[CANCER RESEARCH 34, 1503-1515, June 1974]

Mechanism of Reaction, Tissue Distribution, and Inhibition ofArylhydroxamic Acid Acyltransferase1

Charles M. King

Division of Cancer Research, Department of Medicine, Michael Reese Medical Center and the University of Chicago Pritzker School of Medicine,Chicago, Illinois 60616

SUMMARY

The enzyme of rat liver that can transform /V-hydroxy-/V-2-fluorenyIacetamide into a reactive derivative capable ofintroducing fluorenylamine groups into nucleic acids hasbeen shown to be a sulfhydryl-dependent enzyme with amolecular weight of approximately 28,000. A 30-fold purification of the enzyme from 105,000 x g supernatants of liverhas been achieved by precipitation with ammonium sulfateand gel nitration on Sephadex G-100. Evidence that thismechanism of activation involves transfer of the /V-acetylgroup to the oxygen of the hydroxylamine came fromexperiments that showed that O-methylation of /V-hydroxy-/V-2-fluorenylacetamide prevented activation of the hydrox-amic acid.

Distribution studies demonstrated considerable acyltrans-ferase activity in kidney, stomach, small intestine, andcolon; lung and spleen were less active; and blood, brain,and muscle were essentially without activity.

Tissue distribution studies and protein purification experiments utilizing ammonium sulfate precipitation and gelfiltration techniques disclosed that the enzyme responsiblefor the activation of /V-hydroxy-/V-2-fluorenylacetamidewas inseparable from the enzyme that transfers the acetylgroup of /V-hydroxy-/V-2-fluorenylacetamide to 4-aminoazobenzene.

Acyltransferase-catalyzed activation of /V-hydroxy-/V-2-fluorenylpropionamide demonstrated that the activationof hydroxamic acids was not restricted to acetylatedderivatives.

Hepatic acyltransferase-catalyzed formation of fluo-renylamine-substituted nucleic acid was inhibited by botharylamines and arylacetamides. This inhibition, consideredwith previous reports of the inhibition of A/-hydroxy-/V-2-fluorenylacetamide hepatocarcinogenesis by acetanilideand /7-hydroxyacetanilide, suggest that acyltransferase-catalyzed activation of arylhydroxamic acids may be involvedin the formation of liver tumors.

INTRODUCTION

The carcinogenic arylhydroxamic acid, N-hydroxy-FAA,is a derivative of 1 of a group of arylamines that can inducetumors in a wide spectrum of tissues in a number of species(36, 43, 44). Arylamines are believed to initiate the formation of tumors by modification of tissue macromolecules(36). These modifications are thought to arise by N-hydroxylation of the amine followed by some furthermetabolic activation of the carcinogen (36, 44).

In synthetic studies designed to identify possible metabolites of arylhydroxamic acids that might be responsible forthe modification of nucleic acid and protein, the Millersandtheir collaborators (24, 25, 34) demonstrated that esterifi-cation of N-hydroxy-FAA yielded a reactive species thatcould combine with both nucleic acids and protein in neutral solutions. Kriek (20, 21) showed that N-hydroxy-FAcould combine with nucleic acid at low pH but that relatively little reaction took place at pH 7.O.

The unstable nature of metabolic derivatives capable ofreaction with tissue macromolecules generally precludestheir isolation by conventional techniques. In order to detectsuch reactive metabolites, it has usually been necessary togenerate the intermediates in situ, trap the activated molecules by reaction with an acceptor molecule, and identify theadducts formed in this reaction. Using this approach,studies with soluble preparations from rat liver, a targetorgan of N-hydroxy-FAA, disclosed that this compoundcould be activated by enzymatic esterification on incubationwith 3'-phosphoadenosine 5'-phosphosulfate, or ATP andMg' ", to give FAA-substituted nucleic acid (4, 5, 16, 17).

An alternative pathway for the activation of N-hydroxy-FAA, also carried out by soluble preparations of rat liverbut without the need for exogenous cofactors. involved theremoval of the ;V-acetyl moiety of the hydroxamic acid andresulted in the formation of FA-substituted nucleic acid (4,16, 17). Initially, it was considered that this mechanism ofactivation was possibly the result of the deacetylation ofN-hydroxy-FAA and the subsequent reaction of the freeN-hydroxy-FA with nucleic acid or protein present in theincubation system (16, 17).

'These studies were supported by a grant from the Jules J. Reingold

Trust and by NIH Research Grant ÇA 13I79 from the National CancerInstitute. Presented in part at the 63rd Annual Meeting of the AmericanAssociation for Cancer Research. Boston. Mass.. May 4, 1972 (19).

Received December 17, 1973; accepted March 7. 1974.

2The abbreviations used are: N-hydroxy-FAA. .V-hydroxy-A'-

2-fluorenylacetamide; N-hydroxy-FA, /V-2-fluorenylhydroxylamine; FAA,/V-2-fluorenylacetamide: FA, iV-2-fluorenylamine; N-methoxy-FAA, /V-methoxy-/V-2-fluorenylacetamide.

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More recently, however, Bartsch et al. (2) have presentedevidence to support the concept that this activation resultsfrom N—Otransfer of the acetyl group of arylhydroxamicacids to yield reactive /V-acetoxyarenes that are capable ofreaction with tissue nucleophiles.

Data presented in this paper add further support to thishypothesis, describe the distribution of this enzyme in anumber of extrahepatic tissues of the rat, and demonstratethat the enzyme responsible for this activation is anacyltransferase. The inhibition of this enzyme by arylaminesand arylacetamides detailed here suggests that the diminution in hepatocarcinogenicity of N-hydroxy-FAA on concurrent administration of acetanilide or /7-hydroxyacetani-lide reported previously by others (45 47) may result fromthe inhibition of acyltransferase in vivo.

MATERIALS AND METHODS

Chemicals. The following were obtained from the commercial sources indicated: N-hvdroxy-FAA-9-MC (Interna

tional Chemical and Nuclear Corp., Cleveland. Ohio);guanosine-8-MC and acetic anhydride-l'-14C (Amersham-Searle Corp., Arlington Heights, 111.):acetic anhydride-2'-3H (New England Nuclear, Boston, Mass.); [.-/V-formyl-kynurenine, dithiothreitol, iodoacetamide, sodium p-chloromercuribenzoate, yeast tRNA, and ovalbumin (Cal-biochem, Los Angeles, Calif.); /7-phenylazoaniline, p-aminophenol hydrochloride, ^-hydroxyacetanilide, and acetanilide (Eastman Kodak Co., Rochester, N. Y.): FAA,FA, and 2-nitrofluorene (Aldrich Chemical Co., Milwaukee, Wis.); .V-ethylmaleimide, horse heart cytochromec, and guanosine (Sigma Chemical Co.. St. Louis, Mo.):Sephadex G-IO and G-100 (Pharmacia Laboratories, Inc.,Piscataway. N. J.); ammonium sulfate (Schwarz/Mann.Orangeburg, N. Y.); aniline (Fisher Scientific Co., Pittsburgh, Pa.); bovine serum albumin and soybean trypsininhibitor (Pentex, Inc., Kankakee, 111.).

N-Hydroxy-FAA (35), N-hydroxy-FAA-l'-MC (11), N-

hydroxy-FA (38), N-methoxy-FAA (4), and N-methoxy-FAA-9-14C-2'-3H (4) were prepared as described in the

references cited. Radiochemical purity was established bythin-layer chromatography on Eastman No. 6060 silica gelusing chloroform :methanol (97:3) as a solvent for N-hydroxy-FAA (RK 0.54) and benzene:chloroform (4:6) forN-methoxy-FAA (RK 0.24 to 0.31). Radiochromatogramswere evaluated by use of a chromatogram scanner (Brink-mann Instruments, Westbury, N. Y.), and/or by assay ofmethanol extracts of the chromatogram segments. Whennecessary, labeled N-hydroxy-FAA was purified by conversion to its cupric chelate, extraction of the chelate withethanol, and decomposition of the chelate by treatment withhydrogen sulfide (6); N-methoxy-FAA was purified bypreparative thin-layer chromatography.

Ar-Hydroxy-A^-2-fluorenylpropionamide was prepared byreductive acylation of 2-nitrofluorene in a benzene solutionwith H2 over palladium on charcoal in the presence ofpropionic anhydride, as described for the preparation ofN-hydroxy-FAA (35). Alternatively, /V-hydroxy-/V-

2-fluorenylpropionamide could also be prepared by directacylation of N-hydroxy-FA with propionic anhydride inethyl acetate (II). /V-Hydroxy-/V-2-f]uorenylpropionamidewas recrystallized from ethanol: water to yield a compoundmelting at 151-152°and with absorption maxima in 95%

ethanol at 282 nm (e 21,400). 290 nm (t 21,600), and 302 nm(e 18,400) (analyses by Schwartzkopf Microanalytical Laboratory, Woodside, N. Y.).

