The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures

Primary cultures of rat hepatocytes were exposed to [ring-³H]-N-hydroxy-2-acetylaminofluorene(N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts wereisolated and identified by h.p.l.c. The DNA adducts were shownto be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF),N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxy-guanosin-8-yl)-2-acetylaminofluorene(dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene,and N-(guanosin-8-yl)-2-aminofluorene. The removal of theseadducts was measured up to 38 h following the cessation of exposureof the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removedwith a half-life of approximately 10 h, while dG-N²-AAF anddG-C8-AF remained constant for 14 h, followed by a slow rateof removal. The dG-C8-AAF adduct initially composed about 60%of the total DNA adducts of primary hepatocytes in contrastto the 20% found in liver in vivo. The formation of the 3 DNAadducts and the different rates of repair indicate that primarycultured rat hepatocytes may be a valuable system to study initiationof liver carcinogenesis by N-OH-AAF.     

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

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

Quantification of adducts formed in DNA treated with N-acetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene: comparison of trifluoroacetic acid and enzymatic degradation

We have examined two methods of preparation of DNA adducts fromX174 RF DNA modified by [³H]N-acetoxy-2-acetylaminofluorene([³H]NA-AAF) or N-hydroxy-2-amino- fluorene ([³]N-OH-AF). Hydrolysisby enzymes (DNase I, snake venom phosphodiesterase and alkalineor acid phosphatase) and subsequent reverse phase h.p.l.c. ofX174 RF DNA treated with [³H]NA-AAF yielded 73% N-(deoxyguanosin-8-yl)-2-acetylaminofluorene(dG-C8-AAF), 7% 3-(deoxyguanosin-N²-yl)-2acetylaminofluorene(dG-N²-AAF), and a peak of unidentified radioactivity (13%).When [³H]N-OH-AF modified X174 DNA was analyzed, both N-(deoxyguanosin-8-yl)-2-aminofluorene(dG-C8-AF) and a large percentage of the imidazole ring-openedderivative and unidentified products were found. In contrast,when anhydrous trifluoroacetic acid (TFA) was used to degradethese DNAs, we found for the [³H]NA-AAF modified DNA 86% N-(guanin-8-yl)-2-acetylaminofluorene(G-C8-AAF) and 6% 3-(guanin-N²-yl)-2acetylaminofluorene (G-N²-AAF),while for [³H]N-OH-AF modified DNA only the N-(guanin-8-yl)-2-aminofluorene(G-C8-AF) was found. When DNA was prepared from human fibroblaststreated with [³H]NA-AAF, only the G-C8-AF product was obtained.Thus, anhydrous TFA solvolysis followed by reverse phase h.p.l.c.is a rapid and convenient method to obtain quantitative yieldsof DNA adducts formed with acetylamino-fluorene and relatedcompounds: quantification by this method prevents loss of G-N²adducts, the conversion of AAF adducts to AF adducts, and theproduction of ring opened products in guanine residue.     

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.     

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

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

Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA.

N-(Deoxyguanosin-8-yl)-2-(acetylamino)fluorene (AAF-G) adducts in the DNA of bacteriophage M13 can be converted to N-(deoxyguanosin-8-yl)-2-aminofluorene (AF-G) adducts in situ by treatment with 1.0 M NaOH for 45 min at room temperature. The conversion is accompanied by a dramatic increase in the transfection activity of the samples which is correlated with the measured deacetylation of the acetylaminofluorene adduct. The pair of substrates (AAF-G/AF-G) with adducts at identical places in the DNA has been used to study bypass synthesis catalyzed by T7 DNA polymerase, an altered T7 DNA polymerase from which the 3'----5' exonuclease has been genetically removed by an 84 nucleotide deletion (Sequenase 2), T4 DNA polymerase and Escherichia coli DNA polymerase I. All polymerases appear blocked at acetylaminofluorene lesions. Sequenase 2 is apparently able to add nucleotides opposite the acetylaminofluorene lesion but is unable to catalyze further elongation. T7 DNA polymerase, including thioredoxin and with an active 3'----5' exonuclease, is unable to bypass aminofluorene adducts, whereas Sequenase 2 bypasses the lesions readily. The data support the view that the elongation step is rate limiting in synthesis past lesions and that low 3'----5' exonuclease activity allows the priming nucleotide opposite the altered template site to remain in position long enough for elongation past particular adducts.     

