Selenium Stimulates Cadmium Detoxification in Caenorhabditis

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Environmental Processes

Selenium Stimulates Cadmium Detoxification in Caenorhabditis elegans through Thiols-mediated Nanoparticles Formation and Secretion Ling-Li Li, Yin-Hua Cui, Li-Ya Lu, You-Lin Liu, Chun-Jie Zhu, Li-Jiao Tian, Wen-Wei Li, Xing Zhang, Hao Cheng, Jingyuan Ma, Jian Chu, Zhong-Hua Tong, and Han-Qing Yu Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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Environmental Science & Technology

Selenium Stimulates Cadmium Detoxification in Caenorhabditis elegans through Thiols-mediated Nanoparticles Formation and Secretion

Ling-Li Li1,†, Yin-Hua Cui2,†, Li-Ya Lu1, You-Lin Liu1, Chun-Jie Zhu1, Li-Jiao Tian1, Wen-Wei Li1,*, Xing Zhang1, Hao Cheng1, Jing-Yuan Ma3, Jian Chu1, Zhong-Hua Tong1, Han-Qing Yu1,* 1CAS

Key Laboratory of Urban Pollutant Conversion, Department of Applied

Chemistry, University of Science and Technology of China, Hefei 230026, China 2School

of Life Sciences, University of Science and Technology of China, Hefei 230026, China

3Shanghai

Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China

1

ACS Paragon Plus Environment


1

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ABSTRACT

2 3

Antagonism between heavy metal and selenium (Se) could significantly affect their

4

biotoxicity,

5

microbial-mediated antagonistic processes as well as the formed products. In this

6

work, we examined the cadmium (Cd)-Se interactions and their fates in

7

Caenorhabditis elegans through in-vivo and in-vitro analysis and elucidated the

8

machinery of Se-stimulated Cd detoxification. Although the Se introduction induced

9

up to 3-fold higher bioaccumulation of Cd in C. elegans than the Cd-only group, the

10

nematode viability remained at a similar level to the Cd-only group. The relatively

11

lower level of reactive oxygen species in Se & Cd group confirms a significantly

12

enhanced Cd detoxification by Se. The Cd-Se interaction, mediated by multiple thiols

13

including glutathione and phytochelatin, resulted in the formation of less toxic

14

cadmium selenide (CdSe)/ cadmium sulfide (CdS) nanoparticles. The CdSe/CdS

15

nanoparticles were mainly distributed in the pharynx and intestine of nematode, and

16

continuously excreted from the body, which also benefited the C. elegans survival.

17

Our findings shed new light on the microbial-mediated Cd-Se interactions and may

18

facilitate an improved understanding and control of Cd biotoxicity in complicated

19

co-exposure environments.

but

little

is

known

about

the

mechanisms

2


underlying

such

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20

INTRODUCTION

21 22

Heavy metal contamination continues to be a global issue of environmental and health

23

concerns.1, 2 As a highly toxic and bio-accumulative heavy metal, cadmium (Cd) has

24

been widely found in water and soil environments.3-5 Thus, a full recognition and

25

proper control of Cd ecotoxicity is highly desired, which necessitate a deep

26

understanding on the bioaccumulation and biotransformation behaviors of Cd in the

27

environment as well as its interactions with environmental factors.6 Antagonism

28

between Cd and selenium (Se) is one of such interactions that lower Cd toxicity

29

through formation of Cd-Se complex.6, 7 Given the environmental ubiquity of both Cd

30

and Se and the widespread implementation of Se supplement in agriculture, livestock,

31

and healthcare sectors as health-protection strategy, the Cd-Se antagonism might

32

substantially affecting the Cd biogeochemical cycling and its ecotoxicity.

33

The phenomenon of Se-stimulated Cd detoxification have been demonstrated

34

previously and several interaction mechanisms have been suggested, such as

35

promoting synthesis of selenoproteins to ameliorate Cd-induced oxidative damage8

36

and a direct binding between Se with Cd ions.9 However, solid evidences are still

37

lacking because the Cd-Se complex has been studied by in-vitro analysis only, which

38

may not reveal the real reaction processes in vivo due to possible sample destruction

39

or loss during the extraction process. In addition, biomolecule mechanisms of the

40

Cd-Se interactions remain elusive.

3



41

Here, we shed light on the Cd-Se interactions in living organisms through

42

combining in-vivo and in-vitro characterization. Caenorhabditis elelgans (C. elegans)

43

was selected as a model organism to study the Cd-Se interactions for several reasons:

44

(1) its transparent body enables in-vivo visualization of the formed fluorophores; (2) it

45

is ubiquitous in the environment and has been widely used for environmental

46

toxicology study; and (3) it is of biomedical significance due to the well-deciphered,

47

highly homologues genes to human beings.10,

48

transformation and distribution of Cd and Se in C. elegans. The transformation and

49

distribution of the ingested Cd were tracked in-vivo by taking advantage of the

50

photoluminescence of formed cadmium selenide/ cadmium sulfide (CdSe/CdS)

51

nanoparticles. The transformation and distribution of Cd and Se in vivo were tracked

52

by synchrotron X-ray analysis and fluorescence microscopy. The extracted

53

nanoparticles were characterized by laser confocal Raman microspectroscopy, high

54

resolution transmission electron microscopy (HRTEM), inductively coupled

55

plasma-atomic emission spectroscopy (ICP-AES) and Energy Dispersive X-Ray

56

Spectroscopy (EDX). To elucidate the underlying Cd detoxification mechanisms, the

57

dynamics of Cd and Se contents, reactive oxygen species (ROS), thiols groups and

58

corresponding mRNA expression levels, and their correlation with the nanoparticles

59

formation and Cd detoxification processes were also examined.

