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The investigation of trace element pollution is important for the environmental assessment and management of lacustrine ecosystems, especially when these represent critical freshwater resources in densely populated areas. In this context, this study determined the levels of 15 trace elements in muscles of shad, Alosa agone (Scopoli 1786), a commercialized zooplanktivourous fish, from three primary, but currently still poorly studied, Italian lakes, namely, Lake Como, Iseo, and Garda. Research findings show that shads present similar trace element accumulation patterns among lakes, except for arsenic, which occurs at lower levels in Lake Como. Results provide evidence also for mercury biomagnification in fish, whereas all the other selected trace elements undergo bio-dilution through the same trophic chain. Maximum allowable limits for foodstuff were exceeded for chromium and selenium in shads, whereas mercury levels exceeded the European Environmental Quality Standard biota. These results highlight the need for regular monitoring activities of trace elements in the biota of these lakes.

Beer contains a large variety of different molecules, of which some can chelate Ca 2+. Here, we show that in PE/CA-PJ15 cells, an experimental model previously employed to investigate Ca 2+ homeostasis, chemical entities with Ca 2+ -chelating properties can markedly impact intra- and extra-cellular levels of the metal ion. This was consistently shown by commercial beers, as well as aliquots of unfinished product taken during the last step (clarification) of the brewing process. Our data suggest that a large number of molecules with chelating properties, can cross the cell membrane and, thus, potentially lead to important biological effects associated with changes in Ca 2+ homeostasis. In this regard, when PE/CA-PJ15 cells were exposed to H 2 O 2 in order to experimentally prompt an oxidative stress, beer antagonized a non-capacitative Ca 2+ entry that was otherwise elicited by H 2 O 2 in its absence. Higher Ca 2+ -chelating activity of stout, as compared to lager beer, suggest that melanoidins, which are generally present in dark malts, are major chelating agents in dark beers. Further, based on dose-response determinations, we report, for the first time, that Ferulic Acid can bind both intra- and extra-cellular Ca 2+ and, as one of the most abundant hydroxycinnamic acids in malted barley, largely account for Ca 2+ chelation. These results support the notion that beer may be considered a natural source of chemical entities that, based on their binding activity to Ca 2+ and, possibly, other metal ions, might be considered as nutritional supplements, detoxification agents, or antibiotics.

Zooplankton is a key node in many trophic webs, both for food that for persistent organic contaminants that can accumulate in biota. Zooplankton of different size was seasonally sampled for two years in three deep Italian subalpine lakes (Maggiore, Como, Iseo) with the aim of determining the concentrations of perfluoroalkyl substances (PFAS), DDT, and PCB, and assessing the seasonality impacts on contaminants concentrations. In general, Lake Maggiore showed the highest concentrations for each group of contaminants, with mean values of 7.6 ng g&minus, 1 ww for PFAS, 65.0 ng g&minus, 1 dw for DDT, and 65.5 ng g&minus, 1 dw for PCB. When considering the composition pattern, perfluorooctane sulfonate (PFOS) was detected in 96% of the samples and it was the predominant PFAS compound in all of the lakes. pp&rsquo, DDE was the most detected congener among DDTs and their metabolites, while for PCBs, the prevalent group was hexa-CB that constituted 35.4% of the total PCB contamination. A seasonal trend was highlighted for all contaminant groups with concentrations in colder months greater than in spring and summer, it was evident that the contaminant concentrations were more dependent from seasonality than from size, trophic levels, and taxa composition of zooplankton. Principal component analysis showed that one of the main driver for the accumulation of most of the studied contaminants is their lipophilicity, except for perfluorooctanoic acid (PFOA) and octachlorobiphenyl.

