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Abstract

We investigate the biological effects of radiation using adult Drosophila melanogaster as a model organism, focusing on gene expression and lifespan analysis to determine the effect of different radiation doses. Our results support a threshold effect in response to radiation: no effect on lifespan and no permanent effect on gene expression is seen at incident radiation levels below 100 J/kg. We also find that it is more appropriate to compare radiation effects in flies using the absorbed energy rather than incident radiation levels.

Keywords

low dose radiation gene expression Drosophila melanogaster threshold effect

INTRODUCTION

According to the Linear No Threshold hypothesis of radiation damage (LNT) lifespan decrease (or cancer or mutation risk) remains linear as the dose of radiation goes to zero. Data used by the National Academy of Sciences (U.S. National Research Council Committee to Assess Health Risks from Exposure to Low Level of Ionizing Radiation 2006) (Figure 1) are consistent with this hypothesis, but the error bars are so large that this data might just as well support a threshold.

FIGURE 1.

Excess Solid Cancer Risk vs. Radiation Dose, redrawn from original figure published in (U.S. National Research Council Committee to Assess Health Risks from Exposure to Low Level of Ionizing Radiation 2006). Note 1 Sv = 1 Gy.

Although it is reasonable to assume that radiation damage to DNA and other cellular machinery is linear as a function of radiation dosage (as has been used in development of radiation protection standards), actual damage is mitigated by cellular repair mechanisms. These repair mechanisms probably increase initially, then reach a maximum so that, depending on parameters, various non-linear curves are possible. It may even be that there is a protective effect due to increased damage response to small amounts of radiation or other types of biological stress.

The goal of this paper is to investigate the biological effects of radiation using Drosophila melanogaster as a model organism, focusing on gene expression and lifespan analysis to determine the effect of different radiation doses, especially near the threshold. Previous research has been done on the effects of radiation on Drosophila lifespan, including research on flies in different developmental stages (Vaiserman et al. 1999; Vaiserman et al. 2003; Vaiserman et al. 2004a; Vaiserman et al. 2004b; Moskalev et al. 2011; Seong et al. 2011; Koana et al. 2012) and in adults (Gowen and Stadler 1952; Strehler 1962; Sacher 1963; Lamb 1964; Noethel 1965; Baxter and Blair 1967a; b; Lamb and Smith 1969; Giess and Planel 1973; Planel and Giess 1973). Each of these papers supports the idea of a threshold - a model where radiation dose does not affect mortality negatively below a certain dose.

RESULTS

Demography Experiment

A demography experiment was done, as detailed in the methods section. Adult flies were irradiated at age 24–48 hours post-eclosion with incident radiation levels¹ ranging from 0 to 400 J/kg. The results of the demography experiment are shown in Figure 2 and Table 1, as well as supplemental Figure S1. We did not detect any significant difference among the lifespans of flies exposed to radiation less than or equal to 50 J/kg (based on log rank test results, shown in supplemental Tables S2 and S3). However, flies exposed to 100 J/kg or more showed a statistically significant reduction in lifespan compared to controls. Larger incident radiation levels increased the magnitude of the reduction in lifespan. Hence, this result is consistent with a threshold effect on mortality. These results were confirmed by a second demography experiment (shown in supplemental Figure S1 and supplemental Table S1), which we refer to as replicate 2 (to distinguish it from the replicate 1 demographic studies presented in this section).

FIGURE 2.

Survivorship curves for the demography experiment. Incident radiation levels less than or equal to 50 J/kg show no statistically significant differences from control; incident radiation levels greater than or equal to 100 J/kg show a statistically significant difference from control.

FIGURE 3.

Comparison of demography data with Baxter and Blair (1967a) suggests absorbed dose as a more appropriate measure of radiation than incident radiation level. The demography experiment featured in the results section is referred to as replicate 1 and the second experiment is referred to as replicate 2. (A) Comparison of incident radiation level. (B) Comparison of absorbed dose. (C) Data from (B), with log of absorbed dose shown.

FIGURE S1.

Supplemental demography data. (A) Survivorship curves for replicate 2. (B) Mortality rate data for replicate 1. (C) Mortality rate data for replicate 2. Mortality rates were calculated by the R packages Survival (Therneau and Grambsch 2000; Therneau 2012) and epiR (Stevenson et al. 2012). All incident radiation levels less than or equal to 50 J/kg in replicate 2 do not show a statistically significant difference as computed by the log rank test, matching the result from replicate 1.

TABLE 1.

Median and maximum lifespan data.

Incident Radiation Level (J/kg)	Median (Days)	90th Percentile (Days)
Control	48	68
0.1	52	70
1	48	68
10	54	68
50	50	68
100	50	62
200	40	48
400	30	32

TABLE 2.

