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Research Newsletter-Spring/Summer 2008

CANCER CHEMOPROTECTION

An interview with George Bailey, Ph.D.
Distinguished Professor Emeritus
LPI Principal Investigator Emeritus

Q. How did you get interested in cancer chemoprotection?

A. When I first came to Oregon State University in 1978 on sabbatical leave, I was interested in studying the structure of chromosomes with Ken van Holde. The nucleosome structure hadn't been worked out at all, and during the course of my year I read about work on the interaction of carcinogens with chromatin. Nucleosomes are structures in the cell's nucleus that package DNA into chromosomes, and chromatin is made up of DNA and histone proteins that together form chromosomes. So I switched into that area and looked at the interaction of the carcinogen aflatoxin with nucleosomes from steelhead trout that I caught in the Alsea River. Eventually I accepted an offer to join Russ Sinnhuber's group in Food Science at OSU. They had a contract through the National Cancer Institute to use rainbow trout to test a few chemical compounds, most of them food derived, that had just been discovered as having cancer chemoprotective activity. We found that one such compound, indole-3-carbinol from cruciferous vegetables, was very effective. Then we became interested in identifying molecular mechanisms and determining dose-response relationships.

Q. Why use trout as a model of carcinogenesis instead of small rodents?

A. Well, I like trout, and they're interesting from an evolutionary perspective, too. Trout are extremely low cost to purchase and to house. That allows us to use thousands of trout in an experiment to achieve high statistical power in examining dose-response issues. To do that with rats or mice would be too costly.

Q. Does that allow you to make observations on dose-response relationships at low doses of carcinogens?

A. Absolutely! I'll give you an example—the simple question of assessing cancer risk at a high dose and extrapolating that to low doses and low tumor incidences relevant to humans. All the data available for risk assessment come from rodent experiments with very high carcinogen exposures, allowing tumor incidence in the 10-100% range. You can measure that incidence with a reasonable number of animals. But we want to know what dose might give one extra cancer per one million individuals because that's the regulatory target we're reaching for. Unfortunately, that requires extrapolating the high dose data 100,000-fold. Using rainbow trout, we conducted the largest ever dose-response study in any animal model to get information on low doses of carcinogens that are relevant to human exposure. The default assumption had been that the risk is linear below 10% tumor incidence, but in our experiment with 42,000 trout, we found that assumption to be false. We found, instead, a threshold.

Q. Are trout studies relevant biologically to humans?

A. Yes. There's tremendous evolutionary conservatism in biology. For instance, patterns of gene expression and altered gene expression are very similar in liver cancer in both humans and trout. We share with trout a similar set of enzymes to metabolize carcinogens. Trout don't repair DNA damage as well as humans do—they are a bit more sensitive—but that doesn't change the shape of the dose-response curve.

Q. Do you compare responses in trout to rodents?

A. We are looking at the inhibition of cancer by chlorophyll and its derivative, chlorophyllin, using two different carcinogens in rainbow trout. We extrapolate that to rats. With LPI Principal Investigator Dave Williams' transplacental mouse model and Rod Dashwood's rat colon cacinogenesis model, we then looked at whether the cancer chemoprotective mechanisms are similar in rodents and people. So we don't focus exclusively on trout.


fish


Q. Do trout require a special facility?

A. Virtually all the money for our facility and its expansion and improvement came from NIH grants to Russ Sinnhuber, Jerry Hendricks, Dave Williams, and me. The National Institutes of Health has encouraged investigators to develop alternative models for human health research. I served as director of the Marine and Freshwater Biomedical Sciences Center at OSU for 17 years. The center was sustained by about $8 million in grants from the National Institute of Environmental Health Sciences. That support was absolutely critical in developing trout as a model for human carcinogenesis and chemoprotection. Of course, access to a good, clean water supply is critical. Some aquarium fish models, like zebra fish, have become popular because their housing requirements are somewhat easier to meet. Our trout facility at OSU is probably the only one in the world that permits us to do our kind of research.

Q. You mentioned studies with chlorophyll and chlorophyllin in trout. What's the difference between chlorophyll and chlorophyllin?

A. Chlorophyllin is a derivative of chlorophyll, the natural green pigment in plants. It's obtained from natural chlorophyll through a hydrolysis process that converts fat-soluble chlorophyllin into a water-soluble dye that can be used commercially, for instance, as a food coloring agent and deodorant. There's a long history of chlorophyllin use in the health care industry.

