Differential effects of Zincum metallicum on cell models

Jenifer Pendiuk Gonçalves; Maria Luiza Ferreira dos Santos; Gustavo Rodrigues Rossi; Viviana Stephanie Costa Gagosian; Carolina Camargo de Oliveira

doi:10.1016/j.homp.2017.02.004

Homeopathy, Table of Contents

Homeopathy 2017; 106(03): 171-180
DOI: 10.1016/j.homp.2017.02.004

Differential effects of Zincum metallicum on cell models

Jenifer Pendiuk Gonçalves

,

Maria Luiza Ferreira dos Santos

,

Gustavo Rodrigues Rossi

,

Viviana Stephanie Costa Gagosian

,

Carolina Camargo de Oliveira

Subject Editor:

Abstract

Introduction: Zinc is an essential trace element necessary for life. Traditional and complementary medicines use zinc-based formulations to treat different classes of diseases. Basic research on homeopathic preparations of zinc are rare and there are a few published clinical cases describing its effects on patients. The use of cell-based models in drug screening is a reliable source of evidence.

Methods: We sought to investigate experimental end-points using cell-based models to determine the effects of dilutions of Zincum metallicum prepared according to the Brazilian Homeopathic Pharmacopoeia. Murine RAW 264.7 macrophages and melanoma B16-F10 cell lines were cultured according to standard procedures. Cells were treated with either 5c, 6c or 30c Zincum metallicum and control cells with its respective vehicle (5c, 6c, or 30c Lactose). Macrophage activation by CD54 immunolabeling and intracellular reactive oxygen species (ROS) using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) were detected by flow cytometry. Phagocytic capacity (endocytic index) was quantified by light microscopy. Features of melanoma cells were analyzed by colorimetric assays to determine melanin content and cell proliferation rate. All obtained data were submitted to normality test followed by statistical analysis.

Results: Zincum metallicum 6c shifted high ROS-producing macrophages to a low ROS-producing phenotype. Macrophage CD54 expression was increased by Zincum metallicum 5c. No changes in endocytic index were observed. Melanoma cells were not affected by any treatment we tested.

Conclusions: Differing responses and non-linearity were found on macrophages challenged with Zincum metallicum at high dilutions. No changes in melanoma cells were observed. Customised assays using target cells can be useful to investigate high-dilution effects. Other cell types and conditions should be explored.

Highlights

• Zinc met did not change cytokine profile but altered ROS and CD54 in macrophages.

• Melanoma morphology, proliferation, and melanogenesis were not changed by Zinc met.

• Highly diluted compounds effects can be determined by selected in vitro models.

Keywords

Homeopathy - Highly diluted compounds - Macrophages - Melanoma - Zincum metallicum

