INFLUENCE OF DIOCTYL SODIUM SULFOSUCCINATE (DOSS)

ON NON-MAMMALIAN ADIPOGENESIS

A Report of a Senior Study

by

Amelia Grace Brumbaugh

Majors: Biology and Environmental Studies

Maryville College

Spring, 2016

Date approved ____________, by ________________________________

Faculty Supervisor

Date approved ____________, by ________________________________

Division Chair

  iii  

ABSTRACT

Dioctyl Sodium Sulfosuccinate (DOSS) is a commonly used emulsifying agent that is

a primary component of Corexit 9500 oil dispersant, which was used in vast quantities

following the Deepwater Horizon Oil Spill. Recent research has indicated that DOSS may act

as an obesogen in mammals, but the impact on non-mammals has not been established. This

study used both in vitro and in vivo methods to examine the American alligator and zebrafish

to better understand the obesogenic activities of DOSS and the mechanism by which non-

mammalian obesity occurs. The PPARγ-RXRα pathway in the American alligator (Alligator

mississippiensis) was examined. The inductive effects of RXRα variants 1-4 on the PPAR

and RXR response elements were compared after exposure to known obesogens. It was

found that RXRα variant 2 had significantly higher inductive effects on the RXR response

element (RXRE) than other variants (P< 0.0001). No significant transactivation by DOSS or

Corexit occurred on either RXRE or PPRE (P>0.05). Zebrafish (Danio rerio) were exposed

to 4.9mg/L DOSS post hatching. Neither control nor exposed zebrafish showed visible

adipose tissue at one-week post exposure. These findings reveal both similarities and distinct

differences compared to mammalian obesogenesis worthy of further investigation.

  iv  

ACKNOWLEDGMENTS

I would like to thank the members of the Guillette Lab at the Medical University of

South Carolina for supporting me throughout my summer research, including Dr. Satomi

Kohno and Cameron Williams for their mentorship. Additionally, I would like to thank Dr.

Crain for his encouragement and critiques on my ongoing thesis research and writing.

Finally, I would like to thank the Maryville College Natural Science Department for funding

my presentation of this research at the 2016 Oil Spill and Ecosystem Science Conference in

Tampa, Florida. Funding was provided by the Gulf of Mexico Research Initiative and

Maryville College.

  v  

TABLE OF CONTENTS

Page

LIST OF FIGURES vi

CHAPTER I: INTRODUCTION 1

Endocrine Disrupting Compounds 1

Nuclear Hormone Receptors 4

Obesogens 6

Dioctyl Sodium Sulfosuccinate (DOSS) 8

CHAPTER II: MATERIALS AND METHODS 12

In vitro Procedures 13

In vivo Procedure 15

CHAPTER III: RESULTS 17

In vitro Results 17

In vivo Results 21

CHAPTER IV: DISCUSSION 23

APPENDIX 27

WORKS CITED 29

  vi  

LIST OF FIGURES

Figure Page 1. Pathway of a normal endogenous hormone, an agonist endocrine disrupting

compound, and an antagonist endocrine disrupting compound. 2. Effect of obesogenic compounds on the PPARγ-RXRα nuclear hormone

receptor pathway.

3. A comparison of relative retinoid X receptor response element (RXRE) induction after nuclear hormone peroxisome proliferator activator-gamma (PPARγ) and retinoid X receptor-alpha (RXRα) variants 1-4 binding.

4. A comparison of relative peroxisome proliferator activator response element (PPRE) induction of luciferase activities 44 hours after exposure to various test compounds.

5. A comparison of American alligator (Alligator mississippiensis) retinoid X receptor-alpha (RXRα) variant conserved domain amino acid length.

6. Interpolated retinoid X receptor response element induction after exposure to Corexit (Corexit 9500A), DOSS (dioctyl sodium sulfosuccinate), CWAF (Corexit water accommodated fraction of oil), and DMSO (dimethylsulfoxide).

7. Interpolated peroxisome proliferator activated response element (PPRE) induction after exposure to Corexit (Corexit 9500A), DOSS (dioctyl sodium sulfosuccinate), CWAF (Corexit water accommodated fraction of oil), and DMSO (dimethylsulfoxide).

8. Zebrafish (Danio rerio) individual one week after hatching and exposure to dioctyl sodium sulfosuccinate (DOSS) at 40X magnification.

9. Transverse sections of Zebrafish individuals one week after hatching that were exposed to dioctyl sodium sulfosuccinate (DOSS) after hatching.

5

8

17

18

19

20

21

22

22

  1  

CHAPTER I

 

INTRODUCTION

Anthropogenic pollution has led to an abundance of endocrine disrupting compounds

(EDCs) throughout the environment. Obesogens, an EDC subset present in many

environments and human products, has been associated with increased adipocyte

differentiation and misregulation of lipid metabolism due to the improper binding of nuclear

receptors by obesogenic compounds (Grün and Blumberg, 2006). Such compounds influence

gene expression and cell fate by functioning as synthetic ligands and activating nuclear

hormone receptors, often through the peroxisome proliferator-activated receptor (PPARγ)-

retinoid X receptor alpha (RXRα) pathway. Dioctyl Sodium Sulfosuccinate (DOSS), a

common detergent used in many man-made products, has been shown to act as an obesogen

in mammals (Nyankson et. al 2014, Chapman et al. 1985, Temkin et al. 2015). However, the

impact of DOSS on non-mammalian adipogenesis has not been established and the

mechanism through which obesogens function in non-mammalian species is not well

understood.

Endocrine Disrupting Compounds

The growing accumulation of environmental contaminants in numerous ecosystems

has caused alarm in regards to both ecological and human health risks (Diamanti-Kandarakis

et al., 2009). Anthropogenic pollution continues to challenge the livelihood of wildlife

  2  

population structures, reproductive capabilities, and overall wellbeing (Kavlock et al. 1996).

