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Varnost nanomaterialov, narejenih iz naravnih ali umetnih snovi, v živilstvu, industriji, medicini in na drugih področjih, predstavlja velik izziv, saj se nanotehnologija kot mlado in izredno perspektivno področje razvija nadvse hitro, medtem ko nanotoksikologija in nanoekotoksikologija za njo močno zaostajata. V literaturi tako ne zasledimo veliko informacij o oceni tveganja pri uporabi nanomaterialov, a ker se izdelava in uporaba nanomaterialov ter nenazadnje izpostavljenost ljudi nanomaterialom močno povečuje, igra pridobivanje podatkov o morebitni akutni in kronični toksičnosti še toliko pomembnejšo vlogo. Nanokapsul se poslužujemo na številnih področjih z namenom povečanja stabilnosti in podaljšanja roka uporabe bioaktivne učinkovine ter za izboljšanje nadzora njene dostave in sproščanja. V prehranski industriji se v ta namen pogosto poslužujemo naravnih polisaharidov, na primer alginata, iz katerega zasnovani nanodelci sodijo med najbolj okarakterizirane biomateriale za tarčno-specifično dostavo bioaktivnih učinkovin. V magistrski nalogi smo na celični liniji človeškega hepatocelularnega karcinoma, HepG2, izvajali testiranja citotoksičnosti in genotoksičnosti sedmih različnih vrst nanokapsul na osnovi alginata kot potencialnih nosilcev bioaktivnih učinkovin. Tri od sedmih različnih vrst nanokapsul so vsebovale kvercetin kot primer bioaktivne učinkovine. Citotoksičnost smo vrednotili s kolorimetrično metodo MTS za določanje živosti celic, medtem ko smo genotoksičnost vrednotili s testom za zaznavanje poškodb DNK na nivoju posamezne celice, testom komet. Za namen uporabe nanokapsul v prehrani, slednje ne smejo izkazovati toksičnega delovanja. Rezultati naše magistrske naloge so pokazali, da testirane polisaharidne nanokapsule na osnovi alginata za celice HepG2 po 24-urni izpostavitvi niso citotoksične v koncentracijskem območju do 500 µg/mL. Ugotovili smo tudi, da testirane nanokapsule po 4- in 24-urni izpostavitvi ne povzročajo poškodb DNK v celicah HepG2 in zatorej ne delujejo genotoksično v proučevanem testnem sistemu pri danih pogojih. The safety of nanomaterials, made from natural or artificial substances, used in food, various industrial processes, medicine, and other areas, represents a significant challenge because nanotechnology, as a young and up-and-coming field, is developing very quickly, while nanotoxicology and nanoecotoxicology are falling behind. That is why we do not find much information on the risk assessment of nanomaterials in the literature. Still, since the production, use, and ultimately the exposure of people to nanomaterials is increasing significantly, the acquisition of data on potential acute and chronic toxicity plays an even more critical role. Nanoencapsulation carriers are used in many areas to increase the stability and shelf life of the bioactive agent and improve the control of its delivery and release. In the food industry, we often use natural polysaccharides like alginate, whose nanoparticles are among the most characterized biomaterials for target-specific delivery of bioactive agents. In the master's thesis, we tested the cytotoxicity and genotoxicity of seven different types of alginate-based nanocapsules as potential carriers of bioactive agents on human hepatocellular carcinoma cell line, HepG2. Three of the seven different types of nanocapsules contained quercetin as an example of a bioactive agent. Cytotoxicity was evaluated using a colorimetric method for determining cell proliferation and viability, while genotoxicity was assessed using a comet assay for detecting DNA damage at the single cell level. If we want to use nanocapsules in the food industry, the latter must not exhibit toxic activity. The results of our master's thesis showed that the tested nanocapsules are not cytotoxic in the concentration range up to 500 µg/mL for HepG2 cells after 24 hours of exposure. We also found that the tested nanocapsules did not cause DNA damage in HepG2 cells after 4- and 24-hour exposure and therefore had no genotoxic effect in the test system studied at applied conditions.

