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The aim of this study was to investigate the genotoxic potential of low doses of chlorpyrifos (CPF) on blood and bone marrow cells in adult male Wistar rats. CPF was administered by oral gavage at daily doses of 0.010, 0.015, and 0.160 mg/kg of body weight (bw) for 28 consecutive days. Positive control (PC) was administered 300 mg/kg bw/day of ethyl methane sulphonate (EMS) for the final three days of the experiment. Toxic outcomes of exposure were determined with the in vivo micronucleus (MN) assay and alkaline comet assay. The 28-day exposure to the 0.015 mg/kg CPF dose, which was three times higher than the current value of acute reference dose (ARfD), reduced body weight gain in rats the most. The in vivo MN assay showed significant differences in number of reticulocytes per 1000 erythrocytes between PC and negative control (NC) and between all control groups and the groups exposed to 0.015 and 0.160 mg/kg bw/day of CPF. The number of micronucleated polychromatic erythrocytes per 2000 erythrocytes was significantly higher in the PC than the NC group or group exposed to 0.015 mg/kg bw/day of CPF. CPF treatment did not significantly increase primary DNA damage in bone marrow cells compared to the NC group. However, the damage in bone marrow cells of CPF-exposed rats was much higher than the one recorded in leukocytes, established in the previous research. Both assays proved to be successful for the assessment of CPFinduced genome instability in Wistar rats. However, the exact mechanisms of damage have to be further investigated and confirmed by other, more sensitive methods., Istražen je genotoksični potencijal niskih doza klorpirifosa na uzorcima krvi i stanica koštane srži u odraslih mužjaka štakora soja Wistar. Pokusnim je životinjama klorpirifos bio 28 dana oralno apliciran pomoću sonde u dnevnim dozama od 0,010 mg/kg t. m., 0,015 mg/kg t. m. i 0,160 mg/kg t. m. Kao pozitivna kontrola korišten je etil metan sulfonat (EMS) u dozi od 300 mg/kg t. m. tijekom posljednja tri dana pokusa. Toksični ishodi izloženosti klorpirifosu istraženi su primjenom in vivo mikronukleus (MN) testa i alkalnoga komet-testa. Utvrdili smo da je 28-dnevna izloženost klorpirifosu u dozi od 0,015 mg/kg t. m./dan, koja je trostruko viša od važeće vrijednosti akutne referentne doze, u najvećoj mjeri smanjila prirast tjelesne mase štakora. Rezultati MN-testa upućuju na značajne razlike u broju retikulocita na 1000 eritrocita između pozitivne i negativne kontrole te između obiju kontrola i skupina izloženih klorpirifosu u dnevnim dozama 0,015 i 0,160 mg/kg t. m. Broj polikromatskih eritrocita s mikronukleusima na 2000 eritrocita u pozitivnoj kontroli bio je značajno povećan u usporedbi s negativnom kontrolom te s uzorcima krvi štakora izloženih klorpirifosu u dnevnoj dozi od 0,015 mg/kg t. m. Izloženost CPF-u nije uzrokovala statistički značajan porast razine primarnih oštećenja DNA u stanicama koštane srži u usporedbi s razinama spontanih oštećenja DNA, izmjerenima alkalnim komet-testom u negativnoj kontroli. Međutim, razine oštećenja u stanicama koštane srži štakora izloženih klorpirifosu bile su značajno više od onih zabilježenih u leukocitima, koje su poznate iz prethodnih istraživanja. Oba su se testa pokazala uspješnima za procjenu nestabilnosti genoma izazvanih klorpirifosom u Wistar štakora. Međutim, točni mehanizmi oštećenja moraju se dodatno istražiti i potvrditi drugim osjetljivijim metodama.

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.

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.

