JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, Suppl 7, 87-92
www.jpp.krakow.pl
C. LOGUERCIO1, C. TUCCILLO1, A. FEDERICO1, V. FOGLIANO2, C. DEL VECCHIO BLANCO1, M. ROMANO1
ALCOHOLIC BEVERAGES AND GASTRIC EPITHELIAL CELL VIABILITY:
EFFECT ON OXIDATIVE STRESS-INDUCED DAMAGE
Centro Interuniversitario di Ricerca su Alimenti, Nutrizione e Apparato Digerente (CIRANAD) - Dipartimento Medico-Chirurgico
di Internistica Clinica e Sperimentale "F. Magrassi e A. Lanzara", Seconda Universita di Napoli, Italia; 2Dipartimento di Scienza
degli Alimenti, Universita di Napoli "Federico II", Italia
1
Alcohol is known to cause damage to the gastric epithelium independently of gastric acid secretion. Different alcoholic
beverages exert different damaging effects in the stomach. However, this has not been systematically evaluated. Moreover,
it is not known whether the non-alcoholic components of alcoholic beverages also play a role in the pathogenesis of gastric
epithelial cell damage. Therefore, this study was designed to evaluate whether different alcoholic beverages, at a similar
ethanol concentration, exerted different damaging effect in gastric epithelial cells in vitro. Moreover, we evaluated whether
pre-treatment of gastric epithelial cells with alcoholic beverages prevented oxidative stress-induced damage to gastric cells.
Cell damage was assessed, in MKN-28 gastric epithelial cells, by MTT assay. Oxidative stress was induced by incubating
cells with xanthine and xanthine oxidase. Gastric cell viability was assessed following 30, 60, and 120 minutes incubation
with ethanol 17.5-125 mg/ml-1 or different alcoholic beverages (i.e., beer, white wine, red wine, spirits) at comparable
ethanol concentration. Finally, we assessed whether pre-incubation with red wine (with or without ethanol) prevented
oxidative stress-induced cell damage. Red wine caused less damage to gastric epithelial cells in vitro compared with other
alcoholic beverages at comparable ethanol concentration. Pre-treatment with red wine, but not with dealcoholate red wine,
significantly and time-dependently prevented oxidative stress-induced cell damage. Conclusions: 1) red wine is less
harmful to gastric epithelial cells than other alcoholic beverages; 2) this seems related to the non-alcoholic components of
red wine, because other alcoholic beverages with comparable ethanol concentration exerted more damage than red wine;
3) red wine prevents oxidative stress-induced cell damage and this seems to be related to its ethanol content.
K e y w o r d s : alcohol, gastric epithelial cell viability, oxidative stress, red wine, xanthine oxidase, flavonoids
INTRODUCTION
Alcohol is a damaging agent for all cells, from the liver to
nervous system (1-3); however, epidemiological evidences
suggest that moderate alcohol consumption, particularly with red
wine, reduces the mortality due to coronary heart disease and
may have a protective effect also on the liver (4-7). The possible
protective effect of red wine has been attributed to its content in
polyphenols such as flavonoids and resveratrol, dietary
antioxidants able to counteract, both in vitro and in vivo,
oxidative-mediated cell damage (8-10).
Oxidative stress plays a mayor pathogenic role in a number
of gastrointestinal tract diseases (11). In particular, generation of
radical oxygen species (ROS) is involved in the pathogenesis of
gastric damage induced by a number of damaging agents,
including alcohol (12-14).
Different alcoholic beverages exert different damaging
effects in the stomach. However, to our knowledge this has not
been systematically evaluated. Moreover, it is not known
whether the non-alcoholic components of alcoholic beverages
also play a role in the pathogenesis of gastric epithelial cell
damage (15).
Therefore, this study was designed to evaluate whether
different alcoholic beverages, at comparable ethanol
concentration, exerted different damaging effect in gastric
epithelial cells in vitro. Moreover, we evaluated whether pretreatment of gastric epithelial cells with alcoholic beverages
prevented oxidative stress-induced damage to gastric cells.
