Multi-target assessment of advanced oxidation processes-based strategies for indirect potable reuse of tertiary wastewater: Fate of compounds of emerging concerns, microbial and ecotoxicological parameters.

Two advanced oxidation processes (AOPs), namely ozone/H2O2 and UV/H2O2, were tested at pilot scale as zero-liquid-discharge alternative treatments for the removal of microbiological (bacteria and viruses), chemical (compounds of emerging concern (CECs)) and genotoxic responses from tertiary municipal wastewater for indirect potable reuse (IPR). The AOP treated effluents were further subjected to granular activated carbon (GAC) adsorption and UV disinfection, following the concept of multiple treatment barriers. As a reference, a consolidated advanced wastewater treatment train consisting of ultrafiltration, UV disinfection, and reverse osmosis (RO) was also employed. The results showed that, for the same electrical energy applied, the ozone/H2O2 treatment was more effective than the UV/H2O2 treatment in removing CECs. Specifically, the ozone/H2O2 treatment, intensified by high pressure and high mixing, achieved an average CECs removal efficiency higher than UV/H2O2 (66.8% with respect to 18.4%). The subsequent GAC adsorption step, applied downstream the AOPs, further improved the removal efficiency of the whole treatment trains, achieving rates of 98.5% and 96.8% for the ozone/H2O2 and UV/H2O2 treatments, respectively. In contrast, the ultrafiltration step of the reference treatment train only achieved a removal percentage of 22.5%, which increased to 99% when reverse osmosis was used as the final step. Microbiological investigations showed that all three wastewater treatment lines displayed good performance in the complete removal of regulated and optional parameters according to both national and the European Directive 2020/2184. Only P. aeruginosa resulted resistant to all treatments with a higher removal by UV/H2O2 when higher UV dose was applied. In addition, E. coli STEC/VTEC and enteric viruses, were found to be completely removed in all tested treatments and no genotoxic activity was detected even after a 1000-fold concentration. The obtained results suggest that the investigated treatments are suitable for groundwater recharge to be used as a potable water source being such a procedure an IPR. The intensified ozone/H2O2 or UV/H2O2 treatments can be conveniently incorporated into a multi-barrier zero-liquid-discharge scheme, thus avoiding the management issues associated with the retentate of the conventional scheme that uses reverse osmosis. By including the chemical cost associated with using 11-12 mg/L of H2O2 in the cost calculations, the overall operational cost (energy plus chemical) required to achieve 50% average CECs removal in tertiary effluent for an hypothetical full-scale plant of 250 m3/h (or 25,000 inhabitants) was 0.183 €/m3 and 0.425 €/m3 for ozone/H2O2 and UV/H2O2 treatment train, respectively.

Multibiomarker response in the earthworm Eisenia fetida as tool for assessing multi-walled carbon nanotube ecotoxicity.

Carbon nanotubes have received a great attention in the last years thanks to their remarkable structural, electrical, and chemical properties. Nowadays carbon nanotubes are increasingly found in terrestrial and aquatic environment and potential harmful impacts of these nanoparticles on humans and wildlife are attracting increasing research and public attention. The effects of carbon nanotubes on aquatic organisms have been explored by several authors, but comparatively the information available on the impact of these particles on soil organisms is much less. Earthworms have traditionally been considered to be convenient indicators of land use impact and soil fertility. The aim of this work was to study the integrated response of a suite of biomarkers covering molecular to whole organism endpoints for the assessment of multi-walled carbon nanotube (MWCNTs) effects on earthworms (Eisenia fetida) exposed to spiked soil. Results showed that cellular and biochemical responses, such as immune cells morphometric alterations and lysosomal membrane destabilization, acetylcholinesterase inhibition and metallothionein tissue concentration changes, showed high sensitivity to MWCNTs exposure. They can improve our understanding and ability to predict chronic toxicity outcomes of MWCNTs exposure such as reproductive alterations. In this context although more investigation is needed to understand the mechanistic pathway relating the biochemical and cellular biomarker analyzed to reproductive alterations, the obtained results give an early contribution to the future development of an adverse outcomes pathways for MWCNTs exposure.

Apoptotic volume decrease (AVD) in A549 cells exposed to water-soluble fraction of particulate matter (PM10).

