Differential activation of promutagens by alloenzymes of human sulfotransferase 1A2 expressed in Salmonella typhimurium.

Various enzymatically formed sulfuric acid esters are chemically reactive and mutagenic. This metabolic activation pathway is not detected in standard in-vitro mutagenicity test systems. We describe the construction of Salmonella typhimurium TA1538-derived strains expressing alloenzymes *1, *2, *3, *5, *6 of human sulfotransferase 1A2 (SULT1A2). The reference compounds, 1-hydroxymethylpyrene (1-HMP), N-hydroxy-2-acetylaminofluorene (OH-AAF) and 2-hydroxylamino-5-phenylpyridine (OH-APP), were activated to mutagens in these strains. Their activity differed 7- to 16-fold between strains expressing various alloenzymes. It was strongest and weakest in the strains expressing the common alloenzymes, *1 and *2, respectively. The SULT1A2 protein expression levels, and the V(max) and K(m) values with the reference substrate 4-nitrophenol, varied 2.5-, 4-, and 110-fold, respectively, in cytosolic preparations from strains TA1538-SULT1A2*1 and *2. Strains with varying protein levels were constructed via insertion of silent mutations in the 5'-part of the cDNA. TA1538-SULT1A2*1Z and TA1538-SULT1A2*2Y showed equal expression levels of alloenzymes *1 and *2, respectively, which were 3 times above those of TA1538-SULT1A2*1. The mutagenicity of OH-AAF and OH-APP was unchanged in strain TA1538-SULT1A2*1Z versus *1, and moderately increased in TA1538-SULT1A2*2Y versus *2. The influence of the protein level was stronger with 1-HMP. Nevertheless, mutagenic activity of 1-HMP was still 11 times higher in TA1538-SULT1A2*1Z than in TA1538-SULT1A2*2Y. Thus, differences in the properties between alloenzymes can lead to differences in the activation of promutagens. The model compounds were also tested in strains expressing the other ten human SULTs identified. Whereas OH-AAF and OH-APP showed the highest mutagenic activities in strains expressing SULT1A2, 1-HMP was more potent in strains expressing other SULT forms. With the limitation that little is known about the tissue distribution and regulation of SULT1A2, the findings suggest that its polymorphism may affect the individual susceptibility towards procarcinogens, in particular certain aromatic amines and amides.

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.

Sulphonation of N-hydroxy-2-acetylaminofluorene by human dehydroepiandrosterone sulphotransferase.

1. The aim was to determine which human recombinant sulphotransferase (ST) isoform(s) were responsible for the sulphonation and, thus, potential further bioactivation of the classical hepatic procarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2AAF). 2. N-OH-2AAF was incubated together with the cosubstrate 3'-phosphoadenosine-5'-phosphosulphate (PAPS) and either human liver cytosol or recombinant P-form phenolsulphotransferase (P-PST), M-form PST, dehydroepiandrosterone-ST (DHEA-ST) or oestrogen ST (EST). Formation of 3'-phosphoadenosine-5'-phosphate (PAP) from PAPS, measured by HPLC, was used as the assay for determination of sulphoconjugation rates. 3. The liver cytosol produced a 100% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline levels (p < 0.01), corresponding to a rate of 19 pmol/min/mg protein. Recombinant P-PST, however, was without effect. This is in contrast to previous suggestions using crude enzyme preparations. Like P-PST, recombinant M-PST and EST did not sulphonate N-OH-2AAF. On the other hand, recombinant DHEA-ST produced a 161% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline values (p < 0.001). 4. Kinetic studies of N-OH-2AAF sulphonation by DHEA-ST and human liver cytosol gave similar apparent Kms. Interestingly, the Vmax for N-OH-2AAF sulphonation by DHEA-ST was very similar to that of DHEA, the natural substrate for DHEA-ST. 5. This is the first paper to demonstrate the involvement of the human DHEA-ST in the sulphonation of an N-hydroxylated aromatic amide carcinogen.

