Zn stimulates the phospholipids (PL) biosynthesis via the pathways of

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Environmental Processes

Zn stimulates the phospholipids (PL) biosynthesis via the pathways of oxidative and endoplasmic reticulum (ER) stress in the intestine of freshwater teleost yellow catfish Kun Wu, Zhi Luo, Christer Hogstrand, Guanghui Chen, Chuan-Chuan Wei, and Dan-Dan Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02967 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Environmental Science & Technology

1

Zn stimulates the phospholipids (PL) biosynthesis via the pathways of oxidative and

2

endoplasmic reticulum (ER) stress in the intestine of freshwater teleost yellow catfish

3 4

Kun Wua, Zhi Luoa,b*, Christer Hogstrandc, Guang-Hui Chena, Chuan-Chuan Weia,

5

Dan-Dan Lia

6 7

a

8

Fishery College, Huazhong Agricultural University, Wuhan 430070, China

9

b

Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture of P.R.C.,

Collaborative Innovation Center for Efficient and Health Production of Fisheries in

10

Hunan Province, Changde 415000, China

11

c

12

London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK

Diabetes and Nutritional Sciences Division, School of Medicine, King’s College

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

ACS Paragon Plus Environment


34

ABSTRACT:

35

The hypothesis of our study was that waterborne Zn exposure evoked phospholipids

36

(PL) biosynthesis to compensate for the loss of membrane integrity, and the pathways

37

of oxidative stress and ER stress mediated the Zn-evoked changes of PL biosynthesis.

38

Thus, we conducted RNA sequencing RNA-seq to analyse the differences in the

39

intestinal transcriptomes between the control and Zn-treated P. fulvidraco. 56-day Zn

40

exposure increased the intestinal Zn accumulation, and mRNA levels of 816 genes

41

were markedly up-regulated and 263 genes down-regulated. Many DEGs in pathways

42

of PL biosynthesis and protein processing in ER were identified. Their expression

43

profiles indicated that waterborne Zn exposure injured protein metabolism and

44

induced PL biosynthesis, caused oxidative stress and ER stress, and activated the

45

unfolded protein response. Then, using the primary enterocytes, we identified the

46

mechanism of oxidative and ER stress mediating Zn-induced PL biosynthesis, and

47

indicated that the activation of these pathways constituted adaptive mechanisms to

48

reduce Zn toxicity. Our study demonstrated that Zn exposure via the water increased

49

Zn accumulation and PL biosynthesis, and that oxidative stress and ER stress were

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interdependent and mediated the Zn-induced PL biosynthesis of the intestine in the

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freshwater teleost.

52 53 54 55 56 57 58 59 60 61 62 63 64


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GRAPHICAL ABSTRACT

66 67 68

1. INTRODUCTION

69

The pollution of aquatic ecosystems by metals is of concern due to their toxicity and

70

bioaccumulation. Among these metals, zinc (Zn) is an essential trace element for

71

many important biochemical processes in vertebrates including fish, but can be toxic

72

above the threshold concentration in the body. Among aquatic organisms, fish are

73

considered to be the ideal organism for pollution monitoring in aquatic ecosystems,1

74

and unique among the vertebrates because they have two routes of metal acquisition,

75

from the diet and from the water.2 In contrast to marine fish, freshwater fish do not

76

drink considerable volumes of water for osmotic homeostasis, and waterborne Zn is

77

absorbed mainly via the gill.3 The influence of waterborne Zn on gill has been well

78

studied due to its direct involvement in Zn toxicity in freshwater fish.4 The intestine is

79

also very sensitive to a wide range of stressors.5 In our pilot study, we found

80

incidentally that waterborne Zn exposure increased Zn accumulation in the intestine

81

of yellow catfish. We found this interesting and toxicologically relevant because

82

increased Zn accumulation in the intestine could potentially influence the

83

physiological functions of the intestine. However, whether and how waterborne Zn

84

had effects on physiological functions of the intestine are not exactly known in

85

freshwater teleost, and the underlying mechanisms of intestinal homeostatic

86

regulation are not well understood.

87

In fish, the failure to maintain Zn homeostasis results in the damage of cellular

88

structure and intracellular membranes, and impedes a wide range of physiological

89

processes. 6 Therefore, it is meaningful to emphasize that Zn exposure via water can

90

increase intestinal Zn accumulation and in turn initiate the change of intestinal

91

physiological functions. Phospholipids (PL) are the major lipid components of cellular



92

and intracellular membranes.7,8 Despite the importance of PL in maintaining integrity

93

and physiological state of membrane, no studies have addressed the effects of mineral

94

elements (including Zn) on PL metabolism. Since excess Zn in the water can damage

95

cellular structures, we hypothesize that waterborne Zn can affect PL biosynthesis in

96

the intestine.

