Inhibitory effects of four neonicotinoid active ingredients on acetylcholine esterase activity.

There is a great concern about the decline of pollinators, and neonicotinoids emerging bee disorders are assumed to play a significant role. Since changes in learning ability has been observed in honey bees exposed to some acetylcholine esterase (AChE) inhibitors, we therefore, tested in vitro the effect of four neonicotinoids on purified eel AChE. AChE activity was inhibited in a concentration-dependent manner, and calculated IC50 values for thiamethoxam (IC50 = 414 µM) and clothianidin (IC50 = 160 µM) were found to be much higher compared to acetamiprid (IC50 = 75.2 µM) and thiacloprid (IC50 = 87.8 µM). The Lineweaver-Burk reciprocal plots for acetamiprid shows unchanged Vmax and increased Km values with inhibitor concentrations, while analysis of Michaelis-Menten plots shows predominantly competitive mechanism. The inhibition constant value (Ki = 24.3 µM) indicates strong binding of the acetamiprid complex to AChE. Finally, the four tested neonicotinoids are not a uniform group regarding their blocking ability. Our results suggest a previously not established, direct AChE blocking mechanism of neonicotinoids tested, thus the neuronal AChE enzyme is likely among the direct targets of the neonicotinoid insecticides. We conclude, that these AChE inhibitory effects may also contribute to toxic effects on the whole exposed animal.

Multi-biomarkers approach in different organs of Anodonta cygnea from the Dnister Basin (Ukraine).

The aim of this study was to compare environmental quality in two sites in western Ukraine-rural (R) and urbanized (U)-with the usage of the resident bivalve mollusk Anodonta cygnea. The study was realized during three seasons. The metal uptake and a set of biochemical markers were determined. For each season, Cd and metallothioneins (MTs) contents in the digestive gland and gills of the mollusc were higher at the U site, reflecting its chronic pollution. The oxidative stress in the mollusk was observed at the U site during spring and at the R site during summer and autumn according to the differences in Mn-superoxide dismutase and catalase activities, O (2) (*-) production, lipid peroxidation, and glutathione levels. The elevated vitellogenin-like protein levels in the hemolymph and the ethoxyresorufin-O-deethylase activity in the digestive gland in summer-autumn suggested pollutions by organic substances at the R site. The acetylcholinesterase activity was similar in both groups. The centroid grouping analysis of biomarkers and morphological and water indexes demonstrated the clear differentiation of general response in each group in spring and, at the R site, in summer and autumn but its similarity at the U site in summer and autumn.

Responses of biochemical markers in carp Cyprinus carpio from two field sites in Western Ukraine.

The aim of this study was to compare environmental quality in two sites of the river in Western Ukraine, rural (R) and industrial (I) during three seasons via a set of biochemical markers in carp Cyprinus carpio L. Upon comparing the values of the I-site with those of the R-site, we found that Mn- and Cu, Zn-superoxide dismutase (SOD) activities decreased and O(.) production increased; metallothionein (MT) and glutathione levels increased in most cases, and acetylcholinesterase (AChE) activity decreased in summer. This confirms our hypothesis about continuous environmental press at the I-site. The higher activity of catalase and lipid peroxidation (TBARS), as well as the increased levels of vitellogenin-like proteins at the R-site, compare to the I-site in spring reflects the permitting effect of agricultural discharges. According to the results of PCA, the most sensitive biomarkers of pollution are MT, TBARS, and AChE in liver.

Validation of oxidative stress responses in two populations of frogs from Western Ukraine.

Although amphibians are considered to be good bioindicators of environmental pollution, few data are available concerning their biochemical parameters in natural populations. We investigated seasonal (spring, summer, autumn) and spatial (wetlands in rural and urban areas) fluctuations of oxidative stress biomarkers in the liver of frog Rana ridibunda in Western Ukraine. The Centroid grouping analysis demonstrated that despite the fluctuations of separate indices, frogs from an urban site in summer and autumn are differed widely from those at the same site in spring and frogs from the rural site in all three seasons, joined in common set. In summer, suppression of Mn-superoxide dismutase and catalase activity, as well as increase in oxidized glutathione and lipid peroxidation levels demonstrate a collapse of antioxidant defense system in frogs from an urban site. The integrated oxidative stress index confirms this conclusion.

Different responses of biochemical markers in frogs (Rana ridibunda) from urban and rural wetlands to the effect of carbamate fungicide.

Laboratory studies were conducted to determine the effects of carbamate fungicide TATTU (mixture of propamocarb and mancozeb, 0.091 mg L(-1)) on biochemical markers of exposure in Rana ridibunda from clean (reference) and polluted sites. The untreated animals from the polluted site had lower Cu,Zn- and Mn-superoxide dismutase (SOD) and acetylcholinesterase activity, the levels of lipid peroxidation products (TBARS) and protein carbonyls in the liver and vitellogenin-like proteins (Vtg-LP) in the serum, but higher levels of glutathione in the liver in comparison with untreated frogs from the reference site. Catalase activity, superoxide anion and metallothionein levels were the same in both groups. The animals from two sites demonstrate different response on the effect of TATTU during 14 days. In the frogs from polluted site the oxidative damage (the decrease of Mn-SOD activity, lipids and protein oxidative destruction), neurotoxicity (depletion of acetylcholinesterase activity), and endocrine disruption (increase of Vtg-LP level) were revealed. On the other hand, the part of the indices in the animals from the reference site was unchanged after the treatment and the level of metallothionein was elevated demonstrating the satisfactory ability for the adaptation to unfavourable conditions.

Seasonal and spatial comparison of metallothioneins in frog Rana ridibunda from feral populations.

The aim of this study was to elucidate the seasonal and spatial regularity of the properties of metallothioneins (MTs) from the liver and kidney of the frog Rana ridibunda in rural (R) and urban (U) sites in Western Ukraine. This allowed examination of their possibility use in biomonitoring of environmental quality. The positive correlation for Zn and negative correlation for Cu were reflected between their content in the liver and MTs. The content of MTs was higher in summer compared to other seasons and also at the U site compare to the R site. MTs had been comprised of two chromatographic forms (MT-1 and MT-2/MT-2a), with lesser and variable MT-2/2a in frogs from the U site, particularly in the kidney. MTs accumulated about 75% of Cd in the liver. In summary, the ability MTs to elevate content as a stress response, together with the sensitivity of MT-2, may be explored to understand the health status of the frog in each season, reflecting the higher overall anthropogenic impact at the U site.