Using mRNA Transcripts in the African Clawed Frog (Xenopus laevis) Intestine as a Model
to Study the Effects of Endocrine Disrupting Compounds (EDCs)
Tanya Ray Sutton
Introduction: Endocrine Disrupting Compounds (EDCs) are compounds and chemicals that
disrupt normal endocrine function [13]. EDCs can be found anywhere from water to household
products to soil and impact both humans and wildlife alike. The endocrine system uses chemical
signaling by way of hormones to regulate reproduction, development, energy metabolism,
growth, and behavior. EDCs such as Triclosan (used in antibacterial soaps and sanitizers) [5] and
Bisphenol A (used in plastic products) [3] can be found in human urine and cord blood.
Environmentally relevant EDCs include wastewater [12, 4] and perchlorate (found in rocket fuel)
[16]. EDCs can affect a range of biological processes including, but not limited to, reproductive,
neurological and thyroid function [3]. The purpose of this study is to establish sensitivity in
thyroid regulated genes as a way to evaluate for EDC disruption.
Thyroid hormones (THs) are critical to developmental pathways and essential in the normal
function of the cardiovascular, central nervous, digestive, and reproductive systems [13].
Specifically, these hormones are thyroxine (T4) and triiodothyronine (T3). Hormonal regulation
is similar, and in some cases identical, across clades [7]. In amphibians, TH is vital to the
reorganization of body systems during metamorphosis, which involves reprogramming of gene
expression [2]. Evaluation of TH-regulated genes such as fibroblast activation protein alpha
(FAPα), corticotropin releasing hormone binding protein (CRHBP) and thyroid receptor alpha
(TRα) [6] can give insight into effects on developmental processes. Our lab found several
additional genes to be TH responsive (Col1a2, RpS10, TRβ, DIO2, TH/bZIP, FN1, MMP2, Ef1a,
and TIMM50) using a microarray [CITE SEARCY] and will be further evaluated in this study.
Amphibians share similar biological functions with humans such as thyroid and endocrine
function. This gives a beneficial edge to the amphibian model to study the effects of EDC
disruption on hormones such as TH. One readily used amphibian model is the African Clawed
frog (Xenopus laevis). X. laevis have been used in numerous comparative studies as an indicator
species, and have contributed greatly to the understanding of specific changes that occur during
development and growth through metamorphosis. [1, 9,11]
Neogenesis, apoptosis and restructuring of organ systems all occur during metamorphosis
allowing great impacts to occur within a measurable amount of time. This makes amphibians a
great model to test for disruption. [15] This is a unique phenomenon specific to amphibians, yet
processes involved occur throughout development across species. Evaluation of organs such as
the intestines directly impacted by these processes can grant a greater insight to specific effects
of EDCs.
The amphibian intestinal tract goes through a dramatic reconstruction throughout the early stages
of metamorphosis. During the tadpole stage, amphibians are strict herbivores feeding off of plant
matter and algae. As metamorphosis progresses and tadpoles develop into juveniles, then into
adults, they become carnivorous, feeding off of insects and sometimes other amphibians. The
ability to make such a dramatic shift in nutritional intake is directly correlated with the
reconstruction of the intestinal tract. The intensity of this process allows for many opportunities
to evaluate for disruption over a short period of time.
Fold Difference
This particular study will focus on establishing sensitivity of several known thyroid responsive
genes to T3 in tissues (tail, intestine, brain, hind limb) using X. laevis as a model. To date, we
have completed the chosen gene set in the tail. Sensitivity was shown in FAPα (Figure 1) but not
CRHBP (Figure 1) or TRα (Figure 1). Current focus is on establishing if these same thyroid
responsive genes show sensitivity in the intestines of T3 exposed X. laevis. We hypothesize that
all genes previously tested in the tail will show a similar pattern in the intestine.
3.5
3
2.5
2
1.5
1
0.5
0
0
0.1
1
10
50
T3 Dose (nM)
*
200
150
100
*
50
0
0
0.1
1
10
T3 Dose (nM)
50
Figure 1: (clockwise) No statistically
significant change shown in tail tissue for
TRα. Statistically significant change in tail
tissue at 10nM and 50nM concentrations
of T3 at 72 hours. No statistically
significant sensitivity of CRHBP at 48
hours according to the Steel test, however,
a Kruskul-Wallace test showed sensitivity
between treatments.
