Arylamine N-acetyltransferase 1 (NAT1) genotypes in a Lebanese population.

The frequency distributions of human N-acetyltransferase 1 (NAT1*) alleles in various ethnic groups are largely unknown. This lack of information is in contrast to the many studies of ethnic differences in NAT2* alleles and phenotypes. Increasing interest in NAT1 due to its potential roles in carcinogen metabolism and cancer risk makes it desirable to know the distribution of NAT1* alleles in various populations. Using a polymerase chain reaction-restriction fragment length polymorphism genotyping assay, the frequency of NAT1* alleles in a Lebanese population was determined. Of 84 NAT1* alleles assayed, 56% were NAT1*4. Alleles NAT1*3, *10, and *14 were found at frequencies of 0.036, 0.107, and 0.238, respectively. Five additional alleles (6%) differed from previously reported alleles. Nearly 50% of the population were heterozygous for a NAT1*14 allele. The unusually high frequency of NAT1*14 alleles in Lebanese may be useful for epidemiological studies of the effects of the NAT1 polymorphism in this population.

Tissue- and gender-specific expression of N-acetyltransferase 2 (Nat2*) during development of the outbred mouse strain CD-1.

The human N-acetyltransferase (Nat2) genetic polymorphisms have been modeled in mouse strains. We determined the phenotype and genotype of the N-acetyltransferase 2 (Nat2*) gene among outbred CD-1 mice and found a mixed population of heterozygous and rapid and slow homozygous genotypes. Phenotypes determined with p-aminobenzoic acid demonstrated complete concordance of slow and rapid genotype and phenotype. The kidney p-aminobenzoic acid/Nat2-acetylating activity of CD-1 female mice showed a 2.5-fold increase at 80 days of age compared with day 1, whereas males showed a 4.3-fold increase at 25 days and a 5.8-fold increase at 80 days. Immunoblot analysis revealed a 2-fold increase in male kidney Nat immunoreactive protein at 80 days of age, whereas no significant differences were detected in female mice. Likewise, the Nat2 mRNA levels determined by ribonuclease protection assay showed an increase in transcript levels in kidney of male mice during postnatal development, whereas they remained unchanged in females. Gender-associated differences of Nat2 activity, protein, and transcript levels were absent in liver. These observations suggest that the increase in Nat2 enzymatic activity in kidney is accomplished by an increase in transcript. We propose that the observed increase in Nat2 transcript expression in male mice may be a result of androgen regulation during development.

Substrate selectivity of mouse N-acetyltransferases 1, 2, and 3 expressed in COS-1 cells.

Two human acetyl-CoA:arylamine N-acetyltransferases (NAT1 and NAT2) have been identified. Therapeutic and carcinogenic agents that are substrates for these isoenzymes (including isoniazid, sulfamethazine, p-aminobenzoic acid, 5-aminosalicyclic acid, and 2-aminofluorene) have been used to evaluate the role of the N-acetylation polymorphisms of NAT1 and NAT2 in the treatment of disease and differential risk of various cancers among individuals of differing acetylator phenotypes. The mouse is frequently used as a model of the human acetylator polymorphism. As three Nat isoenzymes have been identified in mouse, it is necessary to determine the selectivity of mouse Nats toward common NAT substrates. In the present study, Nat1*, Nat2*8, and Nat3* were expressed in COS-1 cells, and their substrate selectivity was evaluated with various substrates. Under the conditions used, mouse Nat2 had 20-, 2.4-, and 5.4-fold higher catalytic activity for p-aminobenzoic acid, 5-aminosalicylic acid, and 2-aminofluorene, respectively, than Nat1. Isoniazid N-acetylation was catalyzed only by mouse Nat1. For the substrates tested in this study, mouse Nat3 exhibited activity only toward 5-aminosalicylic acid and only at 1/20 the activity shown by Nat2. In addition, p-aminobenzoylglutamate, the first endogenous NAT substrate identified, was selective for mouse Nat2. These results further support the functional analogy of mouse Nat2 and human NAT1.

