Transcript TCE - University of Arizona

TRICHLOROETHYLENE
(TCE)
MW= 131
Cl
H
C =C
Cl
Since 1940s, in the US.
Cl
EPA MCL: 5ppb (38nM)
TCE
• Industrial degreasing agent
• Solvent used in dry cleaning solutions, paint
removers, cosmetics, adhesives, household
cleaners, and spot removers.
• Anesthetic
• Chlorination product (TCAA, DCA)
TCE : WHERE?
• Air : Urban 3 times > than rural.
• Water: rain, surface water, groundwater,
drinking water, and sea water.
• Marine sediments, marine invertebrates,
marine mammals, foods, mother’s milk,
human urine and blood.
• Foods: butter and margarine, cheese,
processed food, cereals.
TCE IN GROUNDWATER
• 93% of the public water system obtained
from groundwater
• Between 9 and 34% of drinking water
supply sources tested in the US have some
TCE contamination. TCAA and DCA are
products of chlorination.
• MCL violations are rare for any extended
period.
• Private wells?
TCE TARGET OF EXPOSURE
• Lung
• Gastrointestinal tract
• Skin
TCE AND CANCER
• IN ANIMALS:
• Liver, kidney, lung tumors and lymphomas
in rats and mice.
TCE AND CANCER
• IN HUMANS:
• Kidney and liver cancers, lymphomas (NonHodgkin’s and Hodgkin’s disease).
• Pancreatic cancer, multiple myeloma,
prostate and skin cancer, leukemia.
CONCLUSION
• TCE IS A PROBABLE CARCINOGEN TO
HUMAN BASED ON LIMITED HUMAN
EVIDENCE AND SUFFICIENT ANIMAL
EVIDENCE (International Agency for
Research on Cancer, 1995)
NONCANCER EFFECTS OF TCE
• CNS : Dizziness, headache, sleepiness, nausea,
confusion, blurred vision, weakness (acute
exposure)
• Reproductive/Developmental: Miscarriage,
cardiac defects, eye malformations, neural tube
and oral cleft defects, abnormal sperm
morphology(mice), hearing and speech
impairment and increase in urinary tract disorders
(children 0-9 years of age).
Congenital Heart Disease
• Affects almost 1% of newborns and may account
for 2-10% of stillbirths and spontaneous abortions
• Has a significant environmental component as only
about 20% of defects have a clear genetic cause
• Can be caused by retinoic acid(+++), TCE (3),
aspirin (2), fetal alcohol (?), cocaine (?)
• Higher in Yuma and transborder areas
(environmental?)
TCE is a Cardiac Teratogen
• Epidemiologic Studies
• Animal Studies (chick, rat, in utero and
drinking water exposure)
• In vitro Experiments (Collagen gel assay)
Cardiac Looping
Epithelial-Mesenchymal Cell Transformation in the Heart
Cushion Tissue Forms AV Valves and Septa
Explant on Collagen Gel
Endothelial Outgrowth
Conditioned media or ECM
Invasion
How do you find genes with
altered expression?
Hypothesis
Molecules with altered expression in
the rat embryo, as a consequence of
maternal exposure to TCE (or As),
can be used to identify actual
effectors of birth defects.
Alternatively, they might prove useful
as specific biomarkers of
environmental exposure.
Technologies
• PCR Select-Subtractive Hybridization
(SSH)
PCR
mRNA
T
C
UP-REG.
mRNA
C
Amplified
T
DOWN-REG.
Amplified
Dot Blot: gC1qBP expression
Dig-labeled gC1qBP cDNA was hybridized to 1 or 2 ug of
total cDNA isolated from unexposed embryo, heart, or
exposed heart tissue (110 ppm)
p137
• GPI-linked protein identified in Caco-2
cells (Ellis et al., 1992), and in several
tissues. Possible function as second
messenger.
• Very conserved among species (human, rat,
mouse, chicken)
The SERCA Family
• Sarco/endoplasmic reticulum Ca+2ATPases,
mediating the uptake of Ca+2 into
intracellular stores.
• Encoded by three genes in higher
vertebrates: SERCA 1, 2, 3.
TCE down-regulated SERCA 2
• The differentially expressed cDNA isolated
from cardiac embryonic tissue is 300 bp.
• Blast analysis indicates 94-95% homology
to the SERCA 2 gene, in the 3’ untranslated
region common to both SERCA 2a and b
transcripts.
SERCA2 TRANSGENIC MICE
• Serca2a Null heterozygous: Ca++ uptake into
the SR. No outward phenotype (Periasamy et al.
1999)
• Serca2a Overexpressed: Calcium transients,
myocardial contractility and relaxation (He et al.,
1997)
• Serca2b Overexpressed: SR calcium transport
function and cardiac contractility (Greene et al.,
2000)
Future Studies
• Utilize transgenic mice (over-expressing or
null heterozygous for SERCA 2) to test their
altered sensitivity to TCE in vitro.
SUMMARY
• Several potential markers of TCE exposure
in the developing heart have been isolated.
• Characterization of function and expression
is in progress.
• Tools for identification of proximal
effectors of TCE induced heart defects.
Microarray Measurement of
Differential Gene Expression
Control cells
Cells +Toxicant
RNA isolation
Reverse
transcription
Cy-3 label
Cy-3
labeled
cDNA
Cy-5 label
Cy-5
labeled
cDNA
Mix cDNAs and apply
to array. Hybridize
under coverslip.
Spots with more Cy3 are genes down-regulated by treatment
Spots with more Cy5 are genes up-regulated by treatment
Mixed spots are genes unaffected by treatment
Future Directions
• Elucidate the molecular pathways used by
TCE to disrupt normal development.
• Use the most sensitive bio markers for
translation into prevention and/or
intervention measures on affected
populations.