Background: Protein hydrolysates from insects are recognized for their biological activities. Black soldier fly larvae (BSFL) have drawn attention due to their antioxidant protein hydrolysates. However, research on bioactive peptides derived from these hydrolysates, particularly their cancer chemopreventive potential, remains limited. This study aims to evaluate the antioxidant, anti-inflammatory, antimutagenic, and anticancer activities of BSFL-derived bioactive peptides and explore the molecular mechanisms. Methods: Alkali-soluble BSFL protein (ASBP) was extracted and hydrolyzed using Alcalase and bromelain under optimized conditions. Antioxidant activity was assessed via FRAP, ABTS, and DPPH assays. The hydrolysate with the highest antioxidant activity was fractionated into molecular weight (MW) groups (>30, 10, and 30 kDa) fraction exhibited the highest antioxidant capacity. In contrast, the ASBP-AH3 (MW < 3 kDa) fraction exhibited significant antimutagenic effects, reduced nitric oxide production, and decreased COLO205 cell viability. Treatment with ASBP-AH3 at its LC₅₀ dose modulated the SKP2/p21/cyclin D1 pathways. Mostly peptides from ASBP-AH3 were composed of hydrophobic and charged amino acids. Conclusions: BSFL-derived bioactive peptides exhibit potential as multifunctional agents for cancer chemoprevention. In vivo studies are required to explore their clinical applications.

Bioactive peptides from black soldier fly larvae (BSFL) protein hydrolysates have gained attention for their health-promoting properties. Our previous study demonstrated the chemopreventive potential of BSFL hydrolysates prepared with Alcalase (ASBP-AH) in colon cancer cells; their in vivo efficacy has not been fully elucidated. This study evaluated the chemopreventive effects of ASBP-AH, processed by spray-drying (ASBP-AHS) or freeze-drying (ASBP-AHF), in a diethylnitrosamine (DEN) and 1,2-dimethylhydrazine (DMH)-induced rat model of early-stage colorectal carcinogenesis. Oral administration of ASBP-AHS or ASBP-AHF significantly reduced aberrant crypt foci (ACF) and downregulated PCNA, COX-2, and NF-κB expression, without affecting apoptosis. Furthermore, both treatments restored microbial species richness and shifted gut microbial diversity disrupted by carcinogen exposure. ASBP-AHS specifically enriched short-chain fatty acid (SCFA)-producing bacteria, while ASBP-AHF favored anti-inflammatory microbial signatures. Likewise, correlation analysis revealed positive associations between microbial changes and SCFA levels, particularly with ASBP-AHS. Peptidomic profiling identified identical peptides in both hydrolysates, including stable pyroglutamyl-containing sequences with potential anti-inflammatory and microbiota-modulating effects. These findings support the in vivo chemopreventive potential of ASBP-AH and its promise as a functional food ingredient for promoting gut health and reducing colorectal cancer risk.

The urinary bladder is a primary target organ for environmental toxicants such as arsenic. The objects of this study were two-fold. First, we constructed a novel 3D urinary bladder mucosa model (3D-UBMM) composed of an overlying epithelium and a supporting subepithelial layer. Primary human bladder urothelial and fibroblast cells were immortalized by introducing the human CDK4^R24C and TERT genes. The construction of the 3D-UBMM involved incorporating immortalized fibroblast cells into a collagen raft, while immortalized urothelial cells were cultured at the air-liquid interface. This 3D-UBMM closely resembles the human 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 (iAs^III) and dimethylarsenic acid (DMA^V). Exposure to iAs^III and DMA^V resulted in increased urothelial necrosis, increased γ-H2AX-positive cells, and reduced P63-positive cells, all in a dose–response manner. These findings affirm that this novel 3D-UBMM resembles the human bladder epithelium and offers a practical in vitro model for evaluating bladder toxicants and carcinogens, identifying mechanisms of carcinogenesis, and supporting hazard identification and risk assessment.

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. LC ₅₀ values were determined for 49 chemicals and compared with reported LC ₅₀ values from 4-hour rat inhalation studies. A significant positive correlation was observed between A549-NRU-derived LC ₅₀ values and in vivo LC ₅₀ values for water-soluble compounds and chemicals containing aldehyde, ketone, alcohol, ether, and epoxide functional groups, suggesting that in vivo LC ₅₀ values may be predictable using the A549-NRU assay. Additionally, A549-NRU LC ₅₀ 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 LC ₅₀ 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 statement: The 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.