Beneficial and Adverse Effects of Natural, Bioactive Dietary Chemicals on Human Health and Food Safety

NARRATIVE: This research project uses animal models of human disease to understand how different components of food may influence human health. Recent studies have included the nonobese diabetic mouse model to study the influence of food contaminants on their ability to modify the development of diabetes. Another animal model mimics the immune response of humans to tissue transplants. These models are important for evaluating the beneficial effects of phytochemicals on diseases that are caused by an overactive immune response. Such diseases include a variety of autoimmune diseases (lupus, MS, type 1 diabetes, rheumatoid arthritis) as well as allergic responses. Studies in progress are evaluating the ability of natural chemicals that bind to the Ah receptor to mimic the effects of TCDD on the induction of a selected type of immune cell, regulatory T cells. These cells play important roles in regulating the immune response and protect against autoimmune disease and allergy. However, overproduction of regulatory T cells also contribute to decreased anti-tumor immunity. <P> APPROACH: The primary approach of this research is to use animal models of human disease. Recent studies have included the nonobese diabetic mouse model to study the influence of food contaminants on their ability to modify the development of diabetes. Other animal models mimic the immune response of humans to tissue transplants to study chemicals that suppress the immune system. Studies in progress are evaluating the ability of natural chemicals that bind to the Ah receptor to mimic the effects of TCDD on the induction of a selected type of immune cell, regulatory T cells. These cells play important roles in regulating the immune response and protect against autoimmune disease and allergy. Overproduction of regulatory T cells also contributes to decreased anti-tumor immunity. Results of these studies are presented at professional meetings. General presentations to the public about food safety related to chemicals and supplements incorporate our research findings. <P>PROGRESS: 2002/10 TO 2007/09<BR>
OUTPUTS: The outputs of the results from this project have been in peer-reviewed journal articles, interviews with local and national magazine, newspaper and television reporters. There has been a great deal of interest in cruciferous vegetables and the risk:benefit of taking indole-3-carbinol or 3,3'-diindolylmethane (major phytochemical components of cruciferous vegetables) as long-term dietary supplements. There has also been a great deal of interest in our research on the cancer prevention associated with intake of white and green teas. <BR> PARTICIPANTS: The Linus Pauling Institute, Dr. Jane Higdon (deceased), Dr. Barbara Delage, Dr. Gayle Orner, Dr. Carole Jubert. Dr. Oriane Carter and Dr. George Bailey. The Environmental Health Sciences Center, Dr. Cliff Pereira, Dr. Jerry Hendricks, Dr. Christiane Lohr, Dr. Michael Schimerlik and Dr. William Baird. Johns Hopkins University Medical School, Dr. John Groopman and Dr. Thomas Kensler.
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IMPACT: 2002/10 TO 2007/09<BR>
The major outcome/impact of these studies has been the generation of a number of pending large extramural grant proposals including: Pending Research Support NIH- R01 ES015251-01-Williams (PI) 12/01/06-11/30/11. NIH/NIEHS Transplacental Lymphomas by PAHs in Mice: Role of Cyp1b1 We seek support to determine if polycyclic aromatic hydrocarbons function as transplacental inducers of lymphoma via bioactivation in fetal thymus by cytochrome P450 1b1. NIH- P42 ES016465-01- Williams (PI) 7/1/08-6/30/13 NIH/NIEHS PAHs: New Technologies and Emerging Health Risks This is a Superfund Basic Research Program grant proposal involving researchers from Oregon State University and Pacific Northwest National Laboratories. The grant consists of 6 research projects and 5 cores. NIH- R01 ES016576-01- Williams/Baird (Co-PIs) 4/1/08-3/31/13 NIH/NIEHS Inhibition of Transplacental Carcinogenesis by Red Raspberry Extract In this proposal Dr. Baird and Dr. Williams will test red raspberry extract and individual components in the maternal diet for their potency in inhibition of PAH transplacental carcinogenesis. NIH/NCI P01 , "Epigenetic Mechanisms of Cancer Chemoprevention- Dr. Roderick Dashwood PI 4/1/08-3/31/13 Dr. Williams is the project #2 Director which focuses on epigenetic mechanisms of transplacental cancer chemoprevention. The total amount of extramural dollars represented by these pending proposals amount to approximately $5 million annually.
