- Email: [email protected]
The role of corticosterone in cadmium-induced thymic atrophy in mice
Toxicology Letters, 12 (1982) 225-229 Elsevier Biomedical Press
THE ROLE OF CORTICOSTERONE ATROPHY IN MICE
225
IN CADMIUM-INDUCED
THYMJC
YASUKO K. YAMADA, NOBORU MURAKAMI*, FUJI0 SHIMIZU and KENTARO KUBOTA Basic Medical Sciences Division, National Instifute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, and *Department of Medicai Chemistry, Tokyo metropolitan Institute for Neuroscienres, Fuchu-city, Tokyo ~~apa~~ (Received January 2Oth, 1982) (Revision received and accepted March ist, 1982)
SUMMARY Serum corticosterone levels were determined at intervals after i.p. injection of 1.8 mg cadmium (Cd)/kg body weight. Thymus weight decreased significantly 3 days after injection. The effect of Cd injection on serum corticosterone levels was almost indistinguishable from that of saline injection. Further, Cd-induced thymic atrophy was observed in adrenaIectomized mice as well as in shy-operated ones. These results suggest that corticosteroid effect is not essential for the induction of thymic atrophy by Cd.
INTRODUCTION
Cd has been reported to affect the immune function, e.g., the susceptibility to bacterial and viral infection, the humoral antibody synthesis and the activity of lymphocytes or macrophages [I]. We have examined histopathologically the effects of Cd on the immune organs and we have shown that significant thymic atrophy and splenomegaly were caused by a single i.p. injection of Cd into mice 121.It is possible that this thymic atrophy induced by Cd injection may result from the stressful stimulus of Cd injection, since artificial stresses lead to hypersecretion of corticosteroid and thymic atrophy [3,4]. However, to our knowledge, the change in corticosteroid levels after Cd exposure has not yet been described. All correspondence should be addressed to: Yasuko K. Yamada, National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305 (Japan). Abbreviation: MT. metahothionein. 0378-4274/82/~-~/$02.75
Q Elsevier Biomedical Press
226
To clarify the relation between corticosteroid and Cd-induced thymic atrophy, we determined serum corticosterone levels after Cd injection and investigated whether or not Cd administration induces thymic atrophy in adrenalectomized mice. MATERIALS
AND METHODS
Animals Male BALB/c mice (Charles River Japan, Inc., Atsugi) were fed commercial pellets (Clea Japan, Tokyo) and distilled water ad lib in a room that was light from 7.00 h to 19.00 h.
Administration of Cd Cadmium chloride solution in physiological saline at a concentration of 0.1 mg Cd/ml was administered in a single i.p. injection (1.8 mg Cd/kg body weight). Control mice were injected i.p. with physiological saline.
Serum corticosterone level g-week-old mice were injected at 10.00 h and were killed at different intervals after injection. Sampling was performed at 13.00 h and 16.00 h, 3 and 6 h after injection, respectively. 1, 3 and 9 days after injection, the mice were killed at 10.00 h. The animals (5 mice/group) were anesthetized by chloroform and blood was taken by heart puncture within 1 min after removing the animals from the cages. Thymuses were removed and weighed. Sera were stored at - 20°C; corticosterone was determined by a modification [5] of the protein-binding method of Murphy [6].
Adrenalectomy 7-week-old mice were anesthetized by i.p. injection of sodium pentobarbiturate (25 mg/kg b.w.) and, after shaving and disinfecting with 70% ethanol, a small incision was made in the center of the dorsal lumbar skin. The right and left dorsal peritonea were separately incised and the adrenal glands grasped with forceps and removed. Then, the peritoneal and skin incisions were closed by suture. In shamoperated mice, the incisions were made and closed without adrenalectomy. 5 days after operation, adrenalectomized and sham-operated mice were injected with Cd or saline. 3 days after injection, mice were killed and the thymus glands and sera were treated in the same way.