C,gH15NO2

Calculated:Found:

C 75.87,C 75.82.

H 5.97.H 6.09,

N 5.53N 5.52

Animals. Adult male Sprague-Dawley-derived rats wereraised and maintained in our animal facilities at 24°instainless steel cages with solid floors covered with soft-woodshavings. Distilled water and Purina laboratory chow wereavailable continuously.

Tissue Preparation. The animals were anesthetized withether prior to removal of tissue samples. Blood was obtainedby direct aspiration from the heart with a syringe andneedle. Serum samples were obtained by centrifugation ofclots formed on standing at room temperature. Liver,spleen, lung, brain, kidney, skeletal muscle, stomach, smallintestine, and colon were removed and placed in chilled 0.05M PP,:NaCl buffer (pH 7.0) which contained 1 m\idithiothreitol. The stomach, small intestine, and colon wereopened and the mucosa! surface was rinsed gently withbuffer. The tissues were then blotted, weighed, minced, andhomogenized with 4 ml of buffer per g of tissue in a glassand Teflon homogenizer. Soluble fractions were preparedby centrifugation of the homogenate at 105,000 x g(average) for 1 hr at 4°.

Assay for the Activation of N-Hydroxy-FAA by Acyltransferase by Introduction of FA Groups into Nucleic Acid.A modification of our earlier method for the detection of theincorporation of FA groups into nucleic acid was used inthese studies (16) unless indicated otherwise. N-Hydroxy-FAA-9-14C (0.042 ^mole, usually 1.0 /uCi/Mmole) in me-thoxyethanol (10 ¿tl)was placed in a 12-ml glass centrifugetube with tRNA from yeast equivalent to 15 A258nmunits,and sufficient buffer so that, after addition of the enzymepreparation, the final volume of 0.8 ml would contain 40Amólesof PP,:NaCl (pH 7.0) and 0.8 jumóleof dithiothreitol. Prior to use in these incubations the soluble RNAwas purified by ion-exchange chromatography on DEAE-cellulose (37), a procedure that usually increased the yield ofFA substitution of the nucleic acid by 50 to 60% ascompared with unpurified RNA. The reaction was initiatedby addition of 0.1 ml of 105,000 x g tissue supernatant or avolume of partially purified enzyme preparation. Thesolution was incubated at 37°for 20 min in air. The reactionwas stopped by addition of an equal volume of buffer-saturated phenol. The aqueous layer was clarified bylow-speed centrifugation, and an aliquot (usually 0.5 ml)was pipeted into a glass filter apparatus containing 5 ml of2% potassium acetate in 95% ethanol over a 24-mm glassfiber filter. The RNA precipitate was collected on the filters

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Arylhydroxamic Acid Acyltransferase

and then washed successively with 70 and 95% ethanol,acetone, and diethyl ether. The dry filters were placed incounting vials and moistened with 0.15 ml of water. Asolubilizer (1 ml, NCS, Amersham-Searle, ArlingtonHeights, 111.)and toluene-based scintillator (10 ml) (10)were added, and the vials were counted in a liquid scintillation counter. Enzyme activity was expressed as nmoles ofFA bound to nucleic acid per 20 mg, wet weight, of tissue inthe 20-min assay, unless specified otherwise.

Formation of 8-( \-2-Fluoreny laminoIguanosine.Replacement of RNA with guanosine as a trapping agent for theactivated derivatives of N-hydroxy-FAA formed on incubation with 105,000 x g supernatants of rat liver yields 8-(7V-2-fluorenylamino)guanosine (2, 17). In the present studiesthe relative abilities of several hydroxamic acids and theirderivatives to yield this guanosine adduct were examined byincubation of guanosine-8-14C (1 mg, 1.0 /uCi/jitmole), the

hydroxamic acid (0.5 or 2.5 /umoles) in methoxyethanol (0.5ml), PP,:NaCl buffer (100 Amóles,pH 7.0), dithiothreitol(2.0 Amóles),and 105,000 x g supernatant of rat liver (0.5ml) in a final volume of 2.5 ml for 1 hr at 37°.The

fluorene: guanosine adduct was recovered by adsorptiononto Sephadex G-10, removal of unreacted guanosine bywashing successively with Tris buffer and water, and elutionof the adduct from the gel with 60% ethanol as describedpreviously (17). The quantity of 8-(A'-2-fluorenylamino)-guanosine formed in these incubations was determined byassay of the 60% ethanol solutions for 14C. Thin-layer

chromatography was used to confirm the identity andpurity of the labeled derivative (17).

Partial Purification of Acyltransferase. The 105,000 x gsupernatant from rat liver was brought to 45% saturationwith ammonium sulfate by addition, at room temperaturewith stirring, of a saturated solution of ammonium sulfatein PP,:NaCl buffer (0.05 M, pH 7.0) which contained 1 mMdithiothreitol. The precipitate was removed and discarded,and the supernatant was then brought to 65% saturation byfurther addition, also at room temperature, of saturatedammonium sulfate. The precipitate formed in 65% saturated ammonium sulfate was washed twice with 65%saturated ammonium sulfate in the PP¡:NaCl buffer andstored at -20°.

The 45 to 65% ammonium sulfate fraction was dissolvedin a minimal volume of PPjiNaCl buffer (0.02 M, pH 7.0)which contained 1 mM dithiothreitol. The slight amount ofinsoluble protein was removed by centrifugation. A portionof the clear supernatant was dialyzed overnight against thebuffer, and the remainder was applied to a column (5 x 75cm) of Sephadex G-100 which had been equilibrated withthe 0.02 M PP¡:NaCl buffer. All Chromatographie operations were carried out at 4°.A flow rate of 70 to 80 ml/hr

was maintained with the aid of a roller pump. Absorption at280 nm was monitored by use of flow cells and a recordingspectrophotometer. Fractions of 10 ml were collected in arefrigerated collector.

Estimation of the Molecular Weight of Acyltransferase byGel Filtration. A column of Sephadex G-100 (1.5 x 51 cm)was maintained at 4°and equilibrated with the 0.02 M

PPjiNaCl buffer (pH 7.0) which contained 1 mM dithio

threitol. A flow rate of 8 ml/hr was established with a rollerpump. Samples of 0.5 ml containing 5% sucrose and (a)cytochrome c (2 mg) and ovalbumin (2 mg), (b) acyltrans-ferase partially purified by fractional precipitation withammonium sulfate, or (c) soybean trypsin inhibitor (2 mg)and bovine serum albumin (5 mg) were applied on 3successive runs in each of 2 series of experiments. Fractionswere collected each 7.5 min. The volume (0.92 to 0.95 ml)and absorbance at 280 nm of each fraction was determined.The location of the acyltransferase in the effluent wasdetermined enzymatically. The point of maximum absorption at 280 nm was determined from the symmetrical profileof each of the protein components and was used as theelution volume of that macromolecule.

Acetyl Transfer between N-Methoxy-2-FAA and FA.The possibility that acyltransferase of rat liver might transfer the acetyl group of N-methoxy-FAA to FA to formFAA was investigated in experiments using N-methoxy-FAA labeled in the fluorene ring with 14Cand in the acetylgroup with 3H. N-Methoxy-FAA-9-14C-2'-3H (0.042Minole; 3H, 6.25 /uCi/^mole; I4C, 1.07 /tCi/Vmole;3H:14C = 5.84) and FA (0.28 /¿mole)in methoxyethanol

(10 /il) were incubated with PPi:NaCl (40 /¿moles,pH7.0), dithiothreitol (0.8 /¿mole),and 105,000 x g supernatant from rat liver (0.1 ml) in a final volume of 0.8 mlfor 20 min at 37°.Control incubations to which no enzyme

source was added were carried out to determine the extentto which the acetyl group of N-methoxy-FAA might betransferred nonenzymatically to FA. At the end of the incubation, FAA (112 A29onmunits) in ethanol (4.8 ml) wasadded to stop the reaction, introduce unlabeled carrier,and precipitate protein. Following centrifugation to removethe precipitated protein, the supernatant was taken to dry-ness under reduced pressure. The residue was dissolvedin a small volume of ethanol and subjected to repeatedchromatography on thin layers of silica gel using benzene:CHC13 (4:6) as solvent. FAA (RF 0.1), which was wellseparated from N-methoxy-FAA (RF 0.3) and FA (RF 0.4),was eluted from the silica gel with ethanol. The specificactivity of the FAA was established following each chro-matographic separation by use of UV and liquid scintillation spectroscopy.

