Biomedical subjects

W W Weber

Publications and source records attributed to W W Weber.

At least 19 recordsLinked to original sources

PubMed1992

DNA adduct levels in congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) is a human and mouse bladder carcinogen. Epidemiological studies have shown that individuals with a slow acetylator phenotype, especially those exposed to high levels of carcinogenic aromatic amines, show an increased susceptibility to bladder cancer. In order to determine if a slow acetylator phenotype results in increased DNA damage, congenic mouse strains C57BL/6J and B6.A-Nat(s), which differ genetically at the acetyltransferase (EC 2.3.1.5) locus as homozygous rapid (Natr/Natr) and homozygous slow (Nat(s)/Nat(s)) acetylators respectively, were continuously administered 4-ABP.HCl (55-300 p.p.m.) in their drinking water for 28 days. The levels of covalently bound N-(deoxyguanosin-8-yl)-4-ABP-DNA adducts, which are believed to be critical for the initiation of tumors, were quantitated in the liver and bladder by 32P-postlabeling analysis. The levels of the hepatic DNA adduct increased with dose in both sexes, but were independent of the mouse acetylator genotype. At comparable doses, however, the levels of DNA adducts were 2-fold higher in the liver of the female as compared to the male animals. The DNA adducts also increased with dose in bladder of the male mice, but in contrast to the liver, the adduct levels were approximately 2-fold lower in the bladder DNA of the female mice. Also in contrast to the liver, the levels of bladder DNA adducts were significantly higher (P < or = 0.03) in the phenotypic rapid acetylator females compared to the slow acetylators at both 75 and 150 p.p.m. doses; the median levels of adducts were 10-20% higher in the phenotypic slow acetylator male bladders compared to their rapid acetylator counterparts. The results of these studies are consistent with the increased carcinogenicity of 4-ABP to the liver of female mice and the bladder of male mice. They further suggest that factors other than acetylator phenotype limit the extent of DNA adduct formation from 4-ABP in these mice.

Acetylation↗

PubMed1992

2-Aminofluorene-DNA adducts in mouse urinary bladder: effect of age, sex and acetylator phenotype.

Formation of urinary bladder DNA-2-aminofluorene adducts in inbred and acetylator congenic mice was measured 3 h after a 60 mg/kg dose of the arylamine carcinogen. The sensitivity of 32P-postlabeling with HPLC analysis permitted quantitation of adducts in individual mouse bladders. Acetylator phenotype was a significant determinant of DNA damage in female mice as slow acetylators had higher levels of bladder DNA adducts than rapids. This correlation is the reverse of that seen with hepatic DNA. Age was also a significant determinant of DNA damage as older mice (20-23 weeks) formed more bladder DNA adducts than young (7 week) mice. The age-related increase in bladder adduct formation was seen in both sexes of all mouse lines. Male B6 mice exposed to 2-aminofluorene at 20-23 weeks of age showed a 26-fold higher level of bladder DNA adducts than males exposed at 7 weeks. In addition to the large increase in total adduct level, the older male B6 mice produced significant amounts of an unidentified, early-eluting adduct peak that had chromatographic properties similar to an aminofluorene-DNA adduct produced through peroxidative activation. These results indicate that age, sex and acetylator phenotype are all important determinants of aromatic amine-bladder DNA adduct formation in mice.

Acetylation↗

PubMed1992

Metabolic, molecular genetic and toxicological aspects of the acetylation polymorphism in inbred mice.

