BIOMARKERS OF NEUROTOXICITY ASSOCIATED WITH FETAL TOBACCO SMOKE EXPOSURE?

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 尼古丁是一种合法的、高度成瘾的精神药物，在吸入烟草烟雾后迅速转运到大脑。香烟（和其他形式的吸烟烟草）被委婉地称为“尼古丁输送装置”，这承认了吸烟的成瘾性  这是烟草业承认的事实。虽然尼古丁是主要的成瘾成分，但香烟烟雾中含有约4000种化合物，包括丙烯醛、丁二烯、二氧化碳（CO2）、一氧化氮（NO）、铅和镉，这些化合物导致烟草消费的明显毒性。大约20%的美国成年人（3600万）是活跃吸烟者。尽管全国广泛宣传怀孕期间吸烟的有害影响，但在美国，大约25%的怀孕妇女继续吸烟。 在全国范围内，社会经济地位较低的妇女的这一比例上升到近50%。仅在美国，每年就有大约100万婴儿在出生前暴露于香烟烟雾中。已知烟草烟雾含有神经毒素，如尼古丁，暴露于尼古丁会诱导行为和认知异常，在毒性损伤停止后持续很长时间。发育中的香烟烟雾暴露与子宫内脑生长的减少以及细胞和分子神经发育的扰动有关，这被认为介导了在人类和啮齿动物模型中观察到的认知和行为问题的增加率。在目前的建议中，我们将测量发育烟草烟雾暴露对后代血清代谢组标志物的影响（在我们实验室开发的一种独特的动物模型中，该模型模拟人类母亲在怀孕期间吸烟），该模型与海马神经化学改变和伴随的持续行为/认知缺陷平行。 具体目标1：研究发育期烟草烟雾暴露对小鼠模型后代整体血清代谢组的影响，该模型模拟妊娠期间母亲吸烟。C57 BL/6小鼠将从妊娠第1天（GD 1）开始在子宫内暴露，并持续至出生后第14天（PN 14）  通过包括海马体在内的大脑亚区域的分化跨越大脑发育开始的间隔。在不同的时间点，包括停药后即刻、攻毒后3天和2月龄（暴露后代的神经认知缺陷可辨别），将对血清进行分级，用于总体代谢组学分析。采用分离代谢物化学类别提取（极性-甲醇提取;中性脂质-氯仿/甲醇提取）的双管齐下方法，然后进行高分辨率/准确度质量测量，结合保留时间图谱和归一化光谱的峰面积定量。工作假设：血清代谢组表达的时间失调在暴露期间将是明显的（存在尼古丁代谢物），并在神经认知缺陷的发展过程中持续存在（不存在尼古丁代谢物）。如果 如果这一假设是正确的，我们将鉴定出直接指示神经发育异常的外周代谢物标记物，这些标记物与脑形态发生期间烟草烟雾暴露导致的蛋白质组改变（特异性目标2）和转录组改变（见初步数据）有关。此外，我们将确定行为和神经认知异常的潜在分子标志物，这些标志物可能广泛适用于药物中毒以外的疾病。 具体目标二：研究发育期烟草烟雾暴露对小鼠模型后代海马体中全球时间代谢组和蛋白质组表达谱的影响，该模型模拟妊娠期间母亲吸烟。将利用作为特定目标1的一部分采集的组织，分析海马组织代谢组和蛋白质组谱。特定目的1和2中的代谢物组分析将采用LC-LIT/FTICR-MS，然后进行原始数据色谱比对、积分和峰强度提取。将通过准确质量、MS/MS裂解模式（CID/IRMPD）和数据库检索（代谢物-HMDB、METLIN和KEGG数据库;蛋白质-NCBInr哺乳动物正向和反向数据库）鉴别感兴趣的光谱特征。工作假设：在发育中的香烟烟雾暴露小鼠模型中，海马蛋白质组和代谢组表达的暂时失调在暴露后立即变得明显，并持续到神经认知缺陷的发展。如果这一假设是正确的，我们将确定脑结构特异性组织生物标志物，这些生物标志物直接指示神经发育异常，并通过脑形态发生期间烟草烟雾暴露引起的外周代谢组学改变来反映。此外，我们将确定与行为和神经认知障碍相关的组织分子标志物，这些标志物在毒性暴露停止后持续存在，并且可能指示表观遗传改变。反过来，这些标记物可以成为未来研究环境神经毒素对大脑，行为和认知能力发育的表观遗传影响的目标。

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. Nicotine, a licit and highly addictive psychotropic drug, is rapidly transported to the brain following tobacco smoke inhalation. The euphemistic reference to cigarettes (and other forms of smoking tobacco) as "nicotine delivery devices" acknowledges the addictive nature of cigarette smoking  a fact conceded by the tobacco industry. Though nicotine is the primary addictive component, cigarette smoke contains approximately 4000 compounds including acrolein, butadiene, carbon dioxide (CO2), nitric oxide (NO), lead and cadmium that contribute to the overt toxicity of tobacco consumption. Approximately 20% of US adults (36 million) are active smokers. Despite extensive national publicity regarding the detrimental effects of tobacco use during pregnancy, cigarette smoking continues in approximately 25% of all pregnancies in the USA. Nationwide, this percentage escalates to nearly 50% for women in lower socioeconomic groups. In the United States alone, approximately one million infants are exposed prenatally to cigarette smoke each year. Tobacco smoke is known to contain neurotoxins, such as nicotine, exposure to which induces behavioral and cognitive abnormalities persisting well past the cessation of the toxic insult. Developmental cigarette smoke exposure is associated with decreased in utero brain growth and perturbations in both cellular and molecular neurodevelopment which are thought to mediate the increased rates of cognitive and behavioral problems that are observed in humans and rodent models. In the current proposal, we will measure the impact of developmental tobacco smoke exposure on serum metabolome markers of offspring (in a unique animal model developed in our laboratory which simulates human maternal smoking during pregnancy) which parallel both hippocampal neurochemical alterations and the accompanying, persistent