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PROGRESS: 2006/01/01 TO 2006/12/31<BR>
Our working hypothesis is that the dietary supplement chlorophyllin might achieve anti-carcinogen protection largely by complexing with carcinogens to reduce their uptake from the GI tract. We were also interested to determine if natural chlorophyll from edible plants might have similar protective properties. The interactions of chlorophyllin (CHL) and natural chlorophyll (Chla)) with two carcinogens were investigated in vitro through complex formation experiments, and in vivo through carcinogen uptake and distribution (pharmacokinetic) studies as well as full tumor studies. We found that Chla and CHL each formed strong non-covalent complexes with the tobacco smoke carcinogen dibenzo(a,l)pyrene (DBP) in vitro, reduced DBP access to liver in vivo in trout, and provided strong, dose-responsive inhibition of tumor formation when fed with DBP. In a companion experiment, CHL and Chla were found to interact strongly with the potently carcinogenic mycotoxin aflatoxin B1 (AFB1) in vitro, to reduce AFB1 uptake and DNA damage in vivo, and to inhibit liver preneoplastic foci and colon aberrant crypt foci when given with AFB1 to the rat. An IRB-approved human intervention study was then initiated to investigate possible effects of CHL and Chla on the uptake and elimination of ultra-low dose (30 ng, 5 nCi) 14C-AFB1 in human volunteers. The protocol used accelerator mass spectrometry to assess the miniscule 14C levels in urine and plasma over time. This study has succeeded in providing the most detailed pharmacokinetic information ever gained for AFB1 in humans. Moreover, results for the first two volunteers in this on-going study demonstrate clearly that modest levels of CHL or Chla given with AFB1 significantly reduce the rate and amount of AFB1 uptake into plasma, and the levels of AFB1 metabolites excreted into urine. The amounts of natural chlorophyll needed to achieve these effects in animals and humans are well within those found in a helping of spinach.
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IMPACT: 2006/01/01 TO 2006/12/31<BR>
Current statistics are that over one third of all citizens in Oregon and the US will suffer cancer within their lifetime. Research in the past 20 years has revealed a strong link between dietary factors and risk of this disease. The links may be positive, where some factors increase cancer risk, or negative, for factors that may protect against cancer. An outstanding example is parts of Africa and Southeast Asia, where as many as 1 in 10 adults may die of liver cancer by age 45, due in part to aflatoxin B1 contamination of the food supply. In the past year we have shown that modest levels of natural chlorophyll in the diet can strongly protect against DNA damage and cancer in experimental animals. This effect is seen in animals as diverse as trout and rats, and against carcinogens ranging from those in tobacco smoke, to broiled meats, to contaminated grains. These results were then translated directly to humans, where modest doses of purified chlorophyll were shown to significantly reduce aflatoxin B1 uptake in the two volunteers studied to date. These are the first studies ever to show that natural chlorophyll can protect against DNA damage and cancer in whole animals, and that it does so by one or more mechanisms that translate directly to humans. Clearly the continued identification of such simple dietary factors that protect against a variety of cancers has great potential to reduce human suffering and medical costs in Oregon, the US, and worldwide.