The aim of this study was to assess DNA damage in Turkish coal miners with the buccal micronucleus cytome (BMCyt assay as the least invasive and therefore most practical method that may find wider application in coal miner biomonitoring. Buccal epithelial cell samples were taken from 54 coal miners and 42 controls from Zonguldak, Turkey to establish their micronucleus (MN), binucleus (BN), condensed chromatin (CC), karyorrhectic (KHC), karyolytic (KYL), nuclear bud (NBUD), and pyknotic (PYC) frequencies. We also analysed the effects of confounding factors such as age, years of work at the mine, smoking, alcohol drinking, and use of protective equipment on differences in MN frequencies. Two miners had confirmed and three suspect pneumoconiosis, whereas 49 displayed normal chest radiographs. MN, BN, KHC, and NBUD frequencies were significantly higher in coal miners than controls. Years of work at the mine also showed a significant effect on buccal MN frequencies in coal miners, but we found no correlation between MN frequencies and age, smoking, and alcohol consumption. In conclusion, BMCyt assay proved itself an accurate and practical screening method, as it can detect DNA damage much earlier than pneumoconiosis develops., Cilj je ovoga istraživanja bio procijeniti oštećenje DNA u turskih rudara primjenom tzv. Cytome inačice mikronukleustesta na bukalnim epitelnim stanicama (engl. buccal micronucleus cytome assay, krat. BMCyt) kao najmanje invazivne i stoga najpraktičnije metode koja bi mogla naći svoju primjenu u biomonitoringu rudara u ugljenokopima. Uzorci bukalnih epitelnih stanica prikupljeni su od 54 rudara i 42 kontrolna ispitanika iz Zonguldaka u Turskoj radi utvrđivanja učestalosti mikronukleusa (MN), binuklearnih stanica (BN), zgusnutoga kromatina (CC), karioreksije (KHC), kariolize (KYL), jezgrinih pupova (NB) i piknoze (PYC). Osim toga, analizirali smo koliko čimbenici poput dobi, godina staža u rudniku, pušenja, pijenja alkohola i uporabe zaštitne opreme utječu na razlike u nalazima učestalosti MN-a. Dva su rudara imala potvrđenu dijagnozu pneumokonioze, u tri je rudara postojala sumnja na nju, a ostalih 49 rudara imalo je uredne rendgenske nalaze. Učestalosti MN-a, BN-a, KHC-a i NB-a bile su značajno više u rudara nego u kontrolnih ispitanika. Godine staža u rudniku također su značajno pridonijele povišenoj učestalosti MN-a u rudara, ali takva korelacija nije utvrđena za dob, pušenje i pijenje alkohola. BMCyt test pokazao se preciznim i praktičnim probirnim testom jer otkriva oštećenje DNA mnogo ranije nego što se pojavi pneumokonioza.

Individuals chronically exposed to low-level ionising radiation (IR) run the risk of harmful and long-term adverse health effects, including gene mutations and cancer development. The search for reliable biomarkers of IR exposure in human population is still of great interest, as they may have a great implementation potential for the surveillance of occupationally exposed individuals. In this context, and considering previous literature, this study aimed to identify mutations in the human interferon alpha-2b (hIFNα-2b) as a potential biomarker of occupational chronic low-dose IR exposure linking low-IR exposure to the effects on haematopoiesis and reduced immunity. The analysis was performed in the genomic DNA of 51 uranium miners and 38 controls from Kazakhstan, and in 21 medical radiology workers and 21 controls from Italy. hIFNα-2b gene mutations were analysed with the real-time polymerase chain reaction (PCR) or Sanger sequencing. However, none of the investigated workers had the hIFNα-2b mutation. This finding highlights the need for further research to identify biomarkers for early detection of health effects associated with chronic low-dose IR exposure., Kronična izloženost niskim razinama ionizirajućega zračenja povezana je s rizikom od dugoročnih štetnih posljedica za zdravlje, što obuhvaća i mutacije gena te nastanak raka. U tijeku je potraga za pouzdanim biopokazateljima izloženosti ionizirajućem zračenju u ljudi, budući da njihova primjena može značajno unaprijediti praćenje profesionalno izloženih osoba. U tom smislu, a s obzirom na ranija saznanja, cilj je ovoga istraživanja bio utvrditi mutacije gena za proizvodnju humanoga interferona alfa-2b (hIFNα-2b gena) kao mogućega biopokazatelja profesionalne kronične izloženosti niskim dozama ionizirajućega zračenja, koje je usto povezano s djelovanjem na hematopoezu i pad imuniteta. Analiziran je genomski DNA 51 rudara u rudnicima uranija te 38 kontrolnih ispitanika iz Kazahstana, odnosno genomski DNA 21 zdravstvenoga radnika na radiologiji i 21 kontrolnoga ispitanika iz Italije. Mutacije hIFNα-2b gena utvrđivane su metodom lančane reakcije polimerazom u stvarnom vremenu (engl. real-time PCR) odnosno sekvenciranjem prema Sangeru, ali se pokazalo da niti jedan radnik nije imao niti jednu od deset traženih mutacija toga gena. Stoga ne preostaje drugo nego i dalje tražiti pouzdane biopokazatelje za rano otkrivanje štetnih zdravstvenih učinaka povezanih s kroničnom izloženosti niskim dozama ionizirajućega zračenja.

