Ingestion and effects of polystyrene nanoparticles in the silkworm Bombyx mori.

Information on the occurrence and effects of nanoplastics in ecosystems worldwide currently represent one of the main challenges from the ecotoxicological point of view. This is particularly true for terrestrial environments, in which nanoplastics are released directly by human activities or derive from the fragmentation of larger plastic items incorrectly disposed. Since insects can represent a target for these emerging contaminants in land-based community, the aim of this study was the evaluation of ingestion of 0.5 µm polystyrene nanoplastics and their effects in silkworm (Bombyx mori) larvae, a useful and well-studied insect model. The ingestion of nanoplastics, the possible infiltration in the tissues and organ accumulation were checked by confocal microscopy, while we evaluated the effects due to the administered nanoplastics through a multi-tier approach based on insect development and behaviour assessment, as endpoints at organism level, and the measurements of some biochemical responses associated with the imbalance of the redox status (superoxide dismutase, catalase, glutathione s-transferase, reactive oxygen species evaluation, lipid peroxidation) to investigate the cellular and molecular effects. We observed the presence of microplastics in the intestinal lumen, but also inside the larvae, specifically into the midgut epithelium, the Malpighian tubules and in the haemocytes. The behavioural observations revealed a significant (p < 0.05) increase of erratic movements and chemotaxis defects, potentially reflecting negative indirect effects on B. mori survival and fitness, while neither effect on insect development nor redox status imbalance were measured, with the exception of the significant (p < 0.05) inhibition of superoxide dismutase activity.

First evidence of protein modulation by polystyrene microplastics in a freshwater biological model.

Microplastics (MPs) are now one of the major environmental problems due to the large amount released in aquatic and terrestrial ecosystems, as well as their diffuse sources and potential impacts on organisms and human health. Still the molecular and cellular targets of microplastics' toxicity have not yet been identified and their mechanism of actions in aquatic organisms are largely unknown. In order to partially fill this gap, we used a mass spectrometry based functional proteomics to evaluate the modulation of protein profiling in zebra mussel (Dreissena polymorpha), one of the most useful freshwater biological model. Mussels were exposed for 6 days in static conditions to two different microplastic mixtures, composed by two types of virgin polystyrene microbeads (size = 1 and 10 µm) each one. The mixture at the lowest concentration contained 5 × 105 MP/L of 1 µm and 5 × 105 MP/L of 10 µm, while the higher one was arranged with 2 × 106 MP/L of 1 µm and 2 × 106 MP/L of 10 µm. Proteomics' analyses of gills showed the complete lack of proteins' modulation after the exposure to the low-concentrated mixture, while even 78 proteins were differentially modulated after the exposure to the high-concentrated one, suggesting the presence of an effect-threshold. The modulated proteins belong to 5 different classes mainly involved in the structure and function of ribosomes, energy metabolism, cellular trafficking, RNA-binding and cytoskeleton, all related to the response against the oxidative stress.

Cellular pathways affected by carbon nanopowder-benzo(α)pyrene complex in human skin fibroblasts identified by proteomics.

One of the crucial and unsolved problems of the airborne carbon nanoparticles is the role played by the adsorbed environmental pollutants on their toxicological effect. Indeed, in the urban areas, the carbon nanoparticles usually adsorb some atmospheric contaminants, whose one of the leading representatives is the benzo(α)pyrene. Herein, we used the proteomics to investigate the alteration of toxicological pathways due to the carbon nanopowder-benzo(α)pyrene complex in comparison with the two contaminants administered alone on human skin-derived fibroblasts (hSDFs) exposed for 8 days in semi-static conditions. The preliminary confocal microscopy observations highlighted that carbon-nanopowder was able to pass through the cell membranes and accumulate into the cytoplasm both when administered alone and with the adsorbed benzo(α)pyrene. Proteomics revealed that the effect of carbon nanopowder-benzo(α)pyrene complex seems to be related to a new toxicological behavior instead of simple additive or synergistic effects. In detail, the cellular pathways modulated by the complex were mainly related to energy shift (glycolysis and pentose phosphate pathway), apoptosis, stress response and cellular trafficking.

