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Rat pituitary adenoma and hyperplasia induced by caffeine administration
Surg Neurol 1983;20:323-31
323
Rat Pituitary Adenoma and Hyperplasia Induced by Caffeine Administration Tatsuhito Yamagami, Hirofumi
Munemitsu,
M.D., Hajime
Handa, M.D., Juji Takeuchi, M.D.,
M.D., Michio Aoki, M.D., and Yuzuru
Kato, M.D.
Department of Neurosurgery and Second Division, Department of Internal Medicine, Kyoto University Medical School, Kyoto, Japan
Yamagami T, Handa H, TakeuchiJ, Munemitsu H, Aoki M, Kato Y. Rat pituitary adenoma and hyperplasia induced by caffeine administration. Surg Neurol 1983;20:323-31. The effect of caffeine by oral administration on female Wistar rats was studied for 12 months. High concentrations (2000/zg/mL) of caffeine caused a decrease in body weight and an increase in the weight of the pituitary gland in these rats. Increased pituitary weight was caused by the growth of a pituitary adenoma or hyperplasia. Pituitary adenomas and instances of hyperplasia were found in 27 of the 40 rats in the caffeine group, compared to 9 of the 30 rats in the control group. The histologic classification used was microadenoma, papillary (or sinusoidal) macroadenoma, and diffuse macroadenomas. These adenomas appeared to be endocrinologically nonfunctioning.
KEYWORDS: Caffeine; Pituitary adenoma; Pituitary tumor; Rat
Caffeine (1,3,7-trimethylxanthine) has long been known to affect D N A metabolism. Caffeine has inhibitory effects on D N A replication in cells damaged by ultraviolet radiation or chemical carcinogens [ 1,11,26,31]. The mutagenicity of caffeine has been established in Escherichia coli and cultured mammalian cells [9,15,16], but its oncogenicity remains controversial. Cole reported an association, greater in women than in men, between coffee drinking and cancer of the lower urinary tract [7]. On the other hand, an antineoplastic action of caffeine in mice has been reported [22,28]. N o m u r a found that the final yields of lung tumors were markedly reduced by caffeine treatment when young adult mice or mouse fetuses were treated with 4-nitroquinoline 1-oxide or urethane [22].
Address reprint requests to: Hajime Handa, M.D., Department of Neurosurgery, Kyoto University Medical School, 54 Shogoin kawahara-cho, Sakyo-ku, Kyoto 606, Japan.
© 1983by ElsevierSciencePublishingCo., Inc.
Estrogen-induced pituitary tumors in rats have been established as an experimental model of prolactinoma, but other methods of induction of pituitary tumors are also being sought. We investigated the effect of caffeine on the pituitary gland in rats and found that caffeine, when given for a long period, may cause hyperplasia or adenoma of the pituitary gland.
Materials and Methods Eighty female Wistar rats, starting at the age of i month, were given a 0.2% (2000/zg/mL) caffeine solution orally ad libitum for 12 months. The mean dose o f caffeine given during this period was 13.5 g per rat. For the control study, 40 rats were given water instead o f caffeine solution. Twelve months later, 40 rats in the caffeine group and all rats in the control group, which was reduced to 30 at the end of the study, were killed. T h e cause of death of 10 control rats during the experiment was not established. After urethane (0.5 mg/100 g body weight) was injected intraperitoneally, body weight was measured accurately and blood was removed by cardiac or jugular vein puncture. The blood was centrifuged immediately at 2000 revolutions/min for 15 minutes, and serum was preserved in a refrigerator at - 20°C for 2 - 4 weeks until the hormonal study. Radioimmunoassay was done by the double-antibody method for determination of basal levels of prolactin and thyroid stimulating hormone (TSH), and by the solid-phase method for determination of levels of growth hormone (GH). The antibodies o f prolactin, TSH, and G H were supplied by Daiichi Radioisotope (Tokyo), CalbiochemBehring Corp (San Diego, Calif.), and Pharmacia (Sweden), respectively. To investigate pituitary prolactin reserve, the sulpiride stimulation test was performed in 24 rats under urethane anesthesia before they were killed. This test was started after the rats were made to fast for 12 hours. 0090-~019/83/S~.00
324
Surg Neurol 1983;20:323-31
Yamagami et a!
To obtain the pituitary gland, the neck region was incised and the thyroid glands were removed, followed by decapitation and removal of the skull; the brain was cut at the first cervical nerve and removed from the skull base; and the pituitary gland was independently removed under an operating microscope. In addition, the abdomen was incised and the adrenal glands and ovaries were removed bilaterally. Fixation was done with Zamboni's fixative for the endocrinologic organs and with 10% formalin solution for the brain. The weight of each organ, after blotting with paper as much as possible, was measured as wet weight. These specimens were again fixed at 4°C for 24-72 hours. Specimens in Zamboni's fixative were washed with cold phosphate buffer solution (pH 7.2, 0.01 M) overnight, and the brain was washed with tap water. Pituitary glands were embedded in paraffin, cut to a thickness of 3-4 ~m, stained with hematoxylin-eosin, and immunohistochemically studied for prolactin, luteinizing hormone (LH), follicle stimulating hormone (FSH), and TSH. The antisera to these hormones were a kind gift from Dr. Parlow of the National Institute of Arthritis, Metabolism, and Digestive Diseases. Prolactin (1:500), LH (1:200), FSH (1:200), Figure 1. Semilogarithmic graph of body weight (abscissa) and pituitary
weight (ordinate). Body weight in the caffeine group (o) was less than that in the control group (o). The caffeine group had a greater number of heavy pituitary glands.
PRL ng/ml
GH ng/ml
TSH ~uU/ml
~.~ 4.1 3.(
0
• • •
0
. . . . . . . . . . ~0000 000000 ~0000 000000 ~000 O00QO@
O0 •
0.3 . . . . .
