The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance. Polyclonal anti-EAAT1 (GLAST) and anti-EAAT2 (GLT-1) were purchased from Abcam , anti-EAAT3 was purchased from Novus Biologicals , and polyclonal anti-glyoxalase 1 was purchased from Santa Cruz Biotechnology Inc. . All other chemicals were purchased from local commercial suppliers.Methylglyoxal (MG), propidium iodide,  (MTT), neutral red, carbenoxolone disodium salt (CBX), aminoguanidine hemisulfate salt (AG), standard glutathione, o-phthaldialdehyde, metaphosphoric acid, L-glutamate, N-methyl-D-glucamine, 4-(2-hydroxyethyl) piperazine-L-ethanesulfonic acid (HEPES), and cell culture materials were purchased from Sigma . Dulbecco's modified Eagle medium (DMEM) and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Gibco BRL . Fetal bovine serum was obtained from Cultilab , and L- . Resultsμg/mL neutral red (NR) for 30 min in 5% CO2/95% air at 37°C. The cells were then rinsed twice with phosphate-buffered saline (PBS) for 5 min each time. The NR dye taken up by viable cells was then extracted with 500 μL of acetic acid/ethanol/water . Absorbance values were measured at 560 nm  glutamate. The incubation was stopped after 7 min for the astrocyte cultures and after 10 min for the C6 cultures by removing the medium and rinsing the cells twice with ice-cold HBSS. Cells were then lysed in a 0.5 M NaOH solution. Sodium-independent uptake was determined using N-methyl-D-glucamine instead of NaCl. Sodium-dependent glutamate uptake was obtained by subtracting the nonspecific uptake from the total uptake to obtain the specific uptake. Radioactivity was measured in a scintillation counter. Results were calculated as nmol/mg protein/min and were expressed as percentages of the control.Glutamate uptake was performed as previously described  with somProtein content was measured by Lowry's method using bovine serum albumin as standard .w/v) SDS, 5% (w/v) β-mercaptoethanol, 10% (v/v) glycerol, and 0.002% (w/v) bromophenol blue) and boiling for 5 min. Protein samples (15 μg per lane), prepared for electrophoresis as described above, were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes using a semidry blotting apparatus  [After 24 h of MG addition, astrocytes or C6 cultures were processed for electrophoresis/Western blotting by directly homogenizing in electrophoresis sample buffer at pH 6.8  . The memt-test or by one-way ANOVA. Statistically significant one-way ANOVA was followed by a post hoc Dunnett's test. Data were considered significant when p < 0.05. All analyses were carried out using the SPSS software package or Prism 5.0 (GraphPad).Data are reported as the mean ± standard error and analyzed statistically by Student's Hypothesizing that astrocytes and C6 glioma cells may present metabolic differences, we measured the activity and content of glyoxalase 1, the rate-limiting enzyme of the glyoxalase system, in both cell types. The relative expressions and activities of glyoxalase 1 were significantly higher in astrocytes than in C6 cells  Figures . TherefoAstrocytes and C6 glioma cells were exposed to increasing concentrations of MG (from 0.1 to 4 mM) for 24 h; cell viability was then evaluated by MTT reduction, neutral red (NR) incorporation, and propidium iodide (PI) uptake assays . No impaThe incubation of astrocyte cultures with 1 mM MG (but not 0.4 mM) for 24 h increased glyoxalase 1 activity . This elConsidering the importance of GSH as a cosubstrate for glyoxalase 1 activity, we measured possible changes in GSH content induced by MG . IncreasIn order to investigate whether the effects of 1 mM MG on glyoxalase 1 activity and GSH content is mediated by glycation, astrocytes were coincubated for 24 h with aminoguanidine (AG), a well-known antiglycation compound. AG per se did not alter glyoxalase 1 activity or GSH content ; howevert-test, p = 0.11). On the other hand, in C6 glioma cells, MG at this concentration induced an increase in glutamate uptake activity  in primary astrocyte and C6 glioma cultures . PI-posiAs these cultures present significant differences in gap junction expression and function, we investigated whether the gap junctions could be involved in MG-induced alterations. In fact, CBX, a gap junction inhibitor was able to block the damage induced by MG (panel f), in astrocytes but not in C6 glioma cells (panel l).Our data showing the direct effect of MG on primary astrocytes indicate that these cells are very resistant to high concentrations of MG, when compared to neurons , possiblWe found that the highest concentrations of MG significantly decreased the cell viability and survival of astrocyte cultures after 24 