[CANCER RESEARCH 28, 234-244, February 1968] The Carcinogenicity of the o-Methoxy Derivatives of N-2-Fluorenylacetamide and of Related Compounds in the Rat

H. R. Gutmann, S. B. Galitski, and W. A. Foley Laboratory ]or Cancer Research, Veterans Administration Hospital, and Department o] Biochemistry, University o] Minnesota, Minneapolis, Minnesota 55417

SUMMARY by the sequential reactions of deacetylation and oxidation. In order to test the idea that the lack of carcinogenicity of the o-amidofluorenols, N- (1-hydroxy-2-fluorenyl) acetamide and INTRODUCTION N-(3-hydroxy-2-fluorenyl)acetamide, is due to the hydrophilic phenolic hydroxyl group, the methylated derivatives, N-(1- Several model studies from this laboratory have shown that methoxy-2-fluorenyl)acetamide and N-(3-methoxy-2-fluorenyl) the o-quinone imines, 2-imino-l,2-fluorenoquinone and 2-imino- acetamide as well as the hydrochlorides of 1-methoxy-2-fluo- 2,3-fluorenoquinone, which are derived from the carcinogen renamine and 3-methoxy-2-fiuorenamine, were prepared, and N-2-fluorenylacetamide by the sequential enzymatic reactions their carcinogenicity was evaluated in the rat. N-(1-Methoxy- of , deacetylation, and oxidation (6, 10, 12, 27, 2-fluorenyl)acetamide and 1-methoxy-2-fluorenamine hydro- 34, 35), form stable adducts with a variety of proteins (17, 18). chloride, when administered orally to male rats for 5 months, However, the relevance of the binding of these o-quinone imines gave a tumor incidence of 27 and 50%, respectively. Approxi- to chemical carcinogenesis has remained obscure largely be- mately one-half of the lesions produced by either com- cause the o-amidofluorenols, 1-OH-AAF 2 and 3-OH-AAF pound were adenocarcinomas of the small intestine. N-(3- were not carcinogenic by oral and intraperitoneal administration Methoxy-2-fluorenyl) acetamide and 3-methoxy-2-fluorenamine or by bladder implantation (15, 21, 26). It might be argued hydrochloride were inactive. The low-to-moderate carcino- that the lack of activity of these o-amidofluorenols is due to the genicity of N-(1-methoxy-2-fluorenyl)acetamide and of 1- presence of the free phenolic hydroxyl group in the molecule. methoxy-2-fluorenamine hydrochloride contrasted sharply with Because a free hydroxyl group decreases lipid solubility by the high carcinogenicity of the isomeric N-(7-methoxy-2- conferring hydrophilic properties on the molecule (2), the fluorenyl) acetamide and of N-2-fiuorenyl acetamide, which were entry of these compounds into the cell may be retarded. In run for comparison under identical conditions. N-(7-Methoxy- addition, the free hydroxyl group may facilitate the formation 2-fluorenyl)acetamide appeared to be specifically active toward of water-soluble conjugates and, thus, the elimination of the the mammary gland. compounds from the cell. These factors would tend to counter- The intracellular removal of the O- of N-(1- act the attainment of the minimum intracellular concentration methoxy-2-fluorenyl)acetamide was demonstrated by means of which may be required for biologic activity. Observations con- N-(1-methoxy-2-fluorenyl)acetamide labeled with 14C in the cerning the enhancement of the carcinogenic activity of a num- methyl group. The formation of N-(1-hydroxy-2-fluorenyl)- ber of hydroxylated compounds by (5, 22, 23) acetamide, indicated by the radioactive tracer experiments, was appear to support this line of reasoning. Once the methylated confirmed by the isolation of N-(1-hydroxy-2-fluorenyl)acet- compound has penetrated the cell membrane, the methyl group from the urine of rats dosed with N-(1-methoxy-2- would presumably be removed by intracellular demethylases fiuorenyl)acetamide. The metabolic O-demethylation of N-(1- and the active compound (or its precursor) would be released. methoxy-2-fluorenyl)acetamide was compatible with the view Based on these considerations, the methylated derivatives of 1- that N-(1-hydroxy-2-fluorenyl)acetamide, when liberated in OH-AAF and of 3-OH-AAF, 1-MeO-AAF and 3-MeO-AAF, situ, may be weakly carcinogenic. However, the striking dif- respectively, were prepared (28). The present report deals with ferences in the carcinogenicities, as well as in the sites of action the evaluation of the carcinogenicity of these compounds in the of the o-methoxy compounds and of N-2-fluorenyl acetamide, make it exceedingly improbable that the carcinogenic activity 2The following abbreviations are used: 1-OH-AAF, N-(1-hy- of N-2-fluorenylacetamide is mediated through N-(1-hydroxy- droxy-2-fluorenyl)acetamide; 3-OH-AAF, N-(3-hydroxy-2-fluo- renyl) acetamide ; 5-OH-AAF, N-(5-hydroxy-2-fluorenyl)acetamide ; 2-fluorenyl)acetamide or through the o-quinone imine, 2-imino- 7-OH-AAF, N-(7-hydroxy-2-fluorenyl)acetamide; 1-MeO-AAF, 1,2-fluorenoquinone, resulting from N-(1-hydroxy-2-fluorenyl) N-(1-methoxy-2-fluorenyl) acetamide ; 1-MeO-14C-AAF, N- (1- methoxy-14C-2-fluorenyl) acetamide; 3-MeO-AAF, N-(3-methoxy- 1 This investigation was supported by USPHS Research Grant 2-fluorenyl)acetamide; 7-MeO-AAF, N-(7-methoxy-2-fluorenyl)- CA-02571 from the National Cancer Institute. acetamide; 1-MeO-AF-HCI, 1-methoxy-2-fluorenamine hydrochlo- Received June 28, 1967; accepted October 5, 1967. ride; 3-MeO-AF-HC1, 3-methoxy-2-fluorenamine hydrochloride.


Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1968 American Association for Cancer Research. Carcinogenicity of Fluorenylacetamide Derivatives rat as compared to the activity of N-2-fluorenylacetamide and N-2-Fluorenylacetamide. N-2-Fluorenylacetamide, m.p. 196- of 7-MeO-AAF, a position isomer of the o-methoxy derivatives 198~ was obtained by acetylating 2-fluorenamine, m.p. 128- of N-2-fluorenylaeetamide. In addition, the o-demethylation in 130~ (19), in the usual manner (30). rive of 1-MeO-AAF which displayed some carcinogenic activity Preparation of 1-MeO-14C-AAF. To a solution of 1-OH-AAF has been investigated. (100 mg, 0.42 mmole), m.p. 210-212~ (25), in 10 ml of 0.5 N potassium hydroxide was added 2.0 mmoles of dimethyl-14C sulfate (specific radioactivity = 1.25 me/mmole) (New England MATERIALS AND METHODS Nuclear Corp., 575 Albany Street, Boston, Mass.). After the Preparation of Compounds. 1-MeO-AAF, m.p. 148-150~ reaction mixture had been stirred for 1 hour, 2.1 mmoles of A~1o~ 2S8 m~ (e, 24,600), and 3-McO-AFF, m.p. 169-171~ unlabeled dimethylsulfate were added, and stirring was con- II~OX tinued for one additional hour. The reaction mixture was cen- were prepared as described previously (28). trifuged and the precipitate was washed twice with 0.5 N po- 1-MeO-AF'HC1. 1-Methoxy-2-nitrofluorene (0.241 gin, 1 tassium hydroxide. The residue was dissolved in 95% ethanol, mmole), m.p. 100-102~ (28), in ethanol (25 ml) and con- and 20 mg of unlabeled 1-MeO-AAF and a small amount of centrated hydrochloric acid (1 ml), was hydrogenated at room Norite A were added. The mixture was heated briefly on the temperature and atmospheric pressure with palladium black steam bath and was then filtered through a layer of eelite. (0.075 gm) (Nutritional Bioehemicals Corp., Cleveland 28, Water was added to the filtrate to incipient turbidity. After Ohio) as a catalyst. After uptake had ceased, the 12 hours at 4~ the 1-MeO-14C-AAF was collected and dried reaction mixture was filtered and the solvent was evaporated over calcium chloride. There was obtained 27.0 mg of 1-MeO -~4 at reduced pressure. The resulting solid was dissolved in hot C-AAF, m.p. 150-151~ (with softening at 138-140~ The distilled water and filtered through eelite into concentrated hy- labeled compound was chromatographed on silica gel GF2s~ drochloric acid (3 ml). The hydrochloride which crystal- with chloroform:methanol (97:3) as a solvent. The radioactiv- lized on cooling in an ice-bath was purified by two reerystalliza- ity profile, obtained by plotting the radioactivity against the tions from dilute hydrochloric acid. 1-MeO-AF" HCI (0.120 distance from the origin, showed a single radioactive peak which gin, 48% yield) decomposed over a range from 205 to 217~ coincided with the fluorescence-quenching spot (tlF = 0.46) Calculated for C14H14NOCl: C, 67.87; H, 5.69; N, 5.65. seen on exposure of the ehromatogram to ultraviolet light Found: C, 67.89 ; H, 5.68; N, 5.59. (2537 A), Authentic 1-MeO-AAF, run concurrently, had the The compound was ehromatographed on Whatman No. 1 paper same II F value. Radioassay of 1-MeO-14C-AAF before and after by the descending technic with 2.4 N hydrochloric acid as the thin-layer chromatography on silica gel GF2~ ~ gave practically ...... 9~ gave a Mllgte orange spot ~ = 0.28) when the t~p identical values for the specific radioactivity (1.07 • 106 developed chromatogram was sprayed with a 1% solution of p-dimethylaminobenzaldehyde in N hydrochloric acid. 1-MeO- 0.9 AF-HC1 was converted to 1-methoxy-2-fluorenamine, m.p. 108.5-109~ (28), in 78% yield by pouring the amine hydro- 0.8 chloride dissolved in 95% ethanol into an aqueous sodium bi- carbonate solution. The precipitate was collected, washed with water, and dried over calcium chloride. 0.7- 3-MeO-AF'HC1. 3-Methoxy-2-nitrofluorene (1.5 gm, 0.67 mmole), m.p. 180-182~ (28), was dissolved in a mixture of 0.& glacial acetic acid (150 ml), ethyl acetate (100 ml), and con- O centrated hydrochloric acid (8 ml). The mixture was hydrogen- t-. ,~ 0.5 ated at room temperature and atmospheric pressure with pal- ladium black (0.45 gm) as a catalyst. After hydrogen uptake q~ had ceased, the precipitated amine hydroehloride was dissolved -~ 0.4 by the addition of warm ethanol (100 ml). The catalyst was removed by filtration through eelite, and the filtrate was con- 0.3 centrated at reduced pressure. Addition of precipitated 3-MeO-AF'HC1 (1.26 gm, 75% yield), which decomposed over 0.2 a range from 200 to 245~ The compound was recrystallized from ethanol-ether (with charcoal). 0.1 Calculated for C~4H~4NOCI" C, 67.87; H, 5.69; N, 5.65 Found' C, 67.82; H, 5.69; N, 5.49 | ! | t L 3-Methoxy-2-fluorenamine was prepared from the 3-MeO- 35 260 Z85 310 335 AF'HC1 as described above for the 1-isomer. The free base, Wav~[| m/u m.p. 168-169~ (28), was obtained in 44% yield after recrystal- lization from 95% ethanol. Chart 1. Tile ultraviolet absorption spectra of authentic N-(1- T-MeO-AAF, 7-MeO-AAF, m.p. 212-213~ was obtained me~hoxy-2-fluorenyl)~cetamide (__) and of N-(1-methoxy- by met hylating N-(7-hydroxy-2-fluorenyl)aeetamide, m.p. 227- 14C-2-fluorenyl)acetamide ( .... ) purified by thin-layer chro- 229~ (4), with dimethyl sulfate (33). matography on silica gel GF254.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1968 American Association for Cancer Research. H. R. Gutmann, S. B. Galitski, and W. A. Foley dpm/mg and 1.02 X l0 s dpm/mg, respectively). The ultra- were chromatographed routinely on silica gel GF2~4 (1 mm violet absorption spectrum of the chromatographed labeled thickness) with chloroform :methanol (97 : 3) as a solvent. compound was superimposable on that of authentic 1-MeO-AAF Animals, Diets, and Administration of Compounds. The rats (Chart 1). Material of lower specific radioactivity (3.10 X 104 used in these studies were purchased from the Holtzman Co., dpm/mg) was obtained from the combined mother liquor and Madison, Wisconsin. The average weights of the male rats wash liquids to which carrier 1-MeO-AAF had been added. The employed in the carcinogenicity tests are listed in Table 1. specific radioactivity of this material (29 rag), m.p. 148-149.5 ~ The female rats which received 1-MeO-AAF intraperitoneally remained virtually unchanged (3.21 X 104 dpm/mg) when the weighed 50 gm initially. The weights of the adult mate rats compound was subjected to thin-layer chromatography on used in the metabolic experiments ranged from 220 to 300 gin. silica gel GF254 with chloroform:methanol (97:3) as a solvent. For the carcinogenicity tests the rats were caged individually The ultraviolet absorption spectra of this material before and at 26~ and food and water were allowed ad libitum. During after thin-layer chromatography were identical and indistin- the period of carcinogen ingestion the food consumption was guishable from the spectrum of authentic 1-MeO-AAF. Prior recorded daily. The weight of the animals was determined at to the administration of 1-MeO-14C-AAF, 4- to 10-mg samples intervals of one week throughout the experiments. The diets

