N-acetylation Of P-aminobenzoic Acid Research Articles

N-Acetylation of p-Aminobenzoic Acid and p-Phenylenediamine in Primary Porcine Urinary Bladder Epithelial Cells and in the Human Urothelial Cell Line 5637

N-Acetyltransferases (NAT) are important enzymes in the metabolism of certain carcinogenic arylamines, as N-acetylation decreases or prevents their bioactivation via N-hydroxylation. To study such processes in the bladder, cell culture models may be used, but metabolic competence needs to be characterized. This study focused on the N-acetylation capacity of two urothelial cell systems, using p-aminobenzoic acid (PABA) and the hair dye precursor p-phenylenediamine (PPD), two well-known substrates of the enzyme NAT1. The constitutive NAT1 activity was investigated using primary cultures of porcine urinary bladder epithelial cells (PUBEC) and in the human urothelial cell line 5637 to assess their suitability for further in vitro studies on PABA and PPD-induced toxicity. N-Acetylation of PABA and PPD was determined by high-performance liquid chromatography (HPLC) analysis in cytosols of the two cell systems upon incubation with various substrate levels for up to 60 min. The primary PUBEC revealed higher N-acetylation rates (2.5-fold for PABA, 5-fold for PPD) compared to the 5637 cell line, based on both PABA conversion to its acetylated metabolite and formation of mono- and diacetylated PPD. The urothelial cell systems may thus be useful as a tool for further studies on the N-acetylation of aromatic amines via NAT1.

Identification and Characterization of Novel Arylamine NAcetyltransferase Small Molecule Inhibitors

Many environmental arylamines play a vital role in the etiology of cancer and require metabolic activation by acetylation. Arylamine N‐acetyltransferase 1 (NAT1), a xenobiotic metabolizing enzyme, catalyzes the N‐acetylation or O‐acetylation of arylamine carcinogens. O‐acetylation, which is preceded by cytochrome P450 N‐hydroxylation, results in nitrenium ions that form DNA adducts. If unrepaired, these adducts can lead to mutagenesis and initiate cancer. Molecular epidemiology studies suggest that NAT1 expression and genetic polymorphisms play an important role in individual risk for many cancers, including breast cancer. Studies also suggest that NAT1 may play an important role in cell growth and survival, as well as in cell invasion, which is a hallmark of metastatic cancer. The purpose of this study is to use NAT1 as a molecular target for the development of small molecule inhibitors for cancer prevention. We utilized in silico screening of 17 million compounds to identify those that interact with NAT active sites. A total of 150 lead compounds were identified, scored and ranked based on their active site association. Sixty of these compounds were tested for their ability to inhibit recombinant human NAT1. The IC50 values of the most efficacious inhibitors (2, 10, 11 and 16 ) of human NAT1 in vitro were >2 mM, and 0.75, 191, and 202 μM, respectively. The most potent inhibitor (10 ) was further tested for inhibition of N‐acetylation in situ in Chinese hamster ovary cells. The IC50 values of inhibitor 10 were 118 μM and 81.6 μM towards the N‐acetylation of p‐aminobenzoic acid and the arylamine carcinogen 4‐aminobiphenyl. We report the in silico identification and verification of compound 10 as a potential new small molecule for cancer prevention. [NIH/NIEHS (T32‐ ES011564) and UofL Clinical and Translational Sciences Program]

Prostate expression of N-acetyltransferase 1 (NAT1) and 2 (NAT2) in rapid and slow acetylator congenic Syrian hamster.