Tirozin kinazni inhibitorji (TKI) so majhne molekule zasnovane za tarčno zdravljenje raka. Neposredno inhibirajo specifične mutirane tirozin kinaze, ki so prekomerno izražene v določenih vrstah rakavih celic. Ker se pojavnost rakavih obolenj povečuje, in ker so ta zdravila uspešna v boju proti določenim vrstam rakavih obolenj, poraba teh zdravil v zadnjih 20 letih nenehno narašča. Posledično narašča tudi količina ostankov teh snovi v vodnem okolju. Trenutne meritve in predvidevanja kažejo, da se ostanki teh zdravil pojavljajo v koncentracijah nekaj ng/L ali manj. Kljub stalni prisotnosti v okolju pa zaradi tarčnega načina delovanja ne predstavljajo resne grožnje posredno izpostavljenim organizmom v okolju, saj ti ne izražajo tarče za ta zdravila. Kljub temu pa so obstoječe raziskave pokazale, da določeni TKI povzročajo genotoksične učinke na netarčnih okoljskih orgnizmih. V magistrskem delu smo na in vitro modelu jetrnih celic rib cebric (Danio rerio ZFL) ocenili toksičnost petih pogosto uporabljenih TKI: erlotinib, sorafenib, regorafenib, dasatinib in nilotinib. Citotoksične učinke posameznih TKI smo ocenili s testom MTS, njihovo genotoksično delovanje pa s testom komet. S testom MTS smo ugotovili od koncentracije in časa odvisen vpliv sorafeniba, regorafeniba in dasatiniba, medtem ko erlotinib in nilotinib nista značilno vplivala na živost celic ZFL niti po daljšem času izpostavitve (72 ur). S testom komet pri erlotinibu genotoksičnega učinka oz. poškodb DNA nismo zaznali, smo pa zaznali povečano količino poškodb DNA pri drugih preizkušenih TKI. Pri sorafenibu (⡥ 0,8 µM), regorafenibu (⡥ 0,16 µM) in dasatinibu (⡥ 0,006 µM) smo poškodbe DNA zaznali le po 24 urni izpostavitvi, pri nilotinibu pa tako pri krajšem (24 ur ⡥ 2 µM) kot pri daljšem času izpostavitve (72 ur 2 in 10 µM). Rezultati nakazujejo, da regorafenib in nilotinib pri ne-citotoksičnih koncentracijah, ki so sicer višje od pričakovanih v okolju, povzročata poškodbe DNA. Tyrosine kinase inhibitors (TKIs) are small molecules designed for targeted cancer therapy. They directly inhibit certain mutated tyrosine kinases that are overexpressed in certain types of cancer cells, preventing the spread of the disease. As the number of cancer cases increases, the consumption of these pharmaceuticals has steadily increased over the past 20 years. As a result, the amount of these substances in the aquatic environment is also increasing. Current measurements and predictions indicate that residues of these drugs occur at concentrations of a few ng/L or even less. Due to the specific mechanism of action of TKIs, they do not pose a serious threat to indirectly exposed environmental organisms because they do not express targets for these drugs. Nevertheless, existing studies have shown that certain TKIs have genotoxic effects on non-target organisms in the environment. In this master’s thesis, we investigated the toxicity of five commonly used TKIs: erlotinib, sorafenib, regorafenib, dasatinib, and nilotinib using an in vitro model of zebrafish liver cells (Danio rerio ZFL). The cytotoxic effects of each TKIs were assessed by the MTS assay, and their genotoxic effects were assessed by the Comet assay. The MTS assay showed dose- and time-dependent effects of sorafenib, regorafenib, and dasatinib, whereas erlotinib and nilotinib did not significantly affect ZFL cell viability even after prolonged exposure (72 hours). No genotoxic effect or DNA damage was detected with erlotinib using the comet assay, but an increased amount of DNA damage was detected with the other TKIs tested. For sorafenib (⡥ 0.8 µM), regorafenib (⡥ 0.16 µM) and dasatinib (⡥ 0.006 µM), DNA damage was detected only after 24 hours of exposure, whereas nilotinib induced DNA damage at both short (24 hours ⡥ 2 µM) and longer exposure (72 hours 2 and 10 µM). The results suggest that regorafenib and nilotinib cause genotoxic effects at non-cytotoxic concentrations that are, however, higher than would be expected in the environment.

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.

Vrste roda Veronica (hrv. čestoslavice) su višegodišnje, zeljaste biljke s raznolikim staništem diljem Europe i Azije. Broje otprilike 450 vrsta od kojih svega 30 obitava u Hrvatskoj. Zbog ljekovitih svojstava koje duguju visokom sadržaju biološki aktivnih spojeva, čestoslavice se od davnina koriste u tradicionalnoj medicini, a također su i predmet zanimanja brojnih znanstvenika. U ovom istraživanju ispitan je toksični učinak hidrolata dviju vrsta roda Veronica, Veronica officinalis L. (hrv. ljekovita čestoslavica) i Veronica saturejoides Vis. subsp. saturejoides (hrv. vriskolika (vriskova) čestoslavica) na genetički materijal stanica karcinoma debelog crijeva (HCT116). Za procjenu genotoksičnog učinka proveden je standardni in vitro alkalni komet test. Dobiveni rezultati pokazali su kako hidrolati navedenih vrsta posjeduju nizak genotoksični učinak na stanice karcinoma debelog crijeva. Primijećena je razlika u genotoksičnoj aktivnosti između vrsta, no utvrđeno je kako je ta razlika statistički zanemariva. Rezultati ovog istraživanja upućuju na mogućnost sigurne primjene hidrolata ovih biljnih vrsta u medicinske i kozmetičke svrhe. Ipak, nužno je provesti daljnja ispitivanja koja će obuhvatiti spektar testova na različitim tipovima stanica, in vitro i in vivo, s ciljem procjene cjelokupne genotoksičnosti hidrolata i njegovih aktivnih spojeva., The species of the genus Veronica are perennial herbaceous plants found throughout Europe and Asia. There are about 450 species, of which only 30 are found in Croatia. Due to their medicinal properties, which are due to the high content of biologically active compounds, this species has been used in traditional medicine since ancient times and are now the subject of interest of many scientists. In this study, the toxic effect of hydrosols of two species of the genus Veronica, Veronica officinalis L. and Veronica saturejoides Vis. subsp. saturejoides, on the genome of colon cancer cells (HCT116) was investigated. A standard alkaline comet assay was performed in vitro to evaluate the genotoxic effect. The obtained results show that the hydrosols of this species have a low genotoxic effect on colon cancer cells. A difference in genotoxic activity was observed between the species, but it was statistically negligible. The results of this study suggest the possibility of safe use of the hydrolates of these plant species for medicinal and cosmetic purposes. Nevertheless, it is necessary to perform further tests on different cell types in vitro and in vivo to evaluate the overall genotoxicity of the hydrosols and their active compounds.

