A. Calisi

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.

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.

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.

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

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

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

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

Earthworm Biomarkers as Tools for Soil Pollution Assessment

Soil pollution has enormously increased during the last decades due to the intensive use of biocides and fertilizers in agriculture, industrial activities, urban waste and atmospheric deposition. Its occurrence is related to the degree of industrialization and intensity of chemical usage. Soil pollution causes decrease in soil fertility, alteration of soil structure, disturbance of the balance between flora and fauna residing in the soil, contamination of the crops, and contamination of groundwater, constituting a threat for living organisms. The most diffusive chemicals occurring in soil are heavy metals, pesticides, petroleum hydrocarbons, polychlorobiphenyl (PCBs), dibenzo-p-dioxins/dibenzofurans (PCDD/Fs). Heavy metals from anthropogenic sources are widely spread in the environment and most of them finally reach the surface soil layers. Heavy metals can enter the soil from different sources, such as pesticides, fertilizers, organic and inorganic amendants, mining, wastes and sludge residues (Capri & Trevisan, 2002). In contrast to harmful organic compounds, heavy metals do not decompose and do not disappear from soil even if their release to the environment can be restricted (Brusseau, 1997). Therefore, the effects of heavy metal contamination on soil organisms and decomposition processes persist for many years. Pesticides are widely used in agriculture for counteracting insects, fungi, rodents or other animals living in or on the crops. They are either directly applied to soil to control soil borne pests or deposited on soil as run off from foliar applications and their concentrations are high enough to affect the soil macro-organisms (Bezchlebova et al., 2007). The pesticides most widely used in the past have been organochlorine pesticides, characterized by high hydrophobicity and persistence. Currently, they have been replaced by less persistent compounds. Organophosphates have become the most widely used pesticides today. They are used for pest control on crops in agriculture and on livestock, for other commercial purposes, and for domestic use. Due to their water solubility, the organophosphate residues in agricultural practices are capable of infiltrating through soil into surface water. As a consequence of their wide diffusion they have been detected in food, ground and drinking water, and natural surface waters (Dogheim et al., 1996; Garrido et al., 2000). Soil pollution by petroleum hydrocarbons usually originates from spills or leaks of storage tanks during fuel supply and discharge operations. Petroleum hydrocarbons include aliphatic and aromatic compounds; some of them are known or suspected human carcinogens, and are classified as priority pollutants. PCBs are persistent soil contaminants due to their

Role of BK Channels in the Apoptotic Volume Decrease in Native Eel Intestinal Cells

High conductance Ca+-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.

Biomonitoring of water and soil quality: a case study of ecotoxicological methodology application to the assessment of reclaimed agroindustrial wastewaters used for irrigation

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.

Metallothionein Induction in the Coelomic Fluid of the Earthworm Lumbricus terrestris following Heavy Metal Exposure: A Short Report

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

Mercury induced haemocyte alterations in the terrestrial snail Cantareus apertus as novel biomarker

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 for mercury 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 1h 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 cells was 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.