Comet Picture Sperm2

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Egypt. J. Exp. Biol. (Zool.), 11(1): 15 – 21 (2015) © The Egy pti an Soc iet y of Exper im ent al Bio logy

ISSN: 2090 - 0511 On Line ISSN: 2090 - 0503 http://www.egyseb.org

R E S E A R C H A R T I C L E

Heba A. El-Ghawet

Ab de la li m A. Gadall ah

AMEL IOR AT IN G ROL E OF J AMB OL AN OF CAPECIT AB INE IN DUCE D

TESTICULAR DAMAGE OF RATS

ABSTRA CT:

Capecitabine (Xeloda, Roche) is a pro-anticancer drug, that is enzymaticallyconverted to 5-fluorouracil in the body. Thepresent work aimed to evaluate theameliorated role of the jambolan fruit-extractagainst testicular damage induced bycapecitabine. Twenty male Wistar albino rats(Rattus norvegicus) weighing approximately120 g were used during experimentation.

An im als were divi ded int o four groups ; cont rol(saline-treated), Jambolan-treatment (400mg/kg of fruit extract), capicitabine-treatment(40 mg /Kg BW for 30 days), and capicitabineand jambolan-treatment. Daily oral treatmentswere carried out for 30 days. At the end ofexperimentation period, the animals weresacrif iced and their testis were incised andprocessed for histopathological investigation,comet and flow cytometric analysis forapoptosis. The present findings revealed thatthe drug-treatment possessed dramatictesticular damage in the form of focaldisorganization of the seminiferous tubuleswith almost missing of sperm, comparatively

decreased spermatogenic cells, sloughing anddegeneration of spermatogenic cells andinterstitial oedematous lesions. Flowcytometric analysis showed apparent increaseof apoptic M1 spermatogenic and ovariancells. Comet assay revealed apparentincrease of detached spermatogenic cells.Jambolan-treatment ameliorated thepathological picture and decreased theincidence of apoptosis. Finally, the authorsconcluded that the jambolan-treatment with itshigher content of antioxidants resolute thetoxicological aspects of the anticancer drugs.

KEY WORDS: Capecitabine, Jambolan, testicular damage,amelioration effect.

CORRESPONDENCE:

Heba A. El-Ghawet

Zoology Department, Faculty of Science,Mansoura University, Egypt

E-mai l :

Ab de la l im A. Gadal la hZoology Department, Faculty of Science,Mansoura University, Egypt

ART IC LE CO DE : 02 .01. 15

INTRODUCTION:

Capecitabine (Xeloda, Roche) is a new

agent, orally-administered chemotherapeuticagent used in the treatment of numerouscancers (Rossi, 2013). It is orallyadministered as a precursor of 5-fluorouracil(5-FU), which is converted to 5-FUpreferentially in human liver and cancer t issue(Miwa et al. , 1998). Hepatic steatosis, a mildmanifestation of nonalcoholic fatty l iverdisease (NAFLD), may occur after treatmentwith 5-FU. This has become a morerecognized complication in the era of hepaticsurgery for colorectal l iver metastases, wherehepatic steatosis is associated with increasedpost-operative morbidity (Zorzi et al. , 2007).Peppercorn et al. (1998) found that 47% of

patients with colorectal l iver metastasestreated with systemic 5-FU and folinic acidhad computed tomography (CT) findingsconsistent with fatty change.

