Polymorphic metabolism of the carcinogen 2-acetylaminofluorene in human liver microsomes

Two distinct phenotypes, slow and fast metabolizers, were observedfor both metabolic activation and detoxification of the modelchemical carcinogen, 2-acetylaminofluorene, in human liver microsomesfrom 28 individuals. We observed that individuals who were fastactivators of the carcinogen were in most cases also fast detoxifiersof the chemical. However, different phenotype patterns existsuggesting that fast activators of a toxin need not also bephenotyped as fast detoxifiers. Studies in this area may helpunderstand whether certain individuals are predisposed to ahigher rate of chemically-induced cancers.     

The carcinogen 2-acetylaminofluorene inhibits activation and nuclear accumulation of cyclin-dependent kinase 2 in growth-induced rat liver

Growth arrest in G(1) is a common cellular response to DNA damage. In the present study, liver regeneration was combined with continuous exposure for 2-acetylaminofluorene (AAF) to study mechanisms of carcinogen-induced growth arrest in vivo. Growth arrest of uninitiated hepatocytes is central for AAF-induced promotion of premalignant lesions in rat liver. To characterize this growth arrest, we examined the activity of cyclin-dependent kinase (Cdk) 2 in unexposed liver and in AAF-exposed liver after growth induction by partial hepatectomy (PH). Rats were fed either a control diet or an AAF-supplemented diet. After 7 d, a two-third PH was performed and the animals were killed after 0, 12, 18, 24, and 36 h. Kinase assays showed that cyclin E- and Cdk2-associated activities were lower in AAF-exposed liver than in unexposed liver after PH. Although the total cellular levels of cyclin E and Cdk2 were similar, cyclin E-Cdk2 assembly was markedly reduced. In unexposed hepatocytes, Cdk2 translocated to the nuclei after PH. Much of the nuclear Cdk2 was in a rapidly migrating form, presumably representing the Thr160-phosphorylated form of Cdk2. In contrast, in AAF-exposed liver both nuclear Cdk2 accumulation and Thr160-phosphorylation of Cdk2 were reduced. Although p53 and p21(waf1/cip1) were induced by AAF, the binding of p21 to cyclin E and Cdk2 was not increased in growth arrested liver. In conclusion, hepatocyte growth arrest caused by AAF exposure was characterized by a lowered Cdk2 activity that was accompanied by a reduced assembly of cyclin E-Cdk2 complexes but not by binding of p21.     

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.     

Decreased sensitivity to phalloidin of normal-looking rat hepatocytes after short-term 2-acetylaminofluorene feeding

Male F344/DuCrj rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 or 3 weeks, and then fed a basal diet for 2 days, 2 weeks, 8 weeks, 22 weeks or 36 weeks. Hepatocytes were isolated from the liver by collagenase perfusion, and their sensitivity to phalloidin, in terms of the formation of multiple cytoplasmic blebs, was examined. The sensitivity of gamma-glutamyltransferase (GGT)-negative hepatocytes decreased on the 22nd and 36th weeks after withdrawal of 2-AAF feeding, and that of GGT-positive cells decreased on the 36th week. Induction of a small number of foci positive for the placental form of glutathione S-transferase (GSTP) was observed in the liver of all rats on the 8th, 22nd and 36th weeks after the withdrawal of the carcinogen. However, the total area of the foci was estimated to account for less than 0.2% of liver tissues even on the 36th week. Therefore, the decrease in phalloidin sensitivity of hepatocytes, particularly of GGT-negative hepatocytes, on the 22nd and 36th weeks after 2-AAF withdrawal is suggested to be a result of a decrease in the sensitivity of otherwise normal-looking hepatocytes, which may be precursors of the cells forming the preneoplastic foci.     

Effects of the tumor promoter 2-acetylaminofluorene and of 4-acetylaminofluorene on rat liver growth

2-Acetylaminofluorene (2-AAF) is a moderately cytotoxic drug which is nevertheless capable of promoting diethylnitrosamine-initiated rat liver carcinogenesis in the absence of detectable hepatotoxicity. 2-AAF does not stimulate net growth of the normal liver, but alters hepatocellular ploidy distributions (fewer binucleated and more diploid cells) in a manner indicative of altered growth control. The non-cytotoxic analogue 4-AAF induces a similar ploidy change, and also stimulates liver growth. The possibility should therefore be considered that both agents may specifically elicit a mononucleating growth response in normal liver, which in the case of 2-AAF is antagonized by a general (cytotoxic) growth inhibition. In the promotion of liver carcinogenesis, 2-AAF may act as a growth stimulant upon cytotoxicity-resistant initiated cells; cytotoxicity per se playing no causative role.     

