Foodborne Disease and Public Health: Summary of an Iranian-American Workshop (2008)

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5 Potential Associations Between Foodborne and Chronic Diseases Moderator: J. Glenn Morris, Jr. T his session of the workshop covered a wide array of topics. Ini- tially, three Iranian speakers—Razieh Yazdanparast, Narges Zali, and Ali G. Moltagh—described studies investigating cancer in Iran that included considerable use of genetic techniques. Volker Mai then discussed a study of intestinal microbiota, including ways in which the microbiota might be affected by environmental factors and ways in which they might affect health. Lu Wang provided a description of the National Institute of Health’s Human Microbiome Project and indicated that a wealth of new information on microbiota may soon emerge. The session ended with a lively discussion focused mainly on intestinal microbiota. SIGNALING PATHWAYS INVOLVED IN CANCER Presenter: Razieh Yazdanparast This presentation focused on the biological effects of 3-hydrogen- kwadaphnin (3-HK). This substance is derived from Dendrostellera lessertii,   The workshop planning committee, in consultation with Iranian counterparts and other participants, extended the scope of the workshop to include recent research findings in the area of cancer and in potential relationships of gastrointestinal microbiota with chronic dis- ease. Both topics were viewed as important areas of research of mutual interest.   Yazdanparast acknowledged financial support from the Research Council of the Univer- sity of Tehran, the Research Council of the Ministry of Health, and the Research Council of the National Research Center for Genetic Engineering and Biotechnology. She also acknowl- edged the assistance of H. Fasehei, M. Abdolmohammadi, H. Sadeghi, M. Mianabadi, A. Sadeghirizi, A. Meshkini, and M. A. Moosavi. 46 

POTENTIAL ASSOCIATIONS 47 a member of the Thymelaeaceae family of Iranian medicinal plants. The presentation also covered signaling pathways involved in 3-HK-induced apoptosis and in the differentiation of the U937 human leukemic cell line. Yazdanparast showed a series of slides that demonstrated the anti- proliferative activity of the plant. In one study, for example, the research- ers induced breast tumors in rats, treated them with a daily dose of the crude extract, and found substantial tumor suppression. When they eval- uated the biological activity of 3-HK versus that of the crude extract with a battery of cell lines, they found that the effects of the chemical and of the crude extract were similar (see Figure 5-1). Importantly, they found that the effect of 3-HK was irreversible and that it showed its effect on prolif- erating cells but not on resting cells. The latter finding led the researchers to investigate the signaling pathways that are influenced by 3-HK. Yazdanparast showed a number of slides that illustrated the results of studies concerning the cause of the cell death and distinguishing between adherent cells and suspended cells. The U937 cell line reacted in two dif- ferent ways to 3-HK: some of the cells went to apoptosis, and some of Control 3-HK Crude FIGURE 5-1 Effects of 3-hydrogenkwadaphnin (3-HK) and crude extract from Dendrostellera lessertii on cell proliferation. SOURCE: R. Yazdanparast, University of Tehran. 5-1 

48 FOODBORNE DISEASE AND PUBLIC HEALTH them were guided to differentiation. Further investigations supported the finding that 3-HK has the ability to affect the cells in both ways. Additional slides illustrated how specific inhibitors blocked the activ- ity of JNK and P38. One conclusion from the studies is that JNK and P38 are involved in apoptosis, which occurs through the FAS/mitochondrial signaling pathway and includes P21 cleavage. Another conclusion is that differentiation involves extracellular signal-regulated kinase (ERK), but caspase is not involved, and P21 is up-regulated rather than cleaved. Yazdanparast is currently involved in studies concerned with how 3-HK works among the apoptotic and differentiating cells with respect to sig- naling elements, focusing on the fate of P21. She expects that this work will lead to the use of 3-HK as an anti-proliferative drug in the treatment of leukemia. MOLECULAR AND GENETIC ASPECTS OF COLORECTAL CANCER Presenter: Narges Zali In her presentation, Narges Zali briefly described her research on the molecular and genetic aspects of detection of two forms of colorectal cancer: hereditary nonpolyposis colorectal cancer (HNPCC) and famil- ial adenomatous polyposis (FAP). By providing a number of definitions throughout her talk, Zali helped the audience become more familiar with the molecular and genetic approaches to understanding diseases that were discussed during the workshop. Hereditary Nonpolyposis Colorectal Cancer HNPCC is an inherited form of cancer that affects primarily the colon and rectum. It is an autosomal dominant cancer-susceptibility syndrome— that is, a copy of the altered gene inherited from either parent is enough to increase cancer risk—for which the gene map locus is on chromosome 2, short arm, band 22 to 21. It is associated with germ line mutations in mismatch repair (MMR) genes. MMR proteins repair problems that arise during DNA replication, and mutations in the MMR genes damage the body’s ability to repair the mistakes made during DNA replication, lead- ing to an increased risk of cancer. For the study that Narges Zali described, all colorectal cancer patients   Narges Zali expressed thanks to the National Academy for Educational Development, especially to Beverly Attallah, and to the National Academies for the opportunity to pre- sent her work. 

