Science from the Sea
The cold Atlantic waters off Maine's Mount Desert Island serve as a home to a variety of marine creatures that provide unique models for scientists seeking to understand life at its cellular level.
For nearly a century, tissues such as the rectal gland of the dogfish shark, the flounder intestine, or the skate's liver and kidney have drawn researchers to the Mount Desert Island Biological Laboratory (MDIBL), which has earned a worldwide reputation in the study of cell membrane transport--the passage of substances and signals into and out of the cell. Understanding these processes holds the key to understanding cell function. Understanding how toxins disrupt these transport systems has major implications for environmental health.
A site by the sea. Founded in 1898, the Mount Desert Island Biological Laboratory overlooks Salsbury Cove.
Photo: MDIBL
Founded in 1898 as an independent teaching laboratory by J.S. Kingsley of Tufts University, the MDIBL and its supply of cold-water species soon attracted distinguished scientists from prominent universities and institutions for summer investigations. This tradition of affiliated, rather than full-time, researchers continues as a hallmark of both the lab and its center.
For the last decade, the MDIBL has served as home to the Center for Membrane Toxicity Studies, one of five NIEHS-funded Marine and Freshwater Biomedical Sciences Centers. Its scientists focus on the basic and very relevant question of how toxic compounds, especially heavy metals, damage and kill cells. Their efforts have yielded some invaluable insights.
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James L. Boyer
Photo: MDIBL |
"One of our major contributions is in understanding how toxic substances interfere with cell-volume regulation," says center director James L. Boyer, chief of digestive diseases at Yale University School of Medicine. "Another area is how these heavy metals interfere with kidney function (salt secretion). A third is in understanding the mechanisms by which toxic substances disrupt hormone and neurotransmitter signaling processes."
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David H. Evans and student researcher
Photo: MDIBL |
Membrane toxicology seemed a logical extension of the MDIBL's work in 1984 when its director at the time, David H. Evans, a professor of zoology at the University of Florida, sought funding from the NIEHS. Sea creatures, especially those from frigid environs, make excellent models for cell membrane research. Because of their high-saline environment, they have developed potent transport mechanisms for ridding their cells of salt and maintaining proper cell volume. "Many of these systems are highly analogous to our own systems--kidneys, livers, eyes, intestines--but [the systems] are easier to study in marine species," Boyer says.
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Ned Ballatori
Photo: MDIBL |
There are several reasons. First, marine species' have structurally simpler organ systems. Second, the waters off Maine, with ambient temperatures of 5°C-10°C, yield species with lower metabolisms and oxygen tension and more stable tissue. "Because these animals live in cold water, many of the normal things we study such as wound healing, cell replication, [and] cell repair from injury occur at a much slower [rate]," says Ned Ballatori, associate professor of toxicology at the University of Rochester School of Medicine and the center's associate director. "Things basically happen on a slower schedule, so we can more easily visualize and observe them."
Group Efforts
The NIEHS first funded the Center for Membrane Toxicity Studies in 1985, charging it to identify the molecular targets of toxic substances with the goal of developing the scientific basis for treatments of heavy metal intoxication. Evans doubled as both lab and center director until 1992. The center is currently funded through 1998, the MDIBL's centennial year.
The center is an integral and important part of the MDIBL. "About one-third of the research that goes on at the laboratory is related to the theme of the center understanding the toxicity of heavy metals and environmental pollutants on these membrane transport systems," says Boyer, who was elected chairman of the MDIBL's board of trustees last year.
A little over half of the NIEHS funding goes to cover administrative costs and the rest provides small grants. "A great deal of research support is provided by the investigators themselves from other institutional grants," Boyer notes. "So, for example, part of my research is supported by funds that I get from NIH through Yale University."
The center accepts research applications annually, with a closing deadline of 1 February. As part of a laboratory-wide project, it also provides summer fellowships to students at high school through postdoctoral levels to work and study with senior investigators. "Not only does it teach them about science, but it teaches them about the environmental problems we have and how you approach them," Boyer says.
Like its parent laboratory, the center attracts researchers from the United States and abroad. Seven groups of investigators have served as the core of its decade-long effort, with other teams coming and going over the years. Currently, the center provides funds to 14 groups, six of which began work there in the past two years.
Center investigators have provided significant contributions to understanding the specific cellular targets where toxic substances cause harm and at what dose ranges. "Rather than just saying heavy metals will kill animals, or cause low fecundity (that is, they can't breed as well or produce sickness) what we can say is, here are the potential sites of action, such as protein receptors, enzymes, or channels," Evans says. The accomplishments of three long-time research teams illustrate the center's efforts.
