Mode-Of-Action Ecotoxicology of Pharmaceuticals

The purpose of this study is to determine if knowledge of the effects of pharmaceuticals in humans can be used to predict their effects in fish. It has recently been realised that human pharmaceuticals are present in the environment, particularly the aquatic environment. This is because humans excrete the drugs they take in their urine and faeces, and thus they enter the sewage system. Although sewage treatment plants remove a high proportion of the drugs entering them in wastewater, complete removal is rarely achieved and hence low concentrations of many pharmaceuticals are present in the effluents that are discharged into rivers. To date, chemists have been able to identify well over 100 different pharmaceuticals in rivers, although this is still only a small proportion of the 3000 different pharmaceuticals in widespread use. Hence, all aquatic organisms are exposed to many different pharmaceuticals, albeit at low concentrations. Intuitively it seems unlikely that these low concentrations would adversely affect aquatic wildlife such as fish, yet recent research on one particular pharmaceutical, a constituent of the contraceptive 'Pill', has questioned that assumption. This pharmaceutical contributes to the feminisation of male fish that has been widely reported. This discovery has raised the possibility that other pharmaceuticals may also be affecting fish. But with so many different pharmaceuticals present in rivers, which ones are of concern, and what effects do they induce? This project addresses these key questions by testing the hypothesis 'Human pharmaceuticals cause the same effects in fish as they do in humans, and these effects occur only if the blood concentrations of the pharmaceuticals in fish reach the same concentrations as those required in humans for the pharmaceuticals to have their effects.' Fish will be exposed to four different classes of pharmaceuticals during the project: analgesics, selective serotonin reuptake inhibitors (SSRIs), angiotensin converting enzyme inhibitors and synthetic glucocorticoids. Plasma levels of the drugs will be measured to establish that therapeutic concentrations - levels that give effects in humans - are reached. Because fish are very similar to humans at the molecular and biochemical levels, it as anticipated that the main, or even only, effect in fish will be the same as that which occurs in people taking these pharmaceuticals. These effects will be measured in the fish, and by additionally using an exposure concentration of 100-fold less than that required to reached therapeutic concentrations, we will establish that the effects are seen only when blood levels of the drugs compare to effective concentrations in humans. If this is the case, it will support our hypothesis and mean that the potential effects of pharmaceuticals on fish can be predicted based on existing knowledge of the drugs. Being able to do so would have two major advantages: it would aid in predicting which pharmaceuticals present in the environment might be of most concern, and it will mean that the likely effects of pharmaceuticals on fish (if any of course), at environmentally relevant concentrations, could be predicted with some degree of confidence. Being able to do so would be a major step forward in understanding whether or not pharmaceuticals in the environment pose any risk to fish.

Current testing of human pharmaceuticals for possible environmental effects is widely accepted to be inadequate. It is proposed that fish, being very similar to mammals at the biochemical and molecular levels, will respond to pharmaceuticals in the same way that humans do; that is, the mode of action in humans is likely to be the main mode of action in fish. Further, it is hypothesised that to exert an effect, the blood concentration in fish will need to be similar to that in patients taking the drugs. This has recently been shown to be true for beta-blockers, but its wider applicability to other classes of pharmaceuticals is unknown. This proposal is for a 'proof of principle' project, which will study representative drugs from four different classes of pharmaceutical (COX inhibitors, SSRIs, ACE inhibitors, and synthetic glucocorticoids). Zebrafish will be exposed to ibuprofen, fluoxetine, quinapril and beclomethasone at concentrations sufficient to establish therapeutic plasma levels, which will be confirmed by LC/MS/MS measurements. Endpoints relevant to their modes of action in humans will be measured. In order to establish that effects are only seen at therapeutic concentrations, fish will also be exposed to a concentration of each drug 100-fold less than that required for therapeutic plasma levels. If, as anticipated, the results support the so called 'read across' hypothesis from mammals to fish, they would provide a very strong driver for the development of an efficient and effective testing strategy for pharmaceuticals, and refinement of the current regulatory testing, which is designed for toxic chemicals, and not those with known biological actions such as pharmaceuticals.

In addition to the academic community, the pharmaceutical industry and regulatory agencies concerned with ecotoxicity testing and environmental risk assessment will benefit from this research. This is a collaborative project with the pharmaceutical company AstraZeneca (AZ), which is investing significantly both in cash and kind, in the project: it would not do so unless it considered it important to resolve the question of mode-of-action ecotoxicology. If this principle can be established, it is likely that the project will have significant impact on all companies engaged in ecotoxicity testing. It is widely recognised that the current strategy for testing pharmaceuticals using standard ecotoxicity tests is inadequate, as very potent chemicals can 'pass' all such routine tests, yet still have significant adverse effects on wildlife at very low concentrations. Thus, regulatory agencies (e.g. Environment Agency, European Medicines Agency) will also be interested in the research and benefit from the clarity we hope the project will bring to this area. Media interest in the 'Pharmaceuticals in the Environment' issue is likely to continue, and thus the general public may benefit from gaining an informed view of the likely risk of drugs present in the environment. Establishing that pharmaceuticals have effects in non-human species only if these contain the specific targets and when plasma concentrations reach levels in the human therapeutic range, allows an intelligent approach to ecotoxicity testing. Tests relevant to the drugs' modes of action would be more appropriate than the standard ecotoxicological endpoints such as growth and survival that are currently in use, and give the industry confidence that their drugs were effectively tested in a manner likely to be most informative, thus providing the information required for a robust environmental risk assessment. It is anticipated that this project will provide weighted evidence that will inform changes in guidelines by regulatory agencies. The researcher employed on this project will develop skills such as communication to different audiences (for example industrial collaborators, scientific conferences, publications) that have wide applicability, as well as specific research skills and knowledge that will be attractive to industry, regulatory bodies and academia. This proposal builds on a long-standing connection of over 16 years between the partners, resulting in 17 joint publications (5 in the past 2 years) that have been cited over 1000 times so far. This demonstrates that we can collaborate effectively to produce world-class science that has very considerable impact. Our extensive links with other pharmaceutical companies will support engagement with the industry in general. It is not anticipated that the result will remain confidential, and will be disseminated through workshops and scientific meetings as well as publications. AZ has extremely good links with the regulatory authorities, and hence many opportunities will exist to keep the regulatory organisations informed. It is unlikely that the project will produce results that are commercially exploitable, as its focus is to provide experimental proof of a concept that may ultimately inform regulatory testing of pharmaceuticals. However, both partner organisations have the knowledge and experience to exploit the research should this be appropriate. The applicants will undertake the impact activities, and also provide opportunities for the post-doctoral fellow to contribute, in order for him/her to gain experience of the wider dissemination of science. Whilst we do not envisage holding any public meetings, we will continue engaging with society as well as the academic community, as we have done in the past; the P.I. in particular has a lot of experience in successful knowledge exchange and ensuring impact, in advising and supporting other scientists, interacting with the media, and giving public lectures.