And the Failure to Detect N-Acetylation of 2-Aminofluorene in the Dog*

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The N- and Ring-Hydroxylation of 2-Acetylaminofluorene and the Failure To Detect N-Acetylation of 2-Aminofluorene in the Dog*

LIONEL A. P0IRIER, JAMES A. MILLER, AND ELIZABETH C. MILLER

(McArdle Memorial Latoratoryfor Cancer Research, Medical School, University of Wiscon.rin, Madison, Wisconthi)

SUMMARY N-Hydroxy-2-acetylaminofluorene (N-hydroxy-AAF), in conjugated form, was identified as a urinary metabolite of 2-acetylaminofluorene (AM') in male mongrel dogs. This metabolite was isolated and characterized in crystalline form. 7-Hydnoxy AM', in conjugated form, and AM? were also found in the urine of dogs fed AAF; no 1-, 3-, or 5-hydroxy-AAF was detected. The ingestion of N-hydroxy-AAF led to the urinary excretion of the same metabolites; however, none of these acetylated metabo lites was detected in the urine of dogs fed 2-aminofluorene, N-hydroxy-2-aminofluorene, 1-hydroxy-AAF, or 3-hydroxy-AAF. Dietary supplementation with calcium pantoth enate and riboflavin and an attempt to induce acetylase activity by feeding 2-amino fluonene for several days did not lead to the urinary excretion of any recognizable acetylated urinary metabolites of 2-aminofluorene. Furthermore, under similar condi tions the specific activities of the acetylated urinary metabolites of 2-(acetyl-1'-C'4) aminofluonene fed in mixtures with unlabeled 2-aminofluorene were not appreciably different from the specific activity of the ingested acetyl-labeled AAF. In a dog fed a single dose of AAF-9-C'4 63 pen cent of the C'4 was excreted in the feces, and 19 per cent of the C'4 was found in the urine during the next 5 days. Approximately 3 per cent of an oral dose of AAF was found as 7-hydnoxy-AAF in the feces collected during the 1st day. No N-hydroxy amides were detected in the urine of dogs after ingestion of the fol lowing amides: acetanilide and its p-vinyl, p-fluoro, and p-ethoxy derivatives; trans 4-acetylaminostilbene; 2-propionylaminofluonene; and 2-n-butyrylaminofluorene. Ad ministration of 2-acetylaminonaphthalene to a dog led to the urinary excretion of very small amounts of this amide and its N-hydroxy metabolite. The synthesis of N-hydroxy-2-aminofluorene, a new compound, is described.

The versatile carcinogenic activity of 2-acetyl 26) . Other aromatic amides and amines such as aminofluorene (AM') has been demonstrated by 2-aminonaphthalene (5, 16), 4,4'-diaminobiphenyl the variety of species and tissues in which this (benzidine) (30, 33'), 4-aminobiphenyl (11, 33) compound can induce malignant tumors (34) . In and 4-acetylaminobiphenyl (18) also exhibit car the dog AAF has been found to be a carcinogen cinogenic activity in the urinary bladder of the for the urinary bladder (26) and the liver (3, 18, dog. Likewise several of these carcinogensâ€”viz., a This investigation was supported by a research training 2-aminonaphthalene, 4,4'-diaminobiphenyl, and grant, CRTY-5002, and by Grant C355 of the National Cancer 4-aminobiphenylâ€”pnoduce tumors of the urinary Institute, United States Public Health Service; by a grant bladder in humans following industrial exposure from the Jane Coffin Childs Memorial Fund for Medical Re (7, 14, 21, 32). Thus, studies on the metabolism of search; and by the Alexander and Margaret Stewart Trust Fund. We are grateful to Mrs. Wai Won Moy for valuable carcinogenic aromatic amines and amides in the technical assistance. 1 The article by Walpole et at. (33) contains further data on Received for publication January 21, 1963. the carcinogenicity of benzidine in the dogs of Spitz et at. (SO). 790

