Influence of Adrenocortical, Androgenic, and Anabolic Hormones on the Development of Carcinoma and Cirrhosis of the Liver in A X C Rats Fed N-2-Fluorenyldiacetamide 1, 2
HARLAN I. FIRMINGER and MELVIN D. REUBER,3, . Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland
SUMMARY
An experimental model was developed to detect the role of various endocrine organs and their hormones in the induction of cirrhosis and carcinoma of the liver. This model uses inbred A X C rats, fed 0.025 percent N-2- fluorenyldiacetamide in a semi- synthetic diet according to a regimen that produces less than 100 percent of tumors in susceptible intact males surviving the induction period of 25 weeks. With this model it was found that: 1) Male rats were far more susceptible to cirrhosis and carcinoma of the liver than female rats. 2) Castration protected male rats against cirrhosis but showed little effect on tumor induction. 3) Simultaneous administration of testosterone restored the susceptibility to cirrhosis in cas- trated males and resulted in both cirrhosis and carcinoma in intact and castrated female rats. 4) Castrated female rats, given testosterone 4 months after the carcinogen was stopped, failed to develop either cir- rhosis or carcinoma. 5) Adrenalectomy seemed to offer some protection against cirrhosis but not against tumors.
Adrenalectomized female animals given testosterone were far more susceptible to tumors than intact females given testosterone. 6) Deoxycorticosterone acetate had a striking protective effect both against cirrhosis and carcinoma in male rats. 7) Norethandrolone, an anabolic hormone with minimal andro- genic activity in the dosage admin- istered, was equally potent to testoster- one in promoting cirrhosis and carcino- ma of the liver in all the groups tested. 8) The presence of anabolic hormone, either testosterone or norethandrolone, affected not only the incidence but also the morphology of the tumors, since poorly differentiated tumors were found only in animals subjected to exogenously administered testosterone or norethandrolone or endogenous testosterone. These findings suggest a close correlation between carcino- genesis and the level of protein metabolism of the liver cells at the time the carcinogen is administered. The possible relationship of protein metabolism to a mechanism of car- cinogenesis is described .- J. Nat. Cancer Inst. 27: 559-595, 1961.
IT IS well known that hepatocellular carcinoma occurs more frequently in men than in women and that it is generally associated with cirrhosis
1 Received for publication March 27, 1961.
2 Aided by grants from the American Cancer Society, Inc.
3 This research was started at the University of Kansas and was completed at the University of Maryland, the present address of the authors.
. The authors are grateful to Dr. Bertram W. Haines for his assistance in preparing the statistical analysis.
(1, 2). A similar association with cirrhosis and preferential sex incidence has also been reported by a number of investigators in rats fed various carcinogens (3-8). Castration of rats fed N-2-fluorenylacetamide has proved to be some protection against cirrhosis and carcinoma of the liver, but the incidence of both lesions could be restored by administration of testosterone propionate during the period the carcinogen was fed (9). Thyroidectomy prevented the development of neoplasms of the liver (10) in rats exposed to N-2-fluorenylacetamide. As might be expected, hypophysectomy protected rats from hepatic carcinogenesis (11-15) with 3’-methyl-4-dimethylaminoazobenzene or N-2-fluorenyldiacetamide, but there are conflicting reports concerning the effect of adrenalectomy (18, 16-18) when azo dyes are used as carcinogens.
It seemed that some of the reported differences could be due to dif- ferences in the procedures used; in particular, the overwhelming dosage of a powerful carcinogen might well mask the role of hormones. Indeed, a number of experiments with 3’-methyl-4-dimethylaminoazobenzene or N-2-fluorenyldiacetamide have failed to reveal even a difference in suscep- tibility between the sexes (13, 19). Consequently the first efforts were to develop a model in which most of the intact male animals lived long enough to develop large tumors, at times with metastases. The dosage was regulated so that somewhat less than 100 percent of the animals developed tumors.
MATERIALS AND METHODS
Inbred A X Crats weighing from 150 to 200 g were used; a few animals weighed slightly over 200 g. They were fed a semisynthetic diet 5 designed by Morris to which N-2-fluorenyldiacetamide (2-FdiAA) was added to make 0.025 percent. The dosage of carcinogen administered to each group of animals was regulated by varying the time on the carcinogen. Previous experiments had revealed the desirability of alternating periods of carcinogen administration with short “rest periods” on the semisyn- thetic diet without carcinogen (9). Throughout the experiment water was available ad libitum.
To define the desired dosage more precisely, the length of time the carcinogen was administered was varied among 4 groups of animals. In each group the rats were fed the carcinogen-containing diet for 4 weeks followed by 1 week of the same diet without carcinogen. At that time the animals were returned to the carcinogen-containing diet for another 4-week period followed by 1 week of diet without carcinogen. Thus the diet was alternated until 8, 10, 12, or 16 weeks of carcinogen feeding had been completed according to the preselected time for the respective groups. After completion of the last period on the carcinogen, the animals were
5 Morris diet No. 272 consisted of 300 g commercial casein, 2275 g skim-milk powder, 6152 g ground, hard spring wheat (12-13.5% protein), 200 g brewer’s yeast, 200 g desiccated liver, N.P. (Wilson Laboratories, Chicago, III.) 140 g sodium chloride, 10 g ascorbic acid, 13 g ferric citrate, 100 g cod-liver oil, and 610 g corn oil.
maintained on the basic diet without carcinogen until their death or until the experiment was terminated at the end of 65 weeks.
All animals were weighed weekly, and an exploratory laparotomy with biopsy of the liver was performed on each animal at 6- to 8-week intervals to trace the development of lesions and determine the earliest appearance of tumors. This provided material for transplantation, which will serve as the basis of a separate report. Also, at the time of death of the animals, autopsies provided additional material for microscopic examina- tion and transplantation.
RESULTS
Previous studies of carcinogenesis of the liver (20-26) as well as those derived from the present experiments all revealed a similar progression of morphologic changes from normal through various hyperplastic paren- chymal proliferations to neoplasia. Simultaneously there was often proliferation of small bile ducts and minimal accompanying fibroblastic activity progressing to apparent isolation of lobules and in more florid instances to isolation of liver cells (figs. 1 and 2). Some larger bile ducts at times showed similar proliferative activity in localized areas, often with the formation of cystic lesions made up of a group of cystic ducts. Ceroid pigment was usually also evident in macrophages in the portal areas.
