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Effect of vitamin D3 derivatives on calcium concentrations in the serum of rats treated with stannous chloride
183
Toxicology Letters, l(l978) 183-186 o Elsevier/North-Holland Biomedical Press
EFFECT OF VITAMIN D3 DERIVATIVES ON CALCIUM CONCENTRATIONS IN THE SERUM OF R>ATS TREATED WITH STANNOUS CHLORIDE
MASAYOSHI YAMAGUCHI, HIDEJI SAT0 and TAKE0
YAMAMOTO
Department of Environmental Health, Shizuoka College of Pharmaceutical Science, Oshika, Shizuoka, 422 (Japan)
2-2-1,
(Received August 8th, 1977) (Accepted September 23rd, 1977)
SUMMARY
Calcium concentrations in the serum of rats were significantly increased by treatment with vitamin D3 (25 pg/lOO g), but this increase was prevented by a single intraperitoneal administration of tin (Sri”, 3.0 mg/lOO g). The decrease in serum calcium levels after the administration of Sn2+ was reversed by treatment with synthetic la-hydroxyvitamin D3 (0.25 pg/lOO g) or la,25dihydroxyvitamin D3 (0.25 pg/lOO g). These results suggest that the decrease in serum calcium levels produced by the administration of Sn2* m.ay be caused by inhibition of metabolism of 25-hydroxyvitamin D3 to lar,25-dihydroxyvitamin DJ in the kidneys.
INTRODUCTION
Recently, it has been reported that the decrease in calcium concentrations in the serum of rats treated with tin compounds is caused by kidney disturbances due to the accumulation of calcium in the kidneys [9]. Vitamin D3 is metabolically activated in the kidneys to exhibit its hypercalcaemic effect [1,2]. If the metabolism of vitamin D3 in the kidneys is inhibited by the administration of tin compounds, its hypercalcaemic effect would not occur in rats treated with tin compounds. Accordingly, the present study was undertaken to examine whether calcium concentrations in serum of rats treated with stannous chloride (SnCl?) were influenced by the administration of synthetic la-hydroxyvitamin D3 or la,25-dihydroxyvitamin D3. METHODS
Male Wistar strain rats, each weighing approximately 120 g, were used in the present experiment. The animals were kept at a room temperature of 25 f 1°C and allowed laboratory chow and tap water ad lib.
184
SnCl, was diluted in distilled water to a final concentration of 3 mg of Sn’+/ml, to which was added 2 drops of 6 N HCl. This solution (pH 1.8) was given by a single intraperitoneal administration (1.0 ml/100 g body weight) to rats; controls were injected with HCl solution (pH 1.8) only. Vitamin D3, la-hydroxyvitamin D3, and la,25_dihydroxyvitamin D3 freshly dissolved in 99.5% (v/v) ethanol solution to concentrations of 250, 25 and 2.5 pg/ml, respectively. These solutions (0.1 ml/100 g body weight) were given to rats by intraperitoneal injection. Control rats received the ethanol vehicle (0.1 ml/100 g). The rats were bled by cardiac puncture under light ether anaesthesia. Blood samples obtained by cardiac puncture were centrifuged immediately after the collection. The serum was separated and analyzed immediately. Determination of calcium was made on O.l-ml aliquots of serum by atomic absorption spectrophotometry (Perkin-Elmer, Model 303) after precipitation with 10% trichloroacetic acid [ 41. RESULTS
The time-course of serum calcium concentrations in rats after a single intraperitoneal administration of vitamin D3 (25 pg/lOO g) is shown in Fig. 1. The serum calcium concentration increased gradually, reaching a maximum at 16 h after administration. Serum calcium concentrations were markedly lower at 72 h after the administration of SnC12 (Sn” 3.0 mg/lOO g) to rats [9]. Vitamin D3 (25 or 250 pg/lOO g) was intraperitoneally injected at 56 h after a single intraperitoneal
0
8
16 Hours
24
ven,ce Vitomin
P5 D3
250 (pg/lOOg)
Fig. 1. Effect of vitamin D, on calcium concentrations in serum of rats. The animals were intraperitoneally given 25 pg vitamin D,/lOO g. Each point represents the mean value of 5 animals. The vertical lines indicate the SE. * - -*, control; -, vitamin D,. Fig. 2. Effect of vitamin D, on calcium concentrations in serum of rats treated with SnCl, The animals were intraperitoneally given vitamin D, at 56 h after the administration of stannous chloride (3 mg Sn2+/100 g). The animals were killed at 16 h after the injection of vitamin D,. Each bar represents the mean value of 5 rats. The vertical lines indicate the SE. 0, control; I, tin.
