Metals-induced disease pilot projects

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Identifying genes that influence lead-induced OA
Principal Investigator: Joanne M. Jordan, M.D., MPH, Herman & Louise Smith Distinguished Professor of Medicine; Professor, Orthopaedics; Adjunct Professor, Epidemiology; Director, Thurston Arthritis Research Center; Chief, Division of Rheumatology, Allergy, & Immunology.
Award: $25,000

Abstract

Millions of people worldwide, are exposed to lead (Pb) at levels that are detrimental to human health. Such exposure is associated with various inflammatory diseases including osteoarthritis (OA). An understudied mechanism that may contribute to Pb-induced disease is cellular dysregulation associated with epigenetic alterations to DNA. Such epigenetic alterations can influence the regulatory mechanisms controlling gene expression. In this application, the team has come together to test a common hypothesis related to Pb exposure. Specifically, we hypothesize that epigenomic alterations, namely dysregulation of DNA methylation, contribute in a significant way to Pb-induced OA. The research will establish the links between Pb exposure, epigenetic alterations and OA. The proposed research crosses disciplines, is highly synergistic, and brings a novel area of metals research to the UNC-CEHS. The identified epigenetic marks may serve as biomarkers of both Pb exposure and disease. This research is directly in line with the mission of the CEHS.

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Inorganic arsenic-induced DNA methylation and risk of urinary bladder cancer
Principal Investigators: Rebecca Fry, PhD, Assistant Professor, Department of Environmental Sciences and Engineering, and Miroslav Styblo, Associate Professor, Department of Nutrition, Adjunct Associate Professor, Department of Environmental Sciences and Engineering

Abstract

Cancer of the urinary bladder is one of the most common forms of cancer associated with chronic exposures to inorganic arsenic (iAs). A mechanism for this association is not clearly established and has historically been hindered by a limited access to relevant target tissues for study. This project builds off of existing research in Mexico and enables analysis of target tissues (exfoliated urinary bladder cells) collected in an iAs-exposed population. In preliminary data, we have identified 17 tumor suppressors that are hypermethylated in peripheral blood lymphocyte DNA from iAs-exposed individuals who were diagnosed with precancerous skin lesions. Of note, one of these novel sites miR126, has been shown independently to be silenced in bladder tumors. Importantly, these sites, if similarly altered in bladder epithelial cells, could represent a hitherto unknown mechanism by which iAs induces its effects in the bladder. In this application, the team of FIRG researchers come together to test a common hypothesis related to iAs exposure. Specifically, we hypothesize that iAs exposure alters DNA methylation of tumor suppressors in bladder epithelial cells in a manner that is consistent with DNA methylation in bladder cancer. We are uniquely positioned to test this hypothesis in a cohort of iAs-exposed individuals in Mexico. These study subjects have been well-characterized for their iAs exposures, for inter-individual differences in iAs metabolism, and genetic polymorphisms affecting iAs metabolism and toxicity. In addition, bladder epithelial cells are currently being collected from this population for arsenic analysis. The DNA from these cells will be used in our project to identify the sites of DNA methylation across the epigenome that are associated with iAs exposure. DNA from bladder tumor specimens from clinical samples already collected by our collaborators at the UNC Lineberger Cancer Center will be used as a positive control. This interdisciplinary research is likely to provide new insights into the molecular mechanism of bladder carcinogenesis associated with exposure to iAs and to establish early epigenetic markers for risk assessment of this exposure.

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Study to determine the cellular response to DNA damage induced by hexavalent chromium
Principal Investigator: Paul Chastain, Research Associate, Department of Pathology and Laboratory Medicine, School of Medicine.
Award: $25,000

Abstract

Chromium and its compounds have long been used in industry and the by-products of its usage have been proven to pose an environmental health risk. In recent cell survival investigations with DT40 cells knocked out in DNA damage repair as well as cell cycle checkpoint pathways exposed to potassium chromate we have discovered heightened sensitivity to Cr[VI] in cells deficient in the homologous recombination damage repair pathway as well as the cell cycle checkpoint pathway directed by the ATR/ATRIP proteins. These results indicate that both recombination and the ATR/ATRIP pathways are required for cell survival after Cr[VI] exposure. Other preliminary studies we have performed have shown Cr-induced reduction of the rate of replication. In this pilot grant proposal, we seek to determine the mechanism by which the cell controls replication after chromium exposure and the role that homologous recombination may play in that control. Armed with this knowledge, we will be better able to understand how the cell protects itself from chromium-induced DNA damage.

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Mapping methylated DNA sites associated with arsenical-induced skin disease
Principal Investigator: Rebecca Fry, Assistant Professor, Department of Environmental Sciences & Engineering, SPH
Award: $30,000

Abstract

Tens of millions of people worldwide are currently exposed to high levels of arsenic. Consumption of drinking water contaminated with inorganic arsenic (iAs) results in arsenicosis or chronic arsenic poisoning. Advanced stages of arsenicosis are associated with cancer of the skin. While the exact basis for iAs-induced skin disease is not known, a potential mechanism is that iAs causes epigenetic alterations that influence the expression levels of tumor-associated genes. We hypothesize that epigenomic alterations, specifically deregulation of DNA methylation, contribute in a significant way to the skin disease manifestations of arsenicosis. We will test this hypothesis by defining the epigenome-wide sites of DNA methylation that are associated with iAs-induced skin disease, using DNA isolated from residents of arsenicosis-endemic areas of Mexico. We will evaluate associations between DNA methylation, iAs exposure and metabolism, and skin disease. This pilot study will identify differential sites of DNA methylation that are associated with susceptibility to iAs-induced skin disease. This research is directly in line with the mission of the CEHS.