Over the past 10 years, much fascinating information has been obtained concerning the biochemistry, genetics, toxicological implications and molecular genetics of the N-acetylation polymorphism in mice. Using C57BL/6J (B6) mice as representative of rapid acetylation and A/J (A) mice as representing slow acetylation, it has been shown that the polymorphism observed in N-acetyltransferase (NAT) activity in liver also occurs in kidney, bladder, blood, and other tissues. The development of congenic acetylator mouse lines derived from B6 and A, have provided the necessary tools to study the role of the acetylation polymorphism, on either the B6 or A genetic background, free of nearly all other genetic differences between these strains. Eliminating genes which modify and complicate the differences due to the acetylator genes make the congenic lines very useful in toxicology studies, particularly those involving carcinogenesis. The molecular genetic basis of the acetylator polymorphism in B6 and A mice involves two Nat genes. Nat-1 encodes a protein termed NAT1 which is identical in rapid and slow acetylator strains. Nat-2, however, differs between rapid and slow strains by a single nucleotide change in the coding region. The corresponding NAT2 proteins differ by a single change at amino acid 99: an hydrophilic asparagine in rapid acetylator NAT2 to an hydrophobic isoleucine in NAT2 from slow acetylators. The mechanistic basis for the differences between rapid and slow acetylation in mice appears to be that NAT2 from the rapid B6 strain is 15-fold more stable at 37 degrees C and is transcribed/translated with a maximal efficiency twice that of the enzyme from slow acetylator A mice. Results discussed in this review indicate that mice provide an excellent system for studying the N-acetyltransferase polymorphism and also are useful for modelling several aspects of the human N-acetyltransferase polymorphism.

Acetylation↗

PubMed1992

Cloned mouse N-acetyltransferases: enzymatic properties of expressed Nat-1 and Nat-2 gene products.

N-Acetylation plays an important role in the metabolism of a wide variety of hydrazine drugs and arylamine drugs and carcinogens. Humans have genetically determined differences in their N-acetyltransferase activities and are phenotypically classified as rapid or slow acetylators. Mice have a similar genetic polymorphism in N-acetyltransferase activity and have been used as models of the human polymorphism in many studies of the toxicology and carcinogenicity of arylamines. Recently, two N-acetyltransferase genes, Nat-1 and Nat-2, were cloned from rapid (C57BL/6J) and slow (A/J) acetylator mouse strains. The genomic clone encoding NAT-1 is identical in rapid and slow acetylator mouse strains, whereas the clone encoding NAT-2 differs between rapid and slow strains by a single base pair, which changes the encoded amino acid from Asn99 in the rapid acetylator strain to Ile99 in the slow acetylator strain. In this report, the N-acetylation polymorphism in mice was investigated by transiently expressing the cloned N-acetyltransferase genes in COS-1 cells. The intronless coding regions of Nat-1 and Nat-2 showed different substrate specificities; isoniazid was a preferred substrate for NAT-1, whereas p-aminobenzoic acid was preferred for NAT-2(99asn) and NAT-2(99ile). All three enzymes acetylated 2-aminofluorene, but none of them acetylated sulfamethazine. Kinetic constants determined for the expressed enzymes with 2-aminofluorene and p-aminobenzoic acid indicated that Km values were not significantly different between the enzymes, although the Vmax value of NAT-2(99asn) was consistently 2-3-fold higher than that of NAT-1 or NAT-2(99ile). Nat-1 and Nat-2 encoded mRNAs of approximately 1.4 kilobases in livers of rapid and slow acetylators. Nat-2 mRNA was more abundant in liver than Nat-1 mRNA. The abundance of Nat-2 mRNA and Nat-1 mRNA was equivalent in both rapid and slow acetylator mouse strain livers. Incubation of transfected COS-1 cell cytosols at 37 degrees showed that the time for decline of NAT activity to 50% of its initial value was 45 hr for NAT-1, 60 hr for NAT-2(99asn), and 4 hr for NAT-2(99ile). This 15-fold difference in the heat stability of the rapid and slow isoforms of NAT activity was also observed in cytosols from rapid and slow acetylator livers. Comparison of the rates of translation of the rapid and slow isoforms of NAT-2 in an in vitro system showed that NAT-2(99asn) was translated at approximately twice the rate of NAT-2(99ile).(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗

PubMed1991

Diverse point mutations in the human gene for polymorphic N-acetyltransferase.

Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variations. Two highly homologous human genes for N-acetyltransferase (NAT; arylamine acetyltransferase, acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5), NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants. One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (codon 94) and a separate G----A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions: Thr for Ile (codon 114) and Arg for Lys (codon 268). Heterozygosity was detected in three NAT2 samples: two were heterozygous for the rapid and one of the allelic variants, and the third was a compound heterozygote of both mutant alleles. The results show conclusively that the genetically variant NAT is encoded by NAT2.

Acetylation↗

PubMed1991

N-acetylation of aromatic amines: genetic polymorphism in inbred rat strains.

The inheritance of rat liver N-acetyltransferase polymorphism was investigated with reciprocal genetic crosses between slow (NSD/N) and rapid (Peth/N) acetylator strains. Rat liver N-acetyltransferase activity was determined using a spectrophotometric assay which measured the amount of arylamine substrate present after incubation with N-acetyltransferase in vitro. Male N-acetyltransferase activities assayed in liver preparations using p-aminobenzoic acid and p-toluidine as substrates indicate bimodality of the parental strains and unimodality of the F-1 generation; limited data suggest trimodality (not significantly different from a 1:2:1 ratio) of the F-2 generation. Reciprocal crosses of WKY/N, another slow acetylator strain, and the Peth/N strain gave results similar to those of the NSD/N x Peth/N cross. Female N-acetyltransferase activities in all strains studied were lower than male N-acetyltransferase activities, but were similarly distributed in the parental and F-1 generations. The male/female N-acetyltransferase activity ratio was substrate- and genotype-dependent. Results show that regulation of the variation of rat liver N-acetyltransferase activity is consistent with autosomal Mendelian inheritance of two major alleles at a single gene locus.

4-Aminobenzoic Acid↗

PubMed1991

Molecular genetic basis of rapid and slow acetylation in mice.

The molecular genetic basis of N-acetylation polymorphism has been investigated in inbred mouse models of the human acetylation polymorphism. Two genomic clones, Nat1 and Nat2, were isolated from a C57BL/6J (B6) mouse (rapid acetylator) genomic library. The Nat1 and Nat2 genes both have intronless coding regions of 870 nucleotides and display greater than 47% deduced amino acid similarity with human, rabbit, and chicken N-acetyltransferases. Amplification of Nat1 and Nat2 from A/J (A) mouse (slow acetylator) genomic DNA by the polymerase chain reaction and subsequent sequencing revealed that Nat1 was identical in B6 and A mice, whereas Nat2 contained a single nucleotide change from adenine in B6 to thymine in A mice. This nucleotide substitution changes the deduced amino acid at position 99 from asparagine in B6 to isoleucine in A mice. Hydropathy analysis revealed that this amino acid change alters the hydropathy of the flanking peptide segment in NAT2 from hydrophilic in the B6 mouse to hydrophobic in the A mouse. The amino acid change occurs in a region of the gene where no polymorphism has yet been reported in human or rabbit NAT2 and may represent an important structural domain for N-acetyltransferase activity. Nat1 and Nat2 have the same 5' to 3' orientation in the B6 mouse; the two genes are separated by approximately 9 kilobases, with Nat1 located 5' of Nat2.

Acetylation↗

PubMed1990

Human N-acetylation genotype determination with urinary caffeine metabolites.

The human acetylation genotype was determined by measuring urinary caffeine metabolites by use of a modification of a previously published HPLC method. The problem of separation of 7-methylxanthine (7X) from 1-methyluric acid (IU) in urine extracts was achieved by adding a phenyl column, in tandem with a C18 reverse-phase column, by means of a methanol:aqueous acetic acid gradient elution system. The urinary molar ratios of (AAMU)/(AAMU + 1U + 1X) and (AAMU)/(1X) were estimated in 20 subjects phenotyped with dapsone, with 100% concordance for the [AAMU]/[1X] ratio. A population study of 42 unrelated individuals exhibited trimodal distribution in acetylation capacity, consistent with the Hardy-Weinberg theory of population genetics. Definitive pedigree analysis of 16 families (75 subjects) resulted in significant similarity between the observed genotypic matings and those expected by classical Mendelian segregation. This noninvasive genotyping method promises to be useful in future investigation of the relationship between the human acetylation polymorphism and clinical disorders.

