LINUS PAULING INSTITUTE SPRING/SUMMER 2005 RESEARCH REPORT
Roderick H. Dashwood, Ph.D.
Q: Much of your research is focused on tea and cancer. How did you get interested in tea?
A: A study by my former professor from England, Costas Ioannides, reported that when rats were given two percent green tea in their drinking water, the tea produced changes in their liver enzymes. What really grabbed my attention was that the liver enzyme most strongly altered in those studies was the same enzyme that metabolically activates heterocyclic amines. We’ve been working with these compounds from cooked meat and cooked fish and know a lot about their chemistry and mutagenicity. One of the important liver enzymes that converts these compounds from an inactive form to their active form is called CYP1A2, which is part of the cytochrome P450 system. That specific isozyme was strongly induced by green tea, suggesting the possibility that tea might activate rather than detoxify these cooked-meat mutagens. We therefore hypothesized that rats fed green tea and given heterocyclic amines might actually be at an increased risk for colon cancer. When we did the experiment, we found that rather than increasing the risk, green tea decreased the risk of developing precancerous lesions in the colon called aberrant crypt foci.
Q: Are these liver enzymes the same in rats and humans?
A: The classification of these cytochrome P450 enzymes in rats and humans is a little tricky and complicated. While the terminology is often the same, actual enzyme activity can vary in the different species. We do know, however, that green tea induced the detoxification of the cooked-meat mutagens, resulting in fewer preneoplastic lesions. We also know that the detoxified metabolites in rats, called glucuronides, are also the primary metabolites that are excreted in people who eat meat. Again, there are some slight differences in chemical structure between the rat and human metabolites, but they are formed by similar metabolic processes.
Q: Based on those results, do you think that tea would likely protect against colon and other cancers in people?
A: I don’t really know. There is excellent evidence for the chemoprotective role of tea against various cancers in animals, but the human epidemiological evidence is less compelling.
Q: How are the cooked-meat mutagens formed in food?
A: During high-temperature cooking, amino acids like L-phenylalanine fuse with creatine or creatinine, which is present in muscle meat. This process may be influenced by the presence of certain types of sugars, as well. Cooking pyrrolyzes these compounds to create new mutagens. They were identified as potent bacterial mutagens in the 1970s by Sugimura and colleagues in Japan, although they were probably first discovered about 60 years ago by a Swedish scientist who was looking at extracts of grilled horsemeat injected into mice that produced tumors.
Q: What kind of teas do you use in your experiments?
A: In our initial studies we found that green tea was more effective than black tea at inhibiting the aberrant crypt foci induced by cooked-meat mutagens. Subsequently, our lab has investigated white tea.
Q: How does white tea differ from the other types of tea?
A: Typically, the tea leaf is picked and withered, followed by a number of other processing steps that oxidize the polyphenols. That’s true for green tea, oolong tea, and black tea. With white tea, there is no withering step or further processing. Also, white teas contain buds and leaves, whereas other teas are mainly leaves, so the dried tea doesn’t look green—it has a pale appearance. This was nicely illustrated in the LPI Fall/Winter 2002 Newsletter article by Jane Higdon.
Q: You mentioned polyphenols. Have you found that these compounds extracted and purified from tea are as effective as whole tea?
A: That is one of the interesting studies that we did early on. We decided to compare white tea with green tea and found that the white teas had greater antimutagenic activity in our assays than the green teas. Then we analyzed white teas using high-performance liquid chromatography. We wanted to know if the greater antimutagenic activity was due to some unique polyphenol in the white tea. While all of the major constituents that we saw in green tea were also present in the white tea, the relative levels were different. After we identified the nine major catechins and caffeine that were in the teas, we made an artificial tea containing those compounds in the same concentrations found in tea. When we tested the reconstituted tea, it had only about 50% of the antimutagenic potency of the complete tea. This tells us that while we’re focusing on the major constituents, minor constituents may be synergizing with the major polyphenols and increasing their antimutagenic activity. Because of these results, we are now comparing complete tea with individual tea polyphenols in animals.
Q: Tea contains antioxidant compounds, but, as you mentioned earlier, also affects liver enzymes. Do you think that the anticancer effect of tea in animals is primarily related to antioxidant properties?
A: That begs the question, is there good evidence in vivo for a role of oxidative damage in cancer? I think that the jury is still out on that question. The available biomarkers used historically for looking at oxidative damage and oxidative stress in vivo are not highly accurate or reliable, partly because of ex vivo artifacts generated during the methodology. We may indeed one day come up with reliable markers of oxidative damage and show definitively that teas have antioxidant properties in vivo. But I think that the anticancer effect of teas is also due to their effect on enzymes in the liver, phase 1 and phase 2 enzymes, and mechanisms related to cell signaling and apoptosis.
