Bioactivation of Quinolines in a Recombinant Estrogen Receptor Transactivation Assay Is Catalyzed by N-Methyltransferases.

Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly N-heterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely used cell line overexpresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after coexposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using liquid chromatography-mass spectrometry, which indicated that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17ß-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, for example, ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

Important Issues in Ecotoxicological Investigations Using Earthworms.

The importance and beneficial effects of earthworms on soil structure and quality is well-established. In addition, earthworms have proved to be important model organisms for investigation of pollutant effects on soil ecosystems. In ecotoxicological investigations effects of various pollutants on earthworms were assessed. But some important issues regarding the effects of pollutants on earthworms still need to be comprehensively addressed. In this review several issues relevant to soil ecotoxicological investigations using earthworms are emphasized and guidelines that should be adopted in ecotoxicological investigations using earthworms are given. The inclusion of these guidelines in ecotoxicological studies will contribute to the better quantification of impacts of pollutants and will allow more accurate prediction of the real field effects of pollutants to earthworms.

Effect of different river flow rates on biomarker responses in common carp (Cyprinus carpio).

The present study investigated effects of different river flow rates on basal activities of selected biomarkers and the occurrence of oxidative stress in the common carp (Cyprinus carpio). Juvenile carp were exposed to different river flow rates (5-120 cm/s) by caging for 3 weeks. After this period, one half of the fish were sacrificed and used for analysis. The other half received a single intraperitoneal injection of 3-methylcholanthrene (3-MC) and after 6 days were sacrificed and used for analysis. In order to investigate whether the physical activity of carp in the environment will influence the condition status of carp, following biomarkers were measured - activities of glutathione S-transferase (GST), catalase (CAT) and ethoxyresorufin-O-deethylase (EROD) and concentration of protein carbonyls (PC). The results showed that different flow rates significantly influenced biochemical biomarkers. The basal activity of GST did not change significantly after exposure to different river flow rates, whereas the activity of CAT increased with increasing river flow rates. The application of 3-MC caused significant increases in GST and CAT activities, but there were no difference between 3-MC control and 3-MC different flow rates. The occurrence of oxidative stress as a result of exposure to increased physical activity, i.e. increased river flow rates, was confirmed by measurement of PC levels - the level of PC increased with increasing river flow rates. Measurement of EROD basal activity showed that at lower river flow rates the EROD activity increased and at higher river flow rates decreased towards control levels demonstrating a close relationship between oxidative stress, PC levels and EROD activity. Obviously, biomarker responses in carp of different condition status can differ substantially. It can be concluded that flow rate may be an important factor in biomonitoring of rivers using biomarkers and since at different locations river water flow rate can vary significantly, the site selection is extremely important for proper design of river biomonitoring studies involving caging.

Application of microcosmic system for assessment of insecticide effects on biomarker responses in ecologically different earthworm species.

Earthworms from different ecological categories--epigeic Eisenia andrei and Lumbricus rubellus, endogeic Octolasion lacteum and anecic Lumbricus terrestris--were exposed in a microcosmic system to three commonly used insecticides. The effects of the insecticides were evaluated by measuring the following molecular biomarkers-the activities of AChE, CES, CAT, GST and the concentration of GSH. The results showed that environmentally relevant doses of organophosphates dimethoate and pirimiphos-methyl significantly affected the measured biomarkers, whereas pyrethroid deltamethrin did not affect the earthworms at the recommended agricultural dose. Considering the ecological category of earthworms, the results were inhomogeneous and species-specific differences in the biomarker responses were recorded. Since the biomarker responses of the investigated earthworm species were different after exposure to organophosphates in a microcosm compared to the exposure via standardized toxicity tests, two types of species sensitivity should be distinguished-physiological and environmental sensitivity. In addition, the hormetic effect of organophosphates on AChE and CES activities was recorded. The detection of hormesis in a microcosm is of great importance for future environmental research and soil biomonitoring, since in a realistic environment pollutants usually occur at low concentrations that could cause a hormetic effect. The results demonstrate the importance of the application of microcosmic systems in the assessment of the effects of environmental pollutants and the necessity of taking into account the possible differences between physiological and environmental species sensitivity.

Biochemical and molecular responses of the ascorbate-glutathione cycle in wheat seedlings exposed to different forms of selenium.

