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Journal of Receptors and Signal Transduction
ISSN: 1079-9893 (Print) 1532-4281 (Online) Journal homepage: https://www.tandfonline.com/loi/irst20
The potential protective roles of zinc, selenium
and glutathione on hypoxia-induced TRPM2
channel activation in transfected HEK293 cells
Dilek Duzgun Ergun, Sefik Dursun, Nural Pastaci Ozsobaci, Ozden Hatırnaz
Ng, Mustafa Naziroglu & Dervis Ozcelik
To cite this article: Dilek Duzgun Ergun, Sefik Dursun, Nural Pastaci Ozsobaci, Ozden Hatırnaz Ng, Mustafa Naziroglu & Dervis Ozcelik (2020) The potential protective roles of zinc, selenium and glutathione on hypoxia-induced TRPM2 channel activation in transfected HEK293 cells, Journal of Receptors and Signal Transduction, 40:6, 521-530, DOI: 10.1080/10799893.2020.1759093
To link to this article: https://doi.org/10.1080/10799893.2020.1759093
Published online: 30 Apr 2020.
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ORIGINAL ARTICLE
The potential protective roles of zinc, selenium and glutathione on
hypoxia-induced TRPM2 channel activation in transfected HEK293 cells
Dilek Duzgun Erguna,c , Sefik Dursunb, Nural Pastaci Ozsobacic, Ozden Hatırnaz Ngd,e, Mustafa Nazirogluf,g and Dervis Ozcelikc
a
Department of Biophysics, Faculty of Medicine, Istanbul Aydin University, Istanbul, Turkey;bDepartment of Biophysics, Faculty of Medicine, Uskudar University, Istanbul, Turkey;cDepartment of Biophysics, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey;dDepartment of Medical Biology, Faculty of Medicine, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey;eDepartment of Genetic, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey;fDepartment of Biophysics, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey;gDrug Discovery Unit, BSN Health, Analyses, Innovation, Consultancy, Organization, Agriculture, Industry LTD. Inc, G€oller B€olgesi Teknokenti, Isparta, Turkey
ABSTRACT
Hypoxia induces cell death through excessive production of reactive oxygen species (ROS) and calcium (Ca2þ) influx in cells and TRPM2 cation channel is activated by oxidative stress. Zinc (Zn), selenium (Se), and glutathione (GSH) have antioxidant properties in several cells and hypoxia-induced TRPM2 channel activity, ROS and cell death may be inhibited by the Zn, Se, and GSH treatments. We investi-gated effects of Zn, Se, and GSH on lipid peroxidation (LPO), cell cytotoxicity and death through inhib-ition of TRPM2 channel activity in transfected HEK293 cells exposed to hypoxia defined as oxygen deficiency.
We induced four groups as normoxia 30 and 60 min evaluated as control groups, hypoxia 30 and 60 min in the HEK293 cells. The cells were separately pre-incubated with extracellular Zn (100mM), Se (150 nM) and GSH (5 mM). Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release and the LDH and LPO levels were significantly higher in the hypoxia-30 and 60 min-exposed cells according to normoxia 30 and 60 min groups. Furthermore, we found that the LPO and LDH were decreased in the hypoxia-exposed cells after being treated with Zn, Se, and GSH according to the hypoxia groups. Compared to the normoxia groups, the current densities of TRPM2 channel were increased in the hyp-oxia-exposed cells by the hypoxia applications, while the same values were decreased in the treatment of Zn, Se, and GSH according to hypoxia group. In conclusion, hypoxia-induced TRPM2 channel activ-ity, ROS and cell death were recovered by the Se, Zn and GSH treatments.
ARTICLE HISTORY Received 21 January 2020 Revised 18 April 2020 Accepted 19 April 2020 KEYWORDS Hypoxia; TRPM2 channel; HEK293; Zn; Se 1. Introduction
Transient Potential Receptor Melastatin-2 (TRPM2) from the TRP subfamily is a nonselective cation channel that is perme-able to divalent calcium (Ca2þ), and magnesium (Mgþ2) ions, univalent sodium (Naþ), and potassium (Kþ) ions [1,2]. It is known that Ca2þ, hydrogen peroxide (H2O2), cyclic adenosine
diphosphoribose (cADPR) and reactive oxygen species (ROS) has an effect on activation and inhibition mechanisms of TRPM2 channels. It is important in terms of understanding the underlying mechanisms behind many diseases, because
it enables influx of Ca2þ ions to intracellular
environ-ment [3–5].
