Eugenia jambolana extract versus N-acetyl cysteine against ...shodhganga.inflibnet.ac.in/bitstream/10603/34666/... · Chapter 6: Experimental Chapter 3 118 significant reduction of

CHAPTER 6: EXPERIMENTAL

CHAPTER 3

Protective effects of Eugenia jambolana extract versus N-acetyl cysteine against cisplatin

induced damage in rat testis

Chapter 6: Experimental Chapter 3

116

3.1 Introduction

“The protection against CIS induced testicular damage as a result of various abovementioned

interventions is primarily based on strengthening mechanisms of cell survival by augmenting

cell’s own internal resources to meet such a challenge (Anand et al., 2014). However, though

antioxidants are reported and recognized as beneficial, all of the previous studies with herbal

extracts had never used any known antioxidant for comparison (Anand et al., 2014). In addition,

despite reports of mortality in animals, various doses of cisplatin (2.5 to 10 mg/kg b.w) were

utilized, both for short term (10 days) and other studies extending even beyond 30 days

(Atessahin et al., 2006b, (Anand et al., 2014). In all such studies, deleterious effects on

spermatogenesis due to cisplatin treatment ranged from partial disruption to complete arrest. The

extent of revival of spermatogenesis following herbal or other interventions similarly varied

widely (Anand et al., 2014). Separate assessment of Leydig cell function in cisplatin treated

animals has never been reported (Anand et al., 2014). Accordingly, the present study was

carefully planned to evaluate the protective effects of fruit pulp of Eugenia jambolana extract, in

comparison with a known antioxidant, N-acetyl cysteine (NAC) in short-term intervention

studies with CIS-treated adult rats (Anand et al., 2014). The molecular mechanisms associated

with cell survival and steroidogenesis were also extensively studied to assess their impact on the

overall testicular function (Anand et al., 2014).”

3.2. Experimental Plan

“Adult male rats (Holtzman strain) weighing 200-220 g were used (Anand et al., 2014). Sixty

animals were divided into six groups of ten animals each (Anand et al., 2014). The animals were

housed under control temperature (25±20C) and photoperiodic conditions (12 hr light:dark cycle)

with food and water ad libitum (Anand et al., 2014). With prior approval from Institutional

Animal Ethical Committee (IAEC), experiments were conducted under strict compliance of the

guidelines issued by the Committee for the purpose of Control and Supervision of Experiments

on Animals (CPCSEA), India. EJE, Cisplatin (Dabur, India) and NAC (Amresco, Ohio, USA)

were procured and administered to animals either alone or in combination as described below

(Anand et al., 2014). The animals were housed with proper husbandry conditions and monitored

every day (Anand et al., 2014).


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Gr 1: Saline (i.p)/alternate day

Gr 2: CIS (5 mg/kg b.w, i.p) single injection on day 1

Gr 3: EJE (25 mg/kg b.w, i.m)/alternate day

Gr 4: CIS (5 mg/kg b.w, i.p) single injection on day 1 + EJE (25 mg/kg

b.w i.m.)/alternate day

Gr 5: NAC (150 mg/kg b.w, i.p) twice a week (day 1 and 4)

Gr 6: CIS (5 mg/kg b.w, i.p) single injection on day 1 + NAC (150 mg/kg

b.w, i.p) twice a week (day 1 and 4)

While one testis from each animal was immediately fixed in buffered formalin, the other testis

was snap frozen in liquid nitrogen and stored at -80°C till used for biochemical and molecular

analysis (Anand et al., 2014). Under mild anaesthesia, blood was collected from the tail vein of

rats on the day of sacrifice; serum was separated and stored at -20°C till assayed for hormones

(Anand et al., 2014).”

3.3 Material and Methods

“The details of the material and protocols used for the experiments have been listed in the

preceding chapter, Chapter 2.”

3.4 Results

3.4.1 Effect on Seminiferous epithelium and hormone levels

“One time cisplatin treatment showed very little effect on the seminiferous epithelium and

spermatogenesis in the histology preparation by the end of 7 days (Fig.1, Anand et al., 2014).

However, significant alterations in the serum reproductive hormone levels were observed. Rise

in follicle stimulating hormone (FSH) was associated with a significant decline in luteinizing

hormone (LH) and testosterone levels (Anand et al., 2014). EJE rather than NAC

supplementation to the CIS treated rats was more effective in restoring the hormones to near

normal levels (Anand et al., 2014). In contrast, NAC supplementation to CIS treated rats

stimulated a rise in FSH but attenuated both LH and testosterone levels (Fig.2A, B, C) (Anand et

al., 2014). While EJE or NAC, given alone, had no significant effect on serum FSH levels, the

inhibitive effect of NAC on serum LH and testosterone was evident in the backdrop of


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significant reduction of both hormones in CIS+NAC as compared with control levels (Fig.2A, B,

C) (Anand et al., 2014).”