Pseudoenzymatic reduction of N-hydroxy-2-acetylaminofluorene to 2-acetylaminofluorene mediated by cytochrome P450.

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) was reduced to 2-acetylaminofluorene by rat liver microsomes in the presence of both NAD(P)H and FAD under anaerobic conditions. The microsomal reduction proceeds as if it were an enzymatic reaction. However, when the microsomes were boiled, the activity was not abolished, but was enhanced. The activity was also observed with cytochrome P450 2B1 alone, without NADPH-cytochrome P450 reductase, in the presence of these cofactors. Hematin also exhibited a significant reducing activity in the presence of both a reduced pyridine nucleotide and FAD. The activities of microsomes, cytochrome P450 2B1 and hematin were also observed upon the addition of photochemically reduced FAD instead of both NAD(P)H and FAD. The microsomal reduction of N-OH-AAF appears to be a non-enzymatic reaction by the reduced flavin, catalyzed by the heme group of cytochrome P450.

Molecular cloning, expression, and characterization of novel human SULT1C sulfotransferases that catalyze the sulfonation of N-hydroxy-2-acetylaminofluorene.

Upon sulfonation, carcinogenic hydroxyarylamines such as N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) can be further activated to form ultimate carcinogens in vivo. Previous studies have shown that a SULT1C1 sulfotransferase is primarily responsible for the sulfonation of N-OH-2AAF in rat liver. In the present study, two novel human sulfotransferases shown to be members of the SULT1C sulfotransferase subfamily based on sequence analysis have been cloned, expressed, and characterized. Comparisons of the deduced amino acid sequence encoded by the human SULT1C sulfotransferase cDNA 1 reveal 63.7, 61.6, and 85.1% identity to the amino acid sequences of rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. In contrast, the deduced amino acid sequence of the human SULT1C sulfotransferase 2 cDNA displays 62.9, 63.1, 63.1, and 62.5% identity to the amino acid sequences of the human SULT1C sulfotransferase 1, rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. Recombinant human SULT1C sulfotransferases 1 and 2, expressed in Escherichia coli and purified to near electrophoretic homogeneity, were shown to cross-react with the antiserum against the rat liver SULT1C1 sulfotransferase and exhibited sulfonating activities with N-OH-2AAF as substrate. Tissue-specific expression of these novel human SULT1C sulfotransferases were examined by employing the Northern blotting technique. The results provide a foundation for the investigation into the functional relevance of these new SULT1C sulfotransferases in different human tissues/organs.

Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase.

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT-PCR).

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.

Homodimeric and heterodimeric aryl sulfotransferases catalyze the sulfuric acid esterification of N-hydroxy-2-acetylaminofluorene.

Three aryl sulfotransferases (ASTs) isolated from rat liver catalyze the sulfuric acid esterification of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2AAF). These three ASTs were separated by high resolution anion exchange chromatography and were designated Q1, Q2, and Q3. Q1 and Q2 had high N-OH-2AAF sulfonation activity, whereas Q3 showed low activity. Reversed phase high performance liquid chromatography/mass spectrometry analysis showed Q1-Q3 to be comprised of 33,945- and 35,675-Da protein subunits. Q1 contained only the 35,675-Da protein subunit, Q2 contained equal quantities of 33,945- and 35,675-Da subunits, and Q3 contained only the 33,945-Da subunit. The subunit compositions of Q1-Q3 were confirmed by immunochemical analysis. Size exclusion high performance liquid chromatography confirmed that the active quaternary structure of the three isoenzymes was dimeric. Analysis of liver cytosols for the relative contributions of Q1-Q3 to total cytosolic N-OH-2AAF sulfotransferase activity indicated the Q1, Q2, and Q3 accounted for 44, 46, and 10% of the activity, respectively. These results demonstrate the existence of both homodimeric and heterodimeric aryl sulfotransferases and show that two ASTs, a homodimer of 35,675-Da subunits and a heterodimer of a 33,945- and a 35,675-Da subunit, are primarily responsible for hepatic N-OH-2AAF sulfotransferase activity.

