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PROTECTIVE EFFECT OF GREEN TEA EXTRACT ON

GENTAMICIN INDUCED GASTRO- AND

HEPATOTOXICITY IN RATS

SARA A KHAN1,2, SHUBHA PRIYAMVADA1, SHEEBA KHAN1,

Md WASIM KHAN1,3, E.R. AGHARIA2 AND Ahad N.K.YUSUFI1*

1DEPARTMENT OF BIOCHEMISTRY, FACULTY OF LIFE SCIENCES,

ALIGARH MUSLIM UNIVERSITY, ALIGARH-202002, UP, INDIA.

2 SVKMS MITHIBAI COLLEGE, BHAKTI VEDANTA MARG, VILE

PARLE (W), MUMBAI-400 056, INDIA.

3DST-INSPIRE FACULTY, CELL BIOLOGY & PHYSIOLOGY

DIVISION, CSIR-INDIAN INSTITUTE OF CHEMICAL BIOLOGY, 4,

RAJA S.C. MULLICK ROAD, KOLKATA-700032, INDIA.

Email: yusufi@lycos.com

ABSTRACT:

Activity of glucose-6-phosphate dehydrogenase increased whereas

that of malic enzyme decreased. However, GT given to GM rats

improved the overall metabolism, enhanced antioxidant defense and

energy metabolism. The BBM enzymes were differentially altered by

GT.

KEY WORD: Antioxidants; Green tea; Gentamicin; Carbohydrate

metabolism, Intestine; Liver.

INTRODUCTION:

A number of environmental variables, including certain chemicals,

metal ions, and antibiotics, were shown to alter the structure and

function of various tissues [1-6]. Gentamicin (GM) is frequently used

in the treatment of severe infections of the abdomen and urinary

tract including bacteremia and endocarditis [7]. However,

nephrotoxicity and ototoxicity limit its long-term clinical use [8]. GM

also accumulates in other tissues, including intestine, liver, heart, and

ear causing multiple adverse effects in these tissues [3, 4, 9-12].

Aminoglycoside antibiotics were also shown to alter mucosal

SJIF 2012: 2.545

UIF 2013: 1.075

Received on:

6th December 2014

Revised on:

10th December 2014

Accepted on:

11th December 2014

Published on:

1st January 2015

Volume No.

Online & Print

8 (2015)

Page No.

52 to 72

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morphology and absorption of nutrients [13-15]. They are known to interact with the

intestinal epithelium, its innervations causing alterations in intestinal motility which

contribute to antibiotic-associated diarrhea and colitis [16]. GM was shown to inhibit the

growth of majority of E. coli and bactericides fragile strains isolated from gastro-intestinal

tract [17]. GM also produced multiple adverse effects in the liver and induced oxidative stress

[2].

In the past decade, the concept of disease prevention via naturally occurring substances has

gained much interest. Since ancient times, green tea (GT) consumption has been shown to

improve health [18]. GT polyphenols, especially catechins, are well known antioxidative

agents and have been held responsible for most of the beneficial health effects ascribed to GT

[19]. It is well established that gastrointestinal (GI) tract plays an important role in the

absorption, metabolism, and conjugation of GT polyphenols [20]. GT polyphenols were found

to inhibit the growth of stomach cancer and gastritis [21]. GT is found to activate intracellular

antioxidant, inhibits pro-carcinogen formation and cancer cell proliferation in the GI tract

[22]. GT catechins improve gut flora by selectively increasing the growth of bifidobacteria and

lactobacilli in the gut wall while decreasing levels of potential pathogens [23] and also

prevent atrophy of the intestinal mucosa [24]. The health promoting properties of GT have

the potential to prevent GI diseases [25]. GT has been shown to reduce inflammation

associated with Crohn’s disease and ulcerative colitis, a type of inflammatory Bowl disease

(IBD) [24, 26-27]. GT is effective in reducing cholesterol and lipid absorption in the GI tract

[28]. It also appears to protect liver from damaging effects of toxic substances and also against

the development of liver tumors [25].

Recently we have shown that GT consumption enhanced cellular energy metabolism and

antioxidant defense mechanism in the liver, kidney, and small intestine [29] and prevented

GM and CP-induced nephrotoxicity and oxidative damage in the rat kidney [5, 30-32].

Considering the potential clinical use GM and the numerous health benefits of GT, we now

hypothesize that GT would prevent GM-induced gastrotoxicity and hepatotoxicity. The results

obtained indicate that GM administration caused selective alterations in the activities of

various enzymes of carbohydrate metabolism, BBM and oxidative stress and increased lipid

peroxidation. However, GT consumption markedly reversed GM-induced alterations by

improving energy metabolism, BBM integrity and by enhancing antioxidant defense

mechanism. The present results suggest that GT consumption can be an option for long-term

clinical use of GM without any toxic side effects.

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MATERIALS AND METHOD:

Chemicals and drugs-

Green tea (Lipton / Kangra-brand) was purchased from commercial sources (Jain Pan House,

New Delhi, India), Gentamicin (Nicholas, Mumbai, India) from the source indicated in

parentheses. All other chemicals used were of analytical grade and were purchased either

from Sigma Chemical Co. (St Louis, MO, USA) or Sisco Research Laboratory, Mumbai, India.

Green Tea Extract-

Green tea extract (GTE) was prepared by adding green tea (30 g) to 500 ml of boiling water,

steeped for 15-20 min. Infusion was cooled to room temperature and then filtered. The tea

leaves were extracted a second time with 500 ml of boiling water and filtered, and the two

filtrates were combined to obtain 3% green tea extract (3 g tea leaves/100 ml H2O). The

resulting clear solution is similar to tea brews consumed by humans. A known amount of

green tea extract (2 X 125 mL= 250 mL/day) was provided in two servings to GT consuming

rats for 25 days and was found to be sufficient. According to the manufacturer’s information,

the antioxidant content was 95 mg/g of GT.