3.4.2 Evaluation of the presence of functional Leydig cells

“Evaluation on the status of functional Leydig cells present in the testis was carried out through

immunolocalization using 3β-hydroxysteroid dehydrogenase (3β-HSD) antibody (Fig.3A) and

also through analysis of gene expression specifically for 3β-HSD and steroidogenic acute

regulatory protein (StAR) (Anand et al., 2014). One time cisplatin treatment significantly

depleted the numbers of functional Leydig cells (Fig.3B) which were restored only after EJE

(Fig.3D) or NAC (Fig.3F) supplementation (Anand et al., 2014). EJE (Fig.3C) or NAC (Fig.3E)

alone demonstrated no significant beneficial effect as the number of functional Leydig cells was

comparable with that of the controls (Fig.3A) (Anand et al., 2014). 3β-HSD, protein (Fig.3G)

and transcript (Fig.3H) levels followed an identical trend with attenuation of expression due to

cisplatin treatment (Anand et al., 2014). Restoration back to normal levels was observed either

with EJE or NAC intervention. While StAR transcripts are favorably upregulated (Fig.3H), its

protein expression (Fig.3G), on the other hand, was comparatively unaffected following EJE or

NAC intervention to CIS treated rats (Tables 3.1, 3.2) (Anand et al., 2014).”

3.4.3 Redox status of testis

“Redox status in the testis was evaluated simultaneously by measuring malondialdehyde, total

glutathione and antioxidant capacity, hemeoxygenase-1 (HO-1) concentration and activities of

antioxidant enzymes (Anand et al., 2014). Expression analysis of few of the antioxidant enzyme

was also studied. A rise in lipid peroxidation following cisplatin treatment coincided with the

increased formation of thiobarbituric acid reactive substances (TBARS, Fig.4A) or HO-1

(Fig.4B) in the testis (Anand et al., 2014). Intervention with either EJE or NAC was able to

contain the rise and to bring it down to control levels (Anand et al., 2014). Cisplatin, on the other

hand, significantly depleted the total glutathione (Fig.5A) and antioxidant capacity (Fig.5B) in

the testis and both parameters were restored after supplementation with either EJE or NAC

(Anand et al., 2014).

The activities of all the antioxidant enzymes, superoxide dismutase (SOD, Fig.6A), catalase

(Fig.6B), glutathione-s-transferase (GST, Fig.7A), glutathione reductase (GR, Fig.7B) and

glutathione peroxidase (GPx, Fig.7C) demonstrated a significant decline in the testis of cisplatin


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treated rats compared to vehicle treated controls (Anand et al., 2014). Complete restoration in

enzyme activities was seen only when EJE or NAC was supplemented to CIS treated rats

(Anand et al., 2014). The efficiency of restoration was not identical when expression analysis of

specific antioxidant enzyme genes was compared with respect to EJE versus NAC. Catalase

protein and transcript levels were attenuated after CIS treatment but found restored after NAC

supplementation. EJE, in contrast, was less effective in the restoration of catalase gene

expression as seen both in western blot and PCR analysis (Fig.6C, D). Besides catalase,

expression analysis of all other antioxidant enzyme genes such as MnSOD, Cu/ZnSOD (Fig. 6C,

D) and GR (Fig. 7D, E), demonstrated a comparable pattern of restoration either with EJE or

NAC supplementation. GST protein expression in western blots too revealed a very much similar

trend (Fig.7 D) (Anand et al., 2014).”

3.4.4 Apoptotic induction of germ and Leydig cells

“The seminiferous epithelium depicted significantly more TdT-mediated

deoxyuridinetriphosphate nick end-labeling (TUNEL) positive germ cells from the CIS treated

group (Fig.8B) compared to vehicle treated controls (Fig.8A) (Anand et al., 2014). This was

further supported from the data on apoptotic index (Fig.8G) representing each category of

treatment (Anand et al., 2014). No apparent increase in germ cell apoptosis was seen with either

EJE (Fig.8C) or NAC (Fig.8E) given alone (Anand et al., 2014). The rise in germ cell apoptosis

in CIS treated rats was significantly brought down following either EJE (Fig.8D, G) or NAC

(Fig.8F,G) supplementation (Anand et al., 2014).