Carbon nanopowder acts as a Trojan-horse for benzo(α)pyrene in Danio rerio embryos.

Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the "particle-specific" effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

Toxicity decrease in urban wastewaters treated by a new biofiltration process.

We carried out a project aimed to evaluate the possible role played by the freshwater zebra mussel (Dreissena polymorpha) in the possible decrease of some environmental pollutants recalcitrant to tradition wastewater treatments. By the help of a pilot-plant built in the largest wastewater treatment plant of Milan (Italy), we tested several waste mixtures in order to measure the chemicals' abatement made by mussels' biofiltration. This study represents the last step of the wider project and it aimed to evaluate if the decrease in the concentration of some urban pollutants measured in wastewaters was followed by a corresponding toxicity reduction. Thus, we performed 7-day exposures under laboratory conditions to test the toxicity of the raw wastewaters and those preliminary filtered by zebra mussels, through the measurement of different end-points of acute and chronic toxicity. Results showed a clear positive effect of mussels' biofiltration mainly to decrease the acute toxicity made by the two tested wastewater mixtures, while the biomarkers' suite used to evaluate the chronic toxicity showed contradictory results.

Does zebra mussel (Dreissena polymorpha) represent the freshwater counterpart of Mytilus in ecotoxicological studies? A critical review.

One of the fundamentals in the ecotoxicological studies is the need of data comparison, which can be easily reached with the help of a standardized biological model. In this context, any biological model has been still proposed for the biomonitoring and risk evaluation of freshwaters until now. The aim of this review is to illustrate the ecotoxicological studies carried out with the zebra mussel Dreissena polymorpha in order to suggest this bivalve species as possible reference organism for inland waters. In detail,we showed its application in biomonitoring, as well as for the evaluation of adverse effects induced by several pollutants, using both in vitro and in vivo experiments. We discussed the advantages by the use of D. polymorpha for ecotoxicological studies, but also the possible limitations due to its invasive nature.

Analysis of the Dreissena polymorpha gill proteome following exposure to dioxin-like PCBs: mechanism of action and the role of gender.

PCBs are a persistent environmental problem due to their high stability and lipophilicity. The non-ortho- and the mono-ortho-substituted PCBs (dioxin-like-PCBs) share a common and well-described toxicity mechanism in vertebrates, initially involving binding to cytosolic AhRs. Invertebrate AhRs, however, show a lack of dioxin binding, and little information is available regarding the mechanism of toxicity of dl-PCBs in invertebrates. In this study, a proteomic approach was applied to analyse the variations in the pattern of the gill proteome of the freshwater mussel Dreissena polymorpha. Mussels were exposed to a mixture of dl-PCBs, and to perform a more in-depth evaluation, we chose to investigate the role of gender in the proteome response by analysing male and female mussels separately. The results revealed significant modulation of the gill tissue proteome: glycolysis and Ca(2+) homeostasis appear to be the main pathways targeted by dl-PCBs. In light of the differences between the male and female gill proteome profiles following exposure to dl-PCBs, further in-depth investigations of the role of gender in the protein expression profiles of a selected biological model are required.

First evidence of protein profile alteration due to the main cocaine metabolite (benzoylecgonine) in a freshwater biological model.

