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Lipid metabolic response to polystyrene particles in nematode Caenorhabditis elegans
Journal Pre-proof Lipid metabolic response to polystyrene particles in nematode Caenorhabditis elegans Yunhan Yang, Huimin Shao, Qiuli Wu, Dayong Wang PII:
S0269-7491(19)34610-X
DOI:
https://doi.org/10.1016/j.envpol.2019.113439
Reference:
ENPO 113439
To appear in:
Environmental Pollution
Received Date: 15 August 2019 Revised Date:
5 October 2019
Accepted Date: 18 October 2019
Please cite this article as: Yang, Y., Shao, H., Wu, Q., Wang, D., Lipid metabolic response to polystyrene particles in nematode Caenorhabditis elegans, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.113439. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphic abstract:
Lipid metabolic response mediated by lipid metabolic sensors MDT-15 and SBP-1 activated a protective function against nanopolystyrene toxicity in nematodes.
1
Lipid metabolic response to polystyrene particles in nematode Caenorhabditis elegans
2 3
Yunhan Yang, Huimin Shao, Qiuli Wu, Dayong Wang*
4 5
Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical
6
School, Southeast University, Nanjing 210009, China
7 8
*
9
E-mail address: [email protected] (D. Wang)
Corresponding author.
10 11
1
12
ABSTRACT
13 14
Nanoplastics can be used in various fields, such as personal care products.
Nevertheless, the
15
effect of nanoplastic exposure on metabolism and its association with stress response remain
16
largely unclear.
17
effect of nanopolystyrene exposure on lipid metabolism and its association with the response
18
to nanopolystyrene.
19
1 µg/L) induced severe lipid accumulation and increase in expressions of mdt-15 and sbp-1
20
encoding two lipid metabolic sensors. Meanwhile, we found that SBP-1 acted downstream
21
of intestinal MDT-15 during the control of response to nanopolystyrene.
22
transcriptional factor SBP-1 activated two downstream targets, fatty acyl CoA desaturase
23
FAT-6 and heat-shock protein HSP-4 (a marker of endoplasmic reticulum unfolded protein
24
response (ER UPR)) to regulate nanopolystyrene toxicity.
25
involved in the activation of ER-UPR in nanopolystyrene exposed nematodes. Moreover,
26
SBP-1 regulated the innate immune response by activating FAT-6 in nanopolystyrene
27
exposed nematodes.
28
nanopolystyrene toxicity was under the control of upstream signaling cascade
29
(PMK-1-SKN-1) in p38 MAPK signaling pathway.
30
molecular basis for potential protective function of lipid metabolic response in
31
nanopolystyrene exposed nematodes.
Using Caenorhabditis elegans as an animal model, we determined the
Exposure (from L1-larave to adult day-3) to 100 nm nanopolystyrene (≥
Intestinal
Both MDT-15 and SBP-1 were
In the intestine, function of MDT-15 and SBP-1 in regulating
Therefore, our data raised an important
32 33
Keywords: Nanopolystyrene, Lipid metabolism, Intestinal response, Caenorhabdis elegans
34 35
Capsule: Lipid metabolic response mediated by lipid metabolic sensors MDT-15 and SBP-1
36
activated a protective function for nematodes against toxicity of nanopolystyrene particles.
37 38
2
39
1. Introduction
40 41
In the recent years, the toxic effects of nanoplastics, plastic particles with the nano-size,
42
on organisms have been widely investigated (Della Torre et al., 2014; Ma et al., 2016; Rist et
43
al., 2017; Chen et al., 2017; Jin et al., 2018; Feng et al., 2018; Huang et al., 2019). The
44
microplastics in the environment can be potentially degraded into smaller nanoplastics
45
(Mattsson et al., 2015). Microplastic and nanoplastic particles have been detected from
46
different environments, such as marine or soil environment (Koelmans et al., 2019;
47
2018; Alimi et al., 2018; Su et al., 2016; Chae and An, 2018).
Ng et al.,
48
Nanopolystyrene is a widely examined nanoplastics, and can be used in several aspects,
49
especially in personal care products. Caenorhabditis elegans has been shown to be sensitive
50
to environmental toxicants, including nanoparticles (Qu et al., 2019c; Leung et al., 2008;
51
Wang, 2018; Hanna et al., 2018; Kim et al., 2019). For example, exposure (from L1-larvae
52
to adult day-1) to 100 nm nanopolystyrene at concentrations ≥ 10 µg/L could decrease
53
locomotion behaviors, such as head thrash and body bend, and 1 mg/L nanopolystyrene (100
54
nm) could further affect the development of D-type GABAergic motor neurons in nematodes
55
(Qu et al., 2019a).
56
nanopolystyrene at concentrations ≥ 10 µg/L caused both the damage on gonad development
57
and reduced the reproductive capacity (Qu et al., 2019d). Moreover, exposure to 1 µm
58
microplastics (5 mg/m2) for 2-day could reduce calcium levels and increase expression of
59
GST-4::GFP in the intestine, implying the induced intestinal damage in nematodes (Lei et al.
60
2018).
61
Exposure (from L1-larvae to adult day-1) of nematodes to 35 nm
In C. elegans, the molecular basis of toxicity induction of toxicants has been
62
well-described (Wang, 2019).
63
nanopolystyrene has been gradually determined in nematodes (Qu et al., 2019b; Shao et al.,
64
2019; Qu et al., 2019e; Qu et al., 2019f).
65
mitogen-activated protein kinase (MAPK) signaling was involved in the control of 100 nm
66
nanopolystyrene toxicity after exposure from L1-larvae to adult day-3 via activation of
67
endoplasmic reticulum unfolded protein response (ER UPR) (Qu et al., 2019b).
68
Based on this research background, the molecular response to
For example, in the intestine, p38
Recently, it was further found that exposure to 100-1000 µg/L polystyrene particles (5 3
69
µm) for six weeks might affect the fatty acid biosynthesis process in mice (Jin et al., 2019).
70
Additionally, exposure to 100-1000 µg/L polystyrene particles (5 µm) for 7-day could induce
71
the alteration in genes related to lipid metabolism in larval zebrafish (Wan, et al., 2019).
72
nematodes, lipid metabolism is a well-described biological process, which is under the control
73
of lipid metabolic sensors (transcriptional factors NHR-80, NHR-49, SBP-1, and MDT-15)
74
(Ashrafi, 2007; Watts, 2009). We hypothesized that a certain association between lipid
75
accumulation and stress response may exist in nanopolystyrene exposed organisms.
76
aims of this study were to investigate the effect of nanopolystyrene exposure on lipid
77
accumulation and the association of lipid accumulation with stress response. We here first
78
examined the lipid metabolic response to nanopolystyrene in nematodes. Moreover, we
79
determined the possible association of this lipid metabolic response with the response to
80
nanopolystyrene.
81
nanopolystyrene in nematodes. More importantly, our data highlight the protection function
82
of this lipid metabolic response in being against the nanopolystyrene toxicity in organisms.
In
The
Our results demonstrated the important lipid metabolic response to
83 84
2. Materials and methods
85 86
2.1. Properties of polystyrene particles
87 88
Nanopolystyrene was purchased from Janus New-Materials Co. (Nanjing, China).
89
Characterizations of nanopolystyrene in K medium were analyzed.
Assay of transmission
90
electron microscopy (TEM) in K medium shows morphology and size of nanopolystyrene
91
(Fig. 1A).
92
Instrument Ltd.) indicated that nanopolystyrene size in K medium was 102.8 ± 4.5 nm.
93
potential of nanopolystyrene in K medium was -9.698 ± 0.966 mV.
94
Raman spectrum of nanopolystyrene. The nanopolystyrene particles showed that the peaks
95
appeared at 1001.54 cm-1 (breathing vibration of benzene ring), at 1031.85 cm-1 (symmetric
96
extension vibration of carbon atoms in benzene ring), at 1201.44 cm−1 and 1450.47 cm−1
97
(asymmetric bending vibration of carbon atoms and hydrogen atoms), and at 1602.13 cm-1
98
(asymmetric stretching vibration of benzene ring carbon atoms) (Fig. 1B).
Analysis of dynamic light scattering (DLS) using Nano Zetasizer (Malvern
4
Zeta
Fig. 1B shows the
Working
99 100
solutions of nanopolystyrene (0.1, 1, 10, and 100 µg/L) were prepared by diluting 1 mg/mL stock solution using K medium.
101 102
2.2. Strain maintenance and exposure
103 104
Wild-type N2, mutant, and transgenic strains were all maintained on nematode growth
105
medium (NGM) plates fed with Escherichia coli OP50 as a food source (Brenner, 1974).
106
Gravid animals were lysed using bleaching mixture solution containing 2% HOCl and 0.45 M
107
NaOH to release eggs from the body in order to collect age-synchronous L1-larvae
108
nematodes.
109
In liquid solutions (1 mL volume) added with OP50, prolonged exposure (from
110
L1-larvae to adult day-3) to nanopolystyrene was carried out (Shao et al., 2019).
111
nanopolystyrene solutions, OP50 was added to the concentration of ~4 x 106 colony-forming
112
units (CFUs).
