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.

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.

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.