Antioxidant capacity of flaxseed products: the effect of in vitro digestion.

This study evaluated the effect of in vitro digestion of flaxseed products on Folin-Ciocalteu reagent reducing substances (FCRRS), its antioxidant capacity and prevention of oxidative DNA damage in human monocyte cell line U937. Flaxseed protein isolate was obtained from defatted flaxseed meal and the protein hydrolysate with high antioxidant capacity was obtained from hydrolysis of the protein isolate with Alcalase in a two factor central composite rotatable design (pH 8.5 and enzyme: substrate 1:90, w/w). The FCRRS content and antioxidant capacity measured by FRAP and ORAC in aqueous and 70 % methanol extracts were the highest in protein hydrolysate, followed by protein isolate, while the defatted meal showed the lowest values. After in vitro gastrointestinal digestion, the FCRRS content of protein isolate and hydrolysate reached similar values, however the hydrolysate had the highest antioxidant capacity, measured by FRAP while the isolate had the highest ORAC values. The defatted meal showed the lowest capacity in all assays (p < 0.05). The hydrolysate did not protect against DNA damage induced by H2O2 in U937 cells under the conditions of the present study. The results suggest that flaxseed protein isolate and hydrolysate are potential functional food ingredients with antioxidant capacity.

Assessment of the ability of seaweed extracts to protect against hydrogen peroxide and tert-butyl hydroperoxide induced cellular damage in Caco-2 cells.

The ability of brown seaweed extracts, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus to protect against tert-butyl hydroperoxide (tert-BOOH) induced stress in Caco-2 cells was investigated. Oxidative stress was determined by measuring alteration in the enzymatic activity of catalase (CAT) and superoxide dismutases (SOD) and cellular levels of glutathione (GSH). L. hyperborea, P. canaliculata and F. serratus significantly protected against tert-BOOH induced SOD reduction but did not protect against the reduction in CAT activity or the increased cellular levels of GSH. The ability of F. serratus and F. vesiculosus to protect against H(2)O(2) and tert-BOOH induced DNA damage was also assessed. The DNA protective effects of the two seaweed extracts was compared to those of three metal chelators; deferoxamine mesylate (DFO), 1,10-phenanthroline (o-phen) and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM). F. serratus and F. vesiculosus significantly protected (P<0.05) against H(2)O(2) (50 µM) induced DNA damage but not tert-BOOH induced damage.

Geographical location has greater impact on carotenoid content and bioaccessibility from tomatoes than variety.

The suggested health benefits of consuming tomatoes and tomato-based products have been attributed, in part, to the carotenoids present in these foods. Therefore, the objectives of the present study were to (i) analyse carotenoid content and bioaccessibility from different tomato (Lycopersicon esculentum L.) types namely cherry, plum, round, and certain tomatoes-on-the-vine; and (ii) determine if geographical location (Ireland vs Spain) influenced the content and bioaccessibility of carotenoids in tomatoes of the same variety. Carotenoid bioaccessibility is defined as the amount of ingested carotenoids that, after digestion, are available for absorption by intestinal cells. Differences were seen in carotenoid content and bioaccessibility between the different tomato types tested. For instance, Irish round high-lycopene tomatoes contained the greatest amounts of lycopene and lutein but lowest levels of beta-carotene compared with the other Irish tomatoes. Furthermore, the content and bioaccessibility of carotenoids that were sourced from Ireland and Spain also varied greatly. Spanish tomatoes were generally superior in the content, bioaccessibility, and micelle content of carotenoids. To conclude, our findings suggest that geographical location, rather than the type of tomato, seems to have a more pronounced effect on carotenoid bioaccessibility from tomatoes.

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.

The effect of lutein, sesamol, ellagic acid and olive leaf extract on lipid oxidation and oxymyoglobin oxidation in bovine and porcine muscle model systems.

