DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems

Using microsomal preparations from rat and human liver, we investigatedthe activation of the anti-estrogen compound tamoxifen (TMX)to form DNA adducts. Pretreatment of rats with phenobarbitalincreased DNA adduct formation by microsomal activation of TMX3- to 6-fold, depending on the cofactors used. When reducednicotinamide-adenine dinucleotide phosphate (NADPH) was usedas a cofactor in human and rat microsomal activation systems,the relative DNA adduct levels were 2.9 and 5.2 x 10^–8respectively and 1-3 TMX-DNA adducts were detected by ²³P-postlabeling;DNA adduct 1 was the same in both microsomal systems. When cumenehydroperoxide (CuOOH) was used as a cofactor, activation ofTMX produced four major DNA adducts and several minor DNA adductsin both rat and human liver microsomes; the relative adductlevels were 11.1 and 23.1 xlO^–8 respectively. TMX-DNAadducts 1, 4, 5 and 6 were similar in both human and rat microsomalsystems with CuOOH as the cofactor. The TMX-DNA adducts formedwith NADPH as the cofactor were clearly different from thoseformed with CuOOH as the cofactor, which implies that the metabolitesleading to the individual DNA adducts were different. Additionof a P450 inhibitor, either n-octylamine or     

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.     

SHORT COMMUNICATION: Production of 8-hydroxy-2'-deoxguanosine in DNA by microsomal activation of tamoxifen and 4-hydroxytamoxifen

Using rat liver microsomal preparations, we have investigatedthe activation of the anti-estrogen compound tamoxifen (TAM)and its metabolite 4-hydroxytamoxifen (4-OH-TAM) to form 8-hydroxy-2'-deoxyguanosine(8-OH-dG) in DNA. When reduced nicotinamide adenine dinucle-otidephosphate (NADPH) was used as a cofactor in microsomal activationof either TAM or 4-OH-TAM, the levels of 8-OH-dG were 3-foldhigher than in microsomes plus cofactor only. In contrast, nosignificant increase in the level of 8-OH-dG was detected inDNA samples from microsomal activation of either TAM or 4-OH-TAMwith cumene hydroperoxide as the cofactor. These results demonstratethat the microsomal activation of TAM and 4-OH-TAM to form 8-OH-dGis dependent upon the cofactor used. The addition of eitherEDTA or catalase to the activation system significantly decreasedthe formation of 8-OH-dG by TAM, but not by 4-OH-TAM. The presenceof either sodium azide, superoxide dismutase or mannitol inhibitedthe formation of 8-OH-dG by both TAM and 4-OH-TAM. Taken togetherthese findings indicate that microsomal activation of TAM and4-OH-TAM with NADPH generates reactive oxygen species whichresult in the formation of 8-OH-dG. We propose that the formationof 8-OH-dG by TAM and its metabolites may contribute to theobserved carcinogenic ffects of TAM     

SHORT COMMUNICATION: Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene

We have employed the P₁-enhanced ³²P-postlabeling procedureto detect the formation DNA of adducts in the white blood cells(WBC) of B6C3F1 mice treated by i.p. injection with benzene.Treatment twice a day with 440 mg/kg benzene for 1–7 daysresulted in the formation of one major (adduct 1) and one minor(adduct 2) DNA adduct in the WBCs of mice. The same DNA adductpattern was also found in the bone marrow (BM) of benzene treatedmice. The relative adduct levels were dependent upon both benzenedose from 100–440 mg/kg and treatment time from 1 to 7days. The relative adduct levels ranged between 0.11 and 1.33adducts in 10⁷ nucleotides for WBCs and 0.16–1.21 adductsin 107 nucleotides for BM. Following treatment with benzene,the levels of DNA adducts formed in WBCs were significantlycorrelated with the levels of DNA adducts formed in BM (r² =0.97, P <0.001). Our results suggest that measurement ofDNA adducts in WBCs may be an indicator of DNA adduct formationin BM following BZ exposure.     

Detection of dopamine DNA adducts: potential role in Parkinson's disease

Oxidation of catecholamines may lead to the formation of O-semiquinonesand o-quinones in catecholaminergic brain tissues, and thesereactive molecules may form DNA or protein adducts. In thisstudy, cultured cells were treated with dopamine (DA) for ²⁴h and ³²P-postlabeling was used to detect DA-DNA adducts. InHL–60 cells, 250µM DA induced 8.5 DNA adducts/108nucleotides; adduct formation was dose-dependent up to 500 µMDA. Addition of H2O2 increased the relative adduct levels ⁷-to 13-fold, but no adducts were detected when DA and ascorbicacid were added simultaneously. In human glioblastoma cell linesU87, U251, SF-763 and SF-767, 1000 µM DA produced 0.98–2.31adducts/108 nucleotides. These results suggest that the formationof DNA adducts by DA may contribute to the development of certainneurodegenerative diseases such as Parkinson's disease.     

