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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 comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

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 tritiumradiobiology 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 tritiumother than tritiated water (HTO). Within these constraints, different responses toHTOexposure, frommolecular 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.

International audience; During the decommissioning and removal of radioactive material in nuclear facilities, fine, tritiated dusts of stainless steel, cement or tungsten are generated that could be accidently released to the environment. However, the potential radio- and ecotoxicological effects these tritiated particles may have are unknown. In this study, stainless steel particles (SSPs) representative of those likely to be tritiated are manufactured by hydrogenation and their tissue-specific bioaccumulation, release (depuration) and subsequent genotoxic response have been studied in the marine mussel, Mytilus galloprovincialis, as a baseline for future assessments of the potential effects of tritiated SSPs. Exposure to 1000 μg L−1 of SSPs and adopting Cr as a proxy for stainless steel revealed relatively rapid accumulation (∼5 h) in the various mussel tissues but mostly in the digestive gland. Over longer periods up to 18 days, SSPs were readily rejected and egested as faecal material. DNA strand breaks, as a measure of genotoxicity, were determined at each time point in mussel haemocytes using single cell gel electrophoresis, or the comet assay. Lack of chemical genotoxicity was attributed to the rapid processing of SSP particles and limited dissolution of elemental components of steel. Further work employing tritiated SSPs will enable radio-toxicology to be studied without the confounding effects of chemical toxicity

International audience; The interactions between carbon-based engineered nanoparticles (ENPs) and organic pollutants might enhance the uptake of contaminants into biota. The present integrated study aimed to assess this potential ‘Trojan Horse’, probing the interactive effects of purpose-made multi-walled carbon nanotubes (MWCNTs), a representative ENP, and benzo[a]pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon (PAH) pollutant, on the marine mussel Mytilus galloprovincialis. Mussels were exposed to MWCNTs and BaP either alone or in various combinations. The co-exposure of BaP with MWCNTs revealed that the presence of MWCNTs enhanced the aqueous concentrations of BaP, thereby reducing the uptake of this pollutant by mussels as evidenced by lowering BaP concentrations in the tissues. Determination of DNA damage (comet assay) showed a concentration-dependent response for BaP alone which was absent when MWCNTs were present. Global gene expression using microarray analyses indicated that BaP and MWCNTs, in combination, differentially activated those genes which are involved in DNA metabolism compared to the exposures of BaP or MWCNTs alone, and the gene expression response was tissue-specific. Mechanisms to explain these results are discussed and relate primarily to the adsorption of BaP on MWCNTs, mediated potentially by van der Waals interactions. The use of a novel approach based on gold-labeled MWCNTs to track their uptake in tissues improved the traceability of nanotubes in biological samples. Overall, our results did not indicate the ‘Trojan Horse’ effects following co-exposure to the contaminants and clearly showed that the adsorption of BaP to MWCNTs modified the uptake of the pollutant in marine mussels.

This review critically evaluates our current regulatory understanding of genotoxicity testing and risk assessment of medical devices. Genotoxicity risk assessment of these devices begins with the evaluation of materials of construction, manufacturing additives and all residual materials for potential to induce DNA damage. This is followed by extractable and/or leachable (EL) studies to understand the worst case and/or clinical exposures, coupled with risk assessment of extractables or leachables. The TTC (Threshold of Toxicological Concern) approach is used to define acceptable levels of genotoxic chemicals, when identified. Where appropriate, in silico predictions may be used to evaluate the genotoxic potentials of identifiable chemicals with limited toxicological data and above the levels defined by TTC. Devices that could not be supported by EL studies are evaluated by in vitro genotoxicity studies conducted in accordance with ISO10993-3 and 33. Certain endpoints such as 'site of contact genotoxicity' that are specific for certain classes of medical devices are currently not addressed in the current standards. The review also illustrates the potential uses of recent advances to achieve the goal of robust genotoxicity assessment of medical devices which are being increasingly used for health benefits. The review also highlights the gaps for genotoxicity risk assessment of medical devices and suggests possible approaches to address them taking into consideration the recent advances in genotoxicity testing including their potential uses in biocompatibility assessment.

International audience; The hypothesis that C fullerene nanoparticles (C) exert an antagonistic interactive effect on the toxicity of benzo[a]pyrene (BaP) has been supported by this investigation. Mussels were exposed to BaP (5, 50 & 100μg/L) and C (C-1mg/L) separately and in combination. Both BaP and C were shown to co-localize in the secondary lysosomes of the hepatopancreatic digestive cells in the digestive gland where they reduced lysosomal membrane stability (LMS) or increased membrane permeability, while BaP also induced increased lysosomal lipid and lipofuscin, indicative of oxidative cell injury and autophagic dysfunction. Combinations of BaP and C showed antagonistic effects for lysosomal stability, mTORC1 (mechanistic target of rapamycin complex 1) inhibition and intralysosomal lipid (5 & 50μg/L BaP). The biomarker data (i.e., LMS, lysosomal lipidosis and lipofuscin accumulation; lysosomal/cell volume and dephosphorylation of mTORC1) were further analysed using multivariate statistics. Principal component and cluster analysis clearly indicated that BaP on its own was more injurious than in combination with C. Use of a network model that integrated the biomarker data for the cell pathophysiological processes, indicated that there were significant antagonistic interactions in network complexity (% connectance) at all BaP concentrations for the combined treatments. Loss of lysosomal membrane stability probably causes the release of intralysosomal iron and hydrolases into the cytosol, where iron can generate harmful reactive oxygen species (ROS). It was inferred that this adverse oxidative reaction induced by BaP was ameliorated in the combination treatments by the ROS scavenging property of intralysosomal C, thus limiting the injury to the lysosomal membrane; and reducing the oxidative damage in the cytosol and to the nuclear DNA. The ROS scavenging by C, in combination with enhanced autophagic turnover of damaged cell constituents, appeared to have a cytoprotective effect against the toxic reaction to BaP in the combined treatments.

