The effect of steviol on differentiated rat PC-12 cells induced by MPP⁺

 

Antoine Al-Achi, Apoorva Daram, Sirisha Ganapuram,

Shreyas Shridhar Deo, Roobina Didarians

 

ABSTRACT:

Background: The 1-methyl-4-phenylpyridinium ion (MPP+) is a neurotoxin. It inhibits mitochondrial complex I by interfering with oxidative phosphorylation in mitochondria. That causes ATP depletion and eventual cell apoptosis. Objective: This study investigates steviol's effect, a major metabolite of stevia obtained from Stevia rebaudiana, on MPP+-induced apoptosis of differentiated rat adrenal pheochromocytoma (PC-12) cells. Methods: The PC12 cells were differentiated into neuronal cells by adding nerve growth factor and then treated with MPP+ and steviol in different concentrations; twenty-four hours later, two in vitro toxicity assays. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Lactate Dehydrogenase Leakage assays (LDH) were conducted to detect the cells’ viability. Results: The present study demonstrated that steviol caused statistically significant cell damage and inhibited the proliferation of the differentiated rat PC-12 cell line in a dose-dependent manner. Furthermore, the results from MTT and LDH assays complemented the outcome of our experiments that steviol acted synergistically in the presence of MPP+ to increase cell death in differentiated rat PC-12 cells. Conclusion: The data obtained from MTT and LDH assays indicated that steviol acted synergistically in the presence of MPP+ free radicals, increasing cytotoxicity. Both assays revealed that steviol aggravated the cytotoxicity produced by MPP+ in differentiated rat PC-12 cells. However, as the MTT assay does not correspond to mitochondrial dysfunction, it may give a poor estimate of cytotoxicity of steviol as compared to the LDH assay that quantifies the damage to the cell membrane, especially when there are small-scale variations in cellular reduction capacity.

 

 

INTRODUCTION:

Stevia rebaudiana Bertoni, also known as sweet leaf, is a perennial shrub belonging to the Asteraceae family. Stevia herb has sweet-tasting diterpenoid glycosides in its leaves with high sweetness potency (200–300 times sweeter than sucrose).¹ Since the use of sucrose has potentially harmful health consequences, stevia acts as a reasonable replacement for sucrose in formulations.² Stevia has attracted scientific and economic interest due to its sweetness and potential therapeutic properties.³

 

This sweetener is exceptional in having a glycemic index value of zero, zero carbohydrates, and zero calories.⁴ South Americans have been using stevia extracts as sweeteners and for the treatment of diabetes for centuries.^5-7 The S. rebaudiana Bertoni leaf contains several sweet compounds, including stevioside.⁸ Stevioside is a diterpenoid glycoside with hydrophilic properties. Its molecular weight is relatively high (804.9 g/mol), making it difficult for the intestine to absorb. Also, gastric juice and enzymes cannot degrade stevioside, but Bacteroides sp., located in the lower GI tract, can convert stevioside into its metabolite aglycone. Steviol (ent-13-hydroxykaur-16-en-19-oic acid) is diterpenic carboxylic alcohol and is a hydrolysis product of stevia glycosides that can be easily absorbed from the GI tract.⁹ Steviol glycosides have been recognized as non-genotoxic food additives when consumed at the rate of up to 4 mg/kg/day (this amount is based on steviol equivalents) by the joint FAO/WHO Expert Committee on Food Additives. In addition, in 2012, the U.S. Food and Drug Administration classified pure steviol glycosides as "generally recognized as safe."¹⁰ However, in vitro studies on different renal and intestinal cultured cell lines have indicated that stevioside at concentrations greater than 1 mM and steviol at greater than 0.1 mM significantly reduced the viability of the cells.¹¹ Furthermore, cytotoxic activities of nine tested steviol derivatives against different cancer cell lines revealed that two of these nine steviol derivatives exhibited potent cytotoxicity and induced apoptosis.¹² 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a compound that may be accidentally produced during the manufacture of desmethylprodine, a synthetic opioid drug similar to morphine and mephredine. The compound MPTP can also be found in laboratory-made illicit drugs such as heroin. The MPTP is biotransformed to the permanently positively charged quaternary nitrogen metabolite 1-methyl-4-phenylpyridinium (MPP+) in the central nervous system (CNS) through reduction to a tetrahydropyridine and following metabolism by monoamine oxide (MAO-B) inside glial cells. MPP+ is transported selectively into dopaminergic neurons via the dopamine transporter, where it causes deleterious effects by inhibiting the mitochondrial electron-transport chain (complex I), which deprives the neuron of its energy source, adenosine triphosphate (ATP). Also, MPP+ leads to the generation of reactive oxygen species (ROS), which damage macromolecular structures and cause mitochondrial dysfunction. Both effects eventually result in neuronal cell death (apoptosis). In addition, the quaternary nitrogen atom in MPP+ prevents the molecule from crossing the blood-brain barrier and being eliminated from the CNS; this leads to accumulation of MPP+ and accelerates the neurotoxic damage.^13,14

