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.

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.

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.

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.

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.

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.

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.

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 CO(2) and HCO(3)(-). 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.

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.

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.