UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Vito KOVAČ OCCURRENCE AND EFFECTS OF OXIDATIVE STRESS INDUCED BY METAL MATERIALS FROM FIXED ORTHODONTIC APPLIANCES DOCTORAL DISSERTATION Ljubljana, 2022 UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Vito KOVAČ OCCURRENCE AND EFFECTS OF OXIDATIVE STRESS INDUCED BY METAL MATERIALS FROM FIXED ORTHODONTIC APPLIANCES DOCTORAL DISSERTATION NASTANEK IN UČINKI OKSIDATIVNEGA STRESA, POVZROČENEGA S KOVINSKIMI MATERIALI NESNEMNIH ORTODONTSKIH APARATOV DOKTORSKA DISERTACIJA Ljubljana, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doctoral dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Based on the Statute of the University of Ljubljana and the decision of the Biotechnical Faculty senate, as well as the decision of the Commission for Doctoral Studies of the University of Ljubljana adopted on 28th September 2020, it has been confirmed that the candidate meets the requirements for pursuing a PhD in the interdisciplinary doctoral programme in Biosciences, scientific field Biotechnology. The research work was performed in the laboratories of the Chair of Biotechnology, Microbiology and Food Safety Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana and in the laboratories of the Chair of Genetics, Animal biotechnology and Immunology, Biotechnical Faculty, University of Ljubljana. The work was funded by the Slovenian Research Agency. Senate of the University of Ljubljana has named assoc. prof. dr. Borut Poljšak as the supervisor and prof. dr. Polona Jamnik as the co-advisor of the doctoral thesis. Na podlagi Statuta Univerze v Ljubljani in po sklepu senata Biotehniške fakultete ter sklepu Komisije za doktorski študij Bioznanosti Univerze v Ljubljani z dne 28.9.2020 je bilo potrjeno, da kandidat izpolnjuje pogoje za opravljanje doktorata znanosti na interdisciplinarnem doktorskem študijskem programu Bioznanosti, znanstveno področje biotehnologija. Za mentorja je bil imenovan izr. prof dr. Borut Poljšak in za somentorico prof. dr. Polona Jamnik. Doktorsko delo je bilo opravljeno v Laboratoriju za proteomiko in Laboratoriju za industrijske mikroorganizme, Katedre za biotehnologijo, mikrobiologijo in varnost živil, Oddelka za živilstvo na Biotehniški fakulteti Univerze v Ljubljani, in v Laboratoriju za celične kulture, Katedre za genetiko, animalno biotehnologijo in imunologijo, Oddelka za zootehniko na Biotehniški fakulteti univerze v Ljubljani. Supervisor (Mentor): assoc. prof. dr. Borut POLJŠAK Univ. of Ljubljana, Faculty of Health Sciences, Department of Sanitary Engineering Co-advisor (Somenotrica): prof. dr. Polona JAMNIK Univ. of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology Committee for the evaluation and the defense (Komisija za oceno in zagovor): Chair (Predsednica): prof. dr. Mojca NARAT Univ. of Ljubljana, Biotechnical Faculty, Department of Animal Science Member (Član): assoc. prof. dr. Iztok PRISLAN Univ. of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology Member (Član): prof. dr. Stjepan ŠPALJ Univ. of Rijeka, Faculty of Dental Medicine, Department of Orthodontics Date of the defense (datum zagovora): PhD student: Vito Kovač II Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 KEY WORDS DOCUMENTATION DN Dd DC UDC 606:616-089.23(043.3) CX Oxidative stress, ROS, metal ions, nanoparticle, orthodontic appliance, Saccharomyces cerevisiae, HGF AU KOVAČ, Vito, M.Sc. AA POLJŠAK, Borut (supervisor), JAMNIK, Polona (co-advisor) PP SI-1000 Ljubljana, Jamnikarjeva 101 PB University of Ljubljana, Biotechnical Faculty, Interdisciplinary Doctoral Programme of Biosciences, Scientific Field Biotechnology PY 2022 TI OCCURRENCE AND EFFECTS OF OXIDATIVE STRESS INDUCED BY METAL MATERIALS FROM FIXED ORTHODONTIC APPLIANCES DT Doctoral Dissertation NO XV, 128, [67] p., 7 tab., 22 fig., 14 ann., 405 ref. LA en AL en/sl AB Misaligned teeth are often corrected with fixed orthodontic appliances. Prolonged stay of the orthodontic appliance in the oral cavity leads to corrosion and wear of the material, from which metal ions and nanoparticles can be released that catalyze reactive oxygen species-generating reactions, which in turn lead to oxidative stress. The objective of this work was to investigate the level of selected systemic oxidative stress parameters during orthodontic treatment, the composition of selected orthodontic alloys, the release of metal ions, and the oxidative consequences that the metal ions may have on the model organism Saccharomyces cerevisiae ( S. cerevisiae). The work also aimed to investigate the effects of nanoparticles on the human gingival cell line (HGF). An increase in systemic oxidative stress levels was detected in the capillary blood of orthodontic patients only after 24 hours, after which oxidative stress parameters normalized. The release of metal ions from orthodontic appliances into artificial saliva is constant, but the metal concentrations released are still below the maximum tolerated daily dose. Only high metal ion concentrations were able to generate large amounts of reactive oxygen species in the yeast S. cerevisiae, which the antioxidant system was unable to regulate adequately, resulting in oxidative stress and its damage to biological molecules. The toxicity of nanoparticles and the ability to generate reactive oxygen species in HGF mainly depend on the type, concentration and properties of nanoparticles. We have shown that metal ions from orthodontic appliances are released at such low concentrations that they cannot induce oxidative stress in the yeast S. cerevisiae, although some change in the antioxidant activity of the enzyme was observed. Of the selected nanoparticles, the WS2 nanoparticle was the least toxic to HGF. III Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 KLJUČNA DOKUMENTACIJSKA INFORMACIJA ŠD Dd DK UDK 606:616-089.23(043.3) KG Oksidativni stres, ROS, kovinski ioni, nanodelci, ortodonski aparati, Saccharomyces cerevisiae, HGF AV KOVAČ, Vito, mag. biotehnol. SA POLJŠAK, Borut (mentor) / JAMNIK, Polona (somentor) KZ SI-1000 Ljubljana, Jamnikarjeva 101 ZA Univerza v Ljubljani, Biotehniška fakulteta, Interdisciplinarni doktorski študijski program Bioznanosti, znanstveno področje Biotehnologija LI 2022 IN NASTANEK IN UČINKI OKSIDATIVNEGA STRESA, POVZROČENEGA S KOVINSKIMI MATERIALI NESNEMNIH ORTODONTSKIH APARATOV TD Doktorksa disertacija OP XV, 128 [67] str., 7 pregl., 22 sl., 14 pril., 405 vir. IJ en JI en/sl AI Malokluzija se pogosto odpravi s nesnemnimi ortodontskimi aparati. Dolgotrajna izpostavljenost ortodontskega aparata v ustnem okolju vodi do korozije in obrabe materiala, iz katerega se lahko sprostijo kovinski ioni in nanodelci, ki katalizirajo reakcije nastanka reaktivnih kisikovih zvrst in posledično povzročijo oksidativni stres. Namen disertacije je bil raziskati sistemske spremembe parametrov oksidativnega stresa med ortodontskim zdravljenjem, sestavo izbranih ortodontskih zlitin, sproščanje kovinskih ionov in oksidativne posledice, ki jih lahko imajo kovinski ioni na modelni organizem Saccharomyces cerevisiae ( S. cerevisiae). Namen disertacije je bil tudi raziskati učinke nanodelcev na celično linijo fibroblastov človeške dlesni (HGF). Povečanje ravni sistemskega oksidativnega stresa je bilo mogoče opaziti v kapilarni krvi ortodontskih pacientov šele po 24 urah, nato pa se parametri oksidativnega stresa normalizirajo. Sproščanje kovinskih ionov iz ortodontskih aparatov v umetno slino je konstantno, vendar so koncentracije sproščenih kovin še vedno pod maksimalno dovoljeno dnevno dozo. Le visoke koncentracije kovinskih ionov so sposobne ustvariti velike količine reaktivnih kisikovih zvrst v kvasovki S. cerevisiae, katerih antioksidativni sistem ni zmožen uravnati, in zato prihaja do oksidativnega stresa in oksidativnih poškodb molekul. Toksičnost nanodelcev in sposobnost ustvarjanja reaktivnih kisikovih zvrsti v celični liniji HGF je odvisna od vrste, koncentracije in lastnosti nanodelcev. Dokazali smo, da se iz nesnemnih zobnih aparatov sproščajo kovinski ioni, čigar koncentracije so prenizke, da bi lahko povečale nivo ROS in inducirale oksidativni stres v kvasovki S. cerevisiae, čeprav so bile opažene nekatere spremembe v aktivnosti antioksidativnih encimov. Izmed izbranih nanodelcev so se nanodelci WS2 izkazali kot najmanj toksični v celični liniji HGF. IV Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 LIST OF TABLES Table 1: Orthodontic alloys and their metal composition (Dentaurum, 2020). ..................... 6 Table 2: Main ROS molecules (Arjunan et al., 2015). ........................................................ 10 Table 3: Saccharomyces cerevisiae strains used in the study. ............................................ 28 Table 4: Metal ion w/v ratios for simulating orthodontic alloys. ........................................ 29 Table 5: Nanoparticles used in the study. ............................................................................ 39 Table 6: Components of orthodontic appliances used in the study with their corresponding metal composition in weight percentages (%) (Kovač et al., 2022). ................................... 48 Table 7: Size and surface charge of used nanoparticles ...................................................... 77 IX Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 LIST OF FIGURES Figure 1: Lipid oxidation (Barrera et al., 2018). ................................................................. 16 Figure 2: Enzymatical antioxidant defense system (Kovač et al., 2021). ........................... 19 Figure 3: The Trx system and Prx mechanism (Lu and Holmgren, 2014). ......................... 21 Figure 4: Research work flow. ............................................................................................ 24 Figure 5: Changes in FORT/FORD oxidative stress parameter during the one week orthodontic treatment. ........................................................................................... 45 Figure 6: The release of metal ions from different parts of fixed orthodontic appliances. . 51 Figure 7: Cell culturability of Wt, ΔSod1 and ΔCtt1 yeast after 24-hour metal treatment. 55 Figure 8: Metabolic activity of Wt, ΔSod1 and ΔCtt1 yeast treated with different metal mixtures. ................................................................................................................ 57 Figure 9: Intracellular ROS level of Wt, ΔSod1 and ΔCtt1 yeast was performed with two different methods, each with a different time point of H2DCFDA dye addition. . 59 Figure 10: Intracellular ROS level in culturable Wt, ΔSod1 and ΔCtt1 yeast cells after the 24-hour metal treatment. ....................................................................................... 61 Figure 11: Influence of 24-h metal treatment on the formation of oxidative lipid damage in yeast cells. ............................................................................................................. 63 Figure 12: In-gel antioxidative activity of SOD (A) and CAT (B) enzyme after metal ion mixture treatment. ................................................................................................. 64 Figure 13: Yeast antioxidant enzyme activity after 24-hour metal mixture treatment. ....... 65 Figure 14: Protein carbonyl content as a result of oxidative protein damage in S. cerevisiae after 24-hout metal mixuture treatment. ................................................................ 68 Figure 15: HGF cell viablility after 24-hour treatment with metal mixtures ..................... 70 Figure 16: Relative cell death of HGF cells after 24-hour metal mixture treatment. .......... 71 Figure 17: HFG cell intracelllular oxidation level after metal mixture tretament. .............. 73 Figure 18: Cell viability of HFG after treatment with three types of TiO2-NPs. ................ 78 Figure 19: Cell viability of HFG after treatment with WS2-NPs. ....................................... 80 Figure 20: Cell viability of HFG after treatment with two different ZnO-NPs. .................. 81 Figure 21: Cell viability of HFG after treatment with different concentrations of Ag-NPs. ............................................................................................................................... 82 Figure 22: Inracellular ROS generation of HGF cells, treated with different types and concentrations of NPs. ........................................................................................... 84 X Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 LIST OF ANNEXES ANNEX A Ethical approval (0120-523/2018/8) .............................................................. 129 ANNEX B Patients consent .............................................................................................. 130 ANNEX C Questionnaire ................................................................................................. 131 ANNEX D ICP-MS operating parameters ....................................................................... 140 ANNEX E Release of metal ions from SS, Ni-Ti, β-Ti and Co-Cr alloys ....................... 141 ANNEX F TEM pictures of TiO2 NP ............................................................................... 144 ANNEX G TEM pictures of AgNP .................................................................................. 145 ANNEX H TEM pictures of ZnONP ............................................................................... 146 ANNEX I TEM pictures of WS2NP ................................................................................. 147 ANNEX J Consent from publisher Hindawi for the re-publication of article in the print and electronic versions of the doctoral dissertation .............................................. 148 ANNEX K Published article Kovač et al., 2019 .............................................................. 149 ANNEX L Consent from publisher MDPI for the re-publication of article in the print and electronic versions of the doctoral dissertation .............................................. 155 ANNEX M Published article Kovač et al., 2020 .............................................................. 156 ANNEX N Published article Kovač et al., 2021 .............................................................. 171 XI Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ABBREVIATIONS AND SYMBOLS O •− 2 - superoxide anion •OH - hydroxyl radical 1O2 - singlet oxygen 3R - Replacement, Reduction, and Refinement 8-OH-dG - 8-hydroxy-2'-deoxyguanosine AD - antioxidant defense Ag - silver Ag-NPs - silver nanoparticles Al - aluminum APS - ammonium persulfate ATP- adenosine-5-triphosphate CAT - catalase CFU - colony forming units CG - control group Co - cobalt Co-Cr - cobalt-chromium Cr - chromium Cu - copper DCF - dichlorofluorescein DLS - dynamic light scattering DNA - deoxyribonucleic acid DNPH - 2,4-dinitrophenylhydrazine DTNB - 5,5'-dithiobis(2-nitrobenzoic acid) DTT - 1,4-dithio- DL -threitol ELG - elgiloy ETC - electron transport chain F - fluorescence Fe - iron FORD - Free oxygen radical defense FORT - Free oxygen radical test FOX - ferrous xylenol orange GPx -glutathione peroxidase GR - glutathione reductase GSH - glutathione GSSG - generating oxidized glutathione H2DCF- DA - 2', 7'-dichlorofluorescein diacetate H2O2 - hydrogen peroxide HGF - human gingiva fibroblast HNE - 4-hydroxy-2-nonenal XII Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 HNO2 - nitrous acid HOCl - hypochlorous acid HOO• - hydroperoxyl hTERT - human telomerase reverse transcriptase ICP-MS - inductively coupled plasma mass spectrometry IF-MoS2 - inorganic fullerene-like molybdenum disulfide IF-WS2 - inorganic fullerene-like tungsten disulfide L• - lipid radical LIP - liable iron pool LO• - lipid alkoxy radical LOO• - lipid peroxyl radical LOOH - lipid hydroperoxide MDA - malondialdehyde Mo - molybdenum MPs - micro particles mRNA - messenger ribonucleic acid NADH - nicotinamide adenine dinucleotide NADPH - reduced nicotinamide adenine dinucleotide phosphate NBT - nitroblue tetrazolium Ni - nickel NiTi - nickel-titanium NO• - nitric oxide NO • 2 - nitrogen dioxide NPs - nanoparticles O •− 2 - superoxide O3 - ozone OD - optycal density OH• - hyroxyl ONOO− - peroxynitrite ONOOH - peroxynitrous acid PBS - phosphate-buffered saline PI - propidium iodide PPB - potassium phosphate buffer Prx - peroxiredoxin PUFA - Polyunsaturated fatty acid REM - remaloy RNS - reactive nitrogen species RO• - akoxyl RO• - akoxyl radical RO2ONO - peroxynitrate ROO• - peroxyl XIII Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ROO• - peroxyl radical ROOH - organic peroxide ROONO - peroxynitrite ROS - reactive oxygen species S. cerevisiae - Saccharomyces cerevisiae SOD - superoxide dismutase SS - stainless steel TBA - thiobarbituric acid TBARS - thiobarbituric acid reactivity assay TEM - transmission electron microscop TEMED - tetramethylethylenediamine TG - treatment group Ti - titanium TiO2 - titanium dioxide TiO2-NP - titanium dioxide nanoparticles TNB - 5-thio(2-nitrobenzoic) acid Trx - thioredoxin TrxR - thioredoxin reductase UV - ultra violet WS2 - tungsten WS2-NPs – tungsten nanoparticles WSL – white spot lesions Wt – wild type YPD - yeast extract-peptone-glucose Zn – zinc ZnO – zinc oxide ZnO-NPs – zinc oxide nanoparticles ZP - zeta potencial β-Ti – beta-titanium XIV Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 1 INRODUCTION 1.1 PROBLEM DESCRIPTION Tooth misalignment, also known as malocclusion, has a tremendous impact on oral and dental health. From a psychological perspective, malocclusions also contribute to a person's sense of well-being and self-esteem (Nguee et al., 2020). The most efficient method to correct the problem is orthodontic treatment. Orthodontic treatment uses the elastic force of arches to move teeth into the desired position. The arches are placed in brackets, which are attached to the dentin of the tooth in various ways using a plastic material. Depending on the severity of the condition, orthodontic treatment lasts an average of 15 to 24 months (Fleming et al., 2010). However, during this time, inflammation-related side effects may occur, including tooth resorption, pulp changes, periodontal tissue inflammation, hypersensitivity reactions due to the release of metal ions from the appliances, and enlarged gums (either due to poor oral hygiene or hypersensitivity reactions) (Talic, 2011). Fixed orthodontic appliances consist of wire archwires, brackets, and bands. These are usually made of orthodontic metal alloys such as stainless steel, nickel-titanium alloys, or cobalt-chromium alloys. All of the above orthodontic parts are exposed in some way to the ever-changing oral environment. The harsh conditions such as changing pH, temperature, biological and enzymatic composition leave their mark on the orthodontic appliance. Electrochemical reactions, mechanical forces and general wear of the material (Sutow et al., 2004) in the oral cavity lead to corrosion, a deterioration process of orthodontic metals (von Fraunhofer, 1997). Microbial communities are also present on the surface of orthodontic appliances and cause the so-called biocorrosion, which further alters the protective surface layers, topography and mechanical properties of the appliance (Kameda et al., 2014). With the structural weakening of the apparatus, the release of metal ions or metal particles may occur. Most of the released metals belong to the transition metal group, i.e., they have unpaired electrons in their outer shell and therefore can actively participate in the so-called Fenton- and Haber-Weiss-like reactions to generate reactive oxygen species (ROS) (Moriwaki et al., 2008). ROS molecules such as superoxide anion (O •− 2 ), hydroxyl radical (•OH), hydroperoxyl radical (HOO•), akoxyl radical (RO•), peroxyl radical (ROO•), hydrogen peroxide (H2O2) and singlet oxygen (1O2) are also products of normal cell metabolism, cell signaling, the consequence of exposure to some external factors (e.g. environmental pollutants) or use of medicinal drugs (Murphy, 2008). When the amount of ROS exceeds the system's ability to protect itself from them, oxidative stress occurs (Kohen and Nyska, 2002), resulting in oxidative damage at the DNA, lipid, and protein levels (Pham-Huy et al., 2008). To ward off the harmful effects of excessive formation of ROS, organisms 1 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 employ a diverse antioxidant defense system consisting of enzymes and low molecular weight molecules. The modern approach to minimizing appliance wear and improving its surface properties is to use nanoparticles (Batra, 2018). They are primarily used as coatings to increase material strength, reduce friction, and have antimicrobial properties, but are also included in ortothodontic resins to reduce enamel demineralization (Zakrzewski et al., 2021). Nanoparticles have a size of less than 100 nm in diameter and a large surface-to-mass ratio, making them highly reactive (Boverhof et al., 2015). Due to their size, they can easily pass through membranes and due to their high chemical reactivity, they could pose a toxic hazard (Saafan et al., 2018). Metallic nanoparticles, such as TiO2 in ZnO (Ghiciuc et al., 2017), exhibit a broad antibacterial spectrum and are considered to be a better bactericidal agent than drugs, mainly because some of them tend to release metal ions and with it, promoting the formation of ROS (Metin-Gürsoy et al., 2017; Morán-Martínez et al., 2018). While antibacterial effect is desirable, the harmful effect to other cells in the oral cavity, gastrointestinal tract or systemic effect are not desirable. The aim of this dissertation was to investigate whether the occurrence of oxidative stress and consequently oxidative damage to molecules could be a consequence of released metal ions or metal nanoparticles from fixed orthodontic appliances. First, we wanted to observe whether the oxidative stress parameters in the capillary blood of volunteers change during orthodontic treatment, and then plan the in vitro metal release experiment to fully understand which and how many metal ions are released from orthodontic alloys. With the obtained results, cell exposures to metal ion combinations were performed on the model organism Saccharomyces cerevisiae and the occurrence of oxidative stress was evaluated. Since the use of nanoparticles in orthodontics is not well defined, we had to define a toxicity range for the selected metal nanoparticles and evaluate their ability to generate ROS in human gingival fibroblast cell line. 2 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 1.2 RESEARCH GOALS The dissertation research objectives were established as follows: - Investigate and observe the changes in selected parameters of oxidative stress in capillary blood of volunteers during treatment with fixed orthodontic appliances. - Determine the type and amount of released metal ions from fixed orthodontic appliance in artificial saliva. - Examine the effect of selected metal ion combinations (Fe, Ni, Cr, Co, Mo and Ti) of orthodontic alloys on the Saccharomyces cerevisiae model organism, either at the cellular or molecular level. - Evaluate possible toxicity of certain metal nanoparticles (TiO2, ZnO, Ag and IF-WS2) on the human gingival fibroblast (HGF) cell line. - Based on the obtained results, we can confirm/debunk that selected combinations of metal ions from orthodontic alloys and selected metal nanoparticles used in orthodontics generate oxidative stress and cause oxidative damage in cells. - According to the toxicity results and changes in oxidative stress parameters we will recommend the least risky nanoparticle for orthodontic use. 3 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2 LITERATURE REVIEW 2.1 FIXED ORTHODONTIC APPLIANCES Fixed orthodontic appliances are used for various movements of teeth along the alveolar bone. The effectiveness of orthodontic treatment is dependent on the susceptibility of the periodontal tissues to the treatment and the characteristic of the fixed orthodontic appliance parts (Proffit et al., 2007). Brackets, archwire, bands and ligatures make up an orthodontic appliance. Different materials, like metal ceramics, polymers and composites, are used to make the parts, each with their own pros and cons (Chen and Thouas, 2015). The usage of ceramic or polymer is more visually appealing, they are quite fragile and tend to deform over a certain amount of time (Oh et al., 2005). Of them, metal alloys are the most employed material, due to good mechanical properties, strength and heat and electric conductivity (Park and Lakes, 2007). Their mayor flaw lies in the tendency to corrosion in the presence of biological liquids. 2.1.1 Biocompatibility Orthodontic treatment with biocompatible appliances is crucial for the treatment efficiency and patient’s safety. Biocompatible materials are considered to be the ones that do not inflict any negative effect on their environment, meaning that they are not toxic, carcinogenic and not capable of inducing allergic reactions (Widu et al., 1999). Another important property of biocompatible materials is that their physiological and mechanical properties do not change under in vivo conditions. In reality, no material suffices all the requirements. When choosing the right material, the emphasis is pointed to the natural occurring elements in the human body. Carefulness is advised, because high concentrations of micronutrients like chromium, cobalt, copper, iron, iodine, manganese, molybdenum, selenium and zinc are toxic and cause mayor medical problems (Chen and Thouas, 2015). The decision on what material to use is also based on the mechanical properties, corrosion resistance, bending and melting properties, esthetics and finally the price (Upadhyay et al., 2006). Biomaterials are in close contact with body liquids which can affect the material surface. In the presence of saliva or other fluids, orthodontic alloys tend to corrode over time, resulting in metal release from the alloy surface and basic weakening of the alloys properties (Mathew and Wimmer, 2011). In the process called corrosion, where there is a physicochemical (electrochemical) interaction between the metal and its environment, changes in the metal properties could happen. The body fluid, that the orthodontic alloys are exposed to, is saliva and it is a very heterogenic media, containing different microorganisms and having a vast array of physiological properties (House et al., 2008). 4 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2.1.2 Orthodontic alloys Stainless steel alloy (SS) and its variants are used for all parts of fixed orthodontic appliance, from brackets, wires, ligatures and bands. The composition of alloy SS is mainly Fe with smaller amounts of Ni, Cr and other elements (Sfondrini et al., 2009) Depending on the structure and chemical composition, five groups of SS alloys can be distinguished, of which the austenitic SS alloy is the most preferred for the production of orthodontic appliance parts (Arango Santander and Luna Ossa, 2015). The reason for the widespread use of alloy SS is its good corrosion resistance, which is due to the contained Cr and the ability to form a passive chromium oxide layer (Cr2O3) on the surface (Chaturvedi and Upadhayay, 2010). The SS alloy also has an excellent formability, strength and elasticity. The cobalt-chromium alloys (Co-Cr) consist of about 40% Co, 20% Cr, and 14% Ni, with Fe and Mo in the lower 5% range. The mechanical properties such as elasticity and strength of Co-Cr alloy are very similar to SS alloy and they are also good against corrosion due to Cr2O3 passivation on the surface (Harini and Kannan, 2020). According to the shape of Ti material, it can be distinguished between α-Ti and β-Ti. The hexagonal α-Ti is often stabilized with aluminum to obtain an alloy with high strength and low weight, while the β-Ti is stabilized with molybdenum (Mo) and vanadium (V) (Park and Kim, 2000). The most common Ti alloys are Ti6Al4V for brackets and NiTi or CuNiTi alloys for arches (Nakajima and Okabe, 1996). The NiTi alloys consist of about 55% Ni and 45% Ti, depending on the manufacturer. The 5% - 6% Cu is sometimes added to increase strength. NiTi alloys have greater flexibility compared to other alloys, such as SS, Co-Cr and β-Ti (Ferreira et al., 2012). The major advantage of NiTi alloy is its "shape memory", which means that the material retains its original shape even after bending. Most backets are made of SS alloy. In addition to iron (Fe), this steel consists of chromium (Cr), nickel (Ni) and sometimes molybdenum (Mo). The proportion of Cr and Ni varies depending on the specific SS alloy (Oh et al., 2004). Less common are titanium brackets, which are made of pure titanium or a titanium alloy (Gioka et al., 2004). In terms of material, orthodontic alloys for arches are extremely diverse. The first arches were made of the alloy SS, but were then replaced by a nickel-titanium alloy (NiTi) due to its better elasticity and easier bending manipulations. Other available options include beta-titanium (β-Ti) and cobalt-chromium (Co-Cr) alloy (Małkiewicz et al., 2018). The metal composition of orthodontic alloys is shown in Table 1. 5 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Table 1: Orthodontic alloys and their metal composition (Dentaurum, 2020). Alloy Fe (%) Ni (%) Cr (%) Co (%) Mo (%) Ti (%) Other SS Residue 8 - 10 17 - 19 / / / Si = 2 - 3 (dentaflex®) Mn ≤ 2 SS Residue 8 - 10.5 17 - 9 / / / Si ≤ 1 (remanium®) Mn ≤ 2 SS Residue 4.5 - 6.5 25 - 28 / 1.3 - 2 / Si ≤ 1 (AISI 1.4460) Mn ≤ 2 Co-Cr 4-6 19 - 23 18 - 22 Residue 3 - 5 / Mn = 1-3 (Elgiloy®) Co-Cr Residue 14 - 16 19 - 21 38 - 42 6 - 8 / Si ≤ 0.5 (remaloy®) Mn ≤ 1 W = 3-5 Ni-Ti / 50 - 60 / / / Residue Fe ≤ 0.5 (rematitan® LITE) β-Ti / / / / 11.5 78 Zr ≤ 6 (rematitan® Sn ≤ 4.5 Special) 2.1.3 Nanotechnology in orthodontics Nanotechnology represents a great opportunity for further improvements in the field of medicine and dentistry, either as a protective layer or as an improvement in the properties of materials, such as strength and durability. Nanoparticles possess advantageous properties such as high surface-to-volume ratio for better interaction with the environment, zeta potential, particle size and shape, surface chemistry, agglomeration, dissolution, and ion release.(Fernando et al., 2018) Due to their unique properties, such as catalytic, optical, and electromagnetic properties, metal nanoparticles (NPs) and nanomaterials are widely used in biological and medical applications (Mamunya et al., 2004), including orthodontics. NPs, especially metal NPs with their physicochemical, mechanical, and antibacterial properties, could have a great impact on the duration of orthodontic treatment, address certain associated problems, and improve oral health (Sharan et al., 2017). One way to apply NPs to orthodontic appliances is through coatings, where the biological properties of the surface layer and the mechanical properties of the alloy could be improved. 2.1.3.1 Antibacterial activity Orthodontic appliances are a good site for bacterial plaque formation. Their configuration promotes food retention and impedes the self-cleaning of oral muscles and saliva and the maintenance of oral hygiene, allowing the formation of plaque (Lundström and Krasse, 1987), a common undesirable side effect of orthodontic treatment. Persistent plaque accumulation leads to a shift in the imbalance between demineralization and remineralization, which in turn leads to white spot lesions (WSL) (Sudjalim et al., 2006). 6 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Antibiotics could be used in this manner, but fear of resistance development outweighs their potential. Resistance to NPs is less likely because their mode of action is based on direct contact with the cell wall of bacteria (Fernando et al., 2018). Ag-NPs have been shown to be a potentially useful substance for biomedical applications because of their high antimicrobial activity and low toxicity to the environment. Their mode of action is not yet well described, but many mechanisms have been proposed (Rizzello and Pompa, 2014). The ability of Ag-NPs to inhibit yeasts, Gram-positive and - due to the thick cell wall - especially Gram-negative bacteria makes them widely applicable in antimicrobial research (Peiris et al., 2017). The mode of action is that they first make membranes permeable to allow more Ag-NPs to enter and release Ag ions (Marambio-Jones and Hoek, 2010), interacting with thiol-containing intracellular proteins (Chen and Schluesener, 2008), generate reactive oxygen species, interact with sulfur in DNA, and thus interfere with replication and cell division (Prabhu and Poulose, 2012). It has also been suggested that the released silver ions inhibit respiratory enzymes at the membrane and eventually cause cell lysis (Bragg and Rainnie, 1974). TiO2-NP are readily available, low toxic, biocompatible, chemically stable and robust nanoparticles. Due to their photocatalytic activity and antibacterial properties, TiO2-NPs are the most studied NPs. The main antibacterial mechanism of TiO2-NP is based on the photocatalytic formation of ROS under UV light (Wu et al., 2009). Toxicity against bacteria has been demonstrated without UV illumination of TiO2-NPs, indicating a mechanism of action other than the production of ROS (Sohm et al., 2015). However, the mode of action is still controversial. It is believed that the cause of bacterial death is membrane depolarization and eventually membrane permeability (Sohm et al., 2015). Similar to TiO2, ZnO is highly photocatalytic, meaning that under UV light, loosely bound oxygen is degraded in the form of reactive oxygen species (ROS) such as hydrogen peroxide (H •− 2O2) and superoxide ions (O2 ) (Bao et al., 2011). These molecules have been shown to damage proteins and DNA, eventually leading to their death (Kirkinezos and Moraes, 2001). Another antibacterial effect is the release of Zn ions into the media, which disrupts transport, amino acid metabolism, and enzyme systems (Li et al., 2011). Changes in membrane permeability, and thus loss of proton motive force and other biological molecules, also occur when ZnNPs adhere to or are integrated into the membrane (Amro et al., 2000). Because of their antimicrobial activity, ZnNPs can reduce adhesion, proliferation, and biofilm inhibition. 2.1.3.2 Reduction of friction Friction is defined as a force that opposes the opposing force of motion (Rossouw et al., 2003) and occurs as resistance to sliding along the orthodontic archwire when the archwire 7 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 is in contact with the brackets, reducing tooth movement and consequently reducing the efficiency of the orthodontic appliance (Prashant et al., 2015). Friction causes material wear in the form of loss of mass, loss of volume, or loss of coating surface (Zheng and Zhou, 2007). During orthodontic movement of the teeth, static and kinetic friction occur alternately as the teeth slide and flex along the orthodontic wire (Kachoei et al., 2016). The main focus in the manufacture of novel orthodontic appliances is to reduce the friction that occurs during orthodontic treatment. Low friction provides better transmission of force to the teeth and thus more effective tooth movement, while high friction results in a less effective mechanism and less tooth movement, may even lead to prolonged treatment and root resorption at the base (Wei et al., 2011). Orthodontic friction is determined by the size, shape, and material (Muguruma et al., 2013) of the brackets, bands, and wires, the ligature (Thorstenson and Kusy, 2002) and angulation (Articolo and Kusy, 1999), and the dynamic interactions between them. For example, a wire with a round cross-section has a smaller contact area with the bracket and therefore generates less friction than a wire with a rectangular cross-section, which generates high friction forces because it is fully seated in the bracket space. In addition to the mechanical properties of the wire, the biological aspect must also be taken into account. For example, saliva, which contains mucin and other peptides such as statherine (Yakubov et al., 2015), is a natural lubricant that reduces friction but also causes debris to accumulate and bacteria to contribute to the formation of biofilms that increase friction (Marques et al., 2010). With the discovery of the dry lubrication properties of inorganic fullerenes of tungsten disulfide nanoparticles (IF-WS2) (Tenne et al., 1992) and molybdenum disulfide nanoparticles (IF-MoS2) (Feldman et al., 1995) came the inspiration to somehow incorporate them into orthodontic appliances. It appears that when the wire and bracket are aligned in parallel, IF nanoparticles on coated wires act as spacers, resulting in lower friction being observed. With increasing angular pressure, some IF nanoparticles delaminate and transform from tubes to sheets, allowing rolling and sliding in the contact area (Joly-Pottuz et al., 2005). The thin layer of nanosheets allows sliding movements between two bodies, the archwire and the bracket (Samorodnitzky-Naveh et al., 2009). 