Reductase activity of aldehyde oxidase toward the carcinogen N-hydroxy-2-acetylaminofluorene and the related hydroxamic acids.

Liver aldehyde oxidase (EC 1.2.3.1) was capable of reducing N-arylacetohydroxamic acids, N-hydroxy-2-acetyl-aminofluorene, N-hydroxy-4-acetylaminobiphenyl and N-hydroxyphenacetin, to the corresponding amides in the presence of an electron donor of the enzyme under anaerobic conditions. When supplemented with an electron donor of the enzyme, a significant reduction of N-hydroxy-2-acetylaminofluorene occurred, which was sensitive to an inhibitor of the enzyme. These observations were made with cytosolic fractions prepared from the livers of rabbits, guinea pigs, rats and mice.

Modulations of hepatic gamma-glutamyltranspeptidase and N-hydroxy-N-2-fluorenylacetamide sulfotransferase activities following treatment of rats with a hepatocarcinogenic regimen: effect of partial hepatectomy.

The feasibility of using biochemical assays of gamma-glutamyltranspeptidase (gamma-GTP) and N-hydroxy-N-2-fluorenylacetamide sulfotransferase (N-OH-2-FAA ST) activities to monitor the effects of treatment of male Sprague-Dawley rats with a two-stage hepatocarcinogenic regimen was investigated. One week after initiation with diethylnitrosamine (200 mg/kg of bw), the rats were treated with 10 oral doses within 2 weeks of N-2-fluorenylacetamide (2-FAA) at 0.05 mmole/kg or vehicle (corn oil) at 5 ml/kg of body weight. After five doses of 2-FAA or corn oil, half of the rats in each group underwent partial (70%) hepatectomy (PH). Three days after completion of 2-FAA treatment, gamma-GTP activity increased approximately 8-fold in the livers of both the nonhepatectomized (-PH) and hepatectomized (+PH) groups. After 17 days, the enzyme activity decreased to the control level in the -PH group but increased 3.1-fold above the control level in the +PH group. After 31, 66, and 87 days, gamma-GTP activity increased only 1.4- to 2.6-fold in the -PH group, whereas that of +PH group increased 15- to 32-fold. N-OH-2-FAA ST activity, determined 3 days after completion of 2-FAA treatment, decreased by approximately 60% in the -PH and +PH groups. After 17 days, the effect of PH became evident in that the losses of N-OH-2-FAA ST activity were smaller (20%) in the -PH than in the +PH group (45.5%). After 31, 66, and 87 days, the respective decreases of 27, 29, and 41% in the +PH group were significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfation of carcinogenic aromatic hydroxylamines and hydroxamic acids by rat and human sulfotransferases: substrate specificity, developmental aspects and sex differences.

Sulfation of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and structurally related hydroxamic acids by rat and human sulfotransferases was studied. There was a clear sex and age difference in the sulfation of N-OH-AAF and the other hydroxamic acids by rat liver cytosols; adult male rats had the highest sulfation activity. Experiments with purified aryl sulfotransferase IV (AST IV) indicated that the high expression of this enzyme in male rat liver may be responsible for these differences. No such sex or age difference was found for the sulfation of aromatic hydroxylamines. In cytosols of adult human livers, sulfation activity towards aromatic hydroxamic acids and hydroxylamines was clearly present, but activities were much lower than in rat liver cytosols. Sulfation activity towards these compounds was also found in fetal and neonatal liver and adrenals. These compounds probably are sulfated by several different sulfotransferases in humans.

Syrian hamster monomorphic N-acetyltransferase (NAT1) alleles: amplification, cloning, sequencing, and expression in E. coli.

