Abstract Inorganic arsenic is a known human carcinogen associated with liver and bladder cancer. Among its species, sodium arsenite (iAsIII) undergoes complex hepatic biotransformation, producing methylated metabolites such as monomethylated (MMA) and dimethylated (DMA) forms. However, its human-relevant mechanisms of metabolism and carcinogenicity remain unclear due to species differences in arsenic biotransformation. To clarify these differences, we employed a humanized-liver (HL) mouse model, in which mouse hepatocytes are replaced with human hepatocytes, to investigate the metabolism and carcinogenic potential of iAsIII. HL and wild type (WT) mice were administrated 50 ppm of iAsIII in drinking water for 4 weeks. Urine, liver, and bladder samples were collected for analysis. Urinary arsenic speciation was determined using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Histopathology and immunohistochemistry were performed on both liver and bladder tissue, and gene expression profiles were analyzed using RNA sequencing and microarray, respectively. Urinary arsenic speciation revealed distinct species-dependent differences. Dimethylarsinic acid (DMAV) was the predominant urinary arsenic metabolite in both HL and WT mice. Total urinary arsenic in HL mice was lower than in WT mice, but the distribution of metabolites differed markedly. The proportion of MMAV in HL mice (26.4%) was substantially higher than in WT mice (4.7%), whereas DMAV accounted for 58.5% in HL and 80.7% in WT mice. This urinary metabolite pattern more closely resembled that observed in humans exposed to inorganic arsenic. Hepatic arsenic methyltransferase (As3MT) expression was significantly elevated in WT mice treated with iAsIII but tended to decrease in HL mice. Gene expression profiles of liver and bladder tissues of HL mice also differed from those of WT mice. Immunohistochemical analysis of proliferation markers is currently underway to evaluate whether iAsIII promotes hepatocellular and urothelial proliferative response. Arsenic content in the liver is also currently under investigation, and pathway analysis of the transcriptomic data using Ingenuity Pathway Analysis (IPA) is ongoing to identify key molecular alterations for further validation. In conclusion, these findings indicate that human hepatocytes exhibit lower methylation capacity, resulting in higher proportions of toxic MMA species, suggesting that humans may be more susceptible to arsenic than mice. Furthermore, the HL mouse model effectively reflects human arsenic metabolism and provides a valuable in vivo platform for elucidating human-relevant metabolic, toxicological, and molecular mechanisms underlying arsenic-induced hepatotoxicity and carcinogenicity. Citation Format: Arpamas Vachiraarunwong, Shugo Suzuki, Masaki Fujioka, Runjie Guo, Guiyu Qiu, Yurina Kawamura, Ikue Noura, Anna Kakehashi, Hideki Wanibuchi, Min Gi. Metabolism and carcinogenic potential of inorganic arsenic in humanized-liver mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3373.

Arsenic is a well-established human carcinogen and a major global environmental health concern. However, the mechanisms underlying arsenic-induced carcinogenesis remain incompletely understood. This review focuses on dimethylarsinic acid (DMAV), a principal urinary metabolite of inorganic arsenic in humans. Using various animal models, we demonstrated that DMAV promotes carcinogenesis in multiple organs, including the urinary bladder, liver, kidney, and thyroid gland. Two-year bioassays confirmed that DMAV functions as a complete carcinogen in the rat bladder. Furthermore, transplacental exposure to DMAV induced lung and liver tumors in male offspring, highlighting increased fetal susceptibility. Mechanistic investigations revealed a central role for oxidative DNA damage and gene dysregulation in DMAV-induced tumorigenesis. We also identified epigenetic alterations, including aberrant DNA methylation and histone modifications, as key contributors to tumor development, with distinct patterns observed between fetal and adult exposures. Collectively, these findings provide robust experimental evidence for the carcinogenic potential of DMAV and clarify its underlying mechanisms. Our work contributed significantly to the classification of DMAV as "probably carcinogenic to humans" (Group 2 A) by the International Agency for Research on Cancer, underscoring its relevance to human health risk assessment.

Occupational cholangiocarcinoma among printing workers in Japan has raised concern regarding the carcinogenic hazards of chlorinated organic solvents, particularly 1,2-dichloropropane (1,2-DCP) and dichloromethane (DCM). Because workers were often exposed to multiple solvents, this study examined whether DCM modifies 1,2-DCP-associated hepatocellular tumor development in mice. Male C3H/HeN mice were administered a corn oil vehicle, 1,2-DCP alone at 500 mg/kg bw, or 1,2-DCP and DCM at 500 mg/kg bw each by oral gavage twice weekly for 52 weeks. Combined exposure significantly increased the incidence of hepatocellular adenomas (HCAs) compared with both the vehicle control and 1,2-DCP-alone groups. Tumor multiplicity was also significantly higher in the 1,2-DCP + DCM group than in the 1,2-DCP-alone group. HCAs from the 1,2-DCP + DCM group showed increased cell proliferative activity, 29 uniquely altered differentially expressed genes, significant upregulation of Gpc3 and Igfbp1, and downregulation of Dcn, consistent with altered tumor-associated molecular features. Canonical pathway analysis indicated suppression of xenobiotic metabolism, bile acid metabolism, and peroxisomal function in treatment-associated HCAs. These findings indicate that DCM co-exposure enhanced 1,2-DCP-associated hepatocellular tumor development and altered tumor-associated molecular features in mice, highlighting the importance of considering combined solvent exposure in chemical carcinogenic risk assessment.

