ZINC, GENE EXPRESSION AND DNA DAMAGE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0201952
• Grant No.: 2005-35200-15439
• Project No.: ORE00739
• Proposal No.: 2004-01859
• Program Code: 31.0
• Project Start Date: Apr 1, 2005
• Project End Date: Mar 31, 2008
• Grant Year: 2005

Project Director
Ho, E.

Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331

Performing Department
HEALTH AND HUMAN SCIENCES

Non Technical Summary
A large body of evidence suggests that a significant percentage of deaths resulting from cancer in the US could be avoided through greater attention to proper and adequate nutrition. Although many dietary compounds have been suggested to contribute to the prevention of cancer, there is strong evidence to support the fact that zinc, a key constituent or co-factor of over 300 mammalian proteins, may be of particular importance in the defense against several chronic diseases. Remarkably, 10% of the U.S. population consumes less than half the recommended dietary allowance for zinc and are at increased risk for zinc deficiency. Zinc plays an important role in controlling gene expression, antioxidant defense and DNA repair. Dietary deficiencies in zinc can contribute to damage to DNA that increase risk for cancer development. The goal of this proposal is to use both genomic strategies (using microarray technology) and functional assays to determine the consequences of zinc deficiency on gene expression, oxidative stress, DNA damage and DNA repair pathways in human cells. The proposed studies provide a unique opportunity to apply molecular techniques directly to the study of optimal zinc nutrition. Elucidation of the molecular mechanisms by which zinc status affects oxidative stress and DNA integrity will address the importance of adequate zinc in cancer prevention and will lead to a novel approach for identifying putative biomarkers of zinc deficiency.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	5010	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
5010 - Food;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

zinc

oxidative stress

dna repair

human health

gene expression

dna

damage

molecular genetics

transcription

nutrient function

cell physiology

mechanism of action

human physiology

human nutrition

antioxidants

risk

nutrient status

gene function

protein binding

protein dna interactions

signal transduction

metabolic pathways

nutrient disease relations

Goals / Objectives
The longterm objective of these studies is to elucidate the importance of zinc in maintaining DNA integrity and identify zinc-dependent mechanisms that protect host cells from cellular DNA damage in vitro and in vivo. Our central hypothesis is that low zinc status will increase the risk for DNA damage by compromising antioxidant systems, transcription factor binding and DNA repair mechanisms. Thus, zinc deficiency will markedly increase the susceptibility of host cells to DNA damage. The objective of this proposal is to use both genomic strategies and functional assays to determine the consequences of zinc deficiency on gene expression, oxidative stress, DNA damage and DNA repair signal pathways in cells. We will characterize the level of DNA damage and susceptibility to DNA-damaging agents with zinc deficiency and identify key zinc-dependent signal pathways and transcription factors involved in regulation of oxidative stress and DNA damage/repair pathways.

Project Methods
To determine the effect of zinc deficiency on gene expression and DNA damage, we will use human peripheral lymphocytes and prostate epithelial cells as candidate cells sensitive to zinc deficiency. We will quantify oxidative stress, DNA damage and susceptibility to DNA damaging agents (ionizing radiation). To further examine possible mechanisms by which zinc deficiency induces oxidative stress and DNA damage, we propose in this aim to examine the gene expression profiles using microarrays and transcription factor binding arrays in cells. Our central hypothesis is that low zinc status will increase the risk for DNA damage. To determine the effect of zinc deficiency on DNA damage in human cells. Cell culture media will be depleted of zinc, employing a chelation strategy using Chelex-100 (Biorad, Hercules, CA) as described in our previous paper. Cells will be exposed to varying levels of low zinc for a 7-day period and monitored for DNA damage by the comet assay and measurement of oxidative base lesions by HPLC. In conditions of zinc deficiency, we would expect an increased sensitivity to oxidative and DNA damaging stresses. Low dose ionizing radiation also induces DNA damage by oxidative stresses. Thus, we hypothesize that normally non-damaging doses of ionizing radiation will be detrimental to cells with zinc deficiency due a compromised ability to combat free radical production and compromised DNA repair. Cells grown in media with varying levels of zinc will be exposed to 0.1 Gy or sham (0 Gy) of ionizing radiation. 0.1 Gy of ionizing radiation is equivalent to a low dose of radiation, as stated by the Department of Energy and is non-damaging in normal cells. Cell growth and viability, as well as apoptosis and DNA damage will be assessed following treatment of low dose ionizing radiation. We hypothesize that low zinc levels will increase the cells sensitivity to ionizing radiation, and thus induce increased DNA damage. We examine DNA damage, apoptosis/necrosis and protein expression of several DNA repair enzymes in response to low dose ionizing radiation in zinc adequate and zinc deficient cells. To further examine possible mechanisms by which zinc deficiency induces oxidative stress and DNA damage, we propose in this section to examine the gene expression profiles using microarrays The rationale for this approach is it allows us to screen for DNA repair and oxidative stress-related genes affected by zinc deficiency and probe for key molecular targets for future use in vivo. We plan to examine gene expression profiles in zinc adequate vs zinc Deficient (0 nM) cells and zinc Adequate vs mildly zinc deficient (10 or 100 nM) cells. Cells will be harvested following 1, 4, 7 and 14 days in respective zinc media to examine early and late responding genes.

