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.     

Binding of N-acetylbenzidine and N,N''-diacetylbenzidine to hepatic DNA of rat and hamster in vivo and in vitro

Benzidine, a potent hepatocarcinogen in rodents, is readilymetabolised to acetylated derivatives. In this study, the covalentbinding of [³H-acetyl]N-acetylbenzidine and [³H-acetyl]N,N'-diacetylbenzidineto liver DNA in rats and hamsters was investigated. Bindingto liver DNA of rats at 1 or 7 days after i.p. injection ofN-acetylbenzidine was 2-fold higher than that observed in theliver DNA of hamsters which had been similarly treated. Analysisof enzymically hydrolysed DNA from both species indicated thepresence of a single adduct which co-eluted with N-(deoxyguanosin-8-yl)-N'-acetylbenzidine. In vitro treatment of rat or hamster liverslices with N-acetylbenzidine also resulted in covalent bindingto hepatic DNA and the identical DNA adduct was detected atlevels comparable to that observed in vivo. When N,N'-diacetylbenzidinewas injected i.p. into rats, a comparatively low level of bindingto liver DNA was observed. Following enzymic hydrolysis, themajor DNA adduct detected by h.p.l.c. analysis was again N-(deoxyguanosin-8-yl)-N'-acetylbenzidineaccompanied by a small amount of N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine.In vitro incubation of N,N'-diacetylbenzidine with rat liverslices resulted in DNA binding levels similar to that observedwith N-acetylbenzidine. In contrast to what was found in vivo,N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was the majoradduct detected in DNA from rat liver slices. These data suggestthat both N-hydroxy-N'-acetylbenzidine and N-hydroxy-N,N'-diacetylbenzidineare proximate carcinogenic species of benzidine, with N-hydroxy-N'-acetylbenzidinethe more important. The low level of N-(deoxyguanosin-8-yl)N,N'-diacetylbenzidineobserved in vivo may be due to its rapid repair. Alternatively,N-sulphonyloxy-N,N'-diacetylbenzidine, which would produce thisadduct on reaction with DNA, may be efficiently detoxified invivo.     

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.     

Rapid release of carcinogen-guanine adducts from DNA after reaction with N-acetoxy-2-acetylaminofluorene or N-benzoyloxy-N-methyl-4-aminoazobenzene

Reaction of N-benzoyloxy-N-methyl-4-aminoazobenzene (N-benzoyloxy-MAB)or N-acetoxy-N-acetyl-2-aminofluorene (N-acetoxy-AAF), modelultimate aromatic amine or amide carcinogens, with [purine-¹⁴C]DNAat pH 7.4 or reaction of N-hydroxy-2-aminofluorene (N-hydroxy-AF)at pH 4.6 resulted in the rapid release of ¹⁴C-containing productsnot precipitable with the DNA. Four such products were obtainedon reaction with N-acetoxy-AAF, two with N-hydroxy-AF, and sixwith N-benzoyloxy-MAB. Depending on the DNA sample used, thetotal amounts of ¹⁴C in these products from the reactions withN-benzoyloxy-MAB or N-acetoxy-AAF ranged from about 5% to asmuch as 30–40% of the amounts in the N-(deoxyguanosin-8-yl)-MABor N-(deoxyguanosin-8-yl)-AAF residues in the DNA from the samereaction mixtures. Reactions with N-acetoxy-[acetyl-³H]AAF showedthat the two major products retained the amide residue fromthe N-acetoxy-AAF. When the reactions were carried out with[guanine-(8-³H; 8-¹⁴C); adenine-(2,8-³H; 8-¹⁴C)]DNA, the twomajor products formed from N-acetoxy-AAF and four products formedfrom N-benzoyloxy-MAB had very low ³H:¹⁴C ratios; these ratioswere those expected for guanine derivatives which had lost the8-³H. Studies on the major DNA adducts N-(deoxyguanosin-8-yl)-MABand N-(deoxyguanosin-8-yl)-AAF indicated that the new adductswere not formed from these nucleosides. The data suggest thatthe two major AAF products and the four MAB adducts studiedare guanine derivatives formed by depurination of N-7 substitutedadducts in the DNA.     

