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

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

DNA damage and histopathological alterations in liver and gills of common bream Abramis brama (L.) as biomarkers of the Danube River pollution

Biomarkers are used in ecosystem health assessment because they provide information about the biological effects of pollution. Aquatic ecosystems are often the ultimate recipients of different pollutants, which may have potentially genotoxic and carcinogenic effects on aquatic organisms. The aim of this study was to assess the impact of untreated urban wastewater during different seasons on the DNA damage level and histopathological alterations in gills and liver of common bream (Abramis brama). This study was carried out at the site Višnjica, situated downstream from Belgrade, which is identified as one of the major hotspots of fecal pollution along the Danube River. Sampling was performed in February, April, August and November 2014. DNA damage in fish tissues was assessed by alkaline comet assay. Histopathological alterations were quantified by combining pathological significance and the extent for each lesion. Subsequently, lesion scores within organs are summed in order to calculate histopathological index. The highest level of DNA damage in both tissues was observed in August. Liver had the lowest level of DNA damage in February, and gills in April. Gills had a significantly higher level of DNA damage in comparison to liver during all months, except in April. Histopathological index of the liver had the highest values in April and of the gills in November. Both tissues had the lowest value of histopathological index in August. In general, histopathological alterations in liver were more severe in comparison to gills during all months, except in November. The presence of DNA damage acted as an early-marker signal in fish, while histopathological alterations reflected state of fish organs when exposed to contaminants during long term pollution. This study confirmed the potential of untreated urban wastewaters to trigger responses on different levels of biological organization in fish

The impact of the Danube River pollution on biomarkers response in the liver and gills of common bream Abramis brama (L., 1758)

The Danube, the second largest river in Europe, is of great importance for all the countries in the basin for production of drinking water, water supply for domestic, agricultural and industrial purposes. Along the stream, the river receives incompletely treated or untreated wastewaters, which deteriorates its quality. Measurements of physico-chemical, chemical and microbiological parameters are considered the basis of monitoring because they cover a wide spectrum of information for proper water management. However, aquatic ecosystems are often exposed to different pollutants which may exhibit harmful effects on different levels of biological organization. Fish are widely used bioindicators in ecogenotoxicological studies, because of their position in the aquatic trophic chain and importance in human diet. This research was conducted on the Danube River locality Višnjica, situated on the right river bank (1162 rkm), one of the main hotspots of fecal pollution. Discharge of the largest waste water collector in the capital of Serbia, Belgrade, which receives domestic and industrial waste, is situated a few km upstream of the sampling site. Sampling was performed in February, April, August and November 2014, with the aim to evaluate the impact of seasonal variation on water quality parameters and response of biomarkers in the common bream (Abramis brama). Water quality was assessed by using basic physico-chemical parameters and microbiological indicators of fecal pollution. In gills and liver of bream, metal and metalloid concentrations were assessed as an accumulation biomarker, comet assay was applied to assess DNA damage as a biomarker of exposure, while histopathological analysis was performed as a biomarker of effect. According to number of Escherichia coli and enterococci critical and excessive level of fecal pollution was present on the site, indicating the presence of high amounts of untreated waste waters. During the entire period gills had higher concentrations of metals and metalloids in comparison to the liver. Both tissues showed the highest metal accumulation and the highest level of DNA damage in August, which may be attributed to increased activity and feeding during summer, but also to low flow rate and water level which may increase pollutant concentrations in the river. The highest level of histopathological alterations in the gills was recorded in November and in liver in April, probably due to higher level of precipitation during these months and introduction of pollutants through the urban and agricultural runoff. In terms of elements concentrations and DNA damage gills were more affected, while in terms of histopathological alterations liver had higher level of histopathological alterations. These results showed that urban wastewaters have a high potential in inducing genotoxic and histopathological effects in fish, and highlighted the urgent need for implementation of wastewater treatment facilities

The impact of the Sava river pollution on biomarkers response in the liver and gills of three cyprinid species

