11.28.01 Chlorophyllin Reduces Aflatoxin Indicators Among People At Risk For Liver Cancer

George S. Bailey, Ph.D.
OSU Distinguished Professor
Department of Environmental and Molecular Toxicology

One in ten adults in the Yangxi delta region of rural China dies of liver cancer. This extraordinary statistic is due to a combination of two risk factors—chronic hepatitis B virus infection and unavoidable dietary intake of the potent liver carcinogen aflatoxin B₁ from moldy corn and other grains. Other regions of the world where these two risk factors co-exist have similarly high incidences, making liver cancer one of the three leading causes of cancer death worldwide. In August of 1997, I traveled with Dr. Thomas Kensler of Johns Hopkins University to the tiny township of Daxin, China, to initiate a clinical intervention trial aimed at reducing this terribly high cancer risk. Our focus in this first intervention study was to determine if the biological effects of dietary aflatoxin B₁ exposure might be reduced by ingestion of chlorophyllin, a food dye supplement that is a water-soluble derivative of the ubiquitous green plant pigment chlorophyll. The design of the study and the two decades of research leading up to it make a classic tale in the translation of basic research from cell cultures to experimental animals and finally to humans. 

The possibility that simple chlorophyll derivatives might aid in cancer prevention arose in the 1980s, when researchers in Italy, Japan, and the U.S. discovered that chlorophyllin could reduce the ability of certain mutagenic chemicals to damage the genes of bacteria and fruit flies. This was particularly interesting because chlorophyllin had several human uses without known toxicity. It has been used, for instance, as a green food dye in some countries, for accelerated wound healing, and for odor control in geriatric patients. 

Indeed, the ability of chlorophyllin to counteract malodorous chemicals may be a clue to its anti-mutagenic action. Hikoya Hayatsu in Japan, Roderick Dashwood (first at Oregon State, then at the University of Hawaii), and Vibeke Breinholt in my laboratory were able to show in the 1980s and 1990s that chlorophyllin had an ability to bind or "sandwich" certain classes of chemical mutagens and carcinogens. These included polyaromatic hydrocarbons found in tobacco smoke, some heterocyclic amines ("cooked meat mutagens"), and aflatoxin B₁—chemicals suspected or known to cause human lung, colon, or liver cancer, respectively. In order to initiate the cancer process, these carcinogens must be first metabolically "activated" into intermediates that can attack DNA and other cell components. The tight binding of chlorophyllin to these mutagens/carcinogens was shown to interfere with this critical activation step and thus seemed to be a principal mechanism for preventing mutations in cultured cells. 

It was not at all clear, however, that chlorophyllin would be effective in whole animals, with their complex processes of gastrointestinal absorption, biodistribution through the bloodstream, re-uptake in critical target organs, and distribution within the target organ to the cells important in the cancer process. It was possible, for instance, that dietarychlorophyllin would never get to liver cells in amounts sufficient to protect against aflatoxin B₁ metabolism. Protection in a whole animal model was investigated for the first time by Dr. Dashwood in his final experiments before departing my lab for Hawaii. In a landmark study, Rod found that rainbow trout fed chlorophyllin at the same time they were fed aflatoxin B₁ had far less aflatoxin-DNA damage in their livers than trout receiving only aflatoxin and that the degree of protection increased with the amount of chlorophyllin fed. Although this experiment did not define exactly how chlorophyllin operated, it was the first published study to indicate the possibility of chlorophyllin protection in any whole animal model. 

Next, we had to determine if reduction of aflatoxin-DNA damage by chlorophyllin would necessarily lead to reduced cancer later in life and, if so, if there was a direct cause-effect correlation. Experiments to address these issues were completed four years later by Vibeke Breinholt in my lab. Her experiments, published in the journal Carcinogenesis in 1995, showed that increasing doses of dietary chlorophyllin did, in fact, provide increasing protection against aflatoxin B₁-initiated liver cancer in trout. She also found that animals receiving low, human-relevant aflatoxin B₁ doses were protected just as well as those receiving very high aflatoxin doses. Moreover, Vibeke found that the degree of eventual cancer protection by dietary chlorophyllin was predicted exactly by its degree of protection against aflatoxin-DNA damage early in the cancer process. That is, aflatoxin-DNA damage in liver served as an early biomarker for predicting chlorophyllin cancer protective effects many months before cancers could actually be detected. The low-cost trout model is still the only established experimental system in which such statistically demanding predictive correlation studies can be carried out. 