Statistical evaluation All results were examined statistically by Student’s t-test. RESULTS
Serum corticosterone
levels of Cd-injected,
saline-injected and intact mice were
221
determined at intervals after injection (Fig. 1A). The levels of serum corticosterone in intact mice were 50.48 + 7.06 &g/100 ml serum, mean f SD) at 13.00 h and 60.80 + 5.76 at 16.00 h. At 10.00 h, the levels observed on 3 separate days were 17.54 + 1.34, 10.54 + 3.26 and 12.38 + 1.77, respectively. In saline-injected mice, serum corticosterone level was generally shown to be the same as that in intact ones. Although a high level was observed 3 h after injection, it was not significant. The levels in Cd-injected mice tended to be only slightly higher than in saline-injected mice from 3 h to 3 days. A significant difference was recognized only 1 day after injection (P < 0.05). The change of thymus weight is shown in Fig. 1B as the ratio of organ-to-body weight. 3 days after injection, the thymus weight of Cd-injected mice significantly decreased compared with that of saline-injected (P < 0.01) or intact (P < 0.05) ones. 37 adrenalectomized and 16 sham-operated mice were prepared for Cd or saline injection. At the time of injection, 5 days after operation, body weights of adrenalectomized and sham-operated mice were 24.9 + 1.7 g and 26.5 f 1.4 g (mean + SD; P < O.OOl), respectively. Mice were divided into 4 groups, adrenalectomized and Cd-injected, adrenalectomized and saline-injected, shamoperated and Cd-injected, sham-operated and saline-injected. Among 28 adrenalectomized and Cd-injected mice, 18 mice (64%) died 1 or 2 days after injection. In other groups, all mice survived. The thymus was enlarged by adrenalectomy compared with sham-operation (P < 0.001 in both Cd- and salineinjected groups) (Table I). In sham-operated and adrenalectomized mice, the A pg/loO
ml
B s.erum
mwg
injection
Fig. 1 (A) of thymus .____a, x---x
The effects of Cd on serum corticosterone level. (B) The effects of Cd on thyrnus weight (ratio to body weight). Each point represents the mean and the vertical bar the standard deviation. injected i.p. with physiological saline; injected i.p. with 1.8 mg Cd/kg b.w.; -._o, , intact.
228 TABLE I THE EFFECTS OF ADRENALECTOMY Operation
Injectiona
ON Cd-INDUCED THYMIC ATROPHY
Thymus ratiob (mg/g)
CorticosteroneC @g/l00 ml serum)
Adrenalectomy
Cd saline
1.90+ 0.4l*d (77”7oe) 2.46 + 0.47
5.07 + 1.61d (n = 10) 2.25 + 0.59 (n = 9)
Sham-operation
Cd saline
1.21 ? 0.13*** (77%) 1.58 + 0.15
23.57 + 10.75 (n = 8) 37.89 + 17.36 (n = 8)
a 1.8 mg Cd/kg b.w. or saline was injected i.p. 5 days after operation. bThymus weight/Body weight, 3 days after injection. ~3 days after injection. dMean + SD. Significantly different from saline injection (*P < 0.05 ***P < 0.001). eThymus ratio of Cd-injected mice/saline-injected mice.
