PAH metabolites in fish bile: From the Seine estuary to Iceland.

Polycyclic aromatic hydrocarbons (PAH) are environmental contaminants that pose significant risk to health of fish. The International Workshop on Integrated Assessment of Contaminant Impacts on the North Sea (ICON) provided the framework to investigate biomarker responses as well as contaminant concentrations side by side in marine ecosystems. Concentrations of the main PAH metabolites 1-hydroxypyrene, 1-hydroxyphenanthren and 3-hydroxybenzo(a)pyrene were determined in bile by HPLC with fluorescence detection. Fish species under investigation were dab (Limanda limanda), flounder (Platichthys flesus) and haddock (Melanogrammus aeglefinus). A contamination gradient was demonstrated from the low contaminated waters of Iceland and off-shore regions of the North Sea towards higher concentrations in coastal areas. Concentrations of PAH metabolites differed primarily according to sampling region and secondarily to species.

Effects of an environmentally relevant concentration of diuron on oyster genitors during gametogenesis: responses of early molecular and cellular markers and physiological impacts.

Genitors of the Pacific oyster Crassostrea gigas were submitted during gametogenesis to a short pulse exposure to the herbicide diuron at a realistic environmental concentration. Histological analysis showed no effect of diuron on gametogenesis course, sex ratio and reproductive effort. A non-significant increase in testosterone and progesterone levels was observed in genitors exposed to the herbicide. At cell level, diuron exposure was shown to modulate the phagocytic activity of circulating hemocytes. The results of a transcriptional analysis showed that diuron affected the expression of genes belonging to functions known to play a major role during oyster gametogenesis such as gene transcription regulation, DNA replication and repair, DNA methylation and cytokinesis. Taking into account the results we previously obtained on the same genitors, this study showed a negative effect of diuron on oyster reproduction by inducing both structural and functional modifications of the DNA.

Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in Atlantic cod (Gadus morhua).

The aim of this study was to characterize concentration- and time-dependent responses in juvenile Atlantic cod (Gadus morhua) following exposure for one and three weeks to the water-soluble fraction (WAF) of three weathered oils: Arabian Light crude oil (ALC), North Sea crude oil (NSC) and ship-diesel. The sum of polycyclic aromatic hydrocarbons (PAH) in water was highest after one week of exposure and within environmentally relevant concentrations. PAH metabolites in bile confirmed exposure to and uptake of PAHs. Hepatic cytochrome P450 1A (CYP1A) gene expression (mRNA quantification) increased dramatically following exposure to all three oil types (fold-change up to 165) and there was a time lag between gene and protein expression. Hepatic CYP1A protein concentration and ethoxyresorufin-O-deethylase (EROD) activity were more variable among individuals and treatments than gene expression. EROD activity in liver and gills increased in fish exposed to WAF from the two crude oils, but not in fish exposed to WAF from diesel. Exposure to diesel appeared to induce oxidative stress to a greater extent than exposure to crude oils. Other biomarkers (glutathione S-transferases, acetylcholine esterase, vitellogenin) did not appear to respond to the exposure and hence did not discriminate among oils. Biomarker responses in cod after exposure to weathered crude oils and diesel suggested that the CYP1A system and oxidative stress markers have the highest potential for discriminating among different oil types and to monitor the environmental consequences of spills.

Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenic but not genotoxic effects.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be present at high levels as mixtures in polluted aquatic environments. Many PAHs are potent mutagens and several are well-known carcinogens. Despite numerous studies on individual compounds, little is known about the toxicity of PAHs mixtures that are encountered in environmental situations. In the present work, zebrafish were continuously fed from 5 days post-fertilisation to 14 months post-fertilisation (mpf) with a diet spiked with fractions of either pyrolytic (PY), petrogenic light oil (LO), or petrogenic heavy oil (HO) origin at three concentrations. A decrease in survival was identified after 3 mpf in fish fed with the highest concentration of HO or LO, but not for PY. All PAH fractions caused preneoplastic and neoplastic disorders in long-term-exposed animals. Target tissues were almost exclusively of epithelial origin, with the bile duct epithelium being the most susceptible to chronic exposure to all PAH fractions, and with germ cells being the second most responsive cells. Significantly higher incidences of neoplasms were observed with increasing PAH concentration and exposure duration. The most severe carcinogenic effects were induced by dietary exposure to HO compared to exposure to LO or PY (45, 30 and 7 %, respectively, after 9 to 10 months of exposure to an intermediate concentration of PAHs). In contrast, earliest carcinogenic effects were detected as soon as 3 mpf after exposure to LO, including the lowest concentration, or to PY. PAH bioactivation and genotoxicity in blood was assessed by ethoxyresorufin-O-deethylase activity quantification and comet and micronuclei assays, respectively, but none of these were positive. Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenotoxic events that impair survival and physiology of exposed fish.

The integrated biomarker response revisited: optimization to avoid misuse.

The growing need to evaluate the quality of aquatic ecosystems led to the development of numerous monitoring tools. Among them, the development of biomarker-based procedures, that combine precocity and relevance, is recommended. However, multi-biomarker approaches are often hard to interpret, and produce results that are not easy to integrate in the environmental policies framework. Integrative index have been developed, and one of the most used is the integrated biomarker response (IBR). However, an analysis of available literature demonstrated that the IBR suffers from a frequent misuse and a bias in its calculation. Then, we propose here a new calculation method based on both a more simple formula and a permutation procedure. Together, these improvements should rightly avoid the misuse and bias that were recorded. Additionally, a case study illustrates how the new procedure enabled to perform a reliable classification of site along a pollution gradient based on biomarker responses used in the IBR calculations.

Study of genetic damage in the Japanese oyster induced by an environmentally-relevant exposure to diuron: evidence of vertical transmission of DNA damage.

Pesticides represent a major proportion of the chemical pollutants detected in French coastal waters and hence a significant environmental risk with regards to marine organisms. Commercially-raised bivalves are particularly exposed to pollutants, among them pesticides, as shellfish farming zones are subject to considerable pressure from agricultural activities on the mainland. The aims of this study were to determine (1) the genotoxic effects of diuron exposure on oyster genitors and (2) the possible transmission of damaged DNA to offspring and its repercussions on oyster fitness. To investigate these points, oysters were exposed to concentrations of diuron close to those detected in the Marennes-Oleron Basin (two 7-day exposure pulses at 0.4 and 0.6 µg L(-1)) during the gametogenesis period. Genomic abnormalities were characterized using two complementary approaches. The Comet assay was applied for the measurement of early and reversible primary DNA damage, whereas flow cytometry was used to assess the clastogenic and aneugenic effect of diuron exposure. Polar Organic Chemical Integrative Samplers (POCIS) were used in exposed and assay tanks to confirm the waterborne concentration of diuron reached during the experiment. The results obtained by the Comet assay clearly showed a higher level of DNA strand breaks in both the hemocytes and spermatozoa of diuron-exposed genitors. The transmission of damaged genetic material to gamete cells could be responsible for the genetic damage measured in offspring. Indeed, flow cytometry analyses showed the presence of DNA breakage and a significant decrease in DNA content in spat from diuron-exposed genitors. The transmission of DNA damage to the offspring could be involved in the negative effects observed on offspring development (decrease in hatching rate, higher level of larval abnormalities, delay in metamorphosis) and growth. In this study, the vertical transmission of DNA damage was so highlighted by subjecting oyster genitors to short exposures to diuron at medium environmental concentrations. The analysis of POCIS showed that oysters were exposed to integrated concentrations as low as 0.2 and 0.3 µg L(-1), emphasizing the relevance of the results obtained and the risk associated to chemical contamination for oyster recruitment and fitness.

