Environmental Homeopathy: Homeopathic Potencies Regulate the Toxicity and Growth of Raphidiopsis raciborskii (Cyanobacteria) and can be Tracked Physico-Chemically. Part 1: Biological Results

Abstract

Introduction Cyanobacteria are microorganisms found in many parts of the world and several genera, such as Raphidiopsis raciborskii, are producers of cyanotoxins. Homeopathic potencies have been found to modulate toxicity in different biological models, and the present study endeavors to discover whether this might also be the case with cyanobacteria.

Objectives Our objective was to investigate the possible effects of homeopathic potencies on the resilience of Artemia franciscana (brine shrimp) embryos to saxitoxin (STX; cyanotoxin) and on controlling the growth of R. raciborskii in vitro.

Method A. franciscana cysts were cultivated in seawater in 96-well plates to evaluate the hatching rate and vitality, plus the gene expression of heat shock proteins (HSPs), after being challenged with R. raciborskii extract containing 2.5 µg/L of STX and treated with different homeopathic potencies. Untreated wells were used as controls (“base-line”). Potencies were chosen from a screening process based on seven selected homeopathic preparations according to the similitude of STX symptoms (Sulphur, Zincum metallicum, Nitric acidum, Plumbum metallicum, Mercurius solubilis, Phosphoric acidum, Isotherapic from R. raciborskii extract; all at 6cH, 30cH and 200cH). Cultures of R. raciborskii maintained in an artificial seawater medium were equally treated with screened homeopathic potencies selected from the same list but specifically for their growth control as a function of time.

Results A 15% lower rate of hatching of A. franciscana cysts was observed after treatment with Nitric acidum 6cH in comparison with baseline (p = 0.05). A complete toxicity reversal was seen after treatment with Isotherapic 200cH, with a 23-fold increase of Hsp 26 gene expression (p = 0.023) and a 24-fold increase of p26 gene expression (p ≤ 0.001) in relation to baseline. Nitric acidum 200cH and Mercurius solubilis 30cH limited the exponential growth of cyanobacteria up to 95% and 85% respectively (p ≤ 0.003) in relation to baseline. Succussed water presented only a transitory 50% inhibition effect.

Conclusion Isotherapic 200cH improved A. franciscana bioresilience to STX; Nitric acidum 200cH and Mercurius solubilis 30cH showed the optimal performance on limiting R. raciborskii growth. The results point to the potential of homeopathic potencies to mitigate environmental problems related to water quality.

Keywords

saxitoxins - Artemia franciscana - bioresilience - water - HSPs - eco-toxicology

Author Attributions

S.N.M. was the main researcher, PhD student, involved in all experimental procedures.

A.A.G.P. was responsible for experimental procedures—toxicity and solvatochromic dyes tests.

R.A.S. was responsible for experimental procedures—gene expression tests.

I.B.S. was responsible for experimental procedures—solvatochromic dyes tests.

A.T. was responsible for discussion of results on physicochemical parameters.

S.J.C. was responsible for experimental design with solvatochromic dyes and discussion of results.

J.S.Y. was co-adviser, providing cyanobacteria standards, and was responsible for the discussion of results.

L.V.B. was the main adviser and was responsible for the coordination of all steps of the study and discussion of results.

