Ergothioneine protects against neuronal injury induced by cisplatin both
in vitro and in vivo
Tuzz-Ying Song
a,⇑
, Chien-Lin Chen
b, Jiunn-Wang Liao
c, Hsiu-Chung Ou
d, Ming-Shiun Tsai
b aDepartment of Nutrition and Health Science, Chungchou Institute of Technology, Changhua, Taiwan, ROC
b
Department and Graduate Program of Bioindustry Technology, Dayeh University, Changhua, Taiwan, ROC
c
Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan, ROC
d
Department of Physical Therapy, Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 17 April 2010 Accepted 27 September 2010 Available online xxxx Keywords: Ergothioneine Cisplatin Avoidance tests Neuron cells Acetylcholinesterase activity Oxidative stressa b s t r a c t
The neuroprotective effects of ergothioneine (EGT) against cisplatin toxicity were investigated both in vitro and in vivo. For in vitro study, two types of neuronal cells, primary cortical neuron (PCN) cells and rat pheochromocytoma (PC12) cells, were incubated with EGT (0.1–10.0
l
M) for 2 h followed by incubation with 0.5l
M cisplatin for 72 h. Results show that cisplatin markedly decreased the prolifera-tion of PC12 cells and strongly inhibited the growth of axon and dendrite of PCN cells, but these effects were significantly prevented by EGT. For in vivo study, CBA mice were orally administered with 2 or 8 mg EGT/kg body weight for 58 consecutive days and were injected i.p. with 5 mg cisplatin/kg body weight on days 7, 9 and 11. We found that EGT significantly restored the learning and memory deficits in mice trea-ted with cisplatin evaluatrea-ted by active and passive avoidance tests. EGT also significantly preventrea-ted brain lipid peroxidation, restored acetylcholinesterase (AChE) activity and maintained glutathione/glutathione disulfide ratio in brain tissues of mice treated with cisplatin. These results demonstrate that EGT protects against cisplatin-induced neuronal injury and enhances cognition, possibly through the inhibition of oxidative stress and restoration of AChE activity in neuronal cells.Ó 2010 Published by Elsevier Ltd.
1. Introduction
Cisplatin is a potent antitumor agent widely used for the
chemotherapy of different solid malignancies (
Thigpen et al.,
1994; Van Basten et al., 1997; Recchia et al., 2001
). However,
cisplatin is characterized by some severe side effects such as
nephrotoxicity, neurotoxicity, ototoxicity and nausea and
vomit-ing, which frequently hampers its chemotherapeutic efficacy
(
Screnci and McKeage, 1999; Sweeney, 2001
). Cisplatin-caused
neurotoxicity occurs in up to 30% of patients and is dose-limiting
for cisplatin.
Dietrich et al. (2006)
pointed out that
chemothera-peutic agents, including carmustine (BCNU), cisplatin and cytosine
arabinoside (cytarabine), are more toxic for the progenitor cells of
the central nervous system (CNS) and for nondividing
oligodendro-cytes than for cancer cells. When administered systemically in
mice, these chemotherapeutic agents are associated with increased
cell death and decreased cell division in the dentate gyrus of the
hippocampus and in the corpus callosum of the potential CNS
(
Dietrich et al., 2006
).
Pedersen et al. (2000)
have reported that
cisplatin induces severe cognitive malformations in mouse and
rat experimental models.
The neurotoxic and cytotoxic effects of cisplatin have been
shown to be related to the production of reactive oxygen species
(ROS) (
Ravi et al., 1995; Rybak et al., 1995
) and high levels of
Pt–DNA binding and apoptosis of dorsal root ganglion (DRG)
neu-rons (
Ta et al., 2006
). Complete degeneration of the spiral ganglion
following exposure to cisplatin has been reported (
Anniko and
So-bin, 1986
). In vivo studies have shown an increase in the cochlear
activity of superoxide dismutase (SOD), H
2O
2, and
malondialde-hyde (MDA), and a decrease in glutathione (GSH) and GSH
reduc-tase activity after exposure to cisplatin, suggesting that PCN cells
cisplatin-induced neurotoxicity is the result of increased oxidative
stress (
Rybak et al., 1995
).
Gabaizadeh et al. (1997)
also indicated
that intraneuronal levels of ROS play a key role in cisplatin-induced
neuronal cell death. Thus, antioxidant molecule will be effective in
preventing cisplatin-induced damage to neurons.
Recently, great efforts have been put forward to the
neuropro-tective effects of dietary food or chemoproneuropro-tective agents to reduce
the toxic effects of cisplatin. These compounds are able to protect
neuronal cells in various in vivo and in vitro models through
differ-ent intracellular targets (
Mendonça et al., 2009; Gerritsen van der
Hoop et al., 1994; Hol et al., 1994; Tredici et al., 1994
).
