Acute nitrite exposure alters the metabolism of thyroid hormones in grass carp (Ctenopharyngodon idellus)

Accepted Manuscript Acute nitrite exposure alters the metabolism of thyroid hormones in grass carp ( Ctenopharyngodon idellus)

Chen Xiao, Zidong Liu, Dapeng Li, Mohamed M. Refaey, Rong Tang, Li Li, Xi Zhang PII:

S0045-6535(17)31170-0

DOI:

10.1016/j.chemosphere.2017.07.119

Reference:

CHEM 19652

To appear in:

Chemosphere

Received Date:

12 April 2017

Revised Date:

17 July 2017

Accepted Date:

24 July 2017

Please cite this article as: Chen Xiao, Zidong Liu, Dapeng Li, Mohamed M. Refaey, Rong Tang, Li Li, Xi Zhang, Acute nitrite exposure alters the metabolism of thyroid hormones in grass carp ( Ctenopharyngodon idellus), Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.07.119

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ACCEPTED MANUSCRIPT 1

Acute nitrite exposure alters the metabolism of thyroid hormones in

2

grass carp (Ctenopharyngodon idellus)

3 4

Chen Xiao12, Zidong Liu1, Dapeng Li1, *, Mohamed M. Refaey2, Rong Tang1, Li

5

Li1, Xi Zhang1

6 7

1

College of Fisheries, Huazhong Agricultural University, Hubei Provincial

8

Engineering Laboratory for Pond Aquaculture, Wuhan, 430070, P. R. China

9

2

Department of Animal Production, Faculty of Agriculture, Mansoura University,

10

Al-Mansoura 35516, Egypt

11

*Correspondence: E-mail: [email protected]; Tel.: +86-027-87282114

12 13

ABSTRACT: Nitrite has the potential to disturb thyroid hormone homeostasis, but

14

little is known about the underlying mechanisms. In the present study, juvenile grass

15

carp (Ctenopharyngodon idellus) were exposed to various concentrations of nitrite (0,

16

0.5, 1, 4, and 16 mg/L, respectively). Serum concentrations of triiodothyronine (T3),

17

thyroxine

18

triiodothyronine (rT3), thyroid-stimulating hormone (TSH), and the activity of

19

iodothyronine deiodinases were assayed at 0, 12, 24, 48, and 96 h after exposure. It

20

was found that acute nitrite exposure significantly altered the TH levels and

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iodothyronine deiodinase activities. The rT3 levels were significantly increased in the

22

treatment groups, whereas the concentrations of T3, FT3, FT4, and TSH decreased 1

(T4),

free

triiodothyronine

(FT3),

free

thyroxine

(FT4),

3,3,5ʹ-

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significantly. The concentration of T4 was elevated in the lower-dose exposure group,

24

but was reduced in the higher-dose exposure group. Increases in type I iodothyronine

25

deiodinase (ID1) and type III iodothyronine deiodinase (ID3) activities were observed

26

in the exposure groups. The activity of type II iodothyronine deiodinase (ID2)

27

decreased at 12 and 24 h after exposure. A decrease of colloid in the thyroid follicles

28

was observed in the exposure group. The results indicate that acute nitrite=-0exposure

29

has the potential to disturb the homeostasis of thyroid hormone metabolism, leading

30

to a hypothyroidism state in the juvenile grass carp.

31

Keywords: Ctenopharyngodon idellus; nitrite; thyroid hormone; iodothyronine

32

deiodinase

33 34

1. Introduction

35

Nitrite is part of the nitrogen cycle in ecosystems, and is generally present at low

36

levels in freshwaters. However, high levels of nitrite have been recorded in intensive

37

aquaculture systems (Meybeck, 1982; Avnimelech et al., 1986; Svobodová, 1991).

38

The main reason for the reported increase in the nitrite level is high density farming,

39

with excess feeding leading to high levels of residual protein based feed and

40

nitrogen excretion. When the residual protein based feed supply and nitrogen

41

excretion outstrips the metabolic capability of indigenous flora in aquaculture water,

42

the nitrite continuously accumulates (Tiedje, 1988). Acute toxicity of nitrite has

43

been well documented in a number of fish species (Hilmy et al., 1987; Chin and

44

Shyong, 1998; Huertas, 2002; Das et al., 2004a; Zhang et al., 2012). In addition, 2

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nitrite disrupts various physiological functions, including ion regulation (Doblander

46

and Lackner 1996; Jensen, 2003), hemoglobin oxygen capacity (Cosby et al 2003;

47

Jensen and Rohde 2010), immune system performance (Cheng et al., 2002; Tseng

48

and Chen, 2004; Chand and Sahoo, 2006; Xian et al 2011), physiological

49

metabolism (Woo and Chiu 1997; Das et al., 2004b; Das et al., 2004c; Ciji et al.,

50

2012), and endocrine regulation (Deane and Woo, 2007; Ciji et al., 2012).

51

Moreover, studies have shown that in Sparus sarba (Rhabdosargus sarba) exposed

52

to 25 and 50 mg/L nitrite, the serum thyroxine (T4) levels decreased by 42 and 68%,

53

respectively, suggesting that nitrite may disrupt the thyroid endocrine system of

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exposed fish (Deane and Woo, 2007).