2.1.3.3 Increase in strength From a mechanical point of view, orthodontic treatment is based on the movement of teeth to the desired position by the elastic strength of the archwires placed in the position of the brackets. Coating orthodontic appliances is one of the ways to mechanically and biologically modify and improve the metallic properties of the orthodontic material used. Reduction of surface roughness, thickness, mechanical and friction properties, corrosion resistance, antibacterial properties and stability are the desired properties that an ideal coating should provide (Arango et al., 2012). 8 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2.2 OXIDATIVE STRESS The term oxidative stress was first introduced by Helmut Sies as an imbalance between prooxidants and antioxidants in favor of the former, which disrupts redox signaling and causes molecular damage (Sies, 2020). The so-called reduction-oxidation (redox) balance can be disturbed either by an overproduction of prooxidants, by a deficiency of antioxidants, or even by both together. Minor fluctuations in the redox balance do not cause major problems because the organism has the ability to adapt to the reaction, while major disturbances lead to irreparable biological damage to the cell and even cell death (Burton and Jauniaux, 2011). Under physiological conditions, the levels of oxidative stress are considered low and the redox balance is only slightly shifted in favor of prooxidants because they are necessary for the normal functioning of the organism. However, when the balance tilts more and more in favor of the prooxidants, organelle damage and deteriorating processes can be observed (Auten and Davis, 2009). 2.2.1 Reactive oxygen species Free radicals, amongst which are also some reactive oxygen species (ROS), have an incomplete outer electron layer and thus possess one or more unpaired electrons, making them highly reactive. They may be covalently bonded to another molecule with an unpaired electron or they may result from bonding with a nonradical molecule by donating an unpaired electron (reduction radical) or by accepting electrons (oxidation radical). The ensuing reaction can trigger a chain event, producing large amounts of ROS (Halliwell, 1991). The term ROS also includes some non-radical molecules, such as hydrogen peroxide (H2O2) and ozone (O3), which are readily converted to radicals (Irani, 2007). The main ROS molecules found in the biological system are presented in Table 2. In biological systems, ROS can be formed from both endogenous and exogenous sources. In normally functioning cells, the mitochondria, peroxisomes, endoplasmic reticulum, and immune cells are the primary endogenous source of ROS (Burton and Jauniaux, 2011). ROS are constantly generated under normal physiological conditions and their homeostasis is constantly monitored and maintained. The theory of free radicals and oxidative stress was established more than half a century ago and initially ROS were considered harmful by-products of aerobic metabolism, but now ROS are considered to play an essential role in various biological processes (Finkel, 2011) from phagocytosis to cellular signal transduction. 9 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Table 2: Main ROS molecules (Arjunan et al., 2015). ROS (Reactive oxygen species) Free radical species Non-radical species O •− 2 H2O2 (superoxide) (hydrogen peroxide) OH• O3 (hyroxyl ) (ozone) HOO• 1O2 (hydroperoxyl) (singlet) RO• ROOH (akoxyl) (organic peroxide) ROO• HOCl (peroxyl) (hypochlorous acid) The sources of ROS can be exogenous or endogenous. Exogenous triggers of ROS include UV radiation, heavy metal ions, O3, toxins, pollutants, pesticides, and insecticides (dos Santos et al., 2018; Mahajan et al., 2018). The major endogenous sites of ROS generation are the mitochondrial electron transport chain (ETC), the endoplasmic reticulum, peroxisomes, membrane NADPH oxidases, and nitric oxide synthase (Rodriguez and Redman, 2005). The most important reaction in aerobic organisms is respiration, in which H2O is formed by the reduction of O2 with four electrons and oxidation of organic molecules occurs. Through a series of enzymatically catalyzed reactions, an energy source in the form of adenosine-5-triphosphate (ATP) is generated by the process of oxidative phosphorylation (Bertini et al., 1994). Electron loss can occur along the enzymes of the mitochondrial electron transfer chain, especially at mitochondrial complex I (NADH-ubiquinone oxidoreductase) and complex III (ubiquinol cytochrome c oxidoreductase) (Cadenas and Davies, 2000). Approximately 1-3% of circulating electrons from ETC are thought to be lost during ATP production (Hamanaka et al., 2013). Cellular components such as the endoplasmic reticulum, cytoplasmic enzymes, and the plasma membrane surface can also be a source of ROS (Sumimoto 2008; Tu and Weissman 2004), as can some enzyme systems: the cytochrome P450 system, the xanthine oxidoreductase system, nitric oxide synthases, and the inflammatory process system. The primary ROS is formed during the four-step reduction process of O2 to H2O (Halliwell and Gutteridge, 2015). The oxygen molecule has two unpaired electrons with parallel spin, called triplet oxygen (3O2), which is harmless unless it is energetically activated. Activation can occur when enough energy is provided to reverse the spin of an unpaired electron, producing singlet oxygen (1O2) or when a single electron has been added to the oxygen molecule, producing a superoxide radical (O •− 2 ), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) (Apel and Hirt, 2004). 10 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 The electrons attack the molecular oxygen and produce a superoxide anion (O •− 2 ) as shown in the Equation 1. O •− 2 is considered a moderately reactive molecule, unable to penetrate the cell membrane due to its negative charge. However, O •− 2 is an important precursor for other ROS generation (Turrens, 2003). Although the redox potential of O •− 2 s considered high, it is not considered a good reducing agent nor a good oxidizing agent, with some molecules such as cytochrome c, ascorbate, and the enzyme superoxide dismutase being exceptions (Collin, 2019). O 3 •− 2 + e− → O2 … (1) Further reduction of O •− 2 with an electron produces H2O2, a moderately reactive, nonradical molecule with a fairly long half-life compared to other ROS (Equation 2). The reaction is plausible only in the presence of the antioxidant enzyme superoxide dismutase (SOD) (Fukai and Ushio-Fukai, 2011). Unlike other ROS, H2O2 is membrane permeable and therefore can act outside its site of generation (Van Breusegem et al., 2001). In this regard, H2O2 is considered an important signaling molecule (Covarrubias et al., 2008). O •− 2 also catalyzes reduction of nitric oxide (NO•) to peroxynitrite (ONOO−) (Galaris and Pantopoulos, 2008). 𝑆𝑆𝑆𝑆𝑆𝑆 O•− 2 + e− + 2H+ �⎯� H2O2 … (2) The reduction of H2O2 generates the most powerful and toxic oxygen radical, the hydroxyl radical (•OH). It is capable of reacting with surrounding molecules and depriving them of their hydrogen atom, turning biological molecules into new radicals. •OH reacts instantly and is not selective towards its targets. The fact that there is no defense mechanism against - OH known to us indicates its harmful properties (Bhattacharjee, 2019). H2O2 + e− + H+ → H2O + OH • … (3) One of the mechanisms for the generation of •OH is the Fenton reaction, first presented in 1894 (Fenton, 1894) and later corrected and completed to the reaction known today (Haber et al., 1934). The Fenton reaction involves ferrous iron (Fe2+) and H2O2 which produce ferric iron (Fe3+) and •OH (Equation 4). Many other elements with high valence such as copper (Cu), zinc (Zn), and aluminum (Al) have the property of transferring electrons and therefore can also participate in the Fenton reaction. When the reaction involves metals other than Fe or Cu, ligands, or peroxides, it is called a Fenton-like reaction (Meyerstein, 2021). In the Haber-Weiss reaction, an addition to the original Fenton reaction, it is assumed that O •− 2 reacts again with H2O2 to form •OH and hydroxyl anion (OH-) (Equation 5). It is important to note that the Haber-Weiss reaction is thermodynamically favored, but not kinetically, so it must be catalyzed by a transition metal. However, from a kinetic standpoint, 11 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 the O •− 2 reduction of Fe3+ to O2 and Fe2+ is more likely to occur than the reduction of H2O2 by O •− 2 (Equation 6) (Das et al., 2015). Fe2+/Cu+ + H2O2 → Fe3+/Cu2+ + OH• + OH− … (4) Fe/Cu O•− 2 + H2O2 �⎯⎯� OH• + OH− + O2 … (5) Fe3+/Cu2+ + O•− 2 → Fe2+/Cu+ + O2 … (6) 2.2.2 Metal ions and ROS Metal ions play an important role in a variety of cellular functions such as electron transfer in respiration, synthesis and repair of DNA, and cellular metabolism. Elements with a partially filled d subshell and the ability to form cations are called transition elements or transition metals (McNaught and Wilkinson, 1997). Transition metals are found in Group 4-11 of the periodic table and have a large number of complex ions in many positively charged oxidation states with different catalytic properties. Some transition metals are essential elements and key components for many metalloproteins involved in the process of oxygen formation and hypoxia detection. Metal ions occur in various oxidation states and as such can undergo a redox reaction, associate with phospholipids to alter membrane stability, and promote lipid peroxidation. In contrast, the non-redox active metals (cadmium, arsenic, and lead) are maintained at physiological concentrations. Since the formation of ROS is closely related to the involvement of redox-active metals, their concentrations are kept strictly at physiological concentrations (Valko et al., 2005). In the human body, iron (Fe) is the most abundant transition metal stored and transported in specific proteins (Ferrali et al., 1992) so Fe homeostasis is under strict control. Fe exists in ferrous (Fe2+) and ferric (Fe3+) ion configuration, and its ability to readily donate or accept electrons makes it an important catalyst in redox reactions (Galaris et al., 2019). Fe regulation prevents free intracellular Fe formation, but under stress conditions regulation fails and Fe can be released from iron-containing molecules into a so-called labile iron pool (LIP). Excess Fe from LIP can then be subjected to Fenton chemistry, forming extremely reactive OH• and since OH• reacts near its formation, Fe consequently functions in a site-specific manner (Chevion, 1988). Copper (Cu) is also an essential nutrient and a co-factor for enzymes (Uriu-Adams and Keen, 2005). It is capable of catalyzing the Fenton reaction even more effectively than Fe, but its abundance in the organism is less than that of Fe (Barbouti et al., 2001). When OH• is formed, it can have devastating consequences for biological molecules. 12 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Nickel (Ni) compounds and salts are a hazard to humans and animals. Ni compounds are reported to cause DNA single-strand breaks, chromosome aberrations, and DNA-protein crosslinks (Chen et al., 2010). The mechanism of Ni damage is thought to be indirect, catalyzing the Haber-Weiss reaction to form OH•. Ni ions, Ni2+/Ni3+, do not efficiently generate ROS from reactive oxygen derivatives because the redox reactions are chemically unfavorable (Datta et al., 1992), but when bound to certain ligands, Ni2+ bound to the ligand can be oxidized to Ni3+, generating ROS (Kasprzak et al., 2003). Chromium (Cr), a naturally occurring metal, occurs in a variety of oxidation states but is most commonly found in trivalent (Cr3+) and hexavalent (Cr6+) forms, with the trivalent form used in dietary supplements and the hexavalent in industrial applications (Bagchi et al., 2002). The Cr6+ is considered more toxic because it can easily pass through cell membranes where it is reduced by ascorbic acid and low molecular weight thiol compounds to Cr5+, Cr4+ and eventually to Cr3+. Internalization of Cr3+ into cells is thought to occur actively by phagocytosis, in contrast to Cr6+, which is taken up passively by nonspecific anion carriers (Fleury et al., 2006). The Cr5+ intermediate of the Cr6+ reduction generates a significant amount of OH• trough Fenton-like reactions (Shi and Dalal, 1990). Although Cr3+ is 1000-fold less toxic than the hexavalent form, it can still cause damage at high concentrations when bound to a ligand. Cobalt (Co) occurs in divalent (Co2+) and trivalent (Co3+) states, the former being rapidly reduced to a divalent state in aqueous media. Co is required in small amounts for the normal biological activity of some proteins. In addition, Co3+ occupies the catalytic site of vitamin B12 (Scharf et al., 2014) and elevated Co2+ concentrations are thought to have a negative effect on mitochondrial respiration. As shown by Foster et al. (2014), Co2+ competes with Fe2+ for iron binding sites in mitochondrial ETC complexes I, II and III iron (Fe-S) clusters. Since Co2+ forms more stable complexes than Fe2+, mismetallization can occur, leading to disruption of oxidative phosphorylation and release of Fe (Salloum et al., 2018). The intracellular environment, filled with free (unbound) Fe, is now confronted with overproduction of ROS by the Fe-catalyzed Fenton reaction. In addition to Fe, Co is another Fenton-inducing metal with a favorable redox potential (Uzunboy et al., 2019). Molybdenum (Mo) is another essential micronutrient and an important cofactor for some metalloenzymes. It occurs in many oxidation states, of which Mo4+ and Mo6+ are the most abundant. The possible involvement of Mo in the formation of ROS is still debated, but many researchers suspect the following two mechanisms: direct involvement in Fenton chemistry or the possibility that Mo is incorporated into specific ROS generating enzymes such as xanthine oxidase (Perkhulyn et al., 2017). Xanthine oxidase catalyzes the oxidation of hypoxanthine to xanthine, producing O •− 2 , and also converts nitrite into nitric oxide, producing ONOO− (Collin, 2019). 13 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2.2.3 Nanomaterials and ROS Nanotechnology is a multidisciplinary science that deals with objects, nanoparticles (NPs), at the nanoscale (from 1 to 100 nm) and seeks to understand the biological processes that occur at this level (Whitesides, 2003). In 2011, the European Commission defined nanomaterial as a natural or manufactured substance that exists in an unbound state, as an aggregate or as an agglomerate (Rauscher et al., 2013). NPs possess advantageous properties such as high surface area to volume ratio for greater interaction with the environment, zeta potential, particle size and shape, surface chemistry, agglomeration, dissolution, and ion release (Fernando et al., 2018). Due to their unique properties, such as catalytic, optical, and electromagnetic properties, metal NPs and nanomaterials are widely introduced into biological and medical applications (Mamunya et al., 2004), one of which is orthodontics. NPs, especially metal NPs with their physicochemical, mechanical, and antibacterial properties, could have a great impact on the duration of orthodontic treatment, remedy certain related problems, and improve oral health (Sharan et al., 2017). As beneficial as the unique properties of NPs may be, they are also considered potentially toxic. The high surface area to volume ratio makes them highly reactive or catalytic (Drasler et al., 2017) and their small size allows easy penetration into the cell membrane (Yin et al., 2012). The presence and nature of metallic NPs and their metal ions can lead to the formation of ROS, a major cause of NP induced cytotoxicity (Yu et al., 2013). Three mechanisms have been proposed for the NPs formation of ROS: the first mechanism is the interaction between NPs and the cell, the second is the dissolution and release of metal ions from the NPs surface, and the third is the formation of prooxidant functional groups on the NPs surface (Wang et al., 2017a). The mechanism of ROS generation is NPs specific and not yet fully understood. Titanium dioxide nanoparticles (TiO2-NPs) are the most common nanomaterial used in industry, mainly for whitening, whether in cosmetics, plastics, paints, personal care products, or food additives (Ray et al., 2009). TiO2-NPs act as absorbers and deflectors for ultraviolet (UV) radiation. UVA Radiation provides enough energy to the valence electrons to cause them to jump into the conduction orbital, leaving electron holes in the valence band. Since it is necessary to fill the valence band, electrons must be removed from the water, resulting in ROS, •OH especially (Yin et al., 2012). On the other hand, Daimon and Nosaka (Daimon and Nosaka, 2007) postulate that the energy from irradiated TiO2-NPs transfers to molecular oxygen to produce 1O2. Under nonirradiated conditions, the ability of TiO2-NPs to generate ROS is still controversial. Like TiO2-NPs, zinc oxide nanoparticles (ZnO-NPs) are also used in various applications due to their semiconducting property and white appearance (Wang, 2008). The ROS generation mechanism of ZnO-NPs and TiO2-NPs is the same as they are both semiconductors and thus have a gap between the valence band and conduction band. In aqueous media, the electrons of water molecules are hindered and generate •OH. ROS can 14 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 also be spontaneously generated on the ZnO-NPs (Saliani et al., 2016). Another mechanism for the generation of ROS by ZnO-NPs involves interaction with mitochondria, whose dysfunction leads to the generation of ROS (Sharma et al., 2012), and another proposed mechanism emphasizes the dissolution of Zn2+ ions, which causes the production of ROS (Lanone and Boczkowski, 2006). Silver (Ag) has long been known to be a bactericidal agent, mainly due to the interactions between silver ions and molecular thiol groups, which affect DNA replication and cell wall structure (Morones et al., 2005). Silver nanoparticles (Ag-NPs) are found in numerous biomedical and industrial applications. Their bactericidal mechanism is based either on interactions between Ag-NPs and cell structures or on the release of Ag+ and generation of ROS. Rohde et al. (2021) showed that not only Ag+ released from NP can induce ROS in a Fenton-like reaction, but that the surface of Ag-NPs also catalyzes the reduction of H2O2 to •OH (He et al., 2012). Tungsten disulfide (WS2) belongs to transition metal dichalcogenides (TMDCs), a family of layered materials with the general formula MX2, where M stands for a transition metal and X for a chalcogen (Appel et al., 2016a). Tungsten disulfide nanoparticles (WS2-NPs) are gaining their reputation as inert, nontoxic, nonmagnetic, and highly resistant to oxidation and thermal degradation (Chang et al., 2006). Currently, their properties are mainly used as dry lubricants due to their effective friction reduction. The two structures of WS2-NPs are mainly in use: 2H-WS2 and IF-WS2 (inorganic fullerene-like). The underlying mechanism by which WS2-NPs induce oxidative stress is direct, in that the NPs generate ROS, or indirect, in that the NPs affect cellular components and cellular processes, which in turn generate ROS (Yang et al., 2014). Yuan et al. (2018) have shown that WS2-NPs, like other semiconductors, can generate ROS under irradiation conditions, and tungstate (W6+) has also been shown to dissociate from the nanoparticles, but it did not generate ROS. 2.2.4 Molecular targets of ROS ROS can have either a positive or a harmful effect on the biological system. In excess, ROS are considered to deteriorate biological molecules and cellular structures. The organism has the ability to reduce ROS and its damage, but oxidative damage accumulates over time and further damages DNA, proteins, or lipids (Valko et al., 2006). When the scales tip in favor of ROS, oxidative stress occurs and cells take countermeasures in the form of activated/silenced antioxidant defense genes, transcription factors, and structural proteins (Birben et al., 2012). 15 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2.2.4.1 Lipid oxidation Cellular components containing polyunsaturated fatty acids (PUFA) such as cell membranes and organelle membranes are very sensitive to oxidation. The PUFA backbone has two or more double bonds with which ROS can react. The more double bonds a PUFA has, the more likely it is to oxidize in the presence of ROS (Su et al., 2019). In addition to lipid peroxidation, ROS can disrupt the lipid bilayer and inactivate certain membrane-bound proteins and generally increase membrane permeability (Birben et al., 2012). The three-step process of lipid oxidation has been described (Halliwell et al., 1993). In the first step, initiation, ROS (•OH, RO• and ROO•, but not H •− 2O2 and O2 ) subtracts hydrogen from the PUFA methyl group and generates a lipid radical (L•). In the second step, propagation, the L• reacts with either O2 to form a highly reactive lipid peroxyl radical (LOO•) or with transition metals (Fe2+) to form lipid alkoxy radical (LO•). Both radicals are able to subtract hydrogen from a new PUFA, generating lipid hydroperoxide (LOOH) and a new L•, resulting in a chain reaction of lipid radical formation (Ayala et al., 2014). In the final step, termination, LOOH decomposes into aldehydes and ketones, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) (Pisoschi and Pop, 2015). Figure 1: Lipid oxidation (Barrera et al., 2018). 2.2.4.2 Nucleic acid oxidation DNA damage caused by free radicals is a source of mutagenesis, carcinogenesis, and aging in cells. The genetic material of biological systems is constantly threatened by ROS damage, 16 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 namely •OH is considered the main threat because it reacts with the purine, pyrimidine, or deoxyribose in the DNA backbone (Dizdaroglu et al., 2002). Other ROS such as H2O2 and O •− 2 are not directly involved in the generation of oxidative DNA damage. ROS induced damage is also considered as single or double strand DNA breaks and DNA crosslinks. These DNA changes can be observed in transcriptional fluctuations, signal transduction, genomic instability, and error-prone replication (Cooke et al., 2003). The best known DNA oxidation product is the base modification 8-hydroxy-2'- deoxyguanosine (8-OH-dG), which is considered a reliable marker of oxidative damage. It is formed when •OH attacks guanine at the C-8 position. Due to the base pairing of adenine and cytosine, 8-OH-dG can cause transversion mutations from A:T to C:C or G:C to T:A (Kohen and Nyska, 2002). Other carbon positions of pyrimidines are also susceptible to ROS attack. Not only nuclear DNA, but also mitochondrial DNA is susceptible to oxidative damage. The fact that mitochondria are the main source of ROS and their ROS oxidative activity is highly localized places them at higher risk for free radical-induced DNA damage. Because mitochondrial DNA lacks the nucleotide excision repair mechanism and is not protected by histones like nuclear DNA, it is extremely vulnerable to oxidative damage (Inoue et al., 2003). 2.2.4.3 Protein oxidation Oxidation of proteins is a covalently modified process in which ROS or secondary byproducts of oxidative stress react with the protein molecule (Shacter, 2000). Amino acids, simple peptides, and proteins are all susceptible to ROS mediated damage. The consequences of protein oxidation are manifested by a loss of protein activity (receptor, enzyme, transport, or structure) and a tendency toward proteolysis or protein denaturation. The basic constituents of proteins, amino acids, are the primary target of ROS, particularly cysteine, methionine, and the aromatic amino acids (tyrosine, phenylalanine, and tryptophan) (Kehm et al., 2021). One of the ROS induced protein damage is protein carbonylation, which occurs either by direct oxidation of protein amino acids, cleavage of the protein backbone, or incorporation of carbonyls into the protein backbone (Dalle-Donne et al., 2003a). Cleavage of the backbone is •OH dependent and generates a carbon-centered radical. In the presence of transition metals and hydroperoxyl radicals (HOO•), further oxidative reactions of a carbon-centered radical occur. Peptide cleavage leads to the formation of alkyl, alkylperoxyl, and alkoxyl radical intermediates, as well as the formation of carbonyl groups, a marker for protein oxidation (Sitte, 2003). It is also possible for carbon-centered radicals to react with each other to form protein-protein crosslinks. 17 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 So-called "primary protein carbonylation" occurs on proline, arginine, lysine, and threonine side chains when the redox reaction is catalyzed by transition metals. The oxidized side chains are stable and represent an effective protein oxidation marker (Dalle-Donne et al., 2003b). Secondary protein carbonylation can also occur when the oxidatively modified lipids or carbohydrates react with the amino acid side chains (Suzuki et al., 2010). 2.2.5 Cellular Antioxidative defense system Because they are constantly exposed to ROS, aerobic organisms have developed an efficient defense system during evolution consisting of defense, neutralization, and repair mechanisms. The overproduction of ROS and its effects are mediated by antioxidants, either enzymatic antioxidants or non-enzymatic antioxidants. Cellular defenses use enzymatic and nonenzymatic antioxidants to regulate the production of free radicals and their metabolites. Depending on the function of the antioxidant defense, the agents can be classified as follows: those that inhibit the formation of ROS, those that bind metals to prevent the formation of ROS, the ROS deteriorating endogenous enzymes, and the radical and nonradical scavengers (Niki, 2014). 2.2.5.1 Enzymatic defense There are four major defense enzymes responsible for maintaining ROS at appropriate levels: Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and the thioredoxin reductase system (Trx). 18 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Cu2+/ZnSOD producing H2O as a byproduct. The Zn ion in SOD contributes to the stability of the protein structure (Patlevič et al., 2016). The same mechanism of dismutation of O •− 2 is also postulated for Mn-SOD, where conversion of redox active Mn3+ to Mn3+ occurs, but only in the mitochondria where the Mn-SOD is found (Abreu and Cabelli, 2010). 2.2.5.1.2 Catalase CAT is responsible for intracellular conversion of H2O2 to H2O and O2. There are many types of CAT enzymes in organisms, but the most common type consists of four subunits, each of which has an Fe3+ heme group in the active sites and each tetramer is bound to NADPH to protect against possible H2O2 inactivation (Kirkman et al., 1999). The two-step catalytic reduction of H2O2 occurs in peroxisomes, where in the first step the heme-Fe3+ reduces H2O2 to H2O, and in the second step the generated heme compound from the first reaction step oxidizes the second H2O2 molecule to H2O and O2 (Putnam et al., 2000). 2.2.5.1.3 Glutathione peroxidase Another enzyme responsible for the degradation of H2O2 is a tetramer with a selenium atom in the active site, glutathione peroxidase (GPx) (Arthur, 2000). GPx exert antioxidant functions in various cellular compartments. GPx1 is present in the cytosol and mitochondria, GPx2 is present in the cytosol and nucleus, Gpx3 is located in the plasma, and Gpx4 is a membrane-bound protein. (He et al., 2017) This selenoprotein uses glutathione (GSH) as a reducing substrate for the degradation of hydrogen peroxides and organic peroxides to water or the corresponding alcohol. The active form of the selenocysteine residue reduces peroxides, and the oxidized selenic acid uses two molecules of GSH to regenerate, generating oxidized glutathione (GSSG) (Deponte, 2013). Both GPx and CAT simultaneously eliminate H2O2, peroxisomal H2O2 is eliminated by CAT, while mitochondrial and cytosolic Cu/Zn-SOD generated H2O2 is eliminated by GPx (Arthur, 2000; Deponte, 2013). 2.2.5.1.4 Glutathione reductase Although not directly involved in ROS defense, glutathione reductase (GR) plays an important role in GSH metabolism and as such is closely related to the glutathione redox system. GR is a homodimeric flavoprotein that is essentially an oxidoreductase, thus requiring NADPH, H+ and GSSG or its function and producing two molecules of GSH and NADP+ (Berkholz et al., 2008). GSH is not only considered a non-enzymatic antioxidant, but is also a necessary substrate for GPx activity. To maintain redox homeostasis, GR must reduce GSSG to GSH via an NADPH-dependent mechanism, thus maintaining the reduced GSH at high concentration (Jozefczak et al., 2012). Recycling of GHS by GR is essential for the GSH-dependent antioxidant system (Rogers et al., 2004). 20 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2.2.5.1.5 Peroxiredoxin Peroxiredoxin (Prx) is a member of the thiol peroxidase family and is closely associated with the Trx system, providing electrons necessary for its function. Because Prx have two Cys residues in their active site, they are often referred to as 2-Cys Prx. The antioxidant mechanism of Prx involves the deprotonated form of Prx reacting with H2O2, ROOH, to release water or ROH. The newly formed molecule forms a disulfide bond with another Prx molecule. Using the Trx system, the two bound Prx molecules are reduced back to their active form (Lu and Holmgren 2014). The scavenging and catalytic ability of Prx is very efficient and comparable to that of CAT and GPx (Manta et al., 2009). 2.2.5.1.6 Thioredoxin reductase When we talk about the redox defense of thioredoxin reductase (TrxR), the whole thioredoxin system should be mentioned, which also consists of NADPH and thioredoxin (Trx) (He et al., 2017). The system provides necessary electrons to a variety of enzymes responsible for DNA synthesis and protection from oxidative stress. The enzyme consists of the prosthetic group FAD, the NADPH-binding site, and a redox-active disulfide site (Du et al., 2012). Electron transfer from NADPH to the redox-active site allows TrxR to catalyze the reduction of Trx via disulfide reductase activity, thereby regulating the dithiol/disulfide balance of the protein (Balsera and Buchanan, 2019). Depending on their location in the cell, we can distinguish between the TrxR1 in the cytosol and the TrxR2 in the mitochondrion, although their function does not differ. The Trx system provides electrons for the enzyme peroxiredoxin (Prx) to degrade H2O2, ROOH and ONOO− (Figure 3)(Lu and Holmgren, 2014). The Trx system and the other thiol-dependent system, the GHS system, are functionally intertwined and appear to compete with each other, but the two systems are thought to work in parallel to reduce ROS accumulation in the cel (Du et al., 2012). Figure 3: The Trx system and Prx mechanism (Lu and Holmgren, 2014). 21 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2.2.5.2 Non-enyzmatic defense Non-enzymatic antioxidants are mainly low-molecular-weight substances, polypeptides, and proteins that are produced by the organism or ingested in the daily diet. They include vitamin A and vitamin C, β-carotene, uric acid, melatonin, and the most important of all, GSH. 2.2.5.2.1 Glutathione The three amino acids glutamine, cysteine, and glycine form the most important molecule for non-enzymatic redox defense, glutathione (GSH) (Townsend et al., 2003). Its reduced form acts as an O •− 2 scavenger, an electron donor for H2O2 degradation by GPx, it modulates glutathionylation of proteins, and acts as a carrier and store of cysteine (Deponte 2013; Dickinson and Forman, 2002). In view of its multifunctional role in the organism, high levels of GSH and lower levels of GSSG are good indicators of normal or abnormal physiological conditions (Ballatori et al., 2009). Under normal physiological conditions, GSH is present in the reduced form and is found throughout the cell. 2.3 MODEL ORGANISM AND CELL LINE 2.3.1 Yeast Saccharomyces cerevisiae Model organisms are irreplaceable tools in basic biology research and clinical trials research (Hunter, 2008). Initially, model organisms such as bacteria and bacteriophages focused on the study of key molecular mechanisms (replication, transcription, protein synthesis, and gene activity), but the field of research soon expanded to include more complex organisms ( Drosophila, Arabidopsis, zebrafish and rodents). Once the biology of model organisms is well understood, they become not less, but even more important for the study of cellular processes because numerous genes and signaling pathways are conserved across species (Hunter, 2008). Model organisms are often chosen because they overcome ethical and experimental constraints, provide a model for developing, optimizing, and standardizing certain analyzes, and are a clear representative of a larger community of species with the same or similar biological processes (Karathia et al., 2011). It should be stressed however that data obtained on model organisms cannot be directly extrapolated on humans due to intra-species differences. Saccharomyces cerevisiae (S. cerevisiae) is the best known, studied, and characterized eukaryotic model organism. Its cellular function and basic organization resembles that of mammalian cells, making it an ideal model for the study of biological processes or pathologies (Karathia et al., 2011). The complete sequence of the yeast genome reveals a well-conserved amino acid sequence and protein function between eukaryotic species (Botstein and Fink, 2011). Because of its high homology with the human genome, 22 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 comparable homology of protein functions, wide range of inexpensive genetic manipulations, ease and cheapness of cultivation and growth, study of multiple processes at once, and nearly complete database, S. cerevisiae is one of the ideal models for the study of oxidative stress response (de la Torre-Ruiz et al., 2015). The formation of ROS on the website ETC and the mechanism of oxidative stress response or defense is similar in yeast as in mammals (Herrero et al., 2008). 2.3.2 Human gingiva fibroblast cell line To comply with the 3Rs (Replacement, Reduction, and Refinement) principle, alternatives to in vivo animal experiments should be considered by using either primary cells or cell lines (Krewski et al., 2010b). A focus should always be on the cell source, passage number, and cultivation method to allow comparison between laboratories. This includes the laboratory plastic used for culturing and assays, as well as growth conditions, morphology, and cell differentiation. Cell lines are generally preferred over primary cells because they are more stable, homogeneous, and generally available, resulting in better replication and comparison of scientific data. However, the advantages of using cell lines come at a price, as they do not differentiate and thus do not fully represent the in vivo situation (Gstraunthaler and Hartung, 2002). Human gingival fibroblasts (HGF) are the most abundant representatives of the oral mucosa and are therefore frequently used in experiments to evaluate toxicity (Mah et al., 2014). Since they are in close proximity to the orthodontic alloys, they are a clinically relevant model. With each cell division, telomeres shorten and so does the lifespan of the cell. By expressing human telomerase reverse transcriptase (hTERT) in gingival fibroblasts, which prevents telomere shortening, a long-lived cell line can be obtained without altering physiology or phenotypic characteristics (Reijnders et al., 2015). 23 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 3 MATERIALS AND METHODS 3.1 RESEARCH WORK FLOW The research was divided into four research sections, which are shown schematically in Figure 4. Figure 4: Research work flow. 3.2 SYSTEMIC OXIDATIVE STRESS PARAMETERS IN PATIENTS DURING ORTHODONTIC TREATMENT WITH FIXED APPLIANCES 3.2.1 Ethical approval Ethical approval was obtained from the National Medical Ethics Committee (Annex A) prior to the start of the study, as well as informed consent from all patients prior to inclusion (Annex B). The study protocol was designed and conducted in accordance with the Declaration of Helsinki for Medical Research Involving Human Subjects (WHO, 2001). 