thymus weight decreased significantly 3 days after Cd injection compared with saline injection. Serum corticosterone of adrenalectomized mice, with or without Cd injection, showed very low values compared with sham-operated mice. DISCUSSION
Previously, we have reported that cortical atrophy of the thymus was caused by a single i.p. injection of Cd [2]. To clarify the relation between this atrophy and corticosteroid, we determined serum corticosterone levels at different intervals after Cd injection. Although the high levels of serum corticosterone were observed 3 and 6 h after Cd injection, this high level was also observed in both saline-injected and intact mice without thymic atrophy. Therefore, this high level of serum corticosterone is not involved in Cd-induced thymic atrophy. This high level of serum corticosterone may be a peak of general circadian corticosterone rhythm [7, 81. Although serum corticosterone level was slightly altered by Cd injection, the change was significant only at 1 day after injection. To clarify the relation between corticosterone and Cd-induced thymic atrophy, Cd was injected into adrenalectomized mice. The adrenalectomy operation was regarded as successful, since serum corticosterone of adrenalectomized mice showed a very low level and the thymus was enlarged. This enlargement has been described previously [9] and may be caused by the deficiency of glucocorticoid, which is reported to induce the lysis of immature thymocytes [lo]. In adrenalectomized mice as well as sham-operated ones, thymic atrophy occurred after Cd administration. These results indicate that corticosterone may not have an essential role in the induction of thymic atrophy by Cd and that this effect of Cd injection on the thymus is not mediated by the adrenocorticosteroid. As a small amount of Cd is proved to accumulate in the thymus after i.p. injection [I 11, it may be possible that
229
Cd directly destroys thymocytes. In addition, there is a possibility that the precursors of thymocytes in the bone marrow are affected by Cd. Adrenalectomized mice were very susceptible to Cd attack. It has been reported that MT plays an important part in Cd intoxication [12] and that the synthesis of MT is induced by glucocorticoid [ 131. Since glucocorticoid levels became lower after adrenalectomy, mice may not be able to resist Cd attack. REFERENCES 1 L.D.
2 3
4
5
6
I 8
Keller, Immunological effects of cadmium, in J.O. Nriagu (Ed.), Cadmium in the Environment, part II: Health Effects, Wiley, New York, 1981, pp. 719-727. Y.K. Yamada, F. Shimizu, R. Kawamura and K. Kubota, Thymic atrophy in mice induced by cadmium administration, Toxicol. Lett., 8 (1981) 49-55. W.C. Engeland, J. Shinsako, C.M. Winget, J. Vernikos-Danellis and M.F. Dallman, Circadian patterns of stress-induced ACTH secretion are modified by corticosterone response, Endocrinology, 100 (1977) 138-147. R.H. Gisler, A.E. Bussard, J.C. MaziC and R. Hess, Hormonal regulation of the immune response, I. Induction of an immune response in vitro with lymphoid cells from mice exposed to acute systemic stress, 2 (1971) 634-645. K. Takahashi, K. Haneda, K. Kobayashi, C. Hayafuji, S. Otani and Y. Takahashi, Development of the circadian adrenocortical rhythm in rats studied by determination of 24- or 48-hour patterns of blood corticosterone levels in individual pups, Endocrinology, 104 (1979) 954-961. B.E.P. Murphy, Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay, J. Clin. Endocrinol. Metab., 27 (1967) 973-990. R. Guillemin, W.E. Dear and R.A. Liebelt, Nyctohemeral variations in plasma free corticosteroid levels of the rat, Proc. Sot. Exp. Biol. Med., 101 (1959) 394-395. K. Takahashi, K. Inoue and Y. Takahashi, Parallel shift in circadian rhythms of adrenocortical activity and food intake in blinded and intact rats exposed to continuous illumination, Endocrinology, 100 (1977) 1097-l 107. P. DePasquale-Jardieu and P.J. Fraker, Further characterization of the role of corticosterone in the loss of humoral immunity in zinc-deficient A/J mice as determined by adrenalectomy, J. Immunol., 124 (1980) 2650-2655. T. Dougherty and A. White, Functional alteration in lymphoid tissue induced by adrenal cortical secretion, Am. J. Anat., 77 (1945) 81-116. K.T. Suzuki, Y.K. Yamada and F. Shimizu, Essential metals and metallothionein in cadmiuminduced thymic atrophy and splenomegaly, Biochem. Pharmacol., 30 (1981) 1217-1222. F.N. Kotsonis and C.D. Klaassen, Metallothionein and its interactions with cadmium, in J.O. Nriagu (Ed.), Cadmium in the Environment, part II: Health Effects, Wiley, New York, 1981, pp. 595-616. K.R. Etzel, S.G. Shapiro and R.J. Cousins, Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone, Biochem. Biophys. Res. Commun. 89 (1979) 1120-l 126.
