Steroid hormone receptors in the rat mammary adenocarcinoma induced by N-hydroxy-N-2-fluorenylacetamide.

The steroid hormone receptors in N-2-fluorenylacetamide (2-FAA)- and N-hydroxy-2-FAA-induced mammary adenocarcinomas in outbred Sprague-Dawley rats were analyzed. Both 8S and 4S estrogen-binding components have been detected in cytosols of these tumors following sucrose gradient sedimentation in low salt. Competitive binding analyses of this binder indicated a specificity profile expected of an estrogen receptor. Both androgen and progesterone receptors were also present in the cytosols of these mammary tumors. While the androgen receptor sedimented in the 8S region of the gradient, the progestin binder appeared only as a 4S moiety under similar conditions. The relative concentrations of these receptors (expressed in fmol/mg protein +/- SE) were: 17 beta-estradiol (28.6 +/- 4.1) greater than 5 alpha-dihydrotestosterone (8.5 +/- 2.2) greater than progesterone (5.0 +/- 1.3). The progesterone receptor was increased at least eightfold in the mammary adenocarcinomas from ovariectomized rats that were treated with diethylstilbestrol for 6 days. Binding equilibrium data indicated Ka = 1.2-1.8 X 10(9) M-1 for the above cytoplasmic hormone receptor complexes (Ka, association constant). Although cytosols prepared from lactating mammary gland contained appreciable quantities of glucocorticoid receptor, only trace amounts were found in the mammary adenocarcinoma.

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

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

Comparative electrophilicity, mutagenicity, DNA repair induction activity, and carcinogenicity of some N- and O-acyl derivatives of N-hydroxy-2-aminoflourene.

N-Myristoyloxy-N-acetyl-2-aminofluorene, N-acetoxy-N-myristoyl-2-aminofluorene, N-myristoyloxy-N-myristoyl-2-aminofluorene, and N-hydroxy-N-myristoyl-2-aminofluorene each yielded a high incidence of sarcomas in male rats within 5 to 7 months after s.c. injection of 64 micronmoles in divided doses. N-Acetoxy-N-acetyl-2-aminofluorene and N-hydroxy-2-acetylaminofluorene, although potent carcinogens at the s.c. site, were less active than the above derivatives with a myristoyl substituent. N-Sulfonoxy-N-acety--2-aminofluorene (purity larger than or equal to 70%) had little or no carcinogenic activity when administered in large amounts by s.c. injection to rats. The low incidence of tumors could have resulted from N-hydroxy-2-acetylaminofluorene or other decompostion products of the N-sulfonozy derivative. Each of the N-acetoxy and N-myristoyloxy derivatives of N-acetyl-2-aminofluorene and of N-myristoyl-2-aminofluorene showed electrophilic activity toward methionine; N-acetoxy-N-acetyl-2-aminofluorene was the most reactive and N-myristoyloxy-N-myristoyl-2-aminofluorine was the least reactive. Each of these esters also induced unscheduled tritiated thymidine incorportation in nondividing cultured human fibroblasts and thus appeared to induce lesions in DNA that lead to repair synthesis. EACH OF THE N-acetoxy derivatives was highly mutagenic for Salmonella typhimurium strains TA98 and TA1538 without tissue activation; neither N-myristoyloxy derivative was mutagenic under these conditions. While there was a qualitative correspondence between several of the above activities of these 2-aminofluorene derivatives, the quantitative differences and the lack of detectable mutagenicity of the 2N-myristoyloxy derivatives for S. typhimurium indicate the need for multiple short-term tests in the qualitative prediction of potential carcinogenic activity.

Multiple sites of action of N-hydroxy-2-acetylaminofluorene rat hepatic nuclear transcription.

This study attempts to identify the site(s) of action of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) in relation to its inhibition of rat hepatic nuclear RNA synthesis. Two hr after N-OH-AAF injection (3 mg/100 g body weight), rat hepatic nuclear synthesis and nucleolar RNA synthesis in vitro were inhibited by 60 and 80%, respectively. When total nuclear RNA polymerases were solubilized and assayed in the presence of alpha-amanitin (3.2 mug/ml), only alpha-amanitin-sensitive activity was reduced (50%) by N-OH-AAF. Diethylamino-ethyl-Sephadex column chromatography confirmed this finding and further demonstrated that RNA polymerase II was the activity selectively inhibited. Since N-OH-AAF dramatically inhibited nucleolar RNA synthesis but had little effect on RNA polymerase I activity, per se, we therefore concluded that, in addition to its direct inhibitory effect on the enzymic function of RNA polymerase II, N-OH-AAF must also cause impairment of the nucleolar DNA template function.

