A novel adverse outcome pathway in aluminum-induced male infertility: Oxidative stress-mediated endoplasmic reticulum stress and endocannabinoid system activation delineated by network toxicology and metabolomics

Aluminum has been recognized as a reproductive toxin, yet the precise mechanisms by which embryonic exposure impairs male offspring development remain largely unclear. The key point is that no systematic adverse outcome pathway (AOP) network has yet been established to link aluminum exposure with reproductive disorders. This study employed an integrated approach-combining network toxicology and non-targeted metabolomics, and in vivo/in vitro experiments-to establish a novel AOP elucidating how aluminum induces spermatogenesis impairment in mice. Mechanistically, in vitro studies using mouse TM3 and primary Leydig cells demonstrated that aluminum chloride (AlCl₃) triggers oxidative stress, leading to endoplasmic reticulum stress (ERS), via the IRE1α/XBP1s pathway. We identified that the transcription factor XBP1s directly binds to and transactivates the promoter of N-acylphosphatidyl-ethanolamine-specific Phospholipase D (Nape-pld). This cascade results in overactivation of the Cannabinoid Receptor 1 (CB1) and the downregulation of key steroidogenic proteins, Luteinizing Hormone Receptor (LHR) and Steroidogenic Acute Regulatory Protein (StAR). These findings provide compelling evidence for our AOP model, wherein aluminum induces ERS via oxidative stress, subsequently impairing testosterone synthesis through the endocannabinoid system (ECS). Our research advances the assessment of heavy metal reproductive risks by offering a robust methodology for identifying key events (KEs) and constructing causal AOP networks.