Toxic Effects Of Koumine And Gelsenicine On The Early-life Stage Of Zebrafish Development

Koumine and gelsenicine are the main alkaloids in the Gelsemium elegans,and recent studies have shown that they exhibit anti-tumor,anti-anxiety and antiinflammatory analgesia effects.However,due to its strong toxicity and the mechanism is not clear,the use of G.elegans has been restricted.This study used the zebrafish model to explore the acute toxicity and developmental neurotoxicity of koumine and gelsenicine.In the acute toxicity studies,it was found that exposure to koumine and gelsenicine caused malformation of zebrafish embryos and larvae.Not only that,high concentrations of koumine and gelsenicine can also seriously affect the hatching rate and heart rate of zebrafish embryos and larvae.In order to study the developmental neurotoxicity of koumine and gelsencine,we observed the behavior of zebrafish embryos and larvae.The results showed that at all concentrations,koumine did not perturb spontaneous coiling.However,the inhibition of the escape response will become more obvious as the time increases.And the spasmodic movements of larvae exposed to 50,75,and 100 mg/L koumine can be clearly observed.Upon exposure to gelsenicine at or above 5 mg/L,the spontaneous behavior was suppressed during embryo development at 20 hpf.Zebrafish larvae exposed to 5,10,50,and 100 mg/L gelsenicine did not respond to touch at 27 and 36 hpf,which are consist with the results of spontaneous coiling test.Also the distance covered by the larvae at 2.5 mg/L was markedly decreased,and the larvae displayed rigid circular movement in their swimming path.At 120 hpf,we observed the paraffin section HE staining of the muscles.The results showed that gelsenicine did not affect the arrangement and integrity of the muscle fibers,indicating that the behavioral changes of the embryos and larvae exposed to gelsenicine were mainly caused by neural system damage.However,the muscle fiber of larvae exposed to koumine was loose and disordered compared with the controls.Based on the above results,phenotypic comparisons between zebrafish embryo exposure to koumine and ache zebrafish mutant were same.Therefore,we suspected that acetylcholinesterase(AChE)is likely to be one of the targets of koumine.Then we tested the activity of AChE,and the neurotransmitters were determined by high performance liquid chromatography-mass spectrometry(LC/MS).Our results directly demonstrate that koumine inhibited the activity of AChE and caused the accumulation of acetylcholine(ACh).However,gelsenicine has no effect on AChE activity,indicating that AChE may not be the target for gelsenicine.Gelsenicine showed different morphological changes and behavioral effects,so it is likely that toxic mechanism of koumine and gelsenicine may also different.The reason for this difference may be due to differences in their chemical structure.