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Numerous chemicals from fruits and vegetables protect against the damage wrought by carcinogens in experimental animal models. Usually these chemicals occur in edible plants at such low levels that doses sufficient for protection are not practically attained even in a balanced diet. Because of their abundance in green vegetables, chlorophyll and its widely used derivative, chlorophyllin, have attracted attention as potential anti-carcinogens. Scientists in our laboratory have studied the chemoprotective effects of chlorophyllin and have recently discovered that natural chlorophyll itself is a potent anticancer agent.

Chlorophyllin is easily and inexpensively made from crude chlorophyll and has been used for decades without known human toxicity as a food dye, a wound-healing accelerant, and for odor control. The anticancer properties of chlorophyllin have been extensively reported in dozens of studies from cell culture to rats, where different chemical carcinogens were used to initiate cancer. From these studies we have learned that chlorophyllin acts primarily as a blocking agent against chemical initiation of carcinogenesis. Simply put, chlorophyllin is most effective when administered along with the carcinogen, thereby blocking cancer-initiating activity. Molecular complexes formed between chlorophyllin and carcinogen molecules are physically too large to be absorbed from the gut. Because fewer carcinogen molecules reach the target organ, less DNA damage occurs, and the chance of tumor development is diminished. Consistent with the blocking mechanism, chlorophyllin is generally much less effective if administered after the carcinogen, i.e., once the carcinogen’s damage has been done.

Importantly, protection by chlorophyllin extends beyond a single chemical carcinogen to include aflatoxins from heavy fungal contamination of grain or nuts; heterocyclic amines, whose primary source of exposure is overcooked meat; and polycyclic aromatic hydrocarbons (PAH), an increasingly abundant class of combustion-derived air pollutants. While aflatoxin B₁ exposure is insignificant in developed countries with safe grain storage methods, it is prevalent in Asia and sub-Saharan Africa, where it contributes to astonishingly high rates of liver cancer. The latter two carcinogen classes represent significant exposures for the U.S. population, given high consumption of grilled meats and huge increases in airborne PAH-bound particulate matter, especially from diesel exhaust.

Significant protection against cancer by chlorophyllin was reported in the Fall/Winter 2002 LPI Research Newsletter (“Chlorophylls and Cancer Prevention: Passing the First Hurdle,” by Dr. George S. Bailey). That article summarized the results of a clinical trial of dietary chlorophyllin supplementation in a human population in eastern China with chronic, unavoidably high aflatoxin exposure and a high incidence of liver cancer. Administration of 100 mg of dietary chlorophyllin (in pill form) thrice daily led to a highly significant 55% reduction in the amount of aflatoxin-DNA adducts (substances connected by a chemical bond; in this case, indicative of DNA damage) in the urine of participants. Elevated urinary output of this hepatic DNA adduct biomarker in humans is clearly associated with increased risk of liver cancer, and diminished levels of aflatoxin-DNA adduct are associated with reduced liver cancer risk in several animal studies. Thus, simple dietary supplementation with chlorophyllin might cut human liver cancer risk in half for people chronically exposed to high levels of dietary aflatoxin. A long-term, 20-year clinical trial is now being conducted by Chinese investigators to evaluate the reduction of liver cancer incidence by chlorophyllin.

In impoverished regions, where diet choices are limited to survival staples and grain storage methods and pollution exposure will only improve with increases in the standard of living, dietary supplementation with chlorophyllin might be the easiest and most effective protection strategy to implement. In the developed world, a diet high in natural chlorophyll from vegetable consumption could offer substantial protection against food- and air-borne carcinogens, in addition to all the other known benefits of a vegetable-rich diet.

Dietary chlorophyll intake comparable to the 300 mg per day of chlorophyllin administered in the Chinese human intervention trial is obtainable by moderate-to-high consumption of green vegetables. Chlorophyll has no known human toxicity, but its protective properties have been little studied. This is likely due to the extraordinary cost of commercially pure chlorophyll (necessary for unambiguous experimental evaluation), or the difficulty and expense of purification in the laboratory.

Chlorophyll is potently anti-mutagenic and was recently shown to induce carcinogendetoxifying enzymes of phase 2 metabolism in cell cultures. A few studies have examined natural chlorophyll as a cancer preventative in animals. In rainbow trout, exposure to 200 parts per million (ppm) of dibenzo[a,l]pyrene (the most carcinogenic PAH known) resulted in hepatic DNA-adduct formation. Adduct formation was reduced 66% by co-exposure to 3000 ppm chlorophyll in the diet, which was nearly identical to the protection we observed by a similar dietary co-exposure to chlorophyllin. Another lab reported that dietary spinach or an equivalent dose of chlorophyll equally inhibited the proliferation of colon cells in rats induced by heme, an ironcontaining pro-oxidant from red meat that is correlated with increased risk of colon cancer. The chlorophyll-containing diets also largely blocked formation of a toxic heme metabolite. The authors speculated that green vegetables may decrease colon cancer risk from dietary heme through the protective effects of chlorophyll.

