Prime Suspect
Scientists call them the COXs--a pair of closely-related cyclooxygenase enzymes that produce prostaglandins, a class of relatively short-lived hormones that mediate many cellular and physiologic processes. COX-1 is said to be responsible for cellular "housekeeping" functions. COX-2 is necessary for the inflammatory process, but can easily go astray. COX-2 has been observed at the scene--and is the prime suspect--of many a cellular "crime." But the exact role of both enzymes is uncertain. Pharmaceutical companies have developed drugs to contain COX-2 at the first sign of trouble. But scientists at the NIEHS would like to know for sure whether one or both enzymes are guilty of causing damage. And they believe the best way to determine that is by alternately removing each enzyme from experimental animals and observing the effects.
NSAIDs and COXs
Prostaglandins have been the subject of considerable interest in the scientific community because of their role in inflammation. Pain and swelling occur in tissues when cells release prostaglandins in response to physical damage or infection, increasing the permeability of the capillary walls of blood vessels and attracting immune system cells to the damaged tissues. With illnesses such as rheumatoid arthritis, however, joints can become chronically inflamed when prostaglandins are released in the absence of any obvious infection or physical damage.
The recent development of synthetic nonsteroidal antiinflammatory drugs (NSAIDs), such as ibuprofen, represented a breakthrough in the treatment of arthritis and other illnesses involving inflammation. NSAIDs are believed to bring relief by suppressing the COX isoforms that produce pain-inducing prostaglandins. However, the suppression of the COXs also negates the enzymes' beneficial role of protecting the gastric mucosa in the digestive system. The result has been that patients taking high dosages of NSAIDs also suffer a greatly increased incidence of stomach ulcers.
For many years, it was believed that there was only one COX enzyme. However, in 1992, researchers discovered that corticosteroids, which are potent inhibitors of inflammation, blocked the expression of a second enzyme with COX-like activity, subsequently named COX-2. COX-1 is expressed in most tissues and mediates functions including gastric cytoprotection, vascular homeostasis, and normal renal maintenance. COX-2 is normally undetectable in tissues, but can be induced at high levels in macrophages and other cell types in response to proinflammatory stimuli.
The presumed function of COX-2 has sent academic and industry researchers in hot pursuit of a drug that targets only COX-2, while leaving COX-1 to maintain normal homeostatic functions. At stake is a $4 billion business that the pharmaceutical industry has built around the NSAID-COX connection.
COXs and Cancer
While they acknowledge the beneficial effects of NSAIDs, NIEHS researchers want to better understand the biological function of the COXs in disease states. One or both of the enzymes is suspected of playing a key role in colon cancer, skin cancer, certain kidney dysfunctions, and failures in immune cell and reproductive function, among other ailments. The NIEHS has assembled a team of scientists, the Carcinogen Metabolism and Molecular Mechanisms Group, headed by Robert Langenbach, to conduct research aimed at clarifying the functions of the COX enzymes.
(left to right)
Pat Chulada, Howard Tiano, Robert Langenbach, Christopher Lee and Burhan Ghanayem.
(Not pictured)
Barbara Davis, Charles Loftin.
Photo Credit: Steve McCaw, Image Associates
Langenbach's interest in the COXs began in the early 1980s with his belief that the one isoform known at the time might be involved in chemically induced bladder cancers, although he hasn't pursued that particular connection. In 1991, however, Langenbach took a sabbatical at the University of North Carolina at Chapel Hill's Department of Pathology, where he learned a technique that would allow him to pursue research on COXs. At UNC, Langenbach worked with researcher Oliver Smithies, who pioneered the revolutionary technique of "knocking out" specific genes by genetically modifying the DNA of a target gene so that it no longer functions, putting the DNA into a stem cell culture, and inserting the modified stem cells into a developing mice blast. Resulting offspring that carry one copy of the nonfunctioning gene are then bred to produce a strain of "knockout" mice that have no functioning copy of the gene. "The beauty of this technique is that we can have absolute inhibition of a particular gene," says Langenbach. "We can then observe changes in the animals' physiology under normal and stressed conditions, and gain a clearer picture of the gene's function."
Langenbach conducted knockout studies of the COX-1 gene, while Scott Morham, a postdoctoral fellow in the Smithies lab, subsequently took on the disruption of the COX-2 gene. The two groups continue to collaborate and have expanded the effort to include investigators nationwide. At the NIEHS, Langenbach's team is breeding colonies of both COX-1 and COX-2 deficient mice for use in a variety of research projects. Biologist Christopher Lee is responsible for maintaining the proper breeding of animals and using molecular biology techniques to identify the COX-deficient mice to ensure that adequate numbers of specific genotypes are available for all studies.