Identification of N-(deoxyguanosin-8-yl)-2-amino-3,4-dimethylimidazo[4,5-f]quinoline (dG-C8-MeIQ) as a major adduct formed by MeIQ with nucleotides in vitro and with DNA in vivo

The N-hydroxylamine of a carcinogenic heterocyclic amine, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ), was reacted with four 2'-deoxynucleoside 3'-monophosphatesafter O-acetylation. ³²P-Postlabeing analysis demonstrated thatthe adduct was formed with only the guanine nucleotide, andthe structure of the compound in the obtained adduct spot wasdetermined to be N-(deoxyguanosin-8-yl)-MeIQ 3',5'-diphosphate(3',5'-pdGp-C8-MeIQ). DNA samples from livers of mice fed MelQwere also ³²P labeled under standard conditions and additionallytreated with nuclease P1 and phosphodiesterase I. A single adductspot was obtained and the structure of the adduct was identifiedas 5'-pdG-C8-MeIQ. Thus, MelQ binds at the C-8 position of guaninein vitro and in vivo, like other heterocyclic amines.     

Immunohistochemical and microfluorometric determination of hepatic DNA adduct removal in rats fed 2-acetylaminofluorene

Biphasic removal of DNA adducts has previously been demonstratedby radioiminunoassay In whole liver DNA from rats chronicallyfed 2-acetylaminofluorene for 28 days. In the present study,removal of N-(deoxyguanosin-8-yl)-2-amlnofluorene was observedin situ by microfluorometry. Frozen liver sections from animalsfed 0.02% 2-acetylaminofluorene for 28 days, followed by a controldiet for 3, 7, 14, 21 and 28 days, were examined immunohisto-chemicallyfor localization of N-(deoxyguanosln-8-yl)-2-aminofluorene withfluorescein-conjugated secondary antiserum. In addition, bileducts and oval cells were stained with antibodies to keratinsusing Texas red-labelled indirect immunofluorescence. Hoechstdye was used to identify DNA in nuclei. During the 28 days onthe control diet, after 28 days of feeding 2-acetylaminofluorene,the DNA adduct concentrations of parenchymal liver cells werereduced by 85%, as compared to animals fed only the carcinogenfor 28 days. Periportal hepatocytes exhibited biphasic (fastand slow) adduct removal. Only fast adduct removal was demonstratedin midzonal and centrilobular hepatocytes, since the adductlevels were below the detectable range in these regions after7 days on the control diet. After 28 days on the control diet,N-(deoxyguanosin-8-yl)-2-aminofluorene was detected in 50% ofperiportal hepatocytes. These results are compatible with thepreviously observed biphasic removal profile determined by radloinimunoassayof whole liver DNA adducts and indicate that penportal hepatocytesremove adducts from two distinct genomic compartments.     

Mechanism of formation and structural characterization of DNA adducts derived from peroxidative activation of benzidine

Although the peroxidative metabolism of benzidine and othercarcinogenic arylamines has been regarded as a significant pathwayin extrahepatic tissues, the mechanism of peroxidase-mediatedcovalent binding to DNA has remained unclear. In this study,we have compared the metabolic activation of benzidine by prostaglandinH synthase, horseradish peroxidase, chloroperoxidase, and lactoperoxidase.All four peroxidases mediated the binding of benzidine to DNAand equimolar amounts of hydrogen peroxide (or arachidonic acid)and benzidine were required for the maximal binding in the systemwith either horseradish peroxidase or prostaglandin H synthase.In reactions containing both synthetic [³H]benzidine diimineand [¹⁴C]benzidine, rapid equilibration of both compounds wasevident through the formation of its charge-transfer complexand the ¹⁴C-associated binding to DNA. However, the total binding(³H and ¹⁴C) correlated with the concentration of benzidinediimine rather than that of the charge-transfer complex. Twomajor and one minor deoxyguanosine adducts (P-I, P-III and P-IV,respectively) were isolated after the enzymatic hydrolysis ofthe benzidine-modified DNA. P-I was identified as N-(deoxyguanosin-8-yl)-benzidine,which was the major ad-duct formed by reaction of benzidinediimine with DNA. P-IV, which was also formed on reaction ofbenzidine diimine with DNA, was consistent with an N-(deoxyguanosin-N²-yl)-benzidinestructure. P-III, which was formed only in the peroxidase incubationswith DNA, was characterized as a novel N,3-(deoxyguanosin-N7,C8-yl)-benzidine derivative. Furthermore, this DNA adduct wasshown to arise by the action of the peroxidase on DNA that hadbeen previously modified by benzidine diimine. These resultsindicate that the two-electron oxidation product of benzidine,benzidine diimine, is the predominant reactive intermediatefor the DNA binding mediated by peroxidases.     