3.2.2 Subjects and cohort design Fifty-four healthy male patients between the ages of 19 and 28 who were diagnosed with dental anomalies (mild crowding and malocclusion) and were undergoing orthodontic 24 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 treatment at the Department of Orthodontics, College Medical Center of Ljubljana, Slovenia, participated in the study. Participation in the study was anonymous and voluntary. Before the start of the study, patients were informed about the purpose and procedure of the study. Withdrawal from the study was possible at any time. The research and sampling did not affect the course of orthodontic treatment. In addition, informed consent was obtained from each person to participate, and personal data were collected and stored in the archive of Assoc. prof. dr. Jasmine Primožič, Ph.D. dent. med, to which only she has access. Before male subjects were enrolled in the study, a questionnaire about their lifestyle habits had to be completed to meet the inclusion/exclusion criteria (Annex C). Subjects with oral pathology (including periodontitis), poor oral hygiene, and known allergies, as well as smokers or subjects undergoing pharmaceutical therapy, including the use of food supplements with antioxidant properties, were excluded. The exclusive criteria were that subjects should not have prosthetic or other metallic materials in their body (implants, pricing, ...) and should not be exposed to metals in their daily life and should not consume synthetic antioxidants. Females were not included because of possible false results due to hormonal fluctuations. The healthy male patients were randomly divided into two groups, the treatment group (TG), which underwent the orthodontic treatment, and the control group (CG), in which the orthodontic appliance was not placed in the oral cavity. The TG consisted of 27 male patients with a mean age of 24.6 ± 1.7 years, while the control group (CG) had the same number of patients with a mean age of 24.7 ± 1.7 years. During the study, patients adhered to a similar diet program and tried to avoid taking antioxidant supplements and alcohol. Extreme exercise and nocturnal living were discouraged. To exclude a possible influence of periodontal inflammation on the measurements of oxidative stress parameters, all participants were instructed on oral hygiene two weeks before the start of the study. 3.2.3 Insertion of the orthodontic appliance The orthodontic appliance used to treat the malocclusion of the TG consisted of SS brackets (Gemini Brackets, 3M Unitek; USA) and two NiTi archwires (3M Unitek; USA). The appliance was inserted by a certified orthodontist. 3.2.4 Capillary blood collection Capillary blood was collected from both TG and CG at four different time points: before orthodontic appliance insertion (time 0), after 6 hours, 24 hours, and after 7 days. The blood collection site was first disinfected with 70% ethanol and lightly massaged for better blood flow. A sterile needle is used to lightly prick the fingertip. The first drop of blood is discarded and the remainder, approximately 100 µl, is collected in a heparinized tube. 25 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 3.2.5 Free oxygen radical test Free oxygen radical test (FORT) is a colorimetric method using the Fenton reaction of transition metals to catalyze the degradation of hydroperoxides (ROOH) (Pavlatou et al., 2009). Capillary blood samples (50 µl) were dissolved in an acidic medium where the ROOH reacts with transition metals to produce RO• and ROO• radicals. In the presence of phenylenediamine derivative (2CrNH2), the RO• and ROO• radicals reacted with the additive. A color change, the intensity of which correlates with the ROOH concentration, can be measured spectrophotometrically at 505 nm using a special instrument FORM PLUS 3000 (Callegari 1930, Italy). 3.2.6 Free oxygen radical defense Free oxygen radical defense (FORD) is based on the antioxidant ability of the sample to quench the chromogen signal (4-amino-N,N-diethylaniline sulfate). The samples are placed in an acidic buffer containing FeCl3 and, depending on the concentration of the antioxidant, the chromogen loses its color proportionally (Pavlatou et al., 2009). The change is visible at 505 nm on the special instrument FORM PLUS 3000 (Callegari 1930, Italy). The values obtained are compared with the standard curve of Trolox, a non-enzymatic antioxidant. 3.3 TYPE AND AMOUNT OF OF METAL IONS RELEASED FROM ORTHODONTIC ALLOYS 3.3.1 Orthodontic materials Both the upper and lower parts of SS, Ni-Ti, Co-Cr and β-Ti archwires were included in the study. Brackets and bands were all made of SS alloy. The surface area of each part (wire, bracket, and band) was measured using a digital caliper CD-AX /APX (Mitutoyo, Japan) and a rough estimate of the surface area was obtained. The detailed description of each orthodontic part used in the study is described in Table 6. 3.3.2 In vitro conditions Artificial saliva was prepared internally to simulate oral conditions. It consisted of 400 mg/mL NaCl, 400 mg/mL KCl, 960 mg/L CaCl2·2H2O, 690 mg/L NaH2PO4·2H2O, 5 mg/L Na2S·9H2O, and 1000 mg/L urea, respectively (Kovač et al., 2021). All of the above chemicals were purchased from Merck (Darmstadt, Germany). Ultrapure water (Milli-Q, 18.2 MΩ cm) from a Direct-Q 5 Ultrapure (Millipore, Watertown, MA, USA) water system was used to dissolve the chemicals, and the pH of the saliva was adjusted to 6.7-6.8 with WTW 330 pH (Weilheim, Germany) to better match oral conditions. 26 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 250 mL of artificial saliva was placed in Teflon bottles (Merck, Germany) and the corresponding orthodontic parts, either two sets of wires, 24 brackets, or 4 molar bands, were immersed in it. A blind test with artificial saliva served as a control. Teflon bottles were used to minimize voids and prevent absorption of metals on the walls of the containers during the experiment. Samples were stored in a dust-free incubator (Kambič, Semič, Slovenia) at 37 °C for 90 days. 3.3.3 Inductively coupled plasma mass spectrometry analysis 3.3.3.1 Released metal ions in saliva Prior to sampling, the Teflon bottles were carefully turned upside down to ensure an even distribution of elements in the sample volume. Saliva samples were collected at baseline, 2 hours, 24 hours, 48 hours, 7 days, 30 days, 60 days, and 90 days. Three samples of 3 mL were taken from each bottle, acidified with 6 μL Suprapur® nitric acid (Merc, Germany) and stored at -20 °C until analysis. 3.3.3.2 Metal alloy composition Approximately 10 mg of each appliance part was dissolved in 5 mL of acid at 90 °C. The SS alloy parts were dissolved in aqua regia (HNO3 + 3HCl), all titanium-based alloys (Ni-Ti and β-Ti) were dissolved in a mixture of HNO3, HF, and HCl (volume ratio 4:2:1), and Co-Cr alloys in pure HCl. All acids were purchased from Merck (Germany). 3.3.3.3 Metal ion concentration measurement The concentrations of metal ions, both of the digested orthodontic appliances and of the released metal ions in the artificial saliva, were determined by inductively coupled plasma mass spectrometry (ICP-MS) on an Agilent 7700x ICP-MS instrument (Tokyo, Japan), using matrix-matched standards for calibration. ICP-MS Measurement parameters are listed in (Appendix D) of the supplemental material. Since there are no certified reference materials for the determination of trace elements in orthodontic appliances, the accuracy of the determination of metal ions in the analyzed samples was evaluated by the spike recovery test. For this purpose, known amounts of the analyzed elements were added to the bracket samples before digestion and the analytical procedure was applied. To verify the accuracy of the determination of the concentrations of metal ions released into the artificial saliva, the samples collected 90 days after incubation were spiked with known amounts of metals and the concentrations were determined using ICP-MS. Good agreement was obtained between the theoretically calculated and measured concentrations (differences did not exceed ±5%), confirming the accurate determination of 27 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 total metal concentrations and concentrations released into the artificial saliva by ICP-MS. In addition to the samples analyzed throughout the experiment, blank samples of the artificial saliva were also analyzed. 3.4 OXIDATIVE STRESS IN YEAST CELL MODEL 3.4.1 Yeast cultures Wild type S. cerevisiae (BY4742) and two yeast mutants lacking certain antioxidative stress defense, ΔSod1 S. cerevisiae (Y06913) and ΔCtt1 S. cerevisiae (Y04718), were all purchased from EUROSCARF (Oberursel, Germany). The genotype of each yeast is listed in Table 3. Table 3: Saccharomyces cerevisiae strains used in the study. Strain Genotype Source Y10000 (Wt) BY4742; MATa; his3Δ1; leu2Δ0; lys2Δ0; EUROSCARF, Oberursel, ura3DΔ0 Germany Y06913 (ΔSod1) BY4741; MATa; ura3Δ0; leu2Δ0; his3Δ1; EUROSCARF, Oberursel, met15Δ0; YJR104c::kanMX4 Germany Y04718 (ΔCtt1) BY4741; MATa; ura3Δ0; leu2Δ0; his3Δ1; EUROSCARF, Oberursel, met15Δ0; YGR088w::kanMX4 Germany Yeast cell cultures were inoculated and maintained in Petri dishes containing solid yeast extract-peptone-glucose (YPD) medium (2% (w/v) glucose, 2% peptone, 1% (w/v) yeast extract, and 2% (w/v) agar (Merck, Darmstadt, Germany). To facilitate growth of yeast cells, they were inoculated into 50 mL of liquid YPD medium (without agar). To achieve repeatable inoculation, the optical density of YPD with yeast cells (OD) at 650 nm had to be between 0.50 and 0.55. Once this was achieved, 20 mL of the cell suspension was transferred to an Erlenmeyer flask containing 180 mL of YPD. Submerged propagation of the yeast biomass occurred at 28 °C with constant shaking (220 RPM, InforsHT, Bottmingen, Switzerland) until it reached early stationary phase. To obtain the yeast at the desired density, the yeast culture had to be transferred to PBS (phosphate buffer salt solution) (Merck, Darmstadt, Germany). Fifty mL of the yeast culture was centrifuged and the pellet containing yeast cells was washed three times with PBS. After the last washing step, 50 mL of the yeast cells suspension in PBS were transferred to a sterile Erlenmayer flask containing 150 mL of PBS. The final concentration of cells was approximately 1x107 cells/mL. The yeast cells were kept in PBS (28 °C, 220 RPM) until treatment with the metal mixture, but not longer than 48 hours. 28 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 On the day of treatment, 10 mL of the yeast culture was aliquoted into an appropriate volume of sterile 50 mL Falcon tubes and sealed with foam plugs. 3.4.2 Metal ion mixtures and yeast treatment The 0.2 M metal ion stock solutions were prepared in accordance with the safety data sheets of Dentaurum (Germany) (Dentaurum, 2020). The aim was to obtain a solution with a similar composition of metal ions as the orthodontic alloys. The selected metal alloys and their metal compositions are listed in Table 4. High-purity salts of FeCl3×6H2O, CrCl3×6H2O, NiCl2×6H2O and CoCl2×6H2O as well as TraceCERT® titanium and molybdenum standards for AAS were dissolved in sterile ddH2O and their pH was adjusted to 7. All chemicals were purchased from Merck (Darmstadt, Germany). Each 0.2 M metal stock solution was used in an appropriate amount to obtain a metal mixture that corresponded to the orthodontic alloy composition, whether SS, Ni-Ti, Co-Cr, or β-Ti alloy. Before treatment, the metal stock solutions were briefly sonicated in an ultrasonic bath (Sonis 3 GT, Iskra Pio, Šentjernej, Slovenia). Table 4: Metal ion w/v ratios for simulating orthodontic alloys. Metal composition (w/v) Orthodontic alloy Fe Ni Cr Co Ti Mo Stainless steel (SS) 72% 10% 18% / / / Cobalt-chromium 18% 15% 20% 40% / 7% (Elgiloy - ELG) Cobalt-chromium 5% 21% 20% 50% / 4% (Remaloy - REM) Nickel-titanium (NiTi) / 55% / / 45% / β-titanium (TiMo) / / / / 78% 12% Falcon tubes containing 10 mL of yeast culture were used for the treatment with metal mixtures. Final concentrations of 1, 10, 100, and 1000 μM in yeast broth were performed for each of the five different metal ion mixtures. Yeast culture, which was not treated served as a control. The treatment lasted 24 h at 28 °C and 220 RPM. 3.4.3 Cell viability 3.4.3.1 Cell culturability After metal treatment, serial dilutions of the treated cell samples were prepared (Koch, 2014) and then twenty 10-µl drops of the sample were placed on a fresh solid YPD plate with some 29 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 spacing and transferred to an incubator at 28°C for 48 hours. After incubation, the colony forming units (CFU) were counted. For each droplet, the number of colonies formed should be between 3 and 30 to be considered countable. The results were expressed as either CFU/mL or as percentages of cell culturability after each metal mixture treatment compared to the control sample. 3.4.3.2 Metabolic activity of the cells For the assessment of cell metabolic activity, BacTiter-Glo™ Microbial Cell Viability Assay (Promega, San Luis Obispo, CA, USA) was used and performed according to the manufacturer's instructions. The kit is capable of determining the number of viable cells in the culture by quantitatively measuring ATP. The reagent provided lyses the cells and the thermostable luciferase reacts with the ATP from the lysed cells to produce a light signal. Hundred µL of the treated cells were added to a well of a white 96-well plate to which the same volume of BacTiter-Glo™ reagent was added. The microplate was immediately placed in a Tecan Spark® Cyto (Maennedorf, Switzerland) microplate reader, mixed for 30 seconds, and after 5 minutes of incubation, the intensity of emitted luminescence and OD were measured at 650 nm. The results were expressed as ratio of luminescence/optical density (L/OD) relative to the untreated control sample 3.4.4 ROS level determination 3.4.4.1 Determination of ROS content The fluorescent dye 2,7-dichlorofluorescein diacetate (H2DCF- DA) (Merck, Germany) was used to measure intracellular ROS (Jakubowski and Bartosz, 1997). The compound is added to cells in the form of diacetate ester, which can cross the cell membrane due to its nonpolarity. Inside the cells, it is deacetylated by nonspecific esterase to 2,7-dichlorodihydrofluorescein (H2DCF). The polarity of the newly formed molecule prevents it from crossing the membrane, so it remains trapped inside the cells. Inside the cell, it becomes the target of ROS and in their presence it is oxidized to dichlorofluorescein (DCF), a fluorescent product whose fluorescence intensity can be measured at an excitation wavelength of 488 nm and an emission wavelength of 520 nm (Tetz et al., 2013). We took 2 mL of the metal-treated cell sample, centrifuged and washed the cell sample three times with 50 mM PBS (pH 7.8). After the third wash, the pellet was resuspended in 500 μL PBS. Hundred μL of this cell suspension was transferred to a tube containing 890 μL PBS and 10 μL of a freshly prepared 1 mM H2DCF- DA solution was added. The dye-containing samples were incubated in the dark at 28 °C and 220 RPM for 30 minutes. After incubation, the cells were washed again, plated on a black 96-well plate in 200 µL volume, and then the 30 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 fluorescence signal (488/520) and OD650 were measured in a designated plate reader. The results were expressed as F/OD relative to the control sample. 3.4.4.1.1 Modification of the protocol In the first approach (protocol I), H2DCF- DA solution was added to the yeast suspension at a final concentration of 10 µM immediately after the addition of the metal ion mixtures. After 24 hours of metal treatment, the yeast cells were washed only once with PBB. Then, the suspension was aliquoted into the appropriate microplate wells and the fluorescence signal and OD (650 nm) were measured using a microplate reader. The results were expressed as F/OD relative to the control sample. The second approach (protocol II) followed the basic protocol. After 24 hours of metal treatment, cells were washed with PBB and then dye was added at a final concentration of 10 µM. After a 30-minute incubation in the dark, the cells were washed again with PBB medium. Then, fluorescence and OD were measured with the same parameters using an appropriate microplate reader. The results were presented as F/OD relative to the control sample. In the third approach, we also followed the basic protocol, but instead of reporting the results as F/OD, we divided the fluorescence intensity with the corresponding cell cultivability. This ensured that the ROS concentration was represented in terms of culturable cells. As such, the results were presented as F/CFU relative to the control sample. 3.4.5 Enzymatic antioxidant defense 3.4.5.1 Cell lysate preparation After 24-hour metal treatment, yeast cells were washed twice with PBS and the pellet was resuspended in lysis buffer consisting of 0.05 M Tris-HCl (pH 8) with cOmplete™, an EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). Cells were homogenized 3 times with zirconium quartz beads (Biospec, USA) on the Bullet Blender tissue homogenizer (Next Advance, USA) at 30 second intervals. After each homogenization step, samples were incubated on ice for the same time. Cell lysates were then centrifuged at 20000 RCF and 4 °C for 20 minutes. Protein concentration in the supernatant was then determined using Bradford assay (Bradford, 1976). The Bradford method is based on the binding of the dye (Comassie Brilliant Blue G-250) to aromatic amino acids. Upon binding, the color shifts from red to blue, changing the absorption maximum of the dye from 465 nm to 595 nm. The principle of the assay was to first add 196 µL of Bradford reagent (Merck, Germany) to a well of a reader plate, to which 31 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 4 µL of an unknown protein sample had to be added. The dye-protein solution was briefly shaken and incubated for 5 minutes. The absorbance could be read within the next hour as the dye-protein solution is very stable. As with any measurement, a calibration curve had to be established by using different concentrations of BSA (Merck, Germany) to which the same amount of Bradford reagent was added. The plate reader measures the absorbance of the sample at 595 nm and the protein concentration of the sample can then be determined from the calibration curve. 3.4.5.2 Superoxide dismutase activity The protocol for SOD activity was based on the antioxdant ability of the SOD enzyme to inhibit the autoxidation of pyrogallol at alkaline pH, which was first described by Marklund and Marklund (1974). When pyrogallol is oxidized, a yellow colored product (purpurogallin) is formed. When SOD is present, the color change decreases according to the activity of SOD. The difference in color change rate can be determined at 420 nm absorbance. Mesa-Herrera et al. (2019) improved and optimized the method for measurement in 96-well plates. Volumes of 176 µL of the reaction buffer (50 mM Tris-cacodylic acid, 1 mM DTPA, pH 8.2) and 20 µL of the protein sample were pipetted into a well of a multi-well plate and incubated at room temperature in a plate reader under gentle shaking conditions. Both blank and control wells were used in the assay. After incubation, 4 µL of 30 mM pyrogallol was added to the wells (except the blank well) and the entire plate was shaken for 1 minute. Kinetic measurement of absorbance at 420 nm was performed every 15 s for 5 min in a multiwell plate reader at room temperature. The increase/decrease in absorbance change was determined by the linear slope of the control and unknown sample. The results are expressed as the percentage activity of the enzyme SOD after treatment compared to the enzyme activity of the control sample. 3.4.5.3 Catalase activity Spectrophotometric determination of CAT activity was first described by Beers and Sizer (1952). As CAT from cell lysates decomposes H2O2, a decrease in absorbance at 240 nm can be observed. The difference in absorbance over time (∆A240) is derived as a measure of CAT activity (Grilo et al., 2020). Samples had to be diluted 50x with PBS, and 100 µL of the diluted samples were pipetted into a well of a UV-transparent 96-well plate (Greiner Bio-One, The Netherlands). To start the assay, 100 µL of a 30 mM H2O2 solution was added. The initial absorbance had to be between 0.520 and 0.550, otherwise H2O2 had to be added to the solution or diluted with PBB. This step ensured a repeatable measurement between replicates. For a control sample, 10 µL of 1 U/mL bovine liver CAT (Merck, Germany) was used. A blank well was also used 32 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 consisting of 200 µL of PBB and 100 µL of 30 mM H2O2 as a blank. Kinetic measurements were performed every 10 s for 5 min at a wavelength of 240 nm. The results were expressed as the percentage activity of the enzyme CAT after treatment compared to the enzyme activity of the untreated control sample. 3.4.5.4 Glutathione peroxidase activity The GPx activity was determined by the assay described by Smith and Levander (2002) with minor modification. The assay relies on the GPx enzyme to degrade peroxides. In the first reaction, GSH is oxidized to GSSG, and in the second reaction, it is converted back to GSH by GR, using NADPH for the reduction reaction. Consequently, the oxidation of NADPH may be a good measure of GPx activity. The adapted assay (Dong et al., 2016) was performed as follows: For a GPx reaction mixture, final concentrations of 6.5 mM EDTA-Na2, 1.3 mM NaN3, 0.5 mM NADPH, 2.5 mM GSH, and 1.7 U/mL GR were dissolved in 65 mM PBS (all from Merck, Germany), with NADPH and GR added as late as possible to the reaction mixture. Then, 47 µL of the cell sample was mixed with 250 μL of the GPx reaction mixture in a well of a multiwell plate, to which 3 μL of 250 μM H2O2 was added to start the reaction. For a blank sample, 50 μL of lysis buffer was used instead of the cell sample. During the addition of H2O2, the change in optical density was measured at 340 nm every 15 s for 5 min. The results obtained were expressed as the percentage activity of GPx enzyme after treatment compared to the enzyme activity of the control sample. 3.4.5.5 Glutathione reductase activity The GR activity assay was described by Smith et al. (1988) and adapted by Glippa et al., (2018). The principle of the assay is the ability of GR to convert GSSG to GSH using NADPH as a substrate. Instead of measuring the rate of NADPH conversion, the assay measures the concentration of GSH formed, which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) present in the reaction mixture. The formed product 5-thio(2-nitrobenzoic acid) (TNB) is colored yellow and its absorbance can be read at 412 nm. For the assay, the buffer (100 mM K-phosphate buffer (pH 7.5) containing 1 mM EDTA) had to be prepared. A 2 mM GSSG solution, 2 mM NADPH and 3 mM DTNB were prepared in the above assay buffer. Twenty μL of the cell sample was pipetted into one well first, followed by DNTB. NADPH was added last, and immediately the plate was shaken for 5 s and absorbance was measured at 412 nm every 20 seconds for 6 min. For blank wells, the cell sample was replaced with the same amount of lysing buffer, and 4 μL (10 U/mL) GR of baker's yeast (Merck, Germany) was used for the positive sample. The results obtained were 33 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 expressed as the percentage activity of the GR enzyme after treatment compared to the enzyme activity of the control sample. 3.4.5.6 TrxR activity The TrxR activity assay was also adapted from Smith and Levander (2002) with minor modifications. TrxR catalyzes the reduction of oxidized thioredoxin, but in the assay 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) was used as a suitable and inexpensive substrate. DTNB is reduced to a thio-bis-nitrobenzoic acid anion (TNB) in an NADPH-dependent reaction. The absorbance change of the formed product TNB was measured at 412 nm. The assay buffer contained 10 mM EDTA-Na2, 5 mM DTBN, 240 μM NADPH, and 0.2 mg/mL bovine serum albumin, all dissolved in 100 mM PBS. The lysate samples were added to the two corresponding wells, each containing 54 µL. To one well containing the lysate samples, 6 µL of 5% EtOH was added and to the second 6 µL of 1.47 mM auranofin, an inhibitor of TrxR (Dong et al., 2016) was added and incubated for 10 minutes at room temperature. After incubation, 250 µL of the assay buffer was added and absorbance measurement started 1 min after buffer addition, due to non-enzymatic reduction of DTNB in the lysate sample. The absorbance change was measured at 412 nm every 15 s for 5 min. The slope of the reaction in the presence of auranofin was subtracted from the slope of the reaction in which auranofin was omitted. The results obtained were expressed as the percentage activity of the TrxR enzyme after treatment compared to the enzyme activity of the control sample. 3.4.5.7 Peroxiredoxine activity Prx activity was determined according to assay described by Ali and Hadwan (2019), also known as ferrous xylenol orange assay (FOX). Prx activity is based on detoxification of peroxides at the expense of either NADPH or NADH. Peroxides are also degraded by CAT and GPx, but due to the addition of sodium azide (NaN3) to inhibit CAT and the absence of the GPx substrate GSH, the FOX method remains a specific and reliable way to detect Prx activity. The FOX assay utilizes the peroxide-dependent oxidation of Fe(II) to Fe(III), which reacts with xylenol orange. The newly formed product is colored blue/purple and its absorption spectrum can be measured at 560 nm. For the assay, the reagents FOX -A and FOX -B had to be prepared. For FOX-A, 25 mM (NH4)2Fe(SO4)2x6H2O in 25 M H2SO4 had to be prepared and for FOX -B, 100 mM sorbitol and 125 µM concentrations in distilled water were prepared. On the day of the experiment, FOX -A and FOX -B were mixed together in a volume ratio of 1:100, as was a standardized 2.1 mM H2O2 solution in 50 mM PBS. 2.1 mM concentration of 1,4-dithio- DL -threitol (DTT) and 10 mM concentration of NaN3 were also freshly prepared each time the assay 34 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 was performed. In a tube, 100 µL of the cell lysate, 200 µL of DTT, 100 µL of NaN3, and 600 µL of PBS were mixed. A standard tube in which the lysate was replaced with additional DTT and a blank tube containing no DTT and no lysate sample were also analyzed. 200 µL of H2O2 solution was added to each tube, mixed vigorously, and incubated for 3 minutes. Then, 50 µL was aliquoted from each tube, mixed with 950 µL of the freshly prepared FOX -A/B reagent, and incubated for 30 minutes at room temperature. The change in absorbance at 560 nm was recorded and evaluated against the standard and blank. The results were expressed as the percentage activity of the PRDX enzyme after treatment, compared to the enzyme activity of the control sample. 3.4.5.8 In-gel enzyme activity of SOD and CAT Native gels require ten times less protein samples than spectrophotometric activity assays and provide a qualitative result compared to the quantitative results of the assays. In addition, the visual representation of activity is more presentable to the general public. The protocol was adopted from Weydert and Cullen (2010) with minor modifications. Reduction of nitroblue tetrazolium (NBT) by the O2 •− is a key factor in the protocol because the enzyme SOD competes with NBT for O2 •−. Since NBT changes color from yellow to blue when reduced, the color inhibition is related to the SOD activity of sample (Spitz and Oberley 2001). The CAT activity assay was similar to the normal spectrophotometric method in which the principle of activity evaluation was the removal of peroxide. The remaining peroxide reduces the yellow potassium ferricyanide to potassium ferricyanide, which reacts with ferric chloride to form a Prussian blue (green-yellow) precipitate. 3.4.5.8.1 Electrophoresis gels A non-denaturing polyacrylamide gel and a 5% stacking gel were prepared for each SOD or CAT activity assay. The difference in gel preparation when evaluating SOD or CAT activity was the separating gel. For SOD activity, a 12% polyacrylamide separation gel was used, and for CAT activity, an 8% separation gel was used. For the native separation gel, 1.5 M Tris-HCl (pH 8.8), 30% acrylamide/8% bis-acrylamide, 10% ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) were mixed in distilled H2O and the volume was pipetted into a 1.0 mm spacer glass plate cassette (Bio-Rad, United States) with the top edge only about 1 cm short. A layer of water was added on top of the gel to cover it completely. Within 1 hour, the running gel cured and the water was carefully removed from the top of the gel. A 5% stacking gel consisting of the same reagents as the separating gel, except 0.5 M Tris-HCl (pH 6.8), was poured on top and a 1.0 mm comb was inserted. It took 1 hour for the stacking gel to cure. The cassette was then removed from the casting stand, wrapped in a towel soaked in water, and allowed to polymerize completely overnight in the margins (2 - 8°C). 35 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 3.4.5.8.2 Electrophoresis buffers Three different buffers had to be prepared: a buffer before electrophoresis, a buffer for electrophoresis, and a buffer for loading the samples. It was an absolute necessity to prepare them fresh and keep them all at < 4 °C. The pre-electrophoresis running buffer ensures that the free persulfate ions are removed from the gels so that they do not interfere with the activity of the antioxidant enzymes. It consisted of 190 mM Tris- HCl and 1.13 mM disodium EDTA. The pH had to be adjusted to pH 8.8 with HCl. The electrophoresis running buffer provided ions to conduct a current and keep the pH relatively constant. It consisted of 50 mM Tris- HCl, 0.3 M glycine, and 2 mM disodium EDTA. The pH was adjusted to pH 8.3 with HCl. The sample loading buffer was prepared by mixing 1.5 M Tris-HCl (pH 6.8), glycerol and 5% bromophenol blue solution at a ratio of 1:1:0.02. 3.4.5.8.3 Loading of samples and electrophoresis After overnight gel polymerization, the combs were removed from the gel assembly, placed in the electrophoresis assembly, and placed in the electrophoresis apparatus (box). The pre-electrophoresis buffer was poured into the reservoir and chamber of the box. To ensure that the pre-electrophoresis proceeded at < 4 °C, the entire electrophoresis box was surrounded with ice. Pre-electrophoresis ran for 1 hour, and the gels ran for 1 hour at 40 mA per gel. This step removed residual APS, TEMED, and incomplete polymerization products that could inactivate native proteins. Cell lysates of known concentration were mixed 1:1 with the loading buffer. 20 µL of the thoroughly resuspended samples were then pipetted into the appropriate wells. After the pre-electrophoresis step, the buffer was removed and replaced with electrophoresis buffer. The gels were then run under the same conditions (40 mA, 4 °C) until the dye line reached the bottom (between 2-3 hours). The gels were then run for an additional 1 hour. 3.4.5.9 SOD in-gel activity staining For SOD activity staining, the dye solution consisted of 2.43 mM NBT, 28 mM TEMED, and 22.4 µM riboflavin 5'-phosphate in 50 mM phosphate buffer (pH 7.8). Each gel was stained in the dye solution for 20 min at room temperature with shaking and in the dark. Then the gels were rinsed twice with water and lightly immersed in the water under fluorescent light or in a light box. Depending on the light intensity (up to 2 hours), the gels 36 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 turned blue/purple in the areas where there was no SOD activity. The gels were then washed with water and left in water under ambient light for better band development. The gel/band images were acquired, and the intensity of each gel/band image was determined using ImageJ, an image analysis software (Java-based image processing software, National Institutes of Health, Bethesda, MD, USA). The activity of the treated lysate samples was compared with the control. 3.4.5.9.1 CAT in-gel activity staining For CAT stain, 2% ferric chloride was prepared in one tube and 2% potassium ferricyanide in distilled water in a second tube. After electrophoresis, the gels were washed three times in distilled water for 10 minutes. After the washing phase, the gel was incubated in a 0.003% H2O2 solution for an additional 10 minutes. The cells were then washed twice for 5 minutes in distilled water. The water was then poured off and the two tubes containing the CAT staining solution were poured onto the gel simultaneously. The staining of the gel took about 30 minutes until achromatic curvatures appeared. All the staining solution was then removed from the dish. The gel/band images were acquired, and the intensity of each gel/band image was determined using ImageJ, an image analysis software (Java-based image processing software, National Institutes of Health, Bethesda, MD, USA). The activity of the treated lysate samples was compared with the untreated control. 3.4.6 Oxidative damages 3.4.6.1 Oxidative lipid damages To evaluate oxidative damage to lipids, the thiobarbituric acid reactivity assay (TBARS) was used. The method was described by Ohkawa et al. (1979) and adapted by Aguilar Diaz De Leon and Borges (2020). The TBARS method uses the reaction between MDA, a product of lipid peroxidation, and thiobarbituric acid (TBA). The reaction product, MDA-TBA2, has a reddish-pink color and absorbs the visible spectrum at 532 nm. After metal treatment, cells were washed twice with PBS and TBARS reagent consisting of 91.8 mM trichloroacetic acid, 2.5 mM thiobarbituric acid, 45.4 μM butylhydroxytoluene (Merck, Darmstadt, Germany) and 25 mM HCl (all from Merck, Germany) was added to the pellet, thoroughly resuspended and homogenized. After homogenization, samples were incubated at 90 °C (Thermomixer R, Eppendorf, Hamburg, Germany) for 30 minutes and then placed on ice for an additional 10 minutes. After ice treatment, butanol was added and the homogenized samples were centrifuged at 10000 RCF for 10 minutes. Fluorescence intensity (515/555 37 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 nm) and OD (650 nm) were measured using a Varioskan™ LUX (Thermofisher, Waltham, MA, USA) microplate reader. Results were expressed as F/OD relative to the untreated control. 3.4.6.2 Oxidative protein damages The method for determinating protein oxidative damages was described by Levine et al. (1990) and modified by Mesquita et al. (2014). The method proposed to use 2,4-dinitrophenylhydrazine (DNPH) to react with the oxidatively formed protein carbonyl groups. For the simplified method, 40 µL of the lysate sample was mixed with 40 µL of DNPH (10 mM in 0.5 M H3PO4) and incubated for 10 min. For the blank sample, the protein sample was replaced with the lysis buffer, and for the positive control, the BSA solution with the same protein concentration as the lysate sample was used. After incubation, 20 μL of 6 M NaOH was added, and after 10 min of incubation, absorbance was measured at 450 nm. Incubation after addition of NaOH was strictly controlled due to the instability of DNPH in alkaline media. Results were expressed as the percentage of protein oxidative damage after treatment with specific metals compared with the control sample. 3.5 OXIDATIVE STRESS IN HGF 3.5.1 HGF cell line The hTERT-immortalized HGF cell line (T0026) was originally purchased from (ABM, Canada) but was kindly provided to us by Prof. Dr. Sue Gibbs (MCBI, Amsterdam UMC, The Netherlands). Fibroblasts were cultured in 10 mL of fibroblast medium consisting of DMEM (Merck, Germany) supplemented with 5% HyClone FetalClone III serum (Cytiva, United States) 1% penicillin-streptomycin (Merck, Germany) in T75 flasks (Merck, Germany) at 37°C and 5% CO2 atmosphere. After reaching confluence, cells were washed with PBS, trypsinized with 2 mL of 0.05% trypsin-EDTA solution (Merck, Germany), and detached from the flask for approximately 10 minutes in the previously described incubator settings. Then, 8 mL of medium was added, a small sample was taken for cell counting, and the cells were centrifuged at 300 RCF for 5 minutes. The supernatant was discarded and the pellet was resuspended in an appropriate amount of the medium so that the final number of cells was 1.0 x 106 per T75 flask. 3.5.2 Nanoparticle characterization and preparation for treatment TiO2-NPs, ZnO-NPs, Ag-NPs and WS2-NPs were purchased from Nanografi Nano Technology (Ankara, Turkey) in different sizes and in the case of TiO2-NPs in different shapes: Anatase, Rutile and a mixture of both forms (Table 5). For the treatment, 10 mg/mL stock suspensions of each NP were prepared in deionized water (MilliQ, Millipore, USA). 38 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Before preparation of working suspensions from NP, stock suspensions from NP were sonicated for 15 minutes in an ultrasonic water bath at 250 W and 50 Hz (Sonis 2GT, Iskra Pio, Slovenia). The prepared stock solutions were used for NP characterization. Characterization of NPs were done by prof. dr. Darko Makovec and asst. prof. dr. Slavko Kralj from the Department for materials synthesis, Institute Jozef Stefan. For the NPs diameter size evaluation, at least 500 NPs were measured with a transmission electron microscope (JOEL, Tokio, Japan). To observe, how NPs behave in an aquatic solution or as in our case, in cell growth medium, dynamic light scattering (DLS) method combined with zeta potential (ZP) measurement were used to measure hydrodynamic diameter of NPs in 100 μg/mL NP solution and to provide information on the aggregation state of nanoparticles in 100 μg/mL NP suspension. The DLS analysis in the liquid phase provides information on the hydrodynamic particle size of the nanoparticles (Lim et al., 2013), whereas ZP provides information of NPs solution stability due to its surface charge (Bhattacharjee, 2016). DLS and the ZP analysis were performed on Litesizer 500 (Anton Paar, Austria). Table 5: Nanoparticles used in the study. Nanoparticles Average Diameter (nm) Catalog number Company TiO2 - Anatase/Rutile 18 NG04SO3506G25 TiO2 - Anatase 28 NG04SO3503G25 TiO2 - Rutile 28 NG04SO3507G25 Nanografi Nano Ag 28-48 NG04EO0105G5 Technology Ag 48-78 NG04EO0103G5 (Ankara, Turkey) ZnO 18 NG04SO3803G25 ZnO 30-50 NG04SO3802G25 WS2 35-75 NG04CO2401G25 To prepare working solutions, which ranged from 1 µg/mL to 1000 µg/mL, the NP stock solutions were appropriately diluted in fibroblast medium. The NP treatment was performed in either 6-well, 12-well or 96-well plates. For this purpose, we used HGF at a concentration of 2.2 × 104 cells/cm2, regardless of the multi-well plate. A volume of 2 mL was added to each well of the multi-well plate for a 6-well plate, 1 mL for a 12-well plate, and 100 μL for a 96-well plate. Freshly seeded cells on a multi-well plate were then placed back into the incubator and left there for 24 hours to attach properly. After attachment, the cells were treated with the working solution NP. 3.5.3 HGF cell viability To test the viability of the cells, several methods were used because some NP interfered with the analytical methods (Mello et al., 2020). 