Understanding the Causes of Aging and Cancer: The Role of the Environment
BN Ames. Division of Biochemistry and Molecular Biology, University of California, Berkeley, California.
Aging appears to be in good part due to the oxidants produced as by-products of normal metabolism by mitochondria [1]. These oxidants are the same mutagens that are produced by radiation, and cause damage to DNA, proteins, and lipids.
The degenerative diseases of aging such as cancer, cardiovascular disease, cataracts, and brain dysfunction, are increasingly found to have, in good part, an oxidative origin [1]. Dietary antioxidants, such as Vitamins C and E and carotenoids, play a major role in minimizing this damage and most of the world's population is receiving inadequate amounts of them, at a great cost to health [1]. The main source of dietary antioxidants is fruits and vegetables (5 portions a day is advised). The quarter of the American population that eats the fewest fruits and vegetables is deficient in the vitamin folic acid (causing broken chromosomes, cancer and birth defects) and Vitamin C and carotenoids (causing the same oxidative damage to DNA as radiation) [1] and has about double the cancer rate of the quarter that eats the most.
The three main causes of cancer are smoking, dietary imbalances (excess fat and calories; inadequate intake of fruits, vegetables, fiber, and calcium), and chronic infections leading to chronic inflammation [2]. Chronic inflammation is a major cause of cancer in the world because it releases powerful oxidants which both stimulate cell division and are mutagens.
The assumption that residues of synthetic industrial chemicals are significant contributors to cancer rates and that we can lower cancer rates by eliminating them is wrong [2,3].
a. Animal cancer tests, which are done at the maximum tolerated dose (MTD) of the test chemical (mostly synthetic chemicals), are misinterpreted to mean that low doses of synthetic chemicals and industrial pollutants are relevant to human cancer. Half of all chemicals tested, whether synthetic or natural, are carcinogenic to rodents [3]. A plausible explanation for the high frequency of positive results is that testing at the MTD frequently can cause chronic cell killing and consequent cell division due to cell replacement, a risk factor for cancer that can be limited to high doses.
b. The vast bulk of chemicals ingested by humans are natural. For example 99.99% of the pesticides we eat are naturally present in plants to ward off insects and other predators [3]. Half of these natural pesticides tested at the MTD are rodent carcinogens [3].
c. Cooking food also generates thousands of chemicals [3]. There are over 1000 chemicals in a cup of coffee. Only 26 have been tested in animal cancer tests and more than half are rodent carcinogens; there are still a thousand chemicals left to test [3]. The amount of rodent carcinogens consumed as pesticide residues in a year is less than the known amount of rodent carcinogens in a cup of coffee [3]. This does not mean coffee is dangerous but that animal cancer tests and worst-case risk assessment, build in enormous safety factors and should not be considered true risks.
References
RL Price. Department of Nutritional Sciences, University of Arizona, Tucson, AZ.
Although very few intoxications of humans in the United States have been directly connected to mycotoxins in foods, their presence is of major concern to industry and to health and regulatory agencies. Livestock have been affected, sometimes in large numbers, by consumption of contaminated feeds. There is also a small pass-through of some mycotoxins into animal foods. Because of economic implications, it is not always feasible to destroy large quantities of contaminated commodities. Decontamination has been studied extensively. Specific criteria for mycotoxin reduction have been established: 1) The toxin must be removed; 2) No toxic residues must remain; 3) Nutritive value and acceptability must be retained; 4) Functional properties of the product should not be affected significantly. Several commercially viable methods for decontamination of aflatoxin-contaminated peanut, corn, and cottonseed products have been used, the most common being ammoniation. Other methods which have been studied are thermal inactivation, irradiation, extraction, separation, adsorption in solution or in vitro, biological control, microbial degradation, and chemical treatments. Destruction of other mycotoxins, which have fewer regulations than aflatoxin, has been less successful. Our laboratory is now investigating combinations of the above treatments, in particular, treatment of contaminated products with alkali and enzymes, either previous to or during extrusion or fermentation to completely destroy aflatoxin and/or fumonisin. Ammoniation continues to be the treatment of choice for aflatoxin but is not effective for fumonisin.
Aflatoxins in Maize: A Scenario in México
E Moreno-Martinez. Programa Universitario de Alimentos, Universidad Nacional Autónoma de México, México, DF.
The aflatoxin problem in México has not been extensively studied, therefore it is not well understood. Because the fungus involved, Aspergillus flavus, was considered to be a storage fungi, initial research focused on the problem as an outgrowth of poor postharvest techniques versus a field issue. Researchers at Purdue University, however, discovered in the early 1970's that aflatoxin production in maize could occur in the field long before the crop is harvested.
Around 1989 there was a severe outbreak of aflatoxin production in the corn fields in the state of Tamaulipas. This situation repeated itself in the course of the subsequent years. The levels of contamination to huge volumes of maize were high enough to render the grain completely unsuitable for any use. The toxin production in both field and postharvest phases occurred due to the following: 1) environmental conditions in Tamaulipas which predispose the maize toward aflatoxin contamination in the field as well as the inadequate management of the crop during the growing season, and 2) the lack of drying equipment for proper preconditioning of the harvested grain.
In order to lessen the aflatoxin contamination of maize, the National Research Institute of Forestry, Agriculture and Animal Husbandry has designed a technological package which takes into account all possible measures maize producers can take in order to avoid or reduce the aflatoxin contamination.
It is significant to note that México produces maize in tropical areas where the relative humidity and temperature are even higher than those found in Tamaulipas, and the aflatoxin problem is by no means a serious one.
In regard to the contamination of maize grain with this dangerous toxin, our experience in México points to the fact that the main source of contamination of our most basic food grain occurs in the field, and primarily in the state of Tamaulipas. Clearly, further research is needed toward finding a solution to this most confounding and deadly problem.
Inhibitory Effect of Carotenoids on Mycotoxin Metabolism and Genotoxicity
E Gonzalez de Mejía. Departamento de Investigacion y Posgrado en Alimentos. Facultad de Quimica. Universidad Autónoma de Queretaro, Queretaro, QRO.
