Elsevier

Homeopathy

Volume 105, Issue 2, May 2016, Pages 148-159
Homeopathy

Original paper
Impact of homeopathic remedies on the expression of lineage differentiation genes: an in vitro approach using embryonic stem cells

https://doi.org/10.1016/j.homp.2015.11.002Get rights and content

Highlights

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    The study reveals the effects of homeopathic remedies taken during pregnancy using ES cells as the model system.

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    Modulations in lineage specific genes were noted but the differences were not significant with respect to solvent control.

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    Protein expression showed normal differentiation as evidenced by α/βMHC and α-actinin marker protein expression.

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    Our study for the first time has shown the use of ES cells in the developmental toxicity testing.

Background

Well-documented studies of the potential effects and safety of homeopathic medicines in pregnancy are required. In this study, specific genes were studied which could serve as biomarkers for specification of three lineages to predict the safety of homeopathic remedies using mouse embryonic stem (ES) cells. Thus, the present work was to study the effects of homeopathic remedies taken during pregnancy using ES cells as the model.

Methods

Mouse ES cells were exposed to 30C potency of Nux Vomica and Sepia, which are homeopathic medicines prescribed for the management of pregnancy related symptoms. Cytotoxicity studies were done using a modified Embryonic Stem cell test (EST). The expression levels of key genes and proteins were analyzed using real time polymerase chain reaction and immunocytochemistry, respectively.

Results

Homeopathic treatment led to modulations in the expression of certain lineage specific genes but this difference was not significant with respect to solvent control and showed normal differentiation as demonstrated by the expression of α/β MHC and α-actinin proteins in the differentiated ES cells.

Conclusions

Our study for the first time has shown the feasibility of using ES cells in the developmental toxicity testing of remedies. The results suggest that they are not associated with developmental toxicity.

Introduction

Homeopathic medicines are considered to be safe because of the belief that at dilutions which are conventionally used for treatment were associated with less adverse effects.1 Homeopathic medicines are prepared by the process of dynamization, in which a miniscule quantity of the original natural medicinal essence is necessary when manufacturing these medicines. These remedies are believed to be safe and were given even to drug sensitive groups like pregnant women, babies and elders. Some conventional medicines such as zantac, emetrol, pepcid, phenergan are prescribed for nausea and vomiting.2 Generally conventional medicines suppress symptoms rather than treating the underlying cause, and when taken during pregnancy does have a threat of various side effects on the developing fetus.

Homeopathy is controversial, science and has evoked scepticism.3, 4 The most serious criticisms related to homeopathy are the lack of understanding of mechanisms behind these remedies.5 It is said that homeopathy works at the cellular level and therefore, researchers need high quality cells/model systems along with powerful techniques to prove the efficacy of homeopathy. To this end various researchers over the years have tried to ascertain the mechanism by which these ultra-diluted preparations (which usually lie beyond the Avogadro's limit) exert their effect. By using DNA microarray based transcriptomic analysis studies have revealed alterations in the gene expression of key signaling pathways.

Saha et al6 showed that in the cervical cancer HeLa cells exposed to Condurango 30C and Hydrastis canadensis 30C there was a significant quantitative as well as qualitative difference in gene expression. Preethi et al also reported differential gene expression when Dalton's lymphoma tumor cells were treated with 200C potencies of Ruta, Carcinosinum, Hydrastis and Thuja.7 In an elegant review. Bellavite et al have discussed research findings of the use of homeopathic remedies for various disease models. They have reported global alterations in gene expression in treated cells using DNA microarray.8 These studies prove beyond a doubt that homeopathic remedies are not placebos but act by modulating gene expression.

Using extremely diluted dynamized dilutions (3C, 5C and 7C) of Apis mellifica in RPEW-1 prostrate cells, Bigagli and coworkers reported modulation of hundreds of genes using global gene profiling microarrays and carried out cluster analysis wherein certain groups of genes were up- or down-regulated with similar expression profiles among treatments; other genes showed opposite regulation profiles at low and high dilutions of Apis mellifica, suggesting a hormetic response.9 Hence using alterations in gene expression as an endpoint to ascertain effect upon exposure, we treated embryonic stem cells with homeopathic remedies Nux Vomica and Sepia, which are routinely prescribed in early pregnancy.10

Nux vomica is the dried, ripe seed of Nux Vomica L. (family, Loganiaceae). Nux Vomica is given to counter symptoms of nausea and constipation during pregnancy.11 Sepia is a product from the fresh ink of Sepia officinalis which is commonly known as cuttlefish. Sepia is prescribed to counteract morning sickness, constipation and headache during pregnancy.11 The safe use of Nux Vomica and Sepia in pregnancy however is not well documented. Moreover, as these homeopathic remedies are over the counter drugs there is no direct regulatory control for their use. It is therefore, necessary to carry out research on these remedies to generate sufficient experimental data for their safe use in pregnancy.

Embryonic stem cell research contributes to a fundamental understanding of how an organism develops and grows, and how tissues are maintained throughout adult life. The development of ES cell lines has provided researchers with the reliable tool to study developmental toxicity and birth defects.12, 13 The effects of conventional medicine taken during pregnancy have been studied using this in vitro EST model. Currently, there are no methods for testing developmental toxicity associated with homeopathy medicines to prove their safe use in pregnancy. In this study, various lineage specific genes were studied which could serve as biomarkers for specification of these three lineages to predict the safety of homeopathic remedies using mouse ES cells as the model system.

Section snippets

Cell lines and culture conditions

The mouse ES cell line D3 was maintained at 37°C and 5% CO2 and routinely passaged three times a week. ES cells were cultured in Dulbecco's modified Eagles Medium (DMEM) (Invitrogen), supplemented with 15% heat inactivated fetal bovine serum (FBS) (Sigma, USA), 2 mM glutamine(Invitrogen), 50 U/ml penicillin and 50 μg/ml streptomycin (Invitrogen), 1% non-essential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol and 1000 U/ml LIF (leukemia inhibitory factor, ESGRO, Chemicon International Inc.,

Determination of the cytotoxicity of homeopathy remedies using ES cells (D3 cells) and adult fibroblast cells (3T3 cells)

For cytotoxicity analysis, MTT assay according to a modified EST model was performed to study the reduction in the viability or in other words cytotoxic effects on D3 and 3T3 cells which represented embryonic and adult cells, respectively. In both cell types, the homeopathic remedies namely Nux Vomica and Sepia, were tested at their different concentrations. Sensitivity of D3 cells showed varied responses upon treatment with the homeopathic remedies. Cell viability in D3 cells treated with 30C

Discussion

Evidence of potential harmful effects of homeopathic medicines is limited and available data is conflicting. This is of particular concern in pregnancy, where need of considering their potential effects on fetal development and drug interactions are of extreme importance. The mechanism of action of these remedies and factors related to their use in pregnancy need extensive studies for safety assessment during this sensitive stage of life. This research article provides the first step in

Conclusion

This study proposes that the expression of different lineage biomarker genes can be useful in examining the side effects (if any) of homeopathy, an important but neglected issue pregnancy. Implementation for the analysis of biomarkers of differentiation for risk management assessment for a select section of sensitive users in needed. Individuals who are more susceptible to any kind of drug exposure, such as pregnant women and their unborn children would be benefited. This would help to safety

Acknowledgment

This study was financially supported by the Department of Biotechnology (BT/PR14096/MED/31/103/2010), Government of India, New Delhi, India.

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