High concentration salt is an aggressive factor for gastric mucosa (1). It has reported to destroy a mucosal barrier and leads to inflammation and damage such as diffuse erosion and degeneration (2). High concentration salt derives a hyperosmotic pressure environment for gastric mucosa, which results in a physicochemical effects to induce cellular damage. Salt-inducible cell injuries were often investigated in botany, because salt is one of the most significant stresses affecting crop yield all over the world. The researchers in botany called the salt inducible cell injury "salt stress". Salt stress is known to impair many cellular functions, such as metabolism, cellular osmotic balance and ion homeostasis (3, 4). The salt spray accumulation on plants in the field is correlated with increased necrosis (3). Another study reported that a salt treatment for plant induced oxidative stress and superoxide production (5, 6). Salt may be an oxidative stress for an animal cell although a cellular structure in the animal cells is different from that of a plant cells: the former doesn't have cell walls while the latter does.

Gastric acid has also been one of most important aggressive factor for gastric mucosa since Karl Schwartz said a famous dictum "no acid, no ulcer" in 1910 (7). The acid has been regarded as a merely necrotizing factor. We have recently reported that a moderate acidic condition exposure involved mitochondrial superoxide production with an electron paramagnetic resonance (EPR) analysis (8). In another words, gastric acid is not only a necrotizing factor but also an oxidative stress inducer. Since acidic environments inhibited mitochondrial electron transport to generate superoxide anion, a salt-derived hyperosmotic environment may also inhibit it.

In this study we elucidated whether salt is an oxidative stress inducer on rat gastric mucosal cells (RGM-1) (9). For this purpose, we measured living gastric epithelial cells' reactive oxygen species (ROS) spectra with an EPR apparatus. Moreover, to clarify whether the salt-induced ROS is derived from mitochondria, we also investigated the salt-induced ROS production in manganese superoxide dismutase overexpressing cells (RGM-MnSOD) (10).


MATERIALS AND METHODS
Materials

2-(5,5-Dimethyl-2-oxo-2 5-[1,3,2]dioxaphosphinan-2-yl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (CYPMPO) (Radical Research Inc., Tokyo, Japan), ß-nicotinamide adenine dinucleotide (NADH) (SIGMA), D-glutamic acid (SIGMA), malic acid (Wako), succinic acid (SIGMA-ALDRICH), diphenyl-1pyrenylphosphine (DPPP) (DOJINDO, Kumamoto, Japan), Tetra Color ONE® cell proliferation assay kit (Seikagaku, Tokyo, Japan) and sodium chloride (Wako) were purchased. High osmotic culture medium was prepared by melting NaCl, and the culture medium was used after filter-sterilized (Millex 0.22 µm, Millipore Co., Billerica, USA).

Cell culture

RGM-1 cultured in DMEM/F12 (Gibco). This culture medium contained 10% inactivated FBS and 1% penicillin/streptomycin. Culture medium for RGM-1 MnSOD also contained antibiotic G418 sulfate solution for being stable MnSOD expression. Gene vector transfected RGM-1 cell line was used as control for gene transfect. The different number in RGM-1 MnSOD (e.g. RGM-1 MnSOD4, RGM-1 MnSOD6) means different clone. The amounts of expressed-MnSOD in RGM-1 MnSOD6 is higher than that in RGM-1 MnSOD4 (11). Each cells showed different MnSOD-expression (10). All cells were cultured in 5% CO2 cell culture incubator at 37°C.

Cell viability test

Cell viability test was examined with the Tetra Color one assay kit according to the manufacturer's instructions. RGM-1 was dispersed in the 96-well dish at 10,000 cells/well and they were incubated for overnight. The medium was replaced to the high osmotic culture medium which contained NaCl 150, 300, 450, 650, and 1000 mM and it was incubated for 0, 1, 3, and 6 hours. After incubation, mediums were replaced to the medium contained 10% Tetra Color One of 100 µL and cells were incubated for 1 hour.

The absorbance of 450 nm was measured by Vario scan plate reader (Thermo Fisher Scientific K. K., Kanagawa, Japan).

Lipid peroxide detection

The detection of lipid peroxide was performed according to previous reports (11, 12). Briefly, cells were dispersed on the culture dish at the concentration of a 30,000 cells/cm2. After cells attached, culture medium was replaced to the medium contained 10 µM DPPP. Cells were incubated for 30 min in the 5% CO2 incubator. Cells were washed twice with PBS and cellular fluorescent images were observed and their intensities were measured with a chilled CCD camera (AxioCam color, ZEISS, Germany) - mounted epi-fluorescence microscope (Axiovert135M, ZEISS) connected to an image analyzing system (AxioVision, ZEISS).