97

The ER, an important organelle, is responsible for folding and assembly of

98

proteins, PL synthesis and Ca2+ homeostasis. The changes in the ER increase the

99

amounts of unfolded/misfolded proteins in the ER, evoking ER stress and increasing

100

the concentration of Ca ion in the cytoplasm.9 To recover the ER homeostasis, cells

101

activate the UPR, a pathway governed by three sensors of ER stress: PERK, IRE1 and

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ATF6 (their full names were shown in Abbreviation list in Supporting Information,

103

SI).8 Together, they evoke gene transcription to increase folding capacity and

104

down-regulate protein synthesis, and induce elimination of unfolded proteins through

105

ER associated degradation (ERAD).10 At present, increasing evidence indicates that

106

the UPR is involved in the regulation of lipid and PL biogenesis.11,12 However, the

107

molecular mechanism linking the UPR to PL biogenesis has not been elucidated. In

108

addition to ER stress, Zn also induces oxidative stress13,14 by accelerating the

109

generation of ROS.15 Moreover, generation of ROS during oxidative stress directly or

110

indirectly (or both) affects ER homeostasis and UPR activation.16,17 Therefore, we

111

reasoned that this ROS-based model would be very helpful in illuminating the

112

undefined mechanism linking ER stress to Zn-induced changes of PL metabolism.

113

Here, we hypothesize that Zn exposure via the water affected PL metabolism,

114

and that the pathways of oxidative stress and ER stress mediated the Zn-induced

115

alterations of PL metabolism. Therefore, using RNA-seq technology, the present

116

study ascertained the effects of waterborne Zn influencing physiological responses of

117

the intestine in Pelteobagrus fulvidraco. Using primary yellow catfish enterocytes, the

118

complex relationship between Zn and oxidative stress/UPR/PL metabolism was

119

investigated. Yellow catfish, an omnivorous freshwater teleost, is widely cultured in

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several Asian countries and used as experimental animals. We reveal an

121

unprecedented role for the ER stress and oxidative stress mediating Zn-induced

122

changes of PL biosynthesis.

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2. MATERIALS AND METHODS


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The study consisted of three experiments. All experiments followed the ethical

125

guidelines of Huazhong Agricultural University.

126

2.1. Expt. 1: Experimental procedures and sample analysis

127

2.1.1. Zn exposure and sampling

128

The experimental procedures for waterborne Zn were described in our parallel

129

study.9 Briefly, 144 healthy yellow catfish (mean body weight: 8.4 ± 0.4 g) were were

130

randomly transferred to 6 fiberglass tanks (300 L water, 24 fish per tank). They were

131

exposed to two nominal Zn concentrations: 0 (control, no extra Zn addition) and 7.69

132

μM (0.5 mg/L, 5% of the 96-h LC5018), respectively, with triplicate tanks for each

133

treatment. Zn was added in the form of ZnSO4·7H2O. Here, the Zn concentnration

134

(0.5 mg/L) was lower than those in some contaminated waters in China, and also

135

lower than these in water quality criteria II according to GB 3838-2002 of China (1.0

136

mg Zn/l, GB3838-2002). All fish were fed twice daily with diets (Zn content: 17.45

137

mg/kg diet). The actural Zn concentrations for two treatments were 0.007 ± 0.001 and

138

0.514 ± 0.070 mg/l, respectively.19 The experiment continued for 56 days, and the

139

entire intestine samples (including anterior, mid- and posterior intestine) were

140

collected for the determination of contents of triglyceride (TG) and metal elements,

141

enzymatic activity and mRNA expression.

142

2.1.2. Measurements of metal, TG content and enzymatic activities

143

The

intestine

Zn

content

was

measured

using

ICP-AES.19

Quality

144

assurance/quality control (QA/QC) procedures included analysis of three method

145

blanks (purified water), three certified reference tissues (DORM-2, LUTS-1 and

146

DOLT-2) and two randomly selected duplicate samples.

147

Activities of G6PD, FAS, 6PGD, ME and ICDH (their full names were listed in

148

SI) were analyzed based on the methods described in our previous study.19 The TG

149

content and activities of Cu, Zn-SOD were measured by using commercial kits

150

(Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).

151

2.2. Transcriptome analysis

152

2.2.1. RNA isolation, library preparation and sequencing

153

Three fish per tank were randomly selected for RNA isolation. Trizol reagent

154

(Invitrogen, CA, USA) was used for total RNA isolation. In total, three fish from each

155

tank and three replicates per treatment (six samples in total) were used for



156

transcriptomic sequencing. The construction of cDNA library and sequencing

157

(Illumina HiSeq 2000 platform, Illumina, CA, USA) were undertaken at the Beijing

158

Genome Institute (BGI, Beijing, China) according to the methods described

159

previously.19

160

2.2.2. De novo assembly and annotation

161

Before assembly, clean reads were obtained from raw data by filtering adaptor

162

sequences and low-quality sequences. They were then de novo assembled into

163

unigenes using Trinity.20 We annotated the transcripts over 200 bp using the BLASTx

164

alignment (e-value < 10-5) between unigenes and databases, including NR

165

(non-redundant protein sequence), non-redundant nucleotide (Nt), Swiss-Prot, KEGG

