Alcohol and Iron Derived Oxidant Stress Impact Epigenetic Regulation

描述（由申请人提供）：长期饮酒，通过几个过程，是许多癌症的危险因素，包括肝癌。铁超载也是肝癌的一个危险因素。基本原理。已知酒精通过多效性影响s-腺苷基蛋氨酸（SAMe）的合成。我们假设铁及其加剧氧化应激产生的潜力也可能通过诱导碳通过转硫途径补充谷胱甘肽（GSH）来扰乱SAMe合成。这种对细胞中甲基供体库的影响有可能影响表观遗传调节，特别是通过减弱DNA甲基化。表观遗传变化导致参与控制增殖、凋亡、DNA完整性、血管生成和其他过程的关键基因的表达改变，这对癌症的进展很重要。因此，我们假设，暴露于铁源性氧化应激引起的谷胱甘肽水平的变化会对同型半胱氨酸库造成压力，从而增强乙醇对SAMe的抑制作用，从而导致依赖于这种甲基供体的表观遗传调节反应的改变。同型半胱氨酸提供产生SAMe的蛋氨酸和产生谷胱甘肽的半胱氨酸。本探索性R21提案的目的是通过关键实验来阐明铁诱导的氧化应激与酒精相关癌症发展的表观遗传调控之间的关系。在此过程中，将生成支持该领域后续R01应用程序的关键数据。我们的研究结果将确定铁和乙醇在多大程度上汇聚影响一个基本的重要途径，并表明这是否反过来可能影响基因调控中涉及遗传变化的过程。具体目的：确定乙醇和铁是否联合影响甲基供体的可用性和肝细胞的表观遗传调控。我们的初步数据与一个可诱导铁蛋白H转基因模型表明，我们可以改变铁在组织特异性方式在体内的可用性。我们还证明了评估肝组织中SAMe和SAH水平的能力，并分析了DNA甲基化的变化。我们将利用这一专业知识在体内测试乙醇和铁单独或共同对以下方面的影响：A)肝脏肿瘤前病变的形成；B) SAMe和GSH的可用性；C)全局和基因启动子特异性（e-cadherin和HAI-2/PB） DNA甲基化；D)氧化应激为4-HNE和羰基加合物形成，氧化为还原性GSH比值，还原应激为NAD+/NADH比值。通过回答铁和乙醇是否影响表观遗传调控的问题，我们增加了对微环境影响细胞的机制的理解。鉴于氧化应激和表观遗传基因调控在癌基因刺激和染色体不稳定中的重要性，这一进展可能最终帮助我们设计预防和治疗乙醇相关癌症发展的策略。

DESCRIPTION (provided by applicant): Chronic alcohol consumption, via several processes, is a risk factor for many cancers, including liver cancer. Iron overload is also a risk factor for liver cancer. RATIONALE. Alcohol is known to have an impact on s-adenosyl methionine (SAMe) synthesis, via pleotropic effects. We posit that iron and its potential to exacerbate oxidant stress generation may also perturb SAMe synthesis through drawing carbon to replenish glutathione (GSH) via the transsulfuration pathway. Such an impact on the methyl donor pool in cells has the potential to affect epigenetic regulation, specifically by attenuating DNA methylation. Epigenetic changes that result in altered expression of critical genes involved in control of proliferation, apoptosis, DNA integrity, angiogenesis and other processes are important to cancer progression. Thus, we hypothesize that changes in GSH levels brought about through exposure to iron derived oxidant stress will stress the pool of homocysteine, potentiating ethanol's inhibition of SAMe leading to alterations in epigenetic regulation reactions that depend on this methyl donor. Homocysteine provides both methionine to produce SAMe and cystathionine to produce GSH. The OBJECTIVE of this exploratory R21 proposal is to conduct key experiments to elucidate the relationship between iron induced oxidant stress and epigenetic regulation in alcohol-related cancer development. In doing so, critical data to support a subsequent R01 application in this area will be generated. Our results will determine the extent to which iron and ethanol converge to impact a fundamentally important pathway and indicate if this in turn may affect processes that are involved with heritable changes in gene regulation. Specific Aim: to determine if ethanol and iron combine to affect methyl donor availability and epigenetic regulation in liver cells in vitro and in vivo. Our preliminary data with a tet-inducible Ferritin H transgenic model indicate we can alter iron availability in a tissue specific manner in vivo. We also demonstrate the capacity to evaluate SAMe and SAH levels in liver tissue, and to analyze alterations in DNA methylation. We will use this expertise in vivo, to test the impact of ethanol and iron independently and together on: A) the formation of pre-neoplastic lesions in the liver; B) SAMe and GSH availability; C) global and gene promoter specific (e-cadherin and HAI-2/PB) DNA methylation; D) oxidant stress as 4-HNE and carbonyl adduct formation, oxidized to reduced GSH ratios, and reductive stress as NAD+/NADH ratios. By answering the question of whether iron and ethanol affect epigenetic regulation, we increase our understanding of the mechanisms by which the microenvironment influences the cell. Given the importance of oxidant stress and epigenetic gene regulation in oncogene stimulation and chromosomal instability, such progress may ultimately help us design strategies to prevent and treat the occurrence of ethanol related cancer development. PUBLIC HEALTH RELEVANCE: Alcohol drinking is known to place the drinker at increased risk for many types of cancer. Individuals that have iron overload disease are at much higher risk of developing liver cancer if they drink alcohol regularly. In addition, how cells handle iron can be influenced by alcohol, making iron an important factor in the development of alcohol associated cancer. Iron acts by causing an increase in a process that damages the cell, called oxidant stress. We think this process may affect a chemical that cells make that is responsible for regulating genes through modifying DNA. We propose to find out if this occurs in liver cells in mice. If this is the case, then we will have identified a mechanism by which iron and oxidant stress may help to drive the formation of cancer in alcohol drinkers. Discovering such a mechanism would give us targets that may help prevent alcohol associated cancer formation.