Isoform-selective inactivation of human arylamine N-acetyltransferases by reactive metabolites of carcinogenic arylamines.

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.

ACB-PCR measurement of K-ras codon 12 mutant fractions in livers of Big Blue rats treated with N-hydroxy-2-acetylaminofluorene.

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.

Induction of -2 frameshift mutations by 2-nitrofluorene, N-hydroxyacetylaminofluorene, and N-2-acetylaminofluorene in reversion assays in Escherichia coli strains differing in permeability and acetyltransferase activity.

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 contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines.

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.

Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats.

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.

Gene- and tissue-specificity of mutation in Big Blue rats treated with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene.

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.

Immunohistochemical visualization of wild-type p53 protein in paraffin-embedded rat liver using tyramide amplification: zonal hepatic distribution of p53 protein after N-hydroxy-2-acetylaminofluorene administration.

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.

Influence of nucleotide excision repair on N-hydroxy-2-acetylaminofluorene-induced mutagenesis studied in lambda lacZ-transgenic mice.

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.

Functional characterization of the rat mdr1b encoded P-glycoprotein: not all inducing agents are substrates.

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.

Impairment of respiration and oxidative phosphorylation by redox cyclers 2-nitrosofluorene and menadione.

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.

Acute and chronic toxicity of aromatic amines studied in the isolated perfused rat liver.

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.

Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors.

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.

Induction of minisatellite DNA rearrangements by genotoxic carcinogens 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.

Involvement of umuDCST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium.

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.

Detection of N-(deoxyguanosin-8-yl)-2-fluorenamine in DNA of peritoneal serosa and liver after intraperitoneal exposure of rats to N-hydroxy-N-2-fluorenylbenzamide or N-hydroxy-N-2-fluorenylacetamide.

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)

Mutability by polycyclic hydrocarbons is improved in derivatives of Escherichia coli WP2 uvrA with increased permeability.

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.

Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes.

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.

Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

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.

Lack of carcinogenicity of 2-aminofluorene, its glucuronide and the glucuronide of N-hydroxy-2-acetylaminofluorene in heterotopic bladder of the rat.

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.