri et al. Carcinogenesis 1994, 15, 2061–2064) thatbile acids alone can produce spots when incubated with T4 polynucleotidekinase and [     

DNA damage induced by the environmental carcinogen butadiene: identification of a diepoxybutane-adenine adduct and its detection by 32P-postlabelling

To date only a few studies have been undertaken on DNA adductsformed by epoxybutene (EB) and diepoxybutane (DEB), the twoactive metabolites of 1,3-butadiene. Our interests have focusedon further investigating DNA alkylation by the two epoxides,especially in relation to the development of a method for humanbiomonitoring. Here, following the reaction of deoxyadenosinemonophosphate and poly(dA-dT)(dA-dT) with DEB and subsequentHPLC, we have identified an adenine adduct. MS analyses indicatethe structure of an adenine adducted by DEB at the N⁶ position.A HPLC/³²P-postlabelling method was developed for its measurementin DNA samples and the adduct was detected in calf thymus DNAand DNA from Chinese hamster ovary cells exposed to DEB. The100% labelling efficiency during postlabelling, the amount ofthe adduct and its elution before the normal nucleotides duringHPLC suggest it could be a suitable indicator of BUT exposure.     

Induction of a DNA adduct detectable by 32P-postlabeling in the dorsolateral prostate of NBL/Cr rats treated with estradiol-17{beta} and testosterone

Treatment with estradiol-17ß and testosterone inducesepithelial dysplasia and, subsequently, adenocarcinoma in thedorsolateral prostate of NBL rats. The purpose of this studywas to determine whether this carcinogenic effect is mediatedby genotoxicity. Analogous to adducts produced by estrogensin the male hamster kidney, a target of estrogen carcinogenicity,induction of DNA adducts detectable by ³²P-postlabeling wasinvestigated in the prostate target tissue. NBL rats were treatedwith separate Silastic tubing implants containing testosteroneand estradiol-17ß. Control animals received emptyimplants. Animals were killed at 8, 16 and 24 weeks after initiationof treatment, and accessory sex glands were sampled for adductanalysis. DNA of the dorsolateral and ventral prostate and thecoagulating gland (= anterior prostate) was isolated and analyzedby nuclease Pl-enhancement of the ³²P-postlabeling assay. DNAadducts were quantitated by Cerenkov counting. An adduct occurredselectively in DNA of the dorsolateral prostate of rats treatedwith estradiol plus testosterone for 16 or 24 weeks with relativeadduct level values of     

7, 12-Dimethylbenz[a]anthracene-deoxyribonucleoside adduct formation in vivo: evidence for the formation and binding of a monohydroxymethyl-DMBA metabolite to rat liver DNA

The polycyclic aromatic hydrocarbon, 7, 12-dimethyl benz[a]anthracene(DMBA) is a potent carcinogen to the female Sprague-Dawley rat,and when administered under conditions that have been shownto produce cancer, results in extensive formation of hydrocarbon-deoxyribonucleosideadducts. Sephadex LH-20 and reverse-phase h.p.l.c. and spectrofluorometricanalysis of these adducts demonstrate that at least one adductresults from the binding of 7, 12-dimethylbenz[a]anthracene-1,2, 3, 4-tetrahydro-3, 4-dihydroxy-1, 2-oxide. In these experiments,employing i.p. administration of the hydrocarbon, a second morepolar adduct was observed. Evidence is presented that this adductresults from the formation of a monohydroxymethyl-methylbenz[a]anthracene-A-ring-diol-epoxide.While both of the monohydroxymethyl-DMBA metabolites have beenshown to bind cellular DNA following their administration thisis the first evidence of monohydroxymethyl-DMBA-deoxyribonucleosideadducts being formed after the administration of DMBA per se.The evidence suggests that this more polar adduct is a 7-hydroxymethyl-12-methylbenz[a]anthracenedeoxyribonucleosideadduct.     

