Nephrotoxic mechanisms of f low-rank coal (lignite) leachates in drinking water

摘要 巴尔干地方性肾病(BEN)是一种退行性疾病，常与尿路上皮癌和 在地理上仅限于塞尔维亚、保加利亚、罗马尼亚、克罗地亚和波斯尼亚的农村地区。本病的病因学 有争议，几个环境因素被认为是导致这种疾病的因素。上新世 褐煤假说将本与接触饮用水中的有机物质联系在一起，这些有机物质是从低等级的水中浸出的 被称为褐煤的煤。这一假设解决了本的有限地理性质，因为大多数村庄 该病的困扰也与褐煤矿床密切相关。我们和其他人已经展示了 美国东南部褐煤沉积物与培养的肾细胞毒性和增加的 终末期肾病(ESRD)的发病率。这项研究的总体目标是测试特定的 假设美国墨西哥湾沿岸地区饮用水中褐煤水萃取物的环境水平 在本样肾毒性体内。目标1将检验饮用水接触褐煤的预测 来自美国东南部的提取物导致剂量依赖的肾毒性。在这项研究中，男性和女性 将SD(S-D)大鼠暴露于其饮用水中煤提取物的环境水平6个月 月份。样本将是被发现对肾细胞有毒的褐煤提取物(多莱特希尔斯，洛杉矶和蒙蒂塞洛， 或发现对肾脏细胞毒性较小的褐煤提取物(德克萨斯州圣米格尔；阿肯色州温泉县)。水 每周将评估摄入量、动物体重和一般健康状况。血液和24小时尿液将于3：00采集 6个月后，对一组动物进行肾功能指标评估(尿渗透压、NAG水平； 血清尿素氮和肌酐)。肾脏将进行毒性、细胞死亡和炎症的大体评估。 并由兽医病理学家进行显微镜检查，其他潜在的靶器官(膀胱、肺、心脏、肝脏、 脾、脑、皮肤、全血计数)。目标2将检验褐煤降解产物的预测， 特别是极性有机化合物，对肾脏细胞具有细胞毒性。在这一目标中，有毒提取物(多莱特希尔斯，洛杉矶； 蒙蒂塞洛，德克萨斯州)将通过固相萃取和两步洗脱过程分离，以产生 非极性组分(溶于正己烷的化合物)和极性组分(溶于甲醇的化合物)。 这些组分在不同的浓度下适用于HK-2和RPTEC/TERT1肾脏 细胞，并进行细胞毒性评估。这项提议的影响是开始将这两个发现联系在一起， 即检测大鼠饮用水中褐煤提取物是否具有肾毒性，并开始了解 这种肾毒性的褐煤成分/机制。

Abstract Balkan endemic nephropathy (BEN) is a degenerative disease that is often associated with urothelial cancer and is geographically confined to rural parts of Serbia, Bulgaria, Romania, Croatia, and Bosnia. The etiology of BEN is debated, and several environmental factors have been considered as contributors to the disease. The Pliocene lignite hypothesis links BEN and exposure to organic material in drinking water that is leached from a low-rank coal known as lignite. This hypothesis addresses the limited geographic nature of BEN, as most of the villages plagued with the disease are also closely associated with lignite deposits. We and others have shown an association of lignite deposits in the southeastern US with renal cell toxicity in culture and with an increased incidence of end-stage renal disease (ESRD).The overall goal of the studies in this work are to test the specific hypothesis that environmental levels of U.S. Gulf Coast region lignite aqueous extracts in drinking water result in BEN-like nephrotoxicity in vivo. Aim 1 will test the prediction that drinking water exposure to lignite coal extracts from the southeastern US results in a dose-dependent nephrotoxicity. In this study, male and female Sprague-Dawley (S-D) rats will be exposed to environmental levels of coal extracts in their drinking water for six months. Samples will be either lignite extracts found to be toxic to renal cells (Dolet Hills, LA and Monticello, TX); or lignite extracts found to be less toxic to kidney cells (San Miguel, TX; Hot Springs County, AR). Water intake, animal weights and general health will be assessed weekly. Blood and 24 hr urine will be collected at 3 and 6 months from a subset of animals and assessed for renal function markers (urine osmolality, NAG levels; serum urea nitrogen and creatinine). Kidneys will be assessed for toxicity, cell death and inflammation grossly and microscopically by a veterinary pathologist as will other potential target organs (bladder, lungs, heart, liver, spleen, brain, skin, complete blood count). Aim 2 will test the prediction that lignite degradation products, specifically polar organic compounds, are cytotoxic to renal cells. In this aim, toxic extracts (Dolet Hills, LA; Monticello, TX) will be separated by solid-phase extraction followed by a two-step elution process to generate a non-polar component (compounds soluble in hexane) and polar component (compounds soluble in methanol). These fractions applied at various concentrations as in our preliminary work to HK-2 and RPTEC/TERT1 renal cells and assessed for cytotoxicity. The impact of this proposal is to begin to tie these two findings together, namely to test whether lignite coal extracts in the drinking water of rats is nephrotoxic and to begin to understand the lignite components/mechanisms mediating this nephrotoxicity.