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.     

Formation and persistence of DNA adducts from the carcinogen N-hydroxy-2-acetylaminofluorene in rat mammary gland in vivo

The rat mammary carcinogen, N-hydroxy-2-acetylamino-fluorene(N-hydroxy-2-AAF), has been proposed to be metabolically activatedby mammary cytosolic N,O-acetyltransferase to a DNA bindingspecies. To test this hypothesis, adult female Sprague-Dawleyderived CD rats were treated, l.p., with 4.0 mg/kg [ring-³H]N-hydroxy-2-AAF.After 4 h, 1, 3, 14, and 28 days, the animals were killed, themammary epithelium DNA was isolated and the carcinogen-deoxyribonucleosideadducts present were analyzed by high pressure liquid chromatography.At each time, only one adduct was detected and it was chromatographicallyidentical to N-(deoxyguanosin-8-yl)-2-aminofluorene. The levelof the adduct was maximal at 4 h (1.5 adducts/10⁶ nucleotides)and then decreased, following first order kinetics with a t_1/2of 14.2 days. The detection of a single non-acetylated aminofluoreneadduct is consistent with N,O-acyltransferase being involvedin the metabolic activation of N-hydroxy-2-AAF in the rat mammarygland.     

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.     

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.     

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.     

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.     

Detection of N-hydroxy-2-acetylaminofluorene--DNA adducts in rat liver measured by radioimmunoassay

Antibodies to imidazole ring opened derivatives of alkali treatedN-(guanosin-8-yl)-2-aminofluorene (1-[6-(2, 5-diamino-4-oxopyrimidinyl-N⁶-riboside)]-3-(2-fluorenyl)ureawere elicited in rabbits by immunization with a conjugate betweenthe ring-opened derivative and bovine serum albumin. The specificityof the antibodies was studied by radio-immunoassay. These antibodiesand antibodies to N-(guanosin-8-yl)-N-acetyl-2-aminofluorenewere used to titrate the adducts formed in liver DNA of ratstreated with N-hydroxy-2-acetylaminofluorene. The ring-openedderivatives represent 10% of the dGuo-C8-adducts.     

Formation of DNA adducts by the carcinogen N-hydroxy-2-naphthylamine

The probable ultimate urinary bladder carcinogen, N-hydroxy-2-naphthylamine (N-OH-2-NA), reacted with nucleic acids and proteins under mildly acidic conditions (pH 5) to form covalently bound derivatives. The extent of reaction was in the order: Polyguanylic acid greater than DNA approximately protein greater than rRNA greater than tRNA greater than polyadenylic acid approximately polyuridylic acid greater than polycytidylic acid. At pH 7, appreciable reaction occurred only with protein. Enzymatic hydrolyses of the DNA, which contained 1.5 naphthyl residues/1,000 nucleotides, yielded 3 nucleoside-arylamine adducts. From chemical, UV, nuclear magnetic resonance, and mass spectrometric analyses, the adducts were identified as 1-(deoxyguanosin-N2-yl)-2-NA, 1-(deoxyadenosin-N6-yl)-2-NA, and a purine ring-opened derivative of N-(deoxyguanosin-8-yl)-2-NA, tentatively identified as 1-[5-(2-6-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-2-(2-naphthyl)urea. Preliminary experiments with a dog given [3H]2-NA suggested the presence of these adducts in vivo. The properties of adducts derived from N-OH-1-NA and N-OH-2-NA and their possible roles in the initiation of carcinogenesis are discussed.     

N-hydroxy-2-acetylaminofluorene inhibition of rat live RNA polymerases.

