Director: Martha M. Moore, Ph.D.
Telephone: 870-543-7050
Toll Free: 800-638-3321
E-mail: martha.morre@fda.hhs.gov

Executive Summary

The Division of Genetic and Reproductive Toxicology (DGRT) conducts applied basic research to address specific high-priority issues regarding genetic and reproductive toxicology. Division research is directed toward developing and validating new methods that can be used for the identification of potentially hazardous food additives, human and animal drugs, biological therapies, and medical devices. In addition, in collaboration with other NCTR scientists, DGRT conducts research to understand the potential toxicity of specific high-priority drugs, dietary supplements, and/or other agents. Genistein, malachite green and some of the drugs used for AIDs therapy are currently undergoing extensive evaluations in cross-division collaborative research efforts.

Currently there are four basic focus areas in the Division research program. Genetic Toxicology research addresses the development of methods to assess the potential for chemicals to negatively impact human genetic material or the function of the genetic material. Reproductive/Developmental Toxicology focuses on methods to understand normal human development and how chemicals might alter normal development. In addition to these two disciplinary research areas, the Division conducts research to understand the effects of dietary supplementation. That research primarily focuses on understanding the physiological and genetic consequences of dietary modulation. Recently the Division initiated a new research focus to utilize new molecular approaches to evaluate genomic damage. While administratively a part of DGRT, the new NCTR Functional Genomics Center will be a resource for all NCTR investigators.

Genetic Toxicology

Genetic toxicology is the investigation of the ability of chemicals to alter the genetic material. The FDA requires that petitioners provide data evaluating the potential genetic toxicity of their products as a part of the product approval process. Because genetic damage is believed to be important in tumor development, this information is used as a part of the evaluation of suspected carcinogens. Regulatory decisions are based not only on the identification of potentially genotoxic substances, but also on an understanding of their mode of action. Research within the Division centers on the development and validation of new methods by which to assess genetic risk. While tissue culture approaches are used to detect potential genotoxicity and to generate hypotheses concerning the basic mechanisms of genotoxicity, the Division specializes in the development and validation of in vivo mammalian systems. An increased understanding of mutational mechanisms, combined with test systems with an increased ability to detect genetic damage, will provide the FDA with better information for making decisions. As new assays are validated, Division scientists work with international scientists to assure harmonization of protocols and the development of guidelines.

Reproductive/Developmental Toxicology

One of the difficult challenges facing the FDA is the identification and regulation of chemicals, food additives, and biological therapies that may produce birth defects. Such defects affect 7% of the population at birth, another 7% have low birth weights, and at least 25% of pregnancies end in spontaneous abortion. The Division specializes in research to understand how toxicants may induce birth defects such as neural tube defects. Current research addresses the role that the vitamin folic acid may play in the normal closure of the neural tube. This research supports current thinking that diet may play a role in the development of normal offspring and that interactions between diet and toxicants may be important in producing certain birth defects.

A well-defined database created over the past 20 years led to the initiation of a project to create and validate a computerized knowledge base using quantitative structure-activity relationships to predict which chemicals might affect the normal reproductive function.

Diet and Nutrition

Dietary restriction can increase the length of a rodent’s life and decrease the frequency of tumors. Division scientists have also determined that decreasing caloric intake alters many physiological processes in rodents and decreases the frequency of mutations. The group has developed many physiological, biochemical, and morphological procedures that can now evaluate dietary modulation. Because of a unique opportunity to collaborate with medical scientists at the University of Tennessee at Memphis, the research program is able to compare the responses seen in rodents with those seen in calorically restricted humans. These dietary restriction studies are nearing completion. Currently, dietary research focuses on understanding the impact of dietary supplementation such as anti-oxidants, genistein, and herbals.

Genomics

International research efforts are providing the scientific and medical community with increased understanding of the genome in both humans and rodents. Utilizing this information, new molecular technologies are being rapidly developed and can be used to evaluate structural and functional changes in the genome of both rodents and humans. The Division is developing a new research focus area to use these technologies and to apply them to fundamental risk assessment questions. While current technologies in the field of genetic and reproductive/developmental toxicology generally evaluate single endpoints, these new genomic technologies will provide the opportunity to detect alterations in a number of different endpoints. In addition to addressing research questions in the areas of genetic and reproductive toxicology, the new NCTR Functional Genomics Center will be a means by which all NCTR scientists can collaborate and utilize this new technology. It will also provide an opportunity to evaluate the multiple adverse health effects of chemicals and to assist with the harmonization of cancer and non-cancer risk assessment methodologies.

FY 2001 Accomplishments and FY 2002 Plans

PI: Aidoo, Anane

Title: The Frequency and Types of Spontaneous Mutations Found in the Hprt and lacI Genes of Lymphocytes from Transgenic Big Blue Rats

Project Number: E0697501, E0697511

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

In vivo assays are used to evaluate whether chemicals have the potential to induce genetic damage (mutations). In order to understand an assay and to use it for hazard identification, one must first understand the frequency and types of mutations that occur spontaneously (without any chemical exposure). This project is designed to evaluate spontaneous mutation in one gene that is in its normal location (Hprt) and one gene (lacI) that has been genetically engineered into a strain of rats. This strain of rats, containing the lacI transgene is called the Transgenic Big Blue Rat. The specific goals of this project are:

1. To determine the frequency of spontaneous mutation at the hprt and lacI loci in pre-weanling, young (four-month-old) and old (18-month-old) Big Blue rats.
2. To determine the types of mutations present in the mutants.
3. To determine if rats fed different diets have different spontaneous mutant frequencies and if the types of mutations are different.

FY 2001 Accomplishments:

This project is complete.

FY 2002 Plans:

Prepare the final manuscripts.

Title: The Use of Antioxidants in Single and in Mixture to Study the Effects of Dietary Vitamins on Genotoxicity Produced in Rats Treated with the Mammary Carcinogen 7,12-dimethylbenz(a)anthracene and the Radio-metic Antitumor Drug Bleomycin

Project Number: E0701401

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Antioxidants have been reported to have beneficial health effects including reducing the risk of cancer. They have also been reported to reduce the risk of mutation. In this project, dimethylbenz(a)anthracene (DMBA) and bleomycin (BM), both known carcinogens and mutagens, will be administered to rats. Animals will also receive antioxidants (singly and as a mixture of vitamin C, E, b -carotene and selenium). It is expected that the animals treated with both the carcinogen/mutagen and the antioxidants will have a lower mutant frequency than those animals treated only with the carcinogen/mutagen. Two different genotoxic endpoints, mutation at the Hprt gene and chromosomal effects (cytokinesis-block micronucleus) will be used.

Once the genotoxicity has been determined, experiments will be undertaken to determine the mechanism underlying the inhibitory action of the dietary antioxidants by determining their effects on:

1. Spectra of induced mutations in Hprt gene in lymphocytes.
2. Oncogene (H-ras, K-ras) and tumor suppressor gene, p53 expression.
3. Programmed cell death (apoptosis).
4. The activities of glutathione peroxidase and glutathione S-transferase during DMBA and BM exposures.

FY 2001 Accomplishments:

This project is complete.

FY 2002 Plans:

Write the final report.

Title: ADDEND: Evaluation of the Effects of Dietary Antioxidant Intake on Behavior, DNA Damage and Expression of Free Radical Scavenging Enzymes During Physical Exercise in Male and Female Fischer 344 Rats Treated with 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine (PhIP)

Project Number: E0706311

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

This addendum will enable the use of both treated and untreated animals. We also intend to include measurement of mitochondrial DNA mutations as an additional end- point to the nuclear DNA mutations. This aspect of the study will make it possible to compare in vivo mutations occurring in both nuclear and mitochondrial DNA, as mutations in both systems contribute to human disease burden.

FY 2001 Accomplishments:

The experiments began; about 64 rats are ready to be sacrificed to determine mutant frequency and types of mutation using RT-PCR and DNA sequencing. Some tissues such as muscle and kidney have been frozen for gene and protein expression, as well as oxidative DNA analysis using flow cytometry.