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.     

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.     

Identification and characterization of the major DNA adduct formed chemically and in vitro from the environmental genotoxin 3-nitrofluoranthene

The genotoxic environmental pollutant 3-nitrofluoranthene (3-NFA)was reduced chemically and allowed to react with calf thymusDNA, yielding one major adduct which was determined to be N-deoxyguanosin-8-yl)-3-amino-fluoran-thenebased on Fast Atom Bombardment Mass Spectrometry (FAB-MS), protonnuclear magnetic resonance, ultra-violet-visible wavelengthlight spectroscopy (UV-VIS), and fluorescence data. Extensivecharacterization of the isolated adduct by tandem mass spectrometry(MS/MS) was necessary to demonstrate definitively that the adductisolated was the dG: C8 adduct, and not the isomeric dG: N2adduct. The extent of modification of the initial calf thymusDNA by chemically reduced 3-NFA was 0.12% (1.2 adducts/10P nucleosides),which was sufficient to allow several hundred micrograms ofthe adduct to be isolated and purified. The chemically synthesizedadduct was utilized as a reference standard for comparison tothe major adduct isolated from xanthine-oxidase-catalyzed reductionof 3-NFA in vitro. The yield from the in vitro biological systemwas 2.4 adducts/10⁵ nucleosides; the adduct isolated possessedthe same mass spectrometric. UV-VIS, and fluorescence characteristicsas the purified standard, and co-eluted with the standard onHPLC. No evidence for other adducts was found, either in vitroor in the chemical synthesis, based on FAB-MS examination ofwhole extracts of the reaction mixture for the presence of ionsrelated to other possible adducts. Therefore, if minor adductswere present they were formed in substantially lesser amountsthan N-(deoxyguanosin-8-yl)-3-aminoflouranthene.     

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes

The potent rat colon carcinogen 2-amino-l-methyl-6-phenylimidazo[4,5-d]pyridine (PhIP), unlike other food-borne heterocyclicamines, does not induce tumors in rat liver. This correlateswith an extremely low level of PhIP-DNA adducts formed in thistissue, and together these observations suggest that PhIP isefficiently detoxified in the liver. In order to identify possibledetoxification mechanisms, we assessed the effect of inhibitionof glucuronidation, glutathione (GSH) conjugation and sulfationon PhIP metabolism and PhlP-induced DNA damage in rat hepatocytes.Hepatocytes isolated from rats pretreated with Aroclor 1254metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIPN3-glucuronide and N-hydroxy-PhIP N²-glucuronide were majorand minor metabolites respectively. ³²P-Postlabeling analysisof DNA from the PhIP-treated hepatocytes indicated the presenceof two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP,and one minor adduct. There was no unscheduled DNA synthesis(UDS) in these cells. However, pretreatment of the hepatocyteswith 1-bromoheptane and buthionine sulfoximine, which depletesGSH and prevents its resynthesis, resulted in a 15-fold increasein the formation of PhIP-DNA adducts, as well as in a high levelof UDS. GSH depletion had no effect on the formation of detectablePhIP metabolites. Hepatocyte pretreatment with D-galactosamine,which inhibits glucuronidation, increased the formation of DNAadducts two-fold and UDS was increased similarly. D-Galactosaminedecreased the formation of the two N-glucuronides of N-hydroxy-PhIPby 50–60%, but had no effect on other metabolites. Pentachlorophenol,which strongly inhibits sulfotransferases, decreased adductformation slightly, but had essentially no effect on UDS oron the formation of PhIP metabolites. These results indicatethat metabolic conjugation pathways involvingGSH and glucuronidationmay play an important role in protecting rat liver against PhIPcarcinogenesis.     