Analytical Methods. Protein was determined by use of amodified Folin method (27) using bovine serum albumin asa standard. 3H and 14C were determined in a PackardTri-Carb scintillation counter using an automatic externalstandardization technique to determine counting efficiency.Spectrophotometric determinations were carried out with aModel 2000 Gilford recording spectrophotometer.

Formamidase activity, i.e., the hydrolysis of the /V-formylgroup of formylkynurenine, was assayed by determining theincrease in absorption at 360 nm (33) on incubation with105,000 x g supernatants from tissues or partially purifiedpreparations of acyltransferase. The enzymatic transfer ofthe 7V-acetyl group from N-hydroxy-FAA to 4-aminoazobenzene was assayed by determining the decreasein absorption at 497 nm of the 4-aminoazobenzene in acidicmedia as described by Booth (3). The buffer used in theassay of both the acetyltransferase and formamidase was

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0.02 M PP,:NaCl (pH 7.0) which contained 1 m\i dithio-threitol.

FA was determined spectrophotometrically by conversionto an azo derivative (9). Folin-Ciocalteu reagent was used inspot tests for the detection of arylamines (40).

RESULTS

Formation of FA-substituted Nucleic Acid following Activation of N-Hydroxy-FAA by Acyltransferase of Rat Liver.A more rapid and convenient assay has been devised for thetransformation of N-hydroxy-FAA by 105,000 x g super-natants of rat liver to active intermediates capable ofintroducing FA groups into nucleic acid (16). Addition ofRNA at the end of the incubation period decreasedincorporation of the label into nucleic acid by more than97%. This reduction in the formation of FA:nucleic acidadducts indicated that the presence of the nucleic acid wasrequired during the incubation and that the fluorene associated with the nucleic acid recovered from the incubationsdid not result from failure of this method to removeunbound labeled fluorene derivatives. Assay by UV spec-troscopy of the RNA precipitates collected following incubation with the liver preparations disclosed that nucleic acidcould be recovered in 95% yield. Incorporation of N-hydroxy-FAA into nucleic acid was essentially linear withrespect to time during the 20-min incubation periodsutilized. Preliminary experiments demonstrated thatPP¡:NaCl was a more effective buffer than either Tris or P,.Incubations containing up to 0.1 ml of 105,000 x gsupernatant of rat liver displayed an incorporation of FAinto nucleic acid which was proportional to the volume ofliver preparation added to the system (Chart 1).

Early experiments showed that extensive loss of activityof the liver enzyme occurred in procedures that involvedprolonged experimental periods. Use of dithiothreitol minimized these losses in activity, and at 1 m.M it actuallyincreased the incorporation of FA into nucleic acids (Table1). At the higher concentration of 10 mM, this sulfhydryl-protecting agent reduced the incorporation of N-hydroxy-FAA, as might be expected if the dithiothreitol were to reactwith the reactive intermediate generated in the incubation(2). In agreement with the data of Bartsch et al. (2), additionof the sulfhydryl-blocking agents, p-chloromercuribenzoate,/V-ethylmaleimide, or iodoacetamide each reduced the incorporation of N-hydroxy-FAA by more than 90% at 1 mvi(Table 1).

In view of these findings, subsequent assays of thisenzyme were carried out on 0.1-ml samples of 105,000 x gsupernatants, the equivalent of 20 mg of tissue, in thepresence of 0.05 M PP¡:NaCl and 1 mM dithiothreitol.

Partial Purification of Rat Liver Acyltransferase. Approximately 10 to 15% of the acyltransferase activity wasrecovered in the precipitate formed on bringing the 105,000x g supernatant to 45% saturation by addition of asaturated solution of ammonium sulfate at room temperature. The major portion (80 to 85%) of the acyltransferaseactivity could be recovered in the precipitate that formed onincreasing the ammonium sulfate concentration from 45 to

—¿�2.0

1.5-

1.0-

Z

< 0.5-

0.05 O.I 0.15VOLUME OF SUPERNATANT (ml)

Chart I. Fluorenylamine substitution of tRNA as a function of ratliver 105,000 x g supernatant. The activation of N-hydroxy-FAA-9-"C

was measured by the introduction of label into tRNA in the presence ofvarying amounts of 105.000 x g supernatant of rat liver as described in"Materials and Methods."

Table 1Dependence of the acvltransferase-cataly:ed formation of

N-hydroxy-FAA :lRNA adducts on the presence of free sulßydryl groups

The formation of N-hydroxy-FAA:tRNA adducts catalyzed by acyltransferase in the 105.000 x g supernatant of rat liver was determined asdescribed in "Materials and Methods."

ReagentNoneDithiothreitolDithiothreitolDithiothreitolp-Chloromercuribenzoatep-Chloromercuribenzoatep-Chloromercuribenzoate,V-Ethylmaleimide,V-Ethylmaleimide/V-EthylmaleimideIodoacetamideIodoacetamideIodoacetamideConcentration(mM)O.I1.010.00.010.11.00.010.11.00.01O.I1.0Relativeformation

ofN-hydroxy-FAA:

tRNAadducts1001281237810529210511043552

65% saturation (Table 2). Attempts to utilize more restricted precipitation limits, to obtain precipitates at lowertemperatures, or to add solid ammonium sulfate generallyyielded less satisfactory results. The specific activity of theacyltransferase was increased approximately 4-fold by fractional precipitation with ammonium sulfate (Table 2), andthe precipitate obtained by this procedure could be storedfrozen indefinitely without loss of activity.

Acyltransferase partially purified by precipitation withammonium sulfate was subjected to chromatography oncolumns of Sephadex G-100 equilibrated with 0.02 MPP¡:NaCl buffer. The elution profile, as determined byabsorbance at 280 nm, gave evidence of 5 major proteinfractions (Chart 2). Acyltransferase activity was recoveredin 85% yield and was confined to an area that overlapped thelast 2 protein peaks to emerge from the column. The specific

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Arylhydroxamic Acid Acyltransferase

Table 2Purification oj arylamine-lransforming enzymes from rat liver

Acyltransferase was purified from the 105,000 x g supernatant of rat liver by fractional precipitation with ammonium sulfate andgel filtration on Sephadex G-IOO as described in "Materials and Methods." The 106 ml of 105.000 x g supernatant contained 2.29 g

of protein: 0.48 g of protein was precipitated on raising the ammonium sulfate concentration from 45 to 65^ of saturation.Acyltransferase was assayed by determining the ability of each preparation to catalyze combination of N-hydroxv-FAA with tRNAand by the acetylation of 4-aminoazobenzene. Formylkynurenine formamidase was assayed by determining the increase in

absorption at 360 nm on hydrolysis of the substrate. The purification factor obtained by use of gel filtration is based on theChromatographie fraction with the highest specific activity.

Acyltransferase

N-Hydroxy-FAA-tRNA

adduci formation

Acetylation of 4-aminoazo

benzeneFormylkynurenine form

amidase

Enzyme preparation

% re- Specific Purifi-covery activity" cation

% re- Specific Purifi-covery activity" cation

% re- Specific Purifi-covery activity' cation

105.000 x £supernatant45to 65% (N H4)2fractionEffluent

fromgellionSO,filtra-10082701.4.32.0513513.93110076620.0180.0640.53513.630100650.540.151.2510.32.3

" nmoles FA bound to tRNA/20 min/mg protein.*Amóles 4-aminoazobenzene acetylated/15 min/mg protein.'A Ajso/min/mg protein.

70

0.6 0.8 i.oELUTION VOLUME (I)

Chart 2. Gel filtration chromatography of acyltransferase. Rat liveracyltransferase partially purified by fractional precipitation with ammonium sulfate was chromatographed on Sephadex G-100 as described in"Materials and Methods." A sample of 10 ml (0.4 g protein) was applied to

this column. Acyltransferase activity is expressed as nmoles FA bound toRNA per ml of effluent per 20-min assay.

activity of the most active Chromatographie fraction was8-fold greater than the samples applied to the column andrepresented an overall 31-fold purification based on thespecific activity of the 105,000 x g supernatant (Table 2).

Estimation of Molecular Weight by Gel Filtration. Anestimate of the molecular weight of liver acyltransferase wasobtained by chromatographing a preparation purified byfractional precipitation with ammonium sulfate on a calibrated column of Sephadex G-100. Four proteins ranging inmolecular weight from 13,400 to 69,000 (42) were used toestablish the characteristics of this column. The datasummarized in Chart 3 were obtained from 2 series ofexperiments, each involving 3 Chromatographie profiles.

zO SQ.

