Environmental Chemicals Modulate Polar Bear (Ursus maritimus

Subscriber access provided by Northern Illinois University

Article

Environmental chemicals modulate polar bear (Ursus maritimus) peroxisome proliferator-activated receptor gamma (PPARG) and adipogenesis in vitro Heli Routti, Roger Lille-Langøy, Mari Katrine Berg, Trine Fink, Mikael Harju, Kurt Kristiansen, Pawel Rostkowski, Marte Rusten, Ingebrigt Sylte, Lene Øygarden, and Anders Goksøyr Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03020 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49

Environmental Science & Technology

1

Environmental chemicals modulate polar bear

2

(Ursus maritimus) peroxisome proliferator-activated

3

receptor gamma (PPARG) and adipogenesis in vitro

4

Heli Routti *,†, Roger Lille-Langøy ‡, Mari K. Berg †,‡, Trine Fink §, Mikael Harju ||, Kurt

5

Kristiansen ┴, Pawel Rostkowski ╡, Marte Rusten ‡,∇, Ingebrigt Sylte ┴, Lene Øygarden †,‡,

6

Anders Goksøyr ‡

7 8

† Norwegian Polar Institute, Fram Centre, 9296 Tromsø, Norway

9

‡ Department of Biology, University of Bergen, 5020 Bergen, Norway

10

§ Department of Health Science and Technology, Aalborg University, 9220 Aalborg, Denmark

11

||

Norwegian Institute for Air Research, Fram Centre, 9296 Tromsø, Norway

12

┴ Department of Medical Biology, Faculty of Health Sciences, UiT - The Arctic University of

13

Norway, 9037 Tromsø, Norway

14

╡ Norwegian Institute for Air Research, 2007 Kjeller, Norway

15

ACS Paragon Plus Environment

1


16

Page 2 of 49

ABSTRACT

17

We studied interactions between polar bear peroxisome proliferator-activated receptor gamma

18

(pbPPARG) and selected compounds using a luciferase reporter assay and predictions through

19

molecular docking. Furthermore, we studied adipogenesis by liver and adipose tissue extracts

20

from a polar bear and three synthetic mixtures of contaminants in murine 3T3-L1 preadipocytes

21

and polar bear adipose tissue-derived stem cells (pbASCs). PCB153 and p,p’-DDE antagonized

22

pbPPARG,

23

tetrabromobisphenol A, and PCB170 had a weak agonistic effect on pbPPARG, while

24

hexabromocyclododecane, bisphenol A, oxychlordane, and endosulfan were weak antagonists.

25

pbPPARG-mediated luciferase activity was suppressed by synthetic contaminant mixtures

26

reflecting levels measured in polar bear adipose tissue, as were transcript levels of PPARG and

27

the PPARG target gene fatty acid binding protein 4 (FABP4) in pbASCs. Contaminant extracts

28

from polar bear tissues enhanced triglyceride accumulation in murine 3T3-L1 cells and pbASCs,

29

whereas triglyceride accumulation was not affected by the synthetic mixtures. Chemical

30

characterization of extracts using non-target methods revealed presence of exogenous

31

compounds that have previously been reported to induce adipogenesis. These compounds

32

included phthalates, tonalide, and nonylphenol. In conclusion, major legacy contaminants in

33

polar bear adipose tissue exert antagonistic effects on PPARG, but adipogenesis by a mixture

34

containing emerging compounds may be enhanced through PPARG or other pathways.

although

their

predicted

receptor-ligand

affinity

was

weak.

