Metabolism and activation of 2-acetylaminofluorene in isolated rat hepatocytes.

The metabolism of 2-acetylaminofluorene (AAF) as well as the activation of AAF to covalently bound and mutagenic intermediates were studied in isolated rat hepatocytes. The cell system readily formed oxidized, deacetylated, and conjugated AAF metabolites. Pretreatments of animals with the inducer beta-naphthoflavone led to increases in phenolic and conjugated as well as covalently protein-bound products. Addition of 4-nitrophenol, a substrate for conjugation, increased the levels of free phenols and inhibited the formation of water-soluble metabolites. At the same time, the rates of covalent protein binding were decreased. Formation of 9-hydroxy-2-acetylaminofluorene could also be demonstrated. The pathway leading to this alicyclic hydroxylated AAF metabolite was not induced by prior beta-naphthoflavone treatment, nor was it inhibited by 4-nitrophenol addition. The cells converted AAF as well as aminofluorene and 2,4-diaminoanisole to mutagenic intermediates which were released into the incubation medium. 2-Aminofluorene was considerably more mutagenic than was AAF in this system. Addition of microsomes increased the mutagenicity of AAF, but not that of 2-aminofluorene or 2,4-diaminoanisole, presumably by deacetylation of N-hydroxy-2-acetylaminofluorene to N-hydroxy-2-aminofluorene.     

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.     

Decreased NADH-oxidoreductase activities as an early response in rat liver to the carcinogen 2-acetylaminofluorene

Reduced nicotinamide adenine dinucleotide (NADH):ferricyanide reductase and DT-diaphorase specific activity in total homogenates of rat liver are markedly decreased as a very early biochemical event of hepatocarcinogenesis induced by the carcinogen 2-acetylaminofluorene (AAF). A 50 to 75% decrease in NADH:ferricyanide reductase was observed after 1 day of AAF (0.025% in the diet) feeding and persisted throughout a 7-week continuum of AAF administration. Carcinogen added directly to cell extracts had no effect. Similar results were obtained with single injections of either AAF or diethylnitrosamine. Xanthine dehydrogenase was also reduced in liver following AAF administration to nearly the same extent as NADH:ferricyanide reductase and DT-diaphorase. Total NADH-cytochrome c reductase and mitochondrial activity as estimated from succinic dehydrogenase were not affected by carcinogen administration relative to basal dietary controls. The reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase that functions in drug detoxification was elevated. With livers of animals fed 4-acetamidophenol, a hepatotoxin chemically related to AAF, small decreases were noted in NADH:ferricyanide reductase, but not in xanthine dehydrogenase nor in DT-diaphorase. Initial lowering of these activities in the livers of the carcinogen-treated animals is preceded by or concomitant with a reduction in the levels of extramitochondrial pyridine nucleotides known from other studies to result from DNA damage.     

Differential over-expression of mdr1 genes in multidrug-resistant rat glioblastoma cell lines selected with doxorubicin or vincristine

We have compared the pharmacological and molecular characteristics of 2 cell lines derived from the C6 rat glioblastoma, and selected for resistance either to doxorubicin (C6 0.5 line) or to vincristine (C6 IV line). Each line displays a preferential 400-fold resistance towards the drug used for selection, the C6 IV line being especially weakly resistant to doxorubicin (13-fold). Verapamil completely restored doxorubicin sensitivity in the C6 IV line as well as vincristine resistance in the C6 0.5 line, but could not completely reverse doxorubicin resistance in the C6 0.5 line or vincristine resistance in the C6 IV line. This suggests that specific mechanisms of resistance against each drug were added to a common P-glycoprotein-mediated multi-drug-resistance mechanism. Doxorubicin efflux was total within 2 hr in the C6 IV line, whereas it remained 8 to 10% of drug in the C6 0.5 line 4 hr after drug removal, despite a more rapid efflux of the drug in the first 30 min. This 2-compartment behavior could be related to a special sub-cellular distribution of doxorubicin in C6 0.5 cells. Northern and Western blot analysis of the mdr I gene and of the P-glycoprotein expressed by the 2 resistant cell lines made it possible to quantify their degree of over-expression; when compared with the C6 wild strain, the C6 0.5 line over-expressed both the mdr1 gene and the P-glycoprotein to a slightly higher level than the C6 IV line. Northern and Western blot analysis also suggested that C6 0.5 cell preferentially over-expressed the mdr I a gene, whereas the C6 IV cells preferentially over-expressed the mdr Ib gene. This differential over-expression was confirmed after polymerase-chain-reaction amplification of the cDNA sequences transcribed from total RNA extracted from the 2 lines. It can be concluded therefore that the mdr Ia gene product is more efficient than the mdr Ib gene product in extruding anti-cancer drugs from the cells; and that the mdr Ib gene product might preferentially extrude vincristine rather than doxorubicin. © 1993 Wiley-Liss, Inc.     