Razvoj industrije i poljoprivrede, kao i globalna urbanizacija, doveli su do pojave velikog broja polutanata u životnoj sredini, među kojima je i izuzetno toksični teški metal kadmijum. Cilj ove disertacije bila je procena larvi gubara (Lymantria dispar, L.) kao potencijalnog bioindikatora zagađenosti kadmijumom. Ispitani su odgovori izabranih fizioloških parametara na različitim nivoima biološke organizacije u prisustvu dve koncentracije metala (50 i 100 μg Cd/g suve hrane). Radi ispitivanja efekta porekla populacije, legla gubara sakupljena su na dve nezagađene lokacije (Kosmaj i Homoljske planine) i dve zagađene (pored Ibarske magistrale i borskog rudnika). Larve sa Kosmaja odlikuje veća osetljivost digestivnih enzima srednjeg creva (proteaza, alkalnih i kiselih fosfataza i nespecifičnih esteraza) na tretmane, naročito na nižu koncentraciju kadmijuma, u odnosu na one uz Ibarsku magistralu. Od ispitanih detoksifikacionih mehanizama u crevu, indukcija Hsp70 značajnija je kod kosmajskih larvi, a sinteza metalotioneina kod ibarskih. Trend sniženja aktivnosti antioksidativnih enzima, superoksid dismutaze i katalaze, uočen je kod obe populacije. U svim grupama tretiranim kadmijumom najveća količina ovog metala eliminiše se putem fecesa, ali intenzivnije u ibarskoj populaciji u odnosu na kosmajsku nakon tretmana sa 50 μg Cd/g suve hrane. Kod homoljskih gusenica tretmani kadmijumom doveli su do povećanja nivoa oštećenja DNK u hemocitama i dozno-zavisnog sniženja njihove vijabilnosti, dok je u populaciji iz Bora zabeleženo samo smanjenje vijabilnosti nakon primene više koncentracije metala. Štetni efekti kadmijuma i visoka cena detoksifikacionih mehanizama odrazili su se i na osobine fitnesa u vidu promena mase larvi, relativne brzine rasta i trajanja razvića kod svih populacija u različitom stepenu. Primena većine ispitanih parametara (specifične aktivnosti tripsina, nelizozomskih kiselih fosfataza i nespecifičnih esteraza, nivoa ekspresije Hsp70, vijabilnosti hemocita i nivoa oštećenja DNK) kao biomarkera zagađenosti životne sredine kadmijumom ograničena je na osetljive populacije gubara koje prethodno nisu bile izložene polutantima, dok bi koncentracija metalotioneina mogla biti podobna kao biomarker kod populacija sa lokacija kontaminiranih tokom dužeg perioda. Industrial and agricultural development as well as global urbanization have caused considerable increase in the environmental pollution including extremely toxic heavy metal cadmium. The aim of this dissertation was to evaluate gypsy moth (Lymantria dispar, L.) larvae as bioindicators of cadmium contamination. Parameters at different levels of biological organization were assessed after chronic exposure of larvae to the metal (50 and 100 μg Cd/g dry food). To investigate the effects of population origin, gypsy moth egg-masses were collected from two unpolluted localities on Kosmaj and Homolje mountains and two polluted sites near the busy Ibar highway and Bor copper mine. Midgut digestive enzymes (proteases, alkaline and acid phosphatases and nonspecific esterases) of Kosmaj larvae were more sensitive to cadmium, especially to lower metal concentration, compared to those of Ibar larvae. Hsp70 induction was the most prominent midgut detoxification mechanism against cadmium in the population from the unpolluted forest, whereas metallothionein synthesis was the most significant in larvae from the polluted site. Activities of antioxidative enzymes, superoxide dismutase and catalase, exhibited a decreasing trend in both populations. The primary route of cadmium elimination was via feces in all metal-fed groups, but after an exposure to 50 μg Cd/g dry food metal content was significantly higher in feces of Kosmaj larvae compared to those from the highway locality. Exposure of caterpillars from the Homolje mountains to the metal led to the higher level of DNA damage in the hemocytes and a dose-dependent decrease in cell viability, whereas in Bor population only hemocyte viability was reduced after the treatment with 100 μg Cd/g dry food. Devastating cadmium effects and energetically expensive detoxification mechanisms affected the fitness traits (larval mass, relative growth rate or development parameters) in all populations. The application of selected parameters (specific activities of trypsin, nonlisosomal acid phosphatases and nonspecific esterases, Hsp70 expression, hemocyte viability and DNA damage level) as biomarkers of environmental cadmium pollution is limited mostly to sensitive populations that have not been previously exposed to pollutants. Metallothionein concentration might be suitable as a biomarker for the populations at locations that have been contaminated for a long period.

