Stanford News

3/19/97

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Marine animals shed light on how drug resistance evolved

Flapping in and out with the rhythm of the waves of Monterey Bay, the gills of the mussel Mytilus californianus filter sea water for tiny bits of food. Along with nutrients, the briny broth is also laced with a deluge of natural and man-made chemicals, some of which could do the mollusk more harm than good. Fortunately, says biologist David Epel, most of the toxins are spit out of the cell, and mostly nutritous food stays inside.

That protection is good news for a mussel, but can be deadly to a person with a drug-resistant disease.

He and members of his lab have found the reason: a built-in defense mechanism in the cells that line the mussel's gills. It is the same protection used by bacteria to repel antibiotic drugs that normally would kill them, the same that cancerous tumors use to eject the powerful drugs used in chemotherapy. Epel and other scientists hope to find ways to thwart this natural skill for drug resistance, so scientists can design better treatments for cancer and infectious diseases.

The defense is provided by a protein in the walls of some cells, called an MDR pump ­ the acronym stands for "multidrug resistance." Studies by Epel and other researchers show that MDR pumps may be present in most, if not all, organisms on the planet. If so, this kind of protection against toxins has been around for millions of years. "This protein is an example of how organisms have evolved a way to protect themselves from potentially harmful substances," Epel said.

Epel, professor of biological sciences at Stanford, maintains his lab at the university's Hopkins Marine Station on Monterey Bay. He gained his renown studying another interaction that occurs at the walls of cells: the act of fertilization. Working with sea urchin eggs, he demonstrated how a successful meeting between egg and sperm triggers that egg to become an embryo.

Over the past decade, he also has become interested in a first encounter of a different kind: that of a fragile embryo ­ or any defenseless creature, for that matter ­ with its hostile sea environment.

"Working at Hopkins, seeing the embryos of marine organisms in their natural environment, I was intrigued by how they defend themselves," Epel said. His curiosity about unguarded embryos has steered him toward experiments with similarly helpless organisms that are stuck on rocks or in mud, or drift with the tides, gobbled by predators and unable to swim away from toxins.

What Epel and his lab members have found is that the mussel and another shoreline creature, a mud-dwelling worm called the fat innkeeper, Urechis caupo, aren't so helpless after all. They showed that these animals have the same sort of transporter protein first discovered 10 years ago, in the membranes of cancer cells. A huge snake-like protein that threads through a membrane a dozen times like a needle through a piece of cloth, the MDR pump uses packets of energy made by the cell, called ATP, to force toxins, such as chemotherapy drugs, out of the cells.

The name "multidrug resistance pump" came from the problem that cancer researchers were trying to understand at the time: how a tumor manages to develop resistance to an array of cancer-fighting drugs. Many such drugs are denied entry into MDR-laden tumor cells, causing a serious obstacle to the treatment of a variety of cancers, said Stanford oncologist Branimir Sakic, who studies MDR pumps in lymphoma, leukemia, and breast, lung and ovarian cancers.

Researchers also found MDR pumps in bacteria, which have always had to defend themselves against naturally occurring antibiotics like actinomycin, a common component of soil. The drug resistance that bacteria can develop using MDR is becoming a serious health threat. Some microbes are capable of rejecting a host of first-line drugs used to treat meningitis, malaria, tuberculosis and other potentially deadly infections.

Epel looked for the MDR pump in mussels and fat innkeeper worms because both manage to thrive in environments similar to those encountered by bacteria and cancer cells: They are confronted not only by naturally-occurring toxins that they could have evolved over time to resist, but by new chemicals introduced by humans. As more is learned about this mechanism, scientists may learn more about how to block disease organisms from developing resistance to medications.

The worms thrive in mud so contaminated by industrial pollutants that few other organisms exist there, Epel said. The mussels are so good at surviving water pollution that they have been used for more a decade in a biomonitoring effort called the California State Mussel Watch Program. By measuring levels of pollutants in dissected mussel tissue, state water monitors use the mollusks to gauge concentrations of toxins in coastal waters.

Several features especially suit the mussels to the task. "Since they are basically stuck in place, they can't swim away from a noxious substance," said Nancy Eufemia, a graduate student in Epel's lab. "And since they eat by filtering water through their gills, they are exposed to whatever is in the water. They act as a monitor for any toxins present there," she said.

However, it turns out that water levels of some chemicals could be underestimated using the Mussel Watch method, Epel said. He and his lab tested eight compounds on the program's list of common pollutants ­ half of them were spit out by the MDR pump, he said.

One thing that puzzles scientists like Epel is the broad spectrum of chemicals, called substrates, that MDR proteins recognize and repel. The list of substrates includes a wide variety of compounds that are very different from each other. Some occur naturally, such as alkaloids in plants or antibiotics in dirt, and some are man-made, such as dyes and pesticides. The only common feature is that all of them are relatively hydrophobic (they don't dissolve easily in water). It may well be that there exists a whole family of MDR pumps ­ each equipped to deal with its own special group of substrates, Epel said.

Epel and his crew can't necessarily predict what all of those chemicals are. Some have been identified by testing them in the lab: Two examples are the pesticide DCPA and an environmental pollutant called pentachlorophenol. The MDR pump in mussels also rejects certain cancer drugs such as doxirubicin and vinblastine, though clearly they do not encounter such substances in the ocean.

Epel has tested a variety of chemicals besides chemotherapy drugs and bona fide water pollutants, only some of which the mussel MDR pump can eject. The majority of substrates that it defends against are probably natural substances, he says.

To determine whether any chemical is an MDR substrate, Epel and his team use a fluorescent dye (which itself is repelled by the pump). They take a sliver of mussel gill tissue and bathe it in a solution containing either the fluorescent dye alone or the dye along with a chemical to be tested. If it is susceptible to MDR, a test chemical will "compete" with the dye, making the tissue glow when examined with a special microscope. As the test chemical is spit out by the pump, the fluorescent dye is forced inside the cell.

In humans, MDR pumps are positioned so as to encounter potentially harmful substances face-to-face ­ in places like the intestine, the placenta, and in the blood that bathes and protects important tissues like the brain and the testes. Epel's research with marine organisms, where the pumps are found in gills, sex organs and the stomach lining, is evidence that MDR pumps do seem to be a good defense against toxins ­ a particularly advantageous one at that. "MDR doesn't even let the things get into the cell," Epel said.

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Alison Davis is a science writing intern at Stanford News Service.

By Alison Davis