Experimental design-

The animal experiments were conducted according to the guidelines of Committee for

Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of

Environment and Forests, Government of India. Adult male Wistar rats (8 rats/group)

weighing 150-180 g fed with standard rat chow (Aashirwaad Industries, Chandigarh, India)

and water ad libitum were conditioned for one week before the start of the experiment.

Initially two groups of rats entered the study after acclimatization (Fig 1).

They were fed on a normal rat chow diet with one group consuming water (control) and the

other consuming GT extract (3% w/v) in drinking water for 25 days as described above. After

15 days the rats were subdivided into two groups each (8 rats/group) and continued to

receive their respective drinking fluids. Nephrotoxicity was induced by intraperitoneal

administration of GM (80 mg/kg bwt/day), in 0.9% saline daily for 10 days to one of the sub-

group designated as GM and TGM. The other sub-group from each group received an

equivalent volume of normal saline for the same period. The rats were sacrificed 24 hours

after the last injection under light ether anesthesia. Blood samples were collected and the

intestines and liver were removed and processed for the preparation of homogenate and

brush-border membrane vesicles (BBMVs) as described below. All the preparations and

analyses of various parameters were carried out simultaneously under similar experimental

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conditions to avoid any day-to-day variations. Body weights of rats were recorded at the start

and completion of the experimental procedure.

Preparation of homogenates:-

After the completion of the experiment, liver and intestine were extracted. The intestines

were washed by flushing them with ice cold buffered saline (1mM Tris-HCl, 9g/L NaCl, pH

7.4). The liver was put in Tris buffered saline (TBS). The washed intestines (from control and

treated groups) were slit in the middle and the entire mucosa was gently scraped with a glass

slide and weighed. A 6.5% homogenate of this mucosa was prepared in 50 mM mannitol, pH

7.0, in a glass Teflon homogenizer (Remi motors, Mumbai, India) with 5 complete strokes. The

homogenate was then subjected to high speed Ultra-Turrex homogenizer (Type T-25, Janke &

Kunkel GMBH & Co. KG. Staufen) for 3 pulses of 30 s each with an interval of 30 s between

each stroke.

Preparation of brush border membrane-

The intestinal BBMV was prepared as described by Kessler et al. [33], using differential

precipitation by CaCl2. Mucosa scraped from 4-5 washed intestines was used for each BBM

preparation. Briefly, the mucosal scrapings were collected in a beaker containing 50 mM

mannitol, 5 mM Tris-HCl, pH 7.5. The mucosal homogenate was diluted with the above

mentioned Tris-mannitol buffer (15 ml/g tissue) and further homogenized using Ultra-Turrex

T25 homogenizer with three pulses of 30s each with 30s interval between each pulse.

Aliquots of mucosal homogenate were saved and quickly frozen for further analysis. CaCl2 was

added to the filtrate, to a final concentration of 10 mM and was kept for 20 min on ice, with

intermittent stirring. The homogenate were then centrifuged at 2000 g (5000 rpm) for 10 min

in a Beckman J2-M1 refrigerated centrifuge using a JA-17 rotor. The pellet was discarded and

the supernatant was recentrifuged at 35000 g (17000 rpm) for 30 min. The pellet was

resuspended in a small volume (1-2 ml) of 50 mM sodium maleate buffer, pH 6.8, with four

complete passes by a loose fitting Dounce homogenizer (Wheaton, USA) and centrifuged at

35,000 g (17,000 rpm) for 30 min in 15 ml corex glass tube using JA-20 rotor. The white outer

fluffy portion of the pellet was resuspended carefully in a small volume of the above

mentioned buffer, leaving the dark brown centre of the pellet undisturbed (mitochondrial

contamination). The suspension thus obtained was homogenized by hand held Douncer. The

BBM suspension was quickly frozen in small aliquots and used for enzyme analysis. In each

experiment, tissues from three to six animals (control and experimental) were pooled to

obtain a sufficient amount of starting material. All the steps involved were strictly carried out

at 0-4º C unless otherwise specified.

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Assay of carbohydrate metabolism enzymes:

The activities of the enzymes involving oxidation of NADH or reduction of NADP were

determined spectrophotometrically on Cintra 5 fixed for 340 nm using 3 ml of assay in a 1-cm

cuvette at room temperature (28-30 °C). The enzyme assays of lactate dehydrogenase (LDH,

E.C.1.1.1.27), malate dehydrogenase (MDH, E.C.1.1.1.37), malic enzyme (ME, E.C.1.1.1.40),

glucose-6-phosphate dehydrogenase (G6PDH, E.C.1.1.1.49), glucose-6-phosphatase (G6Pase,

E.C.3.1.3.3) and fructose-1, 6-bisphosphatase (FBPase, E.C.3.1.3.11) activities were studied as

described by Khundmiri et al [34]. Hexokinase was estimated by the method of Crane and Sols

[35] and the remaining glucose was measured by method of Nelson-Somogyi [36].

2.7 Assay of brush border membrane marker enzymes and lysosomal marker enzymes:

The activities of alkaline phosphatase (ALP), leucine amino peptidase (LAP), γ- glutamyl

transpeptidase (GGTase) and acid phosphatase (ACPase) were determined as described by

Farooq et al [3].

Assay of enzymes involved in free radical scavenging:

Superoxide dismutase (SOD, E.C.1.15.1.1) was assayed by the method of Marklund [37].

Catalase (CAT, E.C.1.11.1. 6) activity was assayed by the method of Giri et al [38].

2.9 Lipid peroxidation and Total –SH group estimation:

Total SH groups were determined by the method of Sedlak and Lindsay [39]. Lipid

peroxidation (LPO) by the method of Ohkawa et al [40].

