Zebrafish embryos as alternative model to study intoxication by Acer pseudoplatanus toxins: Hypoglycin A, Methylenecyclopropylglycine and Methylenecyclopropylacetate

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG) are protoxins synthesised by plants of the Sapindaceae family notably Acer pseudoplatanus. A. pseudoplatanus poisoning is a recognised emerging pasture-associated intoxication leading to atypical myopathy (AM) in equids. Once ingested the protoxins are metabolised into toxic compounds including methylenecyclopropylacetate-CoA (MCPA-CoA) that impairs lipid metabolism and induces a severe rhabdomyolysis syndrome. Currently, there are limited laboratory models for studying AM under controlled conditions, with few existing cell culture models utilising MCPA alone. In this study, zebrafish embryos were used to evaluate the toxicity of AM protoxins (i.e. HGA and MCPrG) and the toxic metabolite MCPA. Zebrafish embryos of 1 day post fertilisation were individually exposed during 72 hours to several concentrations of HGA, MCPrG and MCPA. The experiment was performed in triplicate using 20 larvae per concentration, E3 medium and 3,4 dichloroaniline as negative and positive control, respectively. Every 24 hours, four endpoints based on OECD guidelines were recorded as indicator of lethality as well as sublethal effects to determine the LC50 and EC50 using a four parameters log-logistic model. Markedly, MCPrG did not induce mortality or sublethal effects, even after 72h at 5 mM. Conversely, HGA-induced toxicity appeared after 48 hours with mainly a reduced heartbeat. After 72h of intoxication, the LC50 and EC50 of MCPA were 0.98 and 1.33 µM,
respectively, while those for HGA were 1.66 and 2.07 µM. This study suggests that zebrafish embryos could be used as an alternative intoxication model for studying HGA toxicity, particularly as cell culture models did not exhibit cytotoxicity with this protoxin.