Organic waste compounds (OWC’s) are an environmental concern that can have adverse effects on humans, animals, and the environment. Ethinyl estradiol (EE2) is one common OWC and is an active ingredient in oral birth control pills. The presence of EE2 in aquatic environments and drinking water can be linked to decreased fertility and altered physiology in fish. I am developing Q-PCR biomarkers to detect changes in fish at low concentrations before adverse effects occur at higher concentrations. RNA was extracted from twenty fathead minnows that had not been exposed to OWC’s and will be used to synthesize cDNA. Thus far I have tested Q-PCR primers to measure vitellogenin RNA transcription. A standard curve and melt curve demonstrated proper amplification of a single product. A 96 hour exposure of 40 fathead minnows was conducted using EE2 at 0, 3, 17 and 100ng/L. Three 96 hour exposures of triclosan using 36 fathead minnows was conducted at 0, 0.1, 1 ug/L. Livers were dissected from the fish and are currently stored in the -80°C freezer to be used in the future

Frare, Tara

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Undergraduate ReviewVolume 7 Article 112011Development of Biomarkers for EndocrineDisrupting Compounds in the Fathead Minnow,Pimephales promelasTara FrareFollow this and additional works at: http://vc.bridgew.edu/undergrad_revPart of the Biochemistry, Biophysics, and Structural Biology Commons, and the EnvironmentalChemistry CommonsThis item is available as part of Virtual Commons, the open-access institutional repository of Bridgewater State University, Bridgewater, Massachusetts.Copyright © 2011 Tara FrareRecommended CitationFrare, Tara (2011). Development of Biomarkers for Endocrine Disrupting Compounds in the Fathead Minnow, Pimephales promelas.Undergraduate Review, 7, 45-48.Available at: http://vc.bridgew.edu/undergrad_rev/vol7/iss1/11BRIDGEWATER STATE UNIVERSITY 2011  •  THE UNDERGRADUATE REVIEW  •  45Tara Frare is a Bridgewater State University senior, majoring in biology. This research project was funded by a 2010 Adrian Tinsley Summer Grant, and was researched under the mentorship of Dr. Jonathan Roling. It was presented at the 2010 Undergraduate Research Symposium, as well as the National Conference on Undergraduate Research (NCUR) in March 2011.  Development of Biomarkers for Endocrine Disrupting Compounds in the Fathead Minnow, Pimephales promelasTARA FRAREOrganic waste compounds (OWC’s) are an environmental concern that can have adverse effects on humans, animals, and the environment. Ethinyl estradiol (EE2) is one common OWC and is an active ingredient in oral birth control pills. The presence of EE2 in aquatic environments and drinking water can be linked to decreased fertility and altered physiology in fish.  I am developing Q-PCR biomarkers to detect changes in fish at low concentrations before adverse effects occur at higher concentrations.  RNA was extracted from twenty fathead minnows that had not been exposed to OWC’s and will be used to synthesize cDNA.  Thus far I have tested Q-PCR primers to measure vitellogenin RNA transcription.  A standard curve and melt curve demonstrated proper amplification of a single product.    A 96 hour exposure of 40 fathead minnows was conducted using EE2 at 0, 3, 17 and 100ng/L.  Three 96 hour exposures of triclosan using 36 fathead minnows was conducted at 0, 0.1, 1 ug/L.  Livers were dissected from the fish and are currently stored in the -80�C freezer to be used in the future. Introduction:Many chemicals used in today’s society have adverse effects on animals and the environment.  Some of these chemicals are deposited in our rivers, streams and estuaries via effluents of sewage treatment plants.  Exposure to these chemicals can lead to reproductive changes in animals. We want to monitor these changes before significant reproductive and population alterations occur.  These small changes can be used as biomarkers.   A biomarker is a characteristic of molecular change that can be measured and evaluated as in indicator of a normal or pathogenic biological process.   My research project this summer was to develop biomarkers for endocrine disrupting compounds in the fathead minnow.One class of these chemicals that enter through the effluents are the components in oral contraceptives. These synthetic substances act like hormones in the endocrine system.  The endocrine system produces hormones that regulate many functions of an organism such as growth, tissue formation and reproduction.  When an organism is exposed to synthetic hormones the endocrine system may be come disrupted causing normal growth and reproduction to be altered.  These chemicals are called endocrine disrupting compounds (EDCs).National Conference on Undergraduate Research (NCUR)2011 Presentor46 • THE UNDERGRADUATE REVIEW • 2011  BRIDGEWATER STATE UNIVERSITYOne such EDC is ethinyl estradiol (EE2),  the active ingredient in birth control pills.  The presence of ethinyl estradiol in aquatic environments can be linked to changes in reproductive rates including decreased fertility in fish species (Folmer et al. 2001).  A gene known as vitellogenin is an egg yolk precursor expressed in female fish and  should never be expressed in male fish.  