Lunar Regolith Simulants Elicit Iron-dependent Oxidative Stress Responses in Macrophages

Lunar dust exposure represents a potential environmental and occupational hazard during future Artemis missions; however, the cellular mechanisms underlying regolith-induced toxicity remain incompletely defined. Here, we investigate the biological reactivity of two widely used mare-type lunar regolith simulants (JSC-1A and JSC-2A) in macrophages, key mediators of particulate uptake and clearance. Both simulants exhibited micrometer-scale particle size distributions and basaltic silicate–oxide compositions. Acute cytotoxicity was low relative to crystalline silica, although cell viability declined at higher particle loadings.  At a subcytotoxic deposited dose selected to minimize particle-overload effects, transcriptome-wide RNA sequencing revealed robust and largely overlapping gene-expression programs enriched for oxidative stress, inflammatory signaling, xenobiotic metabolism, and iron homeostasis pathways. JSC-2A induced stronger activation of stress- and metabolism-related networks, including ferroptosis-associated redox programs. Functional assays demonstrated that both simulants significantly increased the intracellular labile Fe²⁺ pool, and this response was suppressed by deferoxamine, confirming iron dependence. In parallel, both simulants strongly induced Hmox1 expression, an effect largely abolished by bafilomycin A1, indicating that lysosomal acidification and endolysosomal iron release are required to activate this oxidative-stress pathway. These responses were accompanied by mitochondrial dysfunction, including reduced ATP production and loss of membrane potential, consistent with ferroptosis-associated redox imbalance. Inductively coupled plasma–atomic emission spectroscopy demonstrated that both simulants release Fe and other metal ions under mildly acidic, lysosome-like conditions, but not at neutral pH, providing a physicochemical basis for intracellular iron mobilization.Collectively, our findings identify acid-dependent iron dissolution, disrupted redox signaling, and mitochondrial impairment as central features of lunar regolith simulant-induced macrophage stress. This work advances mechanistic understanding of space-relevant particulate toxicity and supports improved hazard assessment and countermeasure development for human lunar exploration.