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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (2): 210-227.doi: 10.3736/jcim20120212

• Original Experimental Research • Previous Articles     Next Articles

Potentized homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenite-exposed bacteria Escherichia coli

De Arnab1,Das Durba1,Dutta Suman1,Chakraborty Debrup1,Boujedaini Naoual2,Rahman Khuda-Bukhsh Anisur1()   

  1. 1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
    2. Boiron Laboratory, Lyon, France
  • Received:2011-08-08 Accepted:2011-11-14 Online:2012-02-20 Published:2018-10-09

OBJECTIVE: To examine if potentized homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can reduce sodium arsenite-induced toxicity in Escherichia coli.
METHODS: E. coli were exposed to low arsenite insult after they grew up to log phase in standard Luria-Bertani medium. E. coli were treated with 1 or 2 mmol/L sodium arsenite alone (control), or Ars Alb 30C was added to the medium of a subset of sodium arsenite-treated bacteria (drug-treated), or homeopathically agitated alcohol was added to the medium containing a subset of sodium arsenite-treated bacteria (placebo-treated). A sub-set of untreated E. coli served as the negative control. Glucose uptake, specific activities of hexokinase, lipid peroxidase (LPO), superoxide dismutase (SOD) and catalase, intra- and extra-cellular sodium arsenite content, cell growth, cell membrane potential, DNA damage, intracellular reactive oxygen species (ROS), adenosine triphosphate (ATP) and free glutathione content and expressions of arsB and ptsG gene in normal control, sodium arsenite-treated, drug-treated and placebo-treated E. coli were analyzed. Treatments were blinded and randomized.
RESULTS: In sodium arsenite-treated E. coli, glucose uptake, intracellular ROS, LPO and DNA damage increased along with decrease in the specific activities of hexokinase, SOD and catalase, intracellular ATP and free glutathione contents and cell membrane potential and growth, and there were increases in expression levels of arsB gene and ptsG gene. Ars Alb 30C administration reduced arsenic toxicity in E. coli by inhibiting generation of ROS and increasing tolerance to arsenite toxicity and cell growth.
CONCLUSION: Ars Alb 30C ameliorated arsenic toxicity and DNA damage, validating efficacy of ultra-highly diluted remedies used in homeopathy.

Key words: arsenicals, Escherichia coli, homeopathy, reactive oxygen species, DNA damage, adenosine triphosphate, reverse transcription-polymerase chain reaction

Figure 1

Escherichia coli growth curve under different doses of sodium arsenite Growth curve of Escherichia coli subjected to treatment with different doses of sodium arsenite. The doses of 1 mmol/L and 2 mmol/L were selected from this curve."

"

Primer name Primer sequence (5′-3′)
RT-PCR Real-time RT-PCR
G-3-PDH (house-keeping gene) Forward: CCCACTAACATCAAATGGGG
Reverse: CCTTCCACAATGCAAAGTT
Forward: GAGAACGGGAAGCTTGTCATC
Reverse: CATGACGAACATGGGGGCATC
arsB (arsenic resistance gene) Forward: CTGCACGTCTCACGCTGGGG
Reverse: CGGCGATATCCACCGGCACC
Forward: CCGCCAGCCTGCCGCTTATT
Reverse: CGGCGATATCCACCGGCACC
ptsG (glucose permease gene) Forward: CGGCGCTGACCTGGTTCCTG
Reverse: ACGGAACCGCCTGCTTCTGC
Forward: ACGCTTTGTGCCGATCATTTCTGG
Reverse: AACGCAACTACCGGGTTCTGGTAA

Figure 2

Quantitative data of glucose uptake Glucose uptake (μg/mL per cell) was measured in Escherichia coli cells after 45 min and 90 min, respectively. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. Glucose uptake values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenicum Album 30C."

Figure 3

Data of hexokinase assay Specific activity of hexokinase (μmol/(L·mg·min)) was measured at 45 min and 90 min, respectively, after addition of two different doses of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. The values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenium Album 30C."

Figure 4

Intracellular ATP concentration ATP concentration in Escherichia coli cells (μmol/L per cell) was determined at 45 min and 90 min after exposure of the cells to two different concentrations of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. Intracellular ATP concentration was measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. ATP: adenosine triphosphate; Ars Alb 30C: Arsenium Album 30C."

Figure 5

Analysis of membrane potential by spectroflurimetric method Cell membrane potential of Escherichia coli (mV) was estimated at 45 min and 90 min after exposure of the cells to two different doses of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. Values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenium Album 30C."

Figure 6

Growth of bacteria Growth of Escherichia coli cells (CFU/mL) was determined at 45 min and 90 min after exposure of the cells to two different concentrations of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. Bacterial growth was measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. CFU: colony-forming unit; Ars Alb 30C: Arsenicum Album 30C."

Figure 7

Intracellular arsenic measurement data Arsenic content inside the cell (μg/L per cell) was determined at 45 min and 90 min after exposure of the cells to two different concentrations of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. Arsenic was measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenicum Album 30C."

Figure 8

Extracellular arsenic measurement data Arsenic content in the spent medium (μg/L per cell) was determined at 45 min and 90 min after exposure of the cells to two different concentrations of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. Arsenic was measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenicum Album 30C."

