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5 Laboratory Methods Laboratory data cannot be readily translated into schemes for pre- vention of cancer in humans. The principal shortcoming is the inability of current animal bioassays and short-term tests to identify with cer- tainty the causes of cancer in humans and to predict the extent of risk posed by the various exposures. These limitations were discussed in the first report of the Committee on Diet, Nutrition, and Cancer (National Research Council, 1982~. In Chapter 3 of the first report, the committee explained that substances demonstrated to be carcinogenic in animals are regarded as potential carcinogens for humans. However, it also emphasized that there are major drawbacks in the standard procedure for determining carcinogenicity of compounds, i.e., the bioassay in animals fed the test substance for a major portion of their lifetime. These long- term bioassays lack sensitivity, they may produce false negative results, and, because of the high doses given to animals, extrapola- tion of the results to determine the response of humans exposed to lower doses cannot be accomplished with any degree of certainty (National Research Council, 1982~. There is a dearth of animal models that are both sensitive and rele- vant for assessing the influence of diet and nutrition on cancer in humans. For example, we do not know the extent to which these models mimic metabolism of and responses to carcinogens in humans, nor do we know all the ways in which the nutritional requirements of the animals used in these studies differ from those of humans. Furthermore, animal models need to be standardized so that studies in different labora- tories can be compared. Steps should be taken to rectify these inade- quacies, since animal models have to be used if we are to evaluate the influence of diet and nutrition on different stages of carcinogenesis, e.g., initiation and promotion. The recognition that the active fores of carcinogens are electro- philes that bind to DNA has provided the impetus for studies in which short-tenm assays have been used to detect carcinogens. However, even though there is correspondence between mutagenicity and carcinogen- icity, the short-term tests now available provide only qualitative information and cannot be used to predict cancer risk to humans. Better methods are needed for extrapolating data from animal experiments to determine the contribution of different dietary compo- nents to the causation and prevention of cancer in humans. There is a need for laboratory methods to compare the relative contributions to human cancer made by carcinogens that initiate and carcinogens that promote the growth and development of transformed cells. If exposure 19

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20 DIET, NUTRITION, AND CANCER: DIRECTIONS FOR RESEARCH to dietary initiators is the most important variable, it would be helpful to know the detailed steps involved in their metabolism. For example, is the amount of the activated carcinogen that binds to certain sites within DNA directly correlated with the number of newly initiated cells? If so, which metabolic reactions are critical, and which are the ones most influenced by dietary constituents? How much activated carcinogen is needed to induce tumorigenesis, or is there no threshold? Is the long-tenm average level of critical ONA adducts more or less important than short, intense periods of adduct formation? Answers to fundamental questions such as these are needed to develop methods that can relate the In viva response to initiators to the degree of risk. If exposure to dietary factors that modify the course of initiation is the primary determinant of tumor development, then a different type of methodology may be required. Imaginative and sophisticated pro- cedures will be needed to measure the effects of such modifying fac- tors, because different mechanisms are likely to be responsible for their activity. We need to refine our understanding of the relationship between nutrient intake (dose) and cancer risk (response). Perhaps this concept of dose response could be incorporated into the basis for defining the Recommended Dietary Allowances (RDA). These allowances were initially intended to serve as a guide to nutrient intake that would be sufficient to eliminate certain deficiency diseases in the general population. In recent years, the formulators of the RDA have attempted to define the range of adequate and safe levels of certain nutrients (National Research Council, 1980a). This concept needs to be extended so that ranges of recommended intakes also take into account potential associations between nutrients and chronic diseases such as cancer. Finally, there are no reliable methods for the extrapolation of data from animal studies to determine the response in humans. Since current procedures are probably of little or no value for assessing the risk of nutrient-induced tumor modification, it is likely that an entirely new approach will be required (National Research Council, 1982, Chapter 18~. METHODS FOR IDENTIFYING DIETARY INITIATORS AND MOI)IFIERS OF CARCINO- . . . GENES IS Methods for identifying dietary substances that initiate or modify one or more stages of carcinogenesis are inadequate. Initiators, which attack genetic material, are discussed in Chapter 8. Following the initiation of neoplasia, modifiers may either enhance (i.e., by promot- ing or acting as cocarcinogens) or inhibit the subsequent development of tumors by a variety of mechanisms. Therefore, methods for detecting these activities should be broad enough to detect compounds that operate by different kinds of mechanisms

