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 environmentalmedicine. 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 thiswork is to reviewand 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

Acetylcholinesterase (AChE) is a key enzyme in the nervous system, terminating nerve impulses by catalyzing the hydrolysis of neurotransmitter acetylcholine. AChE is the target site of inhibition by organophosphorus and carbamate pesticides. In particular, organophosphorous pesticides inhibit the enzyme activity by covalently phosphorylating the serine residue within the active site group. They irreversibly inhibit AChE, resulting in excessive accumulation of acetylcholine, leading to the hyperactivities and consequently paralysis of the neural and muscle system. Therefore, monitoring of AChE inhibition is widely used as a biomarker of organophosphorous and carbamate exposure either in aquatic or terrestrial environments. Recently, new insights are emerging in the use of AChE as biomarker in environmental biomonitoring. A number of important contaminants other than carbamate and organophosphorus pesticides have recently been shown to have anticholinesterase properties, including heavy metals, detergents, hydrocarbons, and herbicides. It is also worth noting that not only different compounds may reach levels of significance in terms of anticholinesterase effect, but, moreover, combinations of different chemical classes were shown to be highly synergistic in their ability to inhibit AChE activity. Moreover, evidence for additional function of AChE is recently emerging. Apart from its catalytic function in hydrolyzing acetylcholine, different forms of AChE have been shown to affect cell proliferation, differentiation, and responses to various stresses. In addition, the relationship among the antioxidant defence systems and the AChE inhibition is now emerging. These recent insights open a new future in biological monitoring and environmental assessment to this “old” biomarker. Furthermore, its use in different phyla, either vertebrates or invertebrates, makes AChE a particularly versatile biomarker that can be used to investigate pollutant effects in many trophic levels and in many different environments.

The aim of the presentworkwas 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 1 h in 1 μM Carbaryl. The temporal dynamics of the responses was assessed by measurements at 3, 7 and 14 days 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 tissueswas 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

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

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

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) is a zinc metalloenzyme catalysing the reversible hydration of CO2 to produce H+ and HCO3−. Its activity is virtually ubiquitous in nature. The review focuses on one interesting but less investigated aspect of the biochemistry of this metalloenzyme, encompassing several areas of interest from human health to environmental science: the relationships between carbonic anhydrase and heavy metals.

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. The review focuses on carbonic anhydrase, a ubiquitous metalloenzyme which plays key roles in a wide variety of physiological processes involving CO2 and HCO3-. In the last decade a number of studies have demonstrated the sensitivity of this enzyme to pollutants such as heavy metals and organic chemical pollutants in both humans and wildlife. The review analyses these studies and discusses the potentiality of this enzyme as novel biomarker in environmental monitoring and assessment.

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.

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.

In this review the authors summarize the present insight about cell volume regulation in the European eel (Anguilla anguilla), highlighting findings on the intestinal epithelium, which serves as one of the main osmoregulatory organs in the fish. This tissue is a useful model system for functional studies of epithelia that perform near-isosmotic fluid absorption and has specifically been used as a physiological model for the study of cell volume regulation in epithelia

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 Ca2+ removal. DIDS incubation was also able to affect the RVD response. When colon explants were exposed to H2O2 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 Ca2+-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