Exposure to atmospheric particulate matter (PM) is recognized as a human health risk factor of great concern. The present work aimed to study the cellular mechanisms underlying cytotoxic effects of airborne particulate matter <10 µm in size (PM10), sampled in an urban background site from January to May 2020, on A549 cells. In particular, the study addressed if PM10 exposure can be a main factor in the induction of the Apoptotic Volume Decrease (AVD), which is one of the first events of apoptosis, and if the generation of intracellular oxidative stress can be involved in the PM10 induction of apoptosis in A549 cells. The cytotoxicity of PM10 samples was measured by MTT test on cells exposed for 24 h to the PM10 aqueous extracts, cell volume changes were monitored by morphometric analysis of the cells, apoptosis appearance was detected by annexin V and the induction of intracellular oxidative stress was evaluated by the ROS sensitive CM-H2DCFDA fluorescent probe. The results showed cytotoxic effects ascribable to apoptotic death in A549 cells exposed for 24 h to aqueous extracts of airborne winter PM10 samples characterized by high PM10 value and organic carbon content. The detected reduced cell viability in winter samples ranged from 55% to 100%. Normotonic cell volume reduction (ranging from about 60% to 30% cell volume decrease) after PM10 exposure was already detectable after the first 30 min clearly indicating the ability of PM10, mainly arising from biomass burning, to induce Apoptotic Volume Decrease (AVD) in A549 cells. AVD was prevented by the pre-treatment with 0.5 mM SITS indicating the activation of Cl- efflux presumably through the activation of VRAC channels. The exposure of A549 cells to PM10 aqueous extracts was able to induce intracellular oxidative stress detected by using the ROS-sensitive probe CM-H2DCFDA. The PM10-induced oxidative stress was statistically significantly correlated with cell viability inhibition and with apoptotic cell shrinkage. It was already evident after 15 min exposure representing one of the first cellular effects caused by PM exposure. This result suggests the role of oxidative stress in the PM10 induction of AVD as one of the first steps in cytotoxicity.

Effect of heavy metal exposure on blood haemoglobin concentration and methemoglobin percentage in Lumbricus terrestris.

The earthworm haemoglobin (Hb) is a large extracellular hemoprotein flowing in a closed circulatory system. In spite of the fundamental role of this respiratory pigment in earthworm physiology, little is known about its sensitivity to environmental pollutants. The aim of the present work was to investigate the possible effect of heavy metal (cadmium, copper, mercury) exposure on Hb concentration and oxidation state (methemoglobin formation) in the earthworm Lumbricus terrestris. In addition, the tissue concentration of metallothioneins, a well-known biomarker of heavy metal exposure, was determined as an indicator of metal uptake. The animals were exposed to increasing concentrations of Cd, Cu and Hg utilizing the standard acute toxicity test, "Filter paper test" for 48 h. Exposure to heavy metals (10(-5)-10(-3) M for Cd, 10(-4)-10(-3) M for Hg, and 10(-4)-10(-2) M for Cu) was found to increase haemoglobin concentration in L. terrestris, although the magnitude of such an increase was dependent on the metal. In addition, metal exposure led to the formation of methemoglobin. Compared to other known biological responses to heavy metals, such as metallothionein induction, methemoglobin increase showed a higher sensitivity and a higher percentage variation in exposed organisms, showing to be a possible suitable biomarker of exposure/effect to be included in a multi biomarker strategy in earthworm in soil monitoring assessment.

Controlling micropollutants in tertiary municipal wastewater by O3/H2O2, granular biofiltration and UV254/H2O2 for potable reuse applications.

