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Some aspects of the development, biology and biochemistry of rat hepatomas of different growth rate
SOME ASPECTS OF THE DEVELOPMENT, BIOLOGY AND BIOCHEMISTRY OF RAT HEPATOMAS OF DIFFERENT GROWTH RATE HAROLD P. MORRIS,* HELEN M. DYER,* BILLIE P. WAGNER,* HEIDIKE
MIYAJI~
and MILOSLAV
RECHCIGL,
JR*
Laboratory of Biochemistry*and Laboratory of Pathology,t National CancerInstitute, National InstitutesHealth, Public Health Service,U.S. Departmentof Health, EducationandWelfare,Bethesda,Md. INTRODUCTION TRANSPLANTABLEhepatomas
varying from many to few deviations from normal liver have been developed following the ingestion of several different aromatic amines. Those with few deviations from liver have been termed “minimal deviation” hepatomas by Potter.(r) Some of the experimental conditions used in producing hepatomas of different growth rate include the dietary level and type of carcinogen, type of diet, conditions of ingestion, the total intake of carcinogen, and length of time after withdrawal of the carcinogen till the transplantation of the tumor. The role these various factors play in the carcinogenic process has not yet been determined. This report presents data on the biological characteristics of several transplantable rat hepatomas of different growth rate, the effect of the induction of tryptophan pyrrolase in the intact tumor bearing rat, the influence of ethionine injections on lipid content of liver and tumor, and the catalase activities found in a series of transplantable hepatomas. MATERIALS
AND METHODS
One tumor not of the minimal deviation type developed after ingestion of a protein-free diet containing 0.05 per cent, a high level, of N-2fluorenyldiacetamide [also called 2-diacetylaminofluorene] (ZFdiAA). The diet was given continuously for 33 days to male rats of the ACI/N inbred strain. The carcinogen was withdrawn after 33 days and the rats kept on the protein-free diet for 10 more days. Rat No. 3683 in this experiment was sacrificed 144 months after resumption of a diet containing 12 per cent casein, but free of carcinogen. A rapidly growing poorly differentiated anaplastic carcinoma No. 3683 resulted. The rat ingested 118 mg of 321
322
H. P. MORRIS
et d.
2-FdiAA. Another similar type hepatoma, No. 3924A, was induced by 2Flu in a castrated female ACI/N strain rat previously injected with a cholesterol pellet containing testosterone. This rat ingested 321 mg of 2-FdiAA during continuous feeding for 113 days. The hepatoma from rat No. 3924 was first transplanted 7.5 months after the last ingestion of carcinogen. Hepatomas 3683 and 3924A are rapidly growing tumors and have many biochemical deviations from normal liver, as described by Potter et aL(s) and therefore are not of the minimal deviation type. Of the latter type, the Reuber H3Ys) hepatoma was also induced by using the same carcinogen in an AC1 strain male rat. The carcinogenic diet was fed for four Cweek periods with 1 week without the carcinogen between each 4-week period. This rat was sacrificed 10 months after the last ingestion of the carcinogen. We have estimated that approximately 240 mg of 2FdiAA was ingested. Nine hepatomas including 5123 and 7800 were induced in female and male Buffalo-strain rats respectively, after ingestion of a diet containing N-2-fluorenylphthalamic acid (2-FPA) for a period of approximately 10 months. Hepatoma 5123 was first transplanted 8 months and hepatoma 7800, 1 month after the last intake of 2-FPA. Hepatomas 7316A and B were induced from different nodules in the same liver in a Buffalo-strain female rat after continuous ingestion of a diet containing 2,4,6-trimethylaniline (TMA) for 18 months. 844 mg of TMA was ingested.@) Wet4) consider 2-FdiAA to be rapidly acting while 2-FPA and TMA are more slowly acting liver carcinogens. The amount and rate of ingestion of the rapidly acting carcinogen 2-FdiAA appears to be important during the induction of transplantable hepatomas having rapid growth rates with many enzymic deviations from normal liver, whereas intermittent ingestion of lower levels of 2-FdiAA, but not in total amount ingested, has produced the “minimal deviation” type tumor as in H35.@) Slowly acting carcinogens, on the other hand, can be ingested for longer periods than the rapidly acting carcinogens. Some can be ingested during the entire experimental period up to 18 months as with TMA, and still induce transplantable hepatomas of the “minimal deviation” type.
RESULTS
Table 1 presents data on a series of transplantable “minimal deviation” type hepatomas which have been induced in male and female Buffalostrain rats by the ingestion of a diet containing 0.042 per cent 2-FPA for approximately 10 months. All the tumors, with one possible exception, number 7797, induced by this chemical have been of the “minimal deviation” type.
RAT
HEPATOMAS
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RATE
323
Metastases to the lung occur from most of the primary tumors as shown in the table and are frequently found in host lungs of rats bearing the transplantable tumors. Several months (l-144) may elapse after removing the carcinogen from the diet before the hepatoma develops to a size recognized grossly as a visible nodule, or nodules, in the liver or threatens the life of the host. This long delay may not be essential, however, in the carcinogenic process. TABLE
Induction of Minimal N-2-fluorenylphthalamic Amount ingested
Tumor and sex
Average daily intake
mg
5123 7777 7787 7793b
F F
lrf2g3 1203
;
1183 1215
3.7 4.1 4.0 4.1
7794A and B 7795b 7797 7800
M
1539 1448 1454 1387
5.2 4.9 4.9 4.7
M iti
1
Deviation Hepatomas by Ingestion of Acid for Approximately 10 Months Time without carcinogen
Histologic trpe
mos. 8.1 4.0 3.6 4.5
a a a
3.2 2.3
a a
8Trabecular carcinoma-well differentiated. Wertain sublines during transplantation showed partially a few cells which resemble cholangio cells. CCholangiocarcinoma with bony metaplasia.
Metastases
a
C
a
mucin secreting cells, and
The growth of several hepatomas is illustrated in Fig. 1. A series of growth curves of several hepatomas was obtained by plotting the averaged sums of two caliper measurements of the tumors against time after inoculation. The vertical lines represent the standard error of the mean. Four sublines, A, B, C and D, of hepatoma 5123 were arbitrarily established in the 16th transfer generation. All the tumors described grow more rapidly in male than in female hosts. Hepatoma 5123 subline A in male rats appears to be somewhat larger than does hepatoma 5123 subline D. Hepatoma 7800 grows more slowly than hepatoma 5123. Hepatomas 7316A, 7787 and 7794B, not shown in the chart, grow even more slowly than any of the tumors shown. The Reuber hepatoma, H35,@) appears to have about the same growth rate as hepatoma 7800. This is illustrated in Fig. 2 by the 4 bars on the right side. The tumor measurements illustrated here in this figure were all made on subcutaneous tumors 1.4 months after inoculation.
