32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy

Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)- aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin- N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post- labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients.      

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.     

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

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

Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene

The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.     

Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid

Synthetic oligonucleotides (18-mers) containing either a singledeoxyadenosine residue or a single deoxyguanosine residue weretreated with aristolochic acid I (AAI) or aristolochic acidII (AAII), the main components of theplant carcinogen aristolochicacid (AA). These reactions resulted in the formation of site-specificallyadducted oligonucleotides containing the two known AAI—DNAadducts (dA—AAI, dG—AAI) or the two known AAII—DNAadducts (dA—AAII, dG—AAII) at position 15 from the3'end. Using HPLC chromatography, the oligonucleotides werepurified and subsequently shown to contain the adducts of interestby ³²P-postlabelling. The adducted oligonucleotides were usedas templates in primer (11-mer) extension reactions catalysedby modified bacteriophage T7 DNA polymerase (Sequenase). Regardlessof the type of DNA adduct examined, DNA synthesis was blockedpredominantly (80–90%) at the nucleotide 3' to each adduct,although primer extension to the full length of the templatewas noted with unmodified control templates. However, 15 nucleotideproducts, indicating blocking of DNA synthesis after incorporationof a nucleotide opposite the adduct and translesional synthesisproducts were formed in all cases in different amounts, dependingon the adduct structure. When a 14-mer primer together withhigh dNTP concentrations was used to examine nucleotide incorporationdirectly across from the four different purine adducts we foundthat the deoxyadenosine adducts (dA–AAI and dA–AAII)allowed incorporation of dAMP and dTMP equally well, whereasthe deoxyguanosine adducts (dG–AAI and dG–AAII)allowed preferential incorporation of dCMP. Molecular dynamicsimulations showed that the aristolactam moiety of all adductsexhibit a strong stacking, with the adenine residue at the 3'end of the 14-mer primer. These studies demonstrate that allAA purine adducts provide severe blocks to DNA replication andthat the guanine adducts may not be very efficient mutageniclesions. In contrast, the translesional bypass past adenineadducts of the aristolochic acids suggests a mutagenic potentialresulting from dAMP incorporation by polymerase. AT     

DNA adduct formation of aristolochic acid I and II in vitro and in vivo

Aristolochic acid I (AA I) and aristolochic acid II (AA II),the two main ingredients of the carcinogenic plant extract aristolochicacid (AA), are metabolized to reactive intermediates which bindcovalently to DNA in vitro and in vivo. DNA adduct formationwas analysed by the ³²P-postlabelling assay. In in vitro incubationswith rat liver 9000 g supernatant (S9) and calf thymus DNA (CT-DNA),AA I showed an identical pattern of DNA adducts on thin-layerchromatograms under aerobic and anaerobic conditions, whereasAA II gave rise to DNA adduct formation only anaerobically.The anaerobically obtained DNA adduct pattern by AA II in vitrowas similar to the AA I adduct patterns. Aristolactams I andII, the metabolites of AA I and AA II formed under anaerobicconditions, did not form DNA adducts in the presence of S9 mixand CT-DNA. Incubations with xanthine oxidase, known to enzymaticallyreduce aromatic nitro groups, also activated AA I and AA IIto reactive intermediates, producing almost identical adductpatterns as obtained by S9 mix-mediated metabolism. Activationof AA I by S9 mix in the presence of poly(dG) resulted in theformation of two adducts, one of which was shown to be chromatographicallyindistinguishable from an adduct obtained by reaction with CT-DNA.For the in vivo studies AA I and AA II were administered orallyto male Wistar rats, and DNA from liver, brain, oesophagus,stomach lining, forestomach lining, kidney and bladder was analysedfor DNA adducts by ³²P-postlabelling. The adduct patterns inDNA from forestomach and kidney—target tissues of AA—andDNA from non-target tissues like stomach lining and liver weresimilar to the patterns obtained from the in vitro incubations.In the bladder (also a target tissue) only AA II gave rise toDNA adduct formation. These findings suggest that DNA adductformation by AA I and AA II does not directly correlate withthe initiation of the carcinogenic process and subsequent tumourformation in target tissues in the rat.     

Formation and removal of DNA adducts in target and nontarget tissues of rats administered multiple doses of 2-acetylaminophenanthrene

2-Acetylaminophenanthrene (2-AAP) is carcinogenic for the mammarygland, small intestine and ear duct in rats, but is not hepatocarcinogenic.In order to understand the tissue specificity of tumor induction,the formation and removal of DNA adducts in target and nontargettissues have been compared in rats administered 2-AAP. Maleand female Sprague-Dawley rats were treated i.p. with up tofour weekly doses of 5 mg 2-AAP per kg body weight. ³²P-Post-labelinganalysis of the DNA from all tissues showed two predominantadducts (>85–95% of the total binding) and at leastfour minor adducts. By comparing the results obtained from reactingN-hydroxy-2-aminophenanthrene with DNA at pH 5, the major adductswere identified as N(deoxyuguanosin-N-8-yl)-2-aminophenanthreneand l-(deoxyguanosin-N²-yl)-2-aminophenanthrene. Two minor adducts,N-(deoxy-adenosine-8-yl)-2-aininophenanthrene and l-(deoxyadenosin-iN⁶-yl)-2-aminophenanthrene,were also formed in the in vitro reactions. The distributionof adducts, extent of binding and adduct persistence were similarbetween target and nontarget tissues, which indicates that thetissue specificity of 2-AAP for tumor induction is due to factorshi addition to adduct formation.     

