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.     

Pseudoenzymatic reduction of N-hydroxy-2-acetylaminofluorene to 2-acetylaminofluorene mediated by cytochrome P450

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) was reduced to 2-acetylaminofluorene by rat liver microsomes in the presence of both NAD(P)H and FAD under anaerobic conditions. The microsomal reduction proceeds as if it were an enzymatic reaction. However, when the microsomes were boiled, the activity was not abolished, but was enhanced. The activity was also observed with cytochrome P450 2B1 alone, without NADPH-cytochrome P450 reductase, in the presence of these cofactors. Hematin also exhibited a significant reducing activity in the presence of both a reduced pyridine nucleotide and FAD. The activities of microsomes, cytochrome P450 2B1 and hematin were also observed upon the addition of photochemically reduced FAD instead of both NAD(P)H and FAD. The microsomal reduction of N-OH-AAF appears to be a non-enzymatic reaction by the reduced flavin, catalyzed by the heme group of cytochrome P450.     

Mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxy-phenacetin and their respective deacetylated metabolites in nitroreductase deficient Salmonella TA98FR and TA100FR

The mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetinand their respective deacetylated metabolites, N-hydroxy-2-aminofluoreneand 2-nitrosofluorene, and N-hydroxyphenetidine and p-nitrosophenetolewas determined in nitroreductase deficient Salmonella testerstrains TA 98FR and TA100FR. The mutagenicity of N-hydroxy-2-acetylaminofluorenemediated by either rat liver microsomes or rat liver 105 000g supernatant fractions was no different in either TA98 (nitroreductaseproficient) or TA98FR (nitroreductase deficient). Similarlythe mutagenicity of N-hydroxyphenacetin mediated by hamsterliver microsomes was not affected by either the presence orabsence of nitroreductase activity in TA100. N-Hydroxy-2-aminofluoreneand 2-nitrosofluorene were equipotent direct acting mutagensin both TA98 and TA98FR, as were both N-hydroxyphenetidine andp-nitrosophenetole in TA100 and TA 100FR. Ascorbate (5 mM) andNADPH (1 mM) had no significant effect on the mutagenidty ofeither N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-aminofluorene,or 2-nitrosofluorene in TA98 or TA98FR whereas ascorbate andNADPH markedly inhibited the mutagenicity of both N-hydroxyphenetidineand p-nitrosophenetole in both TA100 and TA100FR. Ascorbateappears to be inhibiting the mutagenicity of N-hydroxyphenetidineand p-nitrosophenetole as a result of the nonenzymatic chemicalreduction of these compounds to non-mutagenic derivatives.     

Metabolism of benzo(e)pyrene by rat liver microsomal enzymes

Carcinogensis, 1, 165–173, February 1980. Page 165, Introduction, lines 18–19: should read "...is the fact that the epoxide group forms part of the bayregion of the hydrocarbon..." Page 166, Paragraph on Microsomal preparations, lines 5–6:should read "Cytochrome P450 contents of the microsomal preparations usedwere: ..." The incorrectly used "cytochrome P450 (P448)" refersto a specific form of cytochrome P450 and not total cytochromeP450 content. Page 171, Discussion, line 3 from bottom of page: should read "...Can efficiently form B(e)P bay region diol expoxides..."     

Metabolism of benzo(e)pyrene by rat liver microsomal enzymes

Metabolites of benzo(e)pyrene (B[e]P) formed upon incubationof [³H]-B[e]P with hepatic microsomes from control and inducedrats have been separated by high-pressure liquid chromatographyand identified by comparison of retention times, absorbanceand fluorescence spectra with those of synthetic standards.The major metabolite produced was B[e]P-4, 5-dihydrodiol, accountingfor 20–30% of the total metabolism depending on the sourceof the microsomes. This was followed by a phenolic metabolite(shown not to be 4-OH-; 9-OH-; or 10-OH-B[e]P). A possible proximatecarcinogenic derivative of B[e]P, B[e]P-9, 10-dihydrodiol, wasidentified, but was found to constitute less than 1% of thetotal metabolites. Similar results were obtained with a purifiedand reconstituted mixed-function oxidase system. In these laterincubations, production of the dihydrodiols was dependent onthe addition of purified epoxide hydrase to the incubation mixtures. These results suggest that formation of the reactive diol-epoxide,9, 10-dihydroxy-11, 12-epoxy-9, 10, 11,-12-tetrahydro-B[e]P,a potential ultimate carcinogenic metabolite of B[e]P, is notfavored by rat liver enzymes. This provides a partial explanationfor the lack of carcinogenicity of B[e]P within the frameworkof the bay region theory of chemical carcinogenesis.     

Selective induction of microsomal 2-acetylaminofluorene N-hydroxylation by dietary 2-acetylaminofluorene in rats

Inbred male ACI/N rats were treated with an intermittent carcinogenicregimen of 2-acetylaminofluorene (2-AAF) until the developmentof hepatomas (40 weeks), and the activation of 2-AAF by liverS-9 and the oxidation of 2-AAF and 2 other drugs by microsomalfunctions were periodically examined. The S-9 activity increasedat the end of 1 feeding cycle (3 weeks 2-AAF diet and 1 weeknormal diet), reaching a maximum of 400% of controls after 2or 3 feeding cycles. It then declined, but the elevated S-9activity (300 to 250% of controls) was sustained until the developmentof hyper-plastic nodules in the livers. The microsomal oxidationof 2-AAF to N-hydroxy-2-AAF was activated by dietary 2-AAF,but the activity of cytosol (S-105) to produce mutagen fromN-hydroxy-2-AAF was not affected. Cytochrome P-450 content andmicrosomal oxidation of aminopyrine and aniline were graduallydecreased below the normal levels by 2-AAF feeding, althoughmicrosomal p-hydroxylation of aniline was temporarily elevatedto about 130% of control at the first or the second feedingcycle. These results indicate that dietary 2-AAF selectivelyinduces microsomal 2-AAF N-hydroxylase which mediates the oxidationof 2-AAF to N-hydroxy-2-AAF, a proximate carcinogenic or mutagenicmetabolite.     

