THE EFFECTS OF TRICHLOROETHYLENE ON HEART DEVELOPMENT
Item Type text; Electronic Dissertation
Authors Makwana, Om
Publisher The University of Arizona.
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Download date 05/10/2021 18:07:07
Link to Item http://hdl.handle.net/10150/204310 THE EFFECTS OF TRICHLOROETHYLENE ON HEART DEVELOPMENT
by
Om Makwana
______
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF CELL BIOLOGY & ANATOMY
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2010 2
THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Om Makwana entitled The Effects of Trichloroethylene on Heart Development and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of
______Date: 11/03/10 Raymond B. Runyan
______Date: 11/03/10 Ornella Selmin
______Date: 11/03/10 Scott Boitano
______Date: 11/03/10 R. Clark Lantz
Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
______Date: 11/03/10 Dissertation Director: Raymond B. Runyan 3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: Om Makwana 4
TABLE OF CONTENTS
LIST OF FIGURES...... 7
LIST OF TABLES...... 9
ABSTRACT...... 10
CHAPTER 1: Background and Literature Review...... 12
1.1 Preamble...... 12
1.2 Introduction...... 14
1.3 Heart Development...... 15
1.4 Epidemiological Studies...... 20
1.5 Animal Studies...... 21
1.6 Effects of TCE on Ca2+ and Blood Flow...... 24
1.7 Metabolism of TCE...... 25
1.8 Summary...... 28
CHAPTER 2: Exposure to Low-Dose Trichloroethylene Alters Shear Stress Gene
Expression and Function in the Developing Chick Heart...... 29
2.1 Introduction...... 29
2.2 Material and Methods...... 31
2.3 Results...... 37
2.4 Discussion...... 46 5
TABLE OF CONTENTS - continued
CHAPTER 3: Exposure to Low-Dose Trichloroethylene Results In Increased
Cytochrome P450 - 2C Subfamily Expression in the Developing Chick
Heart...... 53
3.1 Introduction...... 53
3.2 Material and Methods...... 56
3.3 Results...... 60
3.4 Discussion...... 74
CHAPTER 4: Exposure to Low-Dose Trichloroethylene Alters HNF4a
Function...... 79
4.1 Introduction...... 79
4.2 Material and Methods...... 82
4.3 Results...... 85
4.4 Discussion...... 101
CHAPTER 5: Summary and Conclusions...... 104
5.1 Summary...... 104
5.2 Experimental Results...... 105
5.2.1. TCE Alters Cardiac Function...... 105
5.2.2. TCE Induces CYP2C Expression In the Heart...... 107
5.2.3. HNF4A is an Apparent Proximal Mediator of TCE Effects on Gene
Transcription...... 108 6
TABLE OF CONTENTS - continued
5.2.4. Model...... 110
5.3 Future Directions...... 112
REFERENCES...... 115 7
LIST OF FIGURES
FIGURE 1.1. Critical points during heart development...... 17
FIGURE 1.2. Heart cushion development...... 19
FIGURE 1.3. Metabolic scheme of TCE...... 27
FIGURE 2.1. HH17 Whole Hearts were collected after 24 h exposure of TCE at 8 and 800 ppb...... 38
FIGURE 2.2. HH24 Whole Hearts were collected after 48 h exposure of TCE at 8 and 800 ppb...... 40
FIGURE 2.3. a. KLF2 Antibody Expression in HH17 hearts collected after 24-h exposure to 8 ppb TCE b. Illustration of a HH17 chick heart...... 42
FIGURE 2.4. Half-Width of isolated E18 cardiomyocytes exposed to 8 ppb TCE relative to control cardiomyocytes...... 44
FIGURE 2.5. Sarcomere Length of isolated E18 cardiomyocytes exposed to 8 ppb
TCE relative to control cardiomyocytes...... 45
FIGURE 3.1. Illustration of a HH17 chick heart...... 62
FIGURE 3.2. CYP1A4...... 65
FIGURE 3.3. CYP2C45...... 67
FIGURE 3.4. CYP2H1...... 69
FIGURE 3.5. CYP2C Localization...... 71
FIGURE 3.6. CYP2C Immunoblot...... 73
FIGURE 4.1. Interactome analysis of chick microarray...... 98 8
LIST OF FIGURES - continued
FIGURE 4.2. H9C2 cell exposed to 10PPB TCE for 30 minutes and 1 hour and then processed for CHIP analysis using HNF4a specific antibodies...... 100 9
LIST OF TABLES
TABLE 2.1 Real-time PCR primers...... 36
TABLE 3.1 Real-time PCR primers...... 59
TABLE 3.2a and 3.2b...... 61
TABLE 4.1: Genes up-regulated after 8 ppb TCE exposure...... 87
TABLE 4.2: Genes down-regulated after 8 ppb TCE exposure...... 88
TABLE 4.3: Genes up-regulated after 800 ppb TCE exposure...... 90
TABLE 4.4: Genes down-regulated after 800 ppb TCE exposure...... 91
TABLE 4.5A: Genes up-regulated after TCE exposure...... 93
TABLE 4.5B: Genes down-regulated after TCE exposure...... 94
TABLE 4.6: Most highly linked nodes in interactome analysis of TCE...... 99 10
ABSTRACT
Trichloroethylene (TCE; TRI; C2HCl3) is an organic solvent used as an industrial degreasing agent. Due to its widespread use and volatile nature, TCE is a common environmental contaminant. Trichloroethylene exposure has been implicated in the etiology of heart defects in human populations and animal models. Recent data suggest misregulation of Ca2+ homeostasis in a cardiomyocyte cell line after
TCE exposure (Caldwell, Thorne et al. 2008). We hypothesized that misregulation of Ca2+ homeostasis alters myocyte function and leads to changes in embryonic blood flow. In turn, changes in cardiac flow are known to cause cardiac malformations. To investigate this hypothesis we dosed developing chick embryos in ovo with environmentally relevant doses of TCE (8 ppb and 800 ppb).
We then isolated RNA from embryos at crucial time points in development for real-time PCR analysis of markers for altered blood flow. Based on this analysis, we observed effects on ET-1 (Endothelin-1), NOS-3 (Nitric Oxide Synthase-3) and Krüppel-Like Factor 2 (KLF2) expression relative to TCE exposure.
Additionally, we assessed cardiomyocyte function by isolating chick E18 cardiomyocytes from embryos exposed to TCE in ovo. Cells were measured for rate of contraction after pulsing with extracellular Ca2+ and electrical stimulation at a frequency of 1.0 Hz. These functional data showed an effect on Ca2+ handling in cardiomyocytes exposed to TCE. To investigate an apparent non-monotypic effect in the heart where 8 ppb produced a stronger effect than 800 ppb, we 11
isolated RNA from the developing heart and AV Canal to investigate the expression of several candidate Cytochrome P450s (CYPs) related to TCE metabolism. We observed a significant induction of multiple CYP2 family members in the developing heart after low dose TCE exposure. Together, these data suggest cardio-specificity of TCE as a teratogen and may reflect a requirement for normal calcium regulation of contractile function during organ development. 12
CHAPTER 1: BACKGROUND AND LITERATURE REVIEW
1.1. PREAMBLE
The environmental toxicant trichloroethylene or TCE has been associated to the formation of cardiac defects in children whose mothers had been exposed to
TCE while pregnant. The most common route of exposure of TCE is contaminated drinking water, which is then consumed directly, or absorption can occur through the skin while bathing in contaminated water (Research Council
2006). Mechanistically very little is understood about TCE’s effects on cardiac development. The most promising recent studies point to an alteration in Ca2+ handling capacity in cardiomyocytes and an alteration of embryonic blood flow through the developing heart (Drake, Koprowski et al. 2006; Caldwell, Thorne et al. 2008). Furthermore, work by Hogers el al. (1997) demonstrated that reduced cardiac blood flow can alone lead to morphological congenital heart defects. We propose to examine the hypothesis that TCE produces cardiac teratology primarily by reducing cardiac function during heart development with a resulting perturbation of morphogenesis.
Additionally, the developmental metabolism of TCE is poorly understood.
Recent studies of TCE exposure demonstrate a non-monotypic dose-response curve (Rufer, Hacker et al. 2010) and show that the metabolites of TCE
(trichloroacetic acid and dichloroacetic acid) demonstrate greater developmental toxicity than the parent compound TCE (Selmin, Thorne et al. 2005). To date 13
there are no in vivo studies demonstrating developmental metabolism of trichloroethylene. We propose to use the avian model system to demonstrate in vivo metabolism of trichloroethylene to aid in the understanding of the developmental cardio-toxicity induced after TCE exposure. Together, these approaches will focus on the molecular and cellular understanding of TCE- mediated teratogenesis of the heart. 14
1.2. INTRODUCTION
Trichloroethylene (TCE; TRI; C2HCl3) an organic solvent used as an industrial degreasing agent and can be found in many consumer products, e.g., typewriter correction fluids, paint removers, adhesives, spot removers and cleaning fluids (Page, Check et al. 2001). Due to its widespread use and volatile nature, TCE is a common environmental contaminant. TCE contamination can result from a variety of exposures including: evaporative losses during use; discharge to surface waters and groundwater by industry, commerce, and individual consumers; and leaching from hazardous waste landfills into groundwater (Page, Check et al. 2001). According to an EPA national groundwater survey TCE is the most frequently detected organic solvent in groundwater supplies, and is estimated to be in 9-34% of the drinking water supplies tested in the United States (Page, Check et al. 2001). Recently, TCE has been found in at least 852 of the 1,430 proposed sites for inclusion on the U.S.
EPA National Priorities List (Superfund) around the country (ATSDR 2003).
While data on environmental releases of TCE are limited the EPA Toxic Release
Inventory estimated that approximately 42 million pounds of trichloroethylene was released into the environment in 1994 (Scott and Cogliano 2000). Most of the TCE used in the United States is released into the atmosphere from vapor when used in degreasing operations. TCE can enter surface waters via direct discharges or groundwater through leaching from disposal operations (Scott and 15
Cogliano 2000). TCE exposure through inhalation has been associated with nerve, kidney and liver damage and even death (ATSDR 2003). Ingestion of TCE has been associated with liver and kidney damage, impaired immune system function, and impaired fetal development in pregnant women (ATSDR 2003).
Due to both human health effects and environmental abundance TCE exposure poses a relevant threat as an environmental contaminant.
1.3. HEART DEVELOPMENT
Organogenesis has been defined as the “greatest time of susceptibility to teratogenic exposure” by Hodgson et. al. 1997, since the heart is the first organ to develop in most vertebrates it is particularly susceptible to toxicant exposure.
TCE exposure has been associated with several heart defects in exposed human populations: ventricular septal defects, atrial septal defects and pulmonary stenosis (Goldberg, Lebowitz et al. 1990; Yauck, Malloy et al. 2004). Much of the signaling required for cardiac septal and valve formation occurs relatively early in vertebrate cardiac development. An exploration of the processes and events perturbed by TCE exposure can lead to the identification of specific pathways and molecular interactions most at risk in exposed embryos.
Embryonic heart development begins at gastrulation where cells invading the primitive streak are specified to the heart lineage (Hamilton & Hamburger
Staging - HH 2-3, Hamilton and Hamburger 1951). As embryonic development 16
continues, heart specific pre-cardiac mesodermal cells form two heart fields that give rise to myocardial and endocardial cells (HH 4-6). These two separate cardiac fields then fuse to form the cardiac crescent (HH 9), which distorts towards the midline of the embryo through growth and folding. The cells of the cardiac crescent then fuse across the anterior midline to form a linear heart tube
(HH 12). The heart begins to beat at about the time of this fusion. By HH17 four distinct regions can be observed in the developing heart: atrial region, atrial- ventricular canal, ventricular region and the outflow tract. It is the atrial- ventricular canal (AV canal) region that will eventually form the mitral and tricuspid valves and membranous ventricular septum of the mature heart
(Markwald, Fitzharris et al. 1977). 17
(A) (B) (C) (D)
Fig 1.1. Critical points during heart development. (A) Formation of the cardiac cresent at HH 9. (B) Formation of the linear heart tube at HH 12. (C) Looping of the heart tube shown at HH 24 (D) Sagital section of the four-chambered heart at HH 32. (Adapted from Black and Olson, 1999.) 18
The interstitial cells of the valves and septa are formed via an epithelial- mesenchymal transition of regional endothelium. This finding was first demonstrated by Runyan and Markwald (1983) in the collagen gel culture assay in which AV canal explants were incubated on a collagen gel. After overnight incubation cells could be observed migrating into the collagen gel, with mesenchymal fibroblastic morphology. Experiments showed that this EMT is induced by an interaction between the myocardium and the endothelium.
Subsequent studies showed the growth factor TGFβ to be a critical component of the interaction (Potts and Runyan 1989). Early studies reasoned that if TGFβ was required for the development of the valves and septa then TCE could have a direct role in altering the expression of TGFβ in the developing heart, thus resulting in valve and septal defects (Boyer, Finch et al. 2000). Although early studies involving TCE exposure did demonstrate an effect on TGFβ expression, we believe these effects to be nonspecific cytotoxicity due to the high levels of TCE exposure and the volatilization of the compound under in vitro experimental conditions. Furthermore, subsequent studies (Smith el. al., personal communication) have shown that at molecularly relevant levels of TCE exposure
(8 ppb) there appears to be no effect on TGFβ. This finding was further confirmed by a microarray experiment conducted by our laboratory after 8 ppb
TCE exposure (Makwana et. al., In Preparation). 19
Fig 1.2. Heart cushion development. (A) Early looped heart showing the curvature and overlap of the atrioventricular (AV) canal and the outflow tract HH17. A single atrium and ventricle are separated by the atrioventricular canal. The extracellular matrix (ECM) between the myocardium and the endothelium. The extracellular matrix becomes expanded into cushions in the atrioventricular canal by increased synthesis of matrix molecles from the myocardium. The cushions are populated by mesenchymal cells as a result of epithelial–mesenchymal transition and subsequent cell proliferation. (B) Sagital section of the heart that removes the overlap and emphasizes the epithelial–mesenchymal transition process that takes place in the atrioventricular canal and the proximal portion of the outflow tract. (C) Sagital section of the four-chambered heart (HH 32) showing structures (grey) derived from atrioventricular canal mesenchyme. Structures include the leaflets and chordae tendinae of the mitral and tricuspid valves, the membranous portion of the interventricular septum and the portion of the septum intermedium where the septum primum attaches. (Adapted from Camenisch and Runyan 2009.) 20
1.4. EPIDEMIOLOGICAL STUDIES
TCE was first linked to altered heart development in an epidemiological study that found an odds ratio of 3 for congenital heart disease in children living in an area of the Tucson Valley with TCE groundwater contamination in the range of 100-270 ppb (Goldberg, Lebowitz et al. 1990). The types of defects included defects expected from both myocardial and valvular problems. Although this initial study was controversial for methodological reasons based upon the appropriateness of the selected controls, an independent reevaluation of the raw data validated the conclusion that TCE is a cardio-teratogenic compound (Bove,
Shim et al. 2002). Further a University of Wisconsin epidemiological study demonstrated a correlation between the proximity of maternal residence to TCE emitting sites and increased heart defects in their children in Milwaukee, WI
(Yauck, Malloy et al. 2004). Each of these studies demonstrate that TCE is cardiac specific in its action as a teratogen with regard to human exposure.
