Comparison of comet assay parameters for estimation of genotoxicity by sum of ranking differences

Genotoxic potential of waters in six rivers and reservoirs from Serbia was monitored in different tissues of chub (Squalius cephalus L. 1758) with the alkaline comet assay. The comet assay, or a single cell gel electrophoresis, has a wide application as a simple and sensitive method for evaluating DNA damage in fish exposed to various xenobiotics in the aquatic environment. Three types of cells, erythrocytes, gills and liver cells were used for assessing of DNA damage. Images of randomly selected cells were analyzed with a fluorescence microscope Leica and image analysis by software (Comet Assay IV Image analysis system, PI, UK). Three parameters (tail length - l, tail intensity - i and Olive tail moment – m) were analyzed on 1750 nuclei per cell type. The procedure for sum of ranking differences (SRD) was implemented to compare different types of cells and different parameters for estimation of DNA damage. Regarding our nine different estimations of genotoxicity: tail length, intensity and moment in erythrocytes (rel, rei, rem), liver cells (rll, rli, rlm) and gill cells (rgl, rgi, rgm) SRD procedure has shown that the Olive tail moment and tail intensity are (almost) equally good parameters; the SRD value was lower for the tail moment and tail intensity than for tail length in case of all types of cells. The least reliable parameter was rel; close to the borderline case were rei, rll, and rgl (~5% probability of random ranking)

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

Detection of SARS-CoV-2 RNA in the Danube river in Serbia associated with the discharge of untreated wastewaters

In Serbia less than 13 % of collected municipal wastewaters is being treated before their release in the environment. This includes all municipal wastewater discharges from Belgrade (capital city of Serbia; population 1,700,000). Previous research has identified the impacts of raw wastewater discharges from Belgrade on the Danube River, and this study investigated if such discharges also provided a pathway for SARS-CoV-2 RNA material. Samples were collected during the most critical circumstances that occurred so far within the COVID-19 pandemics in Serbia. Grab and composite samples were collected in December 2020, during the peak of the third wave (in terms of reported cases) at the site which receives the wastewater loads in Belgrade. Grab samples collected upstream and downstream of Belgrade were also analyzed. RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. SARS-CoV-2 RNA (5.97×103 to 1.32×104 copies/L) was detected only in samples collected at the site strongly impacted by the wastewaters where all three applied primer sets gave positive signals. Determined concentrations correspond to those reported in wastewater influents sampled at treatment plants in other countries indicating an epidemiological indicator function of used approach for rivers with high pollution loads in countries with poor wastewater treatment

Application of sos/umuc assay in eco/genotoxicology

Introduction. The SOS/umuC assay is used for assessment of water genotoxicity. Salmonella typhimurium TA1535/pSK1002 is used in this test, but the results can be extrapolated on higher eukaryotic organisms with the introduction of enzymatic S9 fraction in experimental procedure. The test is standardised for the determination of the genotoxic potential of water and wastewater (ISO/DIS 13829, 2000). Aim. Sensitivity of the assay was challenged by using parallel in situ and in vitro approach in evaluation of the genotoxic potential in the basins of significant tributaries of the Danube River: the Sava River and the Velika Morava River. Materials and methods. Within in vitro testing, native water samples were analysed by SOS/umuC test. DNA damage in situ was assessed in bleak (Alburnus alburnus) erythrocytes by the comet and micronucleus assays. The concentration of heavy metals in fish tissue and the data of the physico-chemical parameters measured in water were used as a measure of the pollution pressure at the sites. Results. Results showed that applied in vitro tests with native water samples are less sensitive in comparison with in situ tests. None of 20 investigated samples showed genotoxic potential in SOS/umuC assay while in situ analyses indicated variation of genotoxic potential among the investigated sites. Conclusions. The results of our study point towards low sensitivity of the SOS/umuC test when processing un-concentrated (native) water samples; the results of in vitro tests should be taken with precaution when making predictions on the status of the ecosystem

