Investigation Of The Mechanism Of Lead-induced Transcellular Transport In An In Vitro Blood-brain Barrier Model

BackgroundLead is a persistent and common environmental contaminant. It accumulates in human body and exists for a long time. With its entry into the body, lead can cause toxic injury in erythrocytes, liver, renal cortex, aorta, brain, lungs, spleen, teeth, and bones. Studies of effects of lead on children have demonstrated the presence of irreversible neurological alterations at low concentrations. These findings demonstrate that, based on cellular and molecular evidence, there is no threshold for lead toxicity. A chronic, low-level intoxication of lead is more common in the general population. Although a variety of actions have been taken to decrease the use and distribution of lead in the environment, it remains a significant health hazard.Due to its unique structure, the blood-brain barrier (BBB) is capable of limiting the penetration of a variety of substances from the blood into the brain. The BBB plays an important role in the homeostasis and is generally seen as a defence mechanism that protects the brain against various poisonous molecules that may traverse the BBB. The BBB has long been known to be a target for Pb toxicity. Under acute exposure to high levels of Pb, Pb-induced microvascular damage is prevalent with leaky microvessels, as characteristic opening of the interendothelial tight junctions and by enhanced pinocytotic activity seen in the damaged BBB. Studies show that lead is a substrate for the divalent metal transporter 1 (DMT1/ NRAMP2/ DCT1), and Pb2+ has a higher transport affinity than Fe2+. Data suggest that lead may interfere with iron homeostasis through pathways specific intended for iron or the substitute for Fe2+ to alter the function of iron-binding protein.In the present study, we overcome the limitations of the in vivo research and try to explore the mechanism of the transcellular transportation of lead and the relationship between the transportation and cellular iron homeostasis. This study would offer the theoretical basis for elucidating the molecular mechanism of lead transport in the BBB and taking some effective measures to control lead toxicity. Aim:The aim of the present study was, by co-culture ECV304 cells and C6 cells to establish BBB model in vitro, investigate the possible relationship between transcellular transport of lead and cellular iron homeostasis, and further explore the possible pathway of lead-induced transcellular transport and its regulatory mechanism. Methods:1. A model of transwell was developed for the co-culture ECV304 cells and C6 cells to established BBB model in vitro, and the BBB restrictive characteristic was assessed by permeability of FITC-labeled dextran and measurement of transendotheilal electrical resistance (TEER).2. The electrothermal atomization atomic absorption spectrometer was employed to measure the characteristic of lead transport kinetics, the intracellular distribution of DMT1 (IRE) protein was analysed by immunocytochemical method, and deferoximine (DFX), inducing iron deficiency to study the effects of lead on cellular iron homeostasis, was used.3. Co-culture ECV304 cells transfected with antisense oligonucleotides to DMT1and pcDNA3.0-DMT1 vector respectively and C6 cells was used to establish BBB model in vitro respectively, and then the role of DMT1 (IRE) protein in lead trafficking was investigated.4. The expression of IRP1 and p-ERK1/2 were analysed by western blot method, the interaction between IRP1 and p-ERK1/2 was identified by co-immunoprecipitation and immunocytochemical methods, the role of the interaction between IRP1 and p-ERK1/2 on the regulation of DMT1 (IRE) protein was investigated, and the effect of PD98059 on lead transportation in an in vitro BBB model was evaluated.Results:1. An in vitro BBB model by co-culture ECV304 cells and C6 cells was established.Compare to the BBB with only ECV304 cells, TEER value (232.5?cm2) increased significantly in the co-culture. After 4 days of co-culture, apparent permeability coefficiency was significantly lower than that with the only ECV304 cells. Therefore, the in vitro BBB model by co-culture was suitable for the study of molecular changes of endothelial paracellular permeability and transendothelial transport.2. Transportation of lead through the in vitro BBB had the characteristics