Matthew J. Winter

Pharmaceuticals in the aquatic environment: a critical review of the evidence for health effects in fish.

The authors review the current data on the presence and reported biological effects in fish of some of the most commonly detected pharmaceuticals in the aquatic environment; namely nonsteroidal anti-inflammatory drugs (NSAIDs), fibrates, β-blockers, selective serotonin reuptake inhibitors (SSRIs), azoles, and antibiotics. Reported biological effects in fish in the laboratory have often been shown to be in accordance with known effects of pharmaceuticals in mammals. Water concentrations at which such effects have been reported, however, are generally, between μg L−1 and mg L−1, typically at least 1 order of magnitude higher than concentrations normally found in surface waters (ng L−1). There are exceptions to this, however, as for the case of synthetic oestrogens, which can induce biological effects in the low ng L−1 range. Although generally effect levels for pharmaceuticals are higher than those found in the environment, the risks to wild fish populations have not been thoroughly characterised, and there has been a lack of consideration given to the likely chronic nature of the exposures, or the potential for mixture effects. As global consumption of pharmaceuticals rises, an inevitable consequence is an increased level of contamination of surface and ground waters with these biologically active drugs, and thus in turn a greater potential for adverse effects in aquatic wildlife.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Environmental endocrine disrupters dysregulate estrogen metabolism and Ca2+ homeostasis in fish and mammals via receptor-independent mechanisms

Xenoestrogen endocrine disrupters (EDs) in the environment are thought to be responsible for a number of examples of sexual dysfunction that have recently been reported in several species. There is growing concern that these compounds may also cause abnormalities of the male reproductive tract and reduced spermatogenesis in man. Whilst some effects of EDs may be receptor-mediated, there is growing evidence that these compounds can exert potent effects in vivo by directly interacting with cellular enzyme targets. Here we report on, and review, the effects of alkylphenols and other EDs on two such enzymes: (1) sulfotransferases, which convert active estrogenic steroids to inactive steroid sulfates; and (2) Ca(2+)-ATPases, which are responsible for maintaining low, physiological, intracellular Ca(2+) concentrations. These enzymes are potently inhibited by EDs in both fish and mammalian species. The increased concentrations of active estrogens and the likely cytotoxic effects of elevated concentrations of intracellular Ca(2+) arising from these effects may underlie some of the endocrine disrupting potential of these widespread industrial pollutants.

Quantitative Cross-Species Extrapolation between Humans and Fish: The Case of the Anti-Depressant Fluoxetine

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.

Zebrafish assays as early safety pharmacology screens: Paradigm shift or red herring?

The recent flurry of interest in the potential use of the zebrafish (Danio rerio) in Drug Discovery has also led to the development of a range of assays purported to be useful as early screens in safety pharmacology. The purpose of this commentary is to take stock of the available zebrafish assays in the context of alternative mammalian cell-based assays, and of the validation outcomes to date. In addition, we report the results of a recent survey of the membership of the Safety Pharmacology Society regarding their views on zebrafish assays. The survey data indicate that the preferred way forward would be a collaborative effort between the pharmaceutical/biotechnology industry (as potential/eventual customers), and the zebrafish contract research companies (as suppliers), alongside expert input from academia and regulatory authorities.

Approaches to seizure risk assessment in preclinical drug discovery

Assessment of seizure risk traditionally occurs late in the drug discovery process using low-throughput, resource intensive in vivo assays. Such approaches do not allow sufficient time to mitigate risk by influencing chemical design. Early identification using cheaper, higher throughput assays with lower animal and compound requirements would be preferable. Here we review the current techniques available to assess this issue and describe how they may be combined in a rational step-wise cascade allowing more effective assessment of seizure risk.

Complex physiological traits as biomarkers of the sub-lethal toxicological effects of pollutant exposure in fishes

Complex physiological traits, such as routine aerobic metabolic rate or exercise performance, are indicators of the functional integrity of fish that can reveal sub-lethal toxicological effects of aquatic pollutants. These traits have proved valuable in laboratory investigations of the sub-lethal effects of heavy metals, ammonia and various xenobiotics. It is not known, however, whether they can also function as biomarkers of the complex potential range of effects upon overall functional integrity caused by exposure to mixtures of chemicals in polluted natural environments. The current study used portable swimming respirometers to compare exercise performance and respiratory metabolism of fish exposed in cages for three weeks to either clean or polluted sites on three urban European river systems: the river Lambro, Milan, Italy; the rivers Blythe, Cole and Tame, Birmingham, UK; and the river Amstel, Amsterdam, The Netherlands. The UK and Italian rivers were variously polluted with high levels of both bioavailable heavy metals and organics, and the Amstel by mixtures of bioavailable organics at high concentrations. In both the UK and Italy, indigenous chub (Leuciscus cephalus) exposed to clean or polluted sites swam equally well in an initial performance test, but the chub from polluted sites could not repeat this performance after a brief recovery interval. These animals were unable to raise the metabolic rate and allocate oxygen towards exercise in the second trial, an effect confirmed in successive campaigns in Italy. Swimming performance was therefore a biomarker indicator of pollutant exposure in chub exposed at these sites. Exposure to polluted sites on the river Amstel did not affect the repeat swimming performance of cultured cloned carp (Cyprinus carpio), indicating either a species-specific tolerance or relative absence of heavy metals. However, measurements of oxygen uptake during swimming revealed increased rates of routine aerobic metabolism in both chub and carp at polluted sites in all of the rivers studied, indicating a sub-lethal metabolic loading effect. Therefore, the physiological traits of exercise performance and metabolic rate have potential as biomarkers of the overall sub-lethal toxic effects of exposure to complex mixtures of pollutants in rivers, and may also provide insight into why fish do not colonize some polluted environments.

