Combined effects of γ-irradiation and cadmium exposures on osteoblasts in vitro.

The combined effects of γ-irradiation and cadmium (Cd) exposures on osteoblasts were observed in the present study. Osteoblasts were exposed to γ-irradiation (0.5 Gy) and Cd (0-0.5 µmol/L). Cell viability, alkaline phosphatase (ALP) activity, mineralization ability, cell apoptosis and genes expression of ALP, osteocalcin (OC) and caspase 3 were observed. Low concentrations of Cd exposure had no obvious influence on cell viability, ALP activity and apoptosis. However, low levels of Cd exposure combined with γ-irradiation induced more toxic effects on osteoblasts than those treated with Cd or irradiation alone. High concentrations of Cd combined with irradiation exposure induced more significant inhibition in cell viability, ALP activity and mineralization ability than those exposed to Cd or irradiation alone. Meanwhile, OC and ALP mRNA expression of cells treated with Cd combined with irradiation were down-regulated more significantly than those treated with Cd or irradiation alone. Cd combined with γ-irradiation could obviously enhance osteoblast apoptosis and up-regulated caspase 3 mRNA expression compared with those treated with Cd or irradiation alone. This study indicated that ionizing irradiation can enhance Cd toxic effects on osteoblast viability and differentiation and apoptosis may play an important role in this progress.

Bone-prognostic status after cessation of cadmium exposure for one month in male rats.

This study investigated bone status after decreased cadmium (Cd) exposure in male rats. Sprague-Dawley male rats were randomly divided into three groups. One group was injected subcutaneously with sodium chloride as control. The others were given CdCl2 by subcutaneous injection at doses of 0.5 mg Cd/kg body weight (bw) for 2 months (Cd+2m) and for 3 months (Cd+3m). For the Cd+2m group, the rats were shifted to cessation of Cd injection for 1 month after 2 months' exposure. At month 3, micro-computed tomography (micro-CT) analyses were performed on the proximal tibia and lumbar spine, and urine was collected from all rats. Rats were then killed and blood collected for metabolic-marker measurement and Cd assay. Bone tissues were also collected for bone-mass assay, biomechanical test, and bone-histology analysis. Cd burdens of rats in the Cd+2m and Cd+3m groups were both significantly greater than those in the control group. Cd burdens of rats were lower in the Cd+2m group compared with the Cd+3m group. Bone damage occurred in the Cd+2m and Cd+3m groups compared with the control group (p<0.05), but no significant improvement was found in the Cd+2m group compared with the Cd+3m group. Cd damage to bone could not be reversed over the short term. More attention should be paid to Cd's toxic effects on bone after decreased exposure.

Cadmium stimulates the osteoclastic differentiation of RAW264.7 cells in presence of osteoblasts.

Low level of cadmium exposure may have direct effects on bone. But the probable mechanism is far from clarified. Using a co-culture system, the present study investigated the effects of low level of cadmium exposure on osteoclast differentiation in the presence of osteoblasts. Primary osteoblasts were isolated from calvarial bone of newborn Sprague Dawley rats. Primary osteoblasts and RAW264.7 cells were exposed to cadmium (0-60 nmol/l) in a co-culture system. Then, osteoblast viability was observed by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Osteoclast formation and tartrate-resistant acid phosphatase 5b levels were determined by tartrate-resistant acid phosphatase staining and enzyme-linked immunosorbent assay. Osteoprotegerin and receptor activator of NF-kB ligand mRNA expression in osteoblasts were studied via reverse transcription polymerase chain reaction. Viability of osteoblast was obviously decreased by Cd exposure (P < 0.05). Cadmium significantly stimulated the formation of osteoclasts in co-culture system (7.5-60 nmol/l) compared with the control. The levels of tartrate-resistant acid phosphatase 5b in RAW264.7 cells co-cultured with osteoblasts were significantly enhanced by cadmium exposure compared with that without cadmium. The mRNA expression of receptor activator of NF-kB ligand was upregulated by cadmium at 15 and 60 nmol/l. But cadmium had no obvious influence on osteoprotegerin mRNA expression. This data suggested that osteoblasts might be involved in the progress of cadmium effects on osteoclasts.

Effects of cadmium on bone microstructure and serum tartrate-resistant acid phosphatase 5b in male rats.

