N-acetyltransferases Research Articles

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.

Acetylation genotype and the genetic susceptibility to prostate cancer in a southern European population

Epidemiologic studies have suggested that environmental factors and diet are important risk factors in the pathogenesis of prostate cancer. The N-acetyltransferases (NAT) are important enzymes in activation and inactivation of various carcinogens, including those found in well-cooked meat and cigarette smoke. We analyzed DNA samples from 146 prostate cancer patients and 174 healthy men. We used PCR-RFLP method to analyze NAT 1 and NAT 2 polymorphisms. We did not find statistically significant differences in NAT 1 genotypes frequencies between prostate cancer patients and control group. We observed an association of the slow acetylator genotype, NAT 2*6/NAT2*6 with prostate cancer protection (P=0.017; OR=0.31, 95% CI 0.11--0.84). Multivariate logistic regression analysis confirmed this association (0.030; OR=0.32, 95% CI 0.12--0.89). Our results indicate a role of NAT2 polymorphisms in the carcinogenic pathway of prostate cancer, specifically in a population of Southern Europe.

Structure and Regulation of the Drug-Metabolizing Enzymes Arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NAT) are xenobiotic-metabolizing enzymes responsible for N-acetylation of many arylamines. They are also important for O-acetylation of N-hydroxylated heterocyclic amines. These enzymes play thus an important role in the detoxification and activation of numerous therapeutic drugs and carcinogens. Two closely related polymorphic isoforms (NAT1 and NAT2) have been described in humans and interindividual variations in NAT genes have been shown to be a potential source of adverse drug reaction. In addition, NAT1 and/or NAT2 phenotypes may modulate the risk of certain cancers in people exposed to aromatic amine carcinogens. Recent advances on the regulation of human NAT1 activity has shown that hydroxylamine and/or nitroso intermediates of NAT1 substrates inhibit the enzyme through direct irreversible interaction with its catalytic cysteine residue. Oxidative molecules such as hydrogen peroxide, S-nitrosothiols and peroxynitrite have also been shown to inactivate reversibly or irreversibly the enzyme in a similar manner. In this review, after summarizing the general background on human NAT enzymes, we focus on the recent developments on the regulation of the activity of these drug-metabolizing enzymes by substrate-intermediates and by oxidant molecules. The recent findings reviewed here provide possible mechanisms by which these non genetic determinants inhibit NAT1 activity and thereby may affect drug efficacy/toxicity.

Heterocyclic amines and genotype of N-acetyltransferases as risk factors for prostate cancer

A variety of carcinogenic heterocyclic amines are produced during the cooking of meat at high temperatures. These carcinogens are metabolized by N-acetyltransferases (NAT), which are polymorphic in the population. This study examined associations between prostate cancer (PCa) and the consumption of different kinds of meat, heterocyclic amine intake and NAT genotypes. PCa patients and controls were recruited in the Syracuse, NY area. Levels of meat and heterocyclic amine intakes were determined from validated surveys and NAT genotypes were determined by the sequences of PCR-amplified DNA from buccal swabs. A total of 152 cases and 161 controls were eligible for analysis. There was an association between PCa and history of PCa in the first-degree blood relatives (OR = 4.59, 95% CI 2.21-9.70), and family history of bladder cancer (P < 0.02). However, there was no association with the history of other cancers. There was no association between PCa and either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) intake, or NAT1 and NAT2 genotypes. However, there was a trend of association with MeIQx and with rapid NAT2 and NAT1*10 in combination with PhIP. A new NAT1 allele with a frequency of one out of 544 chromosomes was found in the Caucasian subjects.