PBDEs,


2

Page 3 of 49


35

INTRODUCTION

36

The polar bear (Ursus maritimus) is an apex predator of arctic marine ecosystems that uses sea

37

ice as a platform to hunt its prey, namely ringed seals.1 During ringed seal pupping and the

38

moulting period in spring, polar bears feed excessively and accumulate energy reserves to

39

survive periods with limited access to food.2 In the absence of sea ice, polar bears go through

40

extensive fasting periods, which may be prolonged up to eight months combined with subsequent

41

denning by pregnant females.2,3 During fasting periods polar bears depend almost entirely on

42

energy stored in white adipose tissue.2,4

43

White adipose tissue consists mainly of adipocytes, whose primary function is to store lipids in

44

periods of excess of energy and mobilize lipids during energy deprivation.5,6 The process where

45

undifferentiated mesenchymal cells differentiate into preadipocytes and further into lipid-filled

46

adipocytes is called adipogenesis.5 Adipogenesis and promotion of lipid stores proceeds in two

47

waves, as studied extensively using the murine 3T3-L1 preadipocyte cell line as a model.7,8 The

48

first wave of adipogenesis is induced by an adipogenic cocktail and involves several

49

transcription factors such as CCAAT/enhancer-binding protein (C/EBP) β and –δ, the

50

glucocorticoid receptor, and the cAMP-response element binding protein (CREB). Furthermore,

51

the first wave induces the second wave, during which the cells go through terminal

52

differentiation through activation of the nuclear receptor peroxisome proliferator-activated

53

receptor gamma (PPARG, NR1C1). PPARG, that forms a heterodimer with retinoic X receptor,

54

is the major regulator of adipogenesis and promotion of lipid stores, but it also plays a role in

55

other cell types such as macrophages, and several kidney and bone cell types.7 Endogenous

56

PPARG agonists include fatty acids and their derivatives, and oxidized polyunsaturated lipids.9,10


3


Page 4 of 49

57

Polar bears’ ability to store enough energy and to survive during sea ice free periods is

58

challenged by global climate change and decline of Arctic sea ice, which has already been linked

59

to negative impacts on survival and reproduction.11 Another anthropogenic factor, namely

60

pollution, may also affect polar bear energy homeostasis negatively. The polar bear is among the

61

most polluted arctic species due to its position at the top of the Arctic marine food web.12,13 In

62

particular, persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs),

63

polybrominated diphenyl ethers (PBDEs), and chlorinated pesticides, as well as their

64

metabolites, are found at high concentrations in polar bears.14 Because over 90% of the body

65

burden of POPs in polar bears is stored in white adipose tissue,15 adipose tissue is at special risk

66

for toxic effects of pollutants. Recent in vitro studies suggest that several POPs found in polar

67

bear adipose tissue may affect adipogenesis in mice.16-18 In addition, other synthetic chemicals

68

including organotins, and phthalates can activate PPARG, and many of them also induce

69

adipogenesis in vitro and in vivo.18-22 Furthermore, several studies suggest associations between

70

exposure to POPs among other endocrine disrupting chemicals with common metabolic

71

problems in humans, such as obesity and type 2 diabetes.23-25

72

We hypothesize that contaminants in polar bears may target PPARG and disrupt adipose tissue

73

functions. The aim of this study was to study interactions between polar bear PPARG

74

(pbPPARG) and selected contaminants at concentrations reflecting levels measured in polar bear

75

adipose tissue; the contaminants as single compounds and in mixtures. In order to construct a

76

transactivation assay, we cloned and sequenced pbPPARG from polar bear adipose tissue

77

mRNA. Transactivation of pbPPARG was examined using a luciferase reporter assay. Ligand

78

binding affinity was predicted by a docking and scoring strategy. Furthermore, we investigated

79

the effect of both extracts and synthetic mixtures of POPs on the first and second wave of


4

Page 5 of 49


80

adipogenesis using 3T3-L1 cells. POPs in the extracts were quantified using target analyses

81

whereas the presence of emerging and endogenous compounds was characterized using

82

suspect/non-target analytical screening methods. We also established a method using polar bear

83

adipose tissue-derived stem cells (pbASCs) to study adipogenesis by exposure to extracts and

84

synthetic mixtures in these cells.

85 86

EXPERIMENTAL

87

Preparation of polar bear tissue extracts and synthetic mixtures

88

Preparation of polar bear tissue extracts, quantification of POPs and subsequent preparation of

89

synthetic mixtures is described in detail in supporting information. Briefly, contaminants were

90

extracted from liver and adipose tissue from a healthy 10-year-old male bear from Svalbard,

91

Norway. The samples were homogenized in dry Na2SO4, extracted with acetone/cyclohexane

92

and cleaned by adding the lipid extracts onto a 30 cm long semipermeable membrane device.

