[Show abstract][Hide abstract] ABSTRACT: Illicit drugs represent not only a great social problem but are also considered an environmental problem because their use and, often, abuse release large amounts of parent compounds, and especially their metabolites, into freshwaters. One of the most commonly used drugs is cocaine, which is the second most prevalent drug in Europe (accounting for almost 30% of all cocaine users worldwide). Cocaine is rapidly metabolised in humans to benzoylecgonine (35-54%), ecgonine methyl ester (32-49%) and norcocaine (5%), which are eliminated in the urine and are only partially removed by wastewater treatment plants (WWTPs). Because no studies have previously been carried out to evaluate the possible risks due to cocaine and its metabolites in non-target organisms, we applied a multi-disciplinary approach to investigate the possible environmental risk related to benzoylecgonine (BE), the main metabolite of cocaine. Previous studies carried out by means of a biomarker suite and the redox-proteomic approach showed an imbalance of anti-oxidant enzyme activities and several genotoxic effects to be caused by environmental BE concentrations in the freshwater bivalve Zebra mussel (Dreissena polymorpha). This report presents the results obtained in the last step of this study, based on a proteomics analysis. We analysed the protein expression profile in the gills of Zebra mussels exposed to two different concentrations (0.5 and 1μg/L) of BE for 14 days through 2-DE and mass spectrometry analysis (RP-UPLC ESI-LTQ-Orbitrap). Our results highlight significant changes in some proteins in gill cells whose functions are crucial for overall metabolism. In particular, we detected a probable effect of BE on calcium homeostasis and a consequent imbalance of oxidative stress, as verified for vertebrates.
[Show abstract][Hide abstract] ABSTRACT: Cigarette smoke is a significant independent risk factor for vascular diseases and is a leading cause of structural and functional alterations of the vascular endothelium. In this study, we show protein carbonylation in the human umbilical vein endothelial cell line (ECV-304) exposed to whole-phase cigarette smoke extract. The main carbonylated proteins, including cytoskeletal proteins, glycolytic enzymes, xenobiotic metabolizing and antioxidant enzymes, and endoplasmic reticulum proteins, were identified by means of two-dimensional electrophoresis and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry (redox proteomics). Morphological analyses by fluorescence microscopy evidenced alterations in the microtubule cytoskeleton, especially at longer exposure time to cigarette smoke extract. Morphological analyses by transmission electron microscopy showed vacuolisation of the cytoplasm, alteration of mitochondria ultrastructure, and some enlargement of the perinuclear space. The possible role played by protein carbonylation caused by reactive species contained in cigarette smoke in the cigarette smoke-induced endothelial injury is discussed.
No preview · Article · Feb 2013 · Toxicology Letters
[Show abstract][Hide abstract] ABSTRACT: PCBs are a persistent environmental problem due to their high stability and lipophilicity. The non-ortho- and the mono-ortho-substituted PCBs (dioxin-like-PCBs) share a common and well-described toxicity mechanism in vertebrates, initially involving binding to cytosolic AhRs. Invertebrate AhRs, however, show a lack of dioxin binding, and little information is available regarding the mechanism of toxicity of dl-PCBs in invertebrates. In this study, a proteomic approach was applied to analyse the variations in the pattern of the gill proteome of the freshwater mussel Dreissena polymorpha. Mussels were exposed to a mixture of dl-PCBs, and to perform a more in-depth evaluation, we chose to investigate the role of gender in the proteome response by analysing male and female mussels separately. The results revealed significant modulation of the gill tissue proteome: glycolysis and Ca2 + homeostasis appear to be the main pathways targeted by dl-PCBs. In light of the differences between the male and female gill proteome profiles following exposure to dl-PCBs, further in-depth investigations of the role of gender in the protein expression profiles of a selected biological model are required.
