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D W Hein

Publications and source records attributed to D W Hein.

At least 55 records · Page 3Linked to original sources
PubMed1996

2-Aminofluorene-DNA adduct levels in tumor-target and nontarget organs of rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus.

DNA adduct formation is an important initial event in chemical carcinogenesis. Metabolic activation and deactivation pathways are involved in aromatic amine carcinogenesis, and genetic polymorphism in the N-acetyltransferase 2 (NAT2) gene is associated with differential susceptibility to cancer from aromatic amine chemicals. In the present study, aromatic amine-DNA adduct levels were measured in rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Patr) acetylator Syrian hamsters congenic at the NAT2 locus following a single injection of 2-aminofluorene (60 mg/kg). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (C8-AF), was detected and quantitated by 32P-postlabeling assay at 6, 18, 24, 36, and 48 hr postinjection. Peak levels of C8-AF were achieved at 18-36 hr post-injection in both rapid and slow acetylators. C8-AF levels were significantly higher in tumor-target organs (liver and urinary bladder) than in nontarget organs (heart, colon, and prostate). Significant differences in C8-AF levels between rapid and slow acetylators in liver, heart, colon, and prostate were not observed. However, C8-AF levels in urinary bladder were significantly (four-fold) higher in rapid versus slow acetylators. These results suggest that 2-aminofluorene forms significantly higher levels of DNA adducts in tumor-target organs than in non-target organs and that acetyltransferase polymorphism plays a significant role in 2-aminofluorene-DNA adduct formation in urinary bladder of Syrian hamster.

Acetylation↗
PubMed1996

Effect of acetylator genotype on 3,2'-dimethyl-4-aminobiphenyl induced aberrant crypt foci in the colon of hamsters.

Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in both rodent and human carcinogenesis. The colon carcinogen 3,2'-dimethyl-4-aminobiphenyl (DMAB), like other arylamines, undergoes N-acetylation and O-acetylation by polymorphic acetyltransferase (NAT2). In the present study we characterized ACF in hamster colon for the first time and compared the ability of DMAB to induce ACF in homozygous rapid and slow acetylator congenic Syrian hamsters (Bio 1.5/H-NAT2r and Bio 1.5/H-NAT2s, respectively), differing only at the NAT2 gene locus and other closely linked loci. The animals received DMAB (75 mg/kg body weight s.c.) or vehicle (PBS/DMSO 1:1) as a control, twice weekly for 2 weeks, then once a week for 4 weeks. Ten weeks after the first injection ACF were observed in the DMAB treated hamsters, but not in the controls. However, the number of ACF was three times higher (P=0.016) in the colons of the NAT2r hamsters compared with the colons of the NAT2s hamsters. In the two congenic hamster lines we also studied the induction of ACF with 1,2-dimethylhydrazine (DMH) treatment, a colon carcinogen not metabolized by NAT2. Hamsters given DMH (25 mg/kg body weight s.c.), once a week for 3 weeks, showed ACF induction in the colon after 10 weeks, but there was no difference between the NAT2r and NAT2s hamsters. Further scanning electron microscopic and histological examination of ACF observed with the light microscope, revealed the same gross morphology and therefore confirmed the basis for the scoring of ACF. The ACF in hamster colons were principle similar to the lesions observed in other species.

Acetylation↗
PubMed1996

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.

Acetylation↗
PubMed1995

Determination of human NAT2 acetylator genotype by restriction fragment-length polymorphism and allele-specific amplification.

The human N-acetylation polymorphism, encoded by the NAT2 gene locus, has been associated with higher incidence and/or severity to the adverse effects of therapeutic drugs, and to the carcinogenic actions of environmental and occupational chemicals. In this paper, we describe an efficient method of restriction fragment-length polymorphism and allele-specific amplification analysis which distinguishes between each of 15 (NAT2*4, *5A, *5B, *5C, *6A, *6B, *7A, *7B, *12A, *12B, *13, *14A, *14B, *17, *18) NAT2 alleles that have been identified in human populations. The method should have broad applicability to improvement of drug therapy and to molecular epidemiology investigations of genetic predisposition to cancer and other diseases.