dog may provide clues to the mechanism by which butyrylaminofluorene (173Â°â€”175Â°),@trans4-acetyl these compounds induce bladder tumors in both aminostilbene (231Â°â€”232Â°)(4),5p-vinylacetanilide species.2 (140Â°â€”141Â°) (29), and p-fluoroacetanilide (151Â°â€” In the rat the carcinogenic action of AAF (22) 152Â°) (20). The following compounds were com and of 4-acetylaminobiphenyl (25) appears to pro mercial products: acetanilide (115Â°â€”116Â°)(Baker ceed through the N-hydroxylation of the parent and Adamson, N. F.), p-ethoxyacetanilide (134Â°â€” amide. The dog is known to N-hydroxylate 4-ace 136Â°) (Matheson, Coleman, and Bell), 2-acetyl tylaminobiphenyl (2.5), and both the dog (6, 31) aminonaphthalene (131Â°â€”132Â°)(Aldrich). and the human (31) N-hydroxylate 2-aminonaph N-Hydroxy-2-aminofluorene is a new com thalene. This report presents data on : (a) the pound and was prepared in this laboratony@ from N- and ning-hydroxylation of AAF and related 2-nitrofluorene by an adaptation of the general compounds in the dog and (b) our failure to dem procedure of WillstÃ¤tter and Kubli (40). One gram onstrate the N-acetylation of 2-aminofluorene in of pure 2-nitrofluonene (m.p., 157Â°â€”158Â°C.)(Al this species. drich) was dissolved in 800 ml. of 95 per cent ethanol with heat. The solution was placed in a MATERIALS AND METHODS large ice bath and was saturated with NH3 at 10Â°â€” Compounds.â€”Except where noted the melting 15Â°C.; this required about 45 mm. The solution points (Â°C.) are uncorrected. The respective melt was then saturated with H2S. This step also re ing points and references to the syntheses of the quired about 45 mm. and was followed by the ap compounds prepared in this laboratory were as pearance of crystals of NH4SH. The solution was follows : AAF (192Â°â€”194Â°)(24),2-(acetyl-1'-C'4)- allowed to stand in the ice bath for 20 hours, by aminofluorene (192Â°â€”193Â°)(28),2-aminofluorene which time the ice had melted and the solution was (126Â°â€”127Â°) (19), N-hydroxy-AAF (145Â°â€”147Â°) nearly at room temperature. Two volumes of ethyl (22),@ 2-propionylaminofluorene (203Â°â€”205Â°),@2-n- ether were added, and the precipitate of NH4SH 2 Nomenclature in this paper (Chemical Abstracts nomen was removed by gravity filtration through coarse clature): paper. The filtrate was placed in a large pear 2-acetylaminofluorene (N-2-fluorenylacetamide) 2-propionylaminofluorene (N-2-fluorenylpropionamide) shaped sepanatony funnel fitted with an air-driven 2-n-butyrylaminofiuorene (N-2-fluorenyl-n-butyra stirrer and a tube for the introduction of N2. mide) Under air-free conditions the solution was extract 4-acetylaminobiphenyl (4'-phenylacetanilide) ed repeatedly with 50-ml. volumes of air-free 2-acetylaminaphthalene (N-2-naphthylacetamide) water (held in another sepanatory funnel under N2) 4-acetylaminostilbene (4'-styrylacetanilide) N-hydyoxy-2-acetylamino (N-hydroxy-N-2-fluorenyl until the ether-ethanol layer was only a very light fluorene acetamide) yellow in color and the aqueous extracts were x-hydroxy-2-acetylamino (N-(x-hydroxy)-2-fluorenyl colorless. Strict avoidance of 02 is important at fluorene acetamide) this point, since the dissolved hydroxylamine is 2-aminofluorene (2-fluorenamine) readily oxidized to azoxyfluorene. The ether-etha 4-aminobiphenyl (4-biphenylamine) 2-aminonaphthalene (2-naphthylamine) nol layer was then transferred anaerobically to a 4,4'-diaminobiphenyl (same) distillation apparatus and concentrated in vacuo p-x-acetanilide (4'-x-acetanilide) in a water bath at 40Â°C. When the volume of the 2-nitrofluorene (same) solution reached about 100 ml., the hydroxylamine 3 A modified procedure was employed in our more recent preparations of N-hydroxy-AAF. A solution of pure 2-nitro started to crystallize. The concentration was con fluorene (m.p., 157Â°â€”158Â°C.)(Aldrich) in ethyl acetate was tinued until the solution was filled with crystals. reductively acetylated as previously described (22). The hy The suspension of crystals was then quickly fil drolysis with ammonium hydroxide was carried out at room temperature for 4 hour with vigorous mixing of the two liquid tered in air through hard paper in a small suction phases. After removal of the ethyl acetate in vacuo the residue funnel. The crystals were washed with cold water, was dissolved in ethyl ether and extracted twice with dilute and the funnel and contents were dried in vacuo sodium hydroxide solution. The combined alkaline extract was extracted repeatedly with ethyl ether until the ether extracts over concentrated sulfuric acid in the dark. The were only slightly yellow.The alkaline extract was then acidi 4 The propionyl and n-butyryl derivatives of 2-aminofluo fled in an ice bath to pH 3. At this pH the precipitate of N- rene were prepared by the complete reductive acetylation of hydroxy-AAF was readily filtered by suction on paper sup 2-nitrofluorene with the corresponding anhydride in a manner ported by glass cloth. The precipitate was washed with water analogous to that employed in the preparation of N-hydroxy and dried in vacuoover sulfuric acid. At this stage many of AAF (22). These amides have been described previously the preparations have melted in the desired range (145Â°- (12, 27). 147Â°C.); however, other presumably identical preparations have required crystallization from benzene to attain this melt 5 This compound was generously supplied by Dr. R. A. An ing poinL All the products have given titrations of 98â€”100per dersen of this laboratory. cent theory upon reduction with titanium trichloride (15). 6 J@ A. Miller and G. S. Brenner.

