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Toxicol Sci. 2010 January; 113(1): 169–176.
Published online 2009 October 12. doi:  10.1093/toxsci/kfp249
PMCID: PMC2794334

Decreased Expression of the Voltage-Dependent Anion Channel in Differentiated PC-12 and SH-SY5Y Cells Following Low-Level Pb Exposure

Abstract

Lead (Pb) has been shown to disrupt cellular energy metabolism, which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage-dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5 and 10μM Pb on VDAC expression in vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time reverse transcription-polymerase chain reaction demonstrated a significant decrease in messenger RNA levels for the VDAC1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5 and 10μM Pb reduces VDAC transcription and expression and is associated with reduced cellular ATP levels.

Keywords: voltage-dependent anion channel, lead, energy depression, PC-12 cells, SH-SY5Y cells

The extensive use of lead (Pb) in a wide variety of industrial processes and products has resulted in the widespread distribution of this toxic heavy metal throughout the environment. Despite extensive efforts to eliminate the use of Pb, it continues to be a serious problem in many parts of the United States. Pb has been shown to affect many different biological processes, including metal transport, energy metabolism, neurotransmitter storage and release processes, protein maturation, and genetic regulation (Garza et al., 2006; Lidsky and Schneider, 2003). While high levels of Pb exposure induce a variety of physical and behavioral syndromes, lower levels of Pb exposure during development has been shown to be a risk factor for behavioral syndromes, such as learning disabilities and attention-deficit hyperactivity disorder (Braun et al., 2006; Lidsky and Schneider, 2003). Although there have been many studies devoted to studying the effects of Pb exposure, the molecular mechanisms behind the subtle neurotoxic effects of lower levels of Pb remain largely unknown. However, disrupted energy metabolism is one mechanism that has been proposed as a possible cause of the behavioral abnormalities and brain dysfunction induced by Pb (Sterling et al., 1982).

The voltage-dependent anion channel (VDAC) is an ion channel located in the mitochondrial outer membrane that plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering (Shoshan-Barmatz and Gincel, 2003; Shoshan-Barmatz et al., 2006). Decreased VDAC expression has been shown to result in decreased ATP synthesis and decreased cytosolic ADP and ATP levels in vitro (Abu-Hamad et al., 2006). In addition, VDAC has been shown to be involved in learning and synaptic plasticity in mice (Levy et al., 2003; Weeber et al., 2002), two functions that are disrupted by Pb exposure. Pb also causes decreases in cellular ATP levels in rat brain synaptosomes (Rafalowska et al., 1996). Therefore, we hypothesized that Pb exposure will result in decreased VDAC protein levels and decreased ATP levels in neuronal cells.

In order to investigate the interactions among Pb, VDAC, and ATP, two in vitro cell lines were used, a differentiated rat pheochromocytoma cell line (PC-12 cells) and a differentiated human neuroblastoma cell line (SH-SY5Y cells). PC-12 and SH-SY5Y cells serve as relevant in vitro model systems for primary neuronal cells because they stop dividing, grow long neurites, and show changes in cellular composition associated with neuronal differentiation in response to treatment with nerve growth factor and retinoic acid, respectively (Greene and Tischler, 1976; Vignali et al., 1996).

Cells were differentiated for 4–6 days, and total VDAC protein levels were measured following low-level Pb exposure. We found that total VDAC protein expression levels decreased in both cell lines with 48 h of Pb exposure. Interestingly, hypoxia did not result in a change in VDAC expression, indicating that reduced VDAC expression is not a general response to stress. In addition, we found a concomitant decrease in cellular ATP levels with 48 h of Pb exposure, suggesting a correlation between decreased VDAC expression and decreased cellular ATP levels. Finally, real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the Pb-induced decrease in VDAC protein is at least due in part to decreased transcription of VDAC1. Taken together, these results show that Pb reduces VDAC transcription and protein expression, and this decrease is correlated with a reduction in cellular ATP levels.

MATERIALS AND METHODS

PC-12 and SH-SY5Y cell cultures.