324
H. P. MORRIS et a/. MEASUREMENTS OF TUMORS NO.3663, 5123A,5123D, AND 7600 IN MALES AND FEMALES 6.0 , I I I I I I
7 7.5 e 7.0 ?I2 0
z
-d
-----
3683
?
6.5 5123 D
6.0 5.5
E E 6
5.0
2
4.0
4.5 3.5
22 Ib
3.0
2
2.0
2.5 1.5 I .o
TIME AFTER
30 40 INOCULATION
50 60 (Days)
FIG. 1 Measurements of the size of transplantable hepatomas of different growth rate. The four digit numbers indicate the hepatoma line, the verticle lines the standard error of the mean. 5123 sublines A and D, and 7800 are “minimal deviation” type tumors. COMPARISON OFAVERAGE SIZEf f SUBCUTANEOUS TUMORS 1.4 MONTHS AFTER INOCULATION
TUMOR LINES
FIG. 2 Comparison of the average size (sum of two caliper measurements) of four 5123 sublines, 7800, and H35 hepatomas made 1.4 months after inoculation.
RAT HEPATOMAS
OF DIFFERENT
GROWTH
RATE
325
The numbers over the bars represent the age of the host at the time of inoculation. The upper of the two numbers at the base of each column is the per cent of measurable tumors at 1.4 months and the lower number in the column is the number of tumors measurable at that time. The low percentage means that the very slow growing tumors had not yet reached a measurable size at 1.4 months. The H35 tumors illustrated in the 2 bars on the extreme right were in male and female ACI/N strain rats. All other tumor lines were grown in Buffalo-strain or the F1 hybrid produced by crossing the Buffalo with the inbred Lewis strain (designated LBFl). Tumors are slightly slower in developing in the Fl hybrid and the number of takes is considerably lower than in Buffalo strain rats. Therefore, we have discontinued the use of hybrids. The tumors grow faster when inoculated in rats 1 month of age than in rats 3 months old or older. 5123 t.c. came from the 5123C tumor line grown in tissue culture at McArdle Laboratory and put back in the animal. Growth of 5123 t.c. is somewhat faster than any of the other four 5123 sublines. Extensive studies in many laboratories have been carried out during the last 4 years in attempts to gain an understanding of the lesions or defects which will characterize these recently developed hepatomas as malignant neoplasms. The availability of a large group of transplantable hepatomas developed in our laboratory, we believe, has greatly facilitated this quest which appears to expand in size and complexity as the results of each new experiment become available. TRYPTOPHAN
PYRROLASE
A few of the recent studies completed in our laboratory will now be presented. A survey of the tryptophan pyrrolase (TP) activity before and after induction by L-tryptophan (LT) in intact tumor bearing rats has recently been completed by Dyer et a1.(s) The induction of TP in host liver and hepatoma 5123 growing in “tissue isolated” kidneys has been studied. L-tryptophan was introduced into the kidney artery where it was carried directly into the tumor before it reached the systematic circulation and the host liver. This route of administration resulted in no greater TP activity in either the tumor or the host liver than occurred following intraperitoneal administration of L-tryptophan. This observation appeared to rule out insufficient inducer reaching the tumor via the bloodstream as an explanation for less or no induction of TP in some “minimal deviation” hepatomas compared to host liver (cf. Refs. 7, 8, 9). TP activity in the homogenates of livers of intact tumor bearing rats and 14 hepatomas was then examined by a modification of the kinetic method of Greengard and Feigelson.(lo) with an without the intraperitoneal administration of L-T as inducer. The host livers of rats bearing the various lines of hepatomas always showed greater TP activity after induction although there were 22
326
H. P. MORRIS et al.
variations among individual animals in the absolute amounts of activity both before and after substrate induction. Kidney-transplanted 5123 hepatomas, however, failed to show any increase in TP activity by LT after either intraperitoneal or renal artery routes of administration. Hepatoma H35,(s) on the other hand, transplanted either to the exteriorized kidney or subcutaneously showed increased TP activity after intraperitoneal LT administration. The hepatomas investigated could be divided into 3 (Fig. 3) groups with respect to substrate induction of TP activity. Group 1 included hepatomas in which the TP activity produced less than 1 pm kynurenine/hr/g dry wt., there was no progressive increase in TP with increasing time of incubation of the homegenate in this group, and an erratic response often occurred in the tumor with and without administration of LT to the host. It was questionable whether these hepatomas comprising half the entire group studied had TP activity. All 5123 hepatomas with one exception were in this first group. 5123C showed sufficient activity to place it in Group 2. Group 2 included hepatomas that contained small amounts of TP activity that increased during incubation, and was significantly greater after LT administration to the host. Group 3 included hepatomas with more TP activity than those of Group 2 and occasionally as much as host liver. These hepatomas showed increased TP activity after substrate induction (see Fig. 3). There were no recognizable morphological significant differences between the tumors in the 3 groups or in earlier generations of some of these tumors. All the tumors consisted of liver cells usually with no cholangiomatous areas. The studies of Dyer et aZ.(s) may point to an effect of higher TP activity as having restraining effect on the growth of the tumor. This is not entirely clear as hepatoma 7800 is equivocal for TP while H35 t.c., a faster growing tumor than 7800, is positive for TP. The fact that individual tumor lines were consistent in their TP behavior supports the view that the cause of the variation is inherent in the individual tumors. These data for hepatoma 5123 largely con&m reports by Pitot and his co-workers(r~s~sJr) that TP activity in hepatoma 5123 was not significantly altered in response to intraperitoneal administration of LT to the host, and that the failure of TP induction in this hepatoma was the result of some intrinsic defect within the neoplastic cell itself. Several hepatomas studied by both Dyer et a/.(s) and Pitot et ~1.(7~s~aJr) gave only a slight response to induction of TP by LT in the intact tumor bearing animal. Cho et a/.,(11) however, find no response of the hepatomas to TP induction in adrenalectomized (adx) hosts, although induction of TP occurs in adx non-tumor bearing rats (Lee and Baltzua)). Administration of cortisone (Cho et a/.(11)) to adx tumor bearing rats, however, restored TP induction of LT. This observation suggests the dependence of substrate induction on the presence of corticosteroids in these hepatomas.
c.-
g-
%
/p
H3sl.C.O li3sgen 118 5123 c 7316 A
,UMOLES
7764 7795
8
KYNURENINE/HR/GM.
