acetyl coenzyme a dependent activation of w-hydroxy...

[CANCER RESEARCH 46, 4362-4367, September 1986]

Acetyl Coenzyme A Dependent Activation of W-Hydroxy Derivatives of

Carcinogenic Arylamines: Mechanism of Activation, Species Difference,Tissue Distribution, and Acetyl Donor Specificity1

Atsuko Shinohara, Kazuki Saito,2 Yasushi Yamazoe,3 Tetsuya Kamataki,4 and Kvinchi Rato

Department of Pharmacology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

ABSTRACT

Acetyl coenzyme A dependent activation of 2-hydroxyamino-6-meth-yldipyrido|l,2-<i:3',2'-d|imidazole (N-OH-Glu-P-1) and 3-hydroxy-amino-l-methyl-5//-pyrido(4,3-A)indole (N-OH-Trp-P-2) was investigated using cytosols from hepatic and extrahepatic tissues of variousanimal species in comparison with that of Ar-hydroxy-2-aminofluorene.N-OH-Glu-P-1 and N-OH-Trp-P-2 were metabolized to the reactivespecies capable of binding to transfer RNA through a putative (>-acetylation process by liver cytosols. Kidney, small intestinal mucosa,lung, and bladder from hamsters and rats also mediated the reaction,although their activities were lower than that in the liver. Marked speciesdifferences in the enzymatic activities of livers were observed. Hamstersshowed the highest ability in the activation for N-OH-Glu-P-1 and VOH-Trp-P-2, followed by rats. Rabbits with a rapid acetylator phenotype,which showed a high activity in the Â¿V-acetylationof arylamines, activatedN-OH-Glu-P-1 but scarcely N-OH-Trp-P-2. A rabbit with a slow acetylator phenotype, mice, guinea pigs, and a dog showed marginal ornondetectable activities with N-OH-Glu-P-1 and N-OH-Trp-P-2. A typical nonheterocyclic Af-hydroxyarylamine, Ar-hydroxy-2-aminofluorene

was also activated by the acetyl coenzyme A dependent system to anintermediate which bound to transfer RNA. However, the acetyl-CoAdependent binding of yV-hydroxy-2-aminofluorene was markedly differentfrom those observed with N-OH-Glu-P-1 and N-OH-Trp-P-2 concerningthe order of activities among animal species used. In addition to shortchain acyl coenzyme As, JV-hydroxy-2-acetylaminofluorene also servedas an acetyl donor for the activation of N-OH-Glu-P-1 and N-OH-Trp-P-2 in liver cytosol systems. The formation of N-acetyl-N-OH-Glu-P-l,however, was not detected in the cytosolic system of N-OH-Glu-P-1 withacetyl-CoA, suggesting the direct O-acetylation at the \-lmlro\y groupas a major pathway for the activation of /V-hydroxyarylamines.

INTRODUCTION

Carcinogenic-mutagenic heterocyclic arylamines isolatedfrom the pyrolysates of amino acids and proteins, such as Glu-P-15 and Trp-P-2, have been shown to be metabolically activatedto yV-hydroxy derivatives by mammalian cytochrome P-450systems (1-4), similar to those seen with arylamines and amides(5-7). The W-hydroxyarylamines thus formed are activated byenzymatic processes involving esterification such as aminoacyl-ation to reactive species, which are capable of modifying tissuemacromolecules (8, 9). In previous studies we found the activation of N-OH-Glu-P-1 and N-OH-Trp-P-2 by O-acetyltrans-

Received 3/21/85; revised 8/12/85, 1/2/86,4/23/86; accepted 5/23/86.1This research was supported in part by Grants-in-Aid from the Ministry of

Education, Science and Culture and the Ministry of Health and Welfare, Japan,and by additional grants from the Princess Takamatsu Cancer Research Fundand the Nissan Science Foundation.

2 Present address: Department of Plant Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Chiba 260,Japan.

3To whom requests for reprints should be addressed.4 Present address: Department of Analytical Biochemistry, Faculty of Phar

maceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan.'The abbreviations used are: Glu-P-1, 2-amino-6-methyldipyrido[l,2-a:3',2'-

JJimidazole; N-OH-Glu-P-1, 2-hydroxyamino-6-methyldipyrido| 1,2-a:3',2'-</]-imidazole; Trp-P-2, 3-amino-l-methyl-5//-pyrido[4,3-A]indole; N-OH-Trp-P-2,3-hydroxyamino-l -methyl-5//-pyrido[4,3-ftÂ¡indole; 2-AF, 2-aminofluorene; N-OH-AF, ALhydroxy-2-aminofluorene; N-OH-AAF, yV-hydroxy-2-acetylaminoflu-orene; I )'l 1. dithiothreitol; HPLC, high-performance liquid chromatography.

ferase in bacterial cells (10) and we purified the O-acetyltrans-ferase to be named Ar-hydroxyarylamine:acetyl-CoA O-acetyl-transferase (11). We also found that the acetyl-CoA dependentactivation of N-OH-Glu-P-1 and N-OH-Trp-P-2 takes place inmammalian liver cytosol (8, 12,13).

We reported that the 7V-acetylation activities of heterocyclicarylamines, such as Trp-P-2 and Glu-P-1, by liver cytosols werevery low as compared to that of a well-known carcinogenicary hiin inc, 2-AF (14). The results suggest that a sequentialmetabolic pathway, the 7V-acetylation of the arylamines, the N-hydroxylation of arylamides, and the JV,0-acetyl transfer ofarylhydroxamic acids (5, 15-19), as postulated for 2-AF wouldbe less likely to occur for Glu-P-1 and Trp-P-2. Therefore, theO-acetylation of JV-hydroxyarylamines may be an importantpathway for the metabolic activation of these heterocyclic arylamines.

On the acetylation of arylamines, marked differences havebeen noted in both ,/V-acetyltransferase and A^O-acetyltransfer-ase activities among mammalian species and tissues (20, 21).In addition, genetic polymorphism in these activities has beenrecognized in rabbits, mice, hamsters, and humans (22, 28).The analysis of results with species and tissues in the activationof heterocyclic hydroxylamines, such as N-OH-Glu-P-1 and N-OH-Trp-P-2, by O-acetyltransferase can be important in orderto identify the enzyme(s) responsible for the reaction and toclarify the activation mechanisms of these naturally occurringcarcinogens. In this report, acetyl-CoA dependent activation ofN-OH-Glu-P-1 and N-OH-Trp-P-2 was examined in comparison with that of N-OH-AF using various animal species, tissues,and acyl donors to clarify the characteristics of the metabolicactivation pathway of these ./V-hydroxyarylamines.

