The Wise Laboratory of Environmental and Genetic Toxicology

Chromium Toxicology Studies

Background | Experimental Studies | References | Wise Laboratory Publications | Collaborators | Funding

Background

Hexavalent chromium (Cr(VI)) is known to cause lung cancer in humans. What is unknown is how it causes cancer. Lung cancer exhibits a type of genomic instability called chromosome instability, which is characterized by changes in both chromosome structure and number. We are the first lab to demonstrate that hexavalent chromium causes chromosome instability, DNA double strand breaks and neoplastic transformation in human lung cells (1-8). Now we are investigating how it causes these effects. We hope that by determining how hexavalent chromium causes genomic instability we might identify how to prevent chromium-induced cancers and events that are important to lung cancer progression in general that might lead to new treatment approaches or new ways to avoid lung cancer.

Hexavalent chromium is a major environmental problem (9-16). Estimates now indicate that at least 526 of the hazardous waste sites on the National Priority List contain high levels of hexavalent chromium (4). At one site, soil levels of chromium have reached 19,000 mg/kg, which is extraordinarily high compared to normal levels (range of 0-250 mg/kg) (10, 13 and 16). Atmospheric levels of particulate Cr(VI) compounds can range from 1-100 ng/m3, which can lead to an average lung burden of 2 ug/day (17). Further environmental exposure comes from cigarette smoke; 1 cigarette can produce up to 0.5 ug of Cr (14). Superimposed on the amount of Cr that can come from any one exposure route is the fact that Cr accumulates in the body. Autopsies of Cr-exposed workers have shown that Cr accumulates and persists in the lungs for as long as 20 years after exposure (18). Thus, the general public is exposed to significant levels of Cr.

Experimental Studies

Chromosome Instability Studies

Chromium compounds

We believe that a key event in how hexavalent chromium causes cancer is its ability to cause genomic instability. Below, we present and discuss our evidence that hexavalent chromium induces genomic instability which we see more specifically as chromosome instability with both numerical and structural changes in the chromosomes.

Numerical Chromosome Instability

Our initial work determined that particulate chromate (shown in Figure 1) induced numerical chromosome instability (7). Figure 2A shows a normal metaphase with a normal number of chromosomes, 46. Figure 2B shows a metaphase with particulate chromate-induced chromosome instability (note that there are 92 chromosomes in the picture). Further study shows that there is an increase in both the number of metaphases with too few chromosomes (hypodiploid) and the number of metaphases with twice the number of chromosomes (tetraploid).

Normal metaphase Aneuploid metaphase
Figure 2A. Representative picture of a normal metaphase with 46 chromosomes. Figure 2B. Representative picture of a chromate-induced tetraploid metaphase with 92 chromosomes.

We next began exploring the potential cause of this effect (7). We started by considering the effects on centrosomes, the structures that direct cell division by controlling the mitotic spindle. Centrosome amplification (cells with greater than two centrosomes) is a hallmark of lung cancer and can induce numerical chromosome instability by unequally pulling the chromosomes into the daughter cells. Figure 3A shows a normal cell with the normal number of centrosomes (two). We found that particulate chromate induced centrosome amplification producing cells with four to eighteen centrosomes (Figures 3B-C).

Normal mitotic cell with 2 centrosomes Abnormal mitotic cell with 4 centrosomes Abnormal mitotic cell with 18 centrosomes
Figure 3A. Normal mitotic cell with two centrosomes and bipolar spindles. Figure 3B. Abnormal mitotic cell with four centrosomes and multipolar spindles. Figure 3C. Abnormal mitotic cell with 18 centrosomes and disorganized spindles.

We also considered whether bypass of the spindle assembly checkpoint was playing a role (8). The spindle assembly checkpoint is a set of cellular proteins that operate as a network to regulate and monitor assembly of the mitotic spindle, which allows growing cells to divide into two cells. This checkpoint prevents cell division until all of the chromosomes are attached to the spindles and ready to separate. Bypassing the spindle assembly checkpoint can cause aneuploidy.

We found that particulate chromate does indeed cause spindle assembly checkpoint bypass. Normal cells with a properly functioning checkpoint should stop in metaphase when exposed to demecolchicine. Cells treated with particulate chromate, however, bypass the checkpoint and continue through metaphase into anaphase which is seen as premature anaphase and premature centromere division. Figures 4A and 4B show cells treated with particulate chromate exhibiting premature anaphase (Fig. 4A) or premature centromere division (Fig. 4B).

