Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism. METHODS: Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6. RESULTS: We observed that 10 µmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 µg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 µmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor). CONCLUSIONS: Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

Task model-specific operator skill assessment in routine fetal ultrasound scanning.

PURPOSE: For highly operator-dependent ultrasound scanning, skill assessment approaches evaluate operator competence given available data, such as acquired images and tracked probe movement. Operator skill level can be quantified by the completeness, speed, and precision of performing a clinical task, such as biometry. Such clinical tasks are increasingly becoming assisted or even replaced by automated machine learning models. In addition to measurement, operators need to be competent at the upstream task of acquiring images of sufficient quality. To provide computer assistance for this task requires a new definition of skill. METHODS: This paper focuses on the task of selecting ultrasound frames for biometry, for which operator skill is assessed by quantifying how well the tasks are performed with neural network-based frame classifiers. We first develop a frame classification model for each biometry task, using a novel label-efficient training strategy. Once these task models are trained, we propose a second task model-specific network to predict two skill assessment scores, based on the probability of identifying positive frames and accuracy of model classification. RESULTS: We present comprehensive results to demonstrate the efficacy of both the frame-classification and skill-assessment networks, using clinically acquired data from two biometry tasks for a total of 139 subjects, and compare the proposed skill assessment with metrics of operator experience. CONCLUSION: Task model-specific skill assessment is feasible and can be predicted by the proposed neural networks, which provide objective assessment that is a stronger indicator of task model performance, compared to existing skill assessment methods.

Naringenin promotes SDF-1/CXCR4 signaling pathway in BMSCs osteogenic differentiation.

INTRODUCTION: Naringenin, a dihydro-flavonoid compound that shows chemotactic activity, may have a good application prospect in repairing bone tissue, but its specific mechanism in bone regeneration, especially the osteogenic differentiation of stem cells, needs for a further study. The aim of this study was to investigate the effect of naringenin on the osteogenic differentiation and its roles in the C-X-C chemokine receptor type 4/stromal cell-derived factor 1 (SDF-1/CXCR4) signal pathway of bone marrow-derived mesenchymal stem cells (BMSCs). MATERIAL AND METHODS: BMSCs were harvested from the femurs and tibias of 4-to-6-week-old male Sprague-Dawley rats. Cell Counting kit-8 assay was used to determine cytotoxicity of naringenin. Alkaline phosphatase (ALP) activity was measured in cell's precipitates and alizarin-red staining was performed to determine the osteogenic differentiation capacity of the BMSCs. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay and western blotting were adopted to determine the expression of genes and proteins. RESULTS: The cellular morphology was spindle-shaped, and arranged in radial and whorled patterns. The flow cytometric analysis have confirmed the presence of characteristic surface proteins in the harvested BMSCs. Different concentrations (0-200 µg/ml) of naringenin have no influence on the viability and proliferation rate of the BMSCs. The highest ALP activity was found at culture day 7 and 9 when the concentration of naringenin was 75 and 100 µg/ml. Positive red or dark red stained cells with mineralized nodules can be observed on day 14. The expression of ALP, Runt-related transcription factor 2, CXCR4 and SDF-1a at the gene and protein levels in naringenin-treated cells were significantly higher than those in the control cells. Moreover, AMD3100, an inhibitor of CXCR4, suppressed the expression of the studied genes and proteins. CONCLUSIONS: Naringenin does not show toxic effect on BMSCs. Naringenin promotes the expression of the SDF-1a gene and protein via the SDF-1/CXCR4 signaling pathway. A better understanding of the mechanisms of naringenin action would be helpful for developing specific therapeutic strategies to improve bone regeneration after injuries.

[Naringenin promotes osteogenic differentiation of BMSCs via SDF-1α/CXCR4 signaling axis].