Bisfenoli (BP) so skupina industrijskih kemikalij, ki se uporabljajo pri proizvodnji plastike in epoksi smol. Iz njih izdelujejo najrazličnejše izdelke kot so obloge za konzerve, stekleničke za dojenčke, igrače, papir za račune in mnoge druge, ki se uporabljajo v našem vsakdanjem življenju in jih posledično zasledimo v okolju. Zaradi kopičenja podatkov o nevarnih lastnostih in škodljivih vplivih najpogostejšega izmed BP-jev, bisfenola A (BPA), predvsem motenj endokrinega sistema, je bila njegova uporaba v Evropski uniji postopoma omejena oziroma celo prepovedana, da bi zaščitili zdravje ljudi in okolje. Prepoved uporabe BPA je spodbudila razvoj domnevno varnejših analogov, za katere pa v literaturi ni veliko toksikoloških podatkov. Tako smo v magistrski nalogi proučevali citotoksično in genotoksično aktivnost BPA in njegovih analogov: bisfenol AF (BPAF), bisfenol AP (BPAP), bisfenol C (BPC), bisfenol FL (BPFL), bisfenol S (BPS) na jetrnem in vitro 3D celičnem modelu. Sferoide smo pripravili s prisilno plavajočo metodo, vpliv na živost celic sferoidov po izpostavitvi BP-jem smo merili s testom MTS. S pomočjo mikroskopije, smo spremljali površino, obliko in kompaktnost sferoidov, s testom komet pa smo preučevali genotoksičnost v časovnih izpostavitvah 24 in 96 ur. Statistično značilen vpliv na živost celic v sferoidu po 24 urah so imeli BPFL > BPAF = BPAP, po 96 urah pa BPFL > BPC = BPAP > BPA = BPAF. Mikroskopske analize so pokazale, da sta imela največji vpliv na rast in spremembo površine ter kompaktnosti sferoidov BPC in BPFL po 96-urni izpostavitvi. S testom komet smo pokazali, da sta bila po 24 urah izpostavitve najbolj genotoksična BPAF in BPS (⡥ 10 μM), sledil je BPAP ( ⡥ 40 μM). Po 96 urah izpostavitve je BPAP povzročil poškodbe DNA pri koncentraciji ⡥ 0,1 μM, pri vseh ostalih BP-jih smo poškodbe DNA zaznali pri višjih koncentracijah. Povzamemo lahko, da so za analoge BPA potrebne dodatne študije mehanizmov delovanja za pripravo ustrezne ocene tveganja, saj je toksična aktivnost za posamezne analoge, kljub podobni kemijski strukturi, različna. Bisphenols (BPs) are a group of industrial chemicals used in the manufacture of plastics and epoxy resin. They are found in various products, including the inner linings of cans, baby bottles, toys, thermal paper, and others used in our daily life. Therefore, BPs are present in the environment. The most widely used BP is bisphenol A (BPA), on which most studies have been conducted showing its adverse effects on human health, especially its endocrine disrupting activity. Therefore, the European Union began to restrict and eventually ban the use of BPA to protect human health and the environment. The ban has led to the development of supposedly safer BPA analogues, for which few toxicological data are available. In the master thesis, we investigated the cytotoxic and genotoxic activity of BPA and its analogues (BPAF, BPAP, BPC, BPFL, BPS) in a liver in vitro 3D cell model. In this study, we prepared spheroids by the forced floating method and measured cell viability after exposure to BPs using the MTS assay. We measured the surface area and observed the shape and compactness of the spheroids with microscopy and studied the genotoxic effects with the comet assay at 24 and 96-hour exposure. After 24-hour exposure to BPs, BPFL > BPAF = BPAP statistically significantly decreased the viability of HepG2 and after 96 hours, we observed a similar effect on cell viability in BPFL > BPC = BPAP > BPA = BPAF. BPC and BPFL had the greatest effect on morphological changes in growth, surface area, compactness, and shape of the spheroids that were evident at 96-hour exposure. Of the six BPs tested by the comet assay, BPAF and BPS (⡥ 10 μM) and BPAP (⡥ 40 μM) exhibited stronger genotoxic potential than BPA, BPFL, and BPC after 24 hours of exposure. After 96 hours of exposure, BPAP was the most genotoxic causing/inducing DNA damage at ⡥1 µM. All others BPs studied statistically significantly increased DNA damage at higher concentrations. To summarize, despite their similar chemical structure, the toxic activity of the analogues is not the same, and therefore further studies on the BPA analogues and their molecular activity are needed to provide/make accurate risk assessment.

Bisfenol A (BPA) je organski spoj koji se koristi u proizvodnji epoksidnih smola i polikarbonata koji su glavne komponente predmeta u svakodnevnoj upotrebi. Dosadašnja istraživanja su dokazala njegov toksični učinak na okoliš i ljudsko zdravlje pa je glavni cilj ovog istraživanja bio ispitati fitotoksični i genotoksični učinak BPA na meristemske stanice korjenčića luka (Allium cepa L). Allium test ima niz prednosti u istraživanju kao što su jednostavnost rukovanja, veličina i broj kromosoma koji su praktični za analizu kromosomskih aberacija te povoljna cijena. Nakon naklijavanja (dva dana) korjenčići luka (n = 10) bili su izloženi ili BPA (u koncentracijskom rasponu 1-50 mg/L) ili destiliranoj vodi (kontrola) kroz tri dana. Nakon tri dana, pregledana je morfologija korjenčića, izmjerena duljina korjenčića te su pripremljeni mikroskopski preparati meristemskih stanica korjenčića luka u kojima je praćena pojava kromosomskih oštećenja i mikronukleusa. Dobiveni rezultati statistički su obrađeni t-testom (Excel, MS Office), a razina značajnosti postavljena je na p < 0,05. Trodnevni tretman s BPA doveo je do smanjenja produžnog rasta korjenčića te je već nakon tretmana s najnižom ispitivanom koncentracijom (1 mg/L) zabilježena značajno manja duljina korjenčića u odnosu na kontrolu (4,07 ± 0,33 cm vs. 4,61 ± 0,38 cm; p < 0,05). Također, trodnevni tretman s BPA utjecao je i na izgled korjenčića te su s porastom koncentracije BPA korjenčići postali mekši, nepravilni i uvinuti u odnosu na kontrolu. Mikroskopska analiza meristemskih stanica korjenčića luka pokazala je da je trodnevni tretman s BPA doveo do pojave kromosomski oštećenja. S porastom koncentracije BPA povećala se učestalost pojave kromosomskih oštećenja kao što su gubitak kromosoma, lomovi kromosoma i anafazni mostovi. U interfaznim jezgrama meristemskih stanica luka nakon trodnevnog tretmana s BPA zabilježena je pojava mikronukleusa i jezgrinih pupova te je njihova učestalost rasla s povećanjem koncentracije BPA. Rezultati ovog istraživanja pokazuju da BPA ima fitotoksični i genotoksični, ali i potencijalno mutageni učinak na korjenčiće luka. Bisphenol A (BPA) is an organic compound used for production of epoxy resins and polycarbonates that are main components of items used in everyday life. Investigations conducted by now demonstrated its toxicity. Therefore, the aim of this study was to test phytotoxicity and genotoxicity of BPA on meristem cells of onion (Allium cepa L.) roots. The Allium test has number of advantages such as simple handling, size and number of chromosomes which make analysis of chromosome aberrations easy, as well as affordable price. After two-day germination, roots of onion bulbs (n = 10) were exposed to either BPA (in the concentration range of 1-50 mg/L) or distilled water (control) for three days. After three days, the morphology of the roots was examined, and the length of the roots was measured. Additionally, microscopic slides of meristem cells of the onion roots were prepared, in which chromosome aberrations and micronucleus were monitored. The obtained results were statistically analyzed by a t-test (Excel, MS Office), and the level of significance is set to p < 0.05. Three-day treatment with BPA reduced root length and the lowest BPA concentration (1 mg/L) significantly reduced root length compared to the control (4.07 ± 0.33 cm vs. 4.61 ± 0.38 cm; p < 0.05). Additionally, a three-day treatment with BPA had effect on the roots’ appearance; with an increase in the concentration of BPA the roots became softer, irregular, and twisted in comparison to the control. Analysis of onion roots meristem cells revealed that a three-day treatment with BPA led to the chromosome aberrations. The frequency of chromosome damage such as loss of chromosome, chromosome-breaks and anaphase bridges increased with the increase of BPA concentration. In meristem cells in the interphase treatment with BPA induce formation of micronucleus and nuclear buds, and their frequency increased with an increase in BPA concentration. The results of this study demonstrate that BPA has phytotoxic and genotoxic, but also potentially mutagenic effect on onion roots cells.

