NIEHS Superfund

 

Superfund Research Program

Carcinogenic Metals And Their Interactions With Other Toxicants

Program Investigator Max Costa, Ph.D.

Program Overview

Most Superfund sites are contaminated with mixtures of metals and organic chemicals and a major concern for human exposure to Superfund sites is the possibility of disease, and in particular, cancer induction. Our Superfund Research Program has two major themes: 1) Carcinogenic metals have unique as well as multiple mechanisms in causing cancers and can synergize with other environmental carcinogens (i.e. PAH, PCBs and UV) to induce cancers and developmental toxicities; and 2) Environmental mixtures also affect there actions of metals in microorganisms and sediments. Thus, we are investigating the molecular mechanisms of Ni(II), chromate and arsenite carcinogenesis in detail to add more depth to our knowledge of this important research. In particular, we are investigating how these metals interact with other environmental agents such as UV, PAHs and PCBs and how these agents interact mechanistically with metals to cause cancer. In addition, we are investigating the unique molecular mechanisms by which metals cause cancer. Many of these mechanisms are innovative and differ from the etiological mechanisms of organic carcinogens. These biomedical studies will be complemented by non-biomedical research in bacteria, sediments, and fishes examining the same metals. These studies will help us understand what factors influence the release of toxic metals and their uptake by humans and wildlife. All the major pathways that lead to cancer, including genetic damage, epigenetic modifications, and altered signaling pathways are being studied by the biomedical components. Collaboration among biomedical components is catalyzed by the research support core. There is also extensive collaboration between the ecology and engineering components and between the biomedical and non-biomedical

Our Program includes 2 biomedical and 2 non-biomedical research cores, 1 Research Support Core,
as well as an Administrative Core, and Research Translation Core.

The biomedical components are:
Research Project 1 -
Epigenetic Mechanisms of Ni, As, Chromate and BaP Carcinogenesis

Max Costa, Ph.D. – Project Leader

The environment is known to have a significant impact upon the epigenetic program of gene expression. Our exposure to various toxic and carcinogenic chemicals,derived from Superfund sites, is likely to alter our epigenetic program. The modifications of histone tails, including histone acetylation, methylation, phosphorylation and ubiquitination, are critical components of epigenetically regulated gene expression. Among these modifications, methylation of histone H3 lysine 9 (H3K9) is particularly interesting due to the well-known connection between its occupancy in the promoter region and epigenetic gene silencing. The persistent methylation of H3K9 leads to cytosine methylation in the same nucleosome and loss of gene expression when that nucleosome is in the promoter region of an epigenetically regulated gene. Recent findings have identified a new class of enzymes that specifically demethylate H3K9 when it becomes mono-,di-, or trimethylated. These enzymes belong to the 2-oxoglutarate (2-OG)-Fe(II)-dependent dioxygenase family, which use iron [Fe(II)], oxoglutarate (2-OG), ascorbic acid and oxygen to oxidatively demethylate the N-methyl bond on H3K9 yielding a demethylated product, formaldehyde and CO₂ released from the decarboxylation of oxoglutarate.  Environmental agents that target Fe(II), ascorbic acid, andoxygen are able to affect the activity of these demethylases. For example, Fe(II) is not tightly bound to a two histidine carboxylic acid facial triad inthe Jumonji C (JmjC) domain of these demethylases. Nickel (Ni) or cobalt (Co)ions as well as other metal ions can readily bind in place of iron and inactivate these enzymes. Additionally, other toxic metals such as chromate are known to react avidly with ascorbic acid, leading to depletion of reduced ascorbate which is required for the oxidative demethylation. Arsenic (As) and benzo[a]pyrene (B[a]P) may also affect H3K9 methylation by inducing oxidative stress, which may indirectly deplete reduced ascorbate. Our goals in this grantare to investigate the individual and combined effects of toxic metals and compounds on H3K9 methylation and study the role of these effects in the changes of gene expression pattern and the role of these alterations of H3K9 in cell transformation induced by the metals and compounds. We are studying the global changes of H3K9 methylation by single or combined toxic chemicals and the gene-specific promoter H3K9 methylation changes induced by nickel and other toxic chemicals, using ChIP-on-chip, followed by analysis of gene expression alterations and as a consequence of these acquired epigenetic marks. Finally we will determine the importance of H3K9 promoter methylation inchemically-induced cell transformation.