Hypoxia is defined as the decrease in partial pressure of arterial oxygen (PO2) and it causes cell dysfunction. It has
been reported that expression of some ion channels changes
as a result of hypoxia [6–8]. In addition, involvement of
TRPM2 channels in the hypoxia-induced cell cytotoxicity has been clarified by results of recent studies [9,10].
Increase of free radicals in generation of ROS or decrease in their elimination disturbs the oxidative balance and
creates oxidative stress. ROS interacts with biomolecules such as lipids, carbohydrates and DNA, and it causes
mito-chondrial dysfunction and cell membrane damage [11,12].
Zinc (Zn) is an essential element, component of metalloen-zymes and metalloproteinases, and it has a structural and regulating role in intermolecular interactions. Also, Zn is a co-factor of antioxidant enzyme superoxide dismutase (SOD) and it plays a protective role against deleterious effects of
oxidative stress [13,14]. Protective role of SOD on TRPM2
channel activity and cell cytotoxicity was reported in SH-SY5Y neuroblastoma cells [8]. Selenium (Se) is a trace elem-ent that is necessary for cellular functions. This elemelem-ent is main component of selenoproteins such as selenium-dependent glutathione peroxidase (GSH-Px), iodothyronine deiodinase and thioredoxin reductases. It protects biomole-cules such as lipids, DNA, RNA and TRPM2 channel activity in the cell membrane against the oxidative damage caused by
the ROS [15–17]. GSH protects the cell membrane against
lipid peroxidation through enzymatic reactions containing CONTACTDilek Duzgun Ergun dilekergun@aydin.edu.tr Department of Biophysics, Faculty of Medicine, Istanbul Aydin University, Istanbul 34295, Turkey
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION 2020, VOL. 40, NO. 6, 521–530
thiol group [18,19]. In addition to the dose-dependent bene-ficial effects of Zn, Se, and GSH there are also toxic effects.
Results of recent studies indicated protective actions of Zn, Se and GSH on TRPM2 channel activity, cell cytotoxicity and oxidative stress values in several pathophysiology condi-tions [17,19,20], although the protective effects of Zn, Se and GSH on the values in the hypoxia-induced cells have not been clarified yet. In the current study, we aimed to investi-gate protective effects of Zn, Se and GSH on lipid peroxida-tion, cytotoxicity, and TRPM2 current densities in transfected human embryonic kidney cells (HEK293) exposed to hypoxia.
2. Materials and methods 2.1. Cell culture
HEK293 cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in
the standard culture medium consisting 89% Dulbecco’s
Modified Eagle Medium (Wisent Bioproducts, Quebec,
Canada), 10% heat-activated fetal bovine serum (Wisent Bioproducts, Quebec, Canada), and 1% penicillin-strepto-mycin antibiotic solution (Sigma, Germany). The cells were
incubated in a CO2 incubator containing 5% CO2 and 97%
humidified atmosphere at 37C, checked regularly every day,
and the culture mediums were changed every 2 day. The cells were seeded in T25 flasks (Nest Lab, California, ABD) at a density of 1 105 cells/mL. When the cells covered at least 80% of the culture flasks, they were collected and
subcul-tured using 0.25% Trypsin-EDTA (Wisent Bioproducts,
Quebec, Canada) in laminar flow cabin. The cells were used average between 3 and 15 subcultures in all experiments.
2.2. Exposure system and design
The cells were exposed to two different gas mixtures, namely normoxic and hypoxic. The normoxic gas mixture contains
5% CO2, 20% O2, and equilibrium N2, and the hypoxic gas
mixture contains 5% O2, 5% CO2, and equilibrium N2.
Normoxia conditions were evaluated as the control group. Changes that occurred depending on the time exposed to both normoxia and hypoxia were analyzed. Effects of Zn, Se and GSH on the hypoxia was evaluated by comparing to with each other and hypoxic group. The cells were placed into compartments that were connected to the tubes con-taining the gas mixtures and they were controlled through valves. The gas outlet pressure of the tubes containing the
gas mixtures were adjusted as 0.1–0.2 bars. The gas mixtures
were passed through glass bottles containing 200 mL dis-tilled water for balancing and humidifying before passing through the cell samples. The gas mixtures were passed through 25 cm2cell flasks for 30 min or 60 min.
