Exposure to copper induces oxidative and stress responses and DNA damage in the coral Montastraea franksi.

Copper is a common chemical contaminant in coastal environments, including coral reefs. Ecotoxicological studies have demonstrated that exposure to copper can cause stress and detrimental effects in both host cnidarian and algal symbionts. The objective of this study was to investigate the sublethal effects of copper on the reef-building coral Montastraea franksi, by identifying genes with altered expression in corals exposed to dissolved copper, and by measuring the extent of damage to DNA in response to copper exposure. Corals exposed to 30 µg L(-1) copper for 48 h experienced significant DNA damage and displayed changes in expression patterns of genes that are known to play role cellular and oxidative stress responses. Corals also experienced changes in gene expression of genes that are not already known to play roles in oxidative stress in corals. Our data suggest that these genes may either play roles directly in mediating a stress response, or may be genes acting downstream of the stress response. These include an ETS domain-containing transcription factor related to the ETS1 family of transcription factors, known in mammals to mediate development, disease, and stress response, and two genes that are associated with biomineralization: galaxin, a protein from the organic matrix of the coral skeleton, and a coral-specific gene SCRIP2.

The influence of water quality on the toxicity and degradation of juglone (5-hydroxy 1,4-naphthoquinone).

This study was part of a broader investigation of low molecular weight quinones under consideration as biocides for the control of aquatic nuisance species (ANS). Preliminary investigations identified the 2-ring naphthoquinones as broad spectrum biocides controlling a wide range of aquatic organisms. All biocides were relatively short-lived in saline waters, with half-lives between 5 and 30h. Juglone (5-hydroxy 1,4-naphthoquinone) and plumbagin (5-hydroxy-2-methyl-1,4- naphthoquinone) showed the greatest toxicity against most aquatic organisms. These qualities formed the basis for a patent focusing on these two compounds as biocides for ANS control, with juglone identified as the more cost-effective of the two. Although juglone has been extensively studied as a plant toxin and reducing agent, remarkably little information exists on its use as an aquatic biocide. We describe the toxicity of juglone over the range of water quality parameters likely to be encountered in ballast water, a major vector for ANS. Tests indicated that its molecular stability was enhanced in freshwater and particularly under neutral to acid conditions. This was supported by results of bioassays on the freshwater cladoceran Daphnia magna that indicated enhanced juglone toxicity at pHs of < or =6.7. A low octanol:water partition coefficient for juglone indicated little capacity for these compounds to be adsorbed by suspended particulates and for bioaccumulation. These properties together with their relatively rapid degradation (t1/2 < or =30h), particularly in the marine environment, indicated a low the risk of residual toxicity associated with the release of juglone-treated water.

Seasonal variation and estradiol-dependent elevation of Thames estuary eel Anguilla anguilla plasma vitellogenin levels and comparisons with other United Kingdom estuaries.

Eel Anguilla anguilla plasma vitellogenin was investigated as a biomarker of exposure to environmental compounds with estrogenic activity, along the tidal course of the Thames Estuary, UK. A. anguilla was chosen as a sentinel species because of their wide distribution, robustness in field and laboratory studies and also because they have a characterised normal intersex' condition where the gonad contains both developing male and female gonadal cells termed a Syrski organ. Following laboratory exposure to 17beta-estradiol (intraperitoneal injection), a plasma protein (approx. 211 kDa apparent molecular weight) was detected by monoclonal antibodies to vitellogenin of striped bass (Morone saxatilis). Western and dot blot analyses were developed and vitellogenin was isolated from 17beta-estradiol-treated fish to calibrate the quantification of the blots by image analysis. The limits of sensitivity for the Western and dot blots were 100 and 10 ng vitellogenin/ml, respectively. Levels of vitellogenin in Thames estuary samples were below the detection limits of the Western but not the dot blot, and showed no statistically significant site-specific (10 sites) and seasonal-specific (May, August, November) differences. Values were observed to be low, between 11 and 165 ng/ml, compared with 17-50 mg/ml for 17beta-estradiol-treated eels. Similar low levels of plasma vitellogenin were determined in fish sampled along the Tyne, Wear, Tees or Humber estuaries, or the Weston canal Liverpool, with mean plasma vitellogenin levels varying between 44 and 82 ng/ml. These levels of vitellogenin in A. anguilla plasma were observed to be consistent with the known biology of the eel. Immature females, or fish with syrski organs, reported both lower levels and smaller variation of plasma vitellogenin concentrations whereas the highest plasma vitellogenin concentrations were determined in fish above 45 cm consistent with female fish. These results indicate inter-species variation between the plasma vitellogenin concentrations of A. anguilla and other published fish studies undertaken along the same estuaries.

Hepatic 7-ethoxyresorufin O-deethylase activity in eel (Anguilla anguilla) from the Thames Estuary and comparisons with other United Kingdom Estuaries.

Hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activities (indicative of exposure to polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs)) were measured in eel Anguilla anguilla from the Thames Estuary. Fish were collected from up to 13 sites during November 1997, May and August 1998 and October 1999. Throughout this period no clear seasonal variation could be identified at every site along the Thames. However, during the summer months, fish sampled from sites in the middle to the upper estuary (Woolwich, Greenhithe and West Thurrock) reported up to 3-fold higher EROD activities compared to sites either at the upper reaches (Richmond and Brentford) at the same time of the year, or fish sampled in winter, along the entire length of the estuary. A laboratory exposure experiment demonstrated a 3-fold elevation of EROD activity 2 days after injection with beta-naphthoflavone (beta-NF). However, higher levels of activity could be determined in fish sampled from the Weston canal near the Merseyside. The lowest levels of A. anguilla EROD activity were observed in fish sampled from the upper reaches of the River Tamar, Devon, and were comparable to activities determined in fish from the Wear and Humber estuaries. A. anguilla sampled along the Thames, Tyne and Tees estuaries reported between 2.5- and 7-fold higher EROD activities compared to fish collected from the Tamar. These results indicate that a low to moderate induction of A. anguilla CYP1A had occurred (indicative of low to moderate exposure to PAHs and planar PCBs) in fish collected from the Thames, Tyne, Wear, Tees, Humber and Tamar estuaries. However, the highest level of EROD activity was observed in fish from the Weston Canal (Merseyside).

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goksøyr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.

Increased potential for NAD(P)H-dependent reactive oxygen species production of hepatic subcellular fractions of fish species with in vivo exposure to contaminants.

The present study investigated the proposed involvement of contaminant-stimulated reactive oxygen species (ROS) production in disease processes in fish. NAD(P)H-dependent ROS production of subcellular fractions was determined by the iron/EDTA-mediated oxidation of 2-keto-4-methiolbutyric acid. Hepatic cytosolic NADPH-dependent and microsomal NAD(P)H-dependent ROS production were increased 51-160% (P < 0.05) in rainbow trout (Oncorhynchus mykiss) 15 weeks after a single i.p. injection of polychlorobiphenyl (PCB) (100 mg Clophen A50 kg-1 wet wt.). Hepatic microsomal NADH-dependent ROS production was 114% higher in perch (Perca fluviatilis) from PCB-contaminated Lake Järnsjön compared to clean Lake Vänern, Sweden. Hepatic mitochondrial NADH-dependent, cytosolic NADH-dependent and microsomal NADPH-dependent ROS production were variously elevated up to 160% in flounder (Platichthys flesus) at various sites along two pollution transects near to the ports of Rotterdam and Amsterdam, Netherlands. Overall the data indicate increased potential for ROS production in liver of fish exposed to field pollution, and support the hypothesis of oxidative stress as a mechanism of contaminant-mediated disease in fish.

Detection of DNA strand breaks in isolated mussel (Mytilus edulis L. ) digestive gland cells using the "Comet" assay.

Isolated mussel (Mytilus edulis L.) digestive gland cells were analyzed using the single-cell gel electrophoresis or "comet" assay to assess the ability of potential aquatic contaminants to induce DNA strand breaks (SBs) and to investigate the potential application of this technique as part of an aquatic biomonitoring regime. Freshly prepared cell suspensions from digestive gland were exposed in vitro to hydrogen peroxide (H2O2, 0-200 microM), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX, 0-200 microM), benzo[a]pyrene (BaP, 0-200 microM), 1-nitropyrene (1-NP, 0-250 microM) and nitrofurantoin (NF, 0-1000 microM) for 1 h in the dark at 15 degreesC in the presence of the DNA repair inhibitor cytosine-beta-D-arabinofuranoside (araC). DNA strand breakage was measured using the comet assay. There were significant concentration-dependent increases in the percentage of DNA in the comet tail (mean values+/-SD) for all doses compared with controls (P<0.05) with H2O2 (up to 61.4+/-5.1% at 100 microM), MX (up to 34. 3+/-2.2% at 200 microM), BaP (up to 24.7+/-5.1 at 100 microM), 1-NP (up to 54.7+/-5.0% at 200 microM), and NF (up to 68.1+/-4.5% at 500 microM). There was a decrease (P<0.05) in viability (eosin Y exclusion) of exposed compared with control cells at 200 microM H2O2 and BaP only. This study has demonstrated the potential of the comet assay to detect DNA strand breakage at subcytotoxic concentrations of a range of agents, some of which require metabolic activation. This may provide a sensitive, but nonspecific, molecular biomarker of genotoxicity.

DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring.

This review considers the potential for DNA strand breaks, particularly as measured by the comet assay, to act as a biomarker of genetic toxicity in fish and other aquatic species. The background need for such biomarkers is introduced in relation to carcinogenicity, reproductive effects and other adverse effects of pollution. Sensitive measurements of DNA strand breakage can be achieved, e.g., by alkaline elution, alkaline unwinding or by single cell gel electrophoresis (comet) techniques. The DNA damage can be a reflection, not only of direct strand breakage, but also of alkali-labile sites and of repair enzyme-mediated breakage (i.e., is non-specific). A range of genotoxic chemicals (both with and without the requirement for metabolic activation) give positive effects in various cell types of vertebrate and invertebrate aquatic species, following in vitro and in vivo exposures under laboratory conditions. A limited number of analyses of organisms exposed to polluted waters or sediments in the field have implicated DNA strand breakage as a relatively sensitive, rapid and broad specificity indicator of genotoxic pollutant exposure. The comet assay deserves further exploitation to assess inter-individual and inter-cell variability in response to pollutants and naturally occurring genotoxic stimuli, and to assess the persistence of these effects.