Natural compounds attenuate heavy metal-induced PC12 cell damage

Materials

PC12 cells (rat adrenal pheochromocytoma cells) were obtained from American Type Culture Collection (ATCC, USA). Roswell Park Memorial Institute 1640 (RPMI-1640) medium and foetal bovine serum (FBS) were also purchased from ATCC and AlamarBlue cell viability assay reagent (DAL1100) was obtained from Thermo Fisher Scientific, USA. (H2DCFDA). 2′,7′-Dichlorofluorescin diacetate (H₂DCFDA), a chemically reduced form of fluorescein used as an indicator for ROS production, was purchased from Sigma-Aldrich, USA. The Annexin V-FITC apoptosis staining/detection kit with propidium iodide staining solution, was supplied by BD Biosciences, USA.

Antibodies against the mitochondrial proteins, Bax, Bcl-2, Cytochrome C, Caspase-3 and β-actin, were obtained from Santa Cruz Biotechnology, Santa Cruz, USA. ABTS (2,2’-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]), metal salts and small molecules were purchased from Sigma-Aldrich, USA. All other chemicals used in this study were all analytical grade.

Cell culture, viability and test substances

PC12 cells were cultured in RPMI-1640 medium supplemented with 5% (v/v) FBS in a humidified incubator 5% CO₂ atmosphere at 37°C. The culture medium was changed after cell density had reached 3 × 10⁶ cells/ml. The cell viability of the PC12 cells was evaluated using AlamarBlue cell viability assay methods according to the manufacturer’s instructions.

For sub-culturing, approximately 1 × 10⁴ cells were placed into 96-well plates. Cells were incubated with 100 μl metal salts or natural compounds for 24 hours. The four metal salts, at concentrations of 10, 25, 50, 100, 200, 400, 600, 1000 and 1500 µM, were: cadmium chloride (CdCl₂), mercuric chloride (HgCl₂), cobalt chloride (CoCl₂) and lead chloride (PbCl₂). The six natural compounds, at concentrations of 5, 20, 80, and 320 µg/ml, were: caffeine, gallic acid, resveratrol, epigallocatechin gallate (EGCG), L-ascorbic acid and alpha tocopherol (Vitamin E).

Following incubation with the test substances, the cells were incubated for three hours with 10 μl AlamarBlue reagent. The absorbance was detected at 570nm by a microplate reader. PC12 cells without chemicals were used as the control group and cell viability results were expressed as percentage of control. Half-maximal inhibitory concentration (IC₅₀) for each metal salt was obtained by fitting the cell viability curves to the Hill equation. ¹³

Cell apoptosis, necrosis and ROS production

PC12 cells were incubated with relative IC₅₀ concentrations of metal salts to determine cell damage. CdCl₂, HgCl₂, CoCl₂ and PbCl₂ at 500, 300, 100 and 130 μM, respectively, were incubated with PC12 cells for 24 hours. Thereafter, PC12 cells were washed twice using FBS and the samples were dyed with propidium iodide. The apoptotic and necrotic rates were measured using Annexin V-FITC apoptosis detection kit. The cells were assessed by fluorescence-activated cell sorting (FACS) using the Cell Quest software (BD, Pharmingen).

ROS production was evaluated using H₂DCFDA fluorescence dye as described previously. ¹⁴ PC12 cells were sub-cultured into 6-well plates. Cells were incubated with test substances for 24 hours after which time μM H₂DCFDA was added for 30 minutes in the dark. All samples passed through a 40 μm cell strainer before being loaded into FACS flow cytometry (Calibar; BectonDickinson) quantified at least 1 × 10⁴ cells for each sample.

Antioxidant activity assay

The ABTS method, which is a spectrophotometric technique, was used to evaluate the free radical scavenging abilities of the six natural compounds. ¹⁵ ABTS stock solution was prepared by reacting equal volumes of 7 mM ABTS solution with 2.45 mM potassium persulfate solution. The mixture was mixed and kept in dark for 16 h at room temperature. Before use, the stock solution was diluted with ethanol to give an absorbance of 0.70 ± 0.02 at 734 nm. The test samples (10 μl) at different concentrations were added to 1ml ABTS working solution. The control was the ABTS solution without any test sample. After mixing the samples for 5 minutes, the absorbance (A) of the resulting solution at 734 nm was measured. Inhibition of ABTS radical was calculated using the following equation: ¹⁵

ABTS scavenging effect ( % ) = [ ( A control − A sample ) × 1 00 / A control ]

Western blot assay

PC12 cells were incubated with 130 µM lead chloride and 80 µg/ml EGCG for 24 hours. The cells were collected and western blot performed as previously described. ¹⁶ RIPA buffer containing protease inhibitors was used to collect cells. Samples with equal amounts of proteins were separated on 12% SDSPAGE then the separated proteins were blotted onto PVDF membrane, and probed with selective antibodies to mitochondrial proteins (i.e., Bax, Bcl-2, Cytochrome C, Caspase-3 and β-actin) and incubated for one hour. The intensity of the optical bands was quantified using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, USA).

