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Human arylamine N-acetyltransferases (NATs) are expressed as two polymorphic isoforms, NAT1 and NAT2, that have toxicologically significant functions in the detoxification of xenobiotic arylamines by N-acetylation and in the bioactivation of N-arylhydroxylamines by O-acetylation. NAT1 also catalyzes the N-acetylation of 4-aminobenzoylglutamic acid, a product of folic acid degradation, and is associated with endogenous functions in embryonic development. On the basis of earlier studies with hamster NAT1, hamster NAT2, and human NAT1, we proposed that human NAT2 would be more susceptible than NAT1 to inactivation by N-arylhydroxamic acid metabolites of arylamines. Kinetic analyses of the inactivation of recombinant NAT1 and NAT2 by the N-arylhydroxamic acid, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), as well as the inactivation of NAT2 by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), resulted in second-order inactivation rate constants (k(inact)/K(I)) that were several fold greater for NAT2 than for NAT1. Mass spectrometric analysis showed that inactivation of NAT2 in the presence of the N-arylhydroxamic acids was due to formation of a sulfinamide adduct with Cys68. Treatment of HeLa cells with N-OH-4-AABP and N-OH-AAF revealed that the compounds were less potent inactivators of intracellular NAT activity than the corresponding nitrosoarenes, but unexpectedly, the hydroxamic acids caused a significantly greater loss of NAT1 activity than of NAT2 activity. Nitrosoarenes are the electrophilic products responsible for NAT inactivation upon interaction of the enzymes with N-arylhydroxamic acids, as well as being metabolic products of arylamine oxidation. Treatment of recombinant NAT2 with the nitrosoarenes, 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), caused rapid and irreversible inactivation of the enzyme by sulfinamide adduct formation with Cys68, but the k(inact)/K(I) values for inactivation of recombinant NAT2 and NAT1 did not indicate significant selectivity for either isoform. Also, the IC(50) values for inactivation of HeLa cell cytosolic NAT1 and NAT2 by 4-NO-BP were similar, as were the IC(50) values obtained with 2-NO-F. Treatment of HeLa cells with low concentrations (1-10 microM) of either 4-NO-BP or 2-NO-F resulted in preferential and more rapid loss of NAT1 activity than NAT2 activity. Because of its wide distribution in human tissues and its early expression in developing tissues, the apparent high sensitivity of intracellular NAT1 to inactivation by reactive metabolites of environmental arylamines may have important toxicological consequences.

K-ras codon 12 GGT-->GAT and GGT-->GTT mutations are the most frequently observed K-ras point mutations in human and rodent tumors and therefore are implicated in carcinogenesis for many tissues. Measurement of these mutations in rat models and human tissue could facilitate a more logical extrapolation of rodent tumorigenesis data to human disease. We have developed allele-specific competitive blocker PCR (ACB-PCR) assays for rat K-ras codon 12 GGT-->GTT and GGT-->GAT mutations that parallel the already published assays for human K-ras codon 12 mutations. Liver K-ras codon 12 mutant allele fractions were measured in vehicle-treated and N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-treated Big Blue rats. The average K-ras codon 12 GGT-->GTT mutant fraction (MF) for four control rats was 50 x 10(-6) (95% CI: 27 x 10(-6), 95 x 10(-6)) and for four treated rats was 165 x 10(-6) (95% CI: 87 x 10(-6), 312 x 10(-6)), indicating a 3.3-fold increase with treatment (95% CI: 1.3-8.1). The average MF of K-ras codon 12 GGT-->GAT for control rats was 1320 x 10(-6) (95% CI: 498 x 10(-6), 3500 x 10(-6)) and for treated rats was 8450 x 10(-6) (95% CI: 3180 x 10(-6), 22 400 x 10(-6)), indicating a 6.4-fold increase with treatment (95% CI: 1.6-25.4). These transgenic rats were part of a study that included analysis of liver lacI mutations. Although data from lacI determinations show that this compound induces mostly G-->T mutations, using the ACB-PCR method both K-ras codon 12 GGT-->GTT and GGT-->GAT MFs were significantly increased in treated rats versus control rats. This data raises the possibility that N-OH-AAF may not only induce mutations by a genotoxic mechanism, but also by amplification of both de novo and pre-existing K-ras mutation.

The mutagenicity of 2-nitrofluorene (NF), N-hydroxyacetylaminofluorene (N-OH-AAF), and N-2-acetylaminofluorene (AAF) was measured in strains of Escherichia coli that contain a lacZ allele that reverts by -2 frameshift mutations from CG(5) to CG(4). Mutagenesis was compared in a strain having wild-type permeability and metabolism, a strain with increased permeability caused by a lipopolysaccharide-defective (LPS(d)) mutation, a strain with N- and O-acetyltransferase (NAT/OAT) activity conferred by the Salmonella nat gene on plasmid pYG219, and a strain carrying both an LPS(d) mutation and pYG219. The LPS(d) mutation facilitated the measurement of mutagenicity but was not absolutely required, in that lower levels of mutagenicity were detected in LPS(+) strains. The NAT/OAT activity conferred by pYG219 strongly potentiated the mutagenicity of NF and N-OH-AAF. Surprisingly, AAF was mutagenic in the NAT/OAT LPS(d) strain without an exogenous P450 metabolic activation system. Its activity may be ascribable to the detection of a directly mutagenic impurity by the highly sensitive strain or to a low level of metabolic activation by the bacteria under the assay conditions. The findings add to the evidence that the lacZ allele derived from E. coli strain CC109 is an effective indicator of -2 frameshift mutagenesis and that strains expressing high levels of NAT/OAT activity are highly sensitive in monitoring the mutagenicity of nitroarenes and aromatic amides.

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.

