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Cross-Species Gene Mutation Assay
National Center for Toxicological Research (NCTR) scientists have authored an invited book chapter for an upcoming volume of Molecular Toxicology Protocols (Methods in Molecular Biology series). The chapter details methods for conducting the Pig-a gene mutation assay in mice and rats. The Pig-a assay is a flow-cytometer-based assay that can be used to assess the in vivo mutagenic potential of suspected toxicants. The assay is rapid, relatively inexpensive, and is functional across species (mice-to-human), making it a potentially valuable tool for regulatory safety assessments.
For additional information, please contact Vasily Dobrovolsky, Ph.D., Division of Genetic and Reproductive Toxicology or Robert Heflich, Ph.D., Division of Genetic and Reproductive Toxicology, FDA/NCTR.
Regulatory Research Perspectives
The June 2010 issue of NCTR's scientific journal, Regulatory Research Perspectives, has been posted to FDA.gov. The development of diagnostic assays and the identification of specific biomarkers are two critical elements in personalized medicine. The two articles in this issue describe two examples of research being conducted at NCTR to meet these challenges. The first article describes the use of allele-specific competitive blocker PCR (ACB-PCR) to detect and quantify mutations in the K-RAS gene, and the second article highlights the potential for using epigenetic effects as biomarkers of various pathological states. This issue and all previous issues can be found at: http://www.fda.gov/AboutFDA/CentersOffices/NCTR/WhatWeDo/NCTRPublications/ucm076867.htm.
Pharmacokinetics of Bisphenol A (BPA)
Investigators at the National Center for Toxicological Research (NCTR) have shown that non-human primates exhibit small age-related changes in internal exposure to the bioactive aglycone form of BPA, which has been shown to cause endocrine disruption in experimental animal studies. By comparison, the bioactive aglycone is present in higher concentrations in perinatal rats of the same developmental age as the primate. The data from non-human primate and rodent studies will be combined with human biomonitoring data to develop a physiologically based pharmacokinetic (PBPK) model to improve the prediction of internal exposures of target tissues in human infants and fetuses to the bioactive aglycone form of BPA. BPA is a high-production chemical (>3.7 million metric tons/year) used in the manufacture of numerous food-contact and medical products and has been detected universally in the urine of all ages from infant to adult, with the highest concentrations in infants. Two publications describing this research have been recently accepted (Toxicology and Applied Pharmacology, 2010).
For additional information, please contact Daniel R. Doerge, Ph.D., Division of Biochemical Toxicology, FDA/NCTR.
50th Annual Meeting of the Teratology Society
On June 29, 2010, William Slikker, Jr., Ph.D., Director, NCTR, was a featured speaker at the Sunrise Mini-Education Course on “Imaging Tools for Structural and Functional Teratogenesis Analyses” offered by the Education Committee of the Teratology Society at their annual meeting. Dr. Slikker’s presentation entitled “Imaging tools for structural analysis” addressed various imaging techniques and their applications in risk assessment.
Improved Method for Noninvasive Brain Diagnostics
Investigators from the National Center for Toxicological Research (NCTR), Center for Devices and Radiological Health (CDRH), and Johns Hopkins University have developed a computational pre-processing method to improve the computerized pattern recognition of Magnetic Resonance Scans (MRS) of brain tissue. After processing, brain-tissue scans were categorized into nine descriptive categories with 96% accuracy by pattern recognition, as opposed to a previous accuracy of only 30%. This methodology has significant diagnostic and prognostic potential. For example, astrocytomas and oligodendrogliomas (types of brain tumors) are very difficult to distinguish based on MRS images. However, accurate MRS models based on pre-processed scans were able to identify a group of features in the MRS spectra of oligodendrogliomas that were absent in astrocytomas. These biomarkers may permit unequivocal distinction of the two tumor types. The implications for patient care are significant since the prognosis is 1-2 years for astrocytomas vs. 10-15 years for oligodendrogliomas.
The goal is to expand this technology to additional clinical challenges that would benefit from accurate noninvasive diagnostics, such as Alzheimer’s disease and traumatic brain injury. A manuscript describing this research was recently accepted for publication (Journal of Magnetic Resonance Imaging, 2010).