N-acetyltransferases have an important role in the metabolism of arylamine and hydrazine drugs and carcinogens. Human N-acetylation phenotype may predispose individuals toward a variety of drug and xenobiotic-induced toxicities and carcinogenesis. Syrian hamsters express two N-acetyltransferase isozymes; one varies with acetylator genotype (polymorphic) and has been termed NAT2; the other does not (monomorphic) and has been termed NAT1. The intronless NAT1 coding region was cloned via the polymerase chain reaction from homozygous rapid acetylator and homozygous slow acetylator congenic and inbred hamster genomic DNA templates and sequenced. The NAT1 alleles from the homozygous rapid (NAT1) and homozygous slow (NAT1s) acetylator hamsters differed in one nucleotide, but the mutation is silent with no change in deduced amino acid sequence. To characterize the enzyme products of the NAT1 alleles, we developed a prokaryotic-expression system. The NAT1r and NAT1s alleles were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based plasmid vector pKK223-3 for over-production of recombinant NAT1 in E. coli strain JM105. Induced cultures from selected NAT1-inserted transformants yielded high levels of soluble protein capable of N-acetylation, O-acetylation, and N,O-acetylation. The recombinant NAT1r and NAT1s proteins did not differ in substrate specificity, specific activity, Michaelis-Menten kinetic properties, intrinsic stability, and electrophoretic mobility. Also, the over-expressed NAT1 proteins displayed substrate-specificity and electrophoretic mobilities characteristic of NAT1 isolated from Syrian hamster liver and colon cytosols.

Activation of the carcinogens N-hydroxy-N-2-fluorenylbenzamide and N-hydroxy-N-2-fluorenylacetamide via deacylations and acetyl transfers by rat peritoneal serosa and liver.

Intraperitoneally administered N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) are carcinogenic for rat peritoneum. The potential of peritoneal serosa to activate these compounds via deacylations and acyl transfers was compared to that of liver. N-Deacylations of N-OH-2-FBA and N-OH-2-FAA to N-2-fluorenylhydroxylamine (N-OH-2-FA) were faster by liver than serosa and by microsomes than cytosols. N-Debenzoylations of N-OH-2-FBA were 73- to 123-fold faster than N-deacetylations of N-OH-2-FAA. The esters, N-benzoyloxy-2-FBA and N-acetoxy-2-FAA, were O- and N-deacylated to N-OH-2-FA by liver, and the benzoate by serosa. Inhibition by paraoxon of the above deacylations implicated a serine carboxylesterase. Liver and serosa cytosols catalyzed acetyl CoA-, but not benzoyl CoA-, dependent and iodoacetamide (IAA)-sensitive N-acylation of N-2-fluorenamine (2-FA), implicating an acetyltransferase. In hepatic microsomes this activity was IAA-insensitive and partially inhibited by paraoxon. Liver cytosol, but not microsomes, used N-OH-2-FAA as an acyl donor and neither used N-OH-2-FBA. Liver and serosa catalyzed binding to DNA of N-OH-2-[ring-3H]FBA which was paraoxon-sensitive and increased by acetyl CoA, but not benzoyl CoA. Binding to DNA of N-OH-2-[ring-3H]FAA catalyzed by cytosols was approximately 22-fold greater in liver than in serosa and was IAA-sensitive. Microsome-catalyzed binding of this compound in both tissues was increased approximately 2-fold by acetyl CoA. The results support a two-step activation of N-OH-2-FBA in the liver consisting of esterase-catalyzed N-debenzoylation to N-OH-2-FA and an acyltransferase-catalyzed O-acetylation to the putative electrophile N-acetoxy-2-FA. In the serosa, binding to DNA appears to be due to rapid N-debenzoylation to N-OH-2-FA, a fraction of which is O-acetylated. Whereas activation of N-OH-2-FAA by liver and serosa microsomes may also involve N-OH-2-FA and/or its O-acetate, activation by the cytosols is consistent with N,O-acetyltransfer of N-OH-2-FAA to yield N-acetoxy-2-FA. The study provides first evidence for activation of N-OH-2-FBA by rat liver and of both compounds by peritoneum in vitro.