11

60 61

MATERIALS AND METHODS

62 4


We examined the uptake,

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63

Culturing Procedures of C. elegans under Co-Exposure to Se and Cd. C. elegans

64

(Bristol strain N2, wild type) were obtained from Caenorhabditis Genetics Center

65

(Minneapolis, USA). Prior to the exposure, C. elegans was grown on nematode

66

growth medium in petri dishes at 20 °C. Escherichia coli OP50 was used as the food

67

for C. elegans. Age-synchronized nematodes were prepared by treating the gravid

68

hermaphrodites with hypochlorite following a previous method.12 The L4 larvae were

69

harvested from 90 mm-sized petri plates and washed three times with K medium (i.e.,

70

52 mM NaCl and 32 mM KCl) to remove the excess bacterial cells.

71

The obtained nematode larvae were transferred to a 1000 mL sterilized

72

Erlenmeyer flask that contained 200 mL S-complete medium. To prevent starvation or

73

over-feeding of the nematodes,13, 14 concentrated OP50 solution was added into the

74

culture medium, at a final optical density (OD600) of 0.2~0.25, once every two days.

75

The flasks were placed into an orbital shaker (180 rpm) and incubated for three days

76

at 20 °C in dark. Then, Na2SeO3 was added into the medium to a final concentration

77

of 200 mM and further incubated for 24 hours. Subsequently, 125 μM CdCl2 was

78

spiked into the medium twice at a time interval of 48 hours: the time points of the first

79

and secondary Cd dosage were recorded as 0 h and 48 h, respectively. After further

80

incubation for 144 h, C. elegans were harvested from the medium for products

81

characterization.

82

Determination of C. elegans Survival Fraction and Intracellular ROS Level.

83

During the culturing process, samples were collected to count the numbers of live and

84

dead nematodes using dissecting microscopy (SZX2-TR30 ， Olympus Co., Japan). 5



ROS

levels

were

measured

using

a

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85

Intracellular

fluorescent

probe

of

86

2,7-dichlorofluorescencin-diacetate (H2DCF-DA, molecular probes, Invitrogen).15

87

Specifically, the collected nematodes were washed three times with 10 mM phosphate

88

buffered saline (PBS), and transferred into 1 mL PBS with ice bath for 10 min

89

sonication. The suspension was centrifuged at 6000 rpm and 4 ℃ for 3 min. Aliquots

90

of 100 μL supernatant were added into a 96-well microtiter plate, where each well

91

was preloaded with 10 mM PBS and 50 μM H2DCF-DA. The 96-well microtiter plate

92

was incubated at 22 ℃ for 20 min before fluorescence analysis. The fluorescence was

93

measured by a microplate reader (BioTek, Winooski, USA) at excitation wavelength

94

of 485 nm and emission wavelength of 525 nm.

95

ICP-AES Analysis of the Se and Cd Contents in C. elegans. C. elegans were

96

sampled during the culturing process at given time intervals. The collected samples

97

were cleaned by flotation on a cold 30 % (w/v) sucrose cushion to separate nematodes

98

from bacteria and new generation of nematode larvae. The as-obtained nematodes

99

were washed five times with deionized water and put into a mixture of concentrated

100

HNO3 and HClO4 (v/v = 5:1) for digestion. The Se and Cd contents in the solution

101

were then quantified by ICP-AES (AA800, Perkin Elmer Co., USA) following

102

standard methods.

103

In-vivo Characterization of C. elegans. Synchrotron X-ray (SRX) analysis. The

104

same batches of nematodes samples after flotation treatment were used for SRX

105

analysis. Prior to the analysis, the samples were washed ten times with 30 mM

106

Tris-HCl (pH 7.4), preserved in glutaraldehyde and dehydrated using graded ethanol, 6


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107

and then fixed with liquid nitrogen on metal-free polyimide film and freeze-dried with

108

vacuum freeze dryer (FreeZone 2.5, Labconco Co., USA). The Se element

109

distribution in C. elegans dry samples was determined in the BL15U1 beamline

110

station of Shanghai Synchrotron Radiation Facility, China. The beamline was

111

equipped with a Si/Rh-coated Kirkpatrick-Baez mirror-pair to focus the beam to a 3

112

μm × 5 μm region on the sample. A monochromatic synchrotron-based X-ray with

113

12.8 keV photon energy was used as the excitation beam. C. elegans samples were

114

mounted on X-Z translation stages and the sample platform was moved along the X

115

and Z directions at 5 μm increment for each step. For the elemental mapping, the

116

fluorescence intensities of different elements (Se, K, Ca, Fe, Zn) and Compton

117

scattering at each point were collected by a Silicon Drift Detector for up to 3 s. The

118

effects of the synchrotron radiation beam flux variation on the signal intensity were

119

corrected by normalizing the fluorescence intensity to the incident X-ray intensity.