DNA integrity assessment in hemocytes of soft-shell clams (Mya arenaria) in the Saguenay Fjord (Québec, Canada).

The purpose of this study was to examine the effects of pollution on DNA integrity in the feral soft-shell clam (Mya arenaria) in the Saguenay Fjord. Intertidal clams were collected downstream and upstream of the fjord at sites under anthropogenic pollution. DNA integrity was assessed by following changes in single- and double-stranded breaks, variation in DNA content and micro-nuclei (MN) incidence in hemocytes. The results revealed that clams collected at polluted sites had reduced DNA strand breaks (lower DNA repair activity), increased DNA content variation and MN frequency in hemocytes. The data revealed that DNA content variation was closely related to MN frequency and negatively with DNA strand breaks formation. Water conductivity was also related to reduced MN frequency and DNA content variation, indicating that, in addition to the effects of pollution, the gradual dilution of saltwater could compromise mussel health.

Effects of low-dose exposure to pesticide mixture on physiological responses of the Pacific oyster, Crassostrea gigas.

This study investigated the effects on the physiology of Pacific oyster, Crassostrea gigas, of a mixture of pesticides containing 0.8 µg L(-1) alachlor, 0.6 µg L(-1) metolachlor, 0.7 µg L(-1) atrazine, 0.6 µg L(-1) terbuthylazine, 0.5 µg L(-1) diuron, 0.6 µg L(-1) fosetyl aluminum, 0.05 µg L(-1) carbaryl, and 0.7 µg L(-1) glyphosate for a total concentration of 4.55 µg L(-1) . The total nominal concentration of pesticides mixture corresponds to the pesticide concentrations in the shellfish culture area of the Marennes-Oleron basin. Two varieties of C. gigas were selected on the foreshore, based on their characteristics in terms of resistance to summer mortality, to assess the effects of the pesticide mixture after 7 days of exposure under controlled conditions. The early effects of the mixture were assessed using enzyme biomarkers of nitrogen metabolism (GS, glutamine synthetase), detoxification metabolism (GST, glutathione S-transferase), and oxidative stress (CAT, catalase). Sublethal effects on hemocyte parameters (phagocytosis and esterase activity) and DNA damages (DNA adducts) were also measured. Changes in metabolic activities were characterized by increases in GS, GST, and CAT levels on the first day of exposure for the "resistant" oysters and after 3-7 days of exposure for the "susceptible" oysters. The formation of DNA adducts was detected after 7 days of exposure. The percentage of hemocyte esterase-positive cells was reduced in the resistant oysters, as was the hemocyte phagocytic capacity in both oyster varieties after 7 days of exposure to the pesticide mixture. This study highlights the need to consider the low doses and the mixture of pesticides to evaluate the effects of these molecules on organisms.

Genotoxic and enzymatic effects of fluoranthene in microsomes and freshly isolated hepatocytes from sole (Solea solea).

The fluoranthene (Fluo) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in human food and in marine compartments. However, the existing data on its genotoxicity is poor and controversial. The aim of this study was to assess in vitro the potential genotoxicity of Fluo in sole and its possible effect on CYP450 modulation. Freshly isolated hepatocytes were exposed for 24 h to a range of Fluo concentrations from 0.5 to 50 µM in both culture flasks and microplate wells. The ethoxyresorufin-O-deethylase (EROD) activity was measured as an indicator of the activity of the cytochrome P450 1A1 (CYP1A1). The genotoxic effects were evaluated by measuring both DNA strand breaks and DNA adducts by the alkaline comet assay and the postlabeling technique respectively. Calf thymus DNA was also exposed to Fluo in the presence of sole liver microsomes in order to check for Fluo DNA adduct formation. In sole hepatocytes, Fluo was shown to induce a decrease in the EROD activity in a concentration-dependent manner. A significant genotoxic effect was observed in terms of DNA strand breakage from an exposure concentration of 5 µM: despite a concentration-dependent effect was observed, it did not follow a linear dose-response. The response was similar whatever the way of exposure in flasks or in wells. One reproducible adduct was detected in the hepatocytes exposed to the highest concentrations of Fluo. The formation of Fluo adducts was confirmed by the detection of one reproducible adduct following in vitro exposure of calf thymus DNA to 100 and 200 µM of Fluo in the presence of sole microsomes. These results demonstrate the potential of sole hepatocytes to metabolize Fluo in 24 h into reactive species, able to induce genotoxicity by DNA strand breakage and DNA adduct formation. Moreover, a miniaturized cell exposure system was validated for further experiments using fewer amounts of hepatocytes and contaminants, and allowing exposure to PAH metabolites.

