Integrated biological responses and tissue-specific expression of p53 and ras genes in marine mussels following exposure to benzo(α)pyrene and C60 fullerenes, either alone or in combination.

We used the marine bivalve (Mytilus galloprovincialis) to assess a range of biological or biomarker responses following exposure to a model-engineered nanoparticle, C60 fullerene, either alone or in combination with a model polycyclic aromatic hydrocarbon, benzo(α)pyrene [B(α)P]. An integrated biomarker approach was used that included: (i) determination of 'clearance rates' (a physiological indicator at individual level), (ii) histopathological alterations (at tissue level), (iii) DNA strand breaks using the comet assay (at cellular level) and (iv) transcriptional alterations of p53 (anti-oncogene) and ras (oncogene) determined by real-time quantitative polymerase chain reaction (at the molecular/genetic level). In addition, total glutathione in the digestive gland was measured as a proxy for oxidative stress. Here, we report that mussels showed no significant changes in 'clearance rates' after 1 day exposure, however significant increases in 'clearance rates' were found following exposure for 3 days. Histopathology on selected organs (i.e. gills, digestive glands, adductor muscles and mantles) showed increased occurrence of abnormalities in all tissues types, although not all the exposed organisms showed these abnormalities. Significantly, increased levels of DNA strand breaks were found after exposure for 3-days in most individuals tested. In addition, a significant induction for p53 and ras expression was observed in a tissue and chemical-specific pattern, although large amounts of inter-individual variability, compared with other biomarkers, were clearly apparent. Overall, biological responses at different levels showed variable sensitivity, with DNA strand breaks and gene expression alterations exhibiting higher sensitivities. Furthermore, the observed genotoxic responses were reversible after a recovery period, suggesting the ability of mussels to cope with the toxicants C60 and/or B(α)P under our experimental conditions. Overall, in this comprehensive study, we have demonstrated mussels as a suitable model marine invertebrate species to study the potential detrimental effects induced by possible genotoxicants and toxicants, either alone or in combinations at different levels of biological organisation (i.e. molecular to individual levels).

Investigations to extend viability of a rainbow trout primary gill cell culture.

The primary culture of fish gill cells can provide functional, cell diverse, model in vitro platforms able to tolerate an aqueous exposure analogous to in vivo tissues. The utility of such models could be extended to a variety of longer term exposure scenarios if a method could be established to extend culture viability when exposed to water for longer periods. Here we report findings of a series of experiments to establish increased longevity, as monitored by culture transepithelial electrical resistance (TEER) and concurrent histological developments. Experimental cultures improved TEER during apical freshwater exposure for a mean of twelve days, compared to previous viabilities of up to 3 days. Cultures with larger surface areas and the use of trout serum rather than foetal bovine serum (FBS) contributed to the improvement, while perfusion of the intact gill prior to cell harvest resulted in a significantly faster preparation. Detailed scanning electron microscopy analysis of cultures revealed diverse surface structures that changed with culture age. Cultures grown on membranes with an increased porosity, collagen coating or 3D structure were of no benefit compared to standard membranes. Increased culture longevity, achieved in this study and reported for the first time, is a significant breakthrough and opens up a variety of future experimentation that has previously not been possible. The extended viability facilitates exploration of in vitro chronic or pulse-exposure test paradigms, longer term physiological and environmental monitoring studies and the potential for interactive co-culture with other organoid micro-tissues.

Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner.

Evaluation of the Genotoxic and Physiological Effects of Decabromodiphenyl Ether (BDE-209) and Dechlorane Plus (DP) Flame Retardants in Marine Mussels (Mytilus galloprovincialis).

Dechlorane Plus (DP) is a proposed alternative to the legacy flame retardant decabromodiphenyl ether (BDE-209), a major component of Deca-BDE formulations. In contrast to BDE-209, toxicity data for DP are scarce and often focused on mice. Validated dietary in vivo exposure of the marine bivalve (Mytilus galloprovincialis) to both flame retardants did not induce effects at the physiological level (algal clearance rate), but induced DNA damage, as determined by the comet assay, at all concentrations tested. Micronuclei formation was induced by both DP and BDE-209 at the highest exposure concentrations (100 and 200 µg/L, respectively, at 18% above controls). DP caused effects similar to those by BDE-209 but at lower exposure concentrations (5.6, 56, and 100 µg/L for DP and 56, 100, and 200 µg/L for BDE-209). Moreover, bioaccumulation of DP was shown to be concentration dependent, in contrast to BDE-209. The results described suggest that DP poses a greater genotoxic potential than BDE-209.