55

The thyroid hormones (THs) have widespread biological effects on physiological

56

processes (Power et al., 2001; Crane et al., 2004; Porazzi et al., 2009). In fish, THs

57

play a major role in differentiation, growth, metabolism, salinity adaptation, and

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reproduction (Liu and Chan, 2002; Orozco et al., 2002; Crane et al., 2004). Free

59

triiodothyronine (FT3), free thyroxine (FT4), and the thyroid-stimulating hormone

60

(TSH) are the current front line tests for evaluating thyroid functional status (Yeasmin

61

et al., 2016). The plasma concentrations of free THs, FT4, and FT3, are preferred

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clinically as indices of thyroidal status, because they are not influenced by the degree

63

of protein binding, which can be affected by numerous factors (Lazarus, 2005;

64

Refetoff, 1979).The predominant TH synthesized from the thyroid tissue is T4, and

65

the TSH regulates the synthesis of THs secreted by the thyroid gland. However, T4

66

has little biological activity, whereas T3 has considerable biological activity. The 3

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conversion of T4 to T3 is catalyzed by the iodothyronine deiodinases in the peripheral

68

tissues (Liu et al., 2011). Three types of iodothyronine deiodinase,—type I

69

iodothyronine deiodinase (ID1), type II iodothyronine deiodinase (ID2), and type III

70

iodothyronine deiodinase (ID3)—has beenidentified in fish. Each iodothyronine

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deiodinase can convert THs to more or less active forms. For the effective functioning

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of THs, T4 should be converted to the more active T3 by molecular deiodination in the

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outer ring (ORD), which is catalyzed by ID1 and ID2 (Orozco and Valverde, 2005).

74

The inner ring deiodination (IRD) catalyzed by ID3 produces 3,3,5ʹ-triiodothyronine

75

(rT3), which has no biological activity from T4, and the less active diiodotyrosine (T2)

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from T3 (Gai, 2012). ID1 plays a minimal role in plasma TH homeostasis, this

77

enzyme has a considerable influence on iodine recovery and TH degradation (Van der

78

Geyten et al., 2001; Yu et al., 2010). Thus, ID2 and ID3 play the leading roles in

79

adjusting the content of T3 (Orozeo et al., 2002; Brown et al., 2004).

80

Because of the key role of THs in fish, it is important to identify environmental

81

chemicals that may disturb thyroid function, and then evaluate the risk to aquatic

82

organisms (Brucker-Davis, 1998). Although nitrite can alter the concentrations of TH

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in Sparus sarba, the mechanisms underlying the changes in TH levels are still

84

unclear. We hypothesized that nitrite may alter the morphology of thyroid follicles,

85

change the activities of iodothyronine deiodinases and the peripheral circulating

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content of THs, to ultimately influence the TH metabolism. Therefore, the objective

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of this study was to determine the possible mechanisms for changes in TH levels in

4

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fish exposed to nitrite by investigating the effects of nitrite on TH metabolism in grass

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carp.

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2. Materials and Methods

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2.1 Toxin and fish administration

92

The experimental drug was analytical grade NaNO2 (purity ≥99.0%), which was

93

purchased from the Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The

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nitrite was dissolved in double distilled water as a stock solution. The concentrations

95

required for exposure were achieved by diluting the stock solution into aquarium

96

water.

97

The experimental animals were healthy grass carp (mean mass ± S.D., 65.82 ±

98

5.53 g), which were obtained from Tuanfeng Fish Farm in Wuhan City, China and

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then transported to the laboratory at the College of Fisheries in Huazhong Agricultural

100

University. The fish were acclimated for one week before the start of the experiment.

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During the acclimation and experimental period, fish were maintained in 200 L glass

102

tanks, with a natural photoperiod. Dechlorinated aeration water was used in the

103

experiment to achieve the aquaculture water standard, with water quality parameters

104

as follows, NH4+-N + NO3--N <0.1 mg/L, dissolved oxygen (7.0 ± 0.5 mg/L),

105

temperature (26.0 ± 2°C), and pH (7.0–7.6). Nitrite concentrations were checked daily

106

during the acclimation and experiment, as well as the dissolved oxygen, temperature,

107

and pH. The fish were fed with a commercial diet twice a day, and the supply was

108

stopped during the period from two days before the start to the end of experiment.

5

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Fish were exposed to various concentrations (0, 0.5, 1, 4, and 16 mg/L) of nitrite

110

for 96 hours. During the exposure period, 50% of the exposure solution was renewed

111

every day. There were fifteen grass carp specimens in each group, which were

112

distributed equally among tanks, with three replicate tanks used for each group. Dead

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fish were removed from the tank daily.

114

2.2 Sample collection Blood, liver, and the tissues from the first branchial arch to the third branchial

115 116

arch

of six fish in each group were taken after exposures of 0, 12, 24, 48, and 96 h.

117

At each sampling, the fish were anaesthetized with tricaine methanesulfonate

118

(MS222, Sigma-Aldrich, Louis, MO, USA).

119

from the caudal vein with a syringe.

120

4°C and then was used for hormone assay.

121

nitrogen and then kept at -80°C until assay

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2.3 Hormone content measurement

Blood sample (2 mL) was collected

Serum was centrifuged at 3000 ×g for 15 min at Liver samples were frozen in liquid

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Serum T3, T4, rT3, FT3, FT4 and TSH levels were measured by radioimmunoassay

124

(RIA) using commercial kits purchased from the Beijing North Institute of

125

Biotechnology, China. The RIA kits for hormones were validated for use with serum

126

samples by demonstrating parallelism between a series of diluted and spiked samples

127

in relation to the standard curve. The catalog number (standard limits) of T3, T4, rT3,

128

FT3, FT4 and TSH kits were A01TFB (0-7.5 ng/mL ), A02TFB (0-360 ng/mL),

129

A06TBB (0.075-3.0 ng/mL), A03TFB (1-54 fmol/mL or 0.651-35.15 *10-3 ng/mL),

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A04TFB (6-100 pmol/mL or 4.66-77.7 *10-6 ng/mL), A05TFB (0-50 μ IU /mL),

131

respectively. In addition, T3/T4 ratios were calculated for each individual.