The effect of lutein (100, 200, 300µg/ml), sesamol (500, 1000, 2000µg/ml), ellagic acid (300, 600, 900µg/ml) and olive leaf extract (100, 200, 300µg/ml) on oxymyoglobin oxidation and lipid oxidation in bovine and porcine muscle model systems (25% M. longissimus thoracis et lumborum homogenates) was examined. Radical scavenging activity, using the DPPH assay, and iron-chelating activities of lutein, sesamol, ellagic acid and olive leaf extract were assessed at concentrations ranging from 200 to 1000ppm. The radical scavenging activity was of the order: ellagic acid>sesamol>olive leaf extract>lutein. None of the natural antioxidants examined exhibited iron chelating activity. Following induced lipid oxidation (FeCl(3)/sodium ascorbate addition), lipid oxidation and oxymyoglobin oxidation were measured after 24h at 4°C. In bovine and porcine muscle model systems, lipid oxidation decreased (P<0.001) following addition of each of the natural antioxidants relative to the control and antioxidant potency followed the order: sesamol>ellagic acid>olive leaf extract>lutein. Ellagic acid and olive leaf extract decreased oxymyoglobin oxidation (P<0.001) while sesamol increased oxymyoglobin oxidation in both systems. The natural antioxidants examined may have applications in the development of nutritional enhanced meat products with enhanced shelf-life characteristics.

Micellarisation of carotenoids from raw and cooked vegetables.

The efficiency of carotenoid micellarisation from plant foods can be used as an effective tool for the initial screening of carotenoid bioavailability. Therefore, the objectives of the present study were to assess the effects of cooking on the micellarisation of beta-carotene, lycopene, beta-cryptoxanthin and lutein from courgette (zucchini), red pepper and tomato; and, to a minor extent, investigate uptake of lutein by Caco-2 cells from micellar fractions obtained from raw and cooked courgettes. Both raw and cooked vegetables were subjected to an in vitro digestion procedure. beta-Carotene levels were significantly decreased in the digesta from each vegetable after boiling, grilling, microwave-cooking, or steaming, however all of the cooking methods enhanced beta-carotene transfer to micelles. Carotenoid micellarisation ranged from 1.7% to 100% depending on the food, carotenoid, and the cooking method tested. Grilling and microwave-cooking were generally the most detrimental on the transfer of xanthophyll carotenoids, namely beta-cryptoxanthin, to the micelles. Caco-2 cells absorbed greater amounts of lutein from the micelles of microwave-cooked courgettes than those that were raw, boiled, grilled, or steamed. Depending on the cooking methods used, carotenoid retention as well as micellarisation varied for each carotenoid among the different vegetables and different carotenoids present in each particular food.

Addition of grape seed extract and bearberry to porcine diets: Influence on quality attributes of raw and cooked pork.

The effect of supplementation of pig diets with grape seed extract (GSE) (100, 300, 700mg/kg feed) and bearberry (BB) (100, 300, 700mg/kg feed) for 56 days pre-slaughter, on the oxidative stability and quality of raw and cooked M. longissimus dorsi (LD) was examined. Susceptibility of porcine liver, kidney and heart tissue homogenates to iron-induced (1mM FeSO(4)) lipid oxidation was also investigated. In raw LD steaks, stored in modified atmosphere packs (75% O(2):25% CO(2)) (MAP) for up to 16 days at 4°C, surface lightness (CIE 'L' value), redness (CIE 'a' value), lipid stability (TBARS, mg MDA (malondialdehyde)/kg muscle) and pH were not significantly affected by supplemental GSE or BB. Similarly, the oxidative stability and sensory properties of cooked LD steaks, stored in MAP (70% N(2):30% CO(2)), for up to 28 days at 4°C, were not enhanced by dietary GSE or BB. Iron-induced lipid oxidation increased in liver, kidney and heart tissue homogenates over the 24h storage period and susceptibility to oxidation followed the order: liver>heart>kidney. Dietary GSE or BB did not significantly reduce lipid oxidation in tissue homogenates. Potential reasons for the lack of efficacy of supplemental GSE and BB on pork quality were explored.