Inhibition of sulfotransferase in primary cultures of human hepatocytes affecting metabolism and binding of 2-acetylaminofluorene.

The metabolism and DNA binding of acetylaminofluorene (AAF) was investigated in human hepatocytes that were isolated from donor liver tissue by collagenase perfusion. Hepatocytes were treated with 0.01 microM pentachlorophenol (PCP), as a sulfotransferase inhibitor, to investigate the role of sulfotransferase in human bioactivation of aromatic amines. Concentrations of PCP greater than 0.1 microM resulted in cytotoxicity as noted by detachment of cells and atypical morphology. The metabolites of AAF were identified by HPLC as aminofluorene, 7-OH-AAF, 9-OH-AAF, 5-OH-AAF, N-OH-AAF, 1-OH-AAF and 3-OH-AAF. No consistent alteration in the metabolites produced occurred with PCP treatment compared to controls. PCP treatment increased total DNA binding of AAF metabolites compared with controls, suggesting that sulfotransferase does not activate AAF in human hepatocytes. Inhibition of sulfotransferase in human hepatocytes does not decrease DNA binding of AAF metabolites as noted previously with rat hepatocytes. Therefore, PCP may inhibit a detoxication pathway. This study supports N,O-acyltransferase as the critical enzyme for the formation of the major reactive metabolite in human liver.     

Modulation of cytotoxic and genotoxic effects of 2-acetylaminofluorene in rat and hamster hepatocytes by 3-methylcholanthrene pre-treatment

It is well known that 2-acetylaminofluorene (AAF)-induced livercancer is reduced by simultaneous administration of 3-methylcholanthrene(MC) in the rat, but not in the hamster. The present reportexamines the effects of MC pre-treatment on the metabolism andtoxkity of AAF in monolayer cultures of hepatocytes. Hepatocytesisolated from pre-treated animals of both species metabolizedAAF and 2-aminofluorene (AF) to metabolites mutagenic to Salmonellatyphimurium more efficiently than hepatocytes from control animals.MC-pre-treated rat hepatocytes showed increased responses toAAF-and AF-induced unscheduled DNA synthesis, while MC-pre-treatedhamster hepatocytes were less responsive than the untreatedhepatocytes. Increased cytotoxic effects of AAF were observedin MC-pre-treated rat hepatocytes, whereas AAF was not cytotoxicin hamster hepatocytes from either pre-treated or control animals.MC pre-treatment caused increased rates of formation of C-hydroxylated,N-hydroxylated, water-soluble and covalently macromolecularbound AAF metabolites in both species. No significant effectof MC pre-treatment was seen on the formation of AF from AAF.A large decrease in the ratio between covalently macromolecularbound (activated) metabolites and the sum of C-hydroxylatedand water-soluble (detoxified) AAF metabolites, was seen afterMC pre-treatment of rat hepatocytes, whereas no or only a minordecrease was observed in hamster hepatocytes. This ratio correlatedmuch better with the in vivo carcinogen-icity data than theother parameters such as mutagenicity, DNA repair or covalentmacromolecular binding. Thus, the hypothesis that A AF-inducedliver cancer depends less on the rate at which AAF is activated,but more on the relative proportion of the dose which is activated,is supported by the present data.     

