Maria Giulia Lionetto

Acetylcholinesterase as a Biomarker in Environmental and Occupational Medicine: New Insights and Future Perspectives

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.

Roles of the Cytoskeleton and of Protein Phosphorylation Events in the Osmotic Stress Response in EEL Intestinal Epithelium

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl- absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, Vte, at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na+-K+-2Cl- (NKCC) cotransporter, which correlates with the cellular RVI response.Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress.The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (Vte and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of Vte and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of Vte and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine.It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.

Effect of cadmium on carbonic anhydrase and Na+-K+-ATPase in eel, Anguilla anguilla, intestine and gills

Abstract The effect of the heavy metal cadmium on in vitro activities of Na + -K + -ATPase and carbonic anhydrase (CA) in intestine and gills of the European eel, Anguilla anguilla , was analyzed. Na + -K + -ATPase enzymatic activities, measured in intestinal and branchial homogenates, were inhibited by increasing cadmium concentrations (0.5–50 μ M, after 1 h of incubation) in a dose-dependent manner; the maximal inhibition (about 80%) was observed at 5 μ M and 50 μ M CdCl 2 for the branchial and intestinal Na + -K + -ATPase respectively. Carbonic anhydrase activities, measured in gill homogenate and in cytosolic and brush border membrane fractions isolated from intestinal mucosa, were significantly inhibited by pre-incubation (1 h) with CdCl 2 . Maximal inhibition (about 80%) of branchial CA was noted at approximately 60 μ M; higher concentrations evoked no further significant inhibition. Intestinal CA isoforms, cytosolic and membrane-bound, exhibited lower sensitivity to the heavy metal with respect to the branchial CA activity, since the highest concentration of CdCl 2 tested (600 μ M) produced an inhibition of about 30% and 50% respectively. The results of the present study suggest that cadmium, by inhibiting the activity of CA and Na + -K + -ATPase enzymes in intestine and gills, could alter both acid-base balance and osmoregulation in teleostean fish.

Carbonic Anhydrase as Pollution Biomarker: An Ancient Enzyme with a New Use

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 chemicals 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.

Pollutant-induced alterations of granulocyte morphology in the earthworm Eisenia foetida

Earthworms are considered convenient indicators of land use and soil fertility. Recently the use of biomarkers in earthworms has been increasingly investigated. The aim of this work was to study possible pollutant-induced morphometric alterations in Eisenia foetida granulocytes in view of future applications as a sensitive, simple, and quick biomarker for soil monitoring and assessment applications. Results showed consistent enlargement of earthworm granulocytes induced by exposure to either copper sulfate or methiocarb. The increase of cellular size was time-dependent and was about 100% after 14 days of exposure for both treatments. In order to verify the applicability of morphometric granulocyte alteration, a battery of standardized biomarkers such as lysosomal membrane stability, metallothionein induction, or acetylcholinesterase (AChE) inhibition were also determined. We recommend the use of morphometric alterations of granulocytes as a suitable biomarker of pollutant effect to be included in a multibiomarker strategy including responses at different levels of biological organization.

Biomarker application for the study of chemical contamination risk on marine organisms in the taranto marine coastal area

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 and Zn) and brain and muscular acetylcholinesterase (AChE) activity (specific index of exposure to organophosphate and carbamate pesticides) were measured. None of the two fish species showed significant differences in AChE activity and in pesticide trace level between the anthropogenic impact exposed site and the control group. On the other hand, metallothionein hepatic levels in M. 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 AChE 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.

Hypertonicity Stimulates Cl– Transport in the Intestine of Fresh Water Acclimated EEL, Anguilla Anguilla

Eel intestinal epithelium when bathed symmetrically with normal Ringer solution develops a net Cl– current (short circuit current, Isc) giving rise to a negative transepithelial potential (Vt) at the basolateral side of the epithelium, lower in fresh-water (FW)-acclimated animals with respect to sea-water (SW). The aim of the present work was to study the cell response to hypertonic stress of FW eel intestinal epithelium in relation to Cl– absorption. The hypertonicity of the external bathing solutions produced first a transient increase of Vt and Isc, then followed (after10-15min) by a gradual and sustained increase which reached the maximum value after 40-60 min. The morphometric analysis of the intestine revealed the shrinkage of the cells after 5 min hypertonicity exposure, and then a regulatory volume increase (RVI) response, which parallels the gradual and sustained increase in the electrophysiological parameters. This last phase is inhibited by drugs known to block Cl– absorption in eel intestine, such as luminal bumetanide (10 µM), specific inhibitor of Na+-K+-2Cl– cotransport, or basolateral NPPB (0.5 mM), dichloro-DPC (0.5 mM), inhibitors of basolateral Cl– conductance. Serosal dimethyl-amiloride (100 µM), specific inhibitor of the Na+/H+ antiport, was ineffective on the hyperosmotic response. Bicarbonate revealed a crucial role as a modulator of hypertonicity response, since in bicarbonate-free conditions or in the presence of serosal 0.25 mM SITS, blocker of HCO3– transport systems, the Isc response to hypertonicity was lost. In nominally Ca2+-free conditions the Isc response to hypertonicity was abolished. The same results were obtained by bilateral addition of 100 µM verapamil or 50 µM nifedipine or 1 mM lanthanum, known Ca2+ channel blockers, indicating that extracellular Ca2+ plays a key role for the activation of Cl– current in the response to hypertonic stress. The data show that in the eel intestinal epithelium the hypertonicity of the external medium affects cell volume which in turn might represent the signal to increase the rate of Cl– transport. This response is sustained by the activation of the luminal Na+-K+-2Cl– cotransporter and the functionality of basolateral Cl– channels.

Subtle Effects of Biological Invasions: Cellular and Physiological Responses of Fish Eating the Exotic Pest Caulerpa racemosa

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.

Morphometric alterations in Mytilus galloprovincialis granulocytes: A new biomarker

Bivalve molluscs, particularly marine mussels, are used widely as sentinel organisms in environmental quality monitoring and assessment. Biochemical and cellular responses to pollutant exposure (i.e., biomarkers) increasingly are investigated in mussel tissues and their measurements largely used in marine environmental monitoring. The aim of the present study was to investigate possible pollutant-induced morphometric alterations in Mytilus galloprovincialis granulocytes in view of future applications as a sensitive, simple, and quick biomarker for monitoring and assessment applications. Granulocyte morphometric alterations were determined by image analysis on Diff-Quick® stained cells. For the first time, the rapid alcohol-fixed Diff-Quick stain protocol, utilized in clinical and veterinary applications for immediate interpretation of histological samples, was shown to be suitable for rapid cytological staining of M. galloprovincialis haemocytes. The present study was carried out in standardized laboratory conditions and further validated in field conditions. Results show consistent pollutant-induced enlargement of mussel granulocytes. This was verified by standardized biomarkers such as metallothionein concentrations in the digestive gland or lysosomal membrane stability in laboratory and field exposures. Results further suggest that the observed morphometric alterations can be used as a biomarker of chemical stress. Because no single biomarker generally is adequate for describing the complexity of effects induced by environmental pollutants on the organisms, we propose that morphometric alterations of granulocytes should be used in a biomarker battery in marine environmental monitoring programs such as Mussel Watch.

Potential application of carbonic anhydrase activity in bioassay and biomarker studies

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 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. CA activity was 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, CA was 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 1.785 μM 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.