N-hydroxy-2-acetylaminofluorene inhibition of rat live RNA polymerases.

Administration of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to rats inhibits liver nuclear RNA synthesis. This effect is reflected in an in vitro inhibition of RNA synthesis by isolated whole nuclei. The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from these nuclei. Both nucleolar and nucleoplasmic polymerases are affected. A similar inhibition of the polymerases was demonstrated in intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxyadenylate-deoxythymidylate). Chromatin was prepared from similar nuclear preparations by two methods, differing in the extent to which they remove endogenous polymerase activity. Each chromatin preparation was transcribed with added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase respectively. With either polymerase and either chromatin preparation, no inhibition of the template activity of chromatin isolated from N-OH-AAF-treated animals could be detected. It is concluded that N-OH-AAF is a potent inhibitor of rat liver nuclear RNA synthesis and that the mechanism of this inhibition is inactivation of the RNA polymerases. At the same time, N-OH-AAF leaves the chromatin template, at least quantitatively, intact for the synthesis of RNA. The implications of such an effect of N-OH-AAF on RNA synthesis are discussed.

Alterations in the transcriptional capacity of hepatic DNA-dependent RNA polymerase I and II by N-hydroxy-2-acetylaminofluorene.

Using hepatic RNA polymerases I and II, acetylaminofluorene modification of DNA in vitro results in reduction of RNA elongation but not in an altered frequency of incorporation of [alpha32P]ribonucleotides. In contrast, in vivo modification of RNA polymerases by a single dose of N-2-hydroxy-2-acetylaminofluorene results in an alteration of their capacity for transcribing a normal RNA product but does not affect the product size transcribed from normal or carcinogen-modified templates.

Alteration of hepatocytes by subcarcinogenic exposure to n-2-fluorenylacetamide.

These experiments examined the effects of a single, subcarcinogenic dose of dimethylnitrosamine or N-hydroxyfluorenylacetamide when administered after a subcarcinogenic dietary regimen of N-2-fluorenylacetamide. Control rats that received either carcinogen diet alone or a single dose of carcinogen demonstrated neither hepatic nodules nor hepatocellular carcinomas. Those animals that received dimethylnitrosamine subsequent to carcinogen diet demonstrated many persistent hepatic nodules and 100% hepato-cellular carcinomas. These data support the concept that the nodules produced by subcarcinogenic ingestion of N-2-fluorenylacetamide are not composed simply of normal hepatocytes undergoing compensatory regeneration but consist of cells that have been altered by the carcinogen. One manifestation of this alteration is an increased susceptibility to further carcinogenic evolution.

Biochemical and morphological changes in hepatic nuclear membranes produced by N-hydroxy-2-acetylaminofluorene.

The effect of N-hydroxy-2-acetylaminofluorene on the ultrastructure and synthesis of hepatic neclear membranes was evaluated in partially hepatectomized rats. The incorporation of L-[4,5-3H]leucine into two nuclear membrane fractions increased within 2 hr after hepatic resection and reached a peak at 20 hr. After partial hepatectomy, the decay of radioactivity in nuclear membrane proteins labeled with L-[4,5-3H]leucine revealed similar half-lives for the two membrane fractions when compared to those obtained from sham-operated animals. The protein concentration of the nuclear membrane fraction of higher density decreased sharply within 2 hr after partial hepatectomy and remained low throughout a 20-hr postoperative period. Polyacrylamide gel electrophoresis of both nuclear membrane fractions showed a similar composition. Nine proteins were resolved, varying from 21,000 to 190,000 daltons. The two major protein bands were in the range of 50,000 and 70,000 daltons, respectively. Treatment of partially hepatectomized animals with N-hydroxy-2-acetylaminofluorene showed marked dilation of the nuclear envelope and rough endoplasmic reticulum in situ upon electron microscopic examination. Vacuolization and evagination of the perinuclear membranes were also noticeable in isolated nuclei obtained from carcinogen-treated rats. Inhibition by N-hydroxy-2-acetylaminofluorene of the incorporation of L-[4,5-3H]leucine into the nuclear membranes was dose-dependent and remained depressed throughout a 60-min labeling period. These results suggest that the inhibitory effects on RNA and protein synthesis previously shown to be produced by this arylhydroxylamine hepatocarcinogen may lead to disruption of the morphology and synthesis of the nuclear envelope.

