Procena genotoksičnosti korišćenjem kometa testa u različitim tkivima klena iz rečne vode

Cilj ovog rada je bila analiza oštećenja molekula DNK primenom komet testa (engl. Single Cell Gel Electrophoresis, SCGE) na klenu (Squalius cephalus L.) kao potencijalnom model organizmu za procenu genotoksičnosti vode Kolubarskog basena. Kolubarski basen je bogat nalazištima lignita pa kao takav trpi veliki pritisak rudarskog postrojenja, “Kolubara”, pored već postojećeg uticaja komunalnih voda kao i spiranja sa obradivih površina. Uzorkovanja su vršena mesečno tokom sezone 2011/2012 god. Jedinke klena sa Zlatarskog jezera, Uvac, su korišćene kao prirodna kontrolna populacija. Komet test je osetljiva i relativno jednostavna metoda primenljiva na velikom broju različitih organizama i tkiva. Za detekciju oštećenja DNK molekula u ovom radu korišćena su tri tkiva: krv, jetra i škrge

Endoparaziti i ishrana mrene (barbus barbus linnaeus, 1758) u beogradskom sektoru Dunava (Srbija)

U radu su prikazani rezultati istraživanja crevnog sadržaja rečne mrene (Barbus barbus L. 1758). Tokom dvogodišnjeg perioda studije, 2007-2009, ukupno je prikupljeno 63 jedinki mrena u okviru beogradskog sektora reke Dunav. Cilj je bio da se dobije informacija o inficiranosti mrena crevnim parazitima (endoparaziti, helminti) i utvrdi prisustvo organizama makrozoobentosa u njihovom crevnom sadržaju (Gammaridae, Bivalvia, Gastropoda i Oligochaeta), kao posledica raspoloživosti hrane na području uzorkovanja. Nabrojani organizmi makrozoobentosa imaju veoma značajnu ulogu u ishrani mrena, s obzirom da je ona bentofagna vrsta, i da njena ishrana zavisi od sastava i strukture faune dna. Pojedini akvatični makrobeskičmenjaci (gamarusi, oligohete, školjke, puževi, i neke larve insekata), kojima se hrane mrene, predstavljaju i prelazne domaćine za nekoliko vrsta endoparazita. Pregledom crevnog sadržaja mrena ukupno je nađeno i identifikovano šest vrsta endoparazita (helminta) iz tri filuma (Platyhelminthes, Nematoda i Acanthocephala), sa prevalencom infekcije od 98.41%. Broj endoparazita po jedinki mrene varirao je u rasponu od 6 do 207. Najveća procentualna zastupljenost utvrđena je za parazita iz grupe Acanthocephala – Pomphorhynchus laevis (Müller, 1776), sa prevalencom inficiranosti od 7,73%. Takođe, udeo gamarusa u crevnom sadržaju mrena bio je u interval 31.43% do 46.73%, odnosno u zajednici makrozoobentosa od 1.4% do 16.22%. Ova činjenica ukazuje da su gamarusu bili omiljena hrana mreni i da je to doprinelo visokoj abundanci - parazita Pomphorhynchus laevis u crevnom sadržaju mrene. Uzimajući u obzir, raznovrsnost i bogatstvo faune parazita na području Srbije, neophodno je nastaviti istraživanja parazita što većeg broja slatkovodnih vrsta riba

New spectral templates for rhodopsin and porphyropsin visual pigments

A four-parameter model of spectral sensitivity curves was developed. Empirical equations were designed for A(1)- and A(2)-based visual pigments with the main a-band maximum absorptions (lambda(max)) from 350 nm, near the ultraviolet, up to 635 nm in the far-red part of the spectrum. Subtraction of the alpha-band from the full absorbance spectrum left a "beta-band" described by a lambda(max)-dependent Gaussian equation. Compatibility of our templates with A(1)- and A(2)-based spectra was tested on the electroretinographic (ERG-derived) scotopic action spectra recorded in dogfish shark, eel, Prussian carp and perch. To more precisely estimate the accuracy of our model, we compared it with widely used templates for visual pigments. There was almost no difference between the tested models in fitting the above-mentioned spectral data. One of the advantages of our model is that in the fitting of spectral sensitivity data it uses non-transformed wavelengths and the shape of the curve remains the same for a broad range of lambda(max) values. Compared to multiparameter templates of other authors, our model was designed with fewer (four) parameters, which we believe can bring us closer to understanding the true nature of the absorption curve

