ANNUAL REPORT 2003-04



Center for Environmental Health Sciences
University of California, Davis




Professor Fumio Matsumura
Director






National Institute of
Environmental Health Sciences
ES05707




January 2004

TABLE OF CONTENTS

Center Cores - 5

Center Summary - 7

Center Research Highlights - 9

Administrative Core - 10

Research Cores:

Epidemiology - 12

Molecular Neurotoxicology - 18

Reproductive and Developmental Toxicology - 23

Respiratory Toxicology - 31

Toxicogenomics - 40

Facility Cores:

Analytical Biochemistry - 48

Cell/Tissue Technology - 52

Cellular and Molecular Imaging - 57

Field Studies - 60

Functional Genomics and Molecular Biology - 63

Primate/Animal Models - 67

Community Outreach and Education Program - 69

Pilot Projects - 77


Center Summary

Introduction
The major goal of the NIEHS Center for Environmental Health Sciences at UC Davis is to maintain a strong program in the toxicology of agrochemicals and related xenobiotics, particularly relating to human health and the mechanistic aspects of toxicology. Collectively Center investigators are in charge of almost $12 million per year (direct costs) in research grants. Members include new and experienced scientists, many fields of interest, and strong histories of collaboration. The Pilot Projects Program brings in new investigators and research ideas. The Affiliate Scientist category recognizes interactions with UC Davis faculty through a less formal mechanism than full membership. The CEHS program is reviewed annually by the External Advisory Committee consisting of national experts in environmental health, the Internal Advisory Committee consisting of eminent scientists with administrative experience at UCD, and the Executive Committee composed of CEHS faculty. The University of California, Davis has made a strong commitment of support by providing salaries and benefits for all Center investigators, providing space and equipment for the CEHS, and by providing three environmental health graduate fellowships per year.

Research Cores
To promote focused research efforts, intellectual stimulation and synergistic interactions, the Center maintains five research cores: (1) Epidemiology, (2) Molecular Neurotoxicology, (3) Reproductive and Developmental Toxicology, (4) Respiratory Toxicology, and (5) Toxicogenomics. The Center's multidisciplinary infrastructure encourages research collaborations in areas such as: dioxin exposure and early pregnancy loss; indicators of exposure to hazardous substances; reproductive toxicology and epidemiology; air pollution and asthma; pre-natal low birthweight among Hispanic women in local farmworker communities; development of mouse and other animal models; and a joint program on autism/children's health/neuronal developmental research.

Facilities Cores
The UC Davis campus has a large multidisciplinary pool of technical expertise which is reflected in the Center's six facility cores: (1) Analytical Biochemistry, (2) Cell/Tissue Technology, (3) Cellular and Molecular Imaging, (4) Field Studies, (5) Functional Genomics and Molecular Biology, and (6) Primate/Animal Models. Mechanistic toxicology requires application of many and different skills to the solution of environmental health problems. For example, development of pulmonary fibrosis may be detected and to a certain extent even quantitated at a biochemical level; however, should the need arise to localize precisely the biochemical changes, different techniques such as in situ hybridization for detection of increased gene expression might be used. To address the problem successfully, a biochemist might eventually need to use methods of modern morphology. Similarly, epidemiology studies on human populations reveals valuable information between exposure to chemicals and increased incidence of cancer, and data on individual susceptibilities.


Community Outreach and Education
The Community Outreach and Education Program at the University of California, Davis, seeks to fulfill the following goals: 1) To educate the public about environmental health sciences; 2) To identify and assist local community-based efforts in resolving environmental health problems; 3) To inform the public of significant findings made by CEHS scientists and others in the field of environmental health; 4) To serve as a community resource on issues related to this field by increasing the visibility of NIEHS and environmental health research in general; and 5) To build a local COEP network to achieve these goals. The COEP runs four agriculturally-based outreach programs in Northern California. In addition, the COEP coordinates an environmental toxicology alumni/industry/government outreach program. The COEP also serves as an Environmental Health Information Center for the public, answering questions via phone and email, and disseminating information via websites, listservers, newsletters, reports, videos and annual community events which include: a public symposium, a conference for environmental health scientists, alumni/corporate/state meetings, Earth Day, Picnic Day, and presentations at local community centers, farmers' markets, and K-12 schools.


Pilot Projects
The Pilot Projects Program is intended to:
1. Provide initial support for new investigators with P.I. status (who have not received NIH R01, R29, P01 awards) to establish new lines of research.
2. Allow exploration of innovative new research directions (significant departure from ongoing funded research) by CEHS faculty.
3. Stimulate established investigators outside the CEHS to explore innovative new research projects in environmental health, generally in collaboration with CEHS faculty, and who might offer innovative ideas or special skills which benefit this center.
4. Support technical advances to develop a specific technical expertise that the CEHS needs.

For the 2003-04 year, 11 applications were received and 6 awards were made for a total of $110,519


Research Highlights

Title: New Tool for Analyzing Genetic Changes in the Lung Due to Toxicant Exposure

Investigators at the Davis CEHS have been developing and testing a new microarray tool to monitor genetic changes in the lung when exposed to environmental toxicants. Their research showed differences in expression of several thousand genes in different parts of the lung when exposed to naphthalene (a pulmonary cytotoxicant). This ability to identify which subcompartments of the lung are affected by xenobiotics allows investigators to gain insight into some of the fundamental responses of pulmonary epithelial cells during injury and repair. In future studies, investigators will begin to look at the functions of the gene products for those genes whose expression levels are altered by toxicant exposure.

Publications

Shultz MA, Zhang L, Gu YZ, Baker GL, Fannuchi MV, Padua AM, Gurske WA, Morin D, Penn SG, Jovanovich SB, Plopper CG, Buckpitt AR. 2004 Mar. Gene Expression Analysis in Response to Lung Toxicants: I. Sequencing and Microarray Development. Am J Respir Cell Mol Biol30 (3):296-310.

Wheelock AM, Zhang L, Tran MU, Morin D, Penn S, Buckpitt AR, Plopper CG. 2004 Feb. Isolation of rodent airway epithelial cell proteins facilitates in vivo proteomics studies of lung toxicity. Am J Physiol Lung Cell Mol Physiol286 (2):L399-410.



Title: Identification of a New Gene which Creates a Cell-Protecting Mucin

Much of the layer of cells forming the surface membrane of the skin is protected from environmental insult by a gel-like substance composed of proteins called mucins. CEHS investigators, after a genome-wide search, have identified another mucin gene in humans (MUC19) and mice (Muc19) which plays an important role in forming the protective mucus gel. This is the largest gel-forming mucin gene ever identified and it is very possible that this protein contributes to airway mucus secretion. Further analysis of this mucin gene will hopefully reveal new understanding about the function and regulation of mucus in respiratory diseases such as chronic bronchitis.

Publications

Chen Y, Zhao YH, Kalaslavadi TB, Hamati E, Nehrke K, Le AD, Ann DK, Wu R. 2004 Feb. Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues. Am J Respir Cell Mol Biol30 (2):155-65.

Administrative Core

Overview
The Administrative Core serves as the nerve center of the CEHS with its main activities focusing on grant administration, accounting, and coordination of committees and meetings. Since Center investigators are distributed among 20 different departments in three schools and colleges plus the University Medical Center in Sacramento, coordination of Center activities and reporting is very complex. The Internal Advisory Committee, the Executive Committee, and the External Advisory Committee function under the umbrella of the Administrative Core. These Committees serve to guide the Center and review its cores and their activities and members. Specific duties of the Core include preparing and administering Center budgets; preparing annual reports and competitive/non-competitive renewal applications; assisting investigators and research cores with joint-Center investigator grant applications; acting as a liaison with NIEHS, the general public, UCD administrative offices, and other NIEHS centers; administering the Pilot Projects Program; and coordinating the Education and Training Program which include the CEHS Fellowships, Seminar Program, and Undergraduate Training Program.

Members
Fumio Matsumura, Ph.D., Center Director
Dr. Robert Rice, Ph.D., Associate Director , Department of Environmental Toxicology
Ms. Rebecca Morrison, B.A., Program Analyst, Department of Environmental Toxicology
Ms. Tara Edwards, B.A., Program Assistant, Environmental Toxicology
Mr. Daniel Lewis, Computer Resource Specialist , Environmental Toxicology


Internal Advisory Committee
The Center has 2 internal committees. The Executive Committee is composed of core faculty and the Center Director and Associate Director. The Center is led by the Executive Committee under the direct leadership of the Center Director. The Internal Advisory Committee is composed of members of the UC Davis faculty who are not directly linked with the Center. Committee members are chosen for their recognized scientific and administrative expertise. Their main responsibility is a regular and continuing review of the scientific quality of the Center's research and of the competence and productivity of Center investigators.

Dr. Dallas Hyde, Ph.D., Director , California National Primate Research Center
Dr. Bill Lasley, Ph.D., Professor, Department of Veterinary Medicine Population Health and Reproduction
Dr. Fumio Matsumura, Ph.D., Professor, Departments of Environmental Toxicology and Entomology
Dr. Marion Miller, Ph.D., Professor, Department of Environmental Toxicology
Dr. Kent Pinkerton , Ph.D., Professor, Department of Veterinary Medicine Anatomy, Physiology and Cell Biology
Dr. Charles Plopper, Ph.D., Professor, Department of Veterinary Medicine Anatomy Physiology and Cell Biology
Dr. Robert Rice, Ph.D., Professor, Department of Environmental Toxicology
Dr. Dorothy Woolley , Ph.D., Professor, Department of Neurobiology, Physiology and Behavior

External Advisory Committee
The External Advisory Committee is drawn from a pool of internationally recognized experts in environmental toxicology in the United States. It convenes on an annual basis to review the Center on a scientific and programmatic basis. It is the Committee's duty to discuss their observations and conclusions with the Center Director and the members of the Center. Furthermore, a report is prepared which serves to provide overall guidance to the Center, to bring in outside perspectives, to evaluate individual programs, and to help the Director in troubleshooting. This Committee also serves as the first peer-review team to examine the competitive renewal application.

Dr. David Eaton, Ph.D., Director, Center for Ecogenetics and Environmental Health, University of Washington
Dr. Michael Gallo, Ph.D., Director , Center for Environmental Health Sciences of the Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry--New Jersey
Dr. Raymond Novak, Ph.D., Director, Institute of Environmental Health Sciences, Wayne State University
Dr. Kenneth Ramos, Ph.D., Deputy Director, University of Louisville Health Biosciences Center

Institutional Commitment
For the current 5-year project period (2002-2007), UC Davis is providing approximately $3,000,000 in support for the Center. This includes 10% salary support for all Center faculty, $275,000 in equipment funds from various departments and the Office of Research, almost $500,000 for a new DNA Microarray Facility, and $300,000 for a CEHS Fellowships Program. The University provides all space (office and laboratory) for the Center.
Research Cores

Epidemiology

Overview
The Epidemiology Research Core provides intellectual stimulation for research into the relationship between agricultural exposures and adverse health effects in studies involving human populations. This core actively participates in and stimulates new research ideas, approaches and hypothesis testing, and complements the Field Studies Core which supplies samples, access to human populations, and expertise in exposure assessment in these populations. Most projects generated in this core involve several Center investigators, each providing different areas of expertise. The overall goal of this research is to characterize the exposures and associated determinants of disease among populations exposed to toxic chemicals in the agricultural workplace. To this end, this core encourages and facilitates research to assess the contribution of potentially toxic agrochemical exposures to symptoms or disease in the exposed populations. The research involves interaction of disciplines involved in epidemiologic study of disease occurrence, exposure assessment and characterization of toxic exposures, effects and likely routes of transmission. Each project may involve some or all of these components. The interaction of epidemiology with exposure assessment (industrial hygiene)represents a traditional model in occupational/environmental epidemiologic investigations. The integration of laboratory analyses with epidemiologic data, sometimes referred to as molecular or biochemical epidemiology, is also an important approach to studying the determinants and mechanisms of disease and adverse health effects of exposures. In these studies, the laboratory investigators work with epidemiologists to measureboth markers of exposure in biologic specimens, and markers of effect. For example, measurement of organophosphate metabolites in urine is a marker of exposure, while assessment of urinary endocrine markersof reproductive function is a marker of effect. The investigation of dust exposure and pulmonary pathology in lungs from deceased Hispanic males obtained from coroners' cases includes both biomarkers of exposure (lung dust content and composition) and effect (pathologic changes in the lung).

Core Director and Members
Dr. Ellen B. Gold, Ph.D., Professor, Department of Epidemiology and Preventive Medicine
Dr. Bill L. Lasley, Ph.D., Professor, Department of Population Health
Dr. Marc B. Schenker, M.D., M.P.H., Professor, Department of Epidemiology and Preventive Medicine
Dr. Barry W. Wilson, Ph.D., Professor, Departments of Animal Science and Environmental Toxicology

Key Words
Agricultural Exposures, Epidemiology, Farmworkers, Reproductive Health



Progress
Drs. Gold and Lasley are collaborating on three studies currently. The first is funded by NIEHS and involves assessment of ovarian and thyroid function in women in a community near a site contaminated with perchlorate and solvents, compared to those living in adjacent communities without contamination. The protocol includes questionnaire administration, blood and urine specimen collection for assessment of thyroid and ovarian function, and daily diary information collection. Sampling, enrollment, and data collection is currently underway, with nearly 300 women enrolled thus far, and will continue for at least 2 more years.

The second study on which Drs. Gold and Lasley are collaborating was funded by the California TRDRP and is an assessment in an occupational cohort of ovarian function in relation to active and passive smoke exposure and other demographic and lifestyle factors. Three manuscripts have been drafted and submitted for publication, two of which have been accepted for publication.

The third collaboration of Drs. Gold and Lasley is funded by NIA and involves the assessment of endocrine function in relation to demographic, lifestyle, environmental and medical factors in a multi-ethnic nationwide cohort of midlife women, which has generated several publications thus far.

Dr. Gold has also collaborated with Dr. Buckpitt in the Analytical Biochemistry Core (and others) in a study of neurobehavioral effects of organophosphates (OPs) in children of farm workers. The project aims were: 1) To determine the prevalence at baseline of neurobehavioral and/or neurologic abnormalities in a representative sample of children of farmworker parents and to compare these to the prevalence rates in a sample of children of non-farm worker parents who were of comparable ethnic and socioeconomic background; 2) To determine if any differences in prevalence of neurobehavioral and/or neurologic abnormalities in these children were associated with differences in levels of urinary OP metabolites.

Funds were obtained from NIOSH, UC Mexus and the California Latino Policy Seminar to implement this study. Focus groups were conducted with women in the community to gain insights into their knowledge, their concerns, and impediments to participating in the study. In addition, a parental questionnaire was developed, pre-tested and translated; appropriate translated instruments for measuring neurobehavior in young children, and basic neurologic tests were identified; and bilingual and bicultural interviewers and pediatric nurse practitioners were trained. During the summers of 1996 and 1997, nearly 2000 households were enumerated, and 97% of these were screened for eligibility. Among these, 387 were identified as having eligible children, and 78% of these were interviewed, the children tested and baseline urine samples obtained. Of the 300 families who participated, about 100 each include mothers who performed farm work, and families in which the father performed farm work but the mother did not, and families in which neither parent performed farm work. In addition, Dr. Buckpitt has perfected assay techniques for measuring OP metabolites in urine at low levels with good sensitivity and has assayed all of the urine samples. Assays have been completed and analyzed. No significant differences in most neurobehavioral measures were found among the parental work groups or by OP exposure, although exposed children were more likely to have reduced shoulder elevation strength. Two manuscripts reporting the results of this study have been submitted for publication.

Dr. Gold also collaborated with Drs. Lasley and Wilson in a study of reproductive health among female migrant farm workers, funded by NIOSH and UC Mexus. The project aims were: 1) To conduct a cross-sectional survey, with an in-depth interviewer-administered questionnaire, and a limited prospective study, using daily menstrual calendars, of 400 women aged 18-45 years living in five Migrant Housing Centers in three California agricultural counties; 2) To determine the prevalences of specific adverse reproductive health outcomes, including spontaneous abortions, altered menstrual cycle length and variability, pregnancy complications, and premature births; 3) To compare the prevalences of these reproductive outcomes in women who perform farm work with those who do not, adjusting for confounding variables. A participation rate of 87% for the interview was achieved in the five Migrant Housing Centers that were included in the study, with 360 women completing the interview. In addition, 90% of women completing the interview also completed daily calendars on menstrual function and work activities over the course of the summer season, as well as providing a baseline and post-season urine sample, thus demonstrating excellent compliance. In addition, the study included assessment of cholinesterase level in a drop of blood within a subset of the population, performed by Dr. Barry Wilson's lab.

Approximately 60% of women also provided a baseline fingerstick blood sample for measurement of cholinesterase, and about half of these provided a post-season blood sample. No significant differences in adverse pregnancy outcomes or in delivery complications were observed between women working in the fields and other women. Women performing fieldwork had significantly shorter duration of menstrual bleeding and had shorter menstrual cycle lengths than did non-fieldworkers. Results of the analyses will be submitted for publication by mid-2004.

In the past year, Dr. Lasley has applied his biomarker assays to two separate population-based studies of women (Lohstroh et al 2003; Chen et al 2003a), demonstrating adverse effects of environmental toxicants to women's reproductive health.

In the past, Dr. Lasley collaborated with Dr. Overstreet and epidemiologists and laboratory scientists in the People's Republic of China (PRC) for a study of biomarkers of reproductive function in exposed Chinese men and women. The project aims were: 1) To compare FSH values in men with normal and abnormal semen; 2) To assess the correlation of urinary and serum FSH in men; 3) To assess the validity of urinary FSH as a screening assay for subfertility in men; 4) To assess the feasibility of large-scale laboratory evaluations of reproductive function in the PRC. A collaboration between Drs. Lasley and Overstreet and epidemiologists and laboratory scientists in the PRC enabled access to exposed populations in which reproductive effects may be anticipated. Dr. Overstreet has provided technical support for evaluation of sperm biomarkers in the andrology laboratory within the Analytical Biochemistry Facilities Core and he has provided intellectual contributions to the research effort to develop new endocrine biomarkers that is led by Dr. Bill Lasley.

Dr. Lasley has also provided creative input and technical support for endocrine assays through the Analytical Biochemistry Facilities Core and intellectual contributions through the Reproductive and Developmental Toxicology Research Core. In collaboration with other members of the Analytical Facilities Core, a new urinary biomarker, FSH, for assessing male and female reproductive health was developed, validated and applied. To assess the feasibility of performing large-scale laboratory evaluations in PRC, 72 women in the PRC were evaluated by daily urine samples in a prospective pilot study. The 72 cycles were classified using the assays established at UCD and transferred to PRC. Of the 72 cycles evaluated, 37 cycles (51%) were either abnormal or anovulatory, 10 cycles (14%) had evidence of early fetal loss (EFL), 2 cycles (3%) had confirmed clinical spontaneous abortion, 4 cycles (6%) were determined to have normal pregnancy and only 19 menstrual cycles (26%) were considered normal non-conceptive cycles. Quality control studies carried out at UCD confirmed the accuracy of these assessments. These pilot studies demonstrate that conducting large-scale, population-based studies in the PRC is feasible and that the biomarkers developed at UCD are effective in identifying adverse reproductive outcomes in exposed female populations. Total concentrations of urinary follicle stimulating hormone (FSH) were measured in a group of 44 men and were compared to FSH measurements in serum. On the basis of these and other published data, a urinary FSH value of greater than 2ng/mg creatinine (Cr) was selected as the cutoff point to identify men with elevated serum FSH (greater than 12mIU/ml) or low sperm counts (greater than 20 million/ml). The sensitivity and specificity of this algorithm for detecting elevated serum FSH in a group of 58 agricultural workers in the PRC were 100% and 50%, respectively. The sensitivity and specificity of this algorithm for detecting low sperm counts in a population of 105 infertility patients at UCD were 58% and 76%, respectively. This test may have particular value in identifying populations with no evidence of testicular toxicity, and in which labor-intensive semen studies may not be feasible.

