Orphan Receptors
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Investigating orphan receptors. Kenneth Korach (left) and Cary Weinberger. |
A number of chemicals found in the environment exert their effects by interacting with specific receptor molecules. Receptors are cellular proteins with specific and high affinity binding for effector molecules (ligands). This binding results in a functional response and change. Nuclear hormone receptors recognize steroid hormones (estrogen, progesterone, androgen, etc.), thyroid hormones, vitamins, and retinoic acid. The hormone-receptor complex alters expression of key target genes and leads to functional alterations in cells, tissues, and organs. Understanding receptor binding and mechanism of action is key to interpreting signals from our internal as well as the external environment.
For example, a number of foreign chemicals, such as DDT and polycyclic chlorinated biphenyls, have been reported to interact with the estrogen receptor, eliciting many of the same cellular and physiological effects of the natural female sex hormone estradiol. Although the hormonal activity of these xenobiotic estrogens has been clearly demonstrated, their effects on human health is less clear. Chemicals need to be screened for their potential to interact with nuclear hormone receptors, which in turn may help explain their toxic actions.
Research in the area of receptor-mediated mechanisms is expanding at NIEHS. Cary Weinberger has joined Kenneth Korach's research section at NIEHS to supervise research on orphan receptors. Weinberger will also interact with existing groups at NIEHS that have been studying other receptor systems. These areas are headed by Korach, studying estrogen receptors and estrogen hormone action; Anton Jetten, investigating retinoids and retinoic acid receptors; and George Lucier, studying TCDD and dioxin receptors.
Weinberger comes to NIEHS from a Ligand Pharmaceuticals in San Diego, California. Prior to his work at Ligand Pharmaceuticals, he spent a year at NIH and did postdoctoral training at the Salk Institute in Ronald Evans's laboratory. During that time, Weinberger's studies were highlighted by the initial cloning and expression of the human glucocorticoid receptor gene, description of the relationship of the human GR to the V-erb-A oncogene product, and the identification and expression of novel thyroid receptors.
Identification and characterization of the nuclear hormone receptor family of proteins began several years ago with the initial cloning of the estrogen and glucocorticoid hormone receptors. Since that time, other members of this family were identified for which there are no known ligands and thus were called "orphan receptors." The role of orphan receptors in environmental disease is a promising area. Further characterization of orphan receptors and their endogenous ligands will provide a crucial link to understanding the molecular mechanisms through which exogenous chemicals exert toxic effects and through which natural substances influence physiologic processes.
Low-stringency hybridization has ad-ded about 25 orphan receptors to the list of known nuclear receptors. Molecular techniques that enable researchers to switch functional domains of the proteins offers a conspicuous means for identifying the ligands binding to each orphan receptor. Construction of hybrid receptors, for example, containing the glucocorticoid-receptor DNA binding domain linked to an orphan receptor's putative ligand binding domain identified the first retinoic acid receptor (RAR) gene. Cary Weinberger of NIEHS plans to use this approach to investigate specific classes of environmental chemicals.
Identification of the orphan receptor ligands is falling far behind receptor gene isolations and has directly challenged the resourcefulness of researchers. Determin-ing an orphan receptor's ligand dependency remains the rate-limiting step for further characterizing functional aspects of receptors. However, rapid tests of re-sponse-element binding offer some insights into the assembly of transactivation assays. Some of the newest members of the or-phan receptor class underscore the excitement and perplexities of the initial discovery of receptor ligand-dependency. Two new subfamilies include four members of the peroxisome proliferator-activated receptors (PPAR) as well as three of the so-called retinoid X receptor (RXR) kindred.
