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Arsenic is a toxic metalloid that continues to contaminate the water and food sources of millions of people globally. Among the numerous health effects of arsenic exposure are urothelial toxicity and cancer. In recent years, small extracellular vesicles (SEVs) have been shown to be vital in intracellular communication and have been used in clinical studies as biomarkers of disease. The overall goal of this thesis is to understand the mechanisms of cell communication during arsenic exposure and to develop minimally invasive biomarkers for the toxic responses. The specific objectives are to: a) determine if SEVs released from arsenic exposed urothelial cells are responsible for mediating urothelial toxicity; and b) assess the application of urinary SEVs as novel biomarkers of arsenic exposure in an exposed population. The hypothesis leading this research is that the biology and protein packaging profile of urothelial SEVs are altered following arsenic exposure because of the induction of cell stress signaling pathways. I also hypothesize that urinary SEV proteins can be used as biomarkers of arsenic exposure because they are positively correlated with urinary arsenic concentrations in an exposed population. SVHUC1 human urothelial cells were dosed with sodium meta arsenite (1, 2, and 5 uM) for 48 hours. T24 urothelial carcinoma cells were also grown in parallel to compare for carcinogenicity. A label-free quantitative proteomics approach was used to assess the differentially expressed proteins in the cell lysate and the SEVs extracted from the culture media to determine the mechanistic pathways involved and how well the protein profiles in SEVs correlate with those in the cell lysate. SEVs were isolated from the archived urine samples of participants (n=36) enrolled in the Yellow Knife Health Effects Monitoring Program (YKHEMP) and two potential biomarkers, transforming growth factor beta receptor 1 (TGFBR1) and ribonuclease inhibitor 1 (RNH1), were measured by an enzyme linked immunosorbent assay (ELISA). SEVs in all samples were successfully characterized based on their size (50-200 nm) and positive antibody array for eight protein markers indicating their endosomal biogenesis. The total number of SEVs was not shown to increase following arsenic exposure in the in vitro study. However, the cancerous T24 cells had nearly four times higher numbers of SEVS compared to the non-cancerous SVHUC1 cells. The changes in the protein profiles in SEVs released following arsenic dosage indicated activation of pathways important for cell survival, viability, and migration and inactivation of pathways related to cell death and necrosis which were also observed in the paired cell lysate samples. Comparison between paired SEV and cell lysate samples, however, indicated selective SEV packaging of proteins which may be for the purpose of intracellular communication. Comparative assessment of SEVs from T24 and arsenic exposed SVHUC1 cells showed similar activation of cancer related pathways including those responsible for malignant tumors and increased proliferation rates. From the in vitro study results, we identified 8 potential SEV biomarkers. Of which, TGFBR1 showed the most promising association, having been positively associated with both inorganic arsenic and cadmium concentrations in urine samples. This thesis showed that SEVs are important mediators of arsenic exposure in urothelial cells and highlighted the comparability of SEV and cell lysate analysis. Furthermore, TGFBR1 was identified as a promising biomarker of arsenic exposure for its positive association with increased arsenic both in vitro and in human biomonitoring analysis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24493号 / 医博第4935号 / 新制||医||1063(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 浅野 雅秀, 教授 万代 昌紀, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Bladder cancer is the one of the most common malignancies of the genitourinary system and the overwhelming majority of those cases, approximately 90% in the United States(1), are of the urothelial/transitional cell histologic type. Small cell histologic type of bladder cancer is extremely rare with a mean frequency of 0.7% (1), and due to its rarity, there have not been any large phase III clinical trials in order to establish a definitive treatment regimen. We report here one such case of this rare type of bladder cancer and our approach towards treatment. A 69-year-old man had an incidental finding of microscopic hematuria during routine annual testing performed by his primary care physician. He was referred to a urologist for further evaluation, and in the interim, he began to experience symptoms of nocturia, dysuria and gross hematuria. Cystoscopy revealed a 5 cm sessile mass within the bladder and transurethral resection of the tumor was performed. Histopathological analysis of the tumor revealed muscle invasive poorly differentiated urothelial carcinoma with neuroendocrine features suggestive of small cell carcinoma. Follow-up systemic imaging only revealed multiple lesions in the liver, with the largest solitary liver lesion measuring 4.4 x 3.4 cm and no discrete lung lesions. Patient was started on palliative systemic chemotherapy with carboplatin and etoposide and follow-up imaging demonstrated excellent response after four cycles of treatment; however, follow-up imaging after the completion of 6 cycles of treatment demonstrated disease progression. Patient was referred for consideration of enrollment into any clinical trials; however, unfortunately no trials were found to be available. Patient was subsequently offered systemic treatment with single-agent immunotherapy with pembrolizumab. Due to development of left sided hydronephrosis, nephrostomy tube placement was performed and patient was also started on palliative radiation. Primary small cell carcinoma (SCC) of the bladder is an exceedingly rare malignancy and therefore, data is not readily available in order to guide treatment decisions. The most commonly administered regimen consists of etoposide with a platinum agent, and this regimen is extrapolated from the treatment of SCC of the lung. However, as for patients like ours, who had progression of disease in a short interval and are deemed primary treatment (platinum) refractory, the prognosis certainly becomes far more grim and the treatment choices even more limited. In sharing our treatment approach, we hope to be able to provide insight towards potential future treatment choices for this most-challenging diagnosis, primary small cell carcinoma of the bladder. (1) Blomjous CE, et. al. Small cell carcinoma of the urinary bladder. A clinicopathologic, morphometric, immunohistochemical, and ultrastructural study of 18 cases. Cancer. 1989 Sep 15; 64(6):1347-57.