113
working solutions (0.1, 1, 10, and 100 µg/L) were refreshed daily.
114
particle solutions were sonicated for 30 min (40 kHz, 100W).
For the exposurem three replicates were performed.
In
Nanopolystyrene
Before the use, the
115 116
2.3. Sudan black staining
117 118
After the exposure, the adult nematodes were fixed with paraformaldehyde (1%). After
119
that, the animals were treated with 3 freeze–thaw cycles, followed by dehydration by an
120
ethanol series. The animals were stained with sudan black (50%) overnight (Wu et al., 2016).
121
Thirty animals were examined per treatment.
122 123
2.4. Quantitative real-time polymerase chain reaction (qRT-PCR)
124 125
Total nematode RNAs were extracted with the reagent of Trizol followed by
126
determination of its concentration and purity in a spectrophotometer. Using a cDNA
127
Synthesis kit (Bio-Rad Laboratories), the RNAs were reverse-transcribed. Gene expressions
128
were analyzed using ABI 7500 real-time PCR system with Evagreen (Biotium). Relative 5
129
expression ratio between the examined genes and tba-1 (a reference gene encoding
130
alpha-tubulin) was determined.
131
S1 provides the related information for used primers.
Biological reactions were carried out in triplicate. Table
132 133
2.5. Toxicity assessment
134 135
In this study, two endpoints, locomotion behavior and intestinal reactive oxygen species
136
(ROS), were selected to evaluate the nanpolystyrene toxicity (Qu et al., 2019g).
137
stress activation was reflected by the ROS production (Liu et al., 2019a). Functional state of
138
the motor neurons was reflected by locomotion behavior (Liu et al., 2019b).
139
Oxidative
After nanopolystyrene exposure, the nematodes were first washed with M9 buffer for
140
three times.
141
nematodes were treated with CM-H2DCFDA (1 µM) in darkness for 3-h (Kong et al., 2019).
142
The animals were then mounted on agar pad and examined for both excitation wavelength (at
143
488 nm) and emission filter (at 510 nm) using laser scanning confocal microscope. After the
144
CM-H2DCFDA labeling, the nematodes were further washed with M9 buffer for three times.
145
The fluorescent ROS signals in the intestine were analyzed.
146
normalization with autofluorescence was employed to reflect the activated ROS signals.
147
Fifty animals were analyzed per treatment.
148
To examine the ROS production, both control and nanopolystyrene exposed
Relative fluorescence unit after
To reflect the alteration in locomotion behavior, head thrash and body bend were
149
examined (Shi et al., 2019).
After nanopolystyrene exposure, the nematodes were first
150
washed with M9 buffer in order to remove OP50 from body surface and the remaining
151
particle solutions. The nematodes were randomly picked on the surface of an NGM plate
152
without OP50 feeding.
153
a stereomicroscopy.
154
change for bending direction at body mid-region of nematodes is defined as a head thrash.
155
Forty animals were analyzed per treatment.
After 1-min recovery, the locomotion behaviors were counted under
A change of posterior bulb direction is defined as a body bend.
156 157
2.6. DNA constructs and germline transformation
158 6
A
159
Promoter fragment of ges-1 (intestine-specific), unc-14 (neuron-specific), or mex-5
160
expressed in germline was amplified from genomic DNA by PCR.
After insertion of
161
promoter fragment into vector of pPD95_77, cDNA of mdt-15/R12B2.5a or sbp-1/
162
Y47D3B.7.1 was further subcloned into corresponding vector carrying unc-14, ges-1, or
163
mex-5 promoter. Germline transformation was carried out by coinjecting 10-40 µg/mL
164
testing DNA and 60 µg/mL marker DNA (Pdop-1::rfp) into gonad (Zhao et al., 2019a).
165
Table S2 provides the related information for the used primers.
166 167
2.7. RNA interference (RNAi)
168 169
L1-larvae were fed with E. coli HT115 expressing double-stranded RNA for gene(s)
170
(Zhao et al., 2019b). Once they developed into gravid, the animals were transferred to a
171
fresh RNAi plate in order to obtain the second generation.
172
was employed as a negative control. Transgenic strain VP303/kbIs7[nhx-2p::rde-1] was
173
used for intestine-specific RNAi knockdown of gene(s) (Espelt et al., 2005). qRT-PCR was
174
employed to confirm the RNAi efficiency (data not shown).
HT115 harboring L4440 vector
175 176
2.8. Statistical analysis
177 178
SPSS 12.0 software was employed to perform the statistical analysis.
One-way analysis
179
of variance (ANOVA) was used for analyzing differences between groups.
Two-way
180
ANOVA analysis was used for the examination of multiple factor comparison. Probability
181
level of 0.01 (**) was considered to be statistically significant.
182 183
3. Results
184 185
3.1. Effect of nanopolystyrene exposure on lipid accumulation
186 187 188
After prolonged exposure, 0.1 µg/L nanopolystyrene did not influence lipid accumulation (Fig. 1C).
Different from this, exposure to 1-100 µg/L nanopolystyrene caused the 7
189
noticeable increase in lipid accumulation in nematodes (Fig. 1C).
190
We next examined the effect of nanopolystyrene exposure on four lipid metabolic
191
sensors (NHR-80, NHR-49, SBP-1, and MDT-15). Nanopolystyrene (0.1-100 µg/L) did not
192
obviously influence nhr-49 and nhr-80 expressions (Fig. 1D).
193
(0.1-100 µg/L) increased sbp-1 and mdt-15 expressions (Fig. 1D).
In contrast, nanopolystyrene
194 195
3.2. Tissue-activity of MDT-15 and SBP-1 in regulating nanopolystyrene toxicity
196 197
Using locomotion behavior and intestinal ROS production as the endpoints, we observed
198
that loss-of-function mutation of mdt-15 caused more severe toxicity in nanopolystyrene
199
exposed nematodes compared with nanopolystyrene exposed wild-type nematodes (Fig. S1).
200
Considering the fact that MDT-15 is expressed in neurons, intestine, and reproductive
201
(Hunt-Newbury et al., 2007; Taubert et al., 2006), we next examined the tissue-activity of
202
MDT-15 in regulating nanopolystyrene toxicity.
203
germline did not obviously influence susceptibility of mdt-15 mutant to nanopolystyrene
204
toxicity (Fig. S1).
205
suppressed the toxicity induction in nanopolystyrene exposed mdt-15 mutant nematodes (Fig.
206
S1). Therefore, intestinal MDT-15 was involved in the control of nanopolystyrene toxicity
207
in nematodes.
208
Expression of mdt-15 in neurons or in
Different from this, we found that expression of mdt-15 in intestine
SBP-1 is exclusively expressed in the intestine (McKay et al., 2003).
Using VP303
209
strain, we found the more severe intestinal ROS production in nanopolystyrene exposed
210
nematodes with intestine-specific RNAi knockdown of sbp-1 compared with that in
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nanopolystyrene exposed VP303 strain (Fig. 2A), suggesting that intestine-specific RNAi
212
knockdown of sbp-1 caused the susceptibility to nanopolystyrene toxicity.
213 214
3.3. Genetic interaction between MDT-15 and SBP-1 in regulating the nanopolystyrene
215
toxicity
216 217 218
After nanopolystyrene exposure, mutation of mdt-15 could significantly decrease the sbp-1 expression (Fig. 2B).
Using intestinal ROS production as an endpoint, we further 8
219
observed that intestinal overexpression of MDT-15 caused a resistance to nanopolystyrene
220
toxicity (Fig. 2C).
221
resistance in nematodes overexpressing intestinal MDT-15 to nanopolystyrene toxicity in
222
inducing ROS production (Fig. 2C), which suggested that MDT-15 acted upstream of SBP-1
223
to regulate the nanopolystyrene toxicity.
Furthermore, RNAi knockdown of sbp-1 effectively suppressed this
224 225
3.4. Identification of downstream targets of intestinal SBP-1 in regulating nanopolystyrene
226
toxicity
227 228
Some potential targets for SBP-1 have been identified to be required for the possible
229
control of various biological processes (Ceron et al., 2007; Jo et al., 2009; Nomura et al., 2010;
230
Svensk et al., 2013; MacNeil et al., 2015; Shen et al., 2017; Pradhan et al., 2018; Kniazeva et
231
al., 2004), and some of them are expressed in the intestine (https://www.wormbase.org).
232
Among these intestinal targeted genes, exposure to nanopolystyrene (1 µg/L) significantly
233
increased the expressions of fat-7, fat-6, fat-4, fat-2, hsp-4, and sod-3, and decreased elo-5,
234
acs-2, and hpl-2 expressions in wild-type nematodes (Fig. 3A).
235
knockdown of sbp-1 only decreased fat-2, fat-6, fat-7, and hsp-4 expressions, and increase the
236
acs-2 expression in nanopolystyrene exposed nematodes (Fig. 3B).
237
knockdown of fat-6 or hsp-4 caused susceptibility to nanopolystyrene toxicity in inducing
238
intestinal ROS production (Fig. 3C). In contrast, intestine-specific RNAi knockdown of
239
fat-2, fat-7, or acs-2 did not influence the nanopolystyrene toxicity (Fig. 3C).
