Does the Circadian Clock
Modulate an Organism’s
Response to Toxins?
Katherine Sherman
Mentor: Dr. Jaga Giebultowicz
Co-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
40
20
10
ZT
20
16
12
8
4
0
0
LC50
30
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
Jaga's Special Flies M01C11
0
6
12
Jaga's Special Flies M01C30
Normalized
18
0
6
12
18
0
0
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08/01/07
08/03/07
08/03/07
08/05/07
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08/07/07
08/09/07
08/09/07
08/11/07
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08/13/07
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08/15/07
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6
12
Normalized
18
0
6
12
18
0
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
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HHMI
Dr. Kevin Ahern
Dr. Jaga Giebultowicz
Dr. Louisa Hooven
Eileen Chow
Members of the Giebultowicz lab