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Previous observations showed that Trolox, a synthetic analog of vitamin E, widely used as antioxidant standard in a number of bioassays, can exert a pro-oxidant behavior at higher concentrations (>40 µM) on HeLa cells exposed for 24h, producing an isotonic cell shrinkage. A number of cellular events are known to be triggered by oxidative stress, including impairment of ion transport mechanisms and alteration of cell volume homeostasis. This work aims to investigate the possible mechanisms through which Trolox at high concentrations acts on cell volume homeostasis alteration in HeLa cells. The study was carried out by 1) spectrofluorimetric determination of intracellular oxidative stress in cells charged with CM-H2DCFDA, 2) morphometric analysis of cells observed under optical microscopy for cell volume determination, and 3) spectrofluorimetric and confocal analysis of cells charged with Annexin V/Propidium Iodide for apoptotic induction. HeLa cells exposed for 24h to high Trolox concentrations showed a significant dose-dependent isotonic reduction of cell volume associated to intracellular oxidative stress. The observed isotonic shrinkage was accompanied by apoptosis induction, as demonstrated by Annexin V/Propidium Iodide and was ascribed to Apoptotic Volume Decrease (AVD). The isotonic shrinkage appearance was demonstrated to occur early (after 2h) during the exposure to high Trolox concentrations. It was completely inhibited by pretreatment of the cells with a Cl- channel blocker SITS (0.5 mM). These results indicate that treatment of HeLa cells with high Trolox concentrations induces the activation of volume-regulatory Cl- channels, most likely by an increase in endogenous ROS production, which in turn is able to generate AVD.

Atmospheric particulate matter is a concern in most of the European towns because it has potential negative effects on human health (Davidson et al., 2005; Lelieveld et al., 2015). Although the toxic effects of PM have been correlated with some of its chemical and physical properties, the toxicity mechanisms are not yet fully known. Different in vitro toxicological tests are often necessary to characterise potential health effects and, often, it is found significant correlation only among a few of the possible tests. In addition, contrasting results could be obtained comparing in vitro tests with acellular assays like those used to determine oxidative potential (Steenhof et al., 2011; Van Den Heuvel et al., 2018). The aim of the work was to study the oxidative potential (OP) of PM10, determined with the acellular DTT assay, in relationship with its ecotoxicological and cytotoxicological potential. The study was carried out on aqueous extracts of 10 samples of airborne PM10 randomly selected among the samples collected between 16/09/2017 and 25/12/2017. Samples were collected using a low-volume (2.3 m3 /h) sampler (SWAM, Fai Instruments srl) on 47 mm quartz fibre filters (Whatman) pre-fired at 700 °C for 2 hours in order to reduce contamination. Samples were exposed for 24 hours (starting at midnight) at the Environmental-Climate Observatory of ISAC-CNR in Lecce (Southern Italy), regional station of the Global Atmosphere Watch (GAW) network, characterised as an urban background site (Cesari et al., 2018). The aqueous extraction was performed in an ultrasonic bath for 80 min using 10 ml Milli-Q water. The ecotoxicological potential of PM10 was assessed by the bioluminescence inhibition assay based on the Gram-negative non-pathogenic bacterium Vibrio fischeri (Microtox® test), which physiologically emits light as a results of its metabolic activity. The natural bioluminescence of V. fisheri is inhibited by the exposure to a number of chemical pollutants, including organic and inorganic compounds (Abbass et al., 2018). Different exposure times (5, 15, and 30 mins) were used and inhibition results, obtained with five repetitions, are reported as a net effect corrected using field blanks. The cytotoxicological potential of PM10 was assessed on the same extracts by the MTT assay on the cell line A549. The MTT assay is based on a colorimetric reaction dependent on mitochondrial respiration of the cells and indirectly allows assessing the cellular energy capacity of a cell (Stockert et al., 2012). The MTT assay was applied to the A549 cell line, representative of the alveolar type II pneumocytes of the human lung (Foster et al., 1998). Cell mortality after 24h exposition is evaluated, in relative terms, considering the net effect of PM10 using field blanks for correction. Six repetitions were done. The water-soluble fraction of PM10 was also used for the analysis of the OP, performed with the dithiothreitol assay (DTT), a surrogate for cellular antioxidants, which analyses the rate of DTT depletion catalysed by chemical species present in the PM (Chirizzi et al., 2017). An aliquot of the extracts was diluted with deionised water (1:4 factor). Diluted samples were incubated at 37 °C with DTT (0.1 mM) in 0.1 M potassium phosphate buffer at pH 7.4 for times varying from 5 to 90 min. At designated times (specifically at 5, 10, 15, 20, 30, 45, 60, and 90 min) an aliquot of incubation mixture was picked up and 10% trichloroacetic acid was added to stop the reaction. Then, this reaction mixture was mixed with a solution containing 10 mM DTNB. The concentration of the formed 5-mercapto-2-nitrobenzoic acid was measured by its optical density absorption at 412 nm using a Eon BioTek Microplate Spectrophotometer. The consumption of DTT over time was determined through the linear fitting of the absorbance with the time in which it was made the withdrawal. The DTT depletion rate was used to determine OP values as DTT-activity 21 normalized in terms of sampled air volume (OPV) or in terms of mass of collected aerosols (OPM). The OPV and OPM values were comparable with previous measurements in this area or in other Italian towns (Chirizzi et al., 2017). In all the 10 samples analysed a significant inhibition of the Vibrio fisheri bioluminescence was observed as a results of the exposure of bacteria to the undiluted extracts for 5, 15 and 30 min, suggesting the presence in the PM10 of components able to induce an ecotoxic effect. Four samples (samples n. 2,3,4, and 7) showed a % of inhibition ranging from 30% to 50%, ascribable to a slight toxic effect, while six samples (samples 1,5,6, 8,9, and 10) showed a % of inhibition above 50% after 30 min exposure, suggesting the presence of a toxic effect. The correlation analysis between the sampled mass and the Vibrio fisheri bioluminescence inhibition showed a significant positive correlation (p

