ICANCER RESEARCH 27, 81-87, January 1967]
Effects of Adreno cortico tro pic Hormone and Growth Hormone
on the Metabolism of ]V-Hydroxy-]V-2-fmorenylacetamide
and on Physiologic Parameters
YASUHIKO SHIRASU,1 P. H. GRANTHAM, E. K. WEISBURGER, AND J. H. WEISBURGER
National Cancer Institute,
Bethesda, Maryland
Summary
The effects of large doses of adrenocorticotropic hormone
(ACTH) and of growth hormone on physiologic parameters and
on the metabolism of N-hydroxy-JV^-fluorenylacetamide
(N-OH-FAA) were investigated in Fischer male rats. ACTH, 96
or 80 units, and growth hormone, 12 or 10 mg, were injected s.c.
daily for 10 consecutive days into animals prefed either control
diet or the N-OH-FAA-containing diet (160 ppm) for 4 weeks
prior to the hormonal treatment. ACTH, but not growth hor
mone treatment, led to increased rectal temperature and body
weight loss. Water intake and urine output were elevated by
either hormone. ACTH injections enlarged the adrenals 6- to
7-fold and practically involuted the thymus. ACTH caused a sig
nificant percentile weight increase in the liver, kidney, heart, and
pituitary, but growth hormone did not.
14C-labeled N-OH-FAA was given on the last day of the hor
monal treatment. Urinary excretion of total metabolites, of glucosiduronic acids, and particularly of the glucuronide of N-OHFAA was increased by both hormones and was especially notable
in the carcinogen-prefed animals. These changes suggest that
ACTH and growth hormone decreased dehydroxylation and
deacetylation of N-OH-FAA. The radioactivity bound to liver
proteins was increased, remarkably so in the animals treated with
growth hormone.
The results indicate that ACTH plays a paramount role in
inducing marked physiologic and metabolic changes, eventually
leading to the enhancement of hepatoma formation, as seen in
animals fed carcinogen and implanted with MtT, a functional
pituitary tumor. The contributing role of growth hormone,
however, should not be neglected.
Introduction
The endogenous pituitary hormones from a transplantable
pituitary tumor, Furth MtT/F4, which produces large amounts
of ACTH,2 growth hormone, and prolactin (2, 11, 40), enhanced
1Visiting Scientist, NIH, on leave of absence from the Institute
of Physical and Chemical Research, Tokyo, Japan.
2 The abbreviations used are: ACTH, adrenocorticotropic
hor
mone; N-OH-FAA, jV-hydroxy-.iV-2-fluorenylacetamide
(also N*
hydroxy-2-acetylaminofluorene
;
A"-2-fluorenylacetohydroxamic
acid); FAA, N-2-fhiorenylacetamide
(also 2-acetylaminofluorene);
MtT, the functional transplantable
pituitary
tumor, MtT/F4
of Furth (11, 40); GH, growth hormone.
Received May 4, 1966; accepted July 21, I960.
JANUARY
hepatocarcinogenesis by N-OH-FAA (43), a more proximate and
active carcinogen derived by metabolism from FAA (9, 27, 28,
44). The underlying mechanisms of the rapid hepatoma forma
tion caused by MtT appeared to rest in part on an increased
metabolic rate, indicated by a higher body temperature, and a
lower conversion of N-OH-FAA to detoxified products, due to
decreased dehydroxylation and deacetylation (39).
Bates et al. (3) analyzed the physiologic changes caused by
MtT by employing large doses of exogenous ACTH, growth hor
mone, and prolactin in female Fischer rats and succeeded in re
producing the splanchnomegalic syndrome seen in MtT-bearing
animals. The present experiments aimed to assess the roles of
ACTH and growth hormone, major hormones produced by MtT,
on the physiologic parameters and on the metabolism of N-OHFAA in male Fischer rats. In order to simulate the conditions of
our previous study (39) and also to enhance the metabolic effect,
ACTH and growth hormone were given to the animals prefed
the carcinogen prior to the hormonal treatment, in addition to
the rats fed a control diet.