MATERIALS AND METHODS

Chemicals. [ri/iÂ£-3H]-N-OH-Glu-P-l or [r/ng-3H]-N-OH-Trp-P-2

(1.46 or 0.74 Ci/mmol) was freshly prepared metabolically from[3H]Glu-P-l or [3H]Trp-P-2 using liver microsomes from polychlori-nated biphenyl treated rats in the presence of NADPH (1,4). The N-hydroxylated products were isolated by HPLC and were used afterdilution with the corresponding nonradioactive A'-hydroxyarylaminessynthesized as described previously (29) to the specific activity of SO-SOmCi/mmol. [ri/ig-3H]-N-OH-AF (112 mCi/mmol) and [r/ng-3H]-N-OH-AAF (20.4 Ci/mmol) were generous gifts from Dr. Fred F. Kad-lubar, National Center for Toxicological Research, Jefferson, AR, andDr. Snorri S. Thorgeirsson, National Cancer Institute, Bethesda, MD,respectively. [flcefy/-3H]acetyl-CoA (722.0 mCi/mmol) was obtainedfrom New England Nuclear, Boston, MA; acyl-CoA derivatives, tRNA(type X), and acetohydroxamic acid were from Sigma Chemical Co.,St. Louis, MO; and glass microfiber filter (GF-C, 25 mm) was fromWhatman, Kent, England. Other chemicals were of the highest gradecommercially available.

Synthesis of Af-Acetyl-N-hydroxy-Glu-P-l. N-OH-Glu-P-1 (10 mg),obtained from the reduction of the corresponding nitro derivative asdescribed (29), was dissolved in 10 ml of dry tetrahydrofuran at 0Â°C.

Acetyl chloride in tetrahydrofuran solution was added to the N-OH-Glu-P-1 solution. The reaction was monitored by silica gel thin layer

4362

on June 12, 2019. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

ACETYL-CoA DEPENDENT ACTIVATION OF /V-HYDROXYARYLAMINES

chromatography (Kieselgel 60 F254;Merck Art. 5715) with a solventsystem of chloroform-methanol (20:1). Saturated sodium bicarbonatesolution (10 ml) was added after the consumption of N-OH-Glu-P-1,and the solution was agitated for 30 min. The mixture was extractedtwice with ethyl acetate (10 ml) after the addition of 5 N KOH (1 ml),and then 12 N sulfuric acid was added to change the pH of the solutionto neutral. N-acetyl-N-OH-Glu-P-l in the aqueous phase was extractedwith ethyl acetate and subjected to a preparative thin layer chromatography as described above. The product, N-acetyl-N-OH-Glu-P-l (Rf0.39), was well separated in the thin layer chromatogram from Glu-P-1 (Rf 0.50) and N-acetyl-Glu-P-1 (Rf 0.58). These compounds were alsochecked by the HPLC system described below. The product was reducedto N-acetyl-Glu-P-1 by the treatment with titanium chloride andshowed the absorption maxima at 275 and 361 nm (in methanol),respectively. In addition, the quasimolecular and fragment ions wereobserved at m/z 257 [M + 1]+, 241 [M + 1-16]+, and 199 [M + 1-16-42]* in the mass spectrum which was obtained in a direct inlet chemicalionization mode (Jeol D-300 mass spectrometer). Isobutane was used

as a reagent gas.Preparation of Cytosol. Male Sprague-Dawley rats, male Syrian

golden hamsters, male Hartley guinea pigs, male and female HAI HCAnN x DBA/2N FI mice (6-8 weeks old), male and female NewZealand White rabbits (1.5 years old), and a male Mongolian dog (4months old) were used. The animals were decapitated and livers wereperfused in situ with 50 mM Tris-HCl buffer (pH 7.4) and 1 mM DTT.Tissues were removed and homogenized with 3 volumes of the abovebuffer solution. The cytosol (105,000 x g supernatant fraction) wasprepared as described previously (30). To determine the acetylatorphenotype of the rabbits used, W-acetyltransferase activities of livercytosols were measured using a polymorphic substrate, 2-AF, and amonomorphic substrate, p-aminobenzoic acid (22), as described previously (14). Among five rabbits examined, four animals showed highactivities for 2-AF [12.1 Â±4.1 (SD) nmol/min/mg protein] and oneindividual showed nondetectable activity [<0.05 nmol/min/mg]. Sinceall five rabbits showed similar activities for p-aminobenzoic acid (3.9 Â±0.2 nmol/min/mg), one individual having nondetectable activity for 2-AF was regarded as a slow acetylator.

Activation of JV-Hydroxyarylamines: Nucleic Acid Binding Assay.Nucleic acid binding of AMiydroxyarylamines was assayed by themethod of King (31) with modifications. The standard incubationmixture contained 50 mM Tris-HCl buffer (pH 7.0), 1 mM DTT, tRNA(1 mg/ml), 1 mM acetyl-CoA, 20 /Â¿MA43H]hydroxyarylamine, and

cytosolic protein (0.1 mg/ml) in a final volume of 0.5 ml. Reactionswere started by addition of W-hydroxyarylamine and incubated for 10min at 37Â°C.The reaction was terminated by addition of 1 ml of phenol

saturated with 50 mM Tris-HCl (pH 7.0). The mixture was shaken for5 min and centrifuged at 2500 rpm for 10 min. An aliquot (100 Â¿Â¡llofaqueous layer was pipeted into 5 ml of ethanol containing 2% potassiumacetate over a glass filter. The tRNA precipitate was collected on thefilter and then washed with each 5 ml of 70% ethanol (3 times), ethanol(3 times), acetone, and diethyl ether. The filter was moistened with 150n\ of water in a counting vial. After addition of 1 ml of tissue solubilizer(Protosol; New England Nuclear) and 9 ml of xylene-based scintillator,the radioactivity was measured by a liquid scintillation counter (Beck-man; LS-3800). The recovery of tRNA throughout the precipitationprocedure was 95%. The assay was carried out in duplicate. The bindingactivity is calculated from the radioactivity associated to tRNA recovered from the incubation mixture and is expressed in terms of pmolof arylamine moiety bound to tRNA recovered per min per mg ofprotein (20).