This study investigated the effects of cadmium on bone microstructure and serum tartrate-resistant acid phosphatase 5b (Tracp 5b) in male rats. Sprague-Dawley male rats were divided into three groups that were given CdCl(2) by subcutaneous injection at doses of 0, 0.1 and 0.5 mg/kg body weight (bw) for 12 weeks, respectively. Before killing at the 12th week, microcomputed tomography scanning was performed on the proximal tibia, and urine samples were collected from all of the rats. All rats were then killed, and their blood was collected for biomarkers assay. Bone tissues were dissected for mineral density determinations and histology. The concentration of cadmium in the blood, urine and bone of rats treated with cadmium were significantly higher than in the control group. The bone mineral density, bone mineral concentrations and bone microstructure index of rats treated with cadmium at 0.5 mg/kg bw were clearly lower than in the control rats. Histological investigation also revealed damage to the bone microstructure caused by cadmium. Tracp 5b concentrations in rats treated with cadmium were dose dependently higher than the control. The concentration of cadmium in blood, urine and bone was significantly correlated with Tracp 5b and bone microstructure parameters. Cadmium was shown to induce bone microstructure damage, especially to trabecular bone. The elevated concentrations of serum Tracp 5b suggest that bone resorption mediated via osteoclasts is an important mechanism for the toxic effects of cadmium on bone.

Cadmium induces differentiation of RAW264.7 cells into osteoclasts in the presence of RANKL.

The mechanism of cadmium effects on bone is not fully understood. In this study, we investigated the effects of cadmium on osteoclasts differentiation and the probable mechanism. RAW264.7 cells were exposed to cadmium (0-60 nmol/L) in the presence or absence of receptor-activated nuclear factor κ B ligand (RANKL) for 5 days. Then, the viability, tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP positive multinucleated osteoclasts were observed. Receptor activator of nuclear factor κ B (RANK), tumor necrosis factor receptor associated factor 6 (TRAF6), c-src, c-fos, fos-related antigen 1 (Fra1) expression were determined by reverse transcription polymerase chain reaction. Cadmium increased TRAP activity (20-40%) and TRAP positive cell formation in the presence of RANKL, but had no obvious influence on them without RANKL. RANK, TRAF6, Fra1, c-src and c-fos (at 15-30 nmol/L) expression were enhanced (30-70%) by cadmium in the presence of RANKL, but cadmium had little influence on them in the absence of RANKL. This study demonstrated that cadmium could induce differentiation of osteoclasts precursor into osteoclasts in the presence of RANKL. Even though the changes of gene expression were small, RANKL/RANK and downstream genes may play an important role in cadmium effects on osteoclasts.

Cadmium is more toxic on volume bone mineral density than tissue bone mineral density.

It has been showed that Cd induces low areal bone mineral density, but we do not know the effect of Cd on cubic bone density. This study was aimed to investigate the effects of Cd on volumetric bone mineral density (VBMD) and tissue bone mineral density (TBMD) in male rats. Twenty-four Sprague-Dawley male rats were randomly divided into four groups that were given cadmium chloride by subcutaneous injection at doses of 0, 0.1, 0.5, and 1.5 mg/kg body weight for 8 weeks, respectively. Then, microcomputed tomography scanning was performed on the proximal tibia, and region of interest was reconstructed using microview software. The VBMD, bone volume fraction of rats treated with 1.5 mg Cd/kg, were significantly decreased compared to control (p < 0.01). The trabecular numbers of rats exposed to Cd were all significantly decreased relative to control (p < 0.05). The trabecular separation of rats treated with 1.5 mg Cd/kg was obviously increased compared to control (p < 0.01). However, Cd had no obvious influence on TBMD. Cd induced low VBMD but not TBMD; Cd effect on bone may be related with trabecular bone loss but not with trabecular bone demineralization.

Genistein inhibits osteolytic bone metastasis and enhances bone mineral in nude mice.