Publication History

Received: 25 August 2023

Accepted: 14 December 2023

Article published online:
06 May 2024

• References

• 1 Yunes JS, Cunha N, Proença LAO, Barros LPC, Monressat JM. Cyanobacterial neurotoxins from southern Brazilian freshwaters. Comments on Toxicology, USA. 2023; 9: 103-115
• 2 Lagos N, Onodera H, Zagatto PA, Andrinolo D, Azevedo SM, Oshima Y. The first evidence of paralytic shellfish toxins in the fresh water cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil. Toxicon 1999; 37: 1359-1373
• 3 Vico P, Bonilla S, Cremella B, Aubriot L, Iriarte A, Piccini C. Biogeography of the cyanobacterium Raphidiopsis (Cylindrospermopsis) raciborskii: integrating genomics, phylogenetic and toxicity data. Mol Phylogenet Evol 2020; 148: 106824
• 4 Pfennig N, Widdel F. The bacteria of the sulphur cycle. Philos Trans R Soc Lond B Biol Sci 1982; 298: 433-441
• 5 Diehl F, Ramos PB, Dos Santos JM, Barros DM, Yunes JS. Behavioral alterations induced by repeated saxitoxin exposure in drinking water. J Venom Anim Toxins Incl Trop Dis 2016; 22: 18
• 6 Zagatto PA, Buratini SV, Aragão MA, Ferrão-Filho AS. Neurotoxicity of two Cilindrospermo raciborskii (cyanobacteria) strains to mice, Daphnia, and fish. Environ Toxicol Chem 2012; 31: 857-862
• 7 Vilar MCP, Molica RJR. Changes in pH and dissolved inorganic carbon in water affect the growth, saxitoxins production and toxicity of the cyanobacterium Raphidiopsis raciborskii ITEP-A1. Harmful Algae 2020; 97: 101870
• 8 Magalhães VF, Soares RM, Azevedo SM. Microcystin contamination in fish from the Jacarepaguá Lagoon (Rio de Janeiro, Brazil): ecological implication and human health risk. Toxicon 2001; 39: 1077-1085
• 9 Facey JA, Apte SC, Mitrovic SM. A review of the effect of trace metals on freshwater cyanobacterial growth and toxin production. Toxins (Basel) 2019; 11: 643
• 10 Carvalho CA, Matta SLP, Melo FCSA. et al. Cipó-cravo (Tynnanthus fasciculatus miers – Bignoniaceae): Estudo fitoquímico etoxicológico envolvendo Artemia salina . Revista Eletrônica de Farmácia 2009; 6: 51-57
• 11 Tan J, MacRae TH. Stress tolerance in diapausing embryos of Artemia franciscana is dependent on heat shock factor 1 (Hsf1). PLoS One 2018; 13: e0200153
• 12 King AM, Toxopeus J, MacRae TH. Functional differentiation of small heat shock proteins in diapause-destined Artemia embryos. FEBS J 2013; 280: 4761-4772
• 13 MacRae TH. Stress tolerance during diapause and quiescence of the brine shrimp, Artemia. Cell Stress Chaperones 2016; 21: 9-18
• 14 Han J, Park Y, Shin HH. et al. Effects of dinoflagellate Gymnodinium catenatum on swimming behavior and expression of heat shock protein (hsp) genes in the brine shrimp Artemia franciscana . Harmful Algae 2021; 110: 102146
• 15 Gbotsyo YA, Rowarth NM, Weir LK, MacRae TH. Short-term cold stress and heat shock proteins in the crustacean Artemia franciscana . Cell Stress Chaperones 2020; 25: 1083-1097
• 16 De Graaf J, Amons R, Möller W. The primary structure of artemin from Artemia cysts. Eur J Biochem 1990; 193: 737-750
• 17 Wang S, Sun S. Comparative observations on the cyst shells of seven Artemia strains from China. Microsc Res Tech 2007; 70: 663-670
• 18 Artemia franciscana Kellogg,. 1906. In: GBIF Backbone Taxonomy. Accessed December 4, 2023 , at: https://www.gbif.org/species/5180091
• 19 Svirčev Z, Obradović V, Codd GA. et al. Massive fish mortality and Cylindrospermopsis raciborskii bloom in Aleksandrovac Lake. Ecotoxicology 2016; 25: 1353-1363
• 20 Narayana S, Chitra J, Tapase SR. et al. Toxicity studies of Trichodesmium erythraeum (Ehrenberg, 1830) bloom extracts from Phoenix Bay, Port Blair, Andamans. Harmful Algae 2014; 40: 34-39
• 21 Abatzopoulos TJ, Beardmore J, Clegg JS, Sorgeloos P. Artemia: basic and applied biology. Dordrecht, Springer. 1st ed. Reprint; 2010
• 22 Pinto AAG, Nagai MYO, Coimbra EN. et al. Bio-resilience to mercury chloride of the brine shrimp Artemia Salina after treatment with homeopathic Mercurius corrosivus . Homeopathy 2021; 110: 244-255
• 23 Coimbra Melo EN. Efeito protetor do isoterápico sobre a eclosão de cistos de Artemia salina intoxicadas com arseniato de sódio [PhD thesis]. São Paulo: Graduation Program on Environmental and Experimental Pathology, Universidade Paulista—UNIP; 2020. Accessed on April 22, 2023 at: https://www.unip.br/cursos/pos_graduacao/strictosensu/patologia/teses_defendidas_2020.aspx
• 24 Nagai MO, Pinto AAG, von Ancken AC. et al. Exposure of Artemia salina to glyphosate and bioremediation by isotherapy. Proceedings of the XXXIV GIRI meeting. Int J High Dilution Res. 2022; 21: 13
