The Effects of Adrenalectomy, Hypophysectomy, and Castration on the Urinary Metabolites of 2@Acety1aminofluorene in the Rat*

PRABHAKAR D. LOTLIKAR, MAKOTO EN0M0T0,t ELIZABETH C. MILLER, AND JAMES A. MILLER (McArdle Memorial Laboratory for Cancer Research, Univer8ity of lVisconsin Medical School, Madison, Wisconsin) SUMMARY Adrenalectomized or hypophysectomized young male rats excreted in the urine only about 40 per cent as much N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) after intraperitoneal injection of a test dose of AAF as unoperated rats; similar re sults were obtained with adrenalectomized rats given 2-diacetylaminofluorene. The excretion of N-hydroxy-AAF by adrenalectomized rats given either AAF or 2-dia cetylaminofluorene was restored to about 90 and 75 per cent, respectively, of normal by treatment with cortisone and deoxycorticosterone. Administration of adreno corticotropic hormone for 10 days largely restored the excretion of N-hydroxy-AAF by hypophysectomized rats given injections of AAF. In adrenalectomized-hypoph ysectomized young rats, the excretion of N-hydroxy-AAF after injection of AAF was reduced to 30 per cent of normal. Administration of cortisone and deoxycor ticosterone acetate restored the excretion to 75 per cent of normal; growth hormone had little effect. Thyroidectomy of young rats and replacement therapy with either iodide or thyroid powder had minimal effects on the excretion of metabolites of AAF. Adrenalectomized-hypophysectomized-castrated adult male rats excreted only 40 per cent as much N-hydroxy-AAF as normal rats given injections of AAF. Ad ministration of cortisone and deoxycorticosterone with or without testosterone in creased the excretion of N-hydroxy-AAF by the triply operated rats to 3â€”4times the control level, but did not alter the excretion by unoperated rats. Castration alone or in combination with adrenalectomy caused a 2â€”3-foldincrease in the ex cretion of N-hydroxy-AAF; this increase was entirely prevented by testosterone and was partially prevented by cortisone administration. The above endocrine ablations and replacement therapies had much less influence on the excretion of ring-hydroxy derivatives after administration of AAF. Adren alectomy did not alter the excretion of N-hydroxy-AAF after the administration of this compound, nor the ability of liver homogenates to reduce N-hydroxy-AAF to AAF. It is suggested that one of the roles of adrenal hormones in promoting hepatic carcinogenesis by AAF and 2-diacetylaminofluorene in the rat, as shown by other investigators, may be to promote the formation or maintenance of higher levels of the N-hydroxy derivative.

A number of investigators have shown that under ap thyroid glands reduces the amount of hepatic damage and propriate conditions ablation of the adrenal, pituitary, or the incidence of hepatic tumors induced by the amin oazo dyes or by 2-aminofluorene and its acetylated a This investigation was supported by Grants CA-07175 and derivatives (2â€”5, 8, 10â€”12, 17, 18, 25, 26, 28â€”30, 32, CRTY-5002 of the National Cancer Institute, United States Pub lic Health Service; by a grant from the Jane Coffin Childs Me 33). In pioneer studies in this area Symeonidis (32, morial Fund for Medical Research; and by the Alexander and 33) showed that adrenalectomized rats fed 4-dimethyl Margaret Stewart Trust Fund. We wish to thank Mrs. Diane aminoazobenzene were relatively resistant to liver tu McKechnie for excellent technical assistance. A preliminary mor induction. Similar results were obtained by Chany report of some of these data has been made (20). t Present address : Department of Pathology, University of et a!. (5). Studies by other investigators (10, 11, 17) Tokyo. have been less definitive in demonstrating a require Received for publication June 29, 1964. ment for functional adrenal tissue in hepatic car 1835