PURPOSE: To explore the influence of naringenin on osteogenic differentiation of bone mesenchymal stem cells(BMSCs), and the role of SDF-1α/CXCR4 signaling axis in the osteogenic differentiation by naringenin. METHODS: BMSCs of the rats were isolated,cultured and tested. CCK-8 assay was used to explore the proliferation ability of BMSCs in different concentrations of naringenin, and alkaline phosphatase(ALP) activity was detected. RT-qPCR was used to detect the mRNA expression of ALP, OCN, CXCR4 and SDF-1α in different groups. The expressions of CXCR4 and SDF-1α protein in BMSCs during osteogenic differentiation in different experimental groups were detected by ELISA. SPSS 21.0 software package was used for statistical analysis of the data. RESULTS: The results of cell identification showed that the cultured cells were BMSCs. At 1 d and 3 d, all concentrations of naringenin had no significant effect on the proliferation of BMSCs; and at 5 d, 50 µg/mL of naringenin promoted proliferation of BMSCs；furthermore, at 7 d, all concentrations of naringenin promoted proliferation of BMSCs(P＜0.05). ALP activity value gradually increased in each concentration over time. From the RT-qPCR experiment, the mRNA expression of ALP, OCN, CXCR4 and SDF-1α in the naringenin group and the osteogenic induction group was significantly increased compared with the medium group(P＜0.05). ELISA assay showed that the protein expressions of CXCR4 and SDF-1α increased gradually in the four groups as time went on and the expression of two proteins was the highest in 100 µg/mL naringenin group. CONCLUSIONS: Naringenin can promote the proliferation and osteogenic differentiation of BMSCs. SDF-1α/CXCR4 signaling axis is involved in the osteogenic differentiation of BMSCs by naringenin,particularly in the early stage of BMSCs osteogenic differentiation.

Mitochondrial translocation of estrogen receptor ß affords resistance to oxidative insult-induced apoptosis and contributes to the pathogenesis of endometriosis.

Endometriosis is the major cause of female infertility and has been linked to the action of estrogen and estrogen receptor (ER). A new pool of ERß locates within mitochondria, which regulates the endometriotic cell withstanding external insults, but its effect remains controversial. We hypothesize that mitochondrial estrogen receptor ERß (mtERß) is a pivotal regulator in estradiol-mediated cell protection leading to the endometriotic progression. We observed elevated levels of ERß in the endometriotic tissues. A dramatic increase of ERß in mitochondria (mtERß) was found in the ectopic endometriotic tissues, or the estradiol-primed primary endometriotic cells. We analyzed the mtERß-specific overexpressing clone (mtsERß), which exhibited higher mitochondrial bioenergetics and lower reactive oxygen species (ROS) generation. The mtsERß-overexpressed endometriotic cells displayed an enhanced migration phenotype, whereas significantly attenuated migration by mitochondrial respiratory inhibitor (oligomycin) or ERß deficiency by shERß. Further investigations revealed that ERß directly modulated mitochondrial DNA (mtDNA) gene expression by interacting with mtDNA D-loop and polymerase γ. The mtsERß afforded a resistance to oxidative insult-induced apoptosis through the induction of the ROS scavenger enzyme Mn-superoxide dismutase and anti-apoptotic protein Bcl-2. Collectively, the demonstration of mtERß responses in restoration of mitochondrial bioenergetics and inhibition of mitochondria-dependent apoptotic events provides insight into the pathogenesis of endometriosis, suggesting ERß-selective estrogen receptor modulator may serve as novel therapeutics of endometriosis in the future.

Gland Instance Segmentation Using Deep Multichannel Neural Networks.

OBJECTIVE: A new image instance segmentation method is proposed to segment individual glands (instances) in colon histology images. This process is challenging since the glands not only need to be segmented from a complex background, they must also be individually identified. METHODS: We leverage the idea of image-to-image prediction in recent deep learning by designing an algorithm that automatically exploits and fuses complex multichannel information-regional, location, and boundary cues-in gland histology images. Our proposed algorithm, a deep multichannel framework, alleviates heavy feature design due to the use of convolutional neural networks and is able to meet multifarious requirements by altering channels. RESULTS: Compared with methods reported in the 2015 MICCAI Gland Segmentation Challenge and other currently prevalent instance segmentation methods, we observe state-of-the-art results based on the evaluation metrics. CONCLUSION: The proposed deep multichannel algorithm is an effective method for gland instance segmentation. SIGNIFICANCE: The generalization ability of our model not only enable the algorithm to solve gland instance segmentation problems, but the channel is also alternative that can be replaced for a specific task.