The presence of compounds with antimutagenic properties in several vegetables, fruits and legumes of daily consumption opens the possibility to reduce the risks of cancer or any other mutational process. The carotenoids might modulate genotoxicity in humans and it is believed that this effect is intrinsic to the molecule. Several studies have shown that there exists a dose-dependent decrease in mutagenicity due to the presence of some carotenoids, which may provide a valuable chemopreventive effect in the diet. The purpose of this presentation is to provide evidence of the inhibitory effect of carotenoids against mycotoxin genotoxicity. For instance, the xanthophylls have the ability to inhibit aflatoxin Bl(AFB1) mutagenicity using the Salmonella plate incorporation test (YG1024) due to the alteration in the mycotoxin metabolism. Lutein and xanthophyll pigments inhibit the mutagenicity of AFB1 in a dose-dependent manner. The inhibition on AFB1 (0.5 µg/plate) mutagenicity was 52% for lutein (10 µg/plate) and between 82 and 95% for natural xanthophyllic pigments (2 µg/plate). Lycopene (1x106 M) was able to reduce the cytotoxic effect of T-2 mycotoxin (5x104 M) when both were in contact with cells, either simultaneously or previous to incubation. The protective effect correlated with a reduction of glutathione and an increase of lactate dehydrogenase leakage. The results suggest that the carotenoids present in the diet have a chemoprotective potential against mycotoxins, besides their use as natural colorants in the food industry.
Molecular Epidemiology of Aflatoxin Hepatocarcinogenesis in Qidong, People’s Republic of China
TW Kensler, J-S Wang, JD Groopman, B-C Zhang, Y-R Zhu, S-Y Kuang and G-S Qian. Johns Hopkins School of Public Health, Baltimore, MD, Qidong Liver Cancer Institute, Qidong, P.R.C. and Shanghai Cancer Institute, Shanghai, P.R.C.
Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa. Over the past thirty years there has been extensive efforts to investigate the association between aflatoxin exposure and HCC. These studies have been hindered by the lack of adequate dosimetry data on aflatoxin intake, excretion and metabolism in people, as well as by the general poor quality of world-wide cancer morbidity and mortality statistics. These realities have spurred the efforts to develop new technologies to assess exposure status and risk for aflatoxins and these agents are among the few environmental carcinogens for which quantitative risk assessments have been attempted. One of the goals of these risk assessments has been the development of preventive intervention methods that will try to lower the human health impact from aflatoxin exposures.
HCC is the leading cause of cancer death in Qidong City, Jiangsu Province, P.R.C., resulting in approximately 10% of all adult deaths (1). Moreover, the median age of death from HCC in this region is 50 years. Qidong is located 50 km north of Shanghai on the Changjiang River. Two major risk factors for HCC in this region appear to be infection with hepatitis B virus (HBV) and exposure to aflatoxin B1 (AFB1). Climatic conditions in Qidong, featuring high humidity and average summer temperatures above 30 C, are conducive to mold spoilage of foods and therefore aflatoxin contamination. Food surveys indicate persistent contamination of dietary staples including rice, corn, peanuts and soy with aflatoxin. Aflatoxin has also been postulated as a causative agent for the G T transversion mutation at codon 249 in the p53 gene that is commonly seen in HCC from Qidong (2). However, these studies do not directly determine the relationship between AFB1 exposure and HCC.
In general, the most rigorous test of an association between an agent and disease outcome is found in prospective epidemio-logical studies, where healthy people are followed until the diagnosis of disease. Qian et al. (3) have used urinary aflatoxin biomarkers in a nested case-control study in Shanghai to prospectively evaluate the relationship between aflatoxin exposure and HCC risk. Results from this study demonstrated a dramatic synergistic interaction between aflatoxin and HBV infection in the risk of HCC. In HBV-infected individuals, presence of aflatoxin metabolites in urine resulted in a significant, 2.8-fold increase in relative risk for HCC. Unlike measurements of urinary excretion of aflatoxins, which measure very recent exposures to aflatoxins, the determination of serum levels of aflatoxin-albumin adducts provides a more integrated assessment of aflatoxin exposures over a several month period. A recent longitudinal study in 120 residents of Daxin Township in Qidong demonstrated the consistent presence of aflatoxin-albumin adducts in most participants (4). Neither HBV surface antigen status nor gender modified either baseline mean or the temporal trend. Thus, to directly address the role of aflatoxin in the etiology of HCC, a prospective nested case-control study was instituted in Qidong in 1991. Sera were obtained from 804 healthy HBV surface antigen-positive individuals aged 30-65. Between the years 1993-95, 38 of these individuals developed liver cancer. The serum samples for 34 of these cases were matched by age, gender, residence and time of sampling to 170 controls. The relative risk for HCC among aflatoxin-albumin adduct positive individuals was 2.4 (95% C.I.:1.2,4.7). Because the aflatoxin-albumin adduct has been extensively validated as an exposure and risk biomarker in this population, the modulation of levels of aflatoxin-albumin adducts is currently being used to evaluate the efficacy of the drug oltipraz in a chemo-intervention trial in Daxin Township. Supported in part by NIEHS P01 ES06052.
(1) Yu, S-T. (1995) J. Gastroenterology and Hepatology 10:674-682.
(2) Greenblatt, M.S. et al. (1994) Cancer Res.54:4855-4878.
(3) Qian, G-S., et al. (1994) Cancer Epidemiology, Biomarkers and Prevention
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(4) Wang, J-S. et al. (1996) Cancer Epidemiology, Biomarkers and Prevention
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Paradoxical Effects of the Soy Phytoestrogen, Genistein, on Growth of Human Breast Cancer Cells In Vitro and In Vivo
WG Helferich. Department of Food Science & Human Nutrition, Michigan State University, East Lansing, Ml.
Genistein, found in soy products, is a phytochemical with several potent biological activities. Our research has focused on the effects genistein has on growth of both estrogen (E)-dependent and E-independent human breast cancer cells both in vitro and in vivo. In vitro, genistein enhances the proliferation of E-dependent human breast cancer cells at concentration as low as 200nM achieving similar effects to that of estradiol at 1 nM. Expression of the estrogen-responsive gene, pS2 was also induced in response to treatment with genistein. However, at higher concentrations genistein inhibits growth of both E-dependent and E-independent human breast cancer cells. To evaluate whether these proliferative or anti-proliferative effects observed in vitro could be reproduced in vivo, E-dependent cells were implanted into 4 subcutaneous sites in ovariectomized athymic mice. Negative control animals received AIN-93G diet, the positive control group received a new subcutaneous estradiol (2mg) pellet, and the third group received genistein at 750ppm in the AIN-93G diet. Tumor size was measured weekly. Tumor size was larger in the 750 ppm genistein treated compared to the negative control group indicating that genistein was able to act as an estrogen agonist to stimulate growth of E-dependent breast cancer cell tumors in vivo. Similar studies were conducted using E-independent cells in intact athymic nude mice. However, no change in growth of E-independent breast cancer cells was observed. In summary, genistein can block cell proliferation at high concentrations in vitro, however these effects were not observed in vivo. Additionally, genistein acts as an estrogen agonist resulting in proliferation of E-dependent human breast cancer cells in vitro and enhances growth of MCF-7 cell tumors in vivo implanted into ovariectomized athymic mice.