Observational data suggest that the white seabream Diplodus sargus, a sparid fish of economic and ecological relevance in the Mediterranean Sea, has included the invasive green alga Caulerpa racemosa in its diet. Here we adopted a chemoecological approach to study the trophic relationship between the fish and the exotic pest. We demonstrated that the red pigment caulerpin, the most abundant secondary metabolite of C. racemosa, enters food chains and accumulates in the fish tissues. General biological condition markers associated with fish health and reproductive development were measured and correlated with the caulerpin levels in the fish tissues. Significant correlations among caulerpin tissue load (determined by liquid chromatography-mass spectrometry analysis) and fish condition factor and hepatosomatic index were obtained, suggesting a possible detrimental effect of the dietary exposure to C. racemosa on D. sargus. Glutathione peroxidase and catalase activity were also significantly correlated with caulerpin concentrations in the liver, suggesting a possible interaction between algal metabolites and liver antioxidant mechanisms. Studies on the impact of invasive macroalgae on marine assemblages have been almost exclusively focused on the structural modification of benthic assemblages, through the alteration of the relative importance of some endemic species and the modification of habitat complexity. Here we propose a new mechanism by which invasive algae can impact marine systems, namely the entry of pest metabolites in food webs, with potential detrimental effects on the population dynamics of a single species, alteration of trophic webs and changes in the functioning of coastal ecosystems

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

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 (CCl4). 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 CCl4 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.

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 multimarker approach in native Lumbricus 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.

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 1h 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 aim of the present work was to study the response of a suite of cellular and biochemical markers in the terrestrial snail Cantareus apertus exposed to mercury in view of future use as sensitive tool suitable formercury polluted soil monitoring and assessment. Besides standardized biomarkers (metallothionein, acetylcholinesterase, and lysosomal membrane stability) novel cellular biomarkers on haemolymph cells were analyzed, including changes in the spread cells/round cells ratio and haemocyte morphometric alterations. The animals were exposed for 14 days to Lactuca sativa soaked for 1 h in HgCl2 solutions (0.5 e 1 μM). The temporal dynamics of the responses were assessed by measurements at 3, 7 and 14 days. Following exposure to HgCl2 a significant alteration in the relative frequencies of round cells and spread cellswas evident, with a time and dose-dependent increase of the frequencies of round cells with respect to spread cells. These changes were accompanied by cellular morphometric alterations. Concomitantly, a high correspondence between these cellular responses and metallothionein tissutal concentration, lysosomal membrane stability and inhibition of AChE was evident. The study highlights the usefulness of the terrestrial snail C. apertus as bioindicator organism for mercury pollution biomonitoring and, in particular, the use of haemocyte alterations as a suitable biomarker of pollutant effect to be included in a multibiomarker strategy.

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 g/mL (mean ± SEM, = 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, theMt specific concentration significantly ( < 0.001) increased.TheMt 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.

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 multiwalled 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.

The aim of this work was to assess the diffusive properties of poly(ethylene glycol) diacrylate (PEGDA)-based hydrogels, derived from low MW prepolymers, in view of potential biomedical applications. Several hydrogels were synthesized through UV irradiation of PEGDA solutions for different exposure times. Swelling measurements in distilled water were performed to estimate the yielded crosslink density, while swelling tests at 37 °C in selected media allowed to analyze the mesh size changes induced by various pH and ionic strength (IonS) conditions. The transport of glucose and insulin through thin hydrogel membranes was finally assessed in a modified Ussing chamber at physiological values of pH and IonS (7.4 and 150 mM, respectively). Results showed that the swelling was dependent on the IonS (with swelling reductions up to 20–30% for IonS increases in the range 0–300 mM) and, to a lesser extent, on the pH of the surrounding medium (with swelling increments of about 10% for increasing pH in the range 2.5–11). All hydrogels were also permeable to glucose and insulin, which displayed comparable diffusion coefficients (in the order of 10−6 cm2/s). Specific interactions between glucose and the polymer chains were evidenced by values of the partition coefficient higher than unity. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44380. © 2016 Wiley Periodicals, Inc.

