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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.

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.

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 (

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.

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.

Kolorektalni karcinom najčešći je uzrok smrti nakon bolesti srca i krvožilnog sustava, a podjednako zahvaća muškarce i žene. Liječenje kolorektalnog karcinoma uključuje kirurško liječenje te primjenu kemoterapije i zračenja. THC je glavna aktivna komponenta biljke Cannabis sativa, a u zadnje se vrijeme sve više koristi kao suportivna terapija kemoterapiji. Cilj ovog rada je istražiti citotoksičan, prooksidativan i genotoksičan učinak THC-a. Kao test sustavi korištene su humane stanične linije epitelnih stanica karcinoma jezika (Cal 27), hepatocelularnog karcinoma jetre (Hep G2) i adenokarcinoma epitela debelog crijeva (Caco-2). Stanice su bile izložene djelovanju THC-a tijekom 2 – 48 sati, u rasponu koncentracija 0,33 μg mL-1 do 800 μg mL-1. Dokazano je da THC djeluje citotoksično ovisno o duljini tretmana stanica i primjenjenoj koncentraciji. Prooksidativan je učinak povezan s citotoksičnošću. Na genotoksičan učinak su najotpornije stanice Caco-2, a Cal 27 i Hep G2 pokazuju jasnu ovisnost genotoksičnog učinka THC-a o produljenju vremena inkubacije i povećanju koncentracije. Colorectal cancer is the most common cause of death, after cardiovascular diseases which affects both men and women equally. Colorectal cancer treatment involves a surgical operation with use of chemotherapy and radiation. THC is the major active compound of Cannabis sativa plant which has been increasingly used as a support for chemotherapy. The aim of this study is to determine cytotoxic, pro-oxidative and genotoxic effects of THC. Human cell lines of oral epithelial carcinoma cells (Cal 27), hepatocellular liver carcinoma cells (Hep G2) and colon epithelial adenocarcinoma cells (Caco-2) were used as test systems. Cells were exposed to THC 2 – 48 hours, in concentration range from 0,33 μg mL-1 to 800 μg mL-1. It has been shown that cytotoxicity of THC depends on the duration of cell treatments and concentration used. Pro-oxidative effect was related with cytotoxicity. Caco-2 cells are most resistant to genotoxicity, and Cal 27 and Hep G2 cells show a clear connection of genotoxic effect of THC with the prolonging of incubation time and concentration used.

Od ukupnog broja svih godišnje dijagnosticiranih karcinoma u svijetu, karcinomi probavnog sustava su na vodećoj poziciji. Među njima najvišu pojavnost ima kolorektalni karcinom, što ga svrstava među vodeće zdravstvene probleme kako u svijetu tako i u Hrvatskoj. Irinotekan (CPT-11) je polusintetski analog kamptotecina (CPT) koji je glavni lijek izbora u terapiji metastatskog kolorektalnog karcinoma. Cilj ovog rada je istražiti citotoksičnost, genotoksičnost i svojstvo indukcije slobodnih radikala različitih koncentracija irinotekana. 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 tijekom 2, 4, 24 i 48 sati. Dokazano je da irinotekan djeluje toksično produljenjem vremena tretmana na obje stanične linije. Na stanicama jetre toksičnost raste u ovisnosti o koncentraciji, dok na stanicama crijeva ovisnost o kocentraciji nije dokazana. Također je uočena povezanost citotoksičnog učinka irinotekana s indukcijom slobodnih radikala. S produljenjem vremena inkubacije dolazi do genotoksičnog učinka irinotekana na obje tretirane stanične linije. Of the total number of all yearly diagnosed carcinomas in the world, cancers that affect the digestive system are in a leading position. Among them, the highest incidence is colorectal cancer, which puts it among the leading health problems in the world as well as in Croatia. Irinotecan (CPT-11) is a semi-synthetic analogue of camptothecin (CPT) which is the major drug in the choice of metastatic colorectal cancer therapy. The aim of this work is to investigate the cytotoxicity, genotoxicity and induction of free radicals of different concentrations of irinotecan. As the test system the human hepatocellular carcinoma cell line (HepG2) and the human colon adenocarcinoma cell line (Caco-2) were exposed to irinotecan during 2, 4, 24, and 48 hours. Results have shown that irinotecan cytotoxicity is dependent on the cell line. In liver cells, toxicity is increasing depending on concentrations, while on the colon cells no dependence on the concentration has been demonstrated. Also, the association of cytotoxic effect of irinotecan with the induction of free radicals was observed. With the prolongation of incubation time, the genotoxic effect of irinotecan on both treated cell lines is reached.

