No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Arylamine N-acetyltransferase I
Abstract
Arylamine N-acetyltransferase I (NAT1) is a phase II enzyme that acetylates a wide range of arylamine and hydrazine substrates. The NAT1 gene is located on chromosome 8 and shares homology to NAT genes found in most mammalian species. Gene expression occurs from at least two promoters and a number of tissue-specific transcripts have been identified. The gene is polymorphic with most mutations identified to date producing an unstable protein that is subject to polyubiquitination. The NAT1 protein contains a catalytic triad similar to a number of cysteine proteases and transglutaminases. NAT1 is widely distributed in the body, but the only endogenous substrate identified to date is the folate catabolite p-aminobenzoylglutamate. Recent links between NAT1 genotypes and susceptibility to spina bifida suggests that the enzyme has an important role in folate homeostasis.
... The extended NAT2 gene is much simpler than the multiple alternative transcripts of human NAT1. The wide spread tissue expression of human NAT1 (in most tissues including endocrine tissues, blood cells, neural tissue as well as gut and liver) compared with the more restricted expression of human NAT2 (mainly gut and liver) may underlie the more complex gene organisation (see Minchin et al., 2007). These genes as well as the human pseudogene are in close proximity in the genome in the region 8p22 (Hickman et al., 1994;Franke et al., 1994). ...
... Apparently, healthy individuals with very little NAT1 activity have each been shown to have two defective alleles (Hughes et al., 1998;Payton and Sim, 1998). It was nevertheless shown there was an inverse correlation between NAT1 activity and folate levels in red blood cells (Ward et al., 1992), which opened up an ongoing debate on the relationship between NAT1 and folate (see Minchin et al., 2007;Butcher and Minchin, 2012). ...
... The physiological role of human NAT1 has raised many questions (see Minchin et al., 2007) as apparently healthy adult individuals with two defective human NAT1 alleles exist (Ward et al., 1992;Hughes et al., 1998;Payton and Sim, 1998). It was necessary to establish a model system in order to study further the possible relationship between folate and NAT and to decipher the role of human NAT1 in development. ...
Arylamine N-acetyltransferases (NATs) are polymorphic drug metabolising enzymes, acetylating arylamine carcinogens and drugs including hydralazine and sulphonamides. Slow NAT phenotype increases susceptibility to hydralazine and isoniazid toxicity and to occupational bladder cancer. The 2 polymorphic human NAT loci show linkage disequilibrium. All mammalian Nat genes have an intronless open reading frame and non-coding exons. The human gene products NAT1 and NAT2 have distinct substrate specificities: NAT2 acetylates hydralazine and human NAT1 acetylates p-aminosalicylate (p-AS) and the folate catabolite p-aminobenzoylglutamate (p-abaglu). Human NAT2 is mainly in liver and gut. Human NAT1 and its murine homologue are in many adult tissues and in early embryos. Human NAT1 is strongly expressed in ER positive breast cancer and may contribute to folate and acetylCoA homeostasis. NAT enzymes act through a catalytic triad of Cys, His and Asp with the architecture of the active site modulating specificity. Polymorphisms may cause unfolded protein. The C-terminus helps bind acetylCoA and differs amongst NATs including procaryotic homologues. NAT in Salmonella typhimurium supports carcinogen activation and NAT in mycobacteria metabolises isoniazid with polymorphism a minor factor in isoniazid resistance. Importantly nat is in a gene cluster essential for M. tuberculosis survival inside macrophages. NAT inhibitors are a starting point for novel anti-tuberculous drugs. Human NAT1-specific inhibitors may act in biomarker detection in breast cancer and in cancer therapy. NAT inhibitors for co-administration with 5 aminosalicylate (5-AS) in inflammatory bowel disease has prompted ongoing investigations of azoreductases in gut bacteria which release 5-AS from prodrugs including balsalazide.
... It remains to be determined whether an increase of folate catabolite excretion only occurs after folic acid intake or also after the intake of polyglutamated 5-methyltetrahydofolate from food. Urinary apABG arises from endogenous folate after breakdown and acetylation by arylamine N-acetyltransferase I (NAT1) in the cytosol [38,39], which explains the relatively small increase of apABG in contrast to pABG in the short-term study. NAT1 is widely distributed in the body including the cytoplasm of epithelial cells [39]. ...
... Urinary apABG arises from endogenous folate after breakdown and acetylation by arylamine N-acetyltransferase I (NAT1) in the cytosol [38,39], which explains the relatively small increase of apABG in contrast to pABG in the short-term study. NAT1 is widely distributed in the body including the cytoplasm of epithelial cells [39]. Phenotypes with low or higher than normal acetylation activity in some tissues have been reported, which result in ...
... PLOS ONE | www.plosone.orgpersonal variation of pABG acetylation [39] and may explain inter-individual differences of fasted apABG levels and variations of the apABG increase after the ingestion of folic acid in the shortterm study. For the calculated ratio of pABG to apABG (ratio p/ap ) a median (IQR) value of 0.42 (0.38; 0.49) was found in non-dosed state (Figure 2C, black line) which is similar to previous reports [18,19,40,41]. ...
Folate status, as reflected by red blood cell (RCF) and plasma folates (PF), is related to health and disease risk. Folate degradation products para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (apABG) in 24 hour urine have recently been shown to correlate with blood folate.
Since blood sampling and collection of 24 hour urine are cumbersome, we investigated whether the determination of urinary folate catabolites in fasted spot urine is a suitable non-invasive biomarker for folate status in subjects before and during folic acid supplementation.
Immediate effects of oral folic acid bolus intake on urinary folate catabolites were assessed in a short-term pre-study. In the main study we included 53 healthy men. Of these, 29 were selected for a 12 week folic acid supplementation (400 µg). Blood, 24 hour and spot urine were collected at baseline and after 6 and 12 weeks and PF, RCF, urinary apABG and pABG were determined.
Intake of a 400 µg folic acid bolus resulted in immediate increase of urinary catabolites. In the main study pABG and apABG concentrations in spot urine correlated well with their excretion in 24 hour urine. In healthy men consuming habitual diet, pABG showed closer correlation with PF (r = 0.676) and RCF (r = 0.649) than apABG (r = 0.264, ns and 0.543). Supplementation led to significantly increased folate in plasma and red cells as well as elevated urinary folate catabolites, while only pABG correlated significantly with PF (r = 0.574) after 12 weeks.
Quantification of folate catabolites in fasted spot urine seems suitable as a non-invasive alternative to blood or 24 hour urine analysis for evaluation of folate status in populations consuming habitual diet. In non-steady-state conditions (folic acid supplementation) correlations between folate marker (RCF, PF, urinary catabolites) decrease due to differing kinetics.
... Human N-acetyltransferase 1 (NAT1) metabolizes numerous drugs, environmental carcinogens, and endogenous substrates such as folate. It catalyzes N-acetylation of arylamines or arylhydrazines, O-acetylation of arylhydroxylamines, and N to O acetyl transfer of acetylhydroxamates, resulting in either biological activation or inactivation [1,2]. NAT1 maps to chromosome location, 8p21.3, next to its homologue NAT2 and an inactive pseudogene, NATP. ...
... NAT1 is ubiquitously expressed, whereas NAT2 expression is mainly restricted to liver and intestine. NAT1 consists of a single coding exon and 8 differentially spliced upstream exons with three proposed promoters (Fig. 1), giving rise to multiple 5′UTR mRNA variants reported to have different protein translation efficiencies [2][3][4][5][6][7]. In contrast, NAT2 has only one upstream exon and one promoter [1,7]. ...
N‐acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens, affecting drug response and cancer susceptibility. Two common NAT1 alleles, *10 and *11, have been reported to alter enzyme activity compared to wild‐type NAT1 (*4), but their effects and underlying mechanisms remain uncertain. We measured allelic NAT1 mRNA expression and translation, considering multiple transcription start sites, alternative splicing, and three 3′‐polyadenylation sites in human livers and B lymphocytes. *11 was found to favor usage of the second 3'polyadenylation site, increasing formation of NAT1 mRNA with the 3'UTR of intermediate length (major isoform). This major 3'UTR isoform supported more efficient protein translation than the short 3'UTR isoform, resulting in increased *11 NAT1 enzyme activity. Similarly, NAT1 *10 also increased protein translation efficiency, but without affecting NAT1 mRNA levels or 3′‐UTR polyadenylation site usage. B‐lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 enzyme activity than the reference genotype *4/*4. Moreover, carriers of *10/*10 and *11/*4 (‘fast NAT1 acetylators') were less likely to develop hypersensitivity to NAT1/NAT2 substrate drug sulfamethoxazole, but this was observed only in subjects with slow NAT2 acetylator genotype. In conclusion, NAT1 *10 and *11 significantly increase NAT1 enzyme activity, enabling the classification of carriers into normal and rapid acetylators. Supported by NIH grant R21 AI074399 and U01 GM092655.
... Whether slow release of PAS by the GSR-PAS formulation changes this paradigm at higher doses is uncertain. Our analysis of NAT1 genetics and their relationship to PAS concentrations confirmed the loss of activity associated with the NAT1*14 allele; however, we found no evidence that NAT1*10 is associated with increased activity (26). Interestingly the single patient with a NAT1*10/1*3 genotype had particularly high PAS C max and AUC 0 -12 , similar to two patients with a NAT1*14 allele, suggesting reduced enzyme function. ...
... First it has been shown that NAT1 activity is androgen induced, and this may constitute a risk factor for cancer induction (29). Second it is known that NAT1 is widely expressed in muscle tissue, and males might have a greater muscle bulk than females (26). Early studies of the efficacy of regimens of PAS and streptomycin and PAS, streptomycin, and isoniazid did report better efficacy in females than males, but this was not statistically significant (30). ...
The aim of the study was to examine the relationships between N-acetyltransferase genotypes, pharmacokinetics, and tolerability of granular slow-release (GSR) para-aminosalicylic acid (PAS) in tuberculosis patients. The study was a randomized, two-period, open-label, cross-over design wherein each patient received GSR-PAS 4 g twice daily or GSR-PAS 8 g once daily alternately. The PAS concentration-time profiles were modeled by a one-compartment disposition model with three transit compartments in series to describe its absorption. Patients' NAT1 and NAT2 genotypes were determined by sequencing and restriction enzyme analysis, respectively. The number of daily vomits was modeled by a Poisson probability mass function. Comparisons of other tolerability measures by regimens, gender and genotypes were evaluated by linear mixed effects model. The covariate effects associated with efavirenz, gender, NAT1*3, NAT1*14, and NAT2*5 alleles corresponded to 25%, 37%, -17%, -48% and -27% change, respectively, in oral clearance of PAS. NAT1*10 allele did not influence drug clearance. The time above the minimum inhibitory concentration (MIC) of 1 mg/L was significantly different between the two regimens but not influenced by NAT1 or NAT2 genotypes. Occurrence and intensity of intolerance differed little between regimens. Twice daily 4 g GSR-PAS, but not 8 g once daily ensures concentrations exceeding the MIC (1 mg/L) throughout the dosing interval; PAS intolerance is not related to maximum PAS concentrations over doses studied and was not more frequent after once daily dosing. We confirm that the slow phenotype conferred by NAT1*14 and NAT1* 3 alleles resulted in higher PAS exposure, but found no evidence of increased activity of the NAT1*10 allele.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
... Human N-acetyltransferase 1 (NAT1) metabolizes numerous drugs, environmental carcinogens, and endogenous substrates such as folate. It catalyzes N-acetylation of arylamines or arylhydrazines, O-acetylation of arylhydroxylamines, and N to O acetyl transfer of acetylhydroxamates, resulting in either biological activation or inactivation [1,2]. NAT1 maps to chromosome location, 8p21.3, next to its homologue NAT2 and an inactive pseudogene, NATP. ...
... NAT1 is ubiquitously expressed, whereas NAT2 expression is mainly restricted to liver and intestine. NAT1 consists of a single coding exon and 8 differentially spliced upstream exons with three proposed promoters (Fig. 1), giving rise to multiple 5′UTR mRNA variants reported to have different protein translation efficiencies [2][3][4][5][6][7]. In contrast, NAT2 has only one upstream exon and one promoter [1,7]. ...
N-acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens. NAT1 alleles *10 and *11 have been proposed to alter protein level or enzyme activity compared with wild-type NAT1 *4 and to confer cancer risk, through uncertain pathways. This study characterizes regulatory polymorphisms and underlying mechanisms of NAT1 expression.
We measured allelic NAT1 mRNA expression and translation, as a function of multiple transcription start sites, alternative splicing, and three 3'-polyadenylation sites in human livers (one of which was discovered in this study), B lymphocytes, and transfected cells. In a clinical study of 469 patients with HIV/AIDS treated with the NAT1/NAT2 substrate sulfamethoxazole (SMX), associations were tested between SMX-induced hypersensitivity and NAT1 *10 and *11 genotypes, together with known NAT2 polymorphisms.
NAT1 *10 and *11 were determined to act as common regulatory alleles accounting for most NAT1 expression variability, both leading to increased translation into active protein. NAT1 *11 (2.4% minor allele frequency) affected 3'-polyadenylation site usage, thereby increasing formation of NAT1 mRNA with intermediate length 3'-untranslated region (major isoform) at the expense of the short isoform, resulting in more efficient protein translation. NAT1 *10 (19% minor allele frequency) increased translation efficiency without affecting 3'-untranslated region polyadenylation site usage. Livers and B-lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 immunoreactivity and NAT1 enzyme activity than the reference genotype *4/*4. Patients who carry *10/*10 and *11/*4 (fast NAT1 acetylators) were less likely to develop hypersensitivity to SMX, but this was observed only in individuals who are also carrying a slow NAT2 acetylator genotype.
NAT1 *10 and *11 significantly increase NAT1 protein level/enzyme activity, enabling the classification of carriers into reference and rapid acetylators. Rapid NAT1 acetylator status seems to protect against SMX toxicity by compensating for slow NAT2 acetylator status.
... NATs transfer an acetyl group to a nitrogen acceptor of primary arylamines and hydrazines [59]. The NAT superfamily includes N-acetyltransferase 1 (NAT1), which is expressed in the intestine, bladder, and breast, and N-acetyltransferase 2 (NAT2), which is expressed in the liver and intestine [60]. Both NAT1 and NAT2 are expressed in the placenta, with NAT1 providing a greater contribution to placental acetylation capacity [59]. ...
The purpose of this literature review is to comprehensively summarize changes in the expression of phase II drug-metabolizing enzymes and drug transporters in both the pregnant woman and the placenta. Using PubMed®, a systematic search was conducted to identify literature relevant to drug metabolism and transport in pregnancy. PubMed was searched with pre-specified terms during the period of 26 May 2023 to 10 July 2023. The final dataset of 142 manuscripts was evaluated for evidence regarding the effect of gestational age and hormonal regulation on the expression of phase II enzymes (n = 16) and drug transporters (n = 38) in the pregnant woman and in the placenta. This comprehensive review exposes gaps in current knowledge of phase II enzyme and drug transporter localization, expression, and regulation during pregnancy, which emphasizes the need for further research. Moreover, the information collected in this review regarding phase II drug-metabolizing enzyme and drug transporter changes will aid in optimizing pregnancy physiologically based pharmacokinetic (PBPK) models to inform dose selection in the pregnant population.
... The NAT1 and NAT2 genes share 87.5% of the coding sequence homology and are both polymorphic (Blum et al., 1990;Hein, 2009;Sim et al., 2012;Stanley & Sim, 2008). The NAT1 gene was first identified as a metabolizer of xenobiotics, particularly aromatic amine drugs and mutagens, but later its involvement in acetyl-CoA homeostasis and development was also suggested (Butcher & Minchin, 2012;Minchin et al., 2007;Sim et al., 2008). ...