No preview · Article · Jan 2013 · Comparative Biochemistry and Physiology Part D Genomics and Proteomics
[Show abstract][Hide abstract] ABSTRACT: Illicit drugs have been recognized as emerging environmental pollutants that could represent a potential risk for aquatic communities. Even if many studies have shown the occurrence of several drugs of abuse and their metabolites in freshwaters in the High ng/L to Low μg/L range worldwide, no information on their potentially harmful effects on non-target organisms is available. The aim of this study was to investigate sub-lethal effects induced by the main metabolite of cocaine, the benzoylecgonine (BE), on the freshwater bivalve Dreissena polymorpha. Mussels were exposed under semi-static conditions for 14days to two environmentally relevant BE concentrations (0.5μg/L and 1μg/L) and induced adverse effects were evaluated through the application of a suite of ten different biomarkers. We applied on bivalve hemocytes the single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test) to investigate DNA injuries, while the neutral red retention assay (NRRA) was used to assess BE cytotoxicity. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities, as well as the lipid peroxidation (LPO) and protein carbonyl content (PCC), were measured as oxidative stress indices in zebra mussel homogenates. Significant decrease in lysosomal membrane stability and imbalances of defense enzyme activities were found at both exposure concentrations, suggesting the involvement of oxidative stress in BE toxicity. Significant increases in LPO and PCC, as well as in primary (DNA strand breaks) and fixed DNA damage (apoptotic and micronucleated cell frequency), were found at the highest BE treatment, confirming that adverse effects to macromolecules were due to the increase of BE-induced oxidative stress.
Full-text · Article · Dec 2012 · Science of The Total Environment
[Show abstract][Hide abstract] ABSTRACT: Triclosan (TCS, 5-chloro-2-(2,4-dichlorophenoxy)phenol) is commonly used in several personal care products, textiles, and children's toys. Because the removal of TCS by wastewater treatment plants is incomplete, its environmental fate is to be discharged into freshwater ecosystems, where its ecotoxicological impact is still largely unexplored. Previously, we began a structured multi-tiered approach in order to evaluate TCS toxicity in the freshwater mussel Dreissena polymorpha. The results of our previous studies, based on in vitro and in vivo experiments, highlighted a pronounced cytogenotoxic effect exerted by TCS, and showed that an increase in oxidative stress was likely to be one of its main toxic mechanisms. In this work, in order to investigate TCS toxicity mechanisms in aquatic non-target species in greater depth, we decided to use a proteomic approach, analysing changes in protein expression profiles in gills of D. polymorpha exposed for seven days to TCS. Moreover, thiobarbituric acid reactive substances (TBARS) were measured to investigate further the role played by TCS in inducing oxidative stress. Finally, TCS bioaccumulation in mussel tissues was also assessed, to ensure an effective accumulation of the toxicant. Our results not only confirmed the role played by TCS in inducing oxidative stress, but furthered knowledge about the mechanism exerted by TCS in inducing toxicity in an aquatic non-target organisms. TCS induced significant alterations in protein expression profiles in gills of D. polymorpha. The wide range of proteins affected suggested that this chemical has marked effects on various biological processes, especially those involved in calcium binding or stress response. We also confirmed that the proteomic analysis, using 2-DE and de novo sequencing, is a reliable and powerful approach to investigate cellular responses to pollutants in a non-model organism with few genomic sequences available in databases.
No preview · Article · Mar 2012 · Aquatic toxicology (Amsterdam, Netherlands)
[Show abstract][Hide abstract] ABSTRACT: The origin and distribution of 19 priority pollutant polycyclic aromatic hydrocarbons (PAHs) in core sediments (<63 μm) from the Sundarban mangrove wetland, northeastern part of Bay of Bengal, India, were investigated by gas-chromatography/mass spectrometry (GC/MS). Sedimentary organic carbon and textural properties were also analyzed in order to assess the role of geochemistry on PAH distribution. ∑19PAHs concentration in sediments showed wide variations from 9.4–4222.8 ng/g dry weight, with an overall increase of the high molecular PAHs in the deeper depth profiles. Fluoranthene, chrysene, and pyrene were the most abundant hydrocarbons in sediments and it can be explained by active inputs of soil materials due to frequent strong rainfalls in this tropical climatic zone. A rather exceptional prevalence of high molecular weight PAHs was substantiated: nearly 80% of ∑19PAHs include fluoranthene (18–20%), chrysene (14–16%), and pyrene (10–11%). Carcinogenic compounds were present in moderately high (DBA, BkF, BbF) or negligible and low concentrations (BaP, InP) in a majority of cases. The PAH diagnostic ratios indicated that the PAHs in sediment cores were of pyrolytic origin, and that atmospheric deposition and land runoff may serve as the important pathways for PAHs input to the sediments. The total PAH levels were expressed as the benzo (a) pyrene toxicity equivalents (TEQcarc) making this compound a potential marker for PAH pollution. The baseline data can be used for regular ecological monitoring, considering the industrial and agricultural growth around this important estuarine environment. The results indicated that the PAHs in sediments of Sundarban wetland resulted in low to moderate ecosystem risk.
