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.

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.

Resazurin assay of radiation response in cultured cells.

We describe use of resazurin reduction for measurement of cell response to irradiation as a simple and non-destructive assay that complements the conventional colony forming assay and can readily be applied to both adherent and non-adherent cell cultures. The resazurin method yields data comparable with the colony forming assay as well as to assay of DNA synthesis (BrdU incorporation), giving an OER (oxygen enhancement ratio) of 2.5 at 60% isoeffect level versus 3.1 for the colony forming assay. Intraday and interday precisions for the resazurin assay were 4.1% and 5.2%, respectively.

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.

A simple method for producing a monospecific antiserum against IgM.

To avoid the chemical purification of mastomys IgM, a reliable alternative method for producing a specific anti-mastomys IgM antiserum was developed. It employs the strong cross-reactivity of antisera against mouse serum proteins with mastomys serum proteins. By the immunoelectrophoresis technique, precipitates of mastomys IgM and cross-reacting rabbit anti-mouse IgM were obtained. A rabbit was immunized with these precipitates. Activity against the light chains was removed from the antiserum by passage over an immunoadsorbent column of mastomys IgG. The adsorbed antiserum was found to be specific for determinants on mastomys IgM.

Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

In vivo induction of aryl hydrocarbon hydroxylase in human scalp hair follicles by topical application of a commercial coal tar preparation.

Five low-dose applications of a commercial coal tar preparation on a small scalp skin region resulted in an induction of aryl hydrocarbon hydroxylase (AHH) activity in freshly isolated human hair follicles. Large but reproducible interindividual differences in AHH-inducibility could be detected. The method offers the opportunity to measure AHH-inducibility, which has been correlated to the risk of developing chemical-induced cancer, in vivo in normal epithelium, a cell-type highly relevant for chemical carcinogenesis. Smoking habits did not have any effect on AHH-activity in freshly isolated hair follicles. Therefore the method potentially permits the identification of persons with high and low genetically determined AHH-inducibility.

Formation of trifluoroacetylated protein antigens in cultured rat hepatocytes exposed to halothane in vitro.

Immune responses to novel, halothane metabolite-modified protein antigens (tri-fluoroacetylated proteins; TFA-proteins) have been implicated in the pathogenesis of halothane hepatitis. The aim of the present study was to investigate and characterize expression of TFA-proteins in cultures of rat hepatocytes which were exposed to halothane in vitro. Following exposure to halothane, the hepatocytes were harvested, then subcellular fractions were prepared and were analysed by immunoblotting for expression of antigens recognized by a rabbit anti-TFA antiserum, and by antibodies in sera from two patients with halothane hepatitis. Hepatocytes exposed to halothane in vitro were shown to express novel microsomal protein antigens, which exhibited molecular masses that were identical to the molecular masses of the major TFA-protein antigens expressed in vivo, in livers of halothane-treated rats (100, 80 and 60 kDa). Experiments in which hepatocytes were exposed to halothane in the presence of SKF-525A, or were exposed to deuterated halothane in place of halothane, confirmed that these novel antigens were TFA-modified proteins whose generation required cytochrome P450-mediated metabolism of halothane. The maximal levels of TFA-antigens expressed in vitro were about 30% of the levels expressed in halothane-treated rats in vivo. Maximal expression of the TFA-antigens in vitro occurred when hepatocytes were exposed to halothane at doses which yielded concentrations of the drug in culture medium of about 13 microM. Expression of the antigens in vitro occurred slowly, with an apparent half-time of about 8 hr. Overall, these results demonstrate that the properties of the TFA-antigens expressed in cultured hepatocytes in vitro closely resemble the properties exhibited by the antigens expressed in vivo, in livers of halothane-treated rats.

The effect of beclobric acid and clofibric acid on peroxisomal beta-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes.

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey (Macaca fascicularis) and human liver. Determination of peroxisomal fatty acid beta-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal beta-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 microM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations greater than 300 microM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid beta-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisome-proliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.

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.

Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide.

A method is described to investigate the induction of micronuclei in cultured human keratinocytes after short-term exposure to known clastogenic agents. The cytokinesis-block method was applied to facilitate the scoring of micronucleated cells. Mitomycin C, a direct-acting compound, caused a 5-20-fold increase in micronuclei over the controls at the highest concentration tested (1 microgram/ml). Cyclophosphamide, an agent requiring metabolic activation, did not induce the formation of micronuclei in cultured keratinocytes. However, after pretreatment of the keratinocyte cultures with Aroclor 1254 for 72 h, exposure to cyclophosphamide resulted in a 3-fold increase in micronucleus frequency over the controls. No cytogenetic effect of Aroclor 1254 was observed in control experiments.