Electron paramagnetic resonance (EPR) measurement

The methods of electron paramagnetic resonance (EPR) measurement was described in some previous reports (11, 13). Cells were cultured on the slide glass until confluent. The slide glass was immersed into different NaCl contained medium (150, 300 and 500 mM of NaCl concentration) and it was incubated for 10, 30 and 60 min in the 5% CO2 incubator at 37°C. After the incubation, the slide glass was put on the tissue glass (Radical Research Inc., Tokyo, Japan) and 100 µL of the solution for EPR measurement, which was prepared that the respiratory substrates (5 mM succinic acid, 5 mM malic acid, 5 mM D-glutamic acid, 5 mM NADH) and 10 mM CYPMPO was dissolved in phosphate buffer saline, poured in the tissue glass. Then then the EPR spectra were recorded by using a JEOL-TE X-band spectrometer (JEOL, Tokyo, Japan). All EPR spectra were obtained under the following conditions: 10 mW incident microwave power, 0.1 mT modulation width, 8 min sweep time, 7.5 mT sweep width, 0.1 s time contrast, 333.5 mT center field, and 15 mT scan range. Spectral computer simulation was performed using a Win-Rad Radical Analyzer System (Radical Research).

Statistical analysis

The statistical analysis was calculated on the Origin software. Significant static value (p value) was calculated using ANOVA followed by Scheffe's F-test.


RESULTS
NaCl induced the cell death

The cell viability after NaCl-exposed was determined by cell viability test against the normal rat gastric mucosa cells (RGM-1). Fig. 1 showed the solution of 300 mM NaCl had cytotoxicity, and a cell death was begun after the cells incubation with this solution for 30 min. After incubated for 6 hours, RGM-1 was death completely in the medium contained more 450 mM NaCl. Additionally this data suggested that the cytotoxicity by exposing cells to salt has some biological effects, because in addition to the physicochemical effects, the incubation for several hours is no need to degrade cells.

Fig. 1. Cell viability after salt exposure. Cell viability was evaluated by Tetra Color One assay kit. The different salt concentration medium was made by adding salt to culture medium. The absorbance at 450 nm was measured by plate reader. Cell; RGM-1, N=6, Error bar; S.D.

Salt induced ROS from the cells

The ROS concentration from the cells was determined by EPR measurement using spin-trapping agents (CYPMPO). CYPMPO produced the superoxide adduct. The half-life for the superoxide adduct of CYMPO produced in UV-illuminated hydrogen peroxide (H2O2) solution was approximately 15 min, and the half-life in the biological systems such as hypoxanthine/xanthine oxidase (O2-) is approximately 50 min (14). EPR signals intensities in RGM-1 exposed to different concentration of salt are shown in Fig. 2. Data presented in Fig. 2 showed that the 300 mM NaCl induced ROS from the RGM-1 after incubated for the 10 min. The EPR signal intensity of 500 mM NaCl is not significantly different comparing to 300 mM NaCl.

Fig. 2. The EPR spectra from RGM-1 after salt exposure. The intensity of EPR signals was strong after exposed-salt. This phenomena was begun after incubation for 10 min. Spin-trapping agent; CYPMPO.

MnSOD suppressed the production of reactive oxygen species by a salt

MnSOD is the specific scavenger of superoxide from mitochondrion. We used transgenic MnSOD-expressing RGM-1 stable clone (RGM-1 MnSOD) for clarifying the relation between the mitochondria and salt-induced ROS (10). Fig. 2 showed the EPR spectra when RGM-1 cells were incubated in the various concentration of salt contained culture medium. The results showed the intensity of EPR signals from RGM-1 MnSOD cells was weak compared in that of RGM-1 after exposed-300 mM NaCl contained culture medium for 10 min (Fig. 3). These results indicated that productive ROS by a salt has occurred from the mitochondrion.

Fig. 3. The EPR spectra from RGM-1 and RGM-1 MnSOD after salt exposure. MnSOD suppressed the production of ROS from the cells. Spin-trapping agent; CYPMPO.

MnSOD suppressed the peroxidation by a salt

MnSOD suppressed the ROS production by ROS. Therefore we evaluated the cellular membrane peroxidation using DPPP. The amount of the cellular membrane peroxidation in MnSOD-expressing cell showed lower than in the vector cells (Fig. 4). The difference of fluorescence intensity from DPPP between NaCl 150 mM and NaCl 300 mM in MnSOD-expressing cells was low compared to that in vector cells (Fig. 4b). Therefore MnSOD suppressed the cellular membrane peroxidation with reducing the production of ROS. Surprisingly, MnSOD showed no significant difference in the viability test using the medium containing 300 mM NaCl (Fig. 5), although the excessive amounts of an oxidative stress in the cells was suppressed. These data was suggested that the harmful effects against the human body of a salt are not related to the cell death but to increase in an oxidative stress.

