Among the efforts of the past year one in particular stands out that will affect research planning for years to come. Strangely enough, we discovered that biostatisticians in cancer and other disease areas had truncated their data sets for age-specific disease rates at 80 years of age. The National Cancer Institute had no data above this age level.. We reasoned that the data from the extremely aged would allow testing of hypotheses about the fraction of a population really at lifetime risk of cancer, and perhaps other diseases of significant effect on human mortality such as atherosclerosis.
Despite distinct difficulty getting data from scientific bureaucrats, we now have the data giving the number of deaths for each kind of cancer per living individual in age-groups from 0-4 to 100-104 years in the United States, including males and females, majority and minority populations. The data reveal that the rate of death reaches a maximum at an age specific for each cancer type and subsequently declines toward zero. This maximum has been shifting as we compare populations born in 1860, 1870, etc., suggesting that subpopulations at risk of cancer death become extinct before subpopulations at lower or zero risk. The data point toward the value of studying the genotypes of the extremely aged, specifically with reference to their lifetime mutation rates.
To exploit this data and use it to guide direct studies of cell kinetics and mutation rates in humans, we have undertaken the development of a unified field hypothesis. This effort is in collaboration with the mathematician Professor Stephan Morgenthaler ETH (Lausanne) in Switzerland.
The development of technology to directly measure environmental chemicals and genetic change in humans continues to be our strongest area of contribution. We now need to invest in studies of cell turnover (the process of cellular renewal in all tissues), errors in the programming of which we believe are important in carcinogenesis.
We have developed technology which allows us to measure chemical reaction products with proteins and DNA in human tissue (NIEHS Biomarkers Program). We have developed means to measure the point mutational spectra arising in the human mitochondrial genome (NIEHS Superfund Program, DOE Human Cell Mutagenesis Grant). We have studied chemicals entering humans from the environment: food in the Biomarkers Program, air in the Mutagenic Effects of Air-borne Toxicants Program, and water in the Superfund Program.
Yet we have never assumed that environmental chemicals constitute the primary causes of human genetic change. Two major programs, Endogenous Nitrite Carcinogens in Man (NCI) and Genetics and Toxicology (NIEHS), have focused specifically on either one powerful endogenous mutagen, NO, or the endogenous process of DNA replication to determine responsibility for our genetic change rate. We have cast a net to discover if the patterns of spontaneous mutations in bacterial or human cell studies are recapitulated in human tissues. Recent data suggests that for human mitrochondrial DNA, errors in DNA replication, not exogenous chemicals, are the primary causes of point mutation.
The Air Quality Program has two components. The first is directed at identifying airborne chemicals which are mutagens for human lung cells, and relating these to their sources and atmospheric transformation processes. The second is to assess the potential human damage associated with the emissions from a variety of thermal processes proposed for remediation of Superfund sites.
The Water Quality Program focuses on the behavior of toxic chemicals in the natural environment with particular emphasis on processes that lead to human exposure. The studies also work to determine the effectiveness of remediation technologies to attain high waste destruction efficiencies without the formation of mutagenic by-products.
Our Toxicology and Epidemiology Program focuses on discovering the causes of genetic change leading to human disease. We have developed technology which allows us to measure chemical reaction products with proteins and DNA in human tissue, and have also developed means to measure the point mutational spectra arising in the human mitochondrial genome. These twin technologies are now being used in parallel to try to discover the causes of mutations in people.
The Core Laboratory is a central resource in analytical chemistry for CEHS project participants, providing them with analytical expertise, training, and access to analytical instrumentation. A major goal of the CEHS is to foster collaborative research among combustion engineers, genetic toxicologists, analytical chemists, civil engineers and other investigators in order to solve important problems in human health effects research. The Core Lab is a centrally important resource in making this research collaboration come to fruition.
The chemical analysis protocol most important to our work is called bioassay-directed chemical analysis in which the analytical chemistry work is guided by feedback from human cell mutagenicity results. The bioassay work utilizes quantitative forward mutation assays based on a battery of metabolically-competent cell lines derived from diploid human B-lymphoblastoid cells.
Human tissues are being obtained and analyzed for mutational spectra in mitochondrial and nuclear genes. Tissues from specific organs are being analyzed for similarities and differences regarding mutations in order to determine a mechanism for mutation and disease on an organ by organ basis.
Human cell lines are being used to assess the mutagenicity of extracts and fractions of environmental samples provided by the Center investigators, as well as relevant pure compounds. A primary goal is a bioassay directed identification of the principal mutagens in environmental samples. Achieving this goal requires coordination with the individual investigator and the Core Laboratory in Analytical Chemistry.
The data obtained from the analysis of human tissues and compared to the principal mutagens found in environmental samples from human cell assays will be used to determine the significance of environmental mutagens in human disease.