A comprehensive pilot study was carried out to experimentally assess the potential of newly developed treatment trains integrating two-stage AOPs and biofiltration to reach potable reuse water quality standards from municipal wastewater. The processes consisted of a two-stage AOPs with (carbon or limestone) biofiltration, the first AOP (O3/H2O2) serving as pre-treatment to biofiltration and the second AOP (UV254/H2O2) serving as post-biofiltration finishing step to ensure advanced disinfection. A comprehensive monitoring campaign was put in place resulting from the combination of targeted, non-targeted and suspect screening measurements. It was found that 13 organic micropollutants were detected from a list of 219 suspects although at ng/L level only. For the treatment conditions piloted in this study (O3 = 13 ±â€¯0.5 mg/L, H2O2 = 11 ±â€¯0.4 mg/L for the O3/H2O2 process, and UV = 410 ±â€¯63.5 mJ/cm2, H2O2 = 5 mg/l for the UV254/H2O2 process), it was possible to estimate the overall removal efficacy for each unit process, which was found to follow this order: RO (99%)  > BAC (87%) > O3-H2O2 (78%)  > BAL (67%)  > UV/H2O2 (43%)  > AOP contact chamber (19%)  > UF(0%), with the treatment train integrating two AOPs and granular biofiltration with activated carbon (O3/H2O2 + BAC + UV254/H2O2) showing superior performance with a 99% abatement in total micropollutants. No ecotoxicologically-positive response was generally observed for any of the effluent samples from the tested trains, even when pre-concentration factors up to 100-1000 times were employed to increase the sensitivity of the bioassay methods.

Carbonic anhydrase integrated into a multimarker approach for the detection of the stress status induced by pollution exposure in Mytilus galloprovincialis: A field case study.

The work was addressed to study the sensitivity of the enzyme carbonic anhydrase (CA) to chemical pollution in the hepatopancreas of the bioindicator organism Mytilus galloprovincialis in the context of a multimarker approach in view of ecotoxicological biomonitoring and assessment application. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two areas of interest: Augusta-Melilli-Priolo, an heavy polluted industrial site (eastern Sicily, Italy), and Brucoli (eastern Sicily, Italy) an area not affected by any contamination and selected as a reference site. Mussels in Augusta presented a significant increase in the digestive gland CA activity and gene expression compared to the animals caged in the control site of Brucoli. The CA response in animals from the polluted site was paralleled by proliferation/increase in the size of lysosomes, as assessed by Lysosensor green charged cells, induction of metallothionein, up-regulation of hif-α (hypoxia-inducible factor), metabolic changes associated with protein metabolism, and changes in the condition factor. Biological responses data were integrated with information about sediment chemical analysis and metal residue concentration in animal soft tissues. In conclusion, obtained results highlighted the induction of CAs in the hepatopancreas of Mytilus galloprovincialis following to pollution exposure, and demonstrated its suitability to be integrated into a multimarker approach for the detection and characterization of the stress status induced by pollution exposure in this bioindicator organism.

Antioxidant and oxidative stress related responses in the Mediterranean land snail Cantareus apertus exposed to the carbamate pesticide Carbaryl.

The aim of the present work was to study the alterations of the antioxidant defenses and the overall susceptibility to oxidative stress of the terrestrial snail Cantareus apertus exposed to the carbamate pesticide Carbaryl at a low environmentally realistic concentration. The animals were exposed to Lactuca sativa soaked for 1h in 1µM Carbaryl. The temporal dynamics of the responses was assessed by measurements at 3, 7 and 14days of exposure. C. apertus exposed to Carbaryl activates a number of enzymatic antioxidant responses, represented by the early induction of catalase, glutathione peroxidase, glutathione reductase, followed by a delayed induction of superoxide dismutase. Concomitantly, a derangement of the total oxyradical scavenging of the tissues was observed, suggesting an overall impairment of the tissue capability to neutralize ROS probably resulting from the overall negative balance between enzymatic antioxidant defense capability and oxidative stress intensity. This negative balance exposed the animals to the risk of oxidative stress damages including genotoxic damage. Compared to acetylcholinesterase inhibition, the antioxidant responses developed to Carbaryl exposure at the low concentration utilized showed a greater percentage variation in exposed organisms. The results pointed out the high sensitivity of the antioxidant and oxidative stress related responses to Carbaryl exposure at an environmental realistic concentration, demonstrating their usefulness in environmental monitoring and risk assessment. The study highlights also the usefulness of the terrestrial snail C. apertus as potential bioindicator species for assessing the risk of pesticide environmental contamination.

Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area.