Assay of the Reaction Products. For reaction with N-OH-Glu-P-1,the incubation mixture contained 50 mM Tris-HCl (pH 7.0), 1 mMDTT, 1 mM [3H)acetyl-CoA, 20 JIM N-OH-Glu-P-1, and cytosolic

protein (0.2 mg/ml) in a final volume of 0.1 ml. The reaction wasinitiated by the addition of N-OH-Glu-P-1 and performed at 37'C for

definite times (2, 5, 10, and 20 min). After the reaction was terminatedby the addition of 0.1 ml of acetonitrile, cold N-acetyl-N-OH-Glu-P-lwas added to the mixture as a carrier. The reaction products wereextracted with ethyl acetate (2 ml), evaporated with nitrogen gas, anddissolved in 0.1 ml of acetonitrile. An aliquot (60 /Â¿I)was subjected to

a HPLC (Waters Associates; ALC/GPC 204) equipped with aNucleosilnCig reverse phase column (4 x 300 mm; Macherey-Nagel).A mobile phase of 40% acetonitrile containing 0.07% acetic acid and0.07% acetohydroxamic acid was used at a rate of 1.2 ml/min. Theretention times of N-OH-Glu-P-1, N-acetyl-N-OH-Glu-P-l, Glu-P-1,and N-acetyl-Glu-P-1 were 9.6, 6.7, 11.5, and 9.4 min, respectively.The recovery of N-acetyl-N-OH-Glu-P-l from the HPLC column wasabove 90%. Each fraction of eluent was collected and the radioactivitywas determined by a liquid scintillation counter.

RESULTS

Acetyl-CoA Dependent Binding of /V-Hydroxyarylamines byHamster Liver Cytosol. The tRNA binding of N-OH-Glu-P-1and N-OH-Trp-P-2 was dependent on both acetyl-CoA andliver cytosol of hamsters (Table 1). Previously, Mita et al. (32)reported on the nonenzymatic binding of N-OH-Trp-P-2 toDNA. Actually, both N-OH-Glu-P-1 and N-OH-Trp-P-2 boundto tRNA in the absence of DTT in the incubation mixture.Under the present experimental conditions containing 1 mMDTT, however, the amounts of tRNA binding of these N-hydroxyarylamines were very low without both cytosol andacetyl-CoA (Table 1). When acetyl-CoA was added in theincubations, 30% of complete binding was observed for N-OH-Trp-P-2 without cytosol, indicating that the acetyl-CoA dependent nonenzymatic binding occurred substantially. Non-enzymatic binding of N-OH-AF was much higher than that ofN-OH-Trp-P-2. The amounts of binding were increased markedly by the addition of liver cytosol and acetyl-CoA with theseW-hydroxyarylamines (complete system in Table 1). When aheat denatured cytosol (treated at 100Â°Cfor 10 min) was added

instead of fresh cytosol, these /V-hydroxyarylamines were onlysparsely bound to tRNA. The reaction was inhibited by iodo-acetamide, a sulfliydryl blocking agent, but not by paraoxon, amicrosomal deacetylase inhibitor (33, 34). The amount of Glu-P-1 moiety bound to tRNA measured as the radioactivity increased almost linearly with increases in the amounts of cytosolic protein from hamsters, up to 0.2 mg/ml (Fig. \A). Thus,all experiments described below were carried out at a proteinconcentration of 0.1 mg/ml. The amounts of binding alsodepended on the concentration of N-OH-Glu-P-1 added; theactivity reached a plateau level at about 10 MM(Fig. IB). Similarprofiles were also observed using rat liver cytosol for the activation of N-OH-Glu-P-1 or N-OH-Trp-P-2.

Acetyl-CoA dependent binding of N-OH-Glu-P-1 and N-OH-Trp-P-2 was examined using cytosols of several tissues ofhamsters and rats (Table 2). Although the activity was thehighest in a system with the liver, other tissues also catalyzedthe reaction. The cytosols of kidneys in both hamsters and ratsshowed relatively high activities for N-OH-Glu-P-1. For the

Table 1 Acetyl-CoA dependent binding of N-hydroxyarylamines by liver cytosolfrom hamsters

A complete system consisted of 1 mM acetyl-CoA, cytosolic protein (0. l mg/ml), 20 Â»IMN-hydroxyarylamines, tRNA (l mg/ml), l mM DTT, and 50 mMTris-HCl (pH 7.0) in a final volume of 0.5 ml. The incubation was performed at37" for 10 min. Mean Â±SD from at least three different determinations.

System

Amounts of metabolite bound to tRNA(pmol/min)*

N-OH-Glu-P-1 N-OH-Trp-P-2 N-OH-AF

Complete- Acetyl-CoA- Cytosol- Cytosol and acetyl-CoA32.7

Â±1.00.13 Â±0.030.35 Â±0.130.22 Â±0.077.25

Â±1.40.15 Â±0.082. 16 Â±0.490.64 Â±0.4454.1

Â±12.63.61 Â±1.686.05 Â±1.153.79 Â±0.94

" pmol arylamine moiety bound to tRNA recovered from incubation mixture

per min.

4363



ACETYL-CoA DEPENDENT ACTIVATION OF W-HYDROXYARYLAMINES

1000

(B) 1000(B)

0.25 0.5Cytosolic protein (mg/ml)

10 20N-OH-Glu-P-1 (jjM)

Fig. 1. Effects of concentrations of cytosolic protein (A) and N-OH-Glu-P-1(B) on the acetyl-CoA dependent tRNA binding of N-OH-Glu-P-1 by hamsterliver cytosol. Unless specified, the incubation mixture contained SOm\i TrÃs-HCl(pH 7.0), 1 HIMDTT, tRNA (l mg/ml), l mM acetyl-CoA, cytosolic protein (0.1mg/ml), and 20 MMN-OH-Glu-P-1 in a final volume of 0.5 ml. Data are themeans of duplicate determinations.

