2007 SCIENTIFIC PROGRAM

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SCIENTIFIC PROGRAM

Listing of the ISTERH/NTES/HTES '07 Scientific Program (Role mouse over talk title to view abstract)

KEYNOTE SPEAKER "Mediterranean Diet, Traditional Foods and Health: Critical Components and Mediating Mechanisms" Antonia Trichopoulou, MD, FACP, FASN Nutritional investigations have provided strong indications that a diet that adheres to the principles of the traditional Mediterranean one is associated with longer survival. This could be partly attributed to Mediterranean traditional foods, which this diet implies. Traditional foods are an integral part of the Mediterranean diet and there is a need to investigate them in order to elucidate their role in the beneficial effects of this diet. There is also a need to study traditional foods with a view to enrich and improve our diet and at the same time preserve important elements of our cultural inheritance. Our research team has been working towards this approach with the objective to formulate a multi-faceted framework for the systematic investigation of traditional foods and recipes, aiming primarily at the elucidation of the role of traditional Greek diet on health. The study of traditional foods in Greece started in 1992 and has several aspects, including the determination of their nutrient and non-nutrient value. This multifaceted methodology is currently being expanded to 12 European countries. The analysis of several traditional Greek foods indicated that the majority of them are rich in antioxidants and several trace elements and meet current criteria for a prudent diet. Therefore, the traditional foods of Greece may contribute to the apparent health benefits of the Mediterranean diet. Likewise, the analysis of traditional foods in other European countries may reveal beneficial health effects in some or many of them, since tradition rarely, if ever, honors foods and diets incompatible with good health.

RAULIN AWARD SPEAKER "Zinc Nutriture and Fetal Origins of Disease" Harold H. Sandstead, MD, FACP, FASN Primary zinc deficiency, deemed “highly unlikely” in 1959, is now considered common and known to impair gene expression and health of progeny. The essentiality of zinc for reproduction was established from experiments in animal model systems that began more than 50 years ago and from direct human observations that began 30 years ago. From this research, there is the clear understanding that zinc deficiency increases risk of abortion, teratology, fetal-stunting, prematurity and residual functional abnormalities. That there is a problem in the USA is suggested by similar associations of iron and zinc with food choices, the strong association between serum ferritin concentrations and zinc nutriture (measured by zinc kinetics), and the >25% prevalence of absent iron stores (measured by serum ferritin concentrations) among premenopausal women of childbearing age. Independent of knowledge of zinc, many epidemiological observations found associations between birth weight (and more recently maternal nutrition) and progeny health in later life. Metabolic imprinting that affects later health involves epigenetic mechanisms and zinc is one of several micronutrients essential for these phenomena. Given the world prevalence of zinc deficiency, conservatively estimated at 20%, the contribution of developmental zinc nutrition to human health in later life begs clarification and application. At the same time, research is needed for understanding how zinc nutriture affects metabolic imprinting in humans.

Trace minerals: Modulators of Arterial Function Chair: Dorothy Klimis-Zacas, PhD “Manganese: Regulator of vasomotor tone and arterial glycosaminoglycan metabolism," Dorothy Klimis-Zacas, PhD and Anastasia Z. Kalea, PhD Manganese (Mn), has been reported to influence the genesis and development of cardiovascular disease by participating as a cofactor of several enzymes that protect cells from oxidative stress and aid in the synthesis and maintenance of connective tissue extracellular matrix and structure. Additionally, Mn modulates important intracellular pathways, by activating proteins for cell-signaling, influencing cell surface receptor binding and adhesion, and interacting with ionic-channel receptor complexes. We have documented that Mn is involved in arterial glycosaminoglycan metabolism (GAG) by affecting the total proteoglycan content of the aorta, altering the concentration and molecular structure of arterial heparan sulfate. Manganese supplementation resulted in reduced sulfation of heparan sulfate proteoglycans (HSPGs) and altered galactosyl transferase activity. The reduced expression and undersulfation of HSPGs with Mn supplementation may indicate a decreased ability of vascular cells to interact with biologically active molecules, such as lipoproteins and thus protect the endothelium from lipid deposition and lipoprotein oxidation. Alterations in cell membrane binding to a variety of extracellular ligands may affect signal-transduction pathways and arterial functional properties. We have documented that the presence of dietary Mn suppresses vasoconstriction induced by an alpha-adrenergic agonist and operates through an endothelium-mediated pathway. We also studied the effect of Mn on endothelium-dependent (Ach-induced) and endothelium-independent (SNP-induced) vasodilation as it relates to vascular function and reported that Mn operates in the cyclo-oxygenase pathway by inhibiting the synthesis or activity of a prostanoid-derived vasoconstrictor present at basal and stimulated levels. Thus Mn is crucial in atheroprotection and maintainance of vasomotor tone with implications on Cardiovascular Disease. “The Role of Copper in Nitric Oxide Mediated Vasodilation," Dale A. Schuschke, PhD Dietary copper and cuproenzymes have a significant role in microvascular control mechanisms. Of particular importance is the endothelium-dependent regulation of arteriolar vascular smooth muscle. We have used the rat cremaster muscle model to study the in vivo microcirculatory effects of dietary copper. Our initial results showed that nitric oxide (NO)-mediated dilation is attenuated in microvascular resistance vessels during dietary copper deficiency. This attenuated dilator response is accompanied by an inhibition of copper-zinc superoxide dismutase (Cu,Zn-SOD) activity and the decreased dilation is reversed by the addition of exogenous Cu,Zn-SOD. We have proposed that when this cuproenzyme is inactivated by copper restriction, there is a buildup of superoxide anion (O2-), which then reacts with endothelial cell NO to partially destroy the NO available for diffusion to the vascular smooth muscle. This hypothesis is supported by the observation that during copper deficiency there is an increase in peroxynitrite (ONOO-), the end product of the NO-O2- reaction. Peroxynitrite is also a known inhibitor of endothelial cell Ca2+ mobilization which is required for endothelial NO synthase (eNOS) activity. In a study of endothelial cell NO production, we have shown that acetylcholine-induced Ca2+ mobilization and NO release are both attenuated in resistance arterioles of copper-deficient rats. These results suggest that during copper deficiency, there is not only a build up of O2- which scavenges available NO, but there is also an additional inhibitory effect of elevated ONOO- to reduce NO synthesis. “Selenium Status and Regulation of Vascular Homeostasis," Lorraine M. Sordillo, PhD Evidence suggests an inverse relationship exists between selenium (Se) nutrition and cardiovascular disorders. Several intervention studies are currently underway to assess the benefits of Se supplementation to control a wide variety of condition in which oxidant stress and inflammation are the predominant pathological features, including atherosclerosis. Earlier epidemiological and ecological studies examined the potential benefit of Se administration to control cardiovascular disease; however, they have proven to be inconclusive. A number of underlying factors exist to explain some of the equivocal finding from these prospective studies including identification of specific cellular processes that are affected by Se status and determination of the specific Se-dependent bioactive components that are responsible for the desired response in targeted cellular environments. The importance of Se to health may be related to selenoprotein activities, such as glutathione peroxidase and thioredoxin reductase, that have diverse biological roles. These enzymes can function as potent antioxidants by directly reducing pro-atherogenic reactive oxygen species to less reactive metabolites. Recent evidence also suggests that certain selenoproteins are capable of modifying cellular responses to oxidant challenge by controlling the balanced expression of cytoprotective and proapoptitic factors. The ability of Se to modify the way in which cells respond to oxidant stress is controlled, in part, through the redox regulation of kinases and transcription factors involved in the intracellular signaling process. This paper will define the regulatory role of individual selenoproteins in orchestrating the expression of cytoprotective genes necessary for endothelial cell survival during oxidant stress through their ability to control redox sensitive signaling events. The long-term implications of this work will be the ability to identify foods that can provide the optimal amount of specific biological active selenoproteins needed to control the development of atherosclerosis and other cardiovascular disorders.

