Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop III

Abstract

_____________________________________________________________________

Title: Comparison of DNA Damage Risk from Low-Dose Radiation and Folate
Deficiency.

Authors: Chantal Courtemanche, Arnold C. Huang, Nicole Kerry, Bernice Ng, and Bruce N. Ames.

Institutions: Children’s Hospital Oakland Research Institute, Oakland, California.

Our overall goal is to understand and quantify the real effects of low-dose radiation by measuring direct and specific cellular changes. However, since the background dose of radiation to which most individuals are exposed is well below the levels where significant biological effects, such as mutation or tumor induction, are observed, our novel approach is to compare the consequences of radiation to those of specific nutritional deficiencies. By determining which of these two common stresses at physiologically relevant doses leads to a greater amount of DNA damage, we hope to determine whether low-dose radiation has a significant impact on human health, relative to other better understood risks, such as deficiencies in the vitamins folate (about 10% of U.S. population before recent fortification), B6 (about 10% of U.S. population), and B12 (about 14% of U.S. elderly population), which cause incorporation of uracil in human DNA and consequent double-strand breaks.

Our data thus far strongly suggest that nutritionally relevant levels of folate deficiency are likely to be a greater cellular stress than environmentally relevant levels of radiation. We have established human lymphocytes in cell culture and have either treated them with irradiation or maintained them under folate deficiency conditions. Irradiated cells and folate deficient cells had greatly reduced growth curves above 0.5 Gy and below 24 nM folate, respectively. Cell viability, as measured by trypan blue exclusion, was lowered inversely with increasing amounts of radiation exposure and with increasing folate deficiency in the cell medium. However, apoptosis was only increased when cells were exposed at the highest radiation dose of 5 Gy, but there was an inverse dose response in apoptosis when cells were maintained in varying levels of folate in the medium. We also examined the cell cycle in order to determine in which phase most of the cells were accumulating. At the 1 Gy or higher doses, there was an increase in arrest of the cell cycle in the G2/M phase. Folate deficient cells, on the other hand, showed a different profile. Lymphocytes that were cultured in physiologically relevant levels of folate deficiency for 8 days had a cell cycle arrest in the S phase. Furthermore, this arrest appeared to be dose-dependent on the level of folate deficiency. When 3H-thymidine is added, the radioactivity is rapidly incorporated into the cell, suggesting that DNA repair is active and that uracil misincorporation is the likely cause of the arrest. Additionally, at the low doses of 0.5 Gy or less, we did not detect any increase in DNA double-strand breaks, even after we applied all of our DNA repair enzymes to our assay. We have also done 4 separate experiments to measure gene expression changes using Operon’s Stress/Aging DNA microarray. These studies, in total, suggest that while the cellular responses due to irradiation and folate deficiency are somewhat different, these respective responses occur more readily under moderate folate deficiencies than under even moderately high doses of radiation.

In addition to the basic comparison between radiation effects and folate deficiency effects, we have begun work directed at examining the interactive effects of radiation and folate deficiency. It is likely that these results will be more relevant to the general population because we may be able to identify individuals who are at greater risk to the effects of low-dose radiation or perhaps find ways, such as sufficient nutritional supplementation, to alleviate the effects of radiation. We have established a growth curve for human lymphocytes that have been maintained in folate deficient medium and then irradiated with varying doses of 0,0.25, 0.5, 1, and 5 Gy. The growth rate is lower than the rate for either treatment alone. Cell viability was also slightly lower for folate-deficient cells that were subsequently irradiated than when cells were either folate deficient only or irradiated only. Moreover, we found a higher percentage of cells were apoptotic when they were first folate deficient and then irradiated. For cell cycle analysis, folate-deficient cells displayed a S phase arrest, while folate-deficient and irradiated cells displayed an increase in G2/M phase arrest only at the highest irradiation dose of 5 Gy. Since the combined effect of folate deficiency and irradiation has predominantly the same effect as folate deficiency alone, this suggests that folate deficiency may account for a greater amount of cellular stress than low-dose radiation. We have also nearly completed our initial assessment of gene expression changes using DNA microarrays. These preliminary data seem to indicate that there is an interactive effect between radiation and a nutritional deficiency since we observed effects of radiation on folate deficient cells that are not normally observed for similar radiation doses to normal cells.

For governmental agencies, this research may provide data that is useful for setting public health policies, and for the general public, this research can present radiation risk in the readily understandable terms of equivalent effects from eating few fruits and vegetables and may assist in changing their perception as to what levels of radiation exposure are relevant.