Center for Environmental and Rural Health

Stephen H. Safe, Ph.D.

Department of Veterinary Physiology and Pharmacology

Texas A&M University

College Station, Texas 77843-4466

PH: (409) 845-5988

FAX: (409) 862-4929

Introduction: Environmental health research at Texas A&M University has experienced considerable growth during the past 15 years. Increased numbers of faculty with primary research interests in the environmental health sciences, coupled to nationally recognized training programs in Toxicology, Nutrition, Reproductive Biology, and Genetics have placed Texas A&M University among the better research institutions in the country. The next phase of growth for environmental health research at Texas A&M University requires a more comprehensive level of interdisciplinary coordination and development of centralized core facilities that can facilitate the overall research process, synergize the quality of individual research programs, and help attract young faculty into environmental health-related research. With these objectives in mind, the overall goal of the proposed Center for Environmental and Rural Health (CERH) at Texas A&M University is to foster and promote basic and applied science programs focusing on the impact of environmental factors on human health, particularly as it relates to rural communities in the State of Texas and the nation.

Research Cores: The Center consists of four integrated research cores. The Biostatistics and Epidemiology research core has targeted several interrelated research projects including development of improved models for risk assessment, sampling and environmental exposures and the epidemiology of breast cancer and pregnancy outcomes. Investigators in the Cellular and Molecular Biology/Toxicology (CMBT) core are studying the pathogenesis of environmentally-related diseases at the cellular and molecular level. The Nutrition research core is focused on the role of nutritional and environmental factors on various disease outcomes including colon cancer and immune-mediated inflammatory disease. The Reproductive and Developmental Biology research core is focused on the adverse impacts of environmental toxicants on all aspects of reproduction and development including issues of gametogenesis, conception, maintenance of pregnancy and normal embryonic morphogenesis. Research cores meet every month to discuss both individual and collaborative areas of interest.

Facilities Cores: The CERH supports six Facility cores that service the research programs of core faculty members, their students, postdoctoral fellows and staff. The Biological Mass Spectrometry core facilities utilize state-of-the-art technologies for analysis of peptides and proteins for CERH investigators. The Biostatistics and Computational core provides extensive network and software support, data management and statistical support for ongoing research projects. The DNA Technology core provides technical assistance for designing DNA primers and carries out routine DNA synthesis, sequencing and interpretation of sequence data. The Field Services core assists investigators in development and implementation of sampling strategies, provides analytical services and QA-QC plans for various projects. The Image Analysis core provides a host of non-invasive imaging tools that can be used to probe the role of environmental toxicants and nutritional factors on cellular homeostasis and cytotoxic responses to various agents. The Transgenic laboratory provides comprehensive and integrated support for Center investigators in the generation and characterization of transgenic mice produced by pronuclear injection as well as homologous recombination in a more cohesive and cost-efficient manner.

Community Outreach and Education: The COEP has been operational for 3 months during which significant progress has been made in advancing the goals of the Center. Ongoing outreach activities are focused on training and education in human health and the environment in the Lower Rio Grande Valley and the Brazos County. In collaboration with the School of Rural Public Health, the COEP started using Telecommunication Transmission Video Network to reach the Progresso community and other partners in South Texas. Brochures and handouts have been developed to promote better understanding of the oral presentations and the education of individuals who can transmit environmental and health information in theirs communities. On the local front, efforts include coordination of a weekly TV Show featuring topics on human health and the environment, the production of articles focusing environmental health issues for bilingual education, and collaboration with established local community outreach programs in the Brazos Valley.

Pilot Projects: The CERH provides financial support for an annual Pilot Project Program that funds research projects consistent with the major scientific aims and objectives of the Center. This research also facilitates collaborative interactions among faculty. Four projects were funded in 1998 ($15,000/project) and this seed money will support preliminary research by the investigators and enhance their success in competing for external funding.

Title: Levels of Maternal Serum alpha-fetoprotein (AFP) in Pregnant Women and Subsequent Breast Cancer Risk

Significance: High maternal serum alpha-fetoprotein (APF) levels during pregnancy may be instrumental in reducing the subsequent risk of breast cancer. This hypothesis was tested in a nested case-control study using stored frozen sera accrued between 1959 and 1966 by the University of California at Berkeley Child health and Development Studies (CHDS) group from a cohort of pregnant women. Cases with histologically confirmed breast cancer were identified from California Cancer Registry files covering their date of enrollment in the CHDS until 1994. Controls were selected from the CHDS cohort by using randomized recruitment. Third-trimester maternal serum AFP levels were analyzed by using both a radioimmunoassay and an immunoenzymatic method. After controlling for multiple cofounders in logistic regression models, the authors found an inverse association between high levels of maternal serum AFP (top quartile) during the index pregnancy and the risk of breast cancer. The protective effect of high levels of maternal serum AFP varied by age at first full-term pregnancy (age 20 years or less: odds ratio (OR) = 0.43.95% confidence interval (CI) 0.28-0.65; age 21-23 years: OR = 0.62.95% CI.41-0.92). After age 27 years, the estimated risk exceeded unity (OR = 1.67, 95% CI 1.14-2.45). These study findings suggest that some of the protection against breast cancer conferred by early first-term pregnancy may result from high levels of maternal serum AFP. After age 27 years, a high maternal serum AFP level is not protective and may increase risk.

Reference: Richardson, B.E., Hulka, B.S., Peck, J.L. David, Hughes, C.L., van den Berg, B.J., Christianson, R.E. and Calvin, J.A. Am. J. Epidemiol. 148, 719-727, 1998.

Title: Modification of Near-Active Site Residues in Organophosphorus Hydrolase Reduces Metal Stoichiometry and alters Substrate Specificity.

Significance: Organophosphorus hydrolase (OPH, E.C. 8.1.3.1) is a bacterial enzyme which detoxifies many organophosphorus (OP) neutrotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 of OPH are located near the bimetallic active site of the protein. It has been proposed that these residues influence catalysis by interacting with active site residues and through interaction with substrate in the binding pocket of OPH. We replace the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single site modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site, all three of these altered enzymes possessed only one metal per protein monomer while retaining considerable enzymatic activity for the preferred phosphotriester (PÑO bond) substrate, paraoxon (5-100% k_cat). The three altered enzymes achieved a two- to thirty-fold increase in substrate specificity (k_cat /Km) for demeton S (PÑO bond) an analog for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (DFP, PÑF bond) was substantially decreased for each of these enzymes when compared with that of the native protein. In addition, H257L showed a six-fold increase in specificity for NPPMP, the analog for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and we suggest that changes in metal requirements may be associated with these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site.

Reference: diSioudi, B., Grimsley, J.K., Lai, K. and Wild, J. Biochemistry, (in press), 1999.

Description: The Administrative Core of the Center for Environmental and Rural Health functions to facilitate research, service and outreach activities and to ensure fiscal integrity of the Center. The routine activities coordinated by the Administrative Core include; scheduling of meetings for the Executive Committee and Research Cores, arranging biannual and annual meetings of the Internal and External Advisory boards and coordinate presentation of Research and Facility Core directors’ presentations at the External Advisory Board meetings, coordination of the collaborative visiting speakers program, and the annual CERH thematic scientific conference, development of contacts and interactions with other EHS Centers and NIEHS staff, provide support for collaborative CERH grant proposals, prepare CERH annual report, develop contacts with State and Federal elected officials and their staff to ensure that they are aware of the CERH and its potential services.

Stephen H. Safe, Ph.D., Center Director, Distinguished Professor

Department of Veterinary Physiology and Pharmacology

Kenneth S. Ramos, Ph.D., Deputy Director

Department of Veterinary Physiology & Pharmacology and

Department of Environmental and Occupational Health (School of Rural Public Health)

Laura Young, Administrative Assistant

Department of Veterinary Physiology and Pharmacology

Irma N. Ramos, M.D., Director, Community Outreach and Education Program

Department of Veterinary Physiology and Pharmacology and School of Rural Public Health

Internal Advisory Committee: The Internal Advisory committee consists of Deans from Colleges or Administrative units whose faculty are participants in the Center. This Committee provides advice on scientific and administrative concerns and also serves as an advocate for the CERH within the Texas A&M University System. Members of the Internal Advisory committee include:

Robert Kennedy, Ph.D.

Associate Provost for Graduate Studies

Office of Graduate Studies & Research

Texas A&M University

College Station, Texas 77843-3257

Edward Hiler, Ph.D.

Deputy Vice Chancellor and Associate Dean

College of Agricultural & Life Sciences

Texas A&M University

College Station, Texas 77843-3257

H. Richard Adams, Ph.D.

Dean, College of Veterinary Medicine

Texas A&M University

College Station, Texas 77843-3257

Michael Friedland, Ph.D.

Vice President for Health Affairs and Dean of Medicine

College of Medicine

Texas A&M University

College Station, Texas 77843-3257

Richard Ewing, Ph.D.

Dean, College of Science

Texas A&M University

College Station, Texas 77843-3257

Fuller Bazer, Ph.D.

Director, Institute of Biosciences and Technology

2121 West Holcombe Boulevard

Houston, Texas 77030-1201

External Advisory Committee: The External Advisory Committee was selected to provide critical input regarding the initial scientific content and objective for the CERH. They also serve as an important external review committee that will judge the quality of science by CERH faculty and provide input for changing and improving CERH programs. Members of the External Advisory Committee include:

Daniel Acosta, Ph.D.

Dean, College of Pharmacy

University of Cincinnati

3223 Eden Ave

Cincinnati, OH 45267

Steven D. Clarke, Ph.D.

M.M. Love Chair of Nutritional, Cellular, and Molecular Sciences

Department of Human Ecology

The University of Texas

Austin, TX 78712

Dennis M. Bier, Ph.D.

Director, Children’s Nutrition Research Center

Department of Pediatrics

Baylor College of Medicine

1100 Bates Street

Houston, TX 77030

Roger O. McClellan, Ph.D.

Chemical Industry Institute of Toxicology

P.O. Box 12137

6 Davis Dr.

Research Triangle Park, NC 27709

Edward R. McCabe, Ph.D.

Department. of Pediatrics

University of California at Los Angeles

School of Medicine

Los Angeles, CA 90095

Objectives: The overarching goal of the Biostatistics and Epidemiology Research Core is to develop new biostatistical methods related to environmental and rural health, and to perform epidemiologic studies to examine the relationship between risk factors ad disease. The specific objectives of the members of the research core in the past year have been as follows.

Raymond J. Carroll, Ph.D., Director

Distinguished Professor, Department of Statistics

James A. Calvin, Ph.D., Professor and Department Head

Department of Statistics

Barbara Richardson, Ph.D., Assistant Professor

Department of Veterinary Anatomy and Public Health

Michael Sherman, Ph.D., Assistant Professor

Department of Statistics

Naisyin Wang, Ph.D., Associate Professor

Department of Statistics

Measurement Error

Non-linear Models

Missing Data

Longitudinal Data Analysis

Spatial Modeling

Mixed Linear Models

Receptor Modeling

Chemosmetrics

Breast Cancer Research

Genetic Epidemiology

Progress Report: In addition to the collaborations among members internal to the Biostatistics and Epidemiology Research Core, and especially among the members of the Department of Statistics, the existence of the Center for Environmental and Rural Health has led to the development of three major new collaborations, as well as strengthening and revitalization of a number of past collaborations.

James A. Calvin is currently working with Richard H. Finnell, of the Reproductive and Developmental Biology Core, on the assessment of gene expression data taken at varying times during early neural tube development to aid in determining how genes work in unison to affect development. James Calvin and K. C. Donnelly, of the Cellular and Molecular Biology/Toxicology Core, are nearing completion on a paper addressing the effects of supplemental agents on naturally occurring compounds used in bioremediation.

Due to the interactions promoted by the CERH, James Calvin has also developed a collaborative relationship with Steve Safe. This new work with Dr. Safe entails designing and analyzing experiments to study the benefits of specific compounds on the control of breast cancer precursors, while jointly limiting their impact of ovarian cancer precursors.

Naisyin Wang has been working with Tim Phillips, of the Cellular and Molecular Biology/Toxicology Core, on evaluating the effect of a variety of detoxification processes. Two papers resulted from this collaboration: "Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay " in the Journal of Toxicology and Environmental Health, in press, and "Utilization of electochemically generated ozone in the degradation and detoxification of Benzopyrene" submitted.

Barbara Richardson along with James Calvin has collaborated on 2 papers that have been published. The first, " Beyond the Twinning Effect", was published in the American Journal of Epidemiology 1998. The second paper "Levels of maternal serum Alpha-fetoprotein in pregnant women and subsequent breast cancer" was also published in the American Journal of Epidemiology.

Raymond Carroll has been working on a project involving faculty in the Nutrition and Biostatistics and Epidemiology Research Cores. Associated faculty are Dr. Naisyin Wang in biostatistics and Drs. Robert Chapkin, Joanne Lupton and Nancy Turner in the Nutrition Core. They have cast the problem as involving multivariate mixed models and have developed new statistical methods to investigate relationships between high adduct levels in proximal parts of the colon and whether these are associated with high levels in distill regions. The collaboration has and continues to lead to the development of statistical methods not previously considered in the literature. Our current focus is on relating adduct levels and apoptosis. This involves a mixture of classical random effects modeling and binary random affects modeling, as well as nonparametric versions of both. Two abstracts have been submitted to date, and three papers are nearing completion. In addition, a grant proposal to the National Institutes of Health was submitted in February on this work.