DO0001 2.(
• ''0''.,0
.....
0--
~0000 00100.
0
Control Caffeine
--0-
0
Caffeine
Control Caffeine
Figure 2. Hormonal study. Basal levels of plasma prolactin (PRL), growth hormone (GH), and thyroid stimulating hormone (TSH) were within the normal range in both groups.
and TSH (1:200), diluted with 3% normal rabbit serum, were studied by the indirect enzyme-labeled antibody method of Nakane and Pierce [21]. The t test was used for statistical analysis of the results.
pit.gland wt(mg)
Results
1000"
Body Weight and Pituitary Weight As shown in Figure 1, body weight in the group given caffeine was less than that in the control group (p < 0.01). Abnormally heavy pituitary glands (more than 30 mg) were predominantly found in the group given caffeine.
500 0°
~°
100,
°•
Endocrinologic Findings
•
60' 50' 40"
Basal prolactin, TSH, and G H values in the group given caffeine and in the control group were all within the normal range; there was no statistical difference between the two groups (Figure 2). The sulpiride (or dogmatyl) test for prolactin showed normal responses in 10 control rats and in 14 rats given caffeine. At autopsy, pituitary adenomas were found in 2 of 10 control rats and in 10 of 14 rats given caffeine (Figure 3).
30" 20"
"?~
2
o
10"
Histologic Findings i~o
260
3bo
4ao
s6o
,6o
Tbo body (gr)
wt
Histologic examination disclosed that 22 pituitary adenomas (55%) and 5 instances of hyperplasia (12.5%) were found among the 40 rats of the caffeine group,
Rat Pituitary Adenoma
Surg Neurol 1983,20:323-31
caffeine
control
group
Table 1. P i t u i t a r y T u m o r G r o w t h
J ~
group
PRL
Finding Normal
PRL
ng/ml
ng/ml
I0
Z|
48
|
min.
III
211
Controlgroup Caffeinegroup (n = 30) (n = 40) p value 21
13
Neoplasm Microadenoma Papillary macroadenoma Diffuse macroadenoma
8 3 4 1
22 11 6 5
Hyperplasia
1
5
Neoplasm + hyperplasia
9
27
< 0.02
< 0.005
48
rain.
Figure 3. Sulpiride Cdogmatyl) test for prolactin (PRL). A normal PRL response was confirmed in both groups.
compared to 8 pituitary adenomas (26.7%) and 1 instance of hyperplasia (3.3%) among the 30 rats of the control group (Table 1'). We regarded pituitary glands weighing more than 30 mg (wet weight) as having a high possibility of tumor. The histologic classification used in this study was similar to that used for human pituitary adenomas. T h e presence of macroadenoma was established when the pituitary gland (1) weighed more than 30 mg and (2) was replaced by the adenoma microscopically. As in human pituitary adenomas, papillary or sinusoidal type and diffuse type were seen in rat macroadenomas, according to the cell arrangement. On the other hand, hyperplasia was present when the weight of the pituitary gland was more than 30 mg and the cell population was increased.
Figure 4. Microadenoma. A nest of tumor cells i~ visible in the center. ( x I00.)
325
However, the cellular arrangement and staining characteristics of the hyperplasia were similar to those of the normal gland. Accordingly, we were able to classify pituitary glands into five groups: (1) microadenoma; (2) papillary macroadenoma; (3) diffuse macroadenoma; (4) hyperplasia; and (5) normal. M i c r o a d e n o m a . Figure 4 illustrates a typical microadenoma. A group of tumor cells was seen compressing the surrounding normal pituitary cells. The arrangement of the tumor cells was papillary or diffuse. The weights of pituitary glands with microadenomas were in the normal range (less than 30 mg). Figure 5 shows a higher magnification of the microadenoma in Figure 4. The cytoplasm of the cells was distended and enlarged. The prominent nuclei were also enlarged and pleomorphic. The arrangement of the cells was sinusoidal. Most microadenomas were found off the midline.
326
Surg Neurol 1983;20:323-31
Yamagami et al
Figure 5. Microadenoma. Cytoplasm is distended and enlarged. Nuclei are enlarged and pleomorphic. ~x 400.)
T h r e e rats in the control group (10%) and eleven in the caffeine group (27.5%) had microadenomas; among them, one in the control group and two in the caffeine group showed independent multiple microadenomas in both lateral wings.
cytoplasm was chromophobic, with prominent nuclei. Atypia was rarely seen (Figure 7). Four rats in the control group (13.3%) and six in the caffeine group (15%) had papillary or sinusoidal macroadenomas.
The arrangement o f the cells papillary macroadenomas was gensinusoidal (Figure 6). Higher maguniform cell size. The cells were not shape was cuboidal or polygonal. The
D i f f u s e macroadenoma. Adenoma cells were distributed in a diffuse pattern. The normal anterior lobe was extremely compressed and appeared as a very thin strand. The cell population was high, and the cytoplasm was chromophobic (Figure 8).
Papillary macroadenoma.
in the group with erally papillary or nification revealed pleomorphic; their
Figure 6. Papillary macroadenoma. Cell arrangement is sinusoidaL ( x lOOj
Rat Pituitary Adenoma
Surg Neurol 1983;20:323-31
327
Figure 7. Papillary macroadenoma. Cells are cuboidal or polygonal Cytoplasm is chromophobic, f x 400.)
One rat in the control group (3.3%) and five in the caffeine group (12.5%) had diffuse macroadenomas.
Hyperplasia. The weight of the pituitary gland and the cell population were increased. Cells were smaller than adenoma cells and closely packed. However, cellular arrangement and stained appearance were not distinguishable from that of normal pituitary gland (Figure 9). In one rat, very large cells with eosinophilic cytoplasm were observed sporadically. One rat in the control group (3.3%) and five in the caffeine group (12.5%) had hyperplasia.
