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Pilot Projects
The NIEHS Center Pilot Project Program serves to assist faculty in the collection of sufficient data in order to become highly competitive for independent NIH funding. The goals of this Pilot Project Program are achieved by: providing support for short-term and preliminary research projects, especially in areas where future Center development is desired; exploring new areas of potential research interest to the Center; aiding new investigators with initial support to develop new lines of research; helping established investigators with support to explore new and innovative directions that represent a significant departure from their ongoing research; developing educational tools that enhance Community Outreach and Education; providing stimulation for PI's from other areas on campus to apply their expertise to environmental health research; and the encouragement of collaborative research projects.
Pilot Projects funded in 2006-2007
P.I. Name: John Sullivan, MA
Title: Project CEHRO: Community Environmental Health & Risk Outreach
Amount: $17,500
Abstract: CEHRO: Community Environmental Health & Risk Outreach
provides tools for citizens to comprehend public health risks posed by
Gulf Coast hurricane / tropical storm activity. Reentry / recovery
decisions informed by accurate information more effectively mitigate
adverse environmental health outcomes and promote more efficient public
health delivery. Goals: 1) Increase awareness of health risks from
severe wind / storm surge, protracted flooding to promote effective
action in future disasters; 2) Develop outreach template: overview of
public health threats that also incorporates risk from elements of local
economies, behavioral patterns and natural environments; 3) Evaluate /
refine process for use in additional Gulf Coast communities; 4) Identify
funding for expansion of CEHRO. Short Term Aims: 1) translate research
on environmental health threats and precautions, 2) communicate
applicable tox principles: monitoring, exposure assessment, dose /
health effects, epidemiology: health effects distribution,
susceptibility, vulnerable populations, 3) survey mold, pathogens,
chemical and associated waste detected post-Katrina (speciation / levels
/ projected effects), 4) show how community assets and cumulative risk
affect response, reentry / recovery , 5) produce “tox & risk” manual and
workshop / community Forum event in community, 6) evaluate efficacy via
survey. Design: network with community organizations / form advisory
committee, contact regional agencies for monitoring / exposure data,
specific precautions, incorporate local content in health overview,
recruit workshop participants from organizations and community, produce
community Forum. Methods / Techniques: workshop includes lecture-demo,
drama-based ethnography, risk perception sociometry, community asset
maps, case study, simulation of impact & counter-measures; Forum caps
process. Rationale: Methods, techniques, analogous content used
successfully by investigator in EPA, BAQC (Houston TX), regional
community Forums in Texas. Content / network, needs assessment developed
during NIEHS LA outreach (fall 2005) Public Health Relevance: Project
CEHRO addresses information needs and health issues identified by
community leaders in Louisiana / Texas areas impacted by Katrina & Rita.
Immediate risk of infectious disease, metals poisoning, petrochemical
sludge, plus potential long-term respiratory complications from dust
exacerbated an inherently difficult reentry process. CEHRO: Community
Environmental Health & Risk Outreach provides tools for citizens to
comprehend public health risks posed by Gulf Coast hurricane / tropical
storm activity. Reentry / recovery decisions informed by accurate
information more effectively mitigate adverse environmental health
outcomes and promote more efficient public health delivery. Goals: 1)
Increase awareness of health risks from severe wind / storm surge,
protracted flooding to promote effective action in future disasters; 2)
Develop outreach template: overview of public health threats that also
incorporates risk from elements of local economies, behavioral patterns
and natural environments; 3) Evaluate / refine process for use in
additional Gulf Coast communities; 4) Identify funding for expansion of
CEHRO. Short Term Aims: 1) translate research on environmental health
threats and precautions, 2) communicate applicable tox principles:
monitoring, exposure assessment, dose / health effects, epidemiology:
health effects distribution, susceptibility, vulnerable populations, 3)
survey mold, pathogens, chemical and associated waste detected
post-Katrina (speciation / levels / projected effects), 4) show how
community assets and cumulative risk affect response, reentry / recovery
, 5) produce “tox & risk” manual and workshop / community Forum event in
community, 6) evaluate efficacy via survey. Design: network with
community organizations / form advisory committee, contact regional
agencies for monitoring / exposure data, specific precautions,
incorporate local content in health overview, recruit workshop
participants from organizations and community, produce community Forum.