Number of differentially expressed genes. High counts in 100 and 200 J/kg correspond with lifespan reduction in demography results, low counts in 10 and 50 J/kg correspond with no statistically significant change in demography results.

Incident Radiation Level (J/kg)	Day 2	Day 10	Day 20
0.1	138 Up, 545 Down	0 Up, 2 Down	0 Total
10	1 Up, 5 Down	1 Up, 0 Down	0 Total
50	7 Up, 16 Down	4 Up, 6 Down	0 Total
100	3 Up, 9 Down	6 Up, 1719 Down	5 Up, 1607 Down
200	7 Up, 12 Down	12 Up; 1552 Down	16 Up, 1632 Down

TABLE 3.

Summary of overrepresented KEGG functional pathways. Blue pathways were overrepresented in genes up-regulated from control, red pathways were overrepresented in genes down-regulated from control. The full list of KEGG pathways is available in supplemental Table S4.

Incident Radiation Level (J/kg)	Day 2	Day 10	Day 20
0.1	Amino Acid BiosynthesisCarbon MetabolismCircadian RhythmProtein TurnoverAntioxidant MetabolismDrug MetabolismGlycan Biosynthesis, DegradationLipid MetabolismOxidative PhosphorylationPeroxisomeProtein TurnoverSignaling (Jak-STAT, FoxO)Sugar MetabolismVitamin A, B5, C MetabolismXenobiotics Metabolism

10	Sugar Metabolism	Drug MetabolismSugar MetabolismVitamin A, C MetabolismXenobiotic Metabolism

50	Sugar Metabolism	Drug MetabolismSugar MetabolismVitamin A, C MetabolismXenobiotic MetabolismGlycan degredation Lysosome

100	Sugar Metabolism	Drug MetabolismSugar MetabolismSulfur relay systemVitamin A, B1, C MetabolismXenobiotic Metabolism	Sulfur Relay SystemVitamin B1 Metabolism
	Endocytosis Sugar Metabolism	Amino Acid BiosynthesisCarbon MetabolismCitrate CycleOxidative PhosphorylationPentose Phosphate PathwayProteolysisPurine MetabolismSugar Metabolism	Amino Acid BiosynthesisCarbon MetabolismCitrate CycleOxidative PhosphorylationPentose Phosphate PathwayProteolysisSugar Metabolism

200		Antioxidant MetabolismBase Excision RepairDrug MetabolismSugar MetabolismSulfur Relay SystemVitamin A, B1, C MetabolismXenobiotic Metabolism	Drug MetabolismSugar MetabolismSulfur Relay SystemVitamin A, B1, C Metabolism
	Sugar Metabolism	Amino Acid BiosynthesisCarbon MetabolismCitrate CycleOxidative PhosphorylationPentose Phosphate PathwayProteolysisPurine MetabolismSignaling (Wnt)Sugar Metabolism	Amino Acid BiosyntheisCarbon MetabolismCitrate CycleOxidative PhosphorylationPentose Phosphate PathwayProteolysisPurine MetabolismSugar Metabolism

TABLE S1.

Median and 90^th percentile lifespan results for replicate 2.

Incident Radiation Level (J/kg)	Median (Days)	90th Percentile (Days)
0.1	56	68
1	56	74
10	56	72
100	50	64
200	41	47
400	25	29
600	19	19
800	15	19
1,000	6	6

TABLE S2.

Log rank test results for replicate 1.

J/kg	Control	0.1	1	10	50	100	200	400
Control	−	0.239	0.432	0.103	0.579	0	0	0
0.1		−	0.055	0.803	0.067	0	0	0
1			−	0.015	0.810	0.008	0	0
10				−	0.020	0	0	0
50					−	0.001	0	0
100						−	0	0
200							−	0
400								−

TABLE S3.

Log rank test results for replicate 2.

J/kg	0.1	1	10	100	200	400	600	800	1,000
0.1	−	0.351	0.506	0	0	0	0	0	0
1		−	0.554	0	0	0	0	0	0
10			−	0	0	0	0	0	0
100				−	0	0	0	0	0
200					−	0	0	0	0
400						−	0	0	0
600							−	0	0
800								−	0
1,000									−

TABLE S4.

Overrepresented KEGG pathways.