Comparison of human and rat hepatocyte metabolism and mutagenic activation of 2-acetylaminofluorene

A method has been developed to assess the metabolism and mutagenic activation of carcinogens using human and rodent hepatocytes in vitro. A slicing technique which was especially useful for nonperfusable biopsy and resected surgical human liver tissue was used to prepare the hepatocytes. Metabolites of the model carcinogen 2-acetylaminofluorene (AAF) produced by human and rat hepatocytes were similar and consisted primarily of 2-aminofluorene with ring hydroxylated products at the 1-, 3-, 5/9-, 7-, and 8-positions produced in addition to N-hydroxy-AAF. Sulphate and glucuronide conjugates of ring-hydroxylated metabolites and 2-aminofluorene were detected. Metabolism and cell-mediated Salmonella mutagenicity illustrated interindividual variation with human hepatocytes. Levels of metabolism and mutagenesis were generally higher with human hepatocytes compared to rat hepatocyte results. The increased levels of metabolism and mutagenesis of AAF by human hepatocytes compared to rat hepatocytes probably indicates a different sensitivity to hepatocarcinogenic effects of AAF on humans as compared to rats. Understanding differences and similarities between human and rodent carcinogen activation capabilities should be useful in the extrapolation of rodent carcinogenesis data to humans.     

Expression of multidrug resistance (p-glycoprotein) mdr1 and mdr2 genes in human hepatocellular carcinomas and liver metastases of colonic tumors

Overexpression of P-glycoproteins (P-gp), encoded by multidrug resistance (MDR) genes is responsible for multidrug resistance in animal cells. We analyzed the expression of MDR genes in human hepatocellular carcinomas (HCC) and liver metastases of colon tumors by an RNase protection assay. Our results indicated that both genes were not consistently overexpressed in these tumors, whereas MDR2 is often underexpressed in the metastatic tumors. Invasion of colon tumors to livers decreased MDR1 expression. These data suggest differential regulation mechanisms for the expression of MDR1 and MDR2 genes in these tumors, and a complex drug-resistance mechanism for liver cancers.     

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.     

Differential regulation of DEC2 among hypoxia-inducible genes in endometrial carcinomas

In this study, we demonstrate an important role of activation of the hypoxia-inducible factor-1 (HIF-1) pathway in endometrial carcinogenesis and tumor phenotype development of endometrial carcinoma, and suggest a unique role of the HIF-1-target gene, differentiated embryo chondrocyte 2 (DEC2), in carcinogenesis. Hypoxia caused an increase in HIF-1alpha protein expression in 4 endometrial carcinoma cell lines. The expressions of its 5 target genes - DEC1, DEC2, carbonic anhydrase-9 (CA9), vascular endothelial growth factor (VEGF), and solute carrier family 2, member 1 (SLC2A1) - also reactively increased in most of the cell lines, except for DEC2 in the SNG-M cells. The expression levels of DEC2, CA9, and SLC2A1 were significantly higher in the 4 atypical hyperplasia tissues and 82 endometrial carcinomas compared with those in the 21 normal endometria. Clinicopathological analyses of carcinoma patients revealed a significant correlation of the VEGF and SLC2A1 expression with the status of lymph-vascular involvement and lymph node metastasis. The expression levels of CA9 and VEGF were significantly higher in the tumors of post- as opposed to pre-menopausal patients. The SLC2A1 expression was also related to the FIGO stage, but the DEC2 expression was inversely related to the FIGO grade. The activation of the HIF-1 pathway could be related to endometrial carcinogenesis, and the component, DEC2, could have different expression-regulatory mechanisms and unique roles in carcinogenesis.     

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.     

Increased number of beta-adrenoceptors in hepatocytes from rats treated with 2-acetylaminofluorene

Treatment of rats with chemical carcinogens, including 2-acetylaminofluorene (2-AAF), leads to a strong increase in the hepatic catecholamine-sensitive adenylate cyclase activity. The present study was undertaken to investigate the mechanism for the development of this increase. We report that hepatocytes isolated from rats which had been fed 2-AAF (0.025% w/w) for 8-12 weeks had an increased number of beta-adrenoceptors, as determined by [3H]dihydroalprenolol binding to whole cells and [125I]iodocyanopindolol binding to washed particles. For both ligands the number of binding sites was about 4-fold higher in hepatocytes from 2-AAF-treated rats than in those from controls. The adenylate cyclase activity of the carcinogen-fed animals showed both a general increase manifested in the basal level (2-fold) and in the activities obtained by stimulation with guanine nucleotides (2-3-fold), cholera toxin (1.5-fold), and glucagon (1.3-fold) and a selective, larger increase in the beta-adrenoceptor-linked activity (7-fold increment of the isoproterenol-sensitive activity). The results indicate that the number of hepatocyte beta-adrenoceptors increases during 2-AAF carcinogenesis. This may, at least in part, explain the rise in catecholamine-sensitive adenylate cyclase activity.