Dose-dependent effects of lead on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons★
Shuang Gao, Liguang Sun, Yuanyuan You

China Medical University, Shenyang  110001, Liaoning Province, China
Shuang Gao★, Master, Lecturer, School of Public Health, China Medical University, Shenyang  110001, Liaoning Province, China

Corresponding author: Liguang Sun, Doctor, Professor, Department of Biochemistry, China Medical University, Shenyang  110001, Liaoning Province, China
ydslg@163.com

Abstract
BACKGROUND: Previous studies have suggested that the hippocampus is one of the neurotoxic target sites for lead. However, the molecular mechanisms of action, including the effect of lead on cell-cycle arrest, remain poorly understood.
OBJECTIVE: To investigate the effects of different lead concentrations on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons.
DESIGN, TIME AND SETTING: A randomized, controlled, in vitro experiment was performed at the China Medical University between July 2008 and May 2009.
MATERIALS: Antibodies specific to cyclin D1 and actin were synthesized and purified by Santa Cruz Biotechnology, USA. FACStar flow cytometer was purchased from Becton Dickinson, San Jose, California, USA.
METHODS: Wistar rat hippocampal neurons were primary cultured for 7 days. Neurons in the con-trol group were treated with 0.01 mol/L phosphate buffered saline. Neurons in the 0.2, 1.0, and 10 μmol/L lead acetate groups were subjected to 0.2, 1.0, and 10 μmol/L lead acetate. Subsequently, hippocampal neurons in each group were cultured for 24 hours.
MAIN OUTCOME MEASURES: The effects of lead on cell cycle were measured by flow cytometry, DNA damage was measured using the comet assay, and cyclin D1 expression was measured using Western blot analysis.
RESULTS: Treatment of hippocampal neurons with 0.2 μmol/L lead acetate did not significantly alter cell cycle phase distribution, i.e., sub-G1, S, G0/G1, G2/M, whereas treatment with 1.0 and 10 μmol/L lead acetate significantly increased the percentage of S and sub-G1 phase cells (P < 0.05). Olive tail moment in all lead-treated groups and the percentage of DNA in the tail in 1.0 μmol/L and 10 μmol/L lead acetate groups were significantly greater compared with the control group (P < 0.05). In addition, the percentage of tail DNA was greater in the 0.2 μmol/L lead acetate group compared with the control group (P > 0.05). Following incubation with 0.2, 1.0, and 10 μmol/L lead acetate for 24 hours, cyclin D1 expression gradually decreased with exposure to increasing lead acetate concentrations (1.0–10 μmol/L).
CONCLUSION: Lead exposure to primary cultured rat hippocampal neurons resulted in dose-dependently disturbed cellular homeostasis, including DNA damage, reduced cyclin D1 ex-pression, and stagnation of cell-cycle progression.
Key Words: lead; cell-cycle arrest; DNA damage; cyclin D1; hippocampal neurons; nerve factor; neural regeneration

INTRODUCTION
  
Lead (Pb), an environmental contaminant, adversely affects the central nervous system of humans. Neurotoxic effects, as a result of exposure to low-level environmental Pb, are significant problems in the entire world, especially for children and infants[1-3]. Although numerous studies have shown that Pb accumulates in the brain when blood concentrations are elevated[4], the mechanisms of Pb deposition in brain are not yet fully known. Previous epidemiological studies and animal experiments have demonstrated that intelligence of children and animals exposed to high-level Pb is severely impaired, leading to learning and memory deficits[5]. Animal experimental studies have suggested that the hippocampus is the neurotoxic target for Pb[6]. Cell cycle regulation is a key regulatory mechanism of cell growth[7-9], and many cytotoxic and genotoxic agents have been shown to arrest cell cycle at different phases, resulting in cell death[10-11]. Previous studies have demonstrated that Pb results in cytotoxicity in many cell types[12]. However, the effect of Pb on hippocampal neuronal cell death as a result of altered cell cycle has been scarcely reported. Studies have shown that cyclin D1 protein expression is downregulated, which stops cell-cycle progression at the G1 phase[13-14]. The present study used primary cultured rat hippocampal neurons to 
determine the effects of various Pb doses on hippocampal neurons. Cell cycle changes, DNA damage, and cyclin D1 protein expression were measured to provide useful clues for further study of Pb-induced neurotoxicity.

