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Decrease in calcium content in the femur of rats orally administered stannous chloride
Toxicology Letters, 3 (1979) 7-10 o Elsevier/North-Holland Biomedical Press
7
DECREASE IN CALCIUM CONTENT IN THE FEMUR OF RATS ORALLY ADMINISTERED STANNOUS CHLORIDE
MASAYOSHI YAMAGUCHI,
MITUKO KITADE and SHOJI OKADA
Department of Environmental Shizuoka, 422 (Japan)
Biochemistry,
Shizuoka College of Pharmacy, 2-2-1, Oshika,
(Received September 6th, 1978) (Accepted September 14th, 1978)
SUMMARY
The calcium content in the femur of rats was significantly decreased by the oral administration of stannous chloride (3.0 mg Sn2’/100 g body weight, 6 times at 12-h intervals). This decrease was dose-dependent. Administration of tin did not prevent femoral deposition of subcutaneously injected calcium. Administration of tin to thyroparathyroidectomized rats did not reduce femoral calcium, but lowered serum calcium significantly. The present study suggests that the decrease in femoral calcium produced by the oral administration of tin resulted mainly from the mobilization, induced by parathyroid !rormone, of calcium into blood from bone.
INTRODUCTION
Environmental contamination by tin (Sn) is due largely to canned food. Several cases of intoxication have been observed following the intake of canned food containing high levels of Sn [3]. The toxic effects of Sn on bone metabolism are little understood, although Sn has been known to accumulate in the bone of rats [2]. Recently the authors have reported that the oral administration of stannous chloride inhibits transport of calcium in the intestine [ 51 and stimulates excretion of calcium into the bile of rats [ 61. It was therefore assumed that administration of Sn might lower the Ca content of bone. The present study was therefore undertaken to examine whether the Ca content of the femur of rats was affected by oral administration of SnC12. METHODS
Male Wistar strain rats (loo- 120 g) were kept at a room temperature of 25 f 1°C and maintained on a laboratory chow containing 74% carbohydrate,
8
1.1% calcium and 1.1% phosphate, with tap water ad lib. SnCl, was dissolved in demineralized water containing 2 drops of 6 N HC1/25 ml to a final concentration of 3 mg of Sn2’/ml. This solution (pH 1.8) was administered orally (1.Q ml/100 g body weight) to rats 6 times at 12-h intervals; the control animals were dosed with HCl solution (pH 1.8) without Sn. CaC12 (10 mg Ca*‘/0.5 ml/100 g) dissolved in dem~eralized water was injected subcutaneously 6 times at 12-h intervals immediately after the Sn administration. The thyroparathyroid gland complex was removed with fine forceps under light anesthesia with ether. Sn was administered immediately after thyroparathyroidectomy. The rats were bled by cardiac puncture under light anesthesia with ether 17 h after the last administration of Sn, and the serum was obtained by centrifuging the blood. Serum Ca was determined on 0.1 ml aliquots by atomic absorption spectrophotometry (Perkin-Elmer, Model 303) after precipitation of protein with 10% trichloroacetic acid [4] . The femurs were cleaned of soft tissue and marrow, and their diaphyseal and epiphyseal bone separated, ashed for 24 h at 64O”C, weighed, and dissolved in 1 N HCl. Ca was determined by atomic absorption spectrophotometry [ 11. The data were subjected to an analysis of variance, and the standard error (SE) was calculated from the residual error term. Statistical significance is expressed as P values from Student’s t-test.
g !joo Diaphysis rtl t
Epiphysis
jiiJ
if!so0 -
z
g 300s E 2002 loo- ii n(mg/l~g)
(1)
20 17 120 Hours after tin administ.
Fig. 1. Effect of tin at three dose levels (1, 3, or 5 mg /lOO g) on calcium content in the femur of rats. Stannous chloride was orally administered 6 times at 12-h intervals. The rats were killed 17 h after the last administration of tin. Each point represents the mean vatue of 6 rats. Vertical lines indicate the SE. 0 - - - - 0, diaphysis; e-----e, epiphysis. Fig. 2. Changes of calcium content in the femur of rats after stannous chloride. Stannous chloride was orally administered The rats were killed 17 or 120 h after the last administration the mean value of 6 rats. Vertical lines indicate the SE.0,
the oral administration of 6 times at 12-h intervals. of tin. Each bar represents control; m, tin.