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PROGRESS: 2005/01/01 TO 2005/12/31<BR>
Two studies compared the cancer chemopreventive efficacy of natural chlorophyll (Chl) with its water soluble food dye derivative chlorophyllin (CHL). Rainbow trout fed 160 ppm dibenzo(a,l)pyrene (DBP, found in tobacco smoke and other combustion products), developed tumors in the liver ( 53.5%) and stomach (55.5%). Animals receiving 2000 ppm CHL mixed into the DBP diet developed markedly lower tumor response in liver (24.0%) and stomach (26.5%). Those receiving 2000 ppm natural chlorophyll (Chl) admixed with the DBP diet showed similarly reduced incidence in liver (25.0%) and stomach (29.0%). In a second study, Chl and CHL exhibited a similar and potent ability to reduce liver preneoplastic foci and colon aberrant crypts when administered to F344 rats receiving AFB1. These are the first studies in any animal model to demonstrate potent tumor blocking activity by the ubiquitous green plant phytochemical chlorophyll, and at doses easily within the Chl content of spinach. Mechanistically, our working hypothesis has been that dietary chlorins such as chlorophyllin (CHL) achieve anti-carcinogen protection largely by complexing with carcinogens to reduce their uptake from the GI tract. The interactions of two chlorins (CHL, natural chlorophyll (Chla)) and two carcinogens have been investigated in vitro in selected solvents, and in vivo through pharmacokinetic studies. CHL, but not Chla, was shown to interact in vitro to form a strong non-covalent complex with the carcinogen dibenzo(a,l)pyrene (DBP), yet both chlorins reduce DBP access to liver in vivo in trout. By comparison, only CHL but not Chla interacted with aflatoxin B2 in vitro, and inhibit its uptake and transport to liver in vivo, yet both agents substantially inghibited AFB1-DNA damage to trout liver in vivo. These results indicate that both agents have cancer protective properties, but that the ability to form carcinogen complexes is not a prerequisite for chemoprevention by chlorins in vivo. They also suggest that AFB2 may be an inadequate AFB1 surrogate for investigating pharmacokinetic mechanisms in chemoprevention by chlorophylls. A separate tumor study was initiated in rainbow trout to investigate possible anticarcinogenic properties of tomatine from tomatoes, in collaboration with USDA Albany. That study will terminate in spring 2006. Gene expression effects of aflatoxin B1 exposure were examined by extracting RNA from tumorous and histologically normal adjacent tissue in rainbow trout liver, and conducting microarray analysis on our salmonid ToxGeneChip. Genes down-regulated at least 2-fold in hepatocellular carcinoma included genes involved in normal liver functioning, blood coagulation and leukocyte aggregation, and iron absorption, whereas up-regulated genes included dell proliferation, extracellular matrix, immunoregulation and acute phase response genes. These expression changes strongly resemble those reported in human and rodent hepatocellular carcinoma, suggest a highly invasive and aggressive tumor phenotype in the trout, and support the utility and validity of the trout as a carcinogenesis model.
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
Liver cancer is the third leading cause of cancer death worldwide, the most rapidly increasing cancer in the US. Since this devastating disease is often refractory to surgical or chemotherapeutic intervention, it is especially important to identify means of preventing the disease at the outset, rather than hoping only for improved cure rates. We have determined for the first time that the natural phytochemical chlorophyll is highly effective at blocking liver cancer initiation in rat liver by the potent mycotoxin AFB1, and in the trout by the potent combustion product dibenzo(a,l)pyrene. Chlorophyll protection was as great as that seen by its water soluble derivative chlorophyllin, with 50% tumor reduction at levels a fraction of the chlorophyll content of spinach. This finding has enormous implications for people in parts of Asia and Africa, where as many as 10% of adults may die of aflatoxin-related liver cancer by age 45. These studies were carried out by investigators at Oregon State University Agricultural Experiment Station. For additional information contact George Bailey (541/737-3164; george.bailey@oregonstate.edu)
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PROGRESS: 2004/01/01 TO 2004/12/31<BR>
Our working hypothesis is that dietary chlorins such as chlorophyllin might achieve anti-carcinogen protection largely by complexing with carcinogens to reduce their uptake from the GI tract. The interactions of chlorophyllin (CHL) and natural chlorophyll (Chla)) with two carcinogens were investigated in vitro through complex formation experiments, and in vivo through carcinogen uptake and distribution (pharmacokinetic) studies. We found that only CHL formed a strong non-covalent complex with the carcinogen dibenzo(a,l)pyrene (DBP) in vitro, yet both chlorins reduced DBP access to liver in vivo in trout. By comparison, both CHL and Chla interacted strongly with aflatoxin B1 in vitro, but only CHL inhibited its uptake and transport to liver in vivo. These results indicate that both agents have potential cancer protective properties in the trout model, but that the ability to form carcinogen complexes is not a prerequisite for chemoprevention by chlorins in vivo. Two newly developed trout