An on-line TFC (Turbulent Flow Chromatography) clean up procedures coupled with UHPLC–MS/MS (Ultra High Performance Liquid Chromatography Mass Spectrometry) multi-residue method was developed for the simultaneous determination of 8 perfluroalkyl carboxylic acids (PFCA, from 5 to 12 carbon atoms) and 3 perfluoroalkyl sulfonic acids (PFSA, from 4 to 8 carbon atoms) in environmental solid matrices. Fast sample preparation procedure was based on a sonication-assisted extraction with acetonitrile. Phospholipids in biological samples were fully removed by an off-line SPE purification before injection, using HybridSPE® Phospholipid Ultra cartridges. The development of the on-line TFC clean-up procedure regarded the choice of the stationary phase, the optimization of the mobile phase composition, flow rate and injected volume. The validation of the optimized method included the evaluation of matrix effects, accuracy and reproducibility. Signal suppression in the analysis of fortified extracts ranged from 1 to 60%, and this problem was overcome by using isotopic dilution. Since no certified reference materials were available for PFAS in these matrices, accuracy was evaluated by recoveries on spiked clam samples which were 98–133% for PFCAs and 40–60% for PFSAs. MLDs and MLQs ranged from 0.03 to 0.3 ng g−1 wet weight and from 0.1 to 0.9 ng g−1 wet weight respectively. Repeatability (intra-day precision) and reproducibility (inter-day precision) showed RSD from 3 to 13% and from 4 to 27% respectively. Validated on-line TFC/UHPLC–MS/MS method has been applied for the determination of perfluoroalkyl acids in different solid matrices (sediment, fish, bivalves and bird yolk).

The biomagnification of mercury, polychlorobiphenyls (PCBs), dichlorodiphenyltrichloroethane and its metabolites (DDTs) and perfluoroalkyl acids substances (PFASs) was evaluated in the trophic web of Lake Mergozzo, a small and deep Italian subalpine lake, which has been chosen because it is a protected environment, and discharges into the lake are mostly avoided. Carbon source and relative trophic levels were calculated by using 13C and 15N stable isotopes, respectively, and trophic magnification factors (TMFs) were derived. Zooplankton and thirteen species of fish were collected and analyzed, and the results showed the elevated level of biota contamination from both legacy and emerging pollutants, even if direct discharges were avoided. Concentrations in biota, expressed as sums of compounds, ranged from 0.4 to 60 &micro, g kg&minus, 1 wet weight (ww) for PFASs, from 16 to 1.3 104 &micro, 1 lipid content (lw) for DDTs, from 17 to 1.5 104 &micro, 1 lw for PCBs and from 20.0 to 501 &micro, 1 ww for mercury (Hg). TMFs of this deep, cold lake, with a prevalent pelagic trophic chain, were high and clearly indicated fish biomagnification, except for PFAS. The biomagnification capability of PFAS in a fish-only food web was discussed by using the biomagnification of Hg as a benchmark for assessing their bioaccumulation potential.

Interest in Stable Isotopes Analyses (SIA) is increasing in freshwater ecology to better clarify ecosystems’ functioning. By measuring carbon and nitrogen isotopic signatures, food sources and organism trophic levels can be tracked, providing quantitative estimates of bi-dimensional niches. In order to describe some general patterns of carbon and nitrogen stable isotope signatures in lakes, we applied SIA to zooplankton community in five subalpine lakes sampled in spring and summer along a trophic gradient (from oligotrophy to hypereutrophy). Within zooplankton taxa, temporal variation in food sources and trophic levels were compared to find out taxon-specific patterns. Carbon and nitrogen isotopic signatures differed among the five lakes, reflecting depth, topography, and trophic status of the lakes. Carbon isotopic signatures varied more considerably in deeper and larger lakes (Mergozzo and Pusiano) than in a shallower and smaller lake (Lake Endine). Nitrogen isotopic signatures were generally more enriched in lakes Pusiano and Moro than in Lake Mergozzo, whereas in summer, they were depleted in all lakes. These observations indicate that zooplankton taxa specific trophic roles differed among lakes and in time.

