Patrick E. Hanna

N-acetyltransferases, O-acetyltransferases, and N,O-acetyltransferases: enzymology and bioactivation.

Publisher Summary Acetyltransferases play a central role in the metabolic disposition, detoxication, and bioactivation of a diverse group of drugs—carcinogens and other xenobiotics. Acetylation is a major metabolic pathway for primary aromatic amines (arylamines, ArNH) and hydrazines. In earlier times, acetylation was the principal mechanism for termination of the tuberculostatic action of isoniazid. The importance of human acetylation capacity is further illustrated by the propensity of phenotypically slow acetylator patients to experience methemoglobinemia or drug-induced lupus erythematosus or both when treated either with arylamine drugs or with various agents that contain the hydrazine moiety. The mutagenicity and carcinogenicity of arylamines along with thedocumented environmental and dietary exposure to these agents have stimulated numerous investigations of arylamine metabolism. Thus, the premise that formation of unreactive arylamides by N-acetylation of arylamines is primarily a detoxification process and that oxidative Nhydroxylation is a toxification reaction is supported by a substantial body of evidence. However, the demonstration that arylamine N- acetyltransferases are versatile enzymes that can catalyze the conversion of both N-arylhydroxylamines and N-arylhydroxamic acids to reactive, electrophilic metabolites has broadened the scope of research on acetyltransferases, which are now included among those conjugation systems that play important roles in the bioactivation of xenobiotics.

Human arylamine N-acetyltransferase 1: in vitro and intracellular inactivation by nitrosoarene metabolites of toxic and carcinogenic arylamines.

Arylamines (ArNH 2) are common environmental contaminants, some of which are confirmed risk factors for cancer. Biotransformation of the amino group of arylamines involves competing pathways of oxidation and N-acetylation. Nitrosoarenes, which are products of the oxidation pathway, are electrophiles that react with cellular thiols to form sulfinamide adducts. The arylamine N-acetyltransferases, NAT1 and NAT2, catalyze N-acetylation of arylamines and play central roles in their detoxification. We hypothesized that 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), which are nitroso metabolites of arylamines that are readily N-acetylated by NAT1, would be potent inactivators of NAT1 and that nitrosobenzene (NO-B) and 2-nitrosotoluene (2-NO-T), which are nitroso metabolites of arylamines that are less readily acetylated by NAT1, would be less effective inactivators. The second order rate constants for inactivation of NAT1 by 4-NO-BP and 2-NO-F were 59200 and 34500 M (-1) s (-1), respectively; the values for NO-B and 2-NO-T were 25 and 23 M (-1) s (-1). Densitometry quantification and comparisons of specific activities with those of homogeneous recombinant NAT1 showed that NAT1 constitutes approximately 0.002% of cytosolic protein in HeLa cells. Treatment of HeLa cells with 4-NO-BP (2.5 microM) for 1 h caused a 40% reduction in NAT1 activity, and 4-NO-BP (10 microM) caused a 50% loss of NAT1 activity within 30 min without affecting either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glutathione reductase (GR) activities. 2-NO-F (1 microM) inhibited HeLa cell NAT1 activity by 36% in 1 h, and a 10 microM concentration of 2-NO-F reduced NAT1 activity by 70% in 30 min without inhibiting GAPDH or GR. Mass spectrometric analysis of NAT1 from HeLa cells in which NAT1 was overexpressed showed that treatment of the cells with 4-NO-BP resulted in sulfinamide adduct formation. These results indicated that exposure to low concentrations of nitrosoarenes may lead to a loss of NAT1 activity, thereby compromising a critical detoxification process.

Involvement of thioredoxin in sulfoxide reduction by mammalian tissues

Sulindac, a sulfoxide with antiinflammatory activity, is reduced to the corresponding sulfide by rat hepatic cytosolic enzymes requiring NADPH for maximal activity. This reaction is inhibited by insulin, L-cystine, glutathione disulfide and 5,5′-dithiobis(2-nitrobenzoic acid), all of which are known to interact with the thioredoxin system comprised of NADPH, thioredoxin reductase and thioredoxin. Sodium arsenite, a known inhibitor of thioredoxin reductase, also inhibited sulindac reduction. Rat hepatic cytosolic fractions from which thioredoxin had been removed by chromatography on Sephadex G-50 showed minimal sulfoxide reductase activity; activity could be restored by addition of purified Escherichiacoli thioredoxin or dithiothreitol. These findings are the first demonstration of thioredoxin-dependent sulfoxide reduction by mammalian tissues.

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.

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.

Cyclic Hydroxamic Acid Inhibitors of Prostate Cancer Cell Growth: Selectivity and Structure Activity relationships

Clinical symptoms of prostatitis, prostatodynia, and benign prostatic hyperplasia are relieved by the pollen extract cernilton, and the water‐soluble fraction of this extract selectively inhibits growth of some prostate cancer cells. A cyclic hydroxamic acid, DIBOA, has been isolated from this extract and mimics its cell growth‐inhibitory properties, but the specificity of DIBOA for inhibition of prostate cell growth has not been reported.

N-(carbobenzyloxy)isatin: A slow binding α-keto lactam inhibitor of α-chymotrypsin

Abstract The N-carbobenzyloxy derivative of 2,3-dioxindole (N-Cbz-isatin) (1) has been synthesized and shown to be a slow binding inhibitor of chymotrypsin. Compound 1 does not inhibit porcine pancreatic elastase. The N-tert-butoxycarbonyl derivative of 2,3-dioxindole (2) is a weak inhibitor of chymotrypsin and does not inhibit porcine pancreatic elastase.

Effect of group-selective modification reagents on arylamine N-acetyltransferase activities

Two forms of hamster hepatic arylamine N-acetyltransferase (NAT; EC 2.3.1.5), designated NAT I and NAT II, were purified 200- to 300-fold by sequential 35-50% ammonium sulfate fractionation, Sephadex G-100 gel filtration chromatography, AAB affinity chromatography, DEAE ion exchange chromatography, and P-200 gel filtration chromatography. Treatment of either NAT I or NAT II with N-ethylmaleimide (NEM), a cysteine selective reagent, caused a concentration-dependent loss of enzymatic activities. Acetyl coenzyme A (AcCoA) protected NAT I against inactivation by NEM, whereas both 2-acetylaminofluorene (2-AAF) and AcCoA protected NAT II against inactivation. Incubation of either NAT I or NAT II with phenylglyoxal (PG), an arginine selective reagent, caused a time-dependent and a concentration-dependent loss of both NAT I and NAT II activities; the inactivations followed pseudo first-order kinetics. The reaction order with respect to PG was approximately two for each enzyme, consistent with the expected stoichiometry for the reaction of PG with arginine. The presence of AcCoA provided full protection of NAT I against inactivation by PG. However, neither AcCoA nor 2-AAF provided protection of NAT II against inactivation by PG. Diethylpyrocarbonate (DEPC), a histidine selective reagent, caused time-dependent and concentration-dependent pseudo first-order inactivation of both NAT I and NAT II. Neither AcCoA nor products of NAT-catalyzed reactions protected NAT I and NAT II against inactivation by DEPC. These results suggest that cysteine, arginine and histidine residues are essential to the catalytic activity of both NAT I and NAT II; the cysteine(s) is located at or near the binding site of NAT I and NAT II, and the arginine residue appears to be located in the AcCoA binding site of NAT I. In contrast, the essential arginine residue(s) of NAT II and the essential histidine residue(s) of both NAT I and NAT II are not likely to reside in the binding site of the enzymes.

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.

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.