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.

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.

Toxicity of 4-nonylphenol in spiked sediment to three populations of Chironomus riparius.

Nonylphenols (NPs) are the primary stable metabolites of alkylphenol polyethoxylates (APEs), a family of compounds widely used in industry and in some domestic products. As NPs accumulate in sediments in aquatic environments, the risk to benthic organisms needs to be assessed. In this study 4NP-spiked sediments were tested on larvae of the dipteran Chironomus riparius. First instar larvae obtained from populations at three different sources were used. To spike the sediments, an equilibration procedure between water and sediment was adopted to avoid the use of solvents. Lower 10-d LC50 values were determined for two populations of C. riparius from clean environments (315-465 and 315-350 microg g(-1) d.w., respectively) than those of a strain deriving from a population collected in a polluted river (600-680 microg g(-1) d.w.). Larval growth always decreased with increasing 4NP concentration but without any defined trend. The results of this study suggest that tolerance to the toxicant can be developed in populations of polluted environments and that testing procedures should be standardised.

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.

Multi-biomarker approach to investigate the state of contamination of the R. Lambro/R. Po confluence (Italy) by zebra mussel (Dreissena polymorpha).

This study assesses the response of the zebra mussel (Dreissena polymorpha) to chemical pollution derived from the R. Lambro/R. Po confluence, which is one of the most polluted aquatic environments in Europe. The mussels were tested under laboratory conditions to water sampled in the spring and fall at three sites located upstream or downstream of the confluence or directly in R. Lambro alone. We performed on mussel specimens a biomarker battery composed by eight different assays: single cell gel electrophoresis, apoptosis determination, the micronucleus test and Neutral Red retention, as well as catalase, superoxide dismutase, glutathione peroxidase and glutathione transferase activities. We also evaluated the bioaccumulation of several organic pollutants (PAHs, PCBs, DDTs, HCHs and HCB) to characterize the sampling sites. Significant increases in DNA strand breaks, apoptosis and micronuclei were observed, with no significant seasonal differences. We observed a clear induction of the enzyme activities measured in the spring, but the enzymatic activity trend in the fall was very complex, with several enzymes returning to baseline levels of activity, suggesting a possible seasonal change in chemical mixture characteristics.

In vivo experiments for the evaluation of genotoxic and cytotoxic effects of Triclosan in Zebra mussel hemocytes.

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.

Cytotoxic and genotoxic effects of in vitro exposure to triclosan and trimethoprim on zebra mussel (Dreissena polymorpha) hemocytes.

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 microM, while 0.2, 1, and 5 microM 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.

A multi-biomarker assessment of the impact of the antibacterial trimethoprim on the non-target organism Zebra mussel (Dreissena polymorpha).

A battery of eight biomarkers was applied in the freshwater mussel Dreissena polymorpha to evaluate potential sub-lethal effects of the antimicrobial trimethoprim (TMP, 5-[3,4,5-trimethoxybenzyl]pyrimidine-2,4-diamine). Mussels were exposed for 96 h to increasing concentrations (1, 3, 10 nM) of TMP in in vivo experiments. We determined the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test), the apoptotic frequency (Halo assay) and the lysosomal membrane stability (Neutral Red Retention Assay) in mussel hemocytes. Moreover, to reveal whether the oxidative status was altered, measurements of the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and the phase II detoxifying enzyme glutathione S-transferase (GST) were performed using the cytosolic fraction extracted from a pool of entire mussels. The biomarker battery pointed out only a moderate cyto- and genotoxicity on Zebra mussel hemocytes since only a slight increase in DNA damage was registered by apoptosis induction and MN frequency, while significant differences of lysosomal membrane stability from baseline levels were measured at 3 and 10 nM at the end of exposures only. Finally, TMP seems to have a very low induction capability or even an inhibitory effect on the activities of antioxidant enzymes, but a clear significant induction on GST.

[Self-inflicted dermatitis: a case in pediatric age]. / Dermatiti da autoaggressione: un caso in età pediatrica.

The skin is an organ that has a primary function in the tactile receptivity and also reacts significantly to emotional stimuli. The high visibility of the dermatological diseases makes the skin a privileged target for feelings and actions that reflect behavioural problems. The tendency to self-harm can be expressed with remarkable frequency through dermatological lesions; among them neurotic excoriations are a clinical frame in which patients, unlike for other ''similar'' pathologies, succeed however in recognizing their own role in the development of the cutaneous lesions. This pathology is not frequent in paediatric age; the highest statistical frequency, however, is to be found in the adolescence period of development. The authors describe a multidisciplinary, dermatological and psychological intervention on a child who intensifies a pre-existing dermatological symptom for psychological reasons.

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy).

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.

Integrated use of biomarkers and bioaccumulation data in Zebra mussel (Dreissena polymorpha) for site-specific quality assessment.

