Melatonin Protection against Lead-Induced Changes in Human Neuroblastoma Cell Cultures

Abstract

The nervous system is the primary target for low-levels of lead (Pb) exposure and the developing brain appears to be especially vulnerable to Pb neurotoxicity. Chronic low-level Pb exposure causes growth retardation and intellectual impairment. In the present study the protective effect of melatonin during exposure to low-levels of Pb in human SH-SY5Y neuroblastoma cell cultures was assessed. The cells were exposed to Pb (0.01 to 10 μM) for 48 h. Pb inhibited the proliferation of neuroblastoma cells significantly in a concentration-dependent manner. A 50% inhibition (IC₅₀) of cell proliferation was observed at about 5 μM Pb. Pb decreased (16% to 62%) the levels of total cellular glutathione (GSH) in a concentration (0.1 to 10 μM)-dependent manner. Exposure of cells to Pb (5 μM) for 48 h resulted in an eightfold increase in caspase-3 activity and prostaglandin E₂ (PGE₂) level. Pretreatment with melatonin (10 μM) blocked the effects of Pb on GSH content and caspase-3 activity, and showed significant improvement in reducing the level of PGE₂. The results suggest that some of the neurotoxic effects of Pb may be partly mediated by apoptosis and pretreatment with melatonin can prevent these effects. The present study asserts the neuroprotective effect of melatonin in conditions of Pb-induced toxicity in neuroblastoma cell cultures.

Low levels of chronic lead (Pb) exposure results in altered cognitive function in humans and animals (Beck 1992; Cory-Slechta, Pokora, and Widzowski 1992; Silbergeld 1992; Bellinger and Stiles 1993; Regan 1989), primarily targeting the developing nervous system (Reddy and Zawia 2000; Chetty et al. 2001; Basha et al. 2003). Pb decreases the dopamine levels in mesencephalic cells through cell death by necrosis and apoptosis (Scortegagna and Hanbauer 1997). Caspases, a family of cysteine proteases, are an integral part of the apoptotic pathway that triggers a cascade of proteolytic cleavage in mammalian cells (Cohen 1997). Extensive reviews and recent reports confirm the role of caspases in neuronal cell death during normal development and in neuronal assaults (Mandlekar et al. 2000; Emadadul et al. 2003). Caspases are constitutively expressed as inactive proenzymes that get activated by proapoptotic stimuli (Yan, Liu, and Xu 2002). The basic mechanism includes the activation of caspase-3 precursor protein CPP32 by upstream signals such as the release of mitochondrial cytochrome c, cleavage of specific aspartate residues in proteins that finally converge in apoptosis. Hence, inhibitors of caspase-3 activity inherently carry the potential to protect cells through anti-apoptotic mechanisms (Annunziato et al. 2003).

Reactive oxygen species and the peroxidant/antioxidant ratio leading to cellular damage are causes of cell death in Pb toxicity (Hermes-Lima, Pereira, and Bechara 1991; Bechara et al. 1993; Gurer et al. 1999). Glutathione (GSH) is an intracellular antioxidant that scavenges singlet oxygen and hydroxyl radicals and provides a first line of defense in cells exposed to heavy metals (Wu, Murphy, and Chieueh 1994). The state of oxidative stress in different neurodegenerative disorders appears to be multifactorial with the involvement of environmental and metabolic factors ultimately leading to cell death (Pacifici and Davies 1991; Del Rio et al. 1992; Martin, Austad, and Johnson 1996). Loss of GSH and oxidative damage has been suggested to constitute early signaling events in apoptotic cell death (Kane et al. 1993; Sato et al. 1995). It is documented that Pb exposure induces oxidative stress by modulating the GSH metabolism (Gurer and Ercal 2000). Melatonin, a secretory protein of the pineal gland and a metal chelating agent, has been shown to be a neuroprotective agent in reducing the oxidative stress–induced tissue damage caused by heavy metals (Limson, Nyokong, and Daya 1998). Studies of Sainz et al. (1995, 1998, and 2003) demonstrated that melatonin inhibits the programmed cell death in ovarian cells and thymocytes. In the present study we report the protective effects of melatonin on Pb-induced apoptosis and oxidative stress in human SH-SY5Y neuroblastoma cell cultures.

MATERIALS AND METHODS

Chemicals

RPMI-1640 medium and oxaloacetate, pyruvate, and insulin (OPI) were obtained from GIBCO Life Technologies. Fetal bovine serum, penicillin, streptomycin, and phosphate-buffered saline were procured from CELLGRO, Mediatech. The kits were used for the assays of MTT (methylthiazoletetrazolium), cell proliferation (ATCC, Manassas, VA, USA), glutathione (Dojindo Molecular Technologies, Gaithersburg, MA, USA), prostaglandin E₂ (Amersham Biosciences Piscataway, NJ, USA), and caspase-3 (Biovision, Mountain View, CA, USA). All other chemicals purchased from Sigma and Aldrich were of analytical grade.