Table 1

Average Average Duration of Average No. of rats surviving initial weight Compound administration intake No. of At six At seven At end of weight gain b administered (months) (mmoles/rat) rats used months months experiment a (gin) (gm) None 16 14 14 8 48 353 1-MeO-AAFo 5.3 3.3 15 14 14 9 49 394 1-MeO-AF'HCI 5.3 3.4 14 12 10 10 51 384 3-MeO-AAF 5.3 3.5 14 11 11 9 53 431 3-MeO-AF-HC1 5.3 3.5 14 11 11 6 50 426 7-MeO-AAF 5.1 2.7 14 10 8 2 49 206 AAF 5.3 2.9 15 10 0 51 209 The effect of the oral administration of methoxy derivatives of N-2-fluorenylacetamide, 2-fluorenamine hydrochloride, and of N-2- fluorenylacetamide on body weight and survival time of the Holtzman rat. a The experiment was terminated after 11 months. b Average weight gain -- average final weight -- average initial weight. The final weight refers to the weight of the rats at the end of the carcinogen intake. In the case of the controls, the final weight is the weight after 5.3 months. c 1-MeO-AAF, N-(1-methoxy-2-flu~renyl)acetamide; 1-MeO-AF-ttC1, 1-methoxy-2-fluorenaminehydrochloride; 3-MeO-AAF, N-(3- methoxy-2-fluorenyl)acetamide; 3-MeO-AF'HCI, 3-methoxy-2-fluorenamine hydrochloride; 7-MeO-AAF, N-(7-methoxy-2-fiuorenyl)- acetamide ; AAF, N-2-fluorenylacetamide.

Table 2

Number of rats with tumors 9 Percent Compound No. of Total No. Of ear Of mammary Of At other tumor administered rats used Sex of tumors duct Of liver gland intestine locations incidence b None o 16 M 0 0 0 0 0 0 0 None a 12 F 0 0 0 0 0 0 0 I_MeO_AAFe, a 15 M 4 0 1 (1) 0 2 (2) 1I 27 1-MeO-AAFg 12 F 2 0 0 2 (2) 0 0 17 1-MeO-AF.HC1 e 14 M 7 1 (1) 2 (2) 0 4 (4) 0 50 3-MeO-AAFr 14 M 0 0 0 0 0 0 0 3-MeO-AF.tICle 14 M 0 0 0 0 0 0 0 7-MeO-AAFe 14 M 17 5 (5) 3 (3) 7 (9) 0 0 93 AAFe 15 M 36 12 (16) 9 (11) 9 (9) 0 0 100 Comparison of the carcinogenic activities of methoxy derivatives of N-2-fluorenylacetamide, 2-fluorenamine hydrochloride, and of N-2-fluorenylacetamide in the Holtzman rat. a The numbers in parentheses are the number of tumors. b Percent tumor incidence -- No. of tumor-bearing rats • 100. No. of rats used r These controls were fed the basal diet only. ~These controls were given 10 intraperitoneal injections of 0.9% NaC1 containing 1.75% gum acacia. e These rats received the compound in the amounts stated in Table 1. ! This lesion was a malignant lymphoma involving the lymphnodes in the kidney. g These rats received 10 injections of 1-MeO-AF'HC1 (4.5 mg/100 gm of body weight). The average amount of compound ad- ministered was 0.21 mmole/rat. h For abbreviation key see Table 1.


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which contained the test compounds in a concentration of Table 3 0.030% for N-2-fluorenylacetamide, and of 0.033% for the other Percent of administered radioactivity compounds, were prepared by adding the compounds, dissolved Expt. I a Expt. II a Expt. III a

in the minimum amount of acetone, to the standard 20% ~4C in expired CO2, casein diet used in this laboratory (11). After the acetone had cumulative % of dose: evaporated, the diets were mixed in a mechanical food mixer 6 hr 17.0 5.2 4.5 for 1 hour. Individual batches of the diets were prepared fresh 12 hr 25.6 17.5 15.2 every 10 days and stored at 4~ 18 hr 28.7 20.5 16.4 For the intraperitoneal injection of 1-MeO-AAF, the com- 24 hr 31.1 21.2 pound (4.5 rag/100 gm of body weight) was dispersed in saline 36 hr 32.9 21.9 19.6 42 hr 22.6 20.0 containing 1.75% gum acacia by homogenization in a Potter- 14C in 42-hour urine: Elvejehm type glass homogenizer (21). The suspensions were Water-soluble 19.3 25.9 20.1 injected 3 times weekly until 10 injections had been given. Steam-distillable 0.36 0.92 0.71 1-MeO-14C-AAF was dispersed similarly in 2.0 ml of the saline- Ether-extractable 0.49 3.08 0.52 gum acacia vehicle and administered either intragastrieally by The elimination of 14C from 1-methoxy-14C-2-fluorenylacet- stomach tube or intraperitoneally through a disposable needle amide in the respiratory air and in the urine after a single oral (gauge #23). Appropriate corrections were made for losses of or intraperitoneal dose to the adult Holtzman rat. radioactivity in the syringe and needle or stomach tube. a The number of the experiment corresponds to the number Autopsies were performed routinely on all animals immedi- shown in Chart 3. The weight of compound and the amount of ately after death or at. the termination of the experiments (11 radioactivity administered are given in Chart 3. months). The tumors, the liver, the lungs, and the kidneys were fixed in buffered formalin and the sections (5-6 [,~ thickness) oration of the ether, as well as the radioactivity of the aqueous were stained with hematoxylin-eosin. phase, were measured and are referred to in Table 3 as the Collection of Expired 14C02 and of Urine. Following ad- ether-extractable and the water-soluble radioactivity, respec- ministration of 1-MeO-~4C-AAF, the rats were placed into all- tively. glass metabolism cages (manufactured by Delmar Scientific Radioactivity Measurements. The radioactivity of all samples Laboratories, Maywood, Ill.) which permitted the separate col- was determined in duplicate by liquid scintillation spectrometry lection of respiratory C02, urine, and feces. The air, which in 22-ml low-potassium vials. The radioactivity of samples im- was drawn through the cage by means of a water aspirator, miscible with organic solvents was measured after shaking the was freed of Cr 2 and moisture by passage through a tower sample (0.5 ml to 1.0 ml) with thixotropie gel powder (0.6 gm) (22.5 • 6.5 era) filled with a mixture of Asearite and Drierite. (Cab-O-Sil, Packard Instrument Co., Inc., Downers Grove, Ill.) The expired C02 was collected in 2 traps which were connected and 15 ml of scintillator solution A (15). The radioactivity of in series and which contained 60 and 120 ml of 2 N sodium samples soluble in organic solvents was assayed in 10 ml of hydroxide, respectively. The capacity of the system was con- scintillator solution B (15). All samples were counted with a sidered to be more than adequate for the collection of the re- standard error not exceeding 5%. Corrections for quenching spiratory CO,~ since 60 ml of 2 N sodium hydroxide were found were made by means of the channel ratio procedure. sufficient to absorb the CO 2 exhaled by an adult rat during 7 Identification of 1-OH-AAF in the Urine of Rats Dosed hours. The 2 ?r sodium hydroxide solution in both traps was with 1-MeO-AAF. The male adult rats used in these metabolic replaced every 6 hours, and the radioactivity of the solution experiments were placed into stainless steel metabolism cages in each trap was determined with the use of scintillator solu- which permitted the separation of the urine from the feces. tion A (15). The urine excreted during each 6-hour period was 1-MeO-AAF was administered to these animals by gastric in- also collected and stored at -15~ until it was further processed tubation of a suspension of the compound in the saline-gum as indicated below. The collection and estimation of the 14C02 acacia vehicle. The urine was collected for 48 hours in con- exhaled by the adult rat was also tested by the intragastrie ad- tainers packed in Dry Ice. At the end of the collection period ministration of 4.5 mg (7.95 • 107 dpm) of sodium aeetate-1- the urine was filtered through a layer of cotton, and the filtered 14C in 2.0 ml of the vehicle. The expired 14C02, which was urine was diluted with an equal volume of deionized water. The collected for 24 hours, accounted for 69.1% (5.49 • 107 dpm) mixture was buffered with sodium acetate (1 ~, pH 6) and of the administered sodium acetate-l-14C. incubated at 37~ for 18 hours with /3-glueuronidasea and Distribution of 14C in Rat Urine after Administration of takadiastase (Parke, Davis and Co., Ann Arbor, Mich.) in the I-MeO-14C-AAF. The urinary fractions, collected over a period presence of chloroform as described previously (9). After the of 36-42 hours following the administration of i-MeO-14C-AAF, incubation, the mixture was acidified to pH 2-3 with concen- were combined, filtered through cotton, and acidified to pH trated hydrochloric acid, and the metabolites were partitioned 3-4 with concentrated hydrochloric acid. An aliquot of the by solvent extraction as shown in Chart 2. The fraction con- urine was subjected to steam-distillation. The radioactivity of the distillate was considered to be due to volatile urinary me- 3 Bacterial fl-glucoronidase, Type 1, Lot #23B-612, 40,000 Fish- tabolites of the methyl group, such as formic acid (36). Another man units/gin, and bacterial/~-glucuronidase, Type II, Lot #125B- aliquot of the urine was extracted 3 times with an equal volume 6430, 96,000 Fishman units/gm, were obtained from the Sigma of ether. The radioactivity of the residue remaining after evap- Chemical Co., St. Louis, Missouri.

