Two-Dimensional Layered Nano-MoS₂ Induces Earthworm Immune Cell Apoptosis by Regulating Lysosomal Maintenance and Function: Toward Unbiased Screening and Validation of Suspicious Pathways

Molybdenum-based nanosheets (NSMoS₂) are increasingly applied in various fields and undergoing relevant risk evaluations on subjectively hypothesized toxicity pathways. However, risk assessment should be unbiased and focus on appropriate end points to avoid biased prescreening. Here, we developed an adverse biological outcome screening strategy based on nontargeted functional protein profiles in earthworm (<i>Eisenia fetida</i>) immune cells exposed to NSMoS₂ and their ionic counterpart (Na₂MoO₄). Through this framework, the apoptosis-related processes with distinct mechanisms were rapidly identified and thoroughly validated phenotypically. Specifically, upon exposure to 50 μg Mo/mL Na₂MoO₄, cellular signaling and energy homeostasis were disrupted within the transcription-translation biological chain. The autophagic pathway was activated, which, together with energy deprivation, phenotypically induced significant autophagy that ultimately led to apoptosis. In contrast, NSMoS₂, tested at the same concentration, caused a reprogramming of apoptotic gene and protein expressions. Transcriptome plasticity facilitated the endocytic-adaptive transcriptional profile characterized by cytoskeleton remodeling and lysosome organization/movement under NSMoS₂ exposure. Subcellular dynamics further revealed NSMoS₂-induced lysosomal damage with a time-sensitive physiological window, ultimately mediating apoptosis. These findings provide a mechanistic and visual understanding of the distinct risk profile of NSMoS₂ compared to molybdate, highlighting the importance of integrating nontargeted screening and phenotypic validation in early risk warning.