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.     

Acetylator genotype-dependent expression of arylamine N-acetyltransferase in human colon cytosol from non-cancer and colorectal cancer patients

Human epidemiological studies suggest an association between rapid acetylator phenotype and colorectal cancer. Acetylator genotype-dependent expression by the human colon of arylamine N-acetylation capacity, catalyzed by acetyl coenzyme A-dependent N-acetyltransferase(s) (EC 2.3.1.5) (NAT), may be an important risk factor in the initiation of colorectal cancer. Human colon cytosols from 48 fresh surgical samples were investigated for NAT activity toward p-aminobenzoic acid and the arylamine carcinogens 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Apparent Vmax determinations of NAT activity toward these substrates indicated that 40 of these colons segregated into 3 distinct phenotypes. The distribution of the patients into rapid (5), intermediate (18), or slow (17) acetylators is a ratio that is not significantly different from the expected Hardy-Weinberg distribution of 3:16:21 (chi 2 = 2.206, P = 0.363). Significantly greater mean apparent Vmax levels were found in colons from rapid as compared to intermediate acetylators (1.5-3-fold) (P less than 0.001) and intermediate as compared to slow (2.5-3-fold) (P less than 0.005) acetylator phenotypes for the four arylamine substrates. Apparent Km determinations indicated that human colon NAT from rapid acetylators had a significantly lower affinity for the arylamine substrates (P less than 0.05) compared to intermediate or slow acetylator groups. No difference in apparent Km was detected for the cofactor acetyl coenzyme A between the three acetylator phenotypes. The colon samples were also tested for cytosolic N-hydroxy-2-acetylaminofluorene sulfotransferase activity and found to be monomorphically distributed for this enzyme activity. Of the 40 colon samples, 37 were from individuals of known pathology, 25 with colorectal cancer and 12 with no diagnosed neoplasia. Comparisons between mean apparent Vmax and mean apparent Km levels for each of the acetylator phenotypes indicated no significant differences between non-cancer and colorectal cancer patients. The distribution of rapid, intermediate, and slow acetylator phenotypes among the colon samples derived from colorectal cancer patients was precisely that predicted from published frequencies for the rapid and slow acetylator allele in Americans of African and European ancestry.     

Metabolic activation of carcinogenic heterocyclic aromatic amines by human liver and colon

The metabolic activation of the food-borne rodent carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) was compared with that of the known human carcinogen 4-aminobiphenyl (ABP), using human liver microsomes, human and rat liver cytosols, and human colon cytosol. All of these aromatic amines were readily activated by N-hydroxylation with human liver microsomes (2.3-5.3 nmol/min/mg protein), with PhIP and ABP exhibiting the highest rates of cytochrome P450IA2-dependent N-oxidation, followed by MeIQx, IQ and Glu-P-1. In contrast, while ABP and 2-aminofluorene were readily N-acetylated (1.7-2.3 nmol/min/mg protein) by the polymorphic human liver cytosolic N-acetyltransferase, none of the heterocyclic amines were detectable as substrates (less than 0.05 nmol/min/mg protein). Likewise, only low activity was observed (0.11 nmol/min/mg protein) for the N-acetylation of p-aminobenzoic acid, a selective substrate for the human monomorphic liver N-acetyltransferase. The radiolabeled N-hydroxy (N-OH) arylamine metabolites were synthesized and their reactivity with DNA was examined. Each derivative bound covalently with DNA at neutral pH (7.0), with highest levels of binding observed for N-OH-IQ and N-OH-PhIP. Incubation at acidic pH (5.0) resulted in increased levels of DNA binding, suggesting formation of reactive arylnitrenium ion intermediates. These N-OH arylamines were further activated to DNA-bound products by human hepatic O-acetyltransferase. Acetyl coenzyme A (AcCoA)-dependent, cytosol-catalyzed DNA binding was greatest for N-OH-ABP and N-OH-Glu-P-1, followed by N-OH-PhIP, N-OH-MeIQx and N-OH-IQ; and both rapid and slow acetylator phenotypes were apparent. Rat liver cytosol also catalyzed AcCoA-dependent DNA binding of the N-OH arylamines; and substrate specificities were comparable to human liver, except that N-OH-MeIQx and N-OH-PhIP gave relatively higher and lower activities respectively. Human colon cytosols likewise displayed AcCoA-dependent DNA binding activity for the N-OH substrates. Metabolic activity was generally lower than that found with the rapid acetylator liver cytosols; however, substrate specificity was variable and phenotypic differences in colon O-acetyltransferase activity could not be readily discerned. This may be due, at least in part, to the varied contribution of the monomorphic acetyltransferase, which would be expected to participate in the enzymatic acetylation of some of these N-OH arylamines.(ABSTRACT TRUNCATED AT 400 WORDS)     