While public concern is often directed at contaminants with known strong toxicity,

compounds exhibiting sublethal effects on physiologic and molecular pathways are less

defined, particularly on non-mammalian species. Such sublethal effects include interference

of homeostatic biosynthesis and metabolism or endogenous hormone pathways, which could

lead to altered fecundity and increased susceptibility to disease (Diamanti-Kandarakis et al.,

2009).

As such, the increased incidence of endocrine disrupting compounds (EDCs) in the

environment are of particular concern due to their potential impacts on reproductive and

developmental health in a variety of organisms (Zhang et al., 2014 and Kidd et al., 2007). As

living organisms experience critical windows of physiologic and metabolic development in

utero, susceptibility to the effects of EDCs is highest during embryonic development (Barker,

2012). Indeed, numerous studies have linked EDC early life exposure with predispositions to

several diseases and reproductive complications, including altered fecundity, cardiovascular

disease, and cancer (see Table 1).

EDCs act through multiple mechanisms of action (Guillette and Crain, 2000).

Steroidogenesis is an intricate process that involves numerous enzymatic and precursory

components. Thus, alterations in these components can lead to changes in the endogenous

hormone system. Such alterations include those that impact the duration of hormone

production, hormone concentration, and hormone bioavailability. For instance, EDCs that

affect the receptor-mediated feedback loops in hormone production, such as the

gonadotrophic hormone, can influence the concentration of hormone produced. Additionally,

many EDCs influence gene expression by acting as either agonists or antagonists, meaning

  3  

that they can either mimic or bock endogenous hormones respectively. Other mechanisms of

endocrine disruption include those that can change the normal level of cholesterol availability

or enzymatic activity, which are necessary for steroidogenesis to commence.

Table 1. Various Endocrine Disrupting Compounds from Anthropogenic Sources

Compound Group Endocrine Effect Source

Bisphenol A (BPA)

Plasticizer

Increased incidence of diabetes and coronary heart disease.

Rancière et al. 2015

Detrimental effects on the central immune organs of

developing chicken embryos. Tian et al. 2014

Increased feminization in painted turtles (Chrysemys

picta) following developmental exposure. Jandegian, et al. 2015

Phthalates Plasticizer Di(n-butyl) phthalate exposure has resulted in sperm

production decline and Leydig cell hyperplasia. Mylchreest et al. 2002

High levels of Di-(2-ethylhexyl)-phthalate (DEHP)

have been found in the plasma of individuals with chronic diseases, such as endometriosis.

Cobellis, et al. 2003

Tributyltin

(TBT)

Anti-

fouling agent

Tributyltin activates PPARγ nuclear hormone receptor pathway and leads cell fate to adipogenesis.

Biemann, et al. 2014

Tributyltin chloride suppresses thyroid hormone function.

Sharan, et al. 2014

Atrazine

Herbicide

Atrazine exposure resulted in demasculinization, hermaphroditism, and decreased testosterone levels in Xenopus laevis.

Hayes, et al. 2001

Triflumizole

(TFZ)

Fungicide

Triflumizole activates the PPARγ nuclear hormone receptor pathway in human mesenchymal cells and leads to increased adipogenesis in vivo.

Li et al. 2012.

  4  

Nuclear Hormone Receptors

One mechanism through which endocrine disrupting compounds alter homeostasis of

the endocrine system is by acting as artificial ligands and activating nuclear hormone

receptors. EDCs can alter homeostasis by acting as an agonist or an antagonist (see Figure 1).

  5  

Figure  1.  (a)  Pathway  of  a  normal  endogenous  hormone,  (b)  an  agonist  endocrine  disrupting  compound,  (c)  an  antagonist  endocrine  disrupting  compound.  

a.  

b.  

c.  

  6  

Nuclear hormone receptors, which are part of the steroid receptor superfamily, alter

gene expression after ligand activation by influencing gene transcription. Because nuclear

hormone receptors were likely derived from a common ancestor, many receptors are highly

conserved throughout numerous living organisms (McLachlan, 2001; Tilghman et al, 2010.)

When activated, nuclear receptors undergo structural alterations that engage coactivator

complexes. These complexes induce gene expression by binding to hormone response

elements near the core promoters in target genes and influencing chromatin decompaction.

As many natural and synthetic ligands can activate nuclear hormone receptors,

external factors can affect gene expression in a variety of biological systems. The typical

structure of nuclear hormone receptors includes an amine-terminus region (A/B), a DNA-

binding domain (C), a linking region (D), and a ligand-binding region (E). Differing isoforms

of nuclear hormone receptors originating from a common gene typically differ in the A/B

domain due to the region’s susceptibility to alternative splicing. Thus, alternative splicing in

the A/B domain results in variants of nuclear hormone receptors that may differ in their

promoter specificity and affinity and may result in differential gene expression (Aranda and

Pascual, 2001).

Obesogens

The escalating incidence of diabetes, liver disease, cardiovascular disease, infertility,

and other obesity-related conditions has led to a connection between endocrine disrupting

compounds (EDCs) and increased adipocyte differentiation (Diamanti-Kandarakis et al.,

2009). Under normal physiologic function, adipose tissue is mediated by hormones and

governed by the sympathetic nervous system. However, environmental contaminants from a

variety sources can alter the natural balance of adipose development. These compounds,

  7  

termed obesogens, are an EDC subset that influence adipogenesis and lipid metabolism by

functioning as artificial ligands and binding to certain families of nuclear hormone receptors,

leading to transcriptional activation (Grün and Blumberg, 2006).

Many compounds have been shown to lead to adipose tissue development and

predisposition to obesity, including various phthalates (Cocci et al., 2015), bisphenol A

(Wang et al., 2013), tributyltin (Kirchner et al. 2010), and triflumizole (Li et al. 2012).

Because most obesogens have their influential effect during the development of organisms

but do not result in visible disruption until adulthood, compounds affecting adipose

differentiation are categorized as organizational endocrine disrupting compounds (Guillette

et al. 1995).

