全氟辛烷磺酸类物质对剑尾鱼Cu/Zn-SOD及相关应激基因表达的影响

The effects of perfluorooctane sulfonate on Cu/Zn-SOD and stress-related gene expression in swordtails(Xiphophorus helleri)

  • 摘要: 研究全氟辛烷磺酸类物质(PFOS)暴露对剑尾鱼肝脏Cu/Zn-SOD及相关应激基因表达的影响,探讨筛选导致体内氧化应激反应的敏感生物标志物,并首次克隆和分析剑尾鱼Cu/Zn-SOD的cDNA全序列。实验将剑尾鱼随机分为5组,包括对照组和3.5、7.0、14.0和28.0 mg/L等4个PFOS实验暴露组,同时设置平行组。定量测定了24 h、48 h、96 h和14 d肝脏组织中的Cu/Zn-SODHSP70-1、HSP70-2和GST mRNA表达水平的变化,成功克隆包含794 bp核苷酸和编码154个氨基酸的剑尾鱼Cu/Zn-SOD的cDNA全序列。通过与其他相关鱼类Cu/Zn-SOD的核苷酸序列和氨基酸序列相似性比较发现,核苷酸和氨基酸序列相似性分别达到77%~83%和79%~88%。高浓度PFOS暴露后,剑尾鱼肝脏中与应激相关的各种基因发生不同程度的变化。通过RT-PCR技术检测Cu/Zn-SOD mRNA表达在剂量效应上不显著,Cu/Zn-SOD mRNA表达和SOD活性之间并没有相关性,推断是其他类型的SOD基因在PFOS暴露下发挥更为重要的作用。随着暴露时间的持续延长,Cu/Zn-SOD基因的转录水平变化趋势与SOD活性变化及HSP70-1 mRNA表达相一致,表明HSP70通过提高剑尾鱼体内SOD水平以保护机体免受氧化损伤。HSP70-2 mRNA水平比HSP70-1 mRNA表达更加敏感。HSP70-2变化趋势与Cu/Zn-SOD mRNA表达相关性不强,其相关机制有待进一步研究。GST mRNA表达呈不断上升趋势,这与Nrf2信号传导途径有关,也可能是机体在应对PFOS进入鱼体后的一种防御措施。剑尾鱼在体实验表明,PFOS能够诱导肝脏氧化应激反应,GST mRNA和HSP70-2 mRNA表达可以作为PFOS暴露的敏感生物标志物。

     

    Abstract: The effects of perfluorooctane sulfonate(PFOS) exposure on hepatic Cu/Zn-SOD and stress-related gene expression in swordtails(Xiphophorus helleri) were studied in order to screen the sensitive biomarkers induced oxidative stress in vivo.We cloned and analyzed the swordtail fish Cu/Zn-SOD cDNA of full sequence.Swordtails were randomly divided into five groups, with one control group and four experimental groups(3.5, 7.0, 14.0 and 28.0 mg/L PFOS).Parallel experimental groups were also established.The Cu/Zn-SOD, HSP70-1, HSP70-2 and GST mRNA expression levels in fish livers were determined after 24 h, 48 h, 96 h, and 14 d exposure.The X.helleri Cu/Zn-SOD cDNA was cloned, which had 794 bp nucleotides, encoding 154 amino acids.The comparison among the similarity nucleotide sequence and amino acid sequence of Cu/Zn-SOD in other related fishes showed that nucleotide and amino acid sequence similarity were 77%-83% and 79%-88%, respectively.After exposure to high concentrations of PFOS, a variety of stress-related genes in X.helleri liver changed in different degrees.The expression of Cu/Zn-SOD mRNA in the dose-effect was not significant by RT-PCR, and with no correlation between the SOD activity and the expression of Cu/Zn-SOD mRNA.It may be other types of SOD gene that play more important role under the PFOS exposure.With the exposure time to increase continuously, Cu/Zn-SOD gene changes were consistent with the SOD activity and the expression of HSP70-1 mRNA.Thus HSP70 by increasing the level of swordtail fish SOD protected the body from oxidative damage.HSP70-2 mRNA was more sensitive than the levels of HSP70-1 mRNA expression.The changes of HSP70-2 were not relevant with Cu/Zn-SOD mRNA expression.The underlying mechanism needed further study.GST mRNA expression was constantly increasing, which is related with the Nrf2 signal transduction pathways, and it may also be a form of defense in fish when PFOS entered the body.Swordtails in vivo experiments showed that PFOS could induce oxidative stress in the liver.The expression of GST mRNA and HSP70-2 mRNA could act as sensitive biomarkers of PFOS exposure.

     

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