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Optimization of the Comet Assay for Use in Reptiles Testing the Effects of Genotoxic Agents on Estuarine and Freshwater Reptiles John Spinicchia Chesapeake.

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Presentation on theme: "Optimization of the Comet Assay for Use in Reptiles Testing the Effects of Genotoxic Agents on Estuarine and Freshwater Reptiles John Spinicchia Chesapeake."— Presentation transcript:

1 Optimization of the Comet Assay for Use in Reptiles Testing the Effects of Genotoxic Agents on Estuarine and Freshwater Reptiles John Spinicchia Chesapeake Biological Laboratory Teacher Research Fellowship 2004 PI: Carys Mitchelmore, Ph.D. Picture:

2 Principal Investigator: Carys Mitchelmore, Ph. D. Assistant Professor, Aquatic toxicology and Biomarker development picture: Carys Mitchelmore, 2004.

3 Research Project I worked in the Mitchelmore lab at Chesapeake Biological Lab in Solomons, Maryland as part of the UMCES~MD Sea Grant Teacher Research Fellowship from June-August My research was used to develop the “Comet Assay” of DNA single-strand breaks (SSB) as a novel biomarker in reptiles to assess general genotoxic damage. Pictures: John Spinicchia, 2004.

4 Paracelsus ~A.D “All substances are poisons. There is none that is not a poison. The dose differentiates between the poison and the remedy” Toxicologists refer to this phenomenon as dose response Dose, route of exposure, time of exposure significant There are two types of effects on individual organisms Lethal: easy to detect Sub-lethal: one needs to be creative to detect!

5 Why develop the comet assay? A limited number of analyses of organisms have implicated DNA single strand breaks (SSB) as a broad and sensitive indicator of general genotoxic damage from certain chemical by-products of society. These effects of anthropogenic toxicants can have a profound adverse effect on individuals, populations, genetic diversity and ultimately biodiversity of an ecosystem.

6 What are some sub lethal effects of these highly reactive compounds?* Carcinogenesis: up regulated/uncontrolled cell growth Teratogenesis: birth/developmental defects Clastogenicity: gross chromosome damage Genotoxicity: lethal/non-lethal molecular cell damage (DNA) * PCBs, PAHs, metals(PB, Hg, etc), MX(3-chlor-4-(dichloromethyl)-5-hydroxy-(5H)-furanone {a byproduct of commercial water chlorination}, H 2 O 2 and other radical-producing compounds.

7 Specific genotoxic chemicals cause single strand breaks in DNA molecules The Single Cell Gel Electrophoresis (Comet Assay) can illuminate the extent of the damage Pictures: Edler, Kim, Park, Thielmann, 2002.

8 The comet assay is based on the work of Singh, Tice,et. al.(1988) with minor modifications Picture: Picture: How does the comet assay work? Using any nucleated cell:

9 My Research Assay was developed for use in mammals, insects, fish, birds and has been used in plants and aquatic invertebrates with limited success. There is not (as of summer 2004) a single paper using this particular assay to assess DNA SSB in reptiles. Mechanisms are needed to assess toxicity effects in reptiles. Chelydra serpentina (Snapping Turtle) and Malaclemys terrapin (Diamondback Terrapin) were available at CBL* and important in Chesapeake Bay Ecosystem *Thanks, Chris! Female terrapin Male terrapin

10 The Specifics I used blood drawn from the caudal sinus of freshly captured turtles I incubated the blood with known direct acting genotoxic agents which cause SSB: H 2 O 2, Ethylmethanesulfonate (EMS), and 1-methyl-3-nitrosoguanidine (MNNG) Aligned with values in published literature: electrophoresis rigs Chemical dilution strengths Buffer and pH values Blood plasma osmolarity Compared turtle results with Fundulus heteroclitus and Carpus sp. Required sacrificing fish, but not the reptiles  Analyzed results with Komet 5.5 software

11 My Results Were inconclusive because N numbers were low due to time constraints Results did show positive dose/response correlation Results will be published when we get more results (later this year) N=2, n =3

12 Classroom Project: “Discovering Toxic Response in Chesapeake Bay Organisms” I wanted my students to explore the following questions: What is a toxicant? What types of substances can be toxic? What variables of exposure are required to impact organisms? How can you tell if an organism is affected (if it isn’t readily evident)? How are real scientists addressing these questions?

