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Molecular and Cellular Biochemistry 194: 9398, 1999. 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Anabolic effect of daidzein on cortical bone in tissue culture: Comparison with genistein effect
Ying Hua Gao and Masayoshi Yamaguchi
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka City, Japan
Received 22 October 1997; accepted 23 April 1998

Abstract
The effect of daidzein on cortical bone in vitro was investigated. Femoral-diaphyseal tissues obtained from elderly female rats were cultured for 24 h in Dulbeccos modified Eagles medium (high glucose, 4.5%) supplementation with antibiotics and bovine serum albumin. The experimental cultures contained 107 to 105 M daidzein. The presence of daidzein (106 and 105 M) caused a significant increase of alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in bone tissues. This effect was equal to that of genistein (106 and 105 M). Daidzein (105 M) or genistein (105 M)-induced increase of calcium content and alkaline phosphatase activity in bone tissues was completely prevented by cycloheximide (106 M), an inhibitor of protein synthesis. Anabolic effect of daidzein and genistein on bone components was equal to that of 17-estradiol (108 M). The effect of isoflavohoids was not enhanced by the addition of 17-estradiol. The combination of daidzein and genistein did not have an additive effect. These findings indicate that daidzein has an anabolic effect on bone metabolism in tissue culture in vitro, and that this effect is equal to genistein effect. Isoflavonoids may stimulate bone formation and mineralization. (Mol Cell Biochem 194: 9398, 1999) Key words: daidzein, genistein, estrogen, bone metabolism, cortical bone, tissue culture

Introduction
Bone mass decreases with increasing age [13]. This decrease may be due to increased bone resorption and to decreased bone formation. Osteoporosis with decrease of bone mass is widely recognized as a major public health problem. Malnutrition or undernutrition is often observed in the elderly. Deficiency in both micronutrients and macronutrients appears to be strongly implicated in the pathogenesis and the consequences of hip fracture in the osteoporotic elderly [4, 5]. Nutritional and pharmacological factors are needed to prevent bone loss with increasing age. The chemical compounds that act on bone metabolism as nutrients in food, however, are poorly understood. Daidzein and genistein are a natural isoflavonoid phytoestrogen found in Leguminosae. The biological effect of daidzein or genistein, however, has not been fully clarified. Daidzin, which its hydrolysis produces daidzein, has been

shown to have a strong inhibitory effect on mitochondrial aldehyde dehydrogenase [6]. Genistein has been demonstrated to inhibit protein tyrosine kinases [7, 8]. Recently, it has been shown that genistein may have an influence on bone metabolism [9, 10]. Blair et al. [9] have reported that genistein is not effective on avian osteoclastic activity and on reduction of bone loss in ovariectomized rats. Dietary soybean protein has been shown to prevent bone loss in ovariectomized rats [10]. More recently, it has been found that genistein has a direct effect on bone formation and mineralization in the femoralmetaphyseal tissue culture system [11]. Moreover, genistein has been shown to have a direct inhibitory effect on boneresorbing factor-induced bone resorption in the metaphyseal tissue culture in vitro [12]. Whether daidzein has a direct effect on bone metabolism, however, has not been clarified thus far. Therefore, the present study was undertaken to determine the effect of daidzein on bone metabolism in vitro. It was found that daidzein can directly stimulate bone

Address for offprints: M. Yamaguchi, Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422, Japan

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