Progress 04/01/05 to 03/31/08

Outputs
OUTPUTS: Zinc is an essential mineral that is a component of multiple enzymes and transcription factors. Studies have shown that zinc status is compromised in cancer patients and zinc deficiency results in increased oxidative stress and DNA damage. We have examined the effect of zinc deficiency on DNA damage, global gene expression and transcription factor binding in normal prostate epithelial cells. Using the Affymetrix HG-U133A GeneChip, differential expression of genes involved in cell cycle, apoptosis, transcription and DNA damage response due to zinc deficiency in the prostate were identified. In particular, p53 was up-regulated with zinc deficiency. P53 is a transcription factor that is commonly mutated in many cancers, and contains zinc in its DNA binding site. Despite increased p53 gene and nuclear protein expression, no significant change in p53 binding activity was observed by Transcription Factor Array and EMSA. Furthermore, no changes in gene expression of the downstream p53 targets, p21, GADD45 and BAX was observed. These data indicate that zinc deficiency may compromise DNA integrity in the prostate by impairing the function of p53, possibly through loss of zinc in the DNA binding region. In vivo, similar classes of genes were differentially expressed. We hypothesized that zinc deficiency in vivo causes DNA damage through increases in oxidative stress and compromised oxidant defense. To test this hypothesis, SD rats were fed an adequate zinc diet (MZA 30ppm), or marginal zinc deficient diet (MZD 6ppm) for 6 weeks. Comet assays revealed a significant increase in DNA damage in peripheral blood cells of MZD rats. Oxidative stress, indicated by alterations in plasma F2-isoprostates, was also increased. However, no changes in plasma total antioxidant capacity, ascorbic acid, alpha-tocopherol, and erythrocyte SOD activity were found with MZD. Changes in DNA damage and oxidative stress parameters were brought back to control levels following zinc repletion, indicating that the damage was reversible. PARTICIPANTS: Emily Ho, PI Michelle Yan, graduate student Yang Song, graduate student This project offered training for both graduate students (Yan and Song). TARGET AUDIENCES: Target audience is the nutrition scientific community and general population at large. Information and knowledge was deseminated at national conference presentations/posters, as well as public lectures to the Oregon State University community.

Impacts
In summary, we have established an effective model for zinc deficiency in cell culture using a chelation strategy to remove zinc from tissue culture media. We have shown that zinc deficiency increases oxidative stress and induces single strand breaks (DNA damage) in several cell types, including human prostate epithelial cells and human lymphocytes. We have also found an increased sensitivity to DNA damaging agents with zinc deficiency. We have also identified that key pathways involved in DNA repair, such as p53 pathways are impaired with zinc deficiency through alterations in DNA binding. Finally we have verified these mechanisms are disrupted also in vivo. Together these data strongly suggest that a lack of zinc in the diet may be highly detrimental to the health of cells. Together, these data strongly suggest that zinc plays an essential role in maintaining DNA integrity, ultimately leading to increased risk for cancer development. Impact: Our data suggest an important function of zinc could be to protect the cell from DNA damage and oxidative stress. Zinc deficiency does provide an environment for increased DNA damage, inability to respond stress and risk for cancer. Over 70% of Americans are not getting the recommended level of zinc. This research addresses the importance of getting adequate zinc from zinc-rich foods to improve health. Thus, this research will aid in improving the health of Oregonians and the U.S. population as a whole.

Publications

• Song, Y. and Ho, E. (2008) Effects of zinc deficiency on DNA damage, oxidative stress and oxidant defense in peripheral blood of rats. FASEB J. 22(4) A697.10.
• Ho, E. and Song Y. (2008) Influence of zinc on oxidative stress, DNA integrity and cancer risk. Cell Biol Toxicol. 24: in press.

