Genotoxic and biochemical responses in caged eel (Anguilla anguilla L.) after short-term exposure to harbour waters

European eel (Anguilla anguilla L.) were caged and exposed in situ for 8 and 48 h to the Aveiro offward fishing harbour water (HW) and to clean seawater under laboratory conditions (Control). Eel liver biotransformation (Phase I) was measured as ethoxyresorufin-O-deethylase (EROD) activity, cytochrome P450 (P450) and glutathione S-transferase (GST) activity (Phase II). Genotoxic responses were determined as blood, liver and kidney DNA strand breaks as well as erythrocytic nuclear abnormalities (ENAs). HW failed to significant increase liver EROD, GST activities and ENA frequency. Nevertheless, P450 content was significantly increased after 8 and 48 h exposure. Genotoxicity measured as DNA integrity decrease was found in blood after 8 and 48 h exposure to HW, whereas in liver and kidney, it was observed after 48 h exposure to HW. Blood, kidney and liver genotoxicity may be due to the presence of polycyclic aromatic hydrocarbons (PAHs) which are genotoxic compounds and the main HW organic contaminants.     

Juvenile sea bass (Dicentrarchus labrax L.) DNA strand breaks and lipid peroxidation response following 17beta-estradiol two mode of exposures

Juvenile Dicentrarchus labrax L. (sea bass) were exposed to water diluted 17beta-estradiol (E2) (200 ng/L and 2000 ng/L) and treated with intraperitoneal (i.p) injection E2 (0.5 mg/kg and 5 mg/kg) during 10 days in order to study its genotoxicity and peroxidative damage, measured as gill, blood, liver and kidney DNA integrity decrease using DNA strand breaks assay as well as liver, gill and kidney lipid peroxidation (LPO) respectively. Juvenile sea bass gill DNA integrity was significantly decreased for all E2 exposure conditions. However, no differences were detected either between different exposure routes or tested concentrations. Blood DNA integrity was significantly decreased by E2 5 mg/kg as well as by both water diluted E2 exposure conditions. The highest E2 dose (5 mg/kg) also promoted liver DNA integrity decrease. Liver and gill LPO significantly increased at the highest E2 i.p treatment. An increasing trend of gill and liver LPO, though statistically insignificant, was observed in D. labrax exposed to water diluted E2 in both tested concentrations. The current results demonstrated that DNA damage in juvenile sea bass is affected by the E2 exposure conditions, such as water diluted E2 versus i.p E2 injection since i.p E2 injection promoted higher genotoxicity effect, in terms of affected organs than water diluted E2. Moreover, the organ sensitivity to E2 was different, since gill showed more susceptibility than blood, liver and kidney. Concerning kidney LPO and DNA integrity no differences were found between treated and controls juvenile sea bass groups.     

Anguilla anguilla L. antioxidants responses to in situ bleached kraft pulp mill effluent outlet exposure

This study assesses the antioxidant enzymes activities viz., catalase, glutathione peroxidase, glutathione S-transferase and nonenzymatic antioxidant molecule such as glutathione in Anguilla anguilla L. gill, kidney and liver in response to 8- and 48-h exposure to bleached kraft pulp mill effluent (BKPME). A. anguilla were caged and plunged at three different sites-50 (Site 1), 100 (Site 2) and 2000 m (Site 3) away from the closed BKPME outlet. A significant gill (8 and 48 h) and kidney (48 h) catalase activity decrease was observed at site 2 exposure whereas liver showed a significant increase in catalase activity after 8 and 48 h to site 1 exposure. Glutathione peroxidase (GPX) activity was significantly decreased in gill after 8-h exposure to site 1 and 48-h exposures to sites 1 and 2, respectively. Concerning gill, kidney and liver glutathione S-transferase (GST) activity, a significant gill GST activity decrease after 8 h at site 2 and 48 h at sites 1 and 2 was observed; in kidney, a significant decrease in its activity was observed after 48 h at sites 1 and 2, respectively, whereas in liver, the decrease was significant only at site 2 after 48-h exposure. The in situ BKPME exposure caused a significant total gill and kidney reduced glutathione (GSH) decrease after 8 h at site 2 exposure and after 48 h at site 1 and 2 exposures, respectively. However, a biphasic response was observed in liver, i.e. initial significant increase after 8 h at site 2 followed by a significant decrease after 48 h to the same site exposure. The enzymatic and nonenzymatic antioxidants pattern in gill and kidney, as observed in this study, was different than liver, demonstrating that the liver was more resistant to oxidative damage than gill and kidney. In addition, A. anguilla gill, kidney and liver antioxidants adaptation potentials may serve as a surrogate biomarker to BKPME exposure.     

