Search JIM Advanced Search

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


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.


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.


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.


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."

[1] Khuda-Bukhsh AR . Laboratory research in homeopathy: pro[J]. Integr Cancer Ther, 2006,5(4):320-332
doi: 10.1177/1534735406294794
[2] Kassab S, Cummings M, Berkovitz S, van Haselen R , Fisher P.Homeopathic medicines for adverse effects of cancer treatments[J].Cochrane Database Syst Rev, 2009,(2):CD004845
[3] Khuda-Bukhsh AR . Potentized homeopathic drugs act through regulation of gene-expression; a hypojournal to explain their mechanism and pathways of action in vitro[J]. Complement Ther Med, 1997,59(1):43-46
[4] Boericke W . Pocket manual of homeopathic materia medica and repertory[M]. Delhi: B. Jain Publishers, 2004, 643-645
[5] Gaskin A . Comparative study on Kent’s materia medica[M]. Delhi: B. Jain Publishers, 2005,205
[6] Naser B, Bodinet C, Tegtmeier M, Lindequist U . Thuja occidentalis(Arbor vitae): a review of its pharmaceutical, pharmacological and clinical properties[J]. Evid Based Complement Alternat Med, 2005,2(1):69-78
doi: 10.1093/ecam/neh065
[7] Sunila ES, Hamsa TP, Kuttan G . Effect of Thuja occidentalis and its polysaccharide on cell-mediated immune responses and cytokine levels of metastatic tumor-bearing animals[J]. Pharm Biol, 2011,49(10):1065-1073
doi: 10.3109/13880209.2011.565351
[8] Frenkel M, Mishra BM, Sen S, Yang P, Pawlus A, Vence L, Leblanc A, Cohen L, Banerji P, Banerji P . Cytotoxic effects of ultra-diluted remedies on breast cancer cells[J]. Int J Oncol, 2010,36(2):395-403
[9] Banerji P, Banerji P. Homeopathy: treatment of cancer with the Banerji protocols. In: Bhattacharya A. A compendium of essays on alternative therapy. Rijeka: InTech Europe. 2012.
[10] Mukherjee A, Sikdar S, Bishayee K, Paul A, Ghosh S, Boujedaini N, Khuda-Bukhsh AR . Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro[J]. J Chin Integr Med, 2012,10(12):1451-1459
doi: 10.3736/jcim201212
[11] Pacheco SA, Torres VM, Louro H, Gomes F, Lopes C , Mar?al N, Fragoso E, Martins C, Oliveira CL, Hagenfeldt M, Bugalho-Almeida A, Penque D, Simes T. Effects of occupational exposure to tobacco smoke: is there a link between environmental exposure and disease? J Toxicol Environ Health A. 2013; 76(4-5):311-327.
doi: 10.1080/15287394.2013.757269
[12] Sinha R, Kulldorff M, Gunter MJ, Strickland P, Rothman N . Dietary benzo[a]pyrene intake and risk of colorectal adenoma[J]. Cancer Epidemiol Biomarkers Prev, 2005,14(8):2030-2034
doi: 10.1158/1055-9965.EPI-04-0854
[13] Ji K, Xing C, Jiang F, Wang X, Guo H, Nan J, Qian L, Yang P, Lin J, Li M, Li J, Liao L, Tang J . Benzo[a]pyrene induces oxidative stress and endothelial progenitor cell dysfunction via the activation of the NF-κB pathway[J]. Int J Mol Med, 2013,31(4):922-930
doi: 10.3892/ijmm.2013.1288
[14] Kumar M, Sharma VL, Sehgal A, Jain M . Protective effects of green and white tea against benzo(a)pyrene induced oxidative stress and DNA damage in murine model[J]. Nutr Cancer, 2012,64(2):300-306
doi: 10.1080/01635581.2012.648300
[15] Barnes SL, Singletary KW, Frey R . Ethanol and acetaldehyde enhance benzo[a]pyrene-DNA adduct formation in human mammary epithelial cells[J]. Carcinogenesis, 2000,21(11):2123-2128
doi: 10.1093/carcin/21.11.2123
[16] Kundu S, Sengupta S, Chatterjee S, Mitra S, Bhattacharyya A . Cadmium induces lung inflammation independent of lung cell proliferation: a molecular approach[J]. J Inflamm (Lond), 2009,6:19
doi: 10.1186/1476-9255-6-19
