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Zinc Enhancement of Genistein’s Anabolic Effect on Bone Components in Elderly Female Rats
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Gen. Pharmac. Vol. 31, No. 2, pp. 199–202, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Zinc Enhancement of Genistein’s Anabolic Effect on Bone Components in Elderly Female Rats Ying Hua Gao and Masayoshi Yamaguchi* Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422, Japan [Tel. (Fax): 81-54-264-5580] ABSTRACT. 1. The effect of genistein on the bone components in the femoral-metaphyseal and diaphyseal tissues of elderly female rats was investigated. 2. The oral administration of genistein (100 and 200 mg/kg body weight) for 3 days to rats caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the femoral-metaphyseal and diaphyseal tissues. A dose of 50 mg genistein/kg appreciably increased alkaline phosphatase activity and DNA content in the metaphyseal but not the diaphyseal tissues. 3. Alkaline phosphatase activity, DNA and calcium contents in the femoral-metaphyseal and diaphyseal tissues were significantly elevated by the oral administration of zinc sulfate (5.5 mg Zn/kg) for 3 days. The effect of zinc on increasing bone DNA and calcium contents was synergistically enhanced by the simultaneous administration of genistein (100 mg/kg). 4. The stimulatory effect of zinc sulfate (1025 M) or genistein (1025 M) on bone calcium content was also seen in a culture system using the femoral-metaphyseal and diaphyseal tissues in vitro. This effect was completely prevented by the presence of cycloheximide (1026 M). 5. The present findings suggest that genistein has an anabolic effect on bone components and that the effect is enhanced by zinc, owing to its stimulating effect on bone protein synthesis. gen pharmac 31;2:199–202, 1998. 1998 Elsevier Science Inc. KEY WORDS. Genistein, zinc, bone formation, rat femur, aging
INTRODUCTION Bone mass decreases with increasing age. The decrease is due to increased bone resorption and to decreased bone formation. Pharmacological and nutritional factors are needed to prevent bone loss with increasing age. These factors, however, are not fully clarified. Genistein is a natural isoflavonoid phytoestrogen. The flavonoid is found in Leguminosae. Genistein has been shown to reveal an inhibitory effect on protein thyrosine kinases in vitro (Liu et al., 1994; Spinozzi et al., 1994), although the biological effect of flavonoid has not been fully clarified. More recently, genistein has been demonstrated to have a direct stimulatory effect on bone formation in a tissue culture system in vitro (Yamaguchi and Gao, 1997). Whether genistein has an anabolic effect on the bone metabolism of elderly female rats in vivo, however, remains to be elucidated. On the other hand, zinc, an essential trace element, has been shown to stimulate osteoblastic bone formation and to inhibit osteoclastic bone resorption in vitro and in vivo (Kishi and Yamaguchi, 1994; Yamaguchi, 1995; Yamaguchi and Hashizume, 1994; Yamaguchi et al., 1987). These factors may have a preventive role in osteoporosis. The present study was undertaken to clarify whether the combination of genistein and zinc has an anabolic effect on the bone metabolism of elderly female rats in vivo. The oral administration of genistein was found to have an anabolic effect on bone components in the femoral-metaphyseal and diaphyseal tissues of elderly female rats, and the isoflavonoid effect was found to be synergistically enhanced by zinc administration. The combination of genistein and zinc may have a role in the prevention of bone loss in aged rats.
MATERIALS AND METHODS
Chemicals Dulbecco’s modified Eagle medium (high glucose) was obtained from GIBCO Laboratories (Grand Island, NY, USA). Genistein and cycloheximide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Zinc sulfate and all other chemicals were reagent grade from Sigma Chemical and Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All water used was glass distilled.
Animals and administration procedure Female Wistar rats, weighing 200–220 g (50 weeks old), were obtained from Japan SLC (Hamamatsu, Japan). Animals were fed commercial laboratory chow (solid) containing 1.1% calcium, 1.1% phosphorus and 0.012% zinc and distilled water. Genistein was dissolved in 10% ethanol at concentrations of 10, 20 and 40 mg/ml. Genistein (50, 100 and 200 mg/kg body weight) was orally administered through a stomach tube to rats for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Zinc sulfate was dissolved in 10% ethanol containing genistein (20 mg/ml) at a concentration of 1.1 mg zinc/ml. This solution (5.5 mg zinc/kg and 100 mg genistein/kg) was orally administered to rats through a stomach tube for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Control animals received a vehicle solution (10% ethanol; about 5 ml/kg body weight).