166

(Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous

167

Groups). Blast2GO was used to accomplish Gene Ontology (GO) annotation ,21 and

168

WEGO program for further gene classification .22

169

2.2.3. Differentially expressed genes (DEGs) and Q-PCR validation

170

After obtaining de novo transcriptomic data, the BGISEQ-500 platform (BGI,

171

Beijing, China) was used to quantify the expression levels of genes. 6 independent

172

cDNA libraries (three equivalent quantities of RNA samples from the same tank were

173

combined into one pool, and there were three replicate tanks and accordingly 3 cDNA

174

libraries per treatment) were constructed for RNA-Seq quantification analysis (all

175

reads have been submitted to the Sequence Read Archive at NCBI, Accession

176

Number: SRP116984). The gene expression levels were calculated using the

177

fragments per kilobase of transcript per million mapped reads (FPKM) method23 and

178

DEGs were screened using Noiseq method24 with a value of log2fold-change > 1 and a

179

diverge probability ≥ 0.8. All DEGs were mapped to terms in GO and the KEGG

180

database. In the present study, twelve candidate genes were randomly selected for

181

Q-PCR validation. 19 Primers are given in Table S1 (SI). A set of seven housekeeping

182

genes (β-actin, b2m, elfa, gapdh, rpl7, hprt and tbp) were selected from the

183

literature25 in order to test their transcription stability. The mRNA levels of β-actin

184

and hprt were the most stable, based on the analysis of geNorm software25

185

Accordingly, the gene expression levels were normalized to the geometric mean of the

186

β-actin and hprt. The 2-ΔΔCt method26 was used to calculate the fold changes in

187

relative expression to the control.

188

2.3. Expt. 2: in vitro studies


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2.3.1. Enterocytes culture and treatment

190

Enterocytes were isolated from yellow catfish, based on the published

191

methods27,28. Four pathway inhibitors were used, and they are TPEN (Zn2+ chelator,

192

Sigma, MO, USA), NAC (ROS inhibitor, Selleck, TX, USA), fatostatin (SREBP1

193

inhibitor, Selleck, TX, USA) and STF-083010 (IRE1 inhibitor, Selleck, TX, USA).

194

Ten groups were designed as follows: control, 40 μM Zn, 2 μM TPEN, 1mM NAC,

195

10 μM fatostatin, 50 μM STF-083010, 40 μM Zn + 2 μM TPEN, 40 μM Zn + 1 mM

196

NAC, 40 μM Zn + 10 μM fatostatin and 40 μM Zn + 50 μM STF-083010. The

197

concentrations of Zn and inhibitors were selected according to our pilot trials and to

198

other in vitro studies.9,29-31 Each treatment was performed in triplicates. The cells were

199

collected at 48 h for following analysis.

200

2.3.2. Cell viability, TG and PC content

201

Cell

viability

was

evaluated

by

3-(4,5-dimethylthiazol-2-yl)-2,5-

202

diphenyltetrazolium bromide (MTT) assay method.19 Intracellular TG content was

203

measured as described above. The amount of phosphatidylcholine (PC) was measured

204

using a PC assay kit (Sigma, MO, USA).

205

2.3.3. Zn2+ and Ca2+ measurements

206

Two cell permeant probes, Newport Green DCF (Ex/Em = 505nm/535nm) and

207

Fluo-4 (Ex/Em = 494nm/516nm) were used to measure intracellular Zn2+ and Ca2+

208

concentrations, respectively9,32 Fluorescence was detected and quantified using laser

209

scanning confocal microscopy (Leica, Wetzlar, German).

210

2.3.4. Flow cytometry for the determination of apoptosis and ROS

211

Apoptosis analysis was conducted with 5 μl Annexin V-FITC and 5μl propidium

212

iodide (PI), accompanied with flow cytometry (Beckman, CA, USA) with ModFit 3.0

213

LTTM software.

214

ROS production was measured with 10 μM 2, 7-dichlorofluorescin diacetate

215

(DCFH-DA).33 Fluorescence intensity was measured at the wavelengths of excitation

216

at 490 nm and emission wavelength at 525 nm, respectively.34 For fluorescence

217

intensity

218

(2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α] pyrazin-3-one), a specific

219

chemiluminescence probe for superoxide anion and singlet oxygen, the PBS-washed

220

cells were incubated with 8 μM MCLA for 20 min. Fluorescence intensity was

measurement


of

MCLA


221

measured at the wavelengths of excitation at 430 nm and emission wavelength at 546

222

nm, respectively.

223

2.3.5. Enzymatic activity, MDA content and Q-PCR

224

Activities of G6PD, FAS, 6PGD, ME and ICDH were analyzed following the

225

methods in our previous study.19 The malondialdehyde (MDA) content and activities

226

of total-SOD and Cu, Zn-SOD were measured by commercial kits (Nanjing Jian

227

Cheng Bioengineering Institute, Nanjing, China). Soluble protein content of the

228

homogenates was determined by the method of Bradford.35

229

Q-PCR for the in vitro experiment was performed based on the methods

230

mentioned above, andβ-actin and tbp were used as the control genes according to

231

geNorm software.

