(CANCER RESEARCH 51. 549-555. January 15, 1991]

Acetylator Genotype-dependent Expression of Arylamine /V-Acetyltransferase inHuman Colon Cytosol from Non-Cancer and Colorectal Cancer Patients'

Ward G. Kirlin,2 Fredrick Ogolla,3 Allen F. Andrews,4 Alma Trinidad,5 Ronald J. Ferguson,6 Tokunbo Yerokun,7Mandi Mpezo,8 and David VV.Hein

Departments of Pharmacology, ¡WarehouseSchool of Medicine, Allanta, Georgia 30310 and University of North Dakota School of Medicine, Grand Forks, North Dakota58203 ID. H: H.I

ABSTRACT

Human epidemiological studies suggest an association between rapidacetylator phenotype and colorectal cancer. Acetylator genotype-dependent expression by the human colon of arylamine /V-acetylation capacity,

catalyzed by acetyl coenzyme A-dependent /V-acetyltransferase(s) (EC

2.3.1.5) (NAT), may be an important risk factor in the initiation ofcolorectal cancer. Human colon cytosols from 48 fresh surgical sampleswere investigated for NAT activity toward p-aminobenzoic acid and thearylamine carcinogens 4-aminobiphenyl, 2-aminofluorene, and /3-naph-

thylamine. Apparent Vmaxdeterminations of NAT activity toward thesesubstrates indicated that 40 of these colons segregated into 3 distinctphenotypes. The distribution of the patients into rapid (5), intermediate(18), or slow (17) acetylators is a ratio that is not significantly differentfrom the expected Hardy-Weinberg distribution of 3:16:21 (x2 = 2.206,

P = 0.363). Significantly greater mean apparent Vm,.

HUMAN COLON /V-ACETYLATION POLYMORPHISM

Two human epidemiological reports have suggested an association between the rapid acetylator phenotype and risk ofcolorectal cancer (27, 28). The hypothesis for an associationbetween acetylator phenotype and colorectal cancer suggests anacetylator genotype-dependent expression of arylamine /V-ace-tylation capacity. To test this hypothesis, we have investigatedthe variation in acetyltransferase activity from human colonsamples drawn from a population of colorectal cancer and non-cancer patients.

MATERIALS AND METHODS

Chemicals. ABP, BNA. PABA, ATP, dithiothreitol. EDTA, PMSF.S-AcCoA, and S-AcCoA synthetase (EC 6.2.1.1) were obtained fromSigma Chemical Co., St. Louis, MO. AF and N-OH-AAF were obtainedfrom Chemsyn Science Laboratories, Lenexa, KS. Sodium ['H]acctate

(4.5 Ci/mmol) was obtained from Dupont/New England Nuclear Co..Boston, MA.

Preparation of Cytosol. Forty-eight human colon samples were obtained from surgical samples through the National Disease ResearchInterchange, Philadelphia, PA. Thirty-six colons were from Caucasianpatients, eight were from African-Americans, and four were frompatients of unknown race. Colon samples were snap-frozen, placed at-70°Cwithin l h of removal, and shipped overnight on dry ice. Portions

of the frozen colons were thawed, minced, and homogenized (25% w/v) in ice-cold 20 mM potassium phosphate buffer (pH 7.4). 1 mMdithiothreitol. 1 mM EDTA, and 50 p\t phenylmethylsulfonyl fluoridein a motor-driven homogenizer (Biospect Products, Bartlesville, OK).The unfractionated homogenate, including a mixture of cell types, wassubjected to sequential centrifugation at 10,000 x # for 20 min, followedby 105,000 x g for 60 min. The supernatant (cytosol) was isolated andassayed for enzymatic activities and protein concentration as outlinedbelow.

Measurement of AcCoA-dependent PABA NAT Activity. AcCoA-dependent PABA NAT activity was determined as described previouslyforp-aminosalicylic acid (29) by measuring the disappearance of PABA,as reflected by decreasing Schiffs base formation with p-dimethylami-nobenzaldehyde. Absorbance was measured at 460 nm in a Perkin-Eltner Lambda 3A spectrophotometer. Apparent A'mand Vm,xdeter

minations for the substrate PABA were carried out with initial concentrations in the reaction mixture of 50-500 ¿IMPABA and 1 mM AcCoA.AcCoA apparent Kmand Vmaxwere determined with initial concentrations of 50-1500 n\\ AcCoA in the presence of 222 ^M PABA.

