Food and Chemical Toxicology 67 (2014) 176–186

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate/ foodchemtox

Propolis Attenuates Doxorubicin-Induced Testicular Toxicity in Rats

http://dx.doi.org/10.1016/j.fct.2014.02.0310278-6915/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Address: Faculty of Pharmacy, Cairo University, Kasr El-Eini Street, P.O. 11562, Egypt. Tel.: +20 2 24503383, +20 1001040447; fax: +20 224073411.

E-mail address: halafzaki@gmail.com (H.F. Zaki).

Sherine M. Rizk a, Hala F. Zaki b,⇑, Mary A.M. Mina c

a Department of Biochemistry, Faculty of Pharmacy, Cairo University, Egyptb Department of Pharmacology & Toxicology, Faculty of Pharmacy, Cairo University, Egyptc Department of Histology, Faculty of Medicine, Cairo University, Egypt

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 August 2013Accepted 21 February 2014Available online 1 March 2014

Keywords:Propolis extractDoxorubicinTesticular functionOxidative stressApoptosisInflammation

Doxorubicin (Dox), an effective anticancer agent, can impair testicular function leading to infertility. Thepresent 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 kg�1; p.o.), for 3 weeks. Group III received 18 mg kg�1 total cumulative dose of Dox i.p. GroupIV received Dox and propolis extract. Serum and testicular samples were collected 48 h after the lasttreatment. In addition, the effects of propolis extract and Dox on the growth of solid Ehrlich carcinomain mice were investigated. Dox reduced sperm count, markers of testicular function, steroidogenesisand gene expression of testicular 3b-hydroxysteroid dehydrogenase (3b-HSD), 17b-hydroxysteroid dehy-drogenase (17b-HSD) and steroidogenic acute regulatory protein (StAR). In addition, it increased testic-ular oxidative stress, inflammatory and apoptotic markers. Morphometric and histopathologic studiessupported the biochemical findings. Treatment with propolis extract prevented Dox-induced changeswithout reducing its antitumor activity. Besides, administration of propolis extract to normal ratsincreased serum testosterone level coupled by increased activities and gene expression of 3ß-HSD and17ß-HSD. Propolis extract may protect the testis from Dox-induced toxicity without reducing its antican-cer potential.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The anthracycline doxorubicin (Dox), or Adriamycin, is the anti-cancer drug of choice in the treatment of many solid tumors. Itsapplication, however, carries the risk of serious dose-dependenttoxicity to other non-target tissues including the testis (Malekine-jad et al., 2012; Ahmed et al., 2013). Dox could strikingly impedespermatogenesis leading eventually to infertility (Howell andShalet, 2005; Yeh et al., 2007). The mechanism responsible forDox-induced testicular injury is not yet completely clear, butfindings from several studies strongly suggest oxidative stress, li-pid peroxidation (Atessahin et al., 2006; Yeh et al., 2007) and cel-lular apoptosis (Shinoda et al., 1999) as major causes. Based onthis concept, a variety of antioxidant or anti-apoptotic agents havebeen 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 proveneffective enough to prevent or reverse this adverse effect.

Propolis is an adhesive, resinous substance collected, trans-formed and used by honeybees to seal holes in their honeycombs,smooth out the internal walls, and protect the entrance of intrud-ers. Honeybees collect the resin from cracks in the bark of trees andleaf buds. They bring propolis back to the hive, where it is modifiedand mixed with other substances, including bees’ own wax and sal-ivary secretions (Ghisalberti, 1979). Propolis has been used in folkmedicine all over the world. It has anti-inflammatory, immunoreg-ulatory, bacteriostatic, and antibacterial activities (Tosi et al., 2007;Bueno-Silva et al., 2013). Propolis extract presents low toxicity toexperimental animals, with LD50 higher than 7 g/kg for mice (Dan-tas et al., 2006). It has strong cytoprotective effect against differentexogenous toxic stimuli (Bhadauria and Nirala, 2009; Boutabetet al., 2011). Egyptian propolis is rich in aromatic acids, aromaticacid esters, flavonoids, and some triterpenoids (Abd El Hady andHegazi, 2002). Many of the reported pharmacological effects ofpropolis extract reside in its high content of caffeic acid, caffeicacid phenethyl ester (CAPE) and polyphenolic compounds (Ozturket al., 2012; Akyol et al., 2013; Búfalo et al., 2013; Szliszka and Krol,2013).

To determine whether propolis extract could attenuate Dox-in-duced apoptosis and oxidative stress in testicular tissue, the pres-ent 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 byDox in rat testis. The study was extended to examine the antican-cer effects of Dox when combined with propolis extract using solidEhrlich carcinoma (SEC) model in mice. The results of the currentstudy could clarify the role of propolis extract in prevention of thisserious side effect associated with Dox treatment.

2. Materials and methods

2.1. Animals

Male Wistar albino rats weighing 160–180 g and female Swiss albino miceweighing 20–22 g were provided by the breeding unit of the Egyptian organizationfor biological product and vaccines (Helwan, Egypt). They were housed under con-trolled conditioning (25 ± 1 �C constant temperature, 55% relative humidity, 12 hlighting cycle), and received standard pelleted diet and water ad libitum duringthe study period. The study complies with the Guide for Care and Use of LaboratoryAnimals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Ethics Committee for Animal Experi-mentation at Faculty of Pharmacy, Cairo University.

2.2. Preparation of propolis extract

Fifty grams of the resinous material of Egyptian propolis (obtained from Dakah-lia 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 andthe supernatant was evaporated to complete dryness under vacuum at 40 �C (Fran-chi et al., 2012). The yield of dried residue was about 61.1% (w/w) and it was kept at4 �C for further use. Aqueous suspension of propolis was prepared in 1% gum acaciasuspension, and orally administered to the animals for 3 weeks in a dose of200 mg kg�1 (Bhadauria and Nirala, 2009).