POTENTIAL ASSOCIATIONS 49 SCREENING STRATEGY (1) HNPCC ? FIGURE 5-2  Genetic tests for the detection of hereditary nonpolyposis colorectal cancer (HNPCC). IH-negative stands for immunohistochemistry-negative and means that a specific gene was not expressed. SOURCE: N. Zali, Shaheed Beheshti University of Medical Sciences. fig 5-2 from the hospital-based cancer registry at the Research Center for Gastro- enterology and Liver Diseases were asked to enroll. After receiving the patients’ informed consent for the procedures and for genetic counseling, staff collected blood samples and paraffin-embedded blocks of tumoral tissue that were obtained during surgery. The research team extracted genomic DNA from blood and tissue and ran a series of genetic tests for HNPCC, as shown in Figure 5-2. To help clarify the screening strategy, Narges Zali explained some of the terms used and showed slides illustrating the findings. Immuno- histochemistry (IHC) is a technique to detect the presence of a particular protein in a tissue sample through the use of antibodies to that protein. With IHC, one can analyze the expression of MMR genes by looking for their products, the various MMR proteins. MSI occurs when microsatellites (stretches of short, repeated sequences of DNA) vary in length in an individual. MSI indicates errors occurred in the DNA replication process and thus a deficiency in the MMR repair function. Two techniques are available for the detection of MSIs: (1) the older gel electrophoresis, and (2) the newer and more sensitive fragment analysis. 

50 FOODBORNE DISEASE AND PUBLIC HEALTH If the IHC finds that a specific gene is not expressed, the next step is microsatellite instability (MSI) analysis for that gene and then a search for mutations. If the IHC result is normal and Amsterdam criteria are posi- tive, the next test on the tumor tissue is for MSI, potentially followed by a genetic analysis of the MMR genes. If the patient has a positive high MSI, the next step is to sequence and search for MMR genes (not shown in the figure) using a polymerase chain reaction (PCR). To date, the researchers have found 20 MMR germ line mutations in 18 out of 47 families with colorectal cancer. They have also found several novel mutations. Familial Adenomatous Polyposis The research group is also working to determine the molecular epide- miology of APC/MYH genes among Iranian patients with familial adeno- matous polyposis (FAP). The group uses a clinical-molecular approach in the genetic testing of patients with FAP. If examination of the family tree indicates that the condition is familial, PCR and sequencing are used to determine whether there are point mutations. If such mutations are found, the clinician refers the patient for a colonoscopy and for follow up. For cases in which these mutations are not found, the research group is investigating a new technique called multiplex ligation-dependent probe amplification to search for large deletions in the tumor-suppression gene APC. SCREENING OF HIGH-RISK POPULATIONS FOR COLORECTAL CANCER IN IRAN Presenter: Ali G. Motlagh Motlagh provided background information about colorectal cancer in Iran, briefly described molecular events that are associated with the development of colorectal cancer, discussed the development of technol- ogy in Iran to detect the genetic mutations that lead to colorectal cancer, and suggested useful next steps. Background Information In Iran, colorectal cancer is the third most common cancer among women and the fourth most common cancer among men. Sixty-five per-   Amsterdam criteria are used to identify families that are likely to have hereditary non- polyposis colorectal cancer. 