120

Compton scattering peak was used as an internal standard to compensate the

121

differences in sample thickness and density.16

122

Fluorescence Microscopy. To track the fluorophore formation in vivo and avoid

123

interference by fluorescent substances in the culture, the collected nematodes were

124

washed three times with 30 mM Tris-HCl (pH 7.4). The clean nematode samples were

125

then observed by fluorescence microscopy using an epifluorescence microscope

126

(BX51, Olympus Co., Japan) with U-MWU2 (330-385/400/420 nm) excitation filter.

127

In-situ Raman Spectroscopy. To more accurately identify the chemical structure

128

of the fluorophores formed in C. elegans, in-situ Raman spectrum of the nematodes at 7



Page 8 of 37

129

the fluorophores position was obtained using a Micro-Raman system (Horiba Jobin

130

Yvon Co., France). The excitation wavelength was set at 532 nm without additional

131

irradiations to quench the background fluorescence of cellular matrix.

132

Electron Microscopy and EDX Analysis of Nanoparticles in Disrupted

133

Nematodes. The nematodes harvested after 144-h co-exposure were washed with

134

Tris-HCl solution, and sonicated for 3 min in ice bath using a ultrasonic cell disruptor

135

(Ningbo Scientz Biotechnology Co., China) at 120 W power. Such a sonication

136

treatment allowed a moderate disruption of the nematode body so that the internal

137

synthesized materials, with its pristine structure remained, were exposed to favor

138

in-situ morphological observation. The disrupted nematodes were collected and

139

placed on a copper grid with a carbon amorphous film and dried under ambient

140

conditions. The morphology and the chemical compositions of the samples were

141

characterized

142

JEM-ARM200F, JEOL Co., Japan) fitted with an energy dispersive X-ray analyzer

143

(X-Max 80, Oxford Instruments Co., UK).

using

a

scanning

transmission

electron

microscope

(STEM,

144

Isolation and Purification of the Fluorescent Nanoparticles. The intact

145

nematodes cells as obtained above were crushed using a FastPrep TM-24 (MP

146

Biomedicals Inc., USA) with glass beads (acid-washed, 215-300 μm, pre-cooled on

147

ice) at 6.5 m/s for 30 seconds, followed by 1-min ice-cooling. This step was repeated

148

once to ensure sufficient cell crushing so as to release the intracellular substances. The

149

crushed samples were sonicated for 10 min in ice bath. The suspension was

150

centrifuged at 6000 rpm for 10 min to precipitate out the fragments of nematodes. The 8


Page 9 of 37


151

supernatant was treated with 0.5 % SDS for 10 min at 20 °C. The resulting solution

152

was subjected to further purification treatment or directly used for SDS-PAGE

153

analysis.

154

A part of the SDS-treated solution (200 μL) was treated by SDS-PAGE to

155

separate biogenic nanoparticles from the proteins in the cell matrix. Specifically, the

156

solution was mixed with a loading buffer that contained 2 % (w/v) SDS, 5 % (v/v)

157

mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8, 10 % (v/v) glycerol. The mixture was

158

loaded onto a 12 % SDS-PAGE gel and electrophoresed at 100 V for 90 min. The

159

fluorescent images of the gel were taken using a ScanArray GX scanner with UV

160

irradiation at 365 nm (Bio-Rad Inc., USA). A piece of gel at the fluorescent

161

electrophoretic band area was cut to analyze the Se and Cd contents by ICP-AES.

162

The rest of the SDS-treated solution was further centrifuged at 11000 rpm for 20

163

min to remove sediments and filtered through 0.22-µm Millex-GP Filter Unit. The

164

nanoparticles-containing filtrate was concentrated and washed with ultrapure water

165

using tubular ultrafiltration membrane (MWCO-10000, Merck Millipore Co., USA) to

166

remove the residual chemicals. A fraction of the resulting solution with concentrated

167

nanoparticles was freeze-dried for Raman analysis.

168

To further purify the nanoparticles, the concentrated solution with nanoparticles

169

was treated with 50 μg/mL proteinase K at 37 °C for 10 min, followed by

170

centrifugation at 11000 rpm for 30 min. The supernatant was then filtered through

171

0.22-µm Millex-GP Filter Unit, concentrated and washed with ultrapure water as

172

mentioned above. Finally, the obtained samples were subjected to ICP-AES analysis 9



173

and HRTEM, (JEOL Co., Japan) observation.