In this study, the effective activity of genistein on osteolytic bone metastasis and bone mineral was investigated. Female BALB/c-nu/nu mice were injected with estrogen receptor-negative human breast cancer cells, MDA-MB-231, into left cardiac ventricle to form osteolytic bone metastases, and administered genistein subcutaneously after radiologically small but defined osteolytic metastases had been observed (protocol 1), simultaneously with cancer cells inoculation (protocol 2) and prophylactically 7 days before inoculation of cancer cells (protocol 3). In all protocols, genistein (10mg/kg/day) markedly reduced the number and volume of osteolytic bone metastases assessed by radiography and the number of osteoclasts. Furthermore, histomorphometrical analysis revealed that genistein markedly increased trabecular area (Tb.Ar%), trabecular thickness (Tb.Th) and trabecular number (Tb.N), and decreased trabecular separation (Tb.Sp). These results thus demonstrate that genistein could inhibit osteolytic bone metastases, suppress bone resorption, increase bone mass and improve bone microstructure in bone metastases of breast cancer.

Effects of cadmium on forearm bone density after reduction of exposure for 10 years in a Chinese population.

The main focus of this study was to evaluate long term effects of cadmium on forearm bone mineral density after stopping ingestion of cadmium-polluted rice for 10 years in a Chinese population. A total of 532 persons (338 women and 194 men), living in control, moderately and heavily polluted areas, were included in this study. The residents living in the polluted area ceased ingesting cadmium-polluted rice in 1996. All participants were require to answer a questionnaire and the bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DXA) at the proximal radius and ulna. Samples of urine and blood were collected for determination of cadmium in urine (UCd) and blood (BCd).The BMD of subjects living in the heavily polluted area was significantly lower than that of those living in control area in both men and women (p<0.01). For the people living in the moderately polluted area, only the women's BMD was greatly lower compared to that in the control area (p<0.05). The BMD declined with the increasing BCd and UCd in both sexes, especially in the highest level (BCd >5 microg/L, UCd >10 microg/g crea) groups (p<0.01). It was found that there were significant differences in the prevalence of osteoporosis among the different areas (chi(2)=13.046, p=0.0003) and different UCd groups (chi(2)=4.511, p=0.0337) in women, but not in men (chi(2)=0.962, p=0.3268; chi(2)=1.906, p=0.1675). But a significant difference exists in the prevalence of osteoporosis among different BCd groups in both genders (chi(2)=9.304, p=0.00229, in women; chi(2)=4603, p=0.0319, in men). This study suggested that cadmium could play a long-term role on bone and more attention should be paid to cadmium effects on bone metabolism after reduction of exposure.

[Inhibitory effects of breast cancer cells on proliferation and differentiation of osteoblasts].

BACKGROUND AND OBJECTIVE: The effect of breast cancer cells on the normal function of osteoblasts remains unclear. This study was to investigate the effect of conditioned medium from two types of human breast cancer lines on osteoblastic proliferation, differentiation and mineralization. METHODS: Fetal rat carvarial cells were treated with conditioned medium from MCF-7 estrogen-receptor(ER) positive, or MDA-MB-231 ER negative, breast cancer cells. Proliferation was measured by MTT assay, alkaline phosphatase (ALP) activity was assessed by the p-nitrophenyl phosphate (PNPP) method, and mineralized nodules were confirmed by alizarin red S (ARS) staining and the area of bone nodules was measured to observe the mineralization capacity. RESULTS: When osteoblasts were treated with conditioned medium from MDA-MB-231 or MCF-7, the cells became long and spindle-like, assumed a fibroblastic morphology. Conditioned medium from breast cancer cells inhibited proliferation and differentiation of osteoblasts, compared with those cultured with vehicle control medium. The proliferation inhibition rates of culture with 50% conditioned medium from MDA-MB-231 and MCF-7 cells on osteoblasts were 18.1%, 13.0%, 19.2%, 19.3% and 15.8%, 20.8%, 33.9%, 28.7% on day 1, day 3, day 5, and day 7, respectively (P<0.01). Moreover, incubation with conditioned medium inhibited ALP activity and bone-nodule formation of osteoblasts in a dose-dependent manner. Addition of 50% conditioned medium from MDA-MB-231 and MCF-7 cells inhibited ALP activity by 31.9% and 47.5% (P<0.01), and diminished the area of bone nodules by 89% and 74%, respectively, compared with the vehicle control group. CONCLUSION: Breast cancer cells inhibit proliferation, decrease ALP activity and disrupt the mineralization process of osteoblasts.

