Although it has been reported that silver nanoparticles (Ag-NPs) have strong acute toxic effects to various cultured cells, the toxic effects at noncytotoxic doses are still unknown. We, therefore, evaluated in vitro toxicity of Ag-NPs at noncytotoxic doses in human hepatoma cell line, HepG2, based on cell viability assay, micronucleus test, and DNA microarray analysis. We also used polystyrene nanoparticles (PS-NPs) and silver carbonate (Ag2CO3) as test materials to compare the toxic effects with respect to different raw chemical composition and form of silver. The cell viability assay demonstrated that Ag-NPs accelerated cell proliferation at low doses (< 0.5 mg/L), which was supported by the DNA microarray analysis showing significant induction of genes associated with cell cycle progression. However, only Ag-NPs exposure exhibited a significant cytotoxicity at higher doses (> 1.0 mg/L) and induced abnormal cellular morphology, displaying cellular shrinkage and acquisition of an irregular shape. In addition, only Ag-NPs exposure increased the frequency of micronucleus formation up to 47.9 +/- 3.2% of binucleated cells, suggesting that Ag-NPs appear to cause much stronger damages to chromosome than PS-NPs and ionic Ag+. Cysteine, a strong ionic Ag+ ligand, only partially abolished the formation of micronuclei mediated by Ag-NPs and changed the gene expression, indicating that ionic Ag+ derived from Ag-NPs could not fully explain these biological actions. Based on these discussions, it is concluded that both "nanosized particle of Ag" as well as "ionic Ag+" contribute to the toxic effects of Ag-NPs.

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Previous studies have demonstrated the functional expression, by osteoblasts, of N-methyl-D-aspartate (NMDA) receptors responsible for the promotion of cellular differentiation in bone. We have now evaluated the possible role of the endogenous co-agonist of NMDA receptors, glycine (Gly), in chondrogenesis. In ex vivo organotypic cultures of fetal mouse tibias, proximal and distal cartilaginous primordia were significantly increased in the presence of Gly, with the osteogenic center being unchanged. Exposure to Gly drastically increased mRNA expression of the calcified chondrocyte marker osteopontin, without markedly affecting that of a proliferating chondrocyte marker or a hypertrophic chondrocyte marker, as shown in organotypic cultures by in situ hybridization analysis. Gly significantly increased Ca2+ accumulation, osteopontin mRNA expression, and alkaline phosphatase activity in cultured rat costal chondrocytes, without significantly affecting those in cultured rat calvarial osteoblasts. The increase induced by Gly was significantly prevented by an NMDA receptor channel blocker and an antagonist at the Gly site on NMDA receptors, but not by an inhibitory Gly receptor antagonist or a Gly transporter inhibitor, in cultured chondrocytes. Constitutive mRNA expression was seen for NR1, NR2D, and NR3A subunits of NMDA receptors, but not for Gly receptors and transporters, in cultured chondrocytes. Corresponding immunoreactive proteins were detected for NR1 and NR2D subunits in cartilaginous zones of fetal mouse tibias. Thus, Gly might, at least in part, play a role as a trophic factor in the mechanisms associated with chondral calcification through the Gly site of NMDA receptors functionally expressed by chondrocytes in rodent cartilage.

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Insulin-dependent diabetes mellitus (IDDM; Type I) is characterized with low or no insulin production, which sometimes leads to diabetic osteopenia and osteoporosis. 1,2) In the absence of insulin, insulin-sensitive cells exhibit marked reduction of glucose uptake activity, resulting in increased serum glucose levels and subsequent development of a variety of diabetic complications. Histological and bone marker assessments indicate a low turnover state in bone formation rate and decreased osteoblastic activity in rat models of type I diabetes.3) In addition, there is a report about the close relationship between the bone loss and the fasting blood glucose level.4) The occurrence of hyperglycemic bone loss is controversial because not only high glucose but also other factors including insulin deficiency could mediate bone loss in IDDM patients, however, while little is known about the direct effect of hyperglycemia on bone metabolism. By contrast, in vitro analysis reveals the induction by hyperglycemia of osteoblastic dysfunctions. In human osteosarcoma (MG-63) cells cultured under high glucose conditions (ϭ55 mM), for example, impaired response is seen to parathyroid hormone and to 1,25-dihydroxyvitamin D 3 , an active form of vitamin D 3 , required for the synthesis of the matured osteoblast marker protein osteocalcin. 5,6) Other reports using calvarial osteoblastic cell line MC3T3-E1 cells have demonstrated that high glucose (ϭ15.5 mM) inhibits Ca 2ϩ intake and bone mineralization with an increase in both cellular proliferation and alkaline phosphatase (ALP) activity.7) These findings indicate that elevated extracellular glucose concentrations would directly impair osteoblastic functions resulting in defective mineralization similar to clinical findings. Therefore, these in vitro analyses are useful for the better understanding of mechanisms relevant to osteoblastic malfunctions associated with diabetes mellitus.On the other hand, we have previously reported the possible functional expression of particular GABAergic signaling machineries in cultured rat calvarial osteoblasts. 8,9) GABA is known as one of the most abundant inhibitory amino acid neurotransmitters in the mammalian central nervous system (CNS). In the CNS, GABA is supposed to mediate inhibitory neurotransmission thorough different signaling machineries including GABA synthase, GABA receptors and GABA transporters.10-13) These GABAergic machineries are found not only in the CNS but also in some non-neuronal and peripheral organs such as heart, lung, kidney, adrenal, pancreas, liver, spleen and uterus.10) In addition to these peripheral tissues, the expression of GABA and particular GABAergic signaling molecules is found in bone cells such as osteoblasts 8,9) and chondrocytes. 14-16) Amongst different isoforms of GABA transporters required for signal termination, expression of betaine/GABA transporter-1 (BGT-1) is exclusively detected at mRNA and protein levels with a temperature-, sodium-and chloride-dependent activity of [ 3 H]GABA accumulation i...