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Original Experimental Research
Journal of Chinese Integrative Medicine: Volume 9, 2011   Issue 7
Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate
1. Durba Das (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India )
2. Arnab De (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India )
3. Suman Dutta (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India )
4. Raktim Biswas (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India )
5. Naoual Boujedaini (Boiron Laboratory, Lyon, France )
6. Anisur Rahman Khuda-Bukhsh (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India E-mail: prof_arkb@yahoo.co.in, khudabukhsh_48@rediffmail.com)

Objective: This study examines if homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can elicit ameliorative responses in yeast (Saccharomyces cerevisiae) exposed to arsenate.
Methods: The yeast S. cerevisiae 699 was cultured in a standard yeast extract peptone dextrose broth medium. It was exposed to the final concentration of 0.15 mmol/L arsenate for two intervals, 1 h and 2 h, respectively. The cell viability was determined along with the assessment of several toxicity biomarkers such as catalase (CAT), superoxide dismutase (SOD), total thiol (GSH) and glucose-6-phosphate dehydrogenase (G6PDH), lipid peroxidation, protein carbonylation and DNA damage. Reactive oxygen species (ROS) accumulation, expressions of relevant stress transcription activators like Yap-1 and Msn 2, and mRNA expression of yeast caspase-1 (Yca-1) were also measured.
Results: Treatment of arsenate increased lipid peroxidation, protein carbonylation, DNA damage, ROS accumulation and expressions of Yap-1, Msn 2 and Yca-1 and decreased GSH, G6PDH, CAT and SOD. Ars Alb 30C administration decreased lipid peroxidation, protein carbonylation, DNA damage, ROS formation and Msn 2 and Yca-1 expressions and increased cell viability, GSH, G6PDH, CAT and SOD significantly (P<0.05), except for a slight increase in Yap-1 expression.
Conclusion: Ars Alb 30C triggers ameliorative responses in S. cerevisiae exposed to arsenate.

Received January 18, 2011; accepted April 6, 2011; published online July 15, 2011.
Full-text LinkOut at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao.
    
Correspondence: Anisur Rahman Khuda-Bukhsh, PhD, Professor; Tel: +91-33-25828750-315, +91-33-25828282; E-mail: prof_arkb@yahoo.co.in, khudabukhsh_48@rediffmail.com

  

     The use of complementary and alternative medicine (CAM) in the treatment of various health problems is gaining importance in recent years, mainly due to the unavoidable side effects, potential allergic reactions and drug resistance of orthodox medicines. Therefore, the demand exists for alternative medicines, which, besides having potential for alleviating a wide variety of symptoms, are also holistic in nature and do not produce any adverse side effects.
     Among these CAM practices, homeopathy occupies a central position in many countries, particularly in some European and third-world countries. Homeopathy uses many animals, plants, minerals and synthetic substances in a ultrahighly diluted form and is potentised by conventional homeopathic methods1. The use of such ultrahighly diluted remedies, which may not contain even a single molecule of the original drug substance has been the centre of controversy for a long time, because the mechanism of action is yet to be accepted on strong scientific grounds. Skeptics often ridicule the effects of ultrahighly diluted homeopathic remedies to evoke only psychological placebo effects. Therefore, for validation of the scientific basis of efficacy of such ultrahighly diluted remedies, and for understanding its molecular mechanisms, novel approaches are warranted.
     One of the principles of homeopathy is “similia similibus curentur” or “like cures like”2, which means that the symptoms that are produced by repeated feeding of any particular drug in a normal healthy person (drug proving) can also be cured in a diseased person showing the same or similar set of symptoms, by the same drug. In another homeopathic doctrine (isopathy), the ill effects of a crude toxic substance (say, arsenic) can be combated by the ultrahigh dilution of the same substance (ultrahigh dilution of arsenic or potentized arsenic). Potentized Arsenicum Album 30C (Ars Alb 30C) is an ultrahigh dilution of arsenic trioxide, which is generally used when symptoms of arsenic poisoning (like vomiting, diarrhea, thirst, loss of appetite, burning, etc.) are present in the patient3. Arsenic is a semimetal or metalloid that causes several health hazards including cancer. It produces drastic changes in the biochemical and genetic levels, causes DNA damage and generates reactive oxygen species4-6.
     In homeopathy, the use of potentized Ars Alb is routinely made by clinicians against symptoms of arsenic toxicity. It has been experimentally proven by atomic absorption spectrophotometric studies that arsenic molecules are hardly present in the 12th and upward potencies of Ars Alb, though its efficacy as a remedy remains demonstrable7-10. However, in a recent study11, the existence of some nanoparticles of the original drug substance has been demonstrated in potentized homeopathic drugs diluted above Avogadro’s limit. Potentized Ars Alb 30C and 200C have been used against arsenic intoxication in mice and human subjects with satisfactory remedial outcomes3, 7-9, 12. Betti et al13 and Lahnstein et al14 observed positive effects of Ars Alb treatment in wheat seedling growth. However, till date no research has been carried out to test its efficacy on any lower eukaryote except for the lone report published very recently15. However, while these workers used potencies of Ars Alb in decimal dilutions (like 17×, 18×, 24×, 28×, 30×) and studied their effects on growth of Saccharomyces cerevisiae, in the present study we used much higher dilutions in centesimal scale (that is, 1060 times diluted, much above Avogadro’s limit) and used biochemical and molecular parameters for evaluation. Our primary objective was to ascertain whether such an ultrahighly diluted remedy could produce any response in arsenate-exposed S. cerevisiae, one of the simplest and most well-known representatives of eukaryotic cells, which forms a convenient model organism for evaluating toxic effects in human cells and tissues by extrapolation.