Illicit drugs represent not only a great social problem but are also considered an environmental problem because their use and, often, abuse release large amounts of parent compounds, and especially their metabolites, into freshwaters. One of the most commonly used drugs is cocaine, which is the second most prevalent drug in Europe (accounting for almost 30% of all cocaine users worldwide). Cocaine is rapidly metabolised in humans to benzoylecgonine (35-54%), ecgonine methyl ester (32-49%) and norcocaine (5%), which are eliminated in the urine and are only partially removed by wastewater treatment plants (WWTPs). Because no studies have previously been carried out to evaluate the possible risks due to cocaine and its metabolites in non-target organisms, we applied a multi-disciplinary approach to investigate the possible environmental risk related to benzoylecgonine (BE), the main metabolite of cocaine. Previous studies carried out by means of a biomarker suite and the redox-proteomic approach showed an imbalance of anti-oxidant enzyme activities and several genotoxic effects to be caused by environmental BE concentrations in the freshwater bivalve Zebra mussel (Dreissena polymorpha). This report presents the results obtained in the last step of this study, based on a proteomics analysis. We analysed the protein expression profile in the gills of Zebra mussels exposed to two different concentrations (0.5 and 1 µg/L) of BE for 14 days through 2-DE and mass spectrometry analysis (RP-UPLC ESI-LTQ-Orbitrap). Our results highlight significant changes in some proteins in gill cells whose functions are crucial for overall metabolism. In particular, we detected a probable effect of BE on calcium homeostasis and a consequent imbalance of oxidative stress, as verified for vertebrates.

Biological effects of palytoxin-like compounds from Ostreopsis cf. ovata: a multibiomarkers approach with mussels Mytilus galloprovincialis.

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans. The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na(+)/K(+)-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage). Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na(+)/K(+)-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.

Illicit drugs as new environmental pollutants: cyto-genotoxic effects of cocaine on the biological model Dreissena polymorpha.

The increase in global consumption of illicit drugs has produced not only social and medical problems but also a potential new environmental danger. Indeed, it has been established that drugs consumed by humans end up in surface waters, after being carried through the sewage system. Although many studies to measure concentrations of several drugs of abuse in freshwater worldwide have been conducted, no data have been available to evaluate their potentially harmful effects on non-target organisms until now. The present study represents the first attempt to investigate the cyto-genotoxic effects of cocaine, one of the primary drugs consumed in Western Countries, in the biological model Dreissena polymorpha by the use of a biomarker battery. We performed the following tests on Zebra mussel hemocytes: the single cell gel electrophoresis (SCGE) assay, the apoptosis frequency evaluation and the micronucleus assay (MN test) for the evaluation of genotoxicity and the lysosomal membranes stability test (neutral red retention assay; NRRA) to identify the cocaine cytotoxicity. We exposed the molluscs for 96 h to three different nominal concentrations in water (40 ng L(-1); 220 ng L(-1); and 10 µg L(-1)). Cocaine caused significant (p<0.05) primary DNA damage in this short-term experiment, but it also caused a clear increase in micronucleated cells and a marked rise in apoptosis, which was evident in samples from even the lowest environmental cocaine concentration. Because cocaine decreased the stability of lysosomal membranes, we also highlighted its cytotoxicity and the possible implications of oxidative stress for the observed genotoxic effects.

Multi-biomarker approach to investigate the state of contamination of the R. Lambro/R. Po confluence (Italy) by zebra mussel (Dreissena polymorpha).

This study assesses the response of the zebra mussel (Dreissena polymorpha) to chemical pollution derived from the R. Lambro/R. Po confluence, which is one of the most polluted aquatic environments in Europe. The mussels were tested under laboratory conditions to water sampled in the spring and fall at three sites located upstream or downstream of the confluence or directly in R. Lambro alone. We performed on mussel specimens a biomarker battery composed by eight different assays: single cell gel electrophoresis, apoptosis determination, the micronucleus test and Neutral Red retention, as well as catalase, superoxide dismutase, glutathione peroxidase and glutathione transferase activities. We also evaluated the bioaccumulation of several organic pollutants (PAHs, PCBs, DDTs, HCHs and HCB) to characterize the sampling sites. Significant increases in DNA strand breaks, apoptosis and micronuclei were observed, with no significant seasonal differences. We observed a clear induction of the enzyme activities measured in the spring, but the enzymatic activity trend in the fall was very complex, with several enzymes returning to baseline levels of activity, suggesting a possible seasonal change in chemical mixture characteristics.