Materials
and Methods
Seven-week-old male rats of the Fischer strain obtained from
the NIH Animal Production Section were used. The animals were
divided into 6 groups of 4 animals each. The rats of 3 groups were
fed a semisynthetic control diet, A (39, 43), and the animals of
the other 3 groups were given a carcinogen diet, K, which con
tains 160 ppm N-OH-FAA in Diet A. The diets were adminis
tered for 4 weeks prior to the treatment with hormones. The 1st
of each diet group served as a control without treatment (Groups
A and K). The animals of the 2nd group were given ACTH
(A + ACTH and K + ACTH), and those of the 3rd group were
treated with the bovine growth hormone (A + GH and K +
GH).
The doses of the hormones and schedule of the treatment were
based on the experiment of Bates et al. (3) in female Fischer rats.
The hormones were injected s.c. twice a day, in the morning and
in the late afternoon, for 10 consecutive days. The doses of the
hormones were based on the body weight at the start of the hor
monal regimen. ACTH (Acthar gel, 80 units/ml, Armour Phar
maceutical Company, Kankakee, 111.)was given in daily doses
of 96 and 80 units for rats fed A and K diet, respectively. The
bovine growth hormone was obtained from Dr. Wilhelmi, Emory
University, through the courtesy of the NIH Endocrine Study
Section; daily doses of 12 and 10 mg were administered to the
rats fed A and K diets, respectively.
1967
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81
Yasuhiko
Shirasu, P. H. Grantham, E. K. Weisburger, and J. H. Weisburger
A + GH
A
200
I
l
K + GH
K
A+ACTH
150
*
K + ACTH
èr
•INJECTION PERIODJ
-6
L
-4
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_L
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0
DAYS
CHART 1. Body weight curves of male Fischer rats on various experimental regimens. The 1st points of the curve give weights after
3 weeks on Diets A and K. Hormone injections began after the 4th week, Day 0, and continued for 10 days, the daily dose being
given divided in 2 injections in the morning and the afternoon. On Day 7 all rats were placed on Diet A, and labeled N-OH-FAA
was injected on Day 10 with the last dose of hormone. Hollow symbols, Diet A; filled symbols, Diet K; O
O, A; D
ü,A + GH;
A-—A, A + ACTH; •—• K; •-—-•,
K + GH; A-—A, K + ACTH. N-OH-FAA, AT-hydroxy-Ar-2-fluorenylacetamide;
GH,
growth hormone; ACTH, adrenocorticotropic
hormone.
39.0
A+ACTH
K+ACTH
5
K.*-'
A-
A + GH
K+GH
A
38.0
37.5
INJECTION
37.1
l
-4
-2
i
I
PERIODI
I
I
I
8
DAYS
CHART 2. Rectal temperatures of male Fischer rats on various experimental regimens, during, and for 4 days prior to, injection
of hormones. Symbols are given on Chart 1. Note that groups on carcinogen had a generally lower temperature than controls, but
their temperature rose after Day 7 when all rats were on control diet. Adrenocorticotropic
hormone (ACTH) was pyrogenic. The tem
perature changes in the groups of rats treated with ACTH coincided with the loss in body weight (Chart 1).
82
CANCER
RESEARCH
VOL. 27
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ACTH, Growth Hormone, and N-OH-FAA Metabolism
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The rectal temperature was measured twice a day, in the
morning (9:00-10:00 A.M.)and in the late afternoon (5:00-6:00
P.M.), using an electronic thermocouple.
On the last day of the hormonal treatment, 10 mg/kg of
N-OH-FAA-9-14C (specific activity 5.2 X IO6cpm/mg; 0.89 me/
mmole) suspended in steroid suspending vehicle3 (a gift of the
Cancer Chemotherapy National Service Center, National Cancer
Institute) were injected i.p. immediately after the morning ad
ministration of the hormones. The animals were placed in
metabolism cages, and urine was collected in ice-cold receivers.
For 3 days prior to injection of labeled N-OH-FAA, the rats
which had been fed Diet K were placed on control Diet A to
eliminate free carcinogen.
