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Three novel human NAT2 alleles (NAT2*5D, NAT2*6D, and NAT2*14G) were identified and characterized in a yeast expression system. The common rapid (NAT2*4) and slow (NAT2*5B) acetylator human NAT2 alleles were also characterized for comparison. The novel recombinant NAT2 allozymes catalyzed both N- and O-acetyltransferase activities at levels comparable with NAT2 5B and significantly below NAT2 4, suggesting that they confer slow acetylation phenotype. In order to investigate the molecular mechanism of slow acetylation in the novel NAT2 alleles, we assessed mRNA and protein expression levels and protein stability. No differences were observed in NAT2 mRNA expression among the novel alleles, NAT2*4 and NAT2*5B. However NAT2 5B and NAT2 5D, but not NAT2 6D and NAT2 14G protein expression were significantly lower than NAT2 4. In contrast, NAT2 6D was slightly (3.4-fold) and NAT2 14G was substantially (29-fold) less stable than NAT2 4. These results suggest that the 341T --> C (Ile(114) --> Thr) common to the NAT2*5 cluster is sufficient for reduction in NAT2 protein expression, but that mechanisms for slow acetylator phenotype differ for NAT2 alleles that do not contain 341T --> C, such as the NAT2*6 and NAT2*14 clusters. Different mechanisms for slow acetylator phenotype in humans are consistent with multiple slow acetylator phenotypes.

The genetic control of S-acetylcoenzyme A (AcCoA)-dependent N-acetyltransferase activity (EC 2.3.1.5) was investigated in liver, intestine, kidney, and lung cytosols derived from homozygous rapid acetylator (Bio. 87.20), heterozygous acetylator (Bio. 87.20 X 82.73/H F1), and homozygous slow acetylator (Bio. 82.73/H) Syrian inbred hamsters. AcCoA-dependent N-acetyltransferase activity was highest in hepatic cytosol, followed by intestine, kidney, and lung cytosol. In each of these tissues, cytosolic N-acetyltransferase exhibited an acetylator genotype-dependent activity with highest levels in homozygous rapid, intermediate levels in heterozygous F1 progeny, and lowest levels in homozygous slow acetylators. The ratio of N-acetyltransferase activity between acetylator genotypes was in general substrate dependent but not tissue dependent. Acetylator genotype-dependent N-acetyltransferase activity differences were highest for p-aminobenzoic acid, followed by p-aminosalicylic acid, 2-aminofluorene, and beta-naphthylamine. Expression of isoniazid N-acetyltransferase activity in each tissue was acetylator genotype independent. Determination of Michaelis-Menten kinetic constants in each tissue suggested that p-aminobenzoic acid N-acetyltransferase activity was acetylator genotype-dependent because of catalysis by an isozyme(s) that is both an apparent Km and a Vmax variant. In contrast, the acetylator genotype-independent expression of isoniazid N-acetyltransferase activity in each tissue appeared to result from a common isozyme(s) present in each tissue with equivalent kinetic constants in the two phenotypes. These data suggest that acetylator genotype-dependent expression of AcCoA-dependent N-acetyltransferase activity in extrahepatic tissues may play an important role in hereditary predisposition to toxicity and/or carcinogenesis in extrahepatic organs following exposure to arylamine drugs and foreign chemicals.

Nine mixed agonist-antagonist opioids were evaluated in macaque monkeys for their ability to serve as positive reinforcers and for their discriminative stimulus similarity to etorphine and ethylketazocine. For tests of reinforcing properties, various doses of each drug were substituted for codeine under a fixed-ratio 30 time-out 600 sec schedule of i.v. delivery. Discriminative properties were assessed in separate groups of monkeys for which etorphine and saline, or ethylketazocine and saline, were established as discriminative stimuli for responses maintained under a fixed-ratio 20 schedule of food delivery. Two patterns of reinforcing and discriminative stimulus properties were observed. Buprenorphine, butorphanol, GPA 1657, nalbuphine, propiram and WY 16225 (dezocine) functioned as positive reinforcers and occasioned etorphine-appropriate but not ethylketazocine-appropriate responses. dl-Profadol also functioned as a positive reinforcer; its stereoisomers occasioned etorphine-appropriate but not, in general, ethylketazocine-appropriate responses. In contrast, levallorphan and oxilorphan did not function as positive reinforcers and occasioned ethylketazocine-appropriate but no more than 30% etorphine-appropriate responses. Under these experimental conditions, the reinforcing and discriminative stimulus profiles of the mixed agonist-antagonist opioids paralleled those of etorphine-like (mu) or ethylketazocine-like (kappa) opioid agonists.