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone

receptors that tend to be activated when bound by obesogenic compounds. This receptor has

three isoforms: PPARα, PPARβ/δ, and PPARγ. While all are involved in lipid homeostasis,

PPARγ is primarily responsible for adipogenesis and lipid biosynthesis. It has been suggested

that when an agonist in the cytoplasm of a cell activates PPARγ, it will translocate to the

nucleus in order to heterodimerize with retinoid X receptor (RXR) and bind to peroxisome

proliferator hormone response elements (PPREs), which are found in the promoter regions of

PPAR responsive genes (Grygiel-Górniak, 2014). Thus, if activated by a foreign compound,

the PPARγ-RXRα heterodimer complex can induce gene expression that leads to increased

adipocyte differentiation, alter lipid metabolism, and influence inflammation (see Figure 2).

  8  

Figure 2. Effect of obesogenic compounds on the PPARγ-RXRα nuclear hormone receptor pathway. Agonists of this pathway lead to increased adipogenesis, altered lipid metabolism, inflammation, and related problems.

Dioctyl Sodium Sulfosuccinate (DOSS)

As dioctyl sodium sulfosuccinate (DOSS) has been used in pharmaceutical products

since 1943 and as a component of food and beverage additives since 1965, the FDA has

established this compound as “Generally Recognized as Safe (FDA, 1988; Temkin et al,

2015).” The common usage of this anionic detergent is due to its usefulness as a surfactant

and emulsifying agent, making DOSS a primary component in many food and beverages,

laxative products, and oil dispersants. Indeed, DOSS is such an integral component of

modern food products that many of the most popular household food and beverage items,

  9  

such as skim milk, rely on DOSS as a solubilizing agent (Burdock, 1997; Chapman et al.,

1985). Additionally, because DOSS is a major component of oil dispersants and is present in

human waste, many wildlife species are also often exposed to this compound. However, the

low concentration of DOSS used in most consumer products has not shown adverse health

affects (FDA, 1988).

The spread of DOSS throughout the environment increased after the 2010 Deepwater

Horizon oil spill, during which an estimated 4 million oil barrels were released into the Gulf

of Mexico (Redmond and Valentine, 2012). The environmental devastation caused by this oil

spill prompted the use of approximately 2.1 million gallons of oil dispersant, which degraded

oil into the water column. Currently, there are 23 oil dispersants approved by the

Environmental Protection Agency (EPA) for use in response to oil spills. Corexit9500

(EC9500, EC9500A, and EC9500B), a primary dispersant used after the 2010 Deepwater

Horizon Oil Spill, was applied in unconscionable quantities to surface waters and was the

first instance of oil dispersant application to deep waters. These oil dispersants are composed

of solvents and surfactants in varying ratios, with Corexit dispersants containing

approximately 50% hydrocarbons, 40% glycols and 10% dioctyl sulfosuccinate (DOSS). The

goal of these mixtures was to aid in oil biodegradation and ameliorate the devastation caused

by crude oil (Kleindienst et al., 2015). Thus, Corexit is very effective at promoting dispersal

and environmental degradation of crude oil.

However, limited research regarding the sublethality of Corexit and its components

has been conducted. Recently, the assessment of potential endocrine disrupting effects of

Corexit 9500 led to the discovery that isolated DOSS in oil dispersants acts as a probable

obesogen in mammals (Temkin et al., 2015). While determining that DOSS in Corexit likely

  10  

acts as an obesogen in mammals was a novel finding, a growing body of literature supports

that Corexit may result in adverse health impacts on a variety of species (Almeda et al. 2014,

Goodbody-Gringley et al., 2013, Wise et al., 2014, and Wang et al., 2012). However, the

impact of DOSS on non-mammalian adipocyte differentiation has not been examined. As

several ecosystems have been exposed to various compounds in Corexit, including DOSS,

understanding the sublethal impacts of Corexit and its components on wildlife is crucial for

environmental health.

Sentinel and Model Species

The effects of many of the aforementioned endocrine disrupting compounds were

determined by the use of various common laboratory species and affected wildlife species

(e.g., vom Saal et al, 2007). The use of sentinel species to study the effects of endocrine

disrupting compounds is necessary in order to monitor environmental and human health. In

addition to providing scientifically sound policy for the regulation of endocrine disrupting

compounds and pollutants, understanding the biological mechanisms of compounds involved

in environmental remediation is crucial in determining the most effective mode of

contaminant mitigation. As a number of organisms fill critical niches in their ecosystem and

can function as bioindicators for environmental health, human health and prosperity is highly

dependent upon the preservation and evaluation of environmental wellbeing. As such, many

organisms have been declared sentinel species for their importance in detecting

environmental contaminants and service in indicating potential pathologic and endocrine

disruption risks (Bossart et al., 2011).

The American alligator (Alligator mississippiensis) and the Zebrafish (Danio rerio)

are two commonly used organisms in endocrine disruptor research. Due to its environmental-

  11  

dependent patterns of sex determination, its high trophic level, and its critical role in wetland

ecosystems, the American alligator has become a sentinel wildlife species. As this species’

sex determination varies by temperature and is highly dependent on external influences,

disruption during embryonic development can be more readily examined (Guillette et al.,

2000). The American alligator is a keystone species in some wetland ecosystems, functioning

as both a top predator and an ecological engineer (Ikuko et al., 2012).

Zebrafish (Danio rerio) are a common model species for examining developmental

toxicity. These organisms are popular in biomedical research due to their manageable

husbandry, high reproductive rates, ease of inducing and observing genetic change, and

genetic similarity to higher trophic organisms (Feitsma and Cuppen, 2008). Additionally, as

extensive toxicological studies and genomic resources are readily available, assessment of

EDC impact can be more effectively analyzed. The majority of EDC studies conducted using

zebrafish involve sex steroids and their impact on sexual differentiation and reproduction

(Segner, 2009). However, other forms of endocrine disruptors have been examined, including

environmental obesogens, such as bisphenol-A (BPA) and tributylin (TBT) (Riu et al., 2014

and Tingaud-Sequeira et al., 2011).

