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Folate deficiency and ionizing radiation cause DNA breaks in primary human lymphocytes: a comparison¹

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, and Children'Js Hospital Oakland Research Institute, Oakland, California, USA

²Correspondence: Children'Js Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA. E-mail: bames{at}chori.org

SPECIFIC AIMS

DNA double-strand breaks (DSB), the most serious DNA lesion caused by ionizing radiation, are also caused by several common vitamin or mineral deficiencies such as for folate, B₆, or B₁₂. In the current study, we sought to compare the effects of ionizing radiation and folate deficiency on primary human T lymphocytes by measuring common end points such as DNA break induction, proliferation, cell cycle arrest, apoptosis, and gene expression using microarrays.

PRINCIPAL FINDINGS

1. Both ionizing radiation at relatively high doses and folate deficiency cause DNA breaks, as measured by the Comet assay
Primary human T lymphocytes were cultured in folate-sufficient conditions and irradiated or cultured in varying concentrations of folate. DNA breaks as measured by the alkaline Comet assay, which measures the sum of single-strand breaks (SSB) and DSB, were increased for radiation doses of 1 Gy and higher, although statistical significance was achieved only at 5 Gy (Fig. 1 A). Similarly, cells cultured in low folate showed a dose-dependent increase in DNA breaks (Fig. 1B ). For the conditions used in this study, physiological concentrations of folate deficiency (12 nM) induced as much DNA damage as did 1 Gy of radiation, a relatively high dose.

View larger version (13K): [in this window] [in a new window]   Figure 1. DNA damage score from the alkaline Comet assay. A) Cells were embedded in agarose, then irradiated. B) Cells were harvested after 8 days of culture in varying folate concentrations. Data are mean ± SE of 3 separate experiments. *P <0.05 compared with control. ***P <0.001 compared with control (A, control=0 Gy; B, 3000 nM folate).

2. Folate deficiency and high doses of radiation, but not low doses, inhibit growth of primary human lymphocytes
We asked whether radiation or folate deficiency affects lymphocyte proliferation. Cells irradiated with 1 Gy had a proliferation rate of 50% of that of unirradiated cells; 5 Gy completely abolished lymphocyte proliferation. The proliferation rate of cells cultured in 12 nM folate was 46% of that of control cells and 35% for cells cultured in 6 nM. Lymphocytes cultured in 0 nM folate did not proliferate.

3. Apoptosis is increased by folate deficiency and high doses of ionizing radiation, but not by low doses
Because the growth curves of lymphocytes were lowered by radiation and folate deficiency, we examined whether there was an increase in apoptosis under each condition. For radiation, a significant increase in apoptosis was observed only at the highest dose (5 Gy), where 16% of the cells were apoptotic vs. 8% for unirradiated cells (Fig. 2 A). Linear regression analysis showed a statistically significant dose-dependent increase in apoptosis in the folate-deficient cells relative to the non-folate-deficient cells (Fig. 2B ). The percentage of apoptotic cells was 7% for control cells, 13% in 12 nM folate, and 31% in 0 nM folate.

View larger version (15K): [in this window] [in a new window]   Figure 2. Induction of apoptosis A) 24 h after irradiation or B) after 8 days of culture in varying folate concentrations. The percentage of apoptotic cells was determined using the annexin-V binding assay and flow cytometry. Data are mean ± SE of 3 separate experiments. *P <0.05 compared with control. ***P <0.001 compared with control (A, control=0 Gy; B, 3000 nM folate).

4. Ionizing radiation and folate deficiency differentially affect the cell cycle of lymphocytes
Cell cycle arrest is an early marker of induction of cell cycle checkpoints, DNA damage, and initiation of repair mechanisms. Unirradiated lymphocytes had 6.0% of their cells in G₂/M, whereas the percentage rose to 15.3% after 1 Gy of radiation and 26.8% after 5 Gy. The G₂/M arrest was still evident 72 h after irradiation (data not shown). In contrast to radiation, folate deficiency caused an arrest in the S phase of the cell cycle in an apparently dose-dependent manner. The percentage of cells in the S phase was 34.5% for control cells, 53.4% for the 12 nM culture, and 69.4% for 0 nM.

5. Ionizing radiation and folate deficiency induce differential gene expression changes as measured by DNA microarrays
Radiation and folate deficiency induce DNA breaks, but by different mechanisms; therefore, we investigated how gene expression profiles were affected. For radiation studies, RNA was extracted 1 h after 1 or 5 Gy of radiation. After irradiation, 24 genes were up-regulated, including DNA repair and stress genes, and 23 genes were down-regulated, mostly cell cycle and mitochondrial genes. For folate deficiency studies, lymphocytes were cultured in 0 nM folate (complete deficiency) or 12 nM folate (physiological deficiency) for 10 days before RNA from the samples was extracted. Most of the 24 genes up-regulated in folate deficiency were DNA repair and mitochondrial genes; half of the 14 down-regulated genes were involved in folate metabolism.