Suojin Wang has been motivated by issues of environmental health to consider statistical inference in case–control studies, in collaboration with Raymond Carroll. It has been well–known that large case–control studies can be analyzed by classical logistic regression, but Drs. Wang and Carroll have been studying what happens if the case–control study is not large. They have developed small–sample inferential procedures, called saddlepoint approximations, which are far more accurate than the usual large sample techniques. The paper describing this discovery has been submitted for publication and received positive initial reviews.

Because of the existence of the CERH, Michael Sherman has been able to continue his research in subsampling and analysis of spatial data. In particular, he has greatly benefited from his collaboration with Sherry Bane and Ken Ramos, of the Cellular Molecular Biology/Toxicology Core, in their on going study on links between end stage renal failure and nephrotoxins in drinking water. The CERH has provided startup funding for the group to sample sites for their metal level, (nephrotoxin) in counties of both high and low risk of renal failure.

Cliff Spiegelman continues to work on receptor modeling. Partly through his research efforts the NRC has designated receptor modeling as its 3rd highest priority for immediate air pollution research needs. Together with E. Park at University of Washington and R. Henry USC several papers have been submitted to highly regarded peer reviewed journals on this topic. The CERH has agreed to help support a Master’s project in this area, Jerome Bennett. Spiegelman’s research will benefit from the contributions of the center and Jerome.

Bame, S., Autenrieth, R., Bramson, R., Bruner, M. Donnelly, K.C., Harriss, R. Ramos, K.S., Kunesh, R., Morgan, R., Harbert, G. Richardson, B., Rogers, G., Sherman, M. and Zapata, M. ESRD risk and environmental nephrotoxins. Texas A&M University HSC Research Forum, 1998.

Brown, R.E., Lupton, J.R., Morris, J., Wang, N., Carroll, R.J., Turner, N.D., Davidson, L.A. and Chapkin, R. S. Morphodensitometric analysis of protein kinase C beta II expression in rat colon: relation to in situ cell proliferation and apoptosis. Presented at the Protein Kinase C and Cellular Function Symposium, American Society of Biochemistry and Molecular Biology, Lake Tahoe, CA, October 9, 1998.

Carroll, R. J. Measurement error: a review. In Uncertainties in Radiation Dosimetry and Their Impact on Dose response Analysis, E. Ron, editor. National Cancer Institute Press, 1999.

Carroll, R. J. Risk assessment with subjectively derived doses. In Uncertainties in Radiation Dosimetry and Their Impact on Dose response Analysis, E. Ron, editor. National Cancer Institute Press, 1999.

Carroll, R. J., Freedman, L. S. and Kipnis, V. Measurement error and dietary intake. In Mathematical Models in Experimental Nutrition, A. J. Clifford and H. G. Mueuller, editors, pages 139-146, 1998.

Carroll, R. J., Freedman, L. S., Kipnis, V. and Li, L. A new class of measurement error models, with applications to estimating the distribution of usual intake. Can. J. Statistics, 26, 467-477, 1998.

Carroll, R. J., Maca, J. D. and Ruppert, D. Nonparametric regression splines for generalized linear measurement error models. In Econometrics in Theory and Practice: Festschrift in The Honour of Hans Schneeweiss. Physica Verlag, pages 23-30, 1998.

Hong, M.Y., Chapkin, R.S., Turner, N.D., Galindo, C.D., Carroll, R.J. and Lupton, J.R. Fish oil enhances targeted apoptosis of colonocytes within the first 12 h of carcinogen exposure and results in lower levels of DNA damage compared to corn oil. Faseb J. 12: A656, 1998.

Richardson, B.E., Hulka, B.S., Peck, J.L. David, Hughes, C.L., van den Berg, B.J., Christianson, R.E. and Calvin, J.A. Am. J. Epidemiol. 148, 719-727, 1998.

Wang, C.Y., Wang, S., Gutierrez, R. and Carroll, R. J. Local linear regression for generalized linear models with missing data. Annals of Statistics, 26, 1028-1050, 1998.

Wang, N., Lin, X., Gutierrez, R. G. and Carroll, R. J. Generalized Linear Mixed Measurement Error Models. JASA, 93, 249-261, 1998.

Objectives: The overall goal of the Cellular and Molecular Biology/Toxicology (CMBT) core is to develop and promote cell and molecular biology research initiatives that focus on issues of environmental significance to rural communities. With this goal in mind, the CMBT core has been organized to serve as an intellectual resource to members of the center and to promote collaborative research activities. The research efforts of core members focus on three major areas of activity, namely, receptor biology, molecular detection systems and neuro-endocrinology. The specific research aims of the core are to: (1) identify mechanisms of gene deregulation by environmental chemicals (Busbee, Castiglioni, Ramos, Safe); (2) define molecular mechanisms of toxic action (Busbee, Castiglioni, Derr, Dees, Donnelly, Johnson, Kier, Phillips, Ramos, Russell, Safe, Wild); (3) investigate interactions between environmental chemicals and aging (Busbee, Johnson, Kier); (4) develop biologically-based molecular detection systems that define genetic basis of human disease (Busbee, Castiglioni, Derr, Donnelly, Kier, Phillips, Ramos, Russell, Safe, Wild); (5) develop molecular technologies for environmental decontamination and protection (Donnelly, Phillips, Safe, Wild); and (6) delineate molecular mechanisms of action of environmental estrogens (Busbee, Kier, Safe). Our research efforts are of direct relevance to rural communities where the incidence of exposures to agricultural chemicals, food-borne toxicants, and ambient contaminants is often elevated relative to the general population.

Kenneth Ramos, Ph.D., Core Director, Professor and Interim Head

Departments of Physiology and Pharmacology and Environmental and Occupational Health

David Busbee, Ph.D., Professor

Department of Anatomy and Public Health

Evelyn Castiglioni, Ph.D., Professor and Head

Department of Anatomy and Public Health

Les Dees, Ph.D., Professor

Department of Anatomy and Public Health

James Derr, Ph.D., Assistant Professor

Department of Pathobiology

Kirby Donnelly, Ph.D., Associate Professor

Departments of Anatomy and Public Health, Soil and Crop Sciences,

and Environmental and Occupational Health

Larry Johnson, Ph.D., Professor

Department of Anatomy and Public Health

Ann Kier, D.V.M., Professor and Head

Department of Pathobiology

Timothy Phillips, Ph.D, Professor

Department of Anatomy and Public Health

Steve Safe, D.Phil., Distinguished Professor

Departments of Physiology and Pharmacology,

Biochemistry and Biophysics and Environmental and Occupational Health

David Russell, Ph.D., Professor

Department of Chemistry

Jim Wild, Ph.D., Professor and Head

Department of Biochemistry and Biophysics

Aging

Aryl Hydrocarbon Receptor

Dioxin

Electrophile Response Element

Endocrine Disruption

Gene Regulation

Gene Mutations

Molecular Detoxification

Nitroaromatics

Polycyclic Aromatic Hydrocarbons

Pesticides

Progress Report: Monthly meetings have served to promote scientific interactions and formal collaborations within the core and with other CERH members. Our June through September 98 meetings were designed to discuss technologies and services available within individual facility cores. More recent discussions have centered on research activities that can strengthen collaborative research programs. The major benefit realized by core members during the first few months of operation has been increased access to state-of-the-art technologies that facilitate studies that elucidate fundamental biological events involved in cell determination, growth, differentiation, aging, as well as the onset and progression of diseases that share an environmental etiology. Tangible measures of success include breakthroughs in our understanding of gene regulatory mechanisms impacted by environmental agents, molecular detoxification systems, gene/protein structure and function, neuro-endocrine/toxicant interactions, and genotoxicity of complex polycyclic aromatic hydrocarbon (PAH) mixtures. These research activities often relied upon the use of core facilities. In collaboration with members of the Reproductive Biology and Epidemiology and Biostatistics Research Cores, CMBT members have launched a major initiative in the Lower Rio Grande Valley to investigate at the genetic and epidemiology level the link between environmental exposures and birth defects in this region of Texas. These efforts lead to submission of a major grant to support research infrastructure. In addition to several new and competing RO1 applications by CMBT members, two new grant applications were submitted to expand CERH outreach efforts. Dr. Ramos submitted a request to NIEHS for support of the Texas A&M Environmental Health Initiative along the Texas-Mexico border and Dr. Johnson applied to the National Center for Research Resources to develop an environmental and rural health education partnership between the Center, the TAMU College of Education, and the School of Rural Public Health . Finally, it is also worth noting that as a result of collaborative research activities within the core, Drs. Castiglioni and Ramos were recruited to participate in the Superfund Program Project grant renewal application (in which Drs. Burghardt, Donnelly, Finnell, Phillips, Safe are involved). This application will be submitted to NIEHS in Spring 1999.

Work in Dr. Donnelly’s laboratory has established that risk assessment of genotoxic potential for complex mixtures of polycyclic aromatic hydrocarbons (PAHs) should not be based solely on benzo(a)pyrene (BaP) since metabolism or repair mechanisms can markedly influence toxicological outcomes. This work led to collaborations between Drs. Castiglioni, Donnelly, Ramos and Safe which recently secured funding from ATSDR to study neurological, renal and immunologic effects of complex mixtures. In separate studies, Dr. Castiglioni’s group has shown that lead (Pb) exposure induces the glucose-regulated protein (GRP78) in C6 rat glioma cells, an astrocyte-like cell line that accumulates Pb in culture. Given that Pb strongly binds to GRP78, a molecular chaperone in the endoplasmic reticulum, these investigators suggested that Pb triggers increased GRP78 synthesis to maintain protein trafficking and overcome inhibition of protein function. The novelty of the Pb induction response lead to a collaboration between Drs. Castiglioni and Ramos to define molecular mechanisms of stress protein induction by heavy metals. This work now constitutes the basis for an RO1 submission by these investigators.

Investigators in the Dees laboratory continued to work to define the mechanisms involved in the ability of insulin like growth factor-1 (IGF-1) to initiate the onset of puberty in rats and primates by inducing secretion of luteinizing hormone releasing hormone (LHRH)/luteinizing hormone (LH). In these studies, alcohol was shown to cause significant suppression of IGF-1 in female Rhesus monkeys, an event associated with blockade of increased pulsatile secretion of LH. In other studies using the rat, this group showed changes in the synthesis of specific isoforms of nitric oxide synthase (NOS) within the ovary during pubertal development. A new grant application is expected to be funded (ES 9627-01) during the next cycle to support efforts directed at the elucidation of low level lead effects on the neuro-endocrine axis. These studies will determine if insult occurs pre- or post-natally and what is/are the specific mechanisms of action.

In the Ramos laboratory, efforts during the past year focused on the study of functional interactions between the aryl hydrocarbon receptor (AhR) and transactivation of electrophile response-cis acting elements (EpRE) within the regulatory region of toxicant inducible genes. Of significance was the identification of a negative regulatory mechanism operative in the rat GST-Ya gene that is activated by BaP and related chemicals . Supershift experiments showed that C/EBP-beta binds to both EpRE and AhRE, a finding consistent with the presence of C/EBP sites within those consensus sequences. Surprisingly, the AhR itself was detected in the protein complex binding to the EpRE in the absence of an AhR binding site. These data suggest that C/EBP-beta and the AhR participate in the regulation of rat GST-Ya gene in mammalian cells. Four proteins of Mr 62, 60, 50, and 30 kDa have been found to associate specifically with GST-Ya-EpRE in BaP-treated cells, and specific complexes of Mr 80, 60, 55, 42, 23, and 21 kDa have been identified. Drs. Ramos and Russell have entered into a collaboration using MALDI technology for the more definitive identification of critical transacting factors involved in the response triggered by aromatic hydrocarbons.

Research efforts in the Safe laboratory focused on mechanisms of breast cancer cell growth and development of new AhR based drugs for treatment of this devastating disease. Initial interest in breast cancer research evolved from the study of toxic chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), agents which bind the AhR and exhibit antiestrogenic activity. TCDD also inhibited the induction of several genes by estradiol and studies were designed to determine the molecular mechanism of cross talk between the AhR and estrogen receptor signaling pathways. An important new finding was that GC-rich Spl protein binding sites are critical cis-genomic sites required for ER actions. Using a series of constructs containing wild-type and mutant 5'-flanking sequences from the c-fos promoter, it was shown that a GC-rich motif (5'-GGGGCGTGG) containing an imperfect Sp1-binding site was required for hormone-induced activity. This sequence also bound Sp1 protein, and co-incubation with the estrogen receptor (ER) enhanced Sp1-DNA binding. Ongoing studies in this laboratory have identified several other E2-responsive genes regulated via ER/Sp1 interactions and the differential cell- and gene promoter-specific effects of ER /Sp1 and ER /Sp1 are also being investigated.

Alejandro, N. F. and Ramos, K. S. Benzo[a]pyrene-induced transition of renal glomerular mesangial cells from mesenchymal to epithelial phenotypes involves alterations in Pax-2 and WT1 regulation The Toxicologist 31, 310, 1998.

Alejandro, N.F. and Ramos, K.S. WT1, PAX-2 and E-cadherin expression profiles during epithelialization of glomerular mesangial cells by benzo[a]pyrene. FASEB Journal, 12, A321, 1998.