Analytical procedures *To whom correspondence should be addressed. Received 21 July 1997; accepted 12 January 1998.
The femurs were removed immediately after bleeding and soaked in ice-cold 0.25 M sucrose solution. The femurs were cleaned of soft
200 tissue, and the marrow cells were fairly removed by washing with ice-cold 0.25 M sucrose solution; the marrow cells did not remain in the femoral tissues. The femoral metaphysis (not containing epiphysis) and diaphysis were separated, the periosteum was removed and then the bone tissues were weighed. The metaphyseal or diaphyseal tissues were immersed in 3.0 ml of ice-cold 6.5 mM barbital buffer (pH 7.4), cut into small pieces, homogenized in a Potter-Elvehjem homogenizer with a Teflon pestle and disrupted for 60 sec with an ultrasonic device. The supernatant, centrifuged at 600g for 5 min, was used for measurement of alkaline phosphatase activity. The enzyme analysis was reproducible. The enzyme assay described was carried out under optimal conditions. Alkaline phosphatase activity was determined by the method of Walter and Schutt (1965). The enzyme activity was expressed as micromoles of p-nitrophenol liberated per minute per milligram of protein. Protein concentration was determined by the method of Lowry et al. (1951). To measure DNA content in the femoral tissue, the metaphyseal and diaphyseal fragments were immersed in 4.0 ml of ice-cold 0.1 N NaOH solution, cut into small pieces, homogenized and shaken for 24 hr (Flanagan and Nichols, 1962). After alkali extraction, the samples were centrifuged at 10,000g for 5 min, and the supernatant was collected. DNA content in the supernatant was determined by the method of Ceriotti (1955), and it was expressed as the amount of DNA (mg) per gram (wet weight) of bone tissues. To determine bone calcium in the femoral tissue, the metaphyseal and diaphyseal fragments were dried at 1108C for 12 hr. The bone tissues were digested with nitric acid (3.0 ml) at 1208C for 12 hr. Calcium was determined by atomic absorption spectrophotometry (Yamaguchi et al., 1987). Bone calcium content was expressed as milligrams of calcium per gram of dry bone.
Y. H. Gao and M. Yamaguchi
FIGURE 1. Effect of genistein administration on alkaline phosphatase activity in the femoral-metaphyseal and diaphyseal tissues of rats. Genistein (50, 100 and 200 mg/kg) was orally administered to rats for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Each value represents the mean6 SEM of six rats. *P,0.01, compared with the control value.
kg. The diaphyseal enzyme activity was significantly elevated by doses of 100 and 200 mg genistein/kg. The alteration in DNA content in the metaphyseal and diaphyseal tissues after genistein administration is shown in Figure 2. The metaphyseal DNA content was significantly increased by doses of 50, 100 and 200 mg genistein/kg, whereas the diaphyseal DNA content was appreciably raised by the 100 and 200 mg/kg doses. Doses of 100 and 200 mg genistein/kg caused a significant increase in calcium content in the femoral-metaphyseal and diaphyseal tissues (Fig. 3). A dose of 50 mg genistein/kg did not have an appreciable effect on bone calcium content.
Bone culture experiments The femoral-metaphyseal and diaphyseal tissues from normal rats were removed aseptically. The bone tissue fragments were cultured in a 35-mm dish in 2.0 ml of medium (high glucose) supplemented with 0.25% bovine serum albumin (fraction V) plus antibiotics (100 U penicillin and 100 mg streptomycin/ml of medium) with either vehicle (0.1% ethanol or zinc (1025 M) plus genistein (1025 M) or without or with cycloheximide (1026 M) (Yamaguchi et al., 1987). Cultures were maintained at 378C in a water-saturated atmosphere containing 5% CO2 and 95% air for 24 hr. The culture bone tissues were dried at 1108C for 12 hr, and the bone calcium was determined after the digestion with nitric acid.