Premature anaphase Premature centromere division
Figure 4A. Representative picture of premature anaphase with all sister chromatids completely separated from each other (red circle). Figure 4B. Representative picture of premature centromere division with sister chromatids that are completely separated (red circle) and sister chromatids that are still attached (red arrows).

 

Current investigations are aimed at understanding the molecular events that cause centrosome amplification and spindle assembly checkpoint bypass.

Structural Chromosome Instability

The other type of chromosome instability is structural and we are investigating that too (1-5). Changes in chromosome structure can lead to deletions of genes or rearrangements that increase the levels of some proteins and decrease others. We found that particulate chromate can indeed damage chromosome structure causing chromatid lesions (Figure 5A), isochromatid lesions (Figure 5B), dicentric chromosomes (Figure 5C), and centromere spreading (Figure 5D).

Chromatid Lesion Isochromatid Lesion
Figure 5A. Representative picture of a chromatid lesion (circled) where one arm of the chromosome is broken Figure 5B. Representative picture of an isochromatid lesion (circled) where both arms of the chromosome are broken.
Dicentric Centromere Spreading
Figure 5C. Representative picture of a dicentric (circled) where the ends of two chromosomes are joined together giving it two centromeres. Figure 5D. Representative picture of centromere spreading (circled) where the centromeres appear to have holes in them.

Currently, we are determining if particulate chromate can induce a particular type of structural damage called translocations using a technique called spectral karyotyping. This technique uses fluorescent dyes to paint each pair of chromosomes a different color (Figure 6A). Then by examining each chromosome, we can see when two chromosomes exchange pieces called a translocation. In Figure 6B, we show a translocation between chromosomes 2 and 5.

Painted metaphase Spectral karyotype
Figure 6A. Representative example of a painted metaphase Figure 6B. A spectral karyotype of the metaphase in figure 6A. This karyotype shows a translocation between chromosomes 2 and 5.

We have also begun investigating what causes these chromosome aberrations, focusing on DNA double strand breaks. DNA double strand breaks are a particularly dangerous type of DNA damage that may lead to chromosome damage.

We were the first to discover that particulate chromate induced DNA double strand breaks (6). Figures 7 and 8 show two different measures of such damage. Figure 7A shows DNA fragmentation in a neutral gel. The picture resembles a comet with the tail of the comet consisting of DNA fragments caused by double strand breaks. Figure 7B shows control cell that has not been treated, and the picture therefore displays minimal tail and more closely resembles a moon.

Chromate-induced DNA double strand breaks Untreated control
Figure 7A. Chromate-induced DNA double strand breaks in human lung cells. The "comet tail" consists of broken DNA fragments. Figure 7B. An untreated lung cell. Note the figure looks more like a "moon" than a "comet".

When double strand breaks form, gamma-H2A.X (g-H2A.X) rapidly accumulates on them and labels the break for repair. Use of a g-H2A.X-specific fluorescent antibody, will detect this protein at a double strand break site and appear as a glowing green dot. Each dot represents a double strand break in the DNA. Figure 8A shows the formation of these g-H2A.X foci in human lung cells treated with particulate chromate (note the many green dots). The cells in red are the same cells treated with a stain that only recognizes DNA. Figure 8B shows the absence of these dots, as there are no green dots, but the same cell is positive for the presence of DNA (red stain).

H2A.X formation in chromate-treated cells H2A.X foci formation in untreated control
Figure 8A. Chromate induced DNA double strand breaks in a human lung cell. These are 2 pictures of the same cell. In the picture on the left, the green dots indicate sites of broken DNA. In the picture on the right, the red stain shows all DNA in the cell. Figure 8B. Untreated lung cells. These are 2 pictures of the same cell. In the picture on the left, the absence of green dots indicates no broken DNA. In the picture on the right, the red stain shows all DNA in the cell.

In response to particulate Cr(VI)-induced DNA double strand breaks, various proteins are activated in order to repair the damage. For example, after exposure to Cr(VI), ATM and SMC1 are phosphorylated and MRE11 expression is elevated (Figure 9). Mre11 functions as a damage sensor and sends the signal to the ATM kinase which then phosphorylates downstream proteins such as SMC1, to initiate cell cycle arrest allow time for the cell to repair the damage. When we knock down MRE11 expression by siRNA silencing, double strand break repair is reduced. Deficiencies in double strand break repair can lead to chromosome instability and cancer formation. To investigate the relationship between double strand break repair and carcinogenicity, we developed a model of neoplastic transformation based on human lung epithelial cells. Chronic exposure to particulate chromate leads to neoplastic transformation causing cell loss of contact inhibition and anchorage dependent in human lung epithelial cells (Figure 10).