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

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

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

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

Analysis of polychlorinated biphenyl-DNA adducts by 32P-postlabeling

Previous studies reported that metabolic activation of certainpolychlorinated biphenyls (PCBs) resulted in binding to protein,RNA and DNA fractions. However, the formation of DNA adductshas not been demonstrated nor have methods been optimized forthe detection of such adducts. In the present study we investigatedactivation and binding to DNA of lower chlorinated biphenylsusing ³²P-postlabeling. The incubation of 2-chloro-, 3-chloro-,3, 4-dichloro- and 3, 4, 5-trichlorobiphenyl with calf thymusDNA and liver microsomes from rats treated with pheno-barbitaland 3-methylcholanthrene, followed by oxidation with a peroxidase,produced 1–3 major adducts. Reaction of deoxyguanosine3'-monophosphate with metabolites of the congeneric chlorinatedbiphenyls produced adducts with similar chromatographic mobilityas those with DNA, suggesting that guanine was the preferentialsite of attack. Furthermore butanol and nuclease P1 enrichmentsshowed different adduct recoveries, depending upon the the chloro-biphenyl.Adducts derived from incubations with mono-chlorobiphenyls wererecovered 2- to 3-fold higher with butanol, while the recoveryof di- and tri-chlorobiphenyl adducts was 5- to 7-fold higherwith nuclease P1. DNA adducts formed during the metabolism of3, 4-dichlorobi-phenyl were reduced by the sulfur nucleophiles,glutathione and N-acetyl-L-cysteine, suggesting that reactivesemiquin-one(s) or quinone(s) are involved. In contrast, theaddition of superoxide dismutase increased adduct formation,suggesting that the quinone metabolites are responsible forthe major adducts formed. Our results are consistent with thehypothesis that lower chlorinated biphenyls are metabolicallyactivated to electrophilic quinoid species which bind to DNA.     

Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite

Exposure to pentachlorophenol (PCP) strongly intensifies theformation of mouse hepatic DNA adducts elicited by oral administrationof tamoxifen (TAM), as previously shown by ³²P-postlabeling.To explain this effect, PCP was proposed to interfere with thedetoxication by sulfate conjugation of an as yet unidentifiedhydroxylated proximate TAM metabolite. A comparison of the presentand earlier results shows that the hepatic TAM adduct patternin female ICR mice depended on the route of administration ofTAM (120 µmol/kg), with oral administration primarilyeliciting formation of more polar adducts (termed group I adducts),while after i.p. administration less polar adducts (group II)predominated over group I adducts by a factor of 17.5. All theseadducts were also formed in female Sprague–Dawley ratsafter i.p. dosing with TAM, but total adduct levels were 3.5-to 5-fold higher than in mice. After four daily i.p. treatments,TAM adducts accumulated in mouse liver DNA in a non-linear fashion.Adduct levels were 30–50 times lower in mouse kidney andlung than in liver. The phenolic metabolite 4-hydroxy TAM (120µimol/kg) exclusively led to formation of polar (groupI) hepatic adducts, and this process was stimulated 8-fold bycoadministration of PCP (75 µimol/kg). Co-administrationof PCP with the parent compound led to an 11-fold enhancementof group I adduct formation; simultaneously, levels of groupII adducts were suppressed 6-fold. Another inhibitor of sulfateconjugation, 2,6-dichloro-4-nitrophenol, unlike PCP, had noeffect on group I adducts, but it reduced group II adduct formation2.2-fold. The PCP metabolite 2,3,5,6-tetrachlorohydroquinone(75 µimol/kg) did not significantly affect any major TAMadduct, suggesting that PCP itself was the active compound.Similar to group II TAM adducts, the formation of hepatic safrole–DNAadducts was inhibited in female ICR mice by both sulfotransferaseinhibitors, consistent with the proposal that metabolic     

Lack of bioavailability of dichlorobenzidine from diarylide azo pigments: molecular dosimetry for hemoglobin and DNA adducts