Bioactivation of 3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone: evidence for DNA adduct formation mediated by cytochrome P450 enzymes and peroxidases

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in the urine of salt mining workers occupationally exposed to diesel emissions. We evaluated the role of hepatic cytochrome P450 (CYP) enzymes in the activation of 3-ABA in vivo by treating hepatic cytochrome P450 oxidoreductase (POR)-null mice and wild-type littermates intraperitoneally with 0.2 and 2mg/kg body weight of 3-ABA. Hepatic POR-null mice lack POR-mediated CYP enzyme activity in the liver. Using the (32)P-postlabelling method, multiple 3-ABA-derived DNA adducts were observed in liver DNA from wild-type mice, qualitatively similar to those formed in incubations using human hepatic microsomes. The adduct pattern was also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. DNA binding by 3-ABA in the livers of the null mice was undetectable at the lower dose and substantially reduced (by up to 80%), relative to wild-type mice, at the higher dose. These data indicate that POR-mediated CYP enzyme activities are important for the oxidative activation of 3-ABA in livers, confirming recent results indicating that CYP1A1 and -1A2 are mainly responsible for the metabolic activation of 3-ABA in human hepatic microsomes. No difference in DNA binding was found in kidney and bladder between null and wild-type mice, suggesting that cells in these extrahepatic organs have the metabolic capacity to oxidize 3-ABA to species forming the same 3-ABA-derived DNA adducts, independently from the CYP-mediated oxidation in the liver. We determined that different model peroxidases are able to catalyse DNA adduct formation by 3-ABA in vitro. Horseradish peroxidase (HRP), lactoperoxidase (LPO), myeloperoxidase (MPO), and prostaglandin H synthase (PHS) were all effective in activating 3-ABA in vitro, forming DNA adducts qualitatively similar to those formed in vivo in mice treated with 3-ABA and to those found in DNA reacted with N-hydroxy-3-aminobenzanthrone (N-OH-ABA). Collectively, these results suggest that both CYPs and peroxidases may play an important role in metabolizing 3-ABA to reactive DNA adduct forming species.     

Comparative DNA binding of 7, 12-dimethylbenz[a]anthracene and some of its metabolites in mouse epidermis in vivo as revealed by the 32P-postlabeling technique

The binding of some mouse skin metabolites and related derivativesof the tumor initiator 7,12-dimethylbenz[a]-anthracene (DMBA)was investigated by ³²P-postlabeling analysis after its topicaladministration. DMBA and trans-3,4-dihydro3,4-dihydroxy-DMBA(DMBA-3,4-dihydro-diol) both led to the formation of four DNAadducts, which showed a very similar pattern of spots on thin-layer chromato-grams. With trans-8,9-Ktihdbro-8,9-dihydroxy-7,12-dimethyl-benz[a]anthracene(DMBA-8,9-dihydrodiol) one major adduct was obtained which waschromatographically indistinguishable from one of the DMBA adducts.In contrast, 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA)gave rise to two major adducts which were separable from DMBAadducts. 3-hydroxy-7, 12-dimethyIbenz[a]anthra-cene (3-OH-DMBA)and 7,12-dimethylbenz[a]anthracene-7, 12-epoxide (DMBA-O2) didnot lead to detectable amounts of adducts. Quantitative determinationof DNA binding showed that an initiating dose (i = 100 nmol)of DMBA yielded {small tilde}12 adducts/10⁷ normal nucleotides.Adduct formation with the same dose of DMBA-3,4-dihydrodiolwas 7-8 times higher. At a 4-fold higher dose level, DMBA-8,9-dihydrodiolexhibited a 3- to 6-times weaker binding and 7-OHM-12-MBA aslightly stronger binding than DMBA. Chromatography of the DMBAand DMBA-3,4-dihydrodiol adducts with a solvent containing borateshowed a decreased mobility of two out of four adducts in eachcase. These adducts were also sensitive to oxidation by periodate.The results suggest that two DMBA adducts carried vicinal cis-hydroxylgroups and thus were probably derived from the anti3,4-dihydrodiol1,2-oxide(s)of DMBA. The other two adducts were probably derived from thesyn-stereoisomer(s). When the DNA-modifying capabilities andinitiating activities of the more prominent mouse-skin metabolitesare considered in relation to DMBA, DMBA-3,4-dihydrodiol ispostulated to be a proximate and DMBA-3,4-dihydrodiol-1,2-oxide(s)to be ultimate initiators.     