Relationship between PAH biotransformation as measured by biliary metabolites and EROD activity, and genotoxicity in juveniles of sole (Solea solea).

Polycylic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the marine environment. Their toxicity is mainly linked to the ability of marine species to biotransform them into reactive metabolites. PAHs are thus often detected at trace levels in animal tissues. For biomonitoring purposes, this findings have two main consequences, (i) the determination of the PAH tissue concentration is not suitable for the evaluation of individual exposure to PAHs (ii) it can explain sometimes the lack of correlations obtained with relevant markers of toxicity such as genotoxicity biomarkers. The aim of the present study was to better investigate the link between PAH exposure and genotoxicity in marine flatfish. During a laboratory experiment, juvenile soles were exposed for four weeks to a mixture of three PAHs, namely benzo[a]pyrene, fluoranthene and pyrene, followed by one week of depuration. Fish were exposed via the trophic route to a daily PAH concentration of 120 µg/g food. Fish were sampled at different time points. The bioavailability and the biotransformation of PAHs were assessed by the measurement of biliary metabolites using a sensitive UPLC MS/MS method. The 7-ethoxyresorufine-O-deethylase was also measured in liver subcellular fractions as a biomarker of phase I biotransformation activities. Genotoxicity was assessed in parallel by the measurement of DNA strand breaks in fish erythrocytes by the alkaline comet assay. During this study, the high amount of PAH metabolites produced in sole demonstrated the bioavailability of PAHs and their biotransformation by fish enzymes. A positive correlation was observed between the level of hydroxylated PAH metabolites and genotoxicity as measured by the alkaline comet assay.

Spatial variations in biomarkers of Mytilus edulis mussels at four polluted regions spanning the Northern Hemisphere.

Economic and social developments have taken place at the expense of the health of the environment, both locally and on a global scale. In an attempt to better understand the large-scale effects of pollution and other stressors like climate change on the health status of Mytilus edulis, mussels were collected during the first two weeks of June 2005 at three sites (one pristine and two affected by pollution) located in each of the regions of the Canadian West Coast, the St. Lawrence estuary, the Atlantic East Coast and the northwestern coast of France, covering a total distance of some 11000km. The mussels were analyzed for morphologic integrity (condition factor), gametogenic activity (gonado-somatic and gonad maturation index, vitellogenin(Vtg)-like proteins), energy status (temperature-dependent mitochondrial electron transport activity and gonad lipid stores), defense mechanisms (glutathione S-transferase, metallothioneins, cytochrome P4503A activity and xanthine oxidoreductase-XOR), and tissue damage (lipid peroxidation-LPO and DNA strand breaks). The results showed that data from the reference sites in each region were usually not normally distributed, with discriminant factors reaching the number of regions (i.e. four), except for the biomarkers gonadal lipids, XOR and LPO in digestive gland. The integrated responses of the biomarkers revealed that biomarkers of stress were significantly more pronounced in mussels from the Seine estuary, suggesting that the impacts of pollution are more generalized in this area. Mussels from the Seine estuary and the Atlantic East Coast (Halifax Harbor) responded more strongly for Vtg-like proteins, but was not related to gonad maturation and gonado-somatic indexes, suggesting the presence of environmental estrogens. Moreover, these mussels displayed reduced DNA repair activity and increased LPO. Factorial analyses revealed that energy status, cytochrome P4503A activity and Vtg-like proteins were the most important biomarkers. Adaptation to warmer temperatures was reflected at the energy status levels, mussels from both the polluted and warmer sites displaying increased ratios of mitochondrial activity to lipid stores. Regional observations of biomarkers of energy status, gametogenesis and pollutant-related effects were influenced by nutrition, oxygen availability (eutrophication), and thermal history.

Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea.

Seasonal variations of six mussel (Mytilus galloprovincialis) biomarkers at two sites in the Mediterranean Sea were compared with physiological indices (condition, growth and gonad maturation), environmental parameters (temperature, salinity and turbidity), and chemical contamination levels. The basal levels of acetylcholinesterase (AChE), DNA adducts, benzo[a]pyrene hydroxylase (BPH), heat-shock proteins (HSP70), metallothioneins (MT) and P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) were estimated as early warning signals in caged mussels sampled at Carteau (native site) and La Fourcade (transplantation site) over a 2-year period. The Carteau and La Fourcade mussels have specific chemical contamination profiles but a similar range of values. For example, both are highly contaminated by heavy metals (201 and 258.4 mg kg(-1) dw, respectively) and considered as moderately impacted for polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). However, contamination levels at Carteau are twice as high for PAHs (101.5 mg g(-1) dw) and PCBs (90.2 mg g(-1) dw) than La Fourcade. The seasonal contamination trend at Carteau showed six-fold higher levels of pyrolytic pollutants in winter. Although few tissue lesions were detected in individuals studied at either site, greater parasitic infestation was observed at Carteau. The results of findings from the two Mediterranean pilot studies support the adaptability of transplanted mussels to be used as biomarkers and to establish physiological endpoints for chemical contaminant exposure.

Enzymatic biomarker measurement and study of DNA adduct formation in benzo

The aim of this study was to improve the knowledge on the metabolic pathways involved in benzo[a]pyrene (B[a]P) activation and on the relationship between adduct levels and enzymatic biomarker activities. With this purpose, a model to assess pollutant exposure via food supply has been developed for the sentinel organism, Mytilus galloprovincialis. Mussels were fed for 4 weeks with B[a]P-contaminated feed (50 mg/kg dry weight mussel). Bioaccumulation was studied by determination of B[a]P concentration in whole mussel by GC/MS analysis. Different biomarkers of pollutant exposure were measured to assess the metabolic state of the exposed organisms. CYP1A-like immunopositive protein titration and B[a]P hydroxylase (BPH) activity were assessed as indicators of phase I biotransformation. Glutathione-S-transferase (GST) activity was measured as an indicator of the conjugation activities. Catalase (CAT) and DT-diaphorase (DTD) activities were assessed as potential biomarkers of oxidative stress, whereas acetylthiocholine esterase (AChE) activity was measured as an indication of possible neurotoxicity of B[a]P exposure. DNA adduct levels were determined in digestive gland DNA by applying the 32P-postlabeling technique with nuclease P1 enhancement. For the developed conditions of exposure, B[a]P concentration reached in whole mussel tissues was very high (>500 mg/kg d.w. mussel) and significant B[a]P-induced changes were recorded for each enzymatic biomarkers. BPH and CAT activities were significantly increased by B[a]P exposure, whereas GST in the gills, DTD and AChE were significantly depressed. On the other hand, no change in CYP1A-like immunopositive protein content was observed. Induction and increase with time of bulky B[a]P-related DNA adducts were demonstrated in the digestive gland, although at low levels (0.269+/-0.082 adduct/10e8 dNps at maximum) by the 32P-postlabeling assay. DNA adduct level was significantly correlated with whole mussel tissue B[a]P concentration, so were all the enzymatic biomarkers measured except to GST activity in both gill and digestive gland tissues. BPH, DTD, CAT and AChE displayed a strong correlation with adduct levels. These results demonstrate the neurotoxicity and the genotoxicity of B[a]P exposure in the mussel. The induction of bulky DNA adducts in mussels demonstrates the existence of activation pathways already identified in vertebrates. It validates also the suitability of this model for further studies on B[a]P metabolism in mussels. Our results support the proposal of BPH, AChE, DTD and CAT activities as suitable biomarkers of PAH exposure for these sentinel species.