Monitoring of Toxic Residues in Foods of Animal Origin
L Vázquez-Moreno. Centro de Investigación en Alimentación y Desarrollo A.C., Hermosillo, SON.
The past decades have seen a remarkable scientific revolution in agriculture, in which chemical technology has played a crucial role. Chemicals from a variety of sources may enter the food supply, including some that have short or long term harmful effects in humans. The exposure of animals to environmental contamination or the use of pesticides or drugs in unsuitable forms can leave accumulated residues in the edible tissues. In response, the Mexican Government implemented Official Norms which limit the risk to the consumer population from the contaminants. Monitoring was conducted to obtain information on the frequency and levels of residues occurring in the northern part of México. The monitoring of toxic residues in animal tissues and organs included 155 samples from 1993 to April of 1996. The samples were analyzed for pesticides (both organochlorines and organophosphates), polychlorobiphenyls, heavy metals, antibiotic residues and tissue identification using approved international methods. As expected, copper and cadmium were found in all analyzed samples. Lead and arsenic were detected in a reduced number of samples. The antibiotic residues vary in concentration and type during the years. Our results showed no residue violations or above tolerance concentrations in any of the samples where compounds were detected. This data is being used to evaluate residue trends and to identify problems within the meat industry to propose educational or regulatory actions for correction.
Occupational Exposure to Pesticides and its Relationship with Biological Markers
MC González-Horta, ME Olave-Arreola, C Méndez-Rentería, and L Hernández. Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Chihuahua, CHI.
There are few studies in México on the proportion of the population exposed to pesticides as well as on the intensity and type of exposure. The first part of this study consisted of identifying risk groups based on the type and intensity of exposure to organophosphororus pesticides. This was done by applying a survey on the Work Process in 18 agricultural communities in the state of Chihuahua.
From 2200 workers interviewed, 46.6% of them used agrochemical compounds, and 757 applied the agrochemical in a direct way. The group was stratified according to the protection equipment used while handling pesticides. The categories were High Risk, including all the individuals who never used protection equipment (38.44%), Medium Risk, those who use protection equipment sometimes (34.28%) and Low Risk, those who always used protection equipment. Comparing the education level and the way they learn to use pesticides in the High and Medium Risk groups, no relationship was observed. Independently of the education level, 93% of the population learned to handle pesticides by watching other individuals.
In a pilot study of exposed workers, the activity of acetylcholinesterase in blood was determined. Blood samples were taken before and after exposure to organophosphorus pesticides. No significant difference was observed between a control group (never exposed to pesticides) and workers before pesticide exposure. A significant difference was detected in the activity of acetylcholinesterase in the worker's group before and after exposure to organophosphorus pesticides.
Environmental Estrogens and Human Male Reproductive Problems
SH Safe. Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX.
Carlsen and coworkers1 have analyzed several studies on male sperm counts and their results suggest that during the last 30 to 50 years, there has been a 50% decline in sperm counts. Subsequently, it was hypothesized that the worldwide decrease in sperm counts and related male reproductive problems such as testicular cancer and cryptorchidism may be related to in utero exposure to estrogenic compounds2. Included in the list of possible etiologic agents were xenoestrogens and other endocrine disrupters such as polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), and DDT/DDE. Although there are reports which show that sperm counts have decreased in some regions/countries, other reports have shown that sperm counts have not decreased in various locations in the United States3. Similarly, an increased incidence of cryptorchidism in infants was reported in the United Kingdom whereas no changes were observed in a study of infants in New York4. Testicular cancer has increased in most countries; however, a recent study indicated that while the incidence of testicular cancer has increased in Scandinavia since the 1950s, breast milk DDE levels have steadily decreased since 1965. Moreover, despite the higher incidence of male reproductive problems in Denmark compared to Finland, there is no apparent organochlorine or xenoestrogen gradient between these two countries. The factors responsible for region/country-dependent male reproductive problems are unknown and require further investigation.
Essential-Toxic Metal Interactions
RA Goyer. National Institute of Environmental Health Sciences, Research Triangle Park, NC
The adverse health effects of the toxic metals, lead, cadmium and mercury may be enhanced or decreased by interactions with nutritionally essential metals. Lead competes with calcium, inhibiting the release of neurotransmitters, and interfering with the regulation of cell metabolism by binding to second-messenger calcium receptors, blocking calcium transport via calcium channels and calcium-sodium ATP pumps, and by competing for calcium-binding sites and uptake by mitochondria. Dietary deficiencies of calcium, iron and zinc intensify the effects of lead on cognitive and behavioral development. Children at risk to excess exposure to lead should receive adequate dietary calcium and iron supplementation if iron deficient.
Iron deficiency increases the gastrointestinal absorption of cadmium. It has been shown in experimental animals and humans that cadmium absorption from the intestine is inversely related to blood ferritin levels. Cadmium also competes with zinc for binding sites on metallothionein, which is important for the storage and transport of zinc during development. Selenium protects from mercury vapor and methyl mercury toxicity by preventing damage from free radicals or by forming inactive mercury-selenium complexes.
Dietary Patterns and Nutritional Status of the Mexican Population
H Bourges. National Institute of Nutrition, México, DF.