Irreversible Inactivation of Arylamine N-Acetyltransferases in the Presence of N-Hydroxy-4-Acetylaminobiphenyl: A Comparison of Human and Hamster Enzymes

Arylamine N-acetyltransferases (NATs) catalyze the N-acetylation of arylamines, the O-acetylation of N-arylhydroxylamines, and the conversion of N-(aryl)acetohydroxamic acids to N-acetoxyarylamines. NATs also undergo irreversible inactivation in the presence of N-(aryl)acetohydroxamic acids. We previously established that inactivation of hamster NAT1 by N-hydroxy-2-acetylaminofluorene is the result of sulfinamide adduct formation with Cys68. The purpose of this research was to determine the kinetics of inactivation of hamster NAT1, hamster NAT2, and human NAT1 by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), to identify the amino acids that are modified upon NAT-catalyzed bioactivation of N-OH-4-AABP, to characterize the adducts and to identify factors that influence the propensity of NATs to undergo inactivation by N-arylhydroxamic acids. Mass spectrometric analysis of the NATs, after incubation with N-OH-4-AABP, revealed that the principal adduct of each protein was a (4-biphenyl)sulfinamide. Proteolysis of the adducted NATs caused hydrolysis of the sulfinamides to sulfinic acids. Tandem mass spectrometric analysis of the modified peptides revealed that each NAT isozyme contained a sulfinic acid on the Cys68 side chain. Minor adducts were identified as 4-aminobiphenyl conjugates of tyrosines. Hamster NAT1 was more rapidly inactivated by N-OH-4-AABP than either hamster NAT2 or human NAT1, and it was demonstrated that 4-nitrosoobiphenyl, which forms the sulfinamide adducts, accumulates during incubation of N-OH-4-AABP with hamster NAT2 and human NAT1 but not during incubations with hamster NAT1. Steady state kinetic analysis of the hydrolysis of acetylated NATs revealed that the half-lives of acetylated hamster NAT2 and human NAT1 are 7-8-fold greater than that of acetylated hamster NAT1. These results support the proposal that the mechanism of inactivation of NATs by N-OH-4-AABP involves initial deacetylation to produce N-OH-4-aminobiphenyl, which after oxidative conversion to 4-nitrosobiphenyl reacts with Cys68 to form a sulfinamide. The relatively short half-life of the acetylated form of hamster NAT1 contributes to its greater susceptibility to inactivation by N-OH-4-AABP.

Arylamine N-Acetyltransferases: What We Learn from Genes and Genomes

Arylamine N-acetyltransferases (NATs) are phase II xenobiotic metabolizing enzymes, catalyzing acetyl-CoA-dependent N- and O-acetylation reactions. All NATs have a conserved cysteine protease-like Cys-His-Asp catalytic triad inside their active site cleft. Other residues determine substrate specificity, while the C-terminus may control hydrolysis of acetyl-CoA during acetyltransfer. Prokaryotic NAT-like coding sequences are found in > 30 bacterial genomes, including representatives of Actinobacteria, Firmicutes and Proteobacteria. Of special interest are the nat genes of TB-causing Mycobacteria, since their protein products inactivate the anti-tubercular drug isoniazid. Targeted inactivation of mycobacterial nat leads to impaired mycolic acid synthesis, cell wall damage and growth retardation. In eukaryotes, genes for NAT are found in the genomes of certain fungi and all examined vertebrates, with the exception of canids. Humans have two NAT isoenzymes, encoded by highly polymorphic genes on chromosome 8p22. Syntenic regions in rodent genomes harbour two Nat loci, which are functionally equivalent to the human NAT genes, as well as an adjacent third locus with no known function. Vertebrate genes for NAT invariably have a complex structure, with one or more non-coding exons located upstream of a single, intronless coding region. Ubiquitously expressed transcripts of human NAT1 and its orthologue, murine Nat2, are initiated from promoters with conserved Sp1 elements. However, in humans, additional tissue-specific NAT transcripts may be expressed from alternative promoters and subjected to differential splicing. Laboratory animals have been widely used as models to study the effects of NAT polymorphism. Recently generated knockout mice have normal phenotypes, suggesting no crucial endogenous role for NAT. However, these strains will be useful for understanding the involvement of NAT in carcinogenesis, an area extensively investigated by epidemiologists, often with ambiguous results.