In vitro profiling of toxicity and endocrine disrupting effects of bisphenol analogues by employing MCF-7 cells and two-hybrid yeast bioassay.

The potentially adverse health implications of bisphenol A (BPA) have led to increasing use of alternative bisphenols (BPs). However, little is known about the toxicity of alternative BPs. In this study, the cytotoxicity, genotoxicity, intracellular ROS formation, and Ca2+ fluctuation effects of BPs on MCF-7 cells were evaluated. At the same time, the estrogenic and thyroidal hormone effect potentials of six BPs were also evaluated using two-hybrid yeast bioassay. The results showed that most BPs at 0.01-1 µM significantly increased cell viability in MCF-7 cells and at higher exposure concentrations of 25-100 µM, they caused a significant decrease of cell viability. At the same time, these BPs also at 25-100 µM significantly increased LDH release of MCF-7 cells. In addition, several BPs at 10-50 µM resulted in a significantly concentration-depended increase in DNA-damaging effect on MCF-7 cells and elevated ROS production. Most BPs at 0.0001-10 µM significantly increased intracellular Ca2+ level. These results showed that bisphenol AF (BPAF) and thiodiphenol (TDP) exerted cell biological effect, estrogenic, and thyroidal effect potentials greater than those of BPA. The cytotoxicity and endocrine disrupting effects of other BPs are similar to or slightly lower than those of BPA. Therefore, as potential alternatives to BPA, endocrine disrupting effects and potential health harm of alternative BPs to human can also not be ignored. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 278-289, 2017.

Activation of G protein-coupled receptor 30 by thiodiphenol promotes proliferation of estrogen receptor α-positive breast cancer cells.

Many studies have been shown that environmental estrogen bisphenol A (BPA) can activate nuclear receptor (estrogen receptor alpha, ERα) or membrane receptor (G-protein-coupled receptor, GPR30) in breast cancer cells and exerts genomic or nongenomic actions inducing cell proliferation. 4,4'-thiodiphenol (TDP) as one of BPA derivatives exhibits more potent estrogenic activity than BPA does. However, comparatively little is known about the ways in which TDP interferes with these signaling pathways and produces cell biological changes. This study evaluated the effect of TDP on cell viability, reactive oxygen species (ROS) formation, and intercellular calcium (Ca2+) fluctuation in MCF-7 breast cancer cells. The underlying molecular mechanism of cell proliferation induced by TDP was analyzed by examining the activation of ERα and GPR30-mediated phosphatidylinotidol 3-kinase/protein kinase B (PI3K/AKT) and extracellular-signa1regulated kinase (ERK1/2) signaling pathways. The results showed that exposure to 0.1-10 µM TDP for 24, 48, and 72 h significantly increased viability of MCF-7 cells. At the same concentration range, TDP exposure for 3 and 24 h markedly elevated ROS production and intracellular Ca2+ levels. In addition, 0.01-1 µM TDP significantly increased the expression of ERα, GPR30, p-AKT and p-ERK1/2 protein. Specific protein inhibitors blocked phosphorylation of ERK1/2 and AKT and decreased TDP-induced cell proliferation. These findings show that TDP activated the GPR30-PI3K/AKT and ERK1/2 pathways, and the resulting interaction with ERα stimulated MCF-7 cell proliferation. Our results indicate a novel mechanism through which TDP may exert relevant estrogenic action in ERα positive cancer cells.

Highly efficient removal of lead and cadmium during wastewater irrigation using a polyethylenimine-grafted gelatin sponge.