The role of specific DNA adducts in the induction of micronuclei by N-hydroxy-2-acetylaminofluorene in rat liver in vivo

N-Hydroxy-Z-acetylaminofluorene (N-OH-AAF) was administeredi.p. to male Wistar rats 17 h after partial hepatectomy. Hepatocyteswere analyzed for the presence of micronuclei 7 h, 1, 2, 3 and4 days after injection. N-OH-AAF treatment resulted in a highfrequency of micronucleated hepatocytes at days 3 and 4 (19.5and 19.6 respectively). The frequency of micronucleated hepatocyteswas not increased above control values when hepatocytes wereisolated as early as 7 h, 1 or 2 days after injection. Pretreatmentwith the sulfotransferase inhibitor pentachlorophenol (PCP)45 min before injection of N-OH-AAF almost completely preventedthe formation of micronuclei by N-OH-AAF. Parallel biochemicalstudies indicated that inhibition of sulfation of N-OH-AAF byPCP pretreatment prevented the formation of the N-acetylatedDNA adducts iV-deoxyguanosin-8-yl-AAF and 3-deoxyguanosin-N²-yl-AAFby {small tilde}85%. Total adduct formation to DNA was, however,not lowered because of an increase in the formation of the deacetylatedadduct, N-deoxy-guanosin-8-yl-AAF. The lower frequency of micronucleatedhepatocytes observed in the group pretreated with PCP, did notresult from less proliferative activity in this group as comparedto the group treated with N-OH-AAF alone. Therefore, the decreasein the formation of micronuclei indicates that PCP preventsthe clastogenic damage caused by N-OH-AAF. It is concluded thatthe clastogenicity of N-OH-AAF in rat liver is related to theformation of N-acetylated DNA adducts (i.e. N-deoxyguanosin-8-yl-AAFand/or 3-deoxy-guanosin-N²-yl-AAF) and is not related to theformation of the deacetylated DNA adduct N-deoxyguanosin-8-yl-AF.     

Formation and removal of specific acetylaminofluorene-DNA adducts in mouse and human cells measured by radioimmunoassay.

Rabbit antiserum prepared against N-(guanosin-8-yl)-acetylaminofluorene was utilized in radioimmunoassay to detect formation and removal of C-8 adducts from the DNA of cultured cells exposed to N-acetoxy-2-acetylaminoflorene. The assay was able to quantitate both acetylated and deacetylated C-8 adducts between 0.5 and 5 pmol while the N2 adduct, 3-(deoxyguanosin-N2-yl)acetylaminofluorene, was not detected below 160 pmol. By varying the proportions of acetylated and deacetylated C-8 adducts in the radioimmunoassay, a series of standard curves were developed from which the relative proportion of each adduct could be determined in unknown mixtures. DNA from mouse epidermal cells and human skin fibroblasts exposed to N-acetoxy-2-acetylaminofluorene in culture contained only 3 and 5% respectively, of the C-8 adduct in the acetylated form. Quantitation by radioimmunoassay of total C-8 adducts bound to DNA yielded values approximately 25% lower than total carcinogen binding determined by radiolabeling. When removal of C-8 adducts was followed over a 23-hr, carcinogen-free culture period, mouse and human cells removed 40 and 50%, respectively, of bound acetylated and deacetylated C-8 adducts. These studies demonstrate the versatility of radioimmunoassay as a molecular probe for studies of chemical carcinogens.     

Metabolism and DNA adduct formation of 2-acetylaminofluorene by bladder explants from human, dog, monkey, hamster and rat

Cultured bladder explants from human, dog, monkey, hamster andrat were used to study the metabolism, and DNA-adduct formationof [³H]2-acetylaminofluorene (AAF). After 24 h of incubationwith 1 µM [³H]AAF, the organic-soluble and glucuronidefractions represented 29.0–58.4% and 0.7–10.8%,respectively, of the total amount of radioactive material inthe medium. In the organic-soluble fraction of all species,the presence of both ring-hydroxylated (at the 7-, 9-, 5-, 3-and 1-carbons) metabolites and N-hydroxy-AAF could be demonstrated,with 7-hydroxy-AAF and 9-hydroxy-AAF being the most ubiquitousmetabolic products. In addition, 2-aminofluorene could be detectedin all cases with the highest amounts formed by dog bladderexplants. Species susceptible to aromatic amine-induced bladdercarcinogenesis (human and dog) showed widely varying DNA binding(0.23 ± 0.20 and 1.95 ± 1.2 µmol AAF/moldeoxyribonudeotide, respectively), and in explants of the lesssusceptible species (monkey, hamster and rat) DNA binding wascomparable (0.46 ± 0.21, 0.37 ± 0.07 and 0.34± 0.27, respectively). Enzymatic hydrolysis of the [³H]AAF-DNAto the nucleoside level revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene,together with small amounts (5.9–15.4% of the total) ofits imidazole ring-opened derivative, was the most ubiquitousadduct formed (17.8–47.2% of the total) in all species,except the dog. In dog bladder DNA, N-(deoxyguanosine-8-yl)-2-acetyl-aminofluorenewas the principal DNA adduct (39.0–46.5% of the total)with smaller amounts (15.5–22.2% of the total) in theother species. In addition, small amounts (3.5–8.6% ofthe total) of another adduct, 3-(deoxygaunosin-N²-yl)-2-acetylaminofluorene,could be detected in all cases. It is concluded that bladderexplants of the human, dog, monkey, hamster and rat metabolizeAAF mainly to ring-hydroxylated products, but also form smallamounts of the proximate carcinogenic metabolite N-hydroxy-AAF.Neither the overall binding of AAF to bladder DNA, nor the formationof specific AAF-DNA adducts is correlated with the relativesusceptibilities of these five species to aromatic amine-inducedurinary bladder carcinogenesis.     

