- Email: [email protected]
Oxidative stress caused by TiO2 nanoparticles under UV irradiation is due to UV irradiation not through nanoparticles
Accepted Manuscript Oxidative stress caused by TiO2 nanoparticles under UV irradiation is due to UV irradiation not through nanoparticles Akihiro Moriyama, Ikuho Yamada, Junko Takahashi, Hitoshi Iwahashi PII:
S0009-2797(17)31107-9
DOI:
10.1016/j.cbi.2018.08.017
Reference:
CBI 8391
To appear in:
Chemico-Biological Interactions
Received Date: 12 October 2017 Revised Date:
28 July 2018
Accepted Date: 17 August 2018
Please cite this article as: A. Moriyama, I. Yamada, J. Takahashi, H. Iwahashi, Oxidative stress caused by TiO2 nanoparticles under UV irradiation is due to UV irradiation not through nanoparticles, ChemicoBiological Interactions (2018), doi: 10.1016/j.cbi.2018.08.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT
1
Oxidative stress caused by TiO2 nanoparticles under UV irradiation is due to
2
UV irradiation not through nanoparticles
RI PT
3
4
Akihiro Moriyama1*, Ikuho Yamada1, Junko Takahashi2, Hitoshi Iwahashi1
5
1
6
Gifu 501-1193, Japan
7
2
8
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi,
9
Tsukuba, Ibaraki 305-8566, Japan
M AN U
SC
Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu,
Molecular Composite Medicine Research Group, Biomedical Research Institute,
TE D
10
Keywords:
12
Saccharomyces cerevisiae
13
TiO2 nanoparticles
14
ROS generated by TiO2-NOAA
15
DNA microarray
AC C
EP
11
16
17
*Corresponding author.
18
E-mail address: [email protected] (A. Moriyama). 1
ACCEPTED MANUSCRIPT
19
ABSTRACT Currently, nanoparticles are used in various commercial products. One of the most
21
common nanoparticles is titanium dioxide (TiO2). It has a catalytic activity and UV
22
absorption, and generates reactive oxygen species (ROS). This catalytic activity of TiO2
23
nanoparticles was believed to be capable of killing a wide range of microorganisms. In
24
the environment, the unique properties of TiO2 nanoparticles can be maintained;
25
therefore, the increasing use of TiO2 nanoparticles is raising concerns about their
26
environmental risks. Thus, assessment of the biological and ecological effects of
27
TiO2-NOAAs is necessary.
M AN U
SC
RI PT
20
In this study, we assessed the effect of TiO2-NOAAs for S. cerevisiae using DNA
29
microarray. To compare yeast cells under various conditions, six treatment conditions
30
were prepared (1. adsorbed fraction to TiO2-NOAA under UV; 2. non-adsorbed fraction
31
to TiO2-NOAA under UV; 3. adsorbed fraction to TiO2-NOAA without UV; 4.
32
non-adsorbed fraction to TiO2-NOAA without UV; 5. under UV; and 6. untreated
33
control). The result of the DNA microarray analysis, suggested that yeast cells that are
34
adsorbed by TiO2-NOAA under UV irradiation suffer oxidative stress and this stress
35
response was similar to that by only UV irradiation. We concluded that the effect of
AC C
EP
TE D
28
2
ACCEPTED MANUSCRIPT
36
TiO2-NOAAs on yeast cells under UV irradiation is not caused by TiO2-NOAA but UV
37
irradiation.
39
RI PT
38
1. INTRODUCTION
A nanoparticle is defined as a nano-object with all ext\ernal dimensions in the
41
nanoscale (length range approximately from 1 nm to 100 nm), where the lengths of the
42
longest and the shortest axes of the nano-object do not differ significantly1. Compared
43
with a fine particle, the nanoparticle has a larger specific surface area. Therefore,
44
nanoparticles show greater chemical and physical activities, such as ion release,
45
adsorption ability, and production of reactive oxygen species (ROS)2. The unique
46
properties of nanoparticles are beneficial, and thus, the use of nanoparticles is
47
expanding rapidly.
EP
TE D
M AN U
SC
40
TiO2 nanoparticles are used in various commercial products (e.g., self-cleaning
49
surface coatings, light-emitting diodes, solar cells, disinfectant sprays, sporting goods,
50
drinking water treatment agents, and topical sunscreens) because of their catalytic
51
activity and UV absorption (λ<400 nm)3. They are not dissolved in water, and generate
52
ROS under UV irradiation, apparently enabling them to decompose organic substances
53
and damage the function and structure of various cells.4 The catalytic activity of TiO2
AC C
48
3
ACCEPTED MANUSCRIPT
nanoparticles was believed to be capable of killing a wide range of microorganisms (e.g.
55
gram-negative and gram-positive bacteria, including endospores, as well as fungi, algae,
56
protozoa, and viruses)5.
RI PT
54
In culture medium and the water environment, nanoparticles unexceptionally form
58
structures of their aggregates and agglomerates comprising primary particles. These
59
compounds are defined as NOAAs (nano-objects, and their aggregates and
60
agglomerates greater than 100 nm)6. An aggregate is a particle that comprises strongly
61
bonded or fused single primary particles, and an agglomerate is a collection of weakly
62
bound single primary particles, aggregates, or a mix of these. The name NOAA is made
63
by International Standard Organization1 to share the knowledge that nanoparticles may
64
not be protected for aggregation and agglomeration and can not be alone as single
65
molecule in culture medium and water environment. The unique properties of TiO2
66
nanoparticles can be maintained in the environment, and thus the increasing use of TiO2
67
nanoparticles is raising environmental concerns. An assessment of the biological and
68
ecological effects of TiO2-NOAAs is necessary.
AC C
EP
TE D
M AN U
SC
57
69
In our previous study, we assessed the effect of TiO2-NOAAs on microbes using
70
Saccharomyces cerevisiae and Escherichia coli. The TiO2-NOAAs decomposed
71
methylene blue under UV irradiation, which suggested that the TiO2-NOAAs generated 4
ACCEPTED MANUSCRIPT
ROS under UV irradiation. However, the TiO2-NOAAs did not demonstrate growth
73
inhibition in minimal agar medium under UV irradiation; the addition of TiO2-NOAAs
74
to the medium still permitted colony formation under a UV intensity that inactivates
75
microbes. Moreover, the TiO2-NOAAs adsorbed microbes. These results suggested that
76
the amount of ROS generated by TiO2-NOAAs was insufficient to inactivate microbes,
77
and the TiO2-NOAAs might protect microbes from UV7.
M AN U
SC
RI PT
72
In this study, we assessed the effect of TiO2-NOAAs under UV on S. cerevisiae in
79
more detail. We used DNA microarray analysis for qualitative assessment of gene
80
expression profile and carried out a quantitative assessment using qRT-PCR method.
81
These results suggest the effect of TiO2-NOAAs on yeast cells under UV irradiation is
82
not caused by TiO2-NOAA but UV irradiation.
85
86
EP
84
AC C
83
TE D
78
87
88
89 5
ACCEPTED MANUSCRIPT
90
2. EXPERIMENTAL METHODS
91
2.1 Materials TiO2 nanoparticles were obtained from Ishihara Sangyo Kaisha, Ltd. (Japan). Their
93
crystals were in the anatase form, with a primary particle size of 7 nm, and a specific
94
surface area of 316 m2/g.
SC
RI PT
92
96
M AN U
95
2.2 Yeast strains and growth conditions
S. cerevisiae S288C (MATα SUC2 mal mel gal2 CUP1 [cir+]) was used in this
98
study. The yeast was grown in minimal medium (0.67% Difco™ Yeast Nitrogen Base
99
w/o Amino Acids (Becton, Dickinson and Company, NJ, USA), 2% D(+)-Glucose) at
102
103
EP
101
30 °C. A pre-culture was incubated to stationary phase (2 days).
2.3 TiO2 nanoparticle treatment
AC C
100
TE D
97
One mL of pre-culture (5 × 107 cells) and 19 mL of minimal medium were added to
104
petri dishes (φ 90 mm × 20 mm). They were incubated for 6 h at 30 °C with shaking at 60
105
rpm to produce exponentially growing cells. Then, 5.0 mg of TiO2 nanoparticles were
106
added to the petri dishes, with or without UV irradiation (intensity; 0.01 mW/cm2), and
107
the cells were incubated for a further 2h. Treated yeast cells were divided into the 6
ACCEPTED MANUSCRIPT
adsorbed fraction and non-adsorbed fractions. Each of the yeast under different
109
conditions (Condition 1. adsorbed fraction to TiO2-NOAA under UV irradiation, 2.