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.     

Identification and mutagenicity of metabolites of aristolochic acid formed by rat liver

The rat liver 9000 g supernatant mediated metabolism of thecarcinogenic aristolochic acid, which consists of aristolochicacid I (AAI) and aristolochic acid II (AAII), was investigated.Under anaerobic conditions the major metabolites were the correspondingaristolactams for both AAI and AAII. In contrast under aerobicconditions AAII was not detectably metabolized and the onlymetabolite found for AAI was the O-demethylated derivative aristolochicacid la (AAIa). The metabolites were identified by their u.v.,mass and n.m.r. spectra and by comparison with reference standards.The mutagenic activities of the three metabolites were determinedin Salmonella typhimurium strains TA1537 and TA 100. The aristolactamswere mutagenic in both strains when a metabolizing system waspresent. These results indicate that AAI or AAII and their aristolactamsexert their effect via a common reactive intermediate, probablythe corresponding hydroxylamine. AAIa was only very weakly mutagenicand this metabolite may therefore not be regarded as a majormutagenic metabolite of AAI. These findings suggest that theacids are preferentially metabolized by two totally differentpathways in vitro, namely an oxidative pathway for AAI and areductive pathway for AAII.     

Characterization and properties of the DNA adducts formed from N-methyl-4-aminoazobenzene in rats during a carcinogenic treatment regimen

Chronic oral administration of the carcinogenic aminoazo dyeN-methyl-4-aminoazobenzene (MAB) to rats is known to resultin the induction of liver tumors. In order to assess the roleof carcinogen-DNA adduct formation in MAB hepatocarcinogenesis,male rats were fed 0.06% [3'-³H]MAB in the diet for 1, 3 or5 weeks. Groups were sacrificed at 0, 24 and 72 h after dosing,and DNA was isolated from the liver and from two non-targettissues, the kidney and spleen. Upon enzymatic hydrolysis ofthe DNA, [³H]aminoazo dye-nucleoside adduct levels in thesetissues were determined by h.p.l.c. Rats concurrently administeredunlabeled MAB for 5 weeks and continued on a control diet for9 months developed hepatocellular carcinomas (16/30 animals).No tumors were observed in 21 rats given only control diets.After chronic administration of [³H]MAB, three major MAB-DNAadducts were found in vivo: N-(deoxyguanosin-8-yl)-MAB (C8-dG-MAB),3-(deoxyguanosin-N²-yl)-MAB (N²-dG-MAB) and 3-(deoxyadenosin-N⁶-yl)-MAB(N⁶-dA-MAB). In addition, several minor products were identifiedas: (i) an (8,9)-purine ring-opened derivative of C8-dG-MABthat may represent an intermediate in DNA repair; (ii) N-guanosin-8-yl-MABwhich is present due to trace RNA contamination; (iii) cis isomersof C8-dG-MAB and N-guanosin-8-yl-MAB, formed by photo-illuminationduring analyses; and (iv) N-(guanin-8-yl)-MAB, a deribosylatedproduct resulting from thermal depurination of C8-dG-MAB. Inaddition, N-(deoxyguanosin-8-yl)-4-aminoazobenzene (C8-dG-AB),a major adduct previously detected in mouse liver after a singledose of 4-aminoazobenzene, was found in rat liver but appearedto be present in significant amounts only after chronic treatmentwith MAB. This product co-chromatographed with N⁶-dA-MAB butcould be removed by selective decomposition in 0.1 N NaOH. Forall tissues examined N²-dG-MAB and C8-dG-MAB were the majoradducts observed with each accounting for 40-50% of the totalcarcinogen bound to DNA in rats that were sacrificed immediatelyafter MAB feeding for 1, 3 or 5 weeks. The levels of total MAB-DNAadducts in the liver were 2–10 times greater than in thekidney or spleen and appeared to increase 2- to 3-fold overthe dosing period. However, by 24–72 h after cessationof MAB treatment, hepatic C8-dG-MAB showed a rapid decline tolevels similar to that found in non-target tissues. The minoradducts, N⁶-dA-MAB and C8-dG-AB, exhibited similar behaviorand never accounted for > 5–10% of the total DNA binding.In contrast, hepatic N²-dG-MAB was a persistent lesion throughoutthe treatment regimen; at 72 h after dosing, it accounted for60–90% of the hepatic DNA adducts and was the only adductwhose levels correlated with target tissue specificity aftera complete hepatocarcinogenic dose of MAB.     

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.     