Incident Radiation Level (J/kg)	Day 2	Day 10	Day 20
0.1	Biosynthesis of amino acidsCarbon metabolismCircadian rhythm - flyMetabolic pathwaysPentose phosphate pathwayPropanoate metabolismRibosomeSpliceosomeValine, leucine and isoleucine degradationAmino sugar and nucleotide sugar metabolismAscorbate and aldarate metabolismDrug metabolism - cytochrome P450Drug metabolism - other enzymesFoxO signaling pathwayFructose and mannose metabolismGalactose metabolismGlutathione metabolismGlycerolipid metabolismJak-STAT signaling pathwayLysosomeMetabolic pathwaysMetabolism of xenobiotics by cytochrome P450N-Glycan biosynthesisOther glycan degradationOxidative phosphorylationPantothenate and CoA biosynthesisPentose and glucuronate interconversionsPeroxisomeProtein exportRetinol metabolismRibosomeStarch and sucrose metabolism
10	Amino sugar and nucleotidesugar metabolism	Ascorbate and aldarate metabolismDrug metabolism - cytochrome P450Drug metabolism - other enzymesMetabolism of xenobiotics by cytochrome P450Pentose and glucuronate interconversionsPorphyrin and chlorophyll metabolismRetinol metabolismStarch and sucrose metabolism
50		Ascorbate and aldarate metabolismDrug metabolism - cytochrome P450Drug metabolism - other enzymesMetabolism of xenobiotics by cytochrome P450Pentose and glucuronate interconversionsPorphyrin and chlorophyll metabolismRetinol metabolismStarch and sucrose metabolism
	Amino sugar and nucleotide sugar metabolism	Lysosome
		Other glycan degradation
100	Starch and sucrose metabolism	Ascorbate and aldarate metabolismDrug metabolism - cytochrome P450Drug metabolism - other enzymesMetabolism of xenobiotics by cytochrome P450Pentose and glucuronate interconversionsPorphyrin and chlorophyll metabolismRetinol metabolismStarch and sucrose metabolismSulfur relay systemThiamine metabolism	Sulfur relay systemThiamine metabolism
	Amino sugar and nucleotide sugar metabolism	Biosynthesis of amino acids	Biosynthesis of amino acids
	Endocytosis	Carbon metabolismCitrate cycle (TCA cycle)Fructose and mannose metabolismGlycolysis / GluconeogenesisOxidative phosphorylationPentose phosphate pathwayPhagosomeProteasomePurine metabolismPyruvate metabolismUbiquitin mediated proteolysis	Carbon metabolismCitrate cycle (TCA cycle)Fructose and mannose metabolismGlycolysis / GluconeogenesisOxidative phosphorylationPentose phosphate pathwayPhagosomeProteasomePurine metabolismPyruvate metabolismUbiquitin mediated proteolysis
200		Ascorbate and aldarate metabolismBase excision repairDrug metabolism - cytochrome P450Drug metabolism - other enzymesGlutathione metabolism	Ascorbate and aldarate metabolismDrug metabolism - other enzymesECM-receptor interactionPentose and glucuronate interconversionsPorphyrin and chlorophyll metabolism
200		Metabolism of xenobiotics by cytochrome P450Pentose and glucuronate interconversionsPorphyrin and chlorophyll metabolismRetinol metabolismStarch and sucrose metabolismSulfur relay systemThiamine metabolism	Retinol metabolism
	Amino sugar and nucleotide sugar metabolism	Biosynthesis of amino acids	Biosynthesis of amino acids
		Carbon metabolismCitrate cycle (TCA cycle)Fructose and mannose metabolismGlycolysis / GluconeogenesisOxidative phosphorylationPentose phosphate pathwayPhagosomeProteasomePurine metabolismPyruvate metabolismUbiquitin mediated proteolysisWnt signaling pathway	Carbon metabolismCitrate cycle (TCA cycle)Fructose and mannose metabolismGlycolysis / GluconeogenesisOxidative phosphorylationPentose phosphate pathwayPhagosomeProteasomePurine metabolismPyruvate metabolismUbiquitin mediated proteolysis

TABLE S5.

Mean lifespan for each radiation dose in replicates 1 and 2.

Incident Radiation Level (J/kg)	Replicate	Mean Lifespan (Days)
Control	1	50.8
0.1	1	51.6
1	1	49.2
10	1	53.2
50	1	50.2
100	1	48.5
200	1	38.9
400	1	28.4
0.1	2	55.3
1	2	54.9
10	2	54.9
100	2	50.1
200	2	38.2
400	2	24.7
600	2	18.4
800	2	15.9
1,000	2	5.4

In demography analysis, it is important to consider the mortality rate (Tatar 2005). To further demonstrate the differences between mortality curves for different dosages, we fit the Gompertz-Makeham model of mortality (Makeham 1860) to our data. The results, shown in supplemental Figure S2 and discussed in Appendix 1, support the existence of a threshold behavior at incident radiation levels less than or equal to 50 J/kg.