[17] Myllynen P, Kurttila T, Vaskivuo L , V?h?kangas K.DNA damage caused by benzo(a)pyrene in MCF-7 cells is increased by verapamil, probenecid and PSC833[J]. Toxicol Lett, 2007,169(1):3-12
doi: 10.1016/j.toxlet.2006.11.006
[18] Ghosh S, Bishayee K, Paul A, Mukherjee A, Sikdar S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR . Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation[J]. J Integr Med, 2013,11(2):116-124
doi: 10.3736/jintegrmed2013014
[19] Ganguly A, Basu S, Chakraborty P, Chatterjee S, Sarkar A, Chatterjee M, Choudhuri SK . Targeting mitochondrial cell death pathway to overcome drug resistance with a newly developed iron chelate[J]. PLoS One, 2010,5(6):e11253
doi: 10.1371/journal.pone.0011253
[20] Tietze F . Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues[J]. Anal Biochem, 1969,27(3):502-522
doi: 10.1016/0003-2697(69)90064-5
[21] Banerjee T, Mukhopadhyay R . Structural effects of nogalamycin, an antibiotic antitumour agent, on DNA[J]. Biochem Biophys Res Commun, 2008,374(2):264-268
doi: 10.1016/j.bbrc.2008.07.053
[22] Calabrese EJ . Hormesis and homeopathy: introduction[J]. Hum Exp Toxicol, 2010,29(7):527-529
doi: 10.1177/0960327110369769
[23] Bellavite P, Chirumbolo S, Marzotto M . Hormesis and its relationship with homeopathy[J]. Hum Exp Toxicol, 2010,29(7):573-579
doi: 10.1177/0960327110369771
[24] Soo ET, Yip GW, Lwin ZM, Kumar SD, Bay BH . Heat shock proteins as novel therapeutic targets in cancer[J]. In Vivo, 2008,22(3):311-315
[25] Wagner M, Hermanns I, Bittinger F, Kirkpatrick CJ . Induction of stress proteins in human endothelial cells by heavy metal ions and heat shock[J]. Am J Physiol, 1999,277(5 Pt 1):L1026-L1033
[26] Zheng SL, Sun TH, Xiao H, Qiu XY, Song YF . Low dose benzo(a)pyrene up-regulated the transcription of HSP70 and HSP90 in Eisenia fetida[J]. Ying Yong Sheng Tai Xue Bao, 2008,19(2):401-406
[27] Algire C, Moiseeva O, Deschênes-Simard X, Amrein L, Petruccelli L, Birman E, Viollet B, Ferbeyre G, Pollak MN . Metformin reduces endogenous reactive oxygen species and associated DNA damage[J]. Cancer Prev Res (Phila), 2012,5(4):536-543
doi: 10.1158/1940-6207.CAPR-11-0536
[28] Gavina JM, Rubab M, Zhang H, Zhu J, Nong A, Feng YL . A tool for rapid screening of direct DNA agents using reaction rates and relative interaction potency: towards screening environmental contaminants for hazard[J]. J Environ Monit, 2011,13(11):3145-3155
doi: 10.1039/c1em10511f
[29] Khuda-Bukhsh AR . Towards understanding molecular mechanisms of action of homeopathic drugs: an overview[J]. Mol Cell Biochem, 2003,253(1-2):339-345
doi: 10.1023/A:1026048907739
[30] Chikramane PS, Suresh AK, Bellare JR, Kane SG . Extreme homeopathic dilutions retain starting materials: A nanoparticulate perspective[J]. Homeopathy, 2010,99(4):231-242
doi: 10.1016/j.homp.2010.05.006
[31] Bell IR, Schwartz GE, Boyer NN, Koithan M, Brooks AJ . Advances in integrative nanomedicine for improving infectious disease treatment in public health[J]. Eur J Integr Med, 2013,5(2):126-140
doi: 10.1016/j.eujim.2012.11.002
[32] Bell IR, Koithan M, Brooks AJ . Testing the nanoparticle-allostatic cross-adaptation-sensitization model for homeopathic remedy effects[J]. Homeopathy, 2013,102(1):66-81
doi: 10.1016/j.homp.2012.10.005
[1] Cai-lian Fana, Wan-jun Caib, Meng-nan Ye, Miao Chen, Yi Dai. Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro. Journal of Integrative Medicine, 2022, 20(4): 365-375.
[2] Célia Cristina Malaguti Figueiredo, Amanda da Costa Gomes, Filipe Oliveira Granero, João Luiz Bronzel Junior, Luciana Pereira Silva, Valdecir Farias Ximenes, Regildo Márcio Gonçalves da Silva. Antiglycation and antioxidant activities of the crude extract and saponin fraction of Tribulus terrestris before and after microcapsules release. Journal of Integrative Medicine, 2022, 20(2): 153-162.
