Adrian J. Fretland

Acetylator phenotype and genotype in patients infected with HIV : discordance between methods for phenotype determination and genotype

The acetylator phenotype and genotype of AIDS patients, with and without an acute illness, was compared with that of healthy control subjects (30 per group). Two probe drugs, caffeine and dapsone, were used to determine the phenotype in the acutely ill cohort. Polymerase chain reaction amplification and restriction fragment length polymorphism analysis served to distinguish between the 26 known NAT2 alleles and the 21 most common NAT1 alleles. The distribution (%) of slow:rapid acetylator phenotype seen among acutely ill AIDS patients differed with the probe substrate used: 70:30 with caffeine versus 53:47 with dapsone. Phenotype assignment differed considerably between the two methods and there were numerous discrepancies between phenotype and genotype. The NAT2 genotype distribution was 45:55 slow:rapid. Control subjects, phenotyped only with caffeine, were 67:33 slow:rapid versus 60:40 genotypically. Stable AIDS patients, phenotyped only with dapsone, were 55:45 slow:rapid versus 46:54 genotypically. Following resolution of their acute infections, 12 of the acutely ill subjects were rephenotyped with dapsone. Phenotype assignment remained unchanged in all cases. The distribution of NAT1 alleles was similar in all three groups. It is evident from the amount of discordance between caffeine phenotype and dapsone phenotype or genotype that caution should be exercised in the use of caffeine as a probe for NAT2 in acutely ill patients. It is also clear that meaningful study of the acetylation polymorphism requires both phenotypic and genotypic data.

Tissue distribution of N-acetyltransferase 1 and 2 catalyzing the N-acetylation of 4-aminobiphenyl and O-acetylation of N-hydroxy-4-aminobiphenyl in the congenic rapid and slow acetylator Syrian hamster.

N‐acetyltransferase 1 (NAT1) and 2 (NAT2) enzymes catalyzing both deactivation (N‐acetylation) and activation (O‐acetylation) of arylamine carcinogens such as 4‐aminobiphenyl (ABP) were investigated in a Syrian hamster model congenic at the NAT2 locus. NAT2 catalytic activities (measured with p‐aminobenzoic acid) were significantly (P < 0.001) higher in rapid than slow acetylators in all tissues (except heart and prostate where activity was undetectable in slow acetylators). NAT1 catalytic activities (measured with sulfamethazine) were low but detectable in most tissues tested and did not differ significantly between rapid and slow acetylators. ABP N‐acetyltransferase activity was detected in all tissues of rapid acetylators but was below the limit of detection in all tissues of slow acetylators except liver where it was about 15‐fold lower than rapid acetylators. ABP N‐acetyltransferase activities correlated with NAT2 activities (r2 = 0.871; P < 0.0001) but not with NAT1 activities (r2 = 0.132; P > 0.05). Levels of N‐hydroxy‐ABP O‐acetyltransferase activities were significantly (P < 0.05) higher in rapid than slow acetylator cytosols for many but not all tissues. The N‐hydroxy‐ABP O‐acetyltransferase activities correlated with ABP N‐acetyltransferase activities (r2 = 0.695; P < 0.0001) and NAT2 activities (r2 = 0.521, P < 0.0001) but not with NAT1 activities (r2 = 0.115; P > 0.05). The results suggest widespread tissue distribution of both NAT1 and NAT2, which catalyzes both N‐ and O‐acetylation. These conclusions are important for interpretation of molecular epidemiological investigations into the role of N‐acetyltransferase polymorphisms in various diseases including cancer.

Effects of single nucleotide polymorphisms in human N-acetyltransferase 2 on metabolic activation (O-acetylation) of heterocyclic amine carcinogens