Characterization of a hormone response element in the mouse N-acetyltransferase 2 (Nat2*) promoter.

Multiple variant alleles of the human arylamine N-acetyltransferase genes, NAT1* and NAT2*, alter the capacity of individuals to metabolize arylamines by N-acetylation. Although biochemical and genetic studies have improved our understanding of the molecular basis of the acetylation polymorphism in humans and other mammals, regulation of NAT* gene expression is not understood. In the present study, a segment of the 5'-untranslated region of mouse Nat2* was sequenced and characterized. Primer extension analysis and RNase protection assays exposed multiple transcription initiation sites located 112 to 151 bases upstream of the translational start site. Computer sequence analysis revealed a promoter-like region located within the region 530 bases upstream of the translational start site consisting of TATA boxes, upstream promoter elements such as a CAAT box and Sp1 binding site, regulatory elements such as a palindromic hormone response element (HRE), and enhancer regions such as an AP-1 transcription factor binding site. Transient expression of CAT reporter constructs of the mouse Nat2*-palindromic HRE demonstrated positive regulation of the HSV-thymidine kinase 1 (tk1) promoter and induced the expression of chloramphenicol acetyltransferase (CAT). This induction was initiated by the addition of hormones such as 5alpha-dihydrotestosterone (DHT) or dexamethasone and was entirely dependent on the presence of androgen or glucocorticoid receptors, respectively. Together with recent discoveries regarding the effects of testosterone on the expression of Nat2* in mouse kidney during development, the findings reported in this article suggest that the HRE found in the promoter region of Nat2* is a potential candidate for the mediation of androgenic regulation of Nat2* in mouse kidney.

Activation of heterocyclic amines by combinations of prostaglandin H synthase-1 and -2 with N-acetyltransferase 1 and 2.

Cooking of meats produces several heterocyclic amines which are mutagenic and potentially carcinogenic. We found that metabolic activation of one of these heterocyclic amines, the quinoline derivative 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), can be catalyzed by prostaglandin H synthase (PHS) as well as by CYP1A2. N-Acetyltransferase (NAT) increased IQ-DNA adduct formation by either of these pathways. In sonicate from transiently transfected COS cells, NAT1 increased CYP1A2 catalyzed adduct formation 4-fold while NAT2 increased adduct formation 12-fold. Both expressed human and purified ovine PHS-1 and PHS-2 catalyzed IQ-DNA adduct formation. The presence of NAT1 and NAT2 increased PHS-1 catalyzed adduct formation 2.5- and 4-fold, respectively. PHS-2 catalyzed IQ adduct formation was also enhanced by either NAT. The pyridine derivative, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, also produced by protein pyrolysis, did not form detectable DNA adducts during incubation with PHS. These results indicate that IQ is a substrate for both PHS-1 and PHS-2 and that NAT increases the ability of the resulting IQ metabolites to cause DNA damage. PHS activity, constitutive and induced, as well as NAT polymorphisms should be considered as factors in environmental carcinogenesis.

Prostaglandin H synthases, nonsteroidal anti-inflammatory drugs, and colon cancer.

Members of the structurally diverse class of drugs known as nonsteroidal anti-inflammatory drugs (NSAIDs) have the ability to prevent or reduce the occurrence of colorectal, certain other gastrointestinal, and perhaps other cancers. The anticarcinogenic property of NSAIDs has been shown in epidemiological studies with humans and in experimental carcinogenesis studies with animals. In addition, clinical studies of the human disease familial adenomatous polyposis have demonstrated the efficacy of NSAIDs in mediating regression of colorectal adenomas. The mechanism of the anticarcinogenic effect of these drugs is not known, but most hypotheses have involved the common property of the NSAIDs to inhibit prostaglandin synthase (PHS) enzymes and thereby cause a subsequent reduction in levels of prostaglandins (PG) in tissue. Recent reports have questioned the role of PHS inhibition in the anticarcinogenic activity of NSAIDs by showing that some NSAID-related compounds that are not PHS inhibitors can induce the same anticarcinogenic changes in cell cycle and apoptotic response as the PHS inhibitors. In this review we will examine the evidence that NSAIDs are anticarcinogenic, the evidence supporting PHS as the target of NSAIDs, and the evidence for and against inhibition of PG synthesis as the mechanism of cancer prevention by NSAIDs.