The animals were killed, by removal of blood from the ab
dominal aorta followed immediately by perfusion through the
portal vein with isotonic saline solution, 24 hr after administra
tion of the labeled carcinogen. The procedures for identification
of the urinary metabolites and for the measurement of radioac
tivity bound to the liver protein were as previously reported
(39, 42), with minor modification. Briefly, the free compounds
were removed from the urine by ether extraction at pH 6. The
glucosiduronic acids were hydrolyzed by incubation with bac
terial /3-glucuronidase followed by ether extraction. The sulfuric
acid conjugates were extracted with ether at pH 6, after acid
hydrolysis. After evaluation of the ether, ethanol solutions of
the residues from these extracts were chromatographed on paper
in a cyclohexane solvent system (cyclohexane, i-butanol, acetic
acid, water, 16:4:2:1). Radioautographic technics were em
ployed to locate and quantitate the individual metabolites.
A liver homogenate was prepared with 4 volumes of ice-cold
distilled water per gm of liver, and the proteins were precipitated
with 3.5 volumes of ethanol. After extensive washing in centri
fuge tubes with acetone (4 times), ethanol (once), ether-ethanol
(once), and ether (once), the proteins were dried in vacuo. The
radioactivity of all samples was determined with a liquid scintilla
tion counter. The liver proteins were counted after complete
digestion with Hyamine. Samples from each tissue or fraction
were processed in duplicate or in triplicate. Thus, the averages
shown in Tables 2 and 3 are based on 8 or 12 individual analyses.
The values were self-consistent and also in accord, where appro
priate, with those previously published (cf. Ref. 39, 42).
Tissues were fixed in 10% formalin or Bouin's fixative and
stained with hematoxylin and eosin.
Results
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JANUARY
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PHYSIOLOGIC
CHANGES.At the start of hormonal treatments,
the animals which had been on the carcinogen diet for 4 weeks
weighed about 30 gm less than the controls (Chart 1). ACTH
caused a decrease in the body weight of both carcinogen- and con
trol-diet-fed animals. Growth hormone failed to alter body weight
appreciably. The food intake of the animals treated with ACTH
or growth hormone decreased slightly within 4 days after the
beginning of the injections but thereafter returned to near the
control levels of 11-14 gm of A diet, 7-10 gm/rat/day in K diet
3Steroid suspending vehicle is an injectable sterile solution of
sodium chloride, 9 gm; sodium carboxymethyl cellulose 7 LP, 5
gm; polysorbate 80, 5 ml; benzyl alcohol, 9 ml; and disti'l»dwater
to 1 liter.
1967
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83
Yasuhiko Shirasu, P. H. Grantham, E. K. Weisburger, and J. H. Weisburger
group, respectively. The food intake of the rats treated with
ACTH in either A or K diet group showed no change, whereas
the other groups revealed a decrease in food intake during the
last 24-hr period, after injection of labeled N-OH-FAA. The
average food intake (in gm) jier rat (for each group) during this
period was as follows: Group A, 5.2; K, 6.9; A + ACTH, 12.8;
K + ACTH, 10.0; A + GH, 7.3; and K + GH, 6.O. The water
intake was increased in both ACTH- and growth-hormonetreated groups regardless of the diets. The mean values (in nil)
ix-r day for 3 consecutive days prior to the injection of the labeled
carcinogen were as follows: Group A, 11.3; A + ACTH, 14.5;
A + GH, 14.1; K, 9.3; K + ACTH, 14.4; and K + GH, 12.6.
Corresponding to the water intake, urinary volumes were also
larger with the 2 hormones.
ACTH treatment gave rise to a pronounced elevation in the
rectal temperature of the animals in both diet groups, but es
pecially so in the K diet group. The means of morning and after
noon temperatures are plotted in Chart 2. On the 8th day, the
differences between the temperatures of the ACTH group and
those of the control group were 0.7°Cin A-diet-fed and 0.8°Cin
K-diet-fed animals. Growth hormone, however, caused no sig
nificant rise in the body temperature.
ORGANWEIGHTS.The most marked changes caused by ACTH
were enlarged adrenals and involuted thymuses (Table 1). Also,
ACTH induced a sizable percentile weight increase in the liver,
heart, pituitary, and especially in the kidneys. However, the
spleen weighed less. There were no real changes in the organ
weights attributable to the 2 diets in the 4-week feeding period.
