Combination of Umbelliprenin and Arsenic Trioxide Acts as an Effective Modality Against T-Cell Leukemia/Lymphoma Cells

Adult T-cell leukemia/lymphoma (ATLL) is a serious blood malignancy with distinct geographical distribution. ATLL patients have a short survival time because of intrinsic chemoresistance and severe immunosuppression. To introduce a novel treatment, we investigated whether umbelliprenin (UMB), a natural coumarin derivative, could improve the toxicity of arsenic trioxide (ATO) on ATLL cells. To determine the viability of MT-2 cells upon treatment with different concentrations of UMB and ATO, alamarBlue assay was applied. Cell cycle analysis was carried out by propidium iodide staining and the expression of candidate genes was assessed by quantitative reverse transcription-polymerase chain reaction. Our findings revealed that combination of UMB and ATO induced considerable cytotoxic effects on ATLL cells. Flow cytometry analysis indicated accumulation of MT-2 cells in the sub G1 phase of the cell cycle after combinatorial treatment. In addition, significant downregulation in BMI-1, CD44, c-MYC, and nuclear factor-κB (REL-A) expression was observed after UMB + ATO administration. Agents with low side effects are potential candidates for novel cancer treatments. We demonstrated, for the first time, that combination of UMB and ATO might be regarded as an effective regimen for ATLL treatment.

Kefir as a Prevention of Arsenic-mediated Toxicity in Uterine Female Rats

Background: kefir is a fermented milk product that demonstrates numerous health benefits including antioxidant and immunomodulatory. Aim: to study the protective effect kefir on the expression of estrogen receptor alpha (ERα) in endometrial stromal cells and endometrial thickness on female rats that were exposed to arsenic. Methods: twenty-five female Wistar rats (Rattus norvegicus) were divided into five groups (CRL, As, T1, T2, T3). Control group (given a normal diet), As group (given the normal diet and exposed to arsenic trioxide 2 mg/kgBW/day). The T1; T2; T3 were exposed to arsenic trioxide 2 mg/kgBW/day and treated with different doses of kefir (1.25; 2.5; and 5 mL/kgBW/day, respectively) for 35 days. The rats of group As treated with arsenic trioxide only and group CRL served as control with normal feed in water. Cytological samples were taken after 35 days of treatment and examined every day to see the rat oestrus phase, and the proestrus phase of the oestrous cycle was chosen for termination. Uterine tissue fixed in 10% neutral buffered formalin for tissue preparation. ERα expression in endometrial stromal cells was analized using immunohistochemistry method, endometrial thickness was observed using histopathological methods. Results: significant reduction of ERα expression in endometrial stromal cells and endometrial thickness in female rats exposed to arsenic were observed in groups on treated rats (p ≤ 0.000; 0.009, respectively). Conclusion: the administration of kefir in female Wistar rats exposed to arsenic had shown significantly differences on ERα expressions and endometrial thickness. The smallest dose of kefir (1.25 mL/kgBW/day) could increase ERα expression and endometrial thickness in female Wistar rats with arsenic exposure. Therefore kefir has protective effect related to female reproductive system.

Phytopathological and nutraceutical evaluation of cauliflower plants treated with high dilutions of arsenic trioxide

Introduction: This research aimed at verifying the effects of highly diluted (HD) treatments on cauliflower (Brassica oleracea L.) plants both healthy and inoculated by the fungus Alternaria brassicicola, causing the dark leaf spot disease. In vitro spore germination assays (A), growth chamber experiments (B) and field trials (C) were performed. Material and Methods: (A): spore suspensions were prepared in HD treatments and their inhibiting effect on germination was recorded microscopically after incubation at 25°C for 5 h. (B): the same treatments were tested in plants artificially inoculated with the fungus. The infection level on leaves was blindly evaluated by a previously defined infection scale. (C): the field was divided into plots according to a complete randomized block design. In the first trial (i), plants were artificially inoculated and weekly treated; the infection level was evaluated on cauliflower heads. The second trial (ii) was performed on the same field with the aim to induce a natural infection, mediated by infected crop residues. Measurement endpoints concerned the evaluation of some physiological parameters along with the glucosinolate content on cauliflower heads. Results: (A): arsenic trioxide (As 35x and 35x diluted 1:5000) and Cuprum 5x induced highly significant inhibition of germination rate (-60%) vs. control. (B): As 35x and Cu 3 g/l induced a significant decrease of mean infection level (-50%). (C): in (i), a significant reduction of disease symptoms on heads was recorded for As 35x and Cu 3 g/l (-45%). In (ii) natural fungal infection did not occur due to dry weather conditions; physiological and nutraceutical analyses of healthy heads demonstrated that As 35x induced a significant increase of both head size and glucosinolate content. Discussion: Some evidences on the efficacy of arsenic, at different decimal and centesimal HD, in fungal and viral disease control were previously reported [1]. In the present study the efficacy of HD arsenic in dark leaf spot control in field has been shown for the first time: since fungal inoculation was performed on the leaves before flowering, we can hypothesize that this treatment induced an increase of plant resistance to fungal infection. Conclusions: This research showed the possibility of using HD arsenic in agriculture (“agrohomeopathy”), as it increased both plant resistance to fungal infection and the content of glucosinolates, ie secondary metabolites involved in plant resistance mechanisms [2] and considered as “plant food protection agents” [3]. Acknowledgments: Authors declare there is no conflict of interest. This research has been supported by Marche Region. A grateful acknowledgement to Dr. Leonardo Valenti for his support to this research. The authors thank Laboratoires Boiron srl for the grant awarded to one of the author Dr. Grazia Trebbi. Finally, authors are grateful to Cemon srl for financial support of glucosinolate analyses. We had full access to all the data in this study and we take responsibility for the integrity of the data and the accuracy of the data analysis Keywords: cauliflower, arsenic trioxide, Alternaria brassicicola, glucosinolates References [1] Betti L, Trebbi G, Majewsky V, Scherr C, Shah-Rossi D, Jäger T, et al. Use of homeopathic preparations in phytopathological models and in field trials: a critical review. Homeopathy 2009; 98: 244-266. [2] Ménard R, Larue J-P, Silué D, Thouvenot D. Glucosinolates in cauliflower as biochemical markers for resistance against downy mildew. Phytochemistry 1999; 52: 29-35. [3] Talalay P, Fahey JW. Phytochemicals from Cruciferous plants protect against cancer by modulating carcinogen metabolism. J Nutr. 2001; 131:3027S- 3033S.