Pharmacogenetics, Regulation and Structural Properties of the Drugmetabolizing Enzymes Arylamine N-acetyltransferases

Xenobiotic-metabolizing enzymes (XME) are responsible for the biotransformation of a vast range of compounds. Arylamine N-acetyltransferases (NAT) are XME responsible for the acetylation of many arylamine and heterocyclic amines. They therefore, play an important role in the detoxification and activation of numerous drugs and carcinogens. Two closely related isoforms (NAT1 and NAT2) have been described in humans. NAT2 is present mainly in the liver and gut whereas NAT1 is found in a wide range of tissues. Interindividual variations in NAT genes have been shown to be a potential source of pharmacological and / or pathological susceptibility. In addition, there is now evidence that non genetic factors, such as substrate-dependent inhibition or redox conditions, may also contribute to overall NAT1 activity. This mini-review summarizes current knowledge on human NATs. Recent aspects of NAT gene expression, regulation and structural properties are discussed. Keywords: xenobiotic-metabolizing enzymes, arylamine n-acetyltransferases, catalytic activity, phenotypes, expression, polymorphism

Comparative metabonomics of differential hydrazine toxicity in the rat and mouse

Interspecies variation between rats and mice has been studied for hydrazine toxicity using a novel metabonomics approach. Hydrazine hydrochloride was administered to male Sprague–Dawley rats (30 mg/kg, n = 10 and 90 mg/kg, n = 10) and male B6C3F mice (100 mg/kg, n = 8 and 250 mg/kg, n = 8) by oral gavage. In each species, the high dose was selected to produce the major histopathologic effect, hepatocellular lipid accumulation. Urine samples were collected at sequential time points up to 168 h post dose and analyzed by 1H NMR spectroscopy. The metabolites of hydrazine, namely diacetyl hydrazine and 1,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acid (THOPC), were detected in both the rat and mouse urine samples. Monoacetyl hydrazine was detected only in urine samples from the rat and its absence in the urine of the mouse was attributed to a higher activity of N-acetyl transferases in the mouse compared with the rat. Differential metabolic effects observed between the two species included elevated urinary β-alanine, 3- d-hydroxybutyrate, citrulline, N-acetylcitrulline, and reduced trimethylamine- N-oxide excretion unique to the rat. Metabolic principal component (PC) trajectories highlighted the greater degree of toxic response in the rat. A data scaling method, scaled to maximum aligned and reduced trajectories (SMART) analysis, was used to remove the differences between the metabolic starting positions of the rat and mouse and varying magnitudes of effect, to facilitate comparison of the response geometries between the rat and mouse. Mice followed “biphasic” open PC trajectories, with incomplete recovery 7 days after dosing, whereas rats followed closed “hairpin” time profiles, indicating functional reversibility. The greater magnitude of metabolic effects observed in the rat was supported by the more pronounced effect on liver pathology in the rat when compared with the mouse.

Simultaneous Genotyping of Nine Polymorphisms in Xenobiotic-Metabolizing Enzymes by Multiplex PCR Amplification and Single Base Extension