Growth hormone slightly increased liver and kidney weights,
per 100 gm body weight, and moderately raised the weight of the
adrenals and spleens. The weight of the pituitary in Group A +
GH and that of the thymus in Group K + GH were slightly
decreased. Neither ACTH nor growth hormone altered the
weight of testes.
HISTOPATHOLOGIC
OBSERVATIONS.
The livers of all the groups
fed the carcinogen showed fibrosis in the jxirtal and interlobular
areas. The degenerative changes, namely, vacuolated cytoplasm
and increased numbers of karyolytic nuclei, in the parenchymal
liver cells were more pronounced in Group K + ACTH than in
Group K. The livers of Group A + ACTH revealed more vacuolative change in the cytoplasm of the liver cells in the centrolobular area, compared with Group A. Although a few small foci
of hyperplasia were found in the livers of some rats fed the car
cinogen, there was no indication of enhancement of these changes
at this stage by ACTH or growth hormone. Growth hormone led
to no remarkable effect on the liver morphology in the rats
prefed either A or K diet.
Either prefeeding the carcinogen or hormonal treatment
caused degeneration of tubular epithelium of the kidney. Vacuolar degeneration in the epithelium of the convoluted tubules was
noted in the animals treated with ACTH. However, the pro
nounced degenerative changes in the kidneys observed in MtTbearing animals (11, 39, 43) could not be reproduced by this
short experimental regimen.
The adrenals of the rats treated with ACTH revealed hyper
plasia of the zona fasciculata and many vacuoles in the hyjjertrophic cells in this zone.
URINARYMETABOLITES
OFN-OH-FAA. The total radioactivity
84
TABLE 2
URINARYMETABOLITESAND RADIOACTIVITY
BOUNDTO LIVER
PROTEIN24 HR AFTERINJECTIONOF LABELEDN-OH-FAA«
DOSE6Urine
GROUPAKA
OF
protein
(manióles
/gm)18711418913726819
com
duronic
acid
con
"C273352693870Free
pounds1.02.11.82.31.54.2Glucosijugates5.23.08.76.64.96.9Liver(%/gm)114.6(0.57)3.
acids7.515.113.434.114.240.4Sulfuric
ACTHK
+
ACTHA
+
GHK
+
+ GHURINEVOLUME(ral)e8.45.915.513.312.510.9%
<•
N-OH-FAA, Ar-hydroxy-jV-2-fluorenylacetamide;
ACTH, adrenocorticotropic
hormone; GH, growth hormone.
1 There are water-soluble metabolites of N-OH-FAA which are
not of the nature of those listed here. They form the balance of
activity not accounted for by the sum of the known materials.
Their identity is under continuing investigation.
c Averate volume of urine excreted within 24 hr.
* Numbers in parentheses are % of dose per gm of liver.
from injected labeled carcinogen excreted in the urine was higher
in Group K than in Group A (Table 2). Both ACTH and growth
hormone increased the output of the urinary radioactivity more
so in the A + ACTH group compared with the A + GH group.
In K-diet-fed groups, a greatly enhanced excretion of the urinary
radioactivity was also induced by the 2 hormones.
Radioactivity in feces paralleled that seen previously (39), but
comparative quantitation is unreliable in view of the pronounced
differences in fecal output in the short experimental period. The
larger food intake in the ACTH-groups led to elimination of more
radioactivity with the bulkier stools. The animals injected with
growth hormone had small stools.
Both ACTH and growth hormone increased the urinary
glucosiduronic acid fraction, particularly in the K-diet-fed
groups. The most noticeable change among individual glucuronides due to the hormonal treatments was seen in the glucosidu
ronic acid of N-OH-FAA (Table 3). ACTH and growth hormone
increased this metabolite in A- and in K-diet-fed groups. K diet
alone also raised this compound. The other conjugates, with
7-OH-, 5-OH-, and 3-OH-FAA, also contributed to the elevation
of the total glucuronide fraction, although to a lesser degree than
the N-OH-FAA derivative. An unknown material, with a mo
bility of 0.24-0.37, which appeared between 5- and 3-OH-FAA
on the chromatograms, showed an increase after the treatment
with the 2 hormones, more so in the carcinogen-fed groups.