[3] Mohamed S. Othman, Sofian T. Obeidat, Amal H. Al-Bagawi, Mohamed A. Fareid, Alaa Fehaid, Ahmed E. Abdel Moneim. Green-synthetized selenium nanoparticles using berberine as a promising anticancer agent#br#
. Journal of Integrative Medicine, 2022, 20(1): 65-72.
[4] Olarewaju M. Oluba, Oghenerobor B. Akpor, Feyikemi D. Adebiyi, Sunday J. Josiah, Olayinka O. Alabi, Ayoola O. Shoyombo, Augustine O. Olusola. Effects of co-administration of Ganoderma terpenoid extract with chloroquine on inflammatory markers and antioxidant status in Plasmodium berghei-infected mice. Journal of Integrative Medicine, 2020, 18(6): 522-529.
[5] Gerli E.G. Herr, Fábio Goulart da Silva, Francisco José Cidral-Filho, Fabricia Petronilho, Lucinéia Gainski Danielski, Mariana Pereira de Souza Goldim, Afonso Shiguemi Inoue Salgado, Franciane Bobinski, Daniel Fernandes Martins, Eliane R. Winkelmann. Effects of the use of bioceramic wraps in patients with lower limb venous ulcers: A randomized double-blind placebo-controlled trial. Journal of Integrative Medicine, 2020, 18(1): 27-.
[6] Álisson de Carvalho Gonçalves, Einy Jéssika Siqueira Moreira, Guilherme Vannucchi Portari. Benfotiamine supplementation prevents oxidative stress in anterior tibialis muscle and heart. Journal of Integrative Medicine, 2019, 17(6): 423-429.
[7] Pranay Soni, Rajesh Choudhary, Surendra H. Bodakhe. Effects of a novel isoflavonoid from the stem bark of Alstonia scholaris against fructose-induced experimental cataract. Journal of Integrative Medicine, 2019, 17(5): 374-382.
[8] Fang-yuan Wang, Jian Jia, Huan-huan Song, Cheng-ming Jia, Chang-bo Chen, Jing Ma. Icariin protects vascular endothelial cells from oxidative stress through inhibiting endoplasmic reticulum stress. Journal of Integrative Medicine, 2019, 17(3): 205-212.
[9] Lucky Legbosi Nwidu, Yibala Ibor Oboma. Telfairia occidentalis (Cucurbitaceae) pulp extract mitigates rifampicin-isoniazid-induced hepatotoxicity in an in vivo rat model of oxidative stress. Journal of Integrative Medicine, 2019, 17(1): 46-56.
[10] Bruno José Martins Da Silva, Sandro Wilson Gomes Pereira, Ana Paula Drummond Rodrigues, José Luiz Martins Do Nascimento, Edilene Oliveira Silva. In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum. Journal of Integrative Medicine, 2018, 16(6): 404-410.
[11] Sahar Dehdari, Homa Hajimehdipoor, Somayeh Esmaeili, Rasool Choopani, Seyed Alireza Mortazavi. Traditional and modern aspects of hemorrhoid treatment in Iran: A review. Journal of Integrative Medicine, 2018, 16(2): 90-98.
[12] Kayode E. Adewole, Joseph O. Adebayo. Antioxidant defense system induced by cysteine-stabilized peptide fraction of aqueous extract of Morinda lucida leaf in selected tissues of Plasmodium berghei-infected mice. Journal of Integrative Medicine, 2017, 15(5): 388-397.
[13] Uduak Akpan Okon, Idorenyin Udo Umoren. Comparison of antioxidant activity of insulin, Ocimum gratissimum L., and Vernonia amygdalina L. in type 1 diabetic rat model. Journal of Integrative Medicine, 2017, 15(4): 302-309.
[14] Udhaya Lavinya Baskaran, Evan Prince Sabina. Clinical and experimental research in antituberculosis drug-induced hepatotoxicity: A review. Journal of Integrative Medicine, 2017, 15(1): 27-36.
[15] Yuki Takauji, Kyoko Morino, Kensuke Miki, Mohammad Hossain, Dai Ayusawa, Michihiko Fujii. Chyawanprash, a formulation of traditional Ayurvedic medicine, shows a protective effect on skin photoaging in hairless mice. Journal of Integrative Medicine, 2016, 14(6): 473-479.
Full text