Demonstration of a connection between congenital heart disease and TCE is important for several reasons. This has been a controversial issue for many years (see Boyer, Finch et al. 2000; and Dugard 2000). A recent review by Hardin et al. (2005) challenged the idea that TCE is a teratogen based largely upon inconsistencies in dose response data to various exposure protocols. However, an independent evaluation produced by the National Academy of Sciences (Research
Council 2006) concluded that the data pointed to a likely connection that needs 21
more study. The prevalence of TCE as a common soil and water contaminant requires a better understanding of the consequences of exposure levels. The current maximum EPA exposure limit is 5 ppb and there is pressure to raise the limit due to costs involved in soil and water remediation. For yet poorly understood reasons, TCE does not have a consistent dose response curve. Early studies focused on moderate (100 ppm) to high levels of exposure (1,100 ppm)
(Johnson, Dawson et al. 1998; Collier, Selmin et al. 2003). However, recent studies of gene expression in H9C2 cells suggest that there are biphasic curves with maximum effects between 10-100 ppb and again at 10-100 ppm (Caldwell,
Thorne et al. 2008; Selmin, Thorne et al. 2008). As described by Drake et al.
(2006), concentrations of TCE close to the EPA maximum are sufficient to alter cardiac hemodynamics. Thus an understanding of TCE developmental cardio- toxicity has implications for both our understanding of normal heart development and for public health and environmental remediation.
1.5. ANIMAL STUDIES
To explore the cardio-teratogenicity of TCE, Johnson et al. (1998) conducted a rat study in which 1,146 fetuses were examined after exposure to chronic high doses (up to 1,100 ppm) of TCE in maternal drinking water. They concluded that a strong correlation between TCE exposure and congenital heart defects could be verified in an animal model but was statistically significant only 22
at the highest doses. In contrast, exposure to an equivalent level of TCE (500 mg/ kg) provided by daily gavage in corn oil produced no defects (Fisher, Channel et al. 2001). In vitro studies showed that epithelial-mesenchymal transition by chick heart valve precursors was perturbed in the 50-250 ppm range (Boyer, Finch et al.
2000). Subsequent in vivo work with rat embryos exposed in utero to moderate doses (100 ppm) in maternal drinking water showed that TCE exposure altered expression of genes critical for heart development, including several calcium regulators (Collier, Selmin et al. 2003). Two additional studies have found cardio-teratogenicity in ranges from 8-800 ppb (Drake, Koprowski et al. 2006;
Drake, Koprowski et al. 2006) and 80 ppm (Mishima, Hoffman et al. 2006).
Studies on the rat myocyte H9C2 cell line show an in vitro sensitivity with the gene expression of sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a),
Ryanodine receptor 2 (Ryr2), Calcium/calmodulin-dependent protein kinase type
1 (CamK1) and calcium flux changes between 8 and 800 ppb (Selmin, Thorne et al. 2005; Caldwell, Thorne et al. 2008; Selmin, Thorne et al. 2008). Due to the role of calcium in muscle contraction, the consequences of TCE exposure on the functional and morphological formation of the myocardium may be relevant to understanding TCE mediated cardio-teratogenicity. As calcium is sometimes a second messenger molecule in many cell types, it may have additional relevance to cancer and immunological problems associated with TCE exposure in the adult
(Research Council 2006). 23
Since the initial human investigations into TCE as a cardiac teratogen, several animal models and dosing techniques have been applied with varied results. Rodents have shown diverse responsiveness to dosing, with rats being more susceptible than mice to TCE at respective doses (Watson, Jacobson et al.
2006). Similarly the routes, duration, and concentration of exposures varied severely among the various rodent studies (Watson, Jacobson et al. 2006). The avian model system seems to produce the most consistent results, which are replicable assuming a similar method of exposure (Watson, Jacobson et al. 2006).
Due to controversy over TCE safety levels between the Department of Defense and the EPA, the National Academy of Sciences was asked to prepare an evaluation of TCE entitled-Assessing the Human Heath Risk of
Trichloroethylene: Key Scientific Issues (Chiu, Caldwell et al. 2006). Regarding developmental toxicity, their report concluded that, “Multiple studies in several animal models, including mammalian and avian, suggest that trichloroethylene, or one or more of its metabolites (trichloroacetic acid and dichloroacetic acid), can cause cardiac teratogenesis (Research Council 2006).” Additionally, “of the studies the avian studies were the most convincing and mechanistic studies in birds have been performed,” leading to their recommendation that additional studies involving TCE mediated cardio-teratogenicity be performed in the avian model system (Research Council 2006). 24
1.6. EFFECTS OF TCE ON Ca2+ AND BLOOD FLOW
Considering our earlier findings regarding Serca2a and recent work suggesting misregulation of Ryr2 by TCE, we were especially interested in the functional observations of Drake et al. (2006). Their data showed that doses in the range 8-800 ppb reduced aortic flow in the exposed embryos. However, as these low doses also caused a hypertrophy of the valvular primordia, they attributed the change to physical obstruction of the flow by malformed AV valves.
In support of the previous demonstration of a loss of Serca2 in TCE-exposed rat embryos (Collier, Selmin et al. 2003) and microarray data from P19 cells showing loss of Ryr2 and CamK1 (Selmin, Thorne et al. 2008) an alternative explanation could be that reduced contraction of the myocardium has morphogenetic consequences. Recent studies by Caldwell et al. (2008) show that vasopressin- stimulated Ca2+ ion fluxes in H9C2 myocytes are reduced after 18 hr of exposure to TCE.
Considering the findings of Groenedijk el al. (2004), which showed that changes in flow produced by ligation of a vitelline vessel in the chick embryo produced an alteration of Endothelin-1 (ET-1), Nitric Oxide Synthatse-3 (NOS-3) and Krüppel-Like Factor 2 (KLF2) transcription concomitant with flow-mediated morphological changes in the heart. We reasoned that changes in flow could be measured molecularly by measuring endothelial expression of stress markers known to be indicative of flow. We directly measured the expression of these 25
flow sensitive genes (ET-1, NOS-3 and KLF2) by real-time PCR and showed that low level TCE exposure does alter expression of flow-sensitive genes (Chapter 2).
Furthermore, we were able to confirm TCE induced flow changes by visualizing and video capturing flow in real-time.
1.7. METABOLISM OF TCE
At the core of understanding the toxicity of a compound is understanding its pharmacokinetics. TCE is an inherently complex chemical in terms of metabolism, observed effects, and mode of action (Chiu, Okino et al. 2006). As reported in the recommendations of the National Academies of Sciences report, a better understanding of the metabolic activation in the avian model is required to evaluate interspecies differences, tissue-specific concentrations of trichloroethylene and its metabolites (Research Council 2006). A scheme of the metabolism has been demonstrated by Lash et al. in 2000 (Figure 1.2.). Lash et. al. (2000) showed that TCE undergoes an initial oxidative metabolism, mediated by cytochrome P450 enzymes (CYPs). Specific CYPs are indicated by Lash et. al. (2000) in the metabolism of TCE in adult mice and rats and human liver microsomes, these CYPs are: CYP2E1, CYP1A1, CYP2C9 and CYP2B6. After
Phase I (CYP mediated) oxidative metabolism of TCE the primary metabolites of
TCE formed are Trichloroacetic Acid or TCA, Dichloroacetic Acid or DCA and to a lesser extent Trichloroethanol (TCOH). TCOH can further undergo Phase II 26
metabolism, conducted by the enzyme UDP-glucuronosyltransferase (UGT1A1) that results in the formation of a glucuronide metabolite, TCOH glucuronide.
While both TCA and DCA have demonstrated greater developmental toxicity than
TCE (Collier, Selmin et al. 2003; Johnson, Goldberg et al. 2003) due to their chemical properties they are not easily absorbed and thus their in vivo toxicity poorly understood. 27
TCA
DCA
Fig 1.3. Metabolic scheme of TCE. Primary compound TCE (1) undergoes P-450 mediated oxidative metabolism leading to the production of the primary metabolites TCA and/or DCA. Adapted from Lash et al. EHP 2000 vol. 108 (2). 28
1.8. SUMMARY
While high dose exposure to TCE is relatively uncommon, low dose exposures similar to those explored in this thesis are fairly widespread. The following chapters of this thesis exploit whole embryo in ovo exposure combined with real-time measurement of gene changes, measurement of isolated exposed cardiomycoytes and measures of protein localization and protein expression alteration to explore the consequences of low dose exposure in the hearts of early chick embryos. We show that TCE exposure does alter measures of cardiac flow, reduce the rate of myocyte contraction and demonstrate an alteration in the protein expression of KLF2 (a marker of blood flow). To explore the metabolism of TCE we measured expression of cytochrome P450 enzymes and found that the heart is a unique site of expression of these molecules even before the liver develops. 29
CHAPTER 2: EXPOSURE TO LOW-DOSE TRICHLOROETHYLENE
ALTERS SHEAR STRESS GENE EXPRESSION AND FUNCTION IN THE
DEVELOPING CHICK HEART
2.1. INTRODUCTION
Trichloroethylene (TCE; TRI; C2HCl3) is an organic solvent used as an industrial degreasing agent that can be found in many consumer products (Page,
Check et al. 2001). Due to its widespread use and volatile nature, TCE is a common environmental contaminant. TCE was first linked to altered heart development in an epidemiological study that found an odds ratio of 3 for congenital heart disease in children living in an area of the Tucson Valley with
TCE groundwater contamination in the range of 100–270 ppb. The types of defects found included defects expected from both myocardial and valvular etiologies. A subsequent epidemiological study found a correlation between the proximity of maternal residence to TCE-emitting sites and increased heart defects in children (Yauck, Malloy et al. 2004).
A connection between congenital heart disease and TCE has been a controversial issue for many years (see Boyer, Finch et al. 2000; and Dugard 2000). A recent review by Hardin et al. (2005) challenged the idea that TCE is a teratogen based largely upon inconsistencies in dose-response data to various exposure protocols.
At higher doses, differences in exposure methods (maternal exposure via gavage in an oil carrier or dissolved in drinking water) produced different outcomes 30
(normal vs. teratogenic, respectively) in rat embryos (Fisher, Channel et al. 2001;
Johnson, Goldberg et al. 2003). Previously, we showed that gene expression of a number of molecules, including Serca2, was altered by 100 ppm TCE exposure in maternal drinking water of Sprague–Dawley rats (Collier, Selmin et al. 2003). A subsequent study using the rat H9C2 cardiomyocyte cell line found altered expression of Serca2 and other calcium homeostatic genes with concomitant changes in a calcium flux stimulated with vasopressin (Caldwell, Thorne et al.
2008). More profound changes were seen at 10 ppb than at 1 ppm in this cell line.
Drake et al. (2006) found that low levels (8 ppb) of TCE exposure in chick embryos produced changes in aortic flow that were greater than observed at higher levels (800 ppb). An evaluation produced by the National Research
Council concluded that a connection between TCE and heart defects was plausible but needed more study on low-dose effects and mode of action (Research Council
2006).
Due to calcium’s importance in muscle contraction, the consequences of
TCE exposure on the functional and morphological formation of the myocardium may be relevant to understanding TCE-mediated cardio-teratogenicity. Drake et al. (2006) suggested that the reduced aortic flow was most likely due to obstruction caused by hypertrophy of the valve-forming cardiac cushions. We speculate that a reduction in calcium homeostasis in the myocytes would impair myocyte function and have direct morphogenetic consequences. Hogers el al. 31
(1997) showed that reduced cardiac blood flow, alone, leads to morphological congenital heart defects. Accordingly, we exposed developing chick embryos to low level TCE during a critical stage in cardiac development. We show here, by both molecular markers of cardiac blood flow and measurement of contraction in
TCE-exposed primary cardiomyocytes, that low level TCE exposure perturbs cardiac function in the living embryo. These data argue that one mechanism of
TCE-mediated cardiac teratology is by a reduction of cardiac function during heart development.
2.2. MATERIALS AND METHODS
Material. Trichloroethylene ACS reagent, ≥99.5%, (TCE) was ordered from Sigma–Aldrich Co. (St. Louis, MO) Catalog #251402.
Dosing. TCE was dosed at 8 ppb (60 nM) and 800 ppb (6,000 nM) through injection into stage Hamburger Hamilton (HH)13 eggs as reported by
Drake et al. (2006). Injections were performed using Hamilton Co. (Reno, NV)
800 Series Syringes (Part #7646-01) paired with custom needles (Part #7806-02:
RN NDL 6/PK 22 s/1’’/4L).
Quantitative Real-Time PCR. After dosing, embryos were allowed to develop for 24 or 48 h until reaching stages HH17 and HH24, respectively, for analysis. Pooled samples of 18–21 Hamburger Hamilton (HH) 17 whole hearts were then homogenized in Trizol (# 15596-018, Invitrogen) and processed for 32
RNA isolation using PureLinkTM Micro-to-MidiTM Total RNA Purification
System (# 12183-018, Invitrogen). This protocol was then repeated for pooled samples of stage (HH) 24 whole hearts. Data shown are a representation of three experimental replicates representing a total of 60 whole hearts per treatment for each exposure period (24 and 48 h). cDNA was then transcribed using the iScript cDNA synthesis kit (Bio-Rad, Richmond, CA). cDNA concentration was measured using fluorometry (Turner Biosystems) after staining with Quant-iT™
OliGreen® ssDNA Assay Kit (# O11492, Molecular Probes), equal aliquots of control and experimental cDNA samples were added to triplicate reaction mixtures. Real-time PCRs were carried out using primers in Table 2.1, FastStart
SYBR Green Master (Roche) and the Rotor Gene 3000 System from Corbett
Research. Analysis of the data was carried out using the Rotor Gene 6 software.
All real-time PCR results were then normalized to the housekeeping gene
GAPDH. GAPDH expression was essentially unaltered in TCE-treated and control samples after normalizing total cDNA in each tube, showing that GAPDH expression is unaltered by TCE exposure.