THE ROLE OF CORTICOSTERONE ATROPHY IN MICE
225
IN CADMIUM-INDUCED
THYMJC
YASUKO K. YAMADA, NOBORU MURAKAMI*, FUJI0 SHIMIZU and KENTARO KUBOTA Basic Medical Sciences Division, National Instifute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, and *Department of Medicai Chemistry, Tokyo metropolitan Institute for Neuroscienres, Fuchu-city, Tokyo ~~apa~~ (Received January 2Oth, 1982) (Revision received and accepted March ist, 1982)
SUMMARY Serum corticosterone levels were determined at intervals after i.p. injection of 1.8 mg cadmium (Cd)/kg body weight. Thymus weight decreased significantly 3 days after injection. The effect of Cd injection on serum corticosterone levels was almost indistinguishable from that of saline injection. Further, Cd-induced thymic atrophy was observed in adrenaIectomized mice as well as in shy-operated ones. These results suggest that corticosteroid effect is not essential for the induction of thymic atrophy by Cd.
INTRODUCTION
Cd has been reported to affect the immune function, e.g., the susceptibility to bacterial and viral infection, the humoral antibody synthesis and the activity of lymphocytes or macrophages [I]. We have examined histopathologically the effects of Cd on the immune organs and we have shown that significant thymic atrophy and splenomegaly were caused by a single i.p. injection of Cd into mice 121.It is possible that this thymic atrophy induced by Cd injection may result from the stressful stimulus of Cd injection, since artificial stresses lead to hypersecretion of corticosteroid and thymic atrophy [3,4]. However, to our knowledge, the change in corticosteroid levels after Cd exposure has not yet been described. All correspondence should be addressed to: Yasuko K. Yamada, National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305 (Japan). Abbreviation: MT. metahothionein. 0378-4274/82/~-~/$02.75
Q Elsevier Biomedical Press
226
To clarify the relation between corticosteroid and Cd-induced thymic atrophy, we determined serum corticosterone levels after Cd injection and investigated whether or not Cd administration induces thymic atrophy in adrenalectomized mice. MATERIALS
AND METHODS
Animals Male BALB/c mice (Charles River Japan, Inc., Atsugi) were fed commercial pellets (Clea Japan, Tokyo) and distilled water ad lib in a room that was light from 7.00 h to 19.00 h.
Administration of Cd Cadmium chloride solution in physiological saline at a concentration of 0.1 mg Cd/ml was administered in a single i.p. injection (1.8 mg Cd/kg body weight). Control mice were injected i.p. with physiological saline.
Serum corticosterone level g-week-old mice were injected at 10.00 h and were killed at different intervals after injection. Sampling was performed at 13.00 h and 16.00 h, 3 and 6 h after injection, respectively. 1, 3 and 9 days after injection, the mice were killed at 10.00 h. The animals (5 mice/group) were anesthetized by chloroform and blood was taken by heart puncture within 1 min after removing the animals from the cages. Thymuses were removed and weighed. Sera were stored at - 20°C; corticosterone was determined by a modification [5] of the protein-binding method of Murphy [6].
Adrenalectomy 7-week-old mice were anesthetized by i.p. injection of sodium pentobarbiturate (25 mg/kg b.w.) and, after shaving and disinfecting with 70% ethanol, a small incision was made in the center of the dorsal lumbar skin. The right and left dorsal peritonea were separately incised and the adrenal glands grasped with forceps and removed. Then, the peritoneal and skin incisions were closed by suture. In shamoperated mice, the incisions were made and closed without adrenalectomy. 5 days after operation, adrenalectomized and sham-operated mice were injected with Cd or saline. 3 days after injection, mice were killed and the thymus glands and sera were treated in the same way.