Biofortification aims to increase selenium (Se) concentration and bioavailability in edible parts of crops such as wheat (Triticum aestivum L.), resulting in increased concentration of Se in plants and/or soil. Higher Se concentrations can disturb protein structure and consequently influence glutathione (GSH) metabolism in plants which can affect antioxidative and other detoxification pathways. The aim of this study was to elucidate the impact of five different concentrations of selenate and selenite (0.4, 4, 20, 40 and 400 mg kg-1) on the ascorbate-glutathione cycle in wheat shoots and roots and to determine biochemical and molecular tissue-specific responses. Content of investigated metabolites, activities of detoxification enzymes and expression of their genes depended both on the chemical form and concentration of the applied Se, as well as on the type of plant tissue. The most pronounced changes in the expression level of genes involved in GSH metabolism were visible in wheat shoots at the highest concentrations of both forms of Se. Obtained results can serve as a basis for further research on Se toxicity and detoxification mechanisms in wheat. New insights into the Se impact on GSH metabolism could contribute to the further development of biofortification strategies.

Different sensitivities of biomarker responses in two epigeic earthworm species after exposure to pyrethroid and organophosphate insecticides.

In many studies that investigate the toxic effects of pollutants on earthworms, experiments are performed using only one species of earthworms, most commonly the Eisenia species. However, the differences in sensitivities of different earthworm species could potentially lead to an underestimation of environmental aspects of pollutants. Therefore, the aim of this study was to compare the sensitivity of biomarker responses of Eisenia andrei, an epigeic compost species commonly used in laboratory experiments, with those of Lumbricus rubellus, an epigeic species widely distributed in temperate regions. The earthworms were exposed to the three commonly used insecticides: organophosphates dimethoate (0.03, 0.3, and 3 mg kg(-1)) and pirimiphos-methyl (0.02, 0.2, and 2 mg kg(-1)), as well as pyrethroid deltamethrin (0.01, 0.1, and 0.5 mg kg(-1)), for 1 and 15 days using an artificial soil test. The effects of the pesticides were assessed by measuring the activities of acetylcholinesterase (AChE), carboxylesterase (CES), catalase (CAT), glutathione S-transferase (GST) as well as the concentration of glutathione (GSH). The pesticides caused a significant inhibition of AChE and CES activities and significant changes in activities of CAT, GST, and GSH concentration in both earthworm species. A comparison of biomarker responses between E. andrei and L. rubellus showed significant differences; E. andrei proved to be less susceptible to pesticide exposure than L. rubellus. In addition, the results from the filter-paper contact test mortality experiments showed that lethal concentrations were lower for L. rubellus compared with the E. andrei, further showing a greater sensitivity of L. rubellus. The difference in sensitivities of these epigeic species should be taken into account when conducting toxicity studies.

Potentiation effect of metolachlor on toxicity of organochlorine and organophosphate insecticides in earthworm Eisenia andrei.

Acetylcholinesterase, glutathione-S-transferase and catalase activities were determined in earthworms Eisenia andrei exposed to insecticides (endosulfan, temephos, malathion, pirimiphos-methyl) alone and in a binary combination with the herbicide metolachlor. Metolachlor individually was not acutely toxic, even at high concentrations applied; however, in the treated earthworms metolachlor enhanced the toxicity of endosulfan and temephos by significantly reducing the acetylcholinesterase activity. In binary combination with malathion and pirimiphos-methyl, metolachlor did not increase toxicity. The potentiation character of metolachlor is specific rather than general, and probably depends on the chemical structure of pesticides in the mixture.

Heavy metal(loid) effect on multi-biomarker responses in apex predator: Novel assays in the monitoring of white stork nestlings.

The goal of the present study was to investigate differences in biomarker responses related to metal(loid)s in white stork (Ciconia ciconia) nestling's blood from continental Croatia. To achieve this, a battery of biomarkers that can be affected by environmental pollutants, including metal(loid)s, was assessed (esterase activity, fluorescence-based oxidative stress biomarkers, metallothionein levels, glutathione-dependent enzyme activity). The research was conducted during the white stork breeding season in diverse areas (a landfill, industrial and agricultural sites, and an unpolluted area). White storks' nestlings near the landfill exhibited reduced carboxylesterase (CES) activity, elevated glutathione (GSH) concentration, as well as high Pb content in the blood. Increased As and Hg concentrations in blood were attributable to environmental contamination in agricultural area and an assumed unpolluted area, respectively. Furthermore, agricultural practices appeared to affect CES activity, as well as elevate Se levels. In addition to the successful implementation of biomarkers, present research showed that agricultural areas and a landfill are areas with increased metal(loid) levels possibly causing adverse effects on the white storks. This first-time heavy metal and metalloid analyses in the white stork nestlings from Croatia point to the necessary monitoring and future assessments of pollution impact to prevent irreversible adverse effects.