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2.4 Deiodinase activity assay

133

The liver was homogenized in buffer solution (0.1 M PBS, 1 mM DTT, 2 mM

134

EDTA, pH 7.0) and centrifuged at 12,000 ×g for 20 min, and then the insoluble

135

particles were removed. The homogenates were quickly frozen in liquid nitrogen and

136

stored at –80°C prior to the assay. Enzyme activities were measured using a

137

modification of the radiolabeled iodine release method (Van der Geyten et al., 1998;

138

Coimbra, 2005; Liu et al., 2015; Liu et al., 2016 ).

139

The protein concentration was determined by a Bradford Protein Assay (Bio-rad,

140

Hercules, CA, USA). The activity of ID1 was measured by incubating 200 μL liver

141

homogenate at 37°C for 120 min with 200 μL of

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50,000 cpm of 125I-rT3, 0.1 μM unlabeled rT3, and 15 mM DTT. The activity of ID2

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and ID3 were similar to that of ID1, and were measured by incubating 200 μL of liver

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homogenate at 37°C for 120 min with 50,000 cpm of

145

mM DTT in 200 μL of 0.1 M PBS (pH 7.0); and 150,000 cpm of

146

unlabeled T3, 30 mM DTT in 200 μL of 0.1 M PBS (pH 7.0), respectively. All

147

reactions were stopped by adding 200 μL 5% (w/v) bovine serum albumin (Sigma-

148

Aldrich) successively, and 400 μL 10% (w/v) trichloroacetic acid at 4°C. The

149

radioactivity in the supernatant was counted using a GC-911 γ-counter (Zhong Jia,

150

Tianjin, China) after centrifuging at 3500 ×g for 30 min. Instead of liver homogenate,

7

0.1 M PBS (pH 7.0), containing

125I-T , 4

1 nM unlabeled T4, 30 125I-T , 3

1 nM

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0.1 M PBS was used as a blank control. The iodothyronine deiodinase activity was

152

calculated using the following formula: Iodothyronine deiodinase activity [SCc (cpm) × SA

153

(

)

pmol/fmol × 1000] cpm

= [homogenate volume (μL) × protein content

( )

mg × incubation time (min)] mL

154

Where SCc is sample counts minus blank counts, and SA is total moles (rT3, T4 or T3)

155

in the incubation solution/total counts. Therefore, the units of iodothyronine

156

deiodinase activity are expressed as pmol I– released/mg protein per min.

157

2.5 Thyroid histology

158

The organizations from the first branchial arch and the third branchial arch were

159

severed from the fish and fixed in Bouin’s fixative for 48 h. Following fixation, the

160

samples were washed in water and stored in 70% ethanol. The organizations were

161

embedded in paraffin and serial transverse cross-sections (5 μm) were made using a

162

tissue processor (Leica RM 2135, Germany). Dewaxed and rehydrated sections were

163

stained with hematoxylin and eosin. The photograph was taken by NIS-Element BR

164

3.0 software.

165

2.6 Statistical analysis

166

Experimental data were expressed as a mean ± SD, inputted using EXCEL

167

software and performed using SPSS 13.0 software. The differences between the

168

control group and each exposure group were evaluated by one-way analysis of

169

variance (ANOVA) followed by Duncan’s multiple comparison tests where

8

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differences were found. A value of p<0.05 was considered statistically significant and

171

indicated with *.

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3. Results

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3.1 Mortality

174

No fish died in the control and lower concentration groups (0.5 and 1 mg/L)

175

during the experimental period. Mortalities of 4.76 and 9.52% were found in the 4 and

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16 mg/L groups, respectively, at 96 h after exposure.

177

3.2 Serum TSH level

178

After the administration of nitrite, serum TSH levels decreased significantly as the

179

nitrite concentration increased, with the largest decrease observed 48 h after exposure,

180

before rising again at 96 h after exposure (Figure. 2).

181 182

Figure 1. Serum thyroid-stimulating hormone (TSH) concentration of grass carp,

183

exposed to nitrite (0 mg/L, 0.5 mg/L, 1 mg/L, 4 mg/L, 16 mg/L). The values are

184

expressed as mean±SD. Significant differences obtained by one-way analysis of

185

variance followed by Duncan’s multiple comparison test are indicated from value at 0

186

h: *p<0.05.

187

3.3 Serum levels of thyroid hormones 9

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After the exposure, the T4 level initially displayed an upward trend before later

189

descending. The T4 content increased when the nitrite concentration increased from 0

190

to 1 mg/L. The T4 content rose significantly in the 0.5 mg/L group at 12 and 48 h after

191

exposure. When the nitrite concentration increased further, the T4 content fell

192

significantly (Figure. 2A).

193

The FT4 levels decreased significantly with an increase in the exposure

194

concentration. A significant drop in serum FT4 was found in the 1, 4, and 16 mg/L

195

groups 12 h after exposure; in the 1 and 4 mg/L groups 24 h after exposure; and in the

196

4 and 16 mg/L groups 48 h after exposure There was a tendency for a decrease in the

197

0 to 4 mg/L groups, and an increase in the 16 mg/L group (Figure. 2B).

198

During the nitrite administration, serum T3 levels decreased significantly as the

199

nitrite concentration increased and exposure time lengthened, with the largest

200

decrease observed 96 h after exposure (Figure. 2C).

201

After exposure, serum FT3 displayed a similar trend to that of TSH, with a

202

significant decrease as the nitrite concentration increased. At 24 h after exposure, the

203

serum FT3 levels in the 1, 4, and 16 mg/L groups decreased significantly. At 48 and

204

96 h after exposure, a significant decrease was observed in every nitrite group

205

(Figure. 2D).