Hyperplasia of parenchymal cells seemed the obvious forerunner of hepatocellular carcinoma; hence, for purposes of evaluation of the findings in the livers of each of the experimental animals, they were classified and tabulated as showing:
1) No hyperplasia: This included also animals that showed basophilia of the cyto- plasm of the periportal cells or the earliest hyperplastic change diffusely in the periportal zones.
2) Hyperplasia: These were animals with livers containing multiple small foci or larger areas of hyperplasia which were distinct from the surrounding liver and which often showed somewhat different architectural arrangement from the normal lobule (figs. 3, 4, 5, and 6).
3) Hyperplastic nodules: These were animals in which the hyperplasia had progressed to the formation of clearly defined nodules of parenchymal cells with distinct com- pression of surrounding parenchyma (figs. 7 and 8). The architecture in these nodules varied from well-organized adult cords to a disorderly pattern of growth, at times with atypicality of cells but of such a degree that it was not considered malignant (fig. 9).
4) Small hepatoma: These hepatomas represented the earliest and smallest lesions that could be morphologically accepted as malignant; yet, by definition, they were limited to lesions no more than 5 mm in greatest diameter (fig. 10).
5) Well-developed hepatocellular carcinoma or occasionally cholangiohepatoma: These animals had malignant tumors of parenchymal cells which were well differentiated, poorly differentiated, or anaplastic growths (figs. 12 and 15). Occasionally there were cholangiomatous areas in the hepatocellular carcinomas (fig. 14). Invasion of portal or hepatic veins was frequent and in some animals multiple emboli or metastases were seen in the lungs (figs. 11 and 13). In some animals, multiple implants were also found over the peritoneal surfaces.
For the purposes of tabulation or graphing (table 1), only the most advanced lesion of each liver was used, though in most instances “lesser lesions” accompanied the more advanced hyperplastic or neoplastic lesions. The results of this preliminary study, shown in table 1, reveal that 8 weeks of the carcinogen were inadequate to produce either cirrhosis or tumors. Ten weeks of carcinogen feeding produced cirrhosis and 2 small hepatomas, but 16 weeks on the carcinogen were required for more than 50 percent of intact male rats to develop hepatocellular carcinoma. The earliest small hepatoma in this group appeared 25 weeks after the start of the carcinogen-containing diet. Sixteen weeks of carcinogen feeding were adopted for the model. Unfortunately the procedure of periodic laparotomy and biopsy did not reveal many of the tumors, particularly in their early stages. Hence, data collected in this manner could not be used to determine the latent periods for each of the experi- mental groups.
| Time on carcinogen (weeks) | animals Number of in group | Number of animals with lesions shown | |||||||
|---|---|---|---|---|---|---|---|---|---|
| No hy- perpla- sia | Hyper- plasia | Nod- ules | Small hepa- toma | Hepa- tocellu- lar car- cinoma | Metas- tases | Total animals with liver tumors | Cir- rhosis | ||
| 8 | 14 | 8 | 6 | 0 | 0 | 0 | 0 | 0 | 0 |
| 10 | 10 | 0 | 3 | 5 | 2 | 0 | 0 | 2 | 7 |
| 12 | 10 | 0 | 1 | 4 | 0 | 5 | 1 | 5 | 8 |
| 16 | 11 | 0 | 1 | 3 | 1 | 6 | 1 | 7 | 9 |
*Animals are classified according to the most advanced lesion found. The presence of metastases and/or cirrhosis is also indicated. The average latent period in the 12- and 16-week groups was 51 weeks without signif- icant difference between the two groups.
EVALUATION OF THE ROLE OF CERTAIN ENDOCRINE ORGANS AND HORMONES IN CIRRHOSIS AND HEPATIC CARCINOGENESIS
After selection of the basic procedure, the influence of various endocrine glands or exogenously administered hormones was tested. Endocrine organs were removed 2 weeks before the carcinogenic diet was started. In groups of animals given hormone it was administered simultaneously with the start of the carcinogenic diet, with one notable exception in which a group of castrated female rats was given the carcinogen in the diet and testosterone was not administered until 4 months after the carcinogen was withdrawn.
In 6 or 8 weeks after the start of the experiment, an exploratory lapa- rotomy was performed on each group, with biopsy of the liver of the most advanced lesion visible on the surface of the liver. Biopsies generally consisted of wedge-shaped bits of liver measuring about 3 X 3 X 5 mm
on each edge. This was repeated several times in each animal to deter- mine the time of the earliest appearance of tumor and provide material for microscopic examination and transplantation studies. All animals at the time of death or as soon as possible thereafter were autopsied, and tissue was obtained for transplantation and representative micro- scopic sections. The gross findings at autopsy are summarized in table 2. In those animals in which endocrine organs had been removed, a thorough search, both grossly and microscopically, was made for residual, regenerated, or ectopic endocrine tissue.
The multiplicity of findings in the different experimental groups made it necessary to assign numerical values to each of the items considered in the evaluation for determination of significant differences between the groups. Items included were: type of hyperplastic or neoplastic micro- scopic lesion, the number and size of tumors in the liver, the over-all weight of the liver, and the number of metastases in the lungs with a correction for length of survival. (See Appendix for details of scoring procedure.) With this system, the numerical value for each animal was calculated and comparison of the statistical significance of the differences between the means of the two groups being compared was calculated. When differences between two groups are referred to subsequently in the text, they had P values of 0.01 or less unless otherwise stated. Table 3 gives the mean scores for each group with the ranges and standard errors.
Gonads and Sex Hormones
The first experiment was an attempt to re-evaluate the role of sex and sex hormones in cirrhosis and carcinogenesis. In this experiment nine groups of animals were studied. They consisted of intact males and females, intact males and females given testosterone propionate, castrated males and females, castrated males and females given testos- terone propionate, and finally a group of castrated females in which the administration of testosterone propionate was delayed until 4 months after the carcinogen was stopped. In those animals receiving testos- terone, a single pellet containing 1 part of testosterone propionate and 3 parts of cholesterol was implanted subcutaneously in the interscapular area. The dose consisted of 20 mg of testosterone propionate per 100 g of rat or 80 mg of pellet per 100 g. All surviving animals were killed at the end of 40 weeks.
