Presence of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) in human tissues

One of the mutagenic and carcinogenic heterocyclic amines (HCAs),2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), is presentin cooked foods and we are chronically exposed to this compoundin our daily life. To study the role of HCAs in human carcinogenesis,we analyzed MelQx-DNA adducts in 38 DNA samples obtained fromsurgical and autopsy specimens by the ³²P-postlabeling methodunder adduct-intensification conditions with the modificationof additional digestion with nuclease P1 and phosphodiesteraseI after ³²P-labeling at 5' -hydroxyl termini. This modified³²P-postlabeling method can detect N²-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline 5'-monophos-phate (5'-pdG-C8-MeIQx) at levelsdown to 1/10¹⁰ nucleo-tides. The DNA samples from colon andrectum surgical specimens and a kidney taken at autopsy werefound to contain an adduct spot corresponding to that of standard5'-pdG-C8-MeIQx on TLC at levels of 14, 18 and 1.8 per 10¹⁰nucleotides, respectively. Each adduct spot was extracted fromTLC and identified to be 5'-pdG-C8-MeIQx by HPLC. Thus, MelQx-DNAadducts actually exist in human tissues and this adduct formationmay be involved in human cancer development.     

Identification of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f)quinoxaline 3',5'-diphosphate, a major DNA adduct, detected by nuclease P1 modification of the 32P-postlabeling method, in the liver of rats fed MeIQx

The carcinogenic heterocyclic amine 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline(MeIQx) is widely distributed in cooked foods. The nucleaseP1 method increased the sensitivity of the standard ³²P-postlabelinganalysis about 1000-fold for detection of MeIQx-DNA adducts.The recovery of MeIQx-DNA adducts by the nuclease P1 methodwas determined to be about 50% using liver DNA of a rat treatedwith [¹⁴C]MeIQx intragastrically. By the nuclease P1 methodfive adducts were detected in the liver DNA of rats fed MeIQxand two of them, including the most abundant one, were identifiedas MeIQx-deoxyguanosine adducts by comparison with the adductsformed in in vitro reactions of N-acetoxy-2-amino-3,8-dimethylimidazo[4,5-f)quinoxalinewith the four 2'-deoxyribonucleotides. The most abundant adductin vivo was identified as N²-(deoxyguanosin-8-yl)-MeIQx 3',5'-diphosphate(3',5'-pdGp-C8-MeIQx). MeIQx-DNA adduct levels in human tissuescould be determined by the nuclease P1 modification of the ³²P-postlabelingmethod in combination with HPLC, and thus provide informationon the roles of MeIQx in human carcinogenesis.     

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

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

Altered fidelity of a nucleic acid modifying enzyme, T4 polynucleotide kinase, by safrole-induced DNA damage

Mouse liver DNA adducted with metabolites of the spice constituentsafrole (1-allyl-3, 4-methylenedioxybenzene), when analyzedvia the bisphosphate version of the ³²P-postlabeling assay,exhibits two major adducts, which had been previously identifiedas N²-(trans-isosafrol-3' -yl)2' -deoxyguanosine 3', 5'-bisphosphate(adduct 1) and N²(safrol-1'-yl)2'-deoxyguanosine 3', 5'-bisphosphate(adduct 2). However, analysis of the same DNA preparation bythe dinucleotide/monophosphate version of the assay gave twoadditional spots on PEI-ceihilose TLC whose nature was clarifiedin the present study. Several enzymes (T4 polynucleotide kinase,nuclease P1, venom phosphodiesterase and spleen phosphodiesterase)were utilized to hydrolyze these compounds, and the productsco-chromatographed on PEI-cellulose thin layers with radiolabeledand non-radioactive nucleotides of known structure. The additionalspots were found to be adducted dinucleotides carrying ³²P-labelat both the 5'- and 3'-hydroxyls. T4 polynucleotide kinase-catalyzed3'-phosphorylation was highly specific in that only dinucleosidemonophosphate derivatives of adduct 1, with an unmodified purinein the 3'-position, were susceptible to both 5'- and 3'-phosphorylationby the enzyme. Thus, the structures of the two additional ³²P-labeledsafrole derivatives were pX₁pAp and pX₁pGp where X₁ denotesN²-(trans-isosafrol-3'-yI)2'-deoxyguanosine. The official nameof T4 polynucleotide kinase, ATP: 5'-dephosphopolynucleotide5'-phosphotransferase (EC 2.7.1.78 [EC] ), denotes the specific actionof this enzyme as a 5'-phosphokinase. Although the enzyme has3'-phosphatase activity at acidic pH, no 3'-kinase reactionhas been previously reported. Possible implications for chemicalcarcinogenesis of the finding that carcinogen - DNA adductscan specifically alter the fidelity of protein-nucleotide interactionsare discussed.     

