Project 1: Critical Events In The Transformation of Human Bladder Cells

人的膀胱是对砷致癌最敏感的内脏器官。膀胱通过全身和尿液暴露于无机砷和甲基化代谢物中。砷的甲基化代谢物已被发现比无机砷更有细胞毒性。UROtsa细胞是一种长生不老的人类尿路上皮细胞，已成为一种公认的系统，用于检查砷对膀胱的毒性作用。一年慢性低水平接触亚砷酸盐[1 μ m As(III)]或毒性更大的砷代谢物[一甲基胂酸]；50 nM MMA(III)]转化UROtsa细胞，使其在注射到免疫缺陷小鼠体内形成肿瘤。目前的研究表明，短得多（3个月）暴露于低水平MMA（III）足以使它们在软琼脂中生长并在免疫缺陷小鼠中形成肿瘤。这些结果表明，MMA （III）在暴露于UROtsa细胞的前3个月引起关键的、不可逆的事件。因此，本提案的目标是发现短期、低水平的As（III）或MMA（III）在UROtsa细胞中导致其最终转变为恶性转化细胞的关键事件。为了实现这一目标，我们将对UROtsa细胞进行低水平、短期（0-3）暴露于砷[1微米砷（III）和50纳米MMA(III)]，以确定这些细胞发生恶性转化所需的最小暴露量和时间。一旦确定了这个最小暴露时间点，我们将检查细胞中大分子、信号系统或调控系统的特定变化，从而导致不可逆的变化。由于先前的研究发现低水平暴露于As（III）和MMA（III）会产生活性氧（ROS），我们将确定砷对UROtsa细胞的这些早期影响是否由ROS的产生介导。为了确定低水平砷是否能转化“正常”的人类膀胱细胞，将对人类膀胱细胞的原代培养物进行类似的检查。最后，低水平砷对膀胱细胞转化的关键事件将作为暴露人群中可能的生物标志物进行检查。总的来说，这些研究将阐明低水平砷在人体膀胱模型中的毒性作用，并为砷诱导提供潜在的生物标志物

The human bladder is the most sensitive internal organ to arsenic-induced carcinogenesis. The bladder is exposed to inorganic arsenicals and methylated metabolites both systemically and via the urine. Methylated metabolites of arsenic have been found to be more cytotoxic than inorganic arsenicals. The UROtsa cell, an immortalized human urothelium cell, has become an accepted system for examining the toxic effects of arsenicals to the bladder. Year-long chronic low-level exposure of arsenite [1 microM As(III)] or the more toxic arsenic metabolite [monomethyl-arsonous acid; 50 nM MMA(III)] transformed UROtsa cells such that they formed tumors when injected into immuno-deficient mice. Current studies indicate that much shorter exposures (3 months) to low-level MMA(III) is sufficient to allow them to grown in soft agar and form tumors in immuno-deficient mice. These results indicate that MMA (III) causes critical, irreversible events in UROtsa cells in the first 3 months of exposure. Thus the Goal of this proposal is to find the critical events that short-term, low-level As(III) or MMA(III) causes in UROtsa cells resulting in their eventual transition into malignantly transformed cells. To accomplish this goal we will do low-level, short-term (0-3) exposures of UROtsa cells to arsenicals [1 microM As(III) and 50 nM MMA(III)] to determine the minimal exposure and time required to malignantly transform these cells. Once this minimal exposure time point is established we will examine the cells for changes in specific alterations in macromolecules, signaling systems, or regulatory systems resulting in the irreversible changes. Since previous studies have found reactive oxygen species (ROS) generation by low-level exposure to As(III) and MMA(III), we will determine if these early effects of arsenicals on UROtsa cells are mediated by ROS generation. To determine if low-level arsenicals can transform "normal" human bladder cells, primary cultures of human bladder cells will be similarly examined. Lastly pivotal events in the transformation of bladder cells by low-level arsenicals will be examined as possible biomarkers in exposed populations. Overall, these studies will clarify the toxic effects of low-level arsenic in a human bladder model and provide potential biomarkers for arsenic-induced bladder injury.