dichlorodiphenyldichloroethylene (DDE) in hypertension of Wistar rats

Subscriber access provided by Kaohsiung Medical University

Food Safety and Toxicology

Unravelling the effect of p,p’-dichlorodiphenyldichloroethylene (DDE) in hypertension of Wistar rats Carla Sá, Diogo Pestana, Conceição Calhau, and Ana Faria J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05001 • Publication Date (Web): 10 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018

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 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 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.

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 34

Journal of Agricultural and Food Chemistry

1

Unravelling the effect of p,p’-dichlorodiphenyldichloroethylene (DDE) in

2

hypertension of Wistar rats

3

Carla Sá a,b, Diogo Pestana a,c, Conceição Calhau a,c, Ana Faria a,c,d*

4 5

a

6

Monteiro, 4200-369 Porto, Portugal

7

b

8

Hernâni Monteiro, 4200-369 Porto, Portugal

9

c

CINTESIS, Center for Health Technology and Services Research, Al. Prof. Hernâni

Department of Biochemistry, Faculty of Medicine, University of Porto, Al. Prof.

Nutrition & Metabolism, NOVA Medical School – FCM Universidade Nova de

10

Lisboa, Campo Mártires da Pátria, 130 1169-056 Lisboa, Portugal

11

d

12

Nova de Lisboa, Campo Mártires da Pátria, 130 1169-056 Lisboa, Portugal

13

*Corresponding author:

14

Ana Faria

15

NOVA Medical School – FCM Universidade Nova de Lisboa

16

Campo Mártires da Pátria, 130

17

1169-056 Lisboa, Portugal

18

e-mail: [email protected]

19

tel:+351 21 8803033

20

fax:+351 21 8851920

Comprehensive Health Research Centre NOVA Medical School – FCM Universidade

1 ACS Paragon Plus Environment


21

Page 2 of 34

ABSTRACT

22 23

Hypertension is a multifactorial disease with limited knowledge of the involved

24

mechanisms. p,p’-DDE

25

commonly found in tissues that interferes with endocrine signalling.

26

This study aimed to evaluate the mechanism of hypertension triggered by p,p’-DDE

27

exposure in presence or absence of HF-diet, in rats. The renin-angiotensin system

28

(RAS) was evaluated by qPCR in liver and adipose tissue (AT) and a transcriptome

29

analysis comparing visceral AT of HF-diet and HF/DDE groups was performed. HF

30

diet influenced RAS but p,p’-DDE effect was more evident in liver than in AT

31

(interaction between the diet and p,p′-DDE treatment affected aldosterone receptor and

32

angiotensin converting enzyme 2 expression in liver, p< 0.05 two-way ANOVA). p,p’-

33

DDE induced a decrease in expression of genes involved in retinoid acid biosynthesis

34

pathway (Crabp1 -2.07 fold; p=0.018), eNOS activation (Nos1 -1.64 fold; p=0.012) and

35

regulation and urea cycle (Ass1 -2.07 fold; p=0.02).

36

This study suggested that p,p’-DDE may play a fundamental role in the pathogenesis

37

of hypertension, not exclusively in RAS but also by induction of hyperuricemia and

38

increase oxidative stress which may lead to endoplasmic reticulum stress and vascular

39

injury.

(p,p’-dichlorodiphenyldichloroethylene)

is

a

pollutant

40 41

Keywords: Adipose tissue; Endocrine Disruptor; High-Fat; Liver; Renin Angiotensin

42

System.

43


Page 3 of 34


44

INTRODUCTION

45 46

Hypertension, also known as high blood pressure (BP), is a silent worldwide epidemic

47

and is an important global health challenge due to its high prevalence, estimated in

48

about one billion people, being and this is one of the most the leading main global risks

49

for mortality worldwide 1. Obesity and reduced physical activity, together with excess

50

sodium intake, inappropriate intake of fruits and vegetables and alcohol intake in

51

excess,

52

hypertension, such as obesity, excess sodium intake in food, reduced physical activity,

53

inadequate intake of fruits, vegetables and excess alcohol intake 2. Hypertension is a

54

classical feature of the metabolic syndrome and 1/3 of hypertensive patients has

55

metabolic syndrome. Several pathophysiologic factors are involved in the relationship

56

between hypertension and the other components of the metabolic syndrome, including

57

visceral obesity, insulin resistance, inappropriate activation of the renin angiotensin

58

aldosterone system 3, oxidative stress, inflammation, and recently a possible association

59

with hyperuricemia.

60

Recent epidemiological and experimental studies have suggested that persistent organic

61

pollutants (POPs), organochlorine pesticides, such as dichlorodiphenyltrichloroethane

62

(DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE), semi-volatile and

63

resistant to biological, photolytic and chemical degradation, persists in the

64

environment, ultimately resulting in extended degradation periods, up to a century 4,

65

may contribute to the development of play an important role in the development of

66

various components of metabolic syndromesyndrome. These chemicals are highly

67

lipophilic and therefore tend to accumulate and biomagnify in food chains, either

68

terrestrial or aquatic, resulting in a considerable exposure of living organisms and its

There are some factors contributors which lead to high incidence of



Page 4 of 34

69

accumulation in animal and human tissues 5-6. In addition, these chemicals are present

70

in in several food items 7, especially in and fatty foods which are is considered as the

71

main source of exposure in the general population 3, 8.

72

Both epidemiological and in vivo studies found a positive association between DDE

73

exposure and later adiposity

74

endocrine signalling pathways and cause adverse consequences 11, such as obesity 12,

75

insulin resistance 13, hypertension 14, and cardiovascular disease 6, 15-19. Previous works

76

from our group showed that animals (Wistar rats) exposed to p,p′-DDE, in a context of

77

a standard and a high-fat (HF) diet, increase the predisposition to obesity, and appeared

78

to exacerbate some of the co-morbidities, such as glucose intolerance, hyperuricemia,

79

hypertension and inflammation 20. Indeed, these animals treated with p,p′-DDE have

80

increased blood pressure regardless of the diet

81

demonstrated an association between serum uric acid and hypertension, through the

82

increase in reactive oxygen species and angiotensin II, which causes efferent arteriole

83

constriction

84

hypertension and hyperuricemia on humans, largely due to the implausibility of

85

accessing organs tissues, as adipose tissue (AT) and liver.

