LncRNA NEAT1 promoted MPP+­induced ferroptosis via regulating miR­150­5p/BAP1 pathway in SK­N­SH cells.

As widely reported, dysregulated ferroptosis is closely associated with Parkinson's disease (PD) progression. The goal of the present study was to probe the roles of long non­coding RNA (lncRNA) nuclear enriched assembly transcript 1 (NEAT1) in regulating ferroptosis in PD. PD cell model was constructed by subjecting SK­N­SH cells to 1­methyl­4­phenylpyridinium (MPP+) for 24 h. The RNA levels of NEAT1, miRNA (miR)­150­5p, and BRCA1­associated protein 1 (BAP1) were evaluated using qRT­PCR. The protein levels of glutathione peroxidase 4 (GPX4), BAP1, and solute carrier family 7 member 11 (SLC7A11) were determined using western blot. Cell viability was assessed using 3­(4,5­dimethylthiazolyl2)­2, 5­diphenyltetrazolium bromide (MTT) assay. In addition, fluorescent probe 2,7­dichlorodihydrofluorescein diacetate (DCFH­DA) was employed to determine the ROS level. Moreover, the levels of GSH, MDA, and Fe2+ were also measured. Finally, the interactions among NEAT1, miR­150­5p, and BAP1 were identified by dual luciferase reporter gene assay, and/or RIP assay. Upregulated NEAT1 was observed in PD cell model. Knockdown of NEAT1 elevated viability and GSH level in PD cell model and reduced ROS, MDA, and Fe2+ levels. Moreover, NEAT1 functioned as a sponge to suppress miR­150­5p expression. Moreover, miR­150­5p overexpression suppressed ferroptosis in PD cell model. We subsequently found that miR­150­5p regulated SLC7A11 expression by directly binding to BAP1. miR­150­5p inhibition or BAP1 overexpression mitigated the anti­ferroptosis effect meditated by sh­NEAT1. Taken together, knockdown of NEAT1 mitigated MPP+­induced ferroptosis through regulating BAP1/SLC7A11 axis by sponging miR­150­5p, indicating the potential of NEAT1 as a promising therapeutic target for PD.

Adsorption of Cd(II) from aqueous solutions by rape straw biochar derived from different modification processes.

In order to deal with cadmium (Cd(II)) pollution, three modified biochar materials: alkaline treatment of biochar (BC-NaOH), KMnO4 impregnation of biochar (BC-MnOx) and FeCl3 magnetic treatment of biochar (BC-FeOx), were investigated. Nitrogen adsorption-desorption isotherms, Fourier transform infrared spectroscopy (FTIR), Boehm titration, and scanning electron microscopy (SEM) were used to determine the characteristics of adsorbents and explore the main adsorption mechanism. The results show that manganese oxide particles are carried successfully within the biochar, contributing to micropore creation, boosting specific surface area and forming innersphere complexes with oxygen-containing groups, while also increasing the number of oxygen-containing groups. The adsorption sites created by the loaded manganese oxide, rather than specific surface areas, play the most important roles in cadmium adsorption. Batch adsorption experiments demonstrate a Langmuir model fit for Cd(II), and BC-MnOx provided the highest sorption capacity (81.10 mg g-1). The sorption kinetics of Cd(II) on adsorbents follows pseudo-second-order kinetics and the adsorption rate of the BC-MnOx material was the highest (14.46 g (mg·h)-1). Therefore, biochar modification methods involving KMnO4 impregnation may provide effective ways of enhancing Cd(II) removal from aqueous solutions.

[Effect of Modified Biochars on Soil Cadmium Stabilization in Paddy Soil Suffered from Original or Exogenous Contamination].

To investigate the passivation of different modified biochars on the speciation and availability of cadmium contaminated soil, the modified biochars were treated by different approaches (acid/base treatment, impregnation with manganese oxides, magnetic modification) and biochars(BC) were used as soil passivating agents for soil culture experiments. The result indicated that the content of available cadmium decreased significantly by BC and modified biochars in originally contaminated soil. Compared with CK, the percentage of available cadmium in originally contaminated soil was reduced by more than 50% using impregnable biochars by KMnO4(BC-KMnO4) and basic biochars by NaOH(BC-NaOH). And the content of available cadmium decreased significantly by three modified biochars which were BC-KMnO4, BC-NaOH and FeCl3 magnetization biochars(BC-FeCl3) in exogenously contaminated soil. Particularly, the best performance was observed with BC-KMnO4 that reduced 30% available cadmium in exogenously contaminated soil. However, the passivation of BC was not significant, and the content of available cadmium slightly increased (3.8%-24.5%) by BC-HNO3 in exogenously contaminated soil. Furthermore, the content of exchangeable cadmium was increased by 20.2% with 2.5% BC-HNO3 in exogenously contaminated soil, while significantly decreased by other modified biochars and BC, and the 10% BC-KMnO4 reduced 65.1% exchangeable cadmium in originally contaminated soil. Meanwhile, soil pH was increased significantly by BC, BC-KMnO4 and BC-NaOH, while was reduced by BC-HNO3. The contents of organic carbon and exchangeable base cations in soil were improved by all the treatments. The results of regression analysis showed that the content of available cadmium in originally contaminated soil was significantly negatively correlated with soil pH, soil exchangeable Na+, while the content of available cadmium in exogenously contaminated soil was significantly negatively correlated with soil pH, soil organic carbon, soil exchangeable Mg2+, Na+, K+. Accordingly, the lower available cadmium in contaminated soil may correlate with the increasing content of organic carbon, exchangeable base cations and pH. In summary, the materials of BC-KMnO4 could be used as a superior passivating agent for in situ remediation of cadmium pollution, while the materials of BC-HNO3 could slightly activate cadmium in the soil, leading to some risk in in situ remediation.