Metal salts

Cell viability

Metal salts (CdCl₂, HgCl₂, CoCl₂ and PbCl₂) were added to PC12 cells in concentrations up to 1500 μM and dose-dependent decreases in cell viability were observed in all test groups (Figure 1A–1D). Compared with the other metals, the steepest cell viability curve was observed for CoCl₂ and the flattest for CdCl₂. Likewise, the IC₅₀ for CoCl₂ was 97 μM and for CdCl₂ was 525 μM (Figure  1E).

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Figure 1.

The viability of PC12 cells exposed to various metal salts using the AlamarBlue cell viability assay.

Cell apoptosis, necrosis and ROS production

PC12 cell apoptosis and necrosis were mea sured by Annexin V and propidium iodide double straining techniques. Significant cell apoptosis and necrosis were observed in the metal test groups whereas these two events were rarely seen in the control group (Figure 2A).

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Figure 2.

PC12 cell damage induced by metal salts.

PC12 cells exposed to CdCl₂ and PbCl₂ were the most sensitive to cell apoptosis compared with the other metals. The CdCl₂ and PbCl₂ groups generated approximately 30% apoptotic cells which was approximately two-fold higher than cells incubated with HgCl₂ or CoCl₂ (Figure 2C).

Cell necrosis did not correlate with apoptosis. CoCl₂ exposure generated most necrotic cells followed by CdCl₂ and HgCl₂ (Figure 2D).

ROS positive cells were quantified by using the fluorometric probe H₂DCFDA. Its fluorescent intensity is proportional to intracellular ROS production.^17,18 In this study, ROS production varied across the different metal groups (Figure 2B). It strongly increased to about 40% when PbCl₂ was added to PC12 cells (Figure 2E). Values for the CdCl₂ and HgCl₂ groups were significantly different from the control group but the percentage of ROS positive cells in CoCl₂ group was the lowest and there was no difference from controls (Figure 2E).

Natural compounds

Antioxidant and cell viability

The natural compounds selected (Figure 3A) showed potential antioxidant activity in a dose-dependent manner in the ABTS assay (Figure 3B). Compared with the other natural compounds, EGCG and L-Ascorbic acid showed the strongest effect on inhibiting ABTS+, which may be related to their high hydroxyl content. By contrast, caffeine and alpha tocopherol (Vitamin E), which lack hydroxyl in their structure, were found to be the weakest of all tested substances on ABTS+ scavenging.

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Figure 3.

Natural compounds.

The results of cell viability studies showed that PC12 cells were not affected by any of the natural compounds up to 80 μg/ml. However, with the exception of resveratrol, all natural compounds at 320 μg/ml significantly reduced cell viability compared with controls. Therefore 80 μg/ml was selected as the dose for natural compounds when used for co-incubation with metal salts.

Metal salts combined with natural compounds

Cell apoptosis, necrosis and ROS production

PC12 cells were incubated with IC₅₀ concentration of the four metal salts (CdCl₂, HgCl₂, CoCl₂ and PbCl₂) and 80 μg/ml of all six natural compounds for 24 hours to investigate any potential influence on cell damage (Figures 4A–D). While all six natural compounds tested attenuated cell damage to some extent, EGCG and L-ascorbic acid appeared to be the most effective substances in protecting PC12 cells.

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Figure 4.

PC12 cell damage (apoptosis, necrosis and ROS positive cells) after natural compounds were combined with metal salts.

Western blot analysis

The expression of mitochondrial apoptosis-related proteins was evaluated using Western blot analysis. EGCG protected against lead-induced cell damage (Figure 5A–D). The expression of Bax was upregulated in the lead group compared with the control group. By contrast, the level of Bcl-2 was downregulated in the lead group compared with the control group, resulting in an increase in the ratio of Bax to Bcl-2 which was reduced by the addition of ECOG (Figure 5B). In addition, compared with the control group, there was an obvious activation of intracellular cytochrome C and activation of caspase-3 in the cells incubated with PbCl₂and again these were reduced by the addition of ECOG (Figure 5C and D).

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Figure 5.

(A) Effects of lead chloride [PbCl₂] and epigallocatechin gallate (EGCG) on the expression levels of the Bax, Bcl-2, cytochrome C and caspase-3 determined via Western blot analysis.

Hierarchical clustering

PCA analysis was used to investigate the most influential factors in the data sets.^19,20 EGCG and L-ascorbic acid were located within the lower segment of the PCA analysis both in the cell apoptosis and necrosis indicating potential similar mechanisms for those two compounds against metal salts damage (Figure 6A and 6B). Caffeine, resveratrol and alpha tocopherol (Vitamin E) were closely distributed in the analysis of ROS production (Figure 6C).

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Figure 6.

Principle component analysis. PC1, first principal component, PC2, second principal component. The percentages on the x and y axes denote the amount of variance in the dataset described.