Acetylation↗
PubMed1995

Metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by 16 recombinant human NAT2 allozymes: effects of 7 specific NAT2 nucleic acid substitutions.

Human polymorphic N-acetyltransferase (NAT2) catalyzes the N-acetylation of arylamine carcinogens and the metabolic activation of N-hydroxyarylamine and N-hydroxyarylamide carcinogens by O- and N,O-acetylation, respectively. Rapid and slow acetylator phenotype is regulated at the NAT2 locus, and each has been associated with differential risk to certain cancers relating to carcinogenic arylamine exposures. We examined arylamine N-acetylation, N-hydroxyarylamine O-acetylation, and N-hydroxyarylamide N,O-acetylation catalytic activities of 16 different recombinant human NAT2 alleles expressed in an Escherichia coli JM105 expression system. NAT2 alleles contained nucleic acid substitutions at G191A (Arg64-->Gln), C282T (silent), T341C (Ile114-->Thr), C481T (silent), G590A (Arg197-->Gln), A803G (Lys268-->Arg), G857A (Gly286-->Glu), and various combinations of substitutions in the 870-bp NAT2-coding region. Expression of each NAT2 allele produced equivalent amounts of immunoreactive recombinant NAT2 protein with differential levels of N-, O-, and N,O-acetylation activity. Catalytic activities of each of the recombinant human NAT2 allozymes followed the relative order N-acetylation > O-acetylation > N,O-acetylation. Catalytic activation rates for the metabolic activation of N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl by O-acetylation and N-hydroxy-2-acetylaminofluorene by N,O-acetylation showed very strong correlations to the N-acetylation of 2-aminofluorene. NAT2 alleles with nucleic acid substitution T341C (NAT2*5A,*5B,*5C) expressed recombinant NAT2 allozymes, with the greatest reductions in metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by O- and N,O-acetylation, respectively. NAT2 alleles with nucleic acid substitutions G191A (NAT2*14A,*14B) and G590A (NAT2*6A,*6B) expressed recombinant NAT2 allozymes with more moderate reductions. NAT2 alleles with nucleic acid substitution G857A (NAT2*7A,*7B) expressed recombinant NAT2 allozymes with the smallest but yet significant reductions. NAT2 alleles with nucleic acid substitutions C282T (silent), C481T (silent), and A803G (Lys268-->Arg) expressed recombinant NAT2 allozymes that did not have significant reductions in the metabolic activations of N-hydroxyarylamines and N-hydroxyarylamides. The differential capacity for the metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by recombinant human NAT2 allozymes encoded by polymorphic NAT2 alleles supports the hypothesis that acetylator phenotype may predispose to cancers related to activation of N-hydroxy-arylamine and N-hydroxyarylamide carcinogens.

Arylamine N-Acetyltransferase↗
PubMed1995

Nomenclature for N-acetyltransferases.

A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature. The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals. Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized. Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin. For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

Alleles↗
PubMed1994

Polymorphic arylamine N-acetyltransferase encoding gene (NAT2) from homozygous rapid and slow acetylator congenic Syrian hamsters.

The nucleotide (nt) and deduced amino acid (aa) sequences were determined for polymorphic arylamine N-acetyl-transferase (NAT2) and its gene, NAT2, from homozygous rapid and slow acetylator congenic Syrian hamsters. The slow acetylator (NAT2s) allele contained three point mutations which differed from the rapid acetylator allele (NAT2r); two mutations were silent, and the third mutation resulted in a premature stop codon. The NAT2s allele contained a truncated open reading frame of 726 nt encoding a 242-aa protein, which is 48-aa shorter than NAT2r.

Acetylation↗
PubMed1994

Acetylator genotype-dependent formation of 2-aminofluorene-hemoglobin adducts in rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus.