Ortodontske naprave primjenjuju se već niz godina kao primarni oblik postizanja ortodontskog pomaka i estetski ljepšeg pozicioniranja zubi. Najčešće su izrađene od metala, koji u usnoj šupljini lako korodiraju i ispuštaju ione u slinu. Metalni ioni imaju poznat čitav niz negativnih bioloških aktivnosti te se postavlja pitanje štete li metalni ioni ispušteni od strane ortodontskih naprava pacijentima. Cilj ovog rada bio je ispitati citotoksičan, genotoksičan i prooksidativni efekt metala ispuštenih u slinu iz dijelova ortodontskih naprava. Za mjerenje citotoksičnosti korištena je Neutral Red metoda, za mjerenje genotoksičnosti komet test, a za mjerenje prooksidativnog učinka metode DCFH-DA i linearizacije plazmida φX-174 RF I. Vrijeme eluacije ortodontskih naprava utječe na sastav sline te se mogu izmjeriti različiti biološki efekti na stanicama. Većina eluata djelovala je toksično u cijelom koncentracijskom rasponu neovisno o vremenu eluacije na CAL 27 stanice. Na AGS staničnoj liniji određeno je da niže koncentracije eluata bravica, ligatura, prstenova te svih dijelova izazivaju prooksidacijsko djelovanje, dok više koncentracije eluata istih ortodontskih naprava imaju antioksidacijsko djelovanje. Prooksidacijski učinak određen na plazmidu φX-174 RF I je bio slab. Metali prisutni u eluatima ne izazivaju trajne promjene na genetički materijal stanica te je njihova primjena sigurna. Orthodontic appliances have been used for decades as a primary way of treating orthodontic problems and achieving an aesthetically pleasing look. They are usually made of metal, which can easily corrode and release metal ions into saliva. Metal ions are known for their numerous negative biological effects, so a question, whether ions released by appliances cause damage to patients, rises. The goal of this paper was to test cytotoxic, genotoxic and prooxidative effects of metal ions released by appliance parts into saliva. The results show that a certain amount of those effects on cells is visible, but that it is not worrying to the point of saying that orthodontic appliances are harmful for human health. Cytotoxicity was measured using NR method, prooxidative effects were tasted using the DCFH-DA method and φX-174 RF I plasmid linearization, while genotoxicity was measured with comet assay. The eluation time has an impact on the composition of the saliva and biological effects can be measured. Most of the tested samples had a toxic effect on CAL 27 cell line. Regarding AGS cell line, lower concentrations of samples were shown to have a prooxidative effect, while higher concentrations had an antioxidative effect. The prooxidative effect tested using the φX-174 RF I plasmid linearization was weak. Metals present in the eluates do not cause permanent changes of the genetic material and their application is safe.

