Mechanisms of TCDD-induced Wasting Syndrome (Fumio
Matsumura). We compared the ability of two clonally derived murine preadipocyte
cell lines, 3T3-L1(L1) and 3T3-F442A (F442A), to differentiate
after treatment by TCDD, and found that the former cell line was
clearly suppressed by TCDD but the latter was not. It was initially
postulated that the easiest way to explain the lack of response
to TCDD in F442A cells could be an alteration in aryl hydrocarbon
receptor (AhR) functionality. This hypothesis was tested first,
but no differences were found in the levels or functions of AhR.
To find an alternate explanation for such a differential effect
of TCDD, we tested the action of several diagnostic agents on the
process of adipocyte differentiation of these two cells. No differences
were found between these two lines of cells in the susceptibility
to the antiadipogenic action of 12-0-tetradecanoylphorbol-13-acetate
(TPA), or to TNFa, indicating that the basic biochemical components
engaged in the antiadipogenic actions of these agents in these
two cell lines are similar. In contrast, F442A cells were found
to be more resistant to the antiadipogenic action of EGF or TGFb
than L1 cells which were tested side by side. Based on the knowledge
that TNFa preferentially affects C/EBPa and that TGFb specifically
controls C/EBPb and delta in their antiadipogenic action, we hypothesized
that the major cause for the differential response of these two
similar cell lines could be the insensitivity of C/EBPb and/or
delta of F442A cells to the action of TCDD. We could obtain supporting
data for this hypothesis, showing that in F442A cells the level
of C/EBPb is already high even before the addition of adipocyte
differentiation factors and that TCDD did not cause any significant
changes in the titer of C/EBPb.
Mechanism of Estrogenicity of DDT Analogs in MCF-7cells
(Fumio Matsumura). b-HCH is known to be a poor agonist for the estrogen
receptor (ER), and yet it has been shown to act like an estrogen
in stimulating foci formation in MCF-7 cells. We investigated
the reason for such an action of b-HCH, using a rat prolactin-luci-ferase
reporter system transfected to MCF-7 cells. We found that the
presence of c-Neu (erbB2), ER and ERE is needed for b-HCH to
act estrogenic at the transcription activation level in this
cell line. We compared the actions of b-HCH to that of EGF which
is known to act estrogenic without being an agonist for ER in
this cell and found that their action patterns are quite similar,
the only difference being that the former action is blocked by
an antibody against c-Neu and the latter by both c-Neu and EFG
receptor antibody. We concluded that b-HCH's estrogenic action
in this cell model is mediated through "ligand-independent activation
of ER pathway."
Assessment of the Rhizosphere Competency and Pentachlorophenol-metaboling
Activity of a Pesticide-degrading Strain of Trichoderma Harzianum
Introduced into the Root Zone of Corn Seedlings (Fumio Matsumura). To develop a dependable approach to introduce laboratory selected,
pesticide-degrading microorganisms into soil environments for
the purpose of in situ bioremediation, we tested the possibility
of utilizing plant rhizospheres as the vehicle. We first established
the rhizosphere competency of a strain of the soil fungus Trichoderma
harzianum, a biocontrol species well studied by plant pathologists
to colonize plant rhizosphere in many parts of the world. The
strain we chose, T.h.2023 is resistant to many fungicides, and
it has been shown to metabolize several pesticides. Second, we
found that it readily metabolized pentachlorophenol (PCP), which
is quickly and stochiometrically converted to pentachloroanisole
(PCA) in liquid culture. Taking advantage of this specific feature,
we have developed a sensitive metabolic marker approach that
allowed us to monitor for the continuous presence and activity
of this fungal strain in the corn rhizosphere soil in situ over
time.
Assessment of Four Biochemical Parameters of Adipose
Tissues in Samples Collected from Vietnam War Veterans (Fumio
Matsumura). This study undertakes an epidemiological approach to the study
on the health effects of dioxins with particular emphasis on
causes for development of type II diabetes. Approximately 10
grams of adipose tissue has been removed by liposuction from
313 randomly selected participants of the Air Force Health Study
(AFHS) in an effort to determine whether a relation exists between
2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) exposure and glucose
transporting activity in adipose tissue cells. We are performing
semi-quantitative RT-PCR analysis of expression of Src, C/EBPα NFκB,
and GLUT4 genes in the tissue samples. Preliminary results suggest
that, years after TCDD exposure, Src has an effect on NFkB levels,
but other factors are much more important, and NFkB and C/EBPa
levels account for approximately 25% of GLUT4 expression.