In a previous study, we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells, induced lacI mutants in the liver, and induced much lower frequencies of lacI and hprt mutants in spleen lymphocytes. In the present study, sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants, to determine the mutational specificity of N-OH-AAF in the rat. All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals (P = 0.02 to P < 0.0001). Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats (e.g., G:C --> T:A transversion was the most common mutation in all mutation sets), there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants (P = 0.02) and between the spleen lymphocyte lacI and hprt mutants (P = 0.04). Also, multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12% of mutants from treated rats had major deletions in the hprt gene; no corresponding incidence of large deletions was evident among lacI mutations. All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF. The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes. The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver. Environ. Mol. Mutagen. 37:203-214, 2001. Published 2001 Wiley-Liss, Inc.

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.

P53 protein plays an important role in regulation of the cell cycle. Recently, a role in tumour genesis has also been suggested. The protein is induced after various forms of DNA damage. Immunohistochemical detection of p53 protein showed positive cells in human skin after UV-irradiation, in mouse skin after benzo[a]pyrene treatment and in mouse spleen, thymus and bone after gamma-irradiation. However, no staining was found in mouse and rat liver with traditional immunohistochemical staining methods due to the low amount of p53 present. This seriously hampered studies on the role of p53 in hepatocarcinogenesis. We have developed a more sensitive immunohistochemical method for staining of p53 in paraffin-embedded sections of rat liver using microwave irradiation for antigen retrieval, avidin-biotin complexing and tyramide amplification. A strong, specific fluorescence signal for p53 was found in hepatocytes of rats that had received the hepatocarcinogen N-hydroxy-2-acetylaminofluorene; in control liver no such p53 staining was observed. The fluorescence was located in the nucleus of hepatocytes in zone 1 of the liver. This agrees with the fact that N-hydroxy-2-acetylaminofluorene causes cytotoxicity in this zone.

To study the influence of nucleotide excision repair (NER) on mutagenesis in vivo, ERCC1 +/-, XPA-/-, and wild-type (ERCC1+/+ and XPA+/+, respectively) lambda lacZ-transgenic mice were treated i.p. with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and lacZ mutant frequencies were determined in liver. No significant effect of the treatment on the mutant frequency in wild-type or ERCC1-heterozygous mice was observed. The liver mutant frequency appeared to be significantly increased in treated XPA-/- mice only. To distinguish N-OH-AAF-induced from spontaneous mutations, lacZ mutants derived from treated XPA-/- mice were subjected to DNA-sequence analysis and the spectrum obtained was compared to that established for lacZ mutants in liver of PBS-treated lambda lacZ-transgenic mice of the parent strain 40.6. The N-OH-AAF-induced mutation spectrum appeared to be significantly different from the spontaneous mutation spectrum: the former consisted of mainly (19/22) single bp substitutions targeted at G, of which the majority (12/19) were G:C-->T:A transversions, suggesting that N-(deoxyguanosin-8-yl)-2-aminofluorene [dG-C8-AF], the major DNA adduct in N-OH-AAF-treated mice, is the premutagenic lesion. After analysis of 21 spontaneous mutants, only ten single bp substitutions targeted at G were found, of which five were G:C-->T:A transversions. This study with XPA-/- lambda lacZ-transgenic mice shows that one of the components of NER, that is, the XPA protein, suppresses mutagenesis in vivo.

The multidrug resistance (mdr) genes encode P-glycoproteins, integral membrane proteins which function as drug efflux transporters. Exposure of animals in vivo and cells in vitro to a variety of xenobiotics leads to increased mdr1 gene expression and higher levels of P-glycoprotein. This response may protect cells from the cytotoxic effects of these compounds. In this investigation we functionally expressed the rat mdr1b gene in NIH 3T3 cells and assessed the ability of the encoded P-glycoprotein to protect these cells from the cytotoxicity of xenobiotics known to induce mdr1b expression. In long-term colony survival assays, stably expressed mdr1b conferred resistance to cytotoxic drugs such as colchicine, vinblastine and doxorubicin, but not to 5-fluorouracil nor to the carcinogens aflatoxin B1 and N-hydroxy-acetylaminofluorene. The mdr reversal agent verapamil restored cytotoxicity of colchicine, doxorubicin, actinomycin D, vinblastine and taxol, but had no effect on the sensitivity of these cells to 5-fluorouracil, aflatoxin B1 or N-hydroxy-acetylaminofluorene. In a competitive transport assay, verapamil and, to a lesser extent, colchicine blocked the increased efflux of the fluorescent dye rhodamine 123 from mdr1b-transfected cells, whereas aflatoxin B1 did not compete for this export. These data demonstrate that expression of the rat mdr1b encoded P-glycoprotein can protect cells from a diverse group of compounds previously identified to be mdr substrates, however, other effective inducers of mdr expression, such as aflatoxin B1 and N-hydroxy-acetylaminofluorene, remain potent cytotoxins despite high levels of P-glycoprotein. The fact that compounds which are not themselves substrates can induce P-glycoprotein expression may have implications for pharmacokinetic interactions and chemotherapy.

The present study was designed to investigate the effects of 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2-acetylaminofluorene, on mitochondrial respiration and oxidative phosphorylation. NOF reacts with the NADH:ubiquinone oxidoreductase (complex I) and consumes oxygen in a rotenone-insensitive manner. Unlike menadione, which is able to bypass the rotenone-block and to restore ATP-formation, NOF-induced electron flow was almost completely uncoupled. In normal respiration both redox-cyclers decreased the respiratory control and P/O ratios at low concentrations (2-20 nmol/mg) in NADH-dependent oxidation. With succinate as substrate, only NOF was significantly active. In contrast to NOF, the hydroxamic acid N-hydroxy-2-acetylaminofluorene (N-OH-AAF) impaired mitochondrial energy conversion only at much higher concentrations (80 nmol/mg). At concentrations > 10 nmol/mg, NOF inhibited electron flow through the respiratory chain in NADH- and succinate-dependent oxidation, as determined by dinitrophenolate-uncoupled respiration. The small protective effect of L-cysteine indicates that covalent binding of the nitroso-compound to SH-groups may not explain sufficiently the inhibitory effect of NOF. The results support the notion that redox cyclers impair oxidative phosphorylation by establishing alternative pathways for electron transport in the respiratory chain.