93

Further cleanup was achieved using a high performance liquid chromatography (HPLC) system

94

utilizing gel permeation chromatography and a column packed with activated Florisil,

95

fractionating the sample into three fractions. The first fraction contained neutral compounds such

96

as PCBs, PBDEs and organochlorine pesticides, while the second and third fraction contained

97

MeSO2-PCBs/DDE and hydroxylated (OH) PCBs/phenols, respectively. The volume of the final

98

solvent, dimethyl sulphoxide (DMSO) was adjusted until the concentrations of the extracts were

99

equivalent to 100 times the concentrations measured in polar bear tissues. Three separate

100

synthetic mixtures including 1) 44 neutral POPs, 2) 10 neutral POPs, and 3) 16 MeSO2-

101

metabolites of POPs were composed according to the concentrations and composition found in


5


Page 6 of 49

102

polar bear adipose tissue and dissolved in DMSO (Table S1). The mixture of 10 neutral POPs

103

contained only compounds present at more than 0.1µM in the 44 neutral POP mixture.

104 105

Suspect/Non-target screening

106

Extracts of liver and adipose tissue were prepared for suspect/non-target screening and

107

analyses were conducted by an ultra-high performance liquid chromatography system coupled to

108

a high resolution quadrupole time-of flight mass spectrometer (Agilent 6550) as described in

109

supporting information. The data was processed with various modules part of the Agilent Mass

110

Hunter data processing platform (ver. B.07). The levels of confidence were set as previously

111

proposed.26,27 Briefly, confidence level 1 means that the structure is confirmed by a reference

112

standard. Level 2 refers to probable structure based on a library match and diagnostic evidence,

113

and level 3 means that the structure is tentative. At confidence level 4 the molecular formula is

114

unequivocal, whereas at confidence level 5 the compounds mass is of interest but there is no

115

match with a reference standard or with a tentative candidate. We performed a systematic

116

literature search to find out which compounds identified in the polar bear extracts have

117

previously been reported to enhance adipogenesis. These compounds or closely related

118

compounds were classified as “compounds of special interest” and we made a particular effort

119

for improving their confidence level.

120 121

Identification, cloning, and sequencing of polar bear PPARG

122

Tissue sampling, RNA extraction, cDNA synthesis, and amplification of two distinct isoforms

123

of pbPPARG cloned from liver and adipose tissue is described in supporting information. To


6

Page 7 of 49


124

evaluate the phylogeny of pbPPARG2 cloned from adipose tissue, nuclear receptor amino acid

125

sequences were aligned using Clustal Omega.28

126 127

Luciferase reporter assay (LRA)

128

Construction of a LRA plasmid encoding a fusion protein of the GAL4 DNA-binding domain

129

(DBD) and the ligand-binding domain (LBD) of pbPPARG2 is described in detail in supporting

130

information. Briefly, COS7 cells were transiently co-transfected with pCMX-GAL4-pbPPARG,

131

tk(MH100)x4-luciferase, and pCMV-β-galactosidase at a mass ratio of 1:2:2 using the

132

TransIT®-LT1 Transfection Reagent (Mirus Bio, Madison, WI, USA). The cells were exposed

133

to test chemicals diluted in exposure medium for 24 hours. After exposure, the cells were lysed,

134

and enzymatic activity was measured. Test compounds for agonistic effects included BDE28,

135

BDE47, BDE99, BDE100, and BDE153, hexabromocyclododecane (HBCDD), bisphenol A

136

(BPA), tetrabromobisphenol A (TBBPA), PCB118, PCB138, PCB153, PCB170, p,p’-DDE, and

137

endosulfan. We assessed antagonistic effects of BPA, HBCDD, PCB-153, p,p’-DDE,

138

oxychlordane, and endosulfan by exposing the cells to the test compounds in the presence of 0.5

139

µM of a known PPARG ligand, rosiglitazone (Cayman Chemical, Tallinn, Estonia). We also

140

tested agonistic and antagonistic effects of the mixture of neutral POPs, and the mixture of

141

MeSO2-metabolites of POPs. Functionality of the LRA was established by exposure to

142

rosiglitazone, and antagonists, bisphenol A diglycidylether (BADGE), and GW-9662 (Sigma

143

Aldrich).29-31 All compounds for agonistic effects were tested in the range of (5 x 10-3) – 25 µM.