185
administration of Sn” (3.0 mg/lOO g) to rats, and the animals were killed at 16 h after the treatment with vitamin D3. The decreased serum calcium concentrations caused by the administration of tin were not elevated significantly by treatment with vitamin D3 at both dose levels, suggesting that the hypercalcaemic effect of vitamin D3 probably does not occur in rats treated with Sn2+ (Fig. 2). The effect of vitamin D3 derivatives on serum calcium levels after the administration of Sn2+ was also examined. Synthetic lol-hydroxyvitamin D3 (0.25 pg/lOO g) or la,25-dihydroxyvitamin D3 (0.25 pg/lOO g) was intraperitoneally injected immediately after the administration of SnC12 (Sn2’ 3.0 mg/lOO g), and further doses were injected twice at 24-h intervals. The animals were killed at 72 h after the administration of Sn2’. Serum calcium concentrations in rats treated with Sn2’ were markedly elevated by treatment with la-hydroxyvitamin DJ or la,25-dihydroxyvitamin D3 (Table I).
TABLE I EFFECT OF lor-HYDROXYVITAMIN D, or la,25-DIHYDROXYVITAMIN D, ON CALCIUM CONCENTRATIONS IN THE SERUM OF RATS TREATED WITH STANNOUS CHLORIDE Treatment
Ethanol 1wHydroxyvitamin D, l&,25-Dihydroxyvitamin
Number of rats
D,
5 6 6
Serum calcium (mg/lOO ml) Control
Tin
6.80 5 0.05’ 8.61 + 0.25b 8.59 2 0.30b
6.48 f 0.18 8.35 f 0.54b 8.21 f 0.47b
aMean + SE. bSignificance from the ethanol, P < 0.01 (Student’s t-test).
DISCUSSION
The administration of tin compounds causes accumulation of calcium in the kidneys and a decrease of calcium levels in the serum of rats [9]. The urinary excretion of calcium is markedly decreased by the administration of tin compounds [ 71. These studies indicated that hypocalcaemia after the administration of tin compounds was not related to the excretion of calcium into urine. The hypercalcaemic effect of vitamin D3 was not detected in the rats treated with tin compounds, whereas administration of synthetic la-hydroxyvitamin D3 or lo,25_dihydroxyvitamin D3 caused a significant elevation of calcium concentrations in serum of rats thus treated. Vitamin D3 is metabolized to 25-hydroxyvitamin I), by a hepatic enzyme [4] ; this metabolite is subsequently
186
converted to lcu,25dihydroxyvitamin D3 by a renal mitochondrial enzyme [2], and this active metabolite of vitamin D3 causes striking increases in serum calcium levels [ 51. Accordingly, our results suggest that the action of la,25dihydroxyvitamin D3 was not inhibited by the administration of tin compounds and that vitamin D3 can still be metabolized to 25-hydroxyvitamin D3 in the liver of rats treated with tin compounds. Since renal mitochondrial 1-hydroxylase activity is decreased by calcium ions [ 31, it is possible that the accumulation of calcium in renal mitochondria produced by the administration of tin compounds [ 81 may prevent the conversion of 25-hydroxyvitamin D3 to 25dihydroxyvitamin D3.