86

In this study, and taking into account the endocrine disruption ability of p,p′-DDE and

87

its confirmed presence of p,p′-DDE in animal tissues and their recognized endocrine

88

disruption ability 20, 22, we aimed to evaluate the mechanism of p,p′-DDE exposure in

89

Wistar rats, either in presence or absence of a HF diet, in hypertension in target organs

90

(liver and AT) in Wistar rats. This information provides an advantage to try another

91

targets to treatment of hypertension and hyperuricemia.

21.

9-10.

As an endocrine disruptor it may interfere with

20.

Also, several studies have

There is a paucity of data on underlying mechanisms of DDE in

92


Page 5 of 34


93

MATERIALS AND METHODS

94

Chemicals

95

p,p′-

96

medetomidine, and isoflurance were obtained purchased from Sigma (Sigma-Aldrich,

97

St. Louis, MO, USA), RNA-STAT 60TM Reagent (Amsbio, UK).

Dichlorodiphenyldichloroethylene

(p,p′-DDE,

purity>98%),

ketamine,

98 99

Animal Tissues

100

Liver and adipose tissue were collected from thirty male Wistar rats (Charles River

101

Laboratories, Barcelona, Spain), weighing 267±11.8 g (8 weeks), randomly divided

102

into four groups, subjected to different treatments for a total of 12 weeks: St, Standard

103

group (n=6); DDE, standard with p,p′-DDE group (n=9); HF, High-fat group (n=6);

104

HF/DDE, high-fat with p,p′-DDE group (n=9).The p,p′-DDE exposure treatment was

105

applied in the water with the average concentration of 100 µg/kg/day (2.5 times less

106

than LOAEL (Lowest-Observed-Adverse-Effect Level). p,p′-DDE was first dissolved

107

in ethanol EtOH with the final concentration of 0.01%. In and the concentrations of the

108

stock and final solution were corrected (St/DDE and HF/DDE), according to water

109

intake and animal average weight, in order to maintain the exposure concentration and

110

ethanol volume. In the ccontrol groups, St and HF, water had ethanol, in the same

111

percentage as treated groupsthe same final volume of ethanol (0.01%) was added to the

112

water. Intake of Wwater and chow was assessed every 3-4 dayswere supplied ad libitum

113

and renewed every 3-4 days and the intake assessed. The diets were respectively

114

Standard (St) (Teklad 2014, Harlan Laboratories, Santiago, Spain): 48% carbohydrates

115

(w/w), 14.3% proteins (w/w) and 4% lipids (w/w) and HF-diet (D145 Research Diets,

116

New Brunswick, USA): 41% carbohydrates (w/w), 24% proteins (w/w) and 24% lipids

117

(w/w). Animal handling and housing protocols followed European Union guidelines



Page 6 of 34

118

(86/609/EEC) and Portuguese Act (129/92) for the use of experimental animals. At the

119

end of the 12 weeks, the animals were anaesthetized with a mixture of ketamine (50

120

mg/kg) and medetomidine (1 mg/kg) and maintained with isoflurane. Organs were

121

dissected, pat dried, and weighed, before snap freezfreezeing in liquid nitrogen and

122

and storiedng at -80ºC. until further analysis.

123 124

RNA extraction from tissues

125

RNA was extracted and purified from liver and AT samples using RNA-STAT 60TM

126

Reagent (Amsbio, UK). AT (50-100 mg) samples were homogenized with liquid

127

nitrogen, and liver (50-100 mg) was homogenized using a hand homogenizer. RNA-

128

STAT 60TM (1 mL/50-100 mg tissue) was added to the samples and stored for 5 min at

129

room temperatureRT to permit allow the complete dissociation of nucleoprotein

130

complexes. Next, 0.2 ml of chloroform per 1 ml of RNA-STAT 60TM was added, and

131

shaken vigorously for 15 seconds and stored at room temperatureRT for 2-3 minutes.

132

Samples were centrifuged at 12000 g for 15 minutes at 4ºC. Then, a colourless upper

133

aqueous phase and a lower red phenol chloroform phase appearedthe homogenate

134

separated into two phases: a lower red phenol chloroform phase and the colourless

135

upper aqueous phase. The aqueous phase was transferred, to a fresh tube and mixed

136

with 0.5 ml of isopropanol per 1 ml of the RNA-STAT 60TM, and stored at room

137

temperatureRT for 5-10 minutes and centrifuged at 12000 g for 10 min at 4ºC. RNA

138

precipitated and formed a white pellet at the bottom of the tube. Then, the supernatant

139

was discardedremoved and the RNA pellet was washed with 75% ethanol EtOH by

140

vortexing and subsequently centrifuged at 7500 g for 5 min at 4ºC. The RNA pellet was

141

dried by air-dryingdried for 20 minutes and solved in RNAse free water was added.

142

The purity of RNA (by the 260nm/280nm ratio) purity as well as its and quantification


Page 7 of 34


143

(by absorbance at 260 nm) were assessed with a NanoDrop spectrophotometer

144

(NanoDrop® Thermo Scientific, Wilmington, DE, USA). , and the concentration was

145

determined by absorbance at 260 nm, and RNA quality by the 260/280 ratio.

