Radiation Dose-Rate: Engelward and Yanch Respond

Melzer raises many interesting points regarding our study of low dose-rate radiation (Olipitz et al. 2012). Responding to his letter gives us the opportunity to clarify the rationale behind some of our approaches and interpretations. 
 
Melzer points out that sample sizes in our study varied from 6 to 60. This is absolutely true because it was necessary to adjust sample sizes according to the end point being analyzed. Larger cohorts are required under conditions where there is higher variance, which is the case for the FYDR (fluorescent yellow direct repeat) mice. Smaller cohorts are sufficient when the variance is lower, such as for micronuclei. 
 
Melzer notes that we used transgenic mice for one end point and normal mice for others. In our study, all of the animals were isogenic (C57Bl6), with the only difference being the insertion of the reporter transgene into the FYDR mice. We have not observed any biological impact of this insertion, and the insertion was made in only one of the two copies of chromosome 1, making it even less likely to affect the biology of the animal. Even if there were an impact, this would not compromise the approach because each end point of the study is appropriately internally controlled. Because each end point was evaluated relative to an isogenic control cohort, the approach did not weaken the ability to detect effects but actually strengthened the method. 
 
In his letter, Melzer correctly points out that data of Tanaka et al. (2009) show a statistically significant increase in chromosome aberrations in cells from mice exposed to 1 mGy/day up to a total of 1,000 mGy. However, after exposure to that same dose-rate for a longer period (up to 8,000 mGy), there was no statistically significant change in the number of chromosome aberrations. Furthermore, Tanaka et al. (2009) stated that 
 
Regression coefficients (b4 and b3) in the equations for Dic by FISH at low dose rates of 20 mGy/day and 1 mGy/day at doses less than 8,000 mGy were not statistically significant. 
 
Tanaka et al. also stated that 
 
It remains to be clarified whether the dose–response relationship for Dic+Rc, UA or Dic by FISH was significantly different for dose-rates of 1 mGy/day and 20 mGy/day or whether the linear dose–response relationship at 1 mGy/day was significantly different from the spontaneous level. 
 
Melzer is correct that we cannot rule out the possibility that genetic changes might have been observed if the exposures had been carried out for a longer period. However, because cells have the capacity to repair radiation-induced DNA damage, it is possible that DNA damage would not accumulate with time. 
 
Melzer is correct that a recent report suggested that exposure to radiation from CT (computed tomography) scans affects the risk of cancer in exposed children. An important difference is that CT scans are an acute exposure at a high dose-rate, which is very different from the low dose-rate conditions in our study. Nevertheless, we agree that it is very important to consider the fact that children have increased sensitivity to radiation damage. 
 
One of Melzer’s final points is that inflammation might affect radiation sensitivity. Although we did not test the impact of inflammation in our study, it is important to note that inflammation is a highly genotoxic process itself, leading to levels of DNA damage orders of magnitude higher than levels we calculated in response to the low dose-rate we used. Finally, Melzer raises the issue of internal exposure by ingestion versus external sources. The body handles ingested radionuclides according to the chemical behavior of the element. Although we have examined the effect of radiation dose as delivered by internal or external photon- or beta-emitters, we did not consider the internal pattern of radionuclide uptake in our study.