Immunofluorescent Microscopy. Embryos were collected in Tyrode’s solution, and the thorax region dissected from the embryo. Thoraxes were fixed in a solution of 20°C 80% methanol/20% DMSO and cryosubstituted in 100% ethanol at 20°C for one week. The tissues were then embedded in paraffin and sectioned. After deparaffinization, the sections were rinsed in 1XPBS for 10 min, 33
blocked for 1 h at room temperature with a blocking solution containing 1% bovine serum albumin and 0.1% Tween 20 in PBS. Sections containing the heart were processed using indirect immunofluorescence. Sections were incubated overnight with primary antibody (rabbit anti-LKLF H-60, Santa Cruz
Biotechnology, no. sc-28675) at 4°C in a moist chamber. After several rinses in
1XPBS, Alexa fluor 488 or 546-conjugated goat anti-rabbit secondary antibody
(Molecular Probes, Eugene, OR; A-11008, A-11010) were incubated for 1 h at room temperature in a moist chamber. After rinsing in PBS, the nuclei were stained with TO-PRO-3 (Molecular Probes), and the sections mounted using
Prolong Gold mounting media (Molecular Probes). Sections were analyzed using a Zeiss 510 Meta confocal microscope or with a Deltavision deconvolution microscope.
Cardiomyocyte Isolation. Cells were isolated as described previously
(Li, McNelis et al. 1997; O'Connell, Rodrigo et al. 2007), but as the protocol required larger hearts, embryos were incubated to day E18 after a one-time yolk injection at stage 13 (E2). Briefly, following cervical dislocation of E18 chick embryos, hearts were dissected and cannulated via the aorta. Isolated hearts were perfused for four minutes at 41°C and pH 7.4 with perfusion buffer (113 NaCl,
4.7 KCl, 0.6 Na2HPO4, 1.2 MgSO4, 12 NaHCO3, 10 KHCO3, 10 HEPES and 30
Taurine, all in mM). Heart perfusion was switched to digestion buffer (perfusion buffer plus 0.148 mg/ml liberase blendzyme 2 (or 2 mg/ml Collagenase II from 34
Worthington Biochemical and no trypsin), 0.13 mg/ml of trypsin, 12.5 lM CaCl2) for 1.5–3 min. When the heart was flaccid, digestion was halted, and the heart placed in myocyte stopping buffer 1 (perfusion buffer plus 0.04 ml Bovine Calf
Serum (BCS)/ml buffer and 5 lM CaCl2). The heart was cut into small pieces.
Small pieces of left ventricle were then triturated several times with a transfer pipet and then filtered through a 300-lM nylon mesh filter. Following this, the cells were gravity pelleted, and the supernatant was discarded. Finally, Ca2+ was reintroduced stepwise to a final concentration of 1.0 mM.
Cardiomyocyte Contractility. An inverted Olympus IX-70 microscope was used with a modified chamber to mount the mechanical interrogation apparatus. The chamber has platinum electrodes to electrically stimulate cells and a perfusion line with heater control (Cell lControls HPRE-2) and suction out to maintain a flow rate of ∼1 ml/min. Sarcomere length (SL) was measured with an
Ion Optix MyocamS (250 Hz sampling frequency) attached to a computer. Data were collected through an Ion Optix FSI A/D board and IonWizard software with
SarcLen and SoftEdge modules to determine SL. Individual cardiomyocytes were measured at a stimulus frequency of 1 Hz for a minimum of five minutes.
Approximately 30 s of individual contractions were averaged from each cell after
5 min in the chamber to obtain the average contraction. Diastolic SL, systolic SL, peak change SL and half-height half-width (time greater than 50% of diastolic
SL) were measured for each cell. The contractility measurements were obtained 35
from two independent replicates and a total of 18–20 TCE-exposed or control cells. 36
Table 2.1
Real-Time PCR Primers Primers Forward Sequence Reverse Sequence
Endothelin-1 5’-TGTTCCCTATGGTCTTGGAGGC-3’ 5’-AGGTTTTCTCTGCTGTGGACTGAG-3’
Nitric oxide synthase 3 5’-ATGTCCTCCCCCTACACCAAC-3’ 5’-TGCCTCCTCTTCCTCTTCCAG-3’
Krüppel-like 5’-CCCACGCAAAGAGGATGAAGAC-3’ 5’-GCTTGATGCTGTCCACGAACTG-3’ factor 2
GAPDH 5ʼ-GTGTGCCAACCCCCAATGTCT-3ʼ 5ʼ-CCCATCAGCAGCCTTCA-3ʼ 37
2.3. RESULTS
Altered Gene Expression After TCE Exposure. Shear stress markers, specifically Nitric Oxide Synthase 3 (NOS-3) and Krüppel-like Factor 2 (KLF2), provide a characteristic reduction in expression where blood flow is reduced in the embryo, while Endothelin-1 (ET-1) is a more variable marker (Groenendijk,
Hierck et al. 2004). Reduction in aortic blood flow was seen by Drake et al.
(2006), after TCE exposure, but attributed to obstructed flow from the heart by outflow cushion hypertrophy. As calcium homeostasis is perturbed by TCE in cardiomyocytes, we explored whether changes in shear stress markers in the heart might reflect a reduction in cardiac function. ET-1, NOS-3 and KLF2 were examined by real-time PCR to determine changes in transcript expression after
TCE exposure. Incubated avian embryos were exposed to TCE by injection into the yolk at 48 h of development (HH Stage 13), and these embryos were allowed to develop for another 24 h (to reach HH 17) at which time whole heart tissue was extracted for RNA isolation. Real-time PCR was performed on the isolated RNA and normalized to GAPDH expression. We verified that GAPDH expression was not sensitive to TCE exposure by evaluating GAPDH levels in total cDNA from treated and untreated samples. Results in Fig. 2.1 illustrate that NOS-3 and KLF2 demonstrate a significant decrease in expression after 8 ppb TCE exposure, while
ET-1, KLF2 and NOS-3 remain largely unchanged after 800 ppb TCE exposure for 24 h relative to PBS. The data demonstrate that the 8 ppb levels of exposure 38
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Fig. 2.1. HH17 Whole Hearts were collected after 24-h exposure of TCE at 8 and 800 ppb. Real-time PCR results for Endothelin-1 (ET-1), Nitric Oxide Synthase 3 (NOS-3) and Krüppel-like Factor 2 (KLF2). ET-1 was unchanged while both NOS-3 and KFL2 were significantly reduced at 8 ppb TCE exposure, but not at 800 ppb (*** = P-value \ 0.001) 39
to TCE perturb marker expression to a greater extent than 800 ppb exposure.
To determine whether the shear stress marker genes demonstrate sustained transcriptional regulation by TCE, additional avian embryos were dosed at HH13 and allowed to develop for a further 48 h, at which time whole heart tissue was extracted and processed for RNA isolation. Both NOS-3 and KLF2 had modest but significant decreases in expression with the longer exposure at the 8 ppb TCE dose. There was a lesser but still significant decrease in NOS-3 after 800 ppb
TCE exposure (Fig. 2.2). As with the shorter exposure, ET-1 expression does not significantly change after 8 or 800 ppb TCE exposure when compared to the PBS
Control. Since both NOS-3 and KLF2 appeared to be the most responsive at the 8 ppb TCE dose, all subsequent experiments were done using the 8 ppb TCE dosage. 40
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>?> 9:; <%''= <>>''= A,*) Fig. 2.2. HH24 Whole Hearts were collected after 48-h exposure of TCE at 8 and 800 ppb. Real-time PCR results for Endothelin-1 (ET-1), Nitric Oxide Synthase 3 (NOS-3) and Krüppel-like Factor 2 (KLF2). ET-1 was unchanged while NOS-3 was significantly reduced at both 8 and 800 ppb TCE exposure. Additionally, KLF2 was signif- icantly reduced only after 8 ppb TCE exposure (** = P-value \ 0.01; * = P-value \ 0.05) 41
KLF2 Transcription Factor Protein Expression. While KLF2 expression is widely recognized in endothelial cells as a marker of shear stress, it has been reported in cardiomyocytes, smooth muscle cells, fibroblasts and stem cells (Groenendijk, Van der Heiden et al. 2007; Cullingford, Butler et al. 2008).
Relatively little is known of its localization in the developing embryo. Work by
Groenendijk et al. (2004) showed, by radioactive in situ hybridization, localization of transcripts to the endothelial cells of the AV canal and outflow tract where flow in the heart is most constricted. There was an indeterminate amount of label over the myocardium due to the noise inherent in the radioactive protocol.
To verify the expected distribution in the heart, we stained sections of treated and control hearts with an antibody against KLF2. This antibody was verified in a western blot as recognizing a single molecule of the expected size in embryonic heart cell lysates (data not shown). As shown in Fig. 2.3, KLF2 protein is found in endothelial cells of the AV canal as well as mesenchyme and myocardial cells.
Though protein expression was not strongly reduced in TCE-treated samples, there appeared to be a more specific diminution in the endothelia. 42
a
b
Fig. 2.3. a KLF2 Antibody Expression in HH17 hearts collected after 24- h exposure to 8 ppb TCE. KFL2 Antibody expression can be observed in both the endothelium and myocardium of the developing chick heart in both the control and 8 ppb TCE-treated samples. Staining in TCE-treated endothelia appears to show an increase in extra-nuclear distribution of the protein. L = lumen of heart, ECM = extracellular matrix. b Illustration of a HH17 chick heart. Images showing antibody staining were taken from the atrioventricular canal region of the heart for both endothelial (E) and myocardial (M) tissue. The extracellular matrix of the valve-forming region lies between the two cell layers. A = common atrium, V = common ventricle 43
Altered Half-Width After TCE Exposure. We previously showed
(Caldwell, Thorne et al. 2008) in H2C9 rat cardiomyocyte cells a reduced flux of calcium after vasopressin stimulation in TCE-treated cells. The experiments showing reduced levels of shear stress markers are consistent with a reduction in cardiac function but do not show it directly. To investigate changes in myocyte contraction directly, we dosed developing embryos at stage 13 (E2) with TCE and incubated them until embryonic day 18. Cardiomyocytes were then isolated from the hearts of these embryos by a published protocol (Li, McNelis et al. 1997;
O'Connell, Rodrigo et al. 2007) and then directly measured during contraction in vitro. Cells from exposed and control embryos were paced at 60 Hz and imaged to measure sarcomere parameters during contraction. Cells from embryos exposed to 8 ppb TCE demonstrated a significant increase in the half-width of the rate of contraction when compared to control embryos (Fig. 2.4). This increase in half-width reflects a reduced rate of contraction and is characteristic of reduced cytosolic calcium (Ren, Li et al. 2007). It was observed that the relaxed sarcomere length was unchanged, providing support for the idea that the functional effects resulting from TCE exposure are most likely a result of altered
Ca2+ regulation rather than a morphological change to sarcomere structure (Fig.
2.5). 44
Fig. 2.4. Half-Width of isolated E18 cardiomyocytes exposed to 8 ppb TCE relative to control cardiomyocytes. Half-width of TCE-exposed cardiomyocytes is significantly increased when compared to control cardiomyocytes (*** = P- value \ 0.001) 45
Fig. 2.5. Sarcomere Length of isolated E18 cardiomyocytes exposed to 8 ppb TCE relative to control cardiomyocytes. Sarcomere length is unchanged by TCE exposure 46
2.4 DISCUSSION
The prevalence of TCE as a common soil and water contaminant requires a better understanding of the consequences of environmental exposure. While
TCE has been implicated in other health effects, the most significant developmental problem appears to be a relationship to congenital heart defects.
For reasons that have not been entirely clear, TCE is remarkably cardiac-specific as a teratogen (Goldberg, Lebowitz et al. 1990; Yauck, Malloy et al. 2004;
Research Council 2006). The heart is unique in the developing embryo in that its function is required even as it is still undergoing morphogenesis. The mechanism explored here that perturbation of calcium homeostasis in developing myocytes alters contraction and produces morphological consequences provides an explanation for this specificity.
While there are other potential mechanisms for TCE-mediated teratology, the chain of logic suggesting a perturbation of function extends from the initial observations that Serca2 expression was significantly reduced in rat embryonic hearts after in utero exposure to TCE (Collier, Selmin et al. 2003). Subsequent studies using an in vitro culture model of mouse embryonal carcinoma P19 cells showed that low level exposure to TCE perturbed expression of additional calcium homeostatic molecules (Selmin, Thorne et al. 2008). The embryonic heart requires the regulated release and uptake of intracellular calcium for both function and growth. We reasoned that a perturbation of calcium handling and 47
storage would compromise the ability of the heart to contract normally.
Groenendijk et al. (2005), using a venous clip model, showed that reduced blood flow in the heart produced cardiac defects. Thus, the apparent heart-specific teratogenic effects of TCE might be due to a compromise in cardiac function as a critical stage of heart development. Though previous studies explored TCE- mediated teratology in the chick (Loeber, Hendrix et al. 1988), the studies of
Drake et al. (2006; 2006) established that low-dose TCE could be introduced into the chick embryo by injection into the yolk and calculated exposures that could be related to environmental exposure. Importantly, for the mechanistic hypothesis,
Drake et al. (2006) observed a reduction in aortic blood flow in embryos exposed to low-dose 8 ppb TCE.
The first test of this hypothesis was an in vitro exposure of a rat cardiomyocyte cell line to low levels of TCE (Caldwell, Thorne et al. 2008).
Though these cells are not spontaneously contractile in culture, they demonstrate a calcium flux when exposed to vasopressin. A relatively brief exposure to low levels of TCE produced an approximately 40% reduction in peak calcium as well as reduced release and uptake of calcium as measured by Fura2 fluorescence. The data were consistent with the loss of Serca2a and Ryr2 expression observed with these cells. To explore changes in cardiac contraction in the intact embryo, we turned to molecular markers of function. Groenendijk et al. (2005) showed that expression of shear stress markers in the heart provided a useful measure of 48
changes in cardiac blood flow. We examined the expression of these same shear stress markers ET-1, NOS-3 and KLF2 in heart RNA extracts after injection of
TCE in ovo. TCE exposure resulted in the significant decrease in KLF2 and
NOS-3 mRNA expression in both the HH17 and HH24 whole heart. As the reduced expression of these markers in the heart was previously seen with venous ligation, the data are consistent with the idea that flow is reduced in the heart as well as in the dorsal aorta. Reduced cardiac function is consistent with this observation.
