Biomedical subjects

T D Rustan

Publications and source records attributed to T D Rustan.

7 recordsLinked to original sources

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↗

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↗

PubMed1991

Extrahepatic expression of the N-acetylation polymorphism toward arylamine carcinogens in tumor target organs of an inbred rat model.

An N-acetylation polymorphism is described that is expressed toward arylamine carcinogens in tumor target organs of an inbred rat model. High levels (rapid acetylator phenotype) of arylamine carcinogen N-acetyltransferase activity were observed in kidney, colon, prostate and urinary bladder cytosols derived from Fischer (F-344) inbred rats, the strain most commonly used for tumor bioassay studies and the strain most particularly used in arylamine-induced colon and prostate cancer studies. Significantly lower (slow acetylator phenotype) levels of arylamine carcinogen N-acetyltransferase activity were observed in corresponding tissue cytosols derived from Wistar-Kyoto inbred rats. Intermediate levels of arylamine carcinogen N-acetyltransferase activity significantly different from both the parental strains were observed in F1 hybrids of the parental strains, consistent with codominant expression of two alleles at a single gene locus. The arylamine substrates exhibiting the acetylator phenotype-dependent N-acetyltransferase activities included p-aminobenzoic acid, p-aminosalicylic acid, p-phenetidine, p-aminophenol, 2-aminofluorene, 3,2'-dimethyl-4-aminobiphenyl, beta-naphthylamine and 4-aminobiphenyl, but not procainamide. Highest levels of arylamine carcinogen N-acetyltransferase were expressed consistently in colon cytosol, but expression of the N-acetylation polymorphism toward arylamine carcinogens was observed in each (kidney, colon, prostate and urinary bladder) of the tumor target organs. The expression of the N-acetylation polymorphism in tumor target organs suggests that the inbred rat model will be useful in assessing the role of acetylator phenotype in arylamine-induced cancers of the colon and prostate.

Acetylation↗

PubMed1991

Polymorphic and monomorphic expression of arylamine carcinogen N-acetyltransferase isozymes in tumor target organ cytosols of Syrian hamsters congenic at the polymorphic acetyltransferase locus.

A number of human epidemiological investigations suggest a relationship between acetylator phenotype and the incidence and/or severity of tumors caused by exposure to arylamine carcinogens. Conclusions drawn from these investigations can be compromised by a variety of environmental and other genetic factors. To eliminate variability in these other factors, our laboratory recently completed construction of homozygous rapid (Bio. 82.73/H-Patr), heterozygous intermediate (Bio. 82.73/H-Patr/Pat(s)) and homozygous slow (Bio. 82.73/H-Pat(s)) acetylator congenic hamsters. The purpose of the present study was to assess the utility of this congenic hamster model for investigations into the relationship between acetylator genotype and arylamine carcinogenesis. We report the expression of acetylator genotype-dependent (polymorphic) and acetylator genotype-independent (monomorphic) N-acetyltransferase isozymes in hepatic cytosols. The hepatic polymorphic N-acetyltransferase isozyme isolated from the congenic hamsters expressed clearly acetylator-genotype dependent (Patr greater than Patr/Pat(s) greater than Pat(s)) N-acetylation towards p-aminobenzoic acid, 4-aminobiphenyl, 2-aminofluorene, p-aminophenol, 1-aminopyrene, 5-aminosalicylic acid, beta-naphthylamine, 3,4-dichloroaniline, 3,2'-dimethyl-4-aminobiphenyl and p-phenetidine. Acetylator genotype-dependent N-acetylation for a number of arylamines also was observed in liver, colon, kidney and urinary bladder cytosols derived from the congenic hamster lines, including arylamines highly carcinogenic to hamster colon and urinary bladders. It is concluded that the congenic hamster model will be useful in studies to delineate the role of acetylator genotype in the incidence or severity of arylamine tumors.

Animals↗

PubMed1990

Purification of hepatic polymorphic arylamine N-acetyltransferase from homozygous rapid acetylator inbred hamster: identity with polymorphic N-hydroxyarylamine-O-acetyltransferase.