Meanwhile, intestinal RNAi
Intestine-specific RNAi
240
To confirm the role of FAT-6 and HSP-4 as the downstream targets of intestinal SBP-1 in
241
regulating nanopolystyrene toxicity, we generated the transgenic strain overexpressing
242
intestinal SBP-1.
243
nanopolystyrene toxicity in inducing ROS production (Fig. 3D).
244
knockdown of fat-6 or hsp-4 could effectively inhibit the resistance of transgenic strain
245
overexpressing intestinal SBP-1 to nanopolystyrene toxicity (Fig. 3D), which confirmed that
246
FAT-6 and HSP-4 acted downstream of intestinal SBP-1 to regulate nanopolystyrene toxicity.
Intestinal overexpression of MDT-15 caused the resistance to Moreover, RNAi
247 248
3.5. MDT-15 and SBP-1 were required for the activation of ER UPR in nanopolystyrene 9
249
exposed nematodes
250 251
Our previous study has demonstrated that exposure to nanopolystyrene could result in an
252
obvious activation of ER UPR as indicated by HSP-4::GFP expression (Qu et al., 2019b).
253
Using transgenic strain SJ4005/zcIs4[HSP-4::GFP] as a tool, we found that activation of
254
HSP-4::GFP induced by nanopolystyrene exposure was significantly inhibited by RNAi
255
knockdown of mdt-15 or sbp-1 (Fig. 4A).
256
not obviously influence this activation of HSP-4::GFP in nanopolystyrene exposed nematodes
257
(Fig. 4A). These observations suggested that the MDT-15 and SBP-1 but not the FAT-6 were
258
required for ER UPR activation in nanopolystyrene exposed nematodes.
Different from this, RNAi knockdown of fat-6 did
259 260
3.6. Genetic interaction between FAT-6 and HSP-4 in regulating nanopolystyrene toxicity
261 262
With intestinal ROS production as an endpoint, we found that intestine-specific RNAi
263
knockdown of hsp-4 or fat-6 induced more severe induction of ROS production in
264
nanopolystyrene exposed nematodes compared with nanopolystyrene exposed VP303
265
nematodes (Fig. 4B). Moreover, RNAi knockdown of both hsp-4 and fat-6 caused the more
266
severe toxicity compared with RNAi knockdown of hsp-4 or fat-6 alone in nanopolystyrene
267
exposed nematodes (Fig. 4B), which suggested that HSP-4 and FAT-6 functioned
268
synergistically in the intestine to regulate nanopolystyrene toxicity.
269 270
3.7. Identification of anti-microbial proteins CYP-35A3, CLEC-67, and LYS-7 as downstream
271
targets of intestinal FAT-6 in regulating nanopolystyrene toxicity
272 273
Antimicrobial proteins can act as potential downstream target of FAT-6 in controlling
274
stress response (Anderson et al., 2019), and some of them (irg-4, F49F1.7, cyp-35A3,
275
cyp-35B1, dsh-23, cdr-1, oac-6, clec-67, and lys-7) are expressed in the intestine
276
(https://www.wormbase.org).
277
nanopolystyrene (1 µg/L) exposure could increase cyp-35A3, clec-67, and lys-7 expressions
278
(Fig. 5A).
Among
these
9
intestinal
antimicrobial
genes,
Meanwhile, intestinal RNAi knockdown of fat-6 could significantly decrease the 10
279
expressions of cyp-35A3, clec-67, and lys-7 in nanopolystyrene exposed nematodes (Fig. 5B).
280
Furthermore, intestine-specific RNAi knockdown of cyp-35A3, clec-67, or lys-7 could induce
281
a susceptibility to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 5C),
282
suggesting that CYP-35A3, CLEC-67, and LYS-7 acted as the potential targets of intestinal
283
FAT-6 to regulate nanopolystyrene toxicity.
284 285
3.8. Genetic interaction of MDT-15/SBP-1 with PMK-1, SKN-1, or ATF-7 in regulating
286
nanopolystyrene toxicity
287 288
In nematodes, p38 MAPK signaling acted in the intestine to regulate nanopolystyrene
289
toxicity by activating ER UPR (Qu et al., 2019a). Intestinal overexpression of PMK-1,
290
ATF-7, or SKN-1 induced a resistance to nanopolystyrene toxicity (Fig. S2).
291
production as an endpoint, it was found that RNAi knockdown of mdt-15 or sbp-1 suppressed
292
the resistance of transgenic strain overexpressing intestinal PMK-1 (Fig. S2). Similarly,
293
RNAi knockdown of mdt-15 or sbp-1 also inhibited the resistance of transgenic strain
294
overexpressing intestinal SKN-1 (Fig. S2).
295
mdt-15 or sbp-1 did not influence the resistance of nematodes overexpressing intestinal ATF-7
296
(Fig. S2).
297
PMK-1-SKN-1 to regulate nanopolystyrene toxicity.
Using ROS
Different from these, RNAi knockdown of
Therefore, MDT-1 and SBP-1 acted downstream of signaling cascade of
298 299
4. Discussion
300 301
Recently, some reports have implied that polystyrene particles may potentially induce
302
some metabolic alterations, such as amino acid metabolism, bile acid metabolism, and energy
303
related metabolism (Jin et al., 2019; Kim et al., 2019).
304
demonstrated that the polystyrene microplastics could induce the changes of lipid
305
metabolism-related genes (Wan et al., 2019).
306
nanopolystyrene, we provided the direct evidence that exposure to 100 nm nanopolystyrene
307
(≥ 1 µg/L) caused severe lipid accumulation (Fig. 1C).
308
(1 µg/L) from L1-larvae to adult day-3 could cause the toxicity at various aspects in wild-type 11
A recent report has further
In this study, with the concern on the
Exposure to 100 nm nanopolystyrene
Considering that the 1 µg/L is a predicted environmental
309
nematodes (Shao et al., 2019).
310
concentration for 100 nm nanoplastics (Al-Sid-Cheikh et al., 2018; Lenz et al., 2016), our
311
data implies that long-term exposure to low-dose nanopolystyrene may potentially result in
312
the alteration in lipid metabolism.
313
Our recent studies have suggested that exposure to nanopolystyrene could at least cause
314
intestinal, neuronal, and reproductive toxicities in nematodes (Lei et al., 2018; Shao et al.,
315
2019; Qu et al., 2019a; Qu et al., 2019d).
316
intestine-specific activity of MDT-15, SBP-1, or FAT-6 in regulating nanopolystyrene
317
toxicity (Fig. S1, 2, and 3C).
318
induced a susceptibility to nanopolystyrene in inducing intestinal ROS production (Fig. S1A,
319
2A, and 3C). Meanwhile, we found that mutation of mdt-15 also induced a susceptibility to
320
nanopolystyrene in decreasing locomotion behavior (Fig. S1B). Therefore, on the one hand,
321
MDT-15, SBP-1, and FAT-6 could act in the intestine to regulating the induction of intestinal
322
toxicity of nanopolystyrene.
323
to regulating the induction of neuronal toxicity of nanopolystyrene.
324
In this study, our data suggested the
Mutation or RNAi knockdown of mdt-15, sbp1-1, or fat-6
On the other hand, at least MDT-15 could also act the intestine
In nematodes, NHR-80, NHR-49, SBP-1, and MDT-15 are four lipid metabolic sensors
325
(Ashrafi, 2007; Watts, 2009).
326
MDT-15 is a homolog of mammalian PGC-1.
327
hormone receptors, and NHR-49 is a peroxisome proliferator-activated receptor α (PPARα).
328
For the underlying mechanism of this increased lipid accumulation, we found that the
329
observed lipid accumulation was related to the increase in expressions of mdt-15 and sbp-1
330
genes (Fig. 1D).
331
SBP-1 (Ashrafi, 2007; Watts, 2009).
332
SBP-1 is a sterol response element binding protein (SREBP). NHR-80 and NHR-49 are two nuclear
During the control of fat metabolism, MDT-15 acts as a co-activator for
Transcriptional factors of MDT-15 and SBP-1 regulate lipid metabolism by activating
333
several downstream targets (Ashrafi, 2007; Watts, 2009).
334
accumulation, the targets of FATs proteins (such as FAT-7, FAT-6, and FAT-5) regulate
335
synthesis of monounsaturated fatty acid acylCoAs from saturated fatty acid acylCoAs, fatty
336
acid synthease FASN-1 regulates the process of fatty acid synthesis, and ACS-2 regulates
337
process of mitochondrial β-oxidation of fatty acid (Ashrafi, 2007; Watts, 2009).
338
Nevertheless, among the genes required for the lipid accumulation (Ashrafi, 2007; Watts, 12
During the control of lipid
339
2009), intestinal RNAi knockdown of sbp-1 only decreased expressions of genes (fat-7 and
340
fat-6) encoding fatty acyl CoA desaturases and increased the acs-2 encoding acyl CoA
341
synthase (Fig. 3B), suggesting that some other transcriptional factors may also be affected to
342
activate the other genes required for the lipid accumulation. Mutation of mdt-15, sbp-1, or
343
fat-6 caused the reduced lipid accumulation (Wu et al., 2010; Brock et al., 2007).