206

When the nitrite concentration was increased from 0 to 0.5 mg/L, rT3 levels

207

increased significantly. However, when the nitrite concentration was further increased

208

to 1 mg/L, the concentration of rT3 fell at 24 and 48 h after exposure, with the

209

opposite result observed at 12 and 96 h after exposure. At 12 and 96 h after exposure, 10

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the concentration of rT3 decreased in the two higher nitrite concentration groups. At

211

24 and 48 h after exposure, the concentration of rT3 increased significantly (Figure.

212

2E).

213

During the exposure period, T3 /T4 ratio decreased significantly as the nitrite

214

concentration increased and exposure time lengthened, with the largest decrease

215

observed 96 h after exposure (Figure. 2F).

11

(A)

(B)

(C)

(D)

(E)

(F)

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Figure 2.

217

triiodothyronine (FT3), 3,3,5ʹ-triiodothyronine (rT3) concentration and T3/T4 ratio of

218

grass carp, exposed to nitrite (0 mg/L, 0.5 mg/L, 1 mg/L, 4 mg/L, 16 mg/L). The

219

values are expressed as mean±SD. Significant differences obtained by one-way

220

analysis of variance followed by Duncan’s multiple comparison test are indicated

221

from value at 0 h: *p<0.05.

222

3.4 Deiodinase activity In all treatment groups, the ID1 activity increased significantly in a dose-

223 224

Serum thyroxine (T4), free thyroxine (FT4) triiodothyronine (T3), free

dependent manner as the nitrite concentration increased (Figure. 3A).

225

Nitrite exposure significantly affected the ID2 activity. The ID2 activity was

226

significantly decreased in the 0.5 and 1 mg/L groups after 12 h exposure and in the

227

0.5, 1, and 4 mg/L groups 24 h after exposure (Figure. 3B).

228

At 12 and 96 h after exposure, the activity of ID3 significantly increased in the

229

0.5, 1, and 4 mg/L groups. However, there was no significant difference in ID3

230

activity at the other time points. No significant change in ID3 activity occurred in the

231

16 mg/L nitrite exposure group (Figure. 4C).

(A)

12

(B)

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(C)

232

Figure 3. The activity of type I iodothyronine deiodinase (ID1), type II iodothyronine

233

deiodinase (ID2) and type III iodothyronine deiodinase (ID3) in grass carp, exposed

234

to nitrite (0 mg/L, 0.5 mg/L, 1 mg/L, 4 mg/L, 16 mg/L).The values are expressed as

235

mean±SD. Significant differences obtained by one-way analysis of variance followed

236

by Duncan’s multiple comparison test are indicated from value at 0 h: *p<0.05.

237

3.5 Histopathology

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The thyroid follicles were observed in the 0 and 16 mg/L treatment groups 96 h

239

after exposure. Representative images of the control and treatment groups are shown

240

in Figure. 4. The thyroid follicles in the control group had varying sizes, linked the

241

simple cuboidal epithelium, and were filled with colloid within the lumen. The

242

follicles typically had a slightly oval appearance (Figure. 4A). In the 16 mg/L nitrite

243

exposure group, the amount of colloid was diminished in the thyroid follicles. This

244

phenomenon was widespread, and the image shown in Figure. 4B was typical.

13

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(A)

(B)

245

Figure 5. Histologic section of thyroid in grass carp, exposed with nitrite, A --

246

histologic section of thyroid in 0 mg/L; B -- histologic section of thyroid in 16 mg/L

247

after 96 hours of exposure (CO--colloid).

248

4. Discussion

249

In this study, treatment with nitrite significantly altered T4 levels, increased rT3

250

levels, and decreased TSH, T3, FT4, and FT3 levels. Moreover, significant changes in

251

the histology of thyroid follicle and the activity of iodothyronine deiodinase were also

252

observed after exposure to nitrite. Thus, our results showed that exposure to nitrite

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under laboratory conditions had an adverse effect on the TH metabolism of grass carp.

254

Chemical contaminants have been reported to affect thyroidal hormone function

255

in many fish species (Xu et al., 2002; Brown et al., 2004; Scott and Sloman, 2004;

256

Van der Ven et al., 2006), but the effect of nitrite on THs in fish has received little

257

attention, especially in grass carp (Deane, 2007; Hinther et al., 2012). The

258

histopathology of thyroid follicles is frequently used as an indicator of thyroid

259

function (Brown et al., 2004; Liu et al., 2011). In this study, the thyroid follicles in the

260

control group were filled with colloid within the lumen, whereas the amount of 14

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colloid was diminished in the thyroid follicles 96 h after exposure in the 16 mg/L

262

nitrite exposure group. Severity of colloid depletion is a routinely employed marker

263

for thyroid disruption (Liu et al., 2006).There was a general decrease pattern of

264

colloid area in chemical treatment, the result indicated high concentration nitrite

265

exposure altered TH levels by changing colloid size in thyroid follicles.

266

The changes in TH levels are usually used as direct endpoints to assess the thyroid

267

disruption (Liu et al., 2011). In this study, the T4 levels were increased in the 0.5 and

268

1 mg/L treatment and decreased in 4 and 16 mg/L treatment. The alteration in T4

269

levels may be caused by variations in the colloid size of thyroid follicles, regulation of

270

TSH, and change in ID activity. In fish, T4 is the only TH secreted in the thyroid

271

follicle and is stimulated by TSH (Chiamolera et al., 2009; MacKenzie et al., 2009).

272

In the present study, serum TSH levels decreased significantly with increasing nitrite

273

concentration. The minimum of reduction was appeared at 96 h after exposure which

274

may cause by the adaptation of fish to their environment. Thus, the results indicate

275

that the stress response was induced by nitrite and may contribute to the decrease of

276

TSH levels, leading to a depressed synthesis of T4 in thyroid follicles. In this study,

277

the ID1 activity was significantly increased as the nitrite concentration increased, and

278

the ID2 activity was significantly decreased after exposure in first 24 hours and

279

returned to normal level in last 24 hours. ID1 and ID2 can catalyze the outer ring

280

deiodination of thyroid hormones (Van der Geyten et al., 2001; Yu et al., 2010).