PC-12 cells were obtained from American Type Culture Collection (Rockville, MD). PC-12 cells were cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% horse serum and 5% fetal calf serum, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine (GIBCO BRL, Grand Island, NY) at 37°C in humidified air containing 5% CO2. The culture medium was changed every 2–3 days, and cells were used between passages 6 and 12. For experiments, ~15,000 cells/cm2 were seeded onto collagen-coated six-well plates and then allowed to grow for 2 days in RPMI medium. Cells were differentiated using 100 ng/ml nerve growth factor (NGF-7S; Sigma-Aldrich, Inc., St Louis, MO) in RPMI medium containing 1% horse serum, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine. Under these conditions, the cells developed a neuronal phenotype with neurite outgrowth that was already apparent 24 h after seeding; we always used the cells at day 6 of differentiation. For experiments, differentiated cells were treated with various concentrations of lead acetate dissolved in water in RPMI medium containing 1% horse serum, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine.

SH-SY5Y cells were obtained from American Type Culture Collection. SH-SY5Y cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium (GIBCO BRL) supplemented with 10% fetal bovine serum (Hyclone; Thermo Scientific, Waltham, MA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine in a CO2 incubator maintained at 5% CO2 and at 37°C. The medium was changed every 2 days. Cells were treated with 10μM all-trans retinoic acid (Sigma-Aldrich, Inc.) in the dark for 8–10 days to induce differentiation. For experiments, differentiated cells were treated with different concentrations of lead acetate in DMEM/F12 medium containing 1% horse serum, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine for 1–4 days.

Pb exposure.

Cells were exposed to Pb as follows: A 5mM Pb stock solution was prepared by dissolving the appropriate amount of lead acetate in sterile double-distilled H2O. Experimental Pb concentrations were prepared by dilution of stock solution in appropriate culture medium. For PC-12 cells, experimental Pb concentrations were prepared in serum-free RPMI medium containing 1% horse serum, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine. For SH-SY5Y cells, experimental Pb concentrations were prepared in DMEM/F12 medium containing 1% horse serum, sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine. Differentiated cells were incubated with 0, 1, 5, 10, and 50μM of Pb for different time periods (24, 48, 72, or 96 h) at 37°C, with 0μM Pb samples used as the control group.

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide and Lactate Dehydrogenase assays.

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) and Lactate Dehydrogenase (LDH) assay kits were obtained from Roche Applied Science (Indianapolis, IN; Cat. no. 11 465 007 001 and 1 644 793, respectively). For MTT and LDH assays, ~0.25 × 105 cells/ml were differentiated on collagen-coated 96-well plates with eight replicates per treatment group. Cells were exposed for 24, 48, or 72 h to a graded series of Pb concentrations (0, 1, 5, 10, 50, 100, 250, 500, 1000, and 5000μM Pb). For PC-12 cells, a 2% Triton X-100 solution in assay medium was used for a high control. Cells were incubated for 24, 48, or 72 h at 37°C with 5% CO2 and 90% humidity. General assay protocols included with each kit were followed (n = 8 samples per group for SH-SY5Y and PC-12 cells, respectively). Statistical significance was determined by ANOVA followed by a Dunnet’s post hoc test (p < 0.05, n = 8 samples per group).

Briefly, for the LDH assay, cells were rinsed several times with culture medium prior to Pb exposure. Culture medium was removed, and 100 μl of fresh medium containing the graded Pb concentrations was added to each well. At the appropriate time points, 100 μl of supernatant was removed from each well and transferred into the corresponding wells of a new 96-well plate. LDH activity was determined by adding 100 μl of reaction mixture (included with kit) to each well and incubating for 20 min in the dark at room temperature. Absorbance measurements were made at 490 nm on a microplate reader (Versamax; Molecular Devices, Sunnyvale, CA). Reference wavelength was 650 nm.

For the MTT assay, cells were rinsed several times with culture medium prior to Pb exposure. Culture medium was removed, and 100 μl of fresh medium containing the graded Pb concentrations or Triton X-100 was added to each well (n = 8 for each Pb concentration and Triton X-100). After incubation for appropriate time points, 10 μl of MTT-labeling reagent (included with kit) was added to each well. Plates were incubated with MTT-labeling reagent for 4 h and then 100 μl of solubilizing solution (included with kit) was added to each well. Plates were incubated overnight before being measured. Absorbance of samples was measured using a microplate reader (Versamax; Molecular Devices). The wavelength for measuring formazan product was 575 nm, and reference wavelength was 660 nm.

Hypoxia.