MATERIAL AND METHODS
Chemicals
Absolute ethanol was provided by Sigma Aldrich. Beer,
spirits and wine used were selected from different commercial
types. Beers: Tuborg 5,0% ethanol and Ecu28 11.0% ethanol;
Spirits: whisky 40% ethanol; White wine: Falanghina 11.0%;
Red wines: Toscano 11.5% ethanol; Aglianico 12.0% ethanol;
Solopaca 12.5% ethanol.
DMEM F:12 medium (Dulbecco's modified Eagle's
medium), penicillin, streptomycin, fetal bovine serum (FBS), Lglutamine and trypsin/EDTA were obtained from Life
Technologies Inc., (Gaithersburg, Maryland, USA). MTT [3-
88
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide]
and xanthine and xanthine oxidase were purchased by Sigma
Aldrich. All solvents were for HPLC grade.
Design of the study
1. Preparation of alcoholic samples and dealcoholate red wine
for cell assays
All samples were appropriately diluted with DMEM F:12
medium serum-free before addition to harvested cells to reach
the final concentration (17.5 to 125 mg mL-1) in each well. Red
wines were dealcoholated by room temperature vacuum
evaporation using a Rotavapor (Heidolph mod. WB 2000).
2. Cell culture and experiments of cytotoxicity
MKN-28 cells were derived from a human well differentiated
gastric tubular adenocarcinoma and showed gastric-type
differentiation (16). Cells were grown as monolayers in DMEM
F:12 medium supplemented with 10% fetal calf serum, 1%
antibiotic solution and 1% L-glutamine 200 mM at 37°C in a
humidified atmosphere of 5% CO2 in air. Harvested cells were
plated in 6-well microplates (1 x 104 cells/well) and allowed to
grow to confluence over 24 hours before use. Cells were
incubated with or without alcoholic beverages (17.5 to 125 mg
mL-1 of ethanol) for 30, 60 and 120 minutes.
3. Induction of oxidative stress
Oxidative stress was induced by incubating MKN-28 cells
with xanthine oxidase (XO) (50 mU mL-1) in the presence of its
substrate xanthine (X) (1 mM) for 120 minutes. Exposure of
gastric cells in culture to X-XO causes significant cell injury and
this has been demonstrated to be due to generation of ROS and
in particular of OH produced from H2O2 by the iron catalysed
Fenton reaction (17).
4. Evaluation of cell viability
Cell viability was determined by the MTT assay. Briefly, 200
µL of MTT (5 mg mL-1) were added to each well, and samples
were incubated for 180 minutes at 37°C and centrifuged. After
aspiration of supernatant, in each well was added 1 mL of 1 M
HCl containing isopropanol to solubilized formazan produced.
Absorbance of each sample was analyzed at 540 nm using
Microplate Reader. Cell viability (%) was calculated by dividing
the absorbance of samples obtained from cells incubated with
ethanol, red wine, white wine or beer by the absorbance of
samples obtained from cells incubated with culture medium only
(control) and multiplying this ratio by 100. Data are presented as
the mean of three experiments run in duplicate.
5. Identification of red wine phenolic compounds
Two classes of polyphenols, the flavan-3-ols (catechins and
proanthocyanidins) and the anthocyanins, are the natural
antioxidants present at the highest concentration in wine. For the
separation and structural analysis of individual phenolic
compounds, a liquid chromatography-mass spectrometry (LCMS) (Perkin Elmer Sciex API 3000), which combines the
separation of LC with the selectivity and sensitivity of the MS
detector, was used.
For the anthocyanin moiety analytical separation was
performed on a Supelcosil LC-18 (5 µ) column (250x4.6 mm).
Column temperature was 40°C. The mobile phase (flow rate
0.800 mL/minutes) consisted of acidified water (0.5% of
trifluoroacetic acid, solvent A) and methanol (solvent B).
Separation was obtained by the following gradient: at 0 min 20%
B; at 12 min 60% B; 15 min 70% B; 25 min 70% B; 30 min
100% B; at 35 min 100% B; 38 min 20% B. The compounds
were detected at 520 nm. The samples of red wine and
dealcoholated red wine were diluted 1:5 with acidified water
(0.1% trifluoracetic acid) prior injection.
