Chemosphere 80 (2010) 28–34
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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Toxic effect of heavy metal terbium ion on cell membrane in horseradish
Lihong Wang c, Qing Zhou a,c,*, Bo Zhao b, Xiaohua Huang b,**
a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Environmental Science, Nanjing Normal University, Nanjing 210097, China
c
School of Environmental and Civil Engineering, Jiangnan University, Wuxi 214122, China
b
a r t i c l e
i n f o
Article history:
Received 4 December 2009
Received in revised form 23 March 2010
Accepted 29 March 2010
Available online 21 April 2010
Keywords:
Heavy metals
Terbium
Cell membrane
Fatty acids
Membrane lipid peroxidation
a b s t r a c t
In order to understand the toxic mechanism of terbium ion (Tb(III)) on plants, the subcellular distribution
of Tb(III) in horseradish, the effect of Tb(III) on the composition of the fatty acids in the cell membrane,
the peroxidation of membrane lipid, the morphological character of protoplast, the cellular ultrastructure
in horseradish were investigated using transmission electron microscopic autoradiography, molecular
dynamics simulation, gas chromatography, scanning electron microscopy and transmission electron
microscopy. The results show that Tb(III) could not enter the horseradish cell in the presence of 5 mg L 1
Tb(III) and it was distributed on the cell wall and plasma membrane. The behavior caused the decrease in
the contents of unsaturated fatty acids and then the increase in the peroxidation of membrane lipid.
Thereby the structure of horseradish cell was damaged. The effects of Tb(III) mentioned above were
aggravated in horseradish treated with 60 mg L 1 Tb(III) because Tb(III) could enter the horseradish cell.
It was a possible cytotoxic mechanism of Tb(III) on horseradish.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Many positive effects of rare earth elements (REEs) on the yield
and quality of crops have been observed (Hu et al., 2004). Therefore, REEs have been widely used in agriculture for the growth of
crop. More than 100 crops have been treated with 3 lM to
50 mM REEs using the methods, such as the foliar sprays, seed
pre-treatments, and adding REEs into solid or liquid root fertilizers
(Hu et al., 2004). Moreover, the increase in the use of REEs in the
variety of nonnuclear industries, such as ceramic manufacture,
glass production, metallurgy, and environmental protection also
leads to the environmental pollution of REEs (Wood, 1993; Verplanck et al., 2005), and then the accumulation of REEs in the crops
and soils (Wang et al., 2001; Xu et al., 2003; Tyler, 2004). In China,
the content of REEs accumulated in plants is about 4–168 mg kg 1
(Xiong, 1995). The content of REEs in soil is about 76–629 mg kg 1,
and the maximum content of soluble or available REEs, which can
be utilized by plants is 200 mg kg 1 (Xiong, 1995). The REEs accumulated in the crops and soil can transfer through the food chain to
* Corresponding author at: The Key Laboratory of Industrial Biotechnology,
Ministry of Education, Jiangnan University, Wuxi 214122, China. Tel.: +86 510
85326581, +86 25 85891651.
** Correspondence to: X. Huang, Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Environmental Science, Nanjing Normal University,
Nanjing 210097, China. Tel.: +86 510 85326581, +86 25 85891651.
E-mail addresses: [email protected] (Q. Zhou), [email protected]
(X. Huang).
0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2010.03.040
humans, affecting the food safety and the humans health (Zhu
et al., 1996).
Velasco et al. (1979) have suggested that at the high concentration of REEs (>100 mg kg 1 of cerium sulfate), it could inhibit the
physiological metabolism of plant, pose a potential hazard to the
plant (Velasco et al., 1979). The mechanism of such toxic effect is
still unknown despite the efforts devoted by many researchers
(Diatloff et al., 1995; Zeng et al., 2006; Wang et al., 2008). Our previous reports have indicated that the treatment with REEs leads to
the decrease in the activity of protective enzymes (Guo et al.,
2008a; Wang et al., 2009) and then the inhibition of the physiological processes, such as the photosynthesis (Wang et al., 2009). The
results are consisted of the physiological and biochemical base of
the environmental biological effect of REEs. Cells are the structural
and functional unit of plant, and control all kinds of the physiological and biochemical processes in plant (Buchanan et al., 2000). As
we known, cells require membranes for their existence. Especially,
the plasma membrane has been regarded as the first ‘‘living” structure, which is a target for the toxicity of heavy metal on plants
(Howlett and Avery, 1997). The effect of REEs on the cell membrane has been reported (Zheng et al., 2000; Li et al., 2003a,b),
but the effect mechanism is unclear. Therefore, it is necessary to
investigate the effect mechanism of REEs on the cell membrane
of plant.