Assessment of oxidative damage to DNA, transcriptional expression of key genes, lipid peroxidation and histopathological changes in carp Cyprinus carpio L. following exposure to chronic hypoxic and subsequent recovery in normoxic conditions.

In fish, a complex set of mechanisms deal with environmental stresses including hypoxia. In order to probe the hypothesis that hypoxia-induced stress could be manifested in varieties of pathways, a model species, mirror carp (Cyprinus carpio), were chronically exposed to hypoxic condition (dissolved oxygen level: 1.80 ± 0.6 mg/l) for 21 days and subsequently allowed to recover under normoxic condition (dissolved oxygen level: 8.2 ± 0.5 mg/l) for 7 days. At the end of these exposure periods, an integrated approach was applied to evaluate several endpoints at different levels of biological organisation. These included determination of (i) oxidative damage to DNA in erythrocytes (using modified comet assay), (ii) lipid peroxidation in liver samples by measuring the malondialdehyde production using the 2-thiobarbituric acid [i.e. thiobarbituric acid reactive substances (TBARS) assay] and (iii) histopathological changes in gills. In addition, transcriptional expression of hypoxia-inducible factor 1 α (HIF-1α) and genes involved in the repair of oxidative damage to DNA (i.e. ogg1) and base excision repair (i.e. xrcc1) using reverse transcription polymerase chain reaction in liver samples were also determined. The results suggested significantly enhanced expression of these genes in response to hypoxia compared to concurrent normoxic controls. While the expression of HIF-1α reverted to control values within 7 days exposure to normoxic condition (P < 0.05), the transcriptional expression of the two genes involved in DNA repair process remained significantly high under the recovery period, which complemented the induction of oxidative damage to DNA. Hypoxic groups showed significantly increased values for TBARS level (~2-fold) and histopathological changes in gill tissues compared to both normoxic and recovery groups. Overall, oxidative damage to DNA determined by modified comet assay reflected the observed biological responses in other tissues of the fish. Along with other parameters, this integrated experimental design further strengthens the applications of the comet assay as an important technique to assess stress-induced DNA damage in ecotoxicological studies.

Ecotoxicological impacts of landfill sites: Towards risk assessment, mitigation policies and the role of artificial intelligence.

Waste disposal in landfills remains a global concern. Despite technological developments, landfill leachate poses a hazard to ecosystems and human health since it acts as a secondary reservoir for legacy and emerging pollutants. This study provides a systematic and scientometric review of the nature and toxicity of pollutants generated by landfills and means of assessing their potential risks. Regarding human health, unregulated waste disposal and pathogens in leachate are the leading causes of diseases reported in local populations. Both in vitro and in vivo approaches have been employed in the ecotoxicological risk assessment of landfill leachate, with model organisms ranging from bacteria to birds. These studies demonstrate a wide range of toxic effects that reflect the complex composition of leachate and geographical variations in climate, resource availability and management practices. Based on bioassay (and other) evidence, categories of persistent chemicals of most concern include brominated flame retardants, per- and polyfluorinated chemicals, pharmaceuticals and alkyl phenol ethoxylates. However, the emerging and more general literature on microplastic toxicity suggests that these particles might also be problematic in leachate. Various mitigation strategies have been identified, with most focussing on improving landfill design or leachate treatment, developing alternative disposal methods and reducing waste volume through recycling or using more sustainable materials. The success of these efforts will rely on policies and practices and their enforcement, which is seen as a particular challenge in developing nations and at the international (and transboundary) level. Artificial intelligence and machine learning afford a wide range of options for evaluating and reducing the risks associated with leachates and gaseous emissions from landfills, and various approaches tested or having potential are discussed. However, addressing the limitations in data collection, model accuracy, real-time monitoring and our understanding of environmental impacts will be critical for realising this potential.