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme involved in a number of physiological processes. Its sensitivity to chemical pollutants has been recently recognized. The work was aimed to study the CA sensitivity to metal exposure in the digestive gland of the model organism Mytilus galloprovincialis under in vitro, in vivo and in field conditions, with reference to the functional involvement of CA in the lysosomal response to pollutant exposure. The study was carried out by immunofluorescence confocal microscopy, electrometric assay of CA activity, spectrofluorimetric and confocal analysis of the lysosomal system. Under in vitro exposure to CdCl2 or CuCl2, CA activity was dose-dependently inhibited with an IC50 of 8.7 10-5M for copper and 1.1 10-3M for cadmium. On the other hand, under in vivo chronic exposure to CuCl2 (0.3 10-6 M) or CdCl2 (0.54 10-6 M) for 14 days, CA showed a significant upregulation, paralleled by the increased fluorescence of LysoSensor green charged cells, indicative of lysosome proliferation/increase in size. The metal induced lysosomal activation was prevented by the in vivo exposure to the specific CA inhibitor acetazolamide, demonstrating a key role of CA in the pollutant induced lysosomal activation. The response of CA upregulation paralleled by lysosomal activation was validated in the field by an active biomonitoring approach in coastal marine sites interested by metal contamination. In conclusion, data showed the complexity and multi-aspect nature of the CA sensitivity to metals, which can be CA inhibitors at higher concentrations and modulator of CA expression at lower concentrations typical of chronic exposure. In this condition CA upregulation can be functional to the prolonged increased requirement of H+ under lysosomal activation.

Branchiomma boholense is a sabellid polychaete considered highly invasive in the Mediterranean Sea as in few years it was able to expand its distribution range across the entire basin. Along the Italian coasts, this species has also been reported in venting areas near Castello Aragonese (Ischia Island) where CO2 emissions lower the seawater pH up to 6.00 values. The aim of this study was to test the physiological response of B. boholense to the decrease of seawater pH, measuring the activity of carbonic anhydrase (CA), a zinc metalloenzyme known to be affected by low pH conditions in other taxa. A transplant experiment employing the CO2 vents of Ischia was carried out. Worms were collected from a control pH area in the Mar Grande of Taranto and transplanted in two areas around Ischia, a control and a high pCO2 area. Three sites per area were utilized. Thirty individuals per site were exposed in plastic cages for 30 days. CA activity was measured electrometrically on the tissue homogenate of each specimen. Results of the ANOVA analysis on translocated worms showed no significant differences in CA activity either between individuals exposed to control and acidified conditions, nor among specimens transplanted in different sites within the same area. However, organisms exposed to the acidified area showed a decrease of their wet weight of about 20% compared to animals translocated in control sites. Moreover, CA activity was also investigated in specimens native from normal (Taranto) and high pCO2 environments (Ischia). The analysis of their wet weight revealed a marked decrease (by about 70%) in worms from the acidified area with respect to control animals. Interestingly, protein tissue content showed a significant (50%) increase in specimens native from the vent system with respect to the Taranto control area. Therefore, significant differences in CA activity (U/g wet weight) between specimens naturally exposed to normal and acidified conditions were also observed, with individuals living in acidified environments showing doubled values of enzymatic activity. The results of this study demonstrated that B. boholense is inclined to maintain a great homeostatic capacity when exposed to low pH. However, a marked decrease in body weight was observed under chronic exposure to high pCO2, suggesting the need of further studies to understand if the maintenance of a homeostatic balance come at the expense of other energy demanding physiological processes

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme involved in a number of physiological processes including respiration, pH homeostasis, electrolyte transport, calcification, biosynthetic reactions. The aim of this work was to explore a novel functional role of CA in animals: its involvement in the lysosomal system response to metal exposure. Mytilus galloprovincialis digestive gland was used as experimental model. The in vivo exposure of mussels to CdCl2 was able to evoke the induction of digestive gland CA. This response was paralleled by the activation of the lysosomal compartment (proliferation and/or increase in size of lysosomes), as assessed on LysoSensor Green marked cells by confocal microscopy and spectrofluorimetric analysis. When mussels were in vivo exposed to acetazolamide, specific CA inhibitor, the acidification of the lysosomal compartment was prevented. Moreover, during the co-exposure to cadmium and acetazolamide, the cadmium induced activation of the lysosomal compartment was completely inhibited. In conclusion, our results demonstrated in vivo the physiological contribution of CA to the acidification of the lysosomal compartment in digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction following cadmium exposure could physiologically sustain a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation.

Recently a dual behavior (antioxidant vs pro-oxidant) of some bioactive molecules has emerged. Trolox, a synthetic analog of vitamin E, widely used as antioxidant standard in a number of bioassays, in the presence of peroxidase/H2O2 is known to induce lipid peroxidation. The aim of the present study was to investigate the antioxidant/pro-oxidant properties of Trolox in HeLa cells in relationship to cell volume homeostasis alteration and cytotoxicity. The study was carried out by spectrofluorimetric analysis of cells charged with the ROS-sensitive probe CM-H2DCFDA, morphometric analysis of the cells and MTT test. When the cells were exposed (24h) to increasing Trolox concentrations (from 2.5 to 160 µM) and then charged with CM-H2DCFDA, an antioxidant effect on the endogenous ROS production was observed at low concentrations, but a marked pro-oxidant behavior was evident at higher concentrations. The same dose-dependent dual behavior was observed also in the presence of an exogenous oxidative challenge (H2O2). At higher concentrations Trolox was able to induce a dose-dependent isotonic reduction of cell size in parallel to a reduction of cell vitality. In conclusion, our study demonstrated the dose-dependent antioxidant/pro-oxidant behavior of Trolox in HeLa cells. The pro-oxidant effect was associated to an isotonic cell shrinkage, probable early signal of an apoptotic process ascribable to Apoptotic Volume Decrease, suggesting an alteration effect on the membrane transport mechanisms involved in cell volume homeostasis