FEBRUARY 1968 237

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Incubated urine adjusted to pH 2-3 RESULTS r ted 3timee with ~ual volume,s of ether I Careinogenieity Tests. The data on the toxicity and carcino- I I genicity of the compounds tested are summarized in Tables 1 Aeueous phas~ Ether dis~arde, d concentrated to 50 ml and 2. As judged by weight gain and survival time, neither the extrdcted ~vic~ with ZS ml 0.5 NaOH o-methoxy derivatives of N-2-fluorenylacetamide (1-MeO-AAF I and 3-MeO-AAF) nor those of 2-fluorenamine (1-MeO-AF'HCI I I and 3-MeO-AF'HC1) were toxic for the rat. Thus, rats consum- NaOH Ether acidified (pH?.)with 8N HCL discardr ing 1-MeO-AAF or 1-MeO-AF'HC1 showed a slightly greater extracted twice wlfh ?.6ml dher weight gain (~ 10%) than the controls, and the rats ingesting I 3-MeO-AAF or 3-MeO-AF'HC1 were about 20% heavier than i _ I the control rats. In contrast, a 40% growth inhibition was Ether A~[ur phase washed with tO ml discarded noted in animals ingesting N-2-fluorenylacetamide or 7-MeO- distillr water AAF (Table 1). A similar growth depression (38%) had been I observed previously in rats fed a 20% casein diet containing I I 0.030% N-2-fluorenylacetamide for 75 days ad libitum (11). Ether evapora'f~d Wash liCuid residue ~ontaini~ acids and phenols discarded 1-MeO-AAF and 1-MeO-AF'HCI proved to be carcinogens c,hromatooraph~ on Wlutman No. 1 of low to moderate potency as shown by the tumor incidence or NO. 3 MM paper (Table 2). In contrast, 3-MeO-AAF and 3-MeO-AF-HC1 were inactive. The lesions produced by 1-MeO-AAF or 1-MeO- Chart 2. Fractionation scheme of the urinary metabolites of rats treated with N-(1-methoxy-2-fluorenyl)acetamide or with AF'HC1 on oral feeding were slow-growing, single tumors which, N-(1-hydroxy-2-fluorenyl)acetamide. Prior to the fractionation, on account of their small size, were detected only by careful the urine was subjected to enzymatic of O-glucuronides inspection at autopsy. Since the oral administration of N-2- and O-sulfates as described under Materials and Methods. fluorenylacetamide or of derivatives thereof does not usually give rise to tumors of the small intestine, the relatively high frequency of intestinal neoplasms produced by 1-MeO-AAF taining acids and phenols was spotted on Whatman No.1 paper or 1-MeO-AF'HCI seems noteworthy. These tumors were well- or streaked onto Whatman No. 3MM paper by means of an defined nodular lesions maximally 1.0-1.5 cm in diameter. In automatic streaking device. Chromatography on Whatman No. 1 no instance was the intestinal lumen obliterated by these neo- paper was carried out with the upper phase of a mixture of plasms (Fig. 1). Histologically, the tumors were moderately cyclohexane :tertiary butyl :glacial acetic acid: water well-differentiated adenocarcinomas. There was usually an (16"4:2:1) (35) as the solvent. Chromatograms on Whatman abrupt transition from normal intestinal mucosa to atypical No. 3MM paper were developed with the upper phase of gland shapes with other areas showing a more solidly cellular a mixture of cyclohexane:tertiary butyl alcohol:glacial ace- tumor (Fig. 2). Tumor glands and nests penetrated the muscu- tic acid'water (16"2"1:1) as the solvent. Prior to develop- laris mucosa in all instances, and in some cases the muscularis ment, the chromatograms were equilibrated with the upper and propria as well. Mitoses including abnormal mitotic figures were lower phase of the solvent system for 12-18 hours. 1-OH-AFF also readily observed (Fig. 3). The liver tumors found after was located by spraying the chromatograms either with the ingestion of 1-MeO-AF'HC1 were hepatocellular and cholangio- Folin-Ciocalteu reagent (7) or with the diazotized 7-nitro-2- ]ar carcinomas. The hepatocellular carcinoma compressed the fluorenamine (34). The compound was also detected by its adjacent normal liver and was characterized by lack of bile fluorescence-quenching properties on exposure of the chromato- ducts and absence of a lobular pattern. The cells were arranged grams to ultraviolet light (2537 A). Authentic 1-OH-AAF was in cords several cell layers thick (Fig. 4). The tumor cells were run routinely along with the urinary fraction. The metabolite larger than normal hepatocytes, and the cytoplasm appeared was eluted from the paper by descending chromatography with less structured than the cytoplasm of normal liver cells. Nuclear methanol as a solvent. After solvent evaporation, the residue pleomorphism was prominent and mitotic figures were frequent was further purified by thin-layer chromatography on silica gel (Fig. 5). The cholangiolar type of carcinoma expanded the liver GF254 with chloroform:methanol (9:1) as a solvent (RF = lobe in which it was situated. Only a few hepatocytes were seen 0.46-0.62). The compound was eluted from the gel by 2 suc- between solid cords and ducts of tumor cells in a fibrous stroma cessive extractions with methanol (2 ml). At this stage of the with polymorphonuelear leukoeytes in the lumina of the duets purification, the ultraviolet absorption spectrum of the isolated (Fig. 6). The shape of the cells was irregular in solid areas and metabolite was identical with that of authentic 1-OH-AAF. The columnar in areas containing duet elements. The cytoplasm of amount of metabolite in the methanol extract was estimated the tumor cells was paler than that of normal liver cells, and with the use of the molar extinction coefficient of 1-OH-AAF the nuclei were large and vesicular with prominent eosinophilie at the wavelength of maximal absorption xMeo~Imax 278 m~ nueleoli. The size of the nuclei varied markedly and mitotic (e, 25,500). The recovery of duplicate samples of N-(1-hydroxy- figures were prominent (Fig. 7). The mammary tumors ob- 2-fluorenyl-l-41C)acetamide (25) which were carried through served in the animals injected intraperitoneally with 1-MeO- the purification procedure as a control was 66.8 ___ 4.1%, and AAF were typical adenoeareinomas which had the same histo- this value was used to correct for the losses of 1-OH-AAF dur- logic appearance as the mammary tumors elicited with 7-MeO- ing the isolation. AAF (Fig. 8) or N-2-fluorenylacetamide. Some of the tumors


Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1968 American Association for Cancer Research. Carcinogenicity of FIuorenylacetamide Derivatives contained areas of acinar or duct structure. Others were totally c.~ I00 devoid of these structures and presented only solid masses of - .,.,~_~]~,, ~ d,.,~ ~' B..~ --~ pleomorphic cells. The tumors were subcutaneous and fixed to the overlying skin in all cases. Areas of squamatization were -~. 80 noted in some of the tumors. In most of the lesions, single IIJ i, tumor cells as well as cords Could be seen to extend into the surrounding fat. The lobular pattern of normal mammary ~_ 6o gland tissue was replaced, at least partially, in each of the e- tmnors (Fig. 9). The shape of the cells varied from cuboidal 4o cells to small, irregular cells without definite cytoplasmic boun- daries. Mitotic figures were numerous in all tumors. The malig- nant [ymphoma seen in one of the rats ingesting 1-MeO-AAF was of the large cell type and consisted probably of retieulum cells. They presented a rather monotonous cell pattern with E o j I I I I I I frequent mitoses and destruction of normal lymph node archi- c.) o b 12 18 ~4 3O 3b 4~ tecture. Tumor cells were found perivascularly in the kidney. These cells were identical with those observed in the lymph Tim~ Hou rs nodes. Chart 3. The evolution of respirator" 14C0 2 after the ad- ministration of N-(1-methoxy-14C-2-fluorenyl)acetamide to the 7-MeO-AAF, a position isomer of I-MeO-AAF or 3-MeO-AAF, re:de rat. In Experiments I trod II 4.18 mg (4.27 X l06 dpm) and m which the occupies the extended para posi- 4.16 mg (4.24 • 106 dpm), respectively, of the labeled compound tion, w;ts considerably more carcinogenic for the rat than either were administered by stomach tube. In Experiment III, 6.64 mg 1-MeO-AAF or 1-MeO-AF'HC1 and, on the basis of the tumor (9.50 X 105 dpm) was injected intraperitone,~dly. incidence, was almost as active as N-2-fluorenylaeetamide. This compound had previously been shown to be particularly active besides oxidation to CO~. The amount of the administered toward the mammary gland in the female rat (26) and dis- radioactivity excreted in the 42-hour urine (23.1 +-3.9%) was played, in the present experiments, a similar tissue specificity comparable to that eliminated in the expired :~ir (Table 3). in the male rat. The dat.a indicated that 7-MeO-AAF, unlike Most of the urinary radioactivity (95%) was water soluble N-2-fluorenylacetamide, was not a strong hepatocareinogen in and, in part, was presumably referable to conjugated metabo- ~he male rat. The carcinogenicity of N-2-fluorenylacetamide, lites of 1-MeO-AAF not. extractable by ether. Only 3% of the which was run for comparison, and the distribution of tumors urinary radioactivity, corresponding to 0.7%, of the admin- elicited by this compound was in agreement with the numerous istered radioactivity, was distillable by steam. These data indi- data in the literature (32). The finding that N-2-fluorenylaceta- cated that the elimination of the methyl group of 1-MeO-AAF mide was far more carcinogenic than the o-methoxy compounds, in the form of volatile urinary compounds represented only a 1-MeO-AAF or 1-MeO-AF'ttC1, is relevant, for the subsequent minor metabolic pathway. The ether-extractable radioactivity discussion. was examined for the presence of 1-MeO-14C-AAF by inverse Metabolic Experiments. The purpose of the metabolic ex- isotopic dilution. Carrier 1-MeO-AAF was added to the ether periments was to determine whether and to what extent 1-MeO- extract, of the urine and purified repeatedly by thin-layer AAF would be demethylated in vivo. If 1-MeO-AAF were chroma.tography on silica gel GF.,~, with chloroform:methanol demethylated, a part of the resulting 1-OH-AAF would likely (97'3) and n-hexane:acetone (6:4) or n-heptane:acetone be metabolized to 2-imino-l,2-fluorenoquinone by enzymatic (6:4) as solvents. Since only trace amounts of the urinary deacetyhtion and oxidation in succession. This reaction sequence radioactivity (0.064 and 0.056% in Expts. I and II, Table 3, and the interaction of the o-quinone imine with proteins has respectively) were due to unchanged 1-MeO-14C-AAF, 1-MeO- been investigated in previous studies. AAF was evidently extensively metabolized by the rat. O-Demethylation of 1-MeO-14C-AAF. O-Demethylation of The formation of 1-OIt-AAF in the O-demethylation of 1-MeO-AAF was demonstrated by the appearance of 14CO~. in 1-MeO-AAF was confirmed by the isolation of this o-amido- the expired air after the oral or intraperitoneal administration fluorenol, identified as shown in Chart 4, from the appropriate of 1-MeO-~4C-AAF. The rate of evolution of ~4CO~ is shown urinary fraction of rats dosed with 1-MeO-AAF. After further in Chart 3 and appeared to be independent of the route of purification of the compound by thin-layer chromatography, administr~tion of the compound. Essenlially all of the ~4CO2 the identity of the isolated product, was established by the was exhaled by the rat within 36 hours after the administration ultraviolet, absorption spectrum which, in all cases, was indis- of the labeled compound. The oxidation of the methyl group tinguishable from that of authentic 1-OH-AAF. of 1-MeO-AAF appeared, therefore, to be completed within Besides 1-OH-AAF, the phenolic fraction consistently con- ;32 to 36 hours following the oral or intraperitoneal administra- tained 2 metabolites which reacted intensely with the Folin- tion of 1-MeO-AAF. From 20 to 33% (average, 25.1 ----- 5.2%) Cioealteu reagent or with diazotized 7-nitro-2-fluorenamine of the administered labeled methyl groups were oxidized to and (Chart 4). As judged by the degree of quenching of the fluo- elinfina.ted as ~4CO.). This value is likely to be a low estimate rescence on exposure to ultraviolet light, as well as by the size of the total O-demethylation since there may be other path- of the spots, these compounds were excreted in larger amounts ways for the disposition of the methyl group of 1-MeO-AAF than was 1-OH-AAF. Although the structural identification of

