Arsenic and the Ubiquintin-Lysosomal Pathway
Project Leader:
Bruce A. Stanton Ph.D.
Director, Dartmouth Toxic Metals Research Program
Professor, Department of Physiology
Dartmouth Medical School
Small Fish, Big Questions
Killifish are minnow-sized baitfish common up and down the East Coast. They thrive in estuaries, shallow habitats swept by the tides. Each day, as tides rise and fall, the killifish move from freshwater to saltwater and back. Most freshwater fish cannot do this: Plunked in the salty sea they die. But the killifish has an abundance of a protein in its gills that allows it to expel the extra salt when needed.
Except when the water contains a trace of arsenic. Then the system fails; the killifish dies from an accumulation of salt in its blood.
Puzzling out arsenic's low-level effects is enough of a reason to study killifish, says Bruce Stanton, a molecular toxicologist at Dartmouth Medical School. The level of arsenic that kills killifish is considered “non-toxic.” Whether that level could be harmful for humans is another question. “Is the EPA standard of 10 parts per billion of arsenic in drinking water appropriate?” he asks. “Maybe 10 ppb is fine. These are somewhat arbitrary cut-offs. How can the government make policy decisions based on real science if we don't do experiments?”
But there's more: The same protein that lets the killifish swim in salty water, when lacking in human lungs, causes cystic fibrosis.
Saltwater and Cystic Fibrosis
The protein is called CFTR, for Cystic Fibrosis Transmembrane Conductance Regulator—as the name suggests, it was found in cystic fibrosis patients before it turned up in killifish, yet the fish form is essentially identical to the human protein.
“Cystic fibrosis is a genetic disease caused by a mutation in a single gene, the CFTR gene,” says Stanton, whose lab has focused on the disease for many years. “This gene was cloned in 1989, but now, 20 years later, we still don't know completely what it does. We don't understand why, at the cellular and molecular level, a mutation in that gene causes the disease.”
This much is known. “Normally, CFTR creates the salty fluid that allows you to clear pathogens — like bacteria — out of your lungs,” Stanton says. “Technically speaking, CFTR is an ion channel that secretes salt through the cell membrane, and that makes water go into the airway.” This mechanism helps break up the mucus in your lungs — an important defense against infection. Normally, when you inhale a pathogen or bacteria, it gets stuck in the mucus and CFTR triggers secretion of a salty fluid. Then you cough up and swallow the mucus, digesting and inactivating the bacteria. “A cystic fibrosis patient, because of a mutation in the CFTR gene, is no longer able to secrete salt. You still make mucus, and there's still junk trapped in the mucus. But without the salty fluid, you can't expel it. The mucus accumulates. It gets dehydrated, and your lungs start looking like a pan of overcooked oatmeal. Bacteria grow in that mucus, and you can't get rid of it — it's too sticky, too thick.” Antibiotics cannot completely penetrate the thickened mucus and, since they are never entirely killed off, the bacteria become antibiotic-resistant. People with cystic fibrosis have a life expectancy of 37; the usual cause of death is a lung infection that doesn't respond to antibiotics.
One reason the disease is difficult to study is that it doesn't occur naturally in animals, which often are studied to understand human diseases. “Researchers have made the mutation in the CFTR gene in mice,” Stanton explains, “but the mice didn't get cystic fibrosis. They made it in pigs, too, but so far they didn't get lung disease either.”
Until Stanton started studying killifish, the major way to learn how cystic fibrosis worked was to examine cultured human cells. But do cells in a petri dish work the same way as living lung cells? “There were questions: Is the cell model relevant? They're only cells—and each part of our body is interconnected. That's why these little killifish are such brilliant models to study.”
Blocking Cell Signals
Several years ago, Stanton attended a seminar given by his Dartmouth colleague Joshua Hamilton on how arsenic acts as an endocrine disrupter. The body's endocrine glands release hormones that signal cells to turn on or off certain genes, and thus to make more or less of certain proteins.
“The endocrine system is perfectly and exquisitely timed to create the right order of events,” Stanton explains. Arsenic disrupts this cell signaling, Hamilton found, by blocking or otherwise disabling hormone receptors – the molecules that enable hormones to work within cells.
One example Hamilton talked about was the receptor for the glucocorticoid hormone. Glucocorticoids control many functions in the body, such as blood sugar level, cell growth, inflammation, fetal development—and, in killifish, CFTR in the gills. “The same hormones that regulate the system Josh studies also regulate the system we are studying,” Stanton remembers noting: Could there be a connection?