(2)
9
RESULTS
The effects of Sn administration, at 3 dose levels (1, 3, and 5 mg Sn2’/ 100 g), on the calcium content in the femur are shown in Fig. 1. The Ca content of the femoral diaphysis and epiphysis was significantly decreased even by the 1.0 mg Sn2’/100 g dose. The difference between the two higher doses (3.0 and 5.0 mg Sn2+/100 g) was not significant. The Ca content in the diaphysis and epiphysis was significantly increased by the simultaneous administration of Sn (3.0 mg Sn2’/100 g, orally) and CaCl, (10 mg Ca”/lOO g, subcutaneously), but the serum Ca concentration was not elevated significantly (Table I). TABLE I EFFECT OF CALCIUM CHLORIDE ON CALCIUM CONTENT IN THE FEMUR OF RATS ORALLY ADMINISTERED STANNOUS CHLORIDE Treatment
Number of rats
Serum caiciumb (mg/lOO ml)
Bone calcium (ma/g ash)b ,-_ , Diaphysis
Epiphysis
5 6
10.1 + 0.24 9.2 f 0.15c
416.0 f 11.1 316.5 + 12.0c
405.1 f 5.0 305.8 f 1O.V
456.7 f 7.0d 359.9 * 10.8Cd
429.9 f 342.1 f
None Control Tin
Calcium injectiona Control Tin
5 6
9.8 2 0.10 9.1 + 0.17c
5.1 9.0Cd
‘CaClz (10 mg Ca*+/lOO g) was subcutaneously injected 6 times at 12-h intervals immediately after the administration of SnCl, (3.0 mg Sn3+/100 g). The rats were killed 17 h after the last administration of calcium. bMean + SE. CSignificance from control, P < 0.01 (Student’s t-test). dSignificance from none, P < 0.01 (Student’s t-test). TABLE II CHANGE OF CALCIUM CONTENT IN THE FEMUR OF THYROPARATHYROIDECTOMIZED RATS ORALLY ADMINISTERED STANNOUS CHLORIDE Treatment
Control Tina
Number of rats
Serum calciumb (mg/lOO ml)
Bone calcium (mg/g ash)b Diaphysis
Epiphysis
5 6
6.10 + 0.40 4.90 f 0.17e
408.2 r 4.7 401.6 f 5.9
409.1 * 15.8 519.4 * 21.3c
a SnCl, (3.0 mg Sn* +/lOO g) was orally administered 6 times at 12-h intervals immediately after the thyroparathyroidectomy. The rats were killed 17 h after the last administration. bMean + SE. CSignificance from control, P < 0.01 (Student’s t-test).
10
Administration of tin to thyroparathyroidectomized rats did not decrease the femoral Ca content, while it significantly reduced serum Ca concentration (Table II). Tin (3.0 mg Sn2’/100 g) was given orally 6 times at 12-h intervals; the rats were killed 17 h or 120 h after the last administration of tin. A significant decrease in the calcium content in the diaphysis and the epiphysis of the femur was observed 17 h after the last administration of tin, while the calcium content was recovered 120 h after the tin administration (Fig. 2). DISCUSSION
In the present study, the Ca content of the rat femur was markedly decreased by the oral administration of Sn. The subcutaneous injection of CaCl, caused a significant increase in the Ca content of the femur in both the Sri-administered and the control rats (Table I), suggesting that Sn may not prevent the femoral deposition of Ca. Recently it has been reported that the oral administration of SnCl, (3 mg Sn*‘/lOO g every 12 h) for 3 days significantly inhibits transport of Ca in the intestine [S] and markedly stimulates excretion of Ca in rat bile (61, resulting in a significant decrease in serum Ca [6]. The hypocalcemia thus caused presumably stimulates secretion of parathyroid hormone to maintain Ca homeostasis. In the thyrop~athyroidectomized rats, a significant decrease in femoral Ca was not produced by Sn (Table II). These results suggest that the Sn-induced reduction of Ca in the femur of rats was due mainly to the action of parathyroid hormone. REFERENCES 1 D. Henneman and J.J. Zimmerberg, Lack of effect of chronic lithium chloride on bone composition and metabolism, Endocrinology, 94 (1974) 915-917. 2 R.A. Hiles, Absorption, distribution and excretion of inorganic tin in rats, Toxicol. Appl. Pharmacol., 27 (1974) 366-379. 3 S. Warburton, W. Ulder, R.M. Ewert and W.S. Haynes, Outbreak of food borne illness attributed to tin, Public Health Rep., 77 (1962) 798-300. 4 J.B. Willis, Determination of calcium in blood serum by atomic absorption spectroscopy, Nature (London), 186 (1960) 249-250. 5 M. Yamaguchi, Y. Kubo and T. Yamamoto, Inhibitory effect of tin on intestinal calcium absorption in rats, Toxicol. Appl. Pharmacol., in press (1978). 6 M. Yamaguchi and T. Yamamoto, Effect of tin on calcium content in the bile of rats, Toxicol. Appl. Pharmacol., 45 (1978) 611-616.
