Phytoestrogenic Compounds and Their Synthetic Analogs, Contrary to Estradiol- 17b Stimulates Human Derived Female Cultured Bone Cells in Hyperglycemic Conditions

Abstract

Cultured female-derived human osteoblasts (hObs) responded by different parameters to the phytoestrogens: daidzein (D), glabrene (Gla) and glabridin (Glb), to their synthetic derivatives; carboxy-daidzein (cD) and to estradiol-17β (E 2 ). Since the skeletal protective effects of estrogens are not discernible in diabetic women, we tested the effects of these compounds on hObs grown in growth medium with high glucose (HG; 9.0g/L; 44mM) compared to normal glucose (NG; 4.5g/L; 22mM) using the stimulation of creatine kinase specific activity (CK) and 3 [H] thymidine incorporation into DNA (DNA) as hormonal responsiveness markers. HG slightly increased DNA and CK in hObs.Stimulations by E 2 was abolished and by cD and D was slightly decreased in HG, but not by Gla and Glb in both age groups. Growing hObs in HG upregulated the expression of mRNA of both ER and ERβ in cells from pre-but not from post-menopausal women. Cells from both age groups express also mRNA for 25 hydroxy vitamin D 3 1-α hydroxylase and showed enzymic activity which were down-regulated by HG in both age groups. Whether Gla and Glb act differentially via ERs and/or 1-α hydroxylase is not yet established.Since these compounds are active even in HG, they might be used for treating hyperglycemic/diabetic women. Journal of Steroids & Hormonal Science Materials and Methods ReagentsAll reagents used were analytical grade. Estradiol-17β (E 2 ), daidzein (D) and the creatine kinase (CK) assay kit were purchased from Sigma Chemicals Co. (St. Louis, MO). Carboxy-D (cD) was synthesized by us [20], licorice products: Gla and Glb were produced by us [19]. Cell culturesHuman bones were obtained from biopsies of patients undergoing corrective surgery following accidental injury, hip or knee replacement. All patients (women and men) were healthy, non-osteoporotic and not receiving hormonal replacement treatment. Three groups were defined: Pre-menopausal women, ranging between 37-55 years old, (n=5). Post-menopausal women, ranging between 60-84 years old, (n=5). The non-enzymic method for isolation and culture of human bone cells and their characterization as osteoblasts was described previously [12]. Briefly, samples of the trabecular surface of the iliac crest or long bones were cut into 1mm 3 pieces and extensively and repeatedly washed with phosphate buffered saline (PBS) to remove blood components. The explants, with no enzymatic digestion, were seeded in 100mm diameter tissue culture dishes and incubated in DMEM medium without Ca ++ (to avoid fibroblastic growth [12], containing 10% fetal calf serum (FCS) and antibiotics. Cell outgrowth from the bone explants was apparent after 6-10 days. First passage cells were seeded at a density of 3x10 5 cells per 35mm tissue culture dish in phenol red free DMEM with 10% charcoal stripped FCS and incubated at 37°C in 5% CO 2 . To obtain "high glucose" (HG) conditions, the medium including the FCS, was supplemented with glucose up to a final concentration of 44nM (9.0gm/ liter). Glucose concentration in the regula...

The Influence of Environmental and Genetic Factors on Various Disorders and Diseases

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