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Epigallocatechin gallate effectively ameliorates Fluoride induced oxidative stress, DNA
damage in the liver of rats
Shanmugam Thangapandiyan and Selvaraj Miltonprabu*
Department of Zoology
Annamalai University
Annamalainagar-608002
Tamilnadu, India
* Corresponding author:
Dr. Selvaraj Miltonprabu
Assistant Professor
Department of Zoology
Faculty of Science, Annamalai University
Annamalai Nagar – 608002
Tamil Nadu, India.
Tel: +91 04144 – 238282; Cell : +91 9842325222
Fax: +91 04144 – 238080
Email address: [email protected]
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Abstract:
Environmental exposure to NaF compounds inflicts a great health concern worldwide.
Epigallocatechin gallate (EGCG) is a green tea catechin found in various green tea preparations.
The intention of this study was to investigate the hepato-protective role of EGCG in NaF
intoxicated in rats. Rats were orally treated with Sodium fluoride (NaF) alone (25 mg/kg body
weight (bw)/day) plus EGCG at different doses (20, 40 and 80 mg/kg body weight (bw)/day) for
four weeks. Hepatotoxicity of NaF was determined by the increased levels of serum
hepatospecific markers and total bilirubin along with increased levels of thiobarbituric acid
reactive substances, lipid hydroperoxides, protein carbonyl content and conjugated dienes. The
hepatotoxic nature of NaF was further evidenced by the decreased activity of enzymatic and non-
enzymatic antioxidant levels in liver. NaF administered rats also showed an increased DNA
damage and fragmentation in hepatocytes. Administration of EGCG (40 mg/kg BW) in NaF
intoxicated rats, significantly recuperated the distorted biochemical indices, DNA damage and
pathological changes in the liver tissue. Thus, the results of the present study clearly
demonstrated that the EGCG has strong free radical scavenging, antioxidant and anti genotoxic
properties against NaF induced oxidative hepatic injury in rats.
Key words: EGCG, NaF, free radicals, oxidative stress, antioxidants, liver, rat
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1.Introduction:
Fluoride is widely distributed in nature in many forms and its compounds are being used
extensively. Fluorine is not freely found in nature. Fluorine in drinking water is totally in ionic
form and hence it rapidly, totally and passively pass through the intestinal mucosa and interferes
with major metabolic pathways of the living system. NaF in small doses has remarkable
prophylactic influence by inhibiting dental caries while in higher doses it causes dental and
skeletal fluorosis (Shanthakumari et al., 2004). The fluorosis of human beings is mainly caused
by drinking water, toothpaste, mouth rinses, burning coal, NaF dust and fumes from industries
using NaF containing salt and hydrofluoric acid and drinking tea while that of animals is mainly
by drinking water and supplementing feed additives such as calcium monohydrogen phosphate
containing high levels of NaF (Liu et al., 2003). Intake of high levels of NaF is known to cause
structural changes, altered activities of enzymes in the liver and influence the metabolism of
lipids (Shivarajashankara et al., 2002). Acute poisoning can result death by blocking cell
metabolism since NaF inhibit enzymatic processes, particularly metalloenzymes which are
responsible for vital processes (Birkner et al., 2000).
Recent studies revealed that NaF induces excessive production of oxygen free radicals,
and might cause the depletion in biological activities of some antioxidant enzymes (Chlubek,
2003). Increased free radical generation, lipid peroxidation and disturbed antioxidant defense
systems are observed to mediate the toxic effects of NaF in the liver of rats (Shivarajashankara et
al. 2001).
Efforts to prevent and treat fluorosis by therapeutic measures had limited success because
of their low accessibility and side effects (Chinoy and Memon, 2001). Dietary administration of
phytochemicals is a noble strategy to deal with oxidative stress-related disorders (Surh., 2003).
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Flavonoids are naturally occurring polyphenolic compounds widely distributed in plants, and
various flavonoids have been used as a remedy for the amelioration of heavy metal induced
oxidative hepatic stress (Miltonprabu et al., 2011). EGCG (Fig 1) is the most abundant and the
most biologically active green tea polyphenol. It is a potential antioxidant, owing to their free
radical scavenging property and the ability to chelate transition metal ions. The results of earlier
studies suggested that regular intake of EGCG can reduce oxidative stress, which decreases the
risk of disorders associated with oxidative stress (Vassort and Turan, 2010). Silymarin, a
flavolignan compound extracted from the fruits and seeds of the plant Silibum marianum, was
found effective for protecting against cirrhosis, jaundice and hepatitis traditionally (Comar and
Kirby, 2005). Furthermore, in vitro, in vivo and clinical studies have demonstrated the
antioxidant and hepatoprotective effects of silymarin in animal and human models of
hepatotoxicity (Basiglio et al, 2009).
To the best of our knowledge, Zhen et al. (2007) reported the hepatoprotective nature of
EGCG against Ccl4 induced hepatotoxicity in rats. But there are no reports are available on the
hepatoprotective nature of EGCG against NaF.Therefore in this present investigation we intend
to explore the ameliorative potency and antioxidant efficacy of epigallocatechin gallate along
with silymarin, a well known hepatoprotectant against NaF induced oxidative stress and hepatic
damage and brings hepatic recovery in terms of biochemical, molecular and histological indices.
2. Materials and Methods
2.1. Drug and Chemicals
EGCG was purchased from Sigma Aldrich, USA. NaF and ethidium bromide 2-
thiobarbituric acid, butylated hydroxytoluene, reduced glutathione, 2,2’-dipyridyl, xylenol
orange, 2,2’-dinitro phenyl hydrazine, γ-glutamyl-p-nitroanilide, 5,5’-dithiobis-2-nitrobenzoic
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acid, trichloroacetic acid, phenazine methosulphate, nitroblue tetrazolium, reduced nicotinamide
adenine dinucleotide, 1-chloro 2,4-dintrobenzene were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). All other chemicals and solvents were of certified analytical grade and
purchased from S.D. Fine Chemicals, Mumbai or Hi media Laboratories Pvt. Ltd., Mumbai,
India. Reagent kits were obtained from span Diagnostics, Mumbai, India. Chemical structure of
EGCG is shown in Figure 1.