Summary of four scientific studies on Arsenicum album high dilution effect against Arsenic intoxication in mice

Background: Groundwater arsenic affects millions of people in about 20 countries. In West Bengal (India) and Bangladesh alone over 100 million people are exposed. The arsenic concentration in contaminated groundwater in Bangladesh was above the maximum permissible level of 0.05 mg/l as recommended by WHO for developing countries [1]. Drinking water is not the only source of poisoning. In arsenic contaminated areas, crops, vegetables, cereals, poultry, cattle, etc, also contain traces of arsenic. Chronic arsenic intoxication has been associated with several diseases such as melanosis, leuco-melanosis, hyperkeratosis, oedema, skin cancer… Cazin et al [2], have demonstrated the effect of high dilutions of arsenic compounds. They noted increased arsenic elimination from blood through urine and faeces in intoxicated rats. According to these research, the aim of Khuda Buksh studies [3-4-5] was to investigate whether high dilution Arsenicum album have any effect on arsenic accumulation in different tissues and to understand also how this high dilution could produce a protective effect on all the different organs. Methodology: Firstly, the effect of Arsenicum album 30 cH on the amount of arsenic accumulation was determined by spectrophotometric analysis in four tissues namely liver, kidney and testis in mice intoxicated by arsenic. The protective effect in chronic and acute arsenic intoxicated mice of Arsenicum Album 6cH, 30cH and 200cH has been evaluated using not only the activities of enzymatic and biomarker toxicity (aspartate amino transferase (AST), alanine amino transferase (ALT), acid phosphatase (AcP), alkaline phosphatase (AlkP), lipid peroxidation (LPO) and reduced glutathione (GSH)) but also the cytogenetical parameters (chromosome aberrations (CA), mitotic index (MI), sperm head anomaly (SHA) etc., ). Because, it is well demonstrated that these enzymes biomarkers reflect the degree of hepatotoxicity and oxidative stress caused by arsenic intoxication. Results: Compared to controls, Arsenicum album 30cH induced a significant decrease in accumulation of arsenic in 4 tissues namely liver, spleen, kidney and testis in intoxicated mice. In addition, both Arsenicum album 6cH, 30cH and 200cH reduced chromosome aberrations, sperm head abnormality frequencies and activities of acid and alkaline phosphatases, aspartate and alanine aminotransferases and lipid peroxidation, while mitotic index and activities of glutathione, catalase and succinate dehydrogenase were increased compared to controls. Conclusion: Altogether, theses results provide evidence of protective potentials of the Arsenicum album dilution against acute and chronic arsenic intoxication in mice. They also offer a new hypothesis that the mechanism of the homeopathic dilution could act through regulation of expression of certain genes. This explanation seems to be plausible because all biomarker tests are regulated by specific genetic regulatory mechanisms [6]. keywords: Arsenicum album, arsenic intoxication, enzymatic and biomarker toxicity. References: [1] WHO. WHO Guidelines for Drinking Water Quality, Vol. 2, 2nd edition. Geneva: WHO, 1996, 940–9. [2] Cazin JC, Cazin M, Gaborit JL, Chaoui A, Boiron J, Belon P, et al. A study of the effect of decimal and centesimal dilutions of arsenic on the retention and mobilization of arsenic in the rat. Hum Toxicol 1987;6:315–20. [3] Mitra K, Kundu SN, Khuda-Bukhsh AR. Efficacy of a potentized homoeopathic drug (Arsenicum Album-30) in reducing toxic effects produced by of arsenic trioxide in mice. I. On rate accumulation of arsenic in certain vital organs. Comp Ther Med 1998;6:178–84. [4] Pathikrit Banerjee. Evidences of Protective Potentials of Microdoses of Ultra-high Diluted Arsenic Trioxide in Mice Receiving Repeated Injections of Arsenic Trioxide. eCAM 2009; 1-10. [5] Pathikrit Banerjee, Comparative Efficacy of Two Microdoses of a Potentized Homeopathic Drug, Arsenicum Album, to Ameliorate Toxicity Induced by Repeated Sublethal Injections of Arsenic Trioxide in Mice. Pathobiology 2008;75:156–170. [6]/ Khuda-Bukhsh AR. Potentized homeopathic drugs act through regulation of gene expression: a hypothesis to explain their mechanism and pathways of action in vivo. Comp Ther Med 1997;5:43–6