POTENTIAL ASSOCIATIONS 51 cent of the colorectal cancer in Iran is estimated to be sporadic, 25 percent familial, 4 percent hereditary nonpolyposis colorectal cancer (HNPCC), 1 percent familial adenomatous polyposis (FAP), and 5 percent other. Genetics and the environment both play a role in the development of colorectal cancer, but the particular roles vary according to the type of cancer. In particular, genetic factors are known to play different roles in different types of colorectal cancer. In sporadic types, for instance, low- penetrance genes are more important than high-penetrance genes, while the opposite is true for familial cases. Molecular Events Associated with Colorectal Carcinogenesis Two pathways are known to be important in colorectal carcinogen- esis: the chromosomal instability pathway and the microsatellite instabil- ity (MSI) pathway. It has been proposed that MSIs result from a deficient mismatch repair (MMR) system. MSIs are found in the major tumors of patients with HNPCC as well as in 15 percent of sporadic colorectal cancers. Three methods exist for studying alterations of the MMR mecha- nism: sequencing the involved gene, detection of gene products, and MSI analysis. Motlagh pointed out that testing for MSI is a powerful method to screen for HNPCC and that the finding of MSIs in sporadic tumors sug- gests a more favorable prognosis although the response to chemotherapy is uncertain. A person with two or more relatives who have developed colorectal cancer has a substantially higher risk of developing colorectal cancer himself or herself, and the cancer may occur at a relatively young age. Clinical criteria that include family history and the patient’s cancer his- tory are helpful in identifying those persons who are good candidates for genetic testing. A Project to Develop Technology for the Detection of Genetic Mutation for Colorectal Cancer in Iran Motlagh described a project in Iran aimed at developing and using technologies for detecting the genetic mutations leading to colorectal cancer and at establishing a genetic counseling service for high-risk popu- lations. The project will also establish a gastrointestinal cancer registry database, develop IHC and MSI techniques, and create other genetic- analysis capabilities. Records were reviewed for 600 patients with colorectal cancer from 8 of the 30 provinces of Iran. Software was specially designed for collecting the data, and the data were added to the cancer registry database. Inves- 

52 FOODBORNE DISEASE AND PUBLIC HEALTH tigators obtained additional data in order to be able to perform survival analysis, and they traced pedigrees backward and laterally. Amsterdam II criteria were used to identify HNPCC, and Bethesda guidelines were used in identifying HNPCC-related neoplasms. The investigators conducted various genetic tests and found that most mutations were in MLH1 (a gene that codes for a membrane protein), which is similar to findings from China, Korea, Finland, Sweden, and Spain. The phenotype features that the researchers found may have value in the design of genotype-specific screening. Future Considerations Since colorectal cancer is triggered by interactions of genes with the environment, and since environments differ around the world, achieving a consensus on screening guidelines may be difficult. Based on the most recent update of the non-colorectal, non-neuroendocrine guideline and on recent trials, Motlagh and colleagues suggested that the optimal method for screening a high-risk population for colorectal cancer would include • patient evaluation with Bethesda guidelines; • MSI analysis and IHC testing for MMR protein; • mutation analysis and patient evaluation with Amsterdam guide- lines; and • hypermethylation of MHL1 and BRAF mutation analysis. Because this method would probably not be cost effective, the Iranian group has mapped out the following three-phase effort to refine and make effective a screening method for the Iranian population by using a small sample of patients: 1. Estimate the accuracy of IHC and MSI analysis for the prediction of the MMR mutation, design a computer model for predicting the MMR mutation, conduct a cost-effectiveness analysis to identify the most suit- able screening guidelines for the Iranian population, and search for the best method to distinguish familial colorectal cancer from HNPCC. 2. Conduct a pilot study of a population at high risk for colorectal cancer in Tehran province using colonoscopy and laboratory tests, and estimate the accuracy of these tests for screening the Iranian population for colorectal cancer. 3. Develop a comprehensive program for risk stratification and risk modification among those at high risk for colorectal cancer.  BRAF is a protein that plays a central role in the growth and survival of cancer cells. 

POTENTIAL ASSOCIATIONS 53 DIET, MICROBIOTA, AND CARCINOGENESIS Presenter: Volker Mai Mai provided background information about the intestinal microbiota and its investigation, briefly described some studies in which he has been involved, listed desirable characteristics of future studies, and gave a few closing remarks. Overview Description of the Intestinal Microbiota The intestinal microbiota could be considered an organ. It consists of approximately 1011 to 1012 bacterial cells per gram of stool. This means that humans have 10 times more cells in the form of bacteria than they have human cells. The gut microbiota has three key functions: 1) Metabolic: Examples include the fermentation of non-digestible dietary residue and endogenous mucus, salvage of energy as short-chain fatty acids, production of vitamin K, and the absorption of ions. 2) Trophic: This involves development and homeostasis of the immune system and the control of epithelial cell proliferation and differentiation. 3) Protective: Bacteria may prevent the attachment of pathogens in the intestines and thus reduce the risk of disease. The Study of the Intestinal Microbiota Studying the gut microbiota is difficult for a number of reasons. There is no efficient way to sample from the proximate colon without an initial cleansing, for example. Mai said that both the lumen material and the bac- teria that attach to the epithelium are needed to study the gut microbiota, but that this situation poses challenges. For example, formalin, which is used to preserve biopsy samples, depletes the mucus layer. Furthermore, the proportion of organisms that can be cultured is very low, there are difficulties in speciating bacteria, and the work is very labor intensive. Recently, molecular analysis methods have opened new avenues in the study of gut microflora. Interactions between diet and the microbiota might distort epidemio- logical observations. For example, if flora associated with a low risk of certain diseases colonize the intestine, they may efficiently ferment fiber to create such beneficial end products as butyrate. By contrast, if flora associated with a high risk of those diseases colonize the intestine, they may not ferment the dietary fiber. The movement of intact fiber through 

