| BackgroundPb is one of the most commonly used metals in industry and is highly toxic. Pb can enter the human body mainly through inhalation and gastrointestinal absorption. In cells, Pb can increase the intracellular reactive oxygen species (ROS) levels and reduce the concentration and the activity of antioxidant enzymes, which disturbs the oxidative-antioxidative balance and induces apoptosis. High levels of Pb can cause irreversible damage to the nervous system. However, it was recently discovered that blood Pb at acceptable levels may impact the intellectual development of children. Thus, the neurotoxicity of Pb attracts considerable attention.It is known that cells can influence each other in a co-culture microenvironment. Over the past two decades, classical radiation biology research, which focuses on the irradiated cells themselves as the main target of radiation-induced damage, had been challenged by a significant amount of scientific evidence that clearly shows that radiation-induced bystander signals play important roles in mediating the overall radiobiological response. The radiation-induced bystander effects refer to those responses that occur in cells that were not subject to energy deposition events following ionizing radiation. The bystander effect is characterized by a signaling process from irradiated cells to non-irradiated cells that causes oxidative damage in neighboring cells. Bystander effects have been observed in both in vitro and in vivo toxicological studies. It been proposed that the bystander effect of gene therapy, when the part of the tumor cells transfected with a suicide gene, after start the drug suicide, the transfected cells and the surrounding non-transfected cells were killed.To date, the mechanisms underlying these bystander effects have not been well studied. This mechanism may be related to the active signaling molecule transferred between cells via gap junctions. Gap-junctions, which are composed of six transmembrane connexin subunits arranged as cylindrical channels (-1.5-nm diameter) between connecting adjacent cells, can facilitate the transfer of1-to3-kDa molecules with some dependence on the cell type and physiological status. The involvement of this route of communication among neighboring cells was also demonstrated in neuronal and myocardial ischemia-reperfusion injuries, as well as in liver injuries. Accordingly, it is reasonable to speculate on the damage that occurs not only on the target cells but also on the neighboring cells, and researchers have hypothesized that gap-junctional intercellular communication (GJIC) plays an important role in mediating the bystander effect.Thus, it is unclear whether acceptable blood Pb levels impact the intellectual development by themselves and whether the injured cells transmit the damaged information to neighboring cells. Although Pb-induced apoptosis is a well-reported phenomenon, studies of the bystander effect and the underlying cellular mechanisms are lacking. In this study, we developed a novel co-culture method in which PC12cells were cultured with direct cell-to-cell contact with GFP-labeled PC12(GFP-PC12) cells pretreated with lead acetate. The apoptosis, intracellular ROS generation, and mitochondrial membrane potential (MMP) of PC12cells were subsequently detected. A ROS scavenger and gap junction blocker were used to explore the hypothesis that the Pb-induced oxidative damage can propagate via GJIC.Methods:1. MTT assay The cell viability was determined through the MTT assay. Approximately5x103cells were plated in each well of a96-well plate for24h, and the PC12cells were then exposed to0-100μM lead acetate for24h. Subsequently, the medium was removed, and20μl of5mg/ml MTT (dissolved in PBS) and18μl of DMEM were added to each well. The plate was then incubated at37℃for4h. The medium was removed, and the purple formazan crystals were dissolved in150μl of DMSO. The absorbance of each well was measured at490nm (Power Wave XS; Bio-Tek, Winooski, VT, USA). All of the samples were assayed in six duplicates.2. RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)Lead acetate can induce cell apoptosis. To determine the exact concentration of lead acetate required for the induction of apoptosis, the expression levels of the Bax and Bcl-2genes, which are the important apoptosis-related genes, were detected by RT-PCR. The total RNA was extracted according to the manufacturer’s instructions and was reverse-transcribed using Trizol reagents. The primer exhibited as Table1. The PCR was performed with the following temperature program:denaturation at94℃for5min followed by30cycles of denaturation at94℃for30sec, annealing