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.

New Zealand White rabbits were classified as rapid or slow acetylators by measuring the N-acetylation half-life of i.v. administered sulfadiazine, by pedigree analysis and by determination of liver N-acetyltransferase (NAT) activity in vitro. Slow acetylator rabbits were found to have significantly lower levels of p-aminobenzoic acid NAT activity in vitro than did rapid acetylator rabbits, demonstrating genetic covariation of p-aminobenzoic acid (monomorphic) and isoniazid (polymorphic) NAT activity. Monomorphic and polymorphic NAT activities could not be separated by two extensive purification procedures involving sequential centrifugation, fractional precipitation with ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex or Sephacryl and isoelectric focusing or electrophoresis on polyacrylamide gels. In addition, indistinguishable heat inactivation patterns were demonstrated for the two NAT activities utilizing highly purified NAT preparations at two different temperatures. Each of these findings support the coexistence of monomorphic and polymorphic NAT activities on a common protein.

Acetyl CoA-dependent p-aminobenzoic acid and p-aminosalicylic acid N-acetyltransferase (NAT) activity was determined in peripheral blood and blood cells from homozygous rapid (RR) acetylator (Bio. 87.20) and homozygous slow (rr) acetylator (Bio. 82.73/H) inbred hamsters and in their F1, F2 and backcross progeny. NAT activity was localized primarily in erythrocytes and was acetylator genotype dependent, as highest levels were expressed in homozygous rapid acetylator hamsters, intermediate levels in heterozygous acetylator hamsters and lowest levels in homozygous slow acetylator hamsters. Bio. 87.20 X Bio. 82.73/H F1 progeny expressed a unimodal nonoverlapping distribution of NAT activity intermediate between the RR and rr parentals. F2 generation progeny segregated into three modes (low, intermediate and high) of 21, 42 and 11, which is not significantly different from 1, 2 and 1. Bio. 82.73/H X F1 backcross progeny segregated into two modes (low and intermediate) of 18 and 16, whereas Bio. 87.20 X F1 backcross progeny segregated into two modes (intermediate and high) of 17 and 14, neither of which is significantly different from 1 and 1. These data are consistent with simple autosomal Mendelian inheritance of blood NAT activity by two codominant alleles at a single genetic locus. Partial purification of peripheral blood NAT activity by ion-exchange chromatography yielded separation of two isozymes that both exhibited acetylator genotype-dependent expression with highest activity in RR, intermediate activity in Rr and nondetectable activity in rr genotypes, respectively.

Liver N-acetyltransferase (NAT) preparations (105,000 x g cytosol) were obtained from both sexes of 26 strains of inbred hamsters. Liver NAT activity levels were determined for six arylamine substrates; isoniazid, p-aminobenzoic acid (PABA), p-aminosalicylic acid (PAS), sulfamethazine (SMZ), procainamide and 2-aminofluorene. The N-acetylation of isoniazid, SMZ, procainamide and 2-aminofluorene exhibited a monomorphic expression in the hamster as the genetic variation in NAT activity levels between hamster strains was only about 2-fold for each substrate. In contrast, the N-acetylation of PABA and PAS showed a polymorphic expression in the hamster. Two inbred hamster lines (Bio 1.5 and Bio 82.73) had over 400-fold lower PABA NAT and over 20-fold lower PAS NAT activity levels than did the other inbred strains. In addition, the genetically determined N-acetylation differences between the rapid and slow acetylator hamster strains were also demonstrated in vivo for PABA but not for SMZ. Comparison of Michaelis-Menten kinetic constants of PABA NAT activity in a rapid and slow acetylator strain showed a 20-fold lower Km in the rapid acetylator strain suggesting an intrinsic structural difference in rapid and slow acetylator hamster liver NAT. Thus, the pharmacogenetic expression of the N-acetylation polymorphism is quite unique in the inbred hamster, in that PABA and PAS are genetically polymorphic substrates, whereas isoniazid, SMZ, procainamide and 2-aminofluorene are monomorphic, in direct contrast to man and rabbit.