Trace element nutrition and dietary recommendations Chair: Jeanne Freeland-Graves, PhD “The relevance of trace element nutrition in human health," Sophie Ermidou-Pollet, PhD The relevance of trace element nutrition in human health is obvious from the beginning of the life until the old age. Proper trace element nutrition during pregnancy may be important for maternal health and fetal growth and development. Trace mineral requirements of term infants are generally met by exclusive breast-feeding through about the first 6 months of life. Growth of infants and children is a complex process in which several nutritional factors, including trace elements, play roles and interact, and the extent and consequences of such interactions are still not completely understood. Review of the literature has shown that trace element intakes are particularly important in the prevention of cancer, cardiovascular diseases, age related ocular diseases and in aging. Further research is required to determine the optimal micronutrient combinations and the doses required according to timing of intervention. For long it was not fully accepted that food can have an influence on brain structure, and thus on its function, including cognitive and intellectuals. In fact trace-elements have been directly evaluated in the setting of cerebral functioning. With the human race experiencing a progressive increase in life expectancy, we are facing a growing prevalence of chronic age-related conditions, among which osteoporosis. Trace elements or minerals remain a source for putative new and innovative dietary health intervention in the nutritional prevention of osteoporosis. Moreover, according to a widespread hypothesis, trace elements may protect the body against atherosclerotic diseases, especially in the elderly. Finally, deficiencies of trace elements can have profound effects on the development, health and well being of human subjects. They may have various etiologies: inadequate intake, inherited genetic disorders, malabsorption due to intestinal pathology or reduced bioavailability, excessive urinary and/or fecal excretion. Although some progress has been made in the understanding of dietary interactions that determine biological availability of trace elements, a basis for quantitative treatment of these interactions is still lacking. Trace element supplementation for meeting the requirement is also not so easy to perform. Interactions with other minerals or dietary constituents need consideration in the evaluation. “International dietary standards for trace elements," Jeanne Freeland-Graves, PhD The essentiality and toxicity of trace elements in the human diet have been estimated to be related to mortality and morbidity in over half the world’s population. Problems may arise from inadequate and/or poor quality food supply, cultural and societal norms, and environmental factors such as geography, contamination, parasites, and political and economic situations. In addition, trace element status is influenced by impairments in metabolism (absorption, digestion, transport, utilization, and excretion) and consequences of diseases and chronic health conditions. This impact of micro-minerals on health and society necessitates the formulation of recommendations of dietary standards that can be disseminated to health professionals, consumers, researchers, and agricultural and government agencies and policy makers. Common approaches to assess trace element status have included clinical consequences, nutrient balance, functional indicators (biomarkers) and population-based assessments of nutrient intakes associated with good health. Yet the vast diversity of countries, populations and diets throughout the world make it difficult to identify the most appropriate values for each segment. Priorities among regions of the world may differ, from elimination of hunger and malnutrition and maintenance of biological functions to prevention of chronic diseases via optimal intakes. Variations due to genetic determinants, body composition, stages of life and lifestyle may further complicate recommendations. Clearly, further studies are warranted to develop and compare the multiplicity of dietary standards on a global basis. “Future dietary standards for trace elements," Lindsay Allen, PhD Since the Dietary Recommended Intakes for most trace elements were released for the United States and Canada (FNB/IOM, 2000), information has been acquired that may improve our estimates of future dietary standards for some, but not all, of these nutrients. In the case of iron, there is considerable current interest in potential adverse effects of higher intakes in the form of supplements, which could change the current criteria on which the UL is based. Recommended iron intakes for infants be increased based on lower estimates of iron absorption from breast milk, and there are new data showing that cut-offs for iron status indicators in infants should be changed. The International Zinc Nutrition Consultative Group (IZiNCG) has published a detailed review of recommended dietary zinc intakes, which concludes that zinc requirements are substantially lower than those published by IOM/WHO for several age groups, based primarily on using a different set of studies for estimating endogenous zinc losses. A new model is available for predicting the effects of total dietary zinc and total phytate on zinc absorption. However, there has been relatively little progress finding better indicators of human zinc status. New data from long-term feeding of a high copper diet suggest that the current Upper Level for copper is too high, and should be lowered. Ongoing research is investigating the effect of iron deficiency on copper absorption. WHO has recently increased recommended iodine intakes during pregnancy, a position that has been endorsed by the American Thyroid Association. Regarding silicon, new data show that there is a significant correlation between silicon intake and urinary silicon, which may provide some information on which to estimate requirements in the future.

Health Consequences of Trace Element Deficiencies Chair: Sophie Ermidou-Pollet, PhD “Trace element nutrition: a neglected health issue in developing countries," Mohamed Abdulla, MD According to a recent report by the World Health Organization (WHO), deficiency of trace elements is widespread, but unrecognized. Nearly a third of the current population of the world suffer from the deficiency of iron and iodine alone. In countries with low socio-economic status, it is very likely that deficiencies of other trace elements such as zinc and selenium are very common. A lack of characteristic symptoms and lack of appropriate diagnostic tools are the major reasons that such deficiencies are not recognized. The fourth leading cause of death in many parts of the world in the 90s was due to diseases related to nutritional disorders. Even the fourth projected cause of death in coming yerars would be due to nutrition-related disorders. In spite of this report by the WHO, trace element nutrition as a health issue has a very low priority in most developing countries. In the present study, we have investigated the dietary patterns in the general population of 13 countries. Daily dietary samples from 13 countries - both developed and developing - were collected by a standardized food collection technique. After proper handling of the diets, the contents of major and minor trace elements were analyzed by modern techniques. Blood samples were collected from adults and children in India, Pakistan, Sweden and France in order to analyze the contents of lead. The results showed that the intake of potassium, magnesium, zinc, iron and selenium was low when compared with the recommended levels (RDA) at the time of investigation. The levels of lead in the blood of children in India and Pakistan were 3-5 times more than the levels observed in Sweden. The exposure to other heavy metals such as cadmium and mercury varied to a great extent in different countries. The implication of these findings will be discussed from a public health point of view. “Investigation on the transport of trace elements across barriers in human: Studies of plancental and mammary transfer. Impact on possible deficiencies in infancy," Erich Rossipal, PhD To evaluate the barrier function of the human placenta at the end of gestation, the transfer of 17 trace elements from the mother to the foetus was investigated. To this end, 29 maternal and corresponding umbilical cord sera (UCS) were considered. In addition, the uptake of trace elements by the foetus was evaluated in nine pairs of arterial and venous UCS. Our results indicate that the placenta activates the transfer of selected elements such as Mn 150%, Zn 148%, Ca 120% and Mg 105% compared to the maternal serum content, i.e. 100%. Yet the placenta can also inhibit the transfer, for example of Cu (20%), Pb (50%), Se (55%) and Co (60%). However, the placenta is permeable for Li, Mo and Sr following a concentration gradient and resulting in comparable elemental concentrations in maternal serum and UCS. Our findings also indicate that the placenta may act as an organ of excretion to maintain homeostasis between Zn and Cu. Determination of 14 trace elements in 27 maternal sera and corresponding colostrum samples revealed an activating as well as an inhibiting effect of the mammary gland for several trace elements. In colostrum the concentration of Zn was 15-times higher than in maternal sera. For Mo, Mn, Sn and Ca, these values amounted to 8.1, 2.8, 2.3, and 2.2. An inhibiting transfer effect was observed for Se (47%) and Cu (19%). A concentration gradient mode of action was established for the transfer of Li and Sr. The intensive transfer of Zn by the mammary gland during the period up to the third month post partum results in high concentrations of Zn in mother´s milk. This is obviously designed to cater for an increased need for Zn of newborns and young infants. One may speculate that this period of a high Zn supply may not be sufficiently long to meet the requirements of premature infants. Hence it may cause Zn deficiency in these infants when exclusively breast-fed. Taken together, the active transfer of certain trace elements and the retention of other trace elements across the placenta and mammary gland emphasize the important role of the two organs as regulators to meet exactly the demands of foetus, newborns and infants. “Trace element deficiency treatment: much more than trace element supplementation," Ivana Djujic, PhD Maintaining optimal mineral balance is of great importance in the prevention, mitigation and treatment of numerous diseases. Recent data on nutrient and supplement intake indicates that the vast majority of people suffers from micronutrients malnutrition. Investigations of trace elements in blood and scalp hair conducted on healthy adults from different countries showing that most of tested participants have reduced concentration of at least one element and disturbences in the ratios of many other elements. In order to improve deficient trace elements intake humans use various form of supplements and/or foods with improved nutrient status. To identify the most desirable strategy for reducing risks from sever selenium (Se) and in lower extent other nutrients defficiency in food chain we tested efficiency of dietary supplements with Se in the form of selenite and the Se enriched brevery and bakery yeast, as well as crops biofortified with Se use in human and animal nutrition. Conducted investigations on humans and animals showed that in raising defficient trace elements level any form of dietary supplements was not so efficient as food that contains trace elements in natural forms and beneficial amounts. Such food represents well balanced source of deficient trace elements and has optimized content and composition of wide range nutrients important for the proper functioning of the body. Offered strategy can΄t provide enough trace elements when individual requirements for same nutrients are increased. In such cases, to determine individual needs for trace elements, recognise symptoms of mild trace elements deficiency, and determine the optimal dose and combination of trace elements and other nutrients we utilized informations that offer hair analysis to trace elements, saliva pH measurement and bio-informatical methods. Health effects of such formulated nutritional supplements use overcomed all our expectations. . “Predicting relative concentrations of bioavailable iron using in vitro approaches," Maria Kapsokefalou, PhD There is an unequivocal need for predicting the bioavailability of dietary iron. Bioavailability is a key concept in iron nutrition. Ranging from 1-20%, it is the limiting factor in dietary iron intake and consequently it plays a central role in the primary prevention of iron deficiency. Many studies have identified a variety of dietary factors that enhance or inhibit iron bioavailability. Nevertheless, new measurements and/or prediction of iron bioavailability are continuously required because of new developments in the science of foods and nutrition. For example, interest in novel food ingredients (eg sterols, milk peptides) or in new fortified foods (eg milk products, gluten-free breads) or in local, traditional and ethnic foods have produced a need for new measurements of iron bioavailability. Consequently, there is a continuing need for establishing simple, reliable screening techniques that would allow the comparison of a large number of samples. In vitro procedures, in cell-free or cell model systems, offer advantages but also present limitations. New developments in the use of the in vitro methodologies and examples from the application of these methodologies will be discussed. “Iodine in the food chain of animals and man: intake, balance and requirement," Manfred Anke, PhD The iodine intake of 7 omnivorous and ovolactovegetarian women and men was examined in 21 test teams in Central Europe and Mexico by the duplicate portion technique over 7 succes-sive days. Women in Germany presently consume > 100 µg, men > 120 µg I/day on average, or 1.3 and 1.5 µg/kg body weight/day, respectively. In Mexico, I intakes of women and men amount to 150 and 200 µg I/day or 2.5 and 2.6 µg I/kg body weight and day, resp. Although on average the normative iodine requirement of adults is met (1 µg, recommendation 2 µg, per kg body weight and day), 25% of German adults take in < 100 and 120 µg I/day, resp. Iodine consumption in summer is 40% lower than in winter. It increases by 30% with increas-ing age (20 – 69 years) and 45 % with increasing body weight. In the endemic iodine- and selenium-deficient region of Central Europe, where iodine intake and serum T4 status were normalized by iodinated salt, it was not possible to normalize the free T3 level in the blood serum. The GSH-Px levels of women’s blood serum (170 U/L) were normal. Supplementation of 50 µg Se and 100 µg I/day normalized the serum-free T3 level. A normalization of iodine metabolism is only given if both the iodine and selenium require-ments are met. An increase in the incidence of hyperthyroidism worldwide has been described after iodine supplementation in iodine deficiency regions. Older women, patients with Graves’ or Hashi-moto’s disease, autoimmune thyroid disease or herpetiform dermatitis Duhring require spec-ual attention. The I intake of adults is limited to 7 µg/kg body weight. The main excretory routes for iodine are the kidneys and the breast or udder, which compete with the thyroid for plasma iodine. Urine and milk are good indicators of the iodine status and intake, if fecal iodine excretion of humans (about 10 – 80 µg/day) is taken into consideration. On average, omnivores and vegetarians excrete 81 to 85% of the consumed iodine renally and 15 to 19% fecally. Their apparent iodine absorption rate is 82 to 86%, their iodine balance -4 to +11%. Breast-feeding women without I supplementation excrete 48% of their iodine intake with the milk, 45% with urine and 7% with feces. After I supplementation of the mineral mix-tures fed to animals and of salt for humans, iodine concentrations in cows’ and breast milk increased from 10 µg/L to > 100 µg/L.