344
Meanwhile, nanopolystyrene (1 µg/L) decreased acs-2 expression, but did not affect the
345
fasn-1 expression (Fig. 3A), suggesting that long-term and low-dose exposure to
346
nanopolystyrene may only affect the process of fatty acid β-oxidation in nematodes.
347
these, exposure to nanopolystyrene may also affect some other aspects of lipid metabolism,
348
such as fatty acid elongation and desaturation, since nanopolystyrene (1 µg/L) also increased
349
fat-2 and fat-4 expressions, and decreased elo-5 expression (Fig. 3A).
350
Besides
Besides the lipid accumulation, we further found that MDT-15, SBP-1, and their target
351
FAT-6 were also required for the control nanopolystytyrene toxicity.
Mutation or RNAi
352
knockdown of mdt-15, sbp-1, or fat-6 caused a susceptibility to nanopolystyrene toxicity (Fig.
353
S1, 2, and 3C). Previous studies have suggested the role of FAT-6, SBP-1, and MDT-15 in
354
regulating the response to various stresses or toxicants (Goh et al., 2014; Lee et al., 2015;
355
Wang, 2019; Horikawa and Sakamoto, 2009).
356
MDT-15-SBP-1-FAT-6 was raised to be required for the control of nanopolystyrene toxicity
357
(Fig. 5D).
358
MDT-15-SBP-1-FAT-6-mediated lipid accumulation in being against the nanopolystyrene
359
toxicity (Fig. 5D).
In the intestine, a signaling cascade of
That is, our study further implies the potential protective role of
360
In the intestine, two downstream targets (FAT-6 and HSP-4) were identified for SBP-1
361
(Fig. 3), and acted in parallel pathways in regulating nanopolystyrene toxicity (Fig. 4B).
362
HSP-4 is heat-shock protein, a molecular marker of ER UPR (Bischof et al., 2008). This
363
implies that SBP-1 may activate two different downstream biological events.
364
FAT-6-mediated lipid metabolism and stress response. Another is HSP-4-mediated ER UPR
365
response, which may be not directly associated with the lipid metabolism.
366
cascade (PMK-1-SKN-1/ATF-7) was raised in p38 MAPK signaling pathway require for the
367
control of nanopolystyrene toxicity (Qu et al., 2019b).
368
the p38 MAPK, and SKN-1 and ATF-7 (two transcriptional factors) were targets of PMK-1. 13
One is
A signaling
In this signaling cascade, PMK-1 is
369
In the intestine, we identified a signaling cascade (SKN-1-PMK-1-MDT-15-SBP-1) involved
370
in the activation of ER UPR against the nanopolystyrene toxicity (Fig. 4A and S2).
371
Therefore, MDT-15-SBP-1 signaling cascade acted as an important link between ER UPR
372
response and p38 MAPK signaling in nanopolystyrene exposed nematodes.
373
In this study, three genes (cyp-35A3, clec-67, and lys-7) encoding anti-microbial proteins
374
were identified as downstream targeted genes of fat-6 in regulating nanopolystyrene toxicity
375
(Fig. 5A-5C).
376
expression mediated both the activation of innate immune response and the alteration in lipid
377
metabolism in nanopolystyrene exposed nematodes. FAT-6 has been shown to be involved
378
in regulating the innate immune response in nematodes (Anderson et al., 2019).
379
other hand, the intestinal MDT-15-SBP-1 could potentially activate two different responses
380
(innate immune response and ER UPR response) against the nanopolytyrene toxicity (Fig.
381
5D).
382
On the one hand, this observation suggested that the increase in FAT-6
On the
Together, in this study, we investigated the lipid metabolic response and its association
383
with toxicity regulation in nanopolystyrene exposed nematodes.
After nanopolystyrene
384
exposure, we observed the severe lipid accumulation in nematodes.
385
expression of both MDT-15 and SBP-1, two lipid metabolic sensors, were increased by
386
nanopolystyrene exposure.
387
intestine to induce the protective response to nanopolystyrene by activating HSP-4-mediated
388
ER UPR response and FAT-6-mediated innate immune response.
389
important molecular basis for the lipid metabolic response to nanopolystyrene exposure in
390
organisms.
391
response with the control of nanopolystyrene toxicity.
Meanwhile, the
The MDT-15-SBP-1 signaling cascade further acted in the
Our data provided the
Additionally, our results suggested the close association of lipid metabolic
392 393
Acknowledgements
394 395
This work was supported by the grant from National Natural Science Foundation of China
396
(21577016).
397 398
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Fig. 1. Effect of nanopolystyrene exposure on lipid accumulation.
(A) TEM image of
581
nanopolystyrene particles in K medium.
582
particles.
583
lipid accumulation. (D) Effect of nanopolystyrene on transcriptional expressions of sbp-1,
584
nhr-49, nhr-80, and mdt-15.
(B) Raman spectroscopy of nanopolystyrene
(C) Sudan blacking staining showing the effect of nanopolystyrene exposure on
Bars represent means ± SD.
585
21
**
P < 0.01 vs control.
586 587
Fig. 2. Genetic interaction between MDT-15 and SBP-1 in the intestine to regulate the
588
response to nanopolystyrene.
589
nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means ± SD.
590
**
591
expression in nanopolystyrene exposed nematodes. Bars represent means ± SD.
592
vs wild-type.
593
inducing intestinal ROS production in nematodes overexpressing intestinal MDT-15.
594
represent means ± SD.
595
concentration of nanopolystyrene was 1 µg/L.
(A) Effect of intestine-specific RNAi knockdown of sbp-1 on
P < 0.01 vs control (if not specially indicated). (B) Effect of mdt-15 mutation on sbp-1 **
P < 0.01
(C) Effect of RNAi knockdown of sbp-1 on nanopolystyrene toxicity in
**
P < 0.01 vs control (if not specially indicated).
596
22
Bars
Exposure
597 598
Fig. 3. Identification of downstream targets of intestinal SBP-1 in regulating the response to
599
nanopolystyrene. (A) Effect of nanopolystyrene exposure on gene expressions in wild-type
600
nematodes.
601
RNAi knockdown of sbp-1 on gene expressions in nanopolystyrene exposed nematodes.
602
Bars represent means ± SD.
603
knockdown of fat-2, fat-6, fat-7, acs-2, or hsp-4 on nanopolystyrene toxicity in inducing
604
intestinal ROS production.
605
specially indicated).
606
regulating the nanopolystyrene toxicity in inducing intestinal ROS production.
607
represent means ± SD.
608
concentration of nanopolystyrene was 1 µg/L.
609
Bars represent means ± SD.
**
P < 0.01 vs control.
**
P < 0.01 vs VP303.
Bars represent means ± SD.
(B) Effect of intestinal
(C) Effect of intestinal RNAi
**
P < 0.01 vs control (if not
(D) Genetic interaction between SBP-1 and FAT-6 or HSP-4 in
**
P < 0.01 vs control (if not specially indicated).
23
Bars
Exposure
610 611
Fig. 4. MDT-15 and SBP-1 were required for the activation of ER UPR in nanopolystyrene
612
exposed nematodes. (A) Effect of RNAi knockdown of mdt-15, sbp-1, or fat-6 on activation
613
of HSP-4::GFP in nanopolystyrene exposed nematodes. (B) Genetic interaction between
614
FAT-6 and HSP-4 in the intestine to regulate the nanopolystyrene toxicity in inducing
615
intestinal ROS production. Exposure concentration of nanopolystyrene was 1 µg/L.
616
represent means ± SD.
**
P < 0.01 vs control (if not specially indicated).
617 618
24
Bars
619 620
Fig. 5. Identification of several anti-microbial proteins as downstream targets of intestinal
621
FAT-6 in regulating the response to nanopolystyrene.
622
exposure on gene expressions in wild-type nematodes.
Exposure concentration of
623
nanopolystyrene was 1 µg/L.
**
624
Effect of intestinal RNAi knockdown of fat-6 on gene expressions in nanopolystyrene
625
exposed nematodes.
626
represent means ± SD.
627
cyp-35A3, clec-67, or lys-7 on nanopolystyrene toxicity in inducing intestinal ROS production.
628
Exposure concentration of nanopolystyrene was 1 µg/L.
629
0.01 vs control (if not specially indicated). (D) A diagram showing the molecular basis for
630
lipid metabolic response and its association with toxicity regulation in nanopolystyrene
(A) Effect of nanopolystyrene
Bars represent means ± SD.
P < 0.01 vs control. (B)
Exposure concentration of nanopolystyrene was 1 µg/L. **
P < 0.01 vs VP303.
25
Bars
(C) Effect of intestinal RNAi knockdown of
Bars represent means ± SD.
**
P<
631
exposed nematodes.
632
26
Highlights:
- Nanopolystyrene exposure caused lipid accumulation in nematodes. - Lipid metabolic sensors of MDT-15 and SBP-1 were increased by nanopolystyrene. - Lipid metabolic response was associated with the control of nanopolystyrene toxicity. - MDT-15-SBP-1 signaling induced both ER UPR and innate immune response.