Exposure to alkyl anilines may lead to bladder cancer, which is the second most frequent cancer of the urogenital tract. 3,5-dimethylaniline is highly used in industry. Studies on its primary metabolite 3,5-dimethylaminophenol (3,5-DMAP) showed that this compound causes oxidative stress, changes antioxidant enzyme activities, and leads to death of different mammalian cells. However, there is no in vitro study to show the direct effects of 3,5-DMAP on human bladder and urothelial cells. Selenocompounds are suggested to decrease oxidative stress caused by some chemicals, and selenium supplementation was shown to reduce the risk of bladder cancer. The main aim of this study was to investigate whether selenocompounds organic selenomethionine (SM, 10 μmol/L) or inorganic sodium selenite (SS, 30 nmol/L) could reduce oxidative stress, DNA damage, and apoptosis in UROtsa cells exposed to 3,5-DMAP. 3,5-DMAP caused a dose-dependent increase in intracellular generation of reactive oxygen species, and its dose of 50 μmol/L caused lipid peroxidation, protein oxidation, and changes in antioxidant enzyme activities in different cellular fractions. The comet assay also showed single-strand DNA breaks induced by the 3,5-DMAP dose of 50 μmol/L, but no changes in double-strand DNA breaks. Apoptosis was also triggered. Both selenocompounds provided partial protection against the cellular toxicity of 3,5-DMAP. Low selenium status along with exposure to alkyl anilines can be a major factor in the development of bladder cancer. More mechanistic studies are needed to specify the role of selenium in bladder cancer., Izloženost alkilnim anilinima može uzrokovati rak mokraćnoga mjehura, koji je drugi po redu po učestalosti raka mokraćnospolnog sustava. 3,5-dimetilanilin često se rabi u industrijskoj proizvodnji, a istraživanja njegova primarnog metabolita, 3,5-dimetilaminofenola (3,5-DMAP), pokazuju da on uzrokuje oksidacijski stres i promjene u aktivnosti antioksidacijskih enzima te u konačnici dovodi do smrti raznih stanica u sisavaca. Dosad, međutim, nije provedeno nijedno istraživanje njegovih izravnih učinaka na epitelne stanice mokraćnoga mjehura i bubrega u ljudi. Za spojeve selenija smatra se da smanjuju oksidacijski stres različitih kemikalija te da dopuna prehrane selenijem smanjuje rizik od raka mokraćnoga mjehura. Primarni je cilj ovoga istraživanja bio utvrditi može li organski spoj selenija selenometionin (SM, 10 μmol/L), odnosno anorganski spoj natrijev selenit (SS, 30 nmol/L], smanjiti oksidacijski stres, oštećenje DNA i apoptozu u UROtsa stanicama izloženima 3,5-dimetilaminofenolu. Jednosatna izloženost stanica 3,5-DMAP-u dovela je do povećanja razina reaktivnih kisikovih spojeva (ROS), lipidne peroksidacije, oksidacije bjelančevina te do promjena u aktivnosti antioksidacijskih enzima u staničnoj citoplazmi i jezgri, ovisno o primijenjenoj dozi. Osim toga, komet-testom su utvrđeni jednolančani, ali ne i dvolančani lomovi DNA. Također, 3,5-DMAP uzrokovao je apoptozu stanica. Oba su spoja selenija pružila djelomičnu zaštitu od njegova toksičnoga djelovanja. Nedostatak selenija pri izloženosti alkilnim anilinskim spojevima stoga može odigrati značajnu ulogu u nastanku raka mokraćnog mjehura. Potrebna su daljnja istraživanja mehanizama djelovanja selenija u njegovu sprječavanju.

U većini zemalja svijeta, maligna su oboljenja po učestalosti drugi uzrok smrtnosti stanovništva. O samoj vrsti tumora i njegovoj uznapredovalosti ovisi način liječenja, koji najčešće zahtijeva multimodalni pristup. Irinotekan je jedan od najčešće primjenjivanih citostatika za liječenje metastatskog kolorektalnog karcinoma. Δ9-tetrahidrokanabinol (THC) je glavna aktivna komponenta konoplje čija se uporaba u medicinske svrhe aktivno razmatra posljednjih nekoliko desetljeća. Cilj ovog rada je istražiti citotoksičnost, genotoksičnost i prooksidacijski učinak različitih koncentracija irinotekana i THC-a. Kao test sustavi korištene su humana stanična linija hepatocelularnog karcinoma jetre (HepG2) i humana stanična linija adenokarcinoma epitela debelog crijeva (Caco-2) koje su bile izložene djelovanju irinotekana i THC-a tijekom 2, 4, 24 i 48 sati. Dokazano je da irinotekan i THC djeluju citotoksično ovisno o duljini tretmana stanica i primijenjenoj koncentraciji. Najveći prooksidacijski učinak zabilježen je na obje stanične linije nakon 4 sata, dok genotoksično djelovanje ponajviše uvjetuju više koncentracije irinotekana. In most countries, cancer is the second leading cause of death. The type of tumor and the stage of the disease will depend on the exact treatment, which usually involves a combination of therapies. Irinotecan is the key anti-cancer drug for the treatment of metastatic colorectal cancer. Δ9-tetrahydrocannabinol (THC) is the main active compound in the Cannabis plant which has been increasingly used in medicine over the past decades. The aim of this work is to investigate the cytotoxicity, genotoxicity and pro-oxidative effects of different concentrations of irinotecan and THC. As a test system, the human hepatocellular carcinoma cell line (HepG2) and the human colon adenocarcinoma cell line (Caco-2) were exposed to irinotecan and THC during 2, 4, 24, and 48 hours. It has been shown that the cytotoxicity of irinotecan and THC depends on the duration of cell treatments and concentration used. The highest pro-oxidative effect was observed at 4 hours for both cell lines, while genotoxic activity was mostly caused by higher concentrations of irinotecan.

Svijet je suočen s nedostatkom zdravstveno ispravne vode za piće prvenstveno radi ugrožavanja vodenih okoliša pri čemu je jedan od glavnih izvora onečišćenja nepravilno gospodarenje otpadom. Danas je najčešća metoda zbrinjavanja otpada odlaganje razvrstanog otpada na za to predviđena mjesta koja se redovito moraju kontrolirati jer vrlo lako može doći do procjeđivanja raznih zagađivača u podzemne i površinske vode. Cilj ovog istraživanja bio je odrediti citotoksičnost, genotoksičnost te oksidacijsko oštećenje staničnih makromolekula proteina i lipida uzrokovanog djelovanjem voda s područja odlagališta otpada Piškornica. Kao biološki test sustav korištene su humane stanične linije hepatocelularnog karcinoma jetre Hep G2 te oralnog pločastog karcinoma Cal 27, a vrijeme izloženosti bilo je 24 sata. Rezultati su pokazali da uzorak procjedne vode djeluje citotoksično te izaziva karbonilaciju proteina i lipidnu peroksidaciju. Zbog smanjenog postotka preživljenja (

vrha rada: Mikronukleus test u bukalnim epitelnim stanicama je jednostavna metoda za ijagnosticiranje genetskih oštećenja toksičnih tvari. Cilj ove in vivo studije bio je procijeniti citotoksični i genotoksični učinak pušenja koristeći mikronukleus test. Materijali i metode: Istraživanje je provedeno na 160 ispitanika, 40 nepušača te 120 pušača. Pušači su bili podijeljeni u tri podjednake skupine ovisno o korištenom proizvodu (cigarete, električne cigarete, IQOS). Praćen je učinak pušenja na pojavnost jezgrinih anomalija uključujući mikronukleus, kondenzirani kromatin, karioreksu, kariolizu, piknozu, „slomljeno jaje“ te jezgrin pup. Rezultati: Rezultati dobiveni Kruskal-Wallis testom potvrđuju značajnu razliku u učestalosti stanica s mikronukleusom (P = 0, 011), jezgrinim pupom (P = 0, 012), karioreksom (P ≤ 0, 001), kariolizom (P ≤ 0, 001), kondenziranim kromatinom (P ≤ 0, 001) i binuklearnih stanica (P ≤ 0, 001) u pušača različitih duhanskih proizvoda i nepušača. Zaključak: Korištenje duhanskih proizvoda značajno dovodi do citotoksičnih i genotoksičnih oštećenja u oljuštenim bukalnim stanicama