To quantify the other polyphenols (phenolic acids and
catechin) Prodigy ODS 3 100A C18 (5 µ) column (250x4.6 mm)
and gradient elution with A (water/0.1% formic acid) and B
(methanol/water 0.1% formic acid 80/20) solutions, were used.
Column temperature was 40°C. Separation was obtained by the
following gradient: at 0 min 10% B; at 3 min 30% B; 6 min 35%
B; 20 min 45% B; 22 min 50% B; at 24 min 55% B; 25 min 60%
B; 27 min 80% B; 30 min 90% B; 33 min 10% B at a flow rate
of 1.0 mL/minutes. The compounds were detected at 280 nm.
Statistical analysis
Data are expressed as mean±SD. Significance of differences
was assessed by one way analysis of variance (ANOVA) and
(when the F value was significant) by the Tukey-Krammer test
for multiple comparisons or by the Student's t-test for
comparison between two means. Differences were considered to
be significantly different if p<0.05.
RESULTS
Dose- and time-related effects of ethanol on cell viability
Pure ethanol decreased MKN-28 cell viability in a dose and
time dependent manner, as reported in Fig. 1. Pretreatment with
ethanol at a concentration lower than 35 mg mL-1 did not affect
cell viability, whereas increasing doses of ethanol caused a
progressive reduction of cell viability. At 60 minutes, 85 mg mL1 of ethanol induced a cell injury close to 90%. Fig. 2 compares
data obtained by incubating cells with pure ethanol, red wine,
white wine, spirits and beer, at the same ethanol concentration
(i.e. 125 mg mL-1 of ethanol) for up to 120 min. Cell viability
was differently affected by ethanol and other alcoholic
beverages in a time and dose-depending manner. Pure alcohol,
beer, spirits and, to a lesser extent, white wine reduced cell
Fig. 1. Dose and time dependence of MKN-28 cell viability as
function of ethanol concentration. Cells were incubated for 30,
60 and 120 minutes with ethanol (17,5 to 125 mg mL-1).
Means±SD from three separate experiments run in duplicate. *
p<0.05 vs. control 30 min; # p<0.05 vs. control 60 min; † p<0.05
vs. control 120 min. Black bars: 30 min; grey bars: 60 min; white
bars: 120 min.
89
Fig. 2. MKN-28 cell viability was differently affected by
ethanol and other alcoholic beverages in a time and dosedepending manner. Cells were incubated at the same doses
(125 mg mL-1 of ethanol) and times of observation.
Means±SD from three separate experiments run in duplicate.
* p<0.05 vs. control 30 min; # p<0.05 vs. control 60 min; †
p<0.05 vs. control 120 min. Black bars: 30 min; grey bars: 60
min; white bars: 120 min.
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Fig. 3. Effects of three red wine samples
on MKN-28 cell viability (120 min).
Means±SD from three separate
experiments run in duplicate. (* p<0.05
vs. ethanol treated cells) Black bars:
Ethanol; grey bars: Toscano red wine;
white bars: Aglianico red wine; diagonal
bars: Solopaca red wine.
.
.
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.
Fig. 4. Effects on MKN-28 cells viability
of red wine (whole and dealcoholated)
after incubation of 120 min. Means±SD
from three separate experiments run in
duplicate. (* p<0.05 vs. ethanol treated
cells) Black bars: Ethanol; grey bars: red
wine; white bars: dealcoholated red wine.
viability already at 30 minutes and the damaging effect increased
overtime (Fig. 2; p<0.05 vs. controls). On the other hand, red
wine only slightly reduced cell viability at 30 minutes incubation
whereas no significant damage was observed at longer time
period (Fig. 2).
On the basis of these results, we compared the effects on cell
viability of three different red wines: Toscano, Aglianico and
Solopaca at the same ethanol concentration. As shown in Fig. 3
the effect of the three wines was similar. All of them had a
significantly lower damaging effect on gastric cells as compared
to equal concentration of ethanol (Fig. 3). This result was more
evident at relatively high concentration of ethanol. Also,
Toscano red wine, especially at lower ethanol concentrations,
seemed to be less damaging to cultured cells (p<0.05 vs. ethanol)
compared with Aglianico and Solopaca.
