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

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.

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

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

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.

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

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.

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

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

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

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

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

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

Future WorkANOVA

Further lifespan assays with increased replication.

Measuring mitochondrial ROS levels.

AcknowledgementsDr. Dee DenverDana HoweKristin GafnerElizabeth QuimbaSita PingDr. Kevin AhernHoward Hughes Medical InstituteUniversity Honors CollegeNational Institutes of Health