Full Text

References

References
1 U.S. Food and Drug Administration. The Drug Development Process. 2–3. Available from: http://www.fda.gov/ForPatients/Approvals/Drugs/default.htm.
2 Ahuja V., Sharma S. Drug safety testing paradigm, current progress and future challenges: an overview. J Appl Toxicol [Internet] 2014; 34 (06) 576-594 10.1002/jat.2935.
3 Mahto S.K., Chandra P., Rhee S.W. In vitro models, endpoints and assessment methods for the measurement of cytotoxicity. Toxicol Environ Health Sci [Internet] 2010; 2 (02) 87-93 10.1007/BF03216487.
4 Astashkina A., Mann B., Grainger D.W. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. Pharmacol Ther [Internet] 2012; 134 (01) 82-106 10.1016/j.pharmthera.2012.01.001.
5 Mertz W. The essential trace elements. Science 1981; 213 (4514): 1332-1338.
6 Lee S.R., Noh S.J., Pronto J.R. et al. The critical roles of zinc: beyond impact on myocardial signaling. Korean J Physiol Pharmacol 2015; 19: 389-399 Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553398/.
7 Page-McCaw A., Ewald A.J., Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol [Internet] 2007; 8 (03) 221-233 Available from: http://www.nature.com/doifinder/10.1038/nrm2125.
8 Zehnalek J., Trnkova L., Babula P., Adam V., Kizek R. DNA interaction with zinc(II) ions. Int J Biol Macromol [Internet] 2013; 64: 1-7 10.1016/j.ijbiomac.2013.12.013.
9 Kloubert V., Rink L. Zinc as a micronutrient and its preventive role of oxidative damage in cells. Food Funct 2015; 6: 3195-3204.
10 Kambe T., Hashimoto A., Fujimoto S. Current understanding of ZIP and ZnT zinc transporters in human health and diseases. Cell Mol Life Sci [Internet] 2014; 71 (17) 3281-3295 Available from: http://link.springer.com/10.1007/s00018-014-1617-0.
11 Clausen J., van Wijk R., Albrecht H. Geographical and temporal distribution of basic research experiments in homeopathy. Homeopathy [Internet] 2014; 103 (03) 193-197 Available from: http://www.sciencedirect.com/science/article/pii/S1475491614000204.
12 Gibson D.M. Zincum metallicum, a study. Br Homeopath J [Internet] 1967; 56 (03) 172-176 Available from: http://linkinghub.elsevier.com/retrieve/pii/S0007078567800267.
13 Jack R.A.F. A case of diabetic peripheral neuropathy treated with Zincum met. in low potency. Br Homeopath J [Internet] 1984; 73 (01) 15-19 Available from: http://www.sciencedirect.com/science/article/pii/S0007078584800046.
14 Bădulici S., Chirulescu Z., Chirilă P., Chirilă M., Roşca A. Treatment with zincum metallicum CH5 in patients with liver cirrhosis. Preliminary study. Rom J Intern Med 1994; 32 (02) 215-219.
15 Maliekal T.P. Antineoplastic effects of 4 homoeopathic medicines. Br Homeopath J [Internet] 1997; 86 (02) 90-91 Available from: http://www.sciencedirect.com/science/article/pii/S0007078597801238.
16 Islam F., Khan A., Khuwaja G. et al. Protective effect of Zincum metallicum on rat model of Parkinson's disease. Indian J Res Homoeopath [Internet] 2015; 9 (02) 86 Available from: http://www.ijrh.org/text.asp?2015/9/2/86/159528.
17 Bellavite P., Signorini A., Marzotto M., Moratti E., Bonafini C., Olioso D. Cell sensitivity, non-linearity and inverse effects. Homeopathy [Internet] 2015; 104 (02) 139-160 Available from: http://linkinghub.elsevier.com/retrieve/pii/S1475491615000053.
18 de Oliveira C.C., de Oliveira S.M., Godoy L.M.F., Gabardo J., Buchi D.D.F. Canova, a Brazilian medical formulation, alters oxidative metabolism of mice macrophages. J Infect [Internet] 2006; Jun [cited 2014 Jun 15] 52 (06) 420-432 Available from: http://www.ncbi.nlm.nih.gov/pubmed/16386798.
