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Meeting Report |
Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, CANADA (M.C.A.)
Environmental Health Science Center, University of Rochester, Rochester, New York (T.W.C.)
Northern Ireland Center for Diet and Health, University of Ulster, Coleraine, NORTHERN IRELAND (J.J.(S.)S.)
Address for correspondence: Michael C. Archer, Ph.D., Department of Nutritional Sciences, University of Toronto, 150 College St., Fitzgerald Bldg., Toronto, Ontario, M5S 3E2 CANADA. E-MAIL: m.archer{at}utoronto.ca.
ABSTRACT
The health and resilience of humans and animals is, in large part, determined by the quality and quantity of the diet. This, in turn, may influence an individual’s capability to deal with stress including toxic insult. In addition, there may be specific components of the diet that modulate the toxicity of specific toxicants whether the latter are ingested as food or absorbed via other routes.
Many examples attest to the importance of interactions between dietary components and toxicants after absorption in the body. Such interactions occur at every level of biological organization from the molecular to the whole organism. Some may be synergistic, others antagonistic. Some may involve direct chemical reaction between the nutrient molecule and the toxicant, others may occur by indirect action at the cellular or organ levels. All examples point to the importance of considering diet when measuring the response to toxic agents whether in animals or humans.
In order to foster interaction between the sciences of nutrition and toxicology, The Heinz Institute of Nutritional Sciences is sponsoring a series of workshops. The first of these was held in June, 1999 at the University of Ulster to address evolutionary aspects of nutrition—toxicology (for report see Eur. J. Nutr, 39, 49–52, 2000). In June, 2000, a second workshop was held at the University of Toronto to address genetic aspects, and this is a brief summary of the proceedings.
We are beginning to understand the molecular basis of the regulation of gene expression by dietary factors and how genetic changes can affect response to toxicants. Recent advances in technology and a detailed understanding of disease etiology has led to the ability to study molecular determinants of disease risk. The workshop provided a forum for nutritionists, toxicologists, molecular biologists, epidemiologists and others to discuss common interests and to merge their efforts towards an integrated approach to nutrition—toxicology via genetics and genomics. The first session dealt with the mechanism by which nutrients such as fatty acids (Clarke), amino acids (Jefferson) and metal ions (Cousins) can regulate gene expression. In the second session, there were presentations on the effects of nutritional factors on genes of toxicological significance such as phase I and phase II enzymes of drug metabolism (Guengerich, Goodfellow and Grant) as well as on oxidative DNA damage and its repair (Collins, Weindruch). Session three dealt with gene-nutrient interactions in the development of chronic diseases such as diabetes (Hegele, Berdanier) and cancer (Kim, Ambrosone et al.). New developments such as DNA microarrays (McGlynn) and the use of transgenic and knockout models (Sehayek) were presented in the final session.
Key words: polyunsaturated fatty acids, zinc, methionine, cytochrome P450, acetyltransferase, antioxidants, aging, folate, cholesterol, breast cancer, colon cancer
SESSION 1. MECHANISMS OF NUTRIENT REGULATION OF GENE EXPRESSION
Polyunsaturated Fatty Acid Regulation of Intracellular and Interorgan Fuel Partitioning: A Transcriptional Mechanism
Steven D. Clarke
Division of Nutritional Sciences and the Institute of Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas
Dietary polyunsaturated fatty acids (PUFA), particularly those of the n-3 family, function as fuel partitioners in that they direct glucose away from fatty acid biosynthesis and toward glycogen storage and they direct fatty acids away from triglyceride synthesis and toward oxidation. The net effect of this re-partitioning is a decrease in circulating triglycerides and, in some species, a decrease in fat deposition. PUFA exert their effects on metabolism by inducing genes in liver and skeletal muscle that encode proteins of lipid oxidation (e.g., carnitine palmitoyltransferase-1, acyl-CoA oxidase) and by suppressing genes in liver and adipose tissue that encode proteins of fatty acid biosynthesis (e.g., fatty acid synthase). The up-regulation of oxidative genes occurs in response to an increased binding of the nuclear transcription factor, peroxisome proliferator activated receptor (PPAR) 
, to a hexameric direct repeat sequence located in the 5'-flanking region of the respective genes. DNA binding activity of PPAR is induced as a consequence of a PUFA interaction with a lipid ligand-binding domain within the PPAR protein. In contrast to the oxidative genes, PPAR as no direct effect on the expression of lipogenic genes. Rather, PUFA suppress hepatic lipogenic gene expression by decreasing the nuclear content of the powerful lipogenic transcription factor, sterol regulatory element binding protein-1 (SREBP-1). PUFA control the nuclear content of SREBP-1 in two ways. First, PUFA acutely (<3 h) inhibit the proteolytic release of mature SREBP-1 from its endoplasmic reticulum-anchored precursor. This acute effect of PUFA is followed by an adaptive PUFA-mediated increase in SREBP-1 mRNA degradation and a subsequent decrease in the production of precursor SREBP-1. PUFA also decrease the promoter activity of lipogenic genes by inhibiting the DNA binding activity of the potent enhancing factor, Sp1. The mechanism by which this occurs remains unclear. It is noteworthy that the maximal effect of PUFA on lipid metabolism occurs at a level of intake that is greater than five times the dietary amount needed to prevent the skin lesions characteristic of an essential fatty acid deficiency. These observations suggest that the regulation of gene expression by PUFA and the resulting alterations in cellular metabolism should be considered as part of the criteria utilized in defining the dietary needs for n-6 and n-3 PUFA, and in establishing the optimum dietary ratio for n-6:n-3 fatty acids.
