Investigating the effect of emetic and aversive compounds on Dictyostelium identifies a novel non-sentient model for bitter tastant research

Nausea and vomiting are common but serious side effects associated with many therapeutic drugs. Whilst the physiological mechanisms behind the generation of the vomiting response are well characterised, the range of emetic stimuli that can generate the response are poorly understood. The potential of using Dictyostelium discoideum, a eukaryotic amoeba, as a model for predicting emetic liability was examined in this thesis. The effects of a range of known emetic and aversive compounds on Dictyostelium cell behaviour was investigated, resulting in the identification of a small number that strongly inhibited cell migration in a concentration-dependent and reversible manner. These active compounds included a range of bitter compounds and the pungent taslant, capsaicin. A Dictyostelium mutagenesis screen was then used to identify genes controlling sensitivity to bitter tastants. This screen identified a mutant containing a disrupted grlJ gene as showing partial resistance to phenylthiourea in growth and behavioural changes in movement. GrlJ is a Gprotein coupled receptor that regulates a phenylthiourea-dependent effect by inhibition of a phosphatidylinositol (PIP3) signalling pathway. A search for proteins sharing homology to GrlJ identified an uncharacterised GABAB-like receptor, QaNHA5, involved in the detection of phenylthiourea in Dictyostelium. This thesis has therefore identified Dictyostelium as a potentially useful model for the identification of bitter and pungent tastants. In addition, this thesis has identified the Dictyostelium protein, GrIJ, as well as an uncharacterised human protein, Q8NHA5, involved in the detection of the bitter tastant, phenylthiourea.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Summary Report: Regional Assessment of the Ground-water Resources in Eastern Kankakee and Northern Iroquois Counties

published or submitted for publicationis peer reviewedOpe

Investigating the Effect of Emetic Compounds on Chemotaxis in Dictyostelium Identifies a Non-Sentient Model for Bitter and Hot Tastant Research

Novel chemical entities (NCEs) may be investigated for emetic liability in a range of unpleasant experiments involving retching, vomiting or conditioned taste aversion/food avoidance in sentient animals. We have used a range of compounds with known emetic /aversive properties to examine the possibility of using the social amoeba, Dictyostelium discoideum, for research into identifying and understanding emetic liability, and hence reduce adverse animal experimentation in this area. Twenty eight emetic or taste aversive compounds were employed to investigate the acute (10 min) effect of compounds on Dictyostelium cell behaviour (shape, speed and direction of movement) in a shallow chemotaxic gradient (Dunn chamber). Compound concentrations were chosen based on those previously reported to be emetic or aversive in in vivo studies and results were recorded and quantified by automated image analysis. Dictyostelium cell motility was rapidly and strongly inhibited by four structurally distinct tastants (three bitter tasting compounds - denatonium benzoate, quinine hydrochloride, phenylthiourea, and the pungent constituent of chilli peppers - capsaicin). In addition, stomach irritants (copper chloride and copper sulphate), and a phosphodiesterase IV inhibitor also rapidly blocked movement. A concentration-dependant relationship was established for five of these compounds, showing potency of inhibition as capsaicin (IC50?=?11.9±4.0 µM) > quinine hydrochloride (IC50?=?44.3±6.8 µM) > denatonium benzoate (IC50?=?129±4 µM) > phenylthiourea (IC50?=?366±5 µM) > copper sulphate (IC50?=?1433±3 µM). In contrast, 21 compounds within the cytotoxic and receptor agonist/antagonist classes did not affect cell behaviour. Further analysis of bitter and pungent compounds showed that the effect on cell behaviour was reversible and not cytotoxic, suggesting an uncharacterised molecular mechanism of action for these compounds. These results therefore demonstrate that Dictyostelium has potential as a non-sentient model in the analysis of the molecular effects of tastants, although it has limited utility in identification of emetic agents in general