 
  

1  Materials and methods
1.1  Strain and growth condition 
The yeast S. cerevisiae 699 (MATa ade2-1 trp1-1 leu2-3, 112 his3-11, 15 ura3 can-100) was procured from Bose Institute, Kolkata, India and maintained as a primary culture on yeast extract peptone dextrose (2% glucose, 1% yeast extract, 2% peptone, 2% agar, YEPD) agar slants. Colonies were randomly selected from this primary culture and used for inoculation into the YEPD broth in several containers. They were grown to log phase (106/mL) with mechanical shaking at 30 ℃ and then subdivided into the following sets. (1) Normal control: cells grown in standard medium without any treatment. (2) Arsenate-treated: cells grown in medium supplemented with 0.15 mmol/L arsenate (H3AsO4, Merck, Germany). This dose was selected through a range-finding trials that showed the minimum inhibitory concentration of H3AsO4 for S. cerevisiae to be 3.5 mmol/L. (3) Positive control: potentized alcohol 30C (70% ethanol succussed and diluted) was supplemented in the medium to the arsenate-exposed cells. (4) Drug-treated: the potentized Ars Alb 30C (prepared in 70% alcohol as vehicle) was added to the arsenate-exposed cells. This drug was freshly prepared and supplied by Boiron Laboratory, Lyon, France.
     All the control and treated groups of cells were then grown in the same physical condition mentioned above. Aliquots of cells from each group (106/mL) were taken out at 1 h and 2 h time points and different sets of experiments were carried out thrice with three replicates (n=9) for each group and their mean values (obtained from each of 9 sets of replicates) were compared for statistical analysis.
     The experimental cultures either treated with the homeopathic remedy or its placebo were coded and not known to the observers (blinded) during observation and scoring of the data. The codes were later deciphered to know which of them actually belonged to which of the groups.
1.2  Cell viability test
1.2.1  Methylene blue staining 
Viability was tested by methylene blue staining. Cells were counted by haemocytometer under a compound microscope. Dead cells took up the blue colour but viable cells did not16.


