Induction of DNA repair synthesis in human urothelial cells by the N-hydroxy metabolites of carcinogenic arylamines

Urinary N-hydroxy metabolites of carcinogenic arylamines were investigated for their abilities to induce unscheduled DNA synthesis (UDS) in human urothelial cell lines HCV 29, HU 1734, and HU 1752, and in a primary culture of human urothelial cells. N-Hydroxy-2-aminofluorene (CAS: 53-94-1; N-OH-AF), N-hydroxy-2-acetylaminofluorene (CAS: 53-95-2; N-OH-AAF), and the N-glucuronide of N-OH-AF induced UDS in HCV 29, HU 1734, and HU 1752. N-Hydroxy-4-aminobiphenyl (CAS: 6810-26-0; N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (CAS: 4463-22-3; N-OH-AABP), N-hydroxy-2-aminonaphthalene (CAS: 613-47-8; N-OH-AN), N-hydroxy-2-acetylaminonaphthalene (CAS: 2508-23-8; N-OH-AAN), and the N-glucuronide of N-OH-ABP induced UDS in HCV 29. However, the N-glucuronide of N-OH-AN did not. The O-glucuronide of N-OH-AAF induced UDS in HCV 29 only when beta-glucuronidase was present. Paraoxon inhibited the induction of UDS in HCV 29 by N-OH-AAF and N-acetoxy-2-acetylaminofluorene (CAS: 6098-44-8), but not by N-OH-AF. When examined in a primary culture of human urothelial cells, N-OH-AF, N-OH-AAF, N-OH-ABP, and N-OH-AABP were active, but N-OH-AN, N-OH-AAN, 2-aminonaphthalene (CAS: 91-59-8), 2-aminofluorene (CAS: 153-78-6;), and 4-aminobiphenyl (CAS: 92-67-1) were not. These results demonstrate that human urothelial cells are able to activate both acetylated and non-acetylated N-hydroxy metabolites of carcinogenic arylamines, and they suggest that O-glucuronidation may be a detoxification mechanism for N-arylacethydroxamic acids.     

Induction of repair synthesis of DNA in mammary and urinary bladder epithelial cells by N-hydroxy derivatives of carcinogenic arylamines

Unscheduled DNA synthesis (UDS)-inducing activity was used as a parameter to estimate the abilities of rat mammary epithelial cells and urothelial cells from various species to activate carcinogenic aromatic amine derivatives. The N-hydroxy, N-hydroxy-N-acetyl, N-hydroxy-N-glucuronosyl derivatives of 2-aminofluorene (2-AF) and 4-aminobiphenyl (4-ABP) induced UDS in primary cultures of rat mammary epithelial cells, but 2-AF, the O-glucuronide of N-hydroxy-N-acetyl-2-AF (N-OH-AAF) and 4-ABP did not. Neither the activity of N-OH-AAF, N-hydroxy-N-formyl-2-AF, nor N-acetoxy-N-acetyl-2-AF was significantly altered by paraoxon, an inhibitor of microsomal N-deacetylase. Although N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP) also induced UDS, its N-acetyl derivative, which can not be activated by intramolecular, N,O-acetyltransfer, did not. Similarly, rat urothelial cells were responsive to the UDS-inducing activity of this hydroxylamine, but not the hydroxamic acid. In contrast, dog urothelial cells were responsive to both compounds. The UDS-inducing activity of N-OH-AAF was inhibited by paraoxon in the dog, but not in rat urothelial cells. N-Hydroxy-N,N'-diacetylbenzidine induced UDS in the urothelial cells of dog, rat, and rabbit, and a human urothelial cell line, HCV-29, whereas benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine did not. Co-treatment with 12-O-tetradecanoylphorbol-13-acetate did not enable benzidine to induce UDS in dog urothelial cells. Rat mammary epithelial cells activated N-OH-DMABP by acetyl coenzyme A-dependent O-acetylation and N-OH-AAF by N,O-acetyltransfer. They could not N-deacetylate N-OH-AAF. These results suggest that rat mammary and bladder epithelial cells are capable of activating N-arylhydroxylamine metabolites of these carcinogens, probably by N,O-acetyltransfer and O-acetylation, whereas dog urothelial cells are more likely to activate these metabolites by N-deacetylation and a reaction that has yet to be identified.     

