Food and Chemical Toxicology 67 (2014) 176–186
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Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Propolis Attenuates Doxorubicin-Induced Testicular Toxicity in Rats
Sherine M. Rizk a, Hala F. Zaki b,⇑, Mary A.M. Mina c
a
Department of Biochemistry, Faculty of Pharmacy, Cairo University, Egypt
Department of Pharmacology & Toxicology, Faculty of Pharmacy, Cairo University, Egypt
c
Department of Histology, Faculty of Medicine, Cairo University, Egypt
b
a r t i c l e
i n f o
Article history:
Received 12 August 2013
Accepted 21 February 2014
Available online 1 March 2014
Keywords:
Propolis extract
Doxorubicin
Testicular function
Oxidative stress
Apoptosis
Inflammation
a b s t r a c t
Doxorubicin (Dox), an effective anticancer agent, can impair testicular function leading to infertility. The
present study aimed to explore the protective effect of propolis extract on Dox-induced testicular injury.
Rats were divided into four groups (n = 10). Group I (normal control), group II received propolis extract
(200 mg kg1; p.o.), for 3 weeks. Group III received 18 mg kg1 total cumulative dose of Dox i.p. Group
IV received Dox and propolis extract. Serum and testicular samples were collected 48 h after the last
treatment. In addition, the effects of propolis extract and Dox on the growth of solid Ehrlich carcinoma
in mice were investigated. Dox reduced sperm count, markers of testicular function, steroidogenesis
and gene expression of testicular 3b-hydroxysteroid dehydrogenase (3b-HSD), 17b-hydroxysteroid dehydrogenase (17b-HSD) and steroidogenic acute regulatory protein (StAR). In addition, it increased testicular oxidative stress, inflammatory and apoptotic markers. Morphometric and histopathologic studies
supported the biochemical findings. Treatment with propolis extract prevented Dox-induced changes
without reducing its antitumor activity. Besides, administration of propolis extract to normal rats
increased serum testosterone level coupled by increased activities and gene expression of 3ß-HSD and
17ß-HSD. Propolis extract may protect the testis from Dox-induced toxicity without reducing its anticancer potential.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
The anthracycline doxorubicin (Dox), or Adriamycin, is the anticancer drug of choice in the treatment of many solid tumors. Its
application, however, carries the risk of serious dose-dependent
toxicity to other non-target tissues including the testis (Malekinejad et al., 2012; Ahmed et al., 2013). Dox could strikingly impede
spermatogenesis leading eventually to infertility (Howell and
Shalet, 2005; Yeh et al., 2007). The mechanism responsible for
Dox-induced testicular injury is not yet completely clear, but
findings from several studies strongly suggest oxidative stress, lipid peroxidation (Atessahin et al., 2006; Yeh et al., 2007) and cellular apoptosis (Shinoda et al., 1999) as major causes. Based on
this concept, a variety of antioxidant or anti-apoptotic agents have
been employed to counteract Dox-induced testicular damage
(Jahnukainen et al., 2001; Hou et al., 2005; Malekinejad et al.,
2012). Nevertheless, so far there is still no single agent proven
effective enough to prevent or reverse this adverse effect.
⇑ Corresponding author. Address: Faculty of Pharmacy, Cairo University, Kasr ElEini Street, P.O. 11562, Egypt. Tel.: +20 2 24503383, +20 1001040447; fax: +20 2
24073411.
E-mail address: [email protected] (H.F. Zaki).
http://dx.doi.org/10.1016/j.fct.2014.02.031
0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
Propolis is an adhesive, resinous substance collected, transformed and used by honeybees to seal holes in their honeycombs,
smooth out the internal walls, and protect the entrance of intruders. Honeybees collect the resin from cracks in the bark of trees and
leaf buds. They bring propolis back to the hive, where it is modified
and mixed with other substances, including bees’ own wax and salivary secretions (Ghisalberti, 1979). Propolis has been used in folk
medicine all over the world. It has anti-inflammatory, immunoregulatory, bacteriostatic, and antibacterial activities (Tosi et al., 2007;
Bueno-Silva et al., 2013). Propolis extract presents low toxicity to
experimental animals, with LD50 higher than 7 g/kg for mice (Dantas et al., 2006). It has strong cytoprotective effect against different
exogenous toxic stimuli (Bhadauria and Nirala, 2009; Boutabet
et al., 2011). Egyptian propolis is rich in aromatic acids, aromatic
acid esters, flavonoids, and some triterpenoids (Abd El Hady and
Hegazi, 2002). Many of the reported pharmacological effects of
propolis extract reside in its high content of caffeic acid, caffeic
acid phenethyl ester (CAPE) and polyphenolic compounds (Ozturk
et al., 2012; Akyol et al., 2013; Búfalo et al., 2013; Szliszka and Krol,
2013).