Statistical analyses-

All data are expressed as Mean ± SEM for at least 4-5 different preparations. Statistical

evaluation was conducted by one-way ANOVA and by unpaired student’s t test using SPSS 7.5

software. A probability level of p<0.05 was selected as indicating statistical significance. Most

of the changes between various groups were compared with control values for better

understanding and clarity. However, specific differences and statistical significance between

other groups were evaluated separately e.g. GM vs. TGM.

RESULT:

To address the hypothesis whether green tea (GT) extract would prevent GM induced

intestinal and hepatic toxicity and oxidative damage, the effect of green tea was determined in

detail on the biomarkers enzymes of oxidative damage, brush border membrane and

carbohydrate metabolism in intestinal and liver tissues. In general, there was no significant

difference in body weight and food intake between GM administered and control rats.

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However a slight loss of body weight (~10%) was recorded in rats consuming GT alone and in

combination with GM (Table 1).

Effect of GT on GM induced alterations in biomarker enzymes of BBM and lysosomes:-

To assess the structural integrity of various organelles e.g. plasma membrane (BBM) and

lysosomes, the effect of GM and GT was determined on biomarker enzymes of BBM and

lysosomes in the intestinal and liver homogenates and isolated BBM preparations from

intestine.

a) Effect of GM and GT on biomarkers of BBM and lysosomes in intestinal homogenates:

The activities of alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGTase), leucine

aminopeptidase (LAP) and acid phosphatase (ACPase) were determined under different

experimental conditions in the homogenates of intestine and liver (Table 2). The activities of

these enzymes along with sucrase (a biomarker of intestinal BBM) significantly increased in

intestinal homogenate as reported earlier [7]. GM treatment caused small increase in BBM

enzymes in liver homogenates. GT consumption, however affected these enzymes

differentially in different tissues (Table 2). The activities of ALP (+29%), GGTase (+25%), LAP

(+27%) and sucrase (+44%) significantly increased by GT in the intestine. However, except

the decrease of GGTase (-24%) activity, ALP and LAP activities were not altered in the liver by

GT (Figure 2). Since GT caused increase in all BBM enzyme activities in the intestine, its

consumption by GM rats resulted in further improvement of BBM enzyme activities in

intestinal homogenates. The activities of GGTase and LAP were also higher in liver

homogenate in TGM compared to GM rats. It appeared from the results that GT produces an

overall improvement in BBM enzyme activities in GM treated rats in both the tissues. The

activity of ACPase (a lysosomal enzyme) variably decreased by GM treatment in both liver and

intestine. In contrast, GT increased ACPase activity in the intestine. As a result, ACPase activity

was significantly higher in tissues in TGM compared to GM rats

b) Effect of green tea (GT) on gentamicin (GM) induced alterations on brush border

membrane enzymes in isolated BBM preparation:

The effect of GM/GT on BBM marker enzymes was further analyzed in BBM preparations

isolated from intestine. The data summarized in Table 3, (Figure 2) showed a similar activity

pattern of BBM enzymes as observed in intestinal homogenate, however the magnitude of

effects was different. The activities of ALP (+28%), LAP (+21%) and sucrase (+69%)

increased to a much greater extent whereas GGTase (-18%) decreased by GM treatment in

intestinal BBM (Table 3, Figure 2). Except ALP, (which was decreased), the activity of GGTase

(+24%), LAP (+39%) and sucrase (+82%) profoundly increased by GT ingestion. The

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activities of BBM enzymes except ALP were significantly enhanced upon drinking GT by GM

rats as compared to GM alone rats.

Effect of green tea on GM induced alterations on the enzymes of carbohydrate metabolism

in rat intestinal and hepatic tissues:

The effect of GM, GT, and TGM was determined on the activities of various enzymes of

carbohydrate metabolism in intestine and liver (Tables 4 and 5). GM treatment to control rats

significantly increased hexokinase activity in the intestine (+17%) and liver (+20%). The

activity of LDH (a marker enzyme of anaerobic glycolysis), however profoundly increased in

the intestine (+124%) whereas decreased in the liver (-25%). The effect of GM was

differentially observed on MDH activity (an enzyme of TCA cycle) in the different tissues

studied. GM caused significant decline in MDH activity in the intestine (-40%) whereas

increased in the liver (+32%). As previously reported [34], GT consumption significantly

enhanced metabolic activities in almost all tissues studied. The activity of HK, LDH, and MDH

significantly increased in the intestine and liver by GT. When GM treatment was extended to

GT drinking rats, GM induced decrease in most metabolic enzyme activities, was not only

prevented but they remained significantly higher in TGM compared to either control or GM

treated rats (Table 4). The activity of LDH was especially found to be profoundly enhanced in

the intestine. The activity of MDH, however significantly increased in the liver by GT given to

GM rats.

The effect of GM and GT were also observed on enzymes of gluconeogenesis. GM significantly

increased the activity of glucose-6-phosphatase (G6Pase) and fructose 1, 6, bisphosphatase

(FBPase) in the intestine and to some extent in the liver (Table 5). GT significantly enhanced

the activity of both G6Pase and FBPase in the intestine but lowered in the liver. As a result GT

consumption by GM treated rats resulted in significant increase of both G6Pase and FBPase.

The effect of GT-extract alone and with GM was also determined on the activities of glucose-6-

phosphate dehydrogenase (G6PDH, HMP-shunt) and malic enzyme (ME), source of NADPH

needed in reductive anabolic reactions (Table 5). GM treatment to control rats significantly

increased G6PDH activity in the intestine (+46%) whereas decreased in the liver (-38%). The

activity of ME however, significantly lowered by GM treatment in both the tissues (Table 5).

