Early Stage Toxicity Assessment Of Indole-3-Carbinol In Zebrafish Embryos And Its Effects On Anthroslerosis In Hyperlipidemia Zebrafish Larvae

Objective:Evaluate the toxicity effects of indole-3-carbinol（I3C）on the early life stage development of zebrafish embryos.Methods:Different concentration I3C were added to 4 hour post fertilization（4hpf）healthy zebrafish embryos,Holt buffer solution was used as negative control group,Dimethyl sulfoxide（DMSO）was used as solute control group,the final DMSO concentration was 0.1（v/v%）.Early life stage toxicity drugs（Rapamycin and Simvastatin）were used as positive drug controls.Mortality of zebrafish embryos at 120 hpf was counted and median lethal concentration(LC₅₀)was calculated by bliss method.1/4LC₅₀0 and/or 1/2 LC₅₀0 concentration drugs were used to evaluate the early life stage toxicity at 72 hpf or 96 hpf.（1）The general toxicity:hatching rate,spinal curvature,Yolk-sac abnormality,hemorrhage,eyes edema,uninflated swim-bladder were observed under stereoscopic microscope and body length was measured by Image J software;（2）The cardiotoxicity:heart rate,Pericardial Edema were observed under stereoscopic microscope,Sinus venosus–bulbus arteriosus Distance was measured by Image J software;（3）The neurotoxicity:spontaneous movement and tactile sensitivity test were observed under stereoscopic microscope;（4）Cell apoptosis was detected by acridine orange staining.Results:（1）Median lethal concentration:LC₅₀0 for I3C,rapamycin and simvastatin at 120hpf were 9.9332?μmol/l,1.6161μmol/l and 0.0051μmol/l,respectively.（2）The hatching rate of fertilized eggs in I3C and simvastatin groups at 72hpf were not significantly different from that of the control group.However,the hatching rate of fertilized eggs treated with rapamycin were significantly lower than that of the control group（P<0.01）.No significant morphological abnormalities and hemorrhage were observed in I3C treated groups.With the increase of dose,rapamycin and simvastatin retarded the development of embryos significantly including body length decrease,spinal curvature,Yolk sac absorption delay and uninflated swim bladder.Hemorrhage was only observed in rapamycin treated groups.（3）The cardiotoxicity:There were no significant differences in the heart rate,pericardial edema,SV–BA distance between the I3C treated groups and the control group at 96hpf,whereas rapamycin and simvastatin treated groups induced bradycardia,pericardial edema and incresed SV–BA distance indicating severe cardiotoxicity at early life stage.（4）The neurotoxicity:There were no significant differences in spontaneous movement at 19-27hpf and tactile sensitivity test at 72hpf between I3C,rapamycin,simvastatin treated groups and the control group.（5）The cell apoptosis in I3C group at 96hpf were not significantly different from that of the control group,However,the cell apoptosis treated with rapamycin or simvastatin were significantly increase than that of the control group.Conclusions:Comparison with the early developmental toxicity positive control drugs（rapamycin and simvastatin）,We find that I3C has less toxicity effects on the early development of zebrafish embryos.The positive control drugs simvastatin has the most toxic effect on zebrafish embryos,it can lead zebrafish embryos to death at nanomole concentrtion.However,I3C do not induce retardation,malformation,hemorrhage,pericardial edema,bradycardia,neurotoxicity and cell apoptosis in zebrafish embryos even at micromole concentration.It suggests that I3C can be used in the early stage of development,providing supplementary data for the toxicological effect and safety evaluation of I3C.Objective:This study investigated the effects of indole-3-carbinol on the atheroslerosis in hyperlipidemia zebrafish larvae and its mechanisms.Methods: 5dpf zebrafish larvae were randomly divided into four groups including 4% high-cholesterol diet（HCD）group;4%HCD+DMSO group;4%HCD+I3C 2.5μmol/L group and 4%HCD+I3C 5.0 μmol/l group.The larvae were sacrificed two weeks later.Confocal image analysis and oil Red O staining were used to analysis vascular lipid accumulation.Lipid content（T-CHO,LDL-C,HDL-C,TG）in zebrafish tissue homogenate were detected according to the kits instruction（Nanjing Jiancheng Biological Engineering Institute）.Antioxidant effects including the total antioxidant capacity,total superoxide dismutase activity,glutathione peroxidase activity,inhibition of hydroxyl free radical capacity and the content of malondialdehyde in zebrafish tissue homogenate were analyzed according to the kits instruction（Nanjing Jiancheng Biological Engineering Institute）.Expression levels of autophagy related proteins including: LC3-II/I ratio,p62/SQSTM1,cathepsin D and expression level of Beclin-1,bcl-2,h Vps34,Akt,p-akt（Ser473）,m TOR and p-mtor（Ser2448）in the Class III PI3K/Akt/m TOR signaling pathway were analyzed were analyzed by Western blotting.Results: We found that lipid deposition on vasculature was dose dependently decreased in the I3 C treated groups as compared with that in the4%HCD control group（p<0.01）.Comparing with the 4%HCD control group,I3C-treated did not affect the T-CHO,LDL-C,HDL-C,TG content in zebrafish tissue homogenate（p>0.05）.The antioxidant activity of the I3C-treated groups were significantly enhanced compared with the control group,and the total antioxidant capacity,total superoxide dismutase activity and glutathione peroxidase activity were significantly increased while the content of malondialdehyde in the tissues was reduced.In addition to the antioxidant effect,we demonstrated that I3 C inhibited lipid deposition by inducing autophagy,which was identified by the enhancement of LC3-II,beclin-1,h Vps34 and m-cathepsin D as well as the reduction of P62,Bcl-2,Akt,p-Akt,m TOR,and p-m TOR in HCD fed zebrafish larvae（p<0.05）.Conclusions: I3 C decreases the lipid accumulation on blood vessels wall in hyperlipidemia zebrafish larvae and shows a dosage-dependent relationship.The mechanisms of this effects may partly be related to its increasing the antioxidant activity and promoting the autophagy via the PI3K/Akt/m TOR pathway,instead of lipid-lowering effect.it could be suggested that I3 C has a high potential to prevent and treat atherosclerosis.