Terbium (Tb) is one of the REEs and heavy metal elements. It is
existed in the environment and REEs fertilizer. The radioactive isotope of Tb is easy to be obtained and its radioactive cycle is suitable
for the experiment of transmission electron microscopic radioau-
29
L. Wang et al. / Chemosphere 80 (2010) 28–34
tography (EMARG), which is used for the research on the distribution of REEs in the plant cells. Meanwhile, Tb(III) mainly shows the
toxic effect on plants. Therefore, Tb(III) can be used as a representative element to investigate the effect of REEs pollution (Guo et al.,
2008a; Wang et al., 2008, 2009). Horseradish (Armoracia rusticana)
is an important economic crop, and it is a perennial herb of the
brassicaceae family. It contains specific pungency and lots of nutrients. Therefore, it is widely used as a flavoring ingredient in
America, Europe, Japan, Korea, etc. (Veitch, 2004). Meanwhile,
horseradish is also an important economic crop. It contains a great
deal of horseradish peroxidase that is used as immunoenzymic kits
and organic synthesis for the biotransformation of various drugs
and chemicals (Veitch, 2004). Therefore, the investigation about
REEs ions action on horseradish has typically significance for environmental pollution and food safety.
In this study, horseradish as a model plant, the toxic effect of
Tb(III) on the cell membrane in horseradish were investigated
using the optimizing combination of EMARG, molecular dynamics
simulation, gas chromatography (GC), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results can provide some reference for understanding the cytotoxic
mechanism of REEs on plants. It was suggested that heavy REEs
Table 1
The programmed temperature in the GC measurement.
Temperature (°C)
Retention time (min)
Heating rate (°C min
120
190
220
2
0.1
15
–
10
15
1
)
pose the potential threat to the environmental health and food
safety.
2. Materials and methods
2.1. Materials and treatment
The 160Tb4O7 powders were obtained from Beijing Atom High
Tech Co., Ltd. in China. Their specific activity concentration was
1.2 107 Bq mg 1 and their radiochemical purity was greater than
95%. The 160Tb(III) solution was prepared according to the literature (Akaboshi et al., 2000). The TbCl36H20 powders were purchased from Aldrich Chemical Co. All the other reagents used
were the analytical grade.
Horseradishes were obtained from the Planting Base of Horseradish in Jiangsu province (Dafeng, China). Horseradishes were
planted and cultured in terms of our previous methods (Wang
et al., 2009). The 150 mL 5 or 60 mg L 1 Tb(III) solution was
sprayed once on the leaves of the 7-month-old horseradish for
48 h, while the same amount of deionized water was sprayed on
the leaves of horseradish for the control. All treatments were performed in triplicate. The fresh leaves treated without and with
Tb(III) solution were sampled for the determination of the composition of fatty acids, and the observation of protoplast and subcellular structure.
2.2. Measurement of EMARG of
160
Tb(III) in horseradish leaves
The one leaf was treated for 48 h with Tb(III) solution, in which
the total Tb(III) concentration is 5 or 60 mg L 1 and the radioactivity of 160Tb(III) is 55 lCi mL 1. Then, the leaf was cut as the regular
Fig. 1. The EMARG images of the horseradish treated with (A) 0, (B) 5 and (C and D) 60 mg L
membrane, V: vesicle.
1
Tb(III). Chl: chloroplast, CW: cell wall, PM: plasma membrane, T: vesicle
30
L. Wang et al. / Chemosphere 80 (2010) 28–34
ultra-thin section with 1.5 2 mm size and 60 nm thickness for
the electron microscopic observation with the Reichert Ultracut E
ultramicrotome. The nuclear emulsion (Technical Institute of Physics and Chemistry, Chinese Academy of Science) was coated on the
leaf section prior to the development and fixation. Then the distri-
Table 2
Effect of Tb(III) on membrane lipid fatty acids in horseradish.