19 de Oliveira C.C., de Oliveira S.M., Goes V.M., Probst C.M., Krieger M.A., Buchi D.D.F. Gene expression profiling of macrophages following mice treatment with an immunomodulator medication. J Cell Biochem [Internet] 2008; 104 (04) 1364-1377 Available from: http://www.ncbi.nlm.nih.gov/pubmed/18286468.
20 Guimarães F.S.F., Andrade L.F., Martins S.T. et al. In vitro and in vivo anticancer properties of a Calcarea carbonica derivative complex (M8) treatment in a murine melanoma model. BMC Cancer [Internet] 2010; Jan 10: 113 Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2859384&tool=pmcentrez&rendertype=abstract.
21 de Oliveira S.M., de Oliveira C.C., Abud A.P.R. et al. Mercurius solubilis: actions on macrophages. Homeopathy [Internet] 2011; Oct [cited 2014 Jun 15] 100 (04) 228-236 Available from: http://www.ncbi.nlm.nih.gov/pubmed/21962197.
22 de Oliveira C.C., Abud A.P.R., de Oliveira S.M. et al. Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers. BMC Complement Altern Med [Internet] 2011; 11 (01) 101 Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3222609&tool=pmcentrez&rendertype=abstract.
23 Eruslanov Evgeniy, Kusmartsev Sergei. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol [Internet] 2010 Advanced protocols in oxidative stress II 10.1007/978-1-60761-411-1.
24 Terho P. Flowing Software [Internet]. Turku Centre for Biotechnology; 2013. Available from: http://www.uskonaskel.fi/flowingsoftware/
25 Mosser D.M., Edwards J.P. Exploring the full spectrum of macrophage activation. Nat Rev Immunol [Internet] 2008; 8 (12) 958-969 10.1038/nri2448.
26 Fuentes A.L., Millis L., Vapenik J., Sigola L. Lipopolysaccharide-mediated enhancement of zymosan phagocytosis by RAW 264.7 macrophages is independent of opsonins, laminarin, mannan, and complement receptor 3. J Surg Res [Internet] 2014; 189: 304-312 Available from: http://www.sciencedirect.com/science/article/pii/S0022480414002546.
27 Schneider C.A., Rasband W.S., Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods [Internet] 2012; 9 (07) 671-675 10.1038/nmeth.2089.
28 Bonnekoh B., Wevers A., Jugert F., Merk H., Mahrle G. Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity. Arch Dermatol Res [Internet] 1989; 281: 487-490 10.1007/BF00510085.
29 Hu D.N. Methodology for evaluation of melanin content and production of pigment cells in vitro. Photochem Photobiol [Internet] 2008; 84 (03) 645-649 10.1111/j.1751-1097.2007.00228.x/abstract.
30 Weissgerber T.L., Milic N.M., Winham S.J., Garovic V.D. Beyond bar and line graphs: time for a new data presentation paradigm. PLOS Biol [Internet] 2015; 13 (04) e1002128 Available from: http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002128.
31 Prism – graphpad [Internet]. Available from: http://www.graphpad.com/scientific-software/prism/.
32 Sheikh N.A., Jones L.A. CD54 is a surrogate marker of antigen presenting cell activation. Cancer Immunol Immunother [Internet] 2008; 57 (09) 1381-1390 10.1007/s00262-008-0474-9.
33 Babior B. Phagocytes and oxidative stress. Am J Med [Internet] 2000; 109: 33-44 Available from: http://www.sciencedirect.com/science/article/pii/S0002934300004812.
34 Brüne B., Dehne N., Grossmann N. et al. Redox control of inflammation in macrophages. Antioxid Redox Signal [Internet] 2013; 19 (06) 595-637 10.1089/ars.2012.4785.
35 Juhas U., Ryba-Stanisławowska M., Szargiej P., Myśliwska J. Different pathways of macrophage activation and polarization. Postepy Hig Med Dosw (Online) 2015; 69: 496-502.
36 Fidler I.J. Biological behavior of malignant melanoma cells correlated to their survival in vivo. Cancer Res [Internet] 1975; 35: 218-224 Available from: http://cancerres.aacrjournals.org/content/35/1/218.long.