Chemopro-tective agents such as GSH, methionine and para-aminobenzoic
0278-6915/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.fct.2010.09.030
⇑
Corresponding author. Tel.: +886 4 8311 498; fax: +886 4 8395 316. E-mail address:song77@dragon.ccut.edu.tw(T.-Y. Song).Contents lists available at
ScienceDirect
Food and Chemical Toxicology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m t o x
acid have been used and have shown their efficacies in different
experimental models (
Bohm et al., 1999; Basinger et al., 1990;
Esposito et al., 1993
).
Ergothioneine (2-mercaptohistidine trimethylbetaine; EGT) is
formed in some bacteria and fungi but not in animals (
Melville
et al., 1955
). In humans, ergothioneine is only absorbed through
con-sumption of plant diet, primarily by concon-sumption of edible
mush-rooms. Blood concentrations of EGT in humans have been
estimated to be in the range of 1–4 mg/100 ml blood (46–184
l
M)
and have long half-life in the human body (
Touster, 1951; Melville,
1958
), while the EGT concentrations in bovine and porcine ocular
tissues are reported to be 2.96 ± 0.2 and 8.69 ± 1.57 mmol/mg
tis-sues, respectively (
Shires et al., 1997
).
In vitro studies have shown that EGT is radioprotective and that
it scavenges singlet oxygen, hydroxyl radical, hypochlorous acid
and peroxyl radicals as well as inhibits peroxynitrite-dependent
nitration of proteins and DNA (
Aruoma et al., 1997; Dubost et al.,
2007
). In addition,
Jang et al. (2004)
have reported that EGT is
neu-roprotective because EGT protects rat pheochromocytoma (PC12)
cells from oxidative and nitrosative cell death caused by Ab. In vivo
studies have shown that EGT protects retinal neurons from
N-methyl-
D-aspartate-induced excitatoxicity (
Moncaster et al.,
2002
) and protects against diabetic embryopathy in pregnant rats
(
Guijarro et al., 2002
). EGT also confers cellular homeostasis in
neuronal cells challenged with the prooxidant mixture of
N-acetyl-cysteine/hydrogen peroxide (
Aruoma et al., 1999
). Although EGT
has not been shown to protect against cisplatin-induced neuronal
injury, we hypothesized that it could do so and do it effectively,
based on the literature data and our own preliminary in vitro data.
The study reported here was conducted to explore the protective
effects of EGT against cisplatin-induced neuronal damage both
in vitro and in vivo.
2. Materials and methods 2.1. Chemicals
Chemicals including n-butanol, thiobarbituric acid, 1,1,3,3-tetraethoxypropane and all other reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA).
2.2. Cell culture and cell viability assay
PC12 cells (BCRC 60048), a rat pheochromocytoma, were obtained from Food Industry Research & Development Institute (Hsin Chu, Taiwan) and maintained in 10% fetal bovine serum and 90% RPMI with 4 mML-glutamine containing 1.5 g/L so-dium bicarbonate, 100 IU/ml penicillin and 100
l
g/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The medium was changed every 2 days. Cellswere seeded at a density of 1 105
cells/well onto a 12 well-plate (FALCON, Becton Dickinson, NJ, USA) 24 h prior to drug treatment. Various concentrations of EGT (0.110
l
M) were added to cells 2 h prior to cisplatin treatment. After incubation, cells were washed with phosphate buffered saline (PBS). Viable cell numbers were determined 24, 48 and 72 h after the addition of cisplatin and EGT by means of the Trypan blue exclusion method using a hemocytometer.2.3. Primary cultured of rat cortical neuron (PCN) cells
Cultured cortical cells were prepared from the cerebral cortices of one-day-old Sprague–Dawley rats as previously described (Huang et al., 2000). After the brain was dissected, the blood vessels and meninges were removed under microscope. Then, the cortices were placed in ice-cold DMEM, and minced. The tissue chunks were incubated with papain solution (100 U/ml papain, 0.5 mM EDTA, 0.2 mg/ml cysteine, 1.5 mM CaCl2, DNase I) at 37 °C for 20 min to dissociate the cells. The
reac-tions were terminated by adding heat-inactivated horse serum. After the cell sus-pension was centrifuged at 200g, the pellet was resuspended in DMEM supplemented with 10% horse serum. Cells were plated onto poly-D-lysin-coated Petri dishes, and incubated at 37 °C in a humidified incubator with 5% CO2. Two
hours after plating, the medium was replaced with neurobasal containing B27, 25
l
