Search

Frangula alnus (krušina) ima dugu istoriju korišćenja u tradicionalnoj medicini, a u ovom radu su ispitana njena određena nova svojstva, naročito antibiofilm aktivnost. Pripremljen je etil-acetatni ekstrakt kore krušine koji je hemijski okarakterisan te su ispitane njegova biološke aktivnosti. GC×GC/MS analiza je pokazala da je u ekstraktu dosta zastupljena 4-etoksi-benzoeva kiselina, , dok je LC-MS/MS analiza pokazala da je bogat fenolnim i flavonoidnim jedinjenjima među kojima je najdominantniji emodin koji je odabran za dalje testiranje uz ekstrakt. Pokazano je da ekstrakt ima snažnu antioksidantnu aktivnost za razliku od emodina. MIK vrednosti su pokazale da su svi testirani sojevi Staphylococcus aureus izuzetno osetljivi na delovanje ekstrakta i emodina, posebno klinički izolati. Rezultati ispitivanja antibiofilm aktivnosti su pokazali da ekstrakt i emodin imaju izuzetno snažan efekat na inhibiciju formiranja biofilmova sojeva S. aureus, ali da je efekat na već formirane biofilmove slabiji. Pokazano je da obe test supstance imaju inhibitorni efekat na koncentraciju ekstracelularnih polisaharida, da menjaju količinu eDNK u biofilmu kao i broj ćelija perzistera, ali i deluju na metabolizam ćelija. SEM i AFM mikroskopija su potvrdile antibiofilm efekat ukazujući na promene u morfologiji, strukturi i hrapavosti biofilmova. Dodatno, obe supstance su modulisale ekspresiju icaA, icaD, srrA, srrB gena i RNA III. Rezultati MTT testa su ukazali na jaku citotoksičnu aktivnost ekstrakta i emodina prema HepG2 i HCT 116 ćelijskim linijama, dok na imortalizovanoj MRC-5 liniji nije bilo efekta. Međutim, pokazano je da supstance remete ćelijski ciklus svih linija, kao i da indukuju apoptozu i nekrozu i remete njihov mitohondrijski membranski potencijal. Obe test supstance su ispoljile snažan genotoksični efekat na svim odabranim ćelijskim linijama. Frangula alnus (alder buckthorn) has a long history of use in traditional medicine, thus in this study its certain new properties, especially antibiofilm, were examined. Ethyl-acetate extract of buckthorn bark was prepared, chemically characterized and its biological activities were examined. GC×GC/MS analysis showed that 4-ethoxy-benzoic acid, is abundant in the extract, while LC-MS/MS analysis showed that extract is rich in phenolic and flavonoid compounds, with emodin as dominant one, which was selected for further testing along with extract. It was shown that extract has strong antioxidant activity unlike emodin. MIC values showed that all tested strains of Staphylococcus aureus were extremely sensitive to the action of the extract and emodin, especially clinical isolates. The results of the study of antibiofilm activity showed that the extract and emodin have an extraordinary inhibitory effect on biofilm formation of S. aureus strains, but that the effect on already formed biofilms is less pronounced. It was demonstrated that both test substances have inhibitory effect on the concentration of extracellular polysaccharides, alter the amount of eDNA in the biofilms as well as the number of persister cells, but also affect cell metabolism. SEM and AFM microscopy confirmed the antibiofilm effect, indicating changes in the morphology, structure and roughness of the biofilms. Additionally, both substances modulated the expression of icaA, icaD, srrA, srrB genes and RNA III. The results of the MTT test indicated strong cytotoxic activity of the extract and emodin against HepG2 and HCT 116 cell lines, while there was no effect on the healthy MRC-5 line. However, the substances disturbed the cell cycle of all cell lines, as well as induced apoptosis and necrosis and modulated mitochondrial membrane potential. Both test substances showed a strong genotoxic effect on all selected cell lines.

