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Effect of housing stress on the formation and development of tumors in rats
257
Cancer Letters, 34 (1987) 257-261 Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF HOUSING STRESS ON THE FORMATION AND DEVELOPMENT OF TUMORS IN RATS
ZYGMUNT STEPLEWSKI*, VOGEL Department (U.S.A.)
of Pharmacology,
PATRICIA ROBINSON GOLDMAN and WOLFGANG H. Thomas
Jefferson
University,
Philadelphia,
PA 19107
(Received 10 September 1986) (Revised version received 25 October 1986) (Accepted 29 October 1986)
SUMMARY
Tumor induction by 7,12dimethylbenz[a Janthracene (DMBA) or inoculation with adenocarcinoma cells was studied in rats raised in groups (group G) or individually (group I) as well as in rats raised in groups and switched to individual housing at the beginning of the experiment (group G/I). The number of DMBA-induced tumors did not differ among the three groups of female rats but tumor weight was lowest in group I, highest in group G/I and intermediate in group G. The mean weight of adenocarcinoma cell-induced tumors was highest in group G/I and did not differ between groups I and G. Male rats generally showed higher tumor weights than females, the values becoming significant in group G/I. The results show that housing and sex can significantly affect tumor growth.
INTRODUCTION
In studies on the carcinogenic potential of chemicals or the development of tumors in animals after inoculation with tumor cells, animals are frequently bought from suppliers which group-house their animals. The investigators then continue to house the animals in groups for an acclimation period and use them either group housed or individually housed for the experiment. Since social interactions and different housing conditions can markedly influence the homeostasis of the body [ 2,8,10-121, and the immune system in particular [ 111, it is quite conceivable that such conditions can also influAddress correspondence to: Wolfgang H. Vogel. *On leave from the Department of Physiology, Silesian Medical School, Katowice, Poland. 0304-3835/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
ence tumor formation and development. Studies in male mice showed that social stress can influence growth of certain tumors [ 1,5,14]. To determine whether housing conditions can also affect tumor induction and growth in male and female rats, we investigated different housing conditions on the development of 7,12dimethylbenz[c]anthracene (DMBA)-induced tumors in female rats and adenocarcinoma (AC) cell-induced tumors in male and female rats. Animals were raised after weaning and studied in groups, individually or were raised in groups prior to the experiment and switched to individual housing during the experiment. MATERIALS
AND METHODS
Animals
Adult Sprague-Dawley rats (Perfection Breeder, Douglasville, PA) were used for the DMBA tumor study. Adult Lewis rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were used for the AC tumor cell innoculation studies. In both studies, rats were bred in our laboratory and the offspring selected randomly after weaning for the housing conditions described below. Animals received food and water ad libitum. Tumor
induction
The mammary adenocarcinoma cells obtained from E.F. Miller, University of Pennsylvania, School of Medicine, Philadelphia, PA were cultured in RPM1 1640 medium supplemented with 10% fetal bovine serum [ 141. At the age of about 70 days, animals were injected subcutaneously in the dorsal area with 3 X 10’ tumor cells. This had been previously determined to be the minimal dose to produce tumors in all rats [ 143. All rats were sacrificed after 2 weeks. For the DMBA induced tumors, animals at approximately 50 days of age received DMBA (5 mg/ml/week) by gastric intubation [ 61. This treatment was performed once a week for 5 weeks. Animals were sacrificed after 12 weeks. Tumor pathology
Tumors were examined ted carcinoma. Housing
histologically
[ 141 and found
to be undifferentia-
condition
The experimental design utilized three housing conditions. Animals were raised in groups of four per large cage (group G) after weaning, were raised individually after weaning (group I) or were raised in groups of four after weaning and switched to individual housing during the experiment (group G/I). At sacrifice, no statistically significant differences were observed between the weights of comparable groups within the two experiments. Statistical
evaluation
The statistical evaluations variations (P < 0.05).