1.2.2  Methyl thiazolyl tetrazolium assay  Cells from each group were harvested by centrifugation (7 000×g) for 10 min at 4 ℃ and washed twice with potassium phosphate buffer (pH 7.4). MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (1 mg/mL) was added to the cell pellet and incubated for 2 h with frequent shaking. Supernatant was discarded after centrifugation and dimethyl sulfoxide (DMSO) was added to the cell pellet and mixed well by vortex. After centrifugation, supernatant was collected and the optical density (OD) value was measured at 595 nm. The percentage of viable cells was determined by considering 100% viability in control untreated group.
1.3  Preparation of cell extract  Cells were harvested by centrifugation (7 000×g) for 10 min at 4 ℃ and washed twice with potassium phosphate buffer (pH 7.4). The cell pellets were then resuspended in lysis buffer (50 mmol/L tris-hydrochloric acid, 125 mmol/L sodium chloride, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L ethylene diamine tetraacetic acid (EDTA)) and sonicated in cold condition. Cell debris was removed by centrifugation at 15 000×g for 10 min at 4 ℃.
1.4  Molecular and biochemical methods
1.4.1  Evaluation of lipid peroxidation, protein carbonylation, total thiol content and protein concentration 
Lipid peroxidation was analyzed using the method described by Aust17. The amount of malondialdehyde formed in the total reaction was calculated using extinction coefficient 155 mmol/(L·cm). Carbonyl protein content was measured according to the protocol described by Lushchak et al18 and calculated from the absorbance maximum of reaction product 2,4-dinitrophenylhydrazone measured at 370 nm using an extinction coefficient of 22 mmol/(L·cm). For total thiol (GSH) assay, a modified Ellman’s method19 was followed. Cell extract was added to a reaction mixture of sulfosalicylic acid, sodium phosphate buffer (pH 8.0) and deionized water. 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) solution was added and the absorbance was read at 412 nm. GSH content was calculated using an extinction coefficient of 13.6 mmol/(L·cm) for DTNB at 412 nm. Protein concentration was determined by the Coomassie brilliant blue G-250 dye binding method with bovine serum albumin as the standard20.
1.4.2  Assay of catalase, superoxide dismutase and glucose 6 phosphate dehydrogenase activities  Catalase (CAT) and superoxide dismutase (SOD) activities were measured using the methods of Maehly et al21 and Nishikimi et al22, respectively. Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured by the method of Lushchak et al18.
1.4.3  Comet assay  Cells were suspended in 0.7% low-melting agarose containing 2 mg/mL of zymolase 20T23 and layered over a frosted microscopic slide. The slides, immersed overnight in lysis buffer of pH 10.0, were electrophoresed in a buffer containing 300 mmol/L sodium hydroxide and 1 mmol/L disodium EDTA, pH 13.0 for 20 min (300 mA, 20 V). The slides were then washed thoroughly with a neutralizing buffer (Tris 0.4 mol/L, pH 7.5), stained with ethidium bromide (1 mg/mL) and examined under a fluorescence microscope. The extent of DNA breakage was determined by measuring the comet tail length using the software Motic Image, China.
1.4.4  4′,6-diamidino-2-phenylindole staining  Cells were routinely fixed with 70% ethanol and incubated with 4′,6-diamidina-2-phenylindole (DAPI) for 5 min at dark conditions, and then observed under a fluorescence microscope.
1.4.5  Estimation of reactive oxygen species  Cells were washed thoroughly with sterile deionized water and then incubated for 2 h with 10 μg/mL 2′,7′-dichloro dihydrofluorescein diacetate (H2DCF-DA) in dark conditions. The intracellular reactive oxygen species (ROS) were measured by a flow-cytometer with an excitation wave length of 480 nm.
1.4.6  Western blot  Cell lysates from each set, containing equal amounts of protein, were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Taurus Scientific, USA). The membrane was blocked for 3×30 min with 5% nonfat dry milk dissolved in tris buffer saline (pH 7.4). The membranes were then incubated overnight at 4 ℃ with monoclonal antibody for specific protein. Next, the membrane was further incubated for 2 h with goat anti-rabbit IgG as a secondary antibody. The bound antibody was detected by 5-bromo-4-chloro-3-indolyl-phosphate nitroblue tetrazolium (BCIP-NBT). Band intensity was measured by the TotalLab Software (SPSS Data Editor, version 10).
1.4.7  mRNA expression study by reverse transcription-polymerase chain reaction  Total RNA was isolated from yeast cells using TRIzol reagent (Bangalore Genei, India). Total RNA 1 μg was reversely transcribed to cDNA using oligo-dT, dNTP mix, RNase inhibitor and M-MuLV reverse transcriptase (Chromous Biotech, India). The mixture was incubated at 37 ℃ for 1 h and terminated at 95 ℃ for 2 min. Polymerase chain reaction (PCR) was performed with 5 μL of reverse transcription (RT) product in total volume of 20 μL containing 1.5 units of Taq DNA polymerase (Chromous Biotech, India), 10×PCR buffer, 0.2 mmol/L of dNTPs, and 1 μL of each forward and reverse primers for yeast caspase-1 (Yca-1) (Table 1). These primers were procured from Bioserve, India (temperatures for the forward and reverse primers were at 62 ℃ and 60 ℃, respectively). PCR was performed on an automated thermal cycler (Applied Biosystems, USA) for 35 cycles. Amplified cDNA products were separated on 1.5% agarose gel electrophoresis in tris acetate EDTA (TAE) buffer (10 mmol/L Tris, 0.1 mmol/L EDTA, 0.55 mL acetic acid) with 0.5 μg/mL ethidium bromide and visualized under ultra-violate (UV) trans-illuminator and photographed. Densitometry was performed on a negative image using TotalLab Software. Similarly RT-PCR expression of glucose-3-phosphate dehydrogenase (G3PDH) was also studied for data normalization.

