Radiation Dose-Rate and DNA Damage

In their article, Olipitz et al. (2012) examined signs of DNA damage after chronic exposure of C57Bl6 mice to low-level 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.

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 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. 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
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 stated that
It remains to be clarified whether the doseresponse 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 Environmental Health Perspectives • volume 120 | number 11 | November 2012 A 417