Cellular pathways affected by carbon nanopowder-benzo(α)pyrene complex in human skin fibroblasts identified by proteomics.

One of the crucial and unsolved problems of the airborne carbon nanoparticles is the role played by the adsorbed environmental pollutants on their toxicological effect. Indeed, in the urban areas, the carbon nanoparticles usually adsorb some atmospheric contaminants, whose one of the leading representatives is the benzo(α)pyrene. Herein, we used the proteomics to investigate the alteration of toxicological pathways due to the carbon nanopowder-benzo(α)pyrene complex in comparison with the two contaminants administered alone on human skin-derived fibroblasts (hSDFs) exposed for 8 days in semi-static conditions. The preliminary confocal microscopy observations highlighted that carbon-nanopowder was able to pass through the cell membranes and accumulate into the cytoplasm both when administered alone and with the adsorbed benzo(α)pyrene. Proteomics revealed that the effect of carbon nanopowder-benzo(α)pyrene complex seems to be related to a new toxicological behavior instead of simple additive or synergistic effects. In detail, the cellular pathways modulated by the complex were mainly related to energy shift (glycolysis and pentose phosphate pathway), apoptosis, stress response and cellular trafficking.

Ingestion and effects of polystyrene nanoparticles in the silkworm Bombyx mori.

Information on the occurrence and effects of nanoplastics in ecosystems worldwide currently represent one of the main challenges from the ecotoxicological point of view. This is particularly true for terrestrial environments, in which nanoplastics are released directly by human activities or derive from the fragmentation of larger plastic items incorrectly disposed. Since insects can represent a target for these emerging contaminants in land-based community, the aim of this study was the evaluation of ingestion of 0.5 µm polystyrene nanoplastics and their effects in silkworm (Bombyx mori) larvae, a useful and well-studied insect model. The ingestion of nanoplastics, the possible infiltration in the tissues and organ accumulation were checked by confocal microscopy, while we evaluated the effects due to the administered nanoplastics through a multi-tier approach based on insect development and behaviour assessment, as endpoints at organism level, and the measurements of some biochemical responses associated with the imbalance of the redox status (superoxide dismutase, catalase, glutathione s-transferase, reactive oxygen species evaluation, lipid peroxidation) to investigate the cellular and molecular effects. We observed the presence of microplastics in the intestinal lumen, but also inside the larvae, specifically into the midgut epithelium, the Malpighian tubules and in the haemocytes. The behavioural observations revealed a significant (p < 0.05) increase of erratic movements and chemotaxis defects, potentially reflecting negative indirect effects on B. mori survival and fitness, while neither effect on insect development nor redox status imbalance were measured, with the exception of the significant (p < 0.05) inhibition of superoxide dismutase activity.

First evidence of protein modulation by polystyrene microplastics in a freshwater biological model.

Microplastics (MPs) are now one of the major environmental problems due to the large amount released in aquatic and terrestrial ecosystems, as well as their diffuse sources and potential impacts on organisms and human health. Still the molecular and cellular targets of microplastics' toxicity have not yet been identified and their mechanism of actions in aquatic organisms are largely unknown. In order to partially fill this gap, we used a mass spectrometry based functional proteomics to evaluate the modulation of protein profiling in zebra mussel (Dreissena polymorpha), one of the most useful freshwater biological model. Mussels were exposed for 6 days in static conditions to two different microplastic mixtures, composed by two types of virgin polystyrene microbeads (size = 1 and 10 µm) each one. The mixture at the lowest concentration contained 5 × 105 MP/L of 1 µm and 5 × 105 MP/L of 10 µm, while the higher one was arranged with 2 × 106 MP/L of 1 µm and 2 × 106 MP/L of 10 µm. Proteomics' analyses of gills showed the complete lack of proteins' modulation after the exposure to the low-concentrated mixture, while even 78 proteins were differentially modulated after the exposure to the high-concentrated one, suggesting the presence of an effect-threshold. The modulated proteins belong to 5 different classes mainly involved in the structure and function of ribosomes, energy metabolism, cellular trafficking, RNA-binding and cytoskeleton, all related to the response against the oxidative stress.