Q: What do you mean by apoptosis?
A: Apoptosis is programmed cell death. Before a baby is born, for example, the skin between fingers is removed by programmed cell death. In the case of colorectal cancer, a substance that triggers apoptosis is considered chemoprotective. In a tissue like the gut, where there is continuous turnover of cells throughout your life, apoptosis is needed to prevent cells from remaining in the colon and accumulating DNA damage that can lead to mutations and cancer. So if you can take a preneoplastic cell and cause it to undergo apoptosis, it’s not going to continue dividing and produce a tumor. Cancer cells in culture undergo apoptosis when treated with tea or tea polyphenols, and we are now studying this in animals.
Q: If the substances in tea are relatively poorly absorbed, how can they affect health?
A: Relatively poor absorption may mean that their concentration is, therefore, increased in the gastrointestinal tract. This may explain why there is evidence for their role in preventing, primarily, GI cancers. If you look at all cancers, you don’t find strong evidence that tea is chemoprotective, but if you look by site, there seems to be some evidence from human epidemiological studies for protection against gastric cancer, possibly colon cancer, and perhaps esophageal cancer. Ironically, the interest in tea and cancer was initially focused on the possible causal role for tea in esophageal cancer. This has been discounted because it was not tea itself but the high temperature of consumed fluids that provoked esophageal cancers.
Q: There is enormous variability in the type and amount of tea that people drink. In your experiments, have you looked into the consequences of the strength of the tea or the frequency of consumption?
A: We have looked at the effect of brew time and tea concentration during brewing on antimutagenic activity. If you have a very concentrated brew, and brew that for one minute, would it be equivalent to brewing something for four minutes where the initial concentration of leaves was four-fold lower? If you’re short on money and the tea’s expensive, can you cut back on the number of leaves during brewing but then brew for a longer period of time? As far as we can tell, based on the chromatographic analyses and the antimutagenic activity, there seems to be no extra benefit from using highly concentrated tea or brewing for a shorter period of time. Of course, these issues affect flavor. A lot of tea connoisseurs recommend that you brew with a high concentration of leaves for a short period of time to avoid some of the astringency.
Q: You have mainly discussed the role of tea in chemoprotection against cancer. Do you think that tea might play a role in cancer treatment?
A: That’s a tricky question. Researchers have shown that some cancer cells are indeed more sensitive to apoptosis induction by tea and that tea polyphenols can inhibit a process called angiogenesis, or blood vessel growth, thereby starving early cancers of their blood supply. Much more work needs to be done in this area.
Q: Your most recent paper was on the antimutagenic activity of spearmint tea. Do you know the mechanism that might be involved?
A: Strictly defined, tea is from Camellia sinensis and processed as I mentioned, resulting in white, green, oolong, and black teas. All of those teas have well-characterized polyphenols. Herbal teas are completely different. It is a very broad group that is typically much less well characterized. We brewed the spearmint herbal tea as you would brew a regular tea. We took the dried leaves and brewed for 5 minutes. Then we tested the tea in vitro and in vivo, but we haven’t done any human studies yet. And, just as we have done with the other teas, we began by showing that spearmint tea had very good antimutagenic activity against cooked-meat mutagens in our mutagenicity assay. However, unlike tea from Camellia sinensis, the anticancer mechanism seemed to involve the degradation of activated forms of the carcinogens. This is called electrophile-scavenging, which means that the carcinogen that has been metabolized by liver enzymes is prevented by mint from binding to DNA and causing mutations. We don’t know yet what compounds in spearmint may be responsible. Nonetheless, the results were sufficiently promising to then go on to test spearmint tea in rats. Those studies showed very clearly that mint protected against aberrant crypt formation, which are precancerous lesions in the colon.
Q: You’ve also studied indole-3-carbinol from cruciferous vegetables like Brussels sprouts and broccoli. What did you find?
A: I began those studies about 20 years ago with LPI Principal Investigators George Bailey and David Williams. Indole-3-carbinol is a very powerful inhibitor of tumor formation in rats, mice, and other species, including trout, but it has very interesting protocol-dependent effects. For example, if given to trout before and during exposure to aflatoxin, which is a potent liver carcinogen, it inhibits liver cancer. If you give the aflatoxin first, and then stop treatment and give indole-3-carbinol, the indole-3-carbinol actually promotes liver cancer. This raises questions about the ongoing clinical trials with indole-3-carbinol—it may be a double-edged sword that protects against cancer in some circumstances and promotes cancer in others.
