Does the Circadian Clock Modulate an Organism’s Response to Toxins?

1. Does the Circadian Clock Modulate an Organism’s Response to Toxins? Katherine Sherman Mentor: Dr. Jaga Giebultowicz Co-Mentor: Dr. Louisa Hooven

2. T Entrainment Circadian Clock • Mechanism driving daily rhythms of life processes • Entrained by external stimuli: • Light/dark cycles • Temperature cycles Clock Regulated systems

3. Clock Genes • 6-10% of genes rhythmically expressed in Drosophila melanogaster • Clock controlled genes have diverse functions: • metabolic enzymes • oxidative stress • ion channels • detoxification

4. 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

5. Drosophila melanogaster • Common biological model • Has clock mechanism similar to that of humans http://myrmecos.files.wordpress.com/2008/10/drosophila2s.jpg

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

7. 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

8. Disrupting the Clock • It has been shown removing clock genes can disturb activity rhythms Rhythmic Arrhythmic

9. 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

10. Cyc01 Flies Have a Disrupted Clock • Cyc01lack 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

11. Hypothesis • The clock mechanism can shift to increase an organism’s resistance to a recurring chemical insult Phase Shift in Response to a Stimulus

12. 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

13. 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

14. Expose a large group of CSc and cyc01flies 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

15. 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

16. 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

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

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

19. Conclusion • Our results indicate that propoxur cannot shift the clock at the dose and treatment regimen used

20. 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

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