174

Characterization of the Purified Nanoparticles. Laser confocal Raman

175

microspectroscopy. Raman spectra of the purified samples were obtained using a

176

Micro-Raman system (JY-LABRAM-HR, Horiba Jobin Yvon Co., France). The

177

excitation wavelength was 532 nm and the laser spot size at sample was ~1 μm with

178

×100 objective (NA=0.9). Before analysis, the laser beam was applied for 1-2 minutes

179

in advance to quench the strong background fluorescence of cellular species as

180

possible, so that more clear Raman peak of the synthesized nanoparticles could be

181

obtained. Nematodes obtained under other cultivation conditions, including: Se-only,

182

Cd-only, and the negative control with neither Na2SeO3 nor CdCl2, were also

183

characterized for comparison. In addition, the E. coli OP50-only solution (‘no C.

184

elegans’ group) were used to examine the impacts of the bacteria in the medium on

185

the nanoparticles formation. Another medium without nematodes nor E. coli OP50

186

was used as the abiotic control.

187

HRTEM. A drop of the nanoparticles-containing solution was dripped onto a

188

copper grid with an ultrathin carbon amorphous film (Beijing Xinxing Braim

189

Technology Co., China). After drying under ambient conditions, the HRTEM images

190

and selected area electron diffraction patterns were taken using STEM. Based on the

191

STEM images, the sizes of the nanoparticle were estimated from more than 100

192

particles for statistical accuracy.

193

Quantification of Protein Thiols. The reduced and total thiols in the nematodes

194

were determined using high-performance liquid chromatography (HPLC). Sodium 10


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195

borohydride was used as the reducing agent and 4,4ˊ-dithiodipyridine (4-DPS) as the

196

thiol reagent. The standard curve was determined using a known amount of cysteine.17

197

Prior to the HPLC measurement, nematodes were homogenized in 30 mM Tris-HCl

198

(pH 7.4) for 10 min under ultrasonication as described above.

199

To quantify the reduced thiols concentration, the homogenized nematodes sample

200

was incubated in a solution (pH 4.5) containing 0.36 mM 4-DPS, 0.2 mM

201

ethylenediamine-tetraacetic acid (EDTA), 0.1 M citrate and 6 M urea. After

202

incubation at 20 oC for 30 min, 0.2 M HCl was added to terminate the reaction. The

203

mixture was centrifuged at 13000 rpm for 3 min. The supernatant was analyzed using

204

a HPLC (1260, Agilent Inc., USA) equipped with an Agilent Symmetry C18 (No.

205

WAT045905, 150×4.6 mm, 5 μm) column. An aliquot of 20-μL sample was

206

automatically injected into the column, and 4-TP was eluted isocratically at a flow

207

rate of 0.8 mL/min in 50 mM potassium acetate at pH 4.0. The absorbance at 324 nm

208

was monitored continuously, and the peak areas were integrated using the

209

accompanying software.

210

To determine the total thiols concentration, a fraction of the homogenized

211

nematodes sample (10 μL) was transferred into 140 μL solution (pH 8.3) containing

212

8.0 M urea and 0.5 M Tris-HCl, followed by the addition of 20 μL of freshly-prepared

213

30 % (w/v) alkaline sodium borohydride (dissolved in 1.0 M NaOH). After 30-min

214

reaction in a shaker at 50 oC with occasional shaking, the mixture was added with 10

215

μL 99 % hexanol as an antifoam agent. In the meantime, 76 μL of 6.0 M HCl (final

216

concentration of 1.8 M) was added to eliminate the excess sodium borohydride. After 11



217

continuous shaking for 2 min, the solution pH was adjusted to 4.5 by adding 94 μL of

218

1.5 M sodium citrate buffer (pH 4.5). Then the reduced sample with sodium

219

borohydride (350 μL) was further transferred into 650 μL solution (pH 4.5) containing

220

0.55 mM 4-DPS, 0.2 mM EDTA, 0.1 M citrate and 6 M urea. Finally, the sample was

221

subjected to the HPLC analysis as described above.

222

In addition to sulfhydryl quantification, the total protein content of each sample

223

was determined by BCA Protein Assay Kit (Beyotime Biotechnology Co., China) and

224

was used as a common denominator of the individual samples.

225

RNA and real-time qRT-PCR analysis. Several major genes involved in

226

expressing the thiol-proteins of MTs (mtl-1 and mtl-218), GSH (gcs-1 and gsr-119) and

227

PCs (pcs-120) were determined by real-time qRT-PCR. Total RNA from nematodes

228

was isolated using TRIzol according to manufacturer’s instructions (TAKARA Co.,

229

Japan), followed by purification with Recombinant DNase I. Complementary DNAs

230

were synthesized using PrimeScript RT reagent Kit Perfect Real Time (TAKARA

231

Co., Japan). The qRT-PCR analysis was performed with a Step One™ Real-Time

232

PCR System (Life Technologies Inc., USA) using SYBR Premix Ex Taq™ II

233

(TAKARA Co., Japan). The primers used for qRT-PCR are listed in Supplementary

234

Table 1. The mRNA levels were normalized to the expression of ACT-1, which

235

encodes the actin isoform. The fold change was normalized to that of unexposed

236

(without Cd and Se) C. elegans samples. All experiments were conducted in triplicate.