The intact males and females both lost considerable weight during the initial 4 weeks on the carcinogen, but rapidly regained the lost weight when the carcinogen was removed from the diet during the 5th week. Thereafter the animals continued to gain or maintain their weight despite reinstatement of the carcinogen-containing diet. In groups of intact animals the administration of testosterone had little effect on the weight curves except for a slight delay in the initial weight loss in the females.
The castrated males and females initially lost weight in a manner similar to the intact animals and generally followed the same course
| Group | Num- ber of ani- mals | liver Aver- age weight (g) | Average num- ber of tumors per liver | Usual tumor diameter (cm) | Lung metas- tases | Aver- age surviv- al (weeks) |
|---|---|---|---|---|---|---|
| Castrated males given testosterone | 10 | 35. 5 | More than 3 | Up to 7.0 | Raret | 39 |
| Adrenalectomized males | 15 | 29.5 | More than 3 | Up to 7.0 | Massive | 52 |
| Adrenalectomized fe- males given testos- terone | 12 | 33.2 | More than 3 | Up to 4.5 | Many | 57 |
| Castrated males given norethandrolone | 8 | 27.5 | 3 | 1.5 X 2.5 | Nonet | 37 |
| Adrenalectomized males given 2 mg cortisone | 10 | 26. 6 | 2-3 | 3. 0 | Fewt | 38 |
| Adrenalectomized, cas- trated females given testosterone | 14 | 26.2 | 2-3 | 1.5 X 4.5 | Rare | 54 |
| Intact males (2 groups) | 11 | 20.0 | 2 | 2.0 X 4.5 | Rare | 56 |
| 13 | 15. 5 | 2 | 1.0 X 1.5 | None | 39 | |
| Intact males given testosterone | 10 | 24.1 | 1 | 1.5 X 2.0 | None | 42 |
| Intact males given norethandrolone | 14 | 17.6 | 1 | 1.5 × 2.0 | None | 32 |
| Intact females given norethandrolone | 13 | 18. 8 | 1 | 1.5 X 2.0 | None | 41 |
| Adrenalectomized males given 1 mg cortisone | 10 | 16. 5 | 1 | 1.5 X 1.8 | None | 37 |
| Castrated males | 12 | 15. 1 | 1 | 1.5 × 2.0 | None | 60 |
| Castrated females given norethandrolone | 10 | 16. 3 | 1 | 1.5 X 2.0 | None | 40 |
| Castrated females given testosterone | 8 | 16. 5 | 1 | 1.0 | None | 42 |
| Intact females given testosterone | 13 | 11. 6 | 1 | 1.0 | None | 40 |
| Adrenalectomized males given deoxycortico- costerone acetate | 7 | 11. 4 | None | - | - | 40 |
| Adrenalectomized, cas- trated females | 12 | 10. 4 | None | - | - | 63 |
| Castrated females | 14 | 9. 3 | None | - | - | 58 |
| Adrenalectomized females | 14 | 8. 0 | None | - | - | 60 |
| Intact females | 14 | 8. 7 | None | - | - | 62 |
*Liver weight of control adult animals on laboratory pellets is 10.9 g for males and 7.2 for females. The groups of animals are arranged in an approximate order with the most susceptible at the top and the least or nonsusceptible at the bottom. The order is similar but slightly altered from that obtained with the scoring procedure, as seen in table 4.
tThe animals in these groups died earlier in the experiment, which may have influenced the results.
as intact animals, but the administration of testosterone to these two groups delayed the initial weight loss until the 2d month on the carcinogen. After this loss the animals returned to a more normal weight and con- tinued to gain or maintain their weight.
Text-figures 1 and 2 and tables 2 and 3 confirm the difference in susceptibility of males and females to cirrhosis and hepatocellular car- cinoma (figs. 16 and 19). Castration of the males produced a minimal reduction in the incidence of hepatocellular carcinoma, but the tumors tended to be fewer and smaller than in intact males (fig. 17) and there were no metastases. More striking was the complete absence of cirrhosis.
JOURNAL OF THE NATIONAL CANCER INSTITUTE
| Group | Number of animals | Scores | Standard error | |
|---|---|---|---|---|
| Range | Mean | |||
| Castrated males given testosterone | 10 | 58-513 | 277. 7 | ±45. 1 |
| Adrenalectomized males | 13 | 13-546 | 197. 9 | ±48.2 |
| Adrenalectomized males given 2 mg cortisone | 10 | 22-580 | 185. 7 | ±43.0 |
| Adrenalectomized females given testos- terone | 12 | 17-407 | 161. 5 | ±38.3 |
| Castrated males given norethandrolone | 8 | 47-313 | 145. 0 | ±32. 1 |
| Intact males given testosterone | 10 | 34-423 | 125.0 | ±38. 5 |
| Castrated females given norethandro- lone | 10 | 13-315 | 111 | ±37. 6 |
| Adrenalectomized, castrated females given testosterone | 14 | 4-389 | 105. 0 | ±30.3 |
| Intact males given norethandrolone | 14 | 24-330 | 89.7 | ± 21.5 |
| Intact males | 24 | 12-300 | 76 | ± 15.3 |
| Intact females given norethandrolone | 11 | 33-182 | 65 | ±13.5 |
| Castrated females given testosterone | 9 | 31-200 | 54.7 | ±18.7 |
| Adrenalectomized males given 1 mg cortisone | 10 | 8-216 | 53. 2 | ±6.2 |
| Castrated males | 12 | 4-86 | 39. 4 | ±8.1 |
| Intact females given testosterone | 13 | 10-71 | 30 | ±5.9 |
| Adrenalectomized males given deoxy- corticosterone acetate | 7 | 4-70 | 17 | ±9. 0 |
| Adrenalectomized, castrated females | 13 | 4-30 | 13 | ±2.2 |
| Intact females | 14 | 6-15 | 10 | ±0. 68 |
| Castrated females | 13 | 4-15 | 9. 2 | ±1.0 |
| Adrenalectomized females | 14 | 4-17 | 9 | ±1.3 |
*See Appendix for scoring procedure.