Svrha rada: Usporediti citotoksičnost/genotoksičnost zagrijanih kompozita polimeriziranih preko CAD/CAM overleja na kulturi izoliranih ljudskih limfocita. Materijali i postupci: Mikrohibridni (Z100, 3M ESPE) i nanopunjeni kompozit (Filtek Supreme Ultra, 3M ESPE) zagrijani su u uređaju za zagrijavanje kompozita (Calset, AdDent Inc.) na temperaturama od 37 °C (T1), 54 °C (T2) i 68 °C (T3). Mala količina zagrijanog kompozita stavljena je u cilindrični kalup (promjera 6 mm, debljine 0.65 mm), prekrivena Mylar folijom, sprešana, te osvjetljena izravno, preko CAD/CAM keramički pojačanog polimera (CRP) (LAVA Ultimate, 3M ESPE) ili CAD/CAM litij disilikatnog keramičkog overlaja (LDC)(e.max, Ivoclar/Vivadent) debljine 2 mm. Odmah nakon osvjetljavanja uzorci su stavljeni u staničnu kulturu limfocita izoliranih iz periferne krvi. Citotoksičnost je izmjerena metodom dvojnog bojenja etidijevim bromidom i akridinskom narančastom bojom koja omogućuje određivanje postotka živih stanica te stanica u apoptozi i nekrozi na osnovi njihovih morfoloških obilježja. Genotoksičnost je procijenjena uporabom komet-testa u alkalnim uvjetima. Rezultati: Za Z100, najveći postotak živih stanica zabilježen je na T1 (93,7 %) nakon izravnog osvjetljavanja; slijedi osvjetljavanje preko CRP-a (92,3 %) te LDC-a (91,7 % T1, T3). Za Filtek Supreme Ultra najveći postotak živih stranica zabilježen je nakon osvjetljavanja preko CRP-a (91,2 % T2), LDC-a (90 % T1, T3) te pri izravnom osvjetljavanju (88,7 % T2). Zaključak: Za oba ispitivana materijala, zagrijavanje na T1 i T2 postupak je izbora. S obzirom na genotoksičnost, ne preporučuje se zagrijavanje na T3., Objectives: The aim was to compare cytotoxicity/genotoxicity of pre-heated composites polymerized through CAD/CAM overlays on isolated human peripheral blood lymphocytes. Material and Methods: A microhybrid (Z100, 3M ESPE) and nanofilled composite (Filtek Supreme Ultra, 3M ESPE) were heated in a heating unit (Calset, AdDent Inc.) at different temperatures: 37 oC, 54 oC, and 68 oC. A small amount of heated composite was placed in a cylindrical mold (6mm diameter; 0.65mm thick), covered with a Mylar sheet, pressed and light-cured directly and through 2 mm thick CAD/CAM ceramic-reinforced polymer (CRP)(LAVA Ultimate, 3M ESPE) or CAD/CAM lithium disilicate ceramic (LDC)(e.max, Ivoclar/Vivadent) overlay. After curing, the specimens were immediately placed in a prepared lymphocyte cell culture. Cytotoxicity was assessed using a dye exclusion method by simultaneous staining with ethidium bromide and acridine orange, aimed to determine percentages of viable, apoptotic and necrotic cells. Genotoxicity was studied using alkaline comet assay. Results: For Z100, the highest percentage of viable cells is recorded at T1 (93.7%) after direct light curing, followed by light curing through CRP (92.3%) and through LDC (91.7%T1,T3). For Filtek Supreme Ultra, the highest percentage of viable cells is recorded while curing through CRP (91.0% T2), followed by LDC (90% T1,T3) and direct light curing (88.7%T2). Conclusion: For both tested materials, preheating the procedure at T1 and T2 may be the procedure of choice. In terms of genotoxicity, reheating at T3 may not be suggested.

