The Toxic Effects Of ZnSe/ZnS Quantum Dots On The Embryonic Development Of Gobiocypris Rarus

Quantum dots?QDs?,as a new type of nanomaterial,are widely used in the fields of physics,materials,biology and so on.At present,the most studied are Cd-containing QDs.With the widespread use of quantum dots,it will inevitably enter the water environment,which will have a toxic effect on aquatic organisms and ultimately harm human health.Therefore,it is very important to evaluate the safety of quantum dots.Some researchers have pointed out that ZnSe QDs are less toxic.Whether ZnSe/ZnS QDs produce toxic effects on organisms and whether ZnSe/ZnS QDs are safer than Cd QDs have not been reported.In this study,rare minnows?Gobicypris rarus?were used as experimental objects.The early developmental toxicity of ZnSe/ZnS QDs on rare minnnows'embryos was investigated.Method:This experiment explored the toxic effects of ZnSe/ZnS QDs on the development of rare minnows'embryos.The experimental subjects were fish of unique species in rare minnows.Experimental concentrations were set for the control group,50,100,200,400,and 800 nmol/L QDs concentration groups.The effects of different concentrations of ZnSe/ZnS QDs on embryonic development,spontaneous movement frequency,heart rate,hatching rate,survival rate,total length and malformation rate of embryos at 12,24,48,72 and 96 hpf?hours post fertilization?were examined,through water immersion exposure.Real-time quantitative PCR was used to detect mRNA expression levels of development-related genes?Wnt8a?Mstn?Vezf1?Cp?Gfap?Foxl2?and oxidative stress-related genes?Hsp70,Cyp1a,Cat,Gpx?.At the same time,we measured the enzyme activity of SOD,GPX,Ca²⁺-ATP and the content of MDA.Meanwhile we also detect the extent of 96hpf DNA damage by comet assay.The experiment results showed that the exposure to 36hpf,the frequency of autonomous exercise in each concentration group was lower than that of the control group which is 7.58±3.92 times/min,but the difference was not significant?p>0.05?.Exposure to 48 hpf,800nmol/L QDs concentration group heart rate was 44.17±1.75times/min,which is significantly lower than the control group 52.33±0.67 times/min?p<0.05?.Using probability unit method,the LC₅₀ of 72hpf of rare minnow embryos was 1418.683nmol/L.Exposure to 96 hpf,the hatching rate,body length and deformity rate of embryos in QDs groups from 100 to 800nmol/L were significantly different from those in the control group?p<0.05?.At the same time,they caused a variety of malformations,such as pericardial sac edema,spine bending,tail bending,yolk sac edema and so on.At same time,exposure to 96hpf,the survival rate of each QDs group was lower than that of the control group?97.08±0.80%?,but the difference was not significant?p>0.05?.The Ca²⁺-ATPase activity decreases with the increase of ZnSe/ZnS QDs concentration,and the transportation of Ca²⁺is hindered.Ca²⁺accumulates in cells and causes abnormal skeletal development.In addition,there was no significant difference in the expression of Mstn between high-dose group and control group when it exposed to 72hpf,but the Wnt8a level was significantly up-regulated?p<0.01?compared with the control group,resulting in inconsistent growth rate of bone and muscle,caused bending of the embryonic spine.With the increase of exposure concentration of ZnSe/ZnS QDs,the expression of Vezf1 and the activity of Ca²⁺-ATPase were down-regulated.This shows that ZnSe/ZnS QDs produced some damage to the development of the rare minnow embryo heart.At the same time,they exposed to 48hpf.The heart rate of each concentration group was higher than that of the control group.It could be the reason of the embryo is stimulated by the external environment.Exposure to 12hpf,there was no significant difference in SOD activity and Cat expression in concentration groups compared to the control group,maintaining a certain degree of synchronization,whereas the Gpx expression levels in the 100 and200 nmol/L QDs groups were significantly higher than those in the control group.It is possible that SOD and CAT remove ROS in advance,this lead to a recovery of the embryos'oxidative stress and induce the production of GPX.With the extended exposure time,the GPX enzyme activity in the high-concentration QDs groups?400and 800 nmol/L?was significantly activated,and SOD and CAT enzymes were synergized to remove excess ROS from the embryos.Exposed to 24hpf,SOD activity and MDA content were changed,but there was no significant difference compared with the control group.At this time,the expression levels of Hsp70 and Cyp1a were significantly different from the control group?p<0.05?.It could be the ZnSe/ZnS QDs were the first to induce the expression of antioxidant genes,indicating that the antioxidant enzyme activity and the expression of antioxidant gene mRNA levels were not synchronized.Exposure to 48hpf,the result showed no significant change in SOD activity in QDs groups compared with that in the control group.Both MDA content and Olive tail monment were significantly changed in QDs groups compared those in the control group.At the same time,the mRNA expression of Hsp70,Cyp1a,and Cp in the 800nmol/L group were significantly different from those in the control group?p<0.05?.It could be the reason of ROS was converted into H₂O₂ by SOD,SOD was rapidly restored,and antioxidant-system-related enzymes and genes were rapidly activated.Exposure to 72hpf,the result showed no significant difference in SOD activity and MDA content in QDs groups compared with that in the control group.At this time,the expression of Hsp70 and Cyp1a in the 800 nmol/L group were significantly different from those in the control group?p<0.05?.It shows that when the embryos were subjected to oxidative stress,but the generated ROS did not exceed the body's own scavenging capacity.The antioxidant system maintains its homeostasis.Exposure to 96 hpf,the activity of SOD and the MDA content in the 800nmol/L QDs group were significantly different from those in the control group?p<0.05?,while the expression of Hsp70 and Cyp1a in the 200 nmol/L QDs group were significantly different from those in the control group?p<0.05?.Olive tail length of the high-concentration QDs group was significantly higher than that of the control group?p<0.05?.At this time,the expression level of cat in the 800nmol/L group and Gpx in50nmol/L groups was significantly higher than that in the control group?p<0.05?.The expression of Cp in each concentration groups was not significantly different from that in the control group,but the expression of Cp in the concentrations of 50,100,and400nmol/L QDs groups was lower than that in the control group.It may be that the embryos weren't hatched and the embryonic membrane had a protective effect on the embryos before 72hpf.The membrane restricts the entry of QDs into the embryo.The embryonic incubation loses the protection of the membrane after 72hpf and QDs directly contact embryos,causing changes in the structure of anti-oxidation protein and poor embryo antioxidant capacity.This shows that with the extended exposure time of QDs,the degree of oxidative damage in rare minnow embryos was increasing,resulting in a gradual increase in the malformation rate.In addition,with the increase of QDs exposure concentration,the tailing of embryonic nuclei becomes more serious,which indicates that QDs cause DNA damage to rare minnow embryos.It may be that QDs are small in size,penetrate the cell membrane and enter cells,Excessive ROS production eventually leads to embryonic DNA damage.The exposure of ZnSe/ZnS QDs to a certain degree of toxicity of rare minnows'embryos is mainly manifested in the decrease of heart rate,embryo incubation time delay,altered body length,pericardial edema,bent spine,tail flexion and so on.Changes in Ca²⁺-ATPase and development-related genes can lead to abnormal embryos and oxidative stress.This may be related to the size of the quantum dots,their adsorption,their enrichment,and the release of Se.