DETERMINANTS OF DIETARY RISK OF HETEROCYCLIC AMINES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在过去的15年里，我们一直在研究饮食对癌症的作用。富含蛋白质的食物的烹调、热处理和热解会形成一组结构相关的杂环芳香胺，这些胺在许多分析系统中被发现是有效的诱变剂。这些相同的化合物在雄性和雌性小鼠和大鼠以及100%的非人类灵长类动物的多个器官部位产生肿瘤，给予其中一种杂环胺后，在很短的潜伏期后患上肝癌。鉴于这些非常令人信服的数据，确定这些膳食诱变剂/致癌物对人类癌症发病率的影响程度以及制定限制其影响的策略是很重要的。这个NIH计划项目试图通过以下方式实现这些目标：-确定和量化人类饮食中这些杂环胺的摄入量；-通过分析啮齿类动物长期喂食后的DNA结合、细胞遗传损伤和突变效应，了解这些化合物的慢性毒理学；-通过表征重要的代谢途径（啮齿动物、非人类灵长类动物、人类），了解动物研究与人类的机制相关性，并进一步了解这些杂环胺诱导肿瘤形成的组织特异性本质；-通过评估低剂量摄入这些强效诱变剂的剂量学，了解高剂量动物研究与人类风险评估的剂量相关性；-描述与突变相关的致癌物和DNA加合物的结构特征；-利用CHO细胞和在DNA修复和代谢激活方面具有遗传差异的啮齿动物品系，预测修复和代谢中的个体差异的重要性；-识别和验证可能对人类风险或易感性测定有用的生物标志物；-评估本项目产生的数据和文献数据，以进行定量的癌症风险评估。很明显，AMS资源对NIH资助的项目项目产生了重大影响，因为这项工作的各个方面只能利用加速器质谱法来完成。特别是，AMS允许我们在低剂量下进行代谢和DNA损伤的研究，这影响了我们计划项目资助的每个子项目。摘要：细胞色素p450介导的羟基化和UDP-glucuronosyltransferase （UGT）催化的葡萄糖醛酸化是杂环胺（HAs）等多种外源生物转化的主要代谢途径。研究表明，在人类中，HA PhIP的生物活化高度依赖于细胞色素p4501a2介导的n -羟基化对相应的n -羟基PhIP的作用。随后的n -葡糖醛酸化反应生成反应性较低的n -羟基- phip - n2 -葡糖醛酸和n -羟基- phip - n2 -葡糖醛酸，可通过尿液或胆汁排出，或被转运到肝外组织进行进一步代谢。最近的研究表明，在人类中，n -羟基-PhIP的糖醛酸化是PhIP生物转化的主要途径，而UGT1A1同工酶是n -羟基-PhIP糖醛酸化的主要参与者。此外，几种ugt的多态性表达导致了许多底物的差异代谢。这种UGT表达的个体间差异可能潜在地改变某些个体中致癌物如PhIP的生物活性。因此，了解人类PhIP和n -羟基-PhIP的n -葡萄糖醛酸化尤为重要，因为通过葡萄糖醛酸化未能结合n -羟基-PhIP可能导致进一步的酯化反应活化。这些反应会产生高活性的化合物，可以结合DNA，可能导致突变。由于在动物模型中，PhIP的生物激活已被证明与多种组织中DNA加合物的形成有关，因此通过确定葡萄糖醛酸化对啮齿类动物模型中PhIP的组织特异性生物激活/解毒的作用，可以更好地了解PhIP代谢如何促进整个动物中DNA加合物的形成。利用加速器质谱法，我们可以分析肿瘤靶组织中DNA加合物的形成和膳食中PhIP的相关剂量。我们假设糖醛酸化能力降低的动物和/或组织比UGT活性增加的动物更容易受到PhIP诱导的肿瘤靶组织中DNA加合物的影响。Malfatti, m.a., Ubick E.A.和Felton， J.S.（2005）葡萄糖醛酸化对熟食品致癌物2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶体内生物活性和DNA内聚的影响。致癌作用，出版中。

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. Over the last 15 years we have been studying the role of diet on cancer. The cooking, heat processing, and pyrolysis of protein-rich foods result in the formation of a group of structurally related heterocyclic aromatic amines that have been found to be potent mutagens in a number of assay systems. These same compounds produce tumors at multiple organ sites in both male and female mice and rats and 100 percent of non-human primates given one of these heterocyclic amines developed hepatocarcinomas after a very short latency. Given these very compelling data, it is important to determine the extent to which these dietary mutagens/carcinogens contribute to the human cancer incidence and to devise strategies to limit their impact. This NIH program project attempts to achieve these goals by: - Identifying and quantifying the human intake of these heterocyclic amines in the diet; - Understanding the chronic toxicology of these compounds by analysis of DNA binding, cytogenetic damage and mutational effects following chronic long-term feeding exposure of rodents; - Understanding the mechanistic relevance of animal studies for humans by characterizing important metabolic pathways (rodents, non-human primates, humans) with the additional goal of understanding the nature of the tissue specificity in tumor formation induced by these heterocyclic amines; - Understanding the dose-relevance of high dose animals studies for human risk assessment by assessing the dosimetry from ingestion of these potent mutagens at low doses; - Characterizing the structural features of carcinogens and