"The first thing we did was to determine the physiological effects of deleting these genes," says Langenbach. "Preliminary findings showed high incidences of renal failure, altered inflammatory responses, and fertility difficulties in one or both of the knockouts. These discoveries have opened the door to a host of projects looking at the role of the COXs in a variety of diseases."
Already, certain findings have overturned widely held assumptions about the role of the COXs and NSAIDs. Toxicologist Pat Chulada is leading an effort to define the specific roles of COX-1 and COX-2 in various phases of inflammation. Chulada's approach involves inserting an air pouch beneath the skin of test mice and inflating it over several days, then injecting an irritant into the air pouch to induce an inflammatory response that mimics that of an arthritic joint. "The hypothesis was that COX-2 deficient mice would not be able to mount an inflammatory response," Chulada says. "However, our initial studies show that both COX-1 and COX-2 deficient mice do mount inflammatory responses, although these differ from that of wild-type mice." Chaluda is also examining how the inflammatory process varies when subjected to various carcinogenic and noncarcinogenic stimuli.
Toxicologist Burhan Ghanayem and Chulada also found that, contrary to expectations, COX-1 deficient mice did not show an increase in stomach ulcers. In fact, the COX-1 deficient mice were actually more resistant to NSAID-induced stomach ulcers than the wild type. These results indicate that NSAIDs may reduce inflammation through mechanisms other than or in addition to the suppression of COXs. Further, they bring into question the assumption that a COX-2 suppressing drug would be a more effective antiinflammatory agent with less toxic side effects than current NSAIDs. Critical findings could come when the team compares the health of COX knockouts with NSAID-treated, wild-type mice.
Despite the questions they raise concerning how NSAIDs work, Langenbach sees the research as a win-win situation. "It gives us a model to ask whether NSAIDs have effects in addition to inhibiting the COXs," he says. "Either they work by inhibiting these enzymes, or they work by alternative mechanisms. In either case, it narrows the field in which we have to look for answers."
Chulada is also investigating the role of the COXs in colon cancer development. Recent studies indicate that regular use of NSAIDs not only reduces inflammation, but also decreases the incidence of colon cancer in humans by 40-50%. Scientists have suspected that NSAIDs reduce colon cancer by inhibiting cyclooxygenase and decreasing the production of prostaglandins, but the exact mechanism is unclear. Because NSAIDs decrease the incidence of colon cancer in rodents by as much as 90%, Chulada is cross-breeding COX-1 and COX-2 knockout mice that carry a genetic mutation that predisposes them to multiple intestinal adenomas and monitoring the resulting hybrid mice for onset of cancer and rates of polyp formation in the colon. These mice will also be used to study the genetic and biochemical changes that occur as they develop cancer. "These studies will allow us to determine if, in fact, NSAIDs exert their anticancer effects by inhibiting COX-1 and COX-2 or by altering other biochemical processes," Chulada says. "More importantly, they will help us to identify the mechanisms by which each COX isozyme contributes to colon carcinogenesis."
Molecular biologist Howard Tiano is heading a project to examine the effect of COX-1 and COX-2 deficiency in the development of skin cancer in mice. The studies being conducted
will allow Tiano and other researchers to study the role of the COXs in both the initiation and promotion stages of skin cancer. In the skin model, as well as Chulada's colon model, NSAIDs are being fed to wild-type and COX-1 and COX-2 deficient mice to determine what biochemical functions other than COX suppression are involved in the carcinogenesis process. "Looking at the role of COXs in two different cancers may give us an indication of their possible involvement in a whole range of cancers," says Langenbach.
Selective inhibition. Prostaglandins, thought to be produced by COX-2 during inflammation, are evident (brown pigment) in the macrophages and fibroblasts of air pouch tissue of wild type (WT) and COX-1 knockout mice treated with an inflammatory agent. In tissues of treated COX-2 knockout mice, no protein and minimal prostagladins are produced.
Future Developments
Reproductive biologist Barbara Davis is examining how the COXs affect reproductive capabilities. COX-1 is known to have a major role in the onset of labor, and COX-2 is essential for ovulation. Davis is heading a two-pronged study to determine the effect of genetic deletion of COX genes on the reproductive capabilities of mice and the effect of NSAID consumption on these processes. The research has important implications for whether COX-2 specific inhibitors may have unwanted side effects and for whether pregnant women nearing time of delivery should be taking NSAIDs.
