Novel markers of exposure and pathways of response to Chromium

摘要 Cr（VI）是一种人类致癌物质，具有重大的公共卫生问题，在许多国家和地区， 职业环境。铬（VI）诱导突变，基因拷贝数的变化，并已暴露于 在暴露人群和动物模型中与人类癌症相关。我们新的初步数据 表明Cr（VI）暴露会导致核糖体DNA（rDNA）拷贝数扩增， 核仁（粗略理解的核细胞器，是核糖体RNA（rRNA）转录的位点， 以及无数细胞功能的整合）。核仁功能的一个关键因素是rDNA拷贝数 (rDNA CN）。rDNA CN调节（i）整个基因组的表观遗传状态，（ii）DNA损伤反应，（iii）细胞 周期进展，（iv）染色体分离，和（v）全局遗传稳定性。此外， rDNA阵列、核糖体生物发生和核仁是癌发生的中心。我们的中心医疗 有一种假说认为，铬诱导的rDNA CN的变化是铬诱导的致癌作用的原因。我们的中央 一个基本的假设是，rDNA阵列不是固定的，而是细胞核的遗传动态组成部分， 基因组拷贝数受铬暴露调节。我们的建议检查rDNA的变化， Cr（VI）暴露揭示了一种具有医学和基础相关性的铬毒性新途径。关键要素是 仔细研究铬诱导rDNA-a扩增（Cr-i-rDNA-a）的毒理学，假设驱动 Cr（VI）暴露后rDNA和核仁的功能基因组分析，以及广泛的遗传分析 Cr-i-rDNA-a基因的表达。我们的首要目标是研究铬的毒理学- 在人肺上皮细胞模型中诱导rDNA扩增（Cr-i-rDNA-a）。我们会确定剂量- Cr-i-rDNA-a的响应，绘制Cr-i-rDNA-a中的扩增边界，检查时间曲线， 从Cr（VI）暴露中恢复，并检查铬诱导的CN变化是否是导致Cr- 诱发癌我们的第二个目标是研究铬诱导的核仁应激的功能基因组学 和Cr诱导的转化。检查全基因组对Cr的反应 暴露对于理解铬如何诱导rDNA扩增、核应激和致癌作用是至关重要的。我们 第三个目标是解决铬-rDNA相互作用的遗传决定因素。我们将检测Cr-i-rDNA-a 特异性细胞，定量拷贝数变化的程度，分离受影响的组织，并使用高通量 技术来描述变化。我们的努力将揭示Cr-rDNA相互作用，研究表明， 与NIH的人类健康使命直接相关。rDNA CN的多重效应表明， 用Cr干扰该中枢调节器将对细胞功能产生深远的影响。我们预计 具有强烈环境因素的复杂人类疾病（如癌症）的决定因素， 最终可以追溯到环境引发的基因组rDNA片段的变异。

Abstract Cr(VI) is a human carcinogen of significant public health concern, and a substantial exposure in a number of occupational settings. Cr(VI) induces mutations, changes in gene copy number, and exposure has been associated with humans cancers in exposed populations and animal models. Our novel preliminary data demonstrate that Cr(VI) exposure causes amplification in ribosomal DNA (rDNA) copy number and changes in the nucleolus (the crudely understood nuclear organelle that is the site of ribosomal RNA (rRNA) transcription, and integration of myriad cellular functions). A crucial element of nucleolar function is rDNA copy number (rDNA CN). rDNA CN modulates (i) epigenetic states across the genome, (ii) DNA damage responses, (iii) cell cycle progression, (iv) chromosome segregation, and (v) global genetic stability. Furthermore, disruption of rDNA arrays, ribosome biogenesis, and the nucleolus are central to carcinogenesis. Our central medical hypothesis is that Cr-induced changes in rDNA CN are responsible for Cr-induced carcinogenesis. Our central basic hypothesis is that rDNA arrays are not fixed, but rather a genetically dynamic component of the nuclear genome with copy number that is modulated by Cr exposure. Our proposal examines rDNA changes upon Cr(VI) exposure to reveal a novel pathway of Cr toxicity with medical and basic relevance. Key elements are a careful investigation of the toxicology of Cr-induced-rDNA-amplification (Cr-i-rDNA-a), hypotheses-driven functional genomic analysis the rDNA and the nucleolus upon Cr(VI) exposure, and extensive genetic analyses of Cr-i-rDNA-a using a powerful model organism. Our first aim will investigate the toxicology of Chromium- induced-rDNA-amplification (Cr-i-rDNA-a) in a human lung epithelial cell model. We will determine dose- responses of Cr-i-rDNA-a, map amplification boundaries in Cr-i-rDNA-a, examine temporal profiles and recovery from Cr(VI) exposure, and examine whether Chromium-induced CN changes are responsible for Cr- induced carcinogenesis. Our second aim investigates the functional genomics of Cr-induced nucleolar stress and Cr induced transformation in a human lung epithelial cell model. Examining genome-wide responses to Cr exposure is critical to understand how Cr induces rDNA amplification, nuclelar stress, and carcinogenesis. Our third aim addresses the genetic determinants of Chromium-rDNA interactions. We will examine Cr-i-rDNA-a in specific cells, quantify the extent of copy number change, isolate the affected tissues, and use high-throughput techniques to characterize the changes. Our efforts will shed light on Cr-rDNA interactions, with research that is directly relevant to the human health mission of the NIH. The manifold effects of rDNA CN indicate that perturbing this central regulator with Cr will have profound consequences to cellular function. We anticipate that determinants of complex human diseases with strong environmental components such as cancer will ultimately be traced to environmentally triggered variation in rDNA segments of the genome.