2.3. Experimental procedure
In this study, ten different groups were designed with
HEK293 cells. HEK293 cells were incubated in CO2 incubator
with a medium which was prepared with 100mM Zn (48 h),
150 nM Se (48 h), 5 mM GSH (5 h), and then exposed to hyp-oxic gas mixture for 30 and 60 min. [13,17,19] Experiments
repeated six times for each groups. Zinc sulfate (ZnSO4
-Sigma, Germany), sodium selenite (Na2SeO3-Sigma, Germany)
and L-glutathione reduced (GSH- Sigma, Germany) were used as a chemical agent.
2.4. Transformation of pcDNA3-IRES-EGFP-TRPM2 plasmid
Pre-prepared E. coli DH5a competent cells were used for
bacterial transformation of pcDNA3-IRES-EGFP-TRPM2 plas-mid (TRPM2 gene). The plasplas-mids were transformed into com-petent cells through heat shock. In order to determine the right colony that contains the desired plasmid, 6 different colonies were selected from the petri dishes where reproduc-tion was observed. The plasmids were isolated from each colony using Miniprep Kit (Qiagen, Germany), and following the kit protocol. Quality control was performed by running the isolated plasmids on 1% agarose gel electrophoresis.
When the right colony was determined, a higher amount of plasmids were obtained and plasmids were isolated from
the 200 mL maxi culture using Maxiprep Kit (Sigma,
Germany). Plasmid at a concentration of 1mg/mL was
obtained as a result.
2.5. Transfection: TRPM2 channel protein expression at HEK293 cells
Transfection is required to determine the cells that have TRPM2 channels in HEK293 cells. The method specified for Perfectin Transfection kit (Genlantis, USA) was used for trans-fection. Through this method, green fluorescent protein (pcdGFP) that contained TRPM2 sequence in its structure was transfected for HEK293 cells. The cells were analyzed
under fluorescent microscope 24–48 h after the transfection.
The cells that have TRPM2 channels were green under fluor-escent light, and these transfected cells were used for the experiment. Transfection was repeated for each cell passag-ing [21].
2.6. Po2and PCO2values measurement
Blood gas parameters (PO2 and PCO2) were measured at
each stage of the experiments from 200mL of sample
col-lected with an insulin injector using Blood Gas Analyzer (Radiometer-ABL 800) in order to evaluate normoxia and hypoxia conditional.
2.7. LPO level assay
LPO was measured using the method employed by Buege et al. [22]. The principle of the method is the spectrophoto-metric measurement of the color generated by the reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA), a product of lipid peroxidation. The levels of MDA were deter-mined using the absorbance coefficient of the MDA-TBA
Shimadzu UV-VIS Spectrophotometer), and the concentration of LPO of the cells were expressed as nmol/1 105cells.
2.8. LDH% value determinations
LDH enzyme is a cytoplasmic enzyme that is released into the cell culture medium when the cell membrane is destroyed and shows cytotoxicity, cell death rate. The LDH% activity was measured by spectrophotometer (UV-1800 Shimadzu UV-VIS Spectrophotometer) using cell lysate sam-ples as per the method specified in TOX7 Assay kit (Sigma, Germany).
2.9. Electrobiophysics
The measurements were conducted with the patch clamp technique, in the whole-cell mode, using an EPC10 USB (HEKA, Lamprecht, Germany) amplifier. All recordings were
conducted in room temperature (20–22C). The pipette
solu-tion contained in mM: 145 L-glutamic acid, 8 NaCl, 2 MgCl2,
0.001 CaCl2, 10 Hepes, and 10 EGTA, with the pH was
adjusted to 7.2 using CsOH. The pipettes (Sutte Instrument, USA) made of borasilicate glass were filled with pipette solu-tion. Extacellular bath solution contained in mM: 140 NaCl, 1.2 MgCl2,1.2 CaCl2,5 KCl, 10 Hepes, 10 D-Glucose H2O2 (pH
7.4) (used KOH). Extracellular bath solution containing NMDG contained in mM: 150 NMDG, 1.2 MgCl2, 1.2 CaCl2, 10 Hepes,
10 D-Glucose H2O2. The pH of solution was adjusted to 7.4
using 1 M HCl. 2-Aminoethoxydiphenyl borate (2-APB) solution: 22.5 mg was weighed from 2-APB solution stock and it was dissolved in 1 mL dimethyl sulfoxide (DMSO) to reach the last concentration of 100 mM. Hydrogen Peroxide (H2O2)
solu-tion: 10.22mL was taken from 30% H2O2 solution and
dis-solved in 10 mL extracellular media solution. The cell
membrane potential was set to –60 mV, and current
(I)-volt-age (V), current (I)-time (t), relations were obtained.
