INTRODUCTION:

Cisplatin (cis-diamminedichloroplatinum II) is one of the most potent antitumor agents. Activity has been demonstrated against a variety of tumors, particularly for head and neck, testicular, ovarian, bladder and small cell lung cancers ¹.However, the use of this agent is limited by the development of nephrotoxicity and ototoxicity ² , toxicities which involve alterations in the antioxidant defense systems ³. Cisplatin has also been shown to alter the levels of luteinizing hormone (LH) and FSH, to reduce intratesticular testosterone, and to decrease sperm motility and count ⁴. The nephrotoxicity induced by cisplatin in human and experimental animal has been shown to be protected by the prior treatment of various antioxidans like ebselen⁵, vitamin C⁶ and selenium⁷. Green tea is an excellent source of polyphenol antioxidants, particularly of group known as green tea catechins (GTCs)⁸. Green tea tannin protects cisplatin induced nephropathy in LLC-PK₁ cells and in rats ⁹. Green tea reduces iron-induced lipid peroxidation in brain homogenates as well as in cultured C6 astrocytes and lung cells^{10, 11}. In addition, green tea has also been shown to reduce the formation of the spin-adducts of hydroxyl radicals and hydroxyl radical to induced DNA strand breakage in vitro ¹².

Green tea has been found to have inhibitory effects on the chemical-induced lung tumorigenesis¹³. There is also considerable epidemiological evidence suggesting that the consumption of green tea lowers the risk of several types of cancer incidences as a result of these antioxidant mechanisms¹⁴.

Damage to the testicular germinal epithelium is a potential side effect of cancer therapy, and is of particular concern in case of men of reproductive age having tumors with high cure rates. Therefore, the aim of present study was to evaluate the protective effect of green tea extract on cisplatin induced testicular damage by using serologic, histopathologic, and biochemical analyses.

MATERIAL AND METHODS:

Chemicals: Standardized powdered, ethyl acetate extract of green tea leaves, Camellia sinensis (Theaceae) was gift sample from Cherain Chemicals, Baroda, India with total polyphenolic content 35%. Cisplatin injection was gift sample from Serum Institute of India Ltd., Pune, India. All other chemicals used were of analytical grade.

Animals:

Adult male albino rats (Wistar strain) weighing between 175 - 225 g were used for the study. The animals were fed ad libitum with standard pellet diet and had free access to water. All experiments and protocols described in present report were approved by the Institutional Animal Ethics Committee (IAEC) of M. S. University, Baroda and are in accordance with guidance of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

Fig 1: Chemical Analysis: The fingerprint chromatograms are shown in Fig. 1. Details of the fingerprint analysis are given in Table 1.

Experimental Procedure:

Chemical analysis of green tea extract:

TLC fingerprint profile of the extract was established using HPTLC. For development of TLC fingerprint, 500 mg of powdered green tea extract was extracted with (3x25 ml) of methanol. Extracts were pooled, filtered and concentrated to 25 ml. Suitably diluted stock solution of methanolic extract with gallic acid standard solution and catechin were spotted on a pre-coated Silica gel G60 F254 TLC plate (E.Merck) using CAMAG Linomat IV Automatic Sample Spotter and the plate was developed in the solvent system of Toluene: Ethyl acetate: Formic acid (6: 6: 1). The plate was dried at room temperature and scanned using CAMAG TLC Scanner 3 at UV 254 nm and R_f values, and peak area of the resolved bands were recorded. Relative percentage area of each band was calculated from peak areas. The TLC plate was developed by spraying with 5% methanolic ferric chloride solution for the detection of phenolic compounds.

Groups and Treatment Schedule:

Powdered green tea extract was reconstituted in distilled water. Cisplatin injection was dissolved in sterile water for injection. The animals were divided into four groups each consisting of six rats and received following treatment.

Group I: Control group, received distilled water (3ml /kg /day p.o. for 30 days) followed by sterile water for injection (1ml /kg, i.p.) on 30^th day.

