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  1. 1. Effects of ethanol-induced oxidative stress on antioxidant enzyme gene expression in three tissues of the zebrafish, Danio rerio Under Graduate Senior Thesis Submitted to the Division of Biology at Bard College by Hannah Shapero Annandale-on-Hudson, New York May 2, 2012
  2. 2. 2 TABLE OF CONTENTS Acknowledgements………………………………………………………………………………3 Abstract….………………………………………………………………………………………..4 Introduction……………………………………………………………………………………....5 Oxidative stress……………………………………………….…………………………...5 Sources of ROS…………………………………………………………………………...5 Mechanisms of oxidative DNA damage…………………………………………………..8 Antioxidant Protective Mechanisms……………………………………………………..10 Oxidative Stress and Cancer……………………………………………………………..11 Affect of Ethanol on Oxidative Stress…………………………………………………...13 Danio rerio as a Model…………………………………………………………………...16 Diverse Tissue Analysis……………………………………………………………….....17 Objectives………………………………………………………………………………..18 Materials and Methods………………………………………………………………………....20 Animal Care……………………………………………………………………………...20 Exposure………………………………………………………………………………....20 Dissection………………………………………………………………………………..22 RNA Extraction……………………………………………………………………….....23 Reverse Transcription…………………………………………………………………....23 Quantitative PCR………………………………………………………………………...25 Data Analysis………………………………………………………………………….....25 Results…………………………………………………………………………………………...28 Brain Enzyme Expression Levels………………………………………………………..28 Liver Enzyme Expression Levels………………………………………………………..29 Gonad Enzyme Expression Levels……………………………………………………....30 Discussion…………………………………………………………………………………….....31 Brain Tissue……………………………………………………………………………...31 Liver Tissue……………………………………………………………………………...32 Gonad Tissue………………………………………………………………………….....34 General Conclusions……………………………………………………………………..35 Future Direction……………………………………………………………………….....39 References…………………………………………………………………………………….....41 Tables…………………………………………………………………………………………....44 Figures…………………………………………………………………………………………...45 Appendix………………………………………………………………………………………...49
  3. 3. 3 ACKNOWLEDGEMENTS Board members Philip Johns (Adv.), Assistant Professor of Biology, Bard College Mike Tibbetts, Associate Professor of Biology, Bard College Celia Bland, Writer in Residence, Bard College Lab Technicians Maureen O’Callaghan-Scholl, Lab Manager, Bard College Dwane Decker, Lab Technician, Bard College Diana Gutierrez, Lab Technician, Bard College Peer Editing Katherine Hoople, Assistant Researcher, Astor Services Sarah Taylor, Graduated Student, Bard College Ella Jacobson, Student, Bard College Tyler Martinez, Graduated Student, Vassar College
  4. 4. 4 ABSTRACT All aerobic organisms rely on oxygen for survival. Oxygen is an essential component of many cellular processes including cellular metabolism, intercellular and intracellular signaling and immune system response. Oxygen through generation of reactive oxygen species (ROS) can severely damage with DNA, proteins and other cellular components becoming problematic (Kryston et al. 2011). Organisms have evolved ways to balance the essential and detrimental effects of oxygen within the cellular environment. Antioxidant enzymes are proteins produced by cells to minimize the harmful affects of oxidative damage caused by ROS (Barzilai and Yamamoto, 2004). Ethanol, which is a widely consumed drug, can exacerbate the damaging effects of oxygen through the production of excess ROS involved in ethanol metabolism. This study measured the production levels of 3 key antioxidant enzymes (CuZnSOD, MnSOD and CAT) in the brain, gonads and liver of zebrafish during and after exposure to ethanol. Antioxidant enzyme production will be measured by qPCR of cDNA transcribed from RNA samples extracted directly from tissue. It was found that different tissues show remarkably different expression patterns of antioxidant enzymes under the same conditions of acute alcohol exposure. Brain tissues show no significant change in antioxidant expression levels. The highest expression levels were found in gonad tissue. This study gives insight into the beneficial effects and transcriptional mechanisms of antioxidant enzymes in response to oxidative damage, which may protect DNA from excess reactive oxygen species produced by acute ethanol exposure.
  5. 5. 5 INTRODUCTION The oxygen paradox is possibly the most frustrating aspect of aging and disease research, strongly implicated in cancer tumerogenesis and other age related diseases. The oxygen paradox refers to how oxygen is dangerous to the life forms for which it is essential. Oxygen plays a significant role in many processes of the human body including cellular metabolism, intercellular and intracellular signaling and immune system response. Oxygen through generation of reactive oxygen species (ROS) can react with DNA, proteins and other cellular components and become problematic. (Kryston et al. 2011). There is a narrow nomoxic range of oxygen which must be steadily maintained by the production and function of antioxidant enzymes within cells to minimize the harmful affects of oxidative damage caused by excessive oxygen (hyperoxia) and metabolic demise caused by insufficient oxygen (hypoxia) (Barzilai and Yamamoto 2004) Oxidative Stress: Reactive oxygen species are small, highly reactive, oxygen-containing molecules that can react with and damage complex cellular molecules, particularly in the liver (Wu and Cederbaum 2003). ROS are constantly produced within living cells during normal metabolic processes
  6. 6. 6 within cellular mitochondria and pathological processes such as inflammation as well as during incomplete metabolism of ethanol. At normal physiological levels, ROS play a vital role in regulating signaling pathways and gene expression. Maintaining their production below a toxic threshold is important to normal cellular function (Barzilai and Yamamoto 2004). Approximately 2 x 104 DNA damaging events have been estimated to occur in every cell of the human body every day. A significant portion of this damage is caused by reactive oxygen species, because they are generated as by-products of respiration. ROS are the most abundant toxic agents found in aerobic organisms (Barzilai and Yamamoto 2004). The most commonly detrimental ROS are the hydroxyl radical, the superoxide radical and non-radical hydrogen peroxide (Evans et al. 2003). Cellular targets for these ROS include DNA, lipids and proteins. Modified lipids and proteins are readily removed by the normal turnover of cellular molecules, but damage to DNA is not easily dealt with and must be repaired. Living cells have evolved a number of defenses against damage from ROS and other free radicals. Low molecular weight compounds, known as antioxidants intercept free radicals, becoming radicals themselves; complex antioxidant enzyme reactions such as superoxide dismutase, catalase and glutathione peroxidase limit cellular levels of ROS. Even under normal functional conditions there is a propensity for ROS to evade these defenses, resulting in low levels of damage, which in the short term do not appear detrimental to the cell (Evanset al. 2003). It is when an imbalance arises between ROS-producing factors and antioxidant defenses, which cellular damage occurs.