Acetylation of 2-aminofluorene derivatives by dog hepatic microsomes

Dog urinary bladder is a target organ of carcinogenic arylamines. However, dog hepatic and urothelial cytosols lack acetylation enzymes that are capable of activating N-hydroxy metabolites of arylamines, suggesting that other enzymes may be involved. In the present study, we found that dog liver microsomes were capable of N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and that these activities were inhibited by paraoxon. The 0.25% Triton X-100 extractable fraction of microsomes was resolved on an ion-exchange column into three different proteins that retained these activities. Two of these proteins, designated as enzyme I and enzyme II, were further chromatographed on a Sephacryl S-300 column. As judged from the gel filtration profile, the mol. wt of enzyme I was approximately 180 kDa and that of enzyme II was greater than 700 kDa. SDS-PAGE analysis showed that the subunit weight of enzyme II was approximately 150 kDa. In addition to N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-OH-AAF, these three enzymes were capable of the deacetylation of 2-acetylaminofluorene, N-OH-AAF and 4-nitrophenyl acetate. The ability of these microsomal enzymes to activate N-hydroxylated aromatic amines and the presence of these enzymes in urothelial cells, reported previously, suggests that they may play an etiological role in the carcinogenicity of these agents in the dog.     

Metabolic activation and genotoxicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetin derivatives in Reuber (H4-II-E) hepatoma cells

Derivatives of both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxyphenacetin (N-OH-P) were tested for their ability to cause DNA damage in Reuber (H4-II-E) cells using the alkaline elution technique. Reuber cells are devoid of N-OH-AAF deacylase, N,O-acyltransferase, and sulfotransferase activities. The hydroxamic acids themselves caused very little DNA damage, while N-hydroxy-2-aminofluorine (20 to 100 microM), N-hydroxyphenetidine (20 to 200 microM), and p-nitrosophenetole (10 to 100 microM) all caused dose-dependent damage. The dose-dependent DNA damage caused by N-acetoxy-2-acetylaminofluorene (5 to 25 microM) was completely inhibited by the deacylase inhibitor paraoxon (100 microM). In the presence of both partially purified rabbit liver cytosolic N,O-acyltransferase and guinea pig liver microsomal deacylase, N-OH-AAF was genotoxic. Neither paraoxon nor tRNA had any effect on the DNA damage induced by N-OH-AAF in the presence of N,O-acyltransferase, while paraoxon completely inhibited the damage when N-OH-AAF was incubated in the presence of guinea pig deacylase, and N-OH-P only caused slight DNA damage at higher concentrations of enzyme. In addition, partially purified guinea pig liver deacylase and N-OH-AAF (25 microM) caused 2600 revertants in the Salmonella test system, while only 380 revertants were seen with a 40-fold greater concentration of N-OH-P (1000 microM). The mutagenicity of both N-OH-AAF and N-OH-P was completely inhibited by paraoxon. Thus, it is clear that metabolites of N-OH-AAF formed outside the cell are capable of passing both the cellular and nuclear membranes to cause genotoxicity. Metabolic activation of N-OH-AAF by either the membrane-bound deacylase or the cytosolic N,O-acyltransferase caused genotoxicity via a deacetylation process. Metabolic activation of N-OH-P by guinea pig deacylase caused low levels of DNA damage, whereas activation by N,O-acyltransferase was not sufficient to cause genotoxicity.     

Sulfation of hydroxylamines and hydroxamic acids in liver cytosol from male and female rats and purified aryl sulfotransferase IV.

Sulfation activity towards hydroxamic acids and hydroxylamines was determined in liver cytosols for juvenile and adult males and female rats, as well as in purified rat liver aryl sulfotransferase IV preparations. Sulfation activity towards the hydroxamic acids N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminophenanthrene, N-hydroxy-4-acetylaminobiphenyl, N-hydroxy-4'-fluoro-4-acetylaminobiphenyl, N-hydroxy-2-acetylamino-5-phenylpyridine, was higher in cytosols derived from adult males (two or three times) than in those from adult females and juveniles (both sexes). N-Hydroxy-2-acetylamino-3-methyl-5-phenylpyridine (N-OH-2AAMPP), however, was poorly sulfated by any of the cytosols. Sulfation activity towards the hydroxylamines N-hydroxy-2-aminofluorene, N-hydroxy-2-aminophenanthrene, N-hydroxy-4-aminobiphenyl, N-hydroxy-4'-fluoro-4-aminobiphenyl was much lower. N-Hydroxy-2-amino-5-phenylpyridine (N-OH-2APP), however, was sulfated much better than the other hydroxylamines. No higher sulfation activity in adult male cytosols for hydroxylamines was found, except for N-OH-2APP and N-hydroxy-2-amino-3-methyl-5-phenylpyridine (N-OH-2AMPP). Purified aryl sulfotransferase IV (AST IV) converted all hydroxamic acids; N-OH-2AAMPP was a poor substrate. Of the hydroxylamines only N-OH-2APP and N-OH-2AMPP were conjugated. These results suggest that hydroxylamines and hydroxamic acids are converted by different sulfotransferases in the rat in vivo. They also indicate that AST IV may be the major enzyme responsible for sulfation of a variety of aromatic hydroxamic acids in the male rat liver. The results presented here are discussed in relation to the carcinogenic effects of some of these compounds.     