Combined effect of genistein and zinc on bone metabolism in rats The effect of oral administration of zinc solution containing genistein on bone metabolism in rats is shown in Table 1. The doses of zinc sulfate and genistein were 5.5 mg Zn/kg and 100 mg flavonoid/ kg, respectively. The dose of 5.5 mg zinc/kg was an effective dose in weanling rats, although the effect of zinc on bone metabolism is not
Statistical analysis Data are expressed as the mean6SEM. Statistical differences were analyzed by using Student’s t-test. In addition, we used a multiway analysis of variance with the use of a Duncan’s post hoc test to compare between-treatment groups. P values of less than 0.05 were considered to indicate statistically significant differences. RESULTS
Effect of genistein administration on bone metabolism in rats Genistein (50, 100 and 200 mg/kg body weight) was orally administered to rats for 3 days. The effect of genistein on alkaline phosphatase activity in the femoral-metaphyseal and diaphyseal tissues is shown in Figure 1. The enzyme activity in the metaphyseal tissues was significantly increased by doses of 50, 100 and 200 mg genistein/
FIGURE 2. Effect of genistein administration on DNA content in the femoral-metaphyseal and diaphyseal tissues of rats. Genistein (50, 100 and 200 mg/kg) was orally administered to rats for 3 days; 24 h after the last administration, the animals were sacrificed by bleeding. Each value represents the mean6SEM of six rats. *P,0.01, compared with the control value.
Zinc Enhancement of Genistein’s Anabolic Effect
FIGURE 3. Effect of genistein administration on calcium content in the femoral-metaphyseal and diaphyseal tissues of rats. Genistein (50, 100 and 200 mg/kg) was orally administered to rats for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Each value represents the mean6SEM of six rats. *P,0.01, compared with the control value. shown in elderly rats (50 weeks old). The oral administration of zinc sulfate (5.5 mg Zn/kg) to elderly rats for 3 days caused a significant increase in alkaline phosphatase activity, DNA and calcium content in the femoral-metaphyseal and diaphyseal tissues. These increases were also seen with the administration of genistein (100 mg/ kg). The combined doses of zinc sulfate and genistein had a synergistic effect on DNA and calcium contents in the metaphyseal and diaphyseal tissues, although such effect was not seen in the bone alkaline phosphatase activity. However, the combined effect was not demonstrated by the doses of zinc (2.75 mg/kg) and genistein (50 mg/kg) (data not shown).
Effect of cycloheximide on the genistein- and zinc-induced increase in bone calcium content in tissue culture in vitro Femoral-metaphyseal and diaphyseal tissues from elderly female rats were cultured for 24 hr in medium containing either vehicle (0.1%
201
FIGURE 4. Effect of cycloheximide on the genistein- and zincinduced increase in calcium content in the femoral-metaphyseal and diaphyseal tissues in vitro. The metaphyseal and diaphyseal tissues were cultured for 24 hr in medium containing either vehicle (0.1% ethanol) or zinc (1025 M) plus genistein (1025 M) in the absence or presence of cycloheximide (1026 M). Each value represents the mean6SEM of six rats. *P,0.01, compared with the control value. Open bars, control; solid bars, genistein plus zinc. ethanol) or genistein (1025 M) plus zinc sulfate (1025 M) with an effective concentration. The presence of zinc (1025 M) or genistein (1025 M) alone caused a significant increase in calcium content in the femoral-metaphyseal and diaphyseal tissues (data not shown). The presence of both zinc (1025 M) and genistein (1025 M) produced a remarkable elevation of calcium content in the metaphyseal and diaphyseal tissues in vitro. These combinations had a synergistic effect similar to the in vivo effect. This synergistic effect was completely abolished in the presence of cycloheximide (1026 M), an inhibitor of protein synthesis (Fig. 4). DISCUSSION Bone mass decreases with increasing age. We reported previously that bone components in the femoral tissues of elderly rats were appreciably decreased compared with those of younger rats (Yama-
TABLE 1. The effect of oral administration of genistein and zinc sulfate on bone components in the femoral tissues of rats Treatment Metaphysis: Control None Zinc Genistein None Zinc Diaphysis: Control None Zinc Genistein None Zinc
Alkaline phosphatase (mmol/min/mg protein)
DNA (mg/g wet bone)
Calcium (mg/g dry bone)
1.115 6 0.052 1.550 6 0.088*
1.812 6 0.169 2.401 6 0.072*
195.0 6 10.5 248.9 6 12.9*
1.460 6 0.063* 1.516 6 0.091*
2.590 6 0.056* 3.258 6 0.096**
236.9 6 14.0* 290.6 6 8.3**
1.060 6 0.063 1.407 6 0.049*
1.796 6 0.117 2.339 6 0.103*
182.9 6 9.5 239.2 6 11.7*
1.539 6 0.049* 1.679 6 0.064*
2.341 6 0.060* 2.936 6 0.097**
233.2 6 8.0* 292.2 6 3.1**
Rats were orally administered zinc sulfate (5.5 mg Zn/kg), genistein (100 mg/kg) and zinc sulfate plus genistein for 3 days; 24 hr after the last admnistration, the animals were sacrificed. Each value represents the mean 6 SEM of six rats. *P , 0.01, compared with the control (none) value; **P , 0.01, compared with the value of zinc or genistein alone.
