BIOMARKERS OF NEUROTOXICITY ASSOCIATED WITH FETAL TOBACCO SMOKE EXPOSURE?

University Of Louisville, Louisville KY

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Abstract

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 [unreadable]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) [unreadable]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.