Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors.

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to ß-naphthoflavone (ß-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to ß-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

Temperature related toxicity features of acute acetamiprid and thiacloprid exposure in Daphnia magna and implications on reproductive performance.

This study investigated the potential for elevated temperature to alter the toxicity of acetamiprid (ACE) and thiacloprid (Thia) in the ecotoxicity model Daphnia magna. The modulation of CYP450 monooxygenases (ECOD), ABC transporter activity (MXR) and incident cellular reactive oxygen species (ROS) overproduction was screened in premature daphnids following acute (48 h) exposure to sublethal concentrations of ACE and Thia (0.1-, 1.0 µM) at standard 21 °C and elevated 26 °C temperatures. Delayed outcomes of acute exposures were further evaluated based on the reproduction performance of daphnids monitored over 14 days of recovery. Exposures to ACE and Thia at 21o C elicited moderate induction of ECOD activity, pronounced inhibition of MXR activity and severe ROS overproduction in daphnids. In the high thermal regime, treatments resulted in significantly lower induction of ECOD activity and inhibition of MXR activity, suggesting a suppressed metabolism of neonicotinoids and less impaired membrane transport activity in daphnids. Elevated temperature on its own, caused a three-fold rise in ROS levels in control daphnids, while ROS overproduction upon neonicotinoid exposure was less accentuated. Acute exposures to ACE and Thia caused significant decreases also in the reproduction of daphnids, indicating delayed outcomes even at environmentally relevant concentrations. Both the cellular alterations in exposed daphnids and decreases in their reproductive output post exposures evidenced closely similar toxicity patterns and potentials for the two neonicotinoids. While elevated temperature elicited only a shift in baseline cellular alterations evoked by neonicotinoids, it significantly worsened the reproductive performance of daphnids following neonicotinoid exposures.

Behavioral and biochemical alterations induced by acute clothianidin and imidacloprid exposure in the killer shrimp, Dikerogammarus villosus.

Neonicotinoids are widely used insecticides around the world and are preserved permanently in soils and appear in surface waters posing an increased threat to ecosystems. In the present study, we exposed adult specimens of amphipod Dikerogammarus villosus to environmentally relevant and higher concentrations of two widely used agricultural neonicotinoids, clothianidin (CLO) and imidacloprid (IMI), for 2 days. The acute effects were investigated at the behavioral (immobility time and swimming activity) and biochemical (glutathione S-transferase [GST] and acetylcholine esterase [AchE] activity) levels. All CLO concentrations used (64 nM, 128 nM, 192 nM) significantly decreased the immobility time and swimming activity. In the case of IMI, the immobility time decreased significantly only at the highest concentration applied (977 nM), but the distance travelled by the animals significantly decreased even at lower concentrations (78 nM and 313 nM). The GST enzyme activity did not change in the CLO-treated groups, however, the 626 nM and 977 nM IMI concentrations significantly increased the GST activity. Similarly, to the behavioral level, all CLO concentrations significantly decreased the AchE activity. In contrast, IMI has a significant stimulating effect on the AchE activity at the 313 nM, 626 nM, and 977 nM concentrations. Based on the authors' best knowledge, this is the first study to investigate the effects of CLO and IMI at environmentally-relevant concentrations on D. villosus. Our findings contribute to the understanding of the physiological effects of neonicotinoids.

Physiological and metabolic alterations induced by commercial neonicotinoid formulations in Daphnia magna.