2.3. Experimental design and protocol

Forty male Wistar rats were divided into four groups consisting of ten animalsin each group. Group I: animals received distilled water as a vehicle for 21 days andnormal saline (i.p.) on 8th, 10th, 12th, 15th, 17th and 19th day (normal control). GroupII: animals received propolis extract (200 mg kg�1, p.o.) 5 days/week for 21 days.Group III: control Dox received 18 mg kg�1 total cumulative dose of Dox on six di-vided doses (3 mg kg�1, i.p.) on 8th, 10th, 12th, 15th, 17th and 19th day (Jahnukainenet al., 2001). Group IV: animals received propolis extract (200 mg kg�1, p.o.) 5 days/week for 21 days and Dox (3 mg kg�1, 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 oneweek 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 �Ctill determination of testosterone, luteinizing hormone (LH), and follicle-stimulat-ing hormone (FSH). Serum level of testosterone was assayed using automatedchemiluminescence’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 connec-tive tissue. The left side of testis was rinsed in ice-cold saline, weighed and homog-enized in cold physiological saline to give 10% homogenate (using Potter–Elvejhemglass homogenizer). In addition two sections from the right side of testis were pre-pared using flash freezing technique for PCR and histological processing. Aliquots ofthe testicular homogenate were suitably processed for the assessment of the fol-lowing parameters.

2.5.1. Determination of testicular marker enzymesThe 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 Com-biplus ultracentrifuge. The resulting cytosolic fraction was used for the colorimetricdetermination of the activities of acid phosphatase (ACP), alkaline phosphatase(ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate ami-notransferase (AST) and glucose-6-phosphate dehydrogenase (G6PD) based on theinstructions of kits manufacturers. In addition, sorbitol dehydrogenase (SDH) wasmeasured according to the method of Chauncey et al. (1988). Protein content wasmeasured according to the method of Lowry et al. (1951).

2.5.2. Determination of testicular oxidative stress markersMalondialdehyde (MDA), as an index for lipid peroxidation, was determined in

testicular homogenate by detecting the absorbance of thiobarbituric acid reactivesubstances at 532 nm (Mihara and Uchiyama, 1978). Another aliquot of the testic-ular homogenate was mixed with equal volume with 10% metaphosphoric acid andcentrifuged at 1000g. The resulting protein-free supernatant was used for the deter-mination of reduced glutathione (GSH) according to the method described by Beu-tler et al. (1963).

2.5.3. Determination of the extent of Dox-induced hemorrhageAnother 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 homoge-nate was centrifuged at 3000g for 30 min. One milliliter aliquot of the supernatantfluid was diluted with 5 mL of the homogenizing buffer. The absorbance of homog-enate at 405 nm was determined spectrophotometrically (Niewenhuis and Prozia-leck, 1987).

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 super-natant was stored at �80 �C until assay. Tissue contents of IL-4 and TNF-a were as-sayed 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 mea-sured as previously described by Bradley et al. (1982) to assess neutrophilinfiltration.

2.5.5. Determination of testicular 3b-hydroxysteroid dehydrogenase (3b-HSD) and 17b-hydroxysteroid dehydrogenase (17b-HSD) activities

Testicular 3b-HSD and 17b-HSD activities were measured according to themethods of Talalay (1962) and Jarabak et al. (1962). An aliquot of the testicular tis-sue homogenate was mixed with equal volume of homogenizing fluid containing30% spectroscopic-grade glycerol, 10 mmol potassium phosphate, and 2 mM EDTAfollowed by centrifugation at 10,000g for 30 min in an ultracentrifuge at 4 �C. Onemilliliter of the supernatant was mixed with 100 lmol sodium pyrophosphate buf-fer (pH, 8.9), 0.9 ml bidistilled water, and 30 lg dehydroepiandrosterone (dissolvedin ethanol) making up the incubation mixture to a total of 3 mL. 3b-HSD activitywas measured after the addition of 0.5 lmol NAD+ to the tissue supernatant mix-ture at 340 nm. The same supernatant collected for testicular assay of 3b-HSDwas used for assaying of 17b-HSD activity. One milliliter of the supernatant wasmixed with 440 lmol sodium pyrophosphate buffer (pH 10.2), 25 mg crystallinebovine albumin, and 0.3 lmol testosterone bringing the incubation mixture to a to-tal of 3 mL. Enzyme activity was measured after the addition of NADP+ (1.1 lmol) tothe supernatant mixture at 340 nm.

2.5.6. 3b-HSD; 17b-HSD and steroidogenic acute regulatory protein (StAR) geneexpression assay2.5.6.1. RNA extraction and reverse transcription. Total RNA extraction from testiswas done by using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufac-turer’s instructions. RNA concentration was measured with a spectrophotometer(260/280 ratio of all samples was over 1800). The isolated total RNA was reverse-transcribed into complementary DNA (cDNA) using high capacity cDNA reversetranscription kit (Applied Biosystems, Foster City, CA) according to the manufac-turer’s instructions and all products were stored at �20 �C.

2.5.6.2. Real-Time PCR. Gene expressions were analyzed by quantitative RT-PCRusing the SYBR Green PCR Master MIX (Applied Biosystems, California, USA) withthe ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City,CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the house-keeping 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-ACA-AACTCATCGGCGGTCTT-30 (antisense); for rat StAR, 50-ATGCCTGAGCAAAGCGGTGTC-30 (sense) and 50-CAAGTGGCTGGCGAACTCTATCTG-30 (antisense); for ratGAPDH, 50-CCATGGAGAAGGCTGGGG-30 (sense) and 50-CAAAGTTGTCATGGAT-GACC-30 (antisense). The thermal cycle protocol consisted of initial denaturationat 95 �C for 10 min followed by 40 cycles with 30 s denaturation at 95 �C and 30 sannealing/extension at 60 �C.