PCR and flow cytometric analysis of paclitaxel-inhibited arylamine N-acetyltransferase activity and gene expression in human osteogenic sarcoma cells (U-2 OS)

Human epidemiological studies suggest an association between N-acetyltransferase (NAT) activity and the incidence of bladder and colorectal cancers. In this study, paclitaxel was selected to examine the inhibition of arylamine NAT activity, gene expression and 2-aminofluorene-DNA adduct formation in a human osteogenic sarcoma cell line (U-2 OS). The activity of NAT was determined by high performance liquid chromatography (HPLC) assay for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and nonacetylated AF and PABA. Human osteogenic sarcoma cell cytosols and intact cells were used to examine the NAT activity, gene expression and AF-DNA adduct formation. The results demonstrated that NAT activity percent of NAT in examined cells, gene expression (NAT1 mRNA) and AF-DNA adduct formation in human osteogenic sarcoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The results also demonstrated that paclitaxel decreased the apparent values of Km and Vmax from intact human osteogenic sarcoma cells (U-2 OS). Thus, paclitaxel is an uncompetitive inhibitor of the NAT enzyme.     

Acetylator genotype-dependent metabolic activation of carcinogenic N-hydroxyarylamines by S-acetyl coenzyme A-dependent enzymes of inbred hamster tissue cytosols: relationship to arylamine N-acetyltransferase

A genetic polymorphism in S-acetyl coenzyme A (AcCoA)-dependentN-acetyltransferase has been associated with a differentialrisk for certain cancers in humans. In this study, several tissuesfrom the inbred Syrian hamster with a genetically defined AcCoA-dependentN-acetyltransferase polymorphism (homozygous rapid acetylator,Bio. 87.20; homozygous slow acetylator, Bio. 82.73/H; and heterozygousacetylator, Bio. 87.20 x Bio. 82.73/H F₁), were investigatedfor the relationship of arylamine N-acetyltransferase to theAcCoA-dependent metabolic activation of carcinogenic N-hydroxy(N-OH)-arylamines to bind to DNA (O-acetyltransferase). Thelevels of both 2-aminofluorene (AF) N-acetyltransferase andN-OH-AF O-acetyltransferase activity reflected the N-acetylatorgenotype in liver, intestine, kidney and lung cytosols. A significantacetylator gene—dose response for AF N-acetyltransferaseand N-OH-AF O-acetyltransferase activities was observed in liverand lung cytosols. In contrast, acetylator genotype was notconsistently expressed for the AcCoA-dependent N-acetylationof 4-aminobiphenyl (ABP), nor for the AcCoA-dependent metabolicactivation of N-OH ABP and N-OH-3,2'-dimethyl-4-aminobiphenylin these me tissue cytosols. Two peaks of acetyltransferaseactivity were partially purified by ion exchange FPLC chromatographyfrom the hepatic cytosol of both the homozygous rapid and homozygousslow acetylator hamster. In contrast to unfractionated cytosol,the isozyme(s) eluting first clearly demonstrated levels ofAcCoA-dependent arylamine N-acetyltransferase and N-OH-arylamineO-acetyltransferase activities that were consistent with N-acetylatorgenotype (polymorphic) for all substrates tested. In contrast,the slower eluting isozyme(s) in each acetylator cytosol showedlevels of AcCoA-dependent N-and O-acetyltransferase activitiesthat did not vary with N-acetylator genotype (monomorphic).The AcCoA-dependent O-acetyltransferase activity of both themonomorphic and polymorphic peaks was paraoxon resistant. Thesestudies demonstrate acetylator genotype-dependent control ofAcCoA-dependent metabolic activation of N-OH-arylamines(O-acetylation)by polymorphic isozyme(s) similar to that for AcCoA-dependentN-acetylation of arylamines in the hamster. The polymorphicgenetic control of N-OH arylamine O-acetyltransferase may bean important risk factor for arylamine-induced cancer, in thosespecies and tissues expressing appreciable levels of O-acetyltransferaseactivity.     

