Mev-1 - Oregon State University

Report
The Effects of Vitamin E
on the Lifespan of
Caenorhabditis elegans Strains
with Mitochondrial Mutations
Danika Kusuma
Dr. Dee Denver
Summer of 2010
HHMI
http://www.kiwicrossing.com/drrussell/images/worm.jpg
Background
 Free-radical theory.
 Organisms age because cells accumulate free radical damage over
time.
 Free radicals, reactive oxygen species (ROS)
 Comes from food, mitochondrial dysfunctions, etc.
 Antioxidants
 Unpaired electron reacts with biological structures, causing
damage; antioxidants prevent damage as reducing agents.
Background (con.)
 Vitamin E
 Fat-soluble, several different types.
 Alpha tocopherol: often used as a supplement vitamin and main role is as
an antioxidant although it may have cell signaling properties that are not
well-known.

Used as a supplement vitamin and the main type of vitamin E that is held in the body.
http://www.3dchem.com/imagesofmolecules/vitamine.jpg
http://www.cnsforum.com/content/pictures/imagebank/hirespng/MOA_VITE.png
Nematodes
Model Organism
- Genome sequenced.
- Easily maintained.
- Short generation time
- Short lifespan
Two Strains
-N2 wild type
Caenorhabditis elegans
- mev-1 (genetic mutant
strain of C. elegans)
- mitochondrial
electron transport
chain mutant
- higher ROS levels
and shortened
lifespan
http://www.wormclassroom.org/ce/kahn/C.elegans_L4.jpg
Hypothesis
 High levels of free radical damage result in a decrease
in lifespan.
 PREDICTION:
 mev-1 strain will have a shorter lifespan than N2
wildtype C. elegans.
 Vitamin E reduces ROS damage and increases lifespan.
 PREDICTION:
 The introduction of antioxidant vitamin E to N2
wildtype C. elegans and mev-1 strain will increase
lifespan in both strains.
Methods
 Assay lifespans of two genotypes of nematodes: N2
wildtype and mev-1 strain – after vitamin E
treatments.
Maintain
populations
of both
genotypes
Agesynchronize
populations
Treatment
with
Vitamin E
Random
selection of
nematodes
to monitor
lifespan
Checking
nematodes
for mortality
and
recording
days lived
Approach
Strain
Negative control:
100% ethanol (10
microliters in 10 mL)
200
micrograms/mill
iliter alpha
tocopherol
Negative control:
100% ethanol (10
microliters in 10
mL)
200
micrograms/mill
iliter alpha
tocopherol
2 hours
24 hours
24 hours
2 hours
N2 wild
type
24 worms
24 worms
24 worms
24 worms
Mev-1
24 worms
24 worms
24 worms
24 worms
Monitored
192 worms total.
All vitamin E treatments in 100% ethanol.
One treatment group exposed for 2 hours, another exposed for 24 hours.
Approach (con.)
 Worms first developmentally synchronized.
 Populations of worms suspended in 10 milliliters M9 buffer and then
treated with 10 microliters of each treatment substance.
 Put on a nutator for 2 hours or 24 hours
depending on treatment group.
Maintain
populations
of both
genotypes
Agesynchronize
populations
Treatment
with
Vitamin E
Random
selection of
nematodes
to monitor
lifespan
Checking
nematodes
for mortality
and
recording
days lived
Treatments (con.)
 Individual worms are monitored and lifespan is
assayed.
 Worms are determined alive by twitch response.

Lifespan assay recorded as number of days lived.
Maintain
populations
of both
genotypes
Agesynchronize
populations
Treatment
with
Vitamin E
Random
selection of
nematodes
to monitor
lifespan
Checking
nematodes
for mortality
and
recording
days lived
Results: 2 Hour Treatment
30
Number Days Lived
25
20
15
10
5
0
N2 -C 2hrs
N2 200alpha 2hrs
mev-1 -C 2 hrs
Treatment Group
mev-1 200alpha 2
hrs
Results: 2 Hour Treatment (con.)
30
• “mev-1 strain will have a shorter lifespan
than N2 wildtype C. elegans.”
25
•Between N2-C and mev-1 –C:
p-value= 0.00011
Between N2 treated and mev-1 treated:
p-value= 0.0034
Between N2 –C and N2 treated:
p-value= 0.89
Between mev-1 –C and mev-1 treated:
p-value= 0.023
Number Days Lived
20
15
10
5
0
N2 -C
2hrs
N2
mev-1 -C mev-1
200alpha 2 hrs 200alpha
2hrs
2 hrs
Treatment Group
• Trend shows an increase in lifespan.
Results: 24 Hour Treatment
30
Number Days Lived
25
20
15
10
5
0
N2 -C 24hrs
N2 200alpha 24 hrs
mev-1 -C 24 hrs
Treatment Group
mev-1 200alpha 24
hrs
Results: 24 Hour Treatment (con.)
30
• “mev-1 strain will have a shorter
lifespan than N2 wildtype C. elegans.”
Number Days Lived
25
20
15
10
5
0
N2 -C
N2
mev-1 -C mev-1
24hrs 200alpha 24 hrs 200alpha
24 hrs
24 hrs
Treatment Group
•Between N2-C and mev-1 –C:
p-value= 1.73 × 10-5
Between N2 treated and mev-1
treated:
p-value=4.19 × 10-5
Between N2 –C and N2 treated:
p-value= 0.42
Between mev-1 –C and mev-1 treated:
p-value= 0.59
• Trend shows a decrease in lifespan.
Future Work
 ANOVA
 Further lifespan assays with increased replication.
 Measuring mitochondrial ROS levels.
Acknowledgements
 Dr. Dee Denver
 Dana Howe
 Kristin Gafner
 Elizabeth Quimba
 Sita Ping
 Dr. Kevin Ahern
 Howard Hughes Medical Institute
 University Honors College
 National Institutes of Health

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