Richard Beger, Ph.D. 

Branch chief, Center for Metabolomics
Division of Systems Toxicology
National Center for Toxicological Research
Jefferson , AR

Background:

M.S., Marquette University
Ph.D., Purdue University
FDA Experience - 10 years

Research Interests:

Metabolomics technologies offer noninvasive preclinical and clinical diagnostic potential without added risk of exposure to ionizing radiation. The research initiatives of the Center for Metabolomics focus on FDA Critical Path initiatives to develop translational biomarkers of disease, toxicity, and susceptibility.  The Center also plays a crucial role in the FDA voluntary program to evaluate metabolics data submitted to the Agency with regulatory submission packages.  The metabolomic facility uses state of the art NMR and MS equipment to analyze metabolites in biofluid and tissue samples from preclinical and clinical studies.c The NMR lab is equipped with a Bruker 600 MHz NMR that has a cryoprobe and a magic angle spinning (MAS) probe. The Center fo Metabolomics recently added a Waters UPLC-Qtof Premier mass spectrometer system and an Agilent GC mass spectrometer system that will be used to evaluate lipids in biosamples from toxicity and personalized medicine studies.  Initial studies were focused on the development of translational biomarkers of acute liver and kidney toxicity.  Newer projects are evaluating non-invasive biomarkers of susceptibility to hepatotoxins.

Proposed Research Project for FDA Fellow:

The selected fellow will participate in all phases of metabolomic studies of the Center and will develop an understanding of the role of this emerging science in the regulatory environment.

Selected Recent Publications:

1. Beger R D, Holland R D, Sun J, Schnackenberg L K, Devarajan P, Dent C, Portilla D. Metabonomics of acute renal failure in children during cardiac surgery. Pediatric Nephrology 23, 977-984, 2008
2. Espandiari P, Rosenzweig B, Sun J,Schnackenberg L, Holland R D, Vaidya V S, Herman E H, Miller T J, Knapton A, Goering P, Brown R, Johnson A, Bonventre J V, Thompson K, Pine P S, Beger R D, Weaver J,  J Hanig. Characterization of a Rodent Model for Pediatric Nephrotoxicity.  Tox. Sci.99: 637-648, 2007.
3. Schnackenberg L K, Jones R C, Thyparambil S, Taylor J T,  Han T, Tong  W, Hansen D K,  Fuscoe J C, Edmondson R D, Beger R D, and Dragan Y P. An Integrated Study of Acute Effects of Valproic Acid in the Liver Using Metabonomics, Proteomics, and Transcriptomics Platforms. OMICS. 10: 1-14, 2006.
4. Portilla D, Li S, Nagothu K K, Megyesi J, Schnackenberg L, Safirstein R L, Beger R D. Metabolomic study of cisplatin-induced nephrotoxicity. Kidney Int. 69: 2194-2204, 2006.
5. Beger R D, Schnackenberg L K, Li D, and Dragan Y. Metabonomic Models of Human Pancreatic Cancer using 1D Proton NMR Spectra of Organic Plasma Extracts.  Metabolomics 2: 125-134, 2006.

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Frederick A. Beland, Ph.D. 

Division of Biochemical Toxicology
National Center for Toxicological Research
Jefferson, AR

Background:

B.A., Colorado College
M.S., Montana Sate University
Ph.D., Montana Sate University
FDA Experience - 33 years

Research Interests:

The role of genetic and epigenetic changes in the etiology of cancer. Classically, the development of cancer in human has been viewed as disease driven by the progressive genetic alterations. However, current evidence indicates that not only genetic but also epigenetic alterations are similarly important in carcinogenesis. Presently, cancer is recognized as both genetic and epigenetic disease, which is evident from every aspect of tumor biology, and genetic and epigenetic components cooperate at every stage of cancer development.

It is widely accepted that carcinogenesis is initiated by permanent heritable changes in the genome caused by endogenous and environmental agents. However, initiation alone is not sufficient for tumor formation; rather it is a necessary prerequisite for tumor development. Additionally, genetic alterations alone cannot explain the extremely diverse phenotypic changes observed in preneoplastic cells at the promotion and progression stages of carcinogenesis as well as in neoplastic cells. This has led to a suggestion that evolution of preneoplastic cells during promotion and progression stages may be driven primarily by epigenetic mechanisms. Furthermore, it has been proposed that epigenetic alterations during carcinogenic process may precede and provoke genetic changes suggesting that epigenetic events may be primary events while genetic changes may be a consequence of disrupted epigenomic state.

Many important questions in the field of carcinogenesis remain to be answered. Among them, questions whether or not carcinogens cause epigenetic changes during carcinogenesis and the precise relationship between carcinogen-induced genetic changes and epigenetic alterations in carcinogenic process, are the most important.

In view of these considerations, the goal of these proposal is to identify the exact role of genotoxic and epigenetic alterations in rat liver carcinogenesis induced by a variety of chemicals and drugs (furan, tamoxifen, antiretroviral drugs) that are important to the FDA.

Proposed Research Project for FDA Fellow:

The FDA Fellow participating in this research will employ novel molecular biochemical approaches to evaluate genotoxic and epigenetic changes at early stages of liver carcinogenesis induced by chemicals and drugs of interest to the FDA. Changes in DNA methylation will be determined by direct measurement of the 5-methcytosine in DNA using HPLC-ES-MS/MS and by methylation-specific microarray profiling. The alterations in histone modification profiles will be analyzed by western blot and chromatin immunoprecipitation techniques. The methylation status of differentially methylated DNA fragments will be confirmed by other independent techniques (single nucleotide primer extension, methylation-specific PCR, bisulfite sequencing); b) determining the gene expression profile by microarray technology with subsequent real-time RT-PCR verification. Genotoxic changes will be monitored by assessing DNA adduct formation using HPLC-ES-MS/MS and by mutation analyses. Upon the completion of the study, we expect to define the mechanistic role of genotoxic and epigenetic mechanisms in cancer initiation, and provide a solid background to use these alterations as biomarkers for early detection of potential carcinogenic agents.