281

plays a minimal role in thyroid hormone homeostasis, ID2 is the main iodothyronine

282

deiodinase of T3 production which operates by metabolizing T4 into active T3 (Berry, 15

ID1

ACCEPTED MANUSCRIPT 283

1991; Orozco et al., 2002; Larsen et al., 1981 Liu et al., 2011). These data suggest that

284

the decreased ID2 activity resulting from nitrite exposure contribute to a reduced rate

285

of conversion from T4 to T3, which leads to a decrease in T4 decomposition and T3

286

production (Liu et al., 2011). The depressed composition and decomposition during

287

the period of the experiment resulted in changes in the T4 levels. T4 levels depend on

288

the quantity of composition and decomposition. The depressed TSH levels leads to

289

the decrease in T4 production. On the contrary, the reduced ID2 activities resulting

290

from nitrite exposure contribute to the reduced rate of conversion from T4 to T3,

291

which result in the decrease in T4 decomposition. In lower nitrite groups the quantity

292

of reduced composition is less than the quantity of reduced decomposition, the T4

293

levels show an increased observation and vice versa. During the nitrite administration,

294

T3 /T4 ratio decreased significantly and the largest decrease observed 96 h after

295

exposure. In 0.5 and 1 mg/L groups, the reducion of T3/T4 ratios caused by increased

296

T4 levels and decreased T3 levels; in higher nitrite groups, deceased T4 levels and

297

more seriously decreased T3 levels leaded to the reducion of T3/T4 ratios.

298

Furthermore, the T3/T4 ratio is an index which reflects thyroid function and the action

299

of hormones on the tissues. T3/T4 ratios in grass carp indicated the alteration of

300

peripheral deiodinase activity (Brar et al., 2010). In this study, the reducion of T3/T4

301

ratios may point to nitrite decreased the conversion of T4 into T3 in the

302

peripheral.The result was in a good agreement with the tendency of ID2 actvity. FT4,

303

a free form of T4 in serum, can be transported to target tissues and directly induce

304

biological activity (Lazarus, 2005). The decline of FT4 levels were closely related to 16

ACCEPTED MANUSCRIPT 305

the depressed synthesis of T4 observed in the study. Significant decreases in FT4 and

306

FT3 levels were detected after exposure to nitrite.. The decrease in T3 and FT3 levels

307

is mostly due to changes in the peripheral TH metabolism, especially the decline of

308

ID2 activity. (Liu et al., 2015). In our study, a decline of FT4 may be one of the

309

reasons for the decrease in the FT3 level. Increased rT3 levels were observed in the

310

experiment during the experimental period and the maximum of augment was

311

appeared at 48 h after exposure. These results were due to the increased ID3 activities.

312

ID3 was the main physiological inactivator of THs, and acted by metabolizing T4 and

313

T3 into the inactive compounds, rT3 and T2 (Gereben et al., 2007). ID3 activity was

314

significantly increased at 12 and 96 h after nitrite exposure in 0.5,1 and 4 mg/L group.

315

The increase in ID3 activity means that more T4 was catalyzed to rT3 rather than T3.

316

Thus, a rise in ID3 activity can result in a decrease in FT4 and FT3 levels, and can also

317

increase the rT3 level (Orozco and Valverde, 2005). These data suggest that the

318

observed changes in the activities of ID2 and ID3 could contribute to reduced FT4 and

319

FT3 levels, as well as promoting the rT3 levels following the nitrite treatments. The

320

exposure of grass carp to nitrite significantly altered iodothyronine deiodinase

321

activity.

322

Iodothyronine deiodinases are crucial regulators of the concentrations of

323

peripheral circulating THs in fish (Zhang et al., 2013). The detection of changes in

324

iodothyronine deiodinase activity caused by environmental contaminants can reflect

325

the mechanisms of interference more clearly and suggest a possible pathway toward

326

poisoning (Blanton et al., 2007). Because of its toxicity, nitrite is undesirable in 17

ACCEPTED MANUSCRIPT 327

environment water, especially in aquaculture water. In intensive fish culture, for the

328

preservation of grass carp’s health, the water must maintain low nitrite concentration

329

Conclusion

330

This study suggests that nitrite altered iodothyronine deiodinases activity and

331

thyroid follicle morphology, which in turn resulted in the change of serum THs level.

332

The changes also revealed a disturbance in thyroid hormone synthesis and metabolism

333

in fish exposed to nitrite, leading to a decline in FT4 and FT3 production. Thus, acute

334

nitrite exposure disturbs thyroid hormone homeostasis and results in a hypothyroidism

335

state in juvenile grass carp.

336

Acknowledgments

337

This study was supported by the Earmarked Fund for China Agriculture

338

Research System (Project no. CARS-46), the National Natural Foundation of China

339

(Project no. 31502140), the Fundamental Research Funds for the Central Universities

340

(Project no. 2662015PY119).

341

References

342

Avnimelech Y, Weber B, Hepher B, 1986. Studies in circulated fish ponds: organic

343

matter recycling and nitrogen transformation. Aquac FishManag, 17 (13): 231 -

344

242.

