Oxidative Stress in Alcohol-associated Oral Carcinogenesis

作为一种种族差异显著的癌症，在所有类型的癌症中，口腔癌与饮酒的相关性最强。这种联系取决于酒精的剂量和浓度。我们的初步数据清楚地表明，口服酒精(8%、15%、35%和50%)促进了4-硝基喹啉-1-氧化物(4NQO)处理的小鼠口腔癌的发生。用这些浓度的酒精分别在人体内模拟饮用啤酒、葡萄酒、稀释酒或原酒。在同一动物模型中，Nrf2基因敲除显著增强了癌症的发生，Nrf2基因敲除调控多种抗氧化第11阶段酶的表达。因此，我们假设酒精通过在口腔上皮细胞中产生氧化应激来促进口腔癌的发生，而Nrf2介导的抗氧化反应可能在预防口腔癌中发挥关键作用。我们计划在这项拨款提案中测试我们的假设，具体目标如下： 1.确定酒精是否通过口腔上皮细胞的氧化应激促进4NQO诱导的小鼠口腔癌的发生，以及细胞色素P450-2E1是否是导致酒精引起的氧化应激的主要酶。4NQO处理组小鼠灌胃酒精(35%)，在实验过程中(8，9，12，16，24周)处死。将分析小鼠舌头的组织病理学、氧化损伤和Nrf2介导的抗氧化反应。利用野生型和CYP2E1[-/-]小鼠，我们还将确定酒精引起的口腔上皮细胞氧化应激是否依赖于CYP2E1。 2.利用Nrf2[-/-]和Keap1[-/-]小鼠研究Nrf2在酒精诱导的氧化应激和口腔癌发生中的作用。4NQO处理的野生型和Nrf2[-/-]小鼠给予酒精喂养，分析口腔上皮的组织病理学、氧化损伤和抗氧化反应，以确定Nrf2[-/-]在酒精促进的氧化应激和口腔癌变中的作用。一项对Keap1[-/-]小鼠的短期实验将确定Nrf2的基因激活是否可以抑制酒精促进的氧化应激。 3.探讨Nrf2化学激活剂对酒精所致口腔癌的化学预防作用。4NQ0处理的野生型和Nrf2[-/-]小鼠将被酒精喂养，并用两种来自饮食来源的化学上不同的Nrf2激活剂--萝卜硫醚和~3H-1，2-二硫醇-3-硫酮进行治疗。将分析口腔上皮的组织病理学、氧化损伤和抗氧化反应。

As a cancer of significant racial disparity, oral cancer has the strongest association with alcohol consumption among all types of cancer. Such an association depends on both the dose and the concentration of alcohol. Our preliminary data have clearly shown that oral administration of alcohol (8%, 15%, 35%, and 50%) promoted oral carcinogenesis in 4-nitroquinoline-1-oxide (4NQO)-treated mice. Treatment with alcohol of these concentrations mimicked drinking beer, wine, diluted liquor, or straight liquor in humans, respectively. Gene knockout of Nrf2, which regulates expression of multiple anti-oxidative phase 11 enzymes, dramatically enhanced carcinogenesis in the same animal model. Therefore we hypothesize that alcohol promotes oral carcinogenesis by generating oxidative stress in the oral epithelium, and Nrf2-mediated antioxidative response may play a critical role in preventing oral cancer. We plan to test our hypothesis in this grant proposal with the following specific aims: 1. To determine whether alcohol promotes 4NQO-induced oral carcinogenesis in mice through oxidative stress on the oral epithelium, and whether Cyp2E1 is a major enzyme leading to alcohol-generated oxidative stress. 4NQO-treated mice will be fed with alcohol (35%), and sacrificed at several time points during the experiment (Week 8, 9, 12, 16, 24). Mouse tongue will be analyzed for histopathology, oxidative damage and Nrf2-mediated antioxidative response. Using wild-type and Cyp2E1[-/-] mice, we will also determine whether alcohol-generated oxidative stress in the oral epithelium is dependent on Cyp2E1. 2. To examine the role of Nrf2 in alcohol-promoted oxidative stress and oral carcinogenesis using Nrf2[-/-] and Keap1[-/-] mice. 4NQO-treated wild-type and Nrf2[-/-] mice will be fed with alcohol, and histopathology, oxidative damage, and anti-oxidative response in the oral epithelium will be analyzed to determine the impact of Nrf2[-/-] on alcohol-promoted oxidative stress and oral carcinogenesis. A short-term experiment on Keap1[-/-] mice will determine if genetic activation of Nrf2 may inhibit alcohol-promoted oxidative stress. 3. To investigate whether chemical activators of Nrf2 have chemopreventive effects on alcohol-promoted oral carcinogenesis. 4NQ0-treated wild-type and Nrf2[-/-] mice will be fed with alcohol, and treated with two chemically distinct Nrf2 activators from dietary sources, sulforaphane and 3H-1,2-dithiole-3-thione. Histopathology, oxidative damage and antioxidative response in the oral epithelium will be analyzed.