Selected Recent Publications:

1. Tryndyak, V.P., L. Muskhelishvili, O. Kovalchuk, R. Rodriguez-Juarez, B. Montgomery, S.A. Ross, M.I. Churchwell, F.A. Beland, and I.P. Pogribny. Effect of long-term tamoxifen exposure on genotoxic and epigenetic changes in rat liver: implications for tamoxifen-induced hepatocarcinogenesis. Carcinogenesis, 27, 1713-1720 (2006).
2. Tryndyak, V.P., O. Kovalchuk, L. Muskhelishvili, B. Montgomery, R. Rodriguez-Juarez, S. Melnyk, S.A. Ross, F.A. Beland, and I.P. Pogribny. Epigenetic reprogramming of liver cells in tamoxifen-induced rat hepatocarcinogenesis. Mol. Carcinogenesis, 46, 187-197 (2007).
3. Pogribny, I.P., V.P. Tryndyak, A. Boyki, R. Rodriguez-Juarez, F.A. Beland, and O. Kovalchuk. Induction of microRNAome deregulation in rat liver by long-term tamoxifen exposure. Mutation Res., 619, 30-37 (2007).
4. Kovalchuk, O., V.P. Tryndyak, B. Montgomery, A. Boyko, K. Kutanzi, F. Zemp, A.R. Warbritton, J.R. Latendesse, F.A. Beland, and I.P. Pogribny. Estrogen induced rat breast carcinogenesis is characterized by alterations in DNA methylation, histone modifications, and aberrant microRNA expression. Cell Cycle, 6, 2010-2018 (2007).
5. Pogribny, I.P., T.V. Bagnyukova, V.P. Tryndyak, L. Muskhelishvili, R. Rodriguez-Juarez, O. Kovalchuk, T. Han, J.C. Fuscoe, S.A. Ross, and F.A. Beland. Gene expression of profiling reveals underlying molecular mechanisms of the early stages of tamoxifen-induced rat hepatocarcinogenesis. Tox. Appl. Pharm., 225, 61-69 (2007).
6. Pogribny, I.P., I. Rusyn, and F.A. Beland. Epigenetic aspects of genotoxic and non-genotoxic hepatocarcinogenesis: studies in rodents. Environ. Molecular Mutagenesis, 49, 9-15 (2008).
7. Pogribny, I.P., V.P. Tryndyak, S.A. Ross, and F.A. Beland. Differential expression of microRNAs during hepatocarcinogenesis induced by methyl deficiency in rats. Nutrition Rev. 66 (Suppl. 1), S33-S35 (2008).
8. Bagnyukova, T.V., V.P. Tryndyak, B. Montgomery, M.I. Churchwell, A.R. Karpf, S.R. James, L. Muskhelishvili, F.A. Beland, and I.P. Pogribny. Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene. Carcinogenesis, 29, 638-646 (2008).
9. Tyryndyak, V.P., S.A. Ross, F.A. Beland, and I.P. Pogribny. Down-regulation of the microRNAs miR-34a, miR-127, and miR-200b in rat liver during hepatocarcinogenesis induced by a methyl-deficient diet. Mol. Carcinog., in press.
10. Bagnyukova, T.V., V.P. Tryndyak, L. Muskhelishvili, S.A. Ross, F.A. Beland, and I.P. Pogribny. Epigenetic silencing of the suppressor of cytokine signaling 1 (Socs1) gene is associated with the STAT3 activation and development of hepatocellular carcinoma induced by methyl-deficiency in rats. Cell Cycle, 7, 3202-3210 (2008).

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John F. Bowyer, Ph.D. 

Research Pharmacologist
Division of Neurotoxicology
National Center for Toxicological Research
Jefferson, AR

Background:

FDA Experience - 26 years

Research Interests:

My research over the past ten years has had a primary focus on the investigation of the molecular mechanisms (gene expression) involved in neurotoxic insults, particularly amphetamine and substituted amphetamines, and the recovery from such insults. Amphetamines are a good model to study the physiological and molecular mechanisms behind neurotoxicity because they can affect multiple neurotransmitter systems and regions of the brain without producing widespread non-specific necrosis. As well, a long-term recovery process occurs which can be investigated using molecular biology tools. The development of these techniques has allowed me to apply them to other types of potential neurotoxic insults (evaluating acrylamide as a neurotoxin, e.g.).

Proposed Research Project for FDA Fellow:

Evaluate the effects of both acute and chronic amphetamine (AMPH) exposure on the vasculature of the rat brain. In particular, we propose to examine vasculature within the parenchyma of three brain regions; the striatum, parietal cortex and the combined piriform and amygdaloid nuclear cortices where AMPH-induced neurodegeneration can occur. As well, we also propose to look at the effects of AMPH on the vasculature associated with pial and arachnoid membranes (part of the meninges) and vasculature of the choroid plexus. The vasculature of the meninges is particularly interesting because it may be associated with the subarachnoid hemorrhage that is often reported in humans abusing amphetamine and methamphetamine (METH). We have already conducted one preliminary study looking the initial effects of a one-day exposure to high doses of AMPH that produce hyperthermia and significant neurotoxicity but do not normally lead to stroke or hemorrhage in rat (Bowyer et al., submitted for publication). The results of this study clearly show that AMPH can produce profound changes in genes associated with vascular tone, damage and angiogenesis. We now propose further experiments involving AMPH exposures to determine the alterations in vascular gene expression after; 1) a one-day 4-dose exposure that produces hyperthermia [full time course of effects not just 1st day], 2) a single exposure to a very high dose, 3) a 9 day exposure that does not produce significant hyperthermia and 4) a 1 month exposure to AMPH included in the drinking water.