CONCLUSIONS AND SIGNIFICANCE

DNA damage can result from exposure to both external and endogenous sources of stress. Though a comparison of DNA damage and cancer risk from different stresses is difficult when different kinds of DNA damage are produced, a comparison can be made if a common end point is used such as the induction of DSB. Ionizing radiation causes DSB either directly or indirectly by forming clusters of oxidative damage, which can be converted to DNA breaks during repair. Folate deficiency also causes DSB. A low thymidine/uracil ratio caused by folate deficiency results in uracil being incorporated into DNA during synthesis. The repair of two nearby opposing lesions can cause a DSB.

The population generally is exposed only to very low doses of radiation (<0.1 Gy). Because risk estimates for low doses are hard to assess, they are often extrapolated from effects observed at higher doses. The usefulness of this linear model has been debated as it may overestimate cancer risks. Other models have been proposed in which low-dose radiation is harmless below a certain threshold or may even induce a radioadaptive response. On the other hand, deficiencies in vitamins and minerals are common in the population and may contribute to much of preventable cancer. Before the recent folate fortification of grains, the percentage of the U.S. population that had a very low intake of folate was 50% for women and 25% for men. A folate concentration of 5 to 12 nM is in the range found in plasma of individuals having a low consumption of fruits and vegetables. Folate levels of individuals having a better diet are 30 nM.

As radiation and folate deficiency cause DNA breaks in a different fashion, we used the alkaline Comet assay to simultaneously measure single-strand breaks (SSB) and DSB, thereby allowing comparison between the two. DSB are often considered the most dangerous DNA lesion, although SSB can be converted to DSB when lesions (SSB and/or base lesions) are clustered. Lymphocytes irradiated with 5 Gy were virtually all damaged, as were lymphocytes cultured in 0 nM folate. The cells cultured in a physiological level of folate deficiency (12 nM) sustained damage similar to that of lymphocytes irradiated with 1 Gy, a relatively high dose of radiation. Similar results were obtained using other techniques to measure DNA breaks. Microarray analysis of gene expression showed that shortly after lymphocytes were irradiated, many DNA repair and stress genes were up-regulated, including ATM and GADD45, genes involved in the G₂/M arrest after radiation. Consistent with the kinds of DNA damage induced by radiation, many DSB repair and excision repair genes were up-regulated. When lymphocytes were cultured in folate-deficient conditions, a few excision repair genes were up-regulated and many folate metabolism genes were down-regulated.

Our results show that physiological levels of folate deficiency cause more DNA damage than low-dose radiation in primary T lymphocytes. Because our study was made using cultured cells, results cannot be extrapolated directly to estimate cancer risk. Human exposure to chronic low doses of radiation is likely to have a different outcome than acute cell exposure. Chronic and acute low doses of radiation can induce a radioadaptive response, protecting the cells from damage induced by a subsequent irradiation. This adaptive response has not been observed for folate deficiency. Folate deficiency is known to cause DSB, but our microarray data show that DSB recognition and repair genes are not activated. Moreover, folate deficiency has been shown to impair DNA excision repair and mismatch repair. This further suggests that folate deficiency may be more detrimental than low doses of radiation. In addition, dietary folate deficiency is systemic and is likely to affect most dividing cells.

Note that our study did not address the issue of whether low-dose radiation is harmful to human health; rather, our study found that low doses of radiation had no measurable effects in comparison to conditions of folate deficiency for the cells we used. Although further investigation is needed, these findings suggest that exposure to very low doses of radiation from background or occupational exposure may pose a smaller cancer risk than consuming a poor diet. Many vitamins and minerals are important for DNA integrity. More important, our results suggest that research on the biological effects of low-dose radiation in humans should take into account the nutritional status of the subjects, as folate (and other vitamin and mineral) deficiency could confound the effects of low-dose radiation, or could even have a synergistic effect and increase the sensitivity of cells to radiation.

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic diagram. Commonly found levels of folate deficiency cause more cellular damage than exposure to low doses of ionizing radiation. Damage index was calculated from DNA breaks, proliferation, and apoptosis measurements.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0382fje

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This Article Full Text (PDF) All Versions of this Article: 18/1/209 03-0382fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by COURTEMANCHE, C. Articles by AMES, B. N. Search for Related Content PubMed PubMed Citation Articles by COURTEMANCHE, C. Articles by AMES, B. N.