In the present paper the application of on-line preparation techniques applied to LC–MS for the determination of emerging contaminants in environmental samples are critically reviewed highlighting the advantages but also the limits and the way to overcome them. Recent methods are collected in a detailed table and discussed in the text according to the different classes of compounds. The implementation of on-line SPE made possible the effective development of faster methods by reducing the analysis time and thus increasing the analytical productivity. The on-line SPE method are particularly suitable in studies where only limited sample material is available, as the sample volume can be as low as 1 mL. Most of the multiresidual methods have been developed using wettable and polar embedded reverse phases, sometimes sequentially packed with weak ionic exchangers in order to widen the polarity range of the adsorbents. On-line techniques have been also applied in cleanup of extracts of solid environmental samples, such as sediment, soil and biota, in order to minimize the ionization suppression effects from the matrix. Many applications employed RAM, often coupled to monolithic pre-concentration columns, but there is an increasing number of application of TFC for the determination of emerging pollutants in environmental samples.

Bisphosphonates are the most important class of antiresorptive agents used against osteoclast-mediated bone loss, and, more recently, in oncology. These compounds have high affinity for calcium ions (Ca(2+)) and therefore target bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function. They are extensively used in healthcare, however they are affected by severe side effects; pharmacological properties of bisphosphonates depend on their molecular structure, which is frequently the cause of poor intestinal adsorption and low distribution. In this work we synthesized six novel bisphosphonate compounds having a variably substituted indole moiety to evaluate their extra- and intracellular calcium chelating ability in PE/CA-PJ15 cells. Preliminary in silico and in vitro ADME studies were also performed and the results suggested that the indole moiety plays an important role in cell permeability and metabolism properties.

The evidence that in Northern Italy significant sources of perfluoroalkylacids (PFAA) are present induced the Italian government to establish a Working Group on Environmental Quality Standard (EQS) for PFAA in order to include some of them in the list of national specific pollutants for surface water monitoring according to the Water Framework Directive (2000/60/EC). The list of substances included perfluorooctanoate (PFOA) and related short chain PFAA such as perfluorobutanoate (PFBA), perfluoropentanoate (PFPeA), perfluorohexanoate (PFHxA) and perfluorobutanesulfonate (PFBS), which is a substitute of perfluorooctanesulfonate. For each of them a dossier collects available data on regulation, physico-chemical properties, emission and sources, occurrence, acute and chronic toxicity on aquatic species and mammals, including humans. Quality standards (QS) were derived for the different protection objectives (pelagic and benthic communities, predators by secondary poisoning, human health via consumption of fishery products and water) according to the European guideline. The lowest QS is finally chosen as the relevant EQS. For PFOA a QS for biota was derived for protection from secondary poisoning and the corresponding QS for water was back-calculated, obtaining a freshwater EQS of 0.1μgL-1. For PFBA, PFPeA, PFHxA and PFBS threshold limits proposed for drinking waters were adopted as EQS.

Perfluooctane sulfonate (PFOS) is considered an emerging pollutant because of its wide distribution in both aquatic and terrestrial ecosystems, as well as its potential toxicity to living organisms. Although PFOS environmental levels and the adverse effects on classical model organisms in toxicological studies are well known, including developmental alterations and alteration of oxidative status, its toxicity to free-living species has been seldom investigated. The aim of this study was to assess the potential toxicity of environmental levels of PFOS to yellow-legged gull (Larus michahellis) embryos under field experimental conditions. In a within-clutch experimental design, we injected two PFOS concentrations (100 ng PFOS/g egg weight and 200 ng PFOS/g egg weight) in ovo soon after laying. Eggs were collected when they reached the cracking stage. We investigated the effects of PFOS treatment, laying order and sex on both morphological and biochemical endpoints of embryos. Specifically, we assessed changes in embryo body mass and tarsus length, as well as in liver and brain mass. Moreover, the imbalance of oxidative status was evaluated in both liver and brain from embryos by measuring total antioxidant capacity (TAC) and total oxidant status (TOS), while the levels of protein carbonyl content (PCO) and DNA fragmentation were measured as oxidative and genetic damage endpoints, respectively. The concentrations of PFOS we tested did not significantly alter the morphological endpoints, independently of laying order and sex. Similarly, embryo oxidative status and oxidative and genetic damage were not significantly affected by PFOS in ovo exposure. These findings suggest that current environmental PFOS levels do not affect early development of yellow-legged gull embryos.

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 omega-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, omega-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.

