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 (H(T)PCs). 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 H(T)PC range, whereas no effects were observed at plasma concentrations below the H(T)PCs. 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.

Bioavailability of the imidazole antifungal agent clotrimazole and its effects on key biotransformation genes in the common carp (Cyprinus carpio).

Clotrimazole (CTZ) is a persistent imidazole antifungal agent which is frequently detected in the aquatic environment and predicted to bio-concentrate in fish. Common carp (Cyprinus carpio) were exposed to mean measured concentrations of either 1.02 or 14.63µgl(-1) CTZ for 4 and 10 days, followed by a depuration period of 4 days in a further group of animals. Following each exposure regimen, plasma and liver CTZ concentrations were measured. Mean measured plasma concentrations of CTZ in animals exposed to the lower concentration of CTZ were 30 and 44µgl(-1) on days 4 and 10, respectively, and in the higher concentration were 318 and 336µgl(-1). Mean measured liver levels in the same animals were 514, 1725, 2111 and 7017µgl(-1) suggesting progressive hepatic accumulation. Measurement of CTZ in plasma after depuration suggested efficient elimination within 4 days, but appreciable levels of CTZ remained in the liver after depuration suggesting a degree of persistence in this tissue. In addition we measured responses of a number of key hepatic detoxification gene targets in the liver associated with the transcription factor pregnane X receptor (PXR); namely cyp450s 2k and 3a, glutathione-S-transferases a and p (gsta and p), and drug transporters multidrug resistance protein1 (mdr1), and MDR-related protein2 (mrp2). CTZ is a potent ligand of the PXR in humans and there is some evidence of PXR activation following exposure to CTZ in fish. The highest concentration of CTZ was adopted to explore the potential for alterations to detoxification gene expression in fish at a pharmacologically relevant dose level, and the lower concentration is within the range reported in effluents from waste water treatment works (WWTW). The genes for all biotransformation enzymes were up-regulated after exposure to the higher concentration of CTZ for 10 days, and alterations in expression occurred for the drug transporter genes mdr1 and mrp2 following exposure to the lower concentration of 1.02µgl(-1) CTZ (mean measured concentration). These data support the potential for CTZ to induce alterations in biotransformation and drug transporter genes associated with PXR in fish at concentrations measured in some WWTW effluents.

Comparative aquatic toxicity of propranolol and its photodegraded mixtures: algae and rotifer screening.

Transformation products of pharmaceuticals formed by human metabolism within sewage treatment plant or receiving waters are predicted, in most cases, to be less toxic than the parent compound to common aquatic species. However, there is little available data to demonstrate whether this is generally the case. In the present study, a framework was developed to guide testing of transformation products using phototransformation of the beta-blocker propranolol to test the hypothesis for this particular transformation route. Phototransformation is an important depletion mechanism of some pharmaceuticals in surface waters with fast reaction rate constants at environmentally relevant conditions. Samples of propranolol in deionized water (DIW) and river water (RW) were exposed to a solar simulator (lambda: 295-800 nm) and comparative toxicity of propranolol and its degraded mixtures measured using algal (Pseudokirchneriella subcapitata) and rotifer (Brachionus calyciflorus) screening tests. Results suggested a reduction of toxicity in photodegraded mixtures compared to the parent active pharmaceutical ingredient in all samples tested. Chemical analysis of effect test solutions supported the hypothesis that propranolol was transformed into compounds that appear to be less toxic to the organisms tested under the study conditions. Although the reactions were much faster in RW than in DIW, profiles of transformation products were similar in both matrices at two starting concentrations (1 and 10 mg/L). Results for propranolol implied that the reduction of toxicity using algal and rotifer screening tests was probably due to the production of more hydrophilic and more polar transformation products. Such results will provide useful insights into the environmental risk assessment of pharmaceuticals by taking into account their transformation products.

Environmental health impacts of equine estrogens derived from hormone replacement therapy.

Many factors have been considered in evaluations of the risk-benefit balance of hormone replacement therapy (HRT), used for treating menopausal symptoms in women, but not its potential risks for the environment We investigated the possible environmental health implications of conjugated equine estrogens (CEEs), the most common components of HRT, including their discharge into the environment, their uptake, potency, and ability to induce biological effects in wildlife. Influents and effluents from four U.K. sewage treatment works (STWs), and bile of effluent-exposed fish, were screened for six equine estrogens. In vitro estrogen receptor (ER) activation assays were applied in humans and fish to compare their potencies, followed by in vivo exposures of fish to equine estrogens and evaluation of bioaccumulation, estrogenic responses, and ER gene expression. The equine estrogen equilenin (Eqn), and its metabolite 17beta-dihydroequilenin (17beta-Eqn), were detected by tandem GC-MSMS in all STW influent samples and 83% of STW effluent samples analyzed, respectively, at low concentrations (0.07-2.6 ng/L) and were taken-up into effluent-exposed fish. As occurs in humans, these estrogens bound to and activated the fish ERs, with potencies at ERalpha 2.4-3490% of thatfor 17beta-estradiol. Exposure of fish for 21 days to Eqn and 17beta-Eqn induced estrogenic responses including hepatic growth and vitellogenin production at concentrations as low as 0.6-4.2 ng/L. Associated with these effects were inductions of hepatic ERalpha and ERbeta1 gene expression, suggesting ER-mediated mechanism(s) of action. These data provide evidence for the discharge of equine estrogens from HRT into the aquatic environment and highlight a strong likelihood that these compounds contribute to feminization in exposed wildlife.

Photo-induced environmental depletion processes of beta-blockers in river waters.

In order to improve the understanding of the fate and behaviour of pharmaceuticals in the environment there is a need to investigate in-stream depletion mechanisms, e.g. phototransformation of active pharmaceutical ingredients (APIs) in natural surface waters. In this study, abiotic and biotic degradation of selected beta-blockers was measured simultaneously in non-sterilised and sterilised river waters and deionised water (DIW) under simulated sunlight (lambda: 295-800 nm) and dark conditions, and at environmentally relevant concentrations, i.e.