Center for Environmental Health Sciences
The overriding goal of Center for Environmental Health Sciences (CEHS) is to study the biological effects of exposure to environmental agents in order to understand, and predict, how such exposures affect human health. Three fundamental components influence the physiological effects of environmental exposures: the nature of the exposure, the duration of that exposure, and how well the exposed organism is equipped to deal with the exposure, in other words, the organism's genetic susceptibility. Environmental Health research at MIT encompasses a wide range of disciplines and the CEHS brings together 31 faculty members (28 from MIT plus 3 from Harvard University) who employ a diverse set of research tools to tackle research relevant to the Environmental Health Sciences.
In the past year, the CEHS has undergone another reorganization that reflects the evolving needs of the center membership. This change involved consolidation of six research cores into three, in order to provide a more integrated research program. Additionally, the former Accelerator Mass Spectrometer Facilities Core was consolidated with the Bioanalytical Facilities Core and the Molecular and Cellular Imaging Facilities Core merged into a new Animal Models and Pathology Facilities Core. The creation of this latter core represents an important development of great utility to center members.
The center has core funding from the NIEHS and the CEHS-associated research programs are funded through a variety of sources including NIGMS, NCI, DOE, NSF, ACS and DARPA. The many and varied research programs provide challenging interdisciplinary problems for postdoctoral, graduate, and undergraduate students. Research in the center is organized into three research cores that build on the strengths of the center membership and reflect a vision for the future of environmental health research. These are the Mutation and Cancer Research Core, the Bioengineering for Toxicology Research Core, and the Environmental Systems and Health Research Core. The theme of each core derives from the member research interests and all are linked by the overall focus of the center on defining the biological effects of exposure to environmental agents. The Mutation and Cancer Research Core, directed by Professor Dedon, addresses the relationships between DNA damage, DNA repair, mutation and cancer associated with exposure to environmental and endogenous chemical and physical agents. The Bioengineering for Toxicology Research Core, directed by Professor Griffith, was created to facilitate the development of new experimental tools and analysis methods relevant to environmental influences on human health, with a range of approaches that span the molecular-cellular-systems length scales. The mission of the Environmental Systems and Health Research Core, directed by Professor Hemond, is to understand the relationships that link environmental processes and human health in terms of exposure to chemical agents as well as biota. This is most aptly illustrated by the triad of dependent interactions of aflatoxin, hepatitis virus, and human liver cancer, which has been a research foundation for the center since its inception nearly three decades ago.
The three state-of-the-art facilities cores reflect the new CEHS research directions. The cores are heavily used by center researchers, with each contributing to the research of at least 10 members. Under the direction of Drs. Wishnok, Taghizadeh, and Skipper, the Bioanalytical Facilities Core provides center members with the latest tools, techniques and expertise in the characterization and quantification of chemical substances and modifications of cellular molecules such as DNA and protein. The core operates as a resource for the center, and it allows researchers to use the facilities as a service lab, for supervised analyses, or as fully trained users. The Genomics and Bioinformatics Facilities Core, directed by Professor Sorger and Dr. Fry, provides center members with an integrated facility for microarray fabrication and analysis, database storage, database management, data mining, and modeling. These tools are critical to the goal of moving center research to higher levels of complexity in an attempt to understand the response of the whole organism to environmental influences. The Animal Models and Pathology Facilities Core, directed by Professor Fox, provides center members with the latest technology for the application of animal models to environmental health research, including the generation of genetically engineered mice, embryo rederivation of imported mice, colony management, and preparation and interpretation of murine tissue by histological and image analysis.
Research Cores
Mutation and Cancer Research Core
This research core was created in 2002 to provide an integrated and focused scientific program that addresses the relationships between DNA damage, DNA repair, mutation and cancer associated with exposure to environmental and endogenous chemical and physical agents. One of the central questions here is the degree to which environmental and endogenous processes contribute to the mutational burden of human cells. The specific aims of this core are:
- To further the development of center research projects and programs that address the mechanistic linkages between exposure to environmental and endogenous agents, genetic change and cancer and other diseases
- To promote interaction among the members of the Mutation and Cancer Research Core, and interactions between members of this research core and those of the other two research cores, with the goal of creating new research projects and programs
- To promote the development and acquisition of new technologies in the CEHS facilities cores that will facilitate studies in the Mutation and Cancer Research Core.