Bovine bladder urothelial cell activation of carcinogens to metabolites mutagenic to Chinese hamster V79 cells and Salmonella typhimurium

The ability of bovine bladder urothelial cells to activate genotoxic chemicals to mutagens was examined by cocultivating bladder cells with Chinese hamster V79 cells or Salmonella typhimurium as mutable targets. Activation of test chemicals to mutagenic intermediates by urothelial cells was detected by induction of 6-thioguanine resistance in V79 cells or by induction of histidine revertants in Salmonella. In the bladder cell-mediated V79 cell mutagenesis system, a significant increase in mutation frequency was induced by exposure to 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, and 4-aminobiphenyl were weakly mutagenic to V79 cells with bladder cell activation, while no mutagenic activity was detected with 1-naphthylamine, 2-naphthylamine, or benzidine. Because the mutagenic activity of the aromatic amines was low with V79 cells as the target, a bladder cell-mediated S. typhimurium system was developed for these chemicals. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, 4-aminobiphenyl and 2-naphthylamine were mutagenic to S. typhimurium TA98 and TA100 in the presence of bladder cells but not in their absence. Benzidine was mutagenic to TA98 but not to TA100. The putative noncarcinogen 1-naphthylamine was not mutagenic in the system. In contrast to the V79 data, 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine were not mutagenic with either bacterial strain. Mutagenic responses were related to both the number of bladder cells used for activation and the concentration of test chemical in the Salmonella assay. The data demonstrate that bovine bladder urothelial cells can activate carcinogens from three chemical classes to mutagens and indicate the different sensitivities of V79 cells and S. typhimurium to genotoxic agents.     

Removal of arylamine-DNA adducts in cultured epithelial-cells of rat and human urinary-bladder and mammary-gland

Primary mammary and urinary bladder epithelial cells of the rat, and the human urothelial cell line HCV-29 and mammary cell line MCF-10A were incubated for 3 h with 10 mM hydroxyurea and N-hydroxy-N-acetyl-2-aminofluorene, N-hydroxy-N-acetyl-4-aminobiphenyl or N-hydroxy-3,2'-dimethyl-4-aminobiphenyl. DNA adducts of these carcinogens were detected in the nuclei of all cells, except MCF-10A, semi-quantitatively with a novel immunohistochemical-microdensitometric method. The loss of these adducts in these cells was biphasic and was relatively greater between 0 and 24 h than between 24 and 48 h. The half-life of the adducts of all 3 carcinogens was approximately 20 h. The adduct loss is consistent with repair detected by an unscheduled DNA synthesis system reported previously. These results demonstrate that nonacetylated aromatic amine-DNA adducts can be repaired in the target cells for aromatic amine-induced carcinogenesis.     

Metabolism and binding of benzo(a)pyrene and 2-acetylaminofluorene by short-term organ cultures of human and rat bladder

The ability of organ cultures of normal human and rat bladder to metabolize the polycyclic hydrocarbon, benzo(a)pyrene (BP), and the arylamine, 2-acetylaminofluorene, has been studied. Cultures were maintained for 0 to 6 days in a chemically defined medium before incubation with [3H]BP (0.3 to 0.5 microM) or 2-[14C]acetylaminofluorene (18 to 25 microM) for 24 hr. Ethyl acetate-soluble and water-soluble metabolites were produced from both compounds by both species. The ethyl acetate extracts from [3H]BP-treated human cultures contained 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, and 3-hydroxybenzo(a)pyrene. Rat bladder cultures produced similar metabolites but in slightly different proportions. Ethyl acetate-soluble products of 2-[14C]acetylaminofluorene from human cultures contained 7-hydroxy-2-acetylaminofluorene, 9-hydroxy-2-acetylaminofluorene, 2-aminofluorene, and N-hydroxy-2-aminofluorene. Rat bladder cultures produced similar metabolites, but 2-aminofluorene was found in relatively higher proportion. Hydrolysis by beta-glucuronidase of the water-soluble products produced from both carcinogens gave ethyl acetate-extractable derivatives. These hydrolyzable glucuronide conjugates were relatively more abundant following metabolism of the carcinogens by the rat than by the human cultures. Covalent binding to DNA occurred with [3H]BP in both human (19.7 +/- 13 pmol/mg DNA) and rat cultures (22.8 +/- 8.6 pmol/mg DNA). As with other human tissues, considerable variation (50-fold) was observed between individuals. The results demonstrate that both human and rat bladder epithelium can metabolize known potent carcinogens and, in the case of BP, can effect covalent binding between the products of metabolism and the urothelial cell DNA. In theory, carcinogenesis in the urinary bladder could thus be initiated by carcinogens produced or excreted in the urine without the necessity for their prior metabolism elsewhere in the body.     