7
DECREASE IN CALCIUM CONTENT IN THE FEMUR OF RATS ORALLY ADMINISTERED STANNOUS CHLORIDE
MASAYOSHI YAMAGUCHI,
MITUKO KITADE and SHOJI OKADA
Department of Environmental Shizuoka, 422 (Japan)
Biochemistry,
Shizuoka College of Pharmacy, 2-2-1, Oshika,
(Received September 6th, 1978) (Accepted September 14th, 1978)
SUMMARY
The calcium content in the femur of rats was significantly decreased by the oral administration of stannous chloride (3.0 mg Sn2’/100 g body weight, 6 times at 12-h intervals). This decrease was dose-dependent. Administration of tin did not prevent femoral deposition of subcutaneously injected calcium. Administration of tin to thyroparathyroidectomized rats did not reduce femoral calcium, but lowered serum calcium significantly. The present study suggests that the decrease in femoral calcium produced by the oral administration of tin resulted mainly from the mobilization, induced by parathyroid !rormone, of calcium into blood from bone.
INTRODUCTION
Environmental contamination by tin (Sn) is due largely to canned food. Several cases of intoxication have been observed following the intake of canned food containing high levels of Sn [3]. The toxic effects of Sn on bone metabolism are little understood, although Sn has been known to accumulate in the bone of rats [2]. Recently the authors have reported that the oral administration of stannous chloride inhibits transport of calcium in the intestine [ 51 and stimulates excretion of calcium into the bile of rats [ 61. It was therefore assumed that administration of Sn might lower the Ca content of bone. The present study was therefore undertaken to examine whether the Ca content of the femur of rats was affected by oral administration of SnC12. METHODS
Male Wistar strain rats (loo- 120 g) were kept at a room temperature of 25 f 1°C and maintained on a laboratory chow containing 74% carbohydrate,
8
1.1% calcium and 1.1% phosphate, with tap water ad lib. SnCl, was dissolved in demineralized water containing 2 drops of 6 N HC1/25 ml to a final concentration of 3 mg of Sn2’/ml. This solution (pH 1.8) was administered orally (1.Q ml/100 g body weight) to rats 6 times at 12-h intervals; the control animals were dosed with HCl solution (pH 1.8) without Sn. CaC12 (10 mg Ca*‘/0.5 ml/100 g) dissolved in dem~eralized water was injected subcutaneously 6 times at 12-h intervals immediately after the Sn administration. The thyroparathyroid gland complex was removed with fine forceps under light anesthesia with ether. Sn was administered immediately after thyroparathyroidectomy. The rats were bled by cardiac puncture under light anesthesia with ether 17 h after the last administration of Sn, and the serum was obtained by centrifuging the blood. Serum Ca was determined on 0.1 ml aliquots by atomic absorption spectrophotometry (Perkin-Elmer, Model 303) after precipitation of protein with 10% trichloroacetic acid [4] . The femurs were cleaned of soft tissue and marrow, and their diaphyseal and epiphyseal bone separated, ashed for 24 h at 64O”C, weighed, and dissolved in 1 N HCl. Ca was determined by atomic absorption spectrophotometry [ 11. The data were subjected to an analysis of variance, and the standard error (SE) was calculated from the residual error term. Statistical significance is expressed as P values from Student’s t-test.
g !joo Diaphysis rtl t
Epiphysis
jiiJ
if!so0 -
z
g 300s E 2002 loo- ii n(mg/l~g)
(1)
20 17 120 Hours after tin administ.
Fig. 1. Effect of tin at three dose levels (1, 3, or 5 mg /lOO g) on calcium content in the femur of rats. Stannous chloride was orally administered 6 times at 12-h intervals. The rats were killed 17 h after the last administration of tin. Each point represents the mean vatue of 6 rats. Vertical lines indicate the SE. 0 - - - - 0, diaphysis; e-----e, epiphysis. Fig. 2. Changes of calcium content in the femur of rats after stannous chloride. Stannous chloride was orally administered The rats were killed 17 or 120 h after the last administration the mean value of 6 rats. Vertical lines indicate the SE.0,
the oral administration of 6 times at 12-h intervals. of tin. Each bar represents control; m, tin.
