Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts

A further development of an HPLC method to analyze ³²P-postlabeledDNA adducts is presented. The method is based on on-line detectionof ³²P radioactivity after separation by reversed-phase chromatography.The method has an advantage in that the postlabeling mixturecan be injected directly into the HPLC system without any priorpurification, with the background radioactivity on a low level.The analysis includes the whole range of substances from orthophosphateto non-polar DNA adducts, which makes it possible to analyzenormal nucleotides and ATP together with DNA adducts. The analyticalsystem has a high reproducibility and separates complex mixturesof DNA adducts. The slightly lower sensitivity compared to theTLC method is compensated for by the possibility of injectinglarge amounts of DNA into the system without affecting the analyticalproperties. The system can be applied to different DNA adductsas well as complex mixtures of DNA adducts.     

Effect of N,N-didesmethyltamoxifen upon DNA adduct formation by tamoxifen and alpha-hydroxytamoxifen

Tamoxifen undergoes sequential metabolism to N-desmethyltamoxifen and N,N-didesmethyltamoxifen. Whereas N-desmethyltamoxifen is a major metabolite in humans, nonhuman primates, and rats, appreciable concentrations of N,N-didesmethyltamoxifen are formed in humans and nonhuman primates but not in rats. This difference in the extent of N,N-didesmethyltamoxifen formation may be important because it has been proposed that N,N-didesmethyltamoxifen inhibits the cytochrome P450 (CYP)-catalyzed alpha-hydroxylation of tamoxifen and resultant tamoxifen-DNA adduct formation. To test this hypothesis directly, we compared the extent of tamoxifen-DNA adduct formation in rats co-administered 27micromol N,N-didesmethyltamoxifen per kg body weight and either 27micromol tamoxifen per kg body weight or 27micromol alpha-hydroxytamoxifen per kg body weight daily for 7days. Female Sprague-Dawley rats treated with N,N-didesmethyltamoxifen had a 44% decrease (p >0.05) in CYP 3A2 content (the CYP isoform responsible for tamoxifen alpha-hydroxylation), an 18% decrease (p =0.010) in CYP 3A activity, and higher blood levels of tamoxifen and N-desmethyltamoxifen compared to rats treated with solvent. Total tamoxifen-DNA adduct levels were 4.1-fold higher (p <0.001) in rats given alpha-hydroxytamoxifen as compared to tamoxifen. N,N-Didesmethyltamoxifen treatment caused a 1.2-fold increase in total tamoxifen-DNA adduct levels with both tamoxifen and alpha-hydroxytamoxifen, a difference that was not significant. These results indicate that, with this experimental model, N,N-didesmethyltamoxifen does not impair the metabolism of tamoxifen to a reactive electrophile.     

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study, we compared the extent of DNA adduct formation in female Sprague-Dawley rats treated by gavage with seven daily doses of 54 micromol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague-Dawley rats.     

32P-postlabeling analysis of DNA adducts in rats during estrogen-induced hepatocarcinogenesis and effect of tamoxifen on DNA adduct level.

DNA adduct formation in the liver, pancreas, kidneys and uterus in ethynylestradiol (EE)-induced carcinogenesis and the effect of tamoxifen (TAM) on DNA adduct formation were evaluated in female Wistar JCL rats using the 32P-postlabeling method. Hyperplastic nodules were noted in the liver of all rats 4 months after the first oral administration of 0.075 mg of EE, and hepatocellular carcinoma was detected in 8.1% of rats treated with EE for 12 months. DNA adducts increased in the liver for 4 months, reaching a level of 7.3 adducts/10(7) nucleotides and decreasing thereafter. Formation of DNA adducts was also noted in the pancreas and kidney, but the adduct levels were lower than those in the liver. TAM inhibited estrogen receptors (ER) in liver tissues and completely suppressed the development of hyperplastic nodules or hepatocellular carcinoma but did not affect DNA adduct formation in the liver. In this model, therefore, EE is considered to cause mutations of hepatocytes due to DNA adduct formation without mediation by ER and to induce initiated cells to develop into hepatocellular carcinoma in the presence of ER-mediated hormonal activities.     

