6 1243 PDF
6 1243 PDF
Short communication
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Department of Biochemical Toxicology, Department of Toxicology, Jagiellonian University, Medical College,
Medyczna 9, PL 30-688 Krakw, Poland
Abstract:
Ethylene glycol ethers (EGEs) are a class of chemicals used extensively in the manufacture of a wide range of domestic and indus-
trial products, which may result in human exposure and toxicity. Hematologic and reproductive toxicity of EGEs are well known
whereas their action on neuronal cell viability has not been studied so far. In the present study, we investigated the effects of some
EGEs on cell viability and on the hydrogen peroxide-induced damage in the human neuroblastoma (SH-SY5Y) cells. It has been
found that 2-phenoxyethanol in a concentration-dependent manner (525 mM, 24 h) increased the basal and H O -induced lactate
dehydrogenase (LDH) release and 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) reduction. 2-Butoxyethanol
given alone did not affect LDH release and MTT reduction but concentration-dependently enhanced the cytotoxic effect of H O . 2-
Isopropoxyethanol significantly and concentration-dependently (125 mM) increased the basal LDH release and attenuated MTT
reduction, but did not potentiate the cytotoxic effect of H O . Contrary to this, 2-methoxyethanol did not show a cytotoxic effect
while 2-ethoxyethanol at high concentrations intensified the hydrogen peroxide action. This study demonstrated that among the
EGEs studied, 2-phenoxyethanol showed the most consistent cytotoxic effect on neurons in in vitro conditions and enhanced the hy-
drogen peroxide action. 2-Isopropoxyethanol had also a potent cytotoxic effect, but it did not enhance the hydrogen peroxide action,
whereas 2-butoxyethanol only potentiated cytotoxic effect of H O . It is concluded that the results of the present study should be con-
firmed in in vivo conditions and that some EGEs, especially 2-phenoxyethanol, 2-butoxyethanol and 2-isopropoxyethanol, may be re-
sponsible for initiation or exacerbation of neuronal cell damage.
Key words:
ethylene glycol ethers, neurotoxicity, lactate dehydrogenase, MTT assay, SH-SY5Y cells
dia 24 h after the treatment with EGEs alone or to- 2-Phenoxyethanol at concentrations 5, 10 and 25 mM
gether with hydrogen peroxide. Cell-free culture su- significantly and concentration-dependently increased
pernatants were collected from each well and LDH the basal and H O -induced LDH release (Fig. 1A, B).
activity was determined using a colorimetric method At the same concentrations, this compound inhibited
(Cytotoxicity Detection Kit, Roche Diagnostic GmbH), the reduction of MTT, whereas at concentrations of 10
according to which the amount of colored hydrazone, and 25 mM 2-phenoxyethanol enhanced H O -
formed in the reaction of pyruvic acid with 2,4- induced MTT reduction (Fig. 1C, D). 2-Butoxyethanol
dinitrophenylhydrazine, is inversely proportional to the when given alone at a concentration between 0.125 mM
LDH activity in the sample and was quantified by meas- did not affect the basal LDH release and only in the
uring the absorbance at 490 nm. highest examined concentration (25 mM) inhibited
MTT reduction (Fig. 2A, C). However, this compound
3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetra- in a concentration-dependent manner enhanced the
zolium bromide (MTT) reduction assay cytotoxic effect of H O in the LDH release test (5, 10
and 25 mM) and in the MTT test (10 and 25 mM)
Cell viability was measured, as described previously (Fig. 2B, D). Contrary to 2-butoxyethanol, 2-isopro-
[12], by determining the cellular reducing capacity, poxyethanol exerted a small effect on the H O -
estimated through the extent of MTT reduction to the induced damage, because it did not alter H O action
insoluble intracellular formazan, which depends on in the MTT test and only at the highest concentration
the activity of intracellular dehydrogenases and is in- tested (25 mM) it raised H O -induced LDH release
dependent of changes in the integrity of the plasma (Fig. 3B, D). On the other hand, 2-isopropoxyethanol
membrane. Briefly, culture medium was removed and given alone significantly and concentration-
SH-SY5Y cells were incubated with MTT (0.15 dependently (125 mM) increased the basal LDH release
mg/ml) for 1 h at 37C. MTT was prepared in PBS and MTT reduction (Fig. 3A, C). 2-Methoxyethanol did
and added at a final concentration of 0.15 mg/kg. not show a cytotoxic effect because in the concentra-
Then, the crystals of formazan were dissolved in tion range from 0.1 to 25 mM it had no effect either
DMSO and the absorbance of each sample was meas- on basal or on H O -induced LDH-release and MTT
ured at 570 nm in a plate reader (Multiscan, Labsys- reduction (Tab. 1). Also 2-ethoxyethanol alone did
tem). The results were expressed as a percentage of not induce cytotoxic effect in SH-SY5Y cells, as as-
control cells incubated in the absence of EGEs and sessed by the LDH and MTT assay (Tab. 1). However,
HO. this compound at concentrations of 10 and 25 mM en-
hanced H O -induced LDH release and at the highest
Statistical analysis concentration used (25 mM) it potentiated the action
of H O on MTT reduction.
