Pyrethroid Insecticide Cypermethrin Accelerates Pubertal Onset in

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Pyrethroid Insecticide Cypermethrin Accelerates Pubertal Onset in Male Mice via Disrupting Hypothalamic-Pituitary-Gonadal Axis Xiaoqing Ye, Feixue Li, Jianyun Zhang, Huihui Ma, Dapeng Ji, Xin Huang, Thomas E. Curry, Weiping Liu, and Jing Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02739 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Environmental Science & Technology

Pyrethroid Insecticide Cypermethrin Accelerates Pubertal Onset in Male Mice via Disrupting Hypothalamic-Pituitary-Gonadal Axis

Xiaoqing Ye1, Feixue Li2, Jianyun Zhang1, Huihui Ma1, Dapeng Ji1, 3, Xin Huang1, Thomas E. Curry Jr.4, Weiping Liu1, 3, Jing Liu1, 3, *

1

MOE Key Laboratory of Environmental Remediation and Ecosystem Health,

College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China 2

Zhejiang Key Laboratory of Organ Development and Regeneration, College of Life

and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China 3

Research Center for Air Pollution and Health, College of Environmental and

Resource Sciences, Zhejiang University, Hangzhou 310058, China 4

Department of Obstetrics and Gynecology, Chandler Medical Center, University of

Kentucky, Lexington, KY 40536, USA * Address correspondence to J.L ([email protected])

1

ACS Paragon Plus Environment


Abstract 1

Pyrethroids, a class of insecticides that are widely used worldwide, have been

2

identified as endocrine-disrupting chemicals (EDCs). Our recent epidemiological

3

study reported on an association of increased pyrethroids exposure with elevated

4

gonadotropins levels and earlier pubertal development in Chinese boys. In this study,

5

we further investigated the effects of cypermethrin (CP), one of the most ubiquitous

6

pyrethroid insecticides, on hypothalamic-pituitary-gonadal (HPG) axis and pubertal

7

onset in male animal models. Early postnatal exposure to CP at environmentally

8

relevant doses (0.5, 5 and 50 µg/kg CP) significantly accelerated the age of puberty

9

onset in male mice. Administration of CP induced a dose-dependent increase in serum

10

levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and

11

testosterone in male mice. CP did not affect gonadotropin-releasing hormone (GnRH)

12

gene expression in the hypothalamus, but CP at higher concentrations stimulated

13

GnRH pulse frequency. CP could induce the secretion of LH and FSH as well as the

14

expression of gonadotropin subunit genes [chorionic gonadotropin α (CGα), LHβ and

15

FSHβ] in pituitary gonadotropes. CP stimulated testosterone production and the

16

expression of steroidogenesis-related genes [steroidogenic acute regulatory (StAR)

17

and Cytochrome p 450, family 11, subfamily A, polypeptide 1 (CYP11A1)] in

18

testicular Leydig cells. The interference with hypothalamic sodium channels as well

19

as calcium channels in pituitary gonadotropes and testicular Leydig cells is

20

responsible for CP-induced HPG axis maturation. Our findings established in animal

21

models provide further evidence for the biological plausibility of pyrethroid exposure 2


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22

as a potentially environmental contributor to earlier puberty in males.

23 24

Keywords: Endocrine-disrupting chemicals (EDCs), Pyrethroids, Cypermethrin,

25

Puberty, hypothalamic-pituitary-gonadal (HPG) axis

26

3



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27

Introduction

28

Pyrethroids, a class of synthetic esters derived from the naturally occurring insecticide

29

pyrethrins, have been widely used for pest control for several decades. Among the

30

currently used pesticide classes, pyrethroids are extensively applied to agricultural,

31

residential and public health sites and account for greater than 30% of global

32

insecticide usage.1 Increased human exposure to pyrethroids is thought to occur

33

mainly via residues in diets and indoor residential use.2-5 Unsurprisingly,

34

3-phenoxybenzoic acid (3-PBA), a common metabolite of most pyrethroids, has been

35

widely identified in urine samples of children and adolescents and the detection rate is

36

usually more than 60% in child populations worldwide.2-5

37 38

Although pyrethroids are generally considered to be safer insecticides given their low