International audience; Tritium has been receiving worldwide attention, particularly because of its production and use in existing fission reactors and future nuclear fusion technologies, leading to an increased risk of release in the environment. Linking human health effects to low-dose tritium exposures presents a challenge for many reasons. Among these: biological effects strongly depend on the speciation of tritiated products and exposure pathway; large dosimetric uncertainties may exist; measurements using in vitro cell cultures generally lack a description of effects at the tissue level, while large-scale animal studies might be ethically questionable and too highly demanding in terms of resources. In this context, threedimensional models of the human airway epithelium are a powerful tool to investigate potential toxicity induced upon inhalation of radioactive products in controlled physiological conditions. In this study we exposed such a model to tritiated water (HTO) for 24 h, with a range of activity levels (up to ;33 kBq ll–1 cm–2). After the exposures, we measured cell viability, integrity of epithelial layer and pro-inflammatoryresponse at different post-exposure time-points. We also quantified tritium absorption and performed dosimetric estimates considering HTO passage through the epithelial layer, leading to reconstructed upper limits for the dose to the tissue of less than 50 cGy cumulative dose for the highest activity. Upon exposure to the highest activity, cell viability was not decreased; however, we observed a small effect on epithelial integrity and an inflammatory response persisting after seven days. These results represent a reference condition and will guide future experiments using human airway epithelium to investigate the effects of other peculiar tritiated products.

Contaminants seldom occur in isolation in the aquatic environment. While pollution of coastal and inland water bodies has received considerable attention to date, there is limited information on potential interactive effects between radionuclides and metals. Whether by accidental or controlled release, such contaminants co-exist in aquatic ecosystems and can pose an enhanced threat to biota. Using a range of biological responses, the study aimed to evaluate relative interactive effects on representative freshwater and marine bivalve species.An integrated, multi-biomarker approach was adopted to investigate response to copper (Cu, 18 Overall, using a tissue-specific (i.e. gill and digestive gland) approach, genotoxic response was reflective of exposures where Cu had a slight additive effect onOur study is the first to explore the interactive effects of ionizing radiation (IR) and Cu on two bivalve species representing two ecological habitats. The complexity of IR-metal interactions demonstrate that extrapolation of findings obtained from single stressor studies into field conditions could be misrepresentative of real-world environments. In turn, environmental protective strategies deemed suitable in protecting biota from a single, isolated stressor may not be wholly adequate.

The hypothesis that C

The objective of this paper is to present the results of discussions at a workshop held as part of the International Congress of Radiation Research (Environmental Health stream) in Manchester UK, 2019. The main objective of the workshop was to provide a platform for radioecologists to engage with radiobiologists to address major questions around developing an Ecosystem approach in radioecology and radiation protection of the environment. The aim was to establish a critical framework to guide research that would permit integration of a pan-ecosystem approach into radiation protection guidelines and regulation for the environment. The conclusions were that the interaction between radioecologists and radiobiologists is useful in particular in addressing field versus laboratory issues where there are issues and challenges in designing good field experiments and a need to cross validate field data against laboratory data and vice versa. Other main conclusions were that there is a need to appreciate wider issues in ecology to design good approaches for an ecosystems approach in radioecology and that with the capture of 'Big Data', novel tools such as machine learning can now be applied to help with the complex issues involved in developing an ecosystem approach.

Anthropogenic radionuclides can enter water bodies through accidental or controlled discharges. In order to assess their potential impact, understanding the link between exposure, tissue specific bioaccumulation and radiation dose rate, to biological or biomarker responses in aquatic biota is required. Adopting an integrated, multi-biomarker, multi-species approach, we have investigated potential biological responses induced by short-lived radionuclide, phosphorus-32 (32P, radiophosphorus) in two ecologically important mussel species, the freshwater Dreissena polymorpha (DP) and marine Mytilus galloprovincialis (MG). Adult individuals were exposed to 32P for 10 days, to acquire nominal whole-body average dose rates of 0.10, 1 and 10 mGy d−1, which encompass a screening value of 10 μGy h−1 (0.24 mGy d−1), in accordance with the ERICA tool. Following exposure, a suite of genotoxic biomarkers (DNA damage, γ-H2AX induction and micronucleus [MN] formation) were measured in gill and digestive gland tissues, along with transcriptional expression of selected stress-related genes in both the species (i.e. hsp70/90, sod, cat and gst). Our results demonstrate the relationship between tissue specific dosimetry, where 32P induced a dose-dependent increase, and biological responses independent of species. Gene expression analysis revealed little significant variation across species or tissues. Overall, MG appeared to be more sensitive to short-term damage (i.e. high DNA damage and γ-H2AX induction), particularly in digestive gland. This study contributes to limited knowledge on the transfer and biological impact of radionuclides within differing aquatic systems on a tissue specific level, aiding the development of adequate management and protective strategies.