The hypothetical release of 3, 3'-dichlorobenzidine (DCB) fromtwo insoluble DCB-based diarylide azo pigments and from a solubleazo dye was investigated in female Wistar rats after a 4 weektreatment with 0.2% (w/w) Colour Index Pigment Yellow 13 (PY13)or 0.2% (w/w) Colour Index Pigment Yellow 17 (PY17) in the dietor 0.06% (w/v) Colour Index Direct Red 46 (DR46) in the drinkingwater. Steady-state DCB-hemoglobin adduct levels were determinedby GC/MS with negative chemical ionization as well as DCB-DNAadduct levels in the liver by ³²P-postlabelling and comparedwith the respective adduct levels obtained in animals aftertreatment for 4 weeks with 0.00024, 0.0012 or 0.006% (w/v) DCBin the drinking water. A dose-proportional increase in adductlevels from 8.1 ng/g hemoglobin and 2.6 ng/g DNA (relative adductlevel, RAL, 3.3x10–9) to 160 ng/g hemoglobin and 45.4ng/g DNA (RAL 56.1 x10–9) was observed in the DCB-treatedrats. In rats treated with DR46 total adduct levels of 17.7ng/g hemoglobin and 5.2 ng/g DNA (RAL 6.4x10–9) were determined.No hemoglobin or DNA adducts were found in rats treated withPY17 in the diet, at a limit of detection of 0.1 ng/g hemoglobinand 0.08 ng/g DNA (RAL 0.1x10–9). In animals treated withPY13 in the diet no adducts or only minimal amounts slightlyabove the limit of detection could be identified. Taking intoconsideration that PY13 was contaminated with 0.02% of the respectivesoluble monoazo compound, it is concluded that the small amountsof DCB detected have been released from the contaminating solublemonoazo compound and not from insoluble PY13. The results ofthe present study demonstrate the lack of bioavailability ofDCB from the diarylide azo pigments PY17 and PY13.     

Reactivity of benzo[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxyguanosine adducts

Polycyclic aromatic hydrocarbon (PAH) o-quinones are productsof the dihydrodiol dehydrogenase-catalyzed oxidation of trans-dihydrodiolswhich are proximate carcinogens. The PAH o-quinones are highlyreactive molecules and have the potential to alkylate DNA. Inthis study, the reactivity of [³H](+/–)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene([³H](+/–)-anti-BPDE), [³H]benzo[a]pyrene-7,8-dione ([³H]BPQ)and [³H](+/–)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ([³H](+/–)-B[a]P-diol) with DNA were compared.(+/–)-anti-BPDE reacted equally well with native, deproteinatedand deproteinated/sheared calf thymus DNA. In each case DNAadducts were formed which upon digestion to deoxyribonucleosidescomigrated on reverse-phase (RP)-HPLC with adducts synthesizedby reacting (+/–)-anti-BPDE with oligo-p(dG)₁₀. (+/–)-anti-BPDEalso reacted with plasmid (pGEM-3) DNA to yield multiple adductsone of which comigrated with the (+)-anti-BPDE-deoxyguanosineadduct. Under identical conditions [³H]BPQ reacted preferentiallywith native calf thymus DNA but displayed low reactivity withdeproteinated and deproteinated/sheared calf thymus DNA. RP-HPLCanalysis of deoxyribonucleoside—BPQ adducts indicatedthat the predominant adduct formed comigrated with a standardsynthesized by reacting BPQ with oligo-p(dG)₁₀. BPQ also reactedwith pGEM-3 DNA to yield multiple adducts one of which comigratedwith the BPQ—deoxyguanosine adduct. Reactions between[³H]BPQ and poly(dA), poly(dT), poly(dC) and oligo-p(dG)₁₀ indicatedthat BPQ preferentially formed deoxyguanosine adducts. In thisstudy, [³H]BPQ and [³H](+/–)-anti-BPDE covalently labelednative calf thymus DNA to an equal extent, however, less [³H]BPQwas recovered as deoxyguanosine adducts. By contrast, no covalentmodification of calf thymus DNA, pGEM-3 DNA or oligonucleotideswas observed with [³H](+/–)-B[a]P-diol. These studiesindicate that BPQ has the potential to be genotoxic in vitro;that reactivity is heightened in the presence of protein orcircular DNA and that the major adduct formed is a deoxyguanosineadduct.     