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Oxidative stress in the progression of experimentally induced osteoarthritis
Principal Investigator: Ronald D. Graff, PhD, Assistant Professor of Orthopaedics
Award: $25,000

Abstract

Evidence is accumulating to implicate environmental toxicants and oxidative stress in a complimentary role in osteoarthritis (OA). Epidemiological studies show positive associations between OA severity and blood lead levels as well as selenium insufficiency. The problem with our understanding of the influence of the environment is that the precise link between oxidative stress and OA remains elusive. This compels us to examine the effect of oxidative stress on joint pathology using a murine model of surgically-induced knee instability, to determine if the time-course of disease is altered in animals maintained on a selenium-deficient diet. We will also examine the role of the transcription factor Nrf2, critically important for expression of antioxidant enzymes, by following OA in animals that lack Nrf2, and normal animals fed a potent activator of Nrf2. We hypothesize that oxidative stress will accelerate tissue damage, and conversely that enhancement of antioxidant gene expression will slow progression as observed by MRI and histolopathogy. The natural extension of this pilot data is to explore the mechanisms of heavy metal exposures as oxidant stressors in the progression of OA.

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Metals and Biomarkers of Joint Metabolism in a Community-based Cohort: The Johnston County Osteoarthritis Project
Principal Investigator: Joanne M. Jordan, MD MPH; Associate Professor, Medicine and Orthopaedics, and Adjunct Associate Professor, Epidemiology

Abstract

Accumulating toxicological data suggest that exposure to metals results in skeletal and joint toxicity. Yet, little attention has been directed to metals in relationship to osteoarthritis (OA), the most common cause of arthritis, characterized by profound bone and cartilage disruption. The primary objective is to understand how toxic metals (lead [Pb] and mercury [Hg]) affect OA. This proposal leverages bio-specimens and data from The Johnston County Osteoarthritis Project, a population-based prospective cohort of OA in African-American and White adults, in which associations between Pb, Hg, and selenium (Se) and OA were seen. Using linear regression and analysis of covariance, we will investigate associations between whole blood Pb and toenail Hg and 5 biomarkers of joint tissue metabolism related to OA pathophysiology in 376 men. Together with extant data on 375 women, we will examine factors likely to interfere with mechanisms of metal-associated OA (age, sex, and race) and identify factors (e.g., Se) that may counteract adverse responses to metal exposures. Further, this proposal will provide preliminary data for planned NIH proposals to examine additional metals in OA.

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Effects of Cadmium on DNA Synthesis, Cell Cycle Control and Microsatellite Mutation Rate in Human Cells in Culture
Principal Investigator: Jayne C. Boyer, PhD, assistant professor, Department of Pathology and Laboratory Medicine

Abstract

Cadmium is an environmental human carcinogen that increases oxidative DNA damage and inhibits DNA repair. This study will determine the effects of cadmium on DNA replication and mismatch repair (MMR) in diploid human fibroblasts. DNA fiber immunofluorescence microscopy will be used to determine whether cadmium-induced DNA damage triggers the intra-S checkpoint to inhibit the initiation of new replicons and/or slows the rate of DNA chain elongation in active replicons. Analysis of Chk1 phosphorylation will test whether cadmium inhibits ATR signaling in response to replicative stress. For statistical significance, oxidative DNA damage will be measured and correlated with effects on DNA replication in five different lines of cadmium-treated human fibroblasts. Cadmium has been shown to increase microsatellite mutation rates in yeast and inhibit MMR in human cell extracts. Using a plasmid-based reporter system microsatellite mutation rates will be quantified in five lines of human fibroblasts after chronic exposure to cadmium. These studies will test the hypothesis that cadmium contributes to human carcinogenesis by inhibiting MMR and thereby increasing mutation rates in microsatellites as well as in non-repetitive sequences.

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The role of metabolism in the modulation of toxic and cancer promoting effects of arsenic in human urinary bladder
Principal Investigator: Zuzana Drobná

Abstract

Cancer of the urinary bladder is one of the most common forms of cancer associated with chronic exposure to inorganic arsenic (iAs) from drinking water. In humans the metabolism of iAs yields methylarsenic (MAs) and dimethylarsenic (DMAs) metabolites that contain arsenic in +3 or +5 oxidation state. The key enzyme in this pathway, AsIII-methyltransferase (Cyt19), is expressed in liver, but not in normal human urothelial (UROtsa) cells. Recent studies have shown that methylated trivalent arsenicals, methylarsonous acid (MAsIII) and dimethylarsinous acid (DMAsIII), are more potent than iAs as cytotoxins, genotoxins, and enzyme inhibitors. We have shown that in human bladder cells, MAsIII and DMAsIII are considerably more potent than iAsIII in activating ERK-mediated AP-1 DNA binding, a mechanism that is known to regulate cell proliferation and transcription of genes involved in bladder carcinogenesis (e.g., TGF-a). Notably, preliminary experiments have shown that iAsIII and methylated trivalent arsenicals do induce TGF-a expression by UROtsa cells. Thus the rate of the production and the yields of MAsIII and DMAsIII may be a critical step in cancer promoting effects of iAs. Recombinant wild-type (w/t) and mutated forms of rat and human Cyt19 are now available in our laboratory. The rates and patterns of in vitro methylation reactions catalyzed by these proteins depend strongly on the extent and character of mutation. Using a retroviral vector encoding w/t and mutated forms of Cyt19, we plan to established a genetically modified UROtsa cell lines with modified methylation capacities. We plan to use this cell line to study the role of enzymatic methylation in the modulation of toxic and cancer promoting effects of iAs in human bladder cells.