Profibrotic predictive toxicology in the lung.

Introduction: Fibrosis in the gossamer alveolar capillary membranes of the lung can lead to abnormalities in gas exchange, hypoxemia and death of the individual. These interstitial lung diseases (ILDs) of known or yet undefined etiologies (such as Idiopathic pulmonary fibrosis) highlight the need for predictive, physiologically relevant models for toxicity studies. Three-dimensional (3D) lung organoids derived from animal cells provide an advanced platform that replicates the structural and cellular complexity of lung tissue while reducing whole-animal use. Methods: Mouse lung organoids (MiLO) were used to evaluate pulmonary toxicity caused by environmental toxicants and pharmacologic agents. MiLO were generated from perfused, minced mouse lungs that were digested with collagenase, filtered, depleted of red blood cells, and embedded in Matrigel. Organoids were stained for lineage markers to characterize cellular diversity, including SPC, α-SMA, CD31, F4/80 and ECM proteins collagen I and fibronectin. Gene expression in MiLO and native lung tissue was compared for fibrosis- and viability-related markers. A well-characterized mouse model of cadmium induced lung fibrosis was used as an in vivo benchmark to assess α-SMA expression, airway resistance to methacholine, hydroxyproline content, malondialdehyde levels (MDA), and superoxide dismutase (SOD) activity. For drug-induced fibrosis modeling, cell viability assays defined 20% inhibitory concentrations of nitrofurantoin (NF, 5 μM) and amiodarone (AD, 20 μM), which were then used to treat MiLO for assessment of MDA, invasion area on collagen-coated plates, and expression of fibrotic and signaling markers. Results: MiLOs faithfully recapitulated native lung architecture, extracellular matrix composition, and fibrosis-related gene expression profiles. In vivo cadmium exposure increased α-SMA expression, airway resistance, collagen content, and malondialdehyde (MDA) levels, while reducing superoxide dismutase (SOD) activity. Consistently, Cd- treated MiLOs exhibited increases in COL1A1 deposition, cellular invasion, hydroxyproline content, and oxidative stress. Exposure to nitrofurantoin (NF) or amiodarone (AD) elevated MDA, enhanced invasion, and upregulated fibrogenic and signaling genes including Tgfb1, Col1a1, Acta2, Akt1, Nfkb1, and Mmp9. Environmental toxicant (Cd) and drug (AD or NF) treatments drove the development of hallmark fibrotic features in lung organoids, characterized by increased collagen deposition, oxidative stress, and profibrotic gene activation. Conclusions: These findings demonstrate that mouse lung organoids effectively recapitulate key molecular and pathological aspects of drug- and toxin-induced pulmonary fibrosis and represent a powerful model for mechanistic investigation and preclinical screening of compounds with potential pro-fibrotic effects.