Measurement of AcCoA-dependent ABP, AF, and BNA NAT Activity. AcCoA-dependent ABP, AF, and BNA NAT activities were determined by a modification of the method of Andres et al. (30) as describedpreviously (23). The AcCoA-dependent formation of ['Hjarylamideproduct was measured in the assay utilizing |'H]acetate in a ['HjAcCoAgenerating system. All assays were carried out in duplicate at 37°Cwith

cytosolic protein concentrations of 1-3 mg/ml. Sample radioactivitywas measured in a Beckman LS5801 scintillation counter and enzymeactivity was calculated as reported previously (30). Apparent Km andVm„determinations were carried out with arylamine concentrations of20-500 MMin the presence of 1 mM AcCoA.

Measurement of N-OH-AAF Sulfotransferase Activity. Twenty-onecolon samples were tested for sulfotransferase activity simultaneouslywith NAT activity determinations. Assays were carried out at 37°C

with cytosolic protein concentrations of 0.5-2 mg/ml as describedpreviously (31). The procedure monitors the rate of release of p-nitrophenyl sulfate, which is used as the sulfate donor for the synthesisin situ of 3'-phosphoadenosine 5'-phosphosulfate from adenosine3',5'-diphosphate. Initial concentrations in the reaction mixture were10 mM for /7-nitrophenyl sulfate, 20 ^M for adenosine 3'.5'-diphos-

phate, and 0.5 mM for N-OH-AAF.Measurement of Protein Concentrations. Protein concentrations in

enzyme assays were determined by the dye-binding method of Bradford(32).

Determination of Kinetic Constants of NAT Activities. Michaelis-Menten kinetic constants were determined by ENZFITTER nonlinearregression data analysis (33) and Eadie-Hofstee analysis by linearregression (34). The standard error of the mean of apparent Vma

HUMAN COLON .V-ACETYLATION POLYMORPHISM

Mean ±SD was calculated for apparent Vmaxand K„levels withinthe rapid, intermediate, and slow acetylator groups and compared forsignificant differences between acetylator phenotypes by Newman-Keuls multiple range tests.

RESULTS

Distribution of NAT Michaelis-Menten Kinetic Constants inHuman Colon Cytosol. Human colon cytosols from forty-eightindividuals were tested for NAT activity toward varying concentrations of PABA, ABP, AF, and BNA. The colon sampleswere ordered by NAT apparent Vmaxactivities towards the 4substrates, and 40 samples were found to be consistent in theirrelative magnitude of NAT activity towards all substrates.

The range of apparent Vmaxvalues of these 40 colon sampleswere tested for normal distribution and found to deviate significantly from normality (P < 0.01) for all 4 substrates. Theordered apparent Vm,,xvalues were analyzed for polymorphicdistributions by plotting against ranked normal deviates andthey appeared to segregate into 3 groups, with some overlap ofindividual samples depending on substrate, as depicted inFig. 1.

Each of the 40 colon samples was assigned to slow, intermediate, or rapid groups based on their apparent Vmaxvaluestowards the 4 substrates aided by the graphic analysis of Fig.1. The apparent Vmaxvalues towards the 4 substrates withineach of the 3 groups were retested for normal distribution andnow found not to differ significantly from normality ( P 5*0.14),except for the following: slow group, ABP (P —0.02); intermediate group, BNA (P = 0.002); and rapid group, PABA(P = 0.04).

The apparent Vmaxvalues segregated into a ratio of 17 slow,18 intermediate, and 5 rapid acetylator phenotypes (Fig. 2),which is not significantly different from the expected Hardy-Weinberg distribution of21:16:3(x2 = 2.026, P = 0.363) (Table

1).Significant differences were found in comparing mean appar

ent Vmavlevels between slow and intermediate groups (P <0.005) and between intermediate and rapid groups (P< 0.001).Apparent Km levels ranged from 30 to 400 UM for PABA andfrom 10 to 300 ^M for the arylamine carcinogens, across the40 colons. Using the apparent Vmaxvalues to assign each colonsample to an acetylator phenotype, the mean apparent A'm

values were compared for each substrate (Table 2). Significantdifferences were found in comparing slow to rapid acetylatorsfor all substrates (P < 0.01) and intermediate to rapid (P <0.05) (Table 2).