Core Members:
- Peter C. Dedon, director, associate director Biological Engineering Division, deputy director CEHS, professor of toxicology
- Jeffrey A. Coderre, associate professor of nuclear engineering
- Catherine L. Drennan, Cecil and Ida Green career development assistant professor of chemistry
- Thomas E. Ellenberger, professor of biological chemistry and molecular pharmacology, Harvard Medical School
- Bevin P. Engelward, assistant professor of biological engineering
- John M. Essignmann, professor of toxicology and chemistry
- James G. Fox, professor and director, Division of Comparative Medicine
- David J Hunter, professor of epidemiology and nutrition, Harvard School of Public Health
- Leona D. Samson, professor of biological engineering and toxicology
- David B. Schauer, associate professor of biological engineering
- James L. Sherley, assistant professor of biological engineering
- Steven R Tannenbaum, professor of biological engineering
- Graham C. Walker, professor of biology
- John S Wishnok, senior research scientist in biological engineering
- Gerald N. Wogan, Underwood-Prescott professor of toxicology, emeritus, professor of chemistry, emeritus
- Jacquelyn C. Yanch, professor of nuclear engineering
Bioengineering for Toxicology Research Core
This new research core, created in 2002, builds on productive research collaborations fostered by a new academic structure at MIT. The Biological Engineering Division was formed in 1998 with the aim of strengthening the educational interface between engineering and molecular cell biology, and to meld the Molecular and Systems and Toxicology (MST) research and educational programs with the nascent discipline of biological engineering. The evolution of this academic structure was strongly influenced by several productive research collaborations among the bioengineering and MST faculty, and in turn, the research collaborations have grown since the formation of the Biological Engineering Division. The Bioengineering for Toxicology core was created to facilitate the development of new experimental tools and analysis methods relevant to the scientific foci of the two other research cores with an initial emphasis on activities in the Mutation and Cancer Research Core. Our aim is to develop a range of approaches that span the molecular-cellular-systems length scales to solve problems in toxicology and environmental health. The experimental tools range from tissue-engineered physiological bioreactors that bridge the gap between cell culture, animal models, and humans; and multiphoton imaging methods that allow in situ quantification of events such as single cell apoptosis and DNA recombination by scanning large populations of cells in tissues and tissue models. Analytical methods include statistical (Bayesian) and deterministic models of signal transduction networks and computational models of protein interactions in the context of different cell compartments.
Core Members:
- Linda G. Griffith, Bioengineering for Toxicology Research Core Director, professor of biological engineering
- Peter C. Dedon, professor of biological engineering
- Bevin P. Engelward, assistant professsor of biological engineering
- Douglas A. Lauffenburger, professor of biological engineering, director of Biological Engineering Division
- Leona D. Samson, professor of biological engineering and toxicology
- Ram Sasisekharan, associate professor of biological engineering
- James L. Sherley, assistant professor of biological engineering
- Peter T.C. So, associate professor of mechanical engineering and biological engineering
- Peter K. Sorger, associate professor of biology and biological engineering
- Steven R. Tannenbaum, codirector, director of Biological Engineering Division
- Bruce Tidor, associate professor of biological engineering and electrical engineering and computer science
- Michael B. Yaffe, assistant professor of biology and biological engineering
Environmental Systems and Health Research Core
This new research core, created in 2002, aims to understand the relationships that link environmental processes and human health. This requires improved understanding of the relationships of human health with environmental chemicals and with their processes of transport and transformation. Increasingly, however, a broader view of environment-health linkages is now required, one in which genomics and ecology play an increasing role. The well being of humans is inextricably interconnected with processes that may best be regarded as ecological, with recognition that environmental biota share with chemicals the linkage to human health. In as much as this emerging field cuts across traditional disciplines, the Environmental Systems and Health Research Core brings together researchers with expertise that includes microbiology, microbial ecology, analytical chemistry, hydrology, veterinary science, fluid mechanics, environmental chemistry, and instrumentation. Some members of this core focus on understanding the diversity and movement of genes in the environment (environmental genomics, gene flow); others specialize in chemical transport and measurement; others focus on the physical processes, notably fluid movement, that is central to the transport of both biological and chemical entities. A unifying theme is that of ecology in the broad sense, as projects and researchers address processes that are rooted in the natural environment, yet have implications for the well being of people. Gene flow, for example, can affect the distribution of pathogenicity, the acquisition of antibiotic resistance, or the presence of biodegradative capability in microbial communities. Ecosystem processes govern the nature of coexisting populations at scales from that of microbes to that of continents, with direct effects on humans at all scales.
Core Members
- Harold Field Hemond, Environmental Systems and Health Research core director, professor of civil and environmental engineering
- Sallie W. Chisholm, professor of civil and environmental engineering and biology
- David C. Christiani, professor of environmental health and epidemiology, Harvard School of Public Health and Harvard School of Medicine
- Patricia J. Culligan, associate professor of civil and environmental engineering
- James G. Fox, professor of biological engineering and director of the Division of Comparative Medicine
- Charles F Harvey, assistant professor of civil and environmental engineering
- Heidi M. Nepf, professor of civil and environmental engineering
- Martin F. Polz, assistant professor of civil and environmental engineering
- David B. Schaurer, associate professor of biological engineering
- Bettina M. Voelker, assistant professor of civil and environmental engineering
Core Facilities
Bioanalytical Core Facility
The purpose of the Bioanalytical Facilities Core is to provide center members with state-of-the-art tools and techniques for the characterization and quantification of chemical substances and modifications of cellular molecules such as DNA and protein. The primary objective is to continually increase our abilities and effectiveness via the following aims:
- To assist center members in the purification, identification and quantitation of unknown compounds, synthetic intermediates and products of DNA, and protein damage
- To maintain a broad range of cutting-edge major analytical instruments and assist in the acquisition of new instrumentation to meet the needs of center members
- To keep center members informed about developments in equipment and methods
- To provide expert assistance with method development, experimental design, and data analysis, with an emphasis on promoting collaborative research projects
- To teach students and postdoctoral scientists to become proficient in their own analyses
Major changes in the past year include the incorporation of the Accelerator Mass Spectrometry Facilities Core into the Bioanalytical Core; the relocation of a portion of the core to 500 Technology Square; and the acquisition of several pieces of equipment. On the advice of our External Advisory Committee, we incorporated the former Accelerator Mass Spectrometry Core into the Bioanalytical Facilities Core. This logical change will streamline the Facilities Cores structure in the center and enhance operations. In mid-July, the Bioanalytical Laboratory previously located in Building 16, was moved to the second floor of NE47 at 500 Technology Square. The increase in laboratory space by 400 square feet has allowed the center to acquire a new triple quadrupole mass spectrometer (ABI 3000) from Professor Steven Tannenbaum in the Biological Engineering Division. This expensive instrument is in high demand by center members and represents a major acquisition. Other equipment acquisitions are described below.