Objectives
The evolutionary mechanisms that shape the genetic structure of a population left their mark on genes that metabolize drugs. The Roma are an example of a population in which the migrations, isolation, and multiple founder effects have affected its genetic structure. In this study, we investigated NAT1 and NAT2 genes, members of the xenobiotic‐metabolizing NAT gene family in three Roma groups from Croatia to explore the specificities of the Roma population in relation to other populations.
Materials and Methods
Seven SNPs in the NAT1 gene and seven in the NAT2 gene were genotyped in 439 Roma from Croatia, members of three socio‐culturally different and geographically distant groups (two groups of Vlax/Bayash Roma and one Balkan Roma group). Intra‐ and inter‐population variation was assessed in the Roma and 2504 individuals from the 1000 Genomes project database.
Results
The distribution of haplotypes differed significantly between the Roma groups for NAT2 , but not for NAT1 . Translation of NAT2 diplotypes into acetylation phenotypes showed significant differences between populations. The Roma from Balkan had the highest frequency of slow acetylators among the studied populations. In the overall worldwide sample, population differentiation was higher for NAT2 than for NAT1 haplotypes consistent with pairwise genetic distances that were smaller for NAT1 than for NAT2 . The Ewens–Watterson test results suggest that NAT1 is subjected to directional selection, while NAT2 is evolving neutrally.
Conclusion
The distribution of variations within NAT genes in the Croatian Roma population is similar to that in the surrounding European populations. The significantly different distribution of NAT2 gene haplotypes and consequent phenotypes between the three investigated Roma groups is probably the result of genetic drift due to different demographic history and socio‐cultural isolation. The highest frequency of slow acetylators in Balkan Roma compared to the world populations makes them more prone to develop a NAT gene‐related adverse drug reaction than other populations.
... The polymorphisms can lead to a truncated gene which effectively reduces enzymatic activity and on the other hand sometimes elevated activity may be due to increased mRNA stability. There are single mutations that alone account for a decrease or increase in enzymatic activity (Minchin et al., 2007;Zhu and Hein, 2008). Therefore, the present project was designed to determine some of the allelic variations at the NAT1 locus. ...
The present research project was designed to determine the genetic polymorphism of N-acetyltransferase 1 (NAT1), the acetylation status in the local Pakistani population because of the genetic polymorphism of drug-metabolizing enzymes constitutes an individual's susceptibility and toxicity for drugs. The normal health status of enrolled volunteers was ensured by their baselines hematological and biochemical studies like CBC, blood cell morphology, LFTs, RFTs Lipid profile and plasma proteins. The acetylation capacity was determined genotypically by PCR-based allele-specific amplification assay and restriction fragment length polymorphism (RFLP) for wild-type and variant alleles. The mutual concordance between the phenotypic and genotypic analysis was determined. The target population showed bimodal distribution based on genotyping. Almost 69% Pakistani local population was found to have fast acetylator alleles/genotype and 30% population with slow acetylator allele/genotype. The interethnic distributions of NAT1 alleles show variability among various populations. Studies should be carried on all possible ethnic divisions to get an insight into varied drug responsiveness in different populations.
... was originally characterized for its role in the metabolism of xenobiotics, particularly aromatic amine drugs and mutagens. Apart from its pharmacogenetic significance, NAT1 has more recently been implicated in cell systems relevant to carcinogenesis [1][2][3] . The human NAT1 gene, and its mouse orthologue Nat2, begin their expression during preimplantation development 4-6 and remain transcriptionally active in most embryonic and adult tissues 4,6-11 . ...
Human NAT1 gene for N-acetyltransferase 1 modulates xenobiotic metabolism of arylamine drugs and mutagens. Beyond pharmacogenetics, NAT1 is also relevant to breast cancer. The population history of human NAT1 suggests evolution through purifying selection, but it is unclear whether this pattern is evident in other primate lineages where population studies are scarce. We report NAT1 polymorphism in 25 rhesus macaques (Macaca mulatta) and describe the haplotypic and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified and experimentally demonstrated to compromise enzyme function, mainly through destabilization of NAT1 protein and consequent activity loss. One non-synonymous SNV (c.560G > A, p.Arg187Gln) has also been characterized for human NAT1 with similar effects. Population haplotypic and functional variability of rhesus NAT1 was considerably higher than previously reported for its human orthologue, suggesting different environmental pressures in the two lineages. Known functional elements downstream of human NAT1 were also differentiated in rhesus macaque and other primates. Xenobiotic metabolizing enzymes play roles beyond mere protection from exogenous chemicals. Therefore, any link to disease, particularly carcinogenesis, may be via modulation of xenobiotic mutagenicity or more subtle interference with cell physiology. Comparative analyses add the evolutionary dimension to such investigations, assessing functional conservation/diversification among primates.
... 1. NAT1 se expresa de manera omnipresente y, por lo tanto, puede estar involucrado en la homeostasis y desarrollo, aunque los niveles de expresión varían entre los tipos de células y tejidos [37,42,[44][45][46]. ...
Se estudió el genotipo y fenotipo de Arilamina-N-acetiltransferasa (NAT2) en 100 individuos en tratamiento con isoniazida. Este fármaco es metabolizado por la genéticamente polimorfa NAT2. Los diferentes alelos están relacionados con incremento o disminución de la capacidad de acetilación y en algunos casos disminución de la eficacia y toxicidad of isoniazida. El polimorfismo de NAT2 está también involucrado en el metabolismo de muchos compuestos relevantes en la farmacología y toxicología con diversas consecuencias clínicas.
Métodos: Los individuos incluidos en el estudio recibieron la dosis oral de isoniazida que se les prescribió, y tres horas después de la misma se extrajeron 3 ml de sangre que se utilizaron para la genotipificación (por técnica de PCR-RFLP) y para fenotipificación de la actividad NAT2 (a través de la cuantificación de concentraciones de isoniazida y su metabolito).
Resultados: Se validó una técnica cromatográfica para la determinación simultánea de isoniazida y acetilisoniazida. Se perfeccionó un método para la obtención de
acetilisoniazida a partir de isoniazida a fin de ser utilizada como estándar interno.
Se incluyeron un total de 100 sujetos, de 34±5 (25-44) años, sin antecedentes de alergia ni reacciones adversas a isoniazida. Los mismos recibieron dosis de isoniazida de entre 200 y 300 mg por día vía oral, dando una dosis ajustada a peso de 4,56 ± 0,89 (3,05-5,99) mg/Kg. Se determinó los alelos presentes como: *4 (35,00%), *5 (41,00 %), *6 (21,00 %), y *7 (2,50 %), configurando distintas asociaciones alélicas en diplotipos que predicen un fenotipo rápido (10,00%), intermedio (51,00%), o lento (39,00%). Las concentraciones de isoniazida y su metabolito monoacetilado fueron 3,16 ± 1,21 (1,51-5,83) μg/mL, y 6,57 ± 4,82 (0,10 – 18,24) μg/mL, con un coeficiente metabólico molar Acetilisoniazida/isoniazida (MR) de 2,56 ± 2,23 (0,02-9,04).
Mediante el cálculo de antimodas de la distribución trimodal de MR (Rápido > 5,4, Intermedio 1,2 a 5,4 y Lento < 1,2) se obtuvo una concordancia completa con el fenotipo predicho a partir del genotipo.
Discusión: La distribución alélica de la población local muestra una frecuencia propia y ligeramente diferente a la reportada en otras poblaciones y estudios. Tanto la genotipificación como la fenotipificación permiten la diferenciación de perfiles metabólicos que podrían vincularse al desarrollo de problemas de falta de eficacia y/o toxicidad. El genotipo predice con exactitud la actividad metabólica real.
Conclusiones: el estudio farmacogenético de la NAT2 es una herramienta útil para la diferenciación de comportamientos metabólicos y la eventual individualización terapéutica.
... In this study, NAT1 Ã 3 and NAT1 Ã 4 haplotypes were associated with ALL. In contrast, NAT1 Ã 10, which has been statistically associated with other types of cancer (colon, lung, breast, prostate), was not associated with ALL [31][32][33][34][35]. Krajinovic et al. [9] reported a statistical association between NAT1 Ã 4 and ALL. ...
Acute lymphoblastic leukemia (ALL) is one of the main causes of death in children and is associated with both genetic susceptibility and environmental factors. Genes encoding the arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2) isoenzymes are highly polymorphic among populations. Single-nucleotide polymorphism analysis was performed by real-time polymerase chain reaction from the genomic DNA of 225 healthy subjects and 57 children with ALL diagnoses. Significant associations were found between the development of ALL and the presence of the haplotypes NAT1*3 (Odds ratio [OR], 2.1), NAT1*4 (OR, 1.92), NAT2*6B (OR, 3.30), NAT2*6J (OR, 3.25) and NAT2*7A (OR, 2.45) and the NAT1 rapid (OR, 6.69) and NAT2 slow phenotypes (OR, 2.95). Our results indicate that haplotypes that provide rapid NAT1 and slow NAT2 acetylating phenotypes may influence the development of ALL in children.
... EPHX1 is localized mainly in the microsomal fraction of the endoplasmic reticulum of eukaryotic cells (V aclavíkov a et al., 2015), the decreased expression of EPHX1 caused by MC-LR may affect the function of endoplasmic reticulum, which confirms that the endoplasmic reticulum was involved in MC-LR-mediated toxicity in HepG2 cells (Menezes et al., 2013). NAT1 is a ubiquitously expressed human arylamine N-acetyltransferase (NAT) enzyme that biotransforms various aromatic, heterocyclic amines, drugs, or environmental carcinogens (Dairou et al., 2005;Minchin et al., 2007). In this study, the NAT1 expression rate was also changed after MC-LR exposure, indicating that MC-LR may activate the expression of NAT1, and NAT might play an important role in the activation and detoxification of environmental carcinogens (Sim et al., 2008;Butcher and Minchin, 2012), such as MCs. ...
This study aimed to evaluate the possible effects of microcystin-LR (MC-LR) exposure on the metabolism and drug resistance of human hepatocellular carcinoma (HepG2) cells. For this purpose, we first conducted an experiment to make sure that MC-LR could penetrate the HepG2 cell membrane effectively. The transcriptional levels of phase I (such as CYP2E1, CYP3A4, and CYP26B1) and phase II (such as EPHX1, SULTs, and GSTM) enzymes and export pump genes (such as MRP1 and MDR1) were altered by MC-LR-exposure for 24 h, indicating that MC-LR treatment may destabilize the metabolism of HepG2 cells. Further research showed that the CYP inducers omeprazole, ethanol, and rifampicin inhibited cell viability, in particular, ethanol, a CYP2E1 inducer, induced ROS generation, lipid peroxidation, and apoptosis in HepG2 cells treated with MC-LR. The CYP2E1 inhibitor chlormethiazole inhibited ROS generation, mitochondrial membrane potential loss, caspase-3 activity, and cytotoxicity caused by MC-LR. Meanwhile, the results also showed that co-incubation with the ROS scavenger l-ascorbic acid and MC-LR decreased ROS levels and effectively prevented apoptosis. These findings provide an interesting mechanistic explanation of cellular metabolism associated with MC-LR, i.e., MC-LR-exposure exerted toxicity on HepG2 cells and induced apoptosis of HepG2 cells via promoting CYP2E1 expression and inducing excessive ROS in HepG2 cells.
... As was previously reported from a clinical study, NAT1 genetics and their relationship to serum PAS concentrations confirmed that the NAT1*14 allele is a loss-of-function allele. In contrast, there was no evidence that NAT1*10 is associated with decreased activity (30). These studies suggest that the influence of transporters and NAT must be considered to fully understand the disposition and pharmacokinetics (PK) of PAS. ...
para-Aminosalicylic acid (PAS) is a second-line anti-tuberculosis drug used to treat multidrug-resistant and extensively drug-resistant tuberculosis for more than 60 years. Renal secretion and glomerular filtration are the major pathways for elimination of PAS. We comprehensively studied PAS transport by using cell lines that overexpressed various transporters, and found that PAS acts as a novel substrate of organic anionic polypeptide (OATP1B1), organic cationic transporters (OCT1 and OCT2), and organic anion transporters (OAT1 and OAT3) but not a substrate of any ATP-binding cassette (ABC) transporters. Net PAS uptake was measured, and transport affinity (Km) for OATP1B1, OCT1, OCT2, OAT1 and OAT3 were found to be 50.0, 20.3, 28.7, 78.1 and 100.1 µM, respectively. From the net uptake rates suggested that renal OAT1 and OAT3 play relatively major roles in PAS elimination. The representative inhibitors rifampin for OATP1B1, probenecid for OAT1 and OAT3, and verapamil for OCT1 and OCT2 greatly inhibited PAS uptake, suggesting that PAS is dependent on multiple transporters for uptake. We also evaluated nonsteroidal antiinflammatory drugs (NSAIDs), proton pump inhibitors (PPIs), and metformin for the inhibition of PAS uptake via these transporters. Half-maximal inhibitory concentrations (IC50) were kinetically determined and used to predict drug–drug interactions (DDI) affecting these transporters’ activity toward PAS. We found that rifampin, probenecid, ibuprofen, naproxen, cimetidine, and quinidine each exhibit significant potential for in vivo DDIs with PAS. In this study, PAS was found to be a novel substrate of several transporters, and drugs that inhibit these transporters can reduce PAS elimination.
... The formation of the acetyl-enzyme intermediate requires the stabilisation of charge across a Cys-His-Asp triad. The acetyl transfer occurs following nucleophilic attack of the acetylated intermediate by the arylamine substrate (Minchin et al., 2007). In humans N-acetyltransferase activity is controlled by two genes: NAT1 (expressed in the liver, colon, white blood cells, breast and epithelium) and NAT2 (expressed in the liver and gastrointestinal tract), which are located on chromosome 8. ...
The aim of this research was to select microbial strains capable of transformation of flavonoid compounds containing an amino group. 6‐Aminoflavone (1) was used as a model compound. Biotransformation of 1 by the cultures of Rhodococcus sp. DSM 364 and Gordonia sp. DSM 44456 bacteria led to 6‐acetamidoflavone (2) with the isolated yields of 86% and 93%, respectively. The structure of the product was determined on the basis of spectroscopic data, including NMR spectra (¹H, ¹³C, ¹H-¹H, ¹H-¹³C, and DEPT 135°), a UV and IR spectrum and HR ESI-MS analysis. The developed method of microbial replacement of the amino group in 6-aminoflavone (1) by a acetamido one may find application in chemical industry, being an alternative to the reactions involving acetic anhydride.
... Interestingly, the Nat1/2 KO mice on DSS show higher inflammation activity (MPO activity) than the WT. Sim et al. (30) suggested that NAT enzymes have an endogenous role(s) but are as yet unidentified and Minchin et al. (31) suggested that an appropriate stress, such as DSS, may be required before an endogenous role for the xenobiotic metabolizing NAT enzyme is found. Because the Nat1/2 KO mice show no obvious phenotype (18), yet show greater DSS induced inflammation in the absence of NAT activity, these suggest that NAT enzymes are involved in regulation of mucosal homeostasis, yet further study will be needed. ...
Background:
5-aminosalicylic acid (5-ASA) is a classic anti-inflammatory drug for the treatment of ulcerative colitis. N-acetyltransferase (NAT) enzymes convert 5-ASA to its metabolite N-acetyl-5-ASA, and it is unresolved whether 5-ASA or N-acetyl-5-ASA is the effective therapeutic molecule. We previously demonstrated that colonic production of N-acetyl-5-ASA (NAT activity) is decreased in dextran sulfate sodium-induced colitis. Our hypothesis is that 5-ASA is the therapeutic molecule to improve colitis, with the corollary that altered NAT activity affects drug efficacy. Since varying clinical effectiveness of 5-ASA has been reported, we also ask if NAT activity varies with inflammation in pediatric or adult patients.
