János Györi

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.

NMDA-like receptors in the CNS of molluscs.

The properties of L-glutamate sensitive neurones in the fresh-water snail Lymnaea stagnalis were studied to determine whether or not NMDA type receptors are present in molluscan ganglia. Experiments were carried out in isolated ganglia and with dialysed neurones. In the neuro-secretory light yellow cells (LYC) both glutamate and NMDA (but not other glutamate agonists) induced prolonged depolarization and inward current. The sensitivity of the neurones for NMDA was lower than for glutamate. Both currents are carried by Na- and Ca-ions and have linear I-V characteristics. Mg-ions did not reduce either but glycine potentiated both glutamate and NMDA responses. It is concluded that in the molluscan central nervous system glutamate receptors have some of the characteristics of mammalian NMDA receptors.

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.

Effect of Ag+ on membrane permeability of perfused Helix pomatia neurons.

1. Isolated, non‐identified neurons were voltage clamped using the internal perfusion technique. 2. Ions of Ag+ (1‐100 microM) introduced into the bathing solution activated a steady‐state inward current (IAg) in the soma. The effect of Ag+ was reversible when the concentration of Ag+ was less than 75 microM or the time of application was shorter than 10 min. 3. IAg was observed both in the presence and absence of Na+ ions in the extracellular saline. It could also be activated when Cs+ ions were substituted for Na+ ions. 4. The current‐voltage characteristics were linear in the voltage range ‐100 to 0 mV. The reversal potential in control saline was an average of 1.19 +/‐ 5.1 mV. 5. The application of Ag+ ions induces an elevation of intracellular free Ca2+ concentration by 10‐20 times in both Ca(2+)‐containing and Ca(2+)‐free extracellular salines, as revealed by Fura‐2 measurements. 6. Agents that increase the intracellular free Ca2+ concentration ([Ca2+]i), like thymol, caffeine and dinitrophenol, increased the amplitude of IAg. The effect was additive. Ruthenium Red, which blocks the release of Ca2+ from intracellular stores, decreased the Ag+ effect. 7. It is concluded that extracellularly applied Ag+ ions increase the cytoplasmic free Ca2+ concentration, which in turn activates non‐specific cationic channels. 8. Ag+ ions in 1‐10 microM concentration were able to decrease the voltage‐activated Ca2+ current amplitude. This decrease, however, was due to the increase of [Ca2+]i which caused Ca(2+)‐dependent inactivation.

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.

Effect of Inorganic and Organic Tin Compounds on ACh- and Voltage-Activated Na Currents

Abstract1. Inorganic tin and organotin compounds, occurring in aquatic ecosystems, are toxic and can cause behavioral abnormalities in living organisms. To determine the possible neuronal basis of these actions, the effects of both forms of Sn were studied on identified neurones of the mollusk, Lymnaea stagnalis L.2. SnCl2 caused a dose-dependent decrease in the acetylcholine (Ach)-induced inward current. The effective threshold concentration, measured by a two microelectrode voltage clamp technique, was 0.1 μM, and the maximal effect occurred at 5 μM SnCl2. The depression of the inward current was greater after a 10 min preapplication (20%) than after 3 min treatment (7%).3. The next series of experiments compared the actions of inorganic or organic tin compounds. In whole cell clamp experiments both (CH3)2SnCl2 and (CH3)3SnCl, like inorganic Sn, decreased the amplitude of Ach-induced current. Increasing the duration of the preapplication time resulted in an increase in the effect, but the action was not reversible. SnCl2 treatment caused a concentration-dependent alteration (initial potentiation followed by depression) of the amplitude of INa(V) over the whole voltage range and slightly shifted the I–V curves to the left. In contrast, trimethyl tin decreased the amplitude of INa(V) only at high concentration (100 μM). The activation time course of INa was increased (τ = 0.43 ms in control and 0.55 ms in Sn), but Sn did not alter the inactivation parameters (τ = 3.43 and 3.41 ms).4. These results support earlier findings that agonist- and voltage-activated channels are direct targets of toxic metals. We conclude that tin in both inorganic and organic forms acts at neuronal membranes to modulate synaptic transmission through direct actions on agonist-activated ion channels, and suggest that these actions may be the basis of the altered behavior of animals in tin-polluted environments.