of active transport.Temperature, pH, time and dose all had effect on the transport of lead. With time passed, the transport of lead was significantly increased. The transport of lead was also significantly increased in high dose than that in low dose. Furthermore, the transport of lead was temperature-dependent and transport was higher at 37°C than at 4°C. Those results suggested that transport of lead might be characterized with active transport.3. Abnormal changes of the BBB permeability was mainly caused by the enhanced transcellular transport of lead.After 24 h, treatment of 1μM or 5μM lead could not significantly affect the TEER and apparent permeability coefficiency compared with the control (p>0.05). Treatment of 1μM or 5μM lead had no significant effect on claudin-1,ZO-1and occludin mRNA at four time points (2 h, 4 h, 10 h, 24 h). Treatment of lead enhanced the expression of DMT1 (IRE) protein, especially in the membrane fraction. At different time points, lead caused the increased expression of DMT1 (IRE), especially in the membrane. Results suggested that there was a correlation of enhanced transport of lead and transcellular transport of the BBB. 4. Lead-induced transcellular transport was related with the expression of DMT1 (IRE) protein and its localization in the membrane.Treatment with 30μM FeCl3 had no significant influence on the transport of lead (p>0.05), while treatment with 150μM FeCl3 could decrease the transport of lead at 4 h and 24 h (p<0.05). Both lead and DFX increased the expression of DMT1 (IRE) protein in whole cell and membrane fraction. Iron inhibited the overexpression of DMT1 (IRE) induced with lead. Iron and/or lead did not affect the expression of DMT1 (IRE) mRNA. Antisense oligonucleotides (MA1) produced significant inhibition of the expression of DMT1 (IRE) protein. In an in vitro BBB model, MA1 treatment inhibited lead transport at all time points, and significant inhibition occurred only at 10 h (p<0.05). The recombined plasmids (pcDNA3.0-DMT1) were transfected into ECV304 cells. Cells were subjected to 200μg/ml of G418 antibiotic selection to obtain stable transfected cells. In an in vitro BBB model, stable transfected cells inhibited lead transport at all time points, and significant inhibition occurred only at 4 h and 10 h (p<0.05). There results suggested that DMT1 was the carrier of lead transport and changes of iron homeostasis were mainly caused by the enhanced lead transport.5. The interaction between IRP1 and p-ERK1/2 negatively regulated the expression of DMT1 (IRE) protein.The expression of IRP1 protein was increased by lead in the membrane and cytoplasm fractions at 2 h, and gradually declined with time passing. Compared to the control, lead significantly enhanced phosphorylation of ERK1/2 in the whole cell, but reduced phosphorylation of ERK1/2 in the cytoplasm fraction. Both lead and DFX increased the expressions of IRP1 and p-ERK1/2 proteins. Iron inhibited the overexpression of IRP1 and p-ERK1/2 induced by lead. Confocal microscopy was used to analyze ECV304 cells stained for IRP1 and p-ERK1/2 distribution. Results demonstrated that there was a cytoplasmic distribution of IRP1 as well as the co-localization with p-ERK1/2. Both lead and DFX could reduce the co-localization with p-ERK1/2, but iron increased the area of the co-localization. Results of co-immunoprecipitation showed that lead increased the interacted portion of IRP1, and decreased the interacted portion of p-ERK1/2. PD98059 could increase the expression of DMT1 (IRE) and cytoplasmic IRP1 proteins. In an in vitro BBB model, PD98059 treatment increased lead transport at all time points, and the significant increase occurred only at 10h (p<0.05). These results suggested that the interaction between IRP1 and p-ERK1/2 negatively regulated the expression of DMT1 (IRE) protein.Conclusions:1. DMT1 (IRE) is important in lead transcellular transport in an in vitro BBB model. 2. DMT1 (IRE) is a carrier of lead into brain, and it's localization in the membrane plays an important role in lead transcellular transport.3. DMT1 (IRE) mRNA is regulated through stabilization by the IRE-IRP system.4. There exists an interaction between IRP1 and p-ERK1/2 in ECV304 cells, regulating the expression and function of IRP1 protein.5. ERK inhibitor, PD98059, increases an expression of DMT1, enhancing the lead transport in an in vitro BBB model.