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Table 4

Formation of 1-OH-AAF calculated 1-MeO-AAF From demcthylation From urinary excretion Expt. administered of 1-MeO-14C-AAF of 1-OH-AAF a No. (rag) (mg) (mg) I 11.1 2.6 b 1.4 II 20.0 4.7b 1.1 The formation of N-(1-hydroxy-2-fluorenyl)acetamide (I-OH- o O AAF) after ingestion of 1-methoxy-2-fluorenylacetamide (1-MeO- R~-=0.75 RT=0.75 Ry=0.7~; AAF) estimated from the demethylation of 1-methoxy-14C-2- fluorenylacetamide (1-MeO-14C-AAF) and from the urinary ex- cretion of N-(1-hydroxy-2-fluorenyl)acetamide. a In Experiments I and II, 0.079 and 0.062 mg of 1-OH-AAF, respectively, were isolated from the 48-hr urine. The calculated values are based on tile urinary excretion of 5.5-4-0.7% of intra- peritoneally administered 1-OH-AAF, ranging from 1.9 to 8.2 mg. Thus, in Expt. I, 0.079 mg _ 1.4 mg. 0.055 b The values are based on an average demethylation of 25% as I J- Rt"= 0.41 RI:" 45 determined from the 14CO2 expired after the administration of "4" 1-MeO-14C-AAF (Table 3). 0 _.> 0 o 0 quantities of 1-MeO-AAF. The discrepancy between the cal- RT= 0.?.9 R~-O.29 culated and the isolated quantities of 1-OH-AAF may, in large t- part, be attributed to the metabolism of the o-amidofluorenol o by the rat (12, 31). In order to test this point experimentally, 6 rats were injected intraperitoneally with 1-OH-AAF in doses L- ranging from 1.9 to 8.2 mg. Only 5.5 - 0.7% of the injected compound was recovered unchanged from the 48-hour urine. These data suggested that the major portion of the 1-OH-AAF Con'l'rol ConCrot Exp'l'L. Exld'l. arising in the demethylation of 1-MeO-AAF likewise was not Uri ne, Urine, Urine Urine, eliminated as such by the rat. On the assumption that 5.5% + 1-HO- ?.-,e~,F ( 50)J.[) (lO0/J [ ) of 1-OH-AAF formed in vivo was eliminated in the urine, the excretion of 0.079 and 0.062 mg of the compound would be Chart 4. Representative chromatogram on Whatman No. 1 equivalent to the formation of 1.4 and 1.1 mg of 1-OH-AAF, paper of the acidic/phenolic fraction (Chart 2) obtained from the respectively. As shown in Table 4, these quantities of 1-OH- 48-hour urine of a male rat after the oral administration of N-(1- AAF were considerably less than would be expected from a 25% methoxy-2-fluorenyl)acetamide as described under Materials and demethylation. The calculations suggested that the demethyla- Methods. The solvent was the upper phase of a mixture of cyclo- tion of 1-MeO-AAF yielded, in addition to 1-OH-AAF, rela- hexane :tertiary butyl alcohol:glacial acetic acid:water (16:4:2:1) tively large amounts of other metabolites. (35).

DISCUSSION these metabolites was not further pursued, the solubility char- acteristics, the chromatographic behavior, and the color reac- The O-demethylation of 1-MeO-AAF in vivo, demonstrated tion of these compounds suggested that they were mono- or here, supports the view that the carcinogenicity of 1-MeO-AAF polyhydroxylated phenols derived from 1-MeO-AAF. Since for the rat may be ascribed to the liberation of 1-OH-AAF these mctabolites migrated at a slower rate than 1-OH-AAF, i.~ situ, even though this o-amidofluorenol when fed or injected and since 5-OH-AAF as well as 7-OH-AAF in the solvent sys- is inactive (21, 26). The carcinogenic activity of 1-MeO- tems used here showed a similar chromatographic mobility AF'IIC1 may similarly be the result of the demethylation of this relative to that of 1-OH-AAF (34), it is possible that these o-methoxy compound to 2-amino-l-fluorenol. Since 1-OH-AAF compounds were products of the ring-hydroxylation of 1-MeO- is rapidly metabolized, as shown here in vivo and previously AAF involving the 5 and 7 positions of the fluorene system. in vitro (12, 31), and since the formation of 2-imino-l,2-fluo- In two separate experiments in which 11.1 and 20.0 mg of renoquinone from 1-OH-AAF or from 2-amino-l-fluorenol has 1-MeO-AAF were administered by gastric intubation, 0.079 been reasonably well documented, it is possible that the o-qui- and 0.062 mg of 1-OH-AAF, respectively, were isolated from none imine is the agent ultimately responsible for the carcino- the 48-hour urine. Since the radioactive tracer experiments had genicity of 1-MeO-AAF or of 1-MeO-AF'HC1. Tile lack of car- indicated that at least 25% of an oral dose of 1-MeO-AAF cinogenicity of 3-MeO-AAF and of 3-MeO-AF'HC1 would was demethylated, it was calculated that 2.6 and 4.7 mg of the appear to be inconsistent with the o-quinone imine hypothesis o-amidofluorenol should have been formed from the above considered above as an explanation for the carcinogenicity of