NO HYPERPLASIA
HYPERPLASIA
NODULES
SMALL HEPATOMA
HEPATOMA
TOTAL TUMORS
CIRRHOSIS
24
24
13
21
9.
8
0
1
2
S
10
10
8
3ª + TESTOSTERONE
2
0
o
0
3
14
14
10
ㅎ
2
2
0
0
O
0
13
13
2 +TESTOSTERONE
6
9
4
0
2
I
2
It is interesting that in approximately 50 percent of the castrated male animals there was cirrhosis early in the course, as shown by liver biopsy, but this had completely disappeared by the time of autopsy. The susceptibility to cirrhosis was restored by implantation of a pellet of testosterone propionate subcutaneously at the beginning of the experi- ment, and tumors in these animals were larger, often confluent, and metastasized. This dosage of testosterone is probably greater than physiologic, and the incidence of hepatocellular carcinoma was 100 percent, with 60 percent of the animals showing metastatic lesions. This increased level of testosterone seemed to increase the incidence of cirrhosis and hepatocellular carcinoma in intact males, but statistically the difference was not significant. Curiously, in intact males given testosterone the tumors were not nearly so large, or numerous, nor were the metastases impressive as in the castrated males given testosterone. Although the P value between these two latter groups was only 0.021, the results suggest a slight protective effect of the testis.
| NO HYPERPLASIA HYPERPLASIA | NODULES | SMALL HEPATOMA | HEPATOMA | TOTAL TUMORS | ~ CIRRHOSIS | |
|---|---|---|---|---|---|---|
| CASTRATED d' | 1 . 3 | 2 | 5 | 0 | ||
| CASTRATED +TESTOSTERONE | o 0 | 0 | o | 10 6 | 10 10 | |
| GASTRATED ? | 6 6 | 2 | 0 | 0 | 14 0 | 14 0 |
| GASTRATED ? +TESTOSTERONE | o 0 | I | 4 | 3 I | 8 | 8 |
| CASTRATED ? + DELAYED | 3 3 | 4 | 10 | 10 | ||
| TESTOSTERONE | 0 | 0 | o | 0 | ||
TEXT-FIGURE 2 .- Castrated animals given testosterone.
As might be expected, castration of the females showed little change from the intact females; in both, the changes in the liver were minimal. Interestingly, testosterone pellets in intact females produced cirrhosis and hepatocellular carcinomas (fig. 18) and increased the number of hyperplastic nodules but did not quite produce the same picture that was seen in intact males, which suggested a possible inhibitory effect of the ovaries (P value 0.012). Upon removal of the ovaries and adminis- tration of testosterone, the incidence of cirrhosis and tumors exceeded
that of intact males and rivaled that of castrated males given testosterone, but there was little comparison between those two groups when one con- sidered the size and number of hepatic tumors and the abundance and size of the metastases in the animals. In these respects the findings were more like those of castrated males without testosterone. The castrated males given testosterone were far more impressive. These differences re-emphasize a distinct difference between males and females, even after castration.
The castrated males receiving testosterone died somewhat earlier than the remaining animals and several died as a result of peritoneal hemor- rhage from a tumor. The latter was not observed in the remaining groups. Microscopically, the tumors of the liver were usually well- differentiated hepatocellular carcinomas. In the intact males there was 1 undifferentiated hepatocellular carcinoma and 2 cholangiohepatomas; in intact males receiving testosterone, 2 poorly differentiated hepato- cellular carcinomas; in castrated males given testosterone, 3 poorly differentiated hepatocellular carcinomas, and 1 cholangiohepatoma; and in one castrated female given testosterone an undifferentiated hepato- cellular carcinoma. Thus testosterone also appeared to have some effect on the cellular pattern as it tended to produce more undifferentiated tumors.
In one group of castrated females the administration of testosterone was delayed until 4 months after the diet containing the carcinogen was stopped. In this group neither cirrhosis nor tumors developed, which indicated the necessity of administering testosterone propionate during the period of carcinogen administration. The significance of this finding will be discussed subsequently.
Adrenal Glands Versus Gonads and Testosterone Propionate
Five groups of animals were studied including adrenalectomized males and females, adrenalectomized and castrated females, adrenalectomized and castrated females given testosterone propionate, and adrenalecto- mized females given testosterone without castration. Surviving animals in each group were killed at the end of 65 weeks. A sixth group of adrenalectomized castrated males died off too rapidly and had to be dropped.
Adrenalectomy and/or the 1 percent salt in the drinking water for either sex seemed to protect somewhat against weight loss compared to intact animals. Even these groups tended to lose some weight initially when given carcinogen-containing diet and to regain their weight as they became adapted or were returned to the diet without carcinogen. The administration of the testosterone to adrenalectomized or adrenalecto- mized and castrated females not only abolished the initial weight loss but actually produced a rather notable early weight gain.
The findings in these animals are shown in text-figures 3 and 4 and tables 2 and 3. In this experiment the group of adrenalectomized females given
testosterone died somewhat earlier with extensive tumor involvement so that before 62 weeks all the animals were dead with massive tumors and extensive pulmonary metastases. The adrenalectomized females given testosterone and the adrenalectomized castrated females given testosterone had more tumors, larger tumors, and more metastases than did the cas- trated females receiving the same hormone. The incidence of cirrhosis, however, in these same groups was directly opposite that of the tumors. Microscopically the tumors were all well-differentiated hepatocellular carcinomas.
| HYPERPLASIA NO HYPERPLASIA | NODULES | SMALL HEPATOMA | HEPATOMA | TOTAL TUMORS | CIRRHOSIS | ||
|---|---|---|---|---|---|---|---|
| ADRENALECTOMIZED ? | 4/3 3 | 0/3 | 0 | 0 | 0 | ||
| ADRENALECTOMIZED ? + TESTOSTERONE | 0 0/1 | 10 | 0 | 4/6 4 | 12 | 12 6 | |
| ADRENALECTOMIZED ? +CASTRATION | 2/3 0/1 | 212 | 0/2 | 0 | 12 | 12 0 | |
| 14 14 | |||||||
| ADRENALECTOMIZED ? | 415 | ||||||
| +CASTRATION | |||||||
| + TESTOSTERONE | 1/0 | 1/0 | 2/ | 0 | 2 | 4 | |
TEXT-FIGURE 3 .- Adrenal glands versus gonads and/or testosterone. In this and the next text-figure upper number above each bar represents the animals without adrenal regeneration or ectopic adrenocortical tissue and lower number those with adrenocortical tissue.
