Inheritance of acetylator genotype-dependent arylamine N-acetyltransferase in hamster bladder cytosol

Acetyl coenzyme A-dependent arylamine N-acetyltransferase (EC2.3.1.5 [EC] ) was examined in bladder cytosol derived from inbredSyrian hamsters. Expression of N-acetyltransferase activitytowards p-aminobenzoic acid, p-aminosalicylic acid and 2-aminofluorenewas acetylator genotype-dependent. Highest levels of bladderN-acetyltransferase activity were expressed in homozygous rapidacetylator hamsters (Bio. 87.20), lowest levels in homozygousslow acetylator hamsters (Bio. 82.73/H), and intermediate levelsin Bio. 87.20 x Bio. 82.73/H F₁, generation progeny. The N-acetyltransferaseactivity was acetylator genotype-dependent in both epithelialand non-epithelial bladder tissue. Genetic crosses using p-aminobenzoicacid and p-aminosalicylic acid as substrates indicated thatbladder N-acetyltransferase activity is controlled via simpleautosomal Mendelian inheritance of two codominant alleles ata single genetic locus. Acetylator genotype as assessed by bladderN-acetyltransferase activity was completely concordant withacetylator genotype as assessed by liver N-acetyltransferaseactivity. N-Acetyltransferase in slow acetylator bladder cytosolwas both an apparent K_m and V_max variant compared to N-acetyltransferasein rapid acetylator bladder cytosol. These results suggest thatgenetic control of arylamine N-acetyltransferase in bladderurothelium may be a factor in hereditary predisposition to arylamine-inducedbladder cancer.     

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.     

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.     

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.     

Effect of acetylator genotype on 3, 2'-dimethyl-4-aminobiphenyl induced aberrant crypt foci in the colon of hamsters

Aberrant crypt foci (ACF) are assumed to be preneoplastic lesionsin both rodent and human carcinogenesis. The colon carcinogen3,2'-dimethyl-4-aminobiphenyl (DMAB), like other arylamines,undergoes N-acetylation and O-acetylation by polymorphic acetyltransferase(NAT2). In the present study we characterized ACF in hamstercolon for the first time and compared the ability of DMAB toinduce ACF in homozygous rapid and slow acetylator congenicSyrian hamsters (Bio 1.5/H-NAT2^r and Bio 1.5/H-NAT2^s, respectively),differing only at the NAT2 gene locus and other closely linkedloci. The animals received DMAB (75 mg/kg body weight s.c.)or vehicle (PBS/DMSO 1:1) as a control, twice weekly for 2 weeks,then once a week for 4 weeks. Ten weeks after the first injectionACF were observed in the DMAB treated hamsters, but not in thecontrols. However, the number of ACF was three times higher(P = 0.016) in the colons of the NAT2^r hamsters compared withthe colons of the NAT2^shamsters. In the two congenic hamsterlines we also studied the induction of ACF with 1, 2-dimethythydrazine(DMH) treatment, a colon carcinogen not metabolized by NAT2.Hamsters given DMH (25 mg/kg body weight s.c.), once a weekfor 3 weeks, showed ACF induction in the colon after 10 weeks,but there was no difference between the NAT2^r and NAT2^s hamsters.Further scanning electron microscopic and histological examinationof ACF observed with the light microscope, revealed the samegross morphology and therefore confirmed the basis for the scoringof ACF. The ACF in hamster colons were in principle similarto the lesions observed in other species.     

2-Aminofluorene-DNA adduct formation in acetylator congenic mouse lines

The effect of the acetylator polymorphism on hepatic 2-aminofluorene-DNAadduct formation in mice was studied using two recent developmentsfrom our laboratory. Acetylator congenic mouse lines differingfrom their parental inbred lines in N-acetyltransferase activitywere used to separate the effect of the N-acetyltransferasepolymorphism from effects of differences in other geneticallypolymorphic enzymes. DNA adduct formation was used as an indicatorof arylamine induced DNA damage. Adduct formation was measuredby HPLC analysis of 3 nucleotides from hepatic DNA of treatedanimals. At a high dose (60 mg/kg) of 2-aminofluorene for a3 h exposure, rapid acetylator mice (C57BL/6J) accumulated twicethe adducts of slow acetylators (A/J). In acetylator congenicmice this difference increased so that rapid acetylators withthe slow background (A.B6-Nat¹) had 5- to 7-times the DNA damageof the slow acetylator congenic with the rapid background (B6.A-Nat⁵).It was also found that within each mouse line examined, femaleshad higher levels of adduct formation than males. Acetylatorcongenic mouse lines were useful in distinguishing the effectof acetylator genes from the total genetic background. Similarly,congenics were useful in demonstrating the contribution thatenzymes other than N-acetyltransferase make to differences inadduct formation in inbred mouse lines.     