Full-text · Article · Jan 2012 · Polycyclic Aromatic Compounds
[Show abstract][Hide abstract] ABSTRACT: The increase in global consumption of illicit drugs has produced not only social and medical problems but also a potential new environmental danger. Indeed, it has been established that drugs consumed by humans end up in surface waters, after being carried through the sewage system. Although many studies to measure concentrations of several drugs of abuse in freshwater worldwide have been conducted, no data have been available to evaluate their potentially harmful effects on non-target organisms until now. The present study represents the first attempt to investigate the cyto-genotoxic effects of cocaine, one of the primary drugs consumed in Western Countries, in the biological model Dreissena polymorpha by the use of a biomarker battery. We performed the following tests on Zebra mussel hemocytes: the single cell gel electrophoresis (SCGE) assay, the apoptosis frequency evaluation and the micronucleus assay (MN test) for the evaluation of genotoxicity and the lysosomal membranes stability test (neutral red retention assay; NRRA) to identify the cocaine cytotoxicity. We exposed the molluscs for 96 h to three different nominal concentrations in water (40 ng L(-1); 220 ng L(-1); and 10 μg L(-1)). Cocaine caused significant (p<0.05) primary DNA damage in this short-term experiment, but it also caused a clear increase in micronucleated cells and a marked rise in apoptosis, which was evident in samples from even the lowest environmental cocaine concentration. Because cocaine decreased the stability of lysosomal membranes, we also highlighted its cytotoxicity and the possible implications of oxidative stress for the observed genotoxic effects.
[Show abstract][Hide abstract] ABSTRACT: It has recently been established that the use of proteomics can be a useful tool in the field of ecotoxicology. Despite the fact that the mussel Dreissena polymorpha is a valuable bioindicator for freshwater ecosystems, the application of a proteomic approach with this organism has not been deeply investigated. To this end, several zebra mussel specimens were subjected to a 7-day exposure of two different concentrations (0.1 and 2 μg L⁻¹) of the model pollutant benzo[α]pyrene (B[α]P). Changes in protein expression profiles were investigated in gill cytosolic fractions from control/exposed male and female mussels using 2-DE electrophoresis. B[α]P bioaccumulation in mussel soft tissue was also assessed to validate exposure to the selected chemical. We evaluated overall changes in expression profiles for 28 proteins in exposed mussels, 16 and 12 of which were, respectively, over- and under-expressed. Surprisingly, the comparative analysis of protein data sets showed no proteins that varied commonly between the two different B[α]P concentrations. Spots of interest were manually excised and analysed by MALDI-TOF/TOF mass spectrometry. The most significant proteins that were identified as altered were related to oxidative stress, signal transduction, cellular structure and metabolism. This preliminary study indicates the feasibility of a proteomic approach with the freshwater mussel D. polymorpha and provides a starting point for similar investigations. Our results confirm the need to increase the number of invertebrate proteomic studies in order to increase the following: their representation in databases and the successful identification of their most relevant proteins. Finally, additional studies investigating the role of gender and protein modulation are warranted.
[Show abstract][Hide abstract] ABSTRACT: Mussels (Mytilus galloprovincialis) were exposed to different concentrations of testosterone (T: 20, 200 and 2000ng/L) in a semi-static water regime (1-day dosing intervals) for up to 5 days in an attempt to see whether endogenous steroid levels and steroid metabolism were altered by exogenous exposure to testosterone. Whole tissue levels of total testosterone (free+esterified) sharply increased in a concentration-dependent manner, from 2ng/g in controls to 290ng/g in organisms exposed to the highest concentration. In contrast, levels of free testosterone were only significantly elevated at the high-exposure group (5-fold increase with respect to controls). Increased activity of palmitoyl-CoA:testosterone acyltransferase (ATAT) was detected in organisms exposed to the highest concentration of testosterone, while those exposed to low and medium concentrations showed significant alterations in their polyunsaturated fatty acid profiles. The obtained results suggest that esterification of the excess of T with fatty acids might act as a homeostatic mechanism to maintain endogenous levels of free T stable. Interestingly, a decrease in CYP3A-like activity was detected in T-exposed mussels together with a significant decrease in the metabolism of the androgen precursor androstenedione to dihydrotestosterone (5α-DHT). Overall, the work contributes to the better knowledge of androgen metabolism in mussels.