light yellow microcrystalline mass separated readi ded in about 75 gm. of ground horsemeat (K-9, ly from the paper, and it was relatively stable to K-9 Canning Co., Madison, Wis.). Such feedings oxygen as long as it was kept in the dank in the were repeated 3 and 6 hours after the initial inges refrigerator. The yield was usually about 0.6 gm. tion to provide a total dose of 150 mg. Occasionally The substance had an indefinite melting point; it doses larger on smaller than this were adminis sintered and partly melted with decomposition at tered; these exceptions are specifically mentioned 170Â°â€”175Â°C.in a capillary tube in air or in nitro below. The interval between the administration of gen. Recrystallization of this compound from van two different compounds to the same dog was gen ous solvents under N2 did not yield material with a enally of the order of 2 weeks. Urine collections sharper melting point. Elementary analysis7 gave were made for 24-hour periods following the first the following data: ingestion of the compound. The urine was collect ed in 250-ml. ice-cooled bottles containing 100 mg. Calculated for C13H11NO: C, 79.25; H, 5.95; N, 7.12. of sodium fluoride. If the urine was not analyzed immediately, it was stored at â€”15Â°C. Found: C, 78.75; H, 5.53; N, 6.8.5. Determination of the urinary metabolite8.â€”The The hydroxylamine reacted with benzaldehyde enzymatic release of the conjugated metabolites in absolute ethanol to give a nitrone which melted and the paper partition chromatography of the at 200Â°â€”202Â°C.Elementary analysis7 of this ni metabolites were performed essentially according trone gave the following values: to the methods previously published (8, 23). The 24-hour urine samples were diluted to 1 liter with Calculated for C2oHi@NO: distilled water and brought to pH 6 with a few C, 84.22; H, 5.26; N, 4.91. drops of glacial acetic acid. An 18-ml. aliquot was Found: incubated for 15 hours at 37Â°with 12 mg. of a bac C, 84.33; H, 5.55; N,4.69. tenial $-glucunonidase preparation and 15 mg. of Takadiastase in the presence of 0.25 ml. of chloro Analyses of the hydroxylamine by titration with form. The mixture was then extracted with 60 ml. TiCl3 (15) gave values of 94â€”98per cent of theory. of peroxide-free ethyl ether, and the ether in turn Usually 0.2 mmoles of the compound was reacted was successively extracted with 15 ml. each of 10 under N2 in 30 ml. of dimethylformamide with 40 pen cent Na2CO3 (17), water, 0.5 N HC1, and ml. of 0.04 N TiCl3 at room temperature for 15 water. The ether was evaporated with a stream of minutes. The reaction mixture was stirred mag N2, and the residue dissolved in 0.5 ml. of ether netically. The solution was then back-titrated with and distributed between three paper strips for standard 0.04 N ferric ammonium sulfate. Ten ml. chromatography. Quantitative estimations of the of 10 per cent ammonium thiocyanate was added per cent excretion of each metabolite were ob as an indicator near the end of the titration. The tamed from the spectra of the eluates of the chro titanous ion solution, indicator solution, and reac matograms. When AAF, N-hydroxy-AAF, and tion mixture were kept free of oxygen by a stream 7-hydroxy-AAF were added to normal urine prior of N2. The fernic ion solution was standardized by to incubation, the compounds could be recovered reaction with excess titanous ion and back-titra after chromatography to the extent of 95, 55, and tion with standard 0.02 N K2Cr2O7. 87 pen cent, respectively. The urinary excretions The 1-hydroxy-AAF (m.p., 212Â°â€”213Â°,corr.) reported here are corrected for these recoveries. and the 3-hydroxy-AAF (m.p., 249Â°â€”251Â°,conr.) 1801a.tion of N-hydroxy-AAF from urine.â€”Two wene generously supplied by Dr. T. Lloyd Fletcher dogs were each fed 1 gm. of AM? over a period of of the University of Washington, Seattle. The 4 days, and the urine was collected and pooled. sample of AAF-9-C'4 was kindly furnished by Dr. After enzymatic hydrolysis of the conjugate, the John H. Weisburger of the National Cancer Insti isolation of the N-hydnoxy-AAF was performed tute, Bethesda, Maryland. essentially according to the cupnic chelate proce Animok.â€”Five young adult male mongrel dogs dure of Enomoto et a!. (13). After decomposition weighing 9â€”likg. were used. They were housed in of the cupnic chelate in ethanol by H2S and remov heavy wire-mesh metabolism cages which were al of the cupnic suffide by centrifugation, the washed daily. Dry commercial dog meals (Gaines, ethanolic solution was taken to dryness in vacuo in Fromm) and water were supplied ad libitum. a flash evaporator. The N-hydroxy-AAF was Administration of compounds and collection of quantitatively transferred to a centrifuge tube urine.â€”The dogs were fed gelatin capsules con with about 2 ml. of ethanol, and the product was taming 50 mg. of the compound under test imbed precipitated by the addition of 6 ml. of n-hexane.

7 Spang Microanalytical Laboratory, Ann Arbor, Michigan. The N-hydroxy-AAF was then recrystallized once

from ethanol-hexane and 3 times from ethanol ether solution containing the AM' was then water and finally dried over P@O@invacuo. washed successively with 10 ml. of 0.5 N NaOH Urinary metabolites of @-(acetyl-1'-C'4)amino and 15 ml. of water. The alkaline solution contain fluorene.â€”The specific radioactivities of the un ing the N-hydroxy-AAF was brought to pH 6 by nary metabolites of 2-(acetyl-1 â€˜-C'4)aminofluorene the addition of 15 ml. of 0.5 N HC1 and 7 ml. of were determined in four experiments. In one exper 1 M pH 6 sodium acetate buffer. This solution was iment only acetyl-labeled AM? was administered. then extracted twice with 20 ml. of ether, and the In the other experiments 2-aminofluonene was also ether extracts containing the N-hydroxy-AAF fed, and in two experiments daily supplements of were combined and washed once with 20 ml. of calcium pantothenate (20 mg/kg/day) and ribo water. flavin (2.5 mg/kg/day) were fed. In the first three For the determination of the specific radioac experiments the doses of the carcinogens were tivities of the metabolites each of the ethereal ex divided into three equal portions and fed at 3-hour tracts was evaporated to dryness with a stream of intervals. The unines were collected for 24 hours N2, and the residue was dissolved in 0.5 ml. of after the first portion, diluted with an equal vol ether. The concentrates were applied to three pre ume of water, and brought to pH 6. As many as washed paper strips for rechnomatography in the fourteen 18-ml. aliquots of the diluted unines were same solvent system. The central portion of each incubated with the enzymes, and the metabolites rechnomatographed zone (the most intensely ab were extracted and chromatographed on paper as sorbing region viewed under ultraviolet light) from described above. This provided the large number the three strips was eluted into 5.0 ml. of absolute of strips needed for the location of the metabolites ethanol. The ultraviolet absorption spectra of the and for the accumulation of enough of the metabo solutions were recorded with a Beckman DB spec lites for the spectral and radioactivity measure trophotometen, and the concentrations of the ments. All the paper strips used for chromatogra metabolites were determined from the spectra. phy in these experiments were pre-washed by de Immediately afterwards, 100â€”[email protected] each solu scending chromatography with the upper (organic) tion was plated in triplicate on aluminum planch phase of the solvent system to remove contami ets. The radioactivities of these samples were de nants that absorb ultraviolet light. After chroma termined from duplicate 5-minute readings in a tography of the extracts three strips from each gas-flow end-window Geiger counter. experiment were sprayed with the indicator re A fourth experiment with acetyl-labeled AAF agents (8) to locate the N-hydroxy-AAF and was performed to investigate the possibility of in 7-hydnoxy-AAF zones. The positions of these duction of an acetylase system in the dog by the zones and of AAF on the remaining strips from administration of 2-aminofluorene for several days. which the metabolites were to be eluted were con A dog was fed supplements of calcium pantothe firmed by visual observation under ultraviolet nate and riboflavin for 12 days. On days 7â€”11 light (@@*s2.54ms). 100 mg. of 2-aminofluonene (two 50-mg. cap The portions of the chromatographic strips con sules given 6 hours apart) was administered daily. taming the N-hydnoxy-AAF and AAF (the zones On day 11 the urine was collected for 24 hours and containing these compounds partly overlap) or the examined carefully for acetylated metabolites. On 7-hydroxy-AAF were cut out for elution. Groups day 12 a mixture of 2-(acetyl-1'-C'4)aminofluorene of three to six pieces were placed in glass-stop and 2-aminofluorene (34 and 136 mg., respective pered test tubes, moistened with 0.3 ml. of ethanol, ly) was fed, and the urine was again collected for and allowed to stand overnight at 5Â°C. The next 24 hours. The 24-hour urine sample collected on day 4â€”flml. (depending on the number of strips) the 11th day was treated as follows. It was diluted of ethyl ether were added to each tube, and the with distilled water to 500 ml., and the pH was contents were gently shaken. The eluates from like adjusted to 6 with acetic acid. Fifty ml. of the di tubes were combined, each tube of strips was luted urine was then added to each of ten 125-mI. washed once or twice with 3â€”5ml. of ethyl ether, Erlenmeyen flasks. To each flask was added 0.5 and the washings were added to the eluates. The ml. chloroform, 25 ml. of 1.0 M pH 6 sodium ace separation of the N-hydroxy-AAF and AAF was tate buffer, and 2 ml. of an enzyme solution con accomplished as follows : The combined eluates taming 30 mg. of Takadiastase and 36 mg. of bac were extracted 3 times in a small separatory funnel tenial @3-glucuronidase. The flasks were incubated with 5-mI. portions of 0.5 N NaOH. The combined at 37Â°C. for 15 hours. After the incubation the alkaline extracts were extracted twice with 15-mi. contents of pairs of flasks were combined and ex portions of ether, and the ether layers were added tnacted with 200 ml. of peroxide-free ether. The to the original ether solutions. This combined aqueous layer was discarded. The ether layer was