SH-SY5Y cells were cultured and differentiated as described above, followed by incubation in a controlled atmosphere of 2% oxygen (14 mmHg partial pressure) for 24 h at 37°C. The gas mixture in the incubator during hypoxia was 2% O2, 5% CO2, and 93% N2. It is important to note that 24 h hypoxia had no effect on cell viability, as indicated by a trypan blue exclusion assay.

Western blot analysis.

For all samples, total protein was extracted from cells by homogenizing in a lysis buffer containing 20mM Tris-HCL (pH 7.5), 150mM NaCl, 1mM Na2EDTA, 1mM ethylene glycol tetraacetic acid, 1% Triton, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, and 1mM Na3VO4 (Cell Signaling Technology, Beverly, MA). Additions were made giving final concentrations of 0.5% Na-deoxycholate; 0.5% SDS; 1μM okadaic acid; 1mM phenylmethylsulfonyl (PMSF); 0.1 mg/ml benzamidine; 8 μg/ml calpain inhibitors I and II; and 1 μg/ml each leupeptin, pepstatin A, and aprotinin. Homogenates were sonicated and vortexed for 5 min before centrifugation at 10,000 × g for 20 min. Protein concentration of the supernatant was then determined by Bradford protein assay. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 4–12% gradient Bis-Tris gels criterion precast gel (Bio-Rad Laboratories, Hercules, CA) and transferred to polyvinylidene diflouride (PVDF) membranes for immunoblotting. Sources for antibodies were as follows: total VDAC (Cell Signaling Technology #4866) and p44/42 MAP kinase (extracellular signal-regulated kinase [ERK]) (Cell Signaling Technology #9102).

Gels were transferred to Immun-Blot PVDF membrane (Bio-Rad Laboratories) using Criterion (Bio-Rad Laboratories) transfer cell with plate electrodes for 60 min at 100 V, blocked in 5% milk in tris-buffered saline tween-20 (TBST) (1× Tris-buffered saline, 0.1% Tween 20, and 5% dry milk), and incubated overnight at 4°C. Membranes were washed in TBST and then incubated overnight at 4°C with VDAC antibody. Membranes were washed in TBST and incubated with secondary antibody (Vector Laboratories, Inc., Burlingame, CA; horseradish peroxidase–conjugated anti-rabbit, PI-1000, 1:2000) for 2 h. Membranes were then washed in TBST before imaging with the Fuji LAS-3000 CCD-based imaging system (FujiFilm Life Science, Valhalla, NY). Membranes were reprobed with total ERK to confirm equal protein loading. Band pixel intensity was measured using the Fuji LAS-3000 software. VDAC band intensities from control and treated groups were normalized to total ERK and plotted using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA) software. Statistical significance was determined by t-test (p < 0.05, n = 3 and n = 6 samples per group for SH-SY5Y and PC-12 cells, respectively).

Cellular ATP assay.

The cellular ATP levels for each Pb treatment group were determined using an ATP Bioluminescence Assay Kit HS II (Roche Applied Science). Cells were grown and differentiated on collagen-coated plates as described above. Cells from each treatment group were rinsed with sterile PBS and then collected and diluted to ~107 cells/ml. Two hundred microliters of the cell suspension and 200 μl of the supplied lysis reagent were placed in a 1.5-ml Eppendorf tube for ~5 min. Samples were centrifuged at 10,000 × g for 60 s, and supernatant was transferred to a fresh Eppendorf tube and kept on ice until analysis. For ATP determinations, 50 μl of sample was transferred to a 96-well plate and luminescence was measured using a microplate luminometer (Centro LB 960; Berthold Technologies, Oak Ridge, TN). Briefly, 50 μl of luciferase reagent was added to 50 μl of sample by automated injection. Measurement was started after a 1-s delay and integrated from 1 to 10 s. Luminescence measurements from control and treated groups were normalized to total protein concentration of sample. Statistical significance was determined by ANOVA followed by a Dunnet’s post hoc test (p < 0.05, n = 6 samples per group).

RNA isolation.