144

Antagonistic effects were tested in the range of (9.6 x 10-3) – 50 µM. All compounds were

145

dissolved in DMSO, and DMSO percentages were kept at 1% or below. In PPARG agonism and

146

antagonism assays general cell viability was monitored through stability of β-galactosidase


7


Page 8 of 49

147

activities. To assess cell viability for antagonistic testing, we repeated the experiments in the

148

presence of 10 µM of rosiglitazone. Potential toxicity of rosiglitazone was tested by Cytotoxicity

149

detection kit LDH (LDH; Roche, Oslo, Norway). Effects of solvent (up to 2.5% DMSO) on the

150

metabolic activity (CDFA-AM) and membrane integrity (resazurin reduction) of COS7 cells was

151

perfomed.32

152 153

Modeling of PPARG-ligand interactions

154

Polar bear PPARG ligand binding potential of environmental contaminants was studied using

155

protein-ligand docking and scoring methods described in supporting information. Briefly, 3D X-

156

ray structures of human PPARG (LBD 100% identical) in complex with structurally diverse

157

ligands were used for docking into the LBD of pbPPARG. A molecular mechanical approach

158

was used to calculate the potential for receptor binding of the test compounds to h/pbPPARG

159

LBD. The virtual ligand screening scoring function was used to predict binding of test

160

compounds to PPARG relative to data achieved for the ligands in the X-ray crystal structures.

161

Test compounds with binding scores below -31 were considered as compounds with high

162

putative binding affinities towards pbPPARG.

163 164 165

3T3-L1 differentiation assay

166

Detailed methods for 3T3-L1 assay are described in supporting information. Briefly, to study

167

how extracts and synthetic mixtures affect adipogenesis in 3T3-L1 cells through the first wave of

168

the differentiation process,7 adipogenesis was carried out without the first-wave adipogenic

169

mixture (isobutylmethylxanthine [IBMX], dexamethazone [DEX], and insulin). Effect of tissue


8

Page 9 of 49


170

extracts and synthetic mixtures on differentiation of 3T3-L1 cells through the first wave was

171

determined after exposure to culture medium including 1 µg/ml insulin and test mixtures,

172

DMSO, or rosiglitazone throughout a 10-day growth period. Contaminant extracts and synthetic

173

mixtures dissolved in DMSO were diluted 1:100 (v/v) to Dulbecco´s Modified Eagle′s Medium

174

(DMEM) to give a final concentration of contaminants in the culture medium equivalent to the

175

concentrations found in polar bear tissues, and a solvent concentration of 1% (v/v). To

176

investigate a dose-response effect, the contaminant mixtures were further diluted 1:3, 1:9, and

177

1:27. The DMSO used for negative control in experiments with tissue extracts was subjected to

178

the same extraction and clean-up steps as the polar bear tissue extracts. Rosiglitazone (1 µM in

179

DMSO) was used as positive control. For our maximum adipogenic differentiation control, the

180

culture medium given at day 0 contained 1 µg/ml insulin, 1 µM rosiglitazone, 500 µM IBMX

181

and 1 µM DEX. The content of triglycerides in the 3T3-L1 cells was quantified

182

spectrophotometrically (OD570nm, Adipogenesis Detection Kit Abcam, Cambridge, UK).

183

Cytotoxicity was assessed by measuring the amount of lactate dehydrogenase released into the

184

culture medium using Cytotoxicity Detection Kit Plus (LDH; Roche).