  7. 7. 7 Sources of ROS: Oxidative stress is inflicted on cells both endogenously and exogenously. Intracellular attacks corresponding with a number of regular cell processes such as cell signaling, inflammation and metabolism cause more extensive damage to DNA then environmental factors. Endogenously induced DNA lesions contribute significantly to the accumulation of DNA mutations in cells and tissues. ROS are generally accepted as the major source of oxidative injury in all aerobic organisms, and are constantly being produced as byproducts of normal cellular processes such as mitochondrial respiration and chronic inflammation (Kryston et al. 2011). Mitochondrial electron transport constitutes the majority intracellular source of ROS (Bailey and Cunningham 2002). Mitochondria are also intimately involved in the generation and defense process involved with reactive oxygen species. Mitochondria are both targets of oxidative stress directly and involved in the mechanisms by which oxidative stress-related signals can lead to apoptosis. Formation of ROS within the mitochondria promotes activation of the mitochondrial permeability transition, which in turn leads cells towards pro-apoptotic pathways, and eventually, complete apoptosis. A second common source of ROS is the O2 and H2O2 generated during the respiratory burst of invading neutrophils, macrophages and eosinophils associated with inflammation and defense against infection. Inflammation initiates a cycle of events in which ROS-damaged tissues release cytokines, which promote the infiltration of further inflammatory cells, in a positive feedback loop responsible for chronic infection. Chronic infection, and the associated rise in oxidative
  8. 8. 8 stress can lead to severe tissue damage in the surrounding tissue. Continuous inflammation can lead to cellular damage and cancer (Reuter et al. 2010). Under sustained oxidative stress, significant damage has been shown to occur to surrounding cells and tissue structures (Reuter et al. 2010). Damage may induce somatic mutations and neoplastic transformations within ROS exposed tissue. Mechanisms of oxidative DNA damage: DNA is a main target of oxidative damage. ROS interact with biological molecules and disrupt the normal synthesis and repair processes of DNA. ROS can cause a number of changes to DNA structure, including strand breaks, point mutations and aberrant DNA cross-linking. These can create mutations in both proto-oncogenes and tumor-suppressor genes, all of which promote neoplastic transformation (Reuter et al. 2010). The cumulative effects of the interaction of DNA with ROS are determined by several factors, including DNA repair, transcriptional and functional levels of antioxidant enzymes, and accessibility of free radicals to react with DNA. Highly reactive hydroxyl radicals lead to a number of complex oxidative reactions within the DNA molecule and it’s nucleic acids (Evans et al. 2003). Hydroxyl radicals add to double bonds of heterocyclic DNA bases and remove an H atom from the methyl group of thymine and from each of the C-H bonds of 2’-deoxyribose, Covalent DNA-protein cross-links are shown to form in cells in vivo by exposure to free radical-generating systems (Evans et al. 2003). The mechanism of DNA-protein cross-linking involves the addition of the allyl radical of thymine in DNA to tyrosine in a protein, fallowed by oxidation. Oxidative damage to DNA produces a
  9. 9. 9 number of purine and pyrimidine-derived base lesions (Evans et al. 2003). The removal of these lesions by cellular repair processes is crucial in terms of limiting mutagenesis. DNA mutation is perhaps the most important consequence of lesion persistence (Evans et al. 2003). Cells have multiple systems in place to prevent lesion formation and ensure rapid repair. There is some overlap in substrates of DNA repair systems. Under normal conditions, cellular systems are continually generating ROS, creating a great potential for cellular damage, despite cellular protective mechanisms. DNA is one of the most important cellular targets with which these damaging species interact (Evans et al. 2003). ROS damage various cellular proteins responsible for DNA repair, cell cycle control, cell death etc. This creates a positive feedback loop leading to the accelerated accumulation of oxidative damage to DNA. (Kryston et al. 2011) Every cell of an organism is continuously exposed to exogenous and endogenous agents that damage its DNA. There are many types of lesions, which these harmful exposures can cause the most dangerous DNA lesion is the double-strand break. A single double stranded break can cause apoptosis, gene inactivation or lead to serious chromosomal aberrations (Barzilai and Yamamoto 2004). Cells have evolved a number of mechanisms to deal with this damage by limiting its occurrence and developing repair mechanisms. When considering the role of oxidative DNA damage in disease, it is important to remember that ROS cause damage to many structures besides DNA and these disruptions most likely play a key role in the development of disease. ROS do not need direct access to cellular DNA to have
  10. 10. 10 dramatic cellular effects. Modification to other proteins such as DNA polymerases can be just as mutagenic as disruptions to the DNA itself. (Evans et al. 2003). Antioxidant Protective Mechanisms A variety of protective cellular mechanisms exist to control the balance between production and removal of ROS. These include both endogenous and exofenous as well as enzymatic and non- enzymatic components. Antioxidant enzymes are the most specific and efficient in protecting cells from ROS (Reuter et al. 2010). Superoxide dismutases (SOD’s) and catylase (CAT) are key enzymes for the detection and clearance of ROS, found in all aerobic organisms (Ballesteros et al. 2008). Both endogenous exymatic and non-enzymatic antioxidants participate in the conversion of ROS to harmless substances and for protection of normal cellular function. Antioxidant enzymes have large potential for use in disease treatment, but little is known about their regulation by cells in response to different damaging ROS. Potentially, up-regulation of specific antioxidant enzymes could limit cell damage or even reduce disease risk (Mruk et al. 2002). Removal of free radicals from the cellular environment involves multiple intricate cascades of enzymes. Super oxide dismutases are a specific category of antioxidant enzymes that dismutate superoxide radicals (O2 -) into hydrogen peroxide (H2O2). Hydrogen peroxide is then converted into H2O by catalase. Catalase (CAT) is a heme protein located predominantly in the peroxisome in the inner mitochondrial membrane (Mruk et al. 2002). SODs are categorized and labeled by the transition
  11. 11. 11 metal found at their active site. Manganese-superoxide dismutase (MnSOD) is found most commonly within the mitochondria of eukaryotes and copper-zinc-superoxide dismutase (CuZnSOD) is most abundant in the cytosol. There is a third type of SOD, iron-superoxide dismutase but it was not examined in this study because it is never found in eukaryotes. SODs were the first characterized antioxidant enzymes and are of vital importance in protecting cells from oxidative damage by ROS. Both CuZnSOD and MnSOD are able to dismutate reactive oxygen (O2 -) anions into H2O2 and stable oxygen (Reuter et al. 2010). H2O2 is still reactive and harmful to cells. Catalase is the main enzyme responsible for the breakdown of H2O2 into water (Reuter et al. 2010). This 2-step process neutralizes and removes harmful ROS from the cellular environment and protects cellular structures, including DNA, from damage. The antioxidant defense system is of increasing interest to researchers because of its ability to provide biochemical biomarkers of oxidative stress and damage (Bellesteros et al. 2008.) Oxidative Stress and Cancer: Reactive oxygen species are implicated in various pathological events such as DNA mutations, carcinogenesis, aging, atherosclerosis, radiation damage, inflammation, neurodegenerative diseases, and toxic injuries (Evans et al. 2003). Through a diverse range of mechanism ROS have been clearly implicated in various phases of tumorigenesis (Reuter et al. 2010). ROS are produced readily during normal processes of metabolization and has many negative effects on the body; the most widely studied of which are its implications in tumerogenesis and cancer.
