BIOMARKERS OF NEUROTOXICITY ASSOCIATED WITH FETAL TOBACCO SMOKE EXPOSURE?

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尼古丁是一种合法的高度上瘾的精神药物，在吸入烟草烟雾后迅速传输到大脑。将香烟(和其他形式的吸烟烟草)委婉地称为“尼古丁输送装置”，承认了吸烟的上瘾性质，这是烟草行业承认的事实。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，这些化合物对烟草消费具有明显的毒性。大约20%的美国成年人(3600万)是活跃的吸烟者。尽管在全国范围内广泛宣传了怀孕期间吸烟的有害影响，但在美国，大约25%的孕妇仍在吸烟。 在全国范围内，处于较低社会经济群体的女性的这一比例上升到近50%。仅在美国，每年就有大约100万名婴儿产前暴露在香烟烟雾中。众所周知，烟草烟雾含有尼古丁等神经毒素，暴露在这种毒素中会导致行为和认知异常，直到有毒侮辱停止后仍会持续。发育性香烟烟雾暴露与子宫脑生长减少以及细胞和分子神经发育障碍有关，这被认为是在人类和啮齿动物模型中观察到的认知和行为问题发生率增加的中介因素。在目前的提案中，我们将测量发育性烟草烟雾暴露对子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，该模型模拟怀孕期间人类母亲吸烟)，该模型平行于海马神经化学变化和伴随的持续性行为/认知缺陷。 具体目标1：通过模拟孕期母亲吸烟的小鼠模型，研究发育期吸烟暴露对子代整体血清代谢组的影响。C57BL/6小鼠将从妊娠第1天(GD1)开始在子宫中暴露，一直持续到出生后第14天(PN14)，这一间隔跨越了包括海马体在内的脑亚区的分化，开始了大脑的发育。在不同的时间点，包括立即退出挑战、挑战后3天和2个月大(暴露于暴露的后代的神经认知缺陷是可识别的)，将对血清进行分级，以进行全球代谢组谱分析。利用离散代谢物化学类提取(极性-甲醇提取；中性脂--氯仿/甲醇提取)的双管齐下的方法将随后进行高分辨率/准确的质量测量，并结合保留时间图和来自归一化光谱的峰面积定量。工作假说：在暴露期间，血清代谢体表达的暂时性失调将是明显的(存在尼古丁代谢物)，并持续到神经认知缺陷的发展(尼古丁代谢物不存在)。如果 这一假设是正确的，我们将识别出外周代谢物标记物，这些标记物直接指示神经发育异常，并与大脑形态发生过程中吸烟暴露导致的蛋白质组变化(特定目标2)和转录组变化(见初步数据)相关。此外，我们还将确定潜在的行为和神经认知异常的分子标记物，这些标记物可能广泛适用于药物中毒以外的疾病。 具体目的2：通过模拟孕期吸烟的小鼠模型，研究发育期吸烟暴露对子代海马区全球时间代谢组和蛋白质组表达谱的影响。利用收集的组织作为特定目标1的一部分，将分析海马区组织代谢组和蛋白质组谱。在特定的AIMS 1和2中，代谢组分析将使用LC-LIT/FTICR-MS，随后将使用原始数据色层对齐、整合和提取峰强度。感兴趣的光谱特征将通过准确的质量、MS/MS裂解模式(CID/IRMPD)和数据库搜索(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)来识别。工作假说：在发育性吸烟暴露的小鼠模型中，海马体蛋白质组和代谢组表达的时间性失调在暴露后立即明显，并持续到神经认知缺陷的发展。如果这一假设是正确的，我们将识别出大脑结构特异的组织生物标记物，这些生物标记物直接指示神经发育异常，并反映在大脑形态发生期间吸烟暴露导致的外周代谢组变化。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用后持续存在，并可能指示表观遗传变化。这些标志物反过来可能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.