INFLUE HETEROCYCLIC AMINE METABOLIC POLYMORPH ON BIOACTIVAT PHIP IN HUMAN COLON

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 本研究的主要目的是通过证明 NAT2 和 CYPIA2 表型以及 GSTM1 和 CYPIA1 基因型与在摄入相关水平的 14C-PhIP 的人类中形成的 PhIP-DNA 加合物的水平相关，来证实 PhIP 代谢在结肠癌中的作用。 这些因素对于研究很重要，因为根据啮齿动物研究，它们代表了被认为在 PhIP 代谢中很重要的酶。 这项工作的结果将进一步证明杂环胺（例如 PhIP）与结肠癌有关，因为这些酶在杂环胺的代谢中很活跃。 此外，这项工作将表明基因型或表型是否是 PhIP 对结肠癌易感性的潜在有用标记。 我们收集了人体数据，通过确定 NAT2 和 CYPIA2 表型、GSTM1 和 CYPIA1 基因型以及人体中形成​​的 PhIP-DNA 加合物水平之间的关系，确定 PhIP 代谢在结肠癌中的作用。 这是通过对诊断为结肠癌并计划进行手术的人类受试者施用极低水平的 14C 标记 PhIP 来实现的。迄今为止，来自 10 名受试者的数据表明 SULT、NAT2 和 CYP1A2 表型影响结肠中的加合物水平。计划进行一项更大规模、更具统计意义的研究来验证这些发现。 需要在乳腺癌、前列腺癌和肺癌等其他癌症中进行类似的工作。 在这些领域中，疾病造成的死亡人数都很大，预防策略因疾病病因学和代谢途径领域的知识缺乏而受到限制，而且暴露信息不完整。 研究总结及结果： 环境中致癌物质暴露的最重要来源之一是通过饮食。 流行病学证据表明，饮食中接触杂环胺 (HA) 是发生结肠癌的重要危险因素。 煮熟的肉类含有大量的HA，已被证明会导致实验动物患癌症。 为了更好地了解人体内 HA 生物激活和潜在肿瘤诱导的机制，使用质量最丰富的 HA，2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶 (PhIP) 来评估 PhIP 代谢与 DNA 加合物形成之间的关系。 10 名人类志愿者在手术切除结肠肿瘤前 48-72 小时服用与饮食相关剂量的 14C-PhIP (70-84 µg)。 给药后24小时收集尿液进行代谢物分析，从结肠组织中提取DNA并通过加速器质谱分析DNA加合物。 还对所有 10 名受试者进行了 CYP1A2、NAT2 和 SULT1A1 酶活性表型分析。 在尿液样本中检测到 12 个与 PhIP 相关的放射性峰。 所有 10 名志愿者中最丰富的代谢物被鉴定为 N-羟基-PhIP-N2-葡萄糖醛酸苷。 观察到志愿者之间代谢物水平的差异。 在所有 10 名志愿者中均检测到结肠 DNA 加合物。 每个个体之间的 DNA 加合物水平存在明显的个体差异。 数据分析表明，尿液中 N-羟基-PhIP-N2-葡萄糖苷酸水平较高且被归类为具有快速 CYP1A2 表型的个体的结肠 PhIP-DNA 加合物水平最低。 意义：这些数据表明葡萄糖醛酸化在 N-羟基-PhIP 解毒中发挥着重要作用。 统计分析表明，尿液中 N-羟基-PhIP-N2-葡萄糖苷酸水平与结肠 DNA 加合物水平呈负相关。 尽管很难从小数据集中得出明确的结论，但这项试点研究的结果鼓励使用更大的研究小组进行进一步的调查。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The main objective of this research is to confirm the role of PhIP metabolism in colon cancer by demonstrating that the NAT2 and CYPIA2 phenotype, as well as GSTM1 and CYPIA1 genotype are related to the level of PhIP-DNA adducts formed in humans administered dietary relevant levels of 14C-PhIP. These factors are important to study because they represent enzymes that are thought to be important in PhIP metabolism based on rodent studies. The outcome of this work will be additional proof that heterocyclic amines such as PhIP are involved in colon cancer since these enzymes are active in the metabolism of heterocyclic amines. Additionally, this work will show whether genotype or phenotype are potentially useful markers of PhIP susceptibility for colon cancer. We have collected data in humans to establish the role of PhIP metabolism in colon cancer by determining the relationship between NAT2 and CYPIA2 phenotype, as well as GSTM1 and CYPIA1 genotype and the level of PhIP-DNA adducts formed in humans. This was accomplished by administering very low levels of 14C-labeled PhIP to human subjects with a diagnosis of colon cancer who are scheduled for surgery. Data so far from 10 subjects indicates that SULT, NAT2 and CYP1A2 phenotypes influence adduct levels in the colon. A larger, more statistically significant, study is planned to verify the findings. There is a need to perform similar work in other cancers such as breast cancer, prostate cancer and lung cancer. In each of these areas, the disease toll is great, the prevention strategies are limited by a knowledge deficit in the areas of disease etiology and metabolism pathway and exposure information is incomplete. Study summary and results: One of the most significant sources of environmental exposures to carcinogens is through the diet. Epidemiologic evidence indicates that exposure to heterocyclic amines (HAs) in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of HAs which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of HA bioactivation and potential tumor induction in humans, the most mass abundant HA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of 14C-PhIP (70-84 ¿g) 48-72 h prior to surgery to remove colon tumors. Urine was collected for 24 h after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All ten subjects were also phenotyped for CYP1A2, NAT2, and SULT1A1 enzyme activity. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The most abundant metabolite in all ten volunteers was identified as N-hydroxy-PhIP-N2-glucuronide. Variation in the levels of metabolites between volunteers was observed. Colon DNA adducts were detected in all ten volunteers. Interindividual differences were evident in the levels of DNA adducts between each individual. Analysis of the data showed that individuals with high levels of urinary N-hydroxy-PhIP-N2-glucuronide, and that were categorized as having a rapid CYP1A2 phenotype had the lowest level of colon PhIP-DNA adducts. Significance: These data suggest that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. Statistical analyses showed that levels of urinary N-hydroxy-PhIP-N2-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.