successively extracted with 50 ml. of 10 per cent sprayed with an acidic solution of p-dimethyl sodium carbonate, 50 ml. water, 50 ml. 0.5 N HC1, aminobenzaldehyde; it also reduced the Folin and 50 ml. of water. The ether extracts were then Ciocalteu phenol reagent to give a blue color. The combined and taken to dryness in vacuo in a flash R@(0.74â€”0.82)andabsorptionspectrumof this evaporator. The residue was removed from the zone were identical with those of synthetic N-by flask by three successive 50-ml. rinses with ether. droxy-AAF. The 7-hydroxy-AAF was detected The ether washes were combined in a sepanatory with the Folin-Ciocalteu reagent and identified funnel and washed once with 40 ml. of water. The by the correspondence of its R@and spectrum with ether layer was then divided equally between two those of the authentic compound. Likewise, the pear-shaped flasks, and the ether was removed AAF zone was identified by its RF and spectrum. with a stream of N2. The residue from each flask Neither N-hydnoxy-AAF non 7-hydroxy-AAF was was spotted on three paper strips. After chromatog observed in the urine prior to incubation with raphy three strips were sprayed with the indicator $-glucuronidase and Takadiastase; thus, these reagents, and the appropriate fractions of the re metabolites were excreted as conjugates. When maining three were eluted for spectral analysis as N-hydroxy-AAF was fed to the dog, 12.3 per cent described above. The urine collected after the in of the dose was recovered in the urine as N-hy gestion of acetyl-labeled AAF and 2-aminofluonene droxy-AAF, 1.2 per cent as AAF, and 0.3 per cent was treated in essentially the same manner. How as 7-hydroxy-AAF. No 1-, 3-, or 5-hydroxy-AAF ever, the final ether extract was divided into six could be detected in the urine following the inges portions for drying and application to eighteen tion of AM? or of N-hydroxy-AAF either by paper strips. Prior to the final spectral and radio spraying the paper strips with the phenol and activity measurements a third chromatographic diazo reagents (8) or by spectral examination of purification of the metabolites was performed. eluates of the strips. Any of these urinary metabo Distribution of the metabolites of AAF-9-C'4 in lites should have been readily detected on the urine and feces.â€”Three experiments were pen strips by visual observation under ultraviolet light formed to determine the urinary and fecal distni and with the indicator reagents if they had been bution of the radioactivity from ingested AAF-9- excreted in excess of 0.3 pen cent of the highest C14 in the dog. Single doses of 150 mg. of AAF-9- doses of AAF on N-hydroxy-AAF. The elution and C'4 with specific radioactivities of 6â€”7X 10@ spectrophotometnic analysis of the chromato counts/mm/mg were employed. The excreta were graphic zones in which these metabolites occurred collected for 1, 3, or 5 days in these trials. Appno could have measured as little as 0.2 per cent of pniate aliquots of the daily urine samples were di these doses. Even when 150 mg. of 1- or 3-by luted with ethanol, and 100â€”200Ml.of the diluted droxy-AAF was fed to the dog the chromatograph unines were plated in triplicate on aluminum ic strips showed no recognizable metabolites. Like planchets for radioactivity measurements. The wise, no recognizable N-acetylated urinary metab daily fecal samples were homogenized in a Waring olites could be observed following the ingestion of Blendor with sufficient water or 20 pen cent etha 2-aminofluorene or N-hydroxy-2-aminofluorene by nol to give suspensions which could be accurately the dog. sampled volumetrically. In a search for fecal When 500 mg. of 2-acetylaminonaphthalene metabolites aliquots of a water suspension were was fed, a urinary metabolite was detected that brought to pH 6 with acetic acid and incubated gave a positive test for a hydroxamic acid with the with enzymes, extracted, and the extracts chno acidic p-dimethylaminobenzaldehyde reagent and matographed as described above. Other aliquots of had the same R@ (0.75â€”0.86) as synthetic N-by the fecal suspensions were further diluted with droxy-2-acetylaminonaphthalene.8 This metabo ethanol, and triplicate [email protected] were plated lite was soluble in dilute NaOH and possessed an for radioactivity determinations. absorption spectrum in ethanol similar to that of the synthetic hydnoxyamic acid (maximum at 248 RESULTS mz, inflection at 9254m@i)and distinct from that Observaiions with paper chromatograms.â€”The of 2-acetylaminonaphthalene (maxima at 243 m@ ingestion of AAF by the dog led to the excretion and 9250mgi). The spectral analyses indicated that of AAF, N-hydnoxy-AAF, and 7-hydroxy-AAF in approximately 0.1 pen cent of the dose was excnet the urine at average levels of 0.7, 5.2, and 0.7 per ed as 2-acetylaminonaphthalene and as its N-hy cent, respectively, of the dose (Table 1) during the droxy derivative. No N-hydroxy-2-acetylamino first day following ingestion. N-Hydroxy-AAF was 8 We are indebted to Dr. Raymond R. Brown, Cancer Re easily detected on the chromatograms by its search Division, Department of Surgery, University of Wiscon capacity to form a yellow colon slowly when sin for a sample of this compound.