Atshaves, B.P., Foxworth W.B., Frolov A., Roths J.B., Kier A.B., Oetama B.K., Piedrahita J.A. and Schroeder, F. Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion. Am. J. Physiol., 274. Cell Physiol.43: C633-C644, 1998.

Bame, S., Autenrieth, R., Bramson, R., Bruner, M. Donnelly, K.C., Harriss, R. Ramos, K.S., Kunesh, R., Morgan, R., Harbert, G. Richardson, B., Rogers, G., Sherman, M. and Zapata, M. ESRD risk and environmental nephrotoxins. Texas A&M University HSC Research Forum, 1998.

Barbacci, D.C. and Russell, D.H. Photoinduced Dissociation of Peptides on a Delayed Extraction MALDI Reflectron Time-of-Flight Mass Spectrometer. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Barbacci, D.C., Shields, S.J. and Russell, D.H. Mass Accuracy in Post Source Decay Analysis. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Bielec, P.E., Gallagher, D.S. Womack, J.E. and Busbee, D.L. Homologies between human and dolphin chromosomes detected by heterologous chromosome painting. Cytogenet. Cell Genet. 81:18-26, 1998.

Bielec, P.E., Riggs, P., Womack, J.E. and Busbee, D.L. A high-resolution GBG-banded karotype of the Atlantic bottlenose dolphin, Tursiop trucatus: generation of an idiogram, and NOR localization by fluorescence in situ hybridization. Cytogenet. Cell Genet., 78:6-11, 1997.

Bluhm, B.K. and Russell, D.H. Dissociation Dynamics of C₇H₇CI⁺ - Thermal Activation and Theory, S. Peterman, 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Bluhm, B.K., Gillig, K., Marini, J. and Russell, D.H. Evaluation of an FTICR/IMS Spectrometer for the Characterization of Ion-Moledule Reaction Products. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Bluhm, B.K., Shields S. and Russell, D.H. Understanding the Energetics and Interactions of Cu⁺ with Peptides Containing Basic Residues. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Bondarenko, P., Edmondson, R.D., Cockrill, S., Watkins, L., Macfarlane, R. and Russell, D.H. Fast Screening Method for Human Serum Apolipoprotein Polymorphism Using MALDI-TOF and ESI-TOF MS. The 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Busbee, D., Tizard, I., Stott, J., Ferrick, D. and Ott-Reeves, E. Environmental pollutants and marine mammal health: The potential role of organohalogens and aromatic hydrocarbons in initiation of immune dysfunction. Proceedings of the Int. Whaling Comm., A. Aguilar and P. Reijnders, eds., 1997.

Campbell, B.D., Busbee, D.L. and McDaniel, H.R. Enhancement of immune function in rodents using a proprietary complex mixture of glyconutritionals. Fisher Institute Proceedings, 1(1):12-15, 1997.

Castro-Rivera, E. and Safe, S. Estrogen- and antiestrogen-responsiveness of HEC1A endometrial adenocarcinoma cells in culture. J. Ster. Biochem. Mol. Biol. 64:387-295, 1998.

Chen, I., McDougal, A., Wang, F. and Safe, S. Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 19:1631-1639, 1998.

Chen, Y.H. and Ramos, K. S. Functional interactions between the electrophile and aryl hydrocarbon responsive elements within the GST-Ya promoter in vascular smooth muscle cells. The Toxicologist 31, 309, 1998.

Cook, J.C., Johnson, L., O’Connor, J.C., Biegel, L.B., Krams, C.H., Frame, S.R., Hurtt, M.E. Effects of dietary 17 –estradiol exposure on serum hormone concentrations and testicular parameters in male Crl:CDÒ BR rats. Toxicol. Sci. 44:155-168, 1998.

Crouch, E.A., Miller, S.M., Wilson, V. and Busbee, D.L. An accessory protein to DNA polymerase K exhibits structural and functional similarities to SV40 large T antigen. Mut. Res. Fund. Mol. Mech. Mut., 374:109-123, 1997.

Crow, R.T., Rosenbau, B., Guo, Y., Sulikowski, G.A., Ramos, K. and Smith III, R. Analysis of the mechanism of action of landomycin in a mouse smooth muscle. Regional meeting of the American Association for the Advancement of Science, 1998.

Derr, J.N. Conservation Genetics and Hybridization. In the proceedings of the Congreso Iberoamericano de Razas Autoctonas y Criollas: Analisis Del Programa Nacional De Los Recurso Geneticos Y Pecuarios y Muestra Nacional De Todas Las Razas De Ganado. Tampico, Tamaulipas, Mexico, 1998.

Derr, J.N. Genetic management of bison. Greater Yellowstone Interagency Brucellosis Conference. Executive Committee Meeting, Idaho Falls, Idaho, May, 1998.

Dikler, S., Kelly, J.W. and Russell, D.H. Study of New Heterobifunctional Cross-Linkers and Cross-Linked Peptide Complexes by MALDI. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Duan, R., Porter, W. and Safe, S. Estrogen-induced c-fos protooncogene expression in MCF-7 human breast cancer cells: role of estrogen receptor Sp1 complex formation. Endocrinology 139:1981-1990, 1998.

Fan, Y.Y., Ramos, K.S., and Chapkin, R.S., Modulation of atherogenesis by dietary gamma-linolenic acid. In Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation and Related Diseases, 1998.

Figueroa, I.D., Bekele, H., Kelly, J.W. and Russell, D.H. Conformational Studies of Cyclic Pentapeptides that Adopt a Beta Turn Conformation by H/D Exchange and Mass Spectrometry. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Figueroa, I.D., Torres, O. and Russell, D.H. Effects of the Water Content in the Sample Preparation for MALDI on the Mass Spectra. Anal. Chem., 70, 4527-4533, 1998.

Figueroa, I.D., Torres, O. and Russell, D.H. Studies of Solvent and Analyte Conformational Effects in the Sample Preparation Process for MALDI, 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Gillig, K.J. and Russell, D.H. Quadrupolar Axialization Diagnostics and Ion Mobility Investigations. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Gillig, K.J., Shields, S.J., Marini, J.T. and Russell, D.H. Using Multiple Laser Wavelengths for the Formation of Cationized Peptide Ions, 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Gossett, R., Edmondson, R.D., Jolly, C.A., Cho, T.H., Russell, D.H., Knudsen, J., Keir, A. and Schroeder, F. Structure and Function of Normal and Transformed Murine Acyl-CoA Binding Proteins. J. Biol. Chem., 350, 201-203, 1998.

Gossett, R.E., Edmondson R.D., Jolly, C.A., Cho, T.-H., Russell, D.H., Knudsen J. and Kier A.B., Schroeder F. Structure and function of normal and transformed murine acyl-CoA binding proteins. Arch. Biochem. Biophys., 350(2):201-213, 1998.

Gould, J.C., Leonard, L.S., Maness, S.C., Wagner, B.L., Conner, K., Zacharewski, T., Safe, S., McDonnell, D.P. and Gaido, K.W. Bisphenol A interacts with the estrogen receptor a in a distinct manner from estradiol. Mol. Cell. Endocrinol. 142:203-214, 1998.

Grant, P.G. and Phillips, T.D. Isothermal adsorption of aflatoxin B₁ on HSCAS clay. J. Agric. Food Chem. 46 (2), 599-605, 1998.

Grant, P.G., Lemke, S.L., Dwyer, M.R., and Phillips, T.D. Modified langmuir equation for S-shaped and multisite isotherm plots. Langmuir. 14 (15), 4292-4299, 1998.

Gupta, M., Miggens, J., Parrish, A., Womack, J., Ramos, K.S., Rodriguez, L.V., Goldstein, L.S., Holtzapple, C., Stanker, L. and Safe, S. Ah receptor-independent induction of CYP1A2 gene expression in genetically-inbred mice. Environmental Toxicol. Pharm. 5, 205-213, 1998.

Hallberg, L.M., Tomlinson, J.A. and Ramos, K.S. Inhibition of DNA repair in chemical atherogenesis. The Toxicologist 31, 309, 1998.

Harris, E.D., Qian, Y., Tiffany-Castiglioni, E., Lacy, A., Reddy, M.C.M. A functional analysis of copper homeostasis in cell culture models: A new perspective on internal copper transport. Am. J. Clin. Nutr. 67:988S-995S, 1998.

Hastings, V., Bral, C.M. and Ramos, K.S. Redox regulation of c-Ha-ras by benzo[a]pyrene and related oxidants is mediated through an electrophile responsive element. The Toxicologist, 31, 309, 1998.

Hiney, J.K., Lara, F., Srivastava,V., Dearth, R.K. and W.L. Dees. Effects of ethanol on serum leptin levels and leptin-induced LH release in prepubertal female rats. Endocrine Society, New Orleans, LA, June, 1998.

Hoivik, D.J., Safe, S.H. and Gaido, K.W. Effects of xenobiotics on hormone receptors. In: Toxicant-Receptor Interactions (M.S. Denison and W.G. Helferich, eds.), Taylor & Francis, Bristol, PA, pp. 53-68, 1998.

Holderman, M.T., Miller, K.P. and Ramos, K.S. Protein interactions with the electrophile response element in vascular smooth muscle cells. Abstracts of the Gordon Research Conference on Mechanisms of Toxicity, 1998.

Hong, S.J., Grover, C.A., Safe, S. H., Tiffany-Castiglioni, E., Frye, G.D. Halogenated aromatic hydrocarbons inhibit CA1 field excitatory post-synaptic potentials in rat hippocampal slices. Toxicol. Appl. Pharmacol 148:7-13, 1998.

Hong, S.J., Grover, C.A., Safe, S.H., Tiffany-Castiglioni, E. and Frye, G.D. Halogenated aromatic hydrocarbons suppress CA1 field exictatory postsynaptic potentials in rat hippocampal slices. Toxicol. Appl. Pharmacol. 148:7-13, 1998.

Huang, Y. and D.H. Russell, D.H. Photochemistry and Proton Transfer Reaction Chemistry of Selected Cinnaminic Acid Derivatives in Hydrogen Bonded Environment. Int. J. Mass Spectrom. Ion Proc., 175, 187-204, 1998.

Huebner, H.S. Lemke, S. Ottinger, P. Grant and Phillips, T.D., Equilibrium adsorption of ergotamine by various phyllosilicate clays. Toxicol. Sci. 42:290, 1998.

Huszar, G., Patrizio, P. and Johnson, L. Cytolasmic extrusion and the switch from the creatine kinase B to M. isoform are completed by the commencement of epididymal transport in human and stallion spermatozoa. J. Androl. 19: 11-20, 1998.

Johnson, L., Barnard, J.J., Rodriguez, L., Smith, E.C., Swerdloff, R.S., Wang, X.H. and Wang, C. Ethnic differences in spermatogenic potential in humans. J. Androl. 19:348-357, 1998.

Kerzee, J. K. and Ramos, K. S. De-regulation of c-Ha-ras expression in vascular smooth muscle cells by benzo[a]pyrene and related oxidants. The Toxicologist 31, 358, 1998.

Kerzee, J.K. and Ramos, K.S. Redox regulation of c-Ha-ras in vascular smooth muscle cells: contributions of the AhR signaling Pathway. Abstracts of the Gordon Research Conference on Mechanisms of Toxicity, 1998.

Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van der Saag, P.T., Van der Burg, B. and Gustafsson, J.C. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor b. Endocrinology 139:4252-4263, 1998.

Legare, M.E., Barhoumi, R., Hebert, E., Bratton, G.R., Burghardt, R.C., Tiffany-Castiglioni., E. Analysis of Pb²⁺ entry into cultured astroglia. Toxicol. Sci. 46:90-100, 1998.

Lemke, S.L., Grant, P. G. and Phillips, T.D. Adsorption of zearalenone by organophilic montmorillonite clay. J. Agric. Food Chem. 46(9), 3789-3796, 1998.

Lemke, S.L., McKenzie, K.S., Grant, P.G. and Phillips, T.D. Fumonisin B₁ adsorption onto organo-substituted clays. Toxicol. Sci. 42:290, 1998.

Lemke, S.L., Ottinger, S.E. and Phillips. T.D. Sorption of fumonisin B₁ by nonexchanged and organo-substituted clays. American Chemical Society Meeting, Boston, MA, 1998.

Lu, K. P., Zhang, Y. and Ramos, K. S. Genetic basis of benzo[a]pyrene-induced atherogenesis. The Toxicologist 31, 309, 1998.

Lu, K.P. and Ramos, K.S. Identification of genes differentially expressed in vascular smooth muscle cells following benzo(a)pyrene challenge: Implications for chemical atherogenesis. Biochem. Biophys. Res. Commun. 253, 828-833, 1998.

Mayura, K., Abdel-Wahhab, M.A., McKenzie, K.S., Sarr, A.B., Edwards, J.F., Naguib, K. and Phillips, T.D. Prevention of maternal and developmental toxicity in rats via dietary inclusion of common aflatoxin sorbents: Potential for hidden risks. Toxicol. Sci. 41(2), 175-182, 1998.

McDougal, A. and Safe, S. Induction of 16a-/2-hydroxyestrone metabolite ratios in MCF-7 cells by pesticides, carcinogens and antiestrogens does not predict mammary carcinogens. Environ. Health Persp. 106:203-206, 1998.

