TRICHLOROETHYLENE(TCE)

C =CH

Cl

Cl

Cl

Since 1940s, in the US. EPA MCL: 5ppb (38nM)

MW= 131

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 (Non-Hodgkin’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)

T C

PCR

UP-REG. Amplified

C T

DOWN-REG.

Amplified

mRNA

mRNA

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)

Effects of anti-p137 antibody on Total Numbers of Invading Mesenchymal Cells

0

10

20

30

40

50

60

70

80

90

untreated anti-rat IgG treated anti-p137 treated

Treatment Group

No. of mesenchymal cells

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.

p137 and Ca2+-ATPase Expression after TCE Exposure

02040

6080

100120

140160180

TCE Exposure Concentration

Ca2+-ATPasep137

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 ExpressionControl cells Cells +Toxicant

RNA isolation

Cy-3 label Cy-5 labelReverse transcription

Mix cDNAs and apply to array. Hybridize under coverslip.

Cy-3 labeled cDNA

Cy-5 labeled cDNA

Spots with more Cy3 are genes down-regulated by treatmentSpots with more Cy5 are genes up-regulated by treatmentMixed 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.