México is a cultural mosaic where a wide variety of dietary patterns coexist. After outstanding agricultural achievements, different highly refined cultures developed for over 30 centuries in a geoclimatically heterogeneous environment generating a diversity of eating styles which later blended in different ways and proportions with the Mediterranean and Arabic elements brought by the Spaniards and, secondarily, with the French and Austrian influences of the Hapsburg's empire. The highest expressions of all these dietary patterns constitute the elegant and baroque Mexican cuisine recognized as one of the great culinary styles of the world. In their simpler day to day expression, these patterns have adapted for centuries to the local economy and resources, and in general are ecologically friendly and correspond to what is presently accepted as desirable for health, i.e. they are rich in unrefined cereals, legume seeds, fruits and vegetables and contain limited portions of animal foods resulting in low-fat, low-cholesterol, low-sodium, low-sucrose, high in complex carbohydrate, fiber-rich diets with a reasonable supply of calcium and antioxidant vitamins. Access to these patterns, however, is limited by unfortunate socioeconomic disparities favoring malnutrition among the poorest and more isolated groups. On the other hand, accelerated and chaotic urbanization and "modernizing" influences have in recent years deteriorated the diet of the affluent sectors favoring nutritional excesses and imbalances. An overview of the main dietary patterns in México and available data on the complex combination of poverty-linked deficiencies and ignorance-linked excesses will be presented.
Dietary Modulation of Chemical Toxicity
RW Hart, Angelo Turturro, Julian Leakey, Peter Duffy and Richie Feuers. National Center for Toxicological Research, Jefferson, AR.
Numerous chemical, physical, and biological agents have been shown to alter homeostasis both in vitro and in vivo. Modest modifications in dietary intake have been demonstrated to alter the response of living systems to these agents. While the mechanism by which such changes are introduced is not understood it appears that if caloric intake rises above that required to maintain resting metabolic rate efficiency of a number of biological systems (whose primary function is the maintenance of homeostasis) decreases. For example, formation of free radicals, fatty acid epoxides, and production of the products of lipid peroxidation all increase, the ability to inactivate free radicals and detoxify both exogenous and endogenous agents decreases as does the capacity to repair DNA damage, the fidelity of DNA replication decreases as does apoptosis whereas cellular replication is enhanced. These and a plethora of other defense mechanisms are thus down regulated as caloric intake increases or malnutrition is induced. As a consequence the ability of the organism to modulate toxicity induced by exogenous agents is impaired. Studies in mice, rats and primates have shown that modest modifications in body weight (as little as 15% to 20%) can result in a significant difference (up to 400%) in tumor occurrence, toxicity of various drugs ( up to 20 fold), and MTD (up to 4 fold). Failure to take into consideration differences in body weight among individuals and study groups can in many cases have an equal or greater impact on interpretation of experimental data than differences of strain, age or sex. Therefore, in risk assessment failure to account for such differences may significantly alter the final calculation of hazard for any given compound. The public policy importance of this becomes profound as we attempt to expand risk assessment procedures across cultures with different dietary intake patterns than those of the United States of America.
The Pima Indians of Sonora and Arizona: A Contrast in Obesity and Non-Insulin Dependent Diabetes Mellitus
ME Valencia, E Ravussin, P Bennett, J Esparza and L Schulz. Centro de Investigación en Alimentación y Desarrollo, Hermosillo, Sonora, México, NIDDKD, NIH, Phoenix, AZ and University of Wisconsin, Milwaukee, WI.
The Pima Indians of Arizona have the highest reported prevalences of obesity and non-insulin dependent diabetes mellitus (NIDDM). In contrast, the Pimas of Maycoba, Sonora México, believed to have been separated from this group 700-1000 years ago, are still living under a traditional lifestyle with non-mechanized agriculture, breeding livestock and lumber milling activities. Their diet is quite simple and is based on a few staples. The study to be discussed deals with the possible impact of environmental factors, such as diet and physical activity, on the prevalence of obesity and NIDDM. Statistically designed samples of Mexican Pimas living in Maycoba (same genetics, different environment) and Non-Pimas of Maycoba (same environment., different genetics), are compared to randomly selected Arizona Pimas matched by sex and age. Pima heritage has been established preliminarily by Pima language, history and glutocronology, DNA Fingerprinting to establish genetic distance is being obtained. Preliminary results show that Mexican Pimas are lighter and shorter, with lower body mass indexes and lower plasma cholesterol levels. Their diet show that 24.7% of the energy comes from fat and dietary fiber consumption is approximately 50 g/day. They show high physical activity indexes, which are due to the long hours of physical labor in the fields and long walking distances to and from the Maguechis (farms). In another preliminary subsample of 170 Mexican Pimas, we found 3.5% of diabetes prevalence, which is much lower than in their counterparts in Arizona. These preliminary results show that obesity and NIDDM is less prevalent among people of Pima heritage living a traditional lifestyle than among Pimas of Arizona, suggesting that environment may play an important role. (funded by: NIH-DK 45957).
Dietary Practices and Cancer in Sonora, México
CJ Wyatt. Centro de Investigación en Alimentación y Desarrollo, A.C., Hermosillo, SON.
In 1981, Doll and Peto estimated that approximately one third of the cancers in the United States (Doll and Peto, 1981) was attributed to dietary factors. Information on diet and cancer has increased since that time, and generally supports the earlier estimate. Some of the specific dietary risk factors are; caloric intake, fruit and vegetable consumption, intake of dietary fiber and animal fat, formation of certain chemical compounds during cooking, certain intestinal infections, and alcohol consumption. Comparing the data available for reported cases of stomach and colon cancers between the states of Sonora, México and Arizona, we find some startling differences. In 1991, Sonora reported 40 cases of colon cancer and Arizona reported 1432, and this level appears to be maintained in following years. The situation for stomach cancer is reversed. Arizona, over the last 5 years, reported an average of 214 cases. In Sonora, the incidence of stomach cancer is much higher and appears to be on the increase. The typical Sonoran diet is high in insoluble dietary fiber, high in saturated fat, low in fresh fruit and vegetable consumption, alcohol calories are 4% of the total energy, "carne asada" (charcoal barbecued meat) and processed meats are frequently consumed. The purpose of this paper is to explore some of the dietary practices in Sonora and how these practices may contribute to the incidence of stomach and colon cancers.
Diet and Nutrition and Protection against Environmental Carcinogenesis
JR Marshall. Department of Family and Community Medicine, Arizona Cancer Center, The University of Arizona, Tucson, AZ.
Epidemiologic research has revealed several possible dimensions of diet and environment that might affect cancer risk. Much of this research to date suggests that the effects of these dimensions upon risk are modest.
It has been suspected that the effects of diet and environment are interactive. Thus, whether environmental exposure alters cancer risk might depend upon exposure to different elements of diet. Such interactive effects could be described as synergy or buffering.