On the other hand, several authorsoutlined the reproductive toxicities post-anticancer-treatments. A single doseadministration of 5-fluorouracil (5-FU) toWistar rats for 1, 3, 15, and 30 days-treatments were found to decrease the testesweight, seminiferous tubular diameter, andgerm cell height (D'Souza and Narayana,



Egypt. J. Exp. Biol. (Zool.), 11(1): 15 – 21 (2015)


16

2001). Sperm abnormalities includingamorphous, coiled, double headed, doubletailed and double headed/double tailedspermatozoa which are confirmed by adecrease of testosterone level were detectedin rats received administration of 5-Fluorouracil (5-FU)-treatment (D'Souza,2003). Also, 5-FU treatment was found tocause germinal cell sloughing, tubular atrophy

and a decline in sperm concentration(Narayana et al. , 2000; D'Souza andNarayana, 2002). A decrease of serumprolactin and testosterone levels associatedwith a reduced function of Sertoli cell werealso reported post 5-FU-treatment byTakizawa and Horii (2002). Decreasedtestosterone level was found to be associatedwith degeneration of Leydig cells post-bleomycin, etoposide and cisplatin treatment(Al-Bader and Kilarkaje, 2015).

Jambolan Jambolan, Syzgium cumini Linn. (Family Myrtaceae) is a tree cult ivatedin the gardens of Mansoura University. Thetree flourished their good taste fruits duringSeptember to November. The fruits is rich incompounds containing anthocyanins,glucoside, elagic acid, isoquercetin, kamferol,or antimellin (Ayyanar and Subash-Babu,2012) raff inose, glucose, fructose, citric a cid,mallic acid (Lewis et al. , 1956), gall ic acid,anthrocyanin (Par Jain and Seshadri, 1975)and crude fibre, magnesium, calcium,phosphorus, iron, potassium, cupper, vitamin

A, thi amine, ri boflavi n, niac in, fol ic aci d,ascorbic acid, choline (Ayyanar and Subash-Babu, 2012).

There is no available work concerningthe reproductive toxicity of the anticancer

drug capecitabine and the beneficial effects ofthe crude fruit of jambolan. The present workil lustrates the cytotoxicity of anticancer drugthrough descriptive histological investigationand quantitative flow cytometry, as well as theimpact of phytotherapy of jambolan in r esolutethe toxicological aspects of drugs.

MATERIAL A ND METHODS:

Capeci tabine and appl ied dos e-t reatment :

Capecitabine (Xeloda, Roche) is anorally-administered chemotherapeutic agentused in t he treatment of numerous cancers. Itis a pro-drug, that is enzymatically converted

to 5-fluorouracil in the body. The usedtherapeutic daily dose is 40 mg/kg BW in 0.4ml saline solution, orally administered for onemonth for rat. The dose was calculatedaccording to Paget and Barnes (1964). Thechosen dose was nearly comparable to thehuman effective therapeutic dose (ETD).

Phytotherapy-treatment with Jambolan-frui t :

The jambolan fruits are oval-shapeddark-brown colour and edible in taste. In thepresent study, the fruits were homogenate in

saline solution and dosed orally at doses of400 mg/kg BW.

Exper imental work:

Twenty male Wistar albino rats ( Rattusnorvegicus) weighing approximately 120 gwere used in the present study. All rats werekept under good ventilation and aerated room.Excess standard diet was supplied ad libitumduring the experimental period. They wereallowed free access to water. Animals weredivided into four groups (n = 5). The firstgroup served as control, the second receivedJambolan-treatment (400 mg/kg BW) of fruithomogenized with saline solution), the thirdgroup received capicitabine-treatment (40mg/kg BW), and the fourth group receiveddaily oral doses of jambolan equal to 400 mg/Kg BW for 30 days. At the end of treatment,the animals were sacrificed and their liver andkidney were incised. Parts of the tissues werekept in refrigerator and the other ones werefixed in 10% phosphate buffered formalin (pH7.4) for routine histological investigation.

Histo log ical invest iga t ions: Testis of control and experimental

groups were dissected and fixed in 10%phosphate buffered formalin (pH 7.4),dehydrated in ascending grades of ethylalcohol, cleared in xylene, and mounted inmolten pararplast 58−62°C. Serial 5 µm thicksections were cut and stained withhaematoxylin and eosin (H&E), examinedunder bright f ield l ight microscopy, and thenphotographed.