Statistical evaluation All results were examined statistically by Student’s t-test. RESULTS
Serum corticosterone
levels of Cd-injected,
saline-injected and intact mice were
221
determined at intervals after injection (Fig. 1A). The levels of serum corticosterone in intact mice were 50.48 + 7.06 &g/100 ml serum, mean f SD) at 13.00 h and 60.80 + 5.76 at 16.00 h. At 10.00 h, the levels observed on 3 separate days were 17.54 + 1.34, 10.54 + 3.26 and 12.38 + 1.77, respectively. In saline-injected mice, serum corticosterone level was generally shown to be the same as that in intact ones. Although a high level was observed 3 h after injection, it was not significant. The levels in Cd-injected mice tended to be only slightly higher than in saline-injected mice from 3 h to 3 days. A significant difference was recognized only 1 day after injection (P < 0.05). The change of thymus weight is shown in Fig. 1B as the ratio of organ-to-body weight. 3 days after injection, the thymus weight of Cd-injected mice significantly decreased compared with that of saline-injected (P < 0.01) or intact (P < 0.05) ones. 37 adrenalectomized and 16 sham-operated mice were prepared for Cd or saline injection. At the time of injection, 5 days after operation, body weights of adrenalectomized and sham-operated mice were 24.9 + 1.7 g and 26.5 f 1.4 g (mean + SD; P < O.OOl), respectively. Mice were divided into 4 groups, adrenalectomized and Cd-injected, adrenalectomized and saline-injected, shamoperated and Cd-injected, sham-operated and saline-injected. Among 28 adrenalectomized and Cd-injected mice, 18 mice (64%) died 1 or 2 days after injection. In other groups, all mice survived. The thymus was enlarged by adrenalectomy compared with sham-operation (P < 0.001 in both Cd- and salineinjected groups) (Table I). In sham-operated and adrenalectomized mice, the A pg/loO
ml
B s.erum
mwg
injection
Fig. 1 (A) of thymus .____a, x---x
The effects of Cd on serum corticosterone level. (B) The effects of Cd on thyrnus weight (ratio to body weight). Each point represents the mean and the vertical bar the standard deviation. injected i.p. with physiological saline; injected i.p. with 1.8 mg Cd/kg b.w.; -._o, , intact.
228 TABLE I THE EFFECTS OF ADRENALECTOMY Operation
Injectiona
ON Cd-INDUCED THYMIC ATROPHY
Thymus ratiob (mg/g)
CorticosteroneC @g/l00 ml serum)
Adrenalectomy
Cd saline
1.90+ 0.4l*d (77”7oe) 2.46 + 0.47
5.07 + 1.61d (n = 10) 2.25 + 0.59 (n = 9)
Sham-operation
Cd saline
1.21 ? 0.13*** (77%) 1.58 + 0.15
23.57 + 10.75 (n = 8) 37.89 + 17.36 (n = 8)
a 1.8 mg Cd/kg b.w. or saline was injected i.p. 5 days after operation. bThymus weight/Body weight, 3 days after injection. ~3 days after injection. dMean + SD. Significantly different from saline injection (*P < 0.05 ***P < 0.001). eThymus ratio of Cd-injected mice/saline-injected mice.