Mineral Elements and Molecular Signaling as it Relates to Human Disease Chair: Bernhard Hennig, PhD “Role of zinc in endothelial cell function: implications in atherosclerosis," Bernhard Hennig, PhD Zinc has multiple roles in maintaining the physiological conditions of the cardiovasculature. We hypothesized that a depressed zinc status may be involved in either initiation of vascular endothelial cell injury or inadequate vascular tissue repair. Thus, zinc requirements of the endothelium may be increased during inflammatory conditions that exist in cardiovascular disease. To assess the protective mechanisms of zinc during an endothelial cell (EC) inflammatory response, EC were activated by treatment with linoleic acid or TNF- and zinc deficiency was induced by treatment with the membrane permeable zinc chelator TPEN. We demonstrated that zinc deficiency induced oxidative stress, increased the DNA binding activity of NF-B and AP-1, and increased EC production of IL-6. Peroxisome proliferator activated receptors (PPARs) may have antiatherogenic properties. We found that zinc deficiency decreased PPAR activation and protein expression. In contrast, zinc supplementation markedly increased PPAR activation and expression, which was correlated with downregulation of DNA binding activity of NF-B and AP-1. Our data demonstrate that zinc exhibits potent antioxidant and anti-inflammatory properties. Furthermore, zinc appears to protect against EC activation and inflammation by functioning as a critical component of the PPAR transcription factor complex. A recently completed in vivo study suggests that in an atherosclerotic mouse model PPAR signaling is compromised during zinc deficiency and that adequate dietary zinc is critical for proper function of the anti-diabetic medicine rosiglitazone. In summary, our data suggest that zinc can downregulate the pathways of signal transduction leading to an inflammatory response and to disruption of endothelial cell integrity. Thus, zinc may have critical nutritive and therapeutic roles in inflammatory diseases such as atherosclerosis. “Oxidative stress in tumor progression: intervention by selenium," Peter Brenneisen, PhD Myofibroblasts, pivotal for tumor progression, populate the microecosystem of reactive stroma. The potential involvement of reactive oxygen species in the mesenchymal-mesenchymal transition (MMT) of human dermal (stromal) fibroblasts to myofibroblasts was studied. We addressed the question of whether intervention with micronutrients such as selenium as well as antioxidants may affect this process in stromal cells and the invasive capacity of skin-derived squamous carcinoma cells. Using a broad spectrum of biochemical and molecular biological methods [ref.1], we found that upon treatment with conditioned medium of tumor cells and transforming growth factor beta 1 (TGF1), respectively, the intracellular level of reactive oxygen species (ROS) of fibroblasts was increased via a protein kinase C (PKC)-dependent pathway. The elevated ROS level, assessed as lipid hydroperoxides, initiates a signaling process resulting in both MMT and release of proinvasive signals. Using a filter-based in-vitro invasion assay, a significant increase in invasive capacity of tumor cells was measured, based on a paracrine effect of myofibroblast-derived hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6). Preincubation of fibroblasts with selenite, selenoprotein P or other antioxidants lowered TGF1-initiated lipid peroxidation, subsequently resulting in inhibition of expression of alpha-smooth muscle actin (SMA), a biomarker for myofibroblastic cells, as well as in lowering of the invasive capacity of tumor cells. Taken together, TGF1 is considered to play a major role in the generation of “tumor-educated” fibroblasts, namely myofibroblasts. The novel concept of stromal therapy, meaning the protection of stromal cells against the dominating influence of tumor cells in tumor-stroma interaction by micronutrients and/or antioxidants, may contribute to anti-invasive and anti-metastatic strategies. “Influence of zinc on oxidative stress, DNA integrity and cancer risk," Emily Ho, PhD There is increasing evidence that micronutrient deficiencies may damage DNA and increase cancer risk. A large proportion of the population has inadequate zinc intakes. Zinc is a component of over 300 proteins, including DNA-binding proteins with zinc fingers, Cu/Zn superoxide dismutase and DNA repair proteins such as p53, a zinc protein which is mutated in half of human tumors. It can be hypothesized that insufficient zinc intake can impair antioxidant defenses and compromise DNA repair mechanisms, making the cell highly susceptible to oxidative DNA damage. Using of both genomic strategies (using microarray technology), and functional assays, we have found that zinc deficiency increases DNA damage, oxidative stress, expression of DNA repair enzymes but impairs downstream signaling events in a cell culture model. This data demonstrates that zinc deficiency causes oxidative stress and DNA damage, but important downstream signals leading to proper DNA repair are lost without zinc. In vivo, we have also found that zinc deficiency increases DNA damage in peripheral blood cells, and increases several markers of oxidative stress. Consequently, zinc deficiency not only causes oxidative stress and induces DNA damage, but also compromises the cell’s ability to repair this damage. Zinc deficiency also significantly increases the sensitivity of cells to DNA damaging agents, such as low dose ionizing radiation. Moreover, expression profiles in zinc adequate and zinc deficient cells following irradiation differed markedly, suggesting alterations in transcriptional response with zinc deficiency following exposure to DNA damaging agents. This work strongly suggests that zinc deficiency has a detrimental effect on DNA integrity and emphasizes the importance of good nutrition in the prevention of cancer. “Maintaining copper balance: roles of copper transporters and chaperones," Jesse Bertinato, PhD Copper (Cu) is an essential micronutrient that plays an important role as a catalytic co-factor for a number of metalloenzymes involved in various biochemical processes including cellular respiration, antioxidant defence, development of connective tissue, pigment biosynthesis, iron homeostasis and neurotransmitter production. Cu’s redox chemistry, however, also makes it a potentially toxic metal if not properly utilized. Therefore, cells have evolved elaborate systems to control the uptake, distribution and elimination of Cu. Characterization of Cu transporters that mediate Cu uptake or efflux and the identification of Cu chaperones that deliver Cu to specific cellular targets have greatly improved our understanding of the mechanisms by which cells effectively use Cu and maintain Cu homeostasis. In yeast, Cu transporter 2 (Ctr2) is thought to function as a vacuolar Cu transporter that mobilizes Cu from the vacuole to the cytosol. Cu chaperone for Cu/Zn superoxide dismutase (CCS) inserts Cu into the antioxidant enzyme Cu/Zn superoxide dismutase. Using a transient overexpression approach in COS-7 (monkey kidney) cells we have shown that although the majority of Ctr2 is localized to the outer membrane of cytoplasmic vesicles, a small proportion is present at the plasma membrane where it promotes specific, saturable Cu uptake. We have also demonstrated that CCS protein is upregulated by Cu deficiency and that Cu regulates CCS degradation by the 26 S proteosome. Using a rat model, CCS was determined to be a sensitive indicator of mild Cu deficiency induced by moderately high intakes of zinc. The potential roles of Ctr2 in Cu transport in the context of known Cu-trafficking pathways in mammalian cells and the application of CCS as a biomarker of Cu status will be discussed.