The activities of G6PDH and ME however were differentially altered by GT. The activity of

G6PDH profoundly decreased in the intestine (-60%) and liver (-21%). ME activity on the

other hand was lowered by GT in both the tissues. GT consumption by GM treated rats was

able to restore G6PDH activity and was significantly higher in the liver in TGM compared to

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control or GM rats. The activity of ME was also improved in TGM compared to GM or control

rats.

Effect of GT on GM induced alterations in antioxidant defense parameters in rat intestinal

and hepatic tissues:-

GM induced gastro- and hepatotoxicity can also be determined, at least in part, by

perturbation of antioxidant defense mechanism (Table 6). GM caused significant decrease in

the activity of superoxide dismutase (SOD) in the intestine (-27%) and liver (-39%). The

activity of catalase, also significantly but differentially, declined in both the rat tissues studied.

It was decreased to greater extent in the intestine (-53%) followed by liver (-25%). GM

induced decrease in SOD and catalase activities was associated with increased production of

malondialdehyde (MDA); an end product of lipid peroxidation (LPO) in all the tissues. This

was accompanied by decreased levels of total SH. Taken together, these results indicated that

GM elicited production of free radicals and suppression of antioxidant enzymes caused

significant damage to rat tissues. In contrast, GT significantly enhanced the activity of either

SOD or catalase or both in both the rat tissues. LPO was lowered in the liver (-8%). GT, by

virtue of its known antioxidant properties prevented GM elicited adverse effects on various

parameters of oxidative damage. The activities of both SOD and catalase were also

significantly increased in the liver and intestine in TGM compared to GM treated rats. The

levels of total SH and LPO were also improved indicating a marked protection by GT against

GM induced oxidative damage in both intestine and liver.

DISCUSSION: Digestion and absorption of nutrients are the major functions of the intestinal mucosa. Liver

also contributes significantly to these processes. Thus, any damage to these organs by

environmental contaminants, including aminoglycoside antibiotics, might lead to

malabsorption of food components. Gentamicin (GM) is an effective and widely used

aminoglycoside antibiotic against severe bacterial infections [7]. Although nephrotoxicity is

one of the most important side effect and therapeutical limitation [41], GM was also shown to

cause gastrotoxicity and hepatotoxicity after prolonged clinical use [3]. Several strategies and

mechanisms were utilized to prevent GM-induced nephrotoxicity, however, there have been

no reports regarding protection against GM-induced gastro- and hepatotoxicity. Tea, the

second most widely consumed beverage worldwide since ancient times, is known for its

beneficial health effects. In particular, green tea (GT) has been shown to retard various forms

of cancers due to its antimutagenic and anticarcinogenic properties [18, 22]. It was also found

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to be cardioprotective, neuroprotective, antidiabetic, and antibacterial besides other health

benefits [19, 42]. GT has been shown to inhibit growth of certain cancers of GI tract and liver

and improve gut flora [21, 23, 29]. Recently, we have reported that GT was able to protect

against GM-induced nephrotoxity and oxidative stress in the rat kidney [5]. In the present

studies, we have tested our hypothesis that GT consumption would ameliorate GM-induced

toxic and other adverse effects in the intestine and liver.

GM was administered to control and GT-consuming rats and the activities of various enzymes

of carbohydrate metabolism, brush border membrane, mitochondria and lysosome and

parameters of oxidative stress were determined to understand detailed biochemical

events/cellular response/ mechanism of GM toxicity in the intestine and liver and its possible

protection by GT consumption. As reported earlier [3], the activities of various enzymes

involved in glycolysis, TCA cycle, gluconeogenesis, and HMP shunt pathway were

differentially altered in the intestine and liver by GM treatment. GM caused significant

increase in HK, LDH, G6Pase, FBPase, and G6PDH but decreased MDH and ME activities in the

intestine. However, in the liver, an opposite GM effect was observed where the decrease in

LDH activity was associated with increased MDH and HK activity but decreased G6PDH and

ME activities. Although the actual rates of glycolysis or TCA cycle were not determined, the

results indicate a shift in energy metabolism as an adaptive cellular response both in the

intestine and liver. A marked increase in LDH, and to some extent hexokinase, associated with

decreased MDH activity by GM may indicate that anaerobic glycolysis become major source

of energy in the intestine as reported earlier [1,3] due to GM-induced mitochondrial

dysfunction as reported for kidney [5]. At the same time GM induced upregulation of G6Pase

and FBPase appeared to provide sufficient glucose from other metabolites by gluconeogenesis

to support higher LDH activities in the intestine. Conversely, the increase in MDH and HK

activities associated with decreased LDH activity in the liver by GM indicates that oxidative

metabolism remained the predominant source of energy in the liver.

In addition, the effect of GM was also observed on the activity of G6PDH (HMP-shunt

pathway) and NADP-malic enzyme (ME) in the intestine and liver. The activity of G6PDH,

however, profoundly increased in the intestine, whereas decreased in the liver. While the

activity of NADP –ME, significantly but variably declined in both the tissues. Increased G6PDH

activity, at least, in the intestine seems to increase glucose oxidation by an alternative HMP-

shunt pathway. It appears to be an important cellular response as increased supply of NADPH

would be utilized in reducing anabolic pathways especially lipid biosynthesis (Membrane

lipids, cholesterol, fatty acids/phospholipids) required during repairing the membranes and

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in maintaining high GSH levels required under oxidative stress and inflammatory conditions

to enhance antioxidant defense mechanism [43]. In contrast to GM, GT consumption caused

selective alterations in the activities of various enzymes involved in carbohydrate metabolism

in different rat tissues. The activity of HK, LDH (glycolysis), MDH (TCA cycle), G6Pase and

FBPase (gluconeogenesis) significantly increased in the intestine but decreased in the liver as

reported earlier [30]. GT administration to GM-treated rats resulted in overall improvement

of carbohydrate metabolism in the intestine and liver. The activities of LDH and

gluconeogenic enzymes in GT fed-GM treated rat intestine remained higher whereas the GM

induced reduction in MDH activity was prevented by GT consumption. It appears that GT

might have lowered number of damaged mitochondria or affected macromolecules or may

have increased number of normally active organelles or macromolecules. Thus, GT given to

GM treated rats appears to maintain anaerobic glycolysis in the intestine and oxidative

metabolism in the liver, respectively, to meet energy requirements.