McKenzie, K.S., Kubena, L.F., Denvir, A.J., Rogers, T.D., Hitchens, G.D., Bailey, Harvey, R.B., Buckley, S.A. and Phillips, T.D. Aflatoxicosis in turkey poults is prevented by treatment of naturally-contaminated corn with ozone generated by electrolysis. Poult. Sci. 77:1094-1102, 1998.

Miller, K. P. and Ramos, K. S. Activation of EpRE binding protein by benzo[a]pyrene-3-6-quinone and hydrogen peroxide in vascular smooth muscle cells. The Toxicologist 31, 310, 1998.

Miller, S.D., Crouch, E.A. and Busbee, D.L. An accessory protein of DNA polymerase K declines in function with increased age. Mut. Res. Fund. Mol. Mech. Mut., 374-124-137, 1997.

Mouniemne, R., Burghardt, R.C., Busbee, D.L. and McDaniel, H.R. Enhancement of glutathione levels and protection from chemically initiated glutathione depletion in rat liver cells by glyconutritionals. Fisher Institute Proceedings, 1(1):7-11, 1997.

Park, A-Y, Apuy, J.L., Baldwin, T.O. and Russell, D.H. Structural Characterization of Luciferase from Vibrio Harveyi by Mass Spectrometry Combined with Enzymatic Digestion and Chemical Modification. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Parrish, A. R., Ramos, K. S., Brendel, K. and Gandolfi, A. J. Precision-cut liver slices: Investigation of the molecular response to toxicants. The Toxicologist 31, 64, 1998.

Parrish, A.R., Alejandro, N.F., Bowes III, R.C., and Ramos, K.S. Cytotoxic response profiles of cultured renal epithelial and mesenchymal cells to selected aromatic hydrocarbons. Toxicology In Vitro 12, 219-232, 1998.

Parrish, A.R., Fisher, R., Bral, C.M., Burghardt, R.C., Gandolfi, A.J., Brendel, K., and Ramos, K.S. Benzo(a)pyrene-induced alterations in growth-related gene expression and signaling in precision-cut adult rat liver slices. Toxicol. Appl. Pharmacol., 152, 302-308, 1998.

Pu, L, Foxworth W.B., Kier, A.B., Annan, R.S., Carr, S.A., Edmondson, R., Russell, D., Wood, W.G. and Schroeder, F. Isolation and characterization of 26 and 30kDa rat liver proteins immunoreactive to anti-sterol carrier protein-2 antibodies. Protein. Exp. Purification., 113:337-348, 1998.

Qian, Y., Tiffany-Castiglioni, E. and Harris, E.D. Sequence of a Menkes-type Cu-transporting ATPase from rat C6 glioma cells: Comparison of the rat protein with other mammalian Cu-transporting ATPases. Mol. Cell. Biochem., 181:49-61, 1998.

Qian, Y., Tiffany-Castiglioni, E., Welsh, C.J. and Harris, E.D. Copper efflux from murine microvascular cells requires expression of the Menkes Disease Cu-ATPase. J. Nutrition, 128:1276-1282, 1998.

Ramos, K.S. Altered regulation of the Ha-ras protooncogene in chemical atherogenesis, Toxicology Program, University of Conneticut, Storrs, Connecticut, 1998.

Ramos, K.S. Novel transcription factors in the regulation of gene expression. Pfizer Pharmaceuticals, Groton, Connecticut, 1998.

Ramos, K.S. Redox regulation of c-Ha-ras and GST-Ya genes in vascular cells by environmental chemicals: implications for human atherosclerotic disease. Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, Ohio, 1998.

Ramos, K.S. Redox regulation of c-Ha-ras and osteopontin signaling in vascular smooth muscle cells: implications in chemical atherogenesis. Annual Rev. Pharmacol. Toxicol. 29, 243-265, 1999.

Ramos, K.S. Redox regulation of gene expression in chemical atherogenesis. Texas Tech University Health Science Center, Lubbock, Texas, 1998.

Ramos, K.S. Toxicities mediated through changes in gene expression. Workshop on Cell Signaling Processes Underlying Toxicological Responses, Raleigh-Durham, North Carolina, 1998.

Ramos, K.S., Parrish, A.R., Zhang, Y., Kerzee, J.K. and Alejandro, N.F. The induction of atherosclerotic vascular lesions by benzo[a]pyrene, a tobacco smoke constituent, correlates with enhanced c-Ha-ras p21 expression in vivo. The Toxicologist 42, 31, 1998.

Ramos, K.S., Zhang, Y. and Bral, C.M. Ras activation by benzo[a]pyrene. In: Molecular Biology of the Toxic Response. Chap. 30, pp. 517-530, 1998.

Rooney, A.P. and Derr, J.N. Evaluating the likelihood of a presumed bottleneck in the Bering-Chukchi-Beaufort seas stock of bowhead whales. The International Whaling Commission Report, Muscat, Oman, 1998.

Russell, D.H. and Figueroa, I.D. Combining MALDI and H/D Exchange to Study Conformational Changes of Peptides and Proteins. The 25^th Annual Conference of the Federation of Analytical Chemistry and Spectroscopy Societies, Austin, Texas, October 1998.

Russell, D.H., Gillig, K.J., Barbacci, D.C. Time-of-Flight Mass Spectrometry: the Versatile High Performance Instrument of the Future. The 25^th Annual Conference of the Federation of Analytical Chemistry and Spectroscopy Societies, Austin, Texas, October, 1998.

Safe, S. Hazard and risk assessment of chemical mixtures using the toxic equivalency factor (TEF) approach. Environ. Health Persp. 106(54):1051-1058, 1998.

Safe, S. Interactions between hormone and chemicals in breast cancer. Annu. Rev. Pharmacol. Toxicol. 38:121-158, 1998.

Safe, S. Limitations of the toxic equivalency factor approach for risk assessment of TCDD and related compounds. Teratogenesis, Carcinogenesis and Mutagenesis 17:285-304, 1998.

Safe, S. Male reproductive capacity and exposure to environmental chemicals. In: Hormonal Toxicants in Food, Proceedings. John Wiley, VCH, pp. 253-259, 1998.

Safe, S. Ah Receptor Agonists as Endocrine Disruptors - Antiestrogenic Activity. Symposium on Endocrine Disruptors, International Congress of Toxicology, Paris, France, 1998.

Safe, S. Alternate Mechanisms for Receptor Signaling: Crosstalk Between Estrogen and Dioxin Receptor-Mediated Signaling Pathways. Symposium on Endocrine Disruptors, regional meeting of American Chemical Society, Research Triangle Park, NC, 1998.

Safe, S. Carcinogenicity Studies with PAH Mixtures. III International Congress of Pathophysiology, Laht, Finland, 1998.

Safe, S. Endocrine-Active Agents in the Environment and Human Health - a Critical Evaluation. Symposia on Environmental Chemicals as Endocrine Disrupters, AAAS, Philadelphia, PA, 1998.

Safe, S. Mammalian Estrogen-Responsive Screening Assays. Symposium on Endocrine Disruptors, International Congress of Toxicology, Paris, France, 1998.

Safe, S. Mechanistic Paradigms - Breast Cancer and Exposure to Environmental Estrogens. Conference on Superfund Communities: Who's Exposed and Who's at Risk, Boston, MA, 1998.

Safe, S. Reproductive Effects: Endocrine Disruption. 44th Annual Nestle's Nutrition Workshop on Hazards for Children in the Food Chain, Prague, Czech Republic, 1998.

Safe, S. TCDD-Induced Antiestrogenicity: Crosstalk Between Ah and Estrogen Receptor-Mediated Signaling Pathways. Annual Meeting of the German Society of Pharmacology and Toxicology, Mainz, Germany, 1998.

Safe, S. Toxicology of Endocrine Disruptors. Watershed Heroes Conference, American Farm Bureau Federation, Amana, Iowa, 1998.

Safe, S., Connor, K., Ramamoorthy, K., Moore, M., Wang, F., Mustain, M., Chen, I., Zacharewski, T., Gillesby, B., Joyeux, A., Balaguer, P., Gaido, K., Leonard, L., Manness, S.C. and McDonnell, D.P. Estrogenic activity of hydroxylated polychlorinated biphenyls (PCBs) and their interaction. In: Hormonal Toxicants in Food, Proceedings. John Wiley, VCH, pp. 200-207, 1998.

Safe, S.H. Development, validation and problems with the TEF approach for risk assessment of dioxins and related compounds. J. Animal Sci. 76:134-141, 1998.

Safe, S.H. and Gaido, K. Phytoestrogens and anthropogenic estrogenic compounds. Environ. Toxicol. Chem. 17:119-126, 1998.

Shields, S.J. and Russell, D.H. Evaluation of Post-source Decay for Deprotonated Peptide Ions, 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Shields, S.J. and Russell, D.H. Fragmentation Reactions of [M+Cu]⁺ Peptide Ions Containing N-terminal and/or C-terminal Arginine. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Sinha Hikim, A.P., Wang, C., Lue, Y., Johnson, L., Wang, X-H and Swerdloff, R.S. Spontaneous germ cell apoptosis in humans: evidence of ethnic difference in the susceptibility of germ cells to programmed cell death. J. Clinical Endocrin. And Metab. 83:152-156, 1998.

Srivastava, V, Hiney J.K. and Dees, W.L. Effects of ethanol on the intraovarian IGF-1 system in the prepubertal female rat. Endocrine Society, New Orleans, LA, June, 1998.

Sumner, L.W., Wolf, B.P., Russell, D.H., Grapperhaus, C.A., Musie, G. and Darensbourg, M.Y. Electrospray Ionization Mass Spectrometry of Biomimetic Metallodithiolates: Correlation of ESI Induced Electrochemistry with Structure. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 1998.

Sun, G., Porter, W. and Safe, S. Estrogen-induced retinoic acid receptor a-1 gene expression: role of estrogen receptor-Sp1 complex. Mol. Endocrinol. 12:882-890, 1998.

Tiffany-Castiglioni, E. Astroglia in metal metabolism and toxicity: commentary on a forum paper. NeuroToxicology 19:19-22, 1998.

Wang, F., Hoivik, D., Pollenz, R. and Safe, S. Functional and physical interactions between the estrogen receptor-Sp1 and the nuclear aryl hydrocarbon receptor complexes. Nucl. Acids Res. 26:3044-3052, 1998.

Wang, W., Smith, III, R. and Safe, S. Aryl hydrocarbon receptor-mediated antiestrogenicity in MCF-7 cells: modulation of hormone-induced cell cycle enzymes. Arch. Biochem. Biophys. 356:239-248, 1998.

Ward, T.J., Bielawski, J.P., Davis, S.K., Templeton, J.W. and Derr, J.N. Identification of domestic cattle hybrids in wild cattle and bison species: A general approach using mtDNA markers and the parametric bootstrap. Presented at the annual meeting of the Society for the Study of Evolution, Vancouver, BC, June, 1998.

Willett, K.L., Randerath, K., Zhou, G-D. and Safe, S.H. Inhibition of CYP1A1-dependent activity by the PAH fluoranthene. Biochem. Pharmacol. 55:831-839, 1998.

Wilson, C.L. and Safe, S. Mechanisms of ligand-induced aryl hydrocarbon receptor-mediated biochemical and toxic responses. Toxicologic Pathol. 26:657-671, 1998.

Wolf, B.K., Sumner, L.W., Shields, S.J., Nielsen, K., Gray, K.A., Russell, D.H. Characterization of Proteins Utilized in the Desulfurization of Petroleum Products of Matrix Assist Laser Desorption Ionization Time of Flight Mass Spectrometry. Anal. Biochem., 260, 117-127, 1998.

Yu, W.H., Karanth, S., Walczewska, A., Sower, S.A., Hiney, J.K., Dees, W.L. and McCann, S.M. FSH-releasing factor is lamprey LHRH-III or a closely related peptide. 28^th Annual Meeting for the Society of Neuroscience, Los Angeles, CA, Nov., 1998.

Zacharewski, T. and Safe, S. Antiestrogenic activity of TCDD and related compounds. In: Reproductive and Developmental Toxicology (K.S. Korach, ed.), Marcel Dekker, Inc., New York. pp. 431-448, 1998.

Zhang, A.J., Russell, D.H., Zhu, J. and Burgess, K. A Method for Removal of N-BOC Protecting Groups from Substrates on TFA- sensitive Resins. Tetrahedron Let., 39, 7439-7442, 1998.

Zhao, W. and Ramos, K.S. Cytotoxic response profiles of cultured rat hepatocytes to selected aromatic hydrocarbons. Toxicology In Vitro 12, 175-182, 1998.

Zhao, W. and Ramos, K.S. Modulation of hepatocyte gene expression by the carcinogen benzo(a)pyrene. Toxicology In Vitro 12, 395-402, 1998.

Zhao, W., Parrish, A.R., and Ramos, K.S. Constitutive and inducible expression of cytochrome P450IA1 and IB1 in human vascular endothelial and smooth muscle cells. In Vitro Cell. Develop. Biol. 34, 671-673, 1998.

Objectives: The primary goal of the Nutrition Research Core (NRC) is to promote active collaboration among the core members, and among core members and members of the other Center cores. The individual scientists in the NRC have served as an interface between nutritional problems observed in the field and the molecular mechanisms behind those specific problems. Of particular interest has been the role of nutritional factors in outcomes associated with environmental exposures to toxic chemicals or anticarcinogens, especially as it relates to colonic cancer. Core activities have supported research initiatives of core members and associate members, and through their body of expertise, the faculty has assisted in the performance of other Center projects that together addressed the goal of the Center — improving environmental and rural health.