  12. 12. 12 Recent evidence supports the implication of persistent or chronic oxidative stress and damage in human cancer. No direct mechanism has yet to be determined, but is thought to involve the conversion of unrepaired DNA lesions into mutations, leading to cell transformation and carcinogenesis (Evans et al. 2003). Recent studies make it clear that mutations involving genes for DNA repair, cell growth and apoptosis related proteins plays a role in the promotion of chromosomal instability and malignancy. (Kryston et al. 2011) Carcinogenesis is a complex, multistep process dependant on an accumulation of genetic mutations leading to malignancy. Included in this process is often, activation of proto-oncogenes and inactivation or loss of tumor suppressor genes. Oxidative mechanisms have been shown to have a potential role in all of these steps (Evens et al. 2003). Evidence for this necessary accumulation of mutations, known as the multiple hit hypothesis, relies mainly on the cumulative cancer risk increase with the fourth power of age, associated with an accumulation of DNA damage due to incomplete cellular maintenance and repair. Because cellular systems are continually generative ROS, the potential for damage to cellular targets, such as DNA, is high. These high levels of oxidative DNA lesions along with their propensity for guanine-containing regions are shown to correspond with observed mutations in tumor suppressing genes ras and p53, both of which are implicated in malignant transformation. GCTA transversions have been observed in both the ras oncogene and the p53 tumor suppressor gene (Evenas et al. 2003). Because 8oxydG is a common product of DNA lesions to guanine-containing regions, it is not only an accurate biomarker of oxidative stress.
  13. 13. 13 Oxidative DNA damage is gaining recognition as an important risk factor in various forms of cancer. The transformation relies on persistent oxidative stress in cancer, derived from an environment low in antioxidant enzymes and high in ROS. This damage leads to the irregular activation of transcription factors, such as proto-oncogenes, leading to increased genomic instability and sometimes leading to cellular malignancy (Evans et al., 2003). ROS are important in carcinogenesis. This is supported by the close link between ROS formation and oxidative DNA damage and the implication of DNA damage and mutation in carcinogenesis (Evans et al. 2003). There are numerous reports of oxidative DNA damage being caused by known carcinogens and elevated levels of such damage associated with a wide range of malignant tumors (Evans et al. 2003). Effect of Ethanol on Oxidative Stress: Ethanol (EtOH) is a popular drug widely consumed throughout the world. The consumption of alcohol has been linked to a number of pathological conditions including several forms of cancer, liver failure, brain damage and fetal injury (Rosenberg et al. 2010). Because of the severity of these conditions and the high rate of alcohol consumption, it is worth looking more closely at the processes by which damage occurs and mechanisms by which organisms prevent and repair this damage. An understanding of these mechanisms could yield therapeutic benefits. There are three distinct enzymatic pathways responsible for alcohol metabolism within the liver. Hepatocytes are specialized liver cells capable of detoxification and are responsible for the metabolization of alcohol (Dey and Cederbaum 2006). The cytosolic hepatic alcohol
  14. 14. 14 dehydrogenase (ALDH) pathway is the main pathway responsible for the metaboliaztion of EtOH, accounting for 90% of alcohol oxidation and metabolism within hepatocytes exposed to ethanol (Lassen et al. 2005). This oxidation leads to the production of acetaldehyde, which can accumulate within cells and cause severe damage if not properly and quickly metabolized. Acetaldehydrogenases (ALDH’s) are responsible for detoxification and metabolism of acetaldehyde, making this process of utmost importance to cells. Ethanol has been shown to lead to an increase in ROS within tissues in a number of ways creating a complex positive feedback loop, leading to extensive tissue damage. Ethanol is not stored in the body and therefore whatever is ingested is oxidized. Ethanol metabolism by the ADLH pathway is directly involved in the production of reactive oxygen species, and also creates a cellular environment favorable to oxidative stress which can lead to further ROS production such as hypoxia, endotoxaemia and cytokine release (sergent et al. 2001). At the same time, alcohol reduces the levels of antioxidants present that are constantly working to eliminate ROS (Das and Vasudevan 2007). Alcohol changes the structure of the mitochondria within liver cells, where metabolism takes place. Alcohol damaged mitochondria are enlarged and distorted with distorted or broken cristae (Swift 2003). These changes lead to less effective metabolism by mitochondria, which are intimately involved in the generation and defense against intercellular ROS. In all cells ethanol decreases the capacity for the mitochondrion to carry out protein synthesis, which can lead to further ROS production in a positive feedback loop.