In addition to other studies, postdoctoral fellow Charles Loftin is attempting to breed mice that are deficient in both COX-1 and COX-2. Such doubly deficient mice, when compared to COX-1 and COX-2 deficient mice, would provide useful models for determining the necessity of prostaglandins for basic biological processes.
Langenbach anticipates that the NIEHS research effort involving the COXs will continue for several years, evolving in focus and level of understanding as questions are answered and new ones posed. "The COXs are involved in such crucial biological functions that one line of research invariably informs another," he says. "As we uncover the basic biological functions of COXs in normal and stressed states, their role in specific illnesses should become clear. And that should allow scientists to design drugs that more accurately prevent and treat inflammatory diseases."
John Manuel
NIEHS in Rapid Pursuit of EMF Effects
In the last 20 years, questions have arisen about the potential health hazards of electric and magnetic fields (EMFs), which are created by power lines and most electric equipment including electric blankets, waterbeds, clocks, shavers, televisions, and computers. Because these fields are less intense than the earth's static electric or magnetic fields, it has been thought that they are unlikely to be hazardous to human health. However, public concern was raised by a 1979 study that reported a 2- to 3-fold increase in leukemia risk for children living in homes near electric power lines with configurations that suggested elevated magnetic field exposure. Estimates of exposure were based on wire configuration coding using criteria such as a home's distance from a power line and the size of wires close by. Several followup studies confirmed the link between wire configuration coding and childhood leukemia risk, although its interpretation has been confused by the lack of an association between an actual measurement of the magnetic fields and the risk of the disease.
Scientists and others have continued to debate the potential for adverse health effects from low frequency EMF exposure typical of that from power lines and home appliances. During the last two decades, numerous research studies have been conducted by various scientific disciplines in an effort to learn if and how EMF exposure might affect biological systems. The National Academy of Sciences (NAS) released an initial compilation and analysis of these studies on 31 October 1996 entitled Possible Health Effects of Exposure to Residential Electric and Magnetic Fields. In an extensive review and evaluation of over 500 studies spanning more than 17 years, an expert committee appointed by the NAS's National Research Council concluded that the current literature "does not show conclusive and consistent evidence that exposures to residential electric and magnetic fields produce cancer, adverse neurobehavioral effects, or reproductive and developmental effects." The report does note a need for further research in several areas, including childhood leukemia and EMFs, and outlines a research strategy. In defining this strategy, the report acknowledges the Electric and Magnetic Fields Research and Public Information Dissemination (EMFRAPID) Program. The EMFRAPID Program is a five-year, federally coordinated effort that was mandated by Congress in the 1992 Energy Policy Act (Section 2118) and is funded by both federal and nonfederal funds (at least 50% of total funding). The program is in its fourth year of funding.
According to Gary Boorman, EMFRAPID Program director at the NIEHS, "The NAS report is a critical document that reviews the literature on EMFs primarily through 1994 but includes almost no research from the EMFRAPID Program. The EMFRAPID Program will also prepare a report to Congress that will start where the NAS report left off and focus on the vast amount of new research that has been reported in the past few years, much of which incorporates quality assurance aspects not previously required. It is also worth noting that much of the research called for in the NAS report is being carried out under the EMFRAPID Program."
The goal of the EMFRAPID Program is to evaluate developing technologies and research on the effects of exposure to 60-Hz EMFs produced by the generation, transmission, and use of electric energy on biological systems and to communicate these results to the public sector. The driving force behind the EMFRAPID Program, as stated in the Energy Policy Act, was the "sense of the Congress that remedial action taken by the government on electric and magnetic fields, if and as necessary, should be based on, and consistent with, scientifically valid research." Under this mandate, the director of the NIEHS must report to Congress on whether exposure to EMFs poses a risk to human health, and if so, the significance of the risk. This report is to be an objective scientific judgment based on a synthesis of the available knowledge on both EMFs and biological systems that may be perturbed by these fields during human, animal, in vitro, and theoretical studies.
The Department of Energy (DOE) and the NIEHS are partners in coordinating the EMFRAPID Program. The DOE is responsible for overall administration of the program and for conducting engineering-related research. The NIEHS is responsible for implementation of a research program on possible human health effects of EMFs. The agencies share the responsibility for communicating research findings and scientific knowledge to policymakers and the public. Since the EMFRAPID Program's inception, extramural and intramural research programs, four regional exposure facilities, and an EMF communication program have been established.