Intestinal microvillus membrane is highly specialized to perform transport and enzymatic

functions essential for normal digestion and absorption. Alterations in the membrane

organization or damage to the structure by toxic insult can affect both these functions. The

hydrolytic enzymes such as GGTase, LAP, lactase and sucrase etc., are involved in the terminal

digestion of proteins and carbohydrates and subsequent absorption of amino acids and

glucose. For example, sucrase splits sucrose into glucose and fructose just before their

absorption. Alkaline phosphatase (ALP) converts organic phosphate to inorganic phosphate

needed for phosphorylation of metabolites and ATP formation. Since BBM and other

intracellular organelles such as mitochondria and lysosomes are known GM target [3-4], the

structural/functional integrity of the tissue was assessed by the status of their respective

biomarker enzymes. GM exerted differential effects on ALP, GGTase, LAP (BBM enzymes) in

the intestine and liver. As reported earlier [3], GM significantly increased the activities of ALP

and LAP in intestinal homogenates and BBM preparations. The activity of sucrase, an

intestinal BBM marker enzyme, was also profoundly increased. The activity of ALP, GGTase

and LAP only slightly increased in the liver. In contrast to GM, GT consumption, however,

significantly increased the activities of BBM enzymes in the homogenate and BBM of the

intestine indicating an overall improvement in mucosal BBM integrity. GT consumption given

along with GM treatment caused profound increase in the activities of BBM enzymes GGTase,

LAP and sucrase with the exception of ALP in TGM compared to GM alone in intestinal BBM.

The results convincingly demonstrate that GT consumption not only prevented GM-elicited

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decrease in many of the enzyme activities but they remained significantly higher in TGM

compared to control and much more higher compared to GM-treated rats.

A relationship between oxidative stress and drug induced nephrotoxicity has been well

documented in many experimental animal studies [43-44]. Oxygen-derived free radical (ROS)

mediated damage has been implicated in the pathophysiology of certain gastrointestinal

diseases [45]. Cellular membranes including those of plasma membranes, mitrochondia,

microsomes etc are the main targets of ROS which results in peroxidation of membrane lipids

and proteins causing alterations in their structure and functions [45-47]. However, oxidative

stress can occur as a result of either increased ROS generation and/or decreased activities of

the antioxidant enzymes SOD, catalase and GSH-peroxidase. These enzymes protect the cell

against cytotoxic ROS. GM has been shown to enhance lipid peroxidation (LPO) and decrease

SOD, catalase and GSH-peroxidase activities in the kidney and heart [4-5, 10, 48]. In

agreement with previous observations [3], GM significantly increased LPO and decreased the

activities of SOD and catalase in the liver and intestine indicating GM-induced oxidative

damage. The severity of the damage appeared to be more prominent in the intestine than in

the liver. In recent years, the concept of chemoprevention by naturally occurring dietary

substances has been strengthened. Green tea provides a dietary source of biologically active

compounds (polyphenols) that help prevent a wide variety of diseases [18]. The present

results show that GT significantly enhanced antioxidant defense mechanism albeit

differentially in different tissues. The activity of SOD significantly decreased in the intestine

and liver. However, catalase activity profoundly increased in both intestine and liver

associated with lowering of LPO. Thus, GT consumption offered a significant protection

against GM-induced oxidative damage either by SOD-mediated or catalase mediated

mechanism or involving both mechanisms simultaneously.

The results of present study show that long-term GM administration induces generation of

free radicals that causes oxidative damage to cellular organelles, macromolecules and

especially to mitochondria and plasma membrane. It suppresses the enzymes of the

antioxidant defense mechanism and enhances lipid peroxidation leading to tissue injury to

both liver and intestine. It significantly decreased the activity of MDH but profoundly

increased the activity of the enzymes of anaerobic glycolysis (LDH) and gluconeogenesis in

the intestine in order to increase energy dependency on anaerobic glycolysis due to

mitochondrial dysfunction. However, GM significantly increased the MDH activity in liver to

boost oxidative metabolism in a tissue specific manner. In contrast, GT consumption reduces

GM induced oxidative stress and its adverse effects by virtue of its antioxidant properties

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thus, improving the structural integrity of various organelles/macromolecules and eventually

enhances nutrition and energy metabolism in the intestine and liver as was reported for

kidney [5, 49]. Taken together, we propose that GT may maximize the long-term clinical use of

GM in the treatment of life threatening bacterial infections without harmful side effects.

Moreover the inherent antibacterial properties of GT may exhibit a synergistic/additive

beneficial effect.

ACKNOWLEDGEMENT: Council of Scientific and Industrial Research (CSIR), New Delhi, India is acknowledged for the

award of Junior Research Fellowship (JRF)/ Senior Research Fellowship to SAK, Indian

Council of Medical Research (ICMR), New Delhi, India for the award of JRF/SRF to SP and WK..

Financial support to the department from University Grant Commission (UGC-DRF),

Department of Science and Technology (DST-FIST) and a research grant (58/21/2001-BMS)

from ICMR to ANKY is also gratefully acknowledged.

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metabolism and brush border membrane in rat intestine. Nutr Res. 24 (2004) 407-416.