237

Statistical Analysis. Using Origin 2018b, the results from at least 3 independent

238

groups were analyzed by Analysis of variance (ANOVA) to compare multiple 12


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239

conditions

after

normality

analysis

with

Kolmogorov-Smirnov

test

and

240

homoscedasticity analysis with Levene’s test. Tukey’s test was further performed if

241

the ANOVA was significant. Student’s t-test was applied to compare two conditions

242

(*p < 0.05, **p < 0.01, *** p < 0.001).

243 244

RESULTS AND DISCUSSION

245 246

Se and Cd Accumulation and detoxification in C. elegans. We first examined how

247

the Cd uptake by C. elegans was affected by Se co-exposure. The Cd content in the

248

Cd-only group remained relatively stable over time in spite of the raised Cd exposure

249

concentration at 48 h (Figure 1a). In contrast, the Cd accumulation was drastically

250

stimulated by the presence of Se, reaching 3-fold higher accumulation than the

251

Cd-only group after 108-h exposure (Figure 1a).21 Interestingly, the Se & Cd group

252

had similar survival percentages to the Cd-only group (p > 0.05, Figure 1b) despite of

253

its higher Cd accumulation, suggesting an improved Cd resistance of the nematodes

254

under co-exposure condition. Here, the survival percentages of both groups declined

255

in the later exposure stage, which should be mainly ascribed to the toxic effect of Cd

256

that accelerated the nematode death relative to the unexposed scenario since there was

257

no other growth stress (food supply was sufficient as indicated by the high vitality of

258

bacteria in Figure S1).13, 14 To correlate the Cd resistance to its detoxification process,

259

we further determined the ROS level in the nematode body. ROS generation in living

260

organisms can be significantly triggered under toxic stress such as heavy metal 13



261

exposure,22 thus the ROS level could reflect the Cd toxicity in our study. Our results

262

showed a significantly lower ROS level in Se & Cd group than in the Cd-only group

263

(P < 0.05, Figure 1c), indicating an alleviated Cd toxicity by the presence of Se (i.e.,

264

Se-stimulated Cd detoxification). Nevertheless, the co-exposure also raised the Cd

265

accumulation, which compromised the detoxification effects and resulted in a similar

266

survival percentage as the Cd-only group (Figure 1b). The enhanced Cd detoxification

267

by Se was further validated by the fact that raising the Cd exposure by three times for

268

the Cd-only group (which was supposed to raise the intracellular Cd accumulation to

269

approaching that in the co-exposure group) significantly lowered the survival

270

percentage than the co-exposure group after 108 h (38±8 % vs. 78±8 %, p < 0.001)

271

(Figure S2).

272

Formation and Excretion of Fluorescent Nanoparticles. The process of

273

Se-stimulated Cd uptake coincided with the formation of yellow fluorophore in

274

pharynx and intestine in the Se & Cd group (Figure 2b). Yellow fluorophore was not

275

observed in all the controls (Figure S3), which showed only weak blue-green

276

fluorescence due to auto-fluorescence of C. elegans.23 To identify the formed yellow

277

fluorophore, we closely examined the disrupted nematode of the Se & Cd group at the

278

fracture position. The morphology of the nanoparticles in the disrupted nematode was

279

observed by dark-field STEM and the elemental composition was analyzed by EDX.

280

Our results confirmed the presence of a large number of Cd- and Se- containing

281

nanoparticles (Figure S4). To further reveal the chemical structure of nanoparticles,

282

we measured the in situ Raman spectrum of the yellow fluorophore formed in pharynx 14


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283

(the marked part in Figure 2b). A distinct Raman peak at 203 cm-1, matched well with

284

CdSe-like substances,24, 25 was observed (Figure 2c), indicating a possible formation

285

of fluorescent CdSe-containing nanoparticles. Consistent results were obtained by

286

in-vitro characterization of the extracted fluorophore (Figure S5). The gradually

287

intensified fluorescence and accumulation of nanoparticles in the Se & Cd group were

288

consistent with the increase body contents of Cd (Figure 1a) and Se (Figure S6)

289

during earlier exposure stage (from 0 to 108 h).

290

Notably, the body contents of both elements declined from 108 to 144 h in the Se

291

& Cd group, which are different from the individual element exposure scenario (Cd

292

content in Cd-only group remained relatively unchanged, shown Figure 1a; Se content

293

in Se-only group increased, shown in Figure S6). The μ-SRXRF map showed a

294

consistent trend of Se content decline at later stage in the Se & Cd group (Figure 2a).