Radiation Dose-Rate and DNA Damage http: //dx.doi.org/10.1289/ehp.1205595 In their article, Olipitz et al. (2012) examined signs of DNA damage after chronic exposure of C57Bl6 mice to lowlevel ionizing radiation (3 mGy/day). For 5 weeks, mice were irradiated continuously with 35.5 keV X-rays produced by the decay of iodine-125, yielding an accumulated dose of 105 mGy. The observed effects were compared with those from acute irradiation by X-ray machine at 1,700 mGy/day up to the same accumulated dose. Olipitz et al. (2012) investigated signs of DNA damage using four histological methods. Most prominent of the methods was the expression of functional fluorescent protein as a result of recombination by homology-directed repair in pancreatic cells of transgenic FYDR (fluorescent yellow direct repeat) mice derived from the C57Bl6 strain. The other three methods were carried out using genetically unaltered C57Bl6 mice. The authors investigated DNA base damage in spleno cytes; DNA double strand breaks in bone marrow erythro cytes; and the expression of select genes implicated in cell cycle arrest, tumor suppression, and apoptosis in white blood cells from blood samples. Olipitz et al. (2012) used equal numbers of unirradiated mice as controls. However, sample sizes across the study ranged from 6 to 60 animals. Because of the wide range in animal numbers, non parametric methods should have been used in statistical analyses. A multi variate analysis of variance comprising all observations in the study should have preceded any pairwise comparisons to allow the authors to evaluate the variability of observations within samples compared with the variability among samples (Mickey and Dunn 2009). Furthermore, the use of trans genic mice with one method and unaltered mice with the other three might have increased the variability in observation, reducing the chance of detecting statistically significant differences. The above weaknesses in experi mental design and statistical analysis may have profoundly compromised the authors' ability to discover statistically signifi cant effects of chronic exposure to low-level ionizing radiation.
In the "Discussion" of their paper, Olipitz et al. (2012) stated that Chromosome aberrations offer an alternative approach for detecting chromosome breaks, and using this approach, others have shown that low dose-rate radiation indeed induces aberrations in vitro (although the dose-rate was approximately 10-fold higher than that used in the present study) (Tanaka et al. 2009). However, in the paper cited by Olipitz et al., Tanaka et al. (2009) noted that Significant changes in regression coefficients (b3, b4 and b6) obtained by multiple linear regression analysis, which are the same as a coefficients in the linear regression lines, revealed that doserate effects on the incidence of unstable-type aberra tions were found at dose rates of 1, 20 and 400 mGy/day. One milligray per day equals about onethird of the dose-rate Olipitz et al. (2012) used for chronic exposure. Tanaka et al. (2009) irradiated mice with γ-radiation at 1 mGy/day for more than a year to establish a statistically signifi cant dependence of splenocytic chromosomal aberrations on exposure dose. In mice at 438 days of irradiation (494 days of age), Tanaka et al. observed a mean frequency of dicentric chromosomes more than twice as high (0.38 ± 0.15 per 100 cells) as the spontaneous frequency determined in unirradiated mice of similar age (556 days; 0.17 ± 0.14 per 100 cells). Thus, the findings of Tanaka et al. (2009) suggest that Olipitz et al. (2012) might have detected DNA damage if they had exposed the mice to low-level radiation for a longer time.
In addition, roughly half the accumulated dose Olipitz et al. (2012) used may be effective in children. Results of a recent study suggest that patients subjected to CT (computed tomography) scans as children incur a 3-fold greater risk for developing leukemia and brain cancer at accumulated doses of 50 mGy and 60 mGy, respectively (Pearce et al. 2012).
Finally, in an actual radiological emergency, multiple environ mental factors may inter act synergistically to effect DNA damage. For example, inflammatory responses may stimulate cell division, increasing the likelihood for ionizing radiation to cause DNA strand breaks. Although Olipitz et al. (2012) investigated only the effects of external exposure to ionizing radiation, internal exposure may pose a greater risk to public health in the 50-mile ingestion zone anticipated in U.S. emergency action plans.
The author is a consultant on neural plasticity in regard to central nervous system injuries, mental disorders, and mental disabilities. Melzer raises many interesting points regarding our study of low dose-rate radiation (Olipitz et al. 2012). Responding to his letter gives us the opportunity to clarify the rationale behind some of our approaches and interpretations.

Peter
Melzer points out that sample sizes in our study varied from 6 to 60. This is absolutely true because it was necessary to adjust sample sizes according to the end point being analyzed. Larger cohorts are required under conditions where there is higher variance, which is the case for the FYDR (fluorescent yellow direct repeat) mice. Smaller cohorts are sufficient when the variance is lower, such as for micronuclei.
Melzer notes that we used transgenic mice for one end point and normal mice for others. In our study, all of the animals were isogenic (C57Bl6), with the only difference being the insertion of the reporter transgene into the FYDR mice. We have not observed any biological impact of this insertion, and the insertion was made in only one of the two copies of chromosome 1, making it even less likely to affect the biology of the animal. Even if there were an impact, this would not compromise the approach because each end point of the study is appropriately internally controlled. Because each end point was evaluated relative to an isogenic control cohort, the approach did not weaken the ability to detect effects but actually strengthened the method.
In his letter, Melzer correctly points out that data of Tanaka et al. (2009) show a statistically significant increase in chromosome aberrations in cells from mice exposed to 1 mGy/day up to a total of 1,000 mGy. However, after exposure to that same doserate for a longer period (up to 8,000 mGy), there was no statistically significant change in the number of chromosome aberrations. Furthermore, Tanaka et al. (2009)

stated that
Regression coefficients (b4 and b3) in the equations for Dic by FISH at low dose rates of 20 mGy/day and 1 mGy/day at doses less than 8,000 mGy were not statistically significant. Tanaka et al. also  Melzer is correct that we cannot rule out the possibility that genetic changes might have been observed if the exposures had been carried out for a longer period. However, because cells have the capacity to repair radiation-induced DNA damage, it is possible that DNA damage would not accumulate with time.
Melzer is correct that a recent report suggested that exposure to radiation from CT (computed tomography) scans affects the risk of cancer in exposed children. An important difference is that CT scans are an acute exposure at a high dose-rate, which is very different from the low dose-rate conditions in our study. Nevertheless, we agree that it is very important to consider the fact that children have increased sensitivity to radiation damage.
One of Melzer's final points is that inflammation might affect radiation sensitivity. Although we did not test the impact of inflammation in our study, it is important to note that inflammation is a highly geno toxic process itself, leading to levels of DNA damage orders of magnitude higher than levels we calculated in response to the low dose-rate we used. Finally, Melzer raises the issue of internal exposure by ingestion versus external sources. The body handles ingested radionuclides according to the chemical behavior of the element. Although we have examined the effect of radiation dose as delivered by internal or external photon-or beta-emitters, we did not consider the internal pattern of radio nuclide uptake in our study.