Comet assay:

The comet assay was performed asdescribed by Sasaki et al. (1997), Two

hundred microliters of 106 cells/mlsuspension were mixed with 600 µl of 1% lowmelting point agarose at 37°C (0.75% finalconcentration). The slides were placed on achil led plate to solidify the agarose followedby immersing in freshly prepared lysingsolution (2.5 M NaCl, 10 mM Tris-base, 0.1%sodium sarcosinate, pH = 12.3) in the dark forone hour at room temperature. After this, theslides were placed in alkaline electrophoresisbuffer (0.3 N NaOH, 1 mM EDTA, pH 12.3) for30 minutes, allowing DNA unwinding.Electrophoresis was performed at 20 V and400 mA for 10 minutes. Then were stainedwith propidium iodide (2.5 mg/ml) for 20

minutes and covered with a glass cover slipbefore analysis. The image of theelectrophoresed DNA appears l ike a comet,with undamaged DNA in the head, andfragmented DNA migrating to form a tail.Comet images were captured on an Axioscopefluorescent microscope equipped with astandard Photonics Camera.

Flow cytometric analysis of cell cycle apoptosis:

DNA ploidy and apoptosis wereanalyzed using fluorescence activated cellsorting (FACS) flow cytometer (Becton



El-Ghawet & Gadallah, Ameliorating Role of Jambolan of Capecitabine Induced Testicular Damage of Rats


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Dickinson, Sunnyvale, CA) equipped with a 15mW air-cooled 488 nm argon-ion laser. FL1(FITC) signals were detected through a530/30 nm band-pass fi lter; FL2 (PI) signalswere detected through a 585/42 nm band-pass filter. A total of 20 000 events wererecorded in l ist mode and analyzed using theCell Quest Pro software (Becton Dickinson) atMansoura University Hospital. The testicular

cell populations were gated assuming thelinear forward scatter (FSC) and side scatter(SSC) properties. Biopsies from liver andkidney of studied animals were taken, and cellsuspension was prepared with Tris-EDTAbuffer at pH 7.4 (Sigma-Aldrich Co.). Cellsuspension was fixed in ice-cold 96−100%ethanol (Sigma) at 4°C overnight, centrifugedat 1500 rpm for 10 min, and then re-suspended in PBS containing 50 µg/mL

propidium iodide (PI) (Sigma-Aldrich Co.).The cells were incubated at 37°C for 30 minbefore analysis by flow cytometry. PIfluorescence excitation at 512 nm, with arelatively large Stokes shift, emits at amaximum wavelength of 617 nm. Apoptosiswas indicated by the percentage of cells inG0/G1, S, and G2/M phases of the cell cycle.

RESULTS:

Morphometr ic assessments:

From table 1, capecitabine-treatmentreduced the number of active seminiferoustubules and increased the average of non-active seminiferous tubules with apparentincrease of missing sperm, spermatids, germcell height and mean tubular diameter.

Table 1. Morphometric assessments of testis of capecitabine-treatment with or without jambolan-treatment

Control JB-Treatment CAP-Treatment JB & CAP-Treatment

Total number and % of ST 120 (100%) 118 (100%) 216 (100%) 145 (100%)

Normal (active) seminiferous tubules (ST) 97% 93% 15% 73%

Non-active ST 1% 4% 85% 24%

ST lacking sperm 0% 3% 87% 15%

ST lacking spermatocytes 1% 0% 87% 4%

ST without spermatids 2% 0% 27% 2%

Germ cells height (m) 64 ± 2.6 63 ± 3.1 38 ± 2.8 52 ± 4.5

Mean tubular diameter (m) 230 ± 12.3 228 ± 11.7* 167 ± 9.8** 194 ± 10.8*

Each replicate represent the M ± SE, n = 5, Significance at *P <0.05.