2.5.6.3. Data analysis. Gene expression was normalized to the endogenous controlGAPDH, and fold changes in the genes of interest were determined using the com-parative threshold cycle (Ct) method (Pfaffl, 2001).

2.6. Determination of epididymal sperm count

Spermatozoa in the epididymis were counted according to the method of Yokoiet al. (2003). Briefly, the epididymis was minced with anatomical scissors in 5 ml ofphysiological saline, placed in a rocker for 10 min, and incubated at room temper-ature for 2 min. The supernatant fluid was diluted 1:100 with a solution containing

178 S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186

5 g sodium bicarbonate, 1 ml formalin (35%) and 25 mg eosin per 100 ml of distilledwater. Total sperm number was determined with a hemocytometer. Approximately10 ll of the diluted sperm suspension was transferred to each counting chamberand was allowed to stand for 5 min for counting under a light microscope at200� magnification.

2.7. Histological studies

The testis were removed and kept in 10% formol saline for 24 h, dehydrated inethanol and embedded in paraffin. Sections were cut at 5 micron thickness,mounted on slides and stained with hematoxylin and eosin (for histological andmorphometric studies). Some sections were mounted on positive charged slidesfor 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 isexpressed in various tissues and cells. Fas-L binds to its receptor (Fas) thus inducingapoptosis (Galvao et al., 2010). Immunohistochemistry to active caspase-3 is com-monly used for apoptosis detection (Bressenot et al., 2009). Tissue sections weredeparaffinized, rehydrated and treated with 10% hydrogen peroxide to reduceendogenous peroxidase. For antigen retrieval tissue sections were boiled in citratebuffer pH 6.0 then cooling was allowed. Sections were incubated with the primaryantibodies; Fas L; a rabbit polyclonal antibody (Neo Markers Laboratories, Westing-house, USA) and caspase-3; a rabbit polyclonal antibody (Neo Markers Laboratories,Westinghouse, USA). Then sections were incubated with the secondary antibody abiotinylated goat immunoglobulin followed by streptavidin biotin complex. The siteof the reaction was visualized by adding diaminobenzidine HCl which is convertedinto brown precipitate by peroxidase. Counterstaining was performed usingMeyer’s hematoxylin.

2.9. Morphometric measurements

Using the computer assisted software Leica Qwin 500 LTD image analyzer, thefollowing parameters were measured:

1. Mean diameters of seminiferous tubules in hematoxylin and eosin stained sec-tions were measured using the interactive measuring menu. They were mea-sured in 10 non-overlapping fields at a magnification 100� for each specimen.

2. Mean area percent of Fas-L and caspase-3 immunoreactivities were measuredin immunostained sections using the color detect menu. They were measuredin 10 non-overlapping fields for each specimen of all groups at a magnification400� 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 transplantedsubcutaneously in the right thigh of the lower limb of each mouse. Mice with a pal-pable solid tumor mass that developed within 7 days after implantation were di-vided into four groups, 6 animals each, and received the same doses of propolisextract and Dox, previously mentioned, for 2 weeks. The change in tumor sizewas measured every 3 days using a Vernier caliper and was calculated using the for-mula adapted from Osman et al. (1993).

Tumor volume ðmm3Þ 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 weredone using one way analysis of variance (ANOVA) followed by Tukey–Kramer mul-tiple comparisons test using the software GraphPad InStat. A probability level ofless 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 testic-ular activities of LDH, SDH, ACP and G6PD by 39.55%, 31.52%,37.28 and 30.23%, respectively parallel to increased activities ofALT and AST by 26.83% and 41.97%, respectively as compared tothe normal control group. Treatment of rats by propolis extractattenuated Dox-induced changes in the aforementioned parame-ters and normalized testicular function markers (Table 1).

3.2. Effects on steroidogenesis

Dox resulted in marked decrease in serum testosterone, FSHand LH levels by 83.37%, 52.38% and 45.56%, respectively as com-pared to normal control group (Fig. 1). To ascertain the possiblemechanism of the suppressed testosterone production followingDox administration, the expressions of testicular key androgenicenzymes like 3ß-HSD and 17ß-HSD along with StAR, a prime reg-ulatory 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 of3ß-HSD, 17ß-HSD and StAR by 67.06%, 71.30% and 57.45%, respec-tively as compared to the normal control group (Fig. 2). Adminis-tration of propolis extract prevented Dox-induced decreases inserum levels of testosterone, FSH and LH as well as activities orgene expression of testicular 3ß-HSD, 17ß-HSD and StAR indicat-ing the protective role of propolis on testicular androgenic disor-ders of Dox exposure (Figs. 1 and 2). Moreover, administration ofpropolis extract to normal rats significantly increased serum tes-tosterone level by 28.59 (Fig. 1A) coupled by increased activitiesof testicular 3ß-HSD and 17ß-HSD by 46.68% and 43.41%, respec-tively (Fig. 2A) and increased gene expression of both enzymesby 36.11% and 49.89% (Fig. 2B), respectively as compared to thenormal control group.