M glutamine and 0.5 mM glutamine. On the 4th day in vitro, the medium was changed and replaced with neurobasal/B27 without glutamate. The PCN cells were grown for another 10 days to permit the growth of axon and dendrite. Mor-phological changes were conducted using a phase-contrast inverse microscope (IMT-2, Olympus Co. Ltd., Tokyo, Japan).2.4. Animal experimental design
CBA mice (6–7 weeks old) were used for the experiments. The mice were pro-vided with food and water ad libitum and received i.p. 5 mg cisplatin/kg body weight (bw) with or without oral supplementation of EGT (2 or 8 mg/kg bw, p.o.) or melatonin (10 mg/kg bw, p.o.) in five experimental groups. Group 1: control (0.9% NaCl, i.p., 10 mL/kg bw + 0.9% NaCl, p.o., 10 mL/kg bw); group 2: cisplatin + 0.9% NaCl, p.o.; group 3: cisplatin + melatonin; group 4: cisplatin + EGT (2 mg/kg bw, p.o.); group 5: cisplatin + EGT (8 mg/kg bw, p.o.). Cisplatin, EGT and melatonin were all prepared in saline. Mice were given daily by gastric feeding (10 mL/kg bw) of EGT or melatonin for 58 consecutive days. Cisplatin solution (10 mL/kg bw, i.p.) was administered consecutively on days 7, 9 and 11. Control ani-mals received equal amounts of 0.9% NaCl to replace cisplatin (i.p.) or EGT (p.o.). The experimental schedule is shown inFig. 1. All experimental procedures involving animals were conducted in accordance with National Institutes of Health (NIH) guidelines. This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of the Chung Chou Institute of Technology.
2.5. Passive avoidance test
Passive avoidance was measured using a Gemini Avoidance System (San Diego Instrument, San Diego, CA) which consists of two-compartment shuttle chambers with a constant current shock generator. On an acquisition trial, each mouse was placed into the start chamber, which remained darkened. After 20 s, the chamber light was illuminated and the door was opened for mouse to move into the dark chamber freely. Immediately it entered the dark chamber, the door was closed and an inescapable scrambled electric shock (0.5 mA, 0.5 s) for three times (5 s interval) was delivered through the floor grid. Twenty-four hours later, each mouse was again placed in the start chamber again (retention trial). The interval between the placement in the lighted chamber and the entry into the dark chamber was measured as latency in both acquisition and retention trials (maximum 180 s) (Jhoo et al., 2004). The longer latency time the mouse stays in the light room, the better learning memory the mouse has.
2.6. Active avoidance test
Active avoidance was also measured using a Gemini Avoidance System (San Diego Instrument, San Diego, CA). Mice received five learning trials per day for 3 days. On the first trial, mice were placed individually into the large compartment of the apparatus with the door closed and accommodated there for about 10 s. A light (60 W, 10 s) was switched on alternately in the two compartments and used as a conditioned stimulus (CS). The CS preceded the onset of the unconditioned
Fig. 1. The animal experimental schedule.
stimulus (US) by 5 s. The US was an electric shock (0.3 mA for 5 s) applied to the floor grid. If the animal avoided the US by running into the dark compartment with-in 5 s after the onset of the CS, the microprocessor recorder unit of the shuttle-box recorded an avoidance response. Each mouse was given 5 trials daily for 3 days with a mixed intertrial interval of 20 s. The activity level was also assessed by measuring the number of crossings between the chambers when no shock was present (inter-trial crossing). The recorder unit of automated shuttle-box continuously recorded this parameter during all experimental period (15 trials). The results were ex-pressed as the mean percent avoidance responses for each daily shuttle-box ses-sion. The more avoidance number and less latency time the mouse escapes, the better learning memory the mouse has.
2.7. Preparation of brain tissue homogenates
After completion of treatment with EGT (on day 59), the mice were killed by decapitation under CO2anesthesia. All following procedures were carried out at
0–4 °C. Whole brains, except cerebellum, were homogenized using an Ultra Turax homogenizer in 1:10 (w/v) buffer (5.0 mM Tris base, 150 mM NaCl and 20 mM EDTA, pH 7.5). The homogenates were sonicated for 30 s in a disruptor (Bronson Sonic, NY) and centrifuged at 16,000g for 10 min. The supernatant was kept at
70 °C until use.