Studies have reported a large interindividual variation in susceptibility to health effects caused by exposure to xenobiotic compounds such as drugs or chemical carcinogens. There is evidence that this can be partly explained by the existence of genetic polymorphisms in metabolic enzymes, such as cytochrome P450 (CYP450), N -acetyltransferases (NATs), and glutathione S -transferases (GSTM, GSTT, GSTP) [see, e.g., Refs. (1)(2)(3)(4)]. However, studies investigating associations between genetic polymorphisms and disease have reported conflicting results, probably caused by insufficient statistical power (5). Moreover, the majority of these studies focused on single polymorphisms. Regarding the number of genes implicated in the metabolism of xenobiotics and the large number of polymorphisms present in the human genome (6), these approaches fail to fully determine the role of genetic variation in an individual’s susceptibility to xenobiotic exposures. Such observations underline the need for methodologies that allow for high-throughput, low-cost genotyping of multiple polymorphisms in large populations (7)(8). In this study we describe the development, validation, and application of a cost-effective and rapid method for simultaneous genotyping of nine polymorphisms in five key enzymes involved in metabolism of xenobiotics: CYP1A2, GSTM1, GSTP1, GSTT1, and NAT2. The fragments containing the nine single-nucleotide polymorphisms (SNPs) were amplified in one sevenplex and one duplex PCR reaction (Table 1⇓ ). Primers were obtained from Qiagen. For the sevenplex PCR, a 50-μL reaction mixture was prepared containing PCR buffer, 0.2 mM deoxynucleotide triphosphates, 0.5 mM MgCl2, 1.25 U of Platinum® Taq Polymerase (Invitrogen), and 200 ng of template DNA. The final primer concentrations were 0.22 μM (for GSTP1 * 3 , GSTT1 , NAT2 * 6 , and NAT2 * 7 ), 0.45 μM (for CYP1A2 * 1F and NAT2 * 5 ), and 0.16 μM (for GSTP1 * 2 ). PCR was …

N-acetyltransferase 2 ( NAT2) genotypes, cigarette smoking, and the risk of breast cancer

N-acetyltransferases (NATs) are important catalytic enzymes that metabolize carcinogenic arylamines. NAT2 genotype might modify the role of cigarette smoking, a source of arylamine exposure, in breast cancer. We conducted a nested case-control study to investigate the association between NAT2 genotype, smoking and breast cancer risk among women (110 cases, 113 matched controls) from the CLUE II cohort in Washington County, MD. Compared to women with the slow acetylator genotype, the main effects odds ratios (OR) for NAT2 were 1.4 for the intermediate acetylator genotype (95% confidence limits (CL) 0.7, 2.7) and 3.6 for the homozygous rapid acetylator genotype (95% CL 1.1, 11.4) ( P for trend=0.05). Smoking was associated in the direction of increased breast cancer risk in slow acetylators (e.g., >15 pack-years versus never smokers OR 2.0; 95% CL 0.7, 5.8) but not in rapid acetylators. These associations were not statistically significant in the total study population, but a statistically significant interaction between smoking and NAT2 acetylator status was present in postmenopausal women. The main effect of NAT2 in the direction of increased risk suggests that exposures to NAT2-activated carcinogens other than cigarette smoke may be important in this study population. The results for smoking were consistent with an inactivation role for NAT2 in breast cancer.

Pharmacogenetics in cancer chemotherapy: balancing toxicity and response.

The goal of chemotherapy is the elimination of tumor cells from the host. This is achieved by the use of therapeutic agents that are often more harmful to normal tissues than to the targeted tumor. Many chemotherapeutic agents are designed to damage cell replication machinery either directly at the level of DNA or indirectly, by inhibiting enzymes involved with DNA repair and synthesis. Novel therapeutic agents that exert their effects at signal transduction pathways have advanced chemotherapy; however, a role for the classic chemotherapeutic agents remains. These classic agents are associated with tumor cell resistance, toxicity, and occasionally secondary neoplasia. Current practices for the dosing of therapeutic agents rely on height and body surface measurements or drug monitoring and Bayesian adaptive control. Pharmacogenetics is emerging as an alternate approach to managing chemotherapy that may prevent undertreatment while avoiding overtreatment and associated toxicities. By determining the polymorphic genetic makeup of the host and, in some instances, the altered genetic expression of the tumor, chemotherapy can be tailored for interindividual response and toxicity avoidance. Chemotherapy is particularly applicable to the pharmacogenetic approach to tailored therapy for a number of reasons. The margin of safety is low with chemotherapeutic agents. Some drugs require biotransformation for activation. Drug activation correlates with toxicity. The pathways of drug clearance or inactivation exhibit polymorphic differences. Interindividual, race-specific, and age-related responses to chemotherapeutic agents are common. Last, drug resistance can be inherent to the tumor as a result of the suppression of apoptosis. Variations in response and toxicity to a specific drug can be caused by alterations in drug-metabolizing enzymes or receptor expression. These effects can be classed as pharmacokinetic and pharmacogenetic differences. Some of the genes known to display polymorphic differences include FLT3 receptor tyrosine kinase, FCG3RA IgG FC receptor, thymidylate synthase, methylenetetrahydrofolate reductase, thiopurine S-methyltransferase, dihydropyrimidine dehydrogenase, aldehyde dehydrogenase, glutathione S-transferase, uridine diphosphate glyuronosyl transferases, N-acetyl transferases, cytochrome P450, and the DNA repair enzymes XPD and XRCC1. To be successful a pharmacogenetic approach to individualized chemotherapy must selectively take advantage of a determination of direct enzyme activity, gene expression, and genotype.