Group II: Received distilled water (3ml /kg /day p.o. for 30 days) followed by cisplatin injection (5 mg /kg i.p.) on 30^th day.

Groups III: Green tea extract (100 mg /kg /day p.o. for 30 days) followed by Cisplatin injection (5 mg/kg i.p.) on 30^th day.

Group IV: Green tea extract (100 mg /kg /day p.o. for 30 days) followed by sterile water for injection (1ml /kg, i.p.) on 30^th day.

Fig.2: Cross sections of testes in rats treated with cisplatin and along with green tea extract.

A: Control, B: Cisplatin, C: Cisplatin + GTE, D: GTE alone

A:Control B: Cisplatin

C: Cisplatin + GTE D: GTE alone

Testes from control (A) and green tea extract treated rats (D) shows normal feature of seminiferous epithelium and interstitial tissue. However, testes from a cisplatin treated rats (B) reveals markedly shrunken and empty seminiferous tubules. Administration of green tea extract along with cisplatin (C) restored these changes towards normalcy.

After 5 days of the injection of either cisplatin or vehicle, blood was collected for serological analyses. Epididymis was removed, cleared off the adhering tissues and weighed. The epididymal sperm count was done immediately. The testes was excised under euthanasia in chilled Tris buffer (10 mM pH 7.4) for measurement of tissue markers of oxidative stress the other one was collected for histopathology.

Epididymal sperm count:

Epididymal sperm was collected by slicing the epididymis in 5 mL phosphate buffered saline (pH 7.2). An aliquot of the epididymal sperm suspension was used for spermatozoa count using Neubauer hemocytometer ^{15, 16}.

Serological analyses:

Serum levels of creatinine, urea, and uric acid were determined by using standard kits of Reckon Diagnostic Ltd, India. Testosterone level was estimated by direct chemiluminescent assay (ADVIA CENTAUR).

Biochemical analyses:

The excised testes was then weighed and homogenized in chilled Tris buffer (10 mM, pH 7.4) at a concentration of 10% (w/v). The homogenates were centrifuged at 10,000 × g at 0º C for 20 min using Remi C-24 high speed cooling centrifuge. The clear supernatant was used for the assays of malondialdehyde content as indicator of lipid peroxidation (LP)¹⁷, endogenous antioxidant enzymes, superoxide dismutase (SOD) ¹⁸, catalase (CAT) ¹⁹ and reduced glutathione (GSH)²⁰. The sediment after centrifugation of tissue homogenate was resuspended in ice-cold Tris buffer (10 mM, pH 7.4) to get a final concentration of 10% and was used for the estimation of different membrane bound enzymes such as Na⁺K⁺ATPase ²¹, Ca²⁺ATPase ²² and Mg²⁺ATPase²³and Total proteins²⁴.

Table 1: Details of fingerprint chromatograms of green tea extract after scanning at 254 nm

Extract	Solvent system	No. of spots
Methanolic extract	Toluene: Ethyl acetate: Formic acid (6 : 6 : 1).	8
Rf values	0.03, 0.12, 0.22, 0.35, 0.43, 0.50, 0.63, 0.68
Relative %	3.30, 1.84, 33.03, 15.11, 35.09, 4.99, 1.27, 1.05

Histopathologic examination:

For histotological evaluation, the testes were fixed in 10% formalin, dehydrated and embedded in paraffin. Tissues were then sectioned at 4 μm, stained with haematoxylin and eosin (H&E) and examined for histopathological evidence under Olympus BX40 Photomicroscope.

Statistical analysis

Results of all the above estimations have been indicated in terms of mean ± S.E.M. Difference between the groups was statistically determined by analysis of variance (ANOVA) followed by Tukey –Kramer multiple Comparisons test with the level of significance set at P ≤ 0.05.

RESULTS:

Chemical Analysis:

The fingerprint chromatograms are shown in Fig. 1. Details of the fingerprint analysis are given in Table 1.