One of the useful biological tools for environmental management is the measurement of biomarkers whose changes are related to the exposure to chemicals or environmental stress. Since these responses might vary with different contaminants or depending on the pollutant concentration reached in the organism, the support of bioaccumulation data is needed to prevent false conclusions. In this study, several persistent organic pollutants -- 23 polychlorinated biphenyl (PCB) congeners, 11 polycyclic aromatic hydrocarbons (PAHs), six dichlorodiphenyltricholroethane (DDT) relatives, hexachlorobenzene (HCB), chlorpyrifos and its oxidized metabolite -- and some herbicides (lindane and the isomers alpha, beta, delta; terbutilazine; alachlor; metolachlor) were measured in the soft tissues of the freshwater mollusc Zebra mussel (Dreissena polymorpha) from 25 sampling sites in the Italian portions of the sub-alpine great lakes along with the measure of ethoxyresorufin dealkylation (EROD) and acetylcholinesterase (AChE) activity. The linkage between bioaccumulation and biomarker data allowed us to create site-specific environmental quality indexes towards man-made chemicals. This classification highlighted three different degrees of xenobiotic contamination of the Italian sub-alpine great lakes: a high water quality in Lake Lugano with negligible pollutant levels and no effects on enzyme activities, an homogeneous poor quality for Lakes Garda, Iseo and Como, and the presence of some xenobiotic point-sources in Lake Maggiore, whose ecological status could be jeopardized, also due to the heavy DDT contamination revealed since 1996.

Serum-induced basophil CD63 expression by means of a tricolour flow cytometric method for the in vitro diagnosis of chronic urticaria.

BACKGROUND: Functional autoantibodies against the alpha-chain of the high-affinity IgE receptor (FcepsilonRIalpha) identify a subset of patients with chronic urticaria (CU) due to autoreactivity, as assessed by an in vivo positive response to autologous serum skin test (ASST). We performed a study to standardize the serum-induced basophil activation assay by flow cytometry (FCM) using a new tricolour method, assessing the diagnostic performance of this test in discriminating between ASST+ and ASST- CU patients. METHODS: Sera of 64 CU patients (22 ASST+ CU and 42 ASST- CU) and 10 healthy subjects were tested for their ability to induce basophil CD63 expression when incubated with whole blood of both atopic (DA) and non-atopic donors (DNA). Using a triple-labelled strategy with anti-CD123, anti-HLA-DR and anti-CD63 antibodies, CD63+ basophils were identified on a selected population of CD123+ HLA-DR- cells. In 3 ASST+ CU patients who underwent cyclosporine therapy, the assay was performed before and after treatment. RESULTS: The ASST+ CU sera resulted in a significant higher induction of basophil CD63 expression compared with ASST- CU and healthy donors sera; when whole blood from DA was used, sensitivity and specificity of the assay were 95.5% and 90.5% respectively. ASST+ CU serum activity was significantly decreased during cyclosporine A treatment, in parallel with clinical remission. CONCLUSIONS: Chronic urticaria serum-induced CD63 expression assay performed on DA whole blood by means of our tricolour FCM method could be the most useful tool for identification of a subset of patients with autoimmune CU and may become a promising tool also for monitoring treatment efficacy.

Risk for human health of some POPs due to fish from Lake Iseo.

Risk evaluation for human health was carried out using PCB and PAH concentrations measured in seven fish species from Lake Iseo (Italy) and comparing the experimental data with legal levels and the two new approaches proposed by the US FDA and the US EPA to protect local populations with different food habits. We verified that none of the persistent organic compounds analyzed exceeded the legal limits set by the US FDA, since no specific limit has yet been proposed by the European Union. The two other approaches have pointed out that the acute and chronic effects do not represent any risk for human health, while a possible carcinogenic risk for the local population can arise when 4 and 8 monthly meals are consumed, based on PCB and PAH content in the edible part of fish, respectively.

Polychlorinated biphenyls and chlorinated pesticides in bird eggs from Calabria (southern Italy).

Polychlorinated biphenyl (PCB) and chlorinaed pesticide levels in eggs of two terrestrial (kestrel and sparrowhawk) and two aquatic (heron and mallard) birds were determined in inhabited areas and woodlands of Calabria. The eggs of the two terrestrial raptors were more polluted than those of the aquatic birds. Among the raptors, the kestrel demonstrated the highest level of pollution because it lives close to villages, whereas sparrowhawk lives in forests. The highest PCB and DDT residue concentrations found in kestrel (11.3 and 8.7 mg g-1 lipid, respectively) could cause shell thinning. Aquatic species show a higher DDT/DDE ratio than terrestrial species, probably because DDT is degraded more slowly in aquatic environments. However, point DDT sources seem to be present in this region.