Nanotechnology has become part of our daily life, and it is a priority field for today's innovation leaders. Nanotechnology is a critical element of advanced manufacturing, which is being pursued by a widening circle of industrial countries. Therefore, their residues are expected to reach natural systems, and their potential impact in the environment and on human health through the trophic chain should be assessed. The recent trends for toxicological and ecotoxicological evaluations of nanomaterials (NMs) are aiming at elucidating the mechanisms of action at sublethal doses, in particular in aquatic organisms, including bioaccumulation, alteration of their growth and reproductive cycles, as well as behavioral changes, oxidative stress, and fatty acid metabolism, among others. In these regard, metabolomics has the potential to enhance the knowledge on ecotoxicology and to assess long-term effects at sublethal concentrations of exposure. This chapter presents and discusses recent metabolomics studies to assess the potential risks of NMs residues in the environment.

Statement paper following discussions in March 2019 between the TREE (Transfer-Exposure-Effects) project participants and members of the European Radioecology ALLIANCE working group on ‘transgenerational effects and species radiosensitivity'.

In the present study, we investigated the health status of marine mussels (Mytilus galloprovincialis) caged and deployed at three different sites on the Sardinian coastline characterized by different levels of contamination: Fornelli (F, the reference site), Cala Real (CR), and Porto Torres (PT). A new low density oligonucleotide microarray was used to investigate global gene expression in the digestive gland of mussels. Target genes were selected to cover most of the biological processes involved in the stress response in bivalve mollusks (e.g. DNA metabolism, translation, immune response, cytoskeleton organization). A battery of classical biomarkers was also employed to complement the gene expression analyses. Chemical analysis revealed higher loads of heavy metals (Pb and Cu) and total polycyclic aromatic hydrocarbons (PAHs) at PT compared to the other sites. In mussels deployed at CR, functional genomics analysis of the microarray data rendered 78 differentially expressed genes (DEGs) involved in 11 biological processes. Animals exposed at PT had 105 DEGs that were characterized by the regulation of 14 biological processes, including mitochondrial activity, adhesion to substrate, DNA metabolism, translation, metal resistance, and cytoskeleton organization. Biomarker data (lysosomal membrane stability, lysosomal/cytoplasm volume ratio, lipofuscin accumulation, metallothionein content, micronucleus frequency, and cytoskeleton alteration) were in trend with transcriptomic output. Biomarker data were integrated using the Mussel Expert System (MES), allowing defining the area in which the presence of chemicals is toxic for mussels. Our study provides the opportunity to adopt a new approach of integrating transcriptomic (microarray) results with classical biomarkers to assess the impact of pollutants on marine mussels in biomonitoring programs.

Lysosomal membrane stability (LMS) has been used in various organisms as a very sensitive biomarker of stress. However, despite the abundance of data about regulation of the autophagic process in mammals, in the invertebrates there is only limited mechanistic understanding. Marine mussels (Mytilus galloprovincialis Lam.) are bivalve molluscs, widely used as models in ecotoxicology and as environmental bioindicators of sea water quality. In order to elucidate this fundamental process, in the present study, mussels were exposed for 3 days to a “priority”, ubiquitous environmental contaminant, benzo[a]pyrene (B[a]P) at different concentrations (i.e. 5, 50, 100 μg/L seawater). B[a]P accumulated in lysosomes of digestive tubule epithelial cells (digestive cells) and in enlarged lipid-rich lysosomes (autolysosomes) as detected by immunofluorescence and UV-fluorescence. B[a]P also activated the autophagic process with a marked decrease of LMS and concurrent increase in lysosomal/cytoplasmic volume ratio. Dephosphorylation of mTOR contributes to increased lysosomal membrane permeability and induced autophagy. B[a]P induced a decrease in phosphorylated (active form) mTOR. The probable role of mTOR in cell signalling and the regulation of the cellular responses to the contaminants has been also confirmed in a field study, where there was significant inactivation of mTOR in stressed animals. Statistical and network modelling supported the empirical investigations of autophagy and mTOR; and was used to integrate the mechanistic biomarker data with chemical analysis and DNA damage.