2.8. Determination of GSH/GSSG ratio in brain tissues
GSH/GSSG ratios in brain tissue homogenates were assayed according to the HPLC method ofSchofiled and Chen (1995)with some modification. In brief, after adding100
l
L of 10% perchloric acid to precipitate the protein of brain homogenates (1.0 mL), the supernatants were treated with iodoacetic acid, neutralized with an excess of NaHCO3,and incubated in the dark at 40 °C for 1 h. A volume (0.2 mL)of 3% (v/v) 2,4-dinitrofluorobenzene was added to the reaction mixtures and al-lowed to react at 40 °C for 4 h in the dark. After centrifugation (3000g for 15 min), a portion (25
l
L) of the supernatant was applied onto HPLC column and the GSH/GSSG content was measured at 365 nm.2.9. Determination of thiobarbituric acid-reactive substances (TBARS) in brain tissues Lipid peroxidation in brain tissue homogenates was determined as MDA using the thiobarbituric acid (TBA) assay (Buege and Aust, 1978). Butylated hydroxytolu-ene (10
l
L, 50 mM) was added to the tissue homogenate (1.0 mL) to terminate the peroxidation reaction and then mixed with 1 mL of 7.5% (w/v) cold trichloroacetic acid (TCA) to precipitate proteins. The supernatant was allowed to react with 1 mL of 0.8% (w/v) TBA in a boiling water bath for 45 min. After cooling, levels of MDA were determined at 555 nm with excitation at 515 nm using 1,1,3,3-tetraeth-oxypropane as the standard. MDA levels were expressed as nmol per mg of protein. 2.10. Determination of acetylcholinesterase (AChE) activity and EGT contents in brain tissuesAChE activity in brain tissues was measured by the spectrophotometric method as developed byEllman et al. (1961). For determination of EGT contents, we used the method outlined in detail byDubost et al. (2007)for measuring EGT in mush-rooms. In short, analysis was carried out using an HPLC with separation carried out on two Econosphere C18 columns (Thermo scientific Associates, IL) with each column being 250 4.6 mm, 5
l
m particle size connected in tandem. The isocratic mobile phase was 50 mM sodium phosphate in water with 3% acetonitrile and 0.1% triethylamine adjusted to a pH of 7.3 with a flow rate of 1 ml per min. The injection volume was 20l
l, with the column temperature being ambient. EGT was quantified by monitoring absorbance at 254 nm using the authentic standard. Data were ex-pressed as mmol of EGT per milligram of protein (mmol/mg protein). Protein con-centrations were determined using a standard commercial kit (Bio-Rad Laboratories Ltd.) with serum bovine albumin as standard.2.11. Statistical analysis
Data are expressed as means ± SD and analyzed using one-way ANOVA followed by LSD Test for multiple comparisons of group means. All statistical analyses were performed using SPSS for Windows, version 10 (SPSS Inc.); a P value <0.05 is con-sidered statistically significant.
3. Results
3.1. Effect of EGT on cisplatin-induced cytotoxicity in PC12 cells
To choose an appropriate concentration of cisplatin to induce
PC12 neuron cell damage, we incubated PC12 cells with different
concentrations (0.5, 1.0 and 5.0
l
M) of cisplatin for 24, 48 and
72 h. As shown in
Fig. 2
, cisplatin resulted in a dose-dependent
decrease in the cell number of PC12 cells. In cells treated with
1.0 or 5.0
l
V
cisplatin for 24, 48 and 72 h, cell proliferation was
almost completely inhibited, pretreatment of PC12 cells with
0.1–10.0
l
M EGT for 2 h provided no protective effect at these
concentrations of cisplatin. However, when PC12 cells were
trea-ted with 0.5
l
M cisplatin for 24, 48 and 72 h after pretreatment
with 0.1–10.0
l
M EGT for 2 h, we found that EGT effectively
pre-vented the antiproliferative effect of cisplatin, and the effect of
EGT was concentration- and time-dependent (
Fig. 3
).
3.2. Effects of EGT on morphological changes in primary cultured rat
cortical neuron cells treated with cisplatin
To determine the effect of cisplatin and EGT on cell morphology
and the growth of axons and dendrites, we used primary cultured
rat cortical neuron cells incubated with 0.5
l
M cisplatin. We found
that incubation with cisplatin (0.5
l
M) for 72 h resulted in marked
inhibition of neuritis outgrowth of rat cortical neuron cells
(
Fig. 4
B). However, when the cells were treated with EGT (0.1 to
Fig. 2. The in vitro antiproliferative effect of cisplatin in PC12 cells. Cells were incubated with saline (control) or cisplatin (0.5, 1.0 or 5.0
l
M) for 24, 48 and 72 h. Cell viability was estimated by Trypan blue dye exclusion method. Values (means ± SD of triplicate tests) not sharing a superscript letter are significantly different (P < 0.05).Fig. 3. The in vitro protective effect of ergothioneine (EGT) against cisplatin-induced growth inhibition of PC12 cells. Cells were incubated with 0.5
l
M cisplatin and 0.5, 1.0 or 5.0l
M EGT for 24, 48 and 72 h. Cell viability was estimated by Trypan blue dye exclusion method. Values (means ± SD of triplicate tests) not sharing a superscript letter are significantly different (P < 0.05).10
l
M) for 2 h followed by incubation with 0.5
l
M cisplatin for
72 h, the damage to axon and dendrite induced by cisplatin was
effectively prevented by EGT (
Fig. 4C–F
).