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1964 American Association for Cancer Research. 1836 Cancer Research Vol. 24, November 1964 cinogenesis by the aminoazo dyes, but the possibility MATERIALS AND METHODS of residual functional adrenal tissue makes the inter Animals and diets.â€”Weanling (50-60 gm.) and adult pretation of these data difficult. Griffin et at. (18, 30) (170â€”180gm.) male rats2 were given an 18 per cent casein did, however, obtain a marked inhibition of carcino semipurifled diet (1) and water ad libitum. The adrenal genesis by 3'-methyl-4-dimethylaminoazobenzene in hy ectomized rats3 were given 1 per cent NaCl, the adrenal pophysectomized rats, and this inhibition was most ectomized-hypophysectomized rats both 5 per cent glucose effectively prevented by the combined administration of and 1 per cent NaCl, and the thyroidectomized rats 5 per insulin and adrenocorticotropic hormone. cent calcium lactate in their drinking water. The hypo On extension of these studies, O'Neal et al. (25) ob physectomized and adrenalectomized-hypophysectomized tamed a similar marked inhibition of hepatic carcinogenesis animals were housed in plastic box cages and were given by 2-diacetylarninofluorene' in hypophysectomized rats. glucose in addition to their diet for the first 24 hr. The In this case, however, replacement therapy with insulin other rats were kept in screen-bottomed cages. and ACTH was ineffective in restoring the susceptibility Compounds.â€”Syntheses or sources of the compounds of the liver to tumor induction. Hypophysectomy also employed were as follows : AAF (Mann Research Labora inhibited hepatocarcinogenesis when 2-aminofluorene was tories, New York); N-hydroxy-AAF (27), DAAF (24), applied topically by Bielschowsky et al. (3). Perry (26) and cortisone acetate (U.S.P., Merck) ; deoxycorticosterone found that complete adrenalectomy protected the livers acetate (DOCA) (U.S.P., Nutritional Biochemicals of his male rats from tumor induction by 2-acetylamino Corp.) ; deoxycorticosterone trimethylacetate (DCT) fluorene (AAF), while Reuber (28) concluded that under (Ciba) ; protamine zinc insulin (Eli Lilly and Co.) ; adreno his conditions both adrenalectomy and castration were corticotropic hormone (ACTH) (The National Drug Co.); necessary for inhibition of hepatic carcinogenesis by bovine growth hormone (National Institutes of Health) ;4 DAAF. Recently Weisburger et al. (36) found that rats testosterone (U.S.P., Nutritional Biochemicals Corp.); bearing a transplanted pituitary tumor which secretes thyroid powder (U.S.P., Retort Pharmaceutical Co., somatotropin, ACTH, and mammotropin are more sus Inc., New York) ; and diphosphopyridine nucleotide, ceptible to hepatic carcinogenesis by N-hydroxy-AAF triphosphopyridine nucleotide, adenosine triphosphate, than normal rats. The observation of Goodall (14), that and glucose-6-phosphate (Sigma Chemical Co.). high levels of cortisone increase the incidence of hepatic Administration of compounds and collection of urines.â€” tumors in unoperated rats given topical applications of One-half of the daily dose of cortisone (2 mg/day), DOCA 2-aminofluorene, suggests that the ACTH secreted by the (2 mg/day), ACTH (2 I.U./day) insulin (0.008 unit/day), transplanted pituitary tumor was an important factor in or growth hormone (2 mg/day) was injected sub the experiments of Weisburger et al. (36). cutaneously in 0.2 ml. of 0.9 per cent sodium chloride Bielschowsky and Hall (2, 4), Goodall (14, 15), and solution into weanling rats at 8 A.M. and at 4 P.M. A Reuber (29) have reported a marked inhibition of hepatic single dose of 2 mg. of DCT was injected on the first day; carcinogenesis by 2-aminofluorene or its acetylated deriva thereafter these animals and the controls received in tives in thyroidectomized rats. In these studies, replace jections of 0.9 per cent NaC1 solution. In all cases adult rats received twice the dose of adrenal hormones that was ment therapy with growth hormone (2), cortisone (14), given to the weaning rats. Testosterone (300 pg/rat! potassium iodide (15), or thyroid powder (29) restored day) was injected subcutaneously in 0.9 per cent NaC1 the susceptibility to liver tumor induction. solution in adult rats. Potassium iodide was admin From the data reported herein it appears that the istered as a 0.05 or 0.1 per cent solution in the drinking excretion of N-hydroxy-AAF is much more responsive to water. Thyroid powder was added to the food at levels the alteration of the hormonal balance, particularly with of 0.1 and 0.5 per cent. In the experiment in which the regard to adrenal hormones, than is the excretion of the weanling animals were both adrenalectomized and hypo phenolic metabolites. On the assumption that the urinary physectomized, the hormonal therapy was started on the excretion is a rough reflection of the tissue levels, it ap day after the endocrine ablations; in all other cases, pears that the lower incidences of hepatic tumors in therapy began on the day of the operations. On the fifth to the seventh day, the rats received intraperitoneal in adrenalectomized or hypophysectomized rats fed 2-amino jections of a freshly prepared suspension of AAF (3.0 fluorene or its acetyl derivatives may be due, at least in mg/100 gm of body weight) or DAAF (3.57 mg/100 gm part, to a reduced ability of the rats to form or maintain of body weight) or N-hydroxy-AAF (3.2 mg/100 gm of adequate levels of N-hydroxy-AAF. Other studies from body weight) in a 1.75 per cent gum acacia-0.9 per cent this laboratory have shown that N-hydroxy-AAF is a NaCl solution (22). By this time the tissues should have proximate carcinogenic metabolite of these fluorene corn been depleted of any residual hormone from the ablated pounds (7, 22). organ; likewise the adrenal hormones should have been