Considering the fact that there was a significant reduction both in serum testosterone and the

number of functional Leydig cells after CIS treatment, a fluorescent based kit was further

utilized to confirm TUNEL positivity, if any, among interstitial cells. Besides germ cells,

TUNEL positive cells were also resolved in the interstitium much more in number in CIS treated

(Fig.9B) compared to other, vehicle treated control (Fig.9A), EJE (Fig.9C) or NAC (Fig.9E)

alone, CIS+EJE (Fig.9D) and CIS+NAC (Fig.9F) groups (Anand et al., 2014). In order to make

a quantitative analysis of the extent of apoptotic induction in Leydig cells, the cells were isolated

and evaluated for TUNEL positivity in vitro. Accordingly, a large percentage (~18%) of Leydig

cells were found TUNEL positive in the CIS (Fig.10B, G) treated as compared to control group

(Fig.10A, G). EJE (Fig.10D, G) or NAC (Fig.10F, G) intervention to CIS treated rats

significantly (p<0.001) attenuated the number of TUNEL positive Leydig cells. Given alone,


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EJE (Fig.10C, G) or NAC (Fig.10E, G) had no significant effect on Leydig cell apoptosis

(Anand et al., 2014).”

3.4.5 Leydig cell function in vitro

“Leydig cell function was assessed through hCG-induced testosterone production in vitro

(Fig.11) (Anand et al., 2014). Steroidogenesis leading to testosterone production in Leydig cells

was seen severely curtailed (~50%) in CIS as compared to the vehicle treated control group.

Individual treatments either with EJE or NAC showed little effect on the hCG-induced

testosterone production (Anand et al., 2014). On the other hand, testosterone production

improved in CIS treated rats receiving EJE (69.5% compared to CIS group) or NAC (60.5%

compared to CIS group) supplementation (Anand et al., 2014).”

3.4.6 Expression analysis of apoptotic markers

“Both upstream and downstream markers of extrinsic, intrinsic and other pathways of metazoan

apoptosis were analyzed using testicular tissue. Caspase 3 activity (Fig.12A) and protein

expression in western blots were seen identically raised following CIS treatment but brought

down to near control levels after EJE or NAC intervention (Anand et al., 2014). The expression

of the cleaved form of DNA repair enzyme, poly ADP ribose polymerase (PARP), demonstrated

a very much identical trend (Fig.12B) (Anand et al., 2014). Extrinsic markers, caspase 8 activity

(Fig.13A) and expression analysis of protein (Fig.13B) and transcript (Fig.13C) levels for

caspase 8, Fas and FasL, all demonstrated a beneficial modulation countering the rise in the

activity and expression of these markers when EJE or NAC was intervened after CIS treatement.

Intrinsic markers, caspase 9 activity (Fig.14A) and expression of Bad (Fig.14B), caspase 9

(Fig.14B, C) and Bax (Fig.14B, C) too displayed a similar trend. Bcl-2, the anti-apoptotic

marker, down regulated (Fig.14B, C) after CIS treatment, restored back to control levels after

EJE or NAC intervention (Fig.14B, C) (Anand et al., 2014).

Positive regulation of cisplatin effect by EJE or NAC was also seen with respect to analysis of

markers in other pathways of apoptosis. Anti-apoptotic Akt and pAkt (Fig.14D), down regulated

as a result of CIS treatment recovered after EJE ( Akt, 31.6%, pAkt, 38%) or NAC (Akt/pAkt,

26.5%) supplementation (Table 3.1) (Anand et al., 2014). NFkB which acted as a pro-apoptotic

factor and up-regulated with cisplatin treatment, was countered and brought down by EJE but not

significantly by NAC supplementation (Fig.14D, Table 3.1) (Anand et al., 2014). No significant


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change was observed for JNK or p-JNK either with cisplatin or any other treatments (Fig.14E)

(Anand et al., 2014). The upstream marker c-Jun, in the JNK pathway, however, was over

expressed in CIS treated group but depleted to near control levels with EJE or NAC intervention

(Fig.14E) (Anand et al., 2014).”

3.5 Discussion

“The above findings substantiate that intervention with either antioxidant, NAC or plant extract,

EJE protects the damage induced by cisplatin in the adult testis. The adverse effect of cisplatin is

not only limited to germ cell apoptosis in the seminiferous epithelium but also extends to impact

Leydig cell survival and function in the interstitium too. The molecular mechanism of action

against cell death due to apoptosis is found mainly channeled through favorable modulation of

marker proteins in the pathways of apoptosis under the backdrop of augmentation of

antioxidative defense in the target tissue (Anand et al., 2014).

Testicular dysfunction is a common long-term sequel of cytotoxic chemotherapy used in

treatment of various types of malignancies. However, the degree of dysfunction is proportional

to duration, dose and the nature of the drug used. Cisplatin chemotherapy has been reported to

produce long-lasting adverse effects on spermatogenesis (Petersen et al., 1994, Howel and

Shalet, 2001). As a heavy metal coordination compound, cisplatin produces cross-links,

including the DNA cross-links that are presumably responsible for its antineoplastic effect

(Boekelheide, 2005). Large scale, short and long term studies in animals confirm identical effects

of cisplatin on spermatogenesis. Cisplatin at a dose of 7 mg/kg bw administered to rats for 5 and

50 days has been reported to affect spermatogenesis; severety being directly proportional to the

duration of treatment. The hypospermatogenesis induced after 5 days of CIS treatment is

supported equally by significant decrease in the testis weight (Atessahin et al., 2006). In the

present study, however, no significant change in the testis weight and spermatogenesis (data not

shown) is observed even after 7 days of one time treatment of the drug (5 mg/kg b.w).