Because the results of these experiments suggested that red
wines were somewhat less damaging than solutions containing
equal concentration of ethanol, we valuated whether
dealcoholated red wine caused even less damage to cultured
cells. Both red wines (i.e. with and without alcohol) were less
damaging to cells than ethanol (Fig. 4). No significant
differences as to this damaging effects were observed between
alcoholated and dealcoholated red wine.
Effect of oxidative stress on MKN-28 cell viability after exposition
to ethanol and red wine
We then examined whether pre-treatment with alcoholic
beverages (pure ethanol, red wine, white wine, spirits and beer)
exerted any effect on damage induced by oxidative stress in
MKN-28 gastric cells. We have previously reported (16, 18) that
2 hour incubation with X (1 mM) and XO (50 mU mL-1) caused
a significant dose-dependent (p<0.05) reduction in cell viability.
90
1
1
.
Fig. 6. LC/MS/MS separation of anthocyanins pattern in three
different types of red wine, recorded at 520 nm. 1. Delphinidin3-O-glucoside; 2. Cyanidin-3-O-glucoside; 3. Petunidin-3-Oglucoside; 4. Malvidin-3-O-glucoside; 5. Delphinidin-3-Omalonylglucoside; 6. Malvidin-3-O-acetylglucoside; 7.
Malvidin-3-O-coumarylglucoside.
In order to study the ability of selected alcoholic beverages to
prevent X-XO oxidative stress, MKN-28 cells were preincubated with alcoholic solutions containing different amounts
of ethanol (17.5 to 125 mg mL-1) and then incubated with X-XO
for 2 hours. Sixty and 120 minutes pre-incubation with red wine
significantly prevented X-XO-induced cell injury increasing cell
.
Fig. 5. Effects of red wine
(Aglianico) on oxidative stress
induced by X-XO. Means±SD from
three separate experiments run in
duplicate. * p<0.05 vs. control; #
p<0.05 vs. X-XO. Black bars:
control (untreated cells); grey bars:
control + X-XO; white bars: 85 mg
mL-1 of red wine; diagonal bars: 85
mg mL-1 of red wine + X-XO.
Fig. 7. LC/MS/MS separation of poliphenols pattern in three
different types of red wine, recorded at 280 nm. 1. Gallic acid; 2.
Catechins; 3. Chlorogenic acid + Caffeic acid.
viability from 45% (without pre-treatment) to 65% and 70%
(after 60 and 120 minutes pre-incubation, respectively) (Fig. 5).
On the other hand, white wine, spirits and beer did not prevent
X-XO induced oxidative stress (data not shown).
We then sought to evaluate whether dealcoholated red wine
also exerted any protective effect against oxidative stressinduced cell injury. Pre-treatment with dealcholated red wine did
not prevent oxidative stress-induced damage to gastric cells.
91
Anthocyanins and flavonoids wine composition
Because Toscano red wine was somewhat less damaging to
cells compared with Aglianico and Solopaca, especially at lower
ethanol concentration (Fig. 3), we assessed whether this was
partially due to a different bioactive compound profile.
Anthocyanins profiles of three red wine are shown in Fig. 6.
It should be noted that the anthocyanins composition of
Aglianico clearly differs from that observed in Toscano and
Solopaca wines. Although malvidin-3-O-glucoside and two
acylated derivatives of malvidin (malvidin-3-O-acetylglucoside
and malvidin-3-O-coumarylglucoside) are the main components
present in all wines; in Aglianico, delphinidin-3-O-glucoside,
delphinidin-3-O-malonylglucoside and cyanidin-3-O-glucoside,
not detected in other types of wine, were present. On the other
hand, Toscano and Solopaca contain petunidin-3-O-glucoside,
not detectable in Aglianico.
Gallic acid is the main flavonoid present in red wine. The
presence of high concentration of gallic acid in red wines would
be expected since this phenolic acid is principally formed by
hydrolysis of flavonoid gallate esters, which are largely absent in
white wines, due to the lack of skin extraction. Other poliphenols
identified are catechins and caffeic and chlorogenic acids as
shown in Fig. 7. No significant differences were observed for
these compounds among the different red wines used.