Selected Recent Publications:

1. Bowyer, J.F., Harris, A.J., Delongchamp, R.R., Jakab, R.L., Miller, D.B., Little, A.R. and O’Callaghan, J.P. (2004) Selective changes in gene expression in cortical regions sensitive to amphetamine during the neurodegenerative process. NeuroToxicology, 25, 555-572.
2. Fang, H., Tong, W., Shi, L. Jakab, R.L. and Bowyer, J.F. (2004) Classification of cDNA array genes that have a highly significant discriminative power due to their unique distribution in four brain regions. DNA and Cell Biol.,23, 661-674. 3.
3. Bowyer, J.F., Delongchamp, R.R. and Jakab, R.L. (2004) Glutamate N-methyl-D-aspartate and dopamine receptors have contrasting effects on the limbic versus the somatosensory cortex with respect to amphetamine-induced neurodegeneration. Brain Res.1030, 234-246.
4. 4. Bowyer, J.F. and Schmued, L.C. (2006) Fluoro-Ruby labeling prior to an amphetamine neurotoxic insult shows a definitive massive loss of dopaminergic terminals and axons in the caudate putamen. Brain Res.1030, 234-246.
5. Bowyer, J.F. and Ali, S. (2006) High Doses of Methamphetamine that Cause Disruption of the Blood Brain Barrier in Limbic Regions Produce Extensive Neuronal Degeneration in Mouse Hippocampus. Synapse 60, 521-532.
6. Bowyer, J.F., Pogge, A.R., Delongchamp, R.R., O’Callaghan, J.P., Patel, K.M., Vrana, K.E. and Freeman, W.M. (2007) A Threshold Neurotoxic Amphetamine Exposure Inhibits Parietal Cortex Expression of Synaptic Plasticity-Related Genes. Neuroscience 144, 66-76.
7. John F. Bowyer, J.F., Robinson, B. Ali, S. and Schmued, L.C. (2008) Neurotoxic -related changes in tyrosine hydroxylase, microglia, myelin, and the blood-brain barrier in the caudate-putamen from acute methamphetamine exposure. Synapse, 62, 193-202.
8. Bowyer, J.F., Latendresse, J.R., Delongchamp, R.R., Muskhelishvili, L., Warbritton, A.R., Thomas, M., Tareke, E., McDaniel, L.P., and Doerge, D.R. (2008) The effects of sub-chronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus-pituitary-thyroid axis of male fischer 344 rats. Toxicol. & Appl. Pharmacol., 230, 208-215.
9. Bowyer, J.F., Latendresse, J.R., Delongchamp, R.R., Warbritton, A.R., Thomas, M., Divine, B., and Doerge, D.R. (Submitted, 10-21-2008) Subchronic Acrylamide Exposures that Produce Movement Impairment and Weight Loss Minimally Affect mRNA Expression Levels and Do Not Necessarily Produce Overt Histological Signs of Neurotoxicity in the Striatum, Substantia Nigra and Somatosensory Cortex. (submitted, Toxicol. & Appl. Pharmacol.)
10. MonzyThomas, M., George, N.I., Patterson, T.A. and Bowyer, J.F. (Submitted 11-1-2008) Amphetamine- and environmentally-induced hyperthermia differentially alter the expression of genes regulating vascular tone, trauma and angiogenesis in the meninges and associated vasculature. (submitted, Synapse).

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Deborah K. Hansen, Ph.D. 

Division of Personalized Nutrition and Medicine
National Center for Toxicological Research
Jefferson, AR

Background:

Ph.D., Indiana University
FDA Experience - 24 years

Research Interests:

My research interests are in the area of developmental toxicology, particularly the effects of nutrition on normal and abnormal embryonic development. Furthermore, I’m interested in the interaction of the genetic constitution of the embryo and environmental influences, such as chemical (or drug) exposure.

Proposed Research Project for FDA Fellow:

The purpose of this study is to determine if marginal biotin deficiency has long term consequences in mice. We plan to use at least two different strains of mice that differ in some of the biotin metabolizing genes as well as two different levels of biotin deficiency. We will develop a state of marginal deficiency during pregnancy by feeding chow with egg white added to bind biotin. We will monitor biomarkers of biotin deficiency, such as urinary excretion of 3-HIA, during pregnancy. After birth, mice from each dietary group will be subdivided into 2 subgroups, one of which will receive chow with normal biotin levels and the other will continue to consume chow with egg white added. Mice will be fed these diets until the age of 9 - 12 months. At various times after birth, blood and urine will be collected from mice, and clinical chemistry and metabolomics profiles may be determined. Reproductive capacity of the mice will be tested by breeding them to members of the opposite sex who have been fed control chow. Various behavioral endpoints will also be tested during several life stages. Animals will be sacrificed at various times during the study, and the biotinylation status of histones will be examined. It is anticipated that early effects on glucose utilization and metabolism caused by the decreased activity of the biotin-requiring carboxylase enzymes will have long term consequences on the mice. Such consequences may include the development of diabetes or other metabolic imbalances as well as possibly behavioral abnormalities. Additionally, it is anticipated that biotinylation of histones will be altered possibly leading to altered imprinting of genes during development.

