Aplication of the Comet assay on freshwater mussels Unio pictorum (Linnaeus, 1758), Unio tumidus (Philipsson, 1788) and Sinanodonta woodiana (Lea, 1834) in ecogenotoxicological assesment of aquatic ecosystems

Široka rasprostranjenost slatkovodnih školjki, ishrana filtriranjem, sesilni način života i sposobnost da akumuliraju zagađivače čine ih pogodnim organizmima za procenu nivoa zagađenja spoljašnje sredine i uticaja različitih zagađivača. Komet, test ili elektroforeza pojedinačnih ćelija (eng. single cell gel electrophoresis - SCGE) je osetljiva i brza tehnika za detekciju DNK oštećenja u pojedinačnim ćelijama i predstavlja jednu od osnovnih metoda u praćenju genotoksičnog efekta zagađivača na akvatične organizame. Da bismo odredili osetljivost školjki kao bioindikatora u ekogenotoksikološkim studijama, primenom alkalnog komet testa pratili smo nivo oštećenja molekula DNK u hemocitama slatkovodnih školjki Unio pictorum i U. tumidus (autohtone vrste) i Sinanodonta woodiana (alohtona vrsta) izlaganih sredinskom stresu in situ i ex situ. U okviru in situ proučavanja, aktivnim i pasivnim biomonitoringom, pratio se uticaj sredine na nivo oštećenja DNK molekula. Procena kvaliteta vode na različitim lokalitetima vršena je na osnovu mikrobioloških i fizičko-hemijskih parametara. Pasivni biomonitoring je rađen u okviru istraživanja na reci Velika Morava, na jedinkama vrste S. woodiana i u okviru istraživanja na reci Dunav, na jedinkama roda Unio. Aktivni biomonitoring je rađen sa jedinkama istog roda na lokalitetima Save i Dunava na teritoriji grada Beograda. U okviru ex situ proučavanja, pratio se uticaj citostatika: 5-fluorouracila, cisplatine, etopozida, vinkristin sulfata i imatinib mezilata na nivo oštećenja DNK molekula u hemocitama vrsta roda Unio in vivo, izlaganjem jedinki 72 h, i in vitro, izlaganjem sveže izolovanih hemocita (30 min) ili hemocita u primarnoj kulturi (22 h). Kao model mutagen, korišćen je kadmijum hlorid koji je u svim tretmanima doveo do indukcije oštećenja DNK molekula. Rezultati in situ studija su pokazali da se primenom komet testa na jedinkama vrsta U. pictorum, U. tumidus i S. woodiana može detektovati i kvantifikovati prisustvo genotoksičnog zagađenja...Due to generally wide distribution of considerable number of species, filter feeding, a sessile life form and an ability to accumulate pollutants, freshwater mussels were found to be favourable organisms for estimating the environmental pollution level and the impact of various pollutants. The comet assay or the single cell gel electrophoresis (SCGE) represents a sensitive and rapid technique for detection of DNA damage and thus this method is commonly employed for detection of genotoxic pollution in aquatic ecosystems. To evaluate the sensitivity of freshwater mussels as bioindicators in ecogenotoxicological studies, we studied the level of DNA damage with alkaline comet assay in haemocytes of freshwater mussels Unio pictorum and U. tumidus (autochtonous species) and Sinanodonta woodiana (alochtonous species) exposed to environmental stress in situ and ex situ. For in situ studies, active and passive biomonitoring were employed for the evaluation of the impact of environmental stress on the level of DNA damage. Water quality assessment on studied sites was based on physical, chemical and microbiological parameters. Passive biomonitoring was performed on specimens of S. woodiana collected from the Velika Morava River, and on two species bellonging to genus Unio collected from the Danube River. Active biomonitoring was performed on specimens of the same species exposed in the Sava and Danube rivers in the urban area of the Belgrade city. Ex situ studies of the impact of cytostatics: 5-fluorouracil, cisplatin, etoposide, vincristine sulphate and imatinib mesylate were performed in vivo, by treatment of the whole animals for 72, and in vitro, by treatment of freshly isolated haemocyes for 30 min or by treatment of haemocytes in primary cultures for 22 h. Treatment with cadmium chloride (model mutagen) resulted in significant induction of DNA damage in all treatment conditions..

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

Supplementary data for article : Kolarević, S.; Milovanović, D.; Kračun-Kolarević, M.; Kostić, J.; Sunjog, K.; Martinović, R.; Đorđević, J.; Novaković, I.; Sladić, D.; Vuković-Gačić, B. Evaluation of Genotoxic Potential of Avarol, Avarone, and Its Methoxy and Methylamino Derivatives in Prokaryotic and Eukaryotic Test Models. Drug and Chemical Toxicology 2019, 42 (2), 130–139. https://doi.org/10.1080/01480545.2017.1413108

Supplementary material for: [https://doi.org/10.1080/01480545.2017.1413108]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/322

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

Mapping of the microbiological water quality of surface waters in Serbia overlooked by the National monitoring programme.