 

Parkinson’s disease is a progressive neurodegenerative disorder.^15,16 It has been suggested that excessive apoptosis of neuronal cells plays a role in various human neurodegenerative diseases. Although the etiopathogenesis has not yet been elucidated for neurodegenerative diseases, studies strongly suggest that mitochondrial damage causes the activation of an apoptotic cascade, which subsequently results in the loss of dopaminergic cells. Therefore, regulation and prevention of an apoptotic cascade may be vital to hinder pathological apoptosis in neurodegenerative disorders. Therefore, MPP+ is an inducer of Parkinsonism.¹⁷ In this study, the rat adrenal pheochromocytoma PC-12 cell line (catalog# ATCC-CRL-1721) was used. The PC-12 cell line is broadly utilized as a dopaminergic neuronal model, showing that when nerve growth factor (NGF) is added, the cells are differentiated to nerve cells. The PC-12 cells respond to NGF by slowly shifting from a proliferating chromaffin/pheochromocytoma cell-like phenotype to a nonproliferating, neurite-bearing sympathetic-like neuron.¹⁸ These cells possess the necessary intracellular substrates for the metabolism, synthesis, and transportation of dopamine. The cell also contains the enzymes essential for the decomposition of dopamine, including monoamine oxidase and tyrosine hydroxylase. The membrane receptors in the PC-12 cell line are very similar to those in the cell line found in dopaminergic neurons located in the midbrain region.¹⁹  The PC-12-Adh (ATCC-CRL-1721.1) cell line had not been tested for differentiation potential in response to NGF treatment by the vendor ATCC, therefore in this study, PC-12 (ATCC-CRL-1721) cells were cultured and differentiated since this cell line had been tested and shown to respond to NGF by induction of the neuronal phenotype. The MPP+ is a mitochondrial complex I inhibitor and causes neurotoxicity and apoptotic death. Therefore, to evaluate the effect of steviol (250 and 414 μM) on MPP+-induced apoptosis, two assays were employed: the lactate dehydrogenase (LDH) leakage assay and the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

 

MATERIALS AND METHODS:

Materials:

The materials used in this project are shown in Table 1.

Table 1.

Item	Specifications
PC-12 cell line	ATCC (VA, USA) # CRL-1721
Steviol hydrated (≥93% purity)	Sigma-Aldrich (Milwaukee, USA)
MPP+ iodide (≥98% purity)	Sigma-Aldrich (Milwaukee, USA)
Nerve growth factor (NGF)	Sigma-Aldrich (Milwaukee, USA)
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich (Milwaukee, USA)
Trypan blue solution	Sigma-Aldrich (Milwaukee, USA)
RPMI-1640 medium	ATCC (VA, USA)
Horse serum inactivate (HS)	Sigma-Aldrich (Milwaukee, USA)
Fetal bovine serum	ATCC (VA, USA)
Pen-Strep 10,000 U/mL solution	Gibco (MD, USA)
Bovine serum albumin	Sigma-Aldrich (Milwaukee, USA)
1N HCl: used in-house product
Phosphate buffered saline	Gibco (MD, USA)
LDH assay kit	Sigma-Aldrich (Milwaukee, USA)
Cell culture laminar hood	Manufactured by Labcon
Inverted microscope	Manufactured by Vista Vision Model#0738866
Microplate reader:	Manufactured by Biotek, Model# Synergy H1
Hemocytometer:	Manufactured by Fisher Scientific
Other equipment and chemicals	Thermo Fisher Scientific (Waltham, MA)

