Abstract
Objectives
The aim of our study was to investigate whether N-acetyl-l-cysteine (NAC) could protect stem cells from exfoliated deciduous teeth (SHED) against oxidative damage, during in vitro cultivation, to preserve regenerative potential of these cells. Accordingly, we examined the potential of cell culture supplementation with NAC in prevention of lipid peroxidation, unfavorable changes of total lipids fatty acid composition, and the effects on the activity of antioxidant enzymes.
Material and methods
We analyzed the extent of oxidative damage in SHED after 48 h treatment with different NAC concentrations. Cellular lipid peroxidation was determined upon reaction with thiobarbituric acid. All enzyme activities were measured spectrophotometrically, based on published methods. Fatty acid methyl esters were analyzed by gas-liquid chromatography.
Results
Concentration of 0.1 mM NAC showed the most profound effects on SHED, significantly decreasing levels of lipid peroxidation in comparison to control. This dose also diminished the activities of antioxidant enzymes. Furthermore, NAC treatment significantly changed fatty acid composition of cells, reducing levels of oleic acid and monounsaturated fatty acids and increasing linoleic acid, n-6, and total polyunsaturated fatty acid (PUFA) proportions.
Conclusion
Low dose of NAC significantly decreased lipid peroxidation and altered fatty acid composition towards increasing PUFA. The reduced oxidative damage of cellular lipids could be strongly related to improved SHED survival in vitro.
Clinical relevance
Low doses of antioxidants, applied during stem cells culturing and maintenance, could improve cellular characteristics in vitro. This is prerequisite for successful use of stem cells in various clinical applications.
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References
Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, Geurtsen W (2011) Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth. Calcif Tissue Int 88(2):130–141. https://doi.org/10.1007/s00223-010-9438-0
Karadzic I, Vucic V, Jokanovic V, Debeljak-Martacic J, Markovic D, Petrovic S, Glibetic M (2015) Effects of novel hydroxyapatite-based 3D biomaterials on proliferation and osteoblastic differentiation of mesenchymal stem cells. J Biomed Mater Res Part A 103(1):350–357. https://doi.org/10.1002/jbm.a.35180
Xiao Y, Li X, Cui Y, Zhang J, Liu L, Xie X, Hao H, He G, Kander MC, Chen M, Liu Z, Verfaillie CM, Zhu H, Lei M, Liu Z (2014) Hydrogen peroxide inhibits proliferation and endothelial differentiation of bone marrow stem cells partially via reactive oxygen species generation. Life Sci 112(1–2):33–40. https://doi.org/10.1016/j.lfs.2014.07.016
Esmaeli S, Allameh A, Soleimani M, Rahbarizadeh F, Frouzandeh-Moghadam M (2014) The role of albumin and PPAR-α in differentiation-dependent change of fatty acid profile during differentiation of mesenchymal stem cells to hepatocyte-like cells. Cell Biochem Funct 32(5):410–419. https://doi.org/10.1002/cbf.3031
Das UN (2006) Essential fatty acids: biochemistry, physiology and pathology. Biotechnol J 1(4):420–439. https://doi.org/10.1002/biot.200600012
Kelly GS (1998) Clinical applications of N-acetylcysteine. Altern Med Rev 3(2):114–127
Fan J, Cai H, Yang S, Yan L, Tan W (2008) Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34(+) cells. Comp Biochem Physiol B Biochem Mol Biol 151(2):153–158. https://doi.org/10.1016/j.cbpb.2008.06.008
Song H, Cha MJ, Song BW, Kim IK, Chang W, Lim S, Choi EJ, Ham O, Lee SY, Chung N, Jang Y, Hwang KC (2010) Reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex. Stem Cells 28(3):555–563. https://doi.org/10.1002/stem.302
Martacić JD, Francuski J, Luzajić T, Vuković N, Mojsilović S, Drndarević N, Petakov M, Glibetić M, Marković D, Radovanović A, Todorović V, Filipović MK (2014) Characterization of deciduous teeth stem cells isolated from crown dental pulp. Vojnosanit Pregl 71(8):735–741. https://doi.org/10.2298/VSP1408735D
Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes Massironi SM, Pereira LV, Caplan AI, Cerruti HF (2006) Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs 184(3–4):105–116. https://doi.org/10.1159/000099617
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317. https://doi.org/10.1080/14653240600855905
Cynamon HA, Isenberg JN, Nguyen CH (1984) A rapid method for erythrocyte membrane phospholipid determination. Clin Chim Acta 144(1):65–70. https://doi.org/10.1016/0009-8981(84)90261-4
Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70–77. https://doi.org/10.1016/0003-9861(59)90090-6