A multi-biomarker assessment of the impact of the antibacterial trimethoprim on the non-target organism Zebra mussel (Dreissena polymorpha).

A battery of eight biomarkers was applied in the freshwater mussel Dreissena polymorpha to evaluate potential sub-lethal effects of the antimicrobial trimethoprim (TMP, 5-[3,4,5-trimethoxybenzyl]pyrimidine-2,4-diamine). Mussels were exposed for 96 h to increasing concentrations (1, 3, 10 nM) of TMP in in vivo experiments. We determined the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test), the apoptotic frequency (Halo assay) and the lysosomal membrane stability (Neutral Red Retention Assay) in mussel hemocytes. Moreover, to reveal whether the oxidative status was altered, measurements of the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and the phase II detoxifying enzyme glutathione S-transferase (GST) were performed using the cytosolic fraction extracted from a pool of entire mussels. The biomarker battery pointed out only a moderate cyto- and genotoxicity on Zebra mussel hemocytes since only a slight increase in DNA damage was registered by apoptosis induction and MN frequency, while significant differences of lysosomal membrane stability from baseline levels were measured at 3 and 10 nM at the end of exposures only. Finally, TMP seems to have a very low induction capability or even an inhibitory effect on the activities of antioxidant enzymes, but a clear significant induction on GST.

Cytotoxic and genotoxic effects of in vitro exposure to triclosan and trimethoprim on zebra mussel (Dreissena polymorpha) hemocytes.

Pharmaceuticals and personal care products (PPCPs) have been detected in several aquatic ecosystems for a number of years, but the potential for biological effects in exposed non-target organisms is only now being reported. In this study the potential cellular damage due to two of the main PPCPs found in aquatic environments was investigated by in vitro exposures. Hemolymph samples of the freshwater bivalve Dreissena polymorpha were collected and treated with increasing concentrations of the antibacterial agent Triclosan (TCS) and the antibiotic Trimethoprim (TMP). Doses selected for TCS were 0.1, 0.15, 0.2, and 0.3 microM, while 0.2, 1, and 5 microM for TMP exposures, respectively. We evaluated the potential genotoxicity on hemocytes by the SCGE (single cell gel electrophoresis) assay and apoptosis frequency evaluation, while the cytotoxicity was measured by the lysosomal membranes stability test (NRRA, neutral red retention assay). TCS genotoxicity increased in a dose-dependent manner and this pharmaceutical significantly affects hemocyte functionality due to severe DNA injuries at very low doses. In contrast, TMP seems to be less dangerous than TCS for D. polymorpha because the cytotoxic and the moderate genotoxic effects noticed were obtained only at very high concentration levels.

In vivo experiments for the evaluation of genotoxic and cytotoxic effects of Triclosan in Zebra mussel hemocytes.

In this work, we investigated the possible genotoxic and cytotoxic effects of the antibacterial agent Triclosan in hemocytes of the freshwater bivalve Zebra mussel (Dreissena polymorpha). For this study, we used several biomarkers for in vivo experiments (96h of exposure) carried out at three possible environmental Triclosan concentrations (1, 2, 3nM). We used the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test) and the measure of the apoptotic frequency (Halo assay) to measure the genotoxic potential of Triclosan, and the neutral red retention assay (NRRA) as a measure of lysosomal membrane stability to identify general cellular stress. We observed significant increases in all of the genotoxic biomarkers examined as early as 24h after initial exposure, as well as a clear destabilization of lysosomal membranes (after 48h), indicating that this chemical is potentially dangerous for the entire aquatic biocoenosis. A comparison of these in vivo data with existing data from in vitro experiments allowed us to suggest possible mechanisms of action for Triclosan in this bivalve. Although further studies are needed to confirm the possible modes of action, our study is the first to report on the effects of this widespread antibiotic on freshwater invertebrates.