Policiklični aromatični ugljovodonici (engl. Polycyclic Aromatic Hydrocarbons, PAHs) predstavljaju grupu organskih jedinjenja koja se sastoje od dva ili više kondenzovanih benzenovih prstenova. Istraživanja ukazuju da PAH-ovi imaju kancerogen i mutagen uticaj na ljude i druge žive organizme. Perzistentni su i bioakumulativni i kao takvi predstavljaju opasnost po životnu okolinu. Dodatni problem predstavljaju proizvodi degradacije policikličnih aromatičnih ugljovodonika, koji nastaju tokom prirodne transformacije ili fizičko-hemijskih tretmana za njihovo uklanjanje. Novoformirana jedinjenja često imaju izraženiji toksični uticaj na ljude i druge žive organizme u poređenju sa polaznim jedinjenjem. Zato se posebna pažnja posvećuje razvoju novih metoda za njihovo što bezbednije uklanjanje. Do sada su razvijene metode za analizu sledećih kontaminiranih medijuma: voda, zemljište, vazduh, sedimenti i dr. Kako je zagađenost PAH-ovima u životnoj sredini rasla, uglavnom usled antropogenih faktora, tako su se pojavili i na drugim mestima, poput betona u stambenim zgradama. Cilj istraživanja doktorske disertacije je razvijanje nove, efikasne, neinvazivne metode za uklanjanje PAH-ova iz betona, koja je zasnovana na elektrohemijskoj oksidaciji. Da bi se osigurala bezbednost metode za praktičnu primenu, posebna pažnja posvećena je proceni toksičnosti PAH-a i njihovih produkata razgradnje nastalih tokom procesa elektrooksidacije. Kao laboratorijski model sistem za elektrohemijsku degradaciju PAH-ova koristi se elektrolitička ćelija sa niklenim elektrodama. Praćenje koncentracije PAH-ova u betonu izvedeno je primenom novopostavljene i validirane metode na uređaju tečni hromatograf ultravisokih performansi sa fotodiodnim detektorom (UPLCPDA). Efikasnost uklanjanja za pojedine PAH-ove dostiže vrednost i preko 90 %. Za procenu toksičnosti elektrohemijskog tretmana određuje se citotoksični i genotoksični potencijal na limfocitima i eritrocitima periferne krvi čoveka. Testoviobuhvataju određivanje učestalosti mikronukleusa i proliferacioniog potencijala ćelija, ispitivanje antioksidativne zaštite enzima (katalaze) kao i uticaja na oksidativni stres (određivanjem sadržaja malondialdehida). Rezultati ispitivanja potvrđuju toksičnost početne smeše PAH-ova - pre elektrohmijske oksidacije - uzrokujući značajno povećanje niva DNK oštećenja, smanjujući potencijal ćelija za deobu i indukujući oksidativni stres što se ogleda kroz povećanje malondialdehida u procesu lipidne peroksidacije ćelijskih membrana i povećanju aktivnosti katalaze, enzima antioksidativne odbrane. Procesom elektrohemijske oksidacije PAH-ova nastaju produkti koji ispoljavaju različit stepen citotoksičnosti zavisno od vremena nastanka. Najtoksičniji efekti su utvrđeni u prvih 15 minuta što se manifestuje kroz povećanje učestalosti genomskih ozleda, suprimiranje proliferativnog potencijala i/ili indukciju oksidativnog stresa, kao i značajanu modifikaciju aktivnosti enzima antioksidativne zaštite. Nakon 60 minuta produkti oksidacije su manje citotoksični u odnosu na početno jedinjenje, ali i dalje su toksičniji u poređenju sa netretiranom kontrolom. Polycyclic Aromatic Hydrocarbons (PAHs) are a group of organic compounds consisting of two or more fused benzene rings. Research indicates that PAHs have a carcinogenic and mutagenic effect on humans and other living organisms. They are persistent and bioaccumulative and, as such, pose a threat to the environment. Products that are formed upon natural degradation or physicochemical procedures for their removal represent a particular problem. Newly formed compounds often have a more pronounced toxic effect on humans and other living organisms compared to the initial compound. Consequently, the development of new methods for their safe removal has been the subject of extensive research. So far, methods for efficient PAH removal from the various media were developped, including: water, soil, air, sediments, etc. As the environmental pollution by PAHs is continuously increasing due to the various, mainly anthropogenic factors, they also