Identification of C8-modified deoxyinosine and N2-and C8-modified deoxyguanosine as major products of the in vitro reaction of N-hydroxy-6-aminochrysene with DNA and the formation of these adducts in isolated rat hepatocytes treated with 6-nitrochrysene and 6-aminochrysene

Since 6-nitrochrysene and 6-aminochrysene have shown activityin carcinogenicity bioassays, we have begun an investigationof their metabolic activation pathways and the nature of thecarcinogen—DNA adducts that may be formed. N-Hydroxy-6-aminochrysene(N-hydroxy-AC), a candidate proximate or ultimate carcinogenand the highest polycyclic N-hydroxy arylamine homolog studiedthus far, was prepared by direct chemical synthesis and characterizedby ¹H-n.m.r. spectroscopy. Its rate and extent of reaction withDNA in vitro was 20–30 nmol bound/mg DNA/30 min, whichis 2–10 times greater than has been reported for severalother carcinogenic N-hydroxy arylamines. Three major amino-chrysene-nucleosideadducts were detected in enzymatic hydrolysates of this N-hydroxy-AC-modifiedDNA, and these were isolated and identified by mass and ¹H-n.m.r.spectroscopy as N-(deoxyinosin-8-yl)-6-aminochrysene, 5-(deoxy-guanosin-N²-yl)-6-aminochrysene,and N-(deoxyguanosin-8-yl-6-aminochrysene. These adducts accountedfor 32%, 28%, and 22% respectively, of the total DNA adductsformed. We hypothesize that the deoxyinosine adduct is derivedfrom spontaneous oxidation of the corresponding deoxyadenosineadduct prior to or during DNA isolation and adduct preparation.DNA isolated from Sprague-Dawley rat hepatocytes which had beentreated with [³H]6-aminochrysene or [³H]6-nitrochrysene containedup to 12 pmol adducts/mg DNA (4 adducts per 10⁶ nucleotides).High performance liquid chromatography (h.p.l.c.) analyses ofenzymatic hydrolysates of this DNA indicated that the majorproducts formed co-chromatographed with the C8-deoxyinosineand C8-deoxy-guanosine adducts. N-(Deoxyinosin-8-yl)-6-aminochryseneand N-(deoxyinosin-8-yl)-6-aminochrysene accounted for 45% and30% respectively, of the total DNA adducts formed in these cells.The preferential modification of deoxyadenosine by N-hydroxy-6-aminochryseneand the apparent facile oxidation of this adduct to a deoxyinosinederivative is thus far unique among the reactions of N-hydroxyarylamineswith DNA and would not be predicted on the basis of reactivityalone.     

32P-Postlabelling analysis of aromatic DNA adducts in human oral mucosal cells

Exfoliated mucosal cells were collected from the oral cavityof three groups at high risk for oral cancer: Indian betel nutchewers, Filipino inverted smokers (burning end of cigar inmouth) and Indian Khaini tobacco chewers. DNA was extractedfrom these samples, as well as from samples of exfoliated cellsof Canadian non-smoking controls. DNA was analyzed for the presenceof aromatic DNA adducts using ³²P-postlabel-ling analysis. Fivechromatographically distinct adducts were found in samples fromboth the high risk groups and the nonsmoking controls. Individualadducts were detectable in 30–95% of samples, dependingon the adduct and population group. Estimated levels of specificadducts ranged from non-detectable (prevalence relative to normalnucleotides < 1 x 10^-9) to occasionally > 1x 10^-7. Noadducts were found in high risk groups which did not also appearin control subjects.     