used a Rank Sum Test due to large individual
259
RESULTS
The results of the different housing conditions on tumor weight are shown in Table 1. In the case of DMBA-induced tumors, the occurrence of palpatable tumors was not different among the three groups and no difference in number of tumors was apparent at sacrifice (3.4 + 2.6, 1.3 + 1.0 and 3.9 + 2.8, respectively). However, the three groups did differ significantly in tumor weight. Individually housed animals (group I) showed the lowest tumor weights whereas the animals switched from group to single housing (group G/I) showed the highest tumor weight. Group housed animals (group G) were intermediate. In the AC cell-innoculated tumors, no difference was observed between the fist two groups (groups G and I) but animals switched from group to single housing exhibited larger tumors (group G/I). In addition, males responded more markedly to the change in housing conditions and showed larger tumors than did females, the values becoming significant for the G/I group only. No differences was seen in the time of final occurrence of palpatable tumors between the three groups. DISCUSSION
The tumor can be tumor
above results show clearly the importance of housing conditions on growth. They also show that different tumor induction procedures differentially affected by such conditions. Tumors developing after cell innoculation grow similarly in individually or group housed
TABLE 1 EFFECT OF HOUSING CONDITIONS ON THE GROWTH OF DMBA AND ADENOCARCINOMA CELL INDUCED TUMORS Group G: group-housed; Group I: individually housed; Group G/I: group housed/ individually housed. Values are expressed as means ? S.E.M. and number in parentheses represent number of animals Tumor wt. (g) --
Group G Group I Group G/I 4 Difterence b Difference c Difference d Difference
DMBA-induced tumors
Tumor cell-induced tumors
Female
Female
Male
4.7 ? 5.6 (17) 0.7 ? 1.3 (6)a 13.9 ? 12.8 (10)b.C
5.5 ? 3.7 (6) 3.3 + 3.1 (6) 9.9 + 3.6 (7)b.c --
6.1 ? 2.8 (6) 4.3 + 3.2 (7) 12.5 + 2.1 (13)b*=d
between between between between
Group G and I, P < 0.05. Group G and G/I, P < 0.05. Group I and G/I, P < 0.05. males and females, P < 0.05.
260
animals but grow much faster in animals with an abrupt switch in housing conditions; this latter finding is more pronounced in male than female rats. DMBA-induced tumors grow very slowly in individually housed but faster in group housed animals; however, socially switched animals again show the fastest tumor growth. Our data are in good agreement with those of Sklar and Anisman [ 131 who first discovered that abrupt social change in mice had the most pronounced effect on tumor cell-induced tumor growth. Thus, social and housing conditions can quite selectively affect tumor growth depending on the species used, the sex of the animals or the tumor induction method employed. It is tempting to speculate on the cause of these different housing conditions on tumor growth. The most obvious cause is the stress - or lack among animals. Social stress has been thereof - of social interactions shown to affect the endocrine and immune state of the body. Plasma catecholamine levels increase during social activity in rats [ 101. Social isolation as compared to group housing has been shown to produce behavioral changes and differences in the uptake of [14C]deoxyglucose in mice [ 91. Neurotransmitter amino acids in the brains of rats respond differently to changes in housing conditions [ 41. Abruptly induced isolation reduced dopamine levels markedly in dopamine neurons [2]. The latter finding is of interest since we found a negative relationship between dopamine-stimulated adenylate cyclase and DMBA-induced tumor growth [6,7]. The decrease in dopamine could lead to a decrease in the activity of the enzyme which results in faster tumor growth. A breakdown in social structure has also been found to be detrimental to human health; increased morbidity and mortality and decreased immune function have been reported in humans with insufficient social support systems [3,15]. Although the exact mechanism remains to be elucidated, these findings point to the brain as a major regulatory organ of tumor growth since social stress is first experienced in this organ [ 161. REFERENCES 1 Andervont, E.B. (1973) Influence of environment on mammary cancer in mice. J. Natl. Cancer Inst., 4, 579-581. 