1 The following abbreviations are used (the Chemical Abstracts 2HoltzmanRatCo.,Madison,Wis. nomenclature is given in parentheses) : AAF = 2-acetylamino 5 The endocrine ablations were carried out at the Endocrine fluorene (N.2-fluorenylacetamide); DAAF = 2-diacetylamino Laboratories, Madison, Wis. fluorene (N-2-fluorenyldiacetamide); x-HO-AAF = x-hydroxy-2- 4ThebovinegrowthhormonewaskindlysuppliedbyDr.A.E. acetylaminofluorene (x-hydroxy-2-fluorenylacetamide); DOCA = Wilhelmi of Emory University, Atlanta, Ga., through the Endo deoxycorticosterone acetate; DCT = deoxycorticosterone tn cninology Study Section of the National Institutes of Health, U.S. methylacetate; ACTH = adrenocorticotropic hormone. Public Health Service.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1964 American Association for Cancer Research. LOTLIKAR et al.â€”Urinary Metabolites of 2-AAF 1837 depleted in the hypophysectomized rats. The adrenalec ml. of ethanol; homogenate equivalent to 40 mg. of wet tomized-hypophysectomized and adrenalectomized-hy liver; 100 pmoles of sucrose; and water to a final volume pophysectomized-castrated rats were also given S.C. of 3.0 ml. The reaction flasks (25-mi. Erlenmeyer flasks) injections of 2 ml. of a 5 per cent glucose plus 1 per cent were incubated in a nitrogen atmosphere for 20 mm. at NaCl solution immediately after injection of the car 37Â°C. in a Dubnoff metabolic shaker. The reaction was cinogen. For the collection of 24-hr. urine samples, one stopped by rapidly chilling the flasks and adding 2 ml. of or two rats were housed in each wire mesh stainless steel 2 N NaOH per flask. The contents of each flask were ex metabolism cage. Feces were separated by a wire screen, tracted with 5 ml. of reagent-grade ethyl ether, and the and the urine was collected under toluene in ice-cooled absorption of the ether extract containing AAF was de containers. Water was available ad libitum; no food was termined in the range of 270â€”350mp in a Beckman model given during urine collections. Urine which was not DB spectrophotometer.6 analyzed immediately after collection was stored at â€”15Â° Calculation of statistical significance.â€”For all of the C. In some cases the daily injections of the hormones data in Tables 1â€”5and Charts 1 and 2, the statistical were continued through the 10th to the 21st day, the rats significance of the differences between the values for the received a second intraperitoneal injection of AAF, and unoperated control animals and the values for each of the 24-hr. urine samples were again collected. When the other groups analyzed after the same treatment period second or final urine collections were completed, the rats have been calculated according to the â€œtâ€•test(13). In were killed and the adequacy of the hypophysectomies and addition, the statistical significance of the differences adrenalectomies was confirmed by gross and microscopic between the values for animals subjected to a given opera study. tion and the similarly operated groups treated with re Determination of the urinary metabolites.â€”The urine placement therapy was calculated by the same method. samples were analyzed as described earlier (23) by pro The differences which are significant with a P value of 0.05 cedures adapted from those of Weisburger et at. (39). In or less are indicated in the tables and in the chart legends. brief, the urine was incubated with $-glucuronidase and Taka-diastase, and the ether-extractable metabolites were RESULTS chromatographed on Whatman no. 1 filter paper. After Effect of single endocrine abkztions on the uriruiry excretion elution with ethanol, the ultraviolet spectra of the me of mekibolites of AAF by weanling male rats.â€”Although tabolites were determined with Beckman model DB the levels of the phenolic metabolites of AAF which were spectrophotometer; before spectral analysis the N-hy excreted in the urine after a test dose of AAF were gen droxy-AAF was separated from AAF by extraction with erally not consistently influenced by adrenalectomy, 0.5 N NaOH. All of the data for urinary metabolites hypophysectomy, thyroidectomy, or the replacement ther presented in this paper were corrected by the following apies used, the excretion of N-hydroxy-AAF was markedly recovery data obtained by analyzing the compounds added affected by these treatments (Tables 1â€”3). Normal male to normal urine : N-hydroxy-AAF, 75 per cent; 3-hydroxy rats which received an I.P. injection of AAF 5 or 10 days AAF, 70 per cent; 5-hydroxy-AAF, 96 per cent; and after weaning excreted an average of 4.0 or 3.1 per cent 7-hydroxy-AAF, 63 per cent. Since about 15 per cent of of the dose as N-hydroxy-AAF in 24 hr. (Table 1). Ad the N-hydroxy-AAF is reduced to AAF during chroma renalectomy reduced the excretion of N-hydroxy-AAF to tography,5 the urinary excretion of AAF was corrected by about 40 per cent of normal, and the administration of deducting 15 per cent of the corrected N-hydroxy-AAF cortisone alone or with either deoxycorticosterone acetate excretion from the observed excretion of AAF ; the recovery or deoxycorticosterone trimethylacetate restored the cx of added AAF is 100 per cent. The amounts of 1-hydroxy cretion of N-hydroxy-AAF by adrenalectomized rats to AAF were generally too low to quantitate adequately, 68â€”110per cent of the normal level. At the levels used, and the data on this metabolite have therefore been the two forms of deoxycorticosterone were less effective omitted. than cortisone alone. Hypophysectomy also reduced the Reduction of N-hydroxy-AAF in vitro.â€”Livers from urinary excretion of N-hydroxy-AAF to about 40 per cent decapitated rats were chified in cold 0.25 M sucrose solu of normal, and the normal level of excretion was partially tion, and a 10 per cent homogenate was prepared in 0.25 restored by 10 days' administration of ACTH alone or M sucrose solution in a Potter-Elvehjem-type homogenizer. with insulin. The incubation medium contained 100 pmoles of tris Administration of cortisone or deoxycorticosterone (hydroxymethyl)aminomethane buffer, pH 7.8 ; 10 pmoles trirnethylacetate to normal rats had little influence on the of MgC12; 0.30 pmole of diphosphopyridine nucleotide; levels of N-hydroxy-AAF or of the phenolic metabolites 0.27 pmole of triphosphopyridine nucleotide; 10 pmoles of of AAF excreted in the urine after a test dose of AAF adenosine triphosphate; 6 pmoles of glucose-6-phosphate; (Table 1). 2.09 pmoles (500 pg.) of N-hydroxy-AAF added in 0.1 Seven days after the operation, thyroidectomized rats BRecentlywehavenotedthatonchromatographyofsyntheticexcreted only 60 per cent as much N-hydroxy-AAF from N-hydroxy-AAF (free of AAF) under the usual conditions, ap a dose of AAF as the control rats (Table 2) ; however, this proximately 15 per cent of the material migrating with the RF of difference had only a low level of statistical significance. N-hydroxy-AAF is neutral and has the spectrum characteristic of AAF. This observation was not made in our earlier studies, a Studies on this enzyme system are now being prepared for since the recovery of synthetic N-hydroxy-AAF from the chro publication. The assay system is linear with respect to homogenate matograms was determined spectrophotometnically without prior concentration and time of incubation and is saturated with respect partition of the material between alkali and ethyl ether. to the substrate.

TABLE 1 THE URINARY METABOLITESOF 2-ACETYLAMINOFLUORENEINWEANLINOMALE RATS SUBJECTEDTO ADRENALECTOMYOR HYPOPHYSECTOMY WITH OR WITHOUT REPLACEMENT THERAPY Replacement therapy was given subcutaneously and was started on the day of the endocrine ablations. Cortisone, 1 or 2 mg/rat/ day; DOCA, 2 mg/rat/day; DCT, 2 mg/rat administered only once on the first day; ACTH, 2 I.U./rat/day; and insulin, 0.008 unit! rat/day. AAF was injected intraperitoneally (3 mg/iÂ® gm of body weight). The urine was collected for 24 hr. Values printed in bold-face type are significantly different from the values for the unoperated controls for the same treatment period with a P value of 0.05 or less. Values printed in italics are significantly different from the values for the corresponding operated controls with a P value of 0.05 or less.

DEVIATION)N-H0-AAF3-HO-AAF5-HO.AAF7-H0-AAFAAFControl5 KETABOLITES (% DOSE Â± STANDARD ot WEIGRT â€˜@a IN oupAAF ANALYSES5AVERAGE GAIN ON DAYNo. (ca.)URINARY

@ (27) Â± 0.9 1.3 Â± 1.8 Â± 6.2 0.2 0.2+ 1012 8 (24)12 134.0 3.1 Â± 1.24.9 7.3 Â± 2.68.0 9.8 Â± 4.015 22 Â± 5.70.4 0.4Â±Â±

Cortisone (2 mg.) (10) Â±0.5 Â±0.3 Â±0.1 Â±3.5 0.3 0.1Adrenalectomy5+ DCT5 52 4 (8)â€”8 82.9 2.9 Â±0.55.9 5.4 Â± 1.49.1 8.4 Â± 2.618 16 Â± 2.40.6 0.5Â±Â±