110
non-adsorbed fraction to TiO2-NOAA under UV irradiation, 3. adsorbed fraction to
111
TiO2-NOAA without UV irradiation, 4. non-adsorbed fraction to TiO2-NOAA without
112
UV irradiation, 5. under UV and 6. untreated control) was harvested by centrifugation at
113
2150×g for 3 min at 4 °C (CAX-371; Tomy Seiko Co., LTD., Tokyo, Japan). Under our
114
TiO2 nanoparticles treatment conditions (30 °C with shaking at 60 rpm), many yeasts
115
aggregated with NOAAs. However, we could yield enough yeast cells for DNA
116
microarray in the supernatant phase over the NOAA and yeast agglomerates (Condition
117
2, 4).
118
2.4 RNA extraction
EP
119
TE D
M AN U
SC
RI PT
108
RNA was extracted from cells using a Fast RNA® Pro Red Kit (MP Biomedicals,
121
Santa Ana, CA, USA), according to the manufacturer’s instructions, with the following
122
exceptions: cell disruption was performed for 10 min using a Multi-Beads
123
Shocker®(Yasui Kikai, Osaka, Japan) and chloroform (Wako Pure Chemical Industries)
124
treatment was performed twice. The extracted RNA was purified using a Qiagen
125
RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s
AC C
120
7
ACCEPTED MANUSCRIPT
instructions. The quality of the purified RNA was verified using an Agilent® 2100
127
Bioanalyzer (Agilent Technologies, CA, USA). The RNA concentration was
128
determined using a Micro-volume Spectrophotometer Q5000 (Tomy Seiko Co., LTD).
RI PT
126
129
132
SC
131
2.5 DNA microarray analysis
DNA microarray analysis was carried out to assess the effect of TiO2-NOAAs on
M AN U
130
yeast qualitatively.
Fluorescent cyanine 3-cytidine triphosphate (CTP)-labeled cRNA was synthesized
134
using a Quick Amp Labeling Kit (Agilent Technologies, CA, USA). The CTP-labeled
135
cRNA was used for hybridization to Yeast (V2) Gene Expression Microarray slides
136
(#G4813A016322, Agilent Technologies) at 65 °C for 17 h. The hybridized microarray
137
slides were washed according to the manufacturer’s instructions and scanned using an
138
Agilent DNA Microarray Scanner (#G2565CA, Agilent Technologies) at a resolution of
139
5 µm. The scanned images were analyzed quantitatively using the Agilent Feature
140
Extraction Software version 10.7.3.1 (Agilent Technologies).
AC C
EP
TE D
133
141
Signals detected from each open reading frame (ORF) were normalized using the
142
25th percentile and quantile methods. The genes classified as upregulated or
143
downregulated were those that passed the Student’s t-test (P< 0.05) and exhibited more 8
ACCEPTED MANUSCRIPT
144
than 2-fold higher intensities or more than 0.5-fold lower intensities than that of
145
negative control. The gene expression profiles were characterized according to the categories of the
147
Munich Information Center for Protein Sequences Genome Research Environment
148
Comprehensive Yeast Genome Database (MIPS GenRE CYGD, http://mips.helmholtz-
149
muenchen.de/genre/proj/yeast/index.jsp) and the Saccharomyces Genome Database
150
(SGD, http://www.yeastgenome.org/).The DNA microarray experiments described in
151
this study were MIAME compliant and the raw data has been deposited in the Gene
152
Expression Omnibus (GEO) database under the accession number GSE99660. For each
153
condition, RNA samples were prepared at least four replications.
154
157
158
EP
156
2.6 Quantitative PCR
qRT-PCR was carried out to assess the effect of TiO2-NOAAs on yeast cells
AC C
155
TE D
M AN U
SC
RI PT
146
quantitatively.
The RNA used for DNA microarray analysis was subjected to reverse transcription
159
(RT) at 37 °C for 15 min using a ReverTra Ace® qPCR RT Master Mix (Toyobo, Osaka,
160
Japan). For quantitative PCR amplification, the Power SYBR® Green Master Mix
161
(Applied Biosystems, CA, USA) and StepOnePlus™ real-time PCR System (Applied 9
ACCEPTED MANUSCRIPT
Biosystems) were used, as described in the manufacturer’s instructions (holding at 95 °C
163
for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, and annealing and
164
extension at 60 °C for 2 min). Relative expression levels were calculated from Ct values.
165
Five pairs of primers were designed for the amplification of the genes upregulated under
166
certain conditions (Table 1), based on the results of DNA microarray analysis. All
167
experiments were performed in triplicate.
Table 1. Sequences of oligonucleotide primers used in this study Gene name
F-primer
GRE2 SOD2 GSY1 TPS2
AAGGTCATCGGTTCTGCCAG CTCCCGCAAACGCAAGAAAA CATATGGGCCATCGTCGTCA CGCAGCTGCCCTACAAAATC
171
CCTTGCCGTGCTTTTGGAAA CTCGTCCAGACTGCCAAACT GGTACCTAAATCGCCCGGAG TGGGTCATCGTCCAGATCCT
ATTGCCGAAAGAATGCAAAAGG CGCACAAAAGCAGAGATTAGAAACA
The ACT1 gene was used as a control housekeeping gene because little difference
AC C
170
R-primer
EP
ACT1
TE D
169
M AN U
168
SC
RI PT
162
172
in its gene expression level was observed between the treatment groups and a control
173
from the result of DNA microarray analysis, and it is widely accepted as a positive control
174
to study the yeast stress response8.
175
10
ACCEPTED MANUSCRIPT
176
3. RESULTS AND DISCUSSION
177
3.1 Conditions for TiO2 nanoparticle treatment
179
To assess the effect of TiO2-NOAAs on yeast cells at the RNA level, initially, an
RI PT
178
appropriate intensity of UV irradiation was determined (Fig. 1).
SC
180
M AN U
181
182
183
184
187
EP
186
TE D
185
Figure 1. Viable yeast cell numbers under each UV irradiation intensity. Yeast cells
189
were incubated under the same conditions with TiO2 nanoparticle treatment (as described
190
in the text) under various UV irradiation intensities. To determine their viabilities, yeast
191
cells were serially diluted and incubated on minimal agar media for 2–3 days.
AC C
188
192
11
ACCEPTED MANUSCRIPT
Under the strongest UV intensity, 0.4 mW/cm2 (A), few yeast cells survived, thus
194
their RNA could not be extracted. Under a UV intensity of 0.01 mW/cm2 (B), UV
195
inactivation of the yeast cells was observed, and their RNA could be extracted. Therefore,
196
RNA extraction for the DNA microarray and qRT-PCR was conducted at a UV intensity
197
of 0.01 mW/cm2.
SC
RI PT
193
The treatment concentration of TiO2 nanoparticle was the same as that used in our
199
previous study7. It is also confirmed that when titanium is added to the minimum
200
medium, it aggregates to form NOAA. Furthermore, it was also confirmed that many
201
yeasts are adsorbed to TiO2 nanoparticles when co-culturing yeast cells (Fig. 2).
AC C
202
EP
TE D
M AN U
198
203
Figure 2. A state of agglomerates of TiO2 nanoparticles and yeast. 1 mL of pre-culture
204
(5 × 107 cells), 19 mL of minimal medium and 5.0 mg of TiO2 nanoparticles were added
205
to the petri dishes (φ 90 mm × 20 mm).
206
12
ACCEPTED MANUSCRIPT
3.2 Qualitative assessment of the effect of TiO2-NOAA on yeast by DNA microarray
208
analysis
209
3.2.1 Overview of altered genes in each treatment condition
RI PT
207
In this study, yeast cells from six different treatment conditions (1. adsorbed
211
fraction to TiO2-NOAA under UV irradiation, 2. non-adsorbed fraction to TiO2-NOAA
212
under UV irradiation, 3. adsorbed fraction to TiO2-NOAA without UV irradiation, 4.
213
non-adsorbed fraction to TiO2-NOAA without UV irradiation, 5. under UV and 6.
214
untreated control) were analyzed using a DNA microarray. From the results, 3236, 1545,
215
2914, 1596, and 2493 ORFs that passed the Student’s t-test (P < 0.05) in Conditions 1, 2,
216
3, 4, and 5, respectively, were obtained by the comparison with untreated control of 6
217
(Table 2).