Conflict of Interests:
The authors declare that they have no competing interests.
S0269-7491(19)34610-X
DOI:
https://doi.org/10.1016/j.envpol.2019.113439
Reference:
ENPO 113439
To appear in:
Environmental Pollution
Received Date: 15 August 2019 Revised Date:
5 October 2019
Accepted Date: 18 October 2019
Please cite this article as: Yang, Y., Shao, H., Wu, Q., Wang, D., Lipid metabolic response to polystyrene particles in nematode Caenorhabditis elegans, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.113439. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphic abstract:
Lipid metabolic response mediated by lipid metabolic sensors MDT-15 and SBP-1 activated a protective function against nanopolystyrene toxicity in nematodes.
1
Lipid metabolic response to polystyrene particles in nematode Caenorhabditis elegans
2 3
Yunhan Yang, Huimin Shao, Qiuli Wu, Dayong Wang*
4 5
Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical
6
School, Southeast University, Nanjing 210009, China
7 8
*
9
E-mail address: [email protected] (D. Wang)
Corresponding author.
10 11
1
12
ABSTRACT
13 14
Nanoplastics can be used in various fields, such as personal care products.
Nevertheless, the
15
effect of nanoplastic exposure on metabolism and its association with stress response remain
16
largely unclear.
17
effect of nanopolystyrene exposure on lipid metabolism and its association with the response
18
to nanopolystyrene.
19
1 µg/L) induced severe lipid accumulation and increase in expressions of mdt-15 and sbp-1
20
encoding two lipid metabolic sensors. Meanwhile, we found that SBP-1 acted downstream
21
of intestinal MDT-15 during the control of response to nanopolystyrene.
22
transcriptional factor SBP-1 activated two downstream targets, fatty acyl CoA desaturase
23
FAT-6 and heat-shock protein HSP-4 (a marker of endoplasmic reticulum unfolded protein
24
response (ER UPR)) to regulate nanopolystyrene toxicity.
25
involved in the activation of ER-UPR in nanopolystyrene exposed nematodes. Moreover,
26
SBP-1 regulated the innate immune response by activating FAT-6 in nanopolystyrene
27
exposed nematodes.
28
nanopolystyrene toxicity was under the control of upstream signaling cascade
29
(PMK-1-SKN-1) in p38 MAPK signaling pathway.
30
molecular basis for potential protective function of lipid metabolic response in
31
nanopolystyrene exposed nematodes.
Using Caenorhabditis elegans as an animal model, we determined the
Exposure (from L1-larave to adult day-3) to 100 nm nanopolystyrene (≥
Intestinal
Both MDT-15 and SBP-1 were
In the intestine, function of MDT-15 and SBP-1 in regulating
Therefore, our data raised an important
32 33
Keywords: Nanopolystyrene, Lipid metabolism, Intestinal response, Caenorhabdis elegans
34 35
Capsule: Lipid metabolic response mediated by lipid metabolic sensors MDT-15 and SBP-1
36
activated a protective function for nematodes against toxicity of nanopolystyrene particles.
37 38
2
39
1. Introduction
40 41
In the recent years, the toxic effects of nanoplastics, plastic particles with the nano-size,
42
on organisms have been widely investigated (Della Torre et al., 2014; Ma et al., 2016; Rist et
43
al., 2017; Chen et al., 2017; Jin et al., 2018; Feng et al., 2018; Huang et al., 2019). The
44
microplastics in the environment can be potentially degraded into smaller nanoplastics
45
(Mattsson et al., 2015). Microplastic and nanoplastic particles have been detected from
46
different environments, such as marine or soil environment (Koelmans et al., 2019;
47
2018; Alimi et al., 2018; Su et al., 2016; Chae and An, 2018).
Ng et al.,
48
Nanopolystyrene is a widely examined nanoplastics, and can be used in several aspects,
49
especially in personal care products. Caenorhabditis elegans has been shown to be sensitive
50
to environmental toxicants, including nanoparticles (Qu et al., 2019c; Leung et al., 2008;
51
Wang, 2018; Hanna et al., 2018; Kim et al., 2019). For example, exposure (from L1-larvae
52
to adult day-1) to 100 nm nanopolystyrene at concentrations ≥ 10 µg/L could decrease
53
locomotion behaviors, such as head thrash and body bend, and 1 mg/L nanopolystyrene (100
54
nm) could further affect the development of D-type GABAergic motor neurons in nematodes
55
(Qu et al., 2019a).
56
nanopolystyrene at concentrations ≥ 10 µg/L caused both the damage on gonad development
57
and reduced the reproductive capacity (Qu et al., 2019d). Moreover, exposure to 1 µm
58
microplastics (5 mg/m2) for 2-day could reduce calcium levels and increase expression of
59
GST-4::GFP in the intestine, implying the induced intestinal damage in nematodes (Lei et al.
60
2018).
61
Exposure (from L1-larvae to adult day-1) of nematodes to 35 nm
In C. elegans, the molecular basis of toxicity induction of toxicants has been
62
well-described (Wang, 2019).
63
nanopolystyrene has been gradually determined in nematodes (Qu et al., 2019b; Shao et al.,
64
2019; Qu et al., 2019e; Qu et al., 2019f).
65
mitogen-activated protein kinase (MAPK) signaling was involved in the control of 100 nm
66
nanopolystyrene toxicity after exposure from L1-larvae to adult day-3 via activation of
67
endoplasmic reticulum unfolded protein response (ER UPR) (Qu et al., 2019b).
68
Based on this research background, the molecular response to
For example, in the intestine, p38
Recently, it was further found that exposure to 100-1000 µg/L polystyrene particles (5 3
69
µm) for six weeks might affect the fatty acid biosynthesis process in mice (Jin et al., 2019).
70
Additionally, exposure to 100-1000 µg/L polystyrene particles (5 µm) for 7-day could induce
71
the alteration in genes related to lipid metabolism in larval zebrafish (Wan, et al., 2019).
72
nematodes, lipid metabolism is a well-described biological process, which is under the control
73
of lipid metabolic sensors (transcriptional factors NHR-80, NHR-49, SBP-1, and MDT-15)
74
(Ashrafi, 2007; Watts, 2009). We hypothesized that a certain association between lipid
75
accumulation and stress response may exist in nanopolystyrene exposed organisms.
76
aims of this study were to investigate the effect of nanopolystyrene exposure on lipid
77
accumulation and the association of lipid accumulation with stress response. We here first
78
examined the lipid metabolic response to nanopolystyrene in nematodes. Moreover, we
79
determined the possible association of this lipid metabolic response with the response to
80
nanopolystyrene.
81
nanopolystyrene in nematodes. More importantly, our data highlight the protection function
82
of this lipid metabolic response in being against the nanopolystyrene toxicity in organisms.
In
The
Our results demonstrated the important lipid metabolic response to
83 84
2. Materials and methods
85 86
2.1. Properties of polystyrene particles
87 88
Nanopolystyrene was purchased from Janus New-Materials Co. (Nanjing, China).
89
Characterizations of nanopolystyrene in K medium were analyzed.
Assay of transmission
90
electron microscopy (TEM) in K medium shows morphology and size of nanopolystyrene
91
(Fig. 1A).
92
Instrument Ltd.) indicated that nanopolystyrene size in K medium was 102.8 ± 4.5 nm.
93
potential of nanopolystyrene in K medium was -9.698 ± 0.966 mV.
94
Raman spectrum of nanopolystyrene. The nanopolystyrene particles showed that the peaks
95
appeared at 1001.54 cm-1 (breathing vibration of benzene ring), at 1031.85 cm-1 (symmetric
96
extension vibration of carbon atoms in benzene ring), at 1201.44 cm−1 and 1450.47 cm−1
97
(asymmetric bending vibration of carbon atoms and hydrogen atoms), and at 1602.13 cm-1
98
(asymmetric stretching vibration of benzene ring carbon atoms) (Fig. 1B).
Analysis of dynamic light scattering (DLS) using Nano Zetasizer (Malvern
4
Zeta
Fig. 1B shows the
Working
99 100
solutions of nanopolystyrene (0.1, 1, 10, and 100 µg/L) were prepared by diluting 1 mg/mL stock solution using K medium.
101 102
2.2. Strain maintenance and exposure
103 104
Wild-type N2, mutant, and transgenic strains were all maintained on nematode growth
105
medium (NGM) plates fed with Escherichia coli OP50 as a food source (Brenner, 1974).
106
Gravid animals were lysed using bleaching mixture solution containing 2% HOCl and 0.45 M
107
NaOH to release eggs from the body in order to collect age-synchronous L1-larvae
108
nematodes.
109
In liquid solutions (1 mL volume) added with OP50, prolonged exposure (from
110
L1-larvae to adult day-3) to nanopolystyrene was carried out (Shao et al., 2019).
111
nanopolystyrene solutions, OP50 was added to the concentration of ~4 x 106 colony-forming
112
units (CFUs).
113
working solutions (0.1, 1, 10, and 100 µg/L) were refreshed daily.
114
particle solutions were sonicated for 30 min (40 kHz, 100W).
For the exposurem three replicates were performed.