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1-MeO-AAF and of 1-MeO-AF'HC1. However, if the interaction 2. Albert, A. Selective Toxicity, pp. 348-349. New York: John of the o-quinone imine with a tissue constituent be required Wiley and Sons, Inc., 1965. for carcinogenic activity, such an interaction would very likeh, 3. Andersen, R. E., Enomoto, M., Miller, E. C., and Miller, J. A. proceed through a 1,4-addition mechanism (1). 1,4-Addition of Carcinogenicity and Inhibition of the Walker 256 Tumor in 1,2-fluorenoquinone-2-acetamide to the ~-amino group of the the Rat by trans-4-Acetylaminostilbene, Its N-Hydroxy Me- tabo]ite and Related Compounds. Cancer Res., 2~: 128-143, lysine residues of albumin or of N~-tosyl-DL-lysine benzyl 1964. has been demonstrated (13, 14). The same mechanism would 4. Bielsehowsky, F. A Metabolite of 2-Acetamidofluorene. Bio- presumably apply to the addition of 2-imino-l,2-fluorenoquinone chem. J., 39: 287-289, 1945. to proteins and/or other tissue constituents. However, in the 5. Cook, J. W., and Schoental, R. Carcinogenic Activity of Meta- ease of 2-imino-2,3-ttuorenoquinone which would arise from the bo!ic Products of 3:4-Benzpyrene: Application to :Rats and O-demethvlation of 3-MeO-AAF and from the further metabo- Mice. Brit. J. Cancer, 6: 400-406, 1952. lism of the resulting o-amidofluorenol, a similar ],4-addition 6. Cr-tmer. J. W., Miller, J. A., and Miller, E. C. The Hydroxyla- naechanism cannot be written. Moreover, beside the o-quinone tion of the Carcinogen 2-Acetylaminofluorene by Rat Liver: imine hypothesis, other mechanisms shoul~t be borne in mind Stimulation by Pretreatment in Vivo with 3-Methylcholan- as underlying the action of 1-MeO-AAF and of 1-MeO-AF-HCI. threne. J. Biol. Chem., 235: 250-256, 1960. 7. Folin, O., and Ciocalteu, V. Tyrosine and Tryptophane De- It has been recognized that metabolic or synthetic N-hydroxy- terminations in Proteins. J. Biol. Chem., 73: 627~50, 1927. lation converts a large number of carcinogenic and noncarcino- 8. Gutmann, H. R., Galitski, S. B., and Foley, W. A. N-Hy- genie aromatic to highly potent arylhydroxamic :wids droxy-2-Fluorenylbenzamide, an Arylhydroxamic Acid with (3, 8, 9, 21, 24) which are currently looked upon as proximate High Carcinogenic Activity. Nature, 209: 202-203, 1966. agents in carcinogenesis by aromatic amides (20). The possi- 9. Gutmann, H. R., (]alitski, S. B., and Foley, W. A. The Con- bility cannot be disregarded that the carcinogenieity of 1-MeO- version of Non-Carcinogenic Aromatic Amides to Carcino- AAF and of 1-MeO-AF'IIC1 was the result of the N-hydroxy- genic Arylhydroxamic Acids by Syntheti(" N-Itydroxylation. lation of these compounds. However, examination of paper Can('er Res., :-~,,: 1443-1455, 1967. chromatograms of the appropriate urinary fraction from rats 10. (hltmann, H. R., and Nagasawa, It. T. The Oxidation of o- treated with 1-MeO-AAF showed no compounds which migrated Aminoplmlmls by Cytochrome c and Cytochrome Oxidase. at a faster rate and, consequently, were le.~s polar ~han III. 2,3-Fluorenoquinone from 2-Amino-3-fluorenol and Bind- ing of Quinonoid Oxidation Products to Bovine Serum Al- 1-OI[-AAF and which reacted either with the Folin-Cioealteu bumin. J. Biol. Chem., 235: 3466-3471, 1960. reagent or with acidic p-dimethylaminobenzaldehyde. In 'mal- I I. Gutmann, H. R., and Peters, J. H. The Effect of 2-Acetyl- ogy with the ehromatogral~hie behavior of N-hydroxy-2-fhm- aminoih,orcne on Growth and Composition of the Liver of renyl -~eetamide and the ring-hydroxylated derivatives of the Rat. Cancer Res., 13: 895-899, 1953. N-2-fluorenylacetamide, it would be anticipated ~hat N-hy- 12. Gutrnann, H. R., Seal, U. S., and Irving, C. C. On the Mecha- droxylated derivatives of 1-MeO-AAF would likewise move :l~ nism of Protein Binding of N-2-Fluorenylacetamide. The De- a faster rnte than 1-Ott-AAF or any ring-hydroxylated metab- of N-(l-hydroxy-2-fluorenyl)acetamide and the olites of 1-MeO-AAF. Nevertheless, it is felt that the evidence Effect of Ethionine. Cancer Res., 20: 1072-1078, 1960. is insufficient to discount N-hydroxylation :as playing a part in 13. Irving, C. C., and Gutmann, H. R. Protein Binding of Model the mechanism of action of these compounds. Quinone . II. The Interaction of Some o-Quinone Imides with Crystalline Bovine Serum Albumin. J. Biol. Chem., Even if the activity of 1-MeO-AAF were largely or entirely 234: 2878-2884, 1958. attrit)ut:tble to 1-OH-AAF, the striking differences in the ear- 14. h'ving, C. C., and Gutmann, H. R. Protein Binding of Model cinogenieities and in the sites of action of 1-MeO-AAF :rod of Quinone Imides. III. Preparation of N-(1-hydroxy-2-acet- 1-MeO-AF'HCI versus N-2-fluorenylaeetamide make it exceed- amido-4-fiuorenyl)-DL-lysine. J. Org. Chem., 26: 1859-1861, ingly improbable that the action of N-2-fluorenylaeetamide is 1961. mediated through 1-OH-AAF. As a consequence, it aI)I~ears 15. Irving, C. C.. Gutmann, H. R., and Larson, D. M. Evaluation unlikely that the binding of I-OH-AAF (or of the 2-iinino- of the Careinogenicity of Aminofluorenols by Implantation into the Bladder of the Mouse. Cancer Res.. 28: 1782-1791, 1,2-fluorenoquinone derived therefrom) to tissue proteins in 1963. vitro (29) or in vivo (16) is causally related to the carcinogenic 16. Irving, C. C., and Williard, R. F. On the in Vivo Protein process initiated by N-2-fluorenylacetamide. Binding of 1-Hydroxy-2-acetylaminofluorene-l-C14 in the Rat. Cancer Res., 24: 77-82, 1964. 17. King, C. M., Chang, S. F., and Gutmann, H. R. The Oxida- ACKNOWLEDGMENTS tion of o-Aminophenols by Cytochrome c and Cytochrome Oxidase. V. Inactivation of Catalase and Arginase by 2-Imino- The authors wish to thank Dr. Lee W. Wattenberg for advice 1,2-fluorenoquinone. J. Biol. Chem., 288: 2206-2212, 1963. in the interpretation of the histologic sections, and Mrs. E. Fok 18. King, C. M., Gutmann, H. R., and Chang, S. F. The Oxida- and Mrs. B. Zakis for technical assistance. tion of o-Aminophenols by Cytochrome c and Cytochrome Oxidase. IV. Interaction of 2-Imino-l,2-fluorenoquinone and REFERENCES of 2-Imino-2,3-fluorenoquinone with Bovine Serum Albumin. J. Biol. Chem., 288: 2199-2205, 1963. 1. Adams, R., and Reifschneider, W. Synthesis and Reaction of 19. Kuhn, W. E. 2-Nitrofluorene and 2-Aminofluorene. In: A. It. Quinone Mono- and I)iimides. Bull. Soc. Chim. (France), 23- Blatt (ed.), Organic Syn. Coll. Vol. 2, pp. 447-448. New York: 65, 1958. John Wiley and Sons, 1943.