gene chips were used to examine genetic mechanisms through which the indole-3-carbinol (I3C) dimer DIM acts as a tumor promoter in the trout model. Dietary estradiol (E2), I3C, and DIM all promoted tumor development in trout liver, and all showed very similar profiles of gene induction and suppression. These results provide further evidence that I3C and DIM promotion in this model proceed through an estrogenic pathway. Interestingly, DIM-mediated alterations in gene expression differed in tumor and normal surrounding tissue, and persisted for at least five months after cessation of DIM treatment. This suggests that the genetic effects involved in tumor promotion do not require chronic exposure, and may persist well after cessation of DIM or I3C treatment. A separate short-term study investigated the toxicity of tomatine from tomatoes, in preparation for possible long-term experiments in collaboration with USDA Albany to examine its effects on tumor formation and progression in rainbow trout. Tomatine at up to 2000 ppm fed continuously for four weeks produced no evident toxicity in trout fry.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
Current statistics are that over one third of all citizens in Oregon and the US will suffer cancer within their lifetime. Research in the past 20 years has revealed a strong link between dietary factors and risk of this disease. The links may be positive, where some factors increase cancer risk, or negative, for factors that may protect against cancer. In the past year we have confirmed that I3C and DIM, both now in clinical trials and available as health food supplements, are double edged swords that not only prrotect in some models but enhance in others. The most recent results suggest that these agents can promote liver cancer through estrogenic mechanisms in trout and rats. The significance of this mechanism now needs to be explored in human liver. We have previously found that the food dye chlorophyllin can suppress human liver cancer risk indicators in China by over 50%, and might save as many as 5% of the population in endemic regions from death by this major killer. Our recent results offer evidence that this protection may extend to natural chlorophyll as found in green and leafy vegetables. Clearly the continued identification of such simple dietary factors that protect against a variety of cancers has great potential to reduce human suffering and medical costs in Oregon, the US, and worldwide.
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PROGRESS: 2003/01/01 TO 2003/12/31<BR>
We were successful in securing a 5-year NCI grant ($6.8 million) to support the research costs for ORE00108A, with funding that began on March 17, 2003. In this first year of project ORE00108A, we have improved the method for purification of natural chlorophyll from spinach, and have accumulated enough material to begin the first trout cancer prevention studies. Methods were also developed for the purification of individual chlorins (Ce4, Ce6, Ce4Et) from chlorophyllin (CHL), in order to study their individual roles in cancer prevention by CHL. A 4-month preliminary study was completed showing that feeding CHL to rats for 16 weeks following administration of aflatoxin B1 did not suppress the development of the pre-cancer biomarker GST-P+ foci in the liver. We developed a first-generation trout "tox-gene" chip containing many genes involved in regulation of the cell cycle and apoptosis, which will be useful in investigating mechanisms for tumor suppression in the trout model. The first gene-expression experiments are under way to validate this chip, and a 3400-element trout gene chip obtained from collaborators in Canada. A trout tumor study was commenced to investigate the ability of green tea to suppress stomach cancer, and a rat study was initiated to determine the effects of selected white and green tea constituents to cuppress colon cancer. A tumor study is in progress examining the effects in mice of dietary indole-3-carbinol on fetal lung cancer in a trans-placental cancer model.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
Current statistics are that over one third of all citizens in Oregon and the US will suffer cancer within their lifetime. Research in the past 20 years has revealed a strong link between dietary factors and risk of this disease. This project seeks to understand the mechanisms by which selected plant chemicals (indoles, chlorophylls, tea anti-oxidants) inhibit cancer in various experimental animal and cell culture models, and to extend those findings to biomarker intervention studies with human volunteers. In year one we developed the first large-scale, low cost method to purify chlorophylls for these studies, and the first trout gene chips for examining cancer chemopreventive mechanisms in the trout model. Cancer prevention experiments were initiated to examine teas, indoles, and chlorophylls in the trout, rat, and/or mouse. These studies are expected to greatly improve our understanding of the role of natural chlorophylls, chlorophyll derivatives, indoles, and tea antioxidants in human cancer prevention. Based on biomarker studies in China, chlorophylls alone may reduce liver cancer by over 50%. In addition, the transplacental I3C chemoprotection studies initiated will provide for the first time an indication of the potential for maternal dietary supplementation to mediate the toxicity of chemicals to which the fetus is exposed in utero.