3.3. Effect of EGT on body weights of mice treated with cisplatin
As shown in
Fig. 5
, cisplatin alone significantly decreased body
weights of CBA mice starting on day 14 of the experiment (i.e.,
3 days after completion of cisplatin treatment) until day 58, as
compared to the control mice. However, supplementation with
EGT (2 or 8 mg/kg bw) or melatonin (10 mg/kg bw) resulted in
substantial recovery of body weights, and no significant differences
in body weights were found on days 28 and 58 in these groups of
mice, as compared with the control mice.
3.4. Passive avoidance test
The passive and active avoidance tests were used to evaluate
the memory and learning ability in this animal model.
Step-through latency from light chamber to dark chamber was used as
a marker to evaluate the memory and learning ability in the
pas-sive avoidance test. As shown in
Fig. 6
, all the mice would
imme-diately go into the dark chamber in the first trial (day 17)
because of skototaxis, but the electric shock in the dark chamber
Fig. 4. Neuroprotective effect of ergothioneine (EGT) on cisplatin-induced morphological change of primary cultured rat cortical neuron cells. EGT (0–10
l
M) was applied to neurons 2 h prior to the treatment with 0.5l
M cisplatin. The photomicrographys were taken under a phase contrast microscope (100). (A) Control; (B) neurons treated with cisplatin (0.5l
M); (C–F) neurons treated with 0.5, 1.0, 5.0 and 10.0l
M of EGT prior 2 h cisplatin-treated, respectively.Fig. 5. Body weights in cisplatin-treated mice supplemented with or without ergothioneine (EGT). Two groups of mice were injected i.p. with saline solution (control group) or cisplatin solution (cisplatin group) without administration of test materials. The other cisplatin-treated mice were supplemented with melatonin (+Melatonin, 10 mg/kg/day), low-dosage EGT (+Low EGT, 2 mg/kg/day) and high-dosage EGT (+High EGT, 8 mg/kg/day). Values (means ± SD, n = 9 mice per group) obtained at the same wk not sharing a superscript letter are significantly different (P < 0.05). Asterisks represent significant differences (P < 0.05) from the 0 week cisplatin-treated group.
should intimidate and prevent mice with normal memory ability
from going into the dark chamber the next time. Therefore, the
step-through latency among each group would show significant
difference in the next trial. The results of the second and the third
trials (on days 18 and 19) clearly indicated that mice treated with
cisplatin alone spent significantly shorter period of time staying in
the light chamber than the mice of the control group (P < 0.05).
However, mice treated with low- and high-dosage EGT or with
melatonin stayed significantly longer in the light chamber than
those treated with cisplatin alone (P < 0.05). Although the
differ-ences in step-through latency were not significantly different
among the mice supplemented with EGT or melatonin, the mice
supplemented with high-dosage EGT tended to stay longer than
those with low-dosage EGT and the control mice.
3.5. Active avoidance test
Active avoidance tests were presented as both short memory
retention and long memory retention using the shuttle-box active
avoidance test. The short memory retention session was conducted
for three consecutive days starting on days 14 through 16. As
shown in
Table 1
, all groups of mice in the short memory retention
session showed gradual decrease in the escape latency and
in-crease in the number of crossings on the 2nd day (day 15) and
3rd day (day 16) of the learning session, as compared to those of
the 1st day (day 14). However, no significant differences were
ob-served in the escape latency and number of crossings of all groups
of mice (P > 0.05). In other words, supplementation with either EGT
or melatonin did not significantly improve the short memory
retention.
Fig. 6. Effect of ergothioneine (EGT) on step-through latency of multiple-trail passive-avoidance test in cisplatin-treated mice. The tests were carried out from day17 to day 19. Two groups of mice were injected i.p. with saline solution (control group) or cisplatin solution (cisplatin group) without administration of test materials. The other cisplatin-treated mice were supplemented with melatonin (+Melatonin, 10 mg/kg/day), low-dosage EGT (+Low EGT, 2 mg/kg/day) and high-dosage EGT (+High EGT, 8 mg/kg/day). Values (means ± SD, n = 9 mice per group) not sharing a superscript letter are significantly different (P < 0.05). Asterisks represent significant differences (P < 0.05) from the cisplatin-treated group on day 16.