Adrenocortical regeneration or ectopic adrenocortical tissue was usually tiny and often only microscopic. Eight of 14 adrenalectomized males not receiving hormones showed such adrenocortical tissue. In the females it was seen in 7 of 14 adrenalectomized and 8 of 12 adrenalecto- mized and castrated animals. The administration of testosterone did not alter the frequency of regeneration in that 7 of 12 adrenalectomized rats and 7 of 14 adrenalectomized castrated females bore such tissue. By contrast, the administration of adrenocorticoids after adrenalectomy was very effective in preventing adrenocortical regeneration.
Adrenalectomized Animals Given Corticoids
For comparison with the effects of adrenalectomy alone, three groups of adrenalectomized male rats were studied. The first group was given 1 mg of a saline suspension of cortisone acetate,6 and the second, 2 mg of
6The Upjohn Co., Kalamazoo, Mich., as Cortone Acetate.
cortisone acetate subcutaneously every 3 days for the duration of the experiment. The third group had 75 mg pellets of pure crystalline deoxycorticosterone acetate (DOCA)7 implanted subcutaneously in the interscapular region at the beginning of the experiment and a second such pellet 6 months later. These animals were maintained continuously on 1 percent sodium chloride in their drinking water just as the groups of adrenalectomized animals without corticoid supplementation. The experiment was terminated at 40 weeks.
Again the adrenalectomy and/or intake of 1 percent salt water pre- vented the initial weight loss except in the group given 2 mg of cortisone. In this latter group the weight curve was much like that of intact male animals. By the 12th week the starting weight was regained and from then on there was a progressive weight gain similar to the other groups in this experiment.
The findings are shown in text-figure 4 and tables 2 and 3. Adrenalec- tomized males had extensive liver involvement by tumor (fig. 20) and metastases to the lungs were usually massive (fig. 21). Livers of animals given DOCA were normal (fig. 22). The incidence of tumors in animals receiving 2 mg of cortisone was similar to that in the adrenalectomized animals without hormones, but the gross characteristics more closely resembled those of intact males without hormones.
There was 1 undifferentiated hepatocellular carcinoma in an adrenalec- tomized male and 1 in an adrenalectomized male given the 2 mg dose of cortisone. This total was somewhat smaller than the number of such undifferentiated tumors in the animals receiving testosterone. There were no cholangiohepatomas. Adrenal regeneration or ectopic adreno- cortical tissue was noted in 2 of 10 rats receiving the 1 mg cortisone dose, 1 of 10 receiving the 2 mg cortisone dose, and 1 of 7 given DOCA.
NO HYPERPLASIA
HYPERPLASIA
NODULES
SMALL HEPATOMA
HEPATOMA
G TOTAL TUMORS
G CIRRHOSIS
5/7
ADRENALECTOMIZED
IL
0
Vo
0
I/
6
7
7
ADRENALECTOMIZED d’ +DOCA
410
2/0
0
0
OF
0
7/1
10
10
9
ADRENALECTOMIZED +2mg. CORTISONE
O
0
110
I/O
4
10
10
ADRENALECTOMIZED O” +Img. CORTISONE
I/o
3/0
110
2/0
1/2
5
7 Schering Corp., Bloomfield, N.J., as “Cortate” pellets.
Norethandrolone Versus Testosterone
It also seemed desirable to determine the effects of adrenocortical anabolic hormone. The readily available compound that might be regarded as comparable is norethandrolone.8 It has predominant ana- bolic activity with minimal androgenic activity (27). The norethandro- lone was dispersed in physiologic saline to make a 10 percent suspension. For comparison with testosterone propionate, identical groups of animals were prepared and given 0.1 cc of the suspension containing 1 mg of norethandrolone, subcutaneously every 3 days, for the duration of the experiment. Unfortunately, a group of adrenalectomized males given norethandrolone in this experiment died very early and had to be abandoned.
Intact males given norethandrolone or testosterone had almost identical weight curves with an initial loss followed by progressive rise. Castrated males given either testosterone or norethandrolone showed a delay in weight loss until after the 12th week of the experiment, and norethandrolone produced a somewhat more noticeable initial loss with earlier death of the animals than testosterone.
In females, either intact or castrated, given testosterone propionate there was considerable delay in weight gain, but this period was much shorter in the comparable animals given norethandrolone.
The findings in these groups are given in text-figure 5 and tables 2 and 3, which by comparison with text-figures 1 and 2 reveal surprising cor- respondence between the effects of norethandrolone and those of testosterone. A representative liver is illustrated in figure 23.
In the intact male group given norethandrolone there was 1 cholangio- hepatoma and 1 undifferentiated hepatocellular carcinoma. The re- mainder of the hepatic tumors in this group were well differentiated.
| NO HYPERPLASIA HYPERPLASIA | NODULES | SMALL HEPATOMA | HEPATOMA | TOTAL TUMORS 14 | CIRRHOSIS | |
|---|---|---|---|---|---|---|
| d'+ NORETHANDROLONE | O 0 | . | 3 | 10 2 | ||
| CASTRATED d + MORETHAN DROLONE | 0 o | 0 | 2 | 6 4 | 8 | 8 |
| Q+NORETHANDROLONE | 3 | 7 | 13 | 13 | ||
| 0 I | 2 | |||||
| CASTRATED Q+ NORE THANDROLONE | 2 0 | 2 | 5 3 | 10 | 10 | |
| I |
TEXT-FIGURE 5 .- Animals receiving anabolic hormone.
Kindly supplied by Searle and Co., Chicago, III., as “Nilevar”®.