Dr. Schenker has conducted studies with industrial hygienists on exposure to dust and agrochemicals and associated respiratory outcomes. The project aims were: 1) To describe work practices and exposure to agrochemicals, including agricultural dust, among a group of randomly selected California farm operators; 2) To ascertain the prevalence of respiratory symptoms and disease among California farmers; 3) To establish a cohort of farm operators for follow-up of incident cases of respiratory disease and for more detailed study, including exposure assessment and physiological measurements of respiratory diseases; 4) To conduct nested case-control studies within the larger cohort of farmers, for more specific characterization of respiratory disease and its determinants; 5) To characterize the respiratory health of California farmers in specific commodities such as rice, poultry and grapes, and investigate possible associations between occupational exposures and respiratory health. A cohort of California farm operators was established for assessment of occupational exposure and physiological measurements of respiratory disease. In 1993, 1,947 eligible farmers and farm managers completed a computer-aided telephone interview, which included questions about the farm environment, work activities and a variety of health outcomes. These farmers comprise the baseline group with which further follow-up studies will be conducted. Analysis of respiratory outcomes among this cross-sectional sample has been completed. Persistent wheeze was significantly associated with self-reported dust exposure, and chronic bronchitis and chronic cough were weakly associated with dust exposure. These associations were investigated in more detail in a nested case-control study of respiratory disease among the farmers who participated in the cross-sectional study. This follow-up survey was completed in 1995 on a subset of symptomatic and non-symptomatic farmers from the original survey. Telephone interviews were conducted by the California Dept. of Health Services survey research unit on 777 farm operators throughout California (71% response rate). The field visit component of this follow-up study has also been completed, with visits made to 375 farmers for measurement of pulmonary function and collection of biologic samples. Each field visit included a measurement of spirometry, collection of a blood sample for measurement of hematologic and immunologic markers and training in the use of a portable peak flow meter. A questionnaire was developed which asks about details of all farm-related tasks, including the level of dust exposures, and about history of respiratory disease and current respiratory and allergy symptoms. Analysis of data generated by the substudy are underway. The original cohort of 1,947 farm operators were interviewed again by telephone in January through April of 1998. This information added a longitudinal aspect to the original data, and allowed observation of the incidence of disease, as well as changes in work and exposure patterns over time. Additionally, spouses of the cohort of farm operators were interviewed, and children under 18 enumerated, which allowed for a better characterization of exposures and diseases affecting California farm families. Associated with this investigation is assessment of repeated exposure to dusts and other agricultural toxins and relationship of these exposures to specific commodities and farm tasks. The characterization of dust exposure in a spectrum of California farms was completed by Dr. Mark Nieuwenhuijsen. Samples of both personal and area pollutants were made, and determinants of increased dust exposure have been analyzed for within-farm and between-farm variability. Sampling included total and PM10 dust concentrations, silica and endotoxin determinations. A crop group and task matrix is being developed which will allow characterization of an individual's dust exposure, based on daily farm activities and the setting in which they take place. This matrix will be used for general risk assessment as well as exposure assessment in the specific study of respiratory disease among farm owners and managers.

Additionally, Dr. Schenker collaborated with Dr. Pinkerton in the study of lung tissue from coroner cases of Hispanic males in Fresno county. This project was undertaken to examine the frequency of silica-associated lung disease in autopsy specimens of agricultural workers and residents of the San Joaquin Valley, while developing methods for reproducible, quantitative analysis of histologic lesions and dust content in human lung tissue for epidemiologic studies. Lung tissue sample and epidemiological data collection has been completed, resulting in 117 lung specimens, which have been analyzed by microdissection in Dr. Pinkerton's lab, and samples from most of these have also been sent to Dr. Frank Green in Calgary and Dr. Val Vallyathan at NIOSH, Morgantown, WV. Preliminary results of this study have been presented at several national meetings. Analyses of the lung samples will be completed for morphologic, histologic, mineralogic and quantitative dust load measurements. Specifically, this will include description of dust deposition and morphologic changes, dust concentration and composition, and histologic changes in relation to dust deposition. Analyses will also include changes in relation to dust content, and comparisons of farmworker vs. non-farmworker changes. Assessment of cigarette smoking-related effects will be done. Analysis of results from the full sample will confirm and/or refine model results and contribute to a better understanding of mineral dust effects in the California farmworker.

PUBLICATIONS - Bold type indicates Center investigators and affiliate scientists.

Arrieta D [CEHS fellow], Ramirez A, DePeters E, Bosworth D, Wilson BW. 2003. Sep. Bovine red blood cell ghost cholinesterase as a monitoring standard. Bull Environ Contam Toxicol 71(3): 447-52.

Monso E, Schenker M, Radon K, Saiki C, Mararolas R, McCurdy S, Danuer B, Iversen M, Riu E, Nowak D. 2003 Feb. Region-related risk factors for respiratory symptoms in European and Californian farmers. European Resp J. 21(2):323-31.

O'Connor-Marer P, Schenker MB. 2003. Pesticide Poisoning. In Preventing Occupational Disease and Injury, 2nd ed. Part II Occupational Diseases and Injuries. American Public Health Association.

Randolph JF, Sowers MF, Gold EB, Luborsky J, et al. 2003. Reproductive hormones in the peri-menopause: correlation with menopausal status. J Clin Endocrinol Met. 77:1516-22.

Santoro N, Crawford SL, Allsworth JE, Gold EB, Greendale GA, Korenman S, Lasley BL, McConnell D, McGaffigan P, Midgely R, Schocken M, Sowers MF, Weiss G. 2003. Assessing comparative features of menstrual cycles using urinary hormone assays. Amer J Physiol:Endocrinol Metab. 284:E521-30.

Schenker MB. 2003. Appendix: Biostatistics & Epidemiology. In J. Ladou, ed. Occupational & Environmental Medicine. New York, NY: McGraw-Hill. 3rd edition:818-840.

Susitaival P, Kirk J, Schenker MB. 2003. Atopic symptoms among California veterinarians. Am J of Ind Med. 44:166-71.

Vijayalaxmi, Sasser LB, Morris JE, Wilson BW, Anderson LE. 2003 Apr. Genotoxic potential of 1.6 GHz wireless communication signal: in vivo two-year bioassay. Radiat Res. 159(4): 558-64.




Molecular Neurotoxicology

Overview
The major long-term goal of the Molecular Neurotoxicology Research Core is to understand the mechanisms by which complex environmental mixtures may produce direct and indirect neurotoxicity and their possible influence on increasing the risk of known neurological disorders. Each of the five investigators in this research core has experience in a different aspect of neuroscience and toxicology ranging from mitochondrial genetics (Cortopassi), to growth factor receptor signaling (Matsumura), to Ca2+ signaling (Pessah), to stem cell research (Vulliet), to acetylcholinesterase (Wilson). The current core therefore fosters multidisciplinary interactions that focus specifically on molecular and cellular mechanisms that influence the integrity of the central and peripheral nervous systems.

Core Director and Members
Dr. Isaac N. Pessah, Ph.D., Professor, Department of Molecular Biosciences
Dr. Gino A. Cortopassi, Ph.D., Associate Professor, Department of Molecular Biosciences
Dr. Fumio Matsumura, Ph.D., Professor, Departments of Environmental Toxicology and Entomology
Dr. P. Richard Vulliet, Ph.D., Professor, Department of Molecular Biosciences
Dr. Barry W. Wilson, Ph.D., Professor, Departments of Environmental Toxicology and Animal Science

Key Words
Cholinesterase, Development, Neurotoxicology, Organophosphates, Pesticides

Progress
Work on scorpion toxins begun last year on Parabuthus venom resulted in a significant publication (Inceoglu et al 2003) identifying the differences between prevenom and venom (Pessah and Hammock labs/Cell Culture Core, Genomics & Molecular Core). Work on scorpion venom toxins continued and several new discoveries were published in 2003. The mechanisms by which the potent scorpion toxin maurocalcine (MCa) from Scorpio maurus alter microsomal Ca2+ sequestration and release were elucidated. MCa targets ryanodine receptors with nanomolar potency. MCa is a putative mimic of the II-III loop peptide (termed peptide A (pA)) of alpha1s-dihydropyridine receptor, and both peptides are thought to act at a common site on ryanodine receptor type 1 (RyR1) important for excitation-contraction coupling. Working with an international team of collaborators, the Pessah Lab/Cell Culture Core has identified the molecular mechanisms by which MCa alters the channel gating properties of RyR1, and its influence on ER/SR Ca2+ signaling properties (Chen et al 2003; Esteve et al 2003). These results provide evidence that MCa and pA stabilize distinct RyR1 channel states through distinct mechanisms that allosterically stabilize gating states having proportional conductance. Using various MCa mutants, we identified Arg24 for MCa as a critical residue for binding onto RyR1.

Mechanism of PCB-mediated toxicity (Pessah Lab/Cell Tissue Core). We have published a study (Gafni et al, 2004) that examines mechanisms by which noncoplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and rapamycin alter Ca2+ signaling in intact cerebellar granule neurons. PCB 95 sensitized RyR-mediated responses whereas coplanar 2,4,4',5-tetrachlorobiphenyl (PCB 66), which lacks RyR activity, failed to sensitize these neurons. PCB 95 or rapamycin significantly enhanced Ca2+ responses following N-methyl-D-aspartate (NMDA) and -amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) activation of ionotropic glutamineric receptors. These results indicate that non-coplanar PCBs enhance Ca2+ signals by modifying a functional association of the FKBP12/RyR complex with Ca2+ entry channels within the plasma membrane. A collaboration initiated last year with Dr. P. Lein of the Oregon CEHS, identified RyRs as a critical target of non-coplanar PCBs, inducing apoptosis at less than 1microM in hippocampal neurons (Howard et al. 2003). Taken together these results indicate that RyRs are the principle target of non-coplanar PCBs neurotoxicity in cultured neurons.

Quantitative structure activity relationship for environmentally relevant PCB congeners toward activation of RyR Type 1. (Collaborator: L. G. Hansen, Univ Illinois) [3H]Ryanodine-binding analysis. The simple tube assay takes advantage of the fact that ryanodine binds with nanomolar affinity and in a manner that is dependent on RyR conformation, permitting detection of chemicals that are either channel activators or inhibitors. We have completed a detailed QSAR that includes 60 congeners and several catechol and sulfonyl derivatives (Wong et al, submitted). The key finding is that many of the most environmentally prevalent noncoplanar congeners exhibit high activity towards RyR1 and the rank order follows those reported by other investigators using cellular based assays. Interestingly some of the hydroxylated PCB derivatives exhibit potent activity, whereas the sulfonyl derivatives lack activity in this assay.

Molecular analysis of Leber's Hereditary Optic Neuropathy (LHON) (Cortopassi lab/Functional Genomics and Molecular Biology & Cell Culture Cores). Microarray technology is being used on RNA extracts from cells collected from human patients with several different mitochondrial diseases. The Core observed that the most productive way to sift through the data is to compare mutants and controls for altered genes in multiple tissue types. Those alterations that show up in multiple tissue types are further scrutinized. In the analysis of cells bearing mtDNA mutations that cause LHON, investigators find increases in levels of enzymes that metabolically activate steroid-like polycyclic compounds, which may explain the strong enhancement of the disease with smoking, and also the sex differences in the occurrence of the disease. The Core continues to create new neural models, by introducing mutant mitochondria into SH-SY5Y (neuroblastoma) and NT2 cells, and to differentiate them and observe the effects of the mitochondrial mutation. One apparent additional effect is an increase in histamine-dependent mitochondrial Ca2+ signaling in the mutant LHON cells. These interesting observations are being examined in more detail. We have developed a method that takes advantage of the observation that a central physiological parameter of mitochondrial function, mitochondrial membrane potential (MMP), can be assayed using the TMRM dye in a high-throughput format in neural NT2 cells. The assay detects decreases in membrane potential, which were either the result of pharmacological inhibition of the respiratory chain or protonophores, or also mutation of the mtDNA. Thus this high-throughput assay has the potential to be used for the screening of mitochondrially-active drugs that decrease or increase MMP, or to screen clones or colonies of cells bearing mtDNA mutations that decrease or increase MMP. We hope to use this newly-developed assay as a screening method for drugs which ameliorate the LHON-dependent defect. Another method provides a rapid and quantitative means for measuring mtDNA copy number, using a very fast PCR machine, the Lightcycler. This method was used to determine the differentiation-dependent alteration of mtDNA/nDNA ratio in NT2 cells bearing LHON mutations as they differentiated.

Decreased expression of genes involved in sulfur amino acid metabolism in frataxin-deficient cells (Cortopassi Lab/ Functional Genomics and Molecular Biology & Cell Tissue Cores). Inherited deficiency of the mitochondrial protein frataxin causes neural and cardiac cell degeneration, and Friedreich's ataxia. Five hypotheses for frataxin's mitochondrial function have been generated, largely from work in non-human cells: iron transporter, iron-sulfur cluster assembler, iron-storage protein, antioxidant and stimulator of oxidative phosphorylation. We analyzed gene expression in three human cell types using microarrays, and identified just 48 transcripts whose expression was significantly frataxin-dependent in at least two cell types. Significant decreases in seven transcripts occurred in the sulfur amino acid (SAA) biosynthetic pathway and the iron-sulfur cluster (ISC) biosynthetic pathway to which it is connected. By contrast, we did not observe a single frataxin-dependent transcript that fits with the other four current hypotheses. Quantitative reverse-transcriptase PCR analysis of ISC-S and rhodanese transcripts confirmed that the expression of these genes involved in ISC metabolism was lower in mutants. Amino acid analysis confirmed the defect in SAA metabolism: homocystine, cysteine, cystathionine and serine were significantly decreased in frataxin-deficient cell extracts and mitochondria. An ISC defect was further confirmed by observing decreases in succinate dehydrogenase and aconitase activities, whose activities require ISCs. The ISC-U scaffold protein was specifically decreased in frataxin-deficient cells, suggesting a role for frataxin in its expression or maintenance, and sodium sulfide partially rescued the oxidant-sensitivity of the FRDA cells. Also, multiple transcripts involved in the Fas/TNF/INF apoptosis pathway were up-regulated in frataxin-deficient cells, consistent with a multi-step mechanism of Friedreich's ataxia pathophysiology, and suggesting alternative possibilities for therapeutic intervention.

Redox sensing mechanisms in skeletal and cardiac muscle (Collaborator: J.E. Casida, UC Berkeley; L.A. Blather, Loyola of Chicago /Cell/Tissue & Analytical Cores). NADH and Ca2+ have important regulatory functions in cardiomyocytes related to excitation-contraction coupling and ATP production. To elucidate elements of these functions, we examined the effect of NADH on sarcoplasmic reticulum (SR) Ca2+ release and the mechanisms of this regulation. Physiologic concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2) mediated Ca2+-induced Ca2+ release (CICR) from SR membranes (IC50=120mmol/L) and significantly lowered single channel open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca2+ release. Blockers of electron transport prevented the inhibitory effect of NADH on CICR in isolated membranes and permeabilized cells, as well as on the activity of RyR2 channels reconstituted in lipid bilayer. An endogenous NADH oxidase activity from rat heart co-purified with SR enriched with RyR2. A significant contribution by mitochondria was excluded as NADH oxidation by SR exhibited greater than 9-fold higher catalytic activity (8.8micromol/mg protein/min) in the absence of exogenous mitochondrial complex (MC) I (ubiquinone) or MC III (cytochrome C) electron acceptors, but was inhibited by rotenone and pyridaben (IC50=2-3nmol/L), antimycin A (IC50=13nmol/L), and diphenyleneiodonium (IC50=28mmol/L). Cardiac junctional SR treated with [3H](trifluoromethyl)diazirinyl-pyridaben specifically labeled a single 23kDa PSST-like protein. These data indicate that NADH oxidation is tightly linked to, and essential for, negative regulation of the RyR2 complex and is a likely component of an important physiological negative feedback mechanism coupling SR Ca2+ fluxes and mitochondrial energy production.

Studies on mechanisms of insecticide resistance in Blattella germanica (collaborators: F. Matsumura, M. Miyazaki, K. Kaku, S. Inagaki, K. Ohyama, and D. Y. Dunlap). This work involves research on key ion channels mediating neurotoxic action of organochlorine and pyrethroid insecticides. The key biological material which has been very helpful in studying the functional aspects of these ion channels has been the nervous system from the insecticide-resistant Blattella germanica. This has been a good species for studying the mechanisms of resistance development. Our efforts involve elucidation of two cases of the resistance mechanisms in this species involving target insensitivities to two specific classes of insecticides: i.e., cyclodiene and kdr resistance. While the pattern of the shift of amino acids resulting from mutation which specifically confer resistance to B. germanica turned out to be identical to those simultaneously discovered in Musca domestica and Drosophila melanogaster, we found some differences in DNA base sequences and the nature of the site specific mutations between B. germanica and these two dipteran species. Interestingly, the action mechanism of pyrethroids on the sodium channel resembles the symptoms shown in a human disease called "hyperkelami periodic paralysis" (HYPP). Its site of mutation has been found to be very close to the mutation found in one of the pyrethroid resistance-associated changes in amino acids.

Effects of TCDD (Functional Genomics and Molecular Biology Core). The group found that TCDD when given as single i.p. injection at 100 ug/kg to male C57 mice, causes brain inflammation as assessed by qRT-PCR. The most prominent end-result of TCDD's action was elevation of IL-12 and IL-23 which is accompanied with increased mRNA expression of cox-2 and TNF- alpha. To confirm that the above phenomenon represents the activation of brain specific macrophages, dendritic cells by TCDD, the investigators established an in vitro U937 human macrophage model by treating them with interferone gamma. The results of in vitro studies indicated that TCDD indeed causes up-regulation of IL-12 and IL-23 as well as all of the inflammation markers such as cox-2, TNF-alpha, etc. Other pollutants acting in a similar manner in this in vitro model were DDT and permethrin (both trans- and cis-). Since the above action of TCDD is accompanied with down-regulation of IL-10, there is a possibility that TCDD could cause a shift in Th1/Th2 ratio, meaning that induced activation of brain autoimmune reaction could be a cause for this action of TCDD.

New Research Directions

In 2004, there will be increasing attention placed on how genetic polymorphisms influence susceptibility to insult from environmental chemicals of concern to human health. Particular attention will be focused on the relationship of PCBs and PBDE and heavy metals.

PUBLICATIONS - Bold type indicates Center investigators and affiliate scientists.

Chen L, Esteve E, Sabatier J-M, Ronjat M, De Waard M, Allen PD, Pessah IN. 2003. Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1 (RyR1) revealing a proportional gating mechanism. J Biol Chem. 278:16095-16106.

Esteve, E., Smida-Rezgui, S., Sarkozi, S., Szegedi, C., Regaya, I., Chen, L., Altafaj, X., Rochat, H., Allen, P., Pessah, I., Marty, I., Sabatier, J.M., Jona, I., De Waard, M., Ronjat, M. Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of maurocalcine in skeletal muscle cells. 2003 J. Biol. Chem. 278: 37822 - 37831.

Fessenden, J. D., Perez, C. F., Goth, S. R., Pessah, I. N. and Allen, P. D. Identification of key determinants of ryanodine receptor type 1 required for activation by 4-chloro-m-cresol. 2003 J. Biol. Chem. 278, 28727-35.

Howard, A. S., Fitzpatrick, R., Pessah, I. N., Kostyniak, P., Lein, P. J. PCBs induce caspase-dependent cell death in cultured embryonic rat hippocampal but not cortical neurons via activation of the ryanodine receptor. 2003 Toxicol. Appl. Pharmacol. 190, 72-86.

Inceoglu, B., Lango, J., Jing, J., Chen, L., Doymaz, F., Pessah, I. N., and Hammock, B. D. One scorpion, two venoms: Prevenom of Parabuthus transvaalicus acts as an alternative type of venom with distinct mechanism of action. 2003 Proc. Natl. Acad. Sci. USA 100, 922-927.

Perez, C. F., Voss, A., Pessah, I. N., and Allen, P. D. RyR-1/RyR-3 Chimeras reveal that multiple domains of RyR-1 contribute to restoration of skeletal-type E-C coupling. 2003 Biophys. J. 84, 2655-2663.

Tan G, Napoli E, Taroni F, Cortopassi G. Decreased expression of genes involved in sulfur amino acid metabolism in frataxin-deficient cells. 2003 Hum Mol Genet. 12(14): 1699-711.

Yang, T., Ta, T. A., Pessah I. N., and Allen, P. D. Functional defects in six RyR1 mutations associated with malignant hyperthermia and their impact on skeletal E-C coupling. 2003 J. Biol. Chem. 278, 25722-25730.

Reproductive and Developmental Toxicology

Overview
The Reproductive and Developmental Toxicology Research Core is responsible for activities in several sub-specialty areas. These activities range from the development of new laboratory analytical methods to population-based studies of human health. The research activities in this Core take a multi-disciplinary approach to solving problems which have direct relevance to human reproductive health. These activities involve one or more Facility Core members and usually at least one Facility Core for support. Experimental designs used by Core members include clinical investigations, in-vivo studies with animal models and in-vitro mechanistic studies. Historically this core has been active in both epidemiological and molecular aspects of reproductive toxicology. For the former aspect, the goal is to develop and apply reliable biomarkers/analytical methods for population-based studies. For the latter aspect, the goal is to understand the mechanisms by which xenobiotics adversely affect the most vulnerable processes of both male and female reproduction.

Core Director and Members
Dr. Bill L. Lasley, Ph.D., Professor, Population Health and Reproduction
Dr. Alan J. Conley, Ph.D., Professor, Population Health and Reproduction
Dr. Ellen B. Gold, Ph.D., Professor, Epidemiology and Preventive Medicine
Dr. Marion G. Miller, Ph.D., Professor, Environmental Toxicology

Key Words
Developmental Toxicology, Reproductive Toxicology

Progress
I. Development of new analytical methods (Biomarkers) for use in population-based studies of reproductive hazards.
In order to complement the numerous existing biomarkers for detecting exposures to reproductive hazards the core developed and validated practical methods for detecting adverse effects of environmental hazards on human reproductive health. Last year, under the direction of the Developmental and Reproductive Toxicology Research Core, the Analytical Core developed and validated a new method to detect xenoestrogens in biological samples (Natarajan et al., 2002). This biomarker, which is based on the ability of specific antibodies to remove endogenous steroidal estrogens, employs a cell-based bioassay for detection. This current year, Center investigators applied this assay to the nonhuman primate model using nonylphenol as the xenoestrogen and additional studies have been designed for similar applications to human samples. More recent activities include the study of the bi-products of water chlorination. Specifically, the Core evaluated the toxic action of bromodichloromethane (BDCM) on the human placenta cells in vitro. Two reports from the group demonstrate that BDCM interferes with chorionic gonadotropin secretion as well as the transformation of the cytotrophoblast to the invasive synciotrophoblast. Current activities include in vivo studies of BDCM using the nonhuman primate animal model and will be extended to compounds under investigation by other Cores. In addition, work continues on cycle-stage specific effects of stressors: Several studies of young women have shown that physical stress results in adverse effects on bone health. While the prevailing dogma has been that the mechanism for this effect was alterations in luteal function, more recent studies have challenged this concept. These studies demonstrate that stressors in the luteal-follicular transition can perturb follicle stimulating hormone (FSH) secretion patterns and lead to a delay in follicle recruitment in the next menstrual cycle. In addition, we have shown that stressors as subtle as changes in work shift during the luteal-follicular transition can lead to a delay of ovulation and increased bone resorption in the subsequent luteal phase. Using newly developed urinary assays for FSH and cortisol, the group will investigate both the effect of physical stressors and the influence of cycle-stage of exposure on menstrual function and bone loss in the female macaque.