Histo log ical invest igat ions:

The control and jambolan-treatedtest is revealed the typical features ofnormal structure. The seminiferous tubulesshowed typical arrangement of the

spermatogenic cel ls, includingspermatogonia, spermatocytes andspermatids, as wel l as groupingspermatozoa. Sertol i cel ls are relat ively fewand distr ibuted at fair ly regular intervals.Thin connect ive t issue was seen in betweenthe tubules (Fig. 1 A&B).

Fig. 1 (A&B). Photomicrographs of histological sections of

control testis showing normal seminiferous tubules(NST) with normal arrangement pattern ofspermatogenic cells (Sertoli cell, St; Spermatogonia,

SG, Spermatocytes, SO and sperm, S). The intertubulartissue appears rich of interstitial cells (ISC).

In capecitabine-treatment, the testisshowed focal disorganization of seminiferoustubules with marked depletion of thespermatogenic cell populations. Many of theseminiferous tubules lacked sperm,spermatids and secondary spermatocytes.Exfoliation of the damaged spermatocytes andspermatids were detected within the tubularlumina of many seminiferous tubules. Theintertubular connective tissue become





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hyalinised and showed comparative reductionof interstitial cells (Fig. 2 A-D).

On the other hand, jambolan-treatmentof capecitabine-intoxicated rats revealedmarked amelioration and improvements of thehistologic picture of testis associated withregeneration of spermatogenic cells andflourishing of sperm within tubular lumina(Fig. 2 E-F).

Fig. 2. Photomicrographs of histological sections oftramadol-treated testis.

A&B: Capecitabine-treatment showing inactiveseminiferous tubules with comparative reduction of

spermatogenic cells, decreased germ cell height andoedematous lesions within interstitial cells (OISC).

C-F: Jambolan and capecitabine-treatment showingamelioration, however restricted zone of interstitial

oedematous lesions are still appeared and flourishingof sperm are observed (S).

E. Exfoliation of germ cells within tubular lumina andoedematous interstitial cells).





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Comet assay:

Following applying comet assay, thesingle strand nucleotide /each testicular cells

were detached with increased tail length andDNA concentration, comparing with normalpattern structure of testis (Fig. 3).

Fig. 3. Comet assay of capecitabine-treated testis with or without jambolan-treatment

Flow cytometry of cel l cycle:

From table 2 and figure 4, capecitabine-intoxication revealed a considerable increaseof M1 (subG1 apoptosis) and a decrease in

the other cell cycle phases (M2, M3, and M4). Jambolan-treatment revealed a decrease in apopticcell death and amelioration of the drug toxicity.

Table 2. Flow cytometry of cell cycle of capecitabine-treated testis with or without jambolan-treatment

Control JB-Treatment CAP-Treatment JB & CAP-Treatment

M1 (Sub-G0/G1 apoptosis) 10.6 ± 0.8 8.6 ± 1.5 42.1 ± 3.7 23 ± 3.2

M2 (G0/1phase) 34.1 ± 5.6 36 ± 4.1 24.6 ± 2.8 30.5 ± 4.1

M3 (S phase) 36.7 ± 2.8 35 ± 3.7 27.4 ± 3.1 32 ± 3.7

M4 (G2/M phase) 7.1 ± 0.9 8.4 ± 1.2 10.6 ± 1.8 9.4 ± 1.5

Each replicate represent the M ± SE, n = 5, Significance at * P <0.05.

Fig. 4. Flow cytometry of testicular cells. C & JB. Control and Jambolan-treatment showing normal range of

cell cycle. CAP. Capecitabine-treatment showing increased average of apoptosis. CAP & JB.Combined Capecitabine and jambolan-treatment showing r educed cell damage.





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DISCUSSION:

From the pr esent f indings treatment withthe pro-anticancer drug capecitabine-treatment possessed dramatic testiculardamage in the form of atrophied seminiferoustubules with focal disorganization ofseminiferous tubules with marked depletion ofthe spermatogenic cell populations.