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Laboratory Methods 21 Animal models for liver and skin cancer have been used extensively to study the form of enhancement called tumor promotion. More recent- ly, tumor promotion in the breast and colon has also been studied. Further investigation using these and other animal systems, and speci- fic efforts to adapt such systems to detect promoters in food, would be useful. The continuing development of In vitro systems for studying promotion should also be encouraged, since they may provide insights into mechanisms of action and may serve as useful test systems for detecting promoters. The process of neoplasia can also be affected by cocarcinogens, which enhance the tumor initiation process. Some cocarcinogens may act by increasing-the binding of initiators to ONAe Others may in- crease cell proliferation before or during attack by carcinogens, there- by enhancing carcinogenesis. Further studies are needed to unravel the specific mechanisms of this early stage of carcinogenesis and to identi- fy dietary constituents that act at that time. Studies in laboratory animals have indicated that food contains many inhibitors of carcinogenesis (National Research Council, 1982, Chapter 15~. It is important to identify the full spectrum of compounds in this category. Proposed research for attaining this objective is dis- cussed in Chapter 7. lIETHODS FOR ASSESSING THE EFFECTS OF NUTRIENTS ON CARCINO&ENESIS Methods for assessing the effects of nutrients on carcinogenesis are inadequate, especially those for determining dose-response rela- tionships and the impact of interactions of nutrients with each other and with other dietary substances. Nutrients at certain levels of intake appear to modify rather than initiate carcinogenesis. Methods should enable investigators to define the dose-response relationship between nutrients and various cancers so that basic principles for assessing risk can be formulated. Animal models whose nutritional characteristics can be reliably related to the nutrient requirements of, and metabolism in, humans are also required. Since modification of cancer risk by individual nutrients can almost always be markedly influenced by other nutrients and other dietary components, statistical methods capable of analyzing such complexities must be used. A lack of standardization among selected diets and expert-mental models often hinders the interpretation of data. It would be appro- priate to consider dietary formulations such as the AIN-76 diet (Anonymous, 1977) or suitable variations thereof. Adoption of such standards should facilitate interlaboratory comparisons but should not restrict continued inquiry into varied and diverse dietary protocols that are suitable for exploring the effects of the heterogeneous diets consumed by humans.