Some of the research aims of the core are: 1) Identify diet components that reduce or delay onset of colon carcinogenesis, and thus determine potential diet interventions that ameliorate the impacts of environmental toxins; 2) Evaluate non-invasive biomarkers of colon cancer as a means of monitoring dietary anticarcinogens; 3) Determine how over- and under-consumption of dietary minerals exacerbate the response to environmental toxins; 4) Evaluate how previous nutritional status affects the response to environmental toxins and if the response can be modulated by changes in diet; and 5) Elucidate the cellular and molecular mechanisms by which diet modulates immune-mediated inflammatory disease, and alters host resistance to infectious pathogens.

Members:

Joanne R. Lupton, Director, Professor

Department of Animal Science

Raymond J. Carroll, Associate Member, Distinguished Professor

Department of Statistics

Robert S. Chapkin, Associate Professor

Department of Animal Science

Edward D. Harris, Professor

Department of Biochemistry and Biophysics

Wallace L. McKeehan (Associate member), Professor

Institute of Bioscience and Technology

David N. McMurray, Professor

Department of Medical Microbiology and Immunology

Friedhelm Schroeder, Professor

Department of Veterinary Physiology and Pharmacology

Guoyao Wu, Associate Professor

Department of Animal Science

Key Words:

Environmental carcinogens or mutagens

Phytochemicals

Colon cancer

Prostate cancer

Minerals

Immune responsiveness

Biomarkers

Progress Report: The main theme of the diverse research programs in the Nutrition Research Core (NRC) is how diet affects the prevention or promotion of disease. In order to make responsible diet recommendations; we must understand the mechanisms whereby specific diet components are protective or promotive of diseases. In addition, an understanding of the genetic bases for disease, and how diet affects gene expression will require the cooperation of all the cores. Some of the specific benefits resulting from the Center are described below.

Interaction with the Biostatistics and Computation Facility Core, as well as the Biostatistics and Epidemiology Research Core (BERC) is enabling core members to better design their experiments in order to be able to answer the questions they pose and to properly analyze the acquired results. For example, Dr. G. Wu (NRC) was assisted in the analysis of a complex data set that facilitated the publication of two papers involving maternal nutrition and fetal development. In addition, a grant proposal involving Drs. J. Lupton (NRC), R. Chapkin (NRC), and R. Carroll (primarily BERC) was submitted to NIH as a result of this collaboration. The Nutrition Core also has benefited the research of Drs. R. Carroll and N. Wang (BERC) who have collaborated with Drs. R. Chapkin, J. Lupton and N. Turner (NRC). From working with them, Drs. R. Carroll and N. Wang have developed new statistical methods to investigate relationships between high levels of DNA damage in the proximal colon and whether these are associated with high levels in distal regions. The collaboration is leading to the development of statistical methods not previously considered in the literature. The current project involves a mixture of classical random effects modeling and binary random affects modeling, as well as nonparametric versions of both.

The RDB core is being used by some members of the NRC. Initial discussions with Dr. Piedrahita (CMBC) concerning cloning strategies for development of animal models have occurred with Drs. R. Chapkin and J. Lupton (NRC). Dr. F. Schroeder (NRC), working with Drs. A. Kier (CMBC) and J. Piedrahita, has developed a series of transfected embryonic stem (ES) cell lines in which the various proteins are overexpressed. Gene ablation studies will be performed next. These ES cell lines will then be used to make transgenic mice in collaboration with Dr. A. Kier. Availability of gene manipulated mice will then allow direct testing of the function of these proteins in uptake and/or utilization of environmental toxicants that may modify lipid metabolism. Both Drs. J. Lupton and G. Wu (NRC) have projects for which Dr. N. Ing (RDBC) is a member. Her expertise in measurement of mRNA has enabled Dr. G. Wu to publish a paper on intestinal arginine-metabolizing enzymes in weanling pigs, and her expertise in estrogen receptor expression has resulted in two funded proposals with Drs. J. Lupton and N. Turner (NRC) for which Dr. Ing is a collaborator. Dr. G. Wu also has collaborated with Dr. F. Bazer and L. Jaeger (RDBC) to submit a proposal to NIH and prepare another proposal that will be submitted in January 1999.

Support from the Imaging Core (IC) is enhancing the ability of the NRC members to discern the in situ and in vitro cellular responses to modification of dietary components. Drs. J. Lupton and R. Chapkin (NRC) are working with Dr. R. Burghardt (IC) to determine the effect of fatty acids on mitochondrial function and membrane potential, and how it affects apoptosis.

Members of the NRC are developing transgenic animals in an attempt to better understand the cellular and molecular responses to changes in diet components. Dr. J. Lupton (NRC), in collaboration with Dr. A. Kier and Dr. B. Foxworth (CMBC) have received funding for an interdisciplinary project to develop a transgenic rat model for colon cancer experiments. Thus, our members can perform more definitive research with access to the expertise and abilities of the Transgenic Facility Core.

The biological mass spectrometry core is used by Drs. R. Chapkin and J. Lupton (NRC) to determine the post-translational processing of ras protein (intracellular signaler involved in colon cancer) to assess whether dietary lipids can influence its prenylation and palmitoylation.

Within the Nutrition core, Drs. R. Chapkin and D. McMurray have recently had a NIH grant funded to determine the effect of n-3 PUFAs on T-lymphocyte activation.

Atshaves, B.P., Foxworth, W.B., Frolov, A., Roths, J.B., Kier, A.B., Oetama, B.K., Piedrahita, J.A. and Schroeder, F. Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion. Am. J. Physiol. 274:C633-C644, 1998.

Atshaves, B.P., Petruscu, A., Starodub, O. Roths, J. B., Kier, A.B., and Schroeder, F. Expression and intracellular processing of sterol carrier protein-2/3-oxoacyl CoA thiolase in transfected L-cell fibroblasts. Gordon Res. Conf. on Lip. Metabolism, Meriden, NH, June 28-July 3, 1998.

Atshaves, B.P., Petruscu, A., Starodub, O. Roths, J. B., Kier, A.B., and Schroeder, F. Intracellular processing of sterol carrier protein-2/3-oxoacyl CoA thiolase and lipid metabolism in transfected L-cell fibroblasts. Lost Pines Molecular Biology Conference, Oct. 9-11, Smithville, TX. 1998.

Brown, R.E., Lupton, J.R., Morris, J., Wang, N., Carroll, R.J., Turner, N.D., Davidson, L.A. and Chapkin, R. S. Morphodensitometric analysis of protein kinase C beta II expression in rat colon: relation to in situ cell proliferation and apoptosis. Presented at the Protein Kinase C and Cellular Function Symposium, American Society of Biochemistry and Molecular Biology, Lake Tahoe, CA, October 9, 1998.

Carstens, R.P., McKeehan, W.L. and Garcia-Blanco, M.A. An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor-2 (FGF-R2) pre-mRNA splicing. Mol. Cell. Biol. 4:2205-2217, 1998.

Chapkin R.S. and Lupton, J.R. Cell proliferation and apoptosis in rodent species: Modulation by diet. Presented at the 8^th Annual Research Conference on Colon Cancer Prevention and Dietary Modulation of Cellular and Molecular Mechanisms, American Institute for Cancer Research, September 4, 1998.

Date, M., Matsuzaki, K., Matshushita, M., Sakitani, K., Shibano, K., Okajima, A., Yamamoto, C., Ogata, N, Okumura, T., Seki, T., Kubota, Y., Kan, M., McKeehan, W.L. and Inoue, K. Differential expression of transforming growth factor-b and its receptors in hepatocytes and nonparenchymal cells of rat liver after CC14 administration. J. Hepatol. 28:572-581, 1998.

Frolov, A. and Schroeder, F. Acyl CoA binding protein: ligand-protein interaction as monitored by time resolved fluorescence. J. Biol. Chem.. 273:11049-11055, 1998.

Gossett, R.E., Edmondson, R.D., Jolly, C.E., Cho, T.H., Russell, D.H., Knudsen, J., Kier, A.B. and Schroeder, F. Structure and function of normal and transformed murine acyl CoA binding proteins" Arch. Biochem. Biophys. 350:201-213, 1998.

Harris, E.D. Amyotrophic Lateral Sclerosis: A lesson in deficiency diseases. Nutr. Rev. 56, 81-84, 1998.

Harris, E.D. Cofactors I. Organic In: Encyclopedia of Human Nutrition. (Sadler, M., Caballero, B. and Strain S., eds), Academic Press Ltd., London, pp. 406-419, 1998.

Harris, E.D. Cofactors II. Inorganic In: Encyclopedia of Human Nutrition. (Sadler, M., Caballero, B. and Strain S., eds), Academic Press Ltd., London, 419-431, 1998.

Harris, E.D. Copper Metabolism and Transport: an update. J. Tr. Ele. Exp. Med. 11, 163-176, 1998.

Harris, E.D., Qian, Y. and Reddy, M.C.M. Genes regulating copper metabolism. Molec. Cell. Biochem. 188: 57-62, 1998.

Harris, E.D., Qian, Y., Tiffany-Castiglioni, E., Lacy, A.R., and Reddy, M.C.M. Functional analysis of copper homeostasis in cell culture models. A new perspective on internal copper transport. Am. J. Clin. Nutr. 67, 988S-995S, 1998.

Hong, M.Y., Chapkin, R.S., Turner, N.D., Galindo, C.D., Carroll, R.J. and Lupton, J.R. Fish oil enhances targeted apoptosis of colonocytes within the first 12 h of carcinogen exposure and results in lower levels of DNA damage compared to corn oil. Faseb J. 12: A656, 1998.

Jolly, C.A., Murphy, E.J. and Schroeder, F. Differential influence of rat liver fatty acid binding protein isoforms on phospholipid composition: phosphatidic acid biosynthesis and phospholipid fatty acid remodeling. Biochem. Biophys. Acta. 1390:258-268, 1998.

Luo, Y., Lu, W., Mohamedali, K., Jang, J.-H., Jones, R.B., Gabriel, J.L., Kan, M. and McKeehan, W.L. The glycine box: A determinant of specificity for fibroblast growth factor. Biochemistry 37:16506-16515, 1998.

Lupton, J.R. Turner, N.D., Zoran, D.L., Chang, W.C., Hong, M.Y., Zhang, J., Wu, G. and Chapkin, R.S. Mechanisms by which wheat bran may protect against colon carcinogenesis: the role of butyrate. Presented at the 6^th International Conference on Mechanisms of Antitumorigenesis and Anticarcinogenesis, Arachen, France, October 25, 1998.

Matsubara, A., Kan, M., Feng, S and McKeehan, W.L. Inhibition of Growth of Malignant Rat Prostate Tumor Cells by Restoration of Fibroblast Growth Factor Receptor 2. Cancer Res. 58:1509-1514, 1998.

McKeehan, W.L. American Association for Cancer Research Special Conference. New Research Approaches in the Prevention and Cure of Prostate Cancer: FGF Genes and FGF Receptor Alterations in Prostate Tumorigenesis. Indian Wells, CA, December 2-6, 1998.

McKeehan, W.L. Progress in Prostate Cancer. Houston A&M Club, Houston, TX, April 27, 1998.

McKeehan, W.L. Structure-Function Relationships in the Heparan Sulfate-Fibroblast Growth Factor Complex. MD Anderson Cancer Center, Dept. of Tumor Biology, April 8, 1998.

McKeehan, W.L. The Heparan Sulfate-FGF Receptor Complex: Assembly and Structure-Function Relationships. University of California-Irvine, November 16, 1998.

McKeehan, W.L. The Heparan Sulfate-Fibroblast Growth Factor Receptor Complex in Prostate Tumor Progression. Mayo Clinic, Rochester, MN, October 2, 1998.

McKeehan, W.L., Wang, F. and Kan, M. The heparan sulfate-fibroblast growth factor family: diversity of structure and function. Prog. Nucleic Acid Res. Mol. Biol. 5:135-176, 1998.

Mikio, Kan, Annual Symposium of Japanese Tissue Culture Association. The role of FGF signaling in development, angiogenesis and cance: Regulation of FGF activity by heparin or heparan sulfates. Hiroshima, Japan, Dec. 3-4, 1998.

Mikio, Kan. Regulation of FGF activity by heparin or heparan sulfates. Tokyo University, Tokyo, Japan, December 7-8, 1998.

Pu, L., Foxworth, W.B., Kier, A.B., Murphy, E.J., Wood, W.G. and Schroeder, F. Characterization of 30 kDa SCP-2 related protein from rat liver. Protein Exp. Purification 13:337-348, 1998.

Qian, Y., Tiffany-Castiglioni, E., and Harris, E.D. Sequence of a Menkes-type Cu-transporting ATPase from rat C6 glioma cells: comparison of the rat protein with other mammalian Cu-transporting ATPases. Molec. Cell. Biochem. 181, 49-61, 1998.

Qian, Y., Tiffany-Castiglioni, E., Welsh, J. and Harris, E.D. Copper entry into murine CNS is controlled by a Menkes Cu-ATPase. J. Nutr. 128, 1276-1282, 1998.

Reddy, M.C.M. and Harris, E.D. Multiple transcripts coding for the Menkes gene. Evidence for alternative splicing of Menkes mRNA. Biochem. J. 334, 71-77, 1998.