3.3. Sperm count and morphometric results

Parallel to the observed decrease in testicular and steroidogen-esis markers, Dox reduced sperm count and the diameter of semi-niferous tubules by 52.51% and 28.87%, respectively (Fig. 3A–B)and increased testicular hemorrhage by 1.62 folds (Fig. 3C) as com-pared to normal control group. Such changes were largely attenu-ated in the group receiving propolis extract where sperm countand the diameter of seminiferous tubules were normalized andtesticular hemorrhage was reduced by 38.24% (Fig. 3A–C). More-over, administration of propolis extract to normal rats increasedsperm 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 thenormal structure of seminiferous tubules. They were lined with athick layer of uniformly arranged spermatogenic cells in variablestages of maturation; spermatogonia, spermatocytes, spermatidsand sperms in addition to Sertoli cells. The interstitium containedsmall scattered groups of Leydig cells (Fig. 4A). Testicular sectionsof rats which received propolis extract showed normal seminifer-ous tubules lined with spermatogenic cells at different stages ofmaturation (Fig. 4B).

In Dox-treated rats, sections of the testes showed disruptedspermatogenic structure in many seminiferous tubules, sheddingand degeneration of many spermatogenic cells (Fig. 4C). Many tu-bules showed detached primary spermatocytes with dark pyknoticcondensed nuclei and increased cytoplasmic eosinophilia, fewelongated spermatids and many apoptotic spematids shedded in-side the lumen. Sertoli cells appeared disrupted (Fig. 4D).

Sections of rat testis which received Dox and propolis extractshowed few tubules exhibiting detachment of germ cells fromthe basement membrane (Fig. 4E). Most of the seminiferous tu-bules showed organized epithelium containing various cell types,1ry spermatocyte, spermatids, and sperms. Germinal epitheliumexhibited detachment in some areas, Few spermatocytes showedpyknotic nuclei (Fig. 4F).

Table 1Effect of propolis extract on testicular markers in normal and doxorubicin-treated rats.

Testicular markers (U/mg protein) Control Propolis Doxorubicin Doxorubicin + propolis

LDH 5.07 ± 0.41 4.58 ± 0.26 3.07 ± 0.35a,b 4.86 ± 0.37c

SDH 4.95 ± 0.38 5.14 ± 0.37 3.39 ± 0.29a,b 5.34 ± 0.27c

ACP 130.19 ± 4.78 116.39 ± 6.49 81.65 ± 8.28a,b 110.75 ± 7.60c

ALP 147.83 ± 5.82 154.93 ± 6.39 129.12 ± 5.87b 157.08 ± 7.73c

ALT 79.64 ± 4.77 78.03 ± 3.61 101.01 ± 5.63a,b 74.42 ± 4.84c

AST 79.78 ± 6.38 77.13 ± 3.69 113.26 ± 10.47a,b 83.61 ± 7.61c

G6PD 7.08 ± 0.39 8.95 ± 0.43a 4.94 ± 0.39a,b 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 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-treatedgroup at p < 0.05. c Significantly different from doxorubicin control group at p < 0.05.

S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186 179

3.5. Effect on oxidative stress markers

Administration of Dox, in the current study, increased testicularMDA content by 81.93% parallel to reduced testicular GSH contentby 46.20% as compared to normal rats (Fig. 5). Pretreatment withpropolis extract prevented Dox-induced changes in both MDAand GSH contents (Fig. 5).

3.6. Effect on MPO, TNF-a and IL-4

Dox administration resulted in marked increase in testicularMPO activity and TNF-a content by 61.10% and 115.76%, respec-tively (Fig. 6A–B) parallel to decreased content of IL-4 by 38.37%(Fig. 6C) as compared to normal control group. Such changes wereprevented 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 andFas-L by 7.19 and 17.91 folds, respectively as compared to normalcontrol group (Fig. 7). Pretreatment of rats by propolis extract de-creased 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 showednegative to weak immunostaining for Fas-L (Fig. 8A and B). In Doxtreated group strong positive immunostaining for Fas-L could bedetected in the cytoplasm of spermatogenic cells. Sertoli andmyoid cells showed negative staining (Fig. 8C). Section of testisof a rat that received Dox and propolis extract showed reducedimmunostaining for Fas-L as few spermatogenic cells exhibited po-sitive reaction (Fig. 8D).

3.9. Caspase-3 immunostained sections

In control and propolis extract treated groups, sections showedweak immunostaining for caspase-3 in some spermatogenic cells(Fig. 9A and B). In Dox-treated group strong positive immunostain-ing for caspase-3 could be detected in the cytoplasm of spermato-genic cells. Sertoli and myoid cells showed negative staining(Fig. 9C). Section of testis from a rat treated with Dox and propolis

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 doxorubicincontrol 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 areexpressed 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.

180 S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186

extract showed reduced immunostaining for caspase-3 as fewspermatogenic cells exhibited positive reaction (Fig. 9D).

3.10. Effects on growth of SEC in mice

The results in Fig. 10 showed that the size of tumors was signifi-cantly reduced by Dox to 16.50% of control values at day 20 from thestart of experiment. Moreover, pretreatment with propolis extract

did not significantly affect the efficiency of Dox in reducing thegrowth of SEC. In other words, there was no significant differenceamong groups receiving Dox, propolis extract or their combination.

4. Discussion

Male germ cells are known to be one of the tissues vulnerable toDox toxicity (Hou et al., 2005; Ahmed et al., 2013). The potentially

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 asection 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 darkpyknotic nuclei and increased cytoplasmic eosiophilia (S1). The section showed apoptotic bodies shedded within the lumen (arrows), few sperms (arrows), elongatedspermatids (S2) and disrupted Sertoli cells, (E) Photomicrograph of a section of a rat treated with doxorubicin and propolis extract showing few tubules which exhibiteddetachment of germ cells from the basement membrane (*) and (F) Another photomicrograph from the same group showing seminiferous tubules with organized epitheliumcontaining various cell types, 1ry spermatocyte (S1), spermatids (S2) and sperms (arrows). Germinal epithelium exhibits detachment in some areas. Few spermatocytesshowed pyknotic nuclei (arrows) (H.&E. X 100, 400).