Body weight, testes weight and sperm count:

All animals survived the experimental period. Administration of cisplatin alone significantly reduced body and testes weight as compared to control animals. Administration of green tea extract along with cisplatin restored body and testes weight to normal. Administration of cisplatin alone significantly decreased sperm count while it was significantly increased with green tea extract coadministration (Table 2).

3.3 Serological analyses:

Administration of cisplatin alone significantly increased serum level of creatinine, urea, and uric acid and decreased the level of testosterone as compared to control rats. The administration of green tea extract along with cisplatin significantly restored serum marker levels towards the control value (Table 3).

Biochemical analyses:

Administration of cisplatin alone significantly increases LP while there was a significant decrease in GSH, SOD and CAT levels as compared to control rats. Administration of green tea extract along with cisplatin significantly restored GSH, SOD, CAT and LP levels towards control value (Table 4). Administration of cisplatin alone significantly decreased the levels of membrane bound enzymes like Na⁺K⁺ATPase, Ca²⁺ATPase and Mg²⁺ATPase as compared to control. Administration of green tea extract along with cisplatin significantly restored membrane bound enzyme levels towards control value (Table 4).

Histopathologic findings:

Cisplatin causes vacuolization and fibrinoid debris in the seminiferous tubules. Shrunken seminiferous tubules showed loss of germ cell. Widening of the interstitial space and severe vacuolization were also observed in interstitial tissues. Administration of green tea extract along with cisplatin restored these changes towards normalcy (Fig.2).

DISCUSSION:

Cisplatin generates reactive oxygen species such as superoxide anion and hydroxyl radicals, and stimulates lipid peroxidation ²⁵. It is accepted that both correlate to oxidative stress and cause an imbalance between the generation of oxygen derived radicals and the organism’s antioxidant potential. As a result of an increase in the formation of free radicals in cisplatin-induced toxicity, the balance normally present in cells between radical formation and protection against them is disturbed²⁶. This will lead to oxidative damage of cell components, e.g. proteins, lipids, and nucleic acids²⁷. There are evidences that cisplatin has a profound deleterious effect on the reproductive system and fertility potential in rat ⁴. Cisplatin inhibits activities of antioxidant enzymes (SOD CAT and GSH) and there is depletion of cellular thiols ²⁸ in rat kidney and testes suggesting that cisplatin cytotoxicity results from generation of reactive oxygen species (ROS) ²⁹. The results obtained in this study is in agreement with these previous reports, that the enhancement in lipid peroxidation, and decrease in reduced glutathione and antioxidant enzymes (SOD and catalase) contributes to cisplatin induced oxidative damage. This has resulted in the altered levels of serum creatinine, urea, blood urea nitrogen (BUN) and uric acid which are the diagnostic indicator of toxicity.

In line with our study, it has been found that several antioxidants like butylated hydroxyl anisole (BHA) and glutathione (GSH) prevented the cisplatin induced lipid peroxidation and depletion of glutathione ³⁰ and antioxidant enzymes. Green tea extract contains gallocatechin (GC), epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCg) and epicatechin gallate (ECg). Tea components possess antioxidant, antimutagenic and anticarcinogenic effects and could protect humans against the risk of cancer by environmental agents ³¹. In the present study, treatment with green tea extract offered a significant protection from the cisplatin induced oxidative damage to testes. Green tea extract treatment significantly reduced lipid peroxidation and increased the levels of glutathione, catalase and SOD in cisplatin induced oxidative damage. The reduction in serum levels of creatinine, urea, blood urea nitrogen (BUN) and uric acid indicates protective effect of green tea extract. Na⁺K⁺ATPase, Ca²⁺ATPase and Mg²⁺ATPase are the membrane bound enzymes and the levels of Na⁺K⁺ ATPase, Ca⁺⁺ ATPase and Mg⁺⁺ ATPase were reduced in testes of cisplatin treated rats. Since these

Table 2: Effect of cisplatin alone and along with green tea extract on body weight, testes weight and sperm count