Q: Does this seem to underscore the importance of compounds in food possibly working together, as you found with tea?
A: I think that’s likely. For example, once indole-3-carbinol has been consumed, it’s converted in the stomach to a mixture of compounds that have all sorts of different biological properties, which probably explains why the outcome with indole-3-carbinol is so complicated.
Q: Dr. Bailey and you have also done some anticancer research on chlorophyll that suggests it may have different effects at low doses and high doses. Why might that be?
A: Again, we think that it is related to what we’ve already discussed—the issue of multiple mechanisms. In the case of chlorophyll and chlorophyllin, one of the chemoprotective mechanisms is the molecular “sandwiching” of carcinogens, rendering them less biologically available. It’s a very simple mechanism, but one that seems to hold up from our studies in trout, rats, mice, and even in people. In sufficiently high concentrations, the chlorophyll molecule is able to bind to mutagens in the gut and to reduce their uptake or bioavailability, so that more of the mutagen is going through the gut and then excreted. With low concentrations, other mechanisms seem to be occurring that affect apoptosis and cell-cycle progression. If you have severe damage to DNA in the cell, normally the cell cycle will arrest and the damage will be repaired, or if it can’t be repaired, the cell will undergo apoptosis. With low doses of chlorophyllin, that process seems to get disrupted, and the decision to proceed or become apoptotic gets confused. If you use higher doses, then apoptosis occurs, as it should.
Q: What’s the difference between chlorophyll and chlorophyllin?
A: Both molecules have a structure shaped like a “satellite dish” that traps sunlight energy for photosynthesis. That same dish-shaped structure explains the ability to complex with carcinogens. Chlorophyll, the naturally occurring molecule, has a magnesium atom at the center of its ring structure, whereas chlorophyllin typically has copper. Unlike chlorophyll, chlorophyllin is very water soluble.
Q: Why do you use chlorophyllin instead of chlorophyll?
A: One reason is cost—chlorophyllin is about 1,000 times less expensive. Chlorophylls are very difficult to work with— they are light sensitive and very susceptible to oxidation. It can be difficult to isolate pure chlorophyll from a leaf and keep it as pure chlorophyll without degradation. Chlorophyllin also has very potent antimutagenic activity in the mutagenicity assays, even more so than chlorophyll.
Q: One of your graduate students, Mindy Myzak, conducted experiments with sulforaphane, which is another phytochemical in cruciferous vegetables that might inhibit cancer. What is sulforaphane?
A: Sulforaphane is a very interesting molecule discovered by Dr. Paul Talalay at Johns Hopkins. He was screening cruciferous and other vegetables to identify those with the greatest ability to induce detoxifying enzymes. He found that cruciferous vegetables, especially broccoli, were the most potent inducers of detoxifying enzymes. He then identified one substance in broccoli that had superior activity over the others, which is called sulforaphane. Subsequently, he has shown that broccoli sprouts have about 50 times more sulforaphane than even broccoli crowns.
Q: How does sulforaphane work?
A: Sulforaphane is a potent enzyme inducer, but Mindy became interested in a different mechanism. If you treat cancer cells with sulforaphane, they undergo apoptosis and increase the levels of certain proteins that are known to be tumor suppressors. This has nothing to do with the ability to induce enzymes that detoxify carcinogens, as far as we can tell. Mindy described this work in the LPI Spring/ Summer 2004 Research Report. We are very excited because these studies are leading us into the province of cancer treatment instead of cancer prevention, which was one of Linus Pauling’s interests.
Q: Have you done any work with sulforaphane in animals?
A: The paper we published last year in Cancer Research focused on human colon cancer cells in culture. Our collaborator, LPI Principal Investigator Emily Ho, has some corresponding data in human prostate cancer cells, as well as in benign prostate hyperplasia, which look very promising. Mindy has now done some in vivo experiments and has some very promising data that we haven’t published yet. She finds that many of those molecular changes that are occurring in cancer cells in culture are recapitulated with either short-term or long-term treatment in mice and rats. In fact, she has shown that colon polyps that occur spontaneously in the mouse can be suppressed by sulforaphane through this newly discovered mechanism.
Q: Does cooking destroy sulforaphane, indole-3- carbinol, or some of these other chemoprotective compounds?
A: There are many cooking practices that may affect these compounds, but their metabolites have been detected in human urine, indicating that they were absorbed to some degree.