Immediate biomarker responses to benzo[a]pyrene in polluted and unpolluted populations of the blue mussel (Mytilus edulis L.) at high-latitudes

Immediate biomarker responses of two high-latitude populations of the blue mussel Mytilus edulis to benzo[a]pyrene (B[a]P) were evaluated. Mussels collected from a clean and a polluted site in southwest Iceland were exposed to the nominal dose of 100 microg B[a]P L(-l) for 3 h, after 4 days of acclimatization in clean seawater. To test the sensitivity to the toxicant and immediate biological responses, the following biomarkers were used: DNA single strand breaks, heart rate and feeding rate. All the biomarkers revealed differences between the study sites. Irrespective of the origin of the organisms, the short time exposure to the high B[a]P concentration did not induce DNA single strand breaks or significantly affect the feeding rate. However, the heart rate results showed significantly different responses. The mussels from the polluted site (Reykjavík harbour) increased their heart rate when exposed to B[a]P, while no difference was observed between the heart rate values of the individuals from the clean site (Hvassahraun). The mussels seem to sense the pollutant they have been previously exposed to, and their acute response indicates physiological adaptation to the polluted environment. The results indicate limited sensitivity and temporal predictivity, i.e. transient measurable changes of these biomarkers, as well as showing that the background of the organisms should be considered when evaluating short-term biomarker responses to contaminants.     

Cytotoxicity, genotoxicity and ecotoxicity assay using human cell and environmental species for the screening of the risk from pollutant exposure

For the screening of the risk from environmental contamination, the cytotoxic/genotoxic effects of various model pollutants were determined using an in vivo system comprised of human HeLa cells; the ecotoxicity was also determined using the acute and genotoxicity tests on two aquatic sentinel species widely used in biomonitoring, namely, freshwater crustacean, Daphnia magna and larva of aquatic midge, Chironomus tentans. Nonylphenol (NP), bisphenol A (BPA), bis(2-ethylhexyl) phthalate (DEPH) and paraquat dichloride (PQ) were used as the model pollutants. The results showed that exposure of HeLa cells to NP, BPA and DEHP was sufficient for the expression of noticeable genotoxic and cytotoxic effects. Ecotoxicity results showed that, as expected, D. magna was more sensitive than C. tentans to chemical exposure. BPA may exert a genotoxic effect on D. magna and C. tentans, given that DNA strand breaks increased in both species exposed to this compound, whereas NP-induced DNA damage occurred only in C. tentans. In vivo genotoxic data obtained in aquatic sentinel species could provide valuable information for freshwater quality monitoring. From the results of the present study, the use of cytotoxic, genotoxic and ecotoxic tests using human cell system, as well as, biomonitoring species, seems to be relevant for preliminary evaluation of the human health and ecological effects of pollutants and thus, a promising screening tool for environmental monitoring and risk assessment.     

Tissue distribution and temperature-dependence of Anguilla anguilla L. EROD activity following exposure to model inducers and relationship with plasma cortisol, lactate and glucose levels

Anguilla anguilla L. ethoxyresorufin-O-deethylase (EROD) elevation by 2.7 microM beta-naphthoflavone (BNF) 3 days water exposure, or 4 mg/kg ip exposure, was studied in four different organs--liver, kidney, gills, and intestine. The results demonstrated a significant increase in liver EROD activity for the two previous conditions, whereas kidney EROD activity only increased during the intraperitoneal exposure. A. anguilla was also exposed during 8, 16, and 24 h to water contaminated with 2.7 microM BNF or benzo[a]pyrene (BaP) at 20 degrees C and 25 degrees C. Both compounds significantly increased liver EROD activity from 8 up to 24 h. There was no significant difference in liver EROD activity elevation by both compounds, either at 20 degrees C or 25 degrees C. Liver EROD activity was demonstrated to be one of the first warning systems concerning the presence of polycyclic aromatic hydrocarbons (PAHs) in water. A. anguilla 3 h exposure to diesel oil water-soluble fraction (DWSF) significantly increased plasma cortisol and significantly decreased plasma lactate. A prolonged exposure beyond 3 h, i.e. 4 h, 2, 3, 4, and 6 days to the previous conditions demonstrated a significant liver EROD activity elevation from Day 2 up to 6, and a significant increase in erythrocytic nuclear abnormalities (ENA) at Day 6.     

