Aim: To assess the effects of semi-refined carrageenan (E407a) on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation in peripheral blood mononuclear cells (PBMCs) and LPS-mediated cell membrane alterations in leukocytes.
Methods: Blood samples collected from 8 intact rats were incubated with E407a (10 mg/ml), E407a (50 mg/ml), LPS (1 µg/ml), E407a (10 mg/ml) + LPS (1 µg/ml), E407a (50 mg/ml) + LPS (1 µg/ml) and without those compounds (controls) for 2 h in RPMI 1640 medium enriched with 5% fetal bovine serum. ROS generation in PBMCs obtained from the incubated samples was estimated by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. The impact of E407a, LPS and their mixture on leukocyte cell membranes was evaluated spectrofluorimetrically using the fluorescent probe 2-(2¢-hydroxy-phenyl)-5-phenyl-1,3-oxazole.
Results: Expectedly, incubation with LPS induced ROS generation in PBMCs and decreased the lipid order of cell membranes in leukocytes. E407a alone was found to alter neither ROS production in PBMCs, nor membrane lipid order in leukocytes. Semi-refined carrageenan partially reduced LPS-mediated ROS overproduction in PBMCs and cell membrane alterations in leukocytes.
Conclusion: E407a attenuates LPS-induced alterations of redox homeostasis in rat PBMCs and LPS-mediated modifications of cell membrane lipid order in leukocytes.
Carocho M, Morales P, Ferreira ICFR. Natural food additives: Quo vadis? Trends in Food Science & Technology 2015; 45(2): 284-295. https://doi.org/10.1016/j.tifs.2015.06.007
David S, Shani Levi C, Fahoum L, et al. Revisiting the carrageenan controversy: do we really understand the digestive fate and safety of carrageenan in our foods? Food Funct 2018; 9(3): 1344-1352. https://doi.org/10.1039/C7FO01721A
Genicot-Joncour S, Poinas A, Richard O, et al. The cyclization of the 3,6-anhydro-galactose ring of iota-carrageenan is catalyzed by two D-galactose-2,6-sulfurylases in the red alga Chondrus crispus. Plant Physiol 2009; 151(3): 1609-16. https://doi.org/10.1104/pp.109.144329
McKim JM, Willoughby JA Sr, Blakemore WR, et al. Clarifying the confusion between poligeenan, degraded carrageenan, and carrageenan: A review of the chemistry, nomenclature, and in vivo toxicology by the oral route. Crit Rev Food Sci Nutr 2019; 59(19): 3054-3073. https://doi.org/10.1080/10408398.2018.1481822
Necas J, Bartosikova L. Carrageenan: a review. Veterinarni Medicina 2013; 58: 187-205. https://doi.org/10.17221/6758-VETMED
Tobacman JK, Wallace RB, Zimmerman MB. Consumption of carrageenan and other water-soluble polymers used as food additives and incidence of mammary carcinoma. Med Hypotheses 2001; 56: 589-98. https://doi.org/10.1054/mehy.2000.1208
Borthakur A, Bhattacharyya S, Anbazhagan AN, et al. Prolongation of carrageenan-induced inflammation in human colonic epithelial cells by activation of an NFκB-BCL10 loop. Biochim Biophys Acta 2012; 1822(8): 1300-7. https://doi.org/10.1016/j.bbadis.2012.05.001
Bhattacharyya S, Liu H, Zhang Z, et al. Carrageenan-induced innate immune response is modified by enzymes that hydrolyze distinct galactosidic bonds. J Nutr Biochem 2010; 21(10): 906-13. https://doi.org/10.1016/j.jnutbio.2009.07.002
Bhattacharyya S, Borthakur A, Pant N, et al. Bcl10 mediates LPS-induced activation of NF-κB and IL-8 in human intestinal epithelial cells. American Journal of Physiology—Gastrointestinal and Liver Physiology 2007; 293(2): 429-437. https://doi.org/10.1152/ajpgi.00149.2007
Borthakur A, Bhattacharyya S, Dudeja PK, et al. Carrageenan induces interleukin-8 production through distinct Bcl10 pathway in normal human colonic epithelial cells. American Journal of Physiology—Gastrointestinal and Liver Physiology 2007; 292(3): 829-838. https://doi.org/10.1152/ajpgi.00380.2006