The effects of C

Anthropogenic radionuclides can enter water bodies through accidental or controlled discharges. In order to assess their potential impact, understanding the link between exposure, tissue specific bioaccumulation and radiation dose rate, to biological or biomarker responses in aquatic biota is required. Adopting an integrated, multi-biomarker, multi-species approach, we have investigated potential biological responses induced by short-lived radionuclide, phosphorus-32 (

This study aimed to assess the ecotoxicological effects of the interaction of fullerene (C60) and benzo[a]pyrene (B[a]P) on the marine mussel, Mytilus galloprovincialis. The uptake of nC60, B[a]P and mixtures of nC60 and B[a]P into tissues was confirmed by GC-MS, LC-HRMS and ICP-MS. Biomarkers of DNA damage as well as proteomics analysis were applied to unravel the toxic effect of B[a]P and C60. Antagonistic responses were observed at the genotoxic and proteomic level. Differentially expressed proteins (DEPs) were only identified in the B[a]P single exposure and the B[a]P mixture exposure groups containing 1 mg/L of C60, the majority of which were down-regulated (~52%). No DEPs were identified at any of the concentrations of nC60 (p < 0.05, 1% FDR). Using DEPs identified at a threshold of (p < 0.05; B[a]P and B[a]P mixture with nC60), gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that these proteins were enriched with a broad spectrum of biological processes and pathways, including those broadly associated with protein processing, cellular processes and environmental information processing. Among those significantly enriched pathways, the ribosome was consistently the top enriched term irrespective of treatment or concentration and plays an important role as the site of biological protein synthesis and translation. Our results demonstrate the complex multi-modal response to environmental stressors in M. galloprovincialis.

Low level laser therapy (LLLT) in the visible to near infrared spectral band (390-1100 nm) is absorption of laser light at the electronic level, without generation of heat. It may be applied in a wide range of treatments including wound healing, inflammation and pain reduction. Despite its potential beneficial impacts, the use of lasers for therapeutic purposes still remains controversial in mainstream medicine. Whilst taking into account the physical characteristics of different qualities of lasers, this review aims to provide a comprehensive account of the current literature available in the field pertaining to their potential impact at cellular and molecular levels elucidating mechanistic interactions in different mammalian models. The review also aims to focus on the integral approach of the optimal characteristics of LLLT that suit a biological system target to produce the beneficial effect at the cellular and molecular levels.Recent research articles were reviewed that explored the interaction of lasers (coherent sources) and LEDs (incoherent sources) at the molecular and cellular levels.It is envisaged that underlying mechanisms of beneficial impact of lasers to patients involves biological processes at the cellular and molecular levels. The biological impact or effects of LLLT at the cellular and molecular level could include cellular viability, proliferation rate, as well as DNA integrity and the repair of damaged DNA. This review summarizes the available information in the literature pertaining to cellular and molecular effects of lasers.It is suggested that a change in approach is required to understand how to exploit the potential therapeutic modality of lasers whilst minimizing its possible detrimental effects.

Antarctic seabirds well adapted to extreme environments often deal during their life cycle with sub-optimal conditions and occasionally with severe environmental stress. Climate changes, pollution, habitat loss, increasing human presence can all significantly affect organism’s health status from molecular to individual up to population level. In the present study, erythrocyte nuclear abnormalities (ENAs) and white blood cells (WBCs) differential were investigated in 19 adults of Adelie penguin (Pygoscelisadeliae) breeding at Edmonson Point, Antarctic Specially Protected Area (ASPA n. 165) in the Ross Sea. Micronuclei (MN) accounted for 10.50% of observed abnormalities in penguin erythrocytes while kidney-shaped nucleus (KSN) was the most abundant (20.88%). Heterophils (HE) were the most common WBC (36.93%) in agreement with the generic avian leukocytes profile while eosinophils (EO) were the lowest (7.45%). A low number of lymphocytes were detected resulting in a higher heterophils to lymphocytes ratio. ENAs and H:L ratio are confirmed as reliable indexes of penguin’s health status since they reflect their individual adaptation during breeding season. These baseline data will be useful for future studies as indicators of penguin’s health status mainly as response to environmental changes.

With respect to environmental protection, understanding radionuclide bioconcentration is necessary to relate exposure to radiation dose and hence to biological responses. Few studies are available on tissue specific accumulation of short-lived radionuclides in aquatic invertebrates. Short-lived radionuclides such as 32Phosphorus (32P), although occurring in small quantities in the environment, are capable of concentrating in the biota, especially if they are chronically exposed. In this study, we firstly compared tissue specific bioaccumulation and release (depuration) of 32P in adult marine (Mytilus galloprovincialis, MG) and freshwater bivalve molluscs (Dreissena polymorpha, DP). Secondly, using the Environmental Risk from Ionising Contaminants Assessment and Management (ERICA) tool, we calculated tissue specific doses following determination of radionuclide concentration. Marine and freshwater bivalves were exposed for 10 days to varying 32P concentrations to acquire desired whole body average dose rates of 0.10, 1.0 and 10 mGy d−1. Dose rates encompass a screening dose rate value of 10 μGy h−1 (0.24 mGy d−1), in accordance with the ERICA tool. This study is the first to relate tissue specific uptake and release (via excretion) of 32P from two anatomically similar bivalve species. Results showed highly tissue specific accumulation of this radionuclide and similarity of accumulation pattern between the two species. Our data, which highlights preferential 32P accumulation in specific tissues such as digestive gland, demonstrates that in some cases, tissue-specific dose rates may be required to fully evaluate the potential effects of radiation exposure on non-human biota. Differential sensitivity between biological tissues could result in detrimental biological responses at levels presumed to be acceptable when adopting a ‘whole-body’ approach.

