| Antidepressants accounting for a large proportion of pharmaceutical and personal care products(PPCPs) around the world, which are widely used in the medical industry. However, due to the excessive drug intake with random discharge,the active ingredients of antidepressants and their metabolites excreted from the body have been released into the environment. Many domestic and foreign literatures have reported the presence of antidepressants in various types of environment media,especially in the aquatic environment including the urban sewage and waste water,drinking water, surface water and groundwater. In addition, recent studies have shown that antidepressants can be bioaccumulated in organism, and thus the toxic effects of antidepressants on aquatic organisms has become a hot issue in the field of ecotoxicology. Amitriptyline, fluoxetine and mianserin are commonly used drugs, and typical representatives of tricyclics antidepressants(TCAs), selective serotonin reuptake inhibitors(SSRIs) and tetracyclic antidepressants(Te CAs), respectively, all of which have been detected in the aquatic environment previously. In the present study, exploring zebrafish(Danio rerio) embryos as a tested model, the toxic effects of these three antidepressants on zebrafish embryos/larvae during their early developing stages were evaluated after a short-term exposure to chemicals at environmentally relevant concentrations. The high-throughput transcriptome sequencing(RNA-Seq) and real-time quantitative PCR(q RT-PCR) methods were applied in this study. The results are shown as follows:(1) After a short-term(120 h) exposure to three antidepressants at levels of 0.1μg/L for amitriptyline, 10 μg/L for fluoxetine and 10 μg/L for mianserin, respectively,the body length of zebrafish larvae were significantly shortened in all three antidepressant treatment groups. But, no significant difference of the mortality and the hatching rate of zebrafish embryos/larvae was observed between the control and three treatment groups after exposure.(2) After sequencing of zebrafish RNA samples from the control and threeantidepressant treatment groups on an Illumina Hiseq 2500 platform, RNA-Seq data were analyzed following a data analysis method with reference genome. With quality control, clean reads were achieved for these four RNA samples and their numbers were 15,557,465, 14,979,480, 13,716,939 and 15,450,283, respectively; the mapped ratio of clean reads to the reference sequence was up to 77%; in addition, high saturation of sequencing results was shown, indicating complete coverage of expressed gene in all samples. Moreover, the differentially expressed genes(DEGs)between the control and antidepressants treatment groups were obtained, and the DEG numbers were 32 in amitriptyline treatment group(7 up and 25 down), 34 in fluoxetine treatment group(22 up and 12 down) and 130 in mianserin treatment group(64 up and 66 down). The number of DEGs in mianserin group was the most among three treatment groups and a part of these DEGs could be significant annotated to some GO classification items and KEGG pathways, which were involved in response to chemical stimulation processes, protein hydrolysis process, as well as the changes in various types of enzyme activity in GO classification, and meanwhile in KEGG pathway were associated with some major metabolism pathways including insulin signaling and citric acid cycle. In total, above results suggested that tetracyclic antidepressant mianserin might regulate gene expression in zebrafish larvae at the transcriptional level, and result in the toxic effect on organisms.(3) Fold changes of the expression of 15 selected DEGs from RNA-seq data were evaluated by q RT-PCR and the results indicated a high correlation with the results by RNA-Seq analysis which suggested the RNA-Seq data and analysis results were accurate. Furthermore, after a short-term(120 h) exposure to three antidepressants at environmentally relevant concentrations of 0.1, 1, 10 μg/L of amitriptyline, fluoxetine and mianserin, respectively, the gene expression levels of 6selected DEGs were examined by q RT-PCR, including prostaglandin D2 synthase gene(ptgdsb), dual specificity phosphatase gene(dusp5), two biological clock-related genes(nr1d1 and per2), and two early growth response genes(egr1 and egr4). The results showed that exposure to antidepressants reduced the expression of all thesegenes, implying that antidepressants might affect the nervous and reproductive systems and then down-regulate the ptgdsb expression, or might promote the cell apoptosis by reducing the expression of dusp5. In addition, they also might change the status of circadian rhythms of zebrafish by altering the expression of nr1d1 and per2,or impair the early development of zebrafish embryos by inhibiting the expression egr4 and egr1, which might lead to observed toxic effects on the zebrafish larvae.In summary, the present study revealed that the exposure to three tested antidepressants could significantly affect the early development of zebrafish embryos.The toxic effects of these three antidepressants are to some extend similar, showing similar suppressing effects on body length of fish larvae, as well as similar regulating trends of related gene expression at a transcriptional level. Those results implying that in real environmental scenarios, a long-term exposure to antidepressants at low and environmentally relevant concentrations might affect the early development of the nervous and reproductive systems and disturb the circadian rhythm in zebrafish embryos/larvae, leading to an adverse impact on ontogenetic process of fish which as a consequence would influence the fish populations. Therefore, results from the present study provide evidence for investigating the toxic effects and action mechanisms of pharmaceuticals, a category of emerging environmental organic pollutants, on aquatic organisms, and to propose that the ecotoxicity and risk of waterborne pharmaceutical exposure should be paid more attention. |
PDF Full Text Request