Oxidative Damage of Liver, Kidney and Serum Proteins with Apoptosis of above Tissues in Guinea Pigs Fed on Carbonated Soft Drink
Health damaging effects of carbonated soft drink are largely based on epidemiological observations. The present study aimed to examine the possible physiological and biochemical effects of such drink in animal. Through oxiblot assay, fluorescence activated cell sorting assay and other biochemical investigations; it has been shown that consumption of such drink makes oxidative damages. Extensive such damages at the level of proteins in vivo in the liver, kidney and blood of experimental animal, guinea pigs, which cannot synthesize vitamin C like humans have been observed. These effects are also associated with significant cellular damages. In vitro studies reviled that such drink contains oxidants those are capable of direct protein oxidation. These observations establish physiological and biochemical link between consumption of carbonated soft drink and its deleterious effects in vitamin C deficient animals.
Received: September 06, 2012;
Accepted: January 23, 2013;
Published: May 25, 2013
Carbonated soft drinks have rapidly spread their influence in worlds
consumer market and these are almost a part of our daily food habits in social
life (Teerasong et al., 2010). The main active
ingredients in these carbonated soft drinks are carbonated water, high sugar
or fructose corn syrup, phosphoric acid, caramel color, natural flavors and
caffeine (Encyclopaedia Britannica, 2012). Consumption
of carbonated soft drinks have been implicated with several health damaging
effects including obesity, (Malik et al., 2006)
type II diabetes, (Vartaman et al., 2007) metabolic
syndrome risk, (Yoo et al., 2004) elevated blood
pressure (Raben et al., 2002) osteoporosis (Heaney
and Rafferty, 2001) and hypokalemia (Tsimihodimos et
al., 2009). Excess sugar consumption increases production of destructive
free radicals and lowers level of key antioxidants, as reported by Mohanty
et al. (2000). Type II diabetes resulting from high sugar consumption
is also associated with an increase in free radical generation, leading to damage
of fats, proteins and DNA. Fructose consumption might result an impact on the
development of insulin resistance and cardiovascular diseases if taken in large
volume (Tappy and Le, 2010). Fructose directly stimulates
the endothelial inflammatory process and reduce Nitric Oxide (NO) levels in
human aortic endothelial cells in vitro (Glushakova
et al., 2008). High fructose feeding in rats showed increased level
of oxidative stress markers and increased reactive oxygen species in circulatory
leukocytes (Al-Awwadi et al., 2005). The antioxidant
ability of caffeine has been reported in contrast with its pro oxidant effects
derived from its action mechanism such as systemic release of catecholamines
(Olcina et al., 2006). Phosphoric acid toxicity
has been reported to accelerate mammalian aging (Ohnishi
and Raijaque, 2010) consumption of soft drink has deleterious effects on
oral mucosa in rats due to its phosphoric acid content (Kapicloglu
et al., 2000). It has been reported that phosphoric acid in soft
drink is associated with increased risk for chronic kidney disease (Saldana
et al., 2007). Effect of cola drink on fertility has been reviewed
(Imai et al., 2010).
Though epidemiological studies have been made over the years to investigate
consumption of carbonated soft drinks and their possible effects on human health,
so far studies on the effects on carbonated soft drinks at the biochemical and
cellular level are scanty (Amato et al., 1998;
Milei et al., 2011). Recently effect of carbonated
soft drinks on the activities of alanine amino transferase and aspartate amino
transferase in serum and kidney in rats has been reported (Jeroh
et al., 2012) and effect of coke consumption on lipid peroxide and
alkaline phosphatase activity in serum and liver of animal has been reported
The present study was designed to ascertain the effects of carbonated soft
drink, Carbonated soft drink at the cellular and biochemical levels in guinea
pigs, animal which cannot synthesize vitamin C, like humans and are suitable
for studying oxidative damages. The experimental approach were made to investigate
impact of such drink on proteins, DNA and in cellular apoptosis in experimental
animal. Carbonated soft drink obtained from Indian market was fed to the experimental
animal, guinea pigs for a period of 0 to 60 days.
MATERIALS AND METHODS
Carbonated soft drink: Carbonated soft drink, available in Indian market in 600 mL bottle, was used to feed the animal.