O6-substituted-2'-deoxyguanosine-3'-phosphate adducts detected by 32P past-labeling of styrene oxide treated DNA

³²P post-labeling of DNA reacted with styrene oxide resultedin the detection of six adducts. In order to determine whichof these corresponded to modification at the O⁶ position ofguanine, O⁶-substituted styrene oxide-deoxyguanosine–-3'–monophosphatederivatives were synthesized. The two synthetic isomers werepurified by HPLC and the structures were confirmed by mass spectrometryand ¹H NMR. ³²P post-labeling and co-chromatography with theDNA-styrene–7, 8–oxide reaction products resultedin the assignment of adduct number 4 as O⁶hydroxy-2-phenylethyl)-2'-deoxyguanosine-3',5'-bisphosphate and adduct number 5 as O⁶-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3',5'-bishosphate.     

Formation of DNA adducts by the food mutagen 2-amino-3,4,8-trimethyl-3H-imidazo [4,8-f]quinoxaline (4,8-DiMeIQx) in vitro and in vivo. Identification of a N-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx adduct

The covalent binding of the mutagenic N²-hydroxy metaboilteof the food mutagen 2-amino-3,4,8-trimethyl-3H-lmldazo[4,5-f]quinoxaline(4,8-DiMeIQx) to 2'-deoxy-nudeosides and DNA was investigatedin vitro and in vivo. N²-Hydroxy-4,8-DiMeIQx reacted to a smallextent spontaneously with 2-deoxyguanosine. However, acetylatlonof N²-hydroxy-4,8-DiMeIqx with acetic anhydride to form theN²-acetoxy derivative prior to reaction with 2-deoxyguanosineresulted in much higher yield of adduct. N²-Acetoxy-4,8-DiMeIQxdid not form adducts with 2'-deoxy- adenosine, 2'-deoxycytldlneor 2'-deoxythymldlne. The adduct formed between the N metaboliteof 4,8- DiMeIQx and 2-deoxyguanosine was analysed by mass spectrometryand NMR spectroscopy and the structure of the adduct was shownto be N²-Acetoxy-4,8-DiMeIQx. N²-Acetoxy-4,8-DiMeIQx. reactedwith calf thymus DNA and formed a covalently bound 4,8-DiMeIQxresidue, which could not be removed by repeated precipita tionsor solvent extractions. The 4,8-DiMeIQx-DNA was hydrolysed enzymaticallywith nuclease P1/acid phosphat ase and HPLC analysis showedthat 70% of the bound mutagen was recovered as N²-Acetoxy-4,8-DiMeIQx.An additional minor adduct accounting for     

32P-Postlabeling analysis of adducts generated by peroxidase-mediated binding of N-hydroxy-4-acetylaminobiphenyl to DNA