have occurred in other places, such as concrete in residential buildings. The aim of the doctoral dissertation is to develop novel, efficient, noninvasive method for PAHs removal in concrete, based on electrochemical oxidation. In order to ensure safeness of the method for practical application, particular attention was paid to the estimation of the toxicity of PAHs and their degradation products generated during the electrooxidation process. Electrochemical cell consisting of two nickel electrodes was used for the laboratory model of PAH oxidation in concrete. Concentration of PAHs in concrete was monitored with the novel, particularly developed and validated method, based on ultra performance liquid chromatography (UPLC) with photo-diode array (PDA) detector. The removal efficiency for individual PAHs exceeds 90 %. Estimation of the toxicity of electrochemically treated samples was performed by determination of the cytotoxic and genotoxic potential of human peripheral blood lymphocytes and erythrocytes. The tests included determination the micronucleusfrequency and the proliferative potential of cells, determining the antioxidant protection of enzyme catalase, as well as the impact on oxidative stress (by determining the content of malondialdehyde). The results of the study confirm the toxicity of initial PAHs showing that - before electrochemical oxidation - they significantly increase the level of DNA damage, reduce the potential of cells to divide and induce oxidative stress, which is reflected by the increase of malondialdehyde in the process of lipid peroxidation of cell membranes and the increase of the activity of catalase, an antioxidant defense enzyme. Products which are formed during electrochemical oxidation of PAHs exhibit different degrees of cytotoxicity depending on the time of their formation. The most toxic effects were determined in the first 15 minutes, which is manifested by an increased incidence of genomic lesions, suppression of proliferative potential and/or induction of oxidative stress, as well as substantial modification of the antioxidant enzyme activity. After 60 minutes, the oxidation products are less cytotoxic than the parent compound, but still more toxic compared to the untreated control.

U ovoj doktorskoj disertaciji ispitan je uticaj antitumorskih mononuklearnih i binuklearnih kompleksa zlata(III) (AubipyC, Aubipy(OH)2, Aupy(OAc)2, Auoxo6, Au2bipyC i Au2phen) na Na+/K+-ATPazu, kao i uticaj polioksometalata, 12- volframsilicijumove i 12-volframfosforne kiseline (WSiA i WPA) na acetilholinesterazu (AChE). Primenom različitih fizičkohemijskih metoda (UV/Vis i fluorescentna spektroskopija, cirkularni dihroizam, elektronska paramagnetna rezonantna (EPR) spektroskopija) u kombinaciji sa molekulskim modelovanjem, dobijen je detaljan uvid u njihovu interakciju na molekulskom nivou. Ispitivanjem uticaja ovih jedinjenja na antioksidativne enzime i na nivo malondialdehida, procenjeni su njihovi toksični potencijali, a merenjem učestalosti mikronukleusa i indeksa ćelijske proliferacije procenjeni su njihovi genotoksični i citotoksični efekti... In this doctoral dissertation, the interaction of antitumor mononuclear and binuclear gold(III) complexes (AubipyC, Aubipy(OH)2, Aupy(OAc)2, Auoxo6, Au2bipyC and Au2phen) with Na+/K+-ATPas,e as well as the interaction of polioxotungstates, 12- tungstosilicic acid and 12-tungstophosphoric acid (WSiA and WPA), with acetylholinesterase have been investigated. Various spectroscopic methods (UV/Vis and fluorescent spectroscopy, circular dichroism, electron paramagnetic resonance (EPR) spectroscopy) in combination with docking studies were used in order to obtain detailed insight into their interaction on the molecular level. By investigating the influence of these compounds on the antioxidative enzymes and the level of malondialdehyde, their toxic potentials were evaluated. Furthermore, their genotoxic and cytotoxic effects were estimated by measuring the incidence of micronuclei and the cytokinesis-block proliferation index in human lymphocyte cultures...

