Evaluation of potential genotoxic/cytotoxic effects induced by epoxiconazole and fenpropimorph-based fungicide in bovine lymphocytes in vitro

Potential genotoxic/cytotoxic effects of the epoxiconazole/fenpropimorph-based fungicide were investigated using single cell gel electrophoresis and cytogenetic assays: chromosomal aberrations, sister chromatid exchanges, micronuclei and fluorescence in situ hybridization in cultured bovine lymphocytes. No statistically significant elevations of DNA damage and increases in cytogenetic endpoints were seen. However, evident cytotoxic effect presented as a decrease in mitotic and proliferation indices were recorded after exposure of bovine lymphocytes to the fungicide for 24 and 48 h at concentrations ranging from 3 to 15 µg mL^?1 (P < 0.05, P < 0.01, P < 0.001). Similarly, for 24 h an inhibition in the cytokinesis block proliferation index (CBPI) was obtained after exposure to the fungicide at concentrations ranging from 1.5 to 15 µg mL^?1 (P < 0.01, P < 0.001) in each donor.     

Pure non-dioxin-like PCB congeners suppress induction of AhR-dependent endpoints in rat liver cells

The relative potencies of non-ortho-substituted coplanar polychlorinated biphenyl (PCB) congeners to activate the aryl hydrocarbon receptor (AhR) and to cause the AhR-dependent toxic events are essential for their risk assessment. Since some studies suggested that abundant non-dioxin-like PCB congeners (NDL-PCBs) may alter the AhR activation by PCB mixtures and possibly cause non-additive effects, we evaluated potential suppressive effects of NDL-PCBs on AhR activation, using a series of 24 highly purified NDL-PCBs. We investigated their impact on the model AhR agonist-induced luciferase reporter gene expression in rat hepatoma cells and on induction of CYP1A1/1B1 mRNAs and deregulation of AhR-dependent cell proliferation in rat liver epithelial cells. PCBs 128, 138, and 170 significantly suppressed AhR activation (with IC₅₀ values from 1.4 to 5.6 μM), followed by PCBs 28, 47, 52, and 180; additionally, PCBs 122, 153, and 168 showed low but still significant potency to reduce luciferase activity. Detection of CYP1A1 mRNA levels in liver epithelial cells largely confirmed these results for the most abundant NDL-PCBs, whereas the other AhR-dependent events (CYP1B1 mRNA expression, induction of cell proliferation in confluent cells) were less sensitive to NDL-PCBs, thus indicating a more complex regulation of these endpoints. The present data suggest that some NDL-PCBs could modulate overall dioxin-like effects in complex mixtures.     

Influence of selected anti-cancer drugs on the induction of DNA double-strand breaks and changes in gene expression in human hepatoma HepG2 cells

In chemotherapy, various anti-cancer drugs with different mechanisms of action are used and may represent different risk of undesirable delayed side effects in treated patients as well as in occupationally exposed populations. The aim of the present study was to evaluate genotoxic potential of four widely used anti-cancer drugs with different mechanisms of action: 5-fluorouracil (5-FU), cisplatin (CDDP) and etoposide (ET) that cause cell death by targeting DNA function and imatinib mesylate (IM) that inhibits targeted protein kinases in cancer cells in an experimental model with human hepatoma HepG2 cells. After 24 h of exposure all four anti-cancer drugs at non-cytotoxic concentrations induced significant increase in formation of DNA double strand breaks (DSBs), with IM being the least effective. The analysis of the changes in the expression of genes involved in the response to DNA damage (CDKN1A, GADD45A, MDM2), apoptosis (BAX, BCL2) and oncogenesis (MYC, JUN) showed that 5-FU, CDDP and ET upregulated the genes involved in DNA damage response, while the anti-apoptotic gene BCL2 and oncogene MYC were downregulated. On the contrary, IM did not change the mRNA level of the studied genes, showing different mechanism of action that probably does not involve direct interaction with DNA processing. Genotoxic effects of the tested anti-cancer drugs were observed at their therapeutic concentrations that may consequently lead to increased risk for development of delayed adverse effects in patients. In addition, considering the genotoxic mechanism of action of 5-FU, CDDP and ET an increased risk can also not be excluded in occupationally exposed populations. The results also indicate that exposure to 5-FU, CDDP and ET represent a higher risk for delayed effects such as cancer, reproductive effects and heritable disease than exposure to IM.     