Mechanism of metabolic activation and DNA adduct formation by the human carcinogen diethylstilbestrol: The defining link to natural estrogens

Diethylstilbestrol (DES) is a human carcinogen, based on sufficient epidemiological evidence. DES is mainly metabolized to its catechol, 3′‐hydroxyDES (3′‐OH‐DES), which can further oxidize to DES‐3′,4′‐quinone (DES‐3′,4′‐Q). Similarly to estradiol‐3,4‐quinone, the reaction of DES‐3′,4′‐Q with DNA would form the depurinating 3′‐OH‐DES‐6′‐N3Ade and 3′‐OH‐DES‐6′‐N7Gua adducts. To prove this hypothesis, synthesis of DES‐3′,4′‐Q by oxidation of 3′‐OH‐DES with Ag₂O was tried; this failed due to instantaneous formation of a spiro‐quinone. Oxidation of 3′‐OH‐DES by lactoperoxidase or tyrosinase in the presence of DNA led to the formation of 3′‐OH‐DES‐6′‐N3Ade and 3′‐OH‐DES‐6′‐N7Gua adducts. These adducts were tentatively identified by LC‐MS/MS as 3′‐OH‐DES‐6′‐N3Ade, m/z = 418 [M+H]⁺, and 3′‐OH‐DES‐6′‐N7Gua, m/z = 434 [M+H]⁺. Demonstration of their structures derived from their oxidation by MnO₂ to the DES quinone adducts and subsequent tautomerization to the dienestrol (DIES) catechol adducts, which are identical to the standard 3′‐OH‐DIES‐6′‐N3Ade, m/z = 416 [M+H]⁺, and 3′‐OH‐DIES‐6′‐N7Gua, m/z = 432 [M+H]⁺, adducts. The reaction of DIES‐3′,4′‐Q or lactoperoxidase‐activated 3′‐OH‐DIES with DNA did not produce any depurinating adducts, due to the dienic chain being perpendicular to the phenyl planes, which impedes the intercalation of DIES into the DNA. Enzymic oxidation of 3′‐OH‐DES suggests that the catechol of DES intercalates into DNA and is then oxidized to its quinone to yield N3Ade and N7Gua adducts. These results suggest that the common denominator of tumor initiation by the synthetic estrogen DES and the natural estrogen estradiol is formation of their catechol quinones, which react with DNA to afford the depurinating N3Ade and N7Gua adducts. © 2008 Wiley‐Liss, Inc.     

Morphological transformation and DNA adduct formation by benz[j]aceanthrylene and its metabolites in C3H10T1/2CL8 cells: evidence for both cyclopenta-ring and bay-region metabolic activation pathways

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused polycyclic aromatic hydrocarbon related to 3-methylcholanthrene, has been studied to identify the major routes of metabolic activation in transformable C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts in culture. Previous studies have reported that the major (55% of total) B[j]A metabolite formed by C3H10T1/2 cells was (+/-)-trans-9,10-dihydro-9,10-dihydroxy-B[j]A (B[j]A-9,10-diol), the dihydrodiol in the bay-region ring, with moderate amounts (14% of total) of (+/-)-trans-1,2-dihydro-1,2-dihydroxy-B[j]A (B[j]A-1,2-diol), the cyclopenta-ring dihydrodiol. The morphological transforming activities of three potential intermediates formed by metabolism of B[j]A by C3H10T1/2 cells, (+/-)-anti-trans-9,10-dihydro-9,10-dihydroxy-B[j]A-7,8-oxide (B[j]A-diol-epoxide), B[j]A-9,10-oxide, and B[j]A-1,2-oxide as well as the two B[j]A-dihydrodiols were examined. B[j]A, B[j]A-diol-epoxide, B[j]A-1,2-oxide, and B[j]A-9,10-diol were found to have moderate to strong activities with B[j]A-diol-epoxide the most active compared to B[j]A, while B[j]A-1,2-diol was inactive. B[j]A-9,10-oxide was found to be a weak transforming agent. At 0.5 microgram/ml, the following percentage of dishes with type II or III foci were observed: B[j]A, 59%; B[j]A-diol-epoxide, 75%; B[j]A-1,2-oxide, 25%; and B[j]A-9,10-diol, 17%. DNA adducts of B[j]A, B[j]A-9,10-diol, B[j]A-diol-epoxide, B[j]A-9,10-oxide, and B[j]A-1,2-oxide in C3H10T1/2 cells were isolated, separated, identified, and quantitated using the 32P-postlabeling method. B[j]A forms two major groups of adducts: one group of adducts is the result of the interaction of B[j]A-1,2-oxide with 2'-deoxyguanosine and 2'-deoxyadenosine; the second group of adducts is a result of the interaction of B[j]A-diol-epoxide with 2'-deoxyguanosine and 2'-deoxyadenosine. Qualitative and quantitative analysis of the postlabeling data suggests that B[j]A is metabolically activated by two distinct routes, the bay-region diol-epoxide route and the cyclopenta-ring oxide route, the former being the most significant.     

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.