Measurements were performed by whole cell mode. The data obtained from the Patch Master software was trans-ferred to the Microcal Origin 8.0 program for evaluation. Mean current density was calculated for each experiment group using the Rmembranvalue.
2.10. Statistical analysis
The data obtained from the experiments was evaluated using IBM SPSS 21.0 package program. The statistical evalu-ation was conducted using ANOVA following appropriate transformation to normalized data and equalized variance where necessary. The results were provided as mean (M) ± standard deviation (SD). The level of significance in all statis-tical comparisons was considered as p< .05.
3. Results
3.1. LPO results in the HEK293 cells
Our study demonstrated that the LPO levels of the hypoxia-exposed cells (30 min and 60 min) showed a statistically
significant increase (p< .01) than the normoxia-exposed cells (30 min or 60 min), which are control groups. LPO values of the group exposed to 60 min hypoxia significantly increased
compared to the group exposed to 30 min hypoxia (p< .05)
(Figure 1(a)). The LPO values measured in the hypoxia-exposed cells for 30 min or 60 min after being incubated
with 100mM Zn, 150 nM Se, and 5 mM GSH showed a
signifi-cant decrease (p< .05) when compared to the cells were
only exposed to hypoxia for 30 min or 60 min (Figure 1(b,c)).
3.2. LDH% values in the HEK293 cells
Our study demonstrated that the LDH% values in the hypoxia-exposed cells for 30 min or 60 min showed a significant increase than to the normoxia-exposed cells for 30 min or 60 min, which are control groups, (respectively, p< .01 and p < .001) . The LDH% values measured in the hypoxia-exposed cells for 60 min showed a statistically significant increase compared to the hyp-oxia-exposed cells for 30 min (p< .001) (Figure 2(a–c)).
The LDH% values measured in the hypoxia-exposed cells
for 30 min and 60 min after being incubated with 100mM Zn
showed a significant decrease compared to the cells that were only exposed to hypoxia for 30 min and 60 min (respectively, p< .05 and p < .001). Furthermore, the LDH% values in the hypoxia-exposed cells for 30 min and 60 min after being incubated with 150 nM Se, and 5 mM GSH also showed a highly significant decrease compared to the cells that were only exposed to hypoxia for 30 min and 60 min (respectively, p< .01 and p < .001) (Figure 2(d,e)).
3.3. Effects of H2O2in HEK293 cells
Figure 3 shows the effect of H2O2 on TRPM2 currents in
HEK293 cells exposed to normoxia that is control group, for 30 min or 60 min. No change was observed in TRPM2 current
before H2O2 stimulation in cells exposed normoxia 30 min
and 60 min (Figure 3(a,c)).
Current-time graphics showed that stimulation with
10 mM H2O2 to the normoxia-exposed cells (30 min and
60 min), resulted with increased negative currents as
–0.49 nA and –0.52 nA at TRPM2 channels of the cells. Within
3–5 min, TRPM2 currents reached plateau. 2-APB, NMDG, and
then H2O2, 2-APB, NMDG were applied, respectively, to the
cell media. The biggest negative current value passing through the channel during whole cell were recorded and channel was waited to close again. Current- time, and cur-rent- voltage graphics were created (Figure 3(b,d)).
The mean current density (pA/pF) after whole cell record-ing of the normoxia-exposed cells for 30 min, and 60 min were–74.11 pA/pF, and –91.02 pA/pF (Figure 3(e)).
3.4. Effects of Zn, Se, and GSH on TRPM2 current in the HEK293 cells exposed to hypoxia
In the current study, the protective effects of Zn, Se, and GSH on TRPM2 currents in the normoxic and hypoxic cells were also tested. For this purpose, the channel was stimu-lated with 10 mM H2O2. There was no change in the currents
in TRPM2 channels before stimulation with H2O2 in only
30 min hypoxia, 100mM Zn þ 30 min hypoxia, 150 nM Se þ
30 min hypoxia, and 5 mM GSH þ 30 min hypoxia groups.