U ovoj tezi su ispitani toksični efekti organo-tiofosfatnih insekticida i proizvoda njihove degradacije primenom različitih metoda kojima se mere parametri toksičnosti. Degradacijom organo-tiofosfatnih insekticida, do koje dolazi tokom fizičko-hemijskih procesa za uklanjanje organskih zagađivača iz voda, delovanjem faktora životne sredine, tokom dužeg skladištenja preparata insekticida ili sterilizacijom hrane, nastaju različita jedinjenja koja često poseduju jači toksični potencijal od polaznih insekticida. Proizvodi degradacije nisu ciljani analiti tokom kontrole prisustva insekticida, a i njihova detekcija primenom klasičnih analitičkih metoda je skupa, komplikovana i dugotrojna i ne može da predvidi toksične efekte na osnovu identifikovanog sastava. Znatno pogodnija je primena bioeseja koji detektuju prisustvo toksičnih proizvoda degradacije na osnovu promene odgovarajućih parametara i istovremeno daju informaciju o njihovom toksičnom potencijalu. Za procenu neurotoksičnog potencijala ispitivanih organofosfatnih jedinjenja i njihovu detekciju modifikovan je standardni esej na bazi acetilholinesteraze, koji je unapredjen u cilju snižavanja limita detekcije organofosfata. Konstruisan je i protočni biosenzor sa imobilizovanom acetilholinesterazom koja se može koristiti više puta tokom nekoliko meseci, što ovaj sistem čini ekonomičnijim u odnosu na šaržni esej. U cilju povaćanja osetljivosti standardnog eseja i biosenzora na prisustvo organo-tiofosfata, korišćeno je oksidaciono sredstvo, N-bromo sukcinimid koji tio oblike prevodi u okso koji dovode do pada aktivnosti enzima pri znatno nižim koncentracijama. Testirano je i simultano dejstvo organo-tiofosfata i njihovih okso i izo analoga. U tezi su analizirani diazinon, malation, hlorpirifos, organo-tiofosfati koji se primenjuju kao preparati insekticida, i proizvodi njihove oksidacije, hidrolize i izomerizacije. Organo-tiofosfatni insekticid diazinon je tretiran UV svetlošću, koja se koristi za uklanjanje organskih zagađivača u procesima za prečišćavanje voda. Hromatografski je praćena razgradnja polaznog jedinjenja i nastajanje proizvoda hidrolize tokom 115 minuta ozračivanja diazinona. Ispitano je neurotoksično, prooksidativno, genotoksično i citotoksično dejstvo ozračenih rastvora, kao i samog diazinona i identifikovanog proizvoda njegove fotohemijske degradacije... Toxic effects of organo-thiophosphorous insecticides i their degradation products have been investigated in this dissertation using various methods for the determination of toxicity parameters. Organo-thiophosphorous insecticides can be degraded during physico-chemical processes for the removal of organic pollutants from wastewaters, insecticides storage, food sterilisation, as well as under the influence of environmental factors. The formed degradation products are not the aimed analytes during insecticides control, and their detection using analytical methods is expensive, complicated and longterm. Additionally, toxic effects of the identified compounds cannot be evaluated. Therefore, the application of bioassays, which detect the degradation products on the basis of the modification of appropriate parameters and give the information about their toxic potential, is considerably suitable. The standard acetylcholinesterase assay has been modified for the evaluation of neurotoxic potential of the investigated organophosphorous compounds and the reduction of their limit of detection. Besides, flow injection analysis biosensor based on immobilized acetylcholinesterase has been constructed. The immobilized enzyme can be used more times during several months, making the biosenzor more economical related to the batch system. In order to increase the sensitivity of the biosenzor, N-bromosuccinimide has been used. Actually, this oxidant transforms thio forms of organophosphates to oxo analogs causing the decrease of the enzyme activity at lower concentrations. The simultaneous influence of the organo-thiophosphates and their oxo and iso analogs has been investigated as well. In this thesis, diazinon, malathion, chlorpyrifos-organo-thiophosphates being applied as insecticides, and the products of their oxidation, hydrolysis and isomerisation have been tested. Organo-thiophosphorous insecticide diazinon has been treated with UV light that is applied in purification processes for the removal of organic pollutants from wastewaters. The decomposition of the parent organo-thiophosphate and forming the hydrolysis product during 115 minute irradiation has been monitored using chromatographic methods. It has been investigated neurotoxic, prooxidative, genotoxic and citotoxic effects of the irradiated diazinon solutions, as well as various concentrations of diazinon and the identified product of diazinon photochemical degradation...