Nanodelci imajo velik potencial za uporabo v medicini in farmaciji, predvsem kot sistemi za dostavo zdravilnih učinkovin za boj proti rakavim obolenjem. Prednost je, da izbrano učinkovino/spojino lahko zaščitijo, stabilizirajo in selektivno dostavijo na tarčno mesto. Vendar je pred uporabo nanodelcev potrebno opredeliti njihovo toksično delovanje. Cilj magistrske naloge je bil preveriti morebiten toksičen učinek s poli-glutaminsko kislino (PGA) obdanih nanodelcev (ND) poli-epsilon- kaprolaktona (PCL), ND poli-(mlečne-ko-glikolne) kisline (PLGA) z inkapsuliranimi ND selena (Se) ter z govejim serumskim albuminom (BSA) obdanih ND Se. Po izpostavitvi celic HepG2 raztopini testiranih ND, smo s testom MTT določali citotoksičnost vzorcev, s testom DCFH-DA merili nastanek reaktivnih kisikovih spojin (ROS) in s tem vpliv na oksidativni stres, ter s testom komet preučevali genotoksično delovanje ND. Ugotovili smo, da noben od testiranih vzorcev ne izkazuje toksičnih učinkov na celicah človeškega hepatoma (HepG2). Največji porast ROS in prelomov DNA smo opazili pri celicah, ki so bile izpostavljene ND PCL brez PGA. Nanoparticles have a high potential for use in medicine and pharmacy, especially as nanodelivery systems used in anti-cancer therapy. Their advantage is protection, stabilization and selective delivery of a compound to a target site. Although, potential toxicity of nanoparticles need to be evaluated before their use. The goal of our master thesis was to investigate potential toxic effect of poly-epsilon-caprolactone (PCL) nanoparticles coated with poly-glutamic acid (PGA), poly-(lactic-co-glycolic) acid (PLGA) with encapsulated selenium (Se) nanoparticles and selenium (Se) nanoparticles coated with bovine serum albumin (BSA). After exposure to solutions of tested nanoparticles we evaluated their cytotoxicity with MTT assay, reactive oxygen species (ROS) production and subsequent oxidative stress with DCFH-DA assay and genotoxic effects of nanoparticles with Comet assay. Our results showed no biologically relevant toxic effects on human hepatoma (HepG2) cell line in all tested samples of nanoparticles although slightly increased ROS production and DNA strand breaks was detected in cells, which were exposed to ND PCL without PGA.

An experiment was performed under in vivo conditions on bone marrow cells of Wistar rats. The following doses of levamisole hydrochloride were tested: a therapeutic dose of 2.2 mg/kg bm, a dose of 4.4 mg/kg bm, LD50 -25% mg/kg bm, and LD50 -75% mg/kg bm. We followed the effect of levamisole hydrochloride on kinetics of the cell cycle and the appearance of structural and numeric changes in chromosomes in bone marrow cells. The therapeutic dose of levamisole of 2.2 mg/kg bm exhibited a capability to increase mitotic activity in the observed cells, thus confirming knowledge of the immunostimulative effect of this dose of the medicine under in vivo conditions. The other tested doses of levamisole in this experiment, observed in comparison with the control group, had an opposite effect, namely, they caused a reduction in the mitotic activity of bone marrow cells. All the examined doses in vivo exhibited the ability to induce numeric (aneuploid and polyploid) and structural (lesions, breaks and insertions) chromosomal aberrations. It can be concluded on the grounds of these findings that the examined doses have a genotoxic effect. Eksperiment je izveden u in vivo uslovima na ćelijama kostne srži pacova Wistar soja. Testirane su doze levamizol hidrohlorida i to: terapijska doza od 2.2 mg/kg, t.m., zatim doza od 4,4 mg/kg t.m., LD50 do 25% mg/kg t.m. i LD50 -75% mg/kg t.m.. Praćen je uticaj levamizol hidrohlorida na kinetiku ćelijskog ciklusa i pojavljivanje strukturnih i numeričkih promena na hromozomima u ćelijama kostne srži. Terapijska doza levamizola od 2,2 mg/kg t.m. pokazala je sposobnost povećanja mitotske aktivnosti posmatranih ćelija, tako potkrepljujući saznanja o imunostimulativnom efektu ove doze leka u in vivo uslovima. Ostale testirane doze levamizola u ovom eksperimentu, posmatrano u odnosu na kontrolnu grupu, imale su suprotan efekat, tj. uslovile su smanjenje mitotske aktivnosti ćelija kostne srži. Sve ispitivane doze in vivo pokazuju sposobnost indukcije numeričkih (aneuploidija i polipleidija) i strukturnih (lezije, prekidi i insercije) aberacija hromozoma. Na osnovu toga može da se zaključi da ispitivane doze imaju genotoksični efekat.