Flounder health status in the Seine Bay. A multibiomarker study.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.

Acetylcholinesterase and ethoxyresorufin-o-deethylase in the surgeonfish Acanthurus bahianus around Martinique Island (French West Indies).

Abstract The biochemical indicators of biological effects of pollutants, ethoxyresorufin-edeethylase (EROD) and acetylcholinesterase (AChE), were measured in Acanthurus bahianus from 10 stations around Martinique Island (French West Indies). Strong induction of EROD was demonstrated in the bay of Fort de France in relation to organic pollution. Depression of AChE may suggest that neurotoxic compounds are having some effects along the east coast.

The micronucleus assay in Crassostrea gigas for the detection of seawater genotoxicity.

The micronucleus (MN) test was performed in vivo and in vitro on the oyster Crassostrea gigas to evaluate the genotoxic effect of the marine environment. In vitro tests were carried out on adult and young (spat) specimens exposed to benzo[a]pyrene (BaP: 0.5, 5, 500 and 1000 micrograms.l-1) and an effluent (5, 50, 75 and 100%) of Seine Bay, one of the most highly contaminated sites in France. MN frequency observed after 48 h exposure to the two pollutants was much greater in adults than spats. A preliminary test of the genotoxic effect of BaP (0.05, 0.5, 1 and 500 micrograms.l-1), cupric sulfate (10, 25, 50 and 100 micrograms.l-1) and a paper mill effluent (1, 3, 10 and 30 mg.l-1) was performed in C. gigas heart cells cultured for 6 days. Comparison of the MN assay with the C. gigas larva test showed the clastogenic action of BaP and the toxic effect of cupric sulfate on culture cells as well as the slighter toxic effect of paper mill effluent on spats. An in vivo study was conducted in an oyster-farming area contaminated by cadmium and copper. MN frequency was not very sensitive to a pollution gradient but showed high interindividual variability. The absence of precise criteria for MN identification in mollusks and the identification of highly basophilic spherical inclusions in the cytoplasm of gill tissue hemocytes in oysters during viral infection are handicap for application of the micronuclei assay in the marine environment. Another limitation of the assay is the particularly onerous requirement for manual observation. Optimization of the assay by automated analysis is necessary but can only be achieved if cytologic preparations are of good quality.

Monitoring biological effects of contamination in marine fish along French coasts by measurement of ethoxyresorufin-O-deethylase activity.

The use of bioindicators to evaluate exposure to the biological effects of chemical pollutants in marine organisms constitutes a new tool in the monitoring field. The establishment of a North Sea monitoring network in 1991, involving such international organizations as the North Sea Task Force, the International Council for the Exploration of the Sea, and the Intergovernmental Oceanography Commission, led French researchers to develop an enzymatic biomarker to monitor biological effects within the National Observation Network. The biomarker, ethoxyresorufin-O-deethylase (EROD), dependent on the CP450 system, has been monitored biannually since 1992 in several species of fish (Callionymus lyra, Limanda limanda, Serranus sp., Mullus barbatus) in two coastal sites particularly exposed to industrial and domestic pollution. A rapid method is used to assay EROD enzymatic activity determined along a pollution gradient, and results are interpreted on a microplate reader. The strategy of this approach is to assess the effects on the marine ecosystem during prolonged exposure to specific pollutants such as polyaromatic hydrocarbons, polychlorinated biphenyls, and dioxins.