The action of N-hydroxy-2-acetylaminofluorene on the synthesis of ribosomal and poly (A)-RNA in normal and regenerating liver.

Polyribosomal RNA prepared from normal and regenerating liver was chromatographed on poly(U)-Sepharose to obtain rRNA and poly(A)-RNA. Under the conditions employed for labeling the RNA with [5-3H]orotic acid, poly (A)-RNA from regenerating liver represented 1--3% of the total RNA content, and 14--18% of the total radioactivity in polyribosomal RNA at varying times after partial hepatectomy. The specific activity of poly (A)-RNA was elevated by 50% and 30% only at 2 h and 6 h after partial hepatectomy, respectively. Ribosomal RNA synthesis increased 2.5--4 fold at 2--20 h after operation. Treatment of normal or partially hepatectomized rats with N-hydroxy-2-acetylaminofluorene showed a differential dose-dependent effect on rRNA and poly (A)-RNA synthesis with rRNA synthesis being more sensitive to the inhibitory effect of the carcinogen. No change in the poly(A) content was apparent as a result of N-hydroxy-2-acetylaminofluorene treatment.

An improved method for DNA alkaline gradient analysis and its application to the effect of carcinogens on mouse liver DNA.

An alkaline sodium iodide density gradient technique is described, for use in sedimentation rate centrifugation studies of in vivo induction of single strand breaks in DNA. The combination of this type of gradient with a sensitive fluorometric DNA estimation makes it possible to analyze very small amounts of DNA without any need for labeling the nucleic acid with radioactive thymidine.

Mechanism-based inactivation of N-arylhydroxamic acid N,O-acyltransferase by 7-substituted-N-hydroxy-2-acetamidofluorenes.

N-Arylhydroxamic acid N,O-acyltransferase (AHAT) catalyzes the transfer of the N-acetyl group from N-arylhydroxamic acids to arylamines. In the absence of an arylamine acceptor, AHAT catalyzes the conversion of N-arylhydroxamic acids to reactive electrophilic intermediates that become irreversibly bound to cellular nucleophiles, including those present on AHAT itself. As part of an investigation of the AHAT-catalyzed bioactivation process, a series of 7-substituted analogues of N-hydroxy-2-acetamidofluorene (1) was synthesized and evaluated in vitro as substrates and inactivators of a partially purified hamster hepatic AHAT preparation. All of the compounds functioned as acetyl donors in the AHAT-catalyzed transacetylation of 4-aminoazobenzene (AAB) and all of them were inactivators of AHAT. The inactivation process exhibited apparent first-order kinetics, and the 7-methoxy compound exhibited the largest inactivation rate constant. Quantitative structure-activity analysis provided support for the concept that positively charged species are involved in the inactivation of AHAT by this series of compounds. Results of experiments in which nucleophilic trapping agents such as glutathione, cysteine, methionine, guanosine phosphate, and tRNA were included in incubation mixtures with AHAT and the N-arylhydroxamic acids indicated that electrophiles which diffuse away from the enzyme active site participate in the inactivation process.

Irreversible inhibition of the cytosolic metabolism of N-hydroxy-2-acetylaminofluorene by its glycolyl analog.

The glycolyl hydroxamic acid derivative of 2-aminofluorene was found to be a potent inhibitor of its own metabolism and the metabolism of N-hydroxy-2-acetylaminofluorene by rat liver cytosol. The inhibition was irreversible, as well as time and concentration dependent, which indicates a suicide-inhibition type of metabolism. There was a direct correlation between the inhibition of N-hydroxy-2-acetylaminofluorene disappearance and 2-acetylaminofluorene formation. In contrast, both the glycolyl and acetyl hydroxamic acid derivatives were metabolized to a similar extent by enzymes in the microsomal fraction.

N'-hydroxy-2-aminofluorene: the principal mutagen produced from N-hydroxy-2-acetylaminofluorene by a mammalian supernatant enzyme preparation.