Relevant Publications
Clarke SD: Polyunsaturated fatty acid regulation of gene transcription: a mechanism to improve energy balance and insulin resistance. Brit J Nutr 83:S59–S66, 1999.
Clarke SD, Thuillier P, Baillie R, Sha X: Fatty acid activation of peroxisome proliferator activated receptors (PPARs): its role in gene expression and cell differentiation. Am J Clin Nutr 70:566–577, 1999.
Xu J, Nakamura MT, Cho HP, Clarke SD: Sterol regulatory element binding protein-1 (SREBP-1) expression is suppressed by dietary polyunsaturated fatty acids: a mechanism for coordinate suppression of lipogenic genes by polyunsaturated fats. J Biol Chem 274:23577–23583, 1999.
Modulation of Gene Expression through Actions of Amino Acids on Translation of Messenger RNA
Leonard S. Jefferson
Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania
Recent advances in biomedical research reveal a key role for amino acids as nutritional signals in the regulation of a number of cellular processes. Studies employing a variety of cell types and different tissues demonstrate that one such process affected is the regulation of gene expression through modulation of the translation of messenger RNA (mRNA). The studies show that cells recognize alterations in amino acid availability and respond by either upregulating or downregulating translation initiation, i.e., the process during which initiator methionyl-tRNA (met-tRNAi) and mRNA bind to a 40S ribosomal subunit followed by the joining of a 60S ribosomal subunit to form a translationally competent 80S ribosome. Translation initiation is mediated by over a dozen proteins referred to as eukaryotic initiation factors (abbreviated eIF’s). Binding of met-tRNAi is mediated by eIF2, the met-tRNAi binding protein, and eIF2B, a GDP/GTP exchange factor for eIF2 that is subject to regulation by the phosphorylation status of the -subunit of eIF2 and the 
-subunit of eIF2B. Binding of mRNA is mediated by eIF4F, a complex consisting of eIF4E, the mRNA cap-binding protein, eIF4A, an RNA helicase, eIF4B, a stimulator of eIF4A helicase activity, and eIF4G, a scaffolding protein that binds eIF4E, eIF4A, eIF3, and the poly(A) binding protein, PABP. This step is regulated by the phosphorylation status of eIF4E, eIF4B and eIF4G, as well as by binding of eIF4E to a family of binding proteins (4E-BP1, -2, and -3) that prevents its association with eIF4G. The response of translation initiation to a change in amino acid availability can be general, i.e., affecting the translation of most if not all mRNAs, and/or specific, i.e., affecting the translation of a single class or subset of mRNAs. The general response is mediated through regulation of both the met-tRNAi and mRNA binding steps, whereas the specific response involves the mRNA binding step and an additional regulatory site, i.e., the phosphorylation status of S6, one of the proteins composing the 40S ribosomal subunit. Sufficient availability of amino acids for optimal rates of protein synthesis is characterized by hypophosphorylation of eIF2 allowing for unimpeded eIF2B activity, hyperphosphorylation of 4E-BP1 resulting in its dissociation from eIF4E and thus allowing association of eIF4E with eIF4G to form the active eIF4F complex, and hyperphosphorylation of S6. The increased availability of eIF4E caused by 4E-BP1 phosphorylation results in preferential translation of mRNAs containing highly structured 5'-untranslated regions. Likewise, the hyperphosphorylation status of S6 favors translation of mRNAs containing a 5'-terminal oligopyrimidine tract (TOP). The Ser51 residue of the -subunit of eIF2 is a substrate for four different protein kinases; however, the one involved in mediating the amino acid response is presently unknown as is the mode through which the cell recognizes amino acid sufficiency. Both 4EBP1 and the protein kinase that phosphorylates S6, i.e., S6K1, are downstream in a signal transduction pathway involving a protein kinase referred to as the mammalian target of rapamycin (mTOR), which appears to be a point of convergence of signals generated by the action of hormones such as insulin and those generated by the cell’s recognition of a sufficiency of amino acids. Learning how the cell recognizes a sufficiency of amino acids is presently the objective of intense research. Present evidence, however, suggests multiple recognition sites and multiple signaling pathways.
Relevant Publications
Kimball SR, Shantz LM, Horetsky RL, Jefferson LS: Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6. J Biol Chem 274:11647–11652, 1999.
Shah OJ, Anthony JC, Kimball SR, Jefferson LS: 4E-BP1 and S6K1: translation integration sites for nutritional and hormonal information in muscle. Am J Physiol Endocrinol Metab 279:E715–E729, 2000.
Genes Differentially Regulated by Dietary Zinc
Robert J. Cousins
Center for Nutritional Sciences, Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida
The etiology of zinc deficiency and the possible consequences of excesses of zinc supplementation have not been delineated. Mechanisms for these reside within the general classification of zinc’s functions in biology: catalytic, structural and regulatory. Of these, the latter two have received the most recent attention. The involvement of zinc in gene expression falls primarily in the regulatory category, but could also encompass the structural category through zinc finger domains of regulatory proteins. Zinc may regulate and/or influence gene expression either directly or indirectly. The direct mode is via interaction, in an as yet unclear way, with the metal response element (MRE) transcription factor (MTF-1), which binds to the MRE’s of promoter for metallothionein (MT). To date, MT is the only MRE-regulated gene that has been well characterized. However, MTF-1 knockouts are lethal, while MT knockouts are viable, suggesting MTF-1 regulation goes beyond MT expression. Activation of the MTF-1 system is directly influenced by the zinc intake from the diet. This has been shown recently in humans, where MT expression was measured by competitive RT-PCR. We measured MT mRNA levels in monocytes and cells from a dried blood spot from males given 15 mg Zn/day above their daily zinc intake. The levels were drastically increased for as long as zinc was consumed and diminished thereafter. The method has application for a variety of nutritional and toxicological studies. Zinc transporter characterization and regulation by zinc was discussed. ZnT-1 and ZnT-2 are zinc regulated, but ZnT-4 is not. ZnT’s are important since zinc metabolism is highly regulated, presumably to control function and therefore dictates dietary requirements. Zinc regulation of ZnT expression is believed to be controlled in part by the direct MRE/MTF system. We have used differential mRNA display and recently cDNA array analysis to examine global differences in zinc nutrition on gene expression via either the direct or indirect mode of regulation. Experiments with rats have shown clearly that in the small intestine, cholecystokinin and uroguanylin are upregulated by mild zinc deficiency. The latter has been further confirmed by experiments at the protein level. Murine zinc deficiency has shown expression changes in a myriad of regulatory genes in the thymus. The overall goal is to understand zinc’s contribution to general metabolism and host defense.