Biljni bioaktivni polifenoli su relativno dobro poznati po svojim antioksidativnim, antimutagenim, antikancerogenim, protuupalnim, antiangiogenim, antiulkusnim i antimikrobnim (baktericidnim, fungicidnim te antivirusnim) osobinama. Temeljem preliminarnih istraživanja antigenotoksičnosti ekstrakata različitih biljnih vrsta, uočeno je da one vrste koje su bogate polifenolima imaju značajan antigenotoksični učinak sprječavanjem oksidativnih oštećenja izazvanih citostaticima u kulturi ljudskih limfocita. Ispitano je djelovanje vodenih i alkoholnih ekstrakata vrste Echinacea purpurea L. antigenotoksično u uvjetima in vitro. Rezultati pokazuju konkretan utjecaj vodenih i alkoholnih ekstrakata, kao i najdominantnijih sastavnica E. purpurea, te polifenola (tanina), nakon tretmana stanica mikotoksinima ohratoksinom A i aflatoksinom B1. Nadalje, ispitan je sadržaj polifenola u ekstraktima vrsta Echinacea purpurea L. Moench te određen antioksidativni učinak ekstrakata primjenom elektronske spinske rezonancije i kolorimetrijske metode gašenja slobodnog radikala DPPH. Antioksidativna aktivnost ekstrakata bi mogla biti jedan od pokazatelja antigenotoksičnog učinka s obzirom na mogućnost nastanka oštećenja DNA djelovanjem slobodnih radikala. Antigenotoksični potencijal ekstrakta utvrđen je primjenom alkalnog kometnog testa na ljudskim leukocitima ex situ.

Exposure to radiofrequency electromagnetic fields (RF-EMF) at the operating frequencies of different communication devices can cause various biological effects. However, there is a lack of studies on the oxidative stress response and genotoxicity in the honey bee (Apis mellifera) after exposure to RF-EMF. In this study, we investigated the oxidative stress and DNA damage in honey bee larvae situated in waxcomb cells, exposed to modulated RF-EMF 23 Vm-1. The glutathione S-transferase activity decreased, whereas the catalase activity increased significantly in the honey bee larvae upon RF-EMF exposure. Superoxide dismutase activity, the level of lipid peroxidation, and DNA damage were not statistically altered in exposed honey bee larvae when compared to the control group. These results suggest that the biological effects of modulated RF-EMF in honey bee larvae depend on the exposure design., Izloženost radiofrekvencijskom elektromagnetskom polju (RF-EMF) na radnoj frekvenciji različitih komunikacijskih uređaja može imati razne biološke učinke. Nedostaju istraživanja odgovora na oksidacijski stres i genotoksičnost u medonosne pčele (Apis mellifera) poslije izloženosti RF-EMF-u. U ovom je radu istražen oksidacijski stres i DNA oštećenja u ličinkama medonosnih pčela smještenih u stanicama saća i izloženih modulirajućem RF-EMF 23 Vm-1. Aktivnost glutation S-transferaze smanjena je, dok je aktivnost katalaze značajno povećana u ličinkama medonosnih pčela izloženih RF-EMF-u. Nije bilo statistički značajne promjene u aktivnosti superoksid-dismutaze, razini lipidne peroksidacije i DNA oštećenju između ličinaka medonosne pčele i kontrolne skupine. Ovi rezultati pokazuju da biološki učinci modulirajućeg RF-EMF u ličinkama medonosne pčele ovise o oblikovanju izloženosti.