FY 2002 Plans:

1. The experiments will continue since 46 rats are yet to undergo physical exercise, dietary antioxidant intake and carcinogen treatment.
2. Collection and analysis of data.
3. Results will be presented at scientific meetings and manuscripts will be prepared for publication.

Title: Evaluation of the Effects of Daidzein and Genistein (Hormone Replacement Agents) on the Genotoxic and Carcinogenic Activity of the Model Mammary Carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in Ovariectomized Transgenic Big Blue Rats

Project Number: E0707001, E0707011

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

This project is designed to determine if hormone replacement, in this case the phytoestrogens daidzein and genistein, will alter the risk of mammary tumors. Rats will be exposed to a known mammary carcinogen (DMBA) and given daidzein and/or genistein. In order to model the post-menopausal human situation, the rats will be ovariectomized. In addition to the induction of tumors, other endpoints such as the ability of the DMBA to bind to DNA (DNA adduct analysis) and the frequency and types of mutations induced by DMBA will be investigated.

FY 2001 Accomplishments:

The preliminary work involved in this proposal had been completed. The experiments are still ongoing, and most of the animals were sacrificed to conduct the assays.

FY 2002 Plans:

Experiments will continue and data will be collected for manuscript preparation and for scientific meeting presentations.

PI: Branham, William

Title: Development of a Statistically Robust 3D-QSAR Model to Predict In Vitro Rat Uterine Estrogen Receptor Binding Activity

Project Number: E0290001

Collaborator: None

Strategic Research Goal: Knowledge Base

Objective(s):

There is a Cooperative Research and Development Agreement (CRADA) for this project to develop and validate a statistically robust model for prediction of isolated rat uterine estrogen receptor relative binding affinity (RBA) that could be used as part of a prioritization scheme to identify chemicals for further in vitro/in vivo screening tests.

FY 2001 Accomplishments:

The QSAR and CoMFA models have been completed and delivered to the EPA. These models are currently being evaluated by the EPA under a contract with Battelle Laboratories to validate the models.

FY 2002 Plans:

Any further refinements to these models will be done via contractor.

Title: Development of a Statistically Robust Rat Androgen Receptor (AR) 3D-QSAR Model for Predicting Relative Binding Affinity (RBA) of Untested Chemicals

Project Number: E0290101

Collaborator: None

Strategic Research Goal: Concept-Driven

Objective(s):

To develop and validate a statistically robust 3D-QSAR model to predict in vitro rat androgen receptor (AR) relative binding. Provide an alternative and/or supplemental method to prioritize chemicals for entry into Tier 1 screening under the EPA’s screening and testing program for endocrine disruptors.

FY 2001 Accomplishments:

1. Replicate assays have been completed for 204 chemicals assessing binding to the PanVera Recombinant Androgen Receptor (AR).
2. The data are currently being utilized by R.O.W. computational chemists to model the binding of these chemicals to the AR.
3. The data are being processed and manuscripts are in preparation.

FY 2002 Plans:

The AR data will be used to create both QSAR and CoMFA models. These models will be made available for use by the EPA for assessment of AR binding of a large number of environmental chemicals.

PI: Chen, Tao

Title: Comparison of Mutation Induction and Types of Mutations in the cII Gene of Big Blue Mice Treated with Carcinogens as Neonates and Adults

Project Number: E0709001

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Because cancer is a disease requiring the induction of mutation and the clonal expansion of mutated cells, one would expect that the developing fetus and young infant would be particularly susceptible to carcinogen exposure. This project will be initiated to evaluate this hypothesis. Experiments will be conducted to:

1. Determine the mutant frequencies in the cII gene of Big Blue mice treated at different ages with direct-acting carcinogens.
2. Determine the mutant frequencies in the cII gene of the target tissues from transgenic mice exposed as neonates and adults to different carcinogens that require metabolic activation.
3. Determine the types of mutations produced in the cII genes of the mutants induced in objectives 1 and 2.

FY 2001 Accomplishments:

Protocol approved.

FY 2002 Plans:

Experiments will be initiated in which transgenic animals are exposed as neonates and adults to different carcinogens.

PI: Dobrovolsky, Vasily

Title: Validation of the Mouse Targeted Tk+/- In Vivo System for Use in Mutagenicity Studies

Project Number: E0701801

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

DGRT has developed a new in vivo assay for the evaluation of mutant induction. This assay was modeled after the in vitro mouse lymphoma assay already used internationally for hazard identification. The mouse lymphoma assay uses the thymidine kinase gene (tk) and has been extensively evaluated for its mechanistic basis and shown to detect most, if not all, of the mutational events important to the induction of cancer and other human diseases. This project is designed to further evaluate and validate the in vivo assay. Specific goals of this project are:

1. To expand a colony of transgenic Tk+/- mice using breeding of Tk+/- founders and C57Bl/6 mice, and to transfer the Tk+/- genotype to a C57Bl/6 background.
2. To determine spontaneous mutant frequencies at the Tk and Hprt loci of splenic T-lymphocytes for mice of different ages.
3. To induce mutations in Tk+/- transgenic mice using treatment with the point mutagen ENU (ethyl nitrosourea) and the clastogens BLM and y-radiation, and to measure the kinetics of mutant induction at the Tk and Hprt loci.
4. To breed transgenic Tk+/- parents in an attempt to derive Tk-/- knockout (KO) mice, and study the biological significance of the tk gene in mice.
5. To determine how the Tk-/- genotype may affect mutant frequencies at the Hprt locus.

FY 2001 Accomplishments:

1. The colony of Tk+/- animals was maintained on the site to provide animals for internal research needs as well as for distribution to five other scientific institutions.
2. Mutation frequencies of Hprt and Tk genes were identified in Tk+/- animals following the treatment with ionizing radiation, etoposide and mitomycin C. A manuscript describing these findings was submitted to Environmental and Molecular Mutagenesis Journal (accepted).
3. Age dependent Tk mutant frequencies and types of mutations were identified for Tk+/- males and females.
4. Tk+/- animals were shipped to LRRI for exposure to potential carcinogen butadiene by inhalation, upon the return mutant frequencies were determined for the Hprt and the Tk genes in the exposed animals.
5. Frequencies of Hprt and Tk mutation were determined for butadiene-exposed Tk+/- mice.
6. Tk+/- mice were crossed to Pms2+/- mice to determine Hprt and Tk mutant frequencies in Pms2-deficient mice.
7. Tk+/- animals were submitted to centralized depository, Mutant Mouse Regionall Resource Center (MMRRC), for availability to outside researchers.
8. A chapter, "Analysis of in vivo mutation in the Hprt and Tk genes of mouse lymphocytes", was submitted for publishing in the book Molecular Toxicology Protocols.

FY 2002 Plans:

1. The colony of Tk+/- animals will continue to be maintained for future treatment with agents of interest to FDA.
2. For simplified distribution of Tk-deficient animals to outside researchers, the breeding duties will be transferred to a centralized service at MMRRC).
3. Cooperation with LRRI will start to determine mutational responces in animals exposed in utero to chemicals of interest to FDA.

Title: ADDEND: Validation of the Mouse Targeted Tk+/- In Vivo System for Use in Mutagenicity Studies

Project Number: E0701811

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

In order to insure the health of the colony and to produce and supply Tk+/- animals to other investigators, it is necessary to conduct routine microbiological surveillance of the colony to assure that it remains pathogen free.

FY 2001 Accomplishments:

Microbiological surveillance of Tk+/- animals was performed for continuous monitoring of the health status of the colony. With health reports generated during this period, our Tk+/- mice were accepted by five other outside researchers for independent purposes.

FY 2002 Plans:

The surveillance program will continue until the Tk+/- animals are accepted by MMRRC. If animals are accepted, then the monitoring program may be discontinued and requests for Tk+/- animals from outside investigators will be redirected to MMRRC.

Title: ADDEND: Validation of the Mouse Targeted Tk+/- In Vivo System for Use in Mutagenicity Studies

Project Number: E0701812

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Proposing to breed Tk+/- mice with Pms2+/- mice in order to derive Tk+/- mice that can be used for evaluating LOH mutation and that are also deficient in the Pms2 gene product. Will be using animals bred under the parent protocol E0701801 and another protocol (E0704101). Also extending proposed completion date of master project and associated addenda to 4/30/2004.