Fatty acid composition
Tb(III) concentration
0 mg L
1
5 mg L
1
60 mg L
1
Fatty acid composition
Myristic acid (14:0)
Palmitic acid (16:0)
Palmitoleic acid (16:1)
Margaric acid (17:0)
Stearic acid (18:0)
Oleic acid (18:1)
Linoleic acid (18:2)
Linolenic acid (18:3)
1.53 ± 0.02a
1.95 ± 0.04
20.79 ± 0.02
7.16 ± 0.09
1.65 ± 0.00
3.58 ± 0.06
6.05 ± 0.08
57.29 ± 0.24
2.75 ± 0.03
1.51 ± 0.01
16.88 ± 0.05
7.81 ± 0.03
1.52 ± 0.01
2.92 ± 0.02
9.13 ± 0.06
57.47 ± 0.34
Saturated fatty acid
12.29 ± 0.15
13.59 ± 0.08*
29.36 ± 2.24**
87.71 ± 0.43
86.41 ± 0.47
*
70.6 ± 0.49**
208.34 ± 2.56
210.47 ± 1.87
Unsaturated fatty acid
b
IUFA
16.87 ± 0.08
2.74 ± 0.03
22.14 ± 0.03
7.57 ± 0.07
2.18 ± 0.06
3.53 ± 0.05
4.26 ± 0.06
40.70 ± 0.35
156.29 ± 0.98**
a
Data are the mean ± SD, n = 3, and data are the percentage content of a single
fatty acid in the total fatty acids.
b
Index of unsaturated fatty acid = [16:1% + 18:1% + (18:2%) 2 + (18:3) 3] 100%.
*
Treatment differ from control, P < 0.05.
**
Treatment differ from control, P < 0.02.
bution of rare earth ions was observed with an H-600 TEM (H-600A-2, Hitachi Ltd., Japan) (Feig and Harting, 1992).
2.3. Molecular dynamics simulation
The charge distribution on the surface of H-ATPase, horseradish
peroxidase (HRP), calmodulin, Ca-binding protein (obtained from
http://www.rcsb.org/) in the environment at pH 6.5–7.5 and
0.1 M ionic strength was determined with the commercial molecular simulation software, Discovery Studio 2.1 (Discovery Studio,
Version 2.1, Accelrys Inc., San Diego, CA, 2008, http://www.accelrys.com/) (Rao et al., 2008).
2.4. Lipid extraction and analysis of fatty acids
Fatty acids were extracted and quantified in terms of the previous method (Bligh and Dyer, 1959; Christie, 1989). One gram fresh
horseradish leaves were dried at 105 °C for 5 min, then 10 mL
mixed solution of chloroform and methanol (2/1, v/v) was added
and homogenized. After addition of 2.0 mL water, the chloroform
layer was separated and the solvent was evaporated. Then, 1 mL
toluene and 2 mL 1% H2SO4 in methanol were added. The mixture
was left overnight at 50 °C. Five milliliter 5% sodium chloride was
added and the fatty acid methyl esters formed were extracted with
hexane (2.5 mL) and quantified with GC (Shimadzu GC-2010, Japan) equipped with a flame ionization detector and a capillary column PEG20 M (30 m 0.25 mm). Separation of fatty acid methyl
esters was performed at a programmed temperature shown in
the Table 1. The standard reagents of fatty acids were purchased
Fig. 2. The charge distribution on the surface of (A) H-ATPase, (B) HRP, (C) calmodulin, and (D) Ca-binding protein under the condition of pH 6.5–7.5, ionic strength 0.1 M;
blue represents positive charge, and red represents negative charge. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
L. Wang et al. / Chemosphere 80 (2010) 28–34
from Sigma Company. Peak identification was carried out by comparison with the authentic standards.