Harmful effects of elevated levels of catecholamines are mediated by various mechanisms, including gene transcription and formation of oxidation products. The aim of this study was to see whether the molecular mechanisms underlying the damaging action of adrenaline on DNA are mediated by reactive oxygen species (ROS). To do that, we exposed human whole blood cells to 10 μmol L-1 adrenaline or 50 μmol L-1 H2O2 (used as positive control) that were separately pre-treated or post-treated with 500 μmol L-1 of quercetin, a scavenger of free radicals. Quercetin significantly reduced DNA damage in both pre- and post-treatment protocols, which suggests that adrenaline mainly acts via the production of ROS. This mechanism is also supported by gradual lowering of adrenaline and H2O2-induced DNA damage 15, 30, 45, and 60 min after treatment. Our results clearly show that DNA repair mechanisms are rather effective against ROS-mediated DNA damage induced by adrenaline., Štetni učinci povišenih razina katekolamina posredovani su različitim mehanizmima, uključujući transkripciju gena i formiranje produkata oksidacije. Svrha je ovoga istraživanja bila utvrditi jesu li molekularni mehanizmi koji stoje u osnovi štetnoga djelovanja adrenalina na DNA posredovani reaktivnim vrstama kisika (ROS). Da bismo to postigli, izložili smo humane stanice pune krvi adrenalinu (10 μmol L-1) i vodikovu peroksidu (50 μmol L-1) (pozitivna kontrola), koje su odvojeno predtretirane i post-tretirane kvercetinom (500 μmol L-1), „hvatačem“ slobodnih radikala. Kvercetin je značajno smanjio broj stanica s DNA oštećenjima i u predtretmanu i u post-tretmanu, što je sugeriralo da adrenalin djeluje uglavnom stvaranjem ROSa. Taj je mehanizam podržan i postupnim smanjenjem broja stanica s oštećenjima DNA izazvanim adrenalinom i H2O2 u vremenskim intervalima od 15, 30, 45 i 60 minuta nakon tretmana. Naši rezultati jasno pokazuju da su mehanizmi popravka prilično učinkoviti u odnosu na ROS-posredovana oštećenja DNA izazvana adrenalinom.

U radu je ispitan uticaj odabranih modifikovanih steroida na proliferaciju ćelija humanih malignih tumora i zdravih ćelija, a zatim njihov uticaj na modifikaciju ćelijskog ciklusa, indukovanje apoptoze i genetskih oštećenja u tumorskim ćelijama najsenzitivnijim na ova jedinjenja (MDA-MB-231 ćelijska linija humanog adenokarcinoma dojke, ER-). Ispitana je in vitro citotoksičnost odabranih steroidnih derivata prema ćelijskim linijama MCF-7, MDA-MB-231, PC3, HeLa, HT-29, MRC-5, K562 i A549, i proučavan ćelijski mehanizam koji je u osnovi uočenog antiproliferativnog efekta svakog od ispitivanih derivata. Sva ispitivana jedinjenja indukuju apoptozu MDA-MB-231 ćelija, na različite načine i sa različitim potencijalom. Genotoksikološka studija upotpunjuje rezultate proučavanja efekta ispitivanih steroidnih jedinjenja na biološke sisteme, ukazujući na izostanak genotoksičnosti ovih jedinjenja i njihov biomedicinski potencijal., In this work the effect of selected modified steroids on proliferation of human malignant tumor and healthy cells, as well as their impact on the modification of cell cycle arrest, induction of apoptosis and genetic defects in tumor cells most sensitive to these compounds (MDA-MB-231 cell line of human breast adenocarcinoma, ER-) were examined. We examined the in vitro cytotoxicity of selected steroidal derivatives towards MCF-7, MDA-MB-231, PC-3, HeLa, HT-29, MRC-5, K562 and A549 cell lines, and studied the cellular mechanism that is underlying the antiproliferative activity expressed by these steroidal derivatives. All tested compounds induced apoptosis of MDA-MB-231 cells, in different ways and with different potential. Genotoxicological study complements the results of the study of the effect of tested steroidal compounds on biological systems, pointing out the lack of genotoxicity of these compounds and therefore their biomedical potential.