Core Leadership:
- John S. Wishnok, Bioanalytical Facilities Core director, Biological Engineering Division,
- Paul L. Skipper, Bioanalytical Facilities Core codirector, Biological Engineeering Division
- Koli Taghizadeh, Bioanalytical Facilities Core codirector, Center for Environmental Health Sciences
Usage and Benefits
The Bioanalytical Facilities Core provides state-of-the-art equipment and expertise in the purification, characterization and quantitation of chemicals and modified biomolecules. Center members benefit from the core in several ways. Perhaps the most important is access to technology and expertise not generally available to MIT researchers. Though facets of the Facilities Core are available to researchers not affiliated with the center, CEHS members benefit by receiving top priority for use of equipment and services and they receive a 50 percent discount on all core services. The goal of the core staff is to attend to member needs and promote collaborations, including acquisition of new instruments, development of new analytical methods, and education of students and postdocs. This is illustrated by examples of methods that are now available on a routine basis:
- LC-MS/MS quantitation of ABP-dG adducts in human DNA
- GC-MS quantitation of ABP-hemoglobin adducts
- Enrichment of nitrotyrosine-containing peptides
- LC-MS analysis of 8-oxo-dG oxidation and nitration products
- MS-MS-MS analysis of PAH-adducted peptides
- Sequencing of DNA oligos by exonuclease digestion and MALDI MS
- Peptide mass fingerprinting by MALDI-TOF MS
- Peptide sequencing by ion-trap or tandem quadrupole MS
- Quantitation of nitrate/nitrite in biological fluids
- Characterization and quantitation of mutagens in foods
- Quantitation of modified DNA bases
High demand for the core services led to several major improvements in the past year. A ThermoFinnigan TSQ 7000 tandem quadrupole MS was replaced by an Applied Biosystems/Sciex API 3000 of greater sensitivity, and we acquired a MALDI-TOF instrument shared with the Chemistry Department. The API 3000 was purchased with electrospray and atmospheric-pressure chemical ionization sources; we added a custom nanoelectrospray source. The core has acquired a single-quadrupole LC/MS system and an ion-trap MS with a variety of ion sources including nanoelectrospray, photoionization, and atmospheric-pressure MALDI. The quantitation capabilities of the API 3000 were enhanced by the addition of an automated online-desalting system. A new Agilent 5973 GC-MS with an autosampler and a positive- and negative-ion chemical ionization source was purchased and we have completed construction of two laser-induced fluorescence systems.
Examples of specific member use and benefits include:
- Accelerator mass spectrometry is fundamentally a MS technique for the detection of low-abundance isotopes, with a sensitivity some 1000-times greater than scintillation counting and attomole quantitation of 14C. Several groups make use of the CEHS AMS: Professor Essigmann's project on chemotherapeutic drug development uses AMS to assess drug targeting in cell culture and in murine tumor models; Prof. Dedon's studies of DNA strand breaks use AMS sensitivity to measure the formation of carbonyl termini by derivatization with 14C-reagents; a pharmacokinetic study of acetaminophen in the rat was completed recently in the Tannenbaum lab.
- Professor Engelward's lab has shown that enzymes involved in base excision repair can modulate the susceptibility of microbial cells toward recombination events induced by alkylating agents and by NO delivered by a membrane-based system developed in the core. These experiments showed that recombinational repair can prevent NO-related cytoxicity and the NO can induce recombination, which suggests a mechanism linking inflammation with cancer.
- The Essigmann group is studying the relationships between the structures of specific DNA lesions and the biological endpoints of mutation and cell death. MALDI-TOF MS was used to confirm the structures of synthetic oligos that contain specific DNA lesions formed by chemical carcinogens, oxidants or radiation. The group is also involved in the design of anticancer drugs, and has used LC-MS to characterize the interactions of a potential anti-breast-tumor agent with DNA.
- The Sherley lab has recently proved the immortal strand hypothesis, in which the parent DNA strand is conservatively segregated to the stem cell during asymmetric cell division. GC-MS and LC-MS was used to search for evidence that nucleoside modifications were involved in the selection process.
- Using LC-MS, Professor Tannenbaum's group showed that nitration of human serum albumin by peroxynitrite occurred with high regio-selectivity. Using both ion-trap and tandem quadrupole electrospray MS, they carried out an extensive investigation of the reaction of dG with peroxynitrite and showed that analogous reactions occurred in DNA. They demonstrated that the DNA lesions can be located and tentatively identified with controlled endonuclease digestion followed by MALDI-TOF analysis. They recently designed and synthesized nitrotyrosine-specific affinity probes that show promise for purifying low levels of nitrotyrosine-containing proteins from complex mixtures of peptides from digests, prior to analysis by MS.
- In addition to the NO-related genetic damage, the Wogan group has characterized the DNA reaction products of aflatoxin B1 and developed LC-MS/MS and LC-LIF procedures of adequate sensitivity to measure DNA adduct levels in human tissues and serum albumin. They have recently developed a novel LC-LIF method for detection of DNA adducts of all classes of carcinogens, and— in collaboration with the Tannenbaum group—have developed an LC-MS/MS method for quantitation of the aminobiphenyl-dG adduct in bladder tissue.
