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Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 μg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.

This study addresses a fundamental question in fish welfare: are the anaesthetics used for fish aversive? Despite years of routine general use of many agents, within both scientific research and aquaculture, there is a paucity of information regarding their tolerance and associated behavioural responses by fish. This study examined nine of the most commonly used fish anaesthetic agents, and performed preference tests using adult mixed sex zebrafish (Danio rerio), the most commonly held laboratory fish. Video tracking software quantified swimming behaviour related to aversion for each anaesthetic at 50% of its standard recommended dose compared with clean water in a flow-through chemotaxic choice chamber. Results suggest that several commonly used anaesthetics were aversive, including two of the most commonly recommended and used: MS222 (ethyl 3-aminobenzoate methanesulphate) and benzocaine. For ethical best practice, it is recommended that compounds that are aversive, even at low concentration, should no longer be used routinely for anaesthesia or indeed the first step of humane euthanasia of adult zebrafish. Two agents were found not to induce aversive behavioural responses: etomidate and 2,2,2 tribromoethanol. For the millions of adult zebrafish used in laboratories and breeding worldwide, etomidate appears best suited for future routine humane use.

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.

There is a need to ensure that the care and welfare for fish maintained in the laboratory are to the highest standards. This extends to the use of anaesthetics for both scientific study, humane killing and euthanasia at end of life. An anaesthetic should not induce negative behaviours and fish should not seek to avoid the anaesthetic. Surprisingly little information is available to facilitate a humane choice of anaesthetic agent for fish despite over 100 years of use and the millions of fish currently held in thousands of laboratories worldwide. Using a chemotaxic choice chamber we found different species specific behavioural responses among four closely related fish species commonly held in the laboratory, exposed to three widely used anaesthetic agents. As previously found for zebrafish ( Danio rerio ), the use of MS-222 and benzocaine also appears to induce avoidance behaviours in medaka ( Oryzias latipes ); but etomidate could provide an alternative choice. Carp ( Cyprinus carpio ), although closely related to zebrafish showed avoidance behaviours to etomidate, but not benzocaine or MS-222; and rainbow trout ( Oncorhynchus mykiss ) showed no avoidance to the three agents tested. We were unable to ascertain avoidance responses in fathead minnows ( Pimephales promelas ) and suggest different test paradigms are required for that species.

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.

Significance Climate change impacts on wildlife populations are likely to be accentuated by pollution. Small (inbred) populations may be more vulnerable to these effects, but empirical data supporting these hypotheses are lacking. We present the first substantial empirical evidence, to our knowledge, for interactive effects on population viability of elevated temperature (climate); an endocrine disrupting chemical, clotrimazole (pollution); and inbreeding. Using the zebrafish ( Danio rerio ) as a model, we show these three factors interact to skew population sex ratios toward males and that this interaction can lead to increased risk of extinction. Our results suggest that climate change and pollution impacts are likely to pose significant extinction risks for small, endangered populations exhibiting environmental sex determination and/or differentiation. Endocrine disrupting chemicals (EDCs) are potent environmental contaminants, and their effects on wildlife populations could be exacerbated by climate change, especially in species with environmental sex determination. Endangered species may be particularly at risk because inbreeding depression and stochastic fluctuations in male and female numbers are often observed in the small populations that typify these taxa. Here, we assessed the interactive effects of water temperature and EDC exposure on sexual development and population viability of inbred and outbred zebrafish ( Danio rerio ). Water temperatures adopted were 28 °C (current ambient mean spawning temperature) and 33 °C (projected for the year 2100). The EDC selected was clotrimazole (at 2 μg/L and 10 μg/L), a widely used antifungal chemical that inhibits a key steroidogenic enzyme [cytochrome P450(CYP19) aromatase] required for estrogen synthesis in vertebrates. Elevated water temperature and clotrimazole exposure independently induced male-skewed sex ratios, and the effects of clotrimazole were greater at the higher temperature. Male sex ratio skews also occurred for the lower clotrimazole exposure concentration at the higher water temperature in inbred fish but not in outbred fish. Population viability analysis showed that population growth rates declined sharply in response to male skews and declines for inbred populations occurred at lower male skews than for outbred populations. These results indicate that elevated temperature associated with climate change can amplify the effects of EDCs and these effects are likely to be most acute in small, inbred populations exhibiting environmental sex determination and/or differentiation.

The Adverse Outcome Pathway (AOP) framework represents a valuable conceptual tool to systematically integrate existing toxicological knowledge from a mechanistic perspective to facilitate predictions of chemical-induced effects across species. However, its application for decision-making requires the transition from qualitative to quantitative AOP (qAOP). Here we used a fish model and the synthetic glucocorticoid beclomethasone dipropionate (BDP) to investigate the role of chemical-specific properties, pharmacokinetics, and internal exposure dynamics in the development of qAOPs. We generated a qAOP network based on drug plasma concentrations and focused on immunodepression, skin androgenisation, disruption of gluconeogenesis and reproductive performance. We showed that internal exposure dynamics and chemical-specific properties influence the development of qAOPs and their predictive power. Comparing the effects of two different glucocorticoids, we highlight how relatively similar in vitro hazard-based indicators can lead to different in vivo risk. This discrepancy can be predicted by their different uptake potential, pharmacokinetic (PK) and pharmacodynamic (PD) profiles. We recommend that the development phase of qAOPs should include the application of species-specific uptake and physiologically-based PK/PD models. This integration will significantly enhance the predictive power, enabling a more accurate assessment of the risk and the reliable transferability of qAOPs across chemicals.

