In vitro effects of Zinc in soluble and homeopathic formulations on macrophages and astrocytes

Zinc is an important metal in body homeostasis. Zinc in soluble form (Zn²⁺) and homeopathic Zincum metallicum were tested in macrophages and astrocytes in order to investigate its potential toxic or therapeutic effects. We evaluated cell viability (WST assay), cytokine production such as tumour necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) by enzyme-linked immunosorbent assay (ELISA) and nitric oxide release by Griess reaction. The effect of zinc-depletion and high zinc pre-treatments on the cell adaptation capability was also investigated. In THP-1 macrophage cell line and in human primary macrophages, Zn²⁺ at sub-toxic doses (30 μM) caused stimulation of TNF-α and IL-10 with different dynamics reaching the maximum peak at the zinc concentration 100 μM, before the cell death. Highest doses (300 μM) impaired dramatically cell vitality. Similar effects on cell viability were obtained also in C6 astrocytes, where Zn²⁺ slightly increased the nitric oxide release only in cells activated by one of the pro-inflammatory stimuli used in our cellular model (interferon gamma plus TNF-α). Zinc depletion markedly reduced IL-10 production and cell viability. Zincum metallicum did not cause toxicity in any cell type and showed some small stimulation in WST assay that was statistically significant in a few experimental conditions.

Keywords

Zincum metallicum - Zn²⁺ - Homeopathy - Macrophage - Astrocyte - Cytokine production - Inflammation - Nitric oxide - Hormesis