naphthalene was detected in the urine after a dose served with the Folin-Cioealteu and diazo re of only 131 mg. of the amide. Likewise, no N-hy agents, some may have been obscured by the dnoxy amides were detected by chromatographic normal reducing substances present on the lower analysis of the urine of dogs fed the following half of the chromatograms. amides (highest doses tested in parentheses): I8olatiOfl of N-hydroxy-AAF.â€”Direct proof of acetanilide (500 mg.), p-vinylacetanilide (225 the formation of N-hydnoxy-AAF from AAF by mg.), p-fluoroacetanilide (150 mg.), p-ethoxyacet the dog was provided by the isolation of approxi anilide (500 mg.), trans 4-acetylaminostilbene (100 mately 30 mg. of the hydroxamic acid in crystal line form from urine by the cupnic chelate method. mg.), 2-propionylaminofluorene (300 mg.), and The melting point of the recrystallized material 2-n-butyrylaminofluorene (200 mg.). Our inability was 146Â°â€”147Â°C.,uncorrected, which is identical to detect the N-hydroxy derivatives in the urine with that of synthetic N-hydroxy-AAF. An ap does not preclude the formation of these com proximately 1 :1 mixture of the isolated product pounds in vivo. These tests were carried out before and synthetic N-hydroxy-AAF gave no depression the removal of various interfering substances in of the melting point. The following elementary normal urine by extraction of the ether extract of analysis9 was obtained: urine with sodium carbonate was reported (17). 9 Huffman Microanalytical Laboratories, Wheatridge, Cob Hence, although no phenolic metabolites were ob redo.

TABLE 1 THE URINARY METABOLITES OF 2-ACETYLAMINOFLUORENE (AAF) AND CERTAIN OF ITS DERIVATIVES IN THE Doo DURING THE FIRST DAY FOLLowING INGESTION 0 @ 6@=@ ,Ã¶-cH3

DOSEN-Hydroxy MSTABOLITES, PER CENT OF Coin.ouwn DOSE5 INGESTEDNo. TRIALSDoo NOB.TOTAL (uo.)UaniART AA.FAM' AAF1-Hydroxy AAF3-Hydroxy AAF5-Hydroxy AAF7-Hydroxy

N-Hydroxy-AAF 2 1,5 300(1) 12Â±0.6 123Â±0.7 â€” â€” â€” 0.3# 225(3)

1-Hydroxy-AAF 2 1,4 54(1) â€” â€” - â€” 150(3)

3-Hydroxy-AAF 1 3 150(3) - â€” - â€” - -

2-Aminofluorene 1 3 150(3)jJ â€” â€” â€” â€” â€” â€” 1 3 1oo(2)@@ Less than 0 .04 per cent of each N-acety 1 metabolite

N-Hydroxy-2- aminofluoreneâ€”;iâ€” 1â€”ji:,;â€”3soot 150(3)07Â±04@ â€”52Â±2.7 â€”â€”@ â€”â€” â€”â€” â€”0.7Â±0.4 â€”

B The numbers in parentheses are the number of equal portions in which the dose was administered. t The dosesof AAF wereadministeredin one,two, or three equalportions. @ Standard deviation. Â§â€”= not detected. #Analysisofsecondtrial.In thefirsttrialthepresenceorabsenceof7.hydroxy-AAFcouldnotbedeterminedbecauseof contaminating reducing agents on the chromatographic strips. IISeveralattemptsweremadetofeed2-aminofluoreneindosesof150â€”SÂ®mg.giveninoneorthreeportions.Ingeneral,if a dog received200 mg. within a period of 3 hours, the dose was regurgitated. *5 Analysis of urine from dog fed 100 mg. of 2-aminofluorene per day for 5 days prior to administration of 2-(acetyl-1'-Cl')- aminofluorene (Table 2, Experiment 4). The urine was collected for 24 hours after the fifth dose of 2-aminofluorene and the entire urine sample was used for chromatographic analysis. No metabolites were detected with the spray reagents, and spectral analysis of the material from one-half of the urine sample showed that less than 0 .04 per cent of the dose could have been ex crated as each acetylated metabolite (see â€œStudiesonN-acetyla@ionofe-aminofluoreneâ€•under â€œResultsâ€•).