Table 1
The effects of ergothioneine (EGT) on active avoidance test (shuttle box) of short memory retention session in cisplatin-treated mice: escape latency and number of crossings. Groups Escape latency (sec) Number of crossings
Days Days 14 15 16 14 15 16 Control 6.6 ± 0.6a 2.9 ± 0.8a 2.4 ± 0.6a 1.3 ± 0.9a 3.3 ± 0.6a 4.1 ± 0.6a Cisplatin 5.2 ± 1.2ab 3.8 ± 0.7ab 3.2 ± 0.6a 1.7 ± 1.4a 2.8 ± 0.2ab 3.1 ± 0.6a +Melatonin 5.0 ± 1.2ab 3.1 ± 1.0a 2.9 ± 0.4a 2.1 ± 1.3a 3.4 ± 0.9a 3.8 ± 0.6a +Low EGT 4.6 ± 1.8ab 3.2 ± 0.7a 2.6 ± 0.7a 2.2 ± 1.0a 3.3 ± 0.7a 3.9 ± 0.5a +High EGT 5.6 ± 1.6ab 3.1 ± 0.4a 2.9 ± 1.4a 1.7 ± 1.3a 3.8 ± 0.4a 4.0 ± 0.9a
Two groups of mice were injected i.p. with saline solution (control group) or cisplatin solution (cisplatin group) without administration of test materials. The other cisplatin-treated mice were administered melatonin (+Melatonin, 10 mg/kg/day), low-dosage EGT (+Low EGT, 2 mg/kg/day) and high-dosage EGT (+High EGT, 8 mg/kg/day). Values (means ± SD, n = 9 mice per group) not sharing a superscript letter are significantly different (P < 0.05).
Fig. 7. Effect of ergothioneine (EGT) on step-through latency (A) and mean number of crossings (B) of multiple-trail active-avoidance test in cisplatin-treated mice. The tests were carried out on days14, 28, 42 and 56. Two groups of mice were injected i.p. with saline solution (control group) or cisplatin solution (cisplatin group) without administration of test materials. The other cisplatin-treated mice were supplemented with melatonin (+Melatonin, 10 mg/kg/day), low-dosage EGT (+Low EGT, 2 mg/kg/day) and high-dosage EGT (+High EGT, 8 mg/kg/day). Values (means ± SD, n = 9 mice per group) not sharing a superscript letter are significantly different (P < 0.05).
The long memory retention session was performed on days 14,
28, 42 and 56. As shown in
Fig. 7
A, the mice treated with cisplatin
alone showed a significant increase in the escape latency on the 42
and 56 days (P < 0.05), as compare to those of the control group. In
contrast, mice treated with EGT (2 and 8 mg/kg) or melatonin
(10 mg/kg) showed a significant decrease in the escape latency
(P < 0.05) on days 42 and 56, as compared to those treated with
cis-platin alone. We also found that the mean number of crossings in
mice treated with cisplatin alone was significantly lower than that
of control group (P < 0.05) (
Fig. 7
B). EGT and melatonin
signifi-cantly increased the number of crossings, as compared to those
treated with cisplatin alone (P < 0.05). However, there was no
sig-nificant difference between the low-dosage EGT (2 mg/kg) and
high-dosage EGT (8 mg/kg) groups (P > 0.05).
3.6. Effect of EGT on AChE activity in the brain of cisplatin -treated
mice
In mice treated with cisplatin alone, the brain AChE activity was
significantly increased in comparison with the control group
(
Fig. 8
). Oral administration with EGT (2 or 8 mg/kg) or melatonin
significantly prevented the rise in brain AChE activity induced by
cisplatin (P < 0.05). However, there was no significant difference
in the brain AChE activity between the high-EGT group and the
low-EGT group.
3.7. Effect of EGT on antioxidant status in the brain of cisplatin-treated
mice
As shown in
Table 2
, cisplatin significantly decreased EGT
con-tents in mouse brain tissues (31% lower than the control group,
P < 0.05). Oral administration of EGT significantly restored the loss
of EGT levels induced by cisplatin, and the effect of EGT was dose
dependent, with the high-EGT group completely recovering the
loss of brain EGT levels and reaching a slightly higher level than
that of the control mice. Interestingly, melatonin also completely
recovered the loss of brain EGT contents, and reached a level that
is comparable to that of the high-EGT group, even though the brain
EGT contents in the melatonin group and the high-EGT group are
not significantly different from those in the control group.
As also shown in
Table 2
, cisplatin increased lipid peroxidation
in the brain, as evidenced by a 3.7-fold increase in TBARS. Both EGT
(at 2 and 8 mg/kg bw) and melatonin completely inhibited
cisplatin-induced lipid peroxidation. In contrast, cisplatin
mark-edly decreased the GSH/GSSG ratio (from 14.5 ± 2.7 in the control
group to 4.5 ± 0.5), and treatment with EGT significantly and
dose-dependently prevented the decline of GSH/GSSG ratio.
