Arylamine N-Acetyltransferases in Health and Disease

The following sections are included:

• Contents
• Contributors
• Foreword
• Preface
• Acknowledgements
• In Memoriam

Arylamine N-acetyltransferases (NAT, EC 2.3.1.5) were amongst the first enzymes known to result in a genetically determined response to a drug, in this case isoniazid. We now know there are two functional NAT genes in humans and these encode (HUMAN)NAT1 and (HUMAN)NAT2 enzymes. The NAT genes are each encoded at polymorphic loci. This chapter explores the early studies on phenotypic variation in NAT activity and discusses how genotyping of (HUMAN)NAT1 and (HUMAN)NAT2 has changed since NAT pharmacogenetics were first described 50 years ago. The relationship between clinical and molecular studies on genes and proteins has provided current understanding of the NAT enzymes and their genes in humans, where evidence indicates (HUMAN)NAT1 has an endogenous role in addition to a drug metabolising role. NATs in other animal species, bacteria and fungi, are also introduced and polymorphisms in other species are described. Enzymic and later structural studies established that the acetylation reaction involved the transfer of an acetyl group to a cysteine sulphydryl group, activated as part of a juxtaposed catalytic traid with aspartate and histidine in all NAT enzymes with only one exception in bacteria to date. The body of research on NATs has laid the foundation for an exciting period to come encompassing whole genome analyses and understanding epigenetic control to allow exploitation of NAT biology in order to understand and perhaps treat disease.

The human arylamine N-acetyltransferase type 2 ((HUMAN)NAT2) gene encodes for a xenobiotic metabolising enzyme. NAT2 polymorphisms, which modify acetylation rates, exhibit inter-individual and ethnic variability for drugs and xenobiotics metabolism and act as one of the important factors in explaining variability in cancer incidence and drug toxicity. In addition, a new unbiased and untargeted genome-wide association study (GWAS) has identified (HUMAN)NAT2 as the top signal for insulin resistance (IR), a cardinal feature of diabetes and a major cardiovascular risk factor. Other GWAS studies have also associated (HUMAN) NAT2 polymorphisms with plasma triglyceride levels, detoxification capacity, and urine metabotypes such as the ratio between formate and succinate concentrations. Here we review the links between (HUMAN) NAT2 and cardiometabolic traits including IR, glucose homeostasis and mitochondrial biology. Based on these findings, (HUMAN)NAT2 activation may be a target to improve the function of the metabolic network related to mitochondrial substrate oxidation and biogenesis and insulin responsiveness.

Polymorphic acetylation of drug and xenobiotics is a known cause of interindividual variability in drug pharmacokinetics and in the risk of developing adverse drug events. Acetylation ability is strongly linked to variations in the arylamine N-acetyltransferase 2 (NAT2) gene, and several procedures have been developed to predict acetylation phenotypes based on genetic tests. This chapter analyses the most salient issues inherent to NAT2 genotyping, haplotype reconstruction and phenotype inference, as well as the clinical implications of the polymorphic acetylation of drugs such as aminoglutethimide, amonafide, caffeine, clonazepam, dapsone, hydralazine, isoniazid, metamizole, nitrazepam, phenelzine, phenytoin, procainamide, sulfamethazine, sulfamethoxazole and sulfasalazine. These drugs and/or their metabolites are polymorphically acetylated, and for many of these drugs, acetylation status (either phenotype or genotype) causes pharmacokinetic changes and often adverse drug events. We identify potential areas needing additional research before NAT2 genetic testing can be used for the development of clinical practice pharmacogenomics guidelines.

Human arylamine N-acetyltransferase 1 (NAT1) is one of the two functional NATs in humans that acetylate primary arylamine and hydrazine xenobiotics. There is a growing body of evidence that suggests NAT1 may have an important physiological role in the cell in addition to its seemingly secondary role in xenobiotic metabolism. Specifically, roles in folate homeostasis, epigenetic regulation of gene expression, cell growth and survival, and cancer cell invasion and metastasis have been proposed. This chapter discusses the current evidence linking NAT1 to physiological processes and cancer cell biology, as well as its potential as a diagnostic/prognostic biomarker and therapeutic target.