at60℃for30s, and extension at72℃for5min (n=3). The PCR reaction products were detected through gel electrophoresis and ultraviolet transillumination.3. Flow cytometry analyses3.1. Apoptosis and cell deathAfter induction with lead acetate and co-culture, the cells were rinsed twice with cold PBS and resuspended in binding buffer, and the degree of apoptosis and cell death were then assessed by flow cytometry using an Annexin V-PE/7-AAD kit according to the manufacturer’s instructions. The final suspension was analyzed using a FACS Calibur flow cytometer (BD Biosciences), and the results were analyzed using the CellQuest Pro software (BD Biosciences).3.2. Measurements of mitochondrial membrane potential (△ψm)The△ψm, which has been suggested to be central to the apoptotic pathway (Ly et al.,2003), was measured by flow cytometry using the TMRM detection kit according to the manufacturer’s instructions. PC12cells were incubated in the presence of50nM TMRM for30min at room temperature in the dark and then stored on ice until analysis by flow cytometry. For each sample,10,000particles were analyzed. The final suspension was analyzed using a FACS Calibur flow cytometer, and the results were analyzed using the CellQuest Pro software. The arithmetic mean values of the fluorescence signals in arbitrary units were determined for each sample and graphically represented. All of the experiments were repeated three times.4. ROS detectionDihydroethidium (DHE) was used to measure the ROS generation in cells as previously reported with slight modifications. Briefly, at the end of the treatment period, the cells were washed and incubated with2.5μM DHE (red) at37℃for30min and Hoechst33342(blue) to stain the nucleus for30min. Fluorescence microscopy was used to capture the differences in the red fluorescence intensities. The examination was performed with a laser-scanning confocal microscope (LSM510META; Carl Zeiss, Hamburg, Germany). The images were analyzed with the Image-Pro Plus software (version6.0). The mean fluorescence intensity of each cell was calculated, and the total cell emission signals per field were averaged for data analysis.5. Stable transfection of PC12cells with EF1A-eGFP.To distinguish the normal PC12cells from the lead-induced PC12cells in the direct cell-to-cell contact system, some of the PC12cells were transfected with the EF1A-eGFP vector (GFP-PC12) and selected with400μg/ml G418. The transfections were performed according to the standard protocol from Cyagen. The expression of GFP was identified by fluorescence microscopy and flow cytometry analysis.6. Co-culture system to determine the bystander effects of lead-induced PC12cells6.1Lead acetate-induced PC12-GFP cells co-cultured with PC12-GFP (Pb2+) cellsThe GFP-PC12cells were exposed to different concentrations (0.1μM,1μM,10μM, and100μM) of lead acetate (dissolved in pure water) after incubation for24h. The exposures were performed in normal growth medium. The control cells were treated with5mM NAC, which is commonly used to identify and inhibit ROS, for1h and then exposed to10μM lead acetate for24h (GFP-PC12+NAC+Pb). The cells were harvested with0.025%trypsin and0.02%EDTA (dissolved in PBS and degermed).6.2The bystander effects of PC12-GFP (Pb2+) cells is determined by direct cell to cell contact system.The GFP-PC12cells (1×103cells/ml) were co-cultured with PC12cells (5×103cells/ml) in three groups. First, GFP-PC12cells co-cultured with PC12cells were assumed to be the control group (GFP-PC12+PC12). GFP-PC12(Pb2+) cells co-cultured with PC12cells (GFP-PC12(Pb2+)+PC12) were used to determine the bystander effects of the induced cells. To determine the effect of CBX, an inhibitor of gap junction, on the bystander effect, GFP-PC12(Pb2+) cells were co-cultured with PC12cells in medium with100μM CBX (GFP-PC12(Pb2+)+PC12+CBX), The cells were harvested after24h of co-culture.7. Parachute assay for gap junction functionTo confirm the function of gap junctions, which was hypothesized to underline the bystander effect, in a co-culture system, the parachute assay was used. To avoid overlaps between the green fluorescences of GFP and calcein-AM, which is converted intracellularly into the gap junction-permeable dye calcein. Briefly, before co-culture, GFP-PC12(Pb2+) cells were double-labeled with5μM CM-Dil, a membrane dye that does not spread to coupled cells, and5μM calcein-acetoxymethyl ester, which is converted intracellularly into the gap junction-permeable dye calcein. After incubation for30min, the GFP-PC12(Pb2+) cells were then trypsinized, diluted to500cells/ml, and used as the donor cells. Two groups of PC12cells were plated in12-well plates as receptor