First evidence for the presence of efflux pump in the earthworm Eisenia andrei.

Efflux pumps are transport proteins involved in the extrusion of toxic substrates from cells to the external environment. Activities of efflux pumps have been found in many organisms, however such activity has not been evidenced in earthworms. Adult Eisenia andrei earthworms were exposed to efflux modulators - verapamil (a known inhibitor of efflux pump protein) and dexamethasone (a known inducer of efflux activity) - and the amount of absorbed fluorescent dye rhodamine B was measured. The results showed that verapamil inhibited efflux activity and decreased removal of rhodamine B, whereas dexamethasone induced efflux activity and increased removal of rhodamine B. This is the first evidence of the presence of efflux pump in earthworm Eisenia andrei. Since earthworms are often used as test organisms due to their sensitive reactions towards environmental influences, the discovery of efflux pump activity can contribute to the better understanding of toxicity of certain pollutants.

Blood biomarkers in white stork (Ciconia ciconia) nestlings show different responses in several areas of Croatia.

White stork nestlings can provide quantitative data on the quality of the environment, as they are dependent on their parents that provide locally foraged food. Blood was sampled from the brachial vein (n = 109) and the sampling was performed in parallel with ringing during breeding season 2020 from five areas in eastern Croatia: Lonjsko polje, Jelas polje, Slavonski Brod-east, Podunavlje, and Donje Podravlje. In the present study, for the first time in Croatia, the following enzymatic biomarkers were assessed in white stork nestlings: activities of acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as nonenzymatic biomarkers: levels of glutathione (GSH) and reactive oxygen species (ROS). All endpoints were measured in two blood fractions: plasma and a postmitochondrial fraction (S9). Nestlings from Podunavlje and Donje Podravlje, areas known for intensive agriculture, showed lower AChE and CES activity when compared to the other investigated areas, indicating the presence of inhibitory xenobiotics. Higher oxidative stress was observed in Slavonski Brod-east, an area surrounded by metal and engineering industry, and Podunavlje compared to the other sampling areas. Hence, this study shows the impact of pollutants from the surrounding metal, petroleum, and agricultural industry might have on the biomarkers in white stork nestlings, which are often seen as early-warning signals.

Two types of microplastics (polystyrene-HBCD and car tire abrasion) affect oxidative stress-related biomarkers in earthworm Eisenia andrei in a time-dependent manner.

Microplastics are small plastic fragments that are widely distributed in marine and terrestrial environments. While the soil ecosystem represents a large reservoir for plastic, research so far has focused mainly on the impact on aquatic ecosystems and there is a lack of information on the potentially adverse effects of microplastics on soil biota. Earthworms are key organisms of the soil ecosystem and are due to their crucial role in soil quality and fertility a suitable and popular model organism in soil ecotoxicology. Therefore, the aim of this study was to gain insight into the effects of environmentally relevant concentrations of microplastics on the earthworm Eisenia andrei on multiple levels of biological organization after different exposure periods. Earthworms were exposed to two types of microplastics: (1) polystyrene-HBCD and (2) car tire abrasion in natural soil for 2, 7, 14 and 28d. Acute and chronic toxicity and all subcellular investigations were conducted for all exposure times, avoidance behavior assessed after 48 h and reproduction after 28d. Subcellular endpoints included enzymatic biomarker responses, namely, carboxylesterase, glutathione peroxidase, acetylcholinesterase, glutathione reductase, glutathione S-transferase and catalase activities, as well as fluorescence-based measurements of oxidative stress-related markers and multixenobiotic resistance activity. Multiple biomarkers showed significant changes in activity, but a recovery of most enzymatic activities could be observed after 28d. Overall, only minor effects could be observed on a subcellular level, showing that in this exposure scenario with environmentally relevant concentrations based on German pollution levels the threat to soil biota is minimal. However, in areas with higher concentrations of microplastics in the environment, these results can be interpreted as an early warning signal for more adverse effects. In conclusion, these findings provide new insights regarding the ecotoxicological effects of environmentally relevant concentrations of microplastics on soil organisms.

Demonstration of an aggregated biomarker response approach to assess the impact of point and diffuse contaminant sources in feral fish in a small river case study.