To determine whether propolis extract could attenuate Dox-induced apoptosis and oxidative stress in testicular tissue, the present study was designed to examine the effects of propolis extract
S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186
177
on pathological and molecular biological abnormalities induced by
Dox in rat testis. The study was extended to examine the anticancer effects of Dox when combined with propolis extract using solid
Ehrlich carcinoma (SEC) model in mice. The results of the current
study could clarify the role of propolis extract in prevention of this
serious side effect associated with Dox treatment.
2.5.2. Determination of testicular oxidative stress markers
Malondialdehyde (MDA), as an index for lipid peroxidation, was determined in
testicular homogenate by detecting the absorbance of thiobarbituric acid reactive
substances at 532 nm (Mihara and Uchiyama, 1978). Another aliquot of the testicular homogenate was mixed with equal volume with 10% metaphosphoric acid and
centrifuged at 1000g. The resulting protein-free supernatant was used for the determination of reduced glutathione (GSH) according to the method described by Beutler et al. (1963).
2. Materials and methods
2.5.3. Determination of the extent of Dox-induced hemorrhage
Another portion of the testis from each animal was homogenized in 10 mL of
5 mM Tris HCl (pH 7.0), containing 1 mM MgCl2 and 100 mM CaCl2. The homogenate was centrifuged at 3000g for 30 min. One milliliter aliquot of the supernatant
fluid was diluted with 5 mL of the homogenizing buffer. The absorbance of homogenate at 405 nm was determined spectrophotometrically (Niewenhuis and Prozialeck, 1987).
2.1. Animals
Male Wistar albino rats weighing 160–180 g and female Swiss albino mice
weighing 20–22 g were provided by the breeding unit of the Egyptian organization
for biological product and vaccines (Helwan, Egypt). They were housed under controlled conditioning (25 ± 1 °C constant temperature, 55% relative humidity, 12 h
lighting cycle), and received standard pelleted diet and water ad libitum during
the study period. The study complies with the Guide for Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996) and was approved by the Ethics Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University.
2.2. Preparation of propolis extract
Fifty grams of the resinous material of Egyptian propolis (obtained from Dakahlia Governorate, Egypt) was cut into small pieces and extracted with 600 ml of 80%
(v/v) ethanol at 60 °C for 30 min. After extraction, the mixture was centrifuged and
the supernatant was evaporated to complete dryness under vacuum at 40 °C (Franchi et al., 2012). The yield of dried residue was about 61.1% (w/w) and it was kept at
4 °C for further use. Aqueous suspension of propolis was prepared in 1% gum acacia
suspension, and orally administered to the animals for 3 weeks in a dose of
200 mg kg1 (Bhadauria and Nirala, 2009).
2.3. Experimental design and protocol
Forty male Wistar rats were divided into four groups consisting of ten animals
in each group. Group I: animals received distilled water as a vehicle for 21 days and
normal saline (i.p.) on 8th, 10th, 12th, 15th, 17th and 19th day (normal control). Group
II: animals received propolis extract (200 mg kg1, p.o.) 5 days/week for 21 days.
Group III: control Dox received 18 mg kg1 total cumulative dose of Dox on six divided doses (3 mg kg1, i.p.) on 8th, 10th, 12th, 15th, 17th and 19th day (Jahnukainen
et al., 2001). Group IV: animals received propolis extract (200 mg kg1, p.o.) 5 days/
week for 21 days and Dox (3 mg kg1, i.p.) on 8th, 10th, 12th, 15th, 17th and 19th day.
Animals were weighed and sacrificed by decapitation 48 h after the last treatment.
Treatment schedule was designed to start propolis extract prophylactically one
week before Dox injection and for another 2 weeks concurrently with Dox.
2.4. Analysis of blood samples
Blood samples were collected, and the sera were separated and kept at 20 °C
till determination of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Serum level of testosterone was assayed using automated
chemiluminescence’s immunoassay system (ADVIA Centaur; Bayer Vital, Fernwald,
Germany), while serum LH and FSH were assayed using rat LH and FSH ELISA kits
(Shibayagi Co., Japan) according to manufacturer instructions.
2.5. Analysis of tissue samples
The testes and the epididymis were removed and cleared of adhering connective tissue. The left side of testis was rinsed in ice-cold saline, weighed and homogenized in cold physiological saline to give 10% homogenate (using Potter–Elvejhem
glass homogenizer). In addition two sections from the right side of testis were prepared using flash freezing technique for PCR and histological processing. Aliquots of
the testicular homogenate were suitably processed for the assessment of the following parameters.
2.5.1. Determination of testicular marker enzymes
The first portion of the testicular homogenate was mixed with 0.1 M Tris buffer
(pH 8.1) and centrifuged at 105,000g at 4 °C for 45 min using Dupont Sorvall Combiplus ultracentrifuge. The resulting cytosolic fraction was used for the colorimetric
determination of the activities of acid phosphatase (ACP), alkaline phosphatase
(ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glucose-6-phosphate dehydrogenase (G6PD) based on the
instructions of kits manufacturers. In addition, sorbitol dehydrogenase (SDH) was
measured according to the method of Chauncey et al. (1988). Protein content was
measured according to the method of Lowry et al. (1951).