Arylamine carcinogens induce prostate tumours in rodent models and may contribute to the aetiology of human prostate cancers. N-acetylation and O-acetylation, catalysed by N-acetyltransferase 1 (NAT1) and 2 (NAT2), activate and/or deactivate arylamines to electrophilic intermediates that bind DNA and initiate tumours in target organs. NAT1 and NAT2 are both subject to genetic polymorphism in humans, and molecular epidemiological investigations suggest that NAT1 and/or NAT2 acetylator genotype modifies risk for prostate cancers. A Syrian hamster model congenic at the NAT2 locus was used to investigate the role of acetylator genotype in N- and O-acetylation of aromatic and heterocyclic amine carcinogens in the liver and prostate. A gene dose-response (NAT2*15/*15>NAT2*15/*16A>NAT2*16A/*16A) relationship was observed in liver and prostate cytosol towards the N-acetylation of p-aminobenzoic acid, 2-aminofluorene, beta-napthylamine, 4-aminobiphenyl, and 3,2'-dimethyl-4-aminobiphenyl. NAT1 and NAT2 were separated and partially purified from liver and prostate cytosol. NAT1 and NAT2 in liver and prostate catalysed -acetylation of the arylamines above and O-acetylation of N-hydroxy derivatives of 2-aminofluorene, 4-aminobiphenyl and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Rates were higher in rapid versus slow acetylators when catalysed by NAT2 but not when catalysed by NAT1. Partially purified prostate NAT2 exhibited higher apparent K(m) and V(max) than NAT1. Prostate NAT1 mRNA levels were higher than NAT2 and neither NAT1 nor NAT2 mRNA level differed with NAT2 acetylator genotype. The results provide mechanistic support for a role of NAT1 and/or NAT2 acetylator polymorphism(s) in human prostate cancer risk related to aromatic and/or heterocyclic amine carcinogens.

Cloning, expression, and functional characterization of rapid and slow acetylator polymorphic N-acetyl-transferase encoding genes of the Syrian hamster.

Syrian hamster acetylation capacity is catalysed by two N-acetyltransferase isozymes (NAT1 and NAT2). Hamster NAT2 (polymorphic) displays acetylator-genotype dependent activity resulting in high, intermediate, and low activity levels in homozygous rapid, heterozygous and homozygous slow acetylators, respectively. A lambda gt10 size-selected genomic library was constructed from Eco RI-digested homozygous slow acetylator Bio. 82.73/H-Pats congenic hamster DNA and screened with a hamster NAT1 probe. A 4.2 kb Eco RI insert from a positive clone was subcloned into pUC18 and the intron-free NAT2 coding region was sequenced. The NAT2 coding regions from genomic templates of other homozygous rapid and slow acetylator congenic and inbred hamster lines were amplified by the polymerase chain reaction, cloned, and sequenced. Two NAT2 alleles were found, one (NAT2*15) from each homozygous rapid acetylator line and one (NAT2*16A) from each homozygous slow acetylator line. NAT2*15 contained an 870 bp open reading frame encoding a 290 amino acid protein. NAT2*16A was similar except for two silent (T36C and A633G) and one nonsense (C727T) substitutions yielding a 242 amino acid open reading frame. The NAT2*15 and NAT2*16A alleles were expressed in Escherichia coli JM105 and the recombinant proteins were characterized. Electrophoretic mobilities of the NAT2 15 and NAT2 16A recombinant hamster proteins differed and correlated with the theoretical molecular weights calculated from their respective open reading frames. NAT2 16A exhibited 500-to 1000-fold lower maximum velocities compared to NAT2 15 for N-acetylation of all arylamine and hydrazine substrates tested. NAT2 16A also catalysed the metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides at rates 33- and 23-fold lower than NAT2 15. Intrinsic clearance (Vmax/Km) calculations suggest that N-acetylation of p-aminobenzoic acid and 2-aminofluorene in Syrian hamsters is catalysed primarily by NAT2 (NAT2 15) in rapid acetylators but by NAT1 (NAT1 9) in slow acetylators. These results provide a molecular basis for rapid and slow acetylator phenotype in the Syrian hamster.

Acetylator genotype-dependent N-acetylation of arylamines in vivo and in vitro by hepatic and extrahepatic organ cytosols of Syrian hamsters congenic at the polymorphic acetyltransferase locus.