Ugljenicne nanocevi (CNT) predstavljaju jedinstvene jednodimenzione makromolekule. Obzirom da zahvaljujuci svojoj karakteristicnoj strukturi pokazuju neobicne osobine, te da postoje mogucnosti njihove primene u razlicitim oblastima, to je razvoj i unapreenje metoda za dobijanje novih materijala na bazi ugljenicnih nanocevi od velikog znacaja. Cilj istraživanja u okviru ove disertacije je ispitivanje razlicitih metoda precišcavanja ugljenicnih nanocevi, razvoj nove metode za funkcionalizaciju ugljenicnih nanocevi klasterima srebra cime bi se dobili novi funkcionalizovani nano materijali, te ispitivanje genotoksicnosti ugljenicnih nanocevi. Za precišcavanje ugljenicnih nanocevi primenjene su dve razlicite metode. Prva metoda predstavlja kombinaciju žarenja i tretiranja kiselinom, a druga se zasniva na korišcenju polimera. Za karakterizaciju uzoraka pre i posle precišcavanja korišcena je SEM mikroskopija. U cilju uspešne funkcionalizacije ugljenicnih nanocevi klasterima srebra, primenjena je jednostavna i efikasna jednostepena metoda funkcionalizacije jednoslojnih ugljenicnih nanocevi (SWCNT) i višeslojnih ugljenicnih nanocevi (MWCNT) korišcenjem gama zracenja. Po prvi put, poli(vinil alkohol) (PVA) je uspešno korišcen za redukciju metalnih jona srebra bez prisustva bilo kog drugog redukujuceg agensa. Funkcionalizacija se odvija vezivanjem polimera za površinu CNT i simultanom redukcijom Ag+ jona pod dejstvom gama zracenja. Osim razvoja metode za funkcionalizaciju CNT cilj istraživanja je bio i da se ispita potencijal dva razlicita hemijska puta funkcionalizacije i redukciona sposobnost razlicitih redukcionih vrsta nastalih pod dejstvom gama zracenja. Opticke osobine dobijenih uzoraka i mehanizam funkcionalizacije CNT su ispitani korišcenjem UVvis i FTIR spektroskopije. Funkcionalizovane CNT su vizuelizovane korišcenjem mikroskopskih tehnika TEM i STM. Prisustvo Ag u uzorcima funkcionalizovanih nanocevi je potvreno i korišcenjem EDX tehnike. Da bi se ispitala stabilnost i velicina radioliticki dobijenih klastera srebra kojim su funkcionalizovane CNT korišcena je metoda MALDI TOF masene spektrometrije i vršena su teorijska ab initio izracunavanja. Osim potvrde o stabilnosti dobijenih klastera, MALDI TOF masena spektrometrija ukazuje na odreene klastere posebne stabilnosti što je vezano za elektronsku konfiguraciju klastera, što su potvrdila i teorijska izracunavanja... Carbon nanotubes (CNT) represent unique one-dimensional macromolecules. Since due to its characteristic structure they are showing unusual properties and that there are possibilities of their application in various fields, the development of methods for obtaining new materials based on carbon nanotubes is of great importance. The goal of investigation in this thesis is an investigation of different carbon nanotube purification methods, development of a new method of functionalization of carbon nanotubes with silver clusters with the aim of obtaining new functionalized nanomaterials, and the investigation of genotoxicity of carbon nanotubes. Two different methods of carbon nanotubes purification were applied. First method is a combination of etching and acid treatment, and the other method is based on using of polymer. For characterization of the samples before and after purification, SEM microscopy was used. With the aim of successful functionalization of carbon nanotubes with silver clusters, simple and efficient one-step functionalization method of single walled carbon nanotubes (SWCNT) and multi walled carbon nanotubes (MWCNT) using gamma irradiation was used. For the first time, poly(vinyl alcohol) PVA is successfully utilized to reduce silver (Ag) metal ions without having any additional reducing agents. Functionalization takes place through anchoring of the polymer to the surface of CNT and simultaneous reduction of Ag+ ions under the -irradiation. Beside development of the method for functionalization of CNTs the aim of this investigation was also to investigate the potential of two different chemical routes and the reduction ability of different reduction species produced under the -irradiation. Optical properties of as-prepared samples and CNT functionalization mechanism were investigated using UV–vis and FTIR spectroscopy. Functionalized CNT were visualized using microscopic techniques: transmission electron microscopy (TEM) and scanning tunneling microscopy (STM). Also, the presence of Ag in the samples of functionalized carbon nanotubes was confirmed using energy dispersive X-ray spectroscopy. In order to investigate the size and the stability of radioliticaly produced silver clusters which functionalized CNTs, MALDI TOF mass spectrometry method and theoretical ab initio calculations were applied. Except the information about the stability of obtained clusters, MALDI TOF mass spectrometry indicates some clusters of particular stability, related to the cluster’s electronic configuration, which was also confirmed by theoretical calculations...

