| With the continuous development of society,a large number of pharmaceutical products,such as tetracycline hydrochloride,indomethacin and bezafibrate,have been used for human diseases and livestock industry,and release into environmental media afterwards,which arouse people’s attention to drug contamination issues in the environment.This issue is also one of the major environmental issues facing humankind in the 21st century.Although these pharmaceuticals are used to cure human and animal diseases,they may cause harm to non-target organisms through direct or indirect touch.There have been many reports on the detection of tetracycline hydrochloride(TH),indomethacin(IM)and bezafibrate(BF)in various environmental media and their toxic effects on ecosystem,most of which toxicity researches are based on routine toxicological indicators such as hatchability,teratogenicity,and lethality currently.The research on the potential toxicity and functional mechanism of these pharmaceuticals in vitro and in vivo are still scarce.Therefore,in order to further analyze the toxicity of TH,IM,and BF,this research combined the following two methods to conduct in-depth studies:(1)Using Quantitative Structure Activity Relationship(QSAR)developed by the United States Environmental Protection Agency and based on ToxCast bioactivity evaluation tools to assess the toxicity of selected drugs;(2)By means of in vivo and in vitro toxicity tests reveal the toxicological mechanisms of three pharmaceuticals.The conclusions of this study are as follows:(1)According to the toxicity prediction results of tetracycline hydrochloride,indomethacin and bezafibrate by the iCSS ToxCast Dashboard,hit call value of TH was 7.08%and logAC50 value was-3.38 to 2.02 pM in terms of in vitro toxicity prediction,and it had an effect on nuclear receptors and cell cycle;IM had a hit call value of 2.71%and a logAC50 value of-1.62 to 1.98 μM,and it had toxic effects in various in vitro assays associated with cell cycle,DNAbinding,nuclear receptors and transporters,etc.;however,the hit call value of BF was 3.26%and the logAC50 value was 0.85 to 1.96μM,which only showed an effect on nuclear receptor activity.(2)HEK 293T cells were used as experimental subjects.Comprehensive analysis was performed using comet assay and single cell gel electrophoresis.We found that cell activity in the TH group was significantly decreased with increasing exposure concentration and time,and high concentrations of BF could reduce cells activity significantly.After exposure of the three compounds,damage degree of cells DNA was gradually increased with the increase of drug exposure concentration,and there was a significant concentration-dependent relationship.The detection of key genes in the Pi3K-Akt-mTOR pathway revealed that all drugs could activate the Pi3K-Akt-mTOR signaling pathway in vitro to some extent.(3)After the zebrafish embryos were exposed to different concentrations of TH,IM and BF,high concentration of TH,IM and BF significantly reduced the body weight of zebrafish,which all had a certain degree teratogenicity,and the three compounds caused high.mortality at high concentrations.The expression of key genes in metabolism pathways of three drug was analyzed.We found TH and BF groups may result in developmental toxicity of zebrafish.In the IM group,all genes except cypla and cyp3a65 were significantly down-regulated,which may be due to strong bioaccumulation of IM.The Pearson correlation coefficient between the expression level of drug metabolism-related genes and the developmental toxicity index was analyzed.It was found that the metabolic processes of the three drugs in zebrafish had influence on the development of zebrafish;From the aspect of spatiotemporal expression and tissue specificity,Whole In Situ Hybridization analyzed the developmental marker genes:myod,draculin and tfa expression levels,which displayed that IM and BF promoted zebrafish embryos muscle differentiation,all of pharmaceuticals had certain toxicity effects on hematopoietic development system of zebrafish. |
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