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5. Khan SA, Priyamvada S, Khan S, Khan MW and Yusufi ANK. Protective effect of green tea extract on gentamicin-induced nephrotoxicity and oxidative stress in kidney and other rat tissues. Pharmacol Res. 59(4) (2009a) 254-262.

6. Khan S, Priyamvada S., Khan, SA, Khan MW, Farooq N, Khan F and Yusufi ANK. Effect of trichloroethylene (TCE) toxicity on the enzymes of carbohydrate metabolism, BBM, and oxidative stress in kidney and other rat tissues. Food Chem Toxicol 47 (2009b) 1562-1568.

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14. Faloon WW, Paes IC, Woolfolk D, Nankin H, Wallace K, Haro EN. Effect of neomycin and kanamycin upon intestinal absorption. Annals New York Acad Sci 132: (1966) 879-87.

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16. Goldhill JM, Rosek, Perry WH. Effects of antibiotics on epithelial ion transport in the rabbit distal colon in vitro. J Pharm Pharmacol. 48: (1996) 651-656.

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22. Mukhtar H, Ahmad N. Tea polyphenols: prevention of cancer and optimizing health. Am J Clin Nutr 71(6) (2000) 1698S-1702S.

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24. Safar S, Abdeen S, Dashti H, Khoursheed M, Al-Sayer H, Mathew T, Al Badar A. Effect of green tea in the prevention and reversal of fasting-induced intestinal mucosal damage. Nutr. 19: (2003) 536-540.

25. Suganuma M, Okabe S, Kai Y, Sueoka E, Fujiki H. Green tea and cancer chemoprevention. Mutat Res. 428 (1999) 339-344.

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26. Alic M. Green Tea for remission maintenance in Crohn’s disease? Am J Gastroenterol. 94 (1999) 1710.

27. Sano T and Sasako M. Green tea and gastric cancer. N. Engl. J. Med. 344 (200):675-676.

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30. Khan SA, Priyamvada S, Arivarasu N.A., Khan S and Yusufi A.N.K. Influence of green tea on enzymes of carbohydrate metabolism, antioxiodant defense and plasma membrane in rat tissues. Nutr. 23 (2007) 687-695.

31. Khan SA, Priyamvada S, Khan MW, Khan S, Farooq N, and Yusufi ANK. Studies on the protective effect of green tea against cisplatin induced nephrotoxicity. Pharmacol Res. 60: (2009c) 382-391.

32. Khan SA, Priyamvada S, Yusufi ANK. Protective effect of green tea extract on gentamicin- and cisplatin-induced nephrotoxicity. Chapter 52 in ‘Tea in Health and Disease prevention’, 2012, Elsevier Publication.

33. Kessler M, Acuto O, Storelli C, Murer H , Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim Biophys Acta. 50: (1978) 136-154.

34. Khundmiri SJ, Asghar M, Banday AA, Khan F, Salim S, Levi M, Yusufi ANK. Effect of ischemia and reperfusion on sodium dependent phosphate transport in renal brush border membranes. Biochem Biophys Acta 1716 (2005) 19-28.

35. Crane RK, Sols A. The association of particulate fractions of brain and other tissue homogenates. J Biol Chem. 203: (1953) 273-292.

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37. Marklund S, Marklund G. Involvement of the superoxide anion radical in the auto oxidation of pyrogallol and a convenient assay for superoxide dismutase. European J Biochem. 47, (1974) 469-474.

38. Giri U, Iqbal M, Athar M. Porphyrin - mediated photosensitization has a weak tumor promoting activity in mouse skin: possible role of in-situ generated reactive oxygen species. Carcinogenesis. 17, (1996) 2023-2028.

39. Sedlak J, Lindsay RH. Estimation of total protein bound and non protein bound SH groups in tissue with Ellman’s reagent. Anals of Biochemistry. 25 (1968) 192-20.

40. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anals of Biochemistry 95 (1979) 351-358.

41. Ishikawa Y, Inui K, Hori R. Gentamicin binding to brush border and basolateral membranes isolated from rat kidney cortex. J Pharm Dynam.;8 (1985) 931-941.

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42. Liao S. The medicinal action of androgens and green tea epigallocatechin gallate. Hong Kong Med. 7 (2001) 369-374.

43. Walker PD, Barri Y, Shah SV. Oxidant mechanisms on gentamicin nephrotoxicity. Renal Fail. 21 (1999) 433-442.

44. Devipriya S and Shyamaldevim CS. Protective effect of quercetin in cisplatin-induced cell injury in the rat kidney. Indian J Pharmacol. 31(1999) 422.

45. Nalini S, Ibrahim SA, and Balasubramanian. Effect of oxidant exposure on money intestinal brush-border membrane. Biochim Biophys Acta.. 1147 (1993) 169-176.

46. Szabo C, Cuzzocrea S, Zingarelli B, Connor M, Salzman AL. Endothelial dysfunction in a rat model of endotoxic shock. J Clin Invest. 100 (1997) 723-725.

47. Naqshbandi A, Rizwan S, Khan MW, and Khan F. Dietary flax seed oil supplementation ameliorates the effect of cisplatin on brush border membrane enzymes and antioxidant system in rat intestine. Human Exp Toxicol. 32(4) (2013) 385-394.

48. Priyamvada S, Priyadarshini M, Arivarasu NA, Khan S, Khan SA, Khan MW and Yusufi ANK. Studies on the protective effect of dietary fish oil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney. Prost Leukot Essent Fatty Acids. 78(6) (2008) 369-81.

49. Khan MW, Arivarasu NA, Priyamvada S, Khan SA, Khan S, Yusufi ANK. Protective effect of ω-3 polyunsaturated fatty acids (PUFA) on sodium nitrite induced nephrotoxicity and oxidative damage in rat kidney. J Funct Foods, 2013.