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.     

Development of a 32P-postlabelling method for the analysis of 2'-deoxyguanosine-3'-monophosphate and DNA adducts of methylglyoxal

A ³²P-Postlabelling assay was developed for the analysis ofadducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphatewith the 1,2-dicarbonyl compound methylglyoxal, a major mutagenin several foodstuffs, in particular, instant and brewed coffee.The ³²P-postlabelling reaction was optimized by testing variousparameters such as the kinetics of phosphorylation by T₄ polynucleotidekinase, substrate concentration-dependent labelling efficiencyand the concentration of the various ingredients of the phosphorylationreaction. The sensitivity to the 3'-monophosphate dephosphorylationactivity of nuclease P1 was also studied. Four isomeric reactionproducts were separated by HPLC, structurally characterizedand identified as 3-(2'-deoxy-ß-D-erythro-pentafuranosyl)-6,7-dihydro-6,7-dihydroxy-6-methylimidazo[2,3-b]purine-9(8H)one.The same adducts could be detected from calf thymus DNA thathad been reacted in vitro with methylglyoxal. DNA adducts wereisolated after enzymatic digestion to mononucleotides followedby nuclease P1 digestion of normal nucleotides. The total levelof methylglyoxal-DNA adducts obtained was 5.7 ± 1.7 (n= 15) adducts/10⁶ nucleotides. The ³²P-postlabelling methodwas further validated by the detection of adducts of methylglyoxalin DNA from freshly isolated and stimulated human lymphocytesexposed in vitro. The concentrations of the adducts detectedin these samples were 8.2 ± 0.9 (n = 3) adducts/ 10⁷nucleotides and 1.5 ± 0.1 (n = 3) adducts/10⁶ nucleotidesafter treatment with 1.5 and 3.0 mM methylglyoxal respectively.     

32P-Postlabelling analysis of the DNA adducts formed by aristolochic acid I and II

We report the quantitation of DNA adducts in target and non-targetorgans of male Wistar rats treated orally with five daily doses(10 mg/kg body wt) aristolochic acid I (AAI) or aristolochicacid II (AAII), the major components of the herbal drug aristolochicacid, a forestomach carcinogen In the rat. DNA adducts weredetected and analysed using the nuclease P1-enhanced variationof the Randerath 32 postlabeiling assay. The highest level ofDNA adducts formed was by AAI inthe target organ, forestomach(330 ± 30 adducts/10⁸ nucleotides), but high levels werealso observed in a non-target tissue, the glandular stomach(180 ± 15). Lower amounts of adducts were detected inliver, kidney and urinary bladder epltheliuin. With AAII thebinding Levels were generally lower than the AAII, the highestLevel of adducts being detected in kidney (80 ± 20 adducts/10⁸nucleotides) and lower levels in liver, stomach and urinarybladder epithelia. Adduct patterns similar to those in vivowere observed in two new in vitro assays. Rat faecal bacteriawere shown to be able to activate AM and AAII to reactive species,which were trapped with exogenous calf thymus DNA and analysedby postlabelling. llncuhatlon of AM and AAII in explanted ratstomach held in short-term organ culture resulted In DNA adductformation in the epithelia of both forestomach and glandularstomach. To assign the recently characterized in vitro nucleosideadducts of AII to the bisphosphate derivatives, a new ion-pairH]PLC procedure on a reversed-phase column was developed. Bymonitoring Cerenkov radiation on-line, a good separation ofAII adducts was observed, demonstrating that adducts formedin vivo were chromatographically indistinguishable with thoseformed in vitro, and previously characterized as an aristolactammoiety bound covalently to the exocydlic amino groups of deoxyadenosineand deoxyguanosine.     

Detection and characterization by 32P-postlabelling of DNA adducts induced by a Fenton-type oxygen radical-generating system.