232

2.4. Expt. 3: Bioaccumulation of Zn in tissues

233

Considering that dietary Zn might have the potential influence on the Zn uptake,

234

we further performed an experiment to analyze the relative contribution of dietary Zn

235

and waterborne Zn. 180 yellow catfish (initial mean weight: 10.8 ± 0.3 g) were

236

randomly stocked in 12 tanks and there were 15 fish per tank. The yellow catfish were

237

exposed to four treatments for 8 weeks, three replicates for each treatment. There

238

were two waterborne Zn concentrations: 0 (no extra Zn addition in the water) and 7.69

239

μM (0.5 mg/L, waterborne Zn addition group) waterborne Zn. For each waterborne

240

Zn exposure, fish were fed 6.17 (dietary Zn deficiency group) or 17.45 mg/kg (dietary

241

Zn addition group) Zn, respectively. The group of dietary Zn deficiency, TPEN (Zn

242

chelator) was added at 0.01 g/kg to inhibit dietary Zn uptake. The feed composition

243

and nutrient analysis of diets were shown in Table S6 (SI). The fish were fed diets at a

244

ration of 4% of body weight twice a day. Fish were sampled on days 0, 14, 28, 42 and

245

56, respectively. Gills, intestine and plasma were sampled for the determination of Zn

246

content and mRNA expression of genes involved in Zn uptake and transport.

247

2.5. Statistical analysis

248

Statistical analysis was conducted with SPSS 19.0 software.19 Data were

249

analyzed with one-way ANOVA and Student’s t-test where appropriate. Significant

250

level was set at P < 0.05.

251

3. RESULTS AND DISCUSSION


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3.1. Waterborne Zn had direct effects on Zn accumulation and PL metabolism of

253

the intestine

254

In the freshwater fish, Zn is assimilated both from waterborne and dietary phases.

255

In the present study, Zn exposure via the water significantly increased Zn

256

accumulation (Fig. 1A). 1079 unigenes were identified as DEGs by RNA-seq

257

technology, including 816 up- and 263 down-regulated genes (Fig. S1, SI). The GO

258

(Fig. S2, SI) and KEGG (Fig. S3, SI) database were further used to perform the

259

pathway analysis (For detailed RNA-seq data and validation result, see SI, Table

260

S2-S5, Fig. S4). The results indicated that waterborne Zn exposure impacted the

261

physiological function of the intestine in many aspects and a series of genes and

262

pathways may be of importance for Zn-induced physiological responses. Similar to

263

the results in the in vivo study, intracellular Zn content of primary enterocytes were

264

up-regulated after Zn incubation, and the changes were partly reversed by TPEN (Fig.

265

S5, SI), which to some extent supports the argument. Overall, the present study

266

indicated that waterborne Zn disrupted the Zn balance in the intestine, which thereby

267

led to diverse physiological changes, similar to our study in yellow catfish

268

hepatocytes.33 The present study found an interesting contradiction: Zn increased

269

activities of enzymes involved in fatty acid (FA) synthesis (Fig. 1D) but reduced TG

270

content (Fig. 1B) in the intestine. Considering that FA is not only incorporated into

271

TG, but also for the synthesis of phosphatidic acid (PA), PL precursor, we

272

accordingly analyzed the DEGs involved in PL biosynthesis pathway. The present

273

study found that many genes, such as acc, fas, acsl, gpat, agpat, cds, ck, pap, ept1 and

274

cept1, involved in FA and PL biosynthesis, were up-regulated by waterborne Zn

275

exposure (Fig. 2). The activities of enzymes related to FA synthesis, such as 6pgd, me,

276

icdh and fas, were significantly increased by waterborne Zn exposure (Fig. 1D). These

277

observations indicated that waterborne Zn exposure induced PL biosynthesis.

278

Similarly, several studies pointed out that the synthesis of PL was regulated in

279

response to Zn status in yeast.36,37 Iwanyshyn et al.36 found that the activities of the

280

CDP-diacylglycerol pathway enzymes were positively associated with the

281

cytoplasmic Zn levels. GPAT and AGPAT were key enzymes for the initiation of

282

PL synthesis.7 PC is the main PL and the precursor to SM, PS and PE.12 Some key

283

enzymes: CK, CCT, CHPT1/CEPT1 and PEMT, played important roles in PC

284

biosynthesis.12,38-40 Considering that waterborne Zn increased tissue MDA content

285

(Fig. 1C) and PC is the main membrane PL in cells,12 it is plausible to speculate that



286

increased PC synthesis compensated for the loss of membrane integrity induced by Zn

287

exposure. Moreover, our study found that the expression of DGAT, a crucial enzyme

288

catalyzing formation of TG from DG, was markedly decreased. Thus, it is reasonable

289

to conclude that most of FA participated in the PL biosynthesis process, not in TG

290

biosynthesis. To verify whether increased Zn content directly affected these changes

291

in the intestine, Zn2+ chelator TPEN was used in the in vitro study. Our study

292

indicated that TPEN significantly attenuated the Zn-induced increase of expression of

293

accα, gpat, pap, cds, cct and ect (Fig. 3A), which in turn suppressed the Zn-induced

294

accumulation of PC content and the reduction of TG content (Fig. 3B), as observed

295

here.