Acetylation↗

PubMed1990

2-aminofluorene-hepatic DNA adducts in congenic mouse lines differing in Ah responsiveness.

The influence of beta-naphthoflavone (BNF) pretreatment on 2-aminofluorene (2-AF)-hepatic DNA adduct formation was evaluated in Ah-responsive and non-responsive congenic mouse lines through use of HPLC analysis of 32P-postlabeled nucleotides. C57BL/6J (B6) mice were used as an example of BNF-responsive mice while B6.D-Ahd, a line congenic with B6, was used as the non-responsive line. Induction at the Ah locus with BNF increased adduct levels in hepatic DNA in B6 mice but not in B6.D mice 3 h after a 60 mg/kg i.p. dose of 2-AF. The slow acetylator counterparts of B6 and B6.D, namely B6.A and B6.A.D-NatsAhd (a new congenic line produced from B6.A and B6.D), had lower adduct levels than the rapid acetylators before induction. Although adduct levels in B6.A and B6.A.D were increased following BNF induction, the level of adducts remained below those of induced B6 mice. In the three lines that responded to BNF induction, male mice had a greater relative increase in hepatic DNA adduct levels than females. For all four lines, with or without BNF pretreatment, greater adduct levels were found in the females. These results imply that responsiveness to aromatic hydrocarbon induction, as well as rapid acetylation, may be risk factors in hepatic DNA damage following arylamine exposure. Female mice appear to be more susceptible to such damage than males.

Acetylation↗

PubMed1990

Polymorphic acetylation of arylamines and DNA-adduct formation.

Inbred mouse strains congenic for rapid and slow N-acetyltransferase (NAT) (A.B6, rapid and B6.A, slow) were used to separate the effect of the NAT polymorphism from the influence of other genetically polymorphic enzymes on DNA adduct formation induced by exposure to arylamine carcinogens. Adduct formation was measured by HPLC analysis of 32P-postlabeled nucleotides from DNA of the urinary bladder and liver. Acetylator phenotype was a significant determinant of DNA damage in females as slow acetylators had higher levels of bladder DNA adducts than rapids. This correlation was the reverse of that seen with liver DNA. Older mice (20-23 weeks) formed much higher bladder DNA adduct levels than young mice (7 week). The increase in bladder adduct formation with age was seen in both sexes of all mouse strains. The older male B6 mice showed a 26-fold increase in bladder adducts and the older females showed no more than a 2-fold increase. In addition, the older male B6 mice produced significant amounts of an unidentified, early eluting adduct peak. Biochemical studies of liver NAT and O-acetyltransferase (OAT) activities showed a direct correlation between the levels of liver 2-aminofluorene (AF) NAT activity and levels of liver DNA-adduct formation, but the role of OAT activity in adduct formation in the mouse remains unclear. These results indicate that the NAT phenotype, age and sex are all important determinants of arylamine-DNA adduct formation in mice.

Acetylation↗

PubMed1990

Acetylation.