Selected Recent Publications:

1. Xiao, X., Sun, X.-L., Mackins, J.Y., Tang, Y.-S., Hansen, D.K. and Antony, A.C. Isolation and characterization of a folate receptor mRNA-binding trans-factor from human placenta. J. Biol. Chem., 276:41510-41517, 2001.
2. Tabacova, S., Kimmel, C.A., Wall, K. and Hansen, D. Atenolol developmental toxicity: Animal-to-human comparisons. Birth Defects Research (Part A) 67:181-192, 2003.
3. Hansen, D.K., Streck, R.D. and Antony, A.C. Antisense modulation of the coding or regulatory sequences of folate receptor (Folate Binding Protein-1, [Folbp1]) in mouse embryos leads to neural tube defects. Birth Defects Research (Part A), 67: 475-487, 2003.
4. Mock, D.M., Mock, N.I., Stewart, C., LaBorde, J.B. and Hansen, D.K. Marginal biotin deficiency is teratogenic in CD-1 mice. J. Nutr., 133: 2519-2525, 2003.
5. Xiao, S., Hansen, D.K., Horsley, E.T.M., Tang, Y.-S., Khan, R.A., Stabler, S.A., Jayaram, H.N. and Antony, A.C. Maternal folate-deficiency results in selective up-regulation of folate receptors and heterogeneous nuclear ribonucleoportein-E1 associated with multiple subtle aberrations in fetal tissues. Birth Defects Res. A 73:6-28, 2005.
6. Ferguson, S.A., Berry, K.J., Hansen, D.K., Wall, K.S., White, G. and Antony, A.C. Behavioral effects of prenatal folate deficiency in mice. Birth Defects Res. A 73:249-252, 2005.
7. Sealey, W.S., Stratton, S.L., Mock, D.M. and Hansen, D.K. Molecular changes induced by marginal maternal biotin deficiency in mice. J. Nutr.135:973-977, 2005.
8. Schnackenberg, L.K., Jones, R.C., Thyparambil, S., Taylor, J.T. Taylor, Han, T., Tong,W., Hansen, D.K., Fuscoe, J.C., Edmondson, R.D., Beger, R.D. and Dragan,Y.P. An integrated study of acute effects of valproic acid in the liver using metabonomics, proteomics, and transcriptomics platforms. OMICS 10:1-14, 2006.
9. Center for the Evaluation of Risks to Human Reproduction. NTP-CERHR Expert Panel report on the reproductive and developmental toxicity of genistein. Birth Defects Res. Part B 77:485-638, 2006.
10. Center for the Evaluation of Risks to Human Reproduction. NTP-CERHR Expert Panel report on the reproductive and developmental toxicity of soy formula. Birth Defects Res. Part B 77:280-397, 2006.

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Robert H. Heflich, Ph.D. 

Division of Genetic and Reproductive Toxicology
National Center for Toxicological Research (NCTR)
Jefferson, AR

Background:

Ph.D., Rutgers-The State University of New Jersey
FDA Experience - 27 years

Research Interests:

Dr. Heflich has conducted research in Genetic Toxicology for approximately 30 years. He is presently working on improving FDA regulatory safety assessments by developing rapid, high throughput methods for measuring gene mutation and chromosomal changes.

Proposed Research Project for FDA Fellow:

Nelfinavir (NFV) is a protease inhibitor used in combination with other drugs for the treatment and prophylaxis of HIV-1 infections. During the manufacture of NFV, an error caused a batch of the drug to be contaminated with a relatively low level of ethyl methanesulfonate (EMS). This contamination was not discovered before thousands of patients had been treated with the contaminated NFV. The significance of this exposure is that EMS is a carcinogenic alkylating agent that apparently works through a mutagenic mode of action. The drug company involved in this incident recently submitted a package of data to the FDA/CDER indicating that the genotoxicity of EMS in mice has a nonlinear dose response, exhibiting a threshold dose below which no genotoxicity can be detected. These data, and the argument forwarded by the drug company indicating the absence of risk for the exposed patients, have great significance for regulating mutagenic carcinogens, which have been assumed to have no dose at which some risk for cancer does not exist. The data presented by the company are limited, based mainly on gene mutation responses produced in a single transgenic mouse model. The data also potentially were compromised by an unusually high background mutant frequency in the transgenic mutation assays that were conducted.

In the proposed project, the FDA Fellow will use an in vivo mutation assay that we have recently developed at the NCTR (see references below) to pursue a more thorough investigation of the genotoxicity of EMS. The assay, which uses the endogenous Pig-A gene, has a spontaneous frequency 10-fold below the transgenic system used for the FDA data submission, making it potentially more sensitive. Also, unpublished results indicate that Pig-A mutants are phenotypically neutral in rodent erythroid cells and that mutational responses at low doses accumulate with repeated exposure, making the system ideal for evaluating the effects of chronic exposure to presumed subthreshold doses. Experiments will be conducted in both mice and rats, and responses will be determined for EMS by itself and EMS in combination with NFV. In other experiments, we have found that NFV can potentiate the genotoxicity of another genotoxic agent (AZT), a line of research not pursued by the drug company. The Fellow will be responsible for the design, conduct, interpretation, and reporting the results of the study. The Fellow will gain experience with using targeted research to resolve an FDA regulatory concern.

Selected Recent Publications:

1. Miura, D., V.N. Dobrovolsky, Y. Kasahara, Y. Katsuura, and R.H. Heflich (2008) Environ. Mol. Mutagenesis 49, 614.
2. Miura, D., V.N. Dobrovolsky, R.A. Mittelstaedt et al. (2008) Environ. Mol. Mutagenesis 49, 622.

