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2001 Science Fair  |
The 2001 Superfund
Basic Research Program and Southwest Environmental Health Sciences
Center Annual Science Fair was held on April 27th. There were 54
posters on display during the poster session. This was followed
by a presentation by guest speaker, F. Peter Guengerich, Ph.D.,
entitled, "Reactive Intermediates in Toxciology: Past, Present,
and Future". Dr. Guengerich is the Director of the Center in Molecular
Toxicology at Vanderbilt University School of Medicine, Nashville,
Tennessee.
Poster Abstracts
Variation
in N-Acetyltransferase 2 Activity and 4-Aminobiphenyl Genotoxicity.
CA
McQueen1, B Chau1, RP Erickson1,
RB Tjalkens2 and MA Philbert2. 1University
of Arizona, Tucson, AZ; 2 University of Michigan, Ann
Arbor, MI.
N-Acetyltransferases
catalyze the acetylation of aromatic amines. There are two genes,
NAT1 and NAT2. In humans, polymorphisms in both genes
result in phenotypic variation. Low NAT2 activity is associated
with increased risk of arylamine-induced urinary bladder cancer
and with higher levels of carcinogen-hemoglobin (Hb) adducts. The
hypothesis that lower NAT activity increases the genotoxicity of
aromatic amines was investigated in mice. C57Bl/6J mice are rapid
acetylators with the NAT2*8 allele. A/J mice, slow acetylators,
have the NAT2*9 allele and the congenic B6.A strain has the
NAT2*9 allele on the C57Bl/6J background. All three strains
have the same NAT1 . Adult male mice received a single oral
dose of 120 mg 4-aminobiphenyl (4ABP)/kg or corn oil. After 24h,
livers were harvested and frozen. Histological sections (4-5μm)
were prepared and 4ABP-DNA adducts detected by immunofluoresence.
Increased nuclear fluorescence was observed in all three strains.
There was no significant variation by strain. Hepatic NAT activity
was measured with the murine NAT2 selective substrate p-aminobenzoic
acid (PABA) and with 4ABP. Hepatic PABA NAT activity in C57Bl/6J
was 7.74±0.13 nmol acetylPABA/min/mg, 3.05±0.58 for A/J and 4.19±0.58
for B6.A. No significant differences were observed in 4ABP NAT activity.
Values ranged from 1.1±0.34 to 1.52±024 nmol acetylABP/min/mg. NAT2
genotype correlated with phenotype for PABA acetylation, but there
was no phenotype-genotype correlation with 4ABP. The lack of strain
variation in 4ABP NAT activity was consistent with the comparable
4ABP-DNA adduct levels seen in the three strains. The association
between NAT2 phenotype and 4ABP-Hb adducts in humans was not seen
in mice. Substrate selectivity and amino acid sequence support the
functional analogy of human NAT2 with murine NAT1 and vice versa.
The contribution of murine NAT1 to 4ABP genotoxicity remains to
be elucidated. (ES09812, ES10047 (CAM) and ES08846 (MAP)).
Assessing the Role of Ovarian GSH Levels in Ovotoxicity
in Rats.
PJ
Devine1, IG Sipes2,3, and PB Hoyer1.
Department of 1Physiology, and 2Pharmacology
and Toxicology, and 3Center for Toxicology, The University
of Arizona, Tucson, AZ, USA.
The
diepoxide metabolite (VCD) of the occupational chemical 4-vinylyclohexene
is ovotoxic in rats following repeated exposures. Repeated daily
dosing with VCD for 15d destroys the smallest ovarian follicles.
Since VCD acutely reduced hepatic levels of the antioxidant, glutathione
(GSH), these studies address if reduced GSH levels are involved
in the destruction of ovarian follicles. Thus, immature female Fischer
344 rats (n=3-12 per group) were dosed once or daily for 15d with
VCD (0.57mmol/kg, ip) or the GSH synthesis inhibitor buthionine
sulfoximine (BSO, 2mmol/kg). Animals were killed 2, 6, or 26h following
a single dose, and 2 or 26h following 15d of dosing. Liver and ovarian
tissues were collected for determination of GSH levels by HPLC.
Also, ovaries were collected following repeated dosing and histologically
processed for counting follicles. Following a single dose, both
VCD (51±5% of control) and BSO (42±9% of control) reduced (p<0.05)
hepatic GSH within 2h, but only BSO reduced ovarian GSH (64±5% of
control at 6h, p=0.05). Within 26h, liver and ovarian GSH levels
had returned to control levels with either treatment. Two h after
the 15th dose, BSO (37±5% of control) and VCD (76±4% of control)
reduced hepatic GSH levels (p<0.05), but only BSO decreased ovarian
GSH (60±3% of control). GSH levels in 15d tissues were similar to
controls 26h after the final dose. Oxidized GSH levels were not
affected by BSO or VCD at any time point, suggesting these treatments
did not induce oxidative stress. Ovarian small follicle numbers
were reduced (p<0.05) in 15d VCD-treated rats, whereas BSO did
not affect follicle numbers, even though BSO reduced ovarian GSH
content. Thus, these results support the conclusion that alterations
in GSH levels within small ovarian follicles is not involved in
VCD-induced ovotoxicity, though localized effects within target
follicles cannot be excluded. (ESO06694, ESO8979)
Ovotoxicity Induced in Rats By 4-Vinylcyclohexene
Diepoxide is Associated with Expression and Redistribution of Cellular
BCL-2 Family Members.
Xiaoming
Hu1, Patty Christian1, I Glenn Sipes2,
3, and Patricia B Hoyer1,3. Department of
1Physiology and 2Pharmacology and Toxicology,
3Southwest Environmental Health Sciences Center, University
of Arizona, Tucson, AZ
Previous
studies have shown that ovotoxicity induced in rats by dosing with
4-vinylcyclohexene diepoxide (VCD) is likely via acceleration
of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity
is specific for small pre-antral follicles and is associated with
increased activity of caspase cascades. The present study was designed
to investigate the alteration of expression and distribution of
Bcl-2 family member proteins induced by the treatment of VCD in
rat small ovarian follicles. Female F344 rats were given a single
dose of VCD (80 mg/kg, i.p., 1d; a time when ovotoxicity is not
initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15d; a time
when significant ovotoxicity is underway). Four hours following
the final dose, livers and ovaries were collected. Ovarian small
(25-100 m m) and large (100-250 m m) pre-antral follicles
were isolated, and sub-cellular fractions (cytosolic and mitochondrial)
were prepared. Compared with controls, levels of the pro-apoptotic
protein, Bad, were greater in both cytosolic and mitochondrial fractions
of small pre-antral follicles collected from 15d VCD-treated rats
(cytosol, 1.97± 0.16; mitochondria, 2.20 ± 0.24, VCD/Control,
p<0.05). After 15 days of daily VCD-dosing, total cellular anti-apoptotic
Bcl-xL protein levels were unaffected in small pre-antral
follicles, but its distribution in mitochondrial and cytosolic components
was altered (mitochondria, 0.635± 0.08; cytosol, 1.39±
0.14, VCD/Control, p<0.05). Likewise, VCD did not affect protein
levels of pro-apoptotic Bax in small follicles on d15. However,
consistent with a Bax-mediated mechanism of apoptosis, the relative
ratio of Bax/Bcl-xL in the mitochondrial fraction of
small pre-antral follicles was significantly increased by VCD dosing
(1.62 ± 0.21, VCD/control, p<0.05). Immunofluorescence staining
intensity evaluated by confocal microscopy visualized cytochrome
c protein in the cytosolic compartment in granulosa cells of pre-antral
follicles in various stages of development. Relative to controls,
within the population of small pre-antral follicles, staining intensity
was less (p<0.05) and presumably more diffuse specifically in
stage 1 primary follicles from VCD-treated animals (15d). VCD caused
none of these effects in large pre-antral follicles or liver (not
targeted by VCD). These data provide evidence that apoptosis in
rats (15d of dosing) induced in ovarian small pre-antral follicles
by VCD is associated with increased expression of Bad protein, redistribution
of Bcl-xL protein and cytochrome c from the mitochondria
to the cytosolic compartments, and an increase in the Bax/Bcl-xL
ratio in the mitochondria. These observations are consistent with
the involvement of the Bcl-2 family of genes in VCD-induced acceleration
of atresia.
Alpha-Napthoflavone,
an Aryl Hydrocarbon Receptor Antagonist, Reverses 4-Vinylcyclohexene
Diepoxide-Induced Ovotoxicity in F344 Rats.
K
E Thompson1, I G Sipes2,3, and P B Hoyer1,3.
Departments of 1Physiology and 2Pharmacology
and Toxicology, 3Southwest Environmental Health Sciences
Center, University of Arizona, Tucson, AZ, USA.
Repeated
dosing of rats and mice with 4-vinylcyclohexene diepoxide (VCD)
causes loss of ovarian small preantral follicles. VCD increases
expression of the aryl hydrocarbon receptor (AhR) in small preantral
follicles, but whether this is associated with VCD-induced ovotoxicity
is not known. Thus, this study was designed to investigate the effect
of the AhR antagonist, alpha-napthoflavone (ANF), on VCD-induced
ovotoxicity. F344 rats (d28; n=8-9 rats/treatment) were dosed daily
for 15d with either vehicle control (sesame oil; i.p.), VCD (80
mg/kg), ANF (20 or 80 mg/kg), or VCD and ANF. Ovaries were collected,
prepared for histological evaluation, and assessed for follicle
counts. Treatment with VCD destroyed (p<0.05) a significant number
of primordial (60.6% of control) and primary (64.0% of control)
follicles. Concurrent treatment with VCD and low dose ANF (20 mg/kg)
also resulted in a significant loss of primordial follicles (79.5%
of control; p<0.05); however the high dose of ANF (80 mg/kg)
prevented VCD-induced primordial follicle loss (93.8% of control).
Interestingly, following treatment with the high dose ANF alone,
there were greater numbers of primordial follicles as compared to
control (40.5% above control, p<0.05). As with primordial follicles,
dosing with high dose ANF and VCD protected primary follicles from
ovotoxicity (93.3% of control) and high dose ANF treatment alone
resulted in increased numbers of primary follicles as compared to
control (28.1% above control, p<0.05). Secondary follicle numbers
were unaffected by any treatment. These data show that the AhR antagonist,
ANF, can prevent primordial and primary follicle loss induced by
VCD. Additionally, ANF appears to delay the normal rate of atresia
that occurs in primordial and primary follicles during the dosing
period. These results suggest that the AhR maybe involved in the
physiological control of atresia as well as VCD-induced ovotoxicity.(ES09246)
Effect
of 4-Vinylcyclohexene and its Diepoxide Metabolite on Ovarian Expression
of Microsomal Epoxide Hydrolase in B6C3F1 Mice.
EA
Cannady1, RL Wade3, CA Dyer3, IG
Sipes1, and PB Hoyer2. Departments
of 1Pharmacology/Toxicology and 2Physiology,
The University of Arizona, Tucson, Arizona, USA; 3Department
of Biological Sciences, Northern Arizona University, Flagstaff,
Arizona, USA.
Repeated
daily dosing with 4-vinylcyclohexene (VCH), an industrial chemical,
causes irreversible destruction of ovarian small follicles in mice.
Furthermore, the diepoxide metabolite, VCD, is responsible for this
follicle loss. In vivo, VCD is detoxified by metabolic enzymes
such as microsomal epoxide hydrolase (mEH). The purpose of this
experiment was to investigate whether detoxification of VCD could
occur within the mouse ovary. Therefore, mEH expression in
isolated ovarian fractions from B6C3F1 mice was evaluated
(8 animals/group;n=2) and the effects of dosing with VCH or VCD
were determined. Female B6C3F1 mice (d 28) were given
a single dose or dosed daily (15 d) with VCH (800 mg/kg;i.p.) or
VCD (80 mg/kg;i.p.). Ovaries were removed and follicle fractions
were isolated (Fraction 1/F1, small pre-antral follicles, 25-100 m m; Fraction 2/F2, large pre-antral follicles, 100-250 m
m; Fraction 3/F3, antral follicles, >250 m m). Total RNA
was prepared from the follicles and analyzed by RT-PCR using a real
time lightcycler. mRNA encoding mEH was expressed in all
follicle types. In F1 follicles (those targeted by VCH and VCD)
following a single dose, expression of mEH was increased
by VCH (322%) and VCD (136%). After 15 d of dosing, expression was
increased 530% by VCD, but was largely unaffected by VCH. Following
1 or 15 daily doses with VCH or VCD, expression in non-target fractions
(F2,F3) was not as greatly affected as in F1. Based on steady state
mRNA, expression of mEH is likely most increased in F1 follicles
following a single dose of VCH or 15 doses of VCD. The greater response
in F1 follicles suggests that these selective targets of ovotoxicity
may be those most directly involved in the detoxification of VCD.