During embryogenesis (HUMAN)NAT1 is expressed in oocytes, all foetal tissues and placenta. Wild-type mouse embryos express (MOUSE) Nat2 (the orthologue of (HUMAN)NAT1, which encodes a functionally similar protein) in the developing neural tube, heart and hindbrain. In reporter mice, (MOUSE)Nat2 is transcribed in the developing eye, neuroendocrine system, heart, intestine, kidney, limb buds and vibrissae. (MOUSE)Nat2 deletion has limited developmental consequences; however, attempts to generate mice overexpressing (HUMAN)NAT1 have failed. The role of (HUMAN)NAT1 in birth defects, whose multifactorial nature and rarity necessitate enormous epidemiological studies, remains ambiguous. Either low or high activity may be detrimental: high activity may lower intracellular folate, possibly exacerbating a pre-existing low folate phenotype, while moderately reduced activity may improve cellular folate profiles. Thus, (HUMAN)NAT1 may participate in an integrated network of proteins which maintains cellular folate homeostasis. Its tight regulation is therefore not surprising, nor is the difficulty of overexpressing it in transgenic mice.

Of the three human arylamine N-acetyltransferase (NAT) genes, (HUMAN)NAT1 and (HUMAN)NAT2 code for functional enzymes, namely (HUMAN)NAT1 and (HUMAN)NAT2. (HUMAN)NAT1 is expressed early during development and is ubiquitously expressed during adulthood, whereas (HUMAN)NAT2 expression and enzyme activity is primarily restricted to adult liver and intestine. A similar temporal and spatial distribution of the corresponding orthologues has been found in rodent models regularly used to investigate their expression and endogenous function, and to understand their role in the metabolism of aromatic amines (AAs). While NAT1 is considered to have an additional endogenous role, NATs are defined as xenobiotic-phase II conjugating enzymes, which N- or O-acetylate AAs, heterocyclic aromatic amines (HAAs) and their N-oxidised metabolites using acetyl-CoA as a co-substrate. The substrates are mainly environmental chemicals, including carcinogens. While both human NATs deactivate their substrates, especially AAs, through N-acetylation, (HUMAN)NAT2 appears to be extremely important also in the activation of carcinogenic compounds. Their presence and activity in the organs involved in uptake of arylamines (skin, respiratory tract, gastro-intestinal tract), influences AA and HAA loads and their fates, thereby allowing their distribution throughout the body (blood), metabolism (liver), excretion (bladder, intestine), but also provoking tumour formation in specific organs, particularly in the case of carcinogenic AAs and HAAs.

Since their discovery in the early 1990s, the human arylamine N-acetyltransferase (NAT) genes have been the subject of a tremendous number of molecular anthropological studies describing their nucleotide diversity in a wide range of populations worldwide. While (HUMAN) NAT2 presents a high number of nucleotide substitutions, with seven of them reaching polymorphic frequencies in almost all human populations, and a high level of non-synonymous changes relative to synonymous, (HUMAN)NAT1 is much less diverse, particularly in its coding region. A pseudogene, NATP, the third member of this small gene family, harbours a diversity similar to NAT2. In accordance with that, selective neutrality tests suggest that (HUMAN)NAT1 and (HUMAN)NAT2 evolve under distinct selective regimes. An evolution of (HUMAN)NAT2 under positive population-specific pressures is proposed to be probably linked to the mode of subsistence and/or the chemical environment populations live in, as reflected by climatic zones and biomes; in contrast, for (HUMAN)NAT1, functional constraints determining the strength of purifying selection are generally invoked.