In addition to the development of biomarker assays, the core continues to develop algorithms that can be used for evaluating ovarian function. Recently published manuscripts describe methods for identifying hormone profiles that indicate normal or abnormal ovarian function. One of these reports demonstrates that urinary follicle stimulating hormone profiles are a superior method for identifying the day of ovulation. Two other reports illustrate the differences that can be discerned between highly fecund and less fecund menstrual cycles. These methods compliment the biomarker assays that have been developed to monitor women's reproductive health in population-based studies. Another example of assays developed by the Developmental and Reproductive Toxicology Research Core and transferred to the Analytical Core are those for measurement of chorionic gonadotropin (CG) and ovarian steroids in serum and urine. These assays have been performed for the Epidemiology Core. They have been critical for identifying the adverse effects of TCDD on trophoblast cell function and detecting the adverse effects of environmental hazards such as cigarette smoke on human reproduction. The endocrine laboratory continues to provide technology transfer and training for students from the Neurotoxicology Core. These students were trained in enzyme-immunoassay procedures and provided with critical reagents in order to develop and apply assays for use in population-based studies and service a wide range of epidemiologic studies (see the Epidemiology Core).

While methods for monitoring the reproductive health of women in the workplace have been validated and applied to additional population based human studies in the past, these reports have all used assays that employ the microtiter plate assay format. Our group has begun to convert manual assays to an automated format. This past year, Core investigators have worked with Dr. Dan McConnell at the University of Michigan to convert the enzyme-based biomarker assays for use on an automated fluorescence (Centaur, Corning, Terrytown, NY) analyzer. A report is currently under review that describes this new application and Core members plan to discuss with Corning the ability to make this application available to non-clinical laboratories. The Core successfully converted all of the urinary hormone assays to this format. This platform has been purchased with Center funds and will be made available to all Center faculty. Current activities include the adapting of biomarker assays for exposure from the Hammock laboratory as well as biomarker assays for adverse effects from this Core. Since these same biomarkers and methods of assessment have been validated for the non-human primate model, investigators are currently validating the automated assays for use with nonhuman primate samples because such animal experiments will complement the population-based field studies and permit the identification of both environmental hazards and their mechanism of action.

II. Male reproductive toxicology.
The overall goal of this research is to understand how environmental chemicals adversely affect the male reproductive system. Current studies are exploring the involvement of metabolism in testis and epididymis toxicity and protein targets through which toxicants may act. A continued interest is in the testis specific toxicity of the fungicides benomyl and it hydrolysis product carbendazim, on the Sertoli cell cytoskeleton. In the testis, the Sertoli cell functions to support and provide nourishment and protection to the developing germ cells. It has an extensive cytoskeletal network composed of actin filaments, intermediate filaments and microtubules that serve structural and transport functions. In previous studies, investigators have shown that testicular toxicity seen after benomyl treatment is caused by the benomyl metabolite carbendazim and that carbendazim acts to inhibit the polymerization of tubulin into microtubule polymers. Recent publications have described the loss of alpha-tubulin tyrosination when microtubules are depolymerized. Interestingly, the inhibitory effect of carbendazim on microtubule polymerization seems to be associated with the detyrosinated form of tubulin. One of the intriguing questions about the effects of carbendazim is why are Sertoli cell microtubules and not other microtubules, for example in dividing cells or involved in neuronal transport, uniquely sensitive to the disruptive effects of carbendazim. With this in mind, current studies are exploring the possibility that a microtubule associated protein (MAP) protects the microtubules of most cell types from depolymerization after carbendazim. The hypothesis is that in the Sertoli cell at specific stages of spermatogenesis, this protective MAP is not present.

In studies with nonhuman primate tissue, testes were obtained from 3 month old animals that were part of a study in the Respiratory Toxicology Core. Cell culture methods were explored for optimization of incubation conditions. Techniques for measuring cell proliferation (BRDU staining), apoptosis (TUNEL staining), and cell viability (nuclear staining) were developed. It is anticipated that in the upcoming year a similar cohort of monkeys will be available and pilot studies with thyroid hormone and thyroid hormone antagonists as well as phthalates will be initiated.

Other studies have focused on 1) understanding metabolic mechanisms of epididymal toxicity and 2) assessment of sperm function. To develop methods for assessing sperm mitochondrial function, the well known mitochondrial uncoupling agent pentachlorophenol was used as a model mitochondrial toxicant in flow cytometry studies. Interestingly, pentachlorophenol effectively uncoupled sperm mitochondria even at low 100 nM concentrations. Trichloroethylene has been used to study potential metabolic activation mechanisms in the epididymis. Cytochrome P4502E1 was located in the efferent ducts that connect the testis and the epididymis and TCE adducts were also localized in efferent duct microsomal proteins incubated with TCE. In a recently accepted publication investigators showed that TCE exposure resulted in decreased fertilizing ability as measured by in vitro fertilization and sperm membrane protein oxidation. Another in press paper that resulted in a joint publication with Dr. Bruce Hammock's laboratory described epoxide hydrolase activity and its possible role in xenobiotic and fatty acid metabolism in the different sections of the epididymis. These studies document the possibility that epididymal toxicity could arise via metabolic activation mechanisms.

III. Mechanism of toxic action.
The research goal has been to identify molecular targets of TCDD toxicity in primary human cells that might explain reproductive toxicity in exposed women. The core re-examined the effects of TCDD on gene expression profiles using DNA array analysis on RNA isolated from hLGCs cultured with TCDD (10 nM) or vehicle (DMSO) for 24 hours. RNA was isolated from cells from 8 different patients, 50 mg of each was combined in a pool that was labeled and used with the Affymetrix Human Genome U133A GeneChip®. The results suggested that among all transcripts expressed in hLGCs, the largest fold increase was CYP1B1 (+4-fold) and the largest decrease was topoisomerase (-3-fold). The analysis also indicated that hLGCs expressed other genes of relevance to oxidative metabolism, COMT and mEH, but that these did not change with TCDD. Based on these data, investigators re-examined expression of CYP1B1 and COMT, as well as CYP17 and SET (a phosphatase inhibitor regulating CYP17 phosphorylation and activity) by real-time PCR. The results demonstrate that 24 hrs of TCDD exposure induces CYP1B1 expression in hLGCs, profoundly in some, but not at all in others. This patient-to-patient variability was not obviously related to E2 secretion, even though it varied 10-fold among patients, or to any general patterns of expression of other genes examined. Though others report that 3 mM TCDD induced apoptosis in hLGCs, this group found no evidence of cellular deterioration even with 10 day exposures to TCDD at 10 nM. This variability in response is unlikely to represent the difference between late responders and non-responders because similar inter-individual variation was found in studies conducted after 10 days of TCDD exposure. The findings were further verified by immunoblot analysis for CYP1B1 conducted on hLGCs from two different patients. Cells from one patient showed a clear induction of CYP1B1, the other did not, but CYP1B1 expression was evident in the control cells of both. Collectively, these data indicate that CYP1B1 is expressed in hLGCs and can be induced by TCDD exposure in vitro, but with marked inter-individual variation. The Core confirmed high levels of mEH expression by immunoblot and activity analyses, but did not see marked differences among patients or with TCDD treatment (data not shown). Available evidence provides strong support for the notion that hLGCs possess enzymes necessary for oxidative metabolism of E2-17b as well as PAHs such as benzo[a]pyrene (BaP), but cells recovered from different patients appear to exhibit differences in their capacity at the levels of transcript and enzyme protein expression. Importantly, neither DNA arrays or western immunoblot analyses provided evidence for the expression of CYP1A1 or CYP1A2 basally or after TCDD treatment for 24 hours or 10 days, consistent with the notion that CYP1B1 is likely the major P450 catalyzing oxidative metabolism of E2 and PAH in hLGCs.

Based on the above data, the Core is pursuing studies into oxidative metabolism in hLGCs. Ovaries are the primary sites of estrogen synthesis in non-pregnant women during their reproductive years. Metabolites of estrogen promote oxidative stress and cellular damage by generating reactive oxygen species and DNA-adduct formation. Similar molecular damage also results from toxicants like BaP, and other PAHs. Oxidative metabolism of estrogen and PAHs is initiated by cytochrome P450 (CYP) enzymes including CYP1B1. CYP1B1 expression is inducible in hLGCs by TCDD. Other enzymes, notably mEH and COMT, also influence metabolite turn-over. CYP1B1, mEH and COMT have polymorphic alleles that have been associated with susceptibility to disease in population-based studies. Cigarette smoke is rich in PAHs, and adversely affects reproductive function at the level of the ovary. Smoking induces ovarian oxidative stress and BaP adducts have also been described in granulosa-lutein cells from women smokers. Despite these observations, the enzymes necessary for the oxidative metabolism of either estradiol (E2) or PAHs, CYP1B1, mEH and COMT have not been identified previously nor investigated in primary human granulosa cells. Investigators are pursuing these studies with the assistance of other Center members and core facilities. Drs. Alan Buckpitt and Michael Denison have provided help in developing methods to monitor the production of oxidative metabolites and reactive oxygen species, in addition to exploring ways to study the molecular basis for the patient-to-patient variability in the hLGC response to TCDD. Dr. Buckpitt is a named collaborator on a recent NIH grant application seeking additional funds for this project. These techniques and the data at hand will then be translated to the laboratory macaques where the effects of toxicant exposure and ovarian oxidative metabolism on oocyte quality can be directly assessed.

IV. Epidemiological studies.
Since a major activity within the Reproductive Toxicology Research Core has been directed towards the development of assays for urinary biomarkers of human reproductive health outcomes, Core investigators have also been involved in many of the initial applications of these assays (see the Epidemiology Core) and it would be redundant to re-list all of those here. However, outside collaborations include the California Department of Health Services, where baseline data were obtained from a population of women enrolled in a pre-paid health plan. In addition, collaboration with the Study of Women Across the Nation (SWAN) has brought approximately 1,000 mid-aged women under protocol to determine the effect of life-style, diet and physical activity on ovarian function. Additionally, a collaboration with Harvard School of Public Health researchers has resulted in a study that reveals the adverse effect of shift work on ovarian function. This study also demonstrated that the alterations in ovarian function resulted in increased bone resorption and potentially an increase in the loss of bone.

As reported last year, ovarian function was monitored in a group of women who eat toxicant-contaminated fish from the San Francisco Bay. More than 50% of all intermenstrual intervals were abnormal in the exposed group of women compared to less than 15% of abnormal cycles in the unexposed control group. Most of the abnormalities were associated with prolonged follicular phases and anovulatory intervals that were often accompanied by regular episodes of vaginal bleeding which the women interpreted as normal menstrual periods. The Core is using its cell-based bioassays to measure total Ah-receptor ligand load and analyze blood samples for xenoestrogen using the new methods described earlier (Natarajan et al., 2002). Investigators developed and validated practical methods for detecting adverse effects of environmental hazards on human reproductive health and have used these in a community-based study in women potentially previously exposed to endocrine disruptors but thus far have found no increase in this class of compounds in blood samples. Current studies include the evaluation of follicle stimulating hormone to determine the site of toxic action.

PUBLICATIONS - Bold type indicates Center investigators and affiliate scientists.

Chen JG. Laughlin LS. Hendrickx AG. Natarajan K. Overstreet JW. Lasley BL. The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on chorionic gonadotrophin activity in pregnant macaques. Toxicology. 186(1-2):21-31, 2003.

Chen JG, Qiu, Q, Overstreet JW, Lasley BL. Luteal phase hormone characteristics of non conceptive and conceptive menstrual cycle. Journal of Gynecologic Investigations 10(1):27-31, 2003.

Chen JG. Thirkill TL. Overstreet JW. Lasley BL. Douglas GC. Effect of 2,3,7,8- tetrachlorodi- benzo- p-dioxin (TCDD) on chorionic gonadotropin secretion by human trophoblasts Reproductive Toxicology.17(1):87-93, 2003 .

Chen J, Thirkill TL, Lohstroh PN, Bielmeier SR, Narotsky MG, Best DS, Harrison RA, Natarajan K, Pegram RA, Douglas GC, Lasley BL. Effect of dibromochloromathane on human trophoblast chorionic gonadotropin secretion. (In Press: Toxicologic Sciences, August 2003).

Corbin, C. J., S. M. Mapes, Y. M. Lee and A. J. Conley. Structural and functional differences among purified recombinant mammalian aromatases: Glycosylation, N-terminal sequence and kinetic analysis of human, bovine and the porcine placental and gonadal isozymes. Molecular and Cellular Endocrinology 206:147-157, 2003.

Corbin, C. J., F. M. Moran, J. D. Vidal, J. J. Ford, T. Wise, S. M. Mapes, V. C. Njar, A. M. Brodie and A. J. Conley. Biochemical assessment of limits to estrogen synthesis in porcine follicles. Biology of Reproduction 69:390-397, 2003De Souza MJ, VanHeest J, Demers LM, Lasley BL. Luteal phase deficiency in recreational runners: Evidence for a hypermetabolic state J Clin Endocrin Metab 88(1):337-346,2003.

DuTeaux SB [CEHS fellow], MJ Hengel, DE DeGroot, KA Jelks [CEHS fellow], MG Miller. 2003. Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts. Biol Reprod.69: 771-9.

Golub MS, Hogrefe CE, Germann SL, Lasley BL, Natarajan K, Tarantal A. Effects of exogenous estrogenic agents on pubertal growth and reproductive system maturation in female rhesus monkeys. Toxicological Sciences 74(1):103-113, 2003.

Gravance, C.G., D.L. Garner, M.G. Miller, and T. Berger. 2003. Flow cytometric assessment of changes in rat sperm mitochondrial function after treatment with pentachlorophenol. Toxicol. in Vitro.17:253-7.

Hao J, Janssen WGM, Yang Y, Roberts JA, Mckay H, Lasley BL, Allen PB, Greengard P, Rapp PR, Kordower JH, Hof PR, Morrison JH. Estrogen induces formation of dendritic spines in the hippocampus of young and aged rhesus monkeys. J Comp Neurosci 465:540-550, 2003

Illera, J. C., G. Silvan, M. M. Martinez-Mateos, A. J. Conley, J. Corbin, A. Blass, P. L. Lorenzo and M. Illera. Effect of long-term exposure to combinations of growth promoters in Long Evans rats Part 2: ovarian morphology. Analytica Chimica Acta 483:233-240, 2003.

Lance, V. A., A. J. Conley, S. M. Mapes, C. Steven and A. R. Place. Does Alligator Testis Produce Estradiol? A Comparison of Ovarian And Testicular Aromatase. Biology of Reproduction 69:1201-1207, 2003.

Lohstroh PN, Chen J, Overstreet JW, Ryan L, Xu X, Lasley BL. Bone health is affected by follicular phase length in female rotating shift workers. Environmental Health Perspectives 111:618-622, 2003.

Moran FM. Lohstroh P. VandeVoort CA. Chen J. Overstreet JW. Conley AJ. Lasley BL. Exogenous steroid substrate modifies the effect of 2.3,7,8-tetrachlorodibenzo-p-dioxin on estradiol production of human luteinized granulosa cells in vitro. Biology of Reproduction. 68(1):244-251, 2003.

Moran FM, VandeVoort CA, Overstreet JW, Lasley BL, Conley AJ. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreases estradiol production by altering the enzyme expression and activity of cytochrome P450c17, 20 lyase of human luteinized granulosa cells in vitro. Endocrinology 144(2): 467-473, 2003.

O'Connor KA, Brindle E, Holman DJ, Klein NA, Soules MR, Campbell KL, Kohen F, Munro CJ, Shofer J, Lasley BL, Wood JW. Urinary estrone conjugate and pregnanediol-3-glucuronide enzyme-immunoassays for population research. Clinical Chemistry 49(7):1139-1148, 2003.

Randolph JF, Sowers, MF, Gold EB, Mohr B, Luborsky J, Santoro N, McConnell D, Finkelstein F, Matthews K, Korenman S, Sternfeld BX, Lasley, BL. Reproductive hormones in the early menopausal transition: Relationship to ethnicity, body size and menopausal status. J. Clin Endocrinol Metab. 88:1516-1522, 2003.

Rubenstein, N. M., G. R. Cunha, Y. Z. Wang, K. L. Campbell, A. J. Conley, K. C. Catania, S. E. Glickman and N. J. Place. Variation in ovarian morphology in four species of New World moles with a peniform clitoris. Reproduction 126:713-719, 2003.

Santoro N. Crawford SL. Allsworth JE. Gold EB. Greendale GA. Korenman S. Lasley BL. McConnell D. McGaffigan P. Midgely R. Schocken M. Sowers M. Weiss G. Assessing menstrual cycles with urinary hormone assays. American Journal of Physiology - Endocrinology & Metabolism. 284(3):E521-E530, 2003.

Shideler SE, Gee NA, Laughlin LA, Rapp PR, Morrison JH, Roberts JR, Moran FM, Lasley BL. The contribution of ovarian steroid production to the urinary estrone conjugate measurements in the laboratory macaque (Macaca mulatta). (In Press: Amer. J. Primatology, Sept 2003).

Stanton RJ, DeWitt J, Henshel D, Watkins S, Lasley B. Fatty acid metabolism in neonatal chicken (Gallus domesticus) treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3',4,4',5 pentachlorobiphenol (PCP 126) in ovo. Pharmacology and Toxicology, Part C Vol 136/1 pp 73-84, 2003.

Tang Y, Janssen WGM, Hao J, Roberts JA, McKay H, Lasley B, Allen PB, Greengard P, Rapp P, Kordower JH, Hof PR, Morrison JH. Estrogen replacement increases spinophilin-immunoreactive spine number in the prefrontal cortex of young female rhesus monkeys Cerebral Cortex (In press, October 2003).

Tang Y, Janssen WGM, Roberts, McKay H, Lasley BL, Allen PB, Greengard PE, Rapp PR, Kordower JH, Hof PR, Morrison JH. Effect of estrogen on dendrite spines in the prefrontal cortex and primary visual cortex of young female rhesus monkeys. Neuralscience (In Press, July 2003).

Venners SA, Wang X, Chen C, Wang L, Guang W, Huang A, Ryan L, O'Connor J, Lasley B, Overstreet J. Paternal smoking and pregnancy loss: A prospective study using a biomarker. In Press: Amer J Epidemiology June 2003.

Wang X, Chen C, Wang L, Chen D, Guang W, French J and the Reproductive Health Study Group (Cho S, Huang A, Ryan L, O'Connor J, Lasley BL, Overstreet J, Christiani DC, Wilcox A, Xu X.). Conception, early pregnancy loss and time to clinical pregnancy: a population-based prospective study. Fertility and Sterility 79(3): 577-584, 2003.













Respiratory Toxicology

Overview
The overall goals of this research core are to: 1) promote interdisciplinary research which will elucidate the cellular, metabolic and molecular mechanisms which define and modulate the response of the respiratory system to environmental toxicants; and 2) provide information about the harmful effects of photochemical air pollution and other inhaled toxicants which is useful in understanding the relative susceptibility of humans to harm from exposure to agricultural contaminants whose toxicity results from other metabolic pathways. This group is currently pursuing projects focused on a number of areas of general concern for public health, especially as they relate to cross-contamination of urban and agricultural sectors. The core has six overall objectives: 1) Define the cellular basis of the pulmonary response to toxicant exposure; 2) Identify the metabolic mechanisms for toxicity of xenobiotics which target the lungs; 3) Define the mechanisms regulating the development of tolerance resulting from repeated, long-term exposure; 4) Identify the factors responsible for the varying susceptibility among human subpopulations; 5) Establish the nature of the synergism of exposure to multiple toxicants on acute lung toxicity; 6) Validate biomarkers of respiratory tract exposure and toxicity. Among the intentions of the Core's objectives is to identify the critical steps in the activation and detoxification of environmental pollutants which determine the cellular susceptibility of target lung cells.

Core Director and Members
Dr. Charles G. Plopper, Ph.D., Professor, Anatomy, Physiology and Cell Biology
Dr. Alan R. Buckpitt, Ph.D., Professor, Molecular Biosciences
Dr. Bruce D. Hammock, Ph.D., Professor, Entomology
Dr. Jerold A. Last, Ph.D., Professor, Internal Medicine
Dr. Kent E. Pinkerton, Ph.D., Professor, Anatomy, Physiology and Cell Biology
Dr. Hanspeter Witschi, M.D., Professor, Molecular Biosciences
Dr. Reen Wu, Ph.D., Professor, Internal Medicine

Key Words
Airway, Development, Epoxide Hydrolase, Fibrosis, Lung, Ozone, P450, PM

Progress
During the past year, the Core compared the distribution of CYP2F in the respiratory tract of mouse, rat and rhesus macaques.These studies address the relationship between levels of transcript (mRNA) and 2F protein in the respiratory tract of rodents with tissue susceptibility to injury. Overall, these studies demonstrate a strong association between CYP2F expression levels and susceptibility to naphthalene induced cytotoxicity. Accordingly, these studies were extended to include rhesus macaques. Of all the tissues studied, only the nasal ethmoturbinates contain quantifiable amounts of CYP2F, roughly 10- and 20-fold less than the corresponding tissues in rats and mice, respectively. These results suggest that rhesus macaques may be refractory to naphthalene-induced pulmonary injury.