2.5.4. Measurement of interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-a)
contents and myeloperoxidase (MPO) activity in the rat testis
Testicular homogenate was ultracentrifuged at 105,000g for 1 h, and the supernatant was stored at 80 °C until assay. Tissue contents of IL-4 and TNF-a were assayed using rat IL-4 and TNF-a ELISA kits supplied by Wuhan Eiaab Science Co.,
China and Labs Biotechnology Inc., Canada, respectively. MPO activity was measured as previously described by Bradley et al. (1982) to assess neutrophil
infiltration.
2.5.5. Determination of testicular 3b-hydroxysteroid dehydrogenase (3b-HSD) and 17bhydroxysteroid dehydrogenase (17b-HSD) activities
Testicular 3b-HSD and 17b-HSD activities were measured according to the
methods of Talalay (1962) and Jarabak et al. (1962). An aliquot of the testicular tissue homogenate was mixed with equal volume of homogenizing fluid containing
30% spectroscopic-grade glycerol, 10 mmol potassium phosphate, and 2 mM EDTA
followed by centrifugation at 10,000g for 30 min in an ultracentrifuge at 4 °C. One
milliliter of the supernatant was mixed with 100 lmol sodium pyrophosphate buffer (pH, 8.9), 0.9 ml bidistilled water, and 30 lg dehydroepiandrosterone (dissolved
in ethanol) making up the incubation mixture to a total of 3 mL. 3b-HSD activity
was measured after the addition of 0.5 lmol NAD+ to the tissue supernatant mixture at 340 nm. The same supernatant collected for testicular assay of 3b-HSD
was used for assaying of 17b-HSD activity. One milliliter of the supernatant was
mixed with 440 lmol sodium pyrophosphate buffer (pH 10.2), 25 mg crystalline
bovine albumin, and 0.3 lmol testosterone bringing the incubation mixture to a total of 3 mL. Enzyme activity was measured after the addition of NADP+ (1.1 lmol) to
the supernatant mixture at 340 nm.
2.5.6. 3b-HSD; 17b-HSD and steroidogenic acute regulatory protein (StAR) gene
expression assay
2.5.6.1. RNA extraction and reverse transcription. Total RNA extraction from testis
was done by using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration was measured with a spectrophotometer
(260/280 ratio of all samples was over 1800). The isolated total RNA was reversetranscribed into complementary DNA (cDNA) using high capacity cDNA reverse
transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions and all products were stored at 20 °C.
2.5.6.2. Real-Time PCR. Gene expressions were analyzed by quantitative RT-PCR
using the SYBR Green PCR Master MIX (Applied Biosystems, California, USA) with
the ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City,
CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The sense and anti-sense primers were as follows: for rat 3b-HSD, 50 CCAGTGTATGTAGGCAATGTGGC-30 (sense) and 50 -CCATTCCTTGCTCAGGGTGC-30
(antisense); for rat 17b-HSD, 50 -TTCTGCAAGGCTTTACCAGG-30 (sense) and 50 -ACAAACTCATCGGCGGTCTT-30 (antisense); for rat StAR, 50 -ATGCCTGAGCAAAGCG
GTGTC-30 (sense) and 50 -CAAGTGGCTGGCGAACTCTATCTG-30 (antisense); for rat
GAPDH, 50 -CCATGGAGAAGGCTGGGG-30 (sense) and 50 -CAAAGTTGTCATGGATGACC-30 (antisense). The thermal cycle protocol consisted of initial denaturation
at 95 °C for 10 min followed by 40 cycles with 30 s denaturation at 95 °C and 30 s
annealing/extension at 60 °C.
2.5.6.3. Data analysis. Gene expression was normalized to the endogenous control
GAPDH, and fold changes in the genes of interest were determined using the comparative threshold cycle (Ct) method (Pfaffl, 2001).
2.6. Determination of epididymal sperm count
Spermatozoa in the epididymis were counted according to the method of Yokoi
et al. (2003). Briefly, the epididymis was minced with anatomical scissors in 5 ml of
physiological saline, placed in a rocker for 10 min, and incubated at room temperature for 2 min. The supernatant fluid was diluted 1:100 with a solution containing
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5 g sodium bicarbonate, 1 ml formalin (35%) and 25 mg eosin per 100 ml of distilled
water. Total sperm number was determined with a hemocytometer. Approximately
10 ll of the diluted sperm suspension was transferred to each counting chamber
and was allowed to stand for 5 min for counting under a light microscope at
200 magnification.
2.7. Histological studies
The testis were removed and kept in 10% formol saline for 24 h, dehydrated in
ethanol and embedded in paraffin. Sections were cut at 5 micron thickness,
mounted on slides and stained with hematoxylin and eosin (for histological and
morphometric studies). Some sections were mounted on positive charged slides
for immunohistochemical studies.