Figure 9

arsB expression analysis by RT-PCR A: Arbitrary band intensities of arsB (arsenic resistance gene) were analyzed against the house-keeping gene (G-3-PDH) under different conditions. (a) and (d) represent the expression of house-keeping gene after 1 mmol/L and 2 mmol/L of sodium arsenite addition, respectively; (b) represents the expression of arsB at 45 min after the addition of 1 mmol/L arsenite; (c) represents the expression of arsB at 90 min after the addition of 1 mmol/L arsenite; (e) represents the expression of arsB at 45 min after the addition of 2 mmol/L arsenite; (f) represents the expression of arsB at 90 min after the addition of 2 mmol/L arsenite. Ln1: Control cells; Ln2: Sodium arsenite-treated cells; Ln3: Sodium arsenite plus placebo-treated cells; Ln4: Sodium arsenite plus Ars Alb 30C-treated cells.B: Quantitative results of arsB expression with 1 mmol/L sodium arsenite exposure; C: Quantitative results of arsB expression with 2 mmol/L sodium arsenite exposure. Bars represent average values of intensity. Values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. RT-PCR: reverse transcription-polymerase chain reaction; G-3-PDH: glyceraldehyde-3-phosphate dehydrogenase; Ars Alb 30C: Arsenicum Album 30C."

Figure 10

ptsG expression analysis by RT-PCR A: Arbitrary band intensities of ptsG (glucose permease gene) were analyzed against the house-keeping gene (G-3-PDH) under different conditions. (a) and (c) represent the expression of house-keeping gene after 1 mmol/L and 2 mmol/L of sodium arsenite addition, respectively; (b) represents the expression of ptsG after the addition of 1 mmol/L arsenite; (d) represents the expression of ptsG after the addition of 2 mmol/L arsenite. Ln1: Control cells; Ln2: Sodium arsenite-treated cells; Ln3: Sodium arsenite plus Ars Alb 30C-treated cells; Ln4: Sodium arsenite plus placebo-treated cells. B: Quantitative results of ptsG expression with 1 mmol/L sodium arsenite exposure; C: Quantitative results of ptsG expression with 2 mmol/L sodium arsenite exposure. Bars represent average values of intensity. Values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. RT-PCR: reverse transcription-polymerase chain reaction; G-3-PDH: glyceraldehyde-3-phosphate dehydrogenase; Ars Alb 30C: Arsenicum Album 30C."

Figure 11

arsB expression tested by quantitative real-time RT-PCR Quantitative real-time RT-PCR analysis of arsB (arsenic resistance gene) was measured at 45 min and 90 min, respectively, after addition of two different doses of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. The values were measured in three independent experiments done in triplicate. Mean ± standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. RT-PCR: reverse transcription-polymerase chain reaction; Ars Alb 30C: Arsenicum Album 30C."

Figure 12

ptsG expression tested by quantitative real-time RT-PCR Quantitative real-time RT-PCR analysis of ptsG (glucose permease gene) was measured after addition of two different doses of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. The values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. RT-PCR: reverse transcription-polymerase chain reaction; Ars Alb 30C: Arsenicum Album 30C."

Figure 13

Assay of lipid peroxidation Specific activity of lipid peroxidase (μmol/(L·mg·min)) was measured at 45 min and 90 min, respectively, after addition of two different doses of sodium arsenite to the media. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. The values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenicum Album 30C."

Figure 14

Catalase assay data Specific activity of catalase (μmol/(L·mg·min)) was measured at 45 min and 90 min, respectively, after addition of two different doses of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. The values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenicum Album 30C."

Figure 15

Superoxide dismutase assay data Specific activity of superoxide dismutase (μmol/(L·mg)) was measured at 45 min and 90 min, respectively, after addition of two different doses of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. The values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenicum Album 30C."

Figure 16

Intracellular glutathione concentration data Intracellular glutathione concentration in Escherichia coli cells (μmol/L per 108 cells) was determined at 45 min and 90 min after exposure of the cells to two different concentrations of sodium arsenite. A: 1 mmol/L sodium arsenite-treated cells; B: 2 mmol/L sodium arsenite-treated cells. Intracellular glutathione concentrations were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control. Ars Alb 30C: Arsenicum Album 30C."

Figure 17

DNA damage analysis by comet assay A: Photographs showing the DNA damage by comet assay. (a) and (h) represent the control cells after 1 mmol/L and 2 mmol/L of sodium arsenite addition, respectively; (b) 45 min after the addition of 1 mmol/L sodium arsenite; (c) 45 min after the addition of 1 mmol/L sodium arsenite and placebo; (d) 45 min after the addition of 1 mmol/L sodium arsenite and Ars Alb 30C; (e) 90 min after the addition of 1 mmol/L sodium arsenite; (f) 90 min after the addition of 1 mmol/L sodium arsenite and placebo; (g) 90 min after the addition of 1 mmol/L sodium arsenite and Ars Alb 30C; (i) 45 min after the addition of 2 mmol/L sodium arsenite; (j) 45 min after the addition of 2 mmol/L sodium arsenite and placebo; (k) 45 min after the addition of 2 mmol/L sodium arsenite and Ars Alb 30C; (l) 90 min after the addition of 2 mmol/L sodium arsenite; (m) 90 min after the addition of 2 mmol/L sodium arsenite and placebo; (n) 90 min after the addition of 2 mmol/L sodium arsenite and Ars Alb 30C.B: Quantitative results of 1 mmol/L sodium arsenite exposure. C: Quantitative results of 2 mmol/L sodium arsenite exposure. Bars represent average values of comet tail lengths. Values were measured in three independent experiments done in triplicate. Mean±standard error of mean was plotted. Significance level at *P<0.05, vs normal control and △P<0.05, vs placebo-treated positive control."

Figure 18

DNA gel electrophoresis Images of DNA gel after electrophoresis containing DNA samples of different treatments. A: 1 mmol/L sodium arsenite-treated cells, (B) 2 mmol/L sodium arsenite-treated cells. Ln1: Control cells; Ln2: Sodium arsenite-treated cells; Ln3: Sodium arsenite plus placebo-treated cells; Ln4: Sodium arsenite plus Ars Alb 30C-treated cells."

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