Effects of contaminated sediment from Cork Harbour, Ireland on the cytochrome P450 system of turbot.

Hatchery-reared juvenile turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to inter-tidal sediments collected from polluted sites in Cork Harbour (Whitegate and Agahda) and a reference site at Ballymacoda Co., Cork, Ireland. The potential of the sediment exposure to induce cytochrome P450 activities and CYP1A1 in the fish was assessed. Chemical analysis revealed that the sediments originating from the reference and harbour sites were contaminated principally with PAHs-the harbour sites having double the levels of those at the reference site. Following 3 weeks exposure to the sediments western blotting demonstrated a strong immunogenic response for CYP1A1 in the liver, but not for gill or intestine. P450 activities were generally significantly higher than those exposed to reference site sediment. Liver was the most responsive tissue with significantly greater P450 activities compared with gill and intestinal tissues.

Communication of radiation-induced stress or bystander signals between fish in vivo.

We report data in this paper suggesting that fish irradiated to 0.5 Gy total body dose can release factors into the water that signal other unexposed fish and cause induction of bystander effects expressed as increased cell death in a reporter system. Radiation-induced bystander effects, resulting in the appearance of radiation damage or induction of typical radiation responses in unirradiated cells and tissues are now an established consequence of exposure to low doses of ionizing radiation, however little work has been done in vivo or in species other than humans or mice. In these experiments rainbow trout were irradiated and then paired with unirradiated fish for two hours. Additionally, unirradiated fish were placed in water which had previously been used to hold irradiated fish for 2 h. Sham-irradiated fish and absolute control fish were also examined all using blind protocols. Following a two h incubation period, at these various exposure regimes, the fish were killed by a blow to the head and dissected. Five organs were removed from each fish and tissue explants were cultured using an established technique. After 2 days, the culture medium was harvested and used in a reporter assay to determine whether a bystander effect had been induced. The explants were cultured on in Clonetics growth medium for a further 14 days then fixed for assay of radiation response proteins. The responses varied according to the cell type in the original explants, with the gill and fin showing the most pronounced response. The results suggest that communication signals leading to a typical radiation response can be passed between fish and seem to involve secretion of a chemical messenger into the water.

An assessment of the pollutant status of surficial sediment in Cork Harbour in the South East of Ireland with particular reference to polycyclic aromatic hydrocarbons.

Surface sediment from three polluted sites within Cork Harbour, Ireland, and from a relatively clean reference site were collected and analysed for polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), organotins (OTs), and heavy metals. PAHs were determined to be the most abundant class of contaminant. Concentrations of the sum (Sigma) of the 21 PAHs measured from the Harbour sites (2877.70 ng g(-1), 1000.7 ng g(-1) and 924.40 ng g(-1) dry weight respectively) were significantly higher than that of the sediment from the reference site (528.30 ng g(-1) dry weight). An inner harbour site, Douglas being the more contaminated of the three harbour sites. A similar pattern was observed with the other contaminants however, these compounds, with the exception of the heavy metals, all tended to be detected at concentrations on or below detection limits.

Genotoxicity of field-collected inter-tidal sediments from Cork Harbor, Ireland, to juvenile turbot (Scophthalmus maximus L.) as measured by the Comet assay.

The alkaline single cell gel electrophoresis (SCGE) or Comet assay was employed to test the potential of surficial sediment collected from Cork Harbor, Ireland, to induce DNA damage in turbot (Scophthalmus maximus L.) in a laboratory exposure experiment. Turbot were exposed for 21 days to field-collected sediment from Cork Harbor and from a relatively clean reference site at Ballymacoda and sampled at 0, 7, 14, and 21 days. As a positive control for the sediment exposure experiment, a subsample of the turbot was exposed to cadmium chloride-spiked seawater. DNA damage analysis was performed on epidermal, gill, spleen, liver, and whole blood cell preparations. Liver, gill, and blood were the most sensitive tissues while a lower level of damage was detected in the epidermis and spleen. The blood was determined to be a suitable predictor of DNA damage in the whole organism. Chemical analysis of the sediment indicated that polycyclic aromatic hydrocarbons formed the bulk of the contaminants, with the harbor sites having almost double the levels of those from the reference site. The data indicated that turbot exposed to sediments from Cork Harbor elicited a significant increase in DNA damage in comparison with those exposed to sediment from the reference site and that exposure to the contaminated sediments caused a multi-organ genotoxic response. Results from the study indicate a relationship between the presence of genotoxicants in sediment and DNA damage. This finding was encouraging with regard to the potential use of the Comet assay as part of a marine biomonitoring strategy.

Heat shock protein 70 levels in rainbow trout primary epidermal cultures in response to 2,4-dichloroaniline exposure: a novel in vitro aquatic toxicity marker.

The aim of this work was to investigate the use of the heat shock protein, HSP 70, as a sublethal measurement of ecotoxicity and to identify if the amount of HSP 70 synthesized is proportional to the chemical stress applied. This was achieved by quantifying the HSP 70 levels in primary cultured rainbow trout, Oncorhynchus mykiss (R.), skin epidermal cells in response to 2,4-dichloroaniline (2,4-DCA) exposure. The cellular stress response protects organisms from damage resulting from exposure to a wide variety of stressors including xenobiotics. The use of a HSP 70 polyclonal antibody on rainbow trout primary epidermal skin cultures exposed to 2,4-DCA was investigated as a possible biomarker for environmental stress using an immunocytochemical approach. The epidermis is highly susceptible, as it is the interface between the fish and its aquatic environment. In this study we have developed a simple in vitro system for aquatic-toxicity risk assessment. A method for the quantification of heat shock (stress) protein levels by immunocytochemistry is described. The antibody dilution range enabled the detection and quantification of only the inducible HSP 70 fraction. A 1:2000 dilution was decided upon. This assay was effective in detecting and quantifying the induced HSP 70. There was a direct toxicant concentration-dependent increase in the levels of the cellular stress protein in the primary epidermal cultures. Enhanced localization of HSP 70 in the nuclei of the epidermal cells was observed following exposure to 2,4-DCA. This work indicated the possibility of using heat shock protein induction and subsequent quantification as a sensitive system for aquatic toxicity risk assessment.