Q. Why use chlorophyllin instead of natural chlorophyll?

A. Natural chlorophyll is very unstable, and it deteriorates very quickly after isolation and purification. With the chlorophyllin derivative, through hydrolysis and replacement of magnesium—the metal ion that's naturally found in chlorophyll—with copper, you get a very stable compound that doesn't oxidize or degrade as quickly as chlorophyll. You might think that replacing magnesium with copper would make the compounds more reactive, but the copper is very tightly bound, so it's not available to engage in detrimental reactions.

Q. Have you compared the difference in chemoprotection between the purified compound and the food in which it's found?

A. We can't do that with fish because they are carnivores, not herbivores. They don't digest plant material. Our human studies are all done with purified chlorophyll or chlorophyllin, not with spinach, which is rich in chlorophyll. So that experiment remains to be done. One study that we recently completed in Rod Dashwood's lab used freeze-dried spinach in rats. He got an interesting result in that experiment. When the spinach was fed after colon cancer was initiated in the rats, it had a protective effect against carcinogenesis. It was pretty impressive, and we didn't expect it. We expected to see a protective effect when the spinach was given at the same time as the carcinogen, but that was not observed. So we have to sort it out. Is it because the chlorophyll isn't bioavailable? Was it given appropriately with the carcinogen dose? There are a lot of variables to examine when you get a surprising finding like that.

Q. Are there any side effects or toxicity with either chlorophyll or chlorophyllin?

A. Nobody is aware of any toxicity issues for natural chlorophyll. The FDA allows a level of intake of chlorophyllin that is in the range of 100-150 milligrams per tablet. No toxicity has ever been reported, and we haven't observed any in our short-term studies with humans. In future long-term human trials, there is a potential risk of tumor promotion by chlorophyllin based on the rat colon carcinogenesis model. So, under certain circumstances, at certain doses of chlorophyllin and exposure to certain carcinogens, there may be some risk. Interestingly, we observed protection in both very low and very high doses. In those experiments, chlorophyllin was given after cancer initiation—after the carcinogen was administered.

Q. You've also studied indole-3-carbinol from cruciferous vegetables as a cancer chemoprotective agent. Is it safe and effective?

A. Indole-3-carbinol, or I3C, is the classic example of the double-edged sword in cancer chemoprotection. We were the first to observe this in the early 1980s. When we did our first experiments with I3C, we gave it with the carcinogen aflatoxin to trout and with a different carcinogen, dimethylbenzanthracene, to rodents. I3C helps to block the cancer process when it's given at the same time as the carcinogen. Also, I3C was given to rats in breast cancer experiments because of its anti-estrogen effects. In that model, I3C suppressed mammary tumor development. So there has been tremendous interest in the compound for that reason. In our experiments with trout, we asked if I3C would protect against liver cancer if given over a protracted period. Unfortunately, it promoted liver cancer. Years later we continued that work with rats and saw the same effect. Oddly enough, I3C shows this tumor promotional property because in the liver itself it's estrogenic. The public and the scientific community get very confused by this. It's one of the first examples of a compound that may reduce the risk of prostate cancer or breast cancer in people who are at very high risk of these cancers, but that may be offset by some increased risk for liver cancer. It's a difficult risk-benefit equation.

Q. Does that suggest that people should perhaps avoid I3C supplements and instead eat a good amount of cruciferous vegetables?

A. I would not take I3C supplements, nor would I recommend them. We don't know enough about mechanisms and nuances to gauge risk versus benefit in this situation.

Q. Some years ago you and colleagues at Johns Hopkins were involved in a clinical trial in China to assess the cancer chemoprotective effects of supplemental chlorophyllin. What did you find?

A. This is the study that I felt took the rainbow trout model full cycle for the first time. Liver cancer was discovered in rainbow trout in fish hatcheries in the Pacific Northwest in the early 1960s. That was traced to aflatoxin, a mold toxin that contaminated the cottonseed oil used for part of the fish diet. Building on evidence from rats and then turkeys, it became clear that aflatoxin is an important risk factor for liver cancer in humans. We now know that aflatoxin exposure in certain parts of the world is one of the greatest risk factors for liver cancer, along with hepatitis virus infection. These two risk factors synergize, such that liver cancer is one of the leading causes of cancer death world-wide. We need remediation because exposure to aflatoxin in food in some areas is unavoidable. You can't always see the contamination in foods. There's a vaccination program for protecting against viral hepatitis risk, but that's expensive and not easily available to people at risk in third-world developing countries. Since those people have an unavoidable dietary exposure, we need to find strategies to mitigate cancer risk. There are compounds that occur naturally in foods, as well as some drugs, that will help to block liver cancer in animals exposed to aflatoxin. They do this by increasing the expression of enzymes important in aflatoxin metabolism.