MRE11, phospho-ATM and phospho-SMC1 expression after chromate exposure
Figure 9. Particulate chromate induced increases in protein expression in human lung cells. These pictures are immunoblot images probed with different antibodies.
Chromate-induced focus formation Soft agar colony
Figure 10A. Chronic exposure to particulate chromate leads to neoplastic transformation in human lung epithelial cells. Cells loss contact inhibition forming foci in culture. Figure 10B. Chronic exposure to particulate chromate leads to neoplastic transformation in human lung epithelial cells. Cells became anchorage independent forming colonies on soft agar.

Current efforts are aimed at understanding how particulate chromate-induced DNA strand breaks, both single and double, are repaired and the genetic pathways involved in that repair. We are also trying to determine if double strands breaks are linked to centrosome amplification and SAC bypass.

References

 1. Wise, Sr., J.P., Wise, S.S., and Little, J.E. The Cytotoxicity and Genotoxicity of Particulate and Soluble Hexavalent Chromium in Human Lung Cells. Mutation Research 517:221-229, 2002.

 2. Wise, S.S., Schuler, J.H.C., Katsifis, S.P., and Wise, Sr., J.P. Barium Chromate Is Cytotoxic and Genotoxic to Human Lung Cells. Environmental and Molecular Mutagenesis 42:274-278, 2003.

 3. Wise, S.S., Elmore, L.W., Holt, S.E., Little, J.E., Bryant, B.H., and Wise, Sr., J.P. Telomerase-Mediated Lifespan Extension of Human Bronchial Cells Does Not Affect Hexavalent Chromium-Induced Cytotoxicity or Genotoxicity. Molecular and Cellular Biochemistry 255:103-111, 2004.

 4. Xie, H., Holmes, A.L., Wise, S.S., Gordon, N., and Wise, Sr., J.P. Lead Chromate-Induced Chromosome Damage Requires Extracellular Dissolution to Liberate Chromium Ions But Does Not Require Particle Internalization or Intracellular Dissolution. Chemical Research in Toxicology 17:1362-1367, 2004.

5. Wise, S.S., Holmes, A.L., Ketterer, M.E., Hartsock, W.J., Fomchenko, E., Katsifis, S., Thompson, W.D., and Wise, Sr., J.P. Chromium Is The Proximate Clastogenic Species For Lead Chromate-Induced Clastogenicity In Human Bronchial Cells. Mutation Research 560:79-89, 2004.

 6. Xie, H., Wise, S. S., Holmes, A.L., Xu, B., Wakeman, T., Pelsue, S.C., Singh, N.P., and Wise, Sr., J.P. Carcinogenic Lead Chromate Induces DNA Double-Strand Breaks in Human Lung Cells. Mutation Research 586:160-172, 2005.

7. Holmes, A.L., Wise, S. S., Sandwick, S.J., Lingle, W.L., Negron, V.C., Thompson W.D., and Wise, Sr., J.P. Chronic Exposure to Lead Chromate Causes Centrosome Abnormalities and Aneuploidy in Human Lung Cells. Cancer Research 66(8):4041-4048, 2006.

 8. Wise, S.S., Holmes, A.L., Sandwick, S.J., Thompson, W.D., and Wise, Sr., J.P. Chronic Exposure to Lead Chromate Induces Mitotic Disruption and Chromosome Instability. Chemical Research in Toxicology, In Press.

 9. Agency for Toxic Substances and Disease Research. Top 20 Hazardous Substances: ATSDR/EPA Priority List for 1997. U.S. Department of Health and Human Services Public Health Service/ U.S. Environmental Protection Agency, 1997.

 10. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Chromium, Nickel and Welding, Vol. 49, International Agency for Research on Cancer, Lyons, France, 1990.

 11. National Occupational Exposure Survey, National Institute for Occupational Safety and Health, Cincinnati, Ohio, 1988.

 12. Johnson, B.L. and DeRosa, C.T. The Toxicological Hazard of Superfund Hazardous Waste Sites. Reviews on Environmental Health 12:235-251, 1997.