Research Project 2 -
Molecular Mechanisms of Co-carcinogenic effect of arsenite with UV Radiation

Chuanshu Huang, Ph.D. – Project Leader

Although arsenic is acknowledged to have a co-carcinogenic effect with solar ultraviolet (UV) radiation for skin tumor induction in mice, its action at the cellular and molecular level is not well understood. Our data shows that arsenite inhibits UV-induced cellapoptosis in mouse epidermal Cl41 cells. Apoptosis plays an essential role as aprotective mechanism against neoplastic development in the organism by eliminating genetically damaged cells. Thus, suppression of apoptosis is thought to contribute to tumorigenesis. Our data also demonstrate that arsenite induces activation of phosphatidylinositol 3-kinase (PI-3K) and nuclear factor-kB (NFkB), and has a synergistic effect with UVB on NFkB activation in mouse epidermal Cl41 cells. Considering the important role of PI-3K/Akt/p70S6kand NFkB in the protection of cells from apoptosis, the main hypothesis of this proposal is that activation of PI-3K/Akt/p70S6k and/or NFkB plays an essential role in the arsenite protection of mouse skin epidermal cells from UV-inducedapoptosis. We are investigating this issue in accordance with the following specific aims: 1) To identify the signaling pathway and its downstream gene implicated in arsenite protection of mouse skin cells from UV-induced cell apoptosis in vitro. 2) To determine the effect of arsenite on UV-induced activation of Akt, NFkB, and COX-2 expression in vivo. 3) To test whether arsenite is able to inhibit UV-induced apoptosis and whether NFkB activation is required for this inhibition in vivo. The significance of this research project is that there sults derived from these proposed studies will greatly facilitate the understanding of the molecular mechanisms of co-carcinogenesis of arsenite with UV radiation in mouse skin, which will help to understand cancer development caused by arsenite in human skin. A better understanding of molecular mechanisms of cancer development caused by arsenite in vitro and in vivo will provide valuable information to help design more effective agents for prevention and therapy of cancers caused by arsenite. Such agents may interfere with signaling pathways leading to inhibition of cell apoptosis. We believe that the proposed contribution of the anti-apoptotic effect of arsenite responsible for arsenite skin co-carcinogenic effects with UV radiation is novel and the integrated mechanistic studies in transgenic animal models and cell culture systems are strengths.

Research Project 3 -
Genetic and Epigenetic Mechanisms of Toxicities of Mixtures of Aromatic Hydrocarbon and Metal Contaminants to Aquatic Populations

Isaac Wirgin, Ph.D. – Project Leader

Urban and industrial Superfund sites usually contain complex mixtures of contaminants, including aromatic hydrocarbons and heavy metals. The Hudson River Estuary has individual Superfund sites for PCBs, PCDD/Fs and chromium (Cr) along with some of the highest sediment levels of PAHs of any estuary in the U.S. After decades of debate, the most contaminated locales in the Hudson River PCBs Superfund site are designated for remediation beginning in the summer of 2009. In the late 1970s, Atlantic tomcod Microgadus tomcod from the Hudson River exhibited one of the highest prevalences of tumors (hepatocellular carcinomas) ever observed in a natural population concurrent with a dramatically truncated age structure.  In a combination of field and laboratory studies, we propose to explore the prevalence of tumors and the age structure of the Hudson River tomcod population today, just prior to site remediation, and the mechanistic basis of hepatic neoplasia and earlylife-stage toxicities in its population. In controlled laboratory studies, we are studying the sensitivity of young life-stages of tomcod to a suite of epigenetic alterations at the global and gene-specific levels resulting from exposures to Cr individually and incombination with benzo[a]pyrene and coplanar PCB126. We are also comparing the sensitivities of different individuals, ages, genders, and populations of tomcod to sensitive epigenetic changes. In parallel studies, the magnitude of these epigenetic changes will be compared in tomcod from known contaminated sites in the Hudson River and from a cleaner nearby estuary. Ours studies will complement those from other investigators in this SBRP program by evaluating the across taxa conservation of mechanisms of toxicant-induced epigenetic alterations and the applicability of their results to real-world in vivo environmental exposures of natural populations.

Research Project 4 -
Toxicity and Mobilization of PAHs and Chromium in Soils and Sediments

D. DiToro, Ph.D. – Project Leader

Modern methods for predicting the toxicity of metals at contaminated Superfund sites require that bioavailability be explicitly evaluated. The Biotic Ligand Model (BLM), which accounts for the effects of metal speciation at the site of toxic action (the biotic ligand) is currently being used by the EPA for water column copper criteria and will be the basis for all future criteria. Water column constituents such as inorganic ligands and organic matter (DOC and POC) play an important role in the BLM, competing with the bioticlig and for the metal. The BLM requires a speciation model that can account for partitioning of metal to natural organic carbon, either dissolved (DOC) in the aqueous phase orparticulate (POC) in sediments or soils. These speciation models do not adequately predict metal partitioning in the presence of competing cations (e.g. Ca, Mg), or other metals. Our first specific aim is to remedy this situation.