S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186 181

infertility-causing complication renders protection of testicular tis-sue a critical issue whenever Dox is employed in cancerchemotherapy.

In the present study, we measured several biochemical param-eters related to testicular toxicity and oxidative stress in the testistissue to evaluate the protective effect of propolis extract on Doxtoxicity. In testis, ALP is associated with the division of spermato-genic cells and the transportation of glucose to spermatogeniccells. ACP is one of the markers of dyszoospermia associated withthe denaturation of seminiferous epithelium and phagocytosis ofSertoli cells (Samarth and Samarth, 2009). In the present study,the decreased activities of ALP and ACP were notable manifesta-tions of Dox-induced toxicity. However, treatment with propolisextract maintained the activities of these enzymes almost closeto normal.

LDH and SDH are associated with the maturation of spermato-genic cells, testis and spermatozoa as well as the energy metabo-lism of spermatozoa. Hence, the decline in their activitiesfollowing Dox administration suggests a regression in testiculardevelopment that was reversed by pretreatment with propolisextract.

Similarly, the activities of AST and ALT increase when the mem-brane of spermatozoa is damaged, and the rate of intact acrosomespermatozoa decrease (Yang et al., 2010). The present resultsshowed that Dox-induced elevated activities of AST and ALT in tes-tis were obviously decreased by propolis extract treatment.

In the present study, propolis extract administration signifi-cantly activated G6PD enzyme in normal rats and antagonized itsdecrease in Dox-treated rats. G6PD is another key enzyme of thetesticular tissue (Prasad et al., 1995) that provides reducing equiv-alents 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 diameterof seminiferous tubules was also recorded in Dox-treated rats.High level of testosterone in testis is essential for the normal sper-matogenesis as well as for the maintenance of the structural mor-phology and the normal physiology of seminiferous tubule (Sharpeet al., 1992). To ascertain the possible mechanism of the sup-pressed 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) aswell as StAR, the rate-limiting step in steroidogenesis (Stocco,

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 areexpressed 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.

182 S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186

2001) were studied. In the current study, a marked decrease in theactivities and mRNA expressions of 3b-HSD and 17b-HSD alongwith decrease in mRNA expression of StAR after Dox exposurewere detected. However, propolis treatment significantly reversedall the aforementioned changes induced by Dox. In line with thepresent results, it has been reported that propolis antagonize thedecline in testosterone level and inhibition of 17-ketosteroidreductase associated with aluminum chloride toxicity in male rats(Yousef and Salama, 2009).

In the present study, Dox treated group showed reduced spermcount in addition to degeneration of spermatogenic cells in manytubules. Many apoptotic spermatids appeared within the lumen,few spermatozoa, disrupted Sertoli cells could be observed. Meis-trich et al. (1990) reported that single dose of Dox (5 mg/kg) re-sulted in disruption of spermatogenic cells maturation,alterations of spermatogonia DNA and sperm morphology. Otherinvestigators suggested that Dox reduced the number of Sertolicells and motility of sperms in a dose-dependent manner (Takah-ashi et al., 2011; Brilhante et al., 2012). In addition, Dox adminis-tration caused significant reduction in the diameter ofseminiferous tubules. This could be regarded as a consequence ofmassive germ cell death which is usually followed by a sharp de-cline in testicular morphometric parameters (Vendramini et al.,2010).

The deleterious effects of Dox result chiefly from its inherenttendency to generate free radicals and suppress antioxidant en-zymes in various tissues (Atessahin et al., 2006). The currentstudy confirmed that testicular lipid peroxidation was increasedby Dox as reflected by the increased content of testicular MDAparallel to reduced GSH content. This phenomenon could be atleast partially attributable to the structure of the male germ cellmembrane, which is rich in polyunsaturated fatty acids and isthereby 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 asG6PD is directly associated with GSH metabolism (Prasad et al.,1995). The inherent deficiency of a potent component of thesuperoxide dismutase (SOD) family (SOD2) in testicular tissues,also contributes to the vulnerability of this tissue to Dox-inducedoxidative stress (Kasahara et al., 2002). Treatment with propolisextract reduced Dox-induced alterations in testicular oxidant bal-

ance. The antioxidant effects of propolis extract were previouslyreported (Russo et al., 2006; Capucho et al., 2012; Kamiya et al.,2012).

Bien et al. (2007) found a strong association between oxidativestress and inflammatory response including cytokine release afterDox treatment. Our data showed that Dox causes imbalance in im-mune regulation leading to skewing ratio towards Th1-type cyto-kines (high TNF-a and low IL-4) with a concomitant increase intesticular MPO activity compared to the controls. Pretreatment withpropolis extract reduced Dox-induced inflammatory response in thetestis suggesting that alongside antioxidative mechanisms, propolismay also possess anti-inflammatory properties. Indeed, CAPE, themain component in propolis, decreased the release of the inflamma-tory cytokines from the inflammatory cells and in the same timestimulated production of anti-inflammatory cytokines like IL-10and IL-4 (Korish and Arafa, 2011; Búfalo et al., 2013).