A bioassay approach to determine the dioxin-like activity in sediment extracts from the Danube River: ethoxyresorufin-O-deethylase induction in gill filaments and liver of three-spined sticklebacks (Gasterosteus aculeatus L.)

Sediment samples from the upper Danube River in Germany have previously been characterized as ecotoxicologically hazardous and contaminants in these sediments may contribute to the observed decline of fish populations in this river section. For the investigation of sediment toxicity there is a need for development, standardization and implementation of in vivo test systems using vertebrates. Therefore, the main objective of this study was to apply and evaluate a recently established fish gill EROD assay as a biomarker in sediment toxicity assessment by using extracts of well characterised sediment samples from the upper Danube River. This to our knowledge is the first application of this novel assay to sediment extracts. Sediments from four different sites along the upper Danube River were Soxhlet-extracted with acetone and dissolved in DMSO. Three-spined sticklebacks (Gasterosteus aculeatus L.) were exposed for 48 h to various concentrations of the extracts, to the positive control beta-naphthoflavone or to the solvent. Measurements of EROD activity in gill filaments and liver microsomes followed the exposure. Concentration-dependent induction of EROD in both gill and liver was found for all sediment extracts. The highest EROD-inducing potency was determined for extracts of sediments from the sites "Opfinger See" and "Sigmaringen" and the EROD activities in gill and liver correlated well. The results from the gill and liver assays were in accordance with in vitro results of previous investigations. The EROD activities measured in the present study corresponded with the concentrations of PAHs, PCBs and PCDD/Fs in the sediment samples derived in a previous study. The sticklebacks in this study were in the reproductive phase and a stronger EROD induction was obtained in the females than in the males. Implementation of the EROD assay in testing of sediment extracts gave highly reliable results which make this assay an ecotoxicologically relevant method for assessment of contamination with Ah receptor agonists in sediments.     

Evidence of genetic damage in grass gobies and mussels from the Venice lagoon

The presence of genetic damage has been investigated in two native species of the Venice lagoon: the common mussel Mytilus galloprovincialis and the grass goby Zosterisessor ophiocephalus. Two sampling campaigns were performed in summer 1998 and 1999. Aromatic-like DNA adducts were analysed in selected tissues of gobies and mussels by using the 32P-postlabelling assay. In 1999, micronuclei and other nuclear abnormalities were additionally scored on gill cells and haemocytes of individual mussels whereas inorganic (As, Cd, Cr, Hg, Ni, Pb, Sn) as well as organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and other chlorinated compounds) were measured in the total mussel pulp. Compared to the lagoon inlet area, gobies and mussels from the industrial district (Marghera) showed significant DNA adduct levels and increased frequencies of cytogenetic alterations (evidence of genetic damage was absent or inconsistent in other sites). The substantial levels of aromatic and chlorinated contaminants detected in mussels from Marghera also support the exposure of native organisms to genotoxic agents.     

Naphthalene and beta-naphthoflavone effects on Anguilla anguilla L. hepatic metabolism and erythrocytic nuclear abnormalities

