There’s a children’s song that warns of the consequences if you found a peanut, it was rotten, and you ate it anyway.The song makes light of a real problem that plagues much of the developing world, in countries that cannot afford to inspect food, to store it properly or to toss away food that is found to be moldy.
In the United States, rigorous government inspection makes it difficult to find, and eat, a rotten peanut. But in much of China, Africa, and throughout the tropics, moldy peanuts, corn and other staples of the diet are routinely consumed. In these places, liver cancer is a major killer.
Scientists at OSU studying the relationship between diet and cancer were among the first to isolate the toxins responsible for the plague of liver cancer in developing countries. That same research has now led to the discovery of a simple, inexpensive compound that can block the ability of those toxins to cause cancer in thousands of people around the world.
Such discoveries are often long roads. This one began more than 40 years ago when an unknown disease began sickening rainbow trout in hatcheries throughout the Pacific Northwest.
In the early 1960s, OSU scientists were called in to investigate the unexplained disease. Inside the infected fish, researchers found bulbous tumors erupting from the fishes’ livers. They linked the tumors to the hatchery fish’s diet, and in particular, to a moldy batch of cottonseed meal.
At about the same time, scientists in England were investigating a similar epidemic in turkeys. The unknown Turkey X Disease was eventually linked to moldy peanuts ground into the bird’s feed. The mold, Aspergillus flavus, was the same one found infecting the cottonseed meal fed to trout in Oregon.
Aspergillus flavus is a common and widespread mold in nature. It occurs in soil, decaying vegetation, nuts and grains wherever warm, humid conditions favor its growth. As the investigation continued, however, scientists isolated the real culprit. It was not the A. flavus mold itself, but a chemical produced by the mold, dubbed "A. flavus toxin" or aflatoxin.
Studies of aflatoxin expanded around the world. Cows fed cottonseed meal containing aflatoxin were found to pass the toxic compound to their milk. Aflatoxin turned out to be nearly ubiquitous in foods such as corn, peanuts and a variety of cereal grains grown in warm, humid parts of the world. And people living in these places were found to have a high incidence of liver cancer.
Research continued with aflatoxin and rainbow trout. The OSU team found that exposure of as little as one part per billion of the toxin could promote the development of liver cancer in the fish within a matter of months. Rainbow trout, it turned out, were the perfect subject for studying the relationship between aflatoxin and liver cancer.
Theodore Bunch, Jr., a graduate student in environmental and molecular toxicology at OSU, isolates chlorophyllin. Photo: Steve Dodrill
Even more important, the OSU team discovered that the potency of the aflatoxin greatly increased when combined with other substances. For example, after feeding the fish contaminated food combined with cyclopropenoid fatty acids, a common substance in cottonseed and other plants, the scientists reported, "aflatoxin becomes dynamite. The liver literally explodes."
Subsequent studies of human populations around the world confirmed that not only was aflatoxin an important risk factor for liver cancer, it often did not act alone. Continued exposure to aflatoxin in a person’s diet increased the likelihood of liver cancer by about four times, but aflatoxins in combination with hepatitis B or C increased the risk of liver cancer as much as 60 times.
The OSU scientists asked, if some substances could change aflatoxin into a super-carcinogen, could other substances change it into something harmless?
This question has been a major focus of the Marine-Freshwater Biomedical Sciences Center at OSU since 1965, when construction began on the world’s only trout hatchery devoted to cancer research. Here, rainbow trout continue to help scientists untangle the connections between diet and cancer.
George Bailey is a distinguished professor of environmental and molecular toxicology at OSU, and until recently, the center’s director. Much of his work at OSU has focused on foods we eat that may prevent cancer.
However, cause and effect can be quite tangled when studying living systems. Even with the quick response of rainbow trout as a model, Bailey has sometimes found more complications than clarity in his search for cancer prevention. Bailey has investigated compounds that showed promise in blocking the formation of new cancer, only to find that many of those same compounds could potentially stimulate tumor growth in already established cancers. Each breakthrough seemed to be a double-edged sword.
"There are lots of dead ends on the road to discovery," says Bailey. "You can’t predict where your research will lead you. The way this story has evolved is the way much of science progresses…through serendipity."
Serendipity, in this case, was green.
The idea came from Bailey’s colleague, Rod Dashwood, then a postdoctoral associate studying in Dr. Bailey’s lab. In a chance conversation at a scientific meeting, Dashwood got the idea to try a derivative of one of the most common substances on this green earth…chlorophyll.
Made from natural chlorophyll found in all green plants, the derivative chlorophyllin is widely available as a dietary supplement and an inexpensive food coloring. The researchers theorized that chlorophyllin could interact with aflatoxin, and block its uptake in tissue.
"Atomically, chlorophyll looks like a big, flat electron cloud that can stick tightly to carcinogens with the same general features," explained Bailey. "As it turned out, chlorophyllin made an open-faced sandwich out of aflatoxin. It formed a new complex that was very stable in the stomach acid and could pass easily out of the trout without being digested or absorbed."