Schoer, J., Gallegos, A., Starodub, O., Roths, J.B., Kier, A. B. and Schroeder, F. Lysosomal membrane cholesterol dynamics: role of sterol carrier protein-2 gene products. Gordon Research Conference on Lipoprotein Metabolism, Meriden, NH, June 28-July 3, 1998.

Schroeder, F. Jolly, C.A., Cho, T.-H. and Frolov, A. Fatty acid binding protein isoforms: structure and function. Chem. Phys. Lip. 92:1-25, 1998.

Schroeder, F., Wood, W.G., and Kier, A.B. Lipid Domains and Biological Membrane Function, Chapter 4, in Principles of Cell Physiology (N. Sperelakis, ed.) Academic Press, Inc., Orlando, FL, pp. 61-74, 1998.

Schroeder, F., Frolov, A., Schoer, J.K., Gallegos, A.M., Atshaves, B.P., Stolowich, N.J., Scott, A.I., and Kier, A.B. "Cytosolic cholesterol binding proteins: Intracellular trafficking and membrane domains. (T.Y. Chang, and D.E. Freeman, ed.) in Intracellular Cholesterol Transport. Kluwer Scientific Publishers, New York, November 1998.

Schroeder, F., Kier, A., Frolov, A., Huang, H., Schoer, J., and Ball, J. Membrane domains as sites for internalization of infectious organisms. American Society of Microbiology Meeting, College Station, TX, November 19, 1998.

Starodub, O., Atshaves, B.P., Roths, J.B., Kier, A.B., and Schroeder, F. Expression and Intracellular localization of sterol carrier protein-2 gene products in transfected L-cell fibroblasts. Lost Pines Molecular Biology Conference, Oct. 9-11, Smithville, TX. 1998.

Objectives: Given existing concerns that human populations are being exposed to an increasing number of potentially harmful agents through the environment, the need to understand the mechanisms by which exogenous compounds interact with critical aspects of reproduction and early embryonic development has never been more important. This is true not only in humans, but also in our domestic animals and wildlife populations. The rate at which potentially hazardous reproductive toxicants enter the environment is rapidly outpacing our ability to effectively evaluate their safety. In addition to compounds introduced into the environment by human intervention, naturally occurring toxicants like phytoestrogens in plants and mold can have a significant impact on reproduction and development. There are also reproductive effects of estrogen agonists occurring in the environment that may positively or negatively impact the reproductive well being of human and domestic animal species. Both of these possibilities must be better understood. The research goals and objectives of the Reproduction and Developmental Biology Core (RDB) involves understanding the impact of environmental toxicants on all aspects of reproduction and development, including issues of gametogenesis, conception, pregnancy maintenance, and normal embryonic morphogenesis. The experimental systems used include both domestic (swine, sheep, cattle) and laboratory animals (transgenic and inbred mouse and rat strains), as well as human biological material. By analyzing effects of environmental toxins on reproduction, we assay a very sensitive and responsive system, as well as monitor actions on our posterity.

Richard H. Finnell, Ph.D., Core Director

Department of Veterinary Anatomy and Public Health

James West, Ph.D., Deputy Director

Department of Medical Anatomy and Neurobiology

Louise Abbott, D.V.M., Ph.D.

Department of Veterinary Anatomy and Public Health

Fuller Bazer, Ph.D.

Department of Veterinary Anatomy and Public Health

Robert Burghardt, Ph.D.

Department of Veterinary Anatomy and Public Health

Nancy Ing, D.V.M., Ph.D.

Department of Veterinary Anatomy and Public Health

Laurie Jaeger, D.V.M., Ph.D.

Department of Veterinary Anatomy and Public Health

Jorge Piedrahita, Ph.D.

Department of Veterinary Anatomy and Public Health

Mark Westhusin, Ph.D.

Department of Veterinary Physiology and Pharmacology

Transgenics

Teratogens

Cloning

Pre-implantation

Genetic Sensitivity

Steroid Receptors

Uterine Environment

Signal Transduction

Neuronal Differentiation

Nuclear Transplantation

Progress Report: The Center for Environmental and Rural Health has been a recognized, funded entity for a mere six months at the time of writing this report. Nonetheless, the resources of the Center have created a synergy in research efforts not only between members of this Research Core, but has facilitated the research interactions across Research Core boundaries. The scheduled meetings between RDB Core members have been instrumental in this regard, helping inform members of new developments and opportunities being provided by the core facilities. An example of this type of fostering of research collaboration involves Dr. Louise Abbott and Dr. Rajesh Miranda. Drs. Abbott and Miranda have started to investigate the roles of intrinsic versus extrinsic factors responsible for Purkinje cell and granule cell death in the leaner cerebellum through the use of long-term cerebellar slice culture. Dr. Richard Finnell has also benefited from this mechanism to develop a new collaborative relationship between Dr. Rajesh Miranda. The pilot data that Drs. Finnell and Miranda were able to obtain in an embryonic model of neural tube closure defects has resulted in their successfully competing for local funding-Risk Factors for Neural Tube Defects: Developing Strategies for Prevention-Texas A&M University Interdisciplinary Research Initiative Grant. This new collaboration was written into Dr. Finnell’s renewal (submitted November, 1998) application for his grant entitled: Fetal antiepileptic drug syndrome: a molecular analysis. National Institutes of Dental Research 2R01 DE 11303-05. Dr. R.H. Finnell, P.I.; Dr. J.A. Calvin, Dr. R. Miranda, Co-Investigators.

Another example involves the increased collaborative interaction between Drs. Finnell and Piedrahita. These two members of the RDB Core had been collaborating on a project involving folic acid and the development of neural tube defects. Specifically, they have used homologous recombination in embryonic stem cell technology to successfully knock-out both murine folate binding protein genes (FBP-1 and FBP-2), and have established a breeding colony of heterozygous animals, which have significantly lower serum folic acid levels as well as elevated homocysteine concentrations. While supplemental folic acid has been shown to reduce the risk for neutral tube defects and other congenital malformations in humans, the mechanisms underlying this protective effect are unknown. Drs. Piedrahita and Finnell have been exploring the impact of environmental arsenic as a model toxicant to learn more how a genetically sensitive population-those with genetically-engineered folate transport defects-might respond to the developmental challenge. The focus is on how additional downstream genes might be abnormally regulated under this environmental perturbation. This work is supported by NIEHS grant "Folate receptor knockouts, arsenate and birth defects" ES/HD35396. Dr. R.H. Finnell, P.I.; Dr. J.A. Piedrahita, Co-P.I. Initial observations in this research program concerning the development of craniofacial defects have led to the submission of a new grant application involving members of this Research Core: Folate Receptors and Craniofacial Malformations. R01 ES/DE 09846. Dr. R.H. Finnell, P.I.; Dr. J.A. Piedrahita, Co-P.I.; Dr. R.C. Burghardt, collaborating investigator. The addition of Dr. Burghardt to this program brings state-of-the-art imaging capabilities to bear on the cellular changes occurring in response to the genetic manipulations. With the support of the Center for Environmental and Rural Health grant, we have performed gene targeting and are in the process of performing blastocyst injections with the targeted ES cells for the other important gene in folate transport-the reduced folate carrier gene. This is an extension of the collaborative research program that would not have occurred in the absence of support from the Center.

Drs. Safe and Finnell are working together to determine the relative importance of a newly characterized deletion in the Arnt protein which has shown promise as a prognostic indicator of survival in breast cancer patients. With the use of this genetic marker, we will be able to test the general hypothesis that AhR mediated signaling pathway may be useful as prognostic factors for mammary and potentially other forms of cancer. Drs. Safe and Finnell propose to test this hypothesis as well as determine the effectiveness of using this deleted marker for additional cancer types with respect to survival in several different study populations, both in Texas and Nebraska, as well as in an environmentally challenged foreign population (Baku, Azerbaijan). Funding for this new collaborative effort is being sought through the NIH Superfund Research granting mechanism, as well as through local cancer research funding venues.

Biochemical signaling between the uterus and conceptus (embryo and its associated membranes) is essential for maintenance of ovarian corpora lutea (CL) and their continuous secretion of progesterone which is essential for establishment and maintenance of pregnancy. Dr. Fuller Bazer, in collaboration with several members of the RDB Research Core (Drs. Laurie Jaeger, Nancy Ing, and Robert Burghardt) have been working together in new efforts focused on characterizing the uterine environment with respect to steroid receptor regulation and integrin-mediated attachment, adhesion and signal transduction. Drs. Westhusin, Piedrahita and Burghardt are working to evaluate and develop non-invasive imaging approaches to evaluate oocyte quality in several animal species. These efforts have resulted in sufficient pilot data for submission of both USDA and NIH grant applications. In addition, Dr. Fuller Bazer’s collaborations with Dr. Piedrahita’s laboratory centers around a project involving the isolation of porcine embryonic stem cells. The extent of the collaboration includes shared reagents, expertise, and a steady flow of graduate students moving between the laboratories on a regular basis. Within the last six months, the progress has been so extensive that these two investigators have been able to attract research support from a number of biotechnology firms and are seeking resources to build a new furrowing facility for the transgenic sows. The ability to attract outside resources to the collaborative projects of Drs. Piedrahita and Bazer are due, in no small measure, to the support provided by the CERH.

Drs. Jaeger and Bazer are Co-PIs on Dr. Guoyao Wu’s (member of Nutrition core) funded Center pilot project "Arginine synthesis in the Fetal Pig Small Intestine." The increased level of collaboration fostered by this pilot project facilitated the development of a project directed toward understanding how endogenous and nutritional factors alter development of the small intestine and subsequent fetal somatic growth, and the submission of a new grant application: *Protein Nutrition and Fetal Intestinal Growth,* RO1 DK56241, Dr. L.A Jaeger, PI, Dr. G. Wu, Co-PI, Dr. F.W. Bazer and Dr. N.H. Ing, collaborating investigators. Dr. Jaeger is also utilizing the laser confocal imaging capabilities of the Imaging core to examine regulation of conceptus adhesion molecule expression, resulting in pilot data which has been submitted in a new grant application to the National Science Foundation and in a resubmission of an application to the United States Department of Agriculture. Vital imaging services of the Core are also being utilized to examine effects of estrogenic compounds of cultured porcine trophoblast cells in a project supported by local extramural funds (Texas Higher Education Coordinating Board). Preparations are underway of a new grant application, to be submitted to NIEHS, involving collaborating investigators who are member of the RDB Research Core (R.C. Burghardt, F.W. Bazer, N.H. Ing), currently are in progress.

Drs. Abbott and West have been collaborating on a project to further delineate the effects of alcohol exposure on the early postnatal development of the brain. The study of morphologic cellular changes at the ultrastructural level allows a much clearer understanding of the types of functional changes that occurring in individual cells when they are exposed to toxins such as alcohol. The specific focus of this project is to delineate the changes occurring in cerebellar Purkinje cells within 24-48 hours after acute alcohol exposure that ultimately leads to Purkinje cell death and is supported by a NIH-NIAAA grant, Fetal Alcohol Syndrome - Third Trimester Model, to Dr. West (PI) with Dr. Abbott as a collaborating investigator. This collaboration between Dr. West and Abbott has been strengthened by the infrastructure afforded by the CERH. Additionally, Dr. Abbott is studying the process of cell death using an animal model, the leaner mouse, that exhibits excessive apoptosis of specific cells in the postnatal cerebellum, including Purkinje cells, granule cells and Golgi cells. The direct overlap of Purkinje cell death in this model with the alcohol model being studied by Dr. West provides exciting possibilities for comparison between these two models for similarities and differences by which Purkinje cells undergo cell death. The CERH core facilities have provided excellent resources for the members of Dr. Abbott's lab to design and construct DNA probes to look for mRNA expression for cell death associated proteins as well calcium binding proteins. This work is supported by a NIH-NINDS K08 (K08 NS1681-05) award to Dr. Abbott and forms the basis for a recent R01 proposal submission to NIH-NINDS (November 1, 1998: Ca2+ channel mutations and Purkinje cell function; R01 NS38187).

During the past five years, Dr. Robert Burghardt has developed extensive interactions with members of the Faculty of Toxicology including Drs. Busbee, Phillips, Safe, Tiffany-Castiglioni, Ramos, Donnelly, and Wild (CMB). These collaborative efforts involved the development and adaptation of laser cytometry to mechanistic analysis of underlying cytotoxicity mechanisms in cell types ranging from astrocytes to uterine myometrial cells. Laser cytometric analysis of cellular injury and adaptive mechanisms resulting from exposure to several classes of compounds and more complex mixtures identified in chemical wastes including chlorinated phenols, polychlorinated dibenzopdioxins, and heavy metals have been performed. During the past year, new analytical tools have been developed to characterize frequency encoded intracellular Ca2+ signals and to define and quantify the intracellular Ca2+ pools contributing to intrinsic and hormone-induced Ca2+ oscillations. These methods have been adapted to identify the regulation of oxytocin- and/or vasopressin-induced Ca2+ oscillations in rat liver and human myometrial cells. Using a liver cell model, mechanistic analysis of the cytotoxicity of benzo[a]pyrene (BaP) has exploited the intrinsic fluorescence this polycyclic aromatic hydrocarbon to characterize the uptake and subcellular partitioning of BaP within cytoplasmic membrane systems in several cell types. Relationships between BaP content and altered Ca2+ homeostasis, (including Ca2+ oscillations) and altered membrane functions (intercellular communication, mitochondrial and plasma membrane potential) and reactive oxygen species generation are underway.