The effects of a polycyclic aromatic hydrocarbon (PAH) such as naphthalene (NAP)--an environmental contaminant--and beta-naphthoflavone (BNF)--a model substance (PAH-like compound)--were investigated in European eel (Anguilla anguilla L.) over 3-, 6-, and 9-day exposure (0.1-2.7 microM). Both xenobiotics revealed to be strong biotransformation (phase I) inducers. After 3-day exposure, liver ethoxyresorufin O-deethylase (EROD) activity was significantly increased by all NAP and BNF tested concentrations. At 6 and 9 days, liver EROD activity was significantly induced mainly by the highest NAP and BNF concentrations. Liver cytochrome P450 content was significantly induced after 3-day exposure to 0.9 and 2.7 microM BNF and 9-day exposure to 0.1, 0.3 and 0.9 microM NAP. Liver alanine transaminase (ALT) activity was measured as an indicator of hepatic health condition, revealing a significant decrease after 6-day exposure to 0.9 microM BNF. Genotoxicity measured as erythrocytic nuclear abnormalities (ENA) was detected in all BNF treated fish on day 6, whereas on day 9, ENA frequencies returned to control levels, significantly decreasing at 0.9 microM BNF exposure. Immature erythrocytes (IE) frequency demonstrated a decreasing tendency along the BNF experiment and concomitantly with the above ENA response. The present experimental results elect EROD activity in A. anguilla as a useful short- to medium-term biomarker of exposure to both PAH and PAH-like compounds. However, some problems can emerge in the presence of high xenobiotic concentrations. Concerning genotoxicity, it is hypothesized that ENA response depends on different factors such as the exhaustion of the detoxification process, the balance erythropoiesis/erythrocytic catabolism and the DNA repairing capacity.     

Genotoxic effects of binary mixtures of xenoandrogens (tributyltin, triphenyltin) and a xenoestrogen (ethinylestradiol) in a partial life-cycle test with Zebrafish (Danio rerio)

A partial life-cycle test with the model fish Danio rerio was performed in order to evaluate the genotoxic potential of binary mixtures of xenoandrogenic (tributyltin--TBT; triphenyltin--TPT) and an estrogenic compound (ethinylestradiol--EE2). Five days post-fertilisation larvae were diet-exposed to environmental relevant concentrations of TBT and TPT (25 ng/g-100 ng/g), and water-exposed to ethinylestradiol (3.5 ng/L) for a four-month period; binary mixtures of TBT plus EE2 and TPT plus EE2 were run in parallel. The erythrocytic nuclear abnormalities (ENA) assay in circulating erythrocytes was used to evaluate genotoxicity in the end of the four-month exposure period. A significant increase (p<0.05, Kruskall-Wallis non-parametric ANOVA) in ENA frequency, in comparison with control animals, was observed in those animals exposed to TBT and TPT (the highest doses only), and to EE2 and the binary mixtures, although neither synergistic nor additive effects of the tested compounds were evident. Overall, the results clearly indicate that chronic exposure to low levels of TBT, TPT, EE2 and binary mixtures of TBT plus EE2 and TPT plus EE2 are genotoxic to zebrafish, which may suggest that wild fish populations may be under increased DNA damage in areas contaminated by these endocrine disrupting chemicals.     

Induction of EROD activity in European eel (Anguilla anguilla) experimentally exposed to benzo[a]pyrene and beta-naphthoflavone

The induction of liver ethoxyresorufin O-deethylase (EROD) activity was investigated in the European eel, Anguilla anguilla, collected from a Mediterranean brackish environment and experimentally exposed to benzo[a]pyrene (B[a]P) and beta-naphthoflavone (BNF). Eels were injected intraperitoneally at increasing doses (0.1, 1, 10, and 50 mg/kg wet body weight) using corn oil as a carrier and sacrificed after 7 days. The main objectives of the present study are: (1). to assess of the sensitivity of EROD induction as a biomarker to polycyclic aromatic hydrocarbon (PAH) exposure; (2). to determine an EROD dose-response relationship of the contaminants used; and (3). to compare the efficiency of B[a]P and BNF as inducers of EROD activity. Results showed that both chemicals resulted in a dose-dependent EROD induction, but increases were not linear. EROD activity seemed to reach a plateau at the exposure of 10 mg/kg in both treatment groups; B[a]P was a more potent inducer than BNF was at the higher doses (10 and 50 mg/kg), while the opposite result was observed at the lower ones (0.1 and 1 mg/kg). The greatest induction occurred in eels treated with 10 mg/kg B[a]P, in which a 261-fold increase in EROD activity was observed. Results showed that EROD activity in A. anguilla is significantly induced by B[a]P and BNF exposure, responding to a wide range of concentrations of these contaminants. We infer that this tool may be suited as a diagnostic biomarker for biomonitoring PAHs pollution in Mediterranean brackish environments and further field research is suggested.     

