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

人体膀胱是对砷致癌最敏感的内脏器官。膀胱全身和通过尿液暴露于无机砷和甲基化代谢物。已发现砷的甲基化代谢物比无机砷化合物具有更强的细胞毒性。URotsa细胞是一种永生化的人类尿路上皮细胞，已经成为一种被接受的检测砷对膀胱毒性影响的系统。长达一年的长期低水平亚砷酸盐[1微米As(III)]或毒性更高的砷代谢物[单甲基亚砷酸；50 NM MMA(III)]转化UROtsa细胞，使其在注射到免疫缺陷小鼠体内时形成肿瘤。目前的研究表明，短得多的时间(3个月)暴露于低水平的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)在暴露的前3个月会在UROtsa细胞中引起关键的、不可逆的事件。因此，这项建议的目标是找出短期、低水平的As(III)或MMA(III)在URotsa细胞中引起的关键事件，从而导致它们最终转变为恶性转化的细胞。为了实现这一目标，我们将进行URotsa细胞对砷[1微米As(III)和50 NM MMA(III)]的低水平短期(0-3)暴露，以确定将这些细胞恶性转化所需的最小暴露和时间。一旦确定了这一最小暴露时间点，我们将检查细胞中导致不可逆变化的大分子、信号系统或调节系统中特定变化的变化。由于先前的研究已经发现低水平暴露于As(III)和MMA(III)会产生活性氧物种(ROS)，我们将确定这些砷化合物对URotsa细胞的这些早期效应是否由ROS产生所介导。为了确定低水平的砷是否可以改变“正常”的人类膀胱细胞，我们将对人类膀胱细胞的原代培养进行类似的研究。最后，低水平的砷化合物转化膀胱细胞的关键事件将被视为暴露人群中可能的生物标记物。总体而言，这些研究将阐明低水平砷在人类膀胱模型中的毒性效应，并为砷诱导的潜在生物标志物提供潜在的生物标志物。 膀胱受伤。

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.