For additional information, please contact Jon Wilkes, Ph.D., or Dan Buzatu, Ph.D., Division of Systems Toxicology, FDA/NCTR.
The Toxicology Forum
Five NCTR scientists presented their studies at symposia organized by The Toxicology Forum held July 11-15, 2010 in Aspen, Colorado. NCTR presentations included:
- Tumorigenicity of Acrylamide
- PB/PK Modeling of Acrylamide for Use in Risk Assessment
- Pharmacokinetics of Bisphenol A
- The Role of the Rodent Tumor Mode-of-Action for Naphthalene Toxicity
- Evaluation of Toxicity of Nanoscale Materials
The Toxicology Forum is a nonprofit organization dedicated to providing a forum for open discussion among government, academic, and industry scientists on contemporary toxicology issues of mutual interest. The next biannual meeting of the Toxicology Forum will be held February 1-3, 2011 in Washington, D.C.
For additional information, please contact Paul C. Howard, Ph.D., Associate Director for Scientific Coordination, FDA/NCTR.
AAALAC Triennial Site Visit
The Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) conducted a triennial site visit at the National Center for Toxicological Research (NCTR) on July 13-15, 2010, and reported the finding of "no mandatory items" at the exit briefing. Although formal notification of accreditation status will not be issued until the AAALAC Council meets in the fall 2010, full accreditation for the next three years is expected.
The team commended the NCTR for excellence in documentation, research and support staff training, the occupational safety and health program, the zebrafish facility, animal husbandry, veterinary care, and facility sanitation. The site-visit team was led by an AAALAC Council Member Emeritus, a privilege limited to those institutions that have demonstrated a long-term commitment to achieving and maintaining high standards of laboratory animal care and use.
Assessments included close scrutiny of all animal facilities, procedure laboratories, programs, and records.
For additional information, contact Jeff Carraway, DVM, Veterinary Services, FDA/NCTR.
Crude Oil Bioremediation
During the week of July 19, 2010, Carl Cerniglia, Ph.D., Director, Division of Microbiology, National Center for Toxicological Research (NCTR), consulted with FDA's Center for Food Safety and Applied Nutrition’s Gulf Coast Seafood Laboratory at Dauphin Island, Alabama, concerning research and potential collaborations related to the Deepwater Horizon oil spill. Of particular interest was the recent publication (mBio, 2010) by investigators from the NCTR, the National Institute of Agricultural Biotechnology (Suwon, Republic of Korea), and the National Cancer Institute. The publication discussed the integration of enzyme structural information with metabolite data, biochemical reaction data, and genomic data to give mechanistic insight into the degradation and detoxification of polycyclic aromatic hydrocarbons (PAHs) by microorganisms. PAHs are toxic components of crude oil and have been shown to accumulate in biological systems and persist in the environment. These studies have direct implications in the development of methodologies to remediate oil-contaminated soils and sediments.
For additional information, please contact Carl Cerniglia, Ph.D., Director, Division of Microbiology, FDA/NCTR.
NCTR Summer Student Research Program
The National Center for Toxicological Research (NCTR) concluded its 2010 Summer Student Research Program with oral presentations by the young investigators describing the research that they completed during the summer. This Special Employment Program provides science and mathematics undergraduate and graduate students a 10-week training experience in regulatory science under the guidance and mentorship of NCTR principal investigators. This year, NCTR hosted 20 future scientists/mathematicians from various Arkansas universities, University of Pennsylvania, The Ohio State University, Georgia Institute of Technology, California State University at Long Beach, Jackson State University, Syracuse University, and the University of Oklahoma.
During the week of August 2, 2010, NCTR welcomed two invited speakers for seminars and discussions with the research staff. June Dunnick, Ph.D., National Institute of Health for Environmental Sciences (NIEHS), discussed her studies on the gastrointestinal carcinogenic potential of hydryoxyanthraquinones. Dr. Dunnick is participating in an exchange seminar program between the National Toxicology Program (NTP)/NIEHS and FDA/NCTR. Hydryoxyanthraquinones are believed to be the active compounds in Senna, a medicinal plant used as a laxative.