Induction of human prostaglandin endoperoxide H synthase-2 (PHS-2) mRNA by TCDD.

Numerous transcription response elements (e.g. AP-1, AP-2, GRE, CREB, as well as DRE) have been identified in the transcription regulation region of the PHS-2 gene in both mouse and human. The discovery of a DRE in the region raised the possibility that PHS-2 could be induced by TCDD, a dioxin compound. The time course and dose dependence of TCDD induction of PHS-2 mRNA expression were observed in HUVEC, primary human epithelial cells. In the observed time range (0-24 hours) the steady-state mRNA expression levels of PHS-2, as well as of mRNA for CYP1A1, increased with time at a TCDD dose of 20 nM. At the 24 hour time point, TCDD-treated cells displayed significant dose-dependent elevation of PHS-2 over the range of 0-40 nM TCDD. The increases in PHS-2 mRNA in both the time course and dose dependence experiments were consistent with that of CYP1A1. In contrast, mRNA for PHS-1, the constitutively expressed isoform of PHS, did not show significant changes under the conditions tested. These results are the first to indicate that TCDD can elevate PHS-2 mRNA level in a time and dose dependent manner. Further work needs to be done to learn the molecular mechanism of activation of PHS-2 by TCDD and the relation of TCDD action with other regulatory factors in the control of PHS-2 expression.

Kinetics of arylamine N-acetyltransferase in tissues from human breast cancer.

N-Acetyltransferase activity and Michaelis-Menten kinetic constants were determined in cancerous and non-cancerous breast tissues from 30 female patients with breast cancer. The results derived from tissue cytosol showed that 12 rapid, ten intermediate and eight slow acetylators based on p-aminobenzoic acid and 2-aminofluorene for substrates. The mean apparent Km values for the monomorphic substrate p-aminobenzoic acid and polymorphic substrate 2-aminofluorene were: 55.0 +/- 18.7, 114.0 +/- 30.0, and 137.0 +/- 37.2 microM; and 62.5 +/- 23.7, 166.0 +/- 67.0, and 239.0 +/- 76.6 microM for the slow, intermediate, and rapid enzymes, respectively. Compared to the enzymes from slow acetylators, the rapid acetylators exhibited mean apparent Vmax values eight- and ten-fold greater for p-aminobenzoic acid and 2-aminofluorene, respectively. A similar trend was obtained from the blood cytosols of cancerous patients and healthy volunteers. N-Acetyltransferase activity of breast cancerous and non-cancerous tissues were 1.5- and 2.2-fold different between rapid and slow acetylator with p-aminobenzoic acid and 2-aminofluorene as substrates, respectively. In breast cancerous tissues, 75% and 70% of the cytosolic N-acetyltransferase activity were inhibited under 2 mM of tamoxifen as substrates of 2-aminofluorene and p-aminobenzoic acid, respectively. Similar results were also found in non-cancerous tissues and blood samples from breast cancer patients and healthy volunteers. The effect of 1 mM tamoxifen on the N-acetyltransferase activity from breast cancerous tissues with positive estrogen receptor was 1.6-fold higher than that of negative estrogen receptor. This is the first demonstration to show that anti-estrogen drug can affect N-acetyltransferase activity in breast cancerous tissues. Therefore, this finding may provide a clue to the use of tamoxifen in prevention of human breast cancer.

Evidence for arylamine N-acetyltransferase in the nematode Anisakis simplex.