2-Aminofluorene metabolism and DNA adduct formation by mononuclear leukocytes from rapid and slow acetylator mouse strains

Following exposure of mice to the arylamine carcinogen 2-aminofluorene,DNA-carcinogen adducts can be found in the target tissues liverand bladder, and also in circulating leukocytes. Evidence ispresented here that mouse mononuclear leukocytes (MNL) are capableof metabolizing 2-aminofluorene to DNA-binding metabolites whichgive rise to the adducts found in the MNL. Both lymphocytesand monocytes were able to acetylate arylamines during 18 hof culture. The degree of acetytation was determined by theN-acetyltransferase genotype of the mice as shown through useof acetylator congenic strains which differ only in the Nat-2gene. Cultured MNL from rapid acetylator mice (C57BL/6J andA.B6-Nat¹) produced about twice as much N-acetylaminofluorenefrom 2-aminofluorene and 6- to 8-fold as much N-acetyl-p-amino-benzoicacid from p-aminobenzoic acid as cells from slow acetylatormice (B6.A-Nat⁵ and A/J). Other differences in arylamine metabolismby MNL in culture were observed and shown to be due to geneticfactors, currently unidentified, other than N-acetyltransferase.DNA adduct formation following incubation of MNL with the arylaminecarcinogen 2-aminofluorene was related to both acetylation capacityand to other genetic metabolic factors in the mouse genome.MNL from rapid acetylator mice with the C57BL/6J background(B6) had 3-fold the DNA adduct levels of cells from the correspondingslow acetylator congenic (B6.A-Nat^$). Similarly, MNL from rapidacetylator mice with the A/J background (A.B6-Nat^r) had twicethe DNA adduct levels of those from their corresponding slowcongenic (A). Adduct levels in MNL from C57BL/6J were nearlythe same as those of MNL from A/J, again indicating the involvementof loci other than acetylation in DNA adduct formation. Thefinding of genetically dependent arylamine carcinogen metabolismand DNA adduct formation in cultured MNL suggests the possibilityof using cultured MNL for assessing individual susceptibilityto arylamine-induced DNA damage.     

Mutagenicity and in vitro covalent DNA binding of 2-hydroxyamino-3-methylimidazolo [4,5-f]quinoline

The 2-hydroxyamino-3-methylimidazolo[4,5-f]quinoline (N hydroxy-IQ),a metaholite of the food mutagen-carcinogen IQ, was mutagenicto Salmonella TA98 (nitroreductase deficient). When either rathepatic cytosol, NADPH (1 mM) or ascorbate (0.5 mM) wasaddedto the mutagenicity assay, mutagenicity increased up to 15-,10- and 50-fold respectively. In light of the effects of ascorbateand NADPH, it appears likely that hepatic cytosol may containfactors that protect N-hydroxy-IQ from oxidative decomposition.In contrast, hepatic monooxygenase metabolism of N-hydroxy-IQdecreased mutagenicity. When pentachiorophenol, an inhibitorof O-acetyltransferase and sulfotransferase, was added to themutagenicity assay, a dose-dependent inhibition of N-hydroxyIQ mutagenicity was observed. 2,6-Dichloro-4-nitrophenol, amore specific inhibitor of sulfotransferase than O-acetyltransferase,did not inhibit the mutagenicity of N-hydroxy IQ at concentrationswhich appear to selectively inhibit only bacterial sulfotransferase.The data suggest that bacterial O-acetyltransferase rather thansulfotransferase mutagenically activates N-hydroxy-IQ. N-hydroxy-IQcovalently bound to calf thymus DNA in vitro under non-enzymaticconditionsat pH 7.4. Rat hepatic cytosolic O-acetyltransferase and sulfotransferaseenhanced the covalent binding of N-hydroxy IQ to DNA 30- and5-fold respectively. The data suggest that the mutagenicityof N-hydroxy-IQ is due to the reactivity of N-hydroxy-IQ withDNA and the ability of N-hydroxy-IQ to be further activatedby bacterial O-acetyltransferase.     