Purpose of Study

The goal of this study is to examine the impact of diocytl sodium sulfoccinate

(DOSS) on adipogenesis in non-mammalian vertebrates. By using an important wildlife

species, the American alligator (Alligator mississippiensis), and a well-established study

species, the zebrafish (Danio rerio), the impact of this suspected obesogenic endocrine

disrupting compound can be described by both in vitro and in vivo means.

  12  

CHAPTER II

MATERIALS AND METHODS

Study Overview

To address the impact of dioctyl sodium sulfosuccinate on non-mammalian

adipogenesis, both in vitro and in vivo methods were used in order to assess both molecular

and physiological endpoints. For in vitro methods, a dual-reporter luciferase system was used

to examine the molecular pathway of adipogenesis in the American alligator (Alligator

mississippiensis) via the PPARγ-RXRα nuclear hormone receptor pathway. The role of

different RXRα variants on response element induction was assessed by known obesogens,

rosiglitazone (ROSI) and tributyltin (TBT). Using the RXRα variant that resulted in the

highest induction, a transactivation assay was used to better understand the impact of DOSS

and other oil dispersant components on the PPARγ-RXRα pathway. Next, an in vivo

assessment of DOSS on non-mammalian adipogenesis was conducted using Zebrafish

(Danio rerio). Newly hatched larvae were exposed to DOSS within 12 hours of hatching.

Histology was then used to examine adipose tissue of control and exposed individuals after 7

days of development.  

  13  

In vitro Procedures

Transactivation assays for RXRα Variant Assessment

E. Coli vectors containing PPARγ, RXRα variant 1-4, RXR response element, PPAR

response, and PRL-TK control plasmids were kindly provided by S. Kohno, Medical

University of South Carolina and described by Temkin et al., 2015. After ampicillin resistant

E. Coli were cultured, plasmids were extracted using a GenCatch™ Plasmid DNA Mini-Prep

Kit according to the manufacturer’s protocol (Epoch Life Science). Isolated plasmids were

then purified and adjusted to the needed concentrations: 200ng/µL for receptors, 400 ng/µL

for reporters, and 100ng/µL for the control.

PPARγ-RXRα transactivation assays were conducted using HEK293T cells

maintained in phenol red-free DMEM (Sigma-Aldrich), containing 10% fetal bovine serum

(GE life science) and 1x Penicillin-Streptomycin-Glutamine (Life Technology). Cells were

transfected with FuGENE HD Transfection Reagent (Promega) according to the

manufacturer’s protocol (Promega) in 96-well assay plates with 20,000 cells per well. In

order to compare the inductive effects of RXRα variants 1-4, assay plates were first divided

into eight sections, each with different combinations of an RXRα variant, PPARγ, PRL-TK

control, and either RXR response element or PPAR response element vectors. In total, a

mixture of 141.3µL phenol red-free DMEM (Sigma-Aldrich), 1.5µL PPARγ, 1.5µL of an

RXRα variant (1-4), 1.5µL RXR response element or PPAR response element, 1.5µL of

PRL-TK control, and 2.7µL FuGene transfection reagent was added to each well.

Cells were exposed to 12.2µL DMSO (Dimethyl sulfoxide; Sigma-Aldrich), ROSI

(Rosiglitazone; Sigma-Aldrich), or TBT (Tributyltin; Sigma-Aldrich). Each combination

was plated in triplicate and repeated three times. Cells were lysed 44 hours after treatment,

  14  

and a Dual-Luciferase Reporter Assay (Promega) and microplate luminometer (Perkin

Elmer) were used to detect firefly and Renilla luminescence.

Transactivation Assays for Corexit Assessment

Using the same procedure discussed above, the transactivation process was repeated

by transfecting only the RXRα variant that showed the highest induction, PPARγ, PRL-TK

control, and either the RXR response element or PPAR response element. Transfected cells

were treated with DMSO (1:100, Sigma-Aldrich), DOSS (1:100, Sigma-Aldrich) Corexit

(1:20, Nalco Environmental Solutions), Corexit Water Accommodated Fraction (CWAF;

1:20), or varying concentrations of TBT or ROSI. CWAF, a 1:20:200 mixture of

Corexit9500, MC252 oil, and DMEM cell culture media, was kindly provided by D.

Spyropoulos, Medical University of South Carolina. Dose concentrations of this experiment

were based on the results described by Temkin, et al., 2015.

Statistical Analysis

GraphPad Prism Software (GraphPad Software, La Jolla, CA) was used for all

analyses. After data were normalized relative to DMSO exposed, a one-way ANOVA and a

Bonfferoni post-hoc analysis was used to compare the relative induction of RXRα variants 1-

4. RXRα variant inductive effects on the PPAR response element and the RXR response

element were compared. In order to compare the inductive effects of Corexit, DOSS, CWAF

to TBT and ROSI, these results were first normalized by DMSO and then normalized and

interpolated by TBT or ROSI.

  15  

In vivo Procedure

Embryo Collection

Zebrafish (Danio rerio) were purchased from Aquarium Knoxville and cared for

following the Maryville College IACUC guidelines (see Appendix). Fish were maintained in

10 gallon fish tanks at 27°C-28°C on a 14 hour light/10 hour dark cycle. Each tank contained

both males and females of reproductive age, with up to 20 fish per tank. Fish were fed Tetra

Brand Tropical Flakes Fish Food twice each day. The evening prior to embryo collection,

marbles were placed at the bottom of one tank to ensure embryo recovery. After breeding,

the embryos were collected using a siphon directed over a mesh net. Embryos were

maintained in 250mL glass petri dishes at 27°C-28°C with up to 50 embryos per dish and

were fed frozen brine shrimp twice per day after hatching.