Organochlorine pesticide residues in sediment cores of sunderban wetland, northeastern part of Bay of Bengal, India, and their ecotoxicological significance.

This paper presents the first comprehensive report of the organochlorine pesticide residues (OCs) such as hexachlorocyclohexane isomers (HCHs), dichlorodiphenyltrichloroethane and its six metabolites (DDTs), and hexachlorobenzene (HCB) in core sediments (<63-microm particle size) from the Indian Sunderban wetland. The pooled mean values of the mass fraction of SigmaHCHs, HCB, and SigmaDDTs in the sediments were 0.05-12, 0.05-1.4, and 0.05-11.5 ng g(-1) dry weight, respectively. The vertical distribution of pesticides reveals an erratic pattern. The concentration of four isomers of HCHs reveals a heterogenic distribution where gamma-HCH (lindane) and beta-HCH shared the dominant part. The mass fraction of HCB did not show any sharp spatial variation. The prevailing sequence of DDT metabolites indicates an active degradation of the parent compound in the sediments and/or inputs of already degraded pp'DDT to the region. Peak concentrations of HCH isomers and DDT metabolites have the potential to induce ecotoxicological impact as per the sediment quality guidelines.

Levels and congener profiles of polybrominated diphenyl ethers (PBDEs) in Zebra mussels (D. polymorpha) from Lake Maggiore (Italy).

Several congeners of polybrominated diphenyl ethers (PBDEs) were monitored in 14 different sampling stations of Lake Maggiore, the second largest Italian lake in regard to surface, volume and average depth, using the sentinel-organism Zebra mussel (Dreissena polymorpha). Results revealed a moderate contamination with summation operatorPBDE levels (BDE-17, -28, -47, -66, -71, -85, -99, -100, -138, -153, -154, -183, -190 and -209) ranging from 40 to 447ngg(-1) lipid weight which are similar to those found in environments polluted by deposition or atmospheric transport. The general order of decreasing congener contribution to the total load was BDE-47>-99>-100>-209, which closely reflected patterns observed in mussels collected in freshwater ecosystems worldwide.

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy).

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.

Integrated use of biomarkers and bioaccumulation data in Zebra mussel (Dreissena polymorpha) for site-specific quality assessment.

One of the useful biological tools for environmental management is the measurement of biomarkers whose changes are related to the exposure to chemicals or environmental stress. Since these responses might vary with different contaminants or depending on the pollutant concentration reached in the organism, the support of bioaccumulation data is needed to prevent false conclusions. In this study, several persistent organic pollutants -- 23 polychlorinated biphenyl (PCB) congeners, 11 polycyclic aromatic hydrocarbons (PAHs), six dichlorodiphenyltricholroethane (DDT) relatives, hexachlorobenzene (HCB), chlorpyrifos and its oxidized metabolite -- and some herbicides (lindane and the isomers alpha, beta, delta; terbutilazine; alachlor; metolachlor) were measured in the soft tissues of the freshwater mollusc Zebra mussel (Dreissena polymorpha) from 25 sampling sites in the Italian portions of the sub-alpine great lakes along with the measure of ethoxyresorufin dealkylation (EROD) and acetylcholinesterase (AChE) activity. The linkage between bioaccumulation and biomarker data allowed us to create site-specific environmental quality indexes towards man-made chemicals. This classification highlighted three different degrees of xenobiotic contamination of the Italian sub-alpine great lakes: a high water quality in Lake Lugano with negligible pollutant levels and no effects on enzyme activities, an homogeneous poor quality for Lakes Garda, Iseo and Como, and the presence of some xenobiotic point-sources in Lake Maggiore, whose ecological status could be jeopardized, also due to the heavy DDT contamination revealed since 1996.

New evidences for old biomarkers: effects of several xenobiotics on EROD and AChE activities in Zebra mussel (Dreissena polymorpha).