UNLABELLED: The human acetylation polymorphism has been known for more than three decades since its discovery during the metabolic investigation of the antituberculous hydrazine drug, isoniazid. The trait was originally known as the "isoniazid acetylation polymorphism" but is now usually abbreviated to the "acetylation polymorphism" because the acetylation of numerous hydrazine and arylamine drugs and other chemicals are subject to this trait. A. Individuals phenotype as "slow" acetylators, homozygous for the slow acetylator gene, or "rapid" acetylators either heterozygous or homozygous for the rapid acetylator gene. Differences in individual acetylating capacity are ascribed to differences in the activities of the arylamine acetylating enzymes (isozymic N-acetyltransferase variants) of the liver, intestinal mucosa and certain other tissues. The chromosomal locus of the human gene has not been determined, but linkage analysis in mice indicates that the N-acetyltransferase gene is closely linked to Esterase-1 on mouse chromosome 8. RECOMMENDATION: 1) That the chromosomal locus of the human acetylator gene(s) be determined. B. The acetylator phenotype is a lifelong, stable characteristic of the individual that can be determined by procedures using any of several test agents (eg, caffeine, isoniazid, sulfamethazine, sulfapyridine). All suitable test agents discriminate rapid and slow acetylator phenotypes, whereas caffeine enables homozygous and heterozygous rapid acetylators to be discriminated from each other and from slow acetylators. These procedures can be used with confidence to determine the acetylator status of healthy adults and children but caution is necessary in interpreting this information for infants, in altered physiologic states and in the presence of certain diseases and environmental substances. RECOMMENDATIONs: 1) That investigators should strongly consider the use of the caffeine test for acetylator phenotype determination in human epidemiologic studies of acetylation because of its advantages over other test agents that are available. 2) That efforts to determine the structure of the acetylator genes responsible for the human acetylator polymorphism, and to determine the genes responsible for the hereditary acetylator polymorphisms in animal models for the human trait be continued apace with efforts on the human system. 3) That an improved test to determine the acetylator status utilizing that information and current molecular biology approaches and techniques applied to tissues that are readily available in human subjects (eg, leukocytes) be developed at a high priority. C. The hereditary acetylator status of individuals provides valuable information about their therapeutic, pharmacologic and toxicologic responses and is a prognosticator of unusual susceptibility to toxicity from drugs widely used for the treatment of diverse diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation↗

PubMed1989

2-Aminofluorene-DNA adduct formation in acetylator congenic mouse lines.

The effect of the acetylator polymorphism on hepatic 2-aminofluorene-DNA adduct formation in mice was studied using two recent developments from our laboratory. Acetylator congenic mouse lines differing from their parental inbred lines in N-acetyltransferase activity were used to separate the effect of the N-acetyltransferase polymorphism from effects of differences in other genetically polymorphic enzymes. DNA adduct formation was used as an indicator of arylamine induced DNA damage. Adduct formation was measured by HPLC analysis of 32P-postlabeled nucleotides from hepatic DNA of treated animals. At a high dose (60 mg/kg) of 2-aminofluorene for a 3 h exposure, rapid acetylator mice (C57BL/6J) accumulated twice the adducts of slow acetylators (A/J). In acetylator congenic mice this difference increased so that rapid acetylators with the slow background (A.B6-Natr) had 5- to 7-times the DNA damage of the slow acetylator congenic with the rapid background (B6.A-Nats). It was also found that within each mouse line examined, females had higher levels of adduct formation than males. Acetylator congenic mouse lines were useful in distinguishing the effect of acetylator genes from the total genetic background. Similarly, congenics were useful in demonstrating the contribution that enzymes other than N-acetyltransferase make to differences in adduct formation in inbred mouse lines.

Acetyltransferases↗

PubMed1989

Purification and biochemical characterization of hepatic arylamine N-acetyltransferase from rapid and slow acetylator mice: identity with arylhydroxamic acid N,O-acyltransferase and N-hydroxyarylamine O-acetyltransferase.

An inbred mouse model for the human N-acetylation polymorphism has been used to investigate the biochemical basis for the arylamine N-acetylation polymorphism and the relationship between the cytosolic enzymes arylamine N-acetyltransferase (NAT), arylhydroxamic acid N,O-acyltransferase, and N-hydroxyarylamine O-acetyltransferase. Biochemical studies of partially purified NAT from rapid and slow acetylator mice revealed identical molecular weights of 31,500, activation energies of 21,000 cal/mol, equivalent affinities for acetyl coenzyme A, broad pH optima, the presence of an active site sulfhydryl group, and similar behavior during purification with anion exchange, gel filtration, and hydrophobic interaction chromatography. The enzymes differed in inhibition by hydrogen peroxide and dithiobis(2-nitrobenzoic acid). These observations taken in conjunction with previous investigations indicate that the rapid and slow mouse NAT enzymes are isozymes with minimal structural differences. NATs from rapid and slow acetylator mice were purified more than 10,000-fold by the following sequence of methods: homogenization and fractional centrifugation, protamine sulfate precipitation, and chromatography on DEAE-Trisacryl M, Sephadex G-100, Amethopterin-AH-Sepharose 4B, butyl agarose, and Sephacryl S-200, with a 15-25% recovery. NAT from B6 mice was purified to greater than 95% purity, as judged by silver staining of sodium dodecyl sulfate-polyacrylamide gels. Although only NAT appeared to be subject to a genetic polymorphism as evidenced by N-acetylation activities in liver cytosol, the purified NAT protein possessed arylhydroxamic acid N,O-acyltransferase, N-hydroxyarylamine O-acetyltransferase, and NAT activities. Thus, the cytosolic N-acetyltransferase of mouse liver may catalyze N-, O-, and N,O-acetyltransfer reactions through a common acetylated intermediate of a single protein.