Sulfuric acid conjugates showed only minor changes due to
the hormonal treatments. The major metabolites of this type
were conjugates of 7-hydroxy derivatives of 2-FAA and 2-FA.
There were also several weaker spots in the autoradiographs at
the region of 5-OH-FAA (RP 0.15-0.24), an unknown (RP 0.240.37) between 5-OH- and 3-OH-FAA, 3-OH-FAA (RF 0.37-0.52),
CANCER
RESEARCH
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VOL. 27
ACTH, Growth Hormone, and N-OH-FAA Metabolism
TABLE 3
URINARYMETABOLITES
OFN-OH-FAA0 ASGLUCOSIDURONIC
ACIDS
IN RATS AS FUNCTIONOF CARCINOGEN
PRETREATMENT
ANDHORMONEINJECTION
bly the hepatotoxic effect of carbon tetrachloride. Although bac
tériologie
examination was not performed on the animals treated
with ACTH, an increased body temperature caused by infection
due to hypercorticism can be ruled out. The ACTH-treated ani
mals appeared healthy and showed no loss of appetite during the
COMPOUNDS'7-OH-FAA1.32.11.73.72.24.85-OH-FAA2.44.04.36.93.65.3Unknown'0.300.510.391.70.742.53-OH-FAA0.431.10.971.50.991.5N-OH-FAAd1.44.83.015.33.920.3
OF DOSEor
hormonal treatment. Furthermore, gross and microscopic exami
nations of the lungs and other organs revealed no significant
degree of infection.
Polydipsia and polyuria accompanied by a diabetes mellitusAKA
like condition, observed in MtT-bearing animals (39), may be
ACTHK
+
attributable to both ACTH and growth hormone, which cause
ACTHA
+
disturbances in water metabolism by inhibiting water reabsorpGHK
+
tion through the degenerative tubular epithelium of the kidney,
+ GH%
and also affect carbohydrate metabolism (10, 21). ACTH may
be more responsible than growth hormone for the marked altera
" For abbreviations, see Table 2, footnote a.
tions in organ weight of MtT-bearing animals (43). Nevertheless,
67-OH-FAA, etc.,represents Ar-(7-hydroxy-2-fluorenyl)acetamide. Addition of the amounts of individual metabolites shown in the changes in the rats treated with exogenous pituitary hor
mones were not as severe as those observed in the animals im
the table yields a lower value than that shown for the glucosiduplanted
with MtT, except for the enlarged adrenals and the
ronic acid fraction in Table 2 for 2 reasons. Only the main metabo
completely involuted thymus.
lites are listed. Also, there is radioactivity attributable to other
metabolites (42) or to isotope distributed along the Chromato
There were some sex differences in the response to the exoge
graphie paper strip as well as at the origin and near the solvent
nous hormones, chiefly in relation to body weight. In this study
front.
growth hormone induced no outstanding change in weight of male
c An unknown metabolite with RF0.24-0.37, between 5-OH- and
Fischer rats, whereas it was responsible for notable growth in
3-OH-FAA on chromatograms.
female
Fischer rats in the similar experiments of Bates et al. (3).
d N-OH-FAA does not separate from small amounts of 1-OHHowever, a comparison of the response of body and organ weights
FAA.
in the male and female rats indicates that male animals are more
sensitive to ACTH than female animals. ACTH increased the
1-OH-FAA (Rr 0.52-0.61), and a material (Rr 0.77-0.90) faster
pituitary weight, per 100 gm, in male rats but decreased it in
than N-OH-FAA. These had been observed earlier (42).
female animals. Similar changes in the pituitary weight were ob
The total radioactivity in the liver of all the carcinogen-fed
served in the MtT-bearing animals (43).
groups was lower than that of the corresponding A-diet-fed
ACTH and growth hormone exerted like effects on the urinary
groups (Table 2). ACTH as well as growth hormone increased
metabolites of N-OH-FAA, increasing urinary excretion of total
the total radioactivity in the liver of animals on both diets.