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Jim Kaput, Ph.D. 

Director, Division of Personalized Nutrition and Medicine
National Center for Toxicological Research
Jefferson, AR

Background:

Ph.D., Colorado State University

Research Interests:

Current nutritional and genetic epidemiological methods yield “risk factors” based on population studies. Risk factors, however, are statistical estimates of the percentage reduction in disease in the population if the risk were to be avoided or the gene variant was not present. These measures are often assumed to apply to individuals who are likely to differ in genetic make-up, lifestyle, and dietary patterns than those individuals in the study population. Developing individual risk factors in light of the genetic diversity of human populations, the complexity of foods, culture and lifestyle, and the variety of metabolic processes that lead to health or disease are significant challenges for personalizing dietary advice for healthy or individuals with chronic disease.

The FDA/NCTR Division of Personalized Nutrition and Medicine focus on several aspects of the health to disease continuum using laboratory animals and studies in humans. These questions we seek to answer are: how does one define health (and not just the absence of disease)? What is the full phenotypic range of laboratory animal and human metabolic profiles? Can we assign individuals into genotype – environment bins that will allow for the selection of lifestyles (diet and activity) to optimize health and prevent or delay the onset of chronic diseases? What is the optimum intake of nutrients in children of all ancestral backgrounds to allow them to reach their full physical and mental potential?

The technologies underlying these initiatives are re-sequencing of candidate genes (see reference 10 below), whole genome scans to determine genetic ancestry (for epistasis analyses), gene expression analyses, laboratory animal models, micronutrient analyses, and development of software tools and databases for nutrient and physical activity analyses. About half of the members of our division are statisticians and mathematicians who collaborate on the design and interpretation of high dimensional datasets. We also collaborate with experts at NCTR, nationally, and internationally in other omic technologies.

Proposed Research Projects for FDA Fellow:

1. The development, research, and application of community based participatory research in the Delta (and other) region of Arkansas. The primary projects under this initiative are to analyze micronutrient requirements through intervention of better foods or vitamin supplements in children, which includes genetic analyses. These projects are conducted under a CRADA with the USDA-ARS in Little Rock (http://www.ars.usda.gov/main/site_main.htm?modecode=62-51-05-00) and in collaboration with the Boys, Girls, and Adults Community Development Center (http://www.bgacdc.net) in Marvell, AR. This project is linked to the International Micronutrient Genetic Project that seeks to determine optimal micronutrient intakes for individuals in different ancestral populations throughout the world. (references 1,6-8,10)
2. An association study of obesity and type 2 diabetes (T2DM) in adults of the Delta counties of Arkansas incorporating nutritional and physical activity assessments, candidate gene analyses, and genomic architecture. This project is in the design phase with initiation expected in fall 2009. Collaborators include the USDA – ARS, BGACDC, UAMS – Delta Area Health and Education Center (http://www.uams.edu/deltaahec/), and the Mid-Delta Community Consortium, Inc, a grantee of the Arkansas Rural Development Network (http://adrdnmdcc.com/). This project is linked with the International T2DM initiative that is developing a harmonized protocol for independent but linked population studies in 22 countries. This distributive science initiative will allow for the comparison and identification of genotype – environment interactions that produce health or lead to T2DM. (references 4,5,6-8, 10)
3. The NCTR Healthy Challenge study. A set of intervention studies that use challenges to homeostasis (e.g., oral glucose tolerance test, physical activity challenges, etc) to assess physiological status in various "health" states. This study includes a weight management module that allows for physiological analyses pre- and post weight change. This project is a collaboration with experts within NCTR, academia, and the biotechnology industry. (references 1,5,7-9)
4. Animal models of chronic disease. We are examining gene – nutrient interactions that influence DNA methylation in utero and by early nutrition and developing models to identify causal and modifier genes involved in T2DM. (reference 2,3)

Selected Recent Publications:

1. Kaput J. and Rodriguez RL 2004. Nutritional Genomics: The next frontier in the post-genomic era. Physiological Genomics, 16, 166-177. Invited Review
2. Kaput J. 2004. Diet-Disease Gene Interactions. Nutrition: The International Journal of Applied and Basic Nutrition Sciences. Nutrition 20, 26 – 31. Invited Review.
3. Kaput J, Klein KG, Reyes EJ, Kibbe WA, Visek WJ, and Wolff G. 2004. Identification of Genes Contributing to the Obese Yellow Avy phenotype: Caloric Restriction, Genotype, Diet x Genotype Interactions. Physiological Genomics 18, 316-324.
4. Kaput J*, Jose M. Ordovas*, Lynn Ferguson*, Ben van Ommen*, Raymond L. Rodriguez*, Lindsay Allen*, Bruce N. Ames*, Kevin Dawson*, Bruce German*, Ronald Krauss*, Wasyl Malyj*, et, al. 2005. The Case for Strategic International Alliances to Harness Nutritional Genomics for Public and Personal Health. British Journal of Nutrition 94, 623 – 632. (* indicates primary authors)
5. Kaput J. Perlina A., Hatipolgu B, Bartholomew A, and Nikolsky Y 2007. Nutrigenomics: Applications to Pharmacogenomics and Clinical Medicine. Pharmacogenomics. 8, 369 - 390.
6. Kaput J. 2007. Developing the Promise of Nutrigenomics through Complete Science and International Collaborations. Forum Nutr. 60, 210 – 224
7. Kaput J. 2008. Nutrigenomics Research for Personalized Nutrition and Medicine. Current Opinions in Biotechnology 19, 110 – 120.
8. McCabe-Sellers B, Lovera D, Nuss H, Wise C, Green B, Clark BS, Teitel C, Ning B, Bogle M, and Kaput J. 2008. Personalizing Research through Community Based Participatory Research and Omics Technologies. In press, Omics, A Journal of Integrative Biology, 12, 263 - 272
9. van Ommen B, Heil S, Keijer J, and Kaput J, 2008. Challenging Homeostasis to Define Biomarkers for Individual Nutrition-Related Health. Molecular Nutrition and Food Research. In press.
10. Kaput J, Cotton R, Al-Aama J, Al-Aqeel A, Auerbach AD, Barash C, Bhak J, Bleoo S, Brookes AJ, Cambon-Thomsen A, Chung YJ, Cutting G, Dalgleish R, den Dunnen,JT, Rosemary Ekong R, Simon Flanagan S, Flicek P, Golubenko M, . Greenblatt MS, Lauren Hardman L, Jung J, Katz, M, Laradi S, Lee Y-S, Macrae F, Maglott DR, Marafie M, Matsubara Y, Messiaen L, Möslein G, Oller de Ramirez AM, Parboosingh J, Povey S, Ramesar RS, Roberts L, Richards S, Savige J, Scott R, Seminara D, Shephard E, Solbakk JH, Tavtigian SV, Taylor G, Utsunomiya J, Watson M. The Human Variome Project Planning Meeting: Spain. Human Mutation, submitted