185

Further, we studied the effect of synthetic mixtures on terminal differentiation, a process

186

primarily driven by PPARG.7 Effect of synthetic mixtures of contaminants on terminal

187

differentiation of 3T3-L1 cells was determined by OilRed O33 staining in cells given culture

188

medium added adipogenic cocktail (500 µM IBMX, 250 nM DEX and 5 µg/ml insulin) at day 0,

189

culture medium added 5 µg/ml insulin and the tested mixtures (or controls) at day 2 and culture

190

medium during days 4-10. Synthetic mixtures gave a final concentration equivalent to

191

concentrations in polar bear tissues, and a solvent concentration of 1% (v/v). To study dose-

192

response relationships, the mixtures were further diluted 1:2 and 1:10.


9


Page 10 of 49

193 194

Adipogenic differentiation of polar bear adipose tissue-derived stem cells (pbASCs)

195

Isolation, growth and establishment of suitable conditions for adipogenic differentiation of

196

pbASCs are described in supporting information. pbASCs were seeded in 96-well plates at a

197

density of 6400 cells/well (20,000 cells/cm2) and cultured to 100 % confluency (day -2). After

198

two days adipogenic differentiation was induced in adipogenic medium (culture medium

199

supplemented with 1 µg/mL insulin, 0.45 mM IBMX, 0.1 µM DEX), also containing the tested

200

extract, synthetic mixture, or controls. The adipogenic media was changed day 3, 7 and 10. For

201

exposures, the adipose tissue extract and the mixture of 10 neutral POPs in DMSO were diluted

202

1:100 (v/v) in the adipogenic medium and the highest concentration in the medium was thus

203

equivalent to the concentrations quantified in the polar bear adipose tissue (Table S1).

204

Differentiation control cells were given 1 µM rosiglitazone while solvent control cells were

205

given 1% (v/v) DMSO in addition to adipogenic medium. Undifferentiated control cells were

206

maintained in basal medium (culture medium supplemented with 1 µg/mL insulin and 1%

207

DMSO) without adipogenic medium. Experiments with synthetic mixtures at three different

208

concentration levels were conducted using pbASCs originating from the two polar bears. They

209

were seeded in triplicates, and the experiments were repeated twice. Due to a limited amount of

210

extract, experiments with extract from polar bear adipose tissue were conducted at one

211

concentration level using pbASCs from the 10 year male polar bear, and repeated three times.

212

Controls were seeded in triplicates, while cells receiving the extracts were seeded in duplicates.

213

At day 14, the accumulation of triglycerides in pbASCs was quantified spectrophotometically

214

(OD570nm, Adipogenesis Detection Kit, Abcam).


10

Page 11 of 49


215

Transcriptional activity of PPARG and its target gene FABP4 was studied in pbASCs

216

following exposure to the mixture of 10 neutral POPs for 14 days. Adipogenic differentiation of

217

pbASCs was performed in 12-well plates and otherwise conducted as described above. Detailed

218

methods are given in supporting information.

219 220

Data analyses

221

Statistical analysis was carried out using R version 3.2.2.34 Agonistic luciferase data were

222

normalized over (divided by) solvent control (DMSO) and antagonistic data over positive control

223

(0.5 µM rosiglitazone). Triglyceride levels in 3T3-L1 and pbASCs and transcript levels of

224

PPARG and FABP4 were normalized over levels in solvent control.

225

Effects of contaminants and their mixtures on transactivation of pbPPARG, transcriptional

226

activity of PPARG and FABP4 in pbASCs and triglyceride accumulation in 3T3-L1 cells and

227

pbASCs was investigated using linear models. Linear models were run separately for each

228

compound/mixture. Exposure concentration was used as a categorical explanatory variable and

229

each exposure concentration was compared to the control using treatment contrasts. The null

230

hypothesis was rejected at α=0.05. Diagnostic plots of residuals were used to verify that the

231

model assumptions were met (most importantly constant variance between residuals).35

232

In one model for agonistic effects (BDE153) on pbPPARG transactivation the diagnostic plots

233

of residuals revealed an outlier, which was omitted for further statistical analysis. The omittance

234

of the one extreme value of BDE153 led to a significant positive effect at the 25 µM

235

concentration, but the estimate was likely more robust without this outlier. Relative pbPPARG-

236

mediated luciferase activity for agonistic and antagonistic effects were calculated from β(Cmax test

237

compound)/

β(rosiglitazone) *100 and β(Cmax test compound)/ β(GW9662) *100, respectively. Relative triglyceride


11


test compound)/

β(max

Page 12 of 49

238

accumulation was calculated from β(Cmax

239

difference in fold change between the maximal concentration and the control based on linear

240

models.

change)

*100.