The genomics and phylogeny of N-Acetyltransferase (NAT) genes are described in animals, particularly primates. Surveys have been seeking NAT homologues across the entire spectrum of sequenced animal genomes. NAT genes are not frequently found in less complex animals, but a more universal distribution is evident in vertebrates, including fishes, amphibians, reptiles, birds and mammals. Laboratory birds, such as the pigeon and chicken, were used in early NAT research. Polymorphic strains of rabbit and rodents have also served as models for NAT, and these include various ‘rapid’ and ‘slow’ acetylator inbred, wild-derived inbred, congenic and transgenic lines. The first NAT gene characterised in non-human primates was NAT2 of the rhesus macaque, which bears a polymorphism with unusual effects on enzyme function. NATs appear to be non-essential for survival, as they are missing in dogs: the NATs nevertheless play important roles as the interface between organisms and their chemical environment. Thus, they constitute very good models for evolutionary studies.

Genetically modified mouse models serve as useful in vivo tools to confirm or refute the involvement of specific drug-metabolizing enzymes, including the arylamine N-acetyltransferases (NATs), in drug or xenobiotic metabolism, toxicity and carcinogenesis. Several NAT mouse models have been generated, including congenic lines of naturally polymorphic rapid and slow acetylator strains, targeted knockout mouse models and ‘humanised’ transgenic mice, to interrogate the involvement of NAT enzymes in these processes and during normal physiology and development. While no overt phenotypes have been observed in NAT-knockout mouse strains, several lines of evidence arising from studies conducted using murine and human NAT1/2 transgenic mice have alluded to the existence of potential endogenous roles that are distinct from ‘classical’ metabolism of foreign compounds, and may involve the integration or intersection of different cellular metabolic pathways that affect general health and disease.

Microorganisms can survive highly toxic environments through numerous xenobiotic metabolizing enzymes, including arylamine N-acetyltransferases (NATs). NAT genes are present in bacteria, archaea, protists and fungi. In lower taxa of fungi, NAT genes are found in chytridiomycetes. In Dikarya, NAT genes are present in both basidiomycetes and ascomycetes. Soil fungi are particularly robust against highly toxic phenylamide agrochemicals converted into recalcitrant chloroanilines, which are established NAT substrates. Hence, NATs are currently being investigated relative to the ability of fungi to remediate those compounds. Ascomycetes with NATs appear to possess at least one archetypal homologue with preference for acetyl-CoA and propionyl-CoA. Other NAT homologues have diverged to serve specific needs of different fungi. For example, the malonyl-CoA utilizing NATs of Fusarium species pathogenic on maize and wheat are crucial for defence against host antimicrobials. The comparative investigation of fungal NATs is emerging as a promising research area relevant to agriculture and bioremediation.

This chapter introduces structural and functional features of bacterial arylamine N-acetyltransferases (NATs). It starts by presenting early studies on identification of NAT enzymes and their substrates in Salmonella typhimurium and other eubacteria. It then describes structural and mechanistic features of these enzymes and their comparison with eukaryotic isoforms. Three-dimensional structures, interactions with substrates and catalytic mechanisms are also presented. The third part of the chapter focuses on functional properties of the bacterial NATs and possible consequences in terms of drug resistance. The possible uses of these enzymes as drug targets or remediation tools are also discussed.

Arylamine N-acetyltransferase (NAT) was first identified in humans for its role in metabolising the anti-tubercular drug isoniazid. Later functional NAT proteins were identified in bacteria including Mycobacterium tuberculosis where they were found to N-acetylate a range of arylamines, hydrazines and hydralazines both in vitro and in vivo. From gene deletion and genomic studies in mycobacteria, it became clear that NAT in Mycobacterium bovis Bacillus Calmette–Guérin (BCG) had an endogenous role in cell-wall metabolism, cholesterol degradation and the nat gene was part of an essential gene cluster for intra-cellular survival of the pathogen M. tuberculosis; this has paved the way for validation of NAT and other proteins encoded in the gene cluster as novel therapeutic targets for anti-tubercular drug design and discovery. This chapter reviews the genomic organisation of the nat gene and the function of the NAT protein in mycobacteria, including the location of the nat gene in the genome. The role of nat and its neighbouring genes, their endogenous substrates and the regulation of their expression in fast-growing environmental and slow-growing pathogenic intra-cellular mycobacteria is also covered.