2 Blanc, G., Herve, D., Simon, H., Lisoprawski, A., Glowinski, J. and Tassin, J.P. (1980) Response to stress of mesocortical-frontal dopaminergic neurons in rats after longterm isolation. Nature (London) 284, 265-271. 3 Berkman, L.F. and Syme, S.L. (1979) Social network, host resistance and mortality: a nine year follow-up study of Alameda County residents. Am. J. Epidemiol. 109, 186-215. 4 Cordoleo, F., Yusta, B. and Munoz-Blanco, J. (1984) Changes in neurotransmitter amino acids and proteins in CNS areas of mice subjected to differential housing conditions. Pharmacol. Biochem. Behav., 21, 349-355. 5 Dechambre, R.P. and Gosse, C. (1973) Individual versus group caging of mice with grafted tumors. Cancer Res. 33, 140-144. 6 Goldman, P.R. and Vogel, W.H. (1984) Striatal dopamine-stimulated adenylate
261
7
8
9 10
11 12
13 14
15
16
cyclase activity reflects susceptibility of rats to DMBA-induced mammary tumor development. Carcinogenesis, 5, 971-973. Goldman, P.R. and Vogel, W.H. (1985) Plasma estradiol and prolactin levels and their response to stress in two strains of rats with different sensitivities to DMBA-induced tumors. Cancer Letters, 25, 277-282. Irwin, J., Ahluwalia, P., Zacharko, R.M. and A&man, H. (1986) Central norepinephrine and plasma corticosterone following acute and chronic stress: influence of social isolation and handling. Pharmacol. Biochem. Behav., 24, 1151-1154. McCall, S., VanderWende, C. and Spoerlein, M.T. (1985) “C-Deoxyglucose uptake in discrete areas of brain in differentially housed mice. Neurosci. Abstr., 11, 1263. Pashko, S., DeTurck, K.H. and Vogel, W.H. (1980) Use of the catherized rat in studies on social interactions and plasma catecholamines. Pharmacol. Biochem. Behav., 13, 471-473. Riley, V. (1981) Psychoneuroendocrine influences on immunocompetence and neoplasia. Science, 212,1100-1109. Rosenzweig, M.R. and Bennell, E.L. (1969) Effects of differential environment on brain weights and enzyme activities in gerbils, rats and mice. Dev. Psychobiol. 2, 87-95. Sklar, L.S. and Anisman, H. (1980) Social stress influences tumor growth. Psychosomat. Med., 42, 347-365. Steplewski, Z., Vogel, W.H., Ehya, II., Porvpatich, C. and Smith, J.M. (1985) Effects of restraint stress on inoculated tumor growth and immune response in rats, Cancer Res., 45, 5128-5133. Thomas, P.D., Goodwin, J.M. and Goodwin, J.S. (1985) Effect of social support on stress-related changes in cholesterol level, uric acid level and immune function in an elderly sample. Am. J. Psychiat., 142, 735-817. Vogel, W.H. (1985) Coping, stress, stressors and health consequences. Neuropsychobiology, 13, i29-135.
Cancer Letters, 34 (1987) 257-261 Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF HOUSING STRESS ON THE FORMATION AND DEVELOPMENT OF TUMORS IN RATS
ZYGMUNT STEPLEWSKI*, VOGEL Department (U.S.A.)
of Pharmacology,
PATRICIA ROBINSON GOLDMAN and WOLFGANG H. Thomas
Jefferson
University,
Philadelphia,
PA 19107
(Received 10 September 1986) (Revised version received 25 October 1986) (Accepted 29 October 1986)
SUMMARY
Tumor induction by 7,12dimethylbenz[a Janthracene (DMBA) or inoculation with adenocarcinoma cells was studied in rats raised in groups (group G) or individually (group I) as well as in rats raised in groups and switched to individual housing at the beginning of the experiment (group G/I). The number of DMBA-induced tumors did not differ among the three groups of female rats but tumor weight was lowest in group I, highest in group G/I and intermediate in group G. The mean weight of adenocarcinoma cell-induced tumors was highest in group G/I and did not differ between groups I and G. Male rats generally showed higher tumor weights than females, the values becoming significant in group G/I. The results show that housing and sex can significantly affect tumor growth.