Table 2. The number of the up and downregulated genes in each treatment condition
AC C
219
EP
218
TE D
M AN U
SC
210
≧2-fold
≦0.5-fold
275
425
2. Non-adsorbed fraction of TiO2-NOAA (with UV)
9
109
3. Adsorbed fraction of TiO2-NOAA (without UV)
219
287
4. Non-adsorbed fraction of TiO2-NOAA (without UV)
18
121
5. Under UV
59
223
Treatment conditions
1. Adsorbed fraction of TiO2-NOAA (with UV)
220 13
ACCEPTED MANUSCRIPT
Among them, 275, 9, 219, 18, and 59 genes exhibited more than 2-fold higher
222
intensities, and 425, 109, 287, 121, and 223 genes exhibited more than 0.5-fold lower
223
intensities under Conditions 1, 2, 3, 4, and 5, respectively. The highest number of
224
significantly altered genes was in Conditions 1 (2-fold; 275, 0.5-fold; 425), 3 (2-fold;
225
219, 0.5-fold; 287), and 5 (2-fold; 59, 0.5-fold; 223).
SC
RI PT
221
These results showed that the expressions of genes in yeast cells that were adsorbed
227
by TiO2-NOAAs (Conditions 1 and 3) were altered more than those in yeast cells that
228
were not adsorbed by TiO2-NOAAs (Conditions 2 and 4). Further, a significant number
229
of genes were altered by UV irradiation in the adsorbed fraction (Condition 1). Thus,
230
these results suggested that yeast cells suffer stress from TiO2-NOAA and from UV.
233
234
TE D
EP
232
AC C
231
M AN U
226
235
3.2.2 The genes up and downregulated by TiO2-NOAAs under UV irradiation and
236
their functional distribution
237
The yeast cells in the fraction adsorbed to TiO2-NOAA under UV irradiation
238
showed the highest number of genes with altered expressions among all treatment 14
ACCEPTED MANUSCRIPT
conditions (Table 2; Condition 1).From the DNA microarray analysis, 3236 ORFs were
240
obtained that passed the Student’s t-test (P < 0.05). Among them, 275 genes were
241
upregulated by more than 2-fold and 425 were downregulated by more than 0.5-fold by
242
TiO2-NOAA treatment under UV irradiation. The up and downregulated genes were
243
characterized using MIPS GenRE CYGD. The results showed that the function of
244
upregulated genes were classified with the high probability into the categories of
245
“energy (10.3%)”, “cell rescue (7.4%)” and “metabolism (6.0%)”. Subcategories of
246
“Regulation of glycolysis and gluconeogenesis” and “metabolism of energy reserve (e.g.
247
glycogen, trehalose)” were characterized particularly in the category of “energy” (Fig.
248
3).
AC C
EP
TE D
M AN U
SC
RI PT
239
249
15
ACCEPTED MANUSCRIPT
Figure 3. Functional categories of up-regulated and down-regulated ORFs in
251
TiO2-NOAA treatment under UV irradiation (Condition 1). ORFs up-regulated or
252
down-regulated more than 2 fold were counted. The denominators and numerators in
253
parentheses indicate number of total ORFs and up-regulated or down-regulated ORFs in
254
each category, respectively.
SC
.
M AN U
255
RI PT
250
Trehalose stabilizes the membrane structure and glycogen is a component of the
257
cell wall in S. cerevisiae9, 10. Subcategories of “oxidative stress response” and “oxygen
258
and radical detoxification” were characterized particularly in the category of “cell
259
rescue”.
TE D
256
By contrast, downregulated genes were characterized particularly in the categories
261
of “protein synthesis (16%)”, “transcription (8%)”, and “protein with binding function
262
(6%)” (Fig. 3). Genes involved in protein synthesis are downregulated in response to
263
diverse environmental stresses11. Thus, this result suggested that yeast cells adsorbed by
264
TiO2-NOAAs under UV irradiation were significantly stressed.
265
266
AC C
EP
260
The products of the upregulated genes were localized significantly at the “peroxisome” (Fig. 4).
16
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
267
Figure 4. Locational categories of the gene products upregulated by TiO2-NOAA
269
treatment under UV irradiation (Condition 1). ORFs up-regulated more than 2 fold were
270
counted. The denominators and numerators in parentheses indicate number of total
271
ORFs and up-regulated ORFs in each category, respectively.
EP
272
TE D
268
It is reported that some peroxisomal genes are induced in response to oxidative stress12.
274
Thus, this localization and induction of subcategories related to oxidative stress
275
suggested that yeast cells that were adsorbed by TiO2-NOAA under UV irradiation
276
suffered oxidative stress. Additionally, localization with high probability at
277
“Extracellular/secretion proteins” and “Eukaryotic plasma membrane” were observed.
AC C
273
278
17
ACCEPTED MANUSCRIPT
279
3.2.3 Genes upregulated and downregulated by TiO2-NOAAs without UV
280
irradiation and their functional distribution The expressions of a significant number of genes in yeast cells in the adsorbed
282
fraction to TiO2-NOAAs even without UV irradiation were altered in all treatment
283
conditions (Table 2; Condition 3). From the DNA microarray analysis, 2914 ORFs were
284
obtained that passed the Student’s t-test (P < 0.05). Among them, 219 genes were
285
upregulated by more than 2-fold and 287 genes were downregulated by more than
286
0.5-fold by TiO2-NOAAs treatment without UV irradiation. The functions of the
287
upregulated genes were classified with high probability into the categories of “energy
288
(8.9%)”, “metabolism (5.1%)”, and “cell rescue (5.7%)”. Subcategories of “metabolism
289
of energy reserve (e.g. glycogen, trehalose)” and “regulation of glycolysis and
290
gluconeogenesis” were classified particularly in the category of “energy”, and
291
subcategories of “C-compound and carbohydrate metabolism” was classified
292
particularly in the category of “metabolism” (Fig. 5).
AC C
EP
TE D
M AN U
SC
RI PT
281
18
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
293
Figure 5. Functional categories of the genes upregulated and downregulated by
295
TiO2-NOAA treatment without UV irradiation (Condition 3). ORFs up-regulated or
296
down-regulated more than 2 fold were counted. The denominators and numerators in
297
parentheses indicate number of total ORFs and up-regulated or down-regulated ORFs in
298
each category, respectively.
EP
299
TE D
294
By contrast, the functions of the downregulated genes were classified as
301
“transcription (8.3%)”, “protein synthesis (9.5%)”, and “cell cycle and DNA processing
302
(6.2%)” (Fig. 5). This result showed that the yeast that were adsorbed by TiO2-NOAA
303
without UV irradiation suffered stress. The products of the upregulated genes were
304
localized as “Eukaryotic plasma membrane” (Fig. 6).
AC C
300
305 19
RI PT
ACCEPTED MANUSCRIPT
306
Figure 6. Locational categories of the genes upregulated by TiO2-NOAA
308
treatment under UV irradiation (Condition 3). ORFs up-regulated more than 2 fold were
309
counted. The denominators and numerators in parentheses indicate number of total
310
ORFs and up-regulated ORFs in each category, respectively.
M AN U
SC
307
TE D
311
These results showed that membrane structures of yeast cells that were adsorbed
313
by TiO2-NOAA without UV irradiation were damaged. Taken together, these results
314
imply yeast cells that are adsorbed by TiO2-NOAA without UV irradiation suffer stress
315
at their membrane structures, resulting in the induction of genes related to the synthesis
316
of glycogen and trehalose.
AC C
317
EP
312
318
20
ACCEPTED MANUSCRIPT
319
3.2.4 Genes upregulated and downregulated by UV irradiation and their functional
320
distribution The expressions of a large number of genes in yeast cells under UV without
322
TiO2-NOAAs were altered among all treatment conditions (Table 2; Condition 5). From
323
the DNA microarray analysis, 2493 ORFs were obtained that passed the Student’s t-test
324
(P < 0.05). Among them, 59 genes were upregulated by more than 2-fold and 223 genes
325
were downregulated by more than 0.5-fold by UV irradiation. The functions of the
326
upregulated genes were classified with high probability into the categories of “energy
327
(2.5%)”, “cell rescue (1.7%)”, and “metabolism (3.6%)” (Fig. 7). The subcategory of
328
“metabolism of energy reserve” was characterized particularly in the category of
329
“energy”, and subcategories of “oxygen and radical detoxification” were characterized
330
particularly in the category of “cell rescue”. This result showed that yeast cells that are
331
irradiated by UV suffer oxidative stress. The localizations of the products of the
332
upregulated genes were not found because of the insignificant number of genes in this
333
category.