The zebrafish is rapidly emerging as a promising alternative model for the detection of drug-induced cardiovascular effects. Despite its increasing popularity, the ability of this model to inform the drug development process is often limited by the uncertainties around the quantitative relevance of zebrafish responses compared with nonclinical mammalian species and ultimately humans. In this test of concept study, we provide a comparative quantitative analysis of the cardiovascular responses of zebrafish, rat, dog, and human to three model compounds (propranolol, losartan, and captopril), which act as modulators of two key systems (beta-adrenergic and renin-angiotensin systems) involved in the regulation of cardiovascular functions. We used imaging techniques to generate novel experimental data of drug-mediated cardiovascular effects in zebrafish larvae. These data were combined with a database of interspecies mammalian responses (i.e., heart rate, blood flow, vessel diameter, and stroke volume) extracted from the literature to perform a meta-analysis of effect size and direction across multiple species. In spite of the high heterogeneity of study design parameters, our analysis highlighted that zebrafish and human responses were largely comparable in >80% of drug/endpoint combinations. However, it also revealed a high intraspecies variability, which, in some cases, prevented a conclusive interpretation of the drug-induced effect. Despite the shortcomings of our study, the meta-analysis approach, combined with a suitable data visualization strategy, enabled us to observe patterns of response that would likely remain undetected with more traditional methods of qualitative comparative analysis. We propose that expanding this approach to larger datasets encompassing multiple drugs and modes of action would enable a rigorous and systematic assessment of the applicability domain of the zebrafish from both a mechanistic and phenotypic standpoint. This will increase the confidence in its application for the early detection of adverse drug reactions in any major organ system.

PURPOSE: To evaluate the visual outcomes, efficacy, predictability, and short-term safety of implanting the Sulcoflex (Rayner Intraocular Lenses Ltd) intraocular lens (IOL) to correct residual pseudophakic errors. METHODS: Retrospective study of patients undergoing implantation of the Sulcoflex IOL. Uncorrected (UDVA) and corrected (CDVA) distance visual acuity and refractive outcomes were evaluated. Postoperative follow-up was at 1 week and 1, 3, 6, and 12 months. RESULTS: Fifteen eyes (13 patients) were evaluated. Mean follow-up was 20 months (range: 14 to 30 months). The Sulcoflex aspheric (653L) and toric (653T) IOLs were implanted in 3 and 12 eyes, respectively. Preoperatively, mean logMAR (Snellen) UDVA and CDVA were 0.44 (20/55) and 0.05 (20/22), respectively. At 3 months, all eyes achieved logMAR UDVA of 0.20 (20/32) or better, with 10 (67%) eyes achieving UDVA of 0 (20/20) or better. Preoperative mean spherical and astigmatic errors were 1.07±0.83 diopters (D) and −1.45±0.98 D, respectively. Preoperative mean spherical equivalent refraction was −0.54±1.11 (D). Postoperative mean sphere and astigmatism at 3 months were −0.25±0.38 D and −0.50±0.57 D, respectively. Postoperative mean spherical equivalent refraction at 3 months was −0.15±0.28 D. All patients were within 1.00 D of attempted correction, with 93% within 0.50 D. Linear regression analysis showed good correlation (RFifteen eyes (13 patients) were evaluated. Mean follow-up was 20 months (range: 14 to 30 months). The Sulcoflex aspheric (653L) and toric (653T) IOLs were implanted in 3 and 12 eyes, respectively. Preoperatively, mean logMAR (Snellen) UDVA and CDVA were 0.44 (20/55) and 0.05 (20/22), respectively. At 3 months, all eyes achieved logMAR UDVA of 0.20 (20/32) or better, with 10 (67%) eyes achieving UDVA of 0 (20/20) or better. Preoperative mean spherical and astigmatic errors were 1.07±0.83 diopters (D) and −1.45±0.98 D, respectively. Preoperative mean spherical equivalent refraction was −0.54±1.11 (D). Postoperative mean sphere and astigmatism at 3 months were −0.25±0.38 D and −0.50±0.57 D, respectively. Postoperative mean spherical equivalent refraction at 3 months was −0.15±0.28 D. All patients were within 1.00 D of attempted correction, with 93% within 0.50 D. Linear regression analysis showed good correlation (R 2 =0.72) between attempted versus achieved spherical equivalent refractions. No significant intra- or postoperative complications occurred. CONCLUSIONS: Implantation of the Sulcoflex IOL was found to be an effective and predictable option for enhancing postoperative refractive results and reducing spectacle dependence for distance after surgery. The IOL was well tolerated in all eyes.

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