39 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 3.5.3.1 Resazurin assay Resazurin is a non-toxic, membrane-permeable molecule. It acts as an electron acceptor in cells ETC without interfering with their normal function (Page et al., 1993). When the dye accepts electrons from either mitochondrial reductases or other cytoplasmic enzymes, the blue, nonfluorescent dye is reduced to a pink, highly resorufinated dye. The changes in the fluorescence signal can be observed at an excitation wavelength of 530-560 nm and an emission wavelength of 590 nm (Rampersad, 2012). The method used in this study was described by Elshikh et al (2016). HGF were seeded on a 96-well plate, with each well containing 100 μL of 2.2 × 104 cells/cm2, and allowed to attach in the incubator for 24 hours. The cells were then exposed to NPs with final concentrations ranging from 1 µg/mL to 1000 µg/mL, depending on the type of NPs used. After incubation at 37°C and 5% CO2 for 24 hours, 30 µL of 0.015% resazurin (Merck, Germany) was added to all wells and incubated further for 2.5 hours under the same known conditions. The fluorescence intensity of the samples was then measured at 560/590 nm. Results were expressed as the percentage of fluorescence intensity after a given NP treatment compared to the control sample. 3.5.3.2 Neutral red uptake assay The neutral red assay was proposed by Repettou et al. (2008). The dye penetrates the cell membrane at a physiological pH and concentrates in the lysosomes where it binds to anionic or phosphate groups of the lysosomal matrix (Nemes et al., 1979). There is a low pH gradient in the lysosomal matrix that prevents the dye from escaping the lysosome. This only happens in viable cells because they are able to maintain acidic pH gradients by producing ATP. The dye is easily extracted with an acidified ethanol solution and the absorbance of the extract can be quantified spectrophotometrically. The dye concentration of the extract is proportional to the number of viable cells. HGFs were seeded on a 96-well plate, with each well containing 100 μL of 2.2 × 104 cells/cm2, and allowed to attach in the incubator for 24 hours. The cells were then exposed to NPs with final concentrations ranging from 1 µg/mL to 1000 µg/mL, depending on the type of NPs used. After the 24-hour incubation at 37°C and 5% CO2, 18 µL of neutral red dye (Merck, Germany) was added to all wells and incubated further for 2.5 hours under the same known conditions. After the incubation period, the dye-containing medium was discarded and 80 µL of neutral red dye solvent (50% EtOH, 1% CH3COOH, and 49% ddH2O) was added to each well. The multiwell plate was then incubated for 20 min at room temperature with occasional shaking. Measurement was then performed using a spectrofluorimeter at excitation and emission wavelengths of 530 and 645 nm, respectively. 40 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Results were expressed as the percentage of fluorescence intensity after a given NP treatment compared to the control sample. 3.5.3.3 Coomassie Blue Assay The Coomassie blue stain assay is used to determine total protein content. Under acidic conditions, the Coomassie Brilliant Blue G-250 dye binds to arginine, histidine, phenylalanine, tryptophan and tyrosine residues. Upon binding, there is a color shift from 465 nm to 595 nm (Noble, 2014). Theoretically, the cellular protein amount is related to the number of viable cells. The assay used in the study was performed according to Kononenko et al. (2019). HGF were seeded on a 96-well plate, with each well containing 100 μL of 2.2 × 104 cells/cm2, and allowed to adhere in the incubator for 24 hours. The cells were then exposed to NPs with final concentrations ranging from 1 µg/mL to 1000 µg/mL, depending on the type of NPs used. After the 24-hour incubation at 37°C and 5% CO2, the medium was discarded and 50 μL of 0.05% Coomassie Brilliant Blue G250 (Merck, Germany) (30% MeOH, 10% CH3COOH, 60% ddH2O) was added to each well and incubated for 20 minutes at room temperature. The dye was then discarded, the wells were washed with Dulbecco's phosphate buffered saline (DPBS), and 50 μL 0.1 M NaOH was added to dissolve the dye. A 20-minute incubation followed and absorbance in the wells was measured at 595 nm. Results were expressed as the percentage of absorbance intensity after a given NP treatment compared to the control sample. 3.5.3.4 The trypan blue cellular debris assay The trypan blue dye is well known in the laboratory (trypan blue exclusion assay) as it is used as a dye to distinguish between intact live cells and permeable dead cells and debris, usually routinely used to count live cells on a hemocytometer (Strober, 2015). Live cells do not take up the dye, but dead cells and debris do. The viability assay proposed by Lebeau et al. (2019) and also used in this study suggests using bound dye concentrations to determine cell viability. HGF were seeded on a 6-well plate, with each well containing 2 mL of 2.2 × 104 cells/cm2, and allowed to attach in the incubator for 24 hours. The cells were then exposed to NPs with final concentrations ranging from 1 µg/mL to 1000 µg/mL, depending on the type of NPs used. After incubation at 37°C and 5% CO2 for 24 hours, the well medium containing cell debris and dead cells was carefully collected in a tube and centrifuged at 10000 RCF and room temperature for 2 minutes. The medium was then discarded and 100 μL of a 0.4% trypan blue solution (ThermoFisher, USA) was added, shaken vigorously and stained for 5 min. The samples were then centrifuged again for 2 min at 12000 RCF and the dye was removed. Without disturbing the pellet, a washing step was performed with 500 μL of 99% 41 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2-propanol. To the washed pellet, 100 μL of PBS was added to each of the tubes to extract the dye from the pellet. The pellet was shaken again and heated on the ThermoMixer C dry block heater (Eppendorf, Germany) at 80 °C for 10 min. A final centrifugation step was performed at 12000 RCF for 2 min, and a 40 μL volume of the PBS extract was added to a 96-well plate. The optical density of the extract was measured at a wavelength of 590 nm. Results were expressed as the percentage of absorbance intensity after a given NP treatment compared to the control sample. 3.5.4 ROS level determination The fluorescent probe H2DCF-DA was used to detect the intracellular ROS to evaluate the redox balance in pathological cells. The assay described by El-Hassani and Dupuy (2013) served as a platform for this study. The dye crosses the cell membrane and is converted to a fluorescent product in the presence of ROS. This cleavage of the dye also occurs in dead or senescent cells, so a live/dead ell exclusion assay must be performed simultaneously. The proposed method was to use propidium iodide (PI) to stain the dead and dying cells. PI cannot penetrate the membranes of viable cells, only dead and dying cells, where it binds to nucleic acids. When it attaches to DNA, the fluorescence of PI increases 20-30 fold and the emission and excitation shifts to 535 nm / 617 nm (Rosenberg et al., 2019). Because of the wavelength shift, the use of PI at the same time as H2DCF-DA is possible. HGF were seeded on a 96-well plate, with each well containing 100 μL of 2.2 × 104 cells/cm2, and allowed to adhere for 24 h in the incubator. The cells were then exposed to NPs, the final concentration of which ranged from 1 µg/mL to 1000 µg/mL, depending on the type of NPs used. After incubation at 37°C and 5% CO2 for 24 hours, the cells were washed three times with PBS. After the last washing step, PBS was discarded and 100 µL of 10 µM H2DCF- DA was added to the wells. The multiwell plates were left in dark conditions for 45 min. After incubation, 15 µL of 1 μg/mL PI was added and the plates were immediately placed in the spectrophotometer to obtain fluorescence for intracellular ROS (exc/em = 488/527) and PI (exc/em = 535/617). Results were expressed as the percentage of fluorescence intensity after a given NP treatment compared to the control sample. 3.6 STATISTICAL ANALYSIS Fort he data analysis of the in vivo study, the Statistical Package for Social Sciences Software release 20.0 (SPSS Inc., Chicago, Illinois, USA) was used. To balance the age of each experimental groups, Mann-Whitney U-test was used. After testing the normality of the data with the Shapiro-Wilk test and Q-Q normality plots and the equality of variance among the datasets using a Levene test, nonparametric methods were used for data analysis. A Friedman test was used to assess the significance of the differences in every parameter (FORT, FORD, and FORT/FORD ratio) over the time points within each group. When significant 42 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 interactions were seen, a Bonferroni-corrected Wilcoxon test was used for pairwise comparisons. A Mann-Whitney U-test was used to assess the significance of the differences in every parameter between the two groups within each time point. The results were considered to be significant at p-values below 0.05. Shapiro–Wilk and D’Agostino and Pearson tests were used to analyze the normal distribution of the acquired data. Normally distributed data were analyzed with one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons and non-normally distributed data were analyzed with Kruskal–Wallis test followed by Dunn’s post hoc test. Cutoff for the statistical significance of data was considered when p < 0.05. Visual presentation and statistical analysis were performed with GraphPad Prism (version 8.02 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com). 43 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 4 RESULTS WITH DISCUSSION 4.1 CHANGES IN OXIDATIVE STRESS PARAMETERS IN THE CAPILLARY BLOOD In this study, we sought to evaluate whether oxidative stress in blood samples occurs during orthodontic treatment. The predominance of free radicals over antioxidants may be a trigger for periodontal desies (Panjamurthy et al., 2005). Previous studies have used saliva (Buczko et al., 2017; Portelli et al., 2017; Yamyar and Daokar 2019) or gingival crevicular fluid (Atuǧ Özcan et al., 2014; Chitra et al., 2022) as diagnostic tools to determine oxidative stress parameters. The oral cavity, especially saliva, has numerous antioxidant defense systems and therefore plays an important role in maintaining redox balance. It can be said that saliva acts as the first antioxidant defense, and salivary markers reflect to some extent the condition of the oral cavity (Buczko et al., 2017). Evaluation of individual oxidative markers provides only specific information, whereas estimation of the overall oxidative status and total antioxidant capacity provides a more objective picture of the given prognosis. By measuring the formation of ROS (FORT) and antioxidant potential (FORD) in capillary blood samples, the normal/abnormal physiology of patients can be more accurately estimated at a systhematic level. Blood samples from the TG and CG groups were collected during the first week of orthodontic treatment, and the values FORT and FORD were determined. No significant difference was found between the baseline values of TG and CG before appliance insertion, neither for FORT (p > 0.05) nor for FORD (p > 0.05). The only significant difference in the values of FORT was observed for TG after 24 hours of orthodontic treatment (p < 0.05), but at the next analysis (at 7 days), the value decreased back to the baseline level. Compared to CG, whose FORT values remained almost the same throughout the study, an increase in radical formation is observed after 24 hours of orthodontic treatment. The FORD values did not change significantly in the first week in either group. The results of the analysis of FORT and FORD are shown in Figure 5 as FORT /FORD ratio, which illustrates the balance between radical formation and antioxidant defense. There was no evidence of periodontal disease or inflammation in any subject throughout the study period. 44 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Figure 5: Changes in FORT/FORD oxidative stress parameter during the one week orthodontic treatment. Capilary blood samples of orthodontic patients were taken at different time points. At the 24-hour mark, an significant increase of FORT/FORD ratio was observed in the TG. Results are presented as mean values with standard deviation errors (Kovac et al., 2019). The result suggests that there is a short-term increase in ROS formation after 24 hours in patients undergoing orthodontic treatment. Yamar and Daokar (2019) and Olteanu et al. (2009) also observed a significant increase in salivary oxidative stress markers after 24 hours of orthodontic treatment and a steady decrease in marker levels after 7 days and 90 days. Several other studies (D’Attillio et al., 2004; Olteanu et al., 2009) suggest that the significant increase in oxidative stress markers is due to the release of metal ions, which is highest in the early stages of orthodontic treatment. The obtained results and many other studies demonstrate the long-term effects that orthodontic treatment can have on patients. The longest in vivo experiment to date was conducted by Buczko et al. (2017) who collected saliva samples from orthodontic patients until 24 weeks. They, too, obtained initially higher values for total oxidant status, but after 24 weeks, salivary oxidant status reached the initial basal level obtained before the study. The normalisation of the ROS and antioxidant ratio may be attributed to the organism's adaptation responses or differences in metal ions release within the sampling time period. However, these results should be taken with caution, because the increase in oxidative levels may also be caused by the inflammation of the gingival tissue during orthodontic treatment (Buljan et al., 2012). The mechanical forces alter the periodontal ligament and alveolar bone structure induce local inflammation around the teeth (Shamaa and Mansour 2019). Atuğ Özcan et al. (2014) found no inflammation and pathological changes in healthy tissues during 6 months of orthodontic treatment. In our study, we did not investigate the expression of proinflammatory mediators and we did not perform measurements of metal ions released 45 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 in blood. Other sources of ROS such as oral bacteria, food, chocolate, radiation, and cigarette smoking that affect redox balance could also be responsible for an increase in oxidative markers, but could be neglected based on the inclusion/exclusion criteria used in the study and a regular health examination by an orthodontist. Since the FORT /FORD ratio does not provide any indication of the initial increase in ROS, we assumed that the reason for the occurrence of oxidative stress must be from the orthodontic material, from which metal ions can be released and cause oxidative stress (Kovac et al., 2019). 4.2 METAL ION RELEASE FROM DIFFERENT ORTHODONTIC ALLOYS The orthodontic appliance remains in the oral cavity for about 2 years or until the end of orthodontic treatment (Mavreas and Athanasiou 2008). Based on our previous findings that metallic orthodontic appliances could be the reason for the occurrence of oxidative stress (Kovac et al., 2019), the question arose about the safety of such orthodontic appliances and their permanent use. Although the arches are usually changed every one to three months, the brackets and molar bands are rarely changed throughout the treatment period. In the oral cavity, fixed orthodontic appliances are exposed to constantly changing pH and temperatures, as well as biological and enzymatic environments (Barrett et al., 1993). Electrochemical corrosion, mechanical friction, wear of the appliance, and surface wear are very common in everyday life and lead to increased release of metal ions (Močnik et al., 2017). The biological effects of certain metals and their ions that make up orthodontic appliances have been linked in the past to certain oral health problems such as glossitis, gingivitis, contact stomatitis, multiforme erythema, and gingival hypertrophy (Ortiz et al., 2011). The most commonly used orthodontic alloys for parts of an orthodontic appliance include SS, Ni-Ti, Co-Cr, and β-Ti, all of which are composed of a combination of metals, some of which are considered alergenous or even toxic (Keinan et al., 2010). In vitro studies on the released metal ions from orthodontic alloys are conducted in artificial saliva to mimic in vivo conditions in terms of pH, temperature, and in some cases even saliva composition (Hanawa, 2004). To better understand the metals that make up orthodontic alloys and the concentrations of metal ions they release under laboratory conditions, we designed a study in which parts of the orthodontic appliance were immersed in artificial saliva for 90 days and their metal composition was evaluated. Table 6 shows the metal composition and combined surface area of each orthodontic alloy used in the study. The orthodontic parts (brackets, archwires, and bands) made from the alloy SS had approximately the same weight percent metal composition of Fe (57 ± 2%), Ni (16 ± 2%), and Cr (25 ± 2%), although they were manufactured by different companies. The only notable difference in the composition of the SS alloys was that the SS version also contained 1.9% Mo. The similarity in metal composition was also evident in the two Ni-Ti alloys, 46 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 where the weight fraction of Ni was 73.5 ± 0.3% and that of Ti was 26.5 ± 0.3%. The single β-Ti alloy used in the study consisted of 91.8% Ti and 8.18% Mo. Although the Co-Cr alloys were similar in terms of Co content (50%), they had some differences in terms of the other constituents, which was to be expected since they are marketed as separate products. We used the manufacturer's Data Sheet (Dentaurum, 2020) to compare our alloy compositions to those prescribed. Since there are many different types of SS wires, the exact range cannot be determined. As a rough estimate, the alloy SS should contain between 17-25% Cr, 8-25% Ni, 0.0-6.5% Mo, and the balance Fe (Tian et al., 2017). Analysis of the composition of our Ni-Ti alloy showed a much higher percentage weight ratio (73% Ni and 26% Ti) compared to the manufacturer's data sheet (55% Ni and 45% Ti). One possible explanation for this situation could be the incomplete dissolution of the alloy, but the dissolution liquid obtained was clear and did not contain solid particles. Another explanation is that the metal composition reported by the manufacturers was evaluated by energy dispersive X-ray spectroscopy, where the amount of elements on the surface can be imaged (Shojaei et al., 2022) which means that the metal composition may be different below the surface, as explained by Lazić et al. (2022). Like SS, Co-Cr alloys do not have a specific composition, and data ranges of 40-50% Co, 18-25% Cr, 15-25% Ni, 3-8% Mo, and 1-15% Fe are described (Alobeid et al., 2014; Wepner et al., 2021). 47 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Table 6: Components of orthodontic appliances used in the study with their corresponding metal composition in weight percentages (%) (Kovač et al., 2022). Component Type Specification Number Surface Fe Ni Cr Co Mo Ti of parts (cm2) (%) (%) (%) (%) (%) (%) SS Damon .016 x 0.25 2 5.104 58.3 14.6 27.1 <0.1 <0.1 <0.1 Ormco, USA Biostarter® .016” Ni-Ti Forestadent, 2 4.172 <0.1 73.8 <0.1 <0.1 <0.1 26.2 Germany Ni-Ti rematitan® super elastic .016” 2 4.149 <0.1 73.2 <0.1 <0.1 <0.1 26.8 Dentaurum, Germany Archwire β-Ti rematitan® SPECIAL .032” 2 6.846 <0.1 <0.1 <0.1 <0.1 8.18 91.8 Dentaurum, Germany Elgiloy® .036” Co-Cr Rocky Mountain 2 9.476 6.12 19.9 21.6 49.7 2.65 <0.1 Orthodontics, USA Co-Cr remaloy®.036” Dentaurum, 2 9.217 1.42 26.1 19.4 53.2 <0.1 <0.1 Germany Brackets SS Discovery® 24 14.478 55.4 17.8 24.9 <0.1 1.95 <0.1 Dentaurum, Germany Molar SS W-Fit Form 4 9.881 57 18.2 24.9 <0.1 <0.1 <0.1 bands Forestadent, Germany The release of metal ions from orthodontic alloys during the 90-day incubation in artificial saliva is shown in Figure 6 and the detailed results can be found in Appendix E. During the incubation, samples were taken for analysis at different time points to better evaluate the released kinetics. Overall, the amount of ions released increased during the study with minor fluctuations likely due to minor sampling or measurement errors. It should be noted that only some of the metals were analyzed in the study, so it cannot be ruled out that certain other metals were also released from the material. For more-suitable results, beakers made of Teflon were used to prevent the absorption of metals on the walls of the containers during the experiment. The pH-neutral artificial saliva is not an exact replica of human saliva, whose pH varies and contains different organisms and biological material, but it allowed us to obtain metal release results only. Galeottiet al. (2013) have shown that pH is an important factor in metal release studies. 48 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 In the case of the alloy SS (arches, brackets, and molar bands), the release was mainly Fe ions compared to the other metals studied, remaining almost at the initial level until the 90-day mark, where a slight increase was observed. Since we used upper and lower arches, we also obtained much lower metal concentrations released from the arches compared to the total brackets (24 brackets) and molar bands (4 molar bands). This finding is consistent with the fact that metals are only released from the surfaces and, consequently, with a larger surface area, more ions can be released. When looking at the metal release concentration for both Ni-Ti arcs, we found it interesting that the amount of Ti released was similar for both (Ti ≈ 9.5 ng/mL or 530 ng/cm2), but the amount of Ni released was not comparable (Ni(Dentaurum) ≈ 13 ng/mL or 721 ng/cm2 and Ni(Forestadent) ≈ 116 ng/mL or 6522 ng/cm2). The Ni-Ti(Forestadent) archwire released amost ten times as many Ni ions as Ni-Ti(Dentaurum) at the end of the study. No differences were found between them in the metal composition study, so there must have been a different resonance as to why this situation occurred. It is known that titanium forms a protective oxide layer on the surface of the material that protects against corrosion (Ramazanzadeh et al., 2014) and at the Ti concentrations found in the medium, this could be a sign of deterioration of the protective layer. This was not the case in our study, as Ti concentrations were the same in both arcs. It is believed that surface topography, processing, and finishing techniques used in the fabrication of the sheets are critical to corrosion resistance, as surface roughness and surface imperfections are the main corrosion sites (Hunt et al., 1999). Lazić et al. (2022) stated that the conventional way of manufacturing Ni-Ti wires, unlike modern manufacturing methods, does not ensure a Ni-free zone on the alloy surface, which makes them less stable, less hard, and less resistant to corrosion. For β-Ti, Mo concentrations were undetectable until day 90 of the study, when the concentration was 0.45 ng/mL or 15 ng/cm2. Ti concentrations fluctuated by 1.5 ng/mL or 50 ng/cm2 throughout the study and until day 90, when a peak value of about 8.3 ng/mL or 279 ng/cm2 was detected. Suárez et al. (2011) compared the biocompatibility between SS, Ni-Ti and β-Ti wires and showed that the β-Ti alloy was the most corrosion resistant, followed by Ni-Ti and SS was the least corrosion resistant. The same conclusion can be drawn from our study highlighting the importance of the formation of the passive TiO2 surface layer of β-Ti and Ni-Ti and, to a lesser extent, the passive Cr2O3 oxide layer of SS. Due to the passive surface layer, the β-Ti alloy released the least amount of metals from its surface. The Ti concentration in the media was also lower than that of the Ni-Ti alloy. The release of metal ions depends on the properties of the oxide layer formed, which can vary from alloy to alloy. The corrosion resistance of Ti-containing alloys was evaluated by Schiff et al. (2002), who emphasized that the passive oxide layer of the β-Ti alloy was superior to the oxide layer formed on the Ni-Ti alloy. Huang et al. (2005) hypothesized that the Mo present in the β-Ti alloy forms an additional MoO3 oxide protective layer on the surface, which makes the alloy permanently corrosion resistant. Comparing the corrosion resistance 49 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 of the β-Ti alloy with the alloy SS, whose corrosion protection is based on the passive formation of the Cr2O3 oxide layer, β-Ti with its TiO2 surface layer was less susceptible to corrosion (Castro et al., 2015). The protective layer of β-Ti alloy can be removed by local mechanical forces, which greatly decreases the corrosion resistance of the Ti containing alloy. To overcome this problem, protective coatings or other surface treatments could be applied to dental materials (Velasco-Ibáñez et al., 2020). It is not known whether we have used parts of orthodontic appliances whose surface has been modified. The metal release from both Co-Cr acrh wires gradually increased during the study. The concentrations of Co (30 ng/ml or 730 ng/cm2) and Cr (1.5 ng/ml or 35 ng/cm2) reached similar levels in both wires, but some differences were observed in Fe, Ni, and Mo. This was expected since the two Co-Cr arcs had different metal compositions of these three metals according to our compositional study. For this reason, more Mo (2.54 ng/mL or 61.8 ng/cm2), Fe (13.77 ng/mL or 335 ng/cm2), and Ni (8.55 ng/mL or 208 ng/cm2) were released from Co-CrElgiloy than from Co-Crremaloy, which released more Ni (11 ng/mL or 267 ng/cm2) and less Fe (4.6 ng/mL 112 ng/cm2) and contained no Mo. Comparing the metal release from each arch type throughout the study, it appears that the metal concentrations of the Co-Cr wire increased, indicating metal saturation. Since this occurred at the end of the study, we cannot consider this statement conclusive. 50 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Figure 6: The release of metal ions from different parts of fixed orthodontic appliances. Parts of a fixed orthodontic appliance made of different alloys were immersed in artificial saliva for 90 days. A set of different parts of the orthodontic appliances was used, and three technical replicates were obtained during each incubation time point, after which the metals released from the samples were quantified three times. Results are shown as means with standard deviation. Direct comparison of leached metal concentrations between different studies from other authors is not possible due to differences in study design with respect to the material studied, the immersion media, and the analytical equipment used. In previous studies, combinations of wire, brackets, and tapes were used to determine metal ion release. In the present study, the release of metal ions from brackets, molar bands, and arches was measured separately and, in the case of arches, also according to the composition of their material alloys. This could be considered as a limitation of the study, since it excluded the possible corrosive effect and the related ions released by the friction of the material (Staffolani et al., 1999). With the obtained results, we provided information on the minimum concentrations of metal 51 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ions that can be released from orthodontic alloys only by the pure diffusion process without additional mechanical forces. Our study also did not consider salivary flow rate (Iorgulescu, 2009), pH, and temperature changes (Kuhta et al., 2009) at which even more metal could be released. The effect of low pH on increasing metal release was shown by Kuhta et al. (2009) and the increase in metal release due to mechanical and thermal loading of orthodontic alloys was shown by Arndt (2005). According to Institute of Medicine (US) Panel on Micronutrients (2001), the reported upper intake limits (UL) for each metal are as follows: ULNi =1 mg/d, ULFe = 45 mg/d, ULCr = 0.2 mg/d, ULMo = 2 mg/d, and ULTi = 1.1 mg/d. The fact that orthodontic alloys release metal ions from their surfaces is undeniable, but although a constant release pattern was observed, the amount of metal ions released is still far below the recommended daily levels for ingestion. Comparing our results with the perscribed UL, even when considering the cumulative concentrations, none of the released metal ions studied exceeded the prescribed daily intake concentration. Caution should be exercised when interpreting metal concentrations because even nontoxic concentrations of some metals may have biological effects on the organism (Anderson et al., 2008; Urban et al., 1994) or could induce synergistic effects. For example, Cr is known to be an allergen and Cr6+ is considered toxic and mutagenic (Dayan and Paine 2001). Hypersensitivity to metals, such as Ni, is also present in our population and should not be neglected in orthodontic patients (Santos Genelhu et al., 2005). Hypersensitivity to metals is a type IV delayed immune response, resulting in stomatitis, perioral rashes, loss of taste or metallic taste, burning sensation and soreness of the tongue. Nickel allergies occur more frequently than all other metal allergies combined (Verma and Dhiman, 2015). In addition, delayed nickel allergy reactions are more common in women than in men due to daily contact with nickel in jewelry (Chakravarthi et al., 2012). The European Committee for Standardization has specified in EN 1811:2011 that the release of nickel from products should not exceed 0.5 µg/cm2/week (Tsang, 2016). Extrapolating our data, Ni-Ti(Forestadent) released 2568 µg/cm2/week Ni after only one week, and 0.501 µg/cm2/week Ni after 13 weeks. There is a large market for orthodontic appliances, where each part is made of a different composition of metal alloys. It is the responsibility of the dentist to use the right material for the treatment and safety of the patient. Knowledge of the biocompatibility of the materials is an added advantage to meet the needs of patients and address their potential problems (Kovač et al., 2020). Many adverse oral conditions such as gingival hyperplasia, glossitis, erythema multiforme, and labial desquamation have been associated with the released meteal ions from orthodontic appliances (Nayak et al., 2015). Based on the results obtained, we were able to estimate the type and concentration of released metal ions. This allowed us to prepare specific ion combinations and concentrations for further oxidative stress studies. 52 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 4.3 CAUSATION OF OXIDATIVE STRESS BY METAL IONS IN S. CEREVISIAE Throughout evolution, all eukaryotes, from yeast to vertebrates, share similarities in mitochondrial respiration, antioxidant enzymes, and accumulation of oxidative damage due to metabolic processes and aerobic life. These similarities, along with ease of access, rapid growth, genetic relevance to human disease, and ROS processing, make S. cerevisiae an useful model organism for oxidative stress studies (Lushchak, 2011) In our study, we treated S. cerevisiae cells when they were in the stationary phase. In this phase the yeasts switch from fermentative to mitochondrial respiration, which closely resembles the normal respiration of multicellular eukaryotes, and the transitions cause more ROS to be produced (Vázquez et al., 2017). The cells must cope with increasing amounts of ROS by modulating antioxidant defenses and preventing or repairing potential damage that could result from oxidative processes (Longo et al., 1996). If we were to use yeast cells in the exponential phase, all energy would come from glycolysis rather than oxidative phosphorylation reactions, and the cells would continue to decay after being exposed to the ROS stressor for only a short time (Longo and Fabrizio 2012). Stationary S. cerevisiae, on the other hand, are exposed to the stressor for a longer period of time, so defense and repair mechanisms are upregulated. Using two S. cerevisiae mutants lacking either the antioxidant enzyme SOD (ΔSod1) or CAT (ΔCtt1), we were able to see how the loss of antioxidant defense affects yeast cell culturability. 4.3.1 Culturability As shown above, orthodontic materials do release metal ions from their surfaces, but some authors (Assad et al., 2002; Kao et al., 2007) claim that these released substances are not harmfull, yet other authors (Giudice et al., 2016; Pérez-Navero et al., 2009) state that these metal ions are key producers of free radicals and its mediated toxicity. There are several studies in the literature investigating the biocompatibility of orthodontic materials with respect to the cytotoxicity of metal ions (Hafez et al., 2011; Ortiz et al., 2011; Velasco-Ortega et al., 2010), but only two have used yeast to study the cytotoxic effects of orthodontic metals (Gonçalves et al., 2014; Limberger et al., 2011). Limberger et al. (2011) compared the cytotoxicity of orthodontic materials in yeast and other cell lines, providing the necessary information that the yeast cell model is a reliable model for cytotoxicity studies. In the dissertation study, we treated yeast cells for 24 hours with different metal ion mixtures, detailed presented in Table 4, at concentrations of 1 µM, 10 µM, 100 µM, and 1000 µM. We used metal mixtures of SS, Co-Cr, Ni-Ti, and β-Ti orthodontic alloys becouse they are most commonly used in clinical practise and their nature was previously described by Arndt et al. (2005) and Kusky (2002). Figure 7 shows the cell culturability of Wt, ΔSod1, and ΔCtt1 yeast as CFU/mL cell counts and relative values compared with the untreated control group. A decrease in culturability 53 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 was observed when yeast cells were treated with metal mixtures, although only a significant decrease was observed in all metal mixture treatments at 1000 µM concentrations. Concentrations of SS below 1000 µM did not significantly decrease the culturability of Wt and the mutants. For Wt yeast, all other metal treatments caused a significant decrease in culturability at concentrations below 1000 µM. REM and β-Ti mixtures caused a significant decrease in culturability at 100 µM, and ELG and Ni-Ti mixtures caused a significant decrease at a concentration as low as 10 µM. When the culturability of Wt and the two mutants were compared, a significant difference in culturability was observed, as the CFU/mL of the untreated control samples of the mutant yeast was much lower than that of the untreated control sample of Wt. It appears that, like Wt, the ΔSod1 yeast was affected by all metal mixtures at 1000 µM concentrations, with the exception of 100 µM REM, which also caused a significant decrease in culturability. ΔCtt1, on the other hand, showed the same significant decrease at 1000 µM SS and 1000 µM TiMo as the other yeast cells, but was much more susceptible to ELG and REM, where a significant decrease was observed at 10 µM. Threatening ΔCtt1 with a Ni-Ti mixture had no effect on cell culturability. El Medawar et al. (2002) treated cell lines with only Ni, Ni-Ti, and only Ti and found that 425 µM concentrations of Ni caused a 50% decrease in viability, whereas Ni-Ti and Ti concentrations had no effect on cell viability even at 3750 µM concentrations. Issa et al. (2008) obtained a 50% decrease in viability of HGF at the following concentrations: Co = 705 µM, Ni = 828 µM, and Cr = 1971 µM. Although the individual metal concentrations are useful, they do not provide information on how the cell responds when exposed to multiple ions simultaneously. As described by Terpilowska and Siwicki (2018), certain metal combinations may have a synergistic or even antagonistic effect on cell viability. This is the information that the individual metal treatments do not provide. 