To establish whether the protein expression matched activity levels, airway subcompartments of monkeys exposed to filtered air or to allergen/ozone, conditions resulting in asthma, were incubated with naphthalene or 1-nitronaphthalene to define metabolism of these substrates. In control animals, the rates of formation of water soluble metabolites from naphthalene or 1-nitronaphthalene were approximately 100 times less than those observed previously in rodents. Dihydrodiol was the predominant water soluble metabolite of naphthalene generated at all airways levels while covalently bound metabolites accounted for the greatest percentage of 1-nitronaphthalene metabolites. While prior exposures of animals to HDMA, O3 or a combination had no effect on protein covalent binding of naphthalene metabolites, significantly more reactive 1-nitronaphthalene metabolites were generated in trachea, proximal, and medial airways of exposed monkeys. Reduced glutathione levels were not altered in airway levels of exposed animals. The core concludes that: 1) there are significant quantitative differences between Rhesus and rodents in substrate turnover, 2) the distribution of metabolizing activities for naphthalene but not 1-nitronaphthalene are significantly different for rodents and primates, and 3) prior exposure to allergen or O3 markedly enhances reactive metabolite formation from 1-nitronaphthalene but not naphthalene.

The adducted protein patterns also reflex enzyme activity levels.Three different systems have been utilized to generate proteins which are adducted by reactive naphthalene or nitronaphthalene metabolites: microsomal incubations with naphthalene (mouse), in situ incubations in airways filled with naphthalene (mouse) and in vitro incubations with naphthalene and nitronaphthalene in dissected airways from the rhesus macaque. Proteins were separated by 2D gel electrophoresis, adducted proteins visualized on storage phosphor screens and proteins identified by a combination of MALDI and tandem MS sequencing. Over 45 different proteins including actin, tubulin, heat shock proteins, annexin V, protein disulfide isomerase and peroxiredoxins have been identified as adducts. The heat shock proteins, proteins involved in folding (PDI) and proteins involved in detoxication of ROS (peroxiredoxin) are most interesting to date.

As a foundation for collaborative studies on the impact of toxic exposure on the pre- and postnatal lung, the Core has begun exploring ways to define aspects of airway growth that would be critical to deposition and clearance of inhaled toxicants using airway casts. The goals in developing a casting method for tracheobronchial airways, which will be used for evaluation of changes in architecture with growth, were that the method be applicable equally over a wide range of sizes and have high fidelity for the local alterations in contour in very small spaces. The Core has adapted the approaches used by others and has established a reproducible and flexible casting method for animals ranging in age from one to nine weeks. The lungs are fixed after collapse for 24 hours at high pressure within the thoracic cavity by a small diaphragmatic perforation and filled under negative pressure via tracheal canula with a silicon elastomer (Dow Corning 734 RTV ). The high quality of the surface definition of these casts is emphasized by the core's ability to see the indentations made by the apices of airway epithelial cells and the core's ability to detect the initiation of alveolarization at the terminal bronchiole/alveolar duct junction. Using this method this group has detected three classes of terminations of conducting airways into alveolar ducts. This first class includes direct contact of a larger, more proximal airway with a minimal non-alveolarized bronchiole which exits from this airway at approximately 90 degrees to its axial path. The second class includes a single small bronchiole exiting from a larger bronchiole at approximately a 90-degree angle. The third class involves a small bronchiole with two daughter branches, each exiting at approximately a 45-degree angle and opening into a series of alveolar ducts. The Core also found small areas of indentation in branch points at many proximal airways which constrict the airway size at that point by as much as 50 percent. The group applied this casting method to a wide range of animals including rats, mice and rhesus monkeys.. The ages of mice to which this has been applied ranges from one day to 10 weeks and for rhesus monkeys, five days to three years of age. The differences in body weights is greater than 40-fold between different aged animals. The core now feels that it has a reproducible casting method which will allow it to begin the process of defining how airways grow during postnatal lung development.

The Core's interest in defining the architectural organization of the conducting airways is the result of investigators' recent observations regarding the impact of exposures to an oxidant stressor, the air pollutant ozone, and an allergen, house dust mite, on the growth and reorganization of conducting airways in infant rhesus monkeys. The infants were exposed starting at 30 days of postnatal age and ending at six months of postnatal age to a cyclic protocol that included five days of ozone with or without three days of allergen. These exposure episodes were separated by nine days of filtered air. In the exposed animals, the number of airway branches down the axial path (to trachea respiratory bronchioles) was reduced by an average of four in animals exposed to the cyclic ozone regimen This was true for four different axial paths in four different lobes. The progressive increase in size of proximal bronchi was not altered during the 2.6-fold increase in lung volume with age between 30 days from two to six months. However, in distal bronchioles, diameter decreased at two months and nearly doubled at six months. Length doubled between one and six months. There were marked differences in the size of distal bronchioles as a result of exposure to ozone. This stunting of conducting airways and reversal of alveolarization to consume distal conducting airway branches was consistent in the lungs of animals exposed from two breeding years. The Core subsequently evaluated the impact of this exposure regimen followed by a secession of exposure and an additional six months of exposure to filtered air. While the distal airways continued to grow during the next six months, the stunted characteristics and the loss of the airway generations of branching persisted even in filtered air conditions. This group has recently completed a study of animals exposed from 30 days to 90 days of age with the same regimen. While there was no negative impact on overall lung growth, as measured by total lung volume, or on body size, there was a reduction in airway branching number by two to three generations and a compromise in the overall longitudinal growth of the airways by as much as 15 percent. These changes in defined experimental conditions emphasize how susceptible to perturbation the process of establishing airway architectural organization is during the growth process, especially the tremendous changes in growth that occur in the early postnatal period. Humans, by comparison to monkeys, undergo these changes for at least the first 10 years of postnatal life, while in monkeys, growth appears to be complete by the first two years.

All of the evaluations of airway architecture, which have been conducted to date in the rhesus monkey, have been done of micro dissected lungs by direct measurement of histological sections. While that data emphasizes how critical an understanding of architectural organization of the airways under growth conditions is for appreciation of the biology of the respiratory system, especially under environmental stress conditions, the amount of data which is available and the potential for developing a clear understanding is limited because of the nature of the specimens and the measuring approach. Because this has become such a critical issue for understanding children's health and susceptibility during postnatal growth, the core has done preliminary measurements on the rats used for developing casting methods for infant lungs. The core selected three to four animals at each age for which the casting material stopped at the two to three most proximal alveoli in alveolar ducts. Body weights of these animals had greater than a 40-fold range beginning with an average of 7.2 +/-0.3 g in the one-day-old infants to 296.4 +/- 31.5 g in the 63-day-old adults. The tracheobronchial airways did not change in size at the same rate as the body grew. Airway volume measured from the casts increased only 32-fold between one-day-old animals (0.04 +/-0.01 ml) and adults (1.27 ml +/-0.15 ml). As a percentage of body weight, this ratio was greatest in the early growth phases (7 days to 21 days postnatal ages) -0.60 and was least in the adults (0.42). By directly measuring casts, the core has evaluated changes in size in two proximal airways and a series of terminal bronchioles. The pattern and rate at which proximal airways increase in length, by longitudinal growth, and in diameter, by centrifugal growth, are very different. Additionally, it appears that at the time point during rat lung development when alveolarization has ceased and alveoli become larger, rather than new ones being added, is also the time point when the growth of the conducting airways is established. While this information is useful, it also represents a tremendous amount of effort by one individual to do direct measurements off each cast. This does not provide a global picture of how all lobar bronchi or bronchi of all sizes grow, but it does emphasize they have unique growth patterns. To test whether this was true in other airways, the same measurements were done on the first minor daughter.

Two in vivo studies have been run with epoxide hydrolase inhibitors indicating their ability to reduce inflammation. In one case, spontaneously hypertensive rats develop severe pulmonary inflammation as monitored by several indicators when exposed to tobacco smoke. This inflammatory effect is dramatically reduced when the animals are pretreated with an inhibitor of epoxide hydrolase. This effect is even greater when the inhibitor is given with the natural epoxy eicosanoid. The hypothesis is that the epoxide hydrolase inhibitor blocks the degradation of epoxyeicosanoids which are natural anti-inflammatory chemical mediators. A similar result was obtained in a LPS induced sepsis model in both rats and in mice. Thus epoxide hydrolase inhibitors may be useful therapeutic agents. The SHR work was a collaboration between the laboratories of Kent Pinkerton and Bruce Hammock with analytical chemistry support from the analytical core. This work is being extended to the mechanism of action of nitronaphthalene on the lung in collaboration with Alan Buckpitt. Several herbicides are able to induce epoxide hydrolase, and another class of herbicides are inhibitors of the enzyme.

Other collaborations included: a joint grant application submitted by Drs. Witschi, Last and Gohil to NIH/NCI entitled: "Tobacco Smoke-Induced Mouse Lung Tumors: Gene Expression"; a Research and Training Program in Environmental Toxicology in Chile and Uruguay taught by Drs. Last, Pinkerton, Schenker and Hammock; a study of "Health Effects of Sulfur Dusts Used as Fungicides" by Drs. Lee, Schenker and Lastt (abstract to be presented).

PUBLICATIONS - - Bold type indicates Center investigators and affiliate scientists.

Balasubramaniam V, Tang JR, Maxey A, Plopper CG, Abman SH. Mild hypoxia impairs alveolarization in the endothelial nitric oxide synthase-deficient mouse. Am J Physiol Lung Cell Mol Physiol. 2003 Jun; 284(6): L964-71. Epub 2003 Feb 14.

Bradshaw, T.K., K.M. Kennedy, P.R. Davis, L.L. Lloyd, N. Gwebu, and J.A. Last. 2003. Science first: contributions of a university-industry toxic substances research and teaching program to economic development. The Journal of Higher Education 74:292-320.

Chen CY, Bonham AC, Plopper CG, Joad JP. Neuroplasticity in nucleus tractus solitarius neurons after episodic ozone exposure in infant primates. J Appl Physiol. 2003 Feb; 94(2): 819-27. Epub 2002 Oct 25.

Denton, D.L., C.E. Wheelock, S.A. Murray, L.A. Deanovic, B.D. Hammock and D.E. Hinton. 2003. Joint acute toxicity of esfenvalerate and diazinon to larval fathead minnows (Pimephales promelas). Environ. Toxicol. Chem. 22:336-341.

Evans MJ, Fanucchi MV, Baker GL, Van Winkle LS [CEHS fellow], Pantle LM, Nishio SJ, Schelegle ES, Gershwin LJ, Miller LA, Hyde DM, Sannes PL, Plopper CG. Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. Am J Physiol Lung Cell Mol Physiol. 2003 Oct; 285(4): L931-9. Epub 2003 Jun 27.

Feng, J., G. Shan, B.D. Hammock and I.M. Kennedy. 2003. Fluorescence quenching competitive immunoassay in micro droplets. Biosens. Bioelectron. 18:1055-1063.

Feng, J., G. Shan, A. Maquieira, M.E. Koivunen, B. Guo, B.D. Hammock and I.M. Kennedy. 2003. Functionalized europium oxide nanoparticles used as a fluorescent label in an immunoassay for atrazine. Anal. Chem. 75:5282-5286.

Gohil, K., C. E. Cross, and J.A. Last. 2003. Ozone induced disruptions of lung transcriptosomes. Biochemical and Biophysical Research Communications 305:719-728.

Huang T, You Y, Spoor MS, Richer EJ, Kudva VV, Paige RC, Seiler MP, Liebler JM, Zabner J, Plopper CG, Brody SL. Foxj1 is required for apical localization of ezrin in airway epithelial cells. J Cell Sci. 2003 Dec 15; 116(Pt 24): 4935-45.

Joad JP, Munch PA, Bric JM, Evans SJ, Pinkerton KE, Chen CY, Bonham ACPassive smoke effects on cough and airways in young guinea pigs: role of brainstem substance P. Am J Respir Crit Care Med. 2003 Nov 25 [Epub ahead of print]

Kamita, S.G., A.C. Hinton, C.E. Wheelock, M.D. Wogulis, D.K. Wilson, N.M. Wolf, J.E. Stok, B. Hock and B.D. Hammock. 2003. Juvenile hormone (JH) esterase: Why are you so JH specific? Insect Biochem. Molec. Biol. 33:1261-1273.

Kenyon, N.J., K. Gohil, and J.A. Last. 2003. Susceptibility to ovalbumin-induced airway inflammation and fibrosis in iNOS-deficient mice: mechanisms and consequences. Toxicology and Applied Pharmacology 191:2-11.

Kenyon, N.J., R.W. Ward, and J.A. Last. 2003. Airway fibrosis in a mouse model of airway inflammation, Toxicology and Applied Pharmacology 186:90-100.

Kenyon, N.J., R.W. Ward, G. McGrew, and J.A. Last. 2003. TGF-b1 causes airway fibrosis and increased collagen I and III mRNA in mice. Thorax, 58:772-777.

Kips JC, Anderson GP, Fredberg JJ, Herz U, Inman MD, Jordana M, Kemeny DM, Lotvall J, Pauwels RA, Plopper CG, Schmidt D, Sterk PJ, Van Oosterhout AJ, Vargaftig BB, Chung KF. Murine models of asthma. Eur Respir J. 2003 Aug; 22(2): 374-82. Review.

Larson SD, Schelegle ES, Hyde DM, Plopper CG. The three-dimensional distribution of nerves along the entire intrapulmonary airway tree of the adult rat and the anatomical relationship between nerves and neuroepithelial bodies. Am J Respir Cell Mol Biol. 2003 May; 28(5): 592-9.

Lee, J.K., K.C. Ahn, D.W. Stoutamire, S.J. Gee and B.D. Hammock. 2003. Development of an enzyme-linked immunosorbent assay for the detection of the organophosphorus insecticide acephate. J. Agric. Food Chem. 51:3695-3703.

Lee, H-J., T. Watanabe, S.J. Gee and B.D. Hammock. 2003. Application of an enzyme-linked immunosorbent assay (ELISA) to determine deltamethrin residues in milk. B. Environ. Contam. Tox. 71:14-20.

Maciag A, Bialkowska A, Espiritu I, Powell D, Alvord WG, Kasprzak KS, Anderson LM, Witschi HR. Gestation stage-specific oxidative deoxyribonucleic acid damage from sidestream smoke in pregnant rats and their fetuses. Arch Environ Health. 2003 Apr; 58(4): 238-44.

McElroy, N.R., P.C. Jurs, C. Morisseau and B.D. Hammock. 2003. QSAR and classification of murine and human soluble epoxide hydroalse inhibition by urea-like compounds. J. Med. Chem. 46:1066-1080.

Miller LA, Hyde DM, Gershwin LJ, Schelegle ES, Fanucchi MV, Evans MJ, Gerriets JE, Putney LF, Stovall MY, Tyler NK, Usachenko JL, Plopper CG. The effect of house dust mite aeroallergen and air pollutant exposures during infancy. Chest. 2003 Mar; 123(3 Suppl): 434S. Review. No abstract available.

Miller LA, Plopper CG, Hyde DM, Gerriets JE, Pieczarka EM, Tyler NK, Evans MJ, Gershwin LJ, Schelegle ES, Van Winkle LS [CEHS fellow]. Immune and airway effects of house dust mite aeroallergen exposures during postnatal development of the infant rhesus monkey. Clin Exp Allergy. 2003 Dec; 33(12): 1686-94.

Newman, J.W. [CEHS fellow], C. Morisseau, T.R. Harris and B.D. Hammock. 2003. The soluble epoxide hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate phosphatase activity. PNAS 100:1558-1563.

Nichkova, M., J. Feng, F. Sanchez-Baeza, M.P. Marco, B.D. Hammock, I.M. Kennedy. 2003. Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets. Anal. Chem. 75: 83-90

Park, W.C., Y.A. Cho, Y.J. Kim, B.D. Hammock, Y.T. Lee and H.S. Lee. 2002. Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos. Bull. Korean Chem. Soc. 23:1399-1403.

Phimister AJ, Day KC, Gunderson AD, Wong VJ, Lawson GW, Fanucchi MV, Van Winkle LS [CEHS fellow], Kendall LV, Plopper CG. Detection of viral infection in the respiratory tract of virus antibody free mice: advantages of high-resolution imaging for respiratory toxicology. Toxicol Appl Pharmacol. 2003 Aug 1; 190(3): 286-93.

Plopper CG, Nishio SJ, Schelegle ES. Tethering tracheobronchial airways within the lungs. Am J Respir Crit Care Med. 2003 Jan 1; 167(1): 2-3.

Schelegle ES, Miller LA, Gershwin LJ, Fanucchi MV, Van Winkle LS [CEHS fellow], Gerriets JE, Walby WF, Mitchell V, Tarkington BK, Wong VJ, Baker GL, Pantle LM, Joad JP, Pinkerton KE, Wu R, Evans MJ, Hyde DM, Plopper CG. Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys. Toxicol Appl Pharmacol. 2003 Aug 15; 191(1): 74-85.

Schelegle ES, Walby WF, Alfaro MF, Wong VJ, Putney L, Stovall MY, Sterner-Kock A, Hyde DM, Plopper CG. Repeated episodes of ozone inhalation attenuates airway injury/repair and release of substance P, but not adaptation. Toxicol Appl Pharmacol. 2003 Feb 1; 186(3): 127-42.

Seymour BW, Schelegle ES, Pinkerton KE, Friebertshauser KE, Peake JL, Kurupd VP, Coffman RL, Gershwin LJ Second-hand smoke increases bronchial hyperreactivity and eosinophilia in a murine model of allergic aspergillosis. Clin Dev Immunol. 2003 Mar; 10(1): 35-42.

Shan, G., C. Lipton, S.J. Gee and B.D. Hammock. 2002. Immunoassay, biosensors and other nonchromatographic methods. In: Handbook of Residue Analytical Methods for Agrochemicals (Lee, P.W., ed.), pp. 623-679, John Wiley & Sons, Ltd. Chichester.

Shultz MA, Zhang L, Gu YZ, Baker GL, Fannuchi MV, Padua AM, Gurske WA, Morin D, Penn SG, Jovanovich SB, Plopper CG, Buckpitt AR. Gene Expression Analysis in Response to Lung Toxicants: I. Sequencing and Microarray Development. Am J Respir Cell Mol Biol. 2003 Aug 28 [Epub ahead of print]

Smiley-Jewell SM [CEHS fellow], Plopper CG. Proliferation during early phases of bronchiolar repair in neonatal rabbits following lung injury by 4-ipomeanol. Toxicol Appl Pharmacol. 2003 Oct 1; 192(1): 69-77.

Smith KR, Kim S, Recendez JJ, Teague SV, Menache MG, Grubbs DE, Sioutas C, Pinkerton KE. Airborne particles of the california central valley alter the lungs of healthy adult rats. Environ Health Perspect. 2003 Jun; 111(7): 902-8; discussion A408-9.

Székács, András, Hong T.M. Le, F. Szurdoki, B.D. Hammock. 2003. Optimization and validation of an enzyme immunoassay for the insect growth regulator fenoxycarb. Anal. Chem. ACTA 487:15-29.

Taniai K., A.B. Inceoglu, K. Yukuhiro and B.D. Hammock. 2003. Characterization and cDNA cloning of a clofibrate-inducible microsomal epoxide hydrolase in Drosophila melanogaster. Eur. J. Biochem. 270:4696-4705.

Watanabe, T., C. Morisseau, J.W. Newman [CEHS fellow] and B.D. Hammock. 2003. In vitro metabolism of the mammalian soluble epoxide hydrolase inhibitor, 1-cyclohexyl-3-dodecyl-urea. Drug Metab. Dispos. 31:846-853.

West JA, Van Winkle LS [CEHS fellow], Morin D, Fleschner CA, Forman HJ, Plopper CG. Repeated inhalation exposures to the bioactivated cytotoxicant naphthalene (NA) produce airway-specific Clara cell tolerance in mice. Toxicol Sci. 2003 Sep; 75(1): 161-8. Epub 2003 Jun 12.

Wheelock, C.E., M.E. Colvin, I. Uemura, M.M. Olmstead, J.R. Sanborn, Y. Nakagawa, A.D. Jones and B.D. Hammock. 2002. Use of ab initio calculations to predict the biological potency of carboxylesterase inhibitors. J. Med. Chem. 45:5576-5593.

Wheelock, C.E., Å.M. Karlsson, R. Zhang, J.E. Stok, C. Morisseau, S.E. Le Valley, C.E. Green and B.D. Hammock. 2003. Evaluation of cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes. Anal. Biochem. 315:208-222.

Williams KJ, Cruikshank MK, Plopper CG. Pulmonary heat shock protein expression after exposure to a metabolically activated Clara cell toxicant: relationship to protein adduct formation. Toxicol Appl Pharmacol. 2003 Oct 15; 192(2): 107-18.

Witschi H. Chemoprevention of lung cancer. Methods Mol Med. 2003; 75: 739-54. Review.