Wastewater irrigation is a very important resource for heavy metal pollution in soil and then accumulation in vegetable crops. In this study, a polyethylenimine (PEI)-grafted gelatin sponge was prepared to effectively adsorb heavy metals during wastewater irrigation. Based on the strong water adsorption ability, wastewater remained in the PEI-grafted gelatin sponge for a sufficient time for the heavy metals to interact with the sorbents. The binding capacities of Pb(II) ions and Cd(II) ions on the PEI-grafted gelatin sponge were 66 mg g(-1) and 65 mg g(-1), which were much more than those on the gelatin sponge (9.75 mg g(-1) and 9.35 mg g(-1)). Subsequently, the PEI-grafted gelatin sponge was spread on the surface of soil planted with garlic and then sprayed with synthetic wastewater. The concentrations of cadmium and lead in the garlic leaves were 1.59 mg kg(-1) and 5.69 mg kg(-1), respectively, which were much lower than those (15.78 mg kg(-1) and 27.98 mg kg(-1)) without the gelatin sponge, and the removal efficiencies were 89.9% and 79.7%. The PEI-grafting gelatin sponge could effectively remove heavy metals during wastewater irrigation, which improved the soil environment and reduced human exposure to heavy metals.

In vitro toxicity assessment of sediment samples from Huangpu River and Suzhou River, Shanghai, China.

Sediments are the ultimate sink for many toxic organic contaminants released into aquatic environment. The present study evaluated the toxicity effect of 13 surface sediment samples from Huangpu River and Suzhou River, East China using two-hybrid yeast bioassays for estrogenic and thyroidal effects and H4IIE rat hepatoma cell bioassay for ethoxyresorufin O-deethylase (EROD) activity. Toxicity was expressed as 17ß-estradiol equivalent (E2-EQ), 3,3',5-triiodothyronine equivalent (T3-EQ), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalent (TEQ). At the same time, the causality between the observed EROD activity and concentrations of polycyclic aromatic hydrocarbons (PAHs) was examined. The results showed that the total estrogenic effects in sediments ranged from 0.06 to 1.21 µg E2-EQ kg(-1) dry weight (dw), the thyroidal effects ranged from 4.68 to 69.9 µg T3-EQ kg(-1) dw, and significantly positive correlations were found between lgT3-EQs and lgE2-EQs. The AhR agonist effects varied from 26.5 to 148.3 ng TEQ kg(-1) dw. Chemical analysis-derived TEQs contributed by PAHs ranged from 13.8 to 66.0 ng kg(-1) dw accounting for 27.2-109.9 % with mean of 48.9 % of TEQbio, indicating that PAHs made important contributions to the EROD effects of sediment extracts from the two rivers. The present study would provide meaningful information for further analysis and risk evaluation for organic pollutants in Huangpu River and Suzhou River.

Diethylstilbestrol at environmental levels affects the development of early life stage and target gene expression in Japanese Medaka (Oryzias latipes).

In this study, the biologic effects of DES on the early life and adult life stages of Japanese medaka (Oryzias latipes) were evaluated. At the early life stage, the fertilized eggs were exposed to 1-1000 ng/L diethylstilbestrol (DES) for 15 days and the hatched larvae were continually exposed to the same concentrations for an additional 25 days. Significant adverse effects on hatchability, time to hatching and mortality rate occurred at DES concentrations of 100 and 1000 ng/L, while the abnormality (scoliosis and abdominal swelling) rate was significantly increased at 10 ng/L and above. After exposure, the fish were maintained in charcoal-dechlorinated tap water for a further 30 days. Only the male gonadosomatic index (GSI) at 1000 ng/L was significantly increased. At concentrations greater than 1 ng/L, estrogen receptor α (ERα) mRNA in both sexes and vitellogenin-I (Vtg-I) mRNA in males were significantly down-regulated; while Vtg-I mRNA in females was significantly up-regulated. When sexually mature medaka were exposed to 10 and 1000 ng/L DES for 21 days, only the GSI in females was significantly decreased at 1000 ng/L. At 10 and 1000 ng/L, ERα mRNA in both sexes was significantly down-regulated, while Vtg-I mRNA in males was significantly up-regulated. These findings showed that DES at the environmental concentration of 10 ng/L can affect the early life stage development of medaka and alter liver ERα and Vtg-I gene expression. Therefore, if we only focused on these sensitive toxicity endpoints such as ERα and Vtg-I mRNA expression, DES has a strong estrogenic effect on Japanese medaka.