39

acute toxicity to humans, pyrethroids have been recognized as potential

40

endocrine-disrupting chemicals (EDCs). Both epidemiological investigations and

41

animal

42

hypothalamic-pituitary-gonadal (HPG) axis that regulates reproductive capacity by

43

the production of a variety of hormones, particularly luteinizing hormone (LH),

44

follicle-stimulating hormone (FSH) and androgens. For example, two human studies

45

reported a significantly positive association between serum LH and/or FSH levels and

46

urinary 3-PBA in adult male infertility patients.6, 7 One animal study showed that oral

47

administration of permethrin (a commonly used pyrethroid) at doses of 35 and 70

48

mg/kg caused a significant decline in serum testosterone levels, whereas an increase

studies

implied

that

pyrethroids

could

4


affect

the

male

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49

in LH in adult male mice.8 After exposure of other widely used pyrethroids, such as

50

fenvalerate and cypermethrin (CP), adult male rodents also exhibited decreased

51

testosterone production but increased FSH and LH levels.9,

52

suggested that the reduced production of testosterone resulted in a compensatory

53

response by which the secretion of FSH and LH increased via a negative feedback

54

loop of the HPG axis.8, 10 However, another study demonstrated that subcutaneous

55

treatment with a worldwide used pyrethroid deltamethrin (3 µg/kg/day) for 30 days

56

resulted in a significant increase of serum LH, FSH and testosterone concentrations in

57

adult male rats.11 Therefore, how pyrethroids act on HPG axis in males remains

58

ambiguous.

10

These investigators

59 60

Pubertal development is a key parameter of reproductive health that requires the

61

activation of an intact HPG axis. The onset of puberty is controlled by pulsatile

62

gonadotropin-releasing hormone (GnRH) release from the hypothalamus, which

63

subsequently activates pituitary gonadotropes to secrete gonadotropins, i. e. LH and

64

FSH. Then these gonadotropins drive the production of androgens resulting in the

65

appearance of secondary sexual characteristics in males.12 Since previous studies

66

suggested the disrupting effects of pyrethroids on HPG axis, this class of chemicals

67

may influence pubertal development. More recently, our epidemiological study

68

showed that pyrethroids exposure is positively associated with levels of urinary LH

69

and FSH as well as the risk of being advanced pubertal staging in peripubertal boys.13

70

However, the mechanisms by which pyrethroids affect the HPG axis, secretion of 5



71

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puberty-related hormones and puberty timing in males are still poorly understood.

72 73

CP is one of the top three pyrethroids in use and became one of the most ubiquitous

74

insecticides. According to US Environmental Protection Agency usage data, the

75

annual amount of CP is more than 1 million pounds.14 The residue of CP has been

76

widely detected in indoor environment, food, drinking water and even in human

77

milk.15-17 In this study, we utilized CP as a representative pyrethroid to identify the

78

mechanisms by which it affects the pubertal development using in vivo and in vitro

79

experimental rodent models.

80 81

Materials and Methods

82

Chemicals

83

The standard of CP was obtained from Dr. Ehrenstorfer GmbH, Inc. (Augsburg,

84

Germany). Nimodipine, 1, 2 bis-(2-aminophenoxy) ethane-N, N, N’, N’-tetraacetic

85

acid acetoxymethyl ester (BATPA-AM), ethylenebis (oxyethylenenitrilo) tetraacetic

86

acid (EGTA) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich

87

Corp. (St. Louis, MO). Tetrodotoxin (TTX) was purchased from Ruifang, Inc. (Dalian,

88

China).

89

(DMEM/F12), Minimum Essential Media (MEM), fetal bovine serum (FBS), and

90

trypsine were obtained from Hyclone, Inc. (Logan, UT). Fluo-4AM was obtained

91

from Thermo Fisher Scientific, Inc. (Waltham, MA). Collagenase Ⅱ and DNase were

92

purchased from Toyobo, Inc. (Osaka, Japan) and Bovine Serum Albumin (BSA),

Dulbcco's

modified

eagle's

medium/nutrient

6


mixture

F-12

ham's

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93

D-glucose, ethylenediaminetetraacetic acid (EDTA), sodium pyruvate and NaHCO3

94

were purchased from Sangon, Biotech, Inc. (Shanghai, China).