Primary Tumors Other Than Liver
Other miscellaneous tumors are listed in table 4. Details of the carcino- sarcomas of the salivary glands and the endometrial sarcomas of the uterus have been previously reported (28).
| Group | Tumors | Total number of animals |
|---|---|---|
| Castrated females given noreth- androlone | 3 sarcomas, endometrium 2 carcinosarcomas, salivary gland | 10 |
| Castrated males given testos- terone | 1 carcinosarcoma, salivary gland | 10 |
| Adrenalectomized females | 1 poorly differentiated sarcoma, sub- cutaneous tissue, neck | 14 |
| Adrenalectomized females given testosterone | 2 spindle-cell sarcomas, thoracic wall* 1 ossifying fibroma, groin 1 fibrosarcoma, area of pellet 1 adenocarcinoma, small intestine | 12 |
| Adrenalectomized and castrated females | 1 spindle-cell sarcoma, thoracic wall 1 rhabdomyosarcoma, groin | 12 |
| Adrenalectomized and castrated females given testosterone | 1 osteogenic sarcoma, thoracic wall* 1 poorly differentiated sarcoma, groin, with lung metastases 1 poorly differentiated sarcoma, thora- cic wall | 14 |
| Castrated males | 1 basophil adenoma, pituitary | 12 |
| Castrated females | 1 nodule sparsely granulated basophils, pituitary | 14 |
*Transplanted subcutaneously in A X C rats for 8 generations.
DISCUSSION
The studies of the role of gonads and/or testosterone confirm the pro- carcinogenic effect of the male sex hormone whether endogenous or exog- enous. Comparison of the findings in intact versus castrated animals given testosterone propionate suggests a protective effect of the ovaries and, curiously also, possibly of the testes. Poorly differentiated or un- differentiated tumors were seen only in males or animals receiving anabolic hormone, which corresponded to the findings of Sidransky et al. (29). Studies of the effects of progesterone and estrogens in this model are being carried out (30). A striking finding was the complete protection of castration against cirrhosis in male animals with little change in the over-all incidence of neoplastic lesions, though the latent period was longer, the lesions were smaller, usually solitary, and better differentiated.
Studies of the effect of adrenalectomy revealed a distinctly greater susceptibility to hepatic carcinogenesis and metastases in adrenalectom- ized male rats and in adrenalectomized female rats given testosterone when compared to corresponding intact animals. In female animals, carcinoma began to appear when castration was performed in addition
to adrenalectomy. Yet adrenalectomy or castration alone was insufficient to allow the induction of tumors in female rats. Hence it would seem that the protective effects of the ovaries and adrenal glands in female rats were additive. More well-developed tumors were seen with exog- enously administered testosterone propionate in adrenalectomized than in castrated females, so that the protective effect of the adrenal glands would seem somewhat greater than that of the ovaries, though the P value was only 0.023 between these two groups.
In another experiment both biopsied and nonbiopsied groups of ani- mals were studied (30). The nonbiopsied rats developed a significantly higher percentage of both tumors (P = 0.002) and cirrhosis than did the biopsied animals. This is further evidence that the role of the ad- renal gland in hepatic carcinogenesis is protective.
The protective effect of the adrenal glands was previously reported by Griffin et al. (13) with azo-dye carcinogenesis, but it is contrary to the findings of Symeonidis et al. (16) and of Eversole and Da Vanzo (17, 18). Each of the latter reported a decreased incidence of hepatic tumors in rats after adrenalectomy. Symeonidis further indicated that hepatic tumors could be found when the adrenal cortex regenerated from ectopic foci, as it did in approximately 50 percent of rats. In our ex- periments, also, there was regeneration of adrenocortical tissue in ap- proximately 50 percent of rats in the retroperitoneal fat. But in these experiments there seemed to be no correlation of hepatic tumors with the regeneration of adrenocortical tissue, which suggested that even with regeneration there was a deficiency of the adrenocortical factor or factors concerned in the protection against carcinogensis.
In an attempt to distinguish a cortical factor which might be re- sponsible for the protective effect, mineralocorticoid and glucocorticoid hormones were administered to adrenalectomized animals and the difference in results was striking. Only one of the rats receiving DOCA developed a carcinoma while most of the animals given cortisone, whether in 1 or 2 mg doses every 3 days, developed hepatocellular carcinoma. The protective effect of DOCA in adrenalectomized rats is the one finding that all investigators seem to agree upon, although Eversole suggests it is a factor of dosage. We would be inclined to agree, but not so much on the role of the dosage of deoxycorticosterone as on a dosage of carcinogen which does not swamp all other operative factors.
It is interesting that in addition to the protective effect of DOCA against tumors it also offered complete protection against cirrhosis. Yet it should be pointed out that in all adrenalectomized animals the incidence of cirrhosis of the liver was equal to or less than the incidence of tumors, in contrast to practically all other groups in which cirrhosis almost always equaled or excelled the incidence of tumors, with the outstanding exception of the group of castrated males mentioned.
As an aside it should also be mentioned that the dosages of DOCA or cortisone used were adequate to suppress adrenocortical regeneration in almost all the animals. Curiously, the one animal that developed a
carcinoma, even though given DOCA, was the only animal in the group of 7 which had regenerated adrenocortical tissue. This would seem to be mere coincidence, particularly when the results with this animal are compared to those with the group given 2 mg of cortisone in which 8 of the 9 animals developing tumors failed to regenerate adrenocortical tissue.
The protective effect of DOCA in these experiments is striking, but the mechanism of its action is not clear. One might postulate that it causes a decreased permeability of the liver-cell membrane, which de- creases the amount of carcinogen entering the cell and therefore the development of liver cancer. Kotin (31) has shown that estrogen de- creases the number of carbon particles that enter the liver cell as it decreases the permeability of the cell membrane, and he noted some pro- tective effect against hepatic carcinogenesis by estrogens. Or, one might postulate some competitive or blocking effect comparable to the preferential tight binding of C14-labeled estradiol by rat-liver protein in homogenates as demonstrated by Riegel and Mueller (32).
Probably the most striking finding derived from these experiments is the demonstration of the important procarcinogenic effect of anabolic hormones. It would seem that this is probably dependent on their effect on protein synthesis by liver cells. The Millers (33, 34) have shown that 4-dimethylaminoazobenzene and a number of its derivatives are bound to protein in the hepatic cell. Weisburger et al. (35) demon- strated that radioactivity from labeled N-2-fluorenylacetamide is simi- larly bound by protein of liver cells. Although it has not been specifically demonstrated that N-2-fluorenyldiacetamide is bound to protein, it is assumed that its behavior is entirely similar to N-2-fluorenylacetamide in this respect.