54 FOODBORNE DISEASE AND PUBLIC HEALTH the colon, in turn, might deplete the mucus and increase the risk of a chronic infection. Mai concluded that studying these complex interactions requires multidisciplinary research involving microbiologists, immunolo- gists, epidemiologists, and others. Ongoing Studies In Mai’s laboratory at the University of Florida in Gainesville, the methods of studying the intestinal microbiota range from the use of dena- turing gradient gel electrophoresis to microarrays. The group is using a “shotgun” method that involves collecting stool, extracting DNA, per- forming PCR tests, subcloning, and sequencing. This method should allow the detection of all bacterial species that are present at a concentra- tion of more than 107 bacteria per gram of stool. Cancer mortality data show a number of similarities between the United States and Iran. In both countries, for instance, colorectal cancer is the third-leading cause of cancer deaths for both men and women. In the United States, African Americans of both sexes experience higher inci- dences and higher mortality rates from colorectal cancer than do whites. For this reason, Mai’s group is especially interested in studying the risk of colorectal cancer and the potential contributions of environmental factors to colorectal carcinogenesis among African Americans. If one is interested in preventing cancer, then focusing on environ- mental factors means focusing on those factors that can be changed in order to reduce risk. Current evidence suggests that higher intakes of folate, calcium, vitamin D, vegetables, and perhaps fiber might reduce the risk for colorectal cancer. In contrast, the risks for colorectal cancer appear to be increased by a high body mass index and by the consumption of the nitrosamines in processed meat and the heterocyclic amines in grilled meat. Mai’s laboratory has the long-term goal of developing interventions that modify the intestinal microbiota toward a healthier composition, but he noted that he is aware that dietary factors have not yet been convinc- ingly associated with cancer risk. Diet, Microbiota, and the Prevention of Intestinal Adenomas in ApcMin Mice Mai briefly described a study that looked at the question of whether diet might affect the gut microbiota and, in turn, affect the development of cancer in the intestinal tract. The study used ApcMin mice, which are a standard mouse for studies of human colorectal carcinogesis. The mice have a genetic predisposition to developing intestinal adenomas at an early age. The mice were divided into groups including a high-risk-diet group, a low-risk-diet group, and a set of controls, all of which were fed 

POTENTIAL ASSOCIATIONS 55 ad libitum, plus another group that was fed the control diet but with a 40 percent calorie restriction. The researchers found that both the low-risk diet and the calorie-restricted diet reduced the number of polyps in the mice significantly, but the reductions occurred via different pathways. The full results of the study suggest that specific dietary interventions reduce intestinal carcinogenesis, that diet strongly affects the intestinal micro- biota composition, that some bacteria appear to be associated with lower risk, and that diet and microbiota likely affect immune function. Mai provided detailed information to the workshop attendees by distributing his paper “Intestinal microbiota: A potential diet-responsive prevention target in ApcMin mice” (Mai et al., 2006). Diet and Microbiota Studies in Humans Mai has conducted a pilot study of 52 African Americans and 46 Caucasians to look for associations between diet and microbiota and to explore differences in the microbiota between people in the two groups. One interesting finding was an association between dietary fiber intake and higher amounts of lactic acid bacteria (which are generally thought to be beneficial). African Americans had twice as many Bacteroides as did the Caucasians. An ongoing colonoscopy screening study may offer an opportunity for developing collaborations with the Iranians. In this study investiga- tors are measuring diet, collecting stool samples, recording normal status, and collecting biopsy samples from subjects with the aim of determining if specific parts of the gut microbiota are associated with colorectal cancer risk. Desirable Characteristics of Future Studies Several features will be important in future studies of the human intestinal and fecal microbiota: • An understanding of the standard dynamic of the microbiota needs to be developed because epidemiological studies require assurance that a sample collected at a specific time really is representative of the micro- biota over time. • The use of multiple time points would help clarify dynamics. • Larger studies are needed; to date studies have involved fewer than 10 people. • Studies of different populations would provide valuable informa- tion on variation. 