Methods:
Acute colonic inflammation was induced in C57BL/6 NAT wild-type (WT) or knockout mice, using 3.5% dextran sulfate sodium (w/v) concurrent with 5-ASA treatment. Adult and pediatric rectosigmoid biopsies were collected from control or patients with ulcerative colitis. Tissue was analyzed for NAT and myeloperoxidase activity.
Results:
Dextran sulfate sodium-induced colitis was of similar severity in both NAT WT and knockout mice, and NAT activity was significantly decreased in NAT WT mice. In the setting of colitis, 5-ASA significantly restored colon length and decreased myeloperoxidase activity in NAT knockout but not in WT mice. Myeloperoxidase activity negatively correlated with NAT activity in pediatric patients, but correlation was not observed in adult patients.
Conclusions:
Inflammation decreases NAT activity in the colon of mice and human pediatric patients. Decreased NAT activity enhances the therapeutic effect of 5-ASA in mice. A NAT activity assay could be useful to help predict the efficacy of 5-ASA therapy.
... NAT1 and NAT2 are two members of NAT family, of which NAT1 is distributed in almost all tissues, whereas NAT2 is detected predominantly in the liver. Although divergence exists in the distribution of NAT1 and NAT2, which locate on chromosome 8, the two are involved in metabolizing drugs and other xenobiotics, and are involved in folate catabolism and caffeine metabolism (29). In addition, the overexpression of NAT1 can result increase the survival rates of patients with cancer and has been recommended as a viable target for developing therapy against cancer (30). ...
The present study aimed to investigate the molecular targets for colorectal cancer (CRC). Differentially expressed genes (DEGs) were screened between CRC and matched adjacent noncancerous samples. GENETIC_ASSOIATION_DB_DISEASE analysis was performed to identify CRC genes from the identified DEGs using the Database for Annotation, Visualization and Integrated Discovery, followed by Gene Οntology (GO) and Kyoto Encyclopedia of Genes and Genomes analysis for the CRC genes. A protein‑protein interaction (PPI) network was constructed for the CRC genes, followed by determination and analysis of the hub genes, in terms of the protein domains and spatial structure. In total, 35 CRC genes were determined, including 19 upregulated and 16 downregulated genes. Downregulated N‑acetyltransferase (NAT)1 and NAT2 were enriched in the caffeine metabolism pathway. The downregulated and upregulated genes were enriched in a number of GO terms and pathways, respectively. Cyclin D1 (CCND1) and proliferating cell nuclear antigen (PCNA) were identified as the hub genes in the PPI network. The C‑terminal and N‑terminal domains were similar in PCNA, but different in CCND1. The results suggested PCNA, CCND1, NAT1 and NAT2 for use as biomarkers to enable early diagnosis and monitoring of CRC. These results form a basis for developing therapies, which target the unique protein domains of PCNA and CCND1.
... were first discovered as drug-metabolizing enzymes that catalyze the transfer of an acetyl group from acetyl coenzyme A to the nitrogen of arylamine compounds. There are two isozymes expressed in humans (NAT1 and NAT2) that differ in their tissue distribution and substrate specificity [1, 2]. Because the NATs metabolically activate and detoxify arylamine and heterocyclic amine carcinogens, there has been an extensive interest in their role as risk factors in a range of human cancers [3]. ...
Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and β-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers.
© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
... Human NATs are characterised by differing tissue distributions and patterns of gene expression during development [12][13][14]. In particular, while (HUMAN)NAT2 appears to be a well-established drug metabolising enzyme and is mainly expressed in the liver [15], the selective N-acetylation of the folate catabolites 4ABglu and 4ABA by (HUMAN)NAT1 suggests that this isoform may have an endogenous role in folate homeostasis [16,17]. ...
The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut.
To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated.
Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes.
Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.
... Their protein products have different physiological roles, and despite being structurally similar, differences in key residues result in different substrate profiles/affinities [6,7,9]. NAT1 is ubiquitously expressed and therefore may be involved in homeostasis and development, although the levels of expression vary between cell types and tissues [3,8,[10][11][12]. NAT2 expression is found predominantly in the liver, small intestine and colon tissues, and thus is regarded a typical xenobiotic-metabolizing enzyme [3,8,10,12,13], although basal NAT2 mRNA levels can be found in most tissues [2]. ...
... While (HUMAN)NAT1 is structurally similar to (HUMAN)-NAT2, it is biologically distinct in that it is expressed during embryonic development (as early as the four-cell stage), in placenta during trimester one and throughout pregnancy [9][10][11], and in stem cells. The enzymatic profile of NAT1 expression in different human foetal organs [12] illustrates its widespread tissue distribution, which differs from the much more restricted distribution of (HUMAN)NAT2, and many strands of evidence suggest a link between (HUMAN)NAT1 and folate [13][14][15]. ...
Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.
... and arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) catalyze the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to the amino groups of hydrazine, arylamine drugs, carcinogens, and arylalkylamines (e.g., dopamine and 5-hydroxytryptamine). NATs of humans, mice, and intestinal microorganisms have been characterized in detail (9,10). Human NAT I possibly plays a role in the activation of various carcinogens (11) and in the metabolism of the folate catabolite p-aminobenzoylglutamate (12). ...
N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an
acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence.
The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid
sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and Vmax values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good
substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors
acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).
... An alternative promoter, NATa, originates 51.5 kb upstream of the NAT1 ORF and promotes transcription of Type I transcripts expressed primarily in kidney, lung, liver, and trachea [25,27]. The NAT1 gene is induced following exposure to androgens and NAT1 protein stability is affected by the presence of substrates [28]. ...
N-acetyltransferase 1 (NAT1) is a phase II metabolic enzyme responsible for the biotransformation of aromatic and heterocyclic amine carcinogens such as 4-aminobiphenyl (ABP). NAT1 catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5'-untranslated regions (UTR). NATa mRNA is expressed primarily in the kidney, liver, trachea, and lung while NATb mRNA has been detected in all tissues studied. To determine if differences in 5'-UTR have functional effect upon NAT1 activity and DNA adducts or mutations following exposure to ABP, pcDNA5/FRT plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATa or NATb, the open reading frame, and 888 nucleotides of the 3'-UTR. Following stable transfection of NATb/NAT1*4 or NATa/NAT1*4 into nucleotide excision repair (NER) deficient Chinese hamster ovary cells, N-acetyltransferase activity (in vitro and in situ), mRNA, and protein expression were higher in NATb/NAT1*4 than NATa/NAT1*4 transfected cells (P < 0.05). Consistent with NAT1 expression and activity, ABP-induced DNA adducts and hypoxanthine phosphoribosyl transferase mutants were significantly higher (P < 0.05) in NATb/NAT1*4 than in NATa/NAT1*4 transfected cells following exposure to ABP. These differences observed between NATa and NATb suggest that the 5'-UTRs are differentially regulated.
... Exon 9 contains the entire 870-bp open reading frame, whereas various splicing of the first eight exons make up the 5-untranslated region. NAT1 transcription is driven by two alternative promoters (Minchin et al., 2007). The distal NAT1 promoter, NATa, drives the expression of type I transcripts that begin with exon 1. ...
Arylamine N-acetyltransferase-1 (NAT1) has been associated with disorders involving folate metabolism, such as spina bifida, as well as numerous human cancers. As a result, the transcriptional and post-transcriptional regulation of NAT1 activity has been extensively studied. However, little work has been reported on the epigenetic control of NAT1 expression. Here, we demonstrate that the histone deacetylase inhibitor trichostatin A (TSA) increases NAT1 activity in human cancer cells by increasing transcription from the proximal promoter NATb. A specific Sp1 binding site was identified as essential for optimal induction of NAT1 by TSA. However, TSA did not increase the expression of Sp1 in HeLa cells. Instead, TSA increased the acetylation of histones associated with the NATb promoter. This allowed recruitment of Sp1 to the promoter along with acetylated histones. We propose that NAT1 transcription is partially repressed by the local chromatin condensation in the vicinity of NATb and that histone deacetylase inhibition leads to up-regulation of NAT1 expression via a direct change in chromatin conformation.
... volume 118 | number 12 | December 2010 • Environmental Health Perspectives ortholog of human NAT1(Kawamura et al. 2008;Minchin et al. 2007), to biologically relevant concentrations of Cd (≤ 50 μM). We found that Cd impaired the NATdependent biotransformation of two carcinogenic AAs (4ABP and 2AF) through inhibition of the endogenous Nat2 enzyme with an IC 50 close to 20 μM. ...
Cadmium (Cd) is a carcinogenic heavy metal of environmental concern. Exposure to both Cd and carcinogenic organic compounds, such as polycyclic aromatic hydrocarbons or aromatic amines (AAs), is a common environmental problem. Human arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play a key role in the biotransformation of AA carcinogens. Changes in NAT activity have long been associated with variations in susceptibility to different cancers in relation with exposure to certain AAs.
We explored the possible interactions between Cd and the NAT-dependent biotransformation of carcinogenic AAs.
We exposed purified enzymes, lung epithelial cells, and mouse models to Cd and subsequently analyzed NAT-dependent metabolism of AAs.
We found that Cd, at biologically relevant concentrations, impairs the NAT-dependent acetylation of carcinogenic AAs such as 2-aminofluorene (2-AF) in lung epithelial cells. NAT activity was strongly impaired in the tissues of mice exposed to Cd. Accordingly, mice exposed to Cd and 2-AF displayed altered in vivo toxicokinetics with a significant decrease (~ 50%) in acetylated 2-AF in plasma. We found that human NAT1 was rapidly and irreversibly inhibited by Cd [median inhibitory concentration (IC₅₀) ≈ 55 nM; rate inhibition constant (k(inact)) = 5 × 10⁴ M⁻¹ • sec⁻¹], with results of acetyl coenzyme A (acetyl-CoA) protection assays indicating that Cd-mediated inhibition was due to the reaction of metal with the active-site cysteine residue of the enzyme. We found similar results for human NAT2, although this isoform was less sensitive to inactivation (IC₅₀ ≈ 1 μM; k(inact) = 1 × 10⁴ M⁻¹ • sec⁻¹).
Our data suggest that Cd can alter the metabolism of carcinogenic AAs through the impairment of the NAT-dependent pathway, which may have important toxicological consequences.
... The acetyl-CoA:arylamine N-acetyltransferase 1 (NAT1) is one of the two polymorphic human arylamine N-acetyltransferase (NAT) enzymes which biotransforms various aromatic and heterocyclic amines[1,2]. Aromatic amines (AA) belong to an important class of occupational and environmental pollutants with genotoxic and carcinogenic properties[1]. ...
Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg2+) and organic (CH3Hg+) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC50)=250 nM and kinact=1.4x10(4) M(-1) s(-1) for Hg2+ and IC50=1.4 microM and kinact=2x10(2) M(-1) s(-1) for CH3Hg+). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC50=3 and 20 microM for Hg2+ and CH3Hg+, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.
Studies have reported overexpression of NAT1 gene for xenobiotic metabolizing arylamine N -acetyltransferase type 1 in estrogen receptor positive breast tumors, and this association has been linked to patient chemoresistance and response to tamoxifen. We probed the expression of NAT1 , using quantitative reverse transcription PCR to screen clinically characterized breast cancer tissue cDNA arrays. Primers detecting all NAT1 alternative transcripts were used, and the protocol and results are reported according to consensus guidelines. The clinical information about 166 tumor samples screened is provided, including tumor stage, estrogen and progesterone receptor status and HER2 expression. NAT1 was found to be significantly ( P < 0.001) upregulated in hormone receptor positive vs. negative tumors. No correlation was apparent between NAT1 and tumor stage or HER2 expression. Our findings demonstrate a strong correlation between the expression of NAT1 and steroid hormone receptors in breast tumors, supporting its possible utility as a pharmacogenetic biomarker or drug target. Of the two polymorphic NAT genes, NAT1 is the one primarily expressed in breast tissue, and is subjected to regulation by two differential promoters and more than one polyadenylation signal. Hormonal factors may enhance NAT1 gene expression at the transcriptional or epigenetic level, and tamoxifen has additionally been shown to inhibit NAT1 enzymatic activity. The outcome of tamoxifen treatment is also more favorable in patients with NAT1 overexpressing tumors. The study adds to the growing body of evidence implicating NAT1 in breast cancer and its pharmacological treatment.
In metabolic biotransformation process, foreign compounds are converted into more polar products to facilitate their elimination from the body. As discussed above, phase I activation metabolism involves functionalization reactions, where metabolic reactions catalyzed by activation enzymes introduce functional groups to foreign compounds, resulting in greatly increasing the solubility of parent compounds. The modified lipophilic foreign compounds then undergo phase II enzyme-catalyzed reactions to detoxify metabolites and facilitate their excretion from the body.
Detoxification enzyme-catalyzed reactions are generally considered as detoxification metabolism. Chemical reactions catalyzed by detoxification enzymes include conjugation and non-conjugation enzyme-catalyzed reactions. Conjugation enzymes are such as uridine 5′-diphospho-glucuronosyltransferase, glutathione S-transferase, sulfotransferase, arylamine acetyltransferase, and methyltransferase. Conjugation reactions occur at specific atom such as O, N, C, and S or specific group like carboxylic acid. The functional groups involved in detoxification enzyme-catalyzed conjugation reactions include –OH, SH, –NH2, CH2, –COOH, NHOH, epoxide, halide, and –NO2. Non-conjugation enzymes are such as quinone reductase and epoxide hydrolase, where quinone and epoxide functional groups are involved.
Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics, and their molecular characteristics have been extensively investigated in humans, but not in cynomolgus macaques, non-human primate species important for drug metabolism studies. In this study, cynomolgus NAT1 and NAT2 cDNAs were isolated from livers. NAT1 and NAT2 were characterized by molecular analyses and drug-metabolizing assays. A total of 9 transcript variants were found for cynomolgus NAT1, similar to human NAT1, and contained 1-4 exons with the coding region largely conserved with human NAT1. Genomic organization was similar between cynomolgus macaques and humans. Cynomolgus NAT1 and NAT2 amino acid sequences showed high sequence homology (95% and 89%, respectively) and showed close relationship with human NAT1 and NAT2 in a phylogenetic tree. Cynomolgus NAT2 mRNA was predominantly expressed in liver among the 10 different tissues analyzed, followed by kidney and jejunum. In contrast, cynomolgus NAT1 mRNA showed more ubiquitous expression with relatively more abundant expression in liver, kidney, and jejunum, along with testis. Metabolic assays using recombinant proteins showed that cynomolgus NAT1 and NAT2 metabolized human NAT substrates, including p-aminobenzoic acid, sulfamethazine, isoniazid, and 2-aminofluorene. Interestingly, p-aminobenzoic acid and isoniazid were largely metabolized by NAT1 and NAT2, respectively, in cynomolgus macaques and humans; sulfamethazine, a human NAT2 substrate, was metabolized by both NAT enzymes in cynomolgus macaques. These results suggest molecular and enzymatic similarities of NAT1 and NAT2 between cynomolgus macaques and humans, despite some small differences in substrate specificity of the enzymes.