  15. 15. 15 Damage to cellular proteins and lipids occurs as a result of chronic ethanol consumption. Proteins are targets of free radicals and ROS, Which further compromise the mitochondrial membrane polarization. Membrane polarization is essential for ATP synthesis within the mitochondria (Reuter et al. 2010). Acetaldehyde and aldehyde (oxidized lipids) can bind to proteins and form stable adducts, which can lead to interference with protein function, and in some cases, induce an inappropriate immune response and inflammation. With repeated ethanol exposure, levels of antioxidant enzymes and other, cellular defenses are gradually diminished. This leads to an again increased toxicity as a result of prolonged ethanol exposure. Constructing a better understanding of the mechanisms by which alcohol promotes oxidative stress and the role of free radicals in alcohol-induced tissue injury are clearly important in attempts to look for effective therapies to prevent or diminish the toxic effects of alcohol. (Das and Vasudevan 2007). Alcohol is a major risk factor for disease (Das and Vasudevan 2007). There is a great potential for the usage of oxidatively generated DNA damage biomarkers (such as 8-oxydG and transcriptional levels of key proteins) towards detection and treatment of cancer and other diseases. The exploitation of new ‘cancer biomarkers’ has the potential to contribute significantly to early prognosis and design of more efficient therapeutic treatments especially in oxidative stress-related malignancies. (Kryston et al. 2011)
  16. 16. 16 Danio rerio as a Model: The zebrafish (Danio rerio) is emerging as a promising model organism for experimentation in biochemical research. Zebrafish are inexpensive, low maintenance and share 70-80% genetic homology to humans (Rosenberg et al. 2010). Zebrafish have been shown to react to exposure to oxidative stress on a cellular level. Specifically, alterations in the antioxidant defense system of fish have been observed in response to sub-lethal concentrations of certain harmful substances, which may be due to an adaptive response to ROS (Bellesteros et al. 2008) Dlugos and Rabin (2002) present data that zebrafish are an excellent model for studying genetic interactions with alcohol. In fish, as in humans, sensitivity and response to alcohol are highly influenced by genetics. Zebrafish have been extensively used for biomedical research and as a result a huge database of information is available and the genome has been entirely sequenced (Dlugos and Rabin 2002). Zebrafish are more closely related to humans then c. eligans or Drosophila while still being easy and inexpensive to maintain. This homology makes findings more relevant to human disease research. Fish readily absorb alcohol from their environment and reach steady-state brain-ethanol levels at 90% the concentration of their environment within 15 minutes of exposure (Dlugos and Rabin 2002). These levels can be maintained for over a week constant exposure. Based on this, blood alcohol levels of fish can be assumed without having to do invasive tests. Previous studies have demonstrated behavioral changes induced by acute EtOH exposure during 1 hour and that EtOH-mediated toxicity in zebrafish embryos can be partially attenuated by antioxidants (Reimers et al. 2006). Zebrafish are ideal for modeling the effects of EtOH because
  17. 17. 17 of the simplicity of alcohol delivery into their system from their immediate environment. EtOH is absorbed by the blood vessels of the gill and the skin so that blood alcohol levels reach equilibrium with the external alcohol concentration (Rosenburg et al. 2010). Diverse Tissue Analysis: Little has been done to examine how the effects of oxidative stress may be different between different tissues within a single organism. This is important in understanding cellular and tissue defenses and how they may differ between differentiated cell types. Certain tissues, such as cardiac and brain tissues have been shown to be far more sensitive to oxidative damage. The work of Bellesteros et al. (2008) concluded that SOD and other antioxidant enzymes respond to oxidative stress differently within different organs. This emphasizes the need to evaluate biomarkers in several different organs of fully asses the adaptive response of cellular tissues to ROS (Bellesteros et al. 2008). Ethanol exposure leads to a number of cerebral effects in both zebrafish and humans. Impairment of motor coordination, sensory perception and cognition can be related to oxidative damage to nervous system tissue in the brain (Rosenberg et al. 2010). Sperm are particularly sensitive to oxidative damage because their plasma membranes are enriched with unsaturated fatty acids, which are easily oxidized by ROS (Aiteken and Krausz 2001). The addition of catalase has been shown to partially alleviate these effects. Mutations
  18. 18. 18 within germ-line DNA are more detrimental then in other cells because they are passed directly onto the next generation (Aiteken and Krausz 2001). Based on these facts it is assumable that germ-line cells within the gonads may respond differently to an increased presence of ROS produced by alcohol metabolism. Objectives: Little is known about the effects of EtOH on the adult zebrafish and on the specific pathways it may induce during digestion. Rosenburg et al. (2010) examined the effects of EtOH induced oxidative stress on the brains of adult zebrafish; but the influence of acute EtOH exposure on oxidative stress and antioxidant response pathways that may inhibit cellular damage in different tissues of the adult fish still remain unexamined. This study aims to explore mechanisms of reactive oxygen species production within aerobic tissues induced by moderate exposure to ethanol. Closely observing and quantifying the effects of induced oxidative stress in living tissues may lead to a better understanding of this process and it’s immediate effects to living tissue. This study will focus on the production levels of 3 key antioxidant enzymes (CuZnSOD, MnSOD and CAT) in the brain, gonads and liver of zebrafish during and after exposure to ethanol. Antioxidant enzyme production will be measured by qPCR of cDNA transcribed from RNA samples extracted from tissue. This study aims to investigate the beneficial effects and transcriptional mechanisms of antioxidant enzymes in response to oxidative stress, which may protect DNA from excess reactive oxygen species and lessen the detrimental effects of oxidative stress.
  19. 19. 19 I hypothesize that exposing adult zebrafish to ethanol will cause an increase in oxidative damage to DNA in all tissues; the damage will be greater in tissues where ethanol is metabolized and those most sensitive to reactive oxygen species. This increase in oxidative stress is expected to correlate with an early rise in the production of SOD molecules.
  20. 20. 20 MATERIALS AND METHODS Animal Care: Adult zebrafish (3-6 months) of the wild type (Short Fin- SF) Danio rerio strain between 3 and 6 months old were obtained from a local supplier (Petsmart, Kingston NY). 20 Fish were kept in a 10-gallon tank for 3 days prior to exposure experiments. During this time fish were acclimated at standard conditions. The tank was filled with 10 gallons of water treated with 5mL Aqueon Water Conditioner (Central aquatics, Franklin WI) circulated by a single TopFin Power Filter (Petsmart, Phoenix AZ). Fish were fed once a day with Tetramin Tropical Flakes (Tetra Holding, Blacksberg VA). Fish for each experimental treatment group were randomly selected at the time of the experiment. Exposure: Immediately before exposure, 4 fish were randomly selected and removed from the 10-gallon tank for dissection. This is referred to as 0H and this first group is the control group, with no ethanol exposure. Ethanol was then added to the 10-gallon tank with the remaining 16 fish to a final concentration of 1.0% EtOH by the addition of 10mL 100% EtOH. 4 hours later, 4 more fish were randomly selected and removed for dissection. This is the 4H group, exposed to 4
  21. 21. 21 hours of EtOH. Fish were removed for dissection again at 8 hours (4 hours later). After these fish were removed for the 8H group, all remaining fish were moved to a smaller tank of fresh conditioned water to recreate the initial conditions before ethanol exposure. 4 hours after being moved to the pure water tank 4 more fish were randomly selected for dissection as the 12H group. Remaining fish were returned to population tanks and not used again. Figure 1: Diagrams the experimental procedure for ethanol exposure. 0h refers to the control group, which was removed from the tank (pictured in the middle top) just prior to the addition to ethanol. 4h refers to the group of 4 fish, which were removed and dissected after 4 hours of ethanol exposure. 8h refers to the fish removed after 8 hours of ethanol exposure. After fish were removed for the 8h group, the remaining fish were moved to a new tank of pure whater, represented here as EtOH removal. Time increases moving down the figure from the tank to the clock.
