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1996
Award Recipients |
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Identification of
Genetic Loci Associated with Murine Strain Differences
in Susceptibility to Cadmium-Induced Limb Malformations
Michael D. Collins, Ph.D., David N. Hovland Jr.,
M.S., Environmental Health Sciences, Toxicology,
UCLA |
| Abstract:
The goal of this study is to identify the genetic
factors interacting with an environmental contaminant,
cadmium, which are responsible for the production
of birth defects in mice, to gain insight into the
mechanism of possible human effects. The investigators
are working on two of the specific aims mentioned
in the grant proposal to the SCEHSC. At this point,
they have tested over 700 microsatellite markers
for polymorphism between the sensitive C57BL/6 and
resistant SWV strains of mice. Of those 700 markers,
they have identified over 400 that are polymorphic
between their two strains, allowing them to identify
the parental source of DNA at the location of each
of the markers. They are in the process of selecting
100 of these polymorphic markers to span the genome
of the two strains, at 15-20 cM spacing. They are
also creating a colony of F1 mice, produced by crossing
the C57BL/6 and SWV strains. F2 mice will then be
produced by backcrossing the F1 offspring to the
C57BL/6 strain, and administering cadmium to the
maternal animal at day 9.0 of gestation. The resulting
offspring will be removed by C-section on day 18
of gestation. They will be phenotyped for the presence
or absence of forelimb ectrodactyly and genotyped
for 100 markers to identify regions and the parental
source of the genome that control sensitivity or
resistance to cadmium-induced limb malformations
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Final Report:
This project has been extremely helpful in supporting
an interesting set of experiments to initiate the
use of a genetic approach to explain differential
murine strain susceptibility to cadmium-induced
forelimb malformations. A manuscript entitled "Identification
of a murine locus conveying susceptibility to cadmium-induced
forelimb malformations" and authored by D.
Hovland, R. Cantor, G. Lee, A. Machado and M. Collins
has been provisionally accepted by Genomics pending
revisions. It was revised and resubmitted on 10/15/99.
This manuscript is the first publication by these
authors in a mainstream genetics journal, thus the
seed funding has allowed the environmental teratogenesis
research to expand into the modern genetics field.
The project has also provided the funding for a
doctoral dissertation by Dr. David Hovland, Jr.,
who has gone on to a position at Allergan Pharmaceuticals.
A second doctoral student, Ms. Grace Lee, is currently
working on the project. The project has provided
the underlying foundation for a symposium to be
presented at the Teratology Society Annual Meeting
in June 2000. The symposium is to be entitled "Genetic
susceptibility to teratogenesis," will be chaired
by M. Collins, and will be sponsored by the March
of Dimes.
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Dioxin Exposure
and Carotid Atherosclerosis
James H. Dwyer, Ph.D., Department of Preventive
Medicine, USC |
| Abstract:
The Air Force Health Study is a longitudinal study
of the Ranch Hand cohort and a comparison group.
The Ranch Hands are veterans of the Vietnam War
who were exposed to TCDD as a contaminant in military
herbicides. These two cohorts are examined at five-year
intervals. The hypothesis that TCDD exposure could
lead to increased atherosclerosis stemmed from two
observations. The first is that ischemic heart disease
mortality was directly related to TCDD blood levels
in a cohort of chemical workers involved in the
production of 2,4,5-T in Hamburg Germany. Since
Dr. Dwyer is an investigator in that study, the
Air Force contacted him to help them explore possible
links between TCDD exposure and atherothrombotic
disease. Arterial wall thickness was determined
from computerized edge-detection processing of ultrasound
images of the lumen-intima and media-adventitia
echogenic interfaces. Examinations were conducted
at the Scripps Institute in San Diego where the
veterans were flown for a general examination. |
Final Report:
The goal of the project was the collection of pilot
data on the relation between plasma levels of 2,3,7,8-TCDD
and extent of atherosclerosis in the common carotid
arteries among US Air Force veterans (known as Ranch
Hands) of the Vietnam war. The pilot funds from
the Center allowed the investigators to measure
intimal thickening (intima-media thickness, IMT)
of the common carotid arteries in a sub-sample of
the Ranch Hand and comparison cohorts (n=320). They
found a significant association between serum TCDD
levels and IMT in the older members of the cohort.
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Investigation of
the Potential Synergy Between Environmental Toxins
and Viruses
Deborah Johnson, Ph.D., School of Pharmacy, USC |
| Abstract:
This study is directed towards elucidating the potential
interaction between environmental toxins and viral
infection in which they act synergistically to promote
cell injury. As a model for understanding the effects
of environmental toxins, the investigators will
use the herbicide, diquat, which is capable of producing
injury to tissues by a well-documented oxidative
stress mechanism involving production of oxygen-centered
free radicals and H2O2. They will examine its ability
to enhance the function of the hepatitis B virus
(HBV) protein product, X. The X protein has been
shown to be a potent transactivator of specific
cellular and viral genes. In particular, X has been
shown to activate NF-kB-dependent promoters via
the activation of cellular signaling pathways, including
ras. NF-kB is a key transcription factor in the
expression of cytokines and other components of
the inflammatory process and it has also been shown
to be activated by oxidative stress. Furthermore,
a number of viruses have been shown to modulate
the oxidative state of infected cells. Thus, these
similarities suggest that environmental toxins and
certain viral products will act in a negative synergistic
manner to produce pathological consequences. The
hypothesis is that the activation of NF-kB by the
X protein may enhance the pathophysiologic response
to environmental toxins by synergistic mechanisms.
