TRICHLOROETHYLENE TCE MW 131 Cl H C C

TRICHLOROETHYLENE (TCE) MW= 131 Cl H C =C Cl Since 1940 s, in the US. Cl EPA MCL: 5 ppb (38 n. M)

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) PCR m. RNA T C UP-REG. m. RNA C Amplified T DOWN-REG. Amplified

Dot Blot: g. C 1 q. BP expression Dig-labeled g. C 1 q. BP c. DNA was hybridized to 1 or 2 ug of total c. DNA isolated from unexposed embryo, heart, or exposed heart tissue (110 ppm)

p 137 • 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+2 ATPases, 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 c. DNA 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 2 a and b transcripts.

SERCA 2 TRANSGENIC MICE • Serca 2 a Null heterozygous: Ca++ uptake into the SR. No outward phenotype (Periasamy et al. 1999) • Serca 2 a Overexpressed: Calcium transients, myocardial contractility and relaxation (He et al. , 1997) • Serca 2 b 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 labeled c. DNA Cy-5 labeled c. DNA Mix c. DNAs and apply to array. Hybridize under coverslip. Spots with more Cy 3 are genes down-regulated by treatment Spots with more Cy 5 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.