Relevant Publications
Cousins RJ: A role of zinc in the regulation of gene expression. Proc Nutr Soc 57:307–311, 1998.
Cousins RJ: Nutritional regulation of gene expression. In Shils ME, Olson JA, Shike M, Ross AC (eds): "Modern Nutrition in Health and Disease." Baltimore: Williams & Wilkins, pp 573–584, 1999.
Cousins RJ and Lanningham-Foster L: Regulation of cysteine-rich intestinal protein, a zinc finger protein, by mediators of the immune response. J Infect Dis 182:S81–S84, 2000.
Cousins RJ, McMahon RJ: Integrative aspects of zinc transporters. J Nutr 130:1384S–1387S, 2000.
SESSION 2. NUTRITIONAL FACTORS AND GENES OF TOXICOLOGICAL SIGNIFICANCE
Cytochrome P450 and Phase II Enzymes: Why Are They Important in Considerations of Toxicology, Cancer, Nutrition, and Drug Development?
F. P. Guengerich
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee
The cytochrome P450 (P450) enzymes serve as a prototype for other groups of enzymes involved in the metabolism of xenobiotic chemicals. The current number of human P450 genes is 53. Of these, 
6–8 are responsible for the transformation of >90% of the drugs and carcinogens. A considerable amount of information about these P450s and their substrates, inhibitors and inducers is now available and utilized in drug development in the pharmaceutical industry. Similar information is being developed with Phase II enzymes, including the N-acetyltransferase, epoxide hydrolase, glutathione S-transferase, UDP-glucuronosyltransferase, methyltransferase and sulfotransferase families. There is considerable interest in applying knowledge not only to drug development and prediction of interactions but also to the risk of individuals from carcinogens and to the metabolism of vitamins and food components.
Carcinogenic heterocyclic amines are produced during pyrolysis of meat. Activation involves N-hydroxylation reactions catalyzed by P450 1A2 and subsequent acetylation. Our findings that human P450 1A2 is an order of magnitude more active than rat P450 1A2 with two of the major heterocyclic amines suggests re-evaluation of the risk to humans.
Another P450-related issue in foods is aflatoxin B1 (AFB1), which is activated (and also detoxified) by human P450s 3A4 and 1A2. The activation of AFB1 can be understood in terms of the formation of the 8,9-exo-epoxide. This molecule has a t1/2 of only 1 s in water at neutral pH. With this experimental information, we were able to characterize kinetic constants for glutathione conjugation, hydrolysis and DNA conjugation. Although the rate of non-enzymatic hydrolysis is 1 s-1, the (DNA-catalyzed) rate of DNA adduction is 42 s-1 and helps explain the high level of DNA adduction and carcinogenicity.
An interesting sidelight to the studies utilizing recombinant human P450 systems in bacterial vectors involves amino acid metabolism. Tryptophan is degraded to indole, which is oxidized by P450s to several products. One of these, indoxyl, spontaneously oxidizes in air to indigo. Formation of this blue dye has some potential industrial application; it is also being used in this lab as a phenotypic selection marker in random mutagenesis studies with several P450s.
Natural products contain a variety of chemicals that may affect the course of P450 reactions. Grapefruit juice contains a series of furanocoumarins and possibly other components that are strong inhibitors of P450 3A4. Pharmacokinetic parameters can be markedly affected by a single serving of grapefruit juice (10-fold increase in AUC). Herbal medicines are also of interest. Recently a powerful inducer of P450 3A4 has been identified in St. John’s Wort and may explain some of the observed interactions.
Currently there is considerable interest in the area of pharmacogenomics. A number of advances have been made, and there is considerable potential in this area, including applications involving the enzymes under consideration here. A need exists for more efficient means to analyze the functions of polymorphic variants. The fields of toxicogenomics and environmental genomics are based on the same principles as pharmacogenomics, but are more difficult due to the complexity of endpoints. Issues related to environmental genomics include validation of assays of functional analysis, target validation and causality, etiology, complex mixtures of chemicals, the number of samples available and the complexity of endpoints in the diseases under investigation.
In summary, today we have a considerable understanding of the basic mechanisms by which P450 enzymes activate food-borne and other toxins. Genomic approaches may yield a better understanding on the risks of individuals due to their variations in these enzymes and the environmental exposures.
Relevant Publications
Guengerich FP, Johnson WW: Kinetics of hydrolysis and reaction of aflatoxin B1 exo-8,9-epoxide and relevance to toxicity and detoxication. Drug Metab Rev 31:141–158, 1999.
Guengerich PF: Pharmacogenomics of cytochrome P450 and other enzymes involved in the biotransformation of xenobiotics. Drug Dev Res 49:4–16, 2000.
