Chemoprevention by Black Raspberry of Oral Cancer Induced by Tobacco Carcinogens: Translational Studies

项目摘要 烟草使用仍然是口腔鳞状细胞癌（OSCC）的高风险因素， 强有力的烟草致癌物，包括多环芳烃，如二苯并[a，l]芘 (DB[a，1]P）、苯并[a]芘（B[a]P）和烟草特异性亚硝胺（TSNA）如N '-亚硝基去甲烟碱 （NNN）。为了评估这些致癌物对口腔粘膜的影响，我们开发了一种新的OSCC小鼠模型 使用DB[a，l]P及其峡湾区二醇环氧化物（DB[a，l]PDE）。重要的是，我们发现饮食干预 与冷冻干燥的黑树莓（BRB）粉抑制致癌物诱导的DNA损伤，诱变和 在小鼠口腔中的致癌作用。我们还确定，BRB减少形成和/或增强 在体外修复大的加合物，但是，BRB减少DNA损伤的机制仍然是完全的， 阐明。考虑到DNA加合物结构的多样性，我们将重点讨论BRB对核苷酸的影响， 切除修复（NER）和碱基切除修复（BER）酶。基于丰富的酚类化合物 在BRB例如花青素中，合乎逻辑地提出BRB有助于保持细胞氧化还原平衡，增强氧化还原平衡， 清除，从而防止氧化DNA损伤的功能;我们的数据支持这一主张。此外 与其细胞保护功能相关的是一种特异性的细胞还原等价物-NADPH-， 生物合成反应，包括将核糖核苷酸转化为脱氧核糖核苷酸（dNTP）， DNA修复的组成部分。基于这些数据，我们假设BRB减少DNA损伤（参见。 方案1，显著性）：1）BRB通过保存 关键底物和辅因子，即dNTPs和Mg 2+，同时保持最佳的非氧化环境 有助于DNA修复; 2）BRB通过有效清除活性物质来保持细胞的氧化还原状态 它可以抑制DNA修复酶，从而减少氧化DNA损伤。提出的机制 这些研究需要两个互补但独立的具体目标：目标1A将研究BRB的效果 烟草致癌物-DNA加合物和8-OXO-dG对原代人细胞修复的影响 使用我们建立的测定法用这些加合物转染的口腔角质形成细胞。目的 1B将决定BRB防止野生型（OGG 1 +/+，一种成分， BER酶）和敲除（OGG 1-/-）MEF细胞。适用于两个子目标的同步研究将 评估BRB对dNTP池保存的影响，以及在连续的 经验证的细胞包括原代人口腔角质形成细胞、口腔白斑和OSCC细胞。目标2将 首次确定局部BRB递送对共价DNA加合物形成的影响， 健康吸烟者口腔细胞中的8-OXO-dG。这些结果可以作为未来的框架。 对无法戒烟的吸烟成瘾者以及无法戒烟的非吸烟者或前吸烟者进行化学预防试验。 暴露在环境致癌物中。

Project Abstract Tobacco use remains a high risk factor for oral squamous cell carcinoma (OSCC) resulting from exposure to potent tobacco carcinogens including polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[a,l]pyrene (DB[a,l]P), benzo[a]pyrene (B[a]P) and tobacco-specific nitrosamines (TSNA) such as N’-nitrosonornicotine (NNN). To assess effects of these carcinogens on oral mucosa, we developed a novel OSCC mouse model using DB[a,l]P and its fjord region diol epoxide (DB[a,l]PDE). Importantly, we showed that dietary intervention with freeze-dried black raspberries (BRB) powder inhibited carcinogen-induced DNA damage, mutagenesis and carcinogenesis in the mouse oral cavity. We have also established that BRB reduce formation and/or enhance repair of bulky adducts in vitro, but, the mechanisms by which BRB reduce DNA damage remain to be fully elucidated. Considering the varied structures of DNA adducts, we will focus on BRB effects on the nucleotide excision repair (NER) and base-excision repair (BER) enzymes. Based on the abundant phenolic compounds in BRB e.g. anthocyanins, it is logical to propose that BRB help preserve the cellular redox poise, enhance redox scavenging and thus function to prevent oxidative DNA damage; our data support this proposition. In addition to their cytoprotective functions, a specific cellular reducing equivalent-NADPH-functions in reductive biosynthetic reactions including conversion of ribonucleotides to deoxyribonucleotides (dNTPs) that are essential components for DNA repair. Based on these data, we hypothesize that BRB reduce DNA damage (cf. Scheme 1, Significance) in a multimodal fashion: 1) BRB enhance NER and BER function via preservation of key substrates and cofactors i.e. dNTPs and Mg2+, while maintaining an optimal, non-oxidized environment conducive to DNA repair; 2) BRB preserve the cellular redox status via efficient scavenging of reactive species that can inhibit DNA repair enzymes thereby reducing oxidative DNA damage. The proposed mechanistic studies entail two complementary, yet independent, Specific Aims: Aim 1A will investigate the effects of BRB on repair (NER, BER) of covalent tobacco carcinogen-DNA adducts and 8-OXO-dG in primary human oral keratinocyte cells that have been transfected with these adducts using our established assay. Aim 1B will determine the capacity of BRB to prevent oxidative damage in wild-type (OGG1+/+, a component of BER enzymes) and knockout (OGG1-/-) MEF cells. Concurrent studies, applicable to both subaims, will assess BRB effect on preservation of dNTP pools and key cellular redox poise parameters in a continuum of validated cells ranging from primary human oral keratinocytes, oral leukoplakia and OSCC cells. Aim 2 will determine for the first time the effects of local BRB delivery on formation of covalent DNA adducts and 8-OXO-dG in buccal cells of healthy smokers. The results could serve as the framework for future chemopreventive trials for addicted smokers who are unable to quit as well as non- or former-smokers who are exposed to environmental carcinogens.