Considering the relatively long spermatogenic cycle in the rat (~50days), histological evaluations

of testis against short-term interventions are unlikely to yield any distinguishable signal in this

regard. Still, the observed differential response of the drug on spermatogenesis may be attributed

to a comparatively lower dose of cisplatin, as utilized in the present study (Anand et al., 2014).

Alterations in the hormonal profile are also common in the individuals receiving cispaltin

treatment. Cisplatin-administered to non-seminomatous testicular cancer subjects depicts a rise


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in gonadotropin but fall in serum testosterone levels (Strumberg et al., 2002). In animals too,

adult rats exposed to cisplatin (7-9 mg/kg b.w intravenously) show a significant attenuation in

serum and intratesticular testosterone 7 days after the exposure. The effect is reportedly

reversible following hCG intervention. Gonadotropin levels, both LH and FSH, however, remain

unaffected (Maines and Mayer, 1985, Maines et al., 1990). In the present study, while serum

testosterone and LH have declined, FSH is estimated significantly higher in the CIS treated

group compared to vehicle treated controls. This is for the first time that such a differential

response for gonadotropins was observed after CIS treatment and may represent a part of the

immediate homeostatic mechanism within the gonadal-pituitary loop for maintaining testicular

function close to normal levels, as presently observed (Anand et al., 2014).

Intervention studies to protect the testicular function from cisplatin-induced damage have earlier

been attempted. From herbal extracts (Amin et al., 2008, Azu etal., 2010, Yildrim et al., 2011) to

chemotherapy protecting compounds such as amifostine (Lirdi et al., 2008), to those with

antioxidant properties, like lycopene (Atessahin et al., 2006a) and melatonin (Atessahin et al.,

2006b) are all reportedly tried mostly in animal studies with variable efficacies but without

utilizing a known recognized antioxidant for comparison. In the present work, NAC, a

recognized antioxidant is taken as a case control and the plant extract EJE was simultaneously

intervened to examine the beneficial effects in comparison with the known antioxidant. EJE has

been shown to possess strong antioxidant properties (Anand et al., 2013; Vasi and Austin, 2009).

It is interesting to note that in the restoration of normalcy for reproductive hormone levels, EJE

is found to be more effective than NAC. NAC supplementation to CIS treated rats fails not only

to bring down the FSH but also to restore the declining LH and testosterone anywhere near to

control levels (Fig.2) (Anand et al., 2014).

The cisplatin-induced decline in serum testosterone is reported to be associated with a decreased

number of LH receptors on Leydig cells in which p450 side-chain cleavage activity has also been

found inhibited (Maines et al., 1990). But, falling testosterone levels may not be only due to

inhibition of Leydig cell function but may also be due to a loss in the number of functional

Leydig cells in situ. This aspect is investigated for the first time in the present study utilizing 3β-

HSD antibody as the marker for identifying functional Leydig cells. CIS administration is seen to

severely deplete the number of functional Leydig cells which are restored to near control levels

only after either EJE or NAC supplementation (Fig.3A-F). That cisplatin primarily effect Leydig

cell function is further supported by the fact that the 3β-HSD gene expression (protein and


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transcript levels) is found significantly downregulated but restored only after antioxidant NAC or

EJE intervention (Fig.3G,H). The CIS-induced attenuation in Leydig cell function is further

confirmed from in vitro studies in which isolated Leydig cells are challenged with hCG and later

assayed for efficiency in the form of testosterone released into the medium. EJE administration

to CIS rats is seen to provide a better protection from cisplatin stimulating a higher (~10% more

than NAC) testosterone production (Fig.11). Despite the restoration in the number of functional

Leydig cells, the failure of NAC restoring serum testosterone in the CIS+NAC group (Fig.2C)

may be due to the relative insensitivity of NAC to serum LH which continues to show no

improvement all through (Anand et al., 2014).

Alteration in the redox status in the testicular cells has been consistent with CIS treatment

(Kandemir et al., 2011, Salem et al., 2012). In the present study, it is extensively evaluated by

biochemical measurement of HO-1 protein, TBARS levels, total antioxidant capacity (TAC) and

total glutathione levels in conjunction with evaluation of activity and expression of antioxidant

enzymes. The effect of EJE in restoring the levels of HO-1 protein, TBARS levels (Fig. 4A) and

TAC (Fig. 5B) in the CIS treated rats is comparable with that of NAC supplementation.