Methods:

1. Cell culture and treatment:

1.1 Complete culture medium preparation:

The PC-12 cells were grown and maintained in the growth medium of 84% RPMI-1640 medium, 10% horse serum (donor–grade heated at 56 °C for 30 minutes), 5% fetal bovine serum, and 1% Pen-Strep.

 

1.1.1 Culturing and maintenance of cells:

PC12 cells vial, previously stored under liquid nitrogen conditions, was thawed by gentle agitation in a water bath at 37 ºC for about 2 minutes; the vial was removed from the water bath as soon as the contents were thawed. All of the operations from this point on were carried out under strict aseptic conditions. The vial contents were transferred to a 50-mL centrifuge tube containing 9 mL complete growth medium. Cells were centrifuged at 203 x g for 11 minutes at room temperature (25°C). The supernatant was discarded, and cells were suspended in a 5-mL complete growth medium. Cell clusters were broken up by gently aspirating cells through a 10 mL syringe outfitted with a 22 gauge (11/2 inch) needle, 4 or 5 times. Then the cells were added to a T-25 flask with a vented cap containing 2 mL of complete culture medium. (To avoid excessive alkalinity of the medium during recovery of the cells, the T-25 flask was placed into the incubator for 15 minutes before the addition of the cells, this step allowed the medium to reach its normal pH of 7-7.6). The cells were incubated at 37 °C, and 5% CO2 and 96% relative humidity (RH). The medium was renewed every other day. The cells grew as small, irregularly shaped, loosely adherent or as multi-cell aggregates floating in the growth media (Images 1 and 2).

 

Image 1. High density PC12 Cells in T-25 flask growing in in clusters (10x magnification).

 

Image 2. Low density PC-12 Cells in T-25 flask growing in clusters (10x magnification).

 

1.1.2 Sub-culturing/plating:

As soon as the cells were at the density of 2.5 x 10⁶ viable cells/mL, the cell suspensions were transferred to a 50-mL centrifuge tube and centrifuged for 11 minutes 203 x g at room temperature (25 °C). The supernatant was discarded, and cells were resuspended in a 5-mL complete growth medium. Cell clusters were broken up by gently aspirating cells through a 10-mL syringe outfitted with a 22 gauge (1½inch) needle, 4 or 5 times. Then the cells were seeded into T-25 flasks with a vented cap in the density of 0.7 x 10⁶ cells/mL. The culture flasks were placed in an incubator at 37 °C. The Medium was renewed every other day.

 

2. NGF stock solution (2 x 10⁴ ng/mL) preparation:

The solution of 10 mL PBS and 50 mg of Bovine Serum Albumin (0.5% W/V) were prepared. First, the solution was filtered through a 0.22 μm filter unit, then 5 mL of the filtered solution was added into the vial containing lyophilized NGF (0.1 mg). Finally, the solution was aliquoted into a single-use polypropylene tube and stored at -20 °C.  

 

3. Differentiating medium preparation containing 100 ng/mL nerve growth factor: The culture media composed of RPMI-1640 medium (97.51%), 1% of Horse Serum (donor-grade, heat inactivated), 1% of Pen-Strep solution, 0.5% of NGF (20,000 ng/mL). The differentiating medium was prepared fresh at the time of the use.

 

 

 

Table 2. Well’s content of the 96-well plates.