FIGURE S2.

Gompertz-Makeham mortality (Makeham 1860) fits. (A) Slopes of Log Mortality Rate Fits. The results are consistent with a threshold at 50 J/kg incident radiation level. (B) Intercepts of Log Mortality Rate Fits. The results generally show an earlier start of death at the highest doses of radiation. The variability is likely caused by the fact that the survival curves for the highest doses of radiation in replicate 2 are not exponential (Gompertz-Makeham model (Makeham 1860)) curves-however, these doses are well past the threshold dose. (C) Logarithm of time-independent parameter L in the Gompertz-Makeham mortality (Makeham 1860) fits. (D) Gompertz-Makeham mortality (Makeham 1860) fits for replicate 1. (E) Gompertz-Makeham mortality (Makeham 1860) fits for replicate 2. The demography experiment featured in the results section is referred to as replicate 1 and the second experiment is referred to as replicate 2.

Gene Expression Results

An RNA-sequencing gene expression experiment was performed as described in the methods section. Differentially expressed genes (genes with a statistically significant change in gene expression from control) were determined for each dose of radiation at each days 2, 10 and 20 following irradiation. Gene expression measurements were made for each dose (including control) at each time point, and every measurement had 3 replicates.

The number of differentially expressed genes compared to control in each combination of radiation dose and time point is shown in Table 2. The names of these genes are given in tabs 1–12 of supplemental dataset S1.

These results are consistent with the following conclusions:

The large number of genes changing at days 10 and 20 after incident radiation levels of 100 and 200 J/kg, paired with the lifespan results, suggest a state where the fly is irreparably damaged by the radiation. 1382 genes are in all four of these conditions, which is an 88 percent overlap.

The relatively small number of changes at incident radiation levels of 10 and 50 J/kg at all time points suggests that any damage caused by radiation has been limited and by day 20 has been repaired.

The results for 0.1 J/kg might be an anomaly. It is also possible that it is consistent with a protective effect, such as the effect reported by Seong et al. (2011).

Only a small number of genes change at day 2 in incident radiation levels larger than 0.1 J/kg. This suggests that the effects seen at later days could be due to chronic effects of oxidative stress, possibly from continuous generation of reactive oxygen species as described in Azzam et al. (2012).

In 100 and 200 J/kg at days 10 and 20, the vast majority of differentially expressed genes are down-regulated from control. This is consistent with damage to DNA, possibly from reactive oxygen species generated from the radiation's radiolysis effect on cellular water or mitochondrial DNA translocating to the nucleus (Azzam et al. 2012).

The physiological functions associated with changes in gene expression can be determined using a database of known functions of genes. The significant genes shown in Table 2 were compared to the functional pathway database of the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto 2000; Kanehisa et al. 2012), and the over-represented pathways were computed. Table 3 summarizes the result, and supplemental Table S4 contains the full lists of overrepresented functional pathways. The physiological functions associated with changes include oxidative phosphorylation (oxphos), metabolism of antioxidants and sugars, repair/protein turnover and signaling. These functions are consistent with a response to oxidative damage; this idea is presented in more detail in the discussion section.

Measuring Radiation Dosage

We compare our data with the data in Table I from Baxter and Blair (1967a), another demography experiment with a wide range of incident radiation levels ranging from control to 164,000 Roentgens (approx. 1,640 J/kg). Baxter and Blair's (1967a) experiments and our experiments were done using different gamma ray sources. Baxter and Blair (1967a) used Cobalt-60, which emits photons of 1.17 and 1.33 MeV. We used Cesium-137, which emits photons of 0.66 MeV.

On the surface, our data and Baxter and Blair's (1967a) lifespan results seem to be very different, as evidenced in Figure 3A. However, these two results can be reconciled if we model radiation damage in terms of absorbed dose, instead of the incident radiation level.

For both data sets, we estimated the absorbed dose for each incident radiation level (calculation in Appendix 1). We also scaled the age of death in each dose by the age of death in control flies, or the lowest incident radiation level available in replicate 2 (0.1 J/kg). Figure 3B shows the data from both of our demography experiments with the data of Baxter and Blair (1967a) with the incident radiation levels converted into absorbed doses. Figure 3C shows the result of Figure 3B with the absorbed dose in log scale. The data look consistent, and the shape of Figure 3C resembles Marion Lamb's log-linear/threshold model of radiation damage (Lamb and Smith 1969).

