低剂量全氟辛酸对雄性黑斑蛙生殖毒效应及机理研究

针对新型环境有机污染物全氟辛酸(PFOA)对两栖动物的毒效应问题,以黑斑蛙(Rana nigromaculata)为实验对象,研究了低剂量PFOA暴露对雄性黑斑蛙生殖毒效应及机理.结果表明,在0.001～1mg.L-1低剂量体外暴露20d条件下,PFOA能够显著诱导黑斑蛙精子发生畸形,相对于对照组,精子畸形率呈现明显的剂量-效应关系.酶联免疫试剂盒检测发现,除0.001mg.L-1诱导组外,0.01、0.1和1mg.L-1暴露组的血清雄性激素睾酮和雌性激素雌二醇含量分别显著降低(p<0.01,F=288.7)和上升(p<0.01,F=289.0),说明PFOA作用下黑斑蛙体内性激素水平受到改变.采用荧光定量PCR技术进行进一步的分子生物学检测,发现与性激素水平密切相关的两个基因P450芳香化酶和类固醇激素合成因子(SF-1)均增强表达(0.001mg.L-1诱导组,p<0.05;0.01、0.1和1mg.L-1暴露组,p<0.01,F=140.8).全氟辛酸对两栖类动物的雄性生殖毒效应机理主要是通过介导与性激素分泌相关的两个重要基因P450芳香化酶和类固醇激素合成因子,使其表达上调,从而一方面抑制睾酮的产生,另一方面促进性激素雌二醇的分泌.在0.001mg.L-1的体外暴露剂量下,PFOA就能够明显诱导黑斑蛙精子产生畸形毒性及增强P450芳香化酶和SF-1基因的表达(p<0.05),因此,为保护两栖动物种群,在制订PFOA地表水环境质量标准时应当考虑其下限不能低于0.001mg.L-1.

Reproductive toxic effects and mechanisms of the male frog Rana nigromaculata induced by low dose PFOA

To disclose the toxic effects and mechanisms of perfluorooctanoic acid (PFOA) on amphibians, the male reproductive toxicities of the frog (Rana nigromaculata) induced by low dose PFOA were investigated. After the exposure to PFOA with the concentrations of 0.001, 0.01, 0.1 and 1 mg·L^-1 for 20 days respectively, sperms malformation of Rana nigromaculata was found and the rates were in dose-dependent manners. Enzyme-linked immunosorbent assay showed that with the increasing of PFOA exposure levels, the concentrations of testosterone in serum significantly decreased (p<0.01, F=288.7), while the concentrations of estradiol significantly increased (p<0.01, F=289.0). It was strongly demonstrated that PFOA can change the levels of sex hormones in the frog Rana nigromaculata. Further analysis by RT-PCR clearly proved that the expressions of P450 aromatase gene and steroidogenic factor-1 (SF-1) gene which were closely related to the production of sex hormones in the frog were significantly upregulated. These results firstly reported that PFOA can induce male reproductive toxicity in amphibian. It was also proved that the upregulated expressions of P450 aromatase and SF-1 can inhibit the production of testosterone and promote the secretion of estradiol. The statistics analysis results showed that even in the low dose of 0.001 mg·L^-1, PFOA still can induce sperm malformation and the upregulated expressions of P450 aromatase and SF-1 gene. Thus, it was suggested that for the protection of amphibians the limitation of PFOA concentration for surface water quality criteria should not be less than 0.001 mg·L^-1.