Bioaccumulation and biological effects of hydrogenated cement particles in the marine bivalve, Mytilusgalloprovincialis.

The decommissioning and normal functioning of nuclear facilities can result in the production and release of airborne particles in the environment. Aquatic biota are expected to be exposed to these particles considering that nuclear facilities are often located near water bodies. Aerosols, such as cement dust, can interact with radionuclides as well as with heavy metals, and therefore elicit not only radiological impacts but also chemical toxicity. In the present study, we aimed to determine the effects of hydrogenated cement particles (HCPs) as a first step before evaluating any radiotoxicity of tritiated cement particles in the marine mussels, Mytilus galloprovincialis. Responses at different levels of biological organisation were assessed, including clearance rate (CR), tissue specific accumulation, DNA damage and transcriptional expression of key stress related genes. Acute (5 h) and medium-term, chronic (11 d) exposures to 1000 µg L-1 HCPs showed that bioaccumulation, assessed using Cu as a proxy and determined by inductively coupled plasma mass spectrometry, was time and tissue dependent. The highest levels of Cu were found in the digestive gland (DG) after 11 d. HCP exposure caused changes in the expression of oxidative and other stress-related genes, including mt20 in DG and gst and sod in the gill after 5 h exposure, while an overexpression of hsp70 in the gill was observed after 11 d. Genotoxic effects in haemocytes were observed after 11 d of HCP exposure. Multivariate analysis indicated that oxidative stress is the most probable factor contributing to overall physiological dysfunction. Our results provide a baseline to perform further studies employing tritiated cement particles. Specifically, future work should focus on the DG since only this tissue showed significant bioaccumulation when compared to the negative control.

Changes in expression profiles of genes associated with DNA repair following induction of DNA damage in larval zebrafish Danio rerio.

DNA repair is initiated by transcription of genes in response to specific types of damage. Breaks in DNA strands (single and double) are repaired predominantly through non-homologous end-joining (NHEJ) or homologous recombination (HR), but progression of repair and changes in expression profiles of genes involved are unknown. DNA damage was induced in zebrafish larvae by brief exposure (10min) to hydrogen peroxide (H2O2; 100mM), and induction of DNA strand breaks was assessed by single-cell gel electrophoresis (comet) assay over 24h. H2O2 was selected because it is eliminated rapidly after induction of DNA damage. DNA damage [mean ± standard error of the mean (SEM), % tail DNA] increased significantly immediately after 10-min H2O2 exposure (35.4±3.8; control 17.2±2.0), but damage did not differ from control levels 24h after exposure (9.2±0.4; control 9.9±0.9). At 0-, 1-, 3-, 6-, 12- and 24-h post-exposure, quantitative reverse transcriptase-PCR was conducted to assess expression of selected genes involved in DNA repair including xrcc5, xrcc6 (NHEJ), rad51 (HR) and gadd45a (DNA damage detection). Expression (maximum fold-change ± SEM, triplicate samples of 40 larvae) of each gene increased rapidly (within 6h) after exposure to 100mM of H2O2: 1.8±0.2, rad51; 1.7±0.2, xrcc5 and 1.5±0.1, xrcc6. Acute exposure (200mM of H2O2) caused 10% larval mortality within 2h, upregulated gadd45a (5.0±0.8), but did not change expression of rad51, xrcc5 or xrcc6. Expression profiles (critical exponential model) were similar among genes but differed relative to time and among independent experiments. Results indicate that repair mechanisms are initiated rapidly after DNA damage, that gene expression profiles vary according to potency of H2O2 exposure and that examination of the time course of gene expression changes is necessary to understand the complete gene response over time.

Oxidative DNA damage may not mediate Ni-induced genotoxicity in marine mussels: assessment of genotoxic biomarkers and transcriptional responses of key stress genes.