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.     

The essential role of microsomal deacetylase activity in the metabolic activation, DNA-(deoxyguanosin-8-yl)-2-aminofluorene adduct formation and initiation of liver tumors by N-hydroxy-2-acetylaminofluorene in the livers of infant male B6C3F1 mice

Deacetylation of N-hydroxy-2-acetylaminofluorene. (N-hydroxy-AAF) to N-hydroxy-2-aminofluorene (N-hydroxy-AF) has been proposedas one of the critical metabolic steps in the formation of hepaticDNA adducts and the initiation of liver tumors in 12-day-oldmale B6C3F₁ mice. In this study, the importance of the microsomaldeacetylase activity for N-hydroxy-AAF in the initiation ofhepatocarcinogenesis in these mice was demonstrated by usinga carboxylesterase and amidase inhibitor, bis(p-nitrophenyl)phosphate(BNPP), that is much less toxic in vivo than is paraoxon. Pre-incubationof liver microsomes from 12-day-old male B6C3F₁ mice with 10^–3M BNPP reduced the deacetylase activity by 80% while paraoxoninhibited the deacetylase activity completely at a concentrationof 10_–4 M. Pretreatment of 12-day-old male B6C3F₁ micewith 4 x 75 µg doses of BNPP/g body weight before theadministration of n-hydroxy-AAF reduced the hepatic N-(dGuo-8-yl)-AFadduct levels to 1.09 and 0.68 pmol/mg DNA compared with 2.87and 1.64 pmol/mg DNA for mice treated once with 0.06 or 0.03µmol of N-hydroxy-AAF/g body weight respectively. However,BNPP pretreatments did not affect the levels of the acetylatedDNA adducts, N-(dGuo-8-yl)-AAF and 3-(dGuo-N²-yl)-AAF, formedby these doses of N-hydroxy-AAF. The initiation of liver tumorsby n-hydroxy-AAF was also inhibited by BNPP pretreatment. Thus,for mice that received single doses of 0.12, 0.06 and 0.03 µmolof N-hydroxy-AAF/g body weight, the multiplicities of livertumors at 10 months were reduced by BNPP pretreatments to 5.6,1.0 and 0.3 compared with multiplicities of 11.8, 4.8 and 1.7without pretreatment respectively. On the other hand, BNPP pretreatmentshad no significant inhibitory effects on the levels of the hepaticDNA-N-dGuo-8-yl)-AF adduct or on the liver tumor multiplicitiesinduced by comparable doses of N-hydroxy-AF. It is concludedthat deacetylation of n-hydroxy-AAF to N-hydroxy-AF is essentialfor the metabolic activation, DNA-N-(dGuo-8-yl)-AF adduct formationand liver tumor initiation in infant male B6C3F₁ mice by N-hydroxy-AAF.     

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     

Evidence for N-(deoxyguanosin-8-yl)-1-aminopyrene as a major DNA adduct in female rats treated with 1-nitropyrene

[³H]1-Nitropyrene was administered at a dose of 25 mg/kg byi.p. injection to female Wistar rats. Animals were killed 24hlater and DNA was isolated from kidney, liver and mammary gland,enzymically hydrolysed and analysed by reversephase h.p.l.c.A major adduct peak was detected in DNA from each of the threeorgans. Enzymic hydrolysates of DNA, which had been reactedin vitro with 1-nitropyrene in the presence of xanthine oxidase,were similarly analysed by h.p.l.c. One major adduct peak wasobtained which had the same retention time as the in vivo product.Confirmatory evidence that the in vivo adduct and the in vitroadduct were structurally similar was obtained from the determinationof the pH-dependent solvent partitioning profiles. Further,treatment of the in vivo adduct from liver, kidney or mammarygland DNA hydrolysates and the in vitro adduct with sodium hydroxideresulted in the formation of a more polar product which elutedearlier on h.p.l.c. This behaviour is consistent with scissionof the imidazole ring of a deoxyguanosine adduct.The major DNAadduct formed in vitro following xanthine oxidase reductionof 1-nitropyrene has previously been identified by others asN-(deoxyguanosin-8-yl)-1-amino-pyrene. The present data suggestthat the in vivo 1-nitro-pyrene-DNA adduct has the same structure.     