Table 2 Tissue differences in the acetyl-CoA dependent binding of N-OH-Glu-P-1 and N-OH-Trp-P-2 by cytosol

The incubation mixture contained cytosolic protein (0.1 mg/ml) of each tissueand other components described in Table 1. Pooled cytosol obtained from tenanimals was used for this experiment. Data are the means of duplicate determinations, and values were subtracted with the amounts of nonenzymatic binding(- cytosol) which were 0.31 pmol/min for N-OH-Glu-P-1 and 1.06 pmol/minfor N-OH-Trp-P-2.

Amounts of metabolite bound to tRNA(pmol/min/mg protein)

TissueHamsterLiverLungKidneySmall

intestinalmucosaBladderRatLiverLungKidneySmall

intestinalmucosaBladderN-OH-Glu-P-1

(%) N-OH-Trp-P-2(%)678.524.094.685.712.8177.534.757.722.325.5(100)(3.5)(13.9)(12.6)(1.9)(100)(19.5)(32.5)(12.6)(14.4)120.33.60.313.41.085.68.715.732.40.1(100)(3.0)(0.2)(11.1)(0.8)(100)(10.2)(18.3)(37.9)(O.I)

Table 3 Acyl donor specificity in tRNA binding of N-OH-Glu-P-1 by liver cytosol

The incubation mixture contained each acyl donor at 1 mM and other components described in Table 1. Data are the means of duplicate determinationsusing cytosol which showed approximately the mean activity among four animals.The values were subtracted with the amounts of nonenzymatic binding (â€”cytosol)which were 0.31, 0.25, 0.24, 0.26, and 0.27 pmol/min for acetyl-, propionyl-,butyryl-, and malonyl-CoA and N-OH-AAF, respectively.

Amount bound to tRNA(pmol/min/mg protein)

AcyldonorAcetyl-CoA

n-Propionyl-CoAn-Butyryl-CoAMalonyl-CoAN-OH-AAFHamster566.8

219.941.732.669.2(%)(100)

(38.8)(7.4)(5.8)

(12.2)Rat125.5

45.711.29.0

28.1(%)(100)

(36.4)(8.9)(7.2)

(22.4)

binding of N-OH-Trp-P-2, cytosol of small intestinal mucosaalso showed the high activity.

Comparison of Acyl Donors. To ascertain cofactor specificityfor the activation of yV-hydroxyarylamines, the ability of severalacyl donors was compared. As shown in Table 3, acetyl-CoAwas the most effective followed by propionyl-CoA. Butyryl-CoA and malonyl-CoA showed low activities. N-OH-AAF is aknown acetyl donor for mammalian yV,0-acetyltransferase,which also served as an acetyl donor.

The effects of the concentration of acetyl-CoA or N-OH-AAF on tRNA binding by hamster liver cytosol were examined

1.0 2.0 3.0Acelyl-CoA (mM)

0.2 0.4 0.6 0.8N-OH-AAF (mM)

Fig. 2. Effects of concentrations of acetyl-CoA (A) and N-OH-AAF (A) on thetRNA binding of N-OH-Glu-P-1 by hamster liver cytosol. The incubation mixturecontained 50 min Tris-HCl (pH 7.0), 1 mM DTT, tRNA (1 mg/ml), cytosolicprotein (0.1 mg/ml), 20 UMN-OH-Glu-P-1, and the indicated concentrations ofacetyl donor in a final volume of 0.5 ml. Data are the means of duplicatedeterminations.

(Fig. 2). The amounts of Glu-P-1 moiety bound to tRNAincreased with an increase in the concentration of acetyl-CoAup to 1.0 mM. The amount bound also increased with theincrease in the concentration of N-OH-AAF up to 0.1 mM buttended to decrease in the range higher than 0.1 mM. A similarprofile was observed with rat liver cytosol (data not shown). At1 mM, the amounts of binding were higher with acetyl-CoA asan acetyl donor than with N-OH-AAF (Table 3). At acetyldonor concentrations lower than 0.1 mM, the amounts boundwere higher with N-OH-AAF than that observed with acetyl-CoA in both hamsters and rats.

AAF was reported to be an inhibitor of W.O-acyltransferase(31). In addition, N,O-acyltransferase-dependent methylmer-capto adduct formation of N-OH-AAF was observed to beenhanced by the addition of N-OH-AF (35). In the presentstudy, N-OH-AAF dependent binding of N-OH-Glu-P-1 wasdecreased at higher concentrations of N-OH-AAF (>0.1 HIM).The reason is obscure, but it might be partially attributed to thecompetition between N-OH-Glu-P-1 and liberated N-OH-AFand/or AAF from N-OH-AAF.

Species Differences in the Activation of W-Hydroxyarylaminesby Liver Cytosol. The activities of liver cytosols from severalanimal species were compared. As shown in Table 4, markedspecies differences in the enzyme activity were observed. WhenN-OH-Glu-P-1 was used as a substrate, the highest activity wasseen with hamsters, followed in order by rats and rabbits witha rapid acetylator phenotype, which showed high activity in theN-acetylation of 2-AF. Mice and guinea pigs showed very lowactivities. The slow acetylator rabbit and the dog did not showdetectable activities. In the case of N-OH-Trp-P-2, the amountsof binding were smaller than those found with N-OH-Glu-P-1but the order of activities among the species was similar, exceptthat rabbits and mice showed nondetectable activities.

The activation of N-OH-AF was measured under the sameexperimental conditions to ascertain the ability of the enzymeto activate nonheterocyclic W-hydroxyarylamine. The order ofthe activities among species was not correlated with that seenwith N-OH-Glu-P-1 and N-OH-Trp-P-2 as described in Table4. The highest activity was seen with rapid acetylator rabbitsfollowed in order by hamsters, guinea pigs, mice, rats, and theslow acetylator rabbit. A large difference between rapid andslow acetylator rabbits was observed, and the dog did not showactivity.