Levels and congener profiles of polybrominated diphenyl ethers (PBDEs) in Zebra mussels (D. polymorpha) from Lake Maggiore (Italy).

Several congeners of polybrominated diphenyl ethers (PBDEs) were monitored in 14 different sampling stations of Lake Maggiore, the second largest Italian lake in regard to surface, volume and average depth, using the sentinel-organism Zebra mussel (Dreissena polymorpha). Results revealed a moderate contamination with summation operatorPBDE levels (BDE-17, -28, -47, -66, -71, -85, -99, -100, -138, -153, -154, -183, -190 and -209) ranging from 40 to 447ngg(-1) lipid weight which are similar to those found in environments polluted by deposition or atmospheric transport. The general order of decreasing congener contribution to the total load was BDE-47>-99>-100>-209, which closely reflected patterns observed in mussels collected in freshwater ecosystems worldwide.

Organochlorine pesticide residues in sediment cores of sunderban wetland, northeastern part of Bay of Bengal, India, and their ecotoxicological significance.

This paper presents the first comprehensive report of the organochlorine pesticide residues (OCs) such as hexachlorocyclohexane isomers (HCHs), dichlorodiphenyltrichloroethane and its six metabolites (DDTs), and hexachlorobenzene (HCB) in core sediments (<63-microm particle size) from the Indian Sunderban wetland. The pooled mean values of the mass fraction of SigmaHCHs, HCB, and SigmaDDTs in the sediments were 0.05-12, 0.05-1.4, and 0.05-11.5 ng g(-1) dry weight, respectively. The vertical distribution of pesticides reveals an erratic pattern. The concentration of four isomers of HCHs reveals a heterogenic distribution where gamma-HCH (lindane) and beta-HCH shared the dominant part. The mass fraction of HCB did not show any sharp spatial variation. The prevailing sequence of DDT metabolites indicates an active degradation of the parent compound in the sediments and/or inputs of already degraded pp'DDT to the region. Peak concentrations of HCH isomers and DDT metabolites have the potential to induce ecotoxicological impact as per the sediment quality guidelines.

S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium.

Glutathione (GSH) is one of the most important defense mechanisms against oxidative stress in the respiratory epithelial lining fluid. Considering that GSH secretion in respiratory cells has been postulated to be at least partially electrogenic, and that the mucoregulator S-carbocysteine lysine salt monohydrate (S-CMC-Lys) can cause an activation of epithelial Cl(-) conductance, the purpose of this study was to verify whether S-CMC-Lys is able to stimulate GSH secretion. Experiments have been performed by patch-clamp technique, by high-performance liquid chromatography (HPLC) assay, and by Western blot analysis on cultured lines of human respiratory cells (WI-26VA4 and CFT1-C2). In whole-cell configuration, after cell exposure to 100 microM S-CMC-Lys, a current due to an outward GSH flux was observed, which was inhibitable by 5-nitro-2-(3-phenylpropylamino)-benzoate and glibenclamide. This current was not observed in CFT1-C2 cells, where a functional cystic fibrosis transmembrane conductance regulator (CFTR) is lacking. Inside-out patch-clamp experiments (GSH on the cytoplasm side, Cl(-) on the extracellular side) showed the activity of a channel, which was able to conduct current in both directions: the single channel conductance was 2-4 pS, and the open probability (P(o)) was low and voltage-independent. After preincubation with 100 microM S-CMC-Lys, there was an increase in P(o), in the number of active channels present in each patch, and in the relative permeability to GSH vs Cl(-). Outwardly directed efflux of GSH could also be increased by protein kinase A, adenosine 5'-triphosphate, and cyclic adenosine monophosphate (cAMP) added to the cytoplasmic side (whole-cell configuration). The increased secretion of GSH observed in the presence of S-CMC-Lys or 8-bromoadenosine-3',5'-cyclic monophosphate was also confirmed by HPLC assay of GSH on a confluent monolayer of respiratory cells. Western blot analysis confirmed the presence of CFTR in WI-26VA4 cells. This study suggests that S-CMC-Lys is able to stimulate a channel-mediated GSH secretion by human respiratory cells: electrophysiological and pharmacological characteristics of this channel are similar to those of the CFTR channel.