Environmental Stress and Mineral Homeostasis Chair: James McClung, PhD “Environmental Stress/Physiology Background," Andrew Young, PhD Millions of people live, work and play in regions of the world where the weather is intemperate or the elevation is sufficiently high that ambient oxygen pressure is lower than at sea level. In addition, while relatively few people have traveled in space up to now, increasing numbers of people may engage in space flight as the century proceeds. The environmental stressors of greatest physiological importance are hypoxia, cold, heat, and gravity. When humans are exposed to extremes of these environmental stressors, a variety of physiological responses manifest that assist in re-establishing and/or maintaining homeostasis. Those physiological responses could have implications for dietary micronutrient requirements, e.g., sweating-associated trace element losses. Further, physiological responses to exercise can be a major factor influencing nutritional requirements, and responses to strenuous physical activity can be exacerbated by extreme environmental conditions. Therefore, to begin considering how extreme environments affect dietary trace element requirements, this presentation will identify key environmental factors constituting biologically significant stress for humans. Furthermore, the physiological adjustments elicited by those stressors, especially those exhibited by physically active persons engaging in strenuous activities, will be discussed. “Iron Homeostasis in Environmental Extremes," James McClung, PhD Dietary iron confers biological function through incorporation into a series of proteins, including hemoglobin and myoglobin for the transport and storage of oxygen, and cytochromes for the oxidative production of cellular energy. Iron deficiency is the most prevalent micronutrient deficiency disorder in the world, affecting mainly premenopausal women. Changes in immune function, cognitive performance, and work capacity have been described in humans and animals with suboptimal iron status. Iron status indicators may be affected by a number of environmental conditions, including altitude and exposure to heat. Furthermore, physical activity seems to affect iron status. A recent line of studies has investigated the effect of combat training on iron status in female military personnel. Decrements in iron status indicators, including serum ferritin, transferrin saturation, and red cell width have been reported following eight weeks of military training. This review will focus on the effects of extreme environmental conditions on iron homeostasis, citing recent studies with female military personnel as example of a population that may be sensitive to decrements in iron status. Furthermore, potential nutritional interventions to mitigate the effect of environmental extremes on iron status will be discussed. “Mineral Homeostasis at Altitude: Focus on Antioxidants and Selenium," Andrew Subudhi, PhD Accelerated metabolic rate and free radical production at high altitude increase macronutrient and vitamin requirements; however, data concerning mineral homeostasis at this extreme environment are scarce (Butterfield, 1999). Since minerals are important structural components of many antioxidant enzymes, measurements of specific enzyme activities and oxidative stress can yield insight into mineral status during exposures to high altitude. Particularly, the Cu, Zn isoform of superoxide dismutase (SOD1) and Se dependent cellular glutathione peroxidase (GPx) have been studied during both short-term (< 24 hrs) and prolonged (>24 hrs) exposures to high altitude. In general, antioxidant capacity has been sufficient to counterbalance oxidative stress during short-term exposures. Specifically, acute altitude exposure did not increase whole-body markers of oxidative stress or antioxidant activity of erthyrocyte SOD1 or GPx in individuals eating a well-balanced diet, although repeated short-term exposures tended to deplete antioxidant reserves over time (Subudhi et al., 2001). Prolonged altitude exposure has been associated with increased markers of oxidative stress, but has had equivocal effects on antioxidant enzyme systems. Se status was not compromised during the first 14 days at high altitude, as GPx activity was similar to low altitude conditions and unaffected by Se supplementation (Subudhi et al., 2004). Additionally, SOD1 activity was increased over 13 months at high altitude, suggesting favorable Cu and Zn status (Rawal et al., 1999). To date, research does not indicate a significant disruption in mineral balance at high altitude, yet future studies are needed to determine the effect of altitude on actual plasma and tissue concentrations of various minerals. “Mineral Homeostasis during Spaceflight: Focus on Bone Demineralization," Scott Smith, PhD Bone loss during spaceflight remains a significant challenge to astronaut health on space exploration missions. Associated short-term risks, including renal stone formation, and long-term risks of fractures and increased skeletal fragility, are all matters of concern. Spaceflight-induced bone loss is one of the effects of weightlessness in which nutrition clearly can play a role, either positive or negative. Findings from spaceflight and studies using ground-based analogs of flight (such as bed rest) clearly indicate that bone resorption increases during and after flight. Maintaining vitamin D status remains a challenge for long-duration space travelers, who lack exposure to ultraviolet light in their shielded craft. A great many nutrients besides calcium and vitamin D play a role in bone health, and may play a role in astronaut health during spaceflight. These include excess dietary sodium, excess dietary protein, and insufficient vitamin K. Excess dietary protein and sodium both seem to affect acid/base balance, and subsequently to increase bone resorption. This phenomenon appears to be exacerbated during bed rest. This provides one example of how studying the relationship between nutrition and physiology in spaceflight analogs has proven valuable to space travelers, and has also furthered our understanding of health and disease on Earth. Nutrition—defined as the appropriate nutrient intake and balance of nutrients—is essential for crew health on space exploration missions. Exercise and/or pharmaceutical countermeasures will not mitigate weightlessness-induced bone loss if the nutrients required for bone health are not available (or are available in excess). Much work remains to clearly define the nutritional requirements for space travel, and whether dietary alterations may provide safe and effective countermeasures. “Mineral losses during extreme environmental conditions," Henry C. Lukaski, PhD Minerals are nutrients that are conserved by the body. During exposure to environmental stimuli, such as heat and/or exercise, the excretion of minerals, macro (Na, K, Ca, Mg) and micro (Cu, Fe, Zn), occurs through the body surface in the form of cellular desquamation and sweat, as well as in the urine. Whereas it is well established that urinary excretion of minerals increases following physical activity, compared to no activity, there is uncertainty regarding surface mineral losses during acute, compared to prolonged bouts of exercise, as well as on repeated days in a hot environment. Surface losses of minerals, total of cellular and sweat, have been sampled from various sites of the body including upper and lower arm, chest, back, thigh, and forehead by using different collection devices or materials. These losses are expressed as concentrations of minerals (mg/L or ng/L), are appreciable, and show wide intra- and inter-individual variability. In general, conservation of minerals occurs during prolonged and repeated bouts of exercise in the heat. Methods of sweat collection, sources of contamination, differences by sex, and trends of mineral losses in heat acclimated and unacclimated individuals will be presented. The impact of surface losses of minerals on body mineral retention also will be discussed.

Advances in Analytical Detection of Trace Elements in Biological Tissues Chair: Sophie Ermidou-Pollet, PhD “Ion and electron beams methods for biological trace element characterisation at bioceramics/cells interface," Edouard Jallot, PhD Ion and electron beams methods of analysis are based on X-ray fluorescence induced after interactions between charged particles and atoms present in the samples. They permit to obtain simultaneously multi-elemental maps and concentrations measurements of major, minor and trace elements (ppm) with a spatial resolution from micrometer to nanometer. Micro-PIXE (Particles Induced X-ray Emission), STEM (Scanning Transmission Electron Microscopy) associated to EDXS (Energy Dispersive X-ray Spectroscopy) are useful techniques to determine locally concentrations gradients at biomaterials/cells interface. The prerequisite for bioceramics (calcium phosphates, bioactive glasses) to bond to living bone is the formation of biologically active apatites on their surface in the body. Reactions and bioactivity mechanisms between bioceramics and cells depend on the materials composition in major and trace elements. Knowledge of the local concentration and species of ions released at the ceramics/biological fluids/cells interface is primordial to the understand of the complex interfacial reactions and physico-chemical properties of bioceramics. This work characterises the interface that bioactive ceramics particles develop after immersion into biological fluids with or without cells for different delays (from 1h to 28 days). Results demonstrate materials dissolution, ionic leaching and precipitation of a biomimetic apatite layer at bioceramics surface. This bioactive layer contains biological relevant trace elements. Cells viability can be altered by the presence of bioactive ceramics particles and their features. Determination of intracellular ionic concentrations of cells in a state near the physiological state is of importance to better study cells viability. Use of cryo-methods for cells fixation limit diffusion phenomenon of diffusible ions and preserve the chemical identity of specimens. Then, X-ray analysis of samples allow to study intracellular ions like sodium, potassium, chlorine. Moreover, it is also possible to evaluate the intracellular concentration of magnesium, phosphorous and sulfur at the subcellular level. Thus, the K/Na ratio can be used as a sensitive criteria of viability of cells. “X-ray fluorescence technique on trace element determination in biological samples," Maria Luisa de Carvalho, PhD Elemental composition of tissues and in particular trace elements has gained interest over the last few years, due to the increasing knowledge of the role of some elements and the correlation between abnormal concentration of some elements and pathological effects. Trace elements, although in concentrations of the order of a ug g-1, they play an important role in the organism. Some trace elements are “essential elements” for the organism, which means that the organism can neither grow nor complete its life cycle if the element is not supplied in sufficient quantity. Deficiency or excess of the element leads to specific diseases. On the other hand, between the trace elements are the “toxic elements”, mostly the heavy metals. The toxic effect of the heavy metals is at different levels and can depend on the chemical form of the element. Essential elements can also become toxic at high concentration, and there is a wide range of toxicity, depending on the element and the preference tissue for accumulation. It will be presented studies of toxic elements, e.g. cadmium and its effects in plant cells, Hg in dolphins tissues, Pb elemental profiles in human teeth collected from contemporary miners, fishermen, and patients with renal insufficiency and populations of different pre-historical periods. The used technique for these studies is X-Ray Fluorescence Spectrometry. It is based on the ionization of the atoms of the sample by means of an incident X-Ray beam. The atoms getting back to its fundamental state emit characteristic radiation. The detection of these photons allows identification and quantification of the elements present in the sample. Quantitative calculations for S, Cl, K, Ca, Mn, Fe, Co, Cu, Zn, As, Sr, Ba and Pb in liver, brain and kidney of healthy and cirrhosis disease human samples were performed. Minor and trace element contents of each tissue were obtained in a total of 17 elements. Three groups of elements were considered: P, S, Cl, K, Ca present in relatively high concentrations in all tissues and known as normal constituents of all living matter. They are essential to cellular metabolism. The second group includes Mn, Fe, Cu, Zn, Se, Rb and Sr, which are also essential elements, but may induce toxic effects if present in high concentrations. In the third group we include the toxic elements, Co, Ni, As and Pb which induce normally pathological disorders in the organism, even in small concentrations. Very low concentrations for Ni, As and Sr were found in the analysed samples. Higher concentrations of Co, Fe, Pb and lower values of Zn and Se were found in cirrhosis liver when compared to healthy one. Brain samples from subjects with cirrhosis showed enhanced levels of Pb. In kidney, increased amounts of Zn were found in tissues from subjects with cirrhosis. “Alternative testing methods in food nanoparticles toxicology research for a safe food nanotechnology development," Enrico Sabbioni, PhD The new EU Chemical Policy foresees regulatory actions to prevent health risks of exposure to chemicals. Different kinds of toxicity data (i.e. systemic and topical toxicity, chronic toxicity, carcinogenicity, reproductive toxicity) must be produced to improve the scientific basis of risk assessment along with in vivo data derived from human and animal data and in vitro data. In this context, the European Centre for the Validation of Alternative Methods (ECVAM) has been set up in order to coordinate, at European level, activities to promote the scientific and regulatory acceptance of alternative toxicity testing which are important to biosciences. In particular, advanced approaches that have less or no dependence on animal experimentation (cell cultures) represent the basis of “in house” projects concerning development and validation of mechanistically-based in vitro toxicity testing. In this context, such approaches have a great potential in the emerging area of toxicology research on nanoparticles and nanomaterials. In particular, the possibility that food is designed by shaping molecules and atoms at an extremely small scale – between 1 and 100 nanometers– is opening up a whole universe of new applications such as the development of functional food, nutrient delivery systems and methods for optimizing food appearance, such as colour, flavour and consistency, and the development of food-packaging with enhanced mechanical and thermal properties to ensure better protection of foods from exterior mechanical, thermal, chemical or microbiological effects. Unfortunately, in spite of the great excitement about the potential benefits offered by nanotechnology in food industry a huge health and safety questions remain unsolved. In particular, there is a gap in the current regulation of nanoparticles and there are emerging toxicological evidences to fear that manufactured nanoparticles and nanomaterials could have undesirable health effects. This is pressing researchers and governmental organizations to consider seriously the need to assess possible health risks of nanoscale materials before they become ubiquitous in every aspects of life including the aspects of food packaging, food production and supply chain tracking. In this context, food nanotoxicology research is an urgent need for the assessment of the health impact of nanomaterials relevant to food industry and food processing and it is important that the regulations are tightened up so that nanoparticles are assessed, both in terms of testing and labelling as new chemicals. The aim of this work is to review and to show the potential that in vitro cellular and non-cellular assays can have in food nanotoxicology research in order to take into account the substantial ethical, political, financial and regulatory pressures to reduce, refine and replace animal use with alternative non animal assays (3Rs approach).