• 25 Brasil. ANVISA. Farmacopeia Homeopática Brasileira, 3a. ed. 2011. Accessed April 22, 2023 at: https://www.gov.br/anvisa/pt-br/assuntos/farmacopeia/farmacopeia-homeopatica
• 26 Das D, De A, Dutta S, Biswas R, Boujedaini N, Khuda-Bukhsh AR. Potentized homeopathic drug Arsenicum album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate. J Chin Integr Med 2011; 9: 752-760
• 27 Jäger T, Scherr C, Simon M, Heusser P, Baumgartner S. Effects of homeopathic arsenicum album, nosode, and gibberellic acid preparations on the growth rate of arsenic-impaired duckweed (Lemna gibba L.). ScientificWorldJournal 2010; 10: 2112-2129
• 28 Lahnstein L, Binder M, Thurneysen A. et al. Isopathic treatment effects of Arsenicum album 45x on wheat seedling growth–further reproduction trials. Homeopathy 2009; 98: 198-207
• 29 Calabrese EJ, Giordano J. Ultra low doses and biological amplification: approaching Avogadro's number. Pharmacol Res 2021; 170: 105738
• 30 Madl P, Renati P. Quantum electrodynamics coherence and hormesis: foundations of quantum biology. Int J Mol Sci 2023; 24: 14003
• 31 Nagai MYDO, Mohammad SN, Pinto AAG. et al. Highly diluted glyphosate mitigates its effects on Artemia salina: physicochemical implications. Int J Mol Sci 2023; 24: 9478
• 32 Bonamin LV, Pedro RRP, Mota HMG. et al. Characterization of Antimonium crudum activity using solvatochromic dyes. Homeopathy 2020; 109: 79-86
• 33 Cartwright SJ. Solvatochromic dyes detect the presence of homeopathic potencies. Homeopathy 2016; 105: 55-65
• 34 Cartwright SJ. Interaction of homeopathic potencies with the water soluble solvatochromic dye bis-dimethylaminofuchsone. Part 1: pH studies. Homeopathy 2017; 106: 37-46
• 35 Aparicio ACC, de Oliveira LHS, Silva JS. et al. Interaction between solvatochromic dyes and water sampled from a natural source treated with high dilutions of phosphorus. Homeopathy 2020; 109: 126-132
• 36 Cartwright SJ. Degree of response to homeopathic potencies correlates with dipole moment size in molecular detectors: implications for understanding the fundamental nature of serially diluted and succussed solutions. Homeopathy 2018; 107: 19-31
• 37 Cartwright SJ. Homeopathic potencies may possess an electric field(-like) component: evidence from the use of encapsulated solvatochromic dyes. Homeopathy 2020; 109: 14-22
• 38 Cartwright SJ. Immobilization of solvatochromic dyes on transparent cellulose films: an improved method for the study of homeopathic potencies. Homeopathy 2023; 112: 125-134
• 39 Pinto SAG, Aparicio ACC, Souza JS, Suffredini IB, Cartwright SJ, Bonamin LV. Characterization of physicochemical markers for homeopathic medicines and biological supernatant samples. Proceedings of the XXXIV GIRI meeting. Int J High Dilution Res 2022; 21: 04
• 40 Mohammad SN, Pinto AAG, da Silva RA. et al. Environmental homeopathy: Homeopathic potencies regulate the growth and toxicity of Raphidiopsis raciborskii (cyanobacteria) and can be tracked physico-chemically. Part 2: Physico-chemical models. Homeopathy 2024; (e-pub ahead of print).
• 41 Rourke WA, Murphy CJ, Pitcher G. et al. Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue. J AOAC Int 2008; 91: 589-597
• 42 Ramos PB, Colombo GM, Schmitz MJ. et al. Chemoprotection mediated by açaí berry (Euterpe oleracea) in white shrimp Litopenaeus vannamei exposed to the cyanotoxin saxitoxin analyzed by in vivo assays and docking modeling. Aquat Toxicol 2022; 246: 106148
• 43 Konovalov A, Ryzhkina I, Maltzeva E. et al. Nanoassociate formation in highly diluted water solutions of potassium phenosan with and without permalloy shielding. Electromagn Biol Med 2015; 34: 141-146
• 44 Cairo N. Guia de Medicina Homeopática. São Paulo: Teixeira Publishers.; 21st reprint; 1994
• 45 Rio DC, Ares MJr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010
• 46 Gorham PR, Mclachlav JR, Hammer VT, Kim WK. Isolation and culture of toxic strains of Anabaena flos aquae (Lyngb.) de Bréd. Verh. Int. Verein. Theor. Angew. Limnol. 1964; 15: 796-804
• 47 Brasil. Ministério da Saúde. Orientações técnicas para o monitoramento de cianobactérias/cianotoxinas nos mananciais de abastecimento de água para consumo humano. Brasilia, DF, Brazil; 2016. Available at: https://bvsms.saude.gov.br/bvs/publicacoes/orientacoes_tecnicas_cianobacterias_cianotoxinas_agua.pdf . Consulted on April 13, 2023