AC C
EP
TE D
M AN U
SC
RI PT
321
21
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
334
Figure 7. Functional categories of the genes upregulated by UV irradiation
336
(Condition 5). ORFs up-regulated or down-regulated more than 2 fold were counted.
337
The denominators and numerators in parentheses indicate number of total ORFs and
338
up-regulated or down-regulated ORFs in each category, respectively.
EP
339
TE D
335
By contrast, the functions of the downregulated genes were classified with high
341
probability into the categories of “transcription (3.5%)” and “protein synthesis (3.2%)”
342
(Fig. 7). These results showed that yeast cells that were irradiated by UV suffered stress.
343
These results suggested that yeast cells irradiated by UV suffer oxidative stress
AC C
340
344
because genes involved in the response to oxidative stress were upregulated, and genes
345
involved in protein synthesis were downregulated.
22
ACCEPTED MANUSCRIPT
346
3.3 Quantitative assessment of the effect of TiO2-NOAAs on yeast by qRT-PCR From the result of the DNA microarray analysis, the upregulated genes in this
348
study were mainly involved in response to reserving energy sources and an oxidative
349
stress. In particular, we found that the gene related to reserving energy is induced under
350
condition 3, and the gene related to oxidative stress is induced under condition 1 (Fig.
351
8).
EP
352
TE D
M AN U
SC
RI PT
347
Figure 8. Gene expression of the genes related to “response to oxidative stress”
354
(GO:0006979, left) and “energy reserve metabolic process”(GO:0006112, right) in each
355
conditions. Red color indicates up-regulation. 1. adsorbed fraction to TiO2-NOAA
356
under UV; 2. non-adsorbed fraction to TiO2-NOAA under UV; 3. adsorbed fraction to
357
TiO2-NOAA without UV; 4. non-adsorbed fraction to TiO2-NOAA without UV; 5.
358
under UV; 6. untreated control.
AC C
353
23
ACCEPTED MANUSCRIPT
Thus, we tried to assess the stress levels quantitatively using qRT-PCR. The
360
GRE2 and SOD2 genes were used as indicators of the level of response to oxidative
361
stress, and the GSY1 and TPS2 genes were used as indicators of the levels of reserving
362
energy sources.
RI PT
359
The genes involved in oxidative stress were upregulated in UV treatment
364
Conditions 1 (GRE2; 2.0-fold, SOD2; 1.8-fold), 2 (GRE2; 3.1-fold, SOD2; 2.4-fold),
365
and 5 (GRE2; 3.8-fold, SOD2; 2.8-fold) (Fig. 9). This result suggested yeast cells that
366
are exposed to UV suffer oxidative stress. In addition, oxidative stress was caused under
367
UV not presence of TiO2-NOAA. This shows UV stress is more dominant than that
368
caused by TiO2-NOAA.
371
372
373
M AN U
TE D
EP
370
AC C
369
SC
363
374
375
376 24
ACCEPTED MANUSCRIPT
Figure 9. Relative gene expression levels in each treatment condition. The relative
378
expression levels were calculated by the comparative CT method. The ACT1 gene was
379
used as an endogenous control. 1. adsorbed fraction to TiO2-NOAA under UV; 2.
380
non-adsorbed fraction to TiO2-NOAA under UV; 3. adsorbed fraction to TiO2-NOAA
381
without UV; 4. non-adsorbed fraction to TiO2-NOAA without UV; 5. under UV.
SC
RI PT
377
M AN U
382
Genes that were involved in the response to reserving energy sources were
384
upregulated in the treatment conditions of TiO2-NOAA adsorption Conditions 1 (GSY1;
385
1.9-fold, TPS2; 2.9-fold) and 3 (GSY1; 6.3-fold, TPS2; 5.6-fold). This result suggested
386
that yeast cells that are adsorbed by TiO2-NOAAs suffer stress that induced the
387
synthesis of glycogen and trehalose. Comparing the result of Condition 1 with 3, it is
388
possible that UV suppresses the level of stress that causes reserving energy sources.
389
However, these stress responses are not critical compared to that by oxidative stress
390
response.
EP
AC C
391
TE D
383
392
393
394 25
ACCEPTED MANUSCRIPT
395
4. CONCLUSIONS In this study, we assessed the effects of TiO2-NOAAs on yeast under UV
397
irradiation. The result of the DNA microarray analysis, suggested that yeast cells that
398
are adsorbed by TiO2-NOAA under UV irradiation suffer oxidative stress. However, the
399
quantitative PCR results suggested that the oxidative stress is caused not by the TiO2
400
nanoparticles but by UV. It also suggested that TiO2-NOAAs without UV irradiation
401
damage the membranes of yeast cells, which induces yeast cells to synthesize glycogen
402
and trehalose.
405
406
SC
M AN U
protection of UV irradiation like sun cream, not to cause stress.
TE D
404
Thus, we concluded that the role of TiO2-NOAAs on yeast under UV irradiation is
ACKNOWLEDGEMENTS
EP
403
RI PT
396
This study was partially supported by the Long-range Research Initiative program
408
organized by the Japan Chemical Industry Association. We would like to thank Editage
409
(www.editage.jp) for English language editing.
AC C
407
410
411
412 26
ACCEPTED MANUSCRIPT
REFERENCES
414
1
ISO/TS 80004-2:2015 Nanotechnologies --Vocabulary-- Part 2: Nano-objects
415
2
Horie M., Kato H., Fujita K., Endoh S., Iwahashi H., 2012. In vitro evaluation of
416
cellular response induced by manufactured nanoparticles. Chemical Research in
417
Toxicology. 25 (3), pp 605–619
418
3
419
Dioxide in Water Treatment and in Topical Sunscreen. United States Environmental
420
Protection Agency.
421
4
422
Journal of Photochemistry and Photobiology A: Chemistry 108 (1), 1-35
423
5
424
titanium dioxide: spectrum and mechanism of antimicrobial activity. Applied
425
Microbiology and Biotechnology 90: 1847-1868
426
6
427
characterization of engineered nanoscale materials for toxicologic assessment
428
7
429
(TiO2) nano-objects, and their aggregates and agglomerates greater than 100 nm
430
(NOAA) on microbes under UV irradiation. Chemosphere 143, 123–127
SC
RI PT
413
M AN U
EPA, 2009. External Review Draft, Nanomaterial Case Studies: Nanoscale Titanium
TE D
Andrew M., Stephen L. H., 1997. An overview of semiconductor photocatalysis.
AC C
EP
Howard A. F., Iram B. D., Sajnu V., Alex S., 2011. Photocatalytic disinfection using
ISO/TR
13014:2012
Nanotechnologies
-
Guidance
on
physico-chemical
Yamada I., Nomura K., Iwahashi H., Horie M., 2016. The effect of titanium dioxide
27
ACCEPTED MANUSCRIPT
431
8
432
genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium.
433
Environmental Toxicology and Chemistry, Vol. 20, No. 10, pp. 2353–2360
434
9
435
suggesting that trehalose stabilizes membrane structure in the yeast Saccharomyces
436
cerevisiae. Cellular and molecular biology (Noisy-le-grand). Sep; 41(6): 763-9.
437
10
438
the cell wall in Saccharomyces cerevisiae. Yeast 2002; 19: 131–139.
439
11
440
diverse environmental stresses. Yeast Stress Responses Volume 1 of the series Topics in
441
Current Genetics pp 11-70.
442
12
443
peroxisome biogenesis genes. The EMBO Journal Vol. 19 No. 24 pp. 6770-6777.
RI PT
Momose Y., Iwahashi H., 2001. Bioassay of cadmium using a DNA microarray:
M AN U
SC
Iwahashi H., Obuchi K., Fujii S., Komatsu Y., 1995. The correlative evidence
Akalpita U. A., Narayan B. P., 2002. Glycogen - a covalently linked component of
TE D
Gasch A.P., 2002. The environmental stress response: a common yeast response to
AC C
EP
Eduardo L. H., Wayne L. C., Barbara J., Ian A. G., Alison B., 2000. Stress induces
28
ACCEPTED MANUSCRIPT
►The combination effect of TiO2-NOAAs and UV irradiation on yeast was evaluated. ►The oxidative stress is caused not by the TiO2 nanoparticles. ►TiO2-NOAAs damage the membranes of yeast without UV irradiation.