- Professor Sasisekharan's research is focused on the structure and function of heparin-like glycosaminoglycans. His group has regularly used MALDI-TOF MS to characterize these molecules by carbohydrate-protein complexes.
- Professor Dedon's group has made extensive use of the core in the analysis of DNA damage products by LC-MS and GC-MS and in the development of a sensitive assay for strand breaks and abasic sites. IThey developed sensitive LC/MS methods to quantify the nucleobase deamination products arising from exposure of DNA to NO: dX, dO, dU, and dI. Controlled delivery of NO under biologically relevant conditions revealed the absence of dO and the formation of a significant number of abasic sites presumably derived from NO-mediated depurination. Other studies revealed that dX is a stable lesion in DNA with a half-life of > 2 yr at 37 °C.
- The Hemond group has studied respirable airborne particles, which penetrate deep into the lungs, contain toxins and carcinogens and have been linked epidemiologically to various lung and heart diseases, including lung cancer, asthma and cardiopulmonary distress. The risk of lung cancer is higher in urban areas than in rural areas, although the cause of this difference is not clear. Air samples from Massachusetts and upstate New York were tested for mutagenicity in the h1A1v2 of human cell line. The results indicate that cold weather is significantly correlated with the human cell mutagenicity of respirable particles in the northeastern United States and furthermore show that populations of urban centers in this region are exposed to higher levels of airborne human cell mutagens than in nearby rural areas.
- A recent change that further enhances the benefits to CEHS members was that hiring of Patricia Brown, a technical specialist available ad hoc for software and hardware consulting. To facilitate data-exchange and communication, a web-based network is being developed by Ms. Brown for our current facilities and should include secure-but-accessible data storage and real-time video conferencing.
Genomics and Bioinformatics Core Facility
The Genomics and Bioinformatics Facilities Core was created to provide center members with integrated facilities for microarray fabrication, microarray analysis, database storage, large-scale database management, data mining and data modeling. The core is comprised of the CEHS Bioinformatics Computing Facility tightly integrated with the BioMicro Center. The latter is a joint endeavor of the Center for Environmental Health Sciences, the Department of Biology, the Center for Cancer Research, and Biological Engineering Division that was founded in 2000 as the central biofabrication and bioinformation technology resource at MIT. The specific aims of the Genomics and Bioinformatics Facilities Core are:
- To provide integrated support for both commercial and custom microarrays. Though the current focus of most center members is on mRNA analysis, the capabilities of the core can be extended to the fabrication and analysis of other types of arrays including protein arrays
- To provide strong three-tier architecture for data storage, management and processing
- To provide networks of managed desktop computers for data analysis and mining
- To provide training and educational programs in commercial and open-source bioinformatics software
- To provide center members with technical assistance with programming, database administration and data analysis
- To work with center members in applying their results to the development of data models and quality control procedures, error modeling as well as the development of new technologies
Core Leadership
- Peter K. Sorger, Genomics and Bioinformatics Facilities Core director, Biology Department
- Rebecca C. Fry, Genomics and Bioinformatics Facilities Core Codirector, CEHS
Facilities and Equipment
The Genomics and Bioinformatics Facilities Core consists of the CEHS Bioinformatics Computing Facility and the MIT BioMicro Center. The core provides two areas of focus: biofabrication, and bio-IT. The largest effort in the biofabrication group involves oligo and cDNA based microarrays, while the primary focus of the bio-IT group is the desktop, server and network infrastructure required for database-driven bioinformatics applications. The core is the central provider of experimental and computational facilities for DNA microarraying and for other high-throughput analytic methods in the center. A focus on new technology also helps to ensure that the core remains at the cutting edge of DNA microarray methods. Standardization, quality control and data integrity are major issues in the analysis of DNA microarray data and are areas of active research in the core. Major facilities and services available include:
- Biofabrication/liquid handling equipment for precision automation and experimentation
- Biofabrication: cDNA amplification systems
- Robotic printing for microarray production
- Spotted microarray and Affymetrix GeneChip hybridization equipment
- Spotted Microarray and Affymetrix Genechip Scanning equipment
- Prefabricated arrays and custom arrays
- Microarray experimentation training and support
- Core computing clusters and core workstations
Usage and Benefits
The Genomics and Bioinformatics Facilities Core provides state-of-the-art microarray and data mining technology for the study of cellular responses to environmental agents. The core was created to accommodate the growing needs of center investigators and it benefits center members in several ways. First, the core provides a 50 percent discount on all services for members of the CEHS. Second, the core provides access to technology and expertise not generally available in the surrounding community. In its first year, the core has seen substantial use, with an increase in use projected in the next year. Ten center members are currently using the Genomics and Bioinformatics Facilities Core for studies ranging from creation of whole genome arrays for bacteria to expression profiling with mammalian cells. The following summaries illustrate the center research projects utilizing the core.