Copper in Neurologic and Neurodegenerative Diseases Chair: George J. Brewer, MD “Overview of the role of copper in neurologic and neurodegenerative diseases and potential treatment with tetrathiomolybdate," George J. Brewer, MD In a number of neurodegenerative disease, which include Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis, and prion disease, copper is intimately involved in binding to relevant proteins, aggregate formation, and perhaps in producing damaging oxidant stress. The role of copper in these diseases, to the extent known, will be reviewed. Of these diseases, only in AD has the cumulative evidence become convincing that excess copper is involved with initiation or progression of the disease. In the other talks of this symposium, some of that evidence in AD will be presented. These results indicate that therapy to reduce copper levels may be beneficial in AD, and we propose that tetrathiomolybdate (TM) is the ideal drug for such trials. TM’s mechanism of action in the blood involves formation of a three way complex with copper, albumin, and itself, which renders the relatively mobilizable, and potentially toxic, copper unavailable for cellular uptake. In this manner, TM detoxifies copper in neurologically presenting Wilson’s disease, and lowers the availability of copper in the body in other disease states. For example, by limiting copper availability, TM has been shown to be an effective therapy in a variety of animal models of fibrosis, inflammation, and autoimmunity. These include bleomycin induced pulmonary fibrosis, carbon tetrachloride induced cirrhosis, concanavalin A induced hepatitis, acetaminophen induced liver damage, doxorubicin induced heart damage, immune modulated bovine collagen induced arthritis, autoimmune diabetes in the non-obese diabetic mouse, autoimmune systemic lupus erythematosis, and EAE induced multiple sclerosis. Mechanisms of protection may involve inhibition of damaging cytokines and protection against oxidant damage. We postulate that TM will limit copper availability and toxicity in AD, and clinical trials are planned. “Copper, cholesterol, and cognition: animal and human evidence," D. Larry Sparks, PhD Increased circulating cholesterol levels are associated with serious medical concerns and represent a mounting problem as elevated cholesterol becomes more widespread among the peoples of the world. Besides an obvious increase in coronary artery disease and stroke, emerging data suggest an increased risk of cognitive impairment with increased circulating cholesterol. Furthermore, excess cholesterol promotes production of the Alzheimer’s disease (AD) neurotoxin, amyloid beta (A). We identified a further link between copper, cholesterol and cognition in the cholesterol-fed rabbit model of AD. It was shown that dietary cholesterol induces central production of A and that copper determines if overproduced A is cleared to the blood or accumulates in brain. Levels of copper ion were manipulated by adding trace amounts of the metal to an animal’s distilled drinking water. Memory deficits (80%) were associated with brain A deposits in cholesterol-fed animals administered 0.12 PPM copper water, compared to cholesterol-fed animals drinking unaltered distilled water where there was minimal accumulation of A. Cholesterol and copper may independently influence copper levels in blood and brain. Adding copper to the water of animals fed normal chow increases blood and brain copper levels compared to animals fed normal chow and drinking distilled water. Copper levels are increased in the blood and unchanged in the brain of cholesterol-fed animals drinking distilled water, while levels are decreased in brain and increased in the blood of cholesterol-fed animals drinking copper supplemented water. Copper may inactivate A suggesting low brain copper could enhance A induced degeneration in AD. We have previously reported elevated circulating copper and cholesterol levels in low-function controls (LFC), MCI and AD compared to high-function controls (HFC). We evaluated brain copper, zinc and iron levels in HFC, LFC, MCI and AD, and Parkinson’s (PD) patients with and without dementia. “Excess of non-ceruloplasmin copper in Alzheimer’s Disease patients," Rosanna Squitti, PhD Current hypotheses on Alzheimer Disease (AD) maintain that oxidative stress and metal imbalance may lead to accumulation in the brain of ß-amyloid (Aß), which is the pathological hallmark of AD. Recent evidence suggests that brain Cu may re-distribute outside the neuronal cells leaving them relatively deficient. Circulating Cu also seems to be abnormal. In fact, over the last 6 years our group has collected in vivo evidence in man of serum Cu elevation in AD. Contrasting conclusions from different authors are feeding a debate. However, it seems to us that these conclusions appear different only because different are the approaches taken towards data analyses. Our initial results indicate serum Cu levels were higher in AD patients than in normal elderly controls and vascular dementia patients. Elevated Cu correlated with cognitive deficits of AD. Further studies revealed that the fraction of serum Cu not bound to ceruloplasmin (Cp) was increased in AD. Initially referred to as an “unexplained Cu” in serum, it could now be referred to as non-Cp-Cu (NCC). NCC is the serum pathological marker of Wilson’s disease – the paradigmatic disease of Cu accumulation and intoxication. In AD, NCC is above normal values, similarly, although definitely lower, than in Wilson’s disease. Ultrafiltration experiments revealed that in patients the filtered Cu was 3.7 fold higher than in controls. In a recent study we detected a direct effect of NCC on CSF Cu concentrations (clearance) as well as A and tau proteins. Moreover, the slowing of the brain rhythms typical of AD correlated with NCC abnormalities: temporal delta (r= 0.37, p< 0.001) and alpha 1 (r=-0.30,p=0.005). We tested the hypothesis whether NCC may occur in AD if the liver fails to clear Cu. We found that it correlated with a reduction in liver function, both evaluated as albumin/Prothrombin time ratio (r=-0.36,p=0.02) and free-bilirubin/Albumin-bound-bililirubin (r=-0.37,p=0.05). We attempts to clarify our opinion that contrasting conclusions on serum Cu and cognition could be the result of different data analyses approaches. Cu, Cp and MMSE were collected from 146 AD patients and 165 healthy controls. The correlations between NCC and MMSE stratified for diagnosis were absent in AD and only weak controls. When, instead, was evaluated in the whole population, it was very strong (r=-0.33,p<0.001). “Dietary fat and copper effects on cognition," Martha Clare Morris, Sc.D. There is evidence from epidemiologic and animal studies to suggest that dietary fat composition and trace metals may be related to neurodegenerative diseases of the brain. These hypotheses were investigated in the Chicago Health and Aging Project, a 1993-2002 study of community residents aged 65 years and older that included the administration of four cognitive tests to all participants and clinical evaluations for incident Alzheimer’s disease. Dietary assessment was by food frequency questionnaire. Daily nutrient intakes were computed from food and vitamin supplement sources. Fat composition was associated with incident Alzheimer’s disease and with cognitive decline over 6 years. Saturated and trans fat intakes increased disease risk and cognitive decline, and n-3, n-6, and n-9 fatty acids were protectively associated. Dietary intakes of the trace metals, copper, zinc, and iron (food plus vitamin supplements) were not associated with cognitive decline overall. However, among persons who had high intakes of saturated and trans fats, high copper intake was associated with faster rate of cognitive decline over 6 years. The increase in the rate for the high-fat consumers whose total copper intake was in the top 20% (1.6 mg/d) was the equivalent of 19 years of older age and was highly statistically significant (<0.0001). A similar but weaker association was observed for copper intake from food sources only. There was strong linear relation between copper dose in vitamin supplements and greater cognitive decline. By contrast, there was little evidence of association between cognitive decline and intakes of zinc and iron. These data suggest that high intake of dietary copper among persons who have a high-fat diet may be associated with accelerated loss in cognitive ability. . “Investigations on the role of copper in human neurodegenerative disease using model systems," James Camakaris, PhD Copper plays a key role in the brain due to its requirement by several critical cuproenzymes. Cu is involved in several neurodegenerative diseases. Both Cu deficiency and Cu excess can lead to oxidative stress and severe disease. Alzheimer’s disease (AD) is a severe neurodegenerative disease and is the most common form of dementia in the elderly. Senile plaques contain aggregates of beta amyloid (Abeta) derived from Amyloid Precursor Protein (APP) and are found in the brain of AD patients. Neurofibrillary tangles due to aggregates of Tau protein also contribute to AD pathology. Cu is found in Abeta in plaques and Abeta-Cu oligomers are toxic. Our data suggests that Cu can be effluxed from neurons bound to a cleaved product of APP, probably Abeta. We have also found that Cu regulates the APP promoter which is consistent with APP or an APP cleavage product being involved in Cu homeostasis. The excessive loss of Cu bound to Abeta can lead to a functional Cu deficiency in neurons which would result in oxidative stress and contribute to Alzheimer’s pathology. This is supported by our findings of reduced CuZnSOD activity in N2a neuroblastoma cells expressing familial AD APP mutants. Hence both an intracellular Cu deficiency and an extracellular Cu excess are likely to be major contributors to Alzheimer’s pathology. Our data also shows that mutation of an APP ectodomain Cu binding site results in abnormal processing of APP and accumulation of APP in the membrane. In order to test the role of high and low Cu in AD we are also using a Drosophila model where familial AD mutant APP or mutant Tau is overexpressed in strains which have been genetically engineered to have either high or low Cu by overexpressing either Cu uptake or Cu efflux transporters. The “rough eye” phenotype is being used as an index of neurological damage. Current data using this controlled system suggests that flies with high Cu levels showed an enhancement of neurodegeneration due to mutant Tau. .