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80-90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV activity. In the livers of rats administered only 2AAF (group 1), a general pattern of overall downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action.

Isolation and expression of a cDNA encoding a male-specific rat sulfotransferase that catalyzes activation of N-hydroxy-2-acetylaminofluorene.

A cDNA (ST1C1 cDNA) encoding a N-hydroxyarylamine sulfotransferase (HAST-I) was isolated from a liver cDNA library of a male adult rat and was expressed in COS-1 cells. ST1C1 cDNA (1363 base pairs) encoded a protein of 304 amino acids with a molecular mass of 35,768 daltons, which shared 50.7 and 46.1% sequence identity with rat aryl (ST1A1 (PST-1)) and estrogen (rOST) sulfotransferases, respectively. N-terminal amino acid sequences of three digested polypeptide fragments of HAST-I were completely identical with two portions of the ST1C1 amino acid sequence. The profile of age- and sex-related expression of ST1C1 mRNA was quite consistent with changes in the sulfating activity of N-hydroxyarylamine and HAST contents in rat livers. ST1C1 expressed in COS-1 cells catalyzed a sulfation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) at a rate of 4.98 nmol/mg of protein/min and mediated PAPS (3'-phosphoadenosine-5'-phosphosulfate)-dependent DNA binding of N-OH-AAF. Although ASTIV was believed to be responsible for the activation of N-OH-AAF, ST1A1 encoding an arylsulfotransferase ASTIV, showed only a marginal activity in a sulfation and covalent binding of N-OH-AAF. These data clearly indicate that ST1C1 cDNA codes a new form of a male-dominant sulfotransferase (HAST) responsible for the bioactivation of N-hydroxyarylamines in rat livers.

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils.

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes (PMNL), chiefly neutrophils, elicited with intraperitoneal injections of proteose peptone, was examined. At 10(6) PMNL/ml in media containing halide (X-), 0.14 M Cl- +/- 0.1 mM Br- (without Ca++ and Mg++), addition of 10 nM phorbol myristate acetate (PMA) resulted in generation of superoxide anion and H2O2. Subsequent cetyltrimethylammonium Cl- (Cetac) addition at 0.002% effected myeloperoxidase (MPO) activity release. PMNL treated with PMA and/or Cetac did not metabolize N-OH-2-FAA (30 microM). However, 1-2 pulses of H2O2 (50 microM) after Cetac addition resulted in oxidation of N-OH-2-FAA to N-acetoxy-2-FAA (< 0.5 microM) and 2-nitrosofluorene (2-NOF) (1-2 microM). In the presence of Br- 2-NOF was increased (3-5 microM). The results are consistent with oxidation of N-OH-2-FAA by MPO/H2O2 and MPO/H2O2/X- via two pathways: one electron oxidation leading to N-acetoxy-2-FAA and 2-NOF, and X(-)-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA (10 microM) incubated with PMNL under similar conditions was converted non-enzymatically to 4-OH-2-FAA (< or = 5 microM) and enzymatically to N-OH-2-FAA (< or = 3 microM). In the presence of H2O2, smaller amounts of these products were formed. Formation of N-OH-2-FAA was prevented by paraoxon (0.1 mM) suggesting O-deacetylase activity. However, accountability for N-acetoxy-2-FAA decreased with time, presumably because of binding to cellular macromolecules. With H2O2 addition, 2-NOF (10 microM) was converted to 0.5 or 0.25 microM 2-nitrofluorene by active PMNL or heat-inactivated cell lysates, respectively. Low recoveries of 2-NOF were also attributed to binding. The results suggest that PMNL may be involved in activation of the carcinogenic N-arylhydroxamic acids in vivo.

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.