β is the estimated

241

Dose-response curves for transactivation of pbPPARG were calculated using R library drc

242

version 2.5.12.36 The curves were fitted using a four parameter log-logistic model.36,37 To choose

243

a best-fitting model for compounds showing >20% relative increase/decrease in luciferase

244

activity compared to rosiglitazone/GW9662, we compared three models using a lack-of-fit F-

245

test.38 These models included 1) a four parameter model, 2) a three parameter model where the

246

upper plateau was fixed to the upper asymptote of rosiglitazone (agonistic effects) or the lower

247

plateau to lower asymptote of GW9662 (antagonistic effects), and 3) a two parameter model

248

where the lower and upper asymptotes were fixed to lower and upper plateaus of

249

rosiglitazone/GW9662

250

corresponding to a response midway between the estimates of the lower and upper plateaus

251

(EC50 for agonist, IC50 for antagonist) were determined based on nonlinear least-squares

252

estimates of the parameters of these models. EC/IC50 was calculated only for compounds

253

showing

254

rosiglitazone/GW9662. Dose-response models were also used to determine 20% of minimum

255

response induced by known ligands.

>20%

for

relative

agonistic/antagonistic

increase/decrease

in

effects.

In

luciferase

addition,

activity

concentrations

compared

to

256


12

Page 13 of 49


257

RESULTS AND DISCUSSION

258

Two PPARG isoforms in polar bears

259

PPARG was cloned from liver and adipose tissue, and encoded to 475 and 505 amino acid

260

(AA) proteins respectively (Figure S1). The shorter PPARG cloned from liver tissue (equivalent

261

to hPPARG1), lacks the initial 30 AAs when compared to adipose tissue PPARG (equivalent to

262

hPPARG2). This corresponds well to human PPARG, where the hPPARG1 is 28 AAs shorter

263

than PPARG2. In many vertebrates, PPARG exists as at least two isoforms called PPARG1 and

264

PPARG2. PPARG2 is expressed predominantly in adipose tissue as well as in macrophages,

265

whereas PPARG1 is more widely expressed throughout the body.39,40 Of the two isoforms, the

266

longer PPARG2 is expressed only at very low levels in liver.41 Compared to the PPARG coding

267

sequence predicted from a polar bear genome sequencing (GenBank: XM_008697869)42, the

268

longer adipose tissue PPARG lacked the initial 24 AA.

269

Comparison of PPARG in polar bear to PPARG in other mammals revealed strong

270

conservation, with overall AA identities of 98.2% (human), 99.6% (dog) and 97% (mouse)

271

(Figure S2). The comparison of these PPARG orthologs confirms the variability of the N-

272

terminal A/B-region, and the high degree of conservation (100%) of the DBD (C-region) and

273

hinge (D-region). While the LBD of PPARG from human, dog, and polar bear was identical,

274

three amino acids were substituted in the LBD of mouse PPARG. The high degree of

275

conservation underlines the importance of this receptor and implies that PPARG orthologs would

276

be expected to have similar activation patterns.

277 278

Validation of the transactivation assay


13


Page 14 of 49

279

For the ligand transactivation assay, we used the hinge and LBD domain of PPARG. Thus, the

280

results from the assay should represent both the short and the long pbPPARG forms. In the

281

GAL4-construct system, the risk of cross-reactivity by other cellular pathways is highly

282

reduced,43 and we can thus assume that any activity we saw was due to the activation of

283

pbPPARG-LBD and not nuclear receptors found endogenously in the cell. This assumption is

284

supported by the results of the agonistic and antagonistic effects of known ligands on pbPPARG

285

activation (Table 1, Figure S3). In the transiently transfected COS7 cells, luciferase activities