Stanje kečige (acipenser ruthenus l.) u Srbiji i Mađarskoj

Izlov kečige je tradicionalna i značajna privredna aktivnost i u Srbiji i u Mađarskoj. Osnovni problem vezan za izlov kečige u Srbiji odnosi se na nedostatak validnih podataka o izlovu kečige u poslednjim godinama što je imalo negativan efekat na upravljanje korišćenja ove vrste. Poribljavanje kečigom nije nikada u potpunosti razvijeno u Srbiji dok se u Mađarskoj počelo sa poribljavanjem mlađi kečige od 1980. godine i to sa 10.000-100.000 jedinki/godini. Uzgoj kečige je započet u Mađarskoj od 1990. godine, mada je proizvodnja malog obima. U cilju uspostavljanja boljih planova za upravljanje ovom značajnom vrstom potrebno je uskladiti i koordinisati aktivnosti svih zainteresovanih strana u obe zemlje

Kako poplave modifikuju genotoksični odgovor slatkovodnih riba?

U ovom radu ispitivan je potencijal dve vrste roda Abramis (Abramis bjoerkna i Abramis sapa) kao bioindikatora genotoksičnog potencijala vode reke Save primenom komet testa. Izabrani lokalitet pripada oblasti intezivne poljoprivredne aktivnosti, izložen je otpadnim vodama grada Obrenovca i nalazi se u blizini najveće termoelektrane u Srbiji, “Nikola Tesla”. Uzorkovanje je vršeno od januara do avgusta 2014. godine, obuhvatajući mesec maj kada je došlo do intezivnih poplava u slivu reke Save. Dobijeni rezultati ukazuju da se ispitivane vrste mogu koristiti za procenu genotoksičnog potencijala

Uticaj otpadnih voda na reku Dunav – ekogenotoksikološki aspekt ekspedicije JDS3

Narušavanje kvaliteta površinskih vodotokova otpadnim vodama se odražava na kvalitet akvatičnih ekosistema ali i na kvalitet života i zdravlja ljudi. Variranje u stepenu prerade otpadnih voda najčešće je posledica razlika u nacionalnim zakonskim regulativama što je veoma evidentno u slučaju reke Dunav, u nekim od podunavskih zemalja voda se prerađuje dok se u drugim nikakav tretman ne primenjuje pre ispuštanja. JDS3 (The Joint Danube Survey 3) je bila najveća rečna ekspedicija u 2013. godini koja je obuhvatila svih X sektora reke. Zbog obima uzoraka i različitih analiza, JDS je predstavljao idealnu priliku da se ispita prisustvo genotoksičnog zagađenja u ovoj reci. Komet test je rađen na hemocitama školjki Unio sp. i Sinanodanta woodiana. Najviši nivo genotoksičnog potencijala detektovan je u sektoru VI (Panonska ravan) gde veliki problem predstavljaju otpadne vode koje se bez prerade ispuštaju u Dunav. Ova studija je dala mapu zagađenja i kritičnih tačaka zagađenja duž reke Dunav i ukazala na bitnost prerade otpadnih voda za očuvanje i poboljšanje kvaliteta reke Dunav. Uvođenje molekularnih markera u monitoring kvaliteta voda znatno doprinosi razumevanju prirode i porekla zagađenja