Chemicals and reagents: Sodium Dodecyl Sulphate (SDS), acrylamide, bisacrylamide, Ammonium Persulphate (APS), N,N,N,N-tetramethyl ethylene diamine (TEMED), Coomasie brilliant blue, bovine serum albumin (BSA), 5,5 dithiobis 2-nitrobenzoic acid (DTNB), polyethylene sulphonyl fluoride (PMSF), ethylene diamine tetraacetate (EDTA), potassium chloride, sodium hydroxide, 2,4-dinitrophenylhydrazine (DNPH), hydrocholoric acid, trichloroacetic acid (TCA), ethanol, ethyl acetate, guanidium hydrochloride, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), n-hexane, formalin, paraffin, xylene, ethanol, sodium citrate, triton X-100, fluorescein (Roche), 4, 6'-diamidino-2-phenylindole (DAPI), Protein Reagent (Bio-Rad, USA), sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, Tris-HCl, dithiothreitol (DTT), Tween 20, glycerol, glycine, methanol, skimmed milk, 1° Antibody: Rabbit Anti-DNP Antibody and 2° Antibody: Goat Anti-Rabbit IgG (HRP-conjugated), Electrogenerated Chemiluminescent (ECL) detection kit (Amersham Biosciences), acetic acid, heparin, 1xFluorescence-Activated Cell Sorting (FACS) lysis buffer, 1xFACS binding buffer, annexin V-Flurescein isothiocyanate (FITC), propidium iodide staining solution (PI), Beta-Mercaptoethanol (BME), bromophenol blue, sodium acetate, sodium thiosulphate, glutaraldehyde, silver nitrate, formaldehyde.
All the above reagents were of analytical grade and were purchased from the Sigma Chemical Company, E. Merck Ltd and SRL (India).
Animals: Young male guinea pigs, weighing 450-500 g were used for all
experimental purposes. They were of pure breed with white and black and white
and brown colors. They were housed in well circulated, clean animal house at
comfortable temperature, with 12 h light and 12 h dark conditions. There were
separate cage was used for each guinea pig. All results are of three different
sets of experiments and each set consists of three control and fifteen experimental
animals. Guinea pigs have been used because many of the metabolic characteristics
in guinea pigs are similar to that of humans (Stith and
Das, 1982) including that gunieas pigs cannot smnthesise vitamin C like
humans and are thus suitable animal for the purpose of studying cellular oxidative
Both control and experimental animals for the in vivo studies on the
effect of carbonated soft drink-induced oxidative damage were fed a special
stock diet with following composition: Wheat bran: 70%; Casein: 20%; Sucrose:
8%; AOAC vitamin mixture (without ascorbate): 1%; AOAC Salt mixture: 1%. The
same diet was prepared manually with pure ingredients. This was done as per
procedure of NIH guidelines (NRC, 1985). Both control
and experimental animals were supplemented with vitamin C (2 mg day-1)
along with either water (control group) or carbonated soft drink (experimental
group) to protect the animals from scurvy.
The volumes of carbonated soft drink which were administered orally to guinea
pigs varied from 6-8 mL depending on body weight of the animal. Here volume
of the carbonated soft drink was determined proportionately assuming average
body weight of guinea pigs 500 g compared to 600-1200 mL soft drink consumption
per day by an individual of average 60 kg b.wt. in India. All animal treatment
procedures met the NIH guidelines (NRC, 1985) and were
conducted with institutional animal ethics committee approval.
Oxyblot assay: Oxidative damage of liver tissue and serum proteins were
evidenced by immunoblotting of the dinitrophenylhydrazone derivatives of protein
carbonyls (Misra et al., 2003). Whole liver tissue
weighing 1 g was first minced and suspended in 9 mL of lysis buffer then homogenized,
followed by centrifugation at 16,500 g for 15 min at 4°C. This process of
centrifugation was repeated thrice until a clear whole tissue extract solution
was formed. Lysates were spun to remove all insoluble materials. The protein
concentration was approximately maintained at 50-60 μg for serum and 80-90
μg for liver tissue according to requirement. Protein concentration was
measured using Bio-Rad protein estimation kit. SDS solution (20%) in equal volume
was added to the sample extract and after 5 min incubation, 30 μL of 2,
4-dinitrophenyl-hydrazine (DNPH) solution was added and the reaction mixture
was incubated for 15 min at room temperature. This was neutralized with 22.5
μL of neutralizing solution as per protocols defined in the oxyblot protein
oxidation detection kit (Intergen company, NY, USA). This treated tissue extract
was then separated by SDS-PAGE and blotted on a PVDF (polyvinylidene fluoride)
membrane. The membrane was then incubated with primary antibody, specific to
the DNP-moiety, after blocking with 5% nonfat milk. This step was followed by
incubation with a secondary antibody conjugated with horseradish peroxidase
(goat anti-rabbit IgG) directed against the primary antibody.