Improvement in the activities of all the antioxidant enzymes analysed in the present study

followed an identical trend (Fig.6 and 7). However, total glutathione, fully recovered to control

levels by EJE, is seen almost doubled with NAC intervention (Fig.5A). Such drastic elevation in

glutathione levels is never beneficial and may be considered as one such adverse effects

associated with NAC treatment (van Klaveren, et al., 1997). In the system presently utilized,

NAC-induced high glutathione manifestation resulting either through toxic effects of cysteine or

through other mechanism/s needs further confirmation in future studies. Analysis of catalase

gene expression, however, reveals an opposite trend as NAC supplementation to CIS treated

animals shows superior restoration in protein and transcript levels than EJE (Fig.6C, D) (Anand

et al., 2014).

Strengthening the antioxidant defense is instrumental for augmentation of cell survival both

within the seminiferous tubule and intertubular regions. While cisplatin-induced germ cell

apoptosis has been reported before (Zhang et al., 2001, Seamen et al., 2003), simultaneous

apoptotic induction in the germ (Figs.8,9) along with Leydig cells (Fig.9) is revealed for the first

time in the present study. The findings are further confirmed with the identification of TUNEL

positive cells in vitro (Fig.10) from CIS-treated rats using the streptavidin-horseradish

peroxidase (HRP)-TUNEL assay which, somehow, has not been sensitive enough to detect the


124

same signal in histological sections (Fig.8). Intervention with either EJE or NAC to CIS-treated

rats has successfully countered the rise in the number of germ (Fig.8G) or Leydig cell (Fig.10G)

apoptosis (Anand et al., 2014).

The dynamics of apoptotic induction are primarily regulated by activities and expressions of

marker proteins associated with the pathway of metazoan apoptosis. It is observed that down-

stream apoptotic markers, caspase 3 (activity and expression) and PARP are significantly

upregulated after CIS treatment. But, intervention with EJE or NAC has helped to counter the

rise and bringing it down to control levels (Fig.12A, B). Identical modulation is seen with respect

upstream markers of extrinsic (casapase 8, Fas and FasL, Fig.13) and intrinsic (Caspase 9, Bcl-2,

Bax and Bad, Fig.14A-C) pathway of apoptosis (Anand et al., 2014).

Analyzing the markers for other pathways of apoptosis, again for the first time, it is seen that Akt

and pAkt, being antiapoptotic, are down regulated after CIS treatment but reversed after EJE or

NAC supplementation (Fig.14D). In the present system, cisplatin is seen inducing

overexpression of NFkB which probably acts as an apoptotic inducer but later downregulated by

EJE or NAC supplementation (Fig.14D). The observation is consistent with findings earlier

reported (Kim et al., 2006). NFkB gets activated by different stimuli and appears to be mediated

through the production of reactive oxygen species (ROS). NFkB exists in the cytosol as a pre-

formed trimeric complex. The P50/P65 protein dimer is associated with an inhibitory protein

known as I-kB. Oxidants trigger a change in the cell that results in phosphorylation of the I-kB

subunit. After I-kB is phosphorylated, a process of the proteolytic digestion of this subunit is

activated. When the inhibitor subunit is dislodged from the P60/P65 heterodimer the activator

NFkB can migrate to the nucleus and bind to DNA, thereby initiating transcription (Blackwell

and Christman, 1997, Finkel, 1998) (Anand et al., 2014).

The tyrosine kinase c-Abl plays an important role in stress response to DNA damaging agents. It

belongs to the nonreceptor tyrosine kinases and contains nuclear localization motifs and nuclear

export signals shuttling between the nucleus and cytoplasm. Nuclear import of c-Abl was shown

to be necessary for DNA damage-induced apoptosis (Preyer et al., 2007). It is activated in

response to cisplatin causing activation of c-Jun-N-terminal kinase (JNK)/stress-activated protein

kinase (SAPK) (Kharbanda et al., 1995). Though in the present system, no significant change in

JNK expression is observed following CIS treatment, it does activate upstream marker protein c-

Jun which is, however, favorably modulated by antioxidant NAC or EJE supplementation

(Fig.14E) (Anand et al., 2014).


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In conclusion, this study clearly indicates that CIS-mediated adverse effects in the testicular

function and cell survival are countered both by the antioxidant NAC or EJE possessing similar

properties by augmenting antioxidant defense and favorably modulating molecular pathways

associated with steroidogenesis and cell death by apoptosis. On the basis of the information

obtained, a model representing the mechanism of NAC versus EJE action ameliorating the

adverse effects of cisplatin on rat testis is proposed (Fig.15). The therapeutic application of the

present findings however need further elucidation in future clinical studies (Anand et al., 2014).”


