Chrysanthemum zawadskii extract protects osteoblastic cells from highly reducing sugar-induced oxidative damage.

In this study, Chrysanthemum zawadskii extract (CZE) was investigated to determine its effects on 2-deoxy-D-ribose (dRib)-induced oxidative damage and cellular dysfunction in the MC3T3-E1 mouse osteoblastic cell line. Osteoblastic cells were treated with the highly reducing sugar, dRib, in the presence or absence of CZE. Cell viability, apoptosis and reactive oxygen species (ROS) production were subsequently examined. It was observed that dRib reduced cell survival, while it markedly increased the intracellular levels of ROS and apoptosis. However, pre-treatment of the cells with CZE attenuated all the dRib-induced effects. The antioxidant, N-acetyl-L-cysteine (NAC), also prevented dRib-induced oxidative cell damage. In addition, treatment with CZE resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblast differentiation [ALP, collagen, osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), osteocalcin (OC) and bone morphogenetic protein (BMP)2, BMP4 and BMP7]. In mechanistic studies of the antioxidative potential of CZE, we found that CZE reversed the dRib-induced decrease in the expression of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT)1 and AKT2 genes, which are master regulators of survival-related signaling pathways. CZE also upregulated the gene expression of the antioxidant enzymes, superoxide dismutase (SOD)2, SOD3 and glutathione peroxidase 4 (GPx4), which was inhibited by dRib. Taken together, these results suggest that CZE attenuates dRib-induced cell damage in osteoblastic cells and may be useful for the treatment of diabetes-associated bone disease.

2'-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2'-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites.

5-Aza-2'-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2'-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product.

2-Deoxy-D-ribose induces cellular damage by increasing oxidative stress and protein glycation in a pancreatic beta-cell line.

2-Deoxy-D-ribose (dRib) is a sugar with a high reducing capacity. We previously reported that dRib induced damage in pancreatic beta-cells. The aim of this study was to investigate the mechanism of dRib-induced beta-cell damage. 2-Deoxy-D-ribose provoked cytotoxicity and apoptosis within 24 hours in HIT-T15 cells. Three antiglycating agents-diethylenetriaminepentaacetic acid, aminoguanidine, and pyridoxamine-dose dependently inhibited dRib-triggered cytotoxicity and significantly suppressed apoptosis induced by dRib. 2-Deoxy-d-ribose increased intracellular reactive oxygen species and protein carbonyl levels in a dose-dependent manner. Diethylenetriaminepentaacetic acid and aminoguanidine significantly reduced dRib-induced rises in intracellular reactive oxygen species. All 3 inhibitors decreased the production of intracellular protein carbonyls by dRib. On incubation with albumin, dRib increased dicarbonyl and advanced glycation end product formation. Aminoguanidine and pyridoxamine significantly decreased the dicarbonyl and advanced glycation end product augmentations. These results suggest that both oxidative stress and protein glycation are important mechanisms of dRib-induced damage in a pancreatic beta-cell line.

Kaempferol attenuates 2-deoxy-d-ribose-induced oxidative cell damage in MC3T3-E1 osteoblastic cells.

Reducing sugar, 2-deoxy-D-ribose (dRib), produces reactive oxygen species through autoxidation and protein glycosylation and causes osteoblast dysfunction. Kaempferol, a natural flavonoid, was investigated to determine whether it could influence dRib-induced cellular dysfunction and oxidative cell damage in the MC3T3-E1 mouse osteoblastic cell line. Osteoblastic cells were treated with 30 mM dRib in the presence or absence of kaempferol (10(-9)-10(-5) M) and markers of osteoblast function and lipid peroxidation were subsequently examined. Kaempferol (10(-9)-10(-5) M) significantly inhibited the dRib-induced decrease in growth of MC3T3-E1 osteoblastic cells. In addition, treatment with kaempferol resulted in a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells. Treatment with kaempferol increased osteoprotegerin (OPG) secretion and decreased malondialdehyde (MDA) contents of MC3T3-E1 osteoblastic cells in the presence of 30 mM dRib. Taken together, these results suggest that kaempferol inhibits dRib-induced osteoblastic cell damage and may be useful for the treatment of diabetes-related bone disease.

Myricetin, a naturally occurring flavonoid, prevents 2-deoxy-D-ribose induced dysfunction and oxidative damage in osteoblastic MC3T3-E1 cells.

Myricetin, a naturally occurring flavonoid, was investigated to determine whether it could protect osteoblasts from 2-deoxy-d-ribose induced dysfunction and oxidative damage in the MC3T3-E1 cells. MC3T3-E1 cells were incubated with 2-deoxy-d-ribose and/or myricetin, and markers of osteoblast function and oxidative damage were examined. Compared with control incubation, 2-deoxy-d-ribose significantly (P<0.05) inhibited alkaline phosphatase (ALP) activity, collagen content, and calcium deposition at the concentration of 20 mM. Cellular malondialdehyde (MDA), protein carbonyl, and advanced oxidation protein products contents were significantly (P<0.05) increased in the presence of 2-deoxy-d-ribose (20 mM). Myricetin significantly (P<0.05) increased cell survival, ALP activity, collagen, osteocalcin, osteoprotegerin, and calcium deposition and decreased MDA, protein carbonyl, and advanced oxidation protein products contents of osteoblastic MC3T3-E1 cells in the presence of 20 mM 2-deoxy-d-ribose. These results demonstrate that myricetin attenuates 2-deoxy-d-ribose induced damage, suggesting that myricetin may be a useful dietary supplement for minimizing oxidative injury in diabetes related bone diseases.

