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Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (6): 397-404.doi: 10.3736/jintegrmed2013054

• Research Article • Previous Articles     Next Articles

Homeopathic Thuja 30C ameliorates benzo(a)pyrene-induced DNA damage, stress and viability of perfused lung cells of mice in vitro

Avinaba Mukherjeea, Naoual Boujedainib, Anisur Rahman Khuda-Bukhsha()   

  1. a. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India
    b. Boiron Laboratories, Lyon, France
  • Received:2013-06-06 Accepted:2013-08-20 Online:2013-11-10 Published:2013-11-15

Objective

To examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo(a)pyrene (BaP)-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro.


Methods

Perfused normal lung cells from mice were cultured in 5% Roswell Park Memorial Institute medium and exposed to BaP, a potent carcinogen, at the half maximal inhibitory concentration dose (2.2 μmol/L) for 24 h. Thereafter, the intoxicated cells were either treated with Thuja 30C (used against tumor or cancer) or its vehicle media, succussed alcohol 30C. Relevant parameters of study involving reactive oxygen species (ROS) accumulation, total glutathione (GSH) content, and generations of heat shock protein (hsp)-90 were measured; the cell viability and other test parameters were measured after treatment with either Thuja 30C or its vehicle media. Circular dichroism spectroscopy was performed to examine if Thuja 30C directly interacted with calf thymus DNA as target. For ascertaining if DNA damaged by BaP could be partially repaired and restituted by the remedy, 4′,6-diamidino-2-phenylindole staining was performed.


Results

Thuja 30C increased cell viability of BaP-intoxicated cells significantly, as compared to drug-untreated or drug-vehicle control. A minimal dose of Thuja 30C significantly inhibited BaP-induced stress level, by down-regulating ROS and hsp-90, and increasing GSH content. Thuja 30C itself had no DNA-damaging effect, and no direct drug-DNA interaction. However, it showed quite striking ability to repair DNA damage caused by BaP.


Conclusion

Thuja 30C ameliorates BaP-induced toxicity, stress and DNA damage in perfused lung cells of mice and it apparently has no effect on normal lung cells.

Key words: Thuja 30C, Benzo(a)pyrene, Reactive oxygen species, Oxidative stress, HSP90 heat-shock proteins, In vitro

Figure 1

Effects of Thuja 30C on the viability of BaP-intoxicated mice lung cellsCells were either treated with only Thuja 30C (7 μL per 100 μL culture media) or exposed to 2.2 μmol/L BaP. BaP-exposed cells were simultaneously treated with drug vehicle media (7 μL per 100 μL culture media) and Thuja 30C (1 μL per 100 μL to 7 μL per 100 μL culture media). After 24 h of treatment cell viability was determined by MTT assay. Data are expressed as percentage of viability and each point expressed as mean ± standard deviation (n = 6). **P < 0.01, vs untreated group.BaP: benzo(a)pyrene; MTT: 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenylotetrazolium bromide."

Figure 2

Effects of Thuja 30C treatment on ROS generation ROS generation was quantified fluorimetrically after staining with 2′,7′-dichlorofluorescein diacetate. Data are expressed as percentage of untreated control and expressed as mean ± standard deviation (n = 6). △△P < 0.01, vs BaP group.BaP: benzo(a)pyrene; ROS: reactive oxygen species."

Figure 3

Effects of Thuja 30C treatment on GSH level GSH level was quantified enzymatically. Data are expressed as percentage of untreated control and expressed as mean ± standard deviation (n = 6). △△P < 0.01, vs BaP group.BaP: benzo(a)pyrene; GSH: glutathione."

Figure 4

Effects of Thuja 30C treatment on expression of hsp-90 at protein level Significant hsp-90 upregulation was observed after BaP intoxication. After simultaneous treatment with Thuja 30C, level of hsp-90 was down-regulated. Data are expressed as percentage of untreated control and each point expressed as mean ± standard deviation (n = 6). △△P < 0.01, vs BaP group.BaP: benzo(a)pyrene; hsp: heat shock protein."

Figure 5

Effects of Thuja 30C on DNA damage through interactionThuja 30C-DNA interaction study was done by circular dichroism spectral analysis. DNA damage capacity of the drug itself by interacting with it, if any, was determined by circular dichroism spectroscopy. Uppermost curve in black indicated untreated calf thymus DNA and the curve in green (A) was drug vehicle-treated and red (B) was Thuja 30C-treated."

Figure 6

DNA nicks analysis after DAPI stainingDNA nicks generation was indicated by DAPI. White arrows indicate nicked DNA of the cells.BaP: benzo(a)pyrene; DAPI: 4′,6-diamidino-2-phenylindole."

Figure 7

Proposed mechanistic action of Thuja 30C in combating BaP-induced lung cell toxicityBaP: benzo(a)pyrene; ROS: reactive oxidative species; GSH: glutathione; hsp: heat shock protein."

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