| Background and purpose: In recent years,Graphene oxide(GO)has been widely used in multiple fields including clinical disease treatment,diagnosis,novel drug delivery carriers and biosensors.Due to the widespread use of GO materials,their release into the environment poses a threat to human and aquatic health.The size of nanomaterials significantly affects many cellular parameters,such as oxidative stress,immune response,and cell viability.The size and surface oxidation of GO may affect its properties and related toxicity on different models.Therefore,it is very important to determine the relationship between GO of different sizes and its toxic effects on various organisms.Compared with other vertebrate models,zebrafish is more sensitive to water pollution,has many advantages such as short growth cycle,low cost,and is 87% homologous with the human genome,so the study results can be extrapolated to humans.In this study,zebrafish was used as the study model and we evaluated the toxic effects of different sizes of GO(small size 50-200 nm,S-GO;medium size < 500 nm,M-GO and large size > 500 nm,L-GO)on the embryonic development,swimming behavior and heart rate of zebrafish and cardiotoxicity.Methods: Four hours after fertilization(4 hpf),zebrafish embryos were exposed to different-sized GO particles solutions at different concentrations(0,0.1,1,10,and 100 mg/L)for 120 h,during which the morphological changes of zebrafish were observed and recorded.The survival rate of embryos and larvae was determined after 48-and 120-h exposures to three different size GO solutions,respectively.The hatching rate of the surviving embryos was recorded after 48-,54-,72-,and 96-h exposures.When exposed to 96 h,the heart rate of zebrafish was measured.The body length,blood flow and swimming behavior of zebrafish were recorded after 120-h exposure,respectively.When exposed for 120 h,the transgenic AB strain zebrafish(Lyz: Ds Red,AB strain)with red fluorescent labeled neutrophils and macrophages was used to observe the effect of different sizes of GO on the number of neutrophils and macrophages of zebrafish.After a 120-h exposure,zebrafish(AB)embryos were collected,the changes of nitric oxide synthase(i NOS),acetylcholinesterase(ACh E),total superoxide dismutase(T-SOD)and cysteine protease-3(caspase-3)were detected.The gene expression of apoptotic genes caspase-3,cysteine protease-9(caspase-9),B lymphocyte tumor-2(bcl-2)and bax were determined by real-time fluorescent quantitative PCR.In addition,m RNA expression of genes associated with heart development ?-catenin protein(?-catenin),t-box 6(tbx6)and t-box 16(tbx16)were quantified.Results:(1)All three different GO sizes were collected in the eyes,yolk sac,heart,and tail blood vessels of larvae.At the same time,the developmental toxicity of zebrafish was induced,and the embryo of zebrafish appeared deformity in different degrees,mainly including pericardial edema,yolk sac edema,tail bending,etc.Moreover,the probability of deformities was higher under high concentration exposure.When the GO concentration was 100 mg/L,the incubation rate of S-GO embryos was inhibited(P < 0.05)in the early stage of embryo development(48 h).When the concentrations of M-GO and L-GO were 0.1 and 10 mg/L respectively,the embryo incubation rate was significantly reduced(P < 0.05).When exposed to high concentration(100 mg/L)for 48 and 120 h,the survival rate of zebrafish in the S-GO and L-GO groups was significantly reduced(P < 0.05),while that in the M-GO group was significantly reduced only at 120 h(P < 0.05).When exposed to high concentration(100 mg/L)for 120 h,all three GO sizes significantly inhibited the body length of larvae(P < 0.001).(2)At low concentrations(0.1,1 mg/L),the heart rate of zebrafish in M-GO and L-GO induced heart rate of zebrafish firstly increased(P < 0.05 or P < 0.001),then the heart rate and blood flow were reduced(P < 0.05 or P < 0.01)at high concentrations(100 mg/L).And the abnormal expression of heart development related genes was induced,the expression of both ?-catenin and tbx6 genes in S-GO and M-GO groups was significantly increased(P < 0.05 or P < 0.01),while the expression of tbx6 in M-GO and L-GO groups was up-regulated(P < 0.05).(3)GO promoted the expression of apoptosis-related genes,among which caspase-3,caspase-9,bax and bcl-2 genes in the S-GO group were significantly up-regulated(P < 0.05 or P < 0.01)under high concentration(100mg/L).The expression of caspase-3,bax and bcl-2 in M-GO group were significantly up-regulated(P < 0.05).The expression of bax and bcl-2 in the L-GO group was up-regulated(P < 0.05 or P < 0.01).It also promoted the increase of apoptotic protease caspase-3(P < 0.05 or P < 0.01).(4)GO induced the increase of macrophage number and the activity of i NOS enzyme,in which the number of macrophages in S-GO,M-GO and L-GO increased significantly(P < 0.05)in high concentration(100 mg/L),and the number of macrophages in M-GO and L-GO increased significantly(P < 0.05)in 10 mg/L.In the GO exposure group(0.1-100 mg / L),the i NOS enzyme activity in L-GO increased significantly(P < 0.05 or P < 0.01).In the concentration of 1-100 mg/L,the i NOS enzyme activity in S-GO also increased significantly(P < 0.05 or P < 0.01).(5)Results of detection of zebrafish swimming behavior:(5.1).in the visible light experiment,the activity and swimming distance of fish larvae were increased in the low-concentration groups(0.1,1,and 10 mg/L),while the activity and swimming distance of fish larvae were decreased(P < 0.01 or P < 0.001)in the high-concentration group(100 mg/L)GO treatment group.(5.2).in thedetermination of the time ratio of different swimming speed,compared to the control group,the medium-speed and high-speed time ratios increased in the low concentration groups(0.1,1,10 mg/L),while a low-speed time ratio was induced at the high GO concentration(100 mg/L).(5.3).in response to alternating light-dark photo periods experiment,S-GO activity was significantly reduced(P < 0.05)under high concentration(100 mg/L)in both dark and light environments.In the first and third light stages,M-GO activity was also significantly reduced(P < 0.05)under exposure to high concentrations(100 mg/L).When the GO concentration was 100 mg/L,the activity of the L-GO group decreased significantly(P < 0.05)in the whole light stage.The activity of fish larvae increased significantly in the dark than in the light environment,in addition,in the light environment,the activity of fish larvae decreased gradually than in the previous light period.And ACh E enzyme activity was also abnormally expressed.(6)When the concentration of GO was 100 mg/L,S-GO has more siginifcant toxic effects on behavior,body length and expression of apoptosis related genes than M-GO and L-GO.However,the toxicity of M-GO and L-GO was greater than that of S-GO in terms of hatching rate,heart rate and expression of related enzyme protein in zebrafish embryos exposed to low concentration(0.1 mg/L).Conclusion: Exposure to different-sized GO solution can cause concentrationdependent toxicity to the development of zebrafish embryos and larvae.Generally,the high GO concentration of 100 mg/L significantly changed almost all of the tested endpoints,and the changes of morphology,physiology,biochemistry,and behavior of zebrafish reveal that exposure to GO may induce oxidative stress,immunotoxicity,neurotoxicity,cardiotoxicity and apoptosis of zebrafish.Our results revealed that the comprehensive toxicity of GO was not only closely related to lateral size but also to the exposure concentration,time window,and the tested endpoints. |
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