Micronucleus formation in cultured human keratinocytes: Involvement of intercellular bioactivation.

Micronucleus formation in cultured human keratinocytes was studied after exposure to benzo[a]pyrene, cyclophosphamide and 12-O-tetradecanoylphorbol-13-acetate without the addition of an exogenous metabolizing system. The first two agents need bioactivation by specific isoenzymes of cytochrome P-450 to form genotoxic intermediates. Benzo[a]pyrene induced the micronucleus formation in both uninduced and Aroclor 1254-pretreated cultures. Clastogenic effects of cyclophosphamide were observed only in Aroclor 1254-pretreated cells. The tumour promotor 12-O-tetradecanoylphorbol-13-acetate did not affect the frequency of micronuclei in human keratinocytes. The data indicate that cultured human keratinocytes can be used to study the tissue-specific response to genotoxic agents as well as interindividual variation in biotransformation capacity.

Generation of trifluoroacetylated protein antigens in cultured rat hepatocytes exposed to halothane in vitro.

Previous investigations have implicated an immune response to trifluoroacetylated proteins (TFA-proteins) in the pathogenesis of halothane hepatitis. The objective of this study was to establish conditions for generation of TFA-proteins in hepatocytes exposed to halothane in vitro. Monolayer cultures of rat hepatocytes were incubated in sealed flasks with or without added halothane, then subcellular fractions were prepared by differential centrifugation and analysed by immunoblotting for reactivity with anti-TFA antiserum. The specificity of the antiserum was verified by hapten inhibition with N--TFA-l-lysine. TFA-proteins were generated when hepatocytes were cultured with halothane, but not when hepatocytes were cultured without halothane, and were concentrated in the microsomal fraction. Generation of TFA-proteins was greater when hepatocytes were exposed to an initial halothane concentration of about 0.17 mm-halothane than when hepatocytes were exposed to higher concentrations (0.6 mm and 1.4 mm). The molecular masses of the major TFA-proteins produced in vitro (100, 80 and 60 kDa) were very similar, if not identical, to the molecular masses of the major TFA-proteins produced in livers of rats treated ip with halothane in vivo, as were the kinetics of TFA-protein formation and turnover.

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.

Characteristics of late responses to superior laryngeal nerve stimulation in humans.

To characterize human thyroarytenoid and cricothyroid muscle responses to stimulation of the internal (sensory) and external (motor) branches of the superior laryngeal nerve (SLN), three awake subjects were studied at rest and during muscle activation with stimulation at different current levels. When only the external branch was stimulated, direct cricothyroid muscle responses were obtained without responses in either thyroarytenoid muscle. When only the internal branch was stimulated, no cricothyroid responses were obtained, but two late thyroarytenoid responses occurred (R1 and R2). The R1 response was usually ipsilateral and had a mean onset latency of 18 milliseconds, while the R2 response was bilateral and occurred between 66 and 70 milliseconds. Both responses tended to decrease in latency and increase in amplitude with increased stimulation level. The similarity of R1 to the adductor response and R2 to other late responses is discussed.

Comparison of muscle activation patterns in adductor and abductor spasmodic dysphonia.

Patients with adductor and abductor spasmodic dysphonia were compared with normal controls on speech items particularly difficult for the two patient groups. Intrinsic and extrinsic laryngeal muscles were measured during adduction for voicing and for a glottal stop, and during abduction after a vowel and after a glottal stop. Muscle activity was measured while the patients attempted the speech items, with considerable difficulty, but not during periods of complete voice loss. Muscle activity at rest, activity increases for speech, and percent changes for adduction and abduction did not differ from normal in either patient group. The results demonstrated normal muscle activation levels and changes for speech in the patients, and suggested that symptoms occur when spasmodic bursts intrude on an otherwise normal pattern.

Limitations of electromyography and magnetic stimulation for assessing laryngeal muscle control.

The development of new phonosurgical techniques has increased the level of interest in the field of neurolaryngology. This field requires valid techniques for determining if muscle activation is normal. Laryngeal electromyography is being used more frequently to assess muscle innervation and synkinesis. Further, magnetic stimulation has been introduced as a noninvasive technique for nerve stimulation. Technical limitations that affect the clinical utility of both these techniques are reviewed: 1) difficulties obtaining selective and accurate electromyographic laryngeal muscle recordings, 2) normal variation in movement and muscle activation patterns within and between normal individuals when producing the same speech syllables, and 3) variation in laryngeal muscle response latencies between and within normal subjects during peripheral magnetic stimulation. Given the normal variation in laryngeal electromyography and magnetic stimulation response latencies, these techniques may not yet be reliable or accurate for assessing reinnervation or synkinesis following recurrent laryngeal nerve injury.

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.

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.

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.