Role of propolis in improving male rat fertility affected ...çalışma seminifer tübüllerde, tübüler membran, mitokondri, endoplazmik retikulum, Golgi aygıtı ve çekirdek atrofisi

13

Role of propolis in improving male rat fertility affected

with aluminum chloride cytotoxicity

Aluumlminyum klorid toksisitesinden etkilenmiş erkek sıccedilan fertilitesinin duumlzeltilmesinde propolisin roluuml

Alsayed Ali Mahran1 Ahmed Hassan Abdel-Rahman Al-Rashidy2 Ahmed Mohamed Ahmed

Abd El-Mawla34

Departments of 1Anatomy and Histology 2Pathology Faculty of Medicine and Medical Sciences Taif University Taif 21974 Saudi Arabia

3Department of Pharmacognosy Faculty of Pharmacy Taif University Taif 21974 Saudi Arabia 4Department of Pharmacognosy Faculty of Pharmacy Assiut University Assiut 71526 Egypt

SUMMARY

AIM Aluminum chloride (AlCl3) is commonly used in daily life but it can be induce reproductive toxicity Propolis has been reported to be important antioxidant Therefore the present study aimed to investigate the protective effects of propolis against reproductive toxicity of aluminum chloride (AlCl3) in male rats METHODS Sixty male albino rats were divided into three equal groups the first served as negative control the second received AlCl3 (34 mgkg bw 125 LD50) the third received AlCl3 and treated with propolis (50mgkg bw) Treatment was continued for 70 days RESULTS AlCl3 caused a decrease in body and testes weights and testosterone hormone In addition histological changes as damages within the seminiferous tubules and vascular degeneration of the germ cells and Sertoli cells cytoplasm were observed On the other hand electron microscopy study showed changes in the testis seminiferous tubules such as atrophy of the tubular membrane mitochondria endoplasmic reticulum Golgi apparatus and nucleus Our results revealed that propolis alleviated the reproductive toxic effects of AlCl3 CONCLUSION Treatment with propolis alleviates AlCl3-associated hazards and protects the testicular tissues from AlCl3 toxicity Key words Propolis Aluminum chloride male albino rats testosterone electron microscopy

OumlZET GİRİŞ Aluumlminyum klorid (AlCl3) guumlnluumlk hayatta sıklıkla kullanılmakta fakat reproduumlktif toksisite oluşturabilmektedir Propolisin oumlnemli bir antioksidan olduğu bildirilmiştir Bundan dolayı ccedilalışmamızda erkek sıccedilanlarda aluumlminyum kloride bağlı reproduumlktif toksisiteye karşı propolisin koruyucu etkilerini araştırdık YOumlNTEM Altmış erkek albino sıccedilan uumlccedil eşit gruba ayrıldı İlk grup negatif kontrol grubu olarak ayrılırken ikinci gruba AlCl3 (34 mgkg vuumlcut ağırlığına) uygulandı Uumlccediluumlncuuml gruba ise AlCl3 uygulandıktan sonra propolis (50 mgkg vuumlcut ağırlığına) ile tedavi edildi Tedavi 70 guumln suumlrduuml BULGULAR AlCl3 vuumlcut ve testis ağırlığı ile testosteron hormonunda azalmaya neden oldu Ayrıca seminifer tuumlbuumlllerde hasar germ ve Sertoli huumlcre sitoplazmalarında vaskuumller dejenerasyon şeklinde histolojik değişiklikler goumlzlendi Diğer yandan elektron mikroskopik ccedilalışma seminifer tuumlbuumlllerde tuumlbuumller membran mitokondri endoplazmik retikulum Golgi aygıtı ve ccedilekirdek atrofisi gibi değişiklikler goumlsterdi Sonuccedillarımız propolisin AlCl3rsquouumln reproduumlktif toksik etkilerini hafiflettiğini ortaya koymuştur SONUCcedil Propolisle tedavi AlCl3rsquoe bağlı tehlikeyi hafifletmekte AlCl3 toksisitesinden testikuumller dokuyu korumaktadır Anahtar kelimler Propolis Aluumlminyum klorid erkek albino sıccedilan testosteron elektron mikroskopisi