Branchiomma bairdi is a sabellid polychaete native of the Caribbean Sea and recently introduced in the Mediterranean Sea where the species’s range has rapidly expanded as a consequence of its ability to colonize different environments and substrates. The species has also been reported around Ischia (Naples, Italy), a volcanic Island characterized by numerous submerged CO2 vent systems. In this regard the species occurs at the Castello vent system where intense CO2 emissions along the rocky reef lower the seawater pH up to mean values >7.4. In order to investigate the physiological response of B. bairdi to seawater acidification, worms were collected from the Mar Grande of Taranto, a non-acidified area in the south of Italy, and transported to Ischia in order to perform a transplant experiment into naturally acidified conditions. Three acidified plots were selected around the Castello vent’s area (A4, A5 on the south side; A6 on the north side), and three plots on a control area located at S. Pietro point (mean pH 8.12), approx 4 km from the Castello (C1, C2, C3). Thirty individuals per plot (90 per treatment) were exposed inside plastic cages, to both acidified and control conditions for 30 days. Cages were monitored and pH and temperature were measured in each plot approx. every four days until the end of the experiment. In addition, some individuals sampled both in Taranto and the Castello acidified area were frozen immediately after collection and used as a background condition test. The physiological response of B. bairdi to the acidified treatment was inferred measuring the activity of carbonic anhydrase (CA) a zinc metalloenzyme involved in a wide variety of physiological processes in animals. CA enzymatic activity was measured by electrometric method on the whole body homogenate of the species. CA activity data from the in vivo exposure experiment were analyzed by two-way ANOVA. No significant differences (p>0.05) in CA activity between acidified and control treatment were observed both in the transplanted and background worms, suggesting that this invasive species shows high physiological plasticity and it is well acclimatized to the acidified conditions of the Castello vent system.

The gastrointestinal tract is exposed to a number of pro-oxidant conditions and several gastrointestinal diseases are related to oxidative stress. The preservation of the gastrointestinal redox balance is of pivotal importance for the health status of the organism. It depends on a dynamic system involving antioxidant enzymes, as well as non-enzymatic molecules some of which derive from the diet. This work focuses on the intestinal epithelium and its aim was to explore the intracellular ROS scavenging activity of intracellularly absorbed food antioxidants. Freshly isolated intestinal explants obtained from different experimental animal models were used. They retain the functional and morphological characteristics of the epithelium in vivo. In situ confocal detection on CM-H2DCFDA charged explants experimentally exposed to pro-oxidant conditions was applied. Phenolic phytochemicals, such as hydroxytirosol or anthocyanins, commonly found in the Mediterranean diet, were demonstrated in surface enterocytes to cross the plasma membrane and exert a direct intracellular antioxidant activity. The effect was dose and time dependent and was compared with Trolox, synthetic analog of vitamin E. In conclusion, the gastrointestinal epithelium represents the first interface for the intracellular ROS scavenging activity of food antioxidants, and as such it can benefit directly and earlier than other body tissues of the protective action of food antioxidants against pro-oxidant conditions.

Chloride transport is involved in a wide variety of cellular functions, including regulation of the membrane potential, cell volume, acid base balance, and epithelial fluid absorption and secretion. Alterations in chloride transport mechanisms are related to a number of physio-pathological conditions. The aim of this study was to investigate chloride transport across the plasma membrane of cervical cancer cell lines (HeLa), relying on the halide binding properties of a green (GFP) fluorescent protein variant (YFP) that, if halide complexed, decreases the basal fluorescence intensity. The fluorescence changes of HeLa cells expressing YFP was analyzed by confocal and spectrofluorometric analysis. Our results highlights the presence of a transmembrane halide transport through fluorescence quenching when the transfected cells were exposed to NaI, used as a surrogate ion, under isotonic conditions. This quenching was inhibited by bumetanide, a specific inhibitor of Na+-K+-2Cl- cotransporter, demonstrating the role of such transporter in intracellular halide absorption and allowing cellular fluorescence assay for a functional study of Na+-K+-2Cl-. The kinetic parameters of the transporter were quantified. The present work provides an alternative approach for the measurement and characterization of Cl- and halide transport in living cells expressing YFP-H148Q/I152L based on a non-invasive analysis method.

Colon epithelium is exposed to oxidative stress conditions via exogenous and endogenous routes and a number of colon diseases are associated with the imbalance of the cellular redox system. The aim of the present work was to study the intracellular antioxidant activity of red grape polyphenolic extracts on superficial enterocytes experimentally exposed to prooxidant conditions. In situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15min) was performed. First, the ability of the experimental set up to detect intracellular ROS scavenging activity by intracellularly absorbed antioxidants was demonstrated by a dose-response inhibition of the H2O2 induced intracellular probe fluorescence following pre-incubation with Trolox, synthetic analog of vitamin E. Then, the explants were incubated with grape skin polyphenolic extracts for 1 h producing a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. Results demonstrated that red grape polyphenolic extracts are able to exert an intracellular antioxidant activity on superficial colonocytes, inducing a protection action against pro-oxidant condition. Extracts obtained from mature grapes showed an increased protective activity