h of incubation. Therefore, for the 24 h assays, we chose high concentrations of MG concentrations (0.4 or 1 mM), but these were unable to compromise cell viability during this time. Moreover, similar nonphysiologically elevated concentrations of MG have been used in several other studies and models to investigate the glycation processes mediated by this compound , 24–26. When we evaluated the glyoxalase 1 response to MG treatment, we observed an increase in activity in astrocytes at 1 mM that was not accompanied by any alteration in the enzyme's expression. MG, at this concentration, increased the GSH content and AG was able to block these increments, suggesting that astrocytes possess an important and responsive system to protect against MG toxicity. We are aware that large increases in MG levels  would result in S-(D)-lactoylglutathione accumulation, keeping GSH trapped and decreasing GSH availability . HoweverIn contrast, in C6 glioma cells, we observed a decrease in GSH levels upon MG exposure and no significant differences were observed in glyoxalase 1 activity and content. These findings, again, reinforce the important differences between primary astrocytes and C6 glioma cells, commonly used to investigate glial metabolism , 18. In With regard to glutamate uptake, we found an impairment in glutamate uptake following MG treatment in astrocytes, but not in C6 cells, where an increase in glutamate uptake was observed. Accordingly, the exposure of astrocyte cultures to another glycating agent, glyoxal, caused a reduction in glutamate uptake activity and the formation of a GLT-1 CML adduct . Data frImportantly, astrocytes and C6 glioma cell cultures exhibit differences in glutamate transporter expression. Astrocytes express and demonstrate functional activity, especially of GLAST and GLT-1, although they can also express EAAT3 . On the Coincubation with AG prevented the MG-induced change in glutamate uptake activity in astrocytes and C6 glioma cells cultures, indicating that these alterations are possibly mediated by glycation. These functional alterations in glutamate transporters are not related to glutamate transporter expression, as MG exposure did not modify the protein expressions of GLAST, GLT-1, and EAAT3 by cells. It is possible that MG-induced glycation does not alter protein turnover within 24 h. Moreover, astrocyte glutamate transporters were not affected in an animal model of diabetes , where hThe ability of MG to decrease glutamate uptake in astrocyte-containing preparations is clear; however, the mechanism by which this occurs requires further comprehension. Although, direct glycation appears to be involved in astrocyte cultures, in hippocampal slices, the decrease in glutamate uptake was not prevented by AG incubation . The curOn the other hand, studies in animal models of diabetes and in vitro experiments that have determined the effects of high glucose levels on astrocytes' cultures , 39, as It is important to mention some limitations in this study. Firstly, we used a single commercial source of MG, and possible impurities may compromise findings. Secondly, we are aware that supraphysiological levels of MG limit the relevance of these results. In this context, further experiments using glyoxalase 1 silencing, as performed in other studies , would cA schematic representation of our results in primary astrocytes is shown in MG exposure impairs primary astrocyte viability, as evaluated by different assays. Astrocytes respond acutely to MG elevation by increasing the activity of glyoxalase 1 and GSH levels. This response, in part, may sustain cell viability and survival. In contrast, C6 glioma cells show signs of resistance against MG without significant changes in the glyoxalase system. AG coincubation provided evidence that MG toxicity is mediated by glycation. Glutamate uptake activity is an important target of MG glycation, and a significant decrease in glutamate transport is observed in astrocytes, without associated changes in the protein expression of major glutamate transporters. CBX, a general gap junction inhibitor, prevented the cytotoxicity induced by MG in astrocyte cultures. Therefore, our data reinforce the idea that astrocyte viability depends on gap junctions and that impairments induced by MG involve glutamate excitotoxicity. The susceptibility of astrocytes to MG emphasizes the importance of this compound in the brain reactions to neurodegenerative diseases, where neuronal damage induced by MG may be aggravated by the commitment of the cells responsible for MG clearance.


