Sensitivity assessment of in vitro and in vivo tests in ecogenotoxicology and guidelines formulation for their application instudies of large lowland rivers

metoda uslovljenim efektima (eng. effect based methods), uključujući i genotoksikološke metode, u programe monitoringa i naučna istraživanja na velikim ravničarskim rekama, omogućilo bi sveobuhvatniju procenu stanja ekosistema i odnosa između samog hemijskog i ekološkog statusa. Osnovni ciljevi disertacije su ispitivanje osetljivosti in vitro i in vivo testova u ekogenotoksikologiji i formulisanje smernica za njihovu primenu u istraživanjima velikih ravničarskih reka. Studija je obuhvatila ukupno 17 lokaliteta u slivovima Velike Morave, Save i Dunava. Za procenu genotoksičnog efekta nativnih i koncentrisanih uzoraka rečne vode, in vitro pristup obuhvatio je SOS/umuC test, alkalni komet test na HepG2 i ZFL ćelijama, kao i analizu ćelijskog ciklusa na ZFL ćelijama. In vivo pristup je za procenu genotoksičnosti obuhvatio alkalni, Fpg modifikovani komet test, kao i mikronukleus test na ćelijama krvi Alburnus alburnus (uklija). Dodatno, ispitivana je osetljivost RAPD analize u detekciji genotoksičnog efekta. U in silico pristupu na osnovu literature analiziran je toksični/genotoksični potencijal hemijskih parametara koji su detektovani u vodi i u uklijama, a integrativnim metodama vršena je kvantifikacija pritiska zagađenja. Biotestovi su pokazali različitu osetljivost u detekciji genotoksičnog efekta. U in vitro istraživanju koncentrisani uzorci rečne vode su se pokazali pogodnim za testiranje toksičnosti/genotoksičnosti, a ZFL ćelije kao dobar model sistem. U in vivo istraživanju A. alburnus se pokazao kao pouzdan bioindikator. Najveća osetljivost u detekciji genotoksičnog efekta u ćelijama krvi dobijena je u alkalnom komet testu. In silico analizom ne može se izvršiti potpuna predikcija genotoksičnog efekta ksenobiotika na organizme.The implementation of effect based methods, including genotoxicological methods, in monitoring and research programs on large lowland rivers, could provide comprehensive assessment of ecosystem quality and relationship between chemical and ecological status itself. The main objectives of the dissertation are examination the sensitivity of in vitro and in vivo tests in ecogenotoxicology and guidelines formulation for their application in studies of large lowland rivers. The study included a total of 17 sites in the Velika Morava, Sava and Danube River basins. To assess the genotoxic effect of native and concentrated river water samples, the in vitro approach included the SOS/umuC test, alkaline comet test on HepG2 and ZFL cells, and cell cycle analysis on ZFL cells. The in vivo approach for genotoxicity assessment included the alkaline, Fpg modified comet and micronucleus test on Alburnus alburnus (bleak) blood cells. Also, the sensitivity of RAPD analysis in the detection of genotoxic effects was examined. Within in silico approach, based on the literature, the toxic/genotoxic potential of chemical parameters detected in water and bleaks was analyzed while the quantification of pollution pressure was performed by integrative methods. Bioassays showed different sensitivity in detecting the genotoxic effect. Within in vitro study, concentrated river water samples proved to be a suitable for toxicity/genotoxicity testing, and ZFL cells a good model system. Within in vivo research, bleak proved to be a reliable bioindicator. The highest sensitivity in the detection of genotoxic effect in blood cells was obtained in the alkaline comet test. A complete prediction of the genotoxic effect of xenobiotics on organisms cannot be performed by in silico analysis

