Toxicological Mechanism Of Silver Nanoparticles In Vivo And In Vitro And The Study Of Apex1-mediated BER Signaling Pathway

Silver nanoparticles（AgNPs）are widely used in industry,agriculture and medicine,potentially resulting in adverse effects on natural environment,animals,plants and human health.Studies have shown that AgNPs can cause developmental toxicity,cardiotoxicity,neurotoxicity and cytotoxicity and so on.Most studies have shown that the toxic mechanism of AgNPs was due to ROS accumulated.However,the toxic mechanism of AgNPs in vivo and in vitro is not comprehensive,and the response of organisms to stress caused by AgNPs is rarely seen.In this study,zebrafish was selected as the experimental model in vivo.As one of the important experimental models in vivo,zebrafish have many characteristics,such as high reproductive capacity,transparent embryo,small size,short sexual maturity time and high similarity with human genome.The survival rate of zebrafish embryos exposed to AgNPs at different concentrations under different treatment times were analyzed,and the concentration with the survival rate below LC₅₀ was selected for subsequent research.The developmental toxicity was researched after AgNPs treatment for 72 h.This study found that the AgNPs could decline hatching rate,body length and weight,cause spinal curve,induce cardiac toxicity（abnormal heartbeat,pericardial edema,yolk cyst）,lead to neurotoxicity（deformities,small head,small eyes,spinal neurons abnormal and behavior decreased）.Meanwhile,in this study,the human embryonic kidney cells（HEK293T cells）were used as the model in vitro study.AgNPs exposure can reduce cell activity and affect cell adhesion.Further RNA-seq（transcriptome sequencing）was used for analysis the transcriptional level of zebrafish treated with AgNPs for 72 h,gene ontology（GO）and kyoto encyclopedia of genes and genomes（KEGG）enrichment analyses showed that the differentially expressed genes（DEGs）were enriched in cell cycle,DNA replication,base excision repair,cellular senescence,MAPK,focal adhesion,neuroactive ligand-receptor interaction,ATP binding,DNA replication initiation,ion transport and cell junction in the AgNPs-treated group.Studies have found that AgNPs acute exposure could interfere MAPK and neuroactive ligand-receptor interaction signaling pathway,inhibit cell cycle,DNA replication and BER signaling pathway,affect cellular senescence and induce senescence phenotype of zebrafish or HEK293T cells.Analysis of focal adhesion pathway zebrafish showed that the transcription level of focal adhesion pathway-related genes was induced in the low AgNPs concentration groups,while it was inhibited in the higher AgNPs concentration groups.Focal adhesion plays an important role in heart development.Acute exposure to AgNPs may induce cardiotoxicity by interfering with the transcription level of focal adhesion signaling pathway.AgNPs acute exposure could induce the development toxicity of zebrafish by interfering with MAPK and neuroactive ligand-receptor interaction signaling pathway,inhibiting cell cycle and DNA replication,influencing cellular senescence,focal adhesion and BER signaling pathway.The toxicity mechanism of AgNPs was studied in vitro and in vivo We also found that AgNPs exposure could result in the accumulation of reactive oxygen species（ROS）and malondialdehyde（MDA）,the inhibition of superoxide dismutase（SOD）,catalase（CAT）,and the downregulated expression of SOD,CAT-related genes.Transcriptome analysis showed that down-regulated DEGs were enriched in the oxidative phosphorylation signaling pathway under 4 mg/L AgNPs treatment.Analysis of mitochondrial respiratory chain in vivo and in vitro showed that AgNPs acute exposure could inhibit the activity of mitochondrial respiratory chain complex I-V and down-regulated the expression of mitochondrial respiratory chain complex-related genes.In addition,AgNPs exposure could induce DNA damage and apoptosis in vivo and in vitro,and the expression of mitochondrion-mediated apoptosis signaling pathway-related genes or proteins,such as bax,bcl2,caspase-3,caspase-9,Bax and Bcl2 was significantly regulated after AgNPs exposure.AgNPs acute exposure could induce excessive accumulation of ROS,cause oxidative stress,lead to DNA damage,and induce mitochondrial dysfunction to mediate apoptosis.The accumulation of ROS can induce oxidative stress levels,induce DNA damage repair responses to maintain the organism stability.The RNA-seq analysis found that the transcription levels of apex1（key gene in the BER pathway）and BER downstream genes（xrcc1,lig3,pcna,fen1 and polb）were significantly down-regulated under AgNPs-treated.Meanwhile,fluorescence quantitative PCR analysis showed that the BER pathway-key genes（apex1,lig3,fen1）were significantly inhibited after AgNPs-treated in vivo and in vitro.As one of the main repair pathways of ROS induced oxidative DNA damage,BER signaling pathway plays an important role in organism response to external stress.Apex1regulates BER signaling pathway by controlling protein levels of many enzymes involved in damage repair.We hypothesized that AgNPs could affect DNA damage repair function and induce developmental toxicity,cytotoxicity through apex1 mediated BER signaling pathway.Therefore,CRISPR/Cas9 technology was used to construct the apex1 gene mutants of zebrafish and HEK293T cells.Then the effects of apex1 knockdown on developmental toxicity and oxidative stress were analyzed.Following by the molecular response of BER signaling pathway with the apex1^+/-zebrafish or APEX1KD HEK293T cells under AgNPs exposure were studied.It was found that the mutation of apex1 gene in vivo and in vitro could induce development toxicity,cause oxidative stress and inhibit antioxidant enzymes.AgNPs exposure could aggravate developmental toxicity,oxidative stress levels and up-regulated the protein levels of p53 to induces apoptosis in apex1^+/-zebrafish or APEX1KD HEK293T cells.Analysis of BER signaling pathway showed that the transcription level and protein expression of BER pathway were significantly up-regulated in apex1^+/-zebrafish or APEX1KD HEK293T cells under AgNPs-treated.In conclusion,we hypothesized that AgNPs inhibited the transcription level of apex1 to regulate the expression of BER signaling pathway genes and affect DNA damage repair function in wild-type zebrafish or HEK293T cells.In apex1^+/-zebrafish or APEX1KD HEK293T cells,AgNPs-treated may induce adaptive responses of BER signaling pathway in response to oxidative stress-induced damage.Due to the sensitivity of the apex1 mutated in BER pathway to oxidative stress,it can be used as an experimental model for oxidative stress research,which is helpful to further study the stress response of the body under different oxidative stress.