The present work suggests that a possible mechanism for the lowering of serum calcium levels of rats by tin compounds is a disturbance of vitamin D3 metabolism in the kidneys caused by the accumulation of calcium ions in the kidneys. It is appreciated that different treatment schedules were used in the experiments involving vitamin D3 and those involving its metabolites and that this may have influenced the validity of some of the results. REFERENCES 1 LT. Boyle, J.T. Ohmdahl, R.W. Gray and H.F. DeLuca, Biological activity and metabolism of 24,25dihydroxyvitamin D,, J. Biol. Chem., 248 (1973) 4174-4180. 2 D.R. Fraser and E. Kodicek, Unique biosynthesis by kidney of a biologically active vitamin D metabolite, Nature (London), 228 (1970) 764-766. 3 N. Horiuchi, T. Suda, S. Sasaki, E. Ogata, I. Ezawa and Y. Sano, The regulatory role of calcium in 25-hydroxycholecalciferol metabolism in chick kidney in vitro, Arch. Biothem. Biophys., 171 (1975) 540-548. 4 M. Horsting and H.F. DeLuca, In vitro production of 25-hydroxycholecalciferol, Biochem. Biophys. Res. Commun., 36 (1969), 251-256. 5 Y. Tanaka and H.F. DeLuca, Inhibition of the metabolism of 25-hydroxycholecalciferol by actinomycin D and cycloheximide, Proc. Natl. Acad. Sci. USA, 68 (1971) 605-608. 6.d.B. Willis, Determination of calcium in blood serum by atomic absorption spectroscopy, Nature (London), 16 (1960) 24% --250. 7 M. Yamaguchi, H. Sato and T. Yamamoto, Decrease of calcium concentration in urine of rats treated with stannous chloride, Chem. Pharm. Bull. (Tokyo), 24 (1976) 31993201. 8 M. Yamaguchi, H. Sato and T. Yamamoto, Increase in calcium binding activity in renal cortex of rats treated with stannous chloride, J. Toxicol. Environ. Health, 3 (1977) 411-418. 9 T. Yamamoto, M. Yamaguchi and H. Sato, Accumulation of calcium in kidney and decrease of calcium in serum of rats treated with tin chloride, J. Toxicol. Environ. Health, 1 (1976) 749-756.
Toxicology Letters, l(l978) 183-186 o Elsevier/North-Holland Biomedical Press
EFFECT OF VITAMIN D3 DERIVATIVES ON CALCIUM CONCENTRATIONS IN THE SERUM OF R>ATS TREATED WITH STANNOUS CHLORIDE
MASAYOSHI YAMAGUCHI, HIDEJI SAT0 and TAKE0
YAMAMOTO
Department of Environmental Health, Shizuoka College of Pharmaceutical Science, Oshika, Shizuoka, 422 (Japan)
2-2-1,
(Received August 8th, 1977) (Accepted September 23rd, 1977)
SUMMARY
Calcium concentrations in the serum of rats were significantly increased by treatment with vitamin D3 (25 pg/lOO g), but this increase was prevented by a single intraperitoneal administration of tin (Sri”, 3.0 mg/lOO g). The decrease in serum calcium levels after the administration of Sn2+ was reversed by treatment with synthetic la-hydroxyvitamin D3 (0.25 pg/lOO g) or la,25dihydroxyvitamin D3 (0.25 pg/lOO g). These results suggest that the decrease in serum calcium levels produced by the administration of Sn2* m.ay be caused by inhibition of metabolism of 25-hydroxyvitamin D3 to lar,25-dihydroxyvitamin DJ in the kidneys.
INTRODUCTION
Recently, it has been reported that the decrease in calcium concentrations in the serum of rats treated with tin compounds is caused by kidney disturbances due to the accumulation of calcium in the kidneys [9]. Vitamin D3 is metabolically activated in the kidneys to exhibit its hypercalcaemic effect [1,2]. If the metabolism of vitamin D3 in the kidneys is inhibited by the administration of tin compounds, its hypercalcaemic effect would not occur in rats treated with tin compounds. Accordingly, the present study was undertaken to examine whether calcium concentrations in serum of rats treated with stannous chloride (SnCl?) were influenced by the administration of synthetic la-hydroxyvitamin D3 or la,25-dihydroxyvitamin D3. METHODS
Male Wistar strain rats, each weighing approximately 120 g, were used in the present experiment. The animals were kept at a room temperature of 25 f 1°C and allowed laboratory chow and tap water ad lib.
