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THE EFFECTS OF PHENYLEPHRINE, SODIUM NITROPRUSSIDE, AND HYPOXIA ON THE HEART AND BLOOD VESSELS IN DANIO RERIO A Thesis Presented to The Graduate Faculty at the University of Akron In Partial Fulfillment of the Requirements of the Degree Master of Science Dakota Turner December, 2016 THE EFFECTS OF PHENYLEPHRINE, SODIUM NITROPRUSSIDE, AND HYPOXIA ON THE HEART AND BLOOD VESSELS IN DANIO RERIO Dakota Turner Thesis Approved: Accepted: ________________________________ ____________________________________ Advisor Dean of the College Dr. Brian Bagatto Dr. John Green ________________________________ ____________________________________ Committee Member Dean of the Graduate School Dr. Richard Londraville Dr. Chand Midha ________________________________ ____________________________________ Committee Member Date Dr. Rolando JJ Ramirez ________________________________ Department Chair Dr. Steve Weeks ii ABSTRACT Pharmaceutical medications in waterways are a growing problem worldwide. These include antibiotics, heart medications, and artificial steroids. We examined how a pair of commonly prescribed pharmaceuticals, phenylephrine and sodium nitroprusside, affected the heart and blood vessels in developing zebrafish, Danio rerio. The animals were exposed from 1 day post fertilization (dpf) to 6 dpf. Half of each treatment group was exposed to acute hypoxia on 8 dpf and 15 dpf. On 7 dpf, 8 dpf, 14 dpf, and 15 dpf, heart rate, end diastolic volume, end systolic volume, stroke volume, cardiac output, arterial diameter, and venous diameter were examined via inverted microscope. My results support the concern that pharmaceutical agents can cause physiological changes in locally exposed flora and fauna. Phenylephrine decreased heart rate and vasoconstriction in the veins and hypoxia caused a decrease in heart rate and vasodilation in the arteries. iii TABLE OF CONTENTS Page LIST OF FIGURES ........................................................................................................... vi CHAPTER I. INTRODUCTION ......................................................................................................1 Pharmaceuticals in the Waterways .......................................................................1 Developmental Windows and Deviations .............................................................3 Effects on Vasculature by Phenylephrine and Sodium Nitroprusside ..................3 Environmental Stresses .........................................................................................6 Zebrafish as a Model.............................................................................................7 Aims ..................................................................................................................8 Hypotheses ........................................................................................................8 II. MATERIALS AND METHODS .............................................................................10 Animals ...............................................................................................................10 Chemical Treatment ............................................................................................11 Acute Hypoxia Exposure ....................................................................................11 Video Data Collection ........................................................................................12 ANOVA ..............................................................................................................13 III. RESULTS .................................................................................................................15 Heart Rate ...........................................................................................................15 End Systolic Volume and End Diastolic Volume ...............................................18 iv Stroke Volume ....................................................................................................21 Cardiac Output ....................................................................................................23 Artery Diameter ..................................................................................................25 Vein Diameter .....................................................................................................27 IV. DISCUSSION ...........................................................................................................30 Chronic Phenylephrine Exposure Effects ...........................................................30 Chronic Sodium Nitroprusside Exposure ...........................................................32 Acute Hypoxia Exposure ....................................................................................34 Conclusions .........................................................................................................36 Final Thoughts and Future Directions ................................................................36 REFERENCES ..................................................................................................................38 v LIST OF FIGURES FIGURE Page 1 Timeline of experiment ................................................................................................14 2 Mean standard error of heart rates in Danio rerio ....................................................16 3 Mean standard error of heart rates in Danio rerio ....................................................17 4 Mean standard error of end systolic volume in Danio rerio.....................................18 5 Mean standard error of end systolic volume in Danio rerio.....................................19 6 Mean standard error of end diastolic volume in Danio rerio ...................................20 7 Mean standard error of end diastolic volume in Danio rerio. ..................................21 8 Mean standard error of stroke volume in Danio rerio ..............................................22 9 Mean standard error of stroke volume in Danio rerio ..............................................23 10 Mean cardiac output of untreated, Phe treated, and SNP treated Danio rerio.............24 11 Mean standard error of cardiac output in Danio rerio ..............................................25 12 Mean standard error of tail artery diameters in Danio rerio.....................................26 13 Mean standard error of tail artery diameters in Danio rerio.....................................27 14 Mean standard error of tail vein diameters in Danio rerio .............................28 15 Mean standard error of tail vein diameters in Danio rerio .......................................29 vi CHAPTER I INTRODUCTION In the late 1990’s and early 2000’s, wastewater from cities and towns started gaining attention because it contained measurable amounts of pharmaceutical agents (Grossberger et al., 2014, Bunch et al., 2011, Rocco et al., 2010). While most pharmaceuticals can be removed by water treatment technologies prior to human consumption, the wastewater treatment process does not completely prevent the continuing introduction of pharmaceutical agents into waterways (Rocco et al., 2010). These pharmaceuticals include but are not limited to stimulants, antibiotics, anti- inflammatories, estrogens, and vasodilators (Grossberger et al., 2014). Fish are one example of many species that live in waters contaminated with various pharmaceuticals. Therefore, there are concerns about the physiological impact(s) on fish development and the ability to respond to stress. Pharmaceuticals in the Waterways Humans use a variety of pharmaceuticals every day, including antibiotics, painkillers, high blood pressure medication, opioids, anti-depressants, and artificial hormones (Grossberger et al., 2014; Bunch et al., 2011; Rocco et al., 2010). Pharmaceuticals are expelled from the human body intact or as derivatives and eventually make their way into the environment (Grossberger et al., 2014; Bunch et al., 2011; Rocco et al., 2010). The most commonly discussed pharmaceuticals in the environment are 1 antibiotics, but other pharmaceuticals, such as artificial hormones, are garnering more attention because of bioaccumulation concerns (Grossberger et al., 2014). Bioaccumulation of pharmaceuticals occurs in the local fauna and can cause permanent physiological changes in animals (Grossberger et al., 2014; Bunch et al., 2011; Rocco et al., 2010). The wide use and variety of pharmaceuticals in human populations has had lasting impacts on the local plant and animal life already. Artificial estrogens are chemical compounds that imitate the female sex hormone estrogen, commonly used in artificial birth control (Johnson et al., 2008). The presence of endocrine-disrupting artificial estrogens in the environment has caused developmental feminization of male fish of many species, including those in the Puget Sound. Bioaccumulation of artificial estrogens in the male fish will cause their livers to produce vitellogenin, a protein typically produced by female fish and found in egg yolks, leading to a host of reproductive issues including production of reproductive structures with male and female germ cells and decreased sperm count (Johnson et al., 2008). Another pharmaceutical commonly found in the environment, sildenafil citrate, is a vasodilator commonly sold and used under the brand name Viagra (Rocco et al., 2010). Sildenafil citrate induces DNA degradation and genetic defects that can cause cancer,