(42) .7 Â±0 .5t .3 Â±1 .7 Â± 1.9 Â±4 .5f .4 0.3 0.3+ 1017 4 (10)6 41 1.2 Â±1.16 4.7 Â± 3.76.7 4.8 Â± 4.221 16 Â± 9.20 0.3Â±Â±

0.1+Cortisone (1 mg.)52 (4)â€”32.7 Â± 0.16.6 Â± 0.76.9 Â± 0.625 Â±1.30.7Â±

Cortisone (2 mg.)5 (18) Â± O.8@ Â± 1.lt Â± 2.O@ Â± 4.Ot 0.1 0.4+ 109 2 (4)â€”6 33.1 3.4 Â±0.76.6 7.7 Â± 0.19.0 9.6 Â±1.825 31 Â±2.90.4 0.5Â±Â±

DOCA5 (18) Â±O.4f@ Â± 1.1 Â± 1.5 Â± 5.2 0.2 0.1+ 109 2 (4)12 102.5 2.0 Â± 0.35.6 5.6 Â± 0.66.8 7.0 Â± 0.619 23 Â± 1.10.4 0.4Â±Â±

0.1+DOCA+cortisoneDCT54 (8)132.4 Â± 1.26.1 Â± 2.77.7 Â± 3.519 Â± 5.30.3Â±

0.2+ (12)â€”43.5 Â±[email protected] zE 1.99.0 Â±8.022 Â±7.80.4Â± (2mg.)56

0.1Hypophysectomy5DCT + cortisone (8)â€”63.2 :1:[email protected] Â±2.69.5 Â±1.621 Â±3.50.3Â± (2 mg)54

(26) .7 Â±1 .2t Â± 1.6 .9 Â± 2 .2 Â±4.5 .5 0.2 0.2+ 108 6 (21)0 111 1.6 Â±0.75.3 6.9 Â± 2.45 7.7 Â± 2.618 18 Â± 2.10 0.5Â±Â±

ACTH5 (2) 0.3+ 101 2 (4)4 41.7 8.1 Â± 0.45.7 7.2 Â± 1.98.0 7.3 Â± 1.117 18 Â±0.20.8 0.4Â±

0.4+Insulin5 (3) Â± 1.7 Â± 4.6 Â± 3.2 Â± 11 102 1 (2)1 61.7 1.94.6 5.25.2 6.516 230.5 0.4Â±

ACTH + insulin5 (4) Â±0.1 Â±1.6 Â±1.4 Â± 2.9 0.1 102 2 (4)3 22.3 2.8 Â±0.86.9 7.2 Â±0.99.6 8.0 Â±0.817 55 Â±[email protected] 0.7Â±Â±0.3

* The first number denotes the number of different urine samples analyzed; the number in parentheses denotes the total number of rats studied. t SignificantlydifferentfromthevaluefortheunoperatedcontrolanimalswithaPvalueof0.01orless. @ Significantly different from the value for the corresponding operated control animals with a P value of 0.01 or less.

Administration of 0.1 per cent of potassium iodide in the the carcinogenicity studies of Reuber and Firminger (12, drinking water maintained the excretion of N-hydroxy 28) with adrenalectomized rats utilized DAAF, the urinary AAF by thyroidectomized rats at 87 per cent of the normal metabolites excreted after administration of this carcinogen level, whereas inclusion of 0.1 or 0.5 per cent of thyroid or of AAF were compared (Table 3). In both cases the powder in the diet was without effect. By 21 days, the adrenalectomized rats excreted about 50 per cent as much excretion of N-hydroxy-AAF by the thyroidectomized N-hydroxy-AAF as the unoperated controls. Admin rats was similar to the control rats. istration of cortisone and DOCA restored the excretion of Urinary metabolites of AAF, DAAF, and N-hydroxy N-hydroxy-AAF from AAF and DAAF to 93 and 73 per AAF excreted by adrenalectomizedweanling rats.â€”Since cent, respectively, of the control levels. The levels of the

TABLE 2

THE URINARY METABOLITES OF 2-ACETYLAMINOFLUORENE IN WEANLING MALE RATS SUBJECTED TO THYROIDECTOMY WITH OR WITHOUT REPLACEMENT THERAPY The thyroidectomized rats were given 5 per cent calcium lactate in their drinking water from the day of the operation throughout the experiment. Administration of 0.05 or 0.1 per cent of potassum iodide in the drinking water or of 0.1 or 0.5 per cent of thyroid powder in the diet was started on the day of the operation. AAF, 3 mg/100 gm of body weight, was injected intraperitoneally on the 7th and 21st days. After each dose of AAF, the urine was collected for 24 hr. Values printed in bold-face type are significantly different from the values for the unoperated control animals for the same treatment period with a P value of 0.05 or less. Values printed in italics are significantly different from the values for the corresponding thyroidectomized controls with a P value of 0.05 or less.

DEVIATION)N-HO-AAF3HOAAF5-HO-AAF7-HO-AAFAAFControl7oi METABOLITES (% DOSE Â± STANDARD GaoiwAAF INJECTED ANAL ON DAYNo. VIESAVEIAOE (ox.)URINARY

(6) Â± 1.5 Â±0.6 Â±0.9 Â± 3.5 Â± 0.1Thyroidectomy7 213 3 (6)23 694.7 4.3 Â±1.05.35.9 Â±0.510 9.5 Â±0.520 24 Â±1.40.20.1 Â±0.1

(6) Â±0.8 Â± 2.4 Â± 2.6 Â± 5.7 Â± 0.1+ 213 3 (5)18 572.7 4.9 Â±0.54.0 5.9 Â± 1.38.8 8.9 Â± 2.017 24 Â± 2.40.30.2 Â±0.1

0.05%KI7 (4) Â±0.7 Â±0.1 Â±1.3 Â±0.6 Â± 0.1+ 212 3 (6)9 333.5 5.6 Â± 0.64.9 5.7 Â± 0.48.7 9.7 Â±0.721 19 Â± 2.40.20.1 Â±0.0

0.1% KI7 (4) Â±0.4 Â± 0.1 Â± 0.2 Â± 1.8 Â± 0.1+0.1%thyroid 212 2 (4)6 334.1 3.5 Â±0.74.65.3 Â±0.99.5 9.2 Â± 1.521 21 Â± 1.40.10.1 Â±0.0

(4) Â± 0.4 Â± 1.6 Â± 2.5 Â± 3.7 Â± 0.1+powder7 212 2 (4)â€”1283.0 4.8 Â±1.15.66.0 Â±1.413 8.8 Â±2.318 19 Â± @.90.20.1 Â±0.0

0.5%thyroid (4) Â± 0.2 Â± 0.3 Â± 0.8 Â± 1.6 Â± powder7 212 1 (2)10 402.2 4.37.0 7.611 8.417 250.2 0.10.1

* The first number denotes the number of d@fferent urine samples analyzed; the number in parenthe ses denotes the total number of rats studied.