184
SnCl, was diluted in distilled water to a final concentration of 3 mg of Sn’+/ml, to which was added 2 drops of 6 N HCl. This solution (pH 1.8) was given by a single intraperitoneal administration (1.0 ml/100 g body weight) to rats; controls were injected with HCl solution (pH 1.8) only. Vitamin D3, la-hydroxyvitamin D3, and la,25_dihydroxyvitamin D3 freshly dissolved in 99.5% (v/v) ethanol solution to concentrations of 250, 25 and 2.5 pg/ml, respectively. These solutions (0.1 ml/100 g body weight) were given to rats by intraperitoneal injection. Control rats received the ethanol vehicle (0.1 ml/100 g). The rats were bled by cardiac puncture under light ether anaesthesia. Blood samples obtained by cardiac puncture were centrifuged immediately after the collection. The serum was separated and analyzed immediately. Determination of calcium was made on O.l-ml aliquots of serum by atomic absorption spectrophotometry (Perkin-Elmer, Model 303) after precipitation with 10% trichloroacetic acid [ 41. RESULTS
The time-course of serum calcium concentrations in rats after a single intraperitoneal administration of vitamin D3 (25 pg/lOO g) is shown in Fig. 1. The serum calcium concentration increased gradually, reaching a maximum at 16 h after administration. Serum calcium concentrations were markedly lower at 72 h after the administration of SnC12 (Sn” 3.0 mg/lOO g) to rats [9]. Vitamin D3 (25 or 250 pg/lOO g) was intraperitoneally injected at 56 h after a single intraperitoneal
0
8
16 Hours
24
ven,ce Vitomin
P5 D3
250 (pg/lOOg)
Fig. 1. Effect of vitamin D, on calcium concentrations in serum of rats. The animals were intraperitoneally given 25 pg vitamin D,/lOO g. Each point represents the mean value of 5 animals. The vertical lines indicate the SE. * - -*, control; -, vitamin D,. Fig. 2. Effect of vitamin D, on calcium concentrations in serum of rats treated with SnCl, The animals were intraperitoneally given vitamin D, at 56 h after the administration of stannous chloride (3 mg Sn2+/100 g). The animals were killed at 16 h after the injection of vitamin D,. Each bar represents the mean value of 5 rats. The vertical lines indicate the SE. 0, control; I, tin.
185
administration of Sn” (3.0 mg/lOO g) to rats, and the animals were killed at 16 h after the treatment with vitamin D3. The decreased serum calcium concentrations caused by the administration of tin were not elevated significantly by treatment with vitamin D3 at both dose levels, suggesting that the hypercalcaemic effect of vitamin D3 probably does not occur in rats treated with Sn2+ (Fig. 2). The effect of vitamin D3 derivatives on serum calcium levels after the administration of Sn2+ was also examined. Synthetic lol-hydroxyvitamin D3 (0.25 pg/lOO g) or la,25-dihydroxyvitamin D3 (0.25 pg/lOO g) was intraperitoneally injected immediately after the administration of SnC12 (Sn2’ 3.0 mg/lOO g), and further doses were injected twice at 24-h intervals. The animals were killed at 72 h after the administration of Sn2’. Serum calcium concentrations in rats treated with Sn2’ were markedly elevated by treatment with la-hydroxyvitamin DJ or la,25-dihydroxyvitamin D3 (Table I).
TABLE I EFFECT OF lor-HYDROXYVITAMIN D, or la,25-DIHYDROXYVITAMIN D, ON CALCIUM CONCENTRATIONS IN THE SERUM OF RATS TREATED WITH STANNOUS CHLORIDE Treatment
Ethanol 1wHydroxyvitamin D, l&,25-Dihydroxyvitamin
Number of rats
D,
5 6 6
Serum calcium (mg/lOO ml) Control
Tin
6.80 5 0.05’ 8.61 + 0.25b 8.59 2 0.30b
6.48 f 0.18 8.35 f 0.54b 8.21 f 0.47b
aMean + SE. bSignificance from the ethanol, P < 0.01 (Student’s t-test).