DRY
(
7793
73168
5123
D
WEIGHT
H. P. MORRIS t?t al.
328
2-fold increase in ether extractable lipid following ethionine injections. It is noteworthy that the difference in hepatoma lipid increase following ethionine injections in the two positive hepatoma lines was proportionally somewhat less than the response found in the host liver, and that only two of the five hepatomas tested were responsive to treatment. Studies on the effect of various hormonal treatments of the host on the response of the tumor to ethionine injections are now in progress. TABLE
Effect of Ethionine
Tumor Lines Ethionineinduced Hep.* H35t 5123 D$ 7316 A§ 1193$
I
2
Injections on the Lipid Content of Liver and Tumor
Gen. used Gem 17 Gen. 11 Gen. 33 Gen. 7 Gen. 5
Liver Control 7.8 2 1.9 (3) 5.0 f 0.4 (3) 7.9 f 0.4 (3) 6.8 + 0.3 (3) 8.3 + 1.0 (3)
Tumor Ethionine
26.7kO.3 (3) 16.5+1.9(4) 18.9+ 1.8 (3) 16.3 f 1.2 (3) 18.4+ 1.4 (4)
Control 4.3kO.l 2.5kO.l 2.2kO.5 2.5kO.3 4.6kO.l
Ethionine (3) (3) (3) (3) (3)
3.4kO.2 5.5+0.1 2.OkO.4 4.6F0.2 4.1kO.4
(3) (4) (3) (3) (4)
( )Number of rats. *Ethionine-induced hepatoma, Sidransky, J.N.C.Z. 28, 14:25-1435 (1962). tFdiAA-induced hepatoma. $2FPA-induced hepatoma. §TMA-induced hepatoma.
Hepatoma 7793 which showed the greatest response to TP induction was unresponsive to ethionine injections. The two hepatomas responsive to lipid increase following ethionine injections appeared in group 2 of TP induction studies(s) yet TP induction in these hepatomas appears to be adrenal hormone dependent,(il) because TP induction in the hepatoma by substrate does not occur in the adx rat.(ii) Fatty hepatomas produced by ethionine, therefore, do not seem to be mediated by the same mechanism as TP induction. CATALASE
Nearly a decade ago in the comprehensive review of the Biochemistry the view was expressed that tumor tissue in general had no or negligible amounts of catalase. The development of “minimal deviation” hepatomasc k3~17) with many enzyme systems having quite similar activities to those found in normal rat liveros) indicated to us the need for a reappraisal of catalase activity in a number of the newer type hepatomas. Such studies were also stimulated by the isolation of two ethionme-induced trabecular hepatocellular carcinomas with greatly different
of Cancer (cf. Greenstein
RAT HEPATOMAS
OF DIFFERENT
GROWTH
RATE
329
catalase activities by Rechcigl and Sidransky.(rs) These two lines were established in the first transplant generation. The high catalase line (HC) has catalase activities higher than the catalase activity in normal liver. The low line (LC) has values about 0.1 that found in normal liver. The (HC and LC!) lines are indistinguishable morphologically and have many other enzyme activities in the same range as found for normal liver.(rg) These two lines differed in catalase activity from that found in hepatoma 5123.(is,so) The preliminary examination of catalase activity of a large group of transplantable rat liver tumors shows that three rapidly growing tumors (Novikoff, 3683, and 3924A) have essentially no catalase activity (Fig. 4).
Catalase activities in a large series of transplantable hepatomas of different growth rate. Lines, only, indicate single measurements. The bar at the end of a line indicates range of values obtained for that tumor. In addition, for hepatoma 5123 an average value with its standard error is given.
Considerable catalase activity was found in each of five sublines of 5123, in H35, 7787 the primary, 2nd and 3rd transplant generations: in 7793, and in the two sublines of 7794 (A and B) where considerably higher values were noted in three generations available for analyses. From the values found in these three generations of 7794B it seems possible that the catalase activity of this very slowly growing tumor may be increasing. The HC and LC lines of the ethionine-induced hepatoma are indicated near the
330
H. P. MORRIS
et d.
bottom of Fig. 4.(lQ) These two lines have retained their low and high levels of catalase activity in subsequent transfers. The host liver, of animals bearing the high-catalase activity line, shows depressed catalase activity during growth of the tumor whereas the tumor catalase does not show such depression (Table 3, Rechcigl et aZ.(lQ)). The total catalase units per host liver and variable sizes of hepatoma 5123 have been plotted in Fig. 5.(sQ) It is quite clear from these data that as tumor weight increases the total catalase increases in the tumor but decreases in the host liver. No change has been found in 5123 hepatoma catalase activity of either male or female rats varying in age from 3 to 11 months (Table 3). The TABLE
3
Effect of Sex and Age on Cat&se Activity of Liver, Kidney and Hepatoma 5123(20) Catalase activity
Sex
Age months
ii FM
35 9 3 11 4
FF
Liver P/g
Kidney r/g
Tumor P/f2
177+ 76 137f 195+ 6 116fll 140+ 37 130&
49+2 47e2 47+1 32+5 29&2 30+1
42+3 47+6 47k3 40+2 3752 41f2
Difference in catalase between sexes P = 0.01.
catalase activity values for host liver and kidney of males, however, was significantly higher than for females. The host liver catalase of older male and female rats was somewhat higher than for animals 3 months of age, but host kidney catalase in these studies was not affected by age of the rat. Rechcigl and Pricec2r) have studied factors affecting synthesis and destruction of liver catalase in rats. They have noted a lowering of liver catalase of normal animals on a protein-free diet. A comparison of host liver and hepatoma 5123 catalase activities during a 7.day period when the animals were on a protein free diet is illustrated in Fig. 6.(QQ)Small tumors were selected in this experiment because of the depressing e&t of large tumors on host liver catalase. The data in this figure show no effect on tumor catalase activity per g of tissue, but a striking lowering of liver catalase in hepatoma 5123-bearing rats which was comparable to that of normal animals on a protein-free diet. The decrease of total catalase in starvation is proportional to the decrease in liver size according to Rechcigl and Price.(QQ) They show further
bw
HEP.~TOMAS 0~ DIFF~~
GROWTH k4-rE
331
4
NORMAL
LIVER
t
I-
l
.
1
200 0 0
2
4
6
8
IO
12
14
I8
TUMOR WEIGHT, GM. FIG. 5 Tissue catalase activity of Buffalo-strain female rats bearing hepatoma 5123. T.B. liver refers to the liver of the tumor bearing host.
180
20 0
I
I
I
0
I DAYS
2
I
I
3
4
ON PROTEIN-FREE
I
5
6
7
DIET
FIG. 6 The catalase activity expressed as units per g of tissue in normal female rats, rats bearing hepatoma 5123, and in hepatoma 5123. All rats were fed a proteinfree diet.