Groups	Final body weight (BW, g)	Absolute testes weight (g)	Relative testes weight (per BW, %)	Sperm Count (x 10⁶/mg epididymis)
Group I	227.5±4.23	2±0.085	0.87±0.041	15.7±0.47
Group II	190±2.23^***	1.39±0.032^***	0.66±0.015^**	12.13±0.39^***
Group III	200.8±1.62^NS	1.675±0.041^*	0.74±0.012^NS	13.94±0.22^*
Group IV	227.2±6.27	2.04±0.082	0.91±0.048	15.44±0.58
P value	P<0.0001	P<0.0001	P<0.0001	P<0.0001
F value	22.13	22.38	12.22	14.06

Values are expressed as mean ±SEM. Group II was compared with Group I. Group III was compared with Group II.^*P<0.05, ^**P <0.01, ^***P <0.001, NS = Non significant

Table 3: Effect of cisplatin alone and along with green tea extract on serum levels of creatinine, urea, uric acid and Testosterone

Groups	Creatinine	Urea	Uric acid	Testosterone (ng/ml)
Group I	0.35±0.061	35.7±3.46	0.51±0.06	0.80±0.04
Group II	2.16±0.24^***	112.2±14.78^***	2.86±0.09^***	0.57±0.02^***
Group III	1.13±0.14^***	64.53±9.40^**	1.65±0.19^***	0.70±0.01^*
Group IV	0.31±0.069	35.11±3.84	0.50±0.10	0.86±0.029
P Value	P<0.0001	P<0.0001	P<0.0001	P<0.0001
F Value	34.56	15.76	82.55	16.38

Values are expressed as mean ±SEM. Group II was compared with Group I. Group III was compared with Group II.^*P<0.05, ^**P <0.01, ^***P <0.001, NS = Non significant

Table 4: Effect of administration of cisplatin alone and along with chronic green tea extract (30 days) on biomarkers of the oxidative stress

Groups	L P (nmoles of MDA/mg protein)	GSH (µg of GSH/mg protein)	SOD (Units/mg protein)	CAT (μmoles of H₂O₂ consumed/ min/mg protein)	Na⁺K⁺ATPase (µmoles of inorganic phosphorus liberated/min/mg protein	Ca²⁺ATPase (µmoles of inorganic phosphorus liberated/min/mg protein	Mg²⁺ATPase (µmoles of inorganic phosphorus liberated/min/mg protein
Group I	1.22±0.043	4.15±0.32	4.92±0.97	7.29±0.77	8.50±0.34	4.36±0.51	6.56±0.40
Group II	3.13±0.28^***	2.44±0.2^***	1.63±0.25^**	3.34±0.29^***	4.85±0.39^***	2.70±0.33^*	3.54±0.26^***
Group III	2.14±0.14^**	3.63±0.12^*	3.66±0.13^NS	5.48±0.31^*	7.30±0.15^***	3.77±0.32^NS	5.63±0.20^***
Group IV	1.41±0.15	3.76±0.32	4.22±0.54	6.73±0.51	7.79±0.45	4.31±0.33	5.09±0.25
P value	P<0.0001	P=0.001	P=0.0043	P<0.0001	P<0.0001	P=0.022	P<0.0001
F value	24.07	7.983	6.002	11.73	19.67	4.003	18.68

Values are expressed as mean ±SEM. Group II was compared with Group I. Group III was compared with Group II.^*P<0.05, ^**P <0.01,

^***P <0.001, NS = Non significant

membranes bound enzymes are thiol group containing enzymes, that are lipid dependant³²and hence the restoration of the activities of ATPase enzymes suggest the ability of green tea extract to protect the thiol group from oxidative damage through inhibition of lipid peroxidation.

In conclusion, green tea extract administration during cisplatin therapy reduces the risk of cisplatin induced oxidative damage and the protection can be attributed to a decrease in lipid peroxidation, restoration of activities of antioxidant and membrane bound enzymes.

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Received on 22.07.2009 Modified on 23.08.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 837-841