Grace H. Panter

Adverse reproductive effects in male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone

Recent research has shown that low concentrations of natural and synthetic oestrogens are present in sewage treatment works effluents. However, the effects of these oestrogens on fish, at environmentally relevant concentrations, are unknown. Therefore, male fathead minnows (Pimephales promelas) were exposed to nominal concentrations of 17β-oestradiol (10, 32, 100, 320 and 1000 ng l−1) and molar equivalent concentrations of oestrone (9.9, 31.8, 99.3, 318 and 993 ng l−1) for 21 days, to determine the effects of these chemicals on plasma vitellogenin levels and gonad weight. Exposure to both steroidal compounds resulted in an elevation of plasma vitellogenin levels, in a concentration-related manner. Significant (P 100 ng oestradiol l−1 and 31.8 ng oestrone l−1. This increase in plasma vitellogenin levels was accompanied by an inhibition of testicular growth, which for the highest two concentrations of oestradiol was total. The results from this study indicated that although oestrone induced significant effects at a lower concentration, oestradiol was more potent than oestrone at higher concentrations, causing greater vitellogenin synthesis and testicular inhibition. The key result is that low concentrations of oestradiol and oestrone, similar to the reported concentrations in effluent, have profound effects on male fish.

MEASUREMENT OF VITELLOGENIN, A BIOMARKER FOR EXPOSURE TO OESTROGENIC CHEMICALS, IN A WIDE VARIETY OF CYPRINID FISH

There is increasing concern about man-made chemicals in the aquatic environment that mimic oestrogens because they may disrupt reproductive function. Vitellogenin, a precursor of egg-yolk in fish and other oviparous animals, may be used as a biomarker for “oestrogen” exposure. This study investigated the use of a radioimmunoassay developed to carp (Cyprinus carpio) vitellogenin to measure vitellogenin in other species of fish, especially cyprinids that would be of value for field and laboratory studies on oestrogenic xenobiotics. Of the nine families of fish studied, only vitellogenin from cyprinids (to which the carp belongs) showed good cross-reactivity in the carp vitellogenin radioimmunoassay. Vitellogenin from cyprinids native to Europe that cross reacted in the carp vitellogenin radioimmunoassay included: bream (Abramis brama), roach (Rutilus rutilus), rudd (Scardinius erythropthalmus), gudgeon (Gobio gobio) and minnow (Phoxinus phoxinus). Vitellogenin from cyprinids used widely in ecotoxicology that cross reacted in the carp vitellogenin radioimmunoassay included: fathead minnow (Pimephales promelas), zebrafish (Brachydanio rerio) and goldfish (Carassius auratus). In the cyprinids studies, the concentrations of vitellogenin in mature females were between a few hundred and a thousand microgram per millilitre. Concentrations of plasma vitellogenin in immature females were always greater than 200 ng·m-1, whereas in males (with the exception of the fathead minnow) plasma vitellogenin concentrations were less than 20 ng·ml-1 (and generally, much lower). The results suggest that the structure of vitellogenin is highly conserved within the cyprinid family and that the carp vitellogenin radioimmunoassay may be used to measure the concentrations of vitellogenin in plasma from a wide variety of cyprinids.

The Read-Across Hypothesis and Environmental Risk Assessment of Pharmaceuticals

Pharmaceuticals in the environment have received increased attention over the past decade, as they are ubiquitous in rivers and waterways. Concentrations are in sub-ng to low μg/L, well below acute toxic levels, but there are uncertainties regarding the effects of chronic exposures and there is a need to prioritise which pharmaceuticals may be of concern. The read-across hypothesis stipulates that a drug will have an effect in non-target organisms only if the molecular targets such as receptors and enzymes have been conserved, resulting in a (specific) pharmacological effect only if plasma concentrations are similar to human therapeutic concentrations. If this holds true for different classes of pharmaceuticals, it should be possible to predict the potential environmental impact from information obtained during the drug development process. This paper critically reviews the evidence for read-across, and finds that few studies include plasma concentrations and mode of action based effects. Thus, despite a large number of apparently relevant papers and a general acceptance of the hypothesis, there is an absence of documented evidence. There is a need for large-scale studies to generate robust data for testing the read-across hypothesis and developing predictive models, the only feasible approach to protecting the environment.

Principles of sound ecotoxicology.

We have become progressively more concerned about the quality of some published ecotoxicology research. Others have also expressed concern. It is not uncommon for basic, but extremely important, factors to apparently be ignored. For example, exposure concentrations in laboratory experiments are sometimes not measured, and hence there is no evidence that the test organisms were actually exposed to the test substance, let alone at the stated concentrations. To try to improve the quality of ecotoxicology research, we suggest 12 basic principles that should be considered, not at the point of publication of the results, but during the experimental design. These principles range from carefully considering essential aspects of experimental design through to accurately defining the exposure, as well as unbiased analysis and reporting of the results. Although not all principles will apply to all studies, we offer these principles in the hope that they will improve the quality of the science that is available to regulators. Science is an evidence-based discipline and it is important that we and the regulators can trust the evidence presented to us. Significant resources often have to be devoted to refuting the results of poor research when those resources could be utilized more effectively.