Comparisons of NAT Kinetic Constants between Colon Samples from Non-Cancer and Colorectal Cancer Patients. Of thecolon samples analyzed in Tables 1 and 2, 12 samples werefrom patients with no diagnosed neoplasia and 25 samples werefrom colorectal cancer patients. Distributions of acetylator phenotypes did not differ from expected distributions for eitherpatient group (non-cancer, P = 0.14; colorectal cancer, P =1.00) (Table 1). Comparisons between mean apparent Vma,levels for each acetylator phenotype showed no significantdifferences between non-cancer and cancer patients, with theexception of the substrate PABA (P < 0.005) for the rapidacetylator phenotypes. Comparisons between mean apparentKm levels for each acetylator phenotype also showed no significant differences between non-cancer and cancer patients, withthe exception of the substrate BNA (P < 0.05) for the rapidacetylator phenotypes. However, neither of these differencesare likely to be meaningful, as the sample sizes from bothgroups was only 2.

Colons from Non-Cancer Patients. Within the colons fromnon-cancer patients there was a significant difference in apparent Vmuvof NAT activity between slow and intermediate acetylator groups toward all four substrates ( P < 0.005) andbetween intermediate and rapid acetylator groups (P < 0.01)(Table 1). Apparent ATn,'sdiffered significantly between slow

and rapid acetylator groups for PABA (P < 0.005) and forBNA (P < 0.05) and between intermediate and rapid groupsfor PABA (P < 0.05) (Table 2).

LÜ

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[AF] uMOLAR [BNA] uMOLARFig. 2. Michaelis-Menten saturation curves for NAT activity. Symbols represent the mean apparent Vm„values in nmol/min/mg versus arylamine substrate

concentrations for each acetylator phenotype listed in Tables 1 and 2. Curves are derived from curvilinear regressions of the data.

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HUMAN COLON .V-ACETYLATION POLYMORPHISM

Table 1 \-.-tcetyltransferase velocities in human colon cyfosols

AcetylatorphcnotypeAll

colonsSlowIntermediateRapidNon-cancer

patientsSlowIntermediateRapidApparent

Vm„(nmol/min/mgprotein)''N"171853

72PABA8.26

±2.72'20.12±7.23'48.41

±15.118.58

±2.51''20.30 ±o.-W*38.86

±1.14ABP1.32

±0.561''3.71±1.30'11.80±

5.011.33±

0.03"3.74 ±1.60'9.49

±0.201AF1

.92 ±0.646'5.63±1.54r9.37

±3.512.04

±0.276'

6.11 ±\.S(/8.77±4.60BNA1.40

±0.508'4.02±1.53'11.49

±5.121.33±0.472J

4.04±2.10'9.65

±1.49

Colorectal cancer patientsSlowIntermediateRapid

1310

8.26±2.96*18.03±4.83'

50.95±22.80

1.38 ±0.606"3.44 ±0.825'

9.86 ±3.25

1.98 ±0.659'5.41 ±1.57*7.66 ±0.359

1.46 ±0.525''3.92 ±1.17'

8.84 ±0.793" C'ompared to expected ratios of acetylator phenotype. assuming gene frequencies of 0.72 (slow acctylator) and 0.28 (rapid acctylator). distributions do not differ

iigniFioantly from expected ratios: all colons, 21:16:3 (\~ = 2.026. P = 0.363): non-cancer. 6:5:1 (\- = 3.896. P = 0.143); colorectal cancer. 13:10:2 (\: = 0. P= 1.0).t>\ i . f rv*Mean ±SD.' P< 0.001.d P < 0.005.' P < 0.0005' P 0.20). Meanactivities ±SD of colons from rapid:intermediate:slow acetylators were 1.03 ±0.95:1.78 ±0.88:1.43 ±1.10 nmol/min/mg.Colons from non-cancer patients had mean activities of 1.52 ±

0.77 and those from cancer patients had mean activities of 1.49±1.06.

DISCUSSION

Apparent maximum velocities of NAT activity in the 48colon samples were found not to be normally distributed and40 of these appeared to segregate into three distinct groups.Apparent Vma, levels of NAT activity towards PABA, ABP,AF, and BNA clearly discriminated between slow, intermediate,and rapid acetylator phenotypes in these colon samples. Themean apparent Vmaxactivities towards all four substrates differed significantly between each acetylator phenotype (P <0.005). Two- to 3-fold differences in mean apparent V,mxwereobserved between slow and intermediate acetylator colons and1- to 3-fold differences were observed between intermediate andrapid acetylator samples, dependent on substrate.