High conductance Ca2+-activated K+ channels (BK channels) have previously been demonstrated in the eel intestine. They are specifically activated following a hypotonic stress and sustain Regulatory Volume Decrease (RVD). The aim of the present work was to address the possible role of these channels in the Apoptotic Volume Decrease (AVD) of isolated eel enterocytes, and the possible interaction between BK channels and the progression of apoptosis. The detection of apoptosis was performed by confocal microscopy and annexin V and propidium iodide labelling; cell volume changes were monitored by video imaging. Within a few hours after isolation, enterocytes underwent anoikis (apoptosis induced by detachment from the extracellular matrix). They showed an early normotonic volume decrease (AVD) preceding the appearance of annexin V positivity. AVD occurred in correspondence with an increase in the [Ca2+]i, measured with Fura-2. When the cells were resuspended in high K+ solution or treated with iberiotoxin, AVD was completely abolished. In addition, treatment with high K+ or iberiotoxin significantly inhibited apoptosis progression. It was demonstrated for the first time in native enterocytes that BK channels, which are involved in RVD in these cells, plays also a crucial role in the AVD process and in the progression of apoptosis

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 green alga Caulerpa racemosa var. cylindracea has invaded Mediterranean seabed including marine reserves, modifying the structure of habitats and altering the distributional patterns of associated organisms. However, the understanding of how such invasion can potentially affect functional properties of Mediterranean subtidal systems is yet to be determined. In this study, we show that C. racemosa changes foraging habit of the native white seabream, Diplodus sargus. In invaded areas, we found a high frequency of occurrence of C. racemosa in the stomach contents of this omnivorous fish (72.7 and 85.7%), while the alga was not detected in fish from a control area. We also found a significant accumulation of caulerpin, one of the main secondary metabolites of C. racemosa, in fish tissues. The level of caulerpin in fish tissues was used here as an indicator of the trophic exposure to the invasive pest and related with observed cellular and physiological alterations. Such effects included activation of some enzymatic pathways (catalase, glutathione peroxidases, glutathione S-transferases, total glutathione and the total oxyradical scavenging capacity, 7-ethoxy resorufin O-deethylase), the inhibition of others (acetylcholinesterase and acylCoA oxidase), an increase of hepatosomatic index and decrease of gonadosomatic index. The observed alterations might lead to a detrimental health status and altered behaviours, potentially preventing the reproductive success of fish populations. Results of this study revealed that the entering of alien species in subtidal systems can alter trophic webs and can represent an important, indirect mechanism which might contribute to influence fluctuations of fish stocks and, also, the effectiveness of protection regimes.

Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO2 to HCO3 and H+. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences.

A method for assessing the toxicity of an aqueous matrix, providing for preparing a mixture of an aqueous solution of carbonic anhydrase isoform II and of a sample of the aqueous matrix, to which an aqueous solution comprising carbon dioxide (CO2) is added so as to start the enzymatic reaction of the carbonic anhydrase. The enzymatic activity of the carbonic anhydrase developed in the reaction between carbonic anhydrase and CO2 is then determined and the procedure is repeated with a solution that contains carbonic anhydrase to which the sample of aqueous matrix has not been added. Finally, the percentage of inhibition of carbonic anhydrase is calculated as percentage ratio between the enzymatic activity determined for the mixture with the aqueous matrix sample and the enzymatic activity of the carbonic anhydrase of the solution without the aqueous matrix sample. The inhibition percentage is directly proportional to the toxicity of the examined aqueous matrix.

A method for assessing the toxicity of an aqueous matrix, providing for preparing a mixture of an aqueous solution of carbonic anhydrase isoform II and of a sample of the aqueous matrix, to which an aqueous solution comprising carbon dioxide (CO2) is added so as to start the enzymatic reaction of the carbonic anhydrase. The enzymatic activity of the carbonic anhydrase developed in the reaction between carbonic anhydrase and CO2 is then determined and the procedure is repeated with a solution that contains carbonic anhydrase to which the sample of aqueous matrix has not been added. Finally, the percentage of inhibition of carbonic anhydrase is calculated as percentage ratio between the enzymatic activity determined for the mixture with the aqueous matrix sample and the enzymatic activity of the carbonic anhydrase of the solution without the aqueous matrix sample. The inhibition percentage is directly proportional to the toxicity of the examined aqueous matrix.