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ISSN 2349 – 4425 www.americanij.com The Protective Effect Of Some Natural
Antioxidants Against Azithromycin Induced
Testicular Dysfunction In Rats
El-Dakak, Abeer M. N. H.Ph.D
Special Food and Nutrition Dept., Food Technology Research Institute, Agric. Res. Center, Giza, Egypt Email address: [email protected] Abstract:
Objective: To investigate whethervitamin E, selenium, β-carotene, anthocyanin and green tea extract have a
possible protective effect against azithromycin (AZ)-induced testicular dysfunction. Design: Experimental study.
Setting:Research Institute of Ophthalmology, Giza, Egypt.
Patient(s):3 -week-old weanling male albino rats (n = 54).
Intervention(s):AZ was administered to rats at dose of 20mg/kgbw orally for 3 days every two weeks.
Vitamin E, β-carotene pigment,anthocyanin pigment, green tea extract, Se as sodium selenite and their mixture were administered with AZ by gavage daily for 4 months. Main Outcome Measure(s):Body and reproductive organ weights, spermatozoal activity, fructose contents,
testosterone (T), interstitial cell-stimulating hormone (ICSH), lipid peroxidation and, antioxidant enzyme activities were investigated.Histopathological changes in reproductive organs examined. Result(s):Significant decreases in serum T and ICSH,reproductive organ weights, spermatozoal activity and
fructose content were recorded due to AZ administration, whereas these parameters increased the antioxidants-treated groups. A significant decrease in Testicular MDA level and marked increases in GSH- Px, SOD, and CAT activities were observed in rats treated with different antioxidants. However, the most positive treatment was Se-treatment and the mixture of vitamin E with β-carotene pigment. AZ-treatment led to adverse histopathologic changes in the testes, epididymis cuda, seminal vesicle and prostate, whereas normalhistopathological imageof the studied reproductive parts were observed in groups treated with Se, Vitamin E and β-carotene. Conclusion(s):Antioxidants have a protective effect against testicular toxicity caused by AZ. This protective
effect of the antioxidants seems to be closely involved with the suppressing of oxidative stress. Key words: Azithromycin; Antioxidants; Oxidative stress; spermatozoal activity; Testes.
Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com occurring prior to in-vitro fertilization (IVF) treatment, or 1. INTRODUCTION
when high concentrations of leukocytes are present in the Infertility or sterility is defined as the total inability to semen of these patients, irrespective of microbial evidence reproduce (1); it has been estimated that 7.2% of women and
of infection. Patients on a course of antibiotics often their husbands in Egypt 2012 are infertile according to demonstrate below-average semen parameters. While in WHO, 2013(2). Infertility has an element of time in its
some instances this may be caused by the infection itself, it definition;it can be stated as failure to conceive after one is likely that the antibiotics have a direct effect on sperm year of regular unprotected intercourse with the same function (10). Studies also have shown that antibiotics
partner (3). Infertility can be classified into primary and
e.g.,Cotrimoxazole, dicloxacillin, erythromycin, lincomycin, secondary, A male without biological offspring can be said neomycin, nitrofurantoin, quinolones, tetracycline, and to have ―primary infertility,‖ whereas a male who is unable tylosin;considerably impair sperm motility characteristics to impregnate his partner but who already has biological (8,11) and functional integrity of mature sperm (12).
children is referred to as having ―secondary infertility‖ (4).
Furthermore, perfloxacin produced toxic effect on testicular For healthy young couples, the probability of getting function in animals (13).