  22. 22. 22 Dissection: Fish were randomly chosen and removed from the 10-gallon tank and anesthetized in ice water for 5-10 minutes. A single fish was pined onto the dissecting mat in the prone position with two pins. The dissecting mat was placed on the field of a dissecting microscope. A scalpel was used to cut the skin along the plantar surface from the anal fin along the belly of the fish to the operculum. Dissecting scissors were used to remove the operculum, pectoral fin and pectoral girdle. Forceps and blunt dissection with a scalpel were used to open the thoracic cavity starting from the lower jaw along the cut line to the anal fin. Liver and intestines were carefully removed from the cavity in one piece and placed on the dissecting mat. All extraneous connective tissue was removed from the liver and intestines (left in one piece) using forceps and dissecting scissors. The liver and intestines were then placed in a labeled 2.5mL micro-centrifuge tube and placed on ice. The intestines were used along with the liver because the livers themselves were difficult to identify and too small to yield sufficient RNA. It is assumed that damage to liver tissue is comparable to damage to the intestines as both take part in digestion of EtOH. At this point the gonads can be identified easily within the empty body cavity. Forceps and blunt dissection were used to remove both testes (one from each side of the open cavity). Both were placed together in the same, labeled 2.5mL micro-centrifuge tube and placed on ice. Next the fish was unpinned and the head was removed using a scalpel. The body was removed and the head was pinned back to the dissecting mat ventral side up. Forceps were used to
  23. 23. 23 carefully break the scull and remove as much bone and connective tissue as possible. The brain was carefully removed using forceps and dissecting scissors. Once removed and clean of bone fragment the brain was placed in a labeled 2mL micro-centrifuge tube and placed on ice. The three individual and labeled tubes were placed in liquid nitrogen immediately for storage. This process was repeated for the other 3 fish. Dissection occurred immediately fallowing removal from the tank. RNA Extraction Total RNA extraction was preformed on each sample using mirVana isolation kit (Life Technologies, Grand Island, NY). RNA was extracted and isolated according to the maufacturers protocol (mirVana miRNA isolation kit protocol, Life Technologies Corporation 2011). Tissues were frozen homogenized manually by grinding them thoroughly with a pestle while still frozen. Aqueous layers were separated by chloroform and total RNA was collected and washed on collection filters. Total RNA was eluted in elution solution into 1.5uL microcentrifuge tubes. Nucleic Acid concentration for each sample was measured using a nanodrop. Samples were immediately placed in a -20˚ freezer. Experiments were done previously (data not shown) to determine the mirVana isolation kit to be the most effective method of RNA extraction from frozen tissue. Reverse Transcription: Extracted RNA was used to create cDNA by reverse transcription (rtPCR) using applied biosystems RNA-to-cDNA kit and mastermix according to the manufacturer’s protocol (Applied
  24. 24. 24 Biosystems, Foster City CA). RNA samples were first diluted to comparable concentrations. This was done in standard 96 well plates (Fig 3), which was the standard organization for the rest of the data collection and analysis. PCR (DNAenfine peltier thermal cycler, BioRad, Foster City, CA) settings were programmed according to manufacture’s protocol. 8uL of RNA was used for each reaction, and each sample was transcribed 4 separate times as it appears on the two plates in preparation for qPCR. Nucleic acid concentrations for each sample were measured using nanodrop. Samples were immediately. Figure 2: Diagrams the experimental procedure for RNA extraction, rtPCR and qPCR. There were a number of steps used to measure expression levels of antioxidant enzymes, which are listed in this flow chart.
  25. 25. 25 Quantitative PCR: Quantitative PCR (qPCR) was used run on all samples for each MnSOD, CuZnSOD, CAT and Actin-ß using primers (table 1). Actin-ß was used as to control in differences in overall expression levels as it is a standard housekeeping gene expressed at a relatively constant level. Actin-ß was used as an internal control and each expression level of antioxidant enzyme was normalized to the Actin-ß expression level for the corresponding sample. PCR was run on a BIO RAD CFX96 reaction system (BIO RAD, Hercules CA) using SYBRGreen supermix (Applied Biosystems, Foster City CA) and the corresponding manufacturers manual. Each reaction was prepared by mixing 2.0uL of a premade mix of mix of forward and reverse primers each at 10x, 1.0uL of cDNA template, 10.0uL of 2x SYBRgreen super mix and 7.0uL of DI water. Specific primer sequences are shown below (table 1) and were taken from Jin et al. (2010) whose group looked at expression levels in response to certain toxins. PCR program was run on all samples on two plates for the fallowing program: denaturation for 1 minute at 95˚C, followed by 40 cycles of 15 seconds at 95˚C and one minute at 60˚C. Data Analysis: Quantitative PCR measures the number of cycles of mRNA replication each sample takes to reach a threshold level, called the Ct value, which is measured by fluorescence. The more mRNAs present the fewer cycles it takes to generate the same quantity (threshold) of product. Because each additional cycle represents a doubling in product, a 1-cycle difference in Ct represents a 2-fold difference in starting mRNA. In this way, the relative abundance of mRNA for a specific gene (selected for with specific primers added to the sample) can be measured and
  26. 26. 26 compared to a control. When using qPCR there are two controls, both an internal and an external control. The internal control is a housekeeping gene, Actin-ß , whose expression levels should not be affected by EtOH exposure and should remain constant. The external control is the baseline expression of the target gene (this is the OH group, and is distinct for each measured antioxidant enzyme). This is the Ct value for each SOD before exposure to EtOH under control conditions (table #2). Ct values are normalized to the internal control by subtracting the Actin-ß Ct of the corresponding sample. This value is known as ∆Ct and controls for any variability in cDNA concentration on final expression levels. ∆Ct is divided by the external control of the corresponding untreated expression group (appendix 1). The resulting value is known as ∆ ∆Ct and represents the relative mRNA expression level in control and treatment groups. In untreated groups this value is 1 because it is a value divided by itself. Finally the relative expression is determined by raising 2 to the power of the negative value of ∆ ∆Ct for each sample. This is the RQ-value. This shows the fold-increase in expression of each sample (appendix). For each untreated group the RQ value is 1. There were four biological replicates (4 different dissected fish) measured by QPCR for each treatment group, and the average of these 4 RQ values is what is analyzed. Average RQ value and standard error were calculated and tested for each treatment group. Treatment groups refer to expression levels of the 4 biological replicates of each of the 3 antioxidant enzymes for each separate organ type. RQ-values were analyzed for each treatment group using a one-way ANOVA of the 4 contributing RQ-values calculated for each treatment
  27. 27. 27 group to test for significance. Post-Hok analysis was preformed on treatment groups that proved significant (p<0.05) to determine where the significant variation was coming from and graphs were labeled accordingly (fig 4).
  28. 28. 28 RESULTS Brain Enzyme Expression Levels: Visual inspection of the average expression levels of brain tissues, normalized to a control value of 1.0, shows that none of the antioxidant enzymes increase significantly over a period of 12 hours (Fig.2A). For MnSOD after 4 hours, average RQ was 1.7 times the control level, after 8 hours, average RQ was 1.9 times the control level and after 12 hours it was 2.0 times the control level (table 3A). A one-way ANOVA showed no significant expression differences among EtOH treatments (F =0.9 , P = 0.5). For CuZnSOD after 4 hours, average RQ was 2.2 times the control level, after 8 hours, average RQ was 3.1 times the control level and after 12 hours it was 1.5 times the control level (table 3A). A one-way ANOVA showed no significant expression differences among EtOH treatments (F =0.9 , P = 0.5. For CAT after 4 hours, average RQ was 4.2 times the control level, after 8 hours, average RQ was 5.2 times the control level and after 12 hours it was 2.2 times the control level (table 3A). A one-way ANOVA showed no significant expression differences among EtOH treatments (F = 1.6, P = 0.2).
