Di 2-ethyl hexyl phthalate affects differentiation and matrix mineralization of rat calvarial osteoblasts--in vitro.

Di-2-ethyl hexyl phthalate (DEHP), an industrial plasticizer and a ubiquitous environmental contaminant, is an established endocrine disruptor (ED). Increasing evidences indicate that some EDs interfere with osteoblast differentiation and function. In the present study, we investigated the effects of DEHP on the expression of cell cycle proteins, differentiation markers, Runx2 and its co-activator TAZ in osteoblasts derived from neonatal rat calvaria. A significant decrease in protein levels of cyclin D1 and CDK-2 was found at high dosage of DEHP (100 µM) after 24h treatment. DEHP treatment caused a significant decrease in ALP mRNA. While DEHP treatment significantly decreased the TAZ at mRNA and protein levels, it decreased only the Runx2protein levels. Histochemical localization of ALP, collagen and mineralized nodules studied from cells treated with DEHP (10 and 100 µM) for 21 days revealed a drastic decrease in collagen, ALP and mineralization. In conclusion, DEHP affected differentiation of neonatal rat calvarial osteoblasts and mineralization of matrix secreted by these cells.

Regulation of insulin-like growth factors and their binding proteins by thyroid stimulating hormone in human osteoblast-like (SaOS2) cells.

Thyroid stimulating hormone (TSH) is shown to have definite anabolic effects on skeletal metabolism. Previous studies have demonstrated that Insulin-like growth factors (IGF-I and IGF-II) and their six high affinity binding proteins (IGFBPs 1-6) regulate proliferation and differentiation of bone-forming osteoblasts. The current study was intended to determine whether the anabolic effects of TSH on human osteoblastic (SaOS2) cells are mediated through insulin-like growth factor system components. TSH given at 0.01 ng to 10 ng/ml dose levels for 24 and 48 h significantly increased human osteoblastic (SaOS2) cell proliferation and alkaline phosphatase activity, the differentiation marker. TSH significantly increased IGFs (IGF-I and IGF-II) mRNA expression after 6 and 24 h and their protein levels after 24 and 48 h of treatment, respectively. Unlike the IGFs, the IGFBPs responded differently to TSH treatment. Though there were some inconsistencies in the regulation of stimulatory IGF binding protein-3 and -5 by TSH treatment, there was an overall increase at the mRNA abundance and protein levels. Again, the inconsistency persisted at the regulation of the inhibitory IGFBPs 2, 4, and 6 especially at the level of mRNA expression due to TSH treatment, there is an overall decrease in the levels of IGFBP-2, 4, and 6 in the conditioned media (CM) of SaOS2 cell cultures. The IGFBP proteases which control the availability of IGFs are also regulated by hormones. Pregnancy-Associated Plasma Protein-A (PAPP-A) is responsible for the proteolysis of IGFBP-4. TSH treatment significantly unregulated the expression of PAPP-A both at mRNA and protein levels. In conclusion, TSH promotes human osteoblastic (SaOS2) cell proliferation and differentiation by upregulating IGFs and their stimulatory IGF binding proteins and down regulating the inhibitory IGF binding proteins.

Effects of Aroclor 1254 on femoral bone metabolism in adult male Wistar rats.

Environmental pollutants that disrupt endocrine system might also affect the modeling and remodeling of bone. Environmental factors, irrespective of age and sex contribute for the development of secondary osteoporosis. Polychlorinated biphenyls have adverse effects on various organs including bone. The present study was designed to investigate the effects of PCB (Aroclor 1254) on femur bone and the ameliorative role of vitamin C or E. In this regard, four groups of adult male albino rats were used as control, PCB (2mg/kgb.wt.), PCB+vitamin C (100mg/kgb.wt.) and PCB+vitamin E (50mg/kgb.wt.). The bone formation markers (ALP, Collagen), bone resorption marker (TRAP), antioxidant enzymes (SOD, GPX and GST) and lipid peroxidation in the femur were studied. Aroclor 1254 treatment decreased the ALP activity and collagen, but increased the TRAP activity and lipid peroxidation. While it decreased the SOD and GPX activity, GST was unaltered. Interestingly, simultaneous administration of vitamin C or E prevented the adverse effects of Aroclor 1254 in the femur. In conclusion, the present investigation suggests that Aroclor 1254 induced oxidative stress affects femoral bone metabolism. However, vitamin C or vitamin E protected the femur from the oxidative stress.

Ameliorative effect of vitamins (alpha-tocopherol and ascorbic acid) on PCB (Aroclor 1254) induced oxidative stress in rat epididymal sperm.