2.2. Animals
Healthy male albino Wistar rats (160-180 g) were obtained from the Central Animal
House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital,
Annamalai University, and maintained in an air-conditioned room (25 ± 2º C) with a 12 h
light/12 h dark cycle. Feed and water were provided ad libitum to all the animals. The study
protocols were approved by the Institutional Animal Ethics Committee of Rajah Muthiah
Medical College and Hospital (Reg No. 160/1999/CPCSEA, Proposal number: 952/2012),
Annamalainagar and the study conducted in accordance with the “Guide for the Care and Use of
Laboratory Animals”.
2.3. Preparation of drugs
NaF (25 mg/kg body weight, 1/10 of the oral LD50 value in rats, Chinoy, 1991) was
dissolved in normal saline and administered by intragastric intubation daily for four weeks.
EGCG powder was dissolved in water and given orally 90 min prior to the administration of NaF
daily at a dose of 20, 40 and 80 mg/kg body weight for four weeks. Silymarin was dissolved in
water and given orally 90 min prior to the administration of NaF daily at a dose of 25 mg/kg
body weight for four weeks.
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2.4. Experimental design:
A pilot study was conducted with three different doses of EGCG (20, 40 and 80 mg/kg)
to determine the dose dependent effect in NaF-treated rats. It was observed that EGCG
pretreatment at doses of 20, 40 and 80 mg/kg significantly (P < 0.05) lowered the elevated levels
of AST, ALT, LDH and GGT in serum of NaF -induced rats after 28 days of exposure. Hence,
20, 40 and 80 mg/kg doses were chosen for our study.
The rats were randomly divided into seven groups of six in each group.
Group 1 : Normal rat
Group 2 : Normal + EGCG (40mg/Kg BW)
Group 3 : Normal + NaF (25mg/kg BW)
Group 4 : EGCG (20mg/Kg BW) + NaF (25mg/kg BW)
Group 5 : EGCG (40mg/Kg BW) + NaF (25mg/kg BW)
Group 6 : EGCG (80mg/Kg BW) + NaF (25mg/kg BW)
Group 7 : Silymarin (25mg/kg BW) + NaF (25mg/kg BW)
The total duration of the study was 28 days. Food and water intake were recorded
regularly. After forty-eight hours of administration of the last dose, the rats were anesthetized
with an intramuscular injection of ketamine hydrochloride (25 mg/kg) and sacrificed by cervical
decapitation. Blood was collected for the separation of serum. The liver tissue was dissected out,
weighed and washed in chilled saline solution. Liver tissue was minced and homogenized (10%,
w/v) in 0.025M Tris-HCL buffer, (pH 7.4) using Teflon tissue homogenizer and centrifuged
(8000xg for 10 min). The resulting clear supernatant was used for various enzymatic and non-
enzymatic biochemical assays. For comet assay, liver was cut into small pieces and washed with
Hank’s balanced salt solution. The individual cell suspension was obtained by enzymic digestion
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with collagenase at 37°C. After filtration by 100 and 40 mm mesh, the resulting cell suspension
was centrifuged for 10 min at 3000 rpm. The cell pellet was then suspended in phosphate
buffered saline and used for the estimation of DNA damage.
2.5. Biochemical assays:
2.5.1. Activities of serum marker enzymes:
The activities of serum aspartate aminotransferase (EC. 2.6.1.1), alanine aminotransferase
(EC. 2.6.1.2), alkaline phosphatase (EC.3.1.3.1), lactate dehydrogenase (EC. 1.1.1.27) and total
bilirubin were assayed using commercially available diagnostic kits (Sigma diagnostics (I) Pvt.
Ltd., Baroda, India). Gamma-glutamyl transferase (EC. 2.3.2.2) activity was determined by the
method of Rosalki et al. (1970) using -glutamyl- p-nitroanilide as substrate. Based on Vanden
Berg reaction, serum bilirubin was estimated by the method of Malloy and Evelyn (1937).
2.5.2. Estimation of lipid peroxidation
Lipid peroxidation in the liver was estimated calorimetrically by measuring thiobarbituric
acid reactive substances (TBARS) and hydroperoxides as described by Niehiaus and Samuelsson
(1968) and Jiang et al. (1992), respectively. As a hallmark of protein oxidation, total protein
carbonyl content was determined in the liver by a spectrophotometric method described by
Levine et al. (1990) and expressed as nmol/mg protein.
2.5.3. Determination of non-enzymatic antioxidants
Reduced glutathione (GSH) content in the liver homogenate was determined by the
method of Moron et al. (1979) based on the reaction with Ellman’s reagent (19.8 mg
dithionitrobis benzoic acid in 100 ml of 0.1% sodium citrate). A total sulfhydryl group (TSH)
was measured after the reaction with dithionitrobis benzoic acid using the method of Ellman
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(1959). Vitamin C and vitamin E concentrations were measured by the methods of Omaye et al.
(1979) and Desai (1984), respectively.
2.5.4. Assay of enzymatic antioxidants
Superoxide dismutase (SOD) activity was determined by the method of Kakkar et al. (1984) in
which the inhibition of formation of NADPH-phenazine methosulphate nitroblue tetrazolium
formazon was measured spectrophotometrically at 560 nm. Catalase (CAT) activity was assayed
calorimetrically as described by Sinha (1972) using dichromate-acetic acid reagent. Glutathione
peroxidase activity (GPX) was assayed by the method of Rotruck et al. (1973) based on the
reaction between glutathione remaining after the action of GPX and 5,5’-dithiobis-2-nitrobenzoic
acid to form a complex that absorbs maximally at 412nm. Glutathione S-transferase (GST)
activity was determined by the method of Habig et al. (1974) spectrophotometrically by using
dichloro-2, 4- dinitrobenzene as the substrate. Glutathione reductase (GR) that utilizes NADPH
to convert metabolized glutathione (GSSG) to the reduced form was assayed by the method of
Horn and Burns (1978). The estimation of glucose-6- phosphate dehydrogenase (G6PD) was
carried out by the method of Beutler (1983), where an increase in the absorbance was measured
when the reaction was started by the addition of glucose-6- phosphate. Protein level was
determined by using bovine serum albumin (BSA) as the standard at 560nm by the method of
Lowry et al. (1951).