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20. Miller, E. C., and Miller, J. A. Mechanism of Chemical Car- Enzymatic Oxidations and Binding of Oxidation Products to cinogenesis: Nature of Proximate Carcinogens and Interac- Bovine Serum Albumin. J. Biol. Chem., 234: 1593-1599, 1959. tions with Macromolecules. Pharmacol. Rev., 18: 805-838, 28. Nagasawa, H. T., and Gutmann, H. R. o-Methoxy Derivatives 1966. of the Carcinogen N-2-Fluorenylacetamide. Potential Latent 21. Miller, E. C., Miller, J. A., and Hartmann, It. A. N-Hydroxy- Biological Arylating Agents. J. Med. Chem., 9: 719-725, 1966. 2-acetylaminofluorene: A Metabolite of 2-Acetylaminofluo- 29. Nagasawa, H. T., and Osteraas, A. J., The Biological Arylation rene with Increased Carcinogenic Activity in the Rat. Cancer of Proteins in Vitro by a Metabolite of the Carcinogen N-2- Res., 21: 815-824, 1961. Fluorenylacetamide. Biochem. Pharmacol., 13: 713-723, 1964. 22. Miller, J. A., and Miller, E. C. The Carcinogenicity of 3- 30. Ray, F. E., and Geiser, R. C. Synthesis of 2-Acetylamino- Methoxy-4-aminoazobenzene and Its N-Methyl Derivatives fluorene-9-C14 and 2-Acetylaminofluorene-~-C14. Cancer Res., for Extrahepatic Tissues of the Rat. Cancer Res., 21: 1068- 10: 616-619, 1950. 1072, 1961. 31. Seal, U. S., and Gutmann, H. R. The Metabolism of the Car- 23. Miller, J. A., Miller, E. C., and Finger, G. C. Further Studies cinogen N-(2-fluorenyl)acetamide by Liver Cell Fractions. J. on the Carcinogenicity of Dyes Related to 4-Dimethylamino- Biol. Chem., 23~: 648-654, 1959. azobenzene. The Requirement for an Unsubstituted 2-Position. 32. Weisburger, E. K., and Weisburger, J. H. Chemistry, Car- Cancer Res., 17: 387-398, 1957. cinogenicity and Metabolism of 2-Fluorenamine and Related 24. Miller, J. A., Wyatt, C. S., Miller, E. C., and ttartmann, H. A. Compounds. Adv. Cancer Res., 5: 350-361, 1958. The N-Hyroxylation of 4qAcetylaminobiphenyl by the Rat 33. Weisburger, J. It., and Weisburger, E. K. 2-Acetamido-7- and Dog and the Strong Carcinogenicity of N-Hydroxy-4- methoxyfiuorene. J. Chem. Soc., 758-759, 1954. acetylaminobiphenyl in the Rat. Cancer Res., 21: 1465-1473, 34. Weisburger, J. H., Weisburger, E. K., and Morris, H. P. 1961. Urinary Metabolites of the Carcinogen N-2-Fluorenylacet- 25. Morgan, M. A., and Gutmann, H. R. New Route to -14 amide. J. Natl. Cancer Inst., 17 : 345-361, 1956. Labeled N-(1-hydroxy-2-fluorenyl)acetamide. J. Org. Chem., 35. Weisburger, J. H., Weisburger, E. K., Morris, H. P., and 24: 1163-1165, 1959. Sober, H. A. Chromatographic Separation of Some Metabolites 26. Morris, It. P., Velat, C. A., Wagner, B. P., Dahlgard, M., and of the Carcinogen N-2-Fluorenylacetamide. J. Natl. Cancer Ray, F. E. Studies on Carcinogenicity in the Rat of Deriva- Inst., 17: 363-374, 1956. tives of Aromatic Related to N-2-Fluorenylacetamide. 36. Welch, A. D., and Nichol, C. A. Water-soluble Vitamins Con- J. Natl. Cancer Inst., 2~: 149-180, 1960. cerned with One- and Two-Carbon Intermediates. In: J. M. 27. Nagasawa, H. T., and Gutmann, H. R. The Oxidation of o- Luck (ed.), Ann. Rev. Biochem., Vol. 21, p. 656. Stanford: Aminophenols by Cytochrome c and Cytochrome Oxidase. I. Annual Reviews, Inc., 1952.

Fig. 1. Adenocarcinoma of small intestine from a rat treated with 1-methoxy-2-fluorenamine hydrochloride. This overall view shows normal small intestine at left and pancreas in lower left. Bizarre tumor glands are on extreme right. H & E, enlarged from X 11. Fig. 2. Same specimen as Fig. 1. This shows zone of transition from normal intestine (le/t) to tumor (right). H & E, X 60. Fig. 3. Detail of area from extreme right of Fig. 1. Note bizarre gland shapes and mitoses. Poorly differentiated tumor cells are invading serosal connective tissue. H & E, X 130. Fig. 4. Itepatocellularcarcinoma from rat treated with 1-methoxy-2-fluorenamine hydrochloride. Tumor above compresses normal liver below, tt & E, reduced from X 100. Fig. 5. Detail of tumor from Fig. 4. Note mitoses, pleomorphic nuclei, abundant cytoplasm, and solid cellular pattern. H & E, re- duced from X 400. Fig. 6. Cholangiolarcarcinoma from rat treated with 1-methoxy-2-fluorenamine hydrochloride. Duct-like structures are filled with neutrophils in some areas and by proliferating tumor cells in others. There is an abundant fibrous stroma. H & E, reduced from X 100. Fig. 7. Detail of tumor from Fig. 6. Note neutrophiles, fibrosis, and mitotic figures. H & E, reduced from X 400. Fig. 8. Breast carcinoma from rat treated with N-(7-methoxy-2-fluorenyl)acetamide. Note acinar pattern with irregular duct-like structures in connective tissue. Epithelium lining ducts is very pleomorphic, tt & E, reduced from X 100. Fig. 9. Same specimen as Fig. 8 (lower right) showing two contingent lobules with extreme cellular proliferation. H & E, reduced from • 400.


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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1968 American Association for Cancer Research. The Carcinogenicity of the o-Methoxy Derivatives of N -2-Fluorenylacetamide and of Related Compounds in the Rat

H. R. Gutmann, S. B. Galitski and W. A. Foley

Cancer Res 1968;28:234-244.

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