Column #	Content
1	Differentiated Cells + Fresh media containing NGF (100 ng/mL) (200 μL)
2	Differentiated Cells + Fresh media containing NGF (100 ng/mL)  (198.32 μL) + S1 (1.68 μL of steviol stock solutions (10 mg/mL to obtain 250 μM steviol/well )
3	Differentiated Cells + Fresh media containing NGF (100 ng/mL)  (197.21 μL) + S2 (2.79 μL of steviol stock solution [10 mg/mL] to obtain 414 μM steviol/well)
4	Differentiated Cells + Fresh media containing NGF (100 ng/mL)  (142.96 μL) + 57.04 μL of MPP+ stock solution [10 mg/mL] to obtain 9.6 mM /well)
5	Differentiated Cells + Fresh media containing NGF (100 ng/mL)  (141.28 μL) + S1 (1.68 μL of steviol stock solutions [10 mg/mL] to obtain 250 μM steviol/well )Cells +  57.04 μL of MPP+ stock solution (10 mg/mL) to obtain 9.6 mM /well)
6	Differentiated Cells + Fresh media containing NGF (100 ng/mL)  (140.17μL) + S2 (2.79 μL of steviol stock solution [10 mg/mL] to obtain 414 μM steviol/well)+ 57.04 μL of MPP+ stock solution (10 mg/mL) to obtain 9.6 mM /well)
7	Fresh media containing NGF (100 ng/mL) (200 μL)
8	Fresh media containing NGF (100 ng/mL) (198.32μL) + S1 (1.68 μL of steviol stock solutions [10 mg/mL] to obtain 250 μM steviol/well )
9	Fresh media containing NGF (100 ng/mL) (197.21μL) + S2 (2.79 μL of steviol stock solution (10 mg/mL) to obtain 414 μM steviol/well)
10	Fresh media containing NGF (100 ng/mL)  (142.96 μL) + 57.04 μL of MPP+ stock solution (10mg/mL) to obtain 9.6 mM/well)
11	Fresh media containing NGF (100 ng/mL)  (141.28 μL) + S1 (1.68 μL of steviol stock solutions (10mg/mL) to obtain 250 μM steviol/well )+  57.04 μL of MPP+ stock solution (10 mg/mL) to obtain 9.6 mM /well)
12	Fresh media containing NGF (100 ng/mL) (140.17μL) + S2 (2.79 μL of steviol stock solution (10 mg/mL) to obtain 414 μM steviol/well)+ 57.04 μL of MPP+ stock solution (10 mg/mL) to obtain 9.6 mM /well)

 

 

4. MPP+ iodide solution (10 mg/mL) preparation:

A 100 mg MPP+ iodide powder was dissolved in a 10-mL culture medium (RPMI-1640) until a solution was affected.

 

5. Steviol stock solution (10 mg/mL) preparation:

A 10-mg of steviol powder was dissolved in 1 mL dimethyl sulfoxide (DMSO) until a solution was affected.

 

6. Methyl tetrazolium assay (MTT):

The rat PC12 cells were seeded in collagen IV coated 96-well plates from a cell suspension containing 5 x 10⁴ cells/mL (10,000 cells per well), a total of 6 wells. The plates were incubated at 37 °C with 5% CO2 for 72 hours to allow cell attachment (Image 3). After 72 hours, the culture media was replaced with the medium containing 100 ng/mL of nerve growth factor. Neurites begin to form 3 days after initial treatment. Therefore, the medium was refreshed every other day for seven days for optimal neurite growth (Image 4). The experiment was carried out on the 8th day. On the 8th day, 96 well plates were taken out of the incubator, and the cells in each well were treated, as shown in Table 2. Cell treatments protocol was followed for rows A through H (Table 2). This procedure was repeated for the six 96-well plates for the MTT assay. All the six 96-well plates were incubated in the incubator at 37 °C with 5% CO2 and 82% RH for 24 hours following the treatments. At the end of the incubation period, the media was removed, and the cells were rinsed briefly with 200 μL PBS. The PBS was aspirated, and 200 μL of fresh differentiating media was added to each well. Immediately 20 μL of MTT stock solutions (containing 5 mg MTT in 1 mL PBS) was added to each well to give a concentration of 1 mg MTT per well. The plates were wrapped in foil and incubated for 3 hours at 37 °C. At the end of the incubation period, the media and MTT solution was aspirated, and 200 μL of DMSO was added immediately to dissolve resulting formazan crystals. The plates were left aside for 20 minutes to dissolve the blue MTT formazan. The absorbance was read at a wavelength of 570 nm for the experimental run and 690 nm for the background on a microplate reader.