N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Anisakis simplex, a nematode found in the intestine of the salt water fish Trichiurus lepturus. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activity from a number of Anisakis simplex whole tissue homogenizations was found to be 2.89 +/- 0.52 nmol/min per mg for 2-aminofluorene and 2.54 +/- 0.45 nmol/min per mg for p-aminobenzoic acid. The K(m) and Vmax values obtained were 1.06 +/- 0.69 mM and 9.34 +/- 1.94 nmol/min per mg for 2-aminofluorene, and 2.25 +/- 0.10 mM and 14.44 +/- 0.7 nmol/min per mg for p-aminobenzoic acid. The optimal pH value for the enzyme activity was pH 8.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodoacetamide: at 0.25 mM iodoacetamide, activity was reduced 50% and 1.0 mM iodoacetamide inhibits activity more than 90%. Among a series of divalent cations and salts, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. This is the first demonstration of acetyl CoA/arylamine N-acetyltransferase activity in a nematode and extends the number of phyla in which this activity has been found.

Ahr locus phenotype in congenic mice influences hepatic and pulmonary DNA adduct levels of 2-amino-3-methylimidazo[4,5-f]quinoline in the absence of cytochrome P450 induction.

The potent food mutagen/carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) undergoes metabolic N-hydroxylation by cytochromes P450, including cytochrome P450 1A2, followed by generation of an unstable ester catalyzed by acetyltransferases; promutagenic DNA adducts result. Genetic polymorphisms in these enzymes have been implicated in human cancer risk related to arylamine exposure. We investigated the effects of Ahr locus and acetylator polymorphisms on 32P-postlabeled IQ/DNA adducts in lungs and livers of female C57BL/6 mice congenic for slow acetylation and/or Ahr-nonresponsiveness; some groups were pretreated with beta-naphthoflavone (beta NF), a cytochrome P450 1A inducer. Total adducts in lung were doubled by beta NF pretreatment in Ahr-responsive mice only and consisted of < or = 30% adduct 2 and < or = 60% adduct 3. In contrast, in Ahr-nonresponsive mice, adducts 2 and 3 were each < or= 7% of the total. Livers of noninduced Ahr-responsive mice formed 6-18-fold more adducts than those of nonresponsive mice. This striking difference was not due to altered levels of cyp1a-2, as indicated by specific enzyme assays and immunoblotting, and was not accompanied by a comparable increase in the ability of liver preparations to activate IQ to a mutagen in the Ames test. Pretreatment of responsive mice with beta NF to induce cyp1a-1 and cyp1a-2 led to a reduction in liver adduct levels. Acetylation phenotype also had a significant effect in Ahr-responsive mice, with 3-fold more adducts in slow than in rapid acetylators. These results indicate that in uninduced mice, the normal Ah receptor facilitates formation of IQ/DNA adducts in liver and alters the profile of adducts in lung, via an unknown mechanism, whereas the Ah receptor-dependent enzyme induction reduces adducts in liver, probably due to increased detoxification, but increases them in lung.

Kinetics of acetyl coenzyme A:arylamine N-acetyltransferase from rapid and slow acetylator frog tissues.