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.

Oxidative and Conjugative Xenobiotic Metabolism in Zebrafish Larvae In Vivo

As zebrafish larvae are being increasingly applied to toxicity testing, there is a need to understand the potential for xenobiotic metabolism by these early life-stage organisms. The expression of genes similar to mammalian cytochromes P450 (CYP) 2B6, CYP3A5, and UDP-glucuronosyl transferase (UGT) 1A1, as well as the zebrafish CYP1A was assessed across embryonic development. Activities toward 7-ethoxyresorufin O-deethylase (EROD assay), 7-ethoxycoumarin O-deethylase (ECOD assay), and octyloxymethylresorufin (OOMR assay) were detected at 96 h postfertilization, as was significant phenolic conjugation in the EROD assay (p < 0.001). The induction of CYP1A, the CYP gene zgc:153269, and UGT1A1 after exposure to Aroclor 1254 (100 microg/L, 24 h) was observed, with significant CYP1A induction (p < 0.01). Aroclor exposure also significantly induced EROD activity (p < 0.005), as did coexposure of alpha-naphthoflavone in a dose-dependent manner (p < 0.05; 5 and 10 microM exposures). Inhibition of CYP activity by SKF525A (5 microM) could not be demonstrated because of significant CYP induction as evidenced by OOMR activity (p < 0.05). This study demonstrates that zebrafish larvae express genes similar to mammalian CYP and UGT isoforms throughout early development and have activities toward model CYP substrates. The modulation of these genes and activities by CYP inducers is also reported. The continued use of these model organisms in toxicity testing is supported by this study.

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.

Test concentration setting for fish in vivo endocrine screening assays

Fish in vivo screening methods to detect endocrine active substances, specifically interacting with the hypothalamic-pituitary-gonadal axis, have been developed by both the Organization for Economic Co-operation and Development (OECD) and United States Environmental Protection Agency (US-EPA). In application of these methods, i.e. regulatory testing, this paper provides a proposal on the setting of test concentrations using all available acute and chronic data and also discusses the importance of avoiding the confounding effects of systemic toxicity on endocrine endpoints. This guidance is aimed at reducing the number of false positives and subsequently the number of inappropriate definitive vertebrate studies potentially triggered by effects consequent to systemic, rather than endocrine, toxicity. At the same time it provides a pragmatic approach that maximizes the probability of detecting an effect, if it exists, thus limiting the potential for false negative outcomes.

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna

Highlights ► Daphnia magna have a detectable activity towards ethoxyresorufin. ► This activity is unaffected by 24 h exposure to dimethylsulfoxide or methanol. ► EROD activity in zebrafish is inhibited by 24 h exposure to both DMSO and methanol. ► DMSO and methanol exposures (24 h) decreased expression of CYP and UGT genes in zebrafish larvae. ► A maximum solvent concentration of 0.01% v/v is recommended for use where possible in zebrafish.

Metabolism of ibuprofen in zebrafish larvae.

The application of zebrafish (Danio rerio) larvae to drug discovery assays and toxicity testing, and the occurrence of pharmaceuticals in the environment, has resulted in a need to understand the extent of the metabolic capabilities in the early life stages of this species. The aims of this study were to determine if zebrafish larvae absorbed, metabolized and excreted the model pharmaceutical, ibuprofen. Zebrafish larvae (72 h post fertilization) were exposed to ibuprofen (100 µg/L), 14C-ibuprofen (100 µg/L) or a solvent control (ethanol) for ≤24 h. Water samples and larval extracts were assessed for metabolites of ibuprofen using liquid chromatography mass spectrometry (LC–MS–MS). Fractions from the separation of the samples treated with 14C-ibuprofen were collected after chromatography and analysed for 14C content by scintillation counting. Assessment of larval extracts and water samples by LC–MS–MS at 24 h resulted in the identification of hydroxy-ibuprofen in both water samples and larval extracts (8.2 and 0.08% of the total detected 14C, respectively). A second putative hydroxy-ibuprofen moiety was also observed in water samples at trace levels, and a third minor unknown metabolite was detected in larval extracts only by scintillation counting (0.02% of the total 14C detected). This study provides evidence that zebrafish larvae can metabolize and excrete ibuprofen in a manner known to be cytochrome P450-dependent in mammals, and the similarity to the mammalian pathway supports the use of this system as a surrogate in toxicity and efficacy screening.