Dioctyl Sodium Sulfosuccinate Exposure

After embryo hatching, the larvae of each dish were assigned to be either a control or

treatment group. Treatment groups were dosed with 4.9mg/L dioctyl sodium sulfosuccinate

(Sigma Aldrich, 97%), ten fold less than the literature LC50 for Danio rerio (TechLine

Coating Safety Data Sheet). After one week, half of the control and treatment groups were

anesthetized in 400mg/L Ethyl 3-aminobenzoate (MS222). After fixing the samples in

Bouin’s solution for at least two days and clearing with 70% ethanol, tissues were embedded

in wax by first dehydrating the tissues with an increasing concentration of ethanol and then

exposing the samples to SafeClear clearing agent. Tissues were embedded in paraffin wax by

exposure to increasing pressure under a vacuum and then sectioned into 12 µm segments

using a microtome. Warm water containing gelatin was used to mount the wax ribbons

  16  

containing the tissue onto microscope slides. Both transverse and sagittal sections were

stained with Hematoxylin and Eosin then analyzed qualitatively.

  17  

CHAPTER III

 

RESULTS

 

In vitro Results

Transactivation assays for RXRα Variant Assessment

RXRα variants differed significantly in their inductive effects on the RXR response

element (P<0.0001; Fig. 3). In the RXR response element analysis, the type of test compound

used had a significant impact on response element induction. When exposed to TBT, the

RXR response element had a higher induction than when exposed to ROSI (P<0.0001).

Figure 3. A comparison of relative retinoid X receptor response element (RXRE) induction after nuclear hormone peroxisome proliferator activator-gamma (PPARγ) and retinoid X receptor-alpha (RXRα) variants 1-4 binding. Treatments include DMSO (dimethyl sulfoxide), ROSI (rosiglitazone), and two different concentrations of TBT (tributyltin).

  18  

While induction of the RXR response element was highly dependent on the variant of RXRα,

there were no differences observed among RXR variants on PPAR response element

induction (P=0.8568; Fig. 4). While both TBT and ROSI led to significantly higher induction

than the DMSO control, these test compounds did not lead to significantly different results.

Figure 4. A comparison of relative peroxisome proliferator activator response element (PPRE) induction of luciferase activities 44 hours after exposure to various test compounds. Nuclear hormone peroxisome proliferator activator-gamma (PPARγ) and retinoid X receptor-alpha (RXRα) variants 1-4 binding were used in this system. Treatments include DMSO (dimethyl sulfoxide), ROSI (rosiglitazone), and two different concentrations of TBT (tributyltin).

To assess the structural differences between the variants, a comparison of American

alligator RXRα variant conserved domain amino acid length was constructed (Fig. 5).

DMSORosi

TBT 10-4 M

TBT 10-5 M

0

50

100

150

Treatments

Rel

ativ

e In

duct

ion

(% o

f Max

± S

EM) PPRE

PPRG-RXRA1PPRG-RXRA2PPRG-RXRA3PPRG-RXRA4

ANOVA tableInteractionDoseConstractResidual

SS11491127675748.377930

DF93380

MS127742558249.4974.1

F (DFn, DFd)F (9, 80) = 1.311F (3, 80) = 43.69F (3, 80) = 0.2560

P valueP = 0.2446P < 0.0001P = 0.8568

DMSORosi

TBT 10-4 M

TBT 10-5 M

0

50

100

150

Treatments

Rel

ativ

e In

duct

ion

(% o

f Max

± S

EM) RXRE

PPRG-RXRA1PPRG-RXRA2PPRG-RXRA3PPRG-RXRA4

ANOVA tableInteractionDoseConstractResidual

SS225922401729668658.8

DF93380

MS2510800698898.235

F (DFn, DFd)F (9, 80) = 304.8F (3, 80) = 972.1F (3, 80) = 1201

P valueP < 0.0001P < 0.0001P < 0.0001

ACB

A C AB

A A AB

CD

AB

AC

  19  

Figure 5. A comparison of American alligator (Alligator mississippiensis) retinoid X receptor-alpha (RXRα) variant conserved domain amino acid length. Domains include the activation function 1 (AF1), the DNA binding domain (DBD), and the ligand bind domain (LBD).

The ligand binding domain length for each variant is equal. However, differences can

be seen in the DNA binding domain as well as the activation function 1, which is contained

in the highly variable N-terminal region.

Transactivation Assays for Corexit Assessment

In order to assess the PPARγ-RXRα2 activities of Corexit, CWAF, and DOSS, a dose

response curve of TBT and ROSI was constructed for comparison. Luciferase activity was

showed as percent maximum induction and each transactivation of Corexit, CWAF or DOSS

were transformed into equivalent molar units of TBT and ROSI for RXRE and PPRE,

respectively. As ROSI had little inductive effects on the RXR response element, only a TBT

dose response was observed. To gauge the activity levels of Corexit components, induction

of Corexit, CWAF, and DOSS was interpolated by TBT or ROSI. TBT was used for

114  

104  

115  

AF1  (115)  

77  

96  

77  

DBD  (96)  

212  

212  

212  

LBD  (212)  

RXRα4  

RXRα3  

RXRα2  

RXRα1  

RXRα  Variant  1-­‐4  Conserved  Domain    Amino  Acid  Length  Comparison  

  20  

Corexit (1:200)

Doss(1:1000)

CWAF (1:200)

DMSO (1:1000)

-11

-10

-9

-8

-7

Test Compound

Inte

rpol

ated

[TB

T] (M

)

RXRE Response TBT Dose-Response on RXRE

10-14 10-12 10-10 10-8 10-6

-20

0

20

40

60

80

100

[TBT], M

Ma

xim

um

Ind

uc

tio

n (%

)

(a)  

interpolation of inductive effects on the RXR response element. DMSO was also interpolated

by TBT and was used as a control. All compounds had similar effects and had activities

equivalent to between 10-9 and 10-8 concentration of TBT (P>0.05). A comparison of

inductive effects by Corexit components to the dose response curve of TBT is presented in

Figure 6.