Acetyl coenzyme A dependent activation of N-hydroxy derivatives of carcinogenic arylamines: mechanism of activation, species difference, tissue distribution, and acetyl donor specificity

Acetyl coenzyme A dependent activation of 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) and 3-hydroxyamino-1-methyl-5H-pyrido [4,3-b]indole (N-OH-Trp-P-2) was investigated using cytosols from hepatic and extrahepatic tissues of various animal species in comparison with that of N-hydroxy-2-aminofluorene. N-OH-Glu-P-1 and N-OH-Trp-P-2 were metabolized to the reactive species capable of binding to transfer RNA through a putative O-acetylation process by liver cytosols. Kidney, small intestinal mucosa, lung, and bladder from hamsters and rats also mediated the reaction, although their activities were lower than that in the liver. Marked species differences in the enzymatic activities of livers were observed. Hamsters showed the highest ability in the activation for N-OH-Glu-P-1 and N-OH-Trp-P-2, followed by rats. Rabbits with a rapid acetylator phenotype, which showed a high activity in the N-acetylation of arylamines, activated N-OH-Glu-P-1 but scarcely N-OH-Trp-P-2. A rabbit with a slow acetylator phenotype, mice, guinea pigs, and a dog showed marginal or nondetectable activities with N-OH-Glu-P-1 and N-OH-Trp-P-2. A typical nonheterocyclic N-hydroxyarylamine, N-hydroxy-2-aminofluorene was also activated by the acetyl coenzyme A dependent system to an intermediate which bound to transfer RNA. However, the acetyl-CoA dependent binding of N-hydroxy-2-aminofluorene was markedly different from those observed with N-OH-Glu-P-1 and N-OH-Trp-P-2 concerning the order of activities among animal species used. In addition to short chain acyl coenzyme As, N-hydroxy-2-acetylaminofluorene also served as an acetyl donor for the activation of N-OH-Glu-P-1 and N-OH-Trp-P-2 in liver cytosol systems. The formation of N-acetyl-N-OH-Glu-P-1, however, was not detected in the cytosolic system of N-OH-Glu-P-1 with acetyl-CoA, suggesting the direct O-acetylation at the N-hydroxy group as a major pathway for the activation of N-hydroxyarylamines.     

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.     

Distribution of acetyltransferase activities in the intestines of rapid and slow acetylator rabbits