Figure 8. Diffuse macroadenoma. Cell population is high, cytoplasm is chromophobic, and pituitary weight is markedly increased. ( x 200.)
As a result, pituitary adenomas and instances of hyperplasia were found in 27 of the 40 rats in the caffeine group (67.5%), compared to 9 of the 30 rats in the control group (33.3%). This difference was statistically significant (p < 0.005).
Immunohistochemical Findings In microadenomas of the caffeine group, the adenoma cells were not stained by antiprolactin antibody; however, cells in the normal gland surrounding the tumor were stained (Figure 10).
328
Surg Neurol 1983;20:323-31
Yamagami et al
Figure 9. Hyperplasia. Pituitary weight is slightly more than 30 mg. Cellular arrangement and staining characteristics are only slightly abnormal Cell population is high. ( x 200.)
In macroadenomas, the adenoma cells were not stained by antiprolactin antibody, but the residual compressed normal anterior lobe and stalk were stained (Figure 11). Only two papillary adenomas, which were in rats with mammary or accessory mammary gland tumors, reacted with antiprolactin antibody. In instances of hyperplasia, a reaction to antiprolactin antibody comparable to that in the normal pituitary gland was seen. O f three microadenomas in the control group, two reacted with antiprolactin antibody. O f four papillary adenomas, only one reacted; this rat had a tumor o f the
mammary gland. Table 2 summarizes the response to antiprolactin antibody. Normal cells responded with antibodies to LH, FSH, and TSH, but adenoma cells did not.
Discussion Our results showed that pituitary weight was increased and body weight was decreased in the caffeine group. Wiklund et al stated that female F 344 rats with pituitary weights greater than 19 mg were considered susceptible to tumor development [32]. We judged that rats with
Figure lO. Microadenoma in the caffeine group, lmmunohistochemical study for prolactin. Left." A nest of tumor cells that did not respond to prolactin. Right: Area surrounding normal pituitary cells stained with prolactin. (Methyl green nuclear stain: x 200.)
Rat Pituitary Adenorna
pituitary wet weights greater than 30 mg were susceptible to the development of adenoma or hyperplasia, which causes the greater pituitary weight. T h e cause of decreased body weight [4] might be (1) promotion of lipolysis by phosphodiesterase inhibition in adipose tissue [2,5]; (2) caffeine-induced diarrhea; (3) loss of appetite [20,25]; or (4) a cachectic state resulting from a pituitary tumor. Kihlman et al proposed that cellular damage at the D N A level could occur in any tissue [14]. Spindel et al reported that a single intraperitoneal injection of caffeine resulted in a slight increase of serum prolactin levels, a decrease of serum T S H levels, and an inhibition of pulsatile G H release [30]. In our experiment, oral administration o f caffeine did not cause an increase in serum levels ofprolactin. This difference may be attributable to the experimental method. Pituitary adenomas in rats given caffeine did not yield assayable hormones. This finding is in marked contrast to the prolactinoma induced by estrogen [6,18] or dimethylbenzanthracene [12] administration. The concentration of the caffeine solution used in this study was much greater than that in commercially available tea or coffee. It is well known that the rat is one of the animals in which spontaneous pituitary adenomas develop frequently. Although the incidence differs among the various species, it usually becomes higher with increasing age [23,24,29] (Table 3). The presence of pituitary adenoma is an important factor in rat survival [23]. We did not test whether or not the changes induced by caffeine were reversible; this test would require 18 months of caffeine administration, followed by a 6-month withdrawal period.
F i g u r e 11. Sagittal section of diffuse macroadenoma in the caffeine group. Right: Compressed anterior lobe and part of pituitary stalk, which responded to prolactin. However, macroadenoma cells are not stained. ~× 20.)
Surg Neurol 1983;20:323-31
329
Table 2. Positive Prolactin Stain of Tumor Cell Macroadenoma Group
Microadenorna
Papillary
Diffuse
Hyperplasia
Control Caffeine
2/3 0/10
1"/4 2~/6
0/2
l/1 5/5
~Rats with tumors of mammary or accessory mammary glands.
In human pituitary adenomas no difference in incidence is seen with respect to sex distribution; microadenomas, however, occur predominantly in females. As female sex determinants affect the development o f pituitary adenomas, female rats were used in our experiments. We would like to stress that some environmental factors [8], including caffeine, could induce pituitary adenoma, and that the untoward effects o f caffeine shown in our study should be studied more extensively. The mutagenic effects of caffeine, represented by inhibition of postreplication repair at the D N A level [13,16,17,19], may be related to the development of rat pituitary adenomas or to metabolic changes o f catecholamines by caffeine [ 3, I 0,27 ].
Summary The effect of orally administered caffeine was studied in female Wistar rats for 12 months. Caffeine administration caused a decrease in body weight and an increase in the weight of the pituitary gland in these rats. Increased pituitary weight was caused by the develop-
330
Surg N e u r o l 1983;20:323-31
Yamagami et al
T a b l e 3. Spontaneous Tumor Growth Rate in Rat Anterior Pituitary Gland Author~
Strain
Wolfe (1938)
OM b
Saxton (1944)
Wd OM OM ~
Oberling & Gu6rin (1950) Schulze (1960) Kim (1960) Jacob (1968) Kwa (1969) Festing (1973)
Hansen (1974)
S-D Bethesda-B W/FU F 344 R (Amsterdam) ACI BUF WN ACI/N BUF/N
Maekawa (1975)
F 344/N M 520/N OM/N WN/N ACI/N
Sass (1975)
F 344
Hollander ( 1976)
WAG/Rij BN/Bi
Coleman (1977)
F 344
Incidence (%)
Age (mo)
Sex
12 29 68 60 30 5 52 61 13 30 1.1 5.8 1.8 27 11 3 25-29 15 30 21-25 15-40 5-20 55-75 25 20-40 15- 2 0 40-93 6 21 24 36 96 68 8 23 15
18-30 17-24 17-28
M F F M F
7-13 14-24 > 20 14-20 > 20
M
> 17
F M F F
28 Older Older > 18 < 18 >18 > 18 >18 > 18 > 18 >22 >19
21 31 21 28 > 18
M M F F
M F M F M F M F M
Abbreviations: mo = month; M = male; F = female. ~Studiescited in references 23, 24, and 29.
ment of pituitary adenoma or hyperplasia. These adenomas appeared to be endocrinologically nonfunctioning.