Carbon nanopowder acts as a Trojan-horse for benzo(α)pyrene in Danio rerio embryos.

Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the "particle-specific" effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

New evidences for old biomarkers: effects of several xenobiotics on EROD and AChE activities in Zebra mussel (Dreissena polymorpha).

The biomarker approach is widely used both in vertebrates and invertebrates for environmental biomonitoring, because it can supply an integrated response for multi-xenobiotics contamination. However, the use of biomarkers requires the identification of every possible variation that can influence the biochemical response, because ecosystems are generally subject to a mixture of pollutants, which can create additive, opposite or competitive effects. In recent years, there has been considerable interest in the use of biomarkers within marine bivalves, while very few data are available for freshwater molluscs. The aim of this research was to investigate changes on EROD and AChE activities in the freshwater bivalve Zebra mussel (Dreissena polymorpha) exposed to different pollutants (Arochlor 1260, CB 153 and 126, pp'DDT, chlorpyrifos, carbaryl) at laboratory conditions, in order to standardize the analytical procedures and to highlight eventual interferences on enzyme activities. Chemical concentrations in the mussel soft tissues were analyzed by GC/MS-MS. Main results showed a significant induction of EROD activity when mussels were exposed to 100 ng/l of PCB mixture of Arochlor 1260 and dioxin-like CB 126, but this congener showed also a clear competitive inhibition after 48 h of exposure. Surprisingly, pp'DDT determined a significant decrease of basal EROD activity after only 24 h of exposure, even if it was not possible to discriminate between the effect of the parent compound and that of its metabolites (DDD, DDE). We also found an interaction between the organophosphate insecticide chlorpyrifos, which does not directly decrease the AChE activity, and terbutilazine. This herbicide increased the biotransformation of the organophosphate compound to its oxidized metabolite (oxon), a much stronger AChE inhibitor. The possible use of the oxime Pyridine-2-Aldoxime Methochloride (2-PAM) to bring back the catalytic activity to basal levels was also demonstrated.

Screening of POP pollution by AChE and EROD activities in Zebra mussels from the Italian Great Lakes.

The increase of ethoxyresorufin dealkylation (EROD) and the inhibition of acetylcholinesterase (AChE) as biomarkers have been commonly used in vertebrates for the persistent organic pollutants (POPs) biomonitoring of aquatic environments, but very few studies have been performed for invertebrates. Previous researches demonstrated the interference due to some chemicals on EROD and AChE activities of the freshwater bivalve Zebra mussel (Dreissena polymorpha) in laboratory and field studies, showing its possible use for the screening of POP effects. We investigated the contamination of the Italian sub-alpine great lakes (Maggiore, Lugano, Como, Iseo, Garda) by the biomarker approach on Zebra mussel specimens collected at 17 sampling sites with different morphometric characteristics and anthropization levels. Results showed a homogeneous contamination of AChE inhibitors in Lake Garda, Maggiore, Como and Iseo with values ranging from 0.5 to 3 nmol/min/mg proteins and with an average inhibition of about 66% to controls. The planar compounds pollution, able to activate the EROD activity, seems higher in some sampling stations of Lake Garda, Como and Iseo (2-4 pmol/min/mg proteins) than that measured in Lake Lugano (1.5-3 pmol/min/mg proteins). On the contrary, the enzyme activity in Lake Maggiore showed an interesting opposite effect of AhR-binding compounds and trace metals. Finally, the possible use of Zebra mussel specimens maintained at laboratory conditions as controls against the selection of the less polluted sampling site is discussed.