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     

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.     

32P-Postlabeling analysis of DNA adducts in rat stomach with 1-nitrosoindole-3-acetonitrile, a direct-acting mutagenic indole compound formed by nitrosation

Modification of DNA by a direct-acting mutagen, 1-nitrosoindole-3-acetonitrile,which is formed from indole-3-acetonitrile upon nitrite treatment,was investigated. ³²P-Postlabeling analysis clearly demonstratedthe formation of DNA adducts in the stomach of rats after intragastricadministration of 1-nitrosoindole-3-acetonitrile. The levelof DNA adducts in both the forestomach and glandular stomach2 h after administraction of 100 mg/kg body weight of the compoundwas about one adduct per 10⁷nucleotides. The DNAs of the forestomachand glandular stomach gave six common spots on two-dimensionalchromatography, three of which were also produced by in vitroreaction of this compound with DNA. Thus, 1-nitrosoindole-3-acetonitrilecan form DNA adducts in vivo and in vitro. No DNA adducts weredetected after treatment with the non-nitrosated compound indole-3-acetonitrileboth in vivo and in vitro. These results suggest that 1-nitrosoindole-3-acetonitrilehas the in vivo tumor initiating activity in the stomach.     

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.     

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.     

Carcinogenic aristolochic acids upon activation by DT-diaphorase form adducts found in DNA of patients with Chinese herbs nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of enzymes of rat renal and hepatic cytosolic fractions to activate AA to metabolites forming DNA adducts by the nuclease P1-enhanced version of the (32)P-postlabeling assay. Cytosolic fractions of both these organs generated AA-DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI and 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II were generated from AAII by hepatic cytosol. Qualitatively the same AA-DNA adduct patterns were observed, although at lower levels, upon incubation of AAs with renal cytosol. To define the role of cytosolic reductases in the reductive activation of AA, we investigated the modulation of AA-DNA adduct formation by cofactors, specific inducers or selective inhibitors of the cytosolic reductases, DT-diaphorase, xanthine oxidase (XO) and aldehyde oxidase. The role of the enzymes in AA activation was also investigated by correlating the DT-diaphorase- and XO-dependent catalytic activities in cytosolic sample with the levels of AA-DNA adducts formed by the same cytosolic sample. On the basis of these studies, we attribute most of the cytosolic activation of AA to DT-diaphorase, although a role of cytosolic XO cannot be ruled out. With purified DT-diaphorase, the participation of this enzyme in the formation of AA-DNA adducts was confirmed. The binding orientation of AAI in the active site of DT-diaphorase was predicted by computer modeling based on published X-ray structures. The results presented here are the first report demonstrating a reductive activation of carcinogenic AAs by DT-diaphorase.     

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.     

Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts

Previous work from this laboratory provided strong evidencethat N-sulfooxy-2-aminofluorene is the major ultimate electro-philicand carcinogenic metabolite of N-hydroxy-2-acetyl-aminofluorene(N-hydroxy-AAF) in the livers of infant male B6C3F₁ (C57BL/6Jx C3H/HeJ F₁ mice. Over 90% of the hepatic DNA adducts in thesemice consisted of N-(deoxyguan-osin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)]and<10% were deoxyguanosinyl adducts containing 2-acetylaminofluor-ene(AAF) residues. In the present study hepatic DNA adduct formationand tumor initiation by N-hydroxy-2-aminofluor-ene (N-hydroxy-AF)were examined in these mice. N-(dGuo-8-yl)-AF was the only adductdetected in the hepatic DNA; the level at 9 h after a singlei.p. dose of 0.04 or 0.06 µmol/g body wt of [³H]N-hydroxy-AFwas 1.0 or 1.7 pmol/mg DNA. Pre-treatment with a single i.p.dose (0.04 µmol/g body wt) of the sulfotransferase inhibitorpentachlorophenol (PCP) decreased the DNA adduct level by >80%.Similar levels of this adduct were found by ³²P-postlabelinganalysis of DNA from mice treated with unlabeled N-hydroxy-AF.The liver DNA of in-fant male brachyinorphic B6C3F₂ mice [deficientin 3'-phos-phoadenosine-5'-phosphosulfate (PAPS)] containedonly 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. doseof 0.06 µmol of N-hydroxy-AF/g body wt, while their phenotypi-callynormal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA.A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 µmol/body wt of N-hydroxy-AF in infant male B6C3F mice induced by10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse.Pretreatment with PCP reduced the liver tumor multiplicity ateach dose level by >80%. Essen-tially the same average tumormultiplicities and inhibitions of tumor formation by PCP pretreatmentwere obtained following injections of N-hydroxy-AF or N-hydroxy-AAFat the three lower dose levels. Collectively these data stronglyindicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxyAF in the livers of infant male B6C3F₁ mice. Furthermore, sinceonly N-(dGuo-8-yl)-AF adducts were found in the he atic DNAthese lesions appear to be critical in the initiation of hepatocarcinogenesisin these mice by N-hydroxy-AF.