Antigenotoxic effect of plant extracts

This report reviews our study of antigenotoxic compounds from medicinal and aromatic plants performed over several years. The studies of this type are aimed at understanding the protective mechanisms which may be relevant for the primary prevention of cancer and other mutation-related diseases. Antigenotoxic potential in this study is estimated with prokaryotic and eukaryotic tests measuring spontaneous and induced mutations, recombination, mutagenic repair, chromosomal aberrations and micronuclei. Our results indicate that monoterpenoids from sage act as modulators of DNA repair pathways, whereas sage antioxidants interfere with metabolic activation enzymes. The potential use of sage extracts in cancer prevention is discussed

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.Projekat ministarstva br. 173025 i br. 4300

Seasonal variations of microbiological parameters of water quality of the Velika Morava river Serbia

In this study, we investigated the level of sanitary pollution and organic contamination of the Velika Morava River, the largest river in central Serbia. Samples of water for microbiological analysis were collected at 5 sites, monthly, from April 2010 to February 2011. 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. We also investigated seasonal variations of these groups of bacteria and the factors that could contribute to these differences. Our results showed that the micro- biological quality of the water in the Velika Morava River during different seasons is affected by numerous factors such as unequal loading of wastewaters, solar irradiation, and relations of flow/dilution and rainfall/runoff. [Acknowledgments. This study was financially supported by the Ministry of Education and Science of the Republic of Serbia: projects No 173025, No 177045 and No 043002.

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

Seasonal variations of microbiological parameters of water quality of the Velika Morava river Serbia

In this study, we investigated the level of sanitary pollution and organic contamination of the Velika Morava River, the largest river in central Serbia. Samples of water for microbiological analysis were collected at 5 sites, monthly, from April 2010 to February 2011. 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. We also investigated seasonal variations of these groups of bacteria and the factors that could contribute to these differences. Our results showed that the micro- biological quality of the water in the Velika Morava River during different seasons is affected by numerous factors such as unequal loading of wastewaters, solar irradiation, and relations of flow/dilution and rainfall/runoff.Projekat ministarstva br. 173025, br. 177045 i br. 04300

Ispitivanje antivirusne aktivnosti ekstrakata žalfije Salvia officinalis L. (Lamiaceae)

In the present study, we examined cytotoxicity and extracellular and intracellular antiviral activity of frac­tionated extracts of wild and cultivated sage Salvia officinalis L. (Lamiaceae) in vitro using the WISH-VSV model system. Extracts were obtained by fractionating depigmented ethanol extracts of sage plants with supercritical CO2 at different pressures. Cytotoxicity was determined by examining cellular morphology in situ with the aid of a colorimetric micromethod and by cell staining with trypan blue. The fraction of distilled cultivated sage obtained at CO2 pressure of 300 bars and temperature of 60°C (149/3) was the most cytotoxic, with CTD10 44 μg/ml. That of non-distilled cultivated sage obtained at CO2 pressure of 500 bars and temperature of 100°C (144/5) was the least toxic (CTD10 199 μg/ml). Moreover, 144/5 had an antiviral effect at the intracellular level: when added 5 hours before VSV infection, it caused 100% reduction of CPE at concentrations of 99.5 and 199.0 μg/ml; when added after virus penetration had occurred, the same concentrations caused 35 and 60% reduction, respectively. The obtained results indicate that antiviral activity of 144/5 involves inhibition of the early steps of the virus infective cycle without a direct virucidal effect. Abbreviations: WISH - human amnion epithelial cells, VSV - vesicular stomatitis virus, HSV - herpes simplex virus, CPE - cytopathic effect, IS - selectivity index, TCID50 - tissue culture infective dose, CTD10 - 10% cytotoxic concentrations.U radu je ispitivana antiviralna aktivnost različito frakcionisanih ekstrakata divlje i gajene žalfije Salvia officinalis L. (Lamiaceae) u in vitro uslovima koristeći WISH-VSV model sistem. Ekstrakti su dobijeni frakcionisanjem depigmentisanog etanolnog biljnog ekstrakta pod različitim pritiskom CO2. Citotoksičnost je određivana praćenjem ćelijske morfologije in situ, i bojenjem ćelija sa tripan plavim. Frakcija gajene žalfije dobijena na CO2 pritisku od 300 bara i temperaturi od 60°C (149/3) je pokazala najveću citotoksičnost (CTD10 44 μg/ml). Frakcija nedestilisane gajene žalfije dobijena na CO2 pritisku od 500 bara i temperaturi od 100°C (144/5) je pokazala najmanju toksičnost (CTD10 199 μg/ml). Takođe, frakcija 144/5 je pokazala i antiviralni efekat na intracelularnom nivou: kada se ćelije tretiraju 5 sati pre VSV infekcije, redukcija CPE je bila 100% na koncentraciji od 99.5 i 199.0 μg/ml; kada se ćelije tretiraju posle ulaska virusa u ćeliju na istim koncentarcijama redukcija CPE iz­nosi 35% odnosno 60%. Dobijeni rezultati ukazuju da frakcija 144/5 ima antiviralnu aktivnost koja se ostvaruje krozinhibiciju ranih stupnjeva viralne infekcije bezdirektnog virucidalnog efekta