146 147

Gene expression analysis by real-timeqRT- PCR

148

Purification of RNA was purified assessed with DNase enzyme using the RQ1 RNase-

149

Free DNase kit (Promega Cat. # M6101). cDNA was synthetized from 1 μg of treated

150

mRNA with NZY First-Strand cDNA Synthesis Kit (NZYTech, Portugal). Quantitative

151

real-time polymerase chain reaction (qRT-PCR) was performed in a LightCycler

152

instrument (Roche Applied Science, Indianapolis, ID, USA), using sealed 96-well

153

microplates using awith LightCycler Fast-Start DNA Master SYBR Green kit and a

154

LightCycler instrument (Roche Applied Science, Indianapolis, ID, USA). PCR reaction

155

mixtures (total of 10 μL) contained 5 μL of 2x Fast-start SYBR Green (Roche

156

Diagnostics Ltd), 0.2 μl of each primer (final concentration of 0.2 μM), 3.6 μL of water

157

and 1 μL of cDNA (25 ng/μL). PCR amplification conditions are listed in table 1 as

158

well as the sequence of each primer (Sigma-Aldrich, St. Louis, MO, USA). Primer

159

sequences (Sigma-Aldrich, St. Louis, MO, USA) and the conditions for PCR

160

amplification reactions are reported in Table 1. Primer sequence specific for target

161

genes were design as follows: cDNA sequences were retrieved from Ensembl database

162

for Rattus Norvegicus, pasted in Primer3 software (v.0.4.0) and primers were design to

163

bind only to exon sequences. Additionally, the primers and the amplicon specificity

164

was checked in BLAST® against RefSeq mRNA database for Rattus Norvegicus.

165

Equipment was programed with the following Ccycling conditions parameters (45

166

cycles):were as follows: denaturation (95 °C for 10 min), amplification and

167

quantification (95 °C for 10 s, annealing temperature for 10 s, and 72 °C for 10 s, with



Page 8 of 34

168

a single fluorescence measurement at the end of the 72 °C for 10 s segment) repeated

169

for 45 cycles. A melting curve analysis was performed tTo verify check the specificity

170

of the amplification, a melting curve analysis was performed via monitoring SYBR

171

Green fluorescence from starting at 95 °C 10 s, followed by a 60 s temperature ramp

172

from 60 °C to 97 °C. PCR analysis was performed in duplicate for each samples and

173

the results were normalized for the housekeeping gene (HPRT) after Ddata analysis

174

with were processed and analysed using the LightCycler software (Roche Applied

175

Science), and the results obtained were normalized for one housekeeping gene (HPRT).

176

Data are presented as the mean values of duplicate PCR analysis.

177 178

AT total RNA isolation and microarrays

179

Microarrays were performed in RNA from mesenteric visceral AT of HF and HF/DDE

180

groups as described and presented in literature

181

visceral AT was grinded in liquid nitrogen for total RNA isolation was isolated from

182

mesenteric visceral AT samples in ground in liquid nitrogen, using RNA STAT-60

183

reagent (AMS Bioctechnology, Abingdon, UK). RNA was extracted with chloroform

184

and precipitated with isopropanol, followed by DNaseI treatment to degrade genomic

185

DNA. followed by chloroform extraction and isopropanol precipitation. RNA extracts

186

were treated with DNaseI to avoid contamination with genomic DNA and itsThe RNA

187

was quantified concentration was assesses spectrophotometrically with NanoDrop

188

spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and their its integrity

189

was determined with using the Agilent 2100 Bioanalyzer (Agilent Technologies,

190

Massy, France). Only samples with Only the high-quality RNA wereas further

191

processed and samples from HF and HF/DDE rats were used for the microarray

192

analysis. This was performed. Sample processing and data acquisition were carried out

20.

Briefly, samples from mesenteric


Page 9 of 34


193

by the Genomics Core Lab of the University of Cambridge Biomedical Research Centre

194

(Cambridge, UK), according to Affymetrix protocol (GeneChip Expression Analysis

195

Technical Manual, Affymetrix, Santa Clara, CA, USA)., n amely, the preparation of

196

Bbiotinylated cRNA preparation and its hybridization of in Affymetric Rat GeneChip®

197

Gene 1.0 ST Arrays. Affymetrix Genechip Software was used to convert to CEL files

198

raw image data of were performed according to the recommended Affymetrix protocol

199

(GeneChip Expression Analysis Technical Manual, Affymetrix, Santa Clara, CA,

200

USA). Arrays were scanned arrays. and raw image data were converted to CEL files

201

using Affymetrix Genechip Software.

202 203

Microarray data analysis

204

Agilent’s GeneSpring GX 9 software (Agilent Technologies Inc. Santa Clara, USA)

205

was used to analyse Mmicroarray CEL files under high stringency to prevent false

206

positives. data was performed using Agilent’s GeneSpring GX 9 software (Agilent

207

Technologies Inc. Santa Clara, USA). After importing the data, the CEL files were

208

analysed under high stringency in order to reduce the number of false positives. The

209

algorithms robust multi-array average (RMA) and Plier analysis were used wo different

210

analysis algorithms were used (robust multi-array average (RMA) and Plier analysis)

211

and only the genes with identical expression patterns in both analysis were selected to

212

proceed.whose expression patterns in each of the analysis were identical were taken

213

forward for further study. G A fold change (up or down) of at least 1.25 fold with a p

214

value of 5% (Student’s t-test) in gene expression levels was considered significantene

215

expression levels were considered significantly up- or down- regulated with a fold

216

change of at least 1.25 fold with a p value of 5% (Student’s t-test). Ingenuity pathway

217

analysis software (IPA, Ingenuity® Systems, Redwood City, USA) was used to explore



Page 10 of 34

218

The and analyse pathway and biological analysis of gene expression data was

219

performed using the Ingenuity pathway analysis software (IPA, Ingenuity® Systems,

220

Redwood City, USA) as exposed 20. Reactome version 3.5 was used to further explore

221

pathways of interest.