TABLE 3 THE URINARY METABOLITES OF 2-ACETYLAMINOFLUORENE, 2-DIACETYLAMI NOFLUORENE, AND N-HYDROXY-2-ACETYLAMINOFLUORENE IN WEANLING MALE RATS SUBJECTED TO ADRENALECTOMY WITH OR WITHOUT REPLACEMENT THERAPY The S. C. administration of cortisone and DOCA (2 mg. of each per rat per day) was started on the day of endocrine ablations. The compounds (amounts equimolar to 3 mg. of AAF per 100 gm. of body weight) were injected intraperitoneally on the fifth day. Values printed in bold-face type are significantly different from the values for the unoperated control animals with a P value of 0.05 or less. Values printed in italics are significantly different from the values for the corresponding adrenalectomized control rats with a P value of 0.05or less.

DEVIATION)N-HO-AAF3-HO-AAFS-HO-AAF7-HO-AAFAAFControlAAF NETABOLITES(%DOSEÂ±STANDARD GzoupCospoum INJECTEDAVERAGE WEIGifT GAIN (ou.)URINARY

Â± 0.7 Â± 1.1 Â± 0.7 Â± 4.3 0.2AdrenalectomyAAF DAAF4 4(6)(6)13 144.4 3.5 Â± 1.15.8 5.1 Â± 1.09.8 9.6 Â± 2.422 17 i 4.70.3 0.3Â±Â±0.3

.8 Â±0 .2t Â± 0.7 .5 Â± 1 .lt Â± 3.6 0.1Adrenalectomy DAAF5 5(10)(10)11 101 1.6 Â± 0.75.0 5.5 Â± 1.66 6.9 Â± 1.620 20 Â± 2.80.3 0.2Â±Â±0.2

+ Â± O.5@ Â± 2.2 Â± @.4 Â± 9.2 0.3ControlN-HO-AAF3(6)1425DOCA + cortisoneAAF DAAF6 5(12)(10)â€”5 â€”54.1 5.7 Â± 0.46.5 6.2 Â± 1.410.2 9.4 Â± 2.124 26 Â± 11.60.10.4Â±Â±0.1

0.21.3Â±0.7AdrenalectomyN-HO-AAF3(6)1521 Â± 2.13.2 Â± 0.33.3 Â± 0.23.5 Â±

Â±2.34 .2 Â± 0 .62 .0 Â± 0 .4t4 .8 Â± 0 .61 .3Â±0.1

* The first number denotes the number of different urine samples analyzed; the number in parentheses denotes the total number of rats studied. t Significantly different from the value for the unoperated control animals with a P value of 0.01 or less. @ Significantly different from the value for the corresponding adrenalectomized control rats with a P value of 0.01 or less.

Taka-diastase. In the case of the 3-, 5-, and 7-hydroxy 7-HO-AAF derivatives, 5â€”10per cent appeared not to be conjugated. 40 Effects of multiple en4ocrine ablations on the urinary metabolites of AAF excreted by weanling rats.â€”When both the pituitaries and the adrenals were removed, the cx 20 cretion of N-hydroxy-AAF was reduced to about 30 per cent of the control level (Chart 1). Administration of cortisone and DOCA to these rats restored the urinary Wa ililHO AAF I 20 â€”I- excretion of N-hydroxy-AAF to 75 per cent of the control U Xlii I0 level, and administration of growth hormone with these adrenal hormones had little additional effect. Admin ______istrationofgrowthhormonealonedidnotinfluencethe 2i@. 20 4 excretion of N-hydroxy-AAF by adrenalectomized >.4 I0 hypophysectomized rats. The levels of the phenolic 41i. zo metabolites of AAF were not similarly affected by the 6 ______endocrineablationsorthereplacementtherapiesused. Effects of endocrine ablations on the urinary metabolites of AAF excreted by adult male rats.â€”In contrast to the 4 results with weaning rats, adrenalectomy of adult male rats did not result in a decreased urinary excretion of 2 N-hydroxy-AAF (Table 4). On the other hand, castration alone or in combination with adrenalectomy caused an j@jAjjj ______increase in the N-hydroxy-AAF excretion to 2â€”3 times the CONTROl.I@ ADRENAL.+HYPOPHYS. I control level. This increase was noted in the castrate

CORTI SONE - - â€” + + males when AAF was administered 5, 13, or 18 days after + DOCA the operation, but only in adrenalectomized-castrated