332
H.
P. MORRIS et al.
that in the rat during the first 5 days of starvation the rate of catalase synthesis per gram of liver proceeded as rapidly as in the controls, but because the livers were decreasing in size there was less total catalase being synthesized. It was noted that the rate of catalase per g of liver starts to drop after 5 days of starvation, and continues to go down until the death of the animal. The liver catalase of rats on a protein free diet had a decreased rate of synthesis per g of liver, although there was a relative small decrease in liver size, and the fraction of catalase molecules being destroyed per unit time was in the normal range. These two changes, therefore, resulted in a lowered catalase activity within the liver. It is well known that tumor bearing animals suffer from hypoproteinemia. The effect of a tumor on liver catalase may be mediated through a deficiency in one or more amino’ acids required for catalase synthesis.@) Because the tumor catalase noted above was unaffected by a protein-free diet the tumor may, therefore, have a preferential claim on available metabolic units needed for synthesising catalase. We are still unable to explain the development of hepatomas of variable catalase activity. It is not always related to growth rate of the tumor because the two slowest growing hepatomas in our group of tumors, 7787 and 7794B, have greatly different catalase activity values, but both have similar growth rates. SUMMARY
The results of studies on TP induction, catalase activities, growth studies, and ethionine injections confirm the accumulated evidence from many laboratories that individual transplantable hepatomas, even of the “minimal deviation” type, show wide variation in their enzyme activities, even when the original tumors are induced by the same chemical and under as similar experimental conditions as can be provided. Transplantable hepatomas of widely differing biological and biochemical characteristics have also been produced in different lobes of the same liver of the primary host. The induction of transplantable hepatomas of the “minimal deviation” type has been accomplished by several chemical carcinogens. In case of N-2-3uorenylphthalmic acid, tumors have been induced in rats of both sexes in two entirely separate experiments. Therefore, it is concluded that the first “minimal deviation” hepatoma observed, 5123, is not unique. REFERENCES
1. VAN R. POTTER, Transplantable cancer, the primary standard: Guest editorial, Cancer Research 21, 1331-1333 (1961). 2. VAN R. POTER, H. PITOT, T. ONO and H. P. MORRIS, The comparative enzymology and cell of origin of rat hepatomas. I. Deoxycytidylate deaminase and thymidine degradation, Cancer Research 20, 1255-1261 (1960). 3. M. D. REUBER, A transplantable bile-secreting hepatocellular carcinoma in the rat, J. Nuf. Cancer Inst. 26, 891-897 (1961).
RAT
HEPATOhfAS
OF DIFFERENT
GROWTH
RATE
333
4. H. P. MORRIS and B. P. WAGNER, The development of minimal deviation hepatomas, Proc. VZZZZnt’l. Cancer Congress, Moscow, USSR, July 22-28 (1962). In press. 5. H. M. DYER, P. GIJLLINO and H. P. MORRIS, Tryptophan pyrrolase activity in transplanted “minimal deviation” type hepatomas, Cancer Research 24, 97-104 (1964). 6. P. GULLINO and F. H. GRANTHAM, A method for growing “tissue-isolated” tumors in laboratory animals, J. Natl. Cancer Inst. 27, 679-693 (1961). 7. H. C. PITOT, Biochemical lesions in minimal deviation hepatomas, Proc. VZZZ Znf’l Cancer Congress, Moscow, U.S.S.R., July 22-28 (1962). In press. 8. H. C. PITOT and Y. S. CHO, Studies on the mechanism of enzyme induction in rat liver, Cold Spring Harbor Symposium on Quant. Biol. 26, 371-377 (1961). 9. H. C. PITOT and H. P. MORRIS, Metabolic adaptations in rat hepatomas. II. Tryptophan pyrrolase and tyrosine a-ketoglutarate transaminase, Cancer Research 21, 1009-1014 (1961). 10. 0. GREENGARD and P. FEIGELSON, The activation and induction of rat liver tcyptophan pyrrolase in vivo by its substrate, J. Biol. Chem. 236, 158-161 (1961). 11. Y. S. CHO, H. PITOT and H. P. MORRIS, Metabolic adaptations in rat hepatomas. VI. Substrate-hormone relationships during tryptophan pyrrolase induction in liver and hepatomas, Cancer Research 24,52-58 (1964). 12. N. C. LEE and B. E. BALTZ, Tryptophan pyrrolase induction and the influence of adrenocorticoids, Endocrinology 70, 84-87 (1962). 13. E. FARBER, M. V. SIMPSON and H. TARVER, Studies on ethionine. II. The interference with lipide metabolism, J. Biol. Chem. 182, 91-99 (1950). 14. D. JENSEN, I. L. CHAIKOFF and H. TARVER, The ethionine induced fatty liver: Dosage, prevention, and structural specificity, J. Biol. Chem. 192, 395-403 (1951). 15. E. FARBER and A. SEGOLOFF,Effect of androgens and growth and other hormones in ethionine fatty liver in rats, J. Biol. Chem. 216, 471-477 (1955). 16. J. P. GREENSTEIN, Biochemistry of Cancer; 2nd Ed., Academic Press, New York (1954). 17. H. P. MORRIS, H. SIDRANSKY, B. P. WAGNER and H. M. DYER, Some characteristics of rat hepatoma 5123 induced by ingestion of N-(2-fluorenyl)phthalamic acid, Cancer Research 20, 1252-1254 (1960). 18. H. P. MORRIS, Some growth, morphological, and biochemical characteristics of hepatoma 5123 and other new transplantable hepatomas. In Prog. Exp. Tumor Res. Vol. 3, 370-411 (Karger, Basel/New York) (1963). 19. M. RECHCIGL, JR. and H. SIDRANSKY, Isolation of two lines of transplantable ethionine-induced rat hepatomas of high and low catalase activity from a primary tumor, J. Nut. Cancer Inst. 28, 1411-1423 (1962). 20. M. RECHCIGL, JR., V. E. PUCE and H. P. MORRIS, Studies on the cachexia of tumorbearing animals. II. Catalase activity in the tissues of hepatoma-bearing animals, Cancer Research 22, 874-880 (1962). 21. M. RECHCIGL, JR. and V. E. PRICE, Catalase synthesis and destruction in starvation, Experientia 17, 258-261 (1961). 22. M. RECHCIGL, JR. and V. E. PRICE, The rates and kinetics of enzyme formation and destruction in the living animal. In New Methods of Nutritional Biochem. Vol. 1, 185-197 (1963). 23. M. RECHCIGL, JR., Nutritional factors in the etiology of catalase depression of the tumor-bearing host, Proc. VI International Congress of Nutrition. Edinburgh, August 9-15 (1963).
