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W W Weber

Publications and source records attributed to W W Weber.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1987

Purification, physicochemical, and kinetic properties of liver acetyl-CoA:arylamine N-acetyltransferase from rapid acetylator rabbits.

Cytosolic liver acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from homozygous rapid acetylator rabbits (strain III/J) was purified to homogeneity as judged by gel filtration sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and isoelectrofocusing. The isoelectric point was estimated to be 5.2. The molecular weight was determined to be 33,500 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and 33,000 by Sephacryl S-200 gel filtration. The amino acid composition is reported and 16 tryptic peptides were sequenced by Edmann degradation, including a peptide from which a very specific oligonucleotide probe can be synthesized. The enzyme contained neither amino sugars nor cofactors. A broad pH optimum from pH 5.9 to 8.6 was observed. N-Acetyltransferase activity showed a strong dependency on the salt concentration. From the influence of the basicity of the acceptor amine on the maximum velocity, it was concluded that the formation of the covalent acetyl-enzyme intermediate is the rate-limiting step in the N-acetyltransferase-catalyzed acetylation of amines. The covalent intermediate reacts, then, in a fast step with the acceptor amine, when using aniline derivatives with pKa values ranging from 5.65 to 1.74. However, with the weakly basic 4-nitroaniline, the acetyltransfer from the catalytic intermediate to the amine seems to be rate-limiting. A structure-activity study of 30 aniline derivatives that differ in hydrophobicity, position, size, charge, and number of substituents showed that some ortho-substituted derivatives were not acetylated.

Acetylation↗
PubMed1985

New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines.

Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT; EC 2.3.1.5), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of [3H]acetyl-CoA in the assay using [3H]acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-[3H]acetylarylamine after separation from [3H]acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.

Acetyltransferases↗
PubMed1985

Inheritance of liver N-acetyltransferase activity in the rapid and slow acetylator inbred hamster.

p-Aminobenzoic acid and p-aminosalicylic acid-N-acetyltransferase activities were determined in 105,000 X g liver cytosols from homozygous rapid acetylator (Bio. 4.24, Bio. 41.56 and Bio. 65.67) and homozygous slow acetylator (Bio. 1.5) inbred hamsters. Liver N-acetyltransferase activities were substantially higher in rapid as opposed to slow acetylator hamsters. Genetic crosses yielded F1 generation progeny with unimodal distributions of N-acetyltransferase activity intermediate between those of the rapid and slow acetylator parental strains, and F2 generation progeny with trimodal distributions (low/intermediate/high) of N-acetyltransferase activity not significantly different from a ratio of 1/2/1. Backcross matings yielded bimodal distributions of N-acetyltransferase activity in a ratio (intermediate/parental) not significantly different from 1/1. Each of these findings is consistent with simple autosomal Mendelian inheritance of two codominant alleles at a single gene locus.

4-Aminobenzoic Acid↗
PubMed1985

Biochemical investigation of the basis for the genetic N-acetylation polymorphism in the inbred hamster.

Cytosolic acetyl coenzyme A-dependent N-acetyltransferase enzyme (E.C. 2.3.1.5) from inbred hamsters of each acetylator genotype was partially purified by ion-exchange chromatography. Two distinct N-acetyltransferase enzymes were identified; the first one exhibited a polymorphic expression across acetylator genotypes whereas the second exhibited a monomorphic expression. The monomorphic enzyme had consistently high isoniazid and procainamide N-acetyltransferase activity in homozygous rapid acetylator (Bio. 87.20), homozygous slow acetylator (Bio. 82.73/H), obligate heterozygous rapid acetylator F1 progeny and in each of the three acetylator genotypes of F2 progeny. In contrast, the polymorphic enzyme exhibited marked acetylator genotype-dependent N-acetyltransferase activity. High p-aminobenzoic acid N-acetyltransferase activity was exhibited in the homozygous rapid acetylator parental and F2 generation progeny, intermediate activity was exhibited in the heterozygous rapid acetylator F1 and F2 generation progeny and very low or nondetectable activity was exhibited in homozygous slow acetylator parental and F2 generation progeny. A similar gene dose-response relationship was exhibited by the polymorphic N-acetyltransferase enzyme toward isoniazid and procainamide. These results provide new insight into the biochemical basis for the substrate-dependent polymorphic and monomorphic expression of N-acetylation capacity in the inbred hamster.

4-Aminobenzoic Acid↗
PubMed1984

Acetylation pharmacogenetics: experimental models for human toxicity.

Hereditary acetylation polymorphisms well-suited to experimental pharmacogenetic investigation are now known in three laboratory animal species (rabbit, mouse, and hamster). These animal models provide new evidence for the profound influence of this trait on the metabolic fate of arylamines and hydrazines, and on their pharmacological and toxicological profiles. The rabbit polymorphism most closely resembles that in humans. For the rabbit model, studies have shown that 1) monoacetylhydrazine is a polymorphic substrate for liver N-acetyltransferase in rapid and slow acetylators. This observation, in conjunction with human epidemiological data of others, opposes the commonly held view that rapid acetylators are predisposed to isoniazid (INH)-induced hepatotoxicity. 2) Slow acetylators are much more sensitive than rapid acetylators to the lethal central nervous system toxicity of INH. 3) In hepatocytes in short-term culture and exposed to arylamines and hydrazines, DNA damage is produced by hydralazine in slow acetylator hepatocytes but not in rapid acetylator hepatocytes, whereas hepatocytes from rapid acetylators are more sensitive to toxicity and DNA damage from 2-aminofluorene and benzidine. These investigations in animal models of the acetylation polymorphism provide new insights into human toxicity resulting from environmental arylamines and hydrazines.

Acetylation↗
PubMed1984

Relationship between N-acetylator phenotype and susceptibility toward hydrazine-induced lethal central nervous system toxicity in the rabbit.

Toxicity experiments carried out with rapid and slow acetylator rabbits showed that the slow acetylators exhibited greater susceptibility to lethal central nervous system actions of isoniazid and its metabolite, monoacetylhydrazine. Slow acetylator rabbits receiving a daily 30-mg/kg dose of isoniazid survived an average of 3.4 weeks, whereas rapid acetylators presented no signs of toxicity for the duration of the experiment (12 weeks). Concomitant administration of vitamin B6 (pyridoxine hydrochloride) raised the dose necessary for lethal convulsions by these agents in both rapid and slow acetylators. Inasmuch as isoniazid and other hydrazine drugs and foreign chemicals may produce a vitamin B6 deficiency in humans that can elicit seizures and lethal convulsions at high dose levels, these results suggest that the central nervous system toxicity produced by these agents might be more frequent and more severe in human slow acetylators.

Acetylation↗
PubMed1982

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.

4-Aminobenzoic Acid↗