202 guchi, 1994). The regulatory factors may be important in the therapeutic treatment of osteoporosis with increasing age. In the present study, we found that genistein has an anabolic effect on bone components in the femoral tissues of elderly female rats in vivo. Genistein is a natural flavonoid that is found in Leguminosae, and it is called phytoestrogen. The oral administration of genistein to rats caused a significant increase in alkaline phosphatase activity, DNA and calcium content in the femoral-metaphyseal and diaphyseal tissues. Bone alkaline phosphatase, which is a marker enzyme of osteoblastic cells, is implicated in bone calcification. The present findings, that genistein administration can increase bone alkaline phosphatase activity and bone calcium content, suggest that the flavonoid stimulates bone calcification in vivo. Moreover, DNA content in the bone tissues may be an indicator of the number of bone cells, including osteoblasts and osteocytes. Presumably, genistein administration can stimulate the proliferation of bone cells. More recently, it has been demonstrated that the presence of genistein (1026 to 1024 M) in a bone tissue culture system using femoral-metaphyseal tissues could increase the bone alkaline phosphatase activity, DNA and calcium contents in vitro (Yamaguchi and Gao, 1997), indicating that genistein has a direct effect on bone metabolism. Interestingly, the anabolic effect of genistein on bone components was enhanced by the combination with zinc sulfate; the genistein-induced increases in bone DNA and calcium contents were synergistically enhanced by zinc administration. Zinc has been demonstrated to stimulate bone formation by the presence of newly synthesized protein in osteoblastic cells (Yamaguchi and Hashizume, 1994). The present study showed that the presence of genistein (1025 M) and zinc sulfate (1025 M) in culture medium, with the use of femoral-metaphyseal and diaphyseal tissues, caused a remarkable increase in bone calcium content in vitro. This increase was completely blocked by the coexistence of cycloheximide, an inhibitor of protein synthesis. These results may support the view that the synergistic effect of the combination of genistein and zinc on bone components is partly based on the stimulation of protein synthesis in bone cells. The anabolic effect of genistein on bone components was seen at
Y. H. Gao and M. Yamaguchi the lowest dose (50 mg/kg body weight) used in this experiment. This dose may be a higher level than that of food containing genistein. However, if genistein’s effect is enhanced by zinc, the use of both genistein and zinc may be a useful tool in the prevention of osteoporosis. References Ceriotti G. (1955) Determination of nucleic acids in animal tissues. J. Biol. Chem. 214, 39–77. Flanagan B. and Nichols G., Jr. (1962) Metabolic studies of bone in vitro VI: collagen biosynthesis by surviving bone fragment in vitro. J. Biol. Chem. 237, 3786–3789. Hashizume M. and Yamaguchi M. (1993) Stimulatory effect of b-alanyl-lhistidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells. Mol. Cell. Biochem. 122, 59–64. Kishi S. and Yamaguchi M. (1994) Inhibitory effect of zinc compounds on osteoclast-like cell formation in mouse marrow cultures. Biochem. Pharmac. 48, 1225–1230. Liu Y., Bhalla K., Hill C. and Priest D. G. (1994) Evidence for involvement of tyrosine phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line. Bochem. Pharmac. 48, 1265–1272. Lowry O. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–273. Spinozzi F., Pagliacci M. C., Migliorati G., Moraca R., Grignami F., Riccardi C. and Nicoletti I. (1994) The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells. Leuk. Res. 18, 431–439. Walter K. and Schutt C. (1965) Acid and alkaline phosphatase in serum. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), Vol. 1–2, pp. 856–860. Academic Press, New York. Yamaguchi M. (1994) Alteration of bone metabolism: effect of nutrition. In Pathology of the Aging Rat (Edited by Mohr U., Dungorth D. L. and Capen C. C.), Vol. 2, pp. 499–511. ILSI Press, Washington, DC. Yamaguchi M. (1995) b-Alanyl-l-histidinato zinc and bone resorption. Gen. Pharmac. 