MIYAJI~
and MILOSLAV
RECHCIGL,
JR*
Laboratory of Biochemistry*and Laboratory of Pathology,t National CancerInstitute, National InstitutesHealth, Public Health Service,U.S. Departmentof Health, EducationandWelfare,Bethesda,Md. INTRODUCTION TRANSPLANTABLEhepatomas
varying from many to few deviations from normal liver have been developed following the ingestion of several different aromatic amines. Those with few deviations from liver have been termed “minimal deviation” hepatomas by Potter.(r) Some of the experimental conditions used in producing hepatomas of different growth rate include the dietary level and type of carcinogen, type of diet, conditions of ingestion, the total intake of carcinogen, and length of time after withdrawal of the carcinogen till the transplantation of the tumor. The role these various factors play in the carcinogenic process has not yet been determined. This report presents data on the biological characteristics of several transplantable rat hepatomas of different growth rate, the effect of the induction of tryptophan pyrrolase in the intact tumor bearing rat, the influence of ethionine injections on lipid content of liver and tumor, and the catalase activities found in a series of transplantable hepatomas. MATERIALS
AND METHODS
One tumor not of the minimal deviation type developed after ingestion of a protein-free diet containing 0.05 per cent, a high level, of N-2fluorenyldiacetamide [also called 2-diacetylaminofluorene] (ZFdiAA). The diet was given continuously for 33 days to male rats of the ACI/N inbred strain. The carcinogen was withdrawn after 33 days and the rats kept on the protein-free diet for 10 more days. Rat No. 3683 in this experiment was sacrificed 144 months after resumption of a diet containing 12 per cent casein, but free of carcinogen. A rapidly growing poorly differentiated anaplastic carcinoma No. 3683 resulted. The rat ingested 118 mg of 321
322
H. P. MORRIS
et d.
2-FdiAA. Another similar type hepatoma, No. 3924A, was induced by 2Flu in a castrated female ACI/N strain rat previously injected with a cholesterol pellet containing testosterone. This rat ingested 321 mg of 2-FdiAA during continuous feeding for 113 days. The hepatoma from rat No. 3924 was first transplanted 7.5 months after the last ingestion of carcinogen. Hepatomas 3683 and 3924A are rapidly growing tumors and have many biochemical deviations from normal liver, as described by Potter et aL(s) and therefore are not of the minimal deviation type. Of the latter type, the Reuber H3Ys) hepatoma was also induced by using the same carcinogen in an AC1 strain male rat. The carcinogenic diet was fed for four Cweek periods with 1 week without the carcinogen between each 4-week period. This rat was sacrificed 10 months after the last ingestion of the carcinogen. We have estimated that approximately 240 mg of 2FdiAA was ingested. Nine hepatomas including 5123 and 7800 were induced in female and male Buffalo-strain rats respectively, after ingestion of a diet containing N-2-fluorenylphthalamic acid (2-FPA) for a period of approximately 10 months. Hepatoma 5123 was first transplanted 8 months and hepatoma 7800, 1 month after the last intake of 2-FPA. Hepatomas 7316A and B were induced from different nodules in the same liver in a Buffalo-strain female rat after continuous ingestion of a diet containing 2,4,6-trimethylaniline (TMA) for 18 months. 844 mg of TMA was ingested.@) Wet4) consider 2-FdiAA to be rapidly acting while 2-FPA and TMA are more slowly acting liver carcinogens. The amount and rate of ingestion of the rapidly acting carcinogen 2-FdiAA appears to be important during the induction of transplantable hepatomas having rapid growth rates with many enzymic deviations from normal liver, whereas intermittent ingestion of lower levels of 2-FdiAA, but not in total amount ingested, has produced the “minimal deviation” type tumor as in H35.@) Slowly acting carcinogens, on the other hand, can be ingested for longer periods than the rapidly acting carcinogens. Some can be ingested during the entire experimental period up to 18 months as with TMA, and still induce transplantable hepatomas of the “minimal deviation” type.
RESULTS
Table 1 presents data on a series of transplantable “minimal deviation” type hepatomas which have been induced in male and female Buffalostrain rats by the ingestion of a diet containing 0.042 per cent 2-FPA for approximately 10 months. All the tumors, with one possible exception, number 7797, induced by this chemical have been of the “minimal deviation” type.
RAT
HEPATOMAS
OF DIFFERENT
GROWTH
RATE
323
Metastases to the lung occur from most of the primary tumors as shown in the table and are frequently found in host lungs of rats bearing the transplantable tumors. Several months (l-144) may elapse after removing the carcinogen from the diet before the hepatoma develops to a size recognized grossly as a visible nodule, or nodules, in the liver or threatens the life of the host. This long delay may not be essential, however, in the carcinogenic process. TABLE
Induction of Minimal N-2-fluorenylphthalamic Amount ingested
Tumor and sex
Average daily intake
mg
5123 7777 7787 7793b
F F
lrf2g3 1203
;
1183 1215
3.7 4.1 4.0 4.1
7794A and B 7795b 7797 7800
M
1539 1448 1454 1387
5.2 4.9 4.9 4.7
M iti
1
Deviation Hepatomas by Ingestion of Acid for Approximately 10 Months Time without carcinogen
Histologic trpe
mos. 8.1 4.0 3.6 4.5
a a a
3.2 2.3
a a
8Trabecular carcinoma-well differentiated. Wertain sublines during transplantation showed partially a few cells which resemble cholangio cells. CCholangiocarcinoma with bony metaplasia.
Metastases
a
C
a
mucin secreting cells, and
The growth of several hepatomas is illustrated in Fig. 1. A series of growth curves of several hepatomas was obtained by plotting the averaged sums of two caliper measurements of the tumors against time after inoculation. The vertical lines represent the standard error of the mean. Four sublines, A, B, C and D, of hepatoma 5123 were arbitrarily established in the 16th transfer generation. All the tumors described grow more rapidly in male than in female hosts. Hepatoma 5123 subline A in male rats appears to be somewhat larger than does hepatoma 5123 subline D. Hepatoma 7800 grows more slowly than hepatoma 5123. Hepatomas 7316A, 7787 and 7794B, not shown in the chart, grow even more slowly than any of the tumors shown. The Reuber hepatoma, H35,@) appears to have about the same growth rate as hepatoma 7800. This is illustrated in Fig. 2 by the 4 bars on the right side. The tumor measurements illustrated here in this figure were all made on subcutaneous tumors 1.4 months after inoculation.
