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

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

The impact of different extracts of six Lamiaceae species on deleterious effects of oxidative stress assessed in acellular, prokaryotic and eukaryotic models in vitro

The main objective of this research was to evaluate the impact of methanolic, ethanolic and aqueous extracts of Origanum majorana L., Origanum vulgare L., Teucrium chamaedrys L., Teucrium montanum L., Thymus serpyllum L. and Thymus vulgaris L. (Lamiaceae) on the effects of free radicals using different model systems. The extracts were characterized on the basis of the contents of total phenolics, phenolic acids, flavonoids and flavonols, and also using high-performance liquid chromatography with diode-array detection. Antioxidant activity in vitro was assessed using DPPH assay. The genoprotective properties were tested using plasmid relaxation assay on pUC19 E. coli XL1-Blue, while SOS/umuC assay on Salmonella typhimurium TA1535/pSK1002 and Comet assay on human lung fibroblasts were used to assess the antigenotoxicity of the extracts. Ethanolic extracts had the most phenolics (up to 236.20 mg GAE/g at 0.5 mg/mL), flavonoids (up to 42.47 mg QE/g at 0.5 mg/mL) and flavonols (up to 16.56 mg QE/g at 0.5 mg/mL), and they exhibited the highest DPPH activity (up to 92.16% at 0.25 mg/mL). Interestingly enough, aqueous extracts provided the best protection of plasmid DNA (the lowest IC50 value was 0.17 mg/mL). Methanolic extracts, on the other hand, most efficiently protected the prokaryotic DNA, while all the extracts had a significant impact against genomic damages inflicted on human fibroblasts. O. vulgare extracts are considered to be the most promising in preserving the overall DNA integrity against oxidative genomic damages. Moreover, HPLC-DAD analysis highlighted rosmarinic acid as the most abundant in the investigated samples (551.45 mg/mL in total in all the extracts), followed by luteolin-7-O-glucoside (150.19 mg/mL in total), while their presence correlates with most of the displayed activities. The novelty of this study is reflected in the application of a prokaryotic model for testing the antigenotoxic effects of Lamiaceae species, as no previous reports have yet been published on the genoprotective potential of these species

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

Identification of hotspots of genotoxicological and faecal pollution along the Danube and Sava rivers – the whole river surveys

The level of genotoxic pollution was assessed along the Danube River and its most significant tributary Sava River by measuring the level of DNA damage in aquatic organisms collected from the selected sites. The Danube River survey was conducted within the Joint Danube Survey 3 project in 2013 on 34 sites along the 2285 rkm using mussels (Unio sp.) and fish (Alburnus alburnus) as bioindicators. The Sava River survey was conducted within the Globaqua project in 2015 at 12 sites along 900 rkm using fish (A. alburnus/Alburnoides bipunctatus) as bioindicators. The level of DNA damage was evaluated by the comet assay in haemocytes of mussels and blood cells of fish. The level of faecal pollution along the rivers was studied in parallel with genotoxicological surveys by using the standard indicators, total coliforms, Escherichia coli and Enterococci. Quantification was performed with Colilert/Enterolert Quanti-Tray 2000 and MPN approach. Quantitative PCR (qPCR)-based assays for analysis of human- or animal-associated genetic Bacteroidetes faecal markers have been used for tracking the source of pollution (microbial source tracking -MST). The human-associated BacHum and HF183II, the ruminantassociated BacR and the pig-associated Pig2Bac fecal markers were selected. The hotspots of faecal pollution were detected at both rivers. Presence of pollution was especially evident in the countries in which the legislation related to wastewater treatment and management is not fully implemented. In the case of the Danube River the most critical section of the river was the Pannonian plain (sector VI) while in the case of the Sava River the most affected section was the lower stretch of the river. The results of MST revealed the presence of human-associated fecal markers BacHum and HF183II in the majority of the analyzed samples. High correlation was observed between the standard fecal indicators and human associated faecal markers. Within the Danube survey, the highest levels of DNA damage were recorded in organisms from the section VI, which is under the impact of untreated wastewater discharges. In 2013 the Sava River was characterized with a lower level of both faecal and genotoxic pollution in comparison with the Danube. Similar observations were found within the Sava River survey in 2015 where the level of DNA damage in fish specimens from Sava was lower in comparison with the samples from the Danube. At both rivers detected genotoxic potential was traceable to the deterioration of quality by communal and industrial wastewaters. Acknowledgements: International Commission for the Protection of the Danube River, EU Seventh Framework Program GLOBAQUA (no. 603629-ENV-2013-6.2.1). Ministry of Education, Science and Technological Development of the Republic of Serbia projects no. 173045 and 173025, the bilateral project Serbia and Austria: SER Ev. No. 451-03-01039/2015-09/33

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

Evaluation of genotoxic potential throughout the upper and middle stretches of Adige river basin

In this study a comprehensive genotoxicological survey throughout the upper and middle stretches of Adige river basin is presented. The study was carried out at 7 sites located along the Adige main course and one the most significant tributaries, the Noce creek, both presenting different levels of pollution pressure. To give an insight into the nature of the genotoxic activity we employed the battery of prokaryotic and eukaryotic assays. Mutagenicity in water samples was evaluated by SOS/umuC test in Salmonella typhimurium TA1535/pSK1002. The level of DNA damage as a biomarker of exposure (comet assay) and biomarker of effect (micronucleus assay) and the level of oxidative stress as well (Fpg - modified comet assay) were studied in blood cells of Salmo cenerinus Nardo, 1847 and Salmo marmoratus Cuvier, 1829. Within the applied bioassays, comet assay showed the highest potential for discriminating the sampling sites which are under lesser extent of pressure (sampling sites 1-Barnes at Bresimo and 4-Noce downstream S. Giustina) from the sites under high pressure (sampling sites 5-Noce at Mezzolombardo and 6/7-Adige upstream and downstream municipality of Trento). Significant correlation between the standard and Fpg - modified comet assay indicated that oxidative stress could be a major contributor to observed DNA damage in collected specimens

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

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

Uticaj poplava na mikrobiološki kvalitet vode reke Save na teritoriji grada Obrenovca

U ovom istraživanju praćen je mikrobiološki kvalitet vode reke Save, na lokalitetu nizvodno od grada Obrenovca, pre i nakon velikih poplava koje su se dogodile u maju 2014. godine. Praćeni su indikatori fekalnog zagađenja (ukupni koliformi, Escherichia coli, Enterococcus faecalis) kao i pokazatelji organskog opterećenja (heterotrofi). Dobijeni rezultati pokazuju da je tokom meseca sa najvišim vodostajem (maj) došlo do velikog pada u koncentraciji glavnih pokazatelja fekalnog zagađenja, u prvom redu E. coli. Sa porastom vodostaja tokom perioda poplava uočen je i pad koncentracije heterotrofnih bakterija. S druge strane, detektovan je porast koncentracije ukupnih koliforma tokom poplava. U ovom ispitivanju jasno je pokazano da poplave dovode do promena u trendu variranja koncentracija mikrobioloških parametara kvaliteta vode