Kai Ge, Ph.D., from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/NIH, presented a seminar and held discussions on the role of epigenetic regulation in adipogenesis. Dr. Ge also consulted on mass spectrometry-based proteomic approaches to elucidate epigenetic markers relevant to diabetes and obesity with the NCTR proteomics staff.
Neurotoxicity of Nanoparticles
Investigators from the National Center for Toxicological Research (NCTR) and Wright-Patterson Air Force Base have shown that 25-40 nanometer silver nanoparticles at concentrations of 15–50 micrograms per milliliter associate with the cells on the interior surface of the brain's micro-sized blood vessels (microvessel endothelial cells) and can induce:
- significant cellular toxicity
- alterations in cell form and structure
- alterations in monolayer (layer of cells one cell thick) integrity
- an increase in prostaglandin E2
- tumor necrosis factor α
- interleukin 1β
Primary rat-brain microvessel endothelial cells were used as an in vitro model of the blood brain barrier (BBB) to gain insight into the mechanisms involved in the nanoparticle-induced BBB dysfunction that has been reported. These studies indicate that silver nanoparticles produce an initial cascade of proinflammatory mediators that may contribute to increased BBB permeability, further brain inflammation, and neurotoxicity. Silver nanoparticles are widely used in engineering, manufacturing, and biomedicine. However, their impact on human health has not been adequately evaluated. A manuscript describing this research was recently accepted for publication (Toxicological Sciences, 2010).
For additional information, please contact Merle Paule, Ph.D., Director, Division of Neurotoxicology, FDA/NCTR.
A Proteomics Primer
Scientists from the National Center for Toxicological Research (NCTR) have completed a basic review on the use of the emerging field of proteomics and its techniques in the field of systems biology. Rapid genetic sequencing technologies have transformed “microarrays” and “genomics” into common tools for laboratory study. Nonetheless, the proteins produced from the genome (a full set of genes in a living organism) can be further modified for different functions in the cell. The sum of these proteins, and their resulting cellular functions, are identified as the proteome. The role of the proteome and proteome technology is discussed in an invited chapter for a book to be published under the title, Handbook of Systems Toxicology.
A pre-print of this chapter is available from Donna Mendrick, Ph.D., Director, Division of Systems Biology, FDA/NCTR.
Improvements in Salmonella Identification
Investigators from the National Center for Toxicological Research (NCTR) have shown that using a statistical method (random forest classification) to analyze profiles of genomic DNA fragments produced by pulsed-field gel electrophoresis (PFGE) from eight serotypes of Salmonella enterica improved the accuracy of predicting the relationships between PFGE banding patterns and particular serotypes over the conventional method of hierarchical cluster analysis. Current technology for identifying foodborne pathogens relies on standard microbiological methodology and genomic analyses, such as PFGE, which is the standard typing method for isolates (samples) from Salmonella outbreaks and epidemiological investigations. Because strain identification requires comparison of complex patterns of DNA fragments, this statistical method will improve FDA's ability to classify novel isolates of foodborne pathogens. A manuscript describing this research was recently accepted for publication (Journal of Clinical Microbiology, 2010).
For additional information, please contact Wen Zou, Ph.D., Division of Personalized Nutrition and Medicine, FDA/NCTR.
On August 20, 2010, NCTR organized and hosted an interactive WebEx seminar featuring Bruno Sobral, Ph.D., Virginia Bioinformatics Institute (VBI), Virginia Tech University. VBI is consolidating four large pathogen databases into their PathoSystems Resource Integration Center (PATRIC), which contains approximately 2,300 bacterial genomes and could serve as a resource on foodborne pathogens for FDA’s research community. Discussions are underway between VBI's Cyberinfrastructure Division and FDA Centers interested in developing formal collaborations to work with the PATRIC database.
For additional information, please contact Jim Kaput, Ph.D., Director, Division of Personalized Nutrition and Medicine, FDA/NCTR.