N-Acetyltransferases and Other Genes
Geoffrey H. Goodfellow and Denis M. Grant
Department of Pharmacology, University of Toronto, Hospital for Sick Children, Toronto, Ontario, Canada
A substantial body of experimental and epidemiological literature has documented the relationship between exposure to homocyclic and heterocyclic amines in the diet and the subsequent development of tumors at a variety of tissue sites. In the context of diet, a number of heterocyclic amines are produced in foods during high-temperature cooking and have been shown to be carcinogenic in animals and potential or known substrates in humans. These arylamine compounds require metabolic activation by enzymes of xenobiotic transformation to generate electrophilic intermediates capable of binding to nucleophilic sites on DNA bases and producing mutations that advance cells to a more malignant phenotype. The arylamine N-acetyltransferases NAT1 and NAT2 are among the enzymes involved in the competing detoxifying and activating pathways of arylamine procarcinogen metabolism. NAT2 is the site of classical isoniazid acetylation polymorphism, whereas recent evidence has demonstrated that indeed NAT1 also exhibits genetic variation. Extensive studies have been performed to elucidate the molecular basis and functional consequences of the phenotypic variation of the human NAT1 and NAT2 proteins. The role of the variant human NAT1 and NAT2 proteins as risk factors has also been addressed in numerous epidemiological studies. For instance, an increased risk for bladder cancer has been associated with the slow acetylator phenotype in many studies. However, we matched our bladder cancer patients and controls for smoking, age and gender and observed no differences between the two groups. This suggested that the most important driver of risk for the onset of bladder cancer is exposure and that the metabolic factors may play a more modest modifying role to this risk.
Relevant Publications
Grant DM, Goodfellow GH, Sugamori K, Durette K: Pharmacogenetics of the human arylamine N-acetyltransferases. Pharmacology 61:204–211, 2000.
Hein DW, Doll MA, Fretland AJ, Leff MA, Webb SJ, Xiao GH, Devanaboyina U-S, Nangju NA, Feng Y: Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev 9:29–42, 2000.
Oxidative DNA Damage, Antioxidants and DNA Repair
Andrew R. Collins
Rowett Research Institute, Aberdeen, Scotland
We think of the DNA double helix as inherently stable, but it is not. It is prone to spontaneous breakdown, attack by exogenous and endogenous agents and errors in replication. Among the damaging agents are free radicals or reactive oxygen species, arising during normal respiration, which cause strand breaks and oxidized bases. Oxidative DNA damage has long been considered a likely cause of cancer, partly because of the abundance of its products, notably 8-oxoguanine, in human cell DNA, and the cancer-preventive effect of consuming fruit and vegetables is ascribed to their antioxidant content. However, doubt has recently been cast on the estimates of 8-oxoguanine. It is clear that oxidation of guanine during sample preparation for GC-MS or HPLC analysis is a serious problem, and most of the 8-oxoguanine detected was probably artifact. Recent improvements in technique have led to yields a 100 times less than previously found. New methods (including the comet assay), which use lesion-specific bacterial endonucleases to detect oxidized bases and convert them to breaks, give estimates that are several times lower still—around 0.05 8-oxoguanines per 105 guanines in normal human white blood cells. It is, after all, not surprising if evolution has resulted in protective mechanisms that keep the level of DNA oxidation low; they include a battery of intrinsic antioxidants (catalase, superoxide dismutase, glutathione and its associated enzymes) and very effective DNA repair pathways. The dietary antioxidants such as vitamins C and E, carotenoids and flavonoids, may also play a role. Using the comet assay, we have shown a decrease in endogenous base oxidation and/or increased resistance to damage induced by H2O2 following supplementation with isolated antioxidants and with various foods rich in antioxidants, such as kiwifruit, fried onions and soya milk. However, large-scale intervention trials with ß-carotene have notoriously resulted in either no effect or an increase in the incidence of lung cancer.
There are likely to be individual differences in sensitivity to oxidative stress, which may result from different levels of induction of protective enzymes or from genotypic differences. It is becoming common, in biomonitoring studies, to look for polymorphisms in relevant genes—those involved in phase I and II metabolism, for instance. DNA repair plays a crucial role, but little is known about the factors controlling its activity. We have devised a novel assay based on the comet assay that allows assessment of the repair activity of a cell extract given a substrate containing specific damage. In a pilot study, we found substantial and consistent differences in individuals’ capacity to repair oxidative damage.
It is clear that we need to reconsider the role of endogenous oxidation in the etiology of cancer. The evidence from the study of mutations in human tumors or in model cell culture systems is less than convincing. Human diseases such as diabetes that are associated with high oxidative stress (and elevated oxidative damage to DNA) do not incur increased risk of cancer. A knockout mouse lacking the glycosylase that removes 8-oxoguanine has only slightly elevated levels of DNA oxidation and does not show abnormal incidence of cancer—suggesting that a back-up repair process can take over. If the body is capable of dealing with oxidative DNA damage so effectively, is the further reduction brought about by dietary antioxidants really significant, or should we concentrate on other factors in fruits and vegetables that might be responsible for their cancer-preventive effects?
Relevant Publications
Duthie SJ, Ma A, Ross MA, Collins AR: Antioxidant supplementation decreases oxidative damage in human lymphocytes. Cancer Res 56:1291–1295, 1996.
Mitchell JH, Collins AR: Effects of a soy milk supplement on plasma cholesterol levels and oxidative DNA damage in men—a pilot study. Euro J Nutr 38:143–148, 1999.
Collins AR: Oxidative DNA damage, antioxidants and cancer. BioEssays 21:238–246, 1999.