Reactive oxygen species can give rise to numerous modifications of DNA. We have investigated the formation of such modifications using the nuclease P1 digestion method of the 32P-postlabelling procedure for the detection of DNA damage. Analysis of DNA that had been treated with a Fenton-type system of copper (or iron) ions and H2O2 resulted in the detection of up to ten discrete 32P-labelled spots, displaying chromatographic characteristics similar to aromatic adducts, on PEI-cellulose TLC. Maximum total levels equivalent to 28 adducts/10(8) nucleotides were achieved after 15 min of treatment with Cu2+/H2O2. The formation of adducts was 1.5 times greater if single-stranded rather than double-stranded DNA was employed, suggesting an intrastrand effect. Experiments with 3'-deoxyribonucleotides demonstrated that the adducts detected did not represent base modifications such as 8-hydroxydeoxyguanosine or thymidine glycols. However, treatment of specific dinucleotides (dApdG and dApdA) was found to produce two major adducts that were chromatographically identical by TLC and HPLC to the two major adducts formed in DNA. It is proposed that these species with aromatic adduct-like characteristics are the result of the intrastrand linking of specific adjacent bases in DNA.     

Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells

Samples of DNA irradiated at 405 and/or 365 nm In the presenceof 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelllngassay using three hydrolysis enzymes other than those employedpreviously. These enzymes (deoxyribonucleasel, venom phosphodiesteraseand alkailne phosphatase) liberated 3'-adducted dinudeotidemonophos phate instead of the 5'-modifled dinucleotide monophosphatenormally obtained. The first separalion chromatography (Dl)of samples irradiated in the presence of 8-MOP showed a singlespot above the origin, and the next separation (D2) resolvedthis spot into two components (spots I and II). Double irradiationexperiments in which samples of DNA were first irradiated at405 am before being irradiated at 365 nm showed that spot IIcould be transformed into spot I. The use of 6,4,4'-trimethylangelicin,which induced only photo monoadducts under UVA Irradiation,gave only spot II. These two results indicated that spots Iand II were respectively due to interstrand cross-links andmonoadducts. Dose-effect experiments showed that spots I andII were dose dependent, and low-dose irradiations permittedus to measure one interstrand cross-link and two monoadductsper 10⁸ base pairs.     

Benzo[a]pyrene-globin adducts detected by synchronous fluorescence spectrophotometry: method development and relation to lung DNA adducts in mice.

A simple synchronous fluorescence spectrophotometry (SFS) to detect benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-globin adducts is described. SFS for BPDE-DNA, which measures detached benzo[a]pyrene (B[a]P)-tetrols after acid hydrolysis of DNA, was applied for BPDE-globin adducts in B[a]P-treated C57BL/6 (B6) mice. Unlike DNA samples, globin is not measurable as such after acid hydrolysis because proteins give a background in SFS. Furthermore, proteinase incubation before acid hydrolysis of globin gave too much background even after purification to be useful in this assay. Of several purification procedures tried after acid hydrolysis (protein precipitation, elution through Sep-Pak C18, filtration, ether extraction of tetrols), the lowest background fluorescence was obtained with ether extractions of B[a] moieties. Ether phases were evaporated to dryness and the remainder dissolved in distilled water (1 ml), which was measured by SFS. Compared to DNA, somewhat milder hydrolysis conditions were optimal for globin samples (0.05 M HCl, 1.5 h, + 90 degrees C). Globin samples from B[a]P-treated mice gave a peak at the same wavelength (345 nm excitation) as the hydrolysis products of BPDE-DNA adducts, indicating B[a]P-tetrols and triols in the sample. Less than half of B[a]P measured in globin was from covalently bound BPDE. In mice injected i.p. with 1-160 mg/kg of B[a]P there was a dose-dependent increase in the amount of BPDE adducts in globin and a positive correlation with lung and liver DNA. Globin adducts were a more sensitive indicator of B[a]P exposure than DNA adducts because more globin can be used for the assay. Although both covalently and non-covalently bound BPDE in globin are detected by SFS, this method is the simplest described so far, reproducible and theoretically sensitive enough for human biomonitoring.     