Future studies will determine whether this effect is reflected in
mEH enzyme activity.(ES09246,ES06694,ES07019)
Effects
of Alkylating Agents on SUMO-1 Protein Conjugation in HEK 293 Cells.
Manza,
L. L. and Liebler, D. C. Southwest Environmental Health Sciences
Center, College of Pharmacy, University of Arizona, Tucson, AZ 85721.
The
small ubiquitin-related modifier, SUMO-1 is 101 amino acid ubiquitin-like
protein that follows a conjugation pathway similar to ubiquitin,
but utilizes SUMO specific enzymes. Unlike ubiquitination, which
targets proteins for proteasomal degradation, sumoylation exerts
a variety of effects, such as the nuclear localization of RanGAP1,
the regulation of ubiquitin conjugation of IkB and the transcriptional
activity of p53. Additionally, other studies have demonstrated that
stress conditions such as heat shock, UV, alkylating agents, topoisomerase
inhibitors, and H2O2, increase the levels of SUMO-1 conjugates in
cells. We hypothesize that protein damage by alkylation or oxidation
triggers sumoylation. To investigate the effects of protein alkylation
on SUMO-1 conjugation HEK 293 cells were treated with iodoacetamide,
hydroquinone, benzoquinone, 4-hydroxynonenal, hydrogen peroxide,
and Texas Redâ C5 bromocetamide at doses of 10, 50 and 100
mM or equal volumes of vehicle (water/DMSO/ACN) at 37°C and 95%
air/5% CO2. After 2 hours, the medium was removed and cells were
either trypsinized for determination of viability using Trypan Blue
exclusion or lysed for protein assay and western blot analysis.
Cell viability decreased in a dose dependent mannner for all treatment
groups compared to vehicle controls. Western blots indicated that
formation of the 90 kDa RanGap1 SUMO-1 conjugate as well as higher
molecular weight SUMO-1 conjugates increased with dose while the
low molecular weight SUMO-1 monomer appeared to decrease. The results
suggest that alkylating agents and oxidants stimulate symoylation
of multiple proteins. Identification of chemically modified and
sumoylated proteins is in progress. (Supported in part by NIH grants
ES06694 and ES10056.)
Identification
of Protein Targets and Metabolites of 1,1-Dce in Bile by ESI-MS-MS.
Jones,
J.A.1, Kaphalia, L., Moslen, M.T.2 and Liebler,
D.C.3 Department of Chemistry1 and
Southwest Environmental Health Sciences Center2, College
of Pharmacy, University of Arizona, Tucson, AZ, 85721 and Department
of Pathology2, Medical Branch, University of Texas, Galveston,
TX 77555.
The
hepatotoxin 1,1-dichloroethylene (DCE) undergoes bioactivation to
form 1,1-dichloroethylene epoxide and 2-chloroacetyl chloride. The
latter can covalently bind to glutathione to form S-(2-chloroacetyl)
glutathione, which alkylates cysteinyl sulfhydryls to form GSCOCH2-S-cys-protein
adducts. The epoxide instead forms S-carboxymethylcysteine adducts.
Selective damage to the biliary canalicular membrane is observed
in animals upon exposure to DCE. We hypothesize that this damage
results from alkylation of critical protein targets associated with
the canalicular membrane by electrophilic metabolites of 1,1-DCE.
To date, however, the identity of the protein targets and the site
of modification remain speculative. We are developing a novel, proteomics-based
approach to identify protein targets of xeniobiotics that utilizes
ESI-MS-MS in combination with the data reduction algorithms SALSA,
developed by our research group, and SEQUEST. Model S-carboxymethylated
and GSCOCH2-S-cys-peptide adducts were synthesized
and their MS-MS fragmentation patterns characterized. Fragment ions,
losses from parent ions and ion pairs were identified that corresponded
to the modifications. Rats received 50 m g/kg p.o. of 1,1-DCE
and bile was collected at 15 min intervals for 3.5 h after exposure.
The samples were analyzed by ESI-MS-MS with data dependent scanning
and by SALSA, which identifies MS-MS spectra containing specified
fragmentation patterns. Use of SALSA in combination with Sequest
and de novo sequencing methods has led to the verification
of DCE-metabolites, including a hypothesized cyclic degradation
product of S-(2-chloroacetyl) glutathione not previously
observed in vivo. In addition, a carboxymethylated peptide
was tentatively identified as the C-terminal fragment of phospholipase
A2. This data demonstrates for the first time the ability to locate
small amounts of adducted proteins in complex samples by using MS-MS
fragmentation patterns. This work was supported by NIH grants ES06694
and ES 10056.
Identification
of a PPAR-Responsive Element in the BCL-2 Gene.
B
D Butts, M M Briehl. Department of Pathology, University
of Arizona, Tucson, AZ.
Apoptosis
is a highly regulated cellular process, which, when deregulated,
can lead to many adverse pathologies, including cancer. The transcriptional
regulation of genes which can promote or inhibit apoptosis is one
important mode of apoptotic regulation. One such gene is bcl-2,
whose protein product inhibits apoptosis. In comparison to the extensive
research on the mode of action of Bcl-2, the transcriptional regulation
of bcl-2 is not as comprehensively studied. Peroxisome Proliferator
Activated Receptors (PPARs) are nuclear receptors that have been
known to regulate genes related to lipid metabolism. Recent work
has shown that PPARs may also regulate genes in such fields as carcinogenesis
and apoptosis. In this study, we characterize the functionality
of a PPAR response element (PPRE) found 3' of the coding region
in the human bcl-2 gene. In gel shift experiments, oligos
containing the PPRE from bcl-2 showed a decreased mobility
when incubated with in-vitro translated PPAR gamma, while a mutated
version did not. Reporter assays further demonstrated the functionality
of the bcl-2 PPRE. Cells co-transfected with PPAR gamma and
a bcl-2 PPRE-containing luciferase plasmid showed a 3.5-fold
higher activity when compared to cells co-transfected with the bcl-2
PPRE luciferase plasmid and the empty parent vector of PPAR gamma
(pSG). Interestingly, when cells were transfected with pSG, Northern
blot analysis of bcl-2 showed the presence of the inactive,
3.5 kb bcl-2 beta message. However, when cells were transfected
with PPAR gamma, the 3.5 kb message was absent, while the active,
5.5 kb bcl-2 alpha message was present. Finally, HCA-7 colon
cells transfected with PPAR gamma showed decreased amounts of apoptosis
induced by the bile acid deoxycholic acid, compared to cells transfected
with pSG.
Adaptation
to Oxidative Stress affects Apoptotic Signalling in Dexamethasone-Induced
Apoptosis.
M
M Briehl1, M E Tome1 and E L Jacobson2.
Departments of Pathology1 and Pharmacology and Toxicology2.
University of Arizona. Tucson, AZ, USA
In
this study, we investigated whether glucocorticoid-induced apoptosis
of the WEHI7.2 mouse thymoma cells is subject to redox regulation.
Shortly after treatment with dexamethasone, a synthetic glucocorticoid,
WEHI7.2 cells show an increased production of reactive oxygen species.
This increase occurs prior to increases in phosphatidylserine externalization,
often considered as early event in apoptosis, and well before significant
release of cytochrome c from the mitochondria into the cytosol,
considered the committed step in this pathway for apoptosis. WEHI7.2
variants with an enhanced antioxidant defense through selection
for resistance to hydrogen peroxide or overexpression of catalase
or thioredoxin, are resistant to steroid-induced apoptosis and show
a delay or lack of cytochrome c release into the cytosol. A comparison
of the redox status of the steroid-resistant variants to that of
the sensitive cells has shown that the NADP(H) pool is larger and
more oxidized in the resistant cells. Glucose 6-phosphate dehydrogenase
activity is increased in the variants, including WEHI7.2 cells that
overexpress bcl-2, suggesting that the resistant cells both
produce and use increased amounts of reducing equivalents. Glutathione
S-transferase is also increased in the variants when compared to
that in the parental (sensitive) cells. These data suggest that
the steroid-resistant variants are primed to respond to oxidative
stress and that an early critical event in steroid-induced apoptosis
is subject to redox control.
Tyrosine
Phosphorylation and Calcium Regulation of MEKK4.
Z
E Derbyshire and R R Vaillancourt. Department of Pharmacology
and Toxicology, University of Arizona, Tucson, AZ, USA.
MEKK4
is a recently identified mammalian kinase, though its function in
cellular signaling remains undetermined. Analysis of its amino acid
sequence suggests it should be a serine/threonine kinase. To characterize
the cellular role of MEKK4, identification of co-purifying proteins
would potentially provide insight to its function. To achieve this,
MEKK4 was expressed using a baculovirus expression system with a
hexa-histidine and FLAG monoclonal antibody epitope in order to
attach the protein to a solid Sepharose matrix. This recombinant
protein was incubated with cellular extract and proteins from a
gel were analyzed using proteomic analysis and confirmed by immunoblotting.
Using this process, the calcium regulated protein, annexin II, was
found to associate with MEKK4, suggesting that MEKK4 could be regulated
by calcium as well. Analysis of the sequence of MEKK4 illustrated
three possible sites for tyrosine phosphorylation, a unique observation
as other characterized MEKK proteins are regulated by serine/threonine
phosphorylion. Our studies then were to investigate if and how this
was occurring. Immunoblotting with a phosphotyrosine antibody quickly
verified the tyrsoine phosphorylation of MEKK4. The next process
was to identify the tyrosine kinase responsible for the phosphorylation.
As a 60 kDa band was present in the phosphotyrosine immunoblot,
it was hypothesized that this band could be src. An immunoblot of
a MEKK4 immunoprecipitation with a src antibody was performed and
the presence of src was confirmed. Using kinase-inactive MEKK4 as
a substrate, a src immunoprecipitation showed tyrosine phosphorylation
of MEKK4. Ongoing studies are aimed at determining which MAP kinase
isoform interacts downstream with MEKK4. Our long-term goal is to
identify and characterize the upstream proteins in the cell that
activate a sequential protein kinase cascade for MEKK4 during calcium
exposure.
Modulation
of p110 PITSLRE Kinase Activity by Tyrosine Hydroxylase.
N
A Sachs and R R Vaillancourt. Department of Pharmacology
and Toxicology, University of Arizona, Tucson, AZ USA.
Tyrosine
Hydroxylase (TH) is regulated transiently by the reversible phosphorylation
of serines 8, 19, 31 and 40, through various intracellular transduction
pathways. The physiological significance of phosphorylation of the
four serines remains enigmatic, as pan phosphorylation is not obligatory
for catecholamine biosynthesis. The purpose of our study was to
identify novel interacting proteins in order to characterize the
physiological relevance of the heterogeneous phosphorylation of
TH. To characterize proteins that interact with TH after phosphorylation
on serine 19, we mutated this amino acid to alanine since it was
reported that a serine to alanine mutation promotes a stable interaction
between proteins. For the identification of proteins that interact
with TH, TH(S19A) was co-transformed with a mouse embryo cDNA library
into a host yeast strain. Two clones were identified in the yeast
two-hybrid system as amino acids 147-262 of mouse-derived p130 PITSLRE.
Characterization with the corresponding human cDNA, in the form
of a GST fusion protein, encoding amino acids 1-373 of a splice
variant of the a isoform of p110 PITSLRE (Af067514) demonstrate
an interaction between TH or TH(S19A) and GST-D p110 PITSLRE.
Additionally, in vitro transcribed and translated TH interacts
with immunoprecipitated FLAG-p110 PITSLRE from transiently transfected
COS-7 cells. TH does not serve as a substrate for p110 PITSLRE in
an in vitro kinase assay. Moreover, preliminary data suggest
that TH mediates an increase in PITSLRE kinase phosphorylation through
a cAMP dependent pathway. This is the first report where TH regulates
the activity of a protein kinase.
Akt,
an Upstream Kinase that Phosphorylates MEKK3.
D
G Adams, N A Sachs and R R Vaillancourt. Department of Pharmacology
and Toxicology, The University of Arizona College of Pharmacy, Tucson,
AZ, USA.