• References

• 1 Mayer L.S., Uciechowski P., Meyer S., Schwerdtle T., Rink L., Haase H. Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of pro-inflammatory cytokines by human monocytes. Metallomics 2014; 6: 1288-1295.
• 2 Maret W., Sandstead H.H. Zinc requirements and the risks and benefits of zinc supplementation. J Trace Elem Med Biol 2006; 20: 3-18.
• 3 Dubben S., Honscheid A., Winkler K., Rink L., Haase H. Cellular zinc homeostasis is a regulator in monocyte differentiation of HL-60 cells by 1 alpha,25-dihydroxyvitamin D3. J Leukoc Biol 2010; 87: 833-844.
• 4 Bao B., Prasad A.S., Beck F.W., Godmere M. Zinc modulates mRNA levels of cytokines. Am J Physiol Endocrinol Metab 2003; 285: E1095-E1102.
• 5 von Bülow V., Dubben S., Engelhardt G. et al Zinc-dependent suppression of TNF-alpha production is mediated by protein kinase A-induced inhibition of Raf-1, I kappa B kinase beta, and NF-kappa B. J Immunol 2007; 179: 4180-4186.
• 6 Siebenlist U., Franzoso G., Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol 1994; 10: 405-455.
• 7 Haase H., Ober-Blobaum J.L., Engelhardt G. et al Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes. J Immunol 2008; 181: 6491-6502.
• 8 Sensi S.L., Paoletti P., Bush A.I., Sekler I. Zinc in the physiology and pathology of the CNS. Nat Rev Neurosci 2009; 10: 780-791 http://dx.doi.org/10.1038/nrn2734.
• 9 Chiarini A., Whitfield J., Bonafini C., Chakravarthy B., Armato U., Dal P.I. Amyloid-beta(25-35), an amyloid-beta(1-42) surrogate, and proinflammatory cytokines stimulate VEGF-A secretion by cultured, early passage, normoxic adult human cerebral astrocytes. J Alzheimers Dis 2010; 21: 915-926.
• 10 Beyersmann D., Haase H. Functions of zinc in signaling, proliferation and differentiation of mammalian cells. Biometals 2001; 14: 331-341.
• 11 Jacob C., Maret W., Vallee B.L. Control of zinc transfer between thionein, metallothionein, and zinc proteins. Proc Natl Acad Sci USA 1998; 95: 3489-3494.
• 12 Frederickson C.J., Cuajungco M.P., LaBuda C.J., Suh S.W. Nitric oxide causes apparent release of zinc from presynaptic boutons. Neuroscience 2002; 115: 471-474.
• 13 Krezel A., Hao Q., Maret W. The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling. Arch Biochem Biophys 2007; 463: 188-200.
• 14 Cuajungco M.P., Lees G.J. Zinc metabolism in the brain: relevance to human neurodegenerative disorders. Neurobiol Dis 1997; 4: 137-169.
• 15 Cortese-Krott M.M., Kulakov L., Oplander C., Kolb-Bachofen V., Kroncke K.D., Suschek C.V. Zinc regulates iNOS-derived nitric oxide formation in endothelial cells. Redox Biol 2014; 2: 945-954.
• 16 Pfaender S., Fohr K., Lutz A.K. et al Cellular zinc homeostasis contributes to neuronal differentiation in human induced pluripotent stem cells. Neural Plast 2016; 2016: 3760702 http://dx.doi.org/10.1155/2016/3760702.
• 17 Peters T.L., Beard J.D., Umbach D.M. et al Blood levels of trace metals and amyotrophic lateral sclerosis. Neurotoxicology 2016; 54: 119-126.
• 18 Singla N., Dhawan D.K. Zinc improves cognitive and neuronal dysfunction during aluminium-induced neurodegeneration. Mol Neurobiol 2017; 54: 406-422.
• 19 Kumar H., Katyal J., Gupta Y.K. Low dose zinc supplementation beneficially affects seizure development in experimental seizure models in rats. Biol Trace Elem Res 2015; 163: 208-216 http://dx.doi.org/10.1007/s12011-014-0181-7.
• 20 Mocchegiani E., Giacconi R., Fattoretti P. et al Metallothionein isoforms (I+II and III) and interleukin-6 in the hippocampus of old rats: may their concomitant increments lead to neurodegeneration?. Brain Res Bull 2004; 63: 133-142.
• 21 Orre M., Kamphuis W., Osborn L.M. et al Acute isolation and transcriptome characterization of cortical astrocytes and microglia from young and aged mice. Neurobiol Aging 2014; 35: 1-14.
• 22 Ribeiro S.M., Braga C.B., Peria F.M. et al Effect of zinc supplementation on antioxidant defenses and oxidative stress markers in patients undergoing chemotherapy for colorectal cancer: a placebo-controlled, prospective randomized trial. Biol Trace Elem Res 2016; 169: 8-16.
• 23 Varea E., Alonso-Llosa G., Molowny A., Lopez-Garcia C., Ponsoda X. Capture of extracellular zinc ions by astrocytes. Glia 2006; 54: 304-315.
• 24 Sruthi S., Mohanan P.V. Investigation on cellular interactions of astrocytes with zinc oxide nanoparticles using rat C6 cell lines. Colloids Surf B Biointerfaces 2015; 133: 1-11.
• 25 Haase H., Mazzatti D.J., White A. et al Differential gene expression after zinc supplementation and deprivation in human leukocyte subsets. Mol Med 2007; 13: 362-370.
• 26 Floriańczyk B., Trojanowski T. Inhibition of respiratory processes by overabundance of zinc in neuronal cells. Folia Neuropathol 2009; 47: 234-239.
• 27 Hsieh H., Vignesh K.S., Deepe G.S., Choubey D., Shertzer H.G., Genter M.B. Mechanistic studies of the toxicity of zinc gluconate in the olfactory neuronal cell line Odora. Toxicol In Vitro 2016; 35: 24-30 http://dx.doi.org/10.1016/j.tiv.2016.05.003.
• 28 Ke Q., Li J., Ding J. et al Essential role of ROS-mediated NFAT activation in TNF-alpha induction by crystalline silica exposure. Am J Physiol Lung Cell Mol Physiol 2006; 291: L257-L264.
• 29 Ho E., Ames B.N. Low intracellular zinc induces oxidative DNA damage, disrupts p53, NFkappa B, and AP1 DNA binding, and affects DNA repair in a rat glioma cell line. Proc Natl Acad Sci USA 2002; 99: 16770-16775.
• 30 Kruczek C., Gorg B., Keitel V. et al Hypoosmotic swelling affects zinc homeostasis in cultured rat astrocytes. Glia 2009; 57: 79-92.
• 31 Haase H., Watjen W., Beyersmann D. Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells. Biol Chem 2001; 382: 1227-1234.