A Salmonella typhimurium TA 1538 culture system wasused to monitor the production of mutagen from N-hydroxy-2-acetylaminofluorene by soluble liver enzymes. When benzene was used to extract the mutagen, no mutagenic activity remained in the liver enzyme preparation. The benzene extract contained approximately two-thirds of the total mutagenic activity produced by the liver enzyme preparation. Using thin layer and column chromatography to analyze the benzene extract, we deduced that N-hydroxy-2-aminofluorene accounted for the mutagenic activity which resulted from the incubation of N-hydroxy-2-acetylaminofluorene with soluble liver enzymes.

2-acetylaminofluorene-induced unscheduled DNA synthesis in hepatocytes isolated from 3-methylcholanthrene treated rats.

The dose-dependent induction of unscheduled DNA synthesis (UDS) by 2-acetylaminofluorene (AAF) and N-OH-2-acetylaminofluorene (N-OH AAF) in primary rat hepatocytes isolated from untreated and 3-methylcholanthrene (3-MC) treated rats was investigated. 3-MC treatment was not necessary for the dose-dependent induction of UDS induced by N-OH AAF, suggesting the presence of enzyme levels adequate for its esterification to an active form in isolated primary hepatocytes. Although all doses of AAF increased the level of [3H]thymidine incorporation above that of the control, a dose-dependent response could not be demonstrated, suggesting inadequate constitutive levels of N-hydroxylating enzymes. Treatment of rats with 3-MC 24 h prior to hepatocyte isolation resulted in a dose-dependent induction of UDS by AAF. 3-MC treatment increased the amount of UDS per cell, as well as the percentage of cells induced to repair their DNA.

Synergistic effect of diethylstilbestrol on the mutagenicity of 2-acetylaminofluorene and N-hydroxy-acetylaminofluorene in the Salmonella assay system.

Salmonella typhimurium, TA-1538, was used to investigate the mutagenic potential of N-2-acetylaminofluorene (2-AAF), N-hydroxy-N-2-acetylaminofluorene (N-OH-2-AAF) and diethylstilbestrol (DES) individual and in combination. In the presence of an induced or uninduced rat liver metabolizing system (S-9), the histidine requiring strain of bacteria was reverted to prototrophy by the aromatic amines but not by the synthetic estrogen. However, when DES was combined with 2-AAF or N-OH-I-AAF in the presence of the induced S-9 fraction, the number of revertant colonies was increased 2- to 4-fold above the levels obtained with the aromatic amines alone. The synergistic effect of DES, a non-mutagen, on the mutagenicity of these aromatic amines was observed only when a 3-methylcholanthrene induced rat liver S-9 fraction was used as the source of mammalian enzymes. When uninduced mouse or rat liver S-9 fractions were used in this test system, an inhibitory effect rather than an enhancing effect was observed.

Irreversible inhibition of rat hepatic transacetylase activity by N-arylhydroxamic acids.

Both N-hydroxy-2-acetamidofluorene (N-OH-AAF) and the heterocyclic analogue, 2-(N-hydroxyacetamido)carbazole (N-OH-AAC), were shown to be mechanism-based irreversible inhibitors (suicide inhibitors) of partially purified rat hepatic N-acetyltransferase (NAT) activity. Although N-OH-AAC exhibited an apparent first-order inactivation rate constant (ki) that was 7-fold lower than that of N-OH-AAF, their relative ki/KD values indicate that N-OH-AAC was the more potent and efficient inactivator of transacetylase activity. Inactivation of NAT activity by these N-arylhydroxamic acids appeared to involve contributions by electrophiles that react with the enzyme subsequent to release from the active site and by electrophiles that remain complexed with the active site. The irreversible nature of the enzyme inactivation was demonstrated by the failure to recover transacetylase activity upon either extensive dialysis or gel filtration of preparations that had been subjected to incubation with N-OH-AAF or N-OH-AAC. The use of the nucleophile N-acetylmethionine to trap the electrophilic reactants formed in the transacetylase-catalyzed bioactivation process resulted in a lower rate and extent of formation of methylthio adducts with N-OH-AAC than with N-OH-AAF. The results of this study indicate that N-OH-AAF and N-OH-AAC have potential for use as tools in the investigation of rat hepatic transacetylases.