Exfoliation of the damaged spermatocytes andspermatids were detected within the tubularlumina of many of the seminiferous tubules.The intertubular connective tissue becomehyalinized and showed comparative reductionof interstitial cells. The germ cell height wasmarkedly reduced.

The present f indings agree with the workof D'Souza and Narayana (2001), Narayanaet al. (2000), D'Souza and Narayana (2002)whom repost testicular damage postfluouracil-treatment.

The observed histological picturerevealed oedematous lesion within the

interstitial cells manifesting alteringtestosterone secretion. A decrease of serumprolactin and testosterone levels associatedwith a reduced function of Sertoli cell werealso reported post 5-FU-treatment byTakizawa and Horii (2002). Decreasedtestosterone level was found to be associatedwith degeneration of Leydig cells post-bleomycin, etoposide and cisplatin treatment(Al-Bader and Kilarkaje, 2015).

The blood-testis barrier (BTB) which iscreated by adjacent Sertoli cells near thebasement membrane, serves as a'gatekeeper' to prohibit harmful substancesfrom reaching developing germ cells, most

notably postmeiotic spermatids. It is dividesthe seminiferous epithelium into the basal andadluminal (apical) compartment allowingspermiogenesis, to take place in a specializedmicroenvironment in the apical compartmentbehind the BTB. Recent studies have shownthat numerous drug transporters areexpressed by Sertoli cells. However, many ofthese same drug transporters are alsoexpressed by spermatogonia, spermatocytes,round spermatids, elongating spermatids, andelongated spermatids, suggesting that thedeveloping germ cells are also able toselectively pump drugs 'in' and/or 'out' viainflux or efflux pumps (Su et al., 2011).

The observed finding supported by amarked increase of M1 (subG1 apoptsis) post-drug-treatment. Many anticancer drugs such

as cisplatin, doxorubicin and camptothecinexerted DNA damage such as cisplatin whichinduced DNA damage through caspaseactivation in enucleated cells (cytoplastslacking a cell nucleus) as well as associatedwith rapid induction of cellular reactiveoxygen (Havelka et al. , 2007). Othermechanism involved DNA damage cell cyclearrest (Swift and Golsteyn, 2014).

Phytotherapy belongs to the area ofcomplementary and alternative medicine(CAM) and use of plant in medicinal uses.Jambolan-treatment of capecitabine-intoxicated rats r evealed m arked ameliorationof the histologic picture of testicular t issuesassociated with a marked reduction of DNAdamage. The amelioration of the histologicpicture may be attributed to the antioxidantcapacity including alpha-glucosidase andalpha-amylase inhibitory activities as well asCyanidin, quercetin, ellagic acid (EA),proanthocyanidins, phenolic content catechinand also high-ascorbic acid (Correia et al. ,2012).

Similar amelioration of testiculardamage was detected in several studies. Lycium barbarum polysaccharides was foundto attenuate testicular oxidative stress andprotecting testis-specif ic toxicity indoxorubicin-exposed rats (Xin et al. , 2012). Invitro studies revealed the cytoprotectiveeffects of fruit pulp of Eugenia jambolana (50– 250 µg ml

−1) against hydrogen peroxide

induced Leydig cell toxicity (Anand et al. ,2013).

Oxidative stress occurring duringchemotherapy through liberation of reactiveoxygen species such as cisplatin-induced

nephrotoxicity may be ameliorated bylycopene the major carotenoid present intomatoes, which i s a potent antioxidant (Sahinet al., 2010).

The amelioration of the jambolan fruitsextract- treatment attributed to theantioxidant capacity including alpha-glucosidase and alpha-amylase inhibitoryactivities, as well as Cyanidin, quercetin,ellagic acid (EA), proanthocyanidins, phenoliccontent catechin and also high-ascorbic acid(Correia et al., 2012).

Finally the authors concluded thatduring chemotherapeutic treatment, the

patient must administered fruits and vegetablecontaining antioxidants to ameliorate theirhepatic and renal toxicities.

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