Cell pellets were resuspended in a small volume (< 300 μl) of PBS. Eight milliliters of Trizol Reagent (Invitrogen Corp., Carlsbad, CA) was added and mixed by pipetting to homogenize the cells. After a 10-min incubation at room temperature, 1.6 ml of chloroform was added and the solution was mixed by shaking for 30 s and incubated at room temperature for 3 min. Phase separation was performed by centrifugation at 3200 × g for 30 min. The upper aqueous layer was removed to a tube containing 4 ml of isopropanol and mixed by inversion. Samples were then precipitated overnight at −20°C. RNA was pelleted by centrifugation at 3200 × g for 15 min, the supernatant was removed, and 8 ml of 75% ethanol prepared with diethylpyrocarbonate water was added to wash the pellet. Another centrifugation at 3200 × g for 10 min was performed, the supernatant was removed, and the pellets were air dried for 10 min. Pellets were resuspended in 400 μl of RNase-free water from the Qiagen RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). Hundred microliters of each sample was used for further purification according to the “RNA Cleanup” protocol from the Qiagen RNeasy manual, including the optional DNase treatment. The final elution step was performed with 30 μl of RNase-free water. RNA quantity was determined using a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, LLC, Wilmington, DE). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Nano LabChip (Agilent Technologies, Santa Clara, CA) and reagents to obtain RNA integrity numbers (RINs) and 28S/18S ratios.

Real-time RT-PCR analysis.

A real-time RT-PCR analysis was used to investigate gene expression following Pb exposure in differentiated PC-12 cells. Total RNA was extracted, as described above, from six samples, three control groups, and three groups treated with 5μM Pb for 48 h. Real-time RT-PCR was used to analyze the expression of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NM017008), VDAC1 (NM031353), VDAC2 (NM031354), and VDAC3 (NM031355) genes, and mitogen-activated protein kinase (MAPK) 1/ERK 2 (NM053842) was used for the control or housekeeping gene. Reverse transcription and real-time PCR expression analysis was conducted by SuperArray Bioscience (Frederick, MD).

RNA quality control.

RNA samples were run on the Agilent Bioanalyzer. The integrity of RNA was assessed by looking at 18S and 28S ribosomal RNA peaks and by the RIN. RNA concentrations were measured using the NanoDrop ND1000 Spectrophotometer, and all samples had 260/280 ratios above 2.0 and 260/230 ratios above 1.7, respectively.

Reverse transcription and first-strand complementary DNA synthesis.

Equal amounts of RNA (1 μg) were taken for all samples, and reverse transcription was performed using RT2 First Strand Kit from SuperArray Bioscience. This kit uses Moloney Murine Leukemia Virus reverse transcriptase and a combination of random primers and oligo dT primers. The kit contains optimized DNA removal buffer that prevents false-positive signals due to amplification of genomic contamination and has a built-in external RNA control that verifies lack of enzyme inhibitors and efficient reverse transcription. The total volume of the reaction was 20 μl and was diluted to 100 μl.

Polymerase chain reaction.

PCRs were performed using RT2 qPCR primer assays (SuperArray Bioscience, Frederick, MD) on the ABI 7500 with RT2 Real-Time TM SYBR Green PCR master mix PA-012 (SuperArray Bioscience, Frederick, MD). The total volume of the PCR reaction was 25 μl. The thermocycler parameters were 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Samples consisted of three biological replicates for the control group and treatment group, respectively. Relative changes in gene expression were calculated using the ΔΔCt (threshold cycle) method (Livak and Schmittgen, 2001). PCR for all the genes for each sample was done in triplicate, and the average, SD, and %CV were calculated for all technical replicates. Data were normalized using the MAPK1 gene. Once data had been normalized (ΔCt), the average of the three biological replicates in each group was used to calculate the ΔΔ Ct and fold change (fold change = 2−ΔΔCt). The p value is calculated using a two-tailed Student’s t-test.

RESULTS

Pb Exposure Does Not Induce Cytotoxicity in This In Vitro System

Differentiated SH-SY5Y and PC-12 cells were used to investigate the interactions among Pb, VDAC, and ATP. These two cell lines are established model systems for primary neurons (Greene and Tischler, 1976; Robson and Sidell, 1985; Vignali et al., 1996), and we reasoned that if Pb decreased VDAC expression in both neuronal cell lines, then this would provide evidence that decreased VDAC expression is a neuronal response to Pb exposure. Because recent in vivo studies in our laboratory have demonstrated that low-level Pb exposure during development does not result in neuronal death (Jones et al., 2008), we first established that the Pb concentrations used do not induce cytotoxicity in our in vitro system. Differentiated PC-12 cells were exposed to a graded series of Pb concentrations, and cell viability was assessed using both MTT and LDH assays following 48 or 72 h of exposure. There was no significant cytotoxicity for 1, 5, 10, and 50μM of Pb at either 48 or 72 h (Fig. 1). It should be noted that similar results were observed for SH-SY5Y cells (data not shown).