5

401.5 2

IO'"»3 456

MOLECULAR WEIGHT

Chart 3. Estimation of the molecular weight of acyltransferase by gelfiltration. Acyltransferase partially purified by fractional precipitation withammonium sulfate was chromatographed on a calibrated column ofSephadex G-100 as described in "Materials and Methods." Each set of

points represents a series of 3 consecutive Chromatographie experiments.•¿�,acyltransferase: O, reference proteins. Experiment I; A. referenceproteins. Experiment 2.

The acyltransferase emerged with an apparent molecularweight of 28,000.

Modification of Extrahepatic Macromolecules by Acyltransferase. Under appropriate circumstances, arylaminescan induce tumors in rat tissues other than the liver (36, 43,44). In attempts to assess the role that acyltransferase mightplay in the modification of macromolecules of extrahepatictissues, several possibilities have been considered. Theapparent instability of the reactive derivative generated byacyltransferase, as indicated by failure of the active intermediate to accumulate in incubation systems (16), would seemto preclude the possibility that active intermediates synthesized in the liver might survive sufficiently long that theymight be transported to other tissues. Alternatively, it isconceivable that the relatively small size of acyltransferasemight permit transport of the enzyme from the liver toextrahepatic tissues, particularly if administration of the

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carcinogen led to lysis of liver cells.This latter possibility was approached experimentally by

administering N-hydroxy-FAA i.p. on each of 3 successivedays and then assaying the hepatic and serum acyltransfer-ase levels. The results of this experiment, shown in Table 3,demonstrate that increased quantities of acyltransferase donot appear in serum following administration of largequantities of N-hydroxy-FAA.

Hepatic acyltransferase was lower in animals that received the hydroxamic acid. This observation confirms theearlier report that administration of the acyltransferasesubstrate, N-hydroxy-FAA, does not induce the enzyme (2).

Direct assay of several extrahepatic tissues did revealsubstantial acyltransferase activity, particularly in kidney,stomach, small intestine, and colon (Table 4). No conclusivedifferences between tissues from male and female animalswere noted. Lesser levels of acyltransferase were found inspleen and lung, while serum, blood, muscle, and brainexhibited little, if any, activity. The actual level of acyltransferase in these tissues is best obtained by comparing thequantities of adducts formed per unit of tissue, since thesevalues are independent of the protein concentration ofextracts from the various tissues.

Comparative experiments with acetyl- and ring-labeledN-hydroxy-FAA were carried out with 105,000 x g super-natants of selected tissues to verify that the incorporation ofthe fluorene nucleus into nucleic acid by these preparationswas analogous to the activity observed with liver preparations. Results from each of the tissues assayed showed thatthe fluorene moiety of N-hydroxy-FAA was preferentiallyincorporated into nucleic acid under these conditions (Table5). Absence of the acetyl groups from these adductsdemonstrated that incorporation of N-hydroxy-FAA didnot result from incomplete removal of the hydroxamic acidby the methods used. These data further imply that an FAgroup, rather than an FA A group (16), had been introducedinto the nucleic acids and that the mechanism of activationof N-hydroxy-FAA by these tissues is similar.

Comparison of Acyltransferase with Other Arylamine-metabolizing Enzymes. Bartsch et al. (I, 2) originallyproposed that the introduction of FA groups into nucleic

Table 3Formation of N-hydroxy-FA A-iRNA adducts in vitro following

administration of N-hydroxy-FAAN-Hydroxy-FAA was suspended in 0.9% NaCI solution and injected i.p.

into 3 adult male Sprague-Dawley rats (25 mg/kg) at 9 a.m. on 3consecutive days. The animals were sacrificed 24 hr after the final injection,and the ability of the livers and serum to catalyze combination ofN-hydroxy-FAA with tRNA by acyl transfer was determined as describedin "Materials and Methods." Control animals were given injections of

0.9% NaCI.

Fluorene bound to tRNA (nmoles/20 mg tissue)

TissueLiver

SerumControl2.69

±0.13"

0.014 ±0.001N-Hydroxy-FAA-

treated1.99

±0.600.009 ±0.001

acids by a soluble preparation of rat liver may occur byTV—Otransfer of the acetyl group of N-hydroxy-FAA.Their proposal was based on several lines of evidence. Theyshowed that (a) addition of N-hydroxy-FA to a liver systemcapable of activating N-hydroxy-FAA increases the yield of1- and 3-methyl-mercapto-A'-2-aminofluorene, a degradation product of the FA-methionine adduct formed in theirexperiments; (b) nonenzymatic systems containing aceticanhydride, N-hydroxy-FA, and methionine yielded thesame product; (c) the soluble rat liver enzyme capable oftransferring the /V-acetyl group of arylhydroxamic acids toarylamines, as described by Booth (3), cochromatographedon Sephadex G-75 with the enzyme that activated N-hydroxy-FAA; and (d) the activation of N-hydroxy-FAA byacyltransferase was inhibited by amines (2).

In the present experiments, we have examined moreclosely the possibility that the acetyl transfer enzymedescribed by Booth is identical with the acyltransferaseresponsible for activation of N-hydroxy-FAA. Use ofN-hydroxy-FAA as acetyl donor and 4-aminoazobenzene asacetyl acceptor in Booth's acetyl transfer assay showed the

expected decrease in absorption at 497 nm which ischaracteristic of the loss of free 4-aminoazobenzene. 4-Acetylaminoazobenzene was identified as a product of thisreaction by use of thin-layer chromatography and UVspectroscopy (3).

Parallel assays of the acyltransferase activation enzymeand the acetyl-transferring enzyme described by Booth,following fractional precipitation with ammonium sulfateand gel filtration on Sephadex G-100, disclosed that the 2enzyme activities were recovered in approximately the sameyield by use of these procedures (Table 2). Furthermore, theelution profiles of these 2 enzymes were coincidental (Chart4), and the purification factors of the most active Chromatographie fractions were essentially identical for both enzymes(Table 2).

The relative tissue distribution of the acyltransferase-activating enzyme and the acetyl-transferring enzyme ofBooth is shown in Table 6. These data indicate that there issufficient enzyme in each of the tissues, as determined by thetransfer of the acetyl group to 4-amino-azobenzene, toaccount for the activation of N-hydroxy-FAA.

Although these experiments suggest strongly that theactivation of N-hydroxy-FAA by acyltransferase and thetransfer of acetyl groups of arylhydroxamic acids to arylamines are closely related, if not identical, the questionremains as to whether the acyltransferase can utilize anatural acyl donor to activate arylhydroxylamines. Thisconsideration prompted examination of formylkynurenineformamidase, since guinea pig liver formamidase had beenshown to catalyze transfer of the formyl group of /V-formyl-L-kynurenine to arylamines, including 1- and 2-naphthyla-mine, aniline, and anthranilic acid (39), and could conceivably be involved in the activation of N-hydroxy-FAA.Comparative assays during the partial purification ofacyltransferase disclosed that, unlike the acyltransferase-activating enzyme, most of the formamidase was precipitated with 45% ammonium sulfate (Table 2). The chromato-

1Average values ±S.D.

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Table 4Formation of .\-hydro.\y- FA A:lR N A adduci* by 105,000 x g supernatant* of rat tissues

The combination of N-hydroxy-FAA with tRNA induced by acyltransferase of 105,000 x gsupernatants of rat tissues was determined as described in "Materials and Methods."

N-Hydroxy-FAA bound to tRNA (nmoles)

MaleTissueLiverKidneySmall

intestineColonStomachSpleenLungBrainMuscleBloodSerumPer

20mgtissue2.51(7)°0.46(4)0.34(4)0.33(3)0.23(3)0.15(3)0.13(3)0.06(3)0.04(3)0.04(3)0.01

(6)Per

mgprotein1.56(6)0.29(4)0.54(4)0.47

(3)0.24(3)0.10(2)0.16(3)0.22(3)0.04(3)0.05(2)0.002(6)FemalePer

20mgtissue1.84(3)0.46(3)0.30(3)0.25(2)0.32(3)0.14(3)0.11(3)0.08(3)0.06(3)0.05(3)0.02(2)Permgprotein1.06(2)0.33(3)0.50(3)0.34(2)0.34(3)0.11

(3)0.14(3)0.18(2)0.05(3)0.04(3)0.002(2)

Average values are given. Numbers in parentheses, number of tissues assayed.