26, 1179–1183. Yamaguchi M. and Gao Y. H. (1997) Anabolic effect of genistein on bone metabolism in the femoral-metaphyseal tissues of elderly rats is inhibited by anti-estrogen tamoxifen. Res. Exp. Med. 197, 101–107. Yamaguchi M. and Hashizume M. (1994) Effect of b-alanyl-l-histidinato zinc on protein components in osteoblastic MC3T3-E1 cells: increase in osteocalcin, insulin-like growth factor-I and transforming growth factor-b. Mol. Cell. Biochem. 136, 163–169. Yamaguchi M., Oishi H. and Suketa Y. (1987) Stimulatory effect of zinc on bone formation in tissue culture. Biochem. Pharmac. 36, 4007–4012.
Gen. Pharmac. Vol. 31, No. 2, pp. 199–202, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Zinc Enhancement of Genistein’s Anabolic Effect on Bone Components in Elderly Female Rats Ying Hua Gao and Masayoshi Yamaguchi* Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422, Japan [Tel. (Fax): 81-54-264-5580] ABSTRACT. 1. The effect of genistein on the bone components in the femoral-metaphyseal and diaphyseal tissues of elderly female rats was investigated. 2. The oral administration of genistein (100 and 200 mg/kg body weight) for 3 days to rats caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the femoral-metaphyseal and diaphyseal tissues. A dose of 50 mg genistein/kg appreciably increased alkaline phosphatase activity and DNA content in the metaphyseal but not the diaphyseal tissues. 3. Alkaline phosphatase activity, DNA and calcium contents in the femoral-metaphyseal and diaphyseal tissues were significantly elevated by the oral administration of zinc sulfate (5.5 mg Zn/kg) for 3 days. The effect of zinc on increasing bone DNA and calcium contents was synergistically enhanced by the simultaneous administration of genistein (100 mg/kg). 4. The stimulatory effect of zinc sulfate (1025 M) or genistein (1025 M) on bone calcium content was also seen in a culture system using the femoral-metaphyseal and diaphyseal tissues in vitro. This effect was completely prevented by the presence of cycloheximide (1026 M). 5. The present findings suggest that genistein has an anabolic effect on bone components and that the effect is enhanced by zinc, owing to its stimulating effect on bone protein synthesis. gen pharmac 31;2:199–202, 1998. 1998 Elsevier Science Inc. KEY WORDS. Genistein, zinc, bone formation, rat femur, aging
INTRODUCTION Bone mass decreases with increasing age. The decrease is due to increased bone resorption and to decreased bone formation. Pharmacological and nutritional factors are needed to prevent bone loss with increasing age. These factors, however, are not fully clarified. Genistein is a natural isoflavonoid phytoestrogen. The flavonoid is found in Leguminosae. Genistein has been shown to reveal an inhibitory effect on protein thyrosine kinases in vitro (Liu et al., 1994; Spinozzi et al., 1994), although the biological effect of flavonoid has not been fully clarified. More recently, genistein has been demonstrated to have a direct stimulatory effect on bone formation in a tissue culture system in vitro (Yamaguchi and Gao, 1997). Whether genistein has an anabolic effect on the bone metabolism of elderly female rats in vivo, however, remains to be elucidated. On the other hand, zinc, an essential trace element, has been shown to stimulate osteoblastic bone formation and to inhibit osteoclastic bone resorption in vitro and in vivo (Kishi and Yamaguchi, 1994; Yamaguchi, 1995; Yamaguchi and Hashizume, 1994; Yamaguchi et al., 1987). These factors may have a preventive role in osteoporosis. The present study was undertaken to clarify whether the combination of genistein and zinc has an anabolic effect on the bone metabolism of elderly female rats in vivo. The oral administration of genistein was found to have an anabolic effect on bone components in the femoral-metaphyseal and diaphyseal tissues of elderly female rats, and the isoflavonoid effect was found to be synergistically enhanced by zinc administration. The combination of genistein and zinc may have a role in the prevention of bone loss in aged rats.
