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 ClockRegulated 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

Drosophila melanogaster Common biological model Has clock mechanism similar to that of humans

Propoxur A member of the carbamate type of pesticides Drosophila show rhythmic pattern of susceptibility to propoxur

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

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

Cyc 01 Flies Have a Disrupted Clock Cyc 01 lack the function of the clock gene cyc and are clock deficient To learn how the absence of this gene affects detoxification, cyc 01 are tested alongside flies with a normal clock, CS c

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

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 CS c and cyc 01 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