Mev-1 - Oregon State University

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