Methods / Techniques: workshop includes lecture-demo, drama-based
ethnography, risk perception sociometry, community asset maps, case
study, simulation of impact & counter-measures; Forum caps process.
Rationale: Methods, techniques, analogous content used successfully by
investigator in EPA, BAQC (Houston TX), regional community Forums in
Texas. Content / network, needs assessment developed during NIEHS LA
outreach (fall 2005) Public Health Relevance: Project CEHRO addresses
information needs and health issues identified by community leaders in
Louisiana / Texas areas impacted by Katrina & Rita. Immediate risk of
infectious disease, metals poisoning, petrochemical sludge, plus
potential long-term respiratory complications from dust exacerbated an
inherently difficult reentry process.
P.I. Name: Salama Salama, Pharm D, Ph.D.
Title: Estrogen Metabolites and Estrogen--Metabolizing Gene
Expression in Endometrium
Amount: $17,500
Abstract: Endometrial cancer ranks first in incidence and second
in mortality among gynecological malignancies. Risk factors for
endometrial cancer, including early menarche late menopause, obesity,
and estrogen replacement therapy, operate through estrogen-related
mechanisms. In situ estrogen biosynthesis, catalyzed by aromatase
(CYP19), plays an important role in the malignant transformation and the
promotion of endometrial cancer. The precise mechanism(s) of estrogen
carcinogenicity in the endometrium remain incompletely understood.
Although the common assumption is that estrogen carcinogenicity is
attributed to estrogen receptor-mediated hormonal activity, compelling
evidence suggests that cytochrome P450-mediated metabolic activation of
estrogen is involved. The initial step in estrogen metabolism involves
oxidation by cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) to produce
2-hydroxycatechol estrogen (2-OHE2) and 4-hydroxycatechol estrogen
(4-OHE2), respectively. While 2-OHE2 has antiestrogenic, antiangiogenic,
and antiproliferative effects in various tumor cells, 4-OHE2 is a potent
pro-estrogenic and carcinogenic estrogen metabolite. 4-OHE2 can be
further metabolized into 3,4-quinone with simultaneous production of
reactive oxygen species, which are endogenous initiators of many human
cancers. In extrahepatic tissues, the catechol estrogens are detoxified
primarily by O-methylation catalyzed by catechol-O-methyltransferase (COMT).
Catechol estrogen quinones can be reduced back to catechol estrogens by
NADPH quinone oxidoreductase 1 (NQO1). Thus, COMT and NQO1 are key
enzymes which inactivate carcinogenic estrogen metabolites. Our
preliminary data and recent publications demonstrate that genes involved
in the formation and detoxification of catechol estrogens are expressed
in the endometrial tissues. In addition, we demonstrated that estrogen,
progesterone, and 4-hydroxytamoxifen modulate the expression of CYP1A1,
CYP1B1, and COMT genes in various female’s reproductive tissues. We
hypothesize that regulation of the expression of the genes involved in
estrogen biosynthesis and metabolism is instrumental in the pathogenesis
of endometrial cancer. Endometrial cancer develops as a result of
increased in situ estrogen biosynthesis with concomitant imbalances in
the relative expression of critical enzymes involved in estrogen
activation and detoxification in the endometrial tissues. This imbalance
in estrogen-metabolizing enzymes results in the formation and accretion
of high level of 4-OHE2, which can directly or indirectly induce
mutations and genomic instabilities in critical genes such as oncogenes
or tumor suppressor genes. These genetic effects can cause phenotypic
changes indicative of neoplastic transformation in endometrial tissues.