FIG. 1.
Lower levels of Pb exposure do not generate cytotoxicity in differentiated PC-12 cells. Cells were exposed for 24, 48, or 72 h to a graded series of Pb concentrations (0, 1, 5, 10, 50, 100, 250, 500, 1000, and 5000μM Pb). Triton X-100 was used ...

Low-Dose Pb Exposure Reduces VDAC Expression In Vitro

In order to determine whether lower concentrations of Pb decrease VDAC expression in our in vitro model, differentiated PC-12 cells were exposed to 0, 1, 5, 10, and 50μM of Pb for 24 and 48 h. Western blot analysis using an antibody directed against total VDAC was then used to compare VDAC expression levels between the Pb treatment groups and the no-Pb controls. Our preliminary studies indicated that VDAC begins to decrease within 48 h of exposure to Pb, with large decreases observed at 48 h for the 5 and 10μM Pb concentrations (data not shown). It is important to note that these are relatively low levels of Pb, and the 5–10μM range has been widely used in many in vitro studies examining the effects of Pb on its molecular targets (Cordova et al., 2004; Leal et al., 2002; Zhao et al., 1998). Additional experiments with 5 and 10μM Pb at 48 h confirmed that VDAC is significantly decreased by 44 and 31%, respectively, compared to controls (Fig. 2). In order to determine whether the Pb-induced decrease in VDAC is a general neuronal response to Pb exposure, VDAC expression in differentiated SH-SY5Y cells was quantified following a 10μM Pb exposure for 48 h. There is a significant decrease in VDAC expression as measured by Western blot following Pb exposure in SH-SY5Y cells (Fig. 3). These studies confirm that decreased VDAC is a general response to Pb and is not restricted to a particular neuronal subtype.

FIG. 2.
Pb causes a significant decrease of VDAC at 48 h. Differentiated PC-12 cells were exposed to 5 and 10μM Pb for 48 h, and VDAC expression was quantified by Western blot analysis. (A) At 48 h, 5μM Pb results in a significant decrease in ...
FIG. 3.
Hypoxia fails to decrease VDAC expression in vitro. Differentiated SH-SY5Y cells were exposed to 24 h hypoxia or 10μM Pb for 48 h. A 48-h exposure to 10μM Pb results in a significant decrease in VDAC. In contrast, exposure to 24 h hypoxia ...

Hypoxia Fails to Decrease VDAC Expression In Vitro

In order to rule out the possibility that decreased VDAC expression is a general stress response, cells were exposed to 24 h of hypoxia and VDAC expression measured. Both hypoxia and Pb can interfere with gene expression, cause mitochondrial dysfunction, and result in cellular ATP depletion (Seppet et al., 2009). The 24-h time point for hypoxia was selected because cells still remain viable, although they did show morphological signs of cellular stress, such as slightly shrunken cell bodies (data not shown). Exposing cells for longer than 24 h resulted in cell death. VDAC expression does not change following 24 h of hypoxia (Fig. 3), suggesting that the Pb-induced decrease in VDAC is not part of a general cellular stress response. These results confirm that in our in vitro system, decreased VDAC expression following Pb exposure is not a general cellular response to stress.

Pb Reduces Cellular ATP Levels

Because VDAC expression begins to decrease 48 h following Pb treatment, we would predict that a corresponding decrease in cellular ATP would occur since VDAC plays such a central role in regulating cellular energy metabolism. To determine if the Pb-induced decrease in VDAC corresponds with decreases in cellular ATP levels, ATP levels were measured following Pb exposure in vitro. Differentiated PC-12 cells were incubated with 0, 1, 5, 10, and 50μM of Pb, and cellular ATP levels were quantified using a luciferase-driven bioluminescence assay. ATP levels were compared among the treatment groups and the no-Pb controls. To rule out the possibility that Pb itself could directly interfere with the bioluminescence assay, cells were exposed to the Pb concentrations listed above and cellular ATP levels measured following a short 30-min incubation. After 30 min of exposure, Pb failed to produce a significant difference in cellular ATP levels when compared to no-Pb controls at all the concentrations tested (Fig. 4A), demonstrating that Pb itself does not interfere with bioluminescence assay. In contrast, after 48 h of Pb, cellular ATP levels for all treatment groups are significantly decreased by ~35–45% relative to the no-Pb controls (Fig. 4B). Thus, the observed decreases in cellular ATP levels are correlated with the decreases in VDAC expression after 48 h of exposure to Pb. These results suggest that ATP synthesis may be compromised by the Pb-induced decreases in VDAC.