Acetylation↗

PubMed1988

High-performance liquid chromatographic analysis of 32P-postlabeled DNA-aromatic carcinogen adducts.

The technique of 32P postlabeling of DNA-carcinogen adducts is a useful and extremely sensitive method of detecting and quantitating DNA damage by carcinogens. We have adapted the 32P method to analysis by high-pressure liquid chromatography, making the procedure more rapid and convenient than when thin-layer chromatography is used. Following DNA isolation and hydrolysis, nucleotide-carcinogen adducts are enhanced relative to normal nucleotides by solvent extraction and then labeled with high-specific-activity [gamma-32P]ATP. The resulting 32P-postlabeled nucleotides are resolved by reverse-phase ion-pair HPLC. After as little as 3 h of exposure to carcinogens, DNA adducts can be demonstrated from 1 microgram or less of mouse hepatic DNA. Acetylated and nonacetylated adducts can be resolved from hepatic DNA of mice treated with 2-aminofluorene. Differences in DNA damage as measured by adduct formation were demonstrated between "rapid" and "slow" acetylator mouse strains. Rapid-acetylator C57BL/6J mice had three times the amount of hepatic DNA adducts as slow-acetylator A/J mice 3 h after a 60 mg/kg dose of 2-aminofluorene. 4-Aminobiphenyl and 2-naphthylamine each showed an adduct peak with retention time similar to that of the nonacetylated 2-aminofluorene adduct, while benzidine gave a major adduct that eluted somewhat earlier as would be expected for an acetylated adduct. The alkenylbenzenes, safrole and methyleugenol, also formed DNA adducts detectable by this method. DNA prepared from skin of mice painted with benzo[a]pyrene also contained carcinogen-DNA adducts detectable and resolvable by HPLC analysis following 32P postlabeling. The combination of HPLC with 32P postlabeling appears to be a useful technique for the rapid detection and quantitation of DNA damage caused by several classes of aromatic carcinogens.

Animals↗

PubMed1988

On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J).

A covalent, catalytic intermediate of cytosolic liver acetyl coenzyme A: arylamine N-acetyltransferase (EC 2.3.1.5) from rapid acetylator rabbits (III/J) was isolated and chemically characterized. The active site was further studied using two covalent inhibitors, [2-3H]iodoacetic acid and bromoacetanilide. Inhibition experiments with [2-3H]iodoacetic acid at pH 6.9 showed that the incorporation of 0.7 mol of [2-3H]iodoacetic acid/mol of N-acetyltransferase led to rapid, irreversible loss of enzyme activity. Preincubation of the enzyme with acetyl coenzyme A (acetyl-CoA) completely protected against inactivation by [2-3H]iodoacetic acid. After incubating the N-acetyltransferase with [2-3H]acetyl-CoA in the absence of an acceptor amine, an acetyl-cysteinyl-enzyme intermediate was isolated and characterized. Preincubation of N-acetyltransferase with iodoacetic acid prevented the incorporation of the [2-3H]acetyl group into the enzyme. The product analog, bromoacetanilide, caused a rapid irreversible loss of N-acetyltransferase activity. The reaction was pseudo first-order and saturated at high bromoacetanilide concentrations (KI = 0.67 mM; k3 = 1 min-1). Preincubation of the enzyme with acetyl-CoA prevented inactivation by the inhibitor. The acceptor amine 4-ethylaniline did not prevent inhibition. Incorporation of the inhibitor was directly proportional to the loss of activity showing a 1:1 stoichiometry of enzyme to inhibitor. The target amino acid was identified as cysteine by amino acid analysis of inhibitor-treated enzyme.