||Oxyblot of liver proteins of guinea pigs administered carbonated
soft drink against water as control, (a) after 0 and 30th day of administered
orally for 30 days, L-1: water for 30 days, L-2: Carbonated soft drink for
30 days, (b) Time dependent manner after 0th, 7th, 15th, 30th ,45th and
60th days, of administion, L-1: water as the drink, L-2: Carbonated soft
drink for 7 days, L-3: Carbonated soft drink for 15 days, L-4: Carbonated
soft drink for 30 days, L-5: Carbonated soft drink for 45 days, L-6: Carbonated
soft drink for 60 days
This membrane was then treated with chemiluminescent reagent ECL chemiluminescent
detection kit (GE Healthcare, USA) and the chemiluminescent signals from the
responsive bands captured by autoradiography on a photographic film, as presented
in Fig. 1a, b and Fig. 2a,
Terminal deoxyncleotidyl Transferase (TUNEL) assay for assessing cellular
damage of liver and kidney tissue: The paraffin embedded tissue sections
(5 μm) were deparaffinized, washed and permeabilized. The measurement of
apoptotic cells was carried out using In situ cell death detection kit,
fluorescein (Roche) to exclusively determine cellular apoptosis based
on Terminal deoxynucleotidyl transferase mediated 2'-deoxy Uridene 5' triphosphate
(dUTP) Nick End Labelling (TUNEL) reaction (Banerjee et
al., 2008). After the reaction, the slides were washed with phosphate
buffer saline and DNA fragmentation was detected by labeling with fluorescein
labelled dUTP using terminal deoxynucleotidyl transferase. The cells were examined
using a fluorescence microscope (Olympus Bx40) at an excitation wavelength of
488 nm. Digital images were captured with cool CCD camera (Olympus; magnification,
10x). The nuclei were counted by counter staining with 4', 6'-diamidino-2-phenylindole
(DAPI) at excitation wavelength of 350 nm, as presented in Fig.
3 and 4 .
Fluorescence-activated cell sorting assay (FACS assay): FACS assay (Das
et al., 2011) was conducted to determine oxidative damage. Soon after
the animals were sacrificed; a part of their blood was instantaneously collected
||(a) Oxyblot and (b) Silver stain of serum proteins of guinea
pigs administered carbonated soft drink against water as control, orally
between 0-60 days, of administerated. L-1: water as the drink, L-2: Carbonated
soft drink for 30 days, L-3: Carbonated soft drink for 60 days. Here beyond
30 days, feeding unto 60 days not only brought damages in the serum protein
but also led to protein degradation (Fig. 2b)
The blood was then centrifuged at 250 g for 10 min at room temperature. The
precipitate containing blood cells was then collected and to it 1xFACS lysis
buffer solution was added and it was then again centrifuged at 250 g for 10
min at room temperature. Then cellular precipitate was washed twice with 1x
phosphate buffer saline and was centrifuged at 250 g for 10 min at room temperature.
Finally the blood cells were suspended in 1x FACS binding buffer. From which
100 μL of the solution was transferred in FACS culture tube and to it 3-5
μL of annexin V-FITC (Boehringer Mannheim) and propidium iodide staining
solution (PI) were added parellely according to manufacturers protocols.