• 48 Huo D, Gan N, Geng R. et al. Cyanobacterial blooms in China: diversity, distribution, and cyanotoxins. Harmful Algae 2021; 109: 102106
• 49 Carmichael W. A world overview – one-hundred-twenty-seven years of research on toxic cyanobacteria – where do we go from here?. Adv Exp Med Biol 2008; 619: 105-125
• 50 Godoy RFB, Trevisan E, Battistelli AA, Crisigiovanni EL, do Nascimento EA, da Fonseca Machado AL. Does water temperature influence in microcystin production? A case study of Billings Reservoir, São Paulo, Brazil. J Contam Hydrol 2023; 255: 104164
• 51 Zhao CS, Shao NF, Yang ST. et al. Predicting cyanobacteria bloom occurrence in lakes and reservoirs before blooms occur. Sci Total Environ 2019; 670: 837-848
• 52 Gobler CJ, Burkholder JM, Davis TW. et al. The dual role of nitrogen supply in controlling the growth and toxicity of cyanobacterial blooms. Harmful Algae 2016; 54: 87-97
• 53 Coffer MM, Schaeffer BA, Foreman K. et al. Assessing cyanobacterial frequency and abundance at surface waters near drinking water intakes across the United States. Water Res 2021; 201: 117377
• 54 Nagai MYO, Von Ancken ACB, Bonamin LV. Effects of highly diluted substances on aquatic animals: a review. Water: Multidiscipl Res J 2022; Special Edition: 1-10
• 55 Sinclair JR. Importance of a One Health approach in advancing global health security and the Sustainable Development Goals. Rev Sci Tech 2019; 38: 145-154
• 56 Lubroth J. FAO and the One Health approach. Curr Top Microbiol Immunol 2013; 366: 65-72
• 57 Silva RDS, Chia MA, Barbosa VV. Synergistic effects of temperature and nutrients on growth and saxitoxin content of the cyanobacterium Raphidiopsis raciborskii . J Appl Phycol 2022; 34: 941-952
• 58 Werner VR, Tucci A, da Silva LM. et al. Morphological, ecological, and toxicological aspects of Raphidiopsis raciborskii (Cyanobacteria) in a eutrophic urban subtropical lake in southern Brazil. Iheringia. Série Botânica, Porto Alegre. 2020; 75: e2020018
• 59 Galvanese EF, Padial AA, Aubriot L. Acclimation at high temperatures increases the ability of Raphidiopsis raciborskii (Cyanobacteria) to withstand phosphate deficiency and reveals distinct strain responses. Eur J Phycol 2019; 54: 359-368
• 60 Malitan HS, Cohen AM, MacRae TH. Knockdown of the small heat-shock protein p26 by RNA interference modifies the diapause proteome of Artemia franciscana . Biochem Cell Biol 2019; 97: 471-479
• 61 King AM, MacRae TH. The small heat shock protein p26 aids development of encysting Artemia embryos, prevents spontaneous diapause termination and protects against stress. PLoS One 2012; 7: e43723
• 62 Beristain P, Gajardo G, Bossier P. Species-specific RFLP pattern in the Heat Shock Protein 26 gene (Hsp26): a single-locus tool for species identification and experimental testing of habitat-induced isolation in the New World Artemia species. Mol Ecol Resour 2010; 10: 229-231
• 63 Qiu Z, Bossier P, Wang X, Bojikova-Fournier S, MacRae TH. Diversity, structure, and expression of the gene for p26, a small heat shock protein from Artemia. Genomics 2006; 88: 230-240
• 64 Calderwood SK, Mambula SS, Gray Jr PJ, Theriault JR. Extracellular heat shock proteins in cell signaling. FEBS Lett 2007; 581: 3689-3694
• 65 Carver JA, Rekas A, Thorn DC, Wilson MR. Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function?. IUBMB Life 2003; 55: 661-668
• 66 Yinnon T, Konovalov A. Domains formation mediated by electromagnetic fields in dilute solutions. Annual Conference on the Physics, Chemistry and Biology of Water, Bulgaria, 2014
• 67 Yinnon A, Liu ZQ. Domains formation mediated by electromagnetic fields in very dilute aqueous solutions: Pt-1. Quantum electrodynamic aspects. Water: Multidiscipl Res J 2015; 7: 33-47
• 68 Yinnon A, Liu ZQ. Domains formation mediated by electromagnetic fields in very dilute aqueous solutions: Pt-2. Quantum electrodynamic analyses of experimental data on strong electrolyte solutions. Water: Multidiscipl Res J 2015; 7: 48-69
• 69 Jerman I, Ogrizek L, Periček Krapež V, Jan L. Physicochemical study of the molecular signal transfer of ultra-high diluted antibodies to interferon-gamma. Int J Mol Sci 2023; 24: 11961
• 70 Castillo H, Schoderbek D, Dulal S. et al. Stress induction in the bacteria Shewanella oneidensis and Deinococcus radiodurans in response to below-background ionizing radiation. Int J Radiat Biol 2015; 91: 749-756
• 71 Xue X, Ali YF, Liu C. et al. Geomagnetic shielding enhances radiation resistance by promoting DNA repair process in human bronchial epithelial cells. Int J Mol Sci 2020; 21: 9304