Caloric Intake, Oxidative Stress and Aging
Richard Weindruch
Department of Medicine, VA Hospital, Madison, Wisconsin
Caloric restriction (CR) repeatedly and strongly increases maximum life span in rodent models while retarding the appearance of age-associated pathologic and biologic changes. Although the large majority of rodent studies have initiated CR early in life (1–3 months of age), CR started in mid-adulthood (at 12 months) also extends maximum life span in mice. Two main questions now face gerontologists investigating CR. Question #1: By what mechanisms does CR retard aging and disease processes in rodents? There is increasing evidence that CR may act by reducing oxidative stress, an observation that provides support for the oxidative stress hypothesis of aging. This decrease in oxidative stress with CR appears to be most profound in postmitotic tissues and may be the result of a reduction in mitochondrial production of free radicals. Mechanistic insight was also gained by using oligonucleotide microarrays by which we recently described the gene expression profile of aging skeletal muscle and brain in mice and its alteration by CR. Question #2: Will CR exert similar actions in primates? Two ongoing studies in rhesus monkeys subjected to CR as well as limited human epidemiological data support the notion of human translatability. The available results suggest that CR can be safely carried out in monkeys and that certain physiological effects of CR, which occur in rodents (e.g., decreased blood glucose and insulin levels, improved insulin sensitivity, lowering of body temperature and oxidative damage), are also obtained in rhesus monkeys. Whether life-span extension is achieved in monkeys subjected to CR should become known in about 20 years.
Relevant Publications
Sohal RS, Weindruch R: Oxidative stress, caloric restriction, and aging. Science 273:59–63, 1996.
Lee CK, Klopp RG, Weindruch R, Prolla TA: Gene expression profile of aging and its retardation by caloric restriction. Science 285:1390–1393, 1999.
Lee CK, Weindruch R, Prolla TA. Gene—expression profile of the aging brain in mice. Nat Genet 25:294–297, 2000.
SESSION 3. GENE-NUTRIENT INTERACTIONS IN THE DEVELOPMENT OF CHRONIC DISEASES
Genetics of Type 2 Diabetes in Oji-Cree
Robert A. Hegele
Blackburn Cardiovascular Genetics Laboratory, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
A panel of genetic tests to predict the risk of development of a complex trait, such as atherosclerosis or diabetes, will need to be more effective than either a determination of family history or the assessment of intermediate phenotypes. Our studies in Canadian populations have indicated that a single genetic factor will have only a small effect on an intermediate phenotype of a complex trait at the level of the whole population. Also, the total contribution of genetic factors, while substantial, is usually the aggregate of many small effects. Furthermore, the environment plays an overwhelming role in modulating the expression of genetic susceptibility. The importance of environment is seen in examples of strong monogenic determinants of disease, such as in the case of type 2 diabetes in Oji-Cree due to the HNF1A G319S mutation. The HNF1A G319S mutation is the most specific genetic marker for type 2 diabetes so far identified in any human population and has the hallmarks of the so-called "thrifty allele" for diabetes. It is strongly associated with type 2 diabetes in adolescence. However, its clinical utility is limited, since it is found only in the Oji-Cree of northern Canada. While G319S has enormous positive predictive value in the Oji-Cree, its absence from all other ethnic groups, including other North American first nations, means that it has no diagnostic utility in groups from other genetic backgrounds. Furthermore, the expression of diabetes in carriers of HNF1A G319S depends upon environmental factors, specifically a sedentary lifestyle and a diet rich in saturated fat and sugar. The Oji-Cree HNF1A G319S example indicates that diagnostic tests for diabetes will have to account for ethnic-specific genetic determinants of risk and environmental factors. The complexity of basic science will probably delay the implementation of routine clinical genetic testing. Fortunately, such a delay might provide more time for health care providers and society to consider the possible implications and limitations of the genetic prediction of complex traits.
Relevant Publications
Hegele RA, Cao H, Harris SB, Hanley AJ, Zinman B: The hepatic nuclear factor-1 alpha G319S variant is associated with early-onset type 2 diabetes in Canadian Oji-Cree. J Clin Endocrinol Metab 84:1077–1082, 1999.
Hegele RA, Cao H, Hanley AJ, Zinman B, Harris SB, Anderson CM: Clinical utility of HNF1A genotyping for diabetes in aboriginal Canadians. Diabetes Care 23:775–778, 2000.