Formation of urothelial and hepatic DNA adducts from the carcinogen 2-naphthylamine

The carcinogen, 2-naphthylamine (2-NA), induces tumor formationin the urinary bladder but not the liver of several species,including humans and dogs. Since its proximate carcinogenicmetabolite, N-hydroxy-2-NA, was known to react directly withDNA in vitro to give specific carcinogen-base adducts, we investigatedthe in vivo formation and persistence of (2-NA) - DNA adductsin the bladder and liver and attempted to determine whetheror not these lesions correlated with tissue susceptibility.Male beagle dogs were administered [³H]2-NA and sacrificed after2 or 7 days. The DNA was isolated from the liver and urotheliumand hydrolyzed enzymatically. The (2-NA)-deoxyribonucleosideadducts, which were quantitated by high pressure liquid chromatographicanalysis, were the same as those found in vitro, namely 1-(deoxyguanosin-N²-yl)-2-NA,1-(deoxyadenosin-N⁶-yl)-2-NA, and an imidazole ring-opened derivativeof N-(deoxyguanosin-8-yl)-2-NA. The major difference detectedbetween target and non-target tissues was in the total levelof binding to DNA, which was 4-fold higher in the urotheliumat 2 days and 8-fold higher at 7 days after 2-NA dosing. Analysisof specific adducts suggested that this difference may be dueto the relative persistence of the C-8-guanine adduct in theurothelium as compared to the liver. Similar experiments withthe non-carcinogen, 1-naphthylamine, failed to reveal bindingin urothelial DNA and indicated a 20-fold lower binding levelin hepatic DNA. Evidence for binding of 2-NA to glycogen isalso presented and problems associated with measuring totalradioactivity in glycogen-contaminated DNA fractions are discussed.The data obtained in this study, though from a necessarily limitednumber of animals, are consistent with the hypothesis that theformation and persistence of DNA-carcinogen adducts may be importantin the initiation of the neoplastic process.     

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.     

Persistence of DNA adducts in rat liver and kidney after multiple doses of the carcinogen N-hydroxy-2-acetylaminofluorene

Male and female Sprague-Dawley rats were treated by gastric intubation either 1, 2, 3, or 4 times at biweekly intervals with 10-mg/kg doses of the hepatocarcinogen of N-[ring-3H]-hydroxy-2-acetylaminofluorene. Then either 1 or 14 days following the last treatment, the concentrations of 2-aminofluorene and 2-acetylaminofluorene adducts in liver and kidney DNA were established. 2-Acetylaminofluorene adducts were found in male rat liver DNA only. The C-8-acetylated adduct, N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, was detected only on the day following treatment at a concentration between 1.0 and 2.4 pmol/mg DNA. A second acetylated adduct, 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, was found at both 1 and 14 days after treatment and, as a result, increased in concentration with repeated doses, from 0.2 pmol/mg DNA after one dose to 2.8 pmol/mg DNA after four treatments. The major adduct detected in male rat liver and the only adduct found in female rat liver and in kidney from either sex was N-(deoxyguanosin-8-yl)-2-aminofluorene. This adduct was slowly lost from the DNA and therefore increased in concentration with repetitive treatments as follows: male liver, 4.0 to 9.4 pmol/mg DNA; female liver, 11.4 to 30.6 pmol/mg DNA; male kidney, 1.1 to 1.8 pmol/mg DNA; and female kidney, 1.8 to 17.7 pmol/mg DNA. These data indicate that certain DNA adducts can accumulate in both target and non-target tissues and therefore suggest that persistence of DNA adducts per se is not sufficient for tumor induction.     