286

increased approximately 8-fold when exposed to rosiglitazone, whereas rosiglitazone-induced

287

(0.5 µM) luciferase production was inhibited by the known PPARG-antagonists GW9662 and

288

BADGE (Table 1). EC50 and IC50 for rosiglitazone and GW9662 (Table 1) were comparable to

289

previous studies using GAL4 systems.30,44,45 BADGE exposure led to a similar decrease of

290

luciferase activity as GW9662, although at higher exposure concentrations (Table 1B, Figure

291

S3B). This is in accordance with previous studies that have characterized BADGE as a low-

292

affinity antagonist.29,46 Thus, the pbPPARG LRA appears to produce results comparable to those

293

of previous studies.

294

The relatively constant levels of β-galactosidase activities observed in cells exposed to

295

different concentrations of test compounds (not shown), indicated that the rate of transcription

296

and translation in the COS7 cells was unaffected by the exposures. At 1% and lower DMSO

297

concentration, no significant reduction in metabolic activity (CDFA-AM) or membrane integrity

298

(Resazurin reduction) was seen. Furthermore, antagonistic effects of test compounds were less

299

pronounced in the presence of a higher concentration (10 µM) of rosiglitazone (results not

300

shown). Thus, the viability of the cells appeared not to be severely affected by exposure of test

301

compounds.


14

Page 15 of 49


302 303

Activation and predicted binding of polar bear PPARG by brominated compounds

304

With the exception of bisphenol A and HBCDD, all the tested individual brominated

305

compounds had a weak agonistic effect on pbPPARG (Table 1A, Figure S3A).. The most potent

306

pbPPARG agonist in the test panel was TBBPA, which induced reporter gene transcription 2.5-

307

fold at the highest exposure concentration, which was only about one fifth of the maximal

308

activity induced by rosiglitazone (Table 1A). Of the other brominated flame retardants, all five

309

PBDEs had weak agonistic activity on pbPPARG in our system and the most potent PBDE was

310

BDE-47 (Table 1A). The measured agonistic effects of BDE47 and TBBPA are supported by

311

previous studies which report induction of hPPARG by BDE47 and/or TBBPA in a similar

312

system to ours and HEK293H cells stably expressing GAL4-PPARG-LBD.18,20 Furthermore,

313

TBBPA had agonistic activity in stably-transfected HGELN-cells with full-length hPPARG.47

314

Induction of hPPARG by BDE47 and TBBPA, but not BDE99 and BDE100 as shown in this

315

study, has also been shown by using hPPARG-CALUX assay based on human osteosarcoma

316

(U2OS) cell lines.48 Our results, as well as previous in vitro studies,18,20,48 refutes the hypothesis

317

by Luthe et al.,49 which suggests that PBDEs would not interact with PPARs based on a

318

theoretical structure-activity assessment.

319

In contrast to the other brominated compounds tested in our study, BPA and HBCDD

320

decreased pbPPARG-mediated luciferase activity induced by rosiglitazone (Table 1B, Figure

321

S3B). In a previous study, no BPA-mediated human PPARG antagonism was observed.50 The

322

discrepancy between the results may be related to detailed differences in experimental set-up.

323

Docking of brominated compounds predicted weak receptor-ligand affinity for the brominated

324

compound in this study (binding score < -31). This corresponds to previous in vitro and


15


Page 16 of 49

325

modeling studies which show that brominated compounds have weak binding potency to

326

hPPARG LBD compared to rosiglitazone.47,51 Moreover, the results from the docking analysis

327

were in agreement with the weak agonistic potential of brominated compounds demonstrated in

328

the luciferase reporter gene assays.