MATERIALS AND METHODS
Chemicals Dulbecco’s modified Eagle medium (high glucose) was obtained from GIBCO Laboratories (Grand Island, NY, USA). Genistein and cycloheximide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Zinc sulfate and all other chemicals were reagent grade from Sigma Chemical and Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All water used was glass distilled.
Animals and administration procedure Female Wistar rats, weighing 200–220 g (50 weeks old), were obtained from Japan SLC (Hamamatsu, Japan). Animals were fed commercial laboratory chow (solid) containing 1.1% calcium, 1.1% phosphorus and 0.012% zinc and distilled water. Genistein was dissolved in 10% ethanol at concentrations of 10, 20 and 40 mg/ml. Genistein (50, 100 and 200 mg/kg body weight) was orally administered through a stomach tube to rats for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Zinc sulfate was dissolved in 10% ethanol containing genistein (20 mg/ml) at a concentration of 1.1 mg zinc/ml. This solution (5.5 mg zinc/kg and 100 mg genistein/kg) was orally administered to rats through a stomach tube for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Control animals received a vehicle solution (10% ethanol; about 5 ml/kg body weight).
Analytical procedures *To whom correspondence should be addressed. Received 21 July 1997; accepted 12 January 1998.
The femurs were removed immediately after bleeding and soaked in ice-cold 0.25 M sucrose solution. The femurs were cleaned of soft
200 tissue, and the marrow cells were fairly removed by washing with ice-cold 0.25 M sucrose solution; the marrow cells did not remain in the femoral tissues. The femoral metaphysis (not containing epiphysis) and diaphysis were separated, the periosteum was removed and then the bone tissues were weighed. The metaphyseal or diaphyseal tissues were immersed in 3.0 ml of ice-cold 6.5 mM barbital buffer (pH 7.4), cut into small pieces, homogenized in a Potter-Elvehjem homogenizer with a Teflon pestle and disrupted for 60 sec with an ultrasonic device. The supernatant, centrifuged at 600g for 5 min, was used for measurement of alkaline phosphatase activity. The enzyme analysis was reproducible. The enzyme assay described was carried out under optimal conditions. Alkaline phosphatase activity was determined by the method of Walter and Schutt (1965). The enzyme activity was expressed as micromoles of p-nitrophenol liberated per minute per milligram of protein. Protein concentration was determined by the method of Lowry et al. (1951). To measure DNA content in the femoral tissue, the metaphyseal and diaphyseal fragments were immersed in 4.0 ml of ice-cold 0.1 N NaOH solution, cut into small pieces, homogenized and shaken for 24 hr (Flanagan and Nichols, 1962). After alkali extraction, the samples were centrifuged at 10,000g for 5 min, and the supernatant was collected. DNA content in the supernatant was determined by the method of Ceriotti (1955), and it was expressed as the amount of DNA (mg) per gram (wet weight) of bone tissues. To determine bone calcium in the femoral tissue, the metaphyseal and diaphyseal fragments were dried at 1108C for 12 hr. The bone tissues were digested with nitric acid (3.0 ml) at 1208C for 12 hr. Calcium was determined by atomic absorption spectrophotometry (Yamaguchi et al., 1987). Bone calcium content was expressed as milligrams of calcium per gram of dry bone.
Y. H. Gao and M. Yamaguchi
FIGURE 1. Effect of genistein administration on alkaline phosphatase activity in the femoral-metaphyseal and diaphyseal tissues of rats. Genistein (50, 100 and 200 mg/kg) was orally administered to rats for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Each value represents the mean6 SEM of six rats. *P,0.01, compared with the control value.
kg. The diaphyseal enzyme activity was significantly elevated by doses of 100 and 200 mg genistein/kg. The alteration in DNA content in the metaphyseal and diaphyseal tissues after genistein administration is shown in Figure 2. The metaphyseal DNA content was significantly increased by doses of 50, 100 and 200 mg genistein/kg, whereas the diaphyseal DNA content was appreciably raised by the 100 and 200 mg/kg doses. Doses of 100 and 200 mg genistein/kg caused a significant increase in calcium content in the femoral-metaphyseal and diaphyseal tissues (Fig. 3). A dose of 50 mg genistein/kg did not have an appreciable effect on bone calcium content.