The epidemiologic data offer a limited number of examples of diet-environment interactions, although the implications of these examples are striking. The examples confirm that the interactive component of diet and environmental effects could be extremely powerful.
The leading impediments to identification of diet-environment interactions are imprecision in exposure assessment and statistical imprecision resulting from inadequate sample size. This paper demonstrates that imprecision in exposure assessment, within ranges of epidemiologic plausibility, decisively biases estimates of the interactive effects of exposures. Clearly, therefore, it will be necessary that future research develop means of minimizing imprecision in exposure assessment. At a bare minimum, it will be necessary that epidemiologic inquiries collect detailed data on the nature and structure of imprecision in exposure assessment.
Progress in identification of diet-environment interactions will await development of large, human-based research resources that allow for accurate, unbiased measurement of diet and environmental exposures. Such progress will also await studies in which the imprecision in such exposure assessment is clearly understood.
Developing Dietary Strategies to Reduce Breast Cancer Recurrence
C Thomson, CK Ritenbaugh, D Alberts and J Pierce and S Faerber. Arizona Prevention Center, The University of Arizona, Tucson, AZ.
An estimated 186,000 American women will be diagnosed with breast cancer this year; one in eight will be diagnosed within their lifetime. Current estimates indicate that annually 3% of women treated for stage I or II breast cancer will experience a recurrence of the disease within the first 15 years post-therapy. Several dietary risk factors for breast cancer have been identified including low fruit, vegetable and dietary fiber intake and high dietary fat intake. Case control studies have shown odds ratios for breast cancer as favorable as 0.2 (vegetable index) or 0.3 to 0.4 (broccoli, tomatoes and carrots) internationally. These ratios hold true even when adjusted for confounders such as social class, body weight, and later age at first birth. Many of the nutrients (antioxidants and folate) as well as non-nutrient food components (phytochemicals) found in fruits and vegetables protect against cancer in vitro at several stages of carcinogenesis. These findings have led primarily to the development of clinical trials using supplementation of select nutrient(s) rather than trials implementing a complete dietary intervention which would supplement the diet with a variety of nutrients and phytochemicals in the form of food. The Women's Healthy Eating and Living Study is a prospective, randomized, controlled multi-center clinical trial being conducted in the Southwestern United States to evaluate the efficacy of a high fruit and vegetable, high fiber, low fat diet in improving survival in women previously treated for breast cancer. Women are required to significantly increase their consumption of fruits, vegetables and fiber above baseline intake levels concurrent with a significant reduction in dietary fat intake. Preliminary data indicates compliance at 80% of dietary goals can be achieved using a high intensity counseling protocol.
Diet, Bile Acids and Colon Cancer
D Earnest. Department of Gastroenterology, The University of Arizona, Tucson, AZ
Colorectal cancer is the second most frequent cause of cancer death in the United States for both men and women. During 1995, there were 138,200 new cases and 55,000 people died of this malignancy. Clearly, new approaches to prevention and treatment of colon cancer are needed. Unfortunately, there has been no significant improvement in survival statistics resulting from treatment of patients with advanced disease. Consequently, over the past decade much effort has been placed on developing effective screening programs to locate patients with early cancer and those with adenomatous colon polyps, a recognized cancer precursor. Follow-up programs for those with adenomas generally focus on repeated colon examinations to remove recurring tumors. This approach is expensive and does not provide a method for risk reduction for large population groups.
It is thought that colon cancer is initiated by chronic exposure to low doses of carcinogens in the diet. A number have been identified, such as IQ present in overcooked beef. The expression of induced genetic damage is also thought to be hastened by other environmental factors, including constituents in diet and possibly by bile acids excreted in stool. A large number of studies suggest that colon cancer risk in the population is increased by diets high in animal fat, low in calcium, and low in fiber. Exactly how these nutrients affect cancer risk is unknown.
We have focused on the potential role of deoxycholic acid, a secondary bile acid, that occurs in high concentration in fecal water and which has been shown to be a cancer promoter, to induce DNA damage and which has been proposed to play an important role in promoting colon cancer in humans. Of interest, the concentration of deoxycholic acid in fecal water is increased by high fat, low calcium and low fiber diets. We recently observed that patients treated with ursodeoxycholic acid (Actigall®) for cholestatic liver disease or dissolution of gallstones had significant reductions in the proportion of deoxycholic acid both in bile and in stool. We determined that ursodeoxycholic acid competitively inhibited 7-a hydroxylase in fecal-type bacteria and thereby decreased production of deoxycholic acid from its precursor cholic acid. A subsequent study in F344 rats induced with the carcinogen azoxymethane compared incidence of colon cancer in rats whose diet was supplemented with cholic acid (known cancer promoter) or with the NSAID piroxicam (cancer suppressor) or with ursodeoxycholic acid. As expected, cholic acid promoted and piroxicam suppressed cancer formation. However, ursodeoxycholic acid decreased fecal deoxycholic acid by 50% and completely prevented cancer formation. This presentation will review these studies and discuss ongoing basic and clinical studies evaluating the use of ursodeoxycholic acid as a chemoprevention agent for colon cancer.
Mechanisms of Prevention of Cancer and Chemical Toxicity by Antioxidants
DC Liebler. Department of Pharmacology and Toxicology, College of Pharmacy, TheUniversity of Arizona, Tucson, AZ
A large body of epidemiologic evidence suggests that dietary intake of the antioxidant nutrients vitamin E (alpha-tocopherol) and beta-carotene may reduce risk of certain cancers and of other degenerative diseases. Large, randomized clinical trials are evaluating the effectiveness of alpha-tocopherol and beta-carotene in disease prevention and may provide insight into doses needed to provide antioxidant protection in vivo. Many other studies in animal and in vitro model systems suggest that these same antioxidants play a critical role in protecting cellular components against oxidative damage caused by prooxidant chemicals. These compounds are thought to function in tissues as chain-breaking antioxidants, which scavenge peroxyl radicals responsible for the propagation of oxidative damage. Carotenoids such as beta-carotene function as singlet oxygen quenchers and recent work indicates that topically applied alpha-tocopherol acts as a highly efficient sunscreen to prevent UV-B induced DNA photodamage. These observations suggest that topical antioxidants may provide protection against photosensitized chemical toxicity in the skin. There is a growing interest in other plant-derived natural products as antioxidants with tissue protective properties. These include flavonoids, carotenoids, and diverse plant polyphenols. Little is known about the antioxidant properties of such compounds or their metabolism and disposition in vivo. Further work in this area will expand our understanding of the mechanisms by which certain nutrients prevent chemically induced tissue injury.