The assessment of the exposure of aquatic wildlife to complex environmental mixtures of chemicals originating from both point and diffuse sources and evaluating the potential impact thereof constitutes a significant step towards mitigating toxic pressure and the improvement of ecological status. In the current proof-of-concept study, we demonstrate the potential of a novel Aggregated Biomarker Response (ABR) approach involving a comprehensive set of biomarkers to identify complex exposure and impacts on wild brown trout (Salmo trutta fario). Our scenario used a small lowland river in Germany (Holtemme river in the Elbe river catchment) impacted by two wastewater treatment plants (WWTP) and diffuse agricultural runoff as a case study. The trout were collected along a pollution gradient (characterised in a parallel study) in the river. Compared to fish from the reference site upstream of the first WWTP, the trout collected downstream of the WWTPs showed a significant increase in micronucleus formation, phase I and II enzyme activities, and oxidative stress parameters in agreement with increasing exposure to various chemicals. By integrating single biomarker responses into an aggregated biomarker response, the two WWTPs' contribution to the observed toxicity could be clearly differentiated. The ABR results were supported by chemical analyses and whole transcriptome data, which revealed alterations of steroid biosynthesis and associated pathways, including an anti-androgenic effect, as some of the key drivers of the observed toxicity. Overall, this combined approach of in situ biomarker responses complemented with molecular pathway analysis allowed for a comprehensive ecotoxicological assessment of fish along the river. This study provides evidence for specific hazard potentials caused by mixtures of agricultural and WWTP derived chemicals at sublethal concentrations. Using aggregated biomarker responses combined with chemical analyses enabled an evidence-based ranking of sites with different degrees of pollution according to toxic stress and observed effects.

Oxidative stress elicited by insecticides: a role for the adipokinetic hormone.

Adipokinetic hormones (AKHs) are insect neuropeptides responding to stress situations including oxidative stress. Two insecticides - endosulfan and malathion - were used to elicit oxidative stress conditions in the firebug Pyrrhocoris apterus, and the physiological functions of AKHs and their ability to activate protective antioxidative reactions were studied. The insecticide treatments elicited only a slight increase of the AKH level in CNS, but more intensive increase in haemolymph, which indicates an immediate involvement of AKH in the stress response. The treatment also resulted in a significant increase of catalase activity in the bug's body and depletion of the reduced glutathione pool in the haemolymph, however, co-application of the insecticides with the AKH (80 pmol) reduced the effect. It has also been found that co-application of the insecticides with AKH increased significantly the bug mortality compared to that induced by the insecticides alone. This enhanced effect of the insecticides probably resulted from the stimulatory role of AKH on bug metabolism: the carbon dioxide production was increased significantly after the co-treatment by AKH with insecticides compared to insecticide treatment alone. It was hypothesized that the increased metabolic rate could intensify the insecticide action by an accelerated rate of exchange of metabolites accompanied by faster penetration of insecticides into tissues.

Application of Non-Destructive Methods: Biomarker Assays in Blood of White Stork (Ciconia ciconia) Nestlings.

White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings' physiological condition, biomarkers can be measured in plasma and S9.

Is a liver comparable to a liver? A comparison of different rat-derived S9-fractions with a biotechnological animal-free alternative in the Ames fluctuation assay.

Metabolism has to be considered during the toxicological assessment of chemical and environmental samples because it is an important process in the mammalian liver. It can be assessed in vitro via liver homogenates called S9-fractions, an external metabolic activation system. However, the external metabolic activation systems can vary greatly in their composition due to biological variations among individual animals and animal strains that the S9-fraction are derived as well as the differences in the production treatment. To gain more insight into these variances, three different but commonly used rat-derived S9-fractions were compared in the present study for their variance and performance with a reference compound in the Ames fluctuation assay with Salmonella typhimurium strains TA 98 and TA 100 according to ISO 11350. Severe shortcomings of conventional rat-derived S9-fractions were observed in the present study, such that S9-fractions differed significantly within the same rat strain and for different types of induction procedures in regards to the metabolic capability. An intrinsic mutagenic potential of the three rat-derived S9-fractions were identified in the Ames fluctuation assay with varying S9-fraction concentrations. To address some of the shortcomings of the animal-derived S9-fraction, the present study investigated the use and performance of a biotechnological, animal-free alternative, ewoS9R, in comparison to one of the rat-derived S9-fraction as the others showed a mutagenic potential themselves. Specifically, 12 different chemicals were used as a reference to determine if ewoS9R could serve as an adequate and more consistent replacement of traditional rat-derived metabolic activation systems: 8 pro-mutagenic compounds (i.e., require metabolic activation to show a mutagenic potential), one pro-mutagenic compound but not in the tested strains, one mutagenic compound without metabolic activation and two compounds that are equivocal in the literature. EwoS9R was evaluated as a promising approach in the Ames fluctuation assay with 5 compounds observed to have similar results with both rat-derived S9-fraction and ewoS9R (41%), for 3 compounds ewoS9R was a better metabolization system than the rat-derived S9-fraction (16%). Further research is necessary to determine the full potential of ewoS9R in comparison to rat-derived S9-fractions.

