Osteoarthritis: Insights into Diagnosis, Pathophysiology, Therapeutic Avenues, and the Potential of Natural Extracts.

Osteoarthritis (OA) stands as a prevalent and progressively debilitating clinical condition globally, impacting joint structures and leading to their gradual deterioration through inflammatory mechanisms. While both non-modifiable and modifiable factors contribute to its onset, numerous aspects of OA pathophysiology remain elusive despite considerable research strides. Presently, diagnosis heavily relies on clinician expertise and meticulous differential diagnosis to exclude other joint-affecting conditions. Therapeutic approaches for OA predominantly focus on patient education for self-management alongside tailored exercise regimens, often complemented by various pharmacological interventions primarily targeting pain alleviation. However, pharmacological treatments typically exhibit short-term efficacy and local and/or systemic side effects, with prosthetic surgery being the ultimate resolution in severe cases. Thus, exploring the potential integration or substitution of conventional drug therapies with natural compounds and extracts emerges as a promising frontier in enhancing OA management. These alternatives offer improved safety profiles and possess the potential to target specific dysregulated pathways implicated in OA pathogenesis, thereby presenting a holistic approach to address the condition's complexities.

Carbonic Anhydrase and Biomarker Research: New Insights.

Carbonic anhydrase (CA) is a widespread metalloenzyme with eight genetically distinct families catalyzing the reversible hydration of CO2 to HCO3- and H+ [...].

Intracellular Redox Behavior of Quercetin and Resveratrol Singly and in Mixtures.

Polyphenols have attracted great interest as potent antioxidant compounds and nutraceuticals; however, their antioxidant properties represent a multifaceted phenomenon, including pro-oxidant effects under particular conditions and complex behavior when multiple polyphenols are simultaneously present. Moreover, their intracellular behavior cannot always be predicted from their ability to counteract the production of ROS in acellular assays. The present work aimed to study the direct intracellular redox activity of two polyphenols, resveratrol and quercetin, singly and in mixture in a cellular short-term bioassay under both basal and pro-oxidant conditions. The study was carried out by spectrofluorimetric assessment of the intracellular fluorescence of CM-H2DCFDA-charged HeLa cells under either basal conditions, due to the reactive species associated with the normal cellular oxidative metabolism, or pro-oxidant conditions induced by H2O2 exposure. Under basal conditions, the obtained results showed a significant antioxidant effect of quercetin and a weaker antioxidant effect of resveratrol when used singly, while antagonism of their effect was detected in their equimolar mixtures at all the concentrations used. Under exposure of the cells to H2O2, quercetin exhibited a dose-dependent intracellular antioxidant activity whereas resveratrol manifested a pro-oxidant intracellular activity, while their equimolar mixtures showed an intracellular interaction between the 2 polyphenols, with additive effects at 5 µM and synergic at 25 µM and 50 µM. Thus, the results clarified the direct intracellular antioxidant/pro-oxidant activity of quercetin and resveratrol alone and in their equimolar mixtures in the cell model HeLa cells and highlighted that the antioxidant properties of polyphenols in mixtures at the cellular level depend not only on the nature of the compounds themselves but also on the type of interactions in the cellular system, which in turn are influenced by the concentration and the oxidative status of the cell.

Carbonic Anhydrase Sensitivity to Pesticides: Perspectives for Biomarker Development.

Carbonic anhydrase (CA) is a widespread metalloenzyme playing a pivotal role in several physiological processes. Many studies have demonstrated the in vitro and in vivo sensitivity of CA to the exposure to several classes of pesticides in both humans and wildlife. This review aims to analyze and to discuss the literature available in this field, providing a comprehensive view useful to foresee perspectives for the development of novel CA-based pesticide biomarkers. The analysis of the available data highlighted the ability of several pesticide molecules to interact directly with the enzyme in humans and wildlife and to inhibit CA activity in vitro and in vivo, with possible alterations of key physiological functions. The analysis disclosed key areas of further research and, at the same time, identified some perspectives for the development of novel CA-based sensitive biomarkers to pesticide exposure, suitable to be used in several fields from human biomonitoring in occupational and environmental medicine to environmental monitoring on non-target species.