Species and tissue differences in W.O-acetyltransferase activity have been reported by King and Olive (20). The N,O-acetyltransferase activities of liver cytosols used in the presentexperiments were also measured (Table 4). The assay wascarried out under the same experimental conditions as those

4364



ACETYL-CoA DEPENDENT ACTIVATION OF JV-HYDROXYARYLAMINES

Table 4 Species differences in acetyl-CoA dependent binding by liver cytosol

The incubation mixture for the binding assay of W-hydroxyarylamines contained liver cytosolic protein (0.1 mg/ml), 20 >iMyV-hydroxyarylamine, and othercomponents as described in Table 1. The assay of W,O-acetyltransferase activity was measured as tRNA binding of N-OH-AAF. The incubation mixture was the sameas noted above, except that 20 MMN-OH-AFF was added instead of W-hydroxyarylamine and acetyl-CoA. Data are mean Â±SD from 3 or 4 different preparationsexcept for a slow acetylator rabbit and a dog, for which the data are the means of duplicate determinations. The values were subtracted with the amounts ofnonenzymatic binding (- cytosol) which were 0.31, 1.06, 4.68, and 1.22 pmol/min for N-OH-Glu-P-1, N-OH-Trp-P-2, N-OH-AF, and N-OH-AAF, respectively.

Amounts of metabolite bound to tRNA(pmol/min/mg protein)

A'.O-Acetyltransferaseactivity

(pmol/min/mg protein)

SpeciesHamsterRatRabbit"RapidSlowMouseMaleFemaleGuinea

pigDogN-OH-Glu-P-1656.4

Â±11.2177.5Â±35.875.3

Â±24.8<0.16.3

Â±0.710.8Â±2.51.0Â±1.0<0.1N-OH-Trp-P-285.9

Â±31.062.7Â±23.4<0.1<0.1<0.1<0.10.4

Â±0.4<0.1N-OH-AF1008.8

Â±263.594.9Â±34.65676.4

Â±440.469.9296.0

Â±38.1299.2Â±33.5520.8Â±131.7<0.1N-OH-AAF361.

3Â±87.1114.5Â±59.4218.8

Â±73.5<0.1<0.1<0.11.7

Â±1.2<0.1

* The rabbits which showed polymorphism to the /V-acetylation activity of 2-AF ("Materials and Methods").

for the binding assay to tRNA of Af-hydroxyarylamines exceptthat 20 MM [3H]-N-OH-AAF was added instead of the N-hydroxyarylamine and acetyl-CoA. The highest activity wasobserved in hamsters, followed in order by rapid acetylatorrabbits and rats. The slow acetylator rabbit, mice, and the dogdid not show detectable activities.

Analysis of the Products Formed by the Reaction of N-Hy-droxyarylamines with Acetyl-CoA and Liver Cytosols. To clarifythe pathway of the acetyl-CoA dependent activation of N-hydroxyarylamines, metabolites were analyzed by HPLC. Typical Chromatographie separation of the metabolites formedupon the incubation of N-OH-Glu-P-1 with [3H]acetyl-CoA

and liver cytosols from hamsters is shown in Fig. 3. Radioactivity was not coeluted with synthetic N-acetyl-N-OH-Glu-P-l(peak A) added as a carrier, indicating that the formation of Vacetyl-N-OH-Glu-P-l, if any, was too low to be detected. Glu-P-l, a reduced product of putative N-acetoxy-Glu-P-1 in thepresence of DTT (36), was formed (peak C). The amounts ofGlu-P-1 increased almost linearly with the increase of incuba

ci.01

10 15 20

Elution time (min)Fig. 3. Separation of N-OH-Glu-P-1 and metabolites by HPLC. The incuba

tion was carried out with 50 mm Tris-HCl (pH 7.0), 1 mM DTT, 1 mM [3H]acetyl-CoA, 20 JIM N-OH-Glu-P-1, and cytosolic protein (0.2 mg/ml) fromhamster liver at 37'C for 10 min. Cold synthetic N-acetyl-N-OH-Glu-P-1 was

added as carrier (peak A).

tion time up to 10 min (data not shown). Small amounts ofradioactive product were eluted at peak B, at which the authentic standards of N-OH-Glu-P-1 and N-acetyl-Glu-P-1 wereeluted, indicating that N-acetyl-Glu-P-1 was produced duringincubation. The formation of N-acetyl-Glu-P-1 was not linearwith incubation time but was observed after the appearance ofGlu-P-1, suggesting that N-acetyl-Glu-P-1 is formed mainlythrough A/-acetylation of Glu-P-1. The formation of Glu-P-1and N-acetyl-Glu-P-1 was observed only in the presence of bothacetyl-CoA and cytosol. With liver cytosols from rats andrabbits with rapid acetylator phenotype, Glu-P-1 formation wasobserved but N-acetyl-N-OH-Glu-P-l was not detected. Theamounts of Glu-P-1 formed were the largest with hamstercytosols, followed by rats and rabbits (pmol/min/mg protein:1180 with hamsters, 325 with rats, and 125 with rapid acetylator rabbits). N-Acetyl-Glu-P-1 was not detected with rat cytosols during the period of incubation, since rat hepatic cytosolswere reported to show a low activity of JV-acetylation of heter-ocyclic arylamine (14).

Synthetic N-acetyl-N-OH-Glu-P-l was incubated with hamster cytosol and analyzed by HPLC. After incubation at 37Â°C

for 10 min, formation of Glu-P-1, N-OH-Glu-P-1, or N-acetyl-Glu-P-1 and loss of N-acetyl-N-OH-Glu-P-l by enzymaticprocess were not observed. These results suggest that N-acetyl-N-OH-Glu-P-l is not a good substrate for cytosolic N,O-ncy\-transferase in hamster liver.

DISCUSSION

The present studies clearly showed that A/-hydroxyarylaminesare activated to the reactive species capable of binding to tRNAby a cytosolic enzyme(s) in several animal tissues in the presenceof acetyl-CoA. Recently, acetyl-CoA dependent activation ofseveral N-hydroxyarylamines was reported by us (8, 13) andFlammang et al. (37).

