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.
Hein
D W
DW
Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, Grand Forks 58203.
Doll
M A
MA
Gray
K
K
Rustan
T D
TD
Ferguson
R J
RJ
D000107:Acetylation; D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism*; D000588:Amines / Q000493:pharmacokinetics; D000818:Animals; D001711:Biotransformation / Q000187:drug effects; D002273:Carcinogens / Q000493:pharmacokinetics*; D003106:Colon / Q000201:enzymology* / Q000502:physiology; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005449:Fluorenes / Q000493:pharmacokinetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005838:Genotype; D006881:Hydroxyacetylaminofluorene / Q000493:pharmacokinetics*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008647:Mesocricetus; D008954:Models, Biological; D010261:Paraoxon / Q000494:pharmacology; D011110:Polymorphism, Genetic / Q000235:genetics; D013379:Substrate Specificity
D000588:Amines; D002273:Carcinogens; D005449:Fluorenes; D007527:Isoenzymes; D006881:Hydroxyacetylaminofluorene; C001368:N-hydroxy-2-aminofluorene; D004247:DNA; D000123:Acetyltransferases; D010261:Paraoxon