 13. Burke, T., Fagliano, J., Goldoft, M., Hazen, R.E., Inglewicz, R., and McKee, T. Chromite Ore Processing Residue in Hudson County, New Jersey. Environmental Health Perspectives 92:131-137, 1991.

 14. Smith, C.J., Livingston, S.D., and Doolittle, D.J. An International Literature Survey of "IARC Group I Carcinogens" Reported in Mainstream Cigarette Smoke. Food and Chemical Toxicology 35:1107-1130, 1997.

 15. Damage and Threats caused by Hazardous Materials Sites, EPA/430-9-80/004, U.S. Environmental Protection Agency, Washington DC, 1980.

 16. Falerios, M. Schild, L., Sheehan, P., and Paustenbach, D.J. Airborne Concentrations of Trivalent and Hexavalent Chromium From Contaminated Soils at Unpaved and Partially Paved Commercial/Industrial Sites. Journal of Air and Waste Management Association 42:40-48, 1992.

 17. Singh, J., Pritchard, D.E., Carlisle, D.L., Mclean, J.A., Montaser, A., Orenstein, J.M., and Patierno, S.R. Internalization of Carcinogenic Lead Chromate Particles by Cultured Normal Human Lung Epithelial Cells: Formation of Intracellular Lead-Inclusion Bodies and Induction of Apoptosis. Toxicology and Applied Pharmacology 161:240-248, 1999.

 18. Ishikawa, Y., Nakagawa, K., Satoh, Y., Kitagawa, T., Sugano, H., Hirano, T., and Tsuchiya, E. Characteristics of Chromate Workers' Cancers, Chromium Lung Deposition and Precancerous Bronchial Lesions: An Autopsy Study. British Journal of Cancer 70:160-166, 1994.

    Relevant Wise Laboratory Publications

     1. Wise, S.S., Elmore, L.W., Holt, S.E., Little, J.E., Antonucci, P.G., Bryant, B.H., and Wise, Sr., J.P. Telomerase-Mediated Lifespan Extension of Human Bronchial Cells Does Not Affect Hexavalent Chromium-Induced Cytotoxicity or Genotoxicity. Molecular and Cellular Biochemistry 255:103-111, 2004.

     2. Wise, S.S., Schuler, J.H.C., Katsifis, S.P., and Wise, Sr., J.P. Barium Chromate Is Cytotoxic and Genotoxic to Human Lung Cells. Environmental and Molecular Mutagenesis 42:274-278, 2004.

     3. Wise, S.S., Holmes, A.L., Ketterer, M.E., Hartsock, W.J., Fomchenko, E., Katsifis, S.P., Thompson, W.D., and Wise, Sr., J.P. Chromium Is the Proximate Clastogenic Species for Lead Chromate-Induced Clastogenicity in Human Bronchial Cells. Mutation Research 560:79-89, 2004.

     4. Wise, S.S., Schuler, J.H.C., Holmes, A.L., Katsifis, S.P., Ketterer, M.E., Hartsock, W.J., Zheng T., and Wise, Sr., J.P. A Comparison of Two Carcinogenic Particulate Hexavalent Chromium Compounds: Barium Chromate Is More Genotoxic than Lead Chromate in Human Lung Cells. Environmental and Molecular Mutagenesis 44:156-162, 2004.

     5. Xie, H., Holmes, A.L., Wise, S. S., Gordon, N., and Wise, Sr., J.P. Lead Chromate-Induced Chromosome Damage Requires Extracellular Dissolution to Liberate Chromium Ions but Does Not Require Particle Internalization or Intracellular Dissolution. Chemical Research in Toxicology 17(10):1362-1367, 2004.

     6. Holmes, A.L., Wise, S. S., Xie, H., Gordon, N., Thompson W.D., and Wise, Sr., J.P. Lead Ions Do Not Cause Human Lung Cells to Escape Chromate-Induced Cytotoxicity. Toxicology and Applied Pharmacology 203:167176, 2005.

    7. Xie, H., Wise, S. S., Holmes, A.L., Xu, B., Wakeman, T., Pelsue, S.C., Singh, N.P., and Wise, Sr., J.P. Carcinogenic Lead Chromate Induces DNA Double-Strand Breaks in Human Lung Cells. Mutation Research 586(2):160-172, 2005.