The demonstration that aflatoxin B₁-DNA adducts (substances connected by a chemical bond) in the liver could be used as early biomarkers to predict chlorophyllin-mediated reduction of tumor initiation was critical. This meant that initial studies in humans could concentrate on short-term biomarker reduction rather than liver cancer itself, which takes 20 or more years to develop. However, two final questions remained before such studies could be proposed: 1) Will chlorophyllin results in trout translate to mammals? 2) If so, which biomarkers would be most useful to study human intervention? 

The first question reflects the thinking of many skeptics, who are more inclined to believe findings with rats or mice than with trout. Rod Dashwood addressed this issue in part by showing in 1995 that chlorophyllin treatment protected rats against heterocyclic amine-induced cancer in several organs, including the colon, just as we had found for aflatoxin in the trout. Later on, Tom Kensler and John Groopman carried out a short-term study examining the effects of chlorophyllin co-treatment on liver aflatoxin B₁-DNA adduction in vivo in the rat. They chose for their study a chlorophyllin dose that provided about 50% protection in the trout. Amazingly, this same dose also reduced liver DNA damage in the rat by 50%. These experiments left no doubt that chlorophyllin could protect against carcinogenesis through mechanisms that were not unique to the trout as a model or to aflatoxin as a carcinogen. 

The second question of which biomarker to use was critical. Obviously, it is not feasible to obtain serial liver biopsies on large numbers of healthy people to determine levels of aflatoxin-DNA damage in that organ during the course of a study. Drs. Kensler and Groopman recognized this limitation many years ago and have spent much of their career developing and validating additional biomarkers of aflatoxin exposure in the rat model. In particular, they had shown that most of the aflatoxin B₁ absorbed in one day was excreted as metabolites in the urine. They also had developed highly sensitive immunologic assays to quantify these metabolites. One such metabolite was particularly interesting—the specific aflatoxin B₁-N7-guanyl DNA adduct, which gets largely repaired out of liver DNA and transported into the urine within a few hours of exposure. In the above study, they were able to show that the chlorophyllin-mediated 50% reduction of rat liver aflatoxin-DNA adduct was mirrored by a 50% reduction in the amount of aflatoxin-guanyl DNA repair biomarker appearing in the urine over the next 24 hours. This meant that a fully validated, convenient, and easily accessible biomarker was now at hand to assess the effects of chlorophyllin intervention in aflatoxin-exposed human populations. 

With this information, Drs. Groopman and Kensler successfully applied in 1996 to the National Institute of Environmental Health Sciences for funds to conduct an intervention trial in China. A large number of physicians and technical assistants in China were recruited to help with the study. The study design was a double-blinded, placebo-controlled, biomarker intervention trial, withenough volunteers included to detect a biomarker reduction of 20% or higher with statistical confidence. Among several hundred healthy volunteers determined to have aflatoxin exposure, 180 persons were divided randomly into two groups of 90 each and assigned to receive either a chlorophyllin tablet or a placebo tablet with each meal for four months. Urine and blood samples were taken at the beginning and regularly throughout the four-month treatment process. These were taken by Dr. Kensler to Johns Hopkins University for analysis. All volunteers and their samples were coded throughout the study, so that no one knew who had received chlorophyllin or placebo until the sample analyses were completed and the code was broken.

Technical challenges meant that several years were required to complete the first set of analyses. The code was finally broken in May of 2001, and the results of the study were announced by Dr. Kensler in his presentation at the 2001 LPI "Diet and Optimum Health" conference. Everyone was fascinated, and I was stunned, to hear the result! Volunteers receiving chlorophyllin had a 55% reduction in the urinary aflatoxin exposure biomarker compared to those receiving placebo. This gratifying news carried two implications. First, it showed that what Rod, Vibeke, and I had found in the rainbow trout over a decade earlier was directly translatable to humans. More importantly, this study provides evidence that, for pennies per day, chlorophyllin supplements may cut the liver cancer death rate in aflatoxin-exposed populations in China and elsewhere at least by half. A long-term, 20-year clinical intervention trial will now be needed to determine if this promise can be realized. Our colleagues in China are best positioned to conduct such a study. 

Where does LPI chlorophyll research go from here, and how might this research be targeted to include residents in the U.S., who generally have insignificant aflatoxin exposure? A pending grant application to the National Cancer Institute by Drs. Dashwood, Williams, and myself requests five years of financial support to further investigate chlorophyllin chemoprevention in cultured cells, trout, mice, rats, and in human volunteers on a small scale. We really need to understand exactly how chlorophyllin works and if its mechanisms differ with carcinogen, species, or type of cancer. A second aim is to determine if natural chlorophylls, such as found in spinach and other green leafy vegetables, might have protective activity comparable to chlorophyllin. We will examine colon, mammary, and lung cancer, which are of primary importance to U.S. residents.