Lysosomes are intracellular organelles with an acidic internal pH maintained by a V-ATPase H+ pump. Lysosomal reactions (alterations in lysosomal number and size and lysosomal destabilization) are involved in a number of physiological responses, including xenobiotic and heavy metal sequestration and detoxification. The aim of the study was to investigate the role of carbonic anhydrase, an ubiquitous enzyme catalyzing the hydration of CO2 to HCO2- and H+, in the lysosomal response to heavy metal exposure by using mussel (Mytilus galloprovincialis) digestive cells, characterized by a well developed lysosomal system, as experimental model. CdCl2 was used as reference toxicant. A novel microplate fluorimetric assay was developed on digestive cells in suspension, charged with the fluorescent acidotropic probe Lysosensor™ Green DND-189, for quantitating the number of lysosomes and their pH. In parallel, single cell analysis was performed by confocal microscopy. Digestive cells from Cd exposed animals showed a time and dose dependent increase of the probe fluorescence, suggesting a proliferation of the lysosomal compartment and decrease of the internal pH. This was confirmed by single cell confocal analysis. In animals in vivo exposed to acetazolamide, specific inhibitor of carbonic anhydrase, the metal induced fluorescence increase was dramatically inhibited. In conclusion, results indicate a key role of carbonic anhydrase in the lysosomal responses to metal exposure.

Biomarkers of chemical contamination were measured in M. galloprovincialis sampled in Mar Piccolo of Taranto in order to evaluate the potential ecotoxicological risk in this area exposed to industrial and urban impact. The analysis of a new potential biomarker, the enzymatic activity of carbonic anhydrase, was included in the biomarkers battery utilized and results obtained suggest its applicability in a multimarker approach.

Aim: Carbonic anhydrase is a zinc metalloenzyme catalysing the reversible hydration of CO2. Its activity is virtually ubiquitous in nature and is involved in a wide variety of physiological processes. The aim of the present work was to study the in vitro and in vivo sensitivity of CA to chemical pollutants in Mytilus galloprovincialis, widely used as sentinel organism in marine pollution biomonitoring, with the aim to develop a new biomarker of exposure/effect to chemical contamination. Methods: CA activity was measured applying a simple and low cost electrometric method. Briefly, enzymatic activity units were calculated from the rate of H+ production in the reaction mixture (where CO2 as substrate was present) against a blank containing the specific CA inhibitor acetazolamide. Results: As first step, we demonstrated the presence of a significant CA activity in M. galloprovincialis tissues and its sensitivity to in vitro exposure to several heavy metals. As second step, we demonstrated a significant tissue-specific alteration of CA activity following in vivo Cadmium exposure, with a significant increase in the digestive gland. As third step, immunoprecipitation and western blot analysis on mussel digestive gland revealed a protein that immunoreacts with the antibody at a molecular mass of approximate 42 kDa (the sequencing analysis is in progress). Interestingly the 42 KDa band, ascribed to CA, appeared strongly increased in the digestive gland of cadmium exposed mussel (14 day exposure), thus leading to an induction of CA expression in accordance with the observed enzymatic activity increase. As final step, the laboratory resulted was validated with field experiments. Conclusion: Obtained results represent a good starting point for future potential biomarker application of the CA activity in the sentinel organism M. galloprovincialis.

Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc metalloenzyme catalysing the reversible hydration of CO2. Its activity is virtually ubiquitous in nature and is envolved in a wide variety of physiological processes. Until today, fourteen CA isozymes (CA I-XIV) were characterised in animals, plants and certain bacteria. Pollutants like heavy metals are considered strong CA activity inhibitors, however few works have studied the sensitivity of CA from aquatic organisms to cadmium and comparatively much less studies on invertebrates are available. In Mytilus galloprovincialis (widely used in marine pollution monitoring programmes as sentinel organisms), we previously demonstrated a significant tissue-specific alteration of CA activity following in vivo cadmium (Cd) exposure, with a significant increase in the digestive gland. In order to establish if protein expression was involved in the observed metal activation of the enzymatic activity, in the present work immuno-precipitation and western blot analysis were applied to the tissue extract of control and Cd exposed mussels. Mussels were exposed to 1.8 µM CdCl2 for 14 days. Digestive gland CA activity was measured applying a simple and low cost electrometric method (Caricato et al., ESCPB Congress, 2003). Immunoprecipitation and western blot analysis (using the polyclonal anti-Carbonic Anhydrase II antibody) revealed in mussel digestive gland a protein that immunoreacts with the antibody at a molecular mass of approximate 42 kDa (the sequencing analysis is in progress). 42 kDa corresponds to the molecular weight of native CA VI (Parkkila S. et al., 1997), whose presence in invertebrates has never been revealed before. Interestingly the 42 KDa band, ascribed to CA, appeared strongly increased in the digestive gland of mussel after 14 day exposure, thus leading to a induction of CA expression in accordance with the observed enzymatic activity increase. To our knowledge this is the first time that Cd inducible CA activity and expression are reported. In conclusion, obtained results represent a good starting point for future potential biomarker application of the CA activity in the sentinel organism M. galloprovincialis.

Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc metalloenzyme catalysing the reversible hydration of CO2. Its activity is virtually ubiquitous in nature and is envolved in a wide variety of physiological processes. Until today, fourteen CA isozymes (CA I-XIV) were characterised in animals, plants and certain bacteria. Pollutants like heavy metals are considered strong CA activity inhibitors, however few works have studied the sensitivity of CA from aquatic organisms to cadmium and comparatively much less studies on invertebrates are available. In Mytilus galloprovincialis (widely used in marine pollution monitoring programmes as sentinel organisms), we previously demonstrated a significant tissue-specific alteration of CA activity following in vivo cadmium (Cd) exposure, with a significant increase in the digestive gland. In order to establish if protein expression was involved in the observed metal activation of the enzymatic activity, in the present work immuno-precipitation and western blot analysis were applied to the tissue extract of control and Cd exposed mussels. Mussels were exposed to 1.8 µM CdCl2 for 14 days. Digestive gland CA activity was measured applying a simple and low cost electrometric method (Caricato et al., ESCPB Congress, 2003). Immunoprecipitation and western blot analysis (using the polyclonal anti-Carbonic Anhydrase II antibody) revealed in mussel digestive gland a protein that immunoreacts with the antibody at a molecular mass of approximate 42 kDa (the sequencing analysis is in progress). 42 kDa corresponds to the molecular weight of native CA VI (Parkkila S. et al., 1997), whose presence in invertebrates has never been revealed before. Interestingly the 42 KDa band, ascribed to CA, appeared strongly increased in the digestive gland of mussel after 14 day exposure, thus leading to a induction of CA expression in accordance with the observed enzymatic activity increase. To our knowledge this is the first time that Cd inducible CA activity and expression are reported. In conclusion, obtained results represent a good starting point for future potential biomarker application of the CA activity in the sentinel organism M. galloprovincialis.