54 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 4.3.2 Metabolic activity The cell metabolic activity assay, another assay used to test metal ion toxicity, was also used in the presented study. Due to the small number of biological replicates performed, the results did not show clear differences between yeast cells treated differently. For this reason, we believe that no significant differences were detected when the metabolic activity values for each treated sample were compared with the corresponding untreated control (Figure 8). When the Wt yeast cells with different metal solutions were treated, it was observed that higher concentrations actually increase the mean value of metabolic activity. The metabolic assay measures the ATP present in the cell, the quantification of which should be proportional to the metabolically active viable cells. By observing the metabolic activity of Wt yeast, it does not match the results of the cell culturability assay. Under stress conditions, the membrane and the ETC are the first components to be affected by the toxic metals, hence the observed ATP depletion and the decrease in viability (Chen et al., 2014). On the other hand, both yeast mutants tend to decrease their metabolic activity with increasing metal concentration, especially at 1000 µM concentrations. We also compared the metabolic activity between the untreated yeast cells of Wt and the mutants and found that the baseline metabolic activity of the mutants was at least two times higher than that of the untreated control group of Wt yeast. The reason for this observation might be the in the slow response of the mutant yeast to new metal-induced stressors, so that it requires higher energy consumption to maintain its viability. As explained by Huang et al. (2019) under mild stress conditions, cells use ATP to activate the antioxidant defense system and increase gene transcription, whereas under severe stress, ATP loss occurs due to mitochondrial damage. The ATP increase was explained by Akhova and Tkachenko (2014) as a process in which cells shut down all unnecessary energy-consuming processes and tend to produce more energy to meet the necessary requirements for stress defense. These statements explain why our yeast mutants without antioxidant defenses had much higher metabolic activity than Wt yeast. 56 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 A Stainless steel (SS) B WT Cobalt-Chromium (ELG) WT 200 ΔSod1 200 Δ Δ Sod1 Ctt1 ΔCtt1 150 150 ) ) (% (% D 100 D 100 /OL /OL 50 50 0 0 l l l l l l M M M M M M M M M M M M M M M M M M tro tro tro M M M M M M n µ 0 µ n µ 0 µ n µ 0 µ tro µ tro µ tro µ o 1 o 1 o 1 n 1 0 µ n 1 0 µ n 1 0 µ C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ o 10 µ o 10 µ o 10 µ 100 C 100 C 100 C 100 µ 100 µ 100 µ 100 C 100 C 100 Cobalt-Chromium (REM) WT Nickel-Titanium (Ni-Ti) C D WT Δ 200 Sod1 200 ΔSod1 ΔCtt1 ΔCtt1 150 150 ) ) (% (% D 100 D 100 /O /O L L 50 50 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 o 1 o 1 o 1 C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 β-Titanium (β-Ti ) Untreated contol samples E F WT WT 200 Δ 200 ΔSod1 Sod1 Δ ΔCtt1 Ctt1 150 150 ) ) (% (% D 100 D 100 /O /O L L 50 50 0 0 l l l 1 M M M M M M M M M M M M t1 tro tro tro WT d n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 ΔCt C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ ΔSo 100 C 100 C 100 Figure 8: Metabolic activity of Wt, ΔSod1 and ΔCtt1 yeast treated with different metal mixtures. Yeast cells were treated with SS (A), Co-Cr ELG (B), REM (C), Ni-Ti (D), and β-Ti (E) metal mixtures for 24 hours. The cell metabolic activity is presented as luminescent signal, which is proportional to the number of metabolic active cells in the culture. Values of untreated yeast cells were set at 100% and the treated samples were compared to it. A comparison among untreated control groups of each yeast strain is shown in the graph (F). Results were shown as means with standard mean errors where no significant differences were obtained. 4.3.3 Intracellular ROS level As indicated by the cell viability assessment, the metal mixtures were stressors for the yeast cells. Since all the metals used in the study were transition metals capable of generating ROS through Fenton and Haber-Weiss reactions (Zhao, 2019), we assumed that the reason for the decrease in cell viability was due to the oxidative stress that occurred although other causes are possible. When the metal is present in manageable concentrations, the antioxidant defense system ensures that no oxidative stress damage occurs. Among the endogenous antioxidant enzymes, SOD and CAT are the two major defense enzymes that remove O2•− and H2O2, respectively (Ighodaro and Akinloye 2018). The ΔSod1 yeast lacked the cytosolic SOD, which hindered the scavenging of ETC formed O2•− (Turrens, 2003), and the ΔCtt1 yeast, which lacked the cytosolic CAT, was unable to degrade H2O2 (Farrugia et al., 2012). 57 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 To determine whether metal mixtures had caused oxidative stress in the yeast, an assay was performed to determine the ROS formation. Two different approaches were used to evaluate the intacellularly formed ROS (Figure 9). In the first method, the fluorescent dye was added immediately after the yeast was treated with metal mixtures, and in the second method, the dye was not added until 24 hours after treatment with the metal mixture. As expected, the addition of the dye after treatment with metal mixtures increases the fluorescence signal of all yeast cells, regardless of the metal treatment used. Much higher fluorescence intensity was also observed in the yeast mutants lacking antioxidant defense enzymes, which could be due to the fact that the ROS were probably not efficiently removed and caused higher fluorescence intensity that in the Wt yeasts. It is worth noting that the organism has several isoenzymes of SOD and CAT, so it can thrive under oxidative stress conditions (Ighodaro and Akinloye 2018). The comparison of the different methods in Wt yeast gave different results. When the dye is added immediately after the addition of the metal, an increase in fluorescence is observed in the treatments with 1000 µM SS, ELG and REM, but when the dye is added after the 24-hour treatment, a decreasing trend in fluorescence was observed, and a statistically different decrease was observed in the case of 1000 µM REM and Ni-Ti. The β-Ti treatment of Wt yeast showed no effect on the intracellular formation of ROS. The SS and ELG treatment of ΔSod1 and ΔCtt1 yeast mutants had the same effect as the threat of Wt yeast; after the medium-term addition of the dye, only 1000 µM concentrations showed a statistically detectable increase in the fluorescence signal, while the addition of the dye after the 24-hour treatment resulted in no change in fluorescence compared to the untreated control sample. Similarly, when treated with REM, although the increase was detected at a concentration of 1000 µM, it was not statistically significant. Interestingly, treatment of the mutant yeast with Ni-Ti showed a statistically significant decrease in fluorescence intensity at a concentration of 1000 µM. Just as in Wt yeast, the fluorescence intensity of the mutants was not affected, except for the ΔCtt1 mutant, where a decrease was observed at 100 µM and 1000 µM concentrations. 58 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 A1 A2 Stainless steel (SS) Stainless steel (SS) 250 Wt 250 24h Wt d ΔSod1 d 24h ΔSod1 200 200 ΔCtt1 c a 24h ΔCtt1 ) ) 150 150 (% (% D D /O 100 /O 100 F F 50 50 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 o 1 o 1 o 1 C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 B1 B2 Cobalt-Chromium Elgiloy (ELG) Cobalt-Chromium Elgiloy (ELG) 250 Wt 250 24h Wt d d ΔSod1 200 200 24h ΔSod1 ΔCtt1 a a ) ) 24h ΔCtt1 150 150 (% (% D D /O 100 /O 100 F F 50 50 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 o 1 o 1 o 1 C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 C1 C2 Cobalt-Chromium Remaloy (REM) Cobalt-Chromium Remaloy (REM) 250 Wt 250 24h Wt Δ d Sod1 24h ΔSod1 200 Δ 200 Ctt1 24h ΔCtt1 ) ) 150 150 (% a (% D D /O 100 /O F 100 F 50 50 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M tro tro tro M M M tro tro tro n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 o 1 o 1 o 1 C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 D1 D2 Nickel-Titanium (Ni-Ti) Nickel-Titanium (Ni-Ti) 250 Wt 250 24h Wt ΔSod1 24h ΔSod1 200 200 ΔCtt1 24h ΔCtt1 ) ) 150 150 b (% b b (% D b D /O 100 /O F 100 F 50 50 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M tro tro tro M M M tro tro tro n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 o 1 o 1 o 1 C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 E1 β E2 -Titanium (β-Ti) β-Titanium (β-Ti) 250 Wt 250 24h Wt ΔSod1 24h ΔSod1 200 Δ 200 Ctt1 24h ΔCtt1 ) ) 150 150 (% a (% D c D /O 100 /O 100 F F 50 50 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 o 1 o 1 o 1 C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 Figure 9: Intracellular ROS level of Wt, ΔSod1 and ΔCtt1 yeast was performed with two different methods, each with a different time point of H2DCFDA dye addition. All yeast cells were treated with either SS (A), Co-Cr ELG (B), REM (C), Ni-Ti (D) and β-Ti (E) metal mixtures for 24 hours. Two different protocols to determine the ROS level were performed, each having a different time point of H2DCFDA dye addition. In the first protocol (suffix1), the fluorescent dye was added after 24-hour metal treatment, and in the secod protocol (suffix 2) the fluorescent dye was added simultaniously with the metal mixture. In the cell, the dye is reduced by ROS and the intensity of the fluorescent signal is proportional to the cell ROS level. The results are presented as means with 95% confidence intervals Significant differences are indicated with a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. The results obtained were contradictory, because it appeared that the addition of the dye immediately after the addition of the metal mixture provided information about the ROS 59 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 generated intracellularly by the treatment with the metal mixture, whereas the addition of the dye after the treatment with the 24 metals did not. In fact, the addition of the dye after the 24-hour treatment decreased the fluorescence signal. If the metal mixtures were the stressor resulting in the formation of ROS, the two methods used should show similar results. Since H2DCFDA is not fluorescent, it must first pass through the cell membrane, where it could be cleaved by intracellular esterase’s and oxidized by ROS to become fluorescent (Wang and Roper 2014). This means that the process of converting a non-fluorescent dye into a fluorescent product is only possible in living cells (Tanaka et al., 2020). Valiakhmetov et al. (2019) showed a correlation between ROS fluorescence and viable cell population. After the 24-hour metal treatment, the dye has fewer viable cells for its fluorescence conversion, hence the decreasing fluorescence signature. From our previous culturability measurements, we could see that the culturability of the cells decreased with the increase of the metal concentration. Therefore, we applied the third method, in which we treated the Wt yeast cells for 24 hours, took a small sample and determined the cell culturability, and plotted the obtained fluorescence signal against the number of culturable cells (Figure 10). 60 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 A B Stainless steel (SS) Cobalt-Chromium Elgiloy (ELG) 250 c 250 d d d d 200 200 d d d b ) ) % 150 % 150 U ( U ( /CF 100 /CF 100 F Wt F Wt Δ Δ 50 Sod1 50 Sod1 ΔCtt1 ΔCtt1 0 0 l l l M l l l M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ C 100 µ 100 µ 100 µ 100 C 100 C 100 C 100 µ 100 µ 100 µ 100 C 100 C 100 C D Cobalt-Chromium Remaloy (REM) Nickel-Titanium (Ni-Ti) 250 250 d d 200 c 200 ) d d b ) % 150 % 150 U ( U ( /CF 100 /CF 100 F Wt F Wt Δ Δ 50 Sod1 50 Sod1 ΔCtt1 ΔCtt1 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ C 100 µ 100 µ 100 µ 100 µ 100 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 E β-Titanium (β-Ti) 250 200 )% 150 U ( /CF 100 F Wt Δ 50 Sod1 ΔCtt1 0 l l l M M M M M M M M M M M M tro tro tro n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ o 10 µ o 10 µ o 10 µ C 100 µ 100 µ 100 µ 100 C 100 C 100 Figure 10: Intracellular ROS level in culturable Wt, ΔSod1 and ΔCtt1 yeast cells after the 24-hour metal treatment. Yeast cells were treated with SS (A), Co-Cr ELG (B), REM (C), Ni-Ti (D) and β-Ti (E) metal mixtures for 24-hours. Before the addition of the fluorescent dye H2DCFD, the culturability of each metal treated yeast was obtained. The fluorescence intensity and the CFU count show the ROS level of culturable cells. The results are presented as means with 95% confidence intervals. Significant differences are indicated with a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. Examination of the intracellular ROS in only culturable cells gave a clearer picture of intracellular oxidation level that occurs under metal stress treated conditions. SS Metal mixtures with a concentration of 1000 µM had a significant effect on Wt yeast and ΔSod1, while the ΔCtt1 mutant did not seem to be affected by treatment with the SS mixture. On the other hand, the Co-Cr metal mixtures of ELG and REM had a strong effect on the generation of ROS in the ΔCtt1 mutant, as there was an increase in the fluorescence signal already at 10 µM, which only became more intense with increasing concentration. The Wt and the ΔSod1 mutant also exhibited a significant increase in ROS generation at 100 µM and 1000 µM concentrations. The improved method showed a significant increase in intracellular ROS generation in viable cells after 24 hours of treatment, while there was no change in 61 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 fluorescence intensity when any type of yeast cell was treated with Ni-Ti or β-Ti metal mixture. Compared with other methods, no decrease in fluorescence signal was observed when ROS was probed only in culturable cells. Terpilowska and Siwicki (2019) showed that 1000 µM concentrations of Fe, Cr, Ni, and Mo ions decreased cell viability and induced apoptosis. Caicedo et al. (2008) examined the effects of these metal ions on Jurkat cells and concluded that in terms of inducing apoptosis and DNA damage, Ni was the most detrimental of all, followed by Co, then Mo, then Cr, and finally Fe. Since the Co-Cr metal mixtures contained more of the higher toxic metal ions such as Ni and Co compared to the mixture SS, the increase in intracellularly produced ROS was observed at lower concentrations. If Ni is the most toxic metal ion, the Ni-Ti mixture that contained 45% Ni ions should be the most toxic metal mixture of all, and our results with Wt yeast confirm this. Viability decreased significantly when cells were treated with concentrations as low as 10 µM, but interestingly, no intracellular ROS formation was observed. 4.3.4 Lipid oxidation It is known that certain high concentrations of metal ions actually trigger the formation of ROS in yeast cells. But with an efficient antioxidant defense system, the deleterious effect of ROS overproduction can be prevented. To determine whether the ROS generated by the metal mixture actually causes oxidative damage of molecules and not just increased ROS formation, we used the TBARS assay to examine lipid oxidation damage (Figure 11). Comparing the assessment of intracellular ROS (Figure 10) and the assessment of oxidative damage of lipids, a clear correlation can be seen, i.e., when the formation of ROS was increased, lipids were also oxidatively damaged. SS and the Co-Cr mixture caused lipid oxidation at higher concentrations, at 1000 µM for Wt and even at 100 µM for the mutant. Pallero et al. (2010) also confirmed that the SS mixture, consisting of Fe, Cr, Ni, and Mo ions, increased the formation of ROS in vascular smooth muscle cells. No signs of lipid damage were observed when yeast cells were treated with the Ni-Ti or β-Ti metal mixture. According to previous studies, metal ions compromising, Ni (Chen et al., 2010), Ti (Gholinejad et al., 2019) and Mo (Siddiqui et al., 2015) metal mixtures are capable of inducing ROS formation and lipid oxidation, but this was not revealed in our study. Spalj et al., (2012) showed, that Ni and Ti salts induce ROS formation in gastrointestinal cell lines (Rincic Mlinaric et al., 2019). We also compared lipid oxidation levels between Wt yeast and the two mutant yeasts and found a significant difference between them. It appears that the mutant yeast undergoes mild lipid oxidation even in the absence of metal ions because of the lack of antioxidant enzymes. 62 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 A B Stainless steel (SS) Cobalt-Chromium Elgiloy (ELG) 600 d 600 Wt Wt ΔSod1 ΔSod1 d d d ) 400 d ΔCtt1 ) 400 d ΔCtt1 d (% d (% D a D c /O /O F 200 F 200 0 0 l l l l l l M M M M M M M M M M M M M M M M M M tro tro tro M M M M M M n µ 0 µ n µ 0 µ n µ 0 µ tro µ tro µ tro µ o 1 o 1 o 1 n 1 0 µ n 1 0 µ n 1 0 µ C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ o 10 µ o 10 µ o 10 µ 100 C 100 C 100 C 100 µ 100 µ 100 µ 100 C 100 C 100 C D Cobalt-Chromium Remaloy (REM) Nickel-Titanium (Ni-Ti) 600 600 c Wt Wt ΔSod1 Δ d Sod1 ) 400 ΔCtt1 ) 400 ΔCtt1 (% (% D d d D /O /O F b 200 F 200 0 0 l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 o 1 o 1 o 1 C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 E F β-Titanium (β-Ti) Untreated control samples 600 200 Wt c Wt ΔSod1 b Δ 150 Sod1 ) 400 ΔCtt1 ) ΔCtt1 (% (% D D 100 /O /O F 200 F 50 0 0 l l l 1 M M M M M M M M M M M M t1 tro tro tro Wt d n µ 0 µ n µ 0 µ n µ 0 µ o 1 o 1 o 1 ΔCt C 10 µ 100 µ 10 µ 100 µ 10 µ 100 µ ΔSo 100 C 100 C 100 Figure 11: Influence of 24-h metal treatment on the formation of oxidative lipid damage in yeast cells. Wt, ΔSod1, and ΔCtt1 yeast strains were treated with different concentrations of SS (A), Co-Cr ELG (B) and REM(C), Ni-Ti (D), and β-Ti (E) metal mixtures. The fluorescent product of the TBARS method directly indicates the amount of lipid oxidation damages. Graph (F) compares untreated control groups of different yeast strains relative to the wild type. The results are presented as means with 95% confidence intervals. Significant differences are indicated with a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. 4.3.5 Antioxidative defense We have shown that there is a relationship between the metal ions and the occurrence of oxidative stress in yeast cells. Each organism is equipped with an appropriate antioxidant defense mechanism to maintain redox levels. Because the ΔSod1 and ΔCtt1 yeast mutants do not have the full defense arsenal to successfully remove excess oxygen radicals and their products, intracellularly generated ROS and lipid oxidation damage were observed at lower concentrations than in Wt yeast. This prompted us to investigate the enzymatic antioxidant system in S. cerevisiae, particularly the major enzymes SOD and CAT. When the levels of ROS are too high, the enzymatic activity is increased, which gives a better indication of the occurrence of oxidative stress. Figure 12 shows the results of native electrophoresis. No significant differences in SOD activity were observed when yeast cells were treated with different concentrations of metal mixtures, although some differences in main values were observed. The same was true for the activity of CAT. However, when yeast cells were treated with SS metal mixtures, a decreasing trend in mean values was observed, and the activity 63 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 decrease in activity levels was observed. This observation was contrary to what we expected based on the activity of SOD, as we assumed that with the increase in the activity of SOD, the CAT activity would also increase. The H2O2 could also be converted to H2O by GPx enzyme (Arthur, 2000) and its activity was not affected by the addition of SS and Co-Cr mixture, but a significant increase in activity was observed in the presence of 1000 µM Ni-Ti and β-Ti metal mixture. Since GPx uses GSH as a substrate for its redox reaction, the GR enzyme activity must be consistent with the GSH requirement. The GR activity of 1000 µM SS samples had decreased, whereas the activity was increased after 1000 µM Co-Cr treatment and 10 µM, 100 µM and 1000 µM β-Ti treatment. The thiol-containing enzymes, TrxR and Prx, are also involved in H2O2 removal. The TrxR enzyme requires NADPH as a reducing agent to provide reduced thioredoxin for Prx function (Gromer et al., 2004). While Prx activity also decreased significantly after treatment with 1000 µM SS, other metal mixtures caused no changes in enzymatic activity. TrxR activity also decreased when cells were treated with 1000 µM SS, while metal mixtures of Co-Cr, Ni-Ti and β-Ti had an increasing effect on enzyme activity even at a concentration of 100 µM. SOD CAT 200 200 b ) ) % 150 150 % b a ty ( ty ( ivi 100 ivi SS 100 SS act act D Co-Cr T Co-Cr O A 50 S Ni-Ti 50 C Ni-Ti β-Ti β-Ti 0 0 l l l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro tro tro n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ C 100 µ 100 µ 100 µ 100 µ 100 µ 100 µ 100 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 C 100 C 100 GPx GR 200 200 d a d ) 150 a b % 150 c a ty ( ty% ivi ivi 100 100 ct SS SS act x a Co-Cr R Co-Cr P G 50 G 50 Ni-Ti Ni-Ti β-Ti β-Ti 0 0 l l l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro tro tro n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ C 100 µ 100 µ 100 µ 100 µ 100 C 100 C 100 C 100 C 100 µ 100 µ 100 µ 100 µ 100 C 100 C 100 C 100 TrxR PRX 200 d 200 c c c ) a ) % 150 a 150 (% a ty ( ity ivi v 100 ti 100 ct c SS a SS X a Co-Cr d Co-Cr rxR 50 R 50 T P Ni-Ti Ni-Ti β β -Ti -Ti 0 0 l l l l l l l l M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M tro tro tro tro tro tro tro tro n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ o 10 µ C 100 µ 100 µ 100 µ 100 µ 100 µ 100 µ 100 µ 100 µ 100 C 100 C 100 C 100 C 100 C 100 C 100 C 100 Figure 13: Yeast antioxidant enzyme activity after 24-hour metal mixture treatment. Yeast cell were treated with different concentrations of metal mixtures, and the activity of SOD (A), CAT (B), GPx (C), GR (D), TrxR(E), and Prx (F) was compared to the untreated sample, which enzyme activity was set at 100% for easyer comparison between the enzyme activity changes. The activity results are repsresented as means with 95% confident intervals. Significant differences are indicated with a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. 65 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 When yeast cells were treated with the SS alloy mixture, the increased O2 - dismutation by SOD to H2O2 should be a trigger for CAT, Gpx and Prx to increase their activity, but this was not the case in presented study, where a decrease in enzyme activity was found. The activity of TrxR, a general mediator of the antioxidant defense system (Lopert et al., 2012), was almost completely inhibited. It is possible that majority of the H2O2 generated immediately enters the Fenton and Haber-Weiss reactions, forming OH• in the presence of metal ions. As reported in the previous section the presence of high concentrations of metal ions reduces cell viability and induces intracellular formation of ROS and causes lipid oxidation. The same deleterious effects were seen when cells were treated with a Co-Cr metal mixture, but the antioxidant enzyme defense system did not show the same response. There was a noticeable increase in SOD activity, but this could not be statistically demonstrated. Again, CAT activity showed a dose-dependent decrease after treatment with Co-Cr, Ni-Ti, and β-Ti. The same observation was made by Atli and Canli (2010) who suggested that the inhibition of CAT activity could be due to the binding of the metal ion to the protein - SH groups. Scharf et al. (2014) also described that the Cr ions could replace Fe in the active site of the enzyme and inhibit the activity of CAT. Another explanation of why the activity of CAT might be decreased comes from Bayliak et al. (2006) who attributed the decreasing effect to high H2O2 concentrations, implying that there is a critical H2O2 level above which the CAT enzymes no longer function properly. Our in-gel assay of CAT could prove otherwise, as the CAT enzymes from lysates treated with 1000 µM showed higher electrophoretic mobility, indicating possible oxidation of the enzyme. This was also demonstrated by Rodríguez-Ruiz et al. (2019) who found similar increase in band mobility after H2O2 treatment. Since GPx enzymes require GSH for their reactions and GR obtains it from the conversion of GSSG to GSH, their activity is closely related to the GSH/GSSG ratio, a commonly recognized marker of oxidative stress (Kubrak et al., 2010). There was no increase in GPx activity after SS and Co-Cr treatment, but the activity was increased after Ni-Ti and β-Ti treatment. It was expected that the results for GPx activity would translate to GR activity, but this was not the case in our results. In fact, no clear link could be established between GPx and GR activity. Tandoğan and Ulusu (2008) suggested that Ni ions compete with GSS for the GR ctivity sites and possibly decrease enzyme activity. Ni-Ti treatment, whose Ni ion content was the highest among all metal mixtures, had no effect on GR activity, while SS treatment showed a decrease and Co-Cr and β-Ti treatment showed an increase in GR activity. Like GPx, the enzyme Prx can reduce organic and inorganic H2O2 and often competes with GPx for H2O2 dissociation (Mitozo et al., 2011), but no such competition was observed in our study. Treatment of cells with SS induced inactivation of Prx as well as a decrease in TrxR activity, which provides TrxR with the necessary electron donor for its enzyme reaction. 66 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Terpilowska and Siwicki (2019) treated the cell lines BAKB/3T3 and HepG2 with different concentrations of Cr, Fe, Ni, and Mo and investigated the intracellular ROS level, lipid oxidation, and SOD, CAT and GPx activity. The authors concluded that each of the metal ions at concentrations greater than 200 µM can induce oxidative stress and lipid oxidation and decrease the activity of all antioxidant enzymes. The same result was obtained by Kalaivani et al. (2014), who threated A549 cell line with Ni, and by Siddiqui et al. (2015), who treated L929 cell line with Mo ions. Assessment of Co-Cr metal mixture, Co2+, Cr3+and Cr6+, has different effects on cell viability and enzyme activity. In a cell-free study by Chen et al. (2018) was shown, that 0–50 µM Cr3+ concentrations increased CAT activity and concentrations above 50 µM had a decreasing effect on CAT activity. Feng et al. (2017) and Lazarova et al. (2014) found an increase in SOD and CAT activity when yeast cells were treated with low concentrations of Cr6+ for a short period of time, whereas prolonged Cr exposure and higher concentrations decreased enzyme activity, suggesting that antioxidant enzymes can only handle a certain amount and time of oxidative stressor. Treatment of cells with Co2+, Cr3+, and Cr6+ ions showed a dose-dependent decrease in viability, with Co2+ being the most toxic, followed by Cr6+ and then Cr3+. The reason why the Cr6+ ions were more toxic than Cr3+ is because Cr6+ can easily pass the cell membrane through anion channels, whereas Cr3+ must be taken up by phagocytosis (Wang et al., 2017b). We chose Co2+ and Cr3+ chlorides to simulate Co-Cr alloys in our study because the Co3+ and Cr6+ tend to reduce to lower oxidation states under psychological conditions (Baskey et al., 2017). 4.3.6 Protein oxidation Measuring the carbonyl content of proteins, an end product of protein oxidation in biological samples, is a useful biomarker for evaluating metal-induced oxidative stress because the reaction is irreversible (Lazarova et al., 2014). In our study, it was found that the concentrations of the metal mixtures that induced intracellular ROS generation and lipid oxidation (1000 µM SS, 100 µM, and 1000 µM Co-Cr) also caused oxidative damage to proteins, whereas other metal mixtures had no effect (Firgure 14). These results are consistent with those of Scharf et al. (2014) who also treated cells with Co2+ and Cr3+ ions and found protein carbonylation at concentrations of 100 µM and 1000 µM. Lazarova et al., (2014) also treated yeast cells with 1000 µM Cr3+ concentrations and observed an increase in protein carbonyl content. 67 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 200 d ) a % a e ( 150 ga am 100 e d SS ivat Co-Cr d 50 xi Ni-Ti O β-Ti 0 l l l l M M M M M M M M M M M M M M M M tro tro tro tro n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ n 1 µ 0 µ o 10 µ o 10 µ o 10 µ o 10 µ C 100 µ 100 µ 100 µ 100 µ 100 C 100 C 100 C 100 Figure 14: Protein carbonyl content as a result of oxidative protein damage in S. cerevisiae after 24-hout metal mixuture treatment. Yeast cells were treated with different concentrations of SS, Co-Cr, Ni-Ti, and β-Ti metal mixtures. The change in absorbance spectrum after the DNPH reacted with the protein carbonyl groups and the OD indicates the amount of protein oxidation damages. Graph (F) compares untreated control groups of different yeast strains relative to the wild type. The results are presented as means with 95% confidence intervals. Significant differences are indicated with a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. 4.4 THE EFFECT OF METAL MIXTURES ON HGF CELL LINE The oral mucosa is a mucous membrane consisting of a stratified squamous epithelium – layers of flattened epithelial cells – supported by underlying connective tissue called “lamina propria” (Nanci, 2016). The oral mucosa and gums are the first tissues to come into contact with the released metal ions from orthodontic appliances. The outer epidermal layer of the gingiva is keratinized and protects the inner connective tissue, which consists of gingival fibroblasts. The fibroblasts of the gingiva are responsible for tissue repair and initiating inflammatory responses (Smith et al., 2019). By using human gingival fibroblasts (HGF), we can obtain information about how the cells might respond to certain metals at different concentrations in oral cavity. To confirm that the use of S. cerevisiae as a model organism was justified, we treated the HGF cells with the same metal mixtures for the same period of time with the same concentrations as the yeast cells. As with the yeast cells, we first had to check the viability of the cells (Figure 15). We hoped to obtain more accurate results by using three different methods simultaneously to assess cell viability, but in the case of the Coomassie assay, it was difficult to interpret the results. The Coomassie assay only shows the amount of proteins present; it does not directly describe the viability of the cells. The theory behind this is that the more cells are present, the more proteins are present, and because we discarded the dead cells with the medium, only living cells were dyed. However, for each treatment, we found that the protein content decreased rapidly after treating the HGF cells with low concentration 68 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 mixtures and increased again with increasing treatment concentration. We used only the resazurin assay and neutral red uptake results to interpret the cell viability results. The results presented show that treatment of HGF cells with SS and Co-Cr metal mixtures had no effect on HGF cell viability, while Ni-Ti and β-Ti mixtures had a significant effect on viability at 250 µM for Ni-Ti and at 500 µM for β-Ti. It appears that the resazurin assay is slightly more sensitive than the neutral red assay, as we found significant differences at lower concentrations compared to the neutral red assay. Based on the previous results with yeast cells, we had expected the HGF viability results to be just the opposite, with the SS and Co-Cr mixtures decreasing cell viability and Ni-Ti and β-Ti mixtures having no effect. There still remains the question of what happens to the amount of protein in the samples. By definition, cell viability shows the amount of healthy cells in the sample (Kamiloglu et al., 2020). We used the resazurin assay, which measures viability based on the metabolic activity of the cell to reduce resazurin into a fluorescent product (Riss et al., 2016) and the neutral red uptake assay, which measures the uptake of the dye by functioning lysosomes (Fotakis and Timbrell 2006). It is possible that the viability of the HGF cells did not change because the viability assays only measure the final product colorimetrically, which may have come from less viable cells with greater metabolic functions. This could also explain why we obtained such different results for protein levels. These observations prompted us to use the trypan blue cell debris assay to quantify cell death after treatment with the metal mixture (Figure 16). Trypan blue is commonly used in laboratories to count live cells because the dye is only permeable to dead cells and cell debris (Strober, 1997), but isolating and measuring cell debris could provide important information about the amount of dead cells. 69 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 SS A 200 d ) 150 a (%ty Resazurine assay ili a b b b 100 Neutral red assay ia v Coomassie assay lle 50 C 0 l 1 5 0 l 1 5 0 l 1 5 0 10 50 00 50 00 10 50 00 50 00 10 50 00 50 00 tro 1 2 5 tro 1 2 5 tro 1 2 5 n 100 n 100 n 100 o o o C C C Concentration (μM) Co-Cr B 200 ) d 150 (%ty Resazurine assay ili a c b b 100 d Neutral red assay ia v Coomassie assay lle 50 C 0 l 1 5 0 l 1 5 0 l 1 5 0 10 50 00 50 00 10 50 00 50 00 10 50 00 50 00 tro 1 2 5 tro 1 2 5 tro 1 2 5 n 100 n 100 n 100 o o o C C C Concentration (μM) Ni-Ti C 200 ) 150 (%ty Resazurine assay ili c b 100 d d Neutral red assay ia d d d d d vll d Coomassie assay e 50 C 0 l 1 5 0 l 1 5 0 l 1 5 0 10 50 00 50 00 10 50 00 50 00 10 50 00 50 00 tro 1 2 5 tro 1 2 5 tro 1 2 5 n 100 n 100 n 100 o o o C C C Concentration (μM) β-Ti D 200 ) 150 (%ty Resazurine assay ili d b 100 d d b d Neutral red assay ia d v d ll d Coomassie assay e 50 d C 0 l 1 5 0 l 1 5 0 l 1 5 0 10 50 00 50 00 10 50 00 50 00 10 50 00 50 00 tro 1 2 5 tro 1 2 5 tro 1 2 5 n 100 n 100 n 100 o o o C C C Concentration (μM) Figure 15: HGF cell viablility after 24-hour treatment with metal mixtures HGF cell were treated with SS (A), Co-Cr (B), Ni-Ti (C) and β-Ti (D) metal mixtures for 24-hours and the cell viability was assessed with resazurine assay, neutral red assay, and Coomassie assay. The results are presented as means with 95% confidence intervals. Significant differences are indicated as follows; a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. 70 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 As shown in Figure 16, treatment of HGF with SS and Co-Cr mixtures rapidly induces cell death with increasing concentration. A first significant sign of cell death can be observed at a concentration of 100 µM in the case of SS, at a concentration of 400 µM in the case of Co-Cr and at a concentration of 500 µM in the case of Ni-Ti and β-Ti. The trypan blue cell debris assay showed a significant increase in cell death after treatment of cells with SS and Co-Cr mixtures, while the previous methods showed no change in cell viability. These results suggest that metal mixtures of SS and Co-Cr cause HGF cells to increase their metabolic activity and in some cases even increase lysosome volume (Repetto and Sanz 1993) to maintain the same viability in a smaller cell population. A SS B Co-Cr 300 300 ) d ) % % ( d d ( th 200 d th 200 d ea ea d d d ll d ll d d d d 100 100 e ce a e ce tiv tiv a a el 0 el 0 R R l 1 5 0 l 1 5 0 10 50 00 00 00 00 00 00 10 50 00 00 00 00 00 00 tro 1 2 4 5 6 8 tro 1 2 4 5 6 8 n 100 n 100 o o C C Concentration (μM) Concentration (μM) Ni-Ti ß-Ti C D 300 300 ) ) % % ( ( th 200 th 200 ea ea ll d d d d ll d d 100 b 100 b e ce a e ce a tiv tiv a a el 0 el 0 R R l 1 5 0 l 1 5 0 10 50 00 00 00 00 00 00 10 50 00 00 00 00 00 00 tro 1 2 4 5 6 8 tro 1 2 4 5 6 8 n 100 n 100 o o C C Concentration (μM) Concentration (μM) Figure 16: Relative cell death of HGF cells after 24-hour metal mixture treatment. HGF cell were treated with SS (A), Co-Cr (B), Ni-Ti (C) and β-Ti (D) metal mixtures for 24-hours and the relative procentage of dead cells was assessed with trypan cell debris assay. The results are presented as means with 95% confidence intervals. Significant differences are indicated as follows; a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. 