Calculated for Ci@Hj@NO2: periments 2 and 3 were sufficient to mask possible acetylationsâ€• of the 2-aminofluorene of only ap C, 75.3; H, 5.49; N, 5.85. proximately 1.9 and 0.8 per cent, respectively. Found: C, 75.0; H, 5.0; N, 5.30. D = optical density of the eluate at wavelength selected for determination of B. The probable error (y) of the specific ac Studies on N-acetylation of @â€˜-aminofiuorene.â€”tivity was determined from equation (II): The absence of N-acetylated metabolites on the 1 A2B2c2 A2B2d2 @ chromatograms of the urine from the dog fed p=@-@.@'A2b2+B2a2+ C2 D2 â€˜ (II) 2-aminofluorene raised the question whether the dog could acetylate 2-aminofluorene or its hydroxy where metabolites. Although the data in the literature a = The probable error in the value for the counts/mini (39) have generally indicated that the dog does not planchet; it was determined from the standard devia tion in the determination of the counts/min/planchet. appear to acetylate aromatic amines, Allison et at. b = The probable error in B; it was estimated to be 2.5 per (1) reported the presence of AAF in the urine of cent. dogs fed 2-aminofluorene. Accordingly, this prob C = The probable error in the volume (C) plated on each lem has been further investigated in four expeni planchet; in Experiments 1 and 2, in which different micropipettes were used for each metabolite, it was ments in which the specific activities of ingested estimated as 2 per cent; in Experiment 8, in which the 2-(acetyl-1'-C'4)aminofluorene and of its urinary same micropipette was used in all platings, it was esti metabolites were compared. mated to be 1 per cent. In Experiment 1 only %-(acetyl-1'-C'4)amino d = The probable error in the optical density (D) of the fluorene was administered. 2-Aminofluorene has eluate; it was estimated to be 1.5 per cent. been found as a urinary metabolite of AM? in the Equation (II) was derived from equation (I) by application of dog (1, 26, 35), and the assumption was made that the equation (III) for the probable error p (9): there would be sufficient deacetylation of the @ acetyl-labeled AM? in vivo (34) to provide an ac p = . . . (III) ceptor for unlabeled acetyl groups from the body pool. In the second experiment 2-aminofluorene where 11is a function of the independent variables x, z, w, ... and 2-(acetyl-1 â€˜-C'4)aminofluorene were adminis and r5, r,, r,,, . . . = the probable errors respectively in x, z, to.

tered in a molar ratio of 2 :1 to ensure that su.ffi â€œIn Experiment 1 the extent of replacement of the labeled cient 2-aminofluorene was present in the body to acetyl groups of the ingested AAF by unlabeled acetyb groups accept unlabeled acetyl groups. Experiment 3 was was determined as follows: carried out in a manner similar to that of Allison Percent replacement= 100 Fi â€”s.a.met.1 and Wase (2) in an attempt to enhance acetylation L s.a.jng.J @ by the administration of supplements of calcium where specific activity of metabolite and s.a.%ftQ.= pantothenate and riboflavin for 9days prior to the specific activity of the ingested AAF. administration of a mixture of 2-aminofluorene In Experiments 2 to 4 the extent of acetylation was ex and acetyl-labeled AAF in a molar ratio of 5 :1. pressed as the per cent acetylation of the ingested 2-aminofiuo The results of these experiments are summarized rene and it was calculated as follows: in Table 2. In no instance was there a significant @. S .a . @ decrease in the specific radioactivities of the Per cent acetylation = 100[ ______] N-acetylated urinary metabolites as compared with the activity of the acetyl-labeled AAF fed. In Experiment 1, in which the administered acetyl labeled AM? had a relatively low specific activity, â€” lOOfs.a.i@0. S.O.meg.1 â€” M 1. s.a.mej. i the probable errors'0 of the specific activity meas where urements would not have permitted detection of the replacementâ€• of less than 5 per cent of the s.a.m.g. â€” fraction of acetylated metabolite acetyl groups of AM? or any of its metabolites. S .a . @. The probable errors of the measurements in Ex retaining labeled acetyl group, S .0 â€¢met. . 10 The specific activity (s.a.) of each metabolite was calcu 1 â€” = fraction oi acetylateu metabolite bated from equation (I): s.o.s',9. AB @ s.a. (I) possessing unlabeled acetyl groups, and M = molar ratio of ingested 2-aminofluorene to where A = counts/min/planchet, B â€”absorption coefficient of metabolite, C â€”volume of eluate plated per planchet, and ingested 2-(acetyl-1 â€˜-C'4)aminofluorene

The analyses in the experiments described the whole urine sample. If N-acetylation of 2-ami above were conducted on urine samples collected nofluorene and urinary excretion of the N-acetyl for 24 hours after the ingestion of the compounds. metabolites occurred in our dogs, they did not It was conceivable, however, that the ingestion of exceed the limits of our methods of detection. 2-aminofluorene by the dog might induce the syn Metaboliim of AAF-9-C'4.â€”The three experi thesis of an acetylase for aromatic amines and that ments (Nos. 5â€”7)presented in Chart 1 indicate this might require more than 24 hours to achieve that the radioactivity from a dose of AAF-9-C'4 in a level of enzyme activity sufficient to alter the the dog is excreted primarily in the feces. Although specific activities of the urinary metabolites. the rates of excretion of C'4 in the urine were simi Therefore a fourth experiment was performed in lan in the three experiments, the rate of fecal excre which daily supplements of riboflavin and calcium tien of C'4 in Experiment 6 was unaccountably pantothenate were given for 12 days, and, on days slower than that found in the other two experi 7â€”11, 100 mg. of 2-aminofluorene was fed daily. ments. In the fifth experiment 3.2 per cent of the On day 12 a mixture of 2-aminofluorene and dose of A.A.F was excreted into the feces as 7-hy acetyl-labeled AM' (molar ratio of 5 :1) was ad droxy-AAF; no other N-acetylated metabolite was ministered. Once again no notable decreases were detected. In Experiment 6 the urine was collected