[Study on sperm damage caused by trichloroethylene in male rats].

OBJECTIVE: To study in vitro sperm damage caused by trichloroethylene in male rats. METHODS: Sperms of Sprague-Dawley (SD) rats were collected 4 hours after being contaminated by trichloroethylene of 0, 2, 4, 6, 8, and 10 mmol/L in vitro. Giemsa staining was performed to observe the morphological changes of sperms, and flow cytometer was used to detect the changes in mitochondrial membrane potential. RESULTS: The sperm motilities in 6, 8, and 10 mmol/L trichloroethylene groups decreased significantly compared with that in control group (P <0.01); the sperm aberration rates in 8 and 10 mmol/L trichloroethylene groups were significantly higher than that in control group (P<0.01). With the increase in exposure dose, the proportion of sperms with reduced mitochondrial membrane potential increased, and there were significant differences in sperm apoptosis rate between the 4, 6, 8, and 10 mmol/L trichloroethylene groups and control group (P<0.01). CONCLUSION: In vitro exposure to trichloroethylene can reduce sperm motility and increase the aberration rate and apoptosis rate of sperms in male SD rats.

[Effects of bisphenol A on OCT4 and SOX2 genes expression in mouse embryonic stem cells].

OBJECTIVE: To explore the effects of bisphenol A (BPA) exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells (mESC). METHODS: mESC were cultured, and treated with the doses of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L respectively of BPA and DMSO (the solvent control group)for 24 hours, and three groups of cells were treated with the same method. The morphological changes of mESC in the control and exposure groups were observed through an inverted microscope. Cell counting kit 8 (CCK8) was used to detect the effects of BPA on proliferation of mESC, and based on the results, the half inhibitory concentration (IC50) was calculated. Real-time fluorescent quantitative polymerase chain reaction (RT-QPCR) and western blotting were used to detect the expression of OCT4 and SOX2. RESULTS: BPA had certain toxicity on mESC, the treatment of BPA significantly increased cell toxicity in a concentration-dependent manner, and the IC50 was 4.3×10(-4) mol/L, combined with the BPA exposure concentration of the environment and the related literature, eventually taking the five concentrations of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L as the experimental groups. The mESC morphology were effected after the treatment of BPA for 24 h, compared with the control group, the number of cells decreased, appearing some floating cells, and the cell cloning became irregular and differentiation in the higher concentration groups. The OCT4 mRNA expression level in the 10(-7) mol/L (1.146 ± 0.087), 10(-6) mol/L (1.156 ± 0.030), 10(-5) mol/L (1.158 ± 0.103) and the 10(-4) mol/L (1.374 ± 0.053) dose group were all significantly higher than the control group (1.000 ± 0.000) (t values were -2.384, -2.953, -3.203, -4.021 respectively, P value all < 0.05). Meanwhile, the SOX2 mRNA expression level in the 10(-4) mol/L (1.113 ± 0.052) were higher than the control group (1.000 ± 0.000) (t value was -2.765, P value < 0.05). Moreover, the OCT4 protein expression level in the 10(-5) mol/L (1.360 ± 0.168) and 10(-4) mol/L (1.602 ± 0.151) were all significantly higher than the control group (1.000 ± 0.000) (t values were -3.538, -4.002 respectively, P value all < 0.05), while no obvious change of the SOX2 protein expression level was detected in all treated groups. CONCLUSION: BPA in a certain dose range could upregulate the expression of OCT4 gene in mouse embryonic stem cells while had no significant effect on the expression of SOX2 gene.

[Detection of trace uranium in air with field spectrometry].