Cell Culture

Human SH-SY5Y neuroblastoma cells were obtained from the American Type Culture Collection. The cells were grown in RPMI-1640 medium supplemented with 10% (ν/ν) fetal bovine serum, 50 μg/ml penicillin-streptomycin, and OPI (150 μg/ml oxaloacetate, 50 μg/ml pyruvate, and 0.2 U/ml insulin) in a CO₂ incubator maintained at 5% CO₂ and 37°C. The medium was changed every 3 days and the cells were passed at 80% confluence.

Exposure of Cells to Pb Acetate and Melatonin

Human SH-SY5Y neuroblastoma cells were seeded at 2 × 10⁴ cells per well in a 96-well plate. The cells were allowed to attach and grow for 2 days prior to the treatment. The cells were treated with various concentrations of Pb acetate between 0.01 and 10 μM and the IC₅₀ value was determined. The cells were pretreated with various concentrations of melatonin between 1 and 100 μM to determine the effect of melatonin on cell growth. The cells were exposed to 5 μM (IC₅₀) of Pb in the presence or absence of 10 μM melatonin for 48 h and the level of cell proliferation was determined via the MTT reduction assay.

MTT Assay

Cell proliferation was assessed by the MTT reduction assay as per the manufacturer’s instructions. In brief, 2 × 10⁵ cells per well were seeded into 96-well culture plates and allowed to attach. The medium, containing various concentrations of Pb, was added and the cells were incubated for 48 h. At the end of that 48-h period, 10 μl of MTT reagent was added to the culture, which was then incubated in the dark for 4 h at 37°C, followed by cell lysis with the addition of 100 μl of sodium dodecyl sulfate (SDS) based detergent reagent from the MTT kit. The plate was kept at room temperature in dark for 2 h and the relative amount of MTT reduction was determined based on the absorbance at 570 nm using a plate reader.

Determination of Glutathione Level

In brief, 5 × 10⁵ cells were centrifuged and the suspension was washed with phosphate-buffered saline. The suspended cells were lysed with 10 mM HCl and treated with 5% sulphosalycilic acid. The cells were centrifuged at 8000 × g for 10 min and 20 μl of the enzyme working solution, 140 μl of coenzyme working solution, and 20 μl of either one of the standard solution or the sample solution (cells treated with Pb) were added to the supernatant. The plate was incubated at 37°C for 10 min and 20 μl of the substrate, 5,5′-dithiobis(2-nitrobenzoic acid), working solution was added. The plate was incubated at room temperature for 10 min and the optical density was measured at 415 nm using a microplate reader; then the concentration of GSH was calculated using a standard calibration curve. The reagents used throughout the experiment were prepared according to the manufacturer’s instructions.

Caspase-3 Activity Assay

Briefly, the assay is based on the spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate Asp-Glu-Val-Asp (DEVD)-pNA. After Pb treatment of the cells, 3–5 × 10⁶ cells were collected, re-suspended in 50 μl of chilled cell lysis buffer, incubated for 10 min on ice, and then centrifuged at 10,000 × g for 1 min. Fifty microliters of the reaction buffer containing 10 mM dithiothreitol and 5 μl of a 4 mM DEVD-pNA substrate were added to the supernatant and incubated at 37°C. The optical density was measured at 405 nm after 2 h. The relative increase in caspase-3 activity was determined by comparison with the levels for the untreated control.

Determination of Prostaglandin E₂ Level

The reagents used in this procedure were included in the kit contents. Human SH-SY5Y neuroblastoma cells were plated 10⁴ to 10⁵cells/well in a standard 96-well cell culture plate. The plate was incubated in a humidified air/5% CO₂ chamber at 37°C, and the cells were allowed to attach. The cells were treated with 5 μM Pb and the incubation continued for 48 h, followed by the addition of 20 μl of buffer A (2.5% dodecyltrimethyl ammonium bromide in 0.1 M phosphate buffer pH 7.5). The cells were lysed by simple agitation and 50 μl of the lysate was transferred to a goat anti-mouse immunoglobulin (IgG)–coated plate (from the kit) with 50 μl of lysis reagent containing do-decyltrimethylammonium bromide (prepared as described with 0.1 M phosphate buffer, pH 7.5, containing 0.9% bovine serum albumin and 0.5% kathon). In the next step, 50 μl of diluted prostaglandin E₂ (PGE₂) antibody and 50 μl of diluted conjugate (PGE₂ conjugated to horseradish peroxidase) were added to the plate and incubated at room temperature for 1 h. The cells were washed and added to 150 μl of enzyme substrate (3,3′,5,5′-tetramethylbenzidine) provided with the kit. The contents were mixed thoroughly for exactly 30 min and the reaction was stopped by the addition of 100 μl of 1 M sulfuric acid. The optical density was measured at 450 nm within 30 min and the concentration of total cellular PGE₂ was calculated using appropriate standards.

Statistical Analysis

Data were expressed as the mean ± SD of at least four determinations for each group repeated at least three times on different occasions. The statistical analysis was performed using a one-way analysis of variance (ANOVA) and the statistical significance was assumed at p < .05, using Student’s t test.