296

3.2. Waterborne Zn evoked ER stress and UPR

297

Zn exposure influenced the protein metabolism and processing in ER (Fig. 4).

298

For instance, the expression levels of uggt and bip were markedly elevated. UGGT

299

and BiP are parts of the ER quality control system and preferentially recognizes the

300

unfolded/misfolded proteins.10 Thus, the increased expression of uggt and bip

301

probably reflected the increase of these unfolded/misfolded proteins in the ER. The

302

current study also showed that mRNA levels of perk, atf6 and ire1 were significantly

303

up-regulated (Fig. 4). It has been reported that under ER stress, aberrant proteins

304

rapidly accumulates and accordingly induces the UPR in eukaryotic cells.10 Hence,

305

these events suggested that waterborne Zn caused the UPR in the intestine of yellow

306

catfish. One of the adaptive mechanisms that recover normal physiological state of ER

307

is the upregulation of the folding capacity through induction of molecular

308

chaperones.8 Here we found that expressions of chaperones, such as bip and cnx, were

309

up-regulated, indicating the increased folding capacity in response to Zn exposure.

310

Moreover, Zn increased the expression of many genes from ERAD and ubiquitin

311

proteolysis, including the glycosylase ermani which catalyzes the important step in

312

ERAD.41 The enhanced ERAD was conducive to degrade and remove excess

313

unfolded/misfolded proteins in ER. It has been postulated that activation of UPR

314

increases ERAD efficiency through the IRE1-XBP1 cascade.8,10 As expected,

315

expression levels of ire1 and xbp1 were up-regulated here, which constituted a pivotal

316

link between Zn-induced UPR and ERAD. Similarly, Sriburi et al.42,43 also reported

317

that XBP-1 stimulated the expression of many genes in fibroblasts, including those

318

encoding the protein translational machinery.


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3.3. The activation of IRE1-XBP1 pathway was involved in Zn-induced PL

320

biosynthesis

321

Many enzymes involved in PL biosynthesis, such as GPAT and AGPAT, are

322

related with the ER.7,44 Studies also suggested that PL biosynthesis was induced in a

323

response to ER stress in the yeast.45 Thus, we further explored whether Zn-induced

324

occurrence of ER stress and UPR mediated PL biosynthesis. Here, we found that Zn

325

exposure increased the expression of genes in ER stress and UPR, such as perk, atf6,

326

ire1, bip and cnx (Fig. 3A), indicating their activation. TPEN markedly inhibited the

327

Zn-induced up-regulation of mRNA abundance of grp94, crt, ire1α and perk, further

328

indicating that Zn induced the occurrence of ER stress and UPR in vivo and in vitro.

329

Similarly, van der Sanden et al.46 suggested that the UPR might provide a means to

330

rapidly respond to an increased need for PL in Chinese hamster ovary cells. The

331

present study indicated that STF-083010 (IRE1 inhibitor) significantly suppressed the

332

Zn-induced up-regulation of mRNA expression of ire1α, xbp1, cds, cct and cept (Fig.

333

5A). However, STF-083010 had no suppressed effect on Zn-induced increase of Zn

334

content (Fig. S6, SI) and TG content (Fig. 5B). These findings suggested that CDS,

335

CCT and CEPT, the key regulatory factors involved in the PC biosynthesis, were

336

potential targets of IRE1-XBP1 ptahway, similar to the result by Sriburi et al.42 XBP1

337

is also closely linked with PC synthesis.10 Enforced expression of XBP1 is sufficient

338

to induce PC biosynthesis.42 Other studies indicated that the level and synthesis of

339

CCTa protein, was up-regulated in XBP-1(S)-transduced fibroblasts, and accordingly

340

elevated production of cytidine diphosphocholine (CDP-choline).43 The activation of

341

ER stress and UPR by waterborne Zn exposure has been reported in the yellow catfish

342

liver tissues.9 Other reports pointed out that during a physiological UPR, the

343

IRE1-XBP1 pathway regulated synthesis of PL according to cellular needs for ER

344

membrane components and its activation of IRE1/XBP1 branch of the UPR responded

345

to increased demand for PL in B cells and fibroblasts.12,42 Thus, the XBP1 was

346

activated, presumably to facilitate PL biosynthesis under the ER stress,42,43 which was

347

essential for optimal cellular functions in response to Zn stimulation. In addition,

348

unmitigated ER stress led to apoptosis.10,47,48 Similarly, the present study indicated

349

that inhibition of IRE1 pathway during Zn incubation had a negetive effect on

350

apoptosis (Fig. S7, SI) and cell viability (Fig. S8, SI). Thus, inhibition of IRE1

351

hindered the remedy process of UPR, which likely injured the cells and initiated the

352

apoptosis to eliminate unhealthy cells, as suggested by Sriburi et al.43. Therefore, all



353

these observations above indicated that the UPR, especially the IRE1-XBP1 pathway,

354

played a positive role in alleviating ER stress and maintaining normal functions of ER,

355

at least partly via facilitating PC biosynthesis.