95 96

Animals, cells and exposure

97

CD-1 mice, obtained from Shanghai Laboratory Animal Center, Chinese Academy of

98

Sciences (Shanghai, China), were maintained under a controlled 12-hr light/dark cycle

99

and temperature conditions. All animal procedures were approved by the Zhejiang

100

University Animal Care and Use Committee. Pregnant females were housed

101

individually. Male neonates from each litter were randomly assigned to the different

102

experimental groups and left with the dam. Pups received a daily subcutaneous

103

injection of 0.5 µg/kg, 5 µg/kg, 50 µg/kg CP in peanut oil, or peanut oil alone (vehicle

104

control) beginning in the morning of postnatal day 7 (PND7) and continuing until

105

PND21. During this prepubertal period, the testicular Leydig cell population

106

differentiates and gives rise to the pubertal increase in testosterone levels that are

107

required for sexual maturation and adult fertility.18 Human studies usually used the

108

urinary concentrations of generic metabolites, such as 3-PBA, to reflect the exposure

109

levels of pyrethroids.3, 4, 13 A previous study showed that after 24 h of a single oral

110

administration of CP at dose of 2 mg/kg in adult male mice, urinary 3-PBA levels

111

attained a mean level approximately equivalent to 18 µg/mL.19 Such a urinary

112

concentration of 3-PBA was about 200-20000 times higher than the range of urinary

113

concentrations observed in children.3, 4, 13 In this study, a 4000- and 400-times-lower

114

CP dose (0.5 and 5 µg/kg) we used would be in the range of the relatively moderate 7



115

and high concentrations that have been found in children.3, 4, 13

116 117

Cetrorelix is a GnRH antagonist that has been used to retard the onset of puberty in

118

laboratory animals and precocious patients.20, 21 To block the actions of GnRH

119

receptor, cetrorelix (500 µg/kg) was subcutaneously injected 1 h before the injection

120

of CP (50 µg/kg). To determine the ages of puberty onset, occurrences of preputial

121

separation for a male were observed from PND22.22 To examine puberty-related

122

hormones, pups were sacrificed on PND22 between 08:00 am and 11:00 am and

123

blood was collected. For acute exposure experiments, pups were sacrificed at 24 h

124

after administration with 0.5 to 50 µg/kg CP in the morning of PND 21. The

125

hypothalamus, pituitary and testis were collected for RNA extraction or in situ

126

hybridization. Ten to fifteen replicates for each treatment were used for the in vivo

127

experiments and six replicates were used for the quantification of mRNA levels in the

128

tissues of male mice.

129 130

The primary cultured cells were exposed to fresh serum-free media containing 50

131

µg/L CP for defined periods (3, 6, 12, and 24 hours). To determine mRNA levels or

132

hormone levels, cells were pretreated with vehicle (0.1% DMSO), 10 µM nimodipine,

133

50 µM BAPTA-AM, or 3 mM EGTA for 30 min, and then treated with 50 µg/L CP for

134

24 h. Nimodipine was used to block L-type voltage-gated calcium channels (VGCCs).

135

BAPTA-AM and EGTA were used to chelate intracellular Ca2+ and extracellular Ca2+,

136

respectively. The concentrations of these compounds in this study were set according 8


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137

to previous studies.23 After exposure, cells were collected for RNA and/or protein

138

extraction, or conditioned medium was collected for ELISA/EIA analyses. All

139

chemicals used in cell exposure experiments were dissolved in DMSO and control

140

cells were treated with the same concentration of DMSO (0.1%, v/v). Three replicates

141

were used for each treatment for the in vitro experiments.

142 143

Primary hypothalamic cell cultures

144

The primary hypothalamic cells were isolated from PND21 male mice and cultured as

145

previous described.24 After rapid decapitation, the retrochiasmatic hypothalamus was

146

rapidly dissected and was dispersed in HEPES dissociation buffer containing 4 mg/ml

147

collagenase II, 10 µg/ml DNase, 0.4% BSA, 0.2% D-glucose.21 The explants or cells

148

were incubated in DMEM/F12 medium supplemented with 10% FBS at 37°C in an

149

atmosphere of 5% CO2.