Comparison of Gene Expression Results with Existing Results

The gene expression data of Seong et al. (2011) probably allows the closest comparison to our data.² Seong et al. (2011) found roughly 13% of the genome to be differentially expressed with low dose radiation, with 39 of the changing genes related to aging. They use male flies, 35 days old, whole body tissue, Oregon-R background, irradiated as eggs with 0.2 Gy of Cs-137 radiation. 35 days versus 3–22 days of age is an important difference in methodology, as is irradiation as an egg versus irradiation as an adult fly. The 1,818 differentially expressed genes in their experiment show a statistically significant overlap of 21 genes between genes up-regulated from control in their experiment and genes up-regulated from control in our data for 0.1 J/kg incident radiation level at day 2. The bioconductor package goseq (Young et al. 2010) found no significantly over-represented KEGG pathways in these overlap genes. The data of Seong et al. (2011) also showed a reverse overlap with several of our data points-many of the same genes are differentially expressed in both sets, but the genes are changed from control in the opposite direction. The genes significantly up-regulated from control in Seong's data (Seong et al. 2011) have statistically significant overlaps with the genes significantly down-regulated in our data at 200 J/kg incident radiation level at days 10 and 20 (157 and 159 genes, respectively) and 100 J/kg at days 10 and 20 (166 and 156 genes, respectively). goseq (Young et al. 2010) found only the KEGG (Kanehisa and Goto 2000; Kanehisa et al. 2012) pathway for glutathione metabolism to be significantly overrepresented among these genes — it was found in all four experimental conditions. Interestingly, glutathione has been shown to reduce the effect of oxygen on biological response to radiation in E. coli (Morse and Dahl 1978). Lists of genes in the overlaps stated above are listed in tabs 13–17 of supplemental dataset S1. No statistically significant overlaps were found between our data and the 39 aging-related genes from Seong et al. (2011)

DISCUSSION

Our experiments indicate a threshold effect in response to radiation for Drosophila lifespan and gene expression. The lifespan results, though suggestive, cannot be directly extended to mammals, since Drosophilae generally do not get cancer³. The results of the gene expression analysis are, perhaps, more pertinent since many of the genes in Drosophila have close homologues in mammals. They suggest that the initial effects of radiation on gene expression (incident radiation levels less than or equal to 50 J/kg) are reversed with time, indicating that most of the damage due to these levels of radiation can be fixed by the cell's internal repair mechanisms. In particular we see that between 2 days, 10 days and 20 days gene expression goes back to control for incident radiation levels less than or equal to 50 J/kg. We further find that radiation damage is better measured by absorbed dose rather than by the incident radiation level.

Other laboratories have claimed a protective effect with low doses of radiation (Strehler 1962; Sacher 1963; Lamb 1964; Noethel 1965; Vaiserman et al. 1999; Vaiserman et al. 2003; Vaiserman et al. 2004a; Vaiserman et al. 2004b; Ogura et al. 2009; Moskalev et al. 2011; Seong et al. 2011). Of the studies in that group that present lifespan results, several of these are not comparable to our results. This is due to the irradiation being done at larval stages (Vaiserman et al. 2004b; Moskalev et al. 2011; Seong et al. 2011) or because the experimental conditions were known to be suboptimal (Strehler 1962; Noethel 1965). The results of Gowen and Stadler (1952) seem to support our results, finding a threshold effect in x-rays at dose 12,000 R (approximately 120 J/kg incident radiation level). Another data set from Baxter and Blair (1967a) also support our findings, with a threshold in Co-60 gamma rays at doses 6,800 and 13,700 R (approximately 68 and 137 J/kg incident radiation level). The one data set with comparable methods and a surprisingly different result is the 1963 data set of Sacher (1963), who finds a lifespan extension at doses of 1,500 to 3,000 R (approximately 15 to 30 J/kg incident radiation level) per day (median lifespan of order 40 days) with a 200 kv x-ray machine. Perhaps the difference is due to Sacher's use of daily irradiations instead of our one time irradiation (Sacher 1963).

In our gene expression results, many of the over-expressed pathways in 0.1 J/kg incident radiation level at day 2 show up in the opposite direction in the higher doses. This would be consistent with functions going from protective to harmful as the radiation dose crosses the threshold. The gene expression results do not seem to suggest a significant difference between control and 10 J/kg. Finally, there is a small but statistically significant overlap between 0.1 J/kg incident radiation level at day 2 and the data of Seong et al. (2011). Our data seem to be consistent with the existence of a protective effect, but are not conclusive. It is tempting to speculate that one may see a protective effect in other measures such as behavior.