Genotoxicity assessment of Danube River: in situ and in vitro methods.

Water quality in the Danube River Basin is largely influenced by the inputs of pollutants - particularly excessive nutrients, organic material, and hazardous substances, which affects the whole ecosystem at various levels. The major goal of this study was to evaluate the level of genotoxic potential at the site at Danube River, Novi Banovci, which is under significant anthropogenic pressure of untreated municipal and industrial waste waters. DNA damage in situ was assessed in 5 specimens of white bream (Blicca bjoerkna) erythrocytes, liver and gills cells with comet assay and in erythrocytes with micronucleus test. Additionally, genotoxicity of native water samples collected from the site and upstream and downstream of the site was evaluated by the comet assay on HepG2 cells. Microscopic images of comets were scored using Comet IV Computer Software (Perceptive Instruments, UK) and tail intensity was chosen as parameters to assess the DNA damage. Comet assay on white bream showed that gills were the most affected tissue, while liver showed the smallest values. Micronuclei frequency was low in general, with mean of 1 micronuclei in 5000 scored erythrocytes, with no significant correlation to the values of comet assay in erythrocytes. Comet assay on HepG2 cells showed significant difference between values of negative control samples and samples of water from effluent site and downstream of wastewater discharge. There was no significant difference between negative control and upstream samples of water. Our results showed that great impact of municipal and industrial wastewaters on the river ecosystem. The application of in vitro and in situ tests gives a better insight into genotoxic effects. More importantly, because Serbia still doesn’t have waste water treatment plants, this kind of research points out the imperative for implementation of this facilities

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.Projekat ministarstva br. 173025 i br. 4300

Significance of genotoxicity and toxicity evaluation of freshwater bodies

Fish communities are excellent indicators of biological and ecological integrity due to their continuous exposure to water conditions. Freshwater biodiversity constitute a valu able natural resource but extensive pollution of freshwater can result in biodiversity de cline. For that reason it is important to employ bioassays for purpose of detecting these conditions. In this work we monitored chub and bream species in 2 reservoirs, Uvac and Garasi, and 4 rivers: Dunav, Sava, Pestan and Beljanica. For assessment of metal and metalloid in fish tissues (liver, gills, gonads and muscle) we have used ICP-OES. The comet assay or single cell gel electrophoresis (SCGE) was selected as an in vivo geno toxicity assay, for measuring DNA damage in blood, liver and gills. Histopathological alterations were monitored in liver and gills.The high quality of Uvac water was con firmed by low values of DNA damage in all tissues compared to other sites. An analysis metals in tissues showed a high degree of their differentiation, as well as significant dif ferences in the distribution of tissue elements between the sites tested. The highest con centrations of most of the analyzed metals were found in gills, liver and gonads, and the lowest in muscles at all sites. In chub, blood showed the lowest DNA damage compared to liver and gills, while in all breams DNA damage was the highest in blood cells, follow ing gills and liver. Histopathological analysis performed on bream specimens on the Sava and Danube River revealed a higher degree of alterations in liver compared to the gills