N-acetyltransferase (NAT) activity was determined in 100 frog (Rana tigrina) livers using 2-aminofluorene and p-aminobenzoic acid as substrates. Overall, the liver NAT activity of the 50 females was higher than the liver NAT activity of the 50 males. The activities (mean +/- SD) of NAT from the bladder, blood, colon, and liver of males was 0.30 +/- 0.11, 0.05 +/- 0.03, 0.09 +/- 0.05, and 0.93 +/- 0.56 nmol/min/mg protein for the acetylation of aminofluorene and 0.29 +/- 0.06, 0.36 +/- 0.04, 0.26 +/- 0.02, and 0.32 +/- 0.14 nmol/min/mg protein for the acetylation of p-aminobenzoic acid. In the bladder, blood, colon, and liver from female frogs, the activities obtained were 1.00 +/- 0.41, 0.52 +/- 0.07, 0.08 +/- 0.05, and 1.27 +/- 0.49 nmol/min/mg protein for aminofluorene and 0.34 +/- 0.12, 0.36 +/- 0.04, 0.34 +/- 0.07, and 0.48 +/- 0.21 nmol/min/mg protein for p-aminobenzoic acid. Kinetic constants for arylamine NAT activity in the blood, liver, bladder, and colon from frogs with rapid, intermediate, and slow acetylator activities were determined. KM and Vmax values for aminofluorene were 2- to 6-fold higher for liver than for the other tissues. KM and Vmax values for p-aminobenzoic acid showed a smaller variation among the tissues examined, with values obtained for the liver and bladder being somewhat higher than the values for the blood and colon. An apparent KM difference for aminofluorene was found in the liver from frogs with high and low acetylator activity. Based on the aminofluorene NAT activity of liver, there seems to be a polymorphism in NAT activity with 4 rapid, 21 intermediate, and 75 slow acetylators among the 100 frogs assayed. Distribution of acetylator phenotypes was similar among the 50 males and 50 females in this study. This is the first demonstration of acetyl coenzyme A:arylamine NAT activity in an amphibian and could lead to the development of a frog model for monitoring the effect of pollution of wetland environments on native species.

Activation of 2-aminofluorene by prostaglandin endoperoxide H synthase-2.

Prostaglandin endoperoxide H synthase is the key enzyme in the conversion of arachidonic acid to tissue prostanoids. Two isoforms of prostaglandin endoperoxide H synthase have been identified: PHS-1 is constitutively expressed in most tissues under normal physiological conditions and PHS-2 is expressed in response to inflammatory agents, tumor promotors, and other agents related to mitogenesis. Previous work demonstrated that PHS-1 can activate arylamine carcinogens. We report here that PHS-2 can also activate an arylamine carcinogen to form DNA adducts. This is shown by: (1) use of purified ovine PHS-2 to form DNA adducts; (2) increased DNA adduct formation, PHS-2 mRNA, and PHS-2 protein after treatment of HUVEC cells with the PHS-2 inducer PMA; and (3) transient expression of PHS-2 cDNA in COS-1 cells gave rise to both elevations of PHS-2 enzyme protein and DNA adduct formation. Finally, two PHS inhibitors, aspirin and indomethacin, showed significant inhibition of PHS-2-mediated DNA adduct formation.

DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in colon, bladder, and kidney of congenic mice differing in Ah responsiveness and N-acetyltransferase genotype.

Heterocyclic amines, suspected as cancer initiators, require metabolic activation to exert genotoxicity. The food carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) undergoes activation via N-hydroxylation by cytochrome P450 1A2, followed by O-esterification by N-acetyltransferase. We examined the effects of the Ah locus and acetylator polymorphisms (implicated in human colon and bladder cancer risk) on levels of 32P-postlabeled IQ-DNA adducts in C57BL/6 mice congenic for slow acetylation and/or Ah nonresponsiveness. Some were pretreated with beta-naphthoflavone (beta NF), an inducer of cytochromes P450 1A. Guanine adducts were detected in all organs, the predominant one corresponding to N2-(deoxyguanosine-8-yl)-IQ. In the kidney, beta NF pretreatment reduced total adducts by 50% in Ah-responsive animals (P = 0.021); the Ah or acetylator phenotype did not otherwise affect total adducts. In the colon of Ah-nonresponsive animals, rapid acetylators had 3-fold more adducts than slow acetylators (P = 0.0001, vehicle-pretreated; P = 0.0031, beta NF-pretreated). In Ah-responsive mice of either acetylator phenotype, beta NF pretreatment reduced total adducts in the colon by 70% (P = 0.0003). A significant interaction of phenotypes occurred in the bladder; beta NF-pretreatment caused a 2.5-fold increase in adducts but only in the Ah-responsive, rapid acetylator mice. In sum, these polymorphisms influenced the level of IQ-DNA adducts in the kidney, urinary bladder, and colon in complex ways.