Our study of air samples from major US cities has confirmed that they contain chemicals which are potent mutagens in human cell assays. We have identified the specific chemicals accounting for one third of this activity in the form of four polycyclic aromatic hydrocarbons. We should soon know the identities of most of the remaining mutagenic compounds which appear to be semipolar derivatives of the polycyclic aromatic hydrocarbons.
These mutagenic air pollutants are among the higher molecular weight, less volatile air pollutants. We find that these are disproportionately represented on the smallest air-borne particles which are most likely to reach and reside in the human lung.
Our effort to use electron microscopy to characterize particles from various sources has brought gratifying results. It appears that the approach should allow accurate identification of environmental sources of the particles found in human lungs.
In attempting to find a cause for the 1970-1986 Woburn, Massachusetts childhood leukemia cluster, we have used hydrogeologic methods to show that municipal wells would have drawn some 60% of their volume from the nearby Aberjona River. Sediment core analysis showed that at or about the time of the wells' use, substantial quantities of arsenic and chromium were suspended or dissolved in the river. To discover if these or other metals reached the users of the municipal water supply, we have collected 109 hair samples cut from children from 1938 to 1994. Analysis by neutron activation of unwashed samples has shown marked elevation of a wide variety of toxic metals for the community relative to other US studies. Testing metals in washed hair should now allow us to discern any relationship to the use of municipal water from contaminated wells.
One key observation arising from the use of neutron activation analysis was the discovery of a recent sharp increase in the lanthanide elements in sediments. These highly reactive elements are being used in a wide and growing number of manufacturing processes and automobile catalytic converters. Their properties as strong redox catalysts would seem to make them strong persistent irritants. We will continue to follow this lead in both the Air and Water Quality Programs.
We have at long last observed point mutations as they occur in human tissues and compared them to those arising in human cells in culture. Curiously, the mutations observed (two hotspots in a 100bp mitochondrial sequence) appear to be the same in human cell cultures, human colon and human muscle samples. This may mean that such mutations in humans arise from endogenous chemical reactions or DNA replication error. It may also be that the first sequence studied is not representative of other mitochondrial or nuclear sequences. Reconstruction experiments seem to exclude the possibility of analytical artifact, but at the mutation fractions of about 2 X 10-5 and 2 X 10-6, respectively, interpretive caution of these observations is justified.
Our ability to measure DNA adducts with the methionine 35S post labeling approach, coupled to HPLC, has increased measurement sensitivity as well as allowed for a separation and isolation procedure which integrates with mass spectrometry. This, with continuing advances in the technology to measure certain polycyclic aromatic hydrocarbons adducted to proteins, has greatly advanced our ability to measure the actual amounts of many environmental chemicals which react with the chromatin (DNA and histones) of all human organs. The combination of analytical chemistry and analytical genetics in studying human populations opens new vistas for students and faculty alike.
We may now imagine studies of humans in which both exogenous and endogenous causes of genetic change are considered and analyzed. For instance, the finding that mismatched DNA repair defects were important in the early appearance of colon cancer in certain families followed our earlier discovery that human cell mutants existed with high spontaneous mutation rates which were deficient in this repair pathway. These "mutator" human cells mutate on their own so fast that the low levels of environmental chemicals in the human body are unlikely to contribute to mutations arising in such individuals.
To give students a working understanding of environmental chemical problems, CEHS faculty several years ago created a series of four courses collectively called Chemicals in the Environment, CEHS faculty teach the first three: Sources and Controls (Sarofim), Fate and Transport (Hemond), and Toxicology (Thilly). Professor L. Susskind in the Department of Urban Planning teaches the fourth: Policy and Regulation. These courses continue to be a hit with students and we find both undergraduates and graduates enrolled. In 1991 we received the Sizer Award for Outstanding Contribution to Graduate Teaching at MIT for the series.
TOX 104J required of Civil Engineering IE students is now being revised to take advantage of the undergraduates' greater understanding of molecular biology resulting from the Institute biology requirement.
To promote broader public understanding of our methods and goals, CEHS has teamed up with the Massachusetts Corporation for Educational Telecommunications (MCET). MCET is a not-for-profit, distance-learning organization serving the needs of the K-12 community. It reaches an audience located in 47 states or approximately 1.3 million participants and every school district in Massachusetts. MCET has an annual budget of approximately $9 million funded from various U.S. federal agencies, state agencies and corporations.
On May 3rd we aired a highly successful pilot program, for a five part program series for middle school teachers and students that will increase understanding of how hypothesized connections between the environment and health are explored. The series will highlight the scientific processes used by researchers to prove or disprove hypothetical connections between the environment and disease. We are preparing a proposal to be submitted (along with the pilot video) to the National Institute of Environmental Health Sciences for full funding of the five-part series.
We have been studying the chemical sources, fate and transport, and human exposure in the Aberjona River Watershed here in Massachusetts for eight years. When we began this work we knew we had a special obligation to the people in the several towns included in our work to communicate our aims, our findings and our limitations in interpreting these findings in terms of public health. To do this we regularly hold public meetings to discuss our progress and try to answer questions. Faculty and students present their work and hear what the public thinks soon thereafter.