2.5.5. Estimation of membrane-bound ATPase
The sediment after centrifugation was resuspended in ice cold Tris-HCl buffer (0.1M) pH
7.4. This was used for the estimations of membrane-bound enzymes and protein content. The
membrane bound enzymes such as Na+
/ K+-ATPase, Ca
2+-ATPase and Mg
2+-ATPase activity
were assayed by estimating the amount of phosphorous liberated from the incubation mixture
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containing tissue homogenate, ATP and the respective chloride salt of the electrolytes (Bonting,
1970; Hjerten and Pan, 1983; Ohnishi et al., 1982). The amount of liberated inorganic phosphate
(Pi) was estimated according to the method of Fiske and Subbarow (1925).
2.6. Comet assay
Hepatocytes were isolated from control and experimental groups and were processed for
alkaline comet assay as described previously (Kyoung et al., 2006; Rajagopalan et al., 2003;
Singh, 2000). The slides were immersed in lysis buffer for 1 h at 4 ◦C and equilibrated in
alkaline solution for 20 min, followed by electrophoresis at 18 V, 300 MA (Sub-Cell GT system
with Power Pac basic power supply, Bio-Rad Laboratories Inc., Hercules, CA, USA). After
electrophoresis, the slides were neutralized and stained by ethidium bromide. The images were
captured using a fluorescence microscope (Eclipse TS100, Nikon Instruments Inc., Melville, NY,
USA). Fifty images per slide were analyzed for tail length (TL) and olive tail moment (OTM)
using image analyzer CASP software version 1.2.2.
2.7. DNA fragmentation assay
Agarose gel electrophoresis was performed in order to verify DNA fragmentation (Hebert
et al., 1996). The liver tissue was homogenized using 5 ml of lysis buffer (50 mM Tris HCl, pH
8.0, 10 mM NaCl, 10 mM EDTA, 100 mg/ml proteinase K and 0.5% SDS) and incubated for 1 h
at 50ºC. 10 µl of 100 µg/ml ribonuclease A (RNase A) was added to the mixture and incubated
for an additional 1 h at 50ºC. Tissue samples were treated with 1 ml phenol followed by
extraction with chloroform/isoamyl alcohol. The aqueous phase was treated with 25-50µl of 3 M
sodium acetate (pH 5.2) and one volume of ethanol, shaken gently and left at _20ºC overnight.
The precipitate was collected by centrifugation at 12,000x g for 20 min. The pellet was rinsed
with 1 ml of 70% ethanol and spin for 10 min. The supernatant was discarded and the pellet was
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air dried at room temperature and later dissolved in 0.5-1.0 ml of double distilled water. DNA
was precipitated in cold ethanol at _20ºC and finally dissolved in 0.5 ml of buffer. DNA sample
was loaded in 1.0% agarose gel containing 0.5 µg/ml ethidium bromide, electrophoresed at 80 V
and visualized under UV transilluminator.
2.8. Histopathological studies in the Liver
For qualitative analysis of liver histology, the tissue samples were fixed for 48 h in 10%
formalin-saline and dehydrated by passing successfully in different mixtures of ethyl alcohol–
water, cleaned in xylene and embedded in paraffin. Sections of the tissue (5–6 µm thick) were
prepared by using a rotary microtome and stained with haematoxylin and eosin (H&E) dye,
which was mounted in a neutral deparaffinised xylene medium for microscopic examinations.
2.9. Statistical Analysis
Data are presented as means ± S.D. and were subjected to statistical significance was
evaluated by one way analysis of variance (ANOVA) using SPSS Version 15.0 (SPSS, Cary,
NC, USA) and the individual comparisons were obtained by Duncan’s multiple range test
(DMRT). Values were considered statistically significant when p < 0.05.
3. Results:
3.1. Effect of EGCG on food intake, body weight gain and organ-body weight ratio in control
and experimental rats
Table 1 depicts the effect of NaF and EGCG on food and water intake, body weight gain
and organ-body weight ratio (%) in normal and experimental animals. In NaF treated rats, water
and pellet diet consumption was significantly (P<0.05) decreased with decrease in body weight
gain. A significant (P<0.05) increase in liver body weight ratio was recorded in NaF treated rats.
No significant changes were observed between control and EGCG alone treated rats. All these
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changes induced by NaF intoxication were recovered significantly (P<0.05) on oral
administration with EGCG in a dose dependent manner. Animals treated NaF along with
silymarin significantly (P<0.05) attenuated the NaF induced alterations in the food and water
intake, body weight and organ weight when compared to NaF treated rats.
3.2. Effect of EGCG on hepatic marker
Table 2 illustrates the levels of serum hepatic marker enzymes and bilirubin in control
and experimental rats. Oral administration of NaF caused abnormal liver function in rats. In NaF
treated rats the activities of serum hepatospecific enzymes such as serum aspartate
aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, gamma
glutamyl transferase and the level of bilirubin were significantly (P<0.05) increased, when
compared with control rats. Administration of EGCG (40mg/kg) along with NaF significantly
(P<0.05) decreased the levels of serum hepatic markers and bilirubin when compared to NaF
treated rats. Restoration of hepatic marker enzymes and bilirubin was maximum in the medium
dose level (40mg/kg body weight) of EGCG when compared to other two doses (20 and
80mg/kg). Based on these findings 40mg/kg body weight of EGCG was fixed as a dose and used
for further biochemical investigations. Oral administration of EGCG (40mg/kg body weight)
alone to normal rats did not show any significant effect on hepatic markers. Animals treated NaF
along with silymarin significantly (P<0.05) reduced the NaF induced alterations in serum hepatic
biomarkers and bilirubin when compared to NaF treated rats.
3.3. Effect of EGCG on lipid peroxidation and protein carbonylation
Table 3 shows the changes in the levels of lipid peroxidation product in control and
experimental animals. In NaF treated rats the levels of thiobarbituric acid reactive substances,
hydro peroxides, protein carbonyl content and conjugated dienes were significantly (P<0.05)
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increased. Oral administration of EGCG (40 mg/kg body weight) along with NaF significantly
lowered the levels of lipid peroxidation products and protein carbonylation in liver. Animals
treated NaF along with silymarin significantly (P<0.05) attenuated the NaF induced alterations in
hepatic oxidative stress markers when compared to NaF treated rats.