In this paper, biochar derived from poplar catkins was used as an economical and renewable adsorbent for adsorption organic and inorganic pollutants such as, dyes, organic compounds, and heavy metal ions from wastewater. Mesoporous activated carbonized poplar catkins (ACPCs) were produced from char as a by-product by carbonized poplar catkins (CPCs). With their high surface area, ACPCs exhibited the maximum adsorption capacities of 71.85 and 110.17 mg/g for the removal of inorganic U(VI) and Co(II). Compared other biochars adsorbents, ACPCs can also adsorb organic pollutants with the maximum adsorption capacities of 534, 154, 350, 148 and 384 mg/g for methylene blue (MB), methyl orange (MO), Congo red (CR), chloramphenicol (CAP) and naphthalene. The adsorption of organic pollutants was fitted with pseudo-first order, pseudo-second order, and intra-particle diffusion kinetic models figure out the kinetic parameters and adsorption mechanisms. Langmuir adsorption isotherm was found to be suitable for Co(II) and U(VI) adsorption and thermodynamic studies indicated adsorption processes to be endothermic and spontaneous. The adsorption process includes both outer-sphere surface complexes and hydrogen-bonding interactions. The results showed that biochar derived from poplar catkins was a potential material to remove pollutants in wastewater. Organic pollutants and heavy metal ions management attracts more and more attention of people, because these pollutants are high mobility and long persistence in the environment6. Therefore, it is significant important to develop an efficient and economical strategy for removal of pollutants in waste water8.Water pollution by organic compounds such as polycyclic aromatic hydrocarbon (PAHs), pesticides, antibiotics and dyes and inorganic heavy metal ions, has been causing considerable worldwide concern11. Among these techniques, adsorption has become one of the most widely used techniques for water pollution management due to its outstanding characteristics, such as low cost, wide adaptability, and convenience12. In the past decades, activated carbon has been used to remove pollutants from wastewater but with low regeneration efficiency, which inspire researchers to search for new alternatives with high efficiencies14. In the quest for economic and effective adsorbents, biomass has been evaluated with variable degree of sources16. Low-cost banana and orange peels were carbonized to biochar for removal of dyes from wastewater in Gurusamy Annadurai’s work17. Huang et al. successfully synthesized biomass adsorbent through the pyrolysis of kapok wadding materials for efficiently solving organic pollution18. Physic-chemical characteristics and the adsorption capacity for methylene blue of rice hull ash were systematically studied19. Although these biomasses are cheaper than other absorbents, it still suffers some problems, such as remaining economic value, poor adsorption performance and so on.A number of effective techniques including adsorption, filtration and ion exchange, have been applied to remove inorganic and organic pollutants from waste solutions20. Poplar catkins are lightweight and fly around everywhere in summer season. Nevertheless, the flying poplar catkins are harmful for human body in lots of respects, such as promoting runny nose, affecting respiratory system. Moreover, they can cause skin irritation and insomnia21. Therefore, it is essential to develop an economically technology for utilizing poplar catkins in large scale. Many reports have been focused on dealing with this problem23. Though the poplar catkins can be used as biochar, rarely studied has been focused on its application on heavy metal ion and organic pollution.Poplar alba as a kind of deciduous tree is universally planted in China2 surface area and the powder X-ray diffraction analysis (XRD). Besides, the elemental composition was characterized, including C, H, O, and N. The products were then applied adsorption by batch experiments. The possible adsorption mechanism of the pollutants was investigated through FT-IR analysis.In this study, carbon micro-tube and mesoporous activated samples were prepared by carbonizing the poplar catkin. The as-prepared samples activated carbonized poplar catkins (ACPCs) and carbonized poplar catkins (CPCs) were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), Brunauer–Emmett–Teller (BET)-N25  of CPCs and ACPCs, mainly determined by its chemical nature31, were estimated to be 3.5 and 3.0, respectively. ACPCs had a much lower zeta potential than CPCs, suggesting a more negatively charged surface of ACPCs in water than that of CPCs, which can be caused by oxygen-abundant functional groups on the surface of ACPCs32. The