DNA adducts of the ubiquitous enviromnental contaminant cyclopenta[cd]pyrene

The bioactivation of cydopenta[cd]pyrene (CPP) was investigatedto determine the major DNA adduct-forming metabolite(s) of thiswidespread environmental contaminant and suspect carcinogen.DNA adducts were analyzed by ³²P-postlabeling. Four major andat least seven minor adducts formed when CPP was incubated withcalf thymus DNA in the presence of rat liver microsomal systems.P450 subfamilies IA and IIB both activated CPP as microsomesfrom either phenobarbital- or ß-naphthoflavone-treatedrats produced quantitatively similar and qualitatively identicaladducts. When the epoxide hydrolase inhibitors, 1,1,1-trichloropropene-2,3-oxideor cydohexene oxide were added to the incubatlons, binding increased2.5- to 4-fold, suggesting epoxidation as a mechanism of adductformation in vitro. Sprague-Dawley rats were killed 1,3,7,18,45and 80 days postdosing i.p. with 50 mg/kg CPP. In all tissuesanalyzed, four major and several minor qualitatively Identicaladducts were produced. Binding was highest and most persistentIn lung followed by heart, white blood cells (WBCs) and liver.CPP adducts were detectable at doses from 1 µg/kg to 50mg/kg. Rat lung DNA adducts were cochromatographed with standardizeddeoxyguanosine and deoxyadenosine adducts produced by reactionof CPP-3,4-epoxide in vitro. All rat lung adducts comigratedwith the deoxyguanosine adducts but one was clearly deoxyadenosinederived. Mouse skin DNA adducts from NIH Swiss mice and mouselung DNA adducts from B6C3F1 mice were also analyzed. All adductsfrom either mouse tissue comigrated with rat lung DNA adducts,suggesting CPP-3,4-epoxide was also the major DNA adduct formingspecies in the mouse. CPP-3,4-epoxlde has been suggested tobe the key mediator of the biological activities of CPP. Evidencepresented here strongly suggests CPP-3,4-epoxlde as the majoradduct-forming species of CPP as catalyzed in vitro by rat liverpreparations known to mediate the mutagenic activation of CPP,in the rat in vivo, and in mouse skin and lung, two tissueswith known sensitivity to CPP tumorigenicity.     

32P-Postlabeling analysis of liver DNA adducts in rats chronically fed a choline-devoid diet

Liver DNA, obtained at various time intervals from rats chronicallyfed a choline-devoid diet, was analysed for the presence ofaromatic or alkyl adducts by the ³²P-postlabeling assay. Alkyladducts were not detected. Aromatic DNA adduct lesions wererevealed, but only at levels (1 adduct per 0.5–3x10⁹ nucleotides)which are at the limits of the extremely high sensitivity ofthe method used, levels which remained constant throughout theperiod of feeding. Thus, contamination of the total environmentof the animals with chemical carcinogens does not appear tobe responsible for the genesis of the hepatocellular carcinomasthat develop in rats chronically fed a choline-devoid diet.The diet, therefore, either acts as a complete carcinogen, orpromotes the evolution to cancer of endogenous, ‘spontaneously’initiated liver cells.     

CARCINOGENSIS: Formation of DNA adducts by the co-mutagen norharman with aromatic amines

Norharman, widely distributed in our environment, is alone notmutagenic to Salmonella typhimurium TA98 and TA100 either withor without S9 mix, but becomes mutagenic to S. Typhimurium TA98with S9 mix when non-mutagenic aromatic amines like anilineor o- or m-toluidine are added. Thus norharman has been calleda ‘co-mutagen’. In the present study we examinedwhether or not DNA adducts are formed in DNA of S.typhimuriumTA98 by treatment with norharman and aromatic amines using ³²P-post-labelinganalysis under modified adduct intensification conditions. Whena sample of norharman (8 mg) and aniline (4 mg) was incubatedwith 4 ml of overnight culture of S.typhimurium TA98 in thepresence of 20 ml S9 mix for 6 h at 37°C, three adduct spotswere detected at a total relative adduct labeling (RAL) of 10.8± 2.27/10⁸ nucleotides. Under the same conditions, amixture of norharman (8 mg) and o-toluidine (4 mg) yielded threeadduct spots at a RAL of 3.74 ± 1.71/10⁸ nucleotides.With a combination of norharman and m-toluidine, a single adductspot was seen at a RAL of 0.04 ± 0.01/10⁸ nucleotides.In contrast, norharman with p-toluidine did not produce adductspots. Furthermore, neither norharman nor the aromatic aminesthemselves gave any evidence of adducts. Thus DNA adduct formationby norharman with aromatic amines correlates with the co-mutagenicaction of norharman in S.typhimurium TA98.     

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

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

Persistence, gestation stage-dependent formation and interrelationship of benzo[a]pyrene-induced DNA adducts in mothers, placentae and fetuses of Erythrocebus patas monkeys

Since DNA adducts have been detected in the placentae of pregnantwomen who smoke cigarettes, the importance of these adductsas biomarkers of fetal exposure and risk has been evaluatedusing a non-human primate as a model. Pregnant Erythrocebuspatas monkeys on days 50, 100 or 150 of gestation (term = 160± 5 days) were treated once with 5–50 mg/kg benzo[a]pyrene(B[a]P), p.o. Fetuses were removed by Cesarean section 1–50days after treatment and analyzed for DNA adducts by the nucleaseP₁ version of the ³²P-postlabeling method. B[a]P induced highlevels of DNA adducts in all fetal organs, placentae and maternallivers in all three trimesters of gestation. DNA adduct levelswere higher in mid-gestation compared to early and late gestation.The major adduct detected was 10ß-(deoxyguanosin)-N²-yl-7ß,8     

