Arsenic as an Endocrine Disrupter
Project Leader: Joshua W. Hamilton Ph.D. Senior Scientist Chief Academic & Scientific Officer Marine Biological Laboratory
Project Co-Leader: Jack E. Bodwell, Ph.D. Research Associate Professor, Department of Physiology Dartmouth Medical School
Arsenic as an Endocrine Disruptor
A multitude of effects
Arsenic affects human health in many ways. In humans, long-term exposure in food or drinking water is linked to a host of different cancers including those of the liver, lung, kidney, bladder, and skin; it increases the risks of vascular and heart disease, type 2 diabetes, reproductive and developmental disorders, low birth weights in babies, neurological problems and lower IQ's in children. It can stop a tadpole from metamorphosing into a frog. It can keep the little killifish, a common forager along the tide line, from following its usual daily routine, swimming from freshwater to saltwater and back: Exposed to arsenic, the fish dies in normal salt concentrations. A mouse, if exposed to arsenic, dies if it catches a common flu. And each year the list of serious illnesses associated with arsenic exposure grows, while the concentrations that are recognized to do this get lower and lower.
How can one chemical cause harm in so many different ways, and at doses that seem so small?
Researcher Joshua Hamilton is hoping to answer this question. The chief academic and scientific officer at the Marine Biological Laboratory in Woods Hole, MA, he became director of Dartmouth's Toxic Metals Research Program in 1997, after the program's founder, Dartmouth chemistry professor Karen Wetterhahn, died. Hamilton stepped down as director of the Dartmouth program in 2008, but he continues to collaborate as a member of the program.
Hamilton's early work on killifish gave Dartmouth researcher Bruce Stanton, who is now director of the Dartmouth program, insights on the similarity between arsenic exposure and cystic fibrosis. Both involved the endocrine system, and in particular a group of hormones called glucocorticoids. Hamilton's lab had been the first to report that arsenic is an endocrine disruptor, based on laboratory studies in cultured cells.
“But what we discovered with the killifish,” Hamilton says, “is that this effect of arsenic could have serious consequences to a animal responding to normal environmental challenges.”
Arsenic was clearly an endocrine disruptor in these animals, but not an ordinary one.
Disrupting a complex system
The endocrine system is a complex system in the body that is regulated by hormones. Hormones are important signaling molecules that are produced in one part of the body and have their effects in another part of the body, usually by signaling cells to turn on or off certain genes, and thus to make more or less of certain proteins. Glucocorticoid hormones, which are the major stress hormones, control many functions in the body, such as blood sugar level, cell growth, inflammation, fetal development, and, in killifish, salt tolerance.
Other well-known hormones are the sex steroids: estrogen, progesterone, and testosterone; and hormones like insulin and glucagon that, like glucocorticoids, also regulate blood sugar. An endocrine disruptor is any chemical that has an effect on any part of the endocrine system, which might include altering production, metabolism or release of the hormone, the level or behavior of the specific receptors in cells that are designed respond uniquely to each hormone, or other effects such as how cells respond to hormone stimulation. Endocrine disruptors first made news in the mid-1990s when scientists in Florida discovered that some pollutants were disrupting these sex hormones. Male alligators were showing up with shortened penises—the chemical in this case mimicked the female hormone, estrogen, causing the alligators to be “feminized.” This led to a flurry of interest in endocrine disrupting chemicals in the environment, and most work has focused on chemicals that can mimic the normal hormones by activating or blocking the hormone receptors.
“Arsenic doesn't work like that,” explains Hamilton. “In fact, we know a lot about what arsenic doesn't do. It doesn't mimic a hormone. Nor does it bind to a hormone receptor or block it from binding its normal hormone. It doesn't behave like any other chemical pollutant, such as dioxin, or even like any other toxic metal, such as cadmium or mercury. It doesn't match what we think of as an endocrine disruptor at all.”