Then the solution was gently vortexed and was incubated for 15 min at room temperature
in dark. After the incubation period 400 μL of 1xFACS Ca2+ enriched
binding buffer was added to it to make a total volume of 500 μL and then
it was directly subjected to FACS analysis. FACS Calibur, Becton Dickinson,
San Jose, CA. Data was acquired and analysed using the Cell Quest programme
(Becton Dickinson). Electronic compensation of the instrument was done to exclude
overlapping of the emission spectra. Total 10,000 events were acquired, the
cells were properly gated and dual parameter dot plot of FL2-H linear (X-axis;
Flous-fluorescence) versus FL1-H linear (Y-axis; PI-fluorescence) was observed.
PI and Annexin V-FITC emissions were detected in the FL 1 and FL 2 channels
of FACS Calibur flow cytometer, using emission filters of 623 and 520 nm, respectively.
The percentages of distribution of normal, early apoptosis, late apoptosis and
necrosis cells were calculated and depicted in Fig. 4 and
Assay of carbonyl content: Protein carbonyl was measured by reaction
with 2,4-dinitrophenyl hydrazine (DNPH) following the method of Levine
et al. (1990) similar to that done before in our laboratory (Mukhopadhyay
and Chatterjee, 1994). To solution of pure Bovine Serum Albumin (BSA) 1
mg 100 μL, micro volume of carbonated soft drink was added on a time dependent
manner and was directly subjected to carbonyl formation assay. The dose of carbonated
soft drink was chosen as described above. In time dependant experiments, both
concentration and volume of BSA and volume of carbonated soft drink were kept
fixed at 1 mg 100 μL and 50 μL, respectively and the experimental
time after addition of carbonated soft drink to pure protein were varied between
10-160 min under incubation. Formation of carbonyl was measured at 390 nm spectrophotometrically
(Hitachi model, Fig. 5a). The results were expressed as n
moles of phenylhydrazones formed per milligram protein using a molar extinction
coefficient of 22,000.
Assay of bityrosine formation: To solution of pure Bovine Serum Albumin
(BSA) 1 mg 100 μL, 50 μL of carbonated soft drink was added on a time
dependent manner and was directly used to bityrosine formation estimation (Kaplan
et al., 2003). Here to the solution, 20 mM HEPES buffer, pH 7.5 was
added and the fluorescence measured at 325 nm excitation and 415 nm emission
using a Hitachi fluorescence spectrophotometer, model F-7000. The results were
expressed as n moles of per milligram protein using a molar extinction coefficient
of 13600. Here the experiment was done by varying time between 1 to 140 min,
as presented Fig. 5b.
Measurement of tryptophan loss: To solution 1 mg 100 μL of pure
Bovine Serum Albumin (BSA), 50 μL of carbonated soft drink was added on
a time dependent manner and was directly used to tryptophan loss assay. Tryptophan
loss was measured at 280 nm excitation and 330-360 nm emission (Davies
et al., 1987) using a Hitachi fluorescence spectrophotometer, model
F-7000. The results were expressed as pico moles of per milligram protein using
a molar extinction coefficient of 55,00. Here the time dependent study was done
varying time period 0-160 min and the effect observed has been presented in
Statistical analysis: All values were expressed as Mean±Standard Deviation (SD) of 3 different sets of experiments, each set consisting of 3 controls and 15 experimental animals. Data were analyzed using origin 61 data analysis and graphing software. All statements were significant on based on a probability p≤0.05.
Determination of protein oxidation by carbonated soft drink in vivo
by oxyblot assay: Extensive oxidation of liver tissue proteins (Fig.
1a, b) and serum proteins (Fig. 2a,
b) were observed compared to control through Oxyblot assay
when guinea pigs were orally administered carbonated soft drink for 30 days
(Fig. 1a) and thereafter in a time-dependent manner (Fig.
1b). Besides oxidative damage low levels of serum protein degradations were
also observed apparently due to the observed oxidative damage when animals were
treated with carbonated soft drink for 60 days (Fig. 2b-silver
stain). These observations may be explained due to generation of free radicals,
under the experimental conditions as discussed later.
Determination of carbonated soft drink induced cellular apoptosis in guinea
pigs in vivo through TUNEL assay: Tissue sections from sacrificed
animals fed with carbonated soft drink for 60 days showed discernible apoptosis
of liver (Fig. 3 ) and more significantly in kidney (Fig.