150 151

Primary pituitary cell cultures

152

The primary mice anterior pituitary cells were isolated from PND21 mice as previous

153

described with minor modifications.25 Briefly, anterior pituitary glands were cut into

154

small blocks and digested with HBSS dissociation medium supplemented with 0.3%

155

BSA, 15 mM HEPES, 0.02% EDTA, 0.25% trypsin, 2 µg/mL DNase.25 Cells were

156

suspended in DMEM medium supplemented with 10% FBS, 15 mM HEPES, 110

157

mg/L sodium pyruvate and incubated at 37°C in an atmosphere of 5% CO2.

158 9



159

Primary Leydig cell cultures

160

Purified progenitor Leydig cells were obtained from PND21 mice by collagenase

161

digestion of the testis followed by Percoll density centrifugation of the cell suspension,

162

according to the method of Klinefelter et al.26 Progenitor Leydig cells were harvested

163

from the Percoll gradient at a band between 1.062 and 1.070 g/ml.27 The purity of

164

progenitor Leydig cell fractions was assessed by histochemical staining for

165

3b-hydroxysteroid dehydrogenase (3β-HSD) and was typically 90% enriched. Leydig

166

cells were suspended in DMEM/F-12 medium supplemented with 0.1% BSA, 14 mM

167

NaHCO3 and incubated at 34°C in an atmosphere of 5% CO2.

168 169

Hypothalamic explant incubation

170

For the in vitro study of hypothalamic explants, PND21 mice were used. After

171

decapitation, the hypothalamus was rapidly dissected and incubated as described

172

previously.28 Briefly, each explant was transferred into an individual chamber of

173

24-well plate and incubated in an atmosphere of 5% CO2. Each chamber contained

174

500 µL MEM supplemented with 25 mM glucose, 1 mM Mg2+ and 10 mM glycine.

175

The explants were incubated in the presence or absence of 50, 500 and 5000 µg/L CP

176

for a total period of 3 h. The effects of sodium channels blocker TTX (10 µM) 29, were

177

studied in the presence or absence of maximal effective concentrations of CP (5000

178

µg/L). The incubation medium (500 µL) was collected and renewed every 7.5 min and

179

was kept frozen until assayed.28

180 10


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181

Measurement of GnRH, LH, FSH and Testosterone

182

Serum and conditioned media were collected after exposure to CP alone or in

183

combination with inhibitors as described. GnRH, LH and FSH levels were measured

184

using the ELISA kits according to the manufacturer’s protocol (USCN Life Science

185

Inc., Wuhan, China). Testosterone levels were measured using the EIA kits according

186

to the manufacturer’s protocol (Cayman Chemical, Ann Arbor, MI). Inter and

187

intra-assay coefficient of variation (CVs) for GnRH, LH, FSH and testosterone were

188

less than 10%. The concentrations of hormones in conditioned medium were

189

normalized by cell numbers in each culture.

190 191

Quantification of mRNA

192

Total RNA was isolated from cells or tissues using Trizol reagent (Life Technologies,

193

Carlsbad, CA) according to the manufacturer’s protocol. Synthesis of the first-strand

194

cDNA was performed by reverse transcription of 0.5 µg total RNA using ReverTra

195

Ace ® qPCR RT Kit (Toyobo, Osaka, Japan). Oligonucleotide primer sequences for

196

mouse GnRH, chorionic gonadotropin α (CGα), LHβ, FSHβ, steroidogenic acute

197

regulatory (StAR), Cytochrome p 450, family 11, subfamily A, polypeptide 1

198

(CYP11A1) and L32 were listed in Suppl. Table 1. StAR and CYP 11A1 are

199

important steroidogenesis-related genes. StAR protein facilitates the transfer of

200

cholesterol to the inner mitochondrial membrane where CYP11A1 catalyzes the first

201

and rate-limiting enzymatic step in the biosynthesis of testosterone.30 The real-time

202

PCR reactions were set up with SYBR Green PCR master mix (Toyobo) using a 11



203

Mx3000P Real-time PCR system (Agilent Technologies, Palo Alto, CA), as described

204

previously.31 The relative amount of each gene transcript was calculated using the

205

2−∆∆CT method and normalized to the endogenous reference gene L32.