The homeostasis of cytosolic calcium [Ca2+]c in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+]c, although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 μM) and at high (160 μM) concentrations by measuring the [Ca2+]c by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+]c, whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+]c in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+]c homeostasis by stimulating the movement of the ion from the cytosol to other compartments. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:35–40, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20211

An UHPLC-MS/MS multiresidue method based on an on-line solid phase extraction (SPE) procedure was developed for the simultaneous determination of 9 perfluorinated carboxylates (from 4 to 12 carbon atoms) and 3 perfluorinated sulphonates (from 4 to 8 carbon atoms). This work proposes using an on-line solid phase extraction before chromatographic separation and analysis to replace traditional methods of off-line SPE before direct injection to LC-MS/MS. Manual sample preparation was reduced to sample centrifugation and acidification, thus eliminating several procedural errors and significantly reducing time-consuming and costs. Ionization suppression between target perfluorinated analytes and their coeluting SIL-IS were detected for homologues with a number of carbon atoms less than 9, but the quantitation was not affected. Total matrix effect corrected by SIL-IS, inclusive of extraction efficacy, and of ionization efficiency, ranged between −34 and +39%. The percentage of recoveries, between 76 and 134%, calculated in different matrices (tap water and rivers impacted by different pollutions) was generally satisfactory. LODs and LOQs of this on-line SPE method, which also incorporate recovery losses, ranged from 0.2 to 5.0 ng/L and from 1 to 20 ng/L, respectively. Validated on-line SPE-LC/MS/MS method has been applied in a wide survey for the determination of perfluoroalkyl acids in Italian surface and ground waters.

Publisher Summary The nutritional value of any foods is not limited to their content in essential nutrients or in nutrients generally speaking. Of course, food is a source of energy and essential nutrients, but it is also a mixture of many molecules, some in high, low, or very low amounts. These may interact in various ways and may influence human health in yet unpredictable manners either in the short or long term. For this reason, efforts are being made towards the understanding of the physiological and medical significance of food. The chief constituent of alimentary fat is triacylglycerol. Various triacylglycerol molecules may differ in the proportion and position of the fatty acids they contain. Much work has been done on this subject, especially with reference to polyunsaturated essential fatty acid. However, olive oil contains a number of molecules besides triacylglycerols. This chapter is dedicated to some of these, namely to phenols. Biochemical action of phenols is important for several reasons. Technically speaking, oils can be prepared to contain higher or lower amounts of phenols; moreover, these substances could be used to enrich food or as supplements for the prevention of important illnesses, such as coronary heart disease. The mechanism(s) responsible for the effects of phenols have not yet been fully clarified. Antioxidant activity and free radical scavenging may be relevant in this connection. Yet, other hypotheses have been put forward. For instance, it has been reported that red wine phenols increase cytosolic calcium ([Ca2+]c) in bovine endothelial cells, similarly to bradikinin and to ATP. This chapter reports the effect of extra virgin olive oil phenols on cytosolic calcium [Ca2+]c variations due to nitric oxide (NO).

Ejaculated spermatozoa must undergo a number of modifications before fertilizing the oocyte: among these the capacitation and the acrosome reaction. Calcium signals play an essential role in these functional and structural modifications. Mature spermatozoa have few organelles and a very small cytoplasmic volume but maintain the homeostasis of [Ca 2+ ] c with great accuracy. We study Ca 2+ mobilization in human spermatozoa exposed to FSH and progesterone by measuring the [Ca 2+ ] c with the FURA-2AM method and report for the first time that the exposure to FSH (up to 98 ng/ml) produced an increase of [Ca 2+ ] c to an extent comparable to that observed with 1 μM progesterone. FSH and progesterone increase the spermatozoa [Ca 2+ ] c by acting primarily on calcium entry from the external medium. The effects of the two hormones on [Ca 2+ ] c were similar but not identical; the pre-treatment of progesterone blocks the effects of FSH, but not vice-versa. The increase of [Ca 2+ ] c due to FSH was more sensitive to nifedipine (VOCCs inhibitor) than that of progesterone. The effects of these hormones on calcium homeostasis may be relevant for sperm activation.