Estrogen receptor-ß in mitochondria: implications for mitochondrial bioenergetics and tumorigenesis.

Estrogen enhances mitochondrial function by enhancing mitochondrial biogenesis and sustaining mitochondrial energy-transducing capacity. Shifts in mitochondrial bioenergetic pathways from oxidative phosphorylation to glycolysis have been hypothesized to be involved in estrogen-induced tumorigenesis. Studies have shown that mitochondria are an important target of estrogen. Estrogen receptor-ß (ERß) has been shown to localize to mitochondria in a ligand-dependent or -independent manner and can affect mitochondrial bioenergetics and anti-apoptotic signaling. However, the functional role of mitochondrial ERß in tumorigenesis remains unclear. Clinical studies of ERß-related tumorigenesis have shown that ERß stimulates mitochondrial metabolism to meet the high energy demands of processes such as cell proliferation, cell survival, and transformation. Thus, in elucidating the precise role of mitochondrial ERß in cell transformation and tumorigenesis, it will be particularly valuable to explore new approaches for the development of medical treatments targeting mitochondrial ERß-mediated mitochondrial function and preventing apoptosis.

The levels of PAHs and aryl hydrocarbon receptor effects in sediments of Taihu Lake, China.

A total of 16 priority polycyclic aromatic hydrocarbons (PAHs) in sediment samples from Taihu Lake were analyzed by instruments, and sediment extracts were assayed for aryl hydrocarbon receptor (AhR)-mediated ethoxyresorufin-o-deethylase (EROD) induction using a rat hepatoma cell line (H4IIE). The cause-effect relationship between the observed EROD activity and chemical concentrations of PAHs was examined. Our results showed that sediment extracts could induce significant AhR effects, and the bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents of raw extracts (TEQ(bio)s) ranged from 2.7 to 39.8 pg g(-1) dw. Chemical analysis showed that 16 PAHs were all detected in all samples, and their total concentrations (Σ16PAHs) ranged from 179.8 to 1,669.4 ng g(-1) dw. The abundance of sedimentary PAHs in the three regions (Meiliang Bay, Gonghu Bay, and Xukou Bay) showed a decreasing trend from the inflow region to the outflow region. Chemical analysis-derived TEQs (TEQ(cal)s) contributed by PAHs ranged from 1.6 to 20.7 pg g(-1) dw. The mean contribution rates (CRs) of PAHs to TEQ(bio)s were 48.9%. In Meiliang Bay, EROD effects of 60% samples were caused by PAHs whose CRs were more than 60%, while in most sampling sites of Gonghu Bay and Xukou Bay, the CRs of PAHs to TEQ(bio)s were basically below 40%. In addition, preliminary ecological risk assessment found that PAHs in sediments have very low ecological impact based on the chemical data of PAHs, while the sediments might pose an unacceptable risk to aquatic organisms and their predators based on the data of TEQ(bio). These findings showed that EROD effects of sediment extracts from Taihu Lake were also caused by other compounds, such as dioxins, polychlorinated biphenyls, etc., together.

Long-term exposure investigating the estrogenic potency of estriol in Japanese medaka (Oryzias latipes).