Surface waters in Serbia are under high anthropogenic pressure. One of the major problems is untreated municipal and industrial wastewaters. Unfortunately, Serbia processes only 5 % of wastewaters before discharging. As a consequence, pollutants such as metals and metalloids from industrial wastewaters, pharmaceuticals, compounds from personal care products, etc. directly endure into surface water. Microbial faecal pollution, as an indicator of presence of human or/and animal pathogens, due to health hazard limits water usage for drinking, recreation, irrigation, etc. In regulations, faecal coliforms, with Escherichia coli as dominant representative, are widely used as faecal indicator bacteria. The aim of this study was to investigate the microbiological al water quality in the Republic of Serbia at the sites which are not routinely investigated within the national monitoring program. In this purpose 78 sites situated on canals, mountain springs, as well as lowland rivers were selected. Defined Substrate Technology was used for determination Most Probable Number (MPN) of E. coli using Colilert-18 System. The water classification system, developed for the Danube River (Kavka et al., 2006) was used. The results indicated that more than 47 % of selected sites are under critical (21.79 %) or strong (25.64 %) faecal pollution. On the other hand, about 32 % (32.05 %) of sites are little polluted and 20.51 % of sites showed moderate pollution. The increasing levels of faecal pollution detected on sites situated downstream of settlements indicated discharge of untreated municipal wastewaters directly into surface water. In further research, the focus will be placed on the determination of the origin of pollution by the employment of microbial source tracking technique. Obtained data will be used for modelling and predicting the effect of detected contamination on the water quality of the major water bodies in the Republic of Serbi

Cytotoxicity and antiviral activity of palladium(II) and platinum(II) complexes with 2-(diphenylphosphino)benzaldehyde 1-adamantoylhydrazone

Metal coordination compounds have an important role in the development of novel drugs. Using the resazurin microtitration assay we assessed the cytotoxicity and antiviral activity of the ligand 2-(diphenylphosphino) benzaldehyde 1-adamantoylhydrazone and its Pd(II) and Pt(II) complexes. Cytotoxicity was tested in A549 human lung adenocarcinoma epithelial cells. We observed that the ligand displayed a more pronounced cytotoxic activity than the platinum-based drug, carboplatin. Morphological evaluation of A549 cells treated with the ligand by acridine orange and ethidium bromide double staining revealed the presence of signs of apoptosis. Antiviral activity against poliovirus type 1 was assessed by examination of the cytopathic effect (CPE) in Hep-2 cells. Cells that were exposed to the 19 mu M ligand before infection displayed a maximal significant reduction (by 24.42 +/- 1.49%) of the CPE. This was likely due to the inhibition of virus receptors and prevention of viral adsorption. Treatment with 17 mu M Pt(II) complex after viral infection caused a maximal significant reduction (by 30.52 +/- 3.12%) of the CPE, presumably through an effect on viral replication. The results indicate that the ligand should be viewed as a potential anticancer agent. The ligand and the Pt(II) complex show promising results for further investigation of antiviral activity

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

Effects of 5-Fluorouracil, Etoposide and CdCl2 in Aquatic Oligochaeta Limnodrilus udekemianus Claparede (Tubificidae) Measured by Comet Assay

Genotoxicity of 5-fluorouracil (5-FU), etoposide (ET) and cadmium chloride (CdCl2) was evaluated in Limnodrilus udekemianus, cosmopolitan tubificid species, by alkaline single-cell gel electrophoresis (comet assay). Groups of 50 individuals were exposed in vivo in water-only short-term (96 h) tests to 5-FU (0.004, 0.04, 0.4, 4 and 40 mu M), ET (0.004. 0.04, 0.4 and 4 mu M) and CdCl2 (0.004, 0.04, 0.4, 4 and 40 mu M). Mortality of worms was observed only for CdCl2 (4 and 40 mu M). Cell viability lower than 70 \% was detected for 5-FU (0.4, 4 and 40 mu M), ET (4 mu M) and CdCl2 (0.4 and 4 mu M). All tested substances induced significant increase of DNA damage except 0.004 mu M of ET. L. udekemianus being sensitive to all tested substances indicates that it can be used in ecogenotoxicology studies. Concern should be raised to cytostatics, especially to 5-FU, since concentration of 0.004 mu M induced DNA damage is similar to ones detected in wastewaters.Water Air and Soil Pollution (2015), 226(242

Microbial quality and pollution origin of montenegrin surface waters, the first comprehensive study

Background: Montenegro is characterized by numerous mountain springs and larger streams. Many of them are used as sources of drinking water, irrigation, but also for tourists’ recreation: kayaking, rafting, etc. Unfortunately, there is a lack of wastewater treatment plants as well as information about microbial water quality of many surface and ground waters. Objectives: Taking into consideration issue of scarce data about microbial water quality, the aim of this study was to locate hotspots of faecal pollution and investigate the source of pollution. Within the study, 25 sites were selected while three of them were not included in National monitoring program. Methods: To quantify the most probable number of Escherichia coli we used Colilert-18 System. The water quality based on this parameter was assessed according to Kirshner et al. (2009). For determination of the pollution origin, microbial source tracking method was employed. Indicators of human faecal pollution, markers BacHum and HF183II, and animal faecal pollution, markers BacR and Pig2Bac, were quantified with qPCR. Results: According to most probable number of E. coli, 40% sites were little polluted, 36% moderately, 16% critically and per 4% heavily and excessively polluted. Analysis of genetic faecal markers indicated the prevalence of human-as- sociated faecal pollution at all sites. Considering the results of the study, four sites were classified as hotspots of faecal pollution: downstream Pljevlja (Ćehotina River), downstream Vezišnica (Ćehotina River), wastewater outlet at Podgori- ca (Morača River), and downstream Bijelo Polje (Lim River)