324
H. P. MORRIS et a/. MEASUREMENTS OF TUMORS NO.3663, 5123A,5123D, AND 7600 IN MALES AND FEMALES 6.0 , I I I I I I
7 7.5 e 7.0 ?I2 0
z
-d
-----
3683
?
6.5 5123 D
6.0 5.5
E E 6
5.0
2
4.0
4.5 3.5
22 Ib
3.0
2
2.0
2.5 1.5 I .o
TIME AFTER
30 40 INOCULATION
50 60 (Days)
FIG. 1 Measurements of the size of transplantable hepatomas of different growth rate. The four digit numbers indicate the hepatoma line, the verticle lines the standard error of the mean. 5123 sublines A and D, and 7800 are “minimal deviation” type tumors. COMPARISON OFAVERAGE SIZEf f SUBCUTANEOUS TUMORS 1.4 MONTHS AFTER INOCULATION
TUMOR LINES
FIG. 2 Comparison of the average size (sum of two caliper measurements) of four 5123 sublines, 7800, and H35 hepatomas made 1.4 months after inoculation.
RAT HEPATOMAS
OF DIFFERENT
GROWTH
RATE
325
The numbers over the bars represent the age of the host at the time of inoculation. The upper of the two numbers at the base of each column is the per cent of measurable tumors at 1.4 months and the lower number in the column is the number of tumors measurable at that time. The low percentage means that the very slow growing tumors had not yet reached a measurable size at 1.4 months. The H35 tumors illustrated in the 2 bars on the extreme right were in male and female ACI/N strain rats. All other tumor lines were grown in Buffalo-strain or the F1 hybrid produced by crossing the Buffalo with the inbred Lewis strain (designated LBFl). Tumors are slightly slower in developing in the Fl hybrid and the number of takes is considerably lower than in Buffalo strain rats. Therefore, we have discontinued the use of hybrids. The tumors grow faster when inoculated in rats 1 month of age than in rats 3 months old or older. 5123 t.c. came from the 5123C tumor line grown in tissue culture at McArdle Laboratory and put back in the animal. Growth of 5123 t.c. is somewhat faster than any of the other four 5123 sublines. Extensive studies in many laboratories have been carried out during the last 4 years in attempts to gain an understanding of the lesions or defects which will characterize these recently developed hepatomas as malignant neoplasms. The availability of a large group of transplantable hepatomas developed in our laboratory, we believe, has greatly facilitated this quest which appears to expand in size and complexity as the results of each new experiment become available. TRYPTOPHAN
PYRROLASE
A few of the recent studies completed in our laboratory will now be presented. A survey of the tryptophan pyrrolase (TP) activity before and after induction by L-tryptophan (LT) in intact tumor bearing rats has recently been completed by Dyer et a1.(s) The induction of TP in host liver and hepatoma 5123 growing in “tissue isolated” kidneys has been studied. L-tryptophan was introduced into the kidney artery where it was carried directly into the tumor before it reached the systematic circulation and the host liver. This route of administration resulted in no greater TP activity in either the tumor or the host liver than occurred following intraperitoneal administration of L-tryptophan. This observation appeared to rule out insufficient inducer reaching the tumor via the bloodstream as an explanation for less or no induction of TP in some “minimal deviation” hepatomas compared to host liver (cf. Refs. 7, 8, 9). TP activity in the homogenates of livers of intact tumor bearing rats and 14 hepatomas was then examined by a modification of the kinetic method of Greengard and Feigelson.(lo) with an without the intraperitoneal administration of L-T as inducer. The host livers of rats bearing the various lines of hepatomas always showed greater TP activity after induction although there were 22
326
H. P. MORRIS et al.
variations among individual animals in the absolute amounts of activity both before and after substrate induction. Kidney-transplanted 5123 hepatomas, however, failed to show any increase in TP activity by LT after either intraperitoneal or renal artery routes of administration. Hepatoma H35,(s) on the other hand, transplanted either to the exteriorized kidney or subcutaneously showed increased TP activity after intraperitoneal LT administration. The hepatomas investigated could be divided into 3 (Fig. 3) groups with respect to substrate induction of TP activity. Group 1 included hepatomas in which the TP activity produced less than 1 pm kynurenine/hr/g dry wt., there was no progressive increase in TP with increasing time of incubation of the homegenate in this group, and an erratic response often occurred in the tumor with and without administration of LT to the host. It was questionable whether these hepatomas comprising half the entire group studied had TP activity. All 5123 hepatomas with one exception were in this first group. 5123C showed sufficient activity to place it in Group 2. Group 2 included hepatomas that contained small amounts of TP activity that increased during incubation, and was significantly greater after LT administration to the host. Group 3 included hepatomas with more TP activity than those of Group 2 and occasionally as much as host liver. These hepatomas showed increased TP activity after substrate induction (see Fig. 3). There were no recognizable morphological significant differences between the tumors in the 3 groups or in earlier generations of some of these tumors. All the tumors consisted of liver cells usually with no cholangiomatous areas. The studies of Dyer et aZ.(s) may point to an effect of higher TP activity as having restraining effect on the growth of the tumor. This is not entirely clear as hepatoma 7800 is equivocal for TP while H35 t.c., a faster growing tumor than 7800, is positive for TP. The fact that individual tumor lines were consistent in their TP behavior supports the view that the cause of the variation is inherent in the individual tumors. These data for hepatoma 5123 largely con&m reports by Pitot and his co-workers(r~s~sJr) that TP activity in hepatoma 5123 was not significantly altered in response to intraperitoneal administration of LT to the host, and that the failure of TP induction in this hepatoma was the result of some intrinsic defect within the neoplastic cell itself. Several hepatomas studied by both Dyer et a/.(s) and Pitot et ~1.(7~s~aJr) gave only a slight response to induction of TP by LT in the intact tumor bearing animal. Cho et a/.,(11) however, find no response of the hepatomas to TP induction in adrenalectomized (adx) hosts, although induction of TP occurs in adx non-tumor bearing rats (Lee and Baltzua)). Administration of cortisone (Cho et a/.(11)) to adx tumor bearing rats, however, restored TP induction of LT. This observation suggests the dependence of substrate induction on the presence of corticosteroids in these hepatomas.
c.-
g-
%
/p
H3sl.C.O li3sgen 118 5123 c 7316 A
,UMOLES
7764 7795
8
KYNURENINE/HR/GM.