  29. 29. 29 Liver enzyme Expression Levels: Visual inspections of the average expression levels of liver tissues, normalized to a control value of 1.0, suggest that MnSOD did not significantly increase, but both CuZnSOD and CAT show significant increase in average expression levels normalized to the pretreatment control after either 8 or 4 hours of exposure (Fig. 2B). For MnSOD After 4 hours, average RQ was 1.3 times the control level, after 8 hours, average RQ was 3.4 times the control level and after 12 hours it was 1.4 times the control level (table 3B). A one-way ANOVA showed no significant expression differences among EtOH treatments (F= 3.4, P = 0.06). For CuZnSOD After 4 hours, average RQ was 1.6 times the control level and after 8 hours, average RQ was 2.6 times the control level; but after 12 hours it was only 1.6 times the control level (table 3B). A one-way ANOVA showed a significant expression differences among EtOH treatments (F= 4.6, P = 0.02), and post-hoc analysis revealed that this was entirely due to the difference between the 8-hour treatment and control (Tukey-Kramer q = 2.97, P = 0.016 between 8-hour and control; all other P > 0.3). For CAT After 4 hours, average RQ was 4.8 times the control level but after 8 hours, average RQ was 2.9 times the control level and after 12 hours it was 2.5 times the control level (table 3B). A one-way ANOVA showed a significant expression differences among EtOH treatments (F = 5.04, P = 0.02). Post-hoc analysis revealed that this was entirely due to the difference between the 4-hour treatment and control (Tukey-Kramer q = 3.01, P = 0.012 between 4-hour and control; all other P > 0.3).
  30. 30. 30 Gonad Enzyme Expression Levels: Visual inspection of the average expression levels of gonad tissues, normalized to a control value of 1.0, suggest that MnSOD expression did not inrease, CuZnSOD increased significantly at 4 hours but not at 8 or12 hours and CAT was significantly increased at both 4 and 8 hours of exposre (fig.2C). For MnSODfter 4 hours, average RQ was 4.0 times the control level and after 8 hours, average RQ was 2.9 times the control level; but after 12 hours it was only 2.9 times the control level. A one-way ANOVA showed no significant expression differences among EtOH treatments for MnSOD (F = 2.9, P = 0.07). For CuZnSOD after 4 hours, average RQ was 2.7 times the control level but after 8 hours, average RQ was 2.0 times the control level and after 12 hours it was also 2.0 times the control level (Fig. 2C). A one-way ANOVA showed a significant expression differences among EtOH treatments (F = 3.96, P = 0.04), and post-hoc analysis revealed that this was entirely due to the difference between the 4-hour treatment and control (Tukey-Kramer q = 2.97, P = 0.023 between 8-hour and control; all other P > 0.3). For CAT aftter 4 hours, average RQ was 6.9 times the control level and after 8 hours, average RQ was 7.1 times the control level; but after 12 hours it was only 5.7 times the control level. A one-way ANOVA showed a significant expression differences among EtOH treatments (F = 5.04, P = 0.02), and post-hoc analysis revealed that this was due to the difference between the 8-hour treatment and the control (Tukey-Kramer q = 2.97, P = 0.01 between 8-hour and control); and the 4-hour treatment and the control (Tukey-Kramer q = 2.97, P = 0.02 between 4-hour and control); all other P > 0.3).
  31. 31. 31 DISCUSSION Brain Tissue: Brain tissue showed the lowest relative expression level of all 3 antioxidant enzymes. The highest expression was seen at 8 hours of exposure and only MnSOD showed a significant increase at this time (Figure 4A). This suggests that MnSOD may be the main protective factor, or at least the main acting scavenger of ROS in zebrafish brain tissue. The brain is known to have a relatively low antioxidant denfense system (Rico, et al. 2007). Bellesteros et al (2008) found that enzymatic activity in response to oxidative damage from environmental pollutants are lower in the brain then in other fish organs such as the gills and liver. This may explain, at least in part, why brain tissue is known to be particularly susceptible to oxidative damage (Bellesteros et al. 2008). This is consistent with the findings of the current study and suggests that the low relative expression seen in brain tissue is due to the fish not having a robust defense system. This does not give any information as to whether or not the ethanol treatment lead to oxidative damage within zebrafish brain tissue or not.
  32. 32. 32 Liver Tissue: Ethanol is metabolized by a 2 step process within liver hepacocytes. First, alcohol dehydrogenase (ADH) converts ethanol to acetaldehyde, the first byproduct of ethanol metabolism. Secondly, acetaldehyde is metabolized to acetate by aldehyde dehydrogenase (ALDH) forming acetate (Swift 2003). This process of alcohol metabolism is similar in zebrafish as to in humans, and zebrafish ADH shares a common ancestor with mammalian ADH, which is found in humans (Rico et al. 2007). Increased production of superoxide, H2O2 and hydroxyl radicals have been observed in the livers of EtOH fed rats. Dey and Cederbaum (2006) found that in rat liver, mitochondrial DNA (mtDNA) was affected after just a single dose of EtOH treatment, but cellular DNA was not. The current study looked at cellular DNA and would not have detected any changes in mitochondrial functioning as a result of acute ethanol exposure. Changes in the structure and function of mitochondrial proteins are one of the earliest effects of ethanol metabolization (Das and Vasudevan 207). Given the short exposure times used in the current study it is likely that unobserved damage took place within the mitochondria of hepatocytes. The destruction of mitochondria associated with alcohol metabolization contributes to the development of alcohol induces liver diseases (Hoek 2002). Although treatments were not long enough to show changes in expression levels of cellular DNA there may have been damage that occurred to mtDNA. Increase in expression of mtDNA for transcription factors may have shown an increase in liver tissue.