³²P-Postlabeling analysis of the bisphosphate derivatives wasconducted to characterize the DNA adducts generated from theperoxidase-mediated activation of N-hydroxy-4-acetylaminobiphenyl(N-OH-AABP). Autoradiography of the D1 chromatogram of the postlabeledDNA hydrolysate revealed a major adduct (adduct 1) that migratedat R_f 0.15. An adduct with similar chromatographic characteristicswas also obtained by postlabeling the products generated bychemical interaction of: (i) 2', 6'-dichloro-benzoyloxy-4-acetylaminobiphenylwith the 3'-monophosphate of deoxyguanosine, and (ii) N-acetoxy-4-acetylamino-biphenyl(N-OAc-AABP) with calf thymus DNA. The adduct derived from chemicalreaction exhibited the same mobilities on two-dimensional TLCas that obtained from the peroxidase-mediated DNA binding ofN-OH-AABP. Moreover, on HPLC analyses, these bisphosphate derivativesexhibited identical retention times, suggesting that structurallythey might be the same. Furthermore, adduct 1 was insensitiveto digestion with nuclease P1. In addition to adduct 1, anotherminor adduct (adduct 2) was also detected in the peroxidase-mediatedDNA binding of N-OH-AABP. The adduct 2 in D1 exhibited an R_fof 0.66. Adduct 2 was also observed in the DNA sample chemicallyinteracted with N-OAc-AABP. Both these adducts retained theacetyl moiety, which was confirmed by the presence of radioactivityin the hydrolysate of DNA derived by interaction with N-OAc-[¹⁴C-acetyl]AABP(labeled at the N-acetyl group). Based on proton NMR and MSanalyses of the 5'-phospho analogs of adducts 1 and 2, the structuresof these have been identified as 3-(deoxyguanosin-N²-yl)-4-acetylaminobiphenyl(dG-N2-AABP) and N-(deoxyguanosin-8-yl)-4-acetylaminobiphenyl(dG-C8-AABP). Analyses of the DNA samples obtained from humanuroepithelial cells following exposure to N-OH-AABP revealedprimarily the non-acetylated derivative N-(deoxyguanosin-8-yl)-4-aminobiphenyl(dG-C8-ABP) with trace amounts of dG-N2-AABP. These resultssuggest that in the target cells for 4-aminobiphenyl carcinogenesis,the prevalence of the peroxidase mediated activation reactionof N-OH-AABP is relatively minor compared to the acetyltransferasepathway.     

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.     

32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female Sprague-Dawley rats

The ³²P-postlabeling technique was used to qualitatively establishthe pattern of DNA adduct formation in mammary tissue and liverfollowing administration of 1-nitropyrene to female Sprague-Dawleyrats. 1-Nitropyrene (100 mg/kg b.w.) was administered by gavagein trioctanoin and the rats were sacrificed 24 h later. DNAwas isolated from mammary fat pads and liver, enzymaticallyhydrolyzed to deoxy ribonucleoside-3'-monophosphates and thenconverted to [5'-³²P]3',5'-bisphosphates. The polyethyleneimine-cellulose(PEI-cellulose) TLC ³²P-fingerprints revealed the presence ofmultiple putative adducts in the mammary fat pads and in thelivers. To investigate the role of nitroreduction in the formationof these adducts, calf thymus DNA was incubated with [³H]1-nitropyrenein vitro in the presence of xanthine oxidase. The DNA was isolatedand analyzed by the ³²P-postlabeling technique. A major adductspot was detected and confirmed as N-(deoxyguanosin-8-yl)-1-amino-pyrene.This adduct cochromatographed with a minor in vivo adduct ofDNA obtained from mammary fat pads and livers. However, themajor adducts detected in vivo did not appear to originate fromsimple nitroreduction of 1-nitropyrene. The results of thisstudy suggest that other metabolic pathways, such as ring oxidation,or ring oxidation followed by nitroreduction, may be responsiblefor the putative 1-nitropyrene—DNA adducts observed inmammary fat pads and livers of female Sprague-Dawley rats.     

DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilicand a neurotoxic metabolite of o-tolyl phosphates. In a previouspaper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide is mutagenic in Salmonella typhimurium TA100 and formsDNA adducts in incubations with nucleotides, nucleosides andisolated DNA. In the present study we compare DNA adduct formationusing ³²P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide-treated bacteria (S.typhimurium TA100) and hepatomacells with DNA adducts formed in liver, kidney, lung and heartof tri-o-tolyl phosphate-exposed Fischer 344 male rats. In bothbacteria and hepatoma cells two DNA adducts could be detectedafter treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide. The minor adduct co-chromatographed with syntheticN³-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling.The major DNA adduct was a cytidine adduct, most likely N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate. Male Fischer 344 rats were treated orallyfor 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNAwas isolated from liver, kidney, lung, heart, brain and testes1,4,7 and 28 days after giving the last dose. Analysis by ³²P-postlabellingrevealed that two adducts were present in the DNA isolated fromliver, kidney, lung and heart on the first day after givingthe last dose; DNA adducts were not detected in the brain andtestes. The adduct pattern after in vivo treatment with tri-o-tolylphosphate was identical with that found in bacteria and hepatomacells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin2-oxide, the major adduct being N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate and the minor N³-(o-hydroxybenzyl)deoxyuridine3' monophosphate. Both DNA adducts persisted in the lungs forthe entire observation period, whereas in the kidney only thecytidine adduct could be detected 28 days after the last doseof tri-o-tolyl phosphate. In liver and heart the adducts weredetectable only on the first day after completion of the treatment.The results indicate that in addition to the well establishedneurotoxicity, some o-tolyl phosphates may have a carcinogenicpotential.     

The 32P-post-labeling method in quantitative DNA adduct dosimetry of 2-acetylaminofluorene-induced mutagenicity in Chinese hamster ovary cells and Salmonella typhimurium TA1538

The usefulness of the ³²P-post-labeling/t.l.c. method for quantitativeDNA adduct dosimetry was evaluated. 2-Acetylaminofluorene (2-AAF)-DNAadducts from three systems were characterized qualitativelyand quantitatively by the ³H-radiolabeled technique with subsequentanalysis by h.p.l.c. (pre-labeling method) and by the ³²-post-labelingmethod. Both methods showed N-acetoxyacetylaminofluorene (N-OAc-AAF)reaction products with calf thymus DNA were predominantly N-(deoxyguanosm-8-yl)-2-acetylaminofhiorene(dG-C8-AAF) with some N-(deoxyguanosin-8-yl)-2-amino-fluorene(dG-C8-AF) and N-(deoxyguanosin-N²-yl)-2-acetyl-aminofluorene(dG-N2-AAF). In contrast, Chinese hamster ovary (CHO) cellstreated with [³H]N-OAc-AAF gave 80 or 90% dG-C8-AF adducts and20 or 10% dG-C8-AAF adducts with the post- or pre-labeling method,respectively. Likewise in CHO cells treated with 2-AAF in thepresence of rat liver homogenate, {small tilde}90% dG-C8-AFand 10% dG-C8-AAF adducts were detected using the ³²P-post-labelingmethod. In Salmonella typhimurium strain TA1538 treated with2-AAF or [³H]2-AAF in the presence of a rat liver homogenate,one adduct, dG-C8-AF, was identified. Similar quantitative resultswere also obtained with the two methods. However, the ³²P-post-labelingmethod was more sensitive and also eliminated the use of radiolabeled-mutagentreatments. Quantitative DNA adduct dosimetry was applied toAAF-induced mutagenesis in the S. typhimurium and CHO/HPRT mutationassays. A linear and reproducible relationship existed betweendG-C8-AF levels and AAF-induced mutants in both systems.     