The role of genetic polymorphisms in metabolism of carcinogenic heterocyclic aromatic amines.

More than twenty heterocyclic aromatic amines (HAAs) have been identified in grilled meats, fish, poultry, and tobacco smoke condensate. HAAs are carcinogens and induce tumors at multiple sites in experimental laboratory animals. Because of the widespread occurrence of HAAs in foods, these chemicals may contribute to the etiology of several common human cancers that are associated with frequent consumption of grilled meats including colon, rectum, prostate, and breast. HAAs require metabolism in order to exert their genotoxic effects. Metabolic activation occurs by N-hydroxylation, a reaction catalyzed by cytochromes p450 (CYP). Some N-hydroxy-HAA metabolites may directly react with DNA, but further metabolism by N-acetyltransferases (NATs) or sulfotransferases (SULTs) may occur to form highly reactive N-acetoxy or N-sulfonyloxy esters that readily react with DNA bases. The N-acetoxy ester of the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is detoxified by glutathione S-transferases (GSTs), which catalyze the reduction of the reactive intermediate back to the parent amine. Some HAAs also undergo detoxification through conjugation reactions with the phase II enzymes such as UDP-glucuronosyltransferases (UGTs) or SULTs to form stable, polar products that are readily eliminated. All of these xenobiotic metabolism enzyme systems (XMEs) display common genetic polymorphisms, which may affect protein expression, protein stability, catalytic activity, and thus, the biological potency of these procarcinogens. In this review, the roles of common genetic polymorphisms of XMEs involved in HAA metabolism and cancer risk are discussed.

Risk of colorectal adenomas in relation to meat consumption, meat preparation, and genetic susceptibility in a Dutch population.

We studied the association between meat consumption and colorectal adenomas, and potential influence of genetic susceptibility to heterocyclic aromatic amines (HCAs) formed during meat cooking at high temperatures. We studied HCA concentration in relation to preparation habits among 63 volunteers. Associations of meat consumption, meat preparation habits, and genetic susceptibility with colorectal adenoma risk were investigated among 431 adenoma cases and 433 polyp-free controls recruited at endoscopy. Participants completed a meat consumption and preparation questionnaire and provided blood for DNA isolation. Polymorphisms of N-acetyltransferases (NAT) 1 and 2, sulfotransferase (SULT) 1A1, and glutathione-S-transferases (GST) M1 and T1 were determined. HCAs were present in habitually prepared meat, although meat consumption (7 versus < 5x/week) did not increase the risk of colorectal adenomas (odds ratio (OR) 1.2, 95% confidence interval (CI) 0.8-1.9). Also, presumed unfavorable preparation habits (e.g., use of lid, preference for darkly browned meat) did not increase adenoma risk (OR 0.8 and 0.9, respectively). Only the combination of NAT2 slow acetylation and frequent meat consumption (> 5x/week) slightly increased adenoma risk (OR 1.6, 95% CI 1.1-2.3). In this Dutch population, unfavorable meat consumption and preparation habits did not increase colorectal adenoma risk, and these associations were not influenced by relevant genetic polymorphisms.

Mass spectrometric investigation of the mechanism of inactivation of hamster arylamine N-acetyltransferase 1 by N-hydroxy-2-acetylaminofluorene.