13 Engagement: Day One Activities capture students’ attention, stimulate thinking, and access prior knowledge Student groups are given two 1 L flasks filled with distilled water and two tubes: 1 mg salt and 1mg water soluble dye (like acridine orange) Pour contents of each tube into a flask and dissolve Class discussion: what different ways can we come up with to identify the substances in each flask? How much more solvent than solute is in each flask? (Concept of ppm) Are there substances in the environment that can harm at these concentrations (or less)? Introduce Concept of toxicology with toxicology terms handout Distribute/ complete/discuss SOT handouts p 6&7.

14 Engagement: Day Two Activities capture students’ attention, stimulate thinking, and access prior knowledge Read CBP/Alliance for the Chesapeake pub “Toward a Contaminant- Free Bay” p.1 and “A Toxics Primer” p.2 Students will reflect on the statement in writing: ”No matter where we look in the Bay, we find evidence of some chemical contamination…There are probably no pristine, truly uncontaminated sites left in Chesapeake Bay.” (CBP, 1994, The Chesapeake Bay Toxics Strategy)

15 Exploration (1) Students are given time to think, plan, investigate, and organize collected information Students will use new technology (handout) and Crassostrea virginica to attempt to answer the following: How can scientists use technology to identify genotoxic effects in a large ecosystem like the Chesapeake Bay? Students will: Explore time of exposure and amount of exposure (dose) Practice abstract thinking skills: making serial dilutions Use molecular techniques to identify DNA damage to nucleated hemolymph cells Engage in meaningful homework activities to scaffold lab work: SOT handouts p 8&9. Engage in meaningful class discussion: how do toxicants affect biological levels of organization? SOT graphic organizer p. 4.

16 Exploration (2) Students are given time to think, plan, investigate, and organize collected information Students will use Fast Plants (Brassica rapa) to attempt to answer the following: How can scientists use technology to identify genotoxic effects of every day chemicals on a model organism? Students will: Explore time of exposure and amount of exposure (dose) Practice making serial dilutions Use the scientific method to make inferences and draw conclusions from real data Engage in meaningful homework activities to scaffold lab work: SOT handouts p 8&9. Engage in meaningful class discussion: how do toxicants affect biological levels of organization? SOT graphic organizer p. 4.

17 Explanation Students analyze their exploration, and reflect upon outcomes: understandings are modified and clarified Analyze the collected data through the use of spreadsheet examination, graphing results, and a written lab report in the form of a scientific paper: Introduction, materials and methods, results, and discussion

18 Evaluation Occurs throughout lesson; use of scoring tools targets what students will know and do Evaluation will be ongoing, both formal and informal: Original reflection piece from engagement; Lab report; Performance on selected handout assignments; Student/teacher interaction: students’ ability to answer posed and formal questions

19 Extension Allows students to apply what she/he learned to real world situation(s); solidifies concepts Provide a menu of activities to tie the students’ results into real world issues that have a personal “hook”: A letter to the editor of the local paper encouraging the community to be cognizant of waste production & disposal A storm drain stenciling project More directed research as an after school or science fair project Help to develop WebQuest to find out more info An extensive list of projects and materials on handout

20 I wish to thank the following, without whose help this study would not have been possible: Dr. Carys Mitchelmore and all members of the Mitchelmore lab for direction, patience, and guidance Dr. Chris Rowe and members of the Rowe Lab for providing a “nearly endless” supply of turtles All members of the MD Seagrant/UMCES team My wife for graciously agreeing to “give up my summer”

21 References Mitchelmore, C.L., J.K. Chipman DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mut. Res. 399, Fairbairn, D., Olive, P.L., and O’Neill, K The comet assay: a comprehensive review. Mut. Res. 339, MSDE Teacher developed 5E model for science lessons. Accessed on the World Wide Web at August 4, Edler,L., Kim B.S., Park J., Thielmann H.W The Single Cell Gel Electrophoresis (Comet) Assay as a Means to Estimate the Radiation Sensitivity of Individuals. PowerPoint Presentation. Accessed on the World Wide Web at biostatistics/reports/kievvro.pdf biostatistics/reports/kievvro.pdf July 29, Society of Toxicology Paracelsus goes to School workshop materials. 43 rd annual meeting, Baltimore, MD, March 23, Gilbert, S.G A Small Dose of Toxicology: The Health Effects of Common Chemicals. Washington, D.C.: CRC Press.


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