Two groups established under the Energy Policy Act assist the DOE and the NIEHS in managing, directing, and reporting the scientific portion of the EMFRAPID program. The Interagency Committee on Electric and Magnetic Fields is composed of representatives from 10 federal agencies with responsibilities related to EMFs (the DOE, the NIEHS, the EPA, the Department of Defense, the Occupational Safety and Health Administration, the National Institute of Standards and Technology, the Department of Transportation, the Rural Utilities Service, the Department of Labor, and the Federal Energy Regulatory Commission). This committee is involved in development of the EMF research agenda, the communication plan, and the guidelines for coordinating federal research activities.
The National Electric and Magnetic Field Advisory Committee consists of representatives from public interest groups, organized labor, state governments, academia, and industry. This group advises the DOE and the NIEHS on the design and implementation of the EMFRAPID Program and makes recommendations to the interagency committee.
The NIEHS amd the DOE both administer research programs under the EMF RAPID Program. The DOE oversees engineering projects which focus on research, development, and demonstration of technologies to improve the measurement and characterization of EMFs and on techniques for assessing and managing exposure to these fields. The NIEHS manages a multidisciplinary research program for assessing the health effects related to EMF exposure. This program supports applied and basic biomedical studies as well as projects in engineering and the physical sciences. Issues such as leukemia, brain and breast cancers, neurodegenerative diseases, and reproductive dysfunction are being addressed by NIEHS-supported scientists.
As part of the Energy Policy Act, the NIEHS and the DOE established four regional in vitro 60-Hz magnetic field exposure facilities for use by extramural and intramural scientists. These facilities are located at the Pacific Northwest Laboratories in Richland, Washington; Oak Ridge National Laboratory in Oak Ridge, Tennessee; the National Institute of Occupational Safety and Health in Cincinnati, Ohio; and the Food and Drug Administration Center for Devices and Radiological Health in Rockville, Maryland.
NIEHS, with partial support from EMFRAPID funds has established an EMFRAPID Website. The EMFRAPID program also provides support for an EMF Infoline (1-800-363-2383) which is managed by the EPA. There are other EMF information sources not supported by RAPID funds. NIOSH maintains a technical information service (1-800-356-4674), and the NIEHS provides funds for the Environmental Health Clearinghouse (1-800-NIEHS94 or 1-800-643-4794), which provides information on a variety of envronmental health topics including EMF. Also in cooperation with the DOE and the National Institute for Occupational Health and Safety, two question-and-answer booklets have been published, and both the DOE and the NIEHS have participated in a series of presentations at stakeholders' meetings.
The NIEHS strategy for development of its congressionally mandated report includes a series of three open, public science review symposia and a working group meeting for discussion and critical evaluation of the strength of the scientific evidence concerning human health effects associated with EMF exposure. These meetings will serve as forums for in-depth discussions and evaluations of research studies relating to the interaction of EMFs with biological endpoints and will place special emphasis on the quality of the research studies, the reproducibility of the experimental findings, and the extent to which the scientific evidence supports causal links between EMFs and biological effects. Each symposium will target a different class of studies: theoretical and in vitro findings, epidemiological findings, and in vivo and clinical laboratory findings. Published studies on EMFs, as well as novel data from ongoing research projects sponsored under the EMFRAPID Program, will be available to symposium participants. "The EMF hazard evaluation process we have developed at the NIEHS is intended to open new ground for understanding and acting upon risks of generally high public concern with potentially high economic impact. The process is intended to provide not only competent, open review of the available scientific evidence, but also to serve as a forum for extensive communication of the strengths and weaknesses of this evidence," said Christopher J. Portier, chief of the Laboratory of Computational Biology and Risk Analysis and chairperson of the Risk Assessment Research Committee at the NIEHS. A written report of each meeting will be made available to the public by the NIEHS and public comment on each report will be solicited. The first of these meetings will be held 24-27 March 1997 in Durham, North Carolina.
Following the symposia, a working group meeting will be held comprising a select group of principal authors with specific scientific expertise in EMFs and other fields who will draft a report on the strength of the EMF research data and its implications for human health and disease etiology. This working group will review the draft reports from the science review symposia and conduct an independent and critical evaluation of the literature. The group will submit its report to the NIEHS director. In preparation of the director's report, the NIEHS will solicit comments from the public, advisory groups, and the scientific community on the validity of the conclusions of the various meetings.
Additional information on the EMFRAPID Program is available at the EMFRAPID World Wide Web site located at
www.niehs.nih.gov/emfrapid/home.htm
, or requests can be sent by e-mail to
emf@pbpk.niehs.nih.gov
, or by mail to LCBRA, NIEHS, PO Box 12233, MD A3-06, Research Triangle Park, NC 27709, USA.
Last Update: March 19, 1997