Quantitative cross-species extrapolation between humans and fish: The case of the anti-depressant fluoxetine

This article has been made available through the Brunel Open Access Publishing Fund.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

Development and Analysis of an Adverse Outcome Pathway Network for Human Neurotoxicity

An adverse outcome pathway (AOP) network is an attempt to represent the complexity of systems toxicology. This study illustrates how an AOP network can be derived and analysed in terms of its topological features to guide research and support chemical risk assessment. A four-step workflow describing general design principles and applied design principles were established and implemented. An AOP network linking nine linear AOPs was mapped and made available in AOPXplorer. The resultant AOP network was modelled and analysed in terms of its topological features, including level of degree, eccentricity and betweenness centrality. Several well connected KEs were identified, and cell injury/death was established as the most hyperlinked KE across the network. The derived network expands the utility of linear AOPs to better understand signalling pathways involved in developmental and adult/aging neurotoxicity. The results provide a solid basis to guide the development of in vitro test method batteries, as well as further quantitative modelling of key events (KEs) and key event relationships (KERs) in the AOP network, with an eventual aim to support hazard characterisation and chemical risk assessment

Unexpected removal of the most neutral cationic pharmaceutical in river waters

Contamination of surface waters by pharmaceuticals is now widespread. There are few data on their environmental behaviour, particularly for those which are cationic at typical surface water pH. As the external surfaces of bacterio-plankton cells are hydrophilic with a net negative charge, it was anticipated that bacterio-plankton in surface-waters would preferentially remove the most extensively-ionised cation at a given pH. To test this hypothesis, the persistence of four, widely-used, cationic pharmaceuticals, chloroquine, quinine, fluphenazine and levamisole, was assessed in batch microcosms, comprising water and bacterio-plankton, to which pharmaceuticals were added and incubated for 21 days. Results show that levamisole concentrations decreased by 19 % in microcosms containing bacterio-plankton, and by 13 % in a parallel microcosm containing tripeptide as a priming agent. In contrast to levamisole, concentrations of quinine, chloroquine and fluphenazine were unchanged over 21 days in microcosms containing bacterio-plankton. At the river-water pH, levamisole is 28 % cationic, while quinine is 91–98 % cationic, chloroquine 99 % cationic and fluphenazine 72–86 % cationic. Thus, the most neutral compound, levamisole, showed greatest removal, contradicting the expected bacterio-plankton preference for ionised molecules. However, levamisole was the most hydrophilic molecule, based on its octanol–water solubility coefficient (K ow). Overall, the pattern of pharmaceutical behaviour within the incubations did not reflect the relative hydrophilicity of the pharmaceuticals predicted by the octanol–water distribution coefficient, D ow, suggesting that improved predictive power, with respect to modelling bioaccumulation, may be needed to develop robust environmental risk assessments for cationic pharmaceuticals

<it>Echinacea</it>-induced cytosolic Ca²⁺elevation in HEK293

<p>Abstract</p> <p>Background</p> <p>With a traditional medical use for treatment of various ailments, herbal preparations of <it>Echinacea </it>are now popularly used to improve immune responses. One likely mode of action is that alkamides from <it>Echinacea </it>bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca²⁺. Here, we show that unidentified compounds from <it>Echinacea purpurea </it>induce cytosolic Ca²⁺elevation in non-immune-related cells, which lack CB2 receptors and that the Ca²⁺elevation is not influenced by alkamides.</p> <p>Methods</p> <p>A non-immune human cell line, HEK293, was chosen to evaluate <it>E. purpurea </it>root extracts and constituents as potential regulators of intracellular Ca²⁺levels. Changes in cytosolic Ca²⁺levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP₃) receptor, were tested to determine the mechanism of this Ca²⁺signaling pathway. <it>E. purpurea </it>root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity.</p> <p>Results</p> <p>A rapid transient increase in cytosolic Ca²⁺levels occurs when <it>E. purpurea </it>extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP₃receptor dependent. <it>Echinacea</it>-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca²⁺elevation is sustained after the <it>Echinacea</it>-induced Ca²⁺release from intracellular Ca²⁺stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from <it>E. purpurea </it>root extracts, six induce cytosolic Ca²⁺increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of <it>Echinacea do not </it>explain the observed Ca²⁺response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity.</p> <p>Conclusions</p> <p>Our data indicate that as yet unidentified constituents from <it>Echinacea </it>stimulate an IP₃receptor and phospholipase C mediation of cytosolic Ca²⁺levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.</p