Regulation
of cell death and survival is an essential component of molecular
toxicology that is controlled, in part, by the serine/threonine
protein kinase, Akt. A number of environmental toxicants regulate
stress-activated protein kinase pathways, however, the proteins
that regulate these pathways have not been well characterized. The
protein that phosphorylates and regulates the protein kinase MEKK3,
a kinase known to function in stress-activated protein kinase pathways,
was unknown. In this report, we show that Akt interacts with MEKK3
and phosphorylates two serine residues in the putative amino-terminal
regulatory domain of MEKK3. By using liquid chromatography and electrospray
ionization tandem mass spectrometry, we have identified phosphorylation
of serines 166 and 337 of MEKK3. Phosphorylation of both serines
was localized to the consensus Akt phosphorylation site, RXRXXS/T,
within MEKK3. Activation of Akt by IGF-1 in PC12 cells resulted
in a two-fold increase in MEKK3 phos-phorylation. In addition, recombinant
(His)6FLAG·MEKK3, expressed and purified from Sf9 insect
cells, interacts with endogenous Akt from Sf9 cells, as well as
PC12 cells. Furthermore, precipitated Akt from these cells phosphorylates
recombinant (His)6FLAG·MEKK3. Thus, we have identified
Akt as an upstream kinase that phosphorylates MEKK3. The interaction
between MEKK3 and Akt provides, for the first time, a link between
stress-activated protein kinase pathways regulated by MEKK3 with
cell death and survival pathways regulated by Akt. This work was
supported, in part, by grants from the NIH (AG18041), the Southwest
Environmental Health Sciences Center (P30 ES06694), the Flinn Foundation,
the Pharmaceutical Research and Manufacturers of America Foundation,
and the American Cancer Society (IRG 110T).
Development
and Molecular Characterization of HCT-116 Cell Lines Resistant to
the Tumor Promoter and Multiple Stress Inducer, Deoxycholate.
CL
Crowley-Weber1, CM Payne1,3, M Gleason-Guzman3,
GS Watts3, B Futscher3, C Bernstein1,
H Garewal2,3,4 and H Bernstein1,3. Departments
of Microbiology & Immunology1 and Internal Medicine2,
College of Medicine, University of Arizona, Arizona Cancer Center3,
Tucson Veterans Affairs Medical Center4, Section of Hematology/Oncology,
Tucson, AZ, USA.
Evidence
from live cell bioassays show that the flat mucosa from patients
with colon cancer exhibit resistance to bile salt-induced apoptosis.
Three independent derivative cell lines from the colonic epithelial
cell line HCT-116 were selected that exhibit resistance to bile
salt-induced apoptosis. These were developed as a tissue culture
model of apoptosis resistance. Selection was carried out for resistance
to induction of apoptosis by sodium deoxycholate (NaDOC), the bile
salt found in highest concentrations in human fecal water. Cultures
of HCT-116 cells were serially passaged in the presence of increasing
concentrations of NaDOC. The resulting apoptosis resistant cells
were able to grow at concentrations of NaDOC (0.5 mM) that cause
unselected HCT-116 cells to undergo apoptosis in a few hours. These
cells were then analyzed for changes in gene expression. Combined
data from DNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy,
and confocal microscopy of immunohistochemically stained preparations
indicated underexpression or over-expression of numerous genes at
either the protein or mRNA level. Both novel and previously characterized
genes that may play a role in apoptosis and early stage carcinogenesis
have been identified as upregulated in these cell lines, including
Grp78, Bcl-2, NF-k B, maspin, microsomal GST, Rad 23, pirin,
cofilin, and NOS2. Under-expressed proteins or mRNAs included HSP90,
CD59, elongation factor 2, and ras family member B. The largest
functional category in both the under- and over-expressing groups
was signal transduction proteins. This indicates that signal transduction
pathways provide multiple points for regulatory changes that are
targeted when a cell undergoes chronic stress. Analysis of these
resistant cell lines has suggested potential mechanisms by which
apoptosis resistance may develop in the colonic epithelium of patients
at risk for colon cancer.
Role
of Mitochondrial Complexes I & II, Reactive Oxygen Species and
Arachidonic Acid Metabolism in Deoxycholate-Induced Apoptosis.
CM
Payne1,3, D Washo-Stultz1, H Bernstein1,3,
C Bernstein1 and H Garewal2,3,4. Departments
of Microbiology & Immunology1 and Internal Medicine2,
College of Medicine, University of Arizona, Arizona Cancer Center3,
Tucson Veterans Affairs Medical Center4, Section of Hematology/Oncology,
Tucson, AZ, USA.
Bile
acids are promoters of colon cancer; however, the mechanism(s) of
action of this tumor promoter are largely unknown. Bile acids induce
apoptosis in colon epithelial cells and it is probable that the
modulation of apoptosis contributes, in part, to colon carcinogenesis.
We tested the hypothesis that mitochondria contribute to DOC-induced
apoptosis as an upstream event and that a pro-oxidant state of the
cell favors survival. We found that pretreatment of HT-29 cells
for 2 or 24 hours with 0.01 or 0.1 m M rotenone, an inhibitor
of mitochondrial complex I of the electron transport chain, dramatically
protected against apoptosis induced by 0.5 mM deoxycholate (DOC).
This concentration is representative of the bile salt concentrations
found in the gut of individuals on a high fat diet. We also found
that inhibition of complex II of the mitochondrial electron transport
chain by 0.1 and 1.0 m M thenoyltrifluoroacetone (TTFA) markedly
protected cells against DOC-induced apoptosis. We determined that
rotenone treatment resulted in a reduction in oxidative/nitrosative
stress by using an antibody against nitrotyrosine residues in conjunction
with confocal microscopy. We next determined if the scavenging of
ROS by the lazaroid antioxidants, U-74389G and U-83836E, could protect
against NaDOC-induced apoptosis. Lazaroid treatment resulted in
a sensitization of HT-29 cells to DOC-induced apoptosis. Arachidonic
acid is normally released from membranes and generates ROS and other
products during its metabolism. Inhibitors of arachidonic acid metabolism
(e.g. esculetin, sulindac sulfide, NS-398) also sensitized HT-29
cells to DOC-induced apoptosis. We believe that there are two opposing
pathways that determine the ultimate fate of the cell. On the survival
pathway is the maintenance of a pro-oxidant state with the maintenance
of anti-apoptotic proteins, such as NF-k B, NOS2, COX-2 and
LOX. On the pro-apoptotic pathway is the damage to mitochondria
resulting in the generation of ROS within mitochondria. The exact
mechanisms of colonic epithelial cell death by bile acids are currently
being explored.
The
Growth of Malignant Keratinocytes Depends on Signaling Via the PGE2
Receptor EP1.
EJ
Thompson1, A Gupta2, GT Bowden1.
1Arizona Cancer Center, The University of Arizona,
Tucson AZ; 2James Cancer Hospital and Research Institute,
Ohio State University, Columbus, Ohio.
Recent
discoveries shed light on the importance of prostaglandin production
in development of skin cancer. Work by Fischer's group demonstrates
that skin tumor yield caused by UVB can be decreased by up to 89%
by blocking COX-2 with the drug Celecoxib (Mol Carcinog.25: 231-40).
A similar study by Pentland et al showed that intervention with
oral Celecoxib could decrease new tumor formation by 44% in mice
that already have at least one tumor (Carcinogenesis.20: 1939-44).
These studies demonstrate the importance of COX-2 in the development
of squamous cell carcinoma of the skin. Indeed, there is evidence
from Kanzaki's group that prostaglandin production can be essential
for the growth of human skin tumor cells in culture (Int. J. Cancer
86: 667-71). We have explored growth signaling in a model of skin
tumor progression. Since changes in prostaglandin production have
been implicated in skin carcinogenesis we examined this pathway.
We have treated the benign murine papilloma producing keratinocyte
cell line 308 with ionizing radiation to model tumor progression
in vitro. This produced the progressed malignant variant cell line,
6R90. Another cell line was established from an unusually aggressive
tumor formed by subcutaneous re-injection of 6R90 cells (6RI). Using
enzyme-immunoassay, we found that the malignant variants secrete
more PGE2 than the parental 308 cells. We observed alterations in
the expression of COX-1 and COX-2 by western blot analysis. We also
found that these cells express the PGE2 receptors, EP1 and EP4.
When the cells are grown in the presence of indomethacin, the growth
rate of the malignant variants, but not the 308 cells, is decreased.
This effect can be reversed by addition of PGE2 to the medium. This
growth inhibition can also be rescued by addition of an EP1 agonist
to the medium. Thus, we have shown that skin tumor cells depend
in part on PGE2 signaling via the EP1 receptor for their in vitro
growth.
Microarray
Technology to Profile CpG Island Methylation in Cancer.
B
W Futscher, N Holtan, and R B Isett. Arizona Cancer Center,
University of Arizona, Tucson, AZ, USA.
The
objective of this research project is to adapt microarray-based
technology to the measurement of CpG island methylation in human
cancer cells. CpG islands are ~1kb stretches of DNA that have a
high CG content, are enriched in the dinucleotide 5'-CG -3', are
found at the 5' end of ~50% of all human genes, and participate
in the transcriptional regulation of these genes. The cytosines
in the CpG dinucleotides of CpG islands are unmethylated in normal
tissue; however, CpG islands become aberrantly methylated during
oncogenesis, and this aberrant methylation has been linked to the
transcriptional repression of the associated gene. The target genes
of CpG island methylation are often associated with carcinogenesis
and include tumor suppressors, metastasis suppressors, and DNA repair
genes. In addition, from the limited number of CpG islands and tumors
that have been analyzed to date, it appears that patterns of aberrant
methylation occur in a tumor-specific and stage-specific fashion,
suggesting that CpG island methylation profiles may be useful as
a tumor-specific fingerprint to monitor disease activity and burden.
Thus, a multiplexed assay where the cytosine methylation status
of thousands of CpG islands can be determined simultaneously would
be useful in the molecular profiling of human tumors, and will likely
provide insights into the biology of cancer. To this end we initiated
production of human CpG island microarrays (CGI arrays) as a tool
for determining CpG island methylation profiles in cancer, and from
these profiles identify characteristic patterns of CpG island methylation
that correlate with the tumor's clinical phenotype.
The
Ability of P53 to Reverse the Epigenetic Silencing of Maspin in
Human Breast Cancers.
RJ
Wozniak, MM Oshiro, F Domann, and BW Futscher. Department
of Toxicology and Pharmacology, University of Arizona, Tucson, AZ,
USA. Arizona Cancer Center, University of Arizona, Tucson, AZ, USA.
Maspin
is an anti-metastatic, tumor suppressor gene whose expression is
inappropriately silenced in breast cancer. The transcriptional silencing
of maspin is not due to gene deletion or mutation, indicating that
other regulatory mechanisms are likely at work. We have found that
aberrant cytosine methylation-associated chromatin condensation
of the maspin promoter region is associated with its transcriptional
inactivation in human breast cancer cell lines. In addition, recent
studies in prostate cancer suggest that wild type p53 is a positive
regulator of maspin expression. Based on these observations we set
out to test the hypothesis that enforced over-expression of wild
type p53 can overcome the repressive effects of aberrant promoter
methylation by stimulating chromatin remodeling of the maspin promoter.
Three aberrantly methylated, maspin negative human breast cancer
cell lines were used to test this hypothesis; two were mutant for
p53 (MDA-MB-231 and MDA-MB-435), while one contained wild type expression
of p53 (MCF-7). These three cell lines were used as targets for
adenoviral infection of a vector containing wild type p53 and green
fluorescent protein. Results from the enforced over-expression of
wild type p53 resulted in maspin re-expression in all three cell
lines, compared to non-infected controls. These results suggest
that wild-type p53 can overcome the transcriptionally repressive
effects of aberrant cytosine methylation, and suggests that p53
may participate in chromatin remodeling. Future experiments are
designed to delineate the potential mechanisms by which p53 elicits
these effects.
Microarray
Expression Profiles in Breast Cancer Cells Following Exposure to
Benzo[a]pyrene and B[a]P-Diol Epoxide.
D
F Romagnolo, B D Jeffy, DJ Samuelson, R B Chirnomas, and C M Payne.
Department of Nutritional Sciences, Cancer Biology IDP, Microbiology
and Immunology, The University of Arizona, Tucson, AZ.
Maintenance
of genome integrity in mammalian cells may be compromised by the
concomitant accumulation of DNA damage and loss of repair functions.
Polycyclic aromatic hydrocarbons (PAHs) are classic DNA-damaging
and tumor promoting agents. Benzo[a]pyrene, a prototype PAH, induces
a number of genotoxic responses including cell cycle arrest, oxidative
stress and DNA adduct formation. The objective of this study was
use DNA microarray as a means to assess the genome-wide expression
profile after exposure to benzo[a]pyrene (B[a]P) and its metabolite
7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene
(BPDE). Exposure of MCF-7 cells to benzo[a]pyrene (B[a]P) (1 to
5 mM) and BPDE (50 to 100nM) induced transient S-phase arrest, which
was followed by accumulation in G2/M . In cells treated with B[a]P
and BPDE, we observed segregation of nucleolar material and a drastic
reduction in the number of ribosomal fibrillar centers. To identify
the expression profile in cells treated with B[a]P or BPDE, we analyzed
the expression levels of 1142 genes by GeneMAPTM CancerArray.