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.     

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids

Genes for the 290 amino acid, 33–34 kDa cytosolic acetyltransferases(NAT1^* and NAT2^*) from rat and hamster were cloned and expressedin Escherichia coli. Active clones were selected by a simplevisual test for their ability to decolorize 4-aminoazobenzenein bacterial medium by acetylation. These recombinant acetyltransferaseswere analyzed for: (i) N-acetyltransferase, which was assayedby the rate of acetyl coenzyme A-dependent N-acetylation of2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamicacid acyltransferase, assayed by N, O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene.Both NAT2s showed first order increases in N-acetylation rateswith increasing 2-AF or AAB concentrations between 5 and 100µM, with apparent K_m values of 22–32 and 62–138µM respectively. Although under the same conditions theN-acetylation rates for the two NAT1s declined by >50%, below5 µM 2-AF or AAB, the NAT rate data fit Michaelis-MentenKinetics, and the apparent K_m values were 0.2–0.9 µM.For N, O-acetyltransferase, the apparent K_m values of the NAT1swere 6 µM, while the K_m values of the NAT2s were 20- to70-fold higher. SDS-PAGE/Western blot analysis of the recombinantacetyltransferases gave apparent relative molecular weights(MW_r) of 31 kDa for both NAT1s and rat NAT2 and 29 kDa for hamsterNAT2. Comparable MW_r values were observed for native hamsterliver NAT1 and NAT2 and for rat NAT1 under the same conditions.Although we did not detect NAT2-like activity in rat liver cytosolpreviously, the present data show that the rat NAT2^* gene doescode for a functional acetyltransferase, with properties similarto those of hamster liver NAT2. The data also indicate thatat low substrate concentrations, NAT1 would apparently playthe predominant role in vivo in N-acetylation and N, O-acyltransferof aromatic amine derivatives, including their metabolic activationto DNA-reactive agents.     

Escherichia coli lacZ strains engineered for detection of frameshift mutations induced by aromatic amines and nitroaromatic compounds

Escherichia coli lacZ strains CC107-CC111, which detect specificframeshift mutations, were used to study the mutational specificitiesof 2-nitro-3-methylimidazo[4,5-f]quinoline (NO₂-IQ) and rathepatic S9-activated 2-amino-3-methylimidazo[4,5-f]quinoline(IQ). New constructs were made in which UvrABC-dependent excisionrepair was eliminated (strains DJ3107-DJ3111), followed by introductionof plasmid pYG219 conferring acetyl CoA:arylamine N-acetyltransferase/acetylCoA:arylhydroxylamine O-acetyltransferase (NAT/OAT) activity(strains DJ3207-DJ3211). Sensitivity to mutagens was greatlyenhanced. The mutational specificity of NO₂-IQ was identicalto that of the corresponding amine, IQ. The most prominent mutationscaused by the two compounds were     

Purification and characterization of a rat hepatic acetyltransferase that can metabolize aromatic amine derivatives