In the literature, highly electrophilic W-acetoxyarylaminesare reported to be produced by chemical reaction of jV-hydrox-yarylamines with acetic anhydride or ketene and are subsequently bound to nucleophiles (35, 38). jV-Acetoxyarylaminesare produced enzymatically by A/,O-acetyl transfer of arylhy-droxamic acids such as N-OH-AAF (15, 17, 31, 35, 38, 39).The ultimate metabolites derived from W-hydroxyarylamines inthe present experiments are also assumed to be W-acetoxyar-ylamines formed by O-acetylation

Two pathways would be possible for the acetyl-CoA depend-4365



ACETYL-CoA DEPENDENT ACTIVATION OF JV-HYDROXYARYLAMINES

ent formation of N-acetoxyarylamines from N-hydroxyaryla-mines. One is direct 0-acetylation of the hydroxy group andanother is sequential N-acetylation and N,0-acetyl transfer.When N-OH-Glu-P-1 was incubated with acetyl-CoA and cy-tosols from hamsters, rats, or rabbits with rapid acetylatorphenotype, N-acetyl-N-OH-Glu-P-l was not detected by HPLC(Fig. 3). To ascertain the absence of N-acetyl-N-OH-Glu-P-lin the acetyl-CoA supported system, a portion of the incubationmixture was treated with TiCI3 (40, 41). The amounts of Glu-P-l were increased by the reduction of N-OH-Glu-P-1 but noincrease in the amounts of N-acetyl-GIu-P-1 was observed. Thisresult also supports the lack of N-acetyl-N-OH-Glu-P-l formation (data not shown). In addition, synthetic N-acetyl-N-OH-Glu-P-l was not a substrate for cytosolic N.O-acetyltrans-ferase as was a certain arylhydroxamic acid (37). Thus, thereaction through the formation of arylhydroxamic acid is unlikely to be the case for acetyl-CoA dependent activation of N-OH-Glu-P-1, and the direct 0-acetylation at the N-hydroxygroup is conceivable for the mechanism.

Acetyl-CoA dependent binding of N-OH-AF, but not N,0-acetyl transfer activity of N-OH-AAF, was observed with slowacetylator rabbit, mouse, and guinea pig cytosol (Table 4).Although the N,0-acetyl transfer mechanism could not be excluded, these results suggest the role of a direct 0-acetylationpathway for metabolic activation of N-OH-AF.

Among the animal species used, the highest activity for theactivation of N-OH-Glu-P-1 and N-OH-Trp-P-2 was observedin liver cytosol of hamsters, which showed high activities ofeither N,0-acetyl transfer (Table 4) or N-acetylation of Trp-P-2 and 2-AF (14). Although the rate of N-acetylation of Trp-P-2 by hamster cytosol was about 100-fold slower than that of 2-AF (14), no such marked species difference was observed in theactivation of the corresponding N-hydroxyarylamines, N-OH-Trp-P-2 and N-OH-AF. The rat cytosol, which showed relatively high activities in the activation of ./V-hydroxyarylamines,also exhibited high N,0-acetyl transfer activity (Table 4) butvery low activity in N-acetylation of arylamines (14). Theseresults indicate that the relative activities of several types ofacetyl transfer reactions are different among species, by substrate used and by type of reaction.

In rabbits, genetic polymorphism in both N,0-acetyltransfer-ase and N-acetyltransferase has been reported (17, 22, 26). Wehave noted that similar polymorphism is seen in the N-acetylation of heterocyclic amines such as Trp-P-2 (13, 14). In thepresent experiments, the rapid acetylator rabbits showed muchhigher activities in the activation of N-OH-Glu-P-1 and N-OH-AF than did the slow one (Table 4), indicating that this activation reaction has a similar polymorphism. N-OH-Trp-P-2, however, was not activated by cytosols from all rabbits. The dog,which did not possess detectable activities in either N,0-acetyltransfer or N-acetylation reactions, was also deficient in anyactivity for the activation of N-hydroxyarylamines.

The properties of an enzyme(s) in hamsters and rats catalyzing the activation of N-hydroxyarylamines are consistent withthose of N,0-acetyltransferase (15-17,19,20) or N-acetyltrans-ferase (16, 17, 21, 22), in terms of being inhibited by sulfhydrylinhibitors and unaffected by an esterase inhibitor, paraoxon.

N-Acetyltransferase and arylhydroxamic acid N,0-acetyl-

transferase were reported to be the properties of the sameenzyme in rabbit liver (17). We have recently purified an acetyl-CoA dependent N-hydroxyarylamine activating enzyme to anelectrophoretic homogeneity from hamster liver cytosol, whichalso catalyzes N-acetylation of arylamines and N,0-acetyl transfer of N-OH-AAF (42). In the N-acetylation reaction, a Ping

Pong Bi-Bi mechanism has been postulated, i.e., acetyl residueis first transferred to enzyme to form an acetylated enzymeintermediate and then is transferred from the acetylated enzymeto substrate (43). Intermolecular transfer of acetyl group fromarylhydroxamic acid to arylamines (44, 45) or N-hydroxyarylamines (35,44) by rat tissues has also been noted. The presenceof the enzyme catalyzing various types of acetyl transfer reactions in hamster liver may be explained by postulating that theenzyme accepts an acetyl group from acetyl-CoA or N-OH-AAF to form acetylated enzyme and transfers the acetyl groupto the O-atom of N-hydroxyarylamines or the N-atom of arylamines or N-hydroxyarylamines.

In our recent experiment, we have purified another enzymefrom hamster liver which catalyzes N-acetylation of arylaminesbut not acetyl-CoA dependent activation of N-OH-Glu-P-1.Species and substrate related differences observed in the acet-ylation reactions may be due to the presence of an enzymecatalyzing various types of acetyl transfer reactions with different affinities for acetyl donors and substrates, and/or due tothe presence of multiple acetyltransferases in each animal species.

The present data indicate a possible involvement of the direct0-acetylation pathway in the carcinogenesis of arylamines,especially Glu-P-1 and Trp-P-2. Further detailed studies on the0-acetylation pathway of N-hydroxyarylamines will be neededfor the clarification of species and tissue specificity in carcinogenesis by these heterocyclic arylamines (3, 46, 47) in relationto other metabolic activation and detoxication pathways.

REFERENCES

1. Ishii, K., Yamazoe, Y.. Kamataki, T., and Kato, R. Metabolic activation ofglutamic acid pyrolysis products, 2-amino-6-methyldipyrido[l,2-a:3',2'-d]-imidazole and 2-amino-dipyrido[l,2-a:3',2'-rf]imidazol, by purified cyto-chrome P-450. Chem.-Biol. Interact., 38: 1-13, 1981.

2. Kato, R., Kamataki, T., and Yamazoe, Y. MHydroxylation of carcinogenicand mutagenic aromatic amines. Environ. Health Perspect., 49:21-25,1983.