behavioral/cognitive deficits. Specific Aim 1: Examine the impact of developmental tobacco smoke exposure on the global serum metabolome in offspring from a mouse model which simulates maternal cigarette smoking during pregnancy. C57BL/6 mice will be exposed in utero beginning on gestational day 1 (GD 1) and continuing through postnatal day 14 (PN14)  an interval which spans initiation of brain development through differentiation of brain subregions including the hippocampus. At varying time points, including immediately upon withdrawal of challenge, 3 days post challenge, and 2 months of age (neurocognitive defects in exposed offspring are discernable), serum will be fractionated for global metabolome profiling analysis. A two-pronged approach utilizing discrete metabolite chemical class extractions (polar--methanol extraction; neutral lipids--chloroform/methanol extraction) will be followed by high resolution/accuracy mass measurement coupled with retention time mapping and peak area quantitation from normalized spectra. Working hypothesis: temporal dysregulation of serum metabolome expression will be evident during exposure (nicotine metabolites are present) and persist through the development of neurocognitive deficits (nicotine metabolites not present). If this hypothesis is correct, we will have identified peripheral metabolite markers which are directly indicative of neurodevelopmental abnormalities and which are related to proteomic alterations (Specific Aim 2) and transcriptome alterations (See Preliminary Data) resulting from tobacco smoke exposure during brain morphogenesis. In addition, we will have identified potential molecular markers of behavioral and neurocognitive abnormalities which may be broadly applicable to diseases other than drug intoxication. Specific Aim 2: Examine the impact of developmental tobacco smoke exposure on global temporal metabolome and proteome expression profiles in hippocampus of offspring from a mouse model which simulates maternal cigarette smoking during pregnancy. Utilizing tissue collected as part of Specific Aim 1, hippocampal tissue metabolome and proteome profiles will be analyzed. Metabolome profiling in both Specific Aims 1 and 2 will employ LC-LIT/FTICR-MS followed by raw data chromatographic alignment, integration and extraction of peak intensities. Spectral features of interest will be identified by accurate mass, MS/MS fragmentation pattern (CID/IRMPD), and database searching (metabolites-HMDB, METLIN, and KEGG databases; proteins-NCBInr mammalian forward and reverse databases). Working hypothesis: temporal dysregulation of hippocampal proteome and metabolome expression, in the developmental cigarette smoke exposure mouse model, will be evident immediately after exposure and persist through the development of neurocognitive deficits. If this hypothesis is correct, we will have identified brain structure-specific tissue biomarkers that are directly indicative of neurodevelopmental abnormalities and which are mirrored by peripheral metabolome alterations resulting from tobacco smoke exposure during brain morphogenesis. In addition, we will have identified tissue molecular markers associated with behavioral and neurocognitive impairment which persist after withdrawal of the toxic exposure and which may be indicative of epigenetic alterations. These markers, in turn, could be targets for future investigations into the epigenetic effects of environmental neurotoxins on development of the brain, behavior and cognitive abilities.