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Genetic analysis of circadian responses to low frequency electromagnetic fields in Drosophila melanogaster.

Fedele G, Edwards MD, Bhutani S, Hares JM, Murbach M, Green EW, Dissel S, Hastings MH, Rosato E, Kyriacou CP - PLoS Genet. (2014)

Bottom Line: Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change.In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect.Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

No MeSH data available.


Related in: MedlinePlus

EMF exposure shortens free-running circadian periods in dim blue light.Mean circadian periods (h) +/− sem are shown for the EMF and sham-exposed groups. Note how periods are considerably longer than 24 h. (A–C) period changes in CS flies under static, 50 and 3 Hz field respectively at 300 µT (C–E) period changes in CS flies under 300, 90 and 1000 µT (1 mT) field respectively at 3 Hz. EMF-exposed flies show significant period shortening. For period and N see Table S1. (post-hoc *p<0.05, **p<0.01, ***p<0.001).
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pgen-1004804-g001: EMF exposure shortens free-running circadian periods in dim blue light.Mean circadian periods (h) +/− sem are shown for the EMF and sham-exposed groups. Note how periods are considerably longer than 24 h. (A–C) period changes in CS flies under static, 50 and 3 Hz field respectively at 300 µT (C–E) period changes in CS flies under 300, 90 and 1000 µT (1 mT) field respectively at 3 Hz. EMF-exposed flies show significant period shortening. For period and N see Table S1. (post-hoc *p<0.05, **p<0.01, ***p<0.001).

Mentions: We primarily used 300 µT for our experiments, as this was the intensity used in Yoshii et al., (2009), but we also studied two additional intensities, 90 µT (closer to the Earth's ambient magnetic field) and 1 mT (1000 µT). The minimum frequency possible in the Schuderer apparatus was initially 3 Hz [31] but we also tested 50 Hz (the common frequency in Europe). A subsequent upgrade of the equipment allowed us to also test a static field. Thus the frequencies we used fell within the range of background frequency called the Schumann Resonance [32]. The experimental design was as follows: two groups of flies of the same genotype were studied for seven days under constant dim blue light (LL, hereafter termed pre-exposure) followed by eight days under the same illumination but exposed either to an EMF (EMF exposure) or a sham EMF (sham exposure). The circadian locomotor period was then calculated separately for the pre-exposure and exposure days for each fly and compared (see Methods section for more details). We examined the EMF responses of flies using a standard field intensity of 300 µT with stationary, 3 Hz or 50 Hz frequencies (Figure 1A–C), or using a standard 3 Hz frequency with field intensities of 90, 300 or 1000 µT (1 mT, Figure 1C–E). Irrespective of frequency or intensity of the field, sham-exposed Canton-S (CS) exhibited a lengthening in period between the initial LL pre-exposure and the sham exposure due to the constitutive activation of CRY [26], whereas the EMF-exposed flies showed a significantly shorter period compared to the corresponding sham-exposed flies and to their own pre-exposure (Figure 1, 2A). A three way ANOVA revealed significant effects for EMF frequency (F(2,294) = 37.28, p∼0), exposure to EMF/sham (F(1,294) = 14.81, p<0.001), and for the two-way interaction between pre-exposure and EMF/sham (F(1,294) = 21.73, p<0.01). Importantly, there was no significant three-way interaction (F(2,294) = 1.01, p = 0.36), revealing that a similar pattern is revealed at all three frequencies at 300 µT (Figure 1A–C). Three way ANOVA also revealed significant effects for intensity (F(2, 272) = 23.59, p<0.001) exposure to EMF/sham (F(1,272) = 16.69, p<0.001) and for the pre-exposure x EMF/sham interaction (F(1, 272) = 19.38, p<0.001). There was no significant 3-way interaction (F(2, 272) = 0.04, p = 0.96) showing that the flies were responding in a similar manner to these exposures at 3 Hz (Figure 1C–E, Table S1).