References 1. Ahnstr6m G. Repair processes in germinating seeds: Caffeine enhancement of damage induced by gamma-radiation and alkylating chemicals. Mutat Res 1974;26:99-103. 2. Beavo JA, Rogers NL, Crofford OB, Baird CE, Hardman JG, Sutherland EW, Newmann EV. Effects of phosphodiesterase inhibitors on cyclic AMP levels and on lipolysis. Ann NY Acad Sci 1971;185:129-36. 3. Belier S, Roman L, DeCastro O, Kim KE, Kershbaum A. Effects of coffee ingestion on catecholamine release. Metabolism 1969;18:288-91. 4. Burg AW, Burrows R, Kensler CJ. Unusual metabolism of caffeine in the squirrel monkey. Toxicol Appl Pharmaco11974;28:1626.
5. Cardinali DP. Methylxanthines: Possible mechanisms of action in brain. Trends Pharmacol Sci 1980;1:405-7. 6. Clifton KH, Meyer RK. Mechanism of anterior pituitary tumor induction by estrogen. Anat Rec 1956;125:65-81. 7. Cole P. Coffee-drinking and cancer of the lower urinary tract. Lancet 1971;1:1335-7. 8. Collins WF. Adenomas ofthe pituitary gland--An epidemic? Surg Clin North Am 1980;60:1201-6. 9. Donovan PJ, DiPaolo JA. Caffeine enhancement of chemical carcinogen induced transformation of cultured Syrian hamster cells. Cancer Res 1974;34:2720-7. 10. Enslen M, Milon H, Wiirzner HP. Brain catecholamines and sleep states in offspring of caffeine-treated rats. Experientia 1980;36:1105-6. 11. Fujiwara Y, Kondo T. Caffeine sensitive repair of ultraviolet lightdamaged DNA of mouse L cells. Biochem Biophys Res Commun 1972;47:557-64. 12. Heuson-Stiennon JA, Danguy A, Heuson JC, Pasteels JL. Development of prolactin-secreting pituitary microadenomas in the rat after DMBA administration. In: Faglia G, Giovanelli M,
Rat Pituitary A d e n o m a
13. 14.
15. 16.
17. 18.
19.
20.
MacLeod RM, eds. Pituitary microadenomas. New York: Academic Press, 1980:223-7. Kihlman BA. Effects of caffeine on the genetic material. Mutat Res 1974;26:53-71, Kihlman BA, Sturelid S, Hartley-Asp B, Nilsson K. The enhancement by caffeine of the frequencies of chromosomal aberrations induced in plant and animal cells by chemical and physical agents. Mutat Res 1974;26:105-22. Lee S. Chromosome aberrations induced in cultured human cells by caffeine. Jpn J Genet 1971;46:337-44. Lehmann AR, Kirk-Bell S. Effects of caffeine and theophylline on D N A synthesis in unirradiated and UV-irradiated mammalian cells. Mutat Res 1974;26:73-82. Loprieno N, Barcle R, Baroncelli S. Genetic effects of caffeine. Mutat Res 1974;26:83-7. Lundin PM, Schelin U. Light and electron microscopical studies on the pituitary in stilbol-treated rats. Acta Pathol Microbiol Scand 1962;54:66-74. Mendelson D, Sobels H. The inhibitory effects of caffeine on the material repair of radiation-induced chromosome breaks in Drosophila. Mutat Res 1974;26:123-8. Merkel AD, Wayner MJ, Jolicoeur FB, Mintz R. Effects of caffeine administration on food and water consumption under various experimental conditions. Pharmacol Biochem Behav 1981;14:235-40.
21. Nakane PK, Pierce GB. Enzyme-labeled antibodies: Preparation and application for the localization of antigens. J Histochem Cytochem 1966;14:929-31. 22. Nomura T. Timing of chemically induced neoplasia in mice revealed by the antineoplastic action of caffeine. Cancer Res 1980;40:1332-40.
Surg N e u r o l 1983;20:323-31
331
23. Norman HA, Goodman DG. Neoplastic disease. In: Baker HJ, et al., eds. The laboratory rat. Vol 1. New York: Academic Press 1979;368-71. 24. Oberling C. Gu6rin P, Gudrin M. Les tumeurs hypophysaires spontan~es chez le rat. Bull Assoc Fr Etude Cancer 1950;37:8398. 25. Peters JM, Boyd EM. The influence of sex and age in albino rats given a daily oral dose of caffeine at a high dose level. Can J Physiol Pharmacol 1967;45:305-11. 26. Roberts JJ, Sturrock JE, Ward KN. The enhancement by caffeine of alkylation-induced cell death, mutation and chromosomal ab~ errations in Chinese hamster cells, as a result of inhibition of post~ replication D N A repair. Mutat Res 1974;26:129-43. 27. Robertson D, Fr61ich JC, Carr RK, Watson T, Hollifield JW, Schand DG, Oates JA. Effects of caffeine on plasma renin activity, catecholamines and blood pressure. N EnglJ Med 1978;298:1816. 28. Rothwell K. Dose-related inhibition of chemical carcinogenesis in mouse skin by caffeine. Nature 1974;252:69-70. 29. Schulze E. Spontantumoren der Schiidelh6hle und Genitalorgane bei Sprague-Dawley- und Bethesda-Black-Ratten. Z Krebsforsch 1960;64:78-82. 30. Spindel E, Arnold M, Cusack B, Wurtman J. Effects of caffeine on anterior pituitary function in the rat. J Pharmacol Exp Ther 1980;214:58-62. 31. Swietlinska Z, Z~ik J. Effects of caffeine on chromosome damage induced by chemical mutagens and ionizing radiation in vicia fava and secale cereale. Mutat Res 1974;26:89-97. 32. Wiklund J, Rutledge J, Gorski J. A genetic model fi~r the inheritance of pituitary tumor susceptibility in F 344 rats. Endocrinology 1981;109:1708-14.