AC C
EP
TE D
M AN U
SC
RI PT
►The yeast adsorbed by TiO2-NOAAs need the reserving energy sources.
S0009-2797(17)31107-9
DOI:
10.1016/j.cbi.2018.08.017
Reference:
CBI 8391
To appear in:
Chemico-Biological Interactions
Received Date: 12 October 2017 Revised Date:
28 July 2018
Accepted Date: 17 August 2018
Please cite this article as: A. Moriyama, I. Yamada, J. Takahashi, H. Iwahashi, Oxidative stress caused by TiO2 nanoparticles under UV irradiation is due to UV irradiation not through nanoparticles, ChemicoBiological Interactions (2018), doi: 10.1016/j.cbi.2018.08.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT
1
Oxidative stress caused by TiO2 nanoparticles under UV irradiation is due to
2
UV irradiation not through nanoparticles
RI PT
3
4
Akihiro Moriyama1*, Ikuho Yamada1, Junko Takahashi2, Hitoshi Iwahashi1
5
1
6
Gifu 501-1193, Japan
7
2
8
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi,
9
Tsukuba, Ibaraki 305-8566, Japan
M AN U
SC
Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu,
Molecular Composite Medicine Research Group, Biomedical Research Institute,
TE D
10
Keywords:
12
Saccharomyces cerevisiae
13
TiO2 nanoparticles
14
ROS generated by TiO2-NOAA
15
DNA microarray
AC C
EP
11
16
17
*Corresponding author.
18
E-mail address: [email protected] (A. Moriyama). 1
ACCEPTED MANUSCRIPT
19
ABSTRACT Currently, nanoparticles are used in various commercial products. One of the most
21
common nanoparticles is titanium dioxide (TiO2). It has a catalytic activity and UV
22
absorption, and generates reactive oxygen species (ROS). This catalytic activity of TiO2
23
nanoparticles was believed to be capable of killing a wide range of microorganisms. In
24
the environment, the unique properties of TiO2 nanoparticles can be maintained;
25
therefore, the increasing use of TiO2 nanoparticles is raising concerns about their
26
environmental risks. Thus, assessment of the biological and ecological effects of
27
TiO2-NOAAs is necessary.
M AN U
SC
RI PT
20
In this study, we assessed the effect of TiO2-NOAAs for S. cerevisiae using DNA
29
microarray. To compare yeast cells under various conditions, six treatment conditions
30
were prepared (1. adsorbed fraction to TiO2-NOAA under UV; 2. non-adsorbed fraction
31
to TiO2-NOAA under UV; 3. adsorbed fraction to TiO2-NOAA without UV; 4.
32
non-adsorbed fraction to TiO2-NOAA without UV; 5. under UV; and 6. untreated
33
control). The result of the DNA microarray analysis, suggested that yeast cells that are
34
adsorbed by TiO2-NOAA under UV irradiation suffer oxidative stress and this stress
35
response was similar to that by only UV irradiation. We concluded that the effect of
AC C
EP
TE D
28
2
ACCEPTED MANUSCRIPT
36
TiO2-NOAAs on yeast cells under UV irradiation is not caused by TiO2-NOAA but UV
37
irradiation.
39
RI PT
38
1. INTRODUCTION
A nanoparticle is defined as a nano-object with all ext\ernal dimensions in the
41
nanoscale (length range approximately from 1 nm to 100 nm), where the lengths of the
42
longest and the shortest axes of the nano-object do not differ significantly1. Compared
43
with a fine particle, the nanoparticle has a larger specific surface area. Therefore,
44
nanoparticles show greater chemical and physical activities, such as ion release,
45
adsorption ability, and production of reactive oxygen species (ROS)2. The unique
46
properties of nanoparticles are beneficial, and thus, the use of nanoparticles is
47
expanding rapidly.
EP
TE D
M AN U
SC
40
TiO2 nanoparticles are used in various commercial products (e.g., self-cleaning
49
surface coatings, light-emitting diodes, solar cells, disinfectant sprays, sporting goods,
50
drinking water treatment agents, and topical sunscreens) because of their catalytic
51
activity and UV absorption (λ<400 nm)3. They are not dissolved in water, and generate
52
ROS under UV irradiation, apparently enabling them to decompose organic substances
53
and damage the function and structure of various cells.4 The catalytic activity of TiO2
AC C
48
3
ACCEPTED MANUSCRIPT
nanoparticles was believed to be capable of killing a wide range of microorganisms (e.g.
55
gram-negative and gram-positive bacteria, including endospores, as well as fungi, algae,
56
protozoa, and viruses)5.
RI PT
54
In culture medium and the water environment, nanoparticles unexceptionally form
58
structures of their aggregates and agglomerates comprising primary particles. These
59
compounds are defined as NOAAs (nano-objects, and their aggregates and
60
agglomerates greater than 100 nm)6. An aggregate is a particle that comprises strongly
61
bonded or fused single primary particles, and an agglomerate is a collection of weakly
62
bound single primary particles, aggregates, or a mix of these. The name NOAA is made
63
by International Standard Organization1 to share the knowledge that nanoparticles may
64
not be protected for aggregation and agglomeration and can not be alone as single
65
molecule in culture medium and water environment. The unique properties of TiO2
66
nanoparticles can be maintained in the environment, and thus the increasing use of TiO2
67
nanoparticles is raising environmental concerns. An assessment of the biological and
68
ecological effects of TiO2-NOAAs is necessary.
AC C
EP
TE D
M AN U
SC
57
69
In our previous study, we assessed the effect of TiO2-NOAAs on microbes using
70
Saccharomyces cerevisiae and Escherichia coli. The TiO2-NOAAs decomposed
71
methylene blue under UV irradiation, which suggested that the TiO2-NOAAs generated 4
ACCEPTED MANUSCRIPT
ROS under UV irradiation. However, the TiO2-NOAAs did not demonstrate growth
73
inhibition in minimal agar medium under UV irradiation; the addition of TiO2-NOAAs
74
to the medium still permitted colony formation under a UV intensity that inactivates
75
microbes. Moreover, the TiO2-NOAAs adsorbed microbes. These results suggested that
76
the amount of ROS generated by TiO2-NOAAs was insufficient to inactivate microbes,
77
and the TiO2-NOAAs might protect microbes from UV7.
M AN U
SC
RI PT
72
In this study, we assessed the effect of TiO2-NOAAs under UV on S. cerevisiae in
79
more detail. We used DNA microarray analysis for qualitative assessment of gene
80
expression profile and carried out a quantitative assessment using qRT-PCR method.
81
These results suggest the effect of TiO2-NOAAs on yeast cells under UV irradiation is
82
not caused by TiO2-NOAA but UV irradiation.
85
86
EP
84
AC C
83
TE D
78
87
88
89 5
ACCEPTED MANUSCRIPT
90
2. EXPERIMENTAL METHODS
91
2.1 Materials TiO2 nanoparticles were obtained from Ishihara Sangyo Kaisha, Ltd. (Japan). Their
93
crystals were in the anatase form, with a primary particle size of 7 nm, and a specific
94
surface area of 316 m2/g.
SC
RI PT
92
96
M AN U
95
2.2 Yeast strains and growth conditions
S. cerevisiae S288C (MATα SUC2 mal mel gal2 CUP1 [cir+]) was used in this
98
study. The yeast was grown in minimal medium (0.67% Difco™ Yeast Nitrogen Base
99
w/o Amino Acids (Becton, Dickinson and Company, NJ, USA), 2% D(+)-Glucose) at
102
103
EP
101
30 °C. A pre-culture was incubated to stationary phase (2 days).
2.3 TiO2 nanoparticle treatment
AC C
100
TE D
97
One mL of pre-culture (5 × 107 cells) and 19 mL of minimal medium were added to
104
petri dishes (φ 90 mm × 20 mm). They were incubated for 6 h at 30 °C with shaking at 60
105
rpm to produce exponentially growing cells. Then, 5.0 mg of TiO2 nanoparticles were
106
added to the petri dishes, with or without UV irradiation (intensity; 0.01 mW/cm2), and
107
the cells were incubated for a further 2h. Treated yeast cells were divided into the 6
ACCEPTED MANUSCRIPT
adsorbed fraction and non-adsorbed fractions. Each of the yeast under different
109
conditions (Condition 1. adsorbed fraction to TiO2-NOAA under UV irradiation, 2.