PPAR was initially characterized as being activated by classes of xenobiotics including plasticizers and lipid-lowering drugs. At least two of the four-member PPAR subfamily can be activated by clofibric acid, nafenopin, and a Wyeth-Ayerest compound called 14,643 with potencies in the supramicromolar range. These agents induce acyl coA oxidase, which is responsible for fatty acid ß-oxidation, but their qualifications to lower triglyceride and cholesterol levels are complicated by their possible hepatocarcinogenicity. Endogenously circulating unsaturated fatty acids including oleic and linoleic acids were also found to activate PPAR, while another study has shown that the synthetic arachidonate 5,8,11,14-eicosatetraynoic acid is about 50 times more potent for PPAR than the Wyeth compound. Identification of exogenous and endogenous ligand activators will require researchers to plan and bridge molecular techniques and physiological research approaches for success.
NIH has recently launched a new initiative to investigate bionutrition. Wein-berger has a strong interest in studying the mechanistic action of nutritional agents such as vitamins. Successful correlations of RAR and RXR with the vitamin A metabolites all-trans-retinoic acid and 9-cis-retinoic acid have redirected thinking about molecules such as vitamin E, whose functions have been attributed solely to its antioxidant properties. The antioxidant properties of vitamin E or -tocopherol are especially interesting considering that this molecule is a terpene structurally similar to vitamin A.
One of the classical features of vitamin E deficiency is the inability of rodents to maintain pregnancies. Vitamin E-deficient rodents typically spontaneously resorb 15-day-old fetuses due to suppressed development of mesodermal tissues including the blood islands of the yolk sac and embryonic liver. Other cardinal deficiency signs include inductions of catabolic lysosomal enzymes that produce a muscle wasting from cellular protein and nucleic acid breakdown. These physiological changes may result from the vitamin's actions as a receptor co-activator, perhaps operating like RXR only in the presence of other trans-acting factors to promote gene transcription. The presence of vitamin E-binding polypeptides in liver cytoplasm has been established, although the biochemical evidence was relatively weak. These poly-peptides may be more akin to the cellular retinol-binding proteins, which are thought to function more in a transport role for vitamin A. If vitamin E operates via an identified or unrecognized orphan receptor species, it most likely will require cell culture system for analysis. Such observations, considered with the multiple roles of vitamin A metabolites, may outline future studies of the role of vitamin E and other nutritional factors and environmental chemicals as receptor-transducing signals.
National Worker Training Clearinghouse
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Staff of the National Clearinghouse for Worker Safety and Health Training surround a sculpture of labor leader George Meany (left to right): Katherine Roberts, Jeffrey MacDonald, Betsy Lewis, Joyce Reimherr. |
The NIEHS Superfund Worker Training Program has awarded a new two-year contract to the George Meany Center for Labor Studies in Silver Spring, Maryland, to operate the National Clearinghouse for Worker Safety and Health Training for Hazardous Materials, Waste Operations and Emergency Response. The clearinghouse will support the nationwide NIEHS training program, facilitating the transmission of technical information and curricula developed for safety and health training programs for hazardous waste and emergency personnel.
NIEHS was given major responsibility for initiating a training grants program under the Superfund Amendments and Reauthorization Act of 1986 (SARA). The major objective of this program is to fund nonprofit organizations in developing and delivering training to workers who handle hazardous wastes or who respond to accidental releases of hazardous materials.
Although NIEHS has developed a solid national training program for workers in high-risk occupations, the safety and health problems at toxic waste cleanup sites have substantially increased in extent and severity. After initial delays in beginning remediation at waste sites, the EPA Superfund program has been supplemented with even larger environmental restoration programs by the Departments of Energy and Defense.
In addition to creating a resource library that holds all the curricula created by the NIEHS training program, the clearinghouse at the George Meany Center publishes a monthly news brief and activity report including information about hazardous materials, hazardous wastes and emergency response, and regulatory pro-gress. The clearinghouse also arranges and manages technical workshops related to scientific, administrative, and regulatory issues associated with training for hazardous waste workers and emergency responders.
The George Meany Center for Labor Studies is a residential adult-learning center that provides leadership and technical education for the members, staff, and officers of national and international unions affiliated with the AFL-CIO. The Meany Center is also home to the Railway Work-ers Hazardous Materials Training Pro-gram, which is one of the eighteen cooperative agreement awardees that is supported through the NIEHS Worker Training Program. Further information on the clearinghouse or the NIEHS Worker Training Program can be obtained by calling (301) 431-5425.