RESULTS

Human SH-SY5Y neuroblastoma cells were exposed to different concentrations of Pb (0.1 to 10 μM) for 48 h. At low-concentration (0.1 μM), Pb significantly inhibited the cell proliferation as measured by MTT assay. The data show that Pb reduced the cell proliferation from 98% to 32% in a concentration-dependent manner (Figure 1A ). A 50% decrease in cell proliferation was observed at about 5 μM Pb (IC₅₀). The cells exposed to melatonin (1 to 100 μM) for 48 h did not exhibit any significant alterations in the cell proliferation. The protective effects of melatonin on cell proliferation were tested by preincubating the cells with 10 μM melatonin followed by 5 μM Pb for 48 h (Figure 1B ). The data on cell proliferation and morphology showed that melatonin significantly blocked the effects of Pb (Figures 1B and 2). The intracellular levels of GSH were significantly decreased in the cells exposed to Pb for 48 h and the decrease was concentration dependent (Figure 3A ). Maximum decrease (62%) in GSH levels were observed at 10 μM Pb. The GSH levels were not altered when the cells were pretreated with different concentrations of melatonin (1 to 75 μM) alone for 48 h. However, the decrease in GSH levels was significantly reduced when pretreated with melatonin (Figure 3B ). Figure 4 shows the effects of Pb (5 μM) and melatonin (10 μM) and their combination on caspase-3 activity in neuroblastoma cell cultures. Pb but not melatonin increased the caspase-3 activity by eightfold. However, pretreatment with melatonin significantly blocked the Pb-induced increase in caspase-3 activity. The results (Figure 5) show a negligible (42 pg/ml) level of PGE₂ in the control and melatonin-treated cells. However, an eightfold increase (307 pg/ml) in the release of PGE₂ was observed when the cells were exposed to 5 μM Pb. The pretreatment with melatonin significantly reduced the Pb-induced release of PGE₂.

DISCUSSION

Data on toxicity to low levels of Pb in the central nervous system (CNS) is limited, although previous studies reported Pb-induced alterations in both cellular and animal models (Sandhir and Gill 1995; Adonaylo and Oteiza 1999; Huang and Schneider 2004). In the present study, we have investigated the toxicity of low levels of Pb exposure of human neuroblastoma cells in culture and protection by melatonin. Our results suggest that Pb reduces the proliferation of cells in a concentration-dependent manner. Melatonin, which influences the cell differentiation and growth in a number of cell culture systems, has been shown to prevent in vitro and in vivo apoptosis in thymocytes (Sainz et al. 1995; Roth, Rabin, and Agenello 1997; Mayo et al. 1998). Melatonin alone has no effect on the proliferation of neuroblastoma cells. However, melatonin pre-treatment significantly blocked the effect of Pb on cell proliferation. Apoptosis, also known as programmed cell death, is a crucial biological process involved in maintaining development and tissue homeostasis. Inappropriate stimulation or inhibition of apoptosis can lead to several physiological imbalances and disorders (Thompson and Yu 1985). The consequences of apoptosis in Pb-induced damage to brain may reflect in behavioral, motor, and cognitive impairments. Recently, Feng and Zhang (2004) have demonstrated antiapoptotic effects of melatonin in the PC12 cells during amyloid beta protein induced toxicity.

Caspase-3, a member of the caspase family has been shown to be the key effector of apoptosis (Mandlekar et al. 2000). Pb increased the caspase-3 activity, which was blocked when the cells were pretreated with melatonin. An increase in cell death during Pb exposure could be attributed to increased caspase-3 activity and/or induced oxidative stress as evidenced from a significant reduction in GSH levels. Oxidative stress has been proposed to be one of the molecular mechanisms associated with Pb toxicity. Gurer et al. (1998, 2000) have demonstrated that Pb stimulates oxidative hemolysis in erythrocytes by inhibiting the superoxide dismutase, an antioxidative enzyme that is reversed when the cells were pretreated with succimer, a thiol antioxidant as well as a chelating agent. It has also been shown that taurine, an antioxidant, is effective in treating the Pb toxicity (Gurer et al. 1998). From our results it is evident that the cells pretreated with melatonin were resistant to the effects of Pb, suggesting that melatonin acts as neuroprotecive agent in countering Pb toxicity.

Melatonin is known to bind heavy metals leading to increase in intracellular GSH levels through an up-regulation of GSH-synthesizing enzymes (Limson, Nyokong, and Daya 1998; Todoroki et al. 1998; Martin et al. 2002). It is possible that the metal-chelating effect of melatonin reduced cell damage and increased the production of intracellular GSH levels. Earlier reports have demonstrated that melatonin is an effective free radical scavenger (Hardeland 1997; Reiter 1997). At low concentrations, Pb induced the release of significant levels of PGE₂. Bate et al. (2002) have attributed the activation of cycloxygenase and production of PGE₂ as an essential key step in the process that leads to neuronal loss. It is likely that Pb increased the release of PGE₂ through the activation of the cycloxygenase pathway. However, pretreatment of neuroblastoma cells with melatonin did not elicit significant changes in PGE₂ levels. In conclusion, Pb-induced apoptosis and oxidative stress may contribute to its neurotoxicity and this study demonstrates that melatonin pre-treatment may block these effects of Pb. The data affirm the neuroprotective effect of melatonin during Pb-induced neuronal apoptosis.