Arnold C. Huang1,2, Chantal Courtemanche1,2, Nicole
Kerry1,2, Susan T. Mashiyama1,2, Michael Fenech3,
and Bruce N. Ames1,2
1 University of California, Berkeley, California; 2
Children's Hospital Oakland Research Institute, Oakland, California; 3
CSIRO Division of Health Sciences and Nutrition, Adelaide, Australia
Various micronutrient deficiencies can mimic ionizing radiation in damaging DNA by causing oxidative lesions and/or single- and double-strand DNA breaks. In particular, deficiencies (< 50% of the RDA) of the vitamins folate (was 10% of the U.S. population before the recent supplementation in flour), B12 (4% of the U.S. population), and B6 (10% of the U.S. population) in humans have been shown to cause massive uracil incorporation into DNA, which leads to single-strand breaks and double-strand breaks when there are nearby opposing lesions. Chromosome breaks (i.e., double-strand breaks) have been demonstrated in vivo in humans from folate deficiency. Because double-strand breaks due to the non-uniform deposition of radiation energy are an especially detrimental type of DNA damage from radiation, we propose to put low-dose radiation and folate deficiency on the same scale so as to compare their relative risk to each other. Our general hypothesis is that DNA damage occurring from low-dose radiation is minute compared to that of these deficiencies. By comparing the relative weights of low levels of radiation and physiologically relevant levels of folate deficiency as contributors to human DNA damage, these studies may help in setting occupational safety limits for radiation and protective levels for folate as well as facilitate the Department of Energy's educational efforts by quantifying radiation risk in readily understandable terms such as equivalent effects of not eating enough fruits and vegetables.
A large portion of our research effort has been to develop and improve a variety of assays for measuring DNA damage with greater sensitivity and confidence. These assays have been used to show that human lymphocytes grown in cell cultures, which have been exposed to either moderately low doses of radiation or folate deficiency, undergo numerous cellular changes and have an increased risk of DNA damage.
For irradiated lymphocytes relative to non-irradiated cells, we have used DNA microarrays to analyze the gene expression profiles and have observed that many genes were either up-regulated or down-regulated with irradiation. We have also measured a cell cycle arrest in the G2/M phase by propidium staining of DNA and flow cytometry after exposure to as little as 0.25 Gy. Furthermore, we found that there is a roughly linear relationship between induced micronuclei formation and low-dose X-irradiation between 0.05 and 0.5 Gy.
For folate deficient lymphocytes, DNA microarray experiments revealed changes in the expression of many genes, including several DNA repair genes. Decreased lymphocyte proliferation caused by varying levels of folate deficiency (3 to 24 nM folate) was associated with an arrest in the S phase of the cell cycle. Additionally, folate deficiency was significantly correlated with an increase in uracil content of DNA, as determined by our improved uracil assay using gas chromatography-mass spectrometry. Moreover, micronucleated cell frequency was found to be significantly correlated with folate deficiency in lymphocytes and with the increased DNA uracil content, suggesting that uracil misincorporation is DNA is the major mechanism by which folate deficiency contributes to increased chromosome damage. These results have not only demonstrated the feasibility of our approach, but also have helped us to optimize experimental conditions for future studies to compare low-dose radiation and folate deficiency.
This work was supported by the U.S. Department of Energy Grant DE-FG03-00ER62943.