Metal contamination of short-term snow cover near urban crossroads: correlation analysis of metal content and fine particles distribution

Snow samples were collected near crossroads in Novi Sad, Serbia, during December 2009 to assess metal concentrations (Ca, K, Zn, Fe, Cu, Mn, Al, Pb and Na), fine particle distribution and pH value. The filtered samples of melted snow were analysed, with a focus on particles smaller than a few μm. The most common values of the frequency number distribution curve were either in the range of 0.05-0.07 μm or one order of magnitude higher (0.2-0.5 μm). At examined locations metal concentrations varied from 0.0004 mg L⁻¹ for Pb to 18.9 mg L⁻¹ for Na. Besides Na, which mostly originated from de-icing salt, Ca is found to be the most abundant element in snow indicating the dominant influence of natural factors on snow chemistry. No significant difference was found in concentration of the elements at locations near crossroads with either low or high traffic volume, except for Na and Zn.To investigate how metals were related a correlation analysis was done for the concentrations of metals and with respect to the particle size distribution parameters and pH values. The major influence on the fine particle volume (mass) was concluded to be due to the elements from anthropogenic sources. This conclusion was based on the significant positive correlation between Fe, Zn and Al and the fine particle volume based distribution parameters.     

Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus)

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mre?nica) and exposed in cages for 1 week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.     

Cytogenetic status of human lymphocytes after exposure to low concentrations of p,p'-DDT, and its metabolites (p,p'-DDE, and p,p'-DDD) in vitro

Despite that the use of DDT has been restricted for more than 40 years to malaria affected areas, low doses of this pesticide and its metabolites DDE and DDD can be found in the environment around the world. Although it has been shown that these pollutants induce cell and DNA damage, the mechanisms of their cytogenotoxic activity remains largely unknown. This study looks into their possible genotoxic effects, at doses that can be found in body fluids, on human lymphocytes using the cytokinesis-block micronucleus assay and the comet assay. After exposure for 1, 6, and 24 h compounds p,p′-DDT (0.1 μg mL⁻¹), p,p′-DDE (4.1 μg mL⁻¹), and p,p′-DDD (3.9 μg mL⁻¹) showed increase in DNA damage. The most significant results were observed at exposure period of 24 h where number of micronucleated cells increased from control 2.5 ± 0.71 to 23.5 ± 3.54, 13.5 ± 0.71, and 16.5 ± 6.36 for DDT, DDE, and DDD, respectively. Similar effect was observed using comet test where the percentage of DNA in comets tail increased from control 1.81 ± 0.16 to 17.24 ± 0.55, 11.21 ± 0.56 and 9.28 ± 0.50 for each compound, respectively. At the same time Fpg-comet assay failed to report induction of oxidative DNA damage of these pollutants. Additionally, the type of cell death was determined using diffusion assay and necrosis dominated. Our findings suggest that even at low concentrations, these pesticides could induce cytogenetic damage to human peripheral blood lymphocytes and in that manner have the impact on human health as well.     

Forest Naturalness: Criterion for Decision Support in Designation and Management of Protected Forest Areas