295

The fluorescent microscopic images also showed a fading fluorescence in the

296

nematode at the intestine position in this stage (Figure 2b). The ROS level decreased

297

significantly along with the fluorescence decline (p < 0.05, Figure 1c). Altogether,

298

these results suggest a considerable excretion of the formed fluorescent CdSe

299

containing nanoparticles.26-28 The excreted nanoparticles in the culture medium was

300

validated by Raman spectroscopy, which showed a weak characteristic peak of

301

CdSe-like substances24, 25 (Figure S7). Such nanoparticles excretion would alleviate

302

the total Cd accumulation in the nematodes, and hence were beneficial for the survival

303

of C. elegans under toxic stress.

304

Characteristics of Nanoparticles. The CdSe-containing nanoparticles formation 15



305

was confirmed by a detailed analysis on the chemical composition and structure of the

306

extracted fluorophore, and the reported mild purification process did not unexpectedly

307

produce CdSe containing nanoparticles (Figure S8). To exclude the possible

308

interference by the cellular matrix, the nematode extracts was purified by proteinase K

309

digestion and further washed using tubular ultrafiltration membrane. A comparison of

310

the Cd contents in the purified nanoparticles and in the nematodes collected at 144 h

311

showed that no less than 27.69±9.64 % (n = 3, Table S1) of the accumulated Cd in C.

312

elegans were turned into Cd-containing nanoparticles (the possibly Cd loss during

313

nanoparticles purification was not accounted). In addition, small amount of CdSe

314

containing nanoparticles were also identified in the C. elegans -free culture medium,

315

attributed to the synthetic activity of E. coli OP50 in the medium. Nevertheless, these

316

bacteria-derived nanoparticles accounted for only 6.21±3.16% of the CdSe-containing

317

nanoparticles synthesized in the system (Figure S9).

318

The Raman spectra of the purified nanoparticles from C. elegans revealed two

319

distinct peaks solely in the Se & Cd group (Figure 3a) matching CdSe-like substances

320

(203 cm-1) and CdS (285 cm-1).24, 29, 30 The possibility of their formation induced by

321

laser heating effects can be ruled out because the Raman spectra of the Se and Cd

322

precursors and the biosynthesized samples both remained very stable during a long

323

time laser irradiation (Figure S10). The formation of spherical, crystalline

324

nanoparticles, with an average size of 4.03±0.50 nm (80 particles measured) was

325

clearly illustrated by the HRTEM images (Figure 3b). The resulting nanoparticles

326

possessed clear lattice fringes and diffuse rings with the lattice spacing fully matching 16


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327

the (101) and (002) planes of hexagonal CdSe (cadmoselite, JCPDS 08-0459)

328

(Figures 3c and 3d).

329

In addition to the Cd-Se bond, Cd-S signals were also identified. We enriched the

330

fluorescent nanoparticles in an electrophoretic gel (Figures S5b and S5c) and

331

examined the chemical components by ICP-AES. The result showed a higher content

332

of Cd over Se (Cd/Se ratio= ~1.3:1), consistent with the denser Cd distribution than

333

Se in the STEM observation (Figures S4c and S4d). The excess Cd was likely

334

coordinated with S in the thiol groups to form CdS, as shown by the EDX spectrum

335

(Figure S11).29, 31 The presence of Cd-S bond was verified by the Raman spectrum

336

(Figure 3a). According to the fitting result (Figure S12, baseline drift correction from

337

Figure 3a), the peak at 199.4 cm-1 belongs to CdSe alloy and the weak peak at 193.8

338

cm-1 belongs to surface optical (SO) phonon peak of CdSe, while the peak at 286.8

339

cm-1 belongs to CdS alloy and the peak at 269.2 cm-1 belongs to CdSSO.25 These

340

results suggest that the bio-synthesized nanoparticles mainly consisted of Cd/Se/S

341

alloy and certain amount of CdSe and CdS on the surface. It is likely that the CdS

342

content mainly serves as a structural component of a mixed CdSe1-xSx phase.32 Similar

343

products have also been obtained in other microorganisms such as yeast33,

344

genetically engineered E. coli.35

34

and

345

Mechanisms of CdSe/CdS Nanoparticles Formation and Cd Detoxification.

346

Previous studies suggested that RSH are critically involved in both SeO32- reduction

347

and Cd2+ sequestration.31, 36, 37 Disulfides (-S-S-) would be formed from RSH during

348

SeO32- reduction. Therefore, the contents of both RSH and the total thiol (TSH, the 17



349

sum of RSH and -S-S- groups) would change with the varied RSH generation/

350

consumption rate.38 We found that the contents of RSH and TSH in the Cd-only group

351

were relatively stable (Figure 4), whereas those in the Se & Cd group increased

352

drastically over time (P < 0.01), indicating an obviously stimulated synthesis of RSH

353

for SeO32- reduction, Cd detoxification and CdSe formation in the presence of Se.