Sequence specificity of guanine alkylation and repair

The sequence selectivity of methylation at the O⁶ and N7 positionof guanine by N-methyl-N'-nitrosourea (MNU) and the rate ofremoval of O⁶-methylguanine by O⁶ alkylguanine-DNA alkyltransferase(AGT) was determined using dodecadeoxynucleotides of definedstructure. The extent of guanine adduct formed in self-complementarydodecamers, 5'- TATACGCGTATA-3', 5'-TATACCGGTATA-3' and 5'-TATAGGCCTATA-3'AND 5'-TATAGGCCTATA-3', after methylation with [³H]MNU in arepresentative experiment were, respectively, 10, 19 and 30pmol O⁶-methylguanine/µmol guanine and 97, 189 and 217pmol N7-methylguanine/µmol guanine. The O⁶-methylguanine/N7-molmethylguanineratio remained relatively constant for each dodecamer. A directcomparison between the methylation at guanine with adenine orthymine as the 5'-flanking base was made with two dodecamers,5'-TATACATGTATA-3' and 5'-TATACTAGTATA-3'. When the guanineresidue was preceded 5' by an adenine, the level of O⁶ and N7-alkylationwas, respectively, 2.1-fold and 1.5-fold greater than when guaninewas preceded 5' by a thymine. These date are consistent witha regioselective mechanism for alkylnitrosourea alkylation ofguanine. The methylated dodecamer, 5'-TATACGCGTATA-3' was repairedfaster than 5'-TATACCGGTATA-3' by HT29 extract containing AGTwith a loss in 10min of 0.052 pmol and 0.025 pmol O⁶-methylguanine,respectively. Dodecamers of the structure 5'-dCGCGAATTCm⁶GCG-3'and 5'-dCGCCAATTGm⁶GCG-3' were labeled at the 5' end with ³²Pby the reaction with polynucleotide Kinase and after incubationwith AGT, the methylated and demethylated dodecamers were separatedby reversed-phase HPLC. The amount of demethylated product formedwas greater for the dodecamer containing cytosine as the 5'-flankingbase to O⁶-methylguanine compared to guanine in that same position.A higher extent of alkylation by MNU and a slower rate of repairby AGT for sites in which a guanine or modified guanine is precededby a purine rather than a pyrimidine may explain, at least inpart, mutational hot spots.     

Identification and quantitation of hepatic DNA adducts formed in B6C3F1 mice from 1'-hydroxy-2', 3'-dehydroestragole: comparison of the adducts detected with the l'-3H-labelled carcinogen and by 32P-postlabelling

The identities and levels of DNA adducts formed in mouse liverafter administration of the hepatocarcinogen [l'-³H]1'-hydroxy-2',3'-dehydroestragole obtained by analysis of the ³H-containingadducts were compared with those found by ³²P-postlabellinganalysis. As previously observed the two dia-stereomers of N²-(dehydroestragol-l'-yl)-deoxyguanosinewere the only adducts detected by use of the tritiated carcinogen.Similarly, the unresolved diastereomers of N²-(de-hydroestragol-l'-yl)-deoxyguanosine-3,5-diphosphate were the only adducts detected by the postlabellingprocedure. Analysis by ³²P-postlabelling of defined mixturesof the normal deoxynucleoside-3'-phosphates and synthetic N²-(dehydro-estragol'-l-yl)-deoxyguanosine-3'-phosphateshowed that recovery of the labelled adduct was about 60% ofthat of the normal nucleotides. Likewise, the levels of theadduct in the hepatic DNA from mice treated with l'-hydroxydehydroestra-gole,as determined by ³²P-postlabelling, were generally 60 –80% of those obtained by analysis for the tritiated adducts.Since l'-oxodehydroestragole-deoxyadenosine adducts, the majorproducts obtained on reaction of l'-oxodehydro-estragole withDNA in vitro, were not detected by ³²P-post-labelling in thehepatic DNA from mice treated with 1'-hydroxydehydroestragole,these data provide further evidence that the covalent bindingof l'-hydroxydehydroestragole to liver DNA in vivo does notinvolve thel'-oxo derivative.     

Antibodies to the food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline: useful for immunoassay and immunoaffinity chromatography of biological samples

Monoclonal mouse IgG₁ and IgG₃ antibodies were developed tothe food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline(4,8-DiMeIQx) in order to make specific and sensitive detectionand purification systems suitable for biological samples. Theantibodies were developed with the strategy that cross-reactionwith analogues modified in the N²-position was desirable. Competitiveenzyme-linked immunosorbent assays (ELISA) with 50% inhibitionby 0.4–6 pmol food mutagen were developed. The epitopesrecognized by the antibodies have been characterized by ELISAusing 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx,and other food mutagens. One of the anti-PhIP antibodies onlyrecognizes PhIP and those PhIP-analogues which have minor modificationsin the N²-amino group, whereas the other, 7B7-1, is less stringentand also recognizes several other modified metabolites, includingbulky adducts at the N²-amino group e.g. the major guanine anddeoxyguanosine adducts isolated from PhIP-modified DNA. Theantibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline(4-MeIQx), 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (8-MeIQx),and the corresponding quinolines (4-MeIQ and 8-MeIQ). Two ofthese antibodies only bind analogues with minor modificationsin the free amino group, whereas analogues with major modificationsin this position, including a deoxyguanosine adduct, react withthe third antibody. Urine samples and faecal extracts from ³H-PhIPor 2-¹⁴C-DiMeIQx dosed rats were analysed by these ELISA assays,and high correlations between radioactivity and response inthe ELISA assays were observed. Urine samples and faecal extractsfrom ³H-PhIP-dosed rats were purified on an affinity columncontaining the less stringent anti-PhIP antibody, 7B7-1. Theaffinity column was found by high performance liquid chromatography(HPLC) analysis to concentrate exclusively labelled material.This affinity column also bound PhIP-related materials fromdilute samples of acid hydrolysed PhIP-DNA with high efficiency.Only     

Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32P-postlabelling analysis using different chromatographic procedures

We report the analysis of DNA adducts in the target organ (forestomach)of male Sprague–Dawley rats treated orally with two doses(10 mg/kg body wt) per week for 2 weeks of either aristolochicacid I (AAI), aristolochic acid II (AAII) or the plant extractaristolochic acid (AA). DNA adducts were detected and quantitatedusing the nuclease P1-enhanced version of the ³²P-postlabellingassay. For identification of adducts, reference compounds wereprepared by reaction of enzymatically activated AAI and AAIIwith 3'-purine phosphonucleosides and analysed by the ⁿ-butanolenrichment procedure. These reference compounds were assignedto the previously characterized DNA adducts of AAI [7-(deoxy-guanosin-N²-yl)-aristolactamI = dG-AAI, 7-(deoxyadenosin-N⁶-yl) I = dA-AAI] and AAII [7-(deoxyadenosin-N⁶-yl)-aristolactamII = dA-AAII]. Cross referencing of the carcinogen-modifiednucleoside bisphosphates obtained from forestomach DNA withthe synthetic standard compounds by ion-exchange chromatographyand reversed-phase HPLC demonstrated that the major DNA adductsformed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise,forestomach DNA isolated from AAII-treated rats showed two purinederived adduct spots, the major one being dA-AAII, the minorone being tentatively identified as 7-(deoxyguanosin-N²-yl)-aristolactamII. A minor adduct detected in forestomach DNA of rats treatedwith AAI was found to be chromatographically indistinguishablefrom the adduct identified as dA-AAII, indicating a possibledemethoxylation reaction of AAI. Quantitation of DNA adductsrevealed that in in vitro reactions with 3'-phosphonucleosidesthe adduct levels were approximately one order higher for bothAAI and AAII-derived adducts than in forestomach DNA modifiedwith AAI or AAII in vivo. In vitro as well as in vivo adductionby AAI was more efficient than adduction by AAII. The patternof adduct spots obtained from forestomach DNA of rats treatedwith the plant extract AA reflected the composition of the extractdetermined by HPLC analysis. Irrespective of the aristolochicacid used to induce DNA adducts, deoxyadenosine is the majortarget of modification, pointing to the general importance ofdeoxyadenosine adducts for chemical carcinogenesis of thesenaturally occurring products. This study shows that the combinationof two independent chromatographic systems considerably enhancesthe fidelity of identification of DNA adducts with the ³²P-posthbellingassay.     

DNA adduct formation of the food carcinogen 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) in liver, kidney and colo-rectum of rats

DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)have been measured in the liver, kidney, and colorectum of maleFischer-344 rats given a single oral dose of IQ (20 mg/kg).The pattern and distribution of DNA adducts examined by ³²P-postlabelingwas similar in all tissues. N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]quinoline(dG-C8-IQ) was the principal adduct identified and it accountedfor     