Effect of the flame retardant tris (1,3-dichloro-2-propyl) phosphate (TDCPP) on Na+-K+-ATPase and Cl- transport in HeLa cells.

Tris (1, 3-dichloro-2-propyl) phosphate (TDCPP) is one of the most diffused phosphorus flame retardants in the environment and is highly persistent and abundant in residential dust samples. To date the cellular targets and mechanisms underlying its toxic effects are not completely understood. The aim of this work was to study the effects of TDCPP on ion transport mechanisms fundamental for the cellular ionic homeostasis, such as Na+-K+-ATPase and Cl- transport. HeLa cells were used as experimental model. TDCPP showed a dose-dependent effect on cell viability in cells exposed for 24 h as assessed by MTT test (IC50 = 52.5 µM). The flame retardant was able to exert a dose and time-dependent inhibition on the Na+-K+-ATPase activity. A short-term exposure (1 h) was able to exert a significant inhibition at 75 and 100 µM TDCPP, suggesting that TDCPP is able to directly interfere with the Na+-K+-ATPase phosphate catalytic activity. The sensitivity of the pump to lower TDCPP concentrations increased with the increase of the time of exposure. Following 24 h exposure a significant inhibition of about 40% was evident already at 10 µM and the IC50 value observed was 12.8 ± 6.0 µM. Moreover, TDCPP was also able to impair the NKCC mediated Cl- transport in HeLa cells, as assessed in YFP-H148Q/I152L-expressing HeLa cells. Following 1 h exposure TDCPP significantly inhibited the transport by about 30%. The kinetic analysis demonstrated a noncompetitive mechanism of inhibition. In conclusion, results demonstrated the impairment of ion transport mechanisms fundamental for ion homeostasis by TDCPP on HeLa cells.

The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives.

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.

Cell volume regulation and apoptotic volume decrease in rat distal colon superficial enterocytes.

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 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 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.

Motility of Mytilus galloprovincialis hemocytes: Sensitivity to paracetamol in vitro exposure.

Pharmaceuticals released into the environment (PiEs) represent an environmental problem of growing concern for the health of ecosystems and humans. An increasing number of studies show that PiEs pose a risk to aquatic organisms. The aim of the present work was to contribute to increasing the knowledge of the effects of PiE on marine biota focusing on the effect of paracetamol on the motility of hemocytes in Mytilus galloprovincialis, a bivalve mollusk species widely utilized as bioindicator organism. Hemocytes are the immunocompetent cells of bivalve mollusks. An early and key stage of mollusk immune response is represented by the recruitment and migration of these cells to the site of infection. Therefore, motility is an intrinsic characteristic of these cells. Here, we first characterized the spontaneous cell movement of M. galloprovincialis hemocytes when plated in a TC-treated polystyrene 96-well microplate. Two different cellular morphotypes were distinguished based on their appearance and motility behavior: spread cells and round-star-shaped cells. The two motility morphotypes were characterized by different velocities as well as movement directness, which were significantly lower in round-star-shaped cells with respect to spread cells. The sensitivity of the motility of M. galloprovincialis hemocytes to paracetamol at different concentrations (0.02, 0.2 and 2 mg/L) was investigated in vitro after 1h and 24h exposure. Paracetamol induced alterations in the motility behavior (both velocity and trajectories) of the hemocytes and the effects were cell-type specific. The study of hemocyte movements at the single cell level by cell tracking and velocimetric parameters analysis provides new sensitive tools for assessing the effects of emerging pollutants at the cellular levels in non-target organisms.

Morphological and functional alterations in hemocytes of Mytilus galloprovincialis exposed in high-impact anthropogenic sites.