The physiological responses inferred from our gene expression study (Table 3) are consistent with the oxidative theory of radiation damage (Bokov et al. 2004; Azzam et al. 2012): radiation damages cells by creating reactive oxygen species and other oxidizing agents that damage cell components, in addition to direct damage from the radiation. Perturbations in oxidative metabolism, including oxidative phosphorylation (oxphos) are a known effect of oxidative damage and are associated with chronic inflammation (Azzam et al. 2012). Metabolism of antioxidants is a direct response to oxidizing agents. Vitamins A, B1 and C have been found to have antioxidant properties (Olson 1996; Padayatty et al. 2003; Depeint et al. 2006). Mitochondria are a major target for reactive oxygen species (Azzam et al. 2012); and B vitamins, including vitamin B5, aid the function of mitochondria (Depeint et al. 2006). Reactive oxygen species are known to destroy sugars (Azzam et al. 2012) and amino acids (Stadtman and Levine 2003), which could affect the metabolism and production of sugars and amino acids as well as glycans, which are a type of sugar. Both the pentose phosphate pathway and carbon metabolism are regulated by the reactive oxygen species sensor ATM (Kruger and Ralser 2011); and purine is a known product of the pentose phosphate pathway. The peroxisome is known to have a role in the metabolism of reactive oxygen species (Bonekamp et al. 2009). Reactive oxygen species have been linked to signaling pathways, including Jak-STAT, Wnt and FoxO signaling (Bartosz 2009). C. Elegans with an insulin signaling mutation are resistant to oxidative stress and show increased expression of drug metabolism enzymes, suggesting the involvement of drug metabolism in oxidative stress resistance (Gems and Partridge 2008). Metabolism of xenobiotics is known to induce drug-metabolizing enzymes (Gems and Partridge 2008). Endocytosis and the sulfur relay system are types of transport, which are affected by oxidative damage to lipid bilayers in the cell membrane (Azzam et al. 2012). Wilking et al. (2012) propose a link between oxidative stress and circadian rhythm - several enzymes and antioxidants in the response to oxidative stress oscillate in a circadian rhythm. Repair and replacement mechanisms are also seen in response to the direct damage, including base excision repair, protein turnover and proteolysis.

These oxidative damage responses are almost all seen at days 10 and 20 after level to 100 and 200 J/kg incident radiation, which correspond to the doses in which lifespan is decreased from control. Much of the response is also seen at day 10 after irradiation with 10 and 50 J/kg incident radiation levels; however, the responses are not seen at day 20, suggesting a recovery from the oxidative damage. This is consistent with the threshold effect in lifespan seen at incident radiation levels less than or equal to 50 J/kg. As discussed in Table 2, very few changes are seen at day 2 in any of these four doses, with most of the changes at day 2 related to sugar metabolism - perhaps the sugar destruction happens quickly and forces the metabolism of sugar to decrease initially from lack of availability. Much of the above radiation responses are seen in the lowest dose (0.1 J/kg incident radiation level) at day 2 after irradiation. However, the effects are seen in genes changing in the opposite direction from the other four doses, except for oxidative phosphorylation, which is in the same direction. This could be consistent with a protective response in the lowest dose at day 2. No effect is seen at days 10 and 20 with 0.1 J/kg incident radiation level.

Many questions remain to be answered. Among these: How does one convert from radiation damage levels in Drosophila to those in mammals? How do multiple doses of radiation compare with a single large dose? Do the gene expression results of our experiments carry over to mammals? For example: How similar are the repair mechanisms in the various species?

In spite of the uncertainties, our results provide a strong indication of a very important effect: they contradict the linear no threshold hypothesis (U.S. National Research Council Committee to Assess Health Risks from Exposure to Low Level of Ionizing Radiation 2006).

MATERIALS AND METHODS

Radiation Doses

Two experiments were done. The experiment featured in the results section is called replicate 1, and the second experiment is called replicate 2.

In replicate 1, a demography experiment was done for incident radiation levels (in J/kg) background; 0.1; 1; 10; 50; 100; 200 and 400. The demography for 1 J/kg was an irregular shape and was ignored in the analysis. The gene expression experiment was done concurrently and included three replicates each of background; 0.1; 10; 50; 100 and 200 J/kg at days 2, 10 and 20 after irradiation.

In replicate 2, a demography experiment was done for incident radiation levels (in J/kg) 0.1; 1; 10; 100; 200; 400; 600; 800 and 1,000. The demography for 200; 400; 600 and 800 J/kg were started one week after the other doses.

In the demography data, day 1 was the collection of flies and day 2 was the day of irradiation.

