Tracking of sibling fates reliably identifies the differentiative potential of a single cell taken for PCR analysis, and demonstrates the existence of a variety of distinct and stable states of differentiative commitment. Global amplification of cDNA from single precursor cells, identified by sibling fates, yields a true representation of lineage- and stage-specific gene expression, as confirmed by hybridization to a broad panel of probes. The results provide the first expression mapping of these genes that distinguishes between progenitors in different commitment states, generate new insights and predictions relevant to mechanism, and introduce a powerful set of tools for unravelling the genetic basis of lineage divergence.

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.

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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.

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.

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.

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HighlightsCFA appears to have a low propensity to bioconcentrate and has a plasma half-life of <4 days in carp.CFA increases levels of mRNA of a number of genes known to be regulated by PPARα in mammals.PPARα activation changes levels of mRNA of genes involved with several detoxification/ biotransformation system components in carp.CFA alters levels of mRNA and activity of the inducible β-oxidation pathway enzyme Acox1, a known indicator of peroxisome proliferator exposure.