37 d'Ischia M., Wakamatsu K., Cicoira F. et al. Melanins and melanogenesis: from pigment cells to human health and technological applications. Pigment Cell Melanoma Res [Internet] 2015; 28 (05) 520-544 10.1111/pcmr.12393.
38 Riley P.A. Melanogenesis and melanoma. Pigment Cell Res [Internet] 2003; 16 (05) 548-552 10.1034/j.1600-0749.2003.00069.x/epdf.
39 BCRJ. B16-F10 (BCRJ code 0046) [Internet]. Rio de Janeiro. Available from: http://bcrj.org.br/catalogo/cell/?celula=B16F10-Melanoma-Mouse.
40 Larsen P.O., von Ins M. The rate of growth in scientific publication and the decline in coverage provided by Science Citation Index. Scientometrics [Internet] 2010; 84 (03) 575-603 10.1007/s11192-010-0202-z.
41 Bell I.R., Sarter B., Koithan M. et al. Integrative nanomedicine: treating cancer with nanoscale natural products. Glob Adv Heal Med [Internet] 2014; 3 (01) 36-53 10.7453/gahmj.2013.009.
42 Upadhyay R.P., Nayak C. Homeopathy emerging as nanomedicine. Int J High Dilution Res 2011; 10 (37) 299-310.
43 Chikramane P.S., Kalita D., Suresh A.K., Kane S.G., Bellare J.R. Nanoparticulate hypothesis based on froth flotation. ACS Publ 2012; 28: 15864-15875.
44 Bellavite P., Marzotto M., Olioso D., Moratti E., Conforti A. High-dilution effects revisited. 1. Physicochemical aspects. Homeopathy [Internet] 2014; 103 (01) 4-21 10.1016/j.homp.2013.08.003.
45 Bellavite P., Marzotto M., Olioso D., Moratti E., Conforti A. High-dilution effects revisited. 2. Pharmacodynamic mechanisms. Homeopathy [Internet] 2014; 103 (01) 22-43 10.1016/j.homp.2013.08.002.
46 Bonamin L.V., Endler P.C. Animal models for studying homeopathy and high dilutions: conceptual critical review. Homeopathy [Internet] 2010; 99 (01) 37-50 10.1016/j.homp.2009.11.003.
47 Bonamin L.V., Cardoso T.N., Cunha de Carvalho A., Amaral J.G. The use of animal models in homeopathic research – a review of 2010–2014 PubMed indexed papers. Homeopathy 2015; 104 (04) 283-291.
48 Pascual-Carpe F., Vicente-Ortega V., Campos-Aranda M., Yañez-Gascón J. In vivo treatment of melanoma (B16F10) with a homeopathic agent and with a cytokine (IFN-α). Oncol Res Featur Preclin Clin Cancer Ther [Internet] 2006; 16 (05) 211-216 Available from: http://www.ingentaconnect.com/content/cog/or/2006/00000016/00000005/art00001?token=005719acd6dc972ba573d257025706a23765f24407b4d31745f7c4e75477e4324576b6427382cc304c4c650.
49 Sunila E.M., Girija K., Preethi K.C., Ramadasan K. Effect of homeopathic medicines on transplanted tumors in mice. Asian Pac J Cancer Prev 2007; 8 (03) 390-394.
50 Saha S., Hossain D.M.S., Mukherjee S. et al. Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit. BMC Complement Altern Med [Internet] 2013; 13 (01) 230 Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3856502&tool=pmcentrez&rendertype=abstract.
51 Ferrari de Andrade L., Mozeleski B., Leck A.R. et al. Inhalation therapy with M1 inhibits experimental melanoma development and metastases in mice. Homeopathy [Internet] August 2014; 2015: 1-10 Available from: http://linkinghub.elsevier.com/retrieve/pii/S1475491615000673.
52 Arora S., Aggarwal A., Singla P., Jyoti S., Tandon S. Anti-proliferative effects of homeopathic medicines on human kidney, colon and breast cancer cells. Homeopathy [Internet] 2013; 102 (04) 274-282 Available from: http://linkinghub.elsevier.com/retrieve/pii/S1475491613000416.
53 Frenkel M., Mishra B.M., Sen S. et al. Cytotoxic effects of ultra-diluted remedies on breast cancer cells. Int J Oncol [Internet] 2010; 36: 395-403 Available from: https://www.spandidos-publications.com/ijo/36/2/395.
54 Sunila E.S., Kuttan R., Preethi K.C., Kuttan G. Dynamized preparations in cell culture. Evid Based Complement Altern Med 2009; 6 (02) 257-263.