Compared with the expression levels in untreated cells, transcripts
that were upregulated by both B[a]P and BPDE included members of
the cytochrome P450, AP-1 (Fra-1, Fra-2), and glutathione-S-transferase
families, PARP, and G1/S specific cyclins. In contrast, transcript
levels for BRCA-1, G2/M cyclins, Bcl-2, JunB, and JunD were downregulated.
The treatment with the metabolite BPDE tended to elicit a stronger
response than B[a]P did. RT-PCR analysis for a subset of genes confirmed
the changes in expression levels predicted by DNA microarray.
Regulation
of the BRCA-1 Promoter in Breast Cancer MCF-7 Cells by Benzo[a]pyrene
and its Metabolite BPDE.
B
D Jeffy, D F Romagnolo. Cancer Biology Graduate Interdisciplinary
Program, Department of Nutritional Sciences, University of Arizona,
Tucson, AZ, USA.
The
objective of this study was to investigate potential mechanisms
underlying the negative regulation of BRCA-1 expression by the PAH
benzo[a]pyrene (B[a]P) and its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide
(BPDE). In previous studies, we found that treatment of MCF-7 breast
cancer cells with either B[a]P or BPDE significantly decreased BRCA-1
message and protein levels in a time- and dose-dependent manner.
In this study, in order to determine if loss of BRCA-1 message induced
by B[a]P or BPDE might be occurring via a regulatory mechanism,
we cloned a 1690 bp fragment of the 5¢ BRCA-1 flanking region
into a luciferase reporter vector for transient transfection. We
found that transfection of the construct (pGL3-BRCA-1) into MCF-7
cells followed by treatment with either B[a]P or BPDE caused a dose-dependent
decrease in luciferase activity. However, another ligand of the
AhR, TCDD, did not alter pGL3-BRCA-1 luciferase activity. We previously
reported that B[a]P exerts its negative effects on BRCA-1 message
and protein levels in estrogen receptor positive (ER+) cells but
not in estrogen receptor negative (ER-) cells. In order to further
characterize the relationship between ER status and regulation of
BRCA-1 expression, we transfected pGL3-BRCA-1 into ER- HBL-100 mammary
epithelial and ER- HeLa cervical cancer cells. Treatment of these
transfected cells with B[a]P caused no decrease in luciferase activity.
However, co-transfection of pGL3-BRCA-1 and a plasmid containing
estrogen receptor cDNA (pHEO) followed by treatment with B[a]P did
indeed cause a reduction in reporter activity. These data suggest
that downregulation of BRCA-1 by B[a]P may be occurring via a regulatory
mechanism dependent on ER status.
Effects
of Occupational Levels of Toluene Diisocyanate on Glutathione-Dependent
Enzymes and Glutathione in Human Bronchoepithelial Cells.
R
C Lantz1, R Lemus2, D H Wilson2,
and M H Karol2. 1Dept. of Cell Biology
and Anatomy, Health Sciences Center, University of Arizona, Tucson,
AZ and 2Dept. of Environmental and Occupational Health,
University of Pittsburgh, Pittsburgh, PA.
Toluene
diisocyanate (TDI) is a recognized asthmogen, yet the mechanism
of its toxicity and the molecular reactions involved remain unclear.
We have previously shown a rapid reaction of TDI with glutathione
(GSH), a key cellular antioxidant. The intracellular GSH level is
the final outcome of several processes, one being the uptake of
constituents of the GSH molecule via the glutamyl transpeptidase
(g-GT) pathway. This enzyme plays a key role in the synthesis and
degradation of GSH. Hence, the activity of g-GT may provide a sensitive
indicator of lung injury. Our earlier in vitro studies using the
fluorescent dye, Cell Tracker Green (5-chloromethyl-fluorescein
diacetate, CMFDA), have shown a decrease in intensity of the thiol
staining as a function of the length of TDI exposure in differentiated
and undifferentiated human bronchial epithelium (HBE) cells. Because
CMFDA activation and fluorescence requires functional intracellular
esterases and glutathione-S-transferases (GST), the present study
was undertaken to evaluate the activity of these two enzymes as
well g-GT activity. Differentiated (6-day) air/liquid cultures of
HBE cells were exposed to 20 ppb TDI vapor for 5 or 30 minutes.
Following exposure, cell viability was analyzed, cellular GSH levels
were titrated using the thiol specific fluorophore ThioGlo, and
GST and g-GT enzymatic activities were spectrophotometrically determined.
Cellular esterase activity was additionally assayed by placing cells
in PBS containing 0.5 mM 5-(and-6-)-carboxyfluorescein succinimidyl
ester (CFSE) followed by imaging using confocal microscopy. Exposure
of 6-day cultures to 20 ppb TDI for 5 and 30 minutes was not cytotoxic.
In addition, esterase and GST activities were not affected by TDI
exposure. Therefore, the reduction in CMFD fluorescence detected
following TDI exposure was not due to cytotoxicity, decreased esterase
activity, nor reduced GST activity. TDI exposure of HBE cells resulted
in a 30 % decrease in GSH levels and 35% decrease in g-GT activity.
This reduction of intracellular GSH and inhibition of a GSH-dependent
enzyme can alter cellular redox status. The resulting oxidative
stress may be a significant factor in determining human susceptibility
to TDI-induced lung diseases. Supported by NIEHS Center Grant #ES06694
and NIEHS #05651.
Glucuronidation
of Bisphenol A in Hepatic Microsomes: Age-Dependent Differences.
Kuester,
R.K., Pritchett , J.J., Fontaine, S.M., Solyom, A and Sipes, I.G.
Department of Pharmacology and Toxicology, The University of
Arizona, Tucson, Arizona
Bisphenol
A (BPA) is a phenolic compound with industrial and commercial uses.
In the liver Bisphenol A undergoes extensive metabolism and is eliminated
primarily as a glucuronide conjugate. To understand how age affects
BPA glucuronidation, kinetic constants for BPA-glucuronidation (Vmax
and Km) were determined in incubations of hepatic microsomes isolated
from adult male (77-day old), newborn (4 and 21 day) and fetal (gestational
day 19 [GD-19]) Sprague Dawley rats. Microsomes were incubated with
UDPGA and 14C-BPA (2.6-402 µM) for 2 min. Reaction products
were separated via HPLC and analyzed by scintillation counting.
Under the conditions of these experiments the only metabolite obtained
was the monoglucuronide of BPA. Fetal hepatic microsomes showed
a decreased capacity to glucuronidate BPA as compared to hepatic
microsomes of adult rats (38 versus 72 nmol/min/mg, respectively).
Glucuronidation of BPA was increased in microsomes from livers of
4-day old rats as compared to those of GD-19 rats. Based on Vmax
values, these activities were still lower than those of hepatic
microsomes from adult and 21 day old rats. There were no major differences
in Vmax values between microsomes from 21 and 77-day old rats. For
all age groups the Km values ranged 22 µM to 42 µM. These data demonstrate
that the glucuronsyltransferase activity responsible for BPA metabolism
is developed before GD-19 and increases soon after birth. Although
the activity in GD-19 microsomes was lower than that of adult microsomes,
livers from GD-19 represented 9% of body weight as compared to 4%
for all other groups. An important consideration is whether this
increased liver mass promotes more extensive in vivo conjugation
of BPA by fetal hepatic tissue than is predicted from in vitro studies.
This research was supported in part by the Southwest Environmental
Health Science Center (ES 06694) and the Society of Plastics Industry
Inc.
Hepatoprotection
during Ischemia/Reperfusion in the Fischer 344 Rat: Comparison of
Dimethyl Sulfoxide and Insulin.
CR
Kundavaram, MA Leslie, NJ Young, DC Caretto, PZ Nakazato and JB
Ulreich. University of Arizona, Department Surgery, Tucson,
AZ.
UNOS
lists 16,000 patients nationwide waiting for liver transplants.
Many (12%) will die waiting. Use of non-heart-beating donor organs
or enhancement of viability of livers from heart-beating donors
might reduce the shortage. Warm ischemia causes damage that can
lead to graft failure in recipients. We reported that ischemia/reperfusion
in rat livers caused increased adhering leukocytes, swollen endothelial
cells and phagocytic Kupffer cells. DMSO pretreatment prevented
these effects. Insulin pretreatment has been reported to have protective
effects on rat livers during portal triad clamping and improved
liver function post-transplantation (Morimoto et al.). To compare
the efficacy of DMSO with that of insulin, F344 rats were given
DMSO (2 ml/kg, ip), insulin/20% glucose (15ml/kg, iv) or saline
(control) 30 min. prior to ischemia. Ischemia (60 min.) was followed
by reperfusion (60 min.). Livers were precision-cut and incubated
for 4-48h. Following incubation, assays for K+ retention
and LDH release indicated viability. DMSO pretreatment maintained
K+ levels significantly better than insulin. LDH release
by DMSO pretreated livers was lower than for insulin pretreated
livers. DMSO prevented loss of viability to a greater extent than
insulin. Given prior to organ retrieval, DMSO may preserve organ
viability, thus making more organs available for transplantation.
In cold preservation solutions, DMSO does not have a similar beneficial
effect. In a pilot study using the SELDI (Surface Enhanced Laser
Desorption/Ionization) ProteinChip System which shows differential
expression of proteins, a protein at 8.31 kDa was upregulated in
the presence of DMSO, only under ischemic conditions. The TagIdent
database was used to identify potential candidates for this marker.
Apolipoprotein C-II (pI: 4.71, MW: 8329.12) was the closest matching
candidate. [Supported by ADCRC (JU, PN), UBRP, NIEHS P30-ES-06694
(JU)]
Herbal
Remedies Protect against Chloroform Hepatotoxicity in Precision-Cut
Tissue Slices.
JB
Ulreich, MA Levy, DC Caretto, NJ Young, CR Kundavaram, and PZ Nakazato.
Dept. Surgery & Southwest Environmental Health Sciences Center,
University of Arizona, Tucson, AZ.
By
1990 an estimated 60 million Americans used at least one form of
alternative medicine at an estimated cost of $13.7 billion . Trade
groups estimate that sales of herbal medicines exceeded $4 billion
in 1999. A number of herbs are touted to have beneficial effects
on the liver but scientific evidence of their safety, efficacy and
mechanism of action is limited or lacking. Precision-cut male Fischer
F344 rat liver slices were used to determine whether herbs exhibited
protective effects against chloroform toxicity in vitro.
Three such herbs were included in this study: Schizandra (Schizandra
chinesis), milk thistle (Silybum marianum) and Reishi
mushroom (Ganoderma lucidum). Tinctures were concentrated
4x under nitrogen to remove the alcohol and added to culture medium
at 0, 1, 5, or 10µL/ml. Livers were excised, cored (8mm), precision-cut
(250µ), and incubated at 37oC on titanium carriers in
a rolling incubation system for volatiles for 1h in Waymouth's medium
+/- each herb. After preincubation, chloroform (80µL/L was injected
into some bottles and incubation continued for 2-9h. Potassium and
lactate dehydrogenase content of the tissue slices were determined
as viability indices. All three herbs showed hepatoprotective effects.
Viability (potassium content) of liver slices exposed to herbs with
chloroform, compared to chloroform alone, was 129-192% for Schizandra,
129-187% for milk thistle and 143-184% for Reishi, depending on
dose. Schizandra and milk thistle incubated together had additive
effects. Chaparral (Larrea tridentata), previously reported
by us to be a hepatotoxic herb, had potassium values 61-90% of chloroform
alone. In vivo studies will be conducted to determine whether
there is good correlation with this in vitro model of hepatoprotection.
[Supported by NIEHS Center Grant P30ES06694 (JU), and the UA Undergraduate
Biology Research Program (CK, DC, NY)].
Determining
the Effect of Heavy Metals on Protein Function by Direct Force Measurement.
Phase I: Tethering Proteins to Molecularly Smooth Mica in order
to Study Protein-Protein Interactions.
S
Kim, C Heo, and J E Curry. Department of Soil, Water and
Environmental Science, University of Arizona, Tucson, AZ, USA.
The
overall goal of our work is to directly measure the effect of environmental
contaminants such as heavy metals on protein function. More specifically,
we want to determine the effect of cadmium on cadherin mediated
cell-cell junctions by directly measuring the force between cadherin
fragments tethered to solid supports (molecularly smooth mica).