The use of biomarkers to evaluate the biological effects of chemical pollutants in marine organisms represents a recent tool in the monitoring field responding to the need to detect and assess the effects of chemical contaminants on the biota. The aim of the present work was the field application of the integrated use of acetylcholinesterase (AChE) and antioxidant enzymes (catalase--CAT, glutathione peroxidase--GSH-Px), for detecting the possible exposure/effect induced by chemical pollutants in native marine organisms from a coastal marine area, represented by Salento Peninsula (Italy), that shows a coastline of high environmental value, but under constant urban pressure, including agriculture activities, widely diffused in the whole hinterland. Eight sampling stations were chosen: four not urbanized areas considered "uncontaminated" controls and four clearly exposed to anthropogenic impact. The bioindicator species studied were a sessile invertebrate, Mytilus galloprovincialis, and a benthic teleost fish, Mullus barbatus.AChE activity in M. galloprovincialis revealed significant differences among places; the minimum values observed (3.9+/-1.8 nmolmin(-1)mg(-1)) was about 50% reduced with respect to the maximum found (11.4+/-0.9 nmolmin(-1)mg(-1)). The reduction in AChE activity observed in two control stations could be explained by the leaching of pesticides into the sea from the agricultural lands. Moreover, the inhibition of AChE activity by heavy metals besides pesticides, can also explain the reduction of the enzymatic activity observed in an industrialized and harbour area. In M. galloprovincialis AChE activity showed a significant inverse correlation with catalase activity but not with glutathione peroxidase that did not significantly change in animals sampled from the eight stations. Also in M. barbatus AChE activity showed significant differences among places; it was inversely correlated with liver GSH-Px activity, but not with catalase activity, that did not show any significantly variation in animals sampled in the different stations. In conclusion, the integrated use of AChE and antioxidant enzymes (catalase or glutathione peroxidase) in M. galloprovincialis and M. barbatus, two species living in different compartment of marine coastal ecosystem, can find a useful application within the framework of marine coastal environment monitoring programs for detecting the possible exposure/effect induced by chemical pollutants, including pesticides, on living marine organisms.

Metallothionein induction in the coelomic fluid of the earthworm Lumbricus terrestris following heavy metal exposure: a short report.

Earthworms are useful bioindicator organisms for soil biomonitoring. Recently the use of pollution biomarkers in earthworms has been increasingly investigated for soil monitoring and assessment. Earthworm coelomic fluid is particularly interesting from a toxicological perspective, because it is responsible for pollutant disposition and tissue distribution to the whole organism. The aim of the present work was to study the effect of heavy metal exposure on metallothionein (Mt) induction in the coelomic fluid of Lumbricus terrestris in view of future use as sensitive biomarker suitable for application to metal polluted soil monitoring and assessment. L. terrestris coelomic fluid showed a detectable Mt concentration of about 4.0 ± 0.6 µg/mL (mean ± SEM, n = 10) in basal physiological condition. When the animals were exposed to CuSO4 or CdCl2 or to a mixture of the two metals in OECD soils for 72 h, the Mt specific concentration significantly (P < 0.001) increased. The Mt response in the coelomic fluid perfectly reflected the commonly used Mt response in the whole organism when the two responses were compared on the same specimens. These findings indicate the suitability of Mt determination in L. terrestris coelomic fluid as a sensitive biomarker for application to metal polluted soil monitoring and assessment.

Integrated biomarker analysis in the earthworm Lumbricus terrestris: application to the monitoring of soil heavy metal pollution.

As recently recognized exposure and effect assessment of soil contaminants on soil biota is necessary for decision-making related to ecosystem services and habitat protection, establishment of remediation procedures, or pollution monitoring programs. Therefore, biological approaches to soil monitoring, such as the measurement of biomarkers in soil bioindicator organisms, have recently received increasing attention. The aim of the present work was to assess the performance of a suite of cellular and biochemical biomarkers in native earthworms (Lumbricus terrestris) sampled in heavy metal contaminated sites in view of the validation of this biomarker approach in soil monitoring and assessment. Besides well known and standardized biomarkers such as lysosomal membrane stability, metallothionein tissue concentration and acetylcholinesterase activity, novel potential biomarkers such as changes in blood hemoglobin concentration and granulocyte morphometric alterations were analyzed. Both univariate and multivariate (PCA) statistical analysis applied to the data set revealed that the integrated multi-marker approach in native L. terrestris under field conditions produces a sensitive and cost-effective assessment of heavy metal soil pollution, which could be incorporated as a descriptor of environmental status in future soil biomonitoring programmes.