Table 5Formation oj'fluorenylamine-substiluled ¡K.\A adducts hy 105,000 x g

supernatant* of rat tissuesThe acyltransferase-induced combination of N-hydroxy-FAA with

tRNA which occurs on incubation of 105,000 x g supernatants of rattissues with N-hydroxy-KAA labeled with MC at either position 9 of thefluorene nucleus (N-hydroxy-FAA-9-"C, 1.0 juCi/^mole) or the carbonylcarbon of the acetyl group (N-hydroxy-FAA-l'-"C, 0.46 ^Ci/Mmole) wasdetermined as described in "Materials and Methods."

"C bound (nmoles/20 mg tissue) onincubation with

TissueLiverKidney

SmallintestineColonStomachRing-labeled

N-hydroxy-FAA2.340.44

0.370.340.16Acetyl-labeled

N-hydroxy-FAA0.060.02

0.010.020.01

graphic profile of formamidase also disclosed that it waseluted slightly earlier than the activation enzyme (Chart 4).

Table 6 shows the relative distribution of the formamidase in the tissues of the animals that had been assayed forboth the acyltransferase activation of N-hydroxy-FAA andBooth's enzyme. Little formamidase activity was detected in

tissues other than liver and kidney and therefore could notaccount for the activation of N-hydroxy-FAA in the gastrointestinal tract.

Differentiation between the Acyltransfer and N-Hydroxy-FAA Activation Processes. Activation of arylhydroxamicacids by acyltransfer requires the formation of /V-acyloxy-arylamine derivatives as shown in Reactions 1 and 2 ofChart 5. To test this hypothesis, the metabolism of N-methoxy-FAA, an 0-alkylated derivative of N-hydroxy-FAA, was studied. N-Methoxy-FAA, if deacetylated, couldnot be transformed to /V-acetoxy-/V-2-fluorenylamine, as in

0.8 09EtUTlON VOLUME ( I )

Chart 4. Chromatographie distribution of acyltransferase, as determined by N-hydroxy-FAA activation and acetylation of 4-aminoazobenzene. and formylkynurenine formamidase. Acyltransferase, partially purified by fractional precipitation with ammonium sulfate, was chromato-graphed on Sephadex G-100 as described in "Materials and Methods." The

effluent was assayed for acyltransferase by determining (a) the extent ofadduci formation between N-hydroxy-FAA and nucleic acids, and (b) theacetylation of 4-aminoazobenzene by N-hydroxy-FAA. Formylknurenineformamidase was measured by determining the increase in absorption at360 nm on hydrolysis of the substrate by the effluent. The enzyme activitiesare shown, in arbitrary units, on the ordinate. O, acyltransferase-catalyzedformation of N-hydroxy-FAA:nucleic acid adducts; •¿�.acyltransferase-catalyzed acetylation of 4-aminoazobenzene; A, formylkynurenine formamidase.

Reaction 2, because the oxygen of the hydroxylamine wouldbe blocked by a methyl group.

The activation of N-methoxy-FAA labeled in the fluorenenucleus with UC and in the acetyl group with 3H was studied

by incubation with 105,000 x g supernatant from liver andtRNA as described for N-hydroxy-FAA. Isolation of RNAfrom this system disclosed that less than 0.1% of theN-methoxy-FAA was associated with the nucleic acid(Table 7). In a positive control experiment carried out

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Table 6Relative activities of arylamine-lransforming enzymes oj ral tissues

The 105,000 x g supernatants of tissues from 3 adult male rats wereassayed individually for their ability to induce adduci formation betweenN-hydroxy-FAA and tRNA. catalyze the acetylation of 4-aminoazoben-zene by N-hydroxy-FAA, and hydrolyze formylkynurenine, as described in"Materials and Methods." The average enzyme levels, as based on the wet

weight of the tissues, was determined for each of the 3 enzymes. Forcomparative purposes, the relative capacities of these tissues to carry outeach of these reactions were established by assigning liver, the most activeof the tissues in each case, a value of 100 and expressing the levels in each ofthe other tissues as a percentage of the liver activity.

TissueLiverKidneySmall

intestineColonStomachSpleenLungBrainN-Hydroxy-FAA:tRNAadduciformation100131617122224-Aminoazo-benzeneacetylation1003115373011209Formyl

kynurenineformamidase10028112711

NH*CH3C-ACYirHANSffRASE

OH

(4) CHjC-ACVLTBANSFERASE

Chart 5. Acyltransferase-catalyzed formation of N-hydroxy-FAA:guanosine adducts, and acetylation of 4-aminoazobenzene.

simultaneously, 4.2% of the N-hydroxy-FAA was bound totRNA.

Experiments in which guanosine-8-14C replaced tRNA as

the trapping agent were carried out to examine further thepossible activation of N-methoxy-FAA by the 105,000 x gsupernatant of rat liver. Chromatography of these solutionson Sephadex G-10 disclosed that the radioactivity in the60% ethanol fraction, which would have contained 8-(/V-2-fluorenylamino)guanosine had it been formed, represented only a few percent of that observed in incubationswith N-hydroxy-FAA (Table 8).

It was then necessary to determine whether the inabilityof the enzyme to remove the acetyl group from N-methoxy-FAA, rather than the presence of the alkyl group on theoxygen, accounted for the lack of adduct formation. Acetyltransfer was not readily demonstrated in the assay ofBooth (3).

This question was therefore approached by determiningwhether the acetyl group of N-methoxy-FAA could betransferred to FA on incubation of the 2 compounds with105,000 x g supernatants from rat liver. N-Methoxy-FAA

labeled with 14Cin the fluorene nucleus and with 3H in theacetyl group was used in these experiments, since preliminary experiments showed that significant quantities of FAAwere formed by the reduction of N-methoxy-FAA onincubation in the PP¡:NaCl:dithiothreitol buffer in theabsence of enzyme. Use of the double-label techniquepermitted detection of acetyl transfer in the presence of thisnonenzymatic reduction by determination of the change in3H: 14Cof the FAA isolated from solutions of N-methoxy-

FAA and FA incubated in the presence and absence of105,000 x g supernatants of liver. Results of these experiments, shown in Table 9, indicate that, in the presence ofenzyme, 10% of the tritium but only 6% of the 14C fromN-methoxy-FAA-9-14C-2'-3H appeared in FAA. Consequently, the 3H: 14Cof the FAA was greater than that of theN-methoxy-FAA. In the absence of enzyme, however, the3H:14C in the isolated FAA was essentially that of theN-methoxy-FAA, thereby suggesting that the formation ofFAA in this case occurred without removal of the acetylgroup from the fluorene nucleus.

Inhibition of Acyltransferase by Arylamine Derivatives.Both the ability of added amines to diminish acyl transfer(2, 3) and the identification of 4-acetylaminoazobenzene asa product of the acetyl transfer reaction described abovesuggest that the product of the initial deacetylation ofN-hydroxy-FAA, N-hydroxy-FA, may be displaced byprimary amines so that the subsequent formation of N-acetoxy-yV-2-fluorenylamine is inhibited (Chart 5). Activation of N-hydroxy-FAA by 105,000 x g supernatants of ratliver is decreased in the presence of primary amines (Charts6 and 7). Although they are less effective than the primaryarylamines, the arylacetamides also inhibit adduct formation (Charts 6 and 7), even though they can neither donateacetyl groups to other primary amines (2, 3) nor competewith the hydroxylamine as an acetyl acceptor. This latterconsideration raised the question of whether the arylacetamides might be inhibitory by virtue of contamination withfree amines or by enzymatic deacetylation to more reactivefree amines during the incubations. Several experiments

Table 7Failure of 105,000 x g supernatant of rat liver to activate N-methoxy-FAA

The ability of 105,000 x g supernatant of rat liver to inducecombination of N-methoxy-FAA-9-"C-2'-3H with tRNA was determinedas described for N-hydroxy-FAA in "Materials and Methods." TheN-methoxy-FAA was labeled in the fluorene nucleus with 14C (1.07LiCi/fimo\e) and in the acetyl group with 3H (6.25 >íCi/íimole)to permit

detection of adduct formation with the nucleic acid. A positive control, inwhich N-hydroxy-FAA-9-"C replaced the labeled N-methoxy-FAA, is

included for comparative purposes.

Fluorene derivative bound to tRNA

(nmoles)

IncubationsystemN-Methoxy-FAA

-enzymeN-Methoxy-FAA+enzymeN-Methoxy-FAA+enzyme"N-Hydroxy-FAA

+ enzyme"C0.020.020.011.85'H0.040.030.02

" This experiment was a zero-time control in which the tRNA was

recovered without incubation.

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Table 8Guanosine adduci formation on activation of'\-hydroxy-FA derivatives hy 105,1100 x g

supernatant* of rat liverThe activation of N-hydroxy-FA derivatives by 105,000 x g supernatants of rat liver, as

determined by the formation of guanosine adducts. was carried out as described in "Materials andMethods." Guanosine-8-uC replaced tRNA as the trapping agent in incubations containing the

unlabeled fluorenyl derivatives indicated. 8-(/V-2-Fluorenylamino)guanosine was isolated by adsorption to Sephadex G-10 and elution with 60% ethanol.