DRY
(
7793
73168
5123
D
WEIGHT
H. P. MORRIS t?t al.
328
2-fold increase in ether extractable lipid following ethionine injections. It is noteworthy that the difference in hepatoma lipid increase following ethionine injections in the two positive hepatoma lines was proportionally somewhat less than the response found in the host liver, and that only two of the five hepatomas tested were responsive to treatment. Studies on the effect of various hormonal treatments of the host on the response of the tumor to ethionine injections are now in progress. TABLE
Effect of Ethionine
Tumor Lines Ethionineinduced Hep.* H35t 5123 D$ 7316 A§ 1193$
I
2
Injections on the Lipid Content of Liver and Tumor
Gen. used Gem 17 Gen. 11 Gen. 33 Gen. 7 Gen. 5
Liver Control 7.8 2 1.9 (3) 5.0 f 0.4 (3) 7.9 f 0.4 (3) 6.8 + 0.3 (3) 8.3 + 1.0 (3)
Tumor Ethionine
26.7kO.3 (3) 16.5+1.9(4) 18.9+ 1.8 (3) 16.3 f 1.2 (3) 18.4+ 1.4 (4)
Control 4.3kO.l 2.5kO.l 2.2kO.5 2.5kO.3 4.6kO.l
Ethionine (3) (3) (3) (3) (3)
3.4kO.2 5.5+0.1 2.OkO.4 4.6F0.2 4.1kO.4
(3) (4) (3) (3) (4)
( )Number of rats. *Ethionine-induced hepatoma, Sidransky, J.N.C.Z. 28, 14:25-1435 (1962). tFdiAA-induced hepatoma. $2FPA-induced hepatoma. §TMA-induced hepatoma.
Hepatoma 7793 which showed the greatest response to TP induction was unresponsive to ethionine injections. The two hepatomas responsive to lipid increase following ethionine injections appeared in group 2 of TP induction studies(s) yet TP induction in these hepatomas appears to be adrenal hormone dependent,(il) because TP induction in the hepatoma by substrate does not occur in the adx rat.(ii) Fatty hepatomas produced by ethionine, therefore, do not seem to be mediated by the same mechanism as TP induction. CATALASE
Nearly a decade ago in the comprehensive review of the Biochemistry the view was expressed that tumor tissue in general had no or negligible amounts of catalase. The development of “minimal deviation” hepatomasc k3~17) with many enzyme systems having quite similar activities to those found in normal rat liveros) indicated to us the need for a reappraisal of catalase activity in a number of the newer type hepatomas. Such studies were also stimulated by the isolation of two ethionme-induced trabecular hepatocellular carcinomas with greatly different
of Cancer (cf. Greenstein
RAT HEPATOMAS
OF DIFFERENT
GROWTH
RATE
329
catalase activities by Rechcigl and Sidransky.(rs) These two lines were established in the first transplant generation. The high catalase line (HC) has catalase activities higher than the catalase activity in normal liver. The low line (LC) has values about 0.1 that found in normal liver. The (HC and LC!) lines are indistinguishable morphologically and have many other enzyme activities in the same range as found for normal liver.(rg) These two lines differed in catalase activity from that found in hepatoma 5123.(is,so) The preliminary examination of catalase activity of a large group of transplantable rat liver tumors shows that three rapidly growing tumors (Novikoff, 3683, and 3924A) have essentially no catalase activity (Fig. 4).
Catalase activities in a large series of transplantable hepatomas of different growth rate. Lines, only, indicate single measurements. The bar at the end of a line indicates range of values obtained for that tumor. In addition, for hepatoma 5123 an average value with its standard error is given.
Considerable catalase activity was found in each of five sublines of 5123, in H35, 7787 the primary, 2nd and 3rd transplant generations: in 7793, and in the two sublines of 7794 (A and B) where considerably higher values were noted in three generations available for analyses. From the values found in these three generations of 7794B it seems possible that the catalase activity of this very slowly growing tumor may be increasing. The HC and LC lines of the ethionine-induced hepatoma are indicated near the
330
H. P. MORRIS
et d.
bottom of Fig. 4.(lQ) These two lines have retained their low and high levels of catalase activity in subsequent transfers. The host liver, of animals bearing the high-catalase activity line, shows depressed catalase activity during growth of the tumor whereas the tumor catalase does not show such depression (Table 3, Rechcigl et aZ.(lQ)). The total catalase units per host liver and variable sizes of hepatoma 5123 have been plotted in Fig. 5.(sQ) It is quite clear from these data that as tumor weight increases the total catalase increases in the tumor but decreases in the host liver. No change has been found in 5123 hepatoma catalase activity of either male or female rats varying in age from 3 to 11 months (Table 3). The TABLE
3
Effect of Sex and Age on Cat&se Activity of Liver, Kidney and Hepatoma 5123(20) Catalase activity
Sex
Age months
ii FM
35 9 3 11 4
FF
Liver P/g
Kidney r/g
Tumor P/f2
177+ 76 137f 195+ 6 116fll 140+ 37 130&
49+2 47e2 47+1 32+5 29&2 30+1
42+3 47+6 47k3 40+2 3752 41f2
Difference in catalase between sexes P = 0.01.
catalase activity values for host liver and kidney of males, however, was significantly higher than for females. The host liver catalase of older male and female rats was somewhat higher than for animals 3 months of age, but host kidney catalase in these studies was not affected by age of the rat. Rechcigl and Pricec2r) have studied factors affecting synthesis and destruction of liver catalase in rats. They have noted a lowering of liver catalase of normal animals on a protein-free diet. A comparison of host liver and hepatoma 5123 catalase activities during a 7.day period when the animals were on a protein free diet is illustrated in Fig. 6.(QQ)Small tumors were selected in this experiment because of the depressing e&t of large tumors on host liver catalase. The data in this figure show no effect on tumor catalase activity per g of tissue, but a striking lowering of liver catalase in hepatoma 5123-bearing rats which was comparable to that of normal animals on a protein-free diet. The decrease of total catalase in starvation is proportional to the decrease in liver size according to Rechcigl and Price.(QQ) They show further
bw
HEP.~TOMAS 0~ DIFF~~
GROWTH k4-rE
331
4
NORMAL
LIVER
t
I-
l
.
1
200 0 0
2
4
6
8
IO
12
14
I8
TUMOR WEIGHT, GM. FIG. 5 Tissue catalase activity of Buffalo-strain female rats bearing hepatoma 5123. T.B. liver refers to the liver of the tumor bearing host.
180
20 0
I
I
I
0
I DAYS
2
I
I
3
4
ON PROTEIN-FREE
I
5
6
7
DIET
FIG. 6 The catalase activity expressed as units per g of tissue in normal female rats, rats bearing hepatoma 5123, and in hepatoma 5123. All rats were fed a proteinfree diet.
332
H.