Studies on the metabolic fate of medical drugs, skin care products, cosmetics and other chemicals intentionally or accidently applied to the human skin have become increasingly important in order to ascertain pharmacological effectiveness and to avoid toxicities. The use of freshly excised human skin for experimental investigations meets with ethical and practical limitations. Hence information on xenobiotic-metabolizing enzymes (XME) in the experimental systems available for pertinent studies compared with native human skin has become crucial. This review collects available information of which—taken with great caution because of the still very limited data—the most salient points are: in the skin of all animal species and skin-derived in vitro systems considered in this review cytochrome P450 (CYP)-dependent monooxygenase activities (largely responsible for initiating xenobiotica metabolism in the organ which provides most of the xenobiotica metabolism of the mammalian organism, the liver) are very low to undetectable. Quite likely other oxidative enzymes [e.g. flavin monooxygenase, COX (cooxidation by prostaglandin synthase)] will turn out to be much more important for the oxidative xenobiotic metabolism in the skin. Moreover, conjugating enzyme activities such as glutathione transferases and glucuronosyltransferases are much higher than the oxidative CYP activities. Since these conjugating enzymes are predominantly detoxifying, the skin appears to be predominantly protected against CYP-generated reactive metabolites. The following recommendations for the use of experimental animal species or human skin in vitro models may tentatively be derived from the information available to date: for dermal absorption and for skin irritation esterase activity is of special importance which in pig skin, some human cell lines and reconstructed skin models appears reasonably close to native human skin. With respect to genotoxicity and sensitization reactive-metabolite-reducing XME in primary human keratinocytes and several reconstructed human skin models appear reasonably close to human skin. For a more detailed delineation and discussion of the severe limitations see the Conclusions section in the end of this review.
Arylamine N-acetyltransferases (NATs) are cytosolic conjugating enzymes which add an acetyl group from acetyl Coenzyme A (CoA) to arylamine and arylhydrazines which in general are detoxification reactions. Acetylation of arylhydroxylamines and the transfer of an acetyl group from the O to N group of arylacetohydroxates generally result in activation, particularly of arylamine carcinogens to produce N-acetoxyesters. The polymorphic NAT enzymes were very important in establishing the basics of pharmacogenetics through the metabolism of the antitubercular hydrazine isoniazid. There are now known to be two human isoenzymes: NAT2 responsible for isoniazid metabolism and NAT1, also polymorphic, which is more specific for p-aminosalicylate (pAS) and p-aminobenzoic acid (p-aba) and the folate catabolite, p-aminobenzoylglutamate (p-abaglu). The polymorphism in NAT1 and NAT2 is primarily through a series of SNPs which occur in haplotypes in the single exon coding region of these genes. Amino acid substitutions result in destabilized protein with mutant versions being degraded in the proteasome following ubiquitination. There are NAT enzymes in mammalian (apart from canids), nonmammalian, and also bacterial species. Transgenic mice are helping to unravel the endogenous role of human NAT1 which is widespread in tissues, expressed very early in development and overexpressed in estrogen-receptor positive breast cancer. It is likely that the NAT1 enzyme has a role in acetylating a folate catabolite.Control of expression of NAT genes is beginning to be understood in relation to the splicing patterns of the noncoding exons (NCEs) which appear to be tissue-specific for human NAT1.In all NATs which have been studied the reaction mechanism is through an acetylated cysteine intermediate, with the cysteine being activated through a catalytic triad with histidine and aspartate. The structures of the bacterial and the human enzymes have identified the acetyl CoA binding sites which are subtly different. While the catalytic triads of the various NAT enzymes are superimposable, as is the three-domain core structure, the C-terminus and an interdomain loop in the human enzymes occlude the active-site cleft and this may allow a different range of functions for the bacterial enzymes, particularly from the mycobacteria which, from gene deletion studies, have a role in the formation of cell wall lipids.The availability of structural studies and in silico screening, the potential for using recombinant proteins to identify small molecule inhibitors and substrates, and the availability of transgenic model organisms will allow the role of NAT in toxicology to be addressed at many levels.
Metabolism facilitates the elimination, detoxification and excretion in urine or bile (as biotransformation products) of a myriad of structurally diverse drugs and other chemicals. The metabolism of drugs, non-drug xenobiotics and many endogenous compounds is catalyzed by families of drug metabolizing enzymes (DMEs). These include the hemoprotein-containing cytochromes P450, which function predominantly as monooxygenases, and conjugation enzymes that transfer a sugar, sulfate, acetate or glutathione moiety to substrates containing a suitable acceptor functional group. Drug and chemical metabolism, especially the enzymes that catalyse these reactions, has been the research focus of several groups in Australia for over four decades. In this review, we highlight the role of recent and current drug metabolism research in Australia, including elucidation of the structure and function of enzymes from the various DME families, factors that modulate enzyme activity in humans (e.g. drug-drug interactions, gene expression and genetic polymorphism) and the application of in vitro approaches for the prediction of drug metabolism parameters in humans, along with the broader pharmacological/clinical pharmacological and toxicological significance of drug metabolism and DMEs and their relevance to drug discovery and development, and to clinical practice.
After the functional groups catalyzed by phase I activation enzymes are introduced, lipophilic foreign compounds undergo phase II enzyme-catalyzed reactions, which result in greatly increasing the solubility of parent compounds, thus facilitating their excretion from the body. Foreign compounds that already contain such functional groups can proceed with phase II metabolism without undergoing functionalization reactions. Though phase II reactions are a major defense mechanism, in some cases, the formed conjugates undergo further reactions to yield unstable, reactive metabolites.
The study of drug metabolism places significant importance on the molecular properties of the substrate and the topography of the enzyme's active site. Mechanisms of drug metabolism focus on the different reactions catalyzed within each family of drug‐metabolizing enzymes as well as the chemistry involved in phase I and II reactions. Concepts explored include enzyme‐catalyzed reaction mechanisms, physicochemical properties of substrate molecules that result in favorable sites of metabolism for a given enzyme, and factors that contribute to the regioselectivity and stereoselectivity observed for phase I and II drug‐metabolizing enzymes.
N-acetylation by N-acetyltransferase 1 (NAT1) is an important biotransformation pathway of the human skin and it is involved in the deactivation of the arylamine and well-known contact allergen para-phenylenediamine (PPD). Here, NAT1 expression and activity were analyzed in antigen presenting cells (monocyte-derived dendritic cells, MoDCs, a model for epidermal Langerhans cells) and human keratinocytes. The latter were used to study exogenous and endogenous NAT1 activity modulations. rnWithin this thesis, MoDCs were found to express metabolically active NAT1. Activities were between 23.4 and 26.6 nmol/mg/min and thus comparable to peripheral blood mononuclear cells. These data suggest that epidermal Langerhans cells contribute to the cutaneous N-acetylation capacity. Keratinocytes, which are known for their efficient N-acetylation, were analyzed in a comparative study using primary keratinocytes (NHEK) and different shipments of the immortalized keratinocyte cell line HaCaT, in order to investigate the ability of the cell line to model epidermal biotransformation. N-acetylation of the substrate para-aminobenzoic acid (PABA) was 3.4-fold higher in HaCaT compared to NHEK and varied between the HaCaT shipments (range 12.0–44.5 nmol/mg/min). Since B[a]P induced cytochrome p450 1 (CYP1) activities were also higher in HaCaT compared to NHEK, the cell line can be considered as an in vitro tool to qualitatively model epidermal metabolism, regarding NAT1 and CYP1. The HaCaT shipment with the highest NAT1 activity showed only minimal reduction of cell viability after treatment with PPD and was subsequently used to study interactions between NAT1 and PPD in keratinocytes. Treatment with PPD induced expression of cyclooxygenases (COX) in HaCaT, but in parallel, PPD N-acetylation was found to saturate with increasing PPD concentration. This saturation explains the presence of the PPD induced COX induction despite the high N-acetylation capacities. A detailed analysis of the effect of PPD on NAT1 revealed that the saturation of PPD N-acetylation was caused by a PPD-induced decrease of NAT1 activity. This inhibition was found in HaCaT as well as in primary keratinocytes after treatment with PPD and PABA. Regarding the mechanism, reduced NAT1 protein level and unaffected NAT1 mRNA expression after PPD treatment adduced clear evidences for substrate-dependent NAT1 downregulation. These results expand the existing knowledge about substrate-dependent NAT1 downregulation to human epithelial skin cells and demonstrate that NAT1 activity in keratinocytes can be modulated by exogenous factors. Further analysis of HaCaT cells from different shipments revealed an accelerated progression through the cell cycle in HaCaT cells with high NAT1 activities. These findings suggest an association between NAT1 and proliferation in keratinocytes as it has been proposed earlier for tumor cells. rnIn conclusion, N-acetylation capacity of MoDCs as well as keratinocytes contribute to the overall N-acetylation capacity of human skin. NAT1 activity of keratinocytes and consequently the detoxification capacities of human skin can be modulated by the presence of exogenous NAT1 substrates and endogenous by the cell proliferation status of keratinocytes. rn
There have been important advances in the NATs in the past six years which have been possible as a result of the wealth of information at the genomic, pharmacological, chemical and structural levels. These studies have taken NAT research into new areas which are of particular importance in relation to drug discovery and diagnostics as well as toxicology, including environmental toxicology. The field of NAT research has also impinged on evolution and anthropology as a result of haplotype analyses of the highly polymorphic human NAT genes, two functional genes human NAT1 and NAT2 and a pseudigene encoded on the short arm of chromosome 8 There have been several reviews in that period with the most recent covering much of the information provided in this update (Hein, 2009; Vagena et al., 2008; Butcher and Minchin, 2012; Sim et al., 2012; Sim et al., 2014; Kubiak et al., 2013a; Zhou et al., 2013).
Xenobiotic biotransformation undergoes several stages of simultaneous or successive whose main attractions are the tissues at the interface between the organism and the external environment, namely: digestive, respiratory, kidney and liver. The latter being the most important functionally. The reaction phases constituting the main stages of detoxification, phase I, phase II and phase III, are possible only through the intervention of specific enzyme systems. Given the wide diversity of xenobiotics to which the organism is exposed, there are a multitude of enzymes with various specificities. The biotransformation reactions of xenobiotics are linked linearly rarely, because two or more lanes are often born from a given metabolite. It is therefore understandable that the existence of an enzyme variant defective for one of these reaction pathways can direct the metabolism of a given substance to another track. The latter, usually minor, will therefore important and polymorphisms that concern will guide the fate of metabolites thus formed. The N-acetyltransferases (NATs) is part of enzymes that primarily the conjugation reaction of phase II detoxification of xenobiotics. The polymorphism of NATS is one of the examples of pharmacogenetic variation described, and one of the most documented since its discovery in the early \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\'50s, along with the discovery of the high efficacy of isoniazid (INH) in the treatment of tuberculosis. The work of this thesis aimed to study the profile of the NAT2 acetylation in the Senegalese population in order to distribute them in slow acetylators and rapid acetylators, and determine the kinetics of isoniazid in tuberculous subjects correlated with the results of genotyping. The study of mutations of the NAT2 gene was performed by PCR-direct sequencing and allowed to identify 11 allelic variants in the Senegalese population. The enzymatic activity of NAT2 was determined by using caffeine test and the ratios of major metabolites allowed Senegalese classify fast and slow acetylators. The kinetics of isoniazid used UPLC-MS/MS chromatography.
Esophageal cancer (EC) is one of the most common malignancies in low- and medium-income countries and represents a disease of public health importance because of its poor prognosis and high mortality rate in these regions. The striking variation in the prevalence of EC among different ethnic groups suggests a significant contribution of population-specific environmental and dietary factors to susceptibility to the disease. Although individuals within a demarcated geographical area are exposed to the same environment and share similar dietary habits, not all of them will develop the disease; thus genetic susceptibility to environmental risk factors may play a key role in the development of EC. A wide range of xenobiotic-metabolizing enzymes are responsible for the metabolism of carcinogens introduced via the diet or inhaled from the environment. Such dietary or environmental carcinogens can bind to DNA, resulting in mutations that may lead to carcinogenesis. Genes involved in the biosynthesis of these enzymes are all subject to genetic polymorphisms that can lead to altered expression or activity of the encoded proteins. Genetic polymorphisms may, therefore, act as molecular biomarkers that can provide important predictive information about carcinogenesis. The aim of this review is to discuss our current knowledge on the genetic risk factors associated with the development of EC in different populations; it addresses mainly the topics of genetic polymorphisms, gene-environment interactions, and carcinogenesis. We have reviewed the published data on genetic polymorphisms of enzymes involved in the metabolism of xenobiotics and discuss some of the potential gene-environment interactions underlying esophageal carcinogenesis. The main enzymes discussed in this review are the glutathione S-transferases (GSTs), N-acetyltransferases (NATs), cytochrome P450s (CYPs), sulfotransferases (SULTs), UDP-glucuronosyltransferases (UGTs), and epoxide hydrolases (EHs), all of which have key roles in the detoxification of environmental and dietary carcinogens. Finally, we discuss recent advances in the study of genetic polymorphisms associated with EC risk, specifically with regard to genome-wide association studies, and examine possible challenges of case-control studies that need to be addressed to better understand the interaction between genetic and environmental factors in esophageal carcinogenesis.
The exposure of the skin to medical drugs, skin care products, cosmetics, and other chemicals renders information on xenobiotic-metabolizing enzymes (XME) in the skin highly interesting. Since the use of freshly excised human skin for experimental investigations meets with ethical and practical limitations, information on XME in models comes in the focus including non-human mammalian species and in vitro skin models. This review attempts to summarize the information available in the open scientific literature on XME in the skin of human, rat, mouse, guinea pig, and pig as well as human primary skin cells, human cell lines, and reconstructed human skin models. The most salient outcome is that much more research on cutaneous XME is needed for solid metabolism-dependent efficacy and safety predictions, and the cutaneous metabolism comparisons have to be viewed with caution. Keeping this fully in mind at least with respect to some cutaneous XME, some models may tentatively be considered to approximate reasonable closeness to human skin. For dermal absorption and for skin irritation among many contributing XME, esterase activity is of special importance, which in pig skin, some human cell lines, and reconstructed skin models appears reasonably close to human skin. With respect to genotoxicity and sensitization, activating XME are not yet judgeable, but reactive metabolite-reducing XME in primary human keratinocytes and several reconstructed human skin models appear reasonably close to human skin. For a more detailed delineation and discussion of the severe limitations see the "Overview and Conclusions" section in the end of this review.
Arylamine N-acetyltransferases (NATs) are defined as xenobiotic metabolizing enzymes, adding an acetyl group from acetyl coenzyme A (CoA) to arylamines and arylhydrazines. NATs are found in organisms from bacteria and fungi to vertebrates. Several isoenzymes, often polymorphic, may be present in one organism. There are two functional polymorphic NATs in humans and polymorphisms in NAT2 underpinned pharmacogenetics as a discipline. NAT enzymes have had a role in important metabolic concepts: the identification of acetyl-CoA and endogenous metabolic roles in bacteria and in eukaryotic folate metabolism. In fungi, NAT is linked to formation of unique metabolites. A broad and exciting canvas of investigations has emerged over the past five years from fundamental studies on NAT enzymes. The role of human NAT1 in breast cancer where it is a biomarker and possible therapeutic target may also underlie NAT's early appearance during mammalian fetal development. Studies of NAT in Mycobacterium tuberculosis have identified potential therapeutic targets for tuberculosis whilst the role of NATs in fungi opens up potential toxicological intervention in agriculture. These developments are possible through the combination of genomics, enzymology and structural data. Strong binding of CoA to Bacillis anthracis NAT may point to divergent roles of NATs amongst organisms as does differential control of mammalian NAT gene expression. The powerful combination of phenotypic investigation following genetic manipulation of NAT genes from mice to mycobacteria has been coupled with generation of isoenzyme-specific inhibitors. This battery of molecular and systems biology approaches heralds a new era for NAT research in pharmacology and toxicology.