We show in this paper that the KLF2 marker is expressed in both the endothelium and the myocardium of the developing heart. Although not quantitative, the images shown in Fig. 2.3 suggest that KLF2 expression is both reduced in level and extra-nuclear in TCE-exposed endothelium compared to controls. There may also be a reduction in expression in the myocardium, but the pattern suggests that KLF2 expression is retained in the most epicardial layer of the myocardium. It seems likely that the PCR measurement of KLF-2 reflects changes in both cell types found in the early heart. A potential consequence of the reduction in the transcription factor KLF2 is that this transcription factor is developmentally relevant in modulating vascular development through binding to an ETS family protein member ERG to activate Flk1 expression during vascular development (Meadows, Salanga et al. 2009). Thus, a decrease in KLF2 expression by TCE may function to alter endothelial development. 49
Of the three potential endothelial markers, we observed no significant changes in ET-1. Groenendijk et al. (2005) identified ET-1 as modestly elevated by a reduction in flow, but we found no such effect. Discussions with the laboratory that made this observation suggest that ET-1 is somewhat variable and is not as useful as a marker of flow (Robert Poelmann, personal communication).
While the expression of flow markers is altered by TCE exposure, it is only an indirect measure of the hypothesized mechanism. These studies were extended to look at the effect of TCE on myocyte function. Myocytes were isolated from
TCE-exposed and control embryos on day E18, and the response to a stimulated contraction was measured. Using the sarcomere half-width of contraction rate as a measure of Ca2+ handling, we observed that 8 ppb TCE is sufficient to significantly increase cardiomyocyte half-width relative to control (Fig. 2.4). We did not observe a change in sarcomere length, indicating that the half-width response observed was not a consequence of a TCE-induced sarcomere damage but most likely a result of a TCE-induced alteration of Ca2+ handling. The cells were collected at E18, 16 days after in ovo injection, and although effects on markers showed some recovery at stage 24 relative to stage 17, the data clearly show that the loss of cardiac function persists in the embryo through the majority of the developmental period.
One issue highlighted by these studies is the dose–response effect. One of the major criticisms of human and animal data has been the apparent 50
inconsistency in dose response (Dugard 2000; Fisher, Channel et al. 2001; Hardin,
Kelman et al. 2005). The data reported here show substantially greater perturbation of molecular markers at 8 ppb than at 800 ppb. This is consistent with a number of previous studies. Molecular measures described here are consistent with the functional observations of Caldwell et al. (2008) and Drake et al. (2006; 2006). Recent studies of gene expression in H9C2 cells found a biphasic response with maximum effects between 10 and 100 ppb and again at
10– 100ppm when they measured changes in calcium homeostasis (Caldwell,
Thorne et al. 2008). We previously observed that 50–250 ppm TCE inhibited mesenchymal cell formation in vitro, while Drake et al. (2006; 2006) observed that 8 ppb exposure induced proliferation of these cells in ovo. Differences between loss of cushion tissue mesenchyme at higher doses and a proliferation of cushion mesenchyme suggest an ability to perturb differing developmental pathways at different doses. TCE is a volatile compound that rapidly partitions into headspace above tissue culture media or the plastic of culture dishes, and it has been difficult to work with low doses. In the studies of Mishima and colleagues (2006), they found a loss of 2/3 of the originally dosed TCE after only
1 h in doses ranging from 25–250 ppm. Mishima et al. (2006) reported that TCE was reduced to undetectable levels after 24 h. While the kinetics of loss in the egg are unknown, the deficits produced during in ovo exposure argue for both the toxicity of TCE at specific stages and that it is retained long enough to have an 51
effect. Clearly, we observe significant effects in this animal model near the
Maximum Contaminant Level (MCL) for exposure, while the effects of TCE on markers of function are less severe at higher doses. The dose–response parameters of TCE remain to be explained and are an important component in evaluating the public health consequences of TCE exposure. While preparing the resubmission of this manuscript, Rufer et. al. (2010) independently published the observation that, when 4 nmol of TCE is dosed at embryonic day 2.5, both heart rate and tricuspid velocity (max) were significantly increased in 1- Day Posthatch chickens. Although the dosing parameters vary between the studies, we can infer that a single acute dose of TCE early in development results in a sustained effect on cardiac function. We speculate that an aspect of detoxification or compartmentalization of TCE in cells or tissues is involved, but further work needs to be done to resolve the issue.
Together, the data presented here are consistent with previous data, suggesting that cardiac development is sensitive to low-dose TCE exposure.
Calcium channel genes were seen to be down-regulated by TCE, and this produced an alteration in calcium homeostasis in cultured myocytes. Markers of shear stress were shown to reflect changes in cardiac blood flow (Groenendijk,
Hierck et al. 2005). In this study, we found these same markers to demonstrate reduced flow in the TCE-treated heart and that TCE produces a change in the rate 52
of contraction. Ongoing studies will evaluate whether the metabolism of TCE is responsible for the unusual dose sensitivity seen in these and previous studies. 53
CHAPTER 3: EXPOSURE TO LOW DOSE TRICHLOROETHYLENE
RESULTS IN INCREASED CYTOCHROME P450 - 2C SUBFAMILY
EXPRESSION IN THE DEVELOPING CHICK HEART
3.1. INTRODUCTION
Trichloroethylene (TCE; TRI; C2HCl3) is an organic solvent developed for use as an industrial degreasing agent and can be found in many consumer products, i.e., typewriter correction fluids, paint removers, adhesives, spot removers and cleaning fluids (Page, Check et al. 2001). Due to its widespread use and volatile nature, TCE is a common environmental contaminant. According to an EPA national groundwater survey, TCE is the most frequently detected organic solvent in groundwater supplies and is estimated to be in up to 34% of the nationʼs drinking water supplies (Page, Check et al. 2001). TCE is found in at least 852 of the 1,430 EPA Superfund sites around the country (ATSDR 2003).
TCE was first linked to altered heart development in an epidemiological study that found an odds ratio of 3 for congenital heart disease in children living in an area of the Tucson Valley with TCE groundwater contamination in the range of 100-270 ppb (Goldberg, Lebowitz et al. 1990). Defects found included both myocardial and valvular structures. Although this study was controversial for methodological reasons based upon the appropriateness of the selected controls, an independent reevaluation of the data validated the conclusion that TCE is cardio-teratogenic (Bove, Shim et al. 2002). Further, a Wisconsin epidemiological 54
study shows a correlation between the proximity of maternal residence to TCE emitting sites and increased heart defects in their children (Yauck, Malloy et al.
2004).
A connection between congenital heart disease and TCE has been a controversial issue for many years (see Boyer, Finch et al. 2000; Dugard 2000). A recent review by Hardin et al. (2005) challenged the idea that TCE is a teratogen based largely upon inconsistencies in dose response data to various exposure protocols. However, an independent evaluation produced by the National
Academy of Sciences (Research Council 2006) concluded that TCE or one or more of its metabolites can cause cardiac teratogenesis. They suggest that additional studies of lowest-observed adverse effect level and mode of action was necessary (Research Council 2006). The prevalence of TCE as a soil and water contaminant argues for a better understanding of the consequences of low level exposures. For yet poorly understood reasons, TCE does not have a consistent dose response curve in the cardiovascular system. Early studies focused on moderate to high levels of exposure. However, recent studies of gene expression in H9C2 cells suggest that there are biphasic curves with maximum effects between 10-100 ppb and again at 10-100 ppm (Selmin, Thorne et al. 2005;
Caldwell, Thorne et al. 2008). As described by Drake et al. (2006), concentrations of TCE (8 ppb) close to the EPA maximum contaminant levels (5 ppb) are sufficient to alter cardiac hemodynamics. While non-monotypic dose response 55
curves could reflect issues with metabolism, transport, compartmentalization or a combination of factors, we began to explore this issue by focusing on metabolism.
Lash et al. (2000) demonstrated that in the murine model system, as well as in human liver microsomes, trichloroethylene undergoes cytochrome P450 mediated oxidative metabolism. As seen by Lash et al. (2000), the CYP2 family demonstrates both affinity and substrate specificity for TCE. CYP2E1 has been implicated as the major TCE metabolizing CYP in both human liver microsomes and the murine model systems; while CYP2E1 is shown to be present in low levels in human fetal liver and brain (Carpenter, Lasker et al. 1996;
Brzezinski, Boutelet-Bochan et al. 1999). As we and others (Drake, Koprowski et al. 2006; Mishima, Hoffman et al. 2006; Makwana, King et al. 2010) have found,
TCE effects cardiovascular development prior to both liver and brain development. If metabolic activation of TCE is relevant to teratogenesis, then the existence and localization of cytochrome P450 enzymes in the early embryo is important to identify.
Avian embryos were injected in ovo (Drake, Koprowski et al. 2006) with low levels (8 and 800 ppb) of TCE, and cardiac and extracardiac tissue was analyzed for expression of avian homologues of TCE-metabolizing cytochromes.
The data showed that cytochromes 1A4, 2C45 and 2H1 were found in the early embryonic heart and that expression of mRNA and protein for CYP2H1 was altered by TCE exposure. These enzymes were unique to the heart at an early 56
stage of development as little signal was found in the rest of the embryo
(extracardiac tissue). The expression data recapitulate the non-monotypic dose response seen previously (Drake, Koprowski et al. 2006; Caldwell, Thorne et al.
2008; Makwana, King et al. 2010; Rufer, Hacker et al. 2010). These data showed that the developing heart, a target of TCE toxicity, has a unique ability to metabolize TCE at an early stage in development. The localized expression of cytochromes at this early time may be relevant to the frequency of environmentally-attributed defects found in this organ.
3.2. MATERIALS AND METHODS
Material. Trichloroethylene ACS reagent, ≥ 99.5% (TCE) was obtained from Sigma-Aldrich Co. (St. Louis, MO) Catalog #251402.
Dosing. TCE was dosed at 8 ppb (60 nM) and 800 ppb (6000 nM) through injection into stage Hamburger Hamilton (HH)13 eggs using Hamilton
Co. (Reno, NV) 800 Series Syringes (Part #7646-01) paired with custom needles
(Part #7806-02) with the following specifications: RN NDL 6/PK (22s/1”/4)L.
Methods were as described by Drake et al. (2006) and Makwana et al. (2010).
Quantitative real-time PCR. After dosing embryos were allowed to develop for approximately 24 hrs until reaching stage HH17. Approximately two to three HH17 whole embryos with hearts removed, defined henceforth as
“extracardiac tissue,” were pooled for homogenization in Trizol (# 15596-018,
Invitrogen) and processed for RNA isolation using PureLinkTM Micro-to- 57
MidiTM Total RNA Purification System (# 12183-018, Invitrogen). Additionally, this protocol was repeated for pooled samples of approximately twenty
Hamburger Hamilton (HH) 17 hearts. Data shown are a representation of three experimental replicates representing a total of 9 extracardiac tissues and 60 hearts per treatment for a 24 hour exposure period. Concentration of cDNA was measured using fluorometry (Turner Biosystems) after staining with Quant-iTTM
OliGreen® ssDNA Assay Kit (# O11492, Molecular Probes) and equal aliquots of control and experimental cDNA samples were added to triplicate reaction mixtures. Real-time PCR reactions were carried out using primers in Table 3.1,
FastStart SYBR Green Master (Roche) and the Rotor Gene 3000 System from
Corbett Research. Analysis of the data was carried out using the Rotor Gene 6 software. All real-time PCR results were then normalized to the housekeeping gene GAPDH.
Statistical Analysis. Studentʼs t-test was performed using GraphPad
Prism version 5.0b for Mac OS X, GraphPad Software, San Diego California
USA, www.graphpad.com
Immunofluorescent Microscopy. Embryos were collected in Tyrodeʼs solution and the thorax region dissected from the embryo. Thoraxes were fixed in a solution of 20°C 80% methanol/20% DMSO and cryosubstituted in 100% ethanol at 20°C for one week. The tissues were then embedded in paraffin and sectioned. After deparaffinization, the sections were rinsed in phosphate buffered 58
saline (PBS) for 10 min, blocked for 1 hr at room temperature with a blocking solution containing 1% bovine serum albumin and 0.1% Tween 20 in PBS.
Sections containing the heart were processed using indirect immunofluorescence.
Sections were incubated overnight with primary antibody (goat anti-
CYP2C8/9/18/19 K-21, Santa Cruz Biotechnology, no. sc-23435) at 4°C in a moist chamber. After several rinses in PBS, Alexa fluor 488 or 546-conjugated rabbit anti-goat secondary antibody (Molecular Probes, Eugene, OR; A-11008,
A-11010) were incubated for 1 hr at room temperature in a moist chamber. After rinsing in PBS, the nuclei were stained with TO-PRO-3 (Molecular Probes) and the sections mounted using Prolong Gold mounting media (Molecular Probes).
Sections were analyzed using a Zeiss 510 Meta confocal microscope or with a
Deltavision deconvolution microscope.
Immunoblot Analysis. Relative amounts of protein were determined using immunoblot analysis (Jelinek, Patrick et al. 2009). Protein samples were separated using 10% SDS–PAGE under reducing conditions and then transferred to a nitrocellulose membrane. In brief, blocking buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl, 0.05% Tween 20, and 5% non-fat dry milk) was used to block the nitrocellulose membrane (1 h). Membranes were then incubated in blocking buffer containing the appropriate dilution of primary antibody (4°C overnight). Membranes were rinsed with blocking buffer (3 × 10 min) to remove residual primary antibody and then incubated in blocking buffer containing the 59
appropriate dilution of peroxidase-conjugated goat secondary antibody (45 min).
Membranes were rinsed with blocking buffer (3 × 10 min) to remove residual secondary antibody and enhanced chemiluminescence (ECL) was performed to obtain autoradiograms. Relative molecular weight of sample protein were estimated using semi-log nonlinear regression analysis to determine a fit curve relative to Fisher BioReagents® EZ-RunTM Prestained Rec Protein Ladder.