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.     

Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 ± 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.     

Interaction of nucleophiles with the enzymatically-activated carcinogen, N-hydroxy-2-acetylaminofluorene, and with the model ester, N-acetoxy-2-acetylaminofluorene

A new method was devised to study adduct formation of N-acetyl-L-methionine(N-Ac-met) with activated species of the carcinogen N-hydroxy-2-acetylaminofluorene(N-OH-2-AAF). Following degradation of the adducts, the isomericmethylthio derivatives of 2-acetylaminofluorene (2-AAF) and2-aminofluorene (2-AAF) were separated and quantified by h.p.l.c.With the use of this method the nucleophilicity of N-Ac-mettoward enzymatically produced N-acetoxy and N-sulfonoxy derivativesof 2-AF and 2-AAF, respectively, and toward the synthetic modelester, N-acetoxy-2-acetyl-aminofluorene (N-OAc-2-AAF), was determined.For comparison, the interaction of tRNA and polyguanylic acid(poly G) with the above compounds was measured by standard procedures.tRNA was an efficient acceptor of enzymatically formed N-acetoxyand N-sulfonoxy derivatives as well as of N-OAc-2-AAF. The nucleophilicityof N-Ac-met toward enzymatically formed N-sulfonoxy-2-AAF wascomparable with that of tRNA. However, its reactivity with N-OAc-2-AAFand enzymatically generated N-acetoxy-2-aminofluorene (N-OAc-2-AF)was only 20% of that of tRNA. Poly G was as reactive as tRNAtoward synthetic N-OAc-2-AAF and enzymatically generated N-OAc-2-AFbut was only 25% as efficient as tRNA or N-Ac-met in acceptingenzymatically formed N-sulfonoxy-2-AAF. The difference in theinteraction of the three nucleophiles with N-OAc-2-AAF and N-OAc-2-AFcompared with N-sulfonoxy-2-AAF indicate that N-OAc-2-AAF isnot a general model of the ultimate electrophilic metabolitesof 2-AAF. Studies of the kinetics of the interaction of N-OAc-2-AAFwith N-Ac-met, tRNA and poly G demonstrated dependence of adductformation on nucleophile concentration, indicative of a bimolecularmechanism. Arylamidonium or nitrenium ions are therefore notnecessarily the ultimate electrophilic metabolites of 2-AAFobligatory for interaction with cellular nucleophiles. Therewas no evidence that nitrenium ions are intermediates in thecytosolic reduction of N-OH-2-AAF to 2-AAF.     

Enzymes of the mevalonate pathway in rat liver nodules induced by 2-acetylaminofluorene treatment

Certain enzymes of the mevalonate pathway have been investigatedin persistent liver nodules induced in the rat by 2-acetylaminofluorene.In these nodules the dolichol level was increased 5-fold, theubiquinone-9 content elevated 6-fold and the amount of cholesterolunchanged. Microsomal ß-hydroxy-ß-methylglutaryl-coenzymeA reductase activity was greatly increased compared to controlliver tissue, which was also the case for the cytosolic farnesylpyrophosphate synthase. A significant elevation of all-trans-geranylgeranylpyrophosphate synthase activity in the cytosol was also observed.The branch-point enzyme of microsomal dolichol synthesis, i.e.cis-prenyltransferase, was decreased in the nodules; whereasthe activity of squalene synthase, the terminal regulating enzymeof cholesterol synthesis, remained unchanged. The dolichol speciesin nodular tissue were redistributed towards the longer chainlength species. One factor regulating the chain length of thepolyisoprene products formed in vitro was shown to be the ratioof the concentrations of isopentenyl pyrophosphate: farnesylpyrophosphate employed. Other regulatory factors in the terminalsteps of this biosynthetic pathway appear to determine the amountsand nature of the final isoprenoid compounds formed in vivo.In contrast to the microsomal trans-prenyltransferase activity,which was unchanged, the activity of nonaprenyl-4-hydroxybenzoatetransferase, an enzyme participating in ubiquinone synthesis,was greatly elevated. The alterations observed in the activitiesof enzymes in the mevalonate pathway can at least partiallyexplain the increased levels of dolichol and ubiquinone andthe unchanged level of cholesterol found in liver nodules. Itis reasonable to propose that this modified mevalonate metabolismwill render nodular cells resistant to certain toxic factorsand prone to cell proliferation.     

Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene

Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16(INK4A) gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.     

Role of diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy in the expression of glutathione-S-transferase-P and gamma-glutamyltranspeptidase in the early steps of rat liver carcinogenesis.

Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.