323
Rat Pituitary Adenoma and Hyperplasia Induced by Caffeine Administration Tatsuhito Yamagami, Hirofumi
Munemitsu,
M.D., Hajime
Handa, M.D., Juji Takeuchi, M.D.,
M.D., Michio Aoki, M.D., and Yuzuru
Kato, M.D.
Department of Neurosurgery and Second Division, Department of Internal Medicine, Kyoto University Medical School, Kyoto, Japan
Yamagami T, Handa H, TakeuchiJ, Munemitsu H, Aoki M, Kato Y. Rat pituitary adenoma and hyperplasia induced by caffeine administration. Surg Neurol 1983;20:323-31. The effect of caffeine by oral administration on female Wistar rats was studied for 12 months. High concentrations (2000/zg/mL) of caffeine caused a decrease in body weight and an increase in the weight of the pituitary gland in these rats. Increased pituitary weight was caused by the growth of a pituitary adenoma or hyperplasia. Pituitary adenomas and instances of hyperplasia were found in 27 of the 40 rats in the caffeine group, compared to 9 of the 30 rats in the control group. The histologic classification used was microadenoma, papillary (or sinusoidal) macroadenoma, and diffuse macroadenomas. These adenomas appeared to be endocrinologically nonfunctioning.
KEYWORDS: Caffeine; Pituitary adenoma; Pituitary tumor; Rat
Caffeine (1,3,7-trimethylxanthine) has long been known to affect D N A metabolism. Caffeine has inhibitory effects on D N A replication in cells damaged by ultraviolet radiation or chemical carcinogens [ 1,11,26,31]. The mutagenicity of caffeine has been established in Escherichia coli and cultured mammalian cells [9,15,16], but its oncogenicity remains controversial. Cole reported an association, greater in women than in men, between coffee drinking and cancer of the lower urinary tract [7]. On the other hand, an antineoplastic action of caffeine in mice has been reported [22,28]. N o m u r a found that the final yields of lung tumors were markedly reduced by caffeine treatment when young adult mice or mouse fetuses were treated with 4-nitroquinoline 1-oxide or urethane [22].
Address reprint requests to: Hajime Handa, M.D., Department of Neurosurgery, Kyoto University Medical School, 54 Shogoin kawahara-cho, Sakyo-ku, Kyoto 606, Japan.
© 1983by ElsevierSciencePublishingCo., Inc.
Estrogen-induced pituitary tumors in rats have been established as an experimental model of prolactinoma, but other methods of induction of pituitary tumors are also being sought. We investigated the effect of caffeine on the pituitary gland in rats and found that caffeine, when given for a long period, may cause hyperplasia or adenoma of the pituitary gland.
Materials and Methods Eighty female Wistar rats, starting at the age of i month, were given a 0.2% (2000/zg/mL) caffeine solution orally ad libitum for 12 months. The mean dose o f caffeine given during this period was 13.5 g per rat. For the control study, 40 rats were given water instead o f caffeine solution. Twelve months later, 40 rats in the caffeine group and all rats in the control group, which was reduced to 30 at the end of the study, were killed. T h e cause of death of 10 control rats during the experiment was not established. After urethane (0.5 mg/100 g body weight) was injected intraperitoneally, body weight was measured accurately and blood was removed by cardiac or jugular vein puncture. The blood was centrifuged immediately at 2000 revolutions/min for 15 minutes, and serum was preserved in a refrigerator at - 20°C for 2 - 4 weeks until the hormonal study. Radioimmunoassay was done by the double-antibody method for determination of basal levels of prolactin and thyroid stimulating hormone (TSH), and by the solid-phase method for determination of levels of growth hormone (GH). The antibodies o f prolactin, TSH, and G H were supplied by Daiichi Radioisotope (Tokyo), CalbiochemBehring Corp (San Diego, Calif.), and Pharmacia (Sweden), respectively. To investigate pituitary prolactin reserve, the sulpiride stimulation test was performed in 24 rats under urethane anesthesia before they were killed. This test was started after the rats were made to fast for 12 hours. 0090-~019/83/S~.00
324
Surg Neurol 1983;20:323-31
Yamagami et a!
To obtain the pituitary gland, the neck region was incised and the thyroid glands were removed, followed by decapitation and removal of the skull; the brain was cut at the first cervical nerve and removed from the skull base; and the pituitary gland was independently removed under an operating microscope. In addition, the abdomen was incised and the adrenal glands and ovaries were removed bilaterally. Fixation was done with Zamboni's fixative for the endocrinologic organs and with 10% formalin solution for the brain. The weight of each organ, after blotting with paper as much as possible, was measured as wet weight. These specimens were again fixed at 4°C for 24-72 hours. Specimens in Zamboni's fixative were washed with cold phosphate buffer solution (pH 7.2, 0.01 M) overnight, and the brain was washed with tap water. Pituitary glands were embedded in paraffin, cut to a thickness of 3-4 ~m, stained with hematoxylin-eosin, and immunohistochemically studied for prolactin, luteinizing hormone (LH), follicle stimulating hormone (FSH), and TSH. The antisera to these hormones were a kind gift from Dr. Parlow of the National Institute of Arthritis, Metabolism, and Digestive Diseases. Prolactin (1:500), LH (1:200), FSH (1:200), Figure 1. Semilogarithmic graph of body weight (abscissa) and pituitary
weight (ordinate). Body weight in the caffeine group (o) was less than that in the control group (o). The caffeine group had a greater number of heavy pituitary glands.