Isolated perfused livers from male Wistar rats were used to study acute and chronic toxic effects of carcinogenic aromatic amines. We investigated the hypothesis that aromatic amines can generate reactive oxygen species as part of their metabolism. Concentrations of 200-400 microM of 2-acetylaminofluorene (AAF), N-hydroxy-AAF, trans-4-acetylaminostilbene (AAS), N-hydroxy-AAS, and N-hydroxy-2-acetylaminophenanthrene in the recirculating perfusate were not toxic in a 2-hr exposure time as assessed by LDH efflux into the perfusate, glutathione excretion into bile, and changes of the beta-hydroxybutyrate/acetoacetate ratio in the perfusate. N-Acetoxy-AAF, however, was severely toxic. Menadione served as a positive control. It is concluded that exposures likely to occur in carcinogenicity studies with these aromatic amines will not be acutely toxic. In additional experiments the isolated perfused liver system was used to demonstrate chronic effects generated by feeding the carcinogenic dose of 0.02% AAF for up to 12 weeks. The following alterations were observed in livers from AAF-fed animals. excretion of glutathione into bile is drastically reduced after 5 or more weeks, increasingly less glucose is released into the perfusate, and oxygen consumption is constantly increased by 20% after 3 and more weeks of AAF feeding. Whereas the total glutathione level increased with time in homogenates of such livers, it decreased in the mitochondrial fraction. The results are interpreted as adaptive responses to chronic toxic effects of AAF which may be related to the promoting properties of this carcinogen.

We investigated the role of dosing regimen on ras mutations in chemically induced CD-1 mouse liver tumors. The spectra of ras gene mutations in liver tumors that were induced by 15 daily i.p. injections of 7,12-dimethylbenz[a]anthracene (DMBA), 4-aminoazobenzene (AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-nitrosodiethylamine (DEN) were compared to those previously obtained for tumors induced by a single but higher dose of each carcinogen. The principal assay used was a direct tumor analysis involving sequencing of polymerase chain reaction (PCR)-amplified tumor DNA; additional mutations that were present in only a small fraction of tumor cells were detected using a transfection assay or a PCR-engineered restriction fragment length polymorphism method. Spontaneous liver tumors had a relatively low frequency of ras mutations, all found in Ha-ras codon 61, and most of these mutations were present in only a small fraction of tumor cells. With the exception of multiple-dose DEN, each group of single- and multiple-dose carcinogen-induced tumors exhibited a higher frequency of ras mutations compared with spontaneous tumors. For AAB, N-OH-AAF and DEN, the dosing regimen was found to affect significantly the profile of ras mutations. For each of these carcinogens, the multiple-dose tumor group (versus single-dose group) had fewer Ki-ras and N-ras mutations and more tumors in which the Ha-ras codon 61 (C-->A) mutation was present in a large fraction of cells. Our results demonstrate that the dosing procedure can materially affect the pattern of ras gene mutation in mouse liver tumors.

We investigated whether somatic rearrangements in minisatellite DNA are more frequent in chemically induced mouse liver tumors than they are in spontaneous tumors. CD-1 mouse liver tumors were induced by either a single dose or 15 consecutive daily doses of 7,12-dimethylbenz[alpha]anthracene, 4-aminoazobenzene, N-hydroxy-2-acetyl-aminofluorene or diethylnitrosoamine (DEN). Using DNA fingerprinting analysis, we found that the single- and multiple-dose carcinogen treatments caused a 2- to 5-fold higher frequency of minisatellite DNA rearrangements compared with that found in spontaneous tumors--with the exception of single-dose DEN tumors, which showed no increase in rearrangements. Our results suggest that DNA fingerprinting may be a valuable assay for differentiating certain chemically induced tumors from spontaneous tumors.

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDCST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of the umuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,8-DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2-NF) and 2-amino-3-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDCST substantially lowered the reversion rate induced by 1-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by 1-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.

DNA adduct formation was examined in rat peritoneal serosa, a tumor target for i.p. administered aqueous suspensions of N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), and compared to that in the liver, which is a tumor target for N-OH-2-FAA in the male rat. 32P-Postlabeling analyses showed the presence of a single adduct, N-(deoxyguanosin-8-yl)-2-fluorenamine (dG-C8-FA), from activation of both hydroxamic acids by the serosa and liver in vitro and in vivo. The relatively low levels of dG-C8-FA (60-80 fmol/micrograms DNA) from N-OH-2-FBA in vitro were increased 2.7- and 35-fold upon the addition of acetyl coenzyme A (AcCoA) to the serosal cytosol and hepatic cytosol or microsomes respectively. By contrast, addition of AcCoA led to a decrease (approximately 34%) in the high level of dG-C8-FA (4330 fmol/micrograms DNA) from activation of N-OH-2-FAA by hepatic cytosol and did not alter the levels from activation by hepatic microsomes and serosal cytosols (530 and 78.3 fmol/micrograms DNA respectively). These data and the previously reported hydroxamic acid activation enzyme activities in the serosa and liver indicated that the precursor of dG-C8-FA, N-acetoxy-N-2-fluorenamine, was formed from N-OH-2-FAA chiefly via an intramolecular N,O-acetyltransfer and from N-OH-2-FBA via a two-step sequence of N-debenzoylation and AcCoA-dependent O-acetylation. The levels of dG-C8-FA were approximately 2- to 3-fold higher in the serosal DNA (up to 515 and 1012 fmol/micrograms DNA) after one (30 mumol/rat) and ten or eleven (cumulative dose of approximately 275 mumol/rat) injections of N-OH-2-FBA or N-OH-2-FAA than in the hepatic DNA. This correlated with the carcinogenicities of the hydroxamic acids, but was inversely proportional to the rates and extents of their activation in vitro. Multiple injections affected hepatic enzyme activities related to the activation of the hydroxamic acids in that the cytosolic N-debenzoylation of N-OH-2-FBA increased (approximately 1.7-fold) whereas N-OH-2-FAA acetyltransferase and sulfotransferase activities decreased. The effect of treatment with N-OH-2-FBA was greater than that with N-OH-2-FAA and was greater on the sulfotransferase activity (approximately 88% decrease). The latter suggested that N-OH-2-FBA, although a poor acceptor for an enzymatic sulfate transfer, may be carcinogenic for the rat liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Escherichia coli B, unlike both E. coli K12 and Salmonella typhimurium, is sensitive to the rough-specific phage C21. This sensitivity is probably due to the incomplete lipopolysaccharide core of the E. coli B cells, which confers on them a partial permeability to large molecules. Derivatives of WP2 uvrA, a tryptophan-requiring E. coli B strain, were rendered still more permeable by selecting for C21-resistant clones. The new permeable strains, when tested for mutagenesis induced by polycyclic hydrocarbons, showed a mutagenic response higher than that of the parental strains.

Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAT activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxy-acetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.

The role of 2-aminofluorene and its N-glucuronide and the O-glucuronide of N-hydroxy-2-acetylaminofluorene in the bladder carcinogenesis by 2-aminofluorene were investigated. These compounds were injected into heterotopically transplanted bladders of male rats at a weekly dose of 1 mumol for 20 weeks. The experiment was terminated at the end of 50 weeks. The results showed that none of these compounds were carcinogenic in the heterotopically transplanted bladder. The O-glucuronide was not carcinogenic even when it was administered in a phosphate saline (pH 8.0), that favors the activation of this compound. The N-glucuronide of N-hydroxy-2-aminofluorene, a positive control, produced urothelial tumors. These results are consistent with the hypothesis that the N-glucuronides of hydroxylamines, but not the O-glucuronides of hydroxamic acids, are responsible for bladder carcinogenesis by arylamines.

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.

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.

Reversions induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were measured in the Salmonella/microsome quantitative plate assay using various concentrations of Salmonella typhimurium strain TA1538. The number of induced revertants increased with an increasing number of bacteria/plate, but the variation in reversion frequency was not as great as the variation in bacterial concentration. The effects of bacterial concentration on reversion fixation, phenotypic expression, selection of his+ revertants, and the interaction of the mutagen with bacterial DNA were examined. Suspension cultures of TA1538 were exposed to N-OH-AAF and various dilutions were prepared and assayed for revertants/10(8) bacteria. Reversion frequencies were very dependent on bacterial concentration between approximately 0.2 X 10(8) and 2 X 10(8) bacteria/plate. Revertants/10(8) TA1538 were reduced above about 2 X 10(8) bacteria/plate, indicating that culture conditions limited reversion fixation, expression and/or selection at these concentrations. The number of spontaneous revertants/plate determined both from bacteria exposed to dimethyl sulfoxide in suspension culture and in the Salmonella/microsome assay were not greatly affected by bacterial concentration. To study the effect of bacterial concentration on the interaction of the mutagen with bacterial DNA, various concentrations of TA1538 were exposed to the same dose of N-OH-AAF. Both revertants/10(8) TA1538 and DNA adducts varied inversely with bacterial concentration. The effect of bacterial concentration on both reversion fixation and/or expression and on mutagen binding to DNA may influence reversion frequencies in the Salmonella/microsome assay.

Previous work from this laboratory provided strong evidence that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) in the livers of infant male B6C3F1 (C57BL/6J X C3H/HeJ F1) mice. Over 90% of the hepatic DNA adducts in these mice consisted of N-(deoxyguanosin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)-AF] and less than 10% were deoxyguanosinyl adducts containing 2-acetylaminofluorene (AAF) residues. In the present study hepatic DNA adduct formation and tumor initiation by N-hydroxy-2-aminofluorene (N-hydroxy-AF) were examined in these mice. N-(dGuo-8-yl)-AF was the only adduct detected in the hepatic DNA; the level at 9 h after a single i.p. dose of 0.04 or 0.06 mumol/g body wt of [3H]N-hydroxy-AF was 1.0 or 1.7 pmol/mg DNA. Pretreatment with a single i.p. dose (0.04 mumol/g body wt) of the sulfotransferase inhibitor pentachlorophenol (PCP) decreased the DNA adduct level by greater than 80%. Similar levels of this adduct were found by 32P-postlabeling analysis of DNA from mice treated with unlabeled N-hydroxy-AF. The liver DNA of infant male brachymorphic B6C3F2 mice [deficient in 3'-phosphoadenosine-5'-phosphosulfate (PAPS)] contained only 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. dose of 0.06 mumol of N-hydroxy-AF/g body wt, while their phenotypically normal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA. A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 mumol/g body wt of N-hydroxy-AF in infant male B6C3F1 mice induced by 10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse. Pretreatment with PCP reduced the liver tumor multiplicity at each dose level by greater than 80%. Essentially the same average tumor multiplicities and inhibitions of tumor formation by PCP pretreatment were obtained following injections of N-hydroxy-AF or N-hydroxy-AAF at the three lower dose levels. Collectively these data strongly indicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-AF in the livers of infant male B6C3F1 mice. Furthermore, since only N-(dGuo-8-yl)-AF adducts were found in the hepatic DNA these lesions appear to be critical in the initiation of hepatocarcinogenesis in these mice by N-hydroxy-AF.

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.

Carcinogenicity of N-hydroxy-N-3-fluorenylacetamide for the mammary glands of female inbred F344 rats was examined after systemic and topical administration. The compound given ip produced a 60% mammary tumor incidence but was only marginally active after topical application. Female F344 rats did not develop mammary tumors after topical application of N-hydroxy-N-2-fluorenylacetamide, N-acetoxy-N-2-fluorenylacetamide, or N-acetoxy-N-3-fluorenylacetamide. These results contrasted with those reported earlier for female Sprague-Dawley rats and indicated differences in susceptibility of the mammary glands of these rat strains to tumor induction by these carcinogens.