126

“Fig. 1. Representative testis sections stained with hematoxylin and eosin after (A) saline,

(B) CIS alone one time (single inj. 5mg/kg b.wt.), (C) EJE alone (25 mg/kg b.w), (D) CIS+EJE,

(E) NAC alone (150 mg/kg b.w), and (F) CIS+NAC. No significant change is observed in any of

the treated sections. X400

B C

D E F

A

50 μm 50 μm

50 μm 50 μm

50 μm

50 μm


127

Fig. 2. Serum levels of FSH (A), LH (B) and testosterone (C) on Day 8th of one time (single inj.

5mg/kg b.wt.) cisplatin (CIS) treatment with or without EJE (25 mg/kg b.w)/NAC (150 mg/kg b.w)

supplementation.

* **

##

A

*

*** ***

***

B

** ** *

#

C

Control CIS EJE CIS+EJE NAC CIS+NAC


128

Fig. 3. Detection of the presenc

immunolocalization of 3β-HSD in representative testicular sections from (A) vehicle control, (B)

CIS, (C) EJE alone, (D) CIS+EJE, (E) NAC alone and (F) CIS+NAC. x 400.

B C

D E F

A

50 μm 50 μm 50 μm

50 μm 50 μm 50 μm

3β-HSD

StAR

β-Actin

Cisplatin - + - + - + - + - + - +

EJE - - + + - - - - + + - -

NAC - - - - + + - - - - + +

547 bp

500 bp

539 bp

42 kDa

30 kDa

42 kDa

G H


129

Fig. 4. Evaluation of redox status in testes of CIS treated rats with or without EJE/NAC

supplementation.

***

### ###

A

***

## #

B

** ###

###

** ###


130

Fig. 5. Evaluation of redox status in testes of CIS treated rats with or without EJE/NAC

supplementation.

A

**

# ##

B

***

### ###

### ###

** ###

** ###


131

Fig. 6. Assessment of activities and expression of antioxidant enzymes in CIS treated rats with or

without EJE/NAC supplementation.

A

***

### ### ###

###

B

***

*

###

** ###

###

Cu/ZnSOD

MnSOD

Catalase

β-Actin

CIS - + - + - + - + - + - +

EJE - - + + - - - - + + - -

NAC - - - - + + - - - - + +

C D

277bp

326 bp

970 bp

539 bp

23 kDa

25 kDa

64 kDa

42 kDa

*** ###


132

Fig. 7. Assessment of activities and expression of antioxidant enzymes in CIS treated rats with or

without EJE/NAC supplementation.

A

***

### ### $$

### $$

*** ### $$$

B

***

### ###

## ##

C

**

## ## # #

GST

GR

β-Actin CIS - + - + - + - + - + - +

EJE - - + + - - - - + + - -

NAC - - - - + + - - - - + +

D E

296 bp

539 bp

28 kDa

65 kDa

42 kDa


133

Fig. 8. Analysis of TUNEL positive cells () in testicular sections of rats treated with (A)

saline, (B) CIS, (C) EJE alone, (D) CIS+EJE, (E) NAC alone and (F) CIS+NAC. x 400

B C

D E F

A

50 μm

50 μm

50 μm

50 μm

50 μm

50 μm

***

### ### ###

###

G


134

Fig. 9. Fluorescein based detection of TUNEL positive cells in the seminiferous tubule () and

interstitium ().

A B C

E F D

50

50 μm 50 μm 50 μm

50 μm 50 μm


135

Fig. 10. Assay of Leydig cell TUNEL positivity in vitro. Isolated Leydig cells after 7 days of (A)

vehicle control, (B) CIS alone, (C) EJE alone (D) CIS+EJE, (E) NAC alone and (F) CIS+NAC

treatment.

B A C

D E F

50 μm 50 μm 50 μm

50 μm 50 μm 50 μm

G

***

### ### ###

###


136

Fig. 11. The hCG induced testosterone production from Leydig cells in vitro is stimulated in

CIS+EJE (~60%) versus CIS+NAC (~43%) group.

**

*

*

##

##


137

Fig. 12. (A) Rise in the activity and (B) expression of caspase-3 and cleaved PARP in CIS

treated group is brought down by EJE or NAC supplementation.

***

###

###

### ###

A

Caspase 3

PARP

β-Actin

Cisplatin - + - + - +

EJE - - + + - -

NAC - - - - + +

B 35 kDa

17 kDa

116 kDa

85 kDa

42 kDa


138

Fig. 13. EJE or NAC modulation of extrinsic pathway of apoptosis.

***

### ###

### ###

A

Cisplatin - + - + - + - + - + - +

EJE - - + + - - - - + + - -

NAC - - - - + + - - - - + +

Caspase 8

Fas

Fas L

β-Actin

B C

48 kDa

48 kDa

40 kDa

42 kDa

123 bp

351 bp

238 bp

539 bp


139

Fig. 14. EJE or NAC modulation of intrinsic and other pathways of apoptosis.