Tumble dryer lint has been employed as a surrogate for synthetic and processed (microplastic) fibres discharged to the environment from laundering activities and exposed to marine mussels (Mytilus galloprovinciallis) in controlled experiments for a period of 7 d. A range of biological responses at different levels of organisation were subsequently determined, with copper employed concurrently as a positive control. Physiological changes were assessed from measurements of clearance rate, histopathological effects were evaluated from abnormalities in (or injuries to) gill and digestive gland tissues, and genetic damage was determined by measuring DNA strand breaks using the comet assay. With increasing lint concentration (over the range 56–180 mg L−1) we observed a reduction in mean clearance rate, increasing extents of abnormality in both gills (e.g. deciliation and hypertrophy) and digestive gland (e.g. atrophy and necrosis), and an increase in damage to DNA. The precise causes of these effects are unclear but likely arise from both the fibrous material itself and from chemicals (e.g. additives and metals) that are mobilised from the polymers into seawater or the digestive tract. The latter assertion is consistent with an observed increase in the release of certain trace elements (e.g. zinc) into the exposure medium with increasing lint concentration. Although microfibre concentrations we employed are significantly greater than those typically encountered in the environment, the results indicate the potential for this type of material to exert a range of adverse effects on exposed marine animals.

Temperature is an abiotic factor of particular concern for assessing the potential impacts of radionuclides on marine species. This is particularly true for tritium, which is discharged as tritiated water (HTO) in the process of cooling nuclear institutions. Additionally, with sea surface temperatures forecast to rise 0.5–3.5 °C in the next 30–100 years, determining the interaction of elevated temperature with radiological exposure has never been more relevant. We assessed the tissue-specific accumulation, transcriptional expression of key genes, and genotoxicity of tritiated water to marine mussels at either 15 or 25 °C, over a 7 day time course with sampling after 1 h, 12 h, 3 d and 7d. The activity concentration used (15 MBq L−1) resulted in tritium accumulation that varied with both time and temperature, but consistently produced dose rates (calculated using the ERICA tool) of

Auranofin, an organogold compound classified as an anti-rheumatic agent is under phase 2 clinical trials for re-purposing to treat recurrent epithelial ovarian cancer. We have reported earlier that Breast cancer 1, early onset (BRCA1) mutant ovarian cancer cells exhibit increased sensitivity to auranofin. BRCA1 is a DNA repair protein whose functional status is critical in the prognosis of ovarian cancer. Apart from DNA repair capability of cancer cells, membrane fluidity is also implicated in modulating resistance to chemotherapeutics. We report here that membrane fluidity influences the sensitivity of ovarian cancer cell lines, OVCAR5 and IGROV1, to auranofin. Electron spin resonance (ESR) analysis revealed a more fluidized membrane in IGROV1 compared to OVCAR5. Interestingly, IGROV1 cells were more sensitive to auranofin induced cytotoxicity than OVCAR5. In comparison to OVCAR5, IGROV1 cells also exhibited an increased number of DNA double strand breaks (DSBs) upon auranofin treatment as assessed by 53BP1 immunostaining. Furthermore, correlation analysis demonstrated a strong positive correlation (r=0.856) between membrane fluidity and auranofin sensitivity in these cell lines. Auranofin-treated IGROV1 cells also exhibited increased cellular oxidation and apoptosis. Anti-oxidant, N-acetyl cysteine (NAC) inhibited the cellular oxidation and apoptosis in auranofin-treated ovarian cancer cells suggesting reactive oxygen species (ROS) mediate the anti-cancer properties of auranofin. Overall, our study suggests that auranofin mediates its cytotoxicity via ROS production in ovarian cancer cells which correlates positively with membrane fluidity.

International audience; Accurate dosimetry is critically important for ecotoxicological and radioecological studies on the potential effects of environmentally relevant radionuclides, such as tritium (3H). Previous studies have used basic dosimetric equations to estimate dose from 3H exposure in ecologically important organisms, such as marine mussels. This study compares four different methods of estimating dose to adult mussels exposed to 1 or 15 MBq L-1 tritiated water (HTO) under laboratory conditions. These methods were (1) an equation converting seawater activity concentrations to dose rate with fixed parameters; (2) input into the ERICA tool of seawater activity concentrations only; (3) input into the ERICA tool of estimated whole organism concentrations (woTACs), comprising dry activity plus estimated tissue free water tritium (TFWT) activity (TFWT volume × seawater activity concentration); and (4) input into the ERICA tool of measured whole organism activity concentrations, comprising dry activity plus measured TFWT activity (TFWT volume × TFWT activity concentration). Methods 3 and 4 are recommended for future ecotoxicological experiments as they produce values for individual animals and are not reliant on transfer predictions (estimation of concentration ratio). Method 1 may be suitable if measured whole organism concentrations are not available, as it produced results between 3 and 4. As there are technical complications to accurately measuring TFWT, we recommend that future radiotoxicological studies on mussels or other aquatic invertebrates measure whole organism activity in non-dried tissues (i.e. incorporating TFWT and dry activity as one, rather than as separate fractions) and input this data into the ERICA tool. © 2016 Elsevier Ltd.