2.5. Measurement of SEM
Horseradish protoplasts were isolated according to the previous
procedure (Kovtun et al., 2000; Wang et al., 2005). The isolated
protoplast was fixed for 2 h with 1% glutaraldehyde in 50 mM buffer solution (0.8 M mannitol and 50 mM Tris–HCl, pH 6.8). The
samples were washed several times with the 50 mM buffer solution and then dried supercritically, shadowed with gold, and examined in a JSM-5600LV SEM (JEOL, Japan).
2.6. TEM measurement
The 1.5 2 mm sections from horseradish leaves were cut in
2.5% glutaraldehyde in phosphate buffer, pH 7.4, containing
20 mM sucrose to maintain organellar integrity and fixed for 1 h
at room temperature. They were postfixed in 1% aqueous osmium
tetroxide for 30 min at room temperature, washed in PBS (phosphate-buffered saline, pH 7.4) three times, dehydrated in a 50–
100% ethanol series and embedded in Spurr’s low-viscosity resin
(Spurr, 1969), according to conventional procedures. Ultra-thin
sections were stained with uranyl acetate and lead citrate (Hayat,
1986) and examined using a Jeol 1010 transmission electron
microscope at 75 kV.
31
5 mg L 1 Tb(III) were increased comparing with that of the control
horseradish. The opposite effect was observed in the percentage
contents of unsaturated fatty acids (16:1, 18:1, 18:2, 18:3), especially linolenic acid (18:3). The index of unsaturated fatty acids
(IUFA) in horseradish treated with 60 mg L 1 Tb(III) were 25% lower than that of the control horseradish (Table 2).
3.3. Effect of Tb(III) on structure of horseradish cell
Fig. 3 shows the SEM images of the protoplast in horseradish
treated without and with Tb(III). The protoplast of horseradish
treated without Tb(III) was the spherical with the well-organized
internal structure (Fig. 3A). The protoplast of horseradish treated
with 5 mg L 1 Tb(III) was swelled (Fig. 3B). The ‘‘bleb-like” or
‘‘hole-like” structures on the outside of the protoplast were observed (Fig. 3B). When the concentration of Tb(III) was increased
from 5 mg L 1 to 60 mg L 1, the ‘‘bleb-like” or ‘‘hole-like” structures on the surface of protoplast were increased, and the protoplast was broken, twisted and deformed (Fig. 3C). Fig. 4 shows
the cellular ultrastructure of horseradish treated without and with
2.7. Statistical analysis
The difference between groups was statistically analyzed by the
one-way ANOVA test (Ke et al., 2003).
3. Results
3.1. Distribution of Tb(III) in horseradish cell and charge distribution
on surface of proteins in plasma membrane
Fig. 1 shows the EMARG images of horseradish treated without
and with Tb(III) containing 160Tb(III). It was observed that 160Tb(III)
could not enter the horseradish cell, and they were distributed on
the cell wall and plasma membrane of horseradish treated with
5 mg L 1 Tb(III) containing 160Tb(III) (Fig. 1B). When horseradish
was treated with 60 mg L 1 Tb(III) containing 160Tb(III), most of
160
Tb(III) were still deposited on the cell wall and plasma membrane (Fig. 1C). Many of 160Tb(III) entered the horseradish cell
(Fig. 1C and D), and they were mainly deposited and distributed
on the chloroplast membrane, in the chloroplast, occasionally in
the vacuole and cytoplasm (Fig. 1C and D). Fig. 2 shows the images
of the charge distribution on the surface of H-ATPase, HRP, calmodulin and Ca-binding protein in the plasma membrane. It was observed that a large amount of the negative charges were
distributed on the surface of H-ATPase (Fig. 2A), HRP (Fig. 2B), calmodulin (Fig. 2C), Ca-binding protein (Fig. 2D).
3.2. Effect of Tb(III) on composition of fatty acids in membrane lipid
Table 2 shows the percentage content of fatty acids in the membrane lipid of horseradish treated without and with Tb(III). The
total fatty acids were composed of myristic acid (14:0), palmitic
acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), stearic
acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid
(18:3). It was observed that the composition of fatty acids in horseradish treated with Tb(III) was not changed comparing with that of
the control horseradish. The total percentage contents of saturated
fatty acids (14:0, 16:0, 17:0, 18:0) in horseradish treated with
Fig. 3. The SEM images of protoplast in horseradish treated with (A) 0, (B) 5 and (C)
60 mg L 1 Tb(III).