356

3.4. SREBP1 mediated the regulation of Zn-induced PL biosynthesis

357

SREBP1 is a transcription factor that regulate multiple genes involved in FA

358

synthesis, including acc and fas.49 The present in vivo experiment found that Zn

359

increased the srebp1 mRNA levels. Thus, srebp1 seemed to play key roles in

360

Zn-induced alterations of PL metabolism.49-51 Using the SREBP1 inhibitor fatostatin

361

(F), we found that, compared to the control, the PC content in the Zn-treated group

362

was increased while the PC content in the Zn + F group was relatively stable (Fig. 6C),

363

indicating that srebp1 was involved in Zn-induced PC biosynthesis. Compared to the

364

control, activities of ME, G6PD and FAS, and mRNA levels of srebp1, accα, fas and

365

all the tested genes involved in PC synthesis and PC content were enhanced by Zn

366

incubation (Fig. 6A and 6B), further confirming the results of the transcriptome

367

analysis. ACCα, G6PD and FAS are the crucial enzymes during FA biosynthesis.52

368

SREBP1 inhibitor caused the reversion of Zn-induced increase of fas and accα mRNA

369

expression (Fig. 6A), and increase of G6PD and FAS activities (Fig. 6B), thereby

370

possibly impacting fatty acid synthesis. For genes involved in PL synthesis, compared

371

to single Zn treatment, fatostatin pre-treatment suppressed the changes of gpat, pap

372

and cds. Given the close correlation between the synthetic pathways of fatty acid and

373

PC, SREBP1 plays important roles in PC biosynthesis and SREBP1 activation may be

374

required for providing enough fatty acid to support PC biosynthesis.

375

3.5. The Zn-induced changes of physiological functions was partially attributable

376

to oxidative stress

377

To investigate how Zn exposure induced ER stress, we explored the change of 2+

378

Ca concentration in the enterocytes. However, we did not observe appreciable Ca2+

379

release (Fluo-4) after Zn addition (Fig. S9, SI). Considering that intracellular free

380

radicals, such as ROS, directly or indirectly affect ER homeostasis, and thereby lead

381

to UPR.16,17 We further determine the parameters involved in ROS generation. Here

382

we detected the increased fluorescence intensity of ROS (Fig. S10, SI), increased

383

activities of T-SOD and Cu, Zn-SOD, increased MDA content and increased MCLA

384

fluorescence intensity in the Zn-treated group (Fig. 7C), suggesting the accumulation

385

of intracellular ROS. Cu, Zn-SOD is one of the key free-radical scavengers.53 The


Page 12 of 28

Page 13 of 28


386

upregulated Cu, Zn-SOD activity probably implied the production of cellular

387

superoxide anion radicals in yellow catfish intestines in a response to Zn exposure.

388

Similarly, previous studies also demonstrated that Zn stimulated the production of

389

ROS.14,54 In contrast, Zn incubation seemed to have no effect on ROS production in

390

yellow catfish hepatocytes.33 ROS augments ER stress.55,56 Our study indicated that

391

increasing Zn led to the generation of ROS and oxidative stress, and preincubation

392

with NAC prevented the Zn-evoked ROS generation, indicating that oxidative stress

393

was responsible for the Zn toxicity, in agreement with other studies.54 As a result, the

394

increased expression levels of bip, crt, ire1α and perk in the Zn-treated group was

395

partly reversed in the Zn + N group (Fig. 7A), indicating that Zn-induced ER stress

396

was, at least in part, attributable to generation of ROS. Meantime, NAC attenuated the

397

Zn-induced effects on PC content (Fig. 7B) and expression of genes involved in PL

398

biosynthesis (Fig. 7A), suggesting the involvement of ROS generation in Zn-induced

399

PL biosynthesis. High concentration of ROS can inflict direct damage to proteins and

400

lipids.16,57 In this study, Zn treatment significantly increased intracellular MDA

401

content. MDA has been widely used as a biomarker for lipid peroxidation (LPO).57

402

Taken together, Zn-induced ROS accumulation probably caused the peroxidation,

403

which further impaired proteins, lipids and membrane integrity. Other research also

404

suggests that Zn caused lipid peroxidation and loss of membrane.58,59 The demand for

405

membrane PL components would increase correspondingly and thereby caused a

406

series of adaptive mechanisms, including the activation of IRE1-XBP1 and SREBP1

407

pathways which initiated a cascade of biochemical events that enhanced supplies of

408

PL and alleviated stress. Thus, the present study provided the first evidence that

409

Zn-induced ROS and UPR signaling mutally mediated Zn-induced PL biosynthesis in

410

enterocytes.