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Beverly D. Lyn-Cook, Ph.D. 

Senior Research Scientist
Office of the Associate Director of Regulatory Activities
Women’s Health Research Program
National Center for Toxicological Research Jefferson, AR

Background:

Ph.D., Atlanta University
FDA Experience - 21 years

Research Interests:

Dr. Cook's research areas include epigenomics and nutrition and gender differences in adverse drug reactions and diseases. Although genomics play an important role in adverse drug reactions and the development of diseases, it is also becoming increasingly important to understand the role of nutrition in individuals’ response to drugs and susceptibility to diseases. The preceptor has been involved in research examining the effects of micronutrients, dietary agents and their interaction with polymorphisms in critical genes involved in carcinogenesis, metabolism and drug transport. Her laboratory was one of the first to establish a relationship between changes in methylation status and caloric restriction and methylation changes associated with dietary components found in soy. Her laboratory continues to investigate epigenetic regulation of genes involved in Phase II and Phase III detoxification and transport as it relates to drug toxicity and drug-drug interactions. The laboratory is currently investigating polymorphisms in drug transporter genes and inactivation of critical Phase II enzymes such as UDP-glucuronosyltransferases (UGTs) in liver toxicity and pancreatic cancer resistance to various chemotherapeutic drugs. Often clinical applications of new chemotherapeutic drugs are hindered by their low therapeutic index or lack of efficacy in humans. Research in gender differences in expression of drug transporters as it relates to adverse drug reactions are also being conducted in her laboratory.

Proposed Research Projects for FDA Fellow:

FDA Fellows will have an opportunity to work on the research projects discussed above or to work on a current Office of Women Health project examining sex differences in Systemic Lupus Erythematosus (SLE) and the role of polymorphisms in the prolactin gene on individual response to the dietary supplement, dehydroepiandrosterone (DHEA) therapy. Although there is currently no cure for SLE due to multi-factorial etiology in the pathogenesis of the disease, we postulate that a more complete understanding of gene-drug relationships will improve the efficacy of accepted SLE treatment strategies by directing SLE patients to therapeutic modalities tailored to their genetically-determined characteristics. This project in conducted in collaboration with scientists at East Carolina University.

Selected Recent Publications:

1. Starlard-Davenport, A, Lyn-Cook, B.D. and Radominska-Pandya. 2008. Identification of UDP-glucuronosyltransferase 1A10 in non-malignant and malignant breast tissues. Steroids, 73(6):611-621.
2. Starlard-Davenport, S., Lyn-Cook, B.D, and Radominska-Pandya, A. 2008. Novel identification of UDP-glucuronosyltransferase 1A10 as an estrogen-regulated target gene. J.Steroids, 73(1):139-47.
3. Tareke, E., Lyn-Cook, B.D., Robinson, B. and Ali, S.F. 2008. Acrylamide: A dietary carcinogen formed in vivo? J. Agric. Food Chem. 56(15):6020-6023.
4. Xiao, Y., Word, B., Starlard-Davenport, A., Haefele, A., Lyn-Cook, B.D., and Hammons, G.J. 2008. Age and gender affect DNMT 3a and DNMT 3b expression in human liver. Cell Biology and Toxicology 24(3):265-72
5. Haefele, A, Word, B, Yongmei, X, Hammons, G. and B.D. Lyn-Cook. 2007. Indole-3-carbinol (I3C) modulates expression of DNA methyltransferases 1, 3a, and 3b in pancreatic cancer cells: Effects of gender and a novel (C —› T) polymorphism in the promoter region of DNMT 3b. International J. of Cancer Prevention 2(4):245-255.
6. Ruqing, L., Blower, P.E., Pham, A-N., Fang, J., Dai, Z., Wise, C., Green, B., Teitel, C.H., Ning, B., Ling, W., Lyn-Cook, B.D., Sadee, W., and Huang, Y. 2007 Cystine-glutamate transporter SLC7A11 mediates resistance to geldanamycin but not to 17-AAG. Molecular Pharmacology, 72:1-10.
7. Hammons, G.J. and Lyn-Cook, B.D. 2007. Natural products in neuroprotection: Targeting biotransformation enzymes and DNA methyltransferases. J. Environ. Neurosci. Biomed., 1:13-26.
8. Zhengwen, J., Dragin, N., Jorge-Nerbert, L., Martin, M., Guengerich, F.P., Aklillu, E., Ingelman-Sundberg, M., Hammons, G., Lyn-Cook B.D., Kadlubar, F.F., Saldana, S., Sorter, M., Vinks, A.A., Nassr, N., Richter, O., Jin, L., and Nerbert, D.W. 2006 Search for an association between the human CYP1A2 metabolic phenotype. Pharmacogenetics and Genomics, 18:359-367s.
9. Wilson, W., Pardo-Manuel de Villena, F., Lyn-Cook, B.D., Chatterjee, P.K., Bell, T.A.,Detwiler, D.A., Gilmore, R.C., Valladeras, I.C., et al. 2004 Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15. Genomics 84(4):707-714.
10. Lyn-Cook, B.D., Yan-Sanders, Y., Moore, S., Taylor, S., Word, B. and G.J. Hammons. 2006. Increased Levels of NAD(P)H: Quinone Oxidoreductase 1(NQO1) in Pancreatic Tissues from Smokers and Pancreatic Adenocarcinomas: A Potential Biomarker of Early Damage in the Pancreas. Cell Biology and Toxicology 21:1-8.

