The overall objective of this research project is to understand the mechanism by which chromium(VI) compounds, known human lung carcinogens, affect gene expression. The approaches Dr. Wetterhahn plans to use in attacking this problem are: (1) examine the ability of Cr(VI) to affect gene expression in well-defined cultured human lung cells; (2) determine the ability of Cr(VI) to produce reactive oxygen intermediates (ROIs) and oxidative DNA damage; (3) determine the effects of Cr(VI) on the binding of transcription factors known to be activated by oxidative stress to their DNA recognition sequences; and (4) examine the effects of Cr(VI) on oxidative stress-inducible genes. There is mounting evidence that reactive oxygen species may be involved [in] the """"""""promotion"""""""" phase of carcinogenesis. Intracellular metabolism of Cr(VI) may lead to oxidative stress and this may account for the ability of Cr(VI) to act as a complete carcinogen. Dr. Wetterhahn plans to test the hypothesis that Cr(VI) produces ROIs, and thereby affects signal transduction and expression of oxidative stress genes by altering the interaction of transcription factors with promoter regions of these genes.
The specific aims of the proposed research are: (1) Hypothesis: Cr(VI) produces ROIs and oxidative DNA damage. The ability of Cr(VI) to produce ROIs will be determined in human lung cells. Generation of ROIs by Cr(VI) will be determined using fluorescence spectroscopy to measure the increased cellular oxidation of nonfluorescent 2',7'-di- chlorofluorescin diacetate to the fluorescent 2',7'-dichlorofluorescein. DNA damage in the form of Cr-DNA binding, DNA-protein cross-links, DNA strand breaks and 8-oxo-2'-deoxyguanosine will also be determined. (2) Hypothesis: Cr(VI) will increase levels of nuclear proteins which bind to NF-kB and AP-1 response elements. The effect of Cr(VI) on the interaction of transcription factors known to be induced by oxidative stress, i.e., AP-1 and NF-kB, with their recognition elements will be determined in nuclear extracts from human lung cells treated with Cr(VI). Specific DNA-protein interactions will be determined using gel mobility shift assays. (3) Hypothesis: that concentrations of Cr(VI) which alter activation of transcription factors, will induce expression of oxidative stress responsive genes at the level of transcription. The effects of Cr(VI) on the steady-state mRNA levels and transcription rates of genes known to respond to oxidative stress, i.e., 1) CL100 as an example of a gene that is sensitive to low level oxidant stress, 2) heme oxygenase as an example of a gene that is induced by high level oxidative stress and has AP-1 response elements, 3) urokinase-like plasminogen activator as a gene that is responsive to a variety of changes in signal transduction, has NF-kB response elements and whose changes in steady-state mRNA correlate well with the metastatic potential of several cancers, and 4) the constitutive non-inducible beta-tubulin gene, will be determined following treatment of human lung cells with Cr(VI). The effects of Cr(VI) treatment on the steady-state levels of expression of these genes will be determined by solution hybridization and Northern blot analyses. The effects of Cr(VI) on the rates of transcription of these genes will be determined by nuclear """"""""run-off"""""""" transcription assays. The long-range objective of this research project is to understand the mechanism by which Cr(VI) compounds act as carcinogens. The proposed studies should provide evidence for an oxidative damage pathway for Cr(VI) action on gene expression, provide insight into the mechanism by which Cr(VI) causes cancer, and thus provide fundamental insights into the overall mechanism of induction of the neoplastic process by carcinogens known to induce oxidative stress.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA045735-07A1
Application #
2091987
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1991-09-30
Project End
1998-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
7
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Shumilla, J A; Wetterhahn, K E; Barchowsky, A (1998) Inhibition of NF-kappa B binding to DNA by chromium, cadmium, mercury, zinc, and arsenite in vitro: evidence of a thiol mechanism. Arch Biochem Biophys 349:356-62
Dubrovskaya, V A; Wetterhahn, K E (1998) Effects of Cr(VI) on the expression of the oxidative stress genes in human lung cells. Carcinogenesis 19:1401-7
Martin, B D; Schoenhard, J A; Sugden, K D (1998) Hypervalent chromium mimics reactive oxygen species as measured by the oxidant-sensitive dyes 2',7'-dichlorofluorescin and dihydrorhodamine. Chem Res Toxicol 11:1402-10
Alcedo, J A; Misra, M; Hamilton, J W et al. (1994) The genotoxic carcinogen chromium(VI) alters the metal-inducible expression but not the basal expression of the metallothionein gene in vivo. Carcinogenesis 15:1089-92
Hamilton, J W; Bement, W J; Sinclair, P R et al. (1992) Inhibition of protein synthesis increases the transcription of the phenobarbital-inducible CYP2H1 and CYP2H2 genes in chick embryo hepatocytes. Arch Biochem Biophys 298:96-104
Aiyar, J; De Flora, S; Wetterhahn, K E (1992) Reduction of chromium(VI) to chromium(V) by rat liver cytosolic and microsomal fractions: is DT-diaphorase involved? Carcinogenesis 13:1159-66
Borges, K M; Wetterhahn, K E (1991) Chromium bound to DNA alters cleavage by restriction endonucleases. Chem Res Toxicol 4:638-41
Hamilton, J W; Bement, W J; Sinclair, P R et al. (1991) Heme regulates hepatic 5-aminolevulinate synthase mRNA expression by decreasing mRNA half-life and not by altering its rate of transcription. Arch Biochem Biophys 289:387-92
Borges, K M; Boswell, J S; Liebross, R H et al. (1991) Activation of chromium(VI) by thiols results in chromium(V) formation, chromium binding to DNA and altered DNA conformation. Carcinogenesis 12:551-61
Aiyar, J; Berkovits, H J; Floyd, R A et al. (1991) Reaction of chromium(VI) with glutathione or with hydrogen peroxide: identification of reactive intermediates and their role in chromium(VI)-induced DNA damage. Environ Health Perspect 92:53-62

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