Our laboratory recently reported the successful construction of homozygous rapid (Bio. 82.73/H-Patr) and homozygous slow (Bio. 82.73/H-Pat(s)) acetylator congenic Syrian hamsters. These hamsters are isogenic except for the polymorphic acetylator gene locus (Pat) and perhaps other closely linked loci. The purpose of the present investigation was to assess the expression of acetylator genotype both in vivo and in vitro in a variety of hepatic and extrahepatic organ cytosols. Levels of arylamine N-acetyl-transferase were generally high and in the relative order: liver greater than colon greater than kidney greater than pancreas greater than prostate, urinary bladder, and lung. However, an acetylator gene dose-response was clearly expressed in each tissue, with highest levels in homozygous Patr acetylators, intermediate levels in heterozygous Patr/Pat(s) acetylators, and lowest levels in homozygous Pat(s) acetylators. The magnitude of the acetylator genotype-dependent differences in N-acetyltransferase activity were substrate specific, wherein p-aminobenzoic acid showed the largest differences and p-aminophenol the smallest. The N-acetylation of p-aminobenzoic acid in vivo also reflected acetylator genotype in the congenic hamsters. These results further document the successful construction of rapid and slow acetylator congenic hamsters which should prove very valuable in future studies to assess the role of acetylator genotype in the toxicity and carcinogenicity of arylamine chemicals.

Metabolism of aromatic amines: relationships of N-acetylation, O-acetylation, N,O-acetyltransfer and deacetylation in human liver and urinary bladder.

N-Acetoxyarylamines are reactive metabolites that are implicated in the initiation of the carcinogenic process by some N-substituted aryl compounds. The objective of this study was to explore the relationship between the production of these reactive species and N-acetylation (NAT), a reaction previously demonstrated to be polymorphic in the human. Human liver and urinary bladder mucosa samples were frozen within 4-8 h post mortem. These tissues were assayed for the (i) O-acetylation (OAT) of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP) by acetyl CoA, (ii) intramolecular N,O-acetyltransfer (AHAT) of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), (iii) NAT of 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) by acetyl CoA and (iv) deacetylation of N-OH-AAF. Cytosolic AHAT and OAT showed partial inhibition by paraoxon. The ratio of paraoxon insensitive AHAT to OAT to NAT of PABA to NAT of 2-AF appears to be 1:2:11:22 using freshly made cytosols from frozen livers. Freezing of the cytosol resulted in extensive loss of activities. All four of these cytosolic enzyme activities exhibited a similar polymorphic response. Microsomal deacetylation showed a monomorphic response. Similar to the liver, urinary bladder epithelial cells also catalyzed the same reactions. However, the OAT and AHAT activities were detected mainly in microsomes. These data suggest that phenotypically rapid acetylators have a greater biochemical potential for the metabolic activation of aromatic amines by pathways that involve O-acetylation.

Genetic regulation of aromatic amine N-acetylation in inbred mice.

A survey among 20 inbred mouse strains revealed large variation (up to approximately 20-fold) for the N-acetylation of p-aminobenzoic acid by blood N-acetyltransferase and for the aromatic amine carcinogen benzidine by both liver and blood N-acetyltransferase. Of 20 strains surveyed, three are classified as slow acetylators (A/J, AHe/J, and X/Gf) and 17 are classified as rapid acetylators (AuSsJ, Castaneous, ST/bJ, C57BL/6J, Molossinus, SF, SWR/J, 129/SV, RF/J, RIII/2J, IsCam, SJL/J, Balb/cJ, C3H/HeJ, CBA/J, AKR/J, and DBA/J). The rapid acetylator strains possessed approximately 10 times greater liver benzidine N-acetyltransferase specific activity than the slow acetylator strains. Intercross and backcross matings of A/J and C57BL/6J mice indicate that a single gene with two major alleles is responsible for differences in N-acetyltransferase activity in blood for p-aminobenzoic acid or the alternate aromatic amine carcinogen aminofluorene, and in liver for aminofluorene. Analysis of 11 recombinant inbred strains derived from matings of A/J with C57BL/6J mice support this conclusion and demonstrate the existence of minor modifying genes that segregate independently of the major N-acetyltransferase gene.