Toxicity decrease in urban wastewaters treated by a new biofiltration process.

We carried out a project aimed to evaluate the possible role played by the freshwater zebra mussel (Dreissena polymorpha) in the possible decrease of some environmental pollutants recalcitrant to tradition wastewater treatments. By the help of a pilot-plant built in the largest wastewater treatment plant of Milan (Italy), we tested several waste mixtures in order to measure the chemicals' abatement made by mussels' biofiltration. This study represents the last step of the wider project and it aimed to evaluate if the decrease in the concentration of some urban pollutants measured in wastewaters was followed by a corresponding toxicity reduction. Thus, we performed 7-day exposures under laboratory conditions to test the toxicity of the raw wastewaters and those preliminary filtered by zebra mussels, through the measurement of different end-points of acute and chronic toxicity. Results showed a clear positive effect of mussels' biofiltration mainly to decrease the acute toxicity made by the two tested wastewater mixtures, while the biomarkers' suite used to evaluate the chronic toxicity showed contradictory results.

Does zebra mussel (Dreissena polymorpha) represent the freshwater counterpart of Mytilus in ecotoxicological studies? A critical review.

One of the fundamentals in the ecotoxicological studies is the need of data comparison, which can be easily reached with the help of a standardized biological model. In this context, any biological model has been still proposed for the biomonitoring and risk evaluation of freshwaters until now. The aim of this review is to illustrate the ecotoxicological studies carried out with the zebra mussel Dreissena polymorpha in order to suggest this bivalve species as possible reference organism for inland waters. In detail,we showed its application in biomonitoring, as well as for the evaluation of adverse effects induced by several pollutants, using both in vitro and in vivo experiments. We discussed the advantages by the use of D. polymorpha for ecotoxicological studies, but also the possible limitations due to its invasive nature.

Vaginal transmission of HIV-1 in hu-SCID mice: a new model for the evaluation of vaginal microbicides.

OBJECTIVE: To develop an animal model of vaginal transmission of HIV-1 for the evaluation of vaginal microbicides. DESIGN: Vaginal infection was performed in SCID mice reconstituted with 4 x 107 human peripheral blood lymphocytes (hu-PBL) by non-invasive vaginal administration. The hu-PBL were previously infected in vitro with a non-syncytium (NSI) strain of HIV-1 (SF162) (hu-PBL-SCID). Lymphocyte migration in vivo was examined using fluorescently labelled human lymphocytes. METHODS: The percentage of CD4 T cells, plasma viral load and p24 antigen were evaluated using fluorescent activated cell sorting (FACS), the Amplicor HIV-1 monitor kit and enzyme-linked immunosorbent assay, respectively. Polymerase chain reaction (PCR) analysis was performed on DNA extracted from spleen and lymph nodes. For in vivo migration of labelled lymphocytes, the mice were sacrificed after 4, 24 and 48 h; vaginae and local lymph nodes were removed, snap frozen with OCT, sectioned and examined by fluorescent microscopy and FACS. RESULTS: HIV transmission was established using virus-infected cells inoculated vaginally, as shown by FACS, HIV viral load, p24 and PCR results. Labelled cells were easily located within the vaginal tissues after 4 h. However, few or no cells could be identified after 24 or 48 h at the vaginal level, whereas labelled cells could be detected at the level of regional lymph nodes. CONCLUSIONS: Because of its simplicity and practical features compared with other animal models, the vaginal HIV-infected hu-SCID mouse model may prove useful to test the activity of compounds against cell-associated HIV and, possibly, other sexually transmitted diseases.

Plasma HIV-1 copy number and in vitro infectivity of plasma prior to and during combination antiretroviral treatment.