Fluoranthene-DNA adducts: identification and quantification by an HPLC-32P-postlabeling method

A ³²P-postlabeling procedure for identifying and quantifyingfhioranthene (FA) - DNA adducts has been developed through modificationsof the method of Randerath and collaborators. In this modifiedprocedure, labeled adducts are separated chromatographicallyby high-pressure liquid chromatography (HPLC) and quantifiedby liquid scintillation counting. FA-modified DNA is digestedto nucleotide 3'-monophosphates and nucleotide 3'-monophosphateadducts; unmodified nucleotides are then separated from adductsusing a disposable C₁₈ cartridge. Residual unmodified nucleotides,which reduce the efficiency of ³²P-postlabeling of FA adducts,are removed by brief digestion with nuclease P1. This treatmentselectively dephospborylates unmodified nucleotide 3-monophosphates,while FA adducts are minimally affected. FA adducts are then5-phosphorylated with polynucleotide kinase and [-³²ATP. Prolongedtreatment with nuclease P1 then is employed to remove the unlabeled3-phosphate from adducted diphosphate nucleotides, followingwhich adducts are separated by HPLC and quantified by liquidscintillation counting. Postlabeled microsomally-activated FA-modifiedDNA contained adducts derived from anti- and syn-2,3-dihydroxy-l,10b-epoxy-l,2,3-trihydrofluoranthene.The identity of the major adduct of DNA-bound microsomally-activatedFA was confirmed by this HPLC-P-postlabeling method as an a/tft-2,3-dihydroxy-l,10b-epoxy-fluoranthenenucleotide adduct. Each step in the procedure was optimizedwith respect to experimental conditions, and the recovery ofadducts was determined by analysis of DNA modified with [³H]FA.In repeated analyses of 2–50 µg DNA containing 1.8adducts per 10s nucleotides, 10–15% of total DNA-bound[³H]FA was recovered as the major adduct; recovery was greaterfrom DNA containing higher levels of adducts. The reproducibilityof multiple analyses of the same sample was {small tilde}5%,and multiple analyses at different times were reproducible withinexperimental error. The limit of detection of the method was{small tilde}0.1 finol adduct, representing a binding levelof {small tilde} 3 adducts per 10⁸ nucleotides in 1 µgDNA or {small tilde}1 adduct per 1010 nucleotides in 500 µgDNA. Because the method is not limited with respect to the amountof DNA that can be subjected to analysis, the inherent sensitivityfor adduct detection can be greatly enhanced by analysis oflarger quantities of DNA.     

DNA adducts in lymphocytes and granulocytes of smokers and nonsmokers detected by the 32P-postlabelling assay

The effects of smoking and DNA adduct formation were analysed in isolated human white blood cell populations. As the white cells are composed mainly of granulocytes with a short half-life and T-lymphocytes with a half-life of several years, we isolated the lymphocytes and granulocytes of 11 smokers and 10 nonsmokers to determine any smoking-related DNA adducts by the nuclease-P1-enhanced 32P-postlabelling assay. The differences between the mean lymphocyte DNA adducts/10(8) nucleotides of 31 +/- 5.7 (SE) of smokers were significantly higher (P less than 0.05) than those in the lymphocytes 13 +/- 1.6 (SE) of nonsmokers. The total DNA adducts/10(8) nucleotides obtained from the granulocytes of smokers and nonsmokers was 9.6 +/- 1.9 and 7.6 +/- 1.9 respectively. The plasma cotinine concentrations were in good agreement with the smoking information given by the individual smokers (r = 0.847, P less than 0.001). The DNA adduct levels of the lymphocytes of the 10 smokers correlated with the plasma cotinine concentrations (r = 0.639, P less than 0.05). The variation between the results was explained by the variation among the individuals and the samples, but not by the variation in the parallel determinations. More detailed studies are needed to analyse the source of the individual variations between the smokers' adduct levels, DNA repair, and differences in the metabolism of the compounds in cigarette smoke.     