Lopes AH, Silva RL, Fonseca MD, et al. Molecular basis of carrageenan-induced cytokines production in macrophages. Cell Commun Signal 2020 Sep 7; 18(1): 141. https://doi.org/10.1186/s12964-020-00621-x
Myers MJ, Deaver CM, Lewandowski AJ. Molecular mechanism of action responsible for carrageenan-induced inflammatory response. Mol Immunol 2019; 109: 38-42. https://doi.org/10.1016/j.molimm.2019.02.020
Bhattacharyya S, Gill R, Chen ML, et al. Toll-like receptor 4 mediates induction of the Bcl10-NFkappaB-interleukin-8 inflammatory pathway by carrageenan in human intestinal epithelial cells. J Biol Chem 2008 Apr 18; 283(16): 10550-8. https://doi.org/10.1074/jbc.M708833200
Korneev KV, Atretkhany KN, Drutskaya MS, et al. TLR-signaling and proinflammatory cytokines as drivers of tumorigenesis. Cytokine 2017 Jan; 89: 127-135. https://doi.org/10.1016/j.cyto.2016.01.021
Sokolova EV, Karetin Y, Davydova VN, et al. Carrageenans effect on neutrophils alone and in combination with LPS in vitro. J Biomed Mater Res A 2016; 104(7): 1603-9. https://doi.org/10.1002/jbm.a.35693
Tkachenko AS, Kot YG, Kapustnik VA, et al. Semi-refined carrageenan promotes reactive oxygen species (ROS) generation in leukocytes of rats upon oral exposure but not in vitro. Wien Med Wochenschr 2021; 171(3-4): 68-78. https://doi.org/10.1007/s10354-020-00786-7
Tkachenko AS, Onishchenko AI, Gorbach TV, et al. O-6-methylguanine-DNA methyltransferase (MGMT) overexpression in small intestinal mucosa in experimental carrageenan-induced enteritis. Malay. J. Biochem. Mol. Biol 2018,21 (3); 77-80.
Tkachenko AS, Marakushyn DI, Rezunenko YK, et al. A study of erythrocyte membranes in carrageenan-induced gastroenterocolitis by method of fluorescent probes. HVM Bioflux 2018; 10(2): 37-41.
Tkachenko A, Marakushyn D, Kalashnyk I, et al. A study of enterocyte membranes during activation of apoptotic processes in chronic carrageenan-induced gastroenterocolitis. Med Glas (Zenica) 2018; 15(2): 87-92.
Gubina-Vakyulyk GI, Gorbach TV, Tkachenko AS, et al. Damage and regeneration of small intestinal enterocytes under the influence of carrageenan induces chronic enteritis. Comparative Clinical Pathology, 2015; 24(6): 1473-1477. https://doi.org/10.1007/s00580-015-2102-3
Bhattacharyya S, Xue L, Devkota S, et al. Carrageenan-induced colonic inflammation is reduced in Bcl10 null mice and increased in IL-10-deficient mice. Mediators of Inflammation 2013; 2013: 13. https://doi.org/10.1155/2013/397642
Weiner ML, McKim JM. Comment on "Revisiting the carrageenan controversy: do we really understand the digestive fate and safety of carrageenan in our foods?" by S. David, C. S. Levi, L. Fahoum, Y. Ungar, E. G. Meyron-Holtz, A. Shpigelman and U. Lesmes, Food Funct., 2018, 9, 1344-1352. Food Funct 2019; 10(3): 1760-1762. https://doi.org/10.1039/C8FO01282B
McKim JM, Willoughby JA Sr, Blakemore WR, et al. Clarifying the confusion between poligeenan, degraded carrageenan, and carrageenan: A review of the chemistry, nomenclature, and in vivo toxicology by the oral route. Crit Rev Food Sci Nutr 2018: 1-20. https://doi.org/10.1080/10408398.2018.1481822
McKim JM Jr, Baas H, Rice GP, et al. Effects of carrageenan on cell permeability, cytotoxicity, and cytokine gene expression in human intestinal and hepatic cell lines. Food Chem Toxicol 2016; 96: 1-10. https://doi.org/10.1016/j.fct.2016.07.006
Weiner ML. Food additive carrageenan: Part II: A critical review of carrageenan in vivo safety studies. Critical Reviews in Toxicology 2014; 44: 244-69. https://doi.org/10.3109/10408444.2013.861798
Tkachenko AS, Onishchenko AI, Lesovoy VN, et al. Common food additive E407a affects BCL-2 expression in lymphocytes in vitro. Studia Univ. VG, SSV, 2019; 29(4): 169-76.