Some studies on untreated patients have shown a general correlation between plasma HIV copy number and plasma infectivity in in vitro models. Recent observations also indicate that HIV-RNA level is an important predictor of perinatal transmission and may also have a role in heterosexual transmission. To further analyse the correlation between HIV viral load and plasma infectivity, we studied the relationship between HIV-1 plasma copy number and plasma infectivity prior to and during treatment with antiretroviral combination regimens in HIV-1 infected adults. Plasma infectivity was assessed in vitro by coculture of plasma from HIV-positive patients with PHA-stimulated fresh PBMC from uninfected donors. A positive plasma isolation, in almost all cases (43/45) and irrespective of treatment status, was associated with an HIV viral load higher than 100000 copies per ml, with higher plasma HIV-1 RNA values in isolation-positive samples compared with isolation-negative samples (median values, 710000 vs. 37500 copies per ml, respectively). SI and NSI strains had similarly high viral load values (470000 vs. 790000 copies per ml), but CD4 counts were lower in the SI phenotype group. Our data indicate that low levels of viral load are only exceptionally associated with isolation from plasma in the in vitro model we used. This observation confirms indirectly the presence of an association between viral load and infectivity. The requisite of a high plasma viral load in order to obtain infectivity (i.e. positivity of HIV isolation from plasma) also seems maintained under antiretroviral treatment, adding confidence in the conclusion that reductions in viral load translate into reduction of plasma infectivity. Due to the extreme complexity of factors determining transmission, a very prudent interpretation of the results is essential when information from experimental studies has to be transferred to clinical situations requiring assessment of risks or clinical decisions.

DDT is still a problem in developed countries: the heavy pollution of Lake Maggiore.

The Zebra mussel (Dreissena polymorpha), one of the most widely used bioindicators of persistent organic pollutants, trace metals and radionuclides in several worldwide freshwater ecosystems, has been used to monitor DDT contamination trends in Lake Maggiore since 1996, caused by industrial effluents on a tributary of the River Toce, one of the major affluents of the lake. Dreissena specimens were collected at two sampling sites, one within the Baveno Bay, where the River Toce flows, and the other outside (Villa Taranto). Total DDT levels (3119.6 ng/g lipids at Baveno and 1351.2 ng/g lipids at Villa Taranto) in the soft tissues of the Zebra mussel decreased at both stations by about 30-50% in the first year after the closure of the chemical plant reaching an almost steady-state condition. The high concentrations measured in Zebra mussel specimens of Baveno Bay in 2000 (1947 ng/g lipids) and the percentage of pp'DDE in comparison with total DDT concentration, which showed a slight increase in the last years, clearly indicate that a contamination source is still present, deriving probably from the lacustrine sediments and the River Toce. Data show that the environmental risk is very high within the Baveno Bay and the recovery time should be longer than in the other parts of the lake, where DDT levels in Dreissena are presently two times higher than those measured in the other Italian subalpine lakes.

The PCB pollution of Lake Iseo (N. Italy) and the role of biomagnification in the pelagic food web.

Several models of varying complexity have been used to predict pollutant concentrations in the higher levels of the food web from those in lower levels, but the role of the biomagnification process in aquatic food chains is still controversial. We used the fugacity-based approach to verify the transfer of PCBs through the pelagic food chain of Lake Iseo (N. Italy), sampling several zebra mussel specimens and some fish belonging of different trophic levels. The zebra mussel seems to be a suitable starting species for modelling the bioaccumulation process through the trophic web, not only because its physiological characteristics and population size do not change much with time (as do algae and zooplankton) but also because it takes up toxicants exclusively from the water, as shown by the application of two predictive trophic models commonly used. The data provided by one of those models were in good agreement with our experimental data on fish in Lake Iseo, that show a not negligible uptake from food for the top predator species (pike and perch) with an increase of about three times in comparison with the PCB levels measured in the zebra mussel specimens.

Present status of POP contamination in Lake Maggiore (Italy).