Studies with the center’s rainbow trout showed that when chlorophyllin was ingested at the same time as the aflatoxin, uptake of the toxin was reduced by half or more.
Bailey and his colleagues shared these findings with scientists at Johns Hopkins University, who tested chlorophyllin on laboratory rats, and came up with the same positive results as with trout.
It was time to test chlorophyllin on people.
Lowland China, where warm, humid conditions often promote growth of the mold on stored foods. Photo: Grant Heilman Photography
In the hot, humid lowlands of eastern China near the town of Qidong, peasant farmers tend fields of corn and peanuts along the Yangtze River delta. Qidong has been of interest to epidemiologists for decades. One in 10 adults here die from liver cancer, one-tenth of the population are chronic carriers of hepatitis and 95 percent have been exposed to aflatoxin.
In the mid-1990s, the Johns Hopkins team was in Qidong, studying the connection between hepatitis and aflatoxin. Qidong would be the perfect place to test chlorophyllin’s ability to stop aflatoxin from triggering cancer.
"The Chinese physicians were very interested in the study, and the volunteers, mostly peasant farmers, were dedicated and highly motivated participants," said Thomas Kensler, one of the principle investigators from Johns Hopkins. "We recruited 180 volunteers and all but one stayed with the trial to the end."
Researchers from Johns Hopkins University went to the Yangtze River delta far southeast of this span of the Great Wall to test whether chlorophyllin can block a toxin that promotes liver cancer. The disease kills one in 10 adults in the area. Photo: Andy Duncan
Half the study participants were given tablets of chlorophyllin to take three times a day with meals. The other half received a placebo. Samples of urine and blood taken periodically throughout the four-month study indicated the extent of aflatoxin damage in each of the study participants.
"In clinical trials, you can’t do liver biopsies," said Bailey, "but you can trace biomarkers in urine and blood samples. By examining these fluids, we were able to determine that chlorophyllin had the same protective effect in people as it did in trout and rats. People receiving chlorophyllin had 55 percent less DNA damage related to aflatoxin exposure."
How does it work?
According to Bailey, a great deal of aflatoxin is detoxified by the body and excreted harmlessly. The liver is where that detoxification takes place. But in the process, some of the aflatoxin is metabolized and attacks the DNA of the liver. Some of that damage is repaired by repair enzymes, and then excreted.
"Rats and humans repair the damage better than trout," said Bailey, "which makes trout particularly sensitive to low levels of aflatoxin, and a good model to study the pathways of cancer formation."
Studies of rainbow trout helped OSU Agricultural Experiment Station researcher George Bailey and colleagues confirm chlorophyllin's anti-cancer properties. Photo: Steve Dodrill
Repairing DNA creates a chemical marker in the urine of those—people, rats, or fish—who have been exposed to aflatoxin. With chlorophyllin in the diet, there was less of the marker in the urine, suggesting that there was less aflatoxin damage that needed to be repaired.
"If you want to block the cancer process from the beginning, you have to know what initiates the cancer," says Bailey. "Unfortunately, we don’t know what initiates most human cancers. But we do know that aflatoxin initiates some kinds of cancer, as does tobacco smoke and probably cooked meat mutagens. So chlorophyllin is interesting because it can block the action of compounds that we know start cancer."
A mold on decaying nuts, grains and other foods produces the chemical aflatoxin, which promotes liver cancer. Photo: Eyewire
This brings Bailey to the next twist in the road. Can chlorophyllin suppress cancer that has already started?
"Some chlorophyllin turns you green," says Bailey. "It is visible as a pigment in serum, livers and bile, which means that it is taken up by organs in the body. That means that it may be available to battle carcinogens other than aflatoxin and cancers that have already started."
Not all batches of chlorophyllin have the specific green pigment that is taken up into tissues. It is a minor component, and may not be the important part that blocks the aflatoxin. But it may be an important part for suppressing the development of cells into full-blown cancers.
"So our latest research is focused on that minor component," says Bailey, "to learn if long-term chlorophyllin treatment might play a protective role after exposure to a cancer-causing compound but before a detectable cancer has formed."
The problem that began as an isolated incident in a fish hatchery has led a generation of OSU scientists to investigate ways to prevent one of the world’s deadliest diseases. But the road to discovery winds on.
"It will take a study 20 years or more to learn if the incidence of liver cancer will actually be lowered with the addition of chlorophyllin in the diet," says Bailey. "What we know now from this study is that the level of the carcinogen that causes the cancer was lowered by more that 50 percent. So, for pennies a day we may be able cut in half the incidence of liver cancer in third-world countries."
Kensler concurs. "Compared to Western medicine and chemical intervention, chlorophyllin is remarkably inexpensive, stable and effective, with no side effects.
"It underscores the generic recommendations to eat five servings of fresh vegetables every day. There is a scientific basis to the old advice to eat more greens."