There is at present no mechanistic model for establishing chromium sediment criteria for Superfund sites. In the presence of reduced sulfur, Cr(VI) is reduced to Cr(III) which is very insoluble and not toxic in the normal pH ranges found in sediments. However, Cr(VI), which can exist in the aerobic zone of sediments, is quite soluble and toxic. Our second specific aim is to develop kinetic models for Cr(III) solubilization. This work will provide a better understanding of how Cr(III) potentially transforms to Cr(VI).

It has been hypothesized that the mechanism by which Ni causes toxicity to the HDM enzyme being studied by Dr. Costa’s group is the replacement of Fe by Ni in the active pocket of the enzyme. We are making quantum chemical calculations of there placement Gibbs free energy for Co, Ni, Cu and Zn in order to verify that Ni has the most favorable replacement free energy. This will provide further evidence for the validity of this mechanism and will demonstate the utility of such calculations for other metal-induced enzyme inactiviation investigations.

Research Translational Core

Kevin Farley, Ph.D. – Core Leader

The translation of research outcomes to the regulatory community and to other important stakeholders is an integral part of our grant, which focuses on carcinogenic metals (Ni, Cr, As) and their interactions with other environmental stressors (e.g., PAHs, PCBs, UV radiation). The Research Translation Core (RTC) is serving as an interface between program investigators and government agencies, NGOs and the scientific community addressing issues of both national and local importance as follows: (1) On the local and regional level, agencies are facing difficult issues in implementing scientifically-sound risk assessments at many Superfund sites. Technical support is being provided to EPA Region 2, NOAA, and state agencies through preparation of research summaries, and meetings with agencies personnel to address Superfund-related issues for contaminated sites, particularly for Superfund mega-sites, in the New York-New Jersey area. (2) On the national level, RTC efforts are being directed at needs identified in EPA’s newly-developed Framework for Metals Risk Assessment (U.S. EPA, 2007). Technical support to EPA Office of Research and Development and EPA Office of Water are being provided to support their continuing development of risk assessment methods and modeling approaches for metals. The development of a new interactive risk assessment model for metals, a training seminar, and a training manual will be an integral part of this effort. (3) Communication with government agencies, NGOs and other important stakeholders will be addressed through professional short courses. This initiative will rely on established communication channels of the Manhattan College Institute in Water Pollution Control (MCIWPC). The RTC will also maintain close communications with NIEHS staff.

Molecular and Imaging Core

Wei Dai, Ph.D. – Core Leader

The purpose of the Molecular and Imaging Core Facility is to provide quality-controlled, state-of-the-art service for investigators of the Superfund project. The following two areas comprise the Core Facility: (i) Cell Imaging and (ii) Genomics. The Cell Imaging unit provides training and consultation on, as well as usage of fluorescent and confocal microscopy, and software tools for processing and quantitative analysis of image data. Sophisticated image acquisition and analysis regimes are also available including time-lapse, deconvolution, and FRET. The Genomics unit assists investigators with a variety of needs in real-time PCR set-up and data analysis, detection and quantification of gene-specific DNA methylation, and specialized DNA sequencing. This Core Facility will be complementary to the existing NIEHS Molecular Biology Core, which does not provide “hands-on” bench services or experimental laboratory support as provided herein.

Administrative Core

Max Costa, Ph.D. – Core Leader

This NYU Superfund Research Program (SRP) includes projects from a variety of disciplines housed in three academic institutionsin two states. Thus, it is very important that the SRP Administrative Core provide strong leadership and outstanding administrative support in order to ensure cohesiveness of the entire Program. The NYU SRP Administrative Core, therefore, is designed to provide the support that ensures a cohesive and united Program that meets the goals of the NYU SRP. The Administrative Core represents the hub of coordination of SRP research and all other activities. The Administrative Core assures that meetings of the NYU SRP Internal and External Advisory Committee are held in accordance with the time-line goals. The Core not only supports the coordination of these various meetings, but also supports the communication between the various projects and cores in the program. The Administrative Core is the liaison with the NIEHS and other governmental agencies, providing information to them on the research accomplishments of the NYU SRP.