Phytosterol, squalene, tocopherol content and fatty acid profile of selected seeds, grains, and legumes.

The unsaponifiable lipid fraction of plant-based foods is a potential source of bioactive components such as phytosterols, squalene, and tocopherols. The objective of the present study was to determine the levels of phytosterols, and squalene, as well as tocopherols (alpha and beta + gamma) in selected grains, seeds, and legumes. The method comprised acid hydrolysis and lipid extraction followed by alkaline saponification, prior to analysis by HPLC. In addition, the fatty acid profile of the foods was determined via total lipid extraction, fatty acid derivitisation and GC analysis. In general, beta-sitosterol was the most prevalent phytosterol, ranging in concentration from 24.9 mg/100 g in pumpkin seed to 191.4 mg/100 g in peas. Squalene identified in all foods examined in this study, was particularly abundant in pumpkin seed (89.0 mg/100 g). The sum of alpha- and beta+ gamma-tocopherols ranged from 0.1 mg/100 g in rye to 15.9 mg/100 g in pumpkin seeds. Total oil content ranged from 0.9% (w/w) in butter beans to 42.3% (w/w) in pumpkin seed and the type of fat, in all foods examined, was predominantly unsaturated. In conclusion, seeds, grains, and legumes are a rich natural source of phytosterols. Additionally, they contain noticeable amounts of squalene and tocopherols, and in general, their fatty acid profile is favorable.

Evaluation of the antioxidant potential of grape seed and bearberry extracts in raw and cooked pork.

The effect of grape seed extract (GSE) and bearberry (BB), on lipid oxidation (TBARS, mg malondialdehyde (MDA)/kg muscle), colour (CIE 'a' redness value), pH, microbial status (log(10)CFU colony forming units/g pork) and sensorial properties of cooked pork patties was investigated. GSE (0-1000µg/g muscle) and BB (0-1000µg/g muscle) were added to raw pork (M. longissimus dorsi) patties which were stored in modified atmosphere packs (MAP) (75% O(2):25% CO(2)) for up to 12 days at 4°C. Cooked pork patties were stored in MAP (70% N(2):30% CO(2)) for up to 4 days at 4°C. Mesophilic plate counts and pork pH were unaffected by GSE and BB. GSE and BB addition decreased (P<0.05) lipid oxidation (TBARS) in raw pork patties on days 9 and 12 of storage, relative to controls. Antioxidant activity of GSE and BB was observed in cooked pork patties demonstrating the thermal stability of GSE and BB. The 'a' redness values of raw and cooked pork patties marginally increased with increasing GSE concentration. The sensory properties of cooked pork patties were unaffected by GSE and BB addition. Results obtained demonstrate the potential for using health promoting nutraceuticals in meat and meat products.

Fatty acid profile, tocopherol, squalene and phytosterol content of brazil, pecan, pine, pistachio and cashew nuts.

Nuts contain bioactive constituents that elicit cardio-protective effects including phytosterols, tocopherols and squalene. The objective of the present study was to determine the total oil content, peroxide value, fatty acid composition and levels of tocopherols, squalene and phytosterols in oil extracted from freshly ground brazil, pecan, pine, pistachio and cashew nuts. The total oil content of the nuts ranged from 40.4 to 60.8% (w/w) while the peroxide values ranged from 0.14 to 0.22 mEq O2/kg oil. The most abundant monounsaturated fatty acid was oleic acid (C18:1), while linoleic acid (C18:2) was the most prevalent polyunsaturated fatty acid. The levels of total tocopherols ranged from 60.8 to 291.0 mg/g. Squalene ranged from 39.5 mg/g oil in the pine nut to 1377.8 mg/g oil in the brazil nut. beta-Sitosterol was the most prevalent phytosterol, ranging in concentration from 1325.4 to 4685.9 mg/g oil. In conclusion, the present data indicate that nuts are a good dietary source of unsaturated fatty acids, tocopherols, squalene and phytosterols.

Involvement of calcium in 7beta -hydroxycholesterol and cholesterol-5beta,6beta -epoxide-induced apoptosis.