Genetic variability in deacetylation of 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in inbred strains of mice

Genetic variability in 2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene(N-OH-AAF) deacetylase activities was examined in 19 inbredstrains of mice. AAF deacetylase activities ranged from 0.60to 1.33 nmol/min/mg protein, and there was an {small tilde}2.5-folddifference in AAF deacetylase activity between the fastest (C57BL/6J)and slowest (RIIIS/J) mouse strains. N-OH-AAF deacetylase activitiesranged from 3.28 to 13.24 nmol/min/mg protein, and the differencebetween the fastest (AU/SsJ) and slowest (RIIIS/J) strains was4-fold. N-OH-AAF deacetylase activity was higher (5–13times) than AAF deacetylase activity in all strains examined.Thus, there are genetic differences in AAF and N-OH-AAF deacetylaseactivities; these differences may play an important role inindividual susceptibility to the mutagenic and carcinogeniceffects of the aromatic amides.     

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.     

Biochemical and morphological changes in hepatic nuclear membranes produced by N-hydroxy-2-acetylaminofluorene.

The effect of N-hydroxy-2-acetylaminofluorene on the ultrastructure and synthesis of hepatic neclear membranes was evaluated in partially hepatectomized rats. The incorporation of L-[4,5-3H]leucine into two nuclear membrane fractions increased within 2 hr after hepatic resection and reached a peak at 20 hr. After partial hepatectomy, the decay of radioactivity in nuclear membrane proteins labeled with L-[4,5-3H]leucine revealed similar half-lives for the two membrane fractions when compared to those obtained from sham-operated animals. The protein concentration of the nuclear membrane fraction of higher density decreased sharply within 2 hr after partial hepatectomy and remained low throughout a 20-hr postoperative period. Polyacrylamide gel electrophoresis of both nuclear membrane fractions showed a similar composition. Nine proteins were resolved, varying from 21,000 to 190,000 daltons. The two major protein bands were in the range of 50,000 and 70,000 daltons, respectively. Treatment of partially hepatectomized animals with N-hydroxy-2-acetylaminofluorene showed marked dilation of the nuclear envelope and rough endoplasmic reticulum in situ upon electron microscopic examination. Vacuolization and evagination of the perinuclear membranes were also noticeable in isolated nuclei obtained from carcinogen-treated rats. Inhibition by N-hydroxy-2-acetylaminofluorene of the incorporation of L-[4,5-3H]leucine into the nuclear membranes was dose-dependent and remained depressed throughout a 60-min labeling period. These results suggest that the inhibitory effects on RNA and protein synthesis previously shown to be produced by this arylhydroxylamine hepatocarcinogen may lead to disruption of the morphology and synthesis of the nuclear envelope.     

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.     

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.     

Effect of glutathione depletion on the hepatotoxicity and covalent binding rat liver macromolecules of N-hydroxy-2-acetylaminofluorene

Glutathione plays an important role in the protection of the liver against several hepatotoxins. The hepatocarcinogen N-hydroxy-2-acetylaminofluorene is converted in the rat in vivo to reactive metabolites that bind covalently to cellular macromolecules. These metabolites may also react with glutathione, resulting in the formation of glutathione conjugates and in the detoxification of reactive metabolites. The role of glutathione in detoxification was investigated by depletion of glutathione in the rat in vivo with diethyl maleate. When rats were pretreated with diethyl maleate, 45 min before the administration of N-hydroxy-2-acetylaminofluorene, excretion of 2-acetylaminofluorene:glutathione conjugates in bile was decreased by 60% as compared to controls. However, total covalent binding to rat liver protein was not increased, and total binding to DNA was even decreased (p less than 0.1), apparently at the expense of the acetylated carcinogen-DNA adducts. Formation of deacetylated, 2-aminofluorene adducts to DNA was not affected by diethyl maleate. Pretreatment with diethyl maleate had no major effect on the acute hepatotoxic effects of N-hydroxy-2-acetylaminofluorene. The results indicate that glutathione does not play a vital role in the detoxification of reactive metabolites generated from the carcinogen N-hydroxy-2-acetylaminofluorene, since glutathione is not very effective in competing with macromolecules for trapping of reactive metabolites of N-hydroxy-2-acetylaminofluorene. Thus, 1 mM glutathione did not decrease the covalent binding of 2-acetylaminofluorene-N-sulfate (one of the main reactive metabolites that is formed in vivo) to DNA in vitro, while 10 mM glutathione decreased the covalent binding to RNA by only 20% and to DNA by only 40%.     

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.     

Irreversible inhibition of the cytosolic metabolism of N-hydroxy-2-acetylaminofluorene by its glycolyl analog

The glycolyl hydroxamic acid derivative of 2-aminofluorene was found to be a potent inhibitor of its own metabolism and the metabolism of N-hydroxy-2-acetylaminofluorene by rat liver cytosol. The inhibition was irreversible, as well as time and concentration dependent, which indicates a suicide-inhibition type of metabolism. There was a direct correlation between the inhibition of N-hydroxy-2-acetylaminofluorene disappearance and 2-acetylaminofluorene formation. In contrast, both the glycolyl and acetyl hydroxamic acid derivatives were metabolized to a similar extent by enzymes in the microsomal fraction.     

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.