Witschi H. Induction of lung cancer by passive smoking in an animal model system. Methods Mol Med. 2003; 74: 441-55.

Witschi, H. R. and J.A. Last. 2003. Toxic responses of the respiratory system, p.220-232. In C. D. Klassen and J.B Watkins, (Eds). Casarett and Doull's Essentials of Toxicology. McGraw-Hill Medical Publishing Division, New York.

Zhang, R., K-D. Kang, G. Shan and B.D. Hammock. 2003. Design, synthesis and evaluation of novel P450 fluorescent probes bearing cyanoether. Tetrahedron Letts. 44:4331-4334.

Zhou YM, Zhong CY, Kennedy IM, Pinkerton KE. Pulmonary responses of acute exposure to ultrafine iron particles in healthy adult rats. Environ Toxicol. 2003 Aug; 18(4): 227-35.

Zhou YM, Zhong CY, Kennedy IM, Leppert VJ, Pinkerton KE. Oxidative stress and NFkappaB activation in the lungs of rats: a synergistic interaction between soot and iron particles. Toxicol Appl Pharmacol. 2003 Jul 15; 190(2): 157-69.

Toxicogenomics

Overview
The Core's main goals are: to promote collaborative research activities involving toxicogenomics, such as the identification of various inducible and novel genes, and susceptible genes and signaling pathways involved in toxicant-induced cell injury and repair; to improve understanding of toxic effects of xenobiotics on target cells by applying advanced technologies in genomics, such as the genomic base of cell type specificity and the regulation of gene expression profile associated with xenobiotic metabolism; to advance research activity in determining the genomic base of the neurological and environmental factors that cause neurological diseases and alteration; and to focus efforts on the topics relevant to the Center's theme and ultimately relate the findings to the solution of environmental health problems.

Core Director and Members
Dr. Reen Wu, Ph.D., Professor, Internal Medicine
Dr. Alan R. Buckpitt, Ph.D., Professor, Molecular Biosciences
Dr. Gino A. Cortopassi, Ph.D., Professor, Molecular Biosciences
Dr. Michael S. Denison, Ph.D., Professor, Environmental Toxicology
Dr. Jeffrey P. Gregg, M.D., Assistant Professor, Molecular Pathology
Dr. Bruce D. Hammock, Ph.D., Professor, Entomology
Dr. Hsing-Jien Kung, Ph.D., Professor, UC Davis Medical Center Cancer Center
Dr. Kent C. Lloyd, Ph.D., D.V.M, Professor, Anatomy, Physiology and Cell Biology
Dr. Fumio Matsumura, Ph.D., Professor, Environmental Toxicology and Entomology
Dr. Isaac N. Pessah, Ph.D., Professor, Molecular Biosciences
Dr. Robert H. Rice, Ph.D., Professor, Environmental Toxicology

Key Words
Carcinogenesis, Gene Expression, Genomics, Microarrays, Nuclear Transcription Factors, Proteomics, Transduction

Progress
Investigator collaborations resulted in several publications by the Core including:

A study of TCDD and vitamin A by Drs. Rice and Wu (Krig et al. 2002);

A joint effort with the Cellular and Molecular Imaging Facility Core on how inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys (Schelege et al. 2003);

A training grant application with a priority score of 153 prepared by Drs. Wu, Plopper, Buckpitt, Last, Cross, Pinkerton and Hyde: NIH HL07013, Wu (PI), 7/1/04-6/30/09 , $398,000/year (direct cost), Title: Training in comparative lung biology. As a training grant in Pulmonary Medicine, specific emphasis will be placed on lung comparative biology using cellular and molecular investigative techniques.

A continuation grant by Dr. Plopper for Pulmonary Effects of Environmental Oxidant Pollutants.

Use of the microarray facility of Dr. Jeff Gregg in the application of high density DNA microarray to study smoke- and hydrogen peroxide-induced injury and repair in human bronchial epithelial cells (Yoneda et al. 2003).

Use of Dr. Alan Buckpitt's Analytical Biochemistry Core Lab for work on glutathione levels.

Collaboration by Drs. Buckpitt and Plopper on the following three projects which resulted in several publications on gene expression and toxicity (Shultz et al. 2004 in press; Baker et al. 2004; Wheelock et al. 2004 in press):

Project I. Recent developments in genomics, proteomics and metabolomics allow global assessment of alterations in tissues in response to toxicants. Collectively, these methods hold substantial promise for understanding the reactions of cells to chemical stress. However, with many agents, injury to heterogeneous tissues is highly cell selective necessitating the use of methods capable of discriminating between changes occurring in target vs. non-target cell populations. Studies with the lung are an example. Airway epithelial cells are exquisitely sensitive to oxidants entering via inhalation and to chemicals which are metabolized by P450 monooxygenases. A novel method is presented here for isolation of rodent airway epithelial cell proteins to interrogate changes in the proteome in response to insult. This method reproducibly solubilizes airway epithelial proteins, leaving the underlying basement membrane and smooth muscle intact. Immunoblots show that this method yields epithelial cell-specific proteins in 4.9 to 6.4-fold higher concentrations than microdissected airways, and reduces the yield of non-epithelial cell-specific proteins 12.7 to 17.6-fold in comparison to samples obtained through microdissection of the airways.

Project II. The mechanisms of toxicant-mediated lung injury and repair are influenced by the considerable spatial heterogeneity that exists within the conducting airways of the lungs. As a result of this heterogeneity, significant differences and similarities in gene expression are observed throughout the lungs. RNA-based technologies such as real-time reverse transcription polymerase chain reaction (real time RT-PCR) and cDNA microarray analysis of gene expression provide valuable clues to understanding the mechanisms of toxicant-induced injury. Isolating RNA from lung subcompartments has previously involved considerable time and labor intensive processes that limit the number of animals that could be processed in a day. The aim of this study was to determine if intact, high quality RNA could be preserved in situ, over a period of time, to delay the need to immediately perform site-specific lung subcompartment microdissections and RNA isolations. Rat lungs were inflated with and stored in RNA preservation solution and stored at 4 degrees C for 7 days to 15 weeks. RNA was isolated from lung subcompartments isolated by microdissection. After 7 days to 15 weeks of storage, the RNA was intact, of high quality and could be used for both real time RT-PCR and cDNA microarray analysis. In summary, this simplified technique of in situ RNA preservation and site-specific lung subcompartment microdissection allows the isolation of intact, high quality RNA that may be used with molecular RNA base technologies that will significantly accelerate understanding of pulmonary inquiry and repair mechanisms.

Project III. A key challenge in measuring gene expression changes in the lung in response to site-selective toxicants is differentiating between target and nontarget areas. The toxicity for the cytotoxicant 1-nitronaphthalene is highly localized in the airway epithelium. Target cells comprise but a fraction of the total lung cell mass; measurements from whole lung homogenates are not likely to reflect what occurs at the target site. Additionally, the use of generic microarrays to measure expression in airway epithelium may not provide a good representation of transcripts present at the site of toxic action. cDNA libraries from airway and alveolar subcompartments of rat lung were sequenced for the development of a custom microarray representative of these lung regions. The core identified 7,460 unique rat lung sequences. Nearly 30% of the sequences on this array are not present on the Affymetrix Rat GeneChip 230. A 20,000-element microarray was developed which accurately delineates differences in gene expression between subcompartments. This is the first in a series of papers employing this microarray for detecting gene expression changes during acute injury produced by 1-nitronaphthalene and subsequent repair.

In addition to the above projecs, the Core collaborated on a study of decreased expression of genes involved in sulfur amino acid metabolism in frataxin-deficient cells (Tan, et al., 2003). Inherited deficiency of the mitochondrial protein frataxin causes neural and cardiac cell degeneration, and Friedreich's ataxia. Five hypotheses for frataxin's mitochondrial function have been generated, largely from work in non-human cells: iron transporter, iron-sulfur cluster assembler, iron-storage protein, antioxidant and stimulator of oxidative phosphorylation. The core analyzed gene expression in three human cell types using microarrays, and identified just 48 transcripts whose expression was significantly frataxin-dependent in at least two cell types. Significant decreases in seven transcripts occurred in the sulfur amino acid (SAA) biosynthetic pathway and the iron-sulfur cluster (ISC) biosynthetic pathway to which it is connected. By contrast, the core did not observe a single frataxin-dependent transcript that fits with the other four current hypotheses. Quantitative reverse-transcriptase PCR analysis of ISC-S and rhodanese transcripts confirmed that the expression of these genes involved in ISC metabolism was lower in mutants. Amino acid analysis confirmed the defect in SAA metabolism: homocystine, cysteine, cystathionine and serine were significantly decreased in frataxin-deficient cell extracts and mitochondria. An ISC defect was further confirmed by observing decreases in succinate dehydrogenase and aconitase activities, whose activities require ISCs. The ISC-U scaffold protein was specifically decreased in frataxin-deficient cells, suggesting a role for frataxin in its expression or maintenance, and sodium sulfide partially rescued the oxidant-sensitivity of the FRDA cells. Also, multiple transcripts involved in the Fas/TNF/INF apoptosis pathway were up-regulated in frataxin-deficient cells, consistent with a multi-step mechanism of Friedreich's ataxia pathophysiology, and suggesting alternative possibilities for therapeutic intervention.

Dr. Cortopassi also worked with Dr. Wong to develop a high-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader (Wong and Cortopassi,Biochem Biophys Res Commun, 2002). Mutations in mitochondrial genes cause mitochondrial genetic disease, which is often associated with deficiency of the mitochondrial membrane potential (MMP). The core presents a high-throughput method for measuring MMP in intact neural cells using TMRM, a well-known potentiometric dye, in a 48-well plate format. Addition of known MMP depolarizing agents, FCCP or DNP, resulted in a time- and concentration-dependent decrease in fluorescence, which was saturable, whereas the addition of drugs that affect non-mitochondrial properties did not. A cell line deficient in mtDNA had decreased fluorescence, which was not further depleted by a depolarizing agent. The high-throughput results are similar to those produced by more time-consuming and low-throughput flow cytometry or microscopy methods. This plate-based system could facilitate the identification of cell-permeant small molecules (i.e., drugs) that modify MMP, which could be used to enhance mitochondrial function, and also for screening small populations of neural cells for mutations in nuclear or mtDNA genes that decrease MMP.

New research directions for the core include "bioinformatics" for pathway finding and genome-wide motif searching. In addition, the core is also engaged in both viral and bacterial infection studies. These may not be related to environment. However, the endotoxin (bacterial infection) is relevant to the environmental concern.

PUBLICATIONS - - Bold type indicates Center investigators and affiliate scientists.

Abate-Shen, Cory, Whitney A. Banach-Petrosky, Xiaohui Sun, Kyriakos D. Economides, Nishita Desai, Jeffrey P. Gregg, Alexander D. Borowsky, Robert D. Cardiff, and Michael M. Shen. (2003). Nkx3.1; Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases. Cancer Res. 63: 3886-3890.

Arrieta, D.E. [CEHS fellow], Ontiveros, C.C., Li, W.-W., Garcia, J.H., Denison, M.S., McDonald, J.D., Burchiel, S.W. and Washburn, B.S. (2003) Aryl hydrocarbon receptor-mediated activity of particulate organic matter from the Paso del Norte airshed along the U.S.-Mexico Border, Environ. Health Perspect. 111, 1299-305.

Awasthi PR, French CF, Bedigian R, Sztein J, Sharp J, Lloyd KCK. Frozen sperm as an alternative to shipping of live mice. Contemporary Topics, 2003;42:9-12.

Baston, D.S., Nagy, S.R., Nantz, M., Kurth, M., Springsteel, M. and Denison, M.S. (2003) Identification of novel agonists and antagonists of the Ah receptor signal transduction pathway, Organohalogen compounds 65, 142-145.

Chen KY, Huang LM, Kung HJ, Ann DK, Shih HM. The role of tyrosine kinase Etk/Bmx in EGF-induced apoptosis of MDA-MB-468 breast cancer cells. Oncogene. 2003 Dec 15 [Epub ahead of print]

Chen, Y, Thai, P., Zhao, Y.H., Ho, Y.S., DeSouza, M.M., and Wu, R. Stimulation of airway mucin gene expression by IL-17 through IL-6 paracrine/autocrine loop. J. Biol. Chem. 278: 17036-17043, 2003.

Chen, Y., Zhao, Y.H., Kalaslavadi, T.J., Hamati, E., Nehrke, K., Le, A.D., Ann, D.K., and Wu, R. Genome-wide search of novel gel-forming mucin genes and identification of MUC19/Muc19 as a new glandular tissue-specific mucin gene. Am. J. Respir. Cell Mol. Biol., 30: 155-165, 2004.

Denison, M.S., Han, D.-H., Heath-Pagliuso, S., Scovill, J., Meijer, L., Skaltounis, L., Gillam, E.M.J. and Nagy, S.R. (2003) Activation of the Ah receptor and Ah receptor signal transduction pathway by tryptophan-derived agonists, Organohalogen compounds 65, 90-93.

Denison, M.S. and Nagy, S.R. (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals, Ann. Rev. Pharmacol. Toxicol. 43, 309-334.

Di, Y.P., Zhao, Y.H., Harper, R., and Wu, R. Molecular cloning, and characterization of a human novel gene that is retinoic acid-inducible and encodes a secretory protein specific in upper respiratory tracts, SPURT. J. Biol. Chem. 278:1165-1173, 2003.

Gellar, Sue C., Jeff P. Gregg, Paul Hagerman, David M. Rocke. (2003). Transformation and Normalization of Oligonucleotide Microarray Data. Bioinformatics. 19(14): 1817-23.

Gordon, J.D., Chu, A.C., Chu, M.D., Taylor, C.L., Denison, M.S. and Clark, G.C. (2003) Validation of the Lumi-Cell ER recombinant bioassay for rapid evaluation of chemicals for potential estrogenic activity, Organohalogen Compounds 65, 78-81.

Gwack Y, Nakamura H, Lee SH, Souvlis J, Yustein JT, Gygi S, Kung HJ, Jung JU. Poly(ADP-ribose) polymerase 1 and Ste20-like kinase hKFC act as transcriptional repressors for gamma-2 herpesvirus lytic replication. Mol Cell Biol. 2003 Nov; 23(22): 8282-94.

Han, D.-H., Zhao, B., Baston, D.S., Springsteel, M., Kurth, M., Nantz, M.H. and Denison, M.S., Identification and characterization of novel flavone agonists of the Ah receptor, Organohalogen compounds 2003 65, 126-129.

Izumiya Y, Lin SF, Ellison T, Chen LY, Izumiya C, Luciw P, Kung HJ. Kaposi's sarcoma-associated herpesvirus K-bZIP is a coregulator of K-Rta: physical association and promoter-dependent transcriptional repression. J Virol. 2003 Jan; 77(2): 1441-51.

Izumiya Y, Lin SF, Ellison TJ, Levy AM, Mayeur GL, Izumiya C, Kung HJ. Cell cycle regulation by Kaposi's sarcoma-associated herpesvirus K-bZIP: direct interaction with cyclin-CDK2 and induction of G1 growth arrest. J Virol. 2003 Sep; 77(17): 9652-61.

Jeuken, A., Keser, B.J.G., Khan, E., Brouwer, A., Koeman, Jan and Denison, M.S. (2003) Activation of the Ah Receptor by Extracts of Dietary Herbal Supplements, Vegetables and Fruits, J. Agr. Food Chem. 51, 5478-5487.

Kao, C.Y, Chen, Y., Zhao, Y.H., and Wu, R. ORFeome based search of airway epithelial cell-specific novel human b-defensin genes. Am. J. Respir. Cell Mol. Biol. 29: 71-80, 2003.

Kullman SW, Matsumura F, Hinton DE. Estrogen signaling in trout liver: Estrogen receptor and mitogenic mediated cellular responses. 2003 Environmental Sciences 10 (4): 223-237.

Landry, James P., X.D. Zhu, X.W. Guo, and J.P. Gregg. (2003). Oblique-Incidence Reflectivity Difference and Fluorescence Imaging of Oligonucleotide and IgG Protein Microarrays. Mat. Res. Soc. Symp. Proc. 773: N7.51-N7.56.

Lee LF, Liu JL, Cui XP, Kung HJ. Marek's disease virus latent protein MEQ: delineation of an epitope in the BR1 domain involved in nuclear localization. Virus Genes. 2003 Dec; 27(3): 211-8.

Levy AM, Izumiya Y, Brunovskis P, Xia L, Parcells MS, Reddy SM, Lee L, Chen HW, Kung HJ. Characterization of the chromosomal binding sites and dimerization partners of the viral oncoprotein Meq in Marek's disease virus-transformed T cells. J Virol. 2003 Dec; 77(23): 12841-51.

Liao W, Tang Y, Lin SF, Kung HJ, Giam CZ. K-bZIP of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) binds KSHV/HHV-8 Rta and represses Rta-mediated transactivation. J Virol. 2003 Mar; 77(6): 3809-15.

Long W, Wagner K-U, Lloyd KCK, Binart N, Shillingford JM, Hennighausen L, Jones FE. Impaired differentiation and lactational failure of erbB4-deficient mammary glands identify erbB4 as an obligate mediator of STAT5. Develop 2003;130:5257-5268.

Louie MC, Yang HQ, Ma AH, Xu W, Zou JX, Kung HJ, Chen HW. Androgen-induced recruitment of RNA polymerase II to a nuclear receptor-p160 coactivator complex. Proc Natl Acad Sci U S A. 2003 Mar 4; 100(5): 2226-30. Epub 2003 Feb 14.

Matsumura F. Is the Ah receptor a stress response receptor, mediating cellular defense reactions? J UOEH. 2003 25 Supplement (1): 153-159.

Matsumura F. On the significance of the role of cellular stress response reactions in the toxic actions of dioxin. Biochem Pharmacol. 2003 Aug 15; 66(4): 527-40. Review.

Monk, S.A. [CEHS fellow], Denison, M.S. and Rice, R.H. (2003) Reversible stepwise negative regulation of CYP1A1 in cultured rat epidermal cells, Arch. Biochem. Biophys. 419, 158-169.

Oslund, K.L., Miller, L.M., Usachenko, J.L., Tyler, N.K., Wu, R., and Hyde, D.M. Oxidant-injured airway epithelial cells upregulate thioredoxin but do not produce IL-8. Am. J. Respir. Cell Mol. Biol. In press, 2003(Sept 18).

Rea, Miguel A, Jeff P. Gregg, Qin Qin, Marjorie A. Phillips, Robert H. Rice. (2003). Microarray Analysis of Arsenic-Mediated Gene Expression Changes. Carcinogenesis. 24: 747-756.

Rice RH, Crumrine D, Hohl D, Munro CS, Elias PM (2003) Cross-linked envelopes in nail plate in lamellar ichthyosis. Brit J Dermatol 149:1050-1054.

Serikov, V.R., Choi, H., Chmiel, K.J., Wu, R., and Widdicombe, J.H. Role of ERK in the increase in airway epithelial permeability during leukocyte transmigration. Am. J. Respir. Cell Mol. Biol. In press, 2003(July 3).

Schelege, E.S., Miller, L.A., Gershwin, L.J., Fanucchi, M.V., Van Winkle, L.S. [CEHS fellow], Gerriets, J.E., Walby, W.F., Mitchell, V., Tarkington, BK., Wong, V.J., Baker, G.I., Pantle, L.M., Joad, J.P., Pinkerton, K.E., Wu, R., Evans, M.J., Hyde, D.M., and Plopper, C.G. Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys. Toxicol. Appl. Pharmacol. 191: 74-85, 2003.

Shaw, S.D., Brenner, D., Mahaffey, C.A., De Guise, S., Perkins, C.R., Clark, G.C., Denison, M.S. and Waring, G.T. (2003) Persistent organic pollutants (POPs) and immune function in US Atlantic coast Harbor Seals, Organohalogen compounds 62, 220-223.

Tan G, Napoli E, Taroni F, Cortopassi G. Decreased expression of genes involved in sulfur amino acid metabolism in frataxin-deficient cells. Hum Mol Genet. 2003 Jul 15; 12(14): 1699-711.

Vogel CF, Matsumura F. Interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with induced adipocyte differentiation in mouse embryonic fibroblasts (MEFs) involves tyrosine kinase c-Src. Biochem Pharmacol. 2003 Oct 1; 66(7): 1231-44.

Vogel CF, Sciullo E, Park S, Liedtke C, Trautwein C, Matsumura F. Dioxin increases C/EBPb transcription by activating cAMP/PKA. J Biol Chem. 2003 Dec 18 [Epub ahead of print]

Vogel CF, Zhao Y, Wong P, Young NF, Matsumura F. The use of c-src knockout mice for the identification of the main toxic signaling pathway of TCDD to Induce wasting syndrome. J Biochem Mol Toxicol. 2003; 17(6): 305-15.

Wen-Li M, Lloyd, KCK. Intracytoplasmic Sperm Injection (ICSI). IN: Pease S and Lois C, eds. Mammalian Transgenesis, New Approaches. Principles and Practice Series, Heidelberg: Springer-Verlag, (2003).

Wen-Li M, McGinnis L, Zhu L, Lawitts J, Biggers J, Lloyd KCK. Intracytoplasmic sperm injection (ICSI) enables rescue of valuable mutant mouse strains. Comp Med, 2003;53:265-269.

Yang, Qinghong, Alla Lishanski, Wendy Yang, Sandra Hatcher, Henrietta Seet and Jeffrey P. Gregg. (2003). Allele-Specific Holliday Junction Formation?A New Mechanism of Allelic Discrimination for SNP Scoring. Genome Res. 13: 1754-1764.