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.

Diamondoids are polycyclic saturated hydrocarbons that possess a cage-like carbon skeleton approaching that of diamond. These 'nano-diamonds' are used in a range of industries including nanotechnologies and biomedicine. Diamondoids were thought to be highly resistant to degradation, but their presumed degradation acid products have now been found in oil sands process-affected waters (OSPW) and numerous crude oils. Recently, a diamondoid-related structure, 3-noradamantane carboxylic acid, was reported to cause genetic damage in trout hepatocytes under in vitro conditions. This particular compound has never been reported in the environment but led us to hypothesise that other more environmentally relevant diamondoid acids could also be genotoxic. We carried out in vivo exposures (3 days, semi-static) of marine mussels to two environmentally relevant diamondoid acids, 1-adamantane carboxylic acid and 3,5-dimethyladamantane carboxylic acid plus 3-noradamantane carboxylic acid with genotoxic damage assessed using the Comet assay. An initial screening test confirmed that these acids displayed varying degrees of genotoxicity to haemocytes (increased DNA damage above that of controls) when exposed in vivo to a concentration of 30 μmol L(-1). In a further test focused on 1-adamantane carboxylic acid with varying concentrations (0.6, 6 and 30 μmol L(-1)), significant (P < 0.05%) DNA damage was observed in different target cells (viz. gills and haemocytes) at 0.6 μmol L(-1). Such a level of induced genetic damage was similar to that observed following exposure to a known genotoxin, benzo(a)pyrene (exposure concentration, 0.8 μmol L(-1)). These findings may have implications for a range of worldwide industries including oil extraction, nanotechnology and biomedicine.

Auranofin, a thioredoxin reductase inhibitor and an anti-rheumatic drug is currently undergoing phase 2 clinical studies for repurposing to treat recurrent epithelial ovarian cancer. Previous studies have established that auranofin exerts its cytotoxic activity by increasing the production of reactive oxygen species (ROS). Breast cancer 1, early onset (BRCA1) is a DNA repair protein whose functional status is critical in the prognosis of ovarian cancer. Apart from its key role in DNA repair, BRCA1 is also known to modulate cellular redox homeostasis by regulating the stability of anti-oxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) via direct protein-protein interaction. However, it is currently unknown whether BRCA1 modulates the sensitivity of ovarian cancer cells to auranofin. Here we report that BRCA1-depleted cells exhibited increased DNA double strand breaks (DSBs) and decreased clonogenic cell survival upon auranofin treatment. Interestingly, auranofin induced the expression of Nrf2 in BRCA1-depleted cells suggesting its regulation independent of BRCA1. Furthermore, anti-oxidant agent, N-acetyl cysteine (NAC) protected BRCA1-depleted cells from DNA damage and apoptosis induced by auranofin. Our study suggests that accumulated lethal DSBs resulting from the oxidative damage render BRCA1 deficient cells more sensitive to auranofin despite the activation of Nrf2.

Determination of relative sensitivity of biota following exposures to contaminants including metals is important for environmental protection. Copper (Cu), although biologically essential can be highly toxic to biota if present at higher concentrations in the natural environment. Given its ubiquitous presence within coastal and inland water bodies, we compared Cu-induced genotoxicity in two ecologically important mussel species, the freshwater Dreissena polymorpha (DP) and marine Mytilus galloprovincialis (MG), along with its tissue specific accumulation. Novel biomarker in terms of induction of gamma H2AX (γ-H2AX) foci, along with comet assay and induction of micronuclei (MN) were used to determine DNA damage response (DDR) in these two species following exposure to a range of Cu concentrations (18, 32, 56 μg L−1) for 10 days. Concentration-dependent increases in Cu concentration in gill tissue, as determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), were paralleled by a greater degree of genotoxicity. An induction of γ-H2AX foci was present in all Cu exposure concentrations, proving this technique to be a sensitive and suitable biomarker of genotoxicity in bivalves. The multi-biomarker approach adopted here suggests firstly that in parallel with MG, which is widely used to assess the health of marine and coastal environment, DP is also suitable representative of inland water bodies, and that there is a similar mechanism of action for the induction of genotoxicity between the two species, following exposure to Cu. Secondly, for genotoxicity assessment a battery of responses could simultaneously be assessed in these two bivalve species. Finally, for adequate protection of the environment it is vital to adopt a multi-biomarker, multi-species approach to determine adverse biological effects to gain a holistic understanding of the real threat posed by contaminants to hydrosphere.