2.8. Immunohistochemistry staining for Fas-L, and caspase-3
The pro-apoptotic factor Fas-L is a TNF related type II membrane protein and is
expressed in various tissues and cells. Fas-L binds to its receptor (Fas) thus inducing
apoptosis (Galvao et al., 2010). Immunohistochemistry to active caspase-3 is commonly used for apoptosis detection (Bressenot et al., 2009). Tissue sections were
deparaffinized, rehydrated and treated with 10% hydrogen peroxide to reduce
endogenous peroxidase. For antigen retrieval tissue sections were boiled in citrate
buffer pH 6.0 then cooling was allowed. Sections were incubated with the primary
antibodies; Fas L; a rabbit polyclonal antibody (Neo Markers Laboratories, Westinghouse, USA) and caspase-3; a rabbit polyclonal antibody (Neo Markers Laboratories,
Westinghouse, USA). Then sections were incubated with the secondary antibody a
biotinylated goat immunoglobulin followed by streptavidin biotin complex. The site
of the reaction was visualized by adding diaminobenzidine HCl which is converted
into brown precipitate by peroxidase. Counterstaining was performed using
Meyer’s hematoxylin.
2.9. Morphometric measurements
Using the computer assisted software Leica Qwin 500 LTD image analyzer, the
following parameters were measured:
1. Mean diameters of seminiferous tubules in hematoxylin and eosin stained sections were measured using the interactive measuring menu. They were measured in 10 non-overlapping fields at a magnification 100 for each specimen.
2. Mean area percent of Fas-L and caspase-3 immunoreactivities were measured
in immunostained sections using the color detect menu. They were measured
in 10 non-overlapping fields for each specimen of all groups at a magnification
400 in relation to a standard measuring frame.
2.10. Effects on the growth of SEC in mice
In this experiment, 2 106 Ehrlich ascites carcinoma cells were transplanted
subcutaneously in the right thigh of the lower limb of each mouse. Mice with a palpable solid tumor mass that developed within 7 days after implantation were divided into four groups, 6 animals each, and received the same doses of propolis
extract and Dox, previously mentioned, for 2 weeks. The change in tumor size
was measured every 3 days using a Vernier caliper and was calculated using the formula adapted from Osman et al. (1993).
Tumor volume ðmm3 Þ ¼ 0:52 AB2
where A is the minor tumor axis and B is the major axis.
2.11. Statistical analysis
Data are expressed as mean ± SE. Comparisons between different groups were
done using one way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test using the software GraphPad InStat. A probability level of
less than 0.05 was accepted as statistically significant.
3. Results
3.1. Effects on markers of testicular function
Administration of Dox was associated with reduction in testicular activities of LDH, SDH, ACP and G6PD by 39.55%, 31.52%,
37.28 and 30.23%, respectively parallel to increased activities of
ALT and AST by 26.83% and 41.97%, respectively as compared to
the normal control group. Treatment of rats by propolis extract
attenuated Dox-induced changes in the aforementioned parameters and normalized testicular function markers (Table 1).
3.2. Effects on steroidogenesis
Dox resulted in marked decrease in serum testosterone, FSH
and LH levels by 83.37%, 52.38% and 45.56%, respectively as compared to normal control group (Fig. 1). To ascertain the possible
mechanism of the suppressed testosterone production following
Dox administration, the expressions of testicular key androgenic
enzymes like 3ß-HSD and 17ß-HSD along with StAR, a prime regulatory protein for testosterone biosynthesis in testis were studied.
Dox decreased the activities of 3ß-HSD and 17ß-HSD by 76.53%
and 84.25%, respectively parallel to decreased gene expression of
3ß-HSD, 17ß-HSD and StAR by 67.06%, 71.30% and 57.45%, respectively as compared to the normal control group (Fig. 2). Administration of propolis extract prevented Dox-induced decreases in
serum levels of testosterone, FSH and LH as well as activities or
gene expression of testicular 3ß-HSD, 17ß-HSD and StAR indicating the protective role of propolis on testicular androgenic disorders of Dox exposure (Figs. 1 and 2). Moreover, administration of
propolis extract to normal rats significantly increased serum testosterone level by 28.59 (Fig. 1A) coupled by increased activities
of testicular 3ß-HSD and 17ß-HSD by 46.68% and 43.41%, respectively (Fig. 2A) and increased gene expression of both enzymes
by 36.11% and 49.89% (Fig. 2B), respectively as compared to the
normal control group.
3.3. Sperm count and morphometric results
Parallel to the observed decrease in testicular and steroidogenesis markers, Dox reduced sperm count and the diameter of seminiferous tubules by 52.51% and 28.87%, respectively (Fig. 3A–B)
and increased testicular hemorrhage by 1.62 folds (Fig. 3C) as compared to normal control group. Such changes were largely attenuated in the group receiving propolis extract where sperm count
and the diameter of seminiferous tubules were normalized and
testicular hemorrhage was reduced by 38.24% (Fig. 3A–C). Moreover, administration of propolis extract to normal rats increased
sperm count by 31.20% as compared to normal control group
(Fig. 3A).
