Shellfish toxicity: human health implications of marine algal toxins.

Five major human toxic syndromes caused by the consumption of shellfish contaminated by algal toxins are presented. The increased risks to humans of shellfish toxicity from the prevalence of harmful algal blooms (HABs) may be a consequence of large-scale ecological changes from anthropogenic activities, especially increased eutrophication, marine transport and aquaculture, and global climate change. Improvements in toxin detection methods and increased toxin surveillance programmes are positive developments in limiting human exposure to shellfish toxins.

The effects of salinity on the Manila clam (Ruditapes philippinarum) using the neutral red retention assay with adapted physiological saline solutions.

This study investigated the internal osmotic regulatory capabilities of the Manila clam (Ruditapes philippinarum) following in vivo exposure to a range of salinities. A second objective was to measure the health status of the Manila clam following exposure to different salinities using the neutral red retention (NRR) assay, and to compare results using a range of physiological saline solutions (PSS). On exposure to seawater of differing salinities, the Manila clam followed a pattern of an osmoconformer, although they seemed to partially regulate their circulatory haemolytic fluids to be hyperosmotic to the surrounding aqueous environment. Significant differences were found when different PSS were used, emphasizing the importance of using a suitable PSS to reduce additional osmotic stress. Using PSS in the NRR assay that do not exert additional damage to lysosomal membrane integrity will help to more accurately quantify the effects of exposure to pollutants on the organism(s) under investigation.

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.

A test battery approach for the ecotoxicological evaluation of estuarine sediments.

The purpose of this study was to evaluate the overall sensitivity and applicability of a number of bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening (Tier I) of estuarine sediments. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting results. As sediment is an inherently complex, heterogeneous geological matrix, the toxicity associated with different exposure routes (solid, porewater and elutriate phases) was also assessed. A stimulatory response was detected following exposure of some sediment phases to both the Microtox and algal bioassays. Of the bioassays and endpoints employed in this study, the algal test was the most responsive to both elutriates and porewaters. Salinity controls, which corresponded to the salinity of the neat porewater samples, were found to have significant effects on the growth of the algae. To our knowledge, this is the first report of the inclusion of a salinity control in algal toxicity tests, the results of which emphasise the importance of incorporating appropriate controls in experimental design. While differential responses were observed, the site characterised as the most polluted on the basis of chemical analysis was consistently ranked the most toxic with all test species and all test phases. In terms of identifying appropriate Tier I screening tests for sediments, this study demonstrated both the Microtox and algal bioassays to be more sensitive than the bacterial enzyme assays and the invertebrate lethality assay employing Artemia salina. The findings of this study highlight that salinity effects and geophysical properties need to be taken into account when interpreting the results of the bioassays.

Effect of atorvastatin and fluvastatin on the metabolism of midazolam by cytochrome P450 in vitro.

We have investigated the effects of the statins atorvastatin and fluvastatin on the cytochrome P450 3A4 enzyme (CYP 3A4)-mediated metabolism of midazolam in vitro, using pooled human liver microsomes. Midazolam was metabolised by human hepatic microsomes with a Michaelis-Menten constant (K(m)) of 5.25 (SD 1.2) micromol.l(-1). Atorvastatin was a moderate competitive inhibitor of CYP 3A4 with an inhibitory constant (K(i)) of 12.4 (95% CI 4.65-20.06) micromol.l(-1). Fluvastatin was a weak non-competitive inhibitor of CYP 3A4 with a K(i) of 94.3 (95% CI 55.01-133.5) micromol.l(-1). Both atorvastatin and fluvastatin inhibit the CYP 3A4-mediated metabolism of midazolam in vitro.

Assessing the potential of fish cell lines as tools for the cytotoxicity testing of estuarine sediment aqueous elutriates.

In the present study, we assess the potential of fish cell lines (CHSE, EPC and RTG-2) to be used as screening tools for the ecotoxicological assessment of estuarine sediments. The processing of sediment to a form suitable for in vitro exposure is an inherent problem when using cell cultures. The approach employed in this study was to prepare aqueous elutriate extracts from whole sediments, which were subsequently used to reconstitute powdered media. This procedure allowed the exposure of cell cultures to concentrations of up to and including 100% of the original aqueous sample. Cytotoxicity was assessed using multiple endpoint measurements. Cell viability was quantified using the neutral red and alamar blue colorimetric assays, which specifically assess lysosomal and mitochondrial function, respectively. In addition, the total protein content of the cells was measured using the coomassie blue assay. Initial tests were conducted to ensure that any resultant cytotoxicity was due to sample contaminants and not osmotic stress. In addition, elutriate samples were spiked with a model toxicant to verify the ability of the cell lines to detect and respond to bioavailable contaminants. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting any observed cytotoxicity. A differential response was observed for the cytotoxicity assays following exposure treatments, which emphasises the importance of employing multiple endpoints for the determination of toxicity. Of the three cell lines utilised in this study, RTG-2 cells were the most suitable for the testing of estuarine aqueous elutriate samples on the basis of tolerance to osmolality effects. Slight toxicity was observed following exposure to the aqueous elutriates tested in this study using RTG-2 cells and the alamar blue assay. In order to fully evaluate the overall sensitivity of this cell line, further research is warranted using an extensive range of test sites incorporating more polluted sediments.