GROWTH - - + - + rats studied at the later times. The excretion of 3-hy HORMONE droxy-AAF was also increased to 1.5â€”2times the control level in castrated and castrated-adrenalectomized rats, CHART 1.â€”The urinary metabolites of 2-acetylaminofluorene in weanling male rats subjected to both adrenalectomy and hypo but the levels of 5- and 7-hydroxy-AAF were less affected. physectomy with or without replacement therapy. The replace In the adrenalectomized-castrated rats, administration of ment therapies (2 mg. each of cortisone, DOCA, or growth hor corfisone reduced the excretion of N- and 3-hydroxy-AAF mone per day) were administered subcutaneously starting 24 hr. and in some cases the administration of testosterone after the operations. AAF, 3 mg/100 gm of body weight, was in jected intraperitoneally on the fifth day, and urine was collected and cortisone was more effective. for 24 hr. The results are the averages of three urine collections; Administration of cortisone and DOCA with or without each collection was the pooled urine from two rats. The small bar testosterone to normal rats did not greatly influence the at the top of each large bar denotes the standard deviation. The levels either of the phenolic metabolites of AAF or of urinary excretion of N-hydroxy-AAF by the untreated adrenalec tomized-hypophysectomized animals is significantly different from N-hydroxy-AAF (Table 4). the value for the unoperated control animals at a P value of less When adult male rats were simultaneously adrenalec than 0.01. The urinary excretion of N-hydroxy-AAF by the tomized, hypophysectomized, and castrated, the excre operated rats treated with cortisone and DOCA or with cortisone, tion of N-hydroxy-AAF was reduced to about 40 per DOCA, and growth hormone is significantly different from that of cent of the control level (Chart 2). Administration of the untreated operated animals with a P value less than 0.01. The average weight changes for the rats during the 5-day period cortisone and DOCA with or without testosterone to were as follows : control, +5 gm. ; adrenalectomized-hypophysec these rats increased the urinary level of N-hydroxy-AAF tomized (untreated), â€”4 gm. ; operated + growth hormone, â€”1 to 3â€”4times the control level. Administration of tes gm. ; operated + cortisone + DOCA, â€”6gm. ; operated + corti tosterone alone to these triply operated rats did not cause sone + DOCA + growth hormone, â€”4gm. any significant increase in the excretion of N-hydroxy AAF. In some cases the operated rats with or without hormone therapy excreted more of the phenolic metabolites ring-hydroxy derivatives were not greatly altered by the than the unoperated controls, but the differences were less carcinogen administered or the hormonal status of the dramatic than those observed for the excretion of animals. N-hydroxy-AAF. In contrast to the above results, the excretion of N-hy Enzymatic reduction of N-hydroxy-AAF by weanling rat droxy-AAF after I.P. injection of N-hydroxy-AAF was liver homogenates.â€”The lower excretion of N-hydroxy similar with both control and adrenalectomized rats AAF after administration of AAF to adrenalectomized (Table 3). In control and adrenalectomized rats, es sentially all of the N-hydroxy-AAF excreted, more than rats appears not to be due to an increased capacity of the 20 per cent of that injected, was excreted as conjugates; liver to reduce N-hydroxy-AAF to the amide. As shown less than 0.4 per cent could be extracted with ethyl ether in Table 5, the livers of adrenalectomized rats did not prior to incubation of the urine with $-glucuronidase and differ from those of adrenalectomized rats treated with

TABLE 4 THE URINARY METABOLITES OF 2-ACETYLAMINOFLUORENE IN ADULT MALE RATS SUBJECTED TO ADRENALECTOMY AND CASTRATION WITH OR WITHOUT REPLACEMENT THERAPY The initial weights of the adult male rats were 170â€”180gm. Cortisone and DOCA (4 mg. of each per rat per day) and testosterone (300 gig/rat/day) were injected subcutaneously starting with the day of operation. AAF, 3 mg/100 gm of body weight, was injected intraperitoneally at the time indicated; urine samples were collected for 24 hr. Values printed in bold-face type are significantly different from the values for the unoperated controls for the same treatment period with a P value of 0.05 or less. Values printed in italics are significantly different from the values for the corresponding operated controls with a P value of 0.05 or less.

iLETABOLITZ (% DOSE Â± STANDARD DEVIATION) â€”N-H0-AAF3-HO-AAF5-HO-AAF7-HO-AAFAAFControl5GzouiAAF INJECTED WEIGifT ON DAYNAVERAGE GAIN (Ga.)URINARY

Â±0.1 Â±0.4 Â± 1.1 Â± 1.3 Â± 13 2 (4) 46 1.0 Â±0.1 2.7 Â±0.1 8.9 Â±0.8 17 Â± 0.0 0.1 Â± 0.1 0.1+ 184 3(6)(3)17 580.7 0.9 Â±0.12.3 2.6 Â±0.64.6 8.2 Â± 1.514 13 Â± 2.30.4 0.2 Â±0.1

@0.1+Cortisone+ DOCA52(4)â€” 171 .1 Â±0 .2t3 .6 Â±0 .85 .4 Â±1.514 Â±1.80 .3

@ Â±0.2Castrated5Cortisone+ DOCA 0.22.9 Â±0.25.6 Â±1.219 Â±2.70.4 + testosterone52(4)â€”140.9

@ Â± 0.4 Â± 0.4t Â± 1.2 Â±2.0 13 2 (4) 28 2.2Â±0.5 4.5Â±0.3 11 Â±1.4 20Â±2.5 0.1Â± 0.0 0.1+ 184 2(6)(2)9 561.3 1.8Â±0.54.3 5.0Â±0.47.3 7.7Â±0.118 17Â±2.50.4 0.1Â±0.1

Testosterone5 Â±0.1 Â± O.@ Â±1.4 Â± 1.0 Â± 0.0Adrenalectomized5 132 2(4)(4)15 400.6 0.6 Â± [email protected] Â± 0.54.8 7.4 Â± 1.718 11 Â±1.40.4 0.1 Â±0.1

Â±0.2 Â± 1.3 Â±2.0 Â±2.1 Â± 13 2 (4) 36 1.0 Â±0.0 3.0 Â±0.2 6.1 Â±0.1 14 Â±3.0 0.1 Â±0.0 0.0+ 184 2(6)(2)15 630.7 1.1 Â±0.14.0 2.0 Â± 0.45.8 4.7 Â± 0.516 10 Â±0.90.4 0.1 Â±0.1

Cortisone5 Â±0.3 Â±0.5 Â± 0.8 Â± 4.7 Â± 0.1Adrenalectomized-cas 134 2(6)(4)â€”23â€”290.6 0.8Â±0.12.9 3.9Â±0.36.4 10 Â±O.1@16 21Â±O.Ot0.4 0.5Â±0.2

.9 Â±0 .3 .8 Â± 1 .0 Â±3.6 Â±1.5 .3 Â± trated5 13 2 (4) â€”2 2.4 Â±0.2 4.1 Â±0.3 11 Â±0.0 19 Â±1.0 0.1 Â±0.0 0.0+ 184 2(6)(2)0 300 2.7 Â±02t4 5.7 Â±O.lt6.8 5.0 Â± 0.116 19 Â± 0.50 0.1 Â±0.1

Cortisone5 Â± 0.3 Â± 0.5 Â±1.2 Â± 1.4 Â± 0.0+ 134 2(6)(4)â€”21â€”400.4 1.7Â±0.45.8 3.6Â±0.46.1 6.8Â±O.4@13 23Â±1.00.5 0.2Â±0.2