3. Sugimura, T., and Sato, S. Mutagens-carcinogens in foods. Cancer Res., 43:2415s-2421s, 1983.

4. Yamazoe, Y., Ishii, K., Kamataki, T., Kato, R., and Sugimura, T. Isolationand characterization of active metabolites of tryptophan-pyrolysate mutagenTrp-P-2, formed by rat liver microsomes. Chem.-Biol. Interact., 30: 125-138, 1980.

5. Miller, E. C. Some current perspectives on chemical carcinogens in humansand experimental animals: Presidential Address. Cancer Res., 38: 1479-1496, 1978.

6. Thorgeirsson, S. S., McManus, M. E., and Glowinski, I. B. Metabolicprocessing of aromatic amides. In:Â¡.R. Mitchell and M. G. Horning (eds.),Drug Metabolism and Drug Toxiciry, pp. 183-197. New York: Raven Press,1984.

7. Weisburger, E. K. Mechanisms of chemical carcinogenesis. Annu. Rev.Pharmacol. Toxicol., 18: 395-415, 1978.

8. Yamazoe, Y., Shimada, M., Kamataki, T., and Kato, R. Covalent binding ofN-hydroxy-Trp-P-2 to DNA by cytosolic proline-dependent system.Biochem. Biophys. Res. Commun., 107: 165-172, 1982.

9. Yamazoe, Y., Shimada, M., Shinohara, A., Saito, K., Kamataki, T., andKalo, R. A i prilline and ATP-dependent enzyme in rat hepatic cytosolcatalyzes the covalent binding of 3-hydroxyamino-l-methyl-5//-pyrido[4,3-ejindole to DNA. Cancer Res., 45: 2495-2500, 1985.

10. Saito, K., Yamazoe, Y., Kamataki, T., and Kato, R. Mechanism of activationof proxymate mutagens in Ames' tester strains: the acetyl-CoA dependentenzyme in Salmonella typhimurium TA98 deficient in TA98/1,8-DNP6 catalyzes DNA-binding as the cause of mutagenicity. Biochem. Biophys. Res.Commun., 116:141-147, 1983.

11. Saito, K., Shinohara, A., Kamataki, T., and Kato, R. Metabolic activation ofmutagenic yV-hydroxyarylaminesby O-acetyltransferase in Salmonella typhimurium TA98. Arch. Biochem. Biophys., 239: 286-295, 1985.

12. Kato, R., Saito, k . Shinohara, A., Yamazoe, Y., and Kamataki, T. Acetyl-CoA dependent DNA-binding of mutagenic heterocyclic hydroxylamines:possible route through O-acetylation. Proc. Am. Assoc. Cancer Res., 25:120,1984.

13. Shinohara, A., Saito, K., Yamazoe, Y., Kamataki, T., and Kato, R. DNA-binding of N-hydroxy-Trp-P-2 and N-hydroxy-Glu-P-1 by acetyl-CoA dependent enzyme in mammalian liver cytosol. Carcinogenesis (Lond.), 6:305-307, 1985.

14. Shinohara, A., Yamazoe, Y., Saito, K., Kamataki, T., and Kato, R. Species

4366



ACETYL-CoA DEPENDENT ACTIVATION OF /V-HYDROXYARYLAM1NES

differences in the JV-acetylation by liver cytosol of mutagenic heterocyclicaromatic amines in protein pyrolysates. Carcinogenesis (Lond.). 5:683-686,1984.

15. King, C. M - and Allaben, V\. T. Arylhydroxamic acid acyltransferase. In:W. B. Jakoby (ed.). Enzymatic Basis of Detoxication, Vol. 2, pp. 187-197.New York: Academic Press, 1980.

16. King, C. M., and Glowinski, I. B. Acetylation, deacetylation and acyltransfer.Environ. Health Perspect., 49:43-50, 1983.

17. Glowinski, I. B., Weber, W. W., Fysh, J. M., Vaught, J. B., and King, C. M.Evidence that arylhydroxamic acid JV,O-acyltransferase and the geneticallypolymorphic /V-acetyltransferase are properties of the same enzyme in rabbitliver. J. Biol. Chem., 255: 7883-7890, 1980.

18. King, C. M., and Phillips, B. Enzyme-catalyzed reactions of the carcinogenN-hydroxy-2-fluorenylamide with nucleic acid. Science (Wash. DC), 759:1351-1353, 1968.

19. Allaben, W. T., and King, C. M. Purification of rat liver arylhydroxamic acidtf.O-acyltransferase. J. Biol. Chem., 259: 12128-12134,1984.

20. King, C. M., and Olive, C. W. Comparative effects of strain, species and sexon the acyltransferase- and sulfotransferase-catalyzed activations of N-hy-droxy-2-fluorenylacetamide. Cancer Res., 55: 906-912, 1975.

21. Lower, G. M., Jr., and Bryan, G. T. Enzymatic /V-acetylation of carcinogenicaromatic amines by liver cytosol of species displaying different organ susceptibilities. Biochem. Pharmacol.. 22: 1581-1588, 1973.

22. Glowinski, I. B., Radtke, H. E., and Weber, W. W. Genetic variation in N-acetylation of carcinogenic arylamines by human and rabbit liver. Mol.Pharmacol., 14:940-949, 1978.

23. Glowinski, I. B., and Weber, W. W. Genetic regulation of aromatic amineJV-acetylation in inbred mice. J. Biol. Chem., 257:1424-1430, 1982.

24. Glowinski, I. B., and Weber, W. W. Biochemical characterization of genetically variant aromatic amine W-acetyltransferase in A/J and C57BL/6J mice.J. Biol. Chem., 257: 1431-1437, 1982.

25. Hein, D. W., Smolen, T. N., Fox, R. R., and Weber, W. W. Identification ofgenetically homozygous rapid and slow acetylators of drugs and environmental carcinogens among established inbred rabbit strains. J. Pharmacol. Exp.Ther., 22* 40-44, 1982.

26. McQueen, C. A., Maslansky, C. J., and Williams, G. M. Role of theacetylation polymorphism in determining susceptibility of cultured rabbithepatocytes to DNA damage by aromatic amines. Cancer Res., 43: 3120-3123, 1983.