[Show abstract][Hide abstract] ABSTRACT: HIV-1 non-B subtypes have recently entered Western Europe following immigration from other regions. The distribution of non-B clades and their association with demographic factors, over the entire course of the HIV-1 epidemic, have not been fully investigated in Italy.
We carried out a phylogenetic analysis of HIV-1 pol sequences derived from 3670 patients followed at 50 Italian clinical centres over nearly three decades.
Overall, 417 patients (11.4%) carried non-B subtypes. The prevalence of non-B strains increased from 2.6% in 1980-1992 to 18.9% in 1993-2008 (P<0.0001) in a subset of 2479 subjects with a known year of diagnosis. A multivariate analysis on a subset of 1364 patients for whom relevant demographic data were available indicated that African ethnicity, heterosexual route of infection and year of diagnosis were independently associated with non-B HIV-1 infection (P ≤ 0.0001). All pure subtypes, except for clade K, and seven circulating recombinant forms were detected, accounting for 56.6 and 34.1% of the non-B infections, respectively. The F1 subtype was the most prevalent non-B clade among Europeans and was acquired heterosexually in half of this patient population. Unique recombinant forms accounted for 9.4% of the non-B sequences and showed a B/F1 recombination pattern in one-third of cases.
The circulation of non-B clades has significantly increased in Italy in association with demographic changes. Spread of the F1 subtype and B/F recombinants appears to predominate, which may result in a redistribution of the relative proportions of the different strains, and this could lead to overlapping epidemics. Thus, the HIV-1 landscape in Italy may in future be distinct from that of the rest of Europe.
[Show abstract][Hide abstract] ABSTRACT: In 1996, dichlorodiphenyltrichloroethane (DDT) pollution of industrial origin was discovered in Lake Maggiore. It was caused
by industrial effluents on a tributary of the River Toce, one of the major affluents of the lake in correspondence of Pallanza
Bay. This event is the worst case of environmental pollution that has occurred in Western countries in the last 25years,
not due to agricultural use of DDT, but because of an accidental industrial discharge. Heavy polychlorinated biphenyls (PCBs)
pollution was also noticed in 2002, with concentration levels three to seven times higher than those measured in other Italian
subalpine lakes. In this study, the current DDT and PCBs contamination levels were assessed according to their presence in
zebra mussel (Dreissena polymorpha) specimens sampled in the last 5years (2003–2008) in eight sampling stations of Lake Maggiore, chosen to cover the entire
perimeter of the basin. Moreover, for two stations (Baveno and Pallanza-Villa Taranto) located inside and outside Pallanza
Bay, respectively, it is possible to make comparisons starting from 1996. The results obtained show how Lake Maggiore is still
an ecosystem with a severe environmental risk, more than 10years after the original insecticide discharge. DDT contamination
continues to evolve, and natural events, like lake overturn, floods, and heavy rains, can have a great influence on the insecticide
levels in the lake. By contrast, PCB contamination is absolutely negligible, even if the peak of pollution revealed in 2002
seems to indicate that these pollutants are still present in large quantities in the Lake Maggiore watershed.
No preview · Article · Jul 2010 · Water Air and Soil Pollution
[Show abstract][Hide abstract] ABSTRACT: Nonylphenol (NP) represents the most critical metabolite of alkylphenols (APs) and alkylphenol ethoxylates (APEs), non-ionic surfactants widely used in the formulation of domestic and industrial products. On the basis of in vitro and in vivo animal studies 4-nonylphenol (4-NP) is considered an endocrine disrupting chemical (EDC). The evidence to date indicates that mollusks are able to synthesize sex steroids from the precursor cholesterol and their endocrine pathways are theoretically susceptible to disruption. The aim of this study was to investigate the endocrine modulating potency of 4-NP in the freshwater mussel Dreissena polymorpha by looking at endogenous steroid levels in control and exposed individuals. 4-NP bioaccumulation in mussels tissues and alterations in the activity of enzymes related both to oxidative stress (catalase - CAT- and glutathione peroxidase - GPX-) and phase II metabolism (glutathione-S-transferase - GST-) were also assessed. The results highlighted a build-up of 4-NP in exposed mussels and an overall decrease of 17-beta-estradiol and testosterone levels. On the other hand this chemical at the tested concentrations does not interfere with the antioxidant defense mechanisms in D. polymorpha. The mechanisms by which 4-NP alter steroids levels are unknown and require more in-depth investigations.