P. MORRIS et al.
that in the rat during the first 5 days of starvation the rate of catalase synthesis per gram of liver proceeded as rapidly as in the controls, but because the livers were decreasing in size there was less total catalase being synthesized. It was noted that the rate of catalase per g of liver starts to drop after 5 days of starvation, and continues to go down until the death of the animal. The liver catalase of rats on a protein free diet had a decreased rate of synthesis per g of liver, although there was a relative small decrease in liver size, and the fraction of catalase molecules being destroyed per unit time was in the normal range. These two changes, therefore, resulted in a lowered catalase activity within the liver. It is well known that tumor bearing animals suffer from hypoproteinemia. The effect of a tumor on liver catalase may be mediated through a deficiency in one or more amino’ acids required for catalase synthesis.@) Because the tumor catalase noted above was unaffected by a protein-free diet the tumor may, therefore, have a preferential claim on available metabolic units needed for synthesising catalase. We are still unable to explain the development of hepatomas of variable catalase activity. It is not always related to growth rate of the tumor because the two slowest growing hepatomas in our group of tumors, 7787 and 7794B, have greatly different catalase activity values, but both have similar growth rates. SUMMARY
The results of studies on TP induction, catalase activities, growth studies, and ethionine injections confirm the accumulated evidence from many laboratories that individual transplantable hepatomas, even of the “minimal deviation” type, show wide variation in their enzyme activities, even when the original tumors are induced by the same chemical and under as similar experimental conditions as can be provided. Transplantable hepatomas of widely differing biological and biochemical characteristics have also been produced in different lobes of the same liver of the primary host. The induction of transplantable hepatomas of the “minimal deviation” type has been accomplished by several chemical carcinogens. In case of N-2-3uorenylphthalmic acid, tumors have been induced in rats of both sexes in two entirely separate experiments. Therefore, it is concluded that the first “minimal deviation” hepatoma observed, 5123, is not unique. REFERENCES
1. VAN R. POTTER, Transplantable cancer, the primary standard: Guest editorial, Cancer Research 21, 1331-1333 (1961). 2. VAN R. POTER, H. PITOT, T. ONO and H. P. MORRIS, The comparative enzymology and cell of origin of rat hepatomas. I. Deoxycytidylate deaminase and thymidine degradation, Cancer Research 20, 1255-1261 (1960). 3. M. D. REUBER, A transplantable bile-secreting hepatocellular carcinoma in the rat, J. Nuf. Cancer Inst. 26, 891-897 (1961).
RAT
HEPATOhfAS
OF DIFFERENT
GROWTH
RATE
333
4. H. P. MORRIS and B. P. WAGNER, The development of minimal deviation hepatomas, Proc. VZZZZnt’l. Cancer Congress, Moscow, USSR, July 22-28 (1962). In press. 5. H. M. DYER, P. GIJLLINO and H. P. MORRIS, Tryptophan pyrrolase activity in transplanted “minimal deviation” type hepatomas, Cancer Research 24, 97-104 (1964). 6. P. GULLINO and F. H. GRANTHAM, A method for growing “tissue-isolated” tumors in laboratory animals, J. Natl. Cancer Inst. 27, 679-693 (1961). 7. H. C. PITOT, Biochemical lesions in minimal deviation hepatomas, Proc. VZZZ Znf’l Cancer Congress, Moscow, U.S.S.R., July 22-28 (1962). In press. 8. H. C. PITOT and Y. S. CHO, Studies on the mechanism of enzyme induction in rat liver, Cold Spring Harbor Symposium on Quant. Biol. 26, 371-377 (1961). 9. H. C. PITOT and H. P. MORRIS, Metabolic adaptations in rat hepatomas. II. Tryptophan pyrrolase and tyrosine a-ketoglutarate transaminase, Cancer Research 21, 1009-1014 (1961). 10. 0. GREENGARD and P. FEIGELSON, The activation and induction of rat liver tcyptophan pyrrolase in vivo by its substrate, J. Biol. Chem. 236, 158-161 (1961). 11. Y. S. CHO, H. PITOT and H. P. MORRIS, Metabolic adaptations in rat hepatomas. VI. Substrate-hormone relationships during tryptophan pyrrolase induction in liver and hepatomas, Cancer Research 24,52-58 (1964). 12. N. C. LEE and B. E. BALTZ, Tryptophan pyrrolase induction and the influence of adrenocorticoids, Endocrinology 70, 84-87 (1962). 13. E. FARBER, M. V. SIMPSON and H. TARVER, Studies on ethionine. II. The interference with lipide metabolism, J. Biol. Chem. 182, 91-99 (1950). 14. D. JENSEN, I. L. CHAIKOFF and H. TARVER, The ethionine induced fatty liver: Dosage, prevention, and structural specificity, J. Biol. Chem. 192, 395-403 (1951). 15. E. FARBER and A. SEGOLOFF,Effect of androgens and growth and other hormones in ethionine fatty liver in rats, J. Biol. Chem. 216, 471-477 (1955). 16. J. P. GREENSTEIN, Biochemistry of Cancer; 2nd Ed., Academic Press, New York (1954). 17. H. P. MORRIS, H. SIDRANSKY, B. P. WAGNER and H. M. DYER, Some characteristics of rat hepatoma 5123 induced by ingestion of N-(2-fluorenyl)phthalamic acid, Cancer Research 20, 1252-1254 (1960). 18. H. P. MORRIS, Some growth, morphological, and biochemical characteristics of hepatoma 5123 and other new transplantable hepatomas. In Prog. Exp. Tumor Res. Vol. 3, 370-411 (Karger, Basel/New York) (1963). 19. M. RECHCIGL, JR. and H. SIDRANSKY, Isolation of two lines of transplantable ethionine-induced rat hepatomas of high and low catalase activity from a primary tumor, J. Nut. Cancer Inst. 28, 1411-1423 (1962). 20. M. RECHCIGL, JR., V. E. PUCE and H. P. MORRIS, Studies on the cachexia of tumorbearing animals. II. Catalase activity in the tissues of hepatoma-bearing animals, Cancer Research 22, 874-880 (1962). 21. M. RECHCIGL, JR. and V. E. PRICE, Catalase synthesis and destruction in starvation, Experientia 17, 258-261 (1961). 22. M. RECHCIGL, JR. and V. E. PRICE, The rates and kinetics of enzyme formation and destruction in the living animal. In New Methods of Nutritional Biochem. Vol. 1, 185-197 (1963). 23. M. RECHCIGL, JR., Nutritional factors in the etiology of catalase depression of the tumor-bearing host, Proc. VI International Congress of Nutrition. Edinburgh, August 9-15 (1963).