Optimization of the Comet Optimization of the Comet Assay for Use in ReptilesAssay for Use in Reptiles

Optimization of the Comet Optimization of the Comet Assay for Use in ReptilesAssay for Use in Reptiles

Testing the Effects of Genotoxic Agents on Testing the Effects of Genotoxic Agents on Estuarine and Freshwater ReptilesEstuarine and Freshwater Reptiles

John SpinicchiaJohn SpinicchiaChesapeake Biological LaboratoryChesapeake Biological LaboratoryTeacher Research Fellowship 2004Teacher Research Fellowship 2004

PI: Carys Mitchelmore, Ph.D. PI: Carys Mitchelmore, Ph.D.

Picture: http://cgi.ebay.com/ws/eBayISAPI.dll?ViewItem&category=2312&item=3691846964&rd=1#ebayphotohosting

Principal Investigator:Principal Investigator:Carys Mitchelmore, Ph. D.Carys Mitchelmore, Ph. D.

Principal Investigator:Principal Investigator:Carys Mitchelmore, Ph. D.Carys Mitchelmore, Ph. D.

Assistant Professor, UMCES @CBLAssistant Professor, UMCES @CBL

Aquatic toxicology and Biomarker developmentAquatic toxicology and Biomarker development

picture: Carys Mitchelmore, 2004.

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 2004.

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.

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.

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.

The comet assay is based on the work of Singh, Tice,et. al.(1988) with minor modificationsPicture: Picture: http://www.kineticimaging.com/comet-prep.htm

How does the comet assay work?Using any nucleated cell:

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

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: H2O2, 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

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)

Mean % head/Tail DNA, H2O2

0102030405060708090

100

0 1 2 3 4 5

Treatment

Mea

n %

DN

A

Mean % Head DNA

Mean % Tail DNA

N=2, n =3

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?

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.

Engagement: Day TwoActivities 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)

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.

ExplanationStudents 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

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

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

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”

References Mitchelmore, C.L., J.K. Chipman. 1998. DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mut. Res. 399, 135-147.

Fairbairn, D., Olive, P.L., and O’Neill, K. 1995. The comet assay: a comprehensive review. Mut. Res. 339, 37-59.

MSDE. 2004. Teacher developed 5E model for science lessons. Accessed on the World Wide Web at http://www.mdk12.org/instruction/curriculum/science/5emodel.html August 4, 2004.

Edler,L., Kim B.S., Park J., Thielmann H.W. 2002. 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

www.dkfz-heidelberg.de/ biostatistics/reports/kievvro.pdf July 29, 2004.

Society of Toxicology. 2004. Paracelsus goes to School workshop materials. 43rd annual meeting, Baltimore, MD, March 23, 2004.

Gilbert, S.G. 2004. A Small Dose of Toxicology: The Health Effects of Common Chemicals. Washington, D.C.: CRC Press.