3.4. Effect of EGCG on non-enzymatic antioxidants
Table 4 shows the changes in the levels of hepatic non-enzymatic antioxidants namely reduced
glutathione, total sulfhydryl group, vitamin C and vitamin E in the liver of control and
experimental rats. EGCG alone treated rats shows a significant (P<0.05) increase in the level of
liver total sulfhydryl groups when compared with control rats. A significant (P<0.05) decrease in
the levels of non-enzymatic antioxidants were noticed in rats treated with NaF when compared to
control rats. Treatment of EGCG (40 mg/kg body weight) along with NaF significantly (P<0.05)
increased the levels of non-enzymatic antioxidants to near normal. Animals treated NaF along
with silymarin significantly (P<0.05) attenuated the NaF induced alterations in the non-
enzymatic antioxidants level when compared to NaF treated rats.
3.5. Effect of EGCG on enzymatic antioxidants
Table 5 illustrates the activities of enzymatic antioxidants namely superoxide dismutase,
catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase and glucose-6-
phosphate dehydrogenase in liver of control and experimental rats. A significant (P<0.05)
diminution in the activities of enzymatic antioxidants in NaF treated rats were observed.
Treatment of EGCG along with NaF significantly (P<0.05) increased the levels of these
enzymatic antioxidants in liver. Animals treated NaF along with silymarin significantly (P<0.05)
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editi
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nd p
age
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posi
tion.
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.
attenuated the NaF induced alterations in the enzymatic antioxidants level when compared to
NaF treated rats.
3.6. Effect of EGCG on membrane bound ATPases
Table 6 demonstrates the activities of Na +, K
+, Ca
2+ and Mg
2+ ATPases in liver of control and
experimental rats. A significant decrease (P<0.05) in the activities of Na +, K
+, Ca
2+ and Mg
2+
ATPases were observed in liver of NaF treated rats as compared to normal control rats. Pre oral
administration of EGCG (40mg/kg bw/day) significantly (P<0.05) prevented the decrease in the
activities of ATPases in NaF intoxicated rats. Animals treated NaF along with silymarin
significantly (P<0.05) attenuated the NaF induced alterations in the membrane bound ATPases
when compared to NaF treated rats.
3.7. Effect of EGCG on DNA damage
Figure 2 & 3 displays the level of DNA damage in control and experimental rats. A significant
(P<0.05) increase in different comet assay parameters such as % DNA in tail, tail length, tail
movement and olive tail movement were noted in rats treated with NaF when compared with
normal control rats. Pre oral administration of EGCG with NaF significantly (P<0.05) reduced
the NaF induced DNA damage near to control level. Normal control and epigallocatechin gallate
alone treated rats showed no or minimal DNA migration. Animals treated NaF along with
silymarin significantly (P<0.05) attenuated the NaF induced DNA damage in the hepatocytes
when compared to NaF alone treated rats.
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.
3.8. Effect of EGCG on DNA fragmentation
Figure 4 exhibits the level of DNA damage in the liver of control and experimental rats. A
significant increase in the level of DNA fragmentation was observed in NaF-intoxicated rats
(lane 3) when compared with normal control rats (lane 1). Pre oral administration of EGCG with
NaF considerably reduced the NaF induced DNA fragmentation (lane 4) near to control level.
Normal control and EGCG alone (lane 2) treated rats showed no or minimal DNA fragmentation.
Animals treated NaF along with silymarin also significantly (P<0.05) reduced the NaF induced
DNA fragmentation (lane 5) when compared to NaF treated rats.
3.9. Histopathological changes
Figure 5 exhibits the representative photo microscopic images of intact rat liver observed
in control group (Fig. 5A). The histoarchitectural pattern of liver was almost normal in EGCG
alone administered rats (Fig. 5B).Liver cells of animals exposed to NaF for four weeks induces
several pathological changes such as extensive degeneration of hepatocytes with focal necrosis,
bridging necrosis, inflammation, vacuolization, inflammatory cell infiltration, portal
inflammation and fatty degenerative changes etc., (Figs. 5C and 5D). Administration of EGCG
effectively attenuated the histopathological abnormalities evoked by the exposure to NaF (Fig.
5E). No regressive pathological changes were observed in animals treated with NaF and
silymarin except with some inflammatory cell infiltration and dilation of hepatic sinusoids (Fig.
5F).
4. Discussion:
The liver is a complex organ consisting of well-defined components that function in a
highly coordinated manner. A number of drugs, chemicals, heavy metals have been shown to
alter its structure and function. The degree of hepatic damage by heavy metals depends upon the
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.
nature, the dose, route and duration of exposure. Both acute and chronic intoxication of NaF
have been demonstrated to cause hepatotoxicity with various levels of severity. Animal
experiments were conclusive about metabolic adverse effects and hepatotoxicity of NaF
compounds (Nabavi et al., 2012). EGCG is well documented for the attenuation of oxidant
mediated hepatic damage induced by various xenobiotics. In this context, the present study also
confirmed that administration of EGCG significantly reversed the liver function against the toxic
effect of NaF.
The symptoms of NaF toxicity in rats were the body weight development retardation and
decreased water and food intake. The alterations of organ-liver weight ratio and absolute liver
weight in NaF intoxicated rats were due to tissue damage and reduction in their functions
(Shanthakumari et al., 2004). All these morphological changes observed in NaF administered
rats were effectively attenuated by the treatment with EGCG.
Leakage of hepatospecific enzymes into blood serum have been considered as indicators
of the hepatic dysfunction and damage. The increase in the activities of these enzymes in plasma
is indicative for liver damage and thus causes alteration in liver function. Muthumani and
Miltonprabu (2012) found that cell damage exhibited good correlation with the enzyme leakage.
Hence, cellular damage caused by toxic substances is frequently accompanied by increasing cell
membrane permeability. In our study, the increased activities of serum aspartate
aminotransferase, alanine aminotransferase, serum alkaline phosphatase and lactate
dehydrogenase in serum obviously indicate that liver is susceptible to NaF induced toxicity. This
increase could be attributed to the hepatic damage resulting in increased release of functional
enzymes from biomembranes or its increased synthesis (Muthumani and Miltonprabu, 2012).
Gamma glutamyl transferase has been widely used as an index of liver dysfunction. Recent
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.
studies indicate that serum gamma glutamyl transferase might be useful in studying oxidative
stress- related tissues. The products of the gamma glutamyl transferase reaction may themselves
lead to increased free radical production, particularly in the presence of iron (Lee et al., 2004).