Bone culture experiments The femoral-metaphyseal and diaphyseal tissues from normal rats were removed aseptically. The bone tissue fragments were cultured in a 35-mm dish in 2.0 ml of medium (high glucose) supplemented with 0.25% bovine serum albumin (fraction V) plus antibiotics (100 U penicillin and 100 mg streptomycin/ml of medium) with either vehicle (0.1% ethanol or zinc (1025 M) plus genistein (1025 M) or without or with cycloheximide (1026 M) (Yamaguchi et al., 1987). Cultures were maintained at 378C in a water-saturated atmosphere containing 5% CO2 and 95% air for 24 hr. The culture bone tissues were dried at 1108C for 12 hr, and the bone calcium was determined after the digestion with nitric acid.
Combined effect of genistein and zinc on bone metabolism in rats The effect of oral administration of zinc solution containing genistein on bone metabolism in rats is shown in Table 1. The doses of zinc sulfate and genistein were 5.5 mg Zn/kg and 100 mg flavonoid/ kg, respectively. The dose of 5.5 mg zinc/kg was an effective dose in weanling rats, although the effect of zinc on bone metabolism is not
Statistical analysis Data are expressed as the mean6SEM. Statistical differences were analyzed by using Student’s t-test. In addition, we used a multiway analysis of variance with the use of a Duncan’s post hoc test to compare between-treatment groups. P values of less than 0.05 were considered to indicate statistically significant differences. RESULTS
Effect of genistein administration on bone metabolism in rats Genistein (50, 100 and 200 mg/kg body weight) was orally administered to rats for 3 days. The effect of genistein on alkaline phosphatase activity in the femoral-metaphyseal and diaphyseal tissues is shown in Figure 1. The enzyme activity in the metaphyseal tissues was significantly increased by doses of 50, 100 and 200 mg genistein/
FIGURE 2. Effect of genistein administration on DNA content in the femoral-metaphyseal and diaphyseal tissues of rats. Genistein (50, 100 and 200 mg/kg) was orally administered to rats for 3 days; 24 h after the last administration, the animals were sacrificed by bleeding. Each value represents the mean6SEM of six rats. *P,0.01, compared with the control value.
Zinc Enhancement of Genistein’s Anabolic Effect
FIGURE 3. Effect of genistein administration on calcium content in the femoral-metaphyseal and diaphyseal tissues of rats. Genistein (50, 100 and 200 mg/kg) was orally administered to rats for 3 days; 24 hr after the last administration, the animals were sacrificed by bleeding. Each value represents the mean6SEM of six rats. *P,0.01, compared with the control value. shown in elderly rats (50 weeks old). The oral administration of zinc sulfate (5.5 mg Zn/kg) to elderly rats for 3 days caused a significant increase in alkaline phosphatase activity, DNA and calcium content in the femoral-metaphyseal and diaphyseal tissues. These increases were also seen with the administration of genistein (100 mg/ kg). The combined doses of zinc sulfate and genistein had a synergistic effect on DNA and calcium contents in the metaphyseal and diaphyseal tissues, although such effect was not seen in the bone alkaline phosphatase activity. However, the combined effect was not demonstrated by the doses of zinc (2.75 mg/kg) and genistein (50 mg/kg) (data not shown).