Neonicotinoid insecticides are widely used agents in agriculture to control a broad range of insect pests. Although use of neonicotinoid pesticides has resulted in the widespread contamination of surface waters, sublethal toxicity data of these products in relation to non-target aquatic biota are still poor. Therefore, the objective of this study was to assess the effects of two neonicotinoid pesticides with widespread use on the basic physiological functions: the thoracic limb activity and heart rate of Daphnia magna, and to screen for their potential to affect the cytochrome P450 monooxygenase system (ECOD activity) of daphnids. The considered pesticides were the acetamiprid- and thiacloprid based products Mospilan 20 SG and Calypso 480 SC, respectively. The dose-dependent variation in the three biological endpoints considered were assessed following 24 h exposures. The two neonicotinoid formulations elicited significant depression on the thoracic limb activity and heart rate of daphnids at doses close to the immobility thresholds of formulations (48h-EC50: Mospilan 20 SG = 190 mg L-1; Calypso 480 SC = 120 mg L-1), an effect mainly attributable to the overall drop in the general health status of the organisms. The alterations in the physiological traits were significant at exposures to 190 mg L-1 for Mospilan 20 SG and 48 mg L-1 for Calypso 480 SC. The dose related variation in the ECOD activity of daphnids exposed to the selected neonicotinoid formulations followed a biphasic pattern, with starting effective doses for Mospilan 20 SG of 6.3 mg L-1 (=1/20 of 48h-EC50 for Daphnia neonates), and for Calypso 480 SC of 0.034 mg L-1 (=1/4000 of 48h-EC50). Maximal ECOD activity (2.2 fold increase vs. controls) was induced by Mospilan 20 SG in daphnids exposed to 114 mg L-1 product (=48 h-EC20), and by Calypso 480 SC (1.8 fold increase) at 5.2 mg L-1 dose (=1/20 of 48 h-EC50). Our results outlined significant alterations in the physiological traits and ECOD activity in exposed daphnids at concentrations below the immobility thresholds (48 h-EC50) of the products used as benchmarks to rate their toxicity risks to aquatic biota. Therefore, we think our findings might deserve consideration in the environmental risk evaluation of these products.

Effects of chronic sublethal progestogen exposure on development, reproduction, and detoxification system of water flea, Daphnia magna.

The presence of sex steroid hormones in aquatic ecosystems is of rapidly growing concern worldwide since they can affect the different non-target species including cladocerans. Although data are available on the effects of estrogens on the well-established ecotoxicological model organism Daphnia magna, the molecular or behavioural alterations induced by environmentally relevant concentrations (from a few ng L-1 to a few hundred ng L-1 in average) of progestogens have not been investigated on this species. In the present study, we exposed neonates of D. magna to relevant equi-concentrations (1, 10, 100, 500 ng L-1) of mixtures of four progestogens (progesterone, drospirenone, gestodene, levonorgestrel) in short-term (6 days) and long-term (21 days) experiments. Significant alterations were observed at the molecular, cellular, and individual levels. During the short-term exposure, all of the mixtures increased the gene expression of glutathione S-transferase (GST) detoxification enzyme, moreover, the activity of GST was also significantly increased at the concentrations of 10, 100, and 500 ng L-1. In long-term exposure, the number of days until production of the first eggs was reduced at the 10 ng L-1 concentration compared to control, furthermore, the maximum egg number per individual increased at the concentrations of 1 and 10 ng L-1. Based on the authors' best knowledge, this is the first study to investigate the effects of progestogens in mixtures and at environmentally relevant concentrations on D. magna. Our findings contribute to the understanding of the possible physiological effects of human progestogens. Future research should be aimed at understanding the potential mechanisms (e.g., perception) underlying the changes induced by progestogens.

ATP signaling in the integrative neural center of Aplysia californica.

ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica-a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

Multi-marker approach for the evaluation of environmental impacts of APACS 50WG on aquatic ecosystems.

Neonicotinoids are the most widely used synthetic insecticides in the world. These insecticides are widely distributed in the ecosystem, indicating that more attention should be paid to the potential risks regarding their use in agriculture. Due their intensive use, non-target species in the environment are also exposed to their putative effects. Within acute exposure trials, the time related effect of sublethal dose of the neonicotinoid preparation APACS 50 WG was investigated on swimming behaviour and the multi-xenobiotic resistance system (MXR) activity, as a first line defence pathway of adult Dikerogammarus villosus. Results showed that treated animals manifested an increased swimming activity. Exposed animals were monitored by the rhodamine B accumulation assay, and APACS 50 WG exerted distinct changes in the MXR activity as well. Our results suggested that application of neonicotinoid at a low concentration (3.9 ng/l) contributed to the activation of locomotor activity and at the same concentration range the transmembrane transport mechanisms was altered too.

Neonicotinoids: Spreading, Translocation and Aquatic Toxicity.