DNA adducts formed from the probable proximate carcinogen, N-hydroxy-3,2' -dimethyl-4-aminobiphenyl, by acid catalysis or S-acetyl coenzyme A-dependent enzymatic esterification

The arylamine carcinogen 3,2' -dimethyl-4-aminobiphenyl (DMABP)has been proposed to be metabolically activated to DNA-bindingderivatives through the formation of an N-hydroxy intermediate.In this study, the subsequent activation of N-hydroxy-DMABPthrough acid catalysis or enzymatic esterification was examined.[Ring-³H]N-hydroxy-DMABP was reacted with calf thymus DNA atpH 4.6 for 15 min to yield 370 arylamine residues per 10⁶ nucleotides,while at pH 7.4 the binding was only two residues per 10⁶ nucleotides.The DNA modified under acidic conditions was enzymatically hydrolyzedand analyzed by h.p.l.c. which indicated the presence of threemajor adducts. The products were identified by spectral andchemical properties as N-(deoxyguanosin-8-yl)-DMABP (60–70%),5-(deoxyguanosin-N²-yl)-DMABP (2–3%) and N-(deoxyadenosin-8-yl)-DMABP(1–3%). The same adducts have previously been detectedin the liver and colon of rats administered DMABP or its hydroxamicacid. Incubation of rat hepatic or intestinal cytosol at pH7.4 for 15 min with [ring-³H]N-hydroxy-DMABP in the presenceof S-acetyl coenzyme A (AcCoA) and calf thymus DNA resultedin DNA binding at levels of 30–80 arylamine residues per10⁶ nucleotides. H.p.l.c. analysis of the DNA modified in thepresence of AcCoA indicated the formation of the same adductsdetected in the acid-catalyzed reactions. When arylhydroxamicacid N,O-acyltransferase assays were conducted with rat livercytosol and N-acetyl-N-hydroxy-DMABP as the substrate, bindingto nucleic acids was not observed. Similarly, 3'-phosphoadenosine-5'-phosphosulfate-dependentsulfotransferase-mediated DNA binding could not be demonstrated.These data indicate that in a suitable acidic environment, N-hydroxy-DMABPwill react with DNA to yield the same adducts found in vivo.Under neutral conditions, however, N-hydroxy-DMABP appears toundergo AcCoA-dependent transacetylation to an electrophilicacetoxy ester which will spontaneously react with DNA.     

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.     

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.     

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.     

DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo

The presence of relatively high levels of prostaglandin H synthase(PHS) in the dog urinary bladder and its ability to mediatethe activation of carcinogenic arylamines to DNA-bound productsin vitro suggests the involvement of this enzyme in arylamine-inducedbladder carcinogenesis. Since the PHS-dependent metabolism of2-naphthylamine (2-NA) had been shown to yield both ring- andN-oxidation products in vitro, we compared the reactivity of³H-labeled N-hydroxy-2-naphthylamine (N-OH-2-NA), 2-nitrosonaphthalene,and 2-amino-1-naphthol (2-AN) toward DNA and protein. In thePHS-incubation system, all three derivatives bound at high levelsto protein, but only N-OH-2-NA and 2-AN bound appreciably toDNA. Though ring-oxidation has usually been considered a detoxificationpathway, the covalent binding of [³H]2-AN to DNA was found tooccur readily under aerobic conditions and was enhanced at acidicpH. At pH 5 in air, the reactivity of [³H]2-AN with nucleicacids and protein was in the order: serum albumin > tRNA> poly G > poly C > DNA > poly A > rRNA >poly U. Enzymatic hydrolysis of DNA reacted with [³H]2-AN andsubsequent analysis by h.p.l.c. indicated the presence of severalcarcinogen-nucleoside adducts. The major product was characterizedas N⁴-(deoxyguanosin-N²-yl)-2-amino-1,4-naphthoquinoneimine;and two minor products were tentatively identified as N⁴-(deoxyadenosin-N⁶-yl)-2-amino-1,4-naphthoquinoneimineand a deoxyguanosin-N²-yl adduct of a naphthoquinoneimine dimer.These adducts accounted for 60% of the total DNA binding obtainedby incubation of [³H]2-NA with PHS in vitro and for 20% of the[³H]2-NA bound to dog urothelial DNA in vivo. The remainingadducts were identical to those previously reported as productsof the reaction of N-OH-2-NA with DNA. These results suggestthat a minor proportion of the DNA adducts found in vivo maybe formed by PHS-activation of 2-NA in the target tissue. Furthermore,the reactivity of 2-AN with cellular nucleophiles, presumablythrough formation of 2-imino-1-naphthoquinone or a protonated4-naphthocarbenium ion, indicates that ring-oxidation productsof arylamines and of other carcinogenic aryl compounds shouldbe evaluated as proximate carcinogenic metabolites.     

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.     

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.     

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.