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478. https://doi.org/10.1016/0076-6879(90)86141-H
Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126. https://doi.org/10.1016/S0076-6879(84)05016-3
Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247(10):3170–3175
Radovanović T, Borković Mitić S, Perendija B, Despotović S, Pavlović Z, Cakić PD, Saičić Z (2010) Superoxide dismutase and catalase activities in the liver and muscle of barbel (Barbus barbus) and its intestinal parasite (Pomphoryinchus laevis) from the Danube river, Serbia. Arch Biol Sci 62(1):97–105. https://doi.org/10.2298/ABS1001097R
Rose HG, Oklander M (1965) Improved procedure for the extraction of lipids from human erythrocytes. J Lipid Res 6:428–431
Ristić-Medić D, Suzić S, Vučić V, Takić M, Tepšić J, Glibetić M (2009) Serum and erythrocyte membrane phospholipids fatty acid composition in hyperlipidemia: effects of dietary intervention and combined diet and fibrate therapy. Gen Physiol Biophys 28:190–199
Ates B, Abraham L, Ercal N (2008) Antioxidant and free radical scavenging properties of N-acetylcysteine amide (NACA) and comparison with N-acetylcysteine (NAC). Free Radic Res 42(4):372–377. https://doi.org/10.1080/10715760801998638
Samuni Y, Goldstein S, Dean OM, Berk M (2013) The chemistry and biological activities of N-acetylcysteine. Biochim Biophys Acta 1830(8):4117–4129. https://doi.org/10.1016/j.bbagen.2013.04.016
Francik R, Krośniak M, Sanocka I, Bartoń H, Hebda T, Francik S (2014) Aronia melanocarpa treatment and antioxidant status in selected tissues in Wistar rats. Biomed Res Int 2014:457085. https://doi.org/10.1155/2014/457085
Debeljak Martacic J, Borozan S, Radovanovic A, Popadic D, Mojsilovic S, Vucic V, Todorovic V, Kovacevic Filipovic M (2016) N-acetyl-l-cysteine enhances ex-vivo amplification of deciduous teeth dental pulp stem cells. Arch Oral Biol 70:32–38. https://doi.org/10.1016/j.archoralbio.2016.06.002
Dalvi SM, Patil VW, Ramraje NN (2012) The roles of glutathione, glutathione peroxidase, glutathione reductase and the carbonyl protein in pulmonary and extra pulmonary tuberculosis. J Clin Diagn Res 6(9):1462–1465. https://doi.org/10.7860/JCDR/2012/4410.2533
Todorcević M, Skugor S, Ruyter B (2010) Alterations in oxidative stress status modulate terminal differentiation in Atlantic salmon adipocytes cultivated in media rich in n-3 fatty acids. Comp Biochem Physiol B Biochem Mol Biol 156(4):309–318. https://doi.org/10.1016/j.cbpb.2010.04.010
Kang JX, Wan JB, He C (2014) Concise review: regulation of stem cell proliferation and differentiation by essential fatty acids and their metabolites. Stem Cells 32(5):1092–1098. https://doi.org/10.1002/stem.1620
Russo GL (2009) Dietary n-6 and n-3 polyunsaturated fatty acids: from biochemistry to clinical implications in cardiovascular prevention. Biochem Pharmacol 77(6):937–946. https://doi.org/10.1016/j.bcp.2008.10.020
Kim MH, Kim MO, Kim YH, Kim JS, Han HJ (2009) Linoleic acid induces mouse embryonic stem cell proliferation via Ca2þ/PKC, PI3K/Akt, and MAPKs. Cell Physiol Biochem 23(1-3):53–64. https://doi.org/10.1159/000204090
Lee JH, Tachibana H, Morinaga Y, Fujimura Y, Yamada K (2009) Modulation of proliferation and differentiation of C2C12 skeletal muscle cells by fatty acids. Life Sci 84(13-14):415–420. https://doi.org/10.1016/j.lfs.2009.01.004
Mohammadzadeh F, Mosayebi G, Montazeri V, Darabi M, Fayezi S, Shaaker M, Rahmati M, Baradaran B, Mehdizadeh A, Darabi M (2014) Fatty acid composition of tissue cultured breast carcinoma and the effect of stearoyl-CoA desaturase 1 inhibition. J Breast Cancer 17(2):136–142. https://doi.org/10.4048/jbc.2014.17.2.136
Kilpinen L, Tigistu-Sahle F, Oja S, Greco D, Parmar A, Saavalainen P, Nikkilä J, Korhonen M, Lehenkari P, Käkelä R, Laitinen S (2013) Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality. J Lipid Res 54(3):622–635. https://doi.org/10.1194/jlr.M030650
Funding
This work was supported by the Ministry of Education, Science, and Technological development Republic of Serbia, project grant nos. III41030 and OI175061.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
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Martacic, J., Filipovic, M.K., Borozan, S. et al. N-acetyl-l-cysteine protects dental tissue stem cells against oxidative stress in vitro. Clin Oral Invest 22, 2897–2903 (2018). https://doi.org/10.1007/s00784-018-2377-2
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DOI: https://doi.org/10.1007/s00784-018-2377-2