The observed elevation in the concentration of serum bilirubin in NaF treated rats is also
consistent with the presence of hepatic damage (Nabavi et al., 2012). Administration of EGCG at
a dose of 40mg/kg bw/day has been proved remarkable improvement in hepatic function against
NaF intoxication than the other doses. EGCG have already been reported to exhibit membrane
stabilizing properties against ROS mediated oxidative hepatic injury, mainly by its powerful
hydrogen donating, antioxidant and free radical scavenging properties in a number of invitro
system and invivo models ( Devika and Prince, 2008)
NaF is known to produce oxidative damage in the liver by enhancing peroxidation of
membrane lipids, a deleterious process solely carried out by free radicals (Pieta et al., 2012).
Furthermore, lipid peroxidation causes impaired membrane function, impaired structural
integrity, decrease in fluidity and inactivation of a number of membrane bound enzymes
(Muthumani and Miltonprabu, 2012). Oxidative injury induced by NaF can be monitored in
experimental animals by detecting lipid peroxidative products such as thiobarbituric acid reactive
substances, hydroperoxides and conjugated dienes (Nabavi et al., 2012). In addition to cellular
lipids, studies have shown that cellular proteins may also be affected by free radical
accumulation. The formation of carbonyl derivatives of proteins is suggested to be a useful
measure of oxidative damage to proteins (Muthumani and Miltonprabu, 2012). The carbonyl
derivatives of proteins may result from oxidative modification of amino acid side chains and
reactive oxygen-mediated peptide cleavage. In our study, we observed increased levels of
thiobarbituric acid reactive substances, hydroperoxides, conjugated dienes and protein carbonyl
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.
contents in the liver of the NaF treated group. Administration with EGCG significantly reduced
the levels of lipid peroxidation and protein carbonylation in NaF intoxicated rats revealed the
free radial scavenging ability of EGCG, which could be mainly due to the presence of 8 hydroxyl
groups in a four ring structure and is readily dissolved in water and modified into multifactorial
components such as, stearic (SA), eicosapentaenoic (EPA), and docosahexaenoic acids (DHA)
during EGCG metabolism has been reported to exhibits enhanced ROS scavenging activity and
thereby reduced the NaF induced oxidative stress (Zhong and Shahidi., 2011).
The impairment of the antioxidant defence is considered to be critically involved in NaF
induced toxic effects. Vitamin C is a dietary hydrophilic antioxidant works in concert with
vitamin E, which is a chain breaking antioxidant to prevent the free radical chain oxidation of
lipids. Numerous reports have shown the positive effect of vitamin C as an antioxidant and
scavenge of free radicals (Das et al., 2006). Reduced glutathione is an important antioxidant
defence, which has sulfhydryl group in its peptide largely present in the biological system. The
sulfhydryl group and reduced glutathione interact and form a complex with NaF and thereby
alter NaF distribution and excretion (Chouhan et al., 2010). It is in agreement with our findings,
which showed that the levels of total sulfhydryl groups, reduced glutathione, vitamin C and
vitamin E were significantly depleted, it might also contribute to the development of NaF
induced hepatic damage. Administration of EGCG significantly prevents the depletion of these
non enzymatic antioxidants in NaF intoxicated rat liver. EGCG acts as a powerful scavenger of
many reactive oxygen/nitrogen species (ROS/RNS). Both free radical scavenging and chelating
properties of EGCG are apparently responsible for restoring these levels by reducing the
utilization of non-enzymatic antioxidants consequently lead to improvement of vitamins C, E,
total sulfhydryl groups and reduced glutathione in liver.
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.
Antioxidant enzymes, such as SOD, CAT, GST, GR, and GPX are considered to be the
second line of cellular defense against oxidative stress mediated injury in organs. Assessment of
these antioxidant enzymes is an appropriate indirect way to assess the prooxidant –antioxidant
status in NaF induced toxicity. Among them SOD and CAT mutually functions as important
enzymes in the elimination of ROS and RNS. SOD is an enzyme responsible for the conversion
of superoxide radicals into less harmful products like hydrogen peroxide while CAT brings about
the reduction of hydrogen peroxide and protects tissues from the highly reactive hydroxyl
radicals (Brioukhanov and Netrusoval, 2004). NaF intoxication significantly reduced the activity
of hepatic SOD and CAT in our study which is in line with the finding of Garcia-Montalvo, et al.
(2009) and Izquierdo-Vega, et al. (2008) in NaF treated rats. Glutathione related enzymes such
as GPX, GR and GST function either directly or indirectly acts as antioxidants. GPX is a
selenium containing enzyme that utilizes glutathione in decomposing hydrogen peroxides (H2O2)
to non toxic products. In the present study NaF administration lowered the activities of GPX, GR
and GST in liver. Bruce et al., (1982) reported that the decreased levels of GPX and GST activity
and depleted levels of GSH in NaF treated rats are mainly due to the overproduction of ROS
which is in consonance with our present study. GPX, GST and GR are ‘SH’ dependent enzymes,
and their ‘SH’ groups are inactivated by NaF induced ROS generation, leading to enzyme
inactivation. The inhibition of these enzymes not only reflects oxidative stress, but also exposes
the cells to further oxidative damage, since GPX, GST and GR are the essential enzymes in cell
protection against ROS. EGCG administration significantly up regulated the levels of GPX, GST
and GR by restoring reduced glutathione level and counteracting the free radicals produced by
NaF intoxication. The reduction of G6PD activity in NaF induced rats showed impaired
generation of NADPH which is required for the reduction of GSSG to GSH (Oliver Barbier et
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s Ju
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posi
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.
al., 2010). Interestingly the fact that EGCG administered NaF intoxicated rats shows significant
renewal of these antioxidant defense system by bringing them near to normal levels mainly due
to the strong antioxidant property of EGCG and the presence of its vicinal trihydroxy structure in
which oxygen atoms act as electron donors to from bonds with electrophilic ions and thereby
helps in the recoupment of antioxidant defense system (Higdon and Frei., 2003).
The determination of membrane associated enzyme activities like adenosine
triphosphatases (ATPases) indicates the changes in membranes under pathological conditions
(Kempaiah and Srinivasan, 2006). The maintenance of the cation gradient by the ATPase
enzymes is of fundamental importance and is essential for the regulation of cell growth and
differentiation, which are critical for the normal functioning of the cells. The changes in ionic
concentrations can bring about diverse types of cell injury, which ultimately lead to cell death
(Kane, 1996). Our data indicate a remarkable diminution in the activities of membrane bound
ATPases in the liver of NaF intoxicated rats. Administration of EGCG depleted the elevation of
LPO in liver tissue and sustained the activities of membrane bound enzymes. The preservation of
cellular membrane integrity by EGCG depends on its antioxidant properties that neutralize the
oxidative reactions. The lipophilic nature of EGCG favours its passage through the membrane, and
can accommodate in the lipid bilayer, thereby protecting the radical attack and maintaining the
normal physiology of the membrane. In addition, EGCG reduced the lipid peroxidation by NaF
and improved the levels of endogenous antioxidants involved in membrane protection, which in
turn reduced the NaF induced alterations in membrane bound enzymes as well as ionic gradients
within the cell.