Cytotoxic and genotoxic examinations were performed of the effect of levamisole hydrochloride (2.2 mg/kg bm, 4.4 mg/kg bm, LD50-25% mg/kg bm and LD50-75% mg/kg bm) on bone marrow cells of mice of the BALB/c strain. The effect of levamisole hydrochloride on kinetics of the cellular cycle and the appearance of structural and numerical changes in chromosomes of bone marrow cells were followed. The therapeutic dose of levamisole of 2.2 mg/kg bm showed the ability to increase the mitotic activity of the observed cells, thus confirming knowledge of the immunostimulative effect of this dose of the medicine under in vivo conditions. The other tested doses of levamisole in this experiment, observed in comparison with the control group, had an opposite effect, i.e. they caused a reduction in the mitotic activity of bone marrow cells. All the examined doses in vivo showed the ability of inducing numeric (aneuloid and polyploid) and structural (lesions, breaks and insertions) chromosomal aberrations. On this basis, it can be concluded that the examined doses have a genotoxic effect. Obavljena su citotoksična i genotoksična ispitivanja levamizol hidrohlorida (2.2 mg/kg, t.m., 4,4 mg/kg t.m, LD50-25% mg/kg t.m. i LD50-75% mg/kg t.m) na ćelijama kostne srži miša BALB/c soja. Praćen je uticaj levamizol hidrohlorida na kinetiku ćelijskog ciklusa i pojavu strukturnih i numeričkih promena na hromosomima u ćelijama kostne srži. Terapijska doza levamizola od 2,2 mg/kg t.m. pokazala je sposobnost povećanja mitotske aktivnosti posmatranih ćelija, tako potkrepljujući saznanja o imunostimulativnom efektu ove doze leka u in vivo uslovima. Ostale testirane doze levamizola u ovom eksperimentu, posmatrano u odnosu na kontrolnu grupu, imale su suprotan efekat, tj. uslovile su smanjenje mitotske aktivnosti ćelija kostne srži. Sve ispitivane doze in vivo pokazuju sposobnost indukcije numeričkih (aneuploidije polipleidije) i strukturnih (lezije, prekidi i insercije) aberacija hromosoma. Na osnovu toga može da se zaključi da ispitivane doze imaju genotoksični efekat.

U ovom je radu iznesen pregled najnovijih ihtioloških istraživanja u kojima se autori služe tzv. mikronukleusnim testom. Test se u znanstvenoj literaturi spominje već čitav niz godina kao jedno od osnovnih sredstava za utvrđivanje oštećenja na DNK organizma–modela. Znanstvenici se njime koriste radi dokazivanja genotoksičnih utjecaja različitih tvari na organizme, u ovom slučaju hidrobionte., The purpose of this paper was to give an insight into the most recent ichthiology research in which Micronucleus test is used. This test has been used for some time now as a tool for determining DNA damage in model–organism. Scientists have been using it in order to assess genotoxic effects which certain substancies exhibit towards different organisms, in this case hydrobionts.

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...