Bioterrorism and Food Safety
Investigators from the National Center for Toxicological Research have shown that the thermal stability of ricin (a potential foodborne bioterrorism agent) was similar when added to milk, milk-based infant formula, soy-based infant formula, and fruit juices, but yogurt and blended yogurt-fruit drink enhanced the thermal stability to ricin. This enhanced stability was determined to be due to pH, galactose, and the presence of exopolysaccharides, which may allow other galactose-binding, ribosome-inactivating protein toxins, such as the shiga-like toxins produced by E. coli 0157:H7, to survive standard pasteurization conditions. Since infants and very young children are prone to consume a single type of food as a complete meal (e.g., baby foods or infant formulas), these groups are considered to represent a more vulnerable population for food-borne bioterrorism agents.
For additional information, please contact William Tolleson, Ph.D., and Fred Beland, Ph.D., Division of Biochemical Toxicology, FDA/NCTR.
NCTR Women's Health Research Workshop
The National Center for Toxicological Research hosted an Office of Women’s Health sponsored workshop, “Sex Differences in FDA Regulated Products: Research for the Future” in Little Rock, Arkansas, on September 2, 2010. The workshop, attended by 75 individuals that included FDA scientists, clinical physicians, and industry, addressed the critical data gaps that exist in women’s health research and how these may impact the risk-benefit analysis of FDA regulated products. The four principle issues for discussion included: autoimmune diseases, cardiovascular diseases, adverse drug reactions, and mental health. Although women are disproportionately affected by various conditions and diseases compared to men in terms of incidence, diagnosis, and response to treatment, the biological mechanisms of sex differences are not well understood. The workshop gave NCTR scientists an opportunity to discuss and identify future research needs with a broad audience, and to address the regulatory impact of sex differences in FDA regulated products.
Genetic Variations in Nutritionally Related Chronic Disease
NCTR investigators have used data mining to show that a majority of the genes of the carbohydrate metabolic pathways are associated with chromosomal regions linked to obesity and type 2 diabetes mellitus (T2DM), and that a number of key genes in these pathways contain single nucleotide polymorphisms (single nucleotide variation in the DNA sequence of individuals) that exhibit significant differences in frequencies among diverse ancestral groups. These differences may be among the key genetic variations that explain biological differences observed between individuals to various carbohydrate foods. This study emphasizes the significance of the metabolic pathways genes in the development of disease phenotypes and its differential occurrence across diverse ancestral populations and between individuals. Although health disparities result from a complex set of issues, including socioeconomic status, healthcare, and food access, research is increasingly demonstrating that genetic makeup influences the effects of drugs and nutrients in individuals. This is not a surprising result as a number of conditions are already known to have an ancestral component. A manuscript describing this research was recently published (Biotechnology Journal, 2010).
For more information, contact Vijayalakshmi Varma, Ph.D., Division of Personalized Nutrition and Medicine, FDA/NCTR or Jim Kaput, Ph.D., Director, Division of Personalized Nutrition and Medicine, FDA/NCTR.
RAPID-B in Quality Assurance/Quality Control
Monday, September 13, 2010, NCTR hosted Kraft Foods’ scientists to discuss the potential of the NCTR-developed RAPID-B technology in food quality assurance and quality control for target pathogen detection.
For more information, contact Jon Wilkes, Ph.D., Division of Systems Biology, FDA/NCTR.
Food Safety Tools
Thousands of pathogenic Salmonella strains have been identified, many of which are able to cause foodborne illnesses in humans. NCTR scientists have adapted and combined existing technologies into an accurate and cost-effective strategy to monitor for the presence of virulent Salmonella strains, and/or monitor the progress of cleanup strategies in an impacted environment. The detection strategy combines a targeted microarray — containing 69 of the most common Salmonella-specific virulence genes responsible for the ability of the bacterium to cause infection — with ArrayTrack™ and Microbial Gene Library, a free FDA bioinformatics tool. This detection system could easily be modified to include additional probes for newly described virulence-associated genes. A full description of this system was recently accepted for publication in the Journal of Infectious Diseases in Developing Countries (2010).
For additional information, please contact Carl Cerniglia, Ph.D., and Rajesh Nayak, Ph.D., Division of Microbiology, FDA/NCTR.