56 FOODBORNE DISEASE AND PUBLIC HEALTH • Studies of interventions are needed to determine the potential for modification of the microbiota. • Standardized methods are needed so that comparisons can be made between studies. Closing Remarks In closing, Mai highlighted the following points: • Gut microbiota contribute substantially to intestinal physiology and thus likely to human health. • The complexity of the gut microbiota and its metabolic abilities and dynamics are not yet fully understood. • Microbiota composition is affected by diet. Specific bacteria appear to be associated with carcinogenesis. • Future studies need to test specific hypotheses rigorously. HUMAN MICROBIOME PROJECT Presenter: Lu Wang The Human Microbiome Project is a U.S. National Institutes of Health (NIH) Roadmap project. Roadmap projects have been characterized as high-risk and potentially high-return projects. The Human Microbiome Project has been funded at a level of slightly more than $100 million for a 5-year period. In his presentation, Lu Wang briefly described the project and listed its initiatives. Overview The human microbiome is the collection of genomes of all the microbes that inhabit the human body. Several hundred distinct phyla of microbes live in the human body. As mentioned previously by Mai, the human microbiota is thought to have a profound influence on human health via various effects that it has on human physiology and nutrition, immunity, and development. Current evidence indicates that the human microbiome has different patterns at different anatomical sites. New sequencing technology makes it possible to analyze the genomic content of a human microbiome. The publication The New Science of Metagenomics: Revealing the Secrets of Our Microbial Planet provides a useful perspective on this type of research (NRC, 2007). 

POTENTIAL ASSOCIATIONS 57 The goal of the Human Microbiome Project is to characterize the microbes that inhabit the human body and to examine whether changes in the microbiome can be correlated with disease. In fiscal year 2008, the NIH will award cooperative agreements and grants to support the project. The project’s policy will be to release immediately the data it collects and the resources it develops. The project will address the following questions: • Is there a core microbiome? There is currently a wide divergence of opinions on this matter. • How much variation in the microbiome is there among individu- als? Answering this question may require taking into account the various environments to which people are exposed. • Can changes in microbial populations be connected with factors such as disease, age, diet, antibiotics, and sex? • Can information about an individual’s microbiome be demon- strated to offer any diagnostic or therapeutic benefits? Initiatives of the Human Microbiome Project A broadly based working group of the NIH identified the following seven initiatives that the Human Microbiome Project will support: 1. Developing a reference set of microbial genome sequences and a preliminary characterization of the human microbiome, with a focus on healthy individuals 2. Investigating relationships between changes in the human micro- biome and disease 3. Developing new microbial genomics technologies 4. Developing new tools for the computational analysis of specific types of data 5. Establishing a data analysis and coordinating center with func- tions related to tracking, storage, and distribution of data, coordination of data analyses, the development of data-retrieval tools, coordination of standard development, and establishing a mechanism to display project activities 6. Establishing a central, reasonably priced resource for materials, reagents, cultured organisms, etc. 7. Addressing ethical, legal, and social implications of Human Micro- biome Project research, covering topics such as clinical and health implica- tions, forensic uses of microbiome profiles, bioterrorism and bio-defense applications, and privacy issues 

58 FOODBORNE DISEASE AND PUBLIC HEALTH Closing Remarks The group that is responsible for implementing the Human Micro- biome Project includes four institute directors and representatives from all 27 NIH institutes. The website http://nihroadmap.nih.gov/hmp/ provides further information about the NIH Roadmap Human Micro- biome Project and also posts announcements about new requests for applications. DISCUSSION Moderator: J. Glenn Morris, Jr. The participants spent considerable time discussing studies of the intestinal microbiota and their possible relationship with the development of disease. It was emphasized that this is an exciting new field made pos- sible by technological advances and that the development of new genetic capabilities is leading to major changes in the concept of microbiology. Because the field is very new, there are many questions for which defini- tive answers are not yet available. Topics that sparked special interest include the relationships of the microbiota to the development of chronic disease and the ways that this might be studied, the effects of various fac- tors on the microbiota in the intestinal lumen, the identification of organ- isms that make up the microbiota, and potential future studies. Relationships of the Microbiota to the Development of Chronic Disease Epidemiological evidence indicates that rates of colorectal cancer are much lower in Africa and in some other parts of the developing world than they are in the industrialized countries. Mohammad Reza Zali noted that food in the developing countries tends to contain more bacteria than food in industrialized countries, which could lead the residents of devel- oping countries to have higher numbers of bacteria in their intestinal tracts than do residents of industrialized countries. It also was noted that rates of colon cancer increase when people move to more industrialized countries. Mai explained that it is much more difficult to study the relationships of microbiota in the intestinal lumen with the development of colorectal cancer or with other chronic diseases than it was to study Helicobacter and its association with disease. The stomach environment is essentially ster- ile, except for the presence of Helicobacter itself, which made it relatively easy to isolate and identify the bacterium. The intestines, by contrast, have a population of microorganisms that is vastly more complex. 