U ovom radu cilj istraživanja je bilo ispitivanje primarnih oštećenja DNK izolovanih limfocita čoveka pod uticajem adrenalina i efedrina. Oštećenja DNK su evaluirana primenom in vitro Komet testa. Ispitivan je širok spektar koncentracija adrenalina i efedrina (u rasponu od 0,0005 μM do 500 μM) u različitim vremenskim intervalima (15 min, 60 min, 120 min, 240 min i 24 sata). Najizraženije oštećenje DNK ustanovljeno je nakon 15 min tretmana adrenalinom, pri čemu su sve koncetracije izuzev najniže (0.0005 μM) dovele do povećane migracije DNK. Nakon 60 min, 120 min, 240 min tretmana adrenalinom indukovano je oštećenje DNK u opsegu od 5 do 300 μM. Najslabiji efekat ispoljen je nakon 24 sata, tako da su samo najviše koncetracije adrenalina (150 μM i 300 μM) dovele do povećanog stepena oštećenja DNK. Radi utvrđivanja mogućeg učešća reaktivnih kiseoničnih vrsta (ROS) u indukovanju DNK oštećenja pod dejstvom adrenalina upotrebili smo antiokisanse katalazu (100 IU i 500 IU) i kvercetin (100 μM i 500 μM). Kotretman limfocita adrenalinom (300 μM) i antioksidansima nakon 15 ili 60 minuta doveo je do značajnog smanjenja količine DNK u repovima kometa. Prema tome, može se zaključiti da adrenalin ispoljava svoje genotoksične efekte indukcijom reaktivnih kiseoničnih vrsta i da se neka oštećenja poprave tokom prva četiri sata nakon tretmana adrenalinom. Za razliku od adrenalina, efedrin nije doveo do povećanja migracije DNK u odnosu na negativnu kontrolu tokom različitih vremenskih intervala. Jedino je koncetracija efedrina od 500 μM nakon 15 minuta tretmana indukovala oštećenje DNK koje je bilo statistički značajno. The objectives of these investigations were to investigate primary DNA damage in isolated human lymphocytes exposed to adrenaline and ephedrine. DNA damage was evaluated by the in vitro Comet assay. A broad spectrum of adrenaline and ephedrine concentrations (range from 0.0005 μM to 300 μM) were examined in the Comet assay for various treatment times (15 min, 60 min, 120 min, 240 min and 24 h). The most profound DNA damage was observed after 15 min of adrenaline treatment, as all concentrations tested except the lowest one (0.0005 μM) caused an increase in DNA migration. After 1 h, 2 h and 4 h of treatment, adrenaline induced DNA damage at concentration range from 5 μM to 300 μM. The slightest DNA damage was observed after 24 h of adrenaline treatment, therefore only the highest concentrations of adrenaline (150 μM and 300 μM) caused increased level of DNA damage. In order to evaluate the potential contribution of reactive oxygen species (ROS)-induced DNA damage exposed to adrenaline, we used antoxidants catalase (100 IU and 500 IU) and quercetin (100 μM and 500 μM). Co-treatment of lymphocytes with adrenaline (300 μM) and antioxidants for 15 or 60 min, significantly reduced the quantity of DNA in the comet tails. Therefore, it can be concluded that adrenaline exhibits genotoxic effects mainly through induction of reactive oxygen species and that some of the DNA damage is repaired during the first four hours following the treatment with adrenaline

Introduction The quality of bond between composite and hard dental tissue is most commonly estimated on the basis of strength of bond or absence of marginal gap along restoration border. Marginal index (MI) is a significant indicator of the efficiency of the bond between material and dental tissue because it also shows the values of width and length of marginal gap. The aim of this paper was to estimate quantitative and qualitative features of the bond between composite resin and enamel and dentin, and to determine the values of MI in enamel after application of two techniques of photopolymerization of composite and two composite adhesive systems. Also, the impact of longevity of photopolimerization on genotoxicity of composite adhesives has been investigated. Material and methods Forty Class V cavities were prepared and restored for scanning electron microscope (SEM) analysis of composite bonding to enamel and dentin. Adhesion to enamel and dentin was secured by etch and rinse procedure (adhesive-Adper Single Bond 