As specific molecular target of organophosphate and carbammate pesticides, acetylcholinesterase activity and its inhibition has been early recognized to be a human biological marker of pesticide poisoning, but in the last two decades the interest for acetylcholinesterase has increased a lot in the ecotoxicological field; its determination has become a tool for monitoring environmental contamination and organismal exposure to those compounds in the biota. Furthermore its use in different phyla, either vertebrates or invertebrates makes acetylcholinesterase a particularly versatile biomarker that can be used to investigate pollutant effects at many trophic levels and in different environments.

INTRODUCTION: Bivalve mollusks and specially mussels are widely used as sentinel organisms in marine environmental quality assessment (Goldberg et al., 1978). Pollutant exposure biological responses (i.e. biomarkers) are increasingly investigated in mussel tissues and their measurements largely used in marine environmental monitoring. Mussels emocytes play very important role in mollusk physiology, mainly in the internal defense, and used for general biomarker determination such as lysosomal membrane stability. The aim of the present work was to investigate possible morphological alterations in Mytilus galloprovincialis haemocytes, in particular granulocytes, which represent the main defense system of the organisms, following pollutant exposure either in laboratory or in field conditions. MATERIAL AND METHODS: As regards laboratory exposure, an homogeneous stock of M. galloprovincialis was divided in two groups: the control one and the other one exposed to cadmium chloride (600 g l-1) for 14 days. At time 0, 3, 7 and 14 days haemolymph samples were collected. As regards field exposure an homogeneous stock of M. galloprovincialis was divided in two caged groups, translocated in a reference and in a polluted sites respectively. After 30 days exposure haemolymph samples were collected from both groups. M. galloprovincialis haemolymph samples were stained with Diff Quick (Dade Behring, Newark -USA), a rapid staining solution kit, to date utilized in the diagnostic field (Jorundsson et al, 1999). Diff Quick allowed the staining of haemolymph cells, in particular granulocytes, whose morphology including cytoplasm, nucleus and acidophilic granules appeared clearly defined. Diff Quick stained granulocytes were investigated by optical microscopy and image analysis. RESULTS AND DISCUSSION: Granulocytes from cadmium exposed mussels showed a significant increase of their surface, suggesting an activation of the granulocytes defence system in this organism after pollutant exposure. Moreover, a significant decrease of the perimeter/area ratio of granulocytes bidimentional images was observed, probably due to a cadmium induced reduction of the microfilament and microspine number. Interestingly, these findings were confirmed by results obtained with granulocytes from in field exposed mussels, which also showed a significant increase in their acidophilic granules. Results obtained suggest that the observed cytological alterations in mussel granulocytes can be proposed as potential biomarker of chemical stress exposure suitable to be used in a biomarker battery in marine environmental monitoring programmes. Moreover, the Diff Quick staining protocol proposed for mussel morphological alteration measurements, being rapid, sensitive, inexpensive and easy to be used, responds to the needs of the field biomonitoring application.

Carbonic anhydrase (CA) is a metalloenzyme catalizing the reversible idratation of CO2 in H+ and HCO3-. It is an ubiquitous enzyme in bacteria, plant and animal kingdoms, playing a fundamental role in a number of physiolgical processes. Previous studies demonstrated the sensitivity of carbonic anhydrase activity to dichlorodiphenyl-dichloro-ethane (DDT) exposure in birds [1] and to cadmium exposure in teleost [2]. The aim of the present work was to investigate the in vitro sensitivity of carbonic anhydrase activity to several organic and inorganic chemical compounds, in order to standardize a carbonic anhydrase based bioanalytical method available for monitoring environmental samples. Commercial available CA isozyme II from bovine erythrocytes was utilized for the in vitro bioassay. CA activity was determined by a modification of electrometric method previously described by Wilbur and Anderson (J. Biol. Chem., 257: 12056-12059, 1948): briefly CA activity units were calculated from the rate of H+ production in the reaction mixture (where CO2 as substrate was present) against a blank containing the specific CA inhibitor acetazolamide. [H+] variation in the reaction mixture was followed at 0°C using a Mettler Delta 350 pH-meter. In our experimental set up bovin CA activity was significantly inhibited by nanomolar concentration of heavy metals (Cd, Cu and Hg), organochlorate compounds and carbammate pesticides, showing a dose-response behaviour. Carbonic anhydrase in vitro bioassay can represent a novel tool for rapid and low cost understanding of the toxicity of environmental samples, of bioavailability of pollutants in evironmental matrices, and of their additive or synergistic biological effects when present in mixtures.

Acetylcholinesterase (AChE) is a key enzyme in the nervous system. It terminates nerve impulses by catalysing the hydrolysis of neurotransmitter acetylcholine. As a specific molecular target of organophosphate and carbamate pesticides, acetylcholinesterase activity and its inhibition has been early recognized to be a human biological marker of pesticide poisoning. Measurement of AChE inhibition has been increasingly used in the last two decades as a biomarker of effect on nervous system following exposure to organophosphate and carbamate pesticides in occupational and environmental medicine. The success of this biomarker arises from the fact that it meets a number of characteristics necessary for the successful application of a biological response as biomarker in human biomonitoring: the response is easy to measure, it shows a dose-dependent behavior to pollutant exposure, it is sensitive, and it exhibits a link to health adverse effects. The aim of this work is to review and discuss the recent findings about acetylcholinesterase, including its sensitivity to other pollutants and the expression of different splice variants. These insights open new perspective for the future use of this biomarker in environmental and occupational human health monitoring.