Valproic acid (VPA), cytochalasin D (CD) and 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) each caused abnormal closure of the anterior neuropore in rat embryos cultured in vitro in the absence of an exogenous bioactivation system. Morphological comparisons showed that although all three compounds prevented normal neural tube closure, each did so in a distinctive manner. Modulation of GSH in cultured rat conceptuses was evaluated to determine whether common responses occurred relative to the ability of different chemicals to elicit neural tube defects. Malformation incidence in embryos (10-14 somites) varied widely following exposure to CD (44%), 7-OH-AAF (29%) or VPA (17%). The incidence of CD-elicited malformations was increased by 50% following GSH depletion by L-buthionine-S, R-sulfoximine (BSO) and was decreased by nearly 60% when the cysteine pro-drug 2-oxothiazolidine-4-carboxylate (OTC) was added to the culture medium. GSH modulation also exerted significant effects on the incidence of abnormal neurulation caused by VPA or 7-OH-AAF. A relatively low incidence of open neural tubes produced by VPA or 7-OH-AAF alone was shown to be a function of the state of maturation in the embryos. Conceptuses cultured in the presence of VPA or 7-OH-AAF from an earlier gestational age (6-10 somites) showed 2-3 fold increases in the number of embryos with open neural tubes. Differential alterations in protein and DNA content were observed among embryos and yolk sacs after various treatments indicating possible differences in the site of embryotoxicity. These results demonstrate the role of GSH status on the capacity of three chemically diverse compounds to elicit abnormal neurulation in cultured rat embryos and suggest some possible mechanisms by which normal neurulation may be compromised.

Indole is known to suppress the hepatotoxicity and carcinogenicity of 2-acetylaminofluorene (AAF) in rats and hamsters. For elucidation of the mechanism of its protective role, 2 experiments were conducted using young male rats. In the 1st experiment, the 24-hr biliary excretion of N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-glucuronide was measured after 2 and 4 weeks of dietary administration of 0.03% AAF with or without 1.6% indole. The amount of [9-14C]N-OH-AAF that was excreted as the glucuronide following a single i.p. injection of [9-14C]AAF was lower after 2 weeks in animals fed AAF and indole, as compared to those fed AAF alone [1.5 +/- 1.2% versus 19.6 +/- 3.6% S.E. (p less than 0.001)]. After 4 weeks of AAF administration without indole, the biliary excretion fell to 4.8 +/- 2.1%. This was also significantly higher than that of the animals fed both AAF and indole [1.8 +/- 1.2% (p less than 0.025)]. The suppressive role of indole on the conjugate excretion was also reflected in a decreased biliary excretion of all [9-14C]AAF metabolites in animals treated with indole alone. In the 2nd experiment, the protective action of indole was assessed by survival following daily i.p. injections of N-OH-AAF and Na2SO4 solution. Na2SO4 increased the hepatotoxicity of N-OH-AAF. Indole suppressed the toxicity of N-OH-AAF even in the presence of Na2SO4. This protective role of indole was partially overcome only when excess sulfate was coadministered. These results indicate that indole suppresses the biliary excretion of the O-glucuronide of N-OHAAF during the initial exposure of the animal to the carcinogen, possibly reflecting decreased N-OH-AAF formation. Indole also modifies the metabolism of AAF FOLLOWING N-hydroxylation, perhaps activating N-OH-AAF, depending upon the concentration of sulfate available.

Ethinyl estradiol, the estrogenic component of oral contraceptives, has been shown to enhance the mutagenicity of 2-aminofluorene, 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, and N-acetoxy-2-acetylaminofluorene with strain TA 98 of Salmonella typhimurium and various rat liver activating systems. The magnitude of the enhancement of mutation produced by ethinyl estradiol is dependent upon: the type of mixed-function oxidase inducer of the liver activating system; the structure and concentration of the arylamine; the concentration of ethinyl estradiol; and metabolism of ethinyl estradiol to its catechol, 2-hydroxyethinyl estradiol, by the activating system. Moxestrol, a biologically potent estrogenic derivative of ethinyl estradiol which is not metabolized effectively to its catechol by the mixed-function oxidases, does not enhance the mutagenicity of the above arylamines and related compounds. Both 2-hydroxyethinyl estradiol and 2-hydroxymoxestrol enhance the mutagenicity of 2-aminofluorene and 2-acetylaminofluorene. Neither the estrogens nor their catechols are mutagenic by themselves in this system. In the presence of ethinyl estradiol, a marked inhibition of ring hydroxylation of 2-acetylaminofluorene was demonstrated. Since ring hydroxylation is a well established detoxification pathway of arylamine and arylamide metabolism, the enhancement of mutagenicity by ethinyl estradiol may be the result of a net increase in N-hydroxylation of arylamines and arylamides.

The capacity of the chemical carcinogen 2-acetylaminofluorene (AAF) and its derivates to cause DNA damage in primary mouse hepatocytes from aryl-hydrocarbon responsive C57BL/6 and non-responsive DBA/2 mice was studied using the alkaline elution technique. Low levels of DNA damage were observed after exposure of hepatocytes to either AAF or 2-aminofluorene (AF) (50-100 microM). Quantitation of metabolites produced from AAF in hepatocytes from untreated C57BL/6 and DBA/2 mice using h.p.l.c. showed a similar metabolic profile with respect to C- and N-hydroxylations. After in vivo pretreatment with the potent monooxygenase inducer TCDD (50 micrograms/kg), N-hydroxylation in the C57BL/6- and DBA/2-derived hepatocytes increased 25- and 5-fold, respectively. However, the C-hydroxylation pathways were still responsible for approximately 90% of the metabolism in cells from both strains. This may explain why only a slight increase in the DNA damage was observed in C57BL/6 mouse hepatocytes after incubation with AF or AAF and no increase in DNA damage was seen in the DBA/2 hepatocytes isolated from TCDD treated animals. Both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N- OAc -AAF) caused clear dose-dependent increases in DNA strand breaks (5-100 microM), suggesting that N-hydroxylation was the rate limiting step in the activation process of AAF leading to the DNA damage. Treatment of hepatocytes with paraoxon, an inhibitor of microsomal deacetylase activity, prior to exposure to either N-OH-AAF or N- OAc -AAF completely inhibited the damage caused by N-OH-AAF, while the damage caused by N- OAc -AAF was only partially inhibited. This suggests that these compounds are causing genotoxic effects after deacetylation. In accordance with this, N-hydroxy-2-aminofluorene (N-OH-AF), the deacetylated metabolite of N-OH-AAF, was an effective genotoxic agent, causing DNA strand breaks at low doses. Depletion of cellular glutathione by pretreatment with diethyl maleate, increased the sensitivity of the cells to the damage induced by N-OH-AF. These data indicate that glutathione may play an important role in the detoxification of N-OH-AF in mouse hepatocytes.