Arylamine-hemoglobin adducts are a valuable dosimeter for assessing arylamine exposures and carcinogenic risk. The effects of age, sex, time-course, dose, and acetylator genotype on levels of 2-aminofluorene-hemoglobin adducts were investigated in homozygous rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Pats) acetylator hamsters congenic at the polymorphic (NAT2) acetylator locus. Following administration of a single ip dose of [3H]2-aminofluorene, peak 2-aminofluorene-hemoglobin adduct levels were achieved at 12-18 hr and retained a plateau up to 72 hr postinjection in both rapid and slow acetylator congenic hamsters. 2-Aminofluorene-hemoglobin adduct levels did not differ significantly between young (5-6 weeks) and old (32-49 weeks) hamsters or between male and female hamsters within either acetylator genotype. 2-Aminofluorene-hemoglobin adduct levels increased in a dose-dependent manner (r = 0.95, p = 0.0001) and were consistently higher in slow versus rapid acetylator congenic hamsters in studies of both time-course and dose-effect. The magnitude of the acetylator genotype-dependent difference was a function of dose; 2-aminofluorene-hemoglobin adduct levels were 1.5-fold higher in slow acetylator congenic hamsters following a 60 mg/kg 2-aminofluorene dose (p = 0.0013) but 2-fold higher following a 100 mg/kg 2-aminofluorene dose (p < 0.0001). These results show a specific and significant role for NAT2 acetylator genotype in formation of arylamine-hemoglobin adducts, which may reflect the relationship between acetylator genotype and the incidence of different cancers from arylamine exposures.

Acetylation↗
PubMed1994

Construction of Syrian hamster lines congenic at the polymorphic acetyltransferase locus (NAT2): acetylator genotype-dependent N- and O-acetylation of arylamine carcinogens.

Congenic Bio. 1.5/H-NAT2 Syrian hamster lines were constructed by introducing the NAT2r gene from MHA/SsLak inbred hamsters into a background BIO 1.5 Syrian inbred hamster line. Genetic identity of the Bio. 1.5/H-NAT2 congenic lines and nonidentity with the previously constructed Bio. 82.73/H-Pat congenic lines were determined by "DNA fingerprints" of genomic DNA derived from the different hamster lines. The N-acetylation capacity of the Bio. 1.5/H-NAT2 congenic hamster lines was clearly NAT2-dependent both in vivo and in vitro, with highest levels expressed in Bio. 1.5/H-NAT2r homozygous rapid acetylators, intermediate levels in Bio. 1.5/H-NAT2r/NAT2s heterozygous acetylators, and lowest levels in Bio. 1.5/H-NAT2s homozygous slow acetylators. The NAT2-dependent expression of N-acetyltransferase activity was evident toward p-aminobenzoic acid, 4-aminophenol, 2-aminofluorene, 4-aminobiphenyl, beta-naphthylamine, and 3,2'-dimethyl-4-amino-biphenyl in liver, kidney, colon, lung, and urinary bladder cytosols. The polymorphic acetyltransferase (NAT2) and the monomorphic acetyltransferase (NAT1) were isolated from hepatic cytosols and tested separately for their ability to catalyze arylamine N-acetyltransferase and N-hydroxyarylamine O-acetyltransferase activities. Both arylamine N-acetylation and N-hydroxyarylamine O-acetylation were clearly acetylator genotype-dependent when catalyzed by NAT2, and both were clearly acetylator genotype-independent when catalyzed by NAT1. NAT2/NAT1 activity ratios varied with the particular arylamine substrate acetylated. These studies show an important role for NAT2 acetylator genotype in Syrian hamster carcinogenic arylamine metabolism and confirm its role in the metabolic activation of N-hydroxyarylamines. The Bio. 1.5/H-NAT2 congenic lines provide a new model for investigating the precise role of the NAT2 gene locus in arylamine metabolism and toxicity.

Acetylation↗
PubMed1994

Metabolic activation of aromatic and heterocyclic N-hydroxyarylamines by wild-type and mutant recombinant human NAT1 and NAT2 acetyltransferases.