MATERIALS AND METHODS

Design
A randomized, controlled, in vitro study.
Time and setting
Experiments were performed at the China Medical University between July 2008 and May 2009.
Materials
Healthy Wistar rats, aged 1–6 days and of both genders, were supplied by the Animal Experimental Center, China Medical University (license number SYXK (liao) 2008-0005). Protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[15].
Reagents and equipment are as follows:

Methods
Primary rat hippocampal neuronal culture and Pb exposure
Hippocampal neurons were isolated from neonatal Wistar rat embryos and cultured according to previously described methods[16] with slight modification. Briefly, rat hippocampi were dissected into small pieces and incubated for 20 minutes in Ca2+- and Mg2+-free Hanks-balanced salt solution with 2.5 mg/mL trypsin buffered with 10 mmol/L HEPES. The tissue was transferred to DMEM/F12 medium containing 10% FBS and triturated with a fire-polished Pasteur pipette. After centrifugation for 5 minutes at 800 × g, the cells were re-suspended in DMEM/F12 medium supplemented with 10% FBS, 2% B-27, 0.5 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/L streptomycin. Subsequently, freshly harvested cells were seeded onto sterile 6-well or 96-well poly-L-lysine-coated plastic culture plates with   2 or 0.2 mL mixed cultures. The medium was fully replaced by an equal volume of the previous DMEM/F12 medium without FBS after 24-hour incubation. Following a 48-hour culture, 10 μmol/L cytosine arabinoside was added to the medium to inhibit further proliferation of non-neuronal cells. Half of the medium was replaced by freshly prepared medium of the same composition twice weekly. The cultures were maintained in a humid incubator with 95% air and 5% CO2 at 37 °C.
Control group neurons were treated with 0.01 mol/L phosphate buffered saline (PBS). Neurons in the 0.2, 1.0, and 10 μmol/L Pb acetate groups were subjected to 0.2, 1.0, and 10 μmol/L Pb acetate. Subsequently, hippocampal neurons in each group were cultured for 24 hours.
Cell cycle analysis
DNA content was measured using propidium iodide and flow cytometry to determine cell cycle distribution. Approximately 106 cells from each group were harvested with 2.5 mg/mL trypsin, washed twice in PBS, and fixed at 4 °C for 24 hours with cold 80% ethanol, which was pre-chilled at –20 °C. The cells were then rinsed twice in PBS. Then the cell pellet was re-suspended in 100 μL phosphate citrate buffer (1.6% Na2HPO4?12 H2O, 0.07% sodium citrate, pH 7.8) for 15 minutes at room temperature, and before washing in PBS. One milliliter of propidium iodide solution containing RNase A (10 mg/mL) was added and the cells were incubated in the dark for 30 minutes at 37 °C. The percentage of cells in the various phases of cell cycle was measured using a FACStar flow cytometer and analyzed by lysis II and Cellfit software. Apoptotic cells were considered to be part of the sub-G1 population.
DNA damage measurements
DNA single-strand breaks in treated cells were determined by alkali single-cell gel electrophoresis (Comet assay) according to previously published methods[17-18], with some modifications. The assay was based on analysis of labile DNA damage sites where DNA forms characteristic tails (Comet assay). Normal melting agarose (1%), as well as 0.7% and 0.5% low-melting agarose, was prepared in PBS. Initially,  70 μL 7.0% normal-melting agarose was coated on a frosted glass microscope slide. Cells were then suspended in 0.7% low-melting agarose, and 75 μL of this solution was pipetted onto the first layer of the gel. Finally, the third layer of 80 μL 0.5% low-melting agarose was added. The precipitated sandwich gel was incubated in a cooled cell disruption buffer (2.5 mol/L NaCl, 0.1 mol/L ethylenediamine tetraacetic acid,    0.2 mol/L NaOH, 1% Triton X-100 and 10% dimethyl sulfoxide) for 1.5 hours at 4 °C. After rinsing in cooled distilled water, DNA was unwound in an alkali buffer for 20–30 minutes at 4 °C. Electrophoresis was performed at 25 V and 300 mA for 25 minutes at 4 °C. After the slides were neutralized in 0.4 mol/L Tris-HCl buffer (pH 7.5) for 10 minutes at 4 °C, the cells were stained by ethidium bromide.
To prevent additional DNA damage, all steps were conducted under dimmed light or in the dark. The slides were observed and photographed at 200 × magnification using a fluorescence microscope equipped with a 549 nm excitation filter and a 590 nm barrier filter, which was attached to a video camera. Two slides per dose were used, and a total of 150 cells were randomly captured for each dose. DNA damage was represented by percentage of DNA in the tail (% tail DNA) and olive tail moment (OTM), as determined by CASP software.
Western blot analysis
The harvested cells were ruptured with a Sonifer cell disruptor and centrifuged. The supernatant (total protein) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The membrane was blocked with 5% (mass fraction) free-fat milk, incubated with rabbit anti- cyclin D1 polyantibody (1: 400) for 2 hours, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1: 5 000) for 1 hour at room temperature. Proteins were detected using enhanced chemiluminescence. The results were analyzed using chemiluminescent reagent 5500 and Scion image software. The value of density ratio (cyclin D1 or β-actin) represented the level of each subunit protein.
Main outcome measures
Morphological changes in hippocampal neurons following Pb exposure, OTM, percentage of DNA in the tail (% tail DNA), percentage of cells, and cyclin D1 expression were measured.
Statistical analysis
Measurements were expressed as Mean ± SD. Statistical analysis was performed using one-way analysis of variance with Dunnett’s post-hoc test. A value of P < 0.05 was regarded as statistically significant. Each experiment was repeated three times, and the averaged value of three samples was compared among groups.