Apoptosis plays a causative role to the development of Dox tox-icity in various tissues (Shinoda et al., 1999; Nakamura et al.,2000). In the present study, Dox caused significant increase inthe immunoreactivity of Fas-L and caspase-3 in the cytoplasm ofspermatogenic cells that was confirmed by morphometric mea-surements. Previous studies implicated the Fas system as a possi-ble regulator of germ cell apoptosis in the rat testis especiallyunder some pathological conditions such as thermal stress, hor-mone 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 triggercaspase activation and apoptosis. The current study also elucidatedthe effective suppression of apoptosis and morphometric changesby propolis extract in testis exposed to Dox. This anti-apoptotic ef-fect of propolis has been reported by other investigators (Szliszkaand Krol, 2013; Tsuchiya et al., 2013). Moreover, treatment withpropolis extract prevented Dox-induced decrease in sperm countand increased sperm count of normal rats. In accordance withthe present results, Capucho et al. (2012) reported that propolis ex-tract can increase sperm count of rats.

The observed antioxidant and anti-apoptotic effects of propolisextract raised an important question regarding its influence onDox antitumor activity. Therefore, the effects of propolis andDox as well as their combination were studied on the growth ofSEC in mice. The present experiments showed that Dox anticancer

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).aSignificantly different from normal control group at p < 0.05. bSignificantly different from propolis extract-treated group at p < 0.05. cSignificantly different from Dox controlgroup at p < 0.05.

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 innormal 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 frompropolis extract-treated group at p < 0.05. cSignificantly different from doxorubicin control group at p < 0.05.

S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186 183

effects were not reduced by its combination by propolis extract.The present findings can be explained based on the reported anti-cancer effects of propolis extract (Ozturk et al., 2012; Akyol et al.,2013). Phenolic compounds isolated from propolis extract were re-ported to enhance tumor necrosis factor-related apoptosis inducingligand (TRAIL), a naturally occurring anticancer agent that preferen-tially induces apoptosis in cancer cells and is not toxic to normalcells (Szliszka and Krol, 2013). Moreover CAPE, the main constituentof propolis extract, was shown to enhance docetaxel and paclitaxelcytotoxicity in prostate cancer cells (Tolba et al., 2013).

In conclusion, the present study provided evidence that pre-treatment with propolis could effectively alleviate Dox-inducedtoxicity to testis without affecting its antitumor efficiency. Thesefindings raised the possibility that propolis may be an adjuvanttherapy, potentially protecting the testis from oxidative andapoptotic actions related to Dox, and might help, ultimately, toprevent this devastating adverse effect of Dox in clinical practice.Further studies are needed to verify the role of propolis extractwhen used curatively in management of Dox-induced testiculartoxicity.

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 asection of testis from propolis extract-treated rat showing weak immunostaining for Fas-L in the cytoplasm of some spermatogenic cells (arrows), (C) photomicrograph of asection of rat testis from doxorubicin control group showing strong postive immunostaining for Fas-L in the cytoplasm of spermatogenic cells (arrows). Note negative stainingof 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 asfew 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 forcaspase-3 in the cytoplasm of spermatogenic cells (arrows). Note strong reaction in cytoplasm of myoid cells (arrow heads) (caspase-3 immunostain 400�).

184 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 thegrowth of solid Ehrlich carcinoma in mice. Data are expressed as mean ± SE (n = 6).aSignificantly different from normal control group at p < 0.05. bSignificantlydifferent from propolis extract-treated group at p < 0.05. cSignificantly differentfrom doxorubicin control group at p < 0.05.

S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186 185

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

References

Abd El Hady, F.K., Hegazi, A.G., 2002. Egyptian propolis: 2. Chemical composition,antiviral and antimicrobial activities of east Nile delta propolis. Z. Naturforsch.57, 386–394.

Ahmed, F., Urooj, A., Karim, A.A., 2013. Protective effects of Ficus racemosa stem barkagainst doxorubucin-induced renal and testicular toxicity. Pharmacogn. Mag. 9,130–134.

Akyol, S., Ozturk, G., Ginis, Z., Armutcu, F., Yigitoglu, M.R., Akyol, O., 2013. In vivoand in vitro antineoplastic actions of caffeic acid phenethyl ester (CAPE):therapeutic perspectives. Nutr. Cancer 65 (4), 515–526.

Atessahin, A., Turk, G., Karahan, I., Yolmaz, S., Ceribasi, A.O., Bulmus, O., 2006.Lycopene prevents adriamycin-induced testicular toxicity in rats. Fertil. Steril.85, 1216–1222.

Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the determination ofblood glutathione. J. Lab. Clin. Med. 61, 882–888.

Bhadauria, M., Nirala, S.K., 2009. Reversal of acetaminophen induced sub chronichepatorenal injury by propolis extract in rats. Environ. Toxicol. Pharmacol. 27,17–25.

Bien, S., Riad, A., Ritter, C.A., Gratz, M., Olshausen, F., Westermann, D., et al., 2007.The endothelin receptor blocker bosentan inhibits doxorubicin-inducedcardiomyopathy. Cancer Res. 67, 10428–10435.

Boutabet, K., Kebsa, W., Alyane, M., Lahouel, M., 2011. Polyphenolic fraction ofAlgerian propolis protects rat kidney against acute oxidative stress induced bydoxorubicin. Indian J. Nephrol. 21, 101–106.

Bradley, P.P., Priebat, D.A., Christensen, R.D., Rothstein, G., 1982. Measurement ofcutaneous inflammation: estimation of neutrophil content with an enzymemarker. J. Invest. Dermatol. 78, 206–209.

Bressenot, A., Marchal, S., Bezdetnaya, L., Garrier, J., Guillemin, F., Plénat, F., 2009.Assessment of apoptosis by immunohistochemistry to active caspase-3, activecaspase-7, or cleaved PARP in monolayer cells and spheroid and subcutaneousxenografts of human carcinoma. J. Histochem. Cytochem. 57, 289–300.