Administration of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to rats inhibits liver nuclear RNA synthesis. This effect is reflected in an in vitro inhibition of RNA synthesis by isolated whole nuclei. The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from these nuclei. Both nucleolar and nucleoplasmic polymerases are affected. A similar inhibition of the polymerases was demonstrated in intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxyadenylate-deoxythymidylate). Chromatin was prepared from similar nuclear preparations by two methods, differing in the extent to which they remove endogenous polymerase activity. Each chromatin preparation was transcribed with added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase respectively. With either polymerase and either chromatin preparation, no inhibition of the template activity of chromatin isolated from N-OH-AAF-treated animals could be detected. It is concluded that N-OH-AAF is a potent inhibitor of rat liver nuclear RNA synthesis and that the mechanism of this inhibition is inactivation of the RNA polymerases. At the same time, N-OH-AAF leaves the chromatin template, at least quantitatively, intact for the synthesis of RNA. The implications of such an effect of N-OH-AAF on RNA synthesis are discussed.     

Metabolism of N-hydroxy-2-acetylaminofluorene and N-hydroxy-phenacetin by guinea pig liver microsomal enzymes

Deacylation has been proposed as a mechanism of activation ofarylhydroxamic acids. In the present studies solubilized preparationsfrom guinea pig liver microsomes, a source of high deacylaseactivity, were subjected to gel filtration on Sephacryl S-200.A single peak (peak I) of activity was found when column fractionswere assayed colorimetrically for deacylation of N-hydroxy-2-acetylaminofluorene(N-OH-AAF). Corresponding to this peak were the following activities:binding of [³H-ring]-N-hydroxy-phenacetin (N-OH-P) to tRNA anddeacylation of N-OH-P and N-OH-AAF, measured by the formationof nitrosophenetole (N=O-P) and nitrosofluorene (N=-F), respectively.The binding of [³-ring]-N-OH-AAF to tRNA was catalyzed by peakI, but to a greater extent by a second peak (II). The bindingof both N-OH-P and N-OH-AAF to tRNA was inhibited by paraoxon,an esterase inhibitor. H.p.l.c. analysis revealed that for peakI, the major ether-extractable metabolites of N-OH-P and N-OH-AAFwere the corresponding nitroso derivatives. In the presenceof peak II, little metabolism to organic-extract-able metabolitesoccurred. These data indicate that more than one mechanism isinvolved in the activation of N-OH-P and N-OH-AAF in this system,and that the difference in the activation of these arylhydroxamicacids cannot be explained by differences in the formation ofdeacylated metabolites.     

Microfluorometric determination of DNA adducts in immunofluorescent-stained liver tissue from rats fed 2-acetylaminofluorene

The intensities of immunofluorescence in nuclei stained by an antiserum specific for the DNA adduct N-deoxyguanosin(8-yl)aminofluorene (dG-8-AF), were quantified by microfluorometry in frozen liver sections from male Fischer rats fed 2-acetylaminofluorene (AAF). Results of previous studies demonstrated that dG-8-AF is the predominant adduct (80-100%) formed in livers of rats fed AAF continuously, and that nuclei of hepatocytes and bile duct epithelial cells in rats fed AAF exhibit an adduct-specific immunofluorescence. In the present investigation, nuclear staining for dG-8-AF was quantified by microfluorometry in liver sections from male Fischer rats fed 0.02% AAF continuously for 2, 4, 8, 12, 16, 20, and 28 days. Microfluorometric determinations of the intensities of nuclear immunofluorescence staining within periportal, midzonal, and centrilobular hepatocytes and bile duct epithelial cells revealed that levels of the dG-8-AF adduct increased in these cells during AAF feeding, reaching a plateau by 12 days. However, significant differences were detected in dG-8-AF levels within cells of each lobular area. Nuclei of periportal hepatocytes exhibited the most intense immunofluorescence, nuclei of centrilobular hepatocytes and bile duct epithelial cells emitted the least intense fluorescence, and nuclei of midzonal hepatocytes exhibited an intermediate fluorescence intensity. Quantitation of whole-liver levels of the dG-8-AF adduct by RIA, after extraction of DNA, also revealed that adduct accumulation reached a plateau by 12 days of AAF feeding. Thus, similar profiles of adduct accumulation were obtained by microfluorometric analysis of immunofluorescence staining within frozen liver sections, and by RIA analysis of DNA extracted from whole livers. The periportal concentration of DNA adducts in livers of rats continuously fed a carcinogenic dose of AAF may be an important early event in AAF-induced liver tumorigenesis.     