PRL ng/ml
GH ng/ml
TSH ~uU/ml
~.~ 4.1 3.(
0
• • •
0
. . . . . . . . . . ~0000 000000 ~0000 000000 ~000 O00QO@
O0 •
0.3 . . . . .
DO0001 2.(
• ''0''.,0
.....
0--
~0000 00100.
0
Control Caffeine
--0-
0
Caffeine
Control Caffeine
Figure 2. Hormonal study. Basal levels of plasma prolactin (PRL), growth hormone (GH), and thyroid stimulating hormone (TSH) were within the normal range in both groups.
and TSH (1:200), diluted with 3% normal rabbit serum, were studied by the indirect enzyme-labeled antibody method of Nakane and Pierce [21]. The t test was used for statistical analysis of the results.
pit.gland wt(mg)
Results
1000"
Body Weight and Pituitary Weight As shown in Figure 1, body weight in the group given caffeine was less than that in the control group (p < 0.01). Abnormally heavy pituitary glands (more than 30 mg) were predominantly found in the group given caffeine.
500 0°
~°
100,
°•
Endocrinologic Findings
•
60' 50' 40"
Basal prolactin, TSH, and G H values in the group given caffeine and in the control group were all within the normal range; there was no statistical difference between the two groups (Figure 2). The sulpiride (or dogmatyl) test for prolactin showed normal responses in 10 control rats and in 14 rats given caffeine. At autopsy, pituitary adenomas were found in 2 of 10 control rats and in 10 of 14 rats given caffeine (Figure 3).
30" 20"
"?~
2
o
10"
Histologic Findings i~o
260
3bo
4ao
s6o
,6o
Tbo body (gr)
wt
Histologic examination disclosed that 22 pituitary adenomas (55%) and 5 instances of hyperplasia (12.5%) were found among the 40 rats of the caffeine group,
Rat Pituitary Adenoma
Surg Neurol 1983,20:323-31
caffeine
control
group
Table 1. P i t u i t a r y T u m o r G r o w t h
J ~
group
PRL
Finding Normal
PRL
ng/ml
ng/ml
I0
Z|
48
|
min.
III
211
Controlgroup Caffeinegroup (n = 30) (n = 40) p value 21
13
Neoplasm Microadenoma Papillary macroadenoma Diffuse macroadenoma
8 3 4 1
22 11 6 5
Hyperplasia
1
5
Neoplasm + hyperplasia
9
27
< 0.02
< 0.005
48
rain.
Figure 3. Sulpiride Cdogmatyl) test for prolactin (PRL). A normal PRL response was confirmed in both groups.
compared to 8 pituitary adenomas (26.7%) and 1 instance of hyperplasia (3.3%) among the 30 rats of the control group (Table 1'). We regarded pituitary glands weighing more than 30 mg (wet weight) as having a high possibility of tumor. The histologic classification used in this study was similar to that used for human pituitary adenomas. T h e presence of macroadenoma was established when the pituitary gland (1) weighed more than 30 mg and (2) was replaced by the adenoma microscopically. As in human pituitary adenomas, papillary or sinusoidal type and diffuse type were seen in rat macroadenomas, according to the cell arrangement. On the other hand, hyperplasia was present when the weight of the pituitary gland was more than 30 mg and the cell population was increased.
Figure 4. Microadenoma. A nest of tumor cells i~ visible in the center. ( x I00.)
325
However, the cellular arrangement and staining characteristics of the hyperplasia were similar to those of the normal gland. Accordingly, we were able to classify pituitary glands into five groups: (1) microadenoma; (2) papillary macroadenoma; (3) diffuse macroadenoma; (4) hyperplasia; and (5) normal. M i c r o a d e n o m a . Figure 4 illustrates a typical microadenoma. A group of tumor cells was seen compressing the surrounding normal pituitary cells. The arrangement of the tumor cells was papillary or diffuse. The weights of pituitary glands with microadenomas were in the normal range (less than 30 mg). Figure 5 shows a higher magnification of the microadenoma in Figure 4. The cytoplasm of the cells was distended and enlarged. The prominent nuclei were also enlarged and pleomorphic. The arrangement of the cells was sinusoidal. Most microadenomas were found off the midline.
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Figure 5. Microadenoma. Cytoplasm is distended and enlarged. Nuclei are enlarged and pleomorphic. ~x 400.)
T h r e e rats in the control group (10%) and eleven in the caffeine group (27.5%) had microadenomas; among them, one in the control group and two in the caffeine group showed independent multiple microadenomas in both lateral wings.
cytoplasm was chromophobic, with prominent nuclei. Atypia was rarely seen (Figure 7). Four rats in the control group (13.3%) and six in the caffeine group (15%) had papillary or sinusoidal macroadenomas.
The arrangement o f the cells papillary macroadenomas was gensinusoidal (Figure 6). Higher maguniform cell size. The cells were not shape was cuboidal or polygonal. The
D i f f u s e macroadenoma. Adenoma cells were distributed in a diffuse pattern. The normal anterior lobe was extremely compressed and appeared as a very thin strand. The cell population was high, and the cytoplasm was chromophobic (Figure 8).
Papillary macroadenoma.
in the group with erally papillary or nification revealed pleomorphic; their
Figure 6. Papillary macroadenoma. Cell arrangement is sinusoidaL ( x lOOj
Rat Pituitary Adenoma
Surg Neurol 1983;20:323-31
327
Figure 7. Papillary macroadenoma. Cells are cuboidal or polygonal Cytoplasm is chromophobic, f x 400.)
One rat in the control group (3.3%) and five in the caffeine group (12.5%) had diffuse macroadenomas.