Nutrient-Gene Interactions: Diabetes and the Mitochondrial Genome
Carolyn Berdanier
Department of Nutrition, University of Georgia, Athens, Georgia
Diabetes mellitus is the common phenotype of more than 200 genotypes. Of the population with diabetes mellitus, about 10% have the disease because of one or more failures in the system that produces and releases insulin by the pancreas. These people are said to have type 1 diabetes. Eighty per cent of the population has the disease as a result of mutations in the genes that encode the metabolic components of the glucose oxidizing system, and about 10% has the disease due to mutations in the mitochondrial genome. This genome encodes 13 of the protein subunits of the oxidative phosphorylation (OXPHOS) system. The mitochondrial genome has been completely sequenced and mapped. Major mutations result in major diseases notably involving the CNS; however, when the mutation load is less than 50% of a given gene then diabetes mellitus develops rather than overwhelming CNS disease. Diabetic symptoms are observed in the CNS diseases, but these are less important than the symptoms of epilepsy, myocardial degeneration, muscular dystrophy and so forth. Studying the diabetes due to mutation in the mitochondrial genome is possible through the use of the BHE/Cdb rat. This rat has two mutations in the ATPase 6 gene that affects OXPHOS. The first of these mutations exists in the proton channel and can explain the reduced ATP synthesis efficiency seen in these rats. The second mutation affects the mobility of the ATPase in the inner mitochondrial membrane. Nutrition can affect OXPHOS at both the genomic level and the non-genomic levels. With respect to the latter, nutrients that affect the fluidity of the inner mitochondrial membrane (the fatty acid profile) will affect ATPase movement. If movement is hindered then ATP synthesis efficiency is reduced. Hence, feeding a sucrose-rich diet or a diet containing hydrogenated coconut oil results in a reduction in ATP synthesis efficiency. Some nutrients have genomic effects on OXPHOS. These nutrients are those that are bound by DNA binding proteins. Vitamin A is one of these nutrients. In its gene-active form, retinoic acid, vitamin A influences the expression of the ATPase 6 gene and results in an improvement in OXPHOS and ATP synthesis efficiency. The BHE/Cdb rat has a three fold increased need for dietary vitamin A compared to the Sprague-Dawley rat. The SD rat has a non-mutated mtDNA and normal OXPHOS as well as glucose tolerance. Supplementing the diet of the BHE/Cdb rat with three times as much vitamin A as is recommended by the National Research Council appears to delay the development of impaired glucose tolerance, improves OXPHOS and increases the expression of the ATPase 6 gene. DHEA, triiodothyronine and vitamin D all seem to affect ATPase 6 gene expression, whereas vitamin E does not. The vitamin D effect is tissue-specific while retinoic acid, DHEA and triiodothyronine work in many different tissue types. We suspect that these compounds are gene-active in the mitochondria and that this activity resembles that which occurs in the nucleus. We also suspect that these and perhaps other nutrients can affect the phenotypic expression of the mutated mitochondrial genotype. It is apparent that animals (and perhaps humans) having mutations in the mtDNA may have different nutrient requirements and tolerances than normal animals. In the future, we will need to determine the limits of these variations that are associated with the maintenance of good nutritional health and the delay of onset of nutrition-related degenerative disease.
Relevant Publications
Mathews CE, McGraw RA, Dean R, Berdanier CD: Inheritance of mitochondrial DNA defect and impaired glucose tolerance in BHE/Cdb rats. Diabetologia 42:35–41, 1999.
Kim S-B, Berdanier CD: Oligomycin sensitivity of mt F1F0ATPase in diabetes-prone BHE/Cdb rats. Am J Physiol 277:E702–E707, 1999.
Berdanier CD, Everts HB, Hermoyian C: Role of vitamin A in mitochondrial gene expression. Diabetes Res Clin Prac (in press).
Folate-Gene Interactions in Carcinogenesis
Young-In Kim
Departments of Medicine and Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada
Advances in molecular biology during the past decade have greatly increased the potential for understanding the roles that genes play in the development of chronic diseases. It is now well accepted that environmental factors such as diet interact with genetic predispositions in the development of chronic disease states (e.g. cancer, coronary artery disease, hypertension, diabetes mellitus and obesity). However, the precise nature and magnitude of the gene-environment interactions in the genesis of these diseases have not been clearly elucidated yet. In this regard, folate is an excellent prototype compound by which to study this issue.
Folate, a water-soluble B vitamin, has recently emerged as an important nutritional factor that may modulate carcinogenesis. Epidemiologic studies suggest that folate status is inversely related to the risk of developing several cancers in a dose-dependent manner. For colorectal cancer, epidemiologic studies collectively suggest an approximately 40% reduction in the risk of colorectal cancer in subjects with the highest dietary folate intake compared to those with the lowest. Animal studies conducted in chemical carcinogen and genetic knockout models of colorectal cancer also support a cause and effect relationship between folate deficiency and colorectal cancer and a dose-dependent protective effect of modest supplemental folate levels above the basal dietary requirement. However, these animal studies also suggest that timing, dose and target group of folate supplementation are critical in providing safe and effective chemoprevention. Folate appears to possess dual modulatory effects on carcinogenesis depending on the timing of intervention. Folate deficiency has an inhibitory, whereas folate supplementation has a promoting, effect on progression of established neoplasms. In contrast, folate deficiency in normal epithelial tissues predisposes them to neoplastic transformation, and modest levels of folate supplementation suppresses the development of tumors in normal tissues. It also appears that exceptionally high supplemental folate levels enhance, rather than suppress, carcinogenesis. Collectively, these animal studies suggest that modest levels of folate supplementation should be implemented before the development of any premalignant lesions in the target organ in individuals free of neoplastic foci in order to provide safe and effective chemoprevention.
Folate, as a mediator of the transfer of one-carbon moieties, plays an important role in DNA methylation, DNA synthesis and repair, mutagenesis and cellular proliferation, all of which are mechanistically related to carcinogenesis. Studies in animals and in vitro culture systems have demonstrated that folate deficiency induces DNA strand breaks, impairs DNA repair capacity, increases mutations and causes abnormal patterns of DNA methylation. Some of these effects have been shown to be gene- and site-specific. For example, a highly conserved region (exons 5–8) of the p53 tumor suppressor gene, one of the most commonly implicated genes in carcinogenesis, appears to be particularly susceptible to the DNA damaging and hypomethylating effects of folate deficiency. These molecular effects have been shown to decrease steady-state levels of p53 mRNA. Folate supplementation has been shown to correct these molecular alterations in the p53 gene in a dose-dependent fashion. Because the p53 gene is integrally involved in transcription, DNA repair, genomic stability, senescence, cell cycle control and apoptosis, interruption of p53 integrity and abnormal p53 methylation with resulting decreased steady-state levels of p53 transcript would be particularly prone to promote carcinogenesis. Other molecular effects of folate are under intense investigation in several laboratories. Intervention studies utilizing dietary strategies to prevent cancer have produced conflicting results in most instances. This is mainly because most intervention studies were undertaken empirically without biologically sound rationale or a mechanistic understanding of the molecular and cellular effects of the utilized nutrient. A comprehensive mechanistic understanding of how nutrients can modulate the development and prevention of chronic diseases at the molecular and cellular levels will likely lead to a better dietary strategy to prevent these diseases in humans.