In order to create sites for protein attachment we first coated
the mica surface with an organosilane monolayer of octadecyltriethoxysilane
(OTE). The focus of this study has been to determine if the OTE
monolayer is covalently attached to the molecularly smooth mica
surface. OTE monolayers were self-assembled on bare and water vapor-plasma
treated mica surfaces. The stability of the OTE monolayers on treated
and untreated mica surfaces was studied as a function of relative
humidity using a surface forces apparatus. Measurements of the thickness
of the water layers adsorbed by the OTE were made as a function
of relative humidity. The monolayers self-assembled on plasma treated
mica adsorbed significantly less water compared to the untreated
case. It is thought that the increased stability of the organic
layer self-assembled on plasma treated mica is due in part to covalent
bonding between the silanes and hydroxyl groups on the mica surface
introduced by plasma treatment. This highly stable organic layer
will be useful as a base substrate for our cell adhesion studies.
The
Effects of Hepatoprotectants, DMSO, Aminobenzotriazole And Cyclooxygenase
And Lipooxygenase Inhibitors on Eicosanoid Levels during Chloroform
Induced Hepatic Injury.
C.K.
Begay, A.J. Gandolfi. Department of Pharmacology and Toxicology,
University of Arizona, Tucson, Arizona.
Cytochrome
P-450 inhibitors Dimethyl Sulfoxide (DMSO) and aminobenzotriazole
(ABT) were administered 24 hours after chloroform dosing to determine
their effect on eicosanoids levels in rat plasma and liver. Our
previous studies have demonstrated that the antidote, DMSO protects
against liver injury elicited by chloroform even when given 24 hr
after the toxicant, at a time when the liver injury is taking place
and rapidly developing. Arachidonic acid is a fatty acid that serves
as a substrate for the enzymes leading to the production of potent
substances such as prostaglandins and leukotrienes. Their formation
in the liver contributes to the acute response to inflammation.
DMSO is an anti-inflammatory agent and may affect the production
of prostaglandin and leukotrienes. Cytochrome P-450 is involved
in biotransforming arachidonic acid to its active metabolites and
is inhibited by DMSO. We are now examining if this is one of the
mechanisms of DMSO hepatoprotection. Inhibitors for arachidonic
acid metabolites were studied to determine what route DMSO or ABT
may take in attenuating liver injury. PGE2 and LTB4
are metabolized by the enzymes cyclooxygenase and lipoxygenase,
respectively. Both DMSO and ABT prevented an increase in alanine
aminotransferase (ALT) at 32 and 48 hr after initial toxicant insult.
Plasma PGE2 and LTB4 levels were attenuated
by a single dose of DMSO (2ml/kg) or ABT (30mg/kg) to Sprague Dawley
rats at 48 hr. Indomethacin, a COX-1 inhibitor, and NS-398, a COX-2
inhibitor both prevented an increase in ALT levels at 48 hr after
toxicant ingestion. NDGA, a lipoxygenase inhibitor, Indomethacin,
and NS-398 significantly reduced plasma PGE2 levels 48
hr after chloroform dose. Liver PGE2 was not effected
by DMSO, ABT or any of the eicosanoid inhibitors. These results
show that DMSO and ABT prevented plasma and liver LTB4
formation and provide further support for a role for DMSO as a therapeutic
agent against hepatotoxicants. The lipoxygenase pathway may be a
major factor in the attenuation of liver injury.
SWEHSC
Synthetic Core Laboratory.
Y
Li, B Jagadish, and E A Mash. Department of Chemistry, The
University of Arizona, Tucson, AZ, USA.
The
synthetic core laboratory of the South West Environmental Health
Sciences Center consults with SWEHSC members on the chemical aspects
of their projects and carries out syntheses of labeled and unlabeled
compounds. Recent syntheses in support of SWEHSC projects will be
presented.
IMPACTT:
Integrating Multiple Perspectives for Today and Tomorrow - A Completely
Integrated Environmental Health High School Program.
S
D Hines, R B R Milholland. Southwest Environmental Health
Sciences Center, University of Arizona, Tucson, AZ, USA; Department
of Pharmacology and Toxicology, University of Arizona, Tucson, AZ,
USA.
IMPACTT
is being developed in partnership with the Sunnyside Unified School
District (SUSD) and is a unique, fully integrated environmental
health/environmental science academy, or "school within a school." In this first year of the program, fifty 9th grade students are
obtaining academic credits in science, health, math, English, physical
education, and technology through the context of the umbrella topics
of environmental health and environmental science. Course content
is presented in thematic units through which students make connections
to environmental health and traditional academic subjects. The 9th
grade component consists of five units including Biodiversity, Endangered
Species, Air Quality, Land, and Water.
Fun
Environmental Health Activities: A Web-Based Resource for K-12 Teachers.
S
D Hines, J L King. Southwest Environmental Health Sciences
Center, University of Arizona, Tucson, AZ, USA.
The
Community Outreach and Education Program (COEP) at the Southwest
Environmental Health Sciences Center is continually compiling and
creating engaging, high quality environmental health K-12 classroom
activities. The web is a powerful tool to easily disseminate these
materials to interested teachers. To more fully utilize the Internet,
the COEP has developed a website and promotional materials called "Fun
Environmental
Health Activities" where teachers can access web-based materials
developed by COEP or download lectures and curricula. This poster
shows the breadth of materials and resources available to teachers
through this website.
Microbial
Population Dynamics During 3-Chlorobenzoate Degradation in Soil.
TJ
Gentry, DT Newby, KL Josephson, and IL Pepper. Department
of Soil, Water, and Environmental Science, University of Arizona,
Tucson, AZ, USA.
Changes
in microbial populations during degradation of 3-chlorobenzoate
(3-CB) in soil were evaluated in a laboratory study. Madera sandy
loam was amended with 0, 500, or 1000 μg 3-CB/g dry soil. Selected
microcosms were inoculated with the 3-CB degrader Comamonas testosteroni
BR60. Degraders were enumerated on Medium A containing 3-CB. Isolated
degraders were grouped for identification based on enterobacterial
repetitive intergenic consensus sequence-PCR (ERIC) fingerprints.
In the uninoculated microcosms, degraders increased from undetectable
levels to 6.6 x 107 CFU/g in the 500 μg 3-CB/g microcosms
but no degraders were detected in the 1000 μg 3-CB/g samples.
In both the 500 and 1000 μg 3-CB/g inoculated microcosms, degraders
increased from the initial inoculum of 1.0 x 106 CFU/g
to around 1.5 x 108 CFU/g, and then decreased following
degradation of 3-CB. All degraders isolated from the uninoculated
microcosms produced one of two unique ERIC fingerprints, whereas
all degraders from the inoculated soil had fingerprints identical
to those of C. testosteroni. Bioaugmentation increased the
rate of 3-CB degradation. However, after 18 d when selected microcosms
were re-amended with 500 μg 3-CB/g, degradation was more rapid
in the uninoculated microcosms. The results indicate that bioaugmentation
may increase the rate of 3-CB degradation, but inhibit the development
of indigenous populations that may ultimately be more effective
degraders in the soil environment.
Streamlining
Gene Bioaugmetation: Determining the Relative Importance of Growth
Rates vs. Gene Transfer Rates.
C
J Haney. Department of Microbiology and Immunology.
Soils
co-contaminated with both organic and metal pollutants are difficult
to bioremediate via conventional means due to the high mortality
rate of the introduced remediative organism as a result of the dual
stresses. To get around this problem, it may be possible to transfer
the genes necessary for bioremediation, via conjugation, to the
indigenous soil population, a technique known as gene bioaugmentation.
Using a model system in vitro, the rate at which the resultant
transconjugant population grows will be analyzed to determine whether
the growth rate is more dependent upon growth of the individual
organisms, multiple gene transfer events by the introduced donor,
or gene transfer from transconjugants to indigenous organisms. To
do this, a novel donor system will be engineered using Ralstonia
eutropha JMP134 and it's plasmid, pJP4, which codes for both
mercury resistance and degradation of the organic pesticide 2,4-dichlorophenoxyacetic
acid (2,4-D). A "kill gene" (Gef) will be inserted into
the JMP134 chromosome, making it counterselectable, and green fluorescent
protein (GFP) will be inserted into the plasmid pJP4, allowing for
rapid detection of transconjugants. Gene transfer rates can then
be compared between the donor and recipient by using isolated transconjugants
as donors in separate experiments. The importance of organismal
growth rates in population growth rates will then be assessed by
comparison with pure culture growth rates. This information will
provide information as to how bioremediation via gene bioaugmentation
can be better facilitated.
Application
of a Biodegradation and Transport Model Incorporating Microbial
Lag to Soil Systems of Increasing Heterogeneity and Biological Diversity.
S
K Snyder1, F L Jordan1, L Li1,
R M Maier1, and M L Brusseau1,2. Deparment
of Soil, Water and Environmental Science, Department of Hydrology
and Water Resources, University of Arizona, Tucson, AZ, USA.
A
biodegradation and transport model incorporating microbial lag was
developed, and its predictive capability was examined by comparing
simulation results to an array of experimental results obtained
from systems of increasing complexity. Salicylate was chosen as
the model organic compound because it experiences negligible retardation
and is a known intermediate in the biodegradation pathway of many
common organic contaminants (such as naphthalene and phenanthrene).
The first system consisted of a sterilized, homogeneous quartz sand
inoculated with a pure culture of Pseudomonas putida RB1353,
a strain of bacteria capable of degrading the model organic compound.
The other two systems were soils (of differing textures) containing
indigenous populations capable of degrading salicylate. For all
systems, batch mineralization studies were performed, using the
same population as in the miscible-displacement experiments, to
determine biodegradation and bacterial growth kinetic parameters
(including lag times). Kinetic parameters determined from batch
studies and hydrodynamic parameters obtained from miscible-displacement
experiments performed using conservative tracers were utilized in
the model to allow independent prediction of the experimental results.
Preliminary findings demonstrate that simulation results were significantly
different from the experimental results in the absence of the incorporation
of cell elution and microbial lag into the model. Additionally,
cell elution, substrate biodegradation and transport, and bacterial
growth demonstrated enhanced variability in the systems with indigenous
populations and increased heterogeneity when compared to the single-isolate
system.
The
Development of an Indigenous Phenanthrene Degrading Community during
Long-Term Exposure to Phenanthrene under Saturated Flow Conditions.
A
A Bodour, J-M Wang, M L Brusseau, and R M Maier. Department
of Soil, Water and Environmental Science, University of Arizona,
Tucson, AZ, USA.
The
widespread environmental contamination by polycyclic aromatic hydrocarbons
(PAHs) has led to increased interest in the use of in situ bioremediation
as a cleanup strategy. However, few bioremediation studies have
investigated indigenous populations and population dynamics following
exposure to a PAH. The goal of this study was to examine the temporal
response of an indigenous soil population to long term exposure
to phenanthrene. A column was packed with an uncontaminated loamy
sand soil (organic matter content 2.7%) and saturated conditions
were established with 0.005 M CaCl2. Following saturation,
the column was exposed to a solution saturated with phenanthrene
(~1.2 mg/L). Effluent samples were collected and analyzed for phenanthrene
concentration and microbial counts. Counts were performed on mineral
salts media + phenanthrene (phenanthrene degraders) and R2A
agar (heterotrophic counts). Phenanthrene degraders were separated
according to growth rates. Colonies growing within 3 days were called
fast growers, while slow growers referred to colonies enumerated
on day 14. Unique isolates were chosen and isolated from the fast
and slow growers as well as from the soil at the end of the experiment.
Isolated microorganisms were then grouped either by ERIC analysis
of their genomic DNA or by morphology. For each unique group, 16S
rDNA PCR was performed and then sequencing analysis was used to
identify the isolates. A total of 24 different phenanthrene degraders
were obtained. Of the 24 isolates, only three isolates were found
both in the effluent samples and in the soil from the column. These
results indicate that attached and free-living microbes that compete
for substrate are different. Overall, these results suggest that
a diverse population of microbes participated in phenanthrene degradation
and further, a succession of populations took place.
Influence
of Multiple Bacterial Populations on Phenanthrene Degradation, Bacterial
Cell Elution, and Species Distribution.
B
M Patterson1, M L Brusseau1,2, R M Maier1,
R Frye1. 1Department of Soil Water
and Environmental Science, 2Department of Hydrology and
Water Resources, The University of Arizona, Tucson, AZ, USA.
A
single set of degradation coefficients is typically used when representing
biodegradation in contaminant transport models. Implicit to this
approach is the assumption that only a single degrading isolate
exists, or that the entire community of degraders more typically
present in natural systems has a uniform, constant growth rate and
affinity for the contaminant. This assumption was evaluated through
a miscible displacement experiment conducted using a column packed
with a soil containing an indigenous microbial community comprised
of 24 identified phenanthrene-degrading isolates. Results produced
oscillating phenanthrene concentrations in the column effluent,
indicating potential competitive interactions among the isolates.