Yoneda, K., Chang, M. M.J., Chmiel, K., Chen, Y., and Wu, R. Application of high density DNA microarray to study smoke- and hydrogen peroxide-induced injury and repair in human bronchial epithelial cells. J. Am. Soc. Nephrology, 14: S284-S289, 2003.

Yustein JT, Xia L, Kahlenburg JM, Robinson D, Templeton D, Kung HJ. Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38. Oncogene. 2003 Sep 18; 22(40): 6129-41.

Zhao, B., Nagy, S.R., Baston, D.S., Han, D.-H., Nantz, M., Kurrth, M., Springsteel, M.H. and Denison, M.S., Screening and analysis of novel naphthoflavone ligands for the Ah receptor, Organohalogen compounds 65, 102-105.

Zou E, Matsumura F. Long-term exposure to beta-hexachlorocyclohexane (beta-HCH) promotes transformation and invasiveness of MCF-7 human breast cancer cells. Biochem Pharmacol. 2003 Sep 1; 66(5): 831-40.

Facility Cores

Analytical Biochemistry

Overview
This Core assists other investigators in the development and validation of new methods, provides training to students and postdocs in other laboratories and affords access to equipment and expertise for studies requiring isolation, identification and quantification of metabolites, covalent adducts, and proteins/peptides. It is anticipated that there will be more reliance on this Core in terms of downstream analysis to determine the functional significance of changes noted at the level of gene expression. Emphases in this Core complement capabilities in other service cores and focus on chromatographic purification and quantitative analysis of small molecules, peptides and proteins using a variety of techniques, the transgenic expression of proteins, the development and application of spectrophotometric/fluorimetric /radiometric assays of metabolic enzymes, structural characterization of both small organic metabolites and proteins/peptides using mass spectrometry and NMR spectroscopy and the development of high throughput screens using immunoassay methods.

Core Director and Members
Dr. Bruce D. Hammock, Ph.D., Professor, Department of Entomology
Dr. Alan R. Buckpitt, Ph.D., Professor, Department of Molecular Biosciences
Dr. Jerold A. Last, Ph.D., Professor, Department of Internal Medicine
Dr. Kent C. Lloyd, Ph.D., D.V.M, Professor, Department of Anatomy, Physiology and Cell Biology
Dr. P. Richard Vulliet, Ph.D., D.V.M, Professor, Department of Molecular Biosciences

Equipment and Facilities
The Core offers the following services: Mass spectrometry of proteins and peptides; Mass spectrometric identification of synthetic products and metabolites; Quantitative analysis of molecules based on a variety of chromatograph and mass spectral techniques; HPLC based quantitative analysis of small molecules; Analysis of metabolites in urine and blood; Immunochemical assays; Chemical and biological synthesis of metabolites; In vivo metabolism; Spectrophotometric enzyme assays; Baculovirus expression; 2-D Gel electrophoresis; Protein purification; Peptide design; Peptide purification; Protein expression; Protein characterization; Protein modeling; Immunoassay design.

With the funding for microarray equipment from NCRR, a high throughput spotter, Gene Traffic software and appropriate computers have been purchased. The spotter is capable of spotting 18,000 spits per slide onto 75 slides in less than 8 hrs. The Gene traffic software will be mounted on a server and will allow all users of the facility access to their data from remote locations. Dr. Matthew Bartosiewicz has been recruited from the UC San Diego microarray center to run the new microarray center with funds provided by the School of Veterinary Medicine. The microarray center has three overall goals: 1) to produce high quality microarrays on glass slides using either commercial oligomers (mouse, rat, human) or investigator supplied PCR products, 2) to provide optimized protocols to investigators for RNA isolation and labeling and for data analysis, and 3) to explore new uses of spotted arrays. In this third category, the Center will focus its efforts in investigating the best approaches for examining gene expression profiles in limited tissue samples including those obtained by laser capture. In addition, the possible modification of the equipment for macroarrays of antibodies for protein expression profiling will be examined. To increase the level of analytical support a new liquid chromatograph orthogonal time of flight mass spectrometer (LCT, Micromass) was purchased and installed in April, 2002. This instrument is a state of the art analytical tool what that allows us to perform high resolution LC/MS studies using ESI, AP(C)I ionization technique. The instrument is a dedicated tool for metabolite profiling (metabolome) studies using high speed (4.3 GHz) ADC (analog digital conversation) and high sensitivity dual MC signal detection technique. This equipment has been upgraded with LockSpray unit which allows keeping the ion mass detection error to be maintained below 5 ppm during long term LC/MS experiments, a critical utility for further statistical studies. The Core also added a new Micromass Triple Quadrapole MS, a terabyte data archival and back up system, a 96 well robotic system for sample preparation, and an FT infrared which works in both reflectance and transmission mode.

The purpose of the Analytical Biochemistry Core is to provide advice, training and access to equipment that individuals need for experiments involving metabolism, measurement of small molecules including metabolites in biologic matrices by HPLC, GC, GC/MS and LC/MS, identification of metabolites by mass spectrometry, and separation and analysis of proteins by 2D gel electrophoresis. Custom projects including chemical synthesis are available as well. Investigators are encouraged to send graduate students or postdoctoral fellows to the laboratory for training; following training they have full access to the laboratory for their work. The Core laboratory is available for consultation and is responsible for maintenance of all equipment. In some cases, such as with the analysis of organophosphate metabolites in the urine, these samples are run by laboratory staff. The Core continues to make progress in establishing quantitative methods for measurement of changes in proteins associated with exposure to toxicants and hopes to be able to complete these studies and to validate the methodology (sensitivity, reproducibility, linearity of response) over the coming year. Briefly, this approach involves the use of 2D gel electrophoresis using narrow range IPG strips with Alexa-633 maleimide labeled internal standards for quantitation. Proteins are stained with Ruthenium II tris (bathophenanthroline disulfonate) which provides very sensitive detection on gels. The Core is currently optimizing the protocols for the use of this stain. Once these methods are validated, the Core will provide detailed protocols and access to equipment for any investigators in the Center wishing to use this approach for their work.

In the Analytical Laboratory the instruments are operational; maintenance and service work is done routinely or when necessary to ensure the proper level of experimental data provided by the instruments. The downtime of the instruments was less than 2 weeks in average.



Usage and Benefits
This Core has been used by 25 scientists in this period. These scientists have spent over 600 working days in the laboratory in 2003. The number of analytical experiments performed was more than 8 thousand on the instruments (Ultima, LCT, and GC/MS). Utilization was greater than 2.5 researchers/day ( number of days the laboratory instruments were literally used divided by the theoretical working days decreased by holidays and maintenance days, around 210).

For the support of molecular modeling, basic quantum mechanical computation, data evaluation and data archival, 2 high-performance PC's and molecular modeling software Spartan 02 (upgrade), ChemOffice 7.0 (upgrade) linked to Gaussian98 were purchased and installed.

This Core interacts with all research cores, especially Respiratory Toxicology, Epidemiology and several Center Pilot Projects. The Core provided spectra, interpretation, and immunochemical support. The Core provided immunoassays to analyze paraquat in Dr. Schenker's large epidemiology study of this compound. The Core provided protein purification and expression support for Center investigators and will continue to do so. The Core also provides access to materials and equipment to run analyses on a routine basis. For assays needing high throughput analysis the Core has developed immunoassays. The Core has provided sperm biomarker evaluations for fourteen research projects so far.

Approximately a third of the effort in the Core is spent developing and validating new methodologies, 25% on student training and the remainder in routine sample analysis. Center investigators are encouraged to have students actively involved in the analysis of their samples; where this is not practical, technical help will be provided as part of the service from the Core.

The Core provides analytical chemistry education/training in the area of analytical techniques, particularly in mass spectrometry such as sample preparation, instrumentation, data generation and data evaluation. Training on mass spectrometers has started for 3 graduate students. In the analytical laboratory the instruments work properly, the maintenance and service work is done in time or when it is necessary to ensure the proper level of experimental data provided by the instruments.

Advanced courses in Mechanisms of Toxic Action (ETX 214 Hammock/Denison) and Toxicant and Drug Metabolism (VMB 253) provide didactic background for students and researchers interested in metabolism and analytical applications in toxicology. Core laboratories provide an environment in which graduate students from other investigators' labs receive training in methods development and application of analytical techniques to health-related problems. Training has focused primarily on graduate students in the Pharmacology/Toxicology and Agricultural and Environmental Chemistry graduate groups. In numerous cases, the student projects have resulted in peer-reviewed publications. Finally, the core laboratories have actively recruited undergraduate and high school students from underrepresented groups to provide these individuals with an exposure to laboratory research.

Following is a list of projects conducted in the past year (2003). Most are actively ongoing. Assessment of urinary alkyl phosphates--a collaboration with Marc Schenker on the extraction, derivitization and all analysis of 6 alkylphosphate metabolites in urine. Measurement of glutathione in dissected airways in neonatal animals. Measurement of retinol metabolites with Reen Wu for access to HPLC/UV, all maintenance, and advice on separations. 2D gel electrophoresis separation of proteins with Reen Wu--instruction and access to all equipment. 2D gel electrophoresis for Isaac Pessah which includes instruction and access to all equipment. Isolation of 4-ipomeanol glucuronides from rabbit urine for Charles Plopper in which the Core performed experiments and analysis by HPLC. Measurement of naphthalene metabolism in dissected airways of male compared to female mice for Laura Van Winkle and Charles Plopper which includes instruction, access to equipment, and maintenance of equipment. Measurement of cytochrome P450 monooxygenase activities in dissected airways of smoke-exposed neonatal monkeys for which the Core provided instruction and assistance with incubations and analysis. Support of paraquat epidemiology study for Mark Schenker. Estrogen metabolite studies for Barry Wilson. Development of immunoassays and GLC-MS assays for pyrethroids in support of Barry Wilson. Pharmacokinetic evaluation in support of Alan Buckpitt and Bruce Hammock. Identification of peptide toxins in support of Bruce Hammock and Isaac Pessah. Metabolite profiling for Kent Pinkerton and Alan Buckpitt. Synthetic support for Bruce Hammock. As a separate research effort the Core has addressed the problem of comprehensive metabolite profiling as an indicator of health and exposure to environmental chemicals.



Cell/Tissue Technology

Overview
The Cell/Tissue Technology Core provides Center investigators and their coworkers with training and updating in the techniques of cell, tissue and embryo culture including assistance with experimental design, special facilities and help in obtaining cell lines, organs and tissues. Investigators in the Core also develop special methods of studying cells and make the techniques available to Center Investigators. The Cell/Tissue Technology Core provides investigators and coworkers with (1) basic training in culturing and studying cells and tissues, (2) training in special techniques such as cell cloning, preparation of irradiated feeder layers, suspension and reaggregate cultures, (3) strategic advice in planning experiments and determining their feasibility, (4) cell culture facilities and specialized equipment such as Bioguard hoods needed for culture experiments with toxic agents, (5) facilities to process human and nonhuman primate tissues, (6) target epithelial cell lines, special primary cultures of hepatocytes, myoblasts, spinal cord and cranial neurons, (7) backup storage of valuable lines derived during this work, (8) development of new cell, tissue and embryo culture techniques and (9) direction to appropriate local experts for specialized consultations. In addition, the Cell/Tissue Technology Core serves as a seed bed for developing new ideas, matching collaborators and facilitating communication among its users.

Core Director and Members
Dr. Robert H. Rice, Ph.D., Professor, Department of Environmental Toxicology
Dr. Marion G. Miller, Ph.D., Professor, Department of Environmental Toxicology
Dr. Isaac N. Pessah, Ph.D., Professor, Department of Molecular Biosciences
Dr. Barry W. Wilson, Ph.D., Professor, Departments of Animal Science and Environmental Toxicology

Equipment and Facilities
To supplement existing equipment, the Core now has obtained three single chamber 3-gas culture incubators. This is permitting studies of cell behavior at altered oxygen tension.

Usage and Benefits
During the past year, the Cell Culture Core has serviced 22 research projects from 3 research cores and trained 11 graduate students. The Core has been used by 11 staff and benefitted 12 large competitive grants including 7 from the NIH, 1 from a foundation, and 1 from a California state source.

Collaborations have been fostered in several projects, including proteomic analysis of arsenic action (Rice/Lee), toxicity of the herbicide molinate on nonhuman primate seminiferous tubules (Miller/VandeVoort) and effects of low oxygen on toxicity in human keratinocytes (Pessah/Rice).

Training is an important aspect of the Core's activities. Dr. Wilson leads the graduate course PGG 200L, which teaches basic cell culture techniques to 15-20 students per year as they conduct research projects of their own design. This prepares them to conduct projects independently in their own labs, often leading to continuing interaction with Core laboratories.

FUTURE DIRECTIONS

1. Effects of altered oxygen level. Studies at low oxygen tension by Dr. Pessah's lab have suggested this approach could be generally useful to other Core members and Center investigators. Since culture incubator space for this purpose has been largely unavailable outside the Pessah lab, the Core has obtained a single chamber incubator for each of the three other Core labs to permit wider CEHS use. This is anticipated to benefit studies of responses of rodent and primate testicular cells to reproductive toxicants, human epidermal cells to arsenic and other radical generators, and neuromuscular cells to toxicants. Use of this equipment will be enhanced by the polarographic electrode system of Dr. Wilson for oxygen measurements. Studies of hyperbaric oxygen are also anticipated, which will enhance study of Ah receptor action by Dr. Denison.

2. Methods development

a. Clonal analysis of myogenesis and actions of chemicals. Cloned muscle cells will be tested for their abilities to proliferate and to differentiate in the presence of agricultural and other chemicals. The purposes are to dissect the fusion process and to provide a rapid screen for developmental blockers. This approach may be suitable for heart cells, chondrocytes and other cells capable of exhibiting tissue specific properties from primary clones.

b. Stable transfections for study of gene expression. Efforts are underway to transfect keratinocytes stably using a commercially available retroviral vector. After its optimization, this method promises to be of use in other cell types such as those of the airways. One application envisioned is to examine toxicant effects on promoter response element activity (not possible in many cases in transiently transfected keratinocytes). A second is to examine the long term effect of transfected genes on recipient cells and their toxic responses. Finally, stably transfected siRNA will permit long term suppression of specific genes that can modify cellular toxic response.



Cellular and Molecular Imaging

Overview
The main objectives of the Cellular and Molecular Imaging Facility Core are: (1) to provide service in imaging to scientists affiliated with the Center; (2) to act as a resource by providing information on the latest approaches and techniques; and (3) to offer training to graduate students, postdocs, and junior scientists in using the imaging instruments. The Cellular and Molecular Imaging Facility Core provides equipment, training, services and consulting expertise related to the analysis of normal and abnormal morphology at the whole animal, organ, cell, subcellular, and molecular levels. State-of-the-art techniques available to all CEHS investigators are: cellular imaging with confocal laser scanning microscopy and quantitative ion analysis, histopathology, immunohistochemistry and light, epifluorescence, transmission and scanning electron microscopy, and isolation of lung cells and tissues. The Core also actively assists investigators in developing new methodologies for imaging and cellular quantitation. At times, when services needed go beyond the resources of the Core, investigators facilitate collaboration with other campus groups and/or institutions for Center investigators. The Core's objective is to provide the highest quality tools for histologic and cellular analysis, diagnostic interpretation and consultative expertise for the evaluation of mechanisms of toxicant action.

Core Director and Members
Dr. Kent E. Pinkerton, Ph.D., Professor, Department of Anatomy, Physiology and Cell Biology
Dr. Charles G. Plopper, Ph.D., Professor, Department of Anatomy, Physiology and Cell Biology

Equipment and Facilities
This Core provides local access for tissue isolation and preparation, immunohistochemical analysis, in situ hybridization and imaging of fixed and viable cells. Equipment available includes a variety of instruments to facilitate cell and tissue processing, image analysis, tissue embedding and microtomy for paraffin and epoxy resin embedded cells and tissues.

Usage and Benefits
Technical personnel for the Cellular and Molecular Imaging Facility Core, Ms. Janice Peake and Alison Weir, provide technical support for specialized equipment as well as training to investigators, staff, graduate students and postdoctoral fellows. Ms. Peake has expertise in cell and tissue embedment, microtomy, confocal imaging, morphometric analysis and in situ hybridization. Ms. Weir has expertise in cell and tissue embedment, microtomy, confocal imaging, microdissection and immunohistochemistry. Both provide supervision, training and consultation, with Ms. Weir based at Haring Hall (HH) and Ms. Peake based at the Center for Health and the Environment (CHE) and the California National Primate Research Center (CNPRC). The administration of the Cellular and Molecular Imaging Facility Core is organized to provide access to the facilities at HH, CHE and CNPRC and to provide consultation to investigators to solve research problems. Dr. Pinkerton is the responsible investigator at CHE and CNPRC. Dr. Plopper has the responsibility for Haring Hall on the main campus (including Meyer and Everson Halls). Oversight for the use of the facilities and guidance provided by Ms. Peake and Weir are critical to the success and high level of productivity and shared use of the Cellular and Molecular Imaging Facility Core. Interdisciplinary and interschool-shared use of the Core provides the opportunity to introduce individuals to learning new and novel techniques. This core has trained 18 graduate students in the current project period. The list of users of the Cellular and Molecular Imaging Facility Core and the projects facilitated by the use of the Core are below:

Histochemical localization of soluble epoxide hydrolase (B. Hammock)

Glutathione turnover in target lung cells (A. Buckpitt)

Mechanisms of action of benzoylurea-type insecticides (F. Matsumura)

Calcium-induced lung injury due to inhalation of dusts from the California Central Valley (Kleeman)

Analysis of particle size and composition from the ambient air of Fresno, CA (Sioutas/Smith/Pinkerton)

Lymphocyte profiling of peripheral blood leucocytes in the developing Rhesus macaque infant monkey (K. Pinkerton)

In Situ hybridization as a sensitive new tool for studying pulmonary fibrosis (J. Last/Pinkerton)

Pulmonary fibrosis and mineral content of lung tissues from deceased California farmworkers (M. Schenker)

Neurodegenerative effect of PCBs (F. Matsumura)

Establishment of bronchiolar airways in culture for long-term metabolism and toxicity studies (C. Plopper)

Impact of enhance Clara cell toxicity in neonates on postnatal lung development (C. Plopper)

Mechanisms for alterations in cytochrome P450 monooxygenases by ozone inhalation (C. Plopper)

Lung toxicity to airborne particles (K. Pinkerton)

Expression of cytochrome P450 monooxygenases in perinatal lung and liver development (K. Pinkerton)

Laser capture of cells from regioselective sites of the respiratory tract (C. Plopper)

Metabolic mechanisms of naphthalene toxicity (A. Buckpitt)

Adducted proteins as biomarkers of environmental lung injury (A. Buckpitt)

TCDD effects in c-Src deficient mice (F. Matsumura)

Neuroendocrine cell expression (K. Pinkerton)

Nanoparticles and the environment (A. Navrotsky)

Asthma and the environment (C. Plopper)

Instrumentation for nano particle generation and analysis (A. Wexler)

Health effects of concentrated ambient particles from the Central Valley of California (K. Pinkerton)



Field Studies

Overview
This Core provides a link between agricultural workers and the agricultural environment and bench scientists located in the University. The Field Studies Core supports services and consulting related to agricultural epidemiologic investigations, field exposure assessments and biostatistics. Core personnel develop human populations working in agriculture or with agricultural exposures for epidemiologic investigations of adverse health effects. Human tissues and fluids are collected in epidemiologic investigations from which the exposure and toxicological effects of agro-chemicals are measured. In addition, samples of agricultural dusts and other toxins are collected in the field for analysis by investigators in the research cores. The exact methods vary with the outcome and exposure under investigation, the population being studied and other variables. For example, a cohort study of respiratory disease in farmers continues to provide a basis for focused studies of respiratory and other health outcomes in this population. Biologic specimens collected in the field were sent to the appropriate laboratory-based investigator for analysis. Another study of pulmonary function among paraquat applicators has provided biologic specimens for analysis of paraquat metabolites and their determinants in the population. Still another study is collecting blood samples from female residents who were potentially exposed to endocrine disruptors and comparison groups. The samples are being assayed for environmental estrogens. Exposure assessment investigations involve different approaches to characterizing toxic/chemical exposures in agriculture. These may use personal or area sampling, depending on the specific aims of the study and the amount of specimen required. Sampling may be done cross-sectionally or longitudinally over time as in Center studies of farmers to characterize the effects of season, atmospheric conditions and different work practices on exposure.

Core Director and Members
Dr. Marc B. Schenker , M.D., M.P.H., Professor, Department of Epidemiology and Preventive Medicine
Dr. Ellen B. Gold, Ph.D., Professor, Department of Epidemiology and Preventive Medicine

Equipment and Facilities
The Core maintains an industrial hygiene laboratory equipped for field collection of exposure data and samples. Equipment includes: Cahn microbalance, personal sampling pumps, battery chargers, flow calibrator, inhalable sampling heads (IOM), respirable sampling heads (cyclone), cascade impactors, freezers and miscellaneous material such as assorted filters, belts, tubing, and sprays.

