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M A Doll

Publications and source records attributed to M A Doll.

32 records · Page 2Linked to original sources
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↗
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

Acetyltransferases and susceptibility to chemicals.

Arylamine chemicals inflict a number of toxicities including cancer. Metabolic activation (i.e., oxidation) is required in order to elicit the toxic actions. Acetylation is an important step in the metabolic activation and deactivation of arylamines. N-acetylation forms the amide derivative which is often nontoxic. However, O-acetylation of the N-hydroxyarylamine (following oxidation) yields an acetoxy arylamine derivative which breaks down spontaneously to a highly reactive arylnitrenium ion, the ultimate metabolite responsible for mutagenic and carcinogenic lesions. Human capacity to acetylate arylamine chemicals is subject to a genetic polymorphism. Individuals segregate into rapid, intermediate, or slow acetylator phenotypes by Mendelian inheritance regulated by a single gene encoding for a polymorphic acetyltransferase isozyme (NAT2). Individuals homozygous for mutant alleles are deficient in the polymorphic acetyltransferase and are slow acetylators. A second acetyltransferase isozyme (NAT1) is monomorphic and is not regulated by the acetylator genotype. Several human epidemiological studies suggest an association between slow acetylator phenotype and urinary bladder cancer. In contrast, a few studies suggest a relationship between rapid acetylator phenotype and colorectal cancer. The basis for this paradox may relate to the relative importance of N- versus O-acetylation in the etiology of these cancers. Conclusions drawn from human epidemiological data are often compromised by uncontrolled environmental and other genetic factors. Our laboratory recently completed construction of homozygous rapid, heterozygous intermediate, and homozygous slow acetylator congenic Syrian hamsters to be homologous in greater than 99.975% of their genomes. The availability of these acetylator congenic lines should eliminate genetic variability in virtually all aspects of arylamine carcinogenesis except at the acetylator gene locus. Ongoing studies in these congenic hamster lines should provide unequivocal information regarding the role of genetic acetylator phenotype in susceptibility to arylamine-related cancers.

Acetylation↗
PubMedDate not supplied

Cloning, expression, and functional characterization of two mutant (NAT2(191) and NAT2(341/803)) and wild-type human polymorphic N-acetyltransferase (NAT2) alleles.

The N-acetylation polymorphism segregates individuals into rapid, intermediate, and slow acetylator phenotypes via monogenic inheritance at the NAT2 locus. In a previous study (Arch. Toxicol. 67, 445-452, 1993), we uncovered discrepancies between apparent NAT2 acetylator genotype based on polymerase chain reaction-restriction fragment length polymorphism analysis, in vitro colon arylamine N-acetyltransferase activity, and expected frequency of slow acetylator phenotype in African-Americans, which suggested the presence of not yet defined mutant NAT2 alleles. Two novel NAT2 alleles were discovered after cloning and sequencing of NAT2 polymerase chain reaction products. One allele (NAT2(191)) contained a point mutation at nucleotide 191 [G-->A (Arg-->Gln)], whereas the other allele (NAT2(341/803)) contained two point mutations [341T-->C (Ile-->Thr); 803A-->G (Lys-->Arg)]. The two mutant NAT2 and the NAT2wt alleles were expressed in a prokaryotic expression system. Both the NAT2(191) and NAT2(341/803) mutant alleles expressed functional N-acetyltransferases capable of catalyzing both arylamine N-acetylation and the metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene. However, the NAT2(191) and NAT2(341/803) each exhibited significantly lower N- and O-acetylation capacity and were intrinsically less stable than NAT2wt.

Acetylation↗