Teamwork
Boyer and Ballatori began working together at the MDIBL 15 summers ago. During the 1990s their collaboration has focused on how heavy metals and other compounds affect a cell's ability to regulate its volume. Cells swell normally because, as they take in nutrients, they also absorb water. Cells that lose the power to rid themselves of water can swell until they burst.
"We think we have identified a couple of mechanisms by which chemicals impair the cell's ability to regulate volume," Ballatori says. This evidence indicates that heavy metals and chemicals can directly or indirectly affect a specific membrane channel, through which the cell expels substances, called osmolites, that it uses to regulate its volume. If the channel shuts down and these osmolites cannot exit, the cell also loses its ability to rid itself of water through diffusion.
Mercury, for example, can damage the channel directly. Other chemicals, such as the antifungal agent ketoconazole, work indirectly by altering the cell's adenosine triphosphate (ATP) to adenosine diphosphate (ADP) ratio. ATP provides the cell's energy through its conversion to ADP. "When the ratio drops, the channel closes," Ballatori explains. "So, if the osmolites cannot get out, water can't move. This really can explain the major mechanism by which a lot of chemicals can impair the cell's ability to regulate its volume."
Soon after the center opened, Evans made a discovery related to the issue of whether cadmium causes hypertension. Some studies in rats suggested it could, but the epidemiological evidence was contradictory.
Working with fish aortas, Evans sought to learn what, types of receptors dot the membranes of smooth muscle cells in blood vessels. In the process, he questioned whether heavy metals might affect one of the receptors for the various molecules that carry messages from the endothelium (the layer of cells lining blood vessels) to the smooth muscle cells beneath. The first receptor he looked at was for the neurotransmitter acetylcholine. Evans tested a number of toxic metals and found that he got strong contraction of the smooth muscle cells when he added cadmium. "Bear in mind," Evans says, "that if smooth muscle contracts in blood vessels, you become hypertensive." When Evans blocked the acetylcholine receptors with atropine and added cadmium, he got contractions only about 50% as strong. This "fairly surprising" finding led Evans down two research paths.
First, he sought to determine with which of four forms of the acetylcholine receptor cadmium links. Evans has now found that smooth muscle cells apparently carry only one acetylcholine receptor, but has yet to publish his results identifying the receptor.
Second, Evans addressed the question of what's causing the remaining 50% contraction? Evans has now obtained preliminary evidence that the answer lies in cadmium's effect on the receptor for endothelin, another messenger molecule produced by the endothelium. He and his colleagues are now in the process of characterizing the endothelin receptors on smooth muscle cells.
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John N. Forrest, Jr.
PhotoL Bruce Cassaday |
Discoveries sometimes emerge from the center that at first glance seem to have little to do with human toxicology. John N. Forrest, Jr., professor of medicine at Yale, has been studying the salt, or rectal, gland of the dogfish shark to understand how toxic metals interfere with the signal transduction pathways that regulate salt secretion.
Salt moves in and out of cells through chloride channels in the membrane. Such channels are ubiquitous in cells and important to proper kidney function, but difficult to study in humans. The shark's rectal gland, which serves as a sort of extra kidney, contains enormous numbers of these channels. Forrest and his colleagues have demonstrated that several heavy metals can disrupt a cell's processing of salt, which requires both stimulating and inhibiting signals. Nickel and cobalt, for example, block the pathway by which hormones stimulate chloride transport; cadmium blocks the inhibitory pathway. "The potential for understanding human toxicity is very real," Forrest says. "Whether it will yield the result that will have a direct effect on human toxicity is clearly uncertain."
In the course of their studies, center researchers have discovered a new shark heart hormone, C-type natriuretic peptide, which is controlled by salt secretion. Nickel does not block the hormone. "In other words," says Forrest, "the shark has . . . a way of getting around that form of metal toxicity. And that raises the possibility that there may be a way of getting around toxicity in humans."
How can such findings help to solve the environmental health problems for humans caused by heavy metals and other toxic pollutants? "That's always the hardest question to answer," Evans acknowledges. "The easiest answer is that scientists at the center have discovered sites of action of environmentally relevant toxins like heavy metals. This will allow us, I think, to predict where these pathologies may appear, and also, by knowing more about the particular sites of action, we can figure out ways to avoid or alleviate the problem.
"Even more interesting," says Evans, "when we know the sites of action of these heavy metals, then that metal can be used to study [a] fundamental process. That's kind of going full cycle."
Patrick Young