Hyperplasia. The weight of the pituitary gland and the cell population were increased. Cells were smaller than adenoma cells and closely packed. However, cellular arrangement and stained appearance were not distinguishable from that of normal pituitary gland (Figure 9). In one rat, very large cells with eosinophilic cytoplasm were observed sporadically. One rat in the control group (3.3%) and five in the caffeine group (12.5%) had hyperplasia.
Figure 8. Diffuse macroadenoma. Cell population is high, cytoplasm is chromophobic, and pituitary weight is markedly increased. ( x 200.)
As a result, pituitary adenomas and instances of hyperplasia were found in 27 of the 40 rats in the caffeine group (67.5%), compared to 9 of the 30 rats in the control group (33.3%). This difference was statistically significant (p < 0.005).
Immunohistochemical Findings In microadenomas of the caffeine group, the adenoma cells were not stained by antiprolactin antibody; however, cells in the normal gland surrounding the tumor were stained (Figure 10).
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Figure 9. Hyperplasia. Pituitary weight is slightly more than 30 mg. Cellular arrangement and staining characteristics are only slightly abnormal Cell population is high. ( x 200.)
In macroadenomas, the adenoma cells were not stained by antiprolactin antibody, but the residual compressed normal anterior lobe and stalk were stained (Figure 11). Only two papillary adenomas, which were in rats with mammary or accessory mammary gland tumors, reacted with antiprolactin antibody. In instances of hyperplasia, a reaction to antiprolactin antibody comparable to that in the normal pituitary gland was seen. O f three microadenomas in the control group, two reacted with antiprolactin antibody. O f four papillary adenomas, only one reacted; this rat had a tumor o f the
mammary gland. Table 2 summarizes the response to antiprolactin antibody. Normal cells responded with antibodies to LH, FSH, and TSH, but adenoma cells did not.
Discussion Our results showed that pituitary weight was increased and body weight was decreased in the caffeine group. Wiklund et al stated that female F 344 rats with pituitary weights greater than 19 mg were considered susceptible to tumor development [32]. We judged that rats with
Figure lO. Microadenoma in the caffeine group, lmmunohistochemical study for prolactin. Left." A nest of tumor cells that did not respond to prolactin. Right: Area surrounding normal pituitary cells stained with prolactin. (Methyl green nuclear stain: x 200.)
Rat Pituitary Adenorna
pituitary wet weights greater than 30 mg were susceptible to the development of adenoma or hyperplasia, which causes the greater pituitary weight. T h e cause of decreased body weight [4] might be (1) promotion of lipolysis by phosphodiesterase inhibition in adipose tissue [2,5]; (2) caffeine-induced diarrhea; (3) loss of appetite [20,25]; or (4) a cachectic state resulting from a pituitary tumor. Kihlman et al proposed that cellular damage at the D N A level could occur in any tissue [14]. Spindel et al reported that a single intraperitoneal injection of caffeine resulted in a slight increase of serum prolactin levels, a decrease of serum T S H levels, and an inhibition of pulsatile G H release [30]. In our experiment, oral administration o f caffeine did not cause an increase in serum levels ofprolactin. This difference may be attributable to the experimental method. Pituitary adenomas in rats given caffeine did not yield assayable hormones. This finding is in marked contrast to the prolactinoma induced by estrogen [6,18] or dimethylbenzanthracene [12] administration. The concentration of the caffeine solution used in this study was much greater than that in commercially available tea or coffee. It is well known that the rat is one of the animals in which spontaneous pituitary adenomas develop frequently. Although the incidence differs among the various species, it usually becomes higher with increasing age [23,24,29] (Table 3). The presence of pituitary adenoma is an important factor in rat survival [23]. We did not test whether or not the changes induced by caffeine were reversible; this test would require 18 months of caffeine administration, followed by a 6-month withdrawal period.
F i g u r e 11. Sagittal section of diffuse macroadenoma in the caffeine group. Right: Compressed anterior lobe and part of pituitary stalk, which responded to prolactin. However, macroadenoma cells are not stained. ~× 20.)
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Table 2. Positive Prolactin Stain of Tumor Cell Macroadenoma Group
Microadenorna
Papillary
Diffuse
Hyperplasia
Control Caffeine
2/3 0/10
1"/4 2~/6
0/2
l/1 5/5
~Rats with tumors of mammary or accessory mammary glands.
In human pituitary adenomas no difference in incidence is seen with respect to sex distribution; microadenomas, however, occur predominantly in females. As female sex determinants affect the development o f pituitary adenomas, female rats were used in our experiments. We would like to stress that some environmental factors [8], including caffeine, could induce pituitary adenoma, and that the untoward effects o f caffeine shown in our study should be studied more extensively. The mutagenic effects of caffeine, represented by inhibition of postreplication repair at the D N A level [13,16,17,19], may be related to the development of rat pituitary adenomas or to metabolic changes o f catecholamines by caffeine [ 3, I 0,27 ].
Summary The effect of orally administered caffeine was studied in female Wistar rats for 12 months. Caffeine administration caused a decrease in body weight and an increase in the weight of the pituitary gland in these rats. Increased pituitary weight was caused by the develop-
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Yamagami et al
T a b l e 3. Spontaneous Tumor Growth Rate in Rat Anterior Pituitary Gland Author~
Strain
Wolfe (1938)
OM b
Saxton (1944)
Wd OM OM ~
Oberling & Gu6rin (1950) Schulze (1960) Kim (1960) Jacob (1968) Kwa (1969) Festing (1973)
Hansen (1974)
S-D Bethesda-B W/FU F 344 R (Amsterdam) ACI BUF WN ACI/N BUF/N
Maekawa (1975)
F 344/N M 520/N OM/N WN/N ACI/N
Sass (1975)
F 344
Hollander ( 1976)
WAG/Rij BN/Bi
Coleman (1977)
F 344
Incidence (%)
Age (mo)
Sex
12 29 68 60 30 5 52 61 13 30 1.1 5.8 1.8 27 11 3 25-29 15 30 21-25 15-40 5-20 55-75 25 20-40 15- 2 0 40-93 6 21 24 36 96 68 8 23 15
18-30 17-24 17-28
M F F M F
7-13 14-24 > 20 14-20 > 20
M
> 17
F M F F
28 Older Older > 18 < 18 >18 > 18 >18 > 18 > 18 >22 >19
21 31 21 28 > 18
M M F F
M F M F M F M F M
Abbreviations: mo = month; M = male; F = female. ~Studiescited in references 23, 24, and 29.
ment of pituitary adenoma or hyperplasia. These adenomas appeared to be endocrinologically nonfunctioning.