222 223

Statistical analysis

224

Results are Valuespresented as are expressed as the arithmetic mean ± standard error

225

of the mean. Levene´s test was performed to analyse the distribution and homogeneity

226

of varianceIn a first instance, an exploratory analysis of data was performed to test

227

distribution and homogeneity of variance (Levene´s test). Two-way ANOVA (main

228

effects: diet, p,p’-DDE exposure and their interaction) was used with Bonferroni’s test

229

as post-hoc, according to homogeneity of variance. Correlations were analysed by

230

Pearson correlations. All the analyses were performed using the SPSS Statistics

231

software v22.0 for Mac (IBM, Armonk, NY, USA) and gGraphics and statistical

232

analysis were made using Prism® 6.0 Software (GraphPad Software Inc., La Jolla, CA,

233

USA). The Ddifferences were considered significant when p value < 0.05.

234 235 236 237 238 239 240 241 242 10 ACS Paragon Plus Environment

Page 11 of 34

243


RESULTS

244 245

A. Effect of p,p′-DDE in renin-angiotensin system in adipose tissue

246

The effect of p,p′-DDE in angiotensinogen (AGT), angiotensin II receptor type 1b

247

(AGTR1b), angiotensin I converting enzyme (ACE1), angiotensin II converting

248

enzyme (ACE2), chymase and aldosterone receptor in the AT (Figure 1) was evaluated.

249

Considering the expression of the genes involved in renin angiotensin system in AT,

250

there is a clear effect of the diet in almost all of the genes tested (AGT, AGTR1b, ACE1,

251

chymase and aldosterone receptor) highlighting a HF-diet effect. Except for ACE2,

252

whose expression was not altered, the expression pattern for the tested genes was very

253

similar, a small increase with p,p′-DDE treatment, and increase in the HF-diet groups,

254

showing a clear effect of a HF-diet. With the exception of AGT, a tendency to decrease

255

in the HF/DDE treatment compared to a HF-diet was observed in all groups.

256 257

B. Effect of p,p′-DDE in renin-angiotensin system in liver

258

The gene transcription of AGT, AGTR1b, ACE2 and aldosterone receptor was

259

evaluated in liver (Figure 2). ACE1 and chymase transcription were also evaluated but

260

were not expressed in the liver.

261

Diet seemed to exert effect in the expression of some of the genes involved in renin

262

angiotensin system in the liver. AGT was not significantly affected by the treatments.

263

AGTR1b was affected by the diet, in particular, significantly reduced in the HF-diet

264

groups. ACE2 transcription was significantly affected by an interaction between the

265

diet and p,p′-DDE treatment highlighted by the different effect of p,p′-DDE in the

266

different context of the diet. An interaction between the diet and p,p′-DDE treatment

267

also affected aldosterone receptor expression in the liver. It is worth noticing that in all



Page 12 of 34

268

the tested genes there was a similar pattern: decreased expression in the HF-diet group

269

and a tendency to increase when HF-diet animals were treated with p,p′-DDE.

270 271

C. Comparison of global gene expression between HF and HF/DDE group

272

The analysis of the microarrays Ggene expression microarrays inof mesenteric visceral

273

AT from HF and HF/DDE rats showeddisplayed that 320 and genes were up-regulated

274

and 311 311 were genes that were up- and down-regulated, respectively (at least 1.25-

275

fold change) 20. DetailedThorough data of gene expression (concerning up- and down-

276

regulated) genes was thoroughlycarefully analysed in the hypertension context and it

277

was notice that among the differentially transcribed genes, mainly the down-regulated

278

genes, some of the largest changes induced by HF/DDE were seen in cellular retinoic

279

acid binding protein 1 (Crabp1; -2,07-fold), nitric oxide synthase 1 (Nos1; -1.64-fold)

280

and argininosuccinate synthetase 1 (Ass1; -2.07-fold).

281

Recurring to the Reactome pathway tree built with our microarray data, the selected

282

genes were further analysed to frame these in the changed pathways. It was found that

283

these genes were involved the retinoid acid biosynthesis pathway, eNOS activation and

284

regulation and urea cycle respectively. The resultant analysis is shown in figure 3.

285 286 287 288 289


Page 13 of 34

290


DISCUSSION

291 292

The physiological impact associated with chronic exposure to DDE in hypertension is

293

still poorly understood. According to Park, S. et al 23, high concentrations of DDE were

294

significantly associated with the increased risk of hypertension, but there is no

295

explanation about the mechanisms involved. This is the first study to explore a possible

296

mechanism of DDE in hypertension.

297

The renin-angiotensin system (RAS) conventionally plays a crucial role in the

298

regulation of renal, cardiac, and vascular physiology, and its activation is central to

299

many common pathologic conditions including hypertension and renal disease 24. The

300

“classical” view of the RAS pathway begins with renin (produced by the kidneys)

301

cleaving AGT to Angiotensin I (Ang I). Angiotensin-converting enzyme converts the

302

inactive Ang I to the Angiotensin II (Ang II), that acts on several other organs as a

303

potent vasoconstrictor, vascular growth factor and facilitator of norepinephrine release

304

from sympathetic nerve terminals 25. However, this “classical” view of the endocrine

305

RAS pathway represents an incomplete description of the system, as it is recognized

306

that there are also several tissue (local) RAS that function independently of each other

307

26.

308

Though liver is “classical” known to be the major site for AGT production, RAS

309

components are also expressed in AT making them biologically relevant27-28. Obesity

310

is associated with over activation of both systemic and AT RASs in humans and

311

animals29-30. In our study, AT of Wistar rats exposed to HF-diet increase significantly

312

the expression of AGT, in accordance to other studies in which AT AGT over-

313

expression has been associated to obesity as well as IR31. The transcription levels of



Page 14 of 34

314

AGT in the liver were not altered with the HF-diet but were increased in the presence

315

of p,p′-DDE.

316

The Ang II can exacerbate obesity and may mediate IR by oxidative stress increase via

317

activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and

318

reactive oxygen species (ROS) production as well as stimulating the inflammatory

319

pathways and dysregulating of the secretion of chemokines32. It is recognized as a

320

potent proinflammatory, pro-oxidant, and prothrombotic agent that interferes with

321

several steps of intracellular insulin signalling 24, 33. Also, increased levels of Ang II are

322

closely related to IR which might be linked to chronic kidney disease, and an

323

inappropriate activation of RAS 34.