Our lab’s current research is focused on the rigorous testing of cancer prevention by natural chlorophyll in trout and rats. We recently examined protection by dietary chlorophyll in a rainbow trout multi-organ tumor model. Duplicate groups of 140 juvenile trout were exposed via the diet for four weeks to 224 ppm dibenzo[a,l]pyrene (DBP) alone, or with 1000, 2000, 4000, or 6000 ppm chlorophyll, then returned to the control diet. DBP induced high tumor incidence (number of fish with at least one tumor in each treatment group) in the liver (51%) and stomach (56%), and a low incidence in swim bladder (10%) nine months after initiation. Co-feeding 2000, 4000, or 6000 ppm chlorophyll significantly reduced stomach tumor incidence to 29, 23, and 19%, respectively, and liver tumor incidence to 21, 28, and 26%, respectively. A troubling result from the study was that dietary chlorophyllin given after carcinogen exposure (DBP diet, followed by 2000 ppm chlorophyllin for the duration of the study) had no effect on liver or stomach tumor response but promoted swimbladder tumor incidence to 38%. This finding supports previous evidence that chlorophyllin chemoprevention is not without some potential risk. Post-initiation promotion by chlorophyllin has previously been reported in the rat colon in specific experimental designs. However, in the one study where postinitiation effects of natural chlorophyll on colon carcinogenesis were examined, chlorophyll suppressed rather than promoted pre-cancerous lesions of the rat colon. The available evidence from previous studies and our recent finding in trout suggest that natural chlorophyll may be superior to chlorophyllin as a choice for chemoprevention in humans.

Our finding that chlorophyll substantially reduced tumor formation in trout compelled us to look for similar protection by chlorophyll in rats. We first studied protection against early biomarkers of carcinogenesis. Three groups of seven rats each received five daily doses of 250 ppb aflatoxin B₁ alone, aflatoxin with 250 ppm chlorophyllin, or aflatoxin with natural chlorophyll equivalent to 250 ppm chlorophyllin. The aflatoxin was attached to a radioactive isotope, or radiolabeled, so that we could follow and measure it. Chlorophyllin and chlorophyll strongly reduced liver DNA adduction by 42% and 55%, respectively, and serum albumin adducts by 65% and 71%, respectively. The feces of chlorophyllin- and chlorophyll-treated rats contained 137% and 412% more radiolabel, respectively, than control (aflatoxin only) feces, indicating that chlorophyllin and chlorophyll inhibited aflatoxin uptake from the gut, restricting its distribution to the GI tract. This finding is consistent with chlorophyllin acting as a blocking agent, i.e., by binding the carcinogen and blocking its uptake to the bloodstream. A similar result for chlorophyll suggests that it, too, may protect by this mechanism. We examined whether chlorophyllin and chlorophyll protection might also occur at the metabolic level, after uptake from the gut. Chlorophyllin and chlorophyll were both recently shown by some of our collaborators to induce higher activity levels of the carcinogen-detoxifying enzymes of phase 2 metabolism in cell cultures. However, in the rat liver, neither co-treatment induced activity of the phase 2 enzymes quinone reductase and glutathione S-transferase (GST) above control levels.

A second study was done to examine the protection against late pathophysiological markers in the rat liver and colon. We specifically examined the effect of chlorophyllin and chlorophyll co-treatment on GST-placental form positive staining foci in the liver and aberrant crypt foci in the colon. Both foci types are small populations of cells that have undergone a hyperplastic transformation and, while not yet cancerous, will often become so. Twenty-nine rats in three experimental groups were treated with chlorophyll or chlorophyllin for ten days. At 18 weeks after carcinogen exposure, the chlorophyllin and chlorophyll co-treatments had reduced the percentage of GST-placental form positive foci in the liver by 74% and 77%, respectively, compared to control livers. Chlorophyllin and chlorophyll reduced the mean number of aberrant crypt foci per colon by 63% and 75%, respectively.

Our results show that both chlorophyllin and chlorophyll protect against early biochemical and late pathophysiological biomarkers of aflatoxin carcinogenesis in the rat liver and colon, and against PAH-initiated cancer in the rainbow trout. These studies provide the first demonstration in any animal model of cancer chemoprotection by dietary natural chlorophyll, which may be a less problematic choice for human intervention than its derivative chlorophyllin. While our results are entirely consistent with chlorophyll acting as a blocking agent like chlorophyllin, further experiments are needed to determine the precise inhibitory mechanism by chlorophyll of aflatoxin and dibenzo[a,l]pyrene uptake. The results also support the idea that increased consumption of vegetables with high chlorophyll content may substantially lower cancer rates among human populations at high risk from exposure to aflatoxin and polycyclic aromatic hydrocarbons.