2-ASB/3M ESPE), i.e. by self etch primer/adhesive (Adper Easy One-AEO/3M ESPE). Photopolymerization of composite resin (Filtek Ultimate-3M ESPE) was performed using standard halogen light or gradually, using soft start program. Genotoxyciti of adhesives was invastigated using Comet assay. Results The smallest average MI was determined with gradual light curing and application of ASB/FU material. Qualitative analysis showed better marginal adaptation after application of etch and rinse adhesive technique and gradual light curing, compared to self etch adhesive technique and standard photoactivation. Dentin did not show significant differences in the bond between composite restorations and dentine tissue compared to light curing technique but significant difference were determined with respect to the type of adhesive composite material (adhesive technique). Dentin also 108 showed a better quality of the composite bond with etch and rinse adhesive technique, regardless of light curing technique. Using Comet assay it was concluded that significant lower genotoxicity showed both adhesives after photopolymerization in duration of 40 seconds compared to 10 seconds. Conclusion The best marginal adaptation of composite (lowest MI) to enamel and dentin was obtained with gradual soft start photoactivation in etch and rinse adhesive technique and ASB/ FU material. The best marginal adaptation of composite (lowest MI) for dentin was also determined with etch and rinse adhesive technique and the same material, regardless of photopolymerization technique. Longer duration (40 sek) of photoactivation of adhesives in regard to recommendations of manufactures (10 sek.) contributs to lowering of their genocitotoxicity.

Nanomaterijali zahvaljujući svojim malim dimenzijama i raznovrsnim fizičkohemijskim osobinama imaju velik potencijal za primenu u različitim oblastima života. Među nanočestice sa širokom upotrebom u biomedicini ubraja se fuleren C60. Njegova slaba vodorastvorljivost može se prevazići derivatizacijom, pri čemu fuleren stiče nova svojstva koja utiču na njegovu funkcionalnost. Najpoznatiji vodorastvorni derivati C60 su fulerenoli (С60(ОН)n, n=2-40), koji imaju velik potencijal za primenu u medicini. Taj potencijal je baziran na njihovoj sposobosti da u biološkim sistemima skupljaju slobodne radikale nastale u oksidativnom stresu. Međutim, sa druge strane, fulerenoli imaju sposobnost da pod određenim uslovima deluju prooksidativno. Zbog toga je neophodno definisanje koncentracija i uslova pod kojima fulerenol i njegovi derivati deluju kao antioksidanti odnosno prooksidanti. Taj aspekt je veoma važan zbog uticaja ovih nanočestica na DNK i određuje u velikoj meri njihova genotoksična odnosno antigenotoksična svojstva. Pored uticaja koncentracije nanočestica na oksidativni status, na genotoksičnost utiču i fizičkohemijske karakteristike samih nanočestica kao što su: veličina, oblik, površinsko naelektrisanje, čistoća, rastvorljivost, površinske modifikacije i agregacija, koje je takođe potrebno precizno definisati. Konačno, ispitivanje uticaja novosintentisanih supstanci sa potencijalom za medicinsku aplikaciju na DNK, predstavlja neophodan korak pre faze kliničkih studija. Zbog svega navedenog, primarni cilj ovog rada jeste ispitivanje genotoksičnih odnosno antigenotoksičnih svojstava vodorastvornog fulerenskog derivata fulerenola C60(OH)24, na dva različita sisarska modela. Prvi model je ćelijska kultura ovarijuma kineskog hrčka CHO-K1, a drugi primarna ćelijska kultura humanih limfocita. U radu su evaluirane fizičkohemijske karakteristike nanočestice fulerenola C60(OH)24 u medijumu za ćelijske kulture sa serumom i to u uslovima koji odgovaraju 6 eksperimentalnim uslovima u testovima mikronukleusa i hromozomskih aberacija. Zatim je ispitan uticaj fulerenola na preživljavanje ćelija u cilju utvrđivanja subcitotoksičnih koncentracija fulerenola, potrebnih za ispitivanje genotoksičnosti i antigenotoksičnosti fulerenola. Nakon toga je izvršena evaluacija incidencije biomarkera oštećenja DNK radi utvrđivanja genotoksičnog potencijala fulerenola. U poslednjem segmentu rada