Trace Element Speciation Chair: Yngvar Thomassen, PhD “Challenges in trace metal speciation of workroom air exposures," Yngvar Thomassen, PhD Most compounds of the elements can occur in different physical states, namely solid, gas, liquid, vapour or aerosol. Each of these states can influence or determine the nature of exposure, the uptake or absorption and the toxicological consequences. Many workplace and atmospheric aerosols are composed of a complex mixtures of chemical compounds. Since a number of factors are involved in determining toxicological responses to the chemical exposure this presentation will illustrate how modern technology used in aerosol measurement and characterisation can provide more detailed information about the specific particle-size distribution, time-resolved concentration monitoring of particle-size fractions, chemical composition and morphological characteristics of workroom aerosols collected in metal refineries. Such knowledge would improve our understanding of the basis for the reported differential health risks. Exposure to e.g. nickel oxides and sulfides, which are relatively insoluble in water, figures prominently in the epidemiology of nickel-related lung and nasal cancer. The carcinogenic potency of water-soluble nickel compounds and metallic Ni continues to be debated. For exposure assessment of workers in ongoing epidemiological studies novel chemical leaching schemes have been developed for bulk speciation of nickel and manganese compounds likely to be present in nickel and manganese alloys smelters. Further, single aerosol particle characterisation of aerosols collected from such metal smelters has been performed using modern methods of analysis in order to obtain deeper understanding of particle compositions and physiological behaviour. The aim of this presentation is to discuss the determinant factors and their measurements judged to be important in the understanding and rationalization of the biological chemistry and toxicity of inorganic compounds of the elements with illustrations from a variety of occupational environments. “Analytical challenges to human trace element speciation," Ryszard Lobinski, PhD The measurement of the total trace element concentrations is widely used to assess the elemental status in humans. The recent advances in analytical methodology allow, however, the access to information on the concentration levels of the individual species of an element, referred to as speciation. In particular, the progress in inductively coupled plasma mass spectrometry (ICP MS), used as a detector in chromatography and capillary and gel electrophoresis, have resulted in a considerable number of in-vitro and in-vivo studies of interactions of trace elements in biomolecules. The multiisotopic (including non-metals such as S, P, or Se) detection capability, high sensitivity, tolerance to matrix, and large linearity range regardless of the chemical environment of an analyte make ICP MS a valuable complementary technique to electrospray MS and MALDI MS. The lecture discusses the possibilities of the state-of-the-art hyphenated techniques to the trace element speciation in biological samples of human origin. The examples chosen illustrate, in particular, advances in the three major areas: (i) monitoring of the metal-protein interactions and the determination of metal-protein complexes in-vitro and in-vivo by hyphenated techniques using ICP MS detection in liquid chromatography, gel electrophoresis and capillary electrophoresis; (ii) specific determination and screening for selenium-containing- and selenoproteins and selenoamino acids in blood and serum using two-dimensional affinity chromatography and size-exclusion chromatography with ICP MS detection, and (iii) in-vivo monitoring of the metabolism of metallodrugs containing covalently bound Pt or Ru atom with a focus on ultratrace (0.1 ng/ml detection limits) quantification of the initial drug and the products of its metabolism. “Studies on the biotransformation of arsenic and selenium in humans," Kevin Francesconi, Ph.D. Arsenic and selenium are two metalloids which, in their inorganic forms, have interesting similarities and differences in terms of their toxicity, biotransformation and excretion in humans. Both elements are readily methylated in the body, presumably as a detoxification strategy, and the methylated forms are quickly excreted mainly in the urine. In the case of selenium, the common urine metabolites are methyl selenosugars and trimethylselenonium ion, whereas arsenic metabolic products are dominated by simple mono and dimethylated derivatives. Because selenium is incorporated into essential biomolecules, and only the excess is excreted, the formation of the urine metabolites is strongly dependent on dose and becomes significant only at higher exposure. For arsenic, which has no established essential role, the pattern of urinary metabolites is more constant, and the quantity excreted reflects exposure, even at lower exposure levels. Humans, however, can also show considerable individual variability in the way in which they biotransform both elements. Understanding the reasons for this variability may help explain the observed effects, both beneficial and detrimental, of these elements in population-based studies. Recent results will be discussed from investigations with humans following exposure to inorganic and organic forms of arsenic and selenium. .

Health effects of low dose exposure to toxic metals Chair: Gunnar Nordberg, MD, PhD “Proteomic and metabolomic biomarkers for assessing low dose toxic trace element interactions: an overview," Bruce Fowler, PhD Toxic trace element - specific alterations in target cell protein expression patterns (proteomics) and changes in essential metabolic pathways (metabolomics) have proven extremely valuable for early detection of toxicity. These molecular approaches have been shown to be capable of detecting specific response patterns for both individual elements and mixtures of these elements as a function of dose, duration of exposure and gender. Since these pathways are highly conserved across species, it is possible to make some extrapolations from experimental model systems to humans. This presentation will provide an overview of studies designed to examine low dose biomarker effects from combined exposures to lead, cadmium and arsenic using an in vivo model system. In addition data from similar in vivo and in vitro studies will examine how combined proteomic and metabolomic biomarker approaches have provided useful information on early compound and gender - specific responses from exposures to the semiconductors gallium arsenide and indium arsenide. An overall conclusion from these studies is that translation of findings from modern basic scientific approaches can greatly improve risk assessments for toxic trace elements and help to delineate populations at special risk within the general population. “Neurological and renal effects of low dose exposure to elemental mercury from amalgam in children," Lars Barregard, MD, PhD Dental amalgam constitutes the most common source of exposure to elemental mercury in the general population and the possible health risks have been debated for a long time. Many studies have been performed in adults exposed to low levels of elemental mercury, but until recently few data were available on this topic in children, who might be more vulnerable. In 2006, two large randomized controlled trials were published, comparing neuropsychological (full scale IQ, memory, attention, visuomotor tests) and renal outcomes (e.g. albumin excretion) between children treated with dental amalgam or composites over a 5-year trial period. The studies included more than 1000 children from North East USA or Portugal. These studies failed to demonstrate any statistically significant differences in adverse effects between treatment groups. The increase in urinary mercury in the amalgam-treated groups compared to controls treated with composites was, however, limited, on average about 0.5 – 1 µg/g creatinine. The results of these two trials will be reviewed, together with subsequent studies using this rich data set. The strengths and limitations of the two trials will be discussed as well as children’s exposure to inorganic mercury in general, and the results of some recent observational studies of possible adverse effects. “Health effects of low dose exposure to manganese," Roberto Lucchini, MD PhD Manganese is an essential element for humans and animals and plays an important role in bone mineralization, protein and energy metabolism, metabolic regulation, cellular protection from damaging free radical species, and the formation of glycosaminoglycans. Homeostatic mechanisms regulate the absorption and excretion rates in order to keep manganese concentration within a strict range. Nevertheless, short-term exposure to high doses or prolonged exposure to low doses can determine a manganese overload in the central nervous system, given the slow elimination rate from this organ. In cases of overload, manganese accumulates in the globus pallidus of the basal ganglia where it can cause cellular damage on the GABAergic an dopaminergic pathways. As a consequence, motor function and coordination of fine movements are affected, and mood regulation as well with marked aggressivity. Human exposure to air concentration higher then 1 mg/m3 can determine the clinical picture of manganism, an atypical parkinsonism that shows clinical differences from the typical features of Parkinson’s Disease. After prolonged exposure at much lower levels, manganese may also act as an environmental trigger and favour the onset of typical Parkinsonian disturbances. This can be determined by a damage of the dopaminergic neurons of the substantia nigra - pars compacta, which is located very closely to the globus pallidus and shares various interconnections within the basal ganglia. This hypothesis is presented as based on experimental animal studies and human epidemiological studies on exposed workers and general population resident in the proximities of polluting sources. “Kidney and bone effects of low dose cadmium exposure in Sweden," Agneta Akesson, Ph.D. Cadmium is a toxic environmental pollutant that all are exposed to via foods considered healthy, such as whole grains, vegetables and potatoes. High cadmium exposure is well-known to cause kidney and bone damage but these associations at low-level cadmium exposure need further attention and the critical exposure level in the general population needs to be defined. We investigated health effects of cadmium exposure among 820 women, aged 53 to 64 years, within a population-based women’s health survey in southern Sweden (WHILA) (71% response rate). The area has no known historical cadmium contamination. Specifically, we assessed the associations between cadmium exposure and tubular and glomerular function, forearm bone mineral density, and markers of bone metabolism. We also calculated the benchmark dose, assessed serum 1,25 dihydroxy vitamin D3 and fracture incidence. Associations were evaluated in multiple linear regression analysis including information on the possible confounders or effect modifiers. The exposure levels were generally low; the median urinary cadmium was 0.52 µg/L adjusted to the mean urinary density (1.015 g/mL), corresponding to 0.67 µg/g creatinine and blood cadmium was 0.38 µg/L. Our results showed associations related to negative effects of low level cadmium exposure on tubuli, glumeruli and bone. Cadmium seemed to potentiate diabetes-induced effects on kidney as well as postmenopausal bone resorption. The obtained benchmark doses (95% lower confidence bounds; BMDL) of urinary cadmium were lower (0.4-0.5 µg/L at 5% additional risk) than the critical concentrations previously reported. The BMDL for glomerular effects was only slightly higher (0.55 µg/L) than that for tubular effects. Although these effects were small, they should be considered as early signals of potentially more adverse health effects. “Factors influencing dose-response relationships of cadmium in humans - diabetes, metallothionein and metallothionein antibodies," Taiyi Jin, Ph.D. Cadmium, an environmental pollutant, causes a number of adverse health effects, particularly toward the kidneys. In this study, we focus on the relationships between MT-Ab, urinary cadmium (UCd) and kidney function, in a Chinese diabetic population. The results show that tubular biomarker, urinary  2-microglobulin (UB2M) increased significantly following the elevation of MT-Ab and urinary cadmium; however, glomerular biomarker urinary albumin (UALB) did not display such a pattern. Those patients with tubular but not glomerular damage have highest UB2M，MT-Ab, and UCd levels compared with those subjects without renal dysfunction and those with glomerular but not tubular dysfunction. In both gender subjects, UB2M statistically correlate with UCd (r = 0.25 and 0.24, P < 0.05) and MT-Ab (r = 0.21 and 0.21, P < 0.05), while UCd did not show any significant correlation with MT-Ab (P > 0.05). After adjusting for potential confounding covariates, logistic regression only fasting capillary blood glucose(FCG) had a significant effect on glomerular dysfunction, and the risk increase 2.75 times (95%CI: 1.12-6.74) when FCG was higher that 6.1 mmol/L. However, MT-Ab levels, UCd levels, and smoking habits significantly increase the risk to develop tubular dysfunction within this diabetic population. The odds ratios (OR) for MT-Ab levels, UCd levels, and current smoking were 5.56 (95% CI: 2.25-13.73), 3.34 (95% CI: 1.17-9.53), and 3.51 (95% CI: 1.14-10.80), respectively. This study proved MT-Ab could potentate tubular dysfunction among diabetic subjects, and those patients with high levels MT-Ab were more prone to develop tubular damage.