110
non-adsorbed fraction to TiO2-NOAA under UV irradiation, 3. adsorbed fraction to
111
TiO2-NOAA without UV irradiation, 4. non-adsorbed fraction to TiO2-NOAA without
112
UV irradiation, 5. under UV and 6. untreated control) was harvested by centrifugation at
113
2150×g for 3 min at 4 °C (CAX-371; Tomy Seiko Co., LTD., Tokyo, Japan). Under our
114
TiO2 nanoparticles treatment conditions (30 °C with shaking at 60 rpm), many yeasts
115
aggregated with NOAAs. However, we could yield enough yeast cells for DNA
116
microarray in the supernatant phase over the NOAA and yeast agglomerates (Condition
117
2, 4).
118
2.4 RNA extraction
EP
119
TE D
M AN U
SC
RI PT
108
RNA was extracted from cells using a Fast RNA® Pro Red Kit (MP Biomedicals,
121
Santa Ana, CA, USA), according to the manufacturer’s instructions, with the following
122
exceptions: cell disruption was performed for 10 min using a Multi-Beads
123
Shocker®(Yasui Kikai, Osaka, Japan) and chloroform (Wako Pure Chemical Industries)
124
treatment was performed twice. The extracted RNA was purified using a Qiagen
125
RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s
AC C
120
7
ACCEPTED MANUSCRIPT
instructions. The quality of the purified RNA was verified using an Agilent® 2100
127
Bioanalyzer (Agilent Technologies, CA, USA). The RNA concentration was
128
determined using a Micro-volume Spectrophotometer Q5000 (Tomy Seiko Co., LTD).
RI PT
126
129
132
SC
131
2.5 DNA microarray analysis
DNA microarray analysis was carried out to assess the effect of TiO2-NOAAs on
M AN U
130
yeast qualitatively.
Fluorescent cyanine 3-cytidine triphosphate (CTP)-labeled cRNA was synthesized
134
using a Quick Amp Labeling Kit (Agilent Technologies, CA, USA). The CTP-labeled
135
cRNA was used for hybridization to Yeast (V2) Gene Expression Microarray slides
136
(#G4813A016322, Agilent Technologies) at 65 °C for 17 h. The hybridized microarray
137
slides were washed according to the manufacturer’s instructions and scanned using an
138
Agilent DNA Microarray Scanner (#G2565CA, Agilent Technologies) at a resolution of
139
5 µm. The scanned images were analyzed quantitatively using the Agilent Feature
140
Extraction Software version 10.7.3.1 (Agilent Technologies).
AC C
EP
TE D
133
141
Signals detected from each open reading frame (ORF) were normalized using the
142
25th percentile and quantile methods. The genes classified as upregulated or
143
downregulated were those that passed the Student’s t-test (P< 0.05) and exhibited more 8
ACCEPTED MANUSCRIPT
144
than 2-fold higher intensities or more than 0.5-fold lower intensities than that of
145
negative control. The gene expression profiles were characterized according to the categories of the
147
Munich Information Center for Protein Sequences Genome Research Environment
148
Comprehensive Yeast Genome Database (MIPS GenRE CYGD, http://mips.helmholtz-
149
muenchen.de/genre/proj/yeast/index.jsp) and the Saccharomyces Genome Database
150
(SGD, http://www.yeastgenome.org/).The DNA microarray experiments described in
151
this study were MIAME compliant and the raw data has been deposited in the Gene
152
Expression Omnibus (GEO) database under the accession number GSE99660. For each
153
condition, RNA samples were prepared at least four replications.
154
157
158
EP
156
2.6 Quantitative PCR
qRT-PCR was carried out to assess the effect of TiO2-NOAAs on yeast cells
AC C
155
TE D
M AN U
SC
RI PT
146
quantitatively.
The RNA used for DNA microarray analysis was subjected to reverse transcription
159
(RT) at 37 °C for 15 min using a ReverTra Ace® qPCR RT Master Mix (Toyobo, Osaka,
160
Japan). For quantitative PCR amplification, the Power SYBR® Green Master Mix
161
(Applied Biosystems, CA, USA) and StepOnePlus™ real-time PCR System (Applied 9
ACCEPTED MANUSCRIPT
Biosystems) were used, as described in the manufacturer’s instructions (holding at 95 °C
163
for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, and annealing and
164
extension at 60 °C for 2 min). Relative expression levels were calculated from Ct values.
165
Five pairs of primers were designed for the amplification of the genes upregulated under
166
certain conditions (Table 1), based on the results of DNA microarray analysis. All
167
experiments were performed in triplicate.
Table 1. Sequences of oligonucleotide primers used in this study Gene name
F-primer
GRE2 SOD2 GSY1 TPS2
AAGGTCATCGGTTCTGCCAG CTCCCGCAAACGCAAGAAAA CATATGGGCCATCGTCGTCA CGCAGCTGCCCTACAAAATC
171
CCTTGCCGTGCTTTTGGAAA CTCGTCCAGACTGCCAAACT GGTACCTAAATCGCCCGGAG TGGGTCATCGTCCAGATCCT
ATTGCCGAAAGAATGCAAAAGG CGCACAAAAGCAGAGATTAGAAACA
The ACT1 gene was used as a control housekeeping gene because little difference
AC C
170
R-primer
EP
ACT1
TE D
169
M AN U
168
SC
RI PT
162
172
in its gene expression level was observed between the treatment groups and a control
173
from the result of DNA microarray analysis, and it is widely accepted as a positive control
174
to study the yeast stress response8.
175
10
ACCEPTED MANUSCRIPT
176
3. RESULTS AND DISCUSSION
177
3.1 Conditions for TiO2 nanoparticle treatment
179
To assess the effect of TiO2-NOAAs on yeast cells at the RNA level, initially, an
RI PT
178
appropriate intensity of UV irradiation was determined (Fig. 1).
SC
180
M AN U
181
182
183
184
187
EP
186
TE D
185
Figure 1. Viable yeast cell numbers under each UV irradiation intensity. Yeast cells
189
were incubated under the same conditions with TiO2 nanoparticle treatment (as described
190
in the text) under various UV irradiation intensities. To determine their viabilities, yeast
191
cells were serially diluted and incubated on minimal agar media for 2–3 days.
AC C
188
192
11
ACCEPTED MANUSCRIPT
Under the strongest UV intensity, 0.4 mW/cm2 (A), few yeast cells survived, thus
194
their RNA could not be extracted. Under a UV intensity of 0.01 mW/cm2 (B), UV
195
inactivation of the yeast cells was observed, and their RNA could be extracted. Therefore,
196
RNA extraction for the DNA microarray and qRT-PCR was conducted at a UV intensity
197
of 0.01 mW/cm2.
SC
RI PT
193
The treatment concentration of TiO2 nanoparticle was the same as that used in our
199
previous study7. It is also confirmed that when titanium is added to the minimum
200
medium, it aggregates to form NOAA. Furthermore, it was also confirmed that many
201
yeasts are adsorbed to TiO2 nanoparticles when co-culturing yeast cells (Fig. 2).
AC C
202
EP
TE D
M AN U
198
203
Figure 2. A state of agglomerates of TiO2 nanoparticles and yeast. 1 mL of pre-culture
204
(5 × 107 cells), 19 mL of minimal medium and 5.0 mg of TiO2 nanoparticles were added
205
to the petri dishes (φ 90 mm × 20 mm).
206
12
ACCEPTED MANUSCRIPT
3.2 Qualitative assessment of the effect of TiO2-NOAA on yeast by DNA microarray
208
analysis
209
3.2.1 Overview of altered genes in each treatment condition
RI PT
207
In this study, yeast cells from six different treatment conditions (1. adsorbed
211
fraction to TiO2-NOAA under UV irradiation, 2. non-adsorbed fraction to TiO2-NOAA
212
under UV irradiation, 3. adsorbed fraction to TiO2-NOAA without UV irradiation, 4.
213
non-adsorbed fraction to TiO2-NOAA without UV irradiation, 5. under UV and 6.
214
untreated control) were analyzed using a DNA microarray. From the results, 3236, 1545,
215
2914, 1596, and 2493 ORFs that passed the Student’s t-test (P < 0.05) in Conditions 1, 2,
216
3, 4, and 5, respectively, were obtained by the comparison with untreated control of 6
217
(Table 2).