Andrews, D.L., Kelly, C., Cobb, B.G. and West, J.R. Attenuation of lactate production during ethanol exposure in hypoxic postnatal day 4 rat cerebella in vitro. Alcoholism: Clin. Exp. Res., 22 (3 suppl.):105A, 1998.

Bäckman, C., West, J.R., Mahoney, J.C. and Palmer, M.R. Electrophysiological characterization of cerebellar neurons from adult rats exposed to ethanol during development. Alcoholism: Clin. Exp. Res., 22:1137-1145, 1998.

Barber, R.C., Shaw, G.M., Lammer, E.J., Greer, K.A., Lacey, S.W., Wasserman, C.R. and Finnell, R.H. Lack of association between mutations in the folate receptor alpha gene and spina bifida. Am. J. Med. Gen. 76:310-317, 1998.

Bazer, F.W., Ott, T.L., Spencer, T.E. Maternal recognition of pregnancy: comparative aspects. Trophoblast Res. 12:375-386, 1998.

Chen, W.-J.A. and West, J.R. Alcohol-induced inhibition of cocaine metabolism and the formation of cocaethylene in neonatal rats. Neurotoxicology and Teratology, 20: 565-570, 1998.

Chen, W.-J.A., Parnell, S.E. and West, J.R. Alcohol and nicotine exposure during the brain growth spurt did not alter thyroid status in preweanling rats. Alcoholism: Clin. Exp. Res., 22 (3 suppl.):105A, 1998.

Chen, W.-J.A., Parnell, S.E. and West, J.R., Nicotine decreases blood alcohol concentration in adult rats. Alcoholism: Clin. Exp. Res. , 22 (3 suppl.):109A, 1998.

Finnell, R. Genetic regulation of abnormal neural development. Wayne State University, Institute of Chemical Toxicology, Detroit, MI., October 1998.

Finnell, R. Inactivation of Folate Bendeing Proteins in the Mouse: Impications for Neural Development. FASEB Summer Conference on Folate, B12, and One Carbon Metabolism, Snowmass Village, Colorado, August, 1998.

Finnell, R. Recent Advances on the teratogenicity of Anieopileptic Drugs. Fourth Eilat Conference on New Antiepileptic Drugs, Eilat, Israel, September, 1998.

Finnell, R. Inactivation of the Folate Binding Protein Genes Implications for the role of Homocysteine in Nevral Tube Defects. Second International Conference on Homocysteine Metabolism, Nijmegen, The Netherlands, April, 1998.

Finnell, R. Genetic Susceptibility to Teratogenesis. Teratology Society 38th Annual Meeting, San Diego, CA., June 1998.

Finnell, R. Genetic Regulation of Abnormal Neural Development. University of British Columbia, Department of Medical Genetics, Vancouver, Canada, June 1998.

Finnell, R. Genetic Regulation of Abnormal Neural Development. University of Nebraska Medical Center, Department of Anatomy and Cell Biology, Omaha, NE, June 1998.

Gupta A, Dekaney, C.M., Bazer, F.W., Madrigal, M.M., Jaeger, L.A. Beta transforming growth factors at the porcine conceptus-maternal interface. II. Uterine TGFß activity and expression of immunoreactive TGFßs (TGFß1, TGFß2 and TGFß3) and their receptors (Type I and Type II). Biol. Reprod. 59:911-917, 1998.

Gupta A, Ing NH, Bazer F.W., Bustamante L.S., Jaeger L.A. Beta transforming growth factors at the porcine conceptus-maternal interface. I. Expression of TGFß1, TGFß2 and TGFß3 messenger ribonucleic acids. Biol. Reprod. 59:905-910, 1998.

Hsiao, S.-H., Mahoney, J.C., West, J.R. and Frye, G.D. Development of GABAA receptors on medial septum/diagonal band (MS/DB) neurons after postnatal ethanol exposure. Brain Res., 800: 100-113, 1998.

McAlhany, R.E., Jr., Miranda, R.C. and West, J.R. Interactions between ethanol and tyrosine kinase signaling in the developing cerebellum. Alcoholism: Clin. Exp. Res., 22 (3 suppl.):23A, 1998.

Miller, J.A., Maier, S.E. and West, J.R. Magnitude of deficit in granule cell number varies with age in rats exposed to alcohol during the first two trimesters equivalent. Alcoholism: Clin. Exp. Res., 22:25A, 1998.

Ott, T.L., Yin, J., Wiley, A.A., Kim, H.T., Gerami-Naini, B., Spencer, T.E., Bartol, F.F., Burghardt, R.C. and Bazer, F.W. Effects of the estrous cycle and early pregnancy on uterine expression of Mx protein in sheep (Ovis aries). Biol. Reprod. 59:784-794, 1998.

Pantazis, N..J., West, J.R. and Dai, D. The nitric oxide cyclic GMP pathway plays an essential role in both promoting cell survival of cerebellar granule cells in culture and protecting the cells against ethanol neurotoxicity. J. Neurochem., 70:1826-1838, 1998.

Parnell, S.E., Chen, W.-J.A. and West, J.R. Early postnatal alcohol exposure did not alter cell numbers in locus coeruleus in adult rats. Alcoholism: Clin. Exp. Res., 22 (3 suppl.):56A, 1998.

Piedrahita, J.A., Moore, K., Oetama, B., Lee, C.-K., Scales, N., Ramsoondar, J., Bazer, F. and Ott, T. Generation of transgenic porcine chimeras using primordial germ cell (PGC)-derived colonies. Biol. Reprod. 58:1321-1329, 1998.

Shaw, G.M., Rozen, R., Finnell, R.H., Wasserman, C.R. and Lammer, E.J. Maternal vitamin use, genetic variation of infant methylenetetrahydrofolate reductase and risk for spina bifida. Am. J. Epidem. 148:30-37, 1998.

Singh, B, Ott, T.L., Bazer, F.W., de la Concha-Bermejillo, A. A structural response to pulmonary intravascular macrophages of lentivirus-infected and recombinant ovine interferon tau-treated lambs. Anat. Record 251:472-485, 1998.

Spencer, TE, Ott, TL, Bazer, FW. Expression of interferon regulatory factors one and two in the ovine endometrium: Effects of pregnancy and ovine interferon tau. Biol. Reprod. 58:1154-1162, 1998.

Tuo, W., Brown, W.C., Rogers, E., Zhu, D., Lin, G., Smith, R., Bazer, F.W. Trophoblast IFN-t differentially induces lymphopenia and neutropenia in lambs. J Interferon Cytokine Res. 18:731-737, 1998.

Tysseling, K.A., Thatcher, W.W., Bazer, F.W., Hansen, P.J., Mirando, M.A. Mechanisms regulating prostaglandin F_2a secretion from bovine endometrium. J. Dairy Sci 81:382-389, 1998.

West, J.R., Perrotta, D.M. and Erickson, C.K. Fetal alcohol syndrome: A review for Texas physicians. Texas Medicine, 94 (7):61-67, 1998.

Wu, G, Pond, W.G., Ott, T.L., Bazer, F.W. Maternal dietary protein deficiency decreases amino acid concentrations in fetal plasma and allantoic fluid of pigs. J. Nutr. 128:894-902, 1998.

Description: The Laboratory for Biological Mass Spectrometry (LBMS) is a university level facility that provides modern mass spectrometry capabilities to researchers in the Center for Environmental and Rural Health and other units of the Texas A&M University System. The major focus of the research activities within the LBMS is on the identification and characterization of proteins. The research activities of the LBMS are divided into three components: (1) fundamental ion chemistry and developmental mass spectrometry, (2) collaborative research, and (3) mass spectrometry applications. The research in (1) provides underpinning for developing new methods and mass spectrometry instrumentation and these developments are tested and evaluated by collaborations with biological researchers in advance of commercialization

Research activities involving the LBMS and CERH projects are aimed at identification of proteins that are important to a variety of biological processes. For example, we have developed a rapid, sensitive method for protein identification, including post-translational modification, using matrix-assisted laser desorption ionization (MALDI) high resolution (HR) time-of-flight (TOF) mass spectrometry. This research takes advantage of a unique instrument, a prototype MALDI HR-TOF-MS, specifically designed and constructed for the LBMS by PerSeptive Biosystems, Inc., Houston, Texas. The method involves accurate determinations of protein molecular weights, combined protein proteolysis and MALDI HR-TOF-MS for peptide mapping and protein data base searching for protein identification, and the use of proteolysis and MALDI HR-TOF-MS for determination of post-translational modifications of proteins. This developmental research produced a very powerful analytical method that can be used for analysis of chromatographically purified proteins and even protein mixtures, and in some cases the method can be used for protein identification by using in-gel digest of proteins separated using gel elecrophoresis. Under optimum conditions the analysis can be performed on femtomole amounts of material and it may be possible to push the detection limits to sub-femtomole quantities; however, in most case the experiments requires 1-10 picomole quantities of protein. Although these methods are still under development, they are currently being used in a number of collaborative projects, e.g., to identify fatty acid binding proteins (collaborator Prof. Fred Schroeder) and proteins that bind and activate electrophile response elements in the promoter of xenobiotic regulated genes such as c-Ha-ras and GST-Ya (collaborator Prof. Ken Ramos). In our own research we are using a derivative of this experimental method in combination with H/D exchange chemistry to study protein folding and solvent dependent protein folding.

The LBMS also provides routine low and high resolution mass spectrometry service for CERH research activities as well as both gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry.

David H. Russell, Ph.D., Director

Professor, Department of Chemistry

Lloyd W. Sumner, Ph.D., Associate Director

Research Scientist, Department of Chemistry

Barbara Wolf, Ph.D., Collaborations Coordinator

Assistant Research Specialist, Department of Chemistry

Sharon J. Shields, Staff Scientist

Department of Chemistry

Scott M. Peterman, Laboratory Technician

Department of Chemistry

Joseph Marini, Laboratory Technician

Department of Chemistry

Time-of-flight mass spectrometers: PerSeptive Biosystems, Inc. Voyager Elite XL high resolution matrix-assisted laser desorption ionization (MALDI) This instrument is the cutting-edge in TOF instrumentation for biological research. Mass resolution of greater than 10,000 can be achieved up to m/z ratios of 10,000 with mass measurement accuracy of less than 10 ppm. Analysis requires 1-10 picomoles of sample and in some cases as little as 1-10 femtomoles of sample is sufficient.

Proto-type electrospray ionization (ESI) time-of-flight (TOF) mass spectrometer: This is a unique instrument for direct analysis of solutions containing biological samples. The instrument is especially well-suited for studies of protein-protein and protein-small molecule interactions.

High resolution tandem TOF instrument equipped with photodissociation This instrument is used for developing peptide sequencing using mass spectrometry. Ionization is achieved by using either ESI or MALDI and the fragment ion mass spectrum (used to extract the amino acid sequence) can be obtained using metastable ion, collision-induced dissociation, and/or photodissociation.

Ion mobility-TOF instrument This is a unique instrument that can be used for studies of the size or conformation of proteins and peptides.

Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry: The LBMS is equipped with two FTICR instrument that are used for collaborations and applications research. These two instruments provide unique capabilities for analysis (using ESI and MALDI) of large biomolecules. In addition, one of the ICR systems is equipped with capabilities for ion mobility measurements, determination of volumes of gas-phase ions, similar to gel-electrophoresis.

The major CERH users of the LBMS facilities have been Dr. K. Ramos (identify proteins that bind and activate electrophile response elements in the promoter of xenobiotic regulated genes), Dr. F. Schroeder (studies of fatty acid binding proteins and binding sites), and Dr. R. Chapkin (studies of fatty acid binding proteins and binding sites). Each of these projects involve development of specific methods for a specific protein or class of proteins, and eventually these methods can be modified for more general applications.

Description: The goal of this core is to support four different activities of the CERH. First, we support the research cores by providing statistical support. This support comes in three forms. The first line of support comes from the Help Desk that is maintained in the CERH main offices.

Here we provide support for short-term statistical design and analysis questions. Each investigator in the Center has access to this service at no charge. At the current time we have one graduate student staffing the desk on a no appointment basis for 12 hours per week. The graduate student is supported by the facility core faculty and meets weekly with the core director, as well as keeping a log of all consulting activity. We also support long-term collaborative research by covering 25% of a GAR for those research teams whose work involves more extensive statistical modeling and analysis. Two graduate students are receiving support through this mechanism. Finally, members of the facility core also support research teams through personal interaction with other investigators. This support is funded through individual investigator grants.

The other three activities involve supporting the computational needs of the Center. We have developed a web site that will be used to support the Center's outreach activities. We are finishing an archival storage/retrieval system that will give researchers access to a secure

data storage facility. Finally, we plan to develop an on-line request system for the other facility cores. By using this service, investigators will be able to request services from any facility and the facilities should be better able to track their services and schedule support.