Epidemiological studies have shown that there is a significantly greater proportion of the rapid acetylator phenotype in patients with colorectal tumors than in controls; phenotype-related differences in bioactivation of dietary or environmental amines in the intestinal epithelium have been suggested as a mechanism for this effect. In the present study, we have used hepatic and intestinal cytosols to compare N-acetyltransferase (NAT1 and NAT2), O-acetyltransferase (OAT) and arylhydroxamic acid N,O-acyltransferase (AHAT) distribution in rapid and slow acetylator rabbits. The ratio (rapid/slow) for p-aminobenzoic acid acetylation (a selective substrate for NAT1) was 6 in liver, 1.7-2 in small intestine and 1.3-1.5 in large intestine while the ratio of sulfamethazine acetylation (a selective substrate for NAT2) was 150 in liver, 16-22 in small intestine and 1.8-2.5 in large intestine. The ratios (rapid/slow) for DNA binding of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl and N-hydroxy-4-aminobiphenyl (primarily substrates for OAT) were 82-84 in liver, 13-20 in small intestine and 3.8-5.3 in large intestine and for DNA binding of N-hydroxy-2-acetylamidofluorene (a substrate for AHAT), the ratio was 432 in liver, 32-161 in small intestine and 8.8-13.5 in large intestine. The data show also that NAT1 activity is uniformly distributed along the intestinal tract whereas NAT2 activity is highest in the small intestine. In addition, hepatic and intestinal OAT and AHAT but not NAT1 activities in the rabbit intestine are similarly distributed to activities for NAT2, suggesting that NAT2, OAT and AHAT activities are properties of a single protein in the rapid acetylator phenotype. Moreover, OAT and AHAT activities were much higher in tissues from the rapid than the slow phenotype. The data support the hypothesis that phenotype-dependent metabolic activation of N-OH heterocyclic or aromatic amines to reactive acetoxy metabolites may be involved in the etiology of colorectal cancer.     

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.     

Comparative study on metabolic formation of N-arylformamides and N-arylacetamides from carcinogenic arylamines in mammalian species

The metabolism of carcinogenic arylamines was examined focusing on their N-acylation in mammalian species. When 4-aminobiphenyl, 2-aminonaphthalene, 2-aminofluorene, or 1-aminopyrene was given orally to rabbits, the corresponding N-arylformamides were isolated from the urine together with the corresponding N-arylacetamides. Identification of these N-arylformamides and N-arylacetamides was performed unequivocally by comparing their mass and UV spectra, and thin-layer chromatographic behaviors with those of authentic samples. Such metabolic conversion of the arylamines to the N-arylformamides and N-arylacetamides was also observed in guinea pigs and rats. In addition, carcinogenic nitro compounds such as 4-nitrobiphenyl and 2-nitronaphthalene, which are metabolically reducible to the arylamines, were metabolized to the corresponding N-arylformamides and N-arylacetamides in rabbits. On the other hand, quantitative experiments showed that only minor amounts of the N-arylformamides and N-arylacetamides were excreted in the urine or feces of rats and rabbits given the arylamines. This seems to be due to almost complete further metabolism of these N-acyl derivatives in vivo. Liver cytosols from several mammalian species exhibited a significant N-formylating activity toward the arylamines in the presence of N-formyl-L-kynurenine and N-acetylating activity in the presence of acetyl-CoA. In rabbits, the N-formylating activity was clearly higher than the N-acetylating activity, while the reverse was the case in guinea pigs and hamsters. The experiments with rat liver preparations showed that the liver cytosolic N-formylating and N-acetylating activities are due to formamidase and arylamine acetyltransferase, respectively. Furthermore, enzymatic transfer of the formyl group from one arylamine to another was demonstrated.     

Effect of inhibition of aloe-emodin on N-acetyltransferase activity and gene expression in human malignant melanoma cells (A375.S2)

Arylamine carcinogens and drugs are N-acetylated by cytosolic N-acetyltransferase (NAT), which uses acetyl-coenzyme A as a cofactor. NAT plays an initial role in the metabolism of these arylamine compounds. 2-Aminofluorene is one of the arylamine carcinogens which have been demonstrated to undergo N-acetylation in laboratory animals and humans. Our previous study showed that human cancer cell lines (colon cancer, colo 205; liver cancer, Hep G2; bladder cancer, T24; leukemia, HL-60; prostate cancer, LNCaP; osteogenic sarcoma, U-2 OS; malignant melanoma, A375.S2) displayed NAT activity, which was affected by aloe-emodin in human leukemia cells. The purpose of this study was to determine whether aloe-emodin could affect the enzyme activity and gene expression of NAT at the mRNA and protein levels in malignant human melanoma A375.S2 cells. The results showed that aloe-emodin inhibited NAT1 activity (decreased N-acetylation of 2-aminofluorene) in intact cells in a dose-dependent manner. The effect of aloe-emodin on NAT1 at the protein level was determined by Western blotting and the mRNA levels were examined by polymerase chain reaction (PCR) and cDNA microarray. These results clearly indicate that aloe-emodin inhibits the mRNA expression and enzyme activity of NAT1 in A375.S2 cells.     

Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines.

Epidemiological studies have established the carcinogenic risk of occupational exposure to aromatic amines such as benzidine, beta-naphthylamine, and 4-aminobiphenyl. Metabolic activation of these chemicals to reactive, genotoxic electrophiles, via enzymatic N-oxidation and subsequent conjugation reactions, is necessary for their carcinogenic potential to be realized. Many aromatic amines are mutagenic in prokaryotic test systems, in the presence of exogenous mammalian activating enzymes such as those contained in hepatic 9000 x g supernatant. However, in the Ames (Salmonella typhimurium) assay, induction of mutations by aromatic amines and nitroarenes is also almost completely dependent upon the activity of the endogenous bacterial enzyme, N-acetyltransferase/O-acetyltransferase. The relevance of this assay to the prediction of the carcinogenic potential of aromatic amines in humans is thus restricted by the likelihood that the bacterial and human enzymes possess different substrate specificities. In this paper we report the construction and use of new tester strains of S. typhimurium that express high levels of functional human arylamine N-acetyltransferases, NAT1 and NAT2, retaining characteristic arylamine substrate specificities that are distinct from those of the bacterial enzyme. These new strains support the mutagenic activation of benzidine, 2-aminofluorene and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the Ames test and may provide a new tool for evaluating the carcinogenic potential of aromatic amines.     

Acetyl coenzyme A-dependent metabolic activation of N-hydroxy-3, 2'-dimethyl-4-aminobiphenyl and several carcinogenic N-hydroxy arylamines in relation to tissue and species differences, other acyl donors, and arylhydroxamic acid-dependent acyltransferases

The metabolic activation of several carcinogenic N-hydroxy (N-OH)-arylaminesby cytosolic S-acetyl coenzyme A (AcCoA)-dependent enzymes wasexamined in tissues and species susceptible to arylamine carcinogenesis.Comparisons of the AcCoA-dependent activity were also made withknown cytosolic arylhydroxamic acid-dependent acyltransferasesand with the ability of different acyl donors to mediate thebinding of N-OH-arylamines to DNA. With rat hepatic cytosol,AcCoA-dependent DNA binding was demonstrated for several [³H]N-OH-arylamines,in the order: N-OH-3, 2'-dimethyl-4-aminobiphenyl (N-OH-DMABF),N-OH-2-aminofluorene (N-OH-AF) > N-OH-4-aminobiphenyl >N-OH-N'-acetylbenzidine > N-OH-2-naphthylamine; N-OH-N-methyl-4-amino-azobenzenewas not a substrate. No activity was detected in dog hepaticor bladder cytosol with any of the N-OH-arylamines tested. Usingeither N-OH-DMABP or N-OH-AF and rat hepatic cytosol, activationto DNA-bound products was also detected with acetoacetyl- andpropionyl-CoA but not with folinic acid or six other acyl CoA's.However, p-nitro-phenyl acetate which is known to generate acetyl-enzymeintermediates effectively replaced AcCoA. Subcellular fractionationof rat liver showed that the AcCoA-dependent DNA-binding ofN-OH-DMABP with cytosol was 5 times greater than that obtainedwith the microsomal or mitochondrial/nuclear fractions. Furthermore,the cytosolic activity was insensitive to inhibition by theesterase/deacetylase inhibitor, paraoxon; while the activityof the other subcellular fractions was completely inhibited(>95%). AcCoA-dependent activation of N-OH-DMABP was alsodetected with rat tissue cytosols from intestine, mammary glandand kidney, which like the liver, are targets for arylamine-inducedtumorigenesis. Using N-OH-DMABP, AcCoA-dependent DNA-bindingactivity was also detected in the hepatic cytosols from severalspecies in the order: rabbit > hamster > rat, human >guinea pig > mouse. In contrast, the arylhydroxamic acid,N-OH-N-acetyl-DMABP, was not activated to a DNA-binding metaboliteby the hepatic cytosolic N, O-acyltransferase of any of thesespecies, thus suggesting that the AcCoA-mediated binding ofN-OH-DMABP results from the direct formation of N-acetoxy-DMABP.With N-OH-AF as the substrate, the AcCoA-dependent activationwas in the order: rabbit > guinea pig, hamster > mouse> human, rat. In contrast to the AcCoA-dependent activationof N-OH-AF, only very low N-OH-N-acetyl-4-aminobiphenyl-dependenttransacetylase and N-OH-N-acetyl-2-aminofluorene N, O-acyitransferaseactivity was detected in the hepatic cytosols for the human,guinea pig, and mouse. Selected inhibitors did not discriminatebetween the three acyltransferase activities in rat hepaticcytosol; and up to 40% inhibition was observed with 100 µM4-aminoazobenzene or pentachlorophenol. These studies indicatethat the AcCoA-dependent formation of reactive N-acetoxy arylaminesby cytosolic acetyltransferase(s) could serve as a major metabolicactivation pathway in several species, particularly those whichcannot utilize arylhydrox-amic acids as acyl donors for intramolecularN, O-acyltransfer or for intermolecular transacetylation ofN-OH-arylamines.     