Q: In your tea experiments, you provided tea to animals in concentrations that people drink. Have your experiments with indole-3-carbinol, sulforaphane, and chlorophyllin been done at physiological concentrations? In other words, is the experimental dose comparable to what people would get dietarily by consuming foods that contain these substances?
A: That’s definitely the case with chlorophyllin and indole-3-carbinol. There are lots of studies that used a broad range of concentrations spanning the scope of probable human exposure, as well as pharmacological doses—very high doses that one finds in some supplements from the health-food store. Much less work has been done with sulforaphane in a broad array of doses, although we designed our experiments so that the doses of sulforaphane are comparable to what people would get from eating broccoli.
Q: You mentioned that you’ve begun to look at potential cancer treatment, using cells or animals, with sulforaphane. Is there much evidence that other phytochemicals are useful in treating cancer?
A: Michael Gould of the University of Wisconsin has done some research on monoterpenes, including limonene from citrus peel, and perillyl alcohol. These are being evaluated in preliminary clinical cancer trials. Because of the different mechanisms involved in cancer prevention and cancer treatment, it’s not reasonable to expect all chemoprotective agents to be valuable therapeutic agents. Also, we need to understand the side effects or toxicity, if any, associated with the use of some of these phytochemicals.
Q: The National Toxicology Program of the National Institute of Environmental Health Sciences recently released the 11th annual report on carcinogens. Did you help advise the NIEHS?
A: Yes, I was on the committee of experts. This was part of a federally mandated review process that is often driven by public interest, and anyone can write to request an evaluation of a particular substance. If there is sufficient weight of evidence to move forward, the NTP and NIEHS will ask experts to consult and review documents for the report. In this latest report, the NTP added certain cooked-meat mutagens that we’ve talked about, such as PhIP.
Q: You study these compounds in relation to colon cancer. What are the main causes of colon cancer?
A: A major risk factor for colon cancer is age—people 50-years-old and older are at a greater risk for developing colorectal cancer. However, people who exercise regularly, three times a week or more, and who maintain a healthy body weight throughout life are at a much reduced risk for colorectal cancer. And another risk factor is poor diet. Diet may be responsible for one- to two-thirds of colorectal cancers. Cooked-meat intake seems to be a factor for increased risk, although the type of cooked meat and precisely what it contains are still questions. Consumption of animal fat may be another risk factor, but vegetables may be protective. We don’t yet know for certain if fiber in vegetables actually prevents colon cancer. There seems to be very good evidence that calcium is a chemoprotective agent, and there’s also some evidence for selenium and folate.
Q: Inflammation has been implicated in cancer. What exactly is inflammation?
A: One definition is that inflammation is a process in which host defenses generate reactive oxygen species to kill pathogens.
Q: How would inflammation affect colon cancer risk?
A: There are a number of known inflammatory bowel diseases, such as Crohn’s disease. In these cases, a clear connection between increased inflammation and increased risk of colorectal cancer has been observed. We know that non-steroidal anti-inflammatory drugs (NSAIDs) can protect against colon cancer in people with familial adenomatous polyposis. So that leads us to believe that inflammation plays a role. There are also animal models, some of which we are now using, in which treatment with agents that markedly activate inflammation in the gut through a bowel irritation mechanism greatly increases colon cancer incidence. However, the recent concerns over Vioxx and Celebrex have complicated this story, and we might need to find other ways to control inflammation, perhaps with dietary agents like tea.
Q: You are the head of the LPI Cancer Chemoprotection Program. What is the CCP?
A: The CCP was introduced 5 years ago and has three goals. One is to examine the mechanisms by which dietary factors may be implicated in prevention or treatment of cancer in the broadest context. There are many agents that have already been identified by labs all around the world, but in many cases we are ignorant of the biological mechanisms. We think it’s very important to understand mechanisms thoroughly in order to evaluate the pros and cons. We don’t want to advocate supplements and find out later that they have deleterious consequences, as, for example, was found with beta-carotene and lung cancer in smokers. Another important goal is to identify novel chemoprotective agents in the diet, so we screen for these in terms of antimutagenic activity or antioxidant activity, and we have fully developed assays to examine DNA damage. A third important goal is to educate the public through lectures, the LPI Web site, the Micronutrient Information Center, and the LPI Research Report about the role of diet and lifestyle in cancer prevention.
For information on tea and health, see the Linus Pauling Institute's Micronutrient Information Center.
Last updated May, 2005
Micronutrient Research for Optimum Health
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