345

Barton, B.A., 2002. Stress in fishes: a diversity of responses with particular reference

346

to changes in circulating corticosteroids. Integrative & Comparative Biology,

347

42(3), 517-525. Berry MJ , Kieffer JD , Larsen PR, 1991. Evidence that cysteine

348

not sele-no cysteine, is in the cat alytic sit e of type Ⅱ iodothyronine 18

ACCEPTED MANUSCRIPT deiodinase. Endocrinology, 129: 550

349 350

Brar, N. K., Waggoner, C., Reyes, J. A., Fairey, R., & Kelley, K. M. (2010). Evidence

351

for thyroid endocrine disruption in wild fish in san francisco bay, california, usa.

352

relationships to contaminant exposures.Aquatic Toxicology, 96(3), 203.

353

Brown, S.B., Adams, B.A., Cyr, D.G., Eales, J.G., 2004. Contaminant effects on the

354

teleost fish thyroid. Environmental Toxicology & Chemistry, 23(7), 1680–1701..

355

Brucker-Davis, F, 1998. Effects of environmental synthetic chemicals on thyroid

356

function. Thyroid Official Journal of the American Thyroid Association, 8(9),

357

827-56.

358

Chand RK, Sahoo PK, 2006. Effect of nitrite on the immune response of freshwater

359

Prawn Macrobrachium malcolmsonii and its susceptibility to Aeromonas

360

hydrophila. Aqzaczltzre, 258: 150-156.

361

Cheng W, Liu CH, Chen JC, 2002. Effect of nitrite on interaction between the giant

362

freshwater prawn Macrobrachium rosenbergii and its pathogen Lactococcus

363

garvieae. Diseases of Aquatic Organisms, 50(3), 189-97.

364

Chiamolera, M.I.; Wondisford, F.E., 2009. Minireview: Thyrotropin-releasing

365

hormone and the thyroid hormone feedback mechanism. Endocrinology, 150,

366

1091–1096.

367

Ciji A, Sahu NP, Pal AK, Dasgupta S, Akhtar MS, 2012. Alterations in serum

368

electrolytes, antioxidative enzymes and haematological parameters of Labeo

369

rohita on short-term exposure to sublethal dose of nitrite. Fish Physiology &

370

Biochemistry, 38(5), 1355-1365 19

ACCEPTED MANUSCRIPT 371

Coimbra, A. M., Reis-Henriques, M. A., Darras, V. M., 2005. Circulating thyroid

372

hormone levels and iodothyronine deiodinase activities in Nile tilapia

373

(Oreochromis niloticus) following dietary exposure to Endosulfan and Aroclor

374

1254. Comp Biochem Physiol C Toxicol Pharmacol, 141(1), 8-14.

375

Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK,

376

Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB,

377

Schechter AN, Cannon RO, Gladwin MT, 2003. Nitrite reduction to nitric oxide

378

by deoxyhemoglobin vasodilates the human circulation. Nature Medicine, 9(12),

379

1498-1505.

380

Crane, H.M., Pickford, D.B., Hutchinson, T.H., Brown, J.A., 2004.Developmental

381

changes of thyroid hormones in the fathead minnow, Pimephales promelas.

382

General & Comparative Endocrinology, 139(1), 55-60.Das PC, AyaPPan S, Jena

383

JK, Das BK, 2004a. Nitrite toxicity in Cirrhina mrigala (Ham.): acute toxicity

384

and sublethal effect on selected haematological parameters. Aquaculture, 235:

385

633-644

386

Das PC, Ayyappan S, Jena JK, Das BK, 2004b. Effect of sub-lethal nitrite on selected

387

haematological parameters in fingerling Catla catla (Hamilton). Aquaculture

388

Research, 35(9), 874-880.Das PC, Ayyappan S, Das BK, Jena JK, 2004c. Nitrite

389

toxicity in Indian major carps: sublethal effect on selected enzymes in fingerlings

390

of Catla catla, Labeo rohita and Cirrhinus mrigala. Comparative Biochemistry &

391

Physiology Part C Toxicology & Pharmacology, 138(1), 3-10. Deane EE, Woo

392

NYS, 2007. Impact of nitrite exposure on endocrine, osmoregulatory and 20

ACCEPTED MANUSCRIPT 393

cytoprotective functions in the marine teleost Sparus sarba. Aquatic Toxicology,

394

82 (2): 85-93

395

Doblander C, Lackner R, 1996. Metabolism and detoxification of nitrite by trout hepatocytes. Biochimica Et Biophysica Acta, 1289: 270-274

396 397

Eales, J. G., & Shostak, S, 1985. Free t 4 and t 3 in relation to total hormone, free

398

hormone indices, and protein in plasma of rainbow trout and arctic

399

charr. General & Comparative Endocrinology, 58(58), 291-302.

400

Gereben, B., Zeöld, A., Dentice, M., Salvatore, D., & Bianco, A. C., 2008. Activation

401

and inactivation of thyroid hormone by deiodinases: local action with general

402

consequences. Cellular & Molecular Life Sciences Cmls, , 65(4), 570-590.

403

Geyten, S.V.d., Mol, K.A., Pluymers, W., Kühn, E.R., Darras, V.M., 1998. Changes

404

in plasma T3 during fasting-refeeding in tilapia (Oreochromis niloticus) are

405

mainly regulated through changes in hepatic type II iodothyronine deiodinase.

406

Fish Physiology & Biochemistry, 19(2), 135-143.

407

Hilmy AM, EI-Domiaty NA, Wershana K, 1987. Acute and chronic toxicity of nitrite

408

to Clarias lazera. Comparative Biochemistry & Physiology C Comparative

409

Pharmacology & Toxicology, 86(2): 247-253

410

Hinther, A., Edwards, T. M., Jr, G. L., & Helbing, C. C, 2012. Influence of nitrate and

411

nitrite on thyroid hormone responsive and stress-associated gene expression in

412

cultured rana catesbeiana tadpole tail fin tissue.Frontiers in Genetics, 3(3), 51-51.