In
Nanopolystyrene
Before the use, the
115 116
2.3. Sudan black staining
117 118
After the exposure, the adult nematodes were fixed with paraformaldehyde (1%). After
119
that, the animals were treated with 3 freeze–thaw cycles, followed by dehydration by an
120
ethanol series. The animals were stained with sudan black (50%) overnight (Wu et al., 2016).
121
Thirty animals were examined per treatment.
122 123
2.4. Quantitative real-time polymerase chain reaction (qRT-PCR)
124 125
Total nematode RNAs were extracted with the reagent of Trizol followed by
126
determination of its concentration and purity in a spectrophotometer. Using a cDNA
127
Synthesis kit (Bio-Rad Laboratories), the RNAs were reverse-transcribed. Gene expressions
128
were analyzed using ABI 7500 real-time PCR system with Evagreen (Biotium). Relative 5
129
expression ratio between the examined genes and tba-1 (a reference gene encoding
130
alpha-tubulin) was determined.
131
S1 provides the related information for used primers.
Biological reactions were carried out in triplicate. Table
132 133
2.5. Toxicity assessment
134 135
In this study, two endpoints, locomotion behavior and intestinal reactive oxygen species
136
(ROS), were selected to evaluate the nanpolystyrene toxicity (Qu et al., 2019g).
137
stress activation was reflected by the ROS production (Liu et al., 2019a). Functional state of
138
the motor neurons was reflected by locomotion behavior (Liu et al., 2019b).
139
Oxidative
After nanopolystyrene exposure, the nematodes were first washed with M9 buffer for
140
three times.
141
nematodes were treated with CM-H2DCFDA (1 µM) in darkness for 3-h (Kong et al., 2019).
142
The animals were then mounted on agar pad and examined for both excitation wavelength (at
143
488 nm) and emission filter (at 510 nm) using laser scanning confocal microscope. After the
144
CM-H2DCFDA labeling, the nematodes were further washed with M9 buffer for three times.
145
The fluorescent ROS signals in the intestine were analyzed.
146
normalization with autofluorescence was employed to reflect the activated ROS signals.
147
Fifty animals were analyzed per treatment.
148
To examine the ROS production, both control and nanopolystyrene exposed
Relative fluorescence unit after
To reflect the alteration in locomotion behavior, head thrash and body bend were
149
examined (Shi et al., 2019).
After nanopolystyrene exposure, the nematodes were first
150
washed with M9 buffer in order to remove OP50 from body surface and the remaining
151
particle solutions. The nematodes were randomly picked on the surface of an NGM plate
152
without OP50 feeding.
153
a stereomicroscopy.
154
change for bending direction at body mid-region of nematodes is defined as a head thrash.
155
Forty animals were analyzed per treatment.
After 1-min recovery, the locomotion behaviors were counted under
A change of posterior bulb direction is defined as a body bend.
156 157
2.6. DNA constructs and germline transformation
158 6
A
159
Promoter fragment of ges-1 (intestine-specific), unc-14 (neuron-specific), or mex-5
160
expressed in germline was amplified from genomic DNA by PCR.
After insertion of
161
promoter fragment into vector of pPD95_77, cDNA of mdt-15/R12B2.5a or sbp-1/
162
Y47D3B.7.1 was further subcloned into corresponding vector carrying unc-14, ges-1, or
163
mex-5 promoter. Germline transformation was carried out by coinjecting 10-40 µg/mL
164
testing DNA and 60 µg/mL marker DNA (Pdop-1::rfp) into gonad (Zhao et al., 2019a).
165
Table S2 provides the related information for the used primers.
166 167
2.7. RNA interference (RNAi)
168 169
L1-larvae were fed with E. coli HT115 expressing double-stranded RNA for gene(s)
170
(Zhao et al., 2019b). Once they developed into gravid, the animals were transferred to a
171
fresh RNAi plate in order to obtain the second generation.
172
was employed as a negative control. Transgenic strain VP303/kbIs7[nhx-2p::rde-1] was
173
used for intestine-specific RNAi knockdown of gene(s) (Espelt et al., 2005). qRT-PCR was
174
employed to confirm the RNAi efficiency (data not shown).
HT115 harboring L4440 vector
175 176
2.8. Statistical analysis
177 178
SPSS 12.0 software was employed to perform the statistical analysis.
One-way analysis
179
of variance (ANOVA) was used for analyzing differences between groups.
Two-way
180
ANOVA analysis was used for the examination of multiple factor comparison. Probability
181
level of 0.01 (**) was considered to be statistically significant.
182 183
3. Results
184 185
3.1. Effect of nanopolystyrene exposure on lipid accumulation
186 187 188
After prolonged exposure, 0.1 µg/L nanopolystyrene did not influence lipid accumulation (Fig. 1C).
Different from this, exposure to 1-100 µg/L nanopolystyrene caused the 7
189
noticeable increase in lipid accumulation in nematodes (Fig. 1C).
190
We next examined the effect of nanopolystyrene exposure on four lipid metabolic
191
sensors (NHR-80, NHR-49, SBP-1, and MDT-15). Nanopolystyrene (0.1-100 µg/L) did not
192
obviously influence nhr-49 and nhr-80 expressions (Fig. 1D).
193
(0.1-100 µg/L) increased sbp-1 and mdt-15 expressions (Fig. 1D).
In contrast, nanopolystyrene
194 195
3.2. Tissue-activity of MDT-15 and SBP-1 in regulating nanopolystyrene toxicity
196 197
Using locomotion behavior and intestinal ROS production as the endpoints, we observed
198
that loss-of-function mutation of mdt-15 caused more severe toxicity in nanopolystyrene
199
exposed nematodes compared with nanopolystyrene exposed wild-type nematodes (Fig. S1).
200
Considering the fact that MDT-15 is expressed in neurons, intestine, and reproductive
201
(Hunt-Newbury et al., 2007; Taubert et al., 2006), we next examined the tissue-activity of
202
MDT-15 in regulating nanopolystyrene toxicity.
203
germline did not obviously influence susceptibility of mdt-15 mutant to nanopolystyrene
204
toxicity (Fig. S1).
205
suppressed the toxicity induction in nanopolystyrene exposed mdt-15 mutant nematodes (Fig.
206
S1). Therefore, intestinal MDT-15 was involved in the control of nanopolystyrene toxicity
207
in nematodes.
208
Expression of mdt-15 in neurons or in
Different from this, we found that expression of mdt-15 in intestine
SBP-1 is exclusively expressed in the intestine (McKay et al., 2003).
Using VP303
209
strain, we found the more severe intestinal ROS production in nanopolystyrene exposed
210
nematodes with intestine-specific RNAi knockdown of sbp-1 compared with that in
211
nanopolystyrene exposed VP303 strain (Fig. 2A), suggesting that intestine-specific RNAi
212
knockdown of sbp-1 caused the susceptibility to nanopolystyrene toxicity.
213 214
3.3. Genetic interaction between MDT-15 and SBP-1 in regulating the nanopolystyrene
215
toxicity
216 217 218
After nanopolystyrene exposure, mutation of mdt-15 could significantly decrease the sbp-1 expression (Fig. 2B).
Using intestinal ROS production as an endpoint, we further 8
219
observed that intestinal overexpression of MDT-15 caused a resistance to nanopolystyrene
220
toxicity (Fig. 2C).
221
resistance in nematodes overexpressing intestinal MDT-15 to nanopolystyrene toxicity in
222
inducing ROS production (Fig. 2C), which suggested that MDT-15 acted upstream of SBP-1
223
to regulate the nanopolystyrene toxicity.
Furthermore, RNAi knockdown of sbp-1 effectively suppressed this
224 225
3.4. Identification of downstream targets of intestinal SBP-1 in regulating nanopolystyrene
226
toxicity
227 228
Some potential targets for SBP-1 have been identified to be required for the possible
229
control of various biological processes (Ceron et al., 2007; Jo et al., 2009; Nomura et al., 2010;
230
Svensk et al., 2013; MacNeil et al., 2015; Shen et al., 2017; Pradhan et al., 2018; Kniazeva et
231
al., 2004), and some of them are expressed in the intestine (https://www.wormbase.org).
232
Among these intestinal targeted genes, exposure to nanopolystyrene (1 µg/L) significantly
233
increased the expressions of fat-7, fat-6, fat-4, fat-2, hsp-4, and sod-3, and decreased elo-5,
234
acs-2, and hpl-2 expressions in wild-type nematodes (Fig. 3A).
235
knockdown of sbp-1 only decreased fat-2, fat-6, fat-7, and hsp-4 expressions, and increase the
236
acs-2 expression in nanopolystyrene exposed nematodes (Fig. 3B).
237
knockdown of fat-6 or hsp-4 caused susceptibility to nanopolystyrene toxicity in inducing
238
intestinal ROS production (Fig. 3C). In contrast, intestine-specific RNAi knockdown of
239
fat-2, fat-7, or acs-2 did not influence the nanopolystyrene toxicity (Fig. 3C).
Meanwhile, intestinal RNAi
Intestine-specific RNAi
240
To confirm the role of FAT-6 and HSP-4 as the downstream targets of intestinal SBP-1 in
241
regulating nanopolystyrene toxicity, we generated the transgenic strain overexpressing
242
intestinal SBP-1.