Arylamine N-acetyltransferases (NATs) are expressed in most mammalian tissues. NATs catalyze the N-acetylation of primary arylamines, the O-acetylation of N-arylhydroxylamines, and the N,O-transacetylation of N-arylhydroxamic acids. The latter two reactions result in formation of reactive, electrophilic N-acetoxyarylamines, which are considered to be the ultimate carcinogenic metabolites of certain environmental and dietary arylamines. Incubation of various N-(aryl)acetohydroxamic acids, such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) with hamster NAT1, results in time-dependent, concentration-dependent, and kinetically first-order irreversible inactivation of the enzyme. N-OH-AAF also causes in vivo inactivation of NAT1. The purpose of this research was to investigate the molecular mechanism of NAT1 inactivation by identifying the amino acid residues that undergo covalent modification upon NAT1-catalyzed bioactivation of N-OH-AAF and by characterizing the chemical structures of the adducts. Electrospray ionization quadrupole time-of-flight mass spectrometric analysis of NAT1 that had been incubated with N-OH-AAF revealed that the mass of the major adduct (+195 Da) was consistent with a (2-fluorenyl)sulfinamide modification. The major adduct underwent hydrolysis to yield a protein with a molecular mass that corresponded to a sulfinic acid-modified NAT1. Treatment of NAT1 with 2-nitrosofluorene resulted in a modification (+195 Da) that was identical in mass to that obtained with N-OH-AAF-inactivated enzyme. Matrix-assisted laser desorption-ionization quadrupole time-of flight tandem mass spectrometric (MALDI Q-TOF MS/MS) analysis revealed that the modified residue was the catalytically essential Cys68. MALDI Q-TOF MS/MS sequencing of peptides from protease digests of inactivated NAT1 also identified two minor adducts at Tyr17 and Tyr186, each of which was covalently conjugated with 2-aminofluorene. Thus, the mechanism of inactivation of NAT1 by N-OH-AAF involves NAT1-catalyzed deacetylation to afford N-hydroxy-2-aminofluorene, which after oxidative conversion to 2-nitrosofluorene, forms a sulfinamide adduct by reacting with Cys68. GSH had little effect on the inactivation of NAT1 by N-OH-AAF, although high concentrations of cysteine attenuated both the extent of inactivation and the sulfinamide adduct formation.

Chemical modification of hamster arylamine N-acetyltransferase 2 with isozyme-selective and nonselective N-arylbromoacetamido reagents.

Arylamine N-acetyltransferases (NATs) catalyze a variety of biotransformation reactions, including N-acetylation of arylamines and O-acetylation of arylhydroxylamines. Chemical modification of hamster recombinant NAT2 with 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an affinity label for the enzyme whereas bromoacetanilide inactivates NAT2 through a bimolecular alkylation process. Electrospray ionization quadrupole time-of-flight mass spectrometry analysis of Br-AAF-treated NAT2 showed that a single molecule of 2-acetylaminofluorene had been adducted. Peptide sequencing with tandem mass spectrometry identified the catalytically essential Cys68 as the alkylated amino acid. Br-AAF exhibits similar affinity for hamster NAT1 and NAT2, but is a more effective inactivator of NAT1 because, subsequent to the formation of a reversible enzyme-Br-AAF complex, the rate of alkylation of NAT1 is greater than the rate of alkylation of NAT2. Bromoacetanilide alkylates Cys68 and, to a lesser extent, Cys237 of NAT2; it does not exhibit significant selectivity for either NAT1 or NAT2.

Peroxynitrite Irreversibly Inactivates the Human Xenobioticmetabolizing Enzyme Arylamine N-Acetyltransferase 1 (NAT1) in Human Breast Cancer Cells: A CELLULAR AND MECHANISTIC STUDY

Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (k(inact) = 5 x 10(4) m(-1).s(-1)) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1.

Cytochrome P450 (CYP) 1A2, sulfotransferase (SULT) 1A1, and N-acetyltransferase (NAT) 2 polymorphisms and susceptibility to urothelial cancer.