Production of urothelial tumors in the heterotopic bladder of rats by instillation of N-glucuronosyl or N-acetyl derivatives of N-hydroxy-2-aminofluorene

Male F344 rats which had been implanted with a heterotopic bladder were randomly divided into four groups and their heterotopic bladders were instilled once a week for 30 weeks with 0.5 ml phosphate-buffered saline-dimethyl sulfoxide solution (4:1), or this solution containing 2 mumol N-methyl-N-nitrosourea, 1 mumol N-hydroxy-2-acetylaminofluorene (N-OH-AAF), or 1 mumol N-hydroxy-N-glucuronosyl-2-aminofluorene (N-OH-N-Gl-AF). These bladders were then instilled once a week for an additional 23 weeks with phosphate buffered saline solution without the addition of dimethyl sulfoxide. The animals were killed at the end of 53 weeks. Transitional cell carcinomas were observed in five of 37, 36 of 37, 15 of 35, and 36 of 38 rats of the control, N-methyl-N-nitrosourea, N-OH-AAF, and N-OH-N-Gl-AF groups, respectively. No histological alteration was observed in their natural bladders and no tumor was observed in the liver. As judged by kinetic measurements of the radioactive compounds, N-OH-AAF was removed much faster than N-OH-N-Gl-AF from the fluid of heterotopic bladder. The pH of the fluid in the bladder was between 7.1 and 7.4. The present study demonstrates the carcinogenicity of N-OH-N-Gl-AF and N-OH-AAF for rat bladder.     

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.     

Production of urothelial tumors in the heterotopic bladder of rat by benzidine derivatives

Male Fischer rats which had been implanted with a heterotopic bladder were randomly divided into five groups and their heterotopic bladders were instilled once a week for 20 weeks with 0.5 ml phosphate-buffered saline:dimethyl sulfoxide solution (4:1) or this solution containing 1 mumol benzidine (BZ), N'-hydroxy-N-acetylbenzidine, the N'-glucuronide of N'-hydroxy-N-acetylbenzidine, or the N-glucuronide of N-hydroxy-2-aminofluorene. These bladders were then instilled once a week for an additional 30 weeks with the phosphate-buffered saline without dimethyl sulfoxide. The experiment was terminated at the end of 50 weeks. Transitional cell carcinomas were observed in 1 of 39 (control), 1 of 29 (BZ), 18 of 30 (N'-hydroxy-N-acetylbenzidine), 28 of 28 (N'-hydroxy-N-acetylbenzidine N'-glucuronide), and 24 of 29 (N-hydroxy-2-aminofluorene N-glucuronide) rats. No histological alterations were observed in their natural bladders. These results demonstrate the urothelial carcinogenicity of the N-hydroxy metabolites of BZ and suggest that N'-hydroxy-N-acetylbenzidine N'-glucuronide may play a major role in the initiation of urothelial carcinogenesis by BZ in humans.     

DNA binding of N-hydroxy-Trp-P-2 and N-hydroxy-Glu-P-1 by acetyl-CoA dependent enzyme in mammalian liver cytosol

3-Hydroxyamino-1-methyl-5H-pyrido[4, 3-b]indole and 2-hydroxyamino-6-methyldipyrido[1,2-a:3', 2'-d]imidazole, active metabolites of protein pyrolysatecarcinogens, were further activated to DNA-binding species byan acetyl-CoA dependent enzyme in hepatic cytosol. This activitywas supported also by N-hydroxy-2-acetylaminofluorene insteadof acetyl-CoA, and it was inhibited by iodoacetamide and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) but not by diethyl-p-nitrophenylphosphate.The activity was observed in the cytosols of rats, mice andrapid acetylator rabbits, which possessed N-acetylation activityof 2-aminofluorene and Trp-P-2. On the other hand, the activitycould not be detected in the cytosols of a dog and a slow acetylatorrabbit.     