243
nanopolystyrene toxicity in inducing ROS production (Fig. 3D).
244
knockdown of fat-6 or hsp-4 could effectively inhibit the resistance of transgenic strain
245
overexpressing intestinal SBP-1 to nanopolystyrene toxicity (Fig. 3D), which confirmed that
246
FAT-6 and HSP-4 acted downstream of intestinal SBP-1 to regulate nanopolystyrene toxicity.
Intestinal overexpression of MDT-15 caused the resistance to Moreover, RNAi
247 248
3.5. MDT-15 and SBP-1 were required for the activation of ER UPR in nanopolystyrene 9
249
exposed nematodes
250 251
Our previous study has demonstrated that exposure to nanopolystyrene could result in an
252
obvious activation of ER UPR as indicated by HSP-4::GFP expression (Qu et al., 2019b).
253
Using transgenic strain SJ4005/zcIs4[HSP-4::GFP] as a tool, we found that activation of
254
HSP-4::GFP induced by nanopolystyrene exposure was significantly inhibited by RNAi
255
knockdown of mdt-15 or sbp-1 (Fig. 4A).
256
not obviously influence this activation of HSP-4::GFP in nanopolystyrene exposed nematodes
257
(Fig. 4A). These observations suggested that the MDT-15 and SBP-1 but not the FAT-6 were
258
required for ER UPR activation in nanopolystyrene exposed nematodes.
Different from this, RNAi knockdown of fat-6 did
259 260
3.6. Genetic interaction between FAT-6 and HSP-4 in regulating nanopolystyrene toxicity
261 262
With intestinal ROS production as an endpoint, we found that intestine-specific RNAi
263
knockdown of hsp-4 or fat-6 induced more severe induction of ROS production in
264
nanopolystyrene exposed nematodes compared with nanopolystyrene exposed VP303
265
nematodes (Fig. 4B). Moreover, RNAi knockdown of both hsp-4 and fat-6 caused the more
266
severe toxicity compared with RNAi knockdown of hsp-4 or fat-6 alone in nanopolystyrene
267
exposed nematodes (Fig. 4B), which suggested that HSP-4 and FAT-6 functioned
268
synergistically in the intestine to regulate nanopolystyrene toxicity.
269 270
3.7. Identification of anti-microbial proteins CYP-35A3, CLEC-67, and LYS-7 as downstream
271
targets of intestinal FAT-6 in regulating nanopolystyrene toxicity
272 273
Antimicrobial proteins can act as potential downstream target of FAT-6 in controlling
274
stress response (Anderson et al., 2019), and some of them (irg-4, F49F1.7, cyp-35A3,
275
cyp-35B1, dsh-23, cdr-1, oac-6, clec-67, and lys-7) are expressed in the intestine
276
(https://www.wormbase.org).
277
nanopolystyrene (1 µg/L) exposure could increase cyp-35A3, clec-67, and lys-7 expressions
278
(Fig. 5A).
Among
these
9
intestinal
antimicrobial
genes,
Meanwhile, intestinal RNAi knockdown of fat-6 could significantly decrease the 10
279
expressions of cyp-35A3, clec-67, and lys-7 in nanopolystyrene exposed nematodes (Fig. 5B).
280
Furthermore, intestine-specific RNAi knockdown of cyp-35A3, clec-67, or lys-7 could induce
281
a susceptibility to nanopolystyrene toxicity in inducing intestinal ROS production (Fig. 5C),
282
suggesting that CYP-35A3, CLEC-67, and LYS-7 acted as the potential targets of intestinal
283
FAT-6 to regulate nanopolystyrene toxicity.
284 285
3.8. Genetic interaction of MDT-15/SBP-1 with PMK-1, SKN-1, or ATF-7 in regulating
286
nanopolystyrene toxicity
287 288
In nematodes, p38 MAPK signaling acted in the intestine to regulate nanopolystyrene
289
toxicity by activating ER UPR (Qu et al., 2019a). Intestinal overexpression of PMK-1,
290
ATF-7, or SKN-1 induced a resistance to nanopolystyrene toxicity (Fig. S2).
291
production as an endpoint, it was found that RNAi knockdown of mdt-15 or sbp-1 suppressed
292
the resistance of transgenic strain overexpressing intestinal PMK-1 (Fig. S2). Similarly,
293
RNAi knockdown of mdt-15 or sbp-1 also inhibited the resistance of transgenic strain
294
overexpressing intestinal SKN-1 (Fig. S2).
295
mdt-15 or sbp-1 did not influence the resistance of nematodes overexpressing intestinal ATF-7
296
(Fig. S2).
297
PMK-1-SKN-1 to regulate nanopolystyrene toxicity.
Using ROS
Different from these, RNAi knockdown of
Therefore, MDT-1 and SBP-1 acted downstream of signaling cascade of
298 299
4. Discussion
300 301
Recently, some reports have implied that polystyrene particles may potentially induce
302
some metabolic alterations, such as amino acid metabolism, bile acid metabolism, and energy
303
related metabolism (Jin et al., 2019; Kim et al., 2019).
304
demonstrated that the polystyrene microplastics could induce the changes of lipid
305
metabolism-related genes (Wan et al., 2019).
306
nanopolystyrene, we provided the direct evidence that exposure to 100 nm nanopolystyrene
307
(≥ 1 µg/L) caused severe lipid accumulation (Fig. 1C).
308
(1 µg/L) from L1-larvae to adult day-3 could cause the toxicity at various aspects in wild-type 11
A recent report has further
In this study, with the concern on the
Exposure to 100 nm nanopolystyrene
Considering that the 1 µg/L is a predicted environmental
309
nematodes (Shao et al., 2019).
310
concentration for 100 nm nanoplastics (Al-Sid-Cheikh et al., 2018; Lenz et al., 2016), our
311
data implies that long-term exposure to low-dose nanopolystyrene may potentially result in
312
the alteration in lipid metabolism.
313
Our recent studies have suggested that exposure to nanopolystyrene could at least cause
314
intestinal, neuronal, and reproductive toxicities in nematodes (Lei et al., 2018; Shao et al.,
315
2019; Qu et al., 2019a; Qu et al., 2019d).
316
intestine-specific activity of MDT-15, SBP-1, or FAT-6 in regulating nanopolystyrene
317
toxicity (Fig. S1, 2, and 3C).
318
induced a susceptibility to nanopolystyrene in inducing intestinal ROS production (Fig. S1A,
319
2A, and 3C). Meanwhile, we found that mutation of mdt-15 also induced a susceptibility to
320
nanopolystyrene in decreasing locomotion behavior (Fig. S1B). Therefore, on the one hand,
321
MDT-15, SBP-1, and FAT-6 could act in the intestine to regulating the induction of intestinal
322
toxicity of nanopolystyrene.
323
to regulating the induction of neuronal toxicity of nanopolystyrene.
324
In this study, our data suggested the
Mutation or RNAi knockdown of mdt-15, sbp1-1, or fat-6
On the other hand, at least MDT-15 could also act the intestine
In nematodes, NHR-80, NHR-49, SBP-1, and MDT-15 are four lipid metabolic sensors
325
(Ashrafi, 2007; Watts, 2009).
326
MDT-15 is a homolog of mammalian PGC-1.
327
hormone receptors, and NHR-49 is a peroxisome proliferator-activated receptor α (PPARα).
328
For the underlying mechanism of this increased lipid accumulation, we found that the
329
observed lipid accumulation was related to the increase in expressions of mdt-15 and sbp-1
330
genes (Fig. 1D).
331
SBP-1 (Ashrafi, 2007; Watts, 2009).
332
SBP-1 is a sterol response element binding protein (SREBP). NHR-80 and NHR-49 are two nuclear
During the control of fat metabolism, MDT-15 acts as a co-activator for
Transcriptional factors of MDT-15 and SBP-1 regulate lipid metabolism by activating
333
several downstream targets (Ashrafi, 2007; Watts, 2009).
334
accumulation, the targets of FATs proteins (such as FAT-7, FAT-6, and FAT-5) regulate
335
synthesis of monounsaturated fatty acid acylCoAs from saturated fatty acid acylCoAs, fatty
336
acid synthease FASN-1 regulates the process of fatty acid synthesis, and ACS-2 regulates
337
process of mitochondrial β-oxidation of fatty acid (Ashrafi, 2007; Watts, 2009).
338
Nevertheless, among the genes required for the lipid accumulation (Ashrafi, 2007; Watts, 12
During the control of lipid
339
2009), intestinal RNAi knockdown of sbp-1 only decreased expressions of genes (fat-7 and
340
fat-6) encoding fatty acyl CoA desaturases and increased the acs-2 encoding acyl CoA
341
synthase (Fig. 3B), suggesting that some other transcriptional factors may also be affected to
342
activate the other genes required for the lipid accumulation. Mutation of mdt-15, sbp-1, or
343
fat-6 caused the reduced lipid accumulation (Wu et al., 2010; Brock et al., 2007).