Table 2. The number of the up and downregulated genes in each treatment condition
AC C
219
EP
218
TE D
M AN U
SC
210
≧2-fold
≦0.5-fold
275
425
2. Non-adsorbed fraction of TiO2-NOAA (with UV)
9
109
3. Adsorbed fraction of TiO2-NOAA (without UV)
219
287
4. Non-adsorbed fraction of TiO2-NOAA (without UV)
18
121
5. Under UV
59
223
Treatment conditions
1. Adsorbed fraction of TiO2-NOAA (with UV)
220 13
ACCEPTED MANUSCRIPT
Among them, 275, 9, 219, 18, and 59 genes exhibited more than 2-fold higher
222
intensities, and 425, 109, 287, 121, and 223 genes exhibited more than 0.5-fold lower
223
intensities under Conditions 1, 2, 3, 4, and 5, respectively. The highest number of
224
significantly altered genes was in Conditions 1 (2-fold; 275, 0.5-fold; 425), 3 (2-fold;
225
219, 0.5-fold; 287), and 5 (2-fold; 59, 0.5-fold; 223).
SC
RI PT
221
These results showed that the expressions of genes in yeast cells that were adsorbed
227
by TiO2-NOAAs (Conditions 1 and 3) were altered more than those in yeast cells that
228
were not adsorbed by TiO2-NOAAs (Conditions 2 and 4). Further, a significant number
229
of genes were altered by UV irradiation in the adsorbed fraction (Condition 1). Thus,
230
these results suggested that yeast cells suffer stress from TiO2-NOAA and from UV.
233
234
TE D
EP
232
AC C
231
M AN U
226
235
3.2.2 The genes up and downregulated by TiO2-NOAAs under UV irradiation and
236
their functional distribution
237
The yeast cells in the fraction adsorbed to TiO2-NOAA under UV irradiation
238
showed the highest number of genes with altered expressions among all treatment 14
ACCEPTED MANUSCRIPT
conditions (Table 2; Condition 1).From the DNA microarray analysis, 3236 ORFs were
240
obtained that passed the Student’s t-test (P < 0.05). Among them, 275 genes were
241
upregulated by more than 2-fold and 425 were downregulated by more than 0.5-fold by
242
TiO2-NOAA treatment under UV irradiation. The up and downregulated genes were
243
characterized using MIPS GenRE CYGD. The results showed that the function of
244
upregulated genes were classified with the high probability into the categories of
245
“energy (10.3%)”, “cell rescue (7.4%)” and “metabolism (6.0%)”. Subcategories of
246
“Regulation of glycolysis and gluconeogenesis” and “metabolism of energy reserve (e.g.
247
glycogen, trehalose)” were characterized particularly in the category of “energy” (Fig.
248
3).
AC C
EP
TE D
M AN U
SC
RI PT
239
249
15
ACCEPTED MANUSCRIPT
Figure 3. Functional categories of up-regulated and down-regulated ORFs in
251
TiO2-NOAA treatment under UV irradiation (Condition 1). ORFs up-regulated or
252
down-regulated more than 2 fold were counted. The denominators and numerators in
253
parentheses indicate number of total ORFs and up-regulated or down-regulated ORFs in
254
each category, respectively.
SC
.
M AN U
255
RI PT
250
Trehalose stabilizes the membrane structure and glycogen is a component of the
257
cell wall in S. cerevisiae9, 10. Subcategories of “oxidative stress response” and “oxygen
258
and radical detoxification” were characterized particularly in the category of “cell
259
rescue”.
TE D
256
By contrast, downregulated genes were characterized particularly in the categories
261
of “protein synthesis (16%)”, “transcription (8%)”, and “protein with binding function
262
(6%)” (Fig. 3). Genes involved in protein synthesis are downregulated in response to
263
diverse environmental stresses11. Thus, this result suggested that yeast cells adsorbed by
264
TiO2-NOAAs under UV irradiation were significantly stressed.
265
266
AC C
EP
260
The products of the upregulated genes were localized significantly at the “peroxisome” (Fig. 4).
16
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
267
Figure 4. Locational categories of the gene products upregulated by TiO2-NOAA
269
treatment under UV irradiation (Condition 1). ORFs up-regulated more than 2 fold were
270
counted. The denominators and numerators in parentheses indicate number of total
271
ORFs and up-regulated ORFs in each category, respectively.
EP
272
TE D
268
It is reported that some peroxisomal genes are induced in response to oxidative stress12.
274
Thus, this localization and induction of subcategories related to oxidative stress
275
suggested that yeast cells that were adsorbed by TiO2-NOAA under UV irradiation
276
suffered oxidative stress. Additionally, localization with high probability at
277
“Extracellular/secretion proteins” and “Eukaryotic plasma membrane” were observed.
AC C
273
278
17
ACCEPTED MANUSCRIPT
279
3.2.3 Genes upregulated and downregulated by TiO2-NOAAs without UV
280
irradiation and their functional distribution The expressions of a significant number of genes in yeast cells in the adsorbed
282
fraction to TiO2-NOAAs even without UV irradiation were altered in all treatment
283
conditions (Table 2; Condition 3). From the DNA microarray analysis, 2914 ORFs were
284
obtained that passed the Student’s t-test (P < 0.05). Among them, 219 genes were
285
upregulated by more than 2-fold and 287 genes were downregulated by more than
286
0.5-fold by TiO2-NOAAs treatment without UV irradiation. The functions of the
287
upregulated genes were classified with high probability into the categories of “energy
288
(8.9%)”, “metabolism (5.1%)”, and “cell rescue (5.7%)”. Subcategories of “metabolism
289
of energy reserve (e.g. glycogen, trehalose)” and “regulation of glycolysis and
290
gluconeogenesis” were classified particularly in the category of “energy”, and
291
subcategories of “C-compound and carbohydrate metabolism” was classified
292
particularly in the category of “metabolism” (Fig. 5).
AC C
EP
TE D
M AN U
SC
RI PT
281
18
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
293
Figure 5. Functional categories of the genes upregulated and downregulated by
295
TiO2-NOAA treatment without UV irradiation (Condition 3). ORFs up-regulated or
296
down-regulated more than 2 fold were counted. The denominators and numerators in
297
parentheses indicate number of total ORFs and up-regulated or down-regulated ORFs in
298
each category, respectively.
EP
299
TE D
294
By contrast, the functions of the downregulated genes were classified as
301
“transcription (8.3%)”, “protein synthesis (9.5%)”, and “cell cycle and DNA processing
302
(6.2%)” (Fig. 5). This result showed that the yeast that were adsorbed by TiO2-NOAA
303
without UV irradiation suffered stress. The products of the upregulated genes were
304
localized as “Eukaryotic plasma membrane” (Fig. 6).
AC C
300
305 19
RI PT
ACCEPTED MANUSCRIPT
306
Figure 6. Locational categories of the genes upregulated by TiO2-NOAA
308
treatment under UV irradiation (Condition 3). ORFs up-regulated more than 2 fold were
309
counted. The denominators and numerators in parentheses indicate number of total
310
ORFs and up-regulated ORFs in each category, respectively.
M AN U
SC
307
TE D
311
These results showed that membrane structures of yeast cells that were adsorbed
313
by TiO2-NOAA without UV irradiation were damaged. Taken together, these results
314
imply yeast cells that are adsorbed by TiO2-NOAA without UV irradiation suffer stress
315
at their membrane structures, resulting in the induction of genes related to the synthesis
316
of glycogen and trehalose.
AC C
317
EP
312
318
20
ACCEPTED MANUSCRIPT
319
3.2.4 Genes upregulated and downregulated by UV irradiation and their functional
320
distribution The expressions of a large number of genes in yeast cells under UV without
322
TiO2-NOAAs were altered among all treatment conditions (Table 2; Condition 5). From
323
the DNA microarray analysis, 2493 ORFs were obtained that passed the Student’s t-test
324
(P < 0.05). Among them, 59 genes were upregulated by more than 2-fold and 223 genes
325
were downregulated by more than 0.5-fold by UV irradiation. The functions of the
326
upregulated genes were classified with high probability into the categories of “energy
327
(2.5%)”, “cell rescue (1.7%)”, and “metabolism (3.6%)” (Fig. 7). The subcategory of
328
“metabolism of energy reserve” was characterized particularly in the category of
329
“energy”, and subcategories of “oxygen and radical detoxification” were characterized
330
particularly in the category of “cell rescue”. This result showed that yeast cells that are
331
irradiated by UV suffer oxidative stress. The localizations of the products of the
332
upregulated genes were not found because of the insignificant number of genes in this
333
category.