Rat liver cytosol is capable of N-acetylation of arylamines,O-acetylation of arylhydroxylamines and N, O-acyltransfer ofarylhydroxamic acids. The objective of this study was to characterizethe enzyme(s) responsible for these reactions. A partially purifiedacetyltransferase preparation from rat liver cytosol was usedto produce five mouse monoclonal IgG1s that bound to acetyltransferaseon Western blots and affected one or more of the acetylationreactions. Two immunoaffinity columns were prepared by covalentlycross-linking monoclonal antibodies to protein A - Sepharose.The first column permitted recovery of a single, immunoreactive32 kDa protein that was capable of catalyzing all three reactions,while the second removed all three acetylation activities froma partially purified enzyme preparation and yielded a single,immunoreactive 32 kDa protein on elution. The harsh conditionsnecessary for elution from the latter column precluded recoveryof an active enzyme. Although Western blots from SDS-PAGE atall stages of purification showed a single 32 kDa protein, purificationwas associated with the production of multiple, immunochemicallyreactive peptides with higher pIs. Direct enzymatic assays ofthese immunochemically reactive components after isoelectricfocusing on polyacrylamide gels demonstrated that a single 32kDa, pI 4.5 protein is capable of all three cytosolic acetylationactivities. A second 32 kDa protein, pI 4.8, was able to carryout N-acetylation but not N, O-acetyltransfer. Immunoreactivecomponents with pIs >4.8 that were formed during purificationwere catalytically inactive. However, isoelectric focusing insolution of cytosolic preparations that had been subjected onlyto gel filtration gave a single 32 kD immunoreactive peptidethat was capable of all three acetylation reactions. Bufferconcentration differentially affected the enzymatic activitiesof the enzyme, i.e. as a pH 7.4 buffer was decreased from 50mM sodium pyrophosphate to 2 mM, the ability to N-acetylatearylamines was lowered while the abilities for O-acetylationand N,O-acetyltransfer were unaffected. It has been shown thata single 32 kDa protein carries out all of the acetylation reactionsin rat liver cytosol. Although it cannot be ruled out that othersimilarly sized and closely related enzymes that share antigenicsites are also capable of these acetylation reactions, thesestudies suggest that instabilities of the major peptide responsiblefor these activities, as reflected in changes in isoelectricpoint, may be responsible for changes in the enzymatic potentialsof this peptide.     

Effect of ovariectomy on the in vitro and in vivo activation of carcinogenic N-2-fluorenylhydroxamic acids by rat mammary gland and liver

N-Hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) and its benzamideanalogue N-OH-2-FBA are mammary gland carcinogens in the femaleSprague—Dawley rat. Ovariectomy inhibits tumorigenicityof topically applied N-OH-2-FAA suggesting modulation of carcinogen-activatingenzymes in the gland. This study concerned the activation ofN-OH-2-FBA and N-OH-2-FBA by the mammary gland and liver, achief site of metabolism, from 50-day-old female rats and effectson the activation of ovariectomy performed at 22 days of age.The levels of N-debenzolyation of N-OH-2-FBA to N-hydroxy-N-2-fluor-enamine(N-OH-2-FA), catalyzed by microsomal carboxyl-esterases in mammarygland and liver were similar and increased 1.5- and 1.7-fold,respectively, by ovariectomy. N-Debenzoylating activity in cytosolsof both tissues appeared to be partially of microsomal origin.Mammary gland cytosol contained N-, O- and N,O-acyltransferaseactivities at levels 40–50% those of liver. N-Acyltransferaseactivity was determined via acetyl coenzyme A (AcCoA)-dependentacetylation of 2-FA and a new assay, N-OH-2-FAA-dependent acetylationof 9-oxo-2-FA. The latter activity was decreased in mammarygland by ovariectomy. Microsomal N-acyltransferase activitieswere <36% those of cytosols. AcCoA-dependent binding of N-OH-2-[ring-³H]FBAto DNA, catalyzed by cytosol, was consistent with a two-stepactivation of N-OH-2-FBA involving esterase catalyzed N-debenzoylationto N-OH-2-FA and its O-acyl-transferase-catalyzed acetylationto the electrophilic N-acetoxy-2-FA. O-Acetyltransfer by mammarygland appeared to be rate-limiting since ovariectomy-dependentincreases in N-debenzoylation did not increase binding withS9 fraction. Little or no sulfotransferase-catalyzed bindingof N-OH-2-[ring-³H]FBA-derived N-OH-2-[ring-³H]FA was detectedin the liver or mammary gland cytosol, respectively. The levelof binding of N-OH-2-[ring-³H]FAA to DNA catalyzed by cytosolicN, O-acyltransferase was decreased 23% in mammary gland andincreased 1.2-fold in liver by ovariectomy. ³²P-Postlabelinganalyses indicated a single adduct N-(deoxyguanosin-8-yl)-2-fluorenaminein DNA of both tissues 24 h after one intraperitoneal injectionof N-OH-2-FBA or N-OH-2-FAA. Respective levels were 3.6- and5.5-fold greater in liver than mammary gland. After ovariectomy,the adduct levels from N-OH-2-FBA increased 1.8-fold in mammarygland and from N-OH-2-FAA decreased 50% in both tissues. Thus,the ovariectomy-dependent changes in levels of enzymes activatingN-OH-2-FBA and N-OH-2-FAA were consistent with in vivo DNA adductlevels in the target mammary gland, but not in the liver.     