250
Fold Diffrence
Fold Difference
250
Materials and Methods: X. laevis tadpoles were received at Nieuwkoop and Faber (NF) stage
53 [18]. This is an ideal stage for a thyroid study because the tadpoles have the receptors for
thyroid hormone, but are not actively using TH on their own.[7] Prior to exposure, the tadpoles
were given a 72 hour acclimation period to allow for adjustment to water and room conditions.
Treatment groups were divided into replicates of five with ten tadpoles per treatment, per time
point (Table 1). The exposure included varying levels of T3. The 0nM concentration was used as
a control, the 0.1nM and 1nM concentrations represent low endogenous levels, the 10nM
concentration represents the high endogenous level and the 50nM concentration was used as the
200
150
100
50
0
0
0.
high dose. All treatment groups of T3 were administered with NaOH as the chosen vehicle. It
has been previously demonstrated by our lab that NaOH is neither toxic nor thyroid disrupting.
Table 1: Experimental set up for exposure to varying levels of T3
T3 Doses
48 Hour
72 Hour
0 nM
n=10
n=10
0.1 nM
n=10
n=10
1 nM
n=10
n=10
10 nM
n=10
n=10
50 nM
n=10
n=10
Tadpoles were placed, in pairs, in 800uL artificial pond water due to their social nature. Solitary
housing can cause a stress response, which can have an effect on thyroid function.[7] After each
group of exposures were complete, intestines were collected, stored in RNAlater (a preservative
for RNA extractions) and placed in a -80˚C freezer until RNA was extracted.
Using two separate kits, RNA was extracted from the intestinal tissue and converted to 20 µL of
cDNA according to the manufacturer’s instructions. Real time quantitative polymerase chain
reaction (qPCR) was run on cDNA with iQ SYBR Green master mix. 25µL reactions were run
for 40 cycles at an annealing temperature of 60˚ C on a BioRad iQ5 thermocycler for each gene.
Results were then evaluated using a delta-delta-Ct method and analyzed in JMP 9.0.2 for
significance compared to control within each gene.
Results: We had originally hypothesized that results in the intestine would be nearly identical to
those of tail at 72 hours. However, what we found was a much more sensitive result in many of
the genes (Figure 2-clockwise: CRHBP, FAPα, TRα and Table 2). This suggests that the
intestine of X. laevis may be more sensitive to T3.
*
5
*
4
60
*
50
*
40
3
30
2
*
20
1
10
0
0
0
0.1
1
10
50
3
2.5
2
1.5
1
0.5
0
0
0.1
1
10
50
0
0.1
1
10
50
Figure 2: clockwise- Statistically
significant sensitivity of CRHBP at
10nM and 50nM concentrations of T3 at
72 hours (p= 0.0184 *) Statistically
significant sensitivity of FAPα to 1nM,
10nM and 50nM concentrations of T3 at
72 hours (p= 0.0184 *). No statistically
significant sensitivity of TRα at 72 hours.
Table 2: Results of sensitivity of all genes used in this study to varying nM concentrations of T3
Gene
Col1a2
TH/bZIP
TRβ
DIO2
FN1
T3(nM) Response
No response
No response
10 and 50
10 and 50
No response
Gene
CRHBP
TRα
rpS10
MMP2
FAPα
T3(nM) Response
10 and 50
No response
No response
10 and 50
1, 10 and 50
Alterations seen in mRNA transcript levels show that these genes are sensitive to thyroid
hormone. Demonstrating sensitivity in these genes has implications in human health. FAPα can
serve as either a beneficial or detrimental component in a variety of different cancers. It has been
shown to have an effect on the development of hepatocellular carcinoma [17] and tumor
formation [10]. It has also been suggested that FAPα can be used as a therapeutic measure in
ovarian cancer. [10] CRHBP acts as a regulatory protein in the central nervous system and is
essential in regulating both the adrenal and thyroid axes with focus in development. [14] TRα is
a receptor for thyroid hormone and is found in highest concentrations in cells that proliferate
upon treatment with thyroid hormone [8]. Changes in these genes can alter protein expression
and disrupt normal and essential pathways leading to morphological changes. These changes can
result in an increased risk of significant health problems, such as cancer or thyroid disease. Our
results suggest that these genes may be able to be disrupted by EDCs that affect thyroid function
and can potentially be used to test for EDC effects.
Timeline: Received, exposed, and collected animals January 13-January 19, 2012. RNA
extractions, cDNA synthesis for intestines were completed by July 18, 2012. qPCR for
mentioned genes were completed on October 15, 2012. Entire set of genes expected to be
completed by early January 2013. Long intervals of time between collections, extractions, and
completion of qPCR were due to completion of tail tissue. Then what?
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