Nickel (Ni) is a known carcinogenic and mutagenic compound and an important contaminant of aquatic environments. Ni toxicity and its potential impact on aquatic organisms are, however, not well understood. This study used an integrated approach to evaluate genotoxic effects, tissue-specific accumulation and transcriptional profiles of key genes in mussels, Mytilus galloprovincialis, exposed to a range of concentrations of Ni. The genotoxic effects assessed were total and oxidative DNA damage (DNA strand breaks measured using the enzyme modified comet assay), and induction of micronuclei (MN; clastogenic and/or aneugenic effects) using haemocytes as the target cells. Six genes (pgp, mt10, mt20, sod, hsp70 and gst) were selected for transcriptional analysis in the gills based on their key role in the stress response. Following exposure to sublethal concentrations of Ni (0-3600µgL(-1)) for 5 days, mussel haemocytes showed significant genotoxicity at >1800µgL(-1) (4-fold increase for DNA strand breaks and 3-fold increase for MN induction). There was no significant difference between buffer (control) and enzyme treatments which target oxidised DNA bases (formamidopyrimidine glycosylase or endonuclease IIII). This suggested that, in haemocytes, oxidative DNA damage is not a major mechanism for Ni-induced genotoxicity. The expression of mt20 and gst genes in gill was up-regulated at genotoxic concentrations, whilst pgp expression was markedly up-regulated, particularly at 18µgL(-1) Ni (19-fold increase). Pearson's correlation analysis revealed significant associations between % tail DNA and MN induction in haemocytes (r=0.88, p<0.05), and between Ni accumulation in foot (r=0.47, p<0.05) and digestive gland (r=0.41, p<0.05) and induction of MN in the haemocytes. Our results are the first to suggest that Ni-induced genotoxicity in mussel haemocytes may not be a result of oxidative DNA damage, and that multixenobiotic resistance (MXR) may play an important role in Ni detoxification in this species.

Relative sensitivity of two marine bivalves for detection of genotoxic and cytotoxic effects: a field assessment in the Tamar Estuary, South West England.

The input of anthropogenic contaminants to the aquatic environment is a major concern for scientists, regulators and the public. This is especially relevant in areas such as the Tamar valley in SW England, which has a legacy of contamination from industrial activity in the nineteenth and twentieth centuries. Following on from previous laboratory validation studies, this study aimed to assess the relationship between genotoxic and cytotoxic responses and heavy metal concentrations in two bivalve species sampled from locations along the Tamar estuary. Adult cockles, Cerastoderma edule, and blue mussels, Mytilus edulis, were sampled from five locations in the Tamar and one reference location on the south Devon coast. Bivalve haemocytes were processed for comet and neutral red retention (NRR) assays to determine potential genotoxic and cytotoxic effects, respectively. Sediment and soft tissue samples were analysed for metal content by inductively coupled plasma mass spectrometry. Sediment concentrations were consistent with the physico-chemical nature of the Tamar estuary. A significant correlation (P = 0.05) was found between total metal concentration in sediment and C. edule soft tissues, but no such correlation was found for M. edulis samples. DNA damage was elevated at the site with highest Cr concentrations for M. edulis and at the site with highest Ni and Pb concentrations for C. edule. Analysis of NRR revealed a slight increase in retention time at one site, in contrast to comet data. We conclude that the comet assay is a reliable indicator of genotoxic damage in the field for both M. edulis and C. edule and discuss reasons for the apparent discrepancy with NRR.

In silico prediction of acute chemical toxicity of biocides in marine crustaceans using machine learning.

Biocides are a heterogeneous group of chemical substances intended to control the growth or kill undesired organisms. Due to their extensive use, they enter marine ecosystems via non-point sources and may pose a threat to ecologically important non-target organisms. Consequently, industries and regulatory agencies have recognized the ecotoxicological hazard potential of biocides. However, the prediction of biocide chemical toxicity on marine crustaceans has not been previously evaluated. This study aims to provide in silico models capable of classifying structurally diverse biocidal chemicals into different toxicity categories and predict acute chemical toxicity (LC50) in marine crustaceans using a set of calculated 2D molecular descriptors. The models were built following the guidelines recommended by the OECD (Organization for Economic Cooperation and Development) and validated through stringent processes (internal and external validation). Six machine learning (ML) models were built and compared (linear regression: LR; support vector machine: SVM; random forest: RF; feed-forward backpropagation-based artificial neural network: ANN; decision trees: DT and naïve Bayes: NB) for regression and classification analysis to predict toxicities. All the models displayed encouraging results with high generalisability: the feed-forward-based backpropagation method showed the best results with determination coefficient R2 values of 0.82 and 0.94, respectively, for training set (TS) and validation set (VS). For classification-based modelling, the DT model performed the best with an accuracy (ACC) of 100 % and an area under curve (AUC) value of 1 for both TS and VS. These models showed the potential to replace animal testing for the chemical hazard assessment of untested biocides if they fall within the applicability domain of the proposed models. In general, the models are highly interpretable and robust, with good predictive performance. The models also displayed a trend indicating that toxicity is largely influenced by factors such as lipophilicity, branching, non-polar bonding and saturation of molecules.