 

 

Image 3. PC-12 cells in 96 well plates. 72 hours before treatment (10x magnification).

 

 

 

Image 4. Differentiated PC-12 cells in 96 well plates. 8 days after initial (40x magnification)

 

 

 

 

7. Lactate dehydrogenase leakage assay (LDH):

The rat PC12 cells were treated as described for the MTT assay. The experiment was carried out on the 8th day. On the 8th day, 96 well plates were taken out of the incubator, and the cells in each well were treated as described above (see MTT assay). The above cell treatments protocol was followed for rows A through H. This procedure was repeated for six 96-well plates for the LDH assay. All the six 96-well plates were incubated in the incubator at 37 °C with 5% CO2 and 82% RH for 24 hours following the treatments.  The LDH release in the medium was determined using a commercially available kit from Sigma-Aldrich (TOX7).  At the end of incubation, the well plates were transferred from the incubator into the laminar hood. The media of each well was collected into an individual Eppendorf tube. The Eppendorf tubes were centrifuged at 250 x g for 4 minutes to pellet the cells. A 25 μL of the supernatant from each Eppendorf was transferred into clean flat-bottom plates and proceed with the enzymatic analysis. A 50 μL of the LDH assay mixture was added to each well. The vessel was covered with aluminum foil to protect from light, and then incubated at room temperature for 20 minutes. The 1/10th volume of 1 N HCl was added to each well to terminate the reaction. Absorbance was measured spectrophotometrically at a wavelength of 490 nm. The background absorbance of the multi-well plates was measured at 690 nm.

 

8. The effect of DMSO on the PC12 cells:

8.1 Methyl tetrazolium assay (MTT):

The rat PC12 cells were prepared as described for the MTT assay. The experiment was carried out on the 8th day. On the 8th day, 96 well plates were taken out of the incubator, and the cells in each well were treated, as shown in Table 3. Cell treatments protocol was followed for rows A through H (Table 3). This procedure was repeated for three 96-well plates for the MTT assay. All the three 96-well plates were incubated in the incubator at 37 °C with 5% CO2 and 82% RH for 24 hours following the treatments. At the end of the incubation period, the media was removed, and the cells were rinsed briefly with 200 μL PBS. The PBS was aspirated, and 200 μL of fresh differentiating media was added to each well. Immediately 20 μL of MTT stock solutions (containing 5 mg MTT in 1 mL PBS) was added to each well to give a concentration of 1 mg MTT per well. The plates were wrapped in foil and incubated for 3 hours at 37 °C. At the end of the incubation period, the media and MTT solution was aspirated, and 200 μL of DMSO was added immediately to dissolve resulting formazan crystals. The plates were left aside for 20 minutes to dissolve the blue MTT formazan. The absorbance was read at a wavelength of 570 nm for the experimental run and 690 nm for the background on a microplate reader.

 

8.2 Lactate dehydrogenase leakage assay (LDH):

The rat PC12 cells were processed as shown above (MTT assay). The experiment was carried out on the 8th day. On the 8th day, 96 well plates were taken out of the incubator, and the cells in each well were treated as described above (see LDH assay). The above cell treatments protocol was followed for rows A through H (Table 3; similar to MTT assay shown in section 8.1). In this study, the effect of DMSO on the PC-12 differentiated cells is examined. Steviol is readily soluble (10 mg/mL) in DMSO. DMSO is potentially cytotoxic; hence, to rule out its cytotoxicity on the differentiated PC-12 cells, a separate experiment was designed for that purpose. As noted above, the advantage of conducting this experiment was to evaluate the impact of steviol on the cells’ viability in the presence of DMSO.

 

Table 3. Well’s content in the 96-well plates.