Gelboin and the Millers (36) found that the carcinogens are bound to protein currently being formed. Thus, the finding that delay in ad- ministration of testosterone to castrated females completely prevented cirrhosis and carcinoma takes on added significance and further emphasizes the importance of the rate of protein anabolism at the time the carcinogen is given.
A careful analysis by Farber et al. (37, 38) of the effects of ethionine (a hepatic carcinogen) on protein synthesis and the production of fatty liver in rats revealed a distinct hormonal influence. These findings closely parallel the hormonal relationships in hepatic carcinogenesis of the present experiments and those of others (10-13). Male rats, castrated males given testosterone, and castrated females given testosterone (37) or other anabolic hormones (38) were protected against fatty livers when given ethionine. Growth hormone and thyroxine had some protective effect, but intact and castrated females and castrated males were not protected. Administration of estradiol, deoxycorticosterone, thyrotrophic hormone, prolactin, and crystalline zinc insulin were not protective. Cortisone and adrenocorticotrophic hormone actually intensified the lesion (38). Ranney and Drill (39) confirmed the protective effect of norethandrolone
and showed that progesterone was ineffective. The effect of adrenalec- tomy apparently has not been studied. From this, one can conclude that even blocking of protein synthesis by ethionine can be overcome by hor- mones, particularly androgenic or anabolic hormones.
The recent work of Kochakian (40) suggests that androgens act by altering cellular enzyme activity. In rat liver he showed that the activity of several enzymes concerned with protein metabolism including aspartic- glutamic transaminase, alanine-glutamic transaminase, and d-amino acid oxidase are diminished by castration and restored by testosterone. Thus it would seem likely that androgenic and probably anabolic hormones have their procarcinogenic and procirrhotic effects on the rat liver through their fundamental influence over intracellular enzymes concerned with protein metabolism. In the case of carcinogenesis this means increased protein synthesis and increased carcinogen binding.
It is clear, however, that carcinogen binding represents only the first step in carcinogenesis because, over periods ranging from weeks to months after detectable carcinogen has disappeared (41) and several liver-cell generations later, abnormal hyperplastic areas, nodules, and finally tumors appear. This indicates that a disturbance was introduced into some of the liver cells which was “remembered” long after the carcinogen had dis- appeared and that it had been passed on to the new generations of liver cells.
It has been shown in studies of protein synthesis that labeled amino acids are incorporated early in the microsomal fraction of rat liver (42-44). The bound carcinogen is likewise found principally in the ribonucleo- protein containing microsomal fraction of the liver cells (35, 45, 46). The work of the Millers (33) implies that the carcinogen may be actually incorporated into the newly formed protein and that as a result an ab- normal “protein” is formed. If the carcinogen is bound into the ribo- nucleoproteins it can serve as a template for the subsequent formation of abnormal protein devoid of carcinogen. This nucleoprotein, known to be capable of self-replication, provides an excellent “memory” apparatus for the continued formation of abnormal protein from a template previously made from carcinogen-containing protein (47, 48). Further, the studies of Stent (49) suggest that a single modified strand of ribonucleoprotein may serve as a template for the formation of new deoxyribonucleoprotein. This provides a method of deoxyribonucleoprotein alteration and produc- tion of a heritable induced defect simply by alteration of a single strand of ribonucleoprotein by the carcinogen.
One is readily reminded of the analogy of this hypothesis to the action of viruses which induce tumors in animals and become intimately involved in the deoxyribonucleoprotein and ribonucleoprotein synthetic processes of cells. The viral genome perpetuates itself by substituting itself for the normal nucleoprotein of the same kind and abnormal protein is thereby synthesized (50).
One might also suggest that various types of radiation may act in a similar manner on the heritable nucleoprotein of the cells in producing
the abnormal nucleoprotein which at some later time becomes manifest in an uncontrolled, rapidly growing colony of cells we call cancer.
APPENDIX
Scoring Procedure
To represent adequately the differences between the various groups of animals, it was necessary to attempt conversion of the several path- ological changes to some numerical value. No such arbitrary conversion could hope to satisfy even a majority of investigators, but an educated relative evaluation is probably better than simple incidence figures when so many other parameters are available. The general formula can be written (A + B + C) D = score per animal.
A. The following values were assigned to the various lesions that devel- oped with increasing weight according to the importance of the lesions. Only the most advanced lesion was used in scoring this section.
A = Most advanced lesion
| Number of well-developed hepatomas | ||||||
|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | >4 | |
| Normal liver | 0 | |||||
| Foci or areas of hyperplasia | 5 | |||||
| Nodules of hyperplasia | 10 | |||||
| Small hepatoma | 25 | |||||
| Well-developed hepatoma | 50 | 60 | 70 | 80 | 100 | |
B. To the figure thus arrived at was added an additional sum for metas- tases. As can be seen, the premium awarded for metastases reflects the authors’ healthy respect for this aspect of malignant growths.
B = Metastases
| None | 0 |
| Rare | 100 |
| Few | 150 |
| Many | 200 |
| Massive | 250 |
C. Then a product derived from the weight of the liver in grams times one plus the excess diameter over 1 cm of the largest tumor was divided by 2 and the quotient added to the subtotal of the above. This takes into account the total mass of tissue in the liver and gives special emphasis to tumors more than 1 cm in diameter. It also diminishes the importance of enlargement due to multiple small lesions including lesions that have
not yet reached a definitely malignant state and returns such enlargement to a more nearly normal weight.
C= Weight of liver X (1 + excess of diameter over 1 cm) ☒ 2
D. Finally, to correct for survival time the total score was multiplied by a survival factor which ranged from 1 for 42 weeks or more to 2 for 25 weeks, with straight-line conversion for all intermediate survival times. All animals not surviving the minimal latent period of 25 weeks were not included in the experiments. All animals surviving more than 42 weeks were included with the 42-week group for calculation of the survival factor.