Kinetics of arylamine N-acetyltransferase in tissues from rapid and slow acetylator mice

Kinetic parameters for arylamine N-acetyltransferase activityin liver, blood, and bladder from C57BL/6J and A/J mouse strainswere determined using an improved assay system, and some deviationswere found from previously reported results. In the presentstudies, blood N-acetyltransferase activity with p-aminobenzoicacid and 2-aminofluorene as substrates was 20-and 10-fold greater,respectively, in C57BL/6J than in A/J mice. Urinary bladderpossessed N-acetyltransferase activity for both 2-aminofluoreneand p-aminobenzoic acid which differed 2-fold, and reflectedthe liver and blood phenotype. An apparent K_m difference for2-aminofluorene was observed between C57BL/6J and A/J liverN-acetyltransferase. Contrary to earlier studies, the liverN-acetyltransferase activity differed 3-fold between the A/Jand C57BL/6J mouse strains, with either p-aminobenzoic acidor 2-amino-fluorene as substrates. Dimethylsulfoxide at concentrationsused in the 2-aminofluorene acetylation assay in earlier studies,inhibited the A/J liver N-acetylfransferase to a greater extentthan the C57BL/6J enzyme, which may have con tributed to thelarger difference in liver NAT activity with 2-aminofluorenereported previously.     

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)     

ACCELERATED PAPER: Lack of association between the polyadenylation polymorphism in the NATl (acetyltransferase 1) gene and colorectal adenomas

Smoking and a high intake of red meat are risk factors for colorectaltumors. These effects could be due to aromatic amine carcinogens.Individual susceptibility to aromatic amines has been relatedto acetylation phenotype, which plays a role in the bioactivationof arylamines. Polymorphisms in both N-acetyltransferase genes,NAT1 and NAT2, have been associated with an increased risk ofcolorectal tumors. We studied the NATl*10 fast acetylator allele(1088 T     

N-Acetyltransferase multiplicity and the bioactivation of N-arylhydroxamic acids by hamster hepatic and intestinal enzymes

The mechanism-based inactivation (suicide inactivation) by N-hydroxyphenacetin(NHP) of N-arylhydroxamic acid N,O acyltransferase (AHAT) andp-aminobenzoic acid N-acetyl transferase (PABA NAT) activitiesof a partially purified hamster liver preparation was investigated.The inactivation of both enzyme activities was irreversible,but a partial protection of PABA NAT could be achieved by inclusionof the nucleophile cysteine in the incubation mixture; cysteinedid not reduce the extent of inactivation of AHAT by NHP. HepaticAHAT and PABA NAT activities were separated by affinity chromatography,and the resolved enzyme activities were subjected to incubationin the presence of NHP, N-hydroxy-2acetamidofluorene (N-OH-AAF),and N-hydroxy-4-acetamidobipheny) (N-OH-AABP); AHAT, but notPABA NAT, was inactivated by NHP, N-OH-AAF and N-OH-AABP. Incubationof hamster heptic PABA NAT with radiolabeled N-OH-AAF resultedin the formation of only 15% as much fluorenylamine-tRNA adductas was formed when N-OH-AAF was bioactivated with hamster hepaticAHAT. Hamster intestinal AHAT and PABA NAT activities also wereresolved by affinity chromatography; the intestinal AHAT fractionswere much more effective than the PABA NAT fractions in bioactivatingN-OH-AAF. These results demonstrated that hamster liver andintestine contain at least two arylamine transacetylating activities,one of which is much more effective than the other in the bioactivationof toxic and carcinogenic N-arythydroxamic acids.     

Metabolic activation of heterocyclic aromatic amines catalyzed by human arylamine N-acetyltransferase isozymes (NAT1 and NAT2) expressed in Salmonella typhimurium