3.4. Hematoxylin and eosin stained sections
Examination of testicular sections of control group showed the
normal structure of seminiferous tubules. They were lined with a
thick layer of uniformly arranged spermatogenic cells in variable
stages of maturation; spermatogonia, spermatocytes, spermatids
and sperms in addition to Sertoli cells. The interstitium contained
small scattered groups of Leydig cells (Fig. 4A). Testicular sections
of rats which received propolis extract showed normal seminiferous tubules lined with spermatogenic cells at different stages of
maturation (Fig. 4B).
In Dox-treated rats, sections of the testes showed disrupted
spermatogenic structure in many seminiferous tubules, shedding
and degeneration of many spermatogenic cells (Fig. 4C). Many tubules showed detached primary spermatocytes with dark pyknotic
condensed nuclei and increased cytoplasmic eosinophilia, few
elongated spermatids and many apoptotic spematids shedded inside the lumen. Sertoli cells appeared disrupted (Fig. 4D).
Sections of rat testis which received Dox and propolis extract
showed few tubules exhibiting detachment of germ cells from
the basement membrane (Fig. 4E). Most of the seminiferous tubules showed organized epithelium containing various cell types,
1ry spermatocyte, spermatids, and sperms. Germinal epithelium
exhibited detachment in some areas, Few spermatocytes showed
pyknotic nuclei (Fig. 4F).
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Table 1
Effect of propolis extract on testicular markers in normal and doxorubicin-treated rats.
Testicular markers (U/mg protein)
LDH
SDH
ACP
ALP
ALT
AST
G6PD
Control
5.07 ± 0.41
4.95 ± 0.38
130.19 ± 4.78
147.83 ± 5.82
79.64 ± 4.77
79.78 ± 6.38
7.08 ± 0.39
Propolis
4.58 ± 0.26
5.14 ± 0.37
116.39 ± 6.49
154.93 ± 6.39
78.03 ± 3.61
77.13 ± 3.69
8.95 ± 0.43a
Doxorubicin
Doxorubicin + propolis
a,b
3.07 ± 0.35
3.39 ± 0.29a,b
81.65 ± 8.28a,b
129.12 ± 5.87b
101.01 ± 5.63a,b
113.26 ± 10.47a,b
4.94 ± 0.39a,b
4.86 ± 0.37c
5.34 ± 0.27c
110.75 ± 7.60c
157.08 ± 7.73c
74.42 ± 4.84c
83.61 ± 7.61c
8.02 ± 0.48c
LDH: lactate dehydrogenase; SDH: sorbitol dehydrogenase; ACP: acid phosphatase; ALP: alkaline phosphatase; ALT: alanine transaminase; AST: aspartate transaminase;
G6PD: glucose-6-phosphate dehydrogenase.
Data are expressed as mean ± SE (n = 8–10).
a
Significantly different from normal control group at p < 0.05.
b
Significantly different from propolis extract-treated group at p < 0.05.
c
Significantly different from doxorubicin control group at p < 0.05.
Fig. 1. Effect of propolis extract on testicular contents of: (A) testosterone, (B) follicle stimulating hormone (FSH) and luteinizing hormone (LH) in normal and doxorubicintreated rats. Data are expressed as mean ± SE (n = 8–10). a Significantly different from normal control group at p < 0.05. b Significantly different from propolis extract-treated
group at p < 0.05. c Significantly different from doxorubicin control group at p < 0.05.
3.5. Effect on oxidative stress markers
Administration of Dox, in the current study, increased testicular
MDA content by 81.93% parallel to reduced testicular GSH content
by 46.20% as compared to normal rats (Fig. 5). Pretreatment with
propolis extract prevented Dox-induced changes in both MDA
and GSH contents (Fig. 5).
3.6. Effect on MPO, TNF-a and IL-4
Dox administration resulted in marked increase in testicular
MPO activity and TNF-a content by 61.10% and 115.76%, respectively (Fig. 6A–B) parallel to decreased content of IL-4 by 38.37%
(Fig. 6C) as compared to normal control group. Such changes were
prevented by pretreatment with propolis extract (Fig. 6A–C).
3.7. Effect on caspase-3 and Fas-L
Moreover, Dox increased testicular activities of caspase-3 and
Fas-L by 7.19 and 17.91 folds, respectively as compared to normal
control group (Fig. 7). Pretreatment of rats by propolis extract decreased testicular activities of caspase-3 and Fas-L by 51.15% and
68.58%, respectively as compared to doxorubicin control group
(Fig. 7).
3.8. Fas-L immunostained sections
In control and propolis extract treated groups, sections showed
negative to weak immunostaining for Fas-L (Fig. 8A and B). In Dox
treated group strong positive immunostaining for Fas-L could be
detected in the cytoplasm of spermatogenic cells. Sertoli and
myoid cells showed negative staining (Fig. 8C). Section of testis
of a rat that received Dox and propolis extract showed reduced
immunostaining for Fas-L as few spermatogenic cells exhibited positive reaction (Fig. 8D).