Biomarker response in the earthworm Lumbricus terrestris exposed to chemical pollutants.

Earthworms are important organisms for the soil ecosystem. They are sensitive to toxic chemicals and represent useful bioindicator organisms for soil biomonitoring. Recently the use of biomarkers in earthworms has been increasingly investigated for soil monitoring and assessment purpose. The aim of the preset paper was to analyze the pollutant-induced response of a suite of cellular and biochemical biomarkers in the earthworm Lumbricus terrestris exposed to copper sulphate or methiocarb in OECD soil at the maximal concentrations recommended in agriculture. These responses were compared to lifecycle parameters such as survival, growth and reproduction. Granulocyte morphometric alteration, lysosomal membrane stability, metallothionein concentration, and acetylcholinesterase activity were considered. In either copper sulphate or methiocarb exposure conditions the mean percentage variation of the pollutant-induced molecular and cellular biomarkers was consistent with the whole organism end-point responses. In particular pollutant-induced granulocyte enlargement, detected in either copper sulphate or methiocarb exposed organisms, showed to be a potential general biomarker that may be directly linked to organism health. Compared to the other biological responses to pollutants, it showed high sensitivity to pollutant exposure suggesting its possible applications as a sensitive, simple, and quick general biomarker for monitoring and assessment applications.

Effect of the daily ingestion of a purified anthocyanin extract from grape skin on rat serum antioxidant capacity.

The aim of this work was to study the effect of the daily ingestion of a purified anthocyanin extract from red grape skin on rat serum antioxidant capacity (ORAC) and its safety for the intestinal epithelium. The study was carried out in rats orally administered with the extract for 10 days in either normal physiological conditions or exposed to a pro-oxidant chemical (CCl(4)). The oral administration of the extract significantly (P<0.05) enhanced the ORAC value of the deproteinised serum of about 50 % after 10 days of ingestion. Anthocyanin administration was also able to reverse completely the decrease in the serum ORAC activity induced by the CCl(4) treatment. Experiments with Ussing chamber mounted intestine allowed to exclude any toxicity of the extract for the intestinal epithelium. In conclusion, our results demonstrate that the purified anthocyanin extract from red grape skin enhances the total antioxidant capacity of the serum in either normal physiological condition or during oxidative stress induction, revealing a protective role against the decrease in the serum antioxidant capacity induced by a pro-oxidant compound.

The Na+-K+-2Cl- cotransporter and the osmotic stress response in a model salt transport epithelium.

Epithelia are physiologically exposed to osmotic stress resulting in alteration of cell volume in several aspects of their functioning; therefore, the activation of 'emergency' systems of rapid cell volume regulation is fundamental in their physiology. In this review, the physiological response to osmotic stress, particularly hypertonic stress, was described in a salt-transporting epithelium, the intestine of the euryhaline teleost European eel. This epithelium is physiologically exposed to changes in extracellular osmolarity and represents a good physiological model for functional studies on cellular volume regulation, permitting the study of volume regulated ion transport mechanisms in a native tissue. An absorptive form of the cotransporter, homologue of the renal NKCC2, localized on the apical membrane, was found in the intestine of the euryhaline teleost European eel. This cotransporter accounts for the luminal uptake of Cl-; it operates in series with a basolateral Cl- conductance and presumably a basolateral electroneutral KCl cotransport and in parallel with a luminal K+ conductance. The ion transport model described for eel intestine, based on the operation of an absorptive luminal Na+-K+-2Cl-, is basically the same as the model that has been proposed for the thick ascending limb (cTAL) of the mammalian renal cortex. This paper focuses on the role of Na+-K+-2Cl- cotransport in the responses to hypertonic stress in the eel intestine and the role of cytoskeleton (either actin-based or tubulin based) is discussed.

Hypotonicity induced K+ and anion conductive pathways activation in eel intestinal epithelium.