The work aimed to study the induction of morphological alterations in M. galloprovincialis in the field and its suitability to be integrated into a sensitive, simple, and cost-effective cell-based multimarker approach for the detection of the stress status induced by pollution in coastal marine environments in view of ecotoxicological biomonitoring and assessment application. Cellular morphometric alterations was paralleled by the analysis of standardized biomarkers such as lysosomal membrane destabilization, and genotoxocity biomarkers such as micronuclei and binuclated cells frequencies were investigated. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two multi-impacted coastal sites of the central Mediterranean area, Bagnoli in the eastern Tyrrhenian Sea and Augusta-Melilli-Priolo in the western Ionian Sea. Capo Miseno (NA) for the Tyrrhenian area and Brucoli (ME) for the Ionian area were chosen as control sites. Hemocyte enlargement and filopodial elongation increased frequencies were observed in organisms exposed to the impacted sites. These morphometric alterations showed strong agreement with the lysosomal membrane destabilization and biomarkers of genotoxicity, suggesting their usefulness in detecting the pollutant-induced stress syndrome related to genotoxic damage.

Size-Resolved Redox Activity and Cytotoxicity of Water-Soluble Urban Atmospheric Particulate Matter: Assessing Contributions from Chemical Components.

Throughout the cold and the warm periods of 2020, chemical and toxicological characterization of the water-soluble fraction of size segregated particulate matter (PM) (<0.49, 0.49−0.95, 0.95−1.5, 1.5−3.0, 3.0−7.2 and >7.2 µm) was conducted in the urban agglomeration of Thessaloniki, northern Greece. Chemical analysis of the water-soluble PM fraction included water-soluble organic carbon (WSOC), humic-like substances (HULIS), and trace elements (V, Cr, Mn, Fe, Ni, Cu, Zn, As, Cd and Pb). The bulk (sum of all size fractions) concentrations of HULIS were 2.5 ± 0.5 and 1.2 ± 0.3 µg m−3, for the cold and warm sampling periods, respectively with highest values in the <0.49 µm particle size fraction. The total HULIS-C/WSOC ratio ranged from 17 to 26% for all sampling periods, confirming that HULIS are a significant part of WSOC. The most abundant water-soluble metals were Fe, Zn, Cu, and Mn. The oxidative PM activity was measured abiotically using the dithiothreitol (DTT) assay. In vitro cytotoxic responses were investigated using mitochondrial dehydrogenase (MTT). A significant positive correlation was found between OPmDTT, WSOC, HULIS and the MTT cytotoxicity of PM. Multiple Linear Regression (MLR) showed a good relationship between OPMDTT, HULIS and Cu.

Characterisation of the correlations between oxidative potential and in vitro biological effects of PM10 at three sites in the central Mediterranean.

Atmospheric particulate matter (PM) is one of the major risks for global health. The exact mechanisms of toxicity are still not completely understood leading to contrasting results when different toxicity metrics are compared. In this work, PM10 was collected at three sites for the determination of acellular oxidative potential (OP), intracellular oxidative stress (OSGC), cytotoxicity (MTT assay), and genotoxicity (Comet assay). The in vitro tests were done on the A549 cell line. The objective was to investigate the correlations among acellular and intracellular toxicity indicators, the variability among the sites, and how these correlations were influenced by the main sources by using PMF receptor model coupled with MLR. The OPDTTV, OSGCV, and cytotoxicity were strongly influenced by combustion sources. Advection of African dust led to lower-than-average intrinsic toxicity indicators. OPDTTV and OSGCV showed site-dependent correlations suggesting that acellular OP may not be fully representative of the intracellular oxidative stress at all sites and conditions. Cytotoxicity correlated with both OPDTTV and OSGCV at two sites out of three and the strength of the correlation was larger with OSGCV. Genotoxicity was correlated with cytotoxicity at all sites and correlated with both, OPDTTV and OSGCV, at two sites out of three. Results suggest that several toxicity indicators are useful to gain a global picture of the potential health effects of PM.

Autofluorescence of Model Polyethylene Terephthalate Nanoplastics for Cell Interaction Studies.

