Digestive cell lysosomes as main targets for Ag accumulation and toxicity in marine mussels, <i>Mytilus galloprovincialis</i>, exposed to maltose-stabilised Ag nanoparticles of different sizes

Bioavailability and toxicity of maltose-stabilised AgNPs of different sizes (20, 40 and 100 nm) in mussels were compared with bulk and aqueous forms of the metal through a two-tier experimental approach. In the first tier, mussels were exposed for 3 d to a range of concentrations (0.75, 75, 750 μg Ag/l) in the form of Ag20-Mal, Ag40-Mal, Ag100-Mal, bulk Ag and aqueous Ag (as AgNO₃), as well as to the concentrations of maltose used in the formulation of NPs. Mortality, bioaccumulation, tissue and cell distribution and lysosomal responses were investigated. In the second tier, mussels were exposed for 21 d to Ag20-Mal, Ag100-Mal, bulk Ag and aqueous Ag at the lowest effective concentration selected after <i>Tier 1</i> (0.75 μg Ag/l), biomarkers and toxicopathic effects were investigated. Aqueous Ag was lethal within 3 d at 75 μg Ag/l; Ag NPs or bulk Ag did not produce significant mortality at 750 μg Ag/l. Ag accumulation was limited and metallothionein gene transcription was not regulated although metal accumulation occurred in digestive, brown and stomach epithelial cells and in gut lumen after exposure to AgNPs and aqueous Ag starting at low concentrations after 1 d. Electrondense particles (&lt;10 nm) in lysosomes and residual bodies after exposure to AgNPs contained Ag and S (X-ray). Intralysosomal metal accumulation and lysosomal membrane destabilisation were enhanced after exposure to all the forms of Ag and more marked after exposure to Ag20-Mal than to larger NPs. 21 d exposure to AgNPs provoked digestive cell loss and loss of digestive gland integrity, resulting in atrophy-necrosis in digestive alveoli and oedema/hyperplasia in gills (Ag NP), vacuolisation in digestive cells (aqueous Ag) and haemocyte infiltration of connective tissue (all treatments). Intralysosomal metal accumulation, lysosomal responses and toxicopathic effects are enhanced at decreasing sizes and appear to be caused by Ag⁺ions released from NPs, although the metal was not substantially accumulated.本研究采用双层实验方案，比较了不同粒径（20、40、100 nm）麦芽糖稳定银纳米颗粒（AgNPs）在贻贝体内的生物利用度与毒性，并以该金属的块体态与水相形态作为对照。第一层实验中，贻贝分别以Ag20-Mal、Ag40-Mal、Ag100-Mal、块体银及水相银（以硝酸银AgNO3形式）的形式，在0.75、75、750 μg Ag/l的浓度梯度下暴露3天，同时设置纳米颗粒制剂所用麦芽糖的浓度对照组。该层实验检测了死亡率、生物富集性、组织与细胞分布及溶酶体响应。第二层实验中，以经第一层（Tier 1）实验筛选出的最低有效浓度（0.75 μg Ag/l）为暴露浓度，将贻贝分别以Ag20-Mal、Ag100-Mal、块体银及水相银的形式进行21天暴露，检测其生物标志物与毒理病变效应。结果显示，水相银在75 μg Ag/l浓度下暴露3天内即可导致贻贝死亡；而银纳米颗粒或块体银在750 μg Ag/l浓度下未引发显著死亡率。尽管在低浓度暴露1天后，银纳米颗粒与水相银处理组的贻贝消化细胞、褐色细胞、胃上皮细胞及肠腔中均出现金属富集，但整体银积累量有限，且金属硫蛋白基因转录未发生调控变化。经X射线检测发现，银纳米颗粒处理组贻贝的溶酶体与残余小体中存在粒径<10 nm的电子致密颗粒，其元素成分为银与硫。所有银形态处理组均会加剧溶酶体内金属富集与溶酶体膜稳定性下降，且Ag20-Mal处理组的该效应较大粒径纳米颗粒组更为显著。银纳米颗粒21天暴露可引发消化细胞丢失与消化腺完整性受损，进而导致消化肺泡萎缩坏死、鳃组织水肿/增生（银纳米颗粒组）、消化细胞空泡化（水相银组）以及结缔组织血细胞浸润（所有处理组）。尽管金属整体积累量不高，但溶酶体内金属富集、溶酶体响应及毒理病变效应均随粒径减小而增强，其成因推测为纳米颗粒释放的银离子（Ag+）。