Role of sulfation in the formation of DNA adducts from N-hydroxy-2-acetylaminofluorene in rat liver in vivo. Inhibition of N-acetylated aminofluorene adduct formation by penta-chlorophenol

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is metabolicallyconverted into reactive N, O-esters which are capable of formingcovalent adducts with DNA in rat liver in vivo. The effect ofinhibiting one of the proposed pathways, N-O-sulfation, on DNAadduct formation was studied by using a specific sulfotransferaseinhibitor, pentachlorophenol. Rats were pretreated with pentachlorophenoland, after 45 min, N-OH-AAF was administered. Four hours afterdosing the animals were sacrificed and hepatic DNA was isolated.In DNA from control livers two acetylaminofluorene-and one amino-fluorene-substituteddeoxyguanosine adducts were found. The acetylaminofluorene derivatives,N-(deoxy-guanosin-8-yl)-2-acetylaminofluorene and 3-(deoxy-guanosin-N²-yl)-2-acetylaminofluorene,accounted for 40% of the total binding in the hydrolyzed DNA.The aminofluorene adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene,accounted for the remainder. In rats that were pretreated withpentachlorophenol, total DNA binding was decreased by 26%. Thesame three adducts were found, but the acetylaminofluorene adductswere now only 13% of the total, while the aminofluorene adductaccounted for 87%. The absolute amount of aminofluorene adductwas not altered as compared to control rats. These data demonstratethe involvement of N-O-sulfation in carcinogen - DNA bindingand indicate that at least 70% of the acetylaminofluorene boundto deoxyguanosine in rat liver DNA, in vivo, is formed throughN-O-sulfation of N-OH-AAF.     

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.     

Effect of ovariectomy on the in vitro and in vivo activation of carcinogenic N-2-fluorenylhydroxamic acids by rat mammary gland and liver

N-Hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) and its benzamideanalogue N-OH-2-FBA are mammary gland carcinogens in the femaleSprague—Dawley rat. Ovariectomy inhibits tumorigenicityof topically applied N-OH-2-FAA suggesting modulation of carcinogen-activatingenzymes in the gland. This study concerned the activation ofN-OH-2-FBA and N-OH-2-FBA by the mammary gland and liver, achief site of metabolism, from 50-day-old female rats and effectson the activation of ovariectomy performed at 22 days of age.The levels of N-debenzolyation of N-OH-2-FBA to N-hydroxy-N-2-fluor-enamine(N-OH-2-FA), catalyzed by microsomal carboxyl-esterases in mammarygland and liver were similar and increased 1.5- and 1.7-fold,respectively, by ovariectomy. N-Debenzoylating activity in cytosolsof both tissues appeared to be partially of microsomal origin.Mammary gland cytosol contained N-, O- and N,O-acyltransferaseactivities at levels 40–50% those of liver. N-Acyltransferaseactivity was determined via acetyl coenzyme A (AcCoA)-dependentacetylation of 2-FA and a new assay, N-OH-2-FAA-dependent acetylationof 9-oxo-2-FA. The latter activity was decreased in mammarygland by ovariectomy. Microsomal N-acyltransferase activitieswere <36% those of cytosols. AcCoA-dependent binding of N-OH-2-[ring-³H]FBAto DNA, catalyzed by cytosol, was consistent with a two-stepactivation of N-OH-2-FBA involving esterase catalyzed N-debenzoylationto N-OH-2-FA and its O-acyl-transferase-catalyzed acetylationto the electrophilic N-acetoxy-2-FA. O-Acetyltransfer by mammarygland appeared to be rate-limiting since ovariectomy-dependentincreases in N-debenzoylation did not increase binding withS9 fraction. Little or no sulfotransferase-catalyzed bindingof N-OH-2-[ring-³H]FBA-derived N-OH-2-[ring-³H]FA was detectedin the liver or mammary gland cytosol, respectively. The levelof binding of N-OH-2-[ring-³H]FAA to DNA catalyzed by cytosolicN, O-acyltransferase was decreased 23% in mammary gland andincreased 1.2-fold in liver by ovariectomy. ³²P-Postlabelinganalyses indicated a single adduct N-(deoxyguanosin-8-yl)-2-fluorenaminein DNA of both tissues 24 h after one intraperitoneal injectionof N-OH-2-FBA or N-OH-2-FAA. Respective levels were 3.6- and5.5-fold greater in liver than mammary gland. After ovariectomy,the adduct levels from N-OH-2-FBA increased 1.8-fold in mammarygland and from N-OH-2-FAA decreased 50% in both tissues. Thus,the ovariectomy-dependent changes in levels of enzymes activatingN-OH-2-FBA and N-OH-2-FAA were consistent with in vivo DNA adductlevels in the target mammary gland, but not in the liver.     

