Praseatsook, K. | bioRxiv Science

bioRxiv2025

Construction of a novel 3D urinary bladder mucosa model and its application in toxicity assessment of arsenicals

The urinary bladder is a major target organ for environmental toxicants, including arsenic. The objects of this study were two-fold. First, we constructed a novel 3D urinary bladder mucosa model that incorporated an overlying epithelium and a supporting subepithelial layer, referred to as the 3D-UBMM. Primary human bladder urothelial and fibroblast cells were immortalized by introducing the human CDK4R24C and TERT genes. The subsequent construction of the 3D-UBMM involved incorporating the immortalized fibroblast cells into a collagen raft, while the immortalized urothelial cells were cultured at the air-liquid interface of the raft. This 3D-UBMM closely resembles the human urinary bladder epithelium in terms of morphology and marker protein expression, including uroplakin 1b, P63, and cytokeratin 5. Second, using the 3D-UBMM we investigated the cytotoxicity of sodium arsenite (iAsIII) and dimethylarsenic acid (DMAV). Exposure to iAsIII and DMAV resulted in increased urothelial cell necrosis and increased {gamma}-H2AX-positive cells along with a reduction of P63-positive cells, and each of these effects was induced in a dose-response manner. These findings affirm that this novel 3D-UBMM closely resembles the human urinary bladder epithelial layer, offering a practical in vitro model for the evaluation of the toxicity of arsenic and other bladder carcinogens and the role of cancer-related genes in bladder carcinogenicity. In addition, by identifying mechanisms of carcinogenesis this model will aid in hazard identification and risk assessment of potential bladder carcinogens.

pharmacology and toxicology↗

bioRxiv2025

Developing a Novel In Vitro Toxicity Assay for Predicting Inhalation Toxicity in Rats

The development of alternative in vitro methods for assessing acute inhalation toxicity is a critical step toward reducing animal testing and aligns with the principles of the 3Rs (replacement, reduction, and refinement). In this study, we developed and optimized a neutral red uptake (NRU) assay using human lung adenocarcinoma cells (A549) as a predictive model (A549-NRU) for acute inhalation toxicity. To improve assay efficiency and robustness, we introduced two key modifications: the incubation time was reduced to 15 minutes to enable rapid and high-throughput screening, and for chemicals reactive with polystyrene 6-well glass plates were used to prevent chemical-induced degradation and ensure assay consistency. LC50 values were determined for 49 chemicals and compared with reported LC50 values from 4-hour rat inhalation studies. A significant positive correlation was observed between A549-NRU-derived LC50 values and in vivo LC50 values for water-soluble compounds and chemicals containing aldehyde, ketone, alcohol, ether, and epoxide functional groups, suggesting that in vivo LC50 values may be predictable using the A549-NRU assay. Additionally, A549-NRU LC50 values showed significant negative correlations with molecular weight and octanol-water partition coefficients, indicating that chemicals with higher values tended to be less cytotoxic in vitro. Importantly, the A549-NRU assay demonstrated stronger correlation with in vivo LC50 values than the conventional NRU assay using mouse 3T3 fibroblast cells. These findings support the use of the A549-NRU assay to estimate starting doses for in vivo studies, and potentially as an in vitro alternative for predicting acute inhalation toxicity. Impact statementThe optimized A549-NRU assay demonstrates predictive potential for inhalation toxicity while reducing reliance on animal testing. This model serves as a human-relevant alternative for estimating starting doses for in vivo inhalation toxicity studies.

pharmacology and toxicology↗