more negatively charged surface led to adsorb positively charged pollutant more easily and more oxygen-abundant functional groups resulted in chemisorptions more obvious33. The BET surface area, an important influencing factor for adsorption capacity, gave values on the specific surface area and pore structure of ACPCs and CPCs. Figure 2 adsorption–desorption isotherm measurements. The larger surface areas (351.4 and 191.1 m2/g for ACPCs and CPCs) and slightly smaller average pore diameter (2.15 and 1.96 nm for CPCs and ACPCs) was achieved, indicating better development of the porosity after KOH activation. These values were also higher than many reported biochars as listed in Table Zeta potentials were used to analyze surface properties of CPCs and ACPCs in aqueous solution Fig. . The pHp34. MB surface was positively charged, MO was negatively charged and while CR was neutral. Therefore, the electrostatic attraction force for MB was stronger than MO and CR. The adsorption performances of ACPCs toward organic dyes  were better than CPCs, which attributed to oxygen-containing functional groups and their surface areas. The results were consistent with many reports35. For example, active carbon displayed much higher adsorption capacity toward MB than graphene oxide and carbon nanotubes which was related with its highest surface area in Li’s work35. Chen et al. reported that cotton derived porous carbon oxide possessed the advantage of a higher adsorption capacity than cotton derived porous carbon, which confirmed that oxygen-containing functional groups could enhance adsorption31. Table The adsorption capacities of ACPCs and CPCs with positively charged methylene blue (MB), negatively charged methyl orange (MO) and neutral Congo red (CR) were studied and depicted in Fig. −1 at 303 K. Three kinetic models were used to analyze the mechanism of the adsorption process, i.e. pseudo-first order model  are the adsorption capacities at equilibrium and at time t (min), respectively.The pseudo first-order equation describes adsorption in solid–liquid systems. It expressed as following39:k2 is the rate constant for pseudo second-order adsorption (g mmol−1 min−1).The pseudo second-order rate expression, which was applied for analyzing chemisorption kinetics from liquid solutions, was linearly expressed as2), pseudo-second order kinetic model (0.995–0.998) exhibited much higher correlation coefficients than pseudo-first order kinetic model (0.85–0.89), demonstrating a pseudo-second order kinetic model for the adsorption of MB, MO, and CR onto ACPCs. However, the investigation of diffusion mechanisms and rate controlling procedure were beyond the scope of pseudo-first and pseudo-second order kinetic models, intra-particle diffusion model was applied for further investigation.By comparing the correlation coefficients  and ACPCs44. Cobalt existed in the form of Co2+, Co(OH)+, Co(OH)2, and Co(OH)3− at different pH values  ions, and surface complexation between Co2+, Co(OH)+, and ACPCs is strengthen, thus the Co(II) adsorption capacity increase sharply at pH 6.0–8.5 and Co(OH)2 precipitation begins to form at pH > 8.5.The adsorption capacities of ACPCs toward inorganic Co(II) and U(VI) were also investigated. The adsorption of Co(II) on the ACPCs was highly dependent on pH Fig. . The Co adsorption.The influence of ionic strength toward Co(II) adsorption was also examined within the tested pH ranges Fig. . The ion22+ at pH < 5.0, and more multiple positive charged U(VI) species were observed at pH 5.0–8.0, whereas the negative charged U(VI) species were observed at pH > 8.047. Influence of ionic strength on the adsorption of U(VI) was studied in different NaNO3 solutions. It can be seen that ionic strength has major effect on the U(VI) adsorption, suggesting that an outer-sphere surface complexation dominant adsorption process49.As shown in Fig. 51. qmax (mg/g) represented the maximum adsorption capacity, and b (L/mg) is the constant that corresponds to the heat of adsorption. The Freundlich isotherm model, which allowed for several kinds of adsorption sites on the solid, can be represented by the equation  represented the adsorption capacity when the adsorbate equilibrium concentration was equal to 1, and n expressed the degree of dependence of adsorption with equilibrium concentration.The adsorption isotherms for Co(II) and U(VI) obtained at 303, 313, and 323 K were shown in Fig. Langmuir and Freundlich isotherms parameters for Co(II) and U(VI) adsorption on ACPCs were shown in Table 52:K0 can be calculated by plotting lnKd versus Ce and extrapolating Ce to zero.Thermodynamic parameters  of the Co(II) and