Full-text · Article · Apr 2010 · Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently detected pharmaceuticals in aquatic environments. They are the sixth most sold drugs worldwide and are usually found in significant quantities in municipal effluents. The aim of this study was to assess a first screening evaluation of the cytogenotoxicity of three common NSAIDs (diclofenac, ibuprofen and paracetamol) using an in vitro biomarker approach on the haemocytes of the freshwater bivalve zebra mussel (Dreissena polymorpha). Genotoxicity was evaluated by SCGE (single cell gel electrophoresis) and DNA diffusion assay while cytotoxicity was evaluated by neutral red retention assay (NRRA). The exposure of the haemocytes to increasing concentrations of the three drugs, chosen based on the results of a viability test, revealed high cytogenotoxic potential and allowed the creation of the first toxicity scale for zebra mussel haemocytes (paracetamol < diclofenac < ibuprofen). The present results lay the groundwork for in vivo exposures, which will allow for a better definition of the observed cytogenotoxicity of these molecules in a setting miming real environmental exposure.
No preview · Article · Aug 2009 · Toxicology in Vitro
[Show abstract][Hide abstract] ABSTRACT: Pharmaceuticals and personal care products (PPCPs) have been detected in several aquatic ecosystems for a number of years, but the potential for biological effects in exposed non-target organisms is only now being reported. In this study the potential cellular damage due to two of the main PPCPs found in aquatic environments was investigated by in vitro exposures. Hemolymph samples of the freshwater bivalve Dreissena polymorpha were collected and treated with increasing concentrations of the antibacterial agent Triclosan (TCS) and the antibiotic Trimethoprim (TMP). Doses selected for TCS were 0.1, 0.15, 0.2, and 0.3 µM, while 0.2, 1, and 5 µM for TMP exposures, respectively. We evaluated the potential genotoxicity on hemocytes by the SCGE (single cell gel electrophoresis) assay and apoptosis frequency evaluation, while the cytotoxicity was measured by the lysosomal membranes stability test (NRRA, neutral red retention assay). TCS genotoxicity increased in a dose-dependent manner and this pharmaceutical significantly affects hemocyte functionality due to severe DNA injuries at very low doses. In contrast, TMP seems to be less dangerous than TCS for D. polymorpha because the cytotoxic and the moderate genotoxic effects noticed were obtained only at very high concentration levels.
Full-text · Article · Jul 2009 · Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology
[Show abstract][Hide abstract] ABSTRACT: In this work, we investigated the possible genotoxic and cytotoxic effects of the antibacterial agent Triclosan in hemocytes of the freshwater bivalve Zebra mussel (Dreissena polymorpha). For this study, we used several biomarkers for in vivo experiments (96h of exposure) carried out at three possible environmental Triclosan concentrations (1, 2, 3nM). We used the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test) and the measure of the apoptotic frequency (Halo assay) to measure the genotoxic potential of Triclosan, and the neutral red retention assay (NRRA) as a measure of lysosomal membrane stability to identify general cellular stress. We observed significant increases in all of the genotoxic biomarkers examined as early as 24h after initial exposure, as well as a clear destabilization of lysosomal membranes (after 48h), indicating that this chemical is potentially dangerous for the entire aquatic biocoenosis. A comparison of these in vivo data with existing data from in vitro experiments allowed us to suggest possible mechanisms of action for Triclosan in this bivalve. Although further studies are needed to confirm the possible modes of action, our study is the first to report on the effects of this widespread antibiotic on freshwater invertebrates.
No preview · Article · Dec 2008 · Aquatic toxicology (Amsterdam, Netherlands)