Control of cell volume is a fundamental and highly conserved physiological mechanism, essential for survival under varying environmental and metabolic conditions. Epithelia (such as intestine, renal tubule, gallbladder and gills) are tissues physiologically exposed to osmotic stress. Therefore, the activation of 'emergency' systems of rapid cell volume regulation is fundamental in their physiology. The aim of the present work was to study the physiological response to hypotonic stress in a salt-transporting epithelium, the intestine of the euryhaline teleost Anguilla anguilla. Eel intestinal epithelium, when symmetrically bathed with Ringer solution, develops a net Cl- current giving rise to a negative transepithelial potential at the basolateral side of the epithelium. The eel intestinal epithelium responded to a hypotonic challenge with a biphasic decrease in the transepithelial voltage (V(te)) and the short circuit current (I(sc)). This electrophysiological response correlated with a regulatory volume decrease (RVD) response, recorded by morphometrical measurement of the epithelium height. Changes in the transepithelial resistance were also observed following the hypotonicity exposure. The electrogenic V(te) and I(sc) responses to hypotonicity resulted from the activation of different K+ and anion conductive pathways on the apical and basolateral membranes of the epithelium: (a) iberiotoxin-sensitive K+ channels on the apical and basolateral membrane, (b) apamin-sensitive K+ channels mainly on the basolateral membrane, (c) DIDS-sensitive anion channels on the apical membrane. The functional integrity of the basal Cl- conductive pathway on the basolateral membrane is also required. The electrophysiological response to hypotonic stress was completely abolished by Ca2+ removal from the Ringer perfusing solution, but was not affected by depletion of intracellular Ca2+ stores by thapsigargin.

Cl- absorption in European eel intestine and its regulation.

The intestinal epithelium of the euryhaline teleost fish, Anguilla anguilla, absorbs Cl(-) transepithelially. This gives rise to a negative transepithelial potential at the basolateral side of the epithelium and to a measured short circuit current. Cl(-) absorption occurs via bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransport, localized on the luminal membrane. The cotransport operates in parallel with a luminal K(+) conductance that recycles the ion into the lumen. Cl(-) leaves the cell across the basolateral membrane by way of Cl(-) conductance and presumably via a KCl cotransport. The driving force for this process is provided by the electrochemical sodium gradient across the plasma membrane, generated and maintained by the basolateral Na(+)-K(+)-ATPase. The resulting NaCl absorption process is active and enables marine fish to take up water, thereby compensating for water that was lost passively from the body. Fresh water acclimatized eel also absorb Cl(-) actively, although in smaller quantities, utilizing the same ion transport mechanisms as marine eels. This mechanism is basically the same as the model proposed for the thick ascending limb (cTAL). Cl(-) absorption is regulated by a number of cellular factors, such as HCO(3) (-), pH, Ca(2+), cyclic nucleotides, and cytoskeletal elements. It is sensitive to osmotic stress, and therefore is a good physiological model to study ion transport mechanisms that are activated when osmotic stress induces cell volume regulation. The activation of these various ion transport pathways is dependent on cellular transduction mechanisms in which phosphorylation events (mainly by PKC and MLCK for the hypertonic response) and cytoskeletal elements, either microfilaments or microtubules, seem to play key roles.

Bicarbonate absorption in eel intestine: evidence for the presence of membrane-bound carbonic anhydrase on the brush border membranes of the enterocyte.

Bicarbonate absorptive fluxes through the isolated intestine of the European eel (Anguilla anguilla) were evaluated by the pH-stat method under short-circuited conditions. It was found that bicarbonate absorptive flux was dependent on the luminal Na+ and was inhibited by luminal 4-acetamido-4' stilbene-2-2' disulfonic acid (SITS; 2.5 x 10(-4) M) and luminal acetazolamide (10(-4) M), while luminal amiloride (1 mM) was without effect. Furthermore, by using brush border membrane vesicles (BBMV) isolated from eel intestine, the existence of two carbonic anhydrase (CA) isoforms, one tightly associated to the brush border membrane (BBM) and the other soluble in the cytosol, was demonstrated. The membrane-bound CA differs from the cytoplasmic isoform in that 1) it is relatively resistant to treatment with 0.045% lauryl sulfate sodium salt (SDS); 2) it is less inhibitable by ethoxzolamide and sulfanilamide; and 3) its Kmapp is significantly lower than that of the cytoplasmic isoform. These results suggest that a BBM-bound CA isozyme would play an important role in bicarbonate absorption from the lumen, facilitating the HCO3- transfer through the luminal membrane of the eel enterocyte most likely via a Na+ (HCO3-) or (OH-) cotransport system.