     8. Wise, S.S., Holmes, A.L., Moreland, J.A., Xie, H., Sandwick, S.J., Stackpole, M.M., Fomchenko, E., Teufack, S., May, Jr., A.J., Katsifis, S.P., and Wise, Sr., J.P. Human Lung Cell Growth Is Not Stimulated by Lead Ions after Lead Chromate-Induced Genotoxicity. Molecular and Cellular Biochemistry 279 (1-2):75-84, 2005.

     9. Grlickova-Duzevik E., Wise, S.S., Munroe, R.C., Thompson, W.D., and Wise, J.P., Sr. XRCC1 Protects Against Particulate Chromate-Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells. Toxicological Sciences, 92(1): 96-102, 2006.

     10. Holmes, A.L., Wise, S. S., Sandwick, S.J., Lingle, W.L., Negron, V.C., Thompson W.D., and Wise, Sr., J.P. Chronic Exposure to Lead Chromate Causes Centrosome Abnormalities and Aneuploidy in Human Lung Cells. Cancer Research 66(8):4041-4048, 2006.

     11. Duzevik, E.G., Wise, S.S., Munroe, R.C., Thompson, W.D., and Wise, Sr., J.P. XRCC1 Protects against Particulate Chromate-Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells. Toxicological Sciences 92(2):409-415, 2006.

     12. Wise, S.S., Holmes, A.L., and Wise, Sr., J.P. Particulate and Soluble Hexavalent Chromium Are Cytotoxic and Genotoxic to Human Lung Epithelial Cells. Mutation Research, 610(1-2): 2-7, 2006.

     13. Holmes, A.L., Wise, S. S., Sandwick, S.J., and Wise, Sr., J.P. The Clastogenic Effects of Chronic Exposure to Particulate and Soluble Cr(VI) in Human Lung Cells. Mutation Research, 610(1-2): 8-13, 2006.

    14. Grlickova-Duzevik, E., Wise, S.S., Munroe, R.C., Thompson, W.D., and Wise, J.P., Sr. XRCC1 Protects Cells from Chromate-Induced Chromosome Damage, but Does Not Affect Cytotoxicity. Mutation Research, 610(1-2): 610:31-37, 2006.

     15. Wise, S.S., Holmes, A.L., Xie, H., Thompson, W.D., and Wise, Sr., J.P. Chronic Exposure to Particulate Chromate Induces Spindle Assembly Checkpoint Bypass in Human Lung Cells. Chemical Research in Toxicology, 19(11):1492-1498, 2006.

     16. Savery, L.C., Grlickova-Duzevik, E., Wise, S.S., Thompson, W.D., Hinz, J.M., Thompson, L.H., and Wise, Sr., J.P. Role of the Fancg Gene in Protecting Cells from Particulate Chromate-Induced Chromosome Instability. Mutation Research, 626(1-2): 120-127, 2007.

     17. Camrye, E., Wise, S.S., Milligan, P., Gordon, N., Goodale, B., Stackpole, M., Patzlaff, N., Aboueissa, A., and Wise, Sr., J.P. Ku80 Deficiency Does Not Affect Particulate Chromate-Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells. Toxicological Sciences, 97(2):348-54, 2007.

     18. Stackpole, M.M., Wise, S.S., Duzevik, E.G., Munroe, R.C., Thompson, W.D., Thacker, J., Thompson, L.H., Hinz, J.M., and Wise, Sr., J.P. Homologous Recombination Protects Against Particulate Chromate-Induced Genomic Instability in Chinese Hamster Cells. Mutation Research. 625: 145-154, 2007.

     19. Xie, H., Holmes, A.L., Wise, S.S., Huang, S., Peng, C., and Wise, Sr., J.P. Neoplastic Transformation of Human Bronchial Cells by Lead Chromate Particles. American Journal of Respiratory Cell and Molecular Biology. 37(5): 544-552, 2007.

     20. Xie, H., Wise, S.S., and Wise, Sr., J.P. Deficient Repair of Particulate Chromate-Induced DNA Double Strand Breaks Leads To Neoplastic Transformation. Mutation Research. 649: 230-238, 2008.

     21. Holmes, A.L., Wise, S.S. and Wise, Sr., J.P. Carcinogenicity of hexavalent chromium. Indian Journal of Medical Research, 128: 353-372, 2008.