FY 2001 Accomplishments:

Breeding program for derivation of Tk+/- mice on the Pms2-deficient background has commenced. The first breeding step was done; Tk+/- Pms2+/- mice have been produced in desired quantities.

FY 2002 Plans:

1. Tk+/- animals with Pms2+/-, Pms2+/+ and Pms2-/- backgrounds will be derived.
2. Spontaneous mutant frequencies will be determined for Hprt and Tk genes in TkPms2 mice.
3. Types of mutations will be identified and the significance of the Pms2 gene in induction of loss of heterozygosity mutation induction will be studied.

Title: Evaluation of the Tk-/- Knockout Mouse as a Model of Systemic Lupus Erythematosus

Project Number: E0706901

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Our initial experiments suggest that mice deficient in the thymidine kinase enzyme (Tk-/- animals) may be a useful model for studying the human disease lupus. In this project, we will investigate whether the tk-/- genotype in mice is lupus prone. Particular emphasis will be given to documentation of the putative immune-complex mechanism of the renal disease, and in-depth evaluation of the immune system in Tk-/- mice, seeking comparison with published characteristics of Systemic Lupus Erythematosus in mice and humans.

FY 2001 Accomplishments:

1. 120 mice with Tk+/+, Tk+/- and Tk-/- genotype were produced.
2. Tissues were collected and preserved from all three types of Tk mice. Blood serum samples were analyzed for the concentrations of thymidine nucleoside.
3. Serum samples were analyzed for the presence of autoimmune abnormalities.
4. Tissues were collected from mice of two different ages (three months old and six months old).

FY 2002 Plans:

1. Tissue samples will be examined for pathology in various organs with the emphasis on kidney and salivary gland.
2. The report on Tk-/- phenotype will be prepared and submitted.

PI: Domon, Olen

Title: Evaluation of the Genotoxicity of the Phytoestrogen Coumestrol in Human Lym-phoblast Cells that Differ in the Mutational Status of the p53 Tumor Suppressor Gene

Project Number: E0705501

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

The phytoestrogens are considered to be potentially beneficial dietary supplements, particularly for peri- and post-menopausal women. The phytoestrogen coumestrol will be evaluated in several in vitro gene mutation assays with the following goals:

1. To confirm the ability of coumestrol to break chromosomes. This will be done using the micronucleus assay.
2. To confirm the mutagenicity of coumestrol at the HPRT locus.
3. To determine if coumestrol induces large-scale chromosomal damage such as that detected by the Tk mutation assay.
4. To determine if the toxicity of coumestrol is due to apoptosis.
5. To determine the effect of coumestrol on the ability of cells to grow and divide normally.

FY 2001 Accomplishments:

1. A manuscript entitled, "Evaluation of the genotoxicity of the phytoestrogen, coumestrol, in AHH-1 TK+/- human lymphoblastoid cells," was published in Mutation Research.
2. Continued the molecular analysis of mutants.

FY 2002 Plans:

1. Continue molecular analysis of mutants.
2. Confirm the sequence of the Tk gene in AHH-1 Tk +/- cell line.
3. Determine the nature of the mutations in Tk mutant clones.

Title: ADDEND: Evaluation of the Genotoxicity of the Phytoestrogen Coumestrol in Human Lym-phoblast Cells that Differ in the Mutational Status of the p53 Tumor Suppressor Gene

Project Number: E0705511

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

In the master project, cells in culture were treated with coumestrol and genistein and clones of cells that were made mutant at the thymidine kinase gene were isolated. In this addendum, these mutants will be evaluated to determine the specific types of mutations that were induced by these two phytoestrogens.

FY 2001 Accomplishments:

A manuscript entitled "Evaluation of the genotoxicity of the phytoestrogen, coumestrol, in AHH-1 TK+/- human lymphoblastoid cells", was published in Mutation Research.

FY 2002 Plans:

Experimentation was completed in FY 2001 and a manuscript was published in Mutation Research. A final report will be prepared in FY 2002.

PI: Duffy, Peter

Title: Effect of Different Levels of Caloric Restriction (CR) on Physiological, Meta-bolic, Biochemical, Immunological, Mole-cular, and Body Composition Variables in Rats

Project Number: E0692401

Collaborator: CFSAN

Strategic Research Goal: Concept-Driven

Objective(s):

1. To determine how various levels and durations of CR affect physiological function, enzymes related to intermediary and drug metabolism, hormonal regulation, blood chemistry, etc.
2. To determine the relationship between body fat (BF), fat free mass (FFM), total body water (TBW), and total body electrical conductivity (TOBEC) as a function of strain, age, mass, and nutritional status in rats.
3. To validate and automate the use of a new, non-invasive electromagnetic scanning device to measure BF, FFM, and TBW and to compare the results to a conventional chemical fat extraction technique.
4. To determine if CR alters the relative quantity and disposition of various types of lipids such as cholesterol, phospholipids, free fatty acid, etc., in various tissues, as well as in urine, feces, and blood serum.
5. To develop control data related to CR that can be used by CFSAN to evaluate the toxicity and efficacy of low calorie foods, food additives, and food substitutes.
6. To determine temporal and environmental factors that modulate the effects of CR.
7. To develop experimental methods for utilizing a low level of CR to increase the survival rate and to decrease variablity in the chronic bioassay; to provide the concomitant control data for comparison.
8. To develop control data for a reference purified diet that has been formulated to conform to the long-term nutrient requirements of rodent animal models typically utilized in toxicology and nutrition studies.

FY 2001 Accomplishments:

Survival Study

1. Survival profiles were developed for Sprague Dawley rats on different levels of food intake; AIN-93M (purified) and NIH-31 (cereal) diets used.
2. Dietary Restriction (DR) significantly increased 24-month survival in rats raised on the NIH-31 diet; DR didn’t increase survival in rats raised on the AIN-93M diet.
3. NIH-31 and AIN-93 survival manuscripts were published in the past year.

Mutation Frequency Study

1. Mutation frequency determined for Sprague Dawley rats on different levels of food intake; AIN-93M and NIH-31 diets used.
2. Mutation frequency assays were conducted and data analysis was completed.
3. 40% DR significantly reduced mutation frequency.
4. Manuscript preparation is nearing completion.

Electron Transport and Mitrochondria Study

1. Effects of DR and age on oxidative damage and electron transport were determined in the mitochondria of muscle tissue; AIN-93M and NIH-31 diets used.
2. Assays were completed and the data were partially analyzed.
3. DR significantly reduced oxidative damage in the mitochondria.

Pathology Study

1. Histopathology completed for Sprague Dawley rats on different levels of food intake; AIN-93M and NIH-31 diets were used.
2. Pathology different for four levels of food consumption (NIH-31 diet); DR decreased proliferation of tumors and renal failure in a linear fashion.
3. Pathology different for cereal and purified diets; NIH-31diet associated with high rate of pituitary and liver tumors and low incidence of chronic renal disease. AIN-93M diet associated with high rate of chronic renal disease and a low incidence of pituitary and liver tumors.
4. Final pathology report to be completed in next few months.

Clinical Blood Chemistry Study

1. Developed an interactive database to store and retrieve clinical chemistry data.
2. Clinical blood chemistries completed for Sprague Dawley rats on various levels of food intake; AIN-93M and NIH-31diets were used.
3. Blood parameters such as triglycerides, alanine aminotransferase, creatinine, total protein, total cholesterol, phospholipids, , lymphocytes (%), and white blood cell count were decreased by DR.
4. Blood parameters such as glucose, alkaline phosphatase, hemoglobin, and neutrophils (%) were increased by DR.
5. Data analysis has been partially completed.

Physiological Study

1. Body temperature, activity, energy metabolism, and respiratory quotient continuously monitored in rat food response study; AIN-93M and NIH-31diets used.
2. Data acquisition stage completed and data analysis partially completed; Oracle database developed and maintained for physiological studies.
3. DR was associated with a decrease in metabolism and temperature and an increase in activity (NIH-31 and AIN-93M diets).