Corresponding Author Dr Ahmed Abd El-Mawla Faculty of Pharmacy Department of Pharmacognosy Assiut University Assiut 71526 Egypt E-mail ahmedpha2000yahoocom

Received October 19 2011 accepted November 22 2011 DOI 105455spatula20111122065544 Published online in ScopeMed (wwwscopemedorg) Spatula DD 2011 1(4) 189-198

13

INTRODUCTION

Aluminum is the most abundant metal forming about 8 of the earths crust It is found in combination with oxygen silicon fluorine and other elements in soil rocks clays and gems [1] Aluminum chloride (AlCl3) can be found in food products and in drinking water derived from both natural sources and treatment methods [1] Aluminum components are primary found in corn yellow cheese salt herbs spices tea cosmetics cookware utensils and containers Furthermore it is widely used in food additives and toothpaste [2] and widely used in medicines eg antacids phosphate binders buffered aspirins vaccines and injectable allergens [3-5] Recently increased attention is being paid to aluminum due to its serious effects on the central nervous system energy metabolism and hematology [6 7]

The reproductive toxicology of aluminum in male mice has been studied and noticed that some histological changes including necrosis of spermatocytes and spermatids in the testis of male mice treated with aluminum nitrate [8] In addition the same toxicity on mice testis after the effect of sodium fluoride andor aluminum chloride has been reported [9] A single dose of aluminum chloride (400 mgkg body weight) for 15 days of chronic treatment (200 mgkg body weight) for 60 days administered to male mice caused alterations in the metabolism of testis epididymis and vas deferens that led to poor sperm motility and reduction in fertility rate [10-12]

Propolis a resinous sticky substance that honeybees produce by mixing their own waxes with resins collected from plants It is used by bees to repair and maintain their hives and has been used as a folk medicine from ancient times In modern times it has been found to have a wide range of biological activities such as anti-inflammatory [1314] antibacterial [15 16] anticarcinogenic [17] protection against male infertility [18 19] Propolis is also reported to inhibit the generation of superoxide anion Furthermore propolis has been determined to reverse the consumption of glutathione which is synthesized in the liver and has radical scavenging activity [20] Propolis has been reported to be an important antioxidant [21-24] Therefore the present study aimed to elucidate the possible alleviative effects of propolis against the toxicity of aluminum chloride of reproductive performance structure of testis and testosterone hormone levels of male rats

MATERIALS AND METHODS Propolis sample

Propolis from Al-Shafa region situated 20 km from Taif Saudi Arabia with different dominant floras was used The sample was collected from different sites inside the beehive at March 2010 and was kept in the dark in a freezer The sample has been previously analyzed The HPLC analysis of Al-Shafa propolis revealed presence of high amount of predominant phenolic acids cinnamic coumaric caffeic ferulic sinapic and flavonoids apigenin kaempferol quercetin rutin [24]

Chemicals All chemicals used in this experiment were of

analytical grade and purchased from E-Merck (Darmstadt Germany)

Experimental animals Sixty apparently healthy adult male Wister rats

weighing (190plusmn10 g) were procured from animal house of Jeddah national research center The animals were housed (5 rats per cage) in an animal room and were maintained on standard chow and distilled water provided 13 Animals are caged in groups of 3 and given feed and water 13 After two weeks of acclimation animals are divided into three equal groups each containing 20 animals and treated according to the following experimental protocol first group served as control (untreated) second group were given daily AlCl3 via their drinking water (34 mgkg body weight 125 LD50) for 70 days [1] and the third group were administered AlCl3 as in the second group in addition to propolis at dose of 50 mgkg BW [25] Rats are orally administered their respective doses every day for 70 days The duration of the experiments lasts for 70-day for completion of the spermatogenic cycle and maturation of sperms in epididymis [26]

Animal Rights The conducted research followed the

international ethical standards for the care and use of laboratory animals and was approved by the Ethical Committee for Use of Experimental Animals (King Abdelaziz University Jeddah KSA)

Blood samples Blood samples obtained by collected it by

cardiac puncture initially at the beginning of experiment and finally at the end of experiment Heparin was used as anticoagulant Plasma was