Human epidemiological studies associating chemical exposures to cancer risk often are inconsistently validated across studies. Examples include the effect of smoking on cancer aetiology on cancers other than the lung, such as urinary bladder and breast cancer. Research findings from the laboratory have improved the understanding of arylamine carcinogen metabolism leading to improved design and interpretation of human molecular epidemiology investigations. Laboratory studies that infer and test biological plausibility, including cancer risks modified by differential metabolism of arylamine carcinogens in rapid and slow arylamine N-acetyltransferase (NAT2) acetylators, have been critical for investigating the role of smoking in the aetiology of human cancers. This chapter illustrates these concepts with an example of a cancer in which the role of smoking has largely been validated (urinary bladder cancer) and an example where a consensus has not been reached (breast cancer).

Over recent years, our understanding of the pharmacological roles played by (HUMAN)NAT1 has increased considerably. In this chapter, we evaluate the evidence that (HUMAN)NAT1 has a role in breast cancers, in particular oestrogen-receptor-positive (ER+) tumours. We also examine how a new generation of NAT1-specific inhibitors are being used as chemical tools to evaluate the roles of (HUMAN)NAT1. Colorimetric and fluorescent analogues of a naphthoquinone inhibitor, identified by high-throughput screening, have proved particularly useful, due to their ability to detect the presence of low levels of (HUMAN)NAT1 within tumour cell samples. We also speculate on the possibilities for future research in this field, including an evaluation of in silico studies as an approach to the development of new classes of NAT1-specific inhibitors having improved physicochemical properties and an analysis of the evidence suggesting a possible link between (HUMAN)NAT1 expression levels and folate metabolism in cells.

An N-acetyltransferase (NAT) enzyme has been identified in many mycobacterial species. It has been suggested that the enzyme plays a role in cholesterol degradation and maintaining the homeostasis of acetyl-Coenzyme A, a central metabolite in lipid metabolism. The nat genes from Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette–Guerin (BCG) are identical and are encoded in highly similar operons in both organisms (the nat operon). The other genes of the operon are involved in catabolism of the sterol rings of cholesterol. The gene cluster is essential for the survival of M. bovis BCG within macrophages, where cholesterol is likely to be the fuel for intracellular survival. Strains missing these genes are unable to grow on minimal medium with cholesterol, although they retain the ability to grow on rich media.

NAT inhibitors that are selectively toxic to mycobacteria, therefore, would remove any potential human toxicity caused by inhibition of the human NAT enzymes. The search for novel drugs that can shorten the treatment course for tuberculosis (TB) has become pressing in the light of the shortcomings of the current therapy and the emergence of extensively drug-resistant (XDR) strains. The current anti-tubercular pipeline does not include compounds specifically targeting cholesterol catabolism in mycobacteria or products of the gene cluster encoding NAT. Therefore, the development of novel inhibitors targeting these enzymes would provide new therapeutic options for the treatment of latent and XDR TB.

The history and current status of consensus arylamine N-acetyltransferase (NAT) gene nomenclature is outlined, describing the contributions of the NAT committee over the years. The NAT nomenclature website, hosted by the Democritus University of Thrace, Department of Molecular Biology and Genetics (http://nat.mbg.duth.gr/), is presented. This website consolidates annotated information about prokaryotic and eukaryotic NAT genes, as well as a complete listing of NAT polymorphic alleles in human and other mammalian model species. Detailed instructions about the correct species-specific naming of NAT genes and alleles are also provided on the NAT website. Scientists who wish to name new NAT sequences should follow those guidelines and contact the committee who will approve the official symbols of new NAT genes or alleles. New developments towards a standardised nomenclature across the entire spectrum of pharmacogenetics-associated genes, including human NAT1 and NAT2, will be discussed in view of recent advancements in genomic medicine.

The following section is included:

• Index