Induction of epoxide hydrolase in cultured rat hepatocytes and hepatoma cell lines.

The expression of epoxide hydrolases was studied in cultured rat hepatocytes and hepatoma cell lines. Styrene 7,8-oxide and benzo[a]pyrene 4,5-oxide were used as substrates for microsomal epoxide hydrolase and trans-stilbene oxide for the cytosolic form of this enzyme. In freshly isolated hepatocytes from control rats, microsomal epoxide hydrolase activity was 7.7 and 10.8 nmoles/mg cellular protein/min with benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide as substrates respectively. This enzyme activity increased by more than 2-fold in hepatocytes after 24 hr in culture and remained elevated throughout 96 hr using both substrates. In cultured hepatocytes from rats pretreated in vivo with phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene, both benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide hydrolase activities were increased greater than 1.8 relative to controls. Hepatocytes from 2-acetylaminofluorene-pretreated animals at 24 hr in culture had approximately 9-fold higher activities than control hepatocytes. In marked contrast to microsomal epoxide hydrolase activity, the cytosolic enzyme showed an initial activity of 191 pmoles/mg cellular protein/min in freshly isolated hepatocytes, decreased by 75% after 24 hr in culture, and was barely detectable at 96 hr. A similar trend was apparent in hepatocytes from the pretreated animals. In vitro treatment of hepatocytes with trans-stilbene oxide and phenobarbital increased microsomal epoxide hydrolase, while this activity was refractory to 2-acetylaminofluorene treatment. Styrene 7,8-oxide hydrolase activity was increased in the McA-RH-7777 rat hepatoma cell line by phenobarbital, trans-stilbene oxide and 2-acetylaminofluorene treatment. Similarly, benzo[a]pyrene 4,5-oxide hydrolase activity was also increased in this cell line by treatment with phenobarbital and trans-stilbene oxide but not by 2-acetylaminofluorene. Microsomal epoxide hydrolase activity in rat H4-II-E hepatoma cells was refractory to induction, except by trans-stilbene oxide treatment, which caused a 70% increase in benzo[a]pyrene 4,5-oxide hydrolase activity.

Hepatic N-acetyltransferases: selective inactivation in vivo by a carcinogenic N-arylhydroxamic acid.

N-Hydroxy-2-acetamidofluorene (N-OH-AAF), a carcinogenic N-arylhydroxamic acid, is a selective and irreversible inhibitor of arylamine N-acetyltransferase (NAT) activity in vitro. The present study demonstrates that intraperitoneal administration of N-OH-AAF to hamsters caused an irreversible reduction of the hepatic transacetylase activity that catalyzes the transfer of the acetyl group from N-OH-AAF to 4-aminoazobenzene (AAB), but did not affect the acetyl coenzyme A (CoASAc) dependent NAT that is responsible for acetylation of p-aminobenzoic acid (PABA). A 40% loss of N-OH-AAF:AAB transacetylase activity occurred 4 hr after administration of 50 mg/kg of N-OH-AAF. To determine whether biotransformation of N-OH-AAF is a factor in determining its ability to inactivate N-OH-AAF:AAB transacetylase activity in vivo, the enzyme-inducing agent phenobarbital and the esterase/acylamidase inhibitor bis(p-nitrophenyl)phosphate (BNPP) were administered to the animals prior to the administration of N-OH-AAF. The loss of N-OH-AAF:AAB transacetylase activity was prevented by treatment of the animals with either phenobarbital or with BNPP. The ability of the esterase/acylamidase inhibitor, BNPP, to prevent the N-OH-AAF-mediated loss of transacetylase activity indicates that, in contrast to the inactivation process in vitro, esterase-catalyzed deacetylation of N-OH-AAF may be required for transacetylase inactivation in vivo. It is proposed that in vivo the endogenous acetyl donor, CoASAc, acetylates the enzyme and prevents the deacetylation of N-OH-AAF by NAT, thereby impeding the N-OH-AAF-mediated inactivation process, but facilitating enzyme inactivation by N-hydroxy-2-aminofluorene. The latter proposal was supported by the demonstration that CoASAc inhibited the in vitro inactivation of N-OH-AAF:AAB transacetylase activity by N-OH-AAF.