McKim JM Jr, Wilga PC, Pregenzer JF, et al. The common food additive carrageenan is not a ligand for Toll-Like- Receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model. Food Chem Toxicol 2015; 78: 153-8. https://doi.org/10.1016/j.fct.2015.01.003
Shang Q, Sun W, Shan X, et al. Carrageenan-induced colitis is associated with decreased population of anti-inflammatory bacterium, Akkermansia muciniphila, in the gut microbiota of C57BL/6J mice. Toxicol Lett 2017; 279: 87-95. https://doi.org/10.1016/j.toxlet.2017.07.904
Wu W, Zhen Z, Niu T, et al. κ-Carrageenan enhances lipopolysaccharide-induced interleukin-8 secretion by stimulating the Bcl10-NF-κB Pathway in HT-29 cells and aggravates C. freundii-induced inflammation in mice. Mediators Inflamm 2017; 2017: 8634865. https://doi.org/10.1155/2017/8634865
Doroshenko AO, Posokhov EA, Shershukov VM, et al. Spectral and luminescence properties of derivatives of 2-aryl-[9,10]-phenanthroxazole. Chem. Heterocycl. Comp 1995; 31(4): 492-499. https://doi.org/10.1007/BF01177024
Posokhov YO, Kyrychenko A, Korniyenko Y. Derivatives of 2,5-diaryl-1,3-oxazole and 2,5-diaryl-1,3,4-oxadiazole as environment-sensitive fluorescent probes for studies of biological membranes. Reviews in Fluorescence 2017 (editor C.D. Geddes), Springer Nature Switzerland AG, Chapter 9; 2018. pp 199-230. https://doi.org/10.1007/978-3-030-01569-5_9
Doroshenko AO, Posokhov EA, Verezubova AA, et al. Radiationless deactivation of excited phototautomer form and molecular structure of ESIPT- compounds. Photochem Photobiol Sci 2002; 1: 92-9. https://doi.org/10.1039/b107255m
Doroshenko AO, Posokhov EA, Shershukov VM, et al. Intramolecular proton-transfer reaction in an excited state in a series of ortho-hydroxy derivatives of 2,5-diaryloxazole. High Energy Chemistry 1997; 31(6): 388-394.