A second series of experiments, conducted in a simplified system
comprised of sand and 1,2, or 3 indigenous isolates, examined the
effects of species interactions on phenanthrene degradation and
bacterial cell elution. Bacterial growth rates, density of cells
within the column, and bacterial distribution were also evaluated.
Results show single bacterial species produced relatively stable
cell elution and phenanthrene concentrations in the effluent. Conversely,
the behavior in the multiple species systems indicated synergistic
and antagonistic interactions occurred among the species. These
results illustrate that the dynamics of heterogeneous microbial
communities should be considered when evaluating contaminant biodegradation
and transport in subsurface systems.
An
Ecological Survey of Biosurfactant-Producing Microorganisms in Arid
Southwestern Soils.
A
A Bodour and R M Maier. Department of Soil, Water and Environmental
Science, The University of Arizona, Tucson, AZ, USA.
Biosurfactants
are a unique class of compounds that have been shown to have a variety
of potential applications including; enhanced remediation of organic-
and metal-contaminated sites, enhanced transport of bacteria, enhanced
oil recovery, cosmetic additives, and biological control. However,
little is known about the distribution of biosurfactant-producing
microorganisms in the environment. The goal of this study was to
examine the ecology of surfactant-producing organisms in order to
determine how commonly they occur in pristine and contaminated sites.
A series of contaminated (metals or hydrocarbons) and uncontaminated
soils were collected. Soils were plated on R2A agar and
each unique isolate was grown in mineral salts medium containing
2% glucose to screen for biosurfactant production. Supernatants
from each culture were tested using the drop-collapse method to
determine their ability to reduce surface tension of a solution.
Microorganisms classified as positive for biosurfactant production
from the drop collapse test were then grouped by ERIC or REP analysis
of their genomic DNA. For each unique group of biosurfactant producers,
16S rDNA sequencing was performed to identify the isolates. A total
of 45 biosurfactant-producing isolates were obtained. Of the 45
isolates, the majority were gram positive and were obtained from
heavy metal-contaminated (cadmium and/or lead) or uncontaminated
soils. Overall, these results indicate that biosurfactant-producing
organisms are found in most soils suggesting that in situ production
of biosurfactants in contaminated sites may be feasible.
Metals
Associated with Aquatic Plants in an Acid-Mining Contaminated Stream:
The Role of Metal Plaques.
T
L Corley, and M H Conklin. Department of Hydrology
and Water Resources, The University of Arizona, Tucson, AZ, USA.
Past
acid-mining activities near Globe, Arizona released metal contaminants
into the perennial reach of Pinal Creek. Dissolved Mn is the dominant
metal in Pinal Creek with lower concentrations of Zn, Ni, Cu, Fe,
and Co. Concentrations of these metals were determined in samples
of water speedwell, rabbitfoot grass, algae and moss collected over
a 2.5-year period. Plant concentrations were factors of 100 to over
1,000,000 greater than surface water concentrations. Correlations
between plant concentrations and physical and chemical parameters
of Pinal Creek were examined. Surface water concentrations correlated
with concentrations of all metals except Fe in water speedwell,
all metals in rabbitfoot grass, Mn and Zn in algae, and Mn, Co,
and Cu in moss. Positive correlations with pH were only found for
Zn in rabbitfoot grass and algae, and Mn in water speedwell, algae
and moss. More importantly, Mn concentrations showed positive correlations
with the concentrations of Zn, Ni, Co, Cu, and Fe in water speedwell
and algae, Co in rabbitfoot grass, and Zn, Ni, and Co in moss. The
results strongly suggest Mn-containing metal plaques and extracellular
precipitates have major roles in determining the total metal concentrations
of the Pinal Creek samples.
Reductive
Dissolution of MnO2 by Fe(II): Effects of Chemical Gradients
and Intermediate Phase Structural Information.
John
E. Villinski1, Peggy A. O'Day2, John R. Bargar3
and Martha H. Conklin.1 1Department
of Hydrology and Water Resources, The University of Arizona, Tucson,
AZ, 2Geology Department, Arizona State University, Tempe,
AZ, 3Stanford Synchrotron Radiation Laboratory, PO Box
4349, MS 69, Stanford, CA.
An
in situ, real-time, synchrotron X-ray absorption spectroscopy
study of the reductive dissolution of MnO2 be Fe(II)
at pH 3 was performed in a novel flow-through reaction cell at SSRL.
While the path length was only 7 mm, different results were obtained
from collecting spectra at the inlet (upgradient) and downgradient
portions of the bed. Analysis of XAS spectra indicated that Mn(IV)
reduction occurred without a detectable presence of a reaction intermediate
phase upgradient. Both XANES and pre-edge spectra indicated an intermediate
phase containing tetrahedrally-coordinated Mn, and most likely Fe(III),
was present downgradient. This is consistent with the observed advection
and precipitation of Fe(III). The absorbance of the intermediate
phase reached a constant value after 30 minutes of a 2 hour experiment,
and thus the intermediate phase appears to exert control over the
release of Mn(II) to solution. By probing different regions of a
mineral-solution reaction path spectroscopically, a more complete
picture of the reactions controlling the reductive dissolution is
obtained.
Electrochemical
and Spectroscopic Study of Arsenate Removal from Water using Zerovalent
Iron Media.
J Farrell1, N T Melitas1, J Wang1,
M Conklin2 and P O'Day3. 1Department
of Chemical and Environmental Engineering 2Department
of Hydrology and Water Resources University of Arizona, Tucson,
AZ 85721 3Department of Geological Sciences, Arizona
State University, Tempe, AZ 85287.
This
study investigated the mechanisms involved in removing arsenate
from drinking water supplies using zerovalent iron media. Batch
experiments utilizing iron wires suspended in anaerobic arsenate
solutions were performed to determine arsenate removal rates as
a function of the arsenate solution concentration. Corrosion rates
of the iron wires were determined using Tafel analysis. The removal
kinetics in the batch reactors were best described by a dual rate
model in which arsenate removal was pseudo-first order at low concentrations
and approached zeroth order in the limit of high arsenate concentrations.
This kinetic behavior was attributed to surface saturation effects
with increasing arsenate concentration. The presence of arsenate
decreased iron corrosion rates. However, constant corrosion rates
were attained indicating that the passivation processes had reached
steady state. Tafel analysis did not reveal the electrochemical
reduction of arsenate whereas arsenate complex formation on the
anodic sites of the iron was indicated. Faster arsenate removal
was associated with higher corrosion rates and more completely oxidized
iron. X-ray absorption spectroscopy analyses indicated that all
arsenic associated with the zerovalent iron surfaces was in the
+5 oxidation state. Interatomic arsenic-iron distances determined
from EXAFS analyses were consistent with bidentate corner-sharing
among arsenate tetrahedra and iron octahedra. Results from this
study show that under conditions applicable to drinking water treatment,
arsenate removal by zerovalent iron media involves surface complexation
only, and does not involve reduction to metallic arsenic.
Kinetics
of Soluble Chromium Removal from Contaminated Water by Zerovalent
Iron Media: Corrosion Inhibition Effects.
N
T Melitas, J Farrell and O Chuffe. Department of Chemical
and Environmental Engineering University of Arizona, Tucson, AZ
85721.
Permeable
reactive barriers containing zerovalent iron are being increasingly
employed for in situ remediation of groundwater contaminated with
redox active metals and chlorinated organic compounds. This research
investigated the effect of chromate concentration on its removal
from solution by zerovalent iron. Soluble chromate removal by iron
wires was measured in batch experiments for initial chromium concentrations
ranging from 100 to 10,000 µg/L. Chromate removal was also measured
in columns packed with zerovalent iron filings over this same concentration
range. Electrochemical measurements were made to determine the free
corrosion potential and corrosion rate of the iron reactants. In
both the batch and column reactors, chromium removal rates declined
with increasing chromate concentration. This indicates that simple
first or fractional order kinetic models are not useful for describing
chromate removal kinetics. Corrosion current measurements indicated
that the rate of iron corrosion decreased with increasing chromium
concentrations between 0 and 5,000 µg/L. Analysis of Tafel polarization
diagrams indicated that iron corrosion was hindered by both cathodic
and anodic inhibition. Cathodic inhibition was indicated by a decrease
in the exchange current for the water reduction reaction. Anodic
inhibition was attributed to the buildup of Cr(III)/Fe(III) oxides
at anodic sites on the iron surfaces. Even at the most passivating
concentration of 10,000 µg/L, chromate removal in the column reactors
reached a steady state, indicating that the passivation had also
reached a steady state. Although chromate contributes to iron surface
passivation, the removal rates are still sufficiently fast for iron
barriers to be effective for chromate removal at most environmentally
relevant concentrations.
Catalytic
Hydrodechlorination of Gas-Phase Trichloroethylene on Media Supported
Platinum.
O
Orbay, X Ju, R G Arnold, E A Betterton, W Ela. Department
of Chemical and Environmental Engineering, University of Arizona,
Tucson, AZ, USA.
Trichloroethylene
(TCE) is among the most pervasive contaminants in groundwater and
soil due to widespread historical use and improper disposal practices.
Development of technically feasible, cost effective technologies
for TCE remediation at contaminated sites could save a great deal
of money. Dechlorination of gas-phase TCE using elemental hydrogen
on a platinum catalyst surface was investigated in this research.
Using 5g of 0.5% Pt on an Al2O3 support and
6,423ppmv TCE in N2(g), >99% TCE destruction was obtained
at an estimated residence time of 3s at room temperature. Much faster
reaction rates were available if the column was heated. Ethane production
accounted for more than 90% of the TCE transformed. No partially
halogenated intermediates were observed except for chloroethane.
The advantage of gas-phase hydrodechlorination over aqueous-phase
is in the ease with which H2 can be provided. Mass transfer
limitations are also less likely to limit reaction rate and reaction
products are, on average, less chlorinated than in similar aqueous-phase
systems. Among the problems of this technology is catalyst deactivation.
During these experiments, the activity of the catalyst decreased
due to sintering, coating, poisoning with HCl, or a combination
of these effects. Addition of KOH decreased the observed deactivation
rate. The regeneration of catalyst by O2/H2
treatment is under investigation. Due to its apparent simplicity,
the process offers promise for destruction of chlorinated solvents
in gas streams produced by gas-sparging or soil vapor extraction
operations.
Removing
TCE by Membrane Air Stripping.
He,
T T Nakajima, R G Arnold, W Ela, and E A Betterton. Department
of Chemical and Environmental Engineering, University of Arizona,
Tucson, AZ, USA.
The
use of hydrophobic microporous polypropylene hollow fibers for transferring
TCE from water to gas phase was evaluated in this study. Contaminated
water and (initially) clean air were passed in countercurrent mode
through the hollow fiber reactor. Water was on the shell side (external
to the hollow fibers). The hydrophobic membrane prevents water and
air from mixing but permits the transmembrane transfer of hydrophobic
solutes like TCE. Following transfer to the gas phase, such contaminants
can be conveniently destroyed using a number of catalytic routes
under development in this lab. The membrane air stripping process
offers several advantages over conventional packed-tower air stripping,
including higher overall mass transfer coefficient (KLa),
independence of the transfer surface area from the air or water
flow rates, lower required air flow rate, avoidance of mist development,
elimination of tall structures, and more efficient off-gas treatment
with GAC. A reactor performance model was developed on the basis
of empirical correlations, and engineering economics were considered.
Photo-treatment
of Perchloroethene in Soil Vapor at Mission Uniform and Linen Service,
Tucson, AZ.
BE
Barbaris1*, DS Samorano2, EA Betterton1,
RG Arnold2, WP Ela2, LJ Berry2,
JN Lever1. 1Department of Atmospheric
Sciences (520-621-6832); 2Department of Chemical and
Environmental Engineering (520-621-6044); The University of Arizona,
Tucson, AZ 85721.
Perchloroethene
(PCE) is being scrubbed from contaminated soil vapor by contact
with a 2-popanol/acetone solution (9:1 v/v) and then destroyed by
exposure to sunlight in a photoreactor. The treatment system (scrubber
and photoreactor) has been tried and tested at two landfill locations
over the past two years and is currently operating at the Mission
Uniform and Linen Supply facility, 301 South Park Avenue in Tucson.