Flies Used

Flies used were all male flies of the Canton-S genetic background. In replicate 1, approximately 300 flies were used for each radiation dose (10 vials of 30–32 flies each, before escapes). In replicate 2, approximately 90–120 flies were used for each radiation dose (3–4 vials of 30–32 flies each, before escapes). All flies were kept in a humidified (50%), temperature-controlled incubator with 12 hour on/off light cycle at 25 °C in vials containing standard cornmeal medium, as in Bauer and Antosh et al. (2010). Flies were collected under light anesthesia (carbon dioxide) and randomly divided into treatment groups.

Irradiation methods

Flies were irradiated at the Brown University Irradiator, a Cesium-137 gamma ray source⁴. Exposure rate values were taken in Roentgens (see footnote 1) from initial values given by the manufacturer, corrected for decay, and verified biannually with ion chamber measurements.

Exposure rates varied by 1.2 percent for incident radiation levels greater than or equal to 1,000 R (10 J/kg) because of decay of the radiation source between experiments. We used different exposure rates for the 1 and 10 J/kg incident radiation levels (100 R and 1,000 R) because of the lower limit of exposure time accuracy in the irradiator. However, the longest of these irradiations was 2.77 minutes. The exposure rates changed due to varying the distance between the fly vials and the source.

In replicate 1, the exposure rates and times were 0.1 J/kg (10 R): 0.02 min at 590.6 R/min; 1 J/kg (100 R): 0.17 min at 590.6 R/min; 10 J/kg (1,000 R): 0.70 min at 1435.2 R/min; 50 J/kg (5,000 R): 3.48 min at 1435.2 R/min; 100 J/kg (10,000 R): 6.97 min at 1435.2 R/min; 200 J/kg (20,000 R): 13.93 min at 1435.2 R/min; 400 J/kg (40,000 R): 27.87 min at 1435.2 R/min. Background radiation flies were brought without food to the irradiation room while other flies were irradiated. All samples except 400 J/kg were irradiated between approx. 8:30 – 11:15 am; 400 J/kg was irradiated between approx. 1:30 – 4:00pm.

In replicate 2, the exposure rates and times were 0.1 J/kg (10 R): 0.28 min at 36.1 R/min; 1 J/kg (100 R): 2.77 min at 36.1 R/min; 10 J/kg (1,000 R): 0.69 min at 1452.4 R/min; 100 J/kg (10,000 R): 6.89 min at 1452.4 R/min; 200 J/kg (20,000 R): 13.78 min at 1451.6 R/min; 400 J/kg (40,000 R): 27.56 min at 1451.6 R/min; 600 J/kg (60,000 R): 41.53 min at 1451.6 R/min; 800 J/kg (80,000 R): 55.11 min at 1451.6 R/min; 1,000 J/kg (100,000 R): 68.85 min at 1452.4 R/min. All samples were irradiated between 1:00 and 4:00 pm.

Food was not irradiated. The flies were provided with water, which was injected into the vial stopper. Flies were irradiated at age 24–48 hours.

Demography Analysis

The R packages survival (Therneau and Grambsch 2000; Therneau 2012) and epiR (Stevenson et al. 2012) were used to compute the log rank test p values and to produce the mortality rate plots in the supplement. The R package Survomatic (Pletcher et al. 2000; Bokov and Gelfond 2012) was used to fit the Gompertz-Makeham mortality model (Makeham 1860).

RNA Sequencing

Flies were aged 2, 10 and 20 days after irradiation, then collected using light anesthesia and frozen using liquid nitrogen. Flies were stored at −80 degrees C until processed for sequencing. Sequencing library preparation was performed using the Illumina TruSeq protocol. Samples were multiplexed, 6 samples per lane, and run on an Illumina HiSeq 2000.

Gene Expression Analysis

Samples were aligned using Tophat (version 2) (Trapnell et al. 2009). Analysis was performed using the statistical programming language R (R Core Team 2012) and the open source software project Bioconductor (Gentleman et al. 2004). Gene counts were determined using the bioconductor package easyRNASeq (Delhomme et al. 2012), with annotation coming from the bioconductor package biomaRt (Durinck et al. 2005; Durinck et al. 2009). We used the bioconductor package DESeq to normalize between samples and to compute p values and fold changes for differential expression (Anders and Huber 2010). P values were corrected for multiple hypothesis testing error using the bioconductor package qvalue (Storey and Tibshirani 2003; Dabney 2012) to calculate the false discovery rate. In the qvalue calculation, p values were pooled from all comparisons calculated (15 total comparisons based on 5 radiation doses compared to control at 3 time points). A present/absent cutoff was used — a gene was considered absent in a given comparison if its mean expression was in the bottom quartile in both the radiation condition under consideration and the corresponding control. Only genes marked as present were pooled for the false discovery rate calculation. A gene was labeled as differentially expressed if the gene had false discovery rate less than or equal to 5 percent, had fold change greater than or equal to 1.5, and was present. Overrepresentation of the differentially expressed genes in biological pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto 2000; Kanehisa et al. 2012) was determined using the bioconductor package goseq (Young et al. 2010), which corrects for the effect of gene sequence length on the likelihood of differential expression. In the goseq analysis: (1) both gene names and KEGG pathway genes were converted to Entrez gene names; (2) gene sets with no differentially expressed genes were not tested; (3) the goseq code had to be modified very slightly (changing the command “sapply” to “lapply”) because tests of pathway databases with one pathway were resulting in class-casting errors; (4) the input for a list of all genes was an estimate of all genes in Drosophila; (5) the KEGG pathways were taken from the KEGG website in February 2014; (6) the pathway significance cutoff was a p value of 0.05, which corresponds to a qvalue package corrected false discovery rate of about 0.18. Pathway p values were pooled for the qvalue package false discovery rate calculation.