These studies will determine whether the X- and
oxidant-mediated activation of NF-kB occurs through
independent or dependent pathways. The results from
these pilot studies will allow us in the future
to define, in detail, the common or interacting
pathways that are activated by environmental toxins
and viral protein products that produce pathological
consequences. The HBV X protein will be expressed
in a liver cell line using either stable or transient
transfection. The ability of X to produce an oxidative
stress will be assessed. The oxidative state will
be assessed by measuring both the enzyme catalyzed
oxidation of NADPH and the generation of superoxide
and H2O2. Cells will be further exposed to increasing
concentrations of diquat. The study will determine
whether X expression changes the response of the
cells to diquat-induced injury and oxidative stress.
This key experiment will allow the investigators
to assess whether X and diquat can potentially work
synergistically to create oxidative stress. Finally,
the study will determine whether the X-mediated
activation of NF-kB, which occurs through the activation
of the ras pathway, can be modulated by treating
the X-expressing cells with diquat or antioxidants. |
Final Report:
A Chang liver cell line has been transiently transfected
with an X expression plasmid, and the oxidative
state of the cells was assessed. Since the transfection
efficiencies of this cell line were low (i.e. less
than 5% of the cells are transfected), little change
in the overall oxidative state of the total cell
population was observed. Currently, the investigators
are using alternative approaches for increasing
transfection efficiencies. In addition, they are
creating stable cell lines that can be induced to
express X. Using either approach, they will further
assess the oxidative state of the X-expressing cells
and potential changes that occur when these cells
are exposed to increasing concentrations of diquat.
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Childhood Brain
Tumors, N-Nitroso Compounds, and P53 Mutations
Roberta McKean-Cowdin, Ph.D., Department of Preventive
Medicine, USC |
| Abstract:
The purpose of this project is to examine the relationship
between the development of brain tumors in children,
prenatal exposure to N-nitroso compounds (NOCs)
through maternal diet, and mutations in the p53
tumor suppressor gene. Nitrosamides (a form of NOCs)
are potent neuro carcinogens associated with the
development of brain tumors in animal studies and
NOC precursors are found in foods associated with
increased brain tumor risk in epidemiologic studies.
Specifically, the pilot examines whether transplacental
exposure of the human, fetal brain to NOCs through
maternal dietary intake of nitrites is associated
with GC to AT transitions of the p53 gene found
in childhood astroglial tumors. Tumor tissue for
approximately 20 childhood brain tumor cases previously
evaluated for exposure to NOCs in the West Coast
Childhood Brain Tumor Study will be screened for
mutations in the p53 tumor suppressor gene, using
immunohistochemical analysis and single-strand conformation
polymorphism (SSCP) assay. DNA sequencing will be
completed for exons 5-8. A descriptive analysis
of findings will be completed including: percent
of cases with mutations, types and locations of
mutations, estimated exposure to NOC through maternal
diet, tumor histology and topography. |
| Final Report:
This project was established to evaluate the relationship
between brain tumor occurrence in children, their
mothers' prenatal exposure to dietary nitrites,
and mutations in the p53 tumor suppressor gene.
Nitrite can act as a transplacental neurocarcinogen,
when endogenously converted to certain forms of
nitrosamides in the stomach. Previous data had shown
that high maternal intake of nitrite from cured
meat (>1.3 mg daily) was associated with increased
risk of childhood brain tumor (OR=1.9; 95%CI 1.3,2.6)
and that risk increased with increasing nitrite
level (ptrend=.0003). The investigators further
hypothesized that transplacental exposure to dietary
nitrite may be associated with p53 mutations in
childhood astroglial tumors. Archival tumor tissue
(formalin fixed slides or frozen tissue) was collected
for 15 astroglial cases with high exposure to prenatal,
dietary nitrites determined through in-person interviews
with the mother. Of the 15 samples, 6 anaplastic
astrocytomas [AA] and 7 pilocytic astrocytomas [PA]
contained tumor tissue suitable for analysis. Exons
5 through 8 of the gene were sequenced, revealing
2 mutations (1 AA and 1 PA ) out of 13 samples (15%).
The frequency of p53 mutations in our sample of
AA's (17%) was in the middle of the range reported
for these types of tumors in the literature (0-38%).
However, given the hypothesis, they would have predicted
that the frequency of p53 mutations among AA's with
high dietary exposure to nitrites would exceed 17%.
To their knowledge, p53 mutations have not previously
been reported for any cases of PA. |
| The pilot idea and preliminary results were presented
at the Childhood Brain Cancer Workshop in Minneapolis,
MN in July of 1999. At this meeting, the participants
agreed that the N-nitroso compound hypothesis remains
one of the most compelling potential explanations
of the etiology of childhood brain tumors and that
additional work is needed to understand the genetic
epidemiology of childhood brain tumors. |
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