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5. Cardinali DP. Methylxanthines: Possible mechanisms of action in brain. Trends Pharmacol Sci 1980;1:405-7. 6. Clifton KH, Meyer RK. Mechanism of anterior pituitary tumor induction by estrogen. Anat Rec 1956;125:65-81. 7. Cole P. Coffee-drinking and cancer of the lower urinary tract. Lancet 1971;1:1335-7. 8. Collins WF. Adenomas ofthe pituitary gland--An epidemic? Surg Clin North Am 1980;60:1201-6. 9. Donovan PJ, DiPaolo JA. Caffeine enhancement of chemical carcinogen induced transformation of cultured Syrian hamster cells. Cancer Res 1974;34:2720-7. 10. Enslen M, Milon H, Wiirzner HP. Brain catecholamines and sleep states in offspring of caffeine-treated rats. Experientia 1980;36:1105-6. 11. Fujiwara Y, Kondo T. Caffeine sensitive repair of ultraviolet lightdamaged DNA of mouse L cells. Biochem Biophys Res Commun 1972;47:557-64. 12. Heuson-Stiennon JA, Danguy A, Heuson JC, Pasteels JL. Development of prolactin-secreting pituitary microadenomas in the rat after DMBA administration. In: Faglia G, Giovanelli M,
Rat Pituitary A d e n o m a
13. 14.
15. 16.
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MacLeod RM, eds. Pituitary microadenomas. New York: Academic Press, 1980:223-7. Kihlman BA. Effects of caffeine on the genetic material. Mutat Res 1974;26:53-71, Kihlman BA, Sturelid S, Hartley-Asp B, Nilsson K. The enhancement by caffeine of the frequencies of chromosomal aberrations induced in plant and animal cells by chemical and physical agents. Mutat Res 1974;26:105-22. Lee S. Chromosome aberrations induced in cultured human cells by caffeine. Jpn J Genet 1971;46:337-44. Lehmann AR, Kirk-Bell S. Effects of caffeine and theophylline on D N A synthesis in unirradiated and UV-irradiated mammalian cells. Mutat Res 1974;26:73-82. Loprieno N, Barcle R, Baroncelli S. Genetic effects of caffeine. Mutat Res 1974;26:83-7. Lundin PM, Schelin U. Light and electron microscopical studies on the pituitary in stilbol-treated rats. Acta Pathol Microbiol Scand 1962;54:66-74. Mendelson D, Sobels H. The inhibitory effects of caffeine on the material repair of radiation-induced chromosome breaks in Drosophila. Mutat Res 1974;26:123-8. Merkel AD, Wayner MJ, Jolicoeur FB, Mintz R. Effects of caffeine administration on food and water consumption under various experimental conditions. Pharmacol Biochem Behav 1981;14:235-40.
21. Nakane PK, Pierce GB. Enzyme-labeled antibodies: Preparation and application for the localization of antigens. J Histochem Cytochem 1966;14:929-31. 22. Nomura T. Timing of chemically induced neoplasia in mice revealed by the antineoplastic action of caffeine. Cancer Res 1980;40:1332-40.
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23. Norman HA, Goodman DG. Neoplastic disease. In: Baker HJ, et al., eds. The laboratory rat. Vol 1. New York: Academic Press 1979;368-71. 24. Oberling C. Gu6rin P, Gudrin M. Les tumeurs hypophysaires spontan~es chez le rat. Bull Assoc Fr Etude Cancer 1950;37:8398. 25. Peters JM, Boyd EM. The influence of sex and age in albino rats given a daily oral dose of caffeine at a high dose level. Can J Physiol Pharmacol 1967;45:305-11. 26. Roberts JJ, Sturrock JE, Ward KN. The enhancement by caffeine of alkylation-induced cell death, mutation and chromosomal ab~ errations in Chinese hamster cells, as a result of inhibition of post~ replication D N A repair. Mutat Res 1974;26:129-43. 27. Robertson D, Fr61ich JC, Carr RK, Watson T, Hollifield JW, Schand DG, Oates JA. Effects of caffeine on plasma renin activity, catecholamines and blood pressure. N EnglJ Med 1978;298:1816. 28. Rothwell K. Dose-related inhibition of chemical carcinogenesis in mouse skin by caffeine. Nature 1974;252:69-70. 29. Schulze E. Spontantumoren der Schiidelh6hle und Genitalorgane bei Sprague-Dawley- und Bethesda-Black-Ratten. Z Krebsforsch 1960;64:78-82. 30. Spindel E, Arnold M, Cusack B, Wurtman J. Effects of caffeine on anterior pituitary function in the rat. J Pharmacol Exp Ther 1980;214:58-62. 31. Swietlinska Z, Z~ik J. Effects of caffeine on chromosome damage induced by chemical mutagens and ionizing radiation in vicia fava and secale cereale. Mutat Res 1974;26:89-97. 32. Wiklund J, Rutledge J, Gorski J. A genetic model fi~r the inheritance of pituitary tumor susceptibility in F 344 rats. Endocrinology 1981;109:1708-14.