FIG. 4.
Pb results in reduced levels of cellular ATP. A bioluminescence assay was used to measure cellular ATP levels in differentiated PC-12 cells following exposure to a graded series of Pb concentrations (0, 1, 5, 10, and 50μM Pb). (A) Pb failed to ...

Pb Induces Decreased Gene Transcription of VDAC1

The cellular effects of Pb are widespread and are elicited through a variety of different mechanisms, including altered gene transcription (Garza et al., 2006). Pb is thought to interfere with gene expression by competing for the metal-binding sites of transcription factors, such as zinc finger proteins (Basha et al., 2003; Zawia, 2003). Therefore, the Pb-induced decrease in VDAC protein expression might be due to decreased transcription of the VDAC gene. In order to test this hypothesis, real-time RT-PCR was used to elucidate whether Pb exposure results in decreased gene expression for one or more of the three isoforms of VDAC. Total RNA was extracted from differentiated PC-12 cells following a 48-h exposure to 5μM Pb. Real-time RT-PCR was then used to determine the messenger RNA (mRNA) levels for VDAC1, VDAC2, and VDAC3, relative to the MAPK control gene. MAPK was chosen as a control gene since its expression appeared to be more stable in our in vitro system than other commonly used control genes, such as GAPDH. It is important to note that Pb does not induce a significant difference in mRNA levels between the control gene MAPK and GAPDH, a commonly used housekeeping gene. Figure 5 illustrates that Pb exposure causes a significant decrease (14%) in the levels of VDAC1 mRNA. Pb also induces a decreased trend for mRNA levels of the VDAC2 gene, but this decrease was not statistically significant. In contrast, there was no observed decrease in mRNA levels for VDAC3. In summary, the decreased protein expression of VDAC that is observed following Pb exposure in vitro is due at least in part to decreased transcription of VDAC1.

FIG. 5.
Pb produces significant decreases in mRNA levels for VDAC1. Differentiated PC-12 cells were exposed to Pb, followed by a real-time RT-PCR analysis to determine mRNA levels for VDAC1, VDAC2, and VDAC3. At 48 h, 5μM Pb results in a significant decrease ...

DISCUSSION

The current study demonstrates that Pb exposure results in decreased VDAC expression in differentiated PC-12 and SH-SY5Y cells by 48 h. This is not a general cellular response to stress as 24 h of hypoxia failed to produce any decrease in VDAC expression. These findings suggest that in our in vitro studies, decreased VDAC expression is a specific response to Pb. Furthermore, real-time RT-PCR established that the decrease in VDAC protein expression occurs, at least in part, as a result of the decrease in gene expression for VDAC1. Finally, Pb exposure for 48 h results in a corresponding decrease of cellular ATP levels, suggesting a relationship between decreased ATP levels and decreased VDAC expression.

Involvement of Mitochondrial VDAC in Neuronal Function

VDAC is an ion channel located in the mitochondrial outer membrane and is considered to be essential for the regulation of mitochondrial function (Blachly-Dyson and Forte, 2001). VDAC regulates mitochondrial metabolism by controlling the flow of anions, cations, creatine phosphate, Pi, ATP, ADP, and other metabolites across the mitochondrial outer membrane (Shoshan-Barmatz and Gincel, 2003; Shoshan-Barmatz and Israelson, 2005; Shoshan-Barmatz et al., 2006). Mammals express at least three different VDAC genes: VDAC1, 2, and 3 (Baines et al., 2007; Weeber et al., 2002). VDACs have highly conserved structural and electrophysiological characteristics across plant, yeast, mouse, and human species (Sampson et al., 1997). Our real-time RT-PCR analysis demonstrated that Pb reduced gene expression of the VDAC1 isoform, which has been associated with mitochondrial respiration. It is intriguing to speculate that reduced VDAC expression may impair normal mitochondrial ATP synthesis, thus compromising neuronal activity.