Hepatic microsomal N-glucuronidation and nucleic acid binding of N-hydroxy arylamines in relation to urinary bladder carcinogenesis.

Uridine 5'-diphosphoglucuronic acid-fortified hepatic microsomes from dogs, rats, or humans rapidly metabolized [3H]-N-hydroxy-2-naphthylamine (N-HO-2-NA) to a water-soluble product that yielded 98% of the parent N-hydroxy amine upon treatment with beta-glucuronidase. The metabolite was identified as N-(beta-1-glucosiduronyl)-N-hydroxy-2-naphthylamine from ultraviolet, infrared, and mass spectral analyses of the glucuronide and its nitrone derivative. Incubation of N-hydroxy-1-naphthylamine (N-HO-1-NA), N-hydroxy-4-aminobiphenyl (N-HO-ABP), or the N-hydroxy derivatives of 2-aminofluorene, 4-aminoazobenzene, or N-acetyl-2-aminofluorene with uridine 5'-diphosphoglucuronic acid-fortified hepatic microsomes also yielded water-soluble products. beta-Glucuronidase treatment released 80 to 90% of the [3H]-NHO-1-NA and [3H]-N-HO-ABP conjugates as tritiated ether-extractable derivatives. N-HO-1-NA, N-HO-2-NA, and N-HO-ABP and the glucuronides of these N-hydroxy arylamines were relatively stable and nonreactive near neutral pH. At pH 5, the N-glucuronide of N-HO-2-NA and the presumed N-glucuronides of N-HO-1-NA and N-HO-ABP were rapidly hydrolyzed to the N-hydroxy arylamines that were then converted to reactive derivatives capable of binding covalently to nucleic acids. These data support the concept that arylamine bladder carcinogens are N-oxidized and N-glucuronidated in the liver and that the N-glucuronides are transported to the urinary bladder. The hydrolysis of the glucuronides to N-hydroxy arylamines and the conversion of the latter derivatives to highly reactive electrophilic arylnitrenium ions in the normally acidic urine of dogs and humans may be critical reactions for tumor induction in the urinary bladder.     

