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

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

Ispitivanje osetljivosti testova i pristupa u ekogenotoksikološkim istraživanjima na velikim ravničarskim rekama–studije slučaja Velika Morava i Sava

Velika, Zapadna, Južna Morava, kao i Sava, Kolubara i Barička reka su pod značajnim uticajem komunalnih i industrijskih otpadnih voda. Ciljevi studije su procena genotoksičnog potencijala analiziranih lokaliteta i ispitivanje osetljivosti primenjenih biotestova i pristupa istraživanja. Studija slučaja Velika Morava obuhvatila je veliko ispitivano područje na osam lokaliteta na Velikoj, Zapadnoj i Južnoj Moravi, a u studiji Sava ispitivano je tri prostorno bliska lokaliteta koji su pod različitim pritiscima zagađenja. U in silico pristupu na osnovu literaturnih podataka analiziran je toksični/genotoksični potencijal jedinjenja detektovanih u vodi. Ex situ/in vitro pristup obuhvatio je ispitivanje genotoksičnog potencijala pomoću SOS/umuC testa, kao i primenom alkalnog komet testa na HepG2 ćelijskoj liniji. U in situ/in vivo istraživanju analizirano je DNK oštećenje ćelija krvi vrste Alburnus alburnus (uklija) pomoću alkalnog, Fpg-modifikovanog komet testa i mikronukleus testa. Rezultati su pokazali različitu osetljivost zavisno od upotrebljenih testova i pristupa, ali i različit odgovor kod uklija u zavisnosti od tipa zagađenja na lokalitetima. In silico i ex situ/in vitro pristupi su pokazali niži stepen osetljivosti u odnosu na in situ/in vivo testove, a alkalni komet test je pokazao najveći potencijal u diskriminaciji lokaliteta. Utvrđeno je da uklija može biti pouzdan bioindikator u ekogenotoksikološkim istraživanjima velikih ravničarskih reka

Cytostatics as emerging pollutants – is there a threat for aquatic invertebrates?

Seeking for a better quality of life, consumption of pharmaceuticals is constantly increasing. Pharmaceuticals are designed to be more potent, degradation resistant, to affect protein targets at relatively low doses. Many of them are not selective and became threat to non-target organisms, especially to one living in aquatic ecosystems. Because of the inability of wastewater treatment plants to eliminate these compounds in the end they reach aquatic environments through sewage system. Aquatic invertebrates spend at least a part of their life cycle in the aquatic environment. Mobility is not one of the traits that characterize many species of aquatic invertebrates, especially freshwater mussels and aquatic worms which are almost sedentary organisms. Due to their way of life, this species are under the influence of variety of pollutants via sediment and via water column. Genotoxic effects of cytostatics with different mode of action – alkylation agent (cisplatin - CP), antimetabolite agent (5-fluorouracil – 5-FU), plant alkaloids (etoposide - ETO, vincristine sulphate - VIN) and other neoplastic agent (imatinib mesylate - IM) were studied in vivo and in vitro on haemocytes of two freshwater mussels species Unio sp. (U. pictorum/U. tumidus), and in vivo on haemocytes and coelomocytes of tubificid species Limnodrilus udekemianus. Experiments were organized as short-term treatments (72h for mussels/96h for worms) in static system. Level of DNA damage was evaluated by alkaline single-cell gel electrophoresis (comet assay). Based on our results ranking of cytostatics by their effects on mussels was VIN>5-FU>ETO>CP>IM and on worms was 5-FU>ET. Worms have shown higher sensitivity for the negative effects of 5-FU and ET on the integrity of DNA molecule comparing with mussels. The lowest observed effect concentration (LOEC) for 5-FU was 52 μg/L for mussels and 0.52μg/L for worms. In the case of ET LOEC was 24 mg/L for mussels and 0.024 mg/L for worms. For VIN was detected difference in the response in U. pictorum and U. tumidus – LOEC for U. pictorum was 3.7 μg/L, while for U. tumidus was 36.9 μg/L. Significant damage of DNA wasn’t detected for CP and IM. Although the PEC values for tested cytostatics are lower than the ones used in our study, it must be emphasized that in the environment, organisms are under constant influence of these pollutants and organisms are struggling with the effects of mixture of pharmaceuticals and mixture of different pollutants. Impacts of these mixtures on the aquatic organisms are still unknown, and therefore, further research should consider this fact and the studies should be organized in this direction. Acknowledgements: EU Seventh Framework Programme – Cytothreat (no. 265264). Ministry of Education, Science and Technological Development of the Republic of Serbia project no. 037009

Nivo DNК oštećenja i akumulacije metala u jedinkama Vimba vimba (L., 1758) izloženim neprečišćenim otpadnim vodama u reci Dunav, Srbija.