Ototoxin-induced cellular damage in neuromasts disrupts lateral line function in larval zebrafish.

The ototoxicity of a number of marketed drugs is well documented, and there is an absence of convenient techniques to identify and eliminate this unwanted effect at a pre-clinical stage. We have assessed the validity of the larval zebrafish, or more specifically its lateral line neuromast hair cells, as a microplate-scale in vivo surrogate model of mammalian inner ear hair cell responses to ototoxin exposure. Here we describe an investigation of the pathological and functional consequences of hair cell loss in lateral line neuromasts of larval zebrafish after exposure to a range of well known human and non-human mammalian ototoxins. Using a previously described histological assay, we show that hair cell damage occurs in a concentration-dependent fashion following exposure to representatives from a range of drug classes, including the aminoglycoside antibiotics, salicylates and platinum-based chemotherapeutics, as well as a heavy metal. Furthermore, we detail the optimisation of a semi-automated method to analyse the stereotypical startle response in larval zebrafish, and use this to assess the impact of hair cell damage on hearing function in these animals. Functional assessment revealed robust and significant attenuation of the innate startle, rheotactic and avoidance responses of 5 day old zebrafish larvae after treatment with a number of compounds previously shown to induce hair cell damage and loss. Interestingly, a startle reflex (albeit reduced) was still present even after the apparent complete loss of lateral line hair cell fluorescence, suggesting some involvement of the inner ear as well as the lateral line neuromast hair cells in this reflex response. Collectively, these data provide evidence to support the use of the zebrafish as a pre-clinical indicator of drug-induced histological and functional ototoxicity.

Chronic effects assessment and plasma concentrations of the β-blocker propranolol in fathead minnows (Pimephales promelas)

The presence of many human pharmaceuticals in the aquatic environment is now a worldwide concern, yet little is known of the chronic effects that these bioactive substances may be having on aquatic organisms. Propranolol, a non-specific beta adrenoreceptor blocker (beta-blocker), is used to treat high blood pressure and heart disease in humans. Propranolol has been found in surface waters worldwide at concentrations ranging from 12 to 590ng/L. To test the potential for ecologically relevant effects in fish in receiving waters, short-term (21 days) adult reproduction studies were conducted, in which fathead minnows were exposed to nominal concentrations of propranolol hydrochloride [CAS number 318-98-9] ranging from 0.001 to 10mg/L (measured concentrations typically from 78 to 130%). Exposure of fish to 3.4mg/L (measured) over 3 days caused 100% mortality or severe toxicity requiring euthanasia. The most sensitive endpoints from the studies were a decrease in hatchability (with regard to the number of days to hatch) and a concentration-related increase in female gonadal somatic index (GSI), giving LOEC(hatchability) and LOEC(female GSI) values of 0.1mg/L. Concentration-related decreases in weights of male fish were also observed, with LOEC(male wet weight value) of 1.0mg/L, and the LOEC(reproduction) value was 1.0mg/L. Collectively, these data do not suggest that propranolol was acting as a reproductive toxin. Plasma concentrations of propranolol in male fish exposed to nominal concentrations of 0.1 and 1.0mg/L were 0.34 and 15.00mg/L, respectively, which constitutes 436 and 1546% of measured water concentrations. These compare with predicted concentrations of 0.07 and 0.84mg/L, and thus to a degree support the use of partition coefficient models for predicting concentrations in plasma in fish. In addition, propranolol plasma concentrations in fish exposed to water concentrations of 0.1 and 1.0mg/L were greater than the human therapeutic plasma concentration and hence these data very strongly support the fish plasma model proposed by Huggett et al. [Huggett, D.B., Cook, J.C., Ericson, J.F., Williams, R.T., 2003a. A theoretical model for utilizing mammalian pharmacology and safety data to prioritize potential impacts of human pharmaceuticals to fish. Hum. Ecol. Risk Assess. 9, 1789-1799].

Using data from drug discovery and development to aid the aquatic environmental risk assessment of human pharmaceuticals: concepts, considerations, and challenges.

Over recent years, human pharmaceuticals have been detected in the aquatic environment. This, combined with the fact that many are (by design) biologically active compounds, has raised concern about potential impacts in wildlife species. This concern was realized with two high-profile cases of unforeseen environmental impact (i.e., estrogens and diclofenac), which have led to a flurry of work addressing how best to predict such effects in the future. One area in which considerable research effort has been made, partially in response to regulatory requirements, has been on the potential use of preclinical and clinical pharmacological and toxicological data (generated during drug development from nonhuman mammals and humans) to predict possible effects in nontarget, environmentally relevant species: so-called read across. This approach is strengthened by the fact that many physiological systems are conserved between mammals and certain environmentally relevant species. Consequently, knowledge of how a pharmaceutical works (the “mode-of-action,” or MoA) in nonclinical species and humans could assist in the selection of appropriate test species, study designs, and endpoints, in an approach referred to as “intelligent testing.” Here we outline the data available from the human drug development process and suggest how this might be used to design a testing strategy best suited to the specific characteristics of the drug in question. In addition, we review published data that support this type of approach, discuss the potential pitfalls associated with read across, and identify knowledge gaps that require filling to ensure accuracy in the extrapolation of data from preclinical and clinical studies, for use in the environmental risk assessment of human pharmaceuticals.