Soil vapor is being extracted and treated by contractors using a
conventional granulated carbon absorption system. Soil vapor containing
~200 ppmv PCE is readily obtained for our purposes by
diverting a small flow of soil vapor (1-10 L/min) to the experimental
treatment system. In the experimental system, contaminated soil
vapor is pumped at a rate of 10L/min into the bottom of a 60 cm
x 5 cm countercurrent column packed with ½" ceramic Berl saddles.
PCE is stripped by dissolution in the 2-propanol/acetone mixture
that flowed downward through the scrubber. Field data indicate that
the scrubbing efficiency is consistently above 95 percent. The 2-propanol/acetone
solvent, containing the scrubbed PCE, is pumped to the rooftop photoreactor,
a 3-meter glass tube (2.5 cm diameter), where the PCE is destroyed
by solar-promoted photolysis (Betterton et al., Environ. Sci.
Technol., 2000, 34, 1229 - 1233). The solvent is then returned
to the scrubber to close the loop. The solvent is pre-loaded with
PCE throughout the night and then exposed to sunlight while being
recirculated in the photoreactor during the day (without further
contact with the soil vapor). The effectiveness of the photoreactor
is determined by comparing liquid-phase concentrations of PCE and
other targets in the reactor influent and effluent. PCE concentrations
are typically lowered by 90 percent or more within one hour of exposure.
Results also indicate that the reaction is inhibited by oxygen in
the soil vapor (14%) percent. However, we are able to eliminate
(photoreduce) interfering oxygen and also the PCE by increasing
the exposure time.
Fate
of Endocrine Disrupting Chemicals - During Wastewater Treatment
and Polishing Treatments.
Conroy
O1, Turney KD1, Lansey KE2, and
Arnold RG1. Departments of Chemical and Environmental
Engineering1 and Civil Engineering2 University
of Arizona, Tucson, AZ.
There
is widespread concern over environmental and human health effects
arising from exposure to estrogenic and other endocrine-disrupting
compounds in water. In this study, a competitive binding assay for
the human estrogen receptor, ER-b, was used to assess the presence
of endocrine-disrupting compounds during wastewater treatment and
natural processes that polish wastewater effluents - specifically,
infiltration to groundwater and passage through a constructed wetlands.
Samples were taken from the Roger Road Wastewater Treatment Plant,
the Sweetwater Recharge Facility, and the Constructed Ecosystem
Research Facility in Tucson, AZ. Organic constituents were concentrated
on C18 disks for determination of an EC50 value, the
concentration that displaced 50% of the labeled estrogen from ER-b.
EC50 increased at least three-fold as a consequence of
secondary (bio-tower) wastewater treatment. However, a 35-fold increase
in the EC50 was observed during the infiltration of secondary
effluent to groundwater at 130 feet below land surface. A decrease
in the EC50 (greater endocrine-disrupting potential)
resulted from wetlands treatment.
Up-Regulation
of P68 and GDI in Rabbit Renal Cortical Slices Exposed to Low Level
Arsenic.
X-H
Zheng and A. J. Gandolfi. Department of Pharmacology and
Toxicology, College of Pharmacy, University of Arizona, Tucson,
AZ
Our
previous studies demonstrated rabbit renal cortical slices can be
used to study the low-level toxic effects of arsenic compounds [As(III)
and As(V)]. Using cDNA microarray, we have determined the gene expression
profile in the precision-cut rabbit renal cortical slices exposed
to low-level As(III) and As(V). Although there were common genes
affect by both As(III) and As(V), there were also specific gene
only altered by a specific As chemical species. Among the differential
expressed genes, p68 (the DEAD box protein, which is an established
RNA-dependent ATPase and RNA helicase), is up-regulated in As(III)-treated
slices. GDP-dissociation inhibitor (GDI) is also up-regulated in
As(V)-treated slices. To validate the microarray data, cDNA clones
of p68 and GDI were isolated, and the sequences of the cDNA clones
were confirmed prior to purification and probe labeling. Adult New
Zealand White rabbit renal cortical slices were incubated with (10
nM - 10 m M) As(III) or As(V) in waymouths 752/1 media. At
various time points (2-8 hr) total RNA was isolated from 2 pooled
rabbit renal cortical slices, separated on 1.2 % agarase/formaldehyde
gel, and transferred to nylon membrane. The blots were probed by
(a-32P) dCTP labeled cDNA probes specific for p68 or
GDI. The Northern Analysis revealed up-regulation of p68 in As(III)-treated
slices (- 2-fold from control) that was seen as early as 2 hr. Whereas
the up-regulation of GDI in As(V)-treated slices increased 40 %
by 6 hr. Our findings are in agreement with our earlier cDNA microarrays
studies and help us identify specific marker genes that are indicative
of a low-level exposure to specific chemical forms of arsenic. [NIH
ES 04940]
Low-Level
Arsenite does not Affect the Ubiquitin Conjugating Activity of Rabbit
Renal Slices or Human Embryonic Kidney Cells.
D
S Kirkpatrick, J M Catania, KV Dale, and A J Gandolfi.Department
of Pharmacology and Toxicology, University of Arizona, Tucson, AZ,
USA.
Our
previous work with rabbit renal slices has shown that low-level
arsenite exposures can stimulate an accumulation of ubiquitin (Ub)-protein
conjugates. Additionallly, arsenite exposure (1 m M, 24 hr)
in human embryonic kidney (HEK) cells stimulated a similar increase
in the amount of Ub-conjugated proteins. Increases were detected
in HEK lysates by western blotting with anti-ubiquitin antibodies.
It was hypothesized that the increases in Ub-protein conjugates
seen by western blot result from an arsenic-induced increase in
the levels/activity of the Ub-conjugating enzymes. To investigate
this mechanism, rabbit renal slices and HEK cells were exposed to
nM-m M concentrations of arsenite. Lysates were tested for
their ability to incorporate 125I -labeled ubiquitin
into ubiquitin-protein conjugates. Conjugation of 125I-ubiquitin
to cellular proteins is an ATP-dependent process carried out by
a family of ubiquitin-conjugating enzymes. Labeled samples were
separated by SDS-PAGE and detected by autoradiography. Bands in
the gel corresponding to a molecular weight of greater than 40 kDa
were determined to be cellular protein conjugated by ubiquitin.
Data from these experiments show no significant differences in the
amount of protein-bound 125I-Ub between control slices
and those treated with up to 5 m M arsenite. Similarly, data
from HEK cells treated with concentrations as high as 1 m M
arsenite showed no changes in the ability to incorporate 125I-Ub.
Since these data discount the importance of arsenic-induced increases
in conjugating activity, previously noted increases in conjugated
proteins must be resulting from alternate mechanisms. Among the
possible alternatives are alterations in proteosome activity or
effects on expression of ubiquitin itself. Ongoing work seeks to
identify this mechanism as well as the specific proteins conjugated
by ubiquitin following low-level arsenic exposure. (NIH ES04946,
NIH ES 06694, NIH ES 07091)
Monomethylarsonous
Acid (MMAIII) and Dimethylarsinous Acid (DMAIII)
Toxicity in Hamsters.
JS
Petrick1, WR Cullen3, HV Aposhian1,2.
Department
of Pharmacology & Toxicology1 and Molecular & Cellular Biology2, The University of Arizona, Tucson,
AZ; Department of Chemistry3, University of British Columbia,
Vancouver, BC, Canada.
Recent
toxicological studies have implicated trivalent methylated arsenicals
as toxic intermediates of inorganic arsenic metabolism (Petrick
et al., 2000). Monomethylarsonous acid (MMAIII)
and dimethylarsinous acid (DMAIII) are methylated trivalent
arsenicals produced in the methylation of inorganic arsenic. MMAIII
is significantly more cytotoxic than arsenite in mammalian cell
culture. Cytotoxicity of DMAIII was approximately equal
to that of sodium arsenite (Styblo et al., 1999). Arsenite
is a potent inhibitor of mammalian pyruvate dehydrogenase (PDH)
in vitro. The inhibition of this critical mitochondrial enzyme
by arsenite, MMAIII or DMAIII was studied
in vitro in kidney homogenate of untreated male Golden Syrian
hamsters. Arsenite of MMAIII [as CH3AsI2
or as (CH3AsO)4] inhibited PDH activity in
vitro by 50% at concentrations of 136, 69, and 65 m M,
respectively. DMAIII, as (CH3)2AsI,
did not inhibit PDH at concentrations up to 400 m M. MMAIII
is thus a two fold more potent inhibitor of hamster kidney PDH in
vitro than sodium arsenite. These findings implicate vicinal
dithiol (lipoic acid) binding as a potential mechanism of action
for MMAIII and show that MMAIII is a toxic
intermediate of inorganic arsenic metabolism. (Supported in part
by the NIEHS Superfund Basic Research Program Grant ES-04940 and
SWEHSC P30-ES-06694).
Sodium
Arsenite Enhances AP-1 And NFk B DNA Binding and Induces Stress
Protein Expression in Precision-Cut Rat Lung Slices.
J
B Wijeweera and R C Lantz. Department of Cell Biology and
Anatomy, University of Arizona, Tucson, AZ, USA.
Arsenic
is a known human carcinogen. These studies were designed to examine
the impact of low arsenite concentrations on immediate early gene
expression in precision-cut rat lung slices. Precision-cut lung
slices are a versatile in-vitro system for toxicity studies as they
preserve the architecture and cellular heterogeneity of the lung.
Since 0.1-100 m M arsenite did not compromise slice viability
at 4 hr, effects of arsenite on the expression of c-jun/AP-1, NFk
B, HSP 32, HSP 72, HSP 60 and HSP 90 were studied using these concentrations
of arsenite at 4 hr. Nuclear c-jun was increased by 10 m M
and 100 m M arsenite, while NFk B was not affected. Gel
shift assays indicated that 10 m M arsenite resulted in an
enhanced DNA binding activity of both AP-1 and NFk B. Confocal
microscopic analysis of AP-1 indicated nuclear localization of this
transcription factor mainly in type II epithelial cells and alveolar
macrophages. Nuclear localization of NFk B was lower than that
observed for AP-1 while, most of the NFk B was localized to
cytoplasm of type II epithelial cells and alveolar macrophages.
HSP 32 was increased by 1.0 m M and 10 m M arsenite while,
HSP 72 was increased by only 100 m M arsenite. HSP 60 and HSP
90 were not changed by arsenite. These studies indicate that non-cytotoxic
concentrations of arsenite are capable of affecting signal transduction
pathways and gene expression in the lung.
The
Influence of Previous Arsenic Exposure on the Rate of Arsenite Oxidation
by Soil Microorganisms.
EA
Casarez1, JB McQuaid2, IL Pepper2,
and DE Carter1. 1Department of Pharmacology
and Toxicology; 2Department of Soil, Water, and Environmental
Sciences; University of Arizona, Tucson, AZ, USA.
Arsenic
(As) is ubiquitous in the environment, both as a natural component
of rocks and minerals and as a contaminant from human activities.
Arsenic may be present in different forms, each differing in bioavailability,
mobility, and toxicity. The species of As present is affected by
both biotic and abiotic influences. This research compares the rates
of arsenite (AsIII) oxidation in soils with different histories
of As contamination. We hypothesize that previous exposure to As
has affected the soil microbial population, which will therefore
change how additional As is metabolized. A control soil (no detectable
As) and three soils from a smelter site with low, medium, and high
total As levels were spiked with 0, 100, 500, or 1000ppm As as AsIII.
The soils were monitored for microbial growth and water extractable
As at 0, 1, 3, 7, and 14 days. Arsenic speciation was determined
in the water extracts by high performance liquid chromatography-hydride
generation-atomic fluorescence spectrometry, while changes in the
microbial community were monitored by heterotrophic plate counts
and 16S rDNA denaturing gradient gel electrophoresis. The initial
number and diversity of the soil microorganisms was less in the
soils with histories of higher As contamination. These soils, however,
had faster rates of AsIII disappearance, with a concomitant increase
in arsenate, than soils previously exposed to low or no As. Trends
indicate that the larger the additional AsIII spike, the faster
the rate of AsIII disappearance in pre-exposed soils, while the
opposite is true in the control soil. The decreases in water-extractable
AsIII are biologically mediated since AsIII levels in similarly
treated sterilized soil samples remained constant. The additional
AsIII had no adverse effects on the number or diversity of microorganisms
in the pre-exposed soils after 14d-one microbial population even
became more dominant with increasing AsIII. Although AsIII oxidation
is seen in all the biologically active soils, the rates of the reaction
are correlated with previous As exposure. This may be due to selection
pressures for microorganisms with As resistance and AsIII oxidase
activity.
Arsenite
Cytotoxicity in HK-2 Cells, an Immortalized Human Proximal Tubular
Epithelial Cell Line.