71 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Studies exposing cells to metal ions and metal alloy corrosion products in vitro have shown that the toxic effects of the stressor take the form of a reduction in cell viability, alteration of proliferation, inhibition of enzymes, membrane damage, and impairment of DNA and RNA synthesis (Messer and Lucas, 2000; Takano et al., 2002). A study by Issa et al. (2008) showed that different cell types respond differently to the same metal exposure and that even the response of the same cell types to the stressor can vary, mainly because of differences in passage number and cell density. This makes it very difficult to compare our results with those previously obtained from other studies. Some elements, such as Fe and Ni, are more likely to be released from dental alloys, but not proportionally to the alloy composition (Kovač et al., 2021; Wataha et al., 1991). Issa et al., (2008) found that the viability of HGF cells decreased by 50% after individual treatment with 705.8 μM Co2+, 827.9 μM Ni2+ and 1971 μM Cr3+. The high effective concentrations of Cr3+ indicate the inability of the ion to enter the cell (Shrivastava et al., 2005). This study is consistent with the study by Messer and Lucas (1999) in which they evaluated the toxic effects of some metal ions as follows: Cr6+ > Ni2+ > Cr3+ ≈ Mo6+, while viability was not altered after 1923 μM concentrations of Cr3+ and Mo6+. Similar results were also reported by Hallab et al. (2005) who concluded that Co2+ and Ni2+ were cytotoxic (LC50 < 1000 μM), whereas Cr3+ and Mo6+ posed no toxic hazard at 1000 μM. In our study, we used combinations of metal ions, so we cannot fully compare the cytotoxicity of individual metal ions. Terpilowska et al., (2018) used Fe3+ and Ni2+ to treat BALB/3T3 fibroblasts and examined cell viability, which decreased with increasing concentration. At lower concentrations (100 and 200 μM), they also observed a slight increase in viability, but any concentration above this decreased cell viability. Iron is an essential element for cell metabolism, but at higher concentrations it is thought to alter membrane stability, trigger the production of ROS through the Fenton reaction, and further damage the biological macromolecules. San Miguel et al. (2013) treated HGF with 1000 μM Ni2+ and found a significant decrease in viability, as we did in our study. Ni2+ can also induce the formation of ROS through Fenton and Haber-Weiss reactions and cause lipid oxidation, but it must first be oxidized to Ni3+ (Chen et al., 2003). The ability of Ni2+ to form ROS was put to the test when San Miguel et al. (2013) added shynthetic antioxidants to the metal treatment. The viability of HGF cells in the presence of antioxidants increased and the intracellularly generated ROS decreased, suggesting that Ni2+ ions have a ROS generating potential. The surfaces of β-Ti alloys are considered resistant to corrosion, but prolonged exposure to acids and in the presence of fluoride, as in the oral cavity, tend to release Ti ions (Nakagawa et al., 2002). Mikihira et al., (2010) treated gingival epithelia like cells with Ti ions and found a decrease in cell viability at concentrations greater than 250 μM. Because the treatment lasted only 6 hours, compared with our 24 hours, it is reasonable to speculate that viability might change at lower concentrations if the cells were exposed to the Ti ions for a longer period of time. This was demonstrated by Liao et al, who found that treatment of primary osteoblast cells from rat calvaria with 210 μM Ti ions for 24 hours resulted in a decrease in 72 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Treatment of HGF cells with different metal mixtures produced different results in terms of intracellular ROS production. The first significant differences in ROS production occurred at a concentration of 50 μM for the SS mixture, a concentration of 400 μM for the Co-Cr mixture, a concentration of 400 μM for the Ni-Ti mixture, and a concentration of 100 μM for the β-Ti mixture. Treatment of HGF cells with the metal mixtures SS and Co-Cr increased the ROS production, whereas after the threat with Ni-Ti and β-Ti, ROS production decreased. When we compared the results of intracellular ROS production with the results of the type blue cell debris assay, we find that in the case of SS and Co-Cr, the ROS level increased with cell death, whereas this effect was reversed in the case of Ni-Ti and β-Ti. The results suggest that SS and Co-Cr mixtures induce oxidative stress and cause cell death in the HGF cell line, whereas cell death after treatment with Ni-Ti and β-Ti does not appear to be due to the generation of ROS. The ability to generate intracellular ROS Fe3+, Cr3+, Ni2+ and Mo6+ above a concentration of 200 μM was demonstrated in the BALB/3T3 and HepG2 cell lines in a dose-dependent manner (Terpilowska and Siwicki 2019). Fe3+ induced intracellular formation of ROS at a concentration as low as 10 μM in HeLa cell lines (Poljak-Blazi et al., 2011). The effect of individual metal ion treatments cannot be compared because the metal mixtures may have synergistic or antagonistic effects. This was also shown by Terpilowska and Siwicki (2019) that mixtures of 200 μM Cr3+ and 1000 μM Fe3+ or 1000 μM Cr3+ and another metal ion at a concentration of 200 μM have a synergistic effect on ROS formation. On the other hand, a metal mixture of 200 μM Cr3+ and 1000 μM Ni2+ as well as a mixture of 200 μM Cr3+ and 1000 μM Mo6+ has an antagonistic effect on ROS generation. Also, a study by Patel et al., (2012) showed that Co2+ and Ni2+, alone and in combination, increased ROS production. Assuming that the metal mixtures we prepared did not have synergistic or antagonistic effects, we could obtain some conclusions. The ability to produce ROS in the SS metal mixture of Fe3+, Cr3+ and Ni2+ could be due only to Fe3+ and Cr3+ since Ni2+ could not have induced the production of ROS, as shown in the case of the Ni-Ti mixture of Ni2+ and Ti4+ since no increase in ROS was obtained. The question remains to what extent Cr3+ causes the formation of ROS, because it is known that it cannot freely cross the cell membrane although a study by Fleury et al. (2006) showed that Cr3+ can be taken up by phagocytosis. The ability of Fe3+ to generate ROS via the Fenton reaction has been mentioned in several previous studies (Jia et al., 2012; Keenan et al., 2009). It is important to note that all metals used in the mixture are capable of generating ROS, although the efficiency to generate free radicals varies greatly. For example, the efficiency of Co2+ and Ni2+ in generating ROS is very low due to the high oxidation/reduction potential (Leonard et al., 2004). This brings us to the question of which metal ion is responsible for the generation of ROS in the Co-Cr mixture consisting of Co2+, Cr3+, Fe3+, Ni2+ and Mo6+ ions. As before, we can exclude Cr3+, Ni2+ and Mo6+ ions, leaving Co2+ and Fe3+ ions as potential oxidative stressors. Most of the decrease in viability and formation of ROS can be attributed to Co2+, as it makes up 40% of the Co-Cr mixture compared to the 18% of Fe3+. As mentioned above, the ability of Co2+ to catalyze 74 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 the Fenton reaction is lower compared to Fe3+, so the single fluorescence obtained after Co-Cr treatment was also lower than that of SS. Co2+ has also been shown to induce the formation of ROS and to cause oxidative stress-induced damage to biological molecules (Fleury et al., 2006; Gault et al., 2010; Tripathi et al., 2019). No increase in ROS was observed in the β-Ti mixture composed of Ti and Mo ions, so these two metals could be ruled out as potential ROS generators, although some studies have shown that Ti ions can generate free radicals (Rincic Mlinaric et al., 2019). When comparing the 24-hour effect of metal mixtures between the yeast S. cerevisiae and the HGF cell line, some findings were obtained. Regarding the concentration of metal mixtures, 100 µM concentrations could not affect the viability of HGF cells, but some metal mixtures (Co-Cr) also had a decreasing effect on the culurability of S. cerevisiae at this concentration. Of course, when the concentration of the metal mixture exceeded the 100-µM concentration, the death of HGF cells was observed in all metal mixtures. Assuming that the decrease in culturability/viability was due to the ability of the metals to generate ROS, we found interesting similarities between the yeast and the cell line. Both showed an increase in the levels of ROS when treated with SS or a Co-Cr metal mixture, and a dose-dependent increase in the levels of ROS was observed in the cell line. No increase in ROS levels was observed with the other two metal mixtures, Ni-Ti and β-Ti. Rather, a decrease in the ROS level was observed. This means that the Ni-Ti and β-Ti mixtures do not cause ROS -related decrease in cell viability. The comparison suggests that S. cerevisiae is a useful model organism for oxidative stress studies. 4.5 EFFECT OF NANOPARTICLE EXPOSURE TO HGF CELL LINE 4.5.1 Nanoparticle characteristics The detailed results of the individual measurements of NP are given in Table 7. The main problem with the stability of NPs is their tendency to agglomerate and sediment (Wu et al., 2011). Because of their small size, they have a high surface energy and to reduce the energy distribution, they agglomerate. To solve this problem, nonionic or ionic surfactants are usually added to NP suspensions to form a protective layer around the NP (Rabinow, 2004). Parameters of the medium such as pH, salts and the presence of proteins (serum) can also cause agglomeration (Joris et al., 2013). Sedimentation due to gravity is also common to all NPs. Other factors such as size, density, and zeta potential (ZP) of the NPs can affect sedimentation. For example, the higher the absolute ZP value, the more the NPs tend to settle to the bottom. It should be mentioned that all tested NPs sedimented very quickly. Since the dynamic light scattering (DLS) and ZP measurements had to be performed in suspension, the measurements were not completely accurate because only small NPs dispersed in the solution could be measured, but not the sedimented NPs. After the DLS measurement, the hydrodynamic size of the NPs increased dramatically. 75 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 All three TiO2-NPs samples are very similar after transmission electron microscope (TEM) visualization (Appendix F). Mainly spherical particles ranging in size from 200 nm to several µm are seen, with some larger single crystals. These spherical particles consist of tightly bound small NP crystals and are not expected to be dispersed in the suspension after ultrasonic treatment. In the case of the TiO2 anatase/rutile sample, these small crystals are in the size range of 30-100 nm, in the case of TiO2 anatase in the range of 5 to 100 nm, and in the case of TiO2 rutile in the range of 3-100 nm. The ZP of TiO2-NPs showed negatively charged NPs in both H2O and Fib medium. Most of the Ag-NP material was present in large crystal aggregates. Smaller NP crystals of various sizes were also found among the larger aggregates (Appendix G). When the NPs were placed on a carbon surface for the TEM measurement, the Ag-NPs tended to self-regulate and form clusters in which larger NPs were found in the center of the clusters and the size of the NP decreased toward the edge of the cluster. In the Ag 28-48 nm sample, the size of the NPs ranged from 2-20 nm and in the Ag 48-78 nm sample, the size of the NPs was 3-10 nm. In addition, very small NP were seen in the Ag 48-78 nm sample, which were probably formed from Ag+ ions of the NP solution. The ZP measurement showed negatively charged NP. The two ZnO-NPs look very similar TEM (Appendix H). Again, the smaller NPs tend to agglomerate and form crystals of about 50 nm in size. Smaller agglomerates of larger ones, about 100 nm in size, NP were also seen. After the ZP measurements of H2O, positively charged NP were analyzed, and in the case of fib medium, negatively charged NP were seen. The WS2 sample also showed a tendency to agglomerate into larger structures (Appendix I). All sizes of NPs could be found in the samples, among the larger ones we were larger than 1000 nm and the smaller ones around 200 nm, while no monocrystalline NPs were seen. The ZPs of the WS2-NPs were found to be negatively charged. 76 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Table 7: Size and surface charge of used nanoparticles NP Declared TEM DLS DLS ZP ZP size (nm) H2O Fib-medium H2O Fib-medium (nm) (nm) (nm) TiO2 18 nm Small crystals of 30 nm 1787 nm 853 nm -15.9 -11.2 Anatase/Rutile aggregate to form globular (pH = 7.2) (pH = 8.2) (pH = 7.2) (pH = 8.1) particles (<1000 nm) TiO2 28 nm Small crystals of 5 nm 1919 nm 691 nm -8.3 -11.9 Anatase aggregate to form globular (pH = 7.6) (pH = 7.9) (pH = 7.9) (pH = 7.9) particles (<1000 nm) TiO2 28 nm Small crystals of 3 nm 751 nm 865 nm -18.9 -11.4 Rutile aggregate to form globular (pH = 7.6) (pH = 7.7) (pH = 7.2) (pH = 7.7) particles (<1000 nm) Ag 28-48 nm Small crystals of 2 - 20 nm 582 nm 437 nm -31.2 -12.1 aggregate to form globular (pH = 7.6) (pH = 8.1) (pH = 7.6) (pH = 8.1) particles (< 1000 nm) Ag 48-78 nm Small crystals of 3 - 10 nm 748 nm 1343 nm -25.4 -10.3 aggregate to form globular (pH = 7.6) (pH = 7.8) (pH = 7.6) (pH = 7.8) particles (< 1000 nm) ZnO 18 nm Small crystals of 50 nm 735 nm 7486 nm 24.1 -10.9 aggregate to form globular (pH = 8.1) (pH = 8.1) (pH = 8.1) (pH = 8.1) particles (< 100 nm) ZnO 30-50 nm Small crystals of 50 nm 998 nm 4691 nm 22.2 -11.7 aggregate to form globular (pH = 8.0) (pH = 8.2) (pH = 8.0) (pH = 8.2) particles (< 100 nm) WS2 35-75 nm Small crystals of 50 nm 749 nm 3356 nm -21.6 -11.2 aggregate to form globular (pH = 3.5) (pH = 7.7) (pH = 3.5) (pH = 7.7) particles (< 1000 nm) 4.5.2 Cytotoxicity of NPs Due to their physical and chemical properties, NPs are becoming increasingly important in the research and development of new materials, which include orthodontic materials. However, with the new properties and industrial production of NPs, concerns about possible adverse health effects have also arisen. Therefore, it is first necessary to understand the effects of NPs on living cells in order to recognize them as safe for further use in any applications. As there are more and more NPs of different types, sizes, and shapes, the number of reagents for their safety increases to (Drasler et al., 2017). Based on the 3Rs principle replacement, reduction, and refinement, in vivo safety studies in animals are slowly being replaced by in vitro studies in primary cells or cell lines (Krewski et al., 2010a). TiO2-NPs are the most commonly produced NPs and their use is very versatile, but is mainly used as a white pigment in food additives, medicine, cosmetics, and sunscreens (Hamzeh and Sunahara 2013). Ti materials are widely used in dentistry due to their high corrosion resistance and biocompatibility. The oxide layer formed on the surface of titanium materials is the reason for their excellent properties. However, in the oral environment, tribocorrosion of the layer can occur, reducing the biocompatibility of Ti materials and releasing metal ions 77 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 and abrasion from the surface (Apaza-Bedoya et al., 2017). TiO2-NPs can occur in two crystalline forms: anatase or rutile, and anatase has been shown to be more cytotoxic than rutile (Sayes et al., 2006). In our study, we used anatase, rutile, and a mixture of both to investigate the effects of these NPs on the HGF cell line (Figure 18). TiO A TiO 2 - Anatase/Rutile (18 nm) 2 - Anatase/Rutile (18 nm) 200 150 Resazurin ) d d % d ) Neutral red ( d % 150 th 100 c Commassie ea ility ( 100 ll d 50 ab l vi e ce el 50 tiv 0 C aelR 0 -50 0 l 1 5 0 50 00 50 00 50 00 50 00 10 50 00 00 00 00 00 00 1 1 2 2 3 3 4 tro 1 2 4 5 6 8 n 100 o Concentration (μg/mL) C Concentration (μg/mL) TiO2 - Anatase (28 nm) B TiO2 - Anatase (28 nm) 200 150 d Resazurin ) d % d ) Neutral red ( % 150 th 100 d b Commassie ea a ility ( 100 ll d 50 ab l vi e ce el 50 tiv C 0 aelR 0 -50 0 l 1 5 0 50 00 50 00 50 00 50 00 10 50 00 00 00 00 00 00 1 1 2 2 3 3 4 tro 1 2 4 5 6 8 n 100 Concentration (μg/mL) oC Concentration (μg/mL) TiO2 - Rutile (28 nm) C TiO2 - Rutile (28 nm) 200 150 d Resazurin ) d % d ) Neutral red ( % 150 d th 100 Commassie c ea a ility ( 100 ll d 50 ab l vi e ce el 50 tiv C 0 aelR 0 -50 0 l 1 5 0 50 00 50 00 50 00 50 00 10 50 00 00 00 00 00 00 1 1 2 2 3 3 4 tro 1 2 4 5 6 8 n 100 Concentration (μg/mL) oC Concentration (μg/mL) Figure 18: Cell viability of HFG after treatment with three types of TiO2-NPs. After treating HGF cells with different concentrations of TiO2 Anatase/Rutile (18 nm) (A), TiO2 Anatase (28 nm) (B) and TiO2 Rutile (28 nm) (C) for 24-hours, the resazurine assay, neutral red uptake assay and Coomassie assay were used to assess the cell viability. To obtain the relative cell death after the NP treatment, typan blue cell debris assay was employed. The results are presented as means with 95% confidence intervals. Significant differences are indicated as follows; a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. 78 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 The oral cavity is a portal of entry into the respiratory and gastrointestinal tracts and is the first to be affected by the release of nanoparticles from orthodontic materials. If NPs have an effect on cells of the oral cavity, then it is also worth considering that NPs may migrate to other tissues and have an effect there as well. To test the viability of HGF cells, we used different concentrations of NPs and examined the viability of the cells after 24 hours of treatment of HGF with TiO2-NPs using four different methods. The use of the neutral red uptake assay to determine cell viability was found to be inappropriate because the TiO2-NPs interfered with the assay and gave erroneous results. It was noted by Jalili et al. (2018) and Bessa et al. (2017) that TiO2-NP can interfere with NR assay due to the light reflecting/absorbing of TiO2-NPs at wavelengths used for NR quantification. No change in viability was observed when the resazurin assay was used. Mello et al. (2020) suggest that resazurine assay should be the prefered assay, when assessing NP related cytotoxicity, due to minimal interference of the NP. After treatment of HFG cells with a concentration of 300 µg/mL, an increase in viability was even observed, but this may also be due to the interference of the NPs with the assay. The Coomassie assay indicated the amount of protein in the cells, which was volatile up to the 100 µg/mL concentration of NPs but then decreased significantly. In the case of TiO2 anatase/rutile, the protein concentration was significantly reduced at a concentration of 300 µg/mL, and in the case of TiO2 anatase and TiO2 rutile, the significant decrease was reached at 200 µg/mL. Because each of these three viability assays gave different results, we used the Typan cell debris assay to compare the amount of dead cells after treatment with the untreated cell sample. Dose-dependent cell death was detected in all samples treated with TiO2-NPs. The extent of cell death of the samples treated with TiO2 anatase/rutile was significantly different at 300 µg/mL and higher concentrations compared with the untreated control sample, whereas TiO2 anatase and TiO2 rutile significantly increased cell death at 200 µg/mL and higher. The observations of cell death were consistent with the results of the Coomassie assay, and it could be concluded that high concentrations of TiO2-NP interfered with normal cell activity. Li et al. (2010) also found that the same dose of 200 µg/mL increased cell death in fibroblast cultures. A systematic review by Suárez-López del Amo et al., (2018) reported that TiO2-NPs and TiO2 microparticles (TiO2-MPs) ranging in size from 15 nm to 45 μm were found in animal models and from 100 nm to 54 μm in human tissues as a consequence of particle release from dental materials, as well as dental material wear debris in oral tissues. The Ti particles can cause cytotoxicity (Happe et al., 2019), genotoxicity, inflammation (Pettersson et al., 2017), changes in cytoskeletal structures (Saldaña and Vilaboa 2010). It is difficult to assess the cytotoxicity of NPs, which tend to aggregate into MPs because they are larger than the cell organelles and cannot be taken up by the cell membrane as the NPs below 100 nm do (Singh et al., 2007). Actually, the agglomerated particles should be dispersed before treatment with ultrasound, but in our case, the TiO2-NPs could not be dispersed properly. The agglomerated particles could have a different bioactivity on the cells, but at the same time, these agglomerated crystals are not uniform and usually consist of submicron particles 79 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 and can also release some NPs from their structure into the environment. Li et al., (2022) have shown that after treatment of HGF with TiO2-MPs and TiO2-NPs, TiO2-NPs were found to be more cytotoxic than TiO2-MPs, as they decreased cell viability at 250 µg/mL, in contrast to TiO2-MPs, which significantly decreased cell viability at 1000 µg/mL. Similar results were observed with other cell types such as epithelium (Suárez-López del Amo et al., 2018), fibroblast (Garcia-Contreras et al., 2015), macrophages (Ding et al., 2012) and osteoblasts (Zhang et al., 2020). Lin's group also showed that both NPs and MPs were taken up by the cells, but MPs were distributed only in the cytoplasm, whereas NPs were also found in the nuclei. Importantly, even non-cytotoxic concentrations of NPs and MPs can alter the organization of the cytoskeleton and thus affect the biological behavior of the cell. Cytoskeletal organization regulates cell shape, adhesion, growth, maturation, and migration (Pollard and Cooper 2009; Thievessen et al., 2013). In contrast, Barrak et al. (2020) showed no reduction in viability after treatment of HGF with a mixture of TiO2-NPs and TiO2-MPs, but the average size of MPs was 77.4 ± 9.1 µm, twenty times larger than those we used. WS2 (30-70 nm) WS2 (30-70 nm) 150 150 ) Resazurin % d ) ( c % Neutral red th 100 c 100 ea a Commassie ility ( ll d 50 ab l vi 50 e ce el tiv C 0 aelR 0 -50 0 l 1 5 0 50 00 50 00 50 00 50 00 10 50 00 00 00 00 00 00 1 1 2 2 3 3 4 tro 1 2 4 5 6 8 n 100 o Concentration (μg/mL) C Concentration (μg/mL) Figure 19: Cell viability of HFG after treatment with WS2-NPs. After treating HGF cells with different concentrations of WS2-NPs for 24-hours, we used resazurine assay, neutral red uptake assay and Coomassie assay to assess the cell viability. To obtain the relative cell death after the NP treatment, typan blue cell debris assay was employed. The results are presented as means with 95% confidence intervals. Significant differences are indicated as follows; a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. Due to its high strength and low coefficient of friction, WS2-NPs are gaining interest in dentistry, especially in orthodontics. Therefore, understanding their potential toxicological effects is necessary, but not yet sufficiently explored. Treatment of HGF cells with increasing concentrations of WS2-NPs had no effect on cell viability in all assays up to 400 µg/mL, but the trypan blue cell debris assay showed a dose-dependent increase in dead cells that was significant at a concentration of 500 µg/mL (Figure 19).While Domi et al. (2021) observed no decrease in viability after treatment of A549 cells with a concentration of 160 µg/mL, Domi et al. (2021) and Teo et al., (2014) observed a decrease in cell viability after treatment of cells with a concentration of 400 µg/mL WS2-MPs and WS2-NPs, but this is 80 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 considered mild cytotoxicity. The reason for the mild cytotoxicity of WS2-NPs might be due to the agglomeration of NPs in the presence of the growth medium. Turkez et al. (2014) have shown that high concentrations of NPs disrupt the integrity of cell membranes, leading to cell death, but the actual toxicity of NPs may depend not only on the numerical nominal exposure (concentration) but also on the properties of NPs, such as mass, surface area, and dissolution of ions (Pardo et al., 2014). The exact mechanism of toxicity of WS2-NPs at high concentrations is not defined, but Liu et al. (2017) suggested that proteins in the medium are absorbed onto the WS2-NPs surface and form a protein corona that can reduce cell viability. In our study, the interaction of proteins from fibroblast serum with WS2-NPs may have been the reason why we observed a significant increase in cell death at 500 µg/mL compared with the control sample. From a chemical point of view, there is an intrinsic electron transfer from W to S atoms that makes WS2 hydrophilic and reduces the likelihood of other molecules such as phospholipids being distributed in the membrane (Liu et al., 2017). ZnO (18 nm) ZnO (18 nm) A 150 150 d d d d Resazurin ) d % d ) Neutral red ( d % th 100 100 Commassie ea ility ( ll d 50 ab l vi 50 e ce el tiv C 0 aelR 0 -50 0 5 l 1 5 10 15 20 25 30 35 40 45 50 10 15 20 25 30 35 40 45 50 tro no Concentration (μg/mL) C Concentration (μg/mL) B ZnO (30-50 nm) ZnO (30-50 nm) 150 150 Resazurin )% ) Neutral red ( % th 100 d 100 Commassie ea d d d d d d ility ( ll d ab 50 l vi 50 e ce el tiv C 0 aelR 0 -50 0 5 l 1 5 10 15 20 25 30 35 40 45 50 10 15 20 25 30 35 40 45 50 tro no Concentration (μg/mL) C Concentration (μg/mL) Figure 20: Cell viability of HFG after treatment with two different ZnO-NPs. After treating HGF cells with different concentrations of ZnO (18 nm) (A) and ZnO (30-50 nm) (B) for 24-hours, we used resazurine assay, neutral red uptake assay and Coomassie assay to assess the cell viability. To obtain the relative cell death after the NP treatment, typan blue cell debris assay was employed. The results are presented as means with 95% confidence intervals. Significant differences are indicated as follows; a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. . 81 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Like TiO2-NPs, ZnO-NPs are increasingly used in industrial and commercial applications, especially as pigments and for protection against UV radiation in sunscreens. ZnO-NPs also have good antibacterial, anticorrosive, and antifungal properties, which are in high demand in the field of orthodontics (Service, 2003). To investigate the possibility of toxic damage to HGF cells by ZnO-NPs, we performed various tests to explore the toxic effect (Figure 20). The cell viability data show a strong decrease in viability after treatment with 15 µg/mL and above for both ZnO-NPs used in the study. We also obtained the same results on cytotoxicity as Choudhury et al. (2017) who also found a strong effect of ZnO on the cytoskeleton of the cells. When the used ZnO NPs agglomerate in the aqueous medium, they clump together and form larger crystals. A wide range of cystals of different sizes can bind to a wider range of biomolecules and potentially cause multimodal damage (Rana et al., 2013). Not only MPs and NPs, but also ions could affect cell viability because ZnO-NPs release Zn2+ ions to the environment (Ng et al., 2017; Yin et al., 2010). We found no difference in the cytotoxicity of ZnO-NPs with sizes of 18 nm and 30-50 nm, but these were only the reported sizes, which turned out to be much larger after theoretical characterization. On the other hand, study of Bhattacharya et al. (2016) shows that ZnO NPs of different sizes and shapes have different effects on the cell. Ag (28-48 nm) A Ag (28-48 nm) 150 150 Resazurin )% ) Neutral red ( % th 100 d 100 Commassie ea d a ility ( ll d a a 50 ab l vi 50 e ce el tiv C 0 aelR 0 -50 0 l 1 5 0 50 00 50 00 50 00 50 00 10 50 00 00 00 00 00 00 1 1 2 2 3 3 4 tro 1 2 4 5 6 8 n 100 o Concentration (μg/mL) C Concentration (μg/mL) Ag (48-78 nm) Ag (48-78 nm) B 150 150 d Resazurin )% d ) Neutral red ( d % th 100 d 100 Commassie ea d ility ( ll d d d 50 b ab l vi 50 e ce el tiv C 0 aelR 0 -50 0 l 1 5 50 00 50 00 50 00 50 00 10 15 20 25 30 35 40 45 50 1 1 2 2 3 3 4 tro no Concentration (μg/mL) C Concentration (μg/mL) Figure 21: Cell viability of HFG after treatment with different concentrations of Ag-NPs. After treating HGF cells with different concentrations of Ag (28-48 nm) (A) and Ag (48-78 nm) (B) for 24-hours, we used resazurine assay, neutral red uptake assay and Coomassie assay to assess the cell viability. To 82 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 obtain the relative cell death after the NP treatment, typan blue cell debris assay was employed. The results are presented as means with 95% confidence intervals. Significant differences are indicated as follows; a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. Ag-NPs are a kind of double-edged sword when exposed to humans, as producing both beneficial effects such as antimicrobial effects and harmful effects such as cytotoxicity. Exposure of the HFG cell line to two different Ag-NPs led to different results in cell viability (Figure 21). In the case of the 28-48 nm size NPs, viability slowly decreased with increasing NP concentration, and a significant increase in cell death was observed at 400 µg/mL. In contrast, for the 48-78 size NPs, the effect was much more pronounced, as cell viability dropped below 50% when treated with a concentration of 30 µg/mL, and cell death was significantly increased at a concentration of 15 µg/mL. The different effect of the two AgNPs could not be related to the properties, because both Ag-NPs have similar properties, but when the size was assessed on TEM, very small particles were seen (< 1 nm), which could be the Ag+ ions. The larger presence of Ag+ ions in the 48-78 nm Ag-NPs suspension could be the reason for the strong cytotoxic effect of these NPs. After entering the cell, Ag-NPs and Ag+ ions can have deleterious consequences, ranging from the generation of ROS and oxidative damage to mitochondrial damage and apoptosis (Akter et al., 2018). Depending on the properties, some Ag-NPs are taken up by endocytosis and dissolved into Ag+ ions due to the acidic medium in the lysosomes. Other Ag-NPs diffuse into the cytoplasm, where they are oxidized by cytoplasmic enzymes and release Ag+ ions (Sabella et al., 2014). The released ions interact with mitochondrial proteins, disrupting mitochondrial flux and generate ROS. The cytotoxic mechanism of NPs is still controversial, but several lines of evidence suggest the possible role of ROS and oxidative stress on induced cytotoxic effects (Singh et al., 2007). To investigate the ability of NPs to stimulate the formation of ROS, the dye H2DCFDA was used as an indicator of intracellularly formed ROS (Figure 22). Treatment with TiO2-NP showed a dose increasing effect of intracellularly ROS formation. Even a TiO2-NP concentration of 20 µg/mL resulted in a significant increase in ROS production compared with the control group. As it was already seen in the assessment of cell viability, higher concentrations led to membrane damage, so that the fluorescence signal was also significantly attenuated. The same observation was also reported by Liu et al. (2010). Periasamy et al. (2015) linked the generation of ROS to mitochondrial damage because ROS disrupted the mitochondrial membrane potential, resulting in even more ROS being generated. 83 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 TiO2 - Anatase/Rutile (18 nm) TiO2 - Anatase (28 nm) A B 1000 1000 ) ) % 800 % 800 n ( n ( tio 600 tio 600 ra ra ne ne e 400 e 400 g g S S O 200 O 200 R R 0 0 l l 20 40 60 80 00 50 00 50 20 40 60 80 00 50 00 50 tro 1 1 2 2 tro 1 1 2 2 n n o o C Concentration (μg/mL) C Concentration (μg/mL) TiO C 2 - Rutile (28 nm) WS D 2 (30-70 nm) 1000 150 ) ) % 800 % n ( n ( 100 tio 600 tio ra ra ne ne e 400 e g 50 g S S O 200 O R R 0 0 l l 20 40 60 80 00 50 00 50 20 40 60 80 00 50 00 50 tro 1 1 2 2 tro 1 1 2 2 n n o o C Concentration (μg/mL) C Concentration (μg/mL) ZnO (18 nm) ZnO (30-50 nm) E F 150 150 ) ) % % n ( n ( 100 100 tio tio ra ra ne ne e e 50 g 50 g S S O O R R 0 0 l 5 l 5 10 15 20 25 30 35 40 10 15 20 25 30 35 40 tro tro n n o o C Concentration (μg/mL) C Concentration (μg/mL) Ag (28-48 nm) Ag (48-78 nm) G H 150 a 150 ) ) % % n ( n ( 100 100 tio tio ra ra ne ne e e 50 g 50 g S S O O R R 0 0 l l 5 20 40 60 80 00 50 00 50 10 15 20 25 30 35 40 tro 1 1 2 2 tro n n o o C Concentration (μg/mL) C Concentration (μg/mL) Figure 22: Inracellular ROS generation of HGF cells, treated with different types and concentrations of NPs. After treating HGF cells with different concentrations of TiO2 (Anatase/Rutile 18 nm) (A), TiO2 (Anatase 28 nm) (B), TiO2 (Rutile 28 nm) (C), WS2 (D), ZnO (18 nm) (E), ZnO (30-50 nm) (F), Ag (28-78 nm) (G) and Ag (48-78 nm) (H) for 24-hours, the fluorescent dye H2DCFDA was added to observe the intacellular ROS level. The results are presented as means with 95% confidence intervals. 84 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 As much as TiO2-NPs are praised for their use as UV light blockers in sunscreens, their property also has a downside, because when photoexcited, they can generate ROS (Konaka et al., 2001). Excitation of valence electrons by UV light leaves positively charged spaces that generate ROS in the presence of aqueous media and O2 (Fujishima et al., 2000). Treating HGF cells with as low as 20 µg/mL concentration of TiO2-NPs has significantly risen the fluorescence intensity of the sample and the fluorescence intensity only enhanced when the NPs concentration was increased. Whether the increase in fluorescence intensity was due to the generation of ROS or only to the interference of TiO2-NPs with the H2-DCFDA assay is not known. The viability results indicate that some kind of cytotoxic effect causes the increased the cell death in the presence of TiO2-NPs. The results of the intracellular oxidation assay suggest that viability decreased because of ROS-induced cytotoxicity, but in this case, viability should decrease before treatment with 250 µg/mL TiO2-NPs. A more plausible explanation could be supported by the results of Wang et al. (2015), who reported that excess TiO2-NPs on the cell membrane caused blockage of ion pathways, disrupting ion exchange and causing cell death. Zhang et al. (2013) showed that there was a correlation between the increased ROS levels and cytotoxicity after the addition of TiO2-NPs to cell lines, but the ROS generation might not only be based on the inherent oxidative capacity of TiO2-NPs, as it might also be related to mitochondrial damage due to the interaction of TiO2-NPs with the cell membrane. So far, many studies suggest various toxic mechanisms of toxicity induced by TiO2-NPs, but a precise mechanism remains to be determined. We could not accurately determine the viability of the HGF cell line after treatment with TiO2-NPs, but based on our previous in vitro study on yeast cell model, we have shown that oxidative damage occurs when the ROS level increases, strongly suggesting that a ROS-dependent mechanism of cell death augmented by TiO2-NPs (Kongseng et al., 2016) could be seen in HGF cell line. Although WS2-NPs showed no reduction in cell viability at 400 µg/mL, the possible ROS generation was still plausible, but as shown by Corazzari et al. (2014), oxidative potential of WS2-NPs is negligible. Treatment of cells with increasing concentrations of WS2-NP did not result in the generation of ROS. The inability to generate ROS was also demonstrated by Corazzari et al. (2014) when treating A549 and by Appel et al. (2016b) when treating epithelial cells with WS2-NPs concentrations. Domi et al. (2021) observed only a slight increase in intracellular oxidation, which may not even be due to the NPs themselves. ZnO-NPs were found to be the most cytotoxic NPs used in our study because they exerted their effects at very low concentrations. The same results were hindered when evaluating the intracellularly generated ROS. The lowest ZnO-NP concentration used (5 µg/mL) did not increase ROS production, nor did the higher concentrations. Interestingly, the production of ROS and cell viability correlated well, with low ZnO-NPs concentrations shown to decrease viability. Studies report that citrate toxicity could be due to the Zn2+ ions released by ZnO NPs or the ability of ZnO- NP to induce the formation of ROS. It is important to note that Zn2+ itself cannot undergo electron reduction to generate ROS, but it can inhibit antioxidant 85 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 molecules and thus induce oxidative stress indirectly (Shen et al., 2013). Like TiO2-NPs, ZnO-NPs are also considered photocatalysts that generate electron-hole pairs in the presence of light energy and consequently convert O2 into •OH (Yin et al., 2010). Whether the intracellularly generated ROS molecules are the cause of cytotoxicity of ZnO-NPs or just a consequence of mitochondrial damage caused by Zn2+ ions is unclear. Ng et al. (2017) concluded that ROS is the cause of cell death induced by ZnO-NP. On the other hand, Choudhury et al. (2017) found that ZnO NPs disperse inside the cell, deforming the cytoskeleton and thus affecting mitochondrial function and membrane protein. This leads to caspase activation and increased ROS production (Ricci et al., 2003). Due to the strong oxidative activity of Ag-NPs and the silver ions released by the NPs from their surfaces, Ag-NPs are widely used as antimicrobial agents in medicine (He et al., 2012). Most of the Ag- NP cellular and biochemical changes in the cell are considered to be caused by ROS. In our study, Ag-NPs with size of 28-48 nm did not increase the formation of ROS and Ag-NPs with size of 48-78 nm decreased the production of ROS. It is generally believed that the formation of ROS is the main mechanism for the toxicity of Ag-NPs, as a correlation between the formation of ROS and cytotoxicity has been established in many studies (Xue et al., 2016). What exactly caused Ag-NPs cytotoxic effect in our study, if not increased formation of ROS, should be investigated in further studies to make a definitive statement. Investigating the effect of NPs on immortalized cell lines gave us insight into the field of nanotoxicology. Although some concentrations of NPs are unlikely to occur in vivo, they provided a dose-response analysis for further toxicological studies. So far, we do not know the amount, size, and shape of NPs used in the field of orthodontics, how they are applied (coatings, adhesives, materials) and how they are released into oral cavity. Despite their increasing popularity, certain questions regarding the toxicity and biocompatibility of NPs have yet to be investigated. Toxicity and biocompatibility are two absolute necessities that must be determined when evaluating safety of orthodontic appliances used in healthcare settings. With the advent of nanotechnology and its applications, concerns have been raised about toxic effects on humans (Allaker, 2010). It is not possible to compare toxic levels of bulk materials with those of nanomaterials because the area-to-volume ratio is greater for nanoparticles, making it more likely that potentially toxic substances will elute or particles will be released. Because metal nanoparticles differ in their toxic-kinetic properties from metals without nanoparticles, additional ADME (absorption, distribution, metabolism, and excretion) information must be obtained (Hagens et al., 2007). Of particular concern is their safety status after a prolonged period of exposure and possible dissolution of NPs in living organisms, whether through the chemical aspects of saliva, erosion, chewing, bacterial accumulation, or some other form of destruction of the NPs. Such released metals NP or metal ions can freely enter the bloodstream and cause toxic damage at the systemic level (Behzadi et al., 2017). Therefore, short-term in vitro studies are not sufficient to fully assess the human health risk. 86 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 The toxicity of NP is not yet fully understood, but depends mainly on NP parameters such as shape, size, surface area, composition and stability. The use of NPs in orthodontics involves direct contact with the teeth and surrounding cells and tissues in the oral cavity. Therefore, the handling of the potentially harmful effects of NPs must be carefully managed. 