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 liver and bladder, and also in circulating leukocytes. Evidence is presented here that mouse mononuclear leukocytes (MNL) are capable of metabolizing 2-aminofluorene to DNA-binding metabolites which give rise to the adducts found in the MNL. Both lymphocytes and monocytes were able to acetylate arylamines during 18 h of culture. The degree of acetylation was determined by the N-acetyltransferase genotype of the mice as shown through use of acetylator congenic strains which differ only in the Nat-2 gene. Cultured MNL from rapid acetylator mice (C57BL/6J and A.B6-Natr) produced about twice as much N-acetylaminofluorene from 2-aminofluorene and 6- to 8-fold as much N-acetyl-p-aminobenzoic acid from p-aminobenzoic acid as cells from slow acetylator mice (B6.A-Nat(s) and A/J). Other differences in arylamine metabolism by MNL in culture were observed and shown to be due to genetic factors, currently unidentified, other than N-acetyltransferase. DNA adduct formation following incubation of MNL with the arylamine carcinogen 2-aminofluorene was related to both acetylation capacity and 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 corresponding slow acetylator congenic (B6.A-Nat(s)). Similarly, MNL from rapid acetylator mice with the A/J background (A.B6-Natr) had twice the DNA adduct levels of those from their corresponding slow congenic (A). Adduct levels in MNL from C57BL/6J were nearly the same as those of MNL from A/J, again indicating the involvement of loci other than acetylation in DNA adduct formation. The finding of genetically dependent arylamine carcinogen metabolism and DNA adduct formation in cultured MNL suggests the possibility of using cultured MNL for assessing individual susceptibility to arylamine-induced DNA damage.

Distribution of 2-aminofluorene and p-aminobenzoic acid N-acetyltransferase activity in tissues of C57BL/6J rapid and B6.A-NatS slow acetylator congenic mice.

The distribution of N-acetyltransferase (NAT) activity in 35 tissues of inbred rapid acetylator C57BL/6J and slow acetylator congenic B6.A-NatS mice was determined by incubation of tissue cytosols with 2-aminofluorene or p-aminobenzoic acid followed by HPLC assay. Tissues examined included the gastrointestinal tract, lymphoid tissues, skin, blood components, and other major organs. NAT activity was found in all tissues examined except blood plasma and seminal vesicles. Peyer's patches had the highest activity with either substrate, and lymphoid tissue, in general, was high in NAT activity as was skin and much of the digestive system. The acetylator polymorphism was apparent in most tissues for both p-aminobenzoic acid and 2-aminofluorene. The difference between rapid and slow acetylator phenotypes was usually greater with p-aminobenzoic acid than with 2-aminofluorene. The presence of NAT in the 33 tissues of rapid and slow acetylator mice, as well as the absence of NAT in plasma and seminal vesicles, was confirmed by immunoblots using an anti-NAT antibody raised in rabbits. These results indicate the widespread distribution of NAT activity and the relative abundance of extrahepatic N-acetylation capacity in the mouse.

DNA-carcinogen adducts in circulating leukocytes as indicators of arylamine carcinogen exposure.