The biomarker approach is widely used both in vertebrates and invertebrates for environmental biomonitoring, because it can supply an integrated response for multi-xenobiotics contamination. However, the use of biomarkers requires the identification of every possible variation that can influence the biochemical response, because ecosystems are generally subject to a mixture of pollutants, which can create additive, opposite or competitive effects. In recent years, there has been considerable interest in the use of biomarkers within marine bivalves, while very few data are available for freshwater molluscs. The aim of this research was to investigate changes on EROD and AChE activities in the freshwater bivalve Zebra mussel (Dreissena polymorpha) exposed to different pollutants (Arochlor 1260, CB 153 and 126, pp'DDT, chlorpyrifos, carbaryl) at laboratory conditions, in order to standardize the analytical procedures and to highlight eventual interferences on enzyme activities. Chemical concentrations in the mussel soft tissues were analyzed by GC/MS-MS. Main results showed a significant induction of EROD activity when mussels were exposed to 100 ng/l of PCB mixture of Arochlor 1260 and dioxin-like CB 126, but this congener showed also a clear competitive inhibition after 48 h of exposure. Surprisingly, pp'DDT determined a significant decrease of basal EROD activity after only 24 h of exposure, even if it was not possible to discriminate between the effect of the parent compound and that of its metabolites (DDD, DDE). We also found an interaction between the organophosphate insecticide chlorpyrifos, which does not directly decrease the AChE activity, and terbutilazine. This herbicide increased the biotransformation of the organophosphate compound to its oxidized metabolite (oxon), a much stronger AChE inhibitor. The possible use of the oxime Pyridine-2-Aldoxime Methochloride (2-PAM) to bring back the catalytic activity to basal levels was also demonstrated.

S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium.

Glutathione (GSH) is one of the most important defense mechanisms against oxidative stress in the respiratory epithelial lining fluid. Considering that GSH secretion in respiratory cells has been postulated to be at least partially electrogenic, and that the mucoregulator S-carbocysteine lysine salt monohydrate (S-CMC-Lys) can cause an activation of epithelial Cl(-) conductance, the purpose of this study was to verify whether S-CMC-Lys is able to stimulate GSH secretion. Experiments have been performed by patch-clamp technique, by high-performance liquid chromatography (HPLC) assay, and by Western blot analysis on cultured lines of human respiratory cells (WI-26VA4 and CFT1-C2). In whole-cell configuration, after cell exposure to 100 microM S-CMC-Lys, a current due to an outward GSH flux was observed, which was inhibitable by 5-nitro-2-(3-phenylpropylamino)-benzoate and glibenclamide. This current was not observed in CFT1-C2 cells, where a functional cystic fibrosis transmembrane conductance regulator (CFTR) is lacking. Inside-out patch-clamp experiments (GSH on the cytoplasm side, Cl(-) on the extracellular side) showed the activity of a channel, which was able to conduct current in both directions: the single channel conductance was 2-4 pS, and the open probability (P(o)) was low and voltage-independent. After preincubation with 100 microM S-CMC-Lys, there was an increase in P(o), in the number of active channels present in each patch, and in the relative permeability to GSH vs Cl(-). Outwardly directed efflux of GSH could also be increased by protein kinase A, adenosine 5'-triphosphate, and cyclic adenosine monophosphate (cAMP) added to the cytoplasmic side (whole-cell configuration). The increased secretion of GSH observed in the presence of S-CMC-Lys or 8-bromoadenosine-3',5'-cyclic monophosphate was also confirmed by HPLC assay of GSH on a confluent monolayer of respiratory cells. Western blot analysis confirmed the presence of CFTR in WI-26VA4 cells. This study suggests that S-CMC-Lys is able to stimulate a channel-mediated GSH secretion by human respiratory cells: electrophysiological and pharmacological characteristics of this channel are similar to those of the CFTR channel.