Danika Kusuma Dr. Dee Denver Summer of 2010 HHMI
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Danika KusumaDr. Dee DenverSummer of 2010HHMI
http://www.kiwicrossing.com/drrussell/images/worm.jpg
The Effects of Vitamin E on the Lifespan of
Caenorhabditis elegans Strains with Mitochondrial
Mutations
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BackgroundFree-radical theory.
Organisms age because cells accumulate free radical damage over time.
Free radicals, reactive oxygen species (ROS)Comes from food, mitochondrial dysfunctions, etc.
AntioxidantsUnpaired electron reacts with biological structures,
causing damage; antioxidants prevent damage as reducing agents.
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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
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http://www.cnsforum.com/content/pictures/imagebank/hirespng/MOA_VITE.png
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NematodesModel Organism- Genome sequenced.- Easily maintained.- Short generation time - Short lifespanTwo 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
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HypothesisHigh 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.
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MethodsAssay lifespans of two genotypes of
nematodes: N2 wildtype and mev-1 strain – after vitamin E treatments.
Maintain populations of both
genotypes
Age-synchroniz
e population
s
Treatment with
Vitamin E
Random selection
of nematode
s to monitor lifespan
Checking nematode
s for mortality
and recording days lived
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ApproachStrain Negative control:
100% ethanol (10 microliters in 10 mL)
2 hours
200 micrograms/milliliter alpha tocopherol
2 hours
Negative control: 100% ethanol (10 microliters in 10 mL)
24 hours
200 micrograms/milliliter alpha tocopherol
24 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.
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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 hoursdepending on treatment group.
Maintain populations of both
genotypes
Age-synchroniz
e population
s
Treatment with
Vitamin E
Random selection
of nematode
s to monitor lifespan
Checking nematode
s for mortality
and recording days lived
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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
Age-synchroniz
e population
s
Treatment with
Vitamin E
Random selection
of nematode
s to monitor lifespan
Checking nematode
s for mortality
and recording days lived
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Results: 2 Hour Treatment
N2 -C 2hrs N2 200alpha 2hrs mev-1 -C 2 hrs mev-1 200alpha 2 hrs
0
5
10
15
20
25
30
Treatment Group
Num
ber D
ays
Live
d
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Results: 2 Hour Treatment (con.)• “mev-1 strain will have a shorter
lifespan than N2 wildtype C. elegans.”
• 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= 0.89Between mev-1 –C and mev-1 treated: p-value= 0.023
• Trend shows an increase in lifespan.
N2 -C 2hrs
N2 200alpha
2hrs
mev-1 -C 2 hrs
mev-1 200alpha
2 hrs
0
5
10
15
20
25
30
Treatment Group
Num
ber D
ays
Live
d
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Results: 24 Hour Treatment
N2 -C 24hrs N2 200alpha 24 hrs mev-1 -C 24 hrs mev-1 200alpha 24 hrs
0
5
10
15
20
25
Treatment Group
Num
ber D
ays
Live
d
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Results: 24 Hour Treatment (con.)• “mev-1 strain will have a
shorter lifespan than N2 wildtype C. elegans.”
• 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= 0.42Between mev-1 –C and mev-1 treated: p-value= 0.59
• Trend shows a decrease in lifespan.
N2 -C 24hrs
N2 200al-pha 24
hrs
mev-1 -C 24 hrs
mev-1 200al-pha 24
hrs
0
5
10
15
20
25
Treatment Group
Num
ber D
ays
Live
d
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Future WorkANOVA
Further lifespan assays with increased replication.
Measuring mitochondrial ROS levels.
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AcknowledgementsDr. Dee DenverDana HoweKristin GafnerElizabeth QuimbaSita PingDr. Kevin AhernHoward Hughes Medical InstituteUniversity Honors CollegeNational Institutes of Health