Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal.
Department of Pathology and Molecular Immunology, Biomedical Sciences Institute of Abel Salazar, University of Porto, 4050-313 Porto, Portugal.
NanoImpact. 2021 Jan;21:100300. doi: 10.1016/j.impact.2021.100300. Epub 2021 Feb 12.
Despite that silver (Ag) is among the most studied nanomaterials (NM) in environmental species and Ag's embryotoxicity is well known, there are no studies on Ag NMs embryotoxicity in soil invertebrates. Previous Full Life Cycle (FLC) studies in Enchytraeus crypticus, a standard soil invertebrate, showed that Ag materials decreased hatching success, which was confirmed to be a hatching delay effect for silver nitrate (AgNO) and mortality for Ag NM300K. In the present study, we aimed to investigate if the impact of Ag takes place during the embryonic development, using histology and immunohistochemistry. E. crypticus cocoons were exposed to a range of concentrations of Ag NM300K (0-10-20-60-115 mg Ag/kg) and AgNO (0-20-45-60-96 mg Ag/kg) in LUFA 2.2 soil, in an embryotoxicity test, being sampled at days 1, 2, 3 and 6 (3, 4, 5 and 7 days after cocoon laying). Measured endpoints included the number of embryonic structures, expression of transferrin receptor (TfR) and L type calcium channels (LTCC) through histological and immunohistochemistry analysis, respectively. Results confirmed that Ag materials affected the embryonic development, specifically at the blastula stage (day 3). The expression and localization of TfR in E. crypticus was shown in the teloblasts cells, although this transcytosis mechanism was not activated. Ag affected calcium (Ca) metabolism during embryonic development: for AgNO LTCC was initially activated, compensating the impact, for Ag NM300K, LTCC was not activated, hence no Ca balance, with irreversible consequences, i.e. terminated embryonic development. An Adverse Outcome Pathway was drafted, integrating the mechanisms here discovered with previous knowledge.
尽管银（Ag）是环境物种中研究最多的纳米材料（NM）之一，并且 Ag 的胚胎毒性是众所周知的，但目前还没有关于土壤无脊椎动物中 Ag NM300K 胚胎毒性的研究。之前在标准土壤无脊椎动物 - 赤子爱胜蚓（E. crypticus）中进行的全生命周期（FLC）研究表明，Ag 材料降低了孵化成功率，这被证实是硝酸银（AgNO）的孵化延迟效应，而对 Ag NM300K 则是致死效应。在本研究中，我们旨在使用组织学和免疫组织化学方法研究 Ag 对胚胎发育的影响。E. crypticus 卵茧在 LUFA 2.2 土壤中暴露于一系列浓度的 Ag NM300K（0-10-20-60-115mg Ag/kg）和 AgNO（0-20-45-60-96mg Ag/kg）中，在胚胎毒性测试中，在 1、2、3 和 6 天（从卵茧放置后的第 3、4、5 和 7 天）进行采样。测量的终点包括胚胎结构数量、通过组织学和免疫组织化学分析分别测量转铁蛋白受体（TfR）和 L 型钙通道（LTCC）的表达。结果证实，Ag 材料影响了胚胎发育，特别是在囊胚阶段（第 3 天）。在原生殖细胞中显示了 E. crypticus 中转铁蛋白受体（TfR）的表达和定位，尽管这种转胞吞作用机制没有被激活。Ag 影响了胚胎发育过程中的钙（Ca）代谢：对于 AgNO，LTCC 最初被激活，补偿了影响；对于 Ag NM300K，LTCC 未被激活，因此没有 Ca 平衡，造成不可逆转的后果，即终止胚胎发育。绘制了一条不良结局途径，将此处发现的机制与先前的知识结合起来。