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

Синтеза и биолошка активност алкилтио и арилтио деривата терц-бутилхинона

Biological activity of 2-tert-butyl-1,4-benzoquinone (TBQ) and its derivatives, 2-tert-butyl-5-(2-propylthio)-1,4-benzoquinone, 2-tert-butyl-5- -(propylthio)-1,4-benzoquinone, 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone, 2-tert-butyl-5-(phenylthio)-1,4-benzoquinone and 2-tert-butyl-6-(phenylthio)- 1,4-benzoquinone, were tested for their antioxidant, antibacterial, toxic, cytotoxic and genotoxic potential. Using the DPPH test, all derivatives showed good antioxidant activity, better than ascorbic acid, and the 2-tert- -butyl-5-(propylthio)-1,4-benzoquinone derivative showed the strongest effect. Better antibacterial potential was observed against Gram-positive bacteria in the broth microdilution method in which the 2-tert-butyl-5-(phenylthio)-1,4- -benzoquinone derivative showed the strongest activity (MIC = 15.6 μM). The results of toxicity tests, using the Brine shrimp test, indicated that the derivatives lose their toxic potential compared to TBQ, except for 2-tert-butyl-6- -(phenylthio)-1,4-benzoquinone, which showed a 3 times stronger effect. Cytotoxicity was assessed by the MTT assay in 24 and 72 h treatments in MRC-5, HS 294T and A549 cell lines in threefold decreasing gradient (11, 33 and 100 μM). Modifications potentiate the cytotoxic effect, and the strongest effect was observed with the 2-tert-butyl-5,6-(ethylendithio)-1,4-benzoquinone derivative. In addition, the genotoxic potential was examined in the MRC-5 cell line using the comet assay. All tested derivatives of TBQ showed a genotoxic effect at all applied subtoxic concentrations. In general, the chemical modifications of TBQ enhanced its biological activity.Испитана је биолошка активност 2-терц-бутил-1,4-бензохинона (TBQ) и његових деривата: 2-терц-бутил-5-(изопропилтио)-1,4-бензохинона, 2-терц-бутил-5-(пропилтио)-1,4-бензохинона, 2-терц-бутил-5,6-(етиленедитио)-1,4-бензохинона, 2-терц-бутил- -5-(фенилтио)-1,4-бензохинона и 2-терц-бутил-6-(фенилтио)-1,4-бензохинона укључујући њихов антиоксидативни, антибактеријски, токсични, цитотоксични и генотоксични потенцијал. Применом DPPH теста, сви деривати су показали добру антиоксидативну активност, бољу од аскорбинске киселине, а најјаче дејство показао је дериват 2-терц-бутил-5-(пропилтио)-1,4-бензохинон. Бољи антимикробни потенцијал је примећен против Грам-позитивних бактерија методом микродилуције у бујону, где је дериват 2-терц-бутил-5-(фенилтио)-1,4-бензохинон показао најјачу активност (MIC = 15,6 μМ). Резултати испитивања токсичности, применом теста на Artemia salina, показују да деривати губе токсични потенцијал у односу на TBQ, осим 2-терц-бутил-6-(фенилтио)- -1,4-бензохинона, који је показао 3 пута јачи ефекат. Цитотоксичност је испитана МТТ тестом у третманима од 24 и 72 h на ћелијским линијама MRC-5, HS 294T и A549 у троструко опадајућем градијенту (11, 33 и 100 μМ). Модификације појачавају цитотоксични ефекат, а најјачи ефекат је примећен код деривата 2-терц-бутил-5,6-(етилендитио)-1,4-бензохинона. Поред тога, генотоксични потенцијал је испитан на ћелијској линији MRC-5 комет тестом. Сви испитивани деривати су показали генотоксични ефекат при свим примењеним субтоксичним концентрацијама. Генерално, хемијске модификације побољшавају биолошку активност 2-терц-бутил-1,4-бензохинона

Detection of double-strand breaks in DNA molecules by the γH2AX assay and analysis of the cell cycle after treatment with potential antitumor agents TBQ and its alkylthio and arylthio derivatives.

2-tert-butyl-1,4-benzoquinone (TBQ) and its alkylthio and arylthio derivatives: 2-tert-butyl-5-(isopropylthio)-1,4-ben- zoquinone, 2-tert-butyl-5-(propylthio)-1,4-benzoquinone, 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone, 2-tert-butyl-5-(phenylthio)-1,4-benzoquinone and 2-tert-butyl-6-(phenylthio)-1,4-benzoquinone were synthe- sized as analogs of biologically active compounds of natural origin with antitumor activity: quinone avarol/avarone. The γH2AX test was used to detect the potential genotoxic effect of TBQ and its derivatives in the HepG2 cell line. In addition, the effect of these substances on the cell cycle was monitored on the same HepG2 model system. Since the phosphorylation of histone H2AX is an early event in the cellular response to DNA double-strand breaks (DSBs), an assay based on the detection of phosphorylated histone H2AX (γH2AX) can be used as a biomarker of genotoxicity and genomic instability. In this work, the γH2AX test and flow cytometry were used to analyze the genotoxic potential and the effect on the cell cycle of TBQ and its derivatives. Double-strand breaks (DSBs) in DNA are detected with γH2AX-specific antibodies, and the cell cycle in the HepG2 cell line is analyzed by flow cytometry. Based on the obtained results, only 2-tert-butyl-5,6- (ethylenedithio)-1,4-benzoquinone induced increased formation of DSBs. Also, the same derivative caused a significantly greater arrest of cells in the G2/M phase of the cell cycle compared to TBQ from about 27% (TBQ) to 34% of the total population with a decrease in the S phase cell population. By forming DSBs, 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone leads to genomic instability of the HepG2 cell line, which results in cell cycle arrest in the G2/M phase

Comet assay – a sensitive method for detection DNA damage and primary monitoring of ecosystem pollution pressure