Biotransformation, stress and genotoxic effects of 17beta-estradiol in juvenile sea bass (Dicentrarchus labrax L.)

The effects of 17beta-estradiol (E(2)) on fish became a matter of concern, since significant levels of this hormone were detected in the aquatic environment released mainly by domestic sewage treatment plants. In this perspective, the current study was focused on E(2) effects upon biotransformation, stress and genotoxic responses of juvenile Dicentrarchus labrax L. (sea bass). Fish were exposed to E(2) during 10 days in two different ways: water diluted (200 ng/L or 2,000 ng/L) and i.p. injected (0.5 mg/kg or 5 mg/kg). A battery of biological responses was evaluated: liver ethoxyresorufin-O-deethylase (EROD) and alanine transaminase (ALT) activities, liver somatic index (LSI), plasma cortisol, glucose and lactate concentrations, as well as erythrocytic nuclear abnormalities (ENA). All the exposure conditions induced endocrine disruption, measured as plasma cortisol decrease, and genotoxicity, measured as ENA increase. Thus, no differences were detected either between different exposure routes or tested concentrations. Concerning liver EROD and ALT activities, as well as plasma glucose and lactate concentrations no differences were found between treated and control groups. LSI was the only parameter to respond differently in the two exposure routes, as only E(2) water diluted induced a significant increase in this hepatic indicator.     

Impact of gamma irradiation on the transformation efficiency for extracellular plasmid DNA

Extracellular DNA is omnipresent in aquatic environments and is thought to be a genetic material for horizontal gene transformation between microorganisms. We studied the impact of gamma irradiation on the transformation efficiency (transformants number per ng of DNA per ml) of extracellular DNA. Plasmid pEGFP as a model extracellular DNA was irradiated by gamma rays. The transformation efficiency decreased with the increase in radiation dose. A total dose of 10Gy is normally not lethal for microorganisms but certainly affects the transformation efficiency of extracellular DNA. The decrease in the efficiency would be induced by strand breaks of extracellular DNA because the yield of both single-strand breaks (SSBs) and double-strand breaks (DSBs) increased with the increase in radiation dose. The relative transformation efficiency of SSBs and DSBs to that of covalently closed circles (CCCs) was 30.3% and 0.2%, respectively. This impact on natural transformation suggests an inability of microorganisms to acquire new characteristics which should be normally acquired.     

Relationships between intertidal clam population and health status of the soft-shell clam Mya arenaria in the St. Lawrence Estuary and Saguenay Fjord (Québec, Canada)

The purpose of this study was to examine the impacts of anthropogenic activity on the health status of intertidal clam populations of the Saguenay Fjord and the St. Lawrence Estuary (Québec, Canada). Clams were collected during low tide at sites subject to direct contamination and at sites far from human activity. Clams were analyzed for tributyltin and dibutyltin total levels and toxic stress (glutathione S-transferase, gonadal lipid peroxidation and DNA strand breaks), immunocompetence (phagocytic activity, hemocyte count and viability), reproduction (gonado-somatic index, gamete maturation, and vitellogenin-like proteins), energy status (temperature-dependent mitochondrial electron transport, and gonad lipids), and individual status (age, condition factor, and growth index). These responses were compared against population characteristics such as live clam density, number of empty shells, and sex ratio. The results show that clam density decreased with distance from the estuary (high salinity level) to upstream of the fjord (low salinity). There was no clear relationship between the number of empty shells and distance or site quality. Clam density values corrected against distance were significantly correlated with hemocyte viability, phagocytic activity, mitochondrial electron transport (MET), DNA damage in gonad, and temperature-dependent mitochondrial electron transport activity. A canonical analysis of the various groups of biomarkers revealed that population metrics were more strongly related with immunocompetence, followed by energy status and temperature-dependent mitochondrial electron transport activity. However, toxic stress biomarkers were strongly associated with energy status and reproduction. This was further confirmed by non-linear modeling using adaptive artificial neural networks (genetic selection and back propagation learning paradigms), where the following parameters were able to predict population parameters with <20% error: gonad maturation and somatic index, MET (at 4 degrees C), gonad LPO, DNA damage, and phagocytic capacity. Intertidal clam populations were influenced by a distance gradient effect (salinity), where immunocompetence, in addition to energy status, was the strongest physiological parameter related to clam population metrics.     