The Chisholm group has a long-standing interest in the ecology of ocean microorganisms. One of these, Prochlorococcus, is a prokaryotic oxygenic photoautotroph that is abundant in vast regions of the world's oceans and is responsible for a significant proportion of global carbon fixation by photosynthesis. To understand how Prochlorococcus spp. have become so successful in the oceanic environment, the Chisholm group is studying their response to changes in environmental parameters under controlled laboratory conditions. Three types of arrays have been used to this point. The first was is a 70mer probe array spotted using core facilities. This array was used to optimize protocols for signal intensity and clarity. With this data, they have now switched to NimbleGen arrays in anticipation of forthcoming Affymetrix arrays. These studies will provide important information about the link between this ocean microorganism and human well-being.
The Dedon group studies of the role of deoxyribose damage in the cellular response to oxidizing agents. Deoxyribose oxidation plays a critical role in the genetic toxicology of oxidative stress, including involvement in complex DNA lesions, crosslinking with DNA repair proteins and the formation of endogenous DNA adducts. The Dedon group is approaching this problem by applying the power of genomics (transcriptional profiling) and genetics (targeted knock-outs) to define role of deoxyribose oxidation products in the responses of S. cerevisiae to oxidative insults. The key feature is the use of a series of deoxyribose-specific DNA-damaging agents (enediyne antibiotics) each of which differs in the proportion of single- and double-strand breaks produced and in the chemical products attached to the resulting strand breaks.
The Samson group makes extensive use of the Genomics and Bioinformatics Facilities Core in studies of gene expression and phenotypic analysis with E. coli, yeast and mice. One of these projects, Transcriptional Responses of Mice to Alkylating Agents, has a major goal of painting an integrated picture of how mammalian cells, in culture and in the intact organism, respond upon exposure to alkylating agents. The specific agents are chosen to represent environmental toxicants as well as those commonly used for chemotherapy. Specifically, they are analyzing the global transcriptional responses of specific tissues in wild type, Mgmt and Aag null mice upon exposure to SN1 and SN2 alkylating agents respectively. They will also analyze the global transcriptional responses of isogenic sets of mouse embryonic fibroblasts upon exposure to alkylating agents.
The Griffith research group has developed a microfabricated, perfused bioreactor that fosters development of 3D architecture of liver when seeded with primary cells isolated from liver. When both parenchymal and nonparenchymal cells are present, fenestrated endothelium with an accompanying high degree of liver function are observed. Griffith is a coinvestigator with Samson, Essigmann, and Sorger on the NIEHS project, Global Responses to Aflatoxin B1 and Alkylating Agents, and as part of this grant is comparing the responses of the liver bioreactor to those of native liver under treatment by toxicants. Transcriptional profiling is used to compare responses of cells maintained standard cell culture methods, perfused bioreactor, to those in vivo.
The Schauer group has recently undertaken a series of studies that make use of the Genomics and Bioinformatics Facilities Core. They are currently using cDNA microarrays to characterize the expression profile of hepatocytes following infection with the cancer-causing bacterium Helicobacter hepaticus. Male A/J mice, which are susceptible to Helicobacter hepatitis and hepatocellular carcinoma, are experimentally infected with H. hepaticus, and are euthanized 6 to 12 months later. RNA is isolated from primary hepatocytes, after perfusion and isolation, and from whole liver tissue. Control RNA is isolated from hepatocytes and liver tissue collected from age-matched, uninfected male A/J mice. The goal of these studies is to ascertain the mechanism by which H. hepaticus infection causes persistent hepatitis and hepatocellular carcinoma.
The core has allowed the Sherley lab to accelerate their efforts to identify genes that are specifically up-regulated during asymmetric cell kinetics. Asymmetric cell kinetics are a characteristic of adult stem cells. Therefore, their hypothesis is that some genes that are specifically up-regulated during asymmetric cell kinetics may also identify adult stem cells. Currently, genes that uniquely identify adult stem cells have not been discovered. The identification of such genes is highly desired in efforts to identify, isolate, and manipulate adult stem cells for new therapies.
Sorger and Samson have undertaken a major study entitled, Standardization Experiments of the Toxicogenomics Research Consortium, the goals of which are to determine sources of variation in gene expression profiling and microarray data analysis, to develop standards by which to minimize variation in gene profiling experiments, and to develop methods to harmonize data across different microarraying platforms.
Research on gene expression analysis in the Essigmann laboratory divides into three areas. In the first, the gene expression response of human cells exposed to aflatoxin B1 is being defined. In parallel, DNA containing enriched DNA adduct pools (e.g., the aflatoxin-FAPY adduct) is introduced into cells by transfection to determine the elements of the transcriptional response that may be specific to individual adducts. The hypothesis tested is that there will be certain gene expression patterns that reflect the level and type of challenge to the cell from this toxin. The second project is focused on a striking biological observation that, while adult mice are refractory to aflatoxin toxicity and carcinogenicity, infant mice have a window of sensitivity to aflatoxin in the first few weeks of life. The constellation of genes that are expressed or not during this critical time period are being examined. The third project concerns the response of cells to cisplatin, which forms DNA lesions that hijack transcription factors. Gene expression changes in E. coli that may result from cellular treatment with the toxin are being probed. In all experiments, Affymetrix arrays are used with the fluidics workstation and the data analyzed using the Spotfire software.
Animal Models and Pathology Facilities Core
The overall objective of the Animal Models and Pathology Facilities Core is to provide center members with state-of-the-art pathology support, transgenic resources and a centrally-managed AAALAC-approved animal holding and surgical facility. The core is staffed with experienced personnel and is equipped with essential equipment to generate genetically engineered mice (GEM), rederive imported mice by embryo transfer rederivation, provide colony management, and prepare and interpret tissue samples by histological and image analysis.