Table 3.1 Real-Time PCR Primers Primers Forward Sequence Reverse Sequence
CYP1A4 5’-GTCAATGCTCGTTTCAGTGCCT-3’ 5’-ATCCTCCCCTGTCCTTTTCTCC-3’
CYP2C45 5’-GGTTTGTGTTGCTTGCCTGC-3’ 5’-TTCACCTCCAGTATGTTCCCTACG-3’
CYP2H1 5’-TGGCTTGAAAGGCAACCTACG-3’ 5’-TTGTCTGCTCAGTATGGAGGAAGG-3’
CYP3A37 5ʼ-CCTGGAATACCGCAAAGGCTT-3ʼ 5ʼ-CCACTGGTGAAGGTTGGAGAGA-3ʼ
UGT1A1 5ʼ-ACTCAATGTCCCAATCCCCCT-3ʼ 5ʼ-TCGGTATGGTCTGTAAATGCCCT-3ʼ
GAPDH 5’-GTGTGCCAACCCCCAATGTCT-3’ 5’-CCCATCAGCAGCCTTCA-3’ 60
3.3. RESULTS
Avian Cytochrome P450s. We identified the avian homologues of mouse and human cytochromes associated with TCE metabolism using NCBI homologene (http://www.ncbi.nlm.nih.gov/homologene) (Table 3.2a) (Lash,
Fisher et al. 2000). The major TCE metabolizing enzyme in adult organisms (mice and rats) and adult human liver microsomes indicated by Lash et al. (2000) was
CYP2E1. Other minor enzymes indicated by Lash et al. (2000) were, CYP1A1,
CYP2C9 and CYP2B6. While the chicken does not express either CYP2E1 or
CYP2B6, it does express CYP2 family members: CYP2H1 and CYP2C45 whose respective human homologues are CYP2C18 and CYP2C9 (Table 3.2). Of these two CYPs Thum et al. (2000) report CYP2C18 expression in adult human heart.
Additionally, the avian homologue of CYP1A1 is CYP1A4 (Table 3.2a). CYP2H1 has been characterized as being closely related to the human CYP2C subfamily
(63.4 % DNA Identity with human) (Mattschoss, Hobbs et al. 1986). We designed
PCR primers for CYP1A4, CYP2C45 and CYP2H1 (Table 3.1) and evaluated expression in both cardiac and extracardiac tissue during development with or without TCE exposure. 61
Table 3.2a P450 Human Murine Chick (% DNA Identity) Major CYP2E1 CYP2E1 - Minor CYP1A1 CYP1A1 CYP1A4 (66.9) Minor CYP2C9 CYP2C CYP2C45 (61.4) Minor CYP2B6 CYP2B2 -
Table 3.2b Cardiac CYP2C18 CYP2C80 CYP2H1 (63.4) 62
Fig 3.1. Illustration of a HH17 chick heart. Images showing antibody staining were taken from the atrioventricular canal region of the heart for both endothelial (E) and myocardial (M) tissue. The extracellular matrix of the valve-forming region lies between the two cell layers. A = common atrium, V = common ventricle 63
CYP1A4 Gene Expression after TCE Exposure. The developing heart of the embryo is seen as a looped structure on the anterior thorax during the organogenesis stage of the development. While the heart initially forms as a linear heart tube, developmental movements during morphogenesis produce a loop with concomitant internal processes of septation to produce a 4 chambered heart.
Previous studies in animal models have shown that the window of sensitivity to
TCE in chick and mouse models coincides with looping and septation (Boyer,
Finch et al. 2000; Collier, Selmin et al. 2003; Johnson, Goldberg et al. 2003;
Drake, Koprowski et al. 2006; Mishima, Hoffman et al. 2006; Makwana, King et al. 2010; Rufer, Hacker et al. 2010). Figure 3.1 depicts a partial image of the chick embryo showing the orientation of the looped heart with a partial dissection showing the endothelial and myocardial cell layers with an intervening extracellular matrix occupied by mesenchymal cells of endothelial origin (Boyer,
Finch et al. 2000). Embryos were collected at this stage of development for the analyses described here.
As the major cytochrome for TCE in mouse and humans is not expressed in the chick, we examined the minor cytochromes. CYP1A4 was previously found to be expressed in isolated chick heart myocytes after TCDD exposure (Gannon,
Gilday et al. 2000). Here, realtime PCR was used to determine CYP1A4 expression after TCE exposure in ovo. Incubated avian embryos were exposed to
TCE by injection into the yolk at 48 hrs of development (HH Stage 13), and 64
embryos were allowed to develop for another 24 hrs (to reach HH 17) at which time extracardiac tissue and heart tissue was extracted for RNA isolation. Real- time PCR data were normalized to GAPDH expression. We verified that GAPDH expression was not sensitive to TCE exposure by evaluating GAPDH levels in equal aliquots of cDNA from treated and untreated samples. Results shown in Fig.
3.2 illustrate that CYP1A4 was detectable but does not demonstrate significantly altered expression after either 8 or 800 ppb TCE exposure in extracardiac tissue or in HH17 heart tissue. We conclude that expression of this cytochrome P450 is not stimulated by relevant doses of TCE at this stage of heart development. 65
A 5
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B 5
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Fig 3.2: CYP1A4. A. Real-time PCR results for CYP1A4 expression in HH17 hearts. B. Real-time PCR results for CYP1A4 expression in HH17 extracardiac tissue. CYP1A4 expression was not significantly altered relative to total RNA (GAPDH) in either HH17 hearts or extracardiac tissue after TCE exposure at 8 and 800 ppb. 66
CYP2C45 Gene Expression after TCE Exposure. Lash et al. (2000) identified an additional minor cytochrome involved in TCE metabolism in humans and mice, CYP2C9. The avian homologue of this enzyme is CYP2C45 as identified in NCBIʼs homologene database (Table 3.2a). CYP2C45 expression was determined by real-time PCR in HH17 extracardiac tissue and HH17 heart
(Fig 3.1). Incubated avian embryos were exposed to TCE as described above.
Results shown in Fig. 3.3 illustrate that although detected by PCR, CYP2C45 expression is not significantly altered after TCE exposure in extracardiac tissue at either the 8 ppb or 800 ppb TCE doses. We conclude that neither 8 ppb or 800 ppb
TCE exposure was sufficient to increase the expression of CYP2C45. 67
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Figure 3.3: CYP2C45. A. Real-time PCR results for CYP2C45 expression in HH17 hearts. B. Real-time PCR results for CYP2C45 expression in HH17 extracardiac tissue. CYP2C45 expression was not significantly altered relative to total RNA (GAPDH) in either HH17 hearts or extracardiac tissue after TCE exposure at 8 and 800 ppb. 68
CYP2H1 Gene Expression after TCE Exposure. In the course of our study we identified an additional CYP2C subfamily member, CYP2C18 whose expression after TCE exposure had not been categorized in Lash et al. (2000).
CYP2C18 expression was previously identified in human heart tissue (Thum and
Borlak 2000). The avian homologue of this enzyme is CYP2H1 as identified in
NCBIʼs homologene database (Table 3.2b). In HH17 heart tissue CYP2H1 demonstrated significantly increased expression at 8 ppb TCE exposure
(Studentʼs t-test: p = 0.0004), while after 800 ppb TCE exposure there was no significant change in expression. We found (Fig. 3.4) that CYP2H1 does not significantly alter expression after TCE exposure in the rest of the embryo at either the 8 ppb or 800 ppb TCE doses. These data demonstrate that 8 ppb TCE is sufficient to produce a significant increase in the expression of CYP2H1 in the developing chick heart. Although the liver bud can be identified by marker expression as early as stage HH17, cellular organization begins around stage
HH25 and albumin expression (as a measure of function) does not begin until stage HH30 (Yanai, Tatsumi et al. 2005). Thus, the liver is insufficiently developed at the stage of collection (HH17) to function as a site of detoxification.
This is confirmed by the lack of response in the extracardiac tissue sample. The significant upregulation of expression in the heart shows this organ to be a primary site of phase I enzyme expression in the early embryo. 69
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Figure 3.4: CYP2H1. A. Real-time PCR results for CYP2H1 expression in HH17 hearts. B. Real-time PCR results for CYP2H1 expression in HH17 extracardiac tissue. CYP2H1 expression was significantly increased relative to total RNA (GAPDH) in HH17 hearts after 8 ppb TCE exposure (*** = p-value = 0.0004), while after 800 ppb TCE exposure there was no significant change in expression. While extracardiac tissue demonstrated no significant change in expression after TCE exposure at 8 and 800 ppb. 70
CYP2C Subfamily Protein Localization. The results above show that a cytochrome P450 enzyme transcript associated with TCE metabolism is expressed in the embryonic heart but not in the rest of the embryo (extracardiac tissue).
CYP2H1 is significantly induced by 8 ppb TCE exposure in the developing heart.
To confirm protein expression in heart tissue and to identify the cells where expression takes place, we undertook an immunostaining procedure on embryonic chick tissue. As we were unable to obtain cytochrome-specific antibodies for the chick, we utilized a cytochrome 2C subfamily specific antibody and confirmed its ability to work in avian tissues. CYP2H1 is an avian member of the CYP2C subfamily (Table 3.2a) (Mattschoss, Hobbs et al. 1986). Embryos were injected with TCE at stage 13 as in the previous experiments and were collected and fixed at stage 17. Sections were stained with anti-CYP2C antibody and photographed.
Cytochrome 2C staining was seen in the myocardium at low magnification (Fig.
3.5). TCE exposure (8 ppb) showed an enhanced signal in the myocardium and endothelium. Many endothelial cells in these sections were essentially unstained, but there was a dramatic upregulation of endothelial expression in the cells lining the atrioventricular canal. This is an area of constriction in the heart that produces high rates of blood flow and endothelial cell proximity to the blood (Makwana,
King et al. 2010). 71
Figure 3.5: CYP2C Localization. CYP2C Subfamily Antibody Expression in HH17 hearts collected after 24 hrs exposure to 8 ppb TCE. CYP2C Antibody expression can be observed in both the endothelium and myocardium of the developing chick heart in both the control and 8 ppb TCE-treated samples. Staining in TCE-treated cardiomyocytes appears to show an increase in peri- nuclear distribution of the protein. E = Endothelial, M = Myocardial. The extracellular matrix of the valve-forming region lies between the two cell layers. A = common atrium, V = common ventricle 72
To confirm the specificity of this response, a western blot of control and treated heart material was performed. As seen in figure 3.6, there was staining of two distinct proteins in the CYP2C family (CYP2C45 and CYP2H1). One of these bands, at MW 60 kDa showed a significant upregulation of intensity with
TCE exposure (CYP2H1). 73
Figure 3.6: CYP2C Immunoblot. CYP2C Subfamily member protein expression in HH17 hearts collected after 24-h exposure to 8 ppb TCE. CYP2C subfamily members can be seen expressed in both control HH17 heart lysate (two distinct bands) while after 8 ppb TCE exposure one band appears to increase in protein expression (CYP2H1). Proteins molecular weights were calculated against the migration distance of the standards to determine an actual protein run size of CYP2H1 to be approximately 60 kDa. 74
3.4. DISCUSSION
TCE exposure during development demonstrates tremendous cardiac specificity for poorly understood reasons. Primary defects associated with human exposures include muscular ventricular septal defects, atrial septal defects, membranous ventricular septal defects and pulmonary stenosis (Yauck, Malloy et al. 2004). Similarly, animal studies show almost identical cardiac defects after
TCE exposure during development. In the avian model system: Loeber et al.
(1988) observed atrial and ventricular septal defects and Drake et al. (2006) observed altered valvuloseptal formation. Rufer et al. (2010) was able to observe the formation of cardiac defects in avian embryos post-hatch. In the murine system, Johnson et al. (2003) observed both valvuloseptal and myocardial defects after delivery of high doses (1100 ppm) of TCE in maternal drinking water. Their data show that TCE exposure during the organogenesis stage of development was critical.
A molecular survey in rat embryos exposed to TCE at 100 ppm in maternal drinking water identified Serca2a as transcriptionally downregulated
(Collier, Selmin et al. 2003). Further studies in mice and in a rat cardiomyocyte cell line highlighted transcriptional regulation of Serca2a and additional mediators of Ca2+ homeostasis by TCE exposure by doses as low as 10 ppb (Selmin, Thorne et al. 2008; Caldwell, Manziello et al. 2010). These results were confirmed in the
H2C9 murine cardiomyocyte cell line by Caldwell et al. (2008) which show 75
alterations in expression of the Ca2+ pumps, Ryr2 and Serca2a, after low dose
TCE exposure with concomitant altered Ca2+ handling. Impaired cardiac output is known to be a mechanism for defective heart development (Hogers, DeRuiter et al. 1997; Groenendijk, Hierck et al. 2005). Reduced calcium fluxes result in reduced cardiac output and thus TCE can produce cardiac defects through altered cardiac function. This was confirmed in recent observations that markers of flow within the avian heart (KLF2 and NOS-3) were reduced after TCE exposure in ovo. Analysis of isolated myocytes from these embryos show TCE to have a persistent effect on myocardial contraction (Makwana, King et al. 2010).
Thus, we and others have shown that TCE is a specific teratogen in the embryonic heart but there is an unexplained aspect to the dose response.
Epithelial mesenchymal transition in vitro is inhibited at 250-50 ppb (Boyer,
Finch et al. 2000). Cushion (valve progenitor) proliferation is stimulated at 80 ppb
(Mishima, Hoffman et al. 2006) and Caldwell et al. (2008) noted a biphasic response curve within a cardiomyocyte cell line. Our published work (Makwana,
King et al. 2010) and that of Drake et al. (2006) show that blood flow is more sensitive to 8 ppb than 800 ppb. However, Rufer et al. (2010) found that 400 ppb
TCE produced more embryo loss than 8 ppb exposure. This suggests that TCE may affect different developmental processes at different exposures or that issues of compartmentalization or metabolism may play a role in the non-monotypic dose response curve. 76
The data described here demonstrate that CYP2H1 is significantly upregulated in the heart after TCE exposure. This is an upregulation that precedes the development of the liver and is consistent with observations that CYP1A4 is upregulated in the embryonic cardiomyocytes by dioxin exposure (Jones and
Kennedy 2009). These observations show that the heart is a site of xenobiotic metabolism in early development. The early expression of these CYPs may indicate that the developing heart is particularly sensitive to environmental xenobiotic exposure that may lead to heart to defects. Localization of expression by antibody suggests that there is both myocardial expression and endothelial expression at a stage when these are the two major cell types in the heart. While both cell types appear to upregulate expression, the strong expression seen in the endothelial cells of the atrioventricular canal is interesting as these are precursors of the heart valves. Additionally, these endothelial cells are arranged in the most constricted part of the developing heart where they may serve a surveillance function. Of course, the avian embryo develops in ovo and the expression of phase I enzymes in the heart could be specific to non-placental animals as maternal protection is not available. Though we have not formally explored this expression in mammals, we note that microarray data from the hearts of TCE- exposed murine embryos show upregulation of several cytochromes (Caldwell,
Manziello et al. 2010). 77
The regional expression of CYP2H1 in the heart suggests the possibility that the basis for the TCE dose response curve lies directly in this tissue. It was found that an oxidative metabolite of TCE, trichloroacetic acid (TCA) was more toxic than TCE itself in a study of heart defects (Johnson, Dawson et al. 1998).