The comet assay is a rapid, sensitive and versatile method for the quantification of DNA
damage in the individual cells (Fairbairn et al., 1995). The levels of % DNA in tail, tail length,
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.
and tail movement significantly increased in the hepatocytes of NaF treated rats when compared
with control group. Previous reports show that lipid peroxidation products of polyunsaturated
fatty acids play a major role in genotoxicity of the cell (Comporti, 1989). Several mutagenic and
genotoxic lipid peroxidation products in particular malondialdehyde (MDA) and 4-hydroxy-2-
nonenal (HNE) have been identified to bind to DNA and to damage it (Eder et al., 2006). A loss
of DNA integrity in the form single strand breaks has been recorded during exposure of NaF.
NaF has a dense negative charge and is biochemically very active thus directly affect DNA due
to strong affinity for uracil and amide bonds by –NH--- F- interaction (Li et al., 1987). Wang et
al., (2004) also reported that NaF can induce the production of free radicals, which can damage
DNA strands directly or by lipid peroxidation initiated by free radicals. The DNA fragmentation
forms a typical ladder pattern of multiple sized nucleosomal nucleotides, a hallmark of apoptosis.
In this study, an increased DNA damage in the hepatocytes of NaF treated rats was observed as
compared to control rats. EGCG administered rats showed decreased DNA damage as compared
to NaF intoxicated rats. Muthumani and Miltonprabu (2012) have demonstrated the ability of
antioxidants/free radical scavenging compounds to protect cellular DNA against damages
induced by heavy metals. The green tea catechins has a strong inhibitory activity against
superoxide formation and reduces both intracellular ROS levels and the rate of H2O2 induced
apoptotic cell death (Zhao et al., 2001). Thus, the antioxidant effect of EGCG could be, at least
in part, responsible for their protective effect against NaF-induced DNA damage.
The histopathological results also strongly support our biochemical findings that, the
EGCG protects against the NaF induced oxidative stress related hepatic injury. The present
investigation shows the altered hepatic histoarchitecture in NaF treated rats such as bridging
necrosis, inflammation cell infiltration, vacuolization, dilation of sinusoidal space and
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inflammation. Administration of EGCG in NaF intoxicated rats showed a significant restoration
of hepatic architecture and brings about the normal histology of liver. This may be due to the
strong antilipoperoxidative ability of EGCG. The presence of eight OH groups in EGCG
facilitates its free radicals scavenging ability by directly bind and eliminate them during NaF
intoxication and thereby limits the lipid peroxidation and protein oxidation in the liver.
Scientific reports showed that catechins possess many of the structural components that
contribute to their antioxidant property. Catechins have a gallate moiety esterified at the 3rd
position of the C ring, the catechol group (3,4,5-trihydroxyl groups) on the B ring and the
hydroxyl groups at the 5th and 7th positions on the A ring (Fig. 1). The potent free radical
scavenging activity of EGCG was attributed to the presence of the C ring gallate group. The
observation was also made that the more hydroxyl groups in the catechin (EGCG) possesses,
more effective free radical scavenger the catechin becomes (Zhao et al., 2001).
Hence, it can be summarized that, the oral supplementation of EGCG effectively protects
the rat liver when compared with the standard drug silymarin in multiple ways from NaF induced
oxidative hepatotoxicity. This ameliorative effect was due to the inhibition of ROS formation,
thus inhibiting the peroxidation of membrane lipids and preventing the leakage of hepatic
markers enzyme, in addition EGCG administration significantly improves the levels of both
enzymatic and non enzymatic antioxidant in liver that further contributes to its hepatoprotective
effect. Moreover the prevention of “SH” groups oxidation in hepatic membrane bound ATPases,
stabilizes the membrane. These findings suggest that EGCG can be used as a potential
therapeutic entity for preventing NaF induced oxidative stress related organ dysfunction in
subjects exposed to NaF in their occupational and environmental sources. The hepatoprotective
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efficacy of EGCG was further conformed by comparing with the positive control silymarin, a
standardized hepatoprotectant against NaF induced hepatotoxicity in rats.
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nly.
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s Ju
st-I
N m
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crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
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ay d
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om th
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cord
.
Acknowledgement
Authors acknowledge Professor and Head, Department of Zoology, Annamalai University and
UGC-SAP for their generous support in this work.
Captions for figures
Fig. 1. Chemical structure of EGCG (C22H18O11).
Fig.2. Representative photomicrographs of comets stained with SYBR green-I at a magnification
of 200X showing the DNA migration pattern in hepatocytes: A) control group shows no DNA
migration, B) EGCG administered group shows no DNA migration, C) NaF treated group shows
extensive DNA migration and D) EGCG administered NaF intoxicated group shows the minimal
DNA migration. E) Silymarin administered NaF intoxicated group shows intact DNA .
Fig.3. Effect of EGCG on DNA damage (in terms of tail lenth, tailmoment, olive tail moment and
% tail area) in the hepatocytes of control and experimental rats. Arbitrary unit = Percentage tail
DNA × tail length; Values are mean ± SD for 6 rats in each group; a, b and c
Values are not sharing
a common superscript letter (a, b and c) differ significantly at P<0.05 (DMRT).
Fig.4. Effect of EGCG on DNA fragmentation in hepatocytes of control and NaF-induced rats.
Lane 1. Control (Normal DNA), Lane 2. Control EGCG (40 mg/kg BW) (Normal DNA), Lane 3.