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.

In vitro cytotoxicity testing of three zinc metal salts using established fish cell lines.

The utilisation of fish cell lines has proven to be a valuable, rapid and cost-effective tool in the ecotoxicological assessment of chemicals and environmental samples. The main objective of this study was to investigate the value of multiple endpoint measurements in evaluating the cytotoxicity of three divalent zinc salts in three established fish cell lines (EPC, CHSE and RTG-2) and the potential for their employment as effective screening tools for zinc contaminated environmental samples. A significant stimulatory effect was detected with the neutral red assay in EPC and RTG-2 cells exposed to the lower doses of some zinc compounds. Significant (p < or = 0.01) lactate dehydrogenase release was detectable only with the highest exposure concentration of ZnCl2. Toxicity ranking based on IC50 values calculated from the neutral red and coomassie blue assay data found that in general, ZnC2 was the most cytotoxic metal compound to the cell lines employed. Differential cell sensitivities were observed to be dependant on the particular compound tested and the endpoint employed. It was found that the use of light microscopy in the identification of cell morphological changes was a valuable adjunct in verifying the results of colorimetric tests. In conclusion, careful consideration should be given to study design and statistics applied and use of a battery style approach is recommended for toxicological screening studies.

Implications of seasonal priming and reproductive activity on the interpretation of Comet assay data derived from the clam, Tapes semidecussatus Reeves 1864, exposed to contaminated sediments.

We explore the use of the clam Tapes semidecussatus Reeves 1864 as an indicator for the presence of potentially genotoxic substances in estuarine sediments. The limitations associated with the interpretation of Comet assay data (expressed as % DNA in tail) in terms of clam reproductive state, size (age) and thermal exposure history following laboratory acclimation are discussed. Hatchery-reared clams, subjected to ambient temperature fluctuations during growth, were exposed in vivo under laboratory conditions for three weeks to sediment samples collected from a polluted site and a "clean" reference site. The DNA damage observed in haemocytes, gill and digestive gland cells was significantly higher in animals exposed to contaminated sediment compared to those exposed to sediment from the reference site. The extent of DNA damage recorded was not correlated with size (age). Spawning was not observed during the experiment. Nevertheless, clams with well-developed gonads showed a statistically higher degree of DNA damage in gill and digestive gland cells- but not haemocytes, demonstrating an increased sensitivity to potential genotoxic compounds, possibly caused by impaired DNA repair capacity due to reproductive activity. Furthermore, the degree of DNA damage in clams exposed to contaminated sediments was higher in autumn and winter compared to spring and summer, suggesting an effect of seasonal priming.

The effects of concurrent atorvastatin therapy on the pharmacokinetics of intravenous midazolam.

Midazolam is a commonly used anaesthetic agent and is metabolised by the 3A4 isoform of the cytochrome P450 enzyme system. Atorvastatin is also metabolised by cytochrome P450 3A4 and, in vitro, atorvastatin inhibits the cytochrome P450 3A4-mediated metabolism of mexazolam. We hypothesised that concurrent administration of atorvastatin and midazolam would result in altered midazolam pharmacokinetics. Fourteen patients scheduled to undergo general anaesthesia for elective surgery were recruited in a matched pair design to receive intravenous midazolam (0.15 mg.kg-1). Of these patients, seven were taking long-term atorvastatin. Atorvastatin patients demonstrated a greater area under the curve (889.4 (standard deviation 388.6) ng-h.ml-1) vs. control patients (629.1 (standard deviation 197.2) ng-h.ml-1) (p < 0.05). Patients taking atorvastatin also demonstrated a decreased clearance (0.18 (standard deviation 0.08) l-kg. h-1) vs. control patients (0.27 (standard deviation 0.08) l-kg.h-1) (p < 0.05). This study suggests that chronically administered atorvastatin decreases the clearance of intravenously administered midazolam.

Detecting genotoxicity using the Comet assay following chronic exposure of Manila clam Tapes semidecussatus to polluted estuarine sediments.

Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-bound contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to pollutants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph, gill and digestive gland from the clam Tapes semidecussatus, using the single cell gel electrophoresis (Comet assay). Clams were exposed for three weeks to sediment samples collected from a polluted site and a 'clean' reference site. The level of DNA damage was assessed using an image analysis package and expressed as Tail Moment. Throughout the study, significant differences in DNA damage were recorded for each tissue type between clams exposed to the two sediment samples. We conclude that the Comet assay is a useful tool for the detection of DNA damage in clams chronically exposed to polluted sediments.