Oxidation of TCE by cytochrome enzymes can lead to TCA production (Larson and Bull 1992; Templin, Stevens et al. 1995). Lumpkin et al. (2003) demonstrate that TCA was able to bind to plasma proteins in mice, rats and humans resulting in an increased half-life of TCA in the bloodstream. This regionally specific metabolism of TCE in the developing heart may imply that the heart is a site of bioaccumulation of the potential oxidative metabolites of TCE: TCA
(trichloroacetic acid) and/or DCA (dichloroacetic acid). If these metabolites bind to proteins in the extracellular matrix (ECM) as well as the blood, they may be retained in the heart where they can act on myocardial function. Several investigators have noted that TCE metabolism can be destructive to the very cytochrome P450s catalyzing the metabolic reaction (Miller and Guengerich
1982; Miller and Guengerich 1983; Ensley 1991). The unusual dose response curve may be due to a combination of production of toxic metabolites and the synthesis or destruction of CYPs. The heart may be susceptible at low doses because it contains competent CYPs to metabolize TCE into TCA. However, at higher doses, CYPs are degraded faster than they can be synthesized and less formation of TCA occurs. This suggests that cardio-specific toxicity is most 78
potent prior to the onset of liver development, as TCA in the heart would be reduced if TCA is produced and bound elsewhere. This suggestion is consistent with the window of toxicity noted by Rufer et al. (2010).
In summary, our findings demonstrated that the early heart is the site of phase I enzyme expression. TCE is sufficient to induce upregulation of these enzymes and the dose sensitivity of the response recapitulates the non-monotypic response seen in a number of laboratories. While the basis for this response curve remains unclear, there are a variety of molecular and physiological markers that confirm this response. Future studies will move upstream to examine transcriptional regulation of perturbed markers in order to resolve a global cell signaling effect of localized cardiac specific metabolism of TCE. 79
CHAPTER 4: EXPOSURE TO LOW-DOSE TRICHLOROETHYLENE ALTERS
HNF4A FUNCTION
4.1. INTRODUCTION
Trichloroethylene (TCE; TRI; C2HCl3) is a man-made organic solvent developed primarily as an industrial degreasing agent but has also historically been used in the extraction of vegetable oil, coffee decaffeination, as a volatile gas anesthetic and as a dry cleaning solvent (Research Council 2006). Due to the widespread use of TCE, the US
EPA declared it the most frequently detected organic solvent in groundwater supplies, where it is estimated to be in up to 34% of the nation’s drinking water supplies (Page,
Check et al. 2001).
TCE exposure is associated with cardiac defects in both epidemiological and animal studies. Several of the more recent animal studies demonstrated that low dose
TCE exposure is sufficient to cause altered blood flow and contractility during heart development (Drake, Koprowski et al. 2006; Makwana, King et al. 2010). Additionally, low dose TCE exposure is able to up-regulate a specific CYP2C subfamily protein isoform in the developing heart, presumably leading to localized cardiac metabolism
(Makwana et al. submitted). TCE toxicity is specific for the early stage of cardiovascular development when valves and septa are forming in the early embryo (Rufer, Hacker et al.
2010). 80
One issue in the analysis of the cardio-specific teratology of TCE is the non- monotonic dose response curve. We found that 8 ppb TCE produced a greater perturbation of markers of blood flow and a greater induction of CYP2H1 than 800 ppb
(Makwana, King et al. 2010; Makwana et al. submitted). This is consistent with observations by Drake et al. (2006) that 8 ppb produced effects on cardiac output and on valvular proliferation that were greater than at 400 ppb. Caldwell et al. (2008) found that
10 ppb produced a greater inhibition of calcium channel gene transcription and a greater loss of vasopressin-stimulated calcium release than higher doses. Rufer et al. (2010) also noted a non-monotonic dose curve in the formation of ventricular septal defects and in cell proliferation in the heart.
Previous studies showed that TCE exposure altered gene transcription in rats, mice and in a rat cell line (Collier, Selmin et al. 2003; Caldwell, Thorne et al. 2008;
Selmin, Thorne et al. 2008). As a part of our studies in chick, we performed a microarray analysis on low dose exposure to TCE (8 ppb). An established protocol of in ovo exposure has proven reliable and produces both heart defects and consistent alterations of cardiac function and cytochrome P450 induction (Drake et al. 2006; Makwana, King et al. 2010; Makwana et al. submitted). While microarray analysis can be useful, the cataloging of gene expression changes often produces a catalog of results of uncertain significance. One useful method of exploring the dataset is to use interactome analysis.
The interactome is a set of data compiled from various biological and biochemical datasets that is based upon measurements of protein interactions, molecular affinities and 81
established pathways. The data reflect known interactions (edges) established between any set of molecules (nodes). A recent interactome based upon nomenclature in the chick but including data from all species was established (Konieczka, Drew et al. 2009). We recently showed that interactome analysis of arsenite exposure in chick hearts was able to dissect the elements of epithelial-mesenchymal transition perturbed by this toxin
(Lencinas, Broka et al. 2010).
Analysis of the TCE interactome from hearts of exposed vs. control embryos identifies a particular set of molecules that appear to be most the highly connected or linked to gene expression changes observed after TCE exposure, a TCE interactome. In many cases these genes are linked to other highly linked genes and their own expression changes with TCE exposure. The most highly-linked gene in the TCE interactome is the transcription factor, HNF4a. Interestingly, HNF4a expression is not directly altered by
TCE exposure but a large number of its interactants are altered. The data suggest that low dose TCE exposure leads to widespread changes in expression of primary and secondary interactants regulated by HNF4a and that it may be a proximal target for TCE perturbation of gene expression. These data identify a possible target of TCE teratogenicity that would not be identified without interactome analysis. Interestingly,
HNF4a is a transcription factor associated with cytochrome p450 expression, liver carcinogenesis and diabetes, all of which have been associated with TCE exposure. 82
4.2. MATERIALS AND METHODS
Materials. Trichloroethylene ACS reagent, ≥99.5% (TCE) was ordered from
Sigma-Aldrich Co. (St. Louis, MO) Catalog #251402. Fertilized white leghorn eggs were obtained from McIntyre Poultry and Eggs, Lakeside, CA and incubated at 37.5°C for approximately 54 hrs or as needed to reach stage 14 (Hamburger and Hamilton, 1951).
Dosing. TCE was dosed at 8 ppb (60 nM) and 800 ppb (6000 nM) through injection into stage Hamburger Hamilton (HH)13 eggs using Hamilton Co. (Reno, NV)
800 Series Syringes (Part #7646-01) paired with custom needles (Part #7806-02) with the following specifications: RN NDL 6/PK (22s/1”/4)L.
Avian microarray. After dosing, embryos were allowed to develop for 48 h until reaching stage HH24. Avian complementary DNA (cDNA) arrays consisting of
~20,000 oligo-DNA elements were printed in duplicate by the Genomic Research
Laboratory in the Steele Children’s Research Center at the University of Arizona. The arrays were utilized as described by Konieczka et al. (2009) and Lencinas et al. (2010).
Total RNA was extracted from pooled samples of 18–21 Hamburger Hamilton (HH) 24 whole hearts using the RNeasy Mini Kit (Qiagen, MD). RNA was concentrated using
RNeasy Mini Elute Cleanup Kit (50) (Qiagen). Sense amplification was performed using
SenseAMP Amplification Kit (Genisphere, PA). The cDNA was then labeled using the 83
SuperScript Indirect cDNA Labeling System (Invitrogen, CA). Labeled cDNA was hybridized to the array slides using the Slide Hyb Hybridization Buffer #1 (Applied
Biosystems, CA). The hybridization process was done at a 50:50 ratio at 42°C for 16 h.
The slides were then scanned using the Applied Precisions Array Worxe (white light scanner). Spot finding was then done with the Soft/ Worxe Tracker 2.8.
A standard wheel design was used for comparing four samples, in which each sample was compared with the others. A dye swap was used for each comparison, making a total of eight microarray chips. To normalize the results, within-chip normalization was performed using the R package OLIN (Optimized-Location and Intensity-dependent
Normalization) (Futschik and Crompton 2005). The false discovery rate was computed for each spot based on intensity- and location-dependent bias. Standard libraries in the R
BioConductor package were used to normalize between array chips (Bolstad, Irizarry et al. 2003). Finally, linear models were fit to the normalized gene expression using the
Limma library, which computes log2 fold change, indicating the quantity and directional change of gene expression, T- and B-statistics, and adjusted p value that takes into account the false discovery rate (Smyth and Speed 2003). Genes differentially expressed by a p < 0.8 value were selected to carry out further analysis using Cytoscape/
BioNetBuilder2.0/jActiveModules software. While the p value selected is higher than the often-used 0.05 value, it is important to note that the 0.08 is the adjusted p value that includes false discovery rate calculations for a more stringent selection. Previous microarray work looking at known components of EMT signaling pathways during chick 84
gastrulation showed that 0.08 appeared to be a better fit than 0.05. The better fit was conveyed by inclusion of known molecular networks under 0.08 p value (Konieczka,
Drew et al. 2009).
Cytoscape analysis. To visualize and characterize data obtained with the avian microarray, we used the chicken interactome downloaded via BioNetBuilder2.0
(Konieczka, Drew et al. 2009) and Cytoscape 2.6.6 (Killcoyne, Carter et al. 2009). The chicken interactome is a genome-wide set of molecular interactions transferred and integrated from interaction data in diverse eukaryotic species. As described by Konieczka et al. (2009), a variety of databases showing both hierarchical regulation and molecular interactions were mined to develop the interactome. Nodes (represented as circles) in the chicken interactome correspond to genes, and edges (connecting lines) represent documented interactions. The Cytoscape plug-in jActiveModules was used with microarray data to identify clusters of genes in the network most representative of TCE treatment. Cytoscape version 2.6.2 and the plug-ins were downloaded from www.cytoscape.org.
85
4.3. RESULTS
Chick Microarray. Microarray studies carried out in by Caldwell et al. (2010) and Selmin et al. (2008) indicate that a large number of genes were affected by TCE in mouse embryonic heart after maternal exposure to TCE in drinking water and P19 embryonal carcinoma cells in tissue culture media. As the chick model enables delivery of TCE in ovo during the critical period of heart development, we examined altered gene expression by microarray after treatment with TCE at 8 or 800 ppb. Control embryos were injected with an equal volume of PBS carrier. Injections were at stage 14
(approximately 48 hrs of development) and heart tissues were collected at 96 hrs later at approximately stage 21. This period of exposure included the interval from an early looped stage of heart development to the near completion of septation and myocardial trabeculation. Procedures for microarray analysis were as previously described (Lencinas et al. 2010) but briefly, hearts were collected, extracted for RNA, copied to cDNA, amplified, labelled and hybridized to a 20,000 element chick oligonucleotide microarray.
After adjustments for dye swap and statistical analysis with a wheel arrangement of treatments and controls, changes in gene expression were evaluated for statistical significance.
Using an adjusted P-value of 0.05, we found that 582 genes were perturbed by 8 ppb TCE exposure and 1286 genes perturbed by 800 ppb when compared to PBS injection into the yolk. At 8 ppb, 250 genes (42%) were significantly downregulated and 86
332 (58%) were upregulated. Tables 4.1 and 4.2 show the 30 most up- or downregulated genes observed with this exposure. 87
TABLE 4.1: Genes up-regulated after 8 ppb TCE exposure Fold Change transmembrane protein 14A 1.23 tissue factor pathway inhibitor (lipoprotein- associated coagulation inhibitor) 1.24 insulin-like growth factor binding protein 7 1.24 similar to junctophilin type 2 1.25 high-mobility group nucleosomal binding domain 2 1.25 similar to RIKEN cDNA 1700012G19 1.26 hypothetical gene supported by CR353580; CR391109 1.28 ribosomal protein S27-like 1.3 titin 1.31 LIM domain only 3 (rhombotin-like 2) 1.32 asp (abnormal spindle) homolog, microcephaly associated (Drosophila) 1.35 cytoskeleton-associated protein 4 1.36 inhibitor of DNA binding 4, dominant negative helix-loop-helix protein 1.38 Yip1 domain family, member 5 1.39 tubulin, alpha 1c 1.39 cofilin 2 (muscle) 1.4 mitochondrial ribosomal protein L44 1.41 nudix (nucleoside diphosphate linked moiety X)- type motif 16-like 1 1.41 dysbindin (dystrobrevin binding protein 1) domain containing 1 1.43 similar to LOC387763 protein 1.48 Tax1 (human T-cell leukemia virus type I) binding protein 3 1.51 proline-rich nuclear receptor coactivator 1 1.51 La ribonucleoprotein domain family, member 5 1.57 casein kinase 1, alpha 1 1.58 similar to urea transporter isoform UT-A1 1.76 heterogeneous nuclear ribonucleoprotein A3 1.77 FXYD domain containing ion transport regulator 6 1.88 titin 2.23 aldolase A 2.27 similar to hypothetical protein LOC776779 3.37 88
TABLE 4.2: Genes down-regulated after 8 ppb TCE exposure Fold Change minichromosome maintenance complex component 10 -3.33 zinc finger, matrin type 2 -1.78 solute carrier family 24 (sodium/potassium/calcium exchanger), member 4 -1.67 basic, immunoglobulin-like variable motif containing -1.62 coiled-coil domain containing 16 -1.6 sperm associated antigen 9 -1.58 kinectin 1 (kinesin receptor) -1.5 cAMP responsive element binding protein 3-like 1 -1.49 RAB21, member RAS oncogene family -1.43 Yip1 domain family, member 1 -1.4 putative homeodomain transcription factor 1 -1.37 pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) -1.34 titin -1.32 TBCC domain containing 1 -1.32 similar to oxidored-nitro domain-containing protein -1.27 similar to ovomucoid precursor - chicken -1.21 PIT 54 protein -1.17 similar to Autoimmune regulator (Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy protein homolog) (APECED protein homolog) -1.14 similar to Hypothetical protein FLJ11506 -1.12 Rho GTPase activating protein 18 -1.1 ribosomal protein L37 -1.1 TatD DNase domain containing 1 -1.06 G protein-coupled receptor 88 -1.05 similar to cytoplasmic dynein intermediate chain 1 -1.03 ring finger protein 20 -1.02 hypothetical protein LOC769325 -1.02 coiled-coil-helix-coiled-coil-helix domain containing 3 -1.01 RAS, dexamethasone-induced 1 -1.01 coiled-coil domain containing 124 -0.99 endoplasmic reticulum protein 29 -0.98 cold shock domain containing E1, RNA-binding -0.97 89
At 800ppb TCE exposure, 321 genes were downregulated (25%) and 965 (75%) were upregulated. Tables 4.3 and 4.4 show the genes with the greatest level of change up or down in expression. 90
TABLE 4.3: Genes up-regulated after 800 ppb TCE exposure Fold Change ribosomal protein S27a 1.29 similar to hypothetical protein LOC127262 1.3 tumor protein, translationally-controlled 1 1.3 eukaryotic translation initiation factor 1 1.32 FUS interacting protein (serine/arginine-rich) 1 1.32 proline-rich nuclear receptor coactivator 2 1.33 huntingtin interacting protein 2 1.33 heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA binding protein 1, 37kDa) 1.35 heat shock 70kDa protein 8 1.35 PDZ domain containing 11 1.36 SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae) 1.36 succinate dehydrogenase complex, subunit D, integral membrane protein 1.37 microtubule-associated protein, RP/EB family, member 1 1.39 activating transcription factor 4 (tax-responsive enhancer element B67) 1.39 arginyl-tRNA synthetase 1.4 phosphoglycerate kinase 1 1.43 four and a half LIM domains 2 1.44 hypothetical gene supported by CR353961; CR354235; CR389862; CR390435 1.47 hypothetical protein 1.48 similar to a-actin 1.49 aurora kinase A 1.49 collagen, type IV, alpha 5 (Alport syndrome) 1.5 ribosomal protein L11 1.5 hemoglobin, zeta 1.59 ATP synthase subunit alpha 1.63 triosephosphate isomerase 1 1.65 ribosomal protein L8 1.68 similar to NPD014 protein (NPD014) 1.72 Ras-related associated with diabetes 1.83 glyceraldehyde-3-phosphate dehydrogenase 2.19 91
TABLE 4.4: Genes down-regulated after 800 ppb TCE exposure Fold Change minichromosome maintenance complex component 10 -2.7 coiled-coil domain containing 16 -1.85 cAMP responsive element binding protein 3-like 1 -1.81 hypothetical protein LOC772138 -1.4 citrate lyase beta like -1.37 similar to oxidored-nitro domain-containing protein -1.32 solute carrier family 24 (sodium/potassium/calcium exchanger), member 4 -1.3 sperm associated antigen 9 -1.23 SET binding protein 1 -1.22 protein O-linked mannose beta1,2-N- acetylglucosaminyltransferase -1.21 PIT 54 protein -1.19 paired-like homeodomain transcription factor 1 -1.19 putative homeodomain transcription factor 1 -1.19 titin -1.17 kinectin 1 (kinesin receptor) -1.15 Yip1 domain family, member 1 -1.11 MOB1, Mps One Binder kinase activator-like 2B (yeast) -1.09 hypothetical protein LOC777053 -1.06 microtubule-associated protein 1 light chain 3 gamma -1.03 similar to collagen, type XXVII, alpha 1 -1 similar to Ectonucleoside triphosphate diphosphohydrolase 2 -0.98 SH3 domain binding glutamic acid-rich protein -0.98 Rho family GTPase 3 -0.98 PRP4 pre-mRNA processing factor 4 homolog B (yeast) -0.97 angiogenic factor with G patch and FHA domains 1 -0.96 TBCC domain containing 1 -0.94 zinc finger, matrin type 2 -0.93 protein phosphatase 1, regulatory (inhibitor) subunit 13B -0.93 EPH receptor A2 -0.91 92
The entire dataset is available in the supplemental data. While there is considerable overlap, there are differences between the sets.