Table 1: Effect of green tea extract (GT) consumption on body weight (in g) of rats with/without GM treatment

Results are expressed as Mean ± SEM

* Significantly different at p< 0.05 from control by one way ANOVA

Groups Before Treatment

After Treatment % change

Control 154.2 ± 11.9 150 ± 10.2 -2.7

GM

158.3 ± 8.3

150 ± 10.2

-5.2

GT

TGM

160 ± 6.1

156.6 ± 4.4

145 ± 5*

140 ± 5

-9.4

-10.6

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Table 2: Effect of green tea extract (GT) consumption on brush border membrane enzymes in intestinal and liver homogenates with/without GM treatment

Results are Mean ± SEM of 3-4 different preparations. *Significantly different at from control, † significantly different at p< 0.05 from GM by one way ANOVA. Values in parentheses represent percent change from control

Table 3: Effect of green tea extract (GT) consumption on brush border membrane enzymes in BBMV isolated from small intestine with/without GM treatment

Results are Mean ± SEM of 3-4 different preparations *Significantly different from control, † significantly different at p< 0.05 from GM by one

way ANOVA. Values in parentheses represent percent change from control

Tissue ALP

(mol/mg

protein/h)

GGTase

(mol/mg

protein/h)

LAP

(mol/mg

protein/h)

ACPase

(mol/mg

protein/h)

Sucrase

(mol/mg

protein/h)

Intestine

Control 0.82 ± 0.02 1.8 ± 0.03 3.7 ± 0.1 1.44 ± 0.02 12.6 ± 0.7

GM 1.13±0.06*

(+38%)

1.93±0.03*

(+7%)

4.3 ± 0.2*

(+16%)

1.4 ± 0.025

(-3%)

16.8 ± 0.65*

(+33%)

GT

TGM

1.1 ± 0.06*

(+34%)

1.04±0.04*

(+27%)

2.25±0.006*

(+25%)

1.8 ± 0.02

4.7 ± 0.2*

(+27%)

4.7±0.16*

(+27%)

1.75 ± 0.08*

(+22%)

1.8 ± 0.06*

(+25%)

18.20 ± 0.2*

(+44%)

18.2± 0.56*

(+44%)

Liver

Control 0.64 ± 0.05 3.4 ± 0.07 0.5 ± 0.01 2.3 ± 0.08

GM 0.63 ± 0.04

(+2%)

3.98 ± 0.2*

(+17%)

0.53±0.02

(+6%)

2.1 ± 0.06

(-9%)

GT

TGM

0.56±0.005*

(-13%)

0.53 ± 0.03

(-17%)

2.6 ± 0.22*

(-24%)

5.2 ± 0.04*†

(+53%)

0.53±0.03

(+6%)

0.6 ± 0.04

(+20%)

2.14 ± 0.02

(-7%)

2.33± 0.033

Groups

ALP

(mol/mg

protein/h)

GGTase

(mol/mg

protein/h)

LAP

(mol/mg

protein/h)

Sucrase

(mol/mg

protein/h)

Control 3.6 ± 0.13 9.14 ± 0.22 34.6 ± 1.6 127.8 ± 2.7

GM 4.6 ± 0.6*

(+28%)

7.5 ± 0.2

(-18%)

42 ± 1.9*

(+21%)

216.0 ± 4.35*

(+69%)

GT

TGM

3.0 ± 0.1*

(-17%)

3.1 ± 0.1*†

(-14%)

11.32 ± 0.3*

(+24%)

11.2 ± 1.3*†

(+23%)

48.2 ± 0.6*

(+39%)

47 ± 3.2*

(+36%)

233.2 ± 18.8*

(+82%)

216.1 ± 7.7*

(+69%)

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Table 4: Effect of green tea extract (GT) consumption on the activity of metabolic enzymes in intestinal and liver homogenates with/without GM treatment

Results are Mean ± SEM of 3-4 different preparations. * Significantly different from control, † significantly different at p< 0.05 from GM by one way

ANOVA. Values in parentheses represent percent change from control Table 5: Effect of green tea extract (GT) consumption on the activity of metabolic enzymes in

intestinal and liver homogenates with/without GM treatment

Tissue Hexokinase

(mol/mgprotein/h)

LDH

(mol/mgprotein/h)

MDH

(mol/mg protein/h)

Intestine

Control 116.8 ± 1.55 3.53 ± 0.41 8.30 ± 0.6

GM 136.3 ± 1.38*

(+17%)

7.9 ± 0.8*

(+124%)

5.0 ± 0.5*

(-40%)

GT

TGM

134.3 ± 1.25*

(+15%)

127.5 ± 1.4

(+9%)

9.7 ± 0.7*

(+175%)

10.52 ± 0.2*†

(+198%)

12.7 ± 0.88*

(+53%)

7.8 ± 1.14†

(-6%)

Liver

Control 16.7 ± 0.44 55.04 ± 2.56 2.6 ± 0.34

GM 20.1 ± 0.9*

(+20%)

41.3 ± 4.8

(-25%)

3.44 ± 0.06*

(+32%)

GT

TGM

21.3 ± 0.7*

(+28%)

22.8 ± 0.4*

(+37%)

71.8 ± 0.45

(+31%)

53.6 ± 0.4

(-3%)

2.9 ± 0.23

(+12%)

3.55 ± 0.4*

(+37%)

Tissue G6Pase

(mol/mg

protein/h)

FBPase

(mol/mg

protein/h)

G6PDH

(mol/mg

protein/h)

ME

(mol/mg

protein/h)

Intestine

Control 0.97 ± 0.08 1.6 ± 0.03 0.67 ± 0.02 0.32 ± 0.06

GM 1.5 ± 0.17*

(+55%)

1.9 ± 0.06*

(+19%)