206 207

In situ hybridization

208

In situ hybridization was performed as described previously.32 Briefly, after treatment

209

with proteinase K and pre-hybridization, slides were incubated with a hybridization

210

solution containing 0.1-0.2 ng/µL of denatured RNA probe at 65 ºC overnight. Signals

211

were developed with BM purple alkaline phosphatase substrate. Sense cRNA probe

212

was used as a control for nonspecific binding. One tissue each from a minimum of

213

three animals per treatment was used for in situ hybridization.

214 215

Determination of intracellular calcium concentration ([Ca2+]i)

216

Intracellular [Ca2+]i was determined as described.33 Briefly, cells were incubated with

217

HEPES-buffered saline containing 2 mM Fluo-4AM with or without 20 mM

218

BAPTA-AM, 0.5 mM EGTA or 10 µM nimodipine. Cells were placed into an Infinite

219

M200 PRO plate reader (Tecan Group, Switzerland) and the kinetic cycle was started

220

with excitation and emission at 490 nm and 525 nm respectively. After fluorescence

221

reached a steady state, CP containing solution was added to the cells immediately

222

prior to measurement for another 60 minutes. Relative fluorescence (∆F/F) was

223

calculated as fluorescence intensities normalized to fluorescence intensity measured at

224

the last time point before CP application. 12


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225 226

Statistical analysis

227

Each experiment of cell treatment was performed in triplicate at least three times. All

228

animal and cellular data were expressed as the mean ± standard deviation (SD).

229

Differences between CP treatments and controls were tested using one-way ANOVA,

230

while differences among co-exposures of CP with inhibitors and corresponding

231

controls were tested using two-way ANOVA. If ANOVA revealed significant effects

232

of treatments, the means were compared by two-tailed unpaired Student’s t test, and

233

p 0.05, data not shown).

239

Pubertal development was accelerated after CP treatment in a dose-dependent manner

240

(Fig. 1A). CP decreased the mean age of puberty onset by 1 day in males dosed with

241

0.5 and 5 µg/kg CP, and by approximately 2 days in males treated with 50 µg/kg CP

242

compared with control animals (Fig. 1A). The circulating levels of LH, FSH and

243

testosterone were measured in male serum on PND22. All experimental doses of CP

244

increased the circulating levels of LH and testosterone (Fig. 1B and D), whereas only

245

5 and 50 µg/kg CP significantly increased serum FSH levels (Fig. 1C). The ontogeny

246

of changes in serum concentrations of gonadotropins and testosterone as well as 13



247

testicular testosterone levels was further determined in males from PND7 through

248

PND22 after daily exposure to 50 µg/kg CP. The testicular weight on PND22 was

249

significantly increased when compared to controls (Suppl. Fig. 1). The timing of the

250

zenith levels of gonadotropins and testosterone in CP-treated animals was

251

approximate 2 to 3 days earlier than controls, indicating that the activation of HPG

252

axis in response to CP was 2 to 3 days earlier than that of untreated mice (Fig. 1E-H).

253 254

Effects of CP on the hypothalamus. To determine whether CP affected the

255

expression of hypothalamic GnRH gene, immature male mice were exposed to 0.5-50

256

µg/kg CP for 24 h. In vivo GnRH mRNA levels had no changes in hypothalamus

257

tissues (Suppl. Fig. 2). In primary cultured hypothalamic cells, in vitro GnRH mRNA

258

levels also had no alteration after exposure to 50 µg/L CP for 3 to 24 h (Suppl. Fig. 3).

259

To investigate whether CP affected GnRH pulse frequency, hypothalamic explants

260

obtained at 21 days were incubated with conditioned media. During a 3-h continuous

261

incubation with 50 µg/L CP, the GnRH interpulse interval (IPI) did not change when

262

compared to control conditions (60.00±0.00 min) (Fig. 2A). However, the GnRH IPI

263

was significantly reduced when incubated with 500 µg/L CP (52.50±3.06 min)

264

(p

Pyrethroid Insecticide Cypermethrin Accelerates Pubertal Onset in

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