Recombinant human NAT1 and polymorphic NAT2 wild-type and mutant N-acetyltransferases (encoded by NAT2 alleles with mutations at 282/857, 191, 282/590, 341/803, 341/481/803, and 341/481) were expressed in Escherichia coli strains XA90 and/or JM105, and tested for their capacity to catalyze the metabolic activation (via O-acetylation) of the N-hydroxy (N-OH) derivatives of 2-aminofluorene (AF), and the heterocyclic arylamine mutagens 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Both NAT1 and NAT2 (including all mutant human NAT2s tested) catalyzed the metabolic activation of each of the N-hydroxyarylamines to products that bound to DNA. Metabolic activation of N-OH-AF was greater than that of the heterocyclic N-hydroxyarylamines. The relative capacity of NAT1 versus NAT2 to catalyze activation varied with N-hydroxyarylamine substrate. N-OH-MeIQx and N-OH-PhIP exhibited a relative specificity for NAT2. These results provide mechanistic support for a role of the genetic acetylation polymorphism in the metabolic activation of heterocyclic amine mutagens and carcinogens.

Acetylation↗
PubMed1994

Molecular genetics of human polymorphic N-acetyltransferase: enzymatic analysis of 15 recombinant wild-type, mutant, and chimeric NAT2 allozymes.

Human polymorphic N-acetyltransferase (NAT2) catalyzes the N-acetylation of arylamine drugs and carcinogens. Human acetylator phenotype is regulated at the NAT2 locus and has been associated with differential risk to certain drug toxicities or cancer. We examined arylamine substrate and acetyl coenzyme A cofactor affinities, and the N-acetyltransferase catalytic activities of the wild-type and 14 different mutant or chimeric human NAT2 alleles expressed in an Escherichia coli JM105 expression system. NAT2 alleles contained nucleic acid substitutions at positions 191(G-->A; Arg64-->Gln), 282(C-->T; silent), 341(T-->C; Ile114-->Thr), 481(C-->T; silent), 590(G-->A; Arg197-->Gln), 803(A-->G; Lys268-->Arg), 857(G-->A; Gly286-->Glu) and various combinations (282/590; 282/803; 282/857; 341/481; 341/803; 341/481/803; 481/803) of the 870 base pair NAT2 coding region. Expression of all 15 NAT2 alleles produced immunoreactive NAT2 protein with N-acetylation activity. NAT2 proteins encoded by alleles with nucleic acid substitutions at positions 191, 341, 590, 282/590, 341/481, 341/803, and 341/481/803 exhibited arylamine N-acetyltransferase maximum velocities significantly (P < 0.001) lower than the wildtype NAT2. Thus, nucleic acid substitutions at positions 191, 341, and 590 either alone or in combination with other silent or conservative amino acid substitutions were sufficient to result in NAT2 proteins with significant reductions in N-acetylation activities. The recombinant NAT2 proteins also showed relative differences in intrinsic stability following incubation at 37 degrees C and 50 degrees C. NAT2 encoded by alleles with nucleotide substitutions at positions 191 and 857 were particularly unstable relative to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl Coenzyme A↗
PubMed1994

Syrian hamster monomorphic N-acetyltransferase (NAT1) alleles: amplification, cloning, sequencing, and expression in E. coli.

N-acetyltransferases have an important role in the metabolism of arylamine and hydrazine drugs and carcinogens. Human N-acetylation phenotype may predispose individuals toward a variety of drug and xenobiotic-induced toxicities and carcinogenesis. Syrian hamsters express two N-acetyltransferase isozymes; one varies with acetylator genotype (polymorphic) and has been termed NAT2; the other does not (monomorphic) and has been termed NAT1. The intronless NAT1 coding region was cloned via the polymerase chain reaction from homozygous rapid acetylator and homozygous slow acetylator congenic and inbred hamster genomic DNA templates and sequenced. The NAT1 alleles from the homozygous rapid (NAT1) and homozygous slow (NAT1s) acetylator hamsters differed in one nucleotide, but the mutation is silent with no change in deduced amino acid sequence. To characterize the enzyme products of the NAT1 alleles, we developed a prokaryotic-expression system. The NAT1r and NAT1s alleles were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based plasmid vector pKK223-3 for over-production of recombinant NAT1 in E. coli strain JM105. Induced cultures from selected NAT1-inserted transformants yielded high levels of soluble protein capable of N-acetylation, O-acetylation, and N,O-acetylation. The recombinant NAT1r and NAT1s proteins did not differ in substrate specificity, specific activity, Michaelis-Menten kinetic properties, intrinsic stability, and electrophoretic mobility. Also, the over-expressed NAT1 proteins displayed substrate-specificity and electrophoretic mobilities characteristic of NAT1 isolated from Syrian hamster liver and colon cytosols.