In the last decade, Lake Maggiore has been subject to heavy DDT contamination due to a chemical plant located near the main influent of the Baveno Bay. The freshwater bivalve zebra mussel (D. polymorpha) was used as a bioindicator of several POPs (DDTs, PCBs, HCB, HCHs) to follow their concentration trend after a heavy flood in autumn 2000. Sampling of mollusc specimens were carried out monthly from April 2001 to October 2002 at two different stations in and outside the bay. Results showed worsening of DDT pollution due to the transport of insecticide from contaminated sediments and soils still present in the closed chemical plant site. Levels of about 4.5 microg/g lipids in soft tissues of specimens from the most contaminated site and 2.0 microg/g lipids outside it were found, which are twice those measured before the flood. HCHs and HCB values were always very low, but it was noticed since winter 2001-2002 a sharp increase of PCB pollution, with values of about 3-4 microg/g lipids, not due to the 2000 flood, but probably to improper discharge or release of contaminated sediments from numerous dams located in the watershed. Concentrations of total dioxin-like PCBs reached dangerous levels for the water community. Fish consumption may be a risk for human health especially for resident population, bearing in mind that fish usually have a higher POP concentration than zebra mussel.

Use of zebra mussel (Dreissena polymorpha) to assess trace metal contamination in the largest Italian subalpine lakes.

Trace metal (Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn) contamination was evaluated in zebra mussels from the lakes Maggiore, Lugano, Como, Iseo and Garda, which are located in the most highly populated and industrialised area in Italy. Zebra mussels from Lake Maggiore contained the highest concentrations (3.44, 1.51, 4.97, 0.158, 5.87, 346 microg g(-1) for Cd, Co, Cr, Hg, Pb, Zn, respectively) of all metals analysed except Cu and Ni. The lowest levels of most metals were in animals from Garda and Lugano (0.78 and 0.60 microg g(-1) for Cd, 2.87 and 2.03 microg g(-1) for Cr, 0.065 and 0.049 microg g(-1) for Hg, 12.1 and 11.9 microg g(-1) for Ni, 1.96 and 2.46 microg g(-1) for Pb, 158 and 163 microg g(-1) for Zn). The most contaminated sites and possible local sources of metals were identified for each lake, and the lakes classified into quality classes concerning metal pollution.

DDT contamination in Lake Maggiore (N. Italy) and effects on zebra mussel spawning.

Zebra mussel (Dreissena polymorpha) is commonly used as contaminant bioindicator in Europe and North America. We used the zebra mussel to follow DDT pollution trends from 1996 to 1997 in Pallanza bay, Lake Maggiore, near the inlet of the River Toce, after a DDT-manufacturing plant discharging residues into a tributary of the River Toce had closed down. DDT contamination fell off sharply outside the bay, but tissue concentrations of the parent compound and residues remained high in molluscs sampled within the bay a year later. Molluscs collected in Pallanza bay in June 1997 released gametes earlier than those sampled at a nearby reference station. Histological studies showed that a significant percentage of these specimens showed marked oocyte degeneration suggesting that DDTs have endocrine-disrupting effects in this species.

POPs in edible clams from different Italian and European markets and possible human health risk.

Human intake of persistent organic pollutants (POPs) occurs primarily through diet and fish and other seafood represent the principal means of contamination. Despite this, few legal limits have been established to protect human health from the various chemicals that are alleged to be carcinogenic or endocrine disruptor compounds. Several pools of edible clams (Tapes philippinarum and Venus gallina) bought in different Italian and European markets were examined in order to perform a risk assessment of some of these contaminants and also to identify possible health issues. POP concentrations in the soft tissues of mollusks varied considerably among source farming sites, particularly in the Venice Lagoon. Some POPs (DDTs, HCB and HCHs) showed very low concentrations and no risk for human health, in line with the limits established by European law. In contrast, PAHs could represent a potential hazard for human health and PCB levels in the most contaminated sites reached or even exceed the Minimal Risk Level as set by the ATSDR for human consumption, even if all the samples did not exceed the PCB limits in edible seafood as set by the FDA.

Analysis of the Dreissena polymorpha gill proteome following exposure to dioxin-like PCBs: mechanism of action and the role of gender.

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 Ca(2+) 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.