As with the previous microarray data in mouse and rat cells, TCE treatment produces a complex view of changes in gene expression and there is a limited overlap between treatments. Table 4.5 shows differences. 93
TABLE 4.5a: Genes up-regulated after TCE exposure Fold Change transmembrane protein 14A 1.23 tissue factor pathway inhibitor (lipoprotein- associated coagulation inhibitor) 1.24 insulin-like growth factor binding protein 7 1.24 similar to junctophilin type 2 1.25 high-mobility group nucleosomal binding domain 2 1.25 similar to RIKEN cDNA 1700012G19 1.26 hypothetical gene supported by CR353580; CR391109 1.28 titin 1.31 LIM domain only 3 (rhombotin-like 2) 1.32 asp (abnormal spindle) homolog, microcephaly associated (Drosophila) 1.35 cytoskeleton-associated protein 4 1.36 inhibitor of DNA binding 4, dominant negative helix- loop-helix protein 1.38 Yip1 domain family, member 5 1.39 tubulin, alpha 1c 1.39 cofilin 2 (muscle) 1.4 mitochondrial ribosomal protein L44 1.41 nudix (nucleoside diphosphate linked moiety X)-type motif 16-like 1 1.41 dysbindin (dystrobrevin binding protein 1) domain containing 1 1.43 similar to LOC387763 protein 1.48 Tax1 (human T-cell leukemia virus type I) binding protein 3 1.51 proline-rich nuclear receptor coactivator 1 1.51 La ribonucleoprotein domain family, member 5 1.57 casein kinase 1, alpha 1 1.58 similar to urea transporter isoform UT-A1 1.76 heterogeneous nuclear ribonucleoprotein A3 1.77 FXYD domain containing ion transport regulator 6 1.88 titin 2.23 aldolase A 2.27 similar to hypothetical protein LOC776779 3.37 similar to hypothetical protein LOC127262 1.3 94
tumor protein, translationally-controlled 1 1.3 eukaryotic translation initiation factor 1 1.32 FUS interacting protein (serine/arginine-rich) 1 1.32 proline-rich nuclear receptor coactivator 2 1.33 huntingtin interacting protein 2 1.33 heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA binding protein 1, 37kDa) 1.35 heat shock 70kDa protein 8 1.35 PDZ domain containing 11 1.36 SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae) 1.36 succinate dehydrogenase complex, subunit D, integral membrane protein 1.37 microtubule-associated protein, RP/EB family, member 1 1.39 activating transcription factor 4 (tax-responsive enhancer element B67) 1.39 arginyl-tRNA synthetase 1.4 phosphoglycerate kinase 1 1.43 four and a half LIM domains 2 1.44 hypothetical gene supported by CR353961; CR354235; CR389862; CR390435 1.47 hypothetical protein 1.48 similar to a-actin 1.49 aurora kinase A 1.49 collagen, type IV, alpha 5 (Alport syndrome) 1.5 ribosomal protein L11 1.5 hemoglobin, zeta 1.59 ATP synthase subunit alpha 1.63 triosephosphate isomerase 1 1.65 ribosomal protein L8 1.68 similar to NPD014 protein (NPD014) 1.72 Ras-related associated with diabetes 1.83 glyceraldehyde-3-phosphate dehydrogenase 2.19
TABLE 4.5b: Genes down-regulated after TCE exposure Fold Change basic, immunoglobulin-like variable motif containing -1.62 RAB21, member RAS oncogene family -1.43 pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) -1.34 similar to ovomucoid precursor - chicken -1.21 95
similar to Autoimmune regulator (Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy protein homolog) (APECED protein homolog) -1.14 similar to Hypothetical protein FLJ11506 -1.12 Rho GTPase activating protein 18 -1.1 ribosomal protein L37 -1.1 TatD DNase domain containing 1 -1.06 G protein-coupled receptor 88 -1.05 similar to cytoplasmic dynein intermediate chain 1 -1.03 ring finger protein 20 -1.02 hypothetical protein LOC769325 -1.02 coiled-coil-helix-coiled-coil-helix domain containing 3 -1.01 RAS, dexamethasone-induced 1 -1.01 coiled-coil domain containing 124 -0.99 endoplasmic reticulum protein 29 -0.98 cold shock domain containing E1, RNA-binding -0.97 hypothetical protein LOC772138 -1.4 citrate lyase beta like -1.37 SET binding protein 1 -1.22 protein O-linked mannose beta1,2-N- acetylglucosaminyltransferase -1.21 paired-like homeodomain transcription factor 1 -1.19 MOB1, Mps One Binder kinase activator-like 2B (yeast) -1.09 hypothetical protein LOC777053 -1.06 microtubule-associated protein 1 light chain 3 gamma -1.03 similar to collagen, type XXVII, alpha 1 -1 similar to Ectonucleoside triphosphate diphosphohydrolase 2 -0.98 SH3 domain binding glutamic acid-rich protein -0.98 Rho family GTPase 3 -0.98 PRP4 pre-mRNA processing factor 4 homolog B (yeast) -0.97 angiogenic factor with G patch and FHA domains 1 -0.96 protein phosphatase 1, regulatory (inhibitor) subunit 13B -0.93 EPH receptor A2 -0.91 96
From these microarray results, we performed an interactome analysis of the microarray results. We compared 8ppb TCE data with PBS (carrier) injected cardiac tissues (48 hrs exposure). The data were imported into Cytoscape for a bioinformatics analysis with jActive Modules and BioNetBuilder. This procedure examines spot intensity and p value and constructs the interactome (See Konieczka, Drew et al. 2009 for description or visit http://err.bio.nyu.edu/cytoscape/bionetbuilder/.) This analysis produced a TCE interactome of 1345 genes. The data was graphed in cytoscape to display the most highly linked genes. Figure 4.1 shows the data in a circular display where we have moved the more highly-linked genes into the center. Linkages between genes are compiled from 7 databases that include known molecular interactions as well as identified direct gene targets (http://err.bio.nyu.edu/cytoscape/bionetbuilder/).
The data were examined to identify the most highly linked genes. These genes are displayed in Table 4.6. HNF4a was identified as the most highly linked gene both in direct linkages and by indirect linkage to additional nodes. Examination of the microarray data showed that while HNF4a was present in the developing heart, its level of expression was unchanged with TCE exposure. However, a relatively large number of genes linked to this molecule were significantly up- or down-regulated by TCE exposure.
HNF4a Transcription factor binding.
HNF4a is not normally associated with expression in the heart. While TCE can affect gene transcription of molecules linked to HNF4a, its own transcription does not have to 97
be altered by TCE to effect function. As its level of expression appears unchanged, we sought indication that the functional activity of this molecule is altered by TCE exposure.
We had previously shown that Serca2a expression is altered by TCE. Examination of the promoter region of Serca2a showed a candidate HNF4a binding site. After an acute low dose exposure of TCE on H9c2 rat cardiomyocytes at two intervals a CHIP analysis was carried out to verify an increased HNF4a expression after TCE exposure (Massie and
Mills 2009). H9C2 cells were fixed, the genomic DNA was sheared and an immunoprecipitation was performed with anti-HNF4a antibodies. The eluted DNA was then measured by quantitative real time PCR to measure changes in HNF4a bound Serca2 promoter region compared to untreated control cells. In Figure 4.2, we show that 10 ppb
TCE exposure after 30 mins and 1hr results in an increase in HNF4a transcription factor binding to this region. This data confirms that HNF4a is found in cardiomyocytes and confirms an alteration of activity consistent with a proximal role for HNF4a in mediating some of the effects of TCE on cardiomyocytes. 98
Fig 4.1. Interactome analysis of chick microarray. Circles represent nodes (molecules) and connecting lines are edges showing an identified interaction in one or more of the core databases. The data are displayed in a circular pattern by Cytoscape and the most highly linked nodes were moved to the center of the circle. HNF4a is shown in light blue and directly linked nodes are shown in dark blue. 99
Name 1' Neighbors 2' Neighbors 1' Sig DN 1' Sig UP HNF4A 159 866 35 55 TRAF6 116 778 12 43 IKBKE 100 621 11 28 14-3-3z 89 670 9 25 14-3-3g 80 555 14 20 EIF1B 63 431 5 16 PRKAB1 56 518 8 13 MAP3K3 54 646 6 17 HSPA8 54 540 4 16
Table 4.6. Mostly highly linked nodes in interactome analysis of TCE. HNF4a shows 159 directly linked (1’Neighbors) nodes in the TCE dataset and indirect linkage to a further 866 nodes (2’Neighbors). The combined 1’ and 2’ linkage represents approximately 76% of the total 1345 nodes found in the TCE interactome. Of the 159 directly linked nodes, 35 were significantly down-regulated and 55 were up-regulated by 8ppb TCE. Thus HNF4a is the most highly linked node and is central to the greatest number of regulated targets in the dataset. Most of the regulated primary targets of HNF4a were also found in the mouse dataset. 100
Fig 4.2. H9C2 cell exposed to 10PPB TCE for 30 minutes and 1 hour and then processed for CHIP analysis using HNF4a specific antibodies. The results show the binding of HNF4alphato the Serca2 proximal promoter region was increased by threefold in TCE exposed myoblasts compared to control cells.
101
4.4. DISCUSSION
Global Effects of TCE Exposure. To determine the effects of TCE exposure during a critical period of cardiogenesis, we performed a microarray assay in which chick embryos were exposed to low dose TCE (8 ppb) for 48 hrs. This interval extends from the first expansion of the atrioventricular cushions through the major events or sepation and trabeculation in the developing heart. Embryonic hearts were then extracted and processed for RNA isolation for use in a chick microarray. Using an adjusted P-value of
0.05, we found that 582 genes were perturbed by 8 ppb TCE exposure and 1286 genes perturbed by 800 ppb when compared to PBS injection into the yolk. At 8 ppb, 250 genes (42%) were significantly downregulated and 332 (58%) were upregulated. Tables
4.1 and 4.2 show the 30 most up- or downregulated genes observed with this exposure.
At 800 ppb TCE exposure, 321 genes were downregulated (25%) and 965 (75%) were upregulated. Tables 4.3 and 4.4 show the genes with the greatest level of change up or down in expression. While there is considerable overlap, there are differences between the sets (Table 4.5). A pattern of effected genes was difficult to assess from our raw output but they remain generally consistent with effects seen at 100ppm in rat embryos
(Collier et al., 2003) and at 10 ppb in mouse embryos (Caldwell et al., 2009). Note that the delivery of TCE in ovo in the chick differs from consumption in maternal drinking water in the mouse. The data show that perturbation of gene expression in the embryo is consistent between these two animal models of exposure. The data confirm the effects of 102
TCE on transcriptional activity at exposure levels near the MCL for TCE in drinking water.
Cytoscape analysis. As direct examination of microarray data is relatively unrewarding, we applied the data to an interactome analysis to discern patterns of pathway alteration. The data identified a subset of linked molecules that were affected by
TCE exposure. The most highly linked molecule in the dataset was HNF4a. While
HNF4a was linked to the largest number of altered transcripts, it was in itself, not altered at the transcriptional level. We found this to be potentially interesting. As one views transcriptional changes with TCE exposure, there must be a point, proximal to the toxicant, where TCE or its metabolite initially acts upon a molecule to alter gene transcription. Such a proximal mediator would not, itself, be expected to be transcriptionally regulated except, perhaps, in an autocrine fashion. HNF4a is linked to the expression of 76% of the nodes in the chick interactome. Furthermore, Rana et al.