And yet its presence perturbs the endocrine system. What is clear from Hamilton's work is that, in the presence of arsenic, hormone-activated receptors behave in unusual ways that can either increase or decrease the hormone response depending on the dose of arsenic and the specific receptor. “It's complicated,” he says, “but we now have evidence that arsenic perturbs many different hormone receptors, making it a very unique endocrine disruptor with widespread effects on the body.”
Hamilton's group is using a variety of experimental models—from fish to frogs to mice to human cell cultures—to discover precisely how arsenic interacts with the endocrine system, and to determine what the consequences are for this disruption. “We think this will go a long way to explaining how arsenic increases disease risk. Look at the long list of diseases arsenic affects: Every one of them has an important hormonal component.”
A puzzling response
Arsenic has been known for thousands of years as a poison. A pellet of pure arsenic the size of a pea will kill a person outright. Murderers throughout history, from ancient Rome until the 19th century (when doctors learned to detect its presence in a dead body which largely began the field of forensics), thought of arsenic as the “King of Poisons and Poison of Kings.” Slipped into an enemy's food or wine, it was colorless, odorless, tasteless, and quick. Potential murderers could even build up tolerance to the poison—and so able to share their enemy's meal—by eating little bits of arsenic over time.
As with other toxic substances, it was thought that “the dose makes the poison.” “That's the basic premise of toxicology,” says Hamilton. “You get a little response from ingesting a little bit of the chemical, and more response with more of the chemical.” Toxicologists routinely prepare dose-response curves showing when a response kicks in and when it becomes dangerous.
“But arsenic doesn't follow this standard dose-response curve,” Hamilton says. “We found that, at extremely low doses, sometimes arsenic will make a hormone receptor work “better”—it will substantially enhance the function of that receptor.” Add a certain amount of a hormone to a cell culture, and you will see a certain response from the hormone receptors. If arsenic is present in the culture, however, you will get two to three times the normal response.
“That's not generally a good idea,” Hamilton says. If you are female, for instance, supercharging your testosterone receptors in this way might make you grow hair on your chest, while too much estrogen signaling in males might feminize them like the Florida alligators.
Turning off a hormone receptor is not a good idea, either. That is what happened when the researchers added a little bit more arsenic to the system, but still well below the level that might cause any signs of toxicity. The arsenic blocked the hormone receptor completely. The cell gave no response to the hormone at all.
Hamilton's group repeated their cell-culture experiments in mice. “It's difficult to directly compare concentrations in cell culture with whole animal levels, but we found that the mouse will respond over a certain dose range that also represents very low doses to them. But in each case, there was a set point where we saw this 'phenotypic switch,' going from enhancement at very low doses to suppression at slightly higher doses and over a very short dose range.”
“That completely goes against the principles of traditional toxicology. It tells us that the body's response to arsenic is both very complex and very dose-dependent. We think that there are two different mechanisms—two completely independent mechanisms—at work, one for enhancement very low doses and another for suppression at slightly higher doses.”
What exactly does he mean by “very low” and “slightly higher” doses? In cell culture and in mice, the enhancement occurs well below the 10 parts per billion (ppb, or 10 micrograms per liter) of arsenic that the current U.S. Environmental Protection Agency regulations consider “safe” in drinking water, the so-called Maximum Contaminant Level or MCL. At slightly higher doses in the range of 50-100 ppb they see the suppression. This level is still low – in fact, until 2006 the U.S. EPA's MCL was 50 ppb, and 50-100 ppb are commonly found concentrations in private wells throughout New England. Ten percent of the private wells in New Hampshire that were tested by Dartmouth researcher Margaret Karagas contained more than 10 ppb; twenty-five percent contained more than 2 ppb.
Says Hamilton, “Based on our experiments in the lab, both in cell culture and in mice, I would be concerned about drinking water containing anything over one part per billion. That's my personal opinion, but I think we have enough data to back it up. From 1 part per billion to 10 parts per billion—the EPA standard—was the range in which we found arsenic to be an endocrine disruptor. We are now trying to determine just what impact this will have on human health and disease risk.”
