URANIUM TOXICOLOGY STUDIES
DU is becoming a wider international concern as a possible health hazard and carcinogen (1-3). Little is currently known about DU mechanisms of effect, but reported data have shown lung cancer (1-3), embryotoxicity and teratogenicity (4), reproductive and developmental damage (5), genomic instability (6) and single strand DNA damage in vitro (7). Given the widespread use of uranium for military applications and the present world-wide deployment of the United States military, it is imperative that we should be able to better define both the risks of DU exposure and the possible mechanisms of carcinogenicity and genotoxicity.
The lack of scientific data concerning the possible health and cancer risks associated with DU are becoming a major issue worldwide (1-3). Fifty-four of the sites on the EPA National Priority List contain unacceptably high levels of uranium contamination (8). Uranium is ubiquitous: occurring naturally, in water and building materials and as a contaminant in phosphate fertilizers, but its military uses are leading to further questions about additional uranium exposure (9-10). Little is also known about threshold doses for frank renal, reproductive or genetic effects, although the existence of such effects is well-documented in the scientific literature. Thus, in keeping with the increased usage of DU, increased knowledge of its mechanisms of action, and the possible environmental and health risks of its use must be more thoroughly investigated.
DU-induced lung cancers occur in human bronchial cells (11). However, despite these observations, the effects of DU in human bronchial cells are unknown. Only two studies have considered the interaction of DU and human bronchial cells (12-13). One study found that soluble DU induced neoplastic transformation of human bronchial cells (12) and the other reported that DU induced lipid peroxidation and micronuclei formation (13). To fully understand how DU causes cancer, it is essential that we study its effects in human bronchial cells, its target cells.
In fact, there are few other data concerning the genotoxic and carcinogenic effects of DU on human cells. Only one other study has considered human cells and that study reported genomic instability, cytotoxicity and micronuclei formation in human osteosarcoma cells (6). Published data demonstrated that DU exposure in vitro can transform immortalized human osteoblast cells (HOS) to the tumorigenic phenotype (14). Recently, it has been reported that this toxic metal can induce leukemia in mice (15).
The Wise Laboratory is investigating the carcinogenicity and genotoxicity of particulate and soluble DU and develops karyotypic and gene expression fingerprints of DU exposure. Our current knowledge of DU carcinogenicity and genotoxicity is clearly inadequate due to an absence of appropriate models of its target cells and very little data about how particulate DU compounds cause their harmful effects. Our research program is significant because it will address these critical shortcomings. When completed it will provide:
1) a better understanding of how DU damages DNA and causes cancer;
2) essential information to better assess the relative risk of exposure to particulate or soluble DU;
3) fingerprints of exposure to better detect soldiers who may have been exposed to harmful levels; and
4) A model of human bronchial cells for further study of DU, other metals, and lung cancer in general.
We first focused our experimental studies on the cytotoxic and clastogenic effects of particulate and soluble depleted uranium in human bronchial fibroblast cells (WTHBF-6) (1). We used uranium trioxide (UO3) and uranyl acetate (UA) as prototypical particulate and soluble DU salts, respectively. After a 24 h exposure, both UO3 and UA induced concentration-dependent cytotoxicity in WTHBF-6 cells. When treated with chronic exposure of up to 72 h, there was an increased degree of cytotoxicity in both UO3 and UA. We assessed the clastogenicity of these compounds at 24 h and found that at concentrations of 0, 0.5, 1, and 5 µg/cm2 UO3, 5, 6, 10, and 15% of metaphase cells exhibit some form of chromosome damage. UA did not induce chromosome damage above background levels. With chronic exposure of 48 and 72 h there were slight increases in chromosome damage induced with UO3 treatment, but no meaningful increase in chromosome damage was observed with UA.
In addition to bronchial fibroblasts, we have examined the cytotoxic and clastogenic effects of UO3 on human bronchial epithelial (BEP2D) cells. BEP2D cells also demonstrate a concentration-dependent cytotoxicity, but a significant increase in chromosome damage is not seen until a 48 h exposure, suggesting a mechanism of toxicity which requires longer exposures to break DNA.
We used immortalized human bronchial epithelial cell lines (BEP2D) to study the transforming potential of particulate DU. We observed focus formation in cells exposed to UO3 for 24 h. (Figure 1)
Normal BEP2D cells
Specifically, after 24 h UO3 treatment with 0, 0.25, 2.5, and 25 μg/cm2 UO3 we observed focus frequencies of 3, 23, 24, and 21 foci, respectively.
The DU transformants also acquired anchorage-independent growth. Figure 2 shows colonies in soft agar formed by DU-transformed cells.
DU-transformed cells formed colonies in soft agar medium.
After 24 h treatment with concentrations of 0, 0.25, 2.5, and 25 μg/cm2 UO3, the percentage of resultant foci that grew colonies in soft agar was 0, 67, 100, and 75, respectively.
In addition, 53% of DU-transformed cell lines tested showed a hypodiploid phenotype (chromosome number less than 44) with a significant increase in metaphases exhibiting a chromosome number ranging from 7 to 43.
We are now focusing on the roles of DNA damage and repair as key factors in uranium's carcinogenicity. We are currently investigating how depleted uranium (DU) causes DNA strand breaks and chromosome aberrations. Efforts are underway to understand how these types of damaging events are sensed, responded to and repaired, considering the roles of gene expression at the RNA and protein levels, and phenotypic changes. We also seek to understand how uranium induces genetic instability. Efforts are also underway to understand how uranium transforms a normal cell into a tumor cell and the chromosomal and gene expression changes that underlie the transformation we observe.