3.9. Caspase-3 immunostained sections
In control and propolis extract treated groups, sections showed
weak immunostaining for caspase-3 in some spermatogenic cells
(Fig. 9A and B). In Dox-treated group strong positive immunostaining for caspase-3 could be detected in the cytoplasm of spermatogenic cells. Sertoli and myoid cells showed negative staining
(Fig. 9C). Section of testis from a rat treated with Dox and propolis
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Fig. 2. Effect of propolis extract on: (A) testicular activities of 3b-hydroxysteroid dehydrogenase (3b-HSD) and 17b-hydroxysteroid dehydrogenase (17b-HSD) as well as (B)
gene expression of 3b-HSD, 17b-HSD and steroidogenic acute regulatory protein (StAR) in normal and doxorubicin-treated rats. Data are expressed as mean ± SE (n = 8–10).
a
Significantly different from normal control group at p < 0.05. b Significantly different from propolis extract-treated group at p < 0.05. c Significantly different from doxorubicin
control group at p < 0.05.
Fig. 3. Effect of propolis extract on: (A) sperm count, (B) diameter of seminiferous tubules and (C) testicular hemorrhage in normal and doxorubicin-treated rats. Data are
expressed as mean ± SE (n = 8–10). a Significantly different from normal control group at p < 0.05. b Significantly different from propolis extract-treated group at p < 0.05.
c
Significantly different from doxorubicin control group at p < 0.05.
extract showed reduced immunostaining for caspase-3 as few
spermatogenic cells exhibited positive reaction (Fig. 9D).
did not significantly affect the efficiency of Dox in reducing the
growth of SEC. In other words, there was no significant difference
among groups receiving Dox, propolis extract or their combination.
3.10. Effects on growth of SEC in mice
4. Discussion
The results in Fig. 10 showed that the size of tumors was significantly reduced by Dox to 16.50% of control values at day 20 from the
start of experiment. Moreover, pretreatment with propolis extract
Male germ cells are known to be one of the tissues vulnerable to
Dox toxicity (Hou et al., 2005; Ahmed et al., 2013). The potentially
S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186
181
Fig. 4. (A) Photomicrograph of a section of rat testis of control group showing seminiferous tubules (*) lined with uniformly arranged spermatogenic cells, (B)
photomicrograph of a section of propolis extract-treated group showing seminiferous tubules (*) lined with uniformly arranged spermatogenic cells, (C) photomicrograph of a
section of rat testis from control doxorubicin group showing many seminifirous tubules exhibiting degeneration and shedding of their lining spermatogenic cells (*), (D)
another photomicrograph from the same group showing shedding of lining spermatogenic cells (*) from basement membrane. 1ry spermatocytes appeared small with dark
pyknotic nuclei and increased cytoplasmic eosiophilia (S1). The section showed apoptotic bodies shedded within the lumen (arrows), few sperms (arrows), elongated
spermatids (S2) and disrupted Sertoli cells, (E) Photomicrograph of a section of a rat treated with doxorubicin and propolis extract showing few tubules which exhibited
detachment of germ cells from the basement membrane (*) and (F) Another photomicrograph from the same group showing seminiferous tubules with organized epithelium
containing various cell types, 1ry spermatocyte (S1), spermatids (S2) and sperms (arrows). Germinal epithelium exhibits detachment in some areas. Few spermatocytes
showed pyknotic nuclei (arrows) (H.&E. X 100, 400).
infertility-causing complication renders protection of testicular tissue a critical issue whenever Dox is employed in cancer
chemotherapy.
In the present study, we measured several biochemical parameters related to testicular toxicity and oxidative stress in the testis
tissue to evaluate the protective effect of propolis extract on Dox
toxicity. In testis, ALP is associated with the division of spermatogenic cells and the transportation of glucose to spermatogenic
cells. ACP is one of the markers of dyszoospermia associated with
the denaturation of seminiferous epithelium and phagocytosis of
Sertoli cells (Samarth and Samarth, 2009). In the present study,
the decreased activities of ALP and ACP were notable manifestations of Dox-induced toxicity. However, treatment with propolis
extract maintained the activities of these enzymes almost close
to normal.
LDH and SDH are associated with the maturation of spermatogenic cells, testis and spermatozoa as well as the energy metabolism of spermatozoa. Hence, the decline in their activities
following Dox administration suggests a regression in testicular
development that was reversed by pretreatment with propolis
extract.
Similarly, the activities of AST and ALT increase when the membrane of spermatozoa is damaged, and the rate of intact acrosome
spermatozoa decrease (Yang et al., 2010). The present results
showed that Dox-induced elevated activities of AST and ALT in testis were obviously decreased by propolis extract treatment.