Непречишћене комуналне и индустријске отпадне воде представљају извор хемијског и микробиолошког загађења водених екосистема и угрожавају све видове њиховог коришћења. Ово истраживање је спроведено у јулу 2017. године, на реци Дунав, локалитету Нови Бановци, који је изложен испусту колектора непречишћених индустријских и комуналних отпадних вода насеља са око 40 000 становника. Вода је узоркована за микробиолошку анализу индикатора фекалног загађења ензимском методом Colilert (IDEXX). Неколико десетина метара од испуста колектора узорковано је 5 јединки шљивара Vimba vimba (L., 1758) за анализу метала у мишићу и јетри методом ICP-OES и нивоа ДНК оштећења у ћелијама крви, јетре и шкрга применом алкалног комет теста. Према нашим сазнањима ово је прва студија у којој је комет тест примењен на ћелијама шљивара. На основу индикатора фекалног загађења (укупни колиформи и E. coli) вода се могла класификовати као критично загађена (III класа). Највиши ниво ДНК оштећења забележен је у ћелијама крви, потом следе шкрге и јетра. Ниво ДНК оштећења у ћелијама крви био је статистички значајно виши у односу на јетру. На основу концентрација Fe и Zn, за које су прописане максимално дозвољене концентрације за рибље производе у лименој амбалажи, месо шљивара је безбедно за исхрану људи

Primena IBR metode (Integrated Biomarker Response) u mikrobiološkoj analizi vodenih ekosistema

UVOD: Mikrobiološki indikatori sanitarnog i organskog zagađenja čine neophodan segment svake procene kvaliteta vode. Međutim, kako se velike količine neprerađenih ili nepropisno prerađenih otpadnih voda ispuštaju u vodotokove teško je razdvojiti sanitarno od organskog zagađenja. Vizuelni prikaz rezultata IBR metodom pokazao se veoma korisnim u analizi međusobnih odnosa različitih biomarkera i ima široku primenu u biomonitoringu. Osim primene na biomarkerima, metoda može da pokaže i uzajamne odnose različitih hemijskih parametara. Metoda IBR do sada nije korišćena za analizu uzajamnih odnosa mikrobioloških parametara. CILJEVI: Primena IBR metode u proceni kvaliteta vode, pri čemu su mikrobiološki parametri fekalnog i organskog zagađenja korišćeni kao “biomarkeri“. METODE: Za potrebe ove studije odabrana su dva seta podataka. Prvi set je dobijen tokom višemesečnog monitoringa na reci Savi tokom 2014. godine, uključujući i period sa poplavom, tj. maj mesec. Od indikatora fekalnog zagađenja praćeni su totalni koliformi, Escherichia coli, Enterococcus faecalis i Clostridium perfringens. Nivo organskog zagađenja određen je na osnovu odnosa heterotrofnih i oligotrofnih bakterija, i indeksa fosfatazne aktivnosti. Drugi set podataka dobijen je tokom višemesečnog monitoringa sezonskog variranja mikrobioloških indikatora na reci Peštan, tokom 2012. godine. Od indikatora fekalnog zagađenja praćeni su totalni koliformi, E. coli i E. faecalis. Nivo organskog zagađenja određen je na osnovu odnosa heterotrofnih i oligotrofnih bakterija. REZULTATI: Vizuelni prikaz rezultata IBR metodom dao je jasan uvid u minimalne i maksimalne vrednosti svakog od indikatora fekalnog i organskog zagađenja tokom različitih meseci. Za prvi set podataka, na osnovu grafika i na osnovu dobijenih IBR vrednosti, jasno se izdvaja mesec u kome je došlo do poplava. Za drugi set podataka očigledno je variranje mikrobioloških parametara tokom različitih sezona. ZAKLJUČAK: Metoda IBR je pogodna za prikaz i analizu kompleksnog seta mikrobioloških podataka

Freshwater mussels in the ecogenotoxicological studies-application of comet assay

