Department of Bioscience and Technology, College of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan 430070, PR China.
Department of Rehabilitation Medicine, People's Hospital of Dongxihu District, Wuhan, 430040, PR China.
Aquat Toxicol. 2019 Oct;215:105269. doi: 10.1016/j.aquatox.2019.105269. Epub 2019 Aug 2.
Blooms of the dominant cyanobacterium Aphanizomenon flosaquae are frequently encountered in natural waters, and their secretion of neurotoxic paralytic shellfish toxins called aphantoxins threatens environmental safety and human health worldwide. The liver is the primary detoxification organ in animals, and its pro- and anti-inflammatory responses are important functions in the detoxification of toxins. Therefore, we investigated the response of these inflammatory factors to aphantoxins in the liver of zebrafish (Danio rerio). A. flosaquae DC-1 was sampled during blooms in Dianchi Lake, China and cultured, and the toxin was extracted and analyzed using high performance liquid chromatography. The primary constituents were gonyautoxins 1 (34.04%) and 5 (21.28%) and neosaxitoxin (12.77%). Zebrafish were injected intraperitoneally with 5.3 μg (low dose) or 7.61 μg (high dose) of saxitoxin equivalents [equivalents (eq.)]/kg body weight of A. flosaquae DC-1 aphantoxins. Hyperemia, the hepatosomatic index (HSI), and physiological and molecular responses of pro- and anti-inflammatory cytokines in the zebrafish liver were investigated at different time points 1-24 h post-exposure. Aphantoxins significantly enhanced hepatic hyperemia and altered the HSI 3-24 h post-exposure, suggesting that inflammation caused morphological changes. Subsequent investigations using the enzyme-linked immunosorbent assay showed that the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6, and IL-8 and anti-inflammatory cytokines IL-10 and transforming growth factor β were higher in the liver of zebrafish exposed to aphantoxins, which indicated physiological inflammatory responses. Further analysis by real-time fluorescence quantitative polymerase chain reaction demonstrated upregulated mRNA expression of these cytokines, suggesting molecular inflammatory responses in the zebrafish liver. These changes showed dose- and time-dependent patterns. These results indicated that aphantoxins induced hyperemia and altered the HSI, and subsequently increased the levels of proinflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 to induce physiological inflammatory responses. These changes activated the anti-inflammatory cytokines IL-10 and TGF-β to suppress inflammatory damage. The induced changes were the result of upregulated mRNA expression of these inflammatory cytokines caused by aphantoxins. Aphantoxins resulted in hepatic immunotoxicity and response by inducing pro-inflammatory cytokines. Zebrafish liver in turn suppressed the inflammatory damage by upregulating the activities of anti-inflammatory cytokines. In the future, these pro- and anti-inflammatory cytokines in the zebrafish liver may be prove to be useful biomarkers of aphantoxins and blooms in nature.
优势蓝藻鱼腥藻的水华在天然水中经常出现，其分泌的神经毒性麻痹性贝类毒素（称为阿片毒素）威胁着全球的环境安全和人类健康。肝脏是动物体内的主要解毒器官，其促炎和抗炎反应是解毒过程中的重要功能。因此，我们研究了这些炎症因子在斑马鱼（Danio rerio）肝脏中对阿片毒素的反应。在中国滇池水华期间采集鱼腥藻 DC-1 并进行培养，使用高效液相色谱法提取和分析毒素。主要成分是 GTX1（34.04%）和 GTX5（21.28%）和 neo-saxitoxin（12.77%）。用鱼腥藻 DC-1 阿片毒素 5.3μg（低剂量）或 7.61μg（高剂量）的雪卡毒素当量[当量（eq.）]/kg 体重经腹腔注射斑马鱼。在暴露后 1-24 小时的不同时间点，研究了斑马鱼肝中促炎和抗炎细胞因子的肝充血、肝体比（HSI）和生理及分子反应。阿片毒素显著增强了肝充血，并在暴露后 3-24 小时改变了 HSI，表明炎症引起了形态变化。随后使用酶联免疫吸附试验（ELISA）发现，暴露于阿片毒素的斑马鱼肝中促炎细胞因子肿瘤坏死因子-α、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和白细胞介素-8（IL-8）以及抗炎细胞因子白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）升高，表明存在生理炎症反应。实时荧光定量聚合酶链反应（PCR）进一步分析表明，这些细胞因子的 mRNA 表达上调，提示斑马鱼肝的分子炎症反应。这些变化呈剂量和时间依赖性。结果表明，阿片毒素诱导肝充血和改变 HSI，随后增加 TNF-α、IL-1β、IL-6 和 IL-8 等促炎细胞因子的水平，诱导生理炎症反应。这些变化激活抗炎细胞因子 IL-10 和 TGF-β 以抑制炎症损伤。阿片毒素通过上调这些炎症细胞因子的 mRNA 表达引起诱导变化。阿片毒素通过诱导促炎细胞因子导致肝免疫毒性和反应。斑马鱼肝脏通过上调抗炎细胞因子的活性来抑制炎症损伤。在未来，这些在斑马鱼肝中的促炎和抗炎细胞因子可能被证明是阿片毒素和自然水华的有用生物标志物。