Progress 04/01/06 to 04/01/07

Outputs
Zinc is a component of over 300 proteins, including DNA-binding proteins with zinc fingers, Cu/Zn superoxide dismutase and DNA repair proteins such as p53, a zinc protein which is mutated in half of human tumors. It can be hypothesized that insufficient zinc intake can impair antioxidant defenses and compromise DNA repair mechanisms, making the cell highly susceptible to oxidative DNA damage. The prostate contains the highest concentration of soff tissue zinc in the body, however its function in the prostate is largely unknown. Zinc concentrations are also known to decrease with the development of prostate cancer. We hypothesize that an important function of zinc in the prostate is to protect the prostate against oxidative stress and DNA damage leading to cancer. Using of both genomic strategies (using microarray technology), and functional assays, we have found that zinc deficiency increases DNA damage, oxidative stress, expression of DNA repair enzymes but impairs downstream signaling events in a cell culture model. This data demonstrates that zinc deficiency causes oxidative stress and DNA damage, but important downstream signals leading to proper DNA repair are lost without zinc. In vivo, using a rat model for both severe and marginal zinc deficiency, we have also found that zinc deficiency increases DNA damage in peripheral blood cells, and increases several markers of oxidative stress. Consequently, zinc deficiency not only causes oxidative stress and induces DNA damage, but also compromises the cell's ability to repair this damage. Zinc deficiency also significantly increases the sensitivity of cells to DNA damaging agents, such as low dose ionizing radiation. We have also found that the activity of several redox-sensitive transcription factors is comprised with zinc deficiency in the prostate. We have utilized transcription factor arrays to screen for other zinc-dependent transcription factor activity. In these studies, zinc deficiency resulted in decreased transcription factor binding of Nrf2, NFkB, Myc and SP1, all which are involved in modulating stress response.

Impacts
Our data suggest an important function of zinc could be to protect the cell from DNA damage and oxidative stress. Zinc deficiency does provide an environment for increased DNA damage, inability to respond stress and risk for cancer. Over 70% of Americans are not getting the recommended level of zinc. This research addresses the importance of getting adequate zinc from zinc-rich foods to improve health. For example, our data suggest that zinc deficiency does provide an environment for increased DNA damage and prostate cancer risk. Based on these preliminary data zinc may function in the prostate to protect from oxidative stress and aid in DNA repair. This research will aid in improving the health of Oregonians and the U.S. population as a whole.

Publications

• Bruno, RS, Song, Y, Leonard, SW, Taylor, A, Traber, MG and Ho, E. (2006) Role of dietary zinc restriction on antioxidant status and oxidative stress. FASEB J 20(5): A625
• Yan, M, Hardin, K and Ho, E. (2006) The effect of zinc status on DNA damage response in prostate epithelial cells. FASEB J 20 (5): A625.
• Bruno, RS, Song, Y, Leonard, SW, Mustacich, DJ, Taylor, AW, Traber, MG and Ho, E. (2007) Dietary zinc restriction in rats alters antioxidant status and increases plasma F(2) isoprostanes.J Nutr Biochem; [Epub ahead of print, Dec 2006]
• Song Y, Chung C, King JC, Brown, KH, and Ho, E. (2006) Alterations in zinc status affects DNA integrity and transcription factor binding in vivo FASEB J 20 (5): A996

Progress 04/01/05 to 03/31/06

Outputs
We have established an effective model for zinc deficiency in cell culture using a chelation strategy to remove zinc from tissue culture media. We have shown that zinc deficiency increases oxidative stress and induces single strand breaks (DNA damage) in several cell types, including human prostate epithelial cells and human lymphocytes. We have also used microarray technology to screen for global gene expression changes with zinc deficiency. Prostate epithelial cells were grown in zinc deficient media for 7 days compared with zinc adequate media. One striking finding is the marked number of genes related to DNA damage and repair that were down-regulated with zinc deficiency despite that fact there is an increase in single strand breaks. This strongly suggests DNA repair mechanism may be dysfunctional. The gene expression changes are consistent with oxidative stress and a decrease in expression in DNA repair genes. We have also found that the activity of several redox-sensitive transcription factors is comprised with zinc deficiency. We have utilized transcription factor arrays to screen for other zinc-dependent transcription factor activity. In these studies, zinc deficiency resulted in decreased transcription factor binding of Nrf2, NFkB, Myc and SP1, all which are involved in modulating stress response. This suggests that zinc deficiency does provide an environment for increased DNA damage and prostate cancer risk. Based on these preliminary data zinc may function in the prostate to protect from oxidative stress and aid in DNA repair. We have also found an increased sensitivity to DNA damaging agents with zinc deficiency. Very low doses of ionizing radiation were administered to zinc deficient and zinc adequate cells. Cells grown in zinc deficient media are died via necrosis and apoptosis much more readily than zinc adequate cells. Gene expression changes were also very different in response to the ionizing radiation stress. Together these data strongly suggest that a lack of zinc in the diet may be highly detrimental to the health of cells.

Impacts
Our data suggest an important function of zinc could be to protect the cell from DNA damage and oxidative stress. Zinc deficiency does provide an environment for increased DNA damage, inability to respond stress and risk for cancer. Over 70% of Americans are not getting the recommended level of zinc. This research addresses the importance of getting adequate zinc from zinc-rich foods to improve health. Thus, this research will aid in improving the health of Oregonians and the U.S. population as a whole.

Publications

• Yan, M, Hardin, K and Ho, E. (2005) The effects of zinc status on gene expression and apoptosis in normal, hyperplasia and cancerous prostate epithelial cells. American Association for Cancer Research, 46: 1221.