Assessing the effects of single and binary exposures of copper and lead on Mytilus galloprovincialis: Physiological and genotoxic approaches.

It is becoming increasingly recognised that contaminants are not isolated in their threats to the aquatic environment, with recent shifts towards studying the effects of chemical mixtures. In this study, adult marine mussels (Mytilus galloprovincialis) were exposed to two aqueous concentrations of the essential trace metal, Cu (5 and 32 µg L-1), and the non-essential metal, Pb (5 and 25 µg L-1), both individually and in binary mixtures. After a 14-day exposure, metal accumulation was determined in the digestive gland, gill and mantle tissues by inductively coupled plasma-mass spectrometry following acid digestion, and a number of biochemical, neurotoxic and physiological markers were assessed. These included measurements of DNA damage using comet assay, total glutathione concentration, acetylcholinesterase (AChE) activity and clearance rate. Metal accumulation was greater in the digestive gland and gill than in the mantle, and based on computed free ion concentrations, was greater for Pb than for Cu. Copper exhibited an inhibitory effect on Pb accumulation but Pb did not appear to affect Cu accumulation. Comet assay results revealed DNA damage (i.e., genotoxic effects) in all treatments but differences between the exposures were not significant (p > 0.05), and there were no significant differences in AChE activities between treatments. The most distinctive impacts were a reduction in clearance rate resulting from the higher concentration of Cu, with and without Pb, and an increase in glutathione in the gill resulting from the higher concentration of Cu without Pb. Multivariate analysis facilitated the development of a conceptual model based on the current findings and previously published data on the toxicity and intracellular behaviour of Cu and Pb that will assist in the advancement of regulations and guidelines regarding multiple metal contaminants in the environment.

Antibiotic Resistance Mediated by Escherichia coli in Kuwait Marine Environment as Revealed through Genomic Analysis.

Antibiotic-resistance gene elements (ARGEs) such as antibiotic-resistance genes (ARGs), integrons, and plasmids are key to the spread of antimicrobial resistance (AMR) in marine environments. Kuwait's marine area is vulnerable to sewage contaminants introduced by numerous storm outlets and indiscriminate waste disposal near recreational beaches. Therefore, it has become a significant public health issue and warrants immediate investigation. Coliforms, especially Gram-negative Escherichia coli, have been regarded as significant indicators of recent fecal pollution and carriers of ARGEs. In this study, we applied a genome-based approach to identify ARGs' prevalence in E. coli isolated from mollusks and coastal water samples collected in a previous study. In addition, we investigated the plasmids and intl1 (class 1 integron) genes coupled with the ARGs, mediating their spread within the Kuwait marine area. Whole-genome sequencing (WGS) identified genes resistant to the drug classes of beta-lactams (blaCMY-150, blaCMY-42, blaCTX-M-15, blaDHA-1, blaMIR-1, blaOKP-B-15, blaOXA-1, blaOXA-48, blaTEM-1B, blaTEM-35), trimethoprim (dfrA14, dfrA15, dfrA16, dfrA1, dfrA5, dfrA7), fluroquinolone (oqxA, oqxB, qnrB38, qnrB4, qnrS1), aminoglycoside (aadA2, ant(3'')-Ia, aph(3'')-Ib, aph(3')-Ia, aph(6)-Id), fosfomycin (fosA7, fosA_6, fosA, fosB1), sulfonamide (sul1, sul2, sul3), tetracycline (tet-A, tet-B), and macrolide (mph-A). The MFS-type drug efflux gene mdf-A is also quite common in E. coli isolates (80%). The plasmid ColRNAI was also found to be prevalent in E. coli. The integron gene intI1 and gene cassettes (GC) were reported to be in 36% and 33%, respectively, of total E. coli isolates. A positive and significant (p < 0.001) correlation was observed between phenotypic AMR-intl1 (r = 0.311) and phenotypic AMR-GC (r = 0.188). These findings are useful for the surveillance of horizontal gene transfer of AMR in the marine environments of Kuwait.