Various environmental and ecotoxicological aspects related to applications of neonicotinoid insecticides are assessed. Dosages of neonicotinoids applied in seed coating materials were determined and are compared to other applications (spray and granule). Environmental levels in soils and affecting factors in translocation are discussed. Excretion of neonicotinoids via guttation from coated maize seeds up to two months upon emergence, as well as cross-contamination of plants emerged from non-coated seeds or weeds nearby have been demonstrated. Contamination of surface waters is discussed in scope of a worldwide review and the environmental fate of the neonicotinoid active ingredients and the formulating surfactant appeared to be mutually affected by each other. Toxicity of neonicotinoid active ingredients and formulations on Daphnia magna completed with some investigations of activity of the detoxifying glutathione S-transferase enzyme demonstrated the modified toxicity due to the formulating agents. Electrophysiological results on identified central neurons of the terrestrial snail Helixpomatia showed acetylcholine antagonist (inhibitory) effects of neonicotinoid insecticide products, but no agonist (ACh-like) effects were recorded. These data also suggested different molecular targets (nicotinergic acetylcholine receptors and acetylcholine esterase enzyme) of neonicotinoids in the snail central nervous system.

The allelochemical tannic acid affects the locomotion and feeding behaviour of the pond snail, Lymnaea stagnalis, by inhibiting peripheral pathways.

(1) The effect of tannic acid (TA), a dominant component of plant allelochemicals, was investigated on the locomotion and feeding of the pond snail, Lymnaea stagnalis. The effect of TA on the neuronal background underlying feeding activity was also analysed. (2) TA affected the spontaneous locomotion and of juvenile snails in a concentration-dependent way. Low (10 µM) TA concentration resulted in an increased (sliding or swimming) activity compared to the control; meanwhile, high (100 µM) TA concentration inhibited the locomotion of the animals. (3) Low (10 µM) TA concentration increased the frequency of sucrose-evoked feeding of intact animals, whereas high (100 µM) TA concentration resulted in significantly longer feeding latency and decreased feeding rate. The feeding changes proved to be partially irreversible, since after 48 h maintained in clear water, the animals tested in 100 µM TA previously still showed lower feeding rate in sucrose. (4) Electrophysiological experiments on semi-intact preparations showed that application of 100 µM TA to the lip area inhibited the fictive feeding pattern of central neurons, the cellular response to sucrose. (5) On isolated CNS preparation, 100 µM TA applied in the bathing solution, however, failed to inhibit the activation of the central feeding (CPG) interneurons following application of extracellular dopamine. Our results suggest that TA affects both afferent and efferent peripheral functions in Lymnaea. TA reduces feeding activity by primarily blocking feeding sensory pathways, and its negative effect on locomotion may imply sensory pathways and/or ciliary activity.

Contamination of the guttation liquid of two common weeds with neonicotinoids from coated maize seeds planted in close proximity.

Neonicotinoid uptake by maize plants emerged from coated seeds and by two common weeds grown in close proximity to coated seeds has been studied. Uptake of thiamethoxam (TMX) and clothianidin (CLO) have been characterized via guttation liquid measurements. The creeping thistle (Cirsium arvense), a well-known maize weed, as well as red poppy or Flanders poppy (Papaver rhoeas) were chosen as model species. The results confirmed that cross-contamination may occur by uptake of the neonicotinoid AIs through soil from neighbouring plants that emerged from coated seeds. Although the levels of these neonicotinoids were substantially lower in the guttation liquid of the weeds than in that of maize plants emerged from coated seeds, the compounds were detected up to 36th day after planting of the maize seeds. The highest peak concentrations of TMX were around 150 and 21 mg L-1, while similar data for CLO were around 70 and 21 mg L-1 for maize and creeping thistle, respectively. Mostly due to its higher guttation intensity significantly lower values were determined for red poppy (0.740 mg L-1).

Neonicotinoid insecticides are potential substrates of the multixenobiotic resistance (MXR) mechanism in the non-target invertebrate, Dreissena sp.