Hypertonicity stimulates Cl(-) transport in the intestine of fresh water acclimated eel, Anguilla anguilla.

Eel intestinal epithelium when bathed symmetrically with normal Ringer solution develops a net Cl(-) current (short circuit current, Isc) giving rise to a negative transepithelial potential (Vt) at the basolateral side of the epithelium, lower in fresh-water (FW)-acclimated animals with respect to sea-water (SW). The aim of the present work was to study the cell response to hypertonic stress of FW eel intestinal epithelium in relation to Cl(-) absorption. The hypertonicity of the external bathing solutions produced first a transient increase of Vt and Isc, then followed (after 10-15 min) by a gradual and sustained increase which reached the maximum value after 40-60 min. The morphometric analysis of the intestine revealed the shrinkage of the cells after 5 min hypertonicity exposure, and then a regulatory volume increase (RVI) response, which parallels the gradual and sustained increase in the electrophysiological parameters. This last phase is inhibited by drugs known to block Cl(-) absorption in eel intestine, such as luminal bumetanide (10 microM), specific inhibitor of Na(+)-K(+)-2Cl(-) cotransport, or basolateral NPPB (0.5 mM), dichloro-DPC (0.5 mM), inhibitors of basolateral Cl(-) conductance. Serosal dimethyl-amiloride (100 microM), specific inhibitor of the Na(+)/H(+) antiport, was ineffective on the hyperosmotic response. Bicarbonate revealed a crucial role as a modulator of hypertonicity response, since in bicarbonate-free conditions or in the presence of serosal 0.25 mM SITS, blocker of HCO(3)(-) transport systems, the Isc response to hypertonicity was lost. In nominally Ca(2+)-free conditions the Isc response to hypertonicity was abolished. The same results were obtained by bilateral addition of 100 microM verapamil or 50 microM nifedipine or 1 mM lanthanum, known Ca(2+) channel blockers, indicating that extracellular Ca(2+) plays a key role for the activation of Cl(-) current in the response to hypertonic stress. The data show that in the eel intestinal epithelium the hypertonicity of the external medium affects cell volume which in turn might represent the signal to increase the rate of Cl(-) transport. This response is sustained by the activation of the luminal Na(+)-K(+)-2Cl(-) cotransporter and the functionality of basolateral Cl(-) channels.

Protective effects of prostaglandins in the isolated gastric mucosa of the eel, Anguilla anguilla.

The protective effect of endogenous prostaglandins on the fish gastric mucosa was evaluated by studying the effect of indomethacin and aspirin, known cyclooxigenase inhibitors, on the mucosal ulceration in the isolated gastric sacs of Anguilla anguilla. Gastric sacs devoid of muscle layers were incubated in the presence of indomethacin (10(-4) mol x l(-1)) or aspirin (10(-4) mol x l(-1)) in different experimental conditions. Both the antiinflammatory drugs produced ulcers, but the effects were more severe in the presence of histamine and in the absence of HCO3- in the incubation bath. The effects of prostaglandin E2 (PGE2) on acid secretion rate (J(H)) and on alkaline secretion rate (J(OH)) were evaluated (with the aid of the pH stat method) in isolated gastric mucosa mounted in Ussing chambers. We found that PGE2 (10(-8)-10(-5) mol x l(-1)) increased JH in a dose-dependent manner. In tissues pretreated with luminal omeprazole (10(-4) mol x l(-1)), PGE2 stimulated gastric alkaline secretion. It was nullified by serosal removal of HCO3- or Na+ and by serosal ouabain (10(-4) mol x l(-1)). These results suggested that prostaglandins also exert their protective effects in fish gastric mucosa. This protection seems partially due to a stimulation of exogenous HCO3- transport from the serosal to the mucosal side. It is likely that this transport is an active transcellular mechanism coupled to Na+ transport.

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla.

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.