聚苯乙烯奈米塑膠對斑馬魚 (Danio rerio) 胚胎的影響

Abstract

塑料是日常生活中使用最廣泛的材料，疏忽的垃圾管理導致環境中的數量不斷增加。值得注意的是，納米塑料（NPs; 直徑從 5 到 100 納米的微小塑料碎片）難以追踪，並已成為 21 世紀的主要污染源。聚苯乙烯 (PS) 是環境中最普遍的納米塑料之一。在實驗室中，PS-NPs 已被證明可以穿透皮膚並積聚在魚胚胎的器官中。然而，人們對 PS-NPs 對魚胚胎皮膚功能（如離子調節和側線感覺）的潛在影響知之甚少。在本研究中，斑馬魚胚胎被用作動物模型來探索 PS-NPs 可能對魚胚胎造成的毒性作用。在本研究的第一部分，斑馬魚胚胎暴露於三種不同大小的 PS-NP（25、75 和 200 nm）中 96 小時或 120 小時。分析了幾類毒性終點，包括發育形態（體長、身體彎曲、體節長度、眼睛大小、心包腔大小和卵黃囊大小）、身體活動（運動活動、視運動反應和強硬誘發反應）和側線感覺（毛細胞數量和形態）。此外，還檢測了神經標記基因（ache、syn2a 和 mbp）和眼睛發育基因(pax6a, pax6b, otx2, 和 rx1)的 mRNA 表達。結果表明，50 mg/L 的 25 nm PS-NPs 在所有的毒性終點均引起顯著的不良反應，表明胚胎發育遲緩和異常，身體活動和感覺毛細胞也顯著受損。在 25 mg/L 的 25 nm PS-NPs 下，只有身體活動和毛細胞顯著受損。在 10 mg/L 的 25 nm PS-NPs 和 75 和 200 nm PS-NPs 的所有濃度下，大多數毒性終點的影響是不顯著的。在本研究的第二部分，斑馬魚胚胎暴露於 25 nm PS-NPs 96 小時，以測試 PS-NPs 對皮膚細胞（離子細胞和角質細胞）離子調節功能的影響。暴露於50 mg/L PS-NPs後，胚胎皮膚細胞的離子（Na+、K+和Ca2+）含量和酸/氨排泄量顯著下降。皮膚角質形成細胞的頂端結構在 10、25 和 50 mg/L 時受損。離子細胞的數量和線粒體活性在 25 和 50 mg/L 時降低。 CellROX 染色顯示的活性氧 (ROS) 水平顯示離子細胞和角質形成細胞都處於氧化壓力狀態。 PS-NPs 降低了抗氧化基因（sod1、sod2、cat 和 gpx1a）的 mRNA 表達，並促進了細胞凋亡相關基因（casp3a）。綜上所述，本研究表明 PS-NPs 可能抑制抗氧化反應並誘導氧化應激，導致線粒體損傷和離子細胞死亡，最終損害皮膚功能，包括離子攝取、pH 調節和氨排泄。本研究揭示了 PS-NPs 可能對魚胚胎造成的潛在風險以及 PS-NPs 可能從分子和細胞到個體水平的毒理學機制。

Plastic is the most widely used material in everyday life, and negligent litter management has resulted in growing amounts in the environment. Notably, nanoplastics (NPs) — minuscule bits of plastic ranging in diameter from 5 to 100 nanometers –are hard to trace and have become a major source of pollution in the twenty-first century. Polystyrene (PS) is one of the most prevalent nanoplastics in the environment. In the laboratory, PS-NPs have been shown to penetrate the skin and accumulate in the organs of fish embryos. However, little is known about the potential impacts of PS-NPs on the skin functions (such as ion regulation and lateral line sensation) of fish embryos. In this study, the zebrafish embryo was used as an animal model to explore the toxic effects that PS-NPs might pose to fish embryos.In the first part of this study, zebrafish embryos were exposed to three distinct sizes of PS-NPs (25, 75, and 200 nm) for 96 h or 120 h. Several categories of endpoints were examined including developmental morphology (body length, body bending, somite length, eye size, pericardial cavity size, and yolk sac size), physical activity (locomotor activity, optomotor response, and touch response), and lateral-line sensory system (hair cell number and morphology). Moreover, the mRNA expression of neuro-marker genes (ache, syn2a, and mbp), and eye development genes (pax6a, pax6b, otx2, and rx1) were examined. The results showed that 50 mg/L of 25 nm PS-NPs caused significant adverse effects in all examined endpoints, suggesting that embryonic development was retarded and disturbed, and physical activity and sensory hair cells were impaired. The effects were not dominant at most endpoints. At 25 mg/L of 25 nm PS-NPs, only physical activity and hair cells were significantly impaired. Marginal effects were shown at 10 mg/L of 25 nm PS-NPs and all concentrations of 75 and 200 nm PS-NPs.In the second part of this study, zebrafish embryos were exposed to 25 nm PS-NPs for 96 h to test the effects of PS-NPs on ion regulation of embryonic skin. After exposure to 50 mg/L PS-NPs, ion (Na+, K+, and Ca2+) contents and acid/ammonia excretion by embryonic skin cells significantly declined. The apical structure of keratinocytes was damaged at 10, 25, and 50 mg/L. The number and mitochondrial activity of ionocytes were reduced at 25 and 50 mg/L. Reactive oxygen species (ROS) levels indicated by CellROX staining showed that both ionocytes and keratinocytes were under oxidative stress. PS-NPs reduced the gene expression of antioxidant enzymes (sod1, sod2, cat, and gpx1a), but promoted apoptosis-related gene (casp3a) expression. Taken together, the present study suggests that PS-NPs suppress antioxidative reactions and induce oxidative stress, significantly resulting in mitochondrial damage and cell death of ionocytes. Finally, it may impair embryonic skin.In conclusion, the present study used molecular and cellular approaches to reveal the potential impacts of PS-NPs on the developmental and physiological function of fish embryos.