The aim of this study was to investigate the possible protective role of vitamins on PCB (Aroclor 1254)-induced spermiotoxicity using qualitative, quantitative and biochemical approaches. Adult male albino rats of Wistar strain were randomly divided into four groups, each group consists of six animals. The control group received corn oil, the second group of rats were administered Aroclor 1254 at a dose of 2 mg/kg bw/day intraperitoneally for 30 days. The third group of rats were treated with Aroclor 1254 along with alpha-tocopherol (50 mg/kg of bw/day) for 30 days, while the fourth group of rats were treated with Aroclor 1254 along with ascorbic acid (100 mg/kg bw/day) orally for 30 days. Twenty-four hours after the last treatment, control and experimental animals were killed by decapitation. Sperm was collected from the cauda epididymal region and its count and motility were detected. Sperm was sonicated and used for the estimation of reactive oxygen species (ROS) [hydroxyl radical (HO(*)) and hydrogen peroxide (H(2)O(2))], non-enzymic antioxidants [alpha-tocopherol, ascorbic acid and reduced glutathione (GSH)], activity of enzymic antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) glutathione reductase (GR) and glutathione-S-transferase (GST)] and lipid peroxidation (LPO). The result of this experiment shows that PCB significantly decreases the level of alpha-tocopherol, ascorbic acid and GSH and the activities of SOD, CAT, GPx, GR and GST with elevated levels of ROS and LPO. In addition, decreased epididymal sperm motility and count were observed. Simultaneous supplementation with alpha-tocopherol and ascorbic acid restored these parameters to that of normal range. In conclusion, alpha-tocopherol and ascorbic acid exhibited protective effect on sperm by inhibiting PCB-induced ROS generation.

Effects of glucose and its modulation by insulin and estradiol on BMSC differentiation into osteoblastic lineages.

It is well known that diabetes affects bone in human and animal models, and leads to osteopenia and osteoporosis. Bone-mineral density and other biochemical markers of bone turnover are very much affected in people with diabetes. Reduced bone mass, occurring with increased frequency in diabetes mellitus, has been attributed to poor glycemic control, but the pathogenic mechanisms remain unknown. High concentrations of glucose (hyperglycemia) in diabetics leads to this complication. Very few in vitro studies using bone-cell lines have been carried out to address this problem. In this study, we examined the effects of different doses of glucose concentration (5.5, 16.5, and 49.4 mmol/L), alone, with insulin (0.6 microg/mL), or with 17beta-estradiol (E2) (10 nmol/L), on rat bone-marrow stromal cells (BMSCs) in the presence of an osteogenic medium. BMSC proliferation and alkaline phosphatase (ALP) were studied after 3 and 7 d of culture, respectively; the area stained for collagen and mineralized nodules was studied after 28 d of culture. With high concentrations of glucose, BMSC proliferation, ALP activity, the number of nodules formed, and the area stained for collagen were greatly reduced. Insulin treatment alone was able to increase [3H]-thymidine uptake or ALP activity, whereas both insulin and estradiol were able to increase the number of mineralized nodules and the area stained for collagen and mineralization. In conclusion, this study suggests that insulin and estradiol are able to contain the deleterious effect of high concentrations of glucose on BMSC-derived osteoblast proliferation and function.

Effect of ethanol on human osteosarcoma cell proliferation, differentiation and mineralization.

The habitual consumption of even moderate quantities of alcoholic beverages is clearly associated with reduced bone mass, increased prevalence of skeletal fracture and also it is the major risk factor for the development of secondary osteoporosis. The present in vitro study was designed to determine the dose response effects of ethanol on osteoblast-like human osteosarcoma cells (SaOS-2) proliferation, differentiation, mineralization and cyto-toxicity. SaOS-2 cells were plated in 48 and 6 well culture plates and exposed to different concentrations of ethanol (1, 10, 100, 200 and 300 mM) for 24, 48 and 72 h. At the end of incubation, proliferation of cells was studied using crystal violet Bioassay. The cell lysate was utilized to determine ALP activity and conditioned media were used to measure LDH activity. Histochemical localization of ALP and mineralized nodules were studied from cells treated with ethanol (10 and 100 mM) for 21 days. At higher doses, there was a significant reduction in cell number, whereas at lower doses there were variable effects. In 24 h treatment, the higher doses showed a significant increase in ALP activity, whereas 48 and 72 h treatments showed an opposite trend. Ethanol treatment caused a dose- and time-dependent increase in LDH activity. Ethanol treatment altered the quality of mineralization at 10 mM dose whereas completely inhibited mineralization at 100 mM dose, despite the presence of serum. In conclusion, the toxic effect of ethanol is reflected on cell proliferation, differentiation and mineralization even at low doses and at extended treatment duration.