324

As previous published 20, the Wistar rats exposed to HF-diet increase weight, impaired

325

response to glucose, developed dyslipidemia, hyperleptinemia, and had a reduction of

326

the anti-inflammatory cytokine IL-10. In our work, it seems that the p,p′-DDE treatment

327

contributes to an increase, but that is not enough to be significant in AGT expression in

328

AT and liver AGT, AGTR1b significantly increased with a HF-diet in AT, as well as

329

ACE1. The inverse was observed in AGTR1b transcription in liver. In the genesis of

330

hypertension, Ang II stimulates the transcription and release of aldosterone, which

331

results in a further rise in BP related to sodium and water retention 35. Our work showed

332

that the expression of aldosterone receptor increased significantly in the presence of a

333

HF-diet in AT, as well as chymase. Chymase Ang II synthesis has emerged as an

334

alternative pathway to ACE, and according to Kumar, R. et al 36, increased chymase

335

levels have been observed in a high-glucose environment, that is in accordance to our

336

previous study which demonstrate that this rats have impaired glucose 20.

337

In liver, ACE 2 and aldosterone receptor raised significantly in the HF/DDE-diet group.

338

ACE2 metabolizes Ang II to Ang 1–7, essentially negatively regulate the RAS. ACE 2


Page 15 of 34


339

expression upregulate mediators of atherogenesis, such as cytokines and adhesion

340

molecules, and enhanced responsiveness to pro-inflammatory stimuli, which leads to

341

atherosclerotic lesions

342

muscle cell hypertrophy, hyperplasia and migration, extracellular matrix production

343

and synthesis of pro-inflammatory mediators

344

aldosterone increase could be related to increased levels of circulating free-fatty acids

345

(FFAs) and oxidative stress 38. It would be very interesting to determine the activity of

346

ACE 2 to support our results however this is a limitation of this study. A previous work

347

from our group 22, showed that addition of p,p′-DDE to feeding raised the fatty acid

348

composition of AT and liver. In addition, there are studies using obese mouse models

349

that have shown upregulation of RAS components and ROS levels38. The presence of

350

p,p′-DDE, free fatty acid accumulation, ectopid lipid accumulation, lipotoxicity,

351

leading to possible disturbance in the fat-liver crosstalk, could explain the observed

352

disturbances 39-40.

353

In a previous study from our group 20, BP of these animals was evaluated by the non-

354

invasive tail-cuff method during the treatment period. Overall, p,p’-DDE exposure was

355

responsible for BP variation, demonstrated by higher systolic BP >140 mmHg in

356

St/DDE and HF/DDE

357

hypertension in these animals 20 and that the changes observed in RAS system seemed

358

weak to explain this outcome, other pathways were hypothesised.

359

Considering that p,p′-DDE interferes with induction of pro-inflammatory cytokines,

360

transcription of genes was analysed. Of notice, HF/DDE treatment induced changes in

361

transcription of genes involved in inflammation, it seems that down-regulates the nitric

362

oxide synthase 1 (Nos1) transcription in HF/DDE mesenteric visceral AT, which could

363

be contributing for this inflammation 41. Nitric oxide is an important signaling molecule

37.

Ang II initiates tissue remodeling via induction of smooth

20.

37.

One explanation for ACE 2 and

Knowing that p,p′-DDE, regardless of HF diet, induce



Page 16 of 34

364

implicated in the regulation of various physiologic events such as inflammation,

365

vascular tone, metabolism, and has been related to mechanism of toxicity, as

366

highlighted in the pathway diagram in figure 3. The down-regulation of gene NO in

367

HF/DDE group seems to support that p,p′-DDE in the presence of HF manifestly

368

induced inflammation, oxidative stress. Argininosuccinate synthetase 1 (Ass1) is the

369

key enzyme responsible for the provision of L-arginine, the substrate of Nos, and this

370

enzyme might play a role in the endothelial nitric oxide production 42, which is down-

371

regulated in HF/DDE group compared with HF group. Therefore, it was hypothesized

372

that Ass1 might contribute to vascular health42, and in our work, the presence of p,p′-

373

DDE might induce vascular injury, increasing ROS. Cellular retinoic acid binding

374

protein 1 (Crabp1) is down-regulated in HF/DDE group, and according to Miller, J. 43,

375

Crapb1 is down-regulated upon induction of adipocyte differentiation, and highlighted

376

an interesting link between Crabp1 and fat accumulation in the AT, through retinoic

377

acid pathway. We can hypothesize that p,p′-DDE exposure led to AT dysfunction,

378

higher C-reactive protein level, increase in plasmatic IL-1β levels and induce

379

endothelial dysfunction44.

380

Of interest was the hyperuricemia observed in HF/DDE Wistar rats (a significant 70%

381

increase in uric acid concentration (0.78±0.05 mg/dL) compared to the other treatments

382

20)

383

hypertension. As highlight in figure 3, Ass1 is also involved in urea cycle, and it is

384

known that uric acid can cause reduction of NO bioavailability, generation of

385

mitochondrial oxidative stress, and is highly associated with more intima media

386

thickness. Hyperuricemia increase ROS and Ang II, which causes efferent arteriole

387

constriction leading to hypertension. Also, ROS is known to significantly contribute to

388

the incidence of hypertension, cardiovascular and kidney diseases

since hyperuricemia and insulin resistance are important risk factors for essential

21.

Although


Page 17 of 34


389

increasing number of studies have been investigating the augmented vascular tone in

390

the hypertension, the exact mechanism remains nuclear. In addition, it is know that

391

endoplasmic reticulum may function as a sensor of oxidative stress in the cell. Under

392

stressful conditions, a disruption of the normal physiological state of ER may occur

393

with the consequent increase in the level of misfolded proteins. The ER stress response

394

is critical for normal cellular homeostasis, and is involved in the pathogenesis of many

395

diseases such as diabetes

396

infections, neurodegeneration, and cancer 47.