Liver and gut mucosa N-acetyltransferase (NAT) cytosol (105,000 x g) was prepared from selected lines of New Zealand White rapid and slow acetylator rabbits bred and housed at the University of Michigan, and from inbred and partially inbred rabbits obtained from The Jackson Laboratory. Liver NAT activity was determined with p-aminobenzoic acid, p-aminosalicyclic acid, procainamide, sulfamethazine, isoniazid and 2-aminofluorene as substrates. Gut mucosal NAT activity was determined with 2-aminofluorene. A gene dose-response relationship was observed for both liver NAT and gut mucosa NAT with all substrates tested. Highest levels were always observed in homozygous rapid acetylator inbred strains (B/J, III/J, IIIC/J, III/DwJ, IIIEP/J and IIIVO/J), lower levels in obligate heterozygous rapid acetylator rabbits and lowest levels in homozygous slow acetylator inbred (ACEP/J, III/cdJ, IIIVO/ahJ, and IIIVO/vptJ) and outbred rabbits. The differences in magnitude of liver NAT activity level between acetylator genotypes was dependent on the substrate employed, progressively increasing in the following order: p-aminobenzoic acid, p-aminosalicyclic acid, procainamide, sulfamethazine, isoniazid, 2-aminofluorene. The determination of kinetic constants for liver p-aminosalicyclic acid NAT activity indicated a 2-fold difference in apparent Vmax between rapid acetylator genotypes and a 30-fold difference between rapid and slow acetylator phenotypes. In addition, the apparent Km for p-aminosalicyclic acid was significantly lower in the slow acetylators than in the rapid acetylators.

p-Aminobenzoic acid and p-aminosalicylic acid-N-acetyltransferase activities were determined in 105,000 X g liver cytosols from homozygous rapid acetylator (Bio. 4.24, Bio. 41.56 and Bio. 65.67) and homozygous slow acetylator (Bio. 1.5) inbred hamsters. Liver N-acetyltransferase activities were substantially higher in rapid as opposed to slow acetylator hamsters. Genetic crosses yielded F1 generation progeny with unimodal distributions of N-acetyltransferase activity intermediate between those of the rapid and slow acetylator parental strains, and F2 generation progeny with trimodal distributions (low/intermediate/high) of N-acetyltransferase activity not significantly different from a ratio of 1/2/1. Backcross matings yielded bimodal distributions of N-acetyltransferase activity in a ratio (intermediate/parental) not significantly different from 1/1. Each of these findings is consistent with simple autosomal Mendelian inheritance of two codominant alleles at a single gene locus.

Cytosolic acetyl coenzyme A-dependent N-acetyltransferase enzyme (E.C. 2.3.1.5) from inbred hamsters of each acetylator genotype was partially purified by ion-exchange chromatography. Two distinct N-acetyltransferase enzymes were identified; the first one exhibited a polymorphic expression across acetylator genotypes whereas the second exhibited a monomorphic expression. The monomorphic enzyme had consistently high isoniazid and procainamide N-acetyltransferase activity in homozygous rapid acetylator (Bio. 87.20), homozygous slow acetylator (Bio. 82.73/H), obligate heterozygous rapid acetylator F1 progeny and in each of the three acetylator genotypes of F2 progeny. In contrast, the polymorphic enzyme exhibited marked acetylator genotype-dependent N-acetyltransferase activity. High p-aminobenzoic acid N-acetyltransferase activity was exhibited in the homozygous rapid acetylator parental and F2 generation progeny, intermediate activity was exhibited in the heterozygous rapid acetylator F1 and F2 generation progeny and very low or nondetectable activity was exhibited in homozygous slow acetylator parental and F2 generation progeny. A similar gene dose-response relationship was exhibited by the polymorphic N-acetyltransferase enzyme toward isoniazid and procainamide. These results provide new insight into the biochemical basis for the substrate-dependent polymorphic and monomorphic expression of N-acetylation capacity in the inbred hamster.