Self-catalyzed irreversible inactivation of rat hepatic aryl sulfotransferase IV by N-hydroxy-2-acetylaminofluorene

Rat hepatic aryl sulfotransferase IV catalyzes the sulfonation of the hepatocarcinogen, N-hydroxy-2-acetylaminofluorene. The resulting reactive N-O-sulfate ester is believed to be the ultimate carcinogenic species responsible for the induction of hepatic neoplasia. Previous studies have shown that dietary administration of either 2-acetylaminofluorene or N-hydroxy-2-acetylaminofluorene to rats is accompanied by a rapid decline in hepatic aryl sulfotransferase activity in vivo. In the present study, preincubation of purified rat hepatic aryl sulfotransferase IV with N-hydroxy-2-acetylaminofluorene resulted in rapid, time-dependent enzyme inactivation. This in vitro inactivation was not reversed by dialysis or gel filtration. Inclusion of excess nucleophile, methionine, resulted in considerable but not complete protection from inactivation. The inactivation was PAPS dependent and blocked by the sulfotransferase inhibitor, pentachlorophenol. The above observations and the apparent pseudo first-order kinetics observed suggest that the inactivation was in part mechanism based. Mechanism-based inactivation of the aryl sulfotransferases has not been previously reported. Furthermore, the results of the present study indicate that the previously reported in vivo decline in rat hepatic aryl sulfotransferase activity may be attributable in part to enzyme inactivation by its own reactive product.     

Correlation between clastogenicity and promotion activity in liver carcinogenesis by N-hydroxy-2-acetylaminofluorene, N-hydroxy-4'-fluoro-4-acetylaminobiphenyl and N-hydroxy-4-acetylaminobiphenyl

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were compared for their initiation and promotion activity in the rat liver using a modified Solt-Farber system. N-OH-AAF, N-OH-FAABP and N-OH-AABP showed comparable initiation capacity when administered to male Wistar rats at a dose of 30, 120 and 120 mumol/kg respectively, 24 h after a two-thirds partial hepatectomy (PH). In contrast, only N-OH-AAF was very effective as promoter when administered to rats previously initiated with diethylnitrosamine. This was evidenced by a high number of large gamma-glutamyltranspeptidase-positive (GGT+) foci occupying a high percentage (22%) of liver volume. N-OH-FAABP was a much weaker promoter, resulting in smaller foci and lower percentage (4%) of GGT+ liver volume. The incomplete carcinogen N-OH-AABP was totally ineffective as promoter in our model. A similar difference was seen in the clastogenicity of these carcinogens in rat liver in vivo as measured by the formation of micronuclei: N-OH-AAF was far more clastogenic than N-OH-FAABP, which in turn was more clastogenic than N-OH-AABP. We have recently shown that N-acetylated deoxyguanosine adducts are responsible for clastogenicity of N-OH-AAF and may be important for promotion. DNA adduct analysis after injection of 120 mumol/kg of tritium-labeled N-OH-FAABP or N-OH-AABP, 24 h after PH, showed that N-acetylated adducts to C8 of deoxyguanosine are also formed from these structurally related liver carcinogens. However, the formation of these adducts from N-OH-FAABP and N-OH-AABP was approximately 8 and approximately 5% of the formation of dG-C8-AAF after injection of 25 mumol/kg N-OH-AAF. These data show that for the structurally related liver carcinogens N-OH-AAF, N-OH-FAABP and N-OH-AABP, clastogenicity does not predict initiating efficacy but correlates with promotion activity. Possibly, N-acetylated adducts to C8 of deoxyguanosine are involved in both clastogenicity and promotion.