NaF control (25 mg/kg BW) (severe DNA fragmentation), Lane 4. NaF (25 mg/kg BW) and
EGCG (40 mg/kg BW) (mild DNA fragmentation). Lane 5. NaF (25 mg/kg BW) and Silymarin
(25 mg/kg BW) (mild DNA fragmentation)
Fig. 5. Representative photomicrographs from the liver of control and experimental rats: A) Section
of control liver showing normal arrangement of sinusoids and hepatocytes (H & E, 40X). B)
Section of liver treated with EGCG alone showing the normal histoarchitectural pattern of
hepatic parenchyma (H & E, 40X) C) Section of liver exposed to NaF showing the extensive
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inflammation, dilated sinusoids, and degeneration of hepatocytes with necrosis, vacuolization
and inflammatory cell infiltration. (H & E, 40X). D) Section of liver treated with NaF showing
severe necrotic changes, severe inflammation, fatty degeneration of hepatocytes and complete
derangement of hepatic cords (H & E, 40X). E) Section of liver treated with NaF and EGCG
showing significant improvement of hepatic histoarchitecture with mild infiltration (H & E,
40X). F) Section of liver treated with Silymarin and NaF showing the almost normal
histoarchitectural pattern of hepatic parenchyma with mild inflammation and sinusoidal
dilation (H & E, 40X).
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s Ju
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N m
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crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
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om th
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ersi
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f re
cord
.
Table 1.
The effect of Fluoride and Epigallocatechin gallate on food and water intake, body weight gain and relative and
absolute liver weight in normal and experimental rats.
Groups
Body Weight (g) Organ weight
Initial (g) Final (g) body weight
change (g)
Food intake
(g/100g body
weight/day)
Water intake
(mL/rat/ day)
Relative liver
weight (%)
Absolute liver
weight (g)
Control 180.88 ± 20.14 190.00 ± 22.25 09.12 ± 2.11a 14.27 ± 0.97
a 20.15 ± 4.46
a 1.78 ± 0.6
a 3.38 ± 0.14
a
EGCG (40 mg/kg) 181.00 ± 19.87 190.00 ± 18.10 09.00 ± 1.77a 10.95 ± 0.81
a 15.45 ± 1.14
a 1.80 ± 0.6
a 3.42 ± 0.12
a
Fluoride (25 mg/kg) 180.00 ± 16.14 166.10 ± 13.61 -14.90 ± 2.53b 8.18 ± 0.70
b 9.42 ± 0.21
b 1.41 ± 0.4
b 2.35 ± 0.09
b
Fluoride (25 mg/kg)
+ EGCG (20mg/kg) 183.00 ± 19.58 173.00 ± 17.73 -10.00 ±1.85
ac 9.58 ± 0.67
bc 10.50 ± 1.06
bc 1.63 ± 0.5
c 2.82 ± 0.10
c
Fluoride (25 mg/kg)
+ EGCG (40 mg/kg) 184.00 ± 19.70 175.00 ± 16.83 -09.00 ± 2.87
ad 9.64 ± 0.78
c 14.08 ± 2.72
c 1.83 ± 0.6
ad
3.21 ± 0.11d
Fluoride (25 mg/kg)
+ EGCG (80 mg/kg) 182.58 ± 20.80 173.08 ± 22.41 -10.50 ± 1.61
ae 10.56 ± 0.83
d 17.23 ± 2.82
a 1.76 ± 0.5
ae 3.05 ± 0.12
e
Fluoride (25mg/kg)
+Silymarin(25mg/kg) 182.00± 20.55 191.40±22.64 09.40±2.09
ae 14.45 ± 0.92
a 21.05±3.91
a 1.74± 0.6
ae 3.18 ± 0.09
df
Values are given as mean ± S.D. from six rats in each group. Values not sharing a common superscript letter (a-f)
differ significantly at P<0.05 (DMRT).
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s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
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cord
.
Table 2.
Changes in the activities of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase
(ALP), lactate dehydrogenase (LDH) and gamma glutamyl transferase (GGT) in control and experimental rats.
Groups Control EGCG
(40 mg/kg)
Fluoride (25
mg/kg)
Fluoride (25
mg/kg) +
EGCG
(20 mg/kg)
Fluoride (25
mg/kg) +
EGCG (40
mg/kg)
Fluoride (25
mg/kg) +
EGCG (80
mg/kg)
Fluoride
(25mg/kg) +
Silymarin
(25mg/kg)
AST (IU/L) 62.79 ± 5.44a
64.83 ± 3.62a 106.20 ± 7.17
b 95.24 ± 5.30
c 77.50 ± 4.23
d 86.39 ± 3.65
e 90.45 ± 4.81f
ALT (IU/L) 25.95 ± 1.96a 24.05 ± 1.70
a 50.51 ± 3.70
b 42.38 ± 3.25
c 29.86 ± 1.92
d 37.79 ± 1.83
e 39.85 ± 2.11
f
ALP (IU/L) 74.61 ± 5.83a 72.59 ± 4.52
a 120.92 ± 9.50
b 103.58 ± 7.73
c 85.57 ± 6.38 93.93 ± 5.83
e 97.51 ± 5.37
f
LDH (IU/L) 110.72 ± 9.81a 112.26 ± 9.93
a 156.24 ± 13.25
b 149.31 ± 12.61
c 136.85 ± 10.56
d 142.45 ± 12.84
e 139.15 ± 9.17
df
GGT (IU/L) 0.61 ± 0.05a 0.64 ± 0.04
a 0.97 ± 0.09
b 0.89 ± 0.07
c 0.72 ± 0.06
d 0.83 ± 0.04
e 0.78 ± 0.04
f
Bilirubin
(mg/dL) 0.45 ± 0.03
a 0.43 ± 0.04
a 0.94 ± 0.07
b 0.86 ± 0.06
c 0.52 ± 0.04
d 0.63 ± 0.05
e 0.58 ± 0.04
f
Values are given as mean ± S.D. from six rats in each group. Values not sharing a common superscript letter (a-f)
differ significantly at P<0.05 (DMRT).
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s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
Table 3.
The changes in the levels of hepatic lipid peroxidation (TBARS) hydroperoxides (LOOH) protein carbonyl (PC)
content and conjugated dienes (CD) in the liver of control and experimental rats.