Melanosis vesicae (or bladder melanosis) is a rare, benign condition referring to the presence of dark pigmented melanin deposits usually within the bladder mucosa. The clinical presentation can be mistaken for primary or metastatic melanoma of the bladder, and thus a histologic assessment is useful for ruling out a malignant diagnosis. All documented cases of melanosis vesicae have presented with urinary symptoms, including hematuria, symptoms of cystitis, incontinence and obstruction. This is a case report presenting a 57-year-old female with complaints of incomplete bladder emptying who met criteria for urinary retention. She underwent in-office cystoscopy and was found to have suspected melanosis vesicae. The diagnosis was later confirmed on pathology following a bladder biopsy. The next phase in care for this patient is planned sacral neuromodulation for treatment of urinary retention. Previous case reports of bladder melanosis have suggested an association with melanin deposition and inflammatory mechanisms and have not demonstrated malignant transformation during follow up. This is, to our knowledge, the first report of documented bladder melanosis in conjunction with urinary retention. Further studies are required to understand the etiology, clinical significance, and clinical correlation of melanosis vesicae with urinary dysfunction.

According to the CDC, the prevalence of diabetes has increased from 3.3% in 2004 to over 10.1% by 2019 (Prevalence of Diagnosed Diabetes). The United States Department of Agriculture (USDA) recommends that total fat intake should be between 20 - 35% of the total calories an individual consumes in a day and yet the Center for Disease Control and Prevention (CDC) reports that on average, total fat consumption makes up 35.8% of a person’s diet (Dietary guidelines, 2020 & CDC, 2021 respectively). According to Parrish, “dietary fat does not have an immediate effect on blood sugar levels, but consuming a meal high in fat can slow digestion and make it more difficult for insulin to work” (Parrish, 2015). Chronic diabetes can result in hypocontractility of the bladder. Contractility of the bladder is controlled by a multitude of receptors, ligands, and kinases. One receptor our group feels contributes is neuropilin 2. Our group has reported expression of neuropilin 2 in the smooth muscle of the bladder and has shown that expression induces cytoskeleton relaxation. Thus, it is thought that if neuropilin 2 expression is reduced, that potentially that hypocontractility of the bladder can be attenuated. In an in vivo model of diabetes using mice on a high fat diet for 5 months, we observed minimal changes in bladder histology, and variable Nrp2 expression. In silico analysis of data from in vivo and in vitro models of diabetes identified Nrp2 transcriptional induction compared to controls and a connection with multiple differentially expressed genes in the Nrp2 signaling pathway linked with biological processes related to a diabetic pathological state. An in vitro model of diabetes which subjected rat bladder contractile cells to high glucose identified significant cytoskeletal changes, increases in Nrp2 expression, and decreased contractility. Knock-down of Nrp2 using siRNA resulted in increased contractility of smooth muscle cells on collagen gels. These data suggest that Nrp2 signaling is altered under diabetic conditions and could be targeted to attenuate diabetes induced bladder hypocontractility. / 2025-07-06T00:00:00Z

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