U ovoj doktorskoj disertaciji ispitan je hemijski sastav i biološke aktivnosti ekstrakata deset samoniklih taksona roda Allium sect. Codonoprasum: A. carinatum subsp. pulchellum, A. carinatum subsp. carinatum, A. fuscum var. gracile, A. fuscum var. fuscum, A. flavum subsp. flavum, A. melanantherum, A. paniculatum subsp. marginatum, A. pallens subsp. tenuiflorum, A. oleraceum i A. rhodopeum, sakupljenih na 27 lokaliteta u Srbiji. Cilj rada bio je da se dobiju podaci o sadržaju biološki aktivnih jedinjenja u ovim, do sada veoma malo ispitanim vrstama roda Allium, i utvrdi njihova potencijalna lekovita vrednost. Analiza hemijskog sastava obuhvatila je: analizu volatilnih komponenti svežih lukovica primenom headspace GC-MS tehnike, kvalitativnu analizu metanolnih ekstrakata primenom tečnohromatografskih metoda (LC-DAD-MS i LC-MS-MS), kvantitativnu analizu odabranih fenolnih jedinjenja LC-MS-MS tehnikom, određivanje sadržaja ukupnih monomernih antocijana i određivanje aktivnosti aliinaze. Ispitivanja bioloških aktivnosti ekstrakata obuhvatila su: određivanje antioksidantne, antiinflamatorne, antimikrobne, antimutagene i genotoksične aktivnosti, kao i ispitivanje uticaja na rast zdravih i tumorskih ćelija i sposobnosti indukcije ćelijske smrti. Sumiranjem dobijenih rezultata može se zaključiti da ispitivani predstavnici roda Allium sect. Codonoprasum predstavljaju bogate izvore biološki aktivnih jedinjenja sa širokim spektrom bioloških aktivnosti. Sa hemotaksonomskog aspekta značajno je da se dimetil-disulfid izdvaja kao najdominantnija i često jedina isparljiva komponenta, da ekstrakti većine vrsta sadrže veliku količinu flavonoida (prvenstveno derivata kvercetina), da se vrste A. pallens i A. oleraceum izdvajaju od ostalih po tome što ne sadrže rutin a sadrže hiperozid, da je vrsta A. rhodopeum siromašna fenolnim jedinjenjima i da su sve vrste, osim vrste A. flavum, bogate antocijanima. Aktivnost aliinaze je visoka u svim ispitivanim vrstama. Većina ispitivanih ekstrakata, izuzev ekstrakata vrsta A. carinatum i A. melanantherum, pokazala je izraženu antioksidantnu aktivnost, dok su ekstrakti vrsta A. flavum, A. rhodopeum, A. oleraceum i A. paniculatum snažni antiinflamatorni agensi. Ekstrakti ispitivanih predstavnika sect. Codonoprasum nisu pokazali antimikrobnu i antimutagenu aktivnost. Takođe, ovi ekstrakti nisu ispoljili genotoksični efekat na ćelije zdravog tkiva (izuzev slabog genotoksičnog efekta ekstrakta nadzemnih delova A. flavum), što ukazuje na bezbednost upotrebe vrsta sect. Codonoprasum kao hrane ili u obliku lekova. Ekstrakti celih biljaka A. paniculatum i A. rhodopeum, kao i ekstrakt nadzemnih delova A. melanantherum pokazali su snažnu antiproliferativnu aktivnost sa povoljnim ne-tumor/tumor koeficijentima i indukovali apoptozu u tumorskim ćelijama, iz čega se može zaključiti da imaju visok potencijal primene u antitumorskoj terapiji. In the present doctoral thesis the chemical composition and biological activities of 10 wild growing taxa of genus Allium sect. Codonoprasum (A. carinatum subsp. pulchellum, A. carinatum subsp. carinatum, A. fuscum var. gracile, A. fuscum var. fuscum, A. flavum subsp. flavum, A. melanantherum, A. oleraceum, A. paniculatum subsp. marginatum, A. pallens subsp. tenuiflorum and A. rhodopeum) were investigated. The samples were collected from 27 locations in Serbia. The aim of the study was to obtain data on the content of biologically active compounds in extracts of these unexplored species of the genus Allium and to determine their potential medicinal value. Phytochemical caracterisation included: headspace GC-MS analysis of fresh bulb volatiles, LC-DAD-MS and LC-MS-MS qualitative analysis of methanol extracts, LC-MS-MS quantitative analysis of 44 selected phenolic compounds in methanol extracts, determination of total monomeric anthocyanins content and alliinase activity. In order to assess the biological potential of methanol extracts, the antioxidant, anti-inflammatory, antimicrobial, antimutagenic, genotoxic and antiproliferative activities of the extracts were studied. Summing up all the results obtained, it can be concluded that species of genus Allium sect. Codonoprasum are rich sources of biologically active compounds with a broad spectrum of biological activities. Dimethyl disulfide is the most dominant and often the only volatile component of most species, which is significant from chemotaxonomic point of view. The methanol extracts of investigated species contain high levels of flavonoids (primarily quercetin derivatives). Specificity of A. pallens and A. oleraceum extracts is that they do not contain rutin, but contain hyperoside, while small quantity of phenolic compounds is characteristic for A. rhodopeum extract. All investigated species, except of A. flavum, are rich in anthocyanins. Alliinase activity was high in all examined species. Most of the extracts, except A. carinatum and A. melanantherum extracts, express considerable antioxidant activity, while extracts of A. flavum, A. rhodopeum, A. paniculatum and A. oleraceum are potent anti-inflammatory agents. The investigated Allium extracts did not show antimicrobial and antimutagenic activity. Also, the extracts did not express genotoxic effect on healthy tissue cells (except the weak genotoxic effects of aerial parts extract of A. flavum), indicating that the use of these species as a food or as a drug is safe. Whole plant extracts of A. paniculatum and A. rhodopeum, as well as aerial parts extract of A. melanantherum showed strong antiproliferative activity (with a favorable non-tumor/tumor ratios) and induced apoptosis in tumor cells, suggesting that these plants have a high potential for application in antitumor therapy.