32
L. Wang et al. / Chemosphere 80 (2010) 28–34
Fig. 4. The TEM images of the subcellular structure in horseradish treated with (A) 0, (B) 5 and (C and D) 60 mg L
membrane, PV: phagocytic vesicle, T: vesicle membrane, V: vesicle.
Tb(III). The arrangement of the cell in the control horseradish was
regular, and the cell wall and cell membrane were clear and integrated (Fig. 4A). Meanwhile, the chloroplast structure was integral
and in ellipse (Fig. 4A). The plasmolysis in the cell of horseradish
treated with 5 mg L 1 Tb(III) (Fig. 4B) was observed. Meanwhile,
the plasma membrane and chloroplast membrane were damaged,
and the lamellar structures of thylakoid were loose and disorderly
(Fig. 4B). The plasmolysis and the damage of the cell membrane
(plasma membrane, tonoplast and chloroplast membrane) in
horseradish treated with 60 mg L 1 Tb(III) (Fig. 4C and D) were
more obvious than that of horseradish treated with 5 mg L 1
Tb(III). The chloroplast membrane was completely broken, the thylakoid was in radial shape, and the matrix was flowed out of the
chloroplast.
4. Discussion
In order to understand the effect of Tb(III) on the cell membrane,
it is necessary to investigate the chemical behavior of Tb(III) in the
plant cell, such as whether Tb(III) can enter the plant cell and how
to be distributed in the plant cell. There are two distinct opinions on
whether REEs could enter the plant cell. One is that REEs cannot enter the cytoplasm in plant, and they are only located in the apoplasm, i.e., cell wall or the outside of plasma membrane (Taylor
and Hall, 1978; Zhou and Liu, 1998; Wei, 2001). On the contrary, another opinion is that REEs can enter the plant cell, and they firmly
bind to the membrane of protoplasm, chloroplast, mitochondrion,
cytoplast, vacuole or karyon in the plant cells (Gschneider and Eyr-
1
Tb(III). Chl: chloroplast, CW: cell wall, PM: plasma
ing, 1990; Zhang et al., 2000, 2001; Gao et al., 2003; Wei et al., 2005;
Guo et al., 2007; Li et al., 2008). The difference depends on the plant
species, experimental conditions, measurement methods and the
concentration of REEs. Our experimental results clearly demonstrated that Tb(III) could not enter the horseradish cell in the presence of 5 mg L 1 Tb(III), while it could enter the horseradish cell
through plasmodesma (Shan et al., 2003) or with the help of some
carriers, such as protein and hormone (Ni, 1995) in the presence of
60 mg L 1 Tb(III). Moreover, Tb(III) could be distributed in the plasma membrane of horseradish treated with the low and high concentration of Tb(III). It was reported that there are some
negatively charged substances on the surface of the plasma membrane (Lodish, 2003), especially proteins. Our results from the
molecular dynamic simulation further demonstrated that the surfaces of proteins (ATPase, HRP, calmodulin, Ca-binding protein) in
the plasma membrane were distributed a large amount of the negative charges (Fig. 2). In terms of the chemical principles, Tb(III)
could interact with these proteins, leading to the density-distribution of Tb(III) on the plasma membrane of horseradish (Fig. 1). The
interaction depends on the concentration of Tb(III). When the concentration of Tb(III) is low, the complex of Tb(III) and proteins in the
plasma membrane, such as HRP could not be formed (Guo et al.,
2008b). Thus the interaction between Tb(III) and these proteins in
the plasma membrane was mainly the electrostatic attraction,
van der waals force and hydrogen bond. There are many amide
groups in these proteins. In terms of the basic principle of chemistry, the N and O atoms in these proteins are the Lewis base as electron-pair donors, and Tb(III) is an acid as electron-pair acceptors
L. Wang et al. / Chemosphere 80 (2010) 28–34
(Yin et al., 2007). Thus, the coordinate covalent bond of Tb–O and/or
Tb–N could be formed in the complex between Tb and these proteins in the presence of the high concentration of Tb(III) (Guo
et al., 2008b). It could be concluded that there was a more strong
interaction between Tb(III) and proteins in the plasma membrane
comparing with that of the treatment with the low concentration
of Tb(III). The difference in the interaction between the proteins
in the plasma membrane of plant led to the difference in the distribution of Tb(III) on the cell membrane.