Low Level Effects of Organophosphates Including Sarin
(Barry Wilson and Peter Spencer). Our Department of Defense project
is in its final stages. Its objective is to study the biochemical
and morphological effects of low levels of sarin, a chemical
warfare agent, diisopropyl fluorophosphate, paraoxon (the active
metabolite of parathion) and pyridostigmine on mice and chickens
in collaboration with Peter Spencer and colleagues at Oregon
Health State University. We are testing whether pyridostigmine
(included as a prophylactic to possible exposure to nerve gases
like sarin) plus the organophosphates will be more or less harmful
to the animals than either given alone. Several experiments with
facilities at CHE have been completed. We found cell culture
and animal evidence indicating an additive (not sparing) effect
of organophosphates and pyridostigmine bromide and the passage
of pyridostigmine bromide across the blood/brain barrier into
the brain of the chicken. Experiments are underway to define
the dose conditions in which sarin causes delayed neuropathy
in the chicken and whether pyridostigmine bromide will influence
the intensity of the effect. Supported by DAMD 17-97-C-7057.
Improving Blood Monitoring of Enzymes as Biomarkers
of Risk from Anticholinergic Pesticides and Chemical Warfare
Agents (Barry
Wilson). Another major problem with using blood cholinesterase
measurements as early warnings of exposure to pesticides and
chemical warfare weapons is the lack of reliable normal ranges
for human values based on modern automated instrumentation. We
are working with the Department of Defense laboratory (CHPPM)
that monitors more than 25,000 individuals per year for blood
cholinesterase, using an accurate but slow pH assay. The major
goals of this project are to determine conversion factors between
the delta pH and Ellman cholinesterase assays, and to assist
with a change to the Ellman assay for DOD. The study is underway,
and we hope to soon have normal human ranges for red blood cell
AChE and plasma BChE to report. Supported by DAMD17-01-1-0772,
1/1/01 12/31/04.
Acetylcholinesterase Values among Defense Laboratory
Workers (Stephen McCurdy). In collaboration with Dr. Raven Reitstetter
(U.S. Army), Dr. Barry Wilson (UC Davis) and UC Davis graduate
student Daniel Arrieta, we analyzed over 1,400 acetylcholinesterase
values among defense laboratory workers potentially exposed to
acetylcholinesterase-suppressing agents. Analysis has been completed,
and we anticipate utilizing the data to publish a set of normal
values. Funding for further work (Barry Wilson, PI) has recently
been confirmed.
National Institute of Justice, Networked Terrorism
Detection System (Barry Wilson, Carey Pope). The major goal of the subproject
is to test prototype sensors for sarin. This project, led by
Dr. Carey Pope (Oklahoma State University), is drawing to a close.
Our role was to run some assays with nerve agents to help standardize
a cholinesterase-measuring device under development. We performed
a few experiments and then were informed that the granting agency
was terminating the work. Supported by MIPT 106-113-2999-33 (MIPT-VETMD-03
Subcon), 3/1/01 2/29/04.
Effect of Pyridostigmine on the Physiologic and Morphologic
Changes Induced by Soman at the Human Neuromuscular Junction
(Barry Wilson and Ricardo Maselli). A project with neurobiologist
and neurologist Ricardo Maselli has been funded by the Department
of Defense. The major goal of the project is to investigate the
protection provided by pyridostigmine against nerve agents. Its
objective is to establish conditions in which pretreatment with
pyridostigmine bromide will ameliorate the effects of nerve agents
such as soman and sarin on isolated human muscle obtained from
volunteer donors undergoing surgeries for other matters. A chamber
has been constructed, a protocol has been devised, and discussions
are underway with the Food and Drug Agency concerning the experimental
protocol and the application of the results. Supported by DAMD
17—02-2-0001, 12/10/01 1/9/05.