 

Table 1  Forward and reverse primer sequences of Yca-1 and G3PDH

Primer

Sequence

Yca-1

Forward: ATGTATCCAGGTAGTGGACGTTACACCTAC

Reverse: CTACATAATAAATTGCAGATTTACGTCAATAGG

G3PDH

Forward: CCCACTAACATCAAATGGGG

Reverse: CCTTCCACAATGCAAAGTT

Yca-1: yeast caspase-1; G3PDH: glucose-3-phosphate dehydrogenas.

1.5  Preparation and source of Ars Alb 30C and placebo 30C  The Ars Alb 30C and placebo 30C (70% ethanol) were prepared by the procedure of homeopathic serial dilutions and agitations as recommended by the French Homeopathic Pharmacopea. This involved a specific type of dilutions accompanied by agitations termed as “succussions/jerks”. A total of 1 mL of 1% arsenic trioxide dissolved in 70% ethyl alcohol (the initial drug substance, called the mother tincture in homeopathy) was diluted with 99 mL of 70% ethanol and the mixture was given 10 mechanical jerks as per the traditional method to produce a potency of 1.1 mL of potency 1 and again mixed with 99 mL of 70% ethanol and agitated similarly to produce potency 2 and so on. The Ars Alb 30C and placebo 30C were procured from Boiron Laboratory, Lyon, France by using 70% ethanol of the same source.
1.6  Statistical analysis  Values were shown as x±sx taken from 9 replicates. Data were analyzed and the significance of difference among different groups was analyzed by one-way analysis of variance. Further, the SNK-q test and Dunnette t test were conducted for analysis of significance of difference between the control group and any drug-treated groups. Results were considered significant at P<0.05 level.

  

2  Results
2.1  Cell viability 
After being exposed to arsenate, the percentage of living yeast cells dropped to 30% compared with the normal control group up to 2 h. Placebo-treated positive control cells also showed reduced viability, whereas significant increase in viable cells up to 5% was noticed in the arsenate plus Ars Alb 30C-treated group (Table 2).

 

Table 2  Cell viability tested by methylene blue staining and MTT assay

(x±sx, %)    

Group

n

Percentage of viable cells

(methylene blue staining)

Percentage of viable cells

(MTT assay)

1 h

2 h

1 h

2 h

Normal control

9

98.270±0.730

98.660±0.667

100.000±0

100.000±0

Arsenate-treated

9

78.417±0.217

73.063±0.130

82.140±0.199

70.310±0.317

Positive control (Arsenate+placebo)

9

78.633±0.376

71.933±0.200

81.540±0.280

70.430±0.296

Drug-treated (Arsenate+Ars Alb 30C)

9

81.567±0.467*

76.240±0.229*

85.977±0.440*

74.467±0.740*

*P<0.05, vs positive control. MTT: methyl thiazolyl tetrazolium.