IncubationsystemExperi

ment12FluorenederivativeincubatedNone.\-Hydroxy-/V-2-fluorenyl-propionamide;V-Hydroxy-/V-2-nuorenyl-propionamideN-Hydroxv-FAAN-Methoxy-FAANone.V-Hydroxy-/V-2-fluorenyl-propionamideA'-Hydroxyl-/V-2-fluorenyl-propionamideN-Hydroxy-FAAN-Methoxy-FAAAmóles

Enzyme_0.50.5

+0.5

+0.5+—2.52.5

+2.5

+2.5+i-

recovereoin 60% etha

nol(dpm)2,2004.40015.800130.5002.5008.2002.20074.800341.0006.600Guanosineaauuct

recovered"(nmoles)759331543

" Identity and purity of the 8-(.V-2-fluorenylamino)guanosine was established by thin-layer

Chromatograph y as indicated.

Table 9Transfer of the acetyl group of N-methoxy-FAA to FA by a 105,000 x g supernatant of rat liverTransfer of the 3H-labeled acetyl group of N-methoxy-FAA-9-"C-2'-3H (3H: "C = 5.8) to FA

was accomplished by incubation with 105.000 x g supernatant of rat liver as described in "Materialsand Methods." Unlabeled carrier FAA was added following incubation, protein was removed byprecipitation with ethanol, and the arylacetamide was recovered by repeated TLC" on silica gel. The

specific activity of the FAA was determined following each Chromatographie separation by use ofliquid scintillation and UV spectroscopy.

3H : 14C ratio of FAA recovered

following % conversion to FAA

Incubationsystem+

Enzyme- Enzyme1st

TLC"9.55.92ndTLC10.15.73rdTLC10.05.73H10.0

9.5"C6.0 9.7

" TLC, thin-layer chromatography.

suggest that these possibilities are unlikely. No Folin-posi-tive material was detectable by spot test of FAA oracetanilide (40). Adduci formation in incubation systemscontaining p-hydroxyacelanilide: N-hydroxy-FAA at amolar ratio of 10 was linear for periods of 2 to 20 min andtherefore gave no indication that inhibitory amines had beenformed during the experiment. Incubation of FAA at thehighest concentration used in these studies for periods of upto 20 min did not give rise to diazotizable amines (9).Comparable analyses of incubations that contained knownquantities of FA disclosed that the liberation of as little as 7nmoles of FA (i.e., less than 0.5% of the FAA incubated)would have been detected by this procedure.

Acyl Group Specificity. Interest in determining whetherthe acyltransferase of rat liver was specific for the acetylgroup was prompted by the question of whether this enzymemight play a role in arylamine carcinogenesis in species such

as the dog, which cannot acetylate arylamines (26), or intissues that might not be exposed to acetylated derivatives.Examination of the ability of the propionyl analog ofN-hydroxy-FAA to acylate 4-aminoazobenzene in the system of Booth (3) gave no evidence that this hydroxamic acidcould act as an acyl donor. /V-Hydroxy-/V-2-fluorenylpro-pionamide inhibited the formation of FA-substituted nucleic acid, but far less so than the amide, FAA (Chart 6).Use of guanosine as a trapping agent, however, gaveclear-cut evidence that /V-hydroxy-/V-2-fluorenylpropiona-mide could be activated by 105,000 x g supernatant of ratliver to yield 8-(Ar-2-fluorenylamino)guanosine (Table 8).

The identity of the adduci was established by comparison onIhin-layer chromatography to the adduci formed belweenN-hydroxy-FAA and guanosine. The amounl of adduciformed was related to the quantity of /V-hydroxy-/V-2-fluorenylpropionamide incubaled and, in Ihe 2 experi-

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zo>- 60

2 5[INHIBITOR]

[N-HYDROXY-FAA]

Chart 6. Inhibition of acyltransferase activation of N-hydroxy-FAAby derivatives of FA and 4-aminoazobenzene. The inhibition of theacyltransferase-catalyzed combination of N-hydroxy-FAA with RNA isplotted as a function of the molar ratio of inhibitor to N-hydroxy-FAA inthe activation system. O, FA: A, 4-aminoazobenzene; D. FAA; •¿�,A'-methoxy-FAA; •¿�/V-hydroxy-A^-fluorenylpropionamide.

Chart 7. Inhibition of acyltransferase activation of N-hydroxy-FAAby derivatives of aniline. The inhibition of the acyltransferase-catalyzedcombination of N-hydroxy-FAA with RNA is plotted as a function of themolar ratio of inhibitor to N-hydroxy-FAA in the activation system. O,aniline; D, acetanilide; •¿�,p-aminophenol; •¿�.p-hydroxyacetanilide.

ments shown here, accounted for 12 to 22% as much adducias formed by activation of N-hydroxy-FAA.

DISCUSSION

The 2 enzyme activities studied here, namely the activation of N-hydroxy-FAA and the transacetylation of 4-aminoazobenzene, appear to involve the same enzyme, asindicated by the similarities in tissue distribution andbehavior in purification procedures involving fractionalprecipitation with ammonium sulfate and gel filtration.Similarly, the activation of N-hydroxy-FAA by 105,000 x gsupernatants of tissues, in the absence of added cofactors, toyield intermediates capable of introducing FA groups intoRNA (5, 16, 17), DNA (5, 17), guanosine (2, 17), protein (5,17), and methionine (1,2) are most probably the result ofthe same mechanism of activation, if not the same enzyme.

Rat liver arylhydroxamic acid acyltransferase is a sulf-hydryl-dependent enzyme, as demonstrated by its inhibitionby reagents that react with sulfhydryl groups, and by theability of dithiothreitol to maintain the activity of theenzyme. Although it is to be expected that free sulfhydryl

groups might compete with other compounds added to trapthe reactive products formed by this enzyme, 0.1 to 1 mMdithiothreitol actually increased the acyltransferase-catalyzed formation of FA-substituted nucleic acid and permitted recovery of the enzyme in yields of greater than 80%from such procedures as dialysis, ammonium sulfate precipitation, and chromatography on Sephadex G-100. With 10mM dithiothreitol, however, adduct formation was decreased. These data are in agreement with those of Booth,who showed that cysteine could stimulate transfer of theacetyl group of /V-hydroxy-/V-4-biphenylacetamide to 4-aminoazobenzene and prevent loss of enzyme activity ondialysis (3). Similarly, by dialyzing against 30 mMcysteine,Bartsch el al. (2) could maintain the ability of rat liveracyltransferase to induce adduct formation of N-hydroxy-FAA with methionine, but this concentration of cysteine didnot stimulate the enzyme when added to fresh preparationsof liver cytosol.

The studies presented here differ from those reported byBartsch et al. (2) in several respects. Present and paststudies (15-19) in our laboratory have utilized nucleicacids or guanosine as trapping agents for the activatedintermediates, whereas methionine has been the usualtrapping agent used by Bartsch et al. (I, 2). Although theseworkers have routinely added NAD+ to their systems,

this cofactor has not been included in our assays, since experiments in our laboratory (C. M. King and B. Phillips,unpublished observations) have not shown that NAD+increased the incorporation of N-hydroxy-FAA into nucleic acid by soluble preparations of rat liver. Similarly,in Booth's study, NAD+ did not alter the acetylation of4-aminoazobenzene by /V-hydroxy-./V-4-biphenylacetamide(3). It is conceivable that the oxidation state of the enzymeand the nature of the trapping agent used in the assaymay be responsible for the minor differences noted betweenthese assay systems.

Bartsch et al. (2) observed that free arylhydroxylaminesenhanced the acyltransferase-catalyzed activation of N-hydroxy-FAA and that methionine adduct formation wasstimulated to a greater extent than was the formation ofguanosine derivatives. It was not necessary to add either thearylhydroxylamine or NAD* to our assay systems in order

to demonstrate acyltransferase activity; experiments currently in progress have shown that acyltransferase purifiedsome 5000-fold from rat liver can activate N-hydroxy-FAAin the presence of PP¡,dithiothreitol, and tRNA added asa trapping agent (C. W. Olive and C. M. King, unpublished observations).

Each report on the intracellular distribution of rat liveracyltransferase has presented evidence which suggests that,after homogenization, this enzyme is not associated withparticulate fractions of the cell (2, 3, 16, 17). The relativelylow molecular weight of approximately 28,000 and the easewith which the enzyme is solubilized leaves open, and makesdifficult, the question of its location in the intact cell. Kriek(22, 23) and Irving and Veazey (13, 14) have shown that theFA group is the major form of N-hydroxy-FAA bound toliver DNA in vivo; and an FAA derivative is the primaryform bound to rRNA.