Carcinogenesis and Diet Restriction
Rodents are the most commonly used animal models for chronic toxicity and carcinogenicity studies. The National Tox-icology Program has typically used two-year studies with both sexes of rats and mice to evaluate the carcinogenic potential of chemicals.
In recent years, the survival of many strains of rats at the end of two-year studies has been less than 40%. This decreased survival is a serious concern to research-ers involved in evaluating the safety and carcinogenic potential of drugs, food additives, pesticides, and other chemicals.
Diet is one of the most important environmental factors that influence survival. Diet restriction of greater than 30% for rats not subjected to chemical treatment lowers body weight, lowers the incidences of body weight-associated tumors, and increases survival at the end of two-year studies. Diet restriction markedly decreases incidences of tumors induced by chemicals and makes the animal irritable and aggressive. Furthermore, chemicals influence energy utilization, and diet restriction may disproportionately influence the physiological processes of animals in chemical treatment groups, thus complicating the interpretation of studies. The NTP/NIEHS is conducting studies with diet modification and diet restriction in combination with chemical treatment. The objectives of these studies are to assess 1) the influence of body weight on the carcinogenic response by comparing an ad libitum fed control group and a diet restriction control group with a high-dose treatment group, 2) the influence of moderate (20%) diet restriction in control and treatment groups on the sensitivity of chemical response, and 3) the influence of diet modification on body weight, chronic diseases, tumor rates, and life span.
The problems associated with increasing body weights and decreasing survival of rats must be resolved in order for long-term carcinogenicity studies to be reliable. Rodent diets did not change substantially during the last 15 years. Researchers want large rodents at low cost, which may be the main reason for selecting breeding colony animals that reproduce early and grow faster thus increasing the body weight of breeding colony animals year after year. If not controlled at the breeding colony, the current selection process of breeders would still lead to higher body weights with higher tumor rates and a shorter life span. Diet restriction to lower body weight may be a treatment of the symptom rather than the cause. Rec-ommendations for permanently resolving the problem include 1) selecting slower-growing breeders in the production col-onies to lower the body weight of the progeny, 2) modifying diets so they are adequate for growth and maintenance but do not contain excesses of growth-enhancing nutrients, and 3) modifying the feeding behavior of rodents by husbandry procedures such as group caging and making food available only during the normal feeding period (night).
Biodegradation As a Remedy
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Steering Committee for the biodegradation conference (back row, left to right): William Suk, James Hunt, Steven Aust, James Tiedje, Robert Arnold, (front row) Martin Alexander, Lily Young, Daniel Abramowicz, Jodi Shann. |
Biological treatment of hazardous wastes may be an effective, practical, and economical way to remediate Superfund sites and other hazardous wastes. Scientists from across the United States and from several other nations discussed the principles of microbial detoxification and transformation of environmental contaminants at the conference "Biodegradation: Its Role in Reducing Toxicity and Exposure to En-vironmental Contaminants," held April 26-28 and hosted by the NIEHS Sup-erfund Basic Research Program. The purpose of the conference was to examine the current state of knowledge, the research needs in biodegradation and treatment, and the role of these processes in reducing the toxicity of and exposure to environmental contaminants. More than 300 registrants participated in the three-day meeting, which included presentations by invited speakers and about 60 posters by representatives from industry, academia, and government. The meeting was divided into three broad topic areas: toxicity reduction, exposure reduction, and microbial ecology. Each area was discussed from a multidisciplinary perspective under the broader context of basic research in bio-degradation.
The session on toxicity reduction included presentations on the genetics of novel biodegradation pathways, the biochemistry and genetics of polychlorinated biphenyl (PCB) metabolism, and degradation by fungal peroxidases. In the session on exposure reduction, presenters discussed mechanisms of treatment by white rot fungus, soil treatment laboratory and field studies, and PCB cleanup. The session on microbial ecology focused on selecting chemical-specific degrading bacteria, enhancing degradation capabilities through molecular biological techniques, and tracking microbial populations in the effective reduction of exposure.