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Igor P. Pogribny, Ph.D. 

Division of Biochemical Toxicology
National Center for Toxicological Research
Jefferson, AR

Background
M.D., Ivano-Frankivsk Medical University
Ph.D., Kyiv National Medical University
Food and Drug Administration, 1992 to present

Research Interests:

The role of genetic and epigenetic changes in the etiology of cancer. Classically, the development of cancer in human has been viewed as disease driven by the progressive genetic alterations. However, current evidence indicates that not only genetic but also epigenetic alterations are similarly important in carcinogenesis. Presently, cancer is recognized as both genetic and epigenetic disease, which is evident from every aspect of tumor biology, and genetic and epigenetic components cooperate at every stage of cancer development.

It is widely accepted that carcinogenesis is initiated by permanent heritable changes in the genome caused by endogenous and environmental agents. However, initiation alone is not sufficient for tumor formation; rather it is a necessary prerequisite for tumor development. Additionally, genetic alterations alone cannot explain the extremely diverse phenotypic changes observed in preneoplastic cells at the promotion and progression stages of carcinogenesis as well as in neoplastic cells. This has led to a suggestion that evolution of preneoplastic cells during promotion and progression stages may be driven primarily by epigenetic mechanisms. Furthermore, it has been proposed that epigenetic alterations during carcinogenic process may precede and provoke genetic changes suggesting that epigenetic events may be primary events while genetic changes may be a consequence of disrupted epigenomic state.

Many important questions in the field of carcinogenesis remain to be answered. Among them, questions whether or not carcinogens cause epigenetic changes during carcinogenesis and the precise relationship between carcinogen-induced genetic changes and epigenetic alterations in carcinogenic process, are the most important.

In view of these considerations, the goal of these proposal is to identify the exact role of genotoxic and epigenetic alterations in rat liver carcinogenesis induced by a variety of chemicals and drugs (furan, tamoxifen, antiretroviral drugs) that are important to the FDA.

Proposed Research Project for FDA Fellow:

The FDA Fellow participating in this research will employ novel molecular biochemical approaches to evaluate genotoxic and epigenetic changes at early stages of liver carcinogenesis induced by chemicals and drugs of interest to the FDA. Changes in DNA methylation will be determined by direct measurement of the 5-methcytosine in DNA using HPLC-ES-MS/MS and by methylation-specific microarray profiling. The alterations in histone modification profiles will be analyzed by western blot and chromatin immunoprecipitation techniques. The methylation status of differentially methylated DNA fragments will be confirmed by other independent techniques (single nucleotide primer extension, methylation-specific PCR, bisulfite sequencing); b) determining the gene expression profile by microarray technology with subsequent real-time RT-PCR verification. Genotoxic changes will be monitored by assessing DNA adduct formation using HPLC-ES-MS/MS and by mutation analyses. Upon the completion of the study, we expect to define the mechanistic role of genotoxic and epigenetic mechanisms in cancer initiation, and provide a solid background to use these alterations as biomarkers for early detection of potential carcinogenic agents.

Recent Joint Publications:

