does the circadian clock modulate an organism’s response to toxins?

Does the Circadian Clock Modulate an Organism’s

Response to Toxins?

Katherine Sherman

Mentor: Dr. Jaga GiebultowiczCo-Mentor: Dr. Louisa Hooven

Circadian Clock• Mechanism driving daily rhythms of life

processes• Entrained by external stimuli:

• Light/dark cycles• Temperature cycles

Entrainment

T

Clock Regulated systems

Clock Genes• 6-10% of genes rhythmically expressed in

Drosophila melanogaster

• Clock controlled genes have diverse functions:

• metabolic enzymes• oxidative stress• ion channels• detoxification

Rationale• The clock may be able to adapt to an

organism’s recurring chemical challenge

Significance• The clock may be involved in the

development of resistance to chemicals in both insects and humans

• This may have implications for the timing of administration of pharmaceuticals to patients

http://www.carolinahealthnotes.com/wp-content/uploads/2008/12/j0398845.jpg

Drosophila melanogaster

• Common biological model• Has clock mechanism similar to that of

humans

http://myrmecos.files.wordpress.com/2008/10/drosophila2s.jpg

Propoxur• A member of the carbamate type of

pesticides• Drosophila show rhythmic pattern of

susceptibility to propoxur

0 4 8 12 16 200

10

20

30

40

ZT

LC50

Drosophila Offer Genetic Tools• Several key proteins involved in clock

mechanism• Mutants exist with an absent or defective

clock protein Feedback loops of clock mechanism

Disrupting the Clock• It has been shown removing clock genes

can disturb activity rhythms Rhythmic Arrhythmic

NormalizedJaga's Special Flies M01C11

0 6 12 18 0 6 12 18 0

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NormalizedJaga's Special Flies M01C30

0 6 12 18 0 6 12 18 0

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Detoxification and Circadian Clock• Detoxification genes and susceptibility to

propoxur show parallel rhythmic activity in Drosophila

• This suggests that the circadian clock modulates the ability to detoxify propoxur

Cyc01 Flies Have a Disrupted Clock• Cyc01 lack the function of the clock gene

cyc and are clock deficient • To learn how the absence of this gene

affects detoxification, cyc01 are tested alongside flies with a normal clock, CSc

http://www.familyhistory101.com/images/main/dna_500.jpg

Hypothesis• The clock mechanism can shift to increase

an organism’s resistance to a recurring chemical insult

Phase Shift in Response to a Stimulus

Predictions• Repeated exposure to propoxur at the

same time daily will decrease sensitivity to propoxur at this time in flies with a normal clock

• In response to repeated exposure to propoxur, the peak of susceptibility in these flies will shift

http://www.creativewatch.co.uk/office-wall-clock-cwc415-creativewatch-large.jpg

Method• Perform dose response tests to determine

sub-lethal dose and LC50• Flies are collected around the clock at 4

hour intervals before and after treatment– Measure detoxification enzyme activity

• Expose a large group of CSc and cyc01 flies to the sub-lethal dose at the same time daily for 4 consecutive days– Keep flies in constant darkness– Treatment performed at 9:00pm– Exposure lasts for a duration of one hour

• Control flies are manipulated in a similar way without exposure to propoxur

• After a two day hiatus a dose response test is performed to determine if the LC50 has changed– Upregulation of genes in response to a toxin

would be expected to subside during this time period

– While a change in the rhythm of the clock would be expected to persist

Results• Two days after the dose response test, the

number of flies that have died are counted• Percent mortality is calculated and

graphed to determine the LC50 for each group of flies

• The average of the four trials performed shows no significant difference in the susceptibilities of any group of flies

• When the LC50s for each group are compared there is no significant difference

Conclusion• Our results indicate that propoxur cannot

shift the clock at the dose and treatment regimen used

Future Work

• One data set compared susceptibility to propoxur between males and females

• This revealed dramatic sex-specific differences in LC50

• We plan to examine whether these differences depend on an intact clock– Test LC50 in males and females with mutated

clock genes

Acknowledgements• HHMI• Dr. Kevin Ahern• Dr. Jaga Giebultowicz• Dr. Louisa Hooven• Eileen Chow• Members of the Giebultowicz lab