A single local injection of 2.5 mumol of N-hydroxy-N-formyl-2-aminofluorene (N-hydroxy-FAF), N-hydroxy-N-acetyl-2-aminofluorene (N-hydroxy-AAF), or N-hydroxy-N-pro-pionyl-2-aminofluorene )N-hydroxy-PAF) to each of the six left mammary glands of female Sprague-Dawley derived CD rats gave a mammary tumor incidence, after 12 months, of 53% for the N-acetyl (42% adenocarcinoma, 11% fibroadenoma), 41% for the N-formyl (8% adenocarcinoma, 11% sarcoma, 22% fibroadenoma), and 33% for the N-propionyl (11% adenocarcinoma, 22% fibroadenoma) derivatives of N-hydroxy-N-2-aminofluorene, Latent periods for malignant tumor appearance (adenocarcinoma or sarcoma) was 210 days, 148 days, and 177 days, respectively, with no malignant tumors occurring in the vehicle-treated animals. In contrast, latent periods for benign tumor appearance (fibroadenoma) was 263 days for control animals, 289 days for the N-hydroxy-AAF, 324 days for the N-hydroxy-FAF, and 317 days for the N-hydroxy-PAF animals. When N-acetyl-2-aminofluorene (AAF) was applied as above there was only an 8% mammary tumor incidence (4% adenocarcinoma, 4% fibroadenoma) with a latent period of 207 days for malignant tumor (adenocarcinoma) and 221 days for benign tumor (fibroadenoma) appearance. Arylhydroxamic acid N, O-acyltransferase activity has been demonstrated in the mammary glands of male, and lactating and non-lactating female Sprague-Dawley derived CD rats by means of nucleic acid binding assay. Mammary gland cytosol catalyzed tRNA adduct formation to a greater extent with N-hydroxy-FAF. AAF was not activated by this enzyme. Ammonium sulfate fractionation demonstrated the presence of two enzymes, one specific for N-hydroxy-FAF (70-80% fraction), the other specific for N-hydroxy-AAF and N-hydroxy-PAF (40-70% fraction). Moreover, gel filtration chromatography of mammary gland cytosol demonstrated the presence of two enzymes of differing acyl specificity. Mammary gland microsomes catalyzed the formation of tRNA adducts, but only with the N-hydroxy-FAF derivative. Assays that tested the mutagenic potential of the arylhydroxamic acids in Salmonella typhimurium TA-1538 with either mammary gland cytosol or microsomes demonstrated the order of mutagenicity to be N-hydroxy-FAF greater than N-hydroxy-AAF greater than N-hydroxy-PAF. A similar order of mutagenicity was demonstrated without an external metabolic activation system. These data demonstrate that the presence of two distinct enzymes in the rat mammary gland that activate arylhydroxamic acids.

Urinary bladder is a target tissue for aromatic amine carcinogens. The intrinsic capacity of this tissue to form DNA-damaging products was investigated in explant cultures of bladder isolated from New Zealand white rabbits. DNA repair, measured by autoradiography, was used as the indicator of DNA damage. DNA repair was induced by 2-aminofluorene (2-AF) and its acetylated derivatives 2-acetyl-aminofluorene and N-hydroxy-2-acetylaminofluorene. A positive response was observed with benzidine (BZD), but no repair was seen in cultures exposed to monoacetyl BZD or diacetyl BZD. These results indicate that rabbit urinary bladder has the ability to biotransform aromatic amines to DNA-damaging products and has the capacity to repair damaged DNA. Unlike liver, where activation of BZD seems to require N-acetylation, acetylated BZD in the bladder appeared to be a detoxification product. The lack of damage by acetylated BZD is consistent with activation of BZD in bladder by prostaglandin synthetase-mediated pathways.

Inbred male Leeds strain rats were given a diet containing 0.05% N-fluoren-4-ylacetohydroxamic acid for up to 7 months. A modified method for the synthesis of this compound is described. Rats were killed at intervals during and after treatment and their liver tissues examined by light and electron microscopy. The treated rats developed a very low incidence of (non-hepatic) tumours and hepatic clear cell foci appeared during the late stages of the experiment. The only significant fine structural changes observed were glycogenosis and alterations in the morphology of the rough endoplasmic reticulum, both of which developed only after prolonged treatment and affected a minority of hepatocytes. These changes are discussed in relation to the fine structural changes elicited by the strongly carcinogenic N-fluoren-2-ylacetamide and the non-carcinogen, N-fluoren-4-ylacetamide.

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.

Slices of liver from CD rats were evaluated by autoradiography after exposure to N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) labeled in the acetyl moiety with tritium. These in vitro experiments showed that the incorporation of the acetyl group of N-hydroxy-AAF was a sulfate-dependent enzymatic process in livers from male rats. Livers from females, and hyperplastic nodules and carcinomas induced in the livers of males, were essentially incapable of carrying out this reaction. The results of these in vitro experiments were compared with those obtained with liver sections from a male CD rat exposed in vivo to the tritiated N-hydroxy-AAF. A close relationship between covalent binding and necrosis was demonstrated by the observation that the acetyl moiety of N-hydroxy-AAF was preferentially incorporated into the periportal area, the same area in which liver necrosis occurs after a single injection of the hydroxamic acid. These data offer a biochemical rationale for the relative sensitivities of these cells to the toxic effects of N-hydroxy-AAF and its parent amide. There were no differences in the incorporation of ring-labeled N-hydroxy-4-acetylaminobiphenyl with respect to the sex of the animal or to the morphological characteristics of the tissue. The inhibition of the formation of these adducts by paraoxon suggests that they resulted from the action of deacetylases in the endoplasmic reticulum.