Genetic analysis of circadian responses to low frequency electromagnetic fields in Drosophila melanogaster.

Fedele G, Edwards MD, Bhutani S, Hares JM, Murbach M, Green EW, Dissel S, Hastings MH, Rosato E, Kyriacou CP - PLoS Genet. (2014)

EMF exposure shortens free-running circadian periods in dim blue light.Mean circadian periods (h) +/− sem are shown for the EMF and sham-exposed groups. Note how periods are considerably longer than 24 h. (A–C) period changes in CS flies under static, 50 and 3 Hz field respectively at 300 µT (C–E) period changes in CS flies under 300, 90 and 1000 µT (1 mT) field respectively at 3 Hz. EMF-exposed flies show significant period shortening. For period and N see Table S1. (post-hoc *p<0.05, **p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256086&req=5

pgen-1004804-g001: EMF exposure shortens free-running circadian periods in dim blue light.Mean circadian periods (h) +/− sem are shown for the EMF and sham-exposed groups. Note how periods are considerably longer than 24 h. (A–C) period changes in CS flies under static, 50 and 3 Hz field respectively at 300 µT (C–E) period changes in CS flies under 300, 90 and 1000 µT (1 mT) field respectively at 3 Hz. EMF-exposed flies show significant period shortening. For period and N see Table S1. (post-hoc *p<0.05, **p<0.01, ***p<0.001).
Mentions: We primarily used 300 µT for our experiments, as this was the intensity used in Yoshii et al., (2009), but we also studied two additional intensities, 90 µT (closer to the Earth's ambient magnetic field) and 1 mT (1000 µT). The minimum frequency possible in the Schuderer apparatus was initially 3 Hz [31] but we also tested 50 Hz (the common frequency in Europe). A subsequent upgrade of the equipment allowed us to also test a static field. Thus the frequencies we used fell within the range of background frequency called the Schumann Resonance [32]. The experimental design was as follows: two groups of flies of the same genotype were studied for seven days under constant dim blue light (LL, hereafter termed pre-exposure) followed by eight days under the same illumination but exposed either to an EMF (EMF exposure) or a sham EMF (sham exposure). The circadian locomotor period was then calculated separately for the pre-exposure and exposure days for each fly and compared (see Methods section for more details). We examined the EMF responses of flies using a standard field intensity of 300 µT with stationary, 3 Hz or 50 Hz frequencies (Figure 1A–C), or using a standard 3 Hz frequency with field intensities of 90, 300 or 1000 µT (1 mT, Figure 1C–E). Irrespective of frequency or intensity of the field, sham-exposed Canton-S (CS) exhibited a lengthening in period between the initial LL pre-exposure and the sham exposure due to the constitutive activation of CRY [26], whereas the EMF-exposed flies showed a significantly shorter period compared to the corresponding sham-exposed flies and to their own pre-exposure (Figure 1, 2A). A three way ANOVA revealed significant effects for EMF frequency (F(2,294) = 37.28, p∼0), exposure to EMF/sham (F(1,294) = 14.81, p<0.001), and for the two-way interaction between pre-exposure and EMF/sham (F(1,294) = 21.73, p<0.01). Importantly, there was no significant three-way interaction (F(2,294) = 1.01, p = 0.36), revealing that a similar pattern is revealed at all three frequencies at 300 µT (Figure 1A–C). Three way ANOVA also revealed significant effects for intensity (F(2, 272) = 23.59, p<0.001) exposure to EMF/sham (F(1,272) = 16.69, p<0.001) and for the pre-exposure x EMF/sham interaction (F(1, 272) = 19.38, p<0.001). There was no significant 3-way interaction (F(2, 272) = 0.04, p = 0.96) showing that the flies were responding in a similar manner to these exposures at 3 Hz (Figure 1C–E, Table S1).

Bottom Line: Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change.In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect.Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

No MeSH data available.


Related in: MedlinePlus