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法，比较了不同尺寸（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，并与该金属的本体态（bulk Ag）及水相态进行对照。第一层级实验中，将贻贝分别以Ag20-Mal、Ag40-Mal、Ag100-Mal、本体银、水相银（以硝酸银AgNO₃形式存在）以及纳米颗粒制剂中所用麦芽糖浓度的溶液暴露3天，暴露浓度梯度为0.75、75、750 μg Ag/l。本阶段检测指标包括死亡率、生物富集量、组织与细胞分布及溶酶体应答反应。第二层级实验中，选取第一层级（Tier 1）筛选出的最低有效浓度（0.75 μg Ag/l），将贻贝分别以Ag20-Mal、Ag100-Mal、本体银及水相银暴露21天，检测生物标志物与毒理病变效应。水相银在75 μg Ag/l浓度下暴露3天内即可导致贻贝死亡；而银纳米颗粒或本体银在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受调控。电镜观察显示，银纳米颗粒处理组的溶酶体与残余小体内存在直径<10 nm的电子致密颗粒，经X射线检测证实其含有银（Ag）与硫（S）元素。所有银形态处理组均可增强溶酶体内金属富集与溶酶体膜不稳定现象，且Ag20-Mal处理组的该效应较更大尺寸的纳米颗粒更为显著。银纳米颗粒21天暴露可引发消化细胞丢失及消化腺结构完整性破坏，具体表现为消化腺泡萎缩坏死、鳃组织水肿/增生（银纳米颗粒处理组）、消化细胞空泡化（水相银处理组），且所有处理组均出现结缔组织的血细胞浸润。溶酶体内金属富集、溶酶体应答反应及毒理病变效应随银纳米颗粒尺寸减小而增强，其机制可能与纳米颗粒释放的银离子（Ag⁺）有关，尽管整体金属并未发生显著富集。