     22. Wise, S.S., Holmes, A.L. and Wise, Sr., J.P. Hexavalent chromium-induced DNA damage and repair mechanisms. Reviews on Environmental Health, 23(1): 39-57, 2008.

     23. Xie, H., Holmes, A.L., Young, J.L., Qin, Q., Joyce, K, Pelsue, S.C., Peng, C., Wise, S.S., Jeevarajan, A., Wallace, W.T., Hammond, D. and Wise, Sr., J.P. Zinc Chromate Induces Chromosome Instability and DNA Double Strand Breaks in Human Lung Cells. Toxicology and Applied Pharmacology., 234: 293–299, 2009.

     24. Holmes, A.L., Wise, S.S., Pelsue, S.C., Aboueissa, A., Lingle, W., Salisbury, S., Gallaher, J. and Wise, Sr., J.P. Chronic Exposure to Zinc Chromate Induces Centrosome Amplification and Spindle Assembly Checkpoint Bypass in Human Lung Fibroblasts. Chemical Research in Toxicology, 23(2): 386-395.

     25. Wise, S.S., Holmes, A.L., Qin, Q., Xie, H., Katsifis, S., Thompson, W.D. and Wise, Sr., J.P. Comparative Genotoxicity and Cytotoxicity of Four Hexavalent Chromium Compounds in Human Bronchial Cells. Chemical Research in Toxicology, 23(2): 365-372, 2010.

    Collaborators 

    The Wise Laboratory is assisted in this work by an important number of collaborators and cooperators. In particular, the following prominent scientists and their teams provide significant support and input:

    Dr. Lynne Elmore is an Assistant Professor in the Department of Pathology of the School of Medicine , at Virginia Commonwealth University Medical Center. She provides expert advice and guidance on creating telomerase-infected cells.

    Dr. John Hinz is a research scientist at the Lawrence Livermore National Labs. He provides expert advice in DNA repair mechanisms.

    Dr. Shawn Holt is an Assistant Professor at the Medical College of Virginia. He provides expert advice and guidance on creating telomerase-infected cells.

    Dr. John Lechner provides expert advice and guidance on the immortalization and culture of human and marine mammal cells.

    Dr. Antonio Musio is a Researcher at the Institute of Biomedical Technologies, CNR, Segrate Italy. Dr. Musio provides expert advice and guidance on the spindle assembly checkpoint and RNAi, antisense and gene expression.

    Dr. Wilma Lingle is Assistant Professor of Pathology, Mayo Clinic Medical School . She provides expert advice and guidance on amplification of centrosomes.

    Dr. Stephen Pelsue is an Associate Professor of Applied Immunology at USM. He provides expert advice and guidance on flow cytometry. He and Dr. Wise are collaborating on a project considering the effects of arsenic exposure on the immune system.

    Dr. Olaf Stemmann is the leader of the Chromosome Segregation and Mitosis Research Group, in the Department of Molecular Cell Biology, Max-Planck Institute of Biochemistry, Martinsried, Germany. He provides expert advice and guidance on chromosome segregation.

    Dr. Larry Thompson is a research scientist at the Lawrence Livermore National Labs. He provides expert advice in DNA repair mechanisms and cell lines deficient in DNA repair genes.

    Dr. Douglas Thompson is a Professor of Epidemiology and Associate Director of the Maine Center for Toxicology and Environmental Health at the University of Southern Maine. He provides expert advice and guidance on statistical analysis and study design and also assists with the marine mammal studies.

    Dr. Ronald Walter is Professor of Chemistry and Biochemistry, and Director of the Xiphophorous Genetic Stock Center at Texas State University at San Marcos. He and Dr. Wise are collaborating on a project considering the effects of hypoxia and environmental metals contamination on fish.

    Dr. Richard Winn is an Associate Professor at the University of Georgia. He provides expert advice and guidance on the care and use of Medaka.

    Dr. Bo Xu is Principal Investigator and Leader of the Molecular Radiobiology Laboratory, Southern Research Institute, at the University of Alabama at Birmingham. He provides expert advice and guidance on cellular checkpoints and ectopic expression constructs and coimmunoprecipitation assays.

    Funding

    This work is generously supported by grant # ES016893 and # ES10838 from the National Institute of Environmental Health Sciences, NASA grant EP-08-01, the U.S. Environmental Protection Agency under the STAR Graduate Fellowship and by the Maine Center for Toxicology and Environmental Health.