Določitev genotoksične aktivnosti kemikalij je pomemben del ocene tveganja za ljudi. Za testiranje genotoksičnosti se najpogosteje uporabljajo in vitro jetrne celične linije, saj imajo ohranjeno določeno stopnjo presnovne aktivnosti ksenobiotikov. Vendar imajo tradicionalni dvodimenzionalni (2D) in vitro celični modeli številne pomanjkljivosti, zato v zadnjih letih raziskovalci razvijajo številne in vitro tridimenzionalne (3D) celične modele, ki zaradi svoje večje kompleksnosti bolje posnemajo in vivo pogoje. Prednosti 3D celičnih modelov pred 2D celičnimi modeli so povrnitev številnih bioloških funkcij, kot so medcelične povezave in povezave celica ¬– zunajcelični matriks, kar vodi do boljše diferenciacije celic. V magistrskem delu smo želeli ovrednotiti občutljivost in specifičnost novorazvitega jetrnega 3D in vitro celičnega modela. V prvem delu smo okarakterizirali in optimizirali pogoje za razvoj in vitro 3D celičnega modela (sferoidi), ki smo ga pripravili iz celične linije humanega hepatocelularnega karcinoma (HepG2 celice). Sferoidi, ki smo jih pripravili s prisilno plavajočo metodo, so bili kompaktni ter enakomerni v obliki, velikosti in rasti. V drugem delu smo sferoide pri starosti 72 ur izpostavili različnim koncentracijam dveh posredno delujočih genotoksičnih spojin, in sicer benzo(a)pirenu (BaP) in 2-amino-1-metil-6-fenilimidazo(4,5-b)piridinu (PhIP) za 24 in 72 ur. Proliferacijo celic, celični cikel ter nastanek dvoverižnih prelomov DNA smo spremljali s pretočno citometrijo. Rezultati kažejo, da spojina BaP vpliva tako na celični cikel kot na proliferacijo celic, medtem ko pri spojini PhIP nismo zaznali razlik. Z analizo prisotnosti dvoverižnih prelomov DNA smo pokazali, da BaP povzroči poškodbe DNA tako po 24-urni kot po 72-urni izpostavitvi že pri koncentraciji 䁥 1 μM. PhIP po 24-urni izpostavitvi povzroči poškodbe DNA pri koncentraciji 200 μM, po 72-urni izpostavitvi pa že pri koncentraciji 䁥 25 μM. Genotoksične učinke BaP in PhIP (z izjemo kratkotrajne izpostavitve sferoidov spojini PhIP) smo zaznali pri nižjih koncentracijah, kot so bili ti zaznani v študijah na in vitro 2D celičnem modelu. Zaključimo lahko, da jetrni in vitro 3D celični model kaže izboljšano občutljivost za zaznavanje citotoksičnega in genotoksičnega delovanja posredno delujočih genotoksičnih spojin. Z našo raziskavo smo tako korak bližje k premostitvi vrzeli med in vivo raziskavami in testi, ki temeljijo na in vitro 2D celičnih modelih. Determination of genotoxic activity of chemicals is an essential part of human risk assessment. For genotoxicity assessment, in vitro hepatic cell lines are most commonly used as they have retained certain level of xenobiotic metabolic activity. However, the traditional two-dimensional (2D) in vitro cell models have numerous limitations. Therefore scientists have in the last few years developed many in vitro three-dimensional (3D) cell models, which due to their higher complexity better imitate in vivo conditions. The advantage of 3D cell models compared to 2D monolayer models is the recovery of several biological functions such as intercellular interactions and cell – extracellular matrix interactions, which lead to better cell differentiation. The objective of this master's thesis was to evaluate sensitivity and specificity of newly developed 3D in vitro cell model. In the first part we optimized the conditions for development of in vitro 3D cell model (spheroids) which was formed from human hepatocellular carcinoma (HepG2) cell line. The spheroids were formed by the forced floating method. They were compact and of uniform shape, size and growth. In the second part, 72 hours old spheroids were exposed to graded concentrations of two indirect acting genotoxic compounds, benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) for 24 and 72 hours. Cell proliferation, cell cycle and induction of DNA double-strand breaks were asessed by flow citometry. The results showed that BaP compound affected both cell cycle and cell proliferation, although no differences were noticed for PhIP. By analysing the presence of DNA double-strand breaks it was shown that BaP caused DNA damage after 24- and 72-hour exposure at concentration 䁥 1 μM. PhIP caused DNA damage at 200 μM after 24-hour exposure and at 䁥 25 μM after 72-hour exposure. Genotoxic effects of BaP and PhIP were (with the exception of short term exposure of spheroids to PhIP) detected at lower concentrations as we reported for in vitro 2D monolayer model. We can conclude that hepatic 3D cell model showed improved sensitivity for detection of cytotoxic and genotoxic activity of indirect acting genotoxic compounds. This research presents a step forward to bridge the gap between in vivo research and tests that are based on 2D monolayer models.

Prehrambena industrija proizvodi veliku količinu otpadne biomase koja predstavlja ekološki rizik, stoga se danas intenzivno istražuju novi načini valorizacije otpadnih materijala. Nusproizvodi poput mahune graška i ljuske luka nedovoljno su iskorišteni izvori bioaktivnih spojeva, među kojima se ističu polifenoli, sekundarni biljni metaboliti koji posjeduju visok antioksidacijski potencijal te pružaju značajnu zaštitu od razvoja raznih kroničnih bolesti. Cilj rada je istražiti biološki učinak ekstrakata mahune graška i ljuske luka na stanične makromolekule te humane stanične linije karcinoma epitela usne šupljine Cal27 i hepatocelularnog karcinoma jetre HepG2 u koncentracijama pri kojima se ovi spojevi mogu naći u realnim uvjetima u probavnom sustavu čovjeka. Rezultati su pokazali da polifenoli sadržani u mahuni graška djeluju protektivno na stanične makromolekule sprečavajući štetne učinke UV zračenja i slobodnih radikala na genetički materijal te oksidacijske promjene na proteinima i lipidima, dok je ljuska luka bogata polifenolima koji u određenim uvjetima mogu ispoljiti prooksidacijsko i genotoksično djelovanje The food industry produces a large amount of waste biomass that poses as an environmental risk, hence today new ways of valorizing waste materials are being intensively researched. By-products such as pea peel and onion skin are underutilized sources of bioactive compounds, such as polyphenols, secondary plant metabolites that have high antioxidant potential and provide significant protection against the development of various chronic diseases. The aim of this study is to determine the biological effect of pea peel and onion skin extracts on cellular macromolecules, human oral epithelial carcinoma cell line Cal27 and hepatocellular liver carcinoma cell line HepG2 in concentrations at which these compounds can be found in the human digestive system. The results showed that polyphenols contained in the pea peel have a protective effect on cellular macromolecules by preventing the harmful effects of UV radiation and free radicals on genetic material and oxidative changes on proteins and lipids, while onion skin is rich in polyphenols that can exert prooxidative and genotoxic effects.