2.2  Lipid peroxidation, protein carbonylation and GSH content  Malonaldehyde (MDA) formation occurring due to lipid peroxidation reaction provides indirect evidence for lipid peroxidase activity in cells. In this experiment, arsenate-intoxicated yeast cells showed higher extent of MDA formation compared with the normal untreated one. The arsenate plus Ars Alb 30C-treated cells showed significantly decreased MDA content, signifying the extent of lipid peroxidation in those cells close to the normal cells (Table 3). Similarly, carbonylated protein formation was minimal in normal untreated yeast cells, whereas sharp increase occurred after 1 and 2 h of arsenate administration (Table 3). In comparison, the arsenate plus Ars Alb 30C-treated cells revealed significantly lower levels of protein carbonylation. In both the cases, no significant difference was noticed between the arsenate-treated cells and the arsenate plus placebo-treated cells. Exposure to arsenate for 1 and 2 h decreased the free GSH content significantly in yeast cells compared with the normal untreated cells. Similar decrease of free GSH was also found in the arsenate plus placebo-treated cells. However, free GSH content was found to be increased significantly in the cells treated with arsenate plus Ars Alb 30C (Table 3).

 

Table 3  Lipid peroxidation, protein carbonylation and GSH content of different groups

(x±sx)   

Group

n

Lipid peroxidation
(nmol/mg protein)

Protein carbonylation
(ng/mg protein)

GSH content
(μmol/L per 106 cells)

1 h

 

2 h

 

1h

 

2 h

 

1 h

 

2 h

 

Normal control

9

0.041±0.001

0.068±0.001

903.3±3.3

968.6±10.6

2.723±0.020

2.790±0.003

Arsenate-treated

9

0.059±0

0.079±0.001

1718.7±3.6

2580.3±17.6

2.070±0.073

2.260±0.004

Positive control (Arsenate+placebo)

9

0.060±0.001

0.078±0.001

1722.7±6.3

2610.3±11.3

2.100±0.050

2.261±0.003

Drug-treated (Arsenate+Ars Alb 30C)

9

0.045±0.001*

0.073±0.001*

1299.3±0.7*

2271.0±6.1*

2.390±0.037*

2.470±0.028*

*P<0.05, vs positive control. GSH: total thiol.

2.3  CAT, SOD and G6PDH activities  After 1 and 2 h of incubation with arsenate, there was a prominent decrease in CAT and SOD activities in the yeast cells. Cells belonging to the arsenate plus placebo-treated positive control group also showed reduced activity for these two enzymes (Table 4). On the other hand, Ars Alb 30C-treated intoxicated cells showed a significant increase in CAT and SOD activities. In case of G6PDH, yeast cells exhibited reduced activity after arsenate treatment for both 1 and 2 h. The placebo-treated positive control cells also showed similar results. However, there was a significant increase in G6PDH activity in the cells of Ars Alb 30C plus arsenate-treated group (Table 4).

 

Table 4  Activities of CAT, SOD and G6PDH of different groups

(x±sx, U/mg protein)    

Group

n

CAT

SOD

G6PDH

1 h

2 h

1 h

2 h

1 h

2 h

Normal control

9

84.51±0.50

82.83±0.92

12.47±0.23

13.14±0.23

37.4±0.4

38.2±0.9

Arsenate-treated

9

70.51±0.25

65.10+0.25

9.11±0.05

10.53±0.03

20.9±0.1

18.9±0.9

Positive control (Arsenate+placebo)

9

70.67±0.16

64.90±0.17

9.2±0.20

10.57±0.01

20.7±0.3

18.8±1.0

Drug-treated (Arsenate+Ars Alb 30C)

9

77.70±0.60*

72.43±0.26*

10.22±0.22*

12.14±0.02*

28.7±1.2*

23.5±0.4*

*P<0.05, vs positive control. CAT: catalase; SOD: superoxide dismutase; G6PDH: glucose 6 phosphate dehydrogenase.

2.4  DNA damage  DNA damage was studied by single cell gel electrophoresis. The nuclei of the untreated normal cells appeared intact and round in shape under the microscope. However, the yeast cells treated with arsenate for 1 and 2 h showed many cells with damaged nuclei implying fragmented DNA, presenting long comet tails of varying sizes. Similarly, the arsenate plus placebo-treated cells also had long comet tails. On the contrary, the Ars alb 30 C-administered intoxicated cells presented short comet tails, signifying less DNA damage and fragmentation in their nuclei (Figure 1 and Table 5). The results were further verified with DAPI staining. No visible fluorescence was found in the untreated control cells (Figure 2A), signifying the normal level of chromatin condensation. Intense fluorescence, which is an indication of chromosome condensation, was observed in the arsenate-treated and the arsenate plus placebo-treated yeast cells after 1 and 2 h of incubation (Figures 2B and 2C). Lesser intensity of fluorescence was noted in the arsenate plus Ars Alb 30C-treated cells (Figure 2D) which signified a lesser extent of chromosome condensation in these cells.
    

