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

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

Cytostatics as emerging pollutants in aquatic environments - risk assessment based on genotoxic effects in haemocytes of freshwater mussels

Pharmaceutical compounds have begun to be considered as dangerous environmental pollutants, due to their widespread occurrence in wastewaters and their potential hazard towards the aquatic ecosystems. The comet assay was used to study acute impacts of most used cytostatic drugs on the haemocytes of widely distributed freshwater mussels Unio pictorum and Unio tumidus. After collection from unpolluted location, the mussels were held on accommodation for 10 days in controlled laboratory conditions before exposure to cytostatics. Groups of 5 mussels were exposed to 5-Fluorouracil, (0.04 – 40 μM), Cisplatin (0.004 – 4 μM), Etoposide (4, 40 and 100 μM) and Imatinib msesylate (0.04 - 40 μM). For positive control treatment with Cd was used (4, 40 and 100 μM), while as negative control mussels were held in control aquarium with clean water. Exposure was performed for 72h in aquaria. Comet assay is one of the major tools for the level of DNA damage assessment in ecogenotoxicology. It is based on single cell gel electrophoresis. In our experiments we performed alkaline version of comet assay described by Singh et al. (1988). Images of 250 nuclei per each concentration of test substance were analyzed with a fluorescence microscope and scored using analysis software (Comet Assay IV Image analysis system, PI, UK). Tail intensity was chosen as relevant measure of DNA damage. Our results indicated significant increase of DNA damage in haemocytes during treatment in selected concentration ranges for all cytostatics with the exception of Imatinib mesylate. Detected LOEC values were as following: 5-Fluorouracil – 52 μg/L, Cisplatin 12 μg/L and Etoposide – 24 mg/L. However, all effective concentrations of 5-FU, CP and ETO are higher than ones measured in surface water but still far below PNEC values which are currently used for the environmental risk assessment. This indicates that acute toxicity data might not be sufficient for prediction of adverse effects of substances, and that genotoxicity data should be also considered for the risk assessment

Opis przypadku — występujący rodzinnie wewnątrzczaszkowo germinoma

Background. Intracranial germinomas (ICG) are uncommon brain neoplasms with extremely rare familial occurance. Since ICG invades hypothalamus and/or pituitary, the endocrine dysfunction is one of the common determinants of these tumors. We presented two brothers with the history of ICG. Patient 1 is a 25-year-old male who had been suffering from the weakness of the right half of his body at the age of 18. Cranial MRI revealed mass lesion in the left thalamus. He underwent neurosurgery, tumor was removed completely. Histopathological (HP) and immunohistochemical analyses verified the diagnosis of pure germinoma. He experienced complete remission of the tumor after a radiation therapy. At the age of 22 the diagnosis of isolated growth hormone deficiency (IGHD) was established and GH replacement was initiated. Patient 2 is a 20-year old boy who was presented with diabetes insipidus at the age of 12. MRI detected tumor in the third ventricle and pineal region. After the endoscopic tumor biopsy the HP diagnosis was pure germinoma. He received chemotherapy followed by radiotherapy, and treated with GH during childhood. At the age of 18 GH replacement was reintroduced. A six month follow-up during the next two years in both brothers demonstrated the IGF1 normalization with no MRI signs of tumor recurrence. Conclusion. To the best of our knowledge so far, only six reports have been published related to familial ICG. The presented two brothers are the first report of familial ICG case outside of Japan. They are treated successfully with GH therapy in adult period. Wstęp: Rozrodczaki wewnątrzczaszkowe (intracranial germinomas, ICG) to rzadkie nowotwory mózgu, a szczególnie rzadko stwierdza się ich występowanie rodzinne. W związku z tym, że ICG zajmuje podwzgórze i/lub przysadkę mózgową, zaburzenia endokrynologiczne są jednym z najczęstszych wyznaczników obecności tych guzów. W pracy przedstawiono dwóch braci z ICG. Pacjent 1 to 25-letni mężczyzna, u którego w wieku 18 lat wystąpiło osłabienie mięśni po lewej stronie ciała. Badanie metodą rezonansu magnetycznego (MRI) czaszki ujawniło masę w lewym wzgórzu. Chorego poddano zabiegowi neurochirurgicznemu, podczas którego guz został całkowicie usunięty. Badania histopatologiczne i immunohistochemiczne potwierdziły rozpoznanie czystej postaci rozrodczaka. Po radioterapii nastąpiła całkowita remisja guza. W wieku 22 lat u chorego zdia­gnozowano izolowany niedobór hormonu wzrostu (isolated growth hormone deficiency, IGHD) i wdrożono terapię zastępczą hormonem wzrostu (growth hormone, GH). Genetyczna analiza molekularna tkanki guza wykazała mutację w eksonie 2 w genie KRAS. Pacjent 2 to 20-letni mężczyzna, u którego w wieku 12 lat stwierdzono moczówkę prostą. W badaniu MRI wykryto guz w okolicy trzeciej komory i szyszynki. Po ocenie histopatologicznej materiału pobranego za pomocą biopsji endoskopowej postawiono diagnozę czystego rozrod­czaka. U chorego zastosowano chemioterapię, a następnie radioterapię, a także podawano GH w okresie dzieciństwa. W wieku 18 lat u chorego wznowiono terapię GH. Sześciomiesięczna obserwacja obu braci w następnych 2 latach wykazała normalizację IGF1 przy braku objawów nawrotu guza w badaniu MRI. Wnioski: Według najlepszej wiedzy autorów dotychczas opublikowano 6 doniesień na temat rodzinnego występowania ICG. Przed­stawieni w niniejszej pracy bracia są pierwszym opisanym przypadkiem rodzinnego ICG poza Japonią. W okresie dorosłym chorzy są leczeni GH z dobrym skutkiem