413

Huertas M, Gisbert E, Rodrguez A, Cardona L, Williot P, Castello-Orvay F, 2002.

414

Acute exposure of Siberian sturgeon (Acipenser baeri, Brandt) yearlings to 21

ACCEPTED MANUSCRIPT 415

nitrite: median-lethal concentration (LC50) determination, haematological

416

changes and nitrite accumulation in selected tissues. Aquatic Toxicology, 57(4):

417

257-266

418

Jensen FB, Rohde S, 2010. Comparative analysis of nitrite uptake and hemoglobin-

419

nitrite reactions in erythrocytes: sorting out uptake mechanisms and oxygenation

420

dependencies. American Journal of Physiology Regulatory Integrative &

421

Comparative Physiology, 298(4): 972-982

422

Jensen FB, 2003. Nitrite disrupts multiple physiological functions in aquatic animals.

423

Comparative Biochemistry & Physiology Part A Molecular & Integrative

424

Physiology, 135(1): 9-24

425

Larsen, P. R., & Berry, M. J., 1995. Nutritional and hormonal regulation of thyroid hormone deiodinases. Annual Review of Nutrition, 15(1), 323-352.

426 427

Lazarus, J. H., 2005. Best practice no 184 screening for thyroid disease in pregnancy. Journal of Clinical Pathology, 58(5): 449-452.

428 429

Li, D., Xie, P., Zhang, X., 2008. Changes in plasma thyroid hormones and cortisol

430

levels in crucian carp (Carassius auratus) exposed to the extracted microcystins.

431

Chemosphere, 74, 13–18.

432

Li,W., Zha, J., Yang, L., Li, Z., Wang, Z., 2011. Regulation of iodothyronine

433

deiodinases and sodium iodide symporter mRNA expression by perchlorate in

434

larvae and adult Chinese rare minnow (Gobiocypris rarus). Marine Pollution

435

Bulletin 63, 350-355.

22

ACCEPTED MANUSCRIPT 436

Liu, F. J., Wang, J. S.,

Theodorakis, C. W., 2006. Thyrotoxicity of sodium arsenate,

437

sodium perchlorate, and their mixture in zebrafish danio rerio. Environmental

438

Science & Technology, 40(10), 3429-36.

439

Liu, S., Chang, J., Zhao, Y., Zhu, G., 2011. Changes of thyroid hormone levels and

440

related gene expression in zebrafish on early life stage exposure to triadimefon.

441

Environmental Toxicology & Pharmacology, 32(3), 472-477.

442

Liu, Y., Chan, W., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.

443 444

Liu, Y.; Wang, J.; Fang, X.; Zhang, H.; Dai, J., 2011. The thyroid-disrupting effects

445

of long-term perfluorononanoate exposure on zebrafish (Danio rerio).

446

Ecotoxicology, 20, 47–55.Liu, Z., Li, D., Wang, Y., Guo, W., Gao, Y. and Tang,

447

R. 2015. Waterborne exposure to microcystin-LR causes thyroid hormone

448

metabolism disturbances in juvenile Chinese rare minnow (Gobiocypris rarus).

449

Environmental Toxicology & Chemistry, 34(9)34(9): 2033-2040

450

Liu, Z., Li, D., Hu, Q., Tang, R., & Li, L. (2016). Effects of exposure to microcystin-

451

lr at environmentally relevant concentrations on the metabolism of thyroid

452

hormones in adult zebrafish (danio rerio). Toxicon,124, 15-25.

453

Mackenzie, D., Jones, R.,

Comparative Endocrinology, 161(1), 83–89.

454 455

Miller, T., 2009. Thyrotropin in teleost fish. General &

Meybeck, M. 1982. Carbon, nitrogen and phosphorus transport by world rivers. American Journal of Science, 282(4): 401–450.

456

23

ACCEPTED MANUSCRIPT 457

Orozco, A., Valverde, R.C., 2005. Thyroid Hormone Deiodination in Fish. Thyroid 15, 799-813.

458 459

Orozco, A., Villalobos, P., Valverde, R.C., 2002. Environmental salinity selectively

460

modifies the outer-ring deiodinating activity of liver, kidney and gill in the

461

rainbow trout. Comparative biochemistry and physiology. Part A, Molecular &

462

integrative physiology, 131(2), 387-395.Power, D.M., Llewellyn, L., Faustino,

463

M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V., Sweeney,

464

G.E., 2001.Thyroid hormones in growth and development of fish. Comparative

465

Biochemistry & Physiology Toxicology & Pharmacology Cbp, 130(4), 447–

466

459.

467

Porazzi, P., Calebiro, D., Benato, F., Tiso, N., Persani, L., 2009. Thyroid gland

468

development and function in the zebrafish model. Molecular & Cellular

469

Endocrinology, 312(1–2), 14-23.

470

Refetoff, S., 1979. Thyroid hormone transport. In: DeGroot L. J. (Ed). Endocrinology, Vol. I. Grune & Stratton, New York, pp. 347-356.

471 472

Seo, H., Refetoff, S., Martino, E.,Vassart, G.,& Brocas, H.,1979. The differential

473

stimulatory effect of thyroid hormone on growth hormone synthesis and estrogen

474

on prolactin synthesis due to accumulation of specific messenger ribonucleic

475

acids..Endocrinology, 104 (104), 1083-90.

476

Scott, G. R., & Sloman, K. A., 2004. The effects of environmental pollutants on

477

complex fish behaviour: integrating behavioural and physiological indicators of

478

toxicity. Aquatic Toxicology, 68(68), 369-392. 24

ACCEPTED MANUSCRIPT 479

Sutija, M., & Joss, J. M., 2006. Thyroid hormone deiodinases revisited: insights from lungfish: a review. Journal of Comparative Physiology B,176(2), 87-92.