thymus weight decreased significantly 3 days after Cd injection compared with saline injection. Serum corticosterone of adrenalectomized mice, with or without Cd injection, showed very low values compared with sham-operated mice. DISCUSSION
Previously, we have reported that cortical atrophy of the thymus was caused by a single i.p. injection of Cd [2]. To clarify the relation between this atrophy and corticosteroid, we determined serum corticosterone levels at different intervals after Cd injection. Although the high levels of serum corticosterone were observed 3 and 6 h after Cd injection, this high level was also observed in both saline-injected and intact mice without thymic atrophy. Therefore, this high level of serum corticosterone is not involved in Cd-induced thymic atrophy. This high level of serum corticosterone may be a peak of general circadian corticosterone rhythm [7, 81. Although serum corticosterone level was slightly altered by Cd injection, the change was significant only at 1 day after injection. To clarify the relation between corticosterone and Cd-induced thymic atrophy, Cd was injected into adrenalectomized mice. The adrenalectomy operation was regarded as successful, since serum corticosterone of adrenalectomized mice showed a very low level and the thymus was enlarged. This enlargement has been described previously [9] and may be caused by the deficiency of glucocorticoid, which is reported to induce the lysis of immature thymocytes [lo]. In adrenalectomized mice as well as sham-operated ones, thymic atrophy occurred after Cd administration. These results indicate that corticosterone may not have an essential role in the induction of thymic atrophy by Cd and that this effect of Cd injection on the thymus is not mediated by the adrenocorticosteroid. As a small amount of Cd is proved to accumulate in the thymus after i.p. injection [I 11, it may be possible that
229
Cd directly destroys thymocytes. In addition, there is a possibility that the precursors of thymocytes in the bone marrow are affected by Cd. Adrenalectomized mice were very susceptible to Cd attack. It has been reported that MT plays an important part in Cd intoxication [12] and that the synthesis of MT is induced by glucocorticoid [ 131. Since glucocorticoid levels became lower after adrenalectomy, mice may not be able to resist Cd attack. REFERENCES 1 L.D.
2 3
4
5
6
I 8
Keller, Immunological effects of cadmium, in J.O. Nriagu (Ed.), Cadmium in the Environment, part II: Health Effects, Wiley, New York, 1981, pp. 719-727. Y.K. Yamada, F. Shimizu, R. Kawamura and K. Kubota, Thymic atrophy in mice induced by cadmium administration, Toxicol. Lett., 8 (1981) 49-55. W.C. Engeland, J. Shinsako, C.M. Winget, J. Vernikos-Danellis and M.F. Dallman, Circadian patterns of stress-induced ACTH secretion are modified by corticosterone response, Endocrinology, 100 (1977) 138-147. R.H. Gisler, A.E. Bussard, J.C. MaziC and R. Hess, Hormonal regulation of the immune response, I. Induction of an immune response in vitro with lymphoid cells from mice exposed to acute systemic stress, 2 (1971) 634-645. K. Takahashi, K. Haneda, K. Kobayashi, C. Hayafuji, S. Otani and Y. Takahashi, Development of the circadian adrenocortical rhythm in rats studied by determination of 24- or 48-hour patterns of blood corticosterone levels in individual pups, Endocrinology, 104 (1979) 954-961. B.E.P. Murphy, Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay, J. Clin. Endocrinol. Metab., 27 (1967) 973-990. R. Guillemin, W.E. Dear and R.A. Liebelt, Nyctohemeral variations in plasma free corticosteroid levels of the rat, Proc. Sot. Exp. Biol. Med., 101 (1959) 394-395. K. Takahashi, K. Inoue and Y. Takahashi, Parallel shift in circadian rhythms of adrenocortical activity and food intake in blinded and intact rats exposed to continuous illumination, Endocrinology, 100 (1977) 1097-l 107. P. DePasquale-Jardieu and P.J. Fraker, Further characterization of the role of corticosterone in the loss of humoral immunity in zinc-deficient A/J mice as determined by adrenalectomy, J. Immunol., 124 (1980) 2650-2655. T. Dougherty and A. White, Functional alteration in lymphoid tissue induced by adrenal cortical secretion, Am. J. Anat., 77 (1945) 81-116. K.T. Suzuki, Y.K. Yamada and F. Shimizu, Essential metals and metallothionein in cadmiuminduced thymic atrophy and splenomegaly, Biochem. Pharmacol., 30 (1981) 1217-1222. F.N. Kotsonis and C.D. Klaassen, Metallothionein and its interactions with cadmium, in J.O. Nriagu (Ed.), Cadmium in the Environment, part II: Health Effects, Wiley, New York, 1981, pp. 595-616. K.R. Etzel, S.G. Shapiro and R.J. Cousins, Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone, Biochem. Biophys. Res. Commun. 89 (1979) 1120-l 126.