In vitro DNA reaction with an ultimate carcinogen model of 4-nitroquinoline-1-oxide: the 4-acetoxyaminoquinoline-1-oxide. Enzymatic degradation of the modified DNA

2-³-H-Labelled 4-acetoxyaminoquinoline-1-oxide (Ac-4 HAQO),the ultimate carcinogen model of 4-nitroquinoline-1-oxide, wasreacted in vitro with native and denatured DNA. We found thatAc-4 HAQO is 2- to 3-fold more reactive than diAc-4 HAQO, anotherultimate carcinogen model of 4 NQO which was previously studied[Galiègue et al. (1980) Biochim. Biophys. Acta, 609,383–391]. Ac-4 HAQO-modified DNA is thermally destabilized:when 1% of the bases of DNA were modified by Ac-4 HAQO, itsmelting temperature decreased 1.2°C. Enzymatic degradationof Ac-4 HAQO-modified native and denatured DNA's to nucleosideswas performed. The hydrolysates were analyzed, first with asimple chromatographic system, and then by h.p.l.c. The compoundsrecovered from the modified polymers were characterized by h.p.l.c.and a variation in their respective amounts as a function ofthe secondary structure of DNA was observed. Especially, theN-(deoxyguanosin-(C⁸-yl)-4-aminoquinoline-1-oxide, the so calleddG III adduct, was recovered from DNA, and its amount was evaluatedto be {small tilde} 3.5-fold greater in the case of denaturedDNA than in the case of native DNA.     

DNA adduct formation and tumorigenesis in mice during the chronic administration of 4-aminobiphenyl at multiple dose levels

Recent studies have demonstrated the presence of DNA adductsfrom 4-aminobiphenyl (4-ABP) in the bladder cells of humans;however, the correlation between the concentration of theseadducts and the tumorigenic response is not clear. To help elucidatethis relationship, we have investigated DNA adduct formationin experimental animals continuously administered 4-ABP. Maleand female BALB/c mice were treated for 28 days with 4-ABP hydro-chloridein their drinking water. DNA adducts in target tissues (liverof females and bladder of males) were identified and quantifiedby ³²P-postlabeling analyses and radio-immunoassays. These resultswere compared to previously reported tumor incidences obtainedfrom the lifetime administration of 4-ABP hydrochloride. Themajor adduct observed in both tissues was N-(deoxyguanosin-8-yl)-4-ABP.In the bladders of both sexes and the livers of female mice,adduct levels increased with dose at low doses, but saturationwas observed at high doses. In the livers of males, the adductlevels were linearly correlated with dose throughout the entiredose range. A comparison between DNA adducts and tumorigenesisindicated a linear correlation between adduct levels and theincidence of liver tumors in female mice. In the bladders ofmale mice, however, the relationship was markedly nonlinear.These data suggest that adduct formation alone is insufficientfor tumorigenesis in the bladder and that other factors suchas cell proliferation are necessary for tumor production.