In trout we discovered that chlorophyllin worked against aflatoxin very effectively by a much simpler mechanism—binding to the aflatoxin so that it does not get absorbed as efficiently into the body. The absorption of the carcinogen is reduced, and more is excreted in the feces. That biophysical process should be independent of species. Those results led to the intervention trial in China where there are people unavoidably exposed to aflatoxin in their diet, especially from contaminated corn and rice. Colleagues at Johns Hopkins and I enrolled 180 people in that trial. Half of them—90 people—took a placebo tablet with each meal for three months. The other 90 people took a 100 milligram tablet of chlorophyllin with each meal for three months. Then we tested their effective aflatoxin uptake. We would have to follow them for over 20 years to observe liver cancer, so we instead measured a cancer biomarker—how much aflatoxin DNA repair products appeared in their urine over a 24-hour period. Chlorophyllin should be effective in reducing the aflatoxin uptake in the supplemented people. Less aflatoxin should get to their livers, less should get activated, less should bind with DNA, and less should get excreted in urine. This is indeed what we found—the people who received the chlorophyllin tablet had a 55% decrease in cancer biomarkers compared to those who received the placebo. So the trout model played an important role in helping to discover a method to attenuate aflatoxin-induced carcinogenesis. It played a role in helping to discover an effective chemoprotective agent that costs pennies a day anywhere around the world.


picture of China


Q. Are those results persuasive enough to recommend that people unavoidably exposed to dietary aflatoxin should take chlorophyllin supplements? Or do you need further clinical trials?

A. There is absolutely no reason that definitive clinical trials shouldn't be done now. The people in China know how to do it, they're equipped to do it, and they don't need anybody else to guide them. It's up to them. In affected areas in China, about 10% of men die from liver cancer by the age of 45. That's a huge death rate from a single cancer, and I believe that could be halved for pennies a day.

Q. Does the Chinese government show much interest in promoting this kind of clinical work?

A. The Chinese government has cooperated a lot in making this population available for these kinds of studies by foreign scientists. And I think they will continue to do that.

Q. Is aflatoxin exposure a concern for people living in the United States?

A. Not in the United States, because we're wealthy enough to afford the Food and Drug Administration and USDA that test our food supply for aflatoxin contamination. The nuts, grains, and seeds that are normally contaminated elsewhere, especially in the developing countries, are tested, the food supply is screened, and the action level is to exclude more than 15 parts per billion of aflatoxin in any food—peanut butter, corn, and other grains.

Q. So we don't need to worry about corn, peanut butter, or roasted peanuts?

A. Not in this country. But in Africa, which is another hot spot for liver cancer, people have high rates of hepatitis viral infection coupled with high rates of aflatoxin exposure from peanuts.

Q. How does indole-3-carbinol protect against liver cancer caused by aflatoxin? Is it a mechanism different from the blocking mechanism you described with chlorophyllin?

A. Yes, it is. There are several possible mechanisms, depending on the animal models you examine. One mechanism involves inducing enzymes in the liver that detoxify aflatoxin. These can be the cytochrome p450s that metabolize aflatoxin B1 into a form that is ten-fold less carcinogenic. We see that in dairy cows and trout. A second protective mechanism is the induction of glutathione transferase isozymes that detoxify the aflatoxin before it can damage DNA. That mechanism does not exist in the trout, but it does exist in the rat and is thought to be important in humans. A third mechanism that we discovered in the trout gets rather little attention, but it's probably more important than most of us think. It involves the binding of I3C metabolites to enzymes that are necessary to activate the carcinogen. With rainbow trout it's easy to show that physiological levels of I3C severely inhibit the activity of the enzyme that converts aflatoxin to its carcinogenic form. That mechanism has been shown to operate in all species that have been examined.

Q. Over twenty years ago you published some studies using a synthetic flavonoid called beta-naphthaflavone and the preservative BHA in preventing liver carcinogenesis. You also investigated nitrosamines. What did you find?

A. Beta-naphthaflavone was just as potent as I3C dose for dose. It's a compound that acts in the trout by the mechanisms I mentioned—it's an inhibitor of the enzyme that activates aflatoxin. In rodents, it induces phase 1 and phase 2 detoxification enzymes. It doesn't induce phase 2 enzymes in trout though. BHA has been shown to be generally quite effective as a cancer chemoprotective compound in rodents. We did some studies in collaboration with Dick Scanlan in the Food Science Department at OSU. We tested I3C in rainbow trout exposed to nitrosamines, which caused liver cancer, and found that I3C effectively inhibited tumor development.

Q. You also looked at the effect of ellagic acid or chlorophyllin in preventing dimethylbenzanthracene-induced cancer in trout. What did you find?