2-Acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) could be activated to mutagens in S. typhimurium using either 9000 g supernatant (S9) or hepatocytes isolated from rats, mice, hamsters or guinea pigs. Their relative mutagenic potency was generally N-OH-AAF greater than AF greater than AAF. Monolayer cultures of hepatocytes exposed to AAF/AF/N-OH-AAF showed evidence of DNA damage measured as unscheduled DNA repair synthesis. The order of activity in rat and hamster was N-OH-AAF greater than AAF greater than AF, in guinea pig and mouse N-OH-AAF greater than AF greater than AAF. Only N-OH-AAF caused observable cytotoxicity, and the rat hepatocytes were the far more sensitive species. Neither the resistance of guinea pig liver nor the greater susceptibility of the rat liver to the carcinogenic effects of AAF and N-OH-AAF could be readily explained by the species differences in activating these compounds to mutagens in Salmonella or to DNA damaging agents in the hepatocytes. It is possible that cytotoxic effects of N-OH-AAF may be of some importance for the observed species differences in the liver carcinogenic effects of AAF and N-OH-AAF.

Acetaminophen (APAP) and 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) each produced a similar incidence of, as well as a qualitatively similar, abnormal closure of the anterior neuropore at similar concentrations when added to the medium of cultured rat embryos. At concentrations producing a 50-65% incidence of abnormal neurulation, the affected embryos displayed relatively complete embryonic development as assessed from measurements of protein, axial rotation, and embryonic length. The neural tube defect produced by these agents consisted of elevated neural folds remaining separated by approximately 45 degrees as well as the presence of a mitotically active neural epithelium. In contrast, the nonacetylated structures, p-aminophenol (PAP) and 7-hydroxyaminofluorene (7-OH-AF), were embryotoxic at concentrations 10-fold lower than the corresponding acetylated compounds; each produced a greater incidence of abnormal axial rotation and a greater decrease in embryonic protein than APAP or 7-OH-AAF. In addition, the embryos exposed to PAP or 7-OH-AF were morphologically and histologically dissimilar to those exposed to the acetylated compounds. The neural folds of the latter remained elevated and in apposition, but lacked complete fusion of the folds of neural epithelium and were accompanied by marked cytotoxicity. Addition of active deacetylase sources (guinea pig liver microsomes or commercially obtained, purified carboxylic-ester hydrolase) to the culture medium of conceptuses exposed to 7-OH-AAF or APAP resulted in an increased embryotoxicity which was indistinguishable from that produced by the nonacetylated compounds alone. The increases in toxicity were effectively blocked by the addition of paraoxon, indicating that catalysis of the deacetylation of APAP and 7-OH-AAF was the causative factor. No evidence could be found for deacetylation of 7-OH-AAF or APAP mediated by the Day 10 conceptus itself. When examined for cytotoxicity in F9 embryonal carcinoma cells, APAP and 7-OH-AAF each produced observable cell death only if reduced glutathione (GSH) had previously been depleted and if a deacetylase source were present; this cytotoxicity was also blocked by addition of paraoxon. The nonacetylated metabolites were directly cytotoxic, although GSH depletion greatly increased the incidence of cell death. Therefore, deacetylation of APAP and 7-OH-AAF produced an increase in generalized embryotoxicity and cytotoxicity relative to abnormal neurulation, suggesting that APAP and 7-OH-AAF are capable of eliciting neural tube defects via a mechanism(s) that is distinguishable from the generalized embryotoxicity or cytotoxicity produced by their nonacetylated counterparts.

The mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetin and their respective deacetylated metabolites, N-hydroxy-2-aminofluorene and 2-nitrosofluorene, and N-hydroxyphenetidine and rho-nitrosophenetole was determined in nitroreductase deficient Salmonella tester strains TA 98FR and TA100FR. The mutagenicity of N-hydroxy-2-acetylaminofluorene medicated by either rat liver microsomes or rat liver 105 000 g supernatant fractions was no different in either TA98 (nitroreductase proficient) or TA98FR (nitroreductase deficient). Similarly the mutagenicity of N-hydroxyphenacetin mediated by hamster liver microsomes was not affected by either the presence or absence of nitroreductase activity in TA100. N-Hydroxy-2-aminofluorene and 2-nitrosofluorene were equipotent direct acting mutagens in both TA98 and TA98FR, as were both N-hydroxyphenetidine and rho-nitrosophenetole in TA100 and TA 100FR. Ascorbate (5 mM) and NADPH (1 mM) had no significant effect on the mutagenicity of either N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-aminofluorene, or 2-nitrosofluorene in TA98 or TA98FR whereas ascorbate and NADPH markedly inhibited the mutagenicity of both N-hydroxyphenetidine and rho-nitrosophenetole in both TA100 and TA100FR. Ascorbate appears to be inhibiting the mutagenicity of N-hydroxyphenetidine and rho-nitrosophenetole as a result of the nonenzymatic chemical reduction of these compounds to non-mutagenic derivatives.