- To generate transgenic animals by pronuclear microinjection of DNA constructs or embryonic stem cell manipulation and provide colony management of GEM for use by center members
- To rederive transgenic and other specialized mouse strains by embryo transfer to assure specific pathogen free status of all mice used in this program
- To freeze and store embryos from GEM being utilized by center members
- To provide histology, pathology, molecular characterization and imaging expertise for extensive phenotyping and assessment of pathological damage of GEM and other animal models as needed
Core Leadership:
- James G. Fox, Animal Models and Pathology Facilities Core director, Division of Comparative Medicine
Facilities and Equipment
Histopathology Service
Histopathology Service staff include Dr. Arlin B. Rogers and research technicians Kathy Cormier, Jeff Bajko, and Ernie Smith.
The histopathology service is part of the DCM's research and service laboratories. Staff members assist in the preparation of tissue specimens for diagnosis and analysis of disease processes under the guidance of a board-certified veterinary pathologist.
Colony Management Service
Colony Management Service staff include Dr. Susan E. Erdman, Jill Goslin, Beth Jacobsen, Erica Jarmon, and Kibibi Rwayitare.
Approximately 125,000 GSF central managed AAALAC approved facilities with the necessary equipment, space and containment suite to conduct transgenic and germ-free work are available to center investigators.
Transgenic Service
Transgenic Service staff include Dr. Melanie Ihrig, Dr. Ingrid Bergin, Dina Rooney, Nate Rogers, Tony Chavaria, and John Mkandawire.
DCM operates a centralized transgenic facility for generation of novel genetically-engineered rodents. The 1,652 square foot facility comprises four rooms in the division's barrier facility located in the sub-basement of Building 68. The DCM staff advises investigators on health status or strain backcrossing that may contribute to the utility of the particular rodent model system.
CEHS Imaging Facility
CEHS Imaging Facility is led by Dr. Elena Gostjeva, an expert in the use of state-of-the-art microscopes and advanced digital imaging methods. Use of the instruments listed below is overseen by Dr. Gostjeva, and she trains lab members of the CEHS faculty in the methods required for preparation and analysis of biological materials for immunohistochemistry, molecular cytogenetics, cytotoxicity, and other related scanning microscopy and imaging methods.
- Nikon Eclipse E800 fluorescence microscope
- Nikon Labophot fluorescence phase-contrast microscope
- Zeiss fluorescence phase-contrast microscope
- CompuCyte LSC laser scanning cytometer
- Zeiss phase-contrast microscope
- Zeiss Axioskop 2 MOT fluorescence phase-contrast microscope
Usage and Benefits
Animals continue to be important models for the study of molecular mechanisms of complex biological processes. The Animal Models and Pathology Facilities Core provides and maintains genetically engineered mice that are increasingly being generated to model specific aspects of human diseases and have proven to be extremely valuable in examining how genetic alterations interact with environmental chemicals and microorganisms to induce disease. The core provides personnel with this expertise as well as centralized laboratory equipment as part of the center's program. Center members benefit from the use the core by receiving substantial discounts on core services and technologies (free of charge up to $2,300–5,000 per user depending on the service), which are otherwise generally available at higher cost to the MIT community.
A Mouse Colony Management (MCM) Program provides skilled technical assistance to facilitate research using mouse models. The core staff advises investigators on breeding paradigms and tracking systems to optimize efficiency of production colonies, as well as providing hands-on services for routine mating, weaning, genotyping and culling. Rapid turnover of research data is assured by close cooperation with the centralized genotyping facility and the core pathology laboratories. MCM personnel routinely perform a wide variety of recovery and post-mortem injection and tissue collection techniques providing the investigative team with options for both assistance and further learning in the development of their mouse model system.
Numerous CEHS research projects utilize animal models, particularly GEM in which the end point is the histopathological assessment of tissue. In the projects generating and using transgenic mouse models, there is a requirement for extensive characterization of tissue phenotypes. Both the technical aspects of obtaining quality sections and expertise in the pathological identification of pathological changes are essential for acquiring meaningful genetic and epigenetic changes that are responsible for lesion development. Unfortunately, such expertise is not always available to biomedical research groups and when absent can lead to the misinterpretation of data. Thus, the Histology and Tissue Processing Facility section can provide essential morphological/histopathological services that will enhance the research programs for many of the CEHS investigators. This core offers services for routine processing, special techniques, technical assistance, training and consultation for center investigators. The core also conducts necropsies at the request of center members, particularly for the dissection of target organs that may require special skills or knowledge of anatomy. Histology processing and accurate diagnosis by trained veterinary pathologists, as well as related techniques such as, immunohistochemistry, autoradiography, in situ hybridization, etc. have become indispensable tools for the study of tissue damage and cellular response to a variety of environmental stimuli. In addition, the more recent use of microdissected paraffin sections in molecular biology studies have made these techniques an essential component of many research projects.
The CEHS Imaging Facility has the equipment and ability to accurately measure the influence of environmental agents on genetic, biochemical and biological processes in cells—whether in single cells or tissues. Sophisticated scanning microscopes and sensitive imaging instrumentation are required for monitoring the presence and location of proteins in cells and tissues (using immunofluoresence or fluroescently tagged proteins), for monitoring the induction of damage to chromosomes (cytogenetics), for monitoring the induction of cell death by apoptosis or necrosis, for monitoring the induction of cell cycle checkpoints, and for monitoring pathological changes in cells and tissues.