The levels of covalently bound arylamine-hemoglobin and DNA adduct formation were used as dosimeters to measure the effect of acetylator genotype and sex on the metabolic conversion of the carcinogen, 2-aminofluorene, to reactive intermediates. A single high dose of 2-aminofluorene (60 mg/kg b.wt. i.p.) was administered to male and female homozygous rapid (Patr/Patr) acetylator hamsters (MHA/SsLaK) and homozygous slow (Pats/Pats) acetylator hamsters (Bio. 82.73/H). By using 32P-postlabeling assay methodology, a sole nonacetylated DNA adduct, which cochromatographed with authentic N-(deoxyguanosin-8-yl)-2-aminofluorene was detected at 3, 6, 12, 18 or 24 hr postdosing in liver and urinary bladder DNA of both rapid and slow acetylator hamsters. The highest levels were detected at 18 hr post 2-aminofluorene injection at which time the average levels of hepatic 2-aminofluorene-DNA adducts were similar between male and female rapid and slow acetylators. By comparison, the levels of 2-aminofluorene-DNA adducts in the urinary bladder at 18 hr were about 4-fold lower than in the liver, and were significantly greater in homozygous rapid than in homozygous slow acetylator counterparts (P less than .01). In both the liver and urinary bladder, the levels of 2-aminofluorene-DNA adducts were independent of sex. In contrast to the DNA adduct data, the levels of 2-aminofluorene-hemoglobin adducts, evaluated by capillary gas chromatography-mass spectrometry, were significantly higher in the homozygous slow acetylators than in homozygous rapid acetylators. However, there again were no differences between males and females.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Toxicity experiments carried out with rapid and slow acetylator rabbits showed that the slow acetylators exhibited greater susceptibility to lethal central nervous system actions of isoniazid and its metabolite, monoacetylhydrazine. Slow acetylator rabbits receiving a daily 30-mg/kg dose of isoniazid survived an average of 3.4 weeks, whereas rapid acetylators presented no signs of toxicity for the duration of the experiment (12 weeks). Concomitant administration of vitamin B6 (pyridoxine hydrochloride) raised the dose necessary for lethal convulsions by these agents in both rapid and slow acetylators. Inasmuch as isoniazid and other hydrazine drugs and foreign chemicals may produce a vitamin B6 deficiency in humans that can elicit seizures and lethal convulsions at high dose levels, these results suggest that the central nervous system toxicity produced by these agents might be more frequent and more severe in human slow acetylators.

Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in both rodent and human carcinogenesis. The colon carcinogen 3,2'-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 hamster colon for the first time and compared the ability of DMAB to induce ACF in homozygous rapid and slow acetylator congenic Syrian hamsters (Bio 1.5/H-NAT2r and Bio 1.5/H-NAT2s, respectively), differing only at the NAT2 gene locus and other closely linked loci. 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 injection ACF were observed in the DMAB treated hamsters, but not in the controls. However, the number of ACF was three times higher (P=0.016) in the colons of the NAT2r hamsters compared with the colons of the NAT2s hamsters. In the two congenic hamster lines we also studied the induction of ACF with 1,2-dimethylhydrazine (DMH) treatment, a colon carcinogen not metabolized by NAT2. Hamsters given DMH (25 mg/kg body weight s.c.), once a week for 3 weeks, showed ACF induction in the colon after 10 weeks, but there was no difference between the NAT2r and NAT2s hamsters. Further scanning electron microscopic and histological examination of ACF observed with the light microscope, revealed the same gross morphology and therefore confirmed the basis for the scoring of ACF. The ACF in hamster colons were principle similar to the lesions observed in other species.