13

obtained by centrifugation and stored in at -20 degCuntil utilized for study At the end of each treatment the animals were weighed on an animal weighing balance (Ohaus USA) and sacrificed by over dose of diethyl ether The testes were dissected out carefully blotted of blood and weighted to the nearest milligram

Light microscopy Small slices of testes were taken and fixed in 10

formalin for 24 hours and were imbedded in paraffin Five-micron-thick sections were routinely stained with hematoxyline and eosin and Mallory trichrome methods to demonstrate the tissue fibroses [27] For histochemical study some paraffin sections were stained with periodic acid-Schiff method to demonstrate carbohydrates [28]

Electron microscopy Testicular tissue was cut into small pieces 1 mm

thick and fixed in 4FG in phosphate buffer solution (pH 72) for 3 h at 4 degC after which the tissues were removed and postfixed in buffered 2 OsO4 for one hour at 4degC Post fixed tissues were rinsed in the buffer and dehydrated at 4degC through a graded series of ethanol Then they were embedded in epon-araldite mixture in labeled beam capsules Ultrathin sections (50 nm thick) were cut collected on naked copper-mesh grids and stained with uranyl acetate for 12 hour and lead citrate for 20-30 min [29]

Estimation of plasma testosterone For all the experimental as well the control

groups the Plasma testosterone was estimated by using of the electrochemiluminescence Immunoassay lsquolsquoECLIArdquo Kit obtained from Roche Diagnostics GmbH D-68298 Mannheim USA

Statistical analysis Data were given as mean plusmn SD Statistical

comparisons between different groups were done using one-way analysis of variance (ANOVA) followed by the TukeyndashKramer multiple comparison test [30] to judge the difference between various groups Significance was accepted at lt005

RESULTS

Body and testis weight The rats group orally administrated AlCl3 for 70

days revealed a highly significant decrease in final body weight and body weight gain (lt001) as compared with the control group On the other hand

there were a significant elevation in final body weight and weight gain in AlCl3 group treated with propolis as compared with untreated propolis (Figure 1) Also there was a significant decrease the testes weight (plt001) in AlCl3 group as compared with the control group and there was a significant increase in final testis weight in AlCl3 group treated with propolis as compared with untreated propolis group (Figure 2)

13 13

Figure 1 Histogram showing the effect of AlCl3 alone and AlCl3+ propolis on the body weight of male rat (IBW=Initial body weight FBW= Final body weight and BG=Body gain)

13 13

Figure 2 Histogram showing the effect of AlCl3 alone and AlCl3+ propolis on the testis weight of male rat (ITW=Initial testis weight FTW=Final testis weight)

Serum testosterone and testicular antioxidant enzymes

The oral administration of AlCl3 for 70 days results in highly significant decrease in serum testosterone concentration (lt005) compared to control group while orally treatment with AlCl3 plus propolis caused a significant elevation in serum testosterone concentration (lt005) as compared with the treated AlCl3 group (Figure 3)

13

13

$

Figure 3 Histogram showing the effect of AlCl3 alone and AlCl3+propolis on serum testosterone hormone

Light microscopic examination

In the present study the light microscopy examination of the testis of the control rats should a normal structure of testis with normal thick connective tissue capsule (tunica albogenia) The testis is mainly composed of the seminiferous tubules containing two types of cells the Sertoli cells resting on the thin basement membrane and the spermatogenic cells which arranged in many layers namely the spermatogonia primary and secondary spermatocytes spermatoids and finally mature spermatozoa The normal intestinal of Leydig lying in between the seminiferous tubules and usually associated with blood capillaries (Figure 4 and 5) The testicular tissues also should high positive reaction for PAS (Figure 6)

Figure 4 Photomicrograph of transverse section of testis of control group showing normal histological structure of seminiferous tubules (SM) interstitial cells of Leydig (L) and blood vessel (BV) (HampE X200

Figure 5 Photomicrograph of transverse Section of testis of control group showing normal histological structure of seminiferous tubules (SM) interstitial cells of Leydig (L) (Mallory X200)

Figure 6 Photomicrograph of transverse section of testis of control group showing normal positive reaction for PAS of seminiferous tubules (SM) interstitial cells of Leydig (L) and blood vessel (BV) (PAS X200)

