Low-Level Arsenic Toxicity in Human Bladder Cells

Abstract

Arsenic is a human bladder carcinogen. Inorganic arsenic and methylated metabolites are excreted from the human body in urine. This study investigates the effects of arsenite [As(III)] and monomethylarsonous acid [MMA(III)] on human urothelial cells (UROtsa). Cytotoxicity studies found that MMA(III) was 20 times more toxic than As(III). In addition, UROtsa cells have the ability to biotransform As(III) to pentavalent and trivalent mono-methylated metabolites.To understand the mechanism of arsenic carcinogenesis, it is necessary to know which arsenicals are carcinogenic. Therefore, non-tumorigenic UROtsa cells were chronically exposed to 0.05 uM MMA(III) and monitored for signs of transformation. MMA(III)-treated cells (URO-MSC) became hyperproliferative after 12 weeks of exposure. Anchorage-independent growth was detected after 24 weeks of exposure to MMA(III). Gene array analysis conducted in URO-MSC cells after 52 weeks of exposure detected expression changes consistent with malignant transformation. Enhanced tumorigenicity in SCID mouse xenografts was also observed after 52 weeks of treatment.URO-MSC cells form squamous cell carcinoma, a histology associated with inflammation, when injected into SCID mice. Induction of cycolooxygenase-2 (COX-2) promotes proliferation, angiogenesis, and survival in cancer cells. To identify a potential mechanism of MMA(III) carcinogenesis, the effects of chronic and acute MMA(III) treatment on COX-2 expression were investigated. Western blot analysis revealed that COX-2 was induced in a time-dependent manner in URO-MSC cells. Acute MMA(III) exposure also increased COX-2 protein. To identify signal transduction pathways responsible for COX-2 induction, pharmacological inhibitors of various signaling pathways were co-administered with 0.05 uM MMA(III) and identified src and extracellular signal regulated protein kinase (ERK) activation to be responsible for COX-2 induction. Thus, MMA(III) causes ligand-independent activation of epidermal growth factor receptor (EGFR), which activates the signal cascade responsible for COX-2 expression. EGFR is elevated in URO-MSC cells. To determine if EGFR is a key mediator of URO-MSC cell tumorigenicity, inhibitors of downstream signal transduction (src, PI3K, and COX-1/-2) were found to reduce URO-MSC cell viability and growth in soft agar. Results from this work not only identify that MMA(III) can induce malignant transformation in human cells but also provides insight into the mechanism of arsenic-induced bladder cancer.