4) cells compared to control as determined by TUNEL assay reconfirming that
the observed increase in cell death in the liver and kidney of the carbonated
soft drink treated animals was apparently associated with the observed oxidative
damage induced by the consumption of such drink, due to presence of high amount
of sugar in such drink.
||Detection of DNA strand breaks as cell death indicator by
TUNEL assay in liver cells of guinea pigs orally administered carbonated
soft drink (10x magnification), Uniform cross section, depict the DNA strand
breaks in apoptotic cells as green fluorescent spots in the control and
experimental liver tissue sections of the guinea pigs fed on water as control
group while others were given carbonated soft drink in a time frame of 7,
15, 30, 45 and 60 days, respectively. T: TUNEL stained and DAPI stained
by D: DAPI stained
Determination of carbonated soft drink induced cellular apoptosis of guinea
pig whole blood cells in vivo as measured by fluorescence-activated
cell sorting (FACS) of annexin V and propidium iodide labeled cells: Whole
blood cells collected from guinea pigs, which were orally administered with
carbonated soft drink for definite experimental periods (as in legands) were
subjected to flow cytometric analysis in order to assess any possible apoptosis
of such cells. Results show that blood cells from treated guinea pigs undergo
a dose-dependent increase in cellular apoptosis compared to control (Fig.
5). Early apoptosis appear between 30 to 60 days. It can be found Table
1 (4th column) that there is increase of apoptotic cell population percentage
compared to control (0-day) from 0.04±0.005 to 30 days (6.75±0.42)
which remained steady till 60th day (5.14±0.22).
||Detection of DNA strand breaks as cell death indicator by
TUNEL assay in kidney cells of guinea pigs exposed to carbonated soft drink
(10x magnification), Uniform cross section, depict the DNA strand breaks
in apoptotic cells as green fluorescent spots in the control and experimental
kidney tissue sections of the guinea pigs which have been given water while
others were given carbonated soft drink for 7, 15, 30, 45 and 60 days, respectively,
T: TUNEL stained and D: DAPI stained
||Percentage distribution of normal, early apoptotic, late apoptotic
and necrotic cells
The annexin V-FITC/PI double staining assay was employed to classify blood
cells in early apoptosis or late apoptosis stages, respectively.
||Determination of carbonated soft drink induced cellular apoptosis
in guinea pig in vivo as measured by FACS of annexin V and propidium
iodide labeled whole blood cells, Annexin V-Flurescein isothiocyanate/ Propidium
Iodide (FITC/PI) stained blood cells was exposed to carbonated soft drink
for a period of 0th, 7th, 15th, 30th, 45th and 60th day. Control: Blood
cells after water intake carbonated soft drink, Experimental group: For
a time frame of 7th, 15th, 30th, 45th and 60th day, respectively. The annexin
V-FITC-/PI-population, Q3-lower left quadrant: Control cells, whereas annexin
V-FITC+/PI- cells Q4-lower right quadrant: Early apoptosis, annexin V-FITC+/PI+
(Q2-upper right quadrant): late apoptosis and annexin V-FITC-/PI+ (Q1-upper
left quadrant) as necrosis
Figure 5 depicts the apoptotic profiles of blood cells isolated
from the guinea pigs which were orally administered with carbonated soft drink
for 0,7,15,30,45 and 60 days, respectively and clearly shows a time-dependent
increase in early as well as late apoptosis in treated animals, with decrease
at later stage.
Evaluation of in vitro oxidative potency of carbonated soft drink:
This study was aimed to determine whether carbonated soft drink contains oxidants
within it or these are generated by secondary metabolic pathway, when consumed,
to cause oxidative damages. A pure protein Bovine Serum Albumin (BSA) was treated
directly with fixed volume of carbonated soft drink and evaluated for oxidation
due to this treatment. Protein carbonyl formation was found to increase in a
time-dependent manner compared to control on addition of carbonated soft drink
to such pure BSA solution (Fig. 6a).
|| Time dependent in vitro assay of protein carbonyl
formation by carbonated soft drink using Bovine Serum Albumin BSA, (a) Protien
carbory formation, (b) Protien bityrosine formation and (c) Protien tryptophan
loss was assessed by standand protocols protein
Bityrosine formation (Fig. 6b) and tryptophan loss (Fig.