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The reason for these differences is not known. If acyl-transferase were a nuclear enzyme, DNA might be alteredpreferentially. Alternatively, the reactive intermediate resulting from the activation of N-hydroxy-FAA by acyl-transferase, /V-acetoxy-TV-fluorenylamine, may be formed

in the cytoplasm and, because the reactive species isuncharged, then be able to cross the nuclear membrane. Bythe same reasoning, charged esters of N-hydroxy-FAAformed by conjugation with sulfate or phosphate outside thenucleus (5, 16, 46) might react more readily with cytoplas-mic nucleic acids than with DNA. Additional experimentsare needed to clarify these points.

The levels of acyltransferase in extrahepatic tissues of therat suggest that this enzyme may be involved in thearylamine-induced formation of tumors in these tissues. Theacyltransferase activities measured in 105,000 x g superna-tants probably represent minimal values, since the presenceof inhibitors or alternate pathways for the metabolism ofN-hydroxy-FAA would tend to decrease the apparent tissuelevels of acyltransferase.

The tissue distribution studies presented here demonstrate that sufficient enzyme, as determined by the trans-acetylation of 4-aminoazobenzene, is present in each of thetissues studied to account for the activation of N-hydroxy-FAA. Some tissues, especially the lung, were more active inthe transacetylation of 4-aminoazobenzene than in theactivation of N-hydroxy-FAA. Although these data implythat transacetylases that cannot acetylate the oxygen ofarylhydroxylarnines (Chart 5, Reaction 2) may exist in thesetissues, our data support the concept that there is 1 liverenzyme that catalyzes both the activation of N-hydroxy-FAA and the acetylation of 4-aminoazobenzene.

Several extrahepatic tissues of the rat, including kidney,mammary gland, and the gastrointestinal tract, have nowbeen shown to possess acyltransferase activity (1,2, 15) andto develop tumors on exposure to carcinogenic aromaticamines (36, 43, 44). No relationship between the sex of theanimal and level of acyltransferase was noted in ourexperiments. This observation may be pertinent to thesex-independent carcinogenic response of the gastrointestinal tract of the Sprague-Dawley rat to arylhydroxamic acids(35). One report of direct assay of isolated sebaceous glandsfor acyltransferase was negative (1), but other data suggestthat an enzyme capable of introducing FA groups intonucleic acid is present in this tissue (12), which is alsosusceptible to arylamine carcinogenesis (35).

The extreme reactivity of /V-acetoxyarylamines (31) andthe inability to synthesize /V-acetoxy-/V-2-fluorenylamine(2) have dictated the use of indirect experiments to determine whether yV-acyloxyarylamines are involved in theactivation of arylhydroxamic acids (2, 19). The failure ofliver acyltransferase to activate N-methoxy-FAA, an 0-alk-ylated derivative of N-hydroxy-FAA, demonstrates thenecessity of the free oxygen for the activation of thishydroxamic acid and further supports the concept that acyltransfer is involved in this mechanism of activation. Experiments with double-labeled N-methoxy-FAA showed thatthe acetyl group could be transferred to FA, thus obviatingthe possibility that adduct formation was prevented by an

inability of the acyltransferase to remove the acyl group.The activation of Ar-hydroxyl-Ar-2-fluorenylpropion-

amide demonstrated conclusively that acyl donors otherthan those possessing acetyl groups are capable of activating arylhydroxylamines. This observation may be important in the formation of bladder tumors in the dog, aspecies that cannot acetylate arylamines (26). Conceivably, bladder cells might extract acyl donors from the urineand utilize them in the activation of the arylhydroxylamines.

Inhibition by arylamines of the acyltransferase-catalyzedactivation of N-hydroxy-FAA had been noted previously (2,3) and has been extended in the present study. The primaryarylamines were more effective in the inhibition of N-hydroxy-FAA than were the arylacetamides; compoundscontaining the fluorenyl moiety were more effective thanwere benzene derivatives. Although arylamines would beexpected to diminish the modification of nucleic acids bycompeting with the arylhydroxylamines for the availableacetyl group, the effective inhibition of N-hydroxy-FAA

activation by the arylacetamides is apparently accomplishedwithout change of structure of the amides. The inhibition byacetanilide and p-hydroxyacetanilide of both the activationof N-hydroxy-FAA by acyl transfer and the hepatocarcino-genic activity of this hydroxamic acid (45 47) raises thepossibility that these processes may be related. Activation ofN-hydroxy-FAA by conjugation with sulfate would notappear to be uniquely involved in the inhibition of carcinogenesis by acetanilide or p-hydroxyacetanilide, sinceadministration of sulfate did not restore the first carcinogenic response although urinary excretion of free sulfateexceeded that of control animals (46, 47).

Furthermore, some biochemical evidence supports theconclusion that administration of acetanilide decreases theacyltransferase-catalyzed formation of adducts betweenN-hydroxy-FAA and nucleic acid in vivo. Pretreatmentwith acetanilide caused a greater decrease in the binding ofN-hydroxy-FAA to DNA than to either ribosomal orsoluble RNA (32). FA groups, probably the products of theactivation of N-hydroxy-FAA by acyltransferase, constitutethe major fluorene structure bound to liver DNA in vivo;FAA moieties are the predominant form of N-hydroxy-FAA bound to rRNA (13, 14, 22, 23) and probably resultfrom conjugation of the hydroxamic acid (4, 5, 16, 17).

The biochemical and biological consequences of theintroduction of FA groups into nucleic acids by acyltransferase may be multifaceted. Although no studies of theeffect of FA substitution on the template activity of nucleicacids are available, FAA substitution has been shown todecrease template activity (7, 8). Introduction of either FAor FAA groups into transforming DNA was associated withan increased frequency of mutations (28 30). FA-sub-stituted nucleic acids have been shown to be labile at neutralpH and 37°(18). Loss of the fluorene nucleus and the

characteristic UV absorption did not, however, restore thedecreased guanine content of the nucleic acid. Such reactions, if they were to occur in vivo, could alter theinformational content of nucleic acid without the continuedpresence of the carcinogen. Furthermore, the absence of the

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bulky fluorenyl group might make these modifications lesseasily recognizable by DN A repair systems of the cell (41).

ACKNOWLEDGMENTS

The participation of Benette Phillips in the initiation of the enzymepurification experiments is gratefully acknowledged. The author wishes tothank, D. J. Donaldson. Z. M. Lori/, and M. R. Thissen for theirassistance in these studies.

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2. Bartsch, H., Dworkin, M., Miller, J. A., and Miller, E. C. Electrophilic /V-Acetoxyaminoarenes Derived from Carcinogenic /V-Hy-droxy-/V-acetylaminoarenes by Enzymatic Deacetylation and Trans

acetylation in Liver. Biochim. Biophys. Acta, 286: 272 298, 1972.3. Booth, J. Acetyl Transfer in Arylamine Metabolism. Biochem. J.. 100:

745-753. 1966.4. DeBaun, J. R., Miller, E. C., and Miller, J. A. /V-Hydroxy-2-

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Rodent Livers Susceptible and Resistant to This Carcinogen. Proc.Soc. Exptl. Biol. Med., 129: 268 273. 1968.

6. Enomoto, M., Lotlikar, P.. Miller. J. A., and Miller. E. C. UrinaryMetabolites of 2-Acetylaminofluorene and Related Compounds in the

Rhesus Monkey. Cancer Res.. 22: 1336 1342, 1962.7. Grunberger. D., Nelson. J. H., Cantor. C. R.. and Weinstein, I. B.

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U. S. 66: 488 494. 1970.8. Grunberger, D., and Weinstein. I. B. Modifications of Ribonucleic

Acid by Chemical Carcinogens. III. Template Activity of Polynucleo-tides Modified by /V-Acetoxyl-2-acetylaminofluorene. J. Biol. Chem.,

246: 1123 1128, 1971.9. Gutmann. H. R.. Kiely, G. E., and Klein, M. Metabolism of

2-Aminofluorene in the Rat. Cancer Res., 12: 350 355. 1950.

10. Hayes, F. N. Technical Bulletin No. I, p. 4. Downers Grove, 111.:Packard Instrument Company. 1963.

11. Irving. C. C. Enzymatic Deacetylation of /V-Hydroxy-2-

acetylaminofluorene by Liver Microsomes. Cancer Res., 26: 139]

1396, 1966.12. Irving, C. C., Janss. D. H., and Russell, L. T. Lack of /V-Hydroxy-2-

acetylaminofluorene Sulfotransferase Activity in the Mammary Glandand Zymbal's Gland of the Rat. Cancer Res., 31: 387 391, 1971.