32P-Postlabelling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA

A ³²P-postlabelling method is reported for the detection of7-alkylguanines, the major adducts formed by the reaction of1,2-alkylepoxides with DNA. Calf thymus DNA was reacted in vitrowith different epoxides (ethylene oxide through octylene oxide)and digested with micrococcal nuclease and spleen phosphodiesteraseto 3'-nucleotides. The adduct enrichment was carried out byan ion-exchange method and adducts were labelled with [     

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

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

A 32P-postlabeling method for simultaneous detection and quantification of exocyclic etheno and propano adducts in DNA

A ³²P-postlabeling method is described that specifically detectsand quantifies the 1,N² adducts derived from acrolein (AdG)and crotonaldehyde (CdG) and 1,N²-ethenodexoxyguanosine (EdG)in DNA. These exocyclic adducts are potential DNA lesions causedby exposure to enals as environmental pollutants and as endogenouscompounds. This method was developed with the use of the syntheticadduct standards of these exocyclic adducts. The assay relieson HPLC for adduct enrichment prior to labeling and for quantitationand identification after labeling. The labeling efficienciesof adducts at the 1 fmol level ranged from 74 to 96%, whereasthey were only 49–60% at the 100 fmol level. This methodcan detect as low as 0.2 fmol of adduct and allows the detectionand quantitative determination of stereolsomers of AdG and CdG.The method was validated by using a sample of enzyme digestsof 180 µg calf thymus DNA spiked with 25 or 75 fmol ofadducts, which is equivalent to 5 or 15 adducts in 10⁸ nucleotides.The recovery rates of these adducts in DNA ranged from 30 to90% at the 25 fmol level and 21 to 55% at the 75 fmol level.Similar to the labeling efficiency, a greater recovery was observedwith a lower amount of adduct in DNA. Overall, this method allowsthe simultaneous identification and quantification of exocycicadducts AdG, CdG and EdG in DNA. Therefore, it provides a potentialtool for studies of the in vivo formation of exocyclic adducts.     

32P-Postlabeling and HPLC separation of DNA adducts formed by diesel exhaust extracts in vitro and in mouse skin and lung after topical treatment

Diesel exhaust extracts contain many carcinogenic compoundswhich have been shown to form polycyclic aromatic hydrocarbon(PAH)— and nitrated PAH—DNA adducts in rodent skinand lung. The aim of this study was to characterize by ³²P-postlabeling,TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formedin vitro and in vivo by diesel extracts. The diesel particleextracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes(B[b,j,k]F) and chrysene. DNA adducts were analyzed in calfthymus DNA incubated in vitro with PAHs activated by S9 mixand in skin and lung DNA from topically treated mice. The maindiesel-derived DNA adduct formed in vitro and in vivo did notco-migrate on HPLC and large TLC plates with ()-r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE)—,B[b]F—, B[j]F—, B[k]F— or chrysene—DNAadduct standards. By co-chromatography DNA adducts formed bychrysene from both in vitro and in vivo samples were identified.Nissan diesel extract containing higher PAH concentrations thanVolkswagen automobile extract formed skin DNA adducts that co-migratedwith chrysene— and anti BPDE—DNA-derived adducts.We conclude that the use of a highly sensitive ³²P-postlabelingmethod combined with HPLC improves the identification of PAHadducts formed by complex mixtures such as diesel exhaust extracts.     

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.      

32P-Postlabelling of DNA adducts formed by allyl glycidyl ether in vitro and in vivo