After 70 days of

treatment of rats with aluminum chloride the testes revealed congestion of interstitial blood vessel disorganized germinal epithelium appearance of many focal areas of degenerative changes in the form of degenerative germinal cells together with few fragmented spermatozoa in the lumen of the seminiferous tubules which appeared to acquired a thick irregular basement membrane Also In many areas of seminiferous tubules the germinal cells spermatogenesis arrest at the spermatoids level with reduction in sperm density (Figure 7 and 8) The testicular tissues showed a low positive reaction with PAS (Figure 9)

13

Figure 7 Photomicrograph of transverse section of testis of AlCl3 treated group showing marked disorganized and necrotic germ cells (arrows) lining of the seminiferous tubules and interstitial oedema (L) with congestion of the interstitial blood vessel (BV) as well as thickening of testicular capsule (C) (HampE X200)

Figure 8 Photomicrograph of transverse section of testis of AlCl3 treated group showing marked disorganized germ cells (arrows) lining of the seminiferous tubules damaged of their basement membranes (head arrow)as well as congested interstitial blood vessel (BV) with marked thickening of its wall (Mallory X200)

The light microscopic examination the rats group received AlCl3 and treated with propolis showed an improvement in histopathological toxicity of testis sections The testis showed almost normal seminiferous tubules epithelium with distinct nuclei and sperm bundles in their lumen with normal basement membrane of that tubules as well as normal interstitial cells of Leydig and lack of congestion of interstitial blood vessels as represented in (Figure 10 and 11) A marked improvement in the positive PAS reaction in the testicular was noticed (Figure 12)

Figure 9 Photomicrograph of transverse section of testis of AlCl3 treated group showing low PAS positive reaction in the disorganized germ cells lining of the seminiferous tubules (SM) and the other testicular tissue (PAS X200)

Figure 10 Photomicrograph of transverse section of testis of AlCl3+propolis treated group showing recovery of histological features of germ cells lining of the seminiferous tubules (SM) as well as the interstitial cells of leydig (L) (HampE X200)

Electron microscopic examination

Electron Microscopic Examination of the testis of control group revealed the normal histological structure of seminiferous tubule with various stages of spermatogenesis and interstitial of dig cells The spermatogonia lie on the basement membrane of seminiferous tubules and have two types A-type and B-type The A-type spermatogonia are characterized by large pale ovoid nuclei with homogenous chromatin and scantly cytoplasmThe B-type cells are slightly smaller than type A and characterized by rounded nuclei with more electron dense nucleoplasm The spermatocytes are two types primary and secondary spermatocytes The primary

13

spermatocytes are characterized b scanty cytoplasm spherical nuclei with finely granular nucleoplasm The secondary spermatocytes have short life span so they are rarely seen among the germinal cells of rat The secondary spermatocytes are characterized by their smaller size than the primary spermatocytes and their spherical nuclei The spermatids appear as rounded cells with large spherical nuclei and lightly stained cytoplasm (Figure 13) The Sertoli Cells are a large cell that extends from the basement membrane to the lumen of the seminiferous tubule and containing infolded and sometimes has more irregular shape nuclei with homogenous nucleoplasm and ovoid Golgi apparatus

Figure 11 Photomicrograph of transverse section of testis of AlCl3+propolis treated group showing recovery of histological features of germ cells lining of the seminiferous tubules (SM) as well as the interstitial cells of Leydig and blood vessels (BV) (Mallory X200)

Figure 12 Photomicrograph of transverse section of testis of AlCl3+propolis treated group showing recovery of PAS positive reaction of the germ cells lining of the seminiferous tubules (SM) and the other testicular tissue (PAS X200)

Figure 13 Photo electron micrograph of the testis of a control rat showing Spermatogonia nuclei (N) mitochondria (arrows) X9000

Figure 14 Photo electron micrograph of rat testis after 70 days of treatment with aluminum chloride Showing spermatogonia nucleus (N) with irregular nuclear membrane (NM) Swollen and vacuolated mitochondria (arrows)dilated endoplasmic reticulum (ER)The cytoplasm appears vacuolated (V) with many lipid droplets (L) X18000