D = Survival factor = 1 + 42 - number of weeks up to 42 17
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PLATES
PLATE 67
FIGURE 1 .- Cirrhosis. Proliferation of small bile ducts, as well as an increase in larger bile ducts, in portal areas. Periportal liver cells have larger nuclei and basophilic staining cytoplasm. Hematoxylin and eosin. X 154 ☒
FIGURE 2 .- Cirrhosis. Proliferation of small bile ducts extends into adjacent lobules and isolates hepatic parenchymal cells. Liver cells, which have large, darkly staining nuclei and increased cytoplasm, represent foci of hyperplasia. Hematoxylin and eosin. X 207 ☒
1
2
PLATE 68
FIGURE 3 .- Focus of hyperplasia. Groups of cells with larger nuclei, more abundant cytoplasm with basophilic material, and frequently double nuclei. They are not clearly demarcated from the uninvolved surrounding parenchyma. Hematoxylin and cosin. X 207 ☒
FIGURE 4 .- Focus of hyperplasia. Arrows point to a focus of hyperplastic liver cells. Hematoxylin and eosin. X 415 ☒
3
4
PLATE 69
FIGURE 5 .- Areas of hyperplasia. Portal areas near top and to right. Adjacent to the portal areas there is an increased number of cells, which are small, closely packed, and contain basophilic material in the cytoplasm. Similar periportal hyperplasia can be seen extending up from the bottom on right. Hematoxylin and eosin. × 156 ☒
FIGURE 6 .- Area of hyperplasia. The focus of hyperplasia increases in size until it becomes an area which is well demarcated from the surrounding liver. Area of hyperplasia occupies upper right corner of photomicrograph. Hematoxylin and eosin. X 207 ☒
5
6
PLATE 70
FIGURE 7 .- - Nodule of hyperplasia. Hepatic cords are less orderly and adjacent hepatic parenchyma is compressed and distorted by spherical growth. Hematoxylin and eosin. X 154 ☒
FIGURE 8 .— Nodule of hyperplasia. There is less well-defined cord structure compared to figure 7. Hematoxylin and eosin. X 207 ☒
7
8
PLATE 71
FIGURE 9 .- Nodule of hyperplasia with atypicality. Cord structure is distorted and irregular. Hyperplastic cells vary in size and nuclei tend to be larger and more vesicular and sometimes vary in staining. There is compression of surrounding liver tissue. Hematoxylin and eosin. X 154 ☒
FIGURE 10 .- Small hepatoma. Cells are large and atypical with variability in size, shape, and staining. Nuclei are large and vesicular with prominent enlarged nucleoli. There are mitotic figures (not shown) and foci of degeneration and necrosis. Adjacent parenchyma is compressed and distorted. Hematoxylin and eosin. × 154 ☒
9
10
PLATE 72
FIGURE 11 .- Well-differentiated hepatocellular carcinoma with invasion of the portal vein. Tumor cells resemble hepatic parenchymal cells with large round or oval nuclei, prominent nucleoli, marginal chromatin lining the nuclear membrane, and abundant eosinophilie or, at times, basophilic cytoplasm. Hematoxylin and eosin. X 207
FIGURE 12 .- Well-differentiated hepatocellular carcinoma. In center the tumor is ar- ranged in cords. On bottom the cells are arranged in islands and trabeculae. Hema- toxylin and eosin. X 154 ☒
FIGURE 13. - Well-differentiated hepatocellular carcinoma, metastatic to the lung. Tumor cells resemble liver cells. Hematoxylin and cosin. X 154 ☒
11
12
13
PLATE 73
FIGURE 14 .- Cholangiomatous area of cholangiohepatoma. Columnar bile-duct epithelial cells with pale cytoplasm, scalloped borders, and basal nuclei without nucleoli form ductlike structures. Hematoxylin and eosin. X 207 ☒
FIGURE 15 .- Anaplastic hepatocellular carcinoma. Cells at top and center have varying sizes and shapes. Their nuclei are crowded and vary in size and staining. The cytoplasm is basophilic. Hematoxylin and eosin. X 207 ☒
14
15
FIGURE 16 .— Intact male. A tan, umbilicated hepatocellular carcinoma 2 X 3 cm in diameter in the median lobe of the liver. There are also many cysts, as well as cirrhosis. Liver weighed 19,6 g.
FIGURE 17 .- Castrated male. A dark, purple, smooth hepatocellular carcinoma 1.5 X 2.5 cm in diameter also in the median lobe. There are few cysts, and cirrhosis of the liver is absent. Liver weighed 17.6 g. This was the largest tumor in the castrated group.
FIGURE 18 .- Intact female receiving testosterone. A 1 cm gray-white hepatocellular carcinoma in median lobe of liver. There are both cirrhosis and multiple small cysts. This is similar to the findings in females given norethandrolone and to those in intact males receiving norethan- drolone or testosterone, except that the tumors in all of these latter groups were slightly larger. Liver weighed 14.5 g.
FIGURE 19 .- - Intact female. Liver surface is smooth with only a few scattered small cysts. Livers of castrated females, castrated and adrenal- ectomized females, and of adrenalectomized females were similar. Liver weighed 9.9 g.
16
17
18
19
FIGURE 20 .- Adrenalectomized male. Large areas of liver are replaced by gray-white hepatocellular carcinoma. Tumor mass on right measures 6.5 cm in greatest dimension. Centrally there is necrosis and hemorrhage. There are scattered small cysts and cirrhosis is present but not well shown. Liver weighed 64.5 g. There were similar findings for adrenalectomized females given testosterone and castrated males given testosterone.
FIGURE 21 .- Adrenalectomized male. Innumerable metastases replacing most of both lungs.
FIGURE 22 .- Adrenalectomized male given deoxycorticosterone. Surfaces are smooth and there are no cysts. Liver resembles that of normal control rats. Liver weighed 8.7 g.
FIGURE 23 .- Castrated male given norethandrolone. Three main tumor masses; largest of the two shown measures 2.5 cm in diameter. Cirrhosis and cysts are present. Liver weighed 48.6 g. This is also representative of adrenalectomized, castrated females given testosterone and adrenalectomized males receiving cortisone (2 mg dose).
FIRMINGER AND REUBER
20
21
22
23