Investigation of Apoptosis and Metabolism as Mechanisms of Trichloroethylene Toxicity During Pregnancy

Abstract

Trichloroethylene (TCE), an organic solvent with multiple industrial uses, is associated with adverse pregnancy outcomes in epidemiological studies. In timed-pregnant Wistar rats, TCE exposure reduces fetal weight. Additionally, the TCE metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) stimulates reactive oxygen species (ROS) generation and apoptosis in HTR-8/SVneo placental cells as mechanisms of toxicity. By using aminooxyacetic acid (AOAA) to decrease cysteine conjugate beta-lyase (CCBL) activity and resulting toxicity of DCVC, prior studies in kidney cells demonstrated that the metabolism of DCVC into 1,2-dichlorovinylthiol (DCVT) by CCBL is critical for producing cell death. N-acetyl-L-cysteine (NAC) is a compound with ROS-scavenging properties and a supplier of an acetyl group that contributes to N-acetylation of DCVC. Because metabolism of DCVC is crucial for TCE toxicity, this dissertation hypothesizes that modification of TCE or DCVC metabolism during pregnancy modulates TCE or DCVC-stimulated apoptosis and toxicity. This dissertation uses AOAA and NAC as modulators of TCE or DCVC-stimulated toxicity. Chapter 2 described TCE-induced decreased fetal weight in timed-pregnant Wistar rats that was prevented by AOAA but not NAC pre/co-treatment. However, AOAA reduced CCBL activity in maternal kidney, but not maternal liver or placenta, suggesting that inhibition of kidney metabolism may have a role in the AOAA effect. Morphometric analysis of the placenta indicated that NAC pre/co-treatment with TCE relative to TCE treatment alone altered placental dimensions consistent with a delayed developmental phenotype. Treatments failed to stimulate any indication of placental apoptosis. Chapter 3 findings showed that TCE exposure stimulates changes in amniotic fluid of the rats. Changes in amniotic fluid of both sexes include decreased adenosine triphosphate, decreased adenosine diphosphate, decreased guanosine diphosphate, and altered pentose phosphate pathway and folate biosynthesis. Chapter 4 described mechanisms of DCVC-stimulated toxicity in the in vitro human trophoblast BeWo cell model. BeWo cells are known to syncytialize, or multinucleate and fuse, in response to forskolin to create the syncytiotrophoblast cell type that is the maternal-fetal interface in vivo. DCVC was found to increase caspase 3/7 activity and nuclear condensation or fragmentation, markers of apoptosis, in unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells. DCVC also stimulated changes consistent with increased ROS production in all three BeWo cell types, including increased hydrogen peroxide abundance and decreased PRDX2 mRNA expression. Chapter 5 showed NAC and AOAA pre/co-treatment modulation of DCVC-stimulated toxicity. NAC either exacerbated or did not affect the DCVC-stimulated response. Because NAC exerted a significant effect on increased CYP3A4 mRNA expression, it is possibile that NAC could have contributed to the CYP3A4-generation of the toxic N-acetyl DCVC sulfoxide (NAcDCVCS) metabolite. AOAA did not modify CCBL activity in BeWo cells regardless of differentiation status. However, syncytialized BeWo cells exhibited higher CCBL activity than unsyncytialized BeWo cells, which could explain increased susceptibility of syncytialized BeWo to DCVC apoptosis compared with unsyncytialized BeWo cells. Chapter 6 revealed that DCVC treatment during syncytialization could either exacerbate or reverse some cellular energy metabolism changes stimulated by syncytialization by itself. Notable reversal effects included ratios within purine metabolism. This research indicates a critical role for CCBL in DCVC- and TCE-stimulated toxicity during pregnancy and raises the possibility that NAC pre/co-treatment with TCE or DCVC could be detrimental. Apoptosis and disrupted energy metabolism could be major mechanisms of TCE or DCVC-stimulated toxicity. Important future work includes detection of reactive TCE metabolites and identification of enzyme targets of TCE.