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

Abstract

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.

Bitter tastant responses in the amoeba Dictyostelium correlate with rat and human taste assays

“…To mimic salty taste, cells were exposed to increasing salt concentrations (potassium phosphate), from standard buffer conditions (24.2 mM potassium) up to a 3.3-fold increase (80 mM), and no gross change was observed in Dictyostelium cell shape following treatment. This is consistent with that reported by Robery et al (2011), where Dictyostelium does not respond to the salty tasting central nervous system depressant lithium chloride (Schiffman and Erickson, 1971) under equivalent conditions. To mimic sour taste (Da Conceicao Neta et al, 2007), cells were transferred to a buffer at pH 5 (from pH 6.3).…”

A novel human receptor involved in bitter tastant detection identified using the model organism Dictyostelium discoideum

“…Our previous study identified a strong and rapid effect of phenylthiourea on Dictyostelium cell movement (Robery et al, 2011), but a molecular mechanism for this effect was unclear. In order to identify this mechanism, we first established conditions necessary for a mutant screen, based upon resistance to a block in growth caused by phenylthiourea in shaking suspension.…”

“…We have previously reported a potent and rapid onset block in Dictyostelium cell behaviour (shape and movement) during chemotaxis for several bitter tastants, including phenylthiourea (Robery et al, 2011). This response was unexpected because Dictyostelium does not contain genes encoding homologues to T2R proteins associated with phenylthiourea detection.…”

“…Our recent research has also reported a simpler organism, the social amoeba Dictyostelium discoideum, to be capable of detecting bitter-tasting compounds (Robery et al, 2011). This organism represents one of the earliest branches from the common ancestor of all eukaryotes (Eichinger et al, 2005).…”

The Rac GEF ZizB regulates development, cell motility and cytokinesis in Dictyostelium

“…Chemotaxis assays were performed as described previously (Robery et al, 2011). In brief, cells were pulsed at a density of 1.7610 6 cells/ml with 30 nM cAMP at 6 minute intervals for 5 hours before plating on coverslips.…”