Chemoprotection by Inducers of Carcinogen Detoxication Enzymes
TW Kensler, T Primiano, J Groopman, Y Zhang, P Talalay and BD Roebuck. Johns Hopkins University, Baltimore, MD and Dartmouth Medical School, Hanover, NH
One of the major mechanisms of chemical protection against carcinogenesis, mutagenesis and other forms of electrophile toxicity is the induction of enzymes involved in electrophile metabolism, particularly phase 2 enzymes such as glutathione S-transferases (GSTs), UDP-glucuronosyl transferases and NAD(P)H:quinone reductase. Furthermore, induction of phase 2 enzymes appears to be a sufficient condition for obtaining chemoprevention and can be achieved in many target tissues by administering any of a diverse array of naturally-occurring and synthetic chemical agents. One class of chemopreventive agents, 1,2-dithiole-3-thiones, was developed on the basis of their potent activity as inducers of GSTs. A substituted dithiolethione, oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione], is an effective inhibitor of aflatoxin B1 -mediated hepatocarcinogenesis in the rat. Oltipraz produces dramatic decreases in the levels of aflatoxin-DNA adducts in the liver as well as in the urinary levels of the depurination product, aflatoxin-N7-guanine. Corresponding increases are seen in the biliary elimination of aflatoxin-glutathione conjugates. Administration of oltipraz results in 3-4 fold increases in hepatic cytosolic GST activities and mRNA levels for some, µ and isoforms. The transcriptional activation of phase 2 enzymes by oltipraz is mediated, at least in part, through the "antioxidant response element" in the 5 flanking region of these genes. Elevations in the levels of some GST isoforms can persist up to 1 week after dosing with oltipraz. Concordantly, intermittent dosing schedules (i.e., once-weekly) are nearly as effective as daily interventions for inhibition of aflatoxin-mediated hepatic tumorigenesis in rats. The protective efficacy of daily and weekly administration of oltipraz to people in Qidong, People's Republic of China, who are at high risk for aflatoxin exposure and subsequent development of hepatocellular carcinoma, is currently under evaluation. In this randomized placebo-controlled trial, levels of aflatoxin biomarkers in blood and urine are being measured over a 4 month period to assess the pharmacodynamic action and possible efficacy of oltipraz.
Although pharmacological agents offer great promise as chemopreventive agents and provide certain practical advantages during the course of their development and evaluation, they are primarily suited for use in well-defined, high-risk populations and are not applicable for use in the general population. Epidemiological studies have also shown that diets containing large quantities of vegetables and, to a lesser extent, fruits are associated with relatively low risks for cancer. Indeed, the realization that dietary factors reduce the incidence of cancer has been a major driving force for the current view that chemoprotection offers an effective means for reducing mortality from cancer. Many classes chemoprotective agents, both nutrients and non-nutrients, are produced by the plant kingdom. The isothiocyanate sulforaphane has been recently identified as the major chemopreventive component of some cultivars of broccoli. Sulforaphane is an effective inhibitor of mammary carcinogenesis in the Huggins model in rats. The anticarcinogenic properties of sulforaphane appear to be related to induced alterations in the metabolism and disposition of carcinogens (i.e., inhibition of phase 1 cytochrome P450 enzymes and induction of phase 2 enzymes). Programs are under development aimed at creating food products fortified with protective phytochemicals. In this way common foods rather than drugs or nutritional supplements may have a prominent role in protection against human cancers in the future. Supported in part by NIH grants CA49316, CA44530, N01-CN-25437 and ES06052.
Induction of Antioxidant Enzymes by Antioxidant Food Additives
L Favreau. Schering Plough Research Institute, Kenilworth, NJ
Phenolic antioxidants are commonly used as food additives and have been shown to have chemopreventive properties, i.e. they protect the cell from the toxic effects of compounds such as carcinogens. Chemopreventive agents have been shown in rodents to induce Phase II drug-metabolizing enzymes such as glutathione S-transferase and quinone reductase.
In order to determine the molecular mechanisms involved in the induction of these enzymes, Northern blots were used to show that mRNA levels become increased after exposure of hepatoma cells to xenobiotics such as 13-naphthoflavone. Therefore regulation of these two genes occurs at the level of transcription. Isolation of each gene and the 5' flanking sequences allowed for the identification of cis-acting regulatory sequences. Using reporter constructs transiently transfected into hepatoma cells, it was found that short sequences of DNA (31-41 nucleotides) contain elements which are responsive to 13-naphthoflavone and t-butylhydroquinone. Using double stranded oligonucleotides containing these Antioxidant Response Elements, nuclear extracts were analyzed for DNA binding proteins. The ARE was found to bind with high affinity and specificity to a nucleoprotein complex from human and rat hepatoma cell lines. However, after treatment of cells with inducers no increase in the intensity of this complex or the induction of a new complex was observed. These observations are consistent with the presence of a constitutive nucleoprotein complex which is modified by inducers to transcriptionally regulate the levels of these two genes. (For a review, see Favreau, L.V. and Pickett, C.B. (1996) "The Antioxidant Response Element" FASEB J. in press)
Vitamin A Potentiation of Chemical-Induced Liver Injury
IG Sipes. Department of Pharmacology and Toxicology, College of Pharmacy, TheUniversity of Arizona, Tucson, AZ
Vitamin A, retinol, is an important dietary component. However, at least in rats, large doses of vitamin A (retinol) dramatically increases the hepatotoxicity of carbon tetrachloride. Experiments were performed to elucidate the mechanism of this potentiation. Hypervitaminosis A was produced by oral administration of retinol, 250,000 IU/kg/day for seven days. CCl4 was then administered at a dose of 0.15 ml/kg, ip.