6c) which are both dependable indicators of protein oxidation also showed
similar time-dependent increase compared to control during incubation of BSA
with fresh carbonated soft drink. This confirmed that carbonated soft drink
contains oxidants which are capable of direct oxidations of proteins.
India is a country with variety of population with different food habits and
genetic variations. Many people may not have proper nutritional diets which
are more prone in rural areas, but are fond of carbonated soft drink particularly
due to hot and humid weather in most calendar months. The data on physiological
and biochemical effects of carbonated soft drinks in animals are scanty (Amato
et al., 1998; Milei et al., 2011;
Jeroh et al., 2012; Awhin,
2012). Most data implicating such drinks to adverse health effects are largely
based on epidemiological studies. The present study was thus aimed to obtain
comprehensive data on the possible health hazards especially with respect to
physiological and biochemical effects of such drink using carbonated soft drink,
available in Indian market on suitable animal model, like guinea pigs, which
represent humans in many metabolic aspects (Stith and Das,
1982) and cannot synthesize Vitamin C like humans and thus very suitable
to study oxidative damages of tissue. Present study confirms that carbonated
soft drink contain oxidants those are capable of oxidative modification of proteins
which can partly explain the oxidative damage of liver, kidney and serum proteins
as have been observed in this study in guinea pigs which were orally administered
with such drink in measured doses.
In oxyblot assay, as described in the present study, extensive oxidation of
liver tissue proteins and serum proteins have been observed under the experimental
conditions in guinea pigs. Beside oxidative damage, protein degradation also
takes place in serum, due to carbonated soft drink consumption. It has been
reported that excess sugar consumption increases production of destructive free
radicals (Mohanty et al., 2000). The high concentration
of sugar as is present in carbonated soft drink under experimental condition
as reported here may be responsible for such oxidation of tissue proteins in
vivo due to generation of free radicals. Carbonated soft drink contains
high sugar. Glucose has been shown to modify the amino group of proteins by
a process of non-enzymatic glycation leading to potentially deleterious effects
on structure and function of proteins (Cussimanio et
In TUNEL assay under the experimental conditions tissue sections of sacrificed
animals fed with carbonated soft drink showed distinct apoptosis of liver and
kidney. It has been reported that damages to proteins and DNA results from excess
sugar consumption (Tappy and Le, 2010). The observed
effect under experimental condition as reported here might be due to high amount
of sugar present in carbonated soft drink.
In this study, it has been found that blood cells from experimental guinea
pigs undergo a dose dependent increase in cellular apoptosis. The increase is
though small but was noted in early apoptosis period between 30-60 days. The
cellular apoptosis decreases at late stage with no significant necrosis of the
cells. Early apoptosis appearance was noted between 30 to 60 days, compared
to control. Reactive oxygen species may cause immediate apoptosis but due to
feeding of subclinical dose of vitamin C (2 mg day-1) to animals
during experimental period to prevent scurvy, such immediate apoptosis might
not occurred. Alternatively, there may be cumulative depletion of other antioxidant
present in the system between 30 to 60 days, which prevented immediate apoptosis.
Certain polysaccharides have shown induced apoptosis of lymphoma cell line (Hattori
et al., 2004). Observations of cellular apoptosis as reported here
might also be due to high sugar content of such drink, in experimental animal
like guinea pigs which are unable to synthesize vitamin C and are susceptible
to oxidative damages and degradations.
In vitro studies as have been presented here show that carbonated soft drink directly furnish the oxidants, which are partly or wholly responsible for the physiological oxidative damage.
In India carbonated soft drink is very popular. But a large number of Indian populations do not get adequate nutrition from their daily diet and may suffer for insufficient antioxidant intake. This population could carry a risk of biochemical damages as observed here due to consumption of such drink in excess.
Carbonated soft drink has been used in this study, obtained from Indian market brings about extensive oxidation of liver and kidney tissue proteins and serum protein in vivo when fed to guinea pigs during the experimental period. Such consumption of carbonated soft drink make discernible apoptosis of liver and kidney tissues in guinea pigs under experimental conditions with early apoptosis of blood cells in a small proportion. In in vitro studies also direct oxidations of proteins have been noted.
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