Tritium: Its relevance, sources and impacts on non-human biota.

Tritium (3H) is a radioactive isotope of hydrogen that is abundantly released from nuclear industries. It is extremely mobile in the environment and in all biological systems, representing an increasing concern for the health of both humans and non-human biota (NHB). The present review examines the sources and characteristics of tritium in the environment, and evaluates available information pertaining to its biological effects at different levels of biological organisation in NHB. Despite an increasing number of publications in the tritium radiobiology field, there exists a significant disparity between data available for the different taxonomic groups and species, and observations are heavily biased towards marine bivalves, fish and mammals (rodents). Further limitations relate to the scarcity of information in the field relative to the laboratory, and lack of studies that employ forms of tritium other than tritiated water (HTO). Within these constraints, different responses to HTO exposure, from molecular to behavioural, have been reported during early life stages, but the potential transgenerational effects are unclear. The application of rapidly developing "omics" techniques could help to fill these knowledge gaps and further elucidate the relationships between molecular and organismal level responses through the development of radiation specific adverse outcome pathways (AOPs). The use of a greater diversity of keystone species and exposures to multiple stressors, elucidating other novel effects (e.g., by-stander, germ-line, transgenerational and epigenetic effects) offers opportunities to improve environmental risk assessments for the radionuclide. These could be combined with artificial intelligence (AI) including machine learning (ML) and ecosystem-based approaches.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem.

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of "comets" positive for 5-bromo-2'-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of "comets" positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site "Saline di Tarquinia". The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising "sentinel organism" to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.

Merging nano-genotoxicology with eco-genotoxicology: an integrated approach to determine interactive genotoxic and sub-lethal toxic effects of C(60) fullerenes and fluoranthene in marine mussels, Mytilus sp.

Whilst there is growing concern over the potential detrimental impact of engineered nanoparticles (ENPs) on the natural environment, little is known about their interactions with other contaminants. In the present study, marine mussels (Mytilus sp.) were exposed for 3 days to C(60) fullerenes (C(60); 0.10-1 mg l(-1)) and a model polycyclic aromatic hydrocarbon (PAH), fluoranthene (32-100 µg l(-1)), either alone or in combination. The first two experiments were conducted by exposing the organisms to different concentrations of C(60) and fluoranthene alone, in order to determine the effects on total glutathione levels (as a measure of generic oxidative stress), genotoxicity (DNA strand breaks using Comet assay in haemocytes), DNA adduct analyses (using (32)P-postlabelling method) in different organs, histopathological changes in different tissues (i.e. adductor muscle, digestive gland and gills) and physiological effects (feeding or clearance rate). Subsequently, in the third experiment, a combined exposure of C(60) plus fluoranthene (0.10 mg l(-1) and 32 µg l(-1), respectively) was carried out to evaluate all endpoints mentioned above. Both fluoranthene and C(60) on their own caused concentration-dependent increases in DNA strand breaks as determined by the Comet assay. Formation of DNA adducts however could not be detected for any exposure conditions. Combined exposure to C(60) and fluoranthene additively enhanced the levels of DNA strand breaks along with a 2-fold increase in the total glutathione content. In addition, significant accumulation of C(60) was observed in all organs, with highest levels in digestive gland (24.90 ± 4.91µg C(60) g(-1) ww). Interestingly, clear signs of abnormalities in adductor muscle, digestive gland and gills were observed by histopathology. Clearance rates indicated significant differences compared to the control with exposure to C(60), and C(60)/fluoranthene combined treatments, but not after fluoranthene exposure alone. This study demonstrated that at the selected concentrations, both C(60) and fluoranthene evoke toxic responses and genetic damage. The combined exposure produced enhanced damage with additive rather than synergistic effects.

Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes.

The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1-5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell-cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6-8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.