[1] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. An explanation on "guiding principles of clinical research on mild cognitive impairment (protocol)". Journal of Chinese Integrative Medicine, 2008, 6(1): 15-21
[2] Yi-ting He, Qing-lin Zha, Jian-ping Yu, Yong Tan, Cheng Lu, Ai-ping Lv. Principal factor analysis of symptoms of rheumatoid arthritis and their correlations with efficacy of traditional Chinese medicine and Western medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 32-36
[3] Jun Cai, Hua Wang, Sheng Zhou, Bin Wu, Hua-rong Song, Zheng-rong Xuan. Effect of Sijunzi Decoction and enteral nutrition on T-cell subsets and nutritional status in patients with gastric cancer after operation: A randomized controlled trial. Journal of Chinese Integrative Medicine, 2008, 6(1): 37-40
[4] Wei Zhang, Xiang-feng Lu, Xiao-mei Zhang, Jian-jun Wu, Liang-duo Jiang. A rat model of pulmonary fibrosis induced by infusing bleomycin quickly through tracheal intubation. Journal of Chinese Integrative Medicine, 2008, 6(1): 60-67
[5] A-gao Zhou, Yong Zhang, Gang Kui, De-Yun Kong, Hai-liang Ge, Qiu-hua Ren, Jia-rong Dong, Sheng Hong, Xu-ming Mao, Yin Wang, Hui-zheng Zhang, Shu-jun Wang. Influence of traditional Chinese compound recipes with different efficacy on body weight, tumor weight and immune function in H22 cancer-bearing mice. Journal of Chinese Integrative Medicine, 2008, 6(1): 77-82
[6] Guo-hong Yuan, Xiao-jing Pang, He-chao Ma. Synergic effects of Danggui Buxue Decoction in reducing toxicity of cytoxan in tumor-bearing mice. Journal of Chinese Integrative Medicine, 2008, 6(1): 83-88
[7] Li Zhou, Hong-xing Zhang, Ling-guang Liu, Wen-jun Wan. Effect of electro-acupuncture at Fenglong (GV 16) on nitric oxide and endothelin in rats with hyperlipidemia. Journal of Chinese Integrative Medicine, 2008, 6(1): 89-92
[8] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. Guiding principles of clinical research on mild cognitive impairment (protocol). Journal of Chinese Integrative Medicine, 2008, 6(1): 9-14
[9] Xin-jun Wang, Ling-ling Wang . A mechanism of endogenous opioid peptides for rapid onset of acupuncture effect in treatment of depression. Journal of Chinese Integrative Medicine, 2010, 8(11): 1014-1017
[10] Bo Wang , Wei Yan , Li-hui Hou, Xiao-ke Wu. Disorder of Tiangui (kidney essence) and reproductive dysfunction in patients with polycystic ovary syndrome. Journal of Chinese Integrative Medicine, 2010, 8(11): 1018-1022