Thyroid Hormone in 7,12-Dimethylbenz(a ... - Cancer Research

Thyroid Hormone in 7,12-Dimethylbenz(a)anthracene-induced
Leukemia in Rats
Alan K. Burnett
Cancer Res 1979;39:4252-4255.
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[CANCER RESEARCH 39. 4252-4255, October 1979)
0008-5472/79/0039-OOOOS02 00
Thyroid Hormone in 7,12-Dimethylbenz(a)anthracene-induced Leukemia in
Rats1
Alan K. Burnett2
The Ben May Laboratory for Cancer Research. University of Chicago, Chicago. Illinois 60637
ABSTRACT
Thyroidectomy reduces the incidence of hormone-depend
ent stem cell leukemia elicited by 4 biweekly doses of 7,12dimethylbenz(a)anthracene
to Long-Evans rats (22% com
pared with 46% in control animals). There is a high incidence
of early death due to aplastic anemia in the thyroidectomized
rats (70%) compared with controls (34%). In established leu
kemia, neither thyroidectomy nor thyroxine excess produces
any lasting benefit. In all experiments, the thyroid status of
each rat is continuously measured by the free thyroxine index.
INTRODUCTION
A stem cell leukemia can be induced rapidly by a series of
P.O. feedings of DMBA3 to Long-Evans rats (8). A remarkable
feature of this leukemia is its rapid regression following hypo-
physectomy in substantial numbers of rats with primary leuke
mia or bearing transplants of leukemia cells (7,11,19).
Which single hormone or combination of hormones under
pituitary control is responsible for this effect is not known.
Prolactin has been found to be unimportant in the induction
time and incidence of this leukemia (1, 20).
The activity of the thyroid gland is under trophic influence of
the pituitary and it is now well established that thyroid hormone
markedly affects the oxidative metabolism of cells (3). Thus, it
seems possible that interference with thyroid gland activity may
be responsible for the pituitary ablative effect on leukemic cell
growth. Previous investigations of the role of the thyroid in the
development and growth of tumors have been sporadic, and
the results have been conflicting. On one hand, some workers
(4, 6, 12, 13, 15, 21 ) found that alteration in the thyroid status
influenced the induction and rate of growth of a variety of
tumors including leukemias, whereas others (14, 18) could
demonstrate little influence of thyroid hormone on tumor
growth.
In none of these studies, however, was the thyroid status
monitored during the period when altered hormone levels were
assumed to be present. This paper, therefore, represents the
first controlled study of the influence of thyroid activity on the
induction and maintenance of a hydrocarbon-induced leukemia
where hormone levels have been carefully monitored through
out the period of experimental observation.
MATERIALS AND METHODS
Animals. Long-Evans rats were studied exclusively. They
' Supported principally by grants from the American Cancer Society and the
Jane Coffins Childs Memorial Fund for Medical Research, by USPHS Research
Grant CA 11603 from the National Cancer Institute, and laterally by The Leukemia
Research Fund
' Present address: Department of Haematology, Glasgow Royal Infirmary,
Glasgow, G4 OSF. Scotland.
' The abbreviation used is: DMBA, 7.12-dimethylbenz(a)anthracene.
Received May 25. 1976; accepted July 19, 1979
were taken from a random-bred closed colony of rats which
has been maintained for 14 years. The rats were housed in
stainless steel or plastic cages in air-conditioned rooms at 25
±1°(S.D.), artificially lit for 12 hr/day. They were fed com
mercial ration [Rockland Mouse/Rat diet (Teklad, Monmouth,
III.) or Diet 41 (William Shearer Ltd., Glasgow, Scotland)]. Tap
water was given freely. Ether was used as anesthetic when
required. Vaginal smears were examined daily.