In the present study, propolis extract administration significantly activated G6PD enzyme in normal rats and antagonized its
decrease in Dox-treated rats. G6PD is another key enzyme of the
testicular tissue (Prasad et al., 1995) that provides reducing equivalents for the hydroxylation of steroids.
In the present study, a significant decline in serum testosterone,
LH and FSH was recorded. In addition disturbance in the diameter
of seminiferous tubules was also recorded in Dox-treated rats.
High level of testosterone in testis is essential for the normal spermatogenesis as well as for the maintenance of the structural morphology and the normal physiology of seminiferous tubule (Sharpe
et al., 1992). To ascertain the possible mechanism of the suppressed testosterone production following Dox administration,
the activities and the mRNA expressions of 3b-HSD and 17b-HSD,
the key enzymes for testicular androgenesis (Jana et al., 2006) as
well as StAR, the rate-limiting step in steroidogenesis (Stocco,
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S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186
Fig. 5. Effect of propolis extract on testicular content of: (A) malondialdehyde (MDA) and (B) reduced glutathione (GSH) in normal and doxorubicin-treated rats. Data are
expressed as mean ± SE (n = 8–10). a Significantly different from normal control group at p < 0.05. b Significantly different from propolis extract-treated group at p < 0.05.
c
Significantly different from doxorubicin control group at p < 0.05.
2001) were studied. In the current study, a marked decrease in the
activities and mRNA expressions of 3b-HSD and 17b-HSD along
with decrease in mRNA expression of StAR after Dox exposure
were detected. However, propolis treatment significantly reversed
all the aforementioned changes induced by Dox. In line with the
present results, it has been reported that propolis antagonize the
decline in testosterone level and inhibition of 17-ketosteroid
reductase associated with aluminum chloride toxicity in male rats
(Yousef and Salama, 2009).
In the present study, Dox treated group showed reduced sperm
count in addition to degeneration of spermatogenic cells in many
tubules. Many apoptotic spermatids appeared within the lumen,
few spermatozoa, disrupted Sertoli cells could be observed. Meistrich et al. (1990) reported that single dose of Dox (5 mg/kg) resulted in disruption of spermatogenic cells maturation,
alterations of spermatogonia DNA and sperm morphology. Other
investigators suggested that Dox reduced the number of Sertoli
cells and motility of sperms in a dose-dependent manner (Takahashi et al., 2011; Brilhante et al., 2012). In addition, Dox administration caused significant reduction in the diameter of
seminiferous tubules. This could be regarded as a consequence of
massive germ cell death which is usually followed by a sharp decline in testicular morphometric parameters (Vendramini et al.,
2010).
The deleterious effects of Dox result chiefly from its inherent
tendency to generate free radicals and suppress antioxidant enzymes in various tissues (Atessahin et al., 2006). The current
study confirmed that testicular lipid peroxidation was increased
by Dox as reflected by the increased content of testicular MDA
parallel to reduced GSH content. This phenomenon could be at
least partially attributable to the structure of the male germ cell
membrane, which is rich in polyunsaturated fatty acids and is
thereby particularly prone to lipid peroxidation (Lenzi et al.,
2002). The present decrease in G6PD activity in testis by Dox,
further increases the risk of oxidative stress in this tissue as
G6PD is directly associated with GSH metabolism (Prasad et al.,
1995). The inherent deficiency of a potent component of the
superoxide dismutase (SOD) family (SOD2) in testicular tissues,
also contributes to the vulnerability of this tissue to Dox-induced
oxidative stress (Kasahara et al., 2002). Treatment with propolis
extract reduced Dox-induced alterations in testicular oxidant bal-
ance. The antioxidant effects of propolis extract were previously
reported (Russo et al., 2006; Capucho et al., 2012; Kamiya et al.,
2012).
Bien et al. (2007) found a strong association between oxidative
stress and inflammatory response including cytokine release after
Dox treatment. Our data showed that Dox causes imbalance in immune regulation leading to skewing ratio towards Th1-type cytokines (high TNF-a and low IL-4) with a concomitant increase in
testicular MPO activity compared to the controls. Pretreatment with
propolis extract reduced Dox-induced inflammatory response in the
testis suggesting that alongside antioxidative mechanisms, propolis
may also possess anti-inflammatory properties. Indeed, CAPE, the
main component in propolis, decreased the release of the inflammatory cytokines from the inflammatory cells and in the same time
stimulated production of anti-inflammatory cytokines like IL-10
and IL-4 (Korish and Arafa, 2011; Búfalo et al., 2013).