Negative currents were detected in the same cells after
stimulation with 10 mM H2O2. The current-time graphics
showed that –0.97 nA, 0.75 nA, 0.51 nA and –0.61 nA
cur-rents, respectively, occurred in the TRPM2 channels of the hypoxia groups (Figure 4(a–e)).
Figure 1. LPO levels in HEK293 cells. (a) The hypoxia-exposed cells according to the normoxia groups. (b) The cells exposed to hypoxia for 30 min after being treated with 100lM Zn, 150 nM Se, and 5 mM GSH according to the hypoxia group. (c) The cells exposed to hypoxia for 60 min after being treated with 100 lM Zn, 150 nM Se, and 5 mM GSH according to the hypoxia group (M ± SD) (n ¼ 6) (p < .05; p < 0.01).
Figure 2. Change of LDH% values in HEK293 cells. (a,b) The hypoxia-exposed cells for 30 min according to normoxia groups. (c) The cells exposed to hypoxia for 60 min relative to the cells exposed to hypoxia for 30 min. (c) The cells exposed to hypoxia for 30 min after being treated with 100lM Zn, 150 nM Se and 5 mM GSH in according to hypoxia group. (d) The cells exposed to hypoxia for 60 min after being treated with 100lM Zn, 150 nM Se and, 5 mM GSH in according to hyp-oxia group (M ± SD) (n ¼ 6) (p < .05; p < 0,01; p < .001).
Figure 3. Effects of normoxia on the TRPM2 current densities (pA/pF) in HEK293 cells. TRPM2 currents in HEK293 cells were stimulated by H2O2(10 mM), and they
were blocked by extracellular TRPM2 antagonist 2-APB (100 mM) in the patch chamber. W.C.: Whole cell. (a) Orginal recordings from HEK293 cells exposed to nor-moxia for 30 min without H2O2. (b) Orginal recordings from HEK293 cells exposed to normoxia for 30 min with H2O2. (c) Orginal recordings from HEK293 cells
exposed to normoxia for 60 min without H2O2. (d) Orginal recordings from HEK293 cells exposed to normoxia for 60 min with H2O2. (e) Current densities of the
groups (M ± SD) (n ¼ 6) (p<.05; p<.001).
The mean current densities in the hypoxia-exposed cells (30 min and 60 min) showed a statistically significant increase compared to the normoxia-exposed cells for 30 min and 60 min (p< .05, p < .01) (Figure 3(e)).
When compared to the mean current density value of the group that was only exposed to hypoxia for 30 min to the hypoxia-exposed (30 min) cells after being incubated with 100mM Zn and 5 mM GSH (p < .05) and 150 nM Se (p < .01) showed a statistically significant decrease (Figure 4(f)).
There was no change at the currents of TRPM2 channels before stimulation with H2O2 in only 60 min hypoxia, 100mM
Zn þ 60 min hypoxia, 150 nM Se þ 60 min hypoxia and,
5 mM GSH þ 60 min hypoxia groups. A negative increase
was observed in the current after stimulation with H2O2 in
the group that was only exposed to hypoxia for 60 min and, the hypoxia-exposed cells for 60 min after being incubated
with 100mM Zn, 150 nM Se and, 5 mM GSH. The
current-time graphics showed that–0.99 nA, 0.78 nA, 0.60 nA and
–0.68 nA currents, respectively, occurred in the TRPM2
chan-nels of the these groups (Figure 5(a–e)). The
hypoxia-exposed cells for 60 min after being incubated with 100mM
Zn, 150 nM Se, and 5 mM GSH showed a statistically
signifi-cantly higher decrease in mean current densities (p< .01)
when compared to the group that was only exposed to hyp-oxia for 60 min (Figure 5(f)). The data indicate that Zn, Se, and GSH have a protective role against oxidative stress, and inhibit TRPM2 channel activity.
4. Discussion
It is well known that TRPM2 currents are activated by H2O2.
The studied on activation and inhibition mechanisms of
TRPM2 channels that enable influx Ca2þ ion to intracellular
environment is important in terms of understanding the
underlying mechanisms behind many diseases [23,24]. There
are numerous studies with action mechanisms of TRPM2 channels at hypoxia [9,10] however, the studies on
hypoxia-oxidative stress and hypoxia-Ca2þ currents on TRPM2
chan-nels at transfected HEK293 cells are inadequate. This study aimed to investigate the effects of hypoxia on TRPM2
cur-rents. In the present study, we show that H2O2-induced
TRPM2 channels are greatly inhibited and therefore the Ca2þ
influx through this channel is reduced in the HEK293 cells incubated with Zn, Se, and GSH.
