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.

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.

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.

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.

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.

Tritiated Steel Micro-Particles: Computational Dosimetry and Prediction of Radiation-Induced DNA Damage for In Vitro Cell Culture Exposures.

Biological effects of radioactive particles can be experimentally investigated in vitro as a function of particle concentration, specific activity and exposure time. However, a careful dosimetric analysis is needed to elucidate the role of radiation emitted by radioactive products in inducing cyto- and geno-toxicity: the quantification of radiation dose is essential to eventually inform dose-risk correlations. This is even more fundamental when radioactive particles are short-range emitters and when they have a chemical speciation that might further concur to the heterogeneity of energy deposition at the cellular and sub-cellular level. To this aim, we need to use computational models. In this work, we made use of a Monte Carlo radiation transport code to perform a computational dosimetric reconstruction for in vitro exposure of cells to tritiated steel particles of micrometric size. Particles of this kind have been identified as worth of attention in nuclear power industry and research: tritium easily permeates in steel elements of nuclear reactor machinery, and mechanical operations on these elements (e.g., sawing) during decommissioning of old facilities can result in particle dispersion, leading to human exposure via inhalation. Considering the software replica of a representative in vitro setup to study the effect of such particles, we therefore modelled the radiation field due to the presence of particles in proximity of cells. We developed a computational approach to reconstruct the dose range to individual cell nuclei in contact with a particle, as well as the fraction of "hit" cells and the average dose for the whole cell population, as a function of particle concentration in the culture medium. The dosimetric analysis also provided the basis to make predictions on tritium-induced DNA damage: we estimated the dose-dependent expected yield of DNA double strand breaks due to tritiated steel particle radiation, as an indicator of their expected biological effectiveness.

Genotoxicity evaluation of medical devices: A regulatory perspective.

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 (E&L) 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 E&L 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.

Bioaccumulation, release and genotoxicity of stainless steel particles in marine bivalve molluscs.

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.

Evaluation of interactive effects of phosphorus-32 and copper on marine and freshwater bivalve mollusks.

PURPOSE: 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. METHODS: An integrated, multi-biomarker approach was adopted to investigate response to copper (Cu, 18 µg L-1), a known environmentally relevant genotoxic metal and differing concentrations of phosphorus-32 (32P; 0.1 and 1 mGy d-1), alone and in combination in marine (Mytilus galloprovincialis) and freshwater (Dreissena polymorpha) mussels. Genetic and molecular biomarkers were determined post-exposure and included DNA damage (as measured by the comet assay), micronuclei (MN) formation, γ-H2AX foci induction and the expression of key stress-related genes (i.e. hsp70/90, sod, cat, gst). RESULTS: 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 on 32P-induced damage across the species (but not all), cell types and dose rates. Multivariate analysis found significant correlations between comet and γ-H2AX assays, across both the tissues. Transcriptional expression of selected genes were generally unaltered in response to contaminant exposures, independent of species or tissues. CONCLUSIONS: Our 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.

From tangled banks to toxic bunnies; a reflection on the issues involved in developing an ecosystem approach for environmental radiation protection.

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.

A 3D In Vitro Model of the Human Airway Epithelium Exposed to Tritiated Water: Dosimetric Estimate and Cytotoxic Effects.

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, three-dimensional 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 µl-1 cm-2). After the exposures, we measured cell viability, integrity of epithelial layer and pro-inflammatory response 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.

Antagonistic cytoprotective effects of C60 fullerene nanoparticles in simultaneous exposure to benzo[a]pyrene in a molluscan animal model.

The hypothesis that C60 fullerene nanoparticles (C60) 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 C60 (C60-1mg/L) separately and in combination. Both BaP and C60 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 C60 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 C60. 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 C60, 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 C60, 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.

Impacts of microplastic fibres on the marine mussel, Mytilus galloprovinciallis.

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.

Effects of fullerene C60 in blue mussels: Role of mTOR in autophagy related cellular/tissue alterations.

The effects of C60 on mTOR (mechanistic Target of Rapamycin) activity in mussel digestive gland were investigated. mTOR is a kinase that senses physiological and environmental signals to control eukaryotic cell growth. mTOR is present in two complexes: the phosphorylated mTORC1 regulates cell growth by activating anabolic processes, and by inhibiting catabolic processes (i.e. autophagy); mTORC2 also modulates actin cytoskeleton organization. Mussels were exposed to C60 (0.01, 0.1 and 1 mg/L) for 72 h. Immunocytochemical analysis using a specific antibody revealed the cellular distribution of C60 in mussel digestive gland, already at the lowest concentration. In exposed mussels, the dephosphorylation of mTORC1 and mTORC2 may explain the C60 effects, i.e. the reduction of lysosomal membrane stability, the enhancement of LC3B protein, and the increase of lysosomal/cytoplasmic volume ratio; as well the cytoskeletal alterations. No oxidative stress was observed. Multivariate analysis was used to facilitate the interpretation of the biomarker data. Finally, a low density oligo-microarray was used to understand the cellular responses to fullerene. Transcriptomics identified a number of differentially expressed genes (DEGs) showing a maximum in animals exposed to 0.1 mg/L C60. The most affected processes are associated with energy metabolism, lysosomal activity and cytoskeleton organization. In this study, we report the first data on the subcellular distribution of C60 in mussel's cells; and on the involvement of mTOR inhibition in the alterations due to nanoparticle accumulation. Overall, mTOR deregulation, by affecting protein synthesis, energy metabolism and autophagy, may reduce the capacity of the organisms to effectively grow and reproduce.

Assessing relative biomarker responses in marine and freshwater bivalve molluscs following exposure to phosphorus 32 (32P): Application of genotoxicological and molecular biomarkers.

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.

An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse 'Trojan Horse' effects.

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.

Antagonistic Interactions between Benzo[a]pyrene and Fullerene (C60) in Toxicological Response of Marine Mussels.

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 Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Biomarkers of DNA damage as well as proteomics analysis were applied to unravel the interactive 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 downregulated (~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.