Molecular mechanisms of metal induced disease Chair: Max Costa, PhD “Changes In Dietary Iron Levels Affect Brain Manganese Accumulation And Distribution," Michael Aschner, MD, PhD Occupational exposure to manganese (Mn) has been associated with the onset of psychological and motor symptoms in some individuals, leading to a phenotype called manganism. Brain Mn accumulation has also been documented in patients with liver failure, those receiving parenteral nutrition or individuals with iron (Fe) deficiency. As it is known that Mn and Fe are transported by the same proteins, we wanted to determine whether changes in dietary Fe levels alter the brain Mn deposition patters. Previous data from magnetic resonance imaging (MRI) and atomic absorption (AA) spectroscopic techniques demonstrated early regionally specific and latter more global bran Mn accumulation in treated populations compared to controls. In the current study, two groups of adult male Sprague-Dawley rats were fed either Fe-deficient (3 mg Fe/kg) or Fe-supplemented (30 mg Fe/kg) chow for 14 weeks. Both groups were also given weekly intravenous injections of 3 mg Mn/kg. Animals were weighed weekly, a measure of general health. At the conclusion of the study, brains were removed and dissected into various regions (cerebellum, brain stem, midbrain, hippocampus, striatum or cortex) and the amount of Mn and Fe determined. Data was compared to control and Mn-treated animals from a previous study. Fe-supplemented animals weighed significantly less than other groups throughout the course of the study (one-way repeated measure ANOVA, p < 0.05). Atomic absorption spectroscopy indicated no difference in brain Mn or Fe levels between the Fe-deficient or Fe-supplemented groups. However, both groups showed an increase in brain Mn levels in the cerebellum, brain stem, striatum and cortex compared to control and Mn-treated cohorts (p < 0.05). Interestingly, there was a trend to decreased brain Fe levels in both Fe-modulated groups. This reached statistical significance in the Fe-deficient populations in the cerebellum, brain stem, midbrain and cortex (p < 0.05) compared to control. These data, which will also be correlated with MRI results, suggest that any change in dietary Fe levels exacerbates brain Mn accumulation, and alters normal brain Fe distribution in multiple discrete brain regions. This work was supported by Dept of Defense, Manganese Research Health W81XWH-05-1-0239 project 04149002 (MA and VF), and NIEHS ES 10563 (MA). “Molecular Carcinogenicity of Nickel Ions targets HIF-1 Prolyl Hydroxylases and Histone-3 Lysine 9 Demethylases,” Max Costa, PhD Both soluble and insoluble Nickel compounds have been implicated as carcinogens. Ni ions compete with Fe transport and are taken up into cells in place of Fe via the Divalent Metal Ion transporter, resulting in a depletion of cellular Fe. HIF-1 alpha transcription factor is stabilized and then activates transcription of its numerous target genes. ( VEGF, Glucose transporters, glycolytic enzymes etc.) The primary mechanism by which Ni ions stabilize and persistently activate HIF-1 alpha is by inhibition of Fe, Oxoglutarate, ascorbic acid dependent dioxygenases that regulate the stability and transcriptional activity of HIF-1 alpha. ( PHD1-3 and FIH) Ni ions are believed to displace and deplete Fe from these enzymes. Hypoxia and Ni ion exposure result in a global loss of histone acetylation and an increase in the methylation of H3K9 resulting in a loss of DHFR and Mlh1 ( mismatch repair) expression in nucleosomes having H3K9 dimethylation as determined by the CHIP assay. Recent experiments have demonstrated the existence of a H3K9 demethylase that shares similarity to the Fe, Oxoglutarate and ascorbic acid dependent dioxygenases. We hypothesized that Ni ions inhibited these demethylases causing enhanced global H3K9 methylation which will lead to DNA methylation and gene silencing. We have expressed recombinant JHDM2A ( H3K9-demethylase) and the 50% inhibitory level for Ni ions is 25 uM. In contrast purified Aconitase is not significantly inhibited at 10mM Ni ions in vitro. Ni ions favor binding the JMJC Fe containing domain of these dioxygenase enzymes and we hypothesize that all of these enzymes represent major targets for Ni ions in the cell. In particular the stabilization of HIF-1 alpha and histone methylation contribute to Ni ion carcinogenicity. “Reprogramming of histone marks by chromium," Michael Schnekenburger, PhD, Li Peng, PhD, and Alvaro Puga, PhD Hexavalent chromium, a carcinogenic metal often found in the environment, is among the top 20 hazardous substances in the Priority List of Hazardous Substances and is commonly found as an environmental co-contaminant with polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P). These two agents co-exist naturally and exposure to their binary mixtures causes multiple adverse health effects that do not show additive or synergistic associations. As an occupational hazard, more than one million workers in the USA alone are exposed to chromium (VI) in the work place. Chromium exposure alters inducible gene expression, forms chromium-DNA adducts and chromium-DNA cross-links, and disrupts transcriptional activator/coactivator complexes. In addition, chromium has been associated with the incidence of birth defects and congenital malformations in wildlife and, to a lesser extent, in humans. We have shown that chromium blocks B(a)P-inducible gene expression by interfering with the assembly of productive transcriptional complexes at the promoters of over 50 different Ah receptor (AHR)-inducible genes involved in a variety of signal transduction pathways. These effects of chromium are the result of the inhibition of critical chromatin remodeling steps necessary for gene transactivation, including release of complexes of DNA methyl transferase-1 (DNMT-1) and histone deacetylase-1 (HDAC1) from the promoters of affected genes, such as the cytochrome P450 Cyp1a1 gene, and subsequent recruitment of p300. Chromium-induced Cyp1a1 repression is inhibited by 5-aza-deoxycytidine, even though no DNA methylation of a canonical CpG island in the promoter of this gene can be detected. Chromatin immunoprecipitation assays show that Cr exposure leads to an inhibition of critical histone marks connected with B(a)P-mediated induction of gene expression, such as histone H3 S10 phosphorylation, H3 K4 tri-methylation, and both H3 and H4 acetylation. These data help develop an understanding of the molecular mechanisms underlying the toxicity associated with environmental chromium exposures and provide an experimental approach to the analysis of epigenetic modifications induced by chromium. Supported by NIEHS grants P30 ES06096, 2R01 ES06273 and 1R01 ES10807. “Arsenic cardiomyopathy and molecular mechanism,” Y. James Kang, DVM, PhD Arsenic trioxide is highly effective in the treatment of acute promyelocytic leukemia (APL). In September 2000, the Trisenox brand of arsenic trioxide for the treatment of relapsed and refractory APL was approved in the United States. A recent clinical report has shown a serious ventricular tachycardia at the therapeutic doses of arsenic trioxide in APL patients. To investigate the cardiotoxic effect of arsenic trioxide, a mouse model of arsenic cardiotoxicity was used. Mice were injected intraperitoneally with arsenic trioxide in 5 mg/kg/d for 30 days, a dose regiment that has been shown to produce plasma concentrations of arsenic within the range of those present in arsenic-treated APL patients. Analysis of myocardial function revealed that arsenic caused a significant decrease in the maximum rate of rise in intraventricular pressure during ventricular contraction (MAX dP/dt), and significant increases in the end diastolic pressure and ventricle minimum diastolic pressure. In response to b-adrenergic stimulation by isoproterenol the arsenic-treated heart did not show increase in MAX dP/dt, which was observed as a stress response in the saline-treated controls. The functional alterations were accompanied by cardiomyopathy as revealed by histopathological and ultrastructural examination. Furthermore, arsenic caused myocardial apoptosis, as determined by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, which was confirmed by caspase-3 activation detected by enzymatic assay. The arsenic cardiotoxic effects can be prevented if metallothionein, a thiol-rich small protein, is elevated in the heart of transgenic mice. Our study thus demonstrates that arsenic trioxide, in a dose that could produce clinically comparable serum concentrations to those observed in humans, causes cardiotoxicity. The arsenic cardiac toxicity is related to its interaction with intracellular thiols. Therefore, elevation of small thiol-protein, metallothionein, greatly attenuated arsenic cardiotoxicity.