DNA adducts that are correlated with mutation; - Predicting the importance of individual differences in repair and metabolism using CHO cells and rodent strains having genetic differences in DNA repair and metabolic activation; - Identifying and validating biomarkers that may be useful for human risk or susceptibility determinations and; - Evaluating data generated from this project and data from the literature to produce quantitative cancer-risk assessment. It is clear that the AMS resource is having a major impact on this NIH funded Program Project, as aspects of this work can only be accomplished utilizing accelerator mass spectrometry. In particular, AMS allows us to conduct studies of metabolism and DNA damage at low dose that impacts every sub-project of our program project grant. Current study abstract: Cytochrome P450-mediated hydroxylation and UDP-glucuronosyltransferase (UGT)-catalyzed glucuronidation are major metabolic pathways in the biotransformation of many xenobiotics including heterocyclic amines (HAs). Studies have shown that, in humans, the bioactivation of the HA PhIP is highly dependent upon cytochrome P4501A2-mediated N-hydroxylation to the corresponding N-hydroxy-PhIP. Subsequent N-glucuronidation results in the formation of the less reactive N-hydroxy-PhIP-N2-glucuronide and N-hydroxy-PhIP-N3-glucuronide, which can be excreted through urine or bile, or can be transported to extrahepatic tissue where further metabolism can occur. Recent studies have shown that, in humans, glucuronidation of N-hydroxy-PhIP is a major pathway in the biotransformation of PhIP and that the UGT1A1 isozyme is a major contributor to N-hydroxy-PhIP glucuronidation. In addition, polymorphic expression of several UGTs has led to differential metabolism of many substrates. This inter-individual variation in UGT expression could potentially alter the bioactivation of pro-carcinogens such as PhIP in certain individuals. Therefore, understanding the N-glucuronidation of PhIP and N-hydroxy-PhIP in humans is especially important because the failure to conjugate N-hydroxy-PhIP by glucuronidation could result in further activation by esterifying reactions. These reactions would result in highly reactive compounds that can bind DNA, potentially causing mutations. Since PhIP bioactivation has been shown to be responsible for the formation of DNA adducts in multiple tissues in animal models, by determining the role glucuronidation has on tissue-specific bioactivation/detoxification of PhIP in rodent models, a better understanding of how PhIP metabolism contributes to DNA adduct formation in whole animals can be established. By using accelerator mass spectrometry we can analyze DNA adduct formation in tumor target tissues and dietary relevant doses of PhIP. We hypothesize that animals and/or tissues with diminished glucuronidation capacity will be more susceptible to PhIP induced DNA adducts in tumor target tissues than animals with increased UGT activity. This work resulted in the following InPress publication: Malfatti, M.A., Ubick E.A. and Felton, J.S. (2005) The impact of glucuronidation on the bioactivation and DNA adduction of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in vivo. Carcinogenesis, in press.