RESULTS

Pb effects on cell cycle
Cell cycle arrest was observed with increasing doses of Pb (Figure 1). In particular, S-phase cell cycle arrest was prominent in hippocampal neurons. Results from Figure 1 demonstrate that treatment with 0.2 μmol/L Pb to hippocampal neurons did not significantly alter cell phase distribution (sub-G1, S, G0/G1, and G2/M), whereas treatment with 1.0 μmol/L and 10 μmol/L Pb significantly increased the percentage of S and sub-G1 phase cells  (P < 0.05).

Pb effects on DNA damage in hippocampal neurons
Compared with the control group, all Pb-treated groups exhibited smaller neuronal nuclei (Figure 2). The Comet assay indicated that OTM in all Pb-treated groups and that the percentage of tail DNA in 1.0 and 10 μmol/L Pb-acetate groups were significantly greater compared with the control group (P < 0.05). In addition, the percentage of tail DNA in the 0.2 μmol/L Pb-acetate group was enhanced, but the statistical difference did not reach a significant level (Figure 3).

Pb effects on cyclin D1 expression
Results from sodium dodecyl sulfate polyacrylamide gel electrophoresis are illustrated in Figure 4. Following treatment with 0.2, 1.0, and 10 μmol/L Pb-acetate for 24 hours, cyclin D1 expression was decreased compared with the control group.

DISCUSSION

Evidence suggests a strong association between enhanced oxidative damage in the hippocampus and learning and memory impairment as a result of chemicals, such as ethanol and industrial solvents[19-20]. Our previous study demonstrated that Pb exposure results in oxidative damage to hippocampal neurons, characterized by the generation of reactive oxygen species, antioxidant expenditure, and malondialdehyde accumulation[21-22]. Oxidative stress is thought to be involved in DNA damage, which elicits a wide variety of cellular events, such as cell cycle arrest, apoptosis, and necrosis[23-25]. DNA damage plays an important role in disease development, such as hereditary deformities, degenerative diseases, and cancer. Many studies have measured DNA damage due to environmental factors under in vitro conditions using various metabolically incompetent indicator organisms, such as bacteria, yeasts, and mammalian cells[26-29]. The present study measured OTM and DNA percentage in the tail as indices of DNA damage. OTM, which is the multiplication of DNA tail length and DNA percentage in the tail, was used to objectively and sensitively reflect the effect of Pb on DNA damage[30]. The present findings confirmed that Pb-induced DNA damage and OTM was a more sensitive measure than the percentage of DNA in the tail. Correlation analysis revealed a positive correlation between reactive oxygen species formation and OTM level, which suggested that reactive oxygen species might play an important role in DNA damage.
Cyclin D1 synthesis is particularly sensitive to translational repression, which makes it an excellent target for rapid regulation of cell cycle[31]. Cyclin D1 synthesis is a specific rate-limiting regulator of G1 phase progression.
In conclusion, the present study has demonstrated that Pb exposure to primary rat hippocampal neurons disturbs cellular homeostasis, and this is characterized by DNA damage, depletion of cyclin D1, and stagnation of cell cycle progression. Results from this study provide a basis for further studies of Pb neurotoxicity.

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 (Edited by Ni H/Qiu Y/Song LP)