To test this hypothesis, we propose the following three specific aims:
1) to examine whether the expressions of the critical enzymes involved
in estrogen biosynthesis (CYP19); activation (CYP1A1, and CYP1B1); and
detoxification (COMT and NQO1) are altered in endometrial cancer tissues
compared with normal endometrium. 2) To assess the capability of
estrogen, 2-OHE2 and 4-OHE2 to induce microsatellite instabilities and
mutations in the tumor suppressor genes (PTEN and TP53), and K-RAS
oncogene in the normal endometrial cells. Furthermore, we will assess
the ability of estrogen and its catechol metabolites to induce
phenotypic changes indicative of the malignant transformation in normal
endometrial cells. 3) To investigate the transcription regulation of
genes involved in biosynthesis (CYP19); activation (CYP1A1 and CYP1B1);
and detoxification (COMT and NQO1) of estrogen in normal endometrial
cells and in a panel of cell lines representative of different stages
and grades of endometrial cancer. Particularly, we will study the effect
of estrogen; progesterone; and tamoxifen on the relative expression of
CYP19, CYP1A1, CYP1B1, COMT, and NQO1 as a potential mechanism whereby
these drugs modify the individual risk for endometrial cancer. The
proposed study will advance our understanding of the pathobiology of
endometrial cancer. Most importantly, understanding the transcription
regulation of genes involved in steroidogenesis or estrogen metabolism
is fundamental to design rational chemopreventive approaches which
attenuate the in situ estrogen biosynthesis and redirect estrogen
metabolism into a non-carcinogenic pathway. In addition, this invaluable
knowledge will have a major impact on several other estrogen-related
malignancies and will lead to a significant improvement in women’s
health.
P.I. Name: Sherif Abdel-Rahman, Ph.D.
Title: Biological and Functional Significance of Polymorphisms in
the MGMT Gene
Amount: $17,500
Abstract: Exposure to methylating agents endogenously through
cellular pathways, or exogenously through environmental exposure,
results in the formation of the highly mutagenic O-6-methylguanine
(O6-meG) lesion. This DNA lesion is repaired by the direct reversal
repair protein O6-Methylguanine-DNA-Methyltransferase (MGMT). Several
single nucleotide polymorphisms (SNPs) in the coding and in the
enhancer-promoter region of the MGMT gene have been reported. While
recent epidemiological studies document associations between these SNPs
and risk for environmental cancers, their functional significance and
their influences on genetic damage induced by methylating agents has not
been systematically characterized. We hypothesize that SNPs in the MGMT
gene alter transcriptional levels and/or the function of the resulting
MGMT protein. This will, in turn, lead to altered repair capacity and
subsequent genetic damage and mutations in individuals exposed to
alkylating agents such as those present in tobacco smoke. To determine
the functional significance of the SNPs in the coding region of the gene
resulting in amino acid changes (cSNPs), we will generate reference and
three variant forms of the MGMT gene corresponding to the L84F, the
I143V and the K178R cSNPs using site-directed mutagenesis and cloning.
Clones will be expressed in MGMT-null Chinese hamster ovary (CHO) cells.
A novel fluorescent-based assay will be used to determine the activity
of the expressed proteins and to examine the hypothesis that the variant
MGMT proteins have reduced capacity to repair O6-meG compared to the
reference protein. To determine changes in promoter activity due to the
SNP in the enhancer region of the gene, we will generate a luciferase
reporter vector under the control of the minimal reporter and enhancer
region of MGMT to examine the hypothesis that the SNP in the enhancer-promotor
region alters baseline promotor activity. To characterize the biological
significance of MGMT polymorphisms, we will use human lymphocytes,
collected through a currently funded project, with one of each of the
three cSNPs that result in amino acid changes or the SNP in the enhancer
region of the MGMT gene. These lymphocytes with the predetermined SNPs
in the MGMT gene will be challenged in vitro with
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a model alkylating
agent. The extent of genetic damage will be determined using the HPRT
cloning gene mutation assay to examine the hypothesis that individuals
with these SNPs in the MGMT gene are more sensitive to the mutagenic
effects of alkylating agents than homozygous reference individuals. Our
study will provide novel mechanistic explanations regarding SNPs in a
key DNA repair gene. This new information will have significant
translational implications in the area of public health and in clinical
settings.
P.I. Name: Bhupendra Kaphalia, Ph.D.