 

Figure 1  Comet assay of cells in different groups (Light microscopy, ×200)

Photographs of ethidium bromide-stained nuclei of control and treated cells under a fluorescence microscope after single cell gel electrophoresis. A: control; B: arsenate; C: arsenate plus placebo; D: arsenate plus Ars Alb 30C.

 

Table 5  Length of comet tails of cells in different groups

(x±sx)    

Group

n

Comet tail length (μm)

1 h

2 h

Normal control

9

79.40±0.30

79.30±0.38

Arsenate-treated

9

146.13±4.84

229.40±1.86

Positive control (Arsenate+placebo)

9

151.53±1.43

231.30±5.30

Drug-treated (Arsenate+Ars Alb 30C)

9

124.10±2.81*

169.03±8.96*

*P<0.05, vs positive control.
    

Figure 2  Chromatin condensation of cells in different groups (Light microscopy, ×400)

Photographs of DAPI-stained cells of different control and treated groups under fluorescence microscope. A: control; B: arsenate; C: arsenate plus placebo; D: arsenic plus Ars Alb 30C. DAPI: 4′,6-diamidino-2-phenylindole.
    
2.5  ROS accumulation  ROS accumulation in cells was determined by fluorescence analysis with fluorescence-activated cell sorting (FACS). A basal level of ROS accumulation was noticed in untreated control cells. After being exposed to arsenate for 1 and 2 h, respectively, intracellular ROS accumulation was increased significantly compared with the normal cells, more so in the 2 h series. The placebo-treated control cells also showed a greater amount of intracellular ROS. In the arsenate-intoxicated cells treated with Ars Alb 30C, ROS generation was found to be at a reduced level (Figure 3).

 

Figure 3  Intracellular ROS accumulation

FACS analysis of intracellular ROS produced by different control and treated yeast cells. A: control; B: arsenate; C: arsenate plus placebo; D: arsenate plus Ars Alb 30C. ROS: reactive oxygen species; FACS: fluorescence-activated cell sorting.


2.6  Expressions of Yap-1 and Msn 2 proteins  Western blot revealed that expression of Yap-1 protein was up-regulated after arsenate exposure for 1 and 2 h. The expression level was somewhat higher in the arsenate plus Ars Alb 30C-treated cells (Figures 4A and 4B). In case of Msn 2, significant increase was noticed after arsenate exposure, whereas the arsenate plus Ars Alb 30C-treated cells showed down-regulation of this protein to a significant extent (Figures 4C and 4D).
    
    

Figure 4  Expressions of Yap-1 and Msn 2 proteins tested by Western blotting

A: Yap-1 expression after 1 h treatment; B: Yap-1 expression after 2 h treatment; C: Msn 2 expression after 1 h treatment; D: Msn 2 expression after 2 h treatment. Ln1: control; Ln2: arsenate; Ln3: arsenate plus placebo; Ln4: arsenate plus Ars Alb 30C.


2.7  Expression of Yca-1 by RT-PCR  Significant increase of Yca-1 expression compared with the normal untreated yeast cells was found after arsenate treatment for 1 and 2 h, but the expression decreased in the arsenate plus Ars alb 30C-administered cells to a significant level (Figures 5A and 5B), while expression of G3PDH housekeeping gene remained alike in each group (Figure 5C).

 

Figure 5  Expression of Yca-1 tested by reverse transcription-polymerase chain reaction

A: Yca-1 expression after 1 h treatment; B: Yca-1 expression after 2 h treatment; C: G3PDH housekeeping gene expression. Ln1: control; Ln2: arsenate; Ln3: arsenate plus placebo; Ln4: arsenate plus Ars Alb 30 C. Yca-1: yeast caspase-1; G3PDH: glucose-3-phosphate dehydrogenase.