(2)
9
RESULTS
The effects of Sn administration, at 3 dose levels (1, 3, and 5 mg Sn2’/ 100 g), on the calcium content in the femur are shown in Fig. 1. The Ca content of the femoral diaphysis and epiphysis was significantly decreased even by the 1.0 mg Sn2’/100 g dose. The difference between the two higher doses (3.0 and 5.0 mg Sn2+/100 g) was not significant. The Ca content in the diaphysis and epiphysis was significantly increased by the simultaneous administration of Sn (3.0 mg Sn2’/100 g, orally) and CaCl, (10 mg Ca”/lOO g, subcutaneously), but the serum Ca concentration was not elevated significantly (Table I). TABLE I EFFECT OF CALCIUM CHLORIDE ON CALCIUM CONTENT IN THE FEMUR OF RATS ORALLY ADMINISTERED STANNOUS CHLORIDE Treatment
Number of rats
Serum caiciumb (mg/lOO ml)
Bone calcium (ma/g ash)b ,-_ , Diaphysis
Epiphysis
5 6
10.1 + 0.24 9.2 f 0.15c
416.0 f 11.1 316.5 + 12.0c
405.1 f 5.0 305.8 f 1O.V
456.7 f 7.0d 359.9 * 10.8Cd
429.9 f 342.1 f
None Control Tin
Calcium injectiona Control Tin
5 6
9.8 2 0.10 9.1 + 0.17c
5.1 9.0Cd
‘CaClz (10 mg Ca*+/lOO g) was subcutaneously injected 6 times at 12-h intervals immediately after the administration of SnCl, (3.0 mg Sn3+/100 g). The rats were killed 17 h after the last administration of calcium. bMean + SE. CSignificance from control, P < 0.01 (Student’s t-test). dSignificance from none, P < 0.01 (Student’s t-test). TABLE II CHANGE OF CALCIUM CONTENT IN THE FEMUR OF THYROPARATHYROIDECTOMIZED RATS ORALLY ADMINISTERED STANNOUS CHLORIDE Treatment
Control Tina
Number of rats
Serum calciumb (mg/lOO ml)
Bone calcium (mg/g ash)b Diaphysis
Epiphysis
5 6
6.10 + 0.40 4.90 f 0.17e
408.2 r 4.7 401.6 f 5.9
409.1 * 15.8 519.4 * 21.3c
a SnCl, (3.0 mg Sn* +/lOO g) was orally administered 6 times at 12-h intervals immediately after the thyroparathyroidectomy. The rats were killed 17 h after the last administration. bMean + SE. CSignificance from control, P < 0.01 (Student’s t-test).
10
Administration of tin to thyroparathyroidectomized rats did not decrease the femoral Ca content, while it significantly reduced serum Ca concentration (Table II). Tin (3.0 mg Sn2’/100 g) was given orally 6 times at 12-h intervals; the rats were killed 17 h or 120 h after the last administration of tin. A significant decrease in the calcium content in the diaphysis and the epiphysis of the femur was observed 17 h after the last administration of tin, while the calcium content was recovered 120 h after the tin administration (Fig. 2). DISCUSSION
In the present study, the Ca content of the rat femur was markedly decreased by the oral administration of Sn. The subcutaneous injection of CaCl, caused a significant increase in the Ca content of the femur in both the Sri-administered and the control rats (Table I), suggesting that Sn may not prevent the femoral deposition of Ca. Recently it has been reported that the oral administration of SnCl, (3 mg Sn*‘/lOO g every 12 h) for 3 days significantly inhibits transport of Ca in the intestine [S] and markedly stimulates excretion of Ca in rat bile (61, resulting in a significant decrease in serum Ca [6]. The hypocalcemia thus caused presumably stimulates secretion of parathyroid hormone to maintain Ca homeostasis. In the thyrop~athyroidectomized rats, a significant decrease in femoral Ca was not produced by Sn (Table II). These results suggest that the Sn-induced reduction of Ca in the femur of rats was due mainly to the action of parathyroid hormone. REFERENCES 1 D. Henneman and J.J. Zimmerberg, Lack of effect of chronic lithium chloride on bone composition and metabolism, Endocrinology, 94 (1974) 915-917. 2 R.A. Hiles, Absorption, distribution and excretion of inorganic tin in rats, Toxicol. Appl. Pharmacol., 27 (1974) 366-379. 3 S. Warburton, W. Ulder, R.M. Ewert and W.S. Haynes, Outbreak of food borne illness attributed to tin, Public Health Rep., 77 (1962) 798-300. 4 J.B. Willis, Determination of calcium in blood serum by atomic absorption spectroscopy, Nature (London), 186 (1960) 249-250. 5 M. Yamaguchi, Y. Kubo and T. Yamamoto, Inhibitory effect of tin on intestinal calcium absorption in rats, Toxicol. Appl. Pharmacol., in press (1978). 6 M. Yamaguchi and T. Yamamoto, Effect of tin on calcium content in the bile of rats, Toxicol. Appl. Pharmacol., 45 (1978) 611-616.