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

Application of sos/umuc assay in eco/genotoxicology

Introduction. The SOS/umuC assay is used for assessment of water genotoxicity. Salmonella typhimurium TA1535/pSK1002 is used in this test, but the results can be extrapolated on higher eukaryotic organisms with the introduction of enzymatic S9 fraction in experimental procedure. The test is standardised for the determination of the genotoxic potential of water and wastewater (ISO/DIS 13829, 2000). Aim. Sensitivity of the assay was challenged by using parallel in situ and in vitro approach in evaluation of the genotoxic potential in the basins of significant tributaries of the Danube River: the Sava River and the Velika Morava River. Materials and methods. Within in vitro testing, native water samples were analysed by SOS/umuC test. DNA damage in situ was assessed in bleak (Alburnus alburnus) erythrocytes by the comet and micronucleus assays. The concentration of heavy metals in fish tissue and the data of the physico-chemical parameters measured in water were used as a measure of the pollution pressure at the sites. Results. Results showed that applied in vitro tests with native water samples are less sensitive in comparison with in situ tests. None of 20 investigated samples showed genotoxic potential in SOS/umuC assay while in situ analyses indicated variation of genotoxic potential among the investigated sites. Conclusions. The results of our study point towards low sensitivity of the SOS/umuC test when processing un-concentrated (native) water samples; the results of in vitro tests should be taken with precaution when making predictions on the status of the ecosystem

Wastewater based epidemiology in countries with poor wastewater treatment - SARS-CoV-2 RNA in surface waters

Background Wastewater-based epidemiology surveillance of COVID-19 and other outbreaks in the future is a challenge for developing countries, as the majority of households are not connected to sewerage systems. In December 2019, we have detected SARS-CoV-2 RNA in the Danube River at a site that is severely affected by wastewater of Belgrade. Considering that rivers are much more complex systems in comparison to wastewater, additional efforts are needed to address all the factors which might influence the adoption of WBE as an alternative of targeting raw wastewater. Objectives The major objective of this study was to provide a more detailed insight in the potential of SARS-CoV-2 surveillance in Serbian surface waters under consideration of epidemiological, microbiological, physico-chemical and hydro-morphological parameters for epidemiological purposes. Methodology Water samples were collected at 12 sites at Sava and Danube Rivers in the Belgrade city area during the fourth COVID-19 wave in Serbia that started in late February 2021. SARS-CoV-2 RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (standard fecal indicator bacteria and microbial faecal source tracking markers), epidemiological, physico-chemical and hydro-morphological parameters were analysed in parallel. Results Out of 44 samples analyzed, 31 were positive for at least one of the target regions of SARS-CoV-2. The results indicated that surveillance of SARS-CoV-2 RNA in surface waters in context with the large amount of epidemiological and environmental metadata can be used as epidemiological early-warning tool in countries with poor wastewater treatment

Variability in DNA damage of chub (Squalius cephalus L.) blood, gill and liver cells during the annual cycle

In this work the genotoxic potential of water in three localities in Serbia, which differ by the nature and degree of pollution, was determined in tissues of European chub (Squalius cephalus L.) on monthly basis over the 2011/2012 year season using the alkaline comet assay. Specimen samples of chub were taken from Special Nature Reserve ``Uvac{''}, as control site, and Pestan and Beljanica Rivers, as polluted sites at Kolubara basin, surrounded with coal mines. Three tissues, blood, gills and liver were used for assessing the level of DNA damage. Analysis was done by software (Comet Assay IV). The control site at Reserve ``Uvac{''} showed the lowest DNA damage values for all three tissues compared to Pestan and Beljanica. Blood has the lowest level of DNA damage in comparison with liver and gills. Decreased damage for all three tissues was observed at summer, while during the spring and autumn damage increased. (C) 2014 Elsevier B.V. All rights reserved.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173045

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3′-methoxyavarone, 4′-(methylamino)avarone and 3′-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3′-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3′-methoxyavarone and 3′-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3033