Mussels are among the most frequently used invertebrate animals in aquatic toxicology to detect toxic exposure in the environment. The presence and activity of a cellular defence system, the multixenobiotic resistance (MXR) mechanism, was also established in these organisms. In isolated gill tissues of dreissenid mussels (D. bugensis) the MXR activity was assayed after treatment by commercially available insecticides (formulated products) which contain neonicotinoids as their active ingredients: Actara (thiamethoxam), Apacs (clothianidin), Calypso (thiacloprid) and Kohinor (imidacloprid), respectively. While applying the accumulation assay method, 0.5 µM rhodamine B was used as model substrate and 20 µM verapamil as model inhibitor of the MXR mechanism. In acute (in vitro) experiments when isolated gills were co-incubated in graded concentrations of insecticides and rhodamine B simultaneously, Calypso and Kohinor treatment resulted increasing rhodamine accumulation. Chemical analysis of gills in vitro incubated in insecticides demonstrated higher tissue concentrations of thiamethoxam, clothianidin and thiacloprid in the presence of verapamil suggesting that the active ingredients of Actara, Apacs and Calypso are potential substrates of the MXR mediated cellular efflux. In contrast, verapamil did significantly alter the accumulated imidacloprid concentrations in gills, suggesting that the active component of Kohinor is not transported by the MXR mechanism. Chronic (in vivo) exposures of the intact animals in lower, 1, 10 mg/L concentration of neonicotinoid products, resulted in a decreased level of both rhodamine accumulation and verapamil inhibition by the 12th-14th days of treatment. These results suggest an enhancement of MXR activity (chemostimulation), building up gradually in the animals exposed to Actara, Apacs and Kohinor, respectively. Neonicotinoid-type insecticides are generally considered as selective neurotoxins for insects, targeting the nicotinic type acetylcholine receptors (nAChRs) in their central nervous system. Our present results provide the first evidences that neonicotinoid insecticides are also able to alter the transmembrane transport mechanisms related to the MXR system.

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.

Diversity of the molecular responses to separate wastewater effluents in freshwater mussels.

The environmental safety of pharmaceutical and personal care products (PPCPs) requires a crucial examination. The aim of this study was to evaluate the responses of biomarkers of stress and toxicity in freshwater mussels to the effect of commonly found PPCPs in wastewater. We treated male mussels Unio tumidus, from an undisturbed site with ibuprofen (IBU, 250 ng L(-1)), triclosan (TCS, 500 ng L(-1)), or estrone (E1, 100 ng L(-1)) for 14 days. Untreated mussels from this site (C) and mussels inhabiting a polluted area (P) were also examined after a similar time of being kept in the laboratory. The consequences of chronic exposure of the mussels in the P-group were reflected in elevated concentrations of oxyradicals (1.4 times), oxidized glutathione (4.3 times), lipofuscin (2.2 times), and DNA-strand breaks in the digestive gland (DG) in comparison to the C-group, higher levels of caspase-3 activity in the DG, and vitellogenin-like proteins in gonads among all studied groups. Exposed mussels demonstrated some common responses with mussels in the P-group: elevated levels of lactate/pyruvate ratio, lipofuscin (IBU and E1), DNA fragmentation (TCS and E1), and caspase-3 activity (TCS and E1). Exposed to PPCPs mussels also showed elevation of ethoxyresorufin-O-deethylase and/or glutathione-S-transferase activity in the DG and a decrease in lysosomal stability in hemocytes (TCS and E1). The TCS group was distinguished by having the highest level of DNA-fragmentation and the lowest concentrations of total glutathione, oxyradicals, lipofuscin, pyruvate, and lactate, reflecting total metabolic depression. These results show that selected PPCPs at low concentrations alter a variety of physiological processes in this animal model system.

The ecotoxicological evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening.

Ecotoxicity of four Cylindrospermopsis raciborskii strains (ACT 9502, ACT 9503, ACT 9504, ACT 9505) isolated from Lake Balaton (Hungary) was evaluated in four aquatic bioassays including the Thamnocephalus platyurus acute lethality test; Daphnia magna acute immobilization assay; D. magna feeding inhibition assay and Danio rerio embryo developmental toxicity assay, assisted by chemical screening for known toxins by HPLC-MS. For reference, we analyzed in parallel the toxin content and toxic effects of two previously characterized toxin-producing strains: the Australian cylindrospermopsin producer AQS C. raciborskii and the anatoxins producer Oscillatoria sp. PCC 6506. Bioassays were used to evaluate the overall toxicity of the hydrophilic bioactive metabolites pool synthesized by the selected cyanobacteria. Chemical screening has proven that the ACT C. raciborskii extracts investigated did not contained cylindrospermopsins and anatoxins. The relative toxicity of the ACT C. raciborskii aqueous extracts observed in each bioassay was comparable to the effects recorded for the anatoxins producer PCC 6506 strain while toxicity values (EC50/LC50) calculated for the AQS extract were in general one order of magnitude lower. Concerning sublethal effects of ACT C. raciborskii extracts to the D. rerio embryogenesis, the general morphological abnormality observed was a significant retardation of development. Overall, our results suggest that C. raciborskii populating Lake Balaton produce metabolites with significant bioactive potencies. Therefore, continued investigation of these unknown compounds is required.