A. Dimethylbenzanthracene is a very potent synthetic carcinogen in rainbow trout that gives us the ability to study cancer in the kidney, stomach, liver, and swim bladder at the same time because it hits all those target organs. We got some curious results with I3C against that carcinogen. The dose-response relationship was particularly interesting. Ellagic acid is found in many kinds of berries—raspberries, blackberries, and black raspberries. In those experiments, we collaborated with Gary Stoner of Ohio State University. Gary had determined that ellagic acid acted as a tumor suppressing agent in esophageal carcinogenesis. That means that it's given after cancer is initiated, and you look at its effects on the further expansion and development of tumor cells. We tested ellagic acid in rainbow trout to see if it was effective as a tumor suppressing agent against stomach carcinogenesis initiated by dimethlybenzanthracene. We found that it was a pretty potent suppressing agent against stomach cancer, although the liver cancers in the same animals initiated by the same carcinogen were not affected. There are problems with ellagic acid, though. It has virtually no bioavailability—it's not taken up by the body, so it's not surprising that it wouldn't work in the liver. And that's why it's been effective in a gastrointestinal model where there's at least some access and ability to interfere with subsequent tumor development.

Q. One of your more recent studies investigated the effect of tomatine in carcinogenesis. What is that compound and where does it come from?

A. Tomatine and tomatadine are flavonoid-type compounds found in tomatoes, especially in green tomatoes. That work came about as a result of a small meeting that I go to every year called Western Regional Project W-2122. One of the members of that project is Mendel Friedman, who works at the USDA facility in Albany, California. Mendel was doing some work with tomatine and tomatadine and found them to be interesting anti-bacterial agents. He suggested that it may be worthwhile to try to increase the amount of these compounds in tomatoes. And I wanted to know about the effects on cancer. Is there any risk? So we planned a trout experiment with Mendel. Tomatine turned out to be a pretty effective blocking agent. We don't know what it will do post-initiation, because we didn't have enough tomatine for that kind of experiment.

Q. You've also done some work in trout with cooked-meat mutagens and carcinogenic polycyclic aromatic hydrocarbons produced by combustion. Did you find any dietary compounds that are chemoprotective?

A. We tested chlorophyllin in trout and saw exactly the same mechanism that we saw against aflatoxin—the ability to form strong complexes between chlorophyllin and the carcinogen, resulting in a reduction in the bioavailability of the carcinogen to the whole animal. We didn't find any effect of chlorophyllin on genes important in carcinogen metabolism. In the early 1980s, I went to a conference in New Orleans, where one of the speakers was Dr. Takashi Sugimura, who discovered the heterocyclic amines or cooked-meat mutagens. He became very interested in our work, so we arranged for my first visit to Japan to try to see if these compounds would cause cancer in rainbow trout. I was particularly interested because nobody had done a chemoprotection experiment at that time with these mutagens. Unfortunately, the four heterocyclic amines that we tested in trout by embryonic microinjection or dietary exposure were not carcinogenic. At the time, we didn't have rodent models that may have produced different results. When Rod Dashwood left my lab and went to Hawaii to begin his own career, he was able to break away from the trout model and tackle the work with rodents. And now he is back at OSU as a principal investigator in LPI.

Q. Based on your knowledge of carcinogenesis and chemoprotection, what can we do to minimize the risk for cancer?

A. When you look at the experimental animal data, it's very clear that many components of fruits and, especially, vegetables are protective in a large number of models. The isothiocyanates in cruciferous vegetables and catechins in tea that Rod has worked with are very effective in many animal models. And now some translational research is being done in humans, especially for esophageal and oral carcinogenesis. The tea catechins look pretty effective for people at high risk. But there is a question for most of us, is there compelling evidence that a diet high in vegetables and fruits, or specific supplements, protects against most cancers? The epidemiological research at present just doesn't support that. So we have to ask, why is this so? Epidemiological studies rely on food recall questionnaires and so on, and many of us don't have a lot of faith in that because human memory is too selective—you're not gathering real data. We're really going to know with more certainty when we have a large enough body of prospective studies with either biomarkers or cancer end points. That's when we'll know the answer. Some risks can be estimated from epidemiological studies. For example, we know that high-calorie, high-fat diets put us at increased risk for endometrial cancer and a number of other cancers.

Q. Do you think that there's any benefit to eating organic food as opposed to food that's been grown with synthetic pesticides?

A. The array of putative cancer chemoprotective phytochemicals is the same in organic or non-organic food. You could avoid the foods that are not organic if you are convinced that you're at some risk from the chemicals that are used in the farming industry to produce the crop. In terms of the environment—the ecosystem—reducing the amount of synthetic pesticides would be a good strategy. To protect the environment we have to pay a cost. And the cost would be, as you know, somewhat lower levels of food production and more blemishes on the food we consume.

Last updated June 2008