Figure 15 Photo electron micrograph of rat testis after 70 days of treatment with AlCl3 and propolis showing recovery spermatid nucleus (N) mitochondria (arrows) endoplasmic reticulum (ER) and Golgi apparatus (G) X15000

13

Electron Microscopic Examination of the testis of rats group treated with AlCl3 for 70 days revealed the seminiferous tubules appeared to have thick basal lamina with fibrous connective tissue with degenerative changes in the germinal cells than in control groups and containing disorganized germinal epithelium The cytoplasm of both of type A and B cells spermatogonia showed swollen and vacuolated mitochondria vascular endoplasmic reticulum and periphery clumped chromatin particles The primary spermatocytes were markedly affected by aluminum chloride toxicity the cells appeared small in size compared to the control group The condensed periphery chromatin and some irregularity of the nuclear membrane were observed The cell cytoplasm appeared vacuolated with lipid droplets Both of Golgi apparatus cisternea and smooth endoplasmic reticulum are dilated and the mitochondria are vacuolated and swollen The spermatid showed generative changes with AlCl3 which appeared in the form an electron dense cytoplasm with dilated and vascular smooth endoplasmic reticulum The Golgi apparatus cisternea were elongated and joined together in groups The mitochondria showed some fragmentations in a vesicular form and more situated periphery (Figure 14)

Electron microscopic examination of the testis of rats group treated AlCl3 combined with propolis for 70 days o the testes revealed an improvement in ultrastructures of the testis which manifested by lack of the generative changes in the seminiferous tubules with normal basal lamina of that tubules as well as normal interstitial cells of Leydig The germinal cells lining of the seminiferous tubules showed marked improvement in their histopathological toxicity caused by the AlCl3 alone particularly in the primary spermatocytes and spermatids (Figure 15)

DISCUSSION

The present results showed that oral administration AlCl3 in rats group caused significant decrease in the body weight gain and weights of testes (lt005) as compared with control group this agreed with the finding of [12 31 32] It was suggested that the reduction in body weight of treated rats with aluminum chloride could be attributed to the decrease in feed consumption and transient diarrhea occurred which resulted in lowering of final body mass of animals in comparison to the controls [33] In addition the

ingestion of aluminum chloride leads to decrease in absolute and relative testes weights and seminal vesicles weight [34] The reported studies [35] noticed that the decrease in the reproductive organs weights could be due to the decrease in testosterone level which was noticed in the present study that may be resulted from the oxidative damage induced in rat testes However it was found that propolis was capable of restoring the body and testes weights in rats group administered AlCl3 and propolis These results were in line with the reported results [19 36] which noticed that the propolis was antagonized and effective in the protection against the reproductive toxicity of AlCl3 in male rats

Testosterone is important hormone that regulates spermatogenesis Our results indicated that AlCl3 induced significant (lt005) decrease in plasma testosterone level as compared to control group These results were in agreement with that reported [37] which demonstrated that the exposure to aluminum lowered plasma and testicular testosterone levels in mice The authors suggested that the severe reduction in male libido and fertility following the aluminum administration might be a result from excessive aluminum accumulation in the testes and low testosterone concentrations However they reported that the high levels of aluminum in aluminum-treated mice were apparent at week 3 before the effects on male libido and fertility proliferatedin addition the others demonistrated also that aluminum administration significantly increased nitric oxide (NO) production and decreased both testicular adenosine 3prime 5prime-cyclic monophosphate (cAMP) and testosterone levels Excessive NO activated inducible NO synthase (iNOS) which may be involved in reproductive toxicity of aluminum and consequently reducing rate and motility of sperm cells increasing their morphological anomalies and suppressing the testosterone secretion in rats testes Moreover these effects of AlCl3 may be attributed to aluminum ability to cross the blood-testis barrier after inducing oxidative stress and lipid peroxidation that disturbs the biological membranes in the testes this in turn leads to the degeneration of the spermatogenic cells that disrupts spermatogenesis and reduces sperm counts [38] The decline in the activity of 17-ketosteroid reductase in animals treated with AlCl3 indicates its adverse effect on production of testosterone hormone which in turn affects on the process of fertility via decreasing testosterone hormone production and sperm production and proliferation and hence motility count and abnormality [36]

