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: http://cgi.ebay.com/ws/eBayISAPI.dll?ViewItem&category=2312&item=3691846964&rd=1#ebayphotohosting
Principal Investigator:
Carys Mitchelmore, Ph. D.
Assistant Professor, UMCES @CBL
Aquatic 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 modifications
Picture: 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: 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

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
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5
Treatment
M
e
a
n

%

D
N
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 isnt 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 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
contaminationThere 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.



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

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 ONeill, 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. 43
rd
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.