Mechanisms underlying activation and detoxification of aristolochic acids in human hepatic and renal cells

马兜铃酸在人肝肾细胞中的激活和解毒机制

基本信息

批准号：
10190228
负责人：
Viktoriya S Sidorenko
金额：
$ 19.94万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-10 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10190228
关键词：
Affect Alleles Area Aristolochia Aristolochic Acids Asians Attention Australia Balkans CRISPR/Cas technology Candidate Disease Gene Carcinogens Cell Extracts Cell Line Cells Chemical Agents China Chronic Kidney Failure Clinical Conflict (Psychology)Country Crystallins Cultured Cells DNA DNA Adducts Development Diagnosis Disease Drug Metabolic Detoxication Engineering Enzymes Europe Experimental Models Exposure to Fibroblasts Frameshift Mutation Genes Goals HepG2 Hepatic Hepatocyte Herb Herbal supplement Human Individual Ingestion Internet Intrahepatic Cholangiocarcinoma Investigation Kidney Kidney Diseases Knock-out Knowledge Light Liver Malignant Neoplasms Malignant neoplasm of urinary bladder Metabolic Biotransformation Metabolism Mutation NQO1 gene Nitroreductases Nutrient Outcome Oxidoreductase Pathway interactions Pharmaceutical Preparations Phenotype Plants Plasmids Poisoning Population Predisposition Primary carcinoma of the liver cells Property Proteins Public Health Quinones Recombinant Proteins Renal Cell Carcinoma Reporting Research Research Design Research Personnel Risk Role Route Scientist Soil System Taiwan Techniques Therapeutic Therapeutic Agents Tissues Toxic effect Toxin Uncertainty Urinary tract Urothelium Variant Xenobiotics base carcinogenicity cytotoxic cytotoxicity design enzyme activity experience exposed human population genetically modified cells genome editing genotoxicity interest kidney cell nephrotoxicity novel novel strategies organ on a chip prevent prophylactic success sulfotransferase tool

项目摘要

Aristolochic acids (AA), principal components of Aristolochia plants used worldwide for medicinal purposes, are potent carcinogens and nephrotoxins. Importantly, a unique mutational signature for AA has been documented in upper urothelial tract cancer, bladder cancer, renal cell carcinoma, hepatocellular carcinoma and intrahepatic cholangiocarcinoma. It is estimated that in China and other Asian countries, where herbal remedies are most widely used, 100 million people are at risk of developing AA-related cancers and/or chronic renal disease. In the US and Europe, herbal supplements containing AA are marketed through the Internet and continue to be used despite warnings to the contrary. Furthermore, in Balkan countries, Aristolochia plants are abundant in farming fields, poisoning soil and crops with AA. Considering all the above, there is an urgent need to understand biotransformation pathways of AA in order to reduce human exposure by devising novel chemical agents that control the activity of enzymes involved in AA metabolism. The limited knowledge of pathways for biotransformation of AA, amplified by the current conflict in this area of research regarding the role of sulfotransferases and nitroreductases in inducing AA toxicities, prevents the development of such strategies. This proposal builds on two important findings we obtained in earlier studies. Using an integrated human “liver- kidney-on-a-chip” system, we reported that activation of AA occurs in the liver as well as in the kidney. We also found that novel reductases might be important for AA metabolism and toxicity. Thus, the objective of this research is to evaluate the role of novel reductases in AA metabolism and toxicity and to resolve a controversy over the involvement of sulfotransferases and nitroreductases in bioactivation of AA in human liver and kidney. To achieve these goals, we employ a targeted CRISPR/CAS9 genome editing approach in human hepatic HepG2 and renal HK-2 cell lines to generate double-allelic, frame-shifting mutations in genes putatively involved in metabolism of AA. Engineered cell lines will be evaluated in terms of their sensitivity to AA and compared with respective parental cells. Mass spectrometric and DNA postlabelling techniques will be applied to quantify the major metabolites of AA and their DNA adducts, respectively. Plasmids expressing corresponding wild-type and catalytically inactive proteins will be used to transform knock-out cell lines in order to verify the involvement of particular enzymatic function in AA toxicities. To support findings in cultured cells, activities of recombinant proteins and cell lysates toward AA and N-hydroxyaristolactams, known metabolites of AA, will be studied. Successful completion of this research will establish novel genes involved in the biotransformaton of AA. This information will inform clinical scientists on design of therapeutics geared to reduce genotoxic and cytotoxic exposure, and will aid in defining individuals at risk of developing AA-related diseases. Given the worldwide exposure to AA, this research has major implications for global public health. Finally, the cell lines generated in our studies will then be available for use in investigations of other human carcinogens, toxins and drugs.

马兜铃酸 (AA) 是全世界药用马兜铃植物的主要成分，重要的是，AA 具有独特的突变特征。记录在上尿路上皮癌、膀胱癌、肾细胞癌、肝细胞癌和估计在中国和其他亚洲国家，草药治疗肝内胆管癌。使用最广泛，一亿人面临患 AA 相关癌症和/或慢性肾病的风险在美国和欧洲，含有 AA 的草药补充剂通过互联网销售。尽管有相反的警告，但仍继续使用马兜铃属植物。 AA在农田中大量存在，污染土壤和农作物，因此迫切需要AA。了解 AA 的生物转化途径，通过设计新型化学物质来减少人类接触 AA 控制参与 AA 代谢的酶活性的药物对 AA 代谢途径的了解有限。 AA 的生物转化，由于当前该研究领域关于 AA 作用的冲突而被放大磺基转移酶和硝基还原酶诱导 AA 毒性，阻碍了此类策略的发展。该提案基于我们在早期研究中使用综合人类“肝脏”获得的两项重要发现。在“肾芯片”系统中，我们报道了 AA 的激活发生在肝脏和肾脏中。发现新型还原酶可能对 AA 代谢和毒性很重要，因此，本研究的目的是。研究旨在评估新型还原酶在 AA 代谢和毒性中的作用并解决争议磺基转移酶和硝基还原酶参与人类肝脏和肾脏中 AA 的生物活化。为了实现这些目标，我们在人类肝脏中采用了靶向 CRISPR/CAS9 基因组编辑方法。 HepG2 和肾 HK-2 细胞系在相关基因中产生双等位基因移码突变将评估工程化细胞系对 AA 的敏感性，并与它们进行比较。将应用质谱和 DNA 后标记技术来量化。分别表达相应野生型和 AA 的主要代谢物及其 DNA 加合物。催化失活蛋白将用于转化敲除细胞系，以验证 AA 毒性中的特殊酶功能支持培养细胞中的发现、重组体的活性。将研究 AA 和 N-羟基马兜铃内酰胺（AA 的已知代谢物）的蛋白质和细胞裂解物。这项研究的成功完成将建立参与 AA 生物转化的新基因。这些信息将为临床科学家提供有关旨在减少基因毒性和细胞毒性的疗法设计的信息鉴于全球范围内的情况，将有助于确定有患 AA 相关疾病风险的个体。暴露于 AA，这项研究对全球公共卫生具有重大影响。我们的研究随后将可用于其他人类致癌物、毒素和药物的研究。