Title: Plasma Lipidomic and Proteomic Changes in Alcohol Liver
Disease
Amount: $17,500
Abstract: Alcoholic liver disease (ALD) is a major health problem
among chronic alcohol abusers and second major cause of deaths from all
the liver diseases. Although a variety of agents or conditions,
including alcohol abuse, cause fatty liver (an early stage and
reversible stage of ALD), subsequent progression to advance and
irreversible stages of ALD (hepatitis, fibrosis and cirrhosis) appears
to be similar, regardless of etiology. The mechanism(s) and metabolic
basis of ALD are not well understood primarily due to the lack of
suitable animal model. The specific and sensitive biomarkers are also
not available for the early detection of ALD. The fatty liver stage thus
offers potential avenues to understand the mechanism(s) and identify
specific and sensitive molecular targets of alcohol abuse and possible
treatment of the ALD. Chronic alcohol abuse impairs hepatic alcohol
dehydrogenase (ADH)-dependent oxidative metabolism of ethanol
disposition (major pathway) and shifts ethanol metabolism predominantly
to nonoxidative pathway. Fatty acid ethyl esters (FAEEs) and
phosphatidylethanol (PEt) are nonoxidative metabolites of ethanol and
found predominantly in the plasma of chronic alcohol abusers. In
preliminary studies, we have found several-fold higher levels of FAEEs
in the plasma of chronic alcohol abusers than those in acutely
intoxicated with alcohol. The hepatocellular toxicity of FAEEs has been
reported in experimental animals and cell culture studies. Based on
common occurrence of fatty liver and nonoxidative metabolites of ethanol
in chronic alcohol abusers, we hypothesize that nonoxidative metabolism
of ethanol and resulting altered lipid homeostasis are the key factors
involved in alcoholic liver injury. Therefore, focus of this pilot
project is to measure altered plasma lipid and protein profiles to
identify specific and sensitive molecular targets and understand the
mechanism(s) and metabolic basis of alcoholic fatty liver. Our results
in this project should also lay the foundation for the development of
biomarkers and new therapies for ALD. The data generated from this
project will be utilized for a translational grant on development of
biomarker(s) of ALD for NIH funding.
P.I. Name: Varatharasa Thiviyanathan, Ph.D.
Title: Structural Basis for Removal of 5-hydroxy Uracil by Human
NEIL2
Amount: $17,500
Abstract: Oxidative DNA damage has been implicated in a variety
of diseases including cancer arthritis and neurodegenerative diseases.
In aerobic organisms, GC to AT transition mutation is the most
frequently observed point mutation resulting from oxidative damage.
Several studies have shown that the oxidized cytosine products are the
major chemical precursors for this transition mutation. NEIL2 is a
recently identified human DNA glycosylase that recofnizes and repair the
oxidized cytosine damages in DNA. Among its substrates, NEIL2 shows
highest activity for 5-hydroxy uracil (5-OH-U). Structure and stability
studies of DNA duplexes containing 5-OHU damages showed that the
presence of 5-OHU residues does not cause significant perturbations at
or near the lesion site in the duplex DNA. These observations raise the
question- how does NEIL2 recognize the damaged residues among the vast
number of normal residues? We will test whether the hydroxyl group at
the 5th position, present in all oxidized cytosine products and lies in
the major grove of the DNA, serves as a marker recognized by the enzyme.
There is no three-dimensional structure of human NEIL2 and no structure
of an enucaryotic Nei protein in complex with damge-containing DNA.
Using homology modeling based on the E.coli structure and site directed
mutagenesis, key residues of NEIL2 required for the substrate binding
and catalytic activity will be identified. Our model and proteolytic
digestion of NEIL2 showed the presence of two distinct domains, - a 198
residue N-terminal domain and a 134 residue C-terminal domain. Structure
of these two domains will be determined by NMR spectroscopic methods.
Our long-term goal is to determine the structure of the full length, 37
KDa NEIL2 and its complex with DNA duplex, to understand how NEIL2
recognizes, binds and repair the oxidative cytosine lesions in DNA
duplex. Successful completion of these studies should provide a picture
of the recognition and repair mechanism of NEIL2 for the removal of
5-OHU and other Cytosine-derived lesions from duplex DNA.