  33. 33. 33 By visual analysis, liver tissue shows generally higher expression levels of all 3 antioxidant enzymes then brain tissue, but lower expression levels then in gonads when compared to baseline pretreatment expression (Figure 4B). Liver tissue shows a steady increase in relative expression levels of all 3 antioxidant enzymes looked at, which peaks at 8 hours. This may have continued to increase if given longer exposure times but fish were removed from the EtOH treatment after 8 hours and by 12 hours the 3 SODs show some recovery. At this time, MnSOD and CAT expression levels are still significantly higher then baseline, pretreatment expression levels; suggesting a small, but significant, cellular response to toxic byproducts of alcohol taking place within liver tissue. Exposure treatments were comparatively short, lasting no more then 8 hours. Changes in gene expression may not take effect that quickly. Koch et al (2004) found no transcriptional modification in response to acute ethanol exposure, but significantly increased transcription of MnSOD after chronic exposure (Koch et al. 2004). A number of intermediate steps exist between initial alcohol exposure and the transcriptional response of cellular DNA (Dey and Cederbaum 2006). This multistep process of metabolization and cellular response to EtOH may be taking longer then 8 hours to activate an actual, significant response that could be detected by measurement of antioxidant enzyme transcription levels. There could still be significant oxidative damage occurring within liver tissue, which would lead to expression of antioxidant enzymes given more time.
  34. 34. 34 Average RQ values calculated for liver tissue show the lowest standard error compared to brain and gonad tissue. Liver tissue samples tended to be larger by mass then brain and gonad tissues (data not shown) and offered the highest yield or RNA. This was controlled for by standardizing RNA concentrations before rtPCR, but may still have had some effect on the accuracy of measured transcriptional levels. Gonad Tissue: Tissue dissected from the gonads of zebrafish show the most interesting results of this study, responding to EtOH exposure with the most immediate and highest increase in expression levels of the 3 antioxidant enzymes. The highest level of expression for gonad tissue was seen at 4 hours of EtOH exposure. Expression levels stayed high and relatively consistent through 8 hours of exposure and then through 4 hours of recover (figure 4C). Gonads show the most consistently significant increases in expression levels when compared to pretreatment controls. Oxidative stress disrupts the fertilizing capacity of sperm and attacks it’s DNA as in other cells. DNA fragmentation in sperm is of great importance because it is known to negatively affect both the fertilizing capacity of sperm and the ability of a resulting embryo to survive to term (Aitken and Krausz 2001); both of which directly affect an organisms reproductive capacity and fitness. Because sperm are so easily damaged by ROS, secretions of the male reproductive tract and of the gonads themselves have been shown (in humans) to contain specialized antioxidants, which surround and protect the germ-line cells within the extracellular space (Aitken and Krausz, 2001). This suggests that gonad tissue has a strong and effective response system to
  35. 35. 35 deal with oxidative damage, and that this defense involves the production of antioxidant enzymes. Female oocytes of zebrafish are bathed in interstitial fluid (similar to the sperm), which contains protective antioxidant enzymes (Lessman 2009). The developed egg remains dormant within the gonad until it is activated by exposure to freshwater at which point it becomes ready for fertilization (Lessman 2009). damages or mutations in the DNA of reproductive cells (found within gonads) lays the groundwork for a developing organism (Lessman 2009) during development. The viability of these reproductive cells is directly related to the fitness of an organism. Protecting these cells is evolutionarily advantageous, which supports the findings of the current study in which there was a significant increase in expression of antioxidant enzymes in as little as 4 hours of EtOH exposure. General Conclusions: This study measured the expression levels of 3 key antioxidant enzymes, but there are a number of other protective mechanisms (including other non enzymatic antioxidant molecules), which the cell utilizes in defense of oxidative damage (Koch et al, 2004). Mechanisms capable of neutralizing ROS include SODs, catalase, two kinds of glutathione (GSH) and a number of vitamins (McDonough 2003). Reduced glutathione (GSH) is an important antioxidant enzyme involved in detoxification of toxic agents within the cell. GSH is particularly important within the mitochondria. GSH was not looked at specifically in the current study and may have had an effect on the outcome of ethanol exposure. Metabolites of Alcohol can have inhibitory effects on gene expression, enhancing the negative effects of ethanol produced ROS (Rico et al. 2007).
  36. 36. 36 Increased production of superoxide, H2O2 and hydroxyl radicals have been observed in the livers of EtOH fed rats. Dey and Cederbaum (2006) found that in rat liver, mitochondrial DNA (mtDNA) was affected after just a single dose of EtOH treatment, but cellular DNA was not. This study looked at cellular DNA exclusively and not mtDNA; which may be why so few significant changes were seen in antioxidant expression levels. Primary damage occurs to the mitochondria and mtDNA, the effects of which could not be seen in the present study because only cellular DNA was examined for transcriptional changes. The most noticeable effect of acute ethanol exposure on liver tissue is a change in the structure of mitochondria (Dey and Cederbaum 2006). They become enlarged and distorted which effects their ability to function and can lead to the greater production of ROS (Das and Vasudevan 2007). EtOH induced oxidative stress has been shown to cause mitochondrial dysfunction and permeability changes, which precede and partially are responsible for liver injury associated with ethanol exposure (Dey and Cederbaum, 2006). Ethanol exposure affects mitochondrial free radical generation and induces expression changes in mitochondrial proteins (Koch et al. 2004). Dysfunctional mitochondria, whose proteins have been affected by ethanol, can lead to increased production of O2- (Koch et al. 2004). This increase in ROS likely causes heightened oxidative damage within associated tissue but could not have been detected in the current study. Mitochondria are constantly exposed to ROS during normal cell functioning because ROS are byproducts of metabolization, which occurs continuously within mitochondria (Das and Vasudevan 2007). Because they are constantly exposed, mtDNA is well protected and
  37. 37. 37 responds with the up regulation of protective mechanisms more quickly then other cellular components (Dey and Cederbaum 2006). Cellular DNA is less directly affected by ROS within the mitochondria and as a result does not need to respond as effectively to oxidative damage caused by distorted or dysfunctional mitochondria. Oxidative damage to the mitochondria interferes with the metabolic process leading to heightened production of ROS. This combined with the increased ROS due to alcohol exposure directly, leads to extensive damage to the mitochondria. More so then other structures, leading to a cascade of mitochondrial produced ROS (Das and Vasudevan 2002). It is important that the mitochondria respond quickly to avoid this cascade production of ROS. Mitochondrial GSH is the main cellular antioxidant responsible for detoxification of ethanol, and was not measured in the current study, but may have shown a more significant change in expression levels had it been analyzed. Acetylcholinesterase (AChE) is another enzyme involved in cellular protection from oxidative damage caused by alcohol, which is known to be expressed in the zebrafish brain (Bertrand et al. 2001). AChE is an enzyme found in zebrafish, encoded by a single gene. AChE is involved in the process of cleaving Ach into acetate during alcohol metabolism and is considered a protective mechanism within zebrafish tissues responding to several environmental contaminants (Rico et al. 2007). AChE expression was not measured in the current study but is likely an important part of the overall mechanism of cellular response to ethanol exposure.