27. Hein, D. W., Kirlin, W. G., Ferguson, R. J., and Weber, W. W. Inheritanceof liver /V-acetyltransferase activity in the rapid and slow acetylator inbredhamster. J. Pharmacol. Exp. Ther., 233: 584-587, 1985.

28. Hein, D. W., Kirlin, W. G., Ferguson, R. J., and Weber, W. W. Biochemicalinvestigation of the basis for the genetic ^-acetylation polymorphism in theinbred hamster. J. Pharmacol. Exp. Ther., 235: 358-364, 1985.

29. Saito, K., Yamazoe, Y., Kamataki, T., and Kato, R. Syntheses of hydroxy-amino, nitroso and nitro derivatives of Trp-P-2 and Glu-P-1, amino acidpyrolysate mutagens, and their direct mutagenicities toward SalmonellatypHimurium TA98 and TA98NR. Carcinogenesis (Lond.), 4: 1547-1550,1983.

30. Yamazoe, Y., Kamataki, T., and Kato, R. Species difference in A'-hydroxyl-ation of tryptophan pyrolysis product in relation to mutagenic activation.Cancer Res., 41:4518-4522, 1981.

31. King, C. M. Mechanism of reaction, tissue distribution, and inhibition ofarylhydroxamic acid acyltransferase. Cancer Res., 34: 1503-1515, 1974.

32. Mita, S., Ishii, K., Yamazoe, Y., Kamataki, T., Kato, R., and Sugimura, T.Evidence for the involvement of/V-hydroxylation of 3-amino-1-methyl-5/Ã-pyrido[4,3-Â¿]indole by cytochrome P-450 in the covalent binding to DNA.Cancer Res., 41: 3610-3614, 1981.

33. Glowinski, I. B., Savage, I .. Lee, MS., and King, C. M. Relation betweennucleic acid adduct formation and deacylation of arylhydroxamic acids.Carcinogenesis (Lond.), 4:67-75, 1983.

34. Shirai, T., Fysh, J. M., Lee, M-S., Vaught, J. B., and King, C. M. Relationshipof metabolic activation of /V-hydroxy-iV-acetylarylamines to biological response in the liver and mammary gland of female CD rat. Cancer Res., 41:4346-4353, 1981.

35. Bartsch, H., Dworkin, M., Miller, J. A., and Miller, E. C. Electrophilic N-acetoxyaminoarenes derived from carcinogenic /V-hydroxy-AT-acetylaminoar-enes by enzymatic deacetylation and transacetylation in liver. Biochim.Biophys. Acta, 286: 272-298, 1972.

36. Saito, K., Shinohara, A., Kamataki, T., and Kato, R. A new assay for \hydroxyarylamine O-acetyltransferase: Reduction of Ar-hydroxyarylaminesthrough /V-aceloxyarylamines. Anal. Biochem., 752: 226-231, 1986.

37. Flammang, T. J., Westra, J. G., Kadlubar, F. F., and Beland, F. A. DNAadducts formed from the probable proximate carcinogen, /V-hydroxy-3,2'-dimethyl-4-aminobiphenyl, by acid catalysis or S-acetyl coenzyme A-depend-ent enzymatic esterification. Carcinogenesis (Lond.), 6: 251-258, 1985.

38. Hashimoto, Y., Shudo, K . and Okamoto, T. Modification of DNA withpotent mutacarcinogenic 2-amino-6-methyldipyrido[l,2-a:3',2'-<f]imidazole

isolated from a glutamic acid pyrolysate: structure of the modified nucleicacid base and initial chemical event caused by the mutagen. J. Am. Chem.Soc., 104:7636-7640, 1982.

39. Beland, F. A., Allaben, W. T., and Evans, F. E. Acyltransferase-mediatedbinding of /V-hydroxyarylamides to nucleic acids. Cancer Res., 40:834-840,1980.

40. Poirier, L. A., Miller, J. A., and Miller, E. C. The N- and ring-hydroxylationof 2-aminofluorene and the failure to detect A'-acetylation of 2-aminofluorenein the dog. Cancer Res., 23:790-800, 1963.

41. Lotlikar, P. D., Miller, E. C., Miller, J. A., and Margreth, A. The enzymaticreduction of the JV-hydroxy derivatives of 2-aminofluorene and related carcinogens by tissue preparations. Cancer Res., 25: 1743-1752, 1965.

42. Saito, K., Shinohara, A., Kamataki, T., and Kato, R. JV-HydroxyarylamineO-acetyltransferase in hamster liver identity with arylhydroxamic acid \.(>acetyltransferase and arylamine W-acetyltransferase. J. Biochem. (Tokyo),99:1689-1697. 1986.

43. Steinberg, M. S . Cohen, S. N., and Weber, W. W. Isotope exchange studieson rabbit liver A'-acetyltransferase. Biochim. Biophys. Acta, 235: 89-98,1971.

44. Lotlikar, P. D., and Luha, L. Enzymic JV-acetylation of /V-hydroxy-2-ami-nofluorene by liver cytosol from various species. Biochem. J., 123: 287-289,1971.

45. Weber, W. W., and Glowinski. I. B. Acetylation. In: W. B. Jakoby (ed.).Enzymatic Basis of Detoxication, Vol. 2, pp. 169-186. New York: AcademicPress, 1980.

46. Matsukura, N., Kawachi, T., Morino, K., Ohgaki, H., Sugimura, T., andTakayama, S. Carcinogenicity in mice of mutagenic compounds from atryptophan pyrolyzate. Science (Wash. DC), 213: 346-347, 1981.

47. Takayama, S., Masuda, M., Mogami, M., Ohgaki, H., Sato, S., and Sugimura, T. Induction of cancers in the intestine, liver and various other organsof rats by feeding mutagens from glutamic acid pyrolysate. Gann, 75: 207-213, 1984.

4367



1986;46:4362-4367. Cancer Res Atsuko Shinohara, Kazuki Saito, Yasushi Yamazoe, et al. Donor SpecificityActivation, Species Difference, Tissue Distribution, and AcetylDerivatives of Carcinogenic Arylamines: Mechanism of

-HydroxyNAcetyl Coenzyme A Dependent Activation of

Updated version

http://cancerres.aacrjournals.org/content/46/9/4362

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/46/9/4362To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



acetyl coenzyme a dependent activation of w-hydroxy...

Documents