Activation of the carcinogen N-hydroxy-N-(2-fluorenyl)benzamide via chemical and enzymatic oxidations. Comparison to oxidations of the structural analogue N-hydroxy-N-(2-fluorenyl)acetamide.

Chemical or enzymatic oxidations of the carcinogen N-hydroxy-N-(2- fluorenyl)benzamide (N-OH-2-FBA) were investigated under the conditions facilitating one-electron oxidation or oxidative cleavage of N-hydroxy-N-(2-fluorenyl)acetamide (N-OH-2-FAA). HPLC methods were developed for separation and quantitation of the above hydroxamic acids and their respective oxidation products. To identify the products of oxidation of N-OH-2-FBA, N-(benzoyloxy)-2-FBA (N-BzO-2-FBA) was synthesized and shown to undergo ortho rearrangement to 1- and 3-BzO-2-FBA. Oxidation of N-OH-2-FBA (4.88 mM) with alkaline K3Fe(CN)6 in benzene was complete and yielded equimolar amounts of 2-nitrosofluorene (2-NOF) and the ester (chiefly N-BzO-2-FBA), indicative of one-electron oxidation to nitroxyl free radical which undergoes bimolecular dismutation. However, one-electron oxidation of N-OH-2-FBA (30 or 10 microM) by horseradish peroxidase/H2O2 at pH 7 or myeloperoxidase/H2O2 at pH 6.5 yielded only approximately 10% as much product as N-OH-2-FAA (30 microM). The addition of 0.1 mM Br- +/- 0.1 M Cl- at pH 4 to 6.5 increased 2-NOF formation in MPO/H2O2-catalyzed oxidations. Simulations of these oxidations with HOCl/Cl- or HOBr/Br- showed that the latter was more efficient, converting N-OH-2-FAA almost completely and less than or equal to 62% of N-OH-2-FBA to 2-NOF. The amounts of the ester (N- and o-BzO-2-FBA), which by itself did not contribute to 2-NOF formation or significant substrate regeneration, indicated that approximately 10% of 2-NOF originated from one-electron oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

Effect of bovine serum albumin on the extent of ortho rearrangement of N-(sulfooxy)-2-fluorenylacetamide and of enzymatically activated N-hydroxy-2-fluorenylacetamide and on the binding of reactive esters to nucleic acids.

We have investigated the effect of the bovine serum albumin (BSA)-catalyzed ortho rearrangement of synthetic and enzymatically generated N-(sulfooxy)-2-fluorenylacetamide (NSF) to the O-sulfate esters on the binding of NSF to transfer ribonucleic acid (tRNA) and to deoxyribonucleic acid (DNA). Binding of synthetic NSF to tRNA and DNA decreased approximately 90 and 70%, respectively, in the presence of BSA. Under these conditions, the ortho rearrangement, a minor reaction in the absence of BSA, was nearly quantitative. The decrease of adduct formation to nucleic acids was not attributable to the competitive binding of NSF to BSA. Binding of NSF, generated by cytosolic sulfonation of the arylhydroxamic acid, N-hydroxy-2-fluorenylacetamide, to tRNA, was diminished approximately 97% in the presence of BSA while the ortho rearrangement of the sulfonated substrate increased from less than 0.5% to approximately 50%. Adduct formation of DNA with N-hydroxy-2-fluorenylacetamide, activated by enzymatic sulfonation, was inhibited approximately 90% in the presence of BSA. In these experiments, the catalytic effect of BSA on the ortho rearrangement of enzymatically sulfonated N-hydroxy-2-fluorenylacetamide was of the same order as observed in the experiments with tRNA. The data obtained on the covalent interaction of DNA with enzymatically activated N-hydroxy-2-fluorenylacetamide indicate that, in addition to NSF, another electrophilic species accounts for binding of activated N-hydroxy-2-fluorenylacetamide to DNA. The data support the view that the reactive electrophile is N-acetoxy-2-fluorenamine, resulting from the N,O-transacetylation of N-hydroxy-2-fluorenylacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)