***

### ### ### ###

A

Cisplatin - + - + - + - + - + - +

EJE - - + + - - - - + + - -

NAC - - - - + + - - - - + +

Bad

Caspase 9

Bcl-2

Bax

β-Actin

B C

132 bp

293 bp

483 bp

539 bp

25 kDa

46 kDa

35 kDa

27 kDa

23 kDa

42 kDa

Akt

pAkt

NFkB

β-Actin


EJE - - + + - -

NAC - - - - + +

D

62 kDa

60 kDa

65 kDa42

kDa

E JNK

pJNK

c-Jun

β-Actin


EJE - - + + - -

NAC - - - - + +

54 kDa

46 kDa

54 kDa

46 kDa

39 kDa

42 kDa


140

Fig. 15. A proposed model depicting the mechanism of NAC versus EJE action ameliorating the

adverse effects of cisplatin on rat testis. EJE modulation of NFκB has been shown (----).

Upregulation (), downregulation () and inhibition ( | ) are indicated.”


141

Table 3.1: Densitometric analysis showing ratio of different protein expression to β-Actin ±

SEM.

Proteins

Protein/β-Actin Ratio

Control

Treatment

Cisplatin (5mg/kg

b.wt)

EJE (25mg/kg

b.wt)

Cisplatin (5mg/kg

b.wt) + EJE (25mg/kg

b.wt)

NAC (150 mg/kg b.wt)

