第一作者：Fangfang Li
通讯作者：李敏杰 副研究员/郭良宏 研究员
通讯单位：中国计量大学能源环境与安全工程学院
DOI:10.1016/j.jhazmat.2024.136947
全氟丁酸（PFBA）是一种新出现的污染物，研究显示它以低活性方式作用于经典雌激素受体（ERs），表现出雌激素效应。本研究旨在揭示PFBA通过雌激素相关受体γ（ERRγ）途径的雌激素干扰效应及其机制。在体内实验中，发现PFBA在斑马鱼卵巢中积累，导致卵巢损伤，干扰了性激素水平，并影响了与雌激素合成和卵泡调节相关的基因表达。在体外实验中，通过细胞增殖实验，发现PFBA在最低观察到有效浓度（LOEC）为10纳米摩尔时，能促进对雌激素敏感的子宫内膜癌细胞Ishikawa的增殖，这一浓度接近人类暴露水平。而细胞增殖可以被ERRγ拮抗剂GSK5182抑制。通过荧光竞争结合实验、分子对接和荧光素酶报告基因实验，证明PFBA可以直接与ERRγ结合，并在10纳米摩尔的LOEC下激活ERRγ的转录活性。此外，PFBA上调了ERRγ下游的增殖相关因子，并受到PI3K/Akt抑制剂LY294002的抑制，后者也抑制了PFBA诱导的细胞增殖。综上所述，研究结果表明，PFBA在与人相关的暴露浓度下具有雌激素效应，并揭示了PFBA通过ERRγ途径的一种新的雌激素效应机制。
Fig. 1. Histopathological observations of the ovary in zebrafish after exposure to PFBA for 28 days. (A) Control; (B-D) PFBA 10, 100, 1000 μg/L; (E) The percentage of stage Ⅰ - Ⅳ oocytes in ovary. 1: perinucleolar oocyte (PO), 2: cortical alveolar oocyte (CAO), 3: early vitellogenic oocyte (EVO), 4: vitellogenic oocyte (VO), AF: atretic follicle. Scale bar: 100 µm. * , p < 0.05 and * *, p < 0.01 indicated significant differences between the control group and treatment groups.
Fig. 2. Estrogen-related hormones levels in serum and genes levels in ovary of zebrafish. (A) the level of E2; (B) the level of T; (C) the level of VTG; (D) E2/T ratio; (E) Heat map of genes expression. Red color means up-regulation and blue color means down-regulation. * , p < 0.05 and * *, p < 0.01.
Fig. 3. Estrogen effects of PFBA on Ishikawa cells and transcriptional activity of ERRγ. (A) Cell proliferation of Ishikawa cells. (B) Transcriptional activity of ERRγ. (C) Cell proliferation of Ishikawa cells after co-exposure to GSK4716 and GSK5182. (D) Cell proliferation of Ishikawa cells after co-exposure to PFBA and GSK5182. GSK4716, ERRγ agonist. GSK5182, ERRγ antagonist. * , p < 0.05, * *, p < 0.01, * ** *, p < 0.0001 means significant difference.
Fig. 4. Competitive binding and binding mode between PFBA and human ERRγ (hERRγ). (A) Relative FP values of GSK4716. (B) Relative FP values of GSK5182. (C) Relative FP values of PFBA. (D) Complex of PFBA and agonistic hERRγ. (E) Complex of PFBA and antagonistic hERRγ.
Fig. 5. The expression of ERRγ, EGF and Cyclin D1 in Ishikawa cells. Ishikawa cells were exposed to 0–200 μM PFBA or 0.1 μM GSK4716 for 48 h. (A) The genes expression of ERRγ, EGF, EGFR and Cyclin D1 in Ishikawa cells. (B) The protein level of ERRγ, EGF and Cyclin D1 by western blot; (C-E) Image J was used to quantify the levels of ERRγ (C), EGF (D) and Cyclin D1 (E). * , p < 0.05; * *, p < 0.01; * ** , p < 0.001; * ** *, p < 0.0001.
Fig. 6. The effect of PFBA on Akt/ERRγ signaling pathway. (A) p-Akt, Akt expression level after PFBA exposure for 48 h in Ishikawa cells. (B) The Akt/ERRγ signaling pathway protein after co-exposure to PFBA (200 μM) and inhibitor of Akt LY294002 (5 μM) for 48 h. (C) The statistical result of B. (D) The effect of inhibitor LY294002 (5 μM) on cell proliferation of Ishikawa induced by GSK4716 (100 nM) for 48 h. (E) The effect of LY294002 (5 μM) on cell proliferation of Ishikawa induced by PFBA (200 μM) for 48 h. * , p < 0.05; * *, p < 0.01; * ** , p < 0.001; ####, p < 0.0001. * means compare with control column and # means compare with PFBA or GSK4716 column.
总的来说，在体内实验中，PFBA在亚慢性暴露28天的情况下，诱导了斑马鱼的卵巢病变，如变形的卵细胞和闭锁的卵泡。同时，PFBA降低了血清中的性激素水平，并伴随着激素相关基因的改变。在体外实验中，PFBA通过调节ERRγ途径中的EGF/Cyclin D1表达，促进了Ishikawa细胞的增殖。此外，PFBA在非常低的（纳摩尔）浓度下直接与ERRγ结合，并诱导了ERRγ的转录活性，这表明ERRγ是PFASs毒性中的敏感受体。最后，PFBA诱导了与细胞增殖相关的蛋白，而Akt抑制剂LY294002抑制了PFBA引起的增加，这表明PFBA可能通过激活Akt蛋白来激活ERRγ途径。总的来说，这表明PFBA在纳摩尔水平上直接和间接地作用于ERRγ，干扰了雌激素平衡，并对生殖系统产生了不利影响，其潜在的健康风险需要进一步评估。这项研究补充了对PFBA的毒性效应和机制的理解，为PFASs的毒理学测试和新兴污染物的评估提供了一个新的靶点。
Fangfang Li, Lu Chen, Sha Shi, Wen-Jun Hong, Minjie Li, Liang-Hong Guo, Perfluorobutanoic acid: A short-chain perfluoroalkyl substance exhibiting estrogenic effects through the estrogen-related receptor γ pathways, Journal of Hazardous Materials, 2025, https://doi.org/10.1016/j.jhazmat.2024.136947
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