At 24 hr after CCl4 administration, plasma GPT activity was increased 7-fold and the extent of centrilobular necrosis was dramatically increased in vitamin A pretreated rats. This large dose of vitamin A did not enhance the biotransformation of CCl4, but did produce a 4-fold increase in the CCl4-induced lipid peroxidation, as assessed by ethane exhalation. Because vitamin A has been shown to activate macrophages, it was hypothesized that this increased lipid peroxidation and liver injury resulted from the release of reactive oxygen species from activated Kupffer cells. To test this hypothesis Kupffer cells from control and vitamin A treated rats were cultured and superoxide anion release was measured following stimulation with phorbal ester. Kupffer cells from vitamin A treated rats released significantly more superoxide anion (p < 0.01) than Kupffer cells from control rats. These Kupffer cells also had enhanced phagocytic activity in vitro towards sheep red blood cells and in vivo towards colloidal carbon. In vivo administration of superoxide dismutase (SOD) and catalase (CAT) 2 hr after CCl4 exposure did not influence CCl4 toxicity in control rats but did block the enhanced ethane exhalation and also the potentiation of CCl4 liver injury in vitamin A treated rats. Administration of methyl palmitate, an inhibitor of Kupffer cell function, did not inhibit CCl4 toxicity in control rats, but did effectively block enhanced ethane exhalation and potentiation of CCl4 injury in vitamin A treated rats.
We conclude that the vitamin A potentiation of CCl4 hepatotoxicity (and that of other chemicals) is mediated in part by reactive oxygen species released from activated Kupffer cells. (Supported by NIH grant ES06095 and NIEHS Center Grant ES06694).
The Nutritional Prevention of Cancer with Selenium 1983 - 1993: A Randomized Clinical Trial
LC Clark1, GF Combs Jr.2 and BW Turnbull3. 1Arizona Cancer Center, College of Medicine, The University of Arizona, Tucson, AZ; 2Division of Nutritional Sciences and 3Statistics Center, Cornell University, Ithaca, NY.
Background. The present study is the first randomized, double-blind prevention trial to test the specific hypothesis that a supplement of the essential nutrient selenium can reduce the risk of cancer.
Methods. The trial randomized a total of 1,312 patients with histories of basal cell or squamous cell carcinomas of the skin to either a daily oral supplement of 200 µg selenium, or a placebo. They were followed with regular semi-annual dermatologic examinations and annual vital status for a total of 8,269 person-years of observations (xx 7.0 years).
Results. No cases of selenium toxicity occurred. Selenium treatment did not affect primary endpoints — incidence of new basal or squamous cell carcinoma of the skin. Selenium-treatment was associated with statistically significant reductions in several secondary endpoints: total and lung cancer mortality, total cancer incidence, colorectal cancer and prostate cancer incidence. Total cancer incidence was 42% lower in the selenium group (p < .001). The consistencies of these associations over time, between study clinics and for the leading cancer sites strongly suggest that, for this cohort of patients, selenium supplementation had substantial health benefits.
Conclusion. Selenium does not show protective effects against non-melanoma skin cancers. However, results support the hypothesis that supplemental selenium can reduce the incidence of, and mortality from, carcinomas of several sites.
Carnitine and its Esters as Biomarkers of Peroxisome Proliferating Agents-Tumorigens
JE Garst. private consultant, Alamogordo, NM.
Peroxisomal proliferating agents (PPA) are structurally diverse, widely used drugs, industrial chemicals, and agrochemicals, that cause hepatomegaly by increasing both the number and size of liver cells. More importantly, they produce hepatic tumors in rodents, but PPA are termed nongenotoxic tumorigens because they do not directly affect DNA. Enhanced gene-expression and reduced apoptosis are suspected of involvement in non-genotoxic tumor formation. Liver tumors may arise because all PPA induce conversion of free l-carnitine, [3-hydroxy-4-(trimethylammonium)-butanoate] (Cn), mostly to O-acetyl-l-carnitine (AcOCn). Since perturbations that diminish free Cl and increase AcOCn have the effect of fostering gene expression and impeding apoptosis, respectively, these perturbations are postulated to define in molecular terms the events underlying non-genotoxic tumor promotion. Strangely PPA can protect against the toxicities of other substances, including acetaminophen. That protection may involve PPA-mediated changes in Cn and/or its esters, since it has become clear that Cn and/or its esters can also protect against a wide variety of toxic substances. Protection by Cn and/or AcOCn arises both from their critical roles in normal and abnormal metabolism and an ability to serve signal and regulatory functions.
Dietary Factors and Risk Assessment Complexity
GW Lucier. National Institute of Environmental Health Sciences (NIEHS) and the National Toxicology Program (NTP), Research Triangle Park, NC.
Recent legislative and government acts have called for improvements in risk assessment and prevention strategies. It is thought that the development of strategies to incorporate all relevent data will strengthen the science base for risk assessment. Increasing knowledge of biology and mechanisms can help in several ways. Programs in mechanism-based toxicology could draw on the tools of molecular biology which can characterize interactions of chemicals with critical target genes, to provide viable approaches for the development of more accurate and inexpensive methods to perform not only the first step in risk assessment, i.e. hazard identification, but also contribute to determining quantitative dose-response relationships and establishing biomarkers for estimation of human exposure. Morever, knowledge of the factors responsible for the existence of sensitive subpopulations should enable us to more accurately estimate the range of expected risks from exposure to a chemical or class of chemicals. These factors include genetic predisposition, age, gender, and nutrition. Although considerable knowledge gaps exist, there is a growing body of scientific evidence that implicates diet and nutition as profound modulators of chemically-mediated diseases. First, trends and geographical incidences of some human diseases are at least, in part, related to diet. For example, breast cancer rates vary tremendously in different countries and diet is now known to play a key role in this variation. Dietary factors such as caloric intake, fat intake, protein intake, ingestion of phytochemicals and dietary intake of synthetic chemicals are likely involved. Secondly, food intake and dietary composition exert a profound influence on the pattern and incidences of cancer in rodents. Moreover, the carcinogenic activity and reproductive toxicity of several chemicals is also modulated by dietary factors. The mechanisms responsible for dietary influences on toxicity is not clear, but there are several possibilities. These include changes in chemical and hormone metabolizing systems, signal transduction pathways, cell proliferation and differentiation and oxidative damage. Approaches for incorporating dietary considerations into risk assessment will require advances in risk assessment methodologies to permit integration of diverse data sets.
Risk Assessment for Oral Exposure to Arsenic
CO Abernathy. Office of Science and Technology, U. S. Environmental Protection Agency, Washington, D C.