The occurrence, fate and behaviour of carbon nanomaterials in the aquatic environment are dominated by their functionalization, association with organic material and aggregation behaviour. In particular, the degradation of fullerene aggregates in the aquatic environment is a primary influence on their mobility, sorption potential and toxicity. However, the degradation and kinetics of water suspensions of fullerenes remain poorly understood. In the present work, first, an analytical method based on liquid chromatography and high-resolution mass spectrometry (LC-HRMS) for the determination of C60 fullerene and their environmental transformation products was developed. Secondly, a series of C60 fullerene water suspensions were degraded under relevant environmental conditions, controlling the salinity, the humic substances content, the pH and the sunlight irradiation. Up to ten transformation products were tentatively identified, including epoxides and dimers with two C60 units linked via one or two adjacent furane-like rings. Fullerenols were not observed under these environmentally relevant conditions. The kinetics of generation of each transformation product were studied with and without simulated sunlight conditions. The ionic strength of the media, its pH and the humic substances content were observed to modulate the kinetics of generation.

Low-level laser therapy (LLLT) is a form of phototherapy used to promote cell proliferation. This study investigates the potential role of LLLT in cellular proliferation of human monocytic leukaemia cells Tamm-Horsfall Protein 1 (THP-1) under in vitro conditions. Cells were irradiated with an 850 nm diode laser and exposed to doses ranging from 0 to 26.8 J/cm 2 . After irradiation, cells were incubated for 12 and 24 h to allow time for proliferation. Comet assay was conducted to evaluate genotoxicity of the irradiated cells. Trypan blue was used to estimate cytotoxicity, which peaked at the highest dose as expected. Preliminary results suggest that cell counts increase at low doses, whereas a decrease in cell number at high doses was noted compared with controls. Comet assay showed no significant difference between irradiated and non-irradiated cells at low doses. In contrast, DNA damage increased at doses ≥8.9 J/cm 2 and was comparable with the 100 μM hydrogen peroxide positive control at the highest fluence. It could be concluded that LLLT has the ability to stimulate the THP-1 cell line to proliferate if supplied with the correct energy and dose.

Biological systems are the ultimate recipients of pollutant-induced damage. Consequently, our traditional reliance on analytical tools is not enough to assess ecosystem health. Biological responses or biomarkers are therefore also considered to be important tools for environmental hazard and risk assessments. Due to historical mining, other anthropogenic activities, and its conservational importance (e.g. NATURA sites, SACs), the Tamar estuary in South West England is an ideal environment in which to examine applications of such biological tools. This review presents a thorough and critical evaluation of the different biological tools used in the Tamar estuary thus far, while also discussing future perspectives for biomarker studies from a global perspective. In particular, we focus on the challenges which hinder applications of biological tools from being more readily incorporated into regulatory frameworks, with the aim of enabling both policymakers and primary stakeholders to maximise the environmental relevance and regulatory usefulness of such tools.

The article addresses the potential role of geosciences in development of civilian nuclear power plants (NPPs). We firstly highlight the fact that the Fukushima accident of 2011 is different from previous major nuclear accidents at the Three Mile Island and Chernobyl in that the former was triggered externally by a natural disaster, that is, a powerful tectonic earthquake followed by tsunami. Following this, we assess the situation of the NPPs to prepare the occurrence of such a tectonic activity through case study of the Fukushima Daiichi NPP. Finally, some policy recommendations are proposed for a wider and innate integration of geosciences into the governance of civilian NPPs

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.

The mechanisms of sublethal toxicity of the antifouling biocide, zinc pyrithione (ZnPT), have not been well-studied. This investigation demonstrates that 14-d sublethal exposure to ZnPT (0.2 or 2 μM, alongside inorganic Zn and sea water controls) is genotoxic to mussel haemocytes but suggests that this is not caused by oxidative DNA damage as no significant induction of oxidised purines was detected by Fpg-modified comet assay. More ecologically relevant endpoints, including decreased clearance rate (CR), cessation of attachment and decreased tolerance of stress on stress (SoS), also showed significant response to ZnPT exposure. Our integrated approach was underpinned by molecular analyses (qRT-PCR of stress-related genes, 2D gel electrophoresis of proteins) that indicated ZnPT causes a decrease in phosphoenolpyruvate carboxykinase (PEPCK) expression in mussel digestive glands, and that metallothionein genes are upregulated; PEPCK downregulation suggests that altered energy metabolism may also be related to the effects of ZnPT. Significant relationships were found between % tail DNA (comet assay) and all higher level responses (CR, attachment, SoS) in addition to PEPCK expression. Principal component analyses suggested that expression of selected genes described more variability within groups whereas % tail DNA reflected different ZnPT concentrations.

In vitro models are emerging tools for reducing reliance on traditional toxicity tests, especially in areas where information is sparse. For studies of fish, this is especially important for extrahepatic organs, such as the intestine, which, until recently, have been largely overlooked in favour of the liver or gill. Considering the importance of dietary uptake of contaminants, the rainbow trout (Oncorhynchus mykiss) intestine-derived cell line RTgutGC was cultured, to test its suitability as a high-throughput in vitro model. Benzo[a]pyrene (B[a]P) is an important contaminant and a model polycyclic aromatic hydrocarbon (PAH). Over 48 h exposure, a range of endpoints and xenobiotic metabolism rates were examined at three different pH levels indicative of the in vitro (pH 7.5) and in vivo mid-gut (pH 7.7) and hind-gut (pH 7.4) regions as a function of time. These endpoints included (i) cell viability: acid phosphatase (APH) and lactate dehydrogenase (LDH) assays; (ii) glucose uptake; (iii) cytochrome P450 enzyme activity: 7-ethoxyresoorufin-O-deethylase (EROD) assay; (iv) glutathione transferase (GST) activity; (v) genotoxic damage determined using the comet assay. Absence of cell viability loss, in parallel with decrease in the parent compound (B[a]P) in the medium and its subsequent increase in the cells suggested active sequestration, biotransformation, and removal of this representative PAH. With respect to genotoxic response, significant differences were observed at both the sampling times and the two highest concentrations of B[a]P. No significant differences were observed for the different pH conditions. Overall, this in vitro xenobiotic metabolism system appears to be a robust model, providing a basis for further development to evaluate metabolic and toxicological potential of contaminants without use of animals.

Release of tritium (3H) in the marine environment is of concern with respect to its potential bioaccumulation and detrimental impact on the biota. Previous studies have investigated the uptake and toxicity of this radionuclide in marine mussels, and the interaction of 3H with dissolved organic ligands and elevated temperature. However, despite the well-established view that toxicity is partly governed by chemical speciation, and that toxic effects of mixture of contaminants are not always additive, there have been no studies linking the prevailing chemistry of exposure waters with observed biological effects and tissue specific accumulation of 3H in combination with other constituents commonly found in natural waters. This study exposed the marine mussel Mytilus galloprovincialis for 14 days to mixtures of 3H (as tritiated water, HTO) and zinc (Zn) at 5 Mbq L-1, and 383, 1913 and 3825 nM Zn, respectively, to investigate (a) 3H and Zn partitioning in soft tissues of mussels, and (b) DNA damage in haemocytes, determined using the single cell gel electrophoresis or the comet assay. Additionally, the extent of association of 3H with dissolved organic carbon (DOC, added as humic acid) over the exposure period was investigated in order to aid the interpretation of biological uptake and effects. Results concluded a clear antagonistic effect of Zn on 3H-induced DNA damage at all Zn concentrations used, likely explained by the importance of Zn in DNA repair enzymes. The interaction of DOC with 3H was variable, with strong 3H-DOC associations observed in the first 3 d of the experiment. The secretion of 3H-binding ligands by the mussels is suggested as a possible mechanism for early biological control of 3H toxicity. The results suggest risk assessments for radionuclides in the environment require consideration of potential mixture effects.

Here we present the findings of a study where 598 isolates of Escherichia coli (351 derived from seawater; 247 derived from the Venus clam, Circenita callipyga) were obtained from Kuwait's marine environment. Isolates were screened for their potential resistance to an array of 23 commonly deployed frontline antibiotics. Results demonstrate the resistant was widespread across all sites with high-levels of resistance (seawater: summer 89–64%; winter 90–57% and biota: summer 77%; winter 88%) observed to at least 1 of the 23 antibiotics tested. Resistance to Ampicillin was by far the most widely observed profile in seawater and biota across both summer and winter seasons, with 55.9 to 70.9% isolates displaying resistance to this antibiotic. This study demonstrates the potential of AMR screening to be used in Kuwait to detect issues related to water quality and the consequences it may pose for human health.

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control.

Extraction of natural gas from shale rock in the United States (US) is one of the landmark events in the 21st century. The combination of horizontal drilling and hydraulic fracturing can extract huge quantities of natural gas from impermeable shale formations, which were previously thought to be either impossible or uneconomic to produce. This review offers a comprehensive insight into US shale gas opportunities, appraising the evolution, evidence and the challenges of shale gas production in the US. The history of US shale gas in this article is divided into three periods and based on the change of oil price (i.e., the period before the 1970s oil crisis, the period from 1970s to 2000, and the period since 2000), the US has moved from being one of the world's biggest importers of gas to being self-sufficient in less than a decade, with the shale gas production increasing 12-fold (from 2000 to 2010). The US domestic natural gas price hit a 10-year low in 2012. The US domestic natural gas price in the first half of 2012 was about $2 per million British Thermal Unit (BTU), compared with Brent crude, the world benchmark price for oil, now about $ 80–100/barrel, or $14–17 per million BTU. Partly due to an increase in gas-fired power generation in response to low gas prices, US carbon emissions from fossil-fuel combustion fell by 430 million ton CO 2 – more than any other country – between 2006 and 2011. Shale gas also stimulated economic growth, creating 600,000 new jobs in the US by 2010. However, the US shale gas revolution would be curbed, if the environmental risks posed by hydraulic fracturing are not managed effectively. The hydraulic fracturing is water intensive, and can cause pollution in the marine environment, with implications for long-term environmental sustainability in several ways. Also, large amounts of methane, a powerful greenhouse gas, can be emitted during the shale gas exploration and production. Hydraulic fracturing also may induce earthquakes. These environmental risks need to be managed by good practices which is not being applied by all the producers in all the locations. Enforcing stronger regulations are necessary to minimize risk to the environment and on human health. Robust regulatory oversight can however increase the cost of extraction, but stringent regulations can foster an historic opportunity to provide cheaper and cleaner gas to meet the consumer demand, as well as to usher in the future growth of the industry.