Relevant Publications
Song J, Medline A, Mason JB, Gallinger S, Kim YI: Effects of dietary folate on intestinal tumorigenesis in the ApcMin mouse. Cancer Res 60:5434–5440, 2000.
Song J, Sohn KJ, Medline A, Ash C, Gallinger S, Kim YI: Chemopreventive effects of dietary folate on intestinal polyps in Apc +/- Msh2 -/- mice. Cancer Res 60:3191–3199, 2000.
Diet, Susceptibility and Risk of Breast and Colon Cancer
Christine B. Ambrosone, Fred F. Kadlubar, Nicholas P. Lang, Peter G. Shields and Jo L. Freudenheim
Division of Molecular Epidemiology, National Center for Toxicology Research, Jefferson, Arkansas
Epidemiology is used to evaluate possible associations between exposures and disease outcome, with the underlying assumption that, for the most part, study populations are homogeneous. However, individuals vary on a biochemical level, and exposure/disease relationships are impacted on by interindividual variability along the continuum from carcinogen metabolism to DNA repair, cell cycle control, apoptosis and immune surveillance. Therefore, risk factors for disease may be modified by variability due to genetic polymorphisms in a number of enzymes related to the exposure under consideration. For example, meat consumption is a probable risk factor for colorectal cancer and a likely risk factor for breast cancer, although study results are often inconsistent. Carcinogens are often formed when meat is cooked and could be the reason for the associations between meat consumption and cancer risk. Heterocyclic amines are pyrolysis products formed when meat is cooked at high temperatures and are colon and mammary gland carcinogens in rodents. They are activated by cytochrome P4501A2 and N-acetyltransferases 1 and 2, each of which is polymorphic with rapid and slow phenotypes. In a study of colorectal cancer, we found that risk of colorectal cancer was greatest among those who preferred their meat cooked well-done and had both rapid CYP1A2 and rapid NAT2 phenotypes. These data were confirmed in a recent study of polyp recurrence, where risk of recurrence was greatest among those in the highest tertile of meat consumption who had rapid NAT2 genotype.
A similar model has been applied to breast cancer, but results have been mixed and possible associations are, to date, unclear. We have also investigated mechanisms of the protective effect of fruit and vegetable consumption on breast cancer risk. There is support in the literature for a role of oxidative stress in breast cancer etiology, and endogenous antioxidant capacity includes superoxide dismutase and glutathione S-transferases. We investigated the role of genetic polymorphisms in MnSOD and GSTM1 in breast cancer risk and the possible modification of risk by consumption of dietary sources of antioxidants. In a case-control study of breast cancer in western New York, we found that individuals who were lacking the GSTM1 allele were not at increased risk for breast cancer, nor was there an effect among low consumers of fruits and vegetables. However, among premenopausal women, we did find that the polymorphism in MnSOD that is thought to alter its structure preventing its transport to the mitochondria increased breast cancer risk more than fourfold. Risk was most pronounced among low consumers of fruits and vegetables and, specifically, dietary sources of ascorbic acid. The identification of associations within susceptible groups may not only target specific populations for cancer prevention, but may also elucidate etiologic pathways with implications for public health. However, in complex diseases such as breast cancer, large studies are needed with innovative hypotheses to evaluate multiple exposures and susceptibility factors.
Relevant Publications
Ambrosone CB, Freudenheim JL, Sinha R, Graham S, Marshall JR, Vena JE, Laughlin R, Nemoto T, Shields PG: Breast cancer risk, meat consumption, and N-acetyltransferase (NAT2) genetic polymorphisms. Int J Cancer 75:825–830, 1998.
Ambrosone CB, Freudenheim JL, Thompson PA, Bowman E, Vena JE, Marshall JR, Graham S, Laughlin R, Nemoto T, Shields PG: Manganese superoxide dismutase (MnSOD) genetic polymorphisms, dietary antioxidants and risk of breast cancer. Cancer Res 59:602–607, 1999.
Ambrosone CB, Coles BF, Freudenheim JL, Shields PG: Glutathione S-transferase (GSTM1) genetic polymorphisms do not affect human breast cancer risk, regardless of dietary antioxidants. J Nutr 129:565–568, 1999
SESSION 4. NEW DEVELOPMENTS
DNA Microarrays: What to Have on Your Chips?
Hugh McGlynn
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland
DNA chips are a revolutionary development in molecular biology. The combination of standard nucleic acid hybridization techniques with innovative high-density microarray technology is proving a winning combination for high throughput genomic analysis. Initially developed to enhance genome-sequencing projects, DNA chips are finding application throughout the field of molecular biology. They have already found application in studying gene expression, discovering new genes, detecting mutations or polymorphisms and mapping genomic libraries. What makes DNA chips so new and innovative is the ability to attach nucleic acids to a solid matrix at precise locations in a densely packed array. This allows simultaneous screening with a large number of probes of a very small sample volume. DNA chip technology has evolved along two major paths. In one method, nucleic acids are immobilized on the chip surface sequentially to form oligonucleotides. The chip is then exposed to a set of labeled probes. The second method uses in situ conventional synthesis techniques or cDNAs as a source of prefabricated oligonucleotides, which are anchored to the chip surface. The "on chip" approach to DNA chip design can be done by photolithography or piezoelectric printing. For example, Affymetrix uses photolithographic masks to direct specific removal of photoactive groups to yield reactive 5'-hydroxy groups capable of binding other nucleotides. Alternatively, piezoelectric printing uses inkjet printer-like technology to direct precise placement of nucleotides that become anchored to a coated surface using standard chemistry. Repeating either process many times yields oligonucleotide chains of 30 and 40–50 nucleotides respectively.