13. Irving, C. C.. and Veazey. R. A. Persistent Binding of 2-Acetylamino-

fluorene to Rat Liver DNA In Vivo and Consideration of theMechanism of Binding of /V-Hydroxy-2-acetylaminofluorene to RatLiver Nuleic Acids. Cancer Res., 29: 1799-1804, 1969.

14. Irving, C. C.. and Veazey, R. A. Differences in the Binding of2-Acetylaminofluorene and Its /V-Hydroxy Metabolite to LiverNucleic Acids of Male and Female Rats. Cancer Res., 31: 19-22,

1971.15. King, C. M., and Olive, C. W. Comparative Effects of Age, Strain and

Species on the Activation of /V-Hydroxy-2-fluorenylacetamide (/V-Hy-droxy-FAA) by Soluble Acyltransferases. Proc. Am. Assoc. Cancer

Res., 14: 4\, 1973.

16. King, C. M., and Phillips, B. Enzyme-Catalyzed Reactions of theCarcinogen /V-Hydroxy-2-fluorenylacetamide with Nucleic Acid.

Science, 159: 1351 1353, 1968.17. King, C. M., and Phillips, B. /V-Hydroxy-2-fluorenylacetamide: Re

action of the Carcinogen with Guanosine. Ribonucleic Acid, Deoxy-

ribonucleic Acid, and Protein following Enzymatic Deacetylation orEsterification. J. Biol., Chem., 244: 6209-6216, 1969.

18. King, C. M.. and Phillips. B. Instability of Fluorenylamine-Sub-

stituted Polynucleotides: Loss of Carcinogen and Production of anAltered Nucleic Acid. Chem.-Biol. Interactions. 2: 267 271, 1970.

19. King, C. M., and Phillips, B. Mechanism of Introduction of Fluorenyl-

amine Substituents into Nucleic Acid by Rat Liver. Proc. Am. Assoc.Cancer Res., 13: 43. 1972.

20. Kriek. E. On the Interaction of A'-2-Fluorenylhydroxylamine with

Nucleic Acids In Vitro. Biochem. Biophys. Res. Commun., 20:

793 799, 1965.21. Kriek, E. Reaction of /V-2-Fluorenylhydroxylamine with Nucleic

Acids In Vitro. In: Carcinogenesis. A Broad Critique. M. D. Anderson

Hospital and Tumor Institute at Houston, pp. 441 446. Baltimore:Williams & Wilkins. 1967.

22. Kriek, E. Difference in Binding of 2-Acetylaminofluorene to Rat LiverDeoxyribonucleic Acid and Ribosomal Ribonucleic Acid In V"o.Biochim. Biophys. Acta, 161: 273-275, 1968.

23. Kriek, E. On the Mechanism of Action of Carcinogenic AromaticAmines. I. Binding of 2-Acetylaminofluorene and /V-Hydroxy-2-acetylaminofluorene to Rat Liver Nucleic Acids In Vivo. Chem.-Biol.

Interactions. /: 3 17. 1969.24. Kriek, E., Miller. J. A.. Juhl, U.. and Miller, E. C. 8-^-2-

Fluorenylacetamido)guanosine, an Arylamidation Reaction Productof Guanosine and the Carcinogen /V-Acetoxy-/V-2-fluorenylacetamide

in Neutral Solution. Biochemistry, 6: 177 182, 1967.25. Lotlikar, P. D., Scribner, J. D., Miller, J. A., and Miller, E. C.

Reactions of Esters of Aromatic /V-Hydroxy Amines and Amides

with Methionine In Vitro: a Model for In Vivo Binding of Amine

Carcinogens to Protein. Life Sci., 5. 1263 1269, 1966.26. Lower, G. M.. Jr.. and Bryan, G. T. Enzymatic /V-Acetylation of

Carcinogenic Aromatic Amines by Liver Cytosol of Species Displaying Different Organ Susceptibilities. Biochem. Pharmacol., 22:1581 1588. 1973.

27. Lowry, O. H., Rosebrough, N. J., Farr. A. L., and Randall. R. J.Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.193: 265 275, 1951.

28. Mäher. V. M.. Miller, E. C.. Miller, J. A., and Szybalski, W.Mutations and Decreases in Density of Transforming DNA Producedby Derivatives of the Carcinogens 2-Acetylaminofluorene and N-Methyl-4-aminoazobenzene. Mol. Pharmacol., 4: 411 -426, 1968.

29. Mäher. V. M., Miller, J. A., Miller, E. C., and Summers, W. C.Mutations and Loss of Transforming Activity of Bacillus subtilisDNA after Reaction with Esters of Carcinogenic /V-Hydroxy Aro

matic Amides. Cancer Res., 30: 1473 1480, 1970.30. Mäher, V. M., and Reuter, M. Mutations and Loss of Transforming

Activity of DNA Produced by Interaction with the O-Glucuronide of/V-Hydroxy-2-aminofluorene (N-OH-AF). Proc. Am. Assoc. Cancer

Res., 12: 72, 1971.31. Marmer, W. N., and Maerker, G. The Preparation and Reactions of

Novel O-Acylhydroxylamines. J. Org. Chem., 37: 3320 3323, 1972.

32. Matsushima, T., and Weisburger, J. H. Inhibitors of ChemicalCarcinogens as Probes for Molecular Targets: DNA as DecisiveReceptor for Metabolite from /V-Hydroxy-/V-2-fluorenylacetamide.Chem.-Biol. Interactions, /. 211 221, 1969.

33. Mehler, A. H., and Knox. W. E. The Conversion of Tryptophan toKynurenine in Liver. I. The Coupled Tryptophan Peroxidase-Oxi-

dase System Forming Formylkynurenine. J. Biol. Chem., 187:431 438, 1950.

34. Miller, E. C., Juhl, U., and Miller, J. A. Nucleic Acid Guanine:

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Reaction with the Carcinogen /V-Acetoxy-2-acetylaminofluorene.Science, 153: 1125 1127, 1966.

35. Miller, E. C., Miller, J. A., and Hartmann, H. A. /V-Hydroxy-2-acetylaminofluorene: A Metabolite of 2-Acetylaminofluorene withIncreased Carcinogenic Activity in the Rat. Cancer Res., 21: 815-824,

1961.36. Miller. J. A. Carcinogenesis by Chemicals: An Overview—G.H. A.

Clowes Memorial Lecture. Cancer Res., 30: 559 576, 1970.37. Monier, R., Stephenson, M. L., and Zamecnik, P. C. The Preparation

and Some Properties of a Low Molecular Weight Ribonucleic Acidfrom Baker's Yeast. Biochim. Biophys. Acta, 43: I 8, 1960.

38. Poirier, L. A., Miller. J. A., and Miller, E. C. The N- andRing-hydroxylation of 2-Acetylaminofluorene and the Failure toDetect /V-Acetylation in the Dog. Cancer Res., 23: 790-800, 1963.

39. Santi, R. S. S., and Hopsu/Havu, V. K. Transformylation ofCarcinogenic Aromatic Amines by Kynurenine Formamidase: ADetoxification Mechanism. Biochem. Pharmacol.. 17: 1110 1113,1968.

40. Stahl, E. (ed.). Thin Layer Chromatography, A Laboratory Handbook, p. 498., New York: Academic Press. Inc., 1965.

41. Stich, H. F., San, R. H., Miller, J. A., and Miller, E. C. VariousLevels of DNA Repair Synthesis in Xeroderma Pigmentosum CellsExposed to the Carcinogens /V-Hydroxy and /V-Acetoxy-2-acetylaminofluorene. Nature New Biol., 238: 9-10, 1972.

Arylhydroxamic Acid Acyltransferase

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1973.45. Weisburger, J. H., Weisburger, E. K., Madison, R. M., Wenk, M. L.,

and Klein, D. S. Effect of Acetanilide and /»-Hydroxyacetanilideonthe Carcinogenicity of /V-2-Fluorenylacetamide and A'-Hydroxy-W-2-fluorenylacetamide in Mice, Hamsters, and Female Rats. J. Nati.Cancer Inst., 51: 235-240, 1973.

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47. Yamamoto, R. S., Williams, G. M., Richardson, H. L., Weisburger,E. K., and Weisburger, J. H. Effect of/J-Hydroxyacetanilide on LiverCancer Induction by /V-Hydroxy-W^-fluoroenylacetamide. CancerRes., 31: 454-457, 1973.

JUNE 1974 1515

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