DISCUSSION
The administration of tin compounds causes accumulation of calcium in the kidneys and a decrease of calcium levels in the serum of rats [9]. The urinary excretion of calcium is markedly decreased by the administration of tin compounds [ 71. These studies indicated that hypocalcaemia after the administration of tin compounds was not related to the excretion of calcium into urine. The hypercalcaemic effect of vitamin D3 was not detected in the rats treated with tin compounds, whereas administration of synthetic la-hydroxyvitamin D3 or lo,25_dihydroxyvitamin D3 caused a significant elevation of calcium concentrations in serum of rats thus treated. Vitamin D3 is metabolized to 25-hydroxyvitamin I), by a hepatic enzyme [4] ; this metabolite is subsequently
186
converted to lcu,25dihydroxyvitamin D3 by a renal mitochondrial enzyme [2], and this active metabolite of vitamin D3 causes striking increases in serum calcium levels [ 51. Accordingly, our results suggest that the action of la,25dihydroxyvitamin D3 was not inhibited by the administration of tin compounds and that vitamin D3 can still be metabolized to 25-hydroxyvitamin D3 in the liver of rats treated with tin compounds. Since renal mitochondrial 1-hydroxylase activity is decreased by calcium ions [ 31, it is possible that the accumulation of calcium in renal mitochondria produced by the administration of tin compounds [ 81 may prevent the conversion of 25-hydroxyvitamin D3 to 25dihydroxyvitamin D3.
The present work suggests that a possible mechanism for the lowering of serum calcium levels of rats by tin compounds is a disturbance of vitamin D3 metabolism in the kidneys caused by the accumulation of calcium ions in the kidneys. It is appreciated that different treatment schedules were used in the experiments involving vitamin D3 and those involving its metabolites and that this may have influenced the validity of some of the results. REFERENCES 1 LT. Boyle, J.T. Ohmdahl, R.W. Gray and H.F. DeLuca, Biological activity and metabolism of 24,25dihydroxyvitamin D,, J. Biol. Chem., 248 (1973) 4174-4180. 2 D.R. Fraser and E. Kodicek, Unique biosynthesis by kidney of a biologically active vitamin D metabolite, Nature (London), 228 (1970) 764-766. 3 N. Horiuchi, T. Suda, S. Sasaki, E. Ogata, I. Ezawa and Y. Sano, The regulatory role of calcium in 25-hydroxycholecalciferol metabolism in chick kidney in vitro, Arch. Biothem. Biophys., 171 (1975) 540-548. 4 M. Horsting and H.F. DeLuca, In vitro production of 25-hydroxycholecalciferol, Biochem. Biophys. Res. Commun., 36 (1969), 251-256. 5 Y. Tanaka and H.F. DeLuca, Inhibition of the metabolism of 25-hydroxycholecalciferol by actinomycin D and cycloheximide, Proc. Natl. Acad. Sci. USA, 68 (1971) 605-608. 6.d.B. Willis, Determination of calcium in blood serum by atomic absorption spectroscopy, Nature (London), 16 (1960) 24% --250. 7 M. Yamaguchi, H. Sato and T. Yamamoto, Decrease of calcium concentration in urine of rats treated with stannous chloride, Chem. Pharm. Bull. (Tokyo), 24 (1976) 31993201. 8 M. Yamaguchi, H. Sato and T. Yamamoto, Increase in calcium binding activity in renal cortex of rats treated with stannous chloride, J. Toxicol. Environ. Health, 3 (1977) 411-418. 9 T. Yamamoto, M. Yamaguchi and H. Sato, Accumulation of calcium in kidney and decrease of calcium in serum of rats treated with tin chloride, J. Toxicol. Environ. Health, 1 (1976) 749-756.