Amino Acid Sequence↗
PubMed1993

Metabolic activation of N-hydroxy-2-aminofluorene and N-hydroxy-2-acetylaminofluorene by monomorphic N-acetyltransferase (NAT1) and polymorphic N-acetyltransferase (NAT2) in colon cytosols of Syrian hamsters congenic at the NAT2 locus.

Acetylator genotype is regulated at the polymorphic acetyltransferase (NAT2) gene locus in humans and other mammals such as Syrian hamsters. Human slow acetylator phenotypes have been associated with increased incidences of urinary bladder cancers, whereas rapid acetylators have been associated with increased incidences of colorectal cancers. The genetic predisposition of rapid acetylators to colorectal cancers suggests localized metabolic activation of arylamine carcinogen metabolites by polymorphic N-acetyltransferase (NAT2) in colon tissues. We tested this hypothesis in Bio. 82.73/H Syrian hamster lines which are congenic at the NAT2 gene locus. Congenic Bio. 82.73/H Syrian hamsters expressed acetylator genotype-dependent N-acetyltransferase activity in colon cytosols toward arylamine carcinogens such as 2-aminofluorene and 4-aminobiphenyl. Partial purification of the hamster colon cytosol by anion exchange chromatography identified two N-acetyltransferase isozymes analogous to those previously described in liver and urinary bladder. One of the isozymes (NAT2) exhibited acetylator genotype-dependent expression for the N-acetylation of each arylamine tested: p-aminophenol; 2-aminofluorene; 4-aminobiphenyl; 3,2'-dimethyl-4-aminobiphenyl; and 2-amino-dipyrido[1,2-a:3',2'd]imidazole as well as for the metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene to form DNA adducts. Although NAT2 catalyzed the metabolic activation of N-hydroxy-2-acetyl-aminofluorene to DNA adducts, the rates were lower, were paraoxon-sensitive, and did not reflect acetylator genotype. A second isozyme (NAT1) also catalyzed the N-acetylation of each arylamine as well as the metabolic activation of N-hydroxy-2-aminofluorene and N-hydroxy-2-acetylaminofluorene to DNA adducts at rates that were independent of acetylator genotype. Metabolic activation of N-hydroxy-2-aminofluorene catalyzed by both NAT1 and NAT2 was resistant to 100 microM paraoxon, an inhibitor of microsomal deacetylases. Metabolic activation of N-hydroxy-2-acetylaminofluorene by NAT1 and NAT2 was partially sensitive to 100 microM paraoxon. Michaelis-Menten kinetic constants were determined for the colon NAT1 and NAT2 isozymes and compared to previous determinations for liver NAT1 and NAT2. For each of the arylamines tested, both apparent Km and apparent Vmax were higher for NAT2 than NAT1. In rapid acetylator hamster colon, NAT2/NAT1 activity ratios were 18 and 13 for the N-acetylation of 2-aminofluorene and 4-aminobiphenyl and 28 for the O-acetylation of N-hydroxy-2-aminofluorene. These results strongly support the role of the polymorphic NAT2 gene locus in the local metabolic activation of N-hydroxyarylamine carcinogens in colon and provide mechanistic support for human epidemiological studies suggesting a predisposition of rapid acetylators to colorectal cancer.