Of course, since we have found that a local Superfund Site is "leaking" fair quantities of chemicals like arsenic into the Aberjona, our presence is regarded as a problem to those who wish to inexpensively develop the site for municipal and commercial use. In order that our observations are available to all in a timely manner, we have agreed with Congressman Edward Markey's staff to meet some weeks before our public presentations to discuss technical progress with representations of the federal EPA, Massachusetts Department of Public Health, Massachusetts Department of Environmental Protection, the custodial and remedial trusts responsible for the particular Superfund Site, as well as elected public officials or their representatives. In our first meeting of this group an assertion was made that the public could not understand our work as we present it and that it was causing undue distress when reported in newspaper articles. My answer was, and is, that our interactions with the public have impressed all of us with their basic understanding, interest, and support for a policy of openness as expressed through our public meetings. Let any one attend one of these open forums and hear the intelligent and informed questions from the audience before positing that the public cannot comprehend the essential elements of our research tasks or fails to differentiate between the testing of hypotheses and findings of fact.
Tangible assistance from the community has taken many forms, including site access, contribution of sample material (including human hair), recollections of historical events not otherwise recorded in the literature, and the identification of potential sources of contamination (repositories of tannery waste, etc.). We believe such outreach is both a responsibility of scientists who work in a community, and a policy that is in the long-term best interests of the research itself.
As we look into the future we see a real need to provide our elected representatives with knowledge about public health and hazardous waste issues as well as our need for basic research support. We know that in a real sense "all pollution is local," but who among us has the means to go into a community and discover the primary causes of mutations leading to cancer, birth defects, or other diseases requiring genetic change? To this end, we have coordinated an endeavor involving 23 Superfund Research Centers and 24 NIEHS centers nationwide. Faculty from these research centers have made presentations to members of Congress or their appropriate aides both in their home districts and in Washington, D.C.
CEHS maintains a broad base of collaborations with researchers from other institutions including: Boston University, California Institute of Technology, Fred Hutchinson Cancer Research Center, Gentest Corp., Harvard School of Public Health, Ohio School of Medicine at Toledo, Tufts University, University of Rochester, University of Michigan. These researchers are directly involved with CEHS research projects and communicate with and share data on a frequent basis. In order to assist these collaborations the Center has actively promoted the use of information technology, including the following projects on the Web:
Dr. John S. Wisknok in collaboration with Hewlett Packard scientists adapted the operating system on the HP 5989B electrospray mass spectrometer to allow open-access operation. In this mode, the instrument can run continually and samples can be introduced and spectra obtained in less than five minutes by users with minimal training. They also have written custom software for: automatic calculation of molecular weights from oligonucleotide sequences and for calculating m/z values for multiple charge states; for controlling Harvard syringe pumps from the HP data system; and for data-dependent automated mode-switching on the TSQ 7000. These programs are available as freeware on the World Wide Web site (http://web.mit.edu/toxms/www/index.htm) that we recently constructed for the mass spectrometry laboratory.
A database has been established to catalogue tissues which have been collected for studies in the Center for Environmental Health Sciences. The database contains information such as: type of tissue, sex, ethnicity, age, pathology, distribution of tissues among CEHS researchers, experimental protocols, and results of experiments. The database has been placed on the World Wide Web for easy access at http://web.mit.edu/cehs/ftp/. The database downloads as a binhex Filemaker Pro file. This file, which is updated on a regular basis, requires a password to open which is available from Dr. John Hanekamp at (617) 253-8096 or hanekamp@mit.edu.
Plans are underway for a web site for all the labs worldwide working on problems involving mutational spectra and related technology. This includes point spectra such as we study and spectra of higher level events in the chromosomal hierarchy. It includes those who would attempt to see the set of human genetic polymorphisms by combining many separate human blood samples. This site would include a chat area to allow questions to be asked and answered interactively.
The CEHS supports the affirmative action goals of the Massachusetts Institute of Technology while maintaining our commitment to hiring solely on qualification of the candidate for the position. This year two of our feasibility studies by female junior faculty members went on to receive direct support as projects in our grants. The majority of our staff, including research specialists, students, and support staff, are women.
The Center has continued to actively recruit graduate students from minority institutions. We, with NIEHS, support undergraduates in summer research internships which have yielded multiple top-notch recruits. In past years we hosted more than a dozen potential applicants from Cheyney College near Philadelphia; one of their number then served a summer internship and has entered our graduate program in toxicology. Our Director, Bill Thilly, along with Dean Ike Colbert, co-authored a grant request to the Sloan Foundation which now provides funds for entering minority graduate students to take the time to fill in their academic backgrounds if necessary before beginning the graduate curriculum.
William G. Thilly