Effect of floods on DNA damage of two cyprinid fish in the Sava River

Freshwater ecosystems are constantly deteriorated due to the multitude of human activities, intensification of the industrial processes, wastewater discharge and agricultural chemicals. Besides, climate change provokes extreme weather and hydrological conditions leading to prolonged periods of droughts and floods. Floods can cause movements of polutants already present in the environment and introduce pathogenic bacteria in the flooded area. Many of the substances that reach environment could exhibit genotoxic potential on the genetic material of aquatic organisms, either alone or in complex mixtures of chemicals. Unfavorable hydrological conditions during 2014 resulted in extensive flooding in May in the Sava River basin. Obrenovac city was one of the most affected settlements on the river with esstimation that nearly 90% of town area was flooded. This led to evacuation of majority of inhabitants from the affected area. Our research was conducted from January to December 2014 on the Sava River, site Duboko, situated downstream of Obrenovac city. This is the area of intensive agricultural activity. Few km upstream the sampling site is the mouth of Kolubara river which is receiving untreated municipal wastewater of the town Obrenovac (50.000 inhabitants) and thermal power plant Nikola Tesla A with belonging ash fild. On monthly basis we have performed the measurements of basic phisico-chemical parameters, sampling water for microbiology analysis and fish tissue for the comet assay. Single Cell Gel Electrophoresis (SGCE) or Comet assay is widley accepted tool in ecogenotoxicology studies, in which DNA damage is measured on the level of a single eukaryotic cell. For the detection and quantification of E. coli and Enterococci we have used enzymathic methods and MPN approach. For comet assay analysis blood, liver and gill tissues were sampled from two cyprinid fishes. The highest water level was observed in May 2014, when flooding occured. Evacuation of inhabitants had led to exclusion of urban wastewater discharge which was refelcted through the lowest concentration of faecal pollution indicator bacteria, E. coli and Enterococci. With return of inhabitants in June their concentration in water started to increase gradually. In contrast, with withdrawl of water from the flooded agricultural land and ash disposal field high amounts of potentialy genotoxic substances were introduced into the Sava river, which was observed in sudden increase of DNA damage level in all three fish tissues during June. This study showed that flood occurence had major impact on the microbiological quality and genotoxic potential of the Sava River. Faecal pollution is mainly under the impact of urban wastewater discharge. On the other hand, genotoxic substances are most likely of industrial and agricultural origin. Acknowledgements This research was funded by project OI173045 „Fishes as water quality indicators in open waters of Serbia” (Ministry of Education, Science and Technological Development of the Republic of Serbia). This research was conducted in the Laboratory at the Chair of Microbiology, Center for genotoxicology and ecogenotoxicology, Faculty of Biology, University of Belgrade, and Institute for Multidisciplinary research, University of Belgrade

Procena genotoksičnog potencijala voda u Srbiji komet testom

Sistemi površinskih voda često su pod pritiskom otpadnih voda iz industrijskih, domaćih i poljoprivrednih izvora. Različiti sastojci prisutni u zagađenim vodama mogu da izazovu biološke promene, koje mogu uticati na populacije i čitave ekosisteme. Mnogi akvatični organizmi, među kojima bitno mesto zauzimaju ribe, imaju sposobnost bioakumulacije genotoksičnih agenasa u tkivima, te se mogu koristiti kao bioindikatori u ekogenotoksikološkim studijama.1,2 Procena genotoksičnosti površinskih voda vršena je na osnovu detekcije oštećenja DNK molekula alkalnim komet testom, testom elektroforeze pojedinačnih ćelija, (engl. SCGE), u krvi, jetri i škrgama klena (Squalius cephalus L.). Ekološki status površinskih voda određivao se na dve akumulacije, Garaši (vodozahvat za pijaću vodu) i latar (Uvac, zaštićeno prirodno dobro), kao i četiri reke u okviru sliva reke Kolubare (područje intenzivnih kopova uglja): Kolubara, Peštan, Beljanica i Bubanj Potok, tokom 2011./2012. godine. Na osnovu analize fizičko-hemijskih i mikrobioloških parametara, izdvojile su se akumulacije, Zlatar i Garaši, u I klasu kvaliteta voda. Sa druge strane, analize vode sa Peštana i Beljanice odgovarale su III i IV klasi kvaliteta voda. Više vrednosti oštećenja DNK molekula su zabeležena na rekama u okviru sliva Kolubare, dok su oštećenja na tkivima klena sa Garaša i latara bile značajno niže. 1. Sunjog, K., Kolarević, S., Kračun-Kolarević, M., et al., 2014, Environ. Toxicol. Pharmacol. 37:967-974. 2. Sunjog, K., Kolarević, S., Kračun-Kolarević, M., et al., 2016, Environ. Pollut. 213:600-607. Zahvalnica: Ovaj rad je finansiran od strane Ministarstva prosvete, nauke i tehnološkog razvoja Republike Srbije, projekat OI173045