Direct additions to culture media of either acetaminophen (APAP) or 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) resulted in abnormal closure of the anterior neuropores of cultured rat embryos in the absence of an exogenous bioactivation system. Concentrations required to produce a 50% incidence of the defect were approximately 500 and 250 microM for APAP and 7-OH-AAF, respectively. Losses of viability were not evident at these concentrations but 7-OH-AAF elicited a somewhat greater effect on growth parameters and generalized embryotoxicity. Transplacental induction with 3-methylcholanthrene (MC) of P450IA1 in subsequently cultured rat embryos did not detectably alter the capacity of APAP or 7-OH-AAF to effect embryotoxicity or neuropore closure. However, additions to the culture medium of exogenous hepatic bioactivating systems (S9) from MC-induced vs phenobarbital (PB)-induced adult rats produced profoundly different effects on neuropore closure. Coincubation with S9 from MC-induced rats reduced the incidence of 7-OH-AAF-elicited abnormal neuropores from 45 to 19%, whereas coincubation with S9 from PB-induced rats increased the incidence to 77%. Coincubation with MC-induced S9 produced no statistically significant effect on APAP-elicited neuropore abnormalities but, with PB-induced S9, resulted in a significant increase from 60 to 86%. Additions of 3-OH-APAP (0.1-0.2 mM) but not N-acetyl-p-benzoquinoneimine (NAPQI, 0.1-0.5 mM) to the culture medium elicited the typical neuropore abnormality. Experiments with APAP and 7-OH-AAF as substrates demonstrated that embryonic enzymes catalyzed their conversion to the corresponding catechols. Considered together, the results provided evidence that embryonic conversion of APAP or 7-OH-AAF to the corresponding catechol metabolites may be instrumental in effecting the abnormal anterior neuropore closure observed after exposure of embryos to the respective parent compounds.

The direct-acting cytotoxic properties of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxy-2-aminofluorene (N-OH-AF) have been determined in repair-proficient (AA8-4) and repair-deficient (UV-5) Chinese hamster ovary cells. Cytotoxicity comparisons indicate that UV-5 cells are considerably more sensitive to exposure to N-OH-AAF than is the parental AA8-4 cell line, i.e., concentrations needed to obtain a D37 for survival of AA8-4 is greater than 5-fold higher than for UV-5. Mutation analysis at the HGPRT locus also indicates the increased sensitivity of UV-5 cells to N-OH-AAF as witnessed by an enhanced induction of 6-thioguanine-resistant colonies at equitoxic doses. Conversely, N-OH-AAF, did not induce a 'UV-mimetic' response when comparing genotoxicity between these two cell lines. Our data coupled with previously published model-building and adduct removal studies (Broyde and Hingerty, 1983; Fuchs and Daune, 1974; Grunberger and Weinstein, 1976; Yamasaki et al., 1977) suggest that the minor DNA adduct species, N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene, may be responsible for the hypermutagenicity witnessed in DNA excision-repair-deficient cells treated with N-OH-AAF.

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%.

In order to investigate rat hepatocyte heterogeneity in asialoglycoprotein transport, rats were pretreated with N-hydroxy-2-acetylaminofluorene (N-OH-AAF, 90 mumol/kg, i.v.) to damage zone 1 hepatocytes, or with carbon tetrachloride (CCl4, 2.1 mmole/kg, p.o.) to damage zone 3 hepatocytes. Twenty-four hours after pretreatment, the asialoglycoprotein dog intestinal alkaline phosphatase (As-ALPase) was injected and plasma disappearance and biliary excretion were measured. In addition, the acinar distribution of the hepatic binding of As-ALPase 10 min after injection in vivo, or after incubation of fixed liver sections with As-ALPase in vitro, was determined by enzyme histochemistry. In control rats, a rapid biexponential plasma disappearance was observed and 6.4 +/- 1.5% of the dose was excreted into bile after 60 min. Hepatocyte binding occurred predominantly in zone 3, both after administration in vivo and after incubation with liver sections in vitro. In rats with zone 1 liver damage, both the half-lives of the first and of the second phase were strongly increased, but biliary excretion did not change significantly. Both in vivo and in vitro the relatively weak binding of As-ALPase in zone 1 of the liver was abolished, whereas binding to zone 3 cells was normal or only slightly decreased. After CCl4-pretreatment histochemically detectable binding to zone 3 cells was completely abolished, leaving only the relatively weak binding in zone 1. Unexpectedly, a normal plasma disappearance and biliary excretion rate were found in these rats. The discrepancy between the pharmacokinetic results, which point to a predominant involvement of zone 1 cells in As-ALPase transport, and the enzyme histochemical studies, which show preferential binding of As-ALPase in zone 3, is discussed.

2-Aminofluorene (AF), 2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were studied for mutagenic activity in S. typhimurium and either liver 9000 g supernatant fractions (S9) or monolayer cultures of hepatocytes isolated from Wistar rats were used as an activation system. All 3 compounds were converted into mutagens excreted into the incubation medium by the cell-culture system, with N-OH-AAF greater than AF greater than AAF. Cultures used 24 h after plating were less efficient in promutagen conversion than were cultures used after 2 h. Phenobarbital, but not 3-methylcholanthrene, pretreatment of the rats caused similar effects on AF, AAF and N-OH-AAF mutagenicity with both S9 and hepatocyte cultures. The mutagenicities of AF and AAF were reduced by the cytochrome-P-450 inhibitors metyrapone and alpha-naphthoflavone, whereas the mutagenicity of N-OH-AAF was increased by using both inhibitors. Further, the microsomal deacetylase inhibitor paraoxon caused only a moderate reduction in N-OH-AAF mutagenicity, but a total inhibition of AAF mutagenicity. No significant effect of paraoxon on AF mutagenicity was seen. With the S9 system, no effect of ascorbate on the mutagenicity of AF, AAF or N-OH-AAF was observed. In contrast, the mutagenicity of all 3 compounds was increased by ascorbate when hepatocyte cultures were used as activation system. Incubation of hepatocyte monolayers in a sulfate-free medium did not change the mutagenicity of AF, AAF or N-OH-AAF. Galactosamine, an inhibitor of glucuronidation in cells, increased the mutagenicity of AF, AAF and N-OH-AAF with hepatocyte cultures. The addition of cofactor for glucuronidation in the S9 system, however, had no effect. A reduction in mutagenicity of AF and AAF, but not that of N-OH-AAF, was observed with the addition of glutathione (GSH) in both the S9 and the hepatocyte systems. On the other hand, no effect of cellular GSH depletion was seen on aromatic-amine mutagenicity in the hepatocyte system. The data indicate that the hepatocyte culture system offers advantages over the conventional liver-sub-fraction activation system as a model, in vivo, for the metabolism of the aromatic amine mutagens/carcinogens.