4.6 STUDY LIMITATIONS AND FUTURE PERSPECTIVES When assessing oxidative stress status, two or more assays should be used to increase validity because each method has its own limitations and no method alone can accurately measure antioxidant status and ROS formation (Poljsak and Jamnik, 2010). In the present study, the FORT and FORD methods were used to determine the ROS/AD ratio. Although changes in the ROS/AD ratio were observed over time, their main cause could not be determined because the observed change in the ROS/AD ratio could be related to many factors (i.e., activation of endogenous antioxidants, inflammation, and metal ions in the blood). We excluded the possibility that periodontal inflammation had an effect on the measured systemic oxidative stress parameters because no signs of inflammation were detected in the patients during the study. However, we could not exclude inflammation as a cause of the increased ROS due to the metal ion release and metal content in the blood. The detection of inflammatory mediators and metal concentrations in blood samples could provide very important information for our study, but for ethical reasons it was not possible to collect consecutive larger venous blood samples throughout the study period. In addition, previous studies (Mikulewicz and Chojnacka, 2010) reported that heavy metal ions (e.g., nickel) are detectable in blood only after long-term exposure. Finding out the exact reason for short-term increase in systemic oxidative stress parameters (ROS/AD levels) in capillary blood was beyond the scope of the present study but real reason of increased ROS/AD ratio would be interesting to determine in further studies as well as the potential of endogenous antioxidative defenses induction in patients In vitro experiments are not able to fully simulate the in vivo environment, such as saliva biological composition, salivary flow rate, temperature fluctuations, pH differences, and microbiological flora (Wendl et al., 2017). For example, corrosion is considered to be the main reason for the release of metal ions, and the extent of corrosion depends on the formation of passive oxide layers on the surfaces of orthodontic materials. These oxide layers effectively prevent corrosion (Eliades et al., 2004), but are strongly influenced by changes in the oral environment, such as pH and temperature level. In our study, a constant pH, temperature and non-shaking conditions were maintained to avoid a potential increase in ion release, but in the in vivo environment, the oxide layers could be degraded more rapidly due to the instability of the environment (Kuhta et al., 2009). 87 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Instead of using primary cells isolated from excised tissue in vivo and proliferating in culture until natural senescence occurs, we used immortalized cell lines. They are produced either by artificial transformation of primary cell cultures with viral vectors to transfer genetic material or by natural transformation and derivation of cancer tissue cells (Nanci, 2016). While primary cells better represent the in vivo environment than immortalised cells, obtaining ethical approval and processing samples for cell isolation is a time-consuming process. In addition, when using primary cells, the amount of samples is very limited and there are large differences between samples because the primary cells come from different individuals (sex, age, disease status, the depth of the removed layer) (Nanci, 2016). On the other hand, imortalized cell lines are ready to use and can be easily cultured indefinitely. However, this comes at a price, as immortalised cell lines may have altered metabolism and phenotype compared to primary cells. Therefore, they may not provide representative in vivo results, thus results should be interpreted with some caution or not directly extrapolated to humans. It should be noted that the metal mixtures may not fully reflect the release of metal ions by orthodontic appliances (Kovač et al., 2022). The release of metals in vivo is very difficult to simulate because many factors influence the release process. We have used specific ratios of metal ions to simulate orthodontic alloys, while these concentration ratios of metal ions may be different in vivo. The use of metal mixtures instead of the released metal ions from our in vitro release study could be considered a limitation. The reason we chose to use metal mixtures was that a large sample volume was needed for a large-scale study and higher metal concentrations were desirable for predicting toxicological effects and risk assessment of metal ions. We could have evaporated the samples from the in vitro release study to obtain higher metal concentrations, but even then they would not have reached the desired concentrations and we would still have less sample volume to test toxic effects in the study. The choice of the HGF cell line may not initially seem reasonable from an in vivo perspective, since fibroblasts among epithelial cells, like keratinocytes, promote connective tissue that is most likely to be the first to come into contact with the released metal ions. However, orthodontic appliances are in the oral cavity for a long period of time, and the continuous release of metal ions eventually reaches the inner layer of the oral mucosa (Milheiro et al., 2016). In orthodontic patients, metal accumulation in the oral mucosa has been observed even 12 months after orthodontic treatment (Orozco-Páez et al., 2021). Although the metal ions released from orthodontic appliances are lower than the concentrations of metal mixtures and Schmalz et al., (1998) indicated that the metal ions released from dental alloys are less cytotoxic compared to the same salt solutions, the potential long-term local toxic effect should not be forgotten. 88 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 5 CONCLUSIONS 1. A short-term increase in systemic oxidative stress parameters (ROS/AD levels) in capillary blood was observed at 24 hours after orthodontic treatment with fixed appliances. 2. All orthodontic alloys released metal ions during a 90-day exposure to artificial saliva, but the final concentrations did not exceed the recommended upper limits for daily metal intake. 3. The β-Ti alloy released the fewest metal ions of all orthodontic alloys tested. 4. All metal mixtures simulating the orthodontic alloys SS, Co-Cr, Ni-Ti, and β-Ti decreased Saccharomyces cerevisiae culturability, but only at high concentrations. The SS and Co-Cr metal mixture induced the formation of ROS and caused oxidative protein and lipid damage at high concentrations, whereas Ni-Ti and β-Ti mixtures did not. 5. The treatment of Saccharomyces cerevisiae with metal mixtures induced a complex response observed as changes in endogenic enzymatic antioxidative defense systems 6. Concentrations of the released metal ions from orthodontic appliances cannot induce oxidative stress and its related damages in the yeast Sacharomyces cerevisiae. 7. All metal mixtures induced HGF cell death at high concentrations, but only SS and Co-Cr metal mixtures were able to generate increased intacellular ROS formation. 8. Characterization of NPs shows aggregation and agglomeration of NPs in aquatic media, meaning that the NPs size increased to micro meter level. 9.Three selected types of TiO2-NPs caused ROS-induced cytotoxicity at high concentrations. WS2-NP did not cause cytotoxicity nor the formation of ROS. The two selected ZnO-NP were found to be cytotoxic already at low concentrations, but their toxic effects could not be associated with the increased formation of ROS. The two selected Ag-NPs gave different cytotoxic results, with the 28-48 nm Ag-NPs being much less cytotoxic compared to the 48-78 nm Ag-NPs. No increase in ROS generaton was detected after threating HGF cells with Ag-NPs. 10. Of the selected nanoparticles, WS2 nanoparticle was the least toxic to human gingival cells and as such has potential to be considered in nano-medical applications. 89 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 6 SUMMARY (POVZETEK) 6.1 SUMMARY Misaligned teeth, also known as malocclusion, have a significant impact on oral and dental health. The most effective method of correcting the problem is orthodontic treatment with fixed orthodontic appliances. However, over time and with the ever-changing conditions in the oral environment, orthodontic appliances are also affected. Electrochemical reactions, mechanical forces and general wear of the material in the oral cavity lead to corrosion, a process of wear of the orthodontic metals. During orthodontic treatment with fixed appliances, subjects are exposed to metals released from the corroded appliances that can in theory increase levels of ROS through metal-catalyzed free radical reactions. If the constantly increasing ROS molecules are not maintained at physiological levels, excessive amounts of ROS generated can lead to oxidative stress that disrupts cellular redox homeostasis and consequently damages lipids, proteins, and DNA. To counteract these harmful effects and intercept overproduced ROS, each cell has a defense system that includes enzymatic and non-enzymatic antioxidant denfenses. Nanotechnology represents a great opportunity for improving dental properties such as strength and durability. The nanoparticles are applied to the orthodontic appliances as a coating, but they can degrade or corrode in the oral cavity during the course of treatment. The lack of knowledge about the safety of nanoparticles after prolonged exposure and the possible dissolution of nanoparticles in living organisms are of concern and should be properly investigated. The objective of this work was to investigate the level of selected systemic oxidative stress parameters during orthodontic treatment, the composition of selected orthodontic alloys, the release of metal ions, and the oxidative consequences that the metal ions may have on the model organism Sacharomyces cerevisiae. The work also aimed to investigate the effects of nanoparticles on the human gingival cell line. Fifty-four male patients with malocclusions who underwent orthodontic treatment had their capillary blood levels ROS and ROS /AD examined. Different parts of orthodontic appliances were analyzed for their metal composition and incubated in artificial saliva for 90 days to quantify metal release. Metal mixtures were prepared according to metal composition and used to treat yeast cells to determine the occurrence of oxidative stress, antioxidant enzyme defense system, and oxidative damage. Selected nanoparticles were prepared at different concentrations and used to treat human gingival fibroblasts to study cell viability and their ability to form ROS. 90 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Our study shows that the induction of systemic oxidative stress in the capillary blood of orthodontic patients occurs only after 24 hours, after which the oxidative stress parameters in capillary blood normalize to the initial level. Whether the increase in systemic oxidative stress levels is due to metal release from orthodontic appliances was not defined. We have shown, that the release of metal ions from orthodontic appliances into artificial saliva is constant, but the metal concentrations released are still below the maximum tolerated daily dose. Of the selected orthodontic alloys, the lowest ceocnentration of metal ions was released from the β-Ti alloy. Based on the metal composition of orthodontic alloys, metal ion mixtures were prepared and used to treat yeast Sacharomyces cerevisiae. Only high metal ion concentrations were able to generate large amounts of reactive oxygen species, which the antioxidant system was unable to regulate adequately, resulting in oxidative stress and its damage to biological molecules. Only SS and Co-Cr metal mixtures caused the formation of reactive oxygen species and caused oxidative damage to lipids and proteins. The comparison between the yeast cell model and the human gingival fibroblast cell line proved the suitability of the yeast Sacharomyces cerevisiae as a model organism for oxidative stress research. NP caracterization showed that NPs aggregate and agglomerate in culture medium and water, increasing their size from the nanometer scale to the micrometer scale. The toxicity of selected nanoparticles and their ability to generate reactive oxygen species in human gingival fibroblasts mainly depends on the type, concentration, and properties of the nanoparticles. All three selected types of TiO2-NPs were cytotoxic at high concentrations, due ROS formation. Both selected ZnO-NPs were found to be cytotoxic even at low concentrations, but their toxic effects could not be linked to increased ROS formation. The two selected Ag-NPs gave different cytotoxic results, with Ag-NPs 28-48 nm being much less cytotoxic compared to Ag-NPs 48-78 nm. After treating HGF cells with Ag-NPs, no increased generation of ROS was detected. Among the selected nanoparticles, the WS2 nanoparticle was the least toxic to human gingival cells, as it did not cause cytotoxicity or ROS formation, and is therefore the most suitable for potential medical use. 6.2 POVZETEK Nepravilno poravnani zobje, znani tudi kot malokluzija, imajo pomemben vpliv na zdravje ustne votline in zob. Tudi iz psihološkega vidika malokluzije vplivajo na človekovo počutje in samozavest (Nguee in sod., 2020). Najučinkovitejša metoda za odpravo težave je ortodontsko zdravljenje z nesnemnimi ortodontskimi aparati, ki se uporabljajo za različne premike zob po alveolarni kosti. Učinkovitost ortodontskega zdravljenja je odvisna od dovzetnosti obzobnih tkiv za zdravljenje in značilnosti delov nesnemnega ortodontskega aparata, ki ga sestavljajo nosilci, loka, obročki in ligature (Proffit et al., 2007). Deli ortodontskega aparata so lahko iz različnih materialov, vendar se zaradi dobrih mehanskih lastnosti, trdnosti ter toplotne in električne prevodnosti najbolj uporabljajo zlitine kovin (Park in Lakes, 2007). Ortodontsko zdravljenje z biokompatibilnimi aparati je ključnega pomena za učinkovitost zdravljenja in varnost pacienta. Za biokompatibilne se štejejo 91 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 materiali, ki ne povzročajo negativnih učinkov na zdraje, kar pomeni, da niso strupeni, rakotvorni in ne morejo povzročiti alergijskih reakcij (Widu in sod., 1999). Druga pomembna lastnost biokompatibilnih materialov je, da se njihove fiziološke in mehanske lastnosti ne spremenijo v pogojih in vivo. Biomateriali so v tesnem stiku s telesnimi tekočinami, ki lahko vplivajo na površino materiala. V prisotnosti sline ali drugih tekočin se ortodontske zlitine sčasoma nagibajo k koroziji, kar povzroči sproščanje kovine s površine zlitine in osnovno oslabitev lastnosti zlitin (Mathew in Wimmer, 2011). V procesu, imenovanem korozija, kjer pride do fizikalno-kemijske (elektrokemične) interakcije med kovino in njenim okoljem, se lahko pojavijo spremembe v lastnostih kovine. Glavna pomanjkljivost kovinskih zlitin je prav v nagnjenosti k koroziji v prisotnosti bioloških tekočin. Nanotehnologija predstavlja dobro priložnost za nadaljnje izboljšave lastnosti medicinskih in dentalnih materialov kot sta trdnost in vzdržljivost. Nanodelci imajo ugodne lastnosti, kot so visoko razmerje med površino in prostornino za boljšo interakcijo z okoljem, zeta potenctial, velikost in oblika delcev, površinska kemija, aglomeracija, raztapljanje in sproščanje ionov (Fernando in sod. 2018). Zaradi svojih katalitičnih, optičnih in elektromagnetnih lastnosti se kovinski nanodelci (NP) pogosto uporabljajo v bioloških in medicinskih aplikacijah (Mamunya in sod. 2004), vključno v ortodontiji. NP, zlasti kovinski NP s svojimi fizikalno-kemijskimi, mehanskimi in antibakterijskimi lastnostmi, bi lahko močno vplivali na trajanje ortodontskega zdravljenja in izboljšali ustno zdravje (Sharan in sod., 2017). Eden od načinov nanašanja NP na ortodontske pripomočke je v obliki prevlek, s katerimi bi lahko izboljšali površinske in mehanske lastnosti kovinskih zlitin. Izraz oksidativni stres je prvi uvedel Helmut Sies kot neravnovesje med prooksidanti in antioksidanti v korist prvih (Sies, 2020). Tako imenovano redukcijsko-oksidacijsko (redoks) ravnovesje se lahko poruši bodisi zaradi prekomernega nastanka prooksidantov, bodisi zaradi pomanjkanja antioksidantov ali celo zaradi obojega hkrati. Manjša nihanja redoks ravnovesja ne povzročajo večjih težav, saj se organizem lahko nanje prilagaja, velika nihanja, zlasti povečano nastajanje reaktivnih kisikovih zvrsti pa vodijo do nepopravljivih bioloških poškodb celičnih komponent in celo celične smrti (Burton in Jauniaux, 2011). V fizioloških pogojih velja, daje redoks ravnovesje le malenkost premaknjeno v korist prooksidantov, saj so ti nujni za normalno delovanje organizma. Ko pa se ravnotežje vedno bolj nagiba v korist prooksidantov, lahko opazimo poškodbe organelov in razgradne procese (Auten in Davis, 2009). Med reaktivne kisikove zvrsti (ang. ROS) spadajo prosti radikali, ki imajo nepopolno zunanjo elektronsko plast in imajo enega ali več neparnih elektronov, kar jih naredi zelo reaktivne. ROS se lahko kovalentno povežejo z drugo molekulo preko neparnega elektrona ali pa lahko nastanejo z dajanjem neparnega elektrona (redukcijski radikal) ali s sprejemanjem elektronov (oksidacijski radikal) (Halliwell, 1991). Izraz ROS vključuje tudi 92 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 nekatere neradikalne molekule, kot sta vodikov peroksid in ozon, ki pa se smatrata kot ROS, saj se zlahka pretvorita v radikale (Irani, 2007). ROS se nenehno proizvajajo v normalnih fizioloških pogojih, njihova homeostaza pa se nenehno spremlja in vzdržuje. Teorija prostih radikalov in oksidativnega stresa je bila ustanovljena pred več kot pol stoletja in sprva so ROS veljali za škodljive stranske produkte aerobne presnove, sedaj pa velja, da imajo ROS bistveno vlogo v različnih bioloških procesih (Finkel, 2011), kot je fagocitoza in prenos celičnih signalov. Primarni ROS nastanejo tekom štiristopenjskega procesa redukcije O2 v H2O (Halliwell in Gutteridge, 2015). Molekula kisika, ki ima dva neparna elektrona, je neškodljiva, razen če se energijsko aktivira. Aktivacija se lahko pojavi, ko je zagotovljena dovoljšna mera energije, da se obrne vrtenje neparnega elektrona, pri čemer nastane singletni kisik (1O2). Ko pa se molekuli kisika dodajajo elektroni, pa nastajajo superoksidni radikal (O2•−), vodikov peroksid (H2O2) in hidroksilni radikal (•OH) (Apel in Hirt, 2004). Kovinski ioni imajo pomembno vlogo pri različnih celičnih funkcijah, kot so prenos elektronov po dihalni verigi, sinteza in popravilo DNA ter celični metabolizem. Kovine z delno napolnjeno d podlupino in zmožnostjo tvorbe kationov imenujemo prehodni elementi ali prehodne kovine (McNaught and Wilkinson, 1997). Prehodne kovine najdemo v skupini 4-11 periodnega sistema in imajo veliko število kompleksnih ionov v številnih pozitivno nabitih oksidacijskih stanjih z različnimi katalitskimi lastnostmi. Nekatere prehodne kovine so bistveni elementi in ključne komponente za številne metaloproteine, vključene v proces tvorbe kisika in odkrivanja hipoksije. Kovinski ioni se pojavljajo v različnih oksidacijskih stanjih in kot taki lahko preidejo v redoks reakcijo, se povežejo s fosfolipidi, spremenijo stabilnost membrane in spodbujajo peroksidacijo lipidov. Ker je nastajanje ROS tesno povezano z vključevanjem redoks aktivnih kovin, se njihove koncentracije vzdržujejo strogo pri fizioloških koncentracijah (Valko in sod., 2005). Eden od mehanizmov nastajanja ROS, v katerega se vključujejo prehodne kovine je Fentonova reakcija, ki je bila prvič predstavljena leta 1894 (Fenton, 1894) in kasneje popravljena in dopolnjena do danes znane reakcije (Haber in sod., 1934). V Fentonovi reakciji železo (Fe2+) katalizira reakcijo pretvorbe H2O2 v •OH. Namesto železa lahko v reakciji sodelujejo tudi druge prehodne kovine z visoko valenco, kot so baker (Cu), cink (Zn) in aluminij (Al). Kadar reakcija vključuje kovine, ki niso Fe ali Cu, ligande ali perokside, se reakcija imenuje Fentonu-podobna reakcija (Meyerstein, 2021). Dodatek k prvotni Fentonovi reakciji je Haber-Weissova reakcija, ki domneva da O2•− ponovno reagira s H2O2 in tvori •OH ter hidroksilni anion (OH-) (Das in sod., 2015). Zaradi svojih edinstvenih lastnosti, kot so katalitične, optične in elektromagnetne lastnosti, se NP in nanomateriali široko uvajajo za biološko in medicinsko uporabo (Mamunya in sod., 2004). NP, zlasti kovinski NP s svojimi fizikalno-kemijskimi, mehanskimi in antibakterijskimi lastnostmi, bi lahko močno vplivali na trajanje ortodontskega zdravljenja, odpravili nekatere z zdravljenjem povezane težave in izboljšali ustno zdravje (Sharan in sod., 2017). Ne glede na to, kako koristne so edinstvene lastnosti, NP veljajo tudi za potencialno 93 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 strupene snovi. Visoko razmerje med površino in prostornino jih naredi zelo reaktivne (Drasler in sod, 2017), njihova majhna velikost pa omogoča enostavno prodiranje skozi celično membrano (Yin in sod., 2012). Prisotnost NP in njihovih kovinskih ionov lahko povzročita nastanek ROS, ki je glavni vzrok njihove citotoksičnosti (Yu in sod., 2013). Da NP lahko ustvarjajo ROS na tri načine: prvi mehanizem je interakcija med NP in celico, drugi je raztapljanje in sproščanje kovinskih ionov s površine NP, tretji pa tvorba prooksidantnih funkcionalnih skupin na površini NP (Yanli Wang in sod., 2017). Mehanizem generiranja ROS, ki je specifičen za posamezen NP, še ni popolnoma razumljen. ROS imajo lahko blagodejen ali škodljiv učinek na biološki sistem. Kadar ROS nastajajo v presežku se smatra, da ROS oslabi biološke molekule in celične strukture. Takrat govorimo o pojavu oksidativnega stresa. Celica oziroma organizem ima sposobnost zmanjšanja ROS in njegovih poškodb, vendar se te oksidativne poškodbe kopičijo in sčasoma dodatno poškodujejo DNA, beljakovine ali lipide (Valko in sod., 2006). Celične komponente, ki vsebujejo polinenasičene maščobne kisline (PUFA), kot je celična membrana in membrane organelov, so zelo občutljive na oksidacijo. Hrbtenica PUFA ima dve ali več dvojnih vezi, s katerimi lahko ROS reagira. Več dvojnih vezi kot ima PUFA, večja je verjetnost, da bo bo prišlo do oksidacija v prisotnosti ROS (Su in sod., 2019). Poleg lipidne peroksidacije lahko ROS poruši tudi lipidni dvosloj in inaktivira nekatere membransko vezane beljakovine ter na splošno poveča prepustnost membrane (Birben in sod., 2012). Poškodbe DNA, ki jih povzročajo prosti radikali, so vir mutageneze, karcinogeneze in staranja celic. Genetski material bioloških sistemov je nenehno ogrožen zaradi poškodb ROS. •OH velja za najbolj reaktiven radikal v bioloških sistemih, ker reagira s purinom, pirimidinom ali deoksiribozo v hrbtenici DNA (Dizdaroglu in sod., 2002). Druga ROS, kot sta H2O2 in O2•−, nista neposredno vključena v nastanek oksidativnih poškodb DNA, ampak le posredno. Poškodbe, ki jih povzroča ROS, so eno- ali dvoverižni prelomi DNA in navzkrižne povezave DNA (Cooke in sod., 2003). Oksidacija beljakovin je kovalentno spremenjen proces, pri katerem ROS ali sekundarni stranski produkti oksidativnega stresa reagirajo z beljakovinsko molekulo (Shacter, 2000). Posledice oksidacije beljakovin se kažejo z izgubo aktivnosti beljakovin (receptorja, encima, transporta ali strukture) in nagnjenostjo k proteolizi ali denaturaciji beljakovine. Osnovne sestavine beljakovin, aminokisline, so primarna tarča ROS, zlasti cistein, metionin in aromatske aminokisline (tirozin, fenilalanin in triptofan) (Kehm in sod., 2021). Ker so aerobni organizmi nenehno izpostavljeni ROS, se je med evolucijo razvil učinkovit obrambni sistem, sestavljen iz obrambnih, nevtralizacijskih in popravljalnih mehanizmov. Prekomerno nastajanje ROS in z njimi povezane škodljive učinke blažijo antioksidanti, bodisi encimski antioksidanti ali neencimski antioksidanti. Obstajajo štirje glavni obrambni encimi, ki so odgovorni za vzdrževanje ROS na za calico neškodljivih ravneh: superoksid dismutaza (SOD), katalaza (CAT), glutation peroksidaza (GPx) in sistem tioredoksin reduktaze (TrxR). SOD katalizira pretvorbo O2•− v O2 in H2O2. CAT je odgovorna za 94 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 znotrajcelično pretvorbo H2O2 v H2O in O2. Drug encim, ki je odgovoren za razgradnjo H2O2 je GPx (Arthur, 2000). Čeprav ni neposredno vključena v obrambo ROS, ima glutation reduktaza (GR) pomembno vlogo pri presnovi GSH in je kot taka tesno povezana z redoks sistemom glutationa. Peroksiredoksin (Prx) je član družine tiol peroksidaze in je tesno povezan s sistemom Trx, ki zagotavlja elektrone, potrebne za njegovo delovanje (Du in sod., 2012). Med neencimske antioksidante spadajo snovi z nizko molekulsko maso, polipeptidi in beljakovine, ki jih organizem proizvaja ali zaužije z vsakodnevno prehrano. Vključujejo vitamin A in vitamin C, β-karoten, sečno kislino, melatonin in GSH. Modelni organizmi so nenadomestljivo orodje v osnovnih bioloških in kliničnih raziskavah (Hunter, 2008), kadar se preučuje škodljive učinke iz okolja na biološke sisteme. Modelni organizmi so pogosto izbrani, ker premagujejo etične in eksperimentalne omejitve, zagotavljajo model za razvoj, optimizacijo in standardizacijo določenih analiz in so jasen predstavnik večje skupnosti vrst z enakimi ali podobnimi biološkimi procesi (Karathia in sod., 2011). Saccharomyces cerevisiae ( S. cerevisiae) je med evkariontskimi organizmi najbolj znan, preučen in karakteriziran model, saj so osnovni celični procesi in celična organizacija podobni celicam sesalcem (Karathia in sod., 2011). Celotno zaporedje genoma kvasovk razkriva dobro ohranjeno zaporedje aminokislin in funkcijo beljakovin med evkariontskimi vrstami (Botstein in Fink, 2011). Zaradi visoke homologije s človeškim genomom, primerljive homologije funkcij beljakovin, širokega nabora poceni genetskih manipulacij, enostavne in poceni pridelave in rasti, preučevanja več procesov hkrati in skoraj popolne baze podatkov, je S. cerevisiae eden izmed idealnih modelnih mikroorganizmov za preučevanje oksidativnega stresa in odziva nanj (de la Torre-Ruiz in sod., 2015). Kar je pa za našo študijo najbolj pomembno pa je to, da je tvorba ROS na ETC ter mehanizem odziva oziroma obrambe na povečan nivo ROS pri kvasovkah podoben kot pri sesalcih (Herrero in sod., 2008). Skladno z načelom 3Rs (zamenjava, zmanjšanje in izboljšanje) alternative poskusom na živalih in vivo uporabljamo primarne celice ali celične linije (Krewski in sod., 2010). Celične linije imajo na splošno prednost pred primarnimi celicami, ker so bolj stabilne, homogene in splošno dostopne, kar ima za posledico boljšo replikacijo in primerjavo znanstvenih podatkov. Vendar imajo prednosti uporabe celičnih linij svojo ceno, saj se ne diferencirajo in tako v celoti ne predstavljajo in vivo razmer (Gstraunthaler in Hartung, 2002). Človeški gingivalni fibroblasti (HGF) so najpogostejši predstavniki ustne sluznice in se zato pogosto uporabljajo v poskusih za oceno toksičnosti (Mah in sod., 2014) pri oralni izpostavljenosti škodljivemu agensu. Ker so v neposredni bližini ortodontskih zlitin, so klinično pomemben model za ugotavljanje vpliva izluževanja kovinskih ionov. Ker se z vsako delitvijo celice se telomeri skrajšajo in s tem tudi življenjska doba celice, smo v študiji smo uporabili imortalizirano celično linijo HFG, ki z izražanjem reverzne transkriptaze človeške telomeraze (hTERT) preprečuje skrajšanje telomere. Tako lahko dobimo dolgoživo celično linijo, kateri se fiziologija in fenotip ne spreminjata (Reijnders in sod., 2015). 95 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Namen disertacije je bil raziskati, ali bi sproščanje kovinskih ionov iz nesnemnih ortodontskih aparatov lahko vodilo do povečanja nivoja ROS in posledično oksidativnih poškodb na modelnem organizmu. Najprej smo opazovali, ali se parametri oksidativnega stresa spreminjajo med ortodontskim zdravljenjem, nato pa smo načrtovali poskus sproščanja kovin in vitro, da bi v celoti razumeli, kateri in koliko kovinskih ionov se sprosti iz ortodontskih zlitin in kako njihova izpostavljenost vpliva na celice. V raziskavi je sodelovalo 54 zdravih moških bolnikov, starih od 19 do 28 let, pri katerih je bila diagnosticirana malokluzija. Zdrave moške bolnike smo naključno razdelili v dve skupini, tarčno skupino (TG), ki je bila podvržena ortodontskemu zdravljenju, in kontrolno skupino (CG), v kateri ortodontski aparat ni bil nameščen v ustni votlini. Ortodontski aparat, ki se uporablja za zdravljenje malokluzije TG, je bil sestavljen iz SS nosilcev. Kapilarna kri je bila odvzeta iz TG in CG v štirih različnih časovnih intervalih: pred vstavitvijo ortodontskega aparata (čas 0), 6 ur po vstavitvi, 24 ur po vstavitvi in po 7 dneh. Z merjenjem tvorbe ROS (FORT test) in antioksidantnega potenciala (FORD test) v vzorcih kapilarne krvi je mogoče stanje oksidativnega stresa v krvi bolnikov natančneje oceniti na sistemski ravni. V tej študiji smo želeli oceniti, ali med ortodontskim zdravljenjem pride do povečanja nivoja ROS v vzorcih kapilarne krvi. Statistično značilna razlika v vrednostih FORT in FORD je bila opažena v TG po 24 urah ortodontskega zdravljenja, vendar se je pri naslednji analizi (po 7 dneh) vrednost znižala nazaj na izhodiščno raven. Na podlagi ugotovitev, da bi lahko bili kovinski aparati vzrok za povečanje nivoja ROS (Kovac in sod., 2019), se je postavilo vprašanje o varnosti tovrstnih ortodontskih aparatov po trajni uporabi (Hanawa, 2004). Da bi bolje razumeli kovine, ki sestavljajo ortodontske zlitine, in koncentracije kovinskih ionov, ki se sproščajo v laboratorijskih razmerah, smo zasnovali študijo, v kateri so bili ortodontski materiali potopljeni v umetno slino za 90 dni. Njihova kovinska sestava je bila ovrednotena z ICP-MS. V primeru zlitine SS (loki, nosilci in obročki) so se sproščali predvsem Fe ioni, koncentracije ostalih preučevanih kovin pa so ostale skoraj na začetni koncentracijski ravni vse do 90 dni, ko je bilo opaziti rahlo povečanje. Ko smo pogledali koncentracijo sproščanja kovine za oba Ni-Ti loka, je bila količina sproščenega Ti pri obeh podobna, vendar količina sproščenega Ni pa ni bila primerljiva, saj je Ni-Ti(Forestadent) sprostil dvakrat več Ni ionov kot Ni-Ti(Dentaurum). Za β-Ti lok koncentracije Mo niso bile zaznavne vse do 90. dneva študije, ko je bila koncentracija 0.45 ng/mL. Koncentracije Ti iz β-Ti loka so nihale za 1.5 ng/mL skozi celoten potek študije vse do 90. dneva, ko je bila zaznana najvišja vrednost približno 8.3 ng/mL. Med študijo se je sproščanje kovin iz obeh Co-Cr žic postopoma povečevalo. Koncentracije Co in Cr so dosegle podobne ravni v obeh žicah, vendar so bile opažene nekatere razlike v Fe, Ni in Mo. S pridobljenimi rezultati smo podali podatke o minimalnih koncentracijah kovinskih ionov, ki se lahko sprostijo iz ortodontskih zlitin izključno s čistim difuzijskim postopkom brez dodatnih mehanskih sil. Dejstvo, da ortodontske zlitine sproščajo kovinske ione s svojih površin je nesporno, a čeprav je bil opažen stalen vzorec sproščanja, je količina sproščenih 96 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 kovinskih ionov še vedno daleč pod dnevno priporočenimi zaužitimi vrednostmi. Če primerjamo naše rezultate s predpisanimi priporočenimi dnevnimi ravnmi za zaužitje, noben od preučenih sproščenih kovinskih ionov ni presegel zgornje predpisane meje dnevne koncentracije vnosa, tudi če upoštevamo kumulativne koncentracije. Na podlagi dobljenih rezultatov smo lahko ocenili vrsto in koncentracijo sproščenih kovinskih ionov. Glede na kovinsko sestavo ortodontskih zlitin smo pripravili mešanice kovinskih ionov za nadaljnje študije oksidativnega stresa. Prikazali smo, da ortodontski materiali sicer sproščajo kovinske ione s svojih površin. S pridobljenimi rezultati smo na modelnem organizmu S. cerevisiae preučili toksičnost in nastanek oksidativnega stresa po izpostavljenosti celic različnim kombinacijam kovinskih ionov v različnih koncentracijah. Z uporabo dveh mutant S. cerevisiae brez antioksidantnega encima SOD (ΔSod1) ali CAT (ΔCtt1) smo lahko prepoznali, kako delna izguba endogene antioksidativne obrambe vpliva na sposobnost preživetja celic. V disertacijski študiji smo celice kvasovk za 24 ur izpostavili različnim mešanicam kovinskih ionov v koncentracijah 1 µM, 10 µM, 100 µM in 1000 µM. Z mešanicami kovin smo posnemali kovinsko sestavo ortodontskih zlitin SS, Co-Cr, Ni-Ti in β-Ti. Pri tretiranju celic kvasovk z naraščajočimi koncentracijami kovinskih mešanic smo opazili zmanjšanje kulturabilnosti, saj pri vseh tretmajih s 1000 µM koncentracijami lahko opazimo statistično značilno zmanjšanje. Ko smo primerjali kulturabilnost Wt in dveh mutant, so opazili pomembno razliko v kulturabilnosti, saj je bila vrednost CFU/mL neobdelanih kontrolnih vzorcev mutant kvasnih celic veliko nižja kot pri netretiranem kontrolnem vzorcu Wt. V predstavljeni študiji je bil uporabljen tudi test celične metabolne aktivnosti, ampak zaradi majhnega števila opravljenih testov rezultati niso pokazali jasnih razlik med različno tretiranimi celicami kvasovk. Ko smo celice kvasovk Wt tretirali z različnimi koncentracijami mešanic kovinskih ionov, je bilo ugotovljeno, da višje koncentracije povečajo povprečno vrednost metabolne aktivnosti. Po drugi strani pa se mutante kvasnih celic nagibajo k zmanjšanju svoje presnovne aktivnosti z naraščajočo koncentracijo kovinskih ionov, zlasti pri 1000 µM koncentracijah. Primerjali smo tudi metabolno aktivnost med netretiranimi celicami kvasovk Wt in mutanti in ugotovili, da je bila izhodiščna metabolna aktivnost mutant vsaj dvakrat višja kot pri netretirani kontrolni skupini kvasovk Wt. Kot je razvidno iz ocene kulturabilnosti celic, so bile kovinske mešanice stresorji za celice kvasovk. Ker so bile vse v študiji uporabljene prehodne kovine, ki so sposobne ustvarjati ROS preko Fentonove in Haber-Weissove reakcije (Zhao, 2019), smo domnevali, da je razlog za zmanjšanje kulturabilnosti celic posledica povečanega znotrajceličnega nivoja ROS. Da bi ugotovili, ali so kovinske mešanice povzročile povečanje znotrajceličnega nivoja ROS v kvasovkah, smo izvedli test znotrajcelične oksidacije za določitev nastalih ROS. Metoda znotrajcelične oksidacije z barvilom H2DCFDA je pokazala znatno povečanje znotrajcelične tvorbe ROS v kulturabilnih celicah po 24 urah tretiranja s SS in Co-Cr, medtem ko ni bilo zaznanih sprememb v intenzivnosti fluorescence, ko je bila katera koli 97 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 vrsta celic kvasovk tretirana s kovinsko mešanico Ni-Ti ali β-Ti. Da bi ugotovili, ali ROS, ki ga ustvari kovinska mešanica, povzroči oksidativne poškodbe molekul in ne le povečano tvorbo ROS, smo uporabili test TBARS za preučitev nastanka