FY 2002 Plans:

1. Survival, histopathology, physiological, and blood chemistry variables will be monitored with age in Fischer 344 rat at different levels of DR; NIH-31 diet used.
2. Proteomic, genomic, and mutation frequency assays will be conducted in Fischer 344 rats to determine age and DR effects.
3. Data acquisition phase will be completed; data analysis will be partially completed.
4. Data analysis and manuscripts (histopathology, mutation frequency and blood chemistry variables) will be completed for Sprague Dawley rats on NIH-31 and AIN-93M diets.

Title: ADDEND: Effect of Different Levels of Caloric Restriction (CR) on Physiological, Meta-bolic, Biochemical, Immunological, Mole-cular, and Body Composition Variables in Rats

Project Number: E0692411

Collaborator: CFSAN

Strategic Research Goal: Concept-Driven

Objective(s):

Requesting 40 additional male Sprague-Dawley rats be added to this protocol to serve as ad libitum controls in a dietary restriction (DR) study.

FY 2001 Accomplishments:

1. Additional animals added to E692401 to increase sample size; not separate study.
2. Data was pooled with main study for analysis (see E0692401 for details).

FY 2002 Plans:

See plans for E0692401 for details.

Title: ADDEND: Task Order #483 & #493 - LIMS Implementation and Review of Heart Rate Variation Analysis Software

Project Number: E0699811

Collaborator: Univ of Tenn at Memphis

Strategic Research Goal: Predictive Toxicology

Objective(s):

Addendum requested to add ADP resources needed for Task Order #483 - Memphis Study: LIMS Implementation.

FY 2001 Accomplishments:

Physiological Studies

1. Physiological studies (resting and exercise) were conducted to monitor metabolism, energy expenditure, and respiratory quotient in obese human patients before gastric bypass surgery and 3, 6, 12, 24 months after the onset of DR.
2. Developed a non-invasive procedure for continuously monitoring temperature, blood pressure, heart rate, and motor activity in humans.
3. Partially completed physiological studies in 43 patients.
4. Completed the development of interactive Oracle database to store and retrieve physiological data.
5. Data has been partially analyzed.
6. DR significantly altered physiological performance in humans and rats in a similar fashion.
7. Temperature, heart rate, and energy expenditure variables were significantly reduced by DR; DR may reverse or delay the aging process.

H-Scan Physiological and Behavioral Assessment Studies

1. A battery of 12 physiological and behavioral biomarkers was tested in obese patients before and after gastric bypass surgery.
2. Visual reaction time with decision, and muscle movement time with decision increased after the onset of DR (negative impact); visual reaction time, auditory reaction time, muscle movement time, alternate button-tapping time, and muscle movement time with decision decreased after the onset of DR (positive impact).
3. Visual accommodation, memory (length of sequence), forced lung expiratory volume, forced lung vital capacity, vibrotactile sensitivity, and highest audible pitch were not altered by DR.
4. Results suggest that H-Scan physiological and behavioral measures may be excellent biomarkers of aging and DR.
5. DR may reverse or delay the aging process; results are similar to rat studies.
6. Data analysis has been partially completed.

FY 2002 Plans

1. Final analysis of data will be completed in the next few months if statistical support is provided.
2. Manuscripts and final report will be prepared for publication.
3. Results will be used to develop beneficial diets to increase longevity in humans.

PI: Feuers, Ritchie

Title: Memphis Study: Evaluation of Calorically Restricted Human Surgical Samples Received from Department of Surgery University of Tennessee, Memphis

Project Number: E0699801

Collaborator: Univ of Tenn at Memphis

Strategic Research Goal: Predictive Toxicology

Objective(s):

To determine whether rodents and humans behave biologically in the same manner when calorically deprived but nutritionally supplemented.

FY 2001 Accomplishments:

Study sampling completed.

FY 2002 Plans:

To complete the analysis of samples and compilation of data.

PI: Fuscoe, James

Title: Development of Glass-slide Based Oligonucleotide Microarrays for Mouse and Human Genes

Project Number: E0711601

Collaborator: UAMS

Strategic Research Goal: Method-Driven

Objective(s):

1. Establish microarray technology for the NCTR Functional Genomics Center.
2. Develop, print, and establish the methodology for using a "rat gene chip" containing approximately 4,000 genes and a "human gene chip" containing approximately 8,300 genes.

FY 2001 Accomplishments:

1. Purchased gene collections for the rat and human.
2. Identified collaborator to print the gene chips.

FY 2002 Plans:

1. Develop and print rat and human gene chips.
2. Validate the quality of the rat and human gene chips.
3. Prepare a large amount of reference rat RNA.
4. Develop a database for handling the large amounts of data that will be generated.
5. Develop statistical methods for interpreting the experimental results.

PI: Hansen, Deborah

Title: Antisense Knockouts of Genes in the Folate Pathway and Effects on Neural Tube Development

Project Number: E0702001

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

The Division specializes in research to understand how toxicants may induce birth defects such as neural tube defects. This research project addresses the role that the vitamin folic acid may play in the normal closure of the neural tube. This research supports current thinking that diet may play a role in the development of normal offspring and that interactions between diet and toxicants may be important in producing certain birth defects. The research addresses fundamental mechanisms by which the enzymes that metabolize folic acid may be involved and whether the addition of folic acid or other vitamins would overcome the toxicity of and result in normal neural tube closure and thus in normal offspring. The specific goals of the project are:

1. To determine if knocking out 5,10-methyltetrahydrofolate (MTHFR) enzyme activity in mouse embryos in vitro produces neural tube defects.
2. To determine if the addition of exogenous 5-methyltetrahydrofolate is able to overcome the lack of MTHFR activity and produce normal closed neural tubes in mouse embryos treated in vitro.
3. To determine if the addition of methionine overcomes the lack of MTHFR activity and results in normal closed neural tubes in mouse embryos treated in vitro.
4. To determine if knocking out methionine synthase (MS) activity in mouse embryos in vitro produces neural tube defects.
5. To determine if the addition of methionine is able to overcome the lack of MS activity and produce closed neural tubes in mouse embryos treated in vitro.
6. To determine if exogenous vitamin B12 is able to overcome the lack of MS activity and produce closed neural tubes in mouse embryos treated in vitro.
7. To determine if knocking out methionine adenosyltransferase (MAT) enzyme activity in mouse embryos in vitro produces neural tube defects.
8. To determine if the addition of exogenous methionine is able to overcome the lack of MAT activity and produce closed neural tubes in mouse embryos treated in vitro.
9. To determine if the addition of exogenous 5-methyltetrahydrofolate is able to overcome the lack of MAT activity and produce closed neural tubes in mouse embryos treated in vitro.

FY 2001 Accomplishments:

1. Experimental work was completed on this project during the last year.
2. The manuscript on micro-injection of homocysteine into the amniotic sac to examine the direct effects of excess methionine on embryonic development was published.
3. The manuscript on modulation of folate binding protein-1 and its regulatory element is still undergoing revision and will be submitted for publication soon.

FY 2002 Plans:

1. Submit manuscript on knock-down of folate binding protein-1 and its regulatory sequence.
2. Submit final report.

Title: Indices of Biotin Nutrition

Project Number: E0703401

Collaborator: None

Strategic Research Goal: Concept-Driven

Objective(s):

To determine the human requirement for biotin in normal individuals and in individuals in certain circumstances in which biotin status may be impaired. We will determine whether biotin of similar severity to that observed in human pregnancy can cause significantly increased rates of fetal malformation in the mouse. In the pilot mouse study, marginal biotin deficiency in mouse dams that caused an increase in 3-HIA excretion similar to that seen in human pregnancy produced 10% incidence of cleft palate in the fetal mouse.

FY 2001 Accomplishments:

A draft manuscript was prepared and is currently being revised. A renewal grant was submitted to NIH, and initial indications are that it will be funded.