Â±0.1+Testosterone52(2)40.9 Â±0.11.8 Â± 0.52.2 Â±0.316 Â±0.50.2

Cortisone+ testos Â±0.4 Â±0.5 Â±0.3 Â±3.5 Â± terone5 134 2(6)(4)â€”6 â€”270.7 1.@ Â± 0.12.6 S.@ Â± 0.45.5 7.4 Â± 0.717 23 Â± 1.00.4 0.4 Â±0.20.2

* The first number denotes the number of different urine samples analyzed; the number in parentheses denotes the total number of rats studied. t Significantly different from the value for the unoperated control animals with a P value of 0.01 or less. @ Significantly different from the value for the corresponding operated control animals with a P value of 0.01 or less.

cortisone and DOCA or from those of normal rats in tions in the amount of this metabolite are of particular their ability to form AAF from N-hydroxy-AAF in vitro. interest since N-hydroxy-AAF is a proximate carcinogenic The extrahepatic tissues have much lower activities for metabolite of AAF (22) ; further, it seems reasonable to the reduction of N-hydroxy-AAF to AAF than does liver.6 expect that the relative amounts of N-hydroxy-AAF in the urine under various conditions may be a rough re DISCUSSION flection of the amount available to the tissues under the From the data presented in this and previous papers same conditions. In the present study the importance (21, 23), the amount of N-hydroxy-AAF excreted in the of the adrenal hormones for maximum excretion of urine after a dose of AAF appears to be much more re N-hydroxy-AAF after a test dose of AAF was the striking sponsive to alterations in experimental conditions than observation. In many experiments cortisone and de the amounts of the other urinary metabolites. Altera oxycorticosterone were administered together to the

TABLE 5 THE REDUCTION OF N-HYDROXY-2-ACETYLAMINOFLUORENE TO 2-ACETYLAMINOFLUORENE BY LIVER HOMOGENATES FROM NORMAL AND ADRENALECTOMIZED RATS There were six weanling male rats per group. Cortisone and DOCA (2 mg. of each per rat per day) were injected subcutane ously for 5 days from the day of adrenalectomy. The rats were f4jjjjjj killed for enzyme assays on the sixth day. The results are cx 5-HO-AAF0 pressed as the mean Â±standard deviation. a â€”lii formed(mpmolesper @1@.. GroupAAF 40 mg. of wet liver per O@ @W 20mm.)Control 4-, @ t; Â±16 II. 53-HO-AAF 4 Adrenalectomy 106 Â± 9 )@45IIlÃIII4 Iâ€˜Â° Adrenalectomy + cortisone + DOCA105 126 Â± 24 !@ N-HO-AAFII @!!2 increased to near-normal or above-normal levels by daily injections of cortisone and deoxycorticosterone. An ab sence of functional testes (as in immature male or adult castrated male rats) seemed to be required to demonstrate IIEE the necessity for adrenal hormones. In the weanling rat the effect of adrenalectomy was reinforced by simultaneous hypophysectomy, and in the adult rat simultaneous @ CONTROL ADRENAL.+ HVPOPHYS.+CASTRATION hypophysectomy was essential to a demonstration of the

CORTISONE - - + + requirement for adrenal hormones. Nevertheless, the + DOCA ability to reverse the effects only with adrenal hormones, TESTOSTERONEâ€” - + â€” + and not with testosterone or growth hormone, implicates the adrenal hormones as a primary requirement. It is CHART 2.â€”The urinary metabolites of 2-acetylaminofluorene in adult male rats subjected simultaneously to adrenalectomy, possible that in our adult rats the presence of the pituitary hypophysectomy, and castration with or without replacement gland permitted sufficient stimulation of accessory adrenal therapy. The replacement therapies (4 mg. each of cortisone and tissue, which was not detectable on careful gross autopsy, DOCA and 300gig.of testosterone) were injected subcutaneously to furnish the rat with adrenal hormones. starting on the day of the operations. AAF, 3 mg/100 gm of body weight, was injected intraperitoneally on the fifth day after From our present knowledge, the metabolic site of in the operations, and the urine was collected for 24 hr. The results volvement of the adrenal hormones appears to be in the are the averages of three analyses ; the urine from one rat was used formation of the N-hydroxy derivative (Chart 3). Thus, for each analysis. The small bar at the top of each large bar adrenalectomy of immature rats did not appreciably im denotes the standard deviation. The urinary excretions of N- hydroxy-AAF by the untreated operated animals, the operated pair the excretion of conjugates of N-hydroxy-AAF when animals treated with cortisone and DOCA, and the operated this derivative was injected. The excretion of AAF and animals treated with cortisone, DOCA, and testosterone, as well of the ring-hydroxy derivatives of AAF was much less as the urinary excretion of 3-hydroxy-AAF and of AAF by the affected by the hormonal manipulations, so that secondary operated animals treated with cortisone and DOCA, are signifi effects due to altered amounts of substrate available for cantly different from the excretions of these compounds by the unoperated control animals with P values of 0.01 or less. The N-hydroxylation seem unlikely. Further, the activities of excretions of N-hydroxy-AAF and of 3-hydroxy-AAF by the oper rat liver homogenates in reducing N-hydroxy-AAF to ated animals treated with cortisone and DOCA and of N-hydroxy AAF were similar whether the immature rats were un AAF by the operated animals treated with cortisone, DOCA, and operated or were adrenalectomized with or without re testosterone are significantly different from those of the untreated placement therapy. A direct test of the effect of operated animals with P values of 0.01 or less. The rats weighed 160-170 vfl@ at the time of the operations. The average weight adrenalectomy on N-hydroxylation by rat liver prepara changes during the 5-day period were as follows: control +18 tions in vitro has not been possible with the very low gm. ; adrenalectomized-hypophysectomized-castrated (untreated) activities obtained with the present N-hydroxylation â€”32gm. ; operated + testosterone, â€”38gm. ; operated + cortisone + DOCA,â€”34gm.;operated+ cortisone+ DOCA+ testos system as applied to rat liver preparations (19).7 terone, â€”32gin. The major objective of these studies was to see if the effects of hormonal alterations on the carcinogenic activity of the fluorene derivatives could be explained through operated rats; where the compounds were administered alterations in metabolic patterns. Some progress ap separately, each appeared to cause an increase in the cx pears to have been made. Although the situation is cretion of N-hydroxy-AAF. Thus, adrenalectomized complex with adult rats, the excretion of N-hydroxy-AAF immature male rats or adrenalectomized-hypophysec was generally greater when adrenal hormones were avail tomized-castrated adult male rats given AAF excreted only 40-50 per cent as much N-hydroxy-AAF as their 7UnpublishedexperimentsbyM. Enomoto,P.D. Lotlikar, unoperated controls ; in each case the level of excretion was J. A. Miller, and E. C. Miller.