0.98 ± 0.05*

(+46%)

0.14 ± 0.008*

(-56%)

GT

TGM

1.2 ± 0.03

(+24%)

1.3 ± 0.023*

(+34%)

1.77 ± 0.014*

(+11%)

1.86 ± 0.105*

(+16%)

0.27 ± 0.046*

(-60%)

0.4 ± 0.015*†

(-40%)

0.2 ± 0.005*

(-38%)

0.32 ± 0.01†

Liver

Control 0.7 ± 0.03 0.32 ± 0.01 0.24 ± 0.0025 0.23 ± 0.06

GM 0.8 ± 0.03*

(+14%)

0.33 ± 0.01

(+3%)

0.15 ± 0.002*

(-38%)

0.20 ± 0.01

(-13%)

GT

TGM

0.56 ± 0.04

(-20%)

0.86 ± 0.02*

(+23%)

0.312 ± 0.02

(-3%)

0.42 ± 0.005†

(31%)

0.19 ± 0.006*

(-21%)

0.28 ± 0.02†

(+17%)

0.18 ± 0.043*

(-22%)

0.19 ± 0.05

(-17%)

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Results are Mean ± SEM of 3-4 different preparations. * Significantly different from control, † significantly different at p< 0.05 from GM by one way

ANOVA. Values in parentheses represent percent change from control Table 6: Effect of green tea extract (GT) consumption on enzymatic and non-enzymatic

antioxidant parameters in intestinal and liver homogenates with/without GM treatment

Results are Mean ± SEM of 3-4 different preparations. * Significantly different from control, † significantly different at p< 0.05 from GM by one way ANOVA. Values in parentheses represent percent change from control

Tissue LPO

(nmol/gmtissue)

Total-SH

(mol/gmtissue)

SOD

(Units/mgprotein)

Catalase

(mol/mgprotein/min)

Intestine

Control 34.7 ± 3.5 2.3 ± 0.05 9.12 ± 1.4 7.78 ± 0.8

GM 40.4 ± 0.08

(+16%)

2.1 ± 0.10

(-9%)

6.7 ± 1.30*

(-27%)

3.65 ± 0.25*

(-53%)

GT

TGM

42.3 ± 0.4

(+22%)

54.6± 0.8*†

(+57%)

2.5 ± 0.10

(+9%)

2.7 ± 0.13

(+17%)

5.11 ± 0.32*

(-44%)

8.02 ± 1.3

(-12%)

11.13 ± 0.42*

(+43%)

12.1 ± 1.15*†

(+56%)

Liver

Control 244.2 ± 10.5 15.44 ± 1.33 25.5 ± 3.4 4.8 ± 0.24

GM 321.4 ± 29.14*

(+32%)

11.4 ± 0.19*

(-26%)

15.5 ± 2.08*

(-39%)

3.6 ± 0.56

(-25%)

GT

TGM

224.0 ± 13.30

(-8%)

196.3 ± 18.1†

(-20%)

6.6 ± 0.30*

(-57%)

10.56 ± 0.4*

(-32%)

22.4 ± 0.17

(-12%)

20± 0.15

(-22%)

13.7 ± 0.62*

(+185%)

13.3 ± 0.27*†

(+177%)

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0

ND

7 days

Control

GT

25 days

Control

GM

GT

TGM 10 days

Sacrifice

Intestine

Liver

Figure 1: Experimental Design (ND normal diet; GT green tea; GM gentamicin; TGM

green tea + GM; = i.p. administration of GM for 10 days)

0

1

2

3

4

5

6

Cont GM GT TGM

Sp

ecif

ic a

ctiv

ity

(μ

mo

l/m

gp

rote

in/h

)

ALP

GGTase

LAP

(A)

*

*

*

*

*

*

*

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LIST OF ABBREVIATIONS

ACPase Acid Phosphatase; an enzyme

ALP Alkaline Phosphatase; an enzyme

ANOVA

ATP

Analysis of variance; statistical tool

Adenosine 5’-triphosphate; energy currency

BBM Brush border membrane; intestinal membrane

BBMV Brush border membrane vesicles

BUN

GT

Blood urea nitrogen; blood parameter

Green Tea

GGTase γ-glutamyl transferase; an enzyme

G6Pase Glucose-6-phosphatase; an enzyme

G6PDH Glucose-6-phosphate dehydrogenase; an enzyme

HK Hexokinase; an enzyme

HMP Hexose monophosphate; an enzyme

LAP Leucine aminopeptidase; an enzyme

0

10

20

30

40

50

60

Cont GM GT TGM

Sp

ecif

ic a

ctiv

ity

(μ

mo

l/m

gp

rote

in/h

)

ALP

GGTase

LAP

(B)

*

*

*

*

*

*†

*†

*

0

50

100

150

200

250

300

Cont GM GT TGM

Sp

ecif

ic a

ctiv

ity

(μ

mo

l/m

gp

rote

in/h

)

Homog

BBM

Figure 2: Effect of green tea extract (GT) consumption on activity of BBM enzymes in Intestine (A)

Homogenate (B) BBM (C) Sucrase in homogenate and BBM with/without GM treatment.

(C)

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LDH Lactate dehydrogenase; an enzyme

LPO Lipid peroxidation; an enzyme

MDH Malate dehydrogenase; an enzyme

ME Malic enzyme; an enzyme

NADPH

NADP+

NADH

Nicotinamide adenine dinucleotide phosphate (reduced);

reducing equivalent

Nicotinamide adenine dinucleotide phosphate

Nicotinamide adenine dinucleotide reduced

Pi

ROS

Inorganic phosphate

Reactive oxygen species

SOD

SH

Superoxide dismutase; an enzyme

Sulphydryl groups

TCA cycle

Tricarboxylic acid cycle