32P-postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents

Carcinogen - DNA adducts were detected and determined by ³²P-postlabelingassay after exposure of mouse or rat tissues in vivo to a totalof 28 compounds comprising 7 arylamines and derivatives, 3 azocompounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons,and 4 methylating agents. DNA was isolated from mouse skin,mouse liver, and rat liver after treatment with the individualcarcinogens, then digested enzymatically to deoxyribonucleoside3'-monophosphates, which were converted to 5'-³²P-labeled deoxyribonucleoside3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [³²P]phosphatetransfer from [-³²P]ATP. The nucleotides were resolved by anion-exchanget.l.c. on polyethyleneimine-cellulose and detected by autoradiography.The determination of low levels of DNA binding of the aromaticcarcinogens entailed the removal of normal nucleotides priorto the resolution of adduct nucleotides. For this purpose, analternative procedure employing reversed-phase t.l.c. was devisedwhich offered advantages for the detection of quantitativelyminor adducts. The procedures described enabled the detectionof 1 aromatic DNA adduct in 10⁸ normal nucleotides, while thelimit of detection of methylated adducts was 1 adduct in 6 x10⁵ nucleotides. The results show that a great number of carcinogen-DNAadducts of diverse structure are substrates for ³²P-labelingby polynucleotide kinase-catalyzed phosphorylation. Becausecovalent DNA adduct formation in vivo appears to be an essentialproperty of the majority of chemical carcinogens, ³²P-postlabelinganalysis of carcinogen -DNA adducts in mammalian tissues mayserve as a test for the screening of chemicals for potentialcarcinogenicity.     

Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-l-methyl-6-phenyUmidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues

The food-borne mutagen 2-amino-l-methyl-6-phenylimidazo[ 4,5-b]pyridine(PhIP) induces tumors in colon of male rats and has been implicatedin the etiology of human cancers, particularly colorectal cancer.This study was conducted to examine: (1) the biliary and/orcirculatory transport of N-hydroxy- PhIP and its N-glucuronides,N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximateand ultimate carcinogenic metabolites of PhIP; (3) the potentialrole of glutathione in modulating PhIP-DNA adduct formation.PhIP-DNA adducts, measured by the ³²P-postlabeling method, werehighest in the pancreas (361 adducts/10⁸ nucleotides or 100%),followed by colon (56%), lung (28%), heart (27%) and liver (2%),at 24 h after a single oral dose of PhIP (220 µmol/kg)to male rats. In each tissue examined, we observed two majoradducts, each of which accounted for 35–45% of the total,and one minor adduct, which represented about 10–20% ofthe total. One of the major adducts was identified as N-(deoxyguanosin-8-yl)-2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine by chromatographic comparisonswith an authentic standard. The major urinary metabolites ofPhIP in these rats were 4'-hydroxy-PhIP and its glucuronideand sulfate conjugates, followed by N-hydroxy-PhIP N3-glucuronide,N-hydroxy-PhIP N-glucuronide and unchanged PhIP. In bile duct-ligatedrats, the urinary excretion of the N-OH-PhIP N3-glucuronidewas increased two-fold, but there was no effect on PhIP-DNAadduct formation in the colon, heart, lung, pancreas or liver.2,6-Dichloro-4-nitrophenol, which strongly inhibits arylsulfo-transferase-mediatedDNA binding in vivo, had no effect on PhIP-DNA adduct levelsin liver or in extrahepatic tissues. Pretreatment of rats withbuthionine sulfoximine, which results in hepatic glutathionedepletion, caused a five-fold increase in adduct formation inthe liver. Intravenous administration (10 µmol/kg) ofN-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels ofPhIP-DNA adducts in each of the extrahepatic tissues examined.Adduct levels ranged from two- to six-fold higher (for N-hydroxy-PhIP)and four- to 28-fold higher (for N-acetoxy-PhIP) as comparedto that after an i.v. dose of the parent compound, indicatingthat these two bioactivated derivatives of PhIP are sufficientlystable to be transported through the circulation to extrahepatictissues. Analyses of whole blood obtained at 2—8 h afteroral administration of [³H]PhIP failed to detect N-hydroxy-PhIP(<0.1% of the radioactivity), however, a decomposition productof N-acetoxy-PhIP was found to account for about 80% of thetotal radioactivity in the blood. These results suggest thattransport of N-acetoxy-PhIP, and perhaps N-hydroxy-PhIP, viathe bloodstream and not biliary transport and deconjugationof N-hydroxy-PhIP N-glucuronides is primarily responsible forPhIP-DNA adduct formation in rat colon and other extrahepatictissues.     

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.