397

It is interesting to note that in the muscle, in p,p′-DDE treatment had an inhibitory

398

effect on BiP and XBP1 expression, even in the presence of HF-diet. Deldicque L. et

399

al 48 also indicate that ingestion of excessive nutrients, and more specifically lipids, can

400

result in endoplasmic reticulum stress in muscle. And this effect in endoplasmic

401

reticulum stress is more pronounced in the presence of p,p′-DDE, which could also

402

enhance this condition (data not shown).

403

This study raises the possibility that p,p′-DDE may play a fundamental role in the

404

pathogenesis of hypertension, as well as in hyperuricemia, particularly in high fat

405

context. Future investigations are required to verify how we can manage the effect of

406

p,p′-DDE, in order to prevent and treat hypertension, hyperuricemia and other

407

metabolic dysfunctions.

45,

obesity, inflammation, cardiovascular diseases

46,

viral

408 409 410

ABBREVIATIONS USED

411

ACE 1 - angiotensin converting enzyme 1

412

ACE 2 - angiotensin converting enzyme 2

413

Agt – Angiotensinogen



414

AGTR1b – angiotensin II receptor type 1b

415

Ang – Angiotensin

416

Ass1 - argininosuccinate synthetase 1

417

AT – adipose tissue

418

BP- blood pressure

419

Crabp -cellular retinoic acid binding protein 1

420

HF– High fat

421

Nos1- nitric oxide synthase 1

422

p,p′-DDE – p,p′-dichlorodiphenyldichloroethylene

423

RAS – Renin–angiotensin system

424

ROS – reactive oxygen species

425

St – Standard group

Page 18 of 34

426 427

ACKNOWLEDGEMENTS

428

This article was supported by FEDER through operation POCI-01-0145-FEDER-

429

007746 funded by the Programa Operacional Competitividade e Internacionalização –

430

COMPETE2020 and by National Funds through FCT - Fundação para a Ciência e a

431

Tecnologia within CINTESIS, R&D Unit (reference UID/IC/4255/2013). DP

432

acknowledges FCT for his Post-Doc grant (SFRH/BPD/109158/2015).

433 434

CONFLICT OF INTEREST

435

The authors declare no competing financial interest.

436 437 438


Page 19 of 34

439


REFERENCES

440 441

1.

Lacruz, M. E.; Kluttig, A.; Hartwig, S.; Loer, M.; Tiller, D.; Greiser, K. H.;

442

Werdan, K.; Haerting, J., Prevalence and Incidence of Hypertension in the General

443

Adult Population: Results of the CARLA-Cohort Study. Medicine 2015, 94 (22),

444

e952.

445

2.

Kumar, J., Epidemiology of hypertension. Nephrology 2013, 56-61.

446

3.

Brauner, E. V.; Raaschou-Nielsen, O.; Gaudreau, E.; Leblanc, A.; Tjonneland,

447

A.; Overvad, K.; Sorensen, M., Predictors of adipose tissue concentrations of

448

organochlorine pesticides in a general Danish population. Journal of exposure science

449

& environmental epidemiology 2012, 22 (1), 52-9.

450

4.

451

organic pollutants and adverse health effects in humans. J Toxicol Env Heal A 2006,

452

69 (21), 1987-2005.

453

5.

454

Grosman, F.; Sanzano, P.; Lo Nostro, F. L.; Miglioranza, K. S., Persistent organic

455

pollutants (POPs) in fish with different feeding habits inhabiting a shallow lake

456

ecosystem. The Science of the total environment 2016, 550, 900-9.

457

6.

458

Faria, A.; Meireles, M.; Marques, C.; Correia-Sa, L.; Cunha, A.; Guimaraes, J. T.;

459

Taveira-Gomes, A.; Santos, A. C.; Domingues, V. F.; Delerue-Matos, C.; Monteiro,

460

R.; Calhau, C., Persistent organic pollutant levels in human visceral and subcutaneous

461

adipose tissue in obese individuals--depot differences and dysmetabolism

462

implications. Environmental research 2014, 133, 170-7.

Li, Q. Q.; Loganath, A.; Chong, Y. S.; Tan, J.; Obbard, J. P., Persistent

Barni, M. F.; Ondarza, P. M.; Gonzalez, M.; Da Cuna, R.; Meijide, F.;

Pestana, D.; Faria, G.; Sa, C.; Fernandes, V. C.; Teixeira, D.; Norberto, S.;



Page 20 of 34

463

7.

Darnerud, P. O.; Atuma, S.; Aune, M.; Bjerselius, R.; Glynn, A.; Grawe, K.

464

P.; Becker, W., Dietary intake estimations of organohalogen contaminants (dioxins,

465

PCB, PBDE and chlorinated pesticides, e.g. DDT) based on Swedish market basket

466

data. Food and chemical toxicology : an international journal published for the

467

British Industrial Biological Research Association 2006, 44 (9), 1597-606.

468

8.

469

Rodriguez, M.; Martin-Olmedo, P.; Fernandez, M. F.; Olea, N., Associations of

470

accumulated exposure to persistent organic pollutants with serum lipids and obesity in

471

an adult cohort from Southern Spain. Environmental pollution 2014, 195, 9-15.

472

9.

473

B., Prenatal exposure to dichlorodiphenyltrichloroethane and obesity at 9 years of age

474

in the CHAMACOS study cohort. American journal of epidemiology 2014, 179 (11),

475

1312-22.

476

10.

477

Prenatal DDT exposure and child adiposity at age 12: The CHAMACOS study.

478

Environ Res 2017, 159, 606-612.

479

11.

480

W. N., Developmental exposure to endocrine disruptors and the obesity epidemic.

481

Reproductive toxicology 2007, 23 (3), 290-6.

482

12.