When horseradish treated with the low concentration of Tb(III)
(5 mg L 1), Tb(III) located on the plasma membrane could directly
interact with HRP on the cell membrane (Ye et al., 2008), and thus
the activity of HRP is decreased (Wang et al., 2009). The decrease
led to the loss of balance between the generation and scavenging
of free radicals in plant, causing the excess accumulation of free
radicals (Ahmad et al., 2008). The excess free radicals can oxidize
the unsaturated fatty acids in the membrane lipid (Barclay and Ingold, 1981; Roozen et al., 1994), causing the peroxidation of membrane lipid (Wang et al., 2009). When the concentration of Tb(III)
was increased to 60 mg L 1, Tb(III) could enter the horseradish cell
(Fig. 1C). Once Tb(III) enters the horseradish cells, trace amounts of
Tb(III) can induce the formation of OH, leading to the autocatalysis
of the chain reaction of free radicals (Benedet and Shibamoto,
2008). Meanwhile, the activity of HRP is decreased, and the decrease is more than that of horseradish treated with 5 mg L 1 of
Tb(III) (Wang et al., 2009). Therefore, the accumulation of free radicals in cells was increased comparing with that of that of horseradish treated with 5 mg L 1 Tb(III), and then the oxidation of
unsaturated fatty acid in the membrane lipid, especially linolenic
acid (18:3) was aggravated (Table 2). Thus the peroxidation of
membrane lipid was increased (Wang et al., 2009). Such toxic effect of Tb(III) on plant was similar to that of heavy metals (Howlett
and Avery, 1997).
The increase in the peroxidation of membrane lipid induced by
the treatment with Tb(III) (Wang et al., 2009) would cause the decrease in the fluidity of the membrane lipid and the damage of the
membrane function, leading to the demolition of the cell. The results from the SEM and TEM measurement demonstrated that
the cell membrane, such as the plasma membrane, tonoplast, and
chloroplast membrane of horseradish treated with Tb(III) was
obviously damaged (Figs. 3 and 4). As we known, cells require
membranes for their existence (Buchanan et al., 2000). Therefore,
the damage of the membranes led to destruction in the structure
of organelle, chloroplast for example (Fig. 4). It was reported that
the treatment with La(III) can lead to the disorganization or delay
of the organization of microtubule and the aggregation of microfilaments (Liu and Hasenstein, 2005). Combined with the results
from the SEM and TEM measurements (Figs. 3 and 4), it was concluded that the damage of Tb(III) to the cellular structure was multi-site, including the cell membrane, cytoskeleton, and organelle.
Thus the ‘‘bleb-like” or ‘‘hole-like” structures on the surface of protoplast were observed in Fig. 3. The damage of the high concentration of Tb(III) to the cellular structure was more serious than that
of the low concentration of Tb(III) because of the higher peroxidation of membrane lipid in horseradish treated with the high concentration of Tb(III).
In conclusion, the present study revealed the toxic effect of the
low and high concentration of Tb(III) on the cell membrane of
plant. The distribution of Tb(III) in the outside or inside of the
horseradish cell caused the oxidation of the unsaturated fatty acids
in the membrane lipid, and then the damage of the cell membrane.
The damage led to the destruction of the cellular structure in
horseradish. The toxic effect of Tb(III) on the structure and function
of the cell membrane in the plant depended on the concentration
of Tb(III). The results can provide some references for understanding the toxic mechanism of REEs on plants.
33
Acknowledgements
The authors are grateful for the financial support of the National
Natural Science Foundation of China (30570323, 20971069), the
Foundation of State Developing and Reforming Committee
(GFZ2071609), and the Natural Science Foundation of Jiangsu Province (BK2009401).
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