Prefabricated nucleotide attachment provides a slightly simplified DNA chip construction method. In this technique nucleic acids can be prefabricated using conventional oligonucleotide synthesis techniques such as controlled pore glass synthesis or using purified single stranded complementary DNA. The nucleic acids or oligopeptides are then placed onto the chips using touch or fine micropipetting. These DNA spotting techniques use surfaces with positively charged coatings such as aminosilane or polylysine, which allow direct printing of nucleic acids onto the chip surface. Each method is subject to drawbacks and benefits. In situ synthesis requires careful monitoring to ensure correct oligonucleotide formation, while the precision and tediousness required for DNA spotting techniques necessitates the use of high throughput robotics. Chips have been used primarily for gene expression studies, allowing the measurement of changes in toto rather than as isolated events. An example of this would be the determination of BRCA1 induced changes in downstream effectors, resulting in upregulation of GADD45 (a DNA damage responsive gene) and the triggering of apoptosis. In summary, DNA microarrays contrary to their name allow macro determination of cellular events in normal and disease tissue and will become the single most important research tool in molecular research.
Relevant Publication
Harkin DP, Bean JM, Miklos D, Song YH, Truong VB, Englert C, Christians FC, Ellison LW, Maheswaran S, Oliner JD, Haber DA: Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell 97:575–586, 1999.
Transgenic and Knockout Models: Novel Insights into the Regulation of Dietary Cholesterol Absorption
Ephraim Sehayek
Laboratory of Molecular Genetics and Metabolism, The Rockefeller University, New York, New York
Dietary cholesterol plays an important role in the regulation of plasma cholesterol levels and the risk of cardiovascular diseases. To exert its metabolic effects, dietary cholesterol must be absorbed from the gastrointestinal tract. The absorption process is critically dependent upon the presence of bile within the intestinal lumen; however, the role of individual biliary lipids and the mechanisms involved in the regulation of the absorption process are poorly understood. We selected the mouse as a paradigm mammalian model to study the regulation of the absorption process. Feeding C57BL/6 wild type mice a high cholesterol diet efficiently suppressed the percentage absorption of dietary cholesterol and efficiently stimulated the excretion of cholesterol into the bile. There was a strong and inverse relationship between biliary cholesterol concentrations and cholesterol absorption rates. To examine whether the increase in biliary cholesterol concentration is responsible for the decrease in absorption rate, we turned to study absorption in mice with liver-specific overexpression of the scavenger receptor class B type I (SRBI), an animal model with increased biliary cholesterol excretion. As expected, the SRBI-Tg mice displayed increased concentrations of cholesterol in their bile. Interestingly, however, the increase in biliary cholesterol concentration was associated with a suppressed cholesterol absorption rate and, again, a strong and inverse relationship between biliary cholesterol concentration and cholesterol absorption rate. To further examine the role of biliary cholesterol excretion we turned to study the absorption in apolipoprotein E knockout (apo E KO) mice. Apo E is known to play a critical role in the disposal of dietary cholesterol, in the form of chylomicron remnants, by the liver. We postulated that the failure of apo E KO mice to dispose of chylomicron remnants from the circulation may decrease the excretion of cholesterol into the bile and affect the suppression of absorption rate upon feeding a high cholesterol diet. Indeed, unlike wild type animals, the apo E KO responded to high cholesterol diet by a failure to stimulate the excretion of cholesterol into the bile that was associated with a failure to suppress the percentage absorption of dietary cholesterol. When combined, the above-described studies provide strong evidence that the excretion of cholesterol into the bile plays an important role in the regulation of dietary cholesterol absorption. Next, we turned to examine the role of biliary phospholipids in the absorption process. For this purpose we studied the absorption of dietary cholesterol in multi-drug resistance 2 gene (mdr2) heterozygous mice that are characterized by a 50% decrease in biliary phospholipid excretion. We found that mdr2 heterozygous animals displayed completely normal rates of dietary cholesterol absorption, suggesting that biliary phospholipids probably play only a minor role in the regulation of dietary cholesterol absorption. Finally, the role of biliary bile acids was examined in cholesterol 7-alpha hydroxylase KO mice. The decrease in bile acid pool and altered bile acid composition in these animals was associated with a severely compromised cholesterol absorption rate. Taken together, these studies (i) exemplify the usefulness of genetically manipulated animal models in dissecting the role of individual biliary lipids in the regulation of dietary cholesterol absorption and (ii) strongly suggest that the regulation of cholesterol excretion into the bile, the bile acid pool size and biliary bile acid composition have important roles in the regulation of dietary cholesterol absorption.
Relevant Publications
Sehayek E, Ono JG, Shefer S, Nguyen LB, Wang N, Batta AK, Salen G, Smith JD, Tall AR, Breslow JL: Biliary cholesterol excretion: a novel mechanism that regulates dietary cholesterol absorption. Proc Natl Acad Sci USA 95:10194–10199, 1998.
Sehayek E, Shefer S, Nguyen LB, Ono JG, Merkel M, Breslow JL: Apolipoprotein E regulates dietary cholesterol absorption and biliary excretion: studies in C57BL/6 apolipoprotein E knockout mice. Proc Natl Acad Sci USA 97:3433–3437, 2000.
In addition to the generous support from the Heinz Institute of Nutritional Sciences, the workshop organizers also gratefully acknowledge support from the National Institute of Environmental Health Sciences and CanTox.
Received November 30, 2000.
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