Untreated municipal wastewaters are one of the major negative contributors on freshwater quality, and consequently on ecosystem balance. Some compounds such as pharmaceuticals, cosmetic products, etc. could be genotoxic and lead to somatic and/or germinative mutations. This mutation should be reflected on organisms’ health and reproductive potential. Comet assay is a widely used test in ecogenotoxicology for detection of primary DNA damage. In that way, this sensitive test could provide information about early warning signs of potential stressors effects before those have an impact on the population or ecosystem level. The scope of our study was to assess the level of DNA damage of Alburnus alburnus specimens’ gill cells and erythrocytes. Three sites on the Sava river were chosen: reference site Zabran suited upstream the municipal discharging; second at the confluence of the Kolubara river and the Sava river; and the third at the confluence of the Barička river and the Sava river. The Kolubara river and the Barička river are chosen as important recipients of untreated wastewaters. From each site, 5 bleak specimens were collected and blood and gills were taken. Tail Intensity (TI%) was chosen as a parameter for evaluating the level of DNA damage. Besides that, cell viability and extremely damaged cells - hedgehogs (HH) were determined. The significant difference in TI% values was recorded in erythrocytes and gills cells in comparison between the Zabran (TI%=14.01±0.61 in erythrocytes, TI%=15.25±1.03 in gills cells) and the second site (TI%=18.53±0.65 in erythrocytes, TI%=22.78±1.89 in gills cells). Correlation between cell viability and HH frequency was not observed. According to the results, we could conclude that the second site is the most affected by pollutants. Also, usage of comet assay on freshwater organisms could be appropriate for preliminary screening of ecosystem state

Antimicrobial activity of 2-tert-butyl-1.4-benzoquinone and its selected alkylthio and arylthio derivatives.

Background: Biologically active compounds, originating from a variety of natural sources: plants, animals and microor- ganisms, have great potential for use as antimicrobial agents. Avarol, a compound originating from the Mediterranean sponge Disidea avara, exhibits a number of biological activities including antimicrobial activity. Considering that, avarol was taken as a model for the synthesis of 2-tert-butyl-1,4-benzoquinone (TBQ) derivatives. Objectives: TBQ was chosen because of its similar chemical structure with avarol who showed strong biological activity but is more accessible and economical than avarol. By selecting the thiol group, we aimed at combining the action of two strong functional groups of natural origin, quinones and thiols, which both have antimicrobial activity. Methods: In this work antimicrobial activity of TBQ and its derivatives: 2-tert-butyl-5-(isopropylthio)-1.4-benzoqui- none, 2-tert-butyl-5-(propylthio)-1.4-benzoquinone, 2-tert-butyl-5,6-(ethylendithio)-1.4-benzoquinone, 2-tert-bu- tyl-5-(phenylthio)-1.4-benzoquinone and 2-tert-butyl-6-(phenylthio)-1.4-benzoquinone was evaluated by the MIC mi- crodilution method on 7 different ATCC bacterial strains. Results: All compounds tested showed stronger antimicrobial activity against Gram positive bacterial strains (Entero- coccus faecalis, Staphylococcus aureus, Bacillus spp.). A strong antimicrobial effect (MIC value less than 100μM) was shown by 2-tert-butyl-5,6- (ethylendithio)-1,4-benzoquinone, 2-tert-butyl-5-(phenylthio)-1.4-benzoquinone deriva- tives and 2-tert-butyl-6-(phenylthio)-1,4-benzoquinone against S.aureus. TBQ, 2-tert-butyl-5-(isopropylthio)-1,4-ben- zoquinone, 2-tert-butyl-5, 6-(ethylendithio)-1,4-benzoquinone and 2-tert-butyl-5-(phenylthio)-1.4-benzoquinone showed strong antimicrobial effect against Bacillus spp. According to our results, chemical modifications of TBQ in- crease its antimicrobial activity while derivative 2-tert-butyl-5,6-(ethylendithio)-1.4-benzoquinone is the best candi- date for further testing

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

Cryopreservation of fish blood – useful tool for assessing genotoxic potential of aquatic ecosystems

One of the major limitations in performing ecogenotoxicological studies is the distance between research field and the laboratory. As some of the methods used in ecogenotoxicology require fresh biological material with intact cell viability, transfer of samples to the laboratory within a few hours after sampling is usually required. To overcome this issue, we have introduced cryopreservation in our research as a possible solution. Cryopreservation is a method which includes preservation of intact, living cells at low temperature for a long time. In natural conditions freezing, forming of ice crystals and dehydration could destroy cell structures. To avoid this consequence, specific compounds were introduced, cryoprotective agents, in the method of cryopreservation. The main characteristic of these compounds is their ability to reduce ice crystal formation in cells at any temperature. We have applied cryopreservation in the evaluation of genotoxic potential along different river streams (the Adige River, the Sava River and the Velika Morava River basin). For this purpose, we focused on the level of DNA damage of cryopreserved fish blood cells (Salmo cenerinus, Salmo marmoratus, Alburnus alburnus) by using the comet assay. To test whether cryopreservation has the impact on cell viability, or that it induces additional DNA damage, we employed preliminary experiments in 4 Abramis brama and 8 A. alburnus specimens. Namely, from every specimen two blood samples were taken: one for analyzing cells viability and the level of DNA damage of fresh blood, and another for observing cell viability and DNA damage of cryopreserved samples. The viability of cell blood was determined by using acridine orange/ethidium bromide differential staining. For analyzing the level of DNA damage alkaline comet assay was used. Obtained results indicated that cryopreserved blood cells had approximately the same viability and the level of DNA damage as nonpreserved blood samples. According to our results, cryopreservation is a very useful method in genotoxicology and could have many benefits: blood samples should not be analyzed immediately after sampling; samples could be transported in liquid nitrogen without concern about additional DNA damage

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)