13

In addition our results showed that treatment of rats with propolis plus AlCl3 alleviates the testosterone levels nearly to the control level This result is in accordance with that reported [36] which demonstrated that the administration of propolis with AlCl3 was significantly (lt001) increased the activity of 17-ketosteroid reductase compared to control This means that propolis increased the process of steroidogenesis and hence testosterone production The others concluded that the presence of propolis with aluminum chloride showed protective effects of the propolis against the reproductive toxicity of aluminum chloride and this may be due to the activity of propolis as antioxidant The biological effects exhibited by propolis could be related to an overall effect of predominant phenolic acids cinnamic coumaric caffeic ferulic sinapic caffeic acid phenethyl ester and flavonoids apigenin kaempferol quercetin rutin flavonol galangin [18 24 39]

The present histopathological study of rats group orally administered AlCl3 showed apparent toxic changes in the testes which noticed as degenerative and necrotic changes of germ cells lining seminiferous tubules as well as interstitial oedema blood congestion and arrest of spermatogenesis mainly at the spermatid level with reduction in sperm density Meanwhile treatment of AlCl3 group with propolis showed noticeable alleviation in histopathological changes induced by AlCl3 in the structures of testis These results were in agreement with published data which found that after 2 weeks of aluminum treatment deleterious effects and histopathological changes of testicular tissues were observed [40] However noticeable spermatogenetic loss was appeared as necroses in the spermatids and spermatozoa in aluminum-treated group at week 5 The impairment caused by aluminum was accompanied primarily by the prolonged accumulation of aluminum in the mice testes The authors suggested that these changes might have also contributed to the reduction in the pregnancy rate The effect of intraperitoneal injection AlCl3 on ratsrsquo testes was studied and observed that the testis has a histological perturbation including severe damage within the seminiferous tubules and vascular degeneration on the spermatogenic and Sertoli cells cytoplasm [41] The germinal epithelium of the seminiferous tubules was thinner in places and spermatids were almost absent sperm numbers was low and there were no sperm in the lumen The protective effect of propolis against the reproductive toxicity of AlCl3 was studied and the data showed the protective effect of propolis against AlCl3

reproductive toxicity and this may be due to the activity of propolis as antioxidant [36] Also reported studies showed a decrease in the plasma and tissue (liver kidney and brain) malondialdehyde (MDA) levels and an increase in the antioxidant enzyme parameters (SOD CAT and GSH-PX) of animals that were administered propolis in association with propetamphos in comparison to the group that was administered propetamphos alone [42] The primary mechanism of this effect of propolis may involve the scavenging of free radicals that cause lipid peroxidation The other mechanism may comprise the inhibition of xanthenes oxidase which is known to cause free radicals to be generated by propolis Propolis decreased the levels of free radicals and increased the antioxidant enzymes in plasma and different tissues of rats treated with AlCl3 [43]

On the other hand the present electron microscopy study of rat treated with AlCl3 revealed changes in the testis seminiferous tubules such as atrophy of the tubular membrane mitochondria endoplasmic reticulum Golgi apparatus and some irregularity of the nuclear membrane The cell cytoplasm appeared vacuolated with lipid droplets Our results agree with those reported which declared that the administration of sodium fluoride (NaF 10 mgkg body weight) together with aluminum chloride (AlCl3 200 mgkg body weight) to mice for 30 days caused degenerative in structure of spermatogensis and formation of giant cells [12] An intraperitoneal injection of aluminum chloride every other day at doses of 15 and 30 mgkg body weight for five weeks showed histological changes including severe damage within the seminiferous tubules such as atrophy of the tubular membrane mitochondria endoplasmic reticulum Golgi apparatus and nucleus and vascular degeneration of the spermatogenic and Sertoli cells cytoplasm [41] In the present study the testis of rats treated with aluminum plus propolis revealed that it regained nearly its normal structure and remarkable restoration of the normal picture of seminiferous tubules was attained The germ cells lining of the seminiferous tubules appeared regular in shape with disappearance of most cytoplasmic vacuoles These results were in accordance with that reported which declared that the propolis extracts was protect sperm membranes from oxidative attack via reducing TBARS formation and LDH release [18] The authors concluded that the protective effect of propolis in human spermatozoa may be correlated with antioxidant capacity and suggested that propolis may protect against male infertility