Apoptosis plays a causative role to the development of Dox toxicity in various tissues (Shinoda et al., 1999; Nakamura et al.,
2000). In the present study, Dox caused significant increase in
the immunoreactivity of Fas-L and caspase-3 in the cytoplasm of
spermatogenic cells that was confirmed by morphometric measurements. Previous studies implicated the Fas system as a possible regulator of germ cell apoptosis in the rat testis especially
under some pathological conditions such as thermal stress, hormone deprivation or environmental toxicants (Nair and Shaha,
2003; Mishra and Shaha, 2005). Moreover, Lizama et al. (2007)
suggested that the germ cells with high levels of Fas could trigger
caspase activation and apoptosis. The current study also elucidated
the effective suppression of apoptosis and morphometric changes
by propolis extract in testis exposed to Dox. This anti-apoptotic effect of propolis has been reported by other investigators (Szliszka
and Krol, 2013; Tsuchiya et al., 2013). Moreover, treatment with
propolis extract prevented Dox-induced decrease in sperm count
and increased sperm count of normal rats. In accordance with
the present results, Capucho et al. (2012) reported that propolis extract can increase sperm count of rats.
The observed antioxidant and anti-apoptotic effects of propolis
extract raised an important question regarding its influence on
Dox antitumor activity. Therefore, the effects of propolis and
Dox as well as their combination were studied on the growth of
SEC in mice. The present experiments showed that Dox anticancer
S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186
183
Fig. 6. Effect of propolis extract on testicular: (A) myeloperoxidase activity (MPO), (B) tumor necrosis factor-alpha (TNF-a) content and (C) interleukin-4 (IL-4) content in
normal and doxorubicin-treated rats. Data are expressed as mean ± SE (n = 8–10). aSignificantly different from normal control group at p < 0.05. bSignificantly different from
propolis extract-treated group at p < 0.05. cSignificantly different from doxorubicin control group at p < 0.05.
Fig. 7. Effect of propolis extract on testicular activities of: (A) caspase-3 and (B) Fas-L in normal and Dox-treated rats. Data are expressed as mean ± SE (n = 8–10).
a
Significantly different from normal control group at p < 0.05. bSignificantly different from propolis extract-treated group at p < 0.05. cSignificantly different from Dox control
group at p < 0.05.
effects were not reduced by its combination by propolis extract.
The present findings can be explained based on the reported anticancer effects of propolis extract (Ozturk et al., 2012; Akyol et al.,
2013). Phenolic compounds isolated from propolis extract were reported to enhance tumor necrosis factor-related apoptosis inducing
ligand (TRAIL), a naturally occurring anticancer agent that preferentially induces apoptosis in cancer cells and is not toxic to normal
cells (Szliszka and Krol, 2013). Moreover CAPE, the main constituent
of propolis extract, was shown to enhance docetaxel and paclitaxel
cytotoxicity in prostate cancer cells (Tolba et al., 2013).
In conclusion, the present study provided evidence that pretreatment with propolis could effectively alleviate Dox-induced
toxicity to testis without affecting its antitumor efficiency. These
findings raised the possibility that propolis may be an adjuvant
therapy, potentially protecting the testis from oxidative and
apoptotic actions related to Dox, and might help, ultimately, to
prevent this devastating adverse effect of Dox in clinical practice.
Further studies are needed to verify the role of propolis extract
when used curatively in management of Dox-induced testicular
toxicity.
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S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186
Fig. 8. (A) Photomicrograph of a section of control rat testis showing negative immunostaining for Fas-L in the cytoplasm of spermatogenic cells, (B) photomicrograph of a
section of testis from propolis extract-treated rat showing weak immunostaining for Fas-L in the cytoplasm of some spermatogenic cells (arrows), (C) photomicrograph of a
section of rat testis from doxorubicin control group showing strong postive immunostaining for Fas-L in the cytoplasm of spermatogenic cells (arrows). Note negative staining
of Sertoli and myoid cells and (D) photomicrograph of a section of testis from rat treated with propolis extract and doxorubicin showing reduced immunostaining for Fas-L as
few spermatogenic cells exhibit positive reaction (arrows) (Fas-L immunostain 400).
Fig. 9. (A) Photomicrograph of a section of control rat testis showing weak immunostaining for caspase-3 in the cytoplasm of some spermatogenic cells (arrows), (B)
photomicrograph of a section of a testis from propolis extract-treated rat showing weak immunostaining for caspase-3 in the cytoplasm of few spermatogenic cells (arrows),
(C) photomicrograph of a section of a rat testis from doxorubicin control group showing positive immunostaining for caspase-3 in the cytoplasm of spermatogenic cells
(arrows) and D. Photomicrograph of a section of a rat testis (group IV) from group treated with doxorubicin and propolis extract showing reduced immunostaining for
caspase-3 in the cytoplasm of spermatogenic cells (arrows). Note strong reaction in cytoplasm of myoid cells (arrow heads) (caspase-3 immunostain 400).
S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186
Fig. 10. Effect of propolis extract on the antitumor action of doxorubicin against the
growth of solid Ehrlich carcinoma in mice. Data are expressed as mean ± SE (n = 6).
a
Significantly different from normal control group at p < 0.05. bSignificantly
different from propolis extract-treated group at p < 0.05. cSignificantly different
from doxorubicin control group at p < 0.05.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
The Transparency document associated with this article can be
found in the online version.
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