Toxic Effect Of Pyrethroids On Danio Rerio And Preliminary Exploration Of Mechanism

Pyrethroid insecticides is one of the the third largest category of pesticides, which is widely used in agriculture and life. Applying pesticides to crops often cause water pollution, and it can bring threat to humans'and animals'safety. With the widespread use of pyrethroids, the attendant security risks have become one of the most concerned problems to people. By studying the toxic effects on model organism zebrafish, and the impact on voltage-gated sodium channel Nav1.2 with typical pyrethroid insecticides, can clear mechanism of pyrethroids and provide data support for safety evaluation of pyrethroids.1. In this paper, the model organism zebrafish(Danio rerio) was selected as the research object, the acute toxicity to zebrafish with three pyrethroids has been studied, the results showed that the 24h-LC50,48h-LC50 and 96h-LC50 of zebrafish with deltamethrin were 2.5 ?g/L,2.2?g/L,1.8?g/L respectively; the 24h-LC50,48h-LC50 and 96h-LC50 of zebrafish with beta-cypermethrin were 6.1?g/L,5.1?g/L,4.7?g/L respectively; the 24h-LC50,48h-LC50 and 96h-LC50 of zebrafish with D-allethrin were 116.3?g/L,95.8?g/L,83.0?g/L respectively. The safe concentration of deltamethrin, beta-cypermethrin, D-allethrin were 0.18?g/L,0.47?g/L,8.30?g/L, respectively. The sensitivity of three pyrethroids to zebrafish is:deltamethrin> beta-cypermethrin> D-allethrin.2. The sodium channel Nav1.2 a subunit gene scnllab of zebrafish is obtained by RT-PCR, the cDNA full-length of ? subunit gene scnllab is 5991bp, encoding 1996 amino acid residues. By Predicting, we found that the scnllab fragment contains typical structure of sodium channel a subunit, including four homologous domains, and each domain has six transmembrane a helix segments (S1?S6), including voltage receptors, selectively pore and the related domain of ion_trans association and channel activation or inactivation gate. Moreover, the eukaryotic expression plasmid pIRESpuro3-scn 1 lab were successfully constructed by cloning a subunit gene scnllab into vector pIRESneo3.3. The sodium channel Nav1.2 ? subunit gene scnlba of zebrafish is obtained by RT-PCR, the cDNA full-length of a subunit gene scnlha is 666bp, encoding 221 amino acid residues. By Predicting, we found that that the scnlba fragment contains typical structure of sodium channel ? subunit, including a single transmembrane domain, an extracellular Ig-like motif. Moreover, the eukaryotic expression plasmid pIRESneo3-scnlba were successfully constructed by cloning (3 subunit gene scnlba into vector pIRESpuro3.4. After the co-transfection of pIRESneo3-scnllab and pIRESpuro3-scnlba in HEK293 cells, the impact of deltamethrin and D-allethrin to zebrafish sodium ion channels has been detected by using the patch clamp technique, the results show that 100?M D-allethrin and 100?M deltamethrin had significant activation to zebrafish sodium ion channel currents, the inactivation of sodium channels was delayed, and the peak current was increased; morever, the activation of deltamethrin on sodium ion channel currents was greater than the activation of D-allethrin on sodium ion channel currents.