Acetanilides↗

PubMed1987

Kinetics of arylamine N-acetyltransferase in tissues from rapid and slow acetylator mice.

Kinetic parameters for arylamine N-acetyltransferase activity in liver, blood, and bladder from C57BL/6J and A/J mouse strains were determined using an improved assay system, and some deviations were found from previously reported results. In the present studies, blood N-acetyltransferase activity with p-aminobenzoic acid and 2-aminofluorene as substrates was 20- and 10-fold greater, respectively, in C57BL/6J than in A/J mice. Urinary bladder possessed N-acetyltransferase activity for both 2-aminofluorene and p-aminobenzoic acid which differed 2-fold, and reflected the liver and blood phenotype. An apparent Km difference for 2-aminofluorene was observed between C57BL/6J and A/J liver N-acetyltransferase. Contrary to earlier studies, the liver N-acetyltransferase activity differed 3-fold between the A/J and C57BL/6J mouse strains, with either p-aminobenzoic acid or 2-aminofluorene as substrates. Dimethylsulfoxide at concentrations used in the 2-aminofluorene acetylation assay in earlier studies, inhibited the A/J liver N-acetyltransferase to a greater extent than the C57BL/6J enzyme, which may have contributed to the larger difference in liver NAT activity with 2-aminofluorene reported previously.

Acetyltransferases↗

PubMed1987

Benzidine activation in the Ames test: roles of hepatic N-acetyltransferase and other cytosolic and microsomal factors.

Benzidine (BZ) is a known animal and human carcinogen, and is mutagenic in the Ames test using strain TA 98. Several workers have shown that hepatic S9 fraction from hamster is much more effective than is rat S9, as an activation system for BZ in the Ames test. We show that rat microsomal fraction inhibits hamster S9 activation of BZ. Hamster microsomal fraction, supplemented with glucose-6-phosphate dehydrogenase (G6PdeH), gives a BZ dose-dependent mutagenic response, in the absence of cytosolic fraction. Rat microsomal fraction, in contrast, gives relatively little activation, under comparable conditions. Activation was enhanced when hamster or rat cytosol was added back to a mixture of hamster microsomes and G6PdeH. When strain TA 98 was replaced by strain TA 98/1,8-DNP6, very little activation of BZ was observed. Partially purified mouse liver acetyltransferase effectively activated BZ to mutagenic products in the presence of acetyl coenzyme A (CoASAc)/hamster microsomes/G6PdeH. Hamster and rat liver cytosol contain a CoASAc-dependent as well as a CoASAc-independent cytosolic activating factor of BZ. Hamster but not rat microsomal activation of BZ is enhanced in the presence of CoASAc. The biochemical mechanisms of BZ activation in the Ames test are discussed in light of these results.

Acetyl Coenzyme A↗

PubMed1987

Genetic variability in deacetylation of 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in inbred strains of mice.

Genetic variability in 2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) deacetylase activities was examined in 19 inbred strains of mice. AAF deacetylase activities ranged from 0.60 to 1.33 nmol/min/mg protein, and there was an approximately 2.5-fold difference in AAF deacetylase activity between the fastest (C57BL/6J) and slowest (RIIIS/J) mouse strains. N-OH-AAF deacetylase activities ranged from 3.28 to 13.24 nmol/min/mg protein, and the difference between the fastest (AU/SsJ) and slowest (RIIIS/J) strains was 4-fold. N-OH-AAF deacetylase activity was higher (5-13 times) than AAF deacetylase activity in all strains examined. Thus, there are genetic differences in AAF and N-OH-AAF deacetylase activities; these differences may play an important role in individual susceptibility to the mutagenic and carcinogenic effects of the aromatic amides.

2-Acetylaminofluorene↗