Posokhov Y, Kyrychenko A. Location of fluorescent probes (2-hydroxy derivatives of 2,5-diaryl-1,3-oxazole) in lipid membrane studied by fluorescence spectroscopy and molecular dynamics simulation. Biophysical Chemistry 2018; 235: 9-18. https://doi.org/10.1016/j.bpc.2018.01.005
Kurad D, Jeschke G, Marsh D. Lipid membrane polarity profiles by high-field EPR. Biophys. J 2003; 85: 1025-1033. https://doi.org/10.1016/S0006-3495(03)74541-X
Bartucci R, Guzzi R, Marsh D, et al. Intramembrane polarity by electron spin echo spectroscopy of labeled lipids. Biophys. J 2003; 84: 1025-1030. https://doi.org/10.1016/S0006-3495(03)74918-2
Ho C, Slater SJ, Stubbs CD. Hydration and order in lipid bilayers. Biochemistry 1995; 34: 6188- 6195. https://doi.org/10.1021/bi00018a023
Binder H, Gawrisch K. Effect of unsaturated lipid chains on dimensions, molecular order and hydration of membranes. J. Phys. Chem., B 2001; 105: 12378- 12390. https://doi.org/10.1021/jp010118h
Noethig-Laslo V, Šentjurc M. Transmembrane polarity profile of lipid membranes. Advances in Planar Lipid Bilayers and Liposomes 2006, Academic Press, Elsevier, Vol. 5, Chapter 13, pp. 365-415. https://doi.org/10.1016/S1554-4516(06)05013-7
Kuzmich NN, Sivak KV, Chubarev VN, et al. TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis. Vaccines (Basel) 2017; 5(4): 34. https://doi.org/10.3390/vaccines5040034
Murdock JL, Núñez G. TLR4: The winding road to the discovery of the LPS Receptor. J Immunol 2016; 197(7): 2561-2. https://doi.org/10.4049/jimmunol.1601400
Wan J, Shan Y, Fan Y, et al. NF-κB inhibition attenuates LPS-induced TLR4 activation in monocyte cells. Mol Med Rep 2016; 14(5): 4505-4510. https://doi.org/10.3892/mmr.2016.5825
Ngkelo A, Meja K, Yeadon M, et al. LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Giα dependent PI-3kinase signalling. J Inflamm (Lond) 2012; 9(1): 1. https://doi.org/10.1186/1476-9255-9-1
Harayama T, Shimizu T. Roles of polyunsaturated fatty acids, from mediators to membranes. J Lipid Res 2020; 61(8): 1150-1160. https://doi.org/10.1194/jlr.R120000800
Catalá Á. Lipid peroxidation modifies the assembly of biological membranes "The Lipid Whisker Model". Front Physiol 2015; 5: 520. https://doi.org/10.3389/fphys.2014.00520
Tkachenko A, Pogozhykh D, Onishchenko A, et al. Gadolinium Orthovanadate GdVO4: Eu3+ Nanoparticles Ameliorate Carrageenan-Induced Intestinal Inflammation. J Pharm Nutr Sci 2021; 11(1): 40-48. https://doi.org/10.29169/1927-5951.2021.11.06
Tkachenko A, Onishchenko A. Oral Intake of Semi-refined Carrageenan by Rats Affects Apoptosis of Lymphocytes. Annals of Colorectal Research, 2020; 8(4): 170-174.
Tkachenko A. Reactive oxygen species (ROS) generation by lymphocytes in rats treated with a common food additive E407a. J Clin Med Kaz 2020; 1(55): 22-26. https://doi.org/10.23950/1812-2892-JCMK-00744
Nacife VP, Soeiro Mde N, Gomes RN, et al. Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo. Cell Struct Funct 2004; 29(2): 27-34. https://doi.org/10.1247/csf.29.27
Ogata M, Matsui T, Kita T, et al. Carrageenan primes leukocytes to enhance lipopolysaccharide-induced tumor necrosis factor alpha production. Infect Immun 1999; 67(7): 3284-9. https://doi.org/10.1128/IAI.67.7.3284-3289.1999
Yermak IM, Volod'ko AV, Khasina EI, et al. Inhibitory Effects of Carrageenans on Endotoxin-Induced Inflammation. Mar Drugs 2020; 18(5): 248. https://doi.org/10.3390/md18050248
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Copyright (c) 2021 Dr. Yevgen Posokhov, Dr. Anatolii Onishchenko, Prof. Tetyana Chumachenko, Prof. Nataliia Makieieva, Dr. Yuliia Kalashnyk-Vakulenko, Dr. Hanna Polikarpova, Dr. Viktoriia Novikova, Dr. Volodymyr Prokopyuk, Prof. Oksana Nakonechna, Dr. Dmytro Chumachenko, Prof. Viktoriya Tkachenko, Dr. Ievgen Meniailov, Maryna Tkachenko, Assoc. Prof. Dr. Anton Tkachenko