In contrast to our previous findings with human bladdercytosols (9) and other studies on human tissue (25, 37-40), theactivities in human colon cytosol suggest a polymorphic distribution of PABA NAT activity. This is in agreement with the

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HUMAN COLON .\-ACETYLATION POLYMORPHISM

2.25

(/)LUr—<

Q

O

oLu

1.75 -t

1.25-t

0.75,-

0.25 --

-0.25 ••

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0.00.5 1.0 1.52.02.53.03.54.04.55.0

SULFOTRANSFERASE(NMOL/MIN/MG PROTEIN)

Fig. 3. Analysis of N-OH-AAF sulfotransfcrasc dislributions. Each symbolrepresents enzyme activity (initial velocities) in nmol/min/mg protein for individual human colon samples versus its ranked normal deviate (sample si/e, 21). fromindividuals with colorectal cancer (•)or no diagnosed neoplasia (O).

report of a wide range of apparent Vmavof PABA NAT activityin human liver surgical samples (41) and a recent study withhuman liver and bladder autopsy samples in which the range ofNAT activities for PABA and AF were comparable within bothtissues but much more widely spread in liver than in bladder(10). These results may reflect differences in NAT isozymestability or tissue-specific expression of NAT activity towardsvarious substrates.

As reviewed previously (25), a large number of in vivo methods are available for distinguishing rapid and slow acetylatorphenotypes. However, they often fail to distinguish the intermediate heterozygous acetylator phenotype. Some early (42-44) and more recent (14, 45-47) in vivo methods resulted inclassification of humans into the three rapid, intermediate, andslow acetylator phenotypes whereas measurement of NAT activities in vitro have heretofore failed to make this classification(9, 10, 37-41). Thus, our findings in human colon representthe first documentation of an acetylator gene dose response inthe expression of human NAT activity in vitro as would beexpected from simple autosomal Mendelian inheritance of twomajor codominant alÃ-elesat a single gene locus. In the inbredhamster model, this locus is designated Pat, with two codominant alÃ-eles(r, rapid acetylator; s, slow acetylator) yieldinghomozygous rapid ( Pat'/Pat') heterozygous intermediate(Pat'/Pat"), and homozygous slow (Pat*/Pat") acetylator gen

otypes. If one assumes the frequency of the major rapid acetylator alÃ-elein Americans of African and European ancestry is0.28 and the corresponding major slow acetylator alÃ-eleis 0.72,then our observed ratios of human colon NAT activities intothe three acetylator phenotypes in our colorectal patients, ournon-cancer patients, and the combined data set are each consistent with the expected Hardy-Weinberg distributions. In

addition, the acetylator genotype expression of arylamine NATactivity in human colons is remarkably similar to recent studiesin hamster colon (24). including the actual levels of arylamineNAT activity measured in each acetylator genotype. The biochemical basis for the polymorphism in NAT activity in theinbred hamster is due to the presence of two distinct isozymes,one of which is acetylator genotype dependent accounting forthe acetylation polymorphism and a second isozyme which isregulated at a gene independent of the Pat locus (48). Kineticdifferences in these two isozymes have been best characterizedin purifications of hamster liver cytosol (49) but also are expressed in hamster bladder cytosol (50).

The kinetic constants reported in this paper describe cytosoliccolon NAT kinetic constants derived from whole homogenates.Thus, the data represent a mixture of tissue and cell types and,most likely, multiple NAT isozymes. This heterogeneity isreflected in the data spread of the kinetic constants determined.The precise number of NAT isozymes and allozymes in humancolon cytosols is unclear. Preliminary data1" suggest that arylamine carcinogens such as AF, ABP, and BNA are /V-acety-lated by two distinct NAT isozymes separable by anion-ex-change chromatography. However, one of the isozymes, whichalso catalyzes PABA, is highly unstable, a finding previouslydocumented for this isozyme in human liver cytosol (38). Asecond, more stable NAT isozyme in human colon cytosol thatalso readily catalyzes the A'-acetylation of arylamine carcinogens has been previously isolated and partially characterized(51). Two human NAT gene loci have been cloned and se-quenced (52-54) and localized to chromosome 8 (54). At thepolymorphic NAT gene locus, a single nucleic acid substitutionresults in the expression of two allozyme forms of polymorphicNAT that differ in a single amino acid yet differ substantiallyin catalytic activity towards polymorphic substrates such as AF(53). Recently, still another mutation at this gene has also beenidentified (55). The polymorphic NAT gene has been amplifiedfrom genomic DNA samples derived from rapid and slowacetylator human colons by the polymerase chain reaction." A

second monomorphic NAT gene locus encodes for NAT(s) thatare regulated independently of the polymorphic NAT gene locus(53, 54). Although some genetic heterogeneity at this locus isto be expected in human populations, it would seem more likelythat they will be expressed as monomorphic (normal) distributions within ethnic groups. Previously studies in the hamstermodel (8, 11, 21, 48-50) suggest that human tissues expresspolymorphic and a monomorphic NAT isozymes that bothcontribute to the A'-acetylation of arylamine carcinogens in

cytosolic preparations. Two NAT isozymes were recently purified from human liver cytosol (56).

