Assessing drug-induced oxidative stress and its interactions with other stressors using a novel transgenic zebrafish model

Pharmaceuticals discharged into the environment are potentially harmful to wildlife as many drug targets are conserved across divergent phyla. Oxidative stress (OS) is a major mechanism by which many pharmaceutical contaminants can induce toxicity. However, this mechanism is relatively poorly understood, particularly regarding multiple stressor interactions. Transgenic zebrafish are an increasingly popular, highly integrated tool for elucidating chemical mixture effects, and in recent years there have been developments in the semi-automation of bioimaging tools to increase throughput using them. However, transgenic models are currently underutilised for studies into physical-chemical interactions and mixture effects of chemicals with different modes of toxicity. In this thesis, the application of the novel OS transgenic zebrafish model EpRE:mCherry (visualising activation of the electrophile response element; EpRE) is developed, together with the Acquifer semi-automated imaging platform. This system is used to screen environmental pharmaceuticals for pro-oxidative action, and assess their interaction with an oestrogenic chemical (EE2) and a physical stressor (temperature). EpRE:mCherry embryo-larvae were exposed to one of a range of pharmaceuticals from 0 – 4 dpf (aqueous exposure) and tissue-specific fluorescence intensity was assessed as a measure of OS. Paracetamol, diclofenac and doxorubicin consistently induced strong OS responses in the TG model, while cisplatin and cyclophosphamide induced responses only during specific exposure windows. The pronephros was generally the most responsive to pharmaceutical-induced OS, followed closely by the liver, except for in response to doxorubicin which had the strongest effect in the gastrointestinal tract. Analytical chemistry techniques confirmed that atenolol, ibuprofen, clarithromycin and clozapine were taken up by exposed larvae, but induced no response in the TG model and so are assumed not to act via the EpRE. Previous studies have investigated the contraceptive hormone ethinyl estradiol (EE2) as part of oestrogenic chemical mixture effects, but there is limited data on its interactive effects with other chemical classes of compounds. In vitro data suggests that oestrogens may have antioxidative properties by upregulating antioxidant enzymes via intracellular signalling pathways, but this has not yet been shown in a whole organism. This was investigated in zebrafish embryo-larvae exposing them to a combination of paracetamol and EE2. However, no effect of EE2 was found on paracetamol- induced OS in the EpRE:mCherry model. Nevertheless, an oestrogen receptor inhibitor ICI 182,780 exhibited potential to exacerbate drug-induced OS, indicating endogenous oestrogen may play an antioxidative role. There is growing evidence that climate change may exacerbate the toxic effect of pollutants and, as poikilotherms, fish are particularly vulnerable to rising temperatures. Incubation of zebrafish at temperatures 2-5°C above standard husbandry temperatures were found to exacerbate drug-induced OS and this was demonstrated for 3 pharmaceuticals from a range of therapeutic classes and potencies (namely, paracetamol, diclofenac and doxorubicin). LC-MS/MS data indicated this interaction is at least partly due to increased uptake of the drug. This indicates the need for future environmental risk assessments to more fully consider the influence of temperature (and other abiotic factors) on chemical toxicity. This is of particular interest as climate change is predicted to result in increased global temperatures and more frequent extreme weather events, potentially increasing the risk of chemicals detected in waterways at concentrations currently considered to be safe. The data presented here also demonstrates the utility of the EpRE:mCherry model and Acquifer as a new screening system for chemical effects analyses and to facilitate more targeted environmental risk assessments.AstraZeneca UK Lt