- Royal Society Working Group on the Health hazards of Depleted Uranium Munitions, Memorandum: The health effects of depleted uranium munitions: a summary, The Royal Society, London England, 2002.
- Bleise, A., Danesi, P.R., and Burkart, W. (2003), Properties, use and health effects of depleted uranium (DU): a general overview. Journal of Environmental Radioactivity, 64:93-112.
- World Health Organization, 54th World Health Assembly (2001) Provisional agenda item 13.10: Health effects of depleted uranium, Report by the Secretariat.
- Bosque, M.A., Domingo, J.L., Llobet, J.M., and Corbella, J., (1992) Embroyotoxicity and Teratogenicity of Uranium in Mice Following Subcutaneous Administration of Uranyl Acetate. Biological Trace Element Research 36:109-118.
- Domingo, J.L. (2001) Reproductive and developmental toxicity of natural and depleted uranium: a review. Reproductive Toxicology 15:603-609.
- Miller, A.C., Brooks, K., Stewart, M., Anderson, B., Lin, S., McClain, D., Page, N. (2003), Genomic instability in human osteoblast cells after exposure to depleted uranium: delayed lethality and micronuclei formation. Journal of Environmental Radioactivity 64:247-259.
- Yazzie, M. Gamble, S.L., Civitello, E.R., and Stearns, D.M. (2003) Uranyl Acetate Causes DNA Single Strand Breaks In Vitro in the Presence of Ascorbate (Vitamin C). Chem. Res. Toxicolo. 16:524-530.
- Agency for Toxic Substances and Disease Research (2003) Toxicological Profile for Uranium., U.S. Department of Health and Human Services Public Health Service/U.S. Environmental Protection Agency.
- McClain, D.E., Benson K.A., Dalton, T.K., Ejnik, J, Emond, C.A., Hodge, S.J., Kalinich, J.F., Landauer, M.A., Miller, A.C., Pellmar, T.C., Steward, M.D., Villa, V., Xu, J. (2001) Biological effects of embedded depleted uranium (DU): summary of the Armed Forces Radiobiology Research Institute research. Science of the Total Environment, 274:115-118.
- Miller, A.C., Fuciarelli, A.F., Jackson, W.E., Ejnik, E.J., Emond, C., Strocko, S., Hogan, J., Pagew, N., and Pellmar, T. (1998) Urinary and serum mutagenicity studies with rats implanted with depleted uranium or tantalum pellets. Mutagenesis 13:643-648.
- Vahakangas, K.H., Smaet, J.M., Metcalf, R.A., Welsh, J.A., Bennett, W.P., Lane, D.P., and Harris, C.C. (1992) Mutations of p53 and ras genes in radon-associated lung cancer from uranium miners. Lancet 339: 576-580.
- Yang, Z.H., Fan, B.X., Lu, Y., Cao, Z.S., Yu, S., Fan F.Y., and Zhu, M.X. (2002) Malignant transformation of human bronchial epithelial cells (BEAS-2B) induced by depleted uranium. AiZheng 21:944-948.
- Ohshima, S., Ying, Xu, and Takahama, M. (1998) Effects of uranium ore dust on cultured human lung cells. Environmental Toxicology and Pharmacology 5:267-271.
- Miller, A.C., Blakely, W.F., Livengood, D., Whittaker, T., Xu, J., Ejnik, J.W., Hamilton, M.M., Parlette, E., John, T.S., Gerstenberg, H.M., and Hsu, H. (1998) Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride. Environ. Health Perspect. 106, 465-471.
- Miller, A.C., Bonait-Pellie, C., Merlot, R.F., Michel, J., Stewart, M., and Lison, P.D. (2005) Leukemic transformation of hematopoetic cells in mice internally exposed to depleted uranium. Mol. Cell Biochem., 279, 97-104.
Relevant Wise Laboratory Publications
LaCerte C., H. Xie, A. Aboueissa, J.P. Wise, Sr. 2010. Particulate Depleted Uranium is Cytotoxic and Clastogenic to Human Lung Epithelial Cells. Mut. Res. 697: 33–37.
Xie, H., C. LaCerte, D. Thompson, and J. P. Wise, Sr. 2010. Depleted Uranium Induces Neoplastic Transformation in Human Lung Epithelial Cells. Chem. Res. Toxicol. 23: 373–378.
Wise, S.S., D. Thompson, A. Aboueissa, M. Mason, and J.P. Wise, Sr. 2007. Particulate Depleted Uranium is Cytotoxic and Clastogenic to Human Lung Cells. Chemical Research in Toxicology. 20: 815-820.
Collaborators and Cooperators
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. AbouEl-Makarim Aboueissa is an Assistant Professor of Mathematics and Statistics at the University of Southern Maine (USM). He provides statistical expertise particularly in the area of handling samples with measurements at or below detection limits.
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. Hongyu Zhao is Ira V. Hiscock Associate Professor of Public Health and Genetics, Yale School of Medicine. He provides expert advice in the design, conduct and analysis in genetic epidemiology and statistical genetics, particularly with respect to microarrays.
This work is generously supported by grant #W911NF-04-1-0240 from the Army Research Office, Department of the Army and by the Maine Center for Toxicology and Environmental Health.