The growth, development, and ERα and Vtg-I gene expressions of Japanese ricefish (Oryzias latipes; medaka) exposed to different concentrations of estriol (E3), including one environmentally relevant concentration, during embryo-adult life stages were evaluated. At the early life stage, fertilized eggs were exposed to 5, 50, 500, 5000ng/L E3 for 15days, and the hatched fry were exposed continuously to the same concentrations for an additional 15days. Exposure to 500 and 5000ng/L E3 resulted in adverse effects on hatchability and time to hatching. At 5000ng/L, the gross abnormality rate was increased and the number of females that hatched was twice that of males. When the fish were exposed to 5-5000ng/L E3 for further 60days, the male hepatosomatic index (HSI) was increased at 5000ng/L. The female gonadosomatic index (GSI) was decreased at 500 and 5000ng/L E3, while the male GSI at 5000ng/L E3 was increased and sex reversal was also found at this concentration. Quantitative RT-PCR showed that the hepatic vitellogenin-I (Vtg-I) genes were up-regulated in females at 500 and 5000ng/L E3 and in males at all E3 concentrations, whereas E3 did not affect estrogen receptor α (ERα) mRNA transcription. These results showed that E3 at environmental concentration of 5ng/L has no adverse effects on growth and development of the Japanese medaka. However, in this study, if we only focused on Vtg gene change in males, E3 had strong estrogenic effects on male medaka under the conditions of these experiments.

Effects of estrone on the early life stages and expression of vitellogenin and estrogen receptor genes of Japanese medaka (Oryzias latipes).

The fertilized eggs of Japanese medaka (Oryzias latipes) were exposed to estrone (E1) at 5-5000 ng L(-1) for 15 d, and the hatched fry were exposed continuously to the same concentrations for the additional 15 d. Adverse effects on hatchability, time to hatching, and gross abnormalities occurred at 50 ng L(-1) or above. Then the fry were divided into a continual exposure group, and a water recovery group. When the fry were exposed to E1 for another 60 d, there was a decrease in the hepatosomatic index (HSI) of males and the influence disappeared in the water recovery group. The gonadosonatic index (GSI) of females at 500 ng L(-1) decreased significantly in another 60 d exposure. While the fry were maintained in dechlorinated tap water for 60 d, a significant decrease in female GSI was observed at 50 ng L(-1) or above. An increased GSI was found in males in both continual exposure and water recovery groups at all E1 treatments. Quantitative RT-PCR showed that vitellogenin-I (Vtg-I) gene expressions in the female liver were significantly down-regulated at 50 ng L(-1) in the continual exposure group, and at 500 ng L(-1) in the water recovery group, while male Vtg-I genes were significantly up-regulated for all E1 treatments. In addition, all E1 treatments caused sex reversal of males. These results suggest that E1 at 5 ng L(-1) or above have unrecoverable impacts on the gonadal growth and development of medaka, even if only early life stages were exposed to E1.

Mixed inhibition of adenosine deaminase activity by 1,3-dinitrobenzene: a model for understanding cell-selective neurotoxicity in chemically-induced energy deprivation syndromes in brain.

Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC(50) of 284 µM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01-0.05%, wt/vol), Nonidet P-40 (0.0015-0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2 nM) did not substantially alter the 1,3-DNB IC(50) value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149-1043 µM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (K(I) = 520 ± 100 µM, n = 1 ± 0.6) and the ADA-adenosine complex (K(IS) = 262 ± 7 µM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10-500 µM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation.

Effects of X-irradiation on mitochondrial DNA damage and its supercoiling formation change.

There have been a small number of reports of radiation-induced mtDNA damage, and mtDNA supercoiling formation change induced by ionizing radiation has not been investigated before. This study evaluated mtDNA damage and supercoiling formation change after X-irradiation. The human breast cancer cell line, MCF-7 cells were used for analysis. Modified supercoiling-sensitive real-time PCR approach was used to evaluate mitochondrial DNA supercoiling formation change and copy number; long-PCR method was applied for the quantification of mtDNA damage. MtDNA damage and formation change induced by high-dose irradiation was persistent in 24h after irradiation and was not significant after low-dose irradiation. MtDNA copy number was slightly increased after high-dose irradiation and a transit increase was observed after low-dose irradiation. This is the first study to evaluate radiation-induced mitochondrial DNA supercoiling formation change using real-time PCR. Combined with data of ROS generation and dynamics of mitochondrial mass, our findings suggested that mtDNA is sensitive to radiation hazards, indicating mitochondrial biogenesis play an important role in radiation-induced cellular response.

The cytotoxic effects of synthetic 6-hydroxylated and 6-methoxylated polybrominated diphenyl ether 47 (BDE47).