cells:one group of cells was incubated with1ml of the donor solution, and the other group of cells was incubated with1ml of the donor solution supplemented with100μM CBX, which inhibits the formation of gap junctions without affecting the activity of connexin. The cells were incubated for approximately4h in5%CO2at37℃and then examined with an inverted fluorescence microscope.8. Statistic The above test needs to be performed at least three times. The results for the between-group tests are presented by "mean±standard deviation". The between-group tests are divided into two cases according to the type of variance. When homogeneity of variance holds, we use one-way analysis of variance and LSD method forpairwise comparison. When heterogeneity of variance holds, we use Welch method and Dunnett-T3for pairwise comparison. And the results for the between-group tests are presented "mean±standard deviation". We use SPSS19.0to analyze the data. The difference is statistically significant if "P<0.05".The main results:1. Lead acetate is toxic to PC12cells dose-dependentty. The toxic can be inhibited by NAC partlyAfter the incubation of PC12cells with different concentrations of lead acetate (0.1-100μM) for24h, the MTT assay, flow cytometry, and RT-PCR were used to detect the effects of lead acetate on PC12cells.1.1Lead acetate is toxic to the viability of PC12cell dose-dependenllyThe cell viability was determined by the MTT assay. The viability of the control cells was normalized to1, and the viabilities of the other cells are expressed as percentages compared with that of the control. The exposure to1μM of lead acetate significantly (0.01<P<0.05) decreased the viability of PC12cells (95.0%±3.8%), whereas exposure to10μM and100μM decreased the viability of PC12cells to69.8%±2.8%and51.4%±3.8%, respectively (P<0.01). This finding shows that the lead acetate-induced cytotoxicity is dependent on the concentration.1.2Effect of lead acetate on Bax and Bcl-2levelsTo determine the exact concentration of lead acetate for use in the subsequent studies, we performed RT-PCR to detect the gene expression levels of Bax and Bcl-2in PC12cells after treatment with lead acetate at different concentrations. All of the RT-PCR results were standardized to the levels observed in normal PC12cells (normalized to1).The RT-PCR analysis demonstrated that the expression of Bax mRNA was significantly (P<0.01,n=3) increased to3.55,3.18, and4.42after treatment with1μM,10μM, and100μM lead acetate, respectively, compared with the untreated PC12cells; however, the0.1μM group did not exhibit a significant difference. However, the differences between these treatment groups were not significant. The expression levels of Bcl-2mRNA were decreased significantly in the10μM and100μM groups (33.3%±0.55%and4.42%±0.14%, respectively, P<0.01, n=3). The analysis of the express ion levels of Bax and Bcl-2mRNA also suggests that a lead concentration of10μM is sufficient to induce cytotoxic effects in PC12cells.1.3Lead acetate is toxic to the apoptosis rate of PC12cells dose-dependently. NAC can inhibit the apoptosis partly.Tthe flow cytometry analysis revealed that the percentage of apoptosis in the untreated PC12cell population was0.40%±0.46%, and significantly higher percentages of PC12cells underwent apoptosis after treatment with0.1μM,1μM,10μM, and100μM(1.33%±0.42%,5.40%±0.51%,38.33%±3.06%, and,46.67%±4.51%, respectively). The percentages of apoptotic cells in the10μM and100μM groups were not significantly different. However, a further study showed that18.67%±2.08%of the cells in the100μM group died, whereas a lower percentage of cells (9.97%±1.95%) died in the10μM group (P<0.01, n=3). Consistent with the MTT and RT-PCR results, a lead acetate concentration of10μM was selected for the subsequent studies. NAC, a commonly used inhibitor of ROS generation, was used to determine the probable mechanism underlying lead-induced apoptosis. The data showed that pre-treatment with5mM NAC decreased the percentage of apoptotic cells to14.33%±1.53%and the percentage of dead cells was to5.13%±0.81%. This result indicates that NAC partly inhibits the apoptosis induced by lead acetate.1.4Effect of lead acetate on ROS generationThe immunofluorescent staining shown that less PC12cells (Hoechst33342, blue) express ROS (red), and the cells exhibit a low fluorescence prior to exposure to lead acetate. After treatment with10μM lead acetate for24h, the ROS generation increased markedly. It is interesting that pre-treatment with5nM NAC for30min can inhibit ROS generation in PC12cells that are exposed to lead acetate.To distinguish the exposed cells and unexposed PC12cells, the former were