In opening remarks, the Chair of the Steering Committee, Lily Young of New York University and Rutgers University, stated that disease prevention and reduction of risk and exposure are fundamentally affected by the degradation and transformations of toxic chemicals mediated by microbial communities both in natural environments and constructed treatment systems. William Suk, director, of the NIEHS Superfund Basic Research Pro-gram, and a member of the steering committee, pointed out that cleanup of contaminated soils, sediments, and groundwaters not only improves the environment, but it is also a means by which human exposure and health risks can be reduced. The conference ended with closing re-marks by Martin Alexander of Cornell University. Alexander addressed the questions yet unanswered about the biodegradation activity of microbes and their interactions with the physical/chemical environment.
New Worker Training Initiative
The U.S. Department of Energy and NIEHS have signed an interagency agreement to develop model worker safety and health training programs for workers involved in waste cleanup at facilities in the nuclear weapons complex. The Con-gress established the worker training program in the National Defense Authoriza-tion Act for fiscal years 1992 and 1993. This authorization appropriates $10 million for worker training. Preference is to be given to current awardees of the NIEHS Worker Education and Training Program.
In the fiscal year 1991 National De-fense Authorization Act, Congress requested that DOE "evaluate the suitability of the training program developed by the Na-tional Institute of Environmental Health Sciences (NIEHS) pursuant to Section 126 of SARA [Superfund Amendments and Reauthorization Act] for training workers involved in environmental restoration and waste management activities of the department." The suitability study found that, in general, the technical quality of the hazardous waste worker health and safety training prepared under the NIEHS program would meet DOE facility needs. The program is cost-effective and provides specialized training to meet the needs of specific segments of the DOE workforce.
The cleanup of the nation's nuclear weapons complex will be the largest and most costly environmental remediation effort ever undertaken. The DOE currently estimates that cleanup at the 14 primary weapons complex sites in 13 states will take more than 30 years, cost more than $100 billion, and employ thousands of scientists, engineers, technicians, and construction workers.
A recent congressional report by the Office of Technology Assessment states that current DOE efforts to protect workers involved in hazardous waste cleanup are "hindered by a lack of emphasis on occupational safety and health issues in Sup-erfund and RCRA procedures." The Office of Technology Assessment found three structural flaws in DOE's worker protection: lack of management commitment to occupational safety and health priorities, insufficient professionally trained staff, and lack of independent oversight and enforcement of DOE safety and health policies.
Applications for supplemental awards to support training programs for nuclear weapons site cleanup workers were submitted to NIEHS during March 1993 and will be evaluated through the regular NIH peer review process. New awards for the program will be made on 1 July 1993, and training will begin at waste sites across the country during September.
DeGray Awarded Walter J. Johnson Prize
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Janice DeGray, co-recipient of the 1993 Walter J. Johnson Prize. |
Janice DeGray, a postdoctoral fellow at NIEHS, has been named the co-recipient of the Walter J. Johnson Prize by the editorial board of the Archives of Biochem-istry and Biophysics. The prize includes an expenses-paid trip to the American Society of Biochemistry and Molecular Biology/American Chemical Society Joint Meeting in San Diego, California, in June to receive the prize and a $5000 cash award.
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G. N. Rao |
The prize is presented by the editorial board every three years to recognize scientists within five years of completion of their doctorates who publish the scientific papers of greatest distinction. De-Gray's paper, "Re-duction of Paraquat and Related Bipyrid-ylium Compounds to Free Radical Me-tabolites by Rat Hepatocytes," was co-authored with NIEHS colleagues D.N. Ramakrishna Rao and Ronald P. Mason. DeGray is in the Free Radical Metabolites Workgroup of the NIEHS Laboratory of Molecular Biophysics.
DeGray's study demonstrates that paraquat and related herbicides are metabolized to free radicals by liver cells. These free radicals are ultimately responsible for the many deaths resulting from accidental poisonings by these chemicals.
Last Update: September 11, 1998