Groups
Control EGCG
(40 mg/kg)
Fluoride
(25 mg/kg)
Fluoride (25
mg/kg) +
EGCG (40
mg/kg)
Fluoride (25mg/kg) +
Silymarin (25mg/kg)
TBARS
8.07 ± 0.09a 8.10 ± 0.07
a 15.50 ± 1.70
b 11.03 ± 0.92
c 11.76 ± 0.81
cd
LOOH
0.85 ± 0.06
a 0.84 ± 0.03
a 1.74 ± 0.23
b 1.07 ± 0.06
c
1.18 ± 0.09d
CD
69.24±4.27a
71.41±4.43a
109.37±9.54b
57.63±3.69c 78.32 ± 5.71
d
PC
2.27 ± 0.05a 2.05 ± 0.01
a 5.90 ± 0.29
b 3.60 ± 0.14
c 3.87 ± 0. 17
d
Values are mean ± SD for 6 rats in each group; a, b, c and d
Values are not sharing a common superscript letter (a-d) differ
significantly at p<0.05 (DMRT).
The levels of TBARS were expressed as nmol/100g wet tissue.
The levels of LOOH and CD were expressed as nmol/100g wet tissue.
The levels of PC were expressed as nmol/mg protein.
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N m
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crip
t is
the
acce
pted
man
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rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
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.
Table 4.
Changes in the levels of vitamin - C, vitamin - E, reduced glutathione (GSH) and total sulfhydryl groups (TSH) in
the liver of control and experimental rats.
Parameters Control EGCG
(40 mg/kg)
Fluoride (25
mg/kg)
Fluoride (25
mg/kg) + EGCG
(40 mg/kg)
Fluoride
(25mg/kg) +
Silymarin
(25mg/kg)
Vitamin - C
(µM/mg) 1.95 ± 0.16
a 1.98 ± 0.18
a 1.56 ± 0.13
b 1.72 ± 0.17
c 1.67 ± 0.09cd
Vitamin - E
(µM/mg) 1.33 ± 0.11
a 1.43± 0.09
a 0.89± 0.09
b 1.23 ± 0.09
c
1.12 ± 0.10e
GSH (µg/mg) 18.54 ± 1.26a 21.79 ± 1.43
b 13.50 ± 1.16
c 20.02 ± 1.20
d 16.25 ± 1.12
e
TSH (µg/mg) 12.01± 1.02a 15.50 ± 0.45
b 09.01± 1.02
c
13.74 ± 0.79 d
11.07± 0.83e
Values are given as mean ± S.D. from six rats in each group. Values not sharing a common letter (a-e) differ
significantly at P<0.05 (DMRT).
Page 34 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
Table 5.
Changes in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione
S-transferase (GST), glutathione reductase (GR) and glucose 6-phosphate dehydrogenase (G6PD) in the liver of
control and experimental rats
Groups Control EGCG (40
mg/kg)
Fluoride (25
mg/kg)
Fluoride
(25 mg/kg) +
EGCG (40
mg/kg)
Fluoride
(25mg/kg) +
Silymarin
(25mg/kg)
SOD 7.73 ± 0.51a
8.37 ± 0.65a
5.32 ± 0.40b
7.28 ± 0.26c
6.48 ± 0.21d
CAT 89.25 ± 4.85a 93.17 ± 6.26
a 54.12 ± 4.25
b 79.30 ± 4.86
c 67.42 ± 4.12
d
GPX 9.65 ± 0.64a 10.71± 0.70
a 5.29 ± 0.40
b 7.90 ± 0.71
c 6.27 ± 0.53
d
GST 8.80 ± 0.50a 9.25 ± 0.56
a 5.73 ± 0.25
b 8.10 ± 0.50
c 7.19 ± 0.21
d
GR 0.45 ± 0.01a 0.47 ± 0.02
a 0.29 ± 0.01
b 0.39 ± 0.06
c 0.31 ± 0.04
d
G6PD 2.10 ± 0.10a 2.15 ± 0.15
a 1.48 ± 0.06
b 1.87 ± 0.11
c 1.65 ± 0.09
d
Values are given as mean ± S.D. from six rats in each group. Values not sharing a common superscript letter (a-d)
differ significantly at P<0.05 (DMRT). SOD –one unit of enzyme activity was taken as the enzyme reaction, which
gave 50% inhibition of NBT reduction in one minute/mg protein; CAT–µmol of H2O2 utilized/min/mg protein;
GPX– µmol of GSH consumed/min/mg protein; GST–µmol of CDNB-GSH conjugate formed/min/mg protein; GR -
µmol of NADPH oxidized/min/mg protein and G6PD - µmol of NADPH formed/min/mg protein.
Page 35 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
Table 6.
Changes in the activities of adenosine triphosphatases (ATPase) in the liver of control and experimental rats.
Groups
Control
EGCG
(40 mg/kg)
Fluoride
(25 mg/kg)
Fluoride (25
mg/kg) + EGCG
(40 mg/kg)
Fluoride
(25mg/kg) +
Silymarin
(25mg/kg)
Total ATPases
(µg Pi liberated/min/mg
protein)
Na+/K
+ATPase
(µg Pi liberated/min/mg
protein)
Ca2+
ATPase
(µg Pi liberated/min/mg
protein)
Mg2+
ATPase
(µg Pi liberated/min/mg
protein)
2.45 ± 0.21a
0.53 ± 0.03a
0.54 ± 0.06a
0.91 ± 0.08a
2.48 ± 0.23a
0.54 ± 0.04a
0.56 ± 0.07a
0.98 ± 0.12a
1.35 ± 0.12b
0.23 ± 0.01b
0.29 ± 0.05b
0.48 ± 0.04b
1.99 ± 0.17c
0.46 ± 0.04c
0.46 ± 0.05c
0.79 ± 0.09c
1.57 ± 0.11d
0.37 ± 0.05d
0.34 ± 0.04d
0.67 ± 0.07d
Values are given as mean ± S.D. from six rats in each group. Values not sharing a common superscript letter (a-d)
differ significantly at P<0.05 (DMRT).
Page 36 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
142x131mm (96 x 96 DPI)
Page 37 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
130x161mm (96 x 96 DPI)
Page 38 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
133x122mm (96 x 96 DPI)
Page 39 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
97x131mm (96 x 96 DPI)
Page 40 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
113x113mm (96 x 96 DPI)
Page 41 of 41C
an. J
. Phy
siol
. Pha
rmac
ol. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
CA
LG
AR
Y o
n 03
/15/
13Fo
r pe
rson
al u
se o
nly.
Thi
s Ju
st-I
N m
anus
crip
t is
the
acce
pted
man
uscr
ipt p
rior
to c
opy
editi
ng a
nd p
age
com
posi
tion.
It m
ay d
iffe
r fr
om th
e fi
nal o
ffic
ial v
ersi
on o
f re
cord
.
