An in-vitro assessment of the effects of Arsenicum album (30CH and 200CH) on leukocytes previously antagonised by arsenic trioxide

Abstract

The therapeutic effects of homoeopathic Arsenicum album potencies were investigated in-vitro, using human cell cultures which were previously antagonised by arsenic trioxide (As2O3). Primary cell culture (peripheral blood mononuclear cells) and a continuous cell line (MT4) were treated with succussed and unsuccussed homoeopathic potencies, 6CH, 30CH and 200CH. This study aimed to verify the homoeopathic law of similars and to determine whether potencies diluted beyond Avogadro’s constant had physiological effects on cells; whether various potencies would cause different effects as proposed by the Arndt-Schultz law; whether succussed and unsuccussed homoeopathic potencies had different effects on the cells; and to establish whether a biotechnological method could be used to evaluate the above. Initial experiments involved isolation and culturing of the peripheral blood mononuclear cells (PBMCs) and the MT4 cell line. Cell titres were determined using the trypan blue dye exclusion assay. The solubilization method of As2O3 was optimized through various dissolution experiments, so as to attain a homogenous arsenical solution. The MTT assay was used to measure the percentage cytotoxicity and the half maximal inhibitory concentration (IC50) caused by the antagonist As2O3 on the PBMCs and the MT4 cell line. The two cell cultures were compared with regard to their susceptibility to As2O3 and their reliability of response. The homoeopathic potencies of Arsenicum album (6CH, 30CH and 200CH) were prepared by initially triturating the As2O3, and then either hand succussing 10 times (succussed) or allowing to diffuse for 30 s (unsuccussed) in sterile distilled water, with the final potencies made up in cell culture media, RPMI. The MTT assay was used to determine the percentage cell viability when the As2O3-antagonised cells were treated with the Arsenicum album potencies. All assays were performed in triplicate. v The As2O3 was found to fully dissolve when 396 mg of dry As2O3 was added to 100 mL of sterile distilled Milli-Q water, which was left to stand for 10 days at 80°C. The cytotoxicity results showed that the PBMCs were not as reliable as the MT4 cells, which showed significant susceptibility to the As2O3. The IC50 of As2O3 on 1 mL of MT4 cells was found to be 5 μM As2O3 (133 μL) for 48 h. The trypan blue dye exclusion assay demonstrated that the viable MT4 cells decreased in number after exposure to the As2O3, with an increase in number of the non-viable cells. Microscopically, the cells were fewer in number and displayed signs of possible blebbing and cell shrinkage, showing potential cell death due to apoptosis. The cell viability results showed that the Arsenicum album 6CH resulted in the lowest absorbance readings and the Arsenicum album 200CH gave the highest readings; this verified the therapeutic effects of homoeopathic remedies when given according to the law of similars; that potencies diluted beyond Avogadro’s constant had stimulating effects; and that the more dilute potencies stimulated recovery in the cells more than the lower potencies, verifying the Arndt-Schultz law. The treatments and the times of exposure were found to be statistically significant determinants of cell viability, whereas succussion did not cause any significant variation in the results. The study thereby provided evidence that a biotechnological method could be used to scientifically evaluate the physiological effects of homoeopathic potencies on human cells; that the homoeopathic potencies did have therapeutic effects; and that succussion was not required in the potentization method in order to produce a curative remedy.