After exposure to estrogenic EDCs, male fish can express the vitellogenin gene.  This causes a feminization of male fish, causing them to no longer be able to reproduce.  This expression of the vitellogenin gene in male fish is used as a biomarker for EE2 exposure.  Because physiological change can be caused by EE2, we want to identify and monitor the effects of low dose exposure before physiological alterations occur.  Detecting these changes at low dose exposure is critical so that more adverse effects do not occur at higher concentrations.  Using biomarkers will enable us to monitor exposure of EE2 in the same low doses observed in the environment.  If these fish are continually exposed to EE2 in the aquatic systems reproduction can become impaired or completely halted. Methods and Materials:General methods for animal exposures: All exposures were completed using fathead minnows.  96 hours exposures were completed with a water exchange at 48 hours. Fish were fed AquaTox fish food daily.  Water was aerated during exposure.  After 96 hours fish were euthanized and weighed. Liver tissue was extracted for future RNA extraction. EE2 Exposure 06/09/2010:An exposure tank was set up using forty aerated 800ml or greater beakers.  EE2 was dissolved in ethanol to 1mg/L.  EE2 was then diluted to the highest concentration of 100ng/L EE2 with 1% ethanol.  Subsequent exposures were prepared by mixing one fifth dilutions from the higher concentration while maintaining 1% ethanol.  Treatment concentrations were divided evenly among ten beakers.  A control exposure was performed at 1% ethanol.   Forty fathead minnows were randomly selected from the BSU aquaculture facility and placed in individual beakers.  After 48 hours a 90% water change was preformed.  Dilutions were prepared as described above.  90% of the water in each beaker was discarded into a waste container, and replaced with fresh dilutions.  After 96 hours fish were euthanized on an RNAse free area.  Liver tissue was dissected out and placed in 800 ul of STAT-60 solution on ice. After samples were collected they were placed in the -80°C freezer.  Fish were weighed were measured after liver dissection.Triclosan Exposure 06/25/2010: An exposure tank was set up using forty aerated 800ml or greater beakers. A stock concentration of triclosan was dissolved to 1g/L. Triclosan was diluted to 10ug/L at 0.02% ethanol.  The subsequent concentrations were prepared using 1/10 dilutions while maintaining 0.02% ethanol.  A control exposure was prepared with  0.02% ethanol.  Forty fathead minnows were randomly selected from the BSU aquaculture facility and placed in individual beakers.  After 48 hours a 90% water change was preformed.  Dilutions were prepared as described above.  90% of the water in each beaker was discarded into a waste container, and replaced with fresh dilutions.  After 96 hours fish were removed from the solutions and euthanized on an RNAse free area.  Liver tissue was dissected out and placed in 800 ul of STAT-60 solution on ice. After samples were collected they were placed in the -80°C freezer.  Fish  were weighed and measured after liver dissection.Triclosan Exposure 07/07/2010:An exposure tank was set up using 12 ten gallon aerated fish tanks. Each aquarium was filled with 20 liters of water. Concentrations remained the same as previously described.      Thirty six fathead minnows were randomly selected from the BSU aquaculture facility three fish were placed in each aquarium.  After 48 hours a 50% water change was preformed. Dilutions were prepared as described above.  At the time of the water change air stones were added to the air hoses to increase the concentration of oxygen in the tanks.  After 96 hours fish were removed from the solutions and euthanized on an RNAse free area.  Liver tissue was dissected out and placed in 800 ul of STAT-60 solution on ice. After samples were collected they were placed in the -80°C freezer.  Fish  were weighed and measured after liver dissection.Triclosan Exposure 07/16/2010:An exposure tank was set up following all methods described in the previous triclosan exposure. The only exception being that air stones were added to the tanks at the onset of the exposure. Water change and dissection procedures also remained constant with the pervious exposure.Testing of 11βHSD primer set: Using Quantitative polymerase chain reaction (Q-PCR) samples of cDNA were run to determine if the primer was BRIDGEWATER STATE UNIVERSITY 2011  •  THE UNDERGRADUATE REVIEW  •  47binding to the correct target gene sequences, and allowing the Q-PCR to amplify the correct target.  Reagents per reaction were 15m moles primer, 1X Sybr Green Supermix (BioRad inc., Hercules, CA), and fathead minnow cDNA. 11βHSD primers were 5’-gCATCggCgAgCAgTTg-3’ and 5-’ CTCCTCgCCggTgATAACgA-3’.   Forty cycles of Q-PCR were performed at 95C for 30 seconds, 60C for thirty seconds, and 72C for thirty seconds using BioRad iQ5 real time PCR machine (BioRad inc. Hercules CA).  After amplification a melt curve was analyzed.Results and Discussion:An exposure of EE2 was successfully completed after each exposure the fish were weighed.  It was determined that there was no significant difference (P≤0.05) in the weights of the fish (Figure1).  