Figure 6. (a) Interpolated retinoid X receptor response element induction after exposure to Corexit (Corexit 9500A), DOSS (dioctyl sodium sulfosuccinate), CWAF (Corexit water accommodated fraction of oil), and DMSO (dimethylsulfoxide). Ratios represent concentration of compounds in the wells of a 96-well assay plate. Compounds were interpolated by effect of TBT (tributyltin) in molar concentration. (b) Dose response of TBT (tributyltin) on the retinoid X receptor response element 44 hours after exposure. Peroxisome proliferation activator gamma (PPARγ) and retinoid X receptor alpha variant 2 (RXRαX2) were used in this system.

A dose response curve was also constructed for TBT and ROSI on the PPAR

response element. Luciferase activity was measured as percent maximum induction and test

compounds were measured in molar units. Induction of the PPAR response element was

assessed by interpolating test compound activity by ROSI. Similarly, DMSO was also

interpolated by ROSI and used as a control. Only the difference between DMSO and CWAF

(b)  

  21  

Corexit (1:200)

Doss(1:1000)

CWAF (1:200)

DMSO (1:1000)

-8.0

-7.5

-7.0

-6.5

-6.0

-5.5

Test Compound

Inte

rpol

ated

[RO

SI] (

M)

PPAR Response TBT and ROSI Dose-Response on PPARE

10-14 10-12 10-10 10-8 10-6 10-4

0

20

40

60

80

100

[TBT or ROSI], MM

axim

um In

duct

ion

(%)

TBTROSI

was significant (P<0.05). Compounds had activities equivalent to between 10-7 and 10-6

concentration of ROSI (Fig. 7).

Figure 7. (a) Interpolated peroxisome proliferator activated response element (PPRE) induction after exposure to Corexit (Corexit 9500A), DOSS (dioctyl sodium sulfosuccinate), CWAF (Corexit water accommodated fraction of oil), and DMSO (dimethylsulfoxide). Ratios represent concentration of compounds in the wells of a 96-well assay plate. Compounds were interpolated by effect of ROSI (rosiglitazone) in molar concentration. (b) Dose response of TBT (tributyltin) and ROSI (rosiglitazone) on the peroxisome proliferated activator response element (PPARE) 44 hours after exposure. Peroxisome proliferation activator gamma (PPARγ) and retinoid X receptor alpha variant 2 (RXRαX2) were used in this system. While RXRα variant activities were dependent on the type of response element, the

effect of Corexit components was similar on both the RXR response element and the PPAR

response element. Compared to DMSO, Corexit test compounds did not correlate closely

with the effects of high ROSI or TBT concentrations.

In vivo Results

The tissues of zebrafish (Danio rerio) individuals did not express any apparent

adipocytes one week after hatching. As adipocytes were not visualized, qualitative

assessment showed no difference in adipogenesis between treatment and control groups. A

  22  

sagittal cross-section of a zebrafish individual after one week of dioctyl sodium

sulfosuccinate exposure is shown in Figure 8.

Figure 8. Zebrafish (Danio rerio) individual one week after hatching and exposure to dioctyl sodium sulfosuccinate (DOSS) at 40X magnification.

Similarly, transverse sections of zebrafish one week after hatching showed no adipose

deposits. Thus, there was no difference between treated and control groups on adipocyte

differentiation (Figure 9).

Figure 9. Transverse sections of Zebrafish individuals one week after hatching that were exposed to dioctyl sodium sulfosuccinate (DOSS) after hatching (A) and individuals that were not exposed after hatching (B). Photographs were taken at 40X magnification.

A.   B.  

  23  

CHAPTER IV

DISCUSSION

Exposure to endocrine disrupting contaminants by humans and wildlife lead to

varying sublethal impacts on both environmental and human health. Therefore,

understanding the mechanisms by which these compounds impact both mammalian and non-

mammalian species is crucial. Additionally, due to the role of obesity in cardiovascular

disease, liver disease, reproductive complications, and other related problems, it is necessary

to characterize the molecular and physiological aspects of increased adipocity. Obesogenic

compounds have been shown to act by influencing the PPARγ-RXRα nuclear hormone

receptor pathway in mammals, but little analysis of this mechanism has been conducted in

non-mammalian species.

As the retinoid X receptor-alpha has several variants that result from alternative

splicing, differences in variant conserved domain regions may have an impact on binding

affinity, thereby altering variant inductive effects on response element activation. Because

the American alligator (Alligator mississippiensis) RXRα variant 2 had the highest inductive

effects on the RXR response element but not the PPAR response element, strong affinities

may be related to the short DNA binding domain of this isoform. These findings indicate that

the RXRα variant 2 in the American alligator may be heavily involved in adipogenesis in this

species, leading to further characterization of this pathway in non-mammalian species.

  24  

Temkin et al. have shown that dioctyl sodium sulfosuccinate (DOSS), a primary

component of Corexit 9500 oil dispersant and numerous other consumer products, activates

the PPARγ-RXRα pathway in mammalian models. However, DOSS did not have a

significant inductive effect on this pathway in the American alligator. While the inductive

effects of Corexit water accommodated fractions of oil (CWAF) samples differed

significantly from the DMSO control on the PPAR response element, there is not evidence to

show that DOSS was the agonist involved. This result indicates a potential unidentified

compound in the crude oil that may act as an obesogen in the American alligator.

To further understand the molecular mechanisms involved in non-mammalian

obesogenesis, future studies should assess the roles of PPARγ and various RXRα variants

individually using G-protein coupled receptor methods in order to determine if non-

mammalian species require a similar PPARγ-RXRα heterodimer complex to induce response

element activation. These non-mammalian analyses will allow for a better understanding of

obesogenic mechanisms and will highlight the differences and similarities of nuclear

hormone receptor pathways among various species. Additionally, because PPAR response

element activation was increased when exposed to Corexit water accommodated fractions,

elements within this crude oil should be further examined for obesogenic activity.