successfully and stably transfected with the EF1A-EGFP vector (GFP-PC12) before induced, and approximately99%of the GFP-PC12cells were found to be GFP-positive, as evaluated using an inverted fluorescence microscope and flow cytometry.To exclude the effect of transfected GFP on ROS generation in PC12cells, fluorescence microscopy was used to capture the fluorescence intensities associated with ROS generation in PC12cells after transfection with GFP. Compared with normal PC12cells (normalized to1), the ROS generation levels in GFP-PC12cells, GFP-PC12+Pb cells, and GFP-PC12+NAC+Pb cells were0.92±0.15,3.97±0.47and1.09±0.16, respectively. These data show that transfected GFP has no effect on ROS generation and that GFP-PC12+Pb cells produced a significantly higher amount of ROS. Pre-treatment with NAC can inhibit the subsequent ROS generation even if the cells are incubated with lead acetate at a later point in time.1.5NAC can inhibit the toxic of lead acetate on mitochondrial membrane potential (△ψm) of PC12cells dose-dependently mainlyFluorescent probes for monitoring△ψm are frequently used for the assessment of mitochondrial function, particularly in the context of oxidative stress and apoptosis research. The△ψm of PC12cells, GFP-PC12cells, GFP-PC12+Pb cells, and GFP-PC12+NAC+Pb cells was determined through FACS analysis using the fluorescent probe TMRM (red). The GFP-PC12+Pb cells expressed the lowest Aym, and the cells in the other groups showed the same intensity compared with the PC12group.2. The bystander effects of GFP-PC12(Pb24) cells are mediated by GJIC and inhibited by CBXAfter successful transfection, the GFP-PC12cells were incubated in normal medium containing10μM lead acetate for24h, and the GFP-PC12(Pb2+) cells were then co-cultured with normal PC12cells.2.1CBX inhibits functional GJIC in the co-culture systemThe parachute assay results demonstrate that functional gap junctions were formed between GFP-PC12(Pb2+) cells and PC12cells. The red fluorescence represents the donor cells, e.g., GFP-PC12(Pb2+) cells, and the green fluorescence demonstrated that the donor cells transmitted the signals to the receptor cells (PC12cells) through functional gap junctions. After incubation with CBX, an inhibitor of functional gap junctions, the donor cells did not convey any signals.2.2The ROS generation in PC12cells in the co-culture system can be inhibited by CBXImages of the immunofluorescent staining and the diagram shown indicates that minimal red fluorescence was detected in the GFP-PC12+PC12cells (normalized to1). However, almost all of the GFP-PC12(Pb2+)+PC12cells expressed a significantly higher level of red fluorescence in the nucleus (3.48±0.59). In contrast, pre-treatment with CBX resulted in a lower level of red fluorescence (1.64±0.42) compared with that expressed by the GFP-PC12(Pb2+)+PC12cells (P<0.01, n=3) and a higher level of fluorescence than that observed in the GFP-PC12+PC12cells (P<0.01,n=3).2.3The apoptosis of PC12cells in the co-culture system can be inhibited by CBXIn the co-culture system, GFP-PC12(Pb2+) cells were GFP(+), whereas the co-cultured PC12cells were GFP(-). To detect the apoptosis of the co-cukured PC12cells, the Annexin V-PE/7-AAD apoptosis detection kit, which stains apoptosis cells a red color, was used. The Annexin V(+)GFP(-) cells represent cells that were affected by the bystander effect and thus need to be further analyzed.The Annexin V(+)GFP(-) PC12cells in the co-culture system endure the highest percentage of apoptosis (43.52%±8.07%). CBX significantly inhibits cell apoptosis (12.19%±1.13%).2.4The changes in the mitochondrial membrane potential (△ψm) of PC12cells in the co-culture system can be inhibited by CBXA FACS analysis was used to measure the△ψm in GFP-PC12+PC12, GFP-PC12(Pb24)+Pb, and GFP-PC12(Pb2+)+Pb+CBX cells using the fluorescent probe TMRM. In the co-culture system, only the TMRM(+)GFP(-) cells were investigated. The selected cells from the GFP-PC12(Pb2+)+Pb group exhibited the lowest△ψm, and the cells in the GFP-PC12(Pb2+)+Pb+CBX group presented a lower intensity compared with the GFP-PC12+PC12group. This finding demonstrates that CBX can inhibit the lead-induced changes in△ψm.Conclusions:1. Lead acetate can induce the mitochondrial pathway of apoptosis in PC12cells.2. In co-culture system, Pb-exposed GFP-PC12cells exerted bystander effects that resulted in increased apoptosis, the generation of ROS, and the collapse of the MMP of the untreated PC12cells.3. The bystander effects, which drive the expansion of the Pb-induced damage to neighboring cells, were determined to be dependent on functional GJIC. |
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