Hormones are cellular products involved in the regulation of a large number of processes in living systems, and which by their actions affect the growth, function and metabo­lism of cells. Considering that hormones are compounds normally present in the organism, it is important to determine if they can, under certain circumstances, lead to genetic changes in the hereditary material. Numerous experimental studies in vitro and in vivo in different systems, from bacteria to mammals, dealt with the mutagenic and genotoxic effects of hormones. This work presents an overview of the research on genotoxic effects of non­steroidal hormones, although possible changes of genetic material under their influence have not still been known enough, and moreover, investigations on their genotoxic influ­ence have given conflicting results. The study results show that mechanisms of genotoxic effect of nonsteroidal hormones are manifested through the increase of oxidative stress by arising reactive oxygen species. A common mechanism of ROS occurence in thyroid hormones and catecholamines is through metabolic oxidation of their phenolic groups. Mani­festation of insulin genotoxic effect is based on production of ROS by activation of NADPH isophorms, while testing oxytocin showed absence of genotoxic effect. Considering that the investigations on genotoxicity of nonsteroidal hormones demonstrated both positive and negative results, the explanation of this discordance involve limitations of test systems themselves, different cell types or biological species used in the experiments, different lev­el of reactivity in vitro and in vivo, as well as possible variations in a tissue-specific expres­sion. Integrated, the provided data contribute to better understanding of genotoxic effect of nonsteroidal hormones and point out to the role and mode of action of these hormones in the process of occurring of effects caused by oxidative stress. Hormoni su ćelijski proizvodi uključeni u regulaciju velikog broja procesa u živim sistemima koji svojim dejstvom utiču na rast, funkciju ili metabolizam ćelija. Obzirom da su hormoni jedinjenja koja su uobičajeno prisutna u organizmu, značajno je utvrditi da li oni mogu pod izvesnim okolnostima dovesti do genetičkih promena na naslednom materijalu. Eksperimentalna ispitivanja u in vitro i in vivo uslovima u različitim sistemima, od bakterija do sisara, bavila su se istraživanjem mutagenih i genotoksičnih efekata hormona. U ovom radu je dat pregled istraživanja genotoksičnih efekata nesteroidnih hormona, pošto još uvek nisu dovoljno poznate moguće promene naslednog materijala pod njihovim uticajem, a i ispitivanja njihovog genotoksičnog dejstva su dala oprečne rezultate. Rezultati studija pokazuju da se mehanizmi genotoksičnog dejstva nesteroidnih hormona ispoljavaju kroz povećanje oksidativnog stresa nastankom reaktivnih vrsta kiseonika (reactive oxygen species - ROS). Uobičajeni mehanizam nastanka ROS kod tireoidnih hormona i kateholamina je putem metaboličke oksidacije njihovih fenolnih grupa. Ispoljavanje genotoksičnog efekta insulina se zasniva na produkciji ROS putem aktivacije NADPH izoformi, dok je ispitivanje oksitocina pokazalo odsustvo genotoksičnog efekta. Uzimajući u obzir da su ispitivanja genotoksičnosti nesteroidnih hormona pokazala i pozitivne i negativne rezultate, objašnjenja ovog neslaganja obuhvataju ograničenja samih test sistema, različite tipove ćelije ili bioloških vrsta upotrebljenih u eksperimentima, različiti nivo reaktivnosti u in vitro i in vivo uslovima, kao i moguće razlike u tkivno specifičnoj ekspresiji. Objedinjeni, navedeni podaci doprinose boljem razumevanju genotoksičnih efekata nesteroidnih hormona i ukazuju na ulogu i načine delovanja ovih hormona u procesu nastanka efekata izazvanih oksidativnim stresom.

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.