Effect of cycloheximide on the genistein- and zinc-induced increase in bone calcium content in tissue culture in vitro Femoral-metaphyseal and diaphyseal tissues from elderly female rats were cultured for 24 hr in medium containing either vehicle (0.1%
201
FIGURE 4. Effect of cycloheximide on the genistein- and zincinduced increase in calcium content in the femoral-metaphyseal and diaphyseal tissues in vitro. The metaphyseal and diaphyseal tissues were cultured for 24 hr in medium containing either vehicle (0.1% ethanol) or zinc (1025 M) plus genistein (1025 M) in the absence or presence of cycloheximide (1026 M). Each value represents the mean6SEM of six rats. *P,0.01, compared with the control value. Open bars, control; solid bars, genistein plus zinc. ethanol) or genistein (1025 M) plus zinc sulfate (1025 M) with an effective concentration. The presence of zinc (1025 M) or genistein (1025 M) alone caused a significant increase in calcium content in the femoral-metaphyseal and diaphyseal tissues (data not shown). The presence of both zinc (1025 M) and genistein (1025 M) produced a remarkable elevation of calcium content in the metaphyseal and diaphyseal tissues in vitro. These combinations had a synergistic effect similar to the in vivo effect. This synergistic effect was completely abolished in the presence of cycloheximide (1026 M), an inhibitor of protein synthesis (Fig. 4). DISCUSSION Bone mass decreases with increasing age. We reported previously that bone components in the femoral tissues of elderly rats were appreciably decreased compared with those of younger rats (Yama-
TABLE 1. The effect of oral administration of genistein and zinc sulfate on bone components in the femoral tissues of rats Treatment Metaphysis: Control None Zinc Genistein None Zinc Diaphysis: Control None Zinc Genistein None Zinc
Alkaline phosphatase (mmol/min/mg protein)
DNA (mg/g wet bone)
Calcium (mg/g dry bone)
1.115 6 0.052 1.550 6 0.088*
1.812 6 0.169 2.401 6 0.072*
195.0 6 10.5 248.9 6 12.9*
1.460 6 0.063* 1.516 6 0.091*
2.590 6 0.056* 3.258 6 0.096**
236.9 6 14.0* 290.6 6 8.3**
1.060 6 0.063 1.407 6 0.049*
1.796 6 0.117 2.339 6 0.103*
182.9 6 9.5 239.2 6 11.7*
1.539 6 0.049* 1.679 6 0.064*
2.341 6 0.060* 2.936 6 0.097**
233.2 6 8.0* 292.2 6 3.1**
Rats were orally administered zinc sulfate (5.5 mg Zn/kg), genistein (100 mg/kg) and zinc sulfate plus genistein for 3 days; 24 hr after the last admnistration, the animals were sacrificed. Each value represents the mean 6 SEM of six rats. *P , 0.01, compared with the control (none) value; **P , 0.01, compared with the value of zinc or genistein alone.
202 guchi, 1994). The regulatory factors may be important in the therapeutic treatment of osteoporosis with increasing age. In the present study, we found that genistein has an anabolic effect on bone components in the femoral tissues of elderly female rats in vivo. Genistein is a natural flavonoid that is found in Leguminosae, and it is called phytoestrogen. The oral administration of genistein to rats caused a significant increase in alkaline phosphatase activity, DNA and calcium content in the femoral-metaphyseal and diaphyseal tissues. Bone alkaline phosphatase, which is a marker enzyme of osteoblastic cells, is implicated in bone calcification. The present findings, that genistein administration can increase bone alkaline phosphatase activity and bone calcium content, suggest that the flavonoid stimulates bone calcification in vivo. Moreover, DNA content in the bone tissues may be an indicator of the number of bone cells, including osteoblasts and osteocytes. Presumably, genistein administration can stimulate the proliferation of bone cells. More recently, it has been demonstrated that the presence of genistein (1026 to 1024 M) in a bone tissue culture system using femoral-metaphyseal tissues could increase the bone alkaline phosphatase activity, DNA and calcium contents in vitro (Yamaguchi and Gao, 1997), indicating that genistein has a direct effect on bone metabolism. Interestingly, the anabolic effect of genistein on bone components was enhanced by the combination with zinc sulfate; the genistein-induced increases in bone DNA and calcium contents were synergistically enhanced by zinc administration. Zinc has been demonstrated to stimulate bone formation by the presence of newly synthesized protein in osteoblastic cells (Yamaguchi and Hashizume, 1994). The present study showed that the presence of genistein (1025 M) and zinc sulfate (1025 M) in culture medium, with the use of femoral-metaphyseal and diaphyseal tissues, caused a remarkable increase in bone calcium content in vitro. This increase was completely blocked by the coexistence of cycloheximide, an inhibitor of protein synthesis. These results may support the view that the synergistic effect of the combination of genistein and zinc on bone components is partly based on the stimulation of protein synthesis in bone cells. The anabolic effect of genistein on bone components was seen at
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