N‐Acetyltransferase 2 (NAT2) catalyzes the O‐acetylation of N‐hydroxy heterocyclic amines such as N‐hydroxy‐2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (NOHMeIQx) and N‐hydroxy‐2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b] pyridine (NOH PhIP) to DNA binding metabolites that initiate mutagenesis and carcinogenesis. NAT2 acetylator phenotype is associated with increased cancer risk. Single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region. Although the effects of these SNPs on N‐acetyltransferase activity have been reported, very little is known regarding their effects on O‐acetylation activity. To investigate the functional consequences of SNPs in the NAT2 coding region on the O‐acetylation of N‐hydroxy heterocyclic amines, reference NAT2*4 and NAT2 variant alleles possessing one were cloned and expressed in yeast (Schizosaccaromyces pombe). T111C, C282T, C481T, C759T, and A803G (K268R) SNPs did not significantly (p > 0.05) modify O‐acetylation catalysis with NOHPhIP or NOHMeIQx. C190T (R64W), G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q) and A845C (K282T) significantly (p < 0.01) reduced the O‐acetylation of both NOHPhIP and NOHMeIQx, whereas G857A (G286E) significantly (p < 0.05) decreased catalytic activity towards the O‐acetylation of NOHMeIQx but not NOHPhIP. These results have important implications towards the interpretation of molecular epidemiological studies of NAT2 genotype and cancer risk.

Effect of nucleotide substitutions in N-acetyltransferase-1 on N-acetylation (deactivation) and O-acetylation (activation) of arylamine carcinogens: implications for cancer predisposition

Genetic polymorphism in N-acetyltransferase-1 (NAT1) is associated with increased risk of various cancers, but epidemiological investigations are compromised by poor understanding of the relationship between NAT1 genotype and phenotype. Human reference NAT1*4 and 12 known human NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Large reductions in the N-acetylation of 4-aminobiphenyl and the O-acetylation of N-hydroxy-2-aminofluorene were observed for recombinant NAT1 allozymes encoded by NAT1*14B, NAT1*15, NAT1*17, NAT1*19, and NAT1*22. Each of these alleles exhibited substantially lower expression of NAT1 protein than the reference NAT1*4 and the other NAT1 alleles. These results show an important effect of the NAT1 genetic polymorphism on the N- and O-acetylation of arylamine carcinogens, suggesting modification of cancer susceptibility following exposures to arylamine carcinogens.

Association between acetylator genotype and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) DNA adduct formation in colon and prostate of inbred Fischer 344 and Wistar Kyoto rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine (HCA) found in cooked meats, causes colon and prostate tumors in male rats. Polymorphic N-acetyltransferase metabolizes N-hydroxy-PhIP to a DNA-reactive form. Liver, colon, and prostate PhIP-DNA adduct levels were compared in male rapid-acetylator Fischer 344 (F344) and slow-acetylator Wistar-Kyoto (WKY) rats fed 0.01 or 0.04% PhIP. Liver PhIP-DNA adduct levels at both PhIP doses, and colon PhIP-DNA adduct levels at the 0.01% PhIP dose were unaffected by acetylator genotype. However, in rats fed 0.04% PhIP, colon PhIP-DNA adduct levels were higher in rapid acetylator F344 rats (P < 0.05). Similarly, prostate PhIP-DNA adduct levels were higher in rapid acetylator F344 rats at both PhIP doses (P < 0.05). The combination of the high-PhIP dose and rapid-acetylator genotype resulted in the highest level of PhIP-DNA adducts in rat colon and prostate.

DNA adduct levels and absence of tumors in female rapid and slow acetylator congenic hamsters administered the rat mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

N‐acetyltransferases (EC 2.3.1.5) catalyze O‐acetylation of heterocyclic amine carcinogens to DNA‐reactive electrophiles that bind and mutate DNA. An acetylation polymorphism exists in humans and Syrian hamsters regulated by N‐acetyltransferase‐2 (NAT2) genotype. Some human epidemiological studies suggest a role for NAT2 phenotype in predisposition to cancers related to heterocyclic amine exposures, including breast cancer. 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is a heterocyclic amine carcinogen prevalent in the human environment and induces a high incidence of mammary tumors in female rats. PhIP‐induced carcinogenesis was examined in female rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus. In both rapid and slow acetylators, PhIP‐DNA adduct levels were highest in pancreas, lower in heart, small intestine, and colon, and lowest in mammary gland and liver. Metabolic activation of N‐hydroxy‐PhIP by O‐acetyltransferase was highest in mammary epithelial cells, lower in liver and colon, and lowest in pancreas. Metabolic activation of N‐hydroxy‐PhIP by O‐sulfotransferase was low in liver and colon and below the limit of detection in mammary epithelial cells and pancreas. Unlike the rat, PhIP did not induce breast or any other tumors in female rapid and slow acetylator congenic hamsters administered high‐dose PhIP (10 doses of 75 mg/kg) and a high‐fat diet.