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Sidestream (SS) smoke, which burns off the tip of a cigarette, is a mixture of 7,000 chemicals (Report of the Surgeon General, 2010), and is the major component of environmental tobacco smoke, which is also called secondhand smoke. Exposure to SS smoke during pregnancy can cause adverse reproductive and embryo developmental effects, which include inability to conceive, spontaneous abortions, congenital defects, increased perinatal mortality, and lower birth weights. With increasing numbers of reports showing health risks associated with tobacco usage, the general public has become interested in harm reduction (HR) cigarettes, such as Marlboro Gold, Advance Lights, and Quest Lights. These cigarettes are marketed as having lower levels of toxins. One of harm reduction ad (Advance Lights) declared that these cigarettes have less of the toxins...and more of the taste. Most analyses done on HR cigarettes have focused on mainstream (MS) smoke, which is actively inhaled by the smoker, and relatively little is known about the toxicity SS smoke from HR products. Surprisingly, my studies using mouse and human embryonic stem cells (ESC) as a model for early embryo development showed that SS smoke from HR cigarettes adversely affected ESC attachment, proliferation, survival, colony growth, and colony attachment. In addition, HR SS smoke was more potent than SS smoke from a conventional brand (Marlboro Red). The chemical composition of SS smoke from HR cigarettes is not well characterized, and no comparisons have been made between HR and conventional brands. In this proposal, I hypothesize that HR SS smoke contains more and/or higher concentrations of toxicants than conventional brand cigarettes. The hypothesis will be tested in three Specific Aims. In Aim #1, SS smoke chemicals will be separated and concentrated based on polarity or charge using solid phase extraction cartridges. Fractions from the cartridges will then be tested in a biological assay system that examines multiple cellular endpoints important during embryo development (i.e., cell attachment, proliferation, apoptosis, migration, cell-to-cell contact, colony attachment, colony growth, and colony movement). The bioassays will be done using novel BioStation CT technology combined with video bioinformatics tools. The BioStation CT is an incubator unit equipped with a microscope, camera, and robotic arm and is capable of taking time-lapse videos for high-content image analysis. Cells will be imaged at specific intervals, and images are converted into time-lapse videos that can later be analyzed using video bioinformatics tools. Video bioinformatics software enables quantitative mining of data from videos images. Various recipes will be used to quantify different cellular endpoints. Currently, some video bioinformatics tools have been developed (i.e., measurement of colony growth, colony movement tracking, and cell migration). More tools are in development and will also be used to analyze data in this proposal. In Specific Aim #2, fractions showing the strongest biological effects in Aim #1 will be purified, and individual chemicals will be identified using gas chromatography-mass spectrometry (GC-MS). The concentration of each chemical will be determined using high performance liquid chromatography (HPLC). GC-MS instrumentation is available in our Analytical Chemistry Instrumentation Facility, managed by doctoral level staff. Once the chemicals are identified, a chemical library will be created by purchasing authentic standards. In Specific Aim #3, the individual chemicals identified in Aim #2 will be tested at varying doses using the BioStation assays. When complete, this study will have established a high-content biological assay system that examines multiple endpoints using video bioinformatics analysis. In addition, I will have identified the individual chemicals in SS smoke that have toxic effects on human ESC and will have made comparisons between SS smoke from HR and conventional cigarettes. This comparison will provide valuable insight into why HR SS smoke is more toxic than SS from conventional brands. |