Yellow gentian (Gentiana lutea L.), a medicinal plant widely used in traditional medicine, displays multiple biological effects, ranging from beneficial to toxic. Since many promising applications have been reported so far, our aim was to evaluate its potential concentration- and time- dependent cytotoxic and genotoxic effects in vitro. To that end we exposed human peripheral blood mononuclear cells to 0.5, 1, and 2 mg/mL of yellow gentian root extract (YGRE) to determine its effects on oxidative stress parameters [pro/antioxidant balance (PAB) and lipid peroxidation], DNA damage (alkaline comet assay and chromosome aberrations), and cell viability (trypan blue exclusion test). Cell viability decreased with increasing concentrations and treatment duration. Only the lowest YGRE concentration (0.5 mg/mL) increased oxidative stress but produced minor DNA damage and cytotoxicity. At higher concentrations, redox parameters returned to near control values. The percentage of chromosome aberrations and percentage of DNA in the comet tail increased with increased YGRE concentration after 48 h and declined after 72 h of treatment. This points to the activation of DNA repair mechanism (homologous recombination), evidenced by the formation of chromosomal radial figures after 72 h of treatment with the highest YGRE concentration of 2 mg/mL. Our results suggest that YGRE, despite induction of cytotoxic and genotoxic effects, activates cell repair mechanisms that counter oxidative and DNA lesions and induce cell death in highly damaged cells. Therefore, observed protective effects of yellow gentian after longer exposure could be a result of activated repair and removal of cells with irreparable damage., Žuta lincura (Gentiana lutea L.), ljekovita biljka koja se često koristi u tradicionalnoj medicini, pokazuje višestruke biološke učinke, od korisnih do toksičnih. Budući da je do sada zabilježeno mnogo mogućih primjena, cilj nam je bio procijeniti potencijalne citotoksične i genotoksične učinke ekstrakta korijena te biljke, koji ovise o njegovoj koncentraciji i vremenu izlaganja in vitro. Mononuklearne stanice ljudske periferne krvi izložili smo ekstraktu korijena žute lincure (YGRE), koncentracije 0,5 mg/mL, 1 mg/mL i 2 mg/mL, da bismo utvrdili njegove učinke na parametre oksidacijskoga stresa [pro/antioksidacijski balans (PAB) i peroksidacija lipida], oštećenja DNA (alkalni komet test i kromosomske aberacije) i preživljavanje stanica (tripan plavo bojenje). Preživljavanje stanica smanjivalo se s povećanjem koncentracije i trajanja izlaganja. Samo najniža koncentracija YGRE-a (0,5 mg/mL) dovela je do povećanja oksidacijskoga stresa, ali je proizvela manja oštećenja DNA i citotoksičnost. Pri višim koncentracijama, redoks parametri vratili su se blizu razine kontrolnih vrijednosti. Postotak kromosomskih aberacija i postotak DNA u repu kometa povećavao se s povećanom koncentracijom YGRE-a nakon 48 sati i smanjivao nakon 72 sata tretmana. To upućuje na aktiviranje mehanizma popravka DNA (homologna rekombinacija), što dokazuje prisutnost kromosomskih radijalnih struktura nakon 72 sata tretmana najvišom koncentracijom YGRE-a od 2 mg/mL. Naši rezultati pokazuju da YGRE, unatoč induciranju citotoksičnih i genotoksičnih učinaka, aktivira mehanizme popravka stanica koji suzbijaju oksidacijske i DNA lezije i induciraju smrt visoko oštećenih stanica. Zaključak je da uočeni zaštitni učinci dužeg izlaganja ekstraktu korijena žute lincure mogu biti rezultat aktivnoga popravka i uklanjanja stanica s nepopravljivim oštećenjima.

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.

Cilj istraživanja: Iako se titanijum (Ti) temeljeni dentalni implantati smatraju bioinertnima, potvrđeno je kao su podložni koroziji i trošenju. Cilj ove in vivo provedene studije bio je procijeniti genotoksičan potencijal dvije vrste dentalni implantata u gingivnim epitelnim stanicama. Materijali i metode: U 78 ispitanika uzeti su uzorci oljuštenih gingivnih stanica tri puta. Prije postavljanja dentalnih implantata (T0), 90 dana nakon implantacije - neposredno prije postavljanja gingiva formera (T1) te 21 dan od postavljanja gingiva formera (T2). DNK oštećenje je procjenjivano mikronukleus testom. Rezultati: Dobiveni rezultati pokazuju kako nakon postavljanja obje vrste ispitivanih dentalnih implantata (Ankylos i Dentium) dolazi do značajnog povećanja broja binuklearnih stanica u odnosu na početne vrijednosti (T0 naspram T1 ; p = 0, 011 i p ≤ 0, 001). Također u odnosu na početne vrijednosti, nakon postavljanja gingiva formera (T0 naspram T2) kod ispitanika dolazi do značajnog povećanja broja stanica s mikronukleusom (p = 0, 007 i p = 0, 022) i binuklearnih stanica (p = 0, 006 i p ≤ 0, 001). Zaključak: Na osnovu dobivenih rezultata može se zaključiti kako titanijum temeljeni dentalni implantati ne izazivaju citotoksične i genotoksične učinke u gingivnim stanicama nakon postavljanja. Uočeni blagi učinci ne mogu se označiti kao biološki relevantni.

Novel three-dimensional (3D) nanohydroxyapatite-PLGA scaffolds with high porosity was developed to better mimic mineral component and microstructure of natural bone. To perform a final assessment of this nanomaterial as a potential bone substitute, its toxicological profile was particularly investigated. Therefore, we performed a comet assay on human monocytes for in vitro genotoxicity investigation, and the systemic subchronic toxicity investigation on rats being per oral feed with exactly administrated extract quantities of the nano calcium hydroxyapatite covered with tiny layers of PLGA (ALBO-OS) for 120 days. Histological and stereological parameters of the liver, kidney, and spleen tissue were analyzed. Comet assay revealed low genotoxic potential, while histological analysis and stereological investigation revealed no significant changes in exposed animals when compared to controls, although the volume density of blood sinusoids and connective tissue, as well as numerical density and number of mitosis were slightly increased. Additionally, despite the significantly increased average number of the Ki67 and slightly increased number of CD68 positive cells in the presence of ALBO-OS, immunoreactive cells proliferation was almost neglected. Blood analyses showed that all of the blood parameters in rats fed with extract nanomaterial are comparable with corresponding parameters of no feed rats, taken as blind probe. This study contributes to the toxicological profiling of ALBO-OS scaffold for potential future application in bone tissue engineering.