Column	Content
1	Differentiated Cells + Fresh media containing NGF (100 ng/mL) (200 μL)
2 to 6	Differentiated Cells + Fresh media containing NGF (100 ng/mL)  (198.32 μL) + S1 (1.68 μL DMSO; equal to the volume of steviol solution in concentration of 250 μM steviol/well)
8	Differentiated Cells + Fresh media containing NGF (100 ng/mL) (200 μL)
7 and 9 to 12	Differentiated Cells + Fresh media containing NGF (100 ng/mL)  (198.32 μL) + S1 (2.79 μL DMSO; equal to the volume of steviol solution in concentration of 414 μM steviol/well)

 

9. Statistical analysis of data:

Statistical evaluation of the data was performed by analysis of variance test (ANOVA) using JMP^Ò Statistical Discovery Software (Version 14.0; SAS Institute, Cary, NC). In addition, Tukey-Kramer HSD post-hoc test was performed after a statistically significant ANOVA. A p-value less than 5% was considered for statistical significance.

 

RESULTS AND DISCUSSION:

This current study aimed to demonstrate the effect of steviol in the presence of MPP+ in differentiated PC-12 cells. We used steviol at concentrations of 250 and 414 μM. These were concentrations within the IC50 range reported by Chen et al.²⁰ The 1-methyl-4 phenylpyridinium ion (MPP+) is a widely used neurotoxin because it causes severe Parkinson’s disease-like syndrome and apoptotic cell death. Differentiated PC-12 cells were exposed to 9.6 mM MPP+ in both the presence and the absence of steviol. In addition, cytotoxicity assays (MTT and LDH) were employed to determine cell viability and mortality. The results obtained from the MTT and LDH assays indicated that there were differences between the groups. Furthermore, all the groups were statistically significantly different from the control (P < 0.0001).

MTT assay:

Differentiated PC-12 cells were exposed to the following conditions (control, steviol 250 μM, steviol 414 μM, MPP+ 9.6 mM, MPP+ 9.6 mM with steviol 250 μM, and MPP+ 9.6 mM with steviol 414 μM). After 24 hours, an MTT assay was performed on the six 96-well plates. The MTT assay is a cell viability assay often used to determine cytotoxicity following exposure to toxic substances. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water-soluble tetrazolium salt converted to insoluble purple formazan by healthy cells. Succinate dehydrogenase inside the mitochondria cleaves the tetrazolium ring. The formazan product is impermeable to the cell membranes, and therefore it accumulates in healthy cells.²¹ The solubilized formazan product absorbance was read at 570 nm against a background of 690 nm using the microplate spectrophotometer system (Synergy H-1). Higher absorbance readings indicated higher survival. The absorbance results were subtracted from the background and then statistically analyzed by ANOVA (Figure 1), followed by post-hoc Tukey-Kramer test (Table 4).

 

LDH assay:

Differentiated PC-12 cells were exposed to the following conditions (control, steviol 250 μM, steviol 414 μM, MPP+ 9.6 mM, MPP+ 9.6 mM with steviol 250 μM, MPP+ 9.6 mM with steviol 414 μM). After 24 hours, the LDH assay was performed on the six 96 well plates.  The cytotoxicity induced by steviol (215 and 414 μM) and MPP+ (9.6 mM) was assessed by LDH leakage into the culture media. The assay is based on the conversion of lactate to pyruvate in the presence of LDH with a reduction of NAD (Nicotinamide adenine dinucleotide). The formation of LDH indicates the cells’ mortality due to the loss of membrane integrity. Therefore, the higher the LDH release, the greater the cell death.²¹ The released LDH absorbance was recorded at 490 nm against a background of 690 nm using the microplate spectrophotometer system (Synergy H-1). Higher absorbance indicated higher numbers of damaged cells. The absorbance results were subtracted from the background and then analyzed by ANOVA (Figure 2), followed by post-hoc Tukey-Kramer test (Table 5) using JMP^Ò Pro software (version 14.0).