Mechanisms of Pb Neurotoxicity

While various mechanisms have been proposed to explain the neurotoxic effects of Pb, the ability of Pb to substitute for other divalent cations, particularly calcium and zinc, at numerous cellular sites has emerged as a common theme for many of its toxic actions (Garza et al., 2006; Lidsky and Schneider, 2003). The substitution of Pb for divalent cations has the potential to affect many different biologically significant processes, including metal transport, energy metabolism, apoptosis, ionic conduction, excitotoxicity, neurotransmitter storage and release processes, altered activity of first and second messenger systems, protein maturation, and genetic regulation (Garza et al., 2006; Lidsky and Schneider, 2003). In addition, Pb is thought to interfere with gene expression by competing for the metal-binding sites of transcription factors, such as zinc finger proteins (Basha et al., 2003; Zawia, 2003). For example, numerous studies have reported that Pb exposure interferes with the DNA binding of the zinc finger transcription factor Sp1 both in vivo and in vitro (Atkins et al., 2003; Basha et al., 2003; Crumpton et al., 2001; Hanas et al., 1999; Zawia et al., 1998). Although it is not known if Sp1 regulates VDAC gene transcription, all three VDAC isoforms contain Sp1-binding sites (Sampson et al., 1997) that could be modified by Pb. Future studies will determine whether Pb exposure decreases VDAC transcription by altering Sp1 binding.

Role of VDAC In Vivo

While this is the first study to demonstrate Pb-induced decreases in VDAC expression within differentiated neuronal cell lines, other studies have also found decreases in ATP concentrations following Pb exposure in vitro (Rafalowska et al., 1996). This suggests that decreases in cellular ATP is a common response to Pb and may be occurring in part through decreased VDAC expression. It is important to note that experiments using VDAC knockout mice have demonstrated that each VDAC isoform appears to have specialized functions. The knockout of VDAC1 and VDAC2 results in reductions of mitochondrial respiratory capacity (Wu et al., 1999), which demonstrates the importance of these proteins in maintaining cellular energy levels. This might explain why our observed Pb-induced decrease in VDAC1 gene expression is correlated with decreased ATP levels and could represent a potential mechanism by which Pb exposure results in decreased ATP concentrations in other in vitro systems.

In contrast, knockout of VDAC3 alone causes male sterility (Sampson et al., 2001). Interestingly, studies utilizing VDAC1- and VDAC3-knockout mice demonstrate that these animals have deficits in learning behavior and synaptic plasticity, and these learning deficits appeared to be specific to each VDAC isoform. VDAC3 appears to be necessary for hippocampus-dependent contextual fear conditioning, whereas both VDAC1- and VDAC3-deficient mice have deficits in cued fear conditioning (Weeber et al., 2002). In addition, VDAC-deficient mice showed deficits in hippocampal short- and long-term synaptic plasticity (Weeber et al., 2002; Levy et al., 2003). There is a large body of evidence linking Pb exposure to learning deficits, and it will be interesting to explore the hypothesis that one mechanism by which Pb could produce these cognitive changes is through alterations in VDAC transcription and expression.

Developmental Pb exposure has also been shown to alter the development of the visual and somatosensory systems in vivo (Fox et al., 2008; Wilson et al., 2000), and we have recently demonstrated that low-level Pb exposure during development results in auditory processing deficits in mice (Jones et al., 2008). The auditory brainstem contains specialized neurons that are extremely active and capable of extremely very fast rates of synaptic activity (Oertel, 1999). High levels of ATP are required for auditory neurons to maintain their high rates of firing (Rowland et al., 2000; von Gersdorff and Borst, 2002). Therefore, the ability of Pb to inhibit VDAC expression could contribute to deficits in cellular energy metabolism and thus compromise the function of neurons with high energy requirements, such as brainstem auditory neurons. Ongoing studies in our laboratory are examining VDAC expression in brainstem auditory neurons following developmental Pb exposure.

The current study demonstrates that VDAC may represent a novel target for Pb in the central nervous system by reducing VDAC transcription and expression in neuronal cells in vitro. In these experiments, this decrease is correlated with a decrease in cellular ATP levels, showing a connection between decreased VDAC expression and decreased cellular ATP levels. The loss of VDAC following Pb exposure could therefore contribute to impaired mitochondrial function and compromise neuronal activity in vivo.

FUNDING

National Institutes of Health, National Center for Research Resources (P20 RR17670 and P20 RR015583 to D.I.L.).

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