Metabolism of aromatic amines: relationships of N-acetylation, O-acetylation, N,O-acetyltransfer and deacetylation in human liver and urinary bladder

N-Acetoxyarylamines are reactive metabolites that are implicatedin the initiation of the carcinogenic process by some N-substitutedaryl compounds. The objective of this study was to explore therelationship between the production of these reactive speciesand N-acetylation (NAT), a reaction previously demonstratedto be polymorphic in the human. Human liver and urinary bladdermucosa samples were frozen within 4–8 h post mortem. Thesetissues were assayed for the (i) O-acetylation (OAT) of N-hydroxy-3,2'-di-methyl-4-aminobiphenyl (N-OH-DMABP) by acetyl CoA, (ii)intramolecular N,O-acetyltransfer (AHAT) of N-hydroxy-2-acetylaminofluorene(N-OH-AAF), (iii) NAT of 2-aminofluorene (2-AF) and p-aminobenzoicacid (PABA) by acetyl CoA and (iv) deacetylation of N-OH-AAF.Cytosolic AHAT and OAT showed partial inhibition by paraoxon.The ratio of paraoxon insensitive AHAT to OAT to NAT of PABAto NAT of 2-AF appears to be 1:2:11:22 using freshly made cytosolsfrom frozen livers. Freezing of the cytosol resulted in extensiveloss of activities. All four of these cytosolic enzyme activitiesexhibited a similar polymorphic response. Microsomal deacetylationshowed a monomorphic response. Similar to the liver, urinarybladder epithelial cells also catalyzed the same reactions.However, the OAT and AHAT activities were detected mainly inmicrosomes. These data suggest that phenotypically rapid acetylatorshave a greater biochemical potential for the metabolic activationof aromatic amines by pathways that involve O-acetylation.     

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.     

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 genotoxicity of aromatic amines in primary hepatocytes isolated from C57BL/6 and DBA/2 mice

The capacity of the chemical carcinogen 2-acetylaminofluorene(AAF) and its derivatives to cause DNA damage in primary mousehepatocytes from aryl-hydrocarbon responsive C57BL/6 and non-responsiveDBA/2 mice was studied using the alkaline elution technique.Low levels of DNA damage were observed after exposure of hepatocytesto either AAF or 2-aminofluorene (AF) (50 –100 µM).Quantitation of metabolites produced from AAF in hepatocytesfrom untreated C57BL/6 and DBA/2 mice using h.p.l.c. showeda similar metabolic profile with respect to C- and N-hydroxylations.After in vivo pretreatment with the potent monooxygenase inducerTCDD (50 µg/kg), N-hydroxylation in the C57BL/6-and DBA/2-derivedhepatocytes increased 25- and 5-fold, respectively. However,the C-hydroxylation pathways were still responsible for 90%of the metabolism in cells from both strains. This may explainwhy only a slight increase in the DNA damage was observed inC57BL/6 mouse hepatocytes after incubation with AF or AAF andno increase in DNA damage was seen in the DBA/2 hepatocytesisolated from TCDD treated animals. Both N-hydroxy-2-acetyl-aminofluorene(N-OH-AAF) and N-acetoxy-2-acetylamino-fluorene (N-OAc-AAF)caused clear dose-dependent increases in DNA strand breaks (5–100 M), suggesting that N-hydroxylation was the ratelimiting step in the activation process of AAF leading to theDNA damage. Treatment of hepatocytes with paraoxon, an inhibitorof microsomal deacetylase activity, prior to exposure to eitherN-OH-AAF or N-OAc-AAF completely inhibited the damage causedby N-OH-AAF, while the damage caused by N-OAc-AAF was only partiallyinhibited. This suggests that these compounds are causing genotoxiceffects after deacetylation. In accordance with this, N-hydroxy-2-aminofluorene(N-OH-AF), the deacetylated metabolite of N-OH-AAF, was an effectivegenotoxic agent, causing DNA strand breaks at low doses. Depletionof cellular glutathione by pretreatment with diethyl maleate,increased the sensitivity of the cells to the damage inducedby N-OH-AF. These data indicate that glutathione may play animportant role in the detoxification of N-OH-AF in mouse hepatocytes.     

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.     

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.     

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.     

Synthesis of N-glucuronides of carcinogenic N-hydroxyarylamines

The N-glucuronides of N-hydroxy-2-aminofluorene, N-hydroxy-4-aminobiphenyl,and N-hydroxy-2-aminonaphthalene were synthesized in good yieldsby reacting the hydroxylamines with glucuronic acid.