 

Figure 1. One-way Analysis of Difference of Absorbance at 570 nm and 690 nm (MTT assay) By Treatment. MTT assay results; R square = 0.820209; p < 0.0001.

Figure 2. One-way Analysis of Difference of Absorbance at 490nm and 690nm (LDH assay) By Treatment. LDH assay results; R square = 0.87071; p < 0.0001.

 

Table 4. MTT Assay: Statistical Comparisons (Tukey Kramer HSD)

Table 5. LDH assay: Statistical comparisons (Tukey-Kramer HSD)

Taken together, the results from the MTT and LDH assays showed that the absence of cells in the sample produced a background baseline reading (absorbance values close to zero). When cells alone are present in the media, the values for absorbance represent a point of natural growth, where the cells were living in harmony with their environment. The addition of DMSO to the cells has caused a slight disturbance from the state of balance, as shown from the MTT assay. The LDH results indicated that DMSO did not cause lysis to cells (in fact, DMSO is used to protect cells from damage caused by freezing); however, a portion of the cells was affected by it, and thus, a lower number of cells were able to secrete normal level of LDH, and therefore, the level of LDH dropped slightly from that expected at optimal natural harmony (cells + media). The addition of steviol to the media resulted in a dose-dependent killing of cells (higher LDH and lower levels of the blue formazan product; steviol at 414 mM vs. 250 mM). This finding is similar to the results demonstrated by Boonkaewwan et al.,^11,22 which indicated that steviol decreased cell viability at concentrations of 200–800 μM. The cytotoxicity of MPP+ (9.6 mM) was evident in both assays. Its cytotoxicity was of the same magnitude as steviol (MTT assay: similar to 250 mM steviol; LDH assay: identical to 414 mM steviol). These findings suggested that steviol induced apoptosis at lower concentrations and cell lysis at higher concentrations, both of which led to cell death. The result that MPP+ is cytotoxic agrees with the documented neurotoxicity of MPP+ reported in the literature.¹⁹

 

Moreover, this observation can be explained by the nature of each assay. The MTT assay is mainly based on the enzymatic conversion of MTT in the mitochondria. In contrast, the LDH assay is based on the release of the enzyme into the culture medium after cell membrane damage.²¹ Thus, the concurrent presence of MPP+ (9.6 mM) with steviol (250 mM or 414 mM) acted synergistically and ultimately killed all cells present in the media.

 

Steviol has been studied for its anticancer potential. The IC50 values reported by Chen et al. were 200–800 μM (100% purity).²⁰ However, steviol has not been studied for its neuroprotective or neurodegenerative properties at the IC50 of 200–800 μM in the presence of MPP+.

 

The cell morphology was changed from neuron phenotype (Image 5) to spherical after 24 hours of treatment with steviol 250 and 414 μM (Images 6-10). This was a visual indication of cell death. However, it should be noted that despite these findings on differentiated PC-12 cells in culture, recent evidence presented by Chappell et al. showed that genotoxic and carcinogenic activities for steviol glycosides were lacking.²³

 

Image 5.  Cells + media 24 hours after incubation in 96 well plates (10x magnification).

 

Image 6. Cells + media + 250 μM steviol 24 hours after treatment in 96 well plates (10x magnification).

 

 

Image 7.  Cells + media +414 μM steviol 24 hours after treatment in 96 well plates (10x magnification).

 

 

Image 8. Cells+media+9.6 mM MPP⁺ 24 hours after treatment in 96 well plates (10x magnification).

 

 

Image 9. Cells+media+9.6 mM MPP⁺ + Steviol 250 μM 24 hours after treatment in 96 well plates (10x magnification).

 

 

Image 10. Cells+media+9.6 mM MPP⁺+Steviol 414 μM 24 hours after treatment in 96 well plates (10x magnification).

 

CONCLUSIONS:

The data obtained from MTT and LDH assays have indicated that steviol acts synergistically in MPP+, increasing its cytotoxicity. Also, the MTT and LDH assay data showed that DMSO did not have statistically significant cytotoxicity on the cells at the concentrations used in this study.

 

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Accepted on 16.05.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):4859-4866.