411

3.6. Zn accumulation in tissues was dominated by waterborne uptake

412

Fish can uptake Zn from diet and from gill-plasma route. A potential problem is

413

that dietary Zn might have the potential influence on the intestinal Zn accumulation,

414

which might further influence the results. In order to clarify the effect, we analyzed

415

the effects of dietary and waterborne Zn exposure on Zn deposition (Fig. S11, SI). No

416

surprisingly, waterborne Zn exposure resulted in significant Zn accumulation in the

417

gill on day 56, similar to the study in rainbow trout.60 However, dietary Zn had no

418

significant effects on gill Zn accumulation. Plasma Zn concentration ranged from 10

419

to 25 μg/ml and increased significantly with time in waterborne Zn groups. Other



420

studies also reported that waterborne Zn exposure increased Zn concentrations in the

421

plasma.61-63 Plasma Zn concentrations are maintained within narrow margins in the

422

fish61 and alterations may mean the occurrence of toxicity.63 Thus, it seemed that Zn

423

exposure via the water evoked the perturbations of plasma Zn and toxicity, whereas

424

the dietary exposure did not. Sappal et al.63 also found that chronic dietary Zn

425

exposure had no significant effect on plasma Zn content in rainbow trout. Similar to

426

the plasma, Zn exposure via the water increased the intestine Zn content much more

427

than dietary exposure did (Fig. S11 A, SI). Previous study in yellow catfish indicated

428

that only an extremely high dietary Zn concentration (213.84 mg/kg) had nearly the

429

same effect on tissues Zn accumulation as 0.35 mg/L waterborne Zn.64 In view of the

430

fact that dietary Zn content we used was much lower than 213.84 mg/kg and belongs

431

to the normal demand range for yellow catfish (dietary Zn requirement for yellow

432

catfish: 17.1-20.9 mg Zn/kg diet)65, our study found that the intestine Zn accumulation

433

came mainly from gill-plasma route. Furthermore, changes of dietary Zn

434

concentration did not result in changes of mRNA levels of znt1 and zip4 on day 56,

435

whereas waterborne Zn exposure increased znt1 expression and decreased zip4

436

expression compared to the group without Zn addition in the water (Fig. S12, SI),

437

indicating that waterborne Zn, but not dietary Zn, influenced mRNA expressions of

438

genes involved in Zn uptake and transport. Taken together, under the present

439

condition, our study strongly indicated that Zn accumulation and changes in the

440

physiological functions (PL biosynthesis) in the intestine were predominantly

441

influenced by waterborne Zn exposure. Our results are in agreement with the opinion

442

that Zn via the water is toxicologically more significant probably because Zn

443

permeates the gill more easily.63

444

In conclusion, for the first time, our data provide evidenced that Zn induced PL

445

biosynthesis by activating the pathways of oxidative stress and ER stress. Our study

446

offered new insights for elucidating the effects and mechanism of Zn toxicology,

447

which will be potential meaningful for incoming Zn risk assessments in aquatic

448

environment.

449

ASSOCIATED CONTENT

450

Supporting information

451

Table S1, primers used for Q-PCR analysis; Table S2, statistics of reads and assembly

452

quality; Table S3, summary of annotation and CDS results; Table S4, summary of


Page 14 of 28

Page 15 of 28


453

sequencing data for each sample; Table S5, alignment statistics of reads align to

454

reference gene; Table S6, feed formulation and proximate analysis of experimental

455

diets ; Figures S1, the correlation between the gene expression profiles of control and

456

Zn-exposed groups; Figures S2, GO functional classification of DEGs; Figures S3,

457

KEGG functional classification of DEGs; Figures S4, comparison of mRNA levels

458

between RNA-seq and Q-PCR results; Figures S5, effect of Zn and/or TPEN on

459

intracellular Zn fluorescence; Figures S6, Effect of Zn and/or inhibitors on

460

intracellular Zn fluorescence; Figures S7, effect of Zn and/or STF-083010 (IRE1

461

inhibitor) on apoptosis; Figure S8, Effect of Zn and/or inhibitors on cell viability;

462

Figures S9, intracellular Ca fluorescence after Zn stimulation; Figures S10, effect of

463

Zn and/or NAC on intracellular ROS generation; Figure S11, effect of waterborne and

464

dietary Zn on tissues Zn accumulation; Figure S12, Effect of waterborne and dietary

465

Zn on mRNA levels.

466

AUTHOR INFORMATION

467

Corresponding Author

468

*Phone:

469

[email protected]; [email protected] (Z. Luo).

470

Notes

471

The authors declare no competing financial interest.

472

ACKNOWLEDGMENTS

473

The authors thank the staff of Aquatic Animal Nutrition and Feed Laboratory of

474

Huazhong Agricultural University for their assistance with excellent sample analysis.