Assessment of adiposity in zebrafish (Danio rerio) via histological techniques was

unable to detect adipose tissue in the developing larvae. Thus, conclusions regarding the

effect of dioctyl sodium sulfosuccinate on zebrafish adipogenesis could not be made between

sagittal or transverse cross-sections of treatment and control groups. This limitation may

have occurred for two primary reasons: young larval stage of samples or inappropriate

methods for measuring adiposity. Previous studies have shown that zebrafish do not reach an

  25  

adult body plan until 28 days post fertilization (Flynn et al., 2009). Though initial adipocyte

deposits begin to develop in the viscera approximately 8 days after fertilization (Flynn et al.,

2009), there was likely very limited development occurring by 7 days after hatching during

this study, resulting in no adipocyte visibility. Additionally, the traditional Hematoxylin and

Eosin histological technique used was ineffective at providing qualitative and quantitative

data for zebrafish adipogenesis. Though H&E staining has been noted as a method to

visualize the general morphological characteristics of adipose tissues, few studies involving

zebrafish have relied entirely on this method (Berry et al., 2014). Many studies that have

used zebrafish as a model also used another stain in addition to H&E to measure adiposity,

such as oil red O staining or Nile red lipophilic staining (Imrie et al, 2010; Flynn et al, 2009).

Future studies regarding zebrafish adipogenesis should utilize alternative means of

examining adipocyte tissue. One such method is quantifying adipocity using Nile Red

fluorescent probe in vivo (Flynn et al., 2009). Additionally, zebrafish exposed to DOSS after

hatching should be examined for increased adiposity after later stages of development.

Examining DOSS exposed specimens after 28 days post fertilization would be especially

valuable due to the adult body plan formed at that stage.

The results of this study indicate a critical role of RXRα variant 2 in the PPARγ-

RXRα nuclear hormone receptor pathway of the American alligator (Alligator

mississippiensis), further characterizing the molecular pathway of obesogenesis in non-

mammalian species. While DOSS did not result in significant PPARγ-RXRα response

element induction in vitro, there is potential for an unidentified obesogenic component of

crude oil due to the activation of the PPAR response element by Corexit water

accommodated fractions of oil (CWAF). These findings vary from those found by Temkin et

  26  

al., which indicate activation of the PPARγ-RXRα pathway by DOSS in mammals. In vivo

assessment of zebrafish exposed to DOSS resulted in no adipocyte visualization. Ultimately,

this study adds to the growing body of literature concerning obesogenic compounds by

expanding the characterization of the PPARγ-RXRα nuclear hormone receptor pathway and

assessing the impact of dioctyl sodium sulfosuccinate and other oil dispersant components.

  27  

APPENDIX

  28  

  29  

WORKS CITED

Almeda, R., Hyatt, C., Buskey, E.J., 2014. Toxicity of dispersant Corexit 9500A and crude oil to marine microzooplankton. Ecotoxicology and Environmental Safety. 106, 76-85.

Aranda, A., Pascual, A., 2001. Nuclear Hormone Receptors and Gene

Expression. Phyiology Review, 81, 1269-1304. Berry R., Church, C., Gericke, M.T., Jeffery, E., Colman, L., Rodeheffer, M.S., 2014.

Methods in Enzymology (MIE): Methods of Adipose Tissue Biology. Methods of Enzymology. 537:47-73.

Biemann, R., Fischer, B., Bluher, M., Santos, A.N, 2014. Tributyltin affects adipogenic cell fate commitment in mesenchymal stem cells by a PPARγ independent mechanism. Chemico-Biological Interactions. 214: 1-9.

Burdock, G.A., 1997. Encyclopedia of Food and Color Additives. Boca Raton, Fla: CRC.

Print. Chapman, R.W., Sillery, J., Fontana, D.D., Matthys, C., Saunders, D.R., 1985. Effect of

oral dioctyl sodium sulfosuccinate on intake-output studies of human small and large intestine. Gastroenterology. 89(3): 489-493.

Cobellis, L., Latini, G., Felice, C.D., Razzi, S., Paris, I., Ruggieri, F., Mazzeo, P., Petraglia,

F., 2003. High plasma concentrations of di-­‐‑(2-­‐‑ethylhexyl)-­‐‑phthalate in women with endometriosis. Human Reproduction. 18(7):1512-1515.

Cocci, P., Mosconi, G., Arukwe, A., Mozzicafreddo, M., Angeletti M., Aretusi, G., Palermo F.A., 2015. Effects of Diisodecyl Phthalate on PPAR:RXR-Dependent Gene Expression Pathways in Sea Bream Hepatocytes. Chemical Research in Toxicology. 28, 935-947.

Diamanti-Kandarkis, E., Bourguignon, J., Giudice L.C., Hauser, R., Prins, G.S., Soto, A.M., Zoeller R.T., Gore, A.C., 2009. Endocrine Disrupting Chemicals: An Endocrine Society Scientific Statement. Endocrine Society. 30(4):293-342.

Feitsma, H., Cuppen, E., 2008. Zebrafish as a Cancer Model. Molecular Cancer Research. 6(5): 685-693.

  30  

Flynn, E.J., Trent, C.M, Rawls, J.F., 2009. Ontogeny and nutritional control of

adipogenesis (Danio rerio). Journal of Lipid Research. 50(8): 1641-1652. Goodbody-Gringley, G., Wetzel, D.L., Gillion, D., Pulster, E., Miller, A., Ritchie, K.B.,

2013. Toxicity of Deepwater Horizon Source Oil and the Chemical Dispersant, Corexit 9500, to Coral Larvae. PLoS ONE. 8, e45574.

Grün, F., Blumberg, B., 2006. Environmental Obesogens: Organotins and

Endocrine Disruption via Nuclear Receptor Signaling. Endocrinology. 147, S50-S55.

Grygiel-Górniak, B., 2014. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical applications: A review. Nutrition Journal. 13, 1-10.

Guillette, L.J., Crain A.D., Rooney, A.A., Pickford, D.B., 1995. Organization versus Activation: The Role of Endocrine-Disrupting Contaminants (EDCs) during Embryonic Development in Wildlife. Environmental Health Perspectives. 103(7): 157-164.