Turning genes on – or off?
While avoiding arsenic-tainted water may be easy for people who can afford to test their wells and buy bottled water if necessary, some people don't have that option. Others are not even aware there's a problem. And many have been exposed to arsenic on a daily basis for years.
In hopes of curing or preventing their diseases, scientists need to know exactly how arsenic interferes with the endocrine system. Hamilton and his colleagues have learned, through a recent series of experiments in mice, that more than just a few genes are affected.
The sequencing of the mouse genome in 2002 has made their work possible. Through the new techniques of functional genomics, they can look at normal patterns of gene expression and see how exposure to arsenic alters them. “We now have 'gene chips' in the lab that contain a copy of every single gene in a mouse,” says Hamilton. “We can treat the mouse, then take out the tissue that we're interested in, and check which genes were affected by the treatment. What genes go up in activity, and what genes go down, for instance, when a mouse is exposed to arsenic in its drinking water?”
Hormones don't always circulate at constant levels in the body. Instead, they are released in pulses. “So if we give a mouse a hormone, we will get a response we can measure,” Hamilton explains. “Glucocorticoid is a stress hormone. When we give the mouse a shot of glucocorticoid, about 2,000 genes respond—that's out of a total of 20,000 in a mouse. When we give the mouse arsenic alone, without the hormone, it affects a couple of hundred genes.
“But when we give arsenic and glucocorticoid together, all the genes that went up in activity with just the hormone now go down, and all the ones that went down with just the hormone now go up. All of them are affected. That's the key point: It profoundly affects every one of those 2,000 genes.”
Arsenic and flu
The tissues Hamilton's group focused on in these studies were the liver and the lungs. Says Hamilton, “The liver is the canary in the coal mine for our bodies. It sees chemicals we ingest first, and normally has the largest response when the body is exposed to a chemical.”
Doctoral candidate Courtney Kozul was especially interested in the lungs, which are a particular target for arsenic effects in human populations that have been studied. The genes affected in the lung, she saw, were completely different than those affected in the same animal's liver. Decoding the function of those genes, Kozul found that the ones in the lungs were part of the immune system. Specifically, they were involved in innate immunity—the most primitive form of an immune response. Babies are born with innate immunity. It includes the lymphocytes and the white blood cells that rush to the site of an infection and start cleaning it up. The second level of immunity—acquired immunity—involves antibodies. This immunity is acquired through exposure: The body has to first be exposed to the disease or allergen, and then it will begin to make antibodies against that threat.
In terms of bacterial infections and flu, the innate immune system might be the more important, and this was the system compromised by arsenic in these mice.
What would happen to these mice, Kozul wondered, if they got the flu?
“If you give healthy mice the flu,” explains Hamilton, “they respond just like we do. They stay home for a few days and feel sick, and then they go back to work.
“But if these mice have been drinking 100 parts per billion of arsenic in their water for as little as five weeks, and then they get the flu—they die. They never mount an appropriate immune response. Their lungs look horrible—they are all full of fluid and are hemorrhaging.”
Kozul and Hamilton submitted an article on the flu experiment to a scientific journal just days before the outbreak of swine flu in Mexico in April 2009. The so-called swine flu, H1N1, is the very same form of flu they used in their experiments, precisely because it is also a “mouse flu” and “human flu” that causes similar effects in several different species. Why were people in Mexico dying from this flu, while those New Yorkers who caught it on vacation there didn't die? What is different? Hamilton suggests that it may be related to arsenic, since parts of Mexico also have high levels of arsenic in their drinking water.
“Because the paradigm that's emerging from our studies is this: Arsenic doesn't cause these diseases. Arsenic makes them worse. You can give mice in the lab arsenic for a lifetime, and they'll be fine until you challenge them with some other stress. If that stressor is sunlight, they'll get skin cancer. If it's the flu—they'll die. Whatever the second agent is, arsenic makes it worse. This may also be why it can influence so many human diseases.”
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