Screening the toxic potential of Cylindrospermopsis raciborskii strains isolated from Lake Balaton, Hungary.

Cylindrospermopsis raciborskii is becoming a major concern among cyanobacteria, due to its potential ability to produce toxic metabolites. We assessed the cytotoxic potential of four C. raciborskii strains (ACT 9502, ACT 9503, ACT 9504 and ACT 9505) isolated from Lake Balaton (Hungary), by lactate dehydrogenase (LDH) leakage measurements and by detecting morphological alterations in CHO-K1 (Chinese Hamster Ovary) cells. The Australian AQS (cylindrospermopsin producer) strain of C. raciborskii and purified cylindrospermopsin (CYN) were used as positive references in both the biochemical and morphological studies. Chemical analysis for known cyanotoxins was performed on aqueous extracts of ACT and AQS strains by the HPLC-MS technique. Comparing threshold values of LDH leakage data, different toxic potentials of cyanobacterial extracts are suggested in short term (3 h) and long (24 h) exposure regimes. In the acute (3 h) experiments the aqueous extract of the ACT 9505 strain proved to be most toxic (EC(50) = 7.4 mg mL(-1)), while after 24 h the ACT 9504 extract was the most effective (EC(50) = 0.65 mg mL(-1)). The extract of the AQS strain and the purified CYN exerted most of their toxic effects after 3 h exposure (EC(50) = 0.74 mg mL(-1), and 0.9 µg mL(-1) respectively). The morphological changes of CHO-K1 cells induced by the crude extracts of the ACT strains included fragmentation of the actin filaments then relocation of the depolymerized actin to the perinuclear region, resulting cell rounding and loss of adhesion. Exposure of CHO-K1 cells to the crude extract of the AQS strain, moreover, resulted cell shrinking and formation of filopodia, i.e. distinctly different cytological alterations from that induced by the ACT extracts and the purified CYN. Chemical analysis of the cyanobacterial crude extracts confirmed the presence of cylindrospermopsin in the extract of the AQS strain (8.5 mg CYN g(-1) dry weight), and none of the presently known cyanotoxins have been analytically confirmed in the extracts of the ACT strains isolated from the Lake Balaton. Although a significant toxicity of all four ACT C. raciborskii strains is confirmed by both biochemical and morphological studies, our results also pointed out the necessity of further studies to identify the toxic, but still unknown metabolic components produced by these cyanobacterial members of the phytoplankton communities.

Vulnerability of biomarkers in the indigenous mollusk Anodonta cygnea to spontaneous pollution in a transition country.

The aim of this study was to estimate the sensitivity of biomarkers of stress and exposure in the bivalve mollusk Anodonta cygnea to spontaneous anthropogenic activities typical for the Western Ukraine. Three sites were examined during spring, summer and autumn: an agricultural site (A); the cooling pond of nuclear power plant (N) and a forestry close to the municipal water inlet (F). Common temporal changes of a battery of biochemical markers in the gills and hemolymph and morphological characteristics were shown by discriminant functional analysis. Classification trees built on the basis of the screened biomarkers demonstrated persistent peculiarities at each site: genotoxicity (nuclear abnormalities) at site A and endocrine disruption (high levels of vitellogenin-like proteins (Vtg-LP) in hemolymph) at site F. Interim local effects were best characterized by metallothionein (MT) concentrations, lipid peroxidation (LPO), activities of glutathione S-transferase and lactate dehydrogenase, and the conditional index of the gills. In autumn, the mollusks from the three sites revealed the highest differences in pollution status: the activation of antioxidant defense and cholinesterase were typical for site A, highest levels of MT related to high levels of Cu and Cd in the water at site B, and a steep increase in the level of Vtg-LP and the decrease of lysosomal membrane stability were recorded at the site selected as reference (F). The biomarker alterations recorded at site F were later related to an emergency event at the municipal dump located nearby. Thus, our case study demonstrated the reliability of using biomarkers of exposure to assess both long-term and accidental environmental pollution loads.

