Masaki Fujioka

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.

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 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.

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.

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment. This study aims to develop a 1-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. In the silico validation using the expression data of the Open TG-GATEs database indicates that this classifier distinguishes GHCs from other compounds with high accuracy. To further assess the model's effectiveness and reliability, we conducted multi-institutional 1-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR 24 h after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present 1-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAs^V, iAs^III, MMA^V, MMA^III, DMA^V, DMA^III, and DMMTA^V). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTA^V exhibited higher cellular uptake than pentavalent arsenicals. Except for MMA^V, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMA^III and DMMTA^V were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMA^V. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMA^V and DMA^III induced necroptosis. MMA^III, DMA^V, and DMMTA^V induced both cell death pathways. In summary, MMA^III exhibited the strongest cytotoxicity, followed by DMMTA^V, iAs^III, DMA^III, iAs^V, DMA^V, and MMA^V. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment, and it also contributes to cancer prevention. This study aims to develop a one-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. This classifier effectively distinguishes GHCs from other compounds, demonstrating 100% sensitivity and over 96% specificity. To further assess the model’s effectiveness and reliability, we conducted multi-institutional one-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR (qPCR) 24 hours after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present one-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

Occupational exposure to aromatic amines (AAs) is an important risk factor for urinary bladder cancer. This study aimed to evaluate the toxicity of AAs and analyze the carcinogenic mechanisms in rat bladder by comprehensive analysis of DNA adducts (DNA adductome). DNA was extracted from the bladder epithelia of rats treated with AAs, including acetoacet-o-toluidine (AAOT) and o-toluidine (OTD), and adductome analysis was performed. Principal component analysis–discriminant analysis revealed that OTD and AAOT observed in urinary bladder hyperplasia could be clearly separated from the controls and other AAs. After confirming the intensity of each adduct, four adducts were screened as having characteristics of the OTD/AAOT treatment. Comparing with the in-house DNA adduct database, three of four candidates were identified as oxidative DNA adducts, including 8-OH-dG, based on mass fragmentation together with high-resolution accurate mass (HRAM) spectrometry data. Therefore, findings suggested that oxidative stress may be involved in the toxicity of rat bladder epithelium exposed to AAs. Consequently, the administration of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, in six-week-old rats fed with 0.6% OTD in their diet resulted in simple hyperplastic lesions in the bladder that were suppressed by apocynin. The labeling indices of Ki67, γ-H2AX, and 8-OHdG were significantly decreased in an apocynin concentration-dependent manner. These findings indicate that oxidative stress may have contributed to the development of urinary cancer induced by OTD.

Arsenic is a known human urinary bladder carcinogen. While arsenic is known to cause aberrant DNA methylation, the mechanism of arsenic-triggered bladder carcinogenesis is not fully understood. The goal of this study was to identify aberrant DNA methylation in rat bladder urothelial carcinoma (UC) induced by dimethylarsinic acid (DMA^V), a major organic metabolite of arsenic. We performed genome-wide DNA methylation and microarray gene expression analyses of DMA^V-induced rat UCs and the urothelium of rats treated for 4 weeks with DMA^V. We identified 40 genes that were both hypermethylated and downregulated in DMA^V-induced rat UCs. Notably, four genes (CPXM1, OPCML, TBX20, and KCND3) also showed reduced expression in the bladder urothelium after 4 weeks of exposure to DMA^V. We also found that CPXM1 is aberrantly methylated and downregulated in human bladder cancers and human bladder cancer cells. Genes with aberrant DNA methylation and downregulated expression in DMA^V-exposed bladder urothelium and in DMA^V-induced UCs in rats, suggest that these alterations occurred in the early stages of arsenic-induced bladder carcinogenesis. Further study to evaluate the functions of these genes will advance our understanding of the role of aberrant DNA methylation in arsenic bladder carcinogenesis, and will also facilitate the identification of new therapeutic targets for arsenic-related bladder cancers.