The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.
The human arylamine N-acetyltransferases first attracted attention because of their role in drug metabolism. However, much of the current literature has focused on their role in the activation and detoxification of environmental carcinogens and how genetic polymorphisms in the genes create predispositions to increased or decreased cancer risk. There are two closely related genes on chromosome 8 that encode the two human arylamine N-acetyltransferases--NAT1 and NAT2. Although NAT2 has restricted tissue expression, NAT1 is found in almost all tissues of the body. There are several single-nucleotide polymorphisms in the protein coding and 3'-untranslated regions of the gene that affect enzyme activity. However, NAT1 is also regulated by post-translational and environmental factors, which may be of greater importance than genotype in determining tissue NAT1 activities. Recent studies have suggested a novel role for this enzyme in cancer cell growth. NAT1 is up-regulated in several cancer types, and overexpression can lead to increased survival and resistance to chemotherapy. Although a link to folate homeostasis has been suggested, many of the effects attributed to NAT1 and cancer cell growth remain to be explained. Nevertheless, the enzyme has emerged as a viable candidate for drug development, which should lead to small molecule inhibitors for preclinical and clinical evaluation.
Arylamine N-acetyltransferases (NATs) are cytosolic conjugating enzymes which transfer an acetyl group from acetylCoenzyme A to a xenobiotic acceptor substrate. The enzyme has an active site cysteine as part of a catalytic triad with histidine and aspartate. NATs have had an important role in pharmacogenetics. Polymorphism in acetylation (and inactivation) of the anti-tubercular agent isoniazid resides in human NAT2, one of two polymorphic human NATs. In humans there is also a third pseudogene and in rodents there are three isozymes. Comparison of human and rodent NAT enzymes and their genes is aiding our understanding of the roles of the individual isoenzymes. This may have clinical importance since human NAT1 is overexpressed in a sub-population of breast cancers and control of expression of the NAT genes is ripe for investigation. The mammalian NAT enzymes are involved in metabolism of drugs and carcinogens but there is growing evidence, including from transgenic mice, that human NAT1 has an endogenous role in folate degradation. Structural studies and intracellular tracking of polymorphic NAT variants, is contributing to appreciation of how individual mutations result in loss of NAT activity. Genome analyses have identified NAT homologues in bacteria including Mycobacterium tuberculosis, in which the NAT enzyme metabolises inactivation of isoniazid. More intriguingly, deletion of the nat gene in mycobacteria, leads to deficits in cell wall synthesis. Structural comparisons of NATs from prokaryotes and eukaryotes, particularly in relation to CoA binding, provide a platform for understanding how the unique NAT protein fold may lend itself to a wide range of functions.
Arylamine N-acetyltransferase (NAT) research has been influenced in recent years by the rapid progress in genomics, proteomics, structural genomics and other cutting-edge disciplines. To keep up with these advancements, the NAT scientific community has fostered collaboration and exchange of know-how between its members. As a specialized event bringing together experts from many different laboratories, the triennial International NAT Workshop has been instrumental in maintaining this culture over the past ten years. The 2007 Workshop took place in Alexandroupolis, Greece, and covered ongoing research on the structure and enzymatic function of human NATs, the prokaryotic and eukaryotic models for NAT, the mechanisms of NAT gene regulation and expression, the frequencies and effects of polymorphisms in the human NAT genes, and the involvement of NATs in multifactorial diseases, including cancer, allergic conditions, endometriosis and endemic nephropathies. Gene nomenclature issues were also addressed and the participants discussed current trends in the field.
The identification, synthesis, and evaluation of a series of naphthoquinone derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 are described. The compounds undergo a distinctive color change (red --> blue) upon binding to these human and mouse NAT isoenzymes driven by a proton transfer event. No color change is observed in the presence of functionally distinct but highly similar isoenzymes which are >70% identical. These molecules may be used as sensors to detect the presence of human NAT1 in cell lysates.
Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme (XME) involved in the biotransformation of many aromatic and heterocyclic amines. This XME plays key roles in both the detoxification and/or bioactivation of numerous drugs and carcinogens. NAT1 is polymorphic and displays a large tissue distribution. NAT1 activity have been extensively studied because of its potential role in the biotransformation of important carcinogens. Several recent studies suggest that NAT1 may have a role in breast cancer progression. Indeed, this XME has been shown to affect the growth and drug resistance of breast cancer cells and appears as a marker in human estrogen receptor positive breast cancer. In addition, it has been shown that this enzyme is inhibited in vivo by cancer drugs such as cisplatin or tamoxifen. Recent published data suggest that NAT1 could be of therapeutic interest for cancer. We provide here an overview on the putative involvement of NAT1 in cancer and its possible role as a drug target.
Xenobiotic metabolizing enzymes like cytochrome P450s and N-acetyltransferase are expressed in keratinocytes and professional antigen-presenting cells. Thus, biotransformation of chemicals applied to the skin can be relevant for their potential to cause skin toxicity and immune responses like allergic contact dermatitis. Considering the keratinocyte cell line HaCaT as a relevant in vitro tool for epidermal biotransformation, we specifically investigated CYP1 (EROD) and N-acetyltransferase 1 (NAT1) activities of three different HaCaT shipments and human primary keratinocytes (NHEK). Solvent treated HaCaT showed EROD levels near the detection limit (0.047 pmol/mg/min), primary keratinocytes (n=4) were in a range between 0 and 0.76 pmol/mg/min. B[a]P (1 microM) induced EROD activities of 19.0+/-0.9 pmol/mg/min (n=11) in HaCaT and 5.8+/-0.5 pmol/mg/min (n=4) in NHEK. N-acetylation activities for para-aminobenzoic acid (PABA) were in average 3.4-fold higher in HaCaT compared to NHEK (8+/-0.5 nmol/mg/min) and varied between the HaCaT shipments (range 12.0-44.5 nmol/mg/min). This was in good agreement with NAT1 promoter P1 dependent mRNA level and N-acetylation of the contact allergen para-phenylenediamine (PPD) under typical cell-based assay conditions. We conclude that HaCaT represent a suitable in vitro model for studying the qualitative contribution of epidermal phase1/phase2 metabolism to toxicological endpoints such as skin sensitization.
Human arylamine N-acetyltransferase 1 (NAT1), a polymorphic xenobiotic metabolising enzyme, has been investigated in relation to susceptibility and prognosis in certain types of cancer. Both human NAT1 and its murine equivalent NAT2 have previously been shown to play roles in the catabolism of folate, which is required for the synthesis of S-adenosylmethionine, the methyl donor for cellular methylation reactions. We have tested whether the expression of mouse Nat2 is subject to epigenetic regulation, specifically CpG methylation in the promoter region, by determining levels of 5-methylcytosine by bisulphite sequencing and methylation-specific PCR. Under normal conditions, methylation levels of the Nat2 promoter were low, and varied in different tissues. However, CpG methylation was significantly increased by dietary folate supplementation, and increased methylation corresponded to decreased use of the core promoter. Functional deletion of the Nat2 gene gave rise to a significant increase in Nat2 methylation, extending our previous observations that folate catabolism is decreased in Nat2 null mice. Mouse NAT2 is likely to influence epigenetic gene control, particularly of its own locus, and this is consistent with recent evidence associating aberrant mouse Nat2/human NAT1 gene expression with certain developmental malformations and cancers.
The single coding exons of the cloned genes encoding two human arylamine N-acetyltransferases (NAT1 and NAT2) were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based phagemid vector pKEN2 for production of the recombinant proteins in Escherichia coli strain XA90. Induction of cultures grown from selected bacterial transformants resulted in the production of substantial quantities of soluble recombinant human NAT1 and NAT2 with identical electrophoretic, immunologic and catalytic properties to those expressed in mammalian cell culture or in human liver. Oligonucleotide-directed mutagenesis of recombinant human NAT2 was then employed to determine the relative importance of 3 highly conserved cysteine residues in the enzyme's catalytic mechanism. Substitution of cysteine with glycine at position 68 of the 290 amino acid protein molecule (Cys68----Gly) resulted in the production of normal quantities of immunoreactive NAT2 which was completely devoid of enzyme activity, suggesting that the sulfhydryl group of Cys68 is directly involved in the transfer of acetate from the essential cofactor CoASAc to acceptor amine substrates. On the other hand, the mutations producing Cys44----Gly and Cys223----Gly led to the production of enzymatically active NAT2 proteins with markedly reduced in vitro stability, suggesting that substitution of either of these amino acids may cause alterations in the tertiary structure of the native enzyme.
N-Acetylation by hepatic arylamine N-acetyltransferase (NAT, EC 2.3.1.5) is a major route in the metabolism and detoxification of numerous drugs and foreign chemicals. NAT is the target of a common genetic polymorphism of clinical relevance in human populations. We have used our recently isolated rabbit cDNA rnat to clone three human NAT genes from human leukocyte DNA. None of the three genomic coding sequences was interrupted by introns. Two genes, designated NAT1 and NAT2, each possessed open reading frames of 870 bp. Both genes have been assigned to human chromosome 8, pter-q11. Following transfection they were transiently expressed in monkey kidney COS-1 cells. NAT1 and NAT2 gave rise to functional NAT proteins, as judged by their NAT enzyme activity with the arylamine substrate sulfamethazine. Western blots with NAT-specific antisera detected proteins of apparent molecular weight of 33 and 31 kD in NAT1- and NAT2-transfected cultures, respectively. The product of NAT2 had an identical apparent molecular weight as that of NAT detected in human liver cytosol. The deduced amino acid sequence of NAT2 also contained 6 peptide sequences which had previously been determined from tryptic peptides of the polymorphic NAT purified from human liver. These data suggest that NAT2 encodes the polymorphic NAT protein. The third gene, NATP, had multiple deleterious mutations and did not encode a functional NAT protein; it most likely represents a pseudogene.
N-acetyl-p-aminobenzoylglutamate is a major urinary metabolite of folic acid. It is formed by acetylation of p-aminobenzoylglutamate following cleavage of the C9-N10 bond of folic acid. Using recombinant human type 1 (NAT1) and type 2 (NAT2) arylamine N-acetyltransferase, we have shown that p-aminobenzoylglutamate is a specific NAT1 substrate. At an acetyl-CoA concentration of 50 microM, the Km for p-aminobenzoylglutamate (pABG) acetylation by recombinant NAT1 was 130 +/- 13 microM. For the human pro-monocytic cell-line U937, the apparent Km was slightly higher (333 +/- 17 microM). Inhibitor studies supported NAT1-dependent acetylation of pABG by U937 cell cytosols. These studies are the first to identify a potential endogenous substrate for human NAT1 and suggest that this enzyme may be important in the cellular clearance of pABG.
The human arylamine N-acetyltransferases NAT1 and NAT2 catalyze the biotransformation of primary aromatic amine or hydrazine drugs and xenobiotics. These enzymes share 81% amino acid sequence identity, yet differ markedly with respect to their acceptor substrate selectivities and intrinsic in vitro stabilities. To define the contribution of large regions of NAT1 and NAT2 polypeptide structure to enzyme integrity and catalytic specificity, we used selected restriction endonuclease digestions and fragment religation into the tac promoter-based phagemid pKEN2 to construct a panel of 18 NAT1/NAT2 hybrid gene vectors for heterologous expression in Escherichia coli. Induction of hybrid gene expression in recombinant transformants of E. coli strain XA90 led to the production of soluble, catalytically active acetylating enzymes in all cases. Chimeric proteins produced in this fashion were then compared to wild-type NAT1 and NAT2 with respect to their enzyme kinetic constants (apparent Km, Vmax, and Vmax/Km) for the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid and sulfamethazine, respectively, and for their in vitro stabilities at 37 degrees C. The ratio of the Vmax/Km for sulfamethazine to that for p-aminosalicylic acid allowed for the unambiguous classification of each enzyme as either NAT1 or NAT2 type, except for one novel chimera possessing a low Michaelis constant and a high maximal velocity for the acetylation of both substrates. A central region (amino acids 112-210) within the 290-residue polypeptide appeared to play a role in determining NAT1- or NAT2-type behavior. On the other hand, the region (residues 47-111) encompassing the putative active site cysteine (Cys68) was important in contributing to a low apparent Km for p-aminosalicylic acid but not for sulfamethazine, while amino acids 211-250 affected Km for sulfamethazine and 251-290 influenced Km for both substrates. Maximal velocities were highest for both substrates when the central 112-210 amino acid region was derived from NAT1. Finally, the region from amino acids 211-250 in NAT2 was important in determining its greater intrinsic enzyme stability than that exhibited by NAT1.
The N-acetyltransferase (NAT) phenotype is an important determinant of individual susceptibility to occupational bladder cancer. N-Acetyltransferases arc known to metabolize aromatic amine bladder carcinogens, but the functional significance of NAT expression in the target organ is unclear. To resolve this issue, polygonal antisera against purified recombinant enzymes and C-terminal peptides of human NAT Type 1 (NAT1) and Type 2 (NAT2) were generated. Western blot analysis of exfoliated cells from human urine, pig bladder homogenate, and human bladder tumor-derived cell lines showed that NAT1 was expressed in all three systems, whereas NAT2 did not appear to be expressed in the bladder. Immunohistochemical analysis of human bladder tumor sections indicated that well-differentiated tumor cells expressed NAT1, with the highest level of expression being found in the umbrella cells that line the bladder lumen. Poorly differentiated tumor regions appeared to express NAT1 at lower levels than did well-differentiated areas. These findings support the hypothesis that aromatic amines are metabolized in the bladder epithelium by NAT1.
The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are responsible for the biotransformation of many arylamine and hydroxylamine xenobiotics. It has been proposed that NATs may act through a cysteine-linked acetyl-enzyme intermediate in a general base catalysis involving a highly conserved arginine residue such as Arg64. To investigate this possibility, we used site-directed mutagenesis and expression of recombinant human NAT1 and NAT2 in Escherichia coli. Sequence comparison with NATs from other species indicated that Arg9 and Arg64 are the only invariant basic residues. Either mutation of the presumed catalytic Cys68 residue or the simultaneous mutation of Arg9 and Arg64 to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg9 or Arg64 with Ala, Met, Gln or Lys exhibited unaltered Km values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg9/Arg64 with lysine in NAT1 altered the Km for arylamine substrates (decreased by 8-14-fold) and for acetyl-CoA (elevated 5-fold), and modified the pH-dependence of activity. Thus, through their positively charged side chains, Arg9 and Arg64 seem to contribute to the conformational stability of NAT1 and NAT2 rather than acting as general base catalysts. Our results also support a mechanism in which Arg9 and Arg64 are involved in substrate binding and transition-state stabilization of NAT1.
Arylamine N-acetyltransferases in humans (NAT1 and NAT2) catalyse the acetylation of arylamines including food derived heterocyclic arylamine carcinogens. Other substrates include the sulphonamide 5-aminosalicylic acid (5-ASA), which is an NAT1 specific substrate; N-acetylation of 5-ASA is a major route of metabolism. NAT1 and NAT2 are both polymorphic.
To investigate NAT expression in apparently healthy human intestines in order to understand the possible role of NAT in colorectal cancer and in the therapeutic response to 5-ASA.
The intestines of four organ donors were divided into eight sections. DNA was prepared for genotyping NAT1 and NAT2 and enzymic activities of NAT1 and NAT2 were determined in cytosols prepared from each section. Tissue was fixed for immunohistochemistry with specific NAT antibodies. Western blotting was carried out on all samples of cytosol and on homogenates of separated muscle and villi after microdissection.