Uticaj poplava na mikrobiološki kvalitet vode reke Save na teritoriji grada Obrenovca

U ovom istraživanju praćen je mikrobiološki kvalitet vode reke Save, na lokalitetu nizvodno od grada Obrenovca, pre i nakon velikih poplava koje su se dogodile u maju 2014. godine. Praćeni su indikatori fekalnog zagađenja (ukupni koliformi, Escherichia coli, Enterococcus faecalis) kao i pokazatelji organskog opterećenja (heterotrofi). Dobijeni rezultati pokazuju da je tokom meseca sa najvišim vodostajem (maj) došlo do velikog pada u koncentraciji glavnih pokazatelja fekalnog zagađenja, u prvom redu E. coli. Sa porastom vodostaja tokom perioda poplava uočen je i pad koncentracije heterotrofnih bakterija. S druge strane, detektovan je porast koncentracije ukupnih koliforma tokom poplava. U ovom ispitivanju jasno je pokazano da poplave dovode do promena u trendu variranja koncentracija mikrobioloških parametara kvaliteta vode

Freshwater mussels in the ecogenotoxicological studies-application of comet assay

Presence of pollutants in aquatic ecosystems can be detected by a range of physiological, histological and molecular responses, including abnormal morphology, alterations of antioxidative status and DNA integrity. The integrity of cellular DNA is continuously attacked by various agents resulting in DNA lesions. Unrepaired DNA lesions may block replication and transcription, potentially leading to cell death or generating mutations. The main objectives of ecogenotoxicology studies are detection of the origin of genotoxic pollution and their effect on individuals and populations. Mussels are commonly employed in the ecogenotoxicological studies. They have several characteristics, such as wide distribution, filter feeding, a sessile life form and an ability to accumulate pollutants, which makes them favorable organisms for estimating the environmental pollution level and the bioavailability of various types of pollutants. Different approaches are used for assessing the conditions of ecosystems, the passive (collection of mussels from selected locations) and the active biomonitoring (subsequent exposure of mussels from unstressed populations at polluted sites). Moreover, in ex situ assessment, the genotoxic effect of different compounds can be detected in laboratory conditions. The comet assay (single cell gel electrophoresis, SCGE), is a sensitive and rapid technique for detection of DNA damage in individual cells based on the migration of denatured DNA during electrophoresis (damaged nuclei form comet-like shapes). It has been accepted as one of the major tools for assessing pollution related genotoxicity in aquatic organisms. In our experiments we performed alkaline version of comet assay described by Singh et al. (1988). Images of 250 nuclei per each concentration of test substance were analyzed with a fluorescence microscope and scored using analysis software (Comet Assay IV Image analysis system, PI, UK). Tail intensity and Olive tail moment are chosen as relevant measure of DNA damage. Active biomonitoring implies translocation of specimens from unstressed populations to selected sites of interest. In active approach issues related to availability of specimens and effects of age, gender and physiological condition are avoided. This approach enables research on highly polluted sites. Specimens of Unio sp. were exposed at selected sites at the Sava and Danube rivers which are under the impact of untreated wastewaters of Belgrade city. Haemolymph was sampled after 7, 14 and 30 days of exposure. Passive biomonitoring implies collection of specimens which inhabit selected sites of interest. Easily performed, suitable when research area is situated far from the laboratory. Study was performed on the Velika Morava River, significant Danubes tributary. Specimens of Sinanodonta woodiana collected monthly on two sites with different pollution pressure. Significant correlation was detected between the level of DNA damage in haemocytes and concentration of haevy metals in water. Seasonal variation in the level of DNA damage was observed. Studies performed ex situ are significant research tool for prediction of the effect of single compounds in environment. Obtained data are useful for risk assessment. Among emerging pollutants, cytostatics represent hot topic as serious threat to non-target organisms due to their cytotoxicity, genotoxicity and mutagenicity. As part of research for Cytotreat project (EU Commision, FP7) we are investigating impact of short term exposure to mostly used drugs: 5-Fluorouracil, Cisplatin, Etoposide Vincristine and Imatinib mesylate with Cadmium chloride as positive control. Data obtained so far indicate that cytistatics can induce increase in DNA damage in concentrations similar to ones measured in wastewater. Our studies indicate that freshwater mussels can be used as reliable bioindicators. Research performed in situ showed that selected species can detect genotoxic pollution present in environment. Results of the research performed ex situ showed harmful effects of environmentally relevant concentrations of cytostatics. Studies performed ex situ on mussels coul be significant research tool for prediction of the effect of single compounds in environment