329 330

Activation and predicted binding of polar bear PPARG by chlorinated compounds

331

The chlorinated compounds included in this study had very weak or no agonistic potential on

332

pbPPARG, however, many of them had antagonistic properties towards pbPPARG (Table 1,

333

Figure S3). PCB153 seems to be a full antagonist with similar efficacy but lower potency

334

compared to that of GW9662 (Table 1B). Furthermore, PCB153 reduced the basal luciferase

335

activity in the absence of rosiglitazone (Table 1A) which suggests that PCB153 is an inverse

336

pbPPARG agonist

337

luciferase reporter gene by PCB153 has also been observed in NIH-3T3 cells transfected with

338

murine PPARG LBD coupled with GAL4.17 Exposure to PCB170 led to a weak increase in

339

pbPPARG-induced luciferase activity (Table 1A). A similar tendency was also observed

340

following PCB138 exposure. The different effects of PCB170 and PCB138 compared to PCB153

341

may be due to their different chlorine-substitution patterns. PCB153 has no vicinal H-atoms,

342

while PCB170 and PCB138 possess vicinal H-atoms in ortho-meta positions. Exposure to

343

PCB118, the only tested PCB that easily acquires a planar configuration, did not lead to any

344

significant changes in luciferase activity.

52

in the luciferase reporter gene assay. Reduction of basal expression of the

345

The chlorinated pesticides p,p’-DDE, oxychlordane and endosulfan showed antagonistic

346

properties towards PPARG (Table 1B, Figure S3B,). p,p’-DDE appears to be a full antagonist

347

with similar efficacy but lesser activity per dosing equivalent than GW9662 (Table 1B).


16

Page 17 of 49


348

Furthermore, and in contrast to PCB153, p,p’-DDE acted as a silent antagonist, as it did not

349

reduce basal luciferase activity in absence of rosiglitazone (Table 1A). Oxychlordane partially

350

decreased rosiglitazone-induced luciferase activity, the efficacy corresponding to one fourth of

351

the maximal GW9662-induced inhibition (Table 1B). Endosulfan seemed to be a partial agonist

352

(Table 1A) that also acted as a competitive antagonist in the presence of the full agonist

353

rosiglitazone (Table 1B).

354

Docking revealed that the chlorinated compounds which showed antagonistic activity did not

355

show strong binding affinity towards pbPPARG LBD (score < -31). PPARG antagonism, as

356

observed in our in vitro results, may be acquired through both covalent and weaker non-covalent

357

modification of LBD.30,53,54 It is likely that most ligands interact with LBD through non-covalent

358

interactions only. A molecule with a rigid and bulky distal phenyl, benzyl, or phenethyl

359

substituent is important for non-covalent PPARG antagonist activity, which is strengthened to

360

full antagonism by a chlorine substituent at the distal ring.54 These rigid molecules prevent helix

361

12 to adopt an active state conformation53,55 which is needed for stable binding of ligand and

362

recruitment of coactivators of PPARG.56 The full antagonists identified in our study, PCB153

363

and p,p’-DDE, are both rigid bulky chlorine substituted molecules. This could explain their

364

antagonistic potential towards pbPPARG in vitro, although they were not predicted as strong

365

binders.

366 367

Synthetic POP mixtures suppress polar bear PPARG

368

Exposure to synthetic mixtures of neutral and MeSO2-POPs suppressed the activity of

369

pbPPARG (Table 1B, Figure S3B). Relative inhibition at highest exposure concentration,

370

corresponding to those identified in polar bear adipose tissue, was 30-50% of the maximal


17


Page 18 of 49

371

inhibition induced by GW9662 (Table 1B). Interestingly, the IC50 for the neutral 10 POP

372

mixture was ~10 µM (concentration of summed compounds), which was slightly lower than the

373

concentrations in adipose tissue of the male polar bear from Svalbard in which the mixture was

374

based on (Table S1). However, 10 µM is within the range of average POP concentrations found

375

in adipose tissue of polar bears from circumpolar subpopulations.14

376

The inhibition at highest exposure concentrations was identical following exposure to the

377

mixture of 44 and 10 neutral POPs. However, the inhibitory effect occurred at a lower

378

concentration with the 44 compound mixture than with the 10 compound mixture (Figure S3B).

379

The latter mixture contains only compounds present at >0.1 µM concentration in the 44 neutral

380

POP mixture. This suggests that the compounds found at

Environmental Chemicals Modulate Polar Bear (Ursus maritimus

Recommend Documents