In the present study we found a significantly lower affinitytowards NAT in the rapid acetylator colons as compared to theslow acetylator colons for each of the arylamine substrates.Rapid acetylator colons also differed significantly from intermediate acetylators in their apparent Km towards these samesubstrates. In contrast, no differences were observed in theaffinity of NAT for the cofactor AcCoA between the threeacetylator phenotypes. This is similar to findings in humanlivers in which NAT activity from rapid acetylators had higherapparent A'msfor both PABA and sulfamethazine than did NAT

activity from slow acetylators (41). Our findings in humanbladder (9) indicated no significant differences in apparent A",,,

for the arylamine carcinogens between rapid and slow acetyla-

10D. Vv. Hein and VV*.G. Kirlin. unpublished data." R. J. Ferguson. L. S. Miller, and D. \V. Hein. unpublished observations.

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HUMAN COLON .V-ACETYLATION POLYMORPHISM

tors. However, as recently reviewed (57), substrate affinities forNAT have consistently been higher in slow acetylators thanrapid acetylators in humans, rabbits, hamsters, and probablymice.

One of the purposes of this study was to determine whetherthere were differences in the distribution of acetylator pheno-types between samples drawn from colorectal cancer patientsand from non-cancer patients. NAT kinetic constants towardseach of the substrates did not differ between acetylator pheno-types drawn from cancer and non-cancer patient samples. Twoepidemiological studies (27, 28) suggest a predisposition amongrapid acetylator individuals to colorectal cancer. We found nosignificant differences in either population of patients fromexpected distributions. In fact, the ratio of acetylator pheno-types in our colorectal cancer patients (13 slow, 10 intermediate, and 2 rapid) is precisely what would be expected in a samplesize of 25 patients assuming alÃ-elefrequencies of 0.72 for theslow acetylator and 0.28 for the rapid acetylator (Table 1). Incontrast with previous studies (27, 28) we did not find a greaterproportion of colorectal cancer patients as rapid acetylatorscompared to non-cancer patients. However, our study was notdesigned to match colorectal cancer patients with age-matchedcontrols and our sample size is small. Clearly, a number of well-controlled epidemiological studies are needed to resolve thisquestion in humans. Recently, one such study reported nodifference between colon cancer patients and controls in thedistribution of rapid and slow acetylator phenotypes (58).

The significance of the present study is that human colonietissue itself expresses the /V-acetylation polymorphism. Inasmuch as the major arylamine-DNA adducts present in carcinogen target tissues are nonacetylated C-8-substituted deoxygu-anosine derivative (59), it might be concluded that /V-acetylationof arylamines by the colon is a detoxification step. However, apotential activation role for acetyltransferase exists involvingthe formation of/V-acetoxy arylamines either by O-acetylationof N-OH-arylamines or by /V,0-acetyltransfer of arylhydrox-amic acids (9-12). Since there is evidence that N-, O-, and N,O-acetyltransferase reactions can be catalyzed by common polymorphic and/or monomorphic NATs (5), the relative contribution of these activation or deactivation steps must be considered.

The role of acetyltransferases in the activation or deactivationof arylamine carcinogens is complex, as is the relative contribution of hepatic and extrahepatic mechanisms in the processleading to initiation of neoplasia. However, the acetylator genotype-dependent expression of levels of NAT by human colontissue may play an important role in the individual variation insusceptibility to colorectal cancer following exposure to arylamine carcinogens.

ACKNOWLEDGMENTS

The assistance of Tonya Savage, Angela Clark, James Perry. CynthiaWoods, Mark Lee. and Jose Rodriquez in the construction of themanuscript is gratefully acknowledged. Appreciation is also expressedto Dr. Fung-Chang Sung and Dr. Gene McGrady for statistical discussion of the data.

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1991;51:549-555. Cancer Res Ward G. Kirlin, Fredrick Ogolla, Allen F. Andrews, et al. and Colorectal Cancer Patients-Acetyltransferase in Human Colon Cytosol from Non-Cancer

NAcetylator Genotype-dependent Expression of Arylamine

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