Is Copper Involved in Carcinogenesis/ Carcinostatics? Chair: Hiroko Kodama, MD, PhD and Norikazu Shimizu, MD, PhD “Carcinogenesis in LEC rat and patients with Wilson disease," Norikazu Shimizu, MD Wilson disease is an autosomal recessive disorder resulting from the defective functioning of copper transport P-type ATPase (ATP7B). It reveals a reduction in the rate of copper incorporation into ceruloplasmin, and a reduction in the biliary excretion of copper. Copper accumulates in the liver, brain, cornea, kidney and others. The major clinical signs and symptoms of the disease are cirrhosis, extrapyramidal signs and Kayser-Fleischer rings. The typical biochemical features of these patients are low serum ceruloplasmin levels and high urinary copper excretion. The hepatic copper content increases remarkably. Prognosis of Wilson disease is very poor without treatment. However, after the introduction of copper chelating agents, the patient’s prognosis improves dramatically. The Long-Evans cinnamon (LEC) rats, which are a mutant inbred strain isolated from Long-Evans rats, develop hepatitis and hepatocellular carcinoma in an autosomal recessive pattern of inheritance. Approximately 40% of LEC rats die of fulminant hepatitis, and remaining 60% of LEC rats survive and develop hepatocellular carcinoma. They also demonstrate many clinical and biochemical features of Wilson disease, including a low serum ceruloplasmin level, a high urinary copper excretion and remarkably increased copper content in the liver. A partial deletion at 3’ end of atp7b, the rat gene homologous to ATP7B, has been identified in the LEC rats. The LEC rats are a rodent model for Wilson disease. Although human hepaticellular carcinoma is a well-recognized complication of cirrhosis, a relationship has not been recognized between hepatocellular carcinoma and Wilson disease. Probably, the Wilson disease patients died before reaching an age at which hepatocellular carcinoma would have developed. Authors expect that the prevalence of liver cancer in Wilson disease may increase as survival improves. The LEC rats will serve as an important model of investigation about the mechanism and prevention for this problem. “The effects of Copper on Carcinoma Development and Angiogenesis," Masaaki Ebara, MD Although hepatitis C virus (HCV) is a major etiologic factor in hepatocellular carcinoma (HCC) in Japan and some European countries, the mechanism leading to HCC has not yet been elucidated. We have reported that (1) Hepatic Cu content increases with progression of hepatic fibrosis but not hepatic Fe and Zn contents. (2) Accumulation of Cu in HCC, especially in small tumors (<40 mm), is greater than that in the surrounding liver parenchyma. (3) Cu exists in the form of Cu-metallothionein (Cu-MT) in HCC, and this Cu-MT produces hydroxyl radicals when H2O2 is produced, according to a study of Long-Evans rats that develop HCC spontaneously. We have suggested that Cu accumulation in the liver parenchyma may be closely related to hepatocarcinogenesis. On the basis of these results, we investigated the relationship between trace metals and the prevalence of HCC in patients with chronic hepatitis or hepatic cirrhosis caused by HCV. We measured the contents of Cu, Fe, and Zn in HCC tissue (n = 112), dysplastic nodules (n = 7), and liver parenchyma in patients with (n = 112) and without (n = 12) HCC. Metals were quantified in thin-needle biopsy specimens using the particle-induced X-ray emission method (PIXE). Cu level in liver parenchyma was higher in patients with HCC than in those without HCC (p < 0.01), while there was no such difference in hepatic Fe. In patients with hepatic cirrhosis, Cu content in the liver parenchyma was higher in the presence of HCC than in its absence (p < 0.05). Multiple regression analysis showed that the only factor significantly associated with the coexistence of HCC in HCV-positive patients with chronic liver disease was the Cu level in the liver parenchyma. In conclusion, hepatic copper overload may contribute to the development of HCC in HCV-positive patients with chronic hepatitis or cirrhosis. However, further studies including a molecular biological approach and elucidation of DNA damage in hepatocytes caused by hydroxyl radicals are required to elucidate the complete mechanism of carcinogenesis. “Copper Transporters and Carcinostatics," Stephen Howell, PhD There is now a large body of evidence to indicate that the copper (Cu) transporters CTR1, ATP7A and ATP7B regulate the cellular pharmacology and cytotoxicity of cisplatin (DDP), and that these proteins can mediate acquired DDP resistance. Cells that have acquired resistance to cisplatin (DDP) demonstrate cross-resistance to copper (Cu) and vice versa. The cross-resistance between DDP and Cu is characterized by parallel changes in Cu and DDP accumulation and altered expression of the Cu efflux transporters ATP7A and ATP7B. Yeast, mouse and human cells engineered to alter the expression of CTR1, ATP7A or ATP7B exhibit altered sensitivity to both Cu and DDP. Detailed studies of uptake and efflux indicate that each protein individually can alter the cellular pharmacology of DDP, and in some cases the DDP analogs carboplatin and oxaliplatin. The major Cu influx transporter CTR1 appears to be the primary transporter mediating the initial influx of DDP. Knockout of CTR1 in either yeast or mammalian cells markedly reduces their ability to accumulate either Cu or DDP. Recent studies have demonstrated that DDP triggers the internalization and degradation of CTR1 via a process that involves macropinocytosis, and that DDP is much more potent than Cu in down-regulating CTR1 expression. While other transporters may also participate in the influx and efflux of the platinum drugs, available evidence supports the concept that DDP enters the cell, is distributed within the cell, and is exported by mechanisms that evolved to manage Cu homeostasis. “Possible association of Wilson’s disease with hepatocellular carcinoma," Yan-Hong Gu, PhD Wilson’s disease (WD) is a hereditary disorder of copper accumulation that results in abnormally high levels of copper in the body, primarily in the liver. Hepatic copper accumulation can lead to chronic active hepatitis, cirrhosis, and fulminant hepatic failure. Although WD causes liver cirrhosis, hepatocelluar carcinoma (HCC) is rare in patients with WD, compared to that of hepatitis C virus-related cirrhotic patients. However, a Long-Evans rat with cinnamon-like coat color (LEC) rat, an animal model for this disease, is known to exhibit HCC, suggesting that copper accumulation in the liver may be associated with carcinoma. There is a low incidence of HCC in patients with WD, but this may be attributable to the significantly shortened life expectancy in untreated patients. The effect of elevated hepatic copper levels on oncogenesis is still unclear. Our studies indicate that the accumulation of copper is not homogeneous in the liver of patients with WD, but is rather 1.4 to 3.6 fold higher in the right lobe than the left lobe. To examine the association between hepatic copper load and the development of carcinoma, we examined the localization of HCC in patients with previously reported WD. Twenty-three patients with WD-associated HCC were reported from 1959 to 2007. All of the patients had hepatic cirrhosis. The mean ages at diagnosis of WD and HCC were 33.8 years (6 to 66) and 37.9 years (12 to 66), respectively. Serological tests for anti-HCV antibody, HCV RNA, or both, were described in 5 of the 23 patients and were negative in all cases. Tests for HBV were performed in 13 of the 23 patients, and only one patient was positive for anti-HBs and anti-HBc antibody. From the reports with description of the position of HCC, HCC was found in the right hepatic lobe in 10 of 15 patients, and in left lobe in 4 of 15 patients. From these results, we will discuss the association of the accumulation of copper in the liver with carcinogenesis.

Zinc and the common cold Chair: Monica Nordberg, PhD “Zinc and the common cold: aspects of solution chemistry," George Eby Common colds were shortened by 7 days in a 1984 clinical trial using zinc gluconate throat lozenges each 2 hours. Between then and now, other double-blind, placebo-controlled clinical trials showed widely varying results. Re-analysis of these trials presents solution chemistry methods to elucidate differences in efficacy. Statistically significant correlation was shown between total daily dosages of positively charged zinc species and reductions in median (p = 0.005) and mean duration (p < 0.02) of common colds in these trials. Positively charged zinc species could shorten colds by 5 to 7 days, neutrally charged species had no effect on duration or severity and negatively charged species could lengthen colds by up to 4.5 days. The biologically closed mouth-nose electric circuit influences the incidence of common cold occurance. “Zinc, ICAM-1, and the common cold," Ananda Prasad, MD, PhD Adults and children in the United States experience two to six colds per year. Evidence that zinc is an effective therapy for colds is inconsistent. We conducted a study to test the efficacy of zinc acetate lozenges in reducing the duration of symptoms of the common cold. We conducted a randomized, double-blind, placebo-controlled trial. Fifty ambulatory volunteers were recruited within 24 h of developing symptoms of the common cold. Participants took one lozenge containing 13.3 mg of zinc as acetate or placebo every 3 hours while awake