In utero NRTIs & mtDNA Changes in Cardiac/Vascular Cells

描述（由申请人提供）： 与核苷类似物逆转录酶抑制剂（NRTI）的临床使用相关的潜在健康风险存在，因为其有效性的分子基础也驱动了其潜在的毒性。目前的研究表明，这种毒性的表现之一是线粒体损伤导致心脏病。本项目的目的是通过评价体外暴露的人类细胞以及经胎盘暴露的小鼠和人类组织中诱导的线粒体tRNA基因突变的类型和频率，确定NRTI诱导的线粒体损伤的性质和程度。将使用改良的ELISA方法，在体外暴露的人淋巴细胞、子宫内暴露的小鼠心脏细胞和淋巴细胞以及子宫内暴露的人婴儿淋巴细胞和脐带组织中，评价这些突变对线粒体功能标志物的影响（线粒体结构的EM研究、mtDNA耗竭研究和OXPHOS/考克斯酶测定）。将进行短期和长期研究，以评估啮齿动物和子宫内暴露儿童的线粒体DNA突变和线粒体功能障碍的持续性。将评价常用的NTRI（AZT、3 TC和d4 T），无论是单独使用还是联合使用。将进行初步研究，以确定在小鼠体内复制人类儿童中发现的AZT血浆水平所需的人体等效剂量。该项目的长期目标是i）确定mtDNA突变的类型和频率与线粒体功能改变之间的关系，以及ii）确定单个NRTI和/或常用NRTI组合诱导有害线粒体损伤的相对效力。阐明NRTI诱导的线粒体损伤的机制将有利于HIV感染患者服用NRTI以控制感染，HIV暴露者服用NRTI预防HIV感染，以及接受围产期NRTI预防HIV感染的儿童。

DESCRIPTION (provided by applicant): Potential health risks associated with the clinical use of nucleoside analogue reverse transcriptase inhibitors (NRTIs) exist because the molecular basis for their effectiveness also actuates their potential toxicities. Current research suggests that one of the manifestations of this toxicity is mitochondrial damage leading to cardiac disease. The purpose of this project is to determine the nature and extent of NRTI-induced damage in mitochondria by evaluating the types and frequencies of mutations induced in mitochondrial tRNA genes of human cells exposed in vitro, and in tissues of mouse and human exposed transplacentally. The impact of these mutations upon markers of mitochondrial function (EM studies of mitochondrial structure, mtDNA depletion studies, and OXPHOS/COX enzyme assays) will be evaluated, using a modified parallelogram approach, in human lymphocytes exposed in vitro, in heart cells and lymphocytes of mice exposed in utero, and in lymphocytes and umbilical cord tissue of human infants exposed in utero. Short-term and long-term studies will be conducted to evaluating the persistence of mitochondrial DNA mutations and mitochondrial dysfunction in rodents and children exposed in utero. Commonly used NTRIs (AZT, 3TC, and d4T), either alone or in combination, will be evaluated. Preliminary studies will be conducted to determine the human-equivalent dose necessary to replicate in vivo in the mouse the plasma levels of AZT found in human children. The long range goals of this project are i) to determine the relationship between the types and frequencies of mtDNA mutations and alterations in mitochondrial function, and ii) to determine the relative potency of individual NRTIs, and/or combinations of commonly used NRTIs, to induce deleterious mitochondrial damage. Elucidation of the mechanisms underlying NRTI-induced mitochondrial damage will benefit HIV-infected patients taking NRTIs to control infection, HIV-exposed individuals who take NRTIs prophylactically to prevent HIV infection, and children receiving perinatal NRTIs to prevent HIV infection.