FY 2002 Plans:

1. Submit manuscript on effects of biotin deficiency during pregnancy in the mouse.
2. Submit final report.

Title: Predictability of Animal Data for Human Developmental Toxicity

Project Number: E0703401

Collaborator: CDER

Strategic Research Goal: Predictive Toxicology

Objective(s):

To determine if the animal tests currently required for pre-market approval of drugs are adequately predicting possible developmental toxicity risk for humans. This is a collaborative project with CDER in which the already-existing data are abstracted and utilized to help determine whether animal data can be used to predict human health. Both the published literature and data in CDER files will be utilized. The specific strategy for conducting this analysis is as follows:

1. To retrieve reports of human data from published literature and FDA files for therapeutic agents for which there are adequate data to indicate either positive effects or no effect.
2. To retrieve reproductive and developmental toxicity study data in laboratory animals from FDA files or directly from pharmaceutical companies on the same products.
3. To extract specific data elements into a database for qualitative and quantitative comparison.
4. To evaluate data using the expertise of pharmacology/toxicology and clinical/epidemiology project participants.
5. To conduct statistical analyses, initially using multiple-regression analyses and correlation approaches, with more sophisticated analyses as the data permit.
6. To draw conclusions about the predictability of animal testing data and recommend design improvements as appropriate.

FY 2001 Accomplishments:

A manuscript comparing pharmacokinetic aspects of altenolol was published by our collaborators. A draft manuscript comparing the data for human and rodent embryotoxicity of altenolol has been prepared and is currently being revised.

FY 2002 Plans:

A manuscript comparing the embyrotoxicity of altenolol in humans and rodents will be submitted for publication. The final report will be submitted.

Title: Mechanism(s) of Folate-Responsive Dysgenesis

Project Number: E0707401

Collaborator: None

Strategic Research Goal: Concept-Driven

Objective(s):

In this project the specific mechanisms by which folate impacts the events that lead to the closure of the neural tube during fetal development will be investigated. The specific aspects and goals of the project are:

1. To determine if there is concordance between the expression of the folate receptor (FBPI) and the most proliferative cohorts of neural tube- and neural crest-cells during defined 12-hour windows on each day of gestation (GD) from GD 5 to GD 15, and to determine if the loss of these cohorts of cells during these windows of anti-folate exposure gives rise to recognizable neural tube defects and neurocristopathies in the fetus at term.
2. To characterize the basal expression of FBPI isoforms; and extent and mechanism of FBPI regulation in the placenta and various fetal tissues on GD 17 among cohorts of dams fed a folate-deficient or folate-replete diet.
3. To determine if sustained quenching of placental cytotrophoblast FBPI by antisense FBPI cDNA over-expression from GD 8 to GD 16 during maternal folate deficiency has an adverse impact on cytotrophoblastic proliferation leading to small placentas and global growth retardation of fetuses.
4. To demonstrate that neural tube closure and neural crest cell function in the whole mouse embyro at GD 8.5 can be perturbed by down-regulating FBPI expression in neural tube cells through the introduction of antisense oligonucleotides to the 43-kDa trans-factor which is required for FBPI transcription.

FY 2001 Accomplishments:

Experimental work was started on the methotrexate and folate deficiency experiments. Female mice were fed diets with various levels of folic acid added for eight weeks prior to breeding. The animals were then bred and continued to consume the same chow during gestation. Fetuses were examined at the end of gestation for effects on weight and the incidence of various malformations. Few malformations were observed in this study, and samples indicated that plasma folate levels were not significantly altered by the treatments. An additional experiment was started to lower the folate levels even further to examine the effects of these very low folate levels on normal embryonic development. Further analysis of the initial diet experiment is on-going.

A second experiment was conducted in which methotrexate was administered at 12- hour intervals during gestation beginning on day 5 of gestation and continuing until day 15. Twelve (12) hours after the methotrexate injection, an osmotic mini-pump containing folinic acid was implanted into the mice to rescue the animals from the effects of methotrexate. Methotrexate was used for its anti-folic acid effects, so the exposure to the anti-folate was only 12 hours long. The purpose of this experiment was to determine if these short periods of anti-folate exposure would alter particular cell types to produce specific malformations. Different malformations were produced by methotrexate exposure and consisted of exencephaly, cleft palate, open eye, micrognathia, and various skeletal anomalies. Fetal weight was adversely affected at a number of different exposures. The data were written up in abstract form and submitted for consideration for the annual meeting of the Teratology Society. The data are also being analyzed further.

FY 2002 Plans:

1. Complete analysis of the methotrexate experiment and the first dietary experiment.
2. Complete the second dietary experiment.

Title: Examination of Embryonic Gene Expression during Neural Tube Closure

Project Number: E0710901

Collaborator: None

Strategic Research Goal: Concept-Driven

Objective(s):

1. To determine which genes may demonstrate altered expression as a result of valproic acid treatment.
2. To determine if this altered expression is related to teratogenicity in general or to valproic acid specifically.
3. To determine if gene expression is altered in subsets of cells that may be involved in teratogenicity.

FY 2001 Accomplishments:

The protocol was approved in early 2002.

FY 2002 Plans:

Experimental work will begin.

PI: Harris, Angela

Title: Modulation of Gene Expression in Chemical Carcinogenesis: Analysis of Aflatoxin B1 Induced Gene Expression in Human Hepatocytes

Project Number: E0704701

Collaborator: None

Strategic Research Goal: Concept-Driven

Objective(s):

This project is a part of the Division's genomic/proteomics focus area. The goal of this project is to utilize the new gene expression technologies to understand which genes are affected by the exposure of human hepatocytes to a known human carcinogen (aflatoxin B1). Because this is a new technology, the experimental approach must include research to define the appropriate experimental parameters. Specific goals are to:

1. Verify aflatoxin B1 effects on steady-state mRNA levels of eight genes previously identified by differential hybridization of a gene filter array to be aflatoxin B1 (AFB1)-responsive in human hepatocytes.
2. Use Northern blot, RT-PCR, and/or RNA protection assay to establish AFB1 time- and dose-response curves for maximal gene expression and also determine the minimum dose at which gene expression can be detected.
3. To identify additional AFB1-induced genes using differential display PCR (DD-PCR) and differential hybridization of a high-density filter array utilizing mRNA from human hepatocytes treated with low, moderate and cytotoxic levels of AFB1.
4. To evaluate selected genes as described for Objective 1.
5. To distinguish genes involved in toxicological response to AFB1 exposure from those that contribute to the carcinogenic response by comparing the gene expression profile of human hepatocytes treated with the hepatotoxic non-carcinogenic chemical, acetaminophen.
6. To compare gene expression of selected genes in human hepatocytes treated with known rat liver chemical carcinogens, including 2-acetylaminofluorene (2-AAF), dimethylnitrosamine (DMN) and methapyrilene.

FY 2001 Accomplishments:

1. Completed the analysis of gene expression data from AFB1- and APAP-treated primary human hepatocytes from five donors and three donors, respectively.
2. Completed the analysis of gene expression data from AFB1 and APAP treated HepG2 cells.
3. Evaluated the effects of culturing matrix on basal gene expression levels of primary rat and human hepatocytes using gene filter arrays.
4. Completed analysis of the culturing matrix data.

FY 2002 Plans:

1. Procure human hepatocytes from three additional male donors to analyze the effects of 2-acetylaminofluorene (2-AAF) and dimethylnitrosamine (DMN). Compare to AFB1 and APAP data.
2. HepG2 cells will be treated as in #1 for comparison.
3. RNA from objectives #1 and #2 will be used to screen approximately 35,000 human genes using filter-array analysis for genes that are differentially expressed after toxicant exposure.
4. RNA from methapyrilene and pyrilamine treated primary rat hepatocytes will be used to screen an additional 10,000 rat genes for differential expression after exposure. Northern blot or RT-PCR analysis will be used to verify differential gene expression.

PI: Heflich, Robert

Title: Effect of Azathioprine in Somatic Cell and Germline Hprt Mutant Frequencies in the Mouse

Project Number: E0709901

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Test the hypothesis that in vivo selection by azathioprine affects both somatic cell and germline Hprt mutant frequencies using the mouse.

FY 2001 Accomplishments:

Protocol prepared and submitted for review.

FY 2002 Plans:

1. Complete review and approval of protocol.
2. Begin Experiment 1: azathioprine range-finding study in male C57Bl/6 mice to establish a treatment protocol that results in a lymphocyte Hprt mutant frequency of >5% with retention of reasonable fertility.