TISSUES N-HYDROXY-AAF ,/@

@ /â€”\ ,=\ ,Ac ______O-GL.UCURONOSYLCONJUOATIQ3(NH@,RO@,AAF)

N-HYDROXY REDUCTION @/â€˜ @RECARCINOGENICTHAN AAF LIVER HOMOGENATE,,/)// STIMULATED BY + @7@,/i:;VERMICROSOMES1ADRENALHORMONES @ â€˜@+ TPNH+ 02 ] IN VIVO? iF I â€˜H URINE ARYL RING - HYDROXYLAT1ON LIVER MICROSOMES + TPNH + 02 @ CARCINOGENIC I-,3-,5-,AND 7-HYDROXY-AAF T 0- GLUCURONOSYL @ HO- CONJUGATION>(Is3@[email protected] HYDROXY- AAF) AND NON-CARCINOGENIC @/â€˜ o- SULFATE TISSUES OF 7-HYDROXY-AAF

CHART 3.â€”The possible site of action of the adrenal hormones in the metabolism of 2- acetylaminofluorene in the rat with reference to the requirement of adrenal hormones for hepatocarcinogenicity. Data on the various metabolic steps are available in the following references: reduction of N-hydroxy-AAF (16, this paper), N-hydroxylation (7, 19, 23, 34), formation of O-glucuronosyl-(N-hydroxy-2-acetylaminofiuorene) (7, 19), ring hydroxylation (6, 31, 35, 37â€”39),andconjugation of ring-hydroxyderivatives (37â€”39).

able. Likewise, the results of Reuber (28), Goodall (14), One puzzling question is why hepatic carcinogenesis is and Perry (26) show that the adrenal hormones are among inhibited by adrenalectomy or hypophysectomy while the factors required for efficient hepatic carcinogenesis. other tumors develop in these animals when they are fed Further, functional testes appear to prevent both the the fluorene carcinogens (3, 25, 26). Each site presum inhibition of hepatic carcinogenesis and the reduced cx ably requires N-hydroxy-AAF or a further metabolite as cretion of N-hydroxy-AAF by adrenalectomized adult rats. a proximate carcinogenic agent (22). Perhaps, when only While Perry (26) did not find castration as such to be limitedamountsof N-hydroxy-AAFareavailablein vivo, required for inhibition of hepatic carcinogenesis in adrenal this carcinogen is more effective in nonhepatic tissues ectomized rats, he did report degeneration of the testicular than in the liver, where it is rapidly subjected to reduction tissue under his conditions. The major difference be to AAF and subsequent ring-hydroxylation (see Chart 3) tween the results of the present metabolic study and the carcinogenicity studies was the additional requirement REFERENCES for hypophysectomy of adult rats to show an impaired excretion of N-hydroxy-AAF. A possible explanation is 1. ANDERSEN,R.A.; ENOMOTO,M.;MILLER,E. C.; ANDMILLER, that, in the long-term carcinogenicity tests, continued J. A. Carcinogenesis and Inhibition of the Walker 256 Tumor administration of the fluorene derivative reduced any in the Rat by trans-4-Acetylaminostilbene, Its N-Hydroxy Metabolite, and Related Compounds. Cancer Res., 24:128â€” effect that could be mediated by stimulation of accessory 43,1964. adrenal tissue. The finding of Reuber (28) that both 2. BIELSCHOWSKY,F.Carcinogenesis in the Thyroidectomized castration and adrenalectomy are required for strong Rat. The Effect of Injected Growth Hormone. Brit. J. Cancer, inhibition of hepatic carcinogenesis by DAAF may cx 12:231â€”33,1958. 3. BIELscnowsxY, F.; BIEr.scnowsxy, M.; ANDFLE@rcnz@R,E.K. plain why other investigators were unable to restore Investigations of the Role of Thyroxine in the Development hepatic carcinogenesis in hypophysectomized rats either of Hepatomas in Hypophysectomized Rats and Pituitary with ACTH and insulin (25) or with cortisone (14). A Dwarf Mice. Brit. J. Cancer, 16:267â€”74, 1962. lack of testosterone may have been the critical factor. 4. BIELSCHOWSKY,F.,ANDHALL, W. H. Carcinogenesis in the Thyroidectomized Rat. Brit. J. Cancer, 7358-66, 1953. The small and inconsistenteffectof thyroidectomyon 5. CHANT,E.; AUJARD,C.; ANDBOY,J. Influence de la SurrÃ©na the urinary level of N-hydroxy-AAF in our experiments lectomie sur la Frequence et le Delai d'Apparition de l'HÃ©pa appears to be at variance with the inhibition of hepatic tome Chez Ia Rat Femelle Adulte. Compt. Rend. Soc. Biol., carcinogenesis in thyroidectomized rats (2, 4, 14, 15, 29). 152:275â€”77,1958. Thyroidectomy causes complex endocrine abnormalities 6. CRAMER,J. W.; MILLER,J. A.; ANDMILLER,E. C. The Hy droxylation of the Carcinogen 2-Acetylaminofluorene by Rat (2, 9), and it is possible that our experiments were of too Liver: Stimulation by Pretreatment in Vivo with 3-Methyl short a duration to be definitive in relation to chronic cholanthrene. J. Biol. Chem., 235250-56, 1960. carcinogenesis experiments. 7. . N-Hydroxylation:A New MetabolicReaction Ob

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1964 American Association for Cancer Research. The Effect of Adrenalectomy, Hypophysectomy, and Castration on the Urinary Metabolites of 2-Acetylaminofluorene in the Rat

Prabhakar D. Lotlikar, Makoto Enomoto, Elizabeth C. Miller, et al.

Cancer Res 1964;24:1835-1844.

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