Brilhante, O., Okada, F.K., Sasso-Cerri, E., Stumpp, T., Miraglia, S.M., 2012. Latemorfofunctional alterations of the Sertoli cell caused by doxorubicinadministered to prepubertal rats. Reprod. Biol. Endocrinol. 10, 79–94.

Bueno-Silva, B., Alencar, S.M., Koo, H., Ikegaki, M., Silva, G.V., Napimoga, M.H., et al.,2013. Anti-inflammatory and antimicrobial evaluation of neovestitol andvestitol isolated from Brazilian red propolis. J. Agric. Food Chem. 61, 4546–4550.

Búfalo, M.C., Ferreira, I., Costa, G., Francisco, V., Liberal, J., Cruz, M.T., et al., 2013.Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-jB and MAPK activation inmacrophages. J. Ethnopharmacol., Epub ahead of print.

Capucho, C., Sette, R., de Souza Predes, F., de Castro Monteiro, J., Pigoso, A.A.,Barbieri, R., et al., 2012. Green Brazilian propolis effects on sperm count andepididymis morphology and oxidative stress. Food Chem. Toxicol. 50, 3956–3962.

Chauncey, B., Leite, M.V., Goldstein, L., 1988. Renal sorbitol accumulation andassociated enzyme activities in diabetes. Enzyme 39, 231–234.

Dantas, A.P., Olivieri, B.P., Gomes, F.H.M., De Castro, S.L., 2006. Treatment ofTrypanosoma cruzi-infected mice with propolis promotes changes in theimmune response. J. Ethnopharmacol. 103, 187–193.

Franchi Jr., G.C., Moraes, C.S., Toreti, V.C., Daugsch, A., Nowill, A.E., Park, Y.K., 2012.Comparison of effects of the ethanolic extracts of Brazilian propolis on humanleukemic cells as assessed with the MTT assay. Evid. Based Complement.Alternat. Med. 2012, 918956.

Galvao, A.M., Ramilo, D.W., Sharzynski, D.J., Lukasik, K., Tramontano, A., Mollo, A.,et al., 2010. Is Fas/Fas ligand system involved in equine corpus luteumfunctional regression? Biol. Reprod. 83, 901–908.

Ghisalberti, E., 1979. Propolis: a review. Bee World 60, 59–84.Hou, M., Chrysis, D., Nurmio, M., Parvinen, M., Eksborg, S., Soder, O., et al., 2005.

Doxorubicin induces apoptosis in germ line stem cells in the immature rat testisand amifostine cannot protect against this cytotoxicity. Cancer Res. 65, 9999–10005.

Howell, S.J., Shalet, S.M., 2005. Spermatogenesis after cancer treatment: damageand recovery. J. Natl. Cancer Inst. Monogr. 2005, 12–17.

Jahnukainen, K., Jahnukainen, T., Salmi, T.T., Svechnikov, K., Eksborg, S., Soder, O.,2001. Amifostine protects against early but not late toxic effects of doxorubicinin infant rats. Cancer Res. 61, 6423–6427.

Jana, K., Jana, S., Samanta, P.K., 2006. Effects of chronic exposure to sodium arseniteon hypothalamo–pituitary–testicular activities in adult rats: possible anestrogenic mode of action. Reprod. Biol. Endocrinol. 4, 9–22.

Jarabak, J., Adams, J.A., Williams-Ashman, H.G., Talalay, P., 1962. Purification of a17beta-hydroxysteroid dehydrogenase of human placenta and studies on itstranshydrogenase function. J. Biol. Chem. 237, 345–357.

Kamiya, T., Izumi, M., Hara, H., Adachi, T., 2012. Propolis suppresses CdCl2-inducedcytotoxicity of COS7 cells through the prevention of intracellular reactiveoxygen species accumulation. Biol. Pharm. Bull. 35, 1126–1131.

Kasahara, E., Sato, E.F., Miyoshi, M., Konaka, R., Hiramoto, K., Sasaki, J., et al., 2002.Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate. Biochem. J. 365, 849–856.

Korish, A.A., Arafa, M.M., 2011. Propolis derivatives inhibit the systemicinflammatory response and protect hepatic and neuronal cells in acute septicshock. Braz. J. Infect. Dis. 15, 332–338.

Lenzi, A., Gandini, L., Lombardo, F., Picardo, M., Maresca, V., Panfili, E., et al., 2002.Polyunsaturated fatty acids of germ cell membranes, glutathione andglutathione-dependent enzyme-PHGPx: from basic to clinic. Contraception65, 301–304.

Lizama, C., Alfaro, I., Reyes, J., Moreno, R., 2007. Up-regulation of CD95 (Apo-1/Fas)is associated with spermatocyte apoptosis during the first round ofspermatogenesis in the rat. Apoptosis 12, 499–512.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem. 193, 265–275.

Malekinejad, H., Janbaz-Acyabar, H., Razi, M., Varasteh, S., 2012. Preventive andprotective effects of silymarin on doxorubicin-induced testicular damagescorrelate with changes in c-myc gene expression. Phytomedicine 19, 1077–1084.

Meistrich, M., van Beek, E., Liang, J., Johnson, S.L., Lu, J., 1990. Low levels ofchromosomal mutations in germ cells derived from doxorubicin-treated stemspermatogonia in the mouse. Cancer Res. 50, 370–374.

Mihara, M., Uchiyama, M., 1978. Determination of malonaldehyde precursor intissues by thiobarbituric acid test. Anal. Biochem. 86, 271–278.

Mishra, P., Shaha, C., 2005. Estrogen induced spermatogenic cell apoptosis occursvia the mitochondrial pathway: role of superoxide and nitric oxide. J. Biol.Chem. 280, 6181–6196.