  38. 38. 38 Rico et al. (2007) measured transcription levels of AChE in response to ethanol exposure and although AChE activity was shown to increase, mRNA levels were significantly decreased. This seemingly contradictory result suggests that an increase in gene activity is not directly related to higher gene expression levels (Rico et al. 2007). This may be true for the gene products measured in the current study as well; no tests were preformed to see if antioxidant enzyme activity was increased. Complex signaling and feedback loops are responsible for regulating transcription, and measuring mRNA by qPCR only looks at one aspect of this process. Rico et al. (2007) suggest that ethanol may regulate AChE post-transcriptionally which explains why higher activity is seen but not higher transcription levels. The same could be true for the 3 antioxidant enzymes examined in the current study. Metabolites of Alcohol can have inhibitory effects on gene expression, enhancing the negative effects of ethanol produced ROS (Rico et al. 2007). Any post transcriptional control of the antioxidant defense system could not be seen in the current study, leaving an incomplete picture of the cellular response to acute alcohol exposure. Relative expression levels of antioxidant enzymes offers important insight into the mechanism associated with response to oxidative damage but it is only a small part of the overall cellular response system which protects DNA from excessive oxidative damage.
  39. 39. 39 Future Direction: General trends from this study show an increase in expression levels in the first 4 and 8 hours of treatment. The levels of increase seen here are minimal and will likely continue to increase steadily if exposure treatments were continued for 12 or even 24 hours. Further experiments are needed to determine if this is the case and at which point expression of antioxidant enzymes peaks or levels off. This study does not look at oxidative damage, but at response to presumed oxidative damage, it would be interesting to have a direct measure of the actual oxidative damage to DNA against which to interpret antioxidant enzyme expression level results. Identification of oxidatively damaged DNA can be used as a reliable indicator of oxidative stress in vivo. 8-oxydG is as a reliable marker of high levels of oxidative stress and damage to tissues associated with cancer (Barzilai and Yamamoto 2004). This alteration in DNA from 8- oxydG is involved in the recognition site utilized by DNA glycosylases to detect damaged guanine bases. These lesions are not lethal to the cell but are highly mutagenic (Barzilai and Yamamoto 2004). The acute alcohol exposure may not have lasted long enough to cause the kind of damage that would elicit a significant enzymatic response, which could be controlled for by directly measuring the level of oxidative DNA damage which occurred. It would be advantageous to have a direct measure of oxidative damage within different tissues to create a more complete picture of how cells are responding to oxidative damage caused by EtOH.
  40. 40. 40 The high standard error seen throughout this experiment can be contributed to individual differences between fish (biological replicates). The internal control of Actin-ß ensured that differences between samples was not due to a difference in RNA concentration or overall expression level differences between fish. There was no control for age or gender of fish, both of which have been shown to have an impact on oxidative damage (Bellesteros et al. 2008). In the future it would be interesting to look at these variables specifically as a possible determinant of relative expression levels of different antioxidant enzymes and the effect of EtOH exposure on diverse tissues of zebrafish. It would be useful to compare baseline expression levels (listed as ∆Ct in appendix 1) between different tissues to create a more complete picture of cellular defense against oxidative damage by antioxidant enzymes. As it is the study looks only at expression levels relative to this standard assuming it is stable between tissues, but this may not be the case. A number of fallow-up experiments are needed to create a complete picture of the cellular defense mechanisms induced within various tissues of zebrafish in response to acute alcohol exposure. The current study establishes a useful protocol and jumping off point for the execution of further experiments.
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  44. 44. 44 FIGURES ANDTABLES Table 1: Sequences of primer pairs used in the quantitative PCR reaction to measure expression levels of corresponding enzymes against the internal control of Actin-ß Gene of Interest Primer name Sequence Annealing temperature Product Length MnSOD MnSOD-F 5’-CCGGACTATGTTAAGGCCATCT-3’ 56.4ºC 123bp MnSOD-R 5’-ACACTCGGTTGCTCTCTTTTCTCT-3’ 58.1ºC CuZnSOD CuZnSOD-F 5’-GTCGCTGGCTTGTGGAGTG-3’ 58.2ºC 113bp CuZnSOD-R 5’-TGTCAGCGGGCTAGTGCTT-3’ 59.3ºC CAT CAT-F 5’-AGGGCAACTGGGATCTTACA-3’ 55.9ºC 499bp CAT-R 5’-TTTATGGGACCAGACCTTGG-3’ 54.3ºC Actin-ß Actin-F 5’-ATGGATGAGGAAATCGCTGCC-3’ 57.7ºC 106bp Actin-R 5’-CTCCCTGATGTCTGGGTCGTC-3’ 59.2ºC
  45. 45. 45 Figure 3: A diagram of the 2 96 well plates, which were run through QPCR for this experiment; and the layout, which was used to organize samples and treatment groups throughout the experiment. Outlines the basic logic behind the experimental design in regard to data analysis.
  46. 46. 46 Figure 4A: Relative antioxidant expression levels in Brain Tissue. Compares antioxidant enzyme expression in brain tissue. Relative expression level (measured by RQ value) of 3 different antioxidant enzymes expressed in brain tissue extracted from Danio rerio after 4 and 8 hours of exposure to a 1% EtOH treatment, 4 hours after removal from EtOH (12H) and a pretreatment control (0H) to which each value is compared for significance of increase. Vertical bars represent standard error. Data taken from appendix 2. 0 1 2 3 4 5 6 7 8 9 10 0H 4H 8H 12H AverageRQValue Length of Treatment MnSOD CuZnSOD CAT
  47. 47. 47 Figure 4B: Relative Antioxidant Expression Levels in Liver Tissue Compares SOD expression in liver tissue. Relative expression level (measured by RQ value) of 3 different SODs expressed in liver tissue extracted from Danio rerio after 4 and 8 hours of exposure to a 1% EtOH treatment, 4 hours after removal from EtOH (12H) and a pretreatment control (0H) to which each value is compared for significance of increase. Vertical bars represent standard error. Data taken from appendix 2. 0 1 2 3 4 5 6 7 8 9 10 0H 4H 8H 12H AverageRQValue Length of Treatment MnSOD CuZnSOD CAT
  48. 48. 48 Figure 4C: Relative Antioxidant Expression Levels in Gonad Tissue Compares SOD expression in gonad tissue. Relative expression level (measured by RQ value) of 3 different antioxidant enzymes expressed in gonad tissue extracted from Danio rerio after 4 and 8 hours of exposure to a 1% EtOH treatment, 4 hours after removal from EtOH (12H) and a pretreatment control (0H) to which each value is compared for significance of increase. Vertical bars represent standard error. Data taken from appendix 2. -1 0 1 2 3 4 5 6 7 8 9 10 0H 4H 8H 12H AverageRQValue Length of Treatment MnSOD CuZnSOD CAT
  49. 49. 49 APPENDIXES Appendix 1: Raw data for expression levels measured by QPCR (given as Ct value) and intermediate values used to determine average RQ values for each sample individually.
  50. 50. 50