NAT1 activity of all cytosols was greater than NAT2 activity. NAT1 and NAT2 activities correlated with the genotypes of NAT1 and NAT2 and with the levels of NAT1 staining determined by western blotting. The ratio of NAT1:NAT2 activities showed interindividual variations from 2 to 70. NAT1 antigenic activity was greater in villi than in muscle. NAT1 was detected along the length of the villi in the small intestine. In colon samples there was less NAT1 at the base of the crypts with intense staining at the tips.
The interindividual variation in NAT1 and NAT2 in the colon could affect how individuals respond to exposure to specific NAT substrates including carcinogens and 5-ASA.
Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560-564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29-131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122-131 in NAT1 model and residues 122-133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.
The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.
The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens. The NATs have been shown to be important in drug detoxification and carcinogen activation, with humans possessing two isoenzymes encoded by polymorphic genes. This polymorphism has pharmacogenetic implications, leading to different rates of inactivation of drugs, including the anti-tubercular agent isoniazid and the anti-hypertensive drug hydralazine. Mice provide a good model for human NAT, allowing genetic manipulation of expression to explore possible endogenous roles of these enzymes. The first three-dimensional NAT structure was resolved for NAT from Salmonella typhimurium, and subsequently the structure of NAT from Mycobacterium smegmatis has been elucidated. These identified a 'Cys-His-Asp' catalytic triad (conserved in all NATs), which is believed to be responsible for the activation of the active site cysteine residue. As more genomic data become available, NAT homologues continue to be found in prokaryotic species, many of which are pathogenic, including Mycobacterium tuberculosis. The discovery of NAT in M. tuberculosis is particularly significant, since this enzyme participates in inactivation of isoniazid in the bacterium, with implications for isoniazid resistance. Structural studies on NAT proteins and phenotypic analyses of organisms (both mice and prokaryotes) following genetic modifications of the nat genes are leading to an understanding of the potentially diverse roles of NAT in endogenous and xenobiotic metabolism. These studies have indicated that NAT, particularly in Mycobacteria, has the potential to be a drug target. Combinatorial chemical approaches, together with in silico structural studies, will allow for advances in the identification of NAT substrates and inhibitors, both as experimental tools and as potential drugs.
Arylamine N-acetyltransferases (NATs) are polymorphic xenobiotic metabolising enzymes, linked to cancer susceptibility in a variety of tissues. In humans and in mice there are multiple NAT isoforms. To identify whether the different isoforms represent inbuilt redundancy or whether they have unique roles, we have generated mice with a null allele of Nat2 by gene targeting. This mouse line conclusively demonstrates that the different isoforms have distinct functions with no compensatory expression in the Nat2 null animals of the other isoforms. In addition, we have used the transgenic line to show the pattern of Nat2 expression during development. Although Nat2 is not essential for embryonic development, it has a widespread tissue distribution from at least embryonic day 9.5. This mouse line now paves the way for the teratological role of Nat2 to be tested.
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.
In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5' non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo.
To test the hypothesis that genetic variation in the metabolism of tobacco carcinogens, such as aromatic amines (AA) and heterocyclic amines (HCA), contributes to pancreatic cancer, we have examined genetic polymorphisms of three key enzymes, i.e. cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 1 and 2 (NAT1 and NAT2), in a hospital-based case-control study of 365 patients with pancreatic adenocarcinoma and 379 frequency-matched healthy controls. Genotypes were determined using PCR-restriction fragment length polymorphism (RFLP) and Taqman methods. Smoking information was collected by personal interview. Adjusted odds ratio (AOR) and 95% confidence interval (CI) was estimated by unconditional multivariate logistic regression analysis. We found that the NAT1 'rapid' alleles were associated with a 1.5-fold increased risk of pancreatic cancer (95% CI: 1.0-2.1) with adjustment of potential confounders. This effect was more prominent among never smokers (AOR: 2.4, 95% CI: 1.4-4.3) and females (AOR: 1.8, 95% CI: 1.0-3.1). Some genotypes were significantly associated with increased risk for pancreatic cancer among smokers, especially heavy smokers (<20 pack years). For example, heavy smokers with the CYP1A2*1D (T-2467delT) delT, CYP1A2*1F(A-163C) C allele, NAT1 'rapid' or NAT2 'slow' alleles had an AOR (95% CI) of 1.4 (0.7-2.3), 1.9 (1.1-3.4), 3.0 (1.6-5.4) and 1.5 (0.8-2.6), respectively, compared with never smokers carrying the non-at-risk alleles. These effects were more prominent in females than in males. The corresponding AOR (95% CI) was 3.1 (1.0-8.0), 3.8 (1.5-10.1), 4.5 (1.6-12.7) and 2.0 (0.8-5.1) for females versus 1.0 (0.4-1.9), 1.1 (0.5-2.4), 2.1 (1.0-4.6) and 1.1 (0.5-2.6) for males. A significant synergistic effect of CYP1A2*1F C allele and NAT1"rapid" alleles on the risk for pancreatic cancer was also detected among never smokers (AOR: 2.9, 95% CI: 1.2-6.9) and among females (AOR: 2.5, 95% CI: 1.1-5.7). These data suggest that polymorphisms of the CYP1A2 and NAT1 genes modify the risk of pancreatic cancer.
The human arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are enzymes responsible for the acetylation of many arylamines and hydrazines, thereby playing an important role in both detoxification and activation of many drugs and carcinogens. Both enzymes show polymorphisms but exhibit key differences in substrate selectivity and tissue expression. In the present study, reverse transcriptase-PCR, Western blotting, and immunohistochemistry were used to investigate the expression of the NATs in human skeletal muscle. Despite the presence of its mRNA, NAT2 enzyme level was below the limit of detection. In contrast, both NAT1 mRNA and enzyme were readily detected in fetal, newborn, and adult muscles. In addition, punctate cytoplasmic and perinuclear NAT1 immunostaining was observed in all tissue sections, the staining being more intense in the fetal tissue. High expression of NAT1 enzyme in fetal muscle was also suggested by Western blotting. Because skeletal muscle accounts for a large proportion of body mass, muscle NAT1 expression may contribute significantly to the total activity in the body. These results further support the involvement of skeletal muscle in the metabolism of xenobiotics.
Patients with breast cancer experience progression and respond to treatment in diverse ways, but prognostic and predictive tools for the oncologist are limited. We have used gene expression data to guide the production of hundreds of novel antibody reagents to discover novel diagnostic tools for stratifying carcinoma patients.
One hundred forty novel and 23 commercial antisera, selected on their ability to differentially stain tumor samples, were used to stain paraffin blocks from a retrospective breast cancer cohort. Cox proportional hazards and regression tree analysis identified minimal panels of reagents able to predict risk of recurrence. We tested the prognostic association of these prospectively defined algorithms in two independent cohorts.
In both validation cohorts, the Kaplan-Meier estimates of recurrence confirmed that both the Cox model using five reagents (p53, NDRG1, CEACAM5, SLC7A5, and HTF9C) and the regression tree model using six reagents (p53, PR, Ki67, NAT1, SLC7A5, and HTF9C) distinguished estrogen receptor (ER)-positive patients with poor outcomes. The Cox model was superior and distinguished patients with poor outcomes from patients with good or moderate outcomes with a hazard ratio of 2.21 (P = .0008) in validation cohort 1 and 1.88 (P = .004) in cohort 2. In multivariable analysis, the calculated risk of recurrence was independent of stage, grade, and lymph node status. A model proposed for ER-negative patients failed validation in the independent cohorts.
A panel of five antibodies can significantly improve on traditional prognosticators in predicting outcome for ER-positive breast cancer patients.
A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid-based analysis to predict patient outcome.
We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section.
The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n=223, 5-year survival=95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n=149).
With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.
Human arylamine N-acetyltransferases (NAT) bioactivate arylamine and heterocyclic amine carcinogens present in red meat and tobacco products. As a result, factors that regulate expression of NATs have the potential to modulate cancer risk in individuals exposed to these classes of carcinogens. Because epidemiologic studies have implicated well-done meat consumption as a risk factor for prostate cancer, we have investigated the effects of androgens on the expression of arylamine N-acetyltransferase type I (NAT1). We show that NAT1 activity is induced by R1881 in androgen receptor (AR)-positive prostate lines 22Rv1 and LNCaP, but not in the AR-negative PC-3, HK-293, or HeLa cells. The effect of R1881 was dose dependent, with an EC(50) for R1881 of 1.6 nmol/L. Androgen up-regulation of NAT1 was prevented by the AR antagonist flutamide. Real-time PCR showed a significant increase in NAT1 mRNA levels for R1881-treated cells (6.60 +/- 0.80) compared with vehicle-treated controls (1.53 +/- 0.17), which was not due to a change in mRNA stability. The increase in NAT1 mRNA was attenuated by concurrent cycloheximide treatment, suggesting that the effect of R1881 may not be by direct transcriptional activation of NAT1. The dominant NAT1 transcript present following androgen treatment was type IIA, indicating transcriptional activation from the major NAT1 promoter P1. A series of luciferase reporter deletions mapped the androgen responsive motifs to a 157-bp region of P1 located 745 bases upstream of the first exon. These results show that human NAT1 is induced by androgens, which may have implications for cancer risk in individuals.
The central role of sequential accumulation of genetic alterations during the development of cancer has been firmly established since the pioneering cytogenetic studies successfully defined recurrent chromosome changes in specific types of tumor. In the course of carcinogenesis, cells experience several genetic alterations that are associated with the transition from a preneoplastic lesion to an invasive tumor and finally to the metastatic state. Tumor progression is characterized by stepwise accumulation of genetic alterations. So does the dominant metastatic clone. Modern molecular genetic analyses have clarified that genomic changes accumulate during the development and progression of cancers. In comparison with the corresponding primary tumor, additional events of chromosomal aberrations (including gains or allelic losses) are frequently found in metastases, and the incidence of combined chromosomal alterations in the primary tumor, plus the occurrence of additional aberrations in the distant metastases, correlated significantly with decreased postmetastatic survival. The deletions at 3p, 4p, 6q, 8p, 10q, 11p, 11q, 12p, 13q, 16q, 17p, 18q, 21q, and 22q, as well as the over-representations at 1q, 8q, 9q, 14q and 15q, have been found to associate preferentially with the metastatic phenotype of human cancers. Among of them, the deletions on chromosomes 8p, 17p, 11p and 13p seem to be more significant, and more detail fine regions of them, including 8p11, 8p21-12, 8p22, 8p23, 17p13.3, 11p15.5, and 13q12-13 have been suggested harboring metastasis-suppressor genes. During the past decade, several human chromosomes have been functionally tested through the use of microcell-mediated chromosome transfer (MMCT), and metastasis-suppressor activities have been reported on chromosomes 1, 6, 7, 8, 10, 11, 12, 16, and 17. However, it is not actually known at what stage of the metastatic cascade these alterations have occurred. There is still controversial with the association between the chromosomal aberrations and the metastatic phenotype of cancer. As the progression of human genome project and the establishment of more and more new techniques, it is hopeful to make clear the genetic mechanisms involved in the tumor metastasis in a not very long future, and provide new clues to predicting and controlling the metastasis.
Acetyltransferase with p-aminobenzoic acid (PABA) as substrate was investigated in the cytosolic fraction of the placenta, liver, adrenals, lungs, kidneys, intestine from human fetuses and the liver, lungs, kidneys and intestinal mucosa from adult subjects. All tissue specimens assayed catalyzed the acetylation of PABA at a significant rate. The activity (expressed as nmol of product formed/min/mg protein; mean ± SE) was 1.10 ± 0.59 in the fetal liver, 0.66 ± 0.04 in the placental and 3.87 ± 0.53 in the adult liver cytosol. Among the fetal tissues, the adrenals had the highest (2.36 ± 0.78) and the gut the lowest activity (0.71 ± 0.11). The acetyltransferase activity (mean ± SE) in the lungs, kidneys and intestinal mucosa from adult subjects was 1.19 ± 0.15; 1.34 ± 0.04 and 3.80 ± 0.34, respectively.
Heterocyclic amines are mammary carcinogens in rats and their N-hydroxy metabolites are substrates for subsequent metabolic activation by N-acetyltransferases (NAT) and sulfotransferases (SULT) in man. We investigated the expression of these enzymes in human breast tissue and the relationship between NAT genotype and NAT mRNA expression or enzyme activity. Immunohistochemical staining of sections of breast tissue identified expression of NAT1 and NAT2 protein in human mammary epithelial cells, but not in the stroma. We also measured the formation of DNA adducts of the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in calf thymus DNA after incubation of their promutagenic N-hydroxy metabolites with mammary cytosols prepared from reduction mammoplasty tissue. Experimental observations gained from use of enzyme cofactors and NAT and/or SULT inhibitors on cytosolic enzyme activity, recombinant NAT1 activity and heterocyclic amine-DNA adduct formation suggest that both NAT1 and SULT1A enzymes contribute significantly to the activation of N-hydroxylated heterocyclic amines in mammary tissue. NAT1 mRNA transcript levels were found to be two- to three-fold higher than mRNA transcripts of the NAT2 gene in reduction mammoplasty tissue and mammary epithelial cells. NAT1-specific p-aminobenzoic acid acetylation activity, but not NAT2-specific sulfamethazine acetylation activity, was detectable in mammary cytosols. There was no association apparent between NAT genotype and the levels of NAT mRNA or NAT enzyme activity, or between NAT1 genotype and IQ-DNA adduct formation mediated by mammary cytosols. Western blot analysis of mammary cytosolic protein showed detectable levels of SULT1A1 and SULT1A3.
In humans, aromatic and heterocyclic amine carcinogens may be acetylated by the expression products of either of the N-acetyltransferase genes, NAT1 or NAT2. This conjugation reaction can result in either activation or detoxication of these carcinogens depending on the tissue involved. Recent studies suggest that polymorphisms in NAT1 or NAT2 may modulate cancer risk. To determine if genetic differences in NAT1 and NAT2 could alter risk of gastric cancer, we tested for the presence of polymorphic N-acetyltransferase alleles (both NAT1 and NAT2) in a preliminary study of 94 gastric adenocarcinoma patients and 112 control subjects from North Staffordshire, England. We used established PCR protocols to genotype for NAT2 and NAT1 alleles (NAT2*4, NAT2*5, NAT2*6, NAT2*7, NAT2*14; NAT1*3, NAT1* 4, NAT1*10, and NAT1*11), and implemented an oligonucleotide ligation assay (OLA) to test for low-activity NAT1 alleles [NAT1*14 (G560A), NAT1*15 (C559T), and NAT1*17 (C190T)]. No significant increased risk was observed for NAT2 acetylation genotypes. However, among all cases, we found that individuals inheriting a variant NAT1 allele, NAT1*10, have a significantly elevated risk for gastric cancer (OR = 2.2, 95% CI 1.2–3.9, P < 0.01). Interestingly, the risk observed for NAT1*10 appears to be solely associated with advanced-stage tumors (OR = 4.8, P < 0.001), suggesting a possible role in progression to advanced disease. This preliminary finding needs confirmation in a larger, detailed epidemiological study. Int. J. Cancer 87:507–511, 2000. © 2000 Wiley-Liss, Inc.