Guillette, L.J., and D.A. Crain. 2000. Environmental Endocrine Disrupters: An Evolutionary Perspective. New York: Taylor & Francis. Print.

Hayes,T.B., Collins, A., Lee, M., Mendoza, M., Noriega, N., Stuart, A.A., Vonk, A., 2001.

Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences. 99(8), 5476-5480.

Imrie D., Sadler K.C., White adipose tissue development in zebrafish is regulated by

both developmental time and fish size. Developmental Dynamics. 239(11): 3013-3023.

Jandegian, C. M., Deem, S. L., Bhandari, R. K., Holliday, C. M., Nicks, D., Rosenfeld, C. S.,

Selcer, K.W., Tillitt, D.E., vom Saal, F.S., Velez-Rivera, V., Yang, Y., Holliday, D. K. 2015. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles (Chrysemys picta). General And Comparative Endocrinology. 216: 77-85.

Kavlock, R. J., Daston, G. P., DeRosa, C., Fenner-Crisp, P., Gray, L. E., Kaattari, S.,

Lucier, G., Luster, M., Mac, M.J., Maczka, C., Miller, R., Moore, J., Rolland, R., Scott, G., Sheehan, D.M., Sinks, T., Tilson, H. A., 1996. Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop. Environmental Health Perspectives, 104(4), 715–740.

  31  

Kirchner, S., Kieu, T., Chow, C., Casey, S., Blumberg, B., 2010. Prenatal exposure to the environmental obesogen tributyltin predisposes multipotent stem cells to become adipocytes. Molecular Endocrinology, 24, 526-539.

Kleindienst, S., Paul, J.H., Joye, S.B., 2015. Using dispersants after oil spills: impacts

on the composition and activity of microbial communities. Nature Reviews Microbiology. 13, 388-396.

Li X, Pham HT, Janesick AS, Blumberg B. 2012. Triflumizole is an obesogen in mice

that acts through peroxisome proliferator activated receptor gamma (PPARγ). Environmental Health Perspectives. 120:1720-1726.

McLachlan, J.A., 2001. Environmental signaling: what embryos and evolution teach

us about endocrine disrupting chemicals. Endocrine Review. 22(3):319-341.

Mylchreest, E., Sar, M., Wallace, D., Paul, Foster, P.M.D., 2002. Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate. Reproductive Toxicology. 16(1):19-28.

Rancière, F., Lyons, J.G., Loh V. H. Y., Botton, J., Galloway, T., Wang, T., Shaw, J.,

Magliano, D.J., 2015. Bisphenol A and the risk of cardiometabolic disorders: a systematic review with meta-analysis of the epidemiological evidence. Environmental Health: A Global Access Science Source. 14(1):1-23.

Redmond, M.C. and Valentine D.L., 2012. Natural gas and temperature structured a

microbial community response to the Deepwater Horizon oil spill. Proceedings of the National Academy of Science. 109, 20292-20297.

Riu, A., McCollum, C. W., Pinto, C. L., Grimaldi, M., Hillenweck, A., Perdu, E., Zalko, D.,

Bernard, L., Laudet V., Balaguer, P., Bondesson M., Gustafsson, J. A., 2014. Halogenated Bisphenol-A Analogs Act as Obesogens in Zebrafish Larvae (Danio rerio). Toxicological Sciences. 139(1), 48–58.

Segner, H., 2009. Zebrafish (Danio rerio) as a model organism for investigating

endocrine disruption. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 149(2):187-195.

Shararn, S., Nikhil, K., Roy, P., 2014. Disruption of thyroid hormone functions by low dose exposure of tributyltin: An in vitro and in vivo approach. General and Comparative Endocrinology. 206: 155-165.

  32  

Temkin, A.M., Bowers, R.B., Magaletta M.E., Holshouser S., Maggl, A., Clana, P., Guillette, L.J., Bowden, J.A., Kucklick, J.R., Baatz, J.E., Spyropoulos, D.D., 2015. Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen. Environmental Health Perspectives. 142(1): 112-119.

Tian, J., Luo, D., She, R., Liu, T., Ding, Y., Yue, Z., Xia, K., 2014. Effects of bisphenol A on

the development of central immune organs of specific-pathogen-free chick embryos. Toxicology and Industrial Health. 30(3):199-205.

Tilghman, S.L., Nierth-Simpson, E.N., Wallace, R., Burow, M.E., McLachlan, J.A., 2010.

Environmental hormones: Multiple pathways for response may lead to multiple disease outcomes. Steroids. 75(8-9):520-530.

Tingaud-Sequeira, A., Ouadah, N., Babin, P.J., 2011. Zebrafish obesogenic test: a tool for screening molecules that target adiposity

Journal of Lipid Research. 52:(9) 1765-1772. Vom Saal, F. S., Akingbemi, B. T., Belcher, S. M., Birnbaum, L. S., Crain, D. A., Eriksen,

M., Zoeller, R. T. (2007). Chapel Hill bisphenol A expert panel consensus statement: Integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reproductive Toxicology. 24(2), 131–138.

Wang, H., Shi, Y., Major, D., Yang, Z., 2012. Lung epithelial cell death induced by oil-

dispersant mixtures. Toxicology In Vitro. 26, 746-751. Wang, J., Sun, B., Hou, M., Pan, X., Li, X., 2013. The environmental obesogen bisphenol

A promotes adipogenesis by increasing the amount of 11β-hydroxysteroid dehydrogenase type 1 in the adipose tissue of children. International Journal of Obesity. 37, 999-1005.

Wise, C.F., Wise, J.T.F., Wise, S.S., Thompson, W.D., Wise, J.P.J., Wise, J.P.S., 2014.

Chemical dispersants used in the Gulf of Mexico oil crisis are cytotoxic and genotoxic to sperm and whale skin cells. Aquatic Toxicology. 152, 335-340.