(2010) showed that, in human cells, HNF4a is competent to upregulate cytochrome P450
2C family expression after xenobiotic exposure. We found that TCE exposure in the avian model system at 8 ppb can result in the up regulation of both CYP2C family RNA and protein expression (Makwana et. al. In submission).
As we examined the literature on HNF4a, we found that its activity can be altered by phosphorylation and by binding to linoleic acid as a ligand. Though yet undefined, we are intrigued by the possibility that the solvent, TCE, could disrupt endogenous interaction between HNF4a and its ligand. Though analysis of linoleic acid and TCE 103
interactions is beyond the scope of this study, we sought to detect altered HNF4a activity after TCE exposure.
CHIP analysis. To confirm functional effects of TCE on HNF4a, a CHIP analysis was conducted in a rat cardiomyocyte cell line after 10 ppb TCE exposure for 30 min and 1 hr respectively. At the end of each interval, the relative binding of the Serca2 promoter region to HNF4a was assessed by real time PCR. In both exposures, HNF4a binding to DNA was increased after TCE exposure. This independent observation in a cell line confirms that HNF4a dose bind to DNA with a higher affinity after TCE exposure. While the data do not show that HNF4a is the only mediator of cardiotoxic effects on the developing heart, they show that HNF4a is present in cardiomyocytes and its activity is altered concomitant with TCE exposure. We note that TCE is associated with liver cancer and with diabetes in human populations and these diseases can be linked to the activity of HNF4a (Research Council 2006).
Future studies will be needed to explore the basis of altered HNF4a binding to the
Serca2 binding site and to determine whether TCE exposure has effects on ligand binding, phosphorylation or even epigenetic modification of its binding domain.
Nevertheless, studies shown here establish an apparent mechanistic explanation for the transcriptional effects of TCE in the developing heart. This explanation can link our previous studies of myocyte function and cytochrome expression with a specific molecular target. Future work will identify the limits of this proposed mechanism. 104
CHAPTER 5: SUMMARY AND CONCLUSIONS
5.1. SUMMARY
Early epidemiological studies described trichloroethylene as a cardiac teratogen in exposed populations in the Tucson Valley (Goldberg, Lebowitz et al.
1990; Bove, Shim et al. 2002). These initial epidemiological studies were further supported by in vivo studies conducted in the rat model system and in vitro studies conducted in the chick model system (Johnson, Dawson et al. 1998; Boyer, Finch et al. 2000). While these early studies demonstrated generalized cytotoxicity after
TCE exposure in vitro and cardiac defects in vivo, they did not provide insights into potential mechanisms involving TCE exposure. Furthermore, methodological differences in subsequent studies (Fisher, Channel et al. 2001) lead to controversy as to the exact teratogenic nature of TCE exposure or whether there was sufficient evidence of teratogenecity at all (Hardin, Kelman et al. 2005).
Additionally, consistent with studies of carcinogenesis in the rodent liver
(ATSDR, 2003) it was argued that the primary metabolite of TCE, TCA
(trichloroacetic acid) produces more severe teratogenic phenotypes (than TCE) when dosed as the primary toxicant (Johnson, Dawson et al. 1998; Selmin,
Thorne et al. 2005), however, the pharmacokinetics of TCA induced toxicity are further complicated by the hydrophilic nature of TCA which would not readily cross cell membranes. The most recent studies involving TCE exposure during development utilized rat H2C9 rat cardiomyocytes (Caldwell, Thorne et al. 2008) 105
as an in vitro model of TCE exposure and chick embryos (Drake, Koprowski et al.
2006; Rufer, Hacker et al. 2010) as an in vivo model of TCE exposure. Both of these studies demonstrated an alteration in Ca2+ handling after TCE exposure and an alteration in cardiac blood flow during embryonic development.
5.2. EXPERIMENTAL RESULTS
5.2.1. TCE ALTERS CARDIAC FUNCTION
Our studies demonstrate that using the chick embryo as an in vivo model system of TCE exposure, low doses of TCE exposure are sufficient to alter shear stress gene expression in developing hearts. These findings are significant because an alteration in shear stress expression in the developing heart due to venous clipping was associated to the development of cardiac defects (Hogers,
DeRuiter et al. 1997; Groenendijk, Hierck et al. 2005). Ventricular septal defects
(VSDs) were most often observed in venous clipped chick embryos; similarly low doses of TCE exposure can result in the formation of VSDs (Rufer, Hacker et al.
2010).
Of the shear stress genes studied, nitric oxide synthase 3 (NOS-3) was shown to be expressed in the endothelial cells of the heart during development and required for proper atrial and ventricular septation (Feng, Song et al. 2002).
Similarly conditional knock out studies of Krüppel like factor 2 (KLF2) demonstrated an embryo lethal high cardiac output state, in which the heart demonstrated an irregular contractile function (Lee, Yu et al. 2006). We 106
demonstrate that low dose TCE exposure was significant to reduce the expression of both NOS-3 and KLF2 in the developing heart. This effect was observed after a single acute dose of TCE was administered in ovo at HH13 and effects on shear stress gene expression could be observed after 24 hrs of exposure (HH17) and 48 hrs of exposure (HH24).
Krüppel like factor 2 (KLF2) protein was expressed in both endothelium and myocardium of the developing chick heart at stage HH17. Low dose exposure of TCE (8 ppb) reduced expression of KLF2 protein in the developing chick heart (Chapter 2; Fig 2.3).
Additionally, we demonstrated that TCE was sufficient to alter cardiomyocyte function by altering cardiomyocyte half-width after 8 ppb TCE exposure (Chapter 2; Fig 2.4) however, this change in half-width was independent of sarcomere length (Chapter 2; Fig 2.5) illustrating that the sarcomere structure were not altered by the TCE treatment only their function.
As the change in cardiomyocyte function was observed in 16 days after initial exposure, the effects of TCE on function are clearly persistent. The combination of marker expression and altered cardiac function provides molecular and functional confirmation of the teratogenicity of TCE exposure at low doses. 107
5.2.2. TCE INDUCES CYP2C EXPRESSION IN THE HEART
To better understand the effects of low doses of TCE (8 ppb) on cardiac development and function we explored a hypothesis on developmental metabolism of TCE. Because heart development precedes liver (primary adult metabolic organ) development, we reasoned that the embryonic heart may be acting as an early metabolic organ. The non-monotonic dose response curve found in Chapter 2 might be produced by differences in TCE metabolism at different doses. After TCE exposure at both 8 and 800 ppb, real-time PCR experiments that showed a significant increase in Cytochrome P450 2H1 enzyme
RNAs in heart after 8 ppb TCE exposure but not at 800 ppb. CYP2H1 and its human and murine homolog have not been previously reported as being involved in TCE metabolism and this is a novel finding. It would seem that while CYP2E1 has been shown to metabolize TCE in adult livers, related family members 2H1 may be involved in the cardiac metabolism of TCE during embryonic development.
Immunohistochemical imaging demonstrated an increase of CYP2H1 proteins in the developing chick heart after 8 ppb TCE exposure (Chapter 3, Fig
3.5). Interestingly, the upregulation of expression occurred in both the myocardium and the endothelium of the atrioventricular canal. This increased protein expression was confirmed with a western blot, in which, a dark band was 108
visible where CYP2H1 protein would be expressed. In contrast the control sample showed the expression of two faint bands, indicating that the antibody could recognize both CYP2C family members, but only CYP2H1 is induced after
8 ppb TCE exposure.
These findings support current data (Drake, Koprowski et al. 2006;
Makwana, King et al. 2010; Rufer, Hacker et al. 2010) that the 8 ppb dose of TCE has greater activity than 800 ppb TCE. The data suggest that TCE would be metabolized to a greater extent at the low dose. Teratogenicity of TCE may be explained by the ability of TCE’s primary metabolite (TCA) ability to bind to plasma proteins, thus extending its half-life (Templin, Stevens et al. 1995).
During heart development TCA could be produced in the cells of developing heart and then be secreted to bind to matrix molecules, leading to cardiac defects.
In combination with the Chapter 2, these data confirm the molecular characterization of the non-monotonic dose curve and demonstrate the sensitivity of the developing heart to low dose TCE exposure.
5.2.3. HNF4A IS AN APPARENT PROXIMAL MEDIATOR OF TCE
EFFECTS ON GENE TRANSCRIPTION
A microarray analysis of low dose exposure shows a significant number of genes whose transcription is altered by TCE. Examination of the microarray data shows a consistency of widespread altered gene expression in the exposed chick 109
heart with mouse embryo hearts exposed through a distinct maternal drinking water protocol (Caldwell, Manziello et al. 2010). The broad nature of perturbed gene expression demonstrates the toxicity of TCE but makes the identification of the proximal element in the teratogenetic pathway difficult to identify. To explore the microarray data, we turned to interactome analysis of the data to identify central elements of perturbed gene regulation. As shown in Chapter 4, we developed a “TCE Interctome” from those perturbed genes from our data that were found in the “ChickNet.” Though not every gene identified in the microarray was found in the ChickNet, we found 1345 genes that were common to both databases and explored the linkages between them. The data pointed to a transcription factor, HNF4a, that was directly or indirectly linked to 76% of the interactome and had the highest number of significantly regulated direct targets.
HNF4a is a transcription factor that is most commonly associated with the liver and is known to mediate transcription of cytochromes (Rana, Chen et al. 2010).
HNF4a is also associated with liver cancer and diabetes, diseases that have been linked to TCE exposure in adult human or animal models (Research Council
2006).
In our analysis of HNF4a, we found that its expression level is unchanged by TCE exposure, suggesting that it is proximal to the teratogenetic pathway and not just an intermediate that is downstream of another transcription factor or complex. As HNF4a is regulated by the ligand, linoleic acid, we can imagine that 110
TCE or a metabolite might perturb its association. HNF4a activity is also regulated by phosphorylation and its post-translational modification might be altered with exposure. Though there remains a need to identify specific HNF4a isoforms found in the embryonic heart and to localize them within tissues, we were able to show that the binding of HNF4a to a specific binding site in Serca2 was perturbed shortly after TCE exposure in a rat myocardial cell model. Though confirmation is continuing in the lab, this data suggests that HNF4a may be a central mediator of many of the effects of TCE on the developing heart. It will be interesting to explore whether it is similarly involved in consequences of TCE exposure in adults.
5.2.4. MODEL
Our findings have culminated to the development of a model for TCE exposure during development. This model integrates the findings of this thesis in a continuum of altered development and function, metabolism and critical gene regulation. While the data may not explain every aspect of TCE toxicity and altered gene regulation, the data put the examination of TCE teratogenicity on a firm molecular basis that can continue to be explored in the future with specific hypotheses and testable experiments. Importantly, the formerly contentious observation that TCE is a potent cardiac-specific teratogen, can be said to be 111
established to a level that was not possible when the data relied upon statistical measures of morphology and a confusing dose response curve.
↓Ca2+ Pumps ↑Half-Width ↑[TCE Metabolites] ↑CYPs
TCE Cardiomyocytes ↓NOS-3 ↓KLF2
↑CHDs ↓FLOW
Model of TCE exposre during heart development. Low doses of TCE expsore (8 ppb) results in an increased expression of CYP RNAs in developing chick hearts. This results in an alteration in cardiomyocyte function illustrated by a decrease in Serca and Ryr and an increase in cardiomyocyte half-width, this may be a result of the formation of TCE metabolites. Altered transcription of calcium channels and other proteins is likely due to proximal effects on HNF4a regulation of gene expression or similar effects on related nuclear receptors. Alteration of cardiomyocyte function leads to a decrease in blood flow through the embyonic heart resulting in a decrease in shear stress genes NOS-3 and KLF2 as markers of flow. Decreased flow can result in increased congenital heart defects.
112
5.3. FUTURE DIRECTIONS
The chick embryo provides us with a useful model system to understanding the basic mechanisms for TCE mediated developmental toxicity.
To better understand human exposure to TCE, future studies must utilize mammalian model systems of exposure. While both the mouse and rat have been used to model TCE for developmental toxicity, it has been suggested that other mammalian systems be utilized to properly assess TCE mediated developmental toxicity. Of these systems, the rabbit has been recommended by Dr. Susan Smith of the University of Wisconsin. Dr. Smith has authored several publications studying the consequences of TCE exposure in chick embryos. Dr. Smith’s reasons that alternative animal models such as the chick have been used to validate human TCE exposure phenotypes; i.e. cardiac defects such as VSDs and valve defects which appear less frequently at low doses in murine systems
(Johnson, Goldberg et al. 2003).
However, because the chick model system lacks the CYP 2 family isoform
E1, it may not be the best model system for understanding the pharmacokinetics of TCE exposure during development. Historically, the best model systems for pharmacokinetics studies have been the dog and pig. However, these systems along with the rhesus macaque are rarely used due to their excessive cost. In spite of that, these systems have demonstrated their usefulness in modeling human 113
developmental phenotypes, as was seen with the earliest thalidomide studies conducted by the US FDA (Cohlan 1963).
Mechanistically, we would hypothesize that mothers exposed to low doses of TCE through ingestion allow their fetus to be exposed to TCE through systemic circulation. Once TCE enters fetal circulation in the earliest stages of development, the cells of the early heart contain CYP 2 family P450s that are capable of metabolizing TCE. These CYPs metabolize the parent compound TCE into TCA and DCA. Due to the acidic non-soluble hydrophilic nature of both
TCA and DCA they remain in the heart cells (endothelial and cardiomyocytes) of the developing embryo leading to cell death or an alteration in cellular programming. Subsequently these cells lose their ability to form the valves and septal structures of the heart leading to the formation of cardiac defects, as seen in exposed populations.
Because of the toxicity demonstrated at low-doses of TCE exposure, the
US EPA is reevaluating the MCL for TCE (5 ppb) and has recommended a
Maximum Contaminant Level Goal (MCLG) for TCE to be 0. In the near future, mechanistic studies in model systems need to examine the localization of HNF4a in the heart and the sufficiency of this molecule to explain the experimental observations on gene expression and cardiac function. As HNF4a is one of a series of nuclear transcription factors that bind hydrophobic ligands, it would be appropriate to compare effects of TCE and its metabolites on related factors. On a 114
wider level, HNF4a may provide a mechanistic explantation for adult diseases such as cancer associated with TCE exposure.
The experiments detailed in this thesis make a useful contribution to the field by putting molecular and cellular components of heart development central to the TCE response and provide a mechanistic explanation for a substantial environmental problem. 115
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