Effects of Fluoridated Water on Pineal Morphology in Male Rats by Aaron Mrvelj Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Biological Sciences Program YOUNGSTOWN STATE UNIVERSITY August, 2017 Effects of Fluoridated Water on Pineal Morphology in Male Rats Aaron Mrvelj I hereby release this Thesis to the public. I understand that this thesis will be made available from the OhioLINK ETD Center and the Maag Library Circulation Desk for public access. I also authorize the University or other individuals to make copies of this thesis as needed for scholarly research. Signature: Aaron Mrvelj, Student Date Approvals: Dr. Mark Womble, Thesis Advisor Date Dr. Jill Tall, Committee Member Date Dr. Jeffrey Coldren, Committee Member Date Dr. Salvatore A. Sanders, Dean of Graduate Studies Date Abstract The pineal gland is a naturally calcifying endocrine organ which produ ces and releases the sleep-promoting hormone melatonin which also serves as an antioxidant. Fluoride is attracted to the calcium in the pineal gland and inhibits the synthesis and activity of melatonin, induces oxidative stress, and causes cellular changes to the neurons of the hippocampus. Morphological changes to the pineal gland have been demonstrated with increased age, exposure to light during a melatonin production period, or sleep deprivation. This study sought to examine the effects of fluoridated water on the morphology of the pineal gland. The effects of a fluoride-free flush were compared to fluoride treatment. Group 1, previously raised on fluoridated tap water served as a control that was sacrificed at the onset of the experiment. The remaining four groups were then subjected to a four-week fluoride-free diet with Group 2 being sacrificed at the end of this period. Group 3 was maintained on fluoride-free food and water while Groups 4 and 5 were switched to fluoridated water for the remaining four weeks. The fluoride-free flush resulted in an increase in the number of supporting cells and pinealocytes and a decrease in the nuclear diameter of pinealocytes, suggesting that the flush encouraged growth of the gland. Fluoride treatment had no effect on the number of supporting cells, but decreased the number of pinealocytes and their nuclear diameter, suggesting that fluoride is detrimental to the pineal gland. iii Table of Contents Abstract......................................................................................................... iii Table of Contents ......................................................................................... iv List of Figures ................................................................................................ v List of Abbreviations ................................................................................... vi The Institutional Animal Care and Use Committee Approval .............. vii Introduction .................................................................................................... 1 Pineal gland .................................................................................................................... 1 Normal pineal morphology ............................................................................................. 2 Melatonin ........................................................................................................................ 6 Altered Pineal Morphology ........................................................................................... 10 Fluoride ......................................................................................................................... 12 Effects of fluoride .......................................................................................................... 15 Hypothesis ..................................................................................................................... 18 Methods.........................................................................................................18 Population ..................................................................................................................... 19 Animal wellness ............................................................................................................ 20 Animal experimental groups ......................................................................................... 20 Tissue processing and staining ..................................................................................... 21 Morphological analysis ................................................................................................ 23 Statistical analyses ........................................................................................................ 25 Results ...........................................................................................................28 Animal wellness ............................................................................................................ 28 Effects of fluoride-free flush .......................................................................................... 31 Effects of fluoride treatment .......................................................................................... 37 Discussion .....................................................................................................48 Conclusions ................................................................................................................... 54 Limitations .................................................................................................................... 55 References .....................................................................................................56 iv List of Figures Figure 1: Neural pathway for signals traveling to the pineal gland ................. 8 Figure 2: The MEL molecular pathway in the suprachiasmatic nucleus ........ 9 Figure 3: Common sources of fluoride exposure ............................................. 15 Figure 4: Graphical representation of the experimental design ..................... 21 Figure 5: Grid reticle with relative dimensions at 400X magnification ......... 24 Figure 6: Photograph of light and dark pinealocytes and supporting cells .. 25 Figure 7: Change from initial body weight per animal for each group ......... 29 Figure 8: Food eaten per animal per week ....................................................... 30 Figure 9: Water drank per animal per week ................................................... 31 Figure 10: Change in the number of cells per unit area over time ................ 34 Figure 11: Change in the number of pinealocytes per unit area over time ... 35 Figure 12: Ratio of pinealocytes to supporting cells over time ....................... 36 Figure 13: Diameter of pinealocyte nuclei ........................................................ 37 Figure 14: Number of cells per unit area for each group ................................ 42 Figure 15: Number of pinealocytes per unit area for each group .................. 44 Figure 16: Ratio of light to dark pinealocyte ................................................... 45 Figure 17: Ratio of pinealocytes to supporting cell by group ......................... 45 Figure 18: Number of light and dark cells in the periphery and deep gland ......... 46 Figure 19: Diameter of pinealocyte nuclei for each group .............................. 48 v List of Abbreviations Melatonin (MEL) Also known as (aka) Micrometer (μm) Milliliter (mL) Gram (g) Hematoxylin and eosin (H&E) Connective tissue capsule (CTC) Retinohypothalamic tract (RHT) Suprachiasmatic nucleus (SCN) Paraventricular nucleus (PVN) Action potential (AP) United States (U.S.) Period (per) Cryptochrome (cry) Clock (clk) Cycle (cyc) Clock-Cycle dimer (Clk-Cyc) Period-Cryptochrome dimer (Per-Cry) Superoxide dismutase (SOD) Glutathione peroxidase (GPx) Glutathione reductase (GR) Catalase (CAT) Reactive oxygen species (ROS) Reactive nitrogen species (RNS) Oxidative stress (OS) Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA) Computerized tomography (CT) Hour (hr) Centers for Disease Control and Prevention (CDC) Pineal proteins (PP) Sodium fluoride (NaF) Lipid peroxidation (LPO) Intraperitoneal (i.p.) Alkaline phosphatase (ALP) Parts per million (ppm) Lethal dose (LD) Environmental Protection Agency (EPA) National Institute of Health (NIH) Water (H2O) Celsius (C) Liter (L) Minute (min) Magnification (X) Analysis of variance (ANOVA) Multivariate analysis of variance (MANOVA) Intrinsically photosensitive retinal ganglion cells (ipRGC) vi n n One University Plaza,Youngscown, Ohio 44555 You5 TATEg U N� I sty�o E� RSw� IT� Y ��������---=� Office of Research 330.941.2377 www.ysu.edu June 26, 2016 Dr. Mark Womble Department of Biological Sciences UNIVERSITY Re: IACUC Protocol# 04-16 Title: Fluoride's effect on sleep and pineal morphology in male rats. Dear Dr. Womble: The Institutional Animal Care and Use Committee of Youngstown State University has reviewed the aforementioned protocol you submitted for consideration and determined it should be unconditionally approved for the period of June 24, 2016 through its expiration date of June 24, 2019. This protocol is approved for a period of three years; however, it must be updated yearly via the submission of an Annual Review-Request to Use Animals form. These Annual Review forms must be submitted to the IACUC at least thirtydays prior to the protocol's yearly anniversary dates of June 24, 2017 and June 24, 2019.