A few examples of specific member use and benefits follow.
The Engelward laboratory has developed one of the first mouse models in which cells that have undergone spontaneous homologous recombination can be detected within tissues of a mouse. This mouse model has provided insights into the relative susceptibility of different cell types to mitotic recombination in mammals. In addition, studies are currently underway to develop technology for detecting recombinant cells within tissues in situ. Engelward's group also used the laser scanning cytometer to monitor recombination events in the mouse model.
Three transgenic mouse strains for the Sherley lab were developed with the L. donovani xanthine phosphoribosyl transferase gene (XPRT). There is no mammalian enzyme with XPRT activity and xanthosine (Xn) exists in very low levels in mammalian tissues. In cell culture models, Sherley's group has shown that guanine nucleotide pools are elevated in mammalian cells that express a XPRT transgene when Xn is added to the culture medium. They propose to control the level of guanine nucleotide pools in the tissues of XPRT-transgenic mice in a controlled fashion by controlling Xn in the diet or by intravenous injection. Sherley's group also made use of the imaging facilties run by Dr. Gostjeva to detect asymmetrical kinetics in p53 mutant mouse cells lines.
K19-TGF b dominant-negative receptor II GEM were generated for the Fox lab. TGF b and its signaling effectors act as key determinants of carcinomatous cell behaviors, such as morphogenesis, growth arrest and apoptosis. To assess the role of TGF b in more detail, a dominant-negative form of the TGF b II receptor (TGF b DNRII) was fused with the mouse cytokeratin 19 (K19) promoter in a DNA construct used to generate the K19-TGF b DNRII transgenic mouse. We plan to assess the histopathological changes in the gastric mucosa of the K19 TGF b DNRII genetically engineered mouse before and after infection with Helicobacter pylori to learn the role of the TGF b signaling pathway in the pathogenesis and carcinogenesis related to H. pylori infection.
The Dedon group employed the fluorescence microscopy facilities of the core in a collaboration with Dr. Gostjeva to develop a yeast comet assay technique to quantify strand breaks and base damage arising in yeast cells treated with a variety of oxidizing agents.
In addition to the use of mouse models for tissue engineering, the Griffith group makes use of the laser scanning cytometer for monitoring liver cell function in a novel bioreactor.
The generation and maintenance of a wide variety of transgenic and knock-out mouse strains, altered in various aspects of DNA alkylation repair, or in their responses to DNA damage, is a crucial part of the Samson labs activities. The imaging facilities are also crucial to the Samson lab, to perform scanning and counting of sister chromatid exchanges in human cells and to visualize and label apoptotic human and mouse cells, and in various mouse tissues.
Much of the research in the Schauer lab relies heavily on the services of this core. As an example, the immunohistochemistry services played a significant part in the characterization of experimental inflammatory bowel disease in lymphocyte (Rag2)-deficient mice with or without adoptive transfer of regulatory T cells.
The Yanch group made use of the imaging facilities to visualize a track of damaged cells produced by a high-LET alpha particle beam that delivers single alpha particles to subcellular locations in cells and thin tissue explants.
Community Outreach and Education Program
The primary goal of the Community Outreach and Education Program (COEP) is to promote community-level scientific literacy with a variety of programs targeted to students and their teachers in grades four through undergraduate. The programs represent extensions of the research activities of the center including the Environmental System and Health Core and the Mutation and Cancer Research Core. Future programs will focus on the Bioengineering and Toxicology Core. Under the direction of Professors Nepf and Culligan, both from the Department of Civil and Environmental Engineering, the COEP is committed to
- developing supplemental materials and activities that augment existing K–12th grade science curricula and which reflect recent advances in environmental health sciences
- providing support and learning enrichment for Boston-area K–12 teachers
- mentoring and training of young scientists and graduate students on the mechanisms and importance of outreach
- engaging CEHS members in these and other COEP activities
The involvement of center members in COEP activities has been promoted along several lines. First, COEP has initiated the NIEHS Summer Teaching Fellows Program that each summer pairs a local K–12 teachers with CEHS members to participate in ongoing research and the development of novel teaching activities. Second, COEP offers funding to encourage CEHS members to participate in the development of outreach activities. Members participating in recent outreach activities include Drs. Gostjeva, Hemond, Schauer, Polz, Harvey, and Yanch. Finally, the COEP has initiated an annual MIT Museum and Outreach Day that involves exhibits developed by center members and family-oriented activities at the MIT Museum.
The COEP is reviewed annually by the Internal Advisory Committee to assess performance in meeting the objectives of the center. Review criteria may include:
- evidence of tangible outcomes (e.g., implementation of new K–12 curricula in specific schools, development of teaching tools such as kits and videotapes)
- feedback from teachers participating in the COEP activities
- numbers of students, teachers and CEHS members participating in COEP activities. The COEP Program is described in detail in section VI-D Detailed Description of Individual Core Units.