Oxidized low-density lipoprotein (oxLDL) is believed to play a central role in the development of atherosclerosis. The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7beta-Hydroxycholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. The exact mechanism through which these oxysterols induce apoptosis remains to be fully elucidated. A perturbation of intracellular calcium homeostasis has been found to trigger apoptosis in many experimental systems. The aim of the present study was to determine the involvement of calcium signaling in 7beta-OH and beta-epoxide-induced apoptosis. To this end, the authors employed the calcium channel blockers verapamil and nifedipine and inhibitors of calpain activation, ALLM and ALLN. Verapamil protected against the decrease in viability induced by 7beta-OH whereas nifedipine had a protective effect in both 7beta-OH and beta-epoxide-treated cells, though these compounds did not restore viability to control levels. Verapamil, nifedipine, and ALLM prevented apoptosis induced by beta-epoxide. None of the compounds employed in the current study protected against 7beta-OH-induced apoptosis. Our results implicate calcium signaling in the apoptotic pathway induced by beta-epoxide and also highlight differences between apoptosis induced by 7beta-OH and beta-epoxide.

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.

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.

Fatty acid profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts and the macadamia nut.

Nuts are high in fat but have a fatty acid profile that may be beneficial in relation to risk of coronary heart disease. Nuts also contain other potentially cardioprotective constituents including phytosterols, tocopherols and squalene. In the present study, the total oil content, peroxide value, composition of fatty acids, tocopherols, phytosterols and squalene content were determined in the oil extracted from freshly ground walnuts, almonds, peanuts, hazelnuts and the macadamia nut. The total oil content of the nuts ranged from 37.9 to 59.2%, while the peroxide values ranged from 0.19 to 0.43 meq O2/kg oil. The main monounsaturated fatty acid was oleic acid (C18:1) with substantial levels of palmitoleic acid (C16:1) present in the macadamia nut. The main polyunsaturated fatty acids present were linoleic acid (C18:2) and linolenic acid (C18:3). alpha-Tocopherol was the most prevalent tocopherol except in walnuts. The levels of squalene detected ranged from 9.4 to 186.4 microg/g. beta-Sitosterol was the most abundant sterol, ranging in concentration from 991.2 to 2071.7 microg/g oil. Campesterol and stigmasterol were also present in significant concentrations. Our data indicate that all five nuts are a good source of monounsaturated fatty acid, tocopherols, squalene and phytosterols.

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.

Phytosterols: lack of cytotoxicity but interference with beta-carotene uptake in Caco-2 cells in culture.

Ingestion of phytosterols has been shown to reduce plasma cholesterol in both animals and humans. The esterified forms of phytosterols are increasingly being incorporated into margarine and fat spreads, which are then marketed as functional foods. The aim was to assess the cytotoxicity and uptake of four phytosterols, beta-sitosterol, campesterol, stigmasterol and stigmastanol, in human intestinal cells in culture. Another aim was to determine if phytosterols would interfere with alpha-tocopherol or beta-carotene uptake by these cells. Human adenocarcinoma Caco-2 cells were supplemented for 24 h with increasing concentrations (0-12.5 microM) of each phytosterol. Cytotoxicity was assessed by neutral red uptake (NRU), lactate dehydrogenase release (LDH) and fluorescein diacetate/ethidium bromide (FDA/EtBr) assays. The phytosterols had no significant effects on Caco-2 cell viability assessed using LDH and FDA/EtBr assays. The highest concentrations of beta-sitosterol and campesterol tested (12.5 microM) resulted in decreased cell viability assessed using the NRU assay. All phytosterols were taken up by Caco-2 cells in culture. The results demonstrate a reduction in the uptake of beta-carotene when Caco-2 cells were supplemented with 20 microM beta-sitosterol. beta-Sitosterol did not interfere with alpha-tocopherol uptake by the cells. In conclusion, Caco-2 cells are a useful model system to study potential interactive effects of phytosterols with fat-soluble dietary components.

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.