483

Mertens, I.; Van Gaal, L., Obesity and persistent organic pollutants: possible

484

obesogenic effect of organochlorine pesticides and polychlorinated biphenyls. Obesity

485

2011, 19 (4), 709-14.

Arrebola, J. P.; Ocana-Riola, R.; Arrebola-Moreno, A. L.; Fernandez-

Warner, M.; Wesselink, A.; Harley, K. G.; Bradman, A.; Kogut, K.; Eskenazi,

Warner, M.; Ye, M.; Harley, K.; Kogut, K.; Bradman, A.; Eskenazi, B.,

Newbold, R. R.; Padilla-Banks, E.; Snyder, R. J.; Phillips, T. M.; Jefferson,

Dirinck, E.; Jorens, P. G.; Covaci, A.; Geens, T.; Roosens, L.; Neels, H.;


Page 21 of 34


486

13.

Magliano, D. J.; Loh, V. H.; Harding, J. L.; Botton, J.; Shaw, J. E., Persistent

487

organic pollutants and diabetes: a review of the epidemiological evidence. Diabetes &

488

metabolism 2014, 40 (1), 1-14.

489

14.

490

pollutants and hypertensive disease. Environmental research 2006, 102 (1), 101-6.

491

15.

492

2006, 368 (9535), 558-9.

493

16.

494

concentrations of persistent organic pollutants and self-reported cardiovascular

495

disease prevalence: results from the National Health and Nutrition Examination

496

Survey, 1999-2002. Environmental health perspectives 2007, 115 (8), 1204-9.

497

17.

498

D. R., Jr., Low dose of some persistent organic pollutants predicts type 2 diabetes: a

499

nested case-control study. Environmental health perspectives 2010, 118 (9), 1235-42.

500

18.

501

mechanisms of environmental disruption of insulin action and glucose homeostasis.

502

Diabetes & metabolism journal 2014, 38 (1), 13-24.

503

19.

504

metabolism and cardiovascular risk. Current diabetes reports 2014, 14 (6), 494.

505

20.

506

Fernandes, V. C.; Correia-Sa, L.; Faria, A.; Guardao, L.; Guimaraes, J. T.; Cooper,

507

W. N.; Sandovici, I.; Domingues, V. F.; Delerue-Matos, C.; Monteiro, R.; Constancia,

508

M.; Calhau, C., Adipose tissue dysfunction as a central mechanism leading to

509

dysmetabolic obesity triggered by chronic exposure to p,p '-DDE. Sci Rep-Uk 2017, 7.

Huang, X.; Lessner, L.; Carpenter, D. O., Exposure to persistent organic

Porta, M., Persistent organic pollutants and the burden of diabetes. Lancet

Ha, M. H.; Lee, D. H.; Jacobs, D. R., Association between serum

Lee, D. H.; Steffes, M. W.; Sjodin, A.; Jones, R. S.; Needham, L. L.; Jacobs,

Sargis, R. M., The hijacking of cellular signaling and the diabetes epidemic:

Kirkley, A. G.; Sargis, R. M., Environmental endocrine disruption of energy

Pestana, D.; Teixeira, D.; Meireles, M.; Marques, C.; Norberto, S.; Sa, C.;



Page 22 of 34

510

21.

Mortada, I., Hyperuricemia, Type 2 Diabetes Mellitus, and Hypertension: an

511

Emerging Association. Current hypertension reports 2017, 19 (9), 69.

512

22.

513

Faria, A.; Calhau, C.; Gomes, A., Endocrine Disruptor DDE Associated with a High-

514

Fat Diet Enhances the Impairment of Liver Fatty Acid Composition in Rats. Journal

515

of agricultural and food chemistry 2015, 63 (42), 9341-8.

516

23.

517

pollutants on hypertension: a meta-analysis. Environmental science and pollution

518

research international 2016, 23 (14), 14284-93.

519

24.

520

receptor blockers in the metabolic syndrome. Circulation 2004, 110 (11), 1507-12.

521

25.

522

heart failure. Expert Opin. Investig. Drugs 2004, 13 (6), 643-652.

523

26.

524

system--an endocrine and paracrine system. Endocrinology 2003, 144 (6), 2179-83.

525

27.

526

L. M., Human adipose tissue expresses angiotensinogen and enzymes required for its

527

conversion to angiotensin II. The Journal of clinical endocrinology and metabolism

528

1998, 83 (11), 3925-9.

529

28.

530

adipose tissue renin-angiotensin system. Hypertension 2000, 35 (6), 1270-7.

531

29.

532

link between obesity, inflammation and insulin resistance. Obesity reviews : an

533

official journal of the International Association for the Study of Obesity 2012, 13 (2),

534

136-49.

Rodriguez-Alcala, L. M.; Sa, C.; Pimentel, L. L.; Pestana, D.; Teixeira, D.;

Park, S. H.; Lim, J. E.; Park, H.; Jee, S. H., Body burden of persistent organic

Prasad, A.; Quyyumi, A. A., Renin-angiotensin system and angiotensin

G. Boerrigter, J. C. B. J., Recent advances in natriuretic peptides in congestive

Lavoie, J. L.; Sigmund, C. D., Minireview: overview of the renin-angiotensin

Karlsson, C.; Lindell, K.; Ottosson, M.; Sjostrom, L.; Carlsson, B.; Carlsson,

Engeli, S.; Negrel, R.; Sharma, A. M., Physiology and pathophysiology of the

Kalupahana, N. S.; Moustaid-Moussa, N., The renin-angiotensin system: a


Page 23 of 34


535

30.

Slamkova, M.; Zorad, S.; Krskova, K., Alternative renin-angiotensin system

536

pathways in adipose tissue and their role in the pathogenesis of obesity. Endocrine

537

regulations 2016, 50 (4), 229-240.

538

31.

539

H.; Wasserman, D. H.; Moustaid-Moussa, N., Overproduction of angiotensinogen

540

from adipose tissue induces adipose inflammation, glucose intolerance, and insulin

541

resistance. Obesity 2012, 20 (1), 48-56.