Cisplatin (5mg/kg

b.wt)+ NAC (150mg/kg

b.wt) 3β-HSD 0.85+0.0088

0.7134 + 0.009*

0.9453 + 0.042 ###

0.951 + 0.031 ###

0.854 + 0.024 #

0.921 + 0.003 ###

StAR 0.9421+0.009

0.8826+ 0.006

1.0334+ 0.047

0.9696+ 0.065 0.9907+ 0.006 0.9256+ 0.003

Cu/Zn SOD

0.8209+0.008

0.5678+ 0.004 ***

0.7392+ 0.033 #

0.9061+ 0.061 ###

0.7755+ 0.004 ##

0.7774+ 0.002 ##

Mn SOD 0.9224+0.008

0.7887+ 0.005

1.0704+ 0.048 ##

1.0725+ 0.072 ##

0.9767+ 0.006 #

0.9454+ 0.003

Catalase 0.9002+0.008

0.6532+ 0.024 ***

0.8214+ 0.037 #

0.8214+ 0.055 #

0.7393+ 0.004 *

0.836+ 0.003 ##

GST 0.4582+0.004

0.3706+ 0.003 ***

0.4352+ 0.020 ##

0.4727+ 0.001 ###

0.5676+ 0.003 ***, ###

0.374+ 0.001 ***

GR 0.8102+0.006

0.7457+ 0.014

0.9422+ 0.008 **, ###

0.9783+ 0.033 **, ###

0.8549+ 0.031 0.8362+ 0.017 #

Caspase 3 0.7670+ 0.007

0.9148+ 0.006 **

0.7336+ 0.006 ###

0.7090+ 0.047 ###

0.656+ 0.004 *, ###

0.584+ 0.002 ***, ###

PARP 0.8421+ 0.021

0.9422+ 0.007 ***

0.8704+ 0.013 ##

0.8612+ 0.001 ##

0.7431+ 0.004 ***, ###

0.8008+ 0.002 ###

Caspase 8 0.6676+ 0.006

0.7778+ 0.005 *

0.7074+ 0.032

0.6542+ 0.044 #

0.6091+ 0.004 ##

0.6583+ 0.002 #

Fas 0.6568+ 0.007

0.8662+ 0.006 ***

0.6183+ 0.028 ###

0.6422+ 0.017 ###

0.6592+ 0.004 ###

0.8188+ 0.003 ***

FasL 0.9699+ 0.009

1.1857+ 0.008 **

0.8067+ 0.036 *, ###

0.8841+ 0.059 ###

0.9158+ 0.005 ###

0.8912+ 0.003 ###

Caspase 9 0.7883+ 0.008

0.9474+ 0.007 **

0.7909+ 0.009 ##

0.7996+ 0.054 ##

0.9863+ 0.006 ***

0.9145+ 0.003 *

Bcl-2 0.8292+ 0.008

0.6894+ 0.004 *

0.8819+ 0.009 ##

0.9419+ 0.063 ###

0.8623+ 0.005 ##

0.8322+ 0.003 #

Bax 0.73+ 0.007

0.918+ 0.006 ***

0.750+ 0.004 ###

0.7788+ 0.001 ***, ###

0.9870+ 0.006 ***, ###

0.8292+ 0.003 ***, ###

Bad 0.39+ 0.004

0.4560+ 0.003

0.4103+ 0.019

0.4571+ 0.031 0.4919+ 0.003 **

0.4248+ 0.002


142

Bak 0.6935+ 0.006

0.8138+ 0.006

0.7614+ 0.035 **

0.8688+ 0.058 ***

0.9335+ 0.005 0.7976+ 0.003

Akt 0.9644+ 0.009

0.7557+ 0.022 ***

1.0205+ 0.046 ###

0.9965+ 0.035 ###

0.9622+ 0.005 ##

0.96+ 0.003 ##

pAkt 1.1001+ 0.011

0.8689+ 0.006 *

1.1232+ 0.051 ##

1.1667+ 0.078 ##

1.0677+ 0.006 #

1.0681+ 0.003 #

NFκB 0.6848+ 0.006

0.8396+ 0.006 **

0.7450+ 0.034

0.7004+ 0.047 #

0.6997+ 0.004 #

0.8777+ 0.003 **

JNK 0.8647+ 0.008

0.8839+ 0.006

0.9654+ 0.044

0.9391+ 0.063 0.9095+ 0.005 0.9272+ 0.003

pJNK 0.6566+ 0.006

0.6168+ 0.004

0.4807+ 0.022

0.4728+ 0.032 0.5922+ 0.003 0.8181+ 0.002

c-Jun 0.3523+ 0.003

0.4868+ 0.003 ***

0.3677+ 0.017 ###

0.4164+ 0.028 *, #

0.4124+ 0.002 #

0.4414+ 0.001 **,#

*p<0.05, **p<0.01, ***p<0.001 compared to untreated control

#p<0.05, ##p<0.01, ###p<0.001 compared to Cisplatin treated sample


143

Table 3.2: Densitometric analysis of RT-PCR showing ratio of gene expression to β-Actin ±

SEM.

Genes

Target Gene/β-Actin Ratio

Control

Treatment

Cisplatin (5mg/kg

b.wt)

EJE (25mg/kg

b.wt)

Cisplatin (5mg/kg b.wt) +

EJE (25mg/kg

b.wt)

NAC (150 mg/kg b.wt)

Cisplatin (5mg/kg

b.wt)+ NAC

(150mg/kg b.wt)

3β-HSD 0.8985+ 0.012

0.6077+ 0.012***

0.9181+ 0.037###

0.8863+ 0.023###

0.9447+ 0.05###

0.8368+ 0.027###

StAR 0.5191+ 0.003

0.4156+ 0.004***

0.4671+ 0.017*, #

0.6038+ 0.015***,

###

0.5497+ 0.007###

0.5305+ 0.001###

Cu/Zn SOD 1.0822+ 0.018

0.6125+ 0.045***

1.2032+ 0.041###

1.0632+ 0.035###

1.1347+ 0.015###

1.1464+ 0.006###

Mn SOD 0.8082+

0.004 0.6347+

0.007*** 0.8112+

0.029### 0.7256+

0.018*, # 0.8381+

0.012###

0.8854+ 0.002*,

###

Catalase 0.7071+

0.003 0.385+ 0.004***

0.5994+ 0.021***,

###

0.4672+ 0.012***,

##

0.6781+ 0.009###

0.7215+ 0.001###

GPx 0.8826+ 0.004

0.6004+ 0.006***

0.6182+ 0.022***

0.6122+ 0.015***

0.5171+ 0.007***,

##

0.5744+ 0.001

GR 0.6056+ 0.004

0.4520+ 0.025***

0.6457+ 0.022###

0.6046+ 0.019###

0.5582+ 0.011###

0.5534+ 0.009###

Caspase 8 0.6214+ 0.003

1.0564+ 0.012***

0.8951+ 0.032***,

###

0.9707+ 0.003***,

#

0.9942+ 0.014***

0.6723+ 0.002###

Fas 0.6145+ 0.005

0.7982+ 0.024**

0.6606+ 0.024##

0.6711+ 0.031##

0.6213+ 0.012###

0.6394+ 0.002##

FasL 0.6315+ 0.015

0.8110+ 0.005**

0.6958+ 0.027#

0.6369+ 0.026##

0.5867+ 0.031###

0.6112+ 0.028###

Caspase 9 0.5778+ 0.018

0.9183+ 0.011***

0.5497+ 0.032###

0.5742+ 0.027###

0.4905+ 0.027*,

###

0.5522+ 0.025###

Bcl-2 0.7367+ 0.032

0.5482+ 0.009***

0.7700+ 0.026###

0.7839+ 0.019###

0.7845+ 0.024###

0.6945+ 0.002*, ##

Bax 0.6058+ 0.003

0.7354+ 0.008***

0.6344+ 0.023###

0.5983+ 0.015###

0.7211+ 0.010***

0.5551+ 0.001###

*p<0.05, **p<0.01, ***p<0.001 compared to untreated control

#p<0.05, ##p<0.01, ###p<0.001 compared to Cisplatin treated sample

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