480 481

Svobodová Z, Vykusová B, Máchová J. Intoxications of Fish, 1991. Diagnostic

482

Preventation and Therapy of Fish Diseases and Intoxicationsis Manual. p.270

483

BN. 80-901087- 0-9.

484

Tiedje, J. M. 1988. Ecolgy of denitrification and dissimilatory nitrate re-duction to

485

ammonium, p. 179-244. In A. J. B. Zehnder (ed.), Biology of anaerobic

486

microorganisms. John Wiley and Sons, New York, N.Y.

487

Tseng I T, Chen J C, 2004. The immune response of white shrimp Litopenaeus

488

vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress. Fish

489

Shell immunol, 17: 325-333

490

Van der Geyten, S., Toguyeni, A., Baroiller, J.F., Fauconneau, B., Fostier, A.,

491

Sanders, J.P., Visser, T.J., Kuhn, E.R., Darras, V.M., 2001. Hypothyroidism

492

induces type I iodothyronine deiodinase expression in tilapia liver. General &

493

Comparative Endocrinology, 124(3), 333-342

494

Van der Geyten S, Mol K, Pluymers W, Kühn E, Darras V. 1998. Changes in plasma

495

T3 during fasting/refeeding in tilapia (Oreochromis niloticus) are mainly

496

regulated through changes in hepatic type II iodothyronine deiodinase. Fish

497

Physiology & Biochemistry, 19(2):135-143.

498

Wedemeyer, G., 1969. Stress-induced ascorbic acid depletion and cortisol production

499

in two salmonid fishes. Comparative Biochemistry & Physiology, 29(3), 1247–

500

1251. 25

ACCEPTED MANUSCRIPT 501

Woo N Y S, Chiu S F, 1997. Metabolic and osmoregulatory responses of the sea bass

502

Lates calcarifer to nitrite exposure. Environmental Toxicology and Water

503

Quality , 12: 257- 264

504

Xian J A, Wang A L, Hao X M, Miao Y T, Li B, Ye C X, Liao S A, 2011. In vitro

505

toxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using

506

flow cytometric analysis. Comparative Biochemistry & Physiology Toxicology

507

& Pharmacology Cbp, 156(2): 75-79

508

Yan, W., Zhou, Y., Yang, J., Li, S., Hu, D., Wang, J., Chen, J., Li, G., 2012.

509

Waterborne exposure to microcystin-LR alters thyroid hormone levels and gene

510

transcription in the hypothalamic-pituitary-thyroid axis in zebrafish larvae.

511

Chemosphere 87, 1301-1307.

512

Yeasmin, S., Hossain, A. A., Yeasmin, T., & Amin, M. R., 2016. Study of serum ft3, ft4 and tsh levels in pregnant women. Medicine today , 27(2): 1-4

513 514

Yu, L., Deng, J., Shi, X., Liu, C., Yu, K., Zhou, B., 2010. Exposure to DE-71 alters

515

thyroid hormone levels and gene transcription in the hypothalamic-pituitary-

516

thyroid axis of zebrafish larvae. Aquatic Toxicology, 97(3), 226-233.

517

Zhang, X., Tian, H., Wang, W., Ru, S. 2013. Exposure to monocrotophos pesticide

518

causes disruption of the hypothalamic-pituitary-thyroid axis in adult male

519

goldfish

520

193(4):158-166.

521

26

(Carassius

auratus).

General

&

Comparative

Endocrinology,

ACCEPTED MANUSCRIPT 3.2 Serum TSH level

Figure 1. Serum thyroid-stimulating hormone (TSH) concentration of grass carp, exposed to nitrite (0 mg/L, 0.5 mg/L, 1 mg/L, 4 mg/L, 16 mg/L). The values are expressed as mean±SD. Significant differences obtained by one-way analysis of variance followed by Duncan’s multiple comparison test are indicated from value at 0 h: *p<0.05.

3.3 Serum levels of thyroid hormones

(A)

(B)

ACCEPTED MANUSCRIPT

Figure 2.

(C)

(D)

(E)

(F)

Serum thyroxine (T4), free thyroxine (FT4) triiodothyronine (T3), free

triiodothyronine (FT3), 3,3,5ʹ-triiodothyronine (rT3) concentration and T3/T4 ratio of grass carp, exposed to nitrite (0 mg/L, 0.5 mg/L, 1 mg/L, 4 mg/L, 16 mg/L). The values are expressed as mean±SD. Significant differences obtained by one-way analysis of variance followed by Duncan’s multiple comparison test are indicated from value at 0 h: *p<0.05.

3.4 Deiodinase activity

ACCEPTED MANUSCRIPT (A)

(B)

(C)

Figure 3. The activity of type I iodothyronine deiodinase (ID1), type II iodothyronine deiodinase (ID2) and type III iodothyronine deiodinase (ID3) in grass carp, exposed to nitrite (0 mg/L, 0.5 mg/L, 1 mg/L, 4 mg/L, 16 mg/L).The values are expressed as mean±SD. Significant differences obtained by one-way analysis of variance followed by Duncan’s multiple comparison test are indicated from value at 0 h: *p<0.05.

3.5 Histopathology

(A)

(B)

Figure 4. Histologic section of thyroid in grass carp, exposed with nitrite, A -histologic section of thyroid in 0 mg/L; B -- histologic section of thyroid in 16 mg/L after 96 hours of exposure (CO--colloid).

ACCEPTED MANUSCRIPT Highlight: 1. Acute nitrite exposure alters iodothyronine deiodinase activities of grass carp. 2. Acute nitrite exposure destroys homeostasis of thyroid hormones in grass carp. 3. High dose of nitrite leads to decline in collide size of thyroid follicles in fish.