542

32.

543

angiotensin system and metabolic disorders: a review of molecular mechanisms.

544

Critical reviews in biochemistry and molecular biology 2012, 47 (4), 379-90.

545

33.

546

aldosterone system, glucose metabolism and diabetes. Trends in endocrinology and

547

metabolism: TEM 2005, 16 (3), 120-6.

548

34.

549

carbohydrate metabolism. Current opinion in nephrology and hypertension 2004, 13

550

(3), 325-32.

551

35.

552

hypertension. In Cardiology, Crawford MH, D. J., eds., Ed. Mosby International:

553

London, 2000; pp 3.1-3.10.

554

36.

555

system: a new paradigm. Trends in endocrinology and metabolism: TEM 2007, 18

556

(5), 208-14.

557

37.

558

Akker, L. H.; Jutten, B.; Cleutjens, J.; Bijnens, A. P.; Corvol, P.; Daemen, M. J.;

Kalupahana, N. S.; Massiera, F.; Quignard-Boulange, A.; Ailhaud, G.; Voy, B.

Kalupahana, N. S.; Moustaid-Moussa, N., The adipose tissue renin-

Giacchetti, G.; Sechi, L. A.; Rilli, S.; Carey, R. M., The renin-angiotensin-

Strazzullo, P.; Galletti, F., Impact of the renin-angiotensin system on lipid and

Calhoun DA, B. S., Oparil S., Etiology and pathogenesis of essential

Kumar, R.; Singh, V. P.; Baker, K. M., The intracellular renin-angiotensin

Sluimer, J. C.; Gasc, J. M.; Hamming, I.; van Goor, H.; Michaud, A.; van den



Page 24 of 34

559

Heeneman, S., Angiotensin-converting enzyme 2 (ACE2) expression and activity in

560

human carotid atherosclerotic lesions. The Journal of pathology 2008, 215 (3), 273-9.

561

38.

562

T.; Yamamoto, Y.; Noguchi, M.; Kusakabe, T.; Tomita, T.; Fujikura, J.; Ebihara, K.;

563

Hosoda, K.; Sakaue, H.; Kobori, H.; Ham, M.; Lee, Y. S.; Kim, J. B.; Saito, Y.;

564

Nakao, K., Adipose tissue-specific dysregulation of angiotensinogen by oxidative

565

stress in obesity. Metabolism: clinical and experimental 2010, 59 (9), 1241-51.

566

39.

567

inflammasome puts obesity in the danger zone. Cell Metab 2012, 15 (1), 10-8.

568

40.

569

Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes.

570

Diabetes research and clinical practice 2014, 105 (2), 141-50.

571

41.

572

Lee, Analysis between nitric oxide synthase 1 (NOS1) and risk of obesity. Mollecular

573

& Cellular Toxicology 2016, 12 (2), 217-222.

574

42.

575

synthetase 1 regulates nitric oxide production and monocyte adhesion under static and

576

laminar shear stress conditions. The Journal of biological chemistry 2011, 286 (4),

577

2536-42.

578

43.

579

Investigating the Role of Crabp1 in Adipose Biology. Case Western Reserve

580

University: 2017.

581

44.

582

oestradiol on endothelium-derived vasoactive factors in human endothelial cells.

583

Toxicology 2011, 285 (1-2), 46-56.

Okada, S.; Kozuka, C.; Masuzaki, H.; Yasue, S.; Ishii-Yonemoto, T.; Tanaka,

Stienstra, R.; Tack, C. J.; Kanneganti, T. D.; Joosten, L. A.; Netea, M. G., The

Esser, N.; Legrand-Poels, S.; Piette, J.; Scheen, A. J.; Paquot, N.,

Hyun Kyung Park, S. K. K., Oh Young Kwon, Joo-Ho Chung, Seong-Kyu

Mun, G. I.; Kim, I. S.; Lee, B. H.; Boo, Y. C., Endothelial argininosuccinate

Miller, J. E.; Theses, O. E.; Center, D.; Pharmacology, C. W. R. U.,

Andersson, H.; Garscha, U.; Brittebo, E., Effects of PCB126 and 17beta-


Page 25 of 34


584

45.

Demirtas, L.; Guclu, A.; Erdur, F. M.; Akbas, E. M.; Ozcicek, A.; Onk, D.;

585

Turkmen, K., Apoptosis, autophagy & endoplasmic reticulum stress in diabetes

586

mellitus. The Indian journal of medical research 2016, 144 (4), 515-524.

587

46.

588

mechanism and therapeutic target for cardiovascular diseases. Acta pharmacologica

589

Sinica 2016, 37 (4), 425-43.

590

47.

591

Crosstalk of Endoplasmic Reticulum (ER) Stress Pathways with NF-kappaB:

592

Complex Mechanisms Relevant for Cancer, Inflammation and Infection.

593

Biomedicines 2018, 6 (2).

594

48.

595

skeletal muscle: origin and metabolic consequences. Exercise and sport sciences

596

reviews 2012, 40 (1), 43-9.

Liu, M. Q.; Chen, Z.; Chen, L. X., Endoplasmic reticulum stress: a novel

Schmitz, M. L.; Shaban, M. S.; Albert, B. V.; Gokcen, A.; Kracht, M., The

Deldicque, L.; Hespel, P.; Francaux, M., Endoplasmic reticulum stress in

597 598



Page 26 of 34

FIGURE CAPTIONS

Figure 1. Relative gene expression in adipose tissue samples: (A) angiotensinogen (AGT), (B) angiotensin II receptor type 1b (AGTR1b), (C) angiotensin I converting enzyme (ACE1), (D) angiotensin II converting enzyme (ACE2), (E) chymase 1, (F) aldosterone receptor. Values are represented as mean ± standard error of mean; twoway ANOVA (main effects: diet, p,p’-DDE exposure and their interaction; p

dichlorodiphenyldichloroethylene (DDE) in hypertension of Wistar rats

Recommend Documents