U(VI) adsorption on ACPCs calculated from isotherms on three different temperature, were investigated to determine whether the adsorption process occurred spontaneously. The value of the Gibbs free energy change (ΔG°) could be achieved by the following equationΔH0) and standard entropy change (ΔS0) could be calculated from the slope and y-intercept to plot of lnKd0 versus 1/T  and spontaneous  adsorption process.The average standard enthalpy change /U(VI)) and organic pollution because of the presence of highly conjugated C-O. An obvious peak at 2903 cm−1 after adsorption organic dyes  clearly indicated the presence of aliphatic C-H stretching from organic dyes54. Similarly, the overlapping bands in the region of 800–600 cm−1 after organic dye adsorption may be ascribed to the out of plane ring deformation of organic dyes55. The results clearly indicated chemisorption was in the process. In the region of 1030–1200 cm−1 after CAP and naphthalene adsorption may be C-N in CAP and naphthalene which indicated successfully sorbed CAP and naphthalene.FT-IR spectra were measured to investigate the adsorption mechanism Fig. . One typIn conclusion, CPCs and ACPCs were successfully fabricated from poplar catkin. The prepared biochars exhibited good to excellent adsorption capacities for both organic pollutant  and heavy metal ions (Co(II) and U(VI)). The adsorption of U(VI) and Co(II) from aqueous solutions onto ACPCs were highly dependent on solution pH and ionic strength, indicating outer-sphere surface complexation dominated the adsorption mechanism. Thermodynamic studies indicated endothermic and spontaneous processes. Kinetic results showed that pseudo-second-order model was better fitted in the removal of dyes process. FT-IR results indicated the participation of the specific functional groups in adsorption interaction. The biochars derived from poplar catkins showed great potential in wastewater management in terms of their facile process, low cost and good ability of adsorption of heavy metals and organic pollution.Analytical pure reagent naphthalene and dyes were purchased from Sinopharm Chemical Reagent Co., Ltd. CAP was purchased from Aladdin Industrial Corporation . Properties of the organic pollutants adsorbates are listed in Table −1 to produced desired ACPCs. CPCs were obtained of the other half dried poplar catkin (~1.0 g) by directly carbonization under same condition with ACPCs. The detailed fabrication process was schematically shown in Fig. The poplar catkins were collected in Shushan in Hefei, Anhui province in China Fig. . The fab−1. The zeta potentials were measured using a Zetasizer Nanosizer ZS instrument . Elemental composition was characterization by Vario EL cube, Elementar, Germany. Ash content of the samples was determined by combusting 0.5 g of adsorbent in a muffle furnace at 650 °C for 2 h.SEM images were taken by a JEOL JSM-7001F microscope. TEM images were obtained using a JEOL-2010 transmission electron microscope with an accelerating voltage of 200 kV. XRD were taken on a Philips X’Pert X-ray diffractometer using Cu Kα radiation (λ = 0.15406 nm). XPS spectra were obtained on an AXIS Ultra DLD spectrometer with an excitation source of Mg Kα (1486.6 eV). FT-IR spectroscopy was performed using a Nicolet Magna-IR 750 spectrometer over a wave number range from 400 to 4000 cm3 or NaOH solutions. The suspensions were then agitated on a shaker for a reaction time of 24 h. The solid phase was separated from the solution phase by centrifugation at 8000 rpm for 10 min.Adsorption experiments of heavy metal ions were performed in polyethylene test tubes and adsorption experiments of organic pollutants were conducted in 10 mL glass bottle by using batch experiment. The pH of the suspension in the range of 2.0–10.0 was adjusted by adding negligible amount of 0.1 or 0.01 mol/L HNOqe) which were achieved by the following equations23:C0 (mg/L) is initial concentration and Ce (mg/L) is final concentration of pollution in the aqueous phase, m (g) is mass of the adsorbent, and V (mL) is the volume adsorption solution. All the experimental data were the averages of duplicate determinations. Error bars represent standard deviation.The concentrations of Co(II) were determined by a spectrophotometric method using Co(II)-Xylenol orange complex at the wavelength of 578 nm and the concentration of U(VI) was analyzed by the Dichlorophosphonoazo III spectrophotometer method at the wavelength of 669 nm. The concentration of organic pollutant was analyzed by UV-vis (Shimadzu UV-2550) spectroscopy (the maximum absorption wavelength for every pollutant was shown in Table Supplementary information