Arylamines are suspected to be the primary causative agent of urothelial cancer in tobacco smoke. In the human liver, arylamines are N-hydroxylated by a cytochrome P450 (CYP)1A2-catalyzed reaction, which produces a substrate for O-esterification that can be catalyzed by N-acetyltransferases (NAT) or sulfotransferases (SULT). Recently, several polymorphisms of CYP1A2, SULT1A1, and NAT2 that affect their activities have been reported. In this study, 306 Japanese patients with urothelial transitional cell carcinoma and 306 healthy controls were compared for frequencies of CYP1A2, SULT1A1, and NAT2 genotypes. The frequencies of NAT2 intermediate or slow acetylator genotype were significantly higher in the urothelial cancer patients than in the healthy control subjects [odds ratio (OR) = 1.49, 95% confidence interval (95% CI) 1.06-2.09, OR = 3.23, 95% CI 1.72-6.08, respectively]. Stratifying by amount of smoking, among subjects who consumed >33.5 pack-years and carried the SULT1A1 *1/*1 or NAT2 slow acetylator genotype, the OR was 1.73 (95% CI 1.01-2.97) whereas it was 7.31 (95% CI 1.90-28.05) in non-smokers who carried the homozygous wild genotype, respectively. The relationships between CYP1A2, SULT1A1, and NAT2 polymorphisms and clinical findings including tumor differentiation, stage, and recurrence rate were analyzed. Only associations between NAT2 genotype and pathological findings were admitted, and the higher OR of NAT2 intermediate and slow acetylator genotype was more likely to present to a low-grade tumor (G1) among heavy-smokers. Our results suggest that SULT1A1 *1/*1 and NAT2 slow acetylator genotypes might modulate the effect of carcinogenic arylamines contained in tobacco smoke, and that the modulation of NAT2 intermediate and slow acetylator genotype has a tendency to present a higher risk for highly differentiated tumors among heavy-smokers.

Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice.

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence.

Synthesis and in vitro Evaluation of Novel Small Molecule Inhibitors of Bacterial Arylamine N‐Acetyltransferases (NATs)

AbstractFor Abstract see ChemInform Abstract in Full Text.

Generation and functional characterization of arylamine N-acetyltransferase Nat1/Nat2 double-knockout mice.

Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a variety of arylamine drugs and carcinogens and may play diametrically opposing roles in enhancing either the detoxification of these chemicals or their metabolic activation into DNA-binding electrophiles. To facilitate the study of these processes, we have generated a Nat1/Nat2 double-knockout mouse model by gene targeting in embryonic stem cells. Nat1/2(-/-) mice were born at the expected frequency and seemed normal and viable with no overt phenotype, indicating that these genes are not critical for development or physiological homeostasis. In wild-type mice, NAT1 and NAT2 transcripts were detectable by RT-PCR in all tissues assayed including liver, kidney, colon, brain, bladder, and spleen. NAT1 and NAT2 transcripts were completely undetectable in the Nat1/2(-/-) mice. The in vitro N-acetylation of p-aminosalicylate was detected at significant levels in liver and kidney cytosols from either wild-type inbred 'rapid acetylator' C57BL/6 mice or from outbred CD-1 mice possessing homozygous rapid, heterozygous, or homozygous 'slow acetylator' Nat2 genotypes. Activity was undetectable in cytosol preparations from Nat1/2(-/-) mice. Nat1/2(-/-) mice also displayed severely compromised in vivo pharmacokinetics of p-aminosalicylate (PAS) and sulfamethazine (SMZ), with a drastically increased plasma area under the curve for PAS and a complete absence of their acetylated metabolites (AcPAS or AcSMZ) from plasma, confirming the functional absence of these enzymes and impaired drug metabolism capacity. This knockout mouse model should be helpful in delineating the role that variation in acetylating enzymes plays in mediating interindividual differences in susceptibility to arylamine-induced chemical toxicity and/or carcinogenesis.