metabolites, of glucosiduronic acids, and especially the glucuronThe radioactivity bound to liver proteins was also less in ide of N-OH-FAA. These changes were enhanced in the carcino
groups on K compared with the A-diet-fed groups. Growth hor
gen-prefed groups. Ring-hydroxylated metabolites were rather
mone increased the protein-bound radioactivity in both groups;
less
affected. Thus, both pituitary hormones seem to decrease
but ACTH, only slightly in the carcinogen-prefed group.
dehydroxylation and deacetylation of N-OH-FAA, yielding
higher levels of N-OH-FAA and lower amounts of detoxified
Discussion
products (see Addendum 2). The in vitro and in vivo transforma
In this study physiologic changes and modification of the tions of N-OH-FAA have been subjects of recent reports (15, 19,
metabolism of the carcinogen N-OH-FAA caused by a functional
26, 39, 42).
Lotlikar et al. (25) observed that administration of ACTH
pituitary tumor, MtT, could be mimicked by employing indi
largely restored the excretion of N-OH-FAA after injection of a
vidual exogenous pituitary hormones. The effect was not as pro
nounced as observed in the MtT-bearing animals, probably be
single dose of FAA in hypophysectomized rats. Cortisone and
cause of the short term of the hormonal treatment. One of the deoxycortisone acted likewise in adrenalectomized-hypophysectomized rats, whereas growth hormone had little effect. Thus,
most striking alterations due to the endogenous pituitary hor
mones supplied by MtT was a marked increase in the body ACTH seems to exert its effect on the metabolism of either
N-OH-FAA or FAA in the direction of maintaining higher levels
temperature, accompanied by loss of body weight despite in
of
N-OH-FAA. The present observation is 1 of the few sizable
creased food intake (39) (see Addendum 1). This condition could
demonstrated effects of ACTH or adrenal hormones on drug
be reproduced by ACTH alone, although there was no significant
metabolism (6, 8, 12, 20, 30, 37). The basic underlying mecha
increase of food intake, again owing to the brief period of treat
ment. Thus, in 1 way ACTH from MtT seems to promote hepa- nism warrants further attention, especially inasmuch as stresstoma formation by increasing the metabolic rate in the rats fed induced alteration in these hormones may, thus, indirectly affect
carcinogen and implanted with this pituitary tumor. From this drug action.
Growth hormone yielded higher levels of protein-bound ac
point of view, the report that high environmental temperature
tivity
in liver than ACTH or controls, reflecting not only the
is an important factor favoring a high incidence of Lückerenal
tumor of the leopard frog is of interest (33). In contrast, Larson higher urinary levels of N-OH-FAA but possibly also a greater
availability of molecular targets. Despite the larger extent of exet al. (24) found that lower body temperature delayed measura
JANUARY 1967
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85
Yasuhiko Shirasu, P. H. Grantham, E. K. Weisburger, and J. H. Weisburger
cretion of radioactivity in the urine in the hormone-supplemented
animals, the activity bound to liver was higher than in controls.
It could be, therefore, that an important part of the hormone
effect is to act in unison with carcinogen and multiply the number
of cellular entities which are in a carcinogen-susceptible state.
One is always impressed with the uniform distribution of car
cinogen throughout the target organ, yet cancer development, in
contrast to toxic action, is a rather localized phenomenon. It
seems as though only certain cells bathed with carcinogen were
actually in a resjionsive condition. Hormones may aid in un
covering such entities. The mechanism is presently unclear, yet
both ACTH, and indirectly adrenal hormones, as well as growth
hormone potentially can act on many molecular targets (17, 21,
22, 29, 32, 38, 45). Additional effort hopefully will discover the
crucial selected area where hormone and carcinogen action meet
and overlap.