344
Meanwhile, nanopolystyrene (1 µg/L) decreased acs-2 expression, but did not affect the
345
fasn-1 expression (Fig. 3A), suggesting that long-term and low-dose exposure to
346
nanopolystyrene may only affect the process of fatty acid β-oxidation in nematodes.
347
these, exposure to nanopolystyrene may also affect some other aspects of lipid metabolism,
348
such as fatty acid elongation and desaturation, since nanopolystyrene (1 µg/L) also increased
349
fat-2 and fat-4 expressions, and decreased elo-5 expression (Fig. 3A).
350
Besides
Besides the lipid accumulation, we further found that MDT-15, SBP-1, and their target
351
FAT-6 were also required for the control nanopolystytyrene toxicity.
Mutation or RNAi
352
knockdown of mdt-15, sbp-1, or fat-6 caused a susceptibility to nanopolystyrene toxicity (Fig.
353
S1, 2, and 3C). Previous studies have suggested the role of FAT-6, SBP-1, and MDT-15 in
354
regulating the response to various stresses or toxicants (Goh et al., 2014; Lee et al., 2015;
355
Wang, 2019; Horikawa and Sakamoto, 2009).
356
MDT-15-SBP-1-FAT-6 was raised to be required for the control of nanopolystyrene toxicity
357
(Fig. 5D).
358
MDT-15-SBP-1-FAT-6-mediated lipid accumulation in being against the nanopolystyrene
359
toxicity (Fig. 5D).
In the intestine, a signaling cascade of
That is, our study further implies the potential protective role of
360
In the intestine, two downstream targets (FAT-6 and HSP-4) were identified for SBP-1
361
(Fig. 3), and acted in parallel pathways in regulating nanopolystyrene toxicity (Fig. 4B).
362
HSP-4 is heat-shock protein, a molecular marker of ER UPR (Bischof et al., 2008). This
363
implies that SBP-1 may activate two different downstream biological events.
364
FAT-6-mediated lipid metabolism and stress response. Another is HSP-4-mediated ER UPR
365
response, which may be not directly associated with the lipid metabolism.
366
cascade (PMK-1-SKN-1/ATF-7) was raised in p38 MAPK signaling pathway require for the
367
control of nanopolystyrene toxicity (Qu et al., 2019b).
368
the p38 MAPK, and SKN-1 and ATF-7 (two transcriptional factors) were targets of PMK-1. 13
One is
A signaling
In this signaling cascade, PMK-1 is
369
In the intestine, we identified a signaling cascade (SKN-1-PMK-1-MDT-15-SBP-1) involved
370
in the activation of ER UPR against the nanopolystyrene toxicity (Fig. 4A and S2).
371
Therefore, MDT-15-SBP-1 signaling cascade acted as an important link between ER UPR
372
response and p38 MAPK signaling in nanopolystyrene exposed nematodes.
373
In this study, three genes (cyp-35A3, clec-67, and lys-7) encoding anti-microbial proteins
374
were identified as downstream targeted genes of fat-6 in regulating nanopolystyrene toxicity
375
(Fig. 5A-5C).
376
expression mediated both the activation of innate immune response and the alteration in lipid
377
metabolism in nanopolystyrene exposed nematodes. FAT-6 has been shown to be involved
378
in regulating the innate immune response in nematodes (Anderson et al., 2019).
379
other hand, the intestinal MDT-15-SBP-1 could potentially activate two different responses
380
(innate immune response and ER UPR response) against the nanopolytyrene toxicity (Fig.
381
5D).
382
On the one hand, this observation suggested that the increase in FAT-6
On the
Together, in this study, we investigated the lipid metabolic response and its association
383
with toxicity regulation in nanopolystyrene exposed nematodes.
After nanopolystyrene
384
exposure, we observed the severe lipid accumulation in nematodes.
385
expression of both MDT-15 and SBP-1, two lipid metabolic sensors, were increased by
386
nanopolystyrene exposure.
387
intestine to induce the protective response to nanopolystyrene by activating HSP-4-mediated
388
ER UPR response and FAT-6-mediated innate immune response.
389
important molecular basis for the lipid metabolic response to nanopolystyrene exposure in
390
organisms.
391
response with the control of nanopolystyrene toxicity.
Meanwhile, the
The MDT-15-SBP-1 signaling cascade further acted in the
Our data provided the
Additionally, our results suggested the close association of lipid metabolic
392 393
Acknowledgements
394 395
This work was supported by the grant from National Natural Science Foundation of China
396
(21577016).
397 398
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Fig. 1. Effect of nanopolystyrene exposure on lipid accumulation.
(A) TEM image of
581
nanopolystyrene particles in K medium.
582
particles.
583
lipid accumulation. (D) Effect of nanopolystyrene on transcriptional expressions of sbp-1,
584
nhr-49, nhr-80, and mdt-15.
(B) Raman spectroscopy of nanopolystyrene
(C) Sudan blacking staining showing the effect of nanopolystyrene exposure on
Bars represent means ± SD.
585
21
**
P < 0.01 vs control.
586 587
Fig. 2. Genetic interaction between MDT-15 and SBP-1 in the intestine to regulate the
588
response to nanopolystyrene.
589
nanopolystyrene toxicity in inducing intestinal ROS production. Bars represent means ± SD.
590
**
591
expression in nanopolystyrene exposed nematodes. Bars represent means ± SD.
592
vs wild-type.
593
inducing intestinal ROS production in nematodes overexpressing intestinal MDT-15.
594
represent means ± SD.
595
concentration of nanopolystyrene was 1 µg/L.
(A) Effect of intestine-specific RNAi knockdown of sbp-1 on
P < 0.01 vs control (if not specially indicated). (B) Effect of mdt-15 mutation on sbp-1 **
P < 0.01
(C) Effect of RNAi knockdown of sbp-1 on nanopolystyrene toxicity in
**
P < 0.01 vs control (if not specially indicated).
596
22
Bars
Exposure
597 598
Fig. 3. Identification of downstream targets of intestinal SBP-1 in regulating the response to
599
nanopolystyrene. (A) Effect of nanopolystyrene exposure on gene expressions in wild-type
600
nematodes.
601
RNAi knockdown of sbp-1 on gene expressions in nanopolystyrene exposed nematodes.
602
Bars represent means ± SD.
603
knockdown of fat-2, fat-6, fat-7, acs-2, or hsp-4 on nanopolystyrene toxicity in inducing
604
intestinal ROS production.
605
specially indicated).
606
regulating the nanopolystyrene toxicity in inducing intestinal ROS production.
607
represent means ± SD.
608
concentration of nanopolystyrene was 1 µg/L.
609
Bars represent means ± SD.
**
P < 0.01 vs control.
**
P < 0.01 vs VP303.
Bars represent means ± SD.
(B) Effect of intestinal
(C) Effect of intestinal RNAi
**
P < 0.01 vs control (if not
(D) Genetic interaction between SBP-1 and FAT-6 or HSP-4 in
**
P < 0.01 vs control (if not specially indicated).
23
Bars
Exposure
610 611
Fig. 4. MDT-15 and SBP-1 were required for the activation of ER UPR in nanopolystyrene
612
exposed nematodes. (A) Effect of RNAi knockdown of mdt-15, sbp-1, or fat-6 on activation
613
of HSP-4::GFP in nanopolystyrene exposed nematodes. (B) Genetic interaction between
614
FAT-6 and HSP-4 in the intestine to regulate the nanopolystyrene toxicity in inducing
615
intestinal ROS production. Exposure concentration of nanopolystyrene was 1 µg/L.
616
represent means ± SD.
**
P < 0.01 vs control (if not specially indicated).
617 618
24
Bars
619 620
Fig. 5. Identification of several anti-microbial proteins as downstream targets of intestinal
621
FAT-6 in regulating the response to nanopolystyrene.
622
exposure on gene expressions in wild-type nematodes.
Exposure concentration of
623
nanopolystyrene was 1 µg/L.
**
624
Effect of intestinal RNAi knockdown of fat-6 on gene expressions in nanopolystyrene
625
exposed nematodes.
626
represent means ± SD.
627
cyp-35A3, clec-67, or lys-7 on nanopolystyrene toxicity in inducing intestinal ROS production.
628
Exposure concentration of nanopolystyrene was 1 µg/L.
629
0.01 vs control (if not specially indicated). (D) A diagram showing the molecular basis for
630
lipid metabolic response and its association with toxicity regulation in nanopolystyrene
(A) Effect of nanopolystyrene
Bars represent means ± SD.
P < 0.01 vs control. (B)
Exposure concentration of nanopolystyrene was 1 µg/L. **
P < 0.01 vs VP303.
25
Bars
(C) Effect of intestinal RNAi knockdown of
Bars represent means ± SD.
**
P<
631
exposed nematodes.
632
26
Highlights:
- Nanopolystyrene exposure caused lipid accumulation in nematodes. - Lipid metabolic sensors of MDT-15 and SBP-1 were increased by nanopolystyrene. - Lipid metabolic response was associated with the control of nanopolystyrene toxicity. - MDT-15-SBP-1 signaling induced both ER UPR and innate immune response.
Conflict of Interests:
The authors declare that they have no competing interests.