MA
Peraza, AJ Gandolfi, and DE Carter. Department of Pharmacology
and Toxicology, University of Arizona, Tucson, AZ.
Arsenic
is an environmental toxicant and a human carcinogen of the skin,
lung, urinary bladder, liver, and kidney. The kidney is a known
target organ of arsenic toxicity and is critical for both in
vivo arsenic biotransformation and elimination. This study investigates
the potential of an immortalized human proximal tubular epithelial
cell line, HK-2, to serve as a representative model for low level
exposures of the human kidney to arsenic. To determine the toxicity
of arsenic in this cell line, HK-2 cells were exposed to sodium
arsenite at concentrations ranging from 100 nM to 1 mM for 24 hours.
Acute cytotoxicity was assessed by determining the leakage of lactate
dehydrogenase (LDH) and the mitochondrial metabolism of the tetrazolium
salt, MTT. Data from these experiments show that significant increases
in cytotoxicity did not occur at 24 hours at concentrations ranging
from 100 nM to 10 µM arsenite. At 24 hours exposure to 25 µM arsenite
caused a release of 40% of total LDH, and maximal release of LDH
(100%) was achieved with 50 µM, 100 µM and 1mM arsenite. Similar
results for the effect of arsenite on mitochondrial MTT metabolism
were observed. Exposure to 10 µM and 25 µM arsenite resulted in
a 20% and 75% reduction of MTT metabolism, respectively. A maximal
decrease (85%) of MTT metabolism occurred in cells exposed to concentrations
of 50 µM and above. Chronic cytotoxicity was assessed by determining
leakage of LDH at similar concentrations of arsenite for periods
of 48 to 168 hours (7 days). Significant increases in LDH leakage
were not observed for cells exposed to 100 nM, 1 µM, or 10 µM arsenite
at any time point up to 7 days. By 48 hours, release of 75% of total
LDH was observed in cells exposed to arsenite at concentrations
of 25 µM, and maximal LDH leakage was observed in cells exposed
to concentrations of 50 µM and above. Ongoing work seeks to determine
arsenic metabolism in HK-2 cells at the sub-cytotoxic concentrations
determined in this study. Current and future results will help determine
the value of this cell line in the characterization of the effect
of low level arsenic exposure on the human kidney. (T32 ES07091;
P42 ES04940).
Health
Effects of Hazardous Materials Exposures.
JL
Burgess, DF Kovalchick, JF Lymp, KB Kyes, and WO Robertson.
College of Public Health, University of Arizona, Tucson, AZ;
University of Washington; Washington Poison Center, Seattle, WA.
Objective:
To study adverse outcomes in individuals exposed to hazardous materials.
Methods: Individuals exposed during hazardous materials incidents
were contacted to complete a questionnaire within 8-40 days, and
medical records were reviewed when available. Logistic regression
analysis adjusting for correlation using GEE was used to identify
predictors of adverse outcome. Results: From 12/97 to 10/99,
87 incidents were reported. 202 (59%) of 339 subjects with contact
information were surveyed. 51 (25%) subjects had persistent medical
symptoms. 18 (9%) left work or school for >2 days. Medical records
were available for 79 (66%) of 119 subjects evaluated in a health
care facility. Medical treatment was reported in 46 (58%) and objective
abnormalities in 57 (72%). For persistent symptoms, dermal exposures
(OR 3.75, 95% CI 1.47-9.59), ³ 3 alcoholic drinks/week (3.16,
1.13-8.80), and present or past use of psychiatric medications (2.68,
1.15-6.28) were significant predictors. For time loss, being divorced,
widowed, or separated (8.36, 1.40-50.1), asthmatic (4.92, 1.29-18.7),
or having initial dermal symptoms (6.77, 1.34-34.2) were significant
predictors. Of patients with medical records, those with preexisting
hypertension (10.6, 1.82-61.3) were more likely to receive medical
treatment or have objective medical findings, while those with inhalation
exposures (0.17, 0.04-0.78) and those decontaminated at the scene
(0.14, 0.03-0.69) were at reduced risk. Conclusions: Both
incident and individual patient factors were associated with persistent
symptoms, time loss, and medical treatment or objective medical
findings.
House
Dust and Inorganic Urinary Arsenic Levels in Two Arizona Mining
Towns.
TA
Hysong, JL Burgess, MK O'Rourke. Department of Community
and Environmental Health Practice and Policy, University of Arizona,
Tucson, AZ, USA.
Residents
of copper mining and smelting towns may have increased risk of arsenic
exposure due to elevated arsenic levels in environmental media.
The influence of arsenic in house dust on urinary biomarker concentrations,
however, is incompletely known. To determine the relationship of
arsenic in house dust to inorganic urinary arsenic concentrations,
a door to door survey was conducted from July to October 1999 in
Hayden and Winkelman, Arizona. A total of 122 households participated,
eight-five of which provided dust samples. Dust was sieved and analyzed
for total arsenic content using x-ray fluorescence. Urine was collected
at first morning void from each household member willing to participate
and analyzed via HG-AAS for total arsenic. Speciation of arsenic
was performed in those samples with total arsenic greater than 10μg/L
(N=106). Forty-seven households had both inorganic urinary arsenic
and house dust samples. In many cases there was more than one urine
sample from each home that provided a dust sample. The generalized
estimating equation was used to determine the relationship between
urinary and house dust arsenic concentrations, allowing adjustment
for the correlation of measurements obtained from the same home.
After adjusting for age and gender, only eating seafood in the past
three days and smoking contributed significantly to inorganic urinary
arsenic concentrations. Arsenic in house dust was not significantly
associated with inorganic urinary arsenic in this adult population.
Results of this study are consistent with others that have reported
no association between house dust and inorganic urinary arsenic
concentrations in adult populations.
Threshold
Affects of TCE Contaminated Maternal Drinking Water on Fetal Heart
Development in the Sprague Dawley Rat.
P
D Johnson, S J Goldberg, B V Dawson. Department of Pediatric
Cardiology, The University of Arizona, Tucson, Arizona, USA
Halogenated
hydrocarbons such as trichloroethylene (TCE) are among the most
common water supply contaminants in the United States and abroad.
Epidemiologic studies have found an association, but not cause and
effect relation, between halogenated hydrocarbon contamination and
increased incidence of congenital cardiac malformations or other
defective birth outcomes. Avian and rat studies demonstrated statistically
significant increases in the number of congenital cardiac malformations
in those treated with high doses of TCE, either via intrauterine
pump or in maternal drinking water, as compared with controls. This
study attempts to determine if there is a threshold dose exposure
to TCE above which the developing heart is more likely to be affected.
Sprague-Dawley
rats were randomly placed in test groups and exposed to various
concentrations of TCE (2.5 ppb, 250 ppb, 1.5 ppm, 1100 ppm) in drinking
water or distilled water (Control group) throughout pregnancy. The
percent abnormal hearts in the treated groups ranged from 0 to 10.48%,
respectively, with controls having 2.1% abnormal hearts. The data
from this study indicate not only that there is a statistically
significant probability overall of a dose response to increasing
levels of TCE exposure but also that this trend begins to manifests
itself at relatively low levels of exposure (i.e. <250ppb). If
maternal rats are exposed to these levels during pregnancy an associated
increased incidence of cardiac malformations in developing rat fetuses
is found.
Handling
of the Mercury-Chelator DMPS (2,3-Dimercapto-1-Propane-Sulfonic
Acid) by the Human Organic Anion Transporter (hOAT1).
F
Islinger1,2, WH Dantzler2, SH Wright2.
1Dept of Physiology, University of Arizona, Tucson,
AZ; 2Dept of Physiology, University of Wurzburg.
Although
DMPS is the preferred clinical antidote for mercury poisoning, the
cellular and molecular basis of its efficacy is unknown. Previous
studies implicated the "classical organic anion secretory pathway" in the renal secretion of DMPS. The basolateral component of this
pathway - hOAT1 - has recently been cloned. We used the Xenopus
oocyte expression system to study directly the interaction of
DMPS and its mercury chelates with hOAT1. Activity of hOAT1 was
assessed by measurement of [3H]PAH uptake (Kt 3.9 ± 1.3 m M). Whereas 100 mM of probenecid and bromsulfopthalein
completely blocked hOAT1-mediated PAH uptake, 1 mM tetraethylammonium,
taurocholate, penicillin G, and 2,3-dimercaptosuccinate had no effect
on PAH transport. Reduced DMPS proved to be a potent inhibitor of
PAH uptake (IC50 of 22 ± 8.4 m M). Because circulating
DMPS is extensively oxidized, we examined the interaction of oxidized
DMPS (DMPSS2-) with hOAT1 and found it to be an effective
inhibitor of PAH uptake (IC50 of 60 ± 13.6 m M).
Titrating 40 m M DMPSH with HgCl2 lead to a systematic
decrease in the inhibition of PAH-Uptake; indeed, 40 mM DMPSH plus
40 mM HgCl2 produced no inhibition. We suggest that hOAT1
supports the entry of both reduced and oxidized DMPS into proximal
renal cells, but does not interact with metal chelates of DMPS.
(ES-06694).
Determinants
of Structural Specificity of the Human Organ Cation Transporter,
hOCT1.
DJ
Bednarczyk, WH Dantzler, SH. Wright. University of Arizona,
Tucson, AZ.
A
stable line of HeLa cells expressing hOCT1 was used to investigate
the structural and chemical characteristics that influence binding
of substrate to the transporter. Expression of hOCT1-mediated transport
was selected for in antibiotic-resistant cells following transient
transfection by monitoring mediated accumulation of the fluorescent
cation, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl] trimethylammonium.
Uptake of [3H]tetraethylammonium into these cells was
linear for five minutes, and the 60 minute accumulation of TEA was
20-fold greater than that observed in wild-type HeLa cells. A series
of n-tetraalkylammonium compounds was used to determine the effect
of increasing lipophilicity on the binding of substrate to hOCT1.
Increasing hydrophobicity (increasing alkyl chain length) was correlated
with decreasing values for IC50s: TEA (IC50
of 165 mM; n=3); tetrapropylammonium (20 mM; 3); tetrabutylammonium
(6.5 mM; 3); and tetrapentylammonium (1.8 mM; 3). The steric influence
of planar, hydrophobic moieties on the binding of substrate to hOCT1
was examined using a series of substituted phenylpyridinium compounds.
Inhibitory effectiveness was again associated with increasing lipophilicity
of these compounds. Such correlative data may provide information
about structural and chemical constraints of the organic cation
binding site. (DK58251)
Characterization
of Molecular Markers of Trichloroethylene Exposure during Heart
Development.
FM
Blachere, PA Thorne, JM Collier(1), PD Johnson, RB Runyan(1), and
O Selmin(2). Department of Pediatrics, Section of Cardiology,(1)
Department of Cell Biology and Anatomy, and (2) Department of Nutritional
Sciences, University of Arizona, Tucson, AZ. Trichloroethylene
(TCE), a halogenated hydrocarbon, is a major contaminant of groundwater
in Tucson, Arizona. Preliminary epidemiological and animal studies
established a correlation between fetal heart defects and maternal
exposure to TCE. In effort to better understand the effects of TCE
on cardiac development, our research has focused on elucidating
the molecular mechanisms that mediate embryonic heart development.
In this study, we have been examining the effects of TCE exposure
on rat embryonic heart in-utero. Utilizing the PCR select subtractive
hybridization method (SSH), differential gene expression was observed
in TCE-treated embryonic hearts. In particular, a number of genes
were found to be negatively regulated, including the GPI-linked
protein p137 and the sarcoplasmic reticulum calcium regulatory protein
Ca+2 ATPase, as a result of TCE exposure. Down regulation of p137
expression was confirmed by RT-PCR and Western analysis in which
protein isolated from heart or embryo exposed to 110ppm TCE was
probed with p137 polyclonal antibodies. In addition, the same antibodies
were shown to inhibit mesenchymal cell invasion using in-vitro collagen
gel assays. Current research is underway to determine how TCE regulates
expression of Ca+2 ATPase during embryonic heart development. Concurrent
to rat in-utero studies, we have also been examining the effects
of TCE exposure on an ATCC rat myocyte cell line (H9c2). RT-PCR
data indicate that H9c2 cells express the TCE-metabolizing enzymes
CYP2E1 and Alcohol dehydrogenase (ADH), both also present in rat
embryo. Further experiments are in progress to evaluate the sensitivity
of the H9c2 at the gene expression level to TCE exposure. Future
work will use the H9c2 cells as an in-vitro system for transfection
and reporter gene studies that will shed light on the molecular
mechanisms of TCE-mediated gene regulation. Supported by: grant
number P42ES04940 from the National Institute of Environmental Health
Sciences, NIH.
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