This was expected, to ensure similar size fish to eliminate differences of fish age. All tissues extracted from the EE2 exposure are currently stored in the -80°C freezer and will be used for extraction of RNA. This RNA will be used to synthesize cDNA.  cDNA samples will be run through Q-PCR to determine if expression of the vitellogenin is seen in fathead minnows exposed to EE2. exposure an additional triclosan exposure was preformed.  Ten gallon aquariums were used for the second exposure to provide more water for the fish and prevent hypoxia. Three fish were placed in each tank.  We eliminated the 10ug/L concentration due to space constraints in the raceway and the extremely high toxicant concentration.  In the second exposure six fish were dead after 48 hours.  At this time we added air stones to the air hoses to increase oxygen concentrations in the water.  After 96 hours the fish were euthanized and liver tissue was extracted. Tissue samples are currently being stored at -80°C for future analysis.   Another triclosan exposure was completed this time using air stones in the tanks at the onset of the exposure to ensure proper oxygenation of the water.   Six fish fatalities were noted during this exposure.  It is uncertain why these fish died. After 96 hours the remaining fish were euthanized and liver tissue was extracted.  Tissue samples are currently being stored at -80°CAfter each exposure the fish were weighed.  It was determined that there was no significant difference (P≤0.05) in the weights of the fish (Figure1).  This was expected, to ensure similar size fish to eliminate differences of fish age.   Testing of primer 11βHSD was done to determine if the primer would correctly amplify the target gene sequences.  Previously isolated fathead minnow cDNA were used for vitellogenin primer testing.  The amplification curve (Figure2a) shows expected sigmoidal amplification of the product.  The standard Figure 1: Average Weights of Fish. Fathead minnows were exposed to EE2 at 0,3,17, 100ng/L. Three triclosan exposures were completed at 0, 0.1, 1ug/L. After liver extraction fish were weighed and measured to ensure consistency between treatments. None of the fish weights were statistically significant than the control fish. n = 10, mean + standard deviation, p < 0.05.Average Weights of Fish    In the first triclosan exposure that was completed we experienced a high mortality rate of the fish during the exposure time. In all 21 fish died. Liver tissue from the remaining fish is currently being stored in the freezer for future RNA extraction.  Because the beaker size may have been too small for the fish, the oxygen levels in the beakers were low.  Due to the outcome of this Figure 2a48 • THE UNDERGRADUATE REVIEW • 2011  BRIDGEWATER STATE UNIVERSITYFigure 2: Determination of efficiency of 11βHSD Development Using Q-PCR Primer. Samples of cDNA were run to determine if the primers were binding to the correct target gene sequences, and allowing the Q-PCR to amplify the correct target.  The amplification curve (Figure 2a) showed expected sigmoidal amplification of the product. The standard curve (Figure 2b) has a 96.9% efficiency rate. The Melt Curve (Figure2c) showed only one product, the gene of interest.curve (Figure2b ) has a 96.9% efficiency rate.  The melt curve (Figure 2c) shows only one product, the gene of interest.  We determined that 11βHSB did successfully bind to the target gene sequence.Conclusions and Future Work:One successful exposure using EE2 was completed. During the fall and spring semester I will continue my research. I will extract RNA from the liver tissue I dissected and use the RNA to synthesize cDNA.  Q_PCR will be run using tissue from the exposed fish, and I will determine if EE2 exposure leads to expression of the vitellogenin gene in the fish. The vitellogenin gene will be cloned and sent out for sequencing in order to verify that the correct gene of interest was amplified. Three exposures of triclosan were completed.  Due to the high mortality rate of the first exposure we will be unable to use any data gathered from the remaining fish tissue because our sample number is low.  This tissue will be used for  primer testing only.  The results of the subsequent triclosan exposures are still unknown and it is uncertain why so many fish died during this exposure. We determined that there was no significant difference in the weights of the fish used in the exposures.  We determined that 11βHSD primers were successfully binding to the correct target gene sequence.  This primer set will be used in the fall when my research continues examining 11bHSD to monitor changes in fish populations.Works CitedFolmer, James John J Tibbetts and Frank P. Delano. “Vitellogenin Gene Expression in Fathead Minnow as an Indicator of Exposure to Endocrine Chemicals (EDCs) in an  Aquatic Environment.”  Life Science and Medicine 62 (2002): 172-85. Print.Figure 2cFigure 2b

Development of Biomarkers for Endocrine Disrupting Compounds in the Fathead Minnow, \u3cem\u3ePimephales promelas\u3c/em\u3e

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Development of Biomarkers for Endocrine Disrupting Compounds in the Fathead Minnow, \u3cem\u3ePimephales promelas\u3c/em\u3e

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