As a natural radioactive element, uranium and its compounds exist as aerosol and transfer in air. In gas phase, uranium can cause various kinds of radioactive damage to human body. The change in its concentration in a local area is related to the exploration and utilization of nuclear energy. Therefore, the development of field method for rapid uranium detection in air sample is very important. In this contribution, the air samples over uranium ores collected by a general pump was absorbed with 2.0 mol x L(-1) nitrate and then reacted with solid reagent kit. When the reaction between trace uranium and chromogenic reagent was finished, the homemade portable photometer was used to measure the absorbance. The results showed that the concentration of uranium in air samples over low grade uranium ores can be successfully determined by the present method and the values agree with that obtained by ICP-MS. The RSD measured by the new method was 1.72%. The application of the new field spectrometry in discriminating uranium ores from other ores has the potential advantages of easy operation, cost-saving and high accuracy.

Role of poly(ADP-ribose) glycohydrolase in the regulation of cell fate in response to benzo(a)pyrene.

Poly(ADP-ribosyl)ation is a crucial regulator of cell fate in response to genotoxic stress. Poly(ADP-ribosyl)ation plays important roles in multiple cellular processes, including DNA repair, chromosomal stability, chromatin function, apoptosis, and transcriptional regulation. Poly(ADP-ribose) (PAR) degradation is carried out mainly by poly(ADP-ribose) glycohydrolase (PARG) enzymes. Benzo(a)pyrene (BaP) is a known human carcinogen. Previous studies in our laboratory demonstrated that exposure to BaP caused a concentration-dependent DNA damage in human bronchial epithelial (16HBE) cells. The role of PARG in the regulation of DNA damage induced by BaP is still unclear. To gain insight into the function of PARG and PAR in response to BaP, we used lentiviral gene silencing to generate 16HBE cell lines with stably suppressed PARG, and determined parameters of cell death and cell cycle following BaP exposure. We found that PARG was partially dependent on PAR synthesis, PARG depletion led to PAR accumulation. BaP-induced cell death was regulated by PARG, the absence of which was beneficial for undamaged cells. Our results further suggested that PARG probably has influence on ATM/p53 pathway and metabolic activation of BaP. Experimental evidences provided from this study suggest significant preventive properties of PAR accumulation in the toxicity caused by BaP.

[Role of poly (ADP-ribose) polymerase 1 on DNA methylation variation induced by B(a)P in human bronchial epithelial cell].

OBJECTIVE: To investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process. METHODS: The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically. RESULTS: The percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01). CONCLUSION: The hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.

Chromium(VI) causes down regulation of biotinidase in human bronchial epithelial cells by modifications of histone acetylation.

Hexavalent chromium (Cr(VI)), a commonly used industrial metal, is a well-known mutagen and carcinogen, and occupational exposure can induce a broad spectrum of adverse health effects, including cancers. Although Cr(VI)-induced DNA damage is thought to be the primary mechanism of chromate genotoxicity and mutagenicity, there is an increasing number of reports showing that epigenetic mechanisms of gene regulation might be a central target of Cr(VI) toxicity. Epigenetic changes, such as changes in phosphorylation, altered DNA methylation status, histone acetylation and signaling pathways, have been observed after chromium exposure. Nevertheless, to better demonstrate the roles of epigenetic modifications in Cr(VI)-induced carcinogenesis, more work needs to be carried out. This study is aimed to investigate changes in biotinidase (BTD) and holocarboxylase synthetase (HCS), two major proteins which maintain homeostasis of the newfound epigenetic modification: histone biotinylation, in cells exposed to Cr(VI). The data showed that Cr(VI) decreased BTD expression at the transcriptional level in human bronchial epithelial cells (16HBE). In addition, using the epigenetic modifiers, 5-Aza-2'-deoxycytidine (Aza) and Trichostatin A (TSA), we found that modifications of histone acetylation reversed the inhibition of BTD, suggesting that Cr(VI) may cause down regulation of BTD by modifications of histone acetylation.

[The effect of DNA methyltransferase 1 low expression on the global genome DNA methylation status of 16HBE cell].

OBJECTIVE: To construct DNA methyltransferase 1 (DNMT1) low expression 16HBE cell line and observe the variation of cell cycle and global genomic DNA methylation. METHODS: The method of Lenti-virus induced RNA interference was applied to introduce four different shRNA fragment into 16HBE cells. Flow cytometry and 5-mC immunofluorescence methods were used to observe the cell cycle and global DNA methylation status of DNMT1 low expression 16HBE cells. RESULTS: The DNMT1 protein relative expression level of 16HBE-shDNMT1-4 cell line was down regulated about 44% (P < 0.05) compared with the control. No obvious differences of cell cycle and global genome DNA methylation status were observed between the 16HBE and 16HBE-shDNMT1. CONCLUSION: The DNMT1 gene low expression cell is successfully constructed, and there are no obvious changes happened on the cell cycle and global genomic DNA methylation.