The purpose of this study was to evaluate the genotoxic potential of components leached from two conventional self-curing glass-ionomer cements (Fuji IX and Ketac Molar), and light-curing, resin modified glass-ionomer cements (Vitrebond, Fuji II LC). Evaluation was performed on human lymphocytes using alkaline and hOGG1 modified comet, and micronucleus assays. Each material, polymerised and unpolymerised, was eluted in extracellular saline (1 cm2 mL-1) for 1 h, 1 day, and 5 days. Cultures were treated with eluates using final dilutions of 10-2, 10-3, and 10-4. Alkaline comet assay did not detect changes in DNA migration of treated cells regardless of the ionomer tested, polymerisation state, and elution duration. Glass ionomers failed to significantly influence micronucleus frequency. No oxidative DNA damage in treated lymphocytes was observed using hOGG1 modified comet assay. Obtained results indicate high biocompatibility of all tested materials used in the study under experimental conditions., Svrha istraživanja bila je procijeniti genotoksični potencijal komponenata koje izlučuju dva konvencionalna samopolimerizirajuća stakleno-ionomerna cementa (Fuji IX i Ketac Molar) te svjetlosno polimerizirajući i smolom modificirani stakleno-ionomerni cementi (Vitrebond, Fuji II LC). Istraživanje je provedeno na ljudskim limfocitima primjenom alkalnog komet testa, komet testa modificiranog hOGG1 enzimom te mikronukleus testa. Svaki materijal, polimerizirani i nepolimerizirani, eluiran je u fiziološkoj otopini (1 cm2 mL-1) tijekom jednog sata, jednog dana i tijekom 5 dana. Kulture limfocita tretirane su eluatima u razrjeđenjima 10-2, 10-3 i 10-4. Alkalnim komet testom nisu zabilježene promjene u migraciji DNA iz tretiranih stanica bez obzira na ispitani ionomer, vrstu polimerizacije i trajanje elucije. Izloženost staklenim ionomerima nije značajno utjecala na učestalost mikronukleusa. Primjenom hOGG1 modificiranog komet testa nije zamijećeno oksidativno oštećenje DNA u tretiranim limfocitima. Dobiveni rezultati upućuju na visoki stupanj biokompatibilnosti svih testiranih materijala koji su se koristili u eksperimentalnim uvjetima.

T-2 toxin is the most toxic type A trichothecene mycotoxin. It is the secondary metabolite of the Fusarium fungi, and is common in grain and animal feed. Toxic effects have been shown both in experimental animals and in livestock. It has been implicated in several outbreaks of human mycotoxicoses. Toxic effects in poultry include inhibition of protein, DNA, and RNA synthesis, cytotoxicity, immunomodulation, cell lesions in the digestive tract, organs and skin, neural disturbances and low performance in poultry production (decreased weight gain, egg production, and hatchability). Concentrations of T-2 toxin in feed are usually low, and its immunosuppressive effects and secondary infections often make diagnosis difficult. If at the onset of the disease, a change in diet leads to health and performance improvements in animals, this may point to mycotoxin poisoning. Regular control of grain and feed samples is a valuable preventive measure, and it is accurate only if representative samples are tested. This article reviews the incidence and toxic effects of T-2 toxin in poultry., T-2 toksin je najtoksičniji predstavnik trikotecenskih mikotoksina tipa A. On je sekundarni produkt metabolizma plijesni roda Fusarium i često je prisutan u žitaricama i hrani za životinje. Štetni učinci uočeni su u eksperimentalnih životinja i životinja u uzgoju. On se povezuje s pojavom bolesti ljudi od mikotoksikoza. Učinci toksina u peradi su višestruki: inhibicija sinteze proteina, DNA i RNA, citotoksični učinak, imunomodulatorni učinak, oštećenje stanica probavnog sustava, organa i kože, živčani poremećaji te pad proizvodnih karakteristika u uzgoju peradi (slabiji prirast, pad nesivosti i valivosti). Koncentracije T-2 toksina u hrani redovito su vrlo malene, a zbog imunosupresivnog djelovanja toksina te istodobne sekundarne infekcije bolest se često teško dijagnosticira. Pri pojavi bolesti promjenom hrane može doći do poboljšanja zdravstvenog stanja, što tako|er upućuje na moguće trovanje mikotoksinima. Redovita kontrola uzoraka žitarica i hrane za životinje jedna je od preventivnih mjera, a detekcija mikotoksina u žitaricama i hrani pouzdana je samo ako se ispituje reprezentativan uzorak. U radu su opisani učestalost i toksični učinci T-2 toksina u peradi.