Inter-estingly, the GSH/GSSG ratio in the melatonin group (9.6 ± 0.1) was
significantly higher than that in the low-EGT group (7.9 ± 1.9)
(P < 0.05).
4. Discussion
EGT is regarded as an antioxidant due to its ability to reduce
the oxidative stress both in vitro (
Jang et al., 2004; Aruoma
et al., 1999
) and in vivo (
Guijarro et al., 2002
). Although several
thiols have been reported to protect against cisplatin toxicity
(
Somani et al., 1995
), it is unclear whether EGT is neuroprotective
against cisplatin toxicity. In this study, we conducted both
in vitro and in vivo experiments to answer this question. Our
in vitro experiments demonstrated that EGT significantly
pre-vented the decrease in cell proliferation of PC12 cells. Cisplatin
also strongly inhibited the growth of axon and dendrite of PCN
cells, and EGT supplementation significantly prevented the
inhibi-tion. Our in vivo experiments in cisplatin-treated CBA mice
con-firmed that EGT significantly prevented brain lipid peroxidation,
and we further demonstrated that EGT restored brain GSH/GSSG
ratios and attenuated the rise in AChE activity. We further
showed that EGT significantly prevented the learning and
mem-ory deficits induced by cisplatin by decreasing the active
avoid-ance time and increasing the successful number of passive and
active avoidance by the electric shock in cisplatin-treated mice.
To the best of our knowledge, this is the first report to document
the neuroprotective effects of EGT on cisplatin-induced memory
and learning impairment in mice.
The protective effect of EGT against cisplatin toxicity is likely
re-lated to the inhibition of lipid peroxidation and to maintaining the
GSH/GSSG ratio in mouse brain. Inhibition of lipid peroxidation can
prevent the alteration of the neuronal membrane, thereby
normal-izing the neuronal glucose transporter, deceased glucose
phos-phorylation, and neuronal cell death (
Pardridge, 1994
). As
expected, the EGT levels were increased in brain tissues of mice
supplemented with EGT, and this free thiol could inhibit lipid
per-oxidation through maintaining the GSH/GSSG ratio by directly
reacting with cisplatin or by reducing GSSG to GSH in brain tissues
of mice treated with cisplatin. It has been shown that endogenous
thiols such as metallothionein and GSH can limit the amount of the
DNA platination by cisplatin (
Sadowitz et al., 2002; Hagrman et al.,
2003
). Indeed,
Deiana et al. (2004)
also have reported that
supple-mentation with EGT not only protects the organs against lipid
per-oxidation but conserves the consumption of endogenous GSH and
alpha-tocopherol. Importantly,
Gründemann et al. (2005)
have
Fig. 8. Effect of ergothioneine (EGT) on the acetylcholinesterase activity in mouse brain tissues. Two groups of mice were injected i.p. with saline solution (control group) or cisplatin solution (cisplatin group) without administration of test materials. The other cisplatin-treated mice were supplemented with melatonin (+Melatonin, 10 mg/kg/day), low-dosage EGT (+Low EGT, 2 mg/kg/day) and high-dosage EGT (+High EGT, 8 mg/kg/day). Values (means ± SD, n = 9 mice per group) not sharing a superscript letter are significantly different (P < 0.05).
Table 2
The effects of ergothioneine (EGT) on antioxidant status in the brain tissue of cisplatin-treated mice.
Groups EGT (mmol/ mg protein)
MDA (nmol MDA/ mg protein) GSH/GSSG ratio Control 35.8 ± 6.9b 100 ± 66b 14.5 ± 2.7d Cisplatin 24.9 ± 6.1a 372 ± 179a 4.5 ± 0.5a +Melatonin 37.9 ± 2.4bc 71 ± 48b 9.6 ± 0.1c +Low EGT 27.1 ± 6.0ab 117 ± 65b 7.9 ± 1.9b +High EGT 37.8 ± 7.5bc 78 ± 64b 10.3 ± 1.0c
Values (means ± SD, n = 9 mice per group) not sharing a superscript letter are sig-nificantly different (P < 0.05).
reported that EGT has an organic cation transporter-1 (OCTN1) in
the neuronal cells that can pass the blood brain barrier (BBB) to
en-ter the central nervous system. Thus, EGT readily enen-ter the
neuro-nal cells to protect neuroneuro-nal neuron cell membrane and the cellular
transport systems by maintain normal membrane fluidity thereby
reducing neuronal cell apoptosis.