Characterization of hamster recombinant monomorphic and polymorphic arylamine N-acetyltransferases: bioactivation and mechanism-based inactivation studies with N-hydroxy-2-acetylaminofluorene.

The purified hamster recombinant arylamine N-acetyltransferases (NATs), rNAT1-9 and rNAT2-70D, were characterized for their capabilities to bioactivate N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA binding reactants and for their relative susceptibilities to mechanism-based inactivation by N-OH-AAF. The rate of DNA adduct formation resulting from rNAT1-9 bioactivation of [14C]N-OH-AAF was more than 30 times greater than that of rNAT2-70D-catalyzed bioactivation of [14C]N-OH-AAF. This result is consistent with substrate specificity data indicating that N-OH-AAF is a much better acetyl donor for hamster NAT1 than NAT2. Previous studies indicated that N-OH-AAF is a mechanism-based inactivator of hamster and rat NAT1. In the presence of N-OH-AAF, both rNAT1-9 and rNAT2-70D underwent irreversible, time-dependent inactivation that exhibited pseudo first-order kinetics and was saturable at higher N-OH-AAF concentrations. The enzymes were partially protected from inactivation by the presence of cofactor and substrates. The limiting rate constants (ki) and dissociation constants (Ki) for inactivation by N-OH-AAF were determined. The second-order rate constants (ki/KI) were 22.1 min-1 mM-1 for rNAT1-9 and 1.0 min-l mM-1 for rNAT2-70D, indicating that rNAT1-9 is approximately 20 times more susceptible than rNAT2-70D to inactivation by N-OH-AAF. The kinetic parameters for rNAT1-9 were nearly identical to values previously reported for partially purified hamster NAT1. Partition ratios were 504 for inactivation of rNAT1-9 by N-OH-AAF and 137 for inactivation of rNAT2-70D. Thus, a turnover of almost 4 times as many N-OH-AAF molecules is required to inactivate each molecule of rNAT1-9 than is needed to inactivate rNAT2-70D. The partition ratio data are consistent with the finding that rNAT1-9 catalyzes a higher rate of DNA adduct formation by N-OH-AAF than rNAT2-70D. The combined results indicate that the recombinant enzymes are catalytically and functionally identical to hamster NATs and, therefore, will be a useful resource for studies requiring purified NATs.

Bioactivation of N-arylhydroxamic acids by rat hepatic N-acetyltransferase. Detection of multiple enzyme forms by mechanism-based inactivation.

Enzymatic N,O-acyltransfer of carcinogenic N-arylhydroxamic acids such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) results in the production of reactive electrophiles that can bond covalently with nucleophiles and also can cause inactivation of acyltransferase activity in a mechanism-based manner. Incubation of partially purified rat hepatic N-acetyltransferases (NAT) with N-OH-AAF resulted in extensive inactivation of N-OH-AAF/4-aminoazobenzene (AAB) N,N-acetyltransferase and acetyl coenzyme A (AcCoA)/procainamide (PA) N-acetyltransferase activities, whereas AcCoA/p-aminobenzoic acid (PABA) N-acetyltransferase activity was inhibited only slightly. Affinity chromatography with Sepharose 6B 2-aminofluorene (2-AF) resulted in the separation of two NAT activities. NAT I primarily catalyzed the AcCoA-dependent acetylation of PABA; NAT II catalyzed, N,N-acetyltransfer (N-OH-AAF/AAB), AcCoA/PA N-acetyltransfer and N-OH-AAF N,O-acyltransfer (AHAT) activities. Most of the AcCoA/2-AF N-acetyltransferase activity eluted in the NAT II fraction. Results of inactivation experiments with N-OH-AAF and the NAT II fractions suggested that one NAT isozyme was responsible for catalyzing the N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransfer reactions and that inactivation of NAT II correlated with the extent of covalent binding to protein. Further purification of the NAT II fractions by chromatofocusing resulted in a 1300-fold purification of the N-OH-AAF/AAB activity and the coelution of N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransferase activities. These studies indicate that N,O-acyltransfer, arylhydroxamic acid-dependent N-acetylation of arylamines (N,N-acetyltransfer), and AcCoA-dependent N-acetylation of PA may be catalyzed by a common enzyme in rat liver, whereas a second enzyme is responsible for the AcCoA-dependent N-acetylation of PABA.