  

3  Discussion
     Although there is a body of evidence for how potentized doses of environmental toxins can have biological effects in animal studies24, to the knowledge of the authors, very limited studies have been carried out to demonstrate whether a potentized homeopathic drug diluted beyond Avogadro’s limit can have any modulatory effects in a primitive form of eukaryote like the yeast25. In the present study, the experiments were designed so as to see if the exposure of the budding yeast S. cerevisiae to sub-lethal doses of arsenate generated ROS and subsequent oxidative stress to the organism and also whether the ultra-highly diluted potentized remedy Ars Alb 30C could successfully combat and help the organism to tide over the stress situation to a more favorable state. The overall results obtained in this study would indicate that the homeopathic remedy clearly altered the parameters, significantly enough to demonstrate that the remedy did play a positive role in ameliorating this unfavorable condition to a much better situation. First, the viability of the organism was more than that of the placebo-treated control, and each of the relevant parameters in respect of toxicity and oxidative stress also provided supportive data. The biochemical data indicated strongly that the oxidative stress generated in the yeast was positively modulated by the homeopathic remedy, as compared with the placebo control. There were also concomitant changes in the molecular parameters which would further confirm this contention. It is known that the generation and accumulation of ROS could lead to carbonylation of protein26. In the present study, carbonylation was also found to be reduced along with decrease in ROS in the drug-exposed yeast cells. Similarly, an increase in CAT and SOD activities is known to be associated with the reduction of oxidative damage by scavenging superoxide radicals27, 28. Further, the generation and accumulation of ROS can make G6PDH a target18. In this study, Ars Alb 30C increased the G6PDH activity in the arsenate-intoxicated yeast cells along with a decrease in lipid peroxidation and an increase in GSH content. Similarly, DNA damage and chromatin condensation have been found to occur in lesser extent in the cells administered with Ars alb 30C. These findings were also supported by the expression pattern of the two stress proteins: Yap-1 and Msn 2. The Yap-1 transcription factor is a functional homologue of mammalian AP-1 in S. cerevisiae29, and the expression of several antioxidant genes is up-regulated by Yap-1 under oxidative stress-inducing conditions. Intoxicated cells administered with Ars alb 30C showed up-regulation of Yap-1 and simultaneous down-regulation of Msn 2. In addition, Yca-1, which is known as a yeast meta-caspase30, showed decreased expression in the Ars Alb 30C-administered intoxicated cells, which in turn denoted its inhibitory effect on the arsenate-induced programmed cell death. Besides, the overall cell viability became increased in the Ars Alb 30C-administered intoxicated cells compared with the placebo-exposed ones. Thus from the whole study it can be concluded that Ars Alb 30C, which is an ultra-highly diluted potentized homeopathic remedy, can manifest its effects on yeast cells in spite of the fact that it belongs to a primitive lower eukaryote group devoid of any centrally localized nervous system. Favorable modulation in some biochemical parameters, change of expression of proteins and genes, decreased DNA damage led the authors to believe that the protective measure exerted by homeopathic drug Ars Alb 30C in the yeast S. cerevisiae is due to its possible gene regulatory actions that could provide them the ability of tolerance and to reduce the arsenate stress. Further studies are warranted to understand more precisely how the initial corrective gene action is triggered and how then the cascading actions of gene expressions are regulated. However, in the meantime, extrapolation of the results of this study on yeast and earlier studies carried out on human victims living in arsenic-contaminated areas of West Bengal3 would convincingly speak for the efficacy of Ars Alb 30C in combating arsenic toxicity in a wide variety of organisms. Therefore, this study has implications for those who are at risk of arsenic exposure through groundwater contamination or other means.

  

4  Acknowledgments
     This work was financially supported by a grant sanctioned to Prof. Anisur Rahman Khuda-Bukhsh, Department of Zoology, University of Kalyani, India by Boiron Laboratory, Lyon, France. The authors express sincere thanks to Dr. Philippe Belon, ExDirector, and Dr. N. Boujedaini, Boiron Laboratory, Lyon, France for their encouragements.

  

5  Competing interests
     The authors declare that they have no competing interests.

  
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This article has been cited by other articles ( Within JCIM & JIM )
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2. Phenotypic evidence of ultra-highly diluted homeopathic remedies acting at gene expression level: a novel probe on experimental phage infectivity in bacteria. 2012, 10(4)
3. Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro. 2015, 13(6)

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