“It turns out that low levels of arsenic do affect CFTR, but not in the same way as Josh found,” Stanton says. “In a normal human lung cell, arsenic causes CFTR to get degraded and thrown into the trashcan of the cell, the lysosome. The result is similar to what happens in people with cystic fibrosis — dehydrated airways, which leads to that oatmeal image I gave you. Our major interest is to try to understand how arsenic does this. We're trying to identify the genes that regulate the degradation of CFTR — the genes that cause it to break down — and to understand how arsenic affects these regulators. We are also interested in understanding if arsenic increases the disease process in CF”
Starting with cultured human lung cells from a deceased cystic fibrosis patient, Stanton's team used gene therapy to repair the gene damage and put a normal copy of the gene back into the cell. “Then we can see how arsenic affects the normal copy.”
That may also help them better understand the mechanisms behind cystic fibrosis. “Cystic fibrosis is still somewhat of a big black box,” says Stanton, “and inside that box, there's a Rube Goldberg device that we can't see. We know that the patient gets lung disease. We know CFTR is involved. But we don't know what the bells and whistles are. Does the ball roll and hit the teakettle? Is CFTR the ball—or the teakettle? If we knew that, we might be able to find a target for a new therapy, to improve the outcomes for patients with cystic fibrosis.”
The Killifish Model
To check if the cultured cell's response is the same as that of a whole organism, Stanton turns to his killifish. “When we repeat the arsenic experiment on killifish, we get exactly the same result. We see the same patterns that in humans lead to the degradation of CFTR and the dumping of it into the trashcan. That means the effect in the lung cell is not something that only happens to a cell in the lab. It's a real effect.
“Using the two models together, we can get a really thorough understanding of how CFTR works and how environmental toxicants like arsenic affect it.”
Killifish are a good model because they are small (easy to keep in tanks in the lab) and common. Stanton catches his experimental subjects off the Maine coast, where he has a summer research lab. “I catch them in a minnow trap. I used to bait the trap with a bagel, but that just brought in eels. Now I use nothing — no bait — and I get hundreds of killifish in a morning.”
Killifish are also experts in CFTR-production. “You can barely detect CFTR in the lungs of humans,” notes Stanton, “but from the gills of a killifish, you get bucketfuls.” The more CFTR, the more salt the fish can withstand. “Killifish are related to salmon. But salmon only go from saltwater to freshwater when they breed. Killifish do it several times a day.”
And they can go from totally freshwater to concentrated saltwater, such as the water in a tide pool, where the salt content increases as the water evaporates in the sun. “They've learned how to survive. Their gills have thousands of cells, and each one pumps salt out like crazy.”
Stanton and his colleagues, Celia Chen of Dartmouth and Joe Shaw at Indiana University, have cloned and sequenced many genes that control these CFTR salt-pumps in killifish. “We have the ability to expose the fish to arsenic and to look at how the genes change.”
With support from the National Institutes of Health, Stanton's killifish are swimming in water containing 2 parts per billion (ppb) up to 1000 ppb of arsenic (the highest levels of arsenic found in well water in New Hampshire is 300 ppb). “After two days in a tank containing arsenic, CFTR is completely eliminated from the fish,” says Stanton. “If they stay in freshwater, they're okay. But if those fish are then put into saltwater, 95 percent will go belly-up”.
“The really strange thing is that, if killifish are in saltwater and you add arsenic, in about 48 hours they'll adapt. We don't know why.”
Stanton and his research team are now identifying the proteins that normally break down CTFR, and looking at arsenic's effects on each one.
“Every protein in your cell gets tagged when it's made with a short peptide called ubiquitin,” he explains; the tag gets its name because it's ubiquitous. “If the ubiquitin tag is not removed, it's like the protein's waving a flag: There's too much of me! Send me to the trash! It's a quality-control mechanism for the cell. But sometimes a protein gets tagged incorrectly. Then there's another class of enzymes that clips the ubiquitin off. That's the second quality-control mechanism. Finally, the process of adding and removing the ubiquitin tag also directs the protein to the appropriate place in the cell. For CFTR, that's the membrane.
“It's not simple — that's why it's fun. There are probably 30 to 40 proteins that bind to CFTR and interact with it and regulate it. So far we've found two, one that sticks ubiquitin onto CFTR, and one that clips it off. We've developed analytical techniques that tell us how well these proteins work. We need to find as many of these partners as we can and ask, “Does arsenic turn any of these on or off? If so, we have a target to develop a new drug to help patients with cystic fibrosis.”