PI: MacGregor, James

Title: An Efficient Regulatory Method for Evaluating Chromosomal Damage

Project Number: E0714001

Collaborator: CDER, CFSAN, CVM, Litron Labs

Strategic Research Goal: Method-Driven

Objective(s):

A collaborative project to evaluate a new method for monitoring chromosomal damage, involving NCTR, CDER, CFSAN, CVM and Litron Laboratories has been initiated. Flow cytometric scoring of micronucleated cells in peripheral blood samples is being compared with traditional microscopic scoring, and the kinetics of micronucleated cell appearance and disappearance is being determined in species of regulatory interest (rat, dog, non-human primate, human). It is expected that the new methodology, by allowing measurement in peripheral blood rather than bone marrow, will permit integration of studies of chromosomal damage into routine toxicological studies and will facilitate evaluation of chromosomal damage in human studies.

FY 2001 Accomplishments:

FY 2002 Plans:

1. Compare flow cytometry method with microscopic scoring in the rat and dog.
2. Determine kinetics of micronucleated cell formation and elimination in the rat and dog.

PI: Manjanatha, Mugimane

Title: ADDEND: Micronucleus and Gene Mutation Analysis in F344 Big Blue Rats Adminis-tered Leucomalachite Green in the Diet for 4, 16, and 32 weeks

Project Number: E0212821

Collaborator: None

Strategic Research Goal: Agent-Driven

Objective(s):

Malachite and leucomalachite green are currently being tested for carcinogenicity under the National Institute of Environmental Health Sciences Interagency Agreement (NIEHS IAG). The objective of this project is to assess the mutagenicity of leucomalachite green in relation to DNA adduct formation in tissues of Big Blue rats.

FY 2001 Accomplishments:

Screened 72 control and leucomalachite green-treated rats for lacI mutations in the liver tissues. Mutant frequency VS doses were plotted and data were presented at the 2001 TSSRC meeting. Hprt mutant frequency was also determined in the spleenic lymphocytes of the treated and control animals. In addition, micronucleus frequency was determined in the bone marrow of treated and control rats. LacI mutational spectrum was developed for 16 week exposure and statistical analysis of mutational data was performed for evaluating any differences between control and treated spectra.

FY 2002 Plans:

Animals exposed to leucomalachite green for 32 weeks will be evaluated for the lacI mutant frequency (MF) in the liver, Hprt MF in the lymphocytes and micronucleus frequency in the bone marrow. If there is any significant MF increase, the mutants will be further characterized for types of mutations. The results will be presented at the next TSSRC meeting at NCTR.

Title: ADDEND: Micronucleus and Gene Mutation Analysis in F344 Big Blue Rats Adminis-tered Leucomalachite Green in the Diet for 4, 16, and 32 weeks

Project Number: E0212831

Collaborator: None

Strategic Research Goal: Agent-Driven

Objective(s):

Malachite and leucomalachite green are currently being tested for carcinogenicity under the NIEHS/NCTR IAG. Recent results from addendum E212821, indicate a two-fold increase in lacI mutations in the livers of Big Blue rats fed leucomalachite green for 16 weeks. The objective of this addendum is to expand the analyses of the remaining rats on E212821 (32-week dose groups) to include additional indicators of hepatic toxicity.

FY 2001 Accomplishments:

The proposal to evaluate 32 weeks-treated-rats for hepatic toxicity began.

FY 2002 Plans:

Pathology services will evaluate hepatic toxicity of rats exposed to 32 weeks of leucomalachite green in diet.

Title: ADDEND: Quantitative and Molecular Analysis of 7,12-dimethylbenz[a]anthracene (DMBA) induced Mutations in the Model Blue Rat: Comparison of Mutagenesis in the Trans-gene lacI with the Endogenous Gene Hprt and Cancer Genes H-ras and p53

Project Number: E0690601

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

1. To determine the mutant frequency and mutation spectrum of the lacI transgene of the Blue Rat following exposure to DMBA in surrogate and target tissues and compare these mutant frequencies and mutational spectra to those determined in Objectives 2 and 3.
2. To determine the mutant frequency and mutation spectrum of the endogenous Hprt reporter gene in T-lymphocytes from the spleens of Fischer 344 and Blue Rats following exposure to DMBA.
3. To induce mammary tumors in Fischer 344 rats and Blue Rats by exposure to DMBA and screen tumor DNA for mutations in the oncogene, H-ras and the tumor suppressor gene, p53.

FY 2001 Accomplishments:

1. This was a long-term project concluded in FY 2000.
2. The technical report will be prepared soon.
3. Several manuscripts generated by this project have been published.

FY 2002 Plans:

This project will not continue. A final manuscript derived from this project is under preparation.

Title: ADDEND: Quantitative and Molecular Analysis of 7,12-dimethylbenz[a]anthracene (DMBA) induced Mutations in the Model Blue Rat: Comparison of Mutagenesis in the Trans-gene lacI with the Endogenous Gene Hprt and Cancer Genes H-ras and p53

Project Number: E0690611

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

NCTR protocol E0690601 was undertaken in order to validate the use of the Big Blue rat as a model for determining the in vivo mutagenicity of potential human toxicants.

FY 2001 Accomplishments:

Results from experiment 1 uncovered unanticipated issues concerning the nature of mutagenic responses in the Big Blue model. These results suggest experiments not included in the original protocol that may resolve these issues. Necessitates using additional animals to complete experiments 1, 2, and 3. However, no work was performed as it was decided not to pursue this project.

FY 2002 Plans:

The technical report will be written.

PI: Mckinzie, Page

Title: Application of the MutEx/ACB-PCR Method of Genotypic Selection to the Detection of K-ras Mutations

Project Number: E0706601

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

The majority of methods for quantitating mutation require the use of selective drugs which allow mutants to grow and prevent normal cells from growing. All of these techniques require extensive cell culture and can be time-consuming and expensive. There are techniques that utilize genotypic selection that allow for a molecular amplification of the rare mutant DNA sequences and thus provide for a direct measurement of mutant frequencies. The Division has several projects in which this new technology is being developed. The specific goals of this project are to:

Establish assays that can provide mechanistic data for chemical risk assessment and aid in establishing the relevance of rodent models for predicting human risk. The proposed research approach is to apply a recently developed method, MutEx/ACB-PCR to the detection of human and rodent k-ras GGTà GAT and GGTà GTT mutations. The assays will then be used to study the chemical induction of these mutations.

FY 2001 Accomplishments:

1. Determined optimized ACB-PCR conditions for human K-ras codon 12 GGT à GAT detection at a sensitivity of 10-5.
2. Determined optimized ACB-PCR conditions for human K-ras codon 12 GGT à GTT detection at a sensitivity of 10-5.

FY 2002 Plans:

1. Make ACB-PCR DNA standards for rat K-ras codon 12 GGT à GAT and GGT à GTT mutations.
2. Optimize ACB-PCR for the rat K-ras DNA standards.
3. Continue with MutEx enrichment for human K-ras codon 12 mutant standards.
4. Begin validation of method with in vitro mutagen exposure experiments.

PI: Mittelstaedt, Roberta

Title: ADDEND: Measurement of H-ras Codon 61 CAA->AAA Mutation in Mouse Liver DNAs using the MutEx/ACB-PCR Genotypic Selection

Project Number: E0704121

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Quantify and identify lacI mutations in liver DNA of mice treated as neonates with 4-aminobiphenyl in order to establish mutation induction and specificity as an early event in hepatic tumorigenesis.

FY 2001 Accomplishments:

Tissue collection is complete and lacI mutational analysis is nearly complete.

FY 2002 Plans:

Publish the results.

Title: ADDEND: Measurement of H-ras Codon 61 CAA->AAA Mutation in Mouse Liver DNAs using the MutEx/ACB-PCR Genotypic Selection

Project Number: E0704141

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Project intended to serve as a recruiting tool for a UAMS Interdisciplinary Toxicology graduate student. Results from these experiments will support experimentation being conducted with neonatal mice in E0704121.

FY 2001 Accomplishments:

The protocol was written and the animals have been ordered.

FY 2002 Plans:

Treat the animals, collect the tissues, and analyze the mutations.