Pilot Projects funded in 2005-2006
P.I. Name: Roberto Garofalo
Project Title: Genetics and Environmental Risk
Factors of Viral Bronchiolitis
Amount Awarded: $17,500
Abstract: The primary goal of this pilot project is to gather
essential preliminary data in support of two separate NIH R01
applications planned for submission within the next twelve months. The
scope of the proposed work addresses the relevant scientific topics of
the Asthma Pathogenesis Core of the NIEHS Center at UTMB, including
examining the effect of combined viral-toxicants exposure and the
application of proteomics technology to investigate airway inflammation.
Epidemiologic studies support the notion that exposure to environmental
tobacco smoke (ETS) is associated with an increased frequency of lower
respiratory tract illness, otitis media, and asthma exacerbations in
children, as well as chronic bronchitis and asthma in adults. It is well
known that these diseases of the respiratory tract are primarily caused
or triggered by viral infections. Respiratory syncytial virus (RSV) is
the single most important viral pathogen causing acute respiratory-tract
infections in infants and children. Severe RSV infections in infancy (bronchiolitis)
have been linked to both the development and the severity of chronic
asthma. Exposure to ETS may occur in up to 60% of the infants with RSV
bronchiolitis in the US, and different studies have suggested that ETS
is a risk factor for the development of severe infection. However,
studies reporting quantifiable measures of exposure to tobacco smoke in
RSV-infected infants have not been performed. Glutathione S-transferases
(GSTs) are cytosolic enzymes involved in major detoxification pathways
of polycyclic aromatic hydrocarbons, including reactive tobacco
metabolic intermediates. The gene locus is polymorphic and certain null
alleles are associated with a complete lack of the enzyme. We
hypothesize that exposure to ETS exacerbates viral-induced airway
disease by enhancing or modifying the pattern of production of
inflammatory cytokines, protein mediators and/or causing oxidative
injury. Moreover, we hypothesize that the effect of ETS on severity of
viral infection is linked to certain GST genotypes, such as the GST M1
null genotype. We propose to conduct a clinical study in which we will
analyze samples of nasal secretions collected from young children with
RSV infections of different severity, who may have been exposed to ETS.
Exposure to ETS will be confirmed by a specific test of the patient’s
urine. Using state-of-the art technology, generally called “proteomics”,
our studies will allow for the first time examining the expression of
protein mediators in airway secretions at a more “global” level and to
discover whether specific protein “signatures“ are associated with
different clinical forms of RSV infection exposure to ETS, and/or GST
genotype. Ultimately, our investigations may identify new strategies to
diagnose, prevent and treat respiratory viral infections as well as
other respiratory diseases that are caused, exacerbated or modified by
exposure to ETS.
P.I. Name: Terumi Midoro-Horiuti
Project Title: Effects of Environmental Estrogens on Allergic
Sensitization and Reaction
Amount Awarded: $17,500
Abstract: Contamination of human milk is widespread due to
inadequately controlled pollution of the environment. Lipid-soluble
pollutants tend to degrade slowly in the environment, to bioaccumulate
and bioconcentrate in the food chain and to have long half-lives in
humans. The high fat content of milk leads to concentrations of some of
the pollutants in breast milk up to 100 times higher than plasma level.
Some of these lipophilic pollutants have estrogen-like activities and
are termed environmental estrogens. Some reports show the positive
relationship between breast feeding and the prevalence of allergic
diseases. We found that exposure to estradiol strongly potentiates the
allergic response in the rat basophilic cell line acting through a
membrane estrogen receptor. The long-term goal of our research is to use
a comprehensive approach to identify possible pathogenic roles for
estrogens or environmental estrogens in developing allergic airway
diseases and to devise ways to interrupt this process.
The general hypothesis to be tested in this project is that
environmental estrogens can modulate the ability of mast cells/basophils
to promote allergic sensitization/reactions. These altered responses may
be due to direct effects of the environmental estrogens or due to
enhancement or antagonism to mother’s endogenous estrogens in breast
milk would reduce the allergic response. We will test the specific
hypotheses that:
1) Environmental estrogens can modulate allergic reactions by
potentiating the release of allergic mediation by IgE cross-linking, and
2) Environmental estrogens can alter allergic sensitization by
modulating the potential of mast cells to present antigens to T cells.
