Protective Effects Of Different Molecular Weight Of Sodium Alginate Against Developmental Toxicity Induced By Cd²⁺ And Pb²⁺ In Zebrafish Embryos

Sodium alginate is a polysaccharide extracted from aquatic macromolecules such as kelp and laver,and is widely used in many fields such as food,medicine,and chemical industry.It has been confirmed that sodium alginate has a wide range of physiological functions that depend its molecular weight.In this work,the alginic acid was degraded by acid hydrolysis and/or oxidation process,and the brown algae polysaccharides with different molecular weight were obtained and their protective effects on the developmental toxicity of Cd²⁺and Pb²⁺in zebrafish embryos were investigated.?1?The sodium alginate was degraded by hydrochloric acid?HCl?,hydrogen peroxide?H₂O₂?alone or combined together.The degradation degree was evaluated by the viscosity measurement.It is found that prolonging the degradation time can not improve the degradation of H₂O₂,and can significantly improve the degradation of HCl;The combination of HCl-H₂O₂ can accelerate the degradation of alginic acid.The functional group of sodium alginate was determined by infrared chromatography.The structure of sodium alginate did not change significantly after degradation.Different degradation conditions only interrupted the internal glycosidic bond of alginic acid to form low molecular weight polysaccharides.The sodium alginate was separated and degraded by a dextran G-50 gel column.The molecular weight of the isolated product was determined by HPLC-ELSD method,and three polysaccharide fractions with average molecular weights of 1575 D,7075 D and 61518 D were obtained.?2?The developmental toxicity evaluation model of Cd²⁺and Pb²⁺exposure was established by using 6 hours?6 hpf?zebrafish eggs after fertilization and exposure to incubation solution containing cadmium chloride(Cd²⁺)and lead nitrate(Pb²⁺).The results showed that with the increase of Cd²⁺and Pb²⁺concentrations,the mortality and malformation rate of zebrafish embryos increased,and the hatching rate decreased,both of which showed obvious developmental toxicity.The median lethal concentration(LC₅₀)of cadmium chloride to zebrafish embryos was 15.86 mg/L,and the LC₅₀ of lead nitrate to zebrafish embryos was 125 mg/L.The isolated sodium alginate with different molecular weights was used in combination with Cd²⁺and Pb²⁺for zebrafish embryos.The results showed that the highest molecular weight of sodium alginate 1575 D,7075 D and 61518D had the strongest protective concentration against zebrafish-induced zebrafish embryo death.5 mg/L,10 mg/L and 50 mg/L,respectively,only 1575 D alginic acid had a protective effect on Pb²⁺-induced zebrafish developmental toxicity.The optimal protective concentration was 25 mg/L.No significant protective effects were observed for 7075 D and 61518 D sodium alginate.?3?The apoptosis of embryos was observed by acridine orange?AO?staining,the level of reactive oxygen species?ROS?in embryos was determined by DCFH-DA probe method,and the level of lipid peroxide malondialdehyde?MDA?was determined by TBA colorimetry.Superoxide dismutase?SOD?activity,Cd²⁺,Pb²⁺content in whole eggs and intact embryos were determined by ICP-MS.The results showed that both Cd²⁺and Pb²⁺could induce apoptosis of zebrafish embryos,and the levels of MDA and ROS increased,and the activity of SOD decreased.Different molecular weight alginic acid can reduce the enrichment of Cd²⁺in whole eggs and embryos of zebrafish,improve the apoptosis of embryonic cells induced by Cd²⁺,decrease the levels of MDA and ROS,increase the activity of SOD,especially the activity of low molecular weight alginic acid.Low molecular weight alginic acid has similar intervention effects on Pb²⁺-induced zebrafish embryo developmental toxicity.The results showed that both Cd²⁺and Pb²⁺could induce embryonic developmental toxicity and oxidative stress damage in zebrafish.After degrading,1575 D low molecular weight sodium alginate could significantly improve the accumulation of Cd²⁺and Pb²⁺in zebrafish eggs and embryos,and significantly improve embryonic oxidation.Stress level,reduce apoptosis,protect against zebrafish embryo developmental toxicity induced by Cd²⁺and Pb²⁺.