The paper presents the results of investigations of the effect of toluene on bone marrow cells of female Wistar rats treated intraperitoneally with toluene for 8 or 11 days, in doses of 0.602 μg/200 g body mass. Cytogenetic analyses were performed on the metaphase figure of chromosomes in order to determine the frequency of structural aberrations – breaks and gaps. The values of the mitotic index and number of poliploid cells were determined. No significant increase was determined in the frequency of breaks and gaps in chromosomes of treated animals in comparison with the controls, which means that, under the experimental conditions, toluene did not exhibit a definite genotoxic effect. However, it has been determined that there was a significant increase in the value of the mitotic index, as well as a significant increase in the number of poliploid cells in both groups of treated rats in comparison with controls. U ovom radu su navedeni rezultati ispitivanja efekata toluena na ćelije kostne srži ženki Wistar pacova, koji su intraperitonealno dobijali toluen tokom 8 i 11 dana, u dozi od 0.602 μg /200 g telesne mase. Citogenetički su analizirane metafazne figure hromozoma da bi se utvrdila učestalost pojavljivanja strukturnih aberacija – prekida i gapova. Utvrđene su vrednosti mitotskog indeksa i broja poliploidnih ćelija. Nije ustanovljeno značajno povećanje učestalosti pojavljivanja prekida i gapova na hromozomima tretiranih jedinki u odnosu na kontrole, što znači da u uslovima ovog eksperimenta toluen nije iskazao nesumnjiv genotoksični efekat. Međutim ustanovljeno je značajno povećanje vrednosti mitotskog indeksa, kao i značajno povećanje broja poliploidnih ćelija u obe grupe tretiranih pacova u odnosu na kontrole.

Cymiazole hydrochloride is the active component of the acaricides and Apichem which are used as varroacide in beekeeping. Since residues of cymiazole hydrochloride were detected in all bee products being used for human nutrition and as alternative medications in human and veterinary medicine the aim of this study was to evaluate the capability of this substance to produce genotoxic effects in peripheral human blood lymphocytes and its possible influence on mitotic activity and cell cycle kinetics. The genotoxic effects were studied by in vitro SCE-test. The results that were obtained for all doses applied in cultures (0.01 mg/ml, 0.1 mg/ml and 1 mg/ml) showed the very significant increases in frequencies of SCEs (p lt 0.001) in comparison to negative controls; i.e. the cymiazole hydrochloricde exhibited genotoxic properties. All experimental concentrations of cymiazole hydrochloride caused statistically highly significant (p lt 0.001) increased mitotic index values (Ml) and proliferation index (PI), too. Thus, the obtained data indicate significant cytomodulatoring effects and genotoxic properties. Cimiazol hidrohlorid je aktivna komponenta akaricida Apitol-a i Apihem-a koji se koriste kao varoacidi u pčelarstvu. Imajući u vidu da su rezidue cimiazol hidrohlorida ustanovljene u svim pčelinjim proizvodima koji su u upotrebi u ljudskoj ishrani, kao i u humanoj i veterinarskoj medicini - kao alternativni medikamenti, cilj ovog proučavanja je bio da se proceni sposobnost ove supstancije da izazove genotoksične efekte u kulturama limfocita periferne krvi čoveka i njen mogući uticaj na mitotsku aktivnost i kinetiku ćelijskog ciklusa. Genotoksični efekti su proučavani primenom SCE-testa in vitro. Rezultati su pokazali statistički vrlo značajno povećanje SCE-a (p lt 0,001) u poređenju sa negativnom kontrolom; tj. cimiazol hidrohlorid je ispoljio genotoksična svojstva. Sve eksperimentalne koncentracije cimiazol hidrohlorida su statistički značajno (p lt 0,001) povisile vrednosti mitotskog indeksa (Ml) i indeksa proliferacije (Pl), takođe. Tako, dobijeni rezultati ukazuju na značajne citomodulirajuće efekte i genotoksična svojstva.

There is a widely accepted view that the endogenous substances, including hormones, do not have any mutagenous effect when present in the usual physiological concentrations. However, beside relative stability and permanence of the genetic material, its changeability is also needed to provide for the biological evolution. Thus, it is possible to expect that certain reactions, due to the complexity of the mechanism of the signal transduction under the effect of hormones, still lead to the creation of reactive derivatives able to inter-react with the DNA molecules thus encouraging the emergence of mutations. This paper gives a survey of the exploration of the hormones' genotoxic effects in various test-systems, namely, from the bacteric through the cell cultures and experiments in vivo upon laboratory guinea-pigs, up to the determination of the mutagenous effects in the people that were treated by hormones. The steroid hormones' effects are described in detail in the literature while, on the other hand, there is not sufficient knowledge yet about possible changes of the genetic material under the influence of the non-steroid hormones. The examinations of the steroid hormones geonotoxicity in the bacterial systems have mainly given negative results. In more complex eukaryotic