Figure 4.Effects of Zn, Se, and GSH on TRPM2 current densities (pA/pF) in HEK293 exposed to hypoxia for 30 min (n ¼ 6). TRPM2 currents in HEK293 cells were stimulated by H2O2(10 mM), and they were blocked by extracellular TRPM2 antagonist 2-APB (100 mM) in the patch chamber. W.C.: Whole cell. (a) Original
record-ings from HEK293 cells exposed to hypoxia for 30 min without H2O2. (b) Original recordings from HEK293 cells exposed to hypoxia for 30 min with H2O2. (c) 100mM
Znþ Hypoxia 30 min (with H2O2). (d) 150 nM Seþ Hypoxia 30 min (with H2O2). (e) 5 mM GSHþ Hypoxia 30 min (with H2O2). (f ) Current densities of the groups
LPO levels and cytoplasmic LDH enzyme which develops as a result of LPO, was measured in the all of groups. Our results demonstrated that the LPO levels and LDH% values in the hypoxia-exposed cells for 30 min or 60 min a statistic-ally significant increase compared to the normoxia-exposed cells for 30 min or 60 min. In consistent with our findings
Yajima et al. [25] reported increased LPO levels and cell
membrane damage under hypoxic conditions in mouse embryonic fibroblast (MEFs) cells. In another study it was determined that lipid hydroperoxidase and plasma LPO con-centrations were increased in individuals who are exposed to intermittent hypoxia [11].
It was showed that under hypoxic conditions nicotina-mide adenine dinucleotide phosphate (NADPH), an oxidative stress enzyme, expression and ROS increase in mesenchymal stromal cells isolated from Sprague-Dawley rat femurs, and as a result, cell viability is decreased due to increased LDH
activity [26]. Results of our study showed LPO levels and
LDH% values increased in parallel with the increase of free oxygen radicals in HEK293 cells are exposed to hypoxia. When oxidant/antioxidant balance is disturbed after the
hyp-oxia, release of pro-apoptotic proteins, which cause
apoptosis increases as a result of the damage to the biomo-lecules such as proteins, lipids, carbohydrates and nucleic acids. This, in return, damages the integrity and selective per-meability of cell membrane, and trigger cellular damage
[27–29]. Increased LDH% values in the hypoxic groups
com-pared to the normoxic groups in our study demonstrates that hypoxia increased cellular damage and cytotoxicity, and therefore cell death. The findings of our study are in line with the literature information on oxidative effects of hyp-oxia. More importantly, however, this study showed that dur-ation of the hypoxia applicdur-ation affects the increase in LPO levels, and LDH% values.
Zn2þ binds to the active domains of the enzymes that are
effective in defense system and enables activation of
enzymes. Furthermore, Zn2þ indirectly reduces the harmful
effects of free radicals together with GHS or as a GSH-Px cofactor [30–32]. Se is an important antioxidant for activation of GSH-Px enzyme, protects the cell against the LPO that damages cell membrane. GSH, contains thiol group, protects the cell membrane against LPO through enzymatic reactions or through its free-radical destroying properties [33,34]. In the current study, it was determined that the LPO levels and Figure 5.Effects of Zn, Se, and GSH on TRPM2 current densities (pA/pF) in HEK293 exposed to hypoxia for 60 min (n ¼ 6). TRPM2 currents in HEK293 cells were stimulated by H2O2(10 mM), and they were blocked by extracellular TRPM2 antagonist 2-APB (100 mM) in the patch chamber. W.C.: Whole cell. (a) Original
record-ings from HEK293 cells exposed to hypoxia for 60 min without H2O2. (b) Original recordings from HEK293 cells exposed to hypoxia for 60 min with H2O2. (c) 100mM
Znþ Hypoxia 60 min (with H2O2). (d) 150 nM Seþ Hypoxia 60 min (with H2O2). (e) 5 mM GSHþ Hypoxia 60 min (with H2O2). (f ) Current densities of the groups
(M ± SD) (n ¼ 6) (p < .01).