COEP Leadership:
- Heidi M. Nepf, director, associate professor of civil and environmental engineering
- Patricia J. Culligan, codirector, associate professor of civil and environmental engineering
- Amy S. Fitzgerald, outreach program coordinator/technical instructor, Edgerton Center
- Luke Higgins, graduate assistant, Department of Chemistry
COEP Programs
Groundwater Pollution Curriculum Package
COEP created a new teacher-ready, student-friendly curriculum package on groundwater pollution and the Superfund program. The curriculum package includes a 45-minute video that walks students through the investigation of a contaminated site and the process by which a site is added to the National Priorities List. A 66-page curriculum guide outlines supporting activities such as hands-on experiments, web-based learning exercises, and suggestions for library and community research. The package is available through the NIEHS COEP Resource Center through the Teacher as Scholars Program and through the Edgerton Center. Portions of the video have already been adopted by the Advanced Technology Environmental Education Center (ATEEC) for use in a new curriculum for junior college students.
Grungy Groundwater
Grungy Groundwater is a hands-on activity that challenges students to discover how water and pollutants move underground and how pollutants impact surface and drinking water supplies. During the four-hour activity, students first explore how fluids travel through different soil types. Then, students build their own models of the underground using the different soil types they have just investigated. In the figure below a student is placing a clay layer over a sand layer. The students use the models to discover how buried and surface contamination enter and travel through the subsurface. Over 500 students took part this year. With cooperation from the Cambridge Public School Science Coordinator, Dr. Melanie Barron, this activity will be integrated into the pollution and ecosystem health unit of every 5th grade class in the CPS system.
The Road to the Double Helix
This activity uses a unique LEGO kit developed by Dr. Kathy Vandiver to visualize the structure of DNA. At the start of the activity students are given a box of LEGO pieces that represent the four nucleotides. Distinctly colored magnets differentiate between adenine, guanine, cytosine, thymine. The polarity of the magnets are arranged such that adenine can only join with thymine and guanine can only join with cytosine. The students use the pieces to create models of DNA and to discover how DNA replicates itself. In the second part of the activity students extract DNA from a variety of fruits.
CEHS Summer Teaching Fellows at MIT
To foster even stronger links between the center and the local teaching community, one or possibly more teachers from the Boston area will be selected each year from a pool of applicants to be NIEHS summer teaching fellows at MIT. The fellows will be given an office within the Center for Environmental Health Sciences and a ten-week stipend. Prior to arriving at MIT the Fellows will work with the COEP directors to identify mutual areas of interest between the teacher and the various center researchers. We will link each fellow with one, or possibly two, specific researchers who will serve as their official hosts.
During the orientation week the fellows will meet with members of each research core and be given tours of each lab. Also, during the first week the Fellows will work with Amy Fitzgerald, the COEP Directors, and the host researcher to define specific goals for the summer. These might include: the participation of the fellow in current, ongoing research; the creation of a hands-on activity for the Edgerton Center (see description of Edgerton Center Activities above); the creation of new classroom activities for the Boston-area schools; and the development of a teacher-training course for the fellow's home district.
Pilot Project Program
The goals of the MIT CEHS Pilot Project Program are:
- to provide initial support for new investigators to establish new lines of research in environmental health
- to allow exploration of possible innovative new directions representing a significant departure from ongoing funded research for established investigators in environmental health sciences
- to stimulate investigators from other areas of endeavor to apply their expertise to environmental health research. These goals are achieved by providing $25,000–$30,000 of funding for four to five novel and innovative research projects related to toxicology and environmental health issues
The center received eleven proposals for review in 2002, of which seven were funded (descriptions below). Each application was reviewed by at least two members of the Internal Advisory Committee and scored using the NIH scale in four areas:
- intrinsic quality of proposed research
- relevance to the stated CEHS mission
- likelihood that the project will lead to formal RO1 or equivalent funding
- likelihood of the project leading to collaborative interactions with center members
In addition to funding of these projects, the center also provided $300,000 of start-up funding to two new junior faculty members at MIT, faculty whose research activities in RNAi (Professor Luke Van Parijs) and proteomics (Professor Forest White) technologies are critical to the future research directions of the center.
Pilot Projects for 2002
"Hsp90, Evolution, and Antifungal Drug Resistance," Professor Susan Lindquist, Whitehead Institute and Department of Biology
"Molecular Markers for Adult Stem Cells," Professor James Sherley, Biological Engineering Division
"Two Technolocial Approaches for Identifying Protein Partners of Family Y Translesion DNA Polymerases and Evaluating the Biological Significance of the Interactions," Professor Graham Walker, Biology
"Arsenic Cycling in a Bangladeshi Village: Groundwater Pumping, Irrigation, Agriculture and Human Ingestion," Professor Charles Harvey, Civil and Environmental Engineering
"Role of Reactive Oxygen Species in the Solar Disinfection of Drinking Water," Professor Bettina Voelker, Civil and Environmental Engineering
"Cellular Responses to Deoxyribose Oxidation in DNA," Professor Peter Dedon, Biological Engineering
"Development of a Lentivirus expression system for use in RNAi Technology," Professor Luke Van Parijs, Biology
Plans for 2003
We await the conclusion of our recent competitive renewal application to the National Institute of Environmental Health Sciences for continuation of the center grant.
We are planning a retreat for center members on January 11 at the Endicott House. This will be the first of what we intend to be an annual CEHS retreat to discuss the science and engineering projects and programs driving center research as well as to promote interactions among center members.
We will be continuing our successful Friday Forum series in which center core directors make monthly research presentations at an event intended to promote interaction among center members in an informal social setting.
Our site will be visited by our highly distinguished External Advisory Committee in the spring/summer of 2003 to gain further input and advice on the growth and development of the MIT CEHS.
More information about the Center for Environmental Health Sciences can be found online at http://mit.edu/cehs/.