Cognitive deficits associated with Alzheimer’s disease (AD) are
thought to be primarily related to the degeneration of cholinergic
neurons in cerebral cortex and hippocampus, resulting in deficits
of cholinergic neurotransmission. AChE is responsible for
acetyl-choline hydrolysis, from both acetyl-cholinergic and non-acetyl-cholinergic
neu-rons of the brain (
Atack et al., 1983
). AChE activity has been shown
to be increased within and around amyloid plaques (
Ulrich et al.,
1990; Morán et al., 1993
), to promote the assembly of amyloid
beta-peptides into fibrils (
Inestrosa et al., 1996
) and to increase
the cytotoxicity of these peptides (
Alvarez et al., 1998
). Treatment
of AD has targeted the inhibition of AChE to increase
concentra-tions of ACh and improve cognitive and behavior symptoms (
Krall
et al., 1999
). Here, we observed that the AChE activity was
in-creased in the brain of cisplatin-treated mice (
Fig. 8
). As indicated
by
Kaizer et al. (2005)
, the elevation of AChE activity may be due to
a direct neurotoxic effect on the plasmatic membrane caused by
increased lipid peroxidation, and that such changes may affect
the integrity and functionality of the cholinergic system. Thus,
we believe that alterations in the lipid membrane could be a
deci-sive factor in changing the conformational state of the AChE
mole-cule, which would result in learning and memory deficits after
cisplatin exposure. Furthermore, our observation that EGT was
capable of preventing the increase of AChE activity suggests that
EGT may play an important role in the maintenance of
acetylcho-line synaptic levels by preventing the compromise of AChE activity.
Thus, the prevention of the restoration in AChE activity may be
an-other mechanistic action by which EGT prevents or improves
learning and memory functions of mice.
The doses of EGT used in the present study (2.0 and 8.0 mg/kg/
day) appear to be achievable in humans, although there is no
esti-mated intake of EGT in humans today. Take the highest EGT dose
(8.0 mg/kg/day) as example: for a mouse whose average body
weight is about 25 g, a daily supplemental level of 8.0 mg/kg bw
translates into 200
l
g EGT/mouse/d (i.e., 8.0 mg/kg bw 0.025
kg bw). This daily consumption level is equivalent to a human
con-sumption level of 40 mg/day, as calculated by the difference in
dai-ly food intake, i.e., 2.5 g (dry weight) for a mouse and 500 g (dry
weight) for a person with an energy intake of 2000 kcal/day (
Liu
et al., 2002
). This intake level (40 mg) can easily be achieved by
consumption of EGT-rich foodstuff, such as mushrooms which
con-tain up to 2.6 mg EGT per g dry weight (
Dubost et al., 2007
). Thus,
an intake of approximately 15 g dry mushroom per day would
achieve an EGT intake of 40 mg. Although little is known about
the safety of EGT consumption, no reports in animal studies have
shown any deleterious effects so far, including the present mouse
study of ours. It should be noted that an early study showed that
some diabetic patients had elevated blood EGT levels (
Fraser,
1950
), and this has led to the speculation that EGT may induce
dia-betes through chelation of zinc (
Epand, 1982
). However, later
experiments have found no statistical significance in blood EGT
levels between diabetics and non-diabetics (
Epand and Epand,
1988
).
Melatonin, which was used as a positive control in the present
study, is the main hormone of the pineal gland has been described
to be a potent scavenger of free radicals and stimulates other
anti-oxidant activities by preventing hydroxyl (OH) and peroxyl (ROO)
radical formation (
Tan et al., 1993; Kaya et al., 1999
). Studies have
demonstrated that melatonin reduces STZ-induced oxidative stress
in diabetic rats (
Montilla et al., 1998
), protects human red bloods
cells from oxidative hemolysis (
Tesoriere et al., 1999
), and inhibits
the vasorelaxant action of peroxynitrite in human umbilical artery
(
Okatani et al., 1999
). In addition, melatonin has been reported to
either ameliorate or prevent the nephrotoxicity of cisplatin (
Hara
et al., 2001; Sener et al., 2000
). Our results also showed that the
administration of melatonin to cisplatin-treated mice significantly
increased the GSH/GSSG ratio and decreased MDA formation in the
brain of mice. These observations support that melatonin functions
as an antioxidant in vivo.
In summary, we demonstrate that EGT administration protects
against cisplatin-induced neurotoxicity both in vitro and in vivo.
The present findings along with the fact that EGT has an OCTN1
in the neuronal cells to facilitate its entrance to the central nervous
system suggest that EGT has the potential to become a useful
neu-roprotective agent in humans. Further studies are warranted.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgement
This study was supported by a grant from the National Science
Council, ROC. (NSC96-2313-B-235-002).
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