PI: Morris, Suzanne

Title: p53 Transgenic Mouse Evaluations of Genistein 28-day and 36-week Studies

Project Number: E0213601

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

The phytoestrogen genistein is a primary component of a high soy diet. There is currently widespread interest in the impact of a high soy diet on human health. While there is some indication that phytoestrogens may improve health in peri- and post-menopausal women, there is concern that these compounds may have the potential to be carcinogens. The study is being conducted to investigate this possibility. Specific goals for the project are:

1. To determine the toxicity of genistein in the C57Bl6/J strain and to select doses for the 36-week studies.
2. To identify the potential carcinogenicity of genistein in the p53 transgenic mouse model.
3. To determine if the potential carcinogenicity of genistein relates to changes in the rates of cell death and cell proliferation.
4. To determine if exposure to genistein results in an increase in the mutant frequency in a reporter gene (Hprt) in the splenic lymphocytes of the p53 mouse.

FY 2001 Accomplishments:

In-life phase, 28-day dose-range-finding study completed; statistical analysis is ongoing with completion expected in early FY 2002.

FY 2002 Plans:

1. Manuscript in preparation from the 28-day dose-range-study.
2. Chronic phase of the experiment cancelled.
3. Technical report to be prepared in late FY 2002.

Title: ILSI/HESI Consortium on Application of Genomics and Proteomics to Mechanism-Based Risk Assessment

Project Number: P00425

Collaborator: CDER

Strategic Research Goal: Knowledge Base

Objective(s):

1. To establish a database for genomics data.
2. To relate the changes in gene expression to in vitro genotoxicity measures that are utilized in hazard assessment.

FY 2001 Accomplishments:

1. Gene-tox studies completed with BPDE and gamma radiation; initial studies with cDNA microarrays completed.
2. Data presented at ILSI workshops in March (San Diego) and May (Washington, DC).
3. Seminars presented at CDER (MOD-1) and FDA (Parklawn) for the FDA; poster presented at the 2001 European Environmental Mutagen Society in Ghent, Belgium; seminar presented at Mario Negri Research Institute in Milan, Italy.

FY 2002 Plans:

1. Analysis of expression changes from TK6 cells exposed to BPDE or gamma radiation as detected by cDNA microarrays is currently underway.
2. One manuscript is in preparation and a second manuscript will follow as the data are analyzed.

PI: Parsons, Barbara

Title: Measurement of H-ras Codon 61 CAA->AAA Mutation in Mouse Liver DNAs using the MutEx/ACB-PCR Genotypic Selection

Project Number: E0704101

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

The majority of methods for quantitating mutations require the use of selective drugs that allow mutants to grow and prevent normal cells from growing. All of these techniques require extensive cell culture and can be time-consuming and expensive. There are techniques that utilize genotypic selection that allow for a molecular amplification of the rare mutant DNA sequences and thus provide for a direct measurement of mutant frequencies. The Division has several projects in which this new technology is being developed. The specific goals of this project are:

1. To quantify somatic mutations in liver DNA of mice treated with 4-aminobiphenyl in order to establish and evaluate MutEx/ACB-PCR genotypic selection as an approach for human risk assessment.
2. To determine whether or not the MutEx/ACB-PCR genotypic selection is sensitive enough to measure the spontaneous frequencies of H-ras codon 61 CAA->AAA mutation in three different mouse models: B6C3F1, C57BL/6, and the Pms2 mismatch repair-deficient, transgenic mouse.

FY 2001 Accomplishments:

1. The 4-aminobiphenyl newborn mouse assays in three different strains of mice (C57BL/6, B6C3F1, and Pms2 mismatch repair-deficient [-/-] mice) have been completed.
2. The ability to measure the background level of spontaneous, oncogenic point mutations in non-tumor tissues was demonstrated.
3. A manuscript entitled, "Prospects for Applying Genotypic Selection of Somatic Oncomutation to Chemical Risk Assessment," was accepted for publication in Mutation Research.
4. A presentation entitled, "Measurement of Rare Point Mutation for Use in Risk Assessment," was presented at the Breakfast Meeting of the New Technologies Interest Group, 32nd Annual Meeting of the Environmental Mutagen Society.
5. A poster entitled, "Detection of spontaneous mouse H-ras mutation by genotypic selection; PNA enrichment of target sequences and analysis by MutEx/ACB-PCR" was presented at the Breakfast Meeting of the New Technologies Interest Group, 32nd Annual Meeting of the Environmental Mutagen Society.
6. A manuscript entitled, "Developing Methods of Genetic Analysis to Improve Cancer Risk Assessment," was accepted for publication in the FDA’s new online journal, Regulatory Research Perspectives.

FY 2002 Plans:

1. Publish the PNA-based, gene-specific enrichment method, a method that is useful for isolating a population of "target" DNA molecules for mutational analyses.
2. Publish a report on the use of ACB-PCR for identifying secondary mutations that occur during mouse liver tumor progression.
3. Analyze the liver DNAs of 4-aminobiphenyl-treated and untreated C57BL/6, B6C3F1 and Pms2 mismatch repair-deficient mice for H-ras codon 61 mutation using PNA selection coupled with MutEx/ACB-PCR.

Title: ADDEND: Measurement of H-ras Codon 61 CAA->AAA Mutation in Mouse Liver DNAs using the MutEx/ACB-PCR Genotypic Selection

Project Number: E0704111

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Due to failure of a freezer, liver tissues being stored collected under the master protocol were thawed. The livers of the one-month post-treatment time point of the newborn mouse assay were destroyed. Additional animals and resources were requested in order to repeat the one-month timepoint of the B6C3F1 newborn mouse assay.

FY 2001 Accomplishments:

Newborn B6C3F1 mice were treated with 4-aminobiphenyl or the dimethylsulfoxide control and liver tissues were harvested one-month post-treatment, thereby replacing the tissues that were lost.

FY 2002 Plans:

The amount of H-ras codon 61 CAA to AAA mutant DNA sequence in these tissues will be measured using a sensitive DNA-based mutation detection method, MutEx/ACB-PCR.

Title: Pms2 Mismatch Repair-Deficient Mouse Breeding Colony

Project Number: S00222

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

The objective is to maintain a breeding colony of Pms2 transgenic mice, which are not available commercially, so this strain can be used in future protocols. Because these animals are mismatch repair-deficient, they accumulate mutation to a greater extent than their mismatch repair-proficient counterparts and, therefore, are a valuable mouse model for mutation research. Heterozygous Pms2+/- animals will be maintained and, as protocols are developed, we will have the capacity to breed the necessary Pms2+/-, mismatch repair-deficient animals.

FY 2001 Accomplishments:

A protocol was prepared describing how a mismatch repair-deficient mouse colony (Pms2 mouse) will be established at NCTR because an animal model that is unable to repair chemically induced DNA damage may provide a sensitive system for genetic toxicology studies.

FY 2002 Plans:

Pms2, mismatch proficient (+/-) breeders will be maintained so that mismatch repair deficient Pms2-/- animals can be bred as needed for other genetic toxicology studies.

PI: Shaddock, Joseph

Title: ADDEND: Lymphocyte Hprt Mutant Frequencies and Types of Mutations in Pms2 Mice Treated as Neonates with Solvent or with 4-aminobiphenyl

Project Number: E0704131

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

Because cancer is a disease requiring the induction of mutation and the clonal expansion of mutated cells, one would expect that the developing fetus and young infant would be particularly susceptible to carcinogen exposure. This project provides information that can be used to evaluate this hypothesis. Experiments will be conducted to quantify and identify the Hprt mutations in spleen lymphocytes of Pms2+/+, Pms2+/-, and Pms2-/- mice treated as neonates with either dimethlysulfoxide (solvent control) or with 4-aminobiphenyl (4-ABP).

FY 2001 Accomplishments:

1. Collection and analyses of Hprt mutant frequencies in 4-ABP-treated and control Pms2 +/+, +/-, and -/- mice was completed.
2. Characterization of Hprt mutant clones and mutant sequencing began.
3. Preliminary data were presented in a poster at the Environmental Mutagen Society (EMS) Meeting in March 2001.

FY 2002 Plans:

The characterization of mutant clones and mutant sequencing will continue until complete and the mutational spectra can be compared between treated and control animals, among animals with different Pms2 genotypes, and presumably different DNA repair capacity.