Metabolism of aromatic amines: relationships of N-acetylation, O-acetylation, N,O-acetyltransfer and deacetylation in human liver and urinary bladder

N-Acetoxyarylamines are reactive metabolites that are implicatedin the initiation of the carcinogenic process by some N-substitutedaryl compounds. The objective of this study was to explore therelationship between the production of these reactive speciesand N-acetylation (NAT), a reaction previously demonstratedto be polymorphic in the human. Human liver and urinary bladdermucosa samples were frozen within 4–8 h post mortem. Thesetissues were assayed for the (i) O-acetylation (OAT) of N-hydroxy-3,2'-di-methyl-4-aminobiphenyl (N-OH-DMABP) by acetyl CoA, (ii)intramolecular N,O-acetyltransfer (AHAT) of N-hydroxy-2-acetylaminofluorene(N-OH-AAF), (iii) NAT of 2-aminofluorene (2-AF) and p-aminobenzoicacid (PABA) by acetyl CoA and (iv) deacetylation of N-OH-AAF.Cytosolic AHAT and OAT showed partial inhibition by paraoxon.The ratio of paraoxon insensitive AHAT to OAT to NAT of PABAto NAT of 2-AF appears to be 1:2:11:22 using freshly made cytosolsfrom frozen livers. Freezing of the cytosol resulted in extensiveloss of activities. All four of these cytosolic enzyme activitiesexhibited a similar polymorphic response. Microsomal deacetylationshowed a monomorphic response. Similar to the liver, urinarybladder epithelial cells also catalyzed the same reactions.However, the OAT and AHAT activities were detected mainly inmicrosomes. These data suggest that phenotypically rapid acetylatorshave a greater biochemical potential for the metabolic activationof aromatic amines by pathways that involve O-acetylation.     

Repair synthesis of DNA induced by the urinary N-hydroxy metabolites of carcinogenic arylamines in urothelial cells of susceptible species

Urinary N-hydroxy metabolites of the bladder carcinogens, 2-aminofluorene and 4-aminobiphenyl, were examined for the induction of unscheduled DNA synthesis (UDS) in urothelial cells of several susceptible species. N-Hydroxy-2-aminofluorene, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, and the N-glucuronides of these two hydroxylamines induced UDS in the urothelial cells of dogs, rats, and rabbits. N-Hydroxy-2-aminonaphthalene, N-hydroxy-2-acetylaminonaphthalene, and the N-glucuronide of the hydroxylamine were not active. The induction of UDS in dog cells by N-OH-AAF or N-acetoxy-2-acetylaminofluorene, but not by N-hydroxy-2-aminofluorene, was inhibited by paraoxon. The microsomal fraction of dog urothelial cells catalyzed the binding of N-OH-AAF to transfer ribonucleic acid; the enzyme activity was completely inhibited by paraoxon, suggesting that N-deacetylase, but not N-,O-acetyltransferase, was responsible for the binding. The O-glucuronide of N-OH-AAF did not induce UDS in the urothelial cells of dogs, rats, or rabbits, nor did it bind to tRNA in the presence of dog urothelial enzymes, which suggest that N-OH-AAF is detoxified by O-glucuronidation. These results are consistent with the hypothesis that nonacetylated, N-hydroxylated metabolites play a major role in arylamine-induced bladder carcinogenesis. The importance of arylacethydroxamic acid metabolites in bladder carcinogenesis for various species may be inversely related to the rate of hepatic O-glucuronidation.     

The S-acetyl coenzyme A-dependent metabolic activation of the carcinogen N-hydroxy-2-aminofluorene by human liver cytosol and its relationship to the aromatic amine N-acetyltransferase phenotype