Polybrominated diphenyl ethers (PBDE) have been widely applied as flame retardants in plastics, polyurethane foam, paints, and synthetic fabrics. The rising PBDE level in human tissues and environment has led to concern about the health impact of exposure to PBDE. The 2,2',4,4'-tetrabromodiphenyl ether (BDE47), the dominant PBDE congener found in the environment and human tissues, has been shown to be an endocrine disruptor. It has also been reported to cause liver and neurodevelopmental toxicity. BDE47 can be metabolized to 6-OH-BDE47 and 6-MeO-BDE47. So far little has been reported on the cytotoxicity of the metabolites. In the present study, the cytotoxicity of the two metabolites was investigated by exposing human hepatoma cell line HepG2 to different doses of 6-OH-BDE47 and 6-MeO-BDE47. The cell viability, cell cycle, apoptosis, DNA damage, micronucleus levels, and oxidative stress response were studied. The results indicated that both metabolites could markedly inhibit the proliferation of HepG2 cells with 6-OH-BDE47 showing a stronger effect, and significantly increase the micronucleus level and apoptosis rate in a dose-dependant manner. Moreover, treatment with 6-OH-BDE47 (≥0.5 µM) resulted in a marked cell cycle block. The SCGE experiments revealed that both metabolites could cause DNA damage in a dose-dependant manner. Analysis of the oxidative stress response showed that 6-OH-BDE47 treatment (≥2.0 µM) significantly increased intracellular ROS levels as indicated by GSH depletion and elevation of SOD level, whereas 6-MeO-BDE47 showed a weaker effect, suggesting that oxidative stress might play a role in the cytotoxic effects. We concluded that 6-OH-BDE47 or 6-MeO-BDE47 exposure was able to induce inhibition of cell viability, increase of apoptosis rate, cell cycle block, and DNA damages, which might involve the alterated oxidative stress response due to the elevated free radicals and impaired antioxidative system.

[Effect of selenium and arsenic on oxidative stress, DNA oxidative damage and repair in HepG2 cells].

OBJECTIVE: To study alone and combined effect of selenium and arsenic on oxidative stress, DNA oxidative damage and repair. METHODS: HepG2 cells were treated with selenium (2.5, 5.0 and 10.0 micromol/L sodium selenite) alone, arsenic (1.56, 3.13, 6.25, 12.5 and 25.0 micromol/L arsenious acid) alone and combined selenium plus arsenic. The quantitative analysis of malondialdehyde (MDA), 8-OHdG and hOGG1 was carried out by fluorometric method, HPLC-EC and Western Blot to represent oxidative stress, DNA oxidative damage and repair, respectively. RESULTS: Under the condition of alone treatment, sodium selenite (5.0 and 10.0 micromol/L) as well as arsenious acid (6.25, 12.5 and 25.0 micromol/L) resulted in significant increased levels of MDA and 8-OHdG, and inhibition of hOGG1 expression in HepG2 cells compared with solvent control (P < 0.05, P < 0.01). Sodium selenite at the relative low dose (2.5 micromol/L) displayed certain anti-oxidative ability (P > 0.05). Combined treatment of sodium selenite (2.5 micromol/L) and arsenious acid (6.25 micromol/L) caused significant lower levels of MDA and 8-OHdG than those of correspondent arsenic alone treatment (P < 0.05). hOGG1 expression showed no difference between combined treatment (2.5 micromol/L of selenium selenite plus 6.25, 12.5 and 25.0 micromol/L of arsenious acid, respectively) and correspondent arsenic alone treatment (P > 0.05). CONCLUSION: Sodium selenite at the concentrations of 5.0, 10.0 micromol/L and arsenious acid at the concentrations of 6.25, 12.5, 25.0 micromol/L induced enhanced oxidative stress and 8-OHdG production, and inhibition of hOGG1 expression, respectively. Selenium at certain concentration (2.5 micromol/L of selenium selenite) has ameliorative effects on oxidative stress and DNA oxidative damage induced by arsenic, but no effect on repair of DNA oxidative damage.