INTRODUCTION
In studies on the carcinogenic potential of chemicals or the development of tumors in animals after inoculation with tumor cells, animals are frequently bought from suppliers which group-house their animals. The investigators then continue to house the animals in groups for an acclimation period and use them either group housed or individually housed for the experiment. Since social interactions and different housing conditions can markedly influence the homeostasis of the body [ 2,8,10-121, and the immune system in particular [ 111, it is quite conceivable that such conditions can also influAddress correspondence to: Wolfgang H. Vogel. *On leave from the Department of Physiology, Silesian Medical School, Katowice, Poland. 0304-3835/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
ence tumor formation and development. Studies in male mice showed that social stress can influence growth of certain tumors [ 1,5,14]. To determine whether housing conditions can also affect tumor induction and growth in male and female rats, we investigated different housing conditions on the development of 7,12dimethylbenz[c]anthracene (DMBA)-induced tumors in female rats and adenocarcinoma (AC) cell-induced tumors in male and female rats. Animals were raised after weaning and studied in groups, individually or were raised in groups prior to the experiment and switched to individual housing during the experiment. MATERIALS
AND METHODS
Animals
Adult Sprague-Dawley rats (Perfection Breeder, Douglasville, PA) were used for the DMBA tumor study. Adult Lewis rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were used for the AC tumor cell innoculation studies. In both studies, rats were bred in our laboratory and the offspring selected randomly after weaning for the housing conditions described below. Animals received food and water ad libitum. Tumor
induction
The mammary adenocarcinoma cells obtained from E.F. Miller, University of Pennsylvania, School of Medicine, Philadelphia, PA were cultured in RPM1 1640 medium supplemented with 10% fetal bovine serum [ 141. At the age of about 70 days, animals were injected subcutaneously in the dorsal area with 3 X 10’ tumor cells. This had been previously determined to be the minimal dose to produce tumors in all rats [ 143. All rats were sacrificed after 2 weeks. For the DMBA induced tumors, animals at approximately 50 days of age received DMBA (5 mg/ml/week) by gastric intubation [ 61. This treatment was performed once a week for 5 weeks. Animals were sacrificed after 12 weeks. Tumor pathology
Tumors were examined ted carcinoma. Housing
histologically
[ 141 and found
to be undifferentia-
condition
The experimental design utilized three housing conditions. Animals were raised in groups of four per large cage (group G) after weaning, were raised individually after weaning (group I) or were raised in groups of four after weaning and switched to individual housing during the experiment (group G/I). At sacrifice, no statistically significant differences were observed between the weights of comparable groups within the two experiments. Statistical
evaluation
The statistical evaluations variations (P < 0.05).
used a Rank Sum Test due to large individual
259
RESULTS
The results of the different housing conditions on tumor weight are shown in Table 1. In the case of DMBA-induced tumors, the occurrence of palpatable tumors was not different among the three groups and no difference in number of tumors was apparent at sacrifice (3.4 + 2.6, 1.3 + 1.0 and 3.9 + 2.8, respectively). However, the three groups did differ significantly in tumor weight. Individually housed animals (group I) showed the lowest tumor weights whereas the animals switched from group to single housing (group G/I) showed the highest tumor weight. Group housed animals (group G) were intermediate. In the AC cell-innoculated tumors, no difference was observed between the fist two groups (groups G and I) but animals switched from group to single housing exhibited larger tumors (group G/I). In addition, males responded more markedly to the change in housing conditions and showed larger tumors than did females, the values becoming significant for the G/I group only. No differences was seen in the time of final occurrence of palpatable tumors between the three groups. DISCUSSION
The tumor can be tumor
above results show clearly the importance of housing conditions on growth. They also show that different tumor induction procedures differentially affected by such conditions. Tumors developing after cell innoculation grow similarly in individually or group housed
TABLE 1 EFFECT OF HOUSING CONDITIONS ON THE GROWTH OF DMBA AND ADENOCARCINOMA CELL INDUCED TUMORS Group G: group-housed; Group I: individually housed; Group G/I: group housed/ individually housed. Values are expressed as means ? S.E.M. and number in parentheses represent number of animals Tumor wt. (g) --
Group G Group I Group G/I 4 Difterence b Difference c Difference d Difference
DMBA-induced tumors
Tumor cell-induced tumors
Female
Female
Male
4.7 ? 5.6 (17) 0.7 ? 1.3 (6)a 13.9 ? 12.8 (10)b.C
5.5 ? 3.7 (6) 3.3 + 3.1 (6) 9.9 + 3.6 (7)b.c --
6.1 ? 2.8 (6) 4.3 + 3.2 (7) 12.5 + 2.1 (13)b*=d
between between between between
Group G and I, P < 0.05. Group G and G/I, P < 0.05. Group I and G/I, P < 0.05. males and females, P < 0.05.