475

This work was supported by the National Natural Science Foundation of China (Grant

476

Nos. 31422056, 31372547).

477

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478

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479

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specific metal accumulation. Aquat. Toxicol. 2000, 50, 245-256.

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(62) Zia, S.; McDonald, D. G. Role of the gills and gill chloride cells in metal uptake

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Chen, Q. L. Different effect of dietborne and waterborne Zn exposure on lipid

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deposition and metabolism in juvenile yellow catfish Pelteobagrus fulvidraco. Aquat.

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Toxicol. 2015, 159, 90-98.

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zinc requirement of juvenile yellow catfish Pelteobagrus fulvidraco, and effects on

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hepatic intermediary metabolism and antioxidant responses. Aquaculture 2011, 319,

663 664

150-155.

665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681



682 683

Fig. 1 Effect of waterborne Zn exposure on Zn (A), TG contents (B), MDA content

684

(C) and enzymatic activities (D) in the intestine of P. fulvidraco on day 56. Values are

685

means ± SEM (n= 3 replicate tanks, six fish were sampled for each tank). Asterisks (*)

686

indicate significant differences between control and Zn-exposed group.

687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704


Page 22 of 28

Page 23 of 28


705 706

Fig. 2 Differentially expressed genes involved in phospholipid biosynthesis. The

707

up-regulated genes (Probability ≥ 0.8, and the absolute value of log2 (Ratio) ≥ 1) are

708

highlighted in red. The down-regulated genes (Probability ≥ 0.8, and the absolute

709

value of log2 (Ratio) ≥ 1) are highlighted in green. The non DEGs are highlighted in

710

grey. FA, fatty acid; PA, phosphatidic acid; DG, diacylglycerol; CL, cardiolipin; SM,

711

sphingomyelin;

712

phosphatidylethanolamine;

713

abbreviations of genes are shown in the Abbreviation List of the Supplementary file.

PC,

phosphatidylcholine; PS,

PI,

phosphatidylserine;

714 715 716 717 718 719 720 721 722


phosphatidylinositol;

PE,

Etn,

The

ethanolamine.


723 724

Fig. 3 Effects of Zn and/or TPEN (Zn2+ chelator) on mRNA levels (A), TG

725

accumulation (B) and PC content (C) in enterocytes of yellow catfish at 48 h. Values

726

are means ± SEM (n = 3). Bars that share different lowercase letters indicate

727

significant differences among groups (p < 0.05).

728 729 730 731 732


Page 24 of 28

Page 25 of 28


733 734

Fig. 4 Differentially expressed genes involved in protein processing in ER. The

735

up-regulated genes (Probability ≥ 0.8, and the absolute value of log2 (Ratio) ≥ 1) are

736

highlighted in red. The down-regulated genes (Probability ≥ 0.8, and the absolute

737

value of log2 (Ratio) ≥ 1) are highlighted in green. The non DEGs are highlighted in

738

grey. G3M9: (Glc)3 (GlcNAc)2 (Man)9 (Asn)1; G1M9: (Glc)1 (GlcNAc)2 (Man)9

739

(Asn)1; M9: (GlcNAc)2 (Man)9 (Asn)1; M8: (GlcNAc)2 (Man)8 (Asn)1; M5,

740

(GlcNAc)2 (Man)5 (Asn)1; Ub, ubiquitin. The abbreviations of genes are shown in

741

the Abbreviation List of the Supplementary file.

742 743 744 745 746 747 748 749 750 751 752 753 754 755 756



757 758

Fig. 5 Effects of Zn and/or STF-083010 (IRE1 inhibitor) on mRNA levels of genes

759

involved in UPR, lipogenesis and PL biosynthesis (A), TG accumulation and PC

760

content (B) in enterocytes of yellow catfish at 48 h. Values are means ± SEM (n = 3).

761

Bars that share different lowercase letters indicate significant differences among

762

groups (p < 0.05).


Page 26 of 28

Page 27 of 28


763 764

Fig. 6 Effects of Zn and/or fatostatin (SREBP1 inhibitor) on mRNA levels of genes

765

involved in lipogenesis, lipolysis and PL biosynthesis (A), enzymatic activities (B),

766

TG accumulation and PC content (C) in enterocytes of yellow catfish. Values are

767

means ± SEM (n = 3). Bars that share different lowercase letters indicate significant

768 769

differences among groups (p < 0.05).

770



771 772

Fig. 7 Effects of Zn and/or NAC (ROS inhibitor) on mRNA levels of genes involved

773

in UPR, ER stress markers, lipogenesis and PL biosynthesis (A), TG accumulation

774

and PC content (B), SOD activities, MDA content and MCLA fluorescent intensity (C)

775

in enterocytes of yellow catfish. Values are means ± SEM (n = 3). Bars that share

776

different lowercase letters indicate significant differences among groups (p < 0.05).

777


Page 28 of 28

Zn stimulates the phospholipids (PL) biosynthesis via the pathways of

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