There have been several reports concerning the involvement
of the pituitary hormones in hepatoma induction. Griffin and his
associates (16, 35, 36) found that ACTH and growth hormone
restored hepatocarcinogenesis by 3'-methyl-4-dimethylaminoazobenzene in hyjiophysectomized rats. However, combined ad
ministration of ACTH and insulin did not reinstate the carcino
genic effect in hypophysectomized animals treated with N-2fluorenyldiacetamide (31). According to Chany and Boy (7),
Goodall (13), and Hoch-Ligeti (18) [see also Lacassagne (23)
and Wahi et al. (41)], cortisone promoted hepatocarcinogenesis,
but Reuber (34) has recently reported that ACTH inhibited
tumor induction with A^-fluorenyldiacetamide. Cortisone like
wise reduced the carcinogenic potency of methylcholanthrene
(1). Possibly a partial explanation for these varied effects rests
on the level of active carcinogen, related in turn to the relative
ini]>ortance of activation versus detoxification reactions (44).
Bielschowsky et al. (4, 5) observed that growth hormone favored
tumor development in normally refractory thyroidectomiz«!
rats treated topically with 2-fluorenamine. Goodall (14) develoj)ed hepatic susceptibility to 2-fluorenamine carcinogenesis
in thyroidectomized rats by iodide treatment, attributing this
effect to a possible increase in growth hormone. Our laboratory
showed that liver tumor induction with N-OH-FAA was en
hanced by pituitary hormones from MtT (43).
In conclusion, this study suggests that ACTH plays a major
role in MtT-bearing animals, inducing considerable physiologic
changes as well as marked modification of the metabolism of
N-OH-FAA, which is involved in faster hepatoma formation.
However, in view of the pronounced changes in the urinary me
tabolites of N-OH-FAA, the higher radioactivity bound to liver
proteins after growth hormone, and the synergism between
pituitary hormones (3), the contributing role of growth hormone
should not be neglected in the interpretation of the MtT effects.
In any case, both ACTH and growth hormone appeared to exert
appreciable additive action with the carcinogen in regard to its
metabolism and also to their joint effect on the liver.
Acknowledgments
We are indebted to L. Mohan and A. Parker for excellent techni
cal support. We are grateful to J. Zuefle for processing the histo
logie material.
86
Addenda
ADDENDUM1. Additional data on this point were obtained fol
lowing a suggestion of Drs. D. Rail and N. Berlin, National Cancer
Institute. The respiratory carbon dioxide production, measured as
barium carbonate, was determined as evidence of increased meta
bolic rate in rats bearing MtT. Control rats with an average body
weight of 166 gm, 4 months old, rectal temperature 37.6°C,pro
duced 2.20 gm of BaCO>/hr; other controls with equal body weight
to MtT group, 205 gm, 15 months old, temperature 37.7°C,yielded
2.00 gm of BaCO3. MtT-bearing rats, 8 weeks after implantation,
body weight 207 gm,4 months old, rectal temperature 38.5°C,gave
2.95 gm of BaCOs. It was also noted that the temperature of the
MtT-bearing rats tended to drop if the rats were deprived of food
even for a few hr.
ADDENDUM2. It has been pointed out that "the pituitary hor
mones could affect urinary levels of Ar-hydroxy-FAA glucuronide
by a number of other mechanisms." One point cited is the rate of
conjugation
of N-OH-FAA with UDP-glucuronic
acid which
might be increased. Hence the compound would be more readily
excreted as a conjugate. Although in vitro dehydroxylation
and
deacetylation have not been studied here, it is believed that the
data obtained with livers from rats bearing MtT, as reported in
prior experiments (39), can be extrapolated to the present series.
Our earlier results showed that the livers from rats with MtT and/
or N-OH-FAA had lower ability both to dehydroxylate
N-OHFAA and to deacetylate the acetyl derivatives. Higher levels of
N-OH-FAA would be available, therefore, for conjugation with
glucuronic acid, and excretion in the urine, as has been found
(Table 3, this paper, and Table 4, Ref. 39).
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87
Effects of Adrenocorticotropic Hormone and Growth Hormone
on the Metabolism of N-Hydroxy-N-2-fluorenylacetamide and on
Physiologic Parameters
Yasuhiko Shirasu, P. H. Grantham, E. K. Weisburger, et al.
Cancer Res 1967;27:81-87.
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