Usage and Benefits
The Core provides support for population-based studies in agricultural and environmental settings that involve collaborations with other Center investigators. These studies include: a study of pregnancy outcome in female Hispanic women residing in the Stockton area (n=~1,100); respiratory function of farmers in the Central Valley (n=2,000); lung tissue from coroner cases of Hispanic males dying in Fresno County (n=110); pesticide exposure and injury among migrant farmworkers (n=1,000); pesticide exposure and neurologic function of farmworkers' children (n=300); paraquat exposure among applicators (n=340); a bi-national health study of Hispanic immigrants to California; and a community-based study of adult women potentially exposed to endocrine disruptors (final n~750). These studies also provide biologic samples for analysis by Center investigators in the Analytical Biochemistry Core, Respiratory Toxicology Core, Reproductive and Developmental Toxicology Core, and Molecular Neurotoxicology Core. Biologic samples collected and/or exposures measured include dust concentration and deposition patterns in human lungs, blood and urine samples for biomarkers of pesticide and herbicide exposure, blood for markers of immune response to agricultural exposures, and urine for biomarkers of reproductive effects (ovulation, conception, fetal loss). Urine samples from female farmworkers have been examined in the Reproductive and Developmental Toxicology Research Core and Analytical Biochemistry Facilities Core, and biomarkers of pesticide exposure and effect are studied in the Molecular Neurotoxicology Research Core. The other major element of this Core is the exposure assessment component. The major activity in this area has been characterization of exposure and specimen collection in the agricultural setting for analysis by other Center investigators. Efforts of the exposure assessment component to date have focused on airborne dust exposures in agriculture and urinary assays for organophosphates and paraquat, with future work planned for assessment of other chemical agents. Agricultural dust samples collected as part of the field studies core activities have been analyzed for composition and toxicity (in-vivo and in-vitro) by Drs. H.P. Witschi, J. Last and R. Southard (Land, Air and Water Resources Department), urinary metabolites of organophosphates have been assayed by Dr. Alan Buckpitt, and paraquat metabolites by Dr. Bruce Hammock.

The Core's innovative epidemiologic techniques and expertise and access to human agricultural populations provide a link between laboratory studies and human populations. Knowledge of levels of agrochemicals and their metabolites in humans, exposure measurements of agrochemicals suspected of causing ill-health, and studies of the toxicological effects of agrochemicals are combined to contribute significantly to understanding the impact of agricultural chemicals on human populations.

Support of Center Investigator Research Programs - The Field Studies Core has supported several large epidemiologic studies involving populations exposed to agricultural and environmental toxicants. Each of these studies has involved collaborations with other Center investigators. These populations include:
The California Farmer Cohort, a population-based study of respiratory and other health effects among California farmers (M. Schenker, PI).
A study of neurotoxic effects of pesticide exposure among a community-based sample of farmworker children (E. Gold, PI).
The study of health, acculturation, reproduction, and environment (SHARE), an investigation of adverse pregnancy outcomes among Latina women in Stockton, California (M. Schenker, PI).
A study of pulmonary pathology in lungs of deceased Hispanic males in Fresno, California (M. Schenker, PI).
Studies of agricultural dust exposure among farmers and farmworkers in California (K. Lee, PI).
A prospective cohort study of occupational injuries among migrant and seasonal farmworkers (S. McCurdy, PI)
A reproductive health study of migrant farmworker women (E. Gold, PI).
A bi-national study of immigration, acculturation, occupation and health among Hispanic immigrants to California (E. Gold, PI).
A community-based health study of adult women potentially exposed to endocrine disruptors (Dr. Gold, PI).

Support of Graduate Student Training - The Core has been involved in graduate student and post-doctoral fellow training in epidemiology and exposure assessment methods. Eight (8) graduate students have worked on projects supported by the Core.



Functional Genomics and Molecular Biology

Overview
The main objective of this service and facility core is to promote the use of modern techniques and approaches in functional genomics and molecular biology among Center investigators, their collaborators, affiliate scientists and pilot project recipients. The Core functions to provide access, training and assistance to students, postdoctoral fellows and staff members with regards to specific molecular techniques and instrumentation, to offer expert advice in experimental design and approaches using these technologies, to assist in data analysis and interpretation and method troubleshooting, to suggest appropriate reference materials and information relative to specific techniques and methodological approaches, to act as a general resource to assist in the integration of molecular biology into existing programs and projects, and to facilitate the education of Center researchers in molecular biological approaches and techniques. More than 60 graduate students and 27 postgrads/docs have received training at least one time in this core. In addition, Dr. Michael Denison offers two formal courses on a regular basis: ETX 214 on "Mechanisms of Toxic Action" (with Dr. Bruce Hammock) and ETX 278 "Molecular Biological Techniques in Toxicology" (with Dr. Robert Rice) each with an average enrollment of about 20 students.The Core offers some specialized samples and materials which are not available commercially, such as specialized plasmid constructs (along with methods for cloning), oligonucleotide primers, specific antibodies (e.g., anti-HSP70, AhR metallothioneins, etc.), markers for cell differentiation, and specific kinase and phosphatase inhibitors. A web-accessible database of available materials is being generated to facilitate dissemination of this information. Although a primary focus is to provide technology transfer to an individual investigator's laboratory, this core can also provide some technical services when instrumentation or facilities are unavailable.

Core Director and Members
Dr. Michael S. Denison, Ph.D., Professor, Department of Environmental Toxicology
Dr. Gino A. Cortopassi, Ph.D., Professor, Department of Molecular Biosciences
Dr. Jeffrey P. Gregg, M.D., Assistant Professor, Department of Molecular Pathology
Dr. Hsing-Jien Kung, Ph.D., Professor , UC Davis Medical Center, Cancer Center
Dr. Kent C. Lloyd, Ph.D., D.V.M, Professor, Department of Anatomy, Physiology and Cell Biology
Dr. Fumio Matsumura, Ph.D., Professor, Departments of Environmental Toxicology and Entomology
Dr. Reen Wu, Ph.D., Professor, Department of Internal Medicine

Equipment and Facilities
This Core maintains the following laboratories: Gene Expression, Molecular Genetics, DNA Microarray, Murine Targeted Genomics, Cancer Molecular Biology, Nucleic Acid and Protein. Gene expression analysis, microarray spotting and analysis as well as the creation of transgenic and knockout mice are examples of services.



Usage and Benefits
This core interacts with all research cores. Some highlights are as follows: With the Reproductive and Developmental Toxicology Core, an analysis of the role of the EGF signal transduction pathway in the etiology of endometriosis resulting from exposure to TCDD and molecular studies with Dr. Miller with regards to her studies on male reproductive toxicology of benomyl and carbendazium and its relationship to tubulin genes as targets. With the Respiratory Toxicology Research Core, a collaborative project with Dr. Reen Wu in the Toxicogenomics Research Core is being conducted, focusing on gene expression patterns associated with effects of inflammatory cytokines on airway epithelial cells since airway inflammation occurs in various environmental pollutants induced injuries. In collaboration with Dr. Jeff Gregg of this facility core, Dr. Reen Wu's lab is also focusing on gene expression pattern associated with smoke-exposed airway epithelial cells. In addition to these studies, both Drs. Charles G. Plopper and Alan Buckpitt of the Respiratory Toxicology Research Core have developed laser capture microdissecting technique to capture specific airway cell types for proteomic and genomic analyses. This core is also developing a method of assessing vitamin C transport through glucose transporters in human and monkey lung epithelial cells in culture. This Core will also continue to examine the relationship between glucose transporter 4 (GLUT 4) gene expression in human adipose tissue among TCDD-exposed veterans of the Vietnam War (in collaboration with Air Force researchers from Operation Ranch Hand) and utilize the Epidemiology Research Core for statistical and other epidemiological analyses and interpretation. The Core has also facilitated analysis of steroid and non-steroid activations of the androgen receptor by the Kung laboratory. Collaborative studies with investigators in the core have not only revealed that paracrine factors such as EGF, IL-6, neuropeptides (bombesin and neurotensin), and IL-8 are able to activate androgen receptor either in the absence of androgen or in the presence of adrenal level of androgen, but that tyrosine kinases are involved in this process. In addition, the Core has contributed to 1) microarray analysis of the gene expression pattern changes upon androgen withdrawal, and after treatment with IL-8 and bombesin (with Gregg), 2) design of RNAi used to block androgen receptor and coactivators to demonstrate their involvement, and 3) the development of chromatin immunoprecipitation technique to study the recruitment of coactivators to the AR complex after the treatment with various ligands.

Some of the ongoing joint projects which will be continued in the future are listed below:

Epidemiology Research Core
a) Application of recombinant cell bioassays for screening of serum for the presence of dioxin-like chemicals and xenoestrogens (with Lasley and Gold).

Molecular Neurotoxicology Research Core
a) Studies on the expression of bak and Bcl 2 genes in PC12 cells during apoptosis induced by 2,2'-dichlorobiphenyl and pyrethroids (with Wilson, Pessah and Pinkerton). b) Calcium homeostasis, intracellular sites of release, receptor activity measurements (with Pessah). c) Expression of ryanodine isoforms in normal and diseased states (with Pessah). d) Heptachlor epoxide alterations of Ca 2+ signaling and phosphoinositide cascade in rat Hepa-1 cells (with Pessah and Matsumura). e) Apoptosis induced by PCBs and pyrethroids in PC12 cells as a model for neurodegeneration (with Pinkerton and Vulliet). f) Coupled analysis of dioxin-like and non-dioxin-like PCB and related chemicals in neuronal cells (with Pessah). The membrane-proximal event accompanying the actions of bombesin and IL-8 on androgen receptor signaling are being examined, with a focus on calcium signaling (with Pessah).

Reproductive and Developmental Toxicology Research Core
a) Sex differences in glucose transporter gene expression and regulation (with Lasley). b) Interactions of nuclear transcription factors with hormone receptors studied using electrophoresis gel mobility shift assay (with Lasley). c) Construction of estrogen, androgen and thyroid hormone-responsive reporter plasmids and stably transfected cell lines (with Lasley). d) Bioassay-based analysis of the presence of xenoestrogens in blood (with Lasley and Gold). e) Etiology of TCDD-induced endometriosis in primates (with Lasley, Hendrickx). f) Molecular mechanisms by which TCDD decreases the bioactivity of HCG from human and primate trophoblasts (with Lasley and Douglas). g) Disruption of testicular tubulin function by benomyl-type fungicides (with Miller, Matsumura). h) Pesticide-induced estrogenic activity (with Lasley).

Respiratory Toxicology Research Core
a) GLUT 4 gene expressions in human lung epithelial cells (with Wu and Phillips). b) Analysis of cytochrome P450 in pulmonary tissue (with Pinkerton). c) Analysis of mucous cell differentiation (with Wu and Plopper). c) Characterization of cigarette smoke-induced lung cell injury and repair (with Wu and Pinkerton). d) Joint training program for pulmonary research fellows (with Wu, Cross, Plopper, Buckpitt and Pinkerton).

Toxicogenomics Research Core
a) Studies on c-erbB2 gene in human breast carcinoma (with Matsumura and Phillips). b) Thymic atrophy caused by type II pyrethroids and determination of PKC isoforms affected (with Pinkerton). c) NfkB response element affected by TCDD among human cell lines in culture (with Wu). d) Regulation of glucose transporters by dioxin-type chemicals (with Rice). e) Analysis of the silencing of CYP1A1 gene expression in keratinocytes (with Rice). f) Estrogenic action of beta-HCH through activation of c-Neu in MCF-7 breast carcinoma cells (with Matsumura). g) Smoke-induced gene expression and its association with lung cancer development (with Wu). h) Analysis of In vitro bromodichloromethane toxicity on human trophoblast cells using DNA microarray analysis (with Lasley). i) DNA microarray analysis of TCDD treated primary human cytotrophoblasts and synctiotrophoblasts (with Denison, Gregg and Douglas). j) DNA microarray analysis of the antiestrogenic effect of TCDD on human ovarian carcinoma cells (with Denison and Gregg). k) DNA microarray analysis of the gene expression pattern changes upon androgen withdrawal, and after treatment with IL-8 and bombesin (with Kung and Gregg).

The Molecular Biology Core Coordinator also provided "hands on" contributions for four collaborative projects. All of these resulted in publications. The first project, a collaboration with Dr. Matsumura's laboratory, explored the use of 3T3-L1 cells as a model for TCDD effects on adipocyte differentiation. The second joint project, with Dr. Pessah, involved the production of species and isoform-specific ryanodine receptor probes that were used to measure mRNA levels in the myopathic heart. The third project between Dr. Denison and Dr. Rice continues to examine the molecular mechanism of CYP1A1 gene silencing in rat keratinocytes. Finally, the coordinator worked with Dr. Lasley to develop a bioassay method to demonstrate the presence of xenoestrogens in human serum.



Primate/Animal Models

Overview
The goals of the Core are not only to provide materials but to identify opportunities and develop collaborations that will expand current Center activities to include studies with primates and primate materials. On request of Center faculty, Core personnel identify the most appropriate California National Primate Research Center (CRPRC) staff member for consultation regarding current or proposed research activities. The specific aim is to optimize the use of the primate model in future Center research activities. While a great deal of information can be gained from experiments conducted with laboratory rodents in in-vivo studies and with tissues and cells in in-vitro studies, there is a growing concern that mechanism of toxic action can be cell-, organ-, species- and even gender-specific. This concept has been appreciated for many decades in terms of reproductive toxicology, but is recently being openly accepted as a tenet in other toxicology sub-disciplines. Immune cell response to toxicants and response of immediate genes when compared between mice and human pathways are caveats regarding the extrapolation of results obtained from the rodent model to the human disease condition. When environmental hazards and their adverse effects are identified by epidemiologists in population-based studies, current understanding of toxicology cautions that conventional animal models may not be appropriate for experiments to investigate mechanisms of toxic action of these substances or to establish safety standards for them. Taken together, the new information at the cell and molecular level dictates that primate models, whenever possible, should be included in the complete design of modern toxicologic investigations.

Core Director and Members
Dr. Bill L. Lasley, Ph.D., Professor, Department of Population Health and Reproduction
Dr. Alan J. Conley, Ph.D., Professor, Department of Population Health and Reproduction
Dr. Charles G. Plopper, Ph.D., Professor, Department of Anatomy, Physiology and Cell Biology

Equipment and Facilities
This Core provides access to materials and experiments that permit Center investigators to assess the potential adverse effects of exposure to agrochemicals in the primate model. In addition, biological specimens and samples of interest at CNPRC will be cataloged, archived, and tracked.

Usage and Benefits
This Core provides several benefits to Center scientists. The broadest of these is the access it provides to collaborations, materials and reagents for investigators that are not currently familiar with primate research. Beyond providing advice and materials, the Core is archiving data that can be accessed by Core faculty for comparing with their own rodent data. The Core is actively developing methods and reagents that will facilitate new research studies using primate materials. Every NIEHS Center Core faculty member has been contacted and invited to meet with and discuss their incorporating primate materials in their laboratories and identifying critical reagents that would be necessary for adapting to primate work. In addition, the Core explores opportunities to work with scientists from other institutions to develop critical reagents. An example of the benefits of such efforts is the current collaboration with scientists at the University of Nebraska and the successful R27 (Grant RR017444) application that will permit the development of macaque-specific reagents for microarrays. These reagents will be archived at the CNPRC and made available along with tissue samples to Center investigators. This Core also facilitated the successful Multicenter Program Project which will investigate species differences in lung disease (Grant P01 ES11617). To date this core has provided the following tissues to Center scientists or affiliates:

- Lung tissues from control and exposed monkeys for R. Wu
- Liver samples from exposed and control monkeys to A. Buckpitt
- Gonadal and adrenal cells for steroidogenic analysis to A. Conley
- Serum from control and exposed monkeys to A. Hendrickx
-Control and exposed embryos to A. Hendrickx
- Blood, urine and tissues for lipid metabolism studies to B. Hammock and A.
Conley
- Urine and blood for reproductive biomarker development to B. Lasley
- Trophoblast cells to E. Bedows
- Ovarian tissue for immunohistochemistry to A. Conley
- Blood and lavage materials for antioxidant analysis to A. Buckpitt and E.
Postlethwait
- Fresh lung tissues for biochemical analysis to M. Miller, A. Buckpitt and E.
Postlethwait



Community Outreach and Education Program

Overview
The Community Outreach and Education Program at the University of California, Davis, seeks to fulfill the following goals: 1) To educate the public (both adults and children) in understanding the basics of environmental health sciences; 2) To identify and assist community-based efforts to resolve environmental health problems; 3) To inform the public of significant findings made by CEHS scientists and others in the field of environmental health; 4) To serve as a community resource on issues related to this field by increasing our visibility; and 5) To build a COEP network to achieve the above goals. The COEP runs four agriculturally-based outreach programs in Northern California. In addition, the COEP coordinates an environmental toxicology alumni/corporate outreach program. The COEP also serves as an Environmental Health Information Center for the public, answering questions via phone and email, and disseminating information via websites, listservers, newsletters, reports, in-house-produced videos and annual community events which include a public symposium, a conference for environmental health scientists, two alumni/corporate/state meetings, Earth Day, UC Davis Picnic Day (annual open house), and presentations at local community centers, farmers' markets, and K-12 schools.

Members
Dr. Fumio Matsumura, Ph.D., COEP Director, Department of Environmental Toxicology
Dr. Faith Boucher, Ph.D., Coordinator, Farmworker Outreach Programs , Department of Environmental Toxicology
Ms. Susan Melton-Piper, B.S., Coordinator, COEP Public Outreach Programs , Department of Environmental Toxicology
Ms. Rebecca Morrison, B.A., COEP Coordinator , Department of Environmental Toxicology
Dr. Marion Miller, Ph.D., Advisor, COEP Primary and Secondary Education, Department of Environmental Toxicology
Dr. Barry Wilson , Ph.D., Molecular Epidemiology Specialist, Departments of Environmental Toxicology and Animal Science
Dr. Patrick O'Connor-Marer, Ph.D., Advisor, COEP Pesticide Education & Training, UC Cooperative Extension
Ms. Jennifer Weber, M.S., Coordinator, COEP Pesticide Education and Training , Integrated Pest Management
Mr. Josue Medellin-Azuara, M.S., Coordinator, COEP Aquatic Toxicology Program , Department of Environmental Toxicology
Ms. Martha Stiles , M.S., Coordinator, COEP Pesticide Safety Literacy for Migrant Farm Worker Families, Department of Biological and Agricultural Engineering

Collaborators from January 2002 to the present

Agricultural/Health Organizations
Endtime Harvest
Tri-County Environmental Health Action
Inter Tribal Council of Arizona, Inc.
Arizona Department of Health Services
University of Arizona Cooperative Extension
California Department of Pesticide Regulation
Integrated Pest Management
Quechan Indian Tribe
Western Center for Agricultural Health and Safety
Western Farmworkers Association
Migrant Education Regions II and XI
Community Medical Centers, Inc.
Del Norte Clinics, Inc.
San Joaquin and Yuba/Sutter County Housing, Public Health and Environmental Health Departments and Agricultural Commissioners
North American Agromedicine Consortium
Family HealthCare Network
Arizona Department of Agriculture
Arizona Structural Control Board
Childhood Injury Prevention of San Diego
California Office of Migrant Services
Yolo County Housing Authority
Yolo/Solano FARMS Program
Yuba City Migrant Housing Center
Lodi Migrant Housing Center
Dixon Migrant Housing Center
Madison Migrant Housing Center
University Cooperative Extension in San Joaquin and Sutter/Yuba Counties
California EPA Office of Environmental Health Hazard Assessment
California Department of Health Services Environmental Health Investigative Unit

Corporations
Canadian Environmental Modeling Center
Merck and Company
Lipomics Technologies

Non-Profit and Other Community/Public Organizations
Davis City Farmer's Market
Punjabi Heritage Society
Punjabi Waves
Sutter County Literacy Program
Lao Family Association
Hmong American Association
WIC Program
National Association for the Advancement of Colored People
American Lung Association
Great Valley Center
Northern California Society of Toxicology
Alliance for Hispanic Advancement
Sacramento Earth Day Network (SAEDN)
Davis Teen Center
Davis Explorit Science Center
East Bay Conservation Corps
Northern California Society of Toxicology
Office of State Senator Martha Escutia

Schools
Woodland High School
Grafton Elementary School, Knights Landing
Dixon High School
Elk Grove High School
Laguna Creek High School
Evergreen Elementary School in West Sacramento
Shirley Rominger Intermediate School located in Winters

University of California Systemwide Offices
UC Berkeley Center for Occupational and Environmental Health Continuing Education
UC Berkeley Children's Hospital of Oakland Research Institute
UC Toxic Substances Research and Teaching Program
UC Merced
UC Berkeley Department of Nutritional Science and Toxicology
Lawrence Livermore National Laboratory

University of California, Davis
Department of Biological and Agricultural Engineering
Department of Environmental Toxicology
Superfund Basic Research Program
NIEHS Center for Children's Environmental Health
Cancer Center
Medical Center in Sacramento
Public Service Research Program
Division of Education
School of Veterinary Medicine
Arboretum
Forensics Master's Program
Department of Molecular Pathology
National Center of Excellence for Nutritional Genomics
Food Animal Residue Avoidance Databank (FARAD)
Department of Food Science and Technology
Department of Medical Pharmacology and Technology
Department of Entomology
Center for Image Processing and Integrated Computing

Highlights

CEHS Science Education and Outreach Internship Program
The CEHS Science Education and Outreach Internship Program conducted a UC Davis course entitled, "After School Discovery Program" with a community environmental health focus. Seventeen UC Davis undergraduates were trained in science outreach. They then taught environmental health workshops at Evergreen Elementary School in West Sacramento and the Shirley Rominger Intermediate School located in Winters. The Evergreen school site had a South East Asian Writing Project for its after school program and it was these children that the UCD undergraduates worked with. These activities took place primarily in the school garden. Overall between 40 to 50 elementary school students at both sites were visited by UC Davis undergraduates on a regular basis through the winter quarter, and an additional 20 students were visited five times in spring quarter. In addition, the students presented exhibits at the Teen Center in Davis to approximately 500 community visitors.