Acetylation↗
PubMed1993

Human acetylator genotype: relationship to colorectal cancer incidence and arylamine N-acetyltransferase expression in colon cytosol.

Polymorphic expression of arylamine N-acetyltransferase (EC 2.3.1.5) may be a differential risk factor in metabolic activation of arylamine carcinogens and susceptibility to cancers related to arylamine exposures. Human epidemiological studies suggest that rapid acetylator phenotype may be associated with higher incidences of colorectal cancer. We used restriction fragment length polymorphism analysis to determine acetylator genotypes of 44 subjects with colorectal cancer and 28 non-cancer subjects of similar ethnic background (i.e., approximately 25% Black and 75% White). The polymorphic N-acetyltransferase gene (NAT2) was amplified by the polymerase chain reaction from DNA templates derived from human colons of colorectal and non-cancer subjects. No significant differences in NAT2 allelic frequencies (i.e., WT, M1, M2, M3 alleles) or in acetylator genotypes were found between the colorectal cancer and non-cancer groups. No significant differences in NAT2 allelic frequencies were observed between Whites and Blacks or between males and females. Cytosolic preparations from the human colons were tested for expression of arylamine N-acetyltransferase activity. Although N-acetyltransferase activity was expressed for each of the arylamines tested (i. e., p-aminobenzoic acid, 4-aminobiphenyl, 2-aminofluorene, beta-naphthylamine), no correlation was observed between acetylator genotype and expression of human colon arylamine N-acetyltransferase activity. Similarly, no correlation was observed between subject age and expression of human colon arylamine N-acetyltransferase activity. These results suggest that arylamine N-acetyltransferase activity expressed in human colon is catalyzed predominantly by NAT1, an arylamine N-acetyltransferase that is not regulated by NAT2 acetylator genotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1993

Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases.

A genetic polymorphism at the NAT2 gene locus, encoding for polymorphic N-acetyltransferase (NAT2), segregates individuals into rapid, intermediate or slow acetylator phenotypes. Both rapid and slow acetylator phenotypes have been associated with increased incidence of cancer in certain target organs related to arylamine exposure, suggesting a role for acetylation in both the activation and deactivation of arylamine carcinogens. A second gene (NAT1) encodes for a different acetyltransferase isozyme (NAT1) that is not subject to the classical acetylation polymorphism. In order to assess the relative ability of NAT1 and NAT2 to activate and deactivate arylamine carcinogens, we tested the capacity of recombinant human NAT1 and NAT2, expressed in Escherichia coli XA90 strains DMG100 and DMG200 respectively, to catalyze the N-acetylation (deactivation) and O-acetylation (activation) of a variety of carbocyclic and heterocyclic arylamine carcinogens. Both NAT1 and NAT2 catalyzed the N-acetylation of each of the 17 arylamines tested. Rates of N-acetylation by NAT1 and NAT2 were considerably lower for heterocyclic arylamines such as 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), particularly those (e.g. IQ) with steric hindrance to the exocyclic amino group. For carbocyclic arylamines such as 4-aminobiphenyl and beta-naphthylamine, the apparent affinity was significantly (P < 0.05) higher for NAT2 than NAT1. NAT1/NAT2 activity ratios and clearance calculations suggest a significant role for the polymorphic NAT2 in the N-acetylation of carbocyclic arylamine carcinogens. Both NAT1 and NAT2 catalyzed acetyl coenzyme A-dependent O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl to yield DNA adducts. NAT1 catalyzed paraoxon-resistant, intramolecular N,O-acetyltransferase-mediated activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl at low rates; catalysis by NAT2 was not readily detectable in the presence of paraoxon. In summary these studies strongly suggest that the human acetylation polymorphism influences both the metabolic activation (O-acetylation) and deactivation (N-acetylation) of arylamine carcinogens via polymorphic expression of NAT2. These findings lend mechanistic support for human epidemiological studies suggesting associations between both rapid and slow acetylator phenotype and cancers related to arylamine exposure.

Acetylation↗
PubMed1992

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.

Acetylation↗