Lead ions close steady-state sodium channels in Helix neurons

Extracellularly applied Pb2+ (1-150 microM) induced an outward current (IPb) in intracellularly perfused snail neurons. The current-voltage relationship of the Pb(2+)-induced current was linear over the potential range of -100 to -40 mV with negative slope conductance. The Pb-induced current was strongly dependent on the Na+ gradient. The IPb in intra- or extracellular K+- and Cl(-)-free or -rich solutions was almost the same as in control external and internal salines. The negative slope of the I-V curve and the decreased conductivity during Pb2+ application suggested that IPb is owing to the blocking of the resting Na conductance. Data obtained from single-channel measurements also supported this conclusion. Patch-clamp data showed that the steady-state Na channel has a conductance of 14 pS and both closed and open time-distributions displayed single exponential character.

Postsynaptic induction of mossy fibre long term depression in developing rat hippocampus

The whole cell configuration of the patch clamp technique was used to study the mechanisms of induction of long term depression (LTD) occurring at the mossy fibre-CA3 synapse between postnatal (P) day 6 and P13. In control conditions, when two pulses were delivered to the mossy fibres with an interval of 50 ms a potentiation of the EPSC evoked by the second pulse associated with a reduction in the number of failures was observed. Tetanization of the mossy fibres induced LTD of the responses to the first and second stimulus without affecting the paired pulse facilitation. Loading the postsynaptic cell with BAPTA prevented the induction of LTD but did not modify the paired pulse facilitation, suggesting that LTD induction occurs at the postsynaptic site.

Action of lead on glutamate-activated chloride currents in Helix pomatia L. neurons.

Summary1. In molluscan neurons glutamate may, on different neurons, evoke either excitation or inhibition. We studied neurons ofHelix pomatia which have hyperpolarizing responses to glutamate and determined the effects of lead on these responses.2. In voltage clamp experiments, the reversal potentials of these glutamate responses indicate that they are due to a conductance increase to chloride ions. Further evidence for this conclusion was obtained by the demonstration that responses to glutamate remained unaffected in experiments with intracellular dialysis with K-free saline in the presence of Na- and K-free extracellular media. In these circumstances, there is effectively no other ion than chloride to carry the current. In isolated neurons the glutamate-evoked chloride current is concentration dependent between 25 and 2500 µM. The current rises over 200 msec and declines in the continued presence of glutamate over a period of about 3 sec.3. Lead (0.5–1.0 µM) potentiated the glutamate-evoked chloride current provided that the channels were not maximally activated. The potentiation was greater if lead was added 30–60 sec before glutamate application.4. These results suggest that potentiation of transmitter-evoked responses by lead must be considered as yet another possible site of action of lead on neurons, and thus this effect must be considered as a part of the mechanism responsible for the neurotoxicity of this heavy metal.

Humoral serotonin and dopamine modulate the feeding in the snail, Helix pomatia

We investigated the effect of elevated levels of humoral 5HT and DA on the feeding latency of Helix pomatia, 1 day, 3 days and 10 days following satiation, by injecting monoamines into the haemocoel. HPLC assay of monoamines showed that both 5HT and DA are present in pmol/ml concentrations in the haemolymph of both starved and non-starved animals. Elevated levels of 5HT and DA were most effective at decreasing the feeding latency 10 days following satiation when DA decreased the feeding latency in a concentration dependent manner between 10(-7) and 10(-5) M whereas 5HT levels decreased the feeding latency only at 10(-6) M but increased it at 10(-5) M. Immunocytochemistry revealed that both 5HT3 and D1 receptor-like immuno-reactivity are present in cell bodies located in the same areas of the buccal ganglia. Our observations suggest that both humoral DA and 5HT mutually modulate the activity of the feeding CPG through neurons which have these receptors.