Multiple variant alleles of the human arylamine N-acetyltransferase genes, NAT1* and NAT2*, alter the capacity of individuals to metabolize arylamines by N-acetylation. Although biochemical and genetic studies have improved our understanding of the molecular basis of the acetylation polymorphism in humans and other mammals, regulation of NAT* gene expression is not understood. In the present study, a segment of the 5'-untranslated region of mouse Nat2* was sequenced and characterized. Primer extension analysis and RNase protection assays exposed multiple transcription initiation sites located 112 to 151 bases upstream of the translational start site. Computer sequence analysis revealed a promoter-like region located within the region 530 bases upstream of the translational start site consisting of TATA boxes, upstream promoter elements such as a CAAT box and Sp1 binding site, regulatory elements such as a palindromic hormone response element (HRE), and enhancer regions such as an AP-1 transcription factor binding site. Transient expression of CAT reporter constructs of the mouse Nat2*-palindromic HRE demonstrated positive regulation of the HSV-thymidine kinase 1 (tk1) promoter and induced the expression of chloramphenicol acetyltransferase (CAT). This induction was initiated by the addition of hormones such as 5alpha-dihydrotestosterone (DHT) or dexamethasone and was entirely dependent on the presence of androgen or glucocorticoid receptors, respectively. Together with recent discoveries regarding the effects of testosterone on the expression of Nat2* in mouse kidney during development, the findings reported in this article suggest that the HRE found in the promoter region of Nat2* is a potential candidate for the mediation of androgenic regulation of Nat2* in mouse kidney.
The reactions of p-nitroacetanilide and p-nitrophenyl acetate with aromatic and aliphatic amines catalyzed by a partially purified preparation of acetyl-coenzyme A:arylamine
acetyltransferase follow "ping-pong" kinetics. At a given concentration of p-nitroacetanilide the identical maximum velocities observed for five substituted anilines, semicarbazide, hydroxylamine, and
hydrazine, suggest that these reactions proceed through the rate-determining formation of a common acetyl-enzyme intermediate
which reacts with the acceptor amine in a fast step. The maximum velocities are directly proportional to the p-nitroacetanilide concentration. p-Nitrophenyl acetate is the most active acyl donor substrate known for the enzyme, with a maximum velocity approximately 140-fold
larger than that of acetyl-CoA. With weakly basic anilines the maximum velocity is different for different acyl acceptors,
suggesting that with these substrates the reaction of the acetyl-enzyme intermediate with the acceptor amine is rate determining
and that the saturation with increasing amine concentration represents binding of the acceptor amine to the acyl-enzyme. With
strongly basic anilines the maximum velocities and the Km for p-nitrophenyl acetate are identical, within experimental error, for all anilines; this is interpreted as evidence for a change
to rate-determining acylation of the enzyme with these more reactive nucleophiles. The increase in reactivity with increasing
aniline basicity suggests the development of a partial positive charge on the attacking nitrogen atom, which may be accompanied
by a relatively small degree of proton removal by a general base catalyst. It is suggested that the wide variation in the
sensitivity of enzyme-catalyzed acyl transfer reactions to the basicity of the nucleophile or leaving group reflects different
degrees of proton removal or addition by general acid-base catalysts in the transition state.
Acetylator phenotype is a common genetic trait in humans as well as other mammals. It results from the presence of several mutations in one of the genes encoding for arylamine N-acetyltransferase. The polymorphism has been associated with several disease states including colorectal cancer. Several epidemiological studies suggest that rapid acetylators are more susceptible to colorectal cancer than slow acetylators. Moreover, individuals that are both rapid acetylators and exhibit a high cytochrome P450 1A2 activity appear to have an even higher risk of colorectal cancer. These observations not only suggest an interesting genetic link to non-familial colon cancer but also suggest that carcinogens that are activated by N-acetyltransferase and cytochrome P450 1A2 may contribute to the etiology of this disease. Heterocyclic amines present in cooked food such as "well done" red meat are carcinogenic in experimental animals forming tumours in several target tissues including the small intestines. We have shown that human polymorphic N-acetyltransferase is present in human colon tissue and that it is capable of activating several heterocyclic amine carcinogens present in cooked food. These studies provide good circumstantial evidence that rapid acetylators may be predisposed to colorectal cancer.
N-acetylation participates in the biotransformation of hydrazine drugs and arylamine carcinogens to cytotoxic and carcinogenic products. Differences in acetylation capacity expressed in several mammalian species, including humans and mice, are associated with differences in toxicity and carcinogenicity from these chemicals. The present study examines the influence of genotype, age and sex on kidney N-acetyltransferase (NAT) activity in C57BL/6J (B6) and A/J inbred mouse strains using p-amino-benzoic acid (PABA) as a substrate. There were no strain differences in kidney PABA NAT activity. However, within these strains, males have greater kidney NAT activity than females. A 2-fold increase in kidney NAT activity of males was evident by 30 days postnatally and persisted into maturity (> 200 days after birth), whereas the kidney NAT activity of females remained unchanged. Castration reduced male kidney NAT to female levels, whereas testosterone replacement restored original levels of activity. Ovariectomized females exhibited the same enzyme activity as intact females. Testosterone increased kidney NAT activity in females, but not in intact males. Estradiol decreased kidney NAT in males, but had no effect on female NAT activity. The data suggest that the increase in kidney NAT activity in male mice that accompanies development is under androgenic control. This idea is further supported by our finding that the kidney NAT activity of androgen-insensitive tfm/y mice is significantly less than the activity of either females or males sharing the same genetic background. These observations may explain, in part, the higher susceptibility of male mice to 2-acetylaminofluorene mutagenicity and carcinogenicity.
Arylamine N-acetyltransferase-1 (NAT1) is a polymorphically expressed enzyme that is widely distributed throughout the body. In the present study, we provide evidence for substrate-dependent regulation of this enzyme. Human peripheral blood mononuclear cells cultured in medium supplemented with p-aminobenzoic acid (PABA; 6 microM) for 24 h showed a significant decrease (50-80%) in NAT1 activity. The loss of activity was concentration-dependent (EC(50) approximately 2 microM) and selective because PABA had no effect on the activity of constitutively expressed lactate dehydrogenase or aspartate aminotransferase. PABA also induced down-regulation of NAT1 activity in several human cell lines grown at confluence. Substrate-dependent down-regulation was not restricted to PABA. Addition of other NAT1 substrates, such as p-aminosalicylic acid, ethyl-p-aminobenzoate, or p-aminophenol to peripheral blood mononuclear cells in culture also resulted in significant (P <.05) decreases in NAT1 activity. However, addition of the NAT2-selective substrates sulfamethazine, dapsone, or procainamide did not alter NAT1 activity. Western blot analysis using a NAT1-specific antibody showed that the loss of NAT1 activity was associated with a parallel reduction in the amount of NAT1 protein (r(2) = 0.95). Arylamines that did not decrease NAT1 activity did not alter NAT1 protein levels. Semiquantitative reverse transcriptase polymerase chain reaction of mRNA isolated from treated and untreated cells revealed no effect of PABA on NAT1 mRNA levels. We conclude that NAT1 can be down-regulated by arylamines that are themselves NAT1 substrates. Because NAT1 is involved in the detoxification/activation of various drugs and carcinogens, substrate-dependent regulation may have important consequences with regard to drug toxicity and cancer risk.
The human arylamine N-acetyltransferases NAT1 and NAT2 catalyse the acetyl-CoA-dependent N- and O-acetylation of primary arylamine and hydrazine xenobiotics and their N-hydroxylated metabolites. We previously used a panel of recombinant NAT1/NAT2 chimaeric proteins to identify linear amino acid segments that have roles in imparting the distinct catalytic specificities to these proteins [Dupret, Goodfellow, Janezic and Grant (1994) J. Biol. Chem. 269, 26830-26835]. These studies indicated that a conserved central region (residues 112-210) distinct from that containing the active-site cysteine residue Cys(68) was important in determining NAT substrate selectivity. In the present study we have refined our analysis through further chimaera generation of this conserved region and by subsequent site-directed mutagenesis of individual amino acids. Enzyme-kinetic analysis of these mutant proteins with the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid (PAS) and sulphamethazine (SMZ) respectively suggests that residues 125, 127 and 129 are important determinants of NAT1-type and NAT2-type substrate selectivity. Modification of Arg(127) had the greatest effect on specificity for PAS, whereas changing Phe(125) had the greatest effect on specificity for SMZ. Selected NAT mutants exhibited K(m) values for acetyl-CoA that were comparable with those of the wild-type NATs, implying that the mutations affected acceptor substrate specificity rather than cofactor binding affinity. Taken together with previous observations, these results suggest that residues 125, 127 and 129 might contribute to the formation of the active-site pocket surrounding Cys(68) and function as important determinants of NAT substrate selectivity.
Enzymes of the arylamine N-acetyltransferase (NAT) family are found in species ranging from Escherichia coli to humans. In humans they are known to be responsible for the acetylation of a number of arylamine and hydrazine drugs, and they are strongly linked to the carcinogenic potentiation of certain foreign substances. In prokaryotes their substrate specificities may vary and members of the gene family have been linked to pathways including amide synthesis during rifamycin production. Here we report the crystal structure at 2.8 A resolution of a representative member of this family from Salmonella typhimurium in the presence and absence of a covalently bound product analog. The structure reveals surprising mechanistic information including the presence of a Cys-His-Asp catalytic triad. The fold can be described in terms of three domains of roughly equal length with the second and third domains linked by an interdomain helix. The first two domains, a helical bundle and a beta-barrel, make up the catalytic triad using a structural motif identical to that of the cysteine protease superfamily.
In humans, aromatic and heterocyclic amine carcinogens may be acetylated by the expression products of either of the N-acetyltransferase genes, NAT1 or NAT2. This conjugation reaction can result in either activation or detoxication of these carcinogens depending on the tissue involved. Recent studies suggest that polymorphisms in NAT1 or NAT2 may modulate cancer risk. To determine if genetic differences in NAT1 and NAT2 could alter risk of gastric cancer, we tested for the presence of polymorphic N-acetyltransferase alleles (both NAT1 and NAT2) in a preliminary study of 94 gastric adenocarcinoma patients and 112 control subjects from North Staffordshire, England. We used established PCR protocols to genotype for NAT2 and NAT1 alleles (NAT2*4, NAT2*5, NAT2*6, NAT2*7, NAT2*14; NAT1*3, NAT1* 4, NAT1*10, and NAT1*11), and implemented an oligonucleotide ligation assay (OLA) to test for low-activity NAT1 alleles [NAT1*14 (G560A), NAT1*15 (C559T), and NAT1*17 (C190T)]. No significant increased risk was observed for NAT2 acetylation genotypes. However, among all cases, we found that individuals inheriting a variant NAT1 allele, NAT1*10, have a significantly elevated risk for gastric cancer (OR = 2.2, 95% CI 1. 2-3.9, P < 0.01). Interestingly, the risk observed for NAT1*10 appears to be solely associated with advanced-stage tumors (OR = 4.8, P < 0.001), suggesting a possible role in progression to advanced disease. This preliminary finding needs confirmation in a larger, detailed epidemiological study.
Heterocyclic amines are mammary carcinogens in rats and their N-hydroxy metabolites are substrates for subsequent metabolic activation by N-acetyltransferases (NAT) and sulfotransferases (SULT) in man. We investigated the expression of these enzymes in human breast tissue and the relationship between NAT genotype and NAT mRNA expression or enzyme activity. Immunohistochemical staining of sections of breast tissue identified expression of NAT1 and NAT2 protein in human mammary epithelial cells, but not in the stroma. We also measured the formation of DNA adducts of the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in calf thymus DNA after incubation of their promutagenic N-hydroxy metabolites with mammary cytosols prepared from reduction mammoplasty tissue. Experimental observations gained from use of enzyme cofactors and NAT and/or SULT inhibitors on cytosolic enzyme activity, recombinant NAT1 activity and heterocyclic amine-DNA adduct formation suggest that both NAT1 and SULT1A enzymes contribute significantly to the activation of N-hydroxylated heterocyclic amines in mammary tissue. NAT1 mRNA transcript levels were found to be two- to three-fold higher than mRNA transcripts of the NAT2 gene in reduction mammoplasty tissue and mammary epithelial cells. NAT1-specific p-aminobenzoic acid acetylation activity, but not NAT2-specific sulfamethazine acetylation activity, was detectable in mammary cytosols. There was no association apparent between NAT genotype and the levels of NAT mRNA or NAT enzyme activity, or between NAT1 genotype and IQ-DNA adduct formation mediated by mammary cytosols. Western blot analysis of mammary cytosolic protein showed detectable levels of SULT1A1 and SULT1A3.
Our aim was to examine the role of NAT1 and NAT2 polymorphisms in human larynx cancer susceptibility. Genotype tests for NAT1 alleles *4, *10 and *11, and NAT2 alleles *4, *5, *6A and *7A, using PCR-RFLP analysis, were performed in 172 healthy Portuguese individuals and 88 patients with squamous cell carcinoma of the larynx. NAT1 and NAT2 genotype frequencies were correlated between patients and control groups, using the chi-square test. Odds ratios and 95% confidence intervals were calculated from 2 x 2 tables with the Fisher's exact model. The statistical analysis of NAT1 and NAT2 genotype frequencies revealed a significant difference of NAT1*10/*11 (p = 0.038) and NAT2*5/*7 (p = 0.003) genotype distribution between cases and controls. We also observed differences concerning tumor location, since NAT1*10/*11 genotype frequency was significantly different when comparing normal control individuals with the glottic subgroup of patients. The present data suggest that NAT1 and NAT2 polymorphisms may be correlated with an increased risk of larynx cancer.
Aromatic and heterocyclic amines require metabolic activation to electrophilic intermediates that initiate carcinogenesis. N-Acetyltransferase 1 (NAT1) and 2 (NAT2) are important enzymes in the biotransformation of these carcinogens and exhibit genetic polymorphism. Human NAT1 and NAT2 alleles are listed at: http://www.louisville.edu/medschool/pharmacology/NAT.html by an international gene nomenclature committee. The high frequency of the NAT1 and NAT2 acetylation polymorphisms in human populations together with ubiquitous exposure to aromatic and heterocyclic amines suggest that NAT1 and NAT2 acetylator genotypes are important modifiers of human cancer susceptibility. For cancers in which N-acetylation is a detoxification step such as aromatic amine-related urinary bladder cancer, NAT2 slow acetylator phenotype is at higher risk. Multiple studies have shown that the urinary bladder cancer risk is particularly high in the slowest NAT2 acetylator phenotype or genotype (NAT2(*)5). In contrast, for cancers in which N-acetylation is negligible and O-acetylation is an activation step such as for heterocyclic amine-related colon cancer, NAT2 rapid acetylator phenotype is at higher risk. Although studies have found associations between NAT1 genotype and various cancers, the findings are less consistent and are not well understood. Since cancer risk requires exposure to aromatic and/or heterocyclic amine carcinogens modified by NAT1 and/or NAT2 acetylator genotype, the results from human epidemiology studies are dependent upon the quality and accuracy of the exposure assessment and genotype determination. Conclusions require understanding the relationship between genotype and phenotype, as well as the role of genetic variation in carcinogen metabolism, DNA repair, and host susceptibility. Investigations have been carried out in rapid and slow acetylator rodent models in which both exposure and genetic variability are tightly controlled. Human NAT1 and NAT2 alleles have been characterized by recombinant expression to further understand the effects of nucleotide polymorphisms on function and phenotype.
N-acetyltransferase-1 (NAT1) and N-acetyltransferase-2 (NAT2) are important in the metabolism of aromatic and heterocyclic amine carcinogens that induce prostate tumors in the rat. We investigated the association of genetic polymorphisms in NAT1 and NAT2, alone and in combination, with human prostate cancer. Incident prostate cancer cases and controls in a hospital-based case-control study were frequency-matched for age, race, and referral pattern. The frequency of slow acetylator NAT1 genotypes (NAT1*14, *15, *17) was 5.8% in controls but absent in cases. In contrast, in comparison with all other NAT1 genotypes the putative rapid acetylator NAT1 genotype (NAT1*10) was significantly higher in prostate cancer cases than controls (OR, 2.17; 95% CI, 1.08-4.33; P = 0.03). Combinations of NAT1*10 with NAT2 slow acetylator genotypes (OR, 5.08; 95% CI, 1.56-16.5; P = 0.008) or with NAT2 very slow (homozygous NAT2*5) acetylator genotypes (OR, 7.50; 95% CI, 1.55-15.4; P = 0.016) further increased prostate cancer risk. The results of this small pilot study suggest increased susceptibility to prostate cancer for subjects with combinations of NAT1*10 and slow (particularly very slow) NAT2 acetylator genotypes. This finding should be investigated further in larger cohorts and in other ethnic populations.
Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.
Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives ( approximately 4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype.
Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion. In addition, substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.
Some carcinogens that initiate rat mammary cancer are substrates of human N-acetyltransferase 1 (NAT1) and variation in NAT1 activity due to environmental or genetic causes may influence human susceptibility to breast cancer. One unexplored potential cause of NAT1 expression variation is polymorphism of transcriptional control sequences. However, the location of the major NAT1 transcription control site is uncertain because earlier publications and current databases report different cDNA structures. To resolve this discrepancy, we used CAP-dependent cDNA cloning to identify 5' ends of NAT1 mRNAs from breast and MCF-7, a mammary adenocarcinoma cell line. Most transcription initiates in a 49-bp region located 11.8 kb upstream of the coding exon. A 79-bp exon located 2.5 kb upstream of the coding exon was found in all 41 of the independent NAT1 cDNA products. Seven of these 41 cDNAs also included other non-coding exons. The structures of NAT1 cDNAs in public databases, as obtained from diverse tissues, reflect a transcription pattern similar to that demonstrated in breast and MCF-7. Genomic fragments spanning the major start region were cloned into a luciferase vector and expressed in MCF-7. Promoter activities were 190-490-fold higher than the vector control and 30-80-fold higher than for a fragment immediately upstream of the coding exon. Our results demonstrate that, in breast, and likely also in other tissues, the major NAT1 mRNA is transcribed from a strong promoter located 11.8 kb upstream of the translated exon, and the mature spliced mRNA includes at least one additional non-coding exon.
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.
The risk of having a child with a neural tube defect (NTD) can be reduced by maternal, periconceptional supplementation with folic acid, but the underlying folate-dependent protective mechanism remains unclear. N-acetyltransferase 1 is involved in acetylation of aromatic and heterocyclic amines and the catabolism of folates. Hence, functional polymorphisms in NAT1, the gene encoding N-acetyltransferase 1, are plausible risk factors for NTDs. Such variants could exert an influence on the risk of NTDs via their role in acetylation or folate catabolism and could act through the maternal or the embryonic genotype.
NAT1 C1095A genotypes and information on maternal, periconceptional multivitamin use and smoking were obtained as part of a family-based study of spina bifida. Associations between spina bifida and the embryonic and maternal NAT1 C1095A genotypes, and potential NAT1 C1095A genotype-exposure interactions were evaluated using log-linear modeling.
The analyses provided no evidence that the embryonic or maternal NAT1 C1095 genotypes influence the risk of spina bifida independently, or through interactions with maternal use of multivitamins. There was evidence that the embryonic, and possibly the maternal, NAT1 C1095A genotype influence the risk of spina bifida via interactions with maternal smoking status.
The genotype for the NAT1 C1095A polymorphism does not appear to be an independent risk factor for spina bifida. However, the results of these analyses provide preliminary evidence that this polymorphism may be associated with the risk of spina bifida in the offspring of women who smoke during early pregnancy.
Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k(cat)/K(AcCoA) exhibited two inflection points at 5.32 +/- 0.13 and 8.48 +/- 0.24, respectively. The pK(a) at 5.32 is virtually identical with the previously reported pK(a) of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Brønsted plot constructed by the correlation of log k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(nuc) = 0.80 +/- 0.1. However, a further increase of the pK(a) from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k(4) value. Analysis of the pH-k(cat)/K(PABA) profile revealed a pK(a) of 5.52 +/- 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 +/- 0.04 on k(cat)/K(PABA). Normal solvent isotope effects of 4.8 +/- 0.1, 3.1 +/- 0.1, and 3.2 +/- 0.1 on the k(cat)/K(b) for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK(a) values <or=5.5 to deprotonation of a tetrahedral intermediate for arylamines with pK(a) values >or=5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catalytic activity. In contrast, an increase in pH-dependent hydrolysis of the acetylated enzyme was not observed over a pH range of 5.2-7.5. On the basis of these observations, a catalytic mechanism for the acetylation of arylamines by NAT2 is proposed.
N-Acetyltransferases 1 and 2 (NAT1 and NAT2), both being highly polymorphic, are involved in the metabolism of aromatic and heterocyclic aromatic amines present in cigarette smoke and red meat cooked by high-temperature cooking techniques. We investigated the effect of differences in acetylation capacity, determined by NAT1 and NAT2 genotypes, on colorectal cancer risk associated with exposure to tobacco smoke or red meat consumption. In this population-based case-control study in Germany, 505 patients with incident colorectal cancer and 604 age- and sex-matched control individuals with genotyping data and detailed risk factor information were included. Genotyping of NAT1 and NAT2 genetic polymorphisms was done using a fluorescence-based melting curve analysis method. The association between genotypes, environmental exposures, and colorectal cancer risk was estimated using multivariate logistic regression. Colorectal cancer risk associated with active smoking was elevated after accumulation of 30(+) pack-years of smoking [odds ratio (OR), 1.4; 95% confidence interval (95% CI), 0.9-2.2] but not significantly modified by either NAT1 or NAT2 genotype. Exposure to environmental tobacco smoke was associated with an increased risk for colorectal cancer only among NAT2 fast acetylators (OR, 2.6; 95% CI, 1.1-5.9 for exposure in childhood and adulthood). Frequent consumption of red meat significantly increased colorectal cancer risk for the group comprising all NAT2 fast acetylators or carriers of the NAT1*10 allele (OR, 2.6; 95% CI, 1.1-6.1) but not among those with "slow" NAT1 and NAT2 genotypes. Our findings indicate that NAT1 and NAT2 genotypes may contribute jointly to individual susceptibility and that heterocyclic aromatic amines may play an important role in colorectal cancer associated with red meat and possibly also exposure to environmental tobacco smoke.
Periconceptional folic acid supplementation reduces the risk of having a child with spina bifida. N-acetyltransferase 1 (NAT1) participates in the catabolism of folates and the acetylation of aromatic and heterocyclic amines. Hence, functional polymorphisms in NAT1, the gene encoding NAT1, could influence the risk of spina bifida via either folate catabolism or acetylation of exogenous agents. Individuals with spina bifida and their parents were genotyped for six NAT1 single nucleotide polymorphisms (SNPs) for which the less common allele is associated with reduced or absent enzyme activity (i.e. 97C>T, 190C>T, 559C>T/560G>A, 640T>G and 752A>T). In addition, a "composite" NAT1 genotype was defined as a function of the genotyped SNPs. Descriptive analyses of the SNPs and of the composite genotype indicated that heterozygous parents were more likely to transmit the common allele than the rare allele to their affected offspring. Furthermore, matings of mothers homozygous for the common allele and heterozygous fathers were more common than the reciprocal matings. Log-linear analyses confirmed that both the maternal (P = 0.008) and offspring (P = 0.003) composite NAT1 genotypes were significantly related to the risk of spina bifida. NAT1 variants that reduce or abolish enzyme activity appear to protect against spina bifida, and to exert their influence via both the maternal and the offspring genotypes. These associations may be attributable to a decrease in either folate catabolism or the conversion of exogenous agents to teratogenic derivatives in women and/or developing embryos with a NAT1 genotype that includes a loss of function allele relative to those who do not.
N-Acetyltransferases (NATs) and sulfotransferases (SULTs) are key phase II metabolizing enzymes that can be involved both in the detoxification and in the activation of many human promutagens and procarcinogens.
We investigated the expression of NATs and SULTs in human prostate and tested their role in the activation the N-hydroxy (N-OH) metabolite of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a dietary carcinogen, to form DNA adducts. Western blotting showed detectable levels of NAT1, SULT1A1 and SULT1A3 with marked inter-individual variation. NAT2 and other SULT enzymes were not detectable. NAT1 was localized by immunohistochemistry to the cytoplasm of epithelial cells. The presence of acetyl Co-enzyme A (acetyl CoA) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS), NAT and SULT cofactors, respectively, significantly increased the level of DNA adducts, detected by P-postlabelling analysis, in calf thymus DNA incubated with N-OH-IQ and prostate cytosolic fractions. The enhancement in the level of DNA adducts in the presence of PAPS correlated with the level of SULT1A1 protein. A single prostate cytosol with the SULT1A1*2/*2 genotype produced less DNA adducts than cytosols with the *1/*2 and *1/*1 genotypes. No significant correlation was observed between NAT1 protein level and the formation of DNA adducts, even in the presence of acetyl CoA.
In conclusion, we demonstrated that NAT1, SULT1A1 and SULT1A3 are present in human prostate and that both enzyme classes significantly contribute to the activation of N-hydroxylated heterocyclic amines to DNA-damaging species in this tissue. Variation in expression levels, in combination with dietary and/or environmental exposure to carcinogens, could be influential in determining individual susceptibility to prostate cancer.
Variable expression of human arylamine N-acetyltransferase 1 (NAT1) due to genetic polymorphism, gene regulation or environmental influences is associated with individual susceptibility to various cancers. Recent studies of NAT1 transcription showed that most mRNAs originate at a promoter, P1, located 11.8 kb upstream of the single open reading frame (ORF) exon. We have now characterized an alternative NAT1 promoter lying 51.5 kb upstream of the NAT1 ORF. In the present study, analysis of human RNAs representing 27 tissue types by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR showed the upstream 51.5 kb promoter, designated P3, to be most active in specific tissues, including kidney, liver, lung, and trachea. All NAT1 P3 mRNAs included 5'-untranslated region (5'-UTR) internal exons of 61 and 175 nucleotides in addition to the 79 nucleotide 5'-UTR exon present in P1 mRNA. CAP-dependent amplification of 5'-P3 mRNA termini defined an 84 bp transcription start region in which most start sites are centrally clustered. The hepatoma-derived HepG2 cell line expressed a high level of P3 mRNA with the same spliced structure and start site pattern as found in normal tissues. A 435-bp minimal promoter was defined by transfection of HepG2 with luciferase expression constructs containing genomic fragments from the P3 start region. These findings imply a fundamental role for P3 in NAT1 regulation and define additional regions for genetic polymorphisms associated with enhanced cancer risk.
Animal studies suggest that lymphomagenesis can be induced by exposure to carcinogenic aromatic and heterocyclic amines found in diet, cigarette smoke and the environment, but human epidemiologic investigations of these exogenous exposures have yielded conflicting results. As part of our evaluation of the role of aromatic and heterocyclic amines, which are metabolized by N-acetyltransferase (NAT) enzymes, in the etiology of non-Hodgkin lymphoma (NHL), we examined NHL risk in relation to genetic variation in NAT1 and NAT2 and exposure to cigarette smoke and dietary heterocyclic amines and mutagens. We genotyped 10 common single nucleotide polymorphisms (SNPs) in NAT1 and NAT2 among 1136 cases and 922 controls from a population-based case-control study in four geographical areas of the USA. Relative risk of NHL for NAT1 and NAT2 genotypes, NAT2 acetylation phenotype, and exposure to cigarette smoke and dietary heterocyclic amines and mutagens was estimated using odds ratios (ORs) and 95% confidence intervals (CIs) derived from unconditional logistic regression models. We observed increased risk of NHL among individuals with the NAT1*10/*10 genotype compared with individuals with other NAT1 genotypes (OR = 1.60, 95% CI = 1.04-2.46, P = 0.03). We also observed increased NHL risk in a dose-dependent model among NAT2 intermediate- and rapid-acetylators compared with slow-acetylators, although only the trend was statistically significant (intermediate: OR = 1.18, 95% CI = 0.97-1.44, P = 0.1; rapid: OR = 1.43, 95% CI = 0.97-2.14, P = 0.07; P for linear trend = 0.03). Compared with non-smokers, NHL risk estimates for current cigarette smoking were increased only among NAT2 intermediate/rapid-acetylators (OR = 2.44, 95% CI = 1.15-5.20, P = 0.02). Our data provide evidence that NAT1 and NAT2 genotypes are associated with NHL risk and support a contributory role for carcinogenic aromatic and/or heterocyclic amines in the multi-factorial etiology of NHL.
Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation. To identify the properties that lead to the reduced cellular activity of certain NAT variants, we introduced one such polymorphism into the human NAT1 ortholog hamster NAT2. The polymorphism chosen was human NAT1*17, which results in the replacement of R64 with a tryptophan residue, and we demonstrate this substitution to cause hamster NAT2 to be constitutively ubiquitylated. Biophysical characterization of the hamster NAT2 R64W variant revealed that its overall protein structure and thermostability are not compromised. In addition, we used steady-state kinetics experiments to demonstrate that the R64W mutation does not interfere with NAT catalysis in vitro. Hence, the constitutive ubiquitylation of this variant is not caused by its inability to be acetylated. Instead, we demonstrate this mutation to cause the hamster NAT2 protein to aggregate in vitro and in vivo. Importantly, we tested and confirmed that the R64W mutation also causes human NAT1 to aggregate in cultured cells. By using homology modeling, we demonstrate that R64 is located at a peripheral location, which provides an explanation for how the NAT protein structure is not significantly disturbed by its mutation to tryptophan. Altogether, we provide fundamental information on why humans harboring certain NAT variants exhibit reduced acetylation capabilities.
Arylamine N-acetyltransferases (NATs) catalyze the acetylation of arylamines, a key step in the detoxification of many carcinogens. The determinants of NAT substrate specificity are not known, yet this knowledge is required to understand why NAT enzymes acetylate some arylamines, but not others. Here, we use NMR spectroscopy and homology modeling to reveal the structural determinants of arylamine acetylation by NATs. In particular, by using chemical shift perturbation analysis, we have identified residues that play a critical role in substrate binding and catalysis. This study reveals why human NAT1 acetylates the sunscreen additive p-aminobenzoic acid and tobacco smoke carcinogen 4-aminobiphenyl, but not o-toluidine and other arylamines linked to bladder cancer. Our results represent an important step toward predicting whether arylamines present in new products can be detoxified by mammalian NATs.
Polymorphisms of cytochrome P4501A2 and N-acetyltransferase genes, smoking, and risk of pancreatic cancerEffectofNAT1andNAT2geneticpolymorphisms on colorectal cancer risk associated with exposure to tobacco smoke and meat consumption
- Mol
- Teratol
- D Li
- L Jiao
- Y Li
- M A Doll
- D W Hein
- M L Bondy
Mol. Teratol., 73, 512–516. Li, D., Jiao, L., Li, Y., Doll, M. A., Hein, D. W., Bondy, M. L., et al. (2006). Polymorphisms of cytochrome P4501A2 and N-acetyltransferase genes, smoking, and risk of pancreatic cancer. Carcinogenesis, 27, 103–111. Lilla,C.,Verla-Tebit,E.,Risch,A.,Jager,B.,Hoffmeister,M.,Brenner, H.,etal.(2006).EffectofNAT1andNAT2geneticpolymorphisms on colorectal cancer risk associated with exposure to tobacco smoke and meat consumption. Cancer Epidemiol. Biomarkers Prev., 15, 99–107. Liu, F., Zhang, N., Zhou, X., Hanna, P. E., Wagner, C. R., Koepp, D
Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: Evidence for the conservation of a cysteine protease catalytic domain and an active-site loop
- Rodrigues-Lima