A genetic polymorphism in the enzymatic N-acetylation of sulfamethazineand other drugs in humans is well known and has been relatedto differential susceptibility to drug toxicities. Carcinogenicaromatic amines such as 2-aminofluorene also undergo N-acetylation,and phenotypic slow acetylator individuals have been suggestedto be at increased risk to arylamine-induced urinary bladdercancer. However, acetyltransferases have also been shown tocatalyze a final metabolic activation step in the conversionof both hydroxamic acid (e.g. N-hydroxy-N-acetyl-2-aminofluoreneN,O-acyltransferase) and N-hydroxy-arylamine (e.g. N-hydroxy-2-aminofluoreneO-acetyltransferase) metabolites to DNA-bound adducts. In thisregard, rapid acetylators have recently been reported to beat higher risk for colorectal cancer. In this study, we examinedthe enzymatic activity of 35 human liver cytosol samples (obtainedsurgically from organ donors) for sulfamethazine and 2-aminofluoreneN-acetyltransferase activities, N-hydroxy-N-acetyl-2-aminofluoreneN,O-acyltransferase activity, and the acetyl coenzyme A (CoA)-dependentO-acetylation of N-hydroxy-2-aminofluorene to form DNA- boundproducts. The results with sulfamethazine indicated that abouttwo-thirds of the human liver samples were of the slow acetylatorphenotype; the same individuals also exhibited levels of 2-aminofluoreneN-acetylation that were consistent with their respective sulfamethazine-N-acetylationactivity. N-Hydroxy-N-acetyl-2-aminofluorene N,O-acyltransferaseactivity was not detected. However, the acetyl CoA-dependentactivation of N-hydroxy-2-aminofluorene was observed for nearlyall of the samples and was consistently higher in the fast acetylatorgroup. These data support the hypothesis that phenotypic rapidacetylator individuals are likely to be at higher risk to aromaticamine-induced cancers in those tissues containing appreciablelevels of N-hydroxy arylamine O-acetyltransferase.     

N-acetyltransferase 2 and bladder cancer: an overview and consideration of the evidence for gene-environment interaction

Genetic polymorphism of the carcinogen metabolizing enzyme N-acetyl transferase 2 (NAT2) may influence susceptibility to bladder cancers related to smoking or to occupational exposure to arylamine carcinogens. This article reviews the results of 21 published case-control studies of NAT2 polymorphism and bladder-cancer risk, with a total of 2700 cases and 3426 controls. The published evidence suggests that NAT2 slow acetylator phenotype or genotype may be associated with a small increase in bladder cancer risk. However, given the possibility of selective publication of results from studies that found an excess risk, the current evidence is not sufficient to conclude that there is a real increase in risk. Only five of the 21 studies reported results separately for the effect of NAT2 on bladder cancer risk in smokers and non-smokers. Although the results suggest that the effect may be greater in smokers than in non-smokers, the possibility of publication bias makes these results difficult to interpret. There was insufficient evidence to assess the joint effect of NAT2 and occupational exposure to arylamines on bladder cancer risk. Even if estimates of the effect of NAT2 from published data are correct, studies with around 3000-5000 cases will be needed to confirm them.     

Acetyl coenzyme A-dependent binding of carcinogenic and mutagenic dinitropyrenes to DNA

Dinitropyrenes are mutagenic and carcinogenic environmental pollutants found in diesel emissions and urban air particulates. In Salmonella typhimurium these compounds appear to be activated to mutagens by sequential nitroreduction and acetylation. We have examined whether or not similar activation pathways occur with mammalian nitroreductases and acetylases. When rat liver cytosol, NADPH and calf thymus DNA were incubated with [4,5,9,10(-3)H]1-nitropyrene, [4,5,9,10(-3)H]1,3-, 1,6- or 1,8-dinitropyrene very low levels of nitrated pyrene binding with DNA were detected. Addition of acetyl coenzyme A (AcCoA) to these incubations increased the binding of dinitropyrenes 20- to 40-fold while the binding of 1-nitropyrene was not affected. The extent of AcCoA-dependent binding of dinitropyrenes reflected the amount of nitroreduction, as measured by aminonitropyrene formation. However, the increase in binding of dinitropyrenes to DNA in the presence of AcCoA did not occur with dog liver cytosol which is known to be deficient in N-acetylases. These results suggest that cytosolic nitroreductases catalyze the formation of N-hydroxy arylamine intermediates which in the case of dinitropyrenes are converted to reactive N-acetoxy arylamines by cytosolic AcCoA-dependent acetylases.