University of Tennessee at Chattanooga UTC Scholar Student Research, Creative Works, and Honors Theses Publications 5-2016 Alkaloids in e-cigarettes: their effects on cell growth and gene regulation Maxwell H. Marlowe University of Tennessee at Chattanooga, [email protected] Follow this and additional works at: https://scholar.utc.edu/honors-theses Part of the Environmental Sciences Commons Recommended Citation Marlowe, Maxwell H., "Alkaloids in e-cigarettes: their effects on cell growth and gene regulation" (2016). Honors Theses. This Theses is brought to you for free and open access by the Student Research, Creative Works, and Publications at UTC Scholar. It has been accepted for inclusion in Honors Theses by an authorized administrator of UTC Scholar. For more information, please contact [email protected]. Alkaloids in E-cigarettes: Their Effects on Cell Growth and Gene Regulation Maxwell H. Marlowe Departmental Honors Thesis The University of Tennessee at Chattanooga Department of Biology, Geology, and Environmental Science Project Director: Dr. Ethan A. Carver Examination Date: April 4th, 2016 Members of Examination Committee: Dr. Margaret J. Kovach Dr. Gretchen E. Potts Mrs. Beverly Kutz Examining Committee Signatures: _________________________________________________________ Project Director _________________________________________________________ Department Examiner _________________________________________________________ Department Examiner _________________________________________________________ Liaison, Departmental Honors Committee _________________________________________________________ Chairperson, University Departmental Honors Committee 1 Table of Contents List of Figures ................................................................................................................................. 4 List of Tables .................................................................................................................................. 5 List of Equations ............................................................................................................................ 5 ABSTRACT .................................................................................................................................... 7 ACKNOWLEDGMENTS ............................................................................................................. 8 GOALS and HYPOTHESIS ......................................................................................................... 9 BACKGROUND INFORMATION ........................................................................................... 10 Cell Cultures ............................................................................................................................. 10 E-Cigarette ............................................................................................................................... 11 E-Cigarette Alkaloids .............................................................................................................. 15 Nicotine ................................................................................................................................. 15 Cotinine ................................................................................................................................. 18 Anabasine ............................................................................................................................. 20 Myosmine .............................................................................................................................. 21 Nicotine CNS Physiology ......................................................................................................... 22 Selected Candidate and Control Genes .................................................................................. 24 CYPIA1 and AhR ................................................................................................................ 25 ALDH3A1 ............................................................................................................................. 26 GPX2 ..................................................................................................................................... 28 SLIT1 .................................................................................................................................... 28 PIR ......................................................................................................................................... 30 CX3CL1 ................................................................................................................................ 31 TLR4 ..................................................................................................................................... 32 CEACAM6............................................................................................................................ 32 Control Genes ....................................................................................................................... 33 Materials and Methods ................................................................................................................ 34 Tissue Culture Maintenance ................................................................................................... 34 Chemical Set-up of Tobacco Alkaloid Exposure Plates........................................................ 35 Cell-Titer Glo Luminescent Viability Assay .......................................................................... 36 Real Time RT-PCR .................................................................................................................. 40 RNA Isolation ....................................................................................................................... 42 Two-Step Quantitative RT-PCR......................................................................................... 43 2 Primer Design ....................................................................................................................... 45 Results ........................................................................................................................................... 46 Statistical Analysis ................................................................................................................... 46 RT-PCR Experiments .............................................................................................................. 46 Cell Proliferation ...................................................................................................................... 48 Nicotine ................................................................................................................................. 49 Cotinine ................................................................................................................................. 50 Myosmine .............................................................................................................................. 52 Anabasine ............................................................................................................................. 54 Gene Expression Results ......................................................................................................... 56 Differential Gene Expression in Nicotine Treated Cell Cultures .................................... 57 Differential Gene Expression in Myosmine Treated Cell Cultures ................................. 59 Differential Gene Expression in Anabasine Treated Cell Cultures ................................. 61 Differential Gene Expression in Cotinine Treated Cell Cultures .................................... 63 Discussion ..................................................................................................................................... 65 Cell-Titer Glo Luminescent Cell Proliferation Assay ........................................................... 65 Differential Gene Expression in Response to Alkaloid Exposure ........................................ 70 Alkaloid Effects on CX3CL1 ............................................................................................... 70 Alkaloid Effects on SLIT1 ................................................................................................... 72 Alkaloid Effects on CEACAM6 .......................................................................................... 74 Alkaloid Effects on ALDH3A1............................................................................................ 77 Alkaloid Effects on PIR ....................................................................................................... 79 Alkaloid Effects on TLR4 .................................................................................................... 83 Limitations .................................................................................................................................... 85 Conclusion .................................................................................................................................... 87 Future Directions ......................................................................................................................... 88 Work Cited ................................................................................................................................... 90 3 List of Figures Figure 1: Functional