1. Tryndyak, V.P., L. Muskhelishvili, O. Kovalchuk, R. Rodriguez-Juarez, B. Montgomery, S.A. Ross, M.I. Churchwell, F.A. Beland, and I.P. Pogribny. Effect of long-term tamoxifen exposure on genotoxic and epigenetic changes in rat liver: implications for tamoxifen-induced hepatocarcinogenesis. Carcinogenesis, 27, 1713-1720 (2006).
2. Tryndyak, V.P., O. Kovalchuk, L. Muskhelishvili, B. Montgomery, R. Rodriguez-Juarez, S. Melnyk, S.A. Ross, F.A. Beland, and I.P. Pogribny. Epigenetic reprogramming of liver cells in tamoxifen-induced rat hepatocarcinogenesis. Mol. Carcinogenesis, 46, 187-197 (2007).
3. Pogribny, I.P., V.P. Tryndyak, A. Boyki, R. Rodriguez-Juarez, F.A. Beland, and O. Kovalchuk. Induction of microRNAome deregulation in rat liver by long-term tamoxifen exposure. Mutation Res., 619, 30-37 (2007).
4. Kovalchuk, O., V.P. Tryndyak, B. Montgomery, A. Boyko, K. Kutanzi, F. Zemp, A.R. Warbritton, J.R. Latendesse, F.A. Beland, and I.P. Pogribny. Estrogen induced rat breast carcinogenesis is characterized by alterations in DNA methylation, histone modifications, and aberrant microRNA expression. Cell Cycle, 6, 2010-2018 (2007).
5. Pogribny, I.P., T.V. Bagnyukova, V.P. Tryndyak, L. Muskhelishvili, R. Rodriguez-Juarez, O. Kovalchuk, T. Han, J.C. Fuscoe, S.A. Ross, and F.A. Beland. Gene expression of profiling reveals underlying molecular mechanisms of the early stages of tamoxifen-induced rat hepatocarcinogenesis. Tox. Appl. Pharm., 225, 61-69 (2007).
6. Pogribny, I.P., I. Rusyn, and F.A. Beland. Epigenetic aspects of genotoxic and non-genotoxic hepatocarcinogenesis: studies in rodents. Environ. Molecular Mutagenesis, 49, 9-15 (2008).
7. Pogribny, I.P., V.P. Tryndyak, S.A. Ross, and F.A. Beland. Differential expression of microRNAs during hepatocarcinogenesis induced by methyl deficiency in rats. Nutrition Rev. 66 (Suppl. 1), S33-S35 (2008).
8. Bagnyukova, T.V., V.P. Tryndyak, B. Montgomery, M.I. Churchwell, A.R. Karpf, S.R. James, L. Muskhelishvili, F.A. Beland, and I.P. Pogribny. Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene. Carcinogenesis, 29, 638-646 (2008).
9. Tyryndyak, V.P., S.A. Ross, F.A. Beland, and I.P. Pogribny. Down-regulation of the microRNAs miR-34a, miR-127, and miR-200b in rat liver during hepatocarcinogenesis induced by a methyl-deficient diet. Mol. Carcinog., in press.
10. Bagnyukova, T.V., V.P. Tryndyak, L. Muskhelishvili, S.A. Ross, F.A. Beland, and I.P. Pogribny. Epigenetic silencing of the suppressor of cytokine signaling 1 (Socs1) gene is associated with the STAT3 activation and development of hepatocellular carcinoma induced by methyl-deficiency in rats. Cell Cycle, 7, 3202-3210 (2008).

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Fatemeh Rafii, Ph.D. 

Division of Microbiology
National Center for Toxicological Research
Jefferson, AR

Background:

B.S., Tehran University
M.S., Washington State University
Ph.D., Washington State University
FDA Experience - 21 years

Research Interests:

Determining the effects of antibiotic exposure on resistance development, metabolic activities and virulence factors of bacterial pathogens.  Elucidating the molecular mechanisms that govern physiological changes occurring in antibiotic-resistant bacteria. Detecting compounds that increase antibiotic potency.

Detecting bacteria from the human gastrointestinal tract that are involved in the metabolism, activation, and detoxification of food additives, nutritional supplements, environmental contaminants and pharmaceutical drugs, including antimicrobial agents.

Proposed Research Project for FDA Fellow:

Transcriptional and translational analysis of gene expression changes triggered by exposure of Clostridium perfringens to fluoroquinolones.

Selected Recent Publications:

1. Rafii, F., M. Park, A. E. Bryant, S. J. Johnson, and R. D. Wagner. (2008). Enhanced production of phospholipase C and perfringolysin O (alpha and theta toxins) in a gatifloxacin-resistant strain of Clostridium perfringens. Antimicrobial Agents and Chemotherapy 52:895-900.
2. Rafii, F., and M. Park. (2008). Detection and characterization of an ABC transporter in Clostridium hathewayi. Archives of Microbiology 190: 417-426.
3. Rafii, F., and M. Park. (2007). Substitutions of amino acids in alpha-helix-4 of gyrase A confer fluoroquinolone resistance on Clostridium perfringens. Archives of Microbiology 187:137-144.
4. Rafii, F., L. D. Jackson, I. Ross, T. M. Heinze, S. M. Lewis, A. Aidoo, L. Lyn-Cook, and M. Manjanatha. (2007). Metabolism of daidzein by fecal bacteria in rats. Comparative Medicine 57:282-286.
5. Rafii, F., and A. R. Shahverdi. (2007). Comparison of essential oils from three plants for enhancement of antimicrobial activity of nitrofurantoin against enterobacteria. Chemotherapy 53:21-25.
6. Rafii, F., and M. Park. (2005). Effects of gyrase mutation on the growth kinetics of ciprofloxacin-resistant strains of Clostridium perfringens. Anaerobe 11:201-205.
7. Rafii, F., M. Park, and J. S. Novak. (2005). Alterations in DNA gyrase and topoisomerase IV in resistant mutants of Clostridium perfringens found after in vitro treatment with fluoroquinolones. Antimicrobial Agents and Chemotherapy 49:488-492.
8. Rafii, F., M. Park, and R. A. Wynne. (2005). Evidence for active drug efflux in fluoroquinolone resistance in Clostridium hathewayi. Chemotherapy 51:256-262.
9. Rafii, F., C. Davis, M. Park, T. M. Heinze and R. D. Beger. (2003). Variations in metabolism of the soy isoflavonoid daidzein by human intestinal microflora from different individuals. Archives of Microbiology 180:11-16.
10. Rafii, F., R. Wynne, T. M. Heinze and D. Paine. (2003). Mechanism of metronidazole resistance in isolates of nitroreductase-producing Enterococcus gallinarum and Enterococcus casseliflavus from the human intestinal tract. FEMS Microbiology Letters 225:195-200.

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