CORE DIRECTOR AND MEMBERS
Michael S. Denison, Ph.D., Core Leader
Professor, Department of Environmental Toxicology
Gino Cortopassi, Ph.D.
Associate Professor, Department of Molecular Biosciences
Hsing-Jien Kung, Ph.D.
Professor, UC Davis Cancer Center
Fumio Matsumura, Ph.D.
Professor, Departments of Environmental Toxicology and Entomology
Reen Wu, Ph.D.
Professor, Department of Internal Medicine
DESCRIPTION
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 advise 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 general a 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. Accordingly, the teaching and training of graduate students and staff is an integral part of the Core's operation. Dr. Denison teaches a graduate course on "Molecular Biological Techniques in Toxicology" to facilitate this process. 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.
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 has provided service in three ways: answering questions, helping to design experiments and troubleshooting; training researchers new to molecular techniques and provided assistance to those unfamiliar with specific molecular techniques and approaches; and collaborating in multi-investigator projects. In addition, Core equipment, specialized supplies and repository (e.g. DNA probes, expression vectors cloning vectors, bacterial strains, restriction and other DNA modification enzymes, DNA sequencing and PCR primers, etc.) have been freely available for the use of Center investigators to supplement equipment and supplies in their own laboratories.
48 graduate students and 20 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" and ETX 298 "Molecular Biological Techniques in Toxicology", each with an average enrollment of about 15 students.
This Core interacts with all research cores. With the Neurotoxicology Research Core, this Core has an ongoing collaboration on the development of markers for apoptosis (Bak and Bcl 2) in PC12 cells affected by PCB congeners. DNA and amino acid sequencing on the ryanodine receptors will continue. With the Reproductive Research Core, there has been work on the subject of: (a) differences in gene expression control between males and females with respect to the actions mechanisms of TCDD; (b) etiology of endometriosis resulting from exposure to TCDD where EGF signal transduction pathway is involved; and (c ) studies on molecular biology of tubulin genes with Dr. Marion Miller in relation to her studies on male reproductive toxicology of benomyl and carbendazium. With Respiratory Toxicology Research Core, a collaborative project with Dr. Reen Wu on Toxicogenomics is being planned. This Core will develop a method of assessing vitamin C transport through glucose transporters in human and monkey lung epithelial cells in culture. With the Toxicogenomics research core, a collaborative work on DDT-induced breast cancer has been initiated. The Core's task is to develop the method to assess c-erbB2 (c-neu) associated protein tyrosine kinase so as to facilitate the study on the action of DDT and beta-BHC on c-erbB2 protein. With the Epidemiology Research Core, Dr. Gino Cortopassi and Dr. Marc Schenker are to initiate the occurrence of Bcl-2 mutation among farm workers heavily exposed to phenoxy herbicides in the future. This Core will study 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 Operation Ranch Hand, Air Force) and in due course, with the Epidemiology Research Core for statistical and other epidemiological analyses and interpretation.
Some of the ongoing joint projects which will be continued in the future are listed below:
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-responsive reporter plasmids and stably
transfected cell lines (with Lasley).
d) Bioassay-based analysis of the presence of xenoestrogens in
blood (with Lasley).
e) Sequencing of testicular ß -tubular gene sensitive to
benomyl (with Miller).
f) Etiology of TCDD-induced endometriosis in primates (with Lasley,
Hendrickx).
g) Molecular mechanisms by which TCDD decreases the bioactivity
of HCG from trophoblasts (with Lasley and Douglass).
h) Disruption of testicular tubulin function by benomyl-type fungicides
(with Miller, Matsumura).
i) Pesticide-induced estrogenic activity (with Lasley).
Molecular Neurotoxicology Research Core
a) Sequencing ryanodine receptor genes (with Pessah).
b) Studies on the expression of bak and KC12 genes in PC12 cells
during apoptosis induced by 2,2'-dichlorobiphenyl and pyrethroids
(with Wilson, Pessah, Pinkerton).
c) Calcium homeostasis, intracellular sites of release, receptor
actively measurements (with Pessah).
d) Expression of ryanodine isoforms in normal and diseased states
(with Pessah, Phillips).
e) Heptachlor epoxide alterations of Ca 2+ signaling and phosphoinositide
cascade in rat Hepa-1 cells (with Pessah and Matsumura).
f) Pyrethroid effects on tau protein production in PC12 cells
as a model for Alzheimer's disease (with Pinkerton and Vulliet).
g) Apoptosis induced by PCBs and pyrethroids in PC12 cells as
a model for neurodegeneration (with Pinkerton and Vulliet).
Cell Biology Carcinogenesis (now renamed Toxicogenomics) Research Core
a) Studies on c-erbB2 gene in human breast carcinoma (with
Matsumura, Phillips).
b) Thymic atrophy caused by type II pyrethroids. 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).
Respiratory Toxicology Research Core
a) GLUT 4 gene expressions in human lung epithelial cells (with
Wu, Phillips).
b) Analysis of Ah receptor activation by sidestream cigarette
smoke (with Pinkerton).
c) Analysis of cytochrome P450 in pulmonary tissue (with Pinkerton).
d) Program project, ES00628 for a continuous effort in Health
effects of oxidant pollutants (with Wu, Plopper, Buckpitt, Pinkerton)
e) Analysis of mucous cell differentiation (with Wu, Plopper).
f) Characterization of cigarette smoke-induced lung cell injury
and repair (with Wu, Pinkerton).
g) Joint training program for pulmonary research fellows (with
Wu, Cross, Plopper, Buckpitt, Pinkerton).
Epidemiology
a) Examined application of recombinant cell bioassays for screening
of serum for the presence of dioxin-like chemicals and xenoestrogens
(with Lasley).
The Molecular Biology Core Coordinator also provided "hands on" contributions for four collaborative projects. All of these resulted in publications (listed below). 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 which were used to measure mRNA levels in the myopathic heart. The third project between Dr. Denison and Dr. Rice examined the molecular mechanism of CYP1A1 gene silencing in rat keratinocytes. Finally, the Coordinator worked with Dr. Rice's laboratory to show that arsenate treatment of cultured human keratinocytes results in the suppression of transcription of involucrin, a marker of terminal differentiation.