13

CONCLUSION The results of the present study showed that

aluminum chloride caused testicular damages and deterioration in testosterone levels The administration of propolis with aluminum chloride showed marked improvement effects against the AlCl3 reproductive toxicity and this may be attributed to the activity of propolis as antioxidant Therefore using diet rich in propolis could be a beneficial way to overcome the reproductive toxicity of aluminum

REFERENCES

1 Sigel H Sigel AE Metal ions in dialysis syndrome and

aluminum intoxication Nephron 1988 311-10 2 Abbasali KM Zhila T Farshad N Developmental toxicity of

aluminum from high doses of AlCl3 in mice J Applied Res 2005 5575-579

3 Cannata JJ Briggs R Junor S Fell Aluminum hydroxide intake real risk of aluminum toxicity Br Med J 1983 2861937-1938

4 Lione A Aluminum toxicity and the aluminum containing medication J Pharmacol Ther 1985 29255-285

5 Jones XC Bennett BG Exposure of man environmental aluminum-an exposure commitment assessment Sci Total Environ 1986 5265-82

6 Graves AB White E Koespsell TD Relfler BV Belle GV Larsom EB The association between aluminum containing products and Alzheimerrsquos disease J Clin Epidemiol 1990 4335-44

7 World Health Organization (Geneva) Environmental Health Criteria 194 Aluminum Printed in Finland 97PLL11539-Vammala-5000 WHO 1997 1-282

8 Liobet JM Corbella J Domingo JL Sirventand JJ Colomina MT Reproductive toxicology of aluminum in male mice Fund Applied Toxicol 1995 2545-51

9 Memon MR Chinoy NJ Effect of sodium fluoride andor aluminum chloride treatments on some organs of male mice [abstract] XVIth National Symposium on Reproductive Biology and Comparative Endocrinology Jan 21-22 University of Kerala Trivandrum India 1998

10 Chinoy NJ Bhattacharya S Effect of single dose of aluminum chloride on some reproductive organs and fertility in male mice Indian J Environ Toxicol 1996 610-13

11 Chinoy NJ Bhattacharya S Effects of chronic administration of aluminum chloride on reproductive functions of testis and some accessory sex organs of male mice Indian J Environ Toxicol 1997 712-5

12 Chinoy J Sorathia P Jhala D Flouride+Aluminum induced toxicity in mice testis with giant cells and its reversal by vitamin C J Fluride 2005 38(2)109-114

13 Khayyal MT El-Ghazaly MA El-Khatib AS Mechanisms involved in the anti-inflammatory effect of propolis extract Drugs Exp Clin Res 1993 19197-203

14 Yoshizumi K Nisshioka N Tsuji T Xanthine oxidase inhibitory activity and hypouricemia effect of propolis in ratsYakgaku Zasshi Mar 2005 125(3)315-21

15 Sforcin JM Kaneno R Funari SRC Absence of seasonal effect on the immunomodulatory action of Brazilian propolis on natural killer activity J Venom Animals Toxins 2002 819-29

16 Oris RO Sforcin JM Funri SR Bankova V Effects of Brazilian and Bulgarian propolis on bactericidal activity of macrophages against salmonella Typhimurium Int Immunolpharmacol 2005 5(2)359-68

17 Aliyazicioglu Y Deger O Ovali E Barlak Y Hosver I Tekelioglu Y Karahan SC Effects of Turkish pollen and propolis extracts on respiratory burst for K-562 cell lines Int Int Immunolpharmacol 2005 5(11)1652

18 Russo A Troncoso N Sanchez F Garbarino JA Vanella A Propels protects human spermatozoa from DNA damage caused by benzo[a] pyrene and exogenous reactive oxygen speciesLife Sci 2006 301-7

19 Yousef MI Kamel KI Hassan MS El-morsy AM Protective role of propolis against reproductive toxicity of triphenyltin in male rabbits Food Chem Toxicol 2010 48(7)1846-1852