Fig. 4. The evaluation of oxidative stress based on lipid peroxide. RGM MnSOD was showed the tendency of reducing lipid peroxidation by NaCl. (a) Ouantification of the intensity of DPPP fluorescent on the pictures. Cells were incubated in the daily culture medium or the medium containing 300 mM of NaCl for 30 min. These pictures were taken by same operation. (b) The difference between the cells exposed 150 mM NaCl and the cells exposed 300 mM. These results were confirmed by twice independent studies and the tendencies of these data showed the same results.

Fig. 5. Cell viability after salt exposure. Cell viability was evaluated by Tetra Color One assay kit. Cells were incubated in the medium containing 300 mM NaCl. This result showed no significant difference between RGM cells and RGM MnSOD cells. The absorbance at 450 nm was measured by plate reader. Cell; RGM-1, RGM-1 vector, RGM-1 MnSOD4, RGM-1 MnSOD6, N=6, Error bar; S.D. * p<0.05.

DISCUSSION
In this study, we demonstrated for the first time that hypertonic salt treatments involved reactive oxygen species (ROS) production in gastric epithelial cells.

Salt is regarded as an aggressive factor inducing gastric mucosal lesions. Salt has been reported to involve hypertonic osmotic pressure to induce necrosis in gastric epithelial cells physically. Although hypertonic NaCl has been reported an oxidative stress inducer in plant cells, there is no report investigating relations between ROS and sodium chloride in animal cells. Salt in soil and atmosphere is a severe problem necessary to find a solution in an agricultural field, while it is one of an aggressive factor for the stomach or other organs. Salt will be an only problem when a patient takes it excessively. Therefore few scientists probably pay attention to the pathogenesis of sodium chloride whether it is an oxidative stress inducer in animal cells.

EPR is an unique technique for determining the amount and the kind of ROS. However, the method of EPR measurement by living cells is not a common technique (15). A cells-attached cover glass was set on a tissue glass for EPR measurement. One of points for obtaining stable EPR spectra for living cells was making the setting time of these glasses shortest. Using this method, we investigated and reported that NSAIDs and bisphosphonate (8, 12, 16) involved ROS production.

Moreover, EPR measurement by a living cells proved that gastric acid is a ROS inducer, nevertheless it has been known as a necrotizing factor (10). In fact, strong acidic environments below pH2 were most likely to be necrotizing factors, while pH3 and/or pH4 environments involved ROS production from gastric epithelial cell mitochondria. In this study, all cells exposed under 1000 mM NaCl environment died within one hour. However, a part of cells exposed under less then concentration of 650 mM NaCl survived for a few hours and generated ROS. We proposed that high concentration of salt such as more than 1000 mM was a necrotizing factor, while moderate concentration of salt was an oxidative stress inducer rather than a necrotizing factor.

MnSOD is the specific scavenger of superoxide produced by mitochondrial electron transport (17). Therefore MnSOD expressing cells (10) are good experimental model to clarify whether intracellular ROS were derived from mitochondria or not (18-22).

In this study, we conclude that salt-induced high osmotic pressure environment inhibited a mitochondrial electron transfer system to involve superoxide anion production using MnSOD-expressing cells. Lysophosphatidylcholines and capsaicin are also known to induce the apoptotic cell death via caspase-3 activation and gastric injury via denervation, respectively (23, 24). The chemicals of gastric injury by superoxide were amplified each other. For example, the combination of superoxide inducers such as NSAIDs (16) and gastric acid (12) or NSAIDs and capsaicin caused severe gastric cell damage (8, 23). Oxidative stress by the salt should be also amplified by other oxidative stressors.

In conclusion, salt is not merely a necrotizing factor for gastric epithelial cells, but also an oxidative stress inducer. Now we are undergoing the study to explain the relations between salt-induced ROS and carcinogenesis in gastric epithelial cells.

Acknowledgements: This work was partially supported by the Japan Society for the Promotion of Science (JSPS) and Grant-in-Aid for Scientific Research (KAKENHI) #24106503, #70272200.
This work in its preliminary from has been presented during 7th International Symposium on Cell/Tissue Injury and Cytoprotection Organoprotection. September 9-11 2012, Honolulu, Hawaii organized by Prof. K. Takeuchi (Kyoto, Japan) and Prof. H. Matsui (Tsukuba, Japan).

Conflict of interests: None declared.


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