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22 DIET, NUTRITION, AND CANCER: DIRECTIONS FOR RESEARCH METHODS FOR IDENTIFYING MARKERS RELATED TO NEOPLASM IN HUMANS One of the most pressing needs is the development of short-term tests that could identify early biological indicators of exposure to dietary constituents that affect the occurrence of neoplasia in humans. Where neoplasia is the sole end point, investigations are severely limited by the long latency period between exposure and ex- pression of neoplasia. Accordingly, attention should be directed toward finding ways to evaluate specific dietary components for their early neoplastic or inhibitory effects in humans and to identify early markers that can be used to predict the likelihood that clinical cancer will develop in humans. For example, the early stages of neoplasia can be detected by the presence of hepatic foci with altered enzymatic activity (Pitot _ al., 1980; Potter, 1981) or altered proliferative compartments in mucosal glands of the large bowel (Lipkin, 1977~. Similar test systems to examine body fluids or other readily accessible tissues for such markers would be valuable if they could be used to identify the stage of carcinogenesis or to predict the occurrence of neoplasia. There are several methods for identifying compounds suspected of damaging genetic material in humans. Of these, cytogenetic studies are the most widely used (Thilly and Liber, 1980), but their utility is limited by their lack of sensitivity. Another such method involves the identification of DNA adducts in human tissues. Studies of sister chromatic exchanges have also been reported, but further research is required to determine the implications of this phenomenon. Studies to detect genetic damage might include the "micronucleus" test (Heddle _ al., 1982~. New procedures for detecting the formation of carcinogens and mutagens in humans are now being developed. For example, recent studies have shown that nitrosoproline is excreted in the urine of humans given oral doses of the precursor compounds (nitrate and proline) (Ohshima et al., 1982~. This is the first test procedure for studying nitrosation in human beings. Once procedures of this type have been developed further, it should be possible to test puta- tive inhibitors of the formation of carcinogenic nitroso compounds for their effectiveness in humans (Newmark and Mergens, 1981~. Bruce et al. (1977) have found a naturally occurring mutagen in human feces, and Hirai _ al. (1982) have established its chemical structure. It is not yet known whether this compound is carcinogenic. Nevertheless, the presence in the lumen of the large bowel of a compound capable of attacking DNA suggests a potential hazard. Therefore, study of dietary factors that alter the amount of this mutagen is warranted. Short-term methods are needed to identify biological indicators of tumor promotion in humans. Possible techniques of this nature are mentioned in Chapter 7. When such systems are developed, they should be used to identify dietary compounds that have the capacity for tumor promotion.

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Laboratory Methods 23 Techniques are needed to identify dietary constituents that pre- vent carcinogens from reaching or reacting with critical target sites in human tissues. When particular constituents have been found to block carcinogenesis in animals, their effects in humans should be investigated . Because these blocking agent s may affect carcinogen- rnetabolizing systems that are tissue enzymes, it will be necessary to select readily available as well as suitable tissues in humans. An alternative strategy i s to admini ster noncarcinogenic prototype compounds that are metabolized in a fashion similar to that of known carcinogens and then to assess their metabolism by measuring blood and urine samples or, possibly, by quantitating volatile metabolites that are exhaled. It may be feasible to study the effects of blocking agent s present in food on carcinogen~netabolizing systems in humans, but initially, considerable efforts will be required to develop appro- priate techniques. Such techniques and studies on blocking agents in animals are discussed in more detail in Chapter 7. RESEARCH RE COM~NDAT IONS Initiators and Modifiers of Carcinogenesi s Better methods based on mechanism of action should be devised to detect carcinogens, promoters, cocarcinogens, and inhibitors in food. Attempts should be made to develop better methods for extrapo- lating to humans risk estimates derived from laboratory studies. The Effects of Nutrients on Carcinogenesis Methods must be developed for evaluating the effects of nutrients on carcinogenesis. Particular emphasis should be given to the follow ing two areas. Methods should be developed to study the dose-response rela- tionship between nutrient s and tumorigenesi s. 0 Methods should be devised and used systematically to evaluate the association between carc'nogenesis and the interaction of nutrients with each other and with nonnutritive dietary constituents. Markers Related to Neoplasia in Humans o Research should be conducted to identify biological markers of exposure to chemicals that cause cancer in humans. o Early biological markers that can forecast the emergence of c linical cancer should be identif fed .

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24 DIET, NUTRITION, AND CANCER: DIRECTIONS FOR RESEARCH Short-ter~Q test systems should be developed to detect the early effects of dietary initiators to which humans are exposed. Thus, tech- niques should be developed to identify and quantify the presence of carcinogen-DNA adducts and to detect alterations in DNA. In addition, attempts should be made to refine cytogenetic procedures. Short-term technique s should be devised to detect the early effects and to quantify the impact of compounds suspected of acting as promoters or cocarcinogens in humans. Methods should be developed for studying the In vivo formation of carcinogens and mutagens in humans. Methods should be developed for the detection of putative in- hibitors in the diet . Thi s should be followed by systematic evaluation of the protective effects of these inhibitors in humans.