Heavy metals are known to in vitro inhibit carbonic anhydrase (CA) activity in a variety of organisms; however, little is known about their in vivo effects on the activity and the expression of this metalloenzyme. The aim of this work was to investigate the in vitro and in vivo sensitivity to cadmium of CA in the digestive gland of Mytilus galloprovincialis. CA activity and protein expression (apparent molecular mass of about 28 kDa) were demonstrated in mussel digestive gland for the first time. CA activity showed week sensitivity to in vitro cadmium exposure, while it was significantly increased (about 40%) following two weeks in vivo exposure. In parallel, CA protein expression appeared significantly enhanced as demonstrated by Western blotting. Laboratory experimental results were confirmed by a field experiment. Mussels exposed for 30 days to an impacted site showed a significant increase of the CA activity and protein expression with respect to animals exposed to the control site in parallel to the increase of the metallothionein tissutal concentration. In conclusion in the present work for the first time CA activity and protein expression have been demonstrated to be enhanced by the exposure to the trace element cadmium in animals.

The aim of this study was to test the effects of short- and long-term exposure to high pCO 2 on the invasive polychaete Branchiomma boholense (Grube, 1878), (Sabellidae), through the implementation of a transplant experiment at the CO 2 vents of the Castello Aragonese at the island of Ischia (Italy). Analysis of carbonic anhydrase (CA) activity, protein tissue content and morphometric characteristics were performed on transplanted individuals (short-term exposure) as well as on specimens resident to both normal and low pH/high pCO 2 environments (long-term exposure). Results obtained on transplanted worms showed no significant differences in CA activity between individuals exposed to control and acidified conditions, while a decrease in weight was observed under short-term acclimatization to both control and low pH, although at low pH the decrease was more pronounced (∼20%). As regard individuals living under chronic exposure to high pCO 2 , the morphometric results revealed a significantly lower (70%) wet weight of specimens from the vents with respect to animals living in high pH/low pCO 2 areas. Moreover, individuals living in the Castello vents showed doubled values of enzymatic activity and a significantly higher (50%) protein tissue content compared to specimens native from normal pH/low pCO 2 . The results of this study demonstrated that B. boholense is inclined to maintain a great homeostatic capacity when exposed to low pH, although likely at the energetic expense of other physiological processes such as growth, especially under chronic exposure to high pCO 2 .

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a hydrophilic analog of vitamin E, is known for its strong antioxidant activity, being a high radical scavenger of peroxyl and alkoxyl radicals. Under particular conditions, Trolox may also exhibit prooxidant properties. The present work aimed at studying the dual antioxidant/prooxidant behavior of Trolox over a wide range of concentrations (from 2.5 to 160 &micro, M) in HeLa cells. In particular, the study addressed the dose-dependent effects of Trolox on the oxidative cell status and vitality of HeLa cells, focusing on the potential role of the vitamin E analog in the induction of one of the first steps of the apoptotic process, Apoptotic Volume Decrease (AVD). In HeLa cells, Trolox showed significant antioxidant activity, expressed as the ability to reduce the endogenous ROS production detected by the ROS-sensitive probe 5-(and-6)-chloromethyl-2&prime, 7&prime, dichlorodihydrofluorescein diacetate (CM-H2DCFDA), at low concentrations (range: 2.5&ndash, 15 &micro, M), but exerted a dose-dependent prooxidant effect at higher concentrations after 24 h exposure. The prooxidant effect was paralleled by the reduction in cell viability due to the induction of the apoptotic process. The dual behavior, antioxidant at lower concentrations and prooxidant at higher concentrations, was evident also earlier after 2 h incubation, and it was paralleled by the isotonic shrinkage of the cells, ascribed to AVD. The use of SITS, known Cl&minus, channel blocker, was able to completely inhibit the Trolox-induced isotonic cell shrinkage, demonstrating the involvement of the vitamin E analog in the alteration of cell volume homeostasis and, in turn, in the AVD induction. In conclusion, the study shed light on the concentration dependence of the Trolox antioxidant/prooxidant activity in HeLa cells and revealed its role in the induction of one of the first events of apoptosis, AVD, at high concentrations.

Environmental pollutants generate harmful conditions for living organisms, including humans. This accounts for the growing interest to early warning tools for detection of adverse biological responses to pollutants in both humans and wildlife. Molecular and cellular biomarkers of pollution meet this requirement. A pollution biomarker is defined as an alteration in a biological response occurring at molecular, cellular or physiological levels which can be related to exposure to or toxic effects of environmental chemicals.Pollution biomarkers have known a growing development in human and environmental biomonitoring representing a valuable tool for early pollutant exposure detection or early effect assessment (exposure/effect biomarkers).The review discusses the recent developments in the use of pollution biomarker in human and environmental biomonitoring and analyzes future perspectives in the application of this tool such as their potentiality for bridging human and environmental issued studies.