lipidnih poškodb in opravili analizo za odkrivanje oksidativnih poškodb beljakovin. Če primerjamo oceno znotrajceličnega nastanka ROS in oceno oksidativnih poškodb lipidov, je med njima razvidna jasna povezava, saj ko je bila tvorba ROS povečana, so bili tudi lipidi oksidativno poškodovani. SS in Co-Cr kovinski mešanici sta povzročili oksidacijo lipidov pri 1000 µM koncentraciji v Wt in celo pri 100 µM pri mutantah. Vsak organizem poseduje antioksidativne obrambne mehanizme za vzdrževanje ravni redoks ravnotežja. Ker mutante kvasovk ΔSod1 in ΔCtt1 nimajo vseh endogenih antioksidativnih encimov za uspešno odstranjevanje presežnkov ROS in njihovih produktov, so znotrajcelično ustvarjene poškodbe ROS opazne pri izpostavljenosti nižjim koncentracijam kovinskih mešanic kot pri Wt. To nas je spodbudilo k raziskovanju encimskega antioksidantnega sistema v S. cerevisiae. Zaradi velike variabilnosti encimske aktivnosti v gelu smo sočasno izvedli še spektrofotometrične meritve encimske aktivnosti povezane z antioksidativno obrambo. Aktivnost SOD se je znatno povečala, ko smo celice tretirali s koncentracijo 1000 µM SS. Druge vrste kovinskih zmesi pri različnih koncentracijah pa niso vplivale na aktivnost SOD. Ko smo ocenili aktivnost CAT, smo opazili znatno zmanjšanje aktivnosti po tretiranju s 1000 µM SS in 1000 µM Ni-Ti. H2O2 lahko pretvori v H2O encim CAT, kot tudi z encim GPx (Arthur, 2000). Dodajanje zmesi SS in Co-Cr ni vplivalo na njegovo aktivnost, vendar smo opazili znatno povečanje aktivnosti v prisotnosti 1000 µM Ni -Ti in β-Ti. Aktivnost GR 1000 µM SS vzorcev se je zmanjšala, medtem ko se je aktivnost povečala po tretiranju s 1000 µM Co-Cr in tretiranju z 10 µM, 100 µM in 1000 µM β-Ti. Medtem ko se je aktivnost Prx tudi znatno zmanjšala po izpostavljenosti s 1000 µM SS, druge kovinske mešanice niso povzročile sprememb v encimski aktivnosti. Aktivnost TrxR se je zmanjšala tudi, ko smo celice obdelali s 1000 µM SS, medtem ko so imele kovinske mešanice Co-Cr, Ni-Ti in β-Ti vse večji učinek na encimsko aktivnost tudi pri koncentraciji 100 µM. Merjenje vsebnosti karbonilov kot oksidativnih poškodb beljakovin, končnem produktu oksidacije beljakovin v bioloških vzorcih, je uporaben biološki označevalec za ocenjevanje oksidativnega stresa, ki ga povzroči kovina, saj je reakcija nepovratna (Lazarova in sod., 2014). V naši študiji je bilo ugotovljeno, da koncentracije kovinskih zmesi, ki povzročajo znotrajcelično tvorbo ROS in oksidacijo lipidov (1000 µM SS, 100 µM in 1000 µM Co-Cr), povzročajo tudi oksidativne poškodbe beljakovin, medtem ko druge kovinske zmesi niso imele učinek na oksidacijo beljakovin. Ustna sluznica in dlesni so prva tkiva, ki pridejo v stik s sproščenimi kovinskimi ioni iz ortodontskih aparatov. Zunanja epidermalna plast dlesni je keratinizirana in ščiti notranje vezivno tkivo, ki je sestavljeno iz fibroblastov dlesni. Z uporabo človeških gingivalnih 98 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 fibroblastov (HGF) lahko pridobimo informacije o tem, kako se celice lahko odzivajo na izbrane kovine v različnih koncentracijah v ustni votlini. Da bi potrdili, da je bila uporaba S. cerevisiae kot modelnega organizma upravičena, smo celice HGF obdelali z istimi kovinskimi mešanicami v istem časovnem obdobju in z enakimi koncentracijami kot celice kvasovk. Predstavljeni rezultati kažejo, da izpostavljenost celic HGF z mešanicami kovin SS in Co-Cr ni vplivala na sposobnost preživetja celic HGF, medtem ko so imele zmesi Ni-Ti in β-Ti pomemben učinek na sposobnost preživetja pri 250 µM za Ni-Ti in pri 500 µM za β-Ti. Vzrok za zmanjšanje sposobnosti preživetja celic in celične smrti je bil nejasen, vendar smo domnevali, da je to lahko posledica sposobnosti kovinskih zmesi za ustvarjanje ROS, ker vse komponente kovinskih zmesi spadajo v skupino prehodnih kovin. Tretiranje celic HGF s kovinskimi mešanicami SS ali Co-Cr poveča proizvodnjo ROS, medtem ko se po izpostavitvi Ni-Ti ali β-Ti kovinskim mešanicam proizvodnja ROS zmanjša. Zaradi svojih fizikalnih in kemijskih lastnosti postajajo NP vse pomembnejši pri raziskavah in razvoju novih materialov, med katere spadajo tudi ortodontski materiali. Z novimi lastnostmi in industrijsko proizvodnjo NP pa so se pojavili tudi pomisleki glede možnih škodljivih učinkov na zdravje. Zato je najprej potrebno razumeti učinke NP na žive celice, da bi jih prepoznali kot varne za nadaljnjo uporabo v kakršni koli aplikaciji. Ustna votlina je vstopno mesti v dihala in gastrointestinalni trakt in je prvo mesto, na katero vpliva sproščanje nanodelcev iz ortodontskih materialov. Če NP vplivajo na celice ustne votline, je potrebno razmisliti tudi o tem, da lahko NP migrirajo v druga tkiva in tam tudi povzročajo škodljive učinke. Uporaba nanodelcev v ortodontiji ni natančno opredeljena. Zato smo morali ovrednotiti razpon toksičnosti za izbrane kovinske nanodelce in oceniti njihovo sposobnost ustvarjanja ROS. Za testiranje sposobnosti preživetja celic HGF smo uporabili različne koncentracije NP in preučili sposobnost preživetja celic po 24 urah tretiranja HGF z NP TiO2-NP, WS2-NP, ZnO-NP in Ag-NP. V primeru tretiranja celic s TiO2 anataze/rutila se je delež umrlih celic znatno povečal pri koncentraciji 300 µg/mL, v primeru TiO2 anataze in TiO2 rutila pa je bilo povečanje deleža mrtvih celic vidno že pri koncentraciji 200 µg/mL. Tretiranje celic HGF z naraščajočimi koncentracijami WS2-NP ni imelo vpliva na sposobnost preživetja celic v vseh testih do 400 µg/mL, vendar je kljub temu bilo opazno povečanje deleža mrtvih celic, ki je bilo statistično značilno pri koncentraciji 500 µg/mL. Podatki o preživetju celic po tretiranju z obema tipoma ZnO-NP kažejo močno zmanjšanje sposobnosti preživetja po tretiranju s 15 µg/mL. Izpostavljenost celične linije HFG dvema različnima Ag-NP je povzročila različne rezultate v analizi sposobnosti preživetja celic. V primeru Ag-NP velikosti 28-48 nm se je sposobnost preživetja počasi zmanjševala z naraščajočo koncentracijo NP, pri 400 µg/mL pa so opazili statistično značilno povečanje deleža mrtvih celic. Nasprotno pa je bil pri Ag-NP velikosti 48-78 nm kjer je učinek veliko bolj izrazit, saj je sposobnost preživetja celic padla pod 50 % pri tretiranju s koncentracijo 30 µg/mL, delež mrtvih celic pa se je statistično značilno povečal pri koncentraciji 15 µg/mL. 99 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Citotoksični mehanizem NP je še vedno sporen in nedorečen, vendar več vrst dokazov kaže na možno vlogo ROS in oksidativnega stresa na induciranje citotoksičnih učinkov (Singh et al., 2007). Da bi raziskali sposobnost NP, da stimulirajo tvorbo ROS, je bilo uporabljeno barvilo H2DCFDA kot indikator znotrajcelično tvorjenih ROS. Tretiranje s TiO2-NP je pokazalo učinek povečanja znotrajceličnega nastanka ROS. Tretiranje celic HGF s samo 20 µg/mL koncentracijo TiO2-NP je znatno povečalo intenzivnost fluorescence izmerjenega vzorca. Intenzivnost fluorescence se je povečala le, ko je bila koncentracija NPs povečana. Obdelava celic z naraščajočimi koncentracijami WS2-NP ni povzročila zaznavne tvorbe ROS. Najnižja uporabljena koncentracija ZnO-NP (5 µg/mL) ni povečala proizvodnje ROS, prav tako ne višje koncentracije. Izpostavljenost Ag-NP z velikostjo 28-48 nm ni povečala tvorbe ROS in Ag-NP z velikostjo 48-78 nm je zmanjšala proizvodnjo ROS v celicah. Naša raziskava je pokazala, da v kapilarni krvi ortodontskih pacientih prihaja do indukcije sistemskega oksidativnega stresa po 24 urah, čigar parametri pa se po 7 dneh zopet normalizirajo na začetno raven. Ali se stopnja sistemskega oksidativnega stresa poveča zaradi sproščanja kovinskih ionov iz ortodontskih aparatov, ni bilo definirano. Izkazalo se je, da je sproščanje kovinskih ionov iz ortodontskega aparata v umetno slino konstantno, vendar so koncentracije sproščenih kovin še vedno pod največjim dovoljenim dnevnim odmerkom. Od izbranih ortodontskih zlitin se je iz zlitine β-Ti sprostilo najmanj kovinskih ionov. Na podlagi kovinske sestave ortodontskih zlitin smo pripravili mešanice kovinskih ionov in jih uporabili za tretiranje kvasovk S. cerevisiae. Le visoke koncentracije kovinskih ionov so lahko ustvarile velike količine ROS, ki jih antioksidativni sistem ni mogel ustrezno regulirati, kar je povzročilo oksidativni stres in z njim povezane poškodbe bioloških molekul. Le SS in Co-Cr kovinski mešanici sta povzročili nastanek reaktivnih kisikovih zvrsti in povzročili oksidativne poškodbe lipidov in proteinov. Primerjava nastanka ROS v kvasnih celicah in celični liniji človeških gingivalnih fibroblastov je dokazala primernost kvasovke S. cerevisiae kot modelnega organizma za raziskave oksidativnega stresa. Karakterizacija NP je pokazala, da NP v gojišču in vodi agregirajo in aglomerirajo, kar poveča njihovo velikost iz nano metrske lestvice na mikro metersko. Toksičnost izbranih nanodelcev in njihova sposobnost tvorjenja reaktivnih kisikovih zvrsti v človeških gingivalnih fibroblastih sta odvisni od vrste, koncentracije in lastnosti nanodelcev. Vse tri izbrane vrste TiO2-NP so bile pri visokih koncentracijah citotoksične, zaradi nastajanja molekul ROS. Ugotovljeno je bilo, da sta oba izbrana ZnO-NP citotoksična že pri nizkih koncentracijah, vendar njunih toksičnih učinkov ni bilo mogoče povezati s povečano tvorbo ROS. Dva izbrana Ag-NP sta dala različne citotoksične rezultate, pri čemer so bili Ag-NP 28-48 nm veliko manj citotoksični v primerjavi z Ag-NP 48-78 nm. Po tretiranju celic HGF z Ag-NP nismo zaznali povečanega nastanka ROS. Izmed izbranih nanodelcev so bili nanodelci WS2 najmanj toksični za človeške gingivalne celice, saj ni povzročil citotoksičnosti niti tvorbe ROS, in so zato najbolj ustrezni za potencialno uporabo v medicinske namene. 100 Kovač V. 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Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ACKNOWLEDGEMENTS Looking back, I realize that I did a lot of work that took many days that stretched into the evenings, many nights that were far too short, and many weekends without the »weekend part«. But all this outweighs the satisfaction of what I have achieved and the thought that all this time I had people by my side who helped me and supported me. My deepest gratitude to my mentor, assoc. prof. dr. Borut Poljšak, cannot be put into words. Thank you, Borut! You provided me with mentorship in the truest sense of the word. Your advice, encouragement, conversations, opportunities, time, and moral support have given a young PhD student more than he could have hoped for. While I still adhered to the original dissertation plan, you gave me the freedom and opportunity to independently research scientific topics and implement them in my work. You recognized my aversion to certain expensive research methods and supported my curiosity with your own resources, for which I am very grateful. One can only wish to be mentored by such a person as you, Borut. The other important person during my research, to whom I am also very grateful, was prof. dr. Polona Jamnik. You put me in charge of the proteomics lab and allowed me to expand my knowledge and learn and develop a wide range of lab techniques. The thought that I could just knock on your office door and ask for help or advice was very welcome. I am very grateful for the many hours you have invested in me and my research. I would also like to thank all of the researchers I have had the opportunity to work with: - Assoc. prof. dr. Jasmina Primožič for the help and support during the clinical research study and for the financial support from the J3-2520 project. - Prof. dr. Radmila Milačič and prof. dr. Jamez Ščančar for the ICP-MS analysis. - Prof. dr. Darko Makovec and assoc. prof. dr. Slavko Kralj for the characterization of NP. - Prof. dr. Sue Gibbs for the HGF donation and research collaboration. - Prof. dr. Mojca Narat for enabeling research in her laboratory. A special thank you goes to Laura for making my 4 year journey so much easier. I would like to take this opportunity to apologize for the late hours, evening meetings, and my mood swings that you had to deal with. Thank you for being there for me. You are simply the best! Thank you mom and dad for supporting me and believing in me. Without the other members of "The Submarine" there would be no happy times during my doctoral studies. The morning coffee, lunch, afternoon drinks, and conversations during the day kept me going when things were difficult for me. I would especially like to mention the help of Matej Šergan, who always had a solution to my problem, for which I am very grateful. Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ANNEX B Patients consent Izjava/soglasje Spoštovani! Vabimo Vas, da se vključite v pilotno študijo, s katero želimo preučiti oksidativni stres kot posledico zdravljenja z nesnemnimi ortodontskimi aparati. Uporaba ortodontskega aparata (kolikor časa je pač potrebno za zdravljenje z ortodontskim aparatom) lahko zaradi izluževanja kovinskih ionov iz aparata poveča delež prostih radikalov v krvi. Kratek opis poteka raziskave: Pred onamestitvijo nesemnega ortodontskega aparata se iz prsta sodelujočega v raziskavi odvzame kapljica kapilarne krvi (50 μL), v kateri se opravi FORT in FORD analiza (določi se celokupen antioksidativen potencial izražen v koncentraciji Troloxa in delež prostih radikalov izražen kot koncentracija H2O2) in 1 mL sline, katera se uporabi za AES analizo (določi se koncentracija kovinskih ionov). Nato udeležencu ortodont namesti nesnemni zobni aparat in po 24, 48, 72 urah ter po 7 dneh se vozrči kapilarna kri (FORT in FORD) in slina (AES). Tako vzorčenje se ponovi vsak mesec in po treh mesecih se vzorčenje zaključi. Ortodontski aparat se sname po uspešnem zdravljenju. Vsak sodelujoči je zaprošen, da izpolni še anonimni vprašalnik o življenjskih navadah, ki vplivajo na stanje oksidativnega stresa. Sodelovanje v raziskavi je anonimno. V izogib upravljanju z osebnimi podatki, bo posameznik sodelujoč v raziskavi ustrezno kodiran, prav tako tudi osebni podatki posameznika. Rezultati bodo obdelani in predstavljeni izključno v sumarni obliki. Rezultatov posameznega udeleženca tako ne bo mogoče pridobiti. Sodelovanje posameznika je prostovoljno in lahko od nje kadarkoli, brez kakršnih koli posledic odstopi. S spodnjim podpisom izkažete strinjanje, da ste pripravljeni sodelovati v predstavljeni raziskavi. S spoštovanjem, Ime in priimek: Podpis Datum: Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ANNEX C Questionnaire Vprašalnik za ugotavljanje oksidativnega stresa I. Splošni podatki o udeležencu (-ki) preizkusa: Šifra udeleženca (-ke) /izpolni vodja testiranja/: 1. Starost: _____ let 2. Spol: M - moški, Ž – ženski II. Splošno počutje in zadovoljstvo 1. Kako ocenjujete svoje splošno počutje oz. zdravstveno stanje? a) sem popolnoma zdrav in sem v dobri kondiciji b) počutim se dobro c) ne počutim se preveč dobro (brez energije…) d) počutim se bolnega 2. Kako ste zadovoljni s svojim življenjem? a) večinoma sem zelo zadovoljen b) zadovoljen sem c) nisem prav zadovoljen, a tudi ne nezadovoljen d) nezadovoljen sem e) večinoma sem zelo nezadovoljen 3. Svoje delo (poklicne, študijske ali druge delovne obveznosti) občutim kot: a) zelo stresno b) stresno c) malo stresno d) nestresno III. Funkcionalno zdravje Prosimo, odgovorite na vprašanja, tako da obkrožite črke a, b ali c pred odgovorom, ki vam najbolj ustreza. Prebava 1. Imate slabo prebavo? . . . . . . . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 2. Vas napenja in imate vetrove? . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 3. Ste po zaužitem obroku zaspani? . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 4. Imate sindrom razdražljivega črevesja (diareja)?. . . . . . . . A) pogosto B) včasih C) redko ali nikoli Srce in obtočila 5. Kakšen je vaš krvni pritisk? . . . . . . . ………………… A) 140/90 ali višji B) med 125/85 in 140/90 C) nižji kot 125/80 6. Se hitro zadihate? . . . . . . . . . . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 7. So vaše roke in noge mrzle? . . . . . . .... . . . A) pogosto B) včasih C) redko ali nikoli 8. Je zdravnik ugotovil, da imate slabo prekrvavitev?. . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 9. Koliko članov vaše družine (starši, stari starši, tete, strici) je umrlo zaradi bolezni srca in ožilja? . . . . . A) trije ali več B) dva C) eden ali nobeden Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Imunski sistem 10. Kolikokrat na leto ste prehlajeni? .... . . . . . . . . . . . . . . . . . . . . A) trikrat ali večkrat B) dvakrat C) enkrat ali nikoli 11. Koliko dni navadno trajajo izraziti simptomi prehlada? . . . . . . . . .A) tri dni ali dlje B) dva dneva C) en dan ali nič 12. Povprečno kolikokrat na leto jemLjete antibiotike?. . . . . . . . . . . . . . . .A) dvakrat ali večkrat B) enkrat C) nikoli 13. Imate pogosta vnetja, ki jih spremLjajo bolečine in pordelost?. . . . . . . . . . . . . . . . . . . . A) da C) ne 14. Imate pogoste alergijske reakcije? . . . . . . . . . . A) da B) občasno C) ne Duševno zdravje 15. Se težje zberete, kot ste se nekoč? . . . . . . . A) pogosto B) včasih C) redko ali nikoli 16. Si težje zapomnite stvari kot pred časom? . . A) pogosto B) včasih C) redko ali nikoli 17. Ste kdaj depresivni? . . . . . ... . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 18. Se vas kdaj polaščajo občutki strahu in tesnobe? . . . . . . . . . . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli Hormonski sistem 19. Imate pogosto močno željo po sladkih prigrizkih ali poživilih? . . . .... . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 20. Bi svoj način življenja označili kot stresen? . . . . . ... . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 21. Morate nov dan začeti s poživilom ali sladkim prigrizkom? . . . . . . ... . . . . . . . A) pogosto B) včasih C) redko ali nikoli Koža, nohti in lasje 22. Imate suho kožo? . . . . . . . . . . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 23. Imate akne? . . . . . . . . .... . . . . . . . . . . .... . . A) pogosto B) včasih C) redko ali nikoli 24. Imate izpuščaje, luskavico ali dermatitis? . A) pogosto B) včasih C) redko ali nikoli 25. So vaši nohti krhki in razpokani? . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 26. Imate mastne oziroma suhe in krhke lase? . A) pogosto B) včasih C) redko ali nikoli Telesna kondicija 27. Povprečno kolikokrat na teden ste telesno dejavni? . . . . . . . . . . . . A) redko ali nikoli B) enkrat C) dvakrat ali večkrat 28. Kolikokrat si med telesnimi napori pretegnete mišice? . . . . . . . . . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 29. Ali ponoči spite manj kot 6 ur in pol? . . . . A) pogosto B) včasih C) redko ali nikoli 30. Kako pogosto ste utrujeni? . . . . . . . . . . . . . A) pogosto B) včasih C) redko ali nikoli 31. Ali sami menite, da imate dobro telesno kondicijo? . . . . . . . . . . . . . . . . . . . A) redko ali nikoli B) včasih C) pogosto Splošna zdravstvena slika 32. Kolikokrat na leto obiščete zdravnika? .... . . . . . . . . . . . . . . . . . . . A) dvakrat ali pogosteje B) enkrat C) nobenkrat Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 33. Povprečno kolikokrat na leto jemLjete zdravila, predpisana z receptom? . . . . . . . . . . . . A) dvakrat ali pogosteje B) enkrat C) nobenkrat 34. Ali. .? . . . . . . . . . . . . . . . . . . . . . . A) ste debeli B) imate prekomerno telesno težo C) imate idealno telesno težo 35. Ste bili kot otrok pogosto bolni? . .... . . . . . A) pogosto B) včasih C) redko ali nikoli 36. Imate kake kronične zdravstvene težave?. . A) da C) ne Točkovalnik ankete o funkcionalnem zdravju Najvišje število možnih točk je 72. Odgovor na vsako vprašanje v anketnem vprašalniku se vrednoti z vrednostmi od 0 do 2 in sicer: - odgovor »pogosto« 2 točki, - odgovor »včasih« 1 točko in - odgovor »redko ali nikoli« 0 točk. Od 0 do 15 točk: Ste funkcionalno zdravi in imate dovolj rezerve za prilagajanje stresnim dogajanjem v življenju. Bodite pozorni na odgovore, pri katerih ste zbrali veliko število točk. Od 16 do 30 točk: Vaše zdravje je nekoliko boljše od povprečnega, a še vedno ni optimalno. Čas je, da naredite korak naprej ter izboljšate način življenja in prehrane. Od 31 do 50 točk: Vaše zdravje je povprečno slabo. Če ne boste nemudoma ukrepali, ga boste spodkopali še bolj in lahko računate na težave, ki se bodo pokazale pozneje v življenju. Obiščite strokovnjaka za prehrano in pazite, kaj pojeste in popijete. Od 51 do 72 točk: Ste »vertikalni bolnik« in kmalu boste postali »horizontalni«, če ne boste v svoj način življenja in prehrane uvedli nekaj sprememb. Posvetujte se s strokovnjakom za prehrano, ta vam bo svetoval, s katero hrano in prehranskimi dodatki boste spet vzpostavili dobro zdravje. 1 Doseženo število točk: 1 Holford, P.: 100 % Zdravi (Prevod dela: 100 % Health).- Ljubljana: MLadinska knjiga, 2000, str. 21 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 IV. Test kazalcev toksemije Prosimo, odgovorite še na naslednja vprašanja. Nekatera so podobna tistim, na katera ste odgovorili v predhodnem testu. Naj vas to ne moti. Ta test je osredotočen na stanje v mesecu tik pred začetkom poizkusa, predhodni pa je zajemal splošno stanje funkcionalnega zdravja udeleženca (-ke). Obkrožite ustrezne odgovore Znaki toksemije Intenzivnost pojava (zastajanja strupov v * enkrat tedensko ali ** 1-2 krat ***manj kot 1 telesu) pogosteje mesečno krat mesečno 1. “Nejasna” glava a) pogosto* b) včasih** c) redko/nikoli** * 2. Nezmožnost zbrati se a) pogosto b) včasih c) redko/nikoli 3. Vzkipljivost a) pogosto b) včasih c) redko/nikoli 4. Vznemirjen želodec a) pogosto b) včasih c) redko/nikoli 5. Kronično zaprtje ali a) pogosto b) včasih c) redko/nikoli pogoste driske 6. Otrdelost v predelu a) pogosto b) včasih c) redko/nikoli ramen (lopatice) 7. Črno (temno) blato a) pogosto b) včasih c) redko/nikoli 8. Smrdeče blato a) pogosto b) včasih c) redko/nikoli 9. Neprijeten telesni vonj a) pogosto b) včasih c) redko/nikoli 10. Kronična utrujenost a) pogosto b) včasih c) redko/nikoli 11. Občutek staranja a) pogosto b) včasih c) redko/nikoli 12. Potreba po veliko spanja a) pogosto b) včasih c) redko/nikoli 13. Nespečnost a) pogosto b) včasih c) redko/nikoli 14. Uvela, postarana koža a) precej b) nekoliko c) malo/nič 15. Gube na obrazu a) precej b) nekoliko c) malo/nič 16. Zasvojenost s a) precej b) nekoliko c) malo/nič sladkarijami, kavo, cigareti, škrobnimi živili… 17. Prenajedanje a) pogosto b) včasih c) redko/nikoli 18. Pomanjkanje teka a) pogosto b) včasih c) redko/nikoli 19. Hranjenje iz navade ali a) pogosto b) včasih c) redko/nikoli “za moč” 20. Glavobol pri zbujanju a) pogosto b) včasih c) redko/nikoli Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 21. Glavobol, ki nastane, ko a) pogosto b) včasih c) redko/nikoli smo izpustili obrok, in mine, ko se najemo 22. Zamašen nos ob zbujanju a) pogosto b) včasih c) redko/nikoli 23. Katar v grlu ob zbujanju a) pogosto b) včasih c) redko/nikoli 24. Kisel, grenak ali slan okus a) pogosto b) včasih c) redko/nikoli v ustih ob zbujanju 25. Krmežljave oči ob a) pogosto b) včasih c) redko/nikoli zbujanju 26. Zamašena ušesa ob a) pogosto b) včasih c) redko/nikoli zbujanju 27. Zadah iz ust (ko se a) pogosto b) včasih c) redko/nikoli zbudimo ali pozneje) 28. Obložen jezik a) pogosto b) včasih c) redko/nikoli 29. Motne, krvave oči a) pogosto b) včasih c) redko/nikoli 30. Vrtoglavost, omotičnost a) pogosto b) včasih c) redko/nikoli Točkovanje: “pogosto” = 2 točki, Vrednotenje rezultatov testa kazalcev toksemije Čim višji je rezultat, tem višja je raven toksemije – zastajanja strupov v organizmu. “včasih” = 1 točka, Dr. Ostan priporoča naslednjo kategorizacijo rezultatov: “redko/nikoli “ = 0 točk. - 0 točk ali 1 točka: brez znakov toksemije, - od 2 do 10 točk: nizka toksemija - od 11 do 24 točk: srednja toksemija - od 25 do 42 točk : visoka toksemija -od 43 do 60 točk: zelo visoka toksemija Zaradi zastajanja strupov v telesu nastajajo mnoge bolezni. Zato je se z višanjem ravni toksemije povečuje možnost obolenj. Skupno točk:______ V. Fizični dejavniki zdravja 1. Koliko vode in svežih sokov popijete dnevno (ne čajev ali drugih napitkov): a) nič ( največ 1 dcl ) b) več kot 0,1 do 0,5 litra c) več kot 0,5 do 1,0 litra d) več kot 1,0 do 1,5 litra e) več kot 1,5 do 2,0 litra f) več kot 2 litra (navedite koliko):________ l 2. Koliko časa dnevno porabite za naslednje aktivnosti v običajnem (delovnem ) dnevu: manj kot 15 0,5 ure 1 uro 1,5 ure 2 uri 2,5 ure ali min več Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Za telesno gibanje (intenzivna x x x x x x hoja, tek, delo na vrtu…) Bivanje na zraku x x x x x x Bivanje na sončni svetlobi (ne x x x x x x v avtu ipd) 3. Sredi dneva si privoščim počitek oz. sprostitev: a) vsak dan /pogosto b) včasih c) redko/nikoli 6. Običajno (če nimam nočne izmene…) grem spat a) pred 22. uro b) med 22. in 23. uro c) med 23. in 24. uro d) med polnočjo in eno uro e) po eni uri zjutraj 7. Običajno spim ____________ ur dnevno. 8. Moje spanje je praviloma: a) dobro (spim globoko in se zbudim spočit-a) b) slabo 9. Ali ste se zadnja 2 dneva ukvarjali s športom. Navedite s katerim, kako intenzivno in koliko časa:___________________________________________________________________________________ _______________________________________________________________________________________ ________________________ 10. Ali kadite? Da, ne Koliko cigaret na dan?______________________ VI. Prehrana 1. Koliko obrokov hrane užijete dnevno: a) en b) dva c) tri d) štiri e) pet f) šest ali več 2. Moj glavni (največji) obrok je: a) zajtrk b) kosilo c) večerja d) kosilo in večerja združena v enem obroku e) drugo : _____________________ 3. Spat grem s polnim želodcem: a) vsak dan /pogosto b) včasih c) redko d) nikoli 4. Moja prehrana je: a) mešana b) vegetarijanska Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 c) veganska (samo hrana rastlinskega izvora) d) presna (samo hrana, ki ni toplotno obdelana) e) drugo (navedite)__________________________________________________ 5. Meso (vključno mesnini izdelki in ribami) jem: a) vsak dan v več obrokih b) vsak dan v enem od obrokov c) večkrat na teden d) redko (enkrat na teden ali redkeje) e) nikoli. 6. Surovo sadje in zelenjavo uživam: a) 5 krat na dan ali več b) 2 - 4 krat na dan c) enkrat dnevno d) nekajkrat na teden e) redko/ ne uživam 7. Surova hrana (sadje, zelenjava, jedrca…) predstavlja v moji prehrani: a) veliko večino ali celoto b) večino c) polovico d) manjši del e) zelo majhen del ali nič 8. Sveže stisnjene sadne ali zelenjavne sokove uživam: a) vsak dan b) nekajkrat tedensko e) včasih f) nikoli (zelo redko) 9. Moja prehrana je: a) pestra, b) enolična/ pomanjkljiva. 10. Običajno jem: a) preveč, b) ravno pravo količino, c) premalo. 11. Sladkarije (npr. čokolado , kekse, slaščice…) uživam: a) vsak dan b) večkrat na teden c) enkrat na teden d) včasih/ redko e) nikoli 12. Kako pogosto se odločate za čistilne prehranske dneve oz. shujševalne kure? Vrsta prehranskega režima Za to vrsto omejevalne prehrane se odločam: 1. Shujševalni dnevi (tudi s a) pogosto b) včasih c) redko/nikoli kuhano hrano) (enkrat mesečno ali pogosteje) Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 2. Čistilni dnevi s surovim a) pogosto b) včasih c) redko/nikoli sadjem in zelenjavo 3. Post ob svežih sokovih ali vodi a) pogosto b) včasih c) redko/nikoli 13. Prehranske dodatke (npr. vitamine, minerale) uživam: a) vsak dan b) vsak teden (enkrat ali nekajkrat) c) včasih/redko d) ne uživam jih. Navedite katere:_______________________________________ VII. Drugi dejavniki zdravja 1. Kako pogosto užijete naslednje snovi? Vrsta snovi Pogostnost Kava b) vsak teden c) d) nikoli a) vsak dan_____skodelic (vsaj enkrat) včasih/redko Črni ali zeleni čaj b) vsak teden c) d) nikoli a) vsak dan_____skodelic (vsaj enkrat) včasih/redko b) vsak teden c) d) nikoli Alkoholne pijače včasih/redko a) vsak dan ____ enot* b) vsak teden c) d) nikoli včasih/redko Kajenje a) vsak dan ____ cigaret Zadrževanje v b) vsak teden c) d) nikoli zakajenem prostoru včasih/redko a) vsak dan_____ ur Zdravila b) vsak teden c) d) nikoli včasih/redko a) vsak dan_____ tablet Dodatki k prehrani b) vsak teden c) d) nikoli včasih/redko a) vsak dan_____ tablet Kontracepcijske DA/NE tablete Opomba: * ena enota alkohola=1dcl vina=0,3 dcl žgane pijače= 3 dcl piva Telesna višina: ______ cm Telesna teža : _______ BMI (teža v kg /(višina v m)2 ) ______ (od 18,5 do 24,9 = normalno; 30 in več = debelost) Ali imate trenutno menstruacijo DA...NE. Datum: ________________________ 2 .Morebitni predlogi, pripombe, komentarji: Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Zahvaljujemo se vam, da ste sodelovali v preizkusu. Prosimo, oddajte vprašalnik enemu od nosilcev študije. Včasih se po preizkusu pojavi potreba, da udeležencu postavimo še kakšno vprašanje. Če nimate nič proti, vas zato prosimo, da napišete svoje ime in priimek ter naslov. Ti podatki bodo na voljo le nosilcem raziskave, rezultati testiranja pa bodo uporabljeni le v študijske namene. Ime in priimek: _____________________________ Naslov: _______________________________________________________________________________________ ________ e-naslov: ____________________________________________________ tel. _____________________________________ Datum: _______________________ Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ANNEX D ICP-MS operating parameters Parameter Type/Value Sample introduction Nebuliser Miramist Spray chamber Scott Skimmer and sampler Ni Plasma conditions Forward power 1550 W Plasma gas flow (Ar) 15.0 L/min Carrier gas flow (Ar) 1.05 L/min Dilution gas flow (Ar) 0.10 L/min Collision gas flow (He) 4.5 mL/min Oct bias -100 V Cell entrance -100 V Cell exit -150 V Deflect -75 V Plate bias -150 V Sample uptake rate 0.3 mL/min Data acquisition parameters Isotopes monitored 47Ti, 52Cr, 56Fe, 59Co, 60Ni, 95Mo Isotopes of internal standards 72Ge, 103Rh Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ANNEX E Release of metal ions from SS, Ni-Ti, β-Ti and Co-Cr alloys Stainless steel archwire Ti Cr Fe Co Ni Mo (Damon - Ormco, USA) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h <0.1 0.36 3.59 <0.01 0.50 <0.05 24 h <0.1 <0.05 2.28 <0.01 <0.05 <0.05 48 h <0.1 0.23 3.00 <0.01 0.36 <0.05 7 days <0.1 0.22 6.14 <0.01 0.21 <0.05 30 days <0.1 0.25 6.36 <0.01 0.23 <0.05 60 days <0.1 0.17 7.41 <0.01 <0.05 <0.05 90 days <0.1 0.58 13.36 <0.01 2.11 <0.05 Ni-Ti archwire (rematitan® super elastic Ti Cr Fe Co Ni Mo - Dentaurum, Germany) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h 0.73 <0.05 <0.1 <0.01 0.73 <0.05 24 h 0.50 <0.05 <0.1 <0.01 1.43 <0.05 48 h 1.87 <0.05 <0.1 <0.01 2.22 <0.05 7 days 1.30 <0.05 <0.1 <0.01 3.01 <0.05 30 days / / / / / / 60 days 2.74 <0.05 <0.1 <0.01 6.10 <0.05 90 days 9.59 <0.05 <0.1 <0.01 12.97 <0.05 Ni-Ti archwire Ti Cr Fe Co Ni Mo (Biostarter® - Forestadent, Germany) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h 0.52 <0.05 <0.1 <0.01 24.59 <0.05 24 h 0.50 <0.05 <0.1 <0.01 41.81 <0.05 48 h 2.28 <0.05 <0.1 <0.01 60.62 <0.05 7 days <0.1 <0.05 <0.1 <0.01 44.23 <0.05 30 days 5.27 <0.05 <0.1 <0.01 94.03 <0.05 60 days 10.07 <0.05 <0.1 <0.01 131.93 <0.05 90 days 9.53 <0.05 <0.1 <0.01 116.66 <0.05 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Ti-Mo archwire (rematitan® SPECIAL Ti Cr Fe Co Ni Mo - Dentaurum, Germany) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h <0.1 <0.05 <0.1 <0.01 <0.05 <0.05 24 h 1.04 <0.05 <0.1 <0.01 <0.05 <0.05 48 h 1.64 <0.05 <0.1 <0.01 <0.05 <0.05 7 days 0.76 <0.05 <0.1 <0.01 <0.05 <0.05 30 days 2.39 <0.05 <0.1 <0.01 <0.05 <0.05 60 days 1.12 <0.05 <0.1 <0.01 <0.05 <0.05 90 days 8.26 <0.05 <0.1 <0.01 <0.05 0.45 Co-Cr-Ni archwire (Elgiloy® Ti Cr Fe Co Ni Mo - Rocky Mountain Orthodontics, USA) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h <0.1 0.16 1.27 6.21 0.90 0.27 24 h <0.1 0.42 4.59 9.43 2.02 0.78 48 h <0.1 0.85 8.29 14.49 3.79 1.28 7 days <0.1 0.52 4.08 13.54 3.37 1.02 30 days <0.1 1.23 11.68 24.74 6.80 1.95 60 days <0.1 1.58 13.84 30.46 8.29 2.47 90 days <0.1 1.66 13.77 31.45 8.55 2.54 Co-Cr-Ni archwire (Remaloy® Ti Cr Fe Co Ni Mo - Dentaurum, Germany) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h <0.1 <0.05 <0.1 8.96 1.86 <0.05 24 h <0.1 0.34 1.42 13.27 3.77 <0.05 48 h <0.1 0.92 6.67 13.38 3.94 <0.05 7 days <0.1 0.42 1.62 17.02 5.06 <0.05 30 days <0.1 1.22 4.74 28.68 10.14 <0.05 60 days <0.1 1.40 6.80 31.56 11.07 <0.05 90 days <0.1 1.15 4.66 28.98 9.09 <0.05 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Stainless steel brackets (Discovery® Ti Cr Fe Co Ni Mo - Dentaurum, Germany) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h <0.1 0.82 8.27 <0.01 1.10 <0.05 24 h <0.1 1.05 13.96 <0.01 1.72 0.15 48 h <0.1 2.64 39.45 <0.01 4.19 0.70 7 days <0.1 1.99 70.07 <0.01 3.89 0.47 30 days <0.1 4.50 126.48 <0.01 10.07 1.25 60 days <0.1 6.36 178.31 <0.01 22.29 1.66 90 days <0.1 6.92 200.25 <0.01 37.03 1.83 Stainless steel molar bands (W Ti Cr Fe Co Ni Mo -Fit Form Forestadent, Germany) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 12 h <0.1 1.01 23.02 <0.01 1.85 <0.05 24 h <0.1 3.73 36.85 <0.01 4.01 <0.05 48 h <0.1 1.52 45.30 <0.01 3.45 <0.05 7 days <0.1 7.01 67.99 <0.01 7.67 <0.05 30 days <0.1 2.72 70.64 <0.01 6.59 <0.05 60 days <0.1 3.13 70.84 <0.01 7.73 <0.05 90 days <0.1 7.44 113.98 <0.01 11.31 <0.05 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ANNEX K Published article Kovač et al., 2019 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ANNEX M Published article Kovač et al., 2020 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 ANNEX N Published article Kovač et al., 2021 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Kovač V. Occurrence and effects of oxidative stress induced by metal materials from fixed orthodontic appliances. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical faculty, 2022 Document Outline KEY WORDS DOCUMENTATION KLJUČNA DOKUMENTACIJSKA INFORMACIJA LIST OF TABLES LIST OF FIGURES ABBREVIATIONS AND SYMBOLS 1 INRODUCTION 1.1 PROBLEM DESCRIPTION 1.2 RESEARCH GOALS 2 LITERATURE REVIEW 2.1 FIXED ORTHODONTIC APPLIANCES 2.1.1 Biocompatibility 2.1.2 Orthodontic alloys 2.1.3 Nanotechnology in orthodontics 2.1.3.1 Antibacterial activity 2.1.3.2 Reduction of friction 2.1.3.3 Increase in strength 2.2 OXIDATIVE STRESS 2.2.1 Reactive oxygen species 2.2.2 Metal ions and ROS 2.2.3 Nanomaterials and ROS 2.2.4 Molecular targets of ROS 2.2.4.1 Lipid oxidation 2.2.4.2 Nucleic acid oxidation 2.2.4.3 Protein oxidation 2.2.5 Cellular Antioxidative defense system 2.2.5.1 Enzymatic defense 2.2.5.1.1 Superoxide dismutase 2.2.5.1.2 Catalase 2.2.5.1.3 Glutathione peroxidase 2.2.5.1.4 Glutathione reductase 2.2.5.1.5 Peroxiredoxin 2.2.5.1.6 Thioredoxin reductase 2.2.5.2 Non-enyzmatic defense 2.2.5.2.1 Glutathione 2.3 MODEL ORGANISM AND CELL LINE 2.3.1 Yeast Saccharomyces cerevisiae 2.3.2 Human gingiva fibroblast cell line 3 MATERIALS AND METHODS 3.1 RESEARCH WORK FLOW 3.2 SYSTEMIC OXIDATIVE STRESS PARAMETERS IN PATIENTS DURING ORTHODONTIC TREATMENT WITH FIXED APPLIANCES 3.2.1 Ethical approval 3.2.2 Subjects and cohort design 3.2.3 Insertion of the orthodontic appliance 3.2.4 Capillary blood collection 3.2.5 Free oxygen radical test 3.2.6 Free oxygen radical defense 3.3 TYPE AND AMOUNT OF OF METAL IONS RELEASED FROM ORTHODONTIC ALLOYS 3.3.1 Orthodontic materials 3.3.2 In vitro conditions 3.3.3 Inductively coupled plasma mass spectrometry analysis 3.3.3.1 Released metal ions in saliva 3.3.3.2 Metal alloy composition 3.3.3.3 Metal ion concentration measurement 3.4 OXIDATIVE STRESS IN YEAST CELL MODEL 3.4.1 Yeast cultures 3.4.2 Metal ion mixtures and yeast treatment 3.4.3 Cell viability 3.4.3.1 Cell culturability 3.4.3.2 Metabolic activity of the cells 3.4.4 ROS level determination 3.4.4.1 Determination of ROS content 3.4.4.1.1 Modification of the protocol 3.4.5 Enzymatic antioxidant defense 3.4.5.1 Cell lysate preparation 3.4.5.2 Superoxide dismutase activity 3.4.5.3 Catalase activity 3.4.5.4 Glutathione peroxidase activity 3.4.5.5 Glutathione reductase activity 3.4.5.6 TrxR activity 3.4.5.7 Peroxiredoxine activity 3.4.5.8 In-gel enzyme activity of SOD and CAT 3.4.5.8.1 Electrophoresis gels 3.4.5.8.2 Electrophoresis buffers 3.4.5.8.3 Loading of samples and electrophoresis 3.4.5.9 SOD in-gel activity staining 3.4.5.9.1 CAT in-gel activity staining 3.4.6 Oxidative damages 3.4.6.1 Oxidative lipid damages 3.4.6.2 Oxidative protein damages 3.5 OXIDATIVE STRESS IN HGF 3.5.1 HGF cell line 3.5.2 Nanoparticle characterization and preparation for treatment 3.5.3 HGF cell viability 3.5.3.1 Resazurin assay 3.5.3.2 Neutral red uptake assay 3.5.3.3 Coomassie Blue Assay 3.5.3.4 The trypan blue cellular debris assay 3.5.4 ROS level determination 3.6 STATISTICAL ANALYSIS 4 RESULTS WITH DISCUSSION 4.1 CHANGES IN OXIDATIVE STRESS PARAMETERS IN THE CAPILLARY BLOOD 4.2 METAL ION RELEASE FROM DIFFERENT ORTHODONTIC ALLOYS 4.3 CAUSATION OF OXIDATIVE STRESS BY METAL IONS IN S. CEREVISIAE 4.3.1 Culturability 4.3.2 Metabolic activity 4.3.3 Intracellular ROS level 4.3.4 Lipid oxidation 4.3.5 Antioxidative defense 4.3.6 Protein oxidation 4.4 THE EFFECT OF METAL MIXTURES ON HGF CELL LINE 4.5 EFFECT OF NANOPARTICLE EXPOSURE TO HGF CELL LINE 4.5.1 Nanoparticle characteristics 4.5.2 Cytotoxicity of NPs 4.6 STUDY LIMITATIONS AND FUTURE PERSPECTIVES 5 CONCLUSIONS 6 SUMMARY (POVZETEK) 6.1 SUMMARY 6.2 POVZETEK 7 REFERENCES ACKNOWLEDGEMENTS ANNEXES