AC C
EP
TE D
M AN U
SC
RI PT
321
21
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
334
Figure 7. Functional categories of the genes upregulated by UV irradiation
336
(Condition 5). ORFs up-regulated or down-regulated more than 2 fold were counted.
337
The denominators and numerators in parentheses indicate number of total ORFs and
338
up-regulated or down-regulated ORFs in each category, respectively.
EP
339
TE D
335
By contrast, the functions of the downregulated genes were classified with high
341
probability into the categories of “transcription (3.5%)” and “protein synthesis (3.2%)”
342
(Fig. 7). These results showed that yeast cells that were irradiated by UV suffered stress.
343
These results suggested that yeast cells irradiated by UV suffer oxidative stress
AC C
340
344
because genes involved in the response to oxidative stress were upregulated, and genes
345
involved in protein synthesis were downregulated.
22
ACCEPTED MANUSCRIPT
346
3.3 Quantitative assessment of the effect of TiO2-NOAAs on yeast by qRT-PCR From the result of the DNA microarray analysis, the upregulated genes in this
348
study were mainly involved in response to reserving energy sources and an oxidative
349
stress. In particular, we found that the gene related to reserving energy is induced under
350
condition 3, and the gene related to oxidative stress is induced under condition 1 (Fig.
351
8).
EP
352
TE D
M AN U
SC
RI PT
347
Figure 8. Gene expression of the genes related to “response to oxidative stress”
354
(GO:0006979, left) and “energy reserve metabolic process”(GO:0006112, right) in each
355
conditions. Red color indicates up-regulation. 1. adsorbed fraction to TiO2-NOAA
356
under UV; 2. non-adsorbed fraction to TiO2-NOAA under UV; 3. adsorbed fraction to
357
TiO2-NOAA without UV; 4. non-adsorbed fraction to TiO2-NOAA without UV; 5.
358
under UV; 6. untreated control.
AC C
353
23
ACCEPTED MANUSCRIPT
Thus, we tried to assess the stress levels quantitatively using qRT-PCR. The
360
GRE2 and SOD2 genes were used as indicators of the level of response to oxidative
361
stress, and the GSY1 and TPS2 genes were used as indicators of the levels of reserving
362
energy sources.
RI PT
359
The genes involved in oxidative stress were upregulated in UV treatment
364
Conditions 1 (GRE2; 2.0-fold, SOD2; 1.8-fold), 2 (GRE2; 3.1-fold, SOD2; 2.4-fold),
365
and 5 (GRE2; 3.8-fold, SOD2; 2.8-fold) (Fig. 9). This result suggested yeast cells that
366
are exposed to UV suffer oxidative stress. In addition, oxidative stress was caused under
367
UV not presence of TiO2-NOAA. This shows UV stress is more dominant than that
368
caused by TiO2-NOAA.
371
372
373
M AN U
TE D
EP
370
AC C
369
SC
363
374
375
376 24
ACCEPTED MANUSCRIPT
Figure 9. Relative gene expression levels in each treatment condition. The relative
378
expression levels were calculated by the comparative CT method. The ACT1 gene was
379
used as an endogenous control. 1. adsorbed fraction to TiO2-NOAA under UV; 2.
380
non-adsorbed fraction to TiO2-NOAA under UV; 3. adsorbed fraction to TiO2-NOAA
381
without UV; 4. non-adsorbed fraction to TiO2-NOAA without UV; 5. under UV.
SC
RI PT
377
M AN U
382
Genes that were involved in the response to reserving energy sources were
384
upregulated in the treatment conditions of TiO2-NOAA adsorption Conditions 1 (GSY1;
385
1.9-fold, TPS2; 2.9-fold) and 3 (GSY1; 6.3-fold, TPS2; 5.6-fold). This result suggested
386
that yeast cells that are adsorbed by TiO2-NOAAs suffer stress that induced the
387
synthesis of glycogen and trehalose. Comparing the result of Condition 1 with 3, it is
388
possible that UV suppresses the level of stress that causes reserving energy sources.
389
However, these stress responses are not critical compared to that by oxidative stress
390
response.
EP
AC C
391
TE D
383
392
393
394 25
ACCEPTED MANUSCRIPT
395
4. CONCLUSIONS In this study, we assessed the effects of TiO2-NOAAs on yeast under UV
397
irradiation. The result of the DNA microarray analysis, suggested that yeast cells that
398
are adsorbed by TiO2-NOAA under UV irradiation suffer oxidative stress. However, the
399
quantitative PCR results suggested that the oxidative stress is caused not by the TiO2
400
nanoparticles but by UV. It also suggested that TiO2-NOAAs without UV irradiation
401
damage the membranes of yeast cells, which induces yeast cells to synthesize glycogen
402
and trehalose.
405
406
SC
M AN U
protection of UV irradiation like sun cream, not to cause stress.
TE D
404
Thus, we concluded that the role of TiO2-NOAAs on yeast under UV irradiation is
ACKNOWLEDGEMENTS
EP
403
RI PT
396
This study was partially supported by the Long-range Research Initiative program
408
organized by the Japan Chemical Industry Association. We would like to thank Editage
409
(www.editage.jp) for English language editing.
AC C
407
410
411
412 26
ACCEPTED MANUSCRIPT
REFERENCES
414
1
ISO/TS 80004-2:2015 Nanotechnologies --Vocabulary-- Part 2: Nano-objects
415
2
Horie M., Kato H., Fujita K., Endoh S., Iwahashi H., 2012. In vitro evaluation of
416
cellular response induced by manufactured nanoparticles. Chemical Research in
417
Toxicology. 25 (3), pp 605–619
418
3
419
Dioxide in Water Treatment and in Topical Sunscreen. United States Environmental
420
Protection Agency.
421
4
422
Journal of Photochemistry and Photobiology A: Chemistry 108 (1), 1-35
423
5
424
titanium dioxide: spectrum and mechanism of antimicrobial activity. Applied
425
Microbiology and Biotechnology 90: 1847-1868
426
6
427
characterization of engineered nanoscale materials for toxicologic assessment
428
7
429
(TiO2) nano-objects, and their aggregates and agglomerates greater than 100 nm
430
(NOAA) on microbes under UV irradiation. Chemosphere 143, 123–127
SC
RI PT
413
M AN U
EPA, 2009. External Review Draft, Nanomaterial Case Studies: Nanoscale Titanium
TE D
Andrew M., Stephen L. H., 1997. An overview of semiconductor photocatalysis.
AC C
EP
Howard A. F., Iram B. D., Sajnu V., Alex S., 2011. Photocatalytic disinfection using
ISO/TR
13014:2012
Nanotechnologies
-
Guidance
on
physico-chemical
Yamada I., Nomura K., Iwahashi H., Horie M., 2016. The effect of titanium dioxide
27
ACCEPTED MANUSCRIPT
431
8
432
genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium.
433
Environmental Toxicology and Chemistry, Vol. 20, No. 10, pp. 2353–2360
434
9
435
suggesting that trehalose stabilizes membrane structure in the yeast Saccharomyces
436
cerevisiae. Cellular and molecular biology (Noisy-le-grand). Sep; 41(6): 763-9.
437
10
438
the cell wall in Saccharomyces cerevisiae. Yeast 2002; 19: 131–139.
439
11
440
diverse environmental stresses. Yeast Stress Responses Volume 1 of the series Topics in
441
Current Genetics pp 11-70.
442
12
443
peroxisome biogenesis genes. The EMBO Journal Vol. 19 No. 24 pp. 6770-6777.
RI PT
Momose Y., Iwahashi H., 2001. Bioassay of cadmium using a DNA microarray:
M AN U
SC
Iwahashi H., Obuchi K., Fujii S., Komatsu Y., 1995. The correlative evidence
Akalpita U. A., Narayan B. P., 2002. Glycogen - a covalently linked component of
TE D
Gasch A.P., 2002. The environmental stress response: a common yeast response to
AC C
EP
Eduardo L. H., Wayne L. C., Barbara J., Ian A. G., Alison B., 2000. Stress induces
28
ACCEPTED MANUSCRIPT
►The combination effect of TiO2-NOAAs and UV irradiation on yeast was evaluated. ►The oxidative stress is caused not by the TiO2 nanoparticles. ►TiO2-NOAAs damage the membranes of yeast without UV irradiation.
AC C
EP
TE D
M AN U
SC
RI PT
►The yeast adsorbed by TiO2-NOAAs need the reserving energy sources.