The data were obtained in three independent experi-
ments, each performed in 34 wells and were pre-
sented as the percentage of control SEM. The sig-
nificance of differences between the means was
Discussion
evaluated by the Duncans test following a one-way
analysis of variance.
This study has demonstrated that the examined EGEs
markedly differ between themselves in their effect on
SH-SY5Y cell damage. Among EGEs under study,
2-phenoxyethanol showed the most consistent
Results
cytotoxic effect on neurons in in vitro conditions. Its
toxicity involved cell membrane disruption, as
Cytotoxic effect of EGEs was examined by LDH re- evidenced by the LDH release and mitochondrial
lease and MTT reduction. There were no fundamental dysfunction, as assessed by the MTT assay. 2-Phen-
differences between these tests. Exposure of SH-SY5Y oxyethanol in a concentration-dependent manner
cells to 2-phenoxyethanol for 24 h produced cytotox- increased both the basal and hydrogen peroxide-
icity, as assessed by LDH release and MTT test. induced damage of the cell membrane and inhibited
the basal and hydrogen peroxide-induced mitochon- hydrogen peroxide, which indicated that this
drial function. The statistically significant cytotoxic compound could be important in intensifying the
effect of this compound was evident at 5 mM concen- effect of some harmful agents, but alone it did not
tration. 2-Isopropoxyethanol already in a concentra- induce neurodegenerative processes. Two remaining
tion of 1 mM caused cell membrane disruption and investigated EGEs, 2-methoxyethanol and 2-ethoxy-
mitochondrial dysfunction, however, it did not intensify ethanol had no clear cytotoxic effect because only
damages caused by hydrogen peroxide. Contrary to 2-ethoxyethanol in a higher concentration enhanced
2-isopropoxyethanol, 2-butoxyethanol alone had no the hydrogen peroxide action. Comparison of the
cytotoxic effect, but strongly enhanced the effect of intensity of action of the investigated monoalkyl- and
160
A B
#
140 *
Fig. 3. Effect of 2-isopropoxyethanol *
LDH release
120 * * *
% of control
on basal (A) and H O -induced (B) 100
LDH release and basal (C) and H O -
80
induced (D) MTT reduction in SH-
60
SY5Y cells. Results are shown as
a percentage of control cells incu- 40
60
40
20
H2O2 - - - - - - + + + + + +
2-isopropoxyethanol - 0.1 1 5 10 25 - 0.1 1 5 10 25 [mM]
Tab. 1. Effect of 2-methoxyethanol and 2-ethoxyethanol on basal and H O -induced LDH release and MTT reduction in SH-SY5Y cells
Vehicle 100.0 1.2 100.0 2.4 Vehicle 100.0 1.1 100.0 2.4
2-methoxyethanol 0.1 mM 100.5 3.4 101.3 2.9 2-ethoxyethanol 0.1 mM 99.0 1.9 95.2 5.9
2-methoxyethanol 1 mM 99.8 3.2 110.2 2.3 2-ethoxyethanol 1 mM 99.5 2.7 106.6 5.1
2-methoxyethanol 5 mM 100.6 2.5 109.5 3.9 2-ethoxyethanol 5 mM 98.0 0.3 108.7 4.3
2-methoxyethanol 10 mM 99.2 1.6 112.4 3.2 2-ethoxyethanol 10 mM 98.4 1.5 106.9 5.5
2-methoxyethanol 25 mM 92.0 3.2 109.1 3.1 2-ethoxyethanol 25 mM 99.4 1.3 101.2 8.1
Vehicle + H O 128.6 2.3* 76.0 3.9* Vehicle + H O 128.0 2.1* 77.0 4.2*
2-methoxyethanol 0.1 mM+ H O 140.8 8.4 72.9 0.6 2-ethoxyethanol 0.1 mM + H O 131.4 7.5 96.9 1.0
2-methoxyethanol 1 mM+ H O 138.2 4.4 70.2 9.2 2-ethoxyethanol 1 mM + H O 131.4 4.2 77.6 4.4
2-methoxyethanol 5 mM+ H O 131.0 2.7 79.5 8.6 2-ethoxyethanol 5 mM + H O 139.7 6.9 74.7 6.8
2-methoxyethanol 10 mM+ H O 128.7 3.8 86.5 4.0 2-ethoxyethanol 10 mM + H O 145.9 5.8 64.4 8.7
2-methoxyethanol 25 mM+ H O 129.4 2.9 86.0 5.0 2-ethoxyethanol 25 mM + H O 162.5 1.2 47.6 3.2
The SH-SY5Y cells were treated with saline, 2-methoxyethanol or 2-ethoxyethanol at concentrations from 0.1 to 25 mM alone or with hydrogen
peroxide (H O , 0.5 mM) for 24 h. Cytotoxic effect was determined by measuring the efflux of lactate dehydrogenase (LDH) into the culture me-
dia and by the MTT assay. The significance of differences between the means was evaluated by Duncans test following a one-way analysis of
variance (* p < 0.05 vs. control culture; p < 0.05 vs. cells treated with H O ; n = 9)
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