James A. Calvin, Ph.D., Director

Professor and Head, Department of Statistics

James Snell, Ph.D., Co-Director

Veterinary Anatomy and Public Health

Virgil Marco, Programmer

Department of Statistics

Chunfeng Chen, Graduate Assistant Research

Department of Statistics

Jeff Morris, Graduate Assistant Research

Department of Statistics

Jerome Bennett, Graduate Assistant Research

Department of Statistics

1. Compaq PC Server which supports the CERH mail system, web site and data archival system.

2. PC in Help Desk office to support statistical consulting activities.

3. HP laserjet printer in Help Desk office to support statistical consulting activities.

The archival storage/retrieval system will be able to storage any electronic file, be it a flat data file, an image file, a word processing document or a spread sheet. Each investigator will have his/her own secure storage hierarchy which will allow oversight over all members of the lab

without requiring that the investigator be the only member of the lab allowed to store data. Files will have generation numbers so that new versions of the same file can be stored without destroying older versions. There will also be keywords and an abstract associated with each file.

The key word mechanism will be searchable on two levels. The investigator can search his/her own data system for files. One can also search the entire CERH system looking for other researchers working in the same area. If another researcher has used the same keyword(s) then contact information will be supplied so that researchers can communicate. Data files will not be able to be retrieved, except by their creator or the investigator running the lab which created them.

The CERH also has its own mail system which is intergrated with each researcher's own regular mail system. Thus, mail can be sent to a PI at lastname@CERH.TAMU.EDU and it will automatically be sent to the address the each PI identifies as the preferable location. Group aliases for research and facility cores, as well as any other recognized group will be established.

This will allow for easier communication for and to teams spread throughout the A&M campus and site in greater Houston.

Statistical support of the Center investigators is the other major focus of this facility. As was outlined above, we are supplying support at three different levels. Through these activities we believe that research teams will be able to perform more complete analyses and design more cost

effective experiments. Texas A&M University does not provide a statistical consulting service and, thus, without the CERH, Center investigators would not have any access to statistical support without creating personal contacts and paying for any and all contacts, regardless of the request. As the Center grows we envision that this aspect of the facility will become the dominant activity of the facility.

Objective: DNA sequence analysis is a multistep process involving oligonucleotide synthesis, template preparation, generation of fluorescently labeled fragments, electrophoretic separation, data acquisition, sequence assembly and functional analysis. The goal of the oligonucleotide synthesis, sequencing and analysis Facility Core is to provide the equipment, expertise and software to support the Center investigators in a multitude of data collection technologies involving nucleic acids. These technologies include; oligonucleotide primer design and synthesis, PCR amplification of template DNAs, construction of genomic and cDNA libraries and automated DNA sequence analysis. Today the state of the art in the application of these technologies covers an enormous range of highly specialized techniques and research strategies. This Service Core will provide an invaluable set of tools for investigators interested in DNA/RNA level analysis that are relevant to environmental and rural health.

The specific research aims of the core include the following:

Provide assistance in the overall strategies, design, and synthesis of oligonucleotides required for PCR and sequencing primers as well as the construction of artificial genes and gene constructs.

Provide an automated and highly reliable quality control system to evaluate newly synthesized oligonucleotides.

Provide the necessary expertise for sample preparation and sequence determination of template DNAs including; PCR amplification, DNA cloning and the generation of labeled fragments for automated sequencing.

Provide a low cost and highly efficient service determining the nucleotide sequence of DNA molecules using both dye terminator and dye primer chemistries coupled with an automated DNA sequencer and/or capillary electrophoresis system.

Provide an automated system for genotyping, mutation detection and sequence assembly .

Provide computer assisted database searching, analysis, and interpretation of DNA sequence data.

J.N. Derr, Ph.D., Director

Department of Veterinary Pathobiology

C.E. Kolenda, Laboratory Manager

Department of Veterinary Pathobiology

Michael McDermont, Laboratory Technician

Department of Veterinary Pathobiology

At present the DNA Technology service core facility housed in the Department of Veterinary Pathobiology and located within the Veterinary Medical Research Building. This facility is associated with the research laboratory of Dr. James Derr. The laboratory has the following permanent equipment; one Applied Biosystems 377 automated DNA/RNA sequencer, one Applied Biosystems model 310 capillary electrophoresis system, a PerSeptive Biosystems Expedite oligosynthesizer with a MOSS attachment, a Beckman T-1000M oligosynthesizer and associated benchtop and hand-held laboratory equipment. In addition, the laboratory has both Internet ready Macintosh and Windows based computers with the most current versions of molecular genetic design and analysis software. This facility produces approximately 50 synthetic oligonucleotides and sequencing in excess of 200 DNA templates per week. For a complete description of this facility please see our WWW Homepage at http://www.cvm.tamu.edu/ vtpb/dna_rna_core.html.

This service core facility is targeted at both the small laboratories that are interested in taking maximum advantage of automated sequencing, genotyping and oligonucleotide synthesis as well as research groups pursuing large-scale cDNA and genome sequencing projects. This facility has helped established more interaction among individual research laboratories by combining oligonucleotide synthesis, DNA sequencing and genotyping in one convenient and centrally located area. In the current funding year, CERH investigators from the Nutrition Research, Reproductive and Developmental Biology, and Cellular and Molecular Biology/Toxicology have taken advantage of the expertise and capabilities of this core facility.

To date, the DNA Technologies Laboratory has provided various services to 11 separate research projects associated with the CERH. In total the CERH includes approximately 30 individual research laboratories. With the cost saving associated with this CERH Center Grant it is anticipated that least two-thirds of the these laboratories will be able to take advantage of the discounted service rates established through this grant.

Description: The conduct of investigations into the impact of environmental contaminants on rural health requires collaborations between a broad range of disciplines. An important component of epidemiologic studies is information to define contaminant concentrations in the environment, while mechanistic studies often require analytical measurements defining the kinetics of metabolite production. In addition, the organized production and evaluation of data from all of these studies requires the establishment of a detailed Quality Assurance/Quality Control (QA/QC) program and the implementation of Standard Operating Procedures for routine laboratory techniques. The primary purpose of the QA/QC program is to improve the precision, accuracy and reproducibility of data generated by Center investigators. The specific aims of the Field Service Facility (FSF) are to:

1. Provide routine analytical support including sample preparation, extraction and standard analytical measurements to all Center investigators.

2. Provide assistance to Center investigators in the development and implementation of a sampling strategy for field investigations conducted in support of epidemiologic studies.

3. Assist in the development and implementation of Quality Assurance Project Plans for all Center research.

The FSF has provided support to investigators in the Cellular & Molecular Biology, Reproductive & Developmental Biology, and Biostatistics and Epidemiology research cores in the CERH. Much of the initial support has been in the areas of preparation of Standard Operating Procedures (SOPs) or standards for calibrating cell culture assays. Fractionation of complex mixtures or preparation of reconstituted samples has been requested by two investigators; and, two studies have required support in field investigations.

K.C. Donnelly, Ph.D., Director

Departments of Veterinary Anatomy and Public Health, Soil and Crop Sciences and

Environmental Occupational Health

L. Y. He, Ph.D., Research Chemist

Department of Veterinary Anatomy and Public Health

1. Bokelman, QA/QC Officer

Department of Veterinary Anatomy and Public Health

1. Field Sampling and Safety Equipment
2. HPLC with PhotoDiode Array Detector
3. Tecator Soxhlet Extractor
4. Greenhouse Space
5. GC/MS and LC/MS-MS (in collaboration with Dr. T. McDonald)

The FSF has provided specific services to 14 separate research projects in the current funding year. These include analytical support to 9 projects, QA/QC support for 3 projects and field services support to 2 projects. Analytical support has included preparation of a reconstituted PAH mixture, analysis of HPLC fractions isolated from a coal tar, preparation of standards for an ATSDR complex mixture study, analysis of groundwater samples treated with a clay sorbent, and preparation of standards for an Endocrine Disruptive Chemicals study. Preparation of Standard Operating Procedures has been conducted for utilization of renal cells and neural cells in culture. These studies will also require assistance in development of a methodology for testing of volatile organic chemicals. Field services have been provided to assist in collection of samples in the El Paso/Big Bend area and Laredo/McAllen area. In addition, an air sampling study was conducted to investigate the impact of haze that migrated to Central Texas from fires in southern Mexico. This project included both a field study and administration of a questionnaire to elementary school children. The FSF has also scheduled sampling for one of the pilot projects of the CERH to assist in collection of drinking water samples in a project investigating End Stage Renal Disease.

Objectives: The purpose of the Image Analysis Core is to provide Center for Environmental and Rural Health investigators with access to microscopy and image analysis services for the evaluation of cellular homeostasis. Specific Aims are to provide instrumentation and technical service for: 1) All aspects of specimen preparation for ultrastructural analysis and immunocytochemistry; 2) Digital imaging, image processing, and image analysis; 3) Quantitative single and multiparameter steady-state analysis of vital fluorescence endpoints within living and/or stabilized cells and tissues; and 4) Quantitative single and multiparameter kinetic analysis of endpoints of cellular homeostasis mechanisms.

The Image Analysis Core works closely with CERH investigators. Since the Center grant was awarded, numerous investigators in the Cellular and Molecular Biology, Reproductive and Developmental Biology, and Nutrition Research Cores have made use of this instrumentation Core. The Image Analysis Laboratory staff are engaged in development and application of new methods to enhance mechanistic assessment of cellular physiology and pathophysiology in support of CERH Investigators. Staff members are also working to acquire new technologies which enhance the aims of this core through proposal submission to the NSF Instrumentation Development Program to develop a new microscopy technology to study oscillatory signals within cells. The technology is relevant to the study of cellular signal transduction, an area of investigation relevant to several CERH investigators. Core staff have also activated an initiative to acquire multiple photon imaging instrumentation. This technology will facilitate the adaptation of mechanistic analysis of cytotoxicity mechanisms developed for use at the individual cell level to the tissue level using precision cut tissue slices.

Robert C. Burghardt, Ph.D., Core Director

Professor, Departments of Veterinary Anatomy & Public Health and Medical Physiology

Rola Barhoumi, Ph.D., Associate Director

Research Scientist, Department of Veterinary Anatomy & Public Health

Youssef Mouneimne, Ph.D. Research Scientist

Department of Veterinary Anatomy & Public Health

Miles Frey, Technician II

Department of Veterinary Pathobiology

Technician II (to be named; candidates being interviewed and selection will be made by January 1, 1999), Department of Veterinary Anatomy & Public Health

Zeiss 10C high resolution Transmission Electron Microscope with top entry stage. Accessories include goniometer stage, cyro- specimen stage, and micro-dose focusing control.

JEOL JSM-25SII mini-column Scanning Electron Microscope equipped with a secondary electron detector.

Meridian ACAS Ultima Interactive Laser Cytometer/Scanning Laser Confocal Microscope. Multi-line, UV/Visible Coherent EnterpriseTM argon ion laser capable of simultaneous excitation with two wavelengths, 3 high quantum efficiency photomultiplier tubes for detection, and an array of detection filter sets to provide fluorochrome versatility. Switching between visible and UV signals (4 µsec) is computer controlled. Laser intensity regulation and duty cycle is computer controlled via 2 acousto-optic modulators and neutral density filters.

Meridian InSIGHT Point Laser Scanning Confocal Microscope. A Zeiss Axioplan microscope interfaced with 100 mW argon ion and 75 mW Krypton ion lasers, capable of direct ocular viewing in real time and real color (i.e., at video rates). An array of detection filter sets on a filter wheel is computer-controlled and integrated with a cooled intensified CCD camera. The scanning system is controlled by galvanometric mirror (X-axis) closed loop servomotor (Y-axis) and stepper motor (Z-axis) and confocal imaging is switchable between selectable pinhole and slit apertures.

Scanalytics CELLscanTM Fluorescence Deconvolution Workstation. The instrument is supported by a Zeiss Axoiplan inverted fluorescence microscope with 100 W mercury source, a cooled CCD camera, widefield image capture, and image deblurring software consisting of a constrained iterative fluorescence deconvolution algorithm.

Photon Technologies International Fluorescence Ratio Spectrometer. This instrument capable of both spectroscopic (cuvette) and fluorescence ratio imaging (Olympus IM-35 inverted microscope). The workstation is supported by instrument control, data acquisition, presentation, and analysis software.

Digital Imaging and Image Analysis Workstation. NIH Image software supported workstation consisting of a Zeiss Axioplan 2 Research Microscope interfaced with a Hamamatsu 3 chip color camera supported by a Power Macintosh G3 Computer, 850 Monitor, a Kodak XLS 8650 PS Digital Printer and Epson Stylus Color Printer, and a Epson, Expression 636 scanner.

Zeiss PMIII Light Microscope equipped for brightfield, phase contrast, fluorescence and Nomarski differential interference contrast microscopy and video imaging.

Bio-Tek FL600FA Fluorescence/Absorbance Reader, supporting flexible kinetic assays with top and bottom probes, a fluorescence excitation and emission range of 300-635 nm and 350-700+ nm respectively and probe diameters of 5, 3, 1.5 and 1.0 mm.

Usage and Benefit: Despite a the initiation of a three phase renovation of the entire Image Analysis Laboratory that was initiated in June 1998 and which remains in progress as of December 1, 1998, the Image Analysis Core has served investigators in the Cellular and Molecular Biology (Ramos, Busbee, Tiffany-Castiglioni, Donnelly, Johnson, Phillips, and Safe), Reproductive and Developmental Biology (Finnell, Abbott, Bazer, Burghardt, Jaeger, and Westhusin), and Nutrition Research Cores (Lupton, Chapk