32P-Postlabelling analysis of allyl glycidyl ether-treated DNA after adduct enrichment on anion-exchange cartridges revealed two major and one minor DNA adducts. The major adducts were shown to originate from alkylation at N-7-guanine and N-1-adenine, respectively, while the minor adduct was at N-3-cytosine. In addition, rearrangement products of the 1-adenine and 3-cytosine adducts to N6-adenine and 3-uracil were indicated. The relative amounts of adenine, cytosine and uracil products appeared to be dependent upon conditions (in particular pH) during sample processing and analysis. When nuclease P1 was used for adduct enrichment the adenine, cytosine and uracil adducts, but not the 7-guanine adduct, were detected. The labelling efficiency of the 7- guanine adduct standard was 40-45%. Total recovery of this adduct from allyl glycidyl ether-modified DNA was 9-12%. The labelling efficiency of the 1-adenine adduct standard was 78-82%. Total recovery of this adduct from DNA was approximately 20% when using anion-exchange chromatography for adduct enrichment and 30-34% when using nuclease P1. Preliminary analysis of DNA from mice treated with allyl glycidyl ether indicated 57 times higher level of the 7-guanine adduct, per unit dose, in skin DNA (120 per 10(8) normal nucleotides) after topical application when compared to liver DNA after i.p. administration. The 1- adenine adduct could not be quantified in liver DNA (due to an interfering background product present in untreated animals) and the level of the 3-cytosine adduct was below the detection limit of the method. After topical application the level of the 1 adenine adduct in skin DNA was approximately 30 per 10(8), using either column or nuclease P1 enrichment. The 3-cytosine adduct was detected in skin, but was not quantified.      

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes,we have investigated the formation of DNA adducts by the antiestrogencompound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen(4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was used as a cofactor in microsomal activation of either4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levelsof 4.6 and 3.1x10^–8, respectively were detected by 32P-postlabeling.The DNA adduct produced by microsomal activation of 4-OH-TAMand TAM was the same. With cumene hydroperoxide (CuOOH) as thecofactor for the microsomal activation of either 4-OH-TAM orTAM, three to six DNA adducts were produced; the relative adductlevels were 8.0 and 20.6x10^–8, respectively. Comparisonof the DNA adduct patterns produced by 4-OH-TAM and TAM showedthat they were distinct However one of the DNA adducts (a) producedby microsomal activation of 4-OH-TAM using CuOOH was the sameas adduct a produced by microsomal activation of 4-OH-TAM withNADPH. Activation of 4-OH-TAM with horseradish peroxidase resultedin the formation of a single DNA adduct and a relative adductlevel of 20.7x10^–8. Rechromatography analysis of thisDNA adduct showed that it was identical to that produced bymicrosomal activation of 4-OH-TAM with NADPH and one of theadducts produced using CuOOH as the cofactor. Ten DNA adductsand a relative adduct level of 15.3x10^–8 were detectedin the liver of female Sprague-Dawley rats treated daily with20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liverof the treated animals was similar to that produced by microsomalactivation of TAM using CuOOH as the co-factor. The principalDNA adduct (no. 6) formed in the livers of rats treated withTAM was the same as the principal DNA adduct formed followingmicrosomal activation of TAM using CuOOH as a cofactor. TheDNA adduct formed following microsomal activation of eitherTAM or 4-OH-TAM using NADPH was also present as one of the adducts(1^*) formed in vivo following TAM treatment These studies demonstratethat 4-OH-TAM can be activated to form DNA adducts and thatit contributes to the formation of DNA adducts in the liverof rats treated with TAM.     

Detection of DNA adducts in human nasal mucosa tissue by 32P- postlabeling analysis

Nasal epithelium is an easily accessible tissue that is potentially useful for human biomonitoring studies aimed at evaluating exposure to airborne carcinogens. We have devised a simple technique, which causes minimum distress to the informed patient, to obtain very small but sufficient biopsies from the inferior or middle turbinate head. DNA adducts were measured by 32P-postlabeling assay in nasal mucosa of nine cigarette smokers (including two subjects who had given up smoking shortly before sampling), two former smokers and 10 non-smoker healthy donors. None of the subjects reported other recent exposures to mutagens or carcinogens. Using the nuclease P1 technique, a mean adduct level of 4.8/10(8) bases and a specific spot pattern, the diagonal radioactive zone, were found in smokers, whereas non-smokers showed a significantly lower global level of DNA adducts, i.e. 1.4/10(8) bases, and no diagonal zone. Another important result was the presence of a significant association between DNA adduct level and the number of cigarettes smoked daily. These preliminary findings suggest that the level of DNA adducts measured from biopsies of the nasal mucosa is a reliable marker of exposure to cigarette smoking and uphold its use in biomonitoring exposures to other airborne DNA binding compounds.