Although numerous experimental studies have demonstrated the carcinogenic potential of multi-walled carbon nanotubes (MWCNTs) in lungs, the underlying molecular mechanisms—especially gene expression changes associated with different tumor types—remain poorly characterized. To elucidate the molecular signatures associated with MWCNT-induced carcinogenesis, we performed microarray-based gene expression profiling of rat lung tumors induced by MWCNT-7, including both adenocarcinoma (ADC) and malignant mesothelioma (MM), as well as ADCs induced by two types of double-walled CNTs (DWCNTs) differing in fiber length (1.5 µm and 7 µm). Hierarchical clustering revealed that the MWCNT-7-induced MM exhibited a gene expression profile distinct from the ADCs. The ADCs induced by the DWCNTs and the ADC induced by MWCNT-7 shared several pathways that were distinct from those of the MWCNT-7 induced MM. The distinct pathways upregulated in the ADCs versus the MM support the conclusion that MWCNT-induced ADCs arise through distinct biological mechanisms compared to MWCNT-induced MMs and identified tumor-type-specific biomarker candidates: complement factor I (CFI) and secreted phosphoprotein 1 (SPP1) for ADCs, and fibronectin 1 (FN1) for MM. In addition, the gene expression profiles of the ADCs induced by the three fiber types indicate that both types of thin flexible DWCNTs used in the present study promoted a number of carcinogenic pathways in the rat lung that were also promoted by MWCNT-7, which is a class 2B carcinogen. These results support the conclusion that DWCNTs are carcinogenic in the rat lung and highlight the importance of further assessments of the potential lung carcinogenicity of inhaled thin flexible CNTs.

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 <3 kDa). The bioactivity of fractionated peptides was evaluated through antioxidant, anti-inflammatory (nitric oxide production in RAW 264.7 cells), antimutagenic (Ames test), and anticancer (CCK-8 assay on HCT 116, COLO205, Cw-2, and Caco-2 cells) assays. Mechanistic insights were obtained via microarray and Western blot analyses. Peptides were identified by LC-MS/MS. Results: The ASBP-Alcalase hydrolysate (ASBP-AH) showed optimal antioxidant activity at 3% (w/w) for 4 h. The ASBP-AH 30 (MW > 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.

The development of alternative in vitro methods for assessing acute inhalation toxicity is essential to reduce animal testing and aligns with the 3Rs principles (replacement, reduction, refinement). In this study, we developed a neutral red uptake (NRU) assay using human lung adenocarcinoma cells (A549) as a predictive model (A549-NRU) for acute inhalation toxicity. The assay incorporates two key features: a 15-minute incubation time to simulate the transient contact of inhaled chemicals with the airway surface under acute inhalation conditions, and the use of both polystyrene plates and glass plates for chemicals reactive with polystyrene. 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 (r = 0.4632, p = 0.0197) as well as chemicals containing aldehyde and ketone (r = 0.9339, p = 0.0007), and alcohol, ether, and epoxide (r = 0.7668, p = 0.0159) functional groups, suggesting that in vivo LC50 values may be predictable using the A549-NRU assay. Importantly, the A549-NRU assay (r = 0.8879, p = 0.1121) demonstrated a stronger correlation with in vivo LC50 values than the conventional NRU assay using mouse 3T3 fibroblast cells (r = 0.4524, p = 0.5476). These findings support the A549-NRU assay as an alternative for predicting acute inhalation toxicity and for estimating starting doses for confirmatory in vivo studies.

Objectives: Our aim was to report integrative evidence for the health risk assessment of ortho-toluidine (OT) in bladder cancer in a mini-review of the recent studies of humans, experimental animals, and OT skin permeability. Methods: Bladder cancer cases were identified in workers in Japan who were occupationally exposed in 2014-2017 to aromatic amines, primarily to the human carcinogen OT. Results: A key epidemiological study of 98 aromatic amine-exposed workers in Japan showed a clear OT exposure-response relationship with a standardized incidence ratio. A rat model experimental study also indicated that OT and acetoaceto-o-toluidine are potent bladder carcinogens. Multiple mechanisms of OT-related bladder cancer have been proposed: metabolic activation to reactive metabolites that bind DNA and proteins, mutagenicity, oxidative DNA damage, chromosomal damage, and cytotoxicity by OT. Recent comprehensive analyses of DNA adducts in rats identified a number of common oxidative DNA adducts, including 8-OHdG, in the rat urothelium and indicated that oxidative stress may play a crucial role in the development of urinary cancer caused by OT. The skin permeability of 6 aromatic amines (o-toluidine, aniline, p-toluidine, o-anisidine, 2,4-dimethylaniline, and o-chloroaniline) was examined with the use of a 3-dimensional (3D) reconstructed human skin model; similar to 70%-80% of the 6 aromatic amines had permeated through the 3D skin within 8 hours. Genotoxic potency testing in a human urothelial cell line using gamma-H2AX, a marker of DNA damage, suggested that OT exhibited strong gamma-H2AX generation. Conclusions: Prolonged dermal exposure to OT along with other genotoxic aromatic amines over many years may contribute to the development of bladder cancer.

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.