The polymorphic acetyltransferase isozyme expressed in homozygous rapid acetylator inbred hamster liver cytosol was purified over 2000-fold by sequential Q-Sepharose fast-flow anion-exchange chromatography, Sephacryl S-200 high-resolution size-exclusion chromatography, Mono Q anion-exchange fast-protein liquid chromatography, and preparative polyacrylamide gel electrophoresis. The isozyme migrated as a single homogeneous monomer following both preparative and sodium dodecyl sulfate-polyacrylamide electrophoresis. The molecular weight was estimated at 34,170 following elution via size-exclusion chromatography and 35,467 following migration via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The homogeneous polymorphic acetyltransferase exhibited a broad substrate specificity; it catalyzed the acetyl coenzyme A-dependent N-acetylation of p-aminobenzoic acid, carbocyclic arylamine carcinogens such as 2-aminofluorene, 4-aminobiphenyl and beta-naphthylamine, and heterocyclic arylamine carcinogens such as 2-aminodipyrido[1,2-a:3'2'd]imidazole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. It also readily catalyzed the acetyl coenzyme A-dependent metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene to DNA adducts but not the metabolic activation (via intramolecular, N,O-acetyltransfer) of N-hydroxy-2-acetylaminofluorene or N-hydroxy-4-acetylaminobiphenyl to DNA adducts. Conversely, the partially purified monomorphic acetyltransferase isozyme from the same hamsters readily catalyzed the metabolic activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl, and rates of metabolic activation of these substrates did not differ between homozygous rapid and slow acetylator liver, intestine, kidney, and lung cytosols. Heat inactivation rates for the purified polymorphic acetyltransferase isozyme were first order and indistinguishable for the acetyl coenzyme A-dependent N-acetylation and O-acetylation activities. The results strongly suggest the expression of a single polymorphic acetyltransferase product of the hamster polymorphic acetyltransferase gene that catalyzes both acetyl coenzyme A-dependent N-acetylation and O-acetylation of arylamine and N-hydroxyarylamine carcinogens but not the metabolic activation of N-hydroxy-N-acetylarylamines (arylhydroxamic acids) via intramolecular N,O-acetyltransfer. Consequently, acetylator genotype-dependent metabolic activation of N-hydroxyarylamines to a DNA adduct in hamster is catalyzed by direct O-acetylation of the hydroxyl group and not via sequential N-acetylation followed by N,O-acetyltransfer.

Acetyltransferases↗

PubMed1985

Human brain tubulin purification: decrease in soluble tubulin with age.

The soluble tubulin of human cerebral cortex, as assessed by [3H]colchicine binding of the 100,000 g supernatant fraction, decreases drastically with age, 75 percent from age 0 to age 90. There is also a considerably lower concentration of high molecular weight proteins in the soluble fraction of postmortem human cerebral cortex than in that of nonhuman species. Human brain tubulin can be polymerized into microtubules with DEAE-dextran. The DEAE-dextran induced microtubules are stable to cold temperature (4 degrees) and calcium. However, in the presence of 1 M glutamate, the microtubules become cold labile and depolymerize at 4 degrees. Thus we have developed a novel method for purifying polymerization competent tubulin from fresh or frozen human cerebral cortex. Human brain tubulin purified by our novel method is very similar to tubulin from the brains of other mammals in molecular weight, amino acid composition, polymerization-depolymerization parameters, and structural dimensions of the microtubules formed.

Adolescent↗

PubMedDate not supplied

Acetylator phenotype-dependent and -independent expression of arylamine N-acetyltransferase isozymes in rapid and slow acetylator inbred rat liver.

Although mouse, hamster, and rabbit models of the human N-acetylation polymorphism have been identified and characterized, many investigations of arylamine toxicity and carcinogenicity are carried out in the rat, particularly the Fischer 344 (F-344) inbred rat. We partially characterized a new rat model of the N-acetylation polymorphism by determining expression of arylamine N-acetyltransferase activities in liver cytosols derived from adult male inbred F-344, WKY, and their F1 hybrid rat strains. Levels of N-acetyltransferase activity differed significantly between the strains for many arylamine substrates, with highest levels in F-344, lowest levels in WKY, and intermediate levels in F1 hybrids of these two parental strains. However, for some other arylamine substrates, levels of N-acetyltransferase activity did not differ significantly between the rat strains. Partial purification of rat liver cytosols from the three strains resulted in identification of two N-acetyltransferase isozymes. The levels of N-acetyltransferase activity of one isozyme differed significantly between strains analogous to the pattern observed in crude cytosol. In contrast, the levels of N-acetyltransferase activity of the second isozyme did not differ between the strains. Based upon these results, the F-344 inbred strain is designated a rapid acetylator phenotype, the WKY inbred strain is designated a slow acetylator phenotype, and F1 hybrids of the two parental strains are designated intermediate acetylator phenotype. The identification of acetylator phenotype-dependent and -independent hepatic N-acetyltransferase isozymes in the inbred rat mimics the biochemical basis for acetylator phenotype-dependent and -independent expressions of N-acetylation in humans and other mammalian species.

Acetylation↗