20 Castaldo S Capasso F Propolis an old remedy used in modern medicine Fitoterapia 2002 731-6

21 Yousef MI Aluminum-induced changes in hemato-biochemical parameters lipid per oxidation and enzyme activities of male rabbits Protective role of ascorbic acid Toxicology 2004a 19947-57

22 Yousef MI Kamel IK Esmail AM Baghdadi HH Antioxidant activities and lipid lowering effects of isoflavone in male rabbits Food Chem Toxicol 2004 421497-1503

23 Mani F Damasceno HC Novelli EL Martins EA Sforcin JM Propolis effect of different concentrations extracts and intake period on seric biochemical variables J Ethnopharmacol 2006 10595-98

24 Abd El-Mawla AMA Osman HEH HPLC analysis and role of the Saudi Arabian propolis in improving the pathological changes of kidney treated with monosodium glutamate Spatula DD 2011 1(3)119-127

25 Park EH Kahng JH Suppressive effects of propolis in rat adjuvant arthritis Arch Pharm Res 1999 22554-558

26 Sarkar M Roy Chaudhury G Chattopadhyay A Biswas NM Effect of sodium arsenate on spermatogenesis plasma gonadotrophins and testosterone in rats Asian J Androl 2003 527-31

27 Drury RAB Wllington Carletons hitological Technique 5th ednoxford Univer 1980 p155-158

28 Hotchkiss RD A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations Arch Biochem 1948 16(1)131-141

29 Reynolds ES Staining of tissue sections for electron microscopy with heavy metals J Cell Biol 1963 17 203-212

30 Ipstein J Poly F Brancroftrsquos introduction to biostatistics 2nd ed harper international New York 1970

31 Sallam SMA Nasser MEA Yousef MSH El-morsy AM Mahmoud SAS Yousef MI Influence of aluminum chloride and ascorbic acid on performance digestibility caecal microbial activity and biochemical parameters of rabbits Research journal of agriculture and biological sciences 2005 1(1)10-16

32 Hala AH Khattab Inas ZA Abdallah Gehan MK Grape seed extract alleviate reproductive toxicity caused by aluminum chloride in male rats Journal of American Science 2010 6(12)1200-1209

33 Cherroret G Capolaghi B Hutin MF Burnel D Effects of postnatal aluminum exposure on biological parameters in the rat plasma Toxicol Lett 1995 78119-125

34 Bataineh H Al-Hamood MH Elbetieha AM Assessment of aggressive sexual behavior and fertility in adult rat following long term ingestion of four industrial metals salts Hum Exp Toxicol 1998 17570-579

35 El-Ashmawy IM Saleh A Salama OM Effects of marjoram volatile oil and grape seed extract on ethanol toxicity in male rats Basic amp Clinical Pharmacology amp Toxicology 2007 101320-327

13

36 Yousef MJ Salama FA Propolis protection from reproductive toxicity caused by aluminum chloride in male rats Food and Chemical Toxicology 2009 47(6)1168-1175

37 Guo C Lu Y Hsu GSW The influence of aluminum exposure on male reproduction and offspring in mice Environ Toxicol Pharmacol 2005 20135-141

38 Latchoumycandane C Chitra KC Mathur PP The effect of methoxychlor on the epididymal antioxidant system of adult rats Reprod Toxicol 2002 16161-172

39 Lee KG Shibamoto T Takeok GR Lee SE Kim JH Park BS Inhibitory effectsof plant-derived flavonoids and phenolic acids on malonaldehyde formation from ethyl arachidonate JAgriculFood Chem 2003 517203-7207

40 Guo C Huang CJ Yeh MS Hsu GSW Aluminum induced suppression of testosterone through nitric oxide production in male mice Environ Toxicol Pharmacol 2005 b 1933-40

41 Khattab FK A histological and ultrastructural study on the testis of rat after treatment with aluminum chloride Ustralian Journal of Basic and Applied Sciences 2007 1(1)63-72

42 Kanbura M Eraslan G Silici S Antioxidant effect of propolis against exposure to propetamphos in rats EcotoxicolEnviron Safety 2009 72900-915

43 Newairy AA Salama AF AF Hussien HM MI Yousef MI Propolis alleviates aluminum induced lipid peroxidation and biochemical parameters in male rats Food Chem Toxicol 2009 471093-1098