This work contributes to fill one of the gaps regarding nanoplastic interactions with biological systems by producing polyethylene terephthalate (PET) model nanoplastics, similar to those found in the marine environment, by means of a fast top-down approach based on mechanical fragmentation. Their size distribution and morphology were characterized by laser diffraction and atomic force microscopy (AFM). Their autofluorescence was studied by spectrofluorimetry and fluorescence imaging, being a key property for the evaluation of their interaction with biota. The emission spectra of label-free nanoplastics were comparable with those of PET nanoplastics labeled with Nile red. Finally, the suitability of label-free nanoplastics for biological studies was assessed by in vitro exposure with Mytilus galloprovincialis hemolymphatic cells in a time interval up to 6 h. The nanoplastic internalization into these cells, known to be provided with phagocytic activity, was assessed by fluorescence microscopy. The obtained results underlined that the autofluorescence of the model PET nanoplastics produced in the laboratory was adequate for biological studies having the potential to overcome the disadvantages commonly associated with several fluorescent dyes, such as the tendency to also stain other organic materials different from plastics, to form aggregates due to intermolecular interactions at high concentrations with a consequent decrease in fluorescence intensity, and to dye desorption from nanoparticles. The results of the autofluorescence study provide an innovative approach for plastic risk assessment.

Development and characterization of a gold nanoparticles glassy carbon modified electrode for dithiotreitol (DTT) detection suitable to be applied for determination of atmospheric particulate oxidative potential.

A gold nanostructured electrochemical sensor based on modified GC electrode for thiols' detection is described and characterized. This sensor is a suitable device for the measurement of the oxidative potential (OP) of the atmospheric particulate matter (PM), considered a global indicator of adverse health effects of PM, as an alternative to the classic spectrophotometric methods. The operating principle is the determination of the OP, through the measurement of the consumption of DTT content. The DTT-based chemical reactivity is indeed a quantitative acellular probe for assessment of the capacity of the atmospheric PM to catalyze reactive oxygen species generation which contributes to the induction of oxidative stress in living organisms and in turn to the outcome of adverse health effects. To make the sensors, glassy carbon electrodes, traditional (GC) and screen printed (SPE) electrodes, have been electrochemically modified with well-shaped rounded gold nanoparticles (AuNPs) by using a deposition method that allows obtaining a stable and efficient modified surface in a very simple and reproducible modality. The chemical and morphological characterization of the nano-hybrid material has been performed by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy coupled with electron dispersive spectroscopy analysis (SEM/EDS). The electrochemical properties have been evaluated by cyclic voltammetry (CV) and chrono-amperometry (CA) in phosphate buffer at neutral pH as requested in DTT assay for OP measurements. The electroanalytical performances of the sensor in DTT detection are strongly encouraging showing low LODs (0.750 µM and 1.5 µM), high sensitivity (0.0622 µA cm-2 µM-1 and 0.0281 µA cm-2 µM-1), wide linear and dynamic ranges extending over 2-4 orders of magnitude and high selectivity. FIA preliminary results obtained on measuring the DTT rate consumption in six PM aqueous extracts samples showed a good correlation with measurements obtained in parallel on the same set of samples by using the classic spectrophotometric method based on the Ellman's reactive use. These results confirm the high selectivity of the method and its suitability for application to be applied in PM oxidative potential measurements.

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 [Ca(2+)](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.

Fluorimetric analysis of copper transport mechanisms in the b104 neuroblastoma cell model: a contribution from cellular prion protein to copper supplying.

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP(C)) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP(C) proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP(C)-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP(C) in mediating copper uptake by clathrin-dependent endocytosis.

Concentration Dependence of the Antioxidant and Prooxidant Activity of Trolox in HeLa Cells: Involvement in the Induction of Apoptotic Volume Decrease.

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 µM) 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',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), at low concentrations (range: 2.5-15 µM), 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- 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.

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.

Effects of short-term and long-term exposure to ocean acidification on carbonic anhydrase activity and morphometric characteristics in the invasive polychaete Branchiomma boholense (Annelida: Sabellidae): A case-study from a CO2 vent system.

The aim of this study was to test the effects of short- and long-term exposure to high pCO2 on the invasive polychaete Branchiomma boholense (Grube, 1878), (Sabellidae), through the implementation of a transplant experiment at the CO2 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 pCO2 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 pCO2, the morphometric results revealed a significantly lower (70%) wet weight of specimens from the vents with respect to animals living in high pH/low pCO2 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 pCO2. 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 pCO2.

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.

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.