Induction of Leukemia. In experiments to assess the effect
of thyroidectomy on the induction of leukemia, a 15% (w/v)
emulsion of DMBA (kindly supplied by Dr. Paul Schurr, The
Upjohn Company, Kalamazoo, Mich.) was injected into the tail
vein of 44- to 54-day-old rats (designated Day 0). This was
repeated on a further 3 occasions at 14-day intervals; the dose
was fixed at 30 mg/kg body weight. In experiments on estab
lished leukemia, the leukemia was induced by p.o. administra
tion of a solution of refined 0.5% (w/v) DMBA in sesame oil, as
described previously (11), to animals ages 28 to 35 days in a
dose of 175 mg/kg body weight, and on 3 further occasions
at 14-day intervals in a dose of 10 mg to each rat.
Diagnosis of Leukemia. The most convenient method of
early diagnosis of this leukemia is its appearance on open liver
biopsy as described earlier (8). The first diagnostic liver biopsy
was performed 14 days after the final exposure to carcinogen
(i.e., Day 56), and on a further 7 occasions at 14-day intervals
thereafter.
Hematological Studies. Blood for hematological procedures
was obtained by cardiac puncture into a heparinized syringe
and examined by conventional hematological techniques.
Erythropoiesis was estimated by 59Fe uptake into bone marrow
2 hr after injection of a dose of 1 juCi/g body weight (17).
Thyroidectomy. Thyroparathyroidectomy was performed by
surgical avulsion. In induction experiments, this preceded the
first dose of carcinogen by 6 to 7 days in all cases. All
thyroparathyroidectomized rats were given 1% calcium lactate
in the drinking water to avoid tetany. Completeness of thyroid
ectomy was assessed by assay of the free thyroxine index.
This index is derived from multiplying the product of the free
thyroxine level (jug/100 ml), based on a competitive binding
technique (16), and the ratio of the uptake of thyroxine onto
resin beads compared with standard sera. Serum from all
animals was assessed at approximately 6 weekly intervals.
Assay of 85 normal rats established a normal range of 5.46
± 1.46 (S.D.). Thyroidectomy was considered incomplete if
the free thyroxine index was greater than 1.0. L-Thyroxine
(Sigma Chemical Co., St. Louis, Mo.) was administered in a
dose of 10 fig/day s.c.
RESULTS
Anatomical and Hematological Effects of Thyroidectomy.
Female rats were used for this study. Thyroidectomy was
performed at age 21 days. The animals became sluggish,
4252 CANCER RESEARCH VOL. 39
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developed coarse fur, and showed retardation of body growth
(Chart 1). Opening of the vaginal plate was not delayed, and
periodicity of the estrus cycle was normal. A mild nonprogres
sive anemia developed around the 35th postthyroidectomy
day. No persistent significant differences in peripheral WBC or
platelet count were noted. At the 77th postthyroidectomy day,
a small but significant reduction (p < 0.05) in spleen size was
observed: 0.18 ± 0.02 versus 0.21 ± 0.01 g/100 g body
weight (10 rats in each group).
Erythropoiesis as measured by 59Fe incorporation into bone
marrow 77 days postthyroidectomy was significantly reduced
( p < 0.05) in the thyroidectomized group (1148 ±324 versus
1773 ±402 cpm, wet weight, bone marrow; 8 rats in each
group).
The Effect of Thyroidectomy on Induction of Leukemia. In
the series of experiments to assess the effect of hypothyroidism
on the induction of leukemia, 21 7 adequately thyroidectomized
rats and 138 intact littermates were given 4 doses of carcino
gen at biweekly intervals. Two major effects were noted.
First, there was a significantly higher incidence of early death
in the thyroidectomized group, compared with controls. Only
45 of the original 219 thyroidectomized animals became "ef
fective," i.e., survived until the day of the first diagnostic liver
biopsy (Day 56), compared with 90 of 138 controls (p <
0.001).
The majority of deaths occurred following the second or third
injection of carcinogen and appeared to be due, in the majority
of cases, to aplastic anemia. This was characterized by loss of
body weight, loss of normal estrus periodicity in females,
reduced body temperature, peripheral blood pancytopenia,
and marrow hypoplasia or aplasia.