Anguilla anguilla L. plasma cortisol, lactate and glucose responses to abietic acid, dehydroabietic acid and retene

Anguilla anguilla L. were exposed to 0, 0.1, 0.3, 0.9 and 2.7 microM abietic (AA), dehydroabietic (DHAA) acids and retene (Re) during 8, 16, 24 and 72 h. The eels plasma cortisol, glucose and lactate were measured. A significant decrease in plasma cortisol was observed at 72 h exposure to 0.9 and 2.7 microM Re. DHAA (0.1 microM) significantly decreased plasma cortisol in eels after 8 and 24 h exposure. However, a significant plasma cortisol increase was found after 16 h, 2.7 microM AA exposure and after 24 h exposure to 0.1 microM and 2.7 microM AA. Furthermore, 72 h exposure to 0.9 microM AA also induced a plasma cortisol increase. A general rise in plasma glucose was detected after all exposure periods to Re. The plasma lactate also increased after 72 h exposure to 2.7 microM AA and after 8 h exposure to 0.1 microM DHAA.     

Endocrine and metabolic changes in Anguilla anguilla L. following exposure to beta-naphthoflavone--a microsomal enzyme inducer

Anguilla anguilla L. were exposed during 24 and 48 h to 2.7 muM beta-naphthoflavone (BNF), a known microsomal enzyme inducer. The BNF effects on thyroid-stimulating hormone (TSH), free triiodothyronine (T3), free thyroxine (T4) and cortisol plasma levels were investigated. Alterations on plasma glucose and lactate levels were also measured as an indication of energy-mobilizing hormones alterations. BNF showed to be able to decrease significantly A. anguilla plasma T4 levels, whereas TSH, T3 and cortisol plasma remained constant. However, plasma glucose levels were significantly increased, demonstrating that intermediary metabolism has been affected. These results demonstrate that BNF a PAH-like compound alters the normal functioning of the hypothalamo-pituitary-thyroid (HPT) axis in A. anguilla.     

Genetic biomonitoring of an urban population exposed to mutagenic airborne pollutants

Biomonitoring studies have increased as a consequence of risks and effects to human health on exposure to environmental contaminants, mainly air pollutants. Genetic biomarkers are useful tools for the early assessment of exposure to occupational and environmental pollution. The objective of the present study was to investigate genotoxic effects on people residing and/or working downwind from an oil refinery in southern Brazil and the mutagenic activity of airborne particulate matter (PM10). Samples of peripheral blood and buccal mucosa cells were evaluated using the single-cell gel electrophoresis assay (comet assay) and the micronucleus (MN) assay, respectively. PM10 samples were collected in the target site and the organic matter extraced with dichloromethane was assessed for mutagenic activity in the Salmonella/microsome assay. The exposed group (n = 37) was compared to a reference group (n = 37) of subjects living in an urban area with limited traffic and industrial influence, located far from the main industrial areas. All PM10 organic extracts showed mutagenic positive responses and the effect decreased in the presence of S9 mix indicating that the predominant compounds present were direct-acting mutagens. The responses of YGs strains are consistent with aromatic amines and nitroarenes being present in the PM10 extracts. The group in the area under the influence of the oil refinery (exposed group) showed significantly higher DNA damage in lymphocytes than the reference group. The MN frequencies in buccal mucosa were very low for both groups and no difference between groups was observed. No association was found between age and tobacco smoking habit and level of DNA damages measured by the comet assay. The results indicate that the comet assay was a sensitive tool to detect DNA damage in subjects under the influence of an oil refinery, with marked genotoxic activity in the atmospheric environment.     