je evaluacijom istih biomarkera analiziran i antigenotoksičan potencijal fulerenola, na ćelijskim kulturama koje su prethodno oštećene poznatim citostatskim agensom mitomicinom C. Rezultati analiza su pokazali da je C60(OH)24 dimenzija od 40-180 nm u zavisnosti od kosolventa, kao i da gradi stabilne nanočestice u medijumu za ćelijsku kulturu sa fetalnim goveđim serumom. Citotoksičnost fulerenola kreće se u opsegu koncentracija od 88,6 μM-443,2 μM. Analizom genotoksičnosti fulerenola testom mikronukleusa i hromozomskih aberacija pokazano je da fulerenol nije bio genotoksičan u ispitivanim ćelijskim kulturama, pri ispitivanim subcitotoksičnim koncentracijama i pri datim vremenskim tretmanima. Ispitivanje antigenotoksičnog potencijala je pokazalo da fulerenol utiče na snižavanje ispitivanih biomarkera oštećenja DNK. Nanomaterials, due to their small dimensions and manifold physicochemical characteristics have great potential for application in various areas of life. One of nanoparticles with wide implementation in biomedicine is fullerene C60. Its weak water solubility can be overcome by derivatization after which fullerene acquires new characteristics, which affect its functionality. The most famous water soluble C60 derivatives are fullerenols (С60(ОН)n, n=2-40), which have great potential for implementation in medicine. This potential is based on their ability to collect free radicals occurring in oxidative stress in biological systems. However, on the other hand, fullerenols are able to act prooxidatively under certain conditions. Thus, it is necessary to define concentrations and conditions under which fullerenol and its derivatives act as antioxidants, i.e. prooxidants. This aspect is very important because of the impact of these nanoparticles to the DNA, which largely defines their genotoxic i.e. antigenotoxic characteristics. Besides the impact of nanoparticles’ concentration to oxidative status, genotoxicity is also affected by physicochemical characteristics of nanoparticles themselves: their size, shape, surface charge, purity, solubility, surface modifications and aggregation, which are all necessary to be precisely defined. Finally, the testing of the impact of newly synthesized substances with potential for medical application to DNA, represents a necessary step before the phase of clinical trials. Due to all the above state, the primary aim of this paper was to test genotoxic i.e. antigenotoxic characteristic of water-soluble fullerene derivative fullerenol C60(OH)24, in two different mammalian models. The first model was an ovarian cell culture of a Chinese hamster CHO-K1, and the other was a primary humane lymphocyte cell culture. The paper evaluated physicochemical characteristics of fullerenol nanoparticle C60(OH)24 in the cell culture medium with serum under the conditions which were in accordance with the experimental conditions in micronuclei and chromosomal 8 aberrations tests. Furthermore, the affect of fullerenol to cell survival was tested with the aim of determination of fullerenol subcytotoxic concentrations, necessary for testing of fullerenol genotoxicity and antigenotoxicity. After that, the DNA damage biomarkers incidence evaluation was performed for determination of fullerenol genotoxicity potential. In the last segment of the paper, the antigenotoxic potential of fullerenol was analyzed by evaluation of the same biomarkers, in cell cultures, which were previously damaged by the known cytostatic agent mitomycin C. The results of the analysis showed that C60(OH)24 dimensions of 40-180nm depended on the cosolvent, and that it crated stable nanoparticles in the cell culture medium with fetal bovine serum. Cytotoxicity of fullerenol varied in the range of concentrations from 88.6 μM-443.2 μM. The analysis of fullerenol genotoxicity by the micronuclei test and chromosomal aberrations test showed that fullerenol was not genotoxic in the tested cell cultures, at given subcytotoxic concentrations and treatment duration. Testing of antigenotoxic potential showed that fullerenol affected the decrease of the tested DNA damage biomarkers.