354

To identify the involved RSH species and their specific roles in the above

355

processes, we examined the content changes of several thiol-capping peptides,

356

including glutathione (GSH), phytochelatin (PCs) and metallothioneins (MTs) during

357

the exposure. These thiol-capping peptides are known for their abilities in

358

metal-binding and elimination of oxidative species. They could be synthesized from

359

cysteine (Cys) under the functioning of different genes (Figure 5a).39

360

Monitoring the relative mRNA expression levels of the relevant genes showed

361

substantially different contents and dynamics of all the corresponding peptides among

362

the different test groups (Figures 5b-5f). Both the total GSH amount and ratio of

363

reduced GSH in the control and Cd-only groups remained relatively stable over time

364

(Figure S13). Considering the significantly increased expression of gcs-1 for GSH

365

production in the Cd-only group relative to the control (Figure 5e), the constant GSH

366

level should be mainly to due to synchronously increased GSH consumption for Cd

367

chelation40 that offset the GSH production. Compared with the Se-free groups, the

368

Se-exposed groups (including the Se-only and Se & Cd group) showed more

369

significant changes in GSH species over time. Specifically, the total GSH in the

370

Se-only group increased substantially at 144 h, which was consistent with elevated 18


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Page 19 of 37


371

expression of gcs-1 (Figure 5e). However, the ratio of reduced GSH decreased to near

372

zero at 144 h, which should be ascribed to rapid consumption of reduced GSH for

373

selenite reduction.36 In contrast, for the Se & Cd group, the total GSH and reduced

374

GSH ratio were both decreased significantly (P < 0.01, Figure S13) in spite of the

375

7-fold elevated expression of gcs-1 (Figure 5e), indicating that GSH was consumed

376

more rapidly than its production. Here, the GSH species were consumed for both Cd2+

377

chelation and selenite reduction, the latter was responsible for the decreased ratio of

378

reduced GSH (Figure S13b). Notably, the enhanced Cd detoxification by Se was

379

compromised by the increased Cd accumulation in the Se & Cd group at the latter

380

stage (Figure 1a), leaving it under a similar toxic stress as the Cd-only group and

381

hence similar expression levels of mtl-1 and mtl-2. The much remarkable increase in

382

the expression of relevant detoxification genes in the Cd-only and Se & Cd groups

383

than the Cd-free ones indicate that their expression were mainly triggered by the Cd

384

uptake.

385

The highest upregulation in pcs-1 and gsr-1 expression for the Se & Cd group

386

coincided with its highest Cd accumulation (nearly 3-fold higher than the Cd-only

387

group) (Figure 1a) and the formation of abundant nanoparticles. Such a correlation

388

could also be seen from the synchronous decline in the pcs-1 expression, Cd

389

accumulation and nanoparticles contents of the Se & Cd group from 108 h to 144 h

390

(Figures 1a and 5f). Altogether, these evidences strongly suggest an essential role of

391

the GSH and PCs in the Se-stimulated Cd detoxification and CdSe/CdS nanoparticles

392

formation. 19



393

With the above findings and literature about Se bioreduction,26, 41 we can propose

394

the following mechanisms of Se-Cd interactions and Cd detoxification in C. elegans

395

(Figure 6). Under co-exposure to Se and Cd, the C. elegans ingests inorganic Se ions

396

(Na2SeO3) and reduces it by RSH to form organoselenium compounds.41 Similar

397

detoxification effect can also be realized by selenomethionine as a more

398

environmentally-relevant form of Se through food-chain transfer (Figure S14). The

399

ingested Cd ions are immediately captured by the thiols-capping peptides (including

400

GSH, PCs and MTs) to form Cd-organic complex. The resulting organocadmium

401

(with abundant Se and S elements) further bind with such complexed and free-form

402

Cd, resulting in the formation of CdSe- and CdS-rich nanoparticles.31, 42 Here, the Se

403

and Cd co-exposure stimulates an over-expression of GSH and PCs to facilitate the

404

nanoparticles formation. Such an enhanced Cd sequestration in turn accelerates the

405

uptake of Cd ions from the medium, thereby giving rise to more nanoparticles

406

formation. The naturally-formed protein capping of the CdSe/CdS nanoparticles

407

confer them a good biocompatibility and much lower toxicity than free Cd ions.43

408

Such biogenic nanoparticles are gradually excreted from the nematode body, which

409

alleviates the total Cd accumulation and benefits the C. elegans survival.

410

Implications of This Work. We identified the CdSe/CdS nanoparticles

411

formation and efflux as an important unrecognized pathway of Se-stimulated Cd

412

detoxification in C. elegans under co-exposure condition. Mining and other industrial

413

activities have resulted in Se- and Cd-enriched surface soils worldwide at

414

concentrations up to several mg·kg−1.44, 45 In addition, Cd is a ubiquitous pollutant in 20


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415

water and soil environments.2 Se supplement has widely implemented in agriculture

416

and livestock sectors,46 which might also contribute to co-existence of Se and Cd in

417

the environment. Therefore, our findings may benefit a better understanding on the Cd

418

biotransformation behaviors and ecotoxicity in such co-exposure environments. This

419

study may also pay the way for more deep understanding on the bioaccumulation and

420

bioconversion behaviors of various heavy metals through food-web transfer in the

421

biosphere as well as a better control on their ecological risks. Lastly, we shed light on

422

the bio-molecule mechanisms of the Cd-Se interactions and nanoparticles formation

423

in. elegans. Since this organism possesses highly homologues genes and biological

424

features relevant to human diseases,10,

425

diagnosis and therapeutic implications for control of metal-related diseases. For

426

example, the Se-enriched functional products, which are widely used as supplements

427

or as food additives to strengthen immunity,46 might be another approach to increase

428

risks of heavy metals accumulation or alter their toxicity in human body, which

429

deserve special attention.