pregnancy per a reproductive cycle is about 20% to 25%. Their cumulative probabilities of conception are 60% within homoerythromycin, Fig.(1), an erythromycin semisynthetic
the first 6 months, 84% within the first year, and 92% within derivative is formed by inserting a methyl-substituted the second year of regular fertility-focused sexual activity nitrogen in place of the carbonyl group at the 9a position of the aglycone ring. The resulting dibasic 15-membered ring Several studies have suggested that human semen macrolide derivative is more appropriately referred to as an quality and fecundity have been declining during the past ‗‗azalide.‘‘ This structural modification gives azithromycin decades(6). Approximately 10-15% of couples demonstrates
several distinct advantages over erythromycin: makes the primary infertility and of these a male factor is identified in compound more stable in acid, significantly increases the approximately 50% of cases (7).Many extrinsic and
environmental factor including the increased use of gastrointestinal tolerability, oral bioavailability and tissue antibiotics have been implicated as potential causes of male penetration, and results in increased activity against gram- negative organisms when compared with erythromycin (14).
inflammatory diseases (PID) and sexually transmitted Its extensive coverage against all the major respiratory infections (STI).In addition personal habits that considered pathogens, excellent safety profiles and desirable tissue risk factors for infertility, i.e., excess alcohol intake, distribution into the infection sites make azithromycin a cigarette smoking and other forms of drug abuse. Therefore, good choice for the treatment of respiratory tract infections Onyije (9) revealed that drugs such as antibiotics, alcohol,
(15).Azithromycin is frequently prescribed for the treatment
tobacco products, illicit drugs and certain medications can of middle ear and upper respiratory tract infections, limit the chances of men having children in the present time, bronchitis, and community-acquired pneumonia (16).In
or even far into the future. recent years, it has been used primarily to prevent bacterial Antibiotics such as amoxicillin and tetracycline are infections in infants and those with weaker commonly prescribed for a multitude of conditions (10), in
most cases these antibiotics are used in treating infections Macrolide antibacterial agents including azithromycin are that are unrelated to infertility. In addition, some patients lipophilic and are extensively distributed in body fluids and requiring assisted conception occasionally show evidence of tissues. They exert their antibacterial effects by reversibly infection on the male reproductive tract. The antibiotics co- binding to the 50s subunit of the bacterial ribosome. This trimoxazole and erythromycin are routinely used by interaction inhibits RNA-dependent protein synthesis by urologists and fertility specialists to treat bacterial infections preventing transpeptidation and translocation reactions (18).
Volume 2 2015 Issue 1 DEC-JAN AIJCSR

ISSN 2349 – 4425 www.americanij.com and produce infertility by two key mechanisms. First, ROS damage the sperm membrane, which in turn reduces sperm's motility and ability to fuse with the oocyte. Secondly, ROS directly damage sperm DNA,compromising the paternal genomic contribution to the embryo. Despite the common association between compromised sperm quality and oxidative damage, men are rarely screened for oxidative stress nor treated for this condition(23).
In more details, free radicals have the ability to directly damage sperm DNA by attacking the purine and pyrimidine bases and the deoxyribosebackbone. Normally, sperm DNA is tightly packaged by protaminesprotecting it from free radical attack. However, infertile men often exhibit deficient protamination, leaving the spermDNA particularly vulnerable to ROS attack (24). Alternatively,
free radicals can initiate apoptosis within the sperm, leading to caspase-mediated enzymatic degradation of the DNA Fig. 1. Azithromycin
Certain macrolide antibiotics have been documented to be hepatotoxic in animals (19) and human subjects in clinical
highunsaturated fatty acids and are sensitive to oxygen studies (20). Macrolide antibiotics are also known to alter
induced damage mediated by lipid peroxidation. The the physiological redox homeostasis leading to oxidative excessive generation of ROS by abnormal spermatozoa and stress and lipid peroxidation(21).
by contaminating leukocytes that has been identified as one Cells require oxygen for certain metabolic processes of the few etiologies for male infertility (26).