2-Deoxy-D-ribose-induced oxidative stress causes apoptosis in human monocytic cells: prevention by pyridoxal-5'-phosphate.

Hyperglycemia-induced oxidative stress plays a crucial role in the pathogenesis of diabetic complications. Although some clinical evidences suggest the use of pyridoxal-5'-phosphate (PLP) in diabetes with nephropathy, the exact mechanism of PLP has not been fully understood. In the present study, the effect of PLP on 2-deoxy-D-ribose (dRib)-induced oxidative damages and apoptosis on human monocytic cells (U937) was investigated. U937 cells exposed to dRib (30 mM) exhibited abnormal properties, including loss pf cell viability, overproduction of reactive oxygen species (ROS), glutathione depletion and some biochemical features of apoptosis. Treatment with PLP at two effective concentrations (100 and 250 microM) strongly inhibited ROS production and glutathione depletion in dRib-treated U937 cells. The extent of Lipid peroxidation and protein oxidation was also decreased in the presence of PLP. In addition, PLP suppressed dRib-induced apoptotic criteria such as sub-G1 apoptotic population and annexin-V staining. The use of N-acetylcysteine (NAC), a thiol antioxidant and GSH precursor, prevented the extent of apoptosis. The results demonstrate that dRib induces the generation of ROS leading to dRib-mediated apoptosis which can be attenuated by PLP through antioxidant mechanisms.

Oxidative-stress-induced apoptosis in PBLs of two patients with Parkinson disease secondary to alpha-synuclein mutation.

Alpha-synuclein has been implicated in the pathology of certain neurodegenerative diseases, including Parkinson disease (PD). Although the precise physiological and pathological role of alpha-synuclein is unclear, overexpression of the protein or its mutants may reduce cell viability. In this study we evaluated the apoptotic response to oxidative stress induced by 2-deoxy-d-ribose (dRib) in peripheral blood lymphocytes (PBLs) of two siblings with Parkinson disease secondary to A53T alpha-synuclein mutation. PBLs exposed to oxidative stress showed a higher percentage of apoptotic cells in PD patients than in controls. However in cells of PD patients, the increase of apoptotic response was lower than in controls, suggesting that cells of PD patients have greater "resistance" to oxidative stress. We conclude that other environmental agents could play a key role in inducing programmed cell death in cells of PD patients with mutant alpha-synuclein.

Polyamine depletion switches the form of 2-deoxy-D-ribose-induced cell death from apoptosis to necrosis in HL-60 cells.

Our previous studies demonstrated that intracellular polyamine depletion blocked HL-60 cell apoptosis triggered by exposure to 2-deoxy-d-ribose (dRib). Here, we have characterized the intracellular events underlying the apoptotic effects of dRib and the involvement of polyamines in these effects. Treatment of HL-60 cells with dRib induces loss of mitochondrial transmembrane potential, radical oxygen species production, intracellular glutathione depletion and translocation of Bax from cytosol to membranes. These effects are followed by cell death. However, the mode of cell death caused by dRib depends on intracellular levels of polyamines. d-Rib-treated cells with normal polyamine levels, progressing through the G(1) into the S and G(2)/M phases, undergo apoptosis, while in polyamine-depleted cells, being blocked at the G(1) phase, cell death mechanisms are switched to necrosis. The present study points to a relationship between the cell cycle distribution and the mode of cell death, and suggests that the level of intracellular spermidine, essential to cell cycle progression, may determine whether a cell dies by apoptosis or necrosis in response to a death stimulus.

The highly reducing sugar 2-deoxy-D-ribose induces apoptosis in human fibroblasts by reduced glutathione depletion and cytoskeletal disruption.

2-deoxy-D-Ribose (dRib), the most reducing sugar, induces apoptosis in normal human fibroblasts, as judged by cytoplasmic shrinkage, chromatin condensation, DNA fragmentation and mitochondrial depolarization. This effect is independent from culture conditions, such as cell density and the presence or absence of serum in the culture milieu, suggesting that dRib-induced apoptosis is cell cycle-independent. dRib was found also to provoke disruption of the actin filament network and detachment from the substratum, while at the same time, interestingly, it increases the expression of several integrins and cell adhesion molecules. Furthermore, dRib was found to reduce the intracellular levels of reduced glutathione (GSH). The apoptotic process was not affected by the macromolecular-synthesis inhibitors cycloheximide and actinomycin D. On the contrary, the antioxidant N-acetyl-L-cysteine (NAC) fully blocks the dRib-induced apoptosis by preventing GSH depletion, while it also inhibits actin-filament-network disruption and mitochondrial depolarization. The above indicate that dRib induces apoptosis in human fibroblasts by a mechanism involving glutathione metabolism and oxidative stress, as well as disturbance of cytoskeletal integrity and cell adhesion.