DNA-carcinogen adducts in leukocytes and putative target tissues (liver and urinary bladder) of C57BL/6J mice were measured by 32P-postlabeling and HPLC analysis after controlled exposure to the arylamine carcinogen 2-aminofluorene (2-AF). After an acute exposure via ip injection, adducts were detected at 3 hr in leukocytes, liver, and bladder. The disappearance of DNA-carcinogen adducts in liver and leukocytes were parallel over the 24-hr period studied. Following a 7-day continuous exposure to 2-AF via drinking water, adduct levels in leukocytes and target tissues were responsive to dose at 30, 100, and 300 ppm. Adduct levels at the highest dose reached 17,000 fmol/mg DNA in leukocytes, 1900 fmol/mg in liver, and 2300 fmol/mg in bladder. Although adduct levels after 7 days were highest in leukocytes, adducts were not detectable in leukocytes 7 days after discontinuing exposure. In contrast, liver and bladder retained approximately 50 and 75% of their respective adduct levels 7 days after exposure was stopped. The results indicate that circulating leukocytes may be useful as indicators of current exposure to arylamine carcinogens. Circulating leukocytes may also be useful as biological monitors of DNA damage in arylamine target tissues during chronic exposure to these compounds. Some important differences in persistence of DNA-carcinogen adducts between leukocytes and target tissues were observed.

Polymorphic N-acetylation of 2-aminofluorene by cell-free colon extracts from inbred mice.

The increased risk of rapid acetylator humans for the development of colorectal cancer has created interest in experimental animal models to study the relationship of N-acetyltransferase phenotype to colon cancer. Colon cytosols from inbred mouse lines were assayed for the ability to N-acetylate 2-aminofluorene to determine if the mouse model of the N-acetyltransferase polymorphism could be used to study this relationship. The results indicate that the colon acetylcoenzyme A: 2-aminofluorene-N-acetyltransferase activity parallels that of the liver. Colon activity from slow acetylator (A and B6.A) mouse lines is significantly lower than that of rapid acetylator (B6, B6.D, and A.B6) lines. p-Aminobenzoic acid N-acetyltransferase activity also differed between colon cytosols from rapid and slow acetylator strains. Isoniazid acetylation in colon and in liver did not differ between phenotypes. Northern blot analysis demonstrated the presence of mRNA for both NAT-1 and NAT-2 in mouse colon as well as in mouse liver. These results indicate that the N-acetyltransferase polymorphism is expressed in mouse colon when 2-aminofluorene or p-aminobenzoic acid is used as substrate and therefore the mouse may be a model for study of the effect of acetylator phenotype on development of colorectal cancer in humans.

DNA adduct levels in congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) is a human and mouse bladder carcinogen. Epidemiological studies have shown that individuals with a slow acetylator phenotype, especially those exposed to high levels of carcinogenic aromatic amines, show an increased susceptibility to bladder cancer. In order to determine if a slow acetylator phenotype results in increased DNA damage, congenic mouse strains C57BL/6J and B6.A-Nat(s), which differ genetically at the acetyltransferase (EC 2.3.1.5) locus as homozygous rapid (Natr/Natr) and homozygous slow (Nat(s)/Nat(s)) acetylators respectively, were continuously administered 4-ABP.HCl (55-300 p.p.m.) in their drinking water for 28 days. The levels of covalently bound N-(deoxyguanosin-8-yl)-4-ABP-DNA adducts, which are believed to be critical for the initiation of tumors, were quantitated in the liver and bladder by 32P-postlabeling analysis. The levels of the hepatic DNA adduct increased with dose in both sexes, but were independent of the mouse acetylator genotype. At comparable doses, however, the levels of DNA adducts were 2-fold higher in the liver of the female as compared to the male animals. The DNA adducts also increased with dose in bladder of the male mice, but in contrast to the liver, the adduct levels were approximately 2-fold lower in the bladder DNA of the female mice. Also in contrast to the liver, the levels of bladder DNA adducts were significantly higher (P < or = 0.03) in the phenotypic rapid acetylator females compared to the slow acetylators at both 75 and 150 p.p.m. doses; the median levels of adducts were 10-20% higher in the phenotypic slow acetylator male bladders compared to their rapid acetylator counterparts. The results of these studies are consistent with the increased carcinogenicity of 4-ABP to the liver of female mice and the bladder of male mice. They further suggest that factors other than acetylator phenotype limit the extent of DNA adduct formation from 4-ABP in these mice.