PI: Valentine, Carrie

Title: The Development of Transgenic Mice Harboring Bacteriophage f X174 with Sites Specific for Detecting Mutations at Guanosine: Cytosine Nucleotides, Small Frameshifts, and Extended Deletions

Project Number: E0700201

Collaborator: None

Strategic Research Goal: Predictive Toxicology

Objective(s):

To find specific mutations in bacteriophage f X174 that render the bacteriophage non-infectious and that will revert to plaque-forming ability only when mutation occurs by specific mechanisms: 1) base substitution at a G:C base pair, or 2) frameshift caused by deletion of one or two nucleotides. An additional objective is to determine the feasibility of using f X174 to detect the deletion of an extended sequence. Phage- harboring these mutations will be used to construct a transgenic mouse model for measuring mutations in vivo.

FY 2001 Accomplishments:

1. Manuscript accepted for publication in Environmental and Molecular Mutagenesis.
2. Forward mutational assay developed for gene A of bacterial virus F X174 carried in mouse cells.
3. Target sites and mutant spectrum identified for the F X174 gene A forward mutational assay in untreated cells and cells treated with the mutagen ethyl nitrosourea (ENU).
4. License offered for forward assay in Federal Register.
5. Frameshift assay developed that detects –1 A deletions.
6. Assay begun on ENU- and solvent-treated animal tissues to evaluate sensitivity using single-burst analysis.

FY 2002 Plans:

Continue sensitivity studies until protocol E0711501 is approved.

FY 2001 Publications

Cada, A.M., Hansen, D.K., Laborde, J.B. and Ferguson, S.A., Minimal behavioral or developmental effects from developmental exposure to St. John's Wort (Hypericum perforatum) in Sprague Dawley rats, Nutritional Neuroscience, 4:135-141. Accepted: 11/4/00 (N/A)

Chen, T., Mittelstaedt, R.A., Aidoo, A., Hamilton, L.P., Beland, F.A., Casciano, D.A. and Heflich, R.H., Hprt and lacI mutant frequency in relation to DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue Rats, Environmental and Molecular Mutagenesis. Accepted: 12/7/00 (E0695801)

Delongchamp, R.R., Valentine, C.R. and Malling, H., Estimation of the average burst size of Phi X174am3, cs70 for use in mutation assays with transgenic mice, Environmental and Molecular Mutagenesis, 37:356-360. Accepted: 4/14/01 (E0709501, S00032)

Desai, V.G., Aidoo, A., Casciano, D.A. and Feuers, R.J., Activity profile of glutathione-dependent enzymes and respiratory chain complexes in rats supplemented with antioxidants and treated with carcinogens, Archives of Biochemistry and Biophysics, 394(2):255-264. Accepted: 7/26/01 (E0701401)

Djuric, Z., Lewis, S.M., Lu, M., Mayhugh, M.A., Tang, N. and Hart, R.W., Effect of Varying Dietary Fat Levels on Rat Growth and Oxidative DNA Damage, Nutrition and Cancer, 39:214-219. Accepted: 2/7/01 (E0260001)

Domon, O.E., McGarrity, L.J., Bishop, M.E., Yoshioka, M., Chen, J.J. and Morris, S.M., Evaluation of the genotoxicity of the phytoestrogen, coumestrol, in AHH-1 TK+/- human lymphoblastoid cells, Mutation Research, 474:129-137. Accepted: 3/1/01 (E0705501)

Duffy, P.H., Feuers, R.J., Seng, J.E., Lewis, S.M., Mayhugh, M.A., Aidoo, A. and Casciano, D.A., The Effects of Different Levels of Dietary Restriction on Aging and Survival in the Sprague Dawley Rat: Implications for Chronic Bioassay Studies, Aging, 13:263-272. Accepted: 12/5/00 (E0692401)

Duffy, P.H., Lewis, S.M., Mayhugh, M.A., Casciano, D.A. and Feuers, R.J., Effect of the AIN-93M Purified Diet and Dietary Restriction on Survival in the Sprague Dawley Rat: Implications for Chronic Studies, The Journal of Nutrition. Accepted: 9/18/01 (E0692401)

Fang, H., Tong, W., Shi, L., Blair, R.M., Perkins, R.G., Branham, W.S., Hass, B.S., Xie, Q., Dial, S.L., Moland, C.L. and Sheehan, D.M., Structure-Activity Relationships for a Large Diverse Set of Natural, Synthetic and Environmental Estrogens, Chem. Res. Toxicol., 14(3):280-294. Accepted: 2/8/01 (E0290001)

Gamboa da Costa, G., Hamilton, L.P., Heflich, R.H., Marques, M.M. and Beland, F.A., DNA adduct formation and mutant induction in Sprague Dawley rats treated with tamoxifen and its derivatives, Carcinogenesis, 22:1307-1315. Accepted: 11/1/00 (E0701101)

Gamboa da Costa, G., Manjanatha, M., Marques, M.M. and Beland, F.A., Induction of lac1 mutations in Big Blue Rats treated with tamoxifen and alpha-hydroxytamoxifen, Cancer Letters, 176:37-45. Accepted: 8/27/01 (E0701101)

Hong, H., Tong, W., Fang, H., Shi, L., Xie, Q., Wu, J., Perkins, R.G., Branham, W.S. and Sheehan, D.M., Prediction of estrogen receptor binding for 58,000 chemicals using an integrated system of a tree-based model with structural alerts, Environmental Health. Accepted: 6/5/01 (E0290001)

Khaidakov, M., Bishop, M.E., Manjanatha, M., Lyn-Cook, L.E., Desai, V.G., Chen, J.J. and Aidoo, A., Influence of dietary antioxidants on the mutagenicity of the model mammary carcinogen 7,12-dimethylbenz[a]-anthracene and the antitumor agent bleomycin in female rats, Mutation Research, 480:163-170. Accepted: 2/2/01 (E0701401)

Khaidakov, M., Manjanatha, M. and Aidoo, A., Molecular analysis of mitochondrial DNA mutations from bleomycin treated rats, Mitochondrian. Accepted: 9/17/01 (E0701401)

Li, J., Feuers, R.J., Buffington, C.K. and Cowan, G.S., Influence of body fat distribution on cardiorespiratory response to exercise testing in bariatric surgical morbidly obese females (Fat distribution and Exercise testing), Journal of Respirology, 6(1):9-13. Accepted: 4/17/01 (E0699801)

Mckinzie, P.B., Delongchamp, R.R., Heflich, R.H. and Parsons, B.L., Prospects for Applying Genotypic Selection of Somatic Oncomutation to Chemical Risk Assessment, Reviews in Mutation Research, 489:47-78. Accepted: 6/22/01 (E0704101)

Monroe, J.J., Manjanatha, M. and Skopek, T.R., Extent of CpG methylation is not proportional to the in vivo spontaneous mutation frequency at transgenic loci in Big Blue rodents, Mutation Research, 476:1-11. Accepted: 1/15/01 (E0690601)

Morris, S.M., Pipkin, J.L., Hinson, W.G., Shaddock, J.G., Tolleson, W.H. and Casciano, D.A., Decreased in vitro interaction between p53 and nuclear stress proteins in the p53-deficient mouse, Electrophoresis, 22:2092-2097. Accepted: 12/2/00 (E0694901)

Parsons, B.L. and Mckinzie, P.B., Developing methods of genetic analysis to improve cancer risk assessment, Regulatory Research Perspectives, 1(2):1-11. Accepted: 9/18/01 (E0704101)

Slikker, W., Desai, V.G., Duhart, H.M., Feuers, R.J. and Imam, S.Z., Hypothermia enhances Bcl-2 expression and protects against oxidative stress induced cell death in chinese hamster ovary cells, Free Radical Biology and Medicine, 31:405-411. Accepted: 5/1/01 (E0698301)

Young, J.F., Wosilait, W.D. and Luecke, R., Analysis of Methyl Mercury Disposition in Humans Utilizing a PBPK Model and Animal Pharmacokinetic Data, Journal of Toxicology and Environmental Health, Part A, 63(1):19-52. Accepted: 10/2/00 (P00393)