Heterocyclic aromatic amines formed during the cooking of meatand meat-derived products can be activated to reactive metaboliteswhich bind to DNA, induce mutations and cause tumors in animals.A principal route of metabolic activation is N-oxidation tohydroxylamines, and their subsequent activation by acetyltransferase-catalyzedO-acetylation. We have used mutagenicity assays to study O-acetylationof heterocyclic arylhydroxylamines by the two isozymes of humanN-acetyltransferase, NAT1 and NAT2, expressed in Salmonellatyphimurium. N-Acetylation was also examined, using an HPLCmethod. In addition, Salmonella strains with endogenous acetyltransferaseand lacking this activating activity were used. Hydroxylaminesof nine heterocyclic aromatic amines, IQ, isoIQ, MeIQ, MeIQx,NI, PhIP, Glu-P-1, Glu-P-2, and Trp-P-2 were generated in situby rat liver S9 mix. The strains expressing human NAT1 and lackingacetyltransferase activity showing little or no ability to activatethese substrates. The strains expressing human NAT2 and Salmonellaacetyltransferase supported to different extents the activationof all the compounds except PhIP and Trp-P-2. N-Acetylationof IQ, MeIQx and PhIP was slow or not detectable. In conclusion,human NAT2 but not NAT1 can O-acetylate heterocyclic hydroxylamines.NAT2 probably plays a key role in the genotoxic effects of theabove heterocyclic amines except for PhIP and Trp-P-2, whichhave NAT2-independent mutagenic activity.     

The role of acetylation in the mutagenicity of the antitumor agent, batracylin

The role of acetylation in the genotoxicity of the heterocyclicamine, batracylin, was evaluated in Salmonella typhimurium strainsexpressing various levels of N-and O-acetyltransferase activity.A significant correlation was observed between batracylin-inducedmutagenicity and bacterial N-acetyltransferase activity. Strainswith the greatest capacity for N-acetylating batracylin (YG1012 and YG 1024) were the most sensitive to the mutagenic effectsof the drug. The number of revertants/nmol batracylin and theformation of acetylbatracylin were     

Enzymatic acetylation and sulfation of N-hydroxyarylamines in bacteria and rat livers

In mammalian hepatic cytosol both acetyltransferase and sulfotransferaseare involved in the activation of N-hydroxy derivatives of arylaminesand arylamides. The role of acetyltransferase is also shownin Salmonella, whereas no rigid evidence- is provided on therole of sulfotransferase in Salmonella. In Ames mutagenesistest without S9-mix, the number of revertants of Salmonellatyphimurium TA98 induced was 10-fold higher with 2-hydroxyamino-3-methyl-imidazo[4,5-f]quinoune(N-hydroxy-IQ) than with 2-hydroxy-amino-6-niethvldipyrido[l,2-a:3',2'-d]imidazole(N-hydroxy-Glu-P-1). The extents of the binding to calf thymusDNA of N-hydroxy-Glu-P-1 were, however, 3.9 to 8.6-fold higherthan that of N-hydroxy-IQ in both acetyl CoA- and PAPS-fortifiedrat hepatic cytosol systems. To understand the mechanism causingthe apparent discrepancy between the results of the mutationand DNA binding, the activating capacities of cytosols of S.typhimuriumTA98 and TA98/1,8-DNP₆ strains on the binding of N-hydroxy-Ghu-P-1and N-hydroxy-IQ have been examined in comparison with thoseof rat livers. Although both N-hydroxyarylamines were activatedby hepatic cytosols in the presence of PAPS, no significantDNA binding of these N-hydroxyarylamines was detected in thepresence of PAPS and either one of the two strains of bacterialcytosols. In addition, both cytosols of TA98 and TA98/1,8-DNP₆strains showed no measurable activity on the sulfation of p-nitrophenol,suggesting no capacity for sulfotransferase-mediated activationof N-hydroxyarylamines in Salmonella. On the contrary, the extentsof the acetyl CoA-dependent binding of N-hydroxy-IQ in cytosolsof TA98, but not of TA98/1,8-DNP₆, were respectively 6- andWold higher than those in hepatic cytosols of male and femalerats, although the extents of the binding of N-hydroxy-Glu-P-1were rather higher in hepatic than in bacterial cytosols. Inaddition, the covalent binding of N-hydroxy-2-acetylaminofhioreneto DNA was detected in hepatic, but not in bacterial cytosob,although the binding of N-hydroxy-2-aminofluorene was detectablein both hepatic and bacterial cytosols in the presence of acetylCoA. These results indicate that the metabolic activating capacitiesof Salmonella and rat liver cytosols differ qualitatively, andthe difference in the substrate specificity of acetyltransferasebetween Salmonella and rat livers may be involved, in part,in the difference of then- DNA damage in bacteria and mammals.