Long and short-term exposure to atmospheric particulate matter (PM) has detrimental effects on human health. The effective mechanisms leading to PM toxicity are still not fully understood, even if it is known that physical-chemical properties, strongly influenced by sources and atmospheric processes, are known to play an important role. In this work, PM10 samples were collected, at an urban background site in southern Italy, to determine cytotoxicity (using MTT test on A549 cells), genotoxicity (using the comet assay), and intracellular oxidative stress on A549 cells exposed for 24 h to aqueous extracts of PM10 samples. Organic carbon (OC) and elemental carbon (EC) content of PM10 and acellular determination of oxidative potential with DTT assay were performed to compare results of acellular and cellular biological assays. Cellular (OSGCV and MTTV) and acellular (OPDTTV) outcomes, normalized in volume, are well correlated (statistically significant results) with carbon content suggesting that combustion sources play an important role in determining cellular oxidative stress and cytotoxicity of PM10. Even if the number of data is limited, genotoxicity results are well correlated (Pearson r &gt, 0.95) with OSGCV and MTTV, and a weaker, but statistically significant correlation was observed with OPDTTV. OSGCV is well correlated with the cell mortality observed with the MTTV test and a lower, but still statistically significant correlation is observed between MTTV and OPDDTV. A statistically significant correlation was found between OPDTTV and OSGCV results. When the outcomes of the cellular and acellular assay are compared normalized in mass (i.e., intrinsic values), the correlations become significantly weaker suggesting that the different sources acting on the site produces particulate matter with different toxicological potential influencing differently the biological tests studied.

Background: Carbonic anhydrase is a metalloenzyme extensively present in nature. In animals, it is involved in several functions such as respiration, pH homeostasis, salt transport, metabolic reactions, calcification, bone resorption. In recent years, the CA wide distribution, relevance in several physiological processes, involvement in several pathological conditions, as well as sensitivity to chemical pollutants has driven the research on CA based biomarkers. Objective: The review is addressed to analyze the potential applicability of CA as biomarker in a variety of fields, from human health to environmental sciences. Results: The main studies in this field are analyzed and discussed in the present review exploring several areas of interest, from clinic to environmental monitoring. The review also details interesting research patents, which have yielded in the last years in this research field. Conclusion: In conclusion, the review highlights that CA is becoming a promising biomarker in several areas of interest and outlines that the research on CA based biomarkers have opened new perspective for translation of advances in basic science into innovative applications and patents.

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme, whose functions in animals span from respiration to pH homeostasis, electrolyte transport, calcification, and biosynthetic reactions. CA is sensitive to trace metals in a number of species. In mussels, a previous study demonstrated CA activity and protein expression to be enhanced in digestive gland by cadmium exposure. The aim of the present work was to investigate the functional meaning, if any, of this response. To this end the study addressed the possible involvement of CA in the lysosomal system response of digestive gland cells to metal exposure. The in vivo exposure to acetazolamide, specific CA inhibitor, significantly inhibited the acidification of the lysosomal compartment in the digestive gland cells charged with the acidotropic probe LysoSensor Green D-189, demonstrating in vivo the physiological contribution of CA to the acidification of the lysosomes. Under CdCl2 exposure, CA activity significantly increased in parallel to the increase of the fluorescence of LysoSensor Green charged cells, which is in turn indicative of proliferation and/or increase in size of lysosomes. Acetazolamide exposure was able to completely inhibit the cadmium induced Lysosensor fluorescence increase in digestive gland cells. In conclusion, our results demonstrated the functional role of CA in the lysosomal acidification of Mytilus galloprovincialis digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction could physiologically respond to a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation.

Colon is exposed to a number of prooxidant conditions and several colon diseases are associated with increased levels of reactive species. Polyphenols are the most abundant antioxidants in the diet, but to date no information is available about their absorption and potential intracellular antioxidant activity on colon epithelial cells. The work was addressed to study the intracellular antioxidant activity of red grape polyphenolic extracts on rat colon epithelium experimentally exposed to prooxidant conditions. The experimental model chosen was represented by freshly isolated colon explants, which closely resemble the functional, and morphological characteristics of the epithelium in vivo. The study was carried out by in situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15 min). The qualitative and quantitative polyphenolic composition of the extracts as well as their in vitro oxygen radical absorbing capacity (ORAC) was determined. The incubation of the explants with the polyphenolic extracts for 1 h produced a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. The results demonstrated the ability of red grape polyphenols to cross the plasma membrane and exert a direct intracellular antioxidant activity in surface colonocytes, inducing a protection against pro-oxidant conditions. The changes in the polyphenol composition due to ripening process was reflected in a more effective antioxidant protection.

The measurement of cellular and sub cellular responses to chemical contaminants (referred to as biomarkers) in living organisms represents a recent tool in environmental monitoring. It answers to the need to detect exposure and to assess effects of pollutants on biota. Biomarkers have recently become an integral component of environmental monitoring programmes of marine environments in several countries as a supplement to the commonly used contaminant monitoring. The review reports and analyzes new insights and perspectives in the biomarker approach, including its recent application to the detection of the impact of biological pollution in marine environment.

The aim of this work was to study spatial and temporal variation in biomarkers in autochthonous Mytilus galloprovincialis sampled inside and outside Mar Piccolo of Taranto, a typical polluted semi-enclosed basin of the Mediterranean Sea characterised by scarce hydrodynamism. Mar Piccolo of Taranto represents a site of Italian National Interest because of the high level of pollution. A battery of biomarkers (lysosomal destabilisation, catalase, metallothioneins, acetylcholinesterase, air survival) was applied to assess pollution-induced stress effects in authoctonous mussels. The responses were analysed comparatively in two different seasons, summer and winter, in order to assess possible changes in the pollutant-induced stress syndrome throughout the year. No significant difference inside and outside Mar Piccolo was observed for metallothioneins. By contrast, the dramatically decreased acetylcholinesterase values and strongly increased catalase activity in organisms taken from Mar Piccolo in winter indicate an increased risk of exposure to anticholinesterase compounds during this season. The results suggest the importance of temporal variability in biomarker responses throughout the year for monitoring possible seasonal changes in the pollutant-induced stress syndrome of organisms living in a certain environment and, in turn, more properly detecting changes in ecotoxicological risks.

