Arsenic (sodium arsenite: NaAsO2) is a potent carcinogen and a known risk factor for the onset of bladder carcinogenesis. The molecular mechanisms that govern arsenic-induced bladder carcinogenesis remain unclear. We used a physiological concentration of NaAsO2 (250 nM: 33 µg/L) for the malignant transformation of normal bladder epithelial cells (TRT-HU1), exposed for over 12 months. The increased proliferation and colony-forming abilities of arsenic-exposed cells were seen after arsenic exposure from 4 months onwards. Differential gene expression (DEG) analysis revealed that a total of 1558 and 1943 (padj < 0.05) genes were deregulated in 6-month and 12-month arsenic-exposed TRT-HU1 cells. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that cell proliferation and survival pathways, such as the MAPK, PI3K/AKT, and Hippo signaling pathways, were significantly altered. Pathway analysis revealed that the enrichment of stem cell activators such as ALDH1A1, HNF1b, MAL, NR1H4, and CDH1 (p < 0.001) was significantly induced during the transformation compared to respective vehicle controls. Further, these results were validated by qPCR analysis, which corroborated the transcriptomic analysis. Overall, the results suggested that stem cell activators may play a significant role in facilitating the arsenic-exposed cells to gain a survival advantage, enabling the healthy epithelial cells to reprogram into a cancer stem cell phenotype, leading to malignant transformation.

Pituitary gonadotropins directly govern ovarian functions, which are in turn regulated by the ovarian steroid hormones. The precise interplay of gonadotropins and steroid hormones is critical for follicle growth and differentiation. Furthermore, autophagy regulates ovarian follicle differentiation. However, how the high-fat-high fructose (HFD-HF) diet regulates gonadotropins and facilitates autophagy-mediated follicular differentiation in the ovary is obscure. We fed prepubertal rats (PND 25) an HFD-HF diet until PND 90. The results showed diminished adenohypophyseal GnRHR, PR, and aromatase expression, whereas AR, ERα, PRLR, and inhibin were augmented, resulting in gonadotropins decline. Interestingly, autophagy biomarkers, Beclin-1, ATG5, ATG12, LC3-II, and LAMP1 were reduced but SQSTM1/p62 was augmented in the ovaries of HFD-HF-fed rats, causing autolysosome to aggregation. The diet altered T, E2, P4, PRL, and their receptors status in the ovary, disturbed estrous cyclicity, and delayed vaginal opening. Ovarian histomorphology exhibited numerous cystic and atretic follicles, along with disturbed follicular maturation and ovulation. Moreover, the reduction of FSHR; steroidogenic proteins; receptor proteins AR, ERβ, PR; and signaling proteins Wnt2 and β-catenin was also noticed in the ovary, whereas PRLR, inhibin, and pGSK3β were augmented. In conclusion, exposure to a prepubertal HFD-HF diet leads to hypogonadotropism and the autophagy-mediated defective differentiation of ovarian follicles, abating fertility in adult rats.

Introduction: Epidemiological, pre-clinical, and clinical studies have linked chronic arsenic (sodium arsenite: NaAsO₂) exposure to a myriad of adverse health dysfunctions including the development of bladder cancer (BCa). However, most of the studies on arsenic-induced BCa have been performed on either unsuitable cell lines or using a higher concentration of arsenic (mM), which does not reflect exposure levels in a real-world setting. We used the telomerase reverse transcriptase (HU-hTERT1) immortalized normal bladder epithelial and a median physiological concentration of NaAsO₂ (250 nM: 33 mg/L), to determine the transcriptomic and phenotypic changes during malignant transformation of HU-hTERT1 cells. Methods: HU-hTERT1 cells were exposed to 250nM arsenic for 12 months. To analyze the effect of arsenic exposure we performed cell viability, cellular proliferation, and clonogenic assays. RNA Seq data was used to perform pathway enrichment analysis, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein-protein interaction (PPI) studies. Results: BCa patients had significantly higher (4-5 fold) levels of arsenic (20-50 mg/L) in their urine when compared to their healthy counterparts ( 400nM) inhibited the HU-hTERT1 growth, whereas concentrations less than 400nM did not adversely affect the viability of these cells. HU-hTER1 cells began forming colonies after six months of chronic NaAsO₂ exposure, with the number of colonies increasing concurrently with the exposure time. GO and KEGG analysis showed a total of 2174 genes that are differentially expressed in arsenic-treated HU-hTER1 transforming cells (0 vs 12 months). The upregulated genes (1392) were involved in cellular response to unfolded protein, response to endoplasmic reticulum stress, proteasome-mediated ubiquitin-dependent protein degradation, autophagy signaling, and tumor angiogenesis(P<0.05). Interestingly, the PPI analysis demonstrated a significant upregulation in histone modification of genes. These genes were enriched for the binding sites of transcription factors that are key regulators of carcinogenic pathways including ataxia-telangiectasia mutated (ATM) kinase and cell cycle regulatory pathways demonstrating a novel link between NaAsO₂ exposure and BCa. Conclusion: An in vitro model of NaAsO₂-induced malignant transformation was developed. Identifying precise molecular events that lead to arsenic-induced malignant transformation of normal urothelial cells would be an important step in defining therapy for BCa. Citation Format: Ajit Kumar Navin, Vaibhav Shukla, Balaji Chandrasekaran, Ashish Tyagi, Uttara Saran, Murali K. Ankem, Chendil Damodaran. Molecular landscape of arsenic-induced bladder carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 745.