260
animals but grow much faster in animals with an abrupt switch in housing conditions; this latter finding is more pronounced in male than female rats. DMBA-induced tumors grow very slowly in individually housed but faster in group housed animals; however, socially switched animals again show the fastest tumor growth. Our data are in good agreement with those of Sklar and Anisman [ 131 who first discovered that abrupt social change in mice had the most pronounced effect on tumor cell-induced tumor growth. Thus, social and housing conditions can quite selectively affect tumor growth depending on the species used, the sex of the animals or the tumor induction method employed. It is tempting to speculate on the cause of these different housing conditions on tumor growth. The most obvious cause is the stress - or lack among animals. Social stress has been thereof - of social interactions shown to affect the endocrine and immune state of the body. Plasma catecholamine levels increase during social activity in rats [ 101. Social isolation as compared to group housing has been shown to produce behavioral changes and differences in the uptake of [14C]deoxyglucose in mice [ 91. Neurotransmitter amino acids in the brains of rats respond differently to changes in housing conditions [ 41. Abruptly induced isolation reduced dopamine levels markedly in dopamine neurons [2]. The latter finding is of interest since we found a negative relationship between dopamine-stimulated adenylate cyclase and DMBA-induced tumor growth [6,7]. The decrease in dopamine could lead to a decrease in the activity of the enzyme which results in faster tumor growth. A breakdown in social structure has also been found to be detrimental to human health; increased morbidity and mortality and decreased immune function have been reported in humans with insufficient social support systems [3,15]. Although the exact mechanism remains to be elucidated, these findings point to the brain as a major regulatory organ of tumor growth since social stress is first experienced in this organ [ 161. REFERENCES 1 Andervont, E.B. (1973) Influence of environment on mammary cancer in mice. J. Natl. Cancer Inst., 4, 579-581. 2 Blanc, G., Herve, D., Simon, H., Lisoprawski, A., Glowinski, J. and Tassin, J.P. (1980) Response to stress of mesocortical-frontal dopaminergic neurons in rats after longterm isolation. Nature (London) 284, 265-271. 3 Berkman, L.F. and Syme, S.L. (1979) Social network, host resistance and mortality: a nine year follow-up study of Alameda County residents. Am. J. Epidemiol. 109, 186-215. 4 Cordoleo, F., Yusta, B. and Munoz-Blanco, J. (1984) Changes in neurotransmitter amino acids and proteins in CNS areas of mice subjected to differential housing conditions. Pharmacol. Biochem. Behav., 21, 349-355. 5 Dechambre, R.P. and Gosse, C. (1973) Individual versus group caging of mice with grafted tumors. Cancer Res. 33, 140-144. 6 Goldman, P.R. and Vogel, W.H. (1984) Striatal dopamine-stimulated adenylate
261
7
8
9 10
11 12
13 14
15
16
cyclase activity reflects susceptibility of rats to DMBA-induced mammary tumor development. Carcinogenesis, 5, 971-973. Goldman, P.R. and Vogel, W.H. (1985) Plasma estradiol and prolactin levels and their response to stress in two strains of rats with different sensitivities to DMBA-induced tumors. Cancer Letters, 25, 277-282. Irwin, J., Ahluwalia, P., Zacharko, R.M. and A&man, H. (1986) Central norepinephrine and plasma corticosterone following acute and chronic stress: influence of social isolation and handling. Pharmacol. Biochem. Behav., 24, 1151-1154. McCall, S., VanderWende, C. and Spoerlein, M.T. (1985) “C-Deoxyglucose uptake in discrete areas of brain in differentially housed mice. Neurosci. Abstr., 11, 1263. Pashko, S., DeTurck, K.H. and Vogel, W.H. (1980) Use of the catherized rat in studies on social interactions and plasma catecholamines. Pharmacol. Biochem. Behav., 13, 471-473. Riley, V. (1981) Psychoneuroendocrine influences on immunocompetence and neoplasia. Science, 212,1100-1109. Rosenzweig, M.R. and Bennell, E.L. (1969) Effects of differential environment on brain weights and enzyme activities in gerbils, rats and mice. Dev. Psychobiol. 2, 87-95. Sklar, L.S. and Anisman, H. (1980) Social stress influences tumor growth. Psychosomat. Med., 42, 347-365. Steplewski, Z., Vogel, W.H., Ehya, II., Porvpatich, C. and Smith, J.M. (1985) Effects of restraint stress on inoculated tumor growth and immune response in rats, Cancer Res., 45, 5128-5133. Thomas, P.D., Goodwin, J.M. and Goodwin, J.S. (1985) Effect of social support on stress-related changes in cholesterol level, uric acid level and immune function in an elderly sample. Am. J. Psychiat., 142, 735-817. Vogel, W.H. (1985) Coping, stress, stressors and health consequences. Neuropsychobiology, 13, i29-135.