This presentation will focus on the adverse health effects observed after exposure to inorganic arsenic (As). For noncarcinogenic effects, the EPA uses the Reference Dose (RfD) for estimating a lifetime "no-effect" level of exposure. In this method, a no-observed-adverse-effect level (NOAEL) is divided by an uncertainty factor (UF) to derive the RfD. Using a Taiwan population, a NOAEL of 0.8 ug/kg/day was selected based on the absence of skin lesions and of possible vascular effects. There was no agreement on the UF. Some members of the RfD Workgroup wanted an UF of 1, while others wanted a 3 or a 10. This disagreement was resolved by the EPA Risk Assessment Council. An UF of 3 was chosen, resulting in an RfD of 0.3 ug/kg/day. However, it was noted that any value from 0.1 to 0.8 ug/kg/day could, with proper justification, be used in risk management decisions.
Unlike most chemicals whose cancer risk is based on animal studies, data on skin cancer prevalence in a human population exposed to As was available. The same Taiwanese population that was used to derive the RfD was used to calculate the carcinogenic potency of As. Among the 40,421 Taiwanese exposed to As, there were three subgroups: 0-0.29, 0.30-0.59 and greater than 0.6 mg As/l of drinking water. A control group of 7,500, exposed to 0 to 0.017 mg As/l were the "nonexposed" and no skin cancers were seen in this group.
A linear-quadratic model was fitted to the skin cancer data in order to estimate risk from ingesting As-containing drinking water. The risk for drinking water containing 1 ug/l is 5 X 10-5 (the 10-4 risk is 2 ug/l). It should be noted that the EPA's lifetime risk estimate for As is a maximum likelihood estimate (MLE) and not an 95% upper bound calculation.
There have been arguments over the use of the Taiwanese data as the basis for a U. S. regulation. Some of the positive aspects are; that a human population was studied, individuals underwent physical examinations, the midpoint of the lowest exposure group was 0.170 mg/l, and that the observed effects were both dose- and age-dependent. On the other hand, weaknesses include the facts that; it was an ecological study, it was assumed that the drinking water was the only source of As exposure, there were other chemicals in the water, and reliability of the analytical method under 100 ug/l. These factors will be discussed in relation to their impact on the risk assessment for As.
Nutritional Modulation of Toxicity: A Research Framework
W Suk. Division of Extramural Research and Training, National Institute of Environmental Health Sciences, Research Triangle Park, NC.
Nutrition holds promise in the amelioration, indeed prevention, of environmentally induced diseases or disorders. Using cancer as a paradigm for environmentally induced disease, nutritional strategies could intercede at a variety of distinct sites, in the multi step/multi event disease process. Further, it is becoming increasingly evident that the timing of exposure during maturation and development is critical in the initiation and subsequent disease/disorder development and progression. Similarly, it stands to reason that there may be critical periods during maturation and development in which intercession by nutritional agents would be of prime importance in the intervention of such processes. Nutritional strategies have cost effective potential to increase the quality of life of many individuals. The ability to modulate human toxicity from environmental agents by nutritional means has ramifications in risk assessment. It is an important research area and one that needs to be pursued from an interdisciplinary perspective. Comments on a research framework will be presented.
ME Cebrián, LM Del Razo and A Albores. Sección de Toxicología Ambiental. CINVESTAV-IPN, México, DF
In several industrialized countries, the risks derived from exposure to environmental contaminants are assessed through a standard process consisting of four basic stages: 1) Hazard identification. 2) Dose-response evaluation. 3) Exposure assessment, and 4) Risk characterization. Very often, international organizations adopt the results of this process to set guidelines meant to be followed by member countries. However, geographical differences in the prevalence and severity of diseases attributed to chemical exposures are known to exist and many explanations have been proposed. One attractive hypothesis relates to differences in nutritional status. An interesting example is related to the effects of nutritional status on the metabolism and excretion of arsenic, since experimental animals with deficient dietary intakes of methionine, choline or proteins, had a decreased excretion of arsenic and increased tissue retention.
In humans, severe health effects resulting from arsenic exposure have been reported mainly in populations of low socioeconomic status or different eating habits. In addition, high chronic arsenic exposure appears to decrease the ability of the body to methylate As. These finding may be of importance for the risk assessment of arsenic, since exposed individuals deficient in those nutrients may have a further impairment in their capacity to methylate, and thereby detoxifying arsenic. However, there have been few attempts to factor nutritional components in the evaluation of the dose response relationship and the hazard identification phases of the risk assessment process. Further research is needed to expand our understanding on how nutritional factors, which vary from toxic to toxic and nutrient to nutrient, quantitatively affects human susceptibility.
Diet and Risk of Human Leukemia
MT Smith1 and N Rothman2. 1School of Public Health, University of California, Berkeley, CA; 2Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.
Benzene is an established cause of acute myeloid leukemia. Recent studies in China suggest that it also causes non-Hodgkin's lymphoma, myelodysplastic syndrome and lymphocytic leukemias. In a study of workers exposed to benzene in Shanghai, we found that low-folate intake caused susceptibility to benzene-induced chromosome aberrations. There are also reports in the literature which suggest that low folate intake is a risk factor for benzene toxicity and leukemia. The most likely mechanism by which benzene exerts its leukemogenic effects is through its conversion to phenolic metabolites (phenol, hydroquinone, catechol and benzenetriol) which are oxidized in the bone marrow to toxic quinones. The evidence for this includes: 1) the fact that a combination of phenol and hydroquinone produces a toxicity in rodents similar to benzene; 2) that the phenolic and quinonoid metabolites of benzene produce a similar pattern of genotoxicity to benzene; 3) that oxygen radicals are formed in the bone marrow of mice exposed to benzene most likely as a result of the redox cycling of quinonoid metabolites; and 4) lack of NAD(P)H quinone-oxidoreductase (NQO1; DT-diaphorase) activity confers susceptibility to benzene toxicity ( Rothman et al. 1996 AACR abstract #1761). Dietary exposure to phenol, hydroquinone and catechol is high. High levels of adducts in blood proteins from these compounds have been found in both human and animal control populations. This suggests that dietary intake of benzene's phenolic metabolites may be involved in causing de novo leukemia. Further, it suggests that lack of NQO1 activity and low folate intake may be susceptibility factors for human leukemia. Future research should examine the role of dietary phenols, quinones and micronutrients in human leukemogenesis.