Availability of Gene Expression Data

The gene expression data is submitted to the Gene Expression Omnibus repository, with accession number GSE47999.

Comparison of Demography Data

We compared with the data from Table I in Baxter and Blair (1967a). We only compared with the data points where the flies were irradiated on day 1, and if there were multiple experiments in the Baxter and Blair (1967a) dataset for a given dose we averaged the results (which were different by 0.3 days at the most). Baxter and Blair (1967a) only give average age of death, so we used average age of death in the comparison.

Comparison of Gene Expression Data

Differentially expressed genes from Seong et al. (2011), Moskalev et al. (2011) and Ogura et al. (2009) were translated to flybase ID numbers using flybase (McQuilton et al. 2012). Genes or probes that had 0 or >1 flybase ID references were not used in the comparison. Fisher's Exact Test was used to calculate the statistical significance of overlaps, with an estimate of N=15,000 total genes in Drosophila. The significance cutoff used was 0.05, after a multiple hypothesis testing correction using the package qvalue (Storey and Tibshirani 2003; Dabney 2012). The bioconductor package goseq (Young et al. 2010) was used to determine pathway over-representation in overlaps with Seong's data (Seong et al. 2011), with the necessary input of “all gene names” coming from the list of genes given by the bioconductor package BiomaRt (Durinck et al. 2005; Durinck et al. 2009). All pathways marked as significant in this analysis had a non-FDR corrected p value of less than 0.007.

Unused Gene Expression Samples

The samples for incident radiation level 1 J/kg in the full experiment were never processed because the lifespan curve looked irregular. The samples for incident radiation level 400 J/kg were never processed because the high radiation effect was clear by 200 J/kg. One sample from control at day 20 after irradiation failed quality check and was discarded.

Footnotes

1

For comparison to ( Baxter and Blair 1967a ) data whose incident irradiation values are reported in Roentgen (R),we take these values as the proportional ionization analogue of ICRU recommended air kerma ( ICRU 2011 ) and convert them to J/kg (or Gy) using 1R = 0.01 J/kg (Gy) (e.g.

1 R = 0.00971 J/kg in water).

2

There are also gene expression-related works in irradiated Drosophila by Ogura et al. (2009) and

,but their experimental methods differ greatly from ours.

3

Drosophila are generally post-mitotic,although there is evidence of neuronal plasticity in the first four days of adult life ( Hirsch and Tompkins 1994;Hirsch et al. 1995;

4

Model 68A Mark 1 Irradiator,J. L. Shepherd & Associates 1010 Arroyo Ave.,San Fernando,CA.

ACKNOWLEDGEMENTS

The authors would like to thank Stephen Helfand and his laboratory for technical assistance,including Rachel Whitaker,Chengyi Chang,Suzanne Hosier,Adam Kroll and Jason Wood. The authors would also like to thank Dr. Reudiger R. Meyer for a valuable discussion related to nomenclature and unit formulation in the Appendix. N.N. was supported by a Mentored Quantitative Research Development Award from the NIH/NIA K25 AG028753 and K25 AG028753-03S1.

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Drosophila Melanogaster Show a Threshold Effect in Response to Radiation

Abstract

Keywords

INTRODUCTION

RESULTS

Demography Experiment

Gene Expression Results

Measuring Radiation Dosage

Comparison of Gene Expression Results with Existing Results

DISCUSSION

MATERIALS AND METHODS

Radiation Doses

Flies Used

Irradiation methods

Demography Analysis

RNA Sequencing

Gene Expression Analysis

Availability of Gene Expression Data

Comparison of Demography Data

Comparison of Gene Expression Data

Unused Gene Expression Samples

Footnotes

1

2

3

4

ACKNOWLEDGEMENTS

References