including the metabolism of xenobiotic and in the process of Actually, a balance between the benefits and risks from certain destructive species referred to as reactive oxygen reactive oxygen species and antioxidants appears to be species (ROS) are generated (22). The cell protects itself
necessary for survival and normal functioning of from these ROSs, by the actions of certain low molecular spermatozoa (27). Increased production of lipid peroxide is
weight substances such as glutathione, vitamin A, E, and C associated with decrease in Zinc as antioxidants .An (the non enzymic antioxidants) and enzymic antioxidants imbalance in this harmony may be responsible for the such as superoxide dismutase (SOD), catalase (CAT) etc. inactivation of hyaluronidase activity thereby leading to Therefore, the objective of this study was to investigate the severity of oligozoospermia(28).Yadavaet al.,(29) observed
effect of azithromycin as antibiotic on the markers of that decrease in total antioxidant power correlates with testicular function like a sperm quality, spermatogenic cell abnormal sperm membrane integrity by ROS and increased density, testosterone level, antioxidant activity and lipid nitric oxide concentration. This suggests that imbalance peroxidation in male rats. between antioxidants and reactive nitrogen species results in Oxidative stress (OS) occurs when the production sperm membrane integrity. of potentially destructive reactive oxygen species (ROS) It has been proposed that bactericidal antibiotics exceeds the bodies own natural antioxidant defenses, can induce cellular death through a common oxidative resulting in cellular damage. OS is a common pathology damage mechanism that relies on the production of seen in approximately half of all infertile men. ROS, defined ROS.Through their various primary targets, antibiotics can as including oxygen ions, free radicals and peroxides are activate cellular respiration, which leads to the formation of generated by sperm and seminal leukocytes within semen superoxide and the release of iron from iron-sulfur clusters. Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com Free iron then activates a chemical reaction, i.e., the Fenton allergies and co-administrated drugs, is important in order to reaction) to produce ROS in the form of hydroxyl radicals minimize the risk of adverse reactions (34). The side effects
(OH•). These radicals can cause cellular death by damaging of antibiotics on spermatogenesis are seldom discussed in proteins, lipids, and DNA, or can cause mutations leading to the literature. The increaseincidence of sterility in man along the development of antibiotic resistance(30).Nguyen,(31)
with increase in number of new therapeutic drugs found that mutant bacteria deficient in the stringent response administrated to patients has made experimental work on exhibited tolerance to a wide range of antibiotics including antibiotics in male reproduction important since these ofloxacin, meropenem, colistin, and gentamicin by medication found to adversely affect male reproductive increasing antioxidant enzyme production and blocking the functions (35).
production of pro-oxidant molecules, thus reducing toxic However, the mechanism(s) by which antibiotics OH•. These mutant bacteria also were more susceptible to exert their antifertility effect in the male are yet to be fully ofloxacin in a mouse infection model. Moreover, Goswami,
elucidated andseveral studies have demonstrated the et al., (32) studied the effect of dietary and cellular
antifertility effects of free radicals and numerous antioxidants on antibacterial effect of commonly used mechanisms of action have been proposed (9, 11,13).Hence,
antibiotics. Dietary supplements such as vitamin C previous investigationssuggested that OS may be an (Ascorbic acid) and E (-tocopherol), having antioxidant important mediator of testicular injury. The present study properties, are prescribed nowdays by the physicians along was therefore designed to investigate the role of some with antibiotics during the course of treatment of infection. e.g., vitamin E, β-carotene, Besides antioxidants such as N-acetylcysteine (NAC) are anthocyanins, polyphenols in green tea extract and selenium used as auxiliary medication in certain pathological on azithromycin (antibiotic) induced alterations which conditions along with the antibiotic therapy (NAC is used as massively used during early age cycles and its effect on a mucolytic agent in combination with clinically relevant male reproductive functions. antibiotics for treatment of lower respiratory tract infection). Therefore it is important to understand the effects of 2. Materials and Methods
antioxidants on theantibacterial action of commonly used 2.1. Materials
antibiotics. Metabolic action of eachantibiotic should be 2.1.1.Lyophilized green tea extract:Ten volumes of
taken in account when applied. boiling water were added to tea (10v/ wt) and allowed Antioxidant mediated protection against ciprofloxacin could standing for 30 min at room temperature and the extracted be through scavenging of ROS generated in the presence of was filtered and lyophilized (36). The lyophilized green tea
antibiotic. Consequently effect of