Second, of the 45 "effective" thyroidectomized animals, 10
42
49
3»
36
34
32
30
28
Hematocrit Platelets
/ *
7 14 21 28 35 42 49 56 63 70 77
day «•
13,000+ Leukocytes
7 14 21 28 35 42 49 56
j
Thyroid Hormone In Hormone-dependent Leukemia
(22%) developed leukemia by Day 140 compared with 41
(46%) of the 90 "effective" controls (p < 0.01) (Table 1).
Since several animals from each group succumbed to the
stress of repeated diagnostic liver biopsy, the incidence of
leukemia was assessed on each biopsy day (Table 1). There
was a statistically significant difference (p < 0.05) on Day 56,
but on each other day the differences failed to achieve signifi
cance. However, the total number of rats developing leukemia
by Days 56, 70, 84, 98, and 112 was significantly greater in
controls compared with thyroidectomized rats (Day 56, p <
0.06; Day 70, p < 0.05; Day 84, p < 0.01 ; Day 98, p < 0.05,
Day 112, p < 0.01).
There were no significant differences in the time taken to
develop leukemia between the groups (thyroidectomy, 91 ±
29 days; controls, 78 ±26 days). The statistical test used in
these experiments was x? with Yates correction for small num
bers.
Effect of Thyroidectomy on Stem Cell Leukemia. None of
the animals with established primary leukemia which were
subjected to thyroidectomy showed any reduction in growth of
leukemia. In every case, peripheral blood values deteriorated,
and liver biopsy on the 14th postoperative day exhibited more
extensive infiltration than preoperatively (Table 2). Survival in
the thyroidectomized animals was shorter (21 ±9.9 days) than
in controls (29.4 ±14.0 days).
Effect of Thyroxine Excess on Stem Cell Leukemia. Of the
20 animals with primary leukemia that received large doses of
thyroxine, 18 showed clear deterioration (Table 3). Peripheral
blood values had deteriorated, and liver histology exhibited
marked deterioration. Two animals, however, showed improve
ment in peripheral blood indices. In these cases, liver biopsy
showed apparent complete regression in one, and partial
28 35 42 49 56 63 70 77
7 14 21 28 35 42 49 56 63 70 77
Chart 1. The effect of thyroidectomy on peripheral blood and body weight. Thyroidectomy performed at 21 days of age (Day 0). Each point is the mean of 8
nalpK
females
OCTOBER 1979 4253
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A. K. Burnett
Table 1
Ellect of thyroidectomy on induction ol stem cell leukemia
Thyroideclomized rats were given DMBA, 30 mg/kg, on 4 occasions at 14-day intervals commencing at age 44 to
54 days Rats surviving until the first diagnostic liver biopsy (Day 56) are termed effective" rats
Thyroidectomy
ControlInitial
1/13ap

The metabolism of DMBA may be retarded in thyroidecto-
mized animals, permitting its toxic effects on the bone marrow
to be enhanced. Hypothyroidism is known to inhibit hepatic
pyridine nucleotide-linked dehydrogenases (10). Administra
tion of DMBA causes an increase in hepatic menadione reductase
(9). Our initial experiments, however, indicate that the
increase in the hepatic level of this enzyme following adminis
tration of carcinogen is no different in hypothyroid animals.
Investigation of this aspect continues.
ACKNOWLEDGMENTS
The author is deeply indebted to Dr. Charles Muggins of the Ben May
Laboratory. University of Chicago, for his kindness and encouragement with this
work. He also wishes to thank Dr. Leslie De Groot, The Thyroid Study Group.
University of Chicago, and Wendy Radcliffe. Department of Biochemistry. Glas
gow Royal Infirmary, Scotland, for facilities to measure thyroid status.
REFERENCES
1. Aida, M. The effect of pituitary hormones on 7.1 2-dimethylbenz(a)anthracene-induced
rat leukemia. Wakayama Med. Rep.. Ã6.57-76. 1974
2. Bird. C. C. Leukemia induced by 7.8.12-trimethylbenz(a)anthracene in rats.
II. Changes in Bone Marrow. J. Nati. Cancer Inst., 48: 429-439. 1972.
3. Burgi, H., and Labhart. A. In: A. Labhart (ed.). The Thyroid Gland, Springer-
Verlag, pp. 135-283. New York: 1974.