Pesticide Safety Literacy Program for Migrant Farm Worker Families
The CEHS Pesticide Safety Literacy Program produced training programs in English and Spanish on "Child Poison Prevention." The presentation includes information on: (1) Dangerous Poisons; (2) Medicines & Vitamins; (3) Household Products; (4) Personal Care Products, Plants, Environmental Toxins; (5) Safe Alternatives; (6) Poisoning Prevention in the Home; (7) Labels and How to Read Them; (8) What To Do in Case of Poisoning; (9) How to Use 9-1-1 and Poison Control Centers. In 2003, over 400 people at local migrant housing centers received training as well as safety latches to install in their homes.

Environmental Health Education for Farmworkers and Farmers
The CEHS Environmental Health Education for Farmworkers and Farmers taught courses on: 1.) the presence of various agricultural chemicals in different phases of farmwork; 2.) pathways whereby agricultural chemicals enter and affect the body; 3.) recognition of acute and chronic pesticide exposure; 4.) first aid for pesticide illness. In addition, workshops were conducted on environmental health topics of interest such as water quality and safety, children's exposure to toxics in the home, childproofing, and asthma related to agricultural dusts. Participants included Mexican, Punjabi, and Hmong farmworkers and farmers in San Joaquin, Solano, and Yuba/Sutter counties, and African-Americans living in agricultural areas. 322 people were trained in 2003.

Rural Health Clinic Outreach
The CEHS Rural Health Clinic Outreach project conducts Pesticide Illnesses and Injuries Workshops for Tribal Health Care and Agricultural Professionals. Participants in 2003 included members from thirteen tribal communities in Arizona, California, and Nevada. Attendees also represented a variety of agencies, services, and programs working directly with tribes on chemical safety, health, and environmental issues, and included health care providers from several Indian Health Services’ facilities.

Aquatic Toxicology Outreach and Training Program
The CEHS Aquatic Toxicology Outreach and Training Program conducted an internship program through the UC Davis Internship and Career Center. Interns were required to participate in the Environmental Health Sciences Rotating Exhibition course (funded through CEHS) held on Friday afternoons to increase their understanding of modern teaching methods and approaches (e.g., inquiry-based learning). Laboratory training was provided to each intern so they were proficient in the test protocols and teaching materials. Interns visited local high schools in teams and organized the classes for toxicity testing. At the end of the quarter, students developed interactive displays on water quality and toxicity issues and presented them to the Davis community at the Teen Center. In addition, project coordinators presented a short course entitled, "Basic Aquatic Toxicity Testing Methods for the Educator and Layperson," during the Society for Environmental Toxicology and Chemistry annual meeting.

CEHS Events

March 11, 2003 - Joint Norcal Society of Toxicology and UC Davis Environmental Toxicology Alumni Reception, 50 attendees.

April 12, 2003 - UC Davis Picnic Day, 400 attendees.

April 26, 2003 - Earth Day on the Sacramento State University campus, 700 attendees.

April 30, 2003 - CEHS Symposium, Protecting Our Children: The Impacts of Environmental actors on Developing Respiratory and Neurological Systems, 77 attendees.

May 30, 2003 - Friends of Environmental Toxicology Day, 76 attendees.

August 25, 2003 - CEHS Fifth Annual NIEHS Conference for Environmental Health Scientists in Napa: Nutritional Toxicology and Metabolomics, the Next Generation, 180 attendees.

November 16-19, 2003 - American Public Health Association Annual Meeting, COEP Resource Booth, San Francisco, 1,000 attendees.

Materials And Publications

Title: Safety Literacy: Child Poison Prevention
Safety Literacy Guide (75-page document); 30-slide PowerPoint training program (3 versions: 1) English, 2) Spanish, 3) Bi-lingual); and a CD entitled, "Safety Literacy: Bilingual Poison Prevention Training"

Grant Applications Submitted

NIEHS. Environmental Justice: Partnerships for Comm. 7/30/04-7/29/08. $900,000. PIs: O'Connor-Marer PG and Schenker MB.

California Policy Research Center. Health Care Access among Retired Farmworkers: Closing the Gaps. 2/1/04-7/31/03. $7,800. PI: Boucher FK.

Great Valley Center. Agricultural Development in the Hmong Community of San Joaquin County. 1/1/04-12/31/04. $66,000. PI: Boucher FK.

NIEHS. Center for Breast Cancer and the Environment at UC Davis. 1/1/03-9/30/10. $9,291,240. PI: Styne D.

NIEHS. Tri-County Environmental Health Action Program. 7/30/03-7/29/07. $971,484. PI: Boucher FK.


CEHS OFFICE OF COMMUNITY OUTREACH AND EDUCATION PROGRAM
ENVIRONMENTAL HEALTH SCIENCES INFORMATION CENTER USAGE TABLE

This is an impartial list which does not include requests for information such as questions about the Friends/Alumni of Environmental Toxicology program, and requests to post jobs via our listservers.

DATE INFORMATION REQUESTED BY WHOM

1-8-2002 Listserve for Northern California UC Davis Office of Research
Science Teachers

1-29-2002 Child advocacy/rehabilitation Northern California resident with lupus

2-15-2002 Effects of malathion on farmworkers UC Davis law student

3-20-2002 Pesticide handouts in Spanish High school teacher and
member of Migrant
Education, Woodland

4-2-2002 Summer program on toxicology for Yadava College, India
students and teachers

4-26-2002 Info on environmental education Coral Reef Alliance

5-11-2002 Request to perform study on peracetic Enviro Tech, Modesto
acid (water chlorination substitute)

8-24-2002 Help with advertising technology classes Sacramento Local Cable
for teens Station

9-11-2002 Herbicides in fertilizer Concerned citizen, Yolo
County

12-12-2002 Questionnaires for pesticide exposures UC Davis NIEHS Children's
Center for Environmental
Health

1-13-2003 Request for website link UC Davis Medical Center
Cancer Center

2-28-2003 Guest speaker for Environmental Chico State University
Advocates Program Community Legal
Information Center

3-10-2003 Bioremediation of petroleum in soil Davis resident (son of
and waterways in China Chinese government official)

3-23-2003 Health standards for exposure to fungi Concerned citizens of
Oxnard, CA

4-10-2003 Connection between Parkinson's and Pesticide inspector from the
environmental exposures California Department of
Toxic Substances

4-29-2003 Health effects of toxic mold Boys and Girls Club of
Galt, California

7-9-2003 Participation in an environmental S. Dakota Black Hills Center
justice grant for American Indian Health

8-18-2003 Toxic potential of odor in new home Homeowner from West
Sacramento

10-2-2003 Conference facilities in Napa UCD Graduate Group in
Epidemiology

12-12-2003 Rural health clinics for migrant Telecommunications group
farmworkers

12-19-2003 Info on Gulf War exposures Attorney from Houston

INDIVIDUAL CEHS MEMBER OUTREACH ACTIVITIES, AWARDS, AND PRESS RELEASES

Ellen Gold

Ellen Gold's research on women and breast cancer was featured in a "Headliner" article in Environmental Health Perspectives. The article entitled, "More Workouts for Women May Mean Less Risk of Breast Cancer" was featured in the January 2003 issue.

Jerold Last

Jerold Last was one of three faculty at UC Davis selected to receive a Distinguished Scholarly Public Service Award for 2002-2003. The UC Davis Academic Senate, representing all tenured faculty at the campus, makes the awards annually to recognize significant contributions to the world, nation, state and community through distinguished public service.

Patrick O'Connor-Marer

Chair of the 2003 Conference on Challenges in Agricultural Health and Safety in San Francisco. Marc Schenker and Kent Pinkerton presented lectures at this conference which was jointly sponsored by the CEHS, the Western Center for Agricultural Health and Safety, the Pacific Northwest Agricultural Safety and Health Center, and the UC Center for Occupational and Environmental Health.

Martha Stiles

Ms. Stiles was interviewed by the Davis Enterprise for a story entitled, "Be safe, not sorry: Program aims to inform parents about proper storage of household products." The article appeared in the "Living" section on April 28, 2003.

Barry Wilson

Dr. Wilson provided a list of Laboratories Approved to Perform Cholinesterase Testing for Occupational Health Surveillance on July 1, 203. His research group reviewed California laboratories and reduced the number of laboratories approved for blood cholinesterase testing from 20 to 9. Dr. Wilson provided copies of the approval to the NIEHS and the UC Division of Agriculture and Natural Resources.








Pilot Projects

Overview
The purpose of the Pilot Projects program is to: 1) Provide initial support for new investigators with P.I. status (who have not received NIH R01, R29, P01 awards) to establish new lines of research. 2. Allow exploration of innovative new research directions (significant departure from ongoing funded research) by CEHS faculty. 3. Stimulate established investigators outside the CEHS to explore innovative new research projects in environmental health. 4. Support technical advances to develop a specific technical expertise that the CEHS needs.

The Pilot Projects program benefits the Center by supporting research that is relevant to the goals and mission of the Center and is relevant to real world health problems. Pilot projects are innovative (often high risk) and provide new directions/dimensions to the repertoire of CEHS research. This program also stimulate use of core facilities and collaborations between Center labs and the recipient lab. Center Pilot Projects also have frequently lead to publications and other tangible results such as new NIH R01 grant funding. The Pilot Project Program has also increased collaborations between the Center and non-Center members and has lead to membership by some outside applicants.

Project Descriptions: Projects funded in the current year

Year of Funding: 2003
Title: 2002, 2003, Achieving Accurate Measurement with Minimal Face Velocity Effect of Passive Sampler
Investigator(s): Kiyoung Lee, Department of Epidemiology and Preventive Med.; Anthony Wexler, Depts. of Mechanical and Aeronautical Engr., Civil and Env. Engr., and Land, Air and Water Resources
Description: This research will focus on providing a reliable passive sampler configuration for measuring exposure to gaseous air pollutant in occupational and environmental health sciences. The objective of this pilot study application is to identify characteristics of passive samplers that minimize the face velocity effect and to provide background information for patent application. The central hypothesis of this research is that adjusting the diffusion path of passive sampler can minimize the face velocity effect on sampling rate. This research application is necessary to prevent release of critical information prior to patent application. The specific aim is to test the hypothesis that geometrical configuration of the passive sampler can minimize the effect of face velocity on sample artifacts. The association between passive sampler configuration and face velocity effect will be evaluated. This research will utilize an innovative and systematic experimental approach to evaluate the face velocity effect. Therefore, the expected outcomes of the research will be produced cost-effectively.

Year of Funding: 2003
Title: Development of a Screen for Nuclear Receptor Activation in Live Vertebrate Embryos and Identification of an Endogenous RXR Ligand
Investigator(s): J. David Furlow, Section of Neurobiology, Physiology and Behavior
Description: The Analytical Biochemistry Facility Core will isolate and identify potential RXR ligands; the Cellular and Molecular Imaging Facility Core was used to obtain high quality immunohistochemical stains of induced GFP expression in transgenic Xenopus laevis spinal cord. The retinoid-X receptor (RXR) serves as a heterodimeric partner for multiple nuclear hormone receptors; however, little is known about its activation pattern during development, and the distribution of endogenous ligands. Therefore, the core sought to visualize activated RXRalpha in live transgenic Xenopus laevis embryos throughout development. The project adopted a nuclear receptor-Gal4 fusion/UAS based reporter system for its assay. Strong activation of the RXRalpha ligand-binding domain was observed in the spinal cord just posterior to the hindbrain. Activation is first detected in neurula stage embryos and persists until swimming tadpole stages. Addition of exogenous ligands, such as 9-cis retinoic acid, expands the activation of RXR throughout the spinal cord but not in the brain. A deletion in the ligand-dependent activation domain (AF2) of RXR prevents reporter gene activation. The results reveal a specific activation pattern for Gal4-RXRalpha specifically in the developing spinal cord and suggest the existence a ligand in this region.

Year of Funding: 2003
Title: Of Mice and Metabolomics: A New Phenotypic Approach to Characterizing Susceptibility to Endocrine Disruption
Investigator(s): Jim L. Spearow (Department of Neurology, Physiology and Behavior); Marion Miller (CEHS Cell/Tissue Technology Facility Core)
Description: The investigators will use an NMR-based metabolomics approach to detect and quantify differences in metabolic profiles between estrogen-resistant and estrogen-sensitive mouse strains in responses to a model endocrine disruptor estradiol (E2). Strains of mice differing dramatically in susceptibility to endocrine will be implanted with silastic implants containing increasing doses of E2. Urine will be collected weekly and liver and testes will be collected at 7 weeks of age. The changes in metabolic profiles will then will be determined by NMR. Multivariate pattern recognition will be used to identify strain-dependent metabolic differences, both due to genetic differences between control animals, and differences in the response to E2. This would be the first application of metabolomic phenotyping that compares strains of mice to resolve a complicated biological phenomenon (genetic susceptibility) of high environmental and human health importance.

Year of Funding: 2003
Title: Endogenous Formation of Halogenated AhR Activators by Leukocyte-Derived Heme Peroxidases
Investigator(s): Jason P. Eiserich, Ph.D., Department of Internal Medicine, Division of Nephrology; Michael Denison (CEHS Functional Genomics and Molecular Biology Facility Core)
Description: Halogenated biomolecules are formed as endogenous products of heme peroxidase catalytic activity, and serve as biomarkers of oxidative processes occurring during inflammatory responses. However, whether these products serve only as dosimeters of oxidative biochemistry, or have inherent biological activity is not known. Based upon the investigators' preliminary studies and the supporting literature, they hypothesize that halogenation reactions catalyzed by mammalian heme peroxidases confers AhR activating activity upon endogenous biological molecules, and that this may modulate the course of inflammation by activating AhR-linked expression of pro- and anti-inflammatory genes.

Year of Funding: 2003
Title: Mechanisms of Macrophage Transformation in Foam Cells by Environmental Pollutants like Dioxins, PCBs, and Airborne Particles
Investigator(s): Christoph Vogel, Department of Environmental Toxicology, Kent Pinkerton, Michael Dunlap, Norm Kado, Paul Kuzmicky, Fumio Matsumura, Michael Denison
Description: Epidemiological data have shown that exposure to atmospheric particulate matter (PM) as well as dioxin-type chemicals which are part of the chemical composition of PMs may cause atherosclerosis and cardiovascular diseases. An early event of atherosclerosis is intimal invasion of monocytes and the formation of foam cells. An indication of foam cell formation is the accumulation of cholesterol which was found after treatment with diesel exhaust particles, chimney soot from wood burning as well as TCDD in U937 macrophages. Scanning electron microscopy confirmed the surface modifications, changes of cell shape, and cell size during differentiation of U937 cells in foam cells induced by TCDD. Further the project found increased expression of typical foam cell markers such as the inflammatory factors Interleukin-1beta, Tumor necrosis factor-alpha, and cyclooxygenase-2, as well as Metalloproteinases (MMP)-3, and MMP-12 after treatment with PM of urban dust, diesel particulate, chimney soot, and their organic extracts.

Year of Funding: 2003
Title: Neuroprotective Effects of Dietary Flavonoids against Environmental Exposure to Xenobiotics
Investigator(s): Heidrun Gross and Alice Wong, Department of Molecular Biosciences
Description: Experiments are being conducted in the CEHS Functional Genomics Facility, and the system used to determine cell viability is located in the Functional Genomics Facility Core. In recent years there has been an increasing awareness that dietary components such as flavonoids play a role in the prevention of chronic diseases. Current studies highlight the potential protective action of dietary flavonoids found in fruit, vegetables and beverages against neurodegenerative disorders. Yet, the mechanisms, modes and sites of action are far from being understood. Onset of neurodegenerative disorders such as Parkinson's and Alzheimer's diseases may involve an environmental component. Exposure to xenobiotics such as rotenone, a widely used pesticide, for example can cause PD like symptoms in rats. The aim of this research project is to test the neuroprotective effects of two abundant dietary flavonoids, (+)catechin and (-)epicatechin and their methylated metabolites in a recognized model for studying sporadic Parkinson's disease. Human neuronal cells (NT2) will be incubated with a test compound before exposure to rotenone, a cell death initiating agent. Protective action of flavonoids will be determined by cell viability of treated compared to control cells.



Project Descriptions: Projects funded in previous years with positive outcomes in the current year

Year of Funding: 2002
Title: Can Bone Marrow Mesenchymal Stem Cells Survive, Proliferate and Differentiate in Rat Livers Injured by a Chemical Toxin?
Investigator(s): Melanie A. Greeley, P. Richard Vulliet and Dennis Wilson, Department of Veterinary Medicine: Molecular Biosciences
Description: 1) To optimize the culture of mesenchymal stem cells from rats to get maximal yield of colony forming units. New culture methods, which will include FACS analysis 3 weeks after primary culture, should result in a more proliferative and plastic cell. 2) To optimize the transduction of mesenchymal stem cells with enhanced Green Fluorescent Protein to maximize transduction efficiency for long term tracking of injected MSC's, transduction was attempted and a low percentage (7-10%) of transduced cells was obtained. Possible problems included poor transfection of the packaging cell line (phoenix cells) with a resulting low viral titer or the possibility that the MSC's were not proliferative enough to allow transduction. With the new culture methods, the resulting cells may be more receptive to the transduction. Improvement of methods to transfect the packaging line will be done and viral titers will be checked with the NIH/3T3 cell line. 3) To assess the ability of mesenchymal stem cells to survive, proliferate and differentiate using a chemically induced model of liver injury. This model will be tried again using the cells cultured using the new method to see if there is engraftment and proliferation. These cells will also be differentiated in vitro and injected into an experimentally induced cirrhotic liver to assess if there is an improvement in liver function.
Positive Outcomes
Investigators have developed a more testable hypothesis that will use a model of chronic liver injury and the pre-differentiated stem cells. The working hypothesis is now: injection of adult bone marrow non-hematopoietic stem cells differentiated in vitro to functional hepatocyte-like cells will improve the morphology and function of an experimentally induced fibrotic/cirrhotic liver in the rat.

Year of Funding: 2002
Title: Heat Shock Protein HSP27 Inhibits UVB-Induced Apoptosis in Human Corneal Epithelial Cells
Investigator(s): : Biao Shi and Rivkah Isseroff, MED:Dermatology; Drs. B. Rice and M. Phillips at the NIEHS Center are giving advice and providing technique for the transfection study.
Description: Ultraviolet B radiation (UVB) is a health risk for various corneal disorders. We hypothesize that the 27 kDa heat shock protein (HSP27) can protects human corneal epithelial cells from UVB-induced damage. In the first year study, investigators found that preconditioning of corneal cells with arsenite or heat shock, which induces HSP27 expression and phosphorylation, reduced UVB-induced cell apoptosis. To further clarify the role of HSP27 in protection of corneal cells from UVB-induced apoptosis, investigators proposed to establish transfected corneal cells that either over- or under-express HSP27, or over-express non-phosphorylatable HSP27. We will determine how changes in HSP27 expression and/or phosphorylation in the transfectants affect cell apoptosis following UVB exposure. Investigators are also manipulating HSP27 level by internalizing HSP27 antibody into cells to neutralize HSP27. In addition, the pilot project is conducting inhibitor studies to determine whether the anti-apoptotic activity against UVB-exposure will be affected in corneal cells when HSP27 expression and/or phosphorylation is inhibited.

Positive Outcomes
1. A research article, UVB Irradiation Induces Changes in the 27 kDa Heat Shock Protein (HSP27) in Human Corneal Epithelial Cells, (Authors: Shi B, Han B, Schwab IR, Isseroff RR) has been submitted for publication. 2. A research article, Stress-preconditioning reduces UVB-induced apoptosis in human corneal epithelial cells through anti-apoptotic activity of 27 kDa heat shock protein (HSP27), (Authors: Shi B, Isseroff RR), is in preparation.3. A poster presentation (Stress-preconditioning reduces UVB-induced apoptosis in human corneal epithelial cells: is the anti-apoptotic activity of 27 kDa heat shock protein involved?) was given in 42nd Annual Meeting of American Society of Cell Biology.

Year of Funding: 2002
Title: The Influence of Airborne Pollutants on Pulmonary Immune Response to Allergens
Investigator(s): Lisa Miller, Department of Anatomy, Physiology and Cell Biology
Description: The objective of this proposal is to determine how airborne pollutants can promote or prime the pulmonary immune response to allergens. Studies in both rodent and non-human primate models of allergic asthma indicate that repeated exposure to ozone can significantly enhance the immune response to aeroallergens. Preliminary data from has indicated that chronic exposure to another airborne pollutant, environmental tobacco smoke, can increase the expression of lymphotactin (a T and natural killer cell chemokine) within airways of BALB/c mice. Although the precise function of lymphotactin is not known, it has been reported to have adjuvant properties in vivo. Because chronic exposure to environmental tobacco smoke has very similar effects to that of ozone during allergic sensitization, we have postulated that ozone exposure can promote the pulmonary immune response to aeroallergens via enhanced expression of lymphotactin. This proposal will utilize lymphotactin-deficient (knock-out) BALB/c mice to determine the functional significance of this chemokine during ozone and allergen co-exposure regimens.