DISCUSSION
Several in vitro studies indicate that ethanol can exert a
differential regulation on cell viability in a dose-depending
manner. High concentrations of ethanol lead to necrotic cell death,
mediated by a high production of reactive oxygen species (ROS)
(19-21). Interestingly, ethanol at low concentration preferentially
causes apoptosis and reduces cell necrosis, probably through a
reduction of intracellular oxidative stress (22-24).
Our data show that the cytotoxic effect of ethanol on MKN28 gastric cells is directly correlated with the time of exposure
and the concentration, but cell viability is differently affected by
ethanol and other alcoholic solutions. This study shows that red
wine causes less injury to gastric epithelial cells in tissue culture
compared to white wine, spirits and beer at comparable ethanol
concentration, thus suggesting that components of red wine
other than ethanol may contribute to its lower damaging effect.
Also, red wine, but not other alcoholic beverages, significantly
counteracted damaging effect of X-XO to gastric epithelial
cells, and this was strictly dependent on its ethanol content. In
fact, this protective effect was not observed with dealcoholated
red wine.
A great attention has been focused on the protective
biochemical function of naturally occurring antioxidants in
biological systems, but the mechanism whereby dietary
antioxidants protect the gastric mucosa against injury is not
completely elucidated. Phenolic compounds have been shown to
exert direct antioxidant effects acting as ROS scavenger,
hydrogen donating compounds, singlet oxygen quenchers and
metal ion chelators (25, 26). It is generally assumed that the
higher is the total polyphenols content of the beverage or food,
the greater is its antioxidant activity (27).
Red wine can be an important dietary source of polyphenols
because contains over 200 different polyphenolic compounds
and a close relationship between total phenolic content and total
antioxidant potential for red wines is documented (28). In this
study, only red wine counteracted the damaging effect of
xanthine-xanthine oxidase generated ROS in gastric epithelial
cells. Interestingly, this protective effect was not observed with
dealcoholated red wine. This indicates that the presence of
ethanol is necessary for this protection against oxidative stress
to occur. We may speculate that this protection exerted by red
wine might be part of the concept of "adaptive cytoprotection",
that is the protective effect exerted by a mild irritant given
before a damaging agent to the gastric mucosa (29). The
amounts of polyphenolic composition is different considerably
in different types of wine, depending on the grape variety,
environmental factor and the wine processing techniques (30,
31). Generally, the phyto phenolic compounds and polyphenols
exhibit strong antioxidizing activity and the inhibitory effect on
breast cancer (32).
We have selected three types of red wine with dissimilar
antioxidant profiles to evaluate their effect on cell viability both
in basal condition and after an oxidative stress. Our results
confirm a variation in polyphenolic content among wine samples
tested; however, the wine composition does not explain the
different effects on cell viability obtained with different wine
samples. Moreover, all types of red wine significantly reduced
the damage brought about by oxidative stress in a similar way.
We focused our attention on anthocyanin moiety, because this
class of compounds differentiates red wine from white wines.
Ghiselli et al., measured the antioxidant activity of whole and
dealcoholated red wine and relative polyphenolic fractions
extracted from an Italian wine and observed that in spite of the
strong loss in antioxidant capacity after dealcoholization, wine
still maintained an appreciable radical scavenging activity (33).
In our study, pre-treatment with dealcholated red wine did not
prevent oxidative stress-induced damage to gastric cells.
In conclusions, red wine is less harmful to gastric epithelial
cells than other alcoholic beverages. This seems related to the
non-alcoholic components of red wine, because other alcoholic
beverages with comparable ethanol concentration exerted more
damage. Red wine prevents oxidative stress-induced cell
damage and this seems to be related to its ethanol content.
Acknowledgement: this work was supported by a grant of
Assessorato all'Agricoltura della Regione Campania.
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R e c e i v e d : October 15, 2009
A c c e p t e d : December 20. 2009
Author's address: Prof. Carmela Loguercio, Via Foria 58,
80131 Naples, Italy; Phone: +39 (0) 81 5666718; Fax: +39 (0)
81 5666718; E-mail: [email protected]