TABLE 2 THE SPECIFIC AcTwITms OF THE URINARY METABOLITES OF 2-(ACETYL-1'-C'4)AMINOFLUORENE IN THE DoG

DAYSOFPRIOR (comxe/@ns/@iMoLx)IngestedAcTIvITIEBX1O' SUPPLEMENTATION FED5 OF DIET WITH (uo.) RATIO OF Exp. 2-AMINO NO.Doo NO.No. Riboflavin . Acetyl AAFIMetaboliteaN-Hydroxy 2-Amin@ 2-Amm@ . . @oAAFS@xcinc plus pan lsbeied fIuorene@ fluorene AAFMoi@a AAF1 tothenatetCOMPOUNDS AAF7-Hydroxy

2 2 60 1.32Â±0.05 1.26Â±0.05 1.33Â±0.04 1.35Â±0.05 3 3 136 34 5/1 1.36Â±0.05 1.33Â±0.05 1.30Â±0.06 1.24Â±0.05 41 39 12598 136139 342/1 5/1O.39Â±O.02#1.22Â±0.040.58Â±0.021.10Â±0.060.37Â±0.031.24Â±0.060.38Â±0.021.15Â±0.06

S All doses were administered in three equal portions at 3-hour intervals. t For Experimentsnos. 3 and 4 the dog was fed calciumpantothenate(20 mg/kg body wt/day) and riboflavin(2.5 mg/kg body wt/day) prior to and on the day of ingestion of the carcinogens. @ 106 mg. daily. Â§Separatedilutionsofacetyl-labeledAAF(1.35X 10@counts/mm/mg)withunlabeledAAFweremadeforeachexperiment. #Probableerror.

found in the specific activities of the urinary each day for 3 days following the ingestion of AM?. metabolites (Table 2). The probable errors of the The usual three metabolites were easily detected specific activities of the metabolites were about the on the 1st and 2d days. No N-hydroxy-AAF and same as in Experiment 3 and indicated again that only very small amounts of AM? and 7-hydroxy less than 1â€”2per cent of the ingested 2-aminofluo AM? could be found in the urine collected on the rene could have been acetylateci. Chromatographic 3d day. Within 5 days following the ingestion of and spectral analysis of the whole urine sample, AM? (Experiment 7) only 81 pen cent of the radio collected on the last day of ingestion of 2-amino activity of the ingested AAF-9-C'4 had been ex fluorene without any AAF, showed that less than creted in the combined urine and feces. Metabo about 0.04 per cent of the dose of 2-aminofluorene bites were still being excreted during the 5th day, appeared in the urine in the form of each of the and on that day the urine and feces contained 0.1 known N-acetyl metabolites of AAF. This calcula and 0.8 pen cent, respectively, of the radioactivity tion was made on the assumption that the small administered. optical densities of the eluates of the paper strips actually represented N-acetylated metabolites. In DISCUSSION fact, however, no AM' on any other acetylated The observations recorded in this paper extend metabolite was detected visually on the chromato the previous findings on the capacity of the dog to grams or by spectrophotometnic examination of N-hydroxylate aromatic amines and amides. The the eluates of the chromatograms obtained from dog is now known to N-hydnoxylate 2-aminonaph

thalene (6, 31), 4-acetylaminobiphenyl (25), and Likewise no 5-hydroxy-AAF and only relatively AM? ; all these compounds are carcinogenic in the low amounts of 7-hydroxy-AAF (in agreement urinary bladder of the dog. Considerable evidence with previous reports [26, 3.5]) were found in the supports the concept that the N-hydnoxy metabo urine of dogs fed AAF. N-Hydroxy-AAF is the bites of AAF and 4-acetylaminobiphenyl are proxi major known urinary metabolite of AM? in the mate carcinogens in the rat (22, 25). The data dog. Other investigators (1, 26, 35) have reported obtained with the dog are thus consistent with the that 1â€”2per cent of a dose of AM? is excreted in view that N-hydroxylation is part of the metabolic the urine as 2-aminofluorene. These known un pathway through which the carcinogenicity of the nary metabolites account for more than one-half aromatic amines and amides is expressed in this of the radioactivity excreted in the urine within species (25). A test of this postulation appears to the first 24 hours after ingestion of AAF-9-C'4. In contrast to the relatively high urinary excretion U:URINE FFECES of metabolites by the rat (36) the dog excretes only ( )=EXP NO. F(7),, 19â€”22per cent of the radioactivity from AM?-9-C'4 in the urine within 3â€”5days. The major share of LL@J@ O@ the C'4 is excreted in the feces, and, except for a small amount of 7-hydroxy-AM?, the nature of the Iâ€” z fecal metabolites is unknown. Weisburger and Li 0 Weisburger (34) have suggested that the fecal metabolites in the rat may include oxidation and Li polymerization products of aminofluorenols. 0@- Of special interest in the present study was our 3 failure to detect the N-acetylation of [email protected] DAYS rene in the dog either by chromatographic and LL@LL@ spectrophotometnic analysis or by dilution of the 0< URINARY METABOLITES (EXP 6) isotope in the urinary metabolites of acetyl-labeled @ n AAF AM? administered with 2-aminofluorene. The @ 0 N-HYDROXY-MF summary of previous literature by Williams (39), (.0 1 our recent experience with 4-aminobiphenyl (25), @@ @w2 7-HYDROXY-AAF Li0 and the present findings all indicate that, if the ci.@I dog can N-acetylate aromatic amine&2 at all, it I can do so only to a small extent which is below the @ I II I III limits of detectability of the analytical methods I 2 3 employed. However, Allison et a!. (1) have report DAYS ed the presumptive N-acetylation of 2-aminofluo CHART 1.â€”The cumulative daily recovery of the radioac rene in the dog. They observed an â€œethersoluble tivity of ingested 2-acetylaminofluorene-9-C't in the urine and conjugated amineâ€•in the urine of dogs fed 2-ami feces of dogs (Experiments 5-7) and the excretion of the nofluorene and found under various conditions urinary metabolites in Experiment 6. Each experiment was that this conjugate appeared to represent 0.09â€”1.5 performed with 150 mg. of the labeled AM given in three per cent (calculated as AM?) of the dose. The high equal portions at 3-hour intervals. Dog no. 2 was used in ex periment 5 and dog no. 3 in Experiments 6 and 7. en excretions were obtained in dogs whose diet was supplemented with calcium pantothenate and require a comparison of the carcinogenicity of each riboflavin. The urinary conjugate was concluded of the above amines and amides with the activity to be AM? on the basis of its absorption spec of the corresponding N-hydroxy metabolite in the trum,'3 but it was not chnomatographed on isolat dog. Such a comparison of the activities of 4-ami ed. Allison et at. (1) found that under various con nobiphenyl and the corresponding hydroxylamine ditions low percentages (0.2â€”2.0)of doses of AM? is now in progress in this laboratory. fed to dogs were excreted in the urine as AM?. If Although the dog is able to metabolize aromatic it is assumed that a similarly low excretion of AM? amines to o-hydroxy derivatives (39), neither 1- non 3-hydroxy-AAF could be detected as a urinary 12 The dog can N-acetylate foreign amino acids and hista.. metabolite of AM?. However, since neither com mine (39). pound could be detected in the urine even after a 13 Allison et at. (1) record an absorption maximum of 275 m@& for ethyl ether solutions of AAF and the conjugate; the in large amount of 1- or 3â€”hydroxy-AM? was fed, no strument employed was not mentioned. We find the absorp conclusions with regard to the o-hydroxylation of tion maximum of AAF in ethyl ether to be 228 ms in the AAF in the dog can be drawn from these data. Beckman DU and DB spectrophotometers.