LDH% values in the hypoxia-exposed cells (30 min or 60 min)
after being incubated with 100mM Zn, 150 nM Se, and 5 mM
GSH show a statistically significant decrease compared to the only hypoxia-exposed (30 min or 60 min) cells. According to our findings, the LPO levels and LDH% values which were increased due to the oxidative damage of hypoxia on the cell decreased significantly in the HEK293 cells were
incu-bated with antioxidant. This data show that Zn2þ, Se, and
GSH concentrations and incubation times were suitable for protective effect against oxidative damage.
An increase in the negative current and mean current densities of TRPM2 channel was observed in the hypoxia-exposed cells for 30 min and 60 min. As a result of the
increase in the TRPM2 channel activity, the Ca2þ current
toward the cell was also increased in hypoxi-exposed cells.
An increase in Ca2þ amount toward the cell triggers Ca2þ
release in intracellular organelles which in return raises the oxidative stress inside the cell, damages the cell membrane
and causes cell death [19,35]. We evaluated LPO and Ca2þ
currents in cells by incubating with Zn2þ, Se, and GSH, which are known to have a protective effect, in order to decrease the oxidative stress induced by hypoxia and reduce the cell death rate. We determined that LPO levels, LDH% values and TRPM2 currents decreased in the cells that were incubated
with Zn2þ, Se, and GSH. Furthermore, we also observed that
the mean current densities was decreased in the cells that
were incubated with Zn2þ when compare to the hypoxia
groups. Extracellular Zn2þ increases ion channel activity or
inhibits the channel through its allosteric modulation effect. It is known that TRPM2 channels have ion selectivity filters in S5 and S6 extracellular domains and that this domain
con-tains 20 different residue areas that interact with Zn2þ
including histidine, cysteine, lysine, aspartate or glutamate.
Zn2þ interacts with the pore domain in the weak channel
domain of the TRPM2 and induces its activation [32,36,37].
Zn2þ acts as an inhibitor for N-methyl-D-aspartic acid
(NMDA) receptor that carries Ca2þ from extracellular area to
the intracellular area. Zn2þ deficiency activates NMDA
recep-tor and increases intracellular Ca2þ concentration. The
increase in Ca2þconcentrations contributes to release of free radicals that cause oxidative stress. Furthermore, Zn2þ inhib-its NADPH oxidase receptor that is a prooxidant enzyme and
induces metallothionein synthesis. Consequently, Zn2þ
defi-ciency activates NADPH which in return increases ROS and nitrogen species [32,33].
Some studies on TRPM2 channel expression in adult rat cardiac fibroblasts report that hypoxic stress has an effect on
the ionic currents passing through cell membranes [38–40].
It was established that hypoxia affects the Ca2þ channels
and consequently increases the Ca2þcurrent, and the current
density of TRPM2 channel than to the normoxic group. Similar to the present results, it was reported that the Ca2þ current through voltage-gated calcium channels in hypoxia-exposed rat primary cortical neurons increased compared to normoxia-exposed cells; however, after 48 h normoxic
recov-ery, Ca2þ current decreased [40]. Another study conducted
on TRPM2 channels in rat cardiac fibroblasts reported that the mean current density in the group that was exposed to
hypoxia showed a significant increase at 70 mV compared to the group that was exposed to normoxia while there was a
significant decrease between 10–40 mV. Furthermore, it was
reported that when the hypoxia-exposed cells were held at
–70 mV, the inward Ca2þ current increased by 297%
com-pared to normoxia, while the outward current at 40 mV increased by 182% [39].
Sarada et al. [41] observed that in comparison to the GSH values of the control group, cytotoxicity and free radical gen-eration decreased, but GSH increased in the cells that were incubated with Se and then exposed to hypoxia. In addition, it was reported that applying Se extracellularly or intracellu-larly to CHO cells has different effects on TRPM2 channel that was induced by H2O2. They reported that application of
intracellular Se is more effective in inhibition of TRPM2 cur-rent when compared to application of extracellular Se [17].
In our study, we used LPO levels and LDH% values to show that hypoxia-induced oxidative stress is decreased in HEK293 cells were incubated with Se. Furthermore, we estab-lished that Se showed an antioxidant effect on TRPM2 chan-nels that reduced activation of the channel along with the
amount of Ca2þ current across the channel. We observed
that the mean current densities are decreased in the groups that were exposed to hypoxia after being incubated with Se compared to the groups that were only exposed to hypoxia. These results demonstrate the antioxidant effects of Se against oxidative stress that induced by hypoxia.