The article analyses the possibilities of developing an integrated indicator and a model of the assessment of forests naturalness using the data from the database of mountainous spruce forests situated in the Western Carpathians of Slovakia. The article presents two variants of such a model, one based on discriminant analysis, while the second one using an additive approach. The analysis of the data from mountainous spruce forests revealed significant indicators of forest naturalness degree: the arithmetic mean of the ratio between crown length and tree height, the deadwood volume, the coverage of grasses, the coverage of mosses and lichens, and the aggregation index. In addition, the coefficient of variation of tree diameters was included in the final model, since its presence in the model had a positive influence on the correctness of the classification of the forest naturalness degree. The correctness of the classification of the proposed discriminant model was 74.5%. For the additive model, the ranges of the values of the integrated indicator were defined for every degree of forest naturalness by taking into account the error ranges of the arithmetic mean values and the percentiles of the values in individual degrees of forest naturalness. The overall correctness of the classification with the additive model was 63.4%. In the second step, the scheme how to apply the classification model of the forest naturalness degree in the decision-making process of designating as a forest protected areas was proposed. In this scheme, the degree of forest naturalness is considered as a basic criterion for the determination of nature-conservation value of forest ecosystems. As further decision-making criteria we identified the possibility to restore, or the possibility to improve the naturalness of less natural forest ecosystems, which are designated as protected; the occurrence of the endangered species; and the occurrence of other natural values.     

Gatekeepers of island communities: exploring the pillars of sustainable development

Sustainable development (SD) is generally recognised as having three dimensions, ecological, economic and social. Yet, its implementation is burdened with resistance and conflict rooted in the short-term ‘business as usual’ development model, opposed to the long-term sustainable benefit of local communities. Hence, the development of strategies to implement SD projects may require further differentiation of these dimensions in relation to the contextual situation in which the project resides. In two studies of SD projects on the Croatian islands, we identify five interlocking spheres of importance, Spiritual, Political, Economic, Educational and Health, in addition to Ecological. Each of these spheres is accessible through gate-keepers, individuals or a group of people who have the authority over the sector and as such, significantly influence public opinion. We suggest that in this particular island context the sustainability of these projects may lie with those gate-keepers. Hence, initiating and maintaining SD projects in these contexts requires a structured and targeted lobbying of these gate-keepers.     

Photosynthetic and biochemical characteristics of invasive species (<Emphasis Type="Italic">Ambrosia artemisiifolia L., Ambrosia trifida L.</Emphasis> and <Emphasis Type="Italic">Iva xanthifolia Nutt.</Emphasis>) depending on soil humidity and phenological phase

Environmental factors like temperature and soil humidity are recognized as influencing factors on photosynthetic response and organic productivity, distribution and biochemical characteristics of plants. Here we present measurements of gas exchange parameters, water-use efficiency (WUE) and nitrogen-use efficiency (NUE) of field-grown invasive species Ambrosia artemisiifolia L., Ambrosia trifida L. and Iva xanthifolia Nutt., in order to define their potentials as colonizers. Biogenic volatile organic compounds (BVOC) in leaves and flowers depending on soil humidity were also investigated.     

Water Quality of Medium Size Watercourse Under Baseflow Conditions: The Case Study of River Sutla in Croatia

The study on medium size river Sutla in Croatia indicated considerable water contamination at specific sites during the baseflow period, probably associated to low flow-rate (0.73-68.8 m3 s(-1)), and consequently low dilution capacity of this river. Various aspects of contamination were observed: increased conductivity to 1,000 microS cm(-1), decreased dissolved oxygen level to 50%, 4-5 degrees C increased water temperature, increased concentrations of several dissolved trace elements (e.g., maximal values of Li: 45.4 microg l(-1); Rb: 10.4 microg l(-1); Mo: 20.1 microg l(-1); Cd: 0.31 microg l(-1); Sn: 30.2 microg l(-1); Sb: 11.8 microg l(-1); Pb: 1.18 microg l(-1); Ti: 1.03 microg l(-1); Mn: 261.1 microg l(-1); and Fe: 80.5 microg l(-1)) and macro elements (e.g., maximal values of Na: 107.5 mg l(-1); and K: 17.3 mg l(-1)), as well as moderate or even critical fecal (E. coli: 4,888 MPN/100 ml; total coliforms: 45,307 MPN/100 ml; enterococci: 1,303 MPN/100 ml) and organic pollution (heterotrophic bacteria: 94,000 cfu/ml). Although metal concentrations still have not exceeded the limits considered as hazardous for aquatic life or eventually for human health, the observed prominent increases of both metal concentrations and bacterial counts in the river water should be considered as a warning and incentive to protect the small and medium size rivers from the future deterioration, as recommended by EU Water Framework Directive.