4. Dargent, M., Viallier, J.. and Guinet. E. Influence de la secretion thyroïdienne
sur la Croissance des tumeurs. Ann. Inst. Pasteur (Paris), 8J. 357-361,
1951.
5. Ford, E., and Muggins, C. Selective destruction in testis induced by 7,12dimethylbenz(a)anthracene.
J. Exp. Med.. 118: 27-40. 1963.
6. Grad, B., Oberleitner. A., and Berenson. J. The effect of thyroxine and
thiouracil on théincidence of lymphogenous leukemia in AKR mice. Proc.
Am. Assoc. Cancer Res.. J. 20-21. 1953.
7. Huggins, C. B., and Oka, H. Regression of stem-cell erythroblastic leukemia
Thyro/d Hormone In Hormone-dependent Leukemia
after hypophysectomy. Cancer Res.. 32 239-242. 1972.
8. Huggins, C. B., and Sugiyama, T. Induction of leukemia in rat by pulse doses
of 7,12-dimethylbenz(a)anthracene. Proc. Nati. Acad. Sei. U. S. A., 55: 74-
81, 1966.
9. Huggins, C. B.. Ueda, N., and Russo, A. Azo dyes prevent hydrocarboninduced
leukemia in the rat Proc. Nati. Acad. Sei. U. S. A.. 75 4524-4527,
1978.
10. Huggins, C., and Yao. F-O. Influence of hormones on liver. 1. Effects of
steroids and thyroxine on pryidine nucleotide-linked dehydrogenases. J.
Exp. Med.. ; 10: 899-919, 1959.
11. Huggins. C. B., Yoshida, H.. and Bird, C. C. Hormone-dependent stem-cell
leukemia evoked by a series of feedings of 7,12-dimethylbenz(a)anthracene
J. Nati. Cancer Inst., 52. 1301-1305. 1974.
12. Juli, J. W., and Huggins, C. B. Influence of hyperthyroidism and of thyroidectomy
on induced mammary cancer. Nature (Lond.), 188: 73. 1960.
13. Kreyberg, L. The influence of intrinsic factors on the development of induced
tumors in animals. Acta Pathol. Microbiol. Scand. Suppl., 37; 317-338,
1938
14. Morimota, S. Influence of thyroidectomy on tar-epithelioma Acta Dermatol.
Kyoto (Engl. Ed.), )9. 185-186, 1932.
15. Morris. D. M.. Upton, C. A., and Wolf, F. F. The influence of the thyroid
gland on the susceptibility of AKR mice to transplanted lymphoid leukemia.
Proc. Am. Assoc. Cancer Res.. 2 36, 1955.
16. Murphy, B. P.. and Jachan, C. The determination of thyroxine by competitive
protein-binding analysis employing an anion-exchange resin and radiothyroxine.
J. Lab. Clin. Med.. 66. 161-167, 1965.
17. Reddi, A. H.. and Huggins. C. B. Formation of bone marrow in fibroblasttransformation
ossicles. Proc. Nati. Acad. Sei. U. S. A., 72 2212-2216.
1975.
18. Solviter, H. A. The effect of complete ablation of thyroid tissue by radioactive
iodine on the survival of tumor-bearing mice. Cancer Res.. ) /. 447-449,
1951.
19. Ueda, N., and Huggins, C B. Regression of experimental leukemias following
hypophysectomy. Proc Am. Assoc. Cancer Res , 16 13, 1975.
20. Welsch. C. W.. Horowitz. S., and Huggins. C. B. Influence of prolactin on
carcinogen-induced leukemagenesis in Long-Evans rats. Cancer Res., 35:
3746-3749. 1975.
21. Wolfson. S. L., Drake, J., and Bass. A. D. Effect of athyreosis on the
induction of rat sarcoma with 3-methylcholanthracene. Growth. 20. 19-28,
OCTOBER 1979 4255
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1956.