Global genomic methylation levels in the liver and gonads of the three-spine stickleback (Gasterosteus aculeatus) after exposure to hexabromocyclododecane and 17-beta oestradiol

Alteration of DNA methylation is a major epigenetic mechanism associated with the effects of nongenotoxic carcinogens. We evaluated the effects of two environmental pollutants, hexabromocyclododecane (HBCD), 17-beta oestradiol (E(2)) as well as 5-aza 2' deoxycytidine (5AdC) on global DNA methylation levels (5-methyl 2' deoxycytidine) in the liver and gonads of the three-spine stickleback (Gasterosteus aculeatus). HBCD at 30 and 300 ng/L of water did not produce statistically significant differences in global genomic methylation in liver of female stickleback. On the other hand, the methylation inhibitor, 5-aza-2'-deoxycytidine, significantly lowered hepatic global methylation levels in these fish by 14% (P<0.05). The naturally occurring oestrogen, 17-beta oestradiol (E(2)) at 100 ng/L also decreased global DNA methylation levels in female liver but this effect was not statistically significant. In contrast, both E(2) and 5AdC caused statistically significant (P<0.001 and P<0.01 respectively) global genomic hypermethylation in the gonads of male sticklebacks although the increase seen in the female gonads was not statistically significant. The male gonad effect though unexplained may potentially be an indirect response to hypomethylation in other tissues (such as the liver) and may have important implications regarding oestrogenic effects in fish. The contrasting effects of HBCD and E(2) on global DNA methylation in stickleback should contribute to the integrated risk assessment of these environmental chemicals.     

What can we learn from monitoring PCBs in the European eel? A Belgian experience

Between 2000 and 2007 pooled muscle tissue samples of the European eel (Anguilla anguilla) from 48 sites in Flanders (Belgium) were analysed for 30 polychlorinated biphenyl (PCB) congeners. There was a large variation between individual sites (range 11-7752 ng/g wet weight (ww) for the sum of the ICES 7 PCBs), eels from the River Meuse basin (mean 1545 ng/g ww) being considerably more polluted than those from the River Scheldt (615) and IJzer (61) basins. Overall, PCB 153, PCB 138 and PCB 180 were the most prominent congeners, however PCB patterns varied between the monitored locations. Analysis of the weight percentage of congeners demonstrates obvious differences in PCB composition between sites, indicating differential sources of pollution. Due to the variation in patterns, atmospheric fallout does not seem to be the main source of the PCB spread, but instead both local and upstream sources linked to industrial activities seem to be the main cause for PCB presence in Flanders. Considering the levels of the Sum 7 PCBs, eels are not compliant with the Belgian legal limits for consumption (75 ng/g ww) in 71% of the sites. Regular consumption of eels from polluted sites leads to a considerable excess of the WHO Acceptable Daily Intake value. Consumption of wild eels should by all means be prevented, as it presents risks for human health, especially for local anglers consuming their catch.     

Genotoxic effects of occupational exposure to lead and influence of polymorphisms in genes involved in lead toxicokinetics and in DNA repair

Lead is still widely used in many industrial processes and is very persistent in the environment. Although toxic effects caused by occupational exposure to lead have been extensively studied, there are still conflicting results regarding its genotoxicity. In a previous pilot study we observed some genotoxic effects in a population of lead exposed workers. Thus, we extended our study analysing a larger population, increasing the number of genotoxicity endpoints, and including a set of 20 genetic polymorphisms related to lead toxicokinetics and DNA repair as susceptibility biomarkers. Our population comprised 148 workers from two Portuguese factories and 107 controls. The parameters analysed were: blood lead levels (BLL) and δ-aminolevulinic acid dehydratase (ALAD) activity as exposure biomarkers, and T-cell receptor (TCR) mutation assay, micronucleus (MN) test, comet assay and OGG1-modified comet assay as genotoxicity biomarkers. Lead exposed workers showed markedly higher BLL and lower ALAD activity than the controls, and significant increases of TCR mutation frequency (TCR-Mf), MN rate and DNA damage. Oxidative damage did not experience any significant alteration in the exposed population. Besides, significant influence was observed for VDR rs1544410 polymorphism on BLL; APE1 rs1130409 and LIG4 rs1805388 polymorphisms on TCR-Mf; MUTYH rs3219489, XRCC4 rs28360135 and LIG4 rs1805388 polymorphisms on comet assay parameter; and OGG1 rs1052133 and XRCC4 rs28360135 polymorphisms on oxidative damage. Our results showed genotoxic effects related to occupational lead exposure to levels under the Portuguese regulation limit of 70μg/dl. Moreover, a significant influence of polymorphisms in genes involved in DNA repair on genotoxicity biomarkers was observed.