In vitro effects of fipronil on neuronal excitability in mammalian and molluscan nervous systems.

The effect of the insecticide fipronil on non-target organisms was studied on rat brain slices and identified giant neurons of the pond snail Lymnaea stagnalis. This compound acts as an antagonist on GABA(A) receptors. Although fipronil has moderate mammalian toxicity, our experiments confirmed that it modifies neuronal excitability in the rat somatosensory cortex. The amplitudes of evoked field potentials increased significantly after 30 min fipronil treatment. Short-term plasticity was examined with paired-pulse stimulation, this phenomenon was not affected by fipronil. On the other hand, the efficacy of LTP-induction was enhanced in the treated slices. Fipronil is highly toxic to freshwater invertebrates, especially molluscs. In Lymnaea stagnalis, the firing pattern of a GABA receptor- containing neuron (RPeD1) was studied. On this neuron, GABA has an excitatory, hypopolarizing effect. Fipronil treatment decreased the action potential frequency in a concentration-dependent manner. On the membrane potential of the cell, it had a slightly hyperpolarizing effect. These experiments confirmed that fipronil toxicity is mediated by GABA receptors in the nervous system of invertebrates as well as vertebrates. These types of experiments may help in establishing tolerance levels of pesticide residues and in finding proper treatment in case of eventual poisonings.

Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells.

In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

Bensultap decreases neuronal excitability in molluscan and mammalian central nervous system.

Electrophysiological experiments were performed on in vitro neuronal preparations from terrestrial snail and rat brain slices, to determine the effect of the insecticide bensultap. Although bensultap has low toxicity in mammals, our results showed that bensultap altered the synaptic transmission in the vertebrate as well as in the invertebrate central nervous system. Bensultap caused a significant decrease of the ACh-induced current. The effect depended on the preapplication time and the concentration of the chemical. Bensultap also had an effect on the kinetic parameters of the ACh-induced current; the desensitization time was altered in a concentration-dependent manner. In the rat brain slice preparations, we observed an increase in the amplitude of the evoked responses after a 30 min treatment. There was no effect on paired-pulse depression, but LTP-induction was significantly inhibited by bensultap. The efficacy of synaptic transmission was modified by bensultap through effects on both input integration and output organization.

Neuroanatomical, immunocytochemical, and physiological studies of the pharyngeal retractor muscle and its putative regulatory neurons playing a role in withdrawal and feeding in the snail, Helix pomatia.

We describe the neurons regulating two separate functions of the pharyngeal retractor muscle (PRM), namely sustained contraction during body withdrawal and rhythmic phasic contractions during feeding, in the snail, Helix pomatia. The distribution of central neurons innervating the PRM is organized into two main units; one in the buccal-cerebral ganglion complex, the other in the subesophageal ganglion complex. Serotonin- (5-HT-), FMRFamide- (FMRFa-), and tyrosine-hydroxylase-immunostained neurons are present among the PRM neurons that densely innervate the PRM. 5HT both decreases and increases the amplitude of the electrically evoked contraction between concentrations of 0.1 microM and 1 microM. Dopamine (DA) only decreases the amplitude of contraction at a 1-microM threshold concentration. In contrast, FMRFa increases the amplitude of the contraction and slightly elevates the tone of the PRM but requires a higher threshold (10 microM). Assay by high-performance liquid chromatography of 5HT and DA in the PRM has shown that the 5HT level decreases during locomotion but increases during feeding, whereas the DA level increases during locomotion but slightly decreases during feeding. Thus, different segments of the PRM are innervated by neurons from different loci within the central nervous system. The segments of the PRM distal to the pharynx are innervated from loci of the subesophageal ganglion complex suggesting that they mediate withdrawal. The proximal segment of the PRM is innervated from cerebral and buccal loci indicating that these neurons mediate the feeding rhythm produced by buccal and cerebral feeding central pattern generators to induce rhythmic phasic contractions in the PRM during feeding.