In the current study, hypoxia-induced TRPM2 channel activities were also decreased by the GSH treatment. There are some reports of interactions between the TRPM2 channel activity and GSH in some physiological/pathophysiological conditions except hypoxia. Similar to the present GSH results on the TRPM2 channel in the HEK293 cells, protective actions of 10 mM extracellular and 2 mM intracellular GSH incuba-tions on the inhibition of TRPM2 current in CHO cells were reported [42]. It was reported that the Ca2þ current across TRPM2 channels is inhibited when the cells are incubated with GSH and the decrease in GSH synthesis is responsible for neuroinflammation [43]. It was established that mean cur-rent density in TRPM2 channels is decreased when hippo-campal pyramidal neurons are treated with N-acetyl-cysteine, a precursor to GSH synthesis; and the mean current density increased when GSH synthesis is inhibited. Furthermore, they
showed that GSH inhibited TRPM2 channel [44].
Our study demonstrated that LPO levels and LDH% values were decreased in the hypoxia-exposed cells after being incubated with GSH compared to the cells that were only exposed to hypoxia. After hypoxia were applied to HEK293
cells for different durations and the amount of Ca2þ current
passing through TRPM2 channels were evaluated, it was
established that the amount of Ca2þ entering the cells
increased when the duration of hypoxia application is also increased. An increase in the duration of hypoxia exposure
might increase Ca2þ current in the cells, which in return
might trigger more Ca2þ release and increase oxidative
stress, possibly damaging cellular membrane permeability and cause irreversible processes in the cell.
The effects of hypoxia on the Ca2þ current can be explained by a shift toward acidity in the intracellular medium. Under hypoxic conditions, inadequacy of oxygen, which is necessary for ATP production in the cell, would cause lactic acid accumulation in the environment. The acidic effect of lactic acid, which is a metabolic acid, would cause damages on the cells and intracellular organelles, and would negatively affect cell functions. Therefore, we can accept that lactic acid causes stress inside the cell [29].
The studies on the effects of hypoxia mainly investigate the effects of chemical hypoxia. In these studies, the out-comes of hypoxic conditions created with different chemicals are not parallel to each other. The hypoxia exposure system used in our study is based on the principle of introducing humidified hypoxic gas directly to the cell medium and therefore different from the other studies [45,46]. The
hyp-oxic environment caused a significant increase in the Ca2þ
current density through TRPM2 channels compared to the normoxia group. Our study demonstrates that hypoxia increases LPO levels and LDH% values and shows that it increases ROS. Based on our findings, we can speculate that
hypoxia possibly increases the amount of Hþions along with
the intracellular ROS, and causes an increase in Ca2þamount
through Ca2þ current. The primary reason for the increase in
Ca2þ current density is the increase in ROS. However, we
think that the increase in Hþ ion also contributes to that
effect.
Conclussion
It was concluded that introduction of Zn, Se, and GSH decreased the hypoxia-induced oxidative stress and the cell death through inhibition of TRPM2 channel in the HEK293
cells. The results of our research conducted on TRPM2 Ca2þ
channels in HEK293 cells should be supported by different cell lines incubated with antioxidants in different concentra-tions and with different duraconcentra-tions. Treatment of Zn, Se, and GSH may be considered as a therapeutic strategy to prevent hypoxia-induced TRPM2 channel activation, oxidative toxicity and cell death in the HEK293 cells.
Acknowledgement
The study was carried out at Patch Clamp Laboratory, Cell Culture Laboratory, and Blood Gas Measurement Laboratory at Department of Biophysics, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, and Department of Genetic, Aziz Sancar Institute of Experimental Medicine, Istanbul University. This study was presented as an oral pres-entation at 7th World Congress of Oxidative Stress, Calcium Signaling and TRP Channels, 20 and 23 April 2018, Alanya, Turkey (http://2018. cmos.org.tr/)
Author contributions
SD designed the study and developed the theory. D.D.E. performed the majority of the experiments, analyzed the data, and wrote the manu-script. N.P.O. contributed to the planning and execution of experiments. O.H.N. shared to experiments. SD, D.O., and M.N. supervised the whole experimental work and revised the manuscript. All authors provided crit-ical feedback and helped shape the research, analysis, and manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Dilek Duzgun Ergun http://orcid.org/0000-0002-6245-6631
Data availability
The data that support the findings of this study are available from the corresponding author, upon reasonable request.
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