11

our findings might also provide valuable

430 431 432 433

AUTHOR INFORMATION †Author

Contributions:

These authors contributed equally to this study.

434 435

*Corresponding authors:

436

Prof. Wen-Wei Li, Fax: +86 551 63601592; E-mail: [email protected] 21



437

Prof. Han-Qing Yu, Fax: +86 551 63601592; E-mail: [email protected]

438 439 440

Notes: The authors declare no competing financial interest.

441 442

ACKNOWLEDGEMENTS

443

The authors thank the National Natural Science Foundation of China (51522812,

444

21590812, and 51821006) for the support of this study. The authors also thank the

445

Prof. Dai-Wen Pang at the Key Laboratory of Analytical Chemistry for Biology and

446

Medicine (MOE), Wuhan University, China for helping nanoparticle purification.

447 448

ASSOCIATED CONTENT

449

Supporting Information Available. Supporting materials and methods, Primer

450

Sequences for qRT-PCR (Table S1), Activities of bacteria at different time point

451

(Figure S1), Survival percentage of C. elegans treated by 3 times of concentrations of

452

Cd (Figure S2), Fluorescence microscopic images of C. elegans with different

453

treatments (Figure S3), Characterization of the biogenic fluorophores in vivo (Figure

454

S4), Properties and chemical contents of the C. elegans extracts (Figure S5), Dynamic

455

changes of element Se in C. elegans during the co-exposure process (Figure S6),

456

Raman spectra of the CdSe containing nanoparticles in culture media (Figure S7),

457

Influence of purification process to CdSe/CdS nanoparticles formation (Figure S8),

458

Raman spectra of two control groups under 532 nm laser excitation and the 22


Page 22 of 37

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459

HRTEM-EDX analysis of the purified nanoparticles in the no C. elegans group

460

(Figure S9), Influence of laser irradiation to Raman spectra detection of bioformed

461

nanoparticles (Figure S10), EDX spectra of the purified nanoparticles from the C.

462

elegans extract (Figure S11), Fitted Raman spectra of purified nanoparticles (Figure

463

S12), Variations of total GSH contents and the ratio of reduced GSH at different time

464

points (Figure S13), Influence of selenomethionine on Cd resistance (Figure S14).

465

The supporting information is available free of charge via the Internet at

466

http://pubs.acs.org/.

467 468

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Figure captions Figure 1 Dynamic changes of Cd elements in C. elegans during co-exposure with Se and the toxic effects. The accumulation of Cd (a) in the nematodes was quantified by ICP-AES. Survival percentage of C. elegans (b) and the relative intracellular level of ROS (c) were record at different time points. Error bars represent the standard error for triplicate samples. The exposure condition (P < 0.05) significantly influenced the intracellular level of ROS. Figure 2 Formation and excretion of the biogenic fluorophores during co-exposure process. In situ μ-SRXRF maps of element Se (a) and light micrographs (inset) of C. elegans in at different time point. The color bar from blue to yellow indicates a raising concentration of Se element. Fluorescence images of C. elegans and the cell extracts (insert) at different cultivation stages (b). The red-shift of color for the extract is likely due to a gradual aggregation of the synthesized nanoparticles. In situ Raman spectrum (c) under 532 nm laser excitation of the pharynx (the marked part in panel b) at 144 h. Figure 3 Characteristics of the purified CdSe/CdS nanoparticles. Raman spectrum under 532 nm laser excitation (a), TEM (b), HRTEM images and selected area electron diffraction pattern (c), and lattice fringe measurement of the purified nanoparticles (d). Control refers to the group without Cd and Se addition. Figure 4 Role of thiol-containing moieties in Cd-Se interactions. Variations of RSH (a) and TSH (b) contents in C. elegans under different Se and Cd exposure schemes. Error bars represent the standard error for triplicate samples. Figure 5 The mRNA expressions of major thiol-capping peptides involved in Cd-Se interactions. Metabolic interconversions of the key thiol-capping peptides (a) and expression of the related genes in C. elegans during the cultivation (b-f). Error bars represent the standard error for triplicate samples. The symbols indicate significance: *p < 0.05, **p < 0.01, *** p < 0.001. Figure 6 Mechanisms of Se-stimulated Cd uptake, nanoparticles formation and secretion in C. elegans. The red bold arrows indicate enhanced pathways under co-exposure. PCS short for PC synthase encoded by pcs-1.

30


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Figure 1

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Figure 2

32


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Figure 3

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Figure 4

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Figure 5

35



Figure 6

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Table of Contents (TOC) Art

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Selenium Stimulates Cadmium Detoxification in Caenorhabditis

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