The Joint Danube Survey 3: Usage of freshwater mussels as bioindicators for detection of the genotoxic pollution

The Joint Danube Survey 3 (JDS3) was the world‟s biggest river research expedition of its kind in 2013. JDS3 catalyzes international cooperation from all 14 of the main Danube Basin countries and the European Commission. The JDS is carried out every six years – JDS1 was in 2001 and JDS2 in 2007. During the JDS3 we assessed the level of genotoxic pollution along the Danube River by measuring the level of DNA damage in the haemocytes of freshwater mussels of Unio sp. and Sinanodonta woodiana with comet assay. The research was performed on 34 out of 68 sites analysed within the JDS3. During research, 2,285 river kilometres were covered with an average distance of 68 km between the sites. The complex data set on concentrations of various substances present in water, suspended particulate matter and sediment on investigated sites gave the opportunity to identify the groups of xenobiotics which mostly affect the studied biomarker – DNA damage. The highest levels of DNA damage were recorded in the section VI (Panonnian Plain), which is under the impact of untreated wastewater discharges. Both positive and negative influence of the large tributaries on the level of genotoxicity in the Danube River was evident. Significant correlation in response was detected between the studied freshwater mussels. The level of DNA damage in mussels correlated with concentrations of compounds from the group of hazardous priority substances (polycyclic aromatic hydrocarbons), persistent organic pollutants (dioxins) and emerging pollutants (Oxazepam, Chloridazon-desphenyl)

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

Androgen receptor expression predicts beneficial tamoxifen response in oestrogen receptor-alpha-negative breast cancer

Background: Although the androgen receptor (AR) is frequently expressed in breast cancer, its relevance in the disease is not fully understood. In addition, the relevance of AR in determining tamoxifen treatment efficiency requires evaluation. Purpose: To investigate the tamoxifen predictive relevance of the AR protein expression in breast cancer. Methods Patients were randomised to tamoxifen 40 mg daily for 2 or 5 years or to no endocrine treatment. Mean follow-up was 15 years. Hazard ratios were calculated with recurrence-free survival as end point. Results: In patients with oestrogen receptor (ER)-negative tumours, expression of AR predicted decreased recurrence rate with tamoxifen (hazard ratio (HR) = 0.34; 95% confidence interval (CI) = 0.14-0.81; P = 0.015), whereas the opposite was seen in the AR- group (HR = 2.92; 95% CI = 1.16-7.31; P = 0.022). Interaction test was significant P &amp;lt; 0.001. Patients with triple-negative and AR+ tumours benefitted from tamoxifen treatment (HR = 0.12; 95% CI = 0.014-0.95 P = 0.044), whereas patients with AR- tumours had worse outcome when treated with tamoxifen (HR = 3.98; 95% CI = 1.32-12.03; P = 0.014). Interaction test was significant P = 0.003. Patients with ER+ tumours showed benefit from tamoxifen treatment regardless of AR expression. Conclusions: AR can predict tamoxifen treatment benefit in patients with ER- tumours and triple-negative breast cancer.Funding Agencies|Swedish research council [A0346701]; Swedish cancer foundation [13 0435]</p