Presence of pollutants in aquatic ecosystems can be detected by a range of physiological, histological and molecular responses, including abnormal morphology, alterations of antioxidative status and DNA integrity. The integrity of cellular DNA is continuously attacked by various agents resulting in DNA lesions. Unrepaired DNA lesions may block replication and transcription, potentially leading to cell death or generating mutations. The main objectives of ecogenotoxicology studies are detection of the origin of genotoxic pollution and their effect on individuals and populations. Mussels are commonly employed in the ecogenotoxicological studies. They have several characteristics, such as wide distribution, filter feeding, a sessile life form and an ability to accumulate pollutants, which makes them favorable organisms for estimating the environmental pollution level and the bioavailability of various types of pollutants. Different approaches are used for assessing the conditions of ecosystems, the passive (collection of mussels from selected locations) and the active biomonitoring (subsequent exposure of mussels from unstressed populations at polluted sites). Moreover, in ex situ assessment, the genotoxic effect of different compounds can be detected in laboratory conditions. 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 (damaged nuclei form comet-like shapes). It has been accepted as one of the major tools for assessing pollution related genotoxicity in aquatic organisms. 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 and Olive tail moment are chosen as relevant measure of DNA damage. Active biomonitoring implies translocation of specimens from unstressed populations to selected sites of interest. In active approach issues related to availability of specimens and effects of age, gender and physiological condition are avoided. This approach enables research on highly polluted sites. Specimens of Unio sp. were exposed at selected sites at the Sava and Danube rivers which are under the impact of untreated wastewaters of Belgrade city. Haemolymph was sampled after 7, 14 and 30 days of exposure. Passive biomonitoring implies collection of specimens which inhabit selected sites of interest. Easily performed, suitable when research area is situated far from the laboratory. Study was performed on the Velika Morava River, significant Danubes tributary. Specimens of Sinanodonta woodiana collected monthly on two sites with different pollution pressure. Significant correlation was detected between the level of DNA damage in haemocytes and concentration of haevy metals in water. Seasonal variation in the level of DNA damage was observed. Studies performed ex situ are significant research tool for prediction of the effect of single compounds in environment. Obtained data are useful for risk assessment. Among emerging pollutants, cytostatics represent hot topic as serious threat to non-target organisms due to their cytotoxicity, genotoxicity and mutagenicity. As part of research for Cytotreat project (EU Commision, FP7) we are investigating impact of short term exposure to mostly used drugs: 5-Fluorouracil, Cisplatin, Etoposide Vincristine and Imatinib mesylate with Cadmium chloride as positive control. Data obtained so far indicate that cytistatics can induce increase in DNA damage in concentrations similar to ones measured in wastewater. Our studies indicate that freshwater mussels can be used as reliable bioindicators. Research performed in situ showed that selected species can detect genotoxic pollution present in environment. Results of the research performed ex situ showed harmful effects of environmentally relevant concentrations of cytostatics. Studies performed ex situ on mussels coul be significant research tool for prediction of the effect of single compounds in environment

Ocena ekološkog potencijala lokaliteta Duboko (Sava) i Višnjica (Dunav) na osnovu zajednice akvatičnih Oligochaeta

Na Savi (Duboko) i Dunavu (Višnjica) vršena je procena ekološkog potencijala na osnovu zajednice akvatičnih oligoheta. Na Savi istraživanje je rađeno tokom 2014. i 2015. godine, a na Dunavu tokom 2014. godine. Na lokalitetu Duboko veća raznovrsnost oligohetne faune bila je zabeležena 2015. godine (11 vrsta), u odnosu na 2014. godine (9 vrsta), a na lokalitetu Višnjica zabeleženo je prisustvo 17 vrsta oligoheta. Na oba lokaliteta familija Tubificidae je bila dominantno zastupljena (>90 %) sa vrstama Limnodrilus hoffmeisteri i L. claparedeanus. Visoka zastupljenost familije Tubificidae u uzorcima ukazuje na veliko organsko zagađenje koje je prisutno na ovim lokalitetima

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

Significance of genotoxicity and toxicity evaluation of freshwater bodies

Fish communities are excellent indicators of biological and ecological integrity due to their continuous exposure to water conditions. Freshwater biodiversity constitute a valu able natural resource but extensive pollution of freshwater can result in biodiversity de cline. For that reason it is important to employ bioassays for purpose of detecting these conditions. In this work we monitored chub and bream species in 2 reservoirs, Uvac and Garasi, and 4 rivers: Dunav, Sava, Pestan and Beljanica. For assessment of metal and metalloid in fish tissues (liver, gills, gonads and muscle) we have used ICP-OES. The comet assay or single cell gel electrophoresis (SCGE) was selected as an in vivo geno toxicity assay, for measuring DNA damage in blood, liver and gills. Histopathological alterations were monitored in liver and gills.The high quality of Uvac water was con firmed by low values of DNA damage in all tissues compared to other sites. An analysis metals in tissues showed a high degree of their differentiation, as well as significant dif ferences in the distribution of tissue elements between the sites tested. The highest con centrations of most of the analyzed metals were found in gills, liver and gonads, and the lowest in muscles at all sites. In chub, blood showed the lowest DNA damage compared to liver and gills, while in all breams DNA damage was the highest in blood cells, follow ing gills and liver. Histopathological analysis performed on bream specimens on the Sava and Danube River revealed a higher degree of alterations in liver compared to the gills