Due to the presence of a large number of different pollutants, monitoring of the surface water quality based solely on the analysis of a limited number of xenobiotics, cannot be considered as reliable. Beside toxic, these agents can exert genotoxic effects, inducing damage in the DNA molecule, which, if not repaired, could lead to mutations and alterations in cells, tissues, organism, whole population and the ecosystem. The surface waters are under the pressure of both anthropogenic and natural sources of pollution. Additionally, extreme hydrological events, such as water scarcity and flooding, may further impair the state of freshwater bodies. Fish may be exposed to harmful substances through water, sediment and food. In ecogenotoxicity studies, gills are used as they represent the first organ in direct contact with water and waterborne pollutants, while the liver, as a key organ that controls many life functions is used as a major organ for metabolic breakdown of xenobiotics. Common bream (Abramis brama), white bream (Blicca bjoerkna) and white-eye bream (Ballerus sapa) are three closely related, benthivorous cyprinids, native for the Sava River. The sampling site Duboko (23 rkm), on the Sava River, is chosen as it is exposed to the untreated wastewater from the town of Obrenovac (more than 70,000 inhabitants), intensive agricultural activity and close proximity to the largest thermal power plant in Serbia (TENTA) and belonging ash field. This study was conducted to assess the impact of multiple stressors during different seasons on different levels of biological organization, subcellular (genotoxic effect) and cellular/tissue level (histopathological effects), in the liver and gills of three bream species. As a biomarker of exposure DNA damage was measured by applying the alkaline comet assay, while histopathological alterations were monitored as a biomarker of effect. In parallel, concentration of metals and metalloids were assessed in gills, liver and muscle. Basic physical (pH, temperature, oxygen concentration, electrical conductivity) and chemical (NO2, NO3-, NH4+, PO43-) parameters were measured on site. Microbiological indicators of faecal pollution, total coliforms (TC), Escherichia coli (EC) and Enterococci (EF) were assessed by using a most probable number approach (MPN). Presumptive Clostridium perfringens (CP) numbers were determined by using membrane filtration and incubation on TSC (Tryptose Sulphite Cycloserine) media. Sampling of fish tissue for comet assay and histopathological analyses was performed in 2014, during winter (January and February), spring (March and early June), and summer (late June, July, and August), once per month, for a total of 52 specimens. Analysis of metals and metalloids was performed only on fish sampled in February, early June and in August, in total 15 specimens. In the mid May extreme hydrological events led to extensive flooding in the studied area. Comet assay was conducted on the liver and gill cells according to Kostić et al. Tail intensity, % of DNA in the comet tail (TI) was used to express the DNA damage level. Histopathological analyses included the use of conventional histological methods, staining of tissues sections using hematoxylin eosine differential staining, and examined under the microscope. The type and the extent of histological alterations were described by using a method developed by Bernet et al. Analysis of metals and metalloids was performed by inductively-coupled plasma optical emission spectrometry (ICP-OES), and included assessment of concentrations of 16 elements (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb, Sr and Zn). To compare the total metal content in different tissues and through different seasons metal pollution index (MPI) was calculated according to equation MPI = (cf1 x cf2 x cf3 x…cfn) 1/n, where cfn = concentration of the metal n in the sample. Statistical analysis of data from the individual months showed the highest DNA damage in gill cells during early June (spring). Gill histopathological index (IG) did not show significant seasonal variations, however it was the lowest during winter, the highest in spring, and slightly decreased in summer. A possible cause of this incidence could be a withdrawal of water which took place in June, after the flooding event that occurred in the middle of May. In liver the highest DNA damage was observed during August. Histopathological index of liver showed significantly higher values in summer in comparison to spring. This could be prescribed to a higher metabolic rate of fish liver during warm seasons and also could be a consequence of processing a large quantities of xenobiotics introduced into the water column due to withdrawal of water after floods and sediment disturbance. According to the MPI, gills were under the highest pressure of metal pollution during spring and summer. Liver was under the highest pressure of metal pollution during winter, while the muscle was the least affected tissue during all three seasons. Overall, gills as the first organ in direct contact with water showed a higher response in terms of DNA damage (molecular level), while the liver as the major organ for processing of xenobiotics both from water and food showed a higher degree of histopathological alterations in comparison to gills (tissue/organ level). Increased response of both biomarkers during spring and summer indicates a joint effect of the flooding event and seasonal changes of climate and hydrological parameters

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 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

The anthropogenic impact on water quality of the river Danube in Serbia: Microbiological analysis and genotoxicity monitoring

The aim of this work was to examine the impact of urban wastewaters on the water quality of the Danube River in Serbia. Samples of water and sediments for microbiological analysis and genotoxicity monitoring were collected from 6 sites during spring and/or autumn 2010. Sanitary analysis, i.e. enumeration of total and fecal coliforms and intestinal enterococci, indicated moderate to critical fecal contamination, while organic load assessment (oligotroph to heterotroph ratio, index of phosphatase activity) revealed the category of moderately polluted water. Mercury-resistant bacteria were detected in all water samples, with high numbers at locations positioned downstream of Belgrade. There was no correlation of the microbiological parameters of the sediment and water samples. Genotoxicity monitoring, performed by the comet assay on hemocytes of mussels Sinanodonta woodiana, indicated a significant increase of DNA damage in mussels collected from the studied sites compared with the control group

Comparative study of the genotoxic response of freshwater mussels Unio tumidus and Unio pictorum to environmental stress

Genotoxic response of freshwater mussels U. tumidus and U. pictorum to environmental stress was studied using comet assay on hemocytes and gill cells. The mussels were acclimated to controlled laboratory conditions for 10 days, and then exposed at 4 sites in the Sava and Danube rivers in the area of the city of Belgrade. Samples of each species were taken after 7, 14, and 30 days of exposure. The mussels sampled immediately after acclimation served as controls. Genotoxic response in both species was induced earlier at sites receiving untreated wastewaters from the city's main collectors (7 days), than at the site receiving only domestic wastewaters from small settlements located upstream from the city (30 days). There was a correlation between the comet tail intensity values in tissues of exposed mussels and the concentrations of zinc, copper, iron, and arsenic at the exposure sites. The genotoxic responses in both tissues of U. pictorum and in hemocytes of U. tumidus were similar, while U. tumidus gill cells failed to exhibit significant genotoxic response at two sites. These findings, together with higher mortality of U. tumidus at the most polluted sites, promote U. pictorum as a model for genotoxicity monitoring in freshwater environments.European Community {[}265264