Effects of cadmium on osteoblasts and osteoclasts in vitro.

Cadmium (Cd) may have direct effects on bone metabolism and the mechanism is not fully understood. To investigate the effects of Cd on bone metabolism, effects of Cd on osteoblasts and osteoclasts in vitro were observed at cellular and molecular levels. Osteoblasts were cultured by sequential enzyme digestion from Sprague-Dawley rats calvarial bone and osteoclasts were isolated from long bones of new-born male and female Sprague-Dawley rats, and then cells were exposed to different concentrations of Cd (0-2.0 µ mol/L for osteoblasts; 0.03 µmol/L for osteoclasts). As for osteoblasts, cell viability, alkaline phosphatase (ALP) activity, and mineralization were determined. Osteoprotegerin (OPG) and receptor activator of NF-kB ligand (RANKL) were studied via reverse transcription-polymerase chain reaction (RT-PCR). For osteoclasts, after exposure to Cd (0.03 µmol/L) for 72 h and 120 h, number of osteoclasts and pits formation was observed. Cd inhibited the viability, ALP activity, mineralization and up-regulated RANKL mRNA expression in osteoblasts. But Cd had no obvious effect on OPG mRNA expression. For osteoclasts, cadmium (0.03 µmol/L) could increase the numbers of osteoclasts (p<0.05) and enhance pits formation (p<0.05). These results suggested that Cd could inhibit bone formation at high concentrations and enhance bone resorption at low level. OPG/RANKL may constitute an important pathway of Cd effects on bone.

[Therapeutic effect of recombinant human parathyroid hormone(1-34) on osteoporosis of ovariectomized rats].

OBJECTIVE: To investigate the therapeutic effect of recombinant human parathyroid hormone(1-34) [rhPTH(1-34)] on osteoporosis of ovariectomized rats. METHODS: The model of osteoporosis was formed after 3 months of ovariectomy with 6-month age of 80 rats. Another 20 rats was control of sham operation. rhPTH(1-34) was subcutaneously injected once daily with 5, 10, 20, 40 micrograms/kg for 3 months. There were 10 rats in each group. The control of therapy included Salmon Calcitonin to 10 rats and Alendronate sodium to 10 rats. The bone weight of dry and ash, bone mineral density, bone biomechanical property, trabecular area, bone mineral deposition and serum alkaline phosphatase, Ca, P and urinary Pyridinoline/creatin (Pyd/Cr) were measured after the end of therapy. RESULTS: When administered to animals as a single subcutaneous injection once daily, rhPTH(1-34) increased obviously bone mass, bone biomechanical property and trabecular area, as well as bone deposition compared with the animals of control group. The bone architecture was ultimately improved by rhPTH(1-34) therapy. CONCLUSIONS: Rats of ovariectomized-induced osteoporosis possess obvious effect of treatment with low dose of rhPTH(1-34) administered once daily.

Modulation of isoflavones on bone-nodule formation in rat calvaria osteoblasts in vitro.

OBJECTIVE: To observe the effects of two main isoflavones, daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro. METHODS: Osteoblasts obtained from newborn Sprague-dawley rat calvaria were cultured for several generations. The second generation cells were cultured in Minimum Essential Medium supplemented with ascorbic acid and Na-beta-glycerophosphate for several days, in the presence of daidzein and genistein, with or without the estrogen receptor antagonist ICI 182780. Number of nodules was counted at the end of the incubation period (day 20) by staining with Alizarin Red S calcium stain. The release of osteocalcin, as a marker of osteoblast activity, was also determined on day 7 and day 12 during the incubation period. RESULTS: Compared with the control, the numbers of nodules were both increased by incubation with daidzein and genistein. 17 beta-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation that daidzein and genistein. The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein, as well as 17 beta-estradiol on day 7 and day 12 (day 12 were higher). The estrogen receptor antagonist ICI 182780 completely blocked the genistein- and 17 beta-estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts. However, the effects induced by daidzein could not be inhibited by ICI 182780. CONCLUSION: These findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts. The effects, like those induced by 17 beta-estradiol, are mediated by the estrogen receptor dependent pathway. Daidzein also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts, but it is not, at least not merely, mediated by the estrogen receptor dependent pathway.