The aim of the work was to evaluate the ecotoxicity of reclaimed agroindustrial wastewaters used for irrigation through ecotoxicological bioassays and biomarkers. The ecotoxicological monitoring was addressed on both treated wastewaters and irrigated soils. Wastewater biomonitoring was performed by the acute test with Daphnia magna, the Microtox® test, and a new in vitro patented method. Soil quality monitoring was performed by the acute and chronic tests with the earthworm Eisenia fetida and biomarker analysis, such as lysosomal membrane stability, general stress biomarker of chemical pollution, and metallothionein, specific biomarker of exposure to heavy metals. Overall the integrated ecotoxicological analysis excluded the presence of ecotoxicity both in the reclaimed waters resulting from tertiary treatment and in the irrigated soils. In particular, the analysis of metallothionein allowed to exclude the accumulation of bioavailable heavy metals in the soil. This study suggests the suitability of ecotoxicological methods for the biomonitoring of water and soil during the reclaimed wastewaters reuse for irrigation, contributing to improving the process of agricultural re-use of wastewater in terms of assessment of the toxicological safety of the waters for the environment, for traders and consumers.

Carbonic anhydrase (CA) (EC 4.2.1.1), a ubiquitous enzyme in bacteria, plant, and animal kingdoms, catalyses the reversible hydration of CO2 to produce H+ and HCO−3 using zinc as cofactor. CA plays a fundamental role in a number of physiological processes, such as respiration, ionic transport, acid–base regulation, and calcification. The aim of the present work was to investigate the sensitivity of this enzyme to heavy metals with a view to possible future applications of CA activity inhibition measurement in biomonitoring as either an in vitro bioassay or a biomarker.CAactivitywas determined by modifying a previously described electrometric method: briefly, CA activity units were calculated from the rate of H+ production in the reaction mixture (where CO2 was present as a substrate) against a blank containing the specific CA inhibitor acetazolamide. As regards the possible application as an in vitro bioassay, the sensitivity to heavy metals (cadmium, mercury, and copper) of the commercially available purified carbonic anhydrase (isozyme II) from bovine erythrocyte was tested in vitro. In our experimental set-up, bovine CA activity was significantly inhibited by micromolar concentrations of heavy metals, showing a dose–response behaviour.As regards the possible application as biomarkers, CAwas investigated in the filter-feeding Mytilus galloprovincialis, widely used in pollution-monitoring programmes as a sentinel organism. Following in vitro and in vivo exposure to 1.785μM cadmium chloride as a reference toxicant, mantle CA activity was significant inhibited. In conclusion, the sensitivity to chemical pollutants and low cost and simplicity of the assay method make CA activity measurement suitable for in vitro bioassay of the toxicity of environmental samples and for field biomarker applications in the sentinel organism M. galloprovincialis.

This work represents a pilot study for monitoring the potential toxicological risk of commercial relevant marine resources along the South coast of Italy by using biomarkers as complementary tool to chemical analysis. The attention was focused on the industrialized area of Salento peninsula, such as Taranto, that, in spite of the presence of the big industry (oil, metal industry), sustains activities related to the sea resources, such as fishery and mussel-culture. The study was carried out in fish, such as Mullus barbatus and Trachurus mediterraneus, two important fish species for the fishery in this area, and in mussels (Mytilus galloprovincialis). As control area S. Maria di Leuca, area of naturalistic interest, was chosen. In fish, liver metallothionein levels (specific index of exposure to heavy metals such as Hg, Cd, Cu, Zn) and brain and muscular acetylcholinesterase activity (specific index of exposure to organophosphate and carbamate pesticides) were measured. None of the two fish species showed significant differences in acetylcholinesterase activity and in pesticide trace level between the anthropogenic impact exposed site and the control group. On the other hand, metallothionein hepatic levels in Mullus barbatus were significantly increased in the organisms coming from Taranto with respect to the organisms coming from the control site, but chemical analysis, routinely performed on edible muscle for the evaluation of chemical quality of fish products, did not reveal high heavy metal concentration in the edible muscle of fish from Taranto. Mussels exposed for one month in the Mar Piccolo of Taranto, an important mussel farming area, showed increase in the level of catalase activity, an oxidative stress index, increase in the levels of metallothioneins and inhibition of acetylcholinesterase activity The need to integrate chemical analysis with the study of biological responses to pollutants (biomarkers) in marine organisms is discussed for a better comprehension of the impact of chemical contaminants on the sea and its resources

Background: The colon epithelium is physiologically exposed to osmotic stress, and the activation of cell volume regulation mechanisms is essential in colonocyte physiology. Moreover, colon is characterized by a high apoptotic rate of mature cells balancing the high division rate of stem cells. Aim: The aim of the present work was to investigate the main cell volume regulation mechanisms in rat colon surface colonocytes and their role in apoptosis. Methods: Cell volume changes were measured by light microscopy and video imaging on colon explants; apoptosis sign appearance was monitored by confocal microscopy on annexin V/propidium iodide labeled explants. Results: Superficial colonocytes showed a dynamic regulation of their cell volume during anisosmotic conditions with a Regulatory Volume Increase (RVI) response following hypertonic shrinkage and Regulatory Volume Decrease (RVD) response following hypotonic swelling. RVI was completely inhibited by bumetanide, while RVD was completely abolished by high K + or iberiotoxin treatment and by extracellular Ca 2+ removal. DIDS incubation was also able to affect the RVD response. When colon explants were exposed to H 2 O 2 as apoptotic inducer, colonocytes underwent an isotonic volume decrease ascribable to Apoptotic Volume Decrease (AVD) within about four hours of exposure. AVD was shown to precede annexin V positivity. It was also inhibited by high K + or iberiotoxin treatment. Interestingly, treatment with iberiotoxin significantly inhibited apoptosis progression. Conclusions: In rat superficial colonocytes K + efflux through high conductance Ca 2+ -activated K + channels (BK channels) was demonstrated to be the main mechanism of RVD and to plays also a crucial role in the AVD process and in the progression of apoptosis.