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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 .

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.

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.

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.

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.

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.

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 fundamental role of this biochemical reaction in diverse biological systems has driven the evolution of several distinct and unrelated families of CAs. Five CA families, referred as ┙-, ┚-, ┛-CA, ├, and ζ-CAs have been identified in animals, plants and bacteria (HewettEmmett and Tashian, 1996; Supuran, 2010). These are the ┙-CAs, present in vertebrates, bacteria, algae and plants; the ┚-CAs, predominantly in bacteria, algae and plants; the ┛CAs, mainly present in archaea and some bacteria; the ├-CAs and ζ-CAs only found in some marine diatoms (Supuran, 2010).

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.

Carbonic anhydrase (CA) is a metalloenzyme catalysing the reversible idratation of CO2 in Hþ and HCO3 . It is an ubiquitous enzyme in bacteria, plant and animal kingdoms, playing a fundamental role in a number of physiological processes. Previous studies demonstrated the sensitivity of CA activity to dichlorodiphenyl-dichloroethane (DDT) exposure in birds and to cadmium exposure in teleosts. The aim of the present work was to develop a new in vitro enzymatic bioassay for detecting toxic chemicals in environmental samples as a cost-effective tool in environmental monitoring. This bioassay uses the commercial available CA isozyme II from bovine erythrocytes whose sensitivity to the main classes of chemical pollutants of importance in water quality research was tested in this work. CA activity was determined by a modification of the electrometric method previously described by Wilbur and Anderson [K.M. Wilbur, G.N. Anderson. J. Biol. Chem., 176, 147 (1948).]: briefly, CA activity units were calculated from the rate of Hþ production in the reaction mixture (where CO2 was present as substrate) against a blank containing the specific CA inhibitor acetazolamide. [Hþ] variation was followed at 0C in the reaction mixture using a Mettler Delta 350 pH-meter. In our experimental set-up bovine CA activity was significantly inhibited by heavy metals (Cd, Cu and Hg), by the organochlorate compound arochlor and by the carbammate pesticides carbaryl in a dose-dependent manner. CA in vitro bioassay can represent a novel tool for rapid and low cost understanding of the toxicity of environmental samples, for assessing bioavailability of pollutants in environmental matrices and their additive or synergistic biological effects when present in mixtures.