occurred from any AAF formed in vivo from 2-ami a useful species for studies on the role of the nofluorene, calculations from the data of Allison N-acetyl group in carcinogenesis with these com et at. suggest that as much as 17â€”49percent of the pounds and their N-acetyl derivatives. Among the administered 2-aminofluorene may have been aromatic amines or their derivatives known to pro N-acetylated by their dogs.'4 If acetylations of this duce bladder tumors in the dog are the following: magnitude had occurred in our experiments, large 2-aminonaphthalene (5, 16), 4,4'-diaminobiphenyl dilutions of isotope in the metabolites derived from (benzidine) (30, 33), 4-aminobiphenyl (11, 33), 9-(acetyl-1'-C'4)aminofluorene should have been AAF (26), 4-acetylaminobiphenyl (18), 4-nitrobi observed when it was administered to dogs with phenyl (10), o-aminoazotoluene (28), and 4-di 2-aminofluorene. Similarly, urinary excretions of methylaminoazobenzene (28). With the exception AAF of the magnitude reported by Allison et a!. of one of the azo dyes only the two acetylated should have been readily detected chromatograph amines, AM? and 4-acetylaminobiphenyl, have ically and spectrophotometnically in our studies. A also induced tumors of the liver in the dog. The possible explanation of the discrepancy may reside dog N-hydroxylates both of these amides and in the previous treatment of the dogs used in the 2-aminonaphthalene. These observations and the experiments of Allison et at. In their experiments fact that 2-aminonaphthalene, 4-aminobiphenyl, the 2-aminofluorene was fed 7 days after a dose of and 4-nitnobiphenyl induce tumors in the urinary AM'. There is no mention of control determina bladder of the dog are consistent with the possi tions for the possible presence of ether-soluble con bility that one of the proximate carcinogens in the jugated amine in the urine collected from the dogs bladder may be a hydroxylamine, whereas in the immediately before the administration of 2-amino liver an N-acetyl hydroxylamine may be required fluorene. Possibly enough previously stored AAF for carcinogenesis. However, the final proximate appeared in the urine following the administration carcinogen may be the same regardless of the tis of the 2-aminofluorene to account for their find sue involved, and the differences cited above may ings. This suggestion is consistent with our finding reside in differences in metabolic pathways on that even after 5 days only 81 per cent of the rates of metabolism in various tissues. Further radioactivity from a dose of AM?-9-C'4 in the dog studies on the metabolism and carcinogenic prop was accounted for in the urine and feces ; on the erties of aromatic amines and amides in the dog 5th day small amounts of radioactivity were still are desirable. being excreted in the urine and feces. In our experi REFERENCES ments with 2-aminofluorene the dogs had not re 1. ALLISON, J. B. ; WANNEMAdRER, R. W. ; and MIGLIARESE, ceived AAF or any of its derivatives for 6â€”15weeks J. F. Diet and the Metabolism of 2-Aminofluorene. J. Nu previously. trition, 52:415â€”25, 1954. The studies in the dog with acetyl-labeled AAF 2. Au@rsoN, J. B., and WASE, A. W. The Effects of Dietary also show that the metabolites N-hydroxy-AAF Riboflavin and Pantothenic Acid on the Metabolism of 2-Aminofluorene. Cancer Res., 12:647â€”50, 1952. and 7-hydroxy-AAF either are formed directly 3. ALLISON,J. B.; WASE, A. W.; LEATHEM,J. H.; and WA! from AAF or arise via derivatives of AAF which RIO, W. W. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. The N- and Ring-Hydroxylation of 2-Acetylaminofluorene and the Failure To Detect N-Acetylation of 2-Aminofluorene in the Dog

Lionel A. Poirier, James A. Miller and Elizabeth C. Miller

Cancer Res 1963;23:790-800.

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