POTENTIAL ASSOCIATIONS 59 It is possible that shifts in the proportions of various microbial groups are related to changes in disease risk. Because the studies of the intestinal microbiota conducted by Mai’s group have so far involved subjects under- going screening colonoscopies rather than patients with cancer, however, his group has not yet been able to show any associations between the microbiota and colon cancer. Effects of Various Factors on the Microbiota in the Intestinal Lumen Cleansing and Antibiotics The preparation of the colon for a colonoscopy has a major effect on the bacteria that can be obtained in a biopsy sample. The bacteria that have the ability to attach to the inner mucus layer and thus remain in the colon may be completely irrelevant to health risks. For this reason, fecal samples may be more useful to a study of the microbiota than are biopsy samples. If it were possible to sample the intestinal contents of a corpse right after a person’s death, one could compare the composition of the microbiota at different parts of the intestine. Clearly, the study of the intestinal microbiota is complicated by the difficulty of obtaining samples from the proximal intestine. It is not clear what effect antibiotics have on the overall composition of the microbiota over time, and the findings will depend in part on the sensitivity of the technique used. Mai suggested that antibiotics have a smaller effect on the intestinal microflora than does colonoscopy. Dietary Change The period of time required to stabilize the microbiota after a drastic dietary change (such as a change from omnivore to vegetarian or the addition of a probiotic substance to one’s diet) has not been studied well. Initial studies suggest that about seven days may be required for the microbial composition to stabilize after it has been seriously disrupted. When the substrate in the intestines changes, the bacteria that are best adapted to use the substrate gain energy and appear to dominate rather quickly. Depending on which bacteria are present in high proportions, the concentrations of compounds present in the intestinal lumen change, and this may influence susceptibility to cancer. It is an extremely complex system, and its investigation has only recently begun to receive support from the NIH. Some participants expressed interest in studying the effects that mal- nutrition has on the composition of the microbiota and how those changes might affect the immune system, potentially resulting in adverse health outcomes. 

60 FOODBORNE DISEASE AND PUBLIC HEALTH Identification of Organisms That Make Up the Microbiota A very large percentage of the microorganisms populating the intes- tine cannot be cultured. Thus the study of the microbiota now gener- ally involves extracting DNA from a stool sample and identifying both anaerobes and aerobes using genetic techniques. The genetic studies have raised questions about the concept of bacterial species. There is much genetic exchange, and the relationship of different molecular signatures to different bacteria is not completely clear. Morris indicated that a tre- mendous revolution is just beginning to occur in our understanding of the composition of microflora of the human gastrointestinal tract. Although Mai focused his discussion on the potential role of bacte- ria in the development of colorectal cancer, his group has not ruled out a potential role for yeasts. However, because the number of yeasts per person is much smaller than the number of bacteria (on the order of 105 or 106 for yeast, compared with 1011 or 1012 for bacteria), few yeasts are found in microscopic studies of the gut microflora. Special methods will be needed to study the involvement of yeasts. Future Directions The evidence suggests that very complex metabolic and biochemical reactions are occurring continually within our intestines and that distur- bances of the microbiota might substantially change a person’s ability to handle specific toxins, metabolic by-products, or other substances in the intestine. This subject lies within the intersection of foodborne disease, the bacteria in the intestines, and the occurrence of chronic disease. Morris said that there is a need to conduct studies in locations outside the United States in order to understand the differences that may arise in different geographic areas. Considering the development that Iran is undergo- ing, Mai suggested that it could be a promising region for the study of a variety of factors that affect the risk of developing colorectal cancer. Such study also would have the benefit of helping identify the environmental causes of cancer that could be controlled.