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene(N-OAc-AAF) have previously been shown to induce dose-dependentDNA strand breaks in primary hepatocytes from mice and rats.In an attempt to determine the relationship between the extentof DNA strand breaks and the formation of specific DNA-carcinogenbound adducts in murine liver, the capability of N-OH-AAF andN-OAc-AAF to induce both DNA single strand breaks and adductformation in in vivo and in primary hepatocytes was measured.N-OH-AAF induced a low level of DNA damage in F344 rats (10mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment.The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene(Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene(Gua-C8-AF) 34% versus 67% and Mguanin-N²-yl)-2-acetylaminofluorene(Gua-N²-AAF) 11% versus 10%, respectively, for rat and mouseliver. An additional unknown adduct (12%) was detected in mouseliver. Dose dependent DNA binding and formation of individualDNA adducts were observed in rat and mouse primary hepatocytesfollowing 1 h exposure to [ring-³H]-N-OH-AAF (0.1-20 µM)and [ring-³-N-OAc-AAF (5–20 /M). The patterns of DNA adductsin mouse and rat primary hepatocytes exposed to N-OH-AAF andN-OAc-AF were similar to those obtained in liver following invivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon(10^–4M) completely inhibited DNA damage induced by N-OH-AAFin mouse and partially in rat hepatocytes while DNA damage causedby N-OAc-AAF was only partially inhibited by paraoxon (10^–4M) in both species. Parallel experiments showed that paraoxon,at low concentration (10^– M), did not alter either thelevel of DNA binding or the pattern of adduct formation in rathepatocytes treated with N-OH-AAF (20 µM). However, at10^–4 M paraoxon partially blocked DNA binding (60%) andthe formation of Gua-C8-AAF (95%) and Gua-N²-AAF (80%) whileGua-C8-AF was increased twofold. In mouse hepatocytes paraoxonpretreatment (10^–4M) inhibited the formation of Gua-C8-AFby 70% following exposure to N-OH-AAF (20 µM). Gua-C8-AAFand Gua-N²-AAF were also inhibited but only at 10^–4M paraoxon.Paraoxon (10^–6 and 10^–4 M) pretreatment induceddosedependent partial inhibition of the covalent binding ofN-OAc-AAF to rat DNA and the formation of all guanine adducts.In the mouse, paraoxon (10^–6 and 10^–4 M) inhibitedthe formation of Gua-C8-AF while it increased Gua-C8-AAF. Theseresults indicate that a positive correlation exists betweenthe extent of DNA strand breaks and the formation of eitherGua-C8-AAF or Gua-C8-AF.     

32P-postlabeling analysis of non-radioactive aromatic carcinogen -- DNA adducts

A newly developed enzymatic ³²P-postlabeling method was appliedto the analysis of DNA's containing non-radioactive arylamine,arylamide, and polycyclic aromatic hydrocarbon adducts. DNAreacted in vitro with N-hydroxy-2-amino-fluorene, N-acetoxy-2-acetylaminofluorene,and 7ß,8-di-hydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene,respectively, as well as DNA preparations from the livers ofrats treated with N-hydroxy-2-acetylaminofluorene and benzo[a]pyrene,respectively, were enzymatically digested to deoxyribonudeoside3'-monophosphates, which were then converted to [5'-³²P]deoxyribonucleoside3',5' -bisphosphates by T4 polynucleotide kinase-catalyzed [³²P]phosphatetransfer from [-³²P]ATP. The ³²P-labeled nucleotides were resolvedby anion-exchange t.l.c. on polyethylenelmine-cellulose anddetected by autoradiography. Aromatic adduct nucleotides werefound to be retained at the origin in aqueous electrolyte solutions,but to migrate as distinct spots in solvents containing 7–8.5M urea. Advantage was taken of this observation to remove ³²P-labelednormal DNA nudeotides from adduct nudeotides. This purificationenabled the detection of a single adduct in 10⁷–10⁸ normalnucleotides. The method appears applicable to the ultrasensitivedetection of a large number of carcinogen - DNA adducts of diversestructure without requiring radioactive carcinogens or specificantibodies.