55 Preethi K., Ellanghiyil S., Kuttan G., Kuttan R. Induction of apoptosis of tumor cells by some potentiated homeopathic drugs: implications on mechanism of action. Integr Cancer Ther [Internet] 2012; 11 (02) 172-182 Available from: http://www.ncbi.nlm.nih.gov/pubmed/21771822.
56 Bishayee K., Sikdar S., Khuda-bukhsh A.R. Evidence of an epigenetic modification in cell-cycle arrest caused by the use of ultra-highly-diluted Gonolobus condurango extract. J Pharmacopuncture 2013; 16 (04) 7-13.
57 Mukherjee A., Boujedaini N., Khuda-Bukhsh A.R. Homeopathic Thuja 30C ameliorates benzo(a)pyrene-induced DNA damage, stress and viability of perfused lung cells of mice in vitro. J Integr Med [Internet] 2013; 11 (06) 397-404 Available from: http://www.ncbi.nlm.nih.gov/pubmed/24299603.
58 Saha S.K., Roy Sourav, Khuda-Bukhsh Anisur R. Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro. J Integr Med [Internet] 2015 (06): 1-12 Available from: http://www.jcimjournal.com/jim/FullText2.aspx?articleID=S2095-4964(15)60201-1.
59 Khuda-Bukhsh A.R., Sikdar S. Condurango 30C induces epigenetic modification of lung cancer-specific tumour suppressor genes via demethylation. Forsch Komplementmed 2015; 22 (03) 172-179.
60 Guimarães F.S.F., Abud A.P.R., Oliveira S.M. et al. Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication. BMC Cancer [Internet] 2009; Jan [cited 2014 Jun 15] 9: 293 Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2749867&tool=pmcentrez&rendertype=abstract.
61 Stafford S.L., Bokil N.J., Achard M.E.S. et al. Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper. Biosci Rep [Internet] 2013; 33 (04) e00049 Available from: http://www.bioscirep.org/content/33/4/e00049.abstract.
62 Martinez F.O., Sica A., Mantovani A., Locati M. Macrophage activation and polarization. Front Biosci [Internet] 2008; 13: 453-461 Available from: http://www.ncbi.nlm.nih.gov/pubmed/20427764.
63 Tan H.-Y., Wang N., Li S., Hong M., Wang X., Feng Y. The reactive oxygen species in macrophage polarization: reflecting its dual role in progression and treatment of human diseases. Oxid Med Cell Longev 2016; 2016: 1-16.
64 Zhu L., Zhao Q., Yang T., Ding W., Zhao Y. Cellular metabolism and macrophage functional polarization. Int Rev Immunol [Internet] 2015; 34 (01) 82-100 Available from: http://www.ncbi.nlm.nih.gov/pubmed/25340307.
65 Olioso D., Marzotto M., Moratti E., Brizzi M., Bellavite P. Effects of Gelsemium sempervirens L. on pathway-focused gene expression profiling in neuronal cells. J Ethnopharmacol [Internet] 2014; 2 (02) 1-10 10.1016/j.jep.2014.02.048.
66 Lopes L., Godoy L.M.F., de Oliveira C.C., Gabardo J., Schadeck R.J.G., de Freitas Buchi D. Phagocytosis, endosomal/lysosomal system and other cellularaspects of macrophage activation by Canova medication. Micron [Internet] 2006; Jan [cited 2014 Jun 15] 37 (03) 277-287 Available from: http://www.ncbi.nlm.nih.gov/pubmed/16364649.
67 Nie F., Yu X.-L., Wang X.-G., Tang Y.-F., Wang L.-L., Ma L. Down-regulation of CacyBP is associated with poor prognosis and the effects on COX-2 expression in breast cancer. Int J Oncol 2010; 37 (05) 1261-1269.
68 Arora S., Aggarwal A., Singla P., Jyoti S., Tandon S. Anti-proliferative effects of homeopathic medicines on human kidney, colon and breast cancer cells. Homeopathy [Internet] 2013; Oct [cited 2014 Jun 15] 102 (04) 274-282 Available from: http://www.ncbi.nlm.nih.gov/pubmed/24050774.
69 Mukherjee A., Boujedaini N., Khuda-Bukhsh A.R. Homeopathic Thuja 30C ameliorates benzo(a)pyrene-induced DNA damage, stress and viability of perfused lung cells of mice in vitro. J Integr Med [Internet] 2013; Nov [cited 2014 Jun 15] 11 (06) 397-404 Available from: http://www.ncbi.nlm.nih.gov/pubmed/24299603.