Free Radicals and Methamphetamine Abuse

描述（由申请人提供）：甲基苯丙胺是滥用最严重的药物之一，导致神经损伤，并与倾向于暴露于HIV的行为有关，HIV是一种穿透中枢神经系统导致神经艾滋病的病毒。由于对神经元的影响，感染艾滋病毒的冰毒使用者通常表现出更严重的神经艾滋病。众所周知，自由基参与大脑病理的增强，但细胞类型和途径受甲基苯丙胺影响，导致自由基介导的压力。这一提议背后的假设是，甲基安非他明将与神经元以外的细胞类型相互作用，特别是关于其诱导和/或调节活性氧和氮中间体的潜力。我们将研究甲基苯丙胺（Meth）对氧化代谢的作用，直接作用于巨噬细胞和其他细胞类型，在体外和体内，在共病因子相互作用的条件下。为此，我们将研究甲基安非他明对细胞系的作用，通过研究药物如何影响氧化破裂的强度和质量，并在ROI和RNI途径中寻找在甲基安非他明暴露后表现出转录模式变化的基因。这种转录变化将通过寻找从感染SIV的经甲氧麻黄碱处理的动物中获得的细胞中获得的类似变化来验证，这些细胞先前已被描述为中枢神经系统疾病参数。本项目的结果将为甲基安非他明参与氧化应激介导的病理提供重要线索，通过作用于中枢神经系统和外周器官的ROI和RNI诱导通路。

DESCRIPTION (provided by applicant): Methamphetamine is one of the most growing drugs of abuse, causing neurological impairments, and associated with behaviors that favor exposure to HIV, a virus that penetrates the CNS leading to neuroAIDS. Meth users infected with HIV usually present a more severe form of neuroAIDS, due to effects on neurons. IT is known that free radicals participate in the enhancement of brain pathology, but the cell types as well as pathways affected by Meth that lead to free-radical mediated stress. The hypothesis behind this proposal is that Meth interacts with cell types other than neurons will be explored, particularly regarding its potential to induce and/or modulate reactive oxygen and nitrogen intermediates. We will investigate the action of methamphetamine (Meth) on oxidative metabolism, directly on macrophages, and on other cell types in vitro, as well as in vivo, in conditions of co-morbid factor interaction. For that we will examine the action of Meth on cell lines, by investigating how the drug affects the intensity and quality of the oxidative burst, and searching for genes within ROI and RNI pathways that exhibit transcriptional pattern changes upon Meth exposure. Such transcriptional changes will be validated by looking for similar changes in cells obtained from Meth-treated animals infected with SIV, which have been previously characterized for CNS disease parameters. The results from this project will offer important clues on the participation of Meth in pathology mediated by oxidative stress by acting on ROI and RNI induction pathways, not only in the Central Nervous System, but also in peripheral organs. PUBLIC HEALTH RELEVANCE: We will investigate the action of methamphetamine (Meth) on oxidative metabolism, directly on macrophages, and on other cell types in vitro, as well as in vivo in conditions of co-morbid factor interaction. The hypothesis behind this proposal is that Meth affects the intensity and quality of the oxidative burst by acting directly on macrophages and/or glial cells, in addition to its action on neurons, contributing to the development of pathology mediated by oxidative stress, not only in the Central Nervous System, but also in peripheral organs. Initially, we will focus on the effect of meth on various human and mouse macrophage (THP1, RAW264.7), microglia (MG5, EOC 2), astrocyte (C8-D1A, CCF-STTG1), and neuronal (SH-SY5Y, SK- N-MC) cell lines, regarding the production of O2_, H2O2, and other reactive oxygen intermediates (ROI), as well as Reactive Nitrogen Intermediates (RNI) using various chromogenic methods. Mitochondria activity upon interaction with meth will be also addressed on cells stimulated with Meth. Following a characterization of the different cell lines regarding production of free radicals and mitochondrial changes in reaction to Meth, we will choose good responders to investigate transcriptional changes on molecules within ROI and RNI pathways, caused by action of Meth. For that we will use multiplex technology, as well as enzymatic checkpoint inhibition strategies. These approaches will help dissect the molecular basis of respiratory burst induction by Meth, and will distinguish whether Meth interferes over mitochodria, peroxisome, or both. Following the in vitro approach using cell lines, we intend to validate findings in the context of pathology. We will use cryopreserved cells and tissues from monkeys infected with SIV (as a model for HIV infection, a common co-morbid condition) subjected to a Meth treatment schedule. We will identify transcriptionally altered oxidative pathways on cryopreserved brain and peripheral tissues derived from SIV-infected Meth-treated monkeys by qRT-PCR. This brings relevance to pathology in humans. The present proposal will identify the basis of oxidative stress in the brain and other organs in Meth users. Meth is one of the most growing street drugs of abuse, exposing users to HIV infection, and potentially facilitating the penetration of virus across the blood brain barrier (BBB) into the brain. In this proposal we will dissect mechanisms by which Meth can induce and modulate free radical production, causing an aggravation on the severity of damage associated with virus presence in the brain tissue, and consequently aggravate AIDS-associated CNS dysfunctions. The results obtained in this proposal will allow the generation of data on direct effects of the drug on cells, relative susceptibility of different cell types to direct actions, and on pathways, aiming a future R01 application on modulation of oxidative stress in Meth abuse in vivo. These will lead to the development of important therapeutic tools for rehabilitating individuals, especially patients presenting co-morbid factors, such as HIV infection.