Evaluation of the Sava and the Danube river genotoxicity by Comet assay on different cyprinid fish

A number of biological consequences of DNA damage can be initiated at the cellular, organ, whole animal, population and community levels. The comet assay, single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for detection of DNA damage in individual cells based on the migration of denatured DNA during electrophoresis, in which damaged nuclei form comet-like shapes. Since, the large rivers are constantly exposed to variety of chemicals we tested genotoxic potential of rivers Sava and Danube in the Belgrade region, on cyprinid fish. DNA damage was monitored in blood, liver and gill cells of Blicca bjoerkna, Ballerus sapa and Abramis brama by comet assay. The specimens of B. bjoerkna and B. sapa were sampled at the site Duboko (Sava river), which is exposed to untreated municipal wastewater, effluents of the Thermal Power Plant and intensive agricultural activity. The specimens of A. brama were sampled at the site Višnjica (Danube river), which is exposed to untreated municipal wastewater from surrounding Belgrade municipalities. Sampling was performed during February and August 2014. Comet IV Computer Software (Perceptive Instruments, UK) was used for the quantification of DNA damage. The increased level of DNA damage obtained in all tissues of B. bjoerkna and B. sapa (Sava river), and A. brama (Danube river) is in correlation with high chemical pollution detected at Duboko and Višnjica sites. Moreover, this study showed the influence of seasonal variations on the level of DNA damage

Cytostatics as emerging pollutants in aquatic environments - risk assessment based on genotoxic effects in haemocytes of freshwater mussels

Pharmaceutical compounds have begun to be considered as dangerous environmental pollutants, due to their widespread occurrence in wastewaters and their potential hazard towards the aquatic ecosystems. The comet assay was used to study acute impacts of most used cytostatic drugs on the haemocytes of widely distributed freshwater mussels Unio pictorum and Unio tumidus. After collection from unpolluted location, the mussels were held on accommodation for 10 days in controlled laboratory conditions before exposure to cytostatics. Groups of 5 mussels were exposed to 5-Fluorouracil, (0.04 – 40 μM), Cisplatin (0.004 – 4 μM), Etoposide (4, 40 and 100 μM) and Imatinib msesylate (0.04 - 40 μM). For positive control treatment with Cd was used (4, 40 and 100 μM), while as negative control mussels were held in control aquarium with clean water. Exposure was performed for 72h in aquaria. Comet assay is one of the major tools for the level of DNA damage assessment in ecogenotoxicology. It is based on single cell gel electrophoresis. In our experiments we performed alkaline version of comet assay described by Singh et al. (1988). Images of 250 nuclei per each concentration of test substance were analyzed with a fluorescence microscope and scored using analysis software (Comet Assay IV Image analysis system, PI, UK). Tail intensity was chosen as relevant measure of DNA damage. Our results indicated significant increase of DNA damage in haemocytes during treatment in selected concentration ranges for all cytostatics with the exception of Imatinib mesylate. Detected LOEC values were as following: 5-Fluorouracil – 52 μg/L, Cisplatin 12 μg/L and Etoposide – 24 mg/L. However, all effective concentrations of 5-FU, CP and ETO are higher than ones measured in surface water but still far below PNEC values which are currently used for the environmental risk assessment. This indicates that acute toxicity data might not be sufficient for prediction of adverse effects of substances, and that genotoxicity data should be also considered for the risk assessment