systems in vitro and in vivo most of the steroid hormones manifest mutagenous effects, though the result may vary depending one the type of cell or the biological species used in the experiment. As for the non-steroid hormones, though they have been studied less, it seems that they do not mostly express mutagenous effects. Today it is clear that the steroid hormones (especially estrogen) are completely cancerigenic and that they are capable of encouraging the process of cancerogenesis both by inducing covalent simulating the cell division (tumor promoters). Široko je prihvaćeno gledište da endogene supstance, uključujući hormone, nemaju mutagene efekte kada su prisutni u uobičajenim fiziološkim koncentracijama. Međutim, pored relativne stabilnosti i stalnosti genetičkog materijala, potrebna je i njegova promenljivost da bi postojala biološka evolucija. Stoga je moguće očekivati da izvesne reakcije, usled kompleksnosti mehanizama transdukcije signala pod dejstvom hormona, ipak dovode do stvaranja reaktivnih derivata sposobnih da interreaguju sa molekulima DNK i potpomognu nastanak mutacija. U ovom radu dat je pregled istraživanja genotoksičnih efekata hormona u različitim test-sistemima: od bakterijskih, preko kultura ćelija, in vivo eksperimenata na laboratorijskim životinjama, do utvrđivanja mutagenih efekata kod ljudi tretiranih hormonima. Efekti steroidnih hormona detaljno su opisani u literaturi, dok se nasuprot tome, još uvek nedovoljno zna o mogućim promenama naslednog materijala pod uticajem nesteroidnih hormona. Ispitivanja genotoksičnosti steroidnih hormona u bakterijskim sistemima uglavnom su dala negativne rezultate. U kompleksnijim eukariotskim sistemima in vitro i in vivo većina steroidnih hormona ispoljava mutagene efekte, mada rezultat može da varira zavisno od tipa ćelije ili biološke vrste upotrebljene u eksperimentu. Sto se tiče nesteroidnih hormona, mada su znatno manje proučavani, stiče se utisak da uglavnom ne ispoljavaju mutagene efekte. Danas je jasno da su steroidni hormoni (naročito estrogeni) kompletni kancerogeni sposobni da podstaknu proces kancerogeneze, kako indukcijom kovalentnih oštećenja DNK koja se mogu prevesti u mutacije (tumor inicijatori) tako i putem stimulacije deobe ćelija (tumor promotori).

The purpose of cytostatic agents is to act exclusively upon tumor cells, and to inhibit growth or induce tumor cell death by impairing their cell cycle progression. However, the majority of these agents are not specific in their action, and subsequently produce toxic effects on healthy tissues causing significant adverse events in both patients and health professionals exposed to these drugs. Various cytogenetic and molecular biology assays play an important role in the assessment of genotoxic effects related to antineoplastic drugs. Within a short period after exposure to a potentially genotoxic agent, these assays are able to assess the level of cellular DNA damage and/or to monitor the dynamics of DNA repair. Sensitive techniques, such as alkaline comet assay, are of special importance in the detection of primary DNA damage occurring in individual cells regardless of the cell cycle phase. The aim of the study was to assess and compare DNA damage that irinotecan and cisplatin induce in peripheral leukocytes, and normal kidney, liver and brain cells of Swiss albino mice. The results show that both cytostatics produce statistically significant DNA damage in normal cells compared to the control group. Compared to irinotecan, cisplatin has a significantly more potent genotoxic effect on these cells, which may be attributed to various mechanisms of action of the studied drugs., Po svojoj namjeni citostatici bi trebali djelovati isključivo na tumorske stanice, te narušavanjem njihovog staničnog ciklusa spriječiti rast ili izazvati smrt tih stanica. Međutim, većina ovih lijekova je u svom djelovanju nespecifična, zbog čega se toksične posljedice odražavaju i na stanicama zdravih tkiva, a rezultat toga su značajne nuspojave u bolesnika i osoba koje su profesionalno izložene tim lijekovima. U procjeni genotoksičnih učinaka antineoplastičnih lijekova značajnu ulogu imaju različiti citogenetični i molekularno-biološki testovi. Pomoću njih u kratkom vremenskom razdoblju nakon izlaganja nekom potencijalno genotoksičnom agensu, možemo procijeniti razinu oštećenja stanične DNA i/ili pratiti dinamiku njenog popravka. Posebnu važnost imaju tehnike poput alkalnog komet testa koje omogućavaju osjetljivo otkrivanje primarnih oštećenja DNA u pojedinačnim stanicama, neovisno o fazi staničnog ciklusa. Cilj našeg istraživanja je bio ustanoviti i usporediti oštećenja DNA koja izazivaju irinotekan i cisplatina na leukocitima periferne krvi, na zdravim stanicama bubrega, jetre i mozga Swiss albino miševa. Sukladno rezultatima istraživanja oba citostatika dovode do statistički značajnih oštećenja DNA spomenutih zdravih stanica u odnosu na kontrolnu skupinu. Međusobno uspoređujući irinotekan i cisplatinu možemo zamijetiti da cisplatina ima statistički značajno jači genotoksični učinak od irinotekana na spomenute stanice, što pripisujemo različitim mehanizmima djelovanja promatranih citostatika.