P.I. Name: Varatharasa Thiviyanathan
Project Title: Structural Studies of Human NEIL2
Amount Awarded: $17,500
Abstract: The goals of this pilot project are to study the
structural features of the oxidatively damaged DNA duplexes and the DNA
binding domain of the human NEIL2, a DNA glycosyalse that is active on
oxidative cytosine lesions in DNA. The overall goal of this project is
to study the repair mechanism of NEIL2, and to understand how this DNA
repair enzyme recognizes the damaged sites in the DNA duplex. Oxidized
cytosine products are the major chemical precursors to the GC to AT
transition mutations. Among the oxidized cytosine products, 5-hydroxy
uracil (5-OH-U) shows the highest mutation frequency. NMR spectroscopy,
UV-monitored melting experiments, and ab-initio calculations will be
used to study the structure, stabilities and backbone dynamics of DNA
duplexes containing 5-OH-U mismatches. Since preliminary results show
that the structure and dynamics of a DNA duplex containing 5-OH-U:A
duplex are so similar to those of a normal DNA duplex, the recognition
mode of NEIL2 remains questionable. NEIL2 shows highest activity for
5-OH-U. The DNA binding domain of NEIL2 that recognizes the 5-OH-U
lesions in the DNA duplex will be identified by site-directed mutagenic
studies and homology modeling based on the known structures of E.coli
homologs. Using labeled proteins and unlabeled DNA duplexes,
interactions between the damaged DNA and NEIL2 will be probed by NMR
spectroscopy.
Publication:
Thiviyanathan, V., Somasunderam, A., Volk, D.E., and Gorenstein, D.G.
2005, 5-Hydroxy Uracil can form stable base-pairs with all four bases in
a DNA duplex. Chemical Communication 3: 400-402.
P.I. Name: Jingwu Xie
Project Title: Activation of the Hedgehog Pathway in
Gastroesophageal Cancers
Amount Awarded: $17,500
Abstract: Gastrointestinal (GI) cancers (including esophageal,
gastric, pancreatic, billary, liver and colon cancer) are among the most
common causes of cancer death worldwide. In the United States, the
incidence of esophageal adenocarcinomas and liver cancers has nearly
quadrupled over last decade. Most of these cancers remain clinically
silent until late in the disease process. Thus, these cancers are often
associated with late diagnosis, poor prognosis and significant
morbidities and a high mortality rate. Lack of specific and sensitive
biomarkers for diagnosis is a major obstacle for early detection of the
primary tumor. Studies from several laboratories, including ours,
indicate that the hedgehog pathway is frequently activated in GI cancers
(except for colon cancer). Based on our preliminary data, we hypothesize
that the cancer stem cells of several types of GI cancers (including
esophageal, gastric, pancreatic and liver cancers) contain activated
hedgehog signaling. Thus, hedgehog signaling activity can be used to
detect GI cancer early. Furthermore, we hypothesize that monitoring
hedgehog signaling activity can be used to assess chemo-drug efficacy
during cancer treatment. To test our hypothesis, we have proposed the
following specific aims: 1) Assess the frequency of hedgehog signaling
activation in surgically removed GI tumor specimens (esophageal cancer,
gastric cancer, pancreatic cancer and liver cancer); 2) Early detection
of GI cancers using hedgehog signaling activity in fine needle
aspiration specimens.
Already, we have submitted a manuscript on hedgehog signaling in GI
cancers, which will not be possible without support of ES06676.
Publications:
Sheng T, Chi S, Zhang X and Xie J. 2006, Regulation of Gli1
localization by the cAMP/ PKA signaling axis through a site near the
nuclear localization signal. J. Biological Chemistry 281(1):9-12 [Epub
2005 Nov. 17]
Wickliffe JK, Galbert LA, Ammenheuser MM, Herring SM, Xie J, Masters OE,
Friedberg EC, Lloyd RS, Ward JB. 2006, 3,4-Epoxy-1-butene, a reactive
metabolite of 1,3-butadiene, induces somatic mutations in Xpc-null mice.
Env. Molec. Mutagen. (47):67-70.
Pilot Project Archives
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