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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 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.

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 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.

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.