Nair, R., Shaha, C., 2003. Diethylstilbestrol induces rat spermatocyte cell apoptosisin vivo through increased expression of spermatogenic cell Fas/Fa-L system. J.Biol. Chem. 278, 6470–6481.

Nakamura, T., Katsuda, S., Takahashi, H., Koh, E., 2000. Fas-mediated apoptosis inadriamycin-induced cardiomyopathy in rats: in vivo study. Circulation 102,572–578.

Niewenhuis, R.J., Prozialeck, W.C., 1987. Calmodulin inhibitors protect againstcadmium-induced testicular damage in mice. Biol. Reprod. 37, 127–133.

186 S.M. Rizk et al. / Food and Chemical Toxicology 67 (2014) 176–186

Osman, A.M., Sayed-Ahmed, M.M., Khayyal, M.T., El-Merzabani, M.M., 1993.Hyperthermic potentiation of cisplatin on solid Ehrlich carcinoma. Tumori 79,268–272.

Ozturk, G., Ginis, Z., Akyol, S., Erden, G., Gurel, A., Akyol, O., 2012. The anticancermechanism of caffeic acid phenethyl ester (CAPE): review of melanomas, lungand prostate cancers. Eur. Rev. Med. Pharmacol. Sci. 16 (15), 2064–2068.

Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucl. Acids Res. 29, 45–50.

Prasad, A.K., Pant, N., Srivastava, S.C., Kumar, R., Srivastava, S.P., 1995. Effect ofdermal application of hexachlorocyclohexane (HCH) on male reproductivesystem of rat. Hum. Exp. Toxicol. 14, 484–488.

Russo, A., Troncoso, N., Sanchez, F., Garbarino, J.A., Vanella, A., 2006. Propolisprotects human spermatozoa from DNA damage caused by benzo[a]pyrene andexogenous reactive oxygen species. Science 78, 1401–1406.

Samarth, R.M., Samarth, M., 2009. Protection against radiationinduced testiculardamage in Swiss albino mice by Mentha piperita (Linn.). Basic Clin. Pharmacol.Toxicol. 104, 329–334.

Sharpe, R.M., Maddocks, S., Millar, M., Saunders, P.T.K., Kerr, J.B., McKinnell, C., 1992.Testosterone and spermatogenesis: identification of stage dependent,androgen-regulated proteins secreted by adult rat seminiferous tubules. J.Androl. 13, 172–184.

Shinoda, K., Mitsumori, K., Yasuhara, K., Uneyama, C., Onodera, H., Hirose, M., et al.,1999. Doxorubicin induces male germ cell apoptosis in rats. Arch. Toxicol. 73,274–281.

Stocco, D.M., 2001. StAR protein and the regulation of steroid hormonebiosynthesis. Annu. Rev. Physiol. 63, 193–213.

Szliszka, E., Krol, W., 2013. Polyphenols isolated from propolis augment TRAIL-induced apoptosis in cancer cells. Evid. Based Complement. Alternat. Med.,Epub ahead of print.

Takahashi, H., Tainaka, H., Umezawa, M., Takeda, K., Tanaka, H., Nishimune, Y., et al.,2011. Evaluation of testicular toxicology of doxorubicin based on microarrayanalysis of testicular specific gene expression. J. Toxicol. Sci. 36, 559–567.

Talalay, P., 1962. Hydroxysteroid dehydrogenases. In: Colowick, S.P., Kaplan, N.O.(Eds.), Method of Enzymology. Academic Press, New York, pp. 512–516.

Tolba, M.F., Esmat, A., Al-Abd, A.M., Azab, S.S., Khalifa, A.E., Mosli, H.A., et al., 2013.Caffeic acid phenethyl ester synergistically enhances docetaxel and paclitaxelcytotoxicity in prostate cancer cells. IUBMB Life 65, 716–729.

Tosi, E.A., Re, E., Ortega, M.E., Cazzoli, A.F., 2007. Food preservative based onpropolis: bacteriostatic activity of propolis polyphenols and flavonoids uponEscherichia coli. Food Chem. 104, 1025–1029.

Tsuchiya, I., Hosoya, T., Ushida, M., Kunimasa, K., Ohta, T., Kumazawa, S., 2013.Nymphaeol-A isolated from Okinawan propolis suppresses angiogenesis andinduces caspase-dependent apoptosis via inactivation of survival signals. Evid.Based Complement. Alternat. Med., Epub ahead of print.

Vendramini, V., Sasso-Cerri, E., Miraglia, M., 2010. Amifostine reduces theseminiferous epithelium damage in doxorubicin-treated prepubertal ratswithout improving the fertility status. Reprod. Biol. Endocrinol. 8, 3–15.

Yang, J., Wu, G., Feng, Y., Lv, Q., Lin, S., Hu, J., 2010. Effects of taurine on malereproduction in rats of different ages. J. Biomed. Sci. 17 (Suppl 1), S9.

Yeh, Y.C., Lai, H.C., Ting, C.T., Lee, W.L., Wang, L.C., Wang, K.Y., et al., 2007. Protectionby doxycycline against doxorubicin-induced oxidative stress and apoptosis inmouse testes. Biochem. Pharmacol. 74, 969–980.

Yokoi, K., Uthus, E.O., Nielsen, F.H., 2003. Nickel deficiency diminishes spermquantity and movement in rats. Biol. Trace Elem. Res. 93, 141–153.

Yousef, M.I., Salama, A.F., 2009. Propolis protection from reproductive toxicitycaused by aluminium chloride in male rats. Food Chem. Toxicol. 47, 1168–1175.