Using a high-throughput method in the micronucleus assay to compare animal-free with rat-derived S9.

This study presents a high-throughput (HTP) micronucleus assay in multi-well plates with an automated evaluation for risk assessment applications. The evaluation of genotoxicity via the micronucleus assays according to international guidelines ISO 21427-2 with Chinese hamster (Cricetulus griseus) V79 cells was the starting point to develop our methodology. A drawback of this assay is that it is very time consuming and cost intensive. Our HTP micronucleus assay in a 48-well plate format allows for the simultaneous assessment of five different sample-concentrations with additional positive, negative and solvent controls with six technical replicates each within a quarter of the time required for the equivalent evaluation using the traditional slide method. In accordance with the 3R principle, animal compounds should be replaced with animal-free alternatives. However, traditional cell culture-based methods still require animal derived compounds like rat-liver derived S9-fraction, which is used to simulate the mammalian metabolism in in vitro assays that do show intrinsic metabolization capabilities. In the present study, a recently developed animal-free biotechnological alternative (ewoS9R) was investigated in the new high-throughput micronucleus assay. In total, 12 different mutagenic or genotoxic chemicals were investigated to assess the potential use of the animal-free metabolization system (ewoS9R) in comparison to a common rat-derived product. Out of the 12 compounds, one compound did not induce micronuclei in any treatment and 2 substances showed a genotoxic potential without the need for a metabolization system. EwoS9R demonstrated promising potential for future applications as it shows comparable results to the rat-derived S9 for 6 of the 9 pro-genotoxic substances tested. The remaining 3 substances (2-Acetamidofluorene, Benzo[a]pyrene, Cyclophosphamide) were only metabolized by rat-derived S9. A potential explanation is that ewoS9R was investigated with an approx. 10-fold lower enzyme concentration and was only optimized for CYP1A metabolization that may be improved with a modified production procedure. Future applications of ewoS9R go beyond the micronucleus assay, but further research is necessary.

Biomarker responses in earthworm coelomocyte extract - Noninvasively collected sample for pesticide effect assessment.

Earthworms are often used as model organisms in ecotoxicological research because of their natural habitat where they can be exposed to many different pollutants, including pesticides. Since a number of them has to be sacrificed for sample collection, it would be useful to develop non-invasive methods and techniques suitable for the analysis of target parameters. The aim of this study is to determine whether the coelomocyte extract, obtained by the non-invasive method, can be used to measure responses of biochemical biomarkers and to establish if it can be used in assessing the effects of pesticides already known to have a negative impact on the earthworms. In the present study Eisenia andrei earthworms were exposed for 48 h to organophosphates dimethoate and pirimiphos-methyl using the filter paper contact test. Following exposure, coelomocyte extracts were prepared and acetylcholinesterase (AChE) and carboxylesterase (CES) activities were measured. The percentage of inhibition of the measured enzymes in the coelomocyte extract was compared with the inhibition of the same enzyme activities in the samples obtained from the whole body homogenate. AChE and CES inhibition was observed at all concentrations for both pesticides in different types of samples. Compared to the coelomocyte extract, the level of AChE inhibition was slightly stronger in the whole body homogenate. Inhibition of CES at the same concentrations in different types of samples did not always coincide, especially in the case of dimethoate, however significant inhibition of CES in coelomocyte extract was recorded. This study indicates the possibility of using the coelomocyte extract for measurement of biochemical biomarkers and assessment of pesticide effects.

Acute toxicities and effects on multixenobiotic resistance activity of eight pesticides to the earthworm Eisenia andrei.

Investigations of deleterious effects on non-target species, including earthworms, have been conducted for a number of pesticides, but there is a need for additional assessments of potential adverse effects. In the present study, the acute toxicity of eight pesticides to the earthworm Eisenia andrei was assessed and compared. The exposures were conducted using the filter paper contact toxicity method. Based on the 48-h LC50 values, one pesticide was classified as supertoxic (combined fungicide containing difenoconazole and fludioxonil), four as extremely toxic (combined herbicide containing pethoxamide and terbuthylazine, combined fungicide containing fluopyram and tebuconazole, fungicide containing pyrimethanil, and combined fungicide containing thiram and carboxin), two as very toxic (combined fungicide containing flutriafol and thiabendazole, and herbicide containing fluroxypyr-meptyl), and one as moderately toxic (insecticide containing thiamethoxam). Additionally, effects of pesticides on the multixenobiotic resistance (MXR) activity were measured. Results showed that four pesticides caused significant effects with a recorded inhibition of the activity, which can consequently lead to a higher toxicity due to longer retention of the pesticides in the cells. Finally, for three chosen pesticides, gene expression of cat, sod, and gst was measured, and significant changes were observed. The obtained results show that earthworms could be significantly affected by pesticides commonly used in agriculture.

Lipopolysaccharides from Microcystis Cyanobacteria-Dominated Water Bloom and from Laboratory Cultures Trigger Human Immune Innate Response.

Massive toxic blooms of cyanobacteria represent a major threat to water supplies worldwide. Here, the biological activities of lipopolysaccharide (LPS) isolated from Microcystis aeruginosa, the most prominent cyanobacteria in water bloom, were studied. LPS was isolated from complex environmental water bloom samples dominated by M. aeruginosa, and from laboratory cultures of non-axenic as well as axenic M. aeruginosa strains PCC7806 and HAMBI/UHCC130. Employing human blood-based in vitro tests, the LPS isolated from complex water bloom revealed the priming of both major blood phagocyte population monocytes and polymorphonuclear leukocytes documented by the increased surface expression of CD11b and CD66b. This was accompanied by a water bloom LPS-mediated dose-dependent induction of tumor necrosis factor α, interleukin-1ß, and interleukin-6 production. In accordance with its priming effects, water bloom LPS induced significant activation of p38 and ERK1/2 kinases, as well as NF-κB phosphorylation, in isolated polymorphonuclear leukocytes. Interestingly, the pro-inflammatory potential of LPS from the axenic strain of M. aeruginosa was not lower compared to that of LPS isolated from non-axenic strains. In contrast to the biological activity, water bloom LPS revealed almost twice higher pyrogenicity levels compared to Escherichia coli LPS, as analyzed by the PyroGene test. Moreover, LPS from the non-axenic culture exhibited higher endotoxin activity in comparison to LPS from axenic strains. Taking the above findings together, M. aeruginosa LPS can contribute to the health risks associated with contamination by complex water bloom mass.

Herbicides diuron and fluazifop-p-butyl affect avoidance response and multixenobiotic resistance activity in earthworm Eisenia andrei.

The usage of pesticides has been steadily increasing over the last decades, and among them herbicides are the most commonly used ones. Despite their main mode of action targeting plant organisms, they can also have adverse effects on non-target animal organisms. In soil ecosystems, earthworms play an important role due to their positive impacts on the soil functioning and they represent good model organisms in soil ecotoxicology. The aim of the present study was to assess the effects of two herbicides on several endpoints at different levels of biological organization in the earthworm Eisenia andrei. Diuron and fluazifop-p-butyl were selected for the investigation and their lethal concentrations were determined: LC50 48 h: 89.087 µg/cm2 for diuron and 6.167 µg/cm2 for fluazifop-p-butyl. Furthermore, measurements of enzymatic biomarkers (catalase (CAT), acetylcholinesterase (AChE), carboxylesterase (CES) and glutathione S-transferase (GST)), multixenobiotic resistance (MXR) activity and gene expression of antioxidative enzymes (only for fluazifop-p-butyl) were conducted. Enzymatic biomarker responses showed no significant differences compared to the control after the exposure to the investigated herbicides, whereas the MXR activity was significantly inhibited. The gene expression level of superoxide dismutase (sod) and glutathione S-transferase (gst) after fluazifop-p-butyl exposure showed a significant increase. Finally, avoidance behavior in soil was assessed and it was determined that both herbicides caused significant avoidance response. The obtained results show that both investigated herbicides significantly affect earthworms on different levels of biological organization. This emphasizes the importance of comprehensive ecotoxicological assessment of herbicide effects on non-target organisms at all organizational levels.