Assessing the impact of benzo[a]pyrene with the in vitro fish gut model: An integrated approach for eco-genotoxicological studies.

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

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

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

An integrated approach to assess the impacts of zinc pyrithione at different levels of biological organization in marine mussels.

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

Exposure to tritiated water at an elevated temperature: Genotoxic and transcriptomic effects in marine mussels (M. galloprovincialis).

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 <20 Gy h-1, i.e. considerably below the recommended guidelines of the IAEA and EURATOM. Despite this, there was significant induction of DNA strand breaks (as measured by the comet assay), which also showed a temperature-dependent time shift. At 15 C, DNA damage was only significantly elevated after 7 d, in contrast to 25 C where a similar response was observed after only 3 d. The transcription profiles of two isoforms of hsp70, hsp90, mt20, p53 and rad51 indicated potential mechanisms behind this temperature-induced acceleration of genotoxicity, which may be the result of compromised defence. Specifically, genes involved in protein folding, DNA double strand break repair and cell cycle checkpoint control were upregulated after 3 d HTO exposure at 15 C, but significantly downregulated when the same exposure occurred at 25 C. This study is the first to investigate temperature efects on radiation-induced genotoxicity in an ecologically relevant marine invertebrate, Mytilus galloprovincialis. From an ecological perspective, our study suggests that mussels (or similar marine species) exposed to increased temperature and HTO may have a compromised ability to defend against genotoxic stress. Abbreviations: HTO, tritiated water; Fpg, formamidopyrimidine glyco- sylase; GoI, gene of interest; LSC, liquid scintillation counting; tDAC, tissue dry activity concentration; TFWT, tissue free water tritium; tTAC, tissue total activity concentration; woTAC, whole organism total activity concentration

Linking genotoxicity and cytotoxicity with membrane fluidity: A comparative study in ovarian cancer cell lines following exposure to auranofin

publisher: Elsevier articletitle: Linking genotoxicity and cytotoxicity with membrane fluidity: A comparative study in ovarian cancer cell lines following exposure to auranofin journaltitle: Mutation Research/Genetic Toxicology and Environmental Mutagenesis articlelink: http://dx.doi.org/10.1016/j.mrgentox.2016.09.003 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved

Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 μl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 μm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological research

Correction: Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

[This corrects the article DOI: 10.1371/journal.pone.0149492.]