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.     

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     

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 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.     

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.     

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.     

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.     

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.     

Use of the 32P-postlabeling method to detect DNA adducts of 2-amino-3-methylimidazolo[4, 5-f]quinoline (IQ) in monkeys fed IQ: identification of the N-(deoxyguanosin-8-yl)-IQ adduct

Eight DNA adducts of 2-amino-3-methylimidazolo[4, 5-f]-quinoline(IQ) were found by the standard ³²P-postlabeling method in liversfrom male Cynomolgus monkeys fed IQ (5 days/week, 3 weeks, 20mg/kg,nasal-gastric intubation). The IQ-DNA adduct fingerprints observedin monkeys were identical to those observed in rats that receivedIQ (0.03%) in the diet for 2 weeks. The C8-guanine-IQ adductwas identified by comigration with the synthetic 3‘, 5’-bisphosphatederivative of N(-deoxyguanosin-8-yl)-Q. DNA modified in vitrowith N-hydroxy-IQ showed seven adducts, including the C8-guanine-IQadduct, that were identical to those found in monkeys and rats.Thus it appears that N-hydroxy-IQ, the reactive metabolite ofIQ, was responsible for all adducts found in vivo, except one.In order to detect adducts in other organs that were presentat lower levels, the intensification (ATP-deficient) methodfor ³²P-postlabeling was used. Five of the adducts detectedunder standard conditions, including the C8-guanine-IQ adduct,were also detected under intensification conditions. The totallevel of DNA-IQ adducts was highest in the liver, followed bythe kidney, colon and stomach, and bladder. The adduct patternswere identical in all organs examined. The results indicatethat IQ is potentially genotoxic in primates and therefore alikely human carcinogen.     

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.     

Activation of 4-hydroxytamoxifen and the tamoxifen derivative metabolite E by uterine peroxidase to form DNA adducts: Comparison with DNA adducts formed in the uterus of Sprague-Dawley rats treated with tamoxifen

Daily intraperitoneal treatment of female Sprague-Dawley ratswith either 5, 10 or 20 mg/kg tamoxifen (TAM) for 1 weeks increasedthe level of peroxidase activity in the uterus 2- to 10-foldcompared to the control level. Using uterine extracts preparedfrom control and TAM treated animals, we investigated the activationof 4-hydroxytamoxifen (4-HO-TAM) and (E, Z)-1, 2-dipheny-1-(4-hydroxyphenyl)-but-1-ene(cis/trans-metabolite E) to form DNA adducts. Activation of4-HO-TAM by uterine extracts prepared from either control orTAM-treated rats produced one major (a) and Two minor DNA (band c) adducts. A similar activation of cis/trans-metaboliteE produced two adducts (d and e). There was good correlationbetween levels of uterine peroxidase activity and levels ofDNA adducts formed by 4-HO-TAM and cis/trans-metabolite E. Activationof 4-HO-TAM and cis/trans-metabolite E with horseradish peroxidase(HRP) produced the same adducts as observed by activation withuterine extract Treatment of Sprague-Dawley rats with 5 and10 mg/kg for 7 days produced eleven DNA adducts in the liverwith no adducts detected in the uterus. However, treatment ofrats with 20 mg/kg of TAM for 7 days produced the same adductpattern in the liver and also one major adduct (1) in the uteruswith a relative adduct level of 6.4 ±4.1x10^–9.Tamoxifen-DNA adduct 1 detected both in the liver and in theuterus of treated rats was similar to adducts produced by activationof 4-HO-TAM with either uterine extract or HRP. The resultsof these studies suggest a general model whereby the tamoxifenmetabolite 4-HO-TAM is further activated in the uterus by peroxidaseenzymes to form DNA adducts.     

Evidence from 32P-postlabeling and the use of pentachlorophenol for a novel metabolic activation pathway of diethylstilbestrol and its dimethyl ether in mouse liver: likely {alpha}-hydroxylation of ethyl group(s) followed by sulfate conjugation

Diethylstilbestrol (DES), a synthetic stilbene estrogen, isa potent developmental toxin and carcinogen in humans and rodents.A number of ³²P-postlabeling studies suggest that genotoxiceffects of DES substantially contribute to these biologicaleffects. The mechanisms involved in DES-mediated genotoxicityare not completely understood, however. As reported here, thestructural resemblance of tamoxifen to DES led to the hypothesisthat DES may be hydroxylated and sulfated at the allylic C2and/or C5 of the ethyl side chains in analogy to     

DNA methylation adduct formation and H-ras gene mutations in progression of N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder tumors caused by a single exposure to N-methyl-N-nitrosourea

After receiving 500 p.p.m. N-butyl-N-(4-hydroxybutyl) nitrosamine(BBN) in their drinking water for an initial 10 weeks, ratswere given a single i.p. injection of N-methyl-N-nitrosourea(MNU) at a dose of 50 mg/kg body wt at week 20 (at a stage whenbladder tumor development had already occurred), and then maintaineduntil they were killed at week 40. Three and six hours afterthe MNU injection, the DNA methylation adducts, O⁶-methyldeoxyguanine(O⁶-medG) and 7-methyldeoxyguanine (7-medG), were immunohistochemicallyrevealed to be markedly more frequent in urothelial preneoplasiasor neoplasias than in normal cells. These adducts were rapidlyrepaired, and although 7-dmeG in tumor cells still persistedafter 72 h, they appeared essentially to have returned to normallevels. At the termination, conversion of transitional cellcarcinomas (TCC) to squamous cell carcinomas (SCC) of the urinarybladder was significantly increased in the BBN + MNU group.The extent of invasion was also significantly greater with theadditional MNU treatment Expression of p21 protein, detectedby immunohistochemistry, was comparable between the groups.Mutations in the H-ras gene were observed in one case each ofthe BBN and BBN + MNU groups, and both cases showed a G:C toA:T transition at codon 12. The present study thus suggestedthat while an additional single treatment with MNU of rats bearingBBN-induced bladder neoplasias is associated with significant,possibly mutation-dependent tumor progression, H-ras mutationsare not necessary events.     

Strain-specific tumorigenesis in mouse skin induced by the carcinogen, 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one, and its relation to DNA adduct formation and persistence

The incidence of skin tumors has been studied in three strains of mice, namely, TO, C57BL, and DBA/2, after treatment with the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one. After either a single dose followed by croton oil promotion or a continual dose of the carcinogen, tumors were observed in the TO and C57BL strains, with the TO mice having the shorter mean latent period. The DBA/2 mice, however, appeared to be resistant to tumor formation by either treatment. To understand the mechanism of resistance, several criteria have been investigated. Metabolism of the carcinogen was assessed in terms of the total DNA adduct formation and the pattern of individual adducts after separation by high-pressure liquid chromatography, and no major differences between the three strains was found. Similarly, the rates of disappearance of the individual adducts when measured over 14 days posttreatment were not strain specific. Persistent binding of the carcinogen after 2 months was found in all three strains and could be reduced markedly if croton oil was administered throughout this period. The ability of the phorbol esters to cause biochemical changes in both sensitive and resistant strains was indicated by the induction of ornithine decarboxylase in each of the three strains after treatment with either croton oil or its active component, 12-O-tetradecanoylphorbol-13-acetate.     

DNA damage as assessed by 32P-postlabelling in three rat strains exposed to dietary tamoxifen: the relationship between cell proliferation and liver tumour formation

Tamoxifen was administered in the diet (420 p.p.m.) to femaleF344 (Fischer), Wistar (LAC-P) and LEW (Lewis) rats to determinefor each strain the early morphological and biochemical changesassociated with the subsequent development of liver cancer.Hepatic DNA damage, as determined by ³²P-postlabelling, showeda cumulative increase with time from 500 adducts/10⁸ nucleotidesat 30 days to almost 3000 adducts/10⁸ nucleotides after 180days, with little difference between strains at this time point.A significant strain difference was found in the number of adductspresent in the Fischer rats at 90 days, compared to the Wistarand Lewis strains. There was a marked strain differences inthe time to development of liver tumours. After 6 months treatment,both Wistar and Lewis rats had tumours while none were seenin the Fischer animals. After 11 months, all of the Wistar andLewis rats had developed liver carcinoma, while the Fischerrats developed liver carcinoma by 20 months. Depression in cellproliferation, relative to age-matched controls, was seen inthe livers of Fischer rats after six months of exposure to tamoxifen,in contrast to an increase in the Wistar and Lewis rats. Thisobservation is consistent with the promotion of foci to tumoursand the subsequent progression of tumours to carcinomas in thelatter two strains. These data may assist in establishing thepossible risk factors, such as extent of DNA damage and increasedliver cell proliferation, to women with long-term prophylacticexposure to tamoxifen.     

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on DNA adduct formation by a mixture of benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung

Dietary phenethyl isothiocyanate (PEITC) and a mixture of dietary PEITC and benzyl isothiocyanate (BITC) inhibit lung tumorigenesis in A/J mice induced by a mixture of the tobacco smoke carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we tested the hypothesis that inhibition of tumorigenesis by these isothiocyanates was due to inhibition of DNA adduct formation. We quantified the following pulmonary DNA adducts: N2-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl]deoxyguanosine (BPDE-N2-dG) from B[a]P; and O(6)-methylguanine (O(6)-mG) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts from NNK. Initial experiments demonstrated that there were no effects of B[a]P on NNK-DNA adduct formation, or vice versa, and established by way of a time course study the appropriate sacrifice intervals for the main experiment. Dietary PEITC, or dietary BITC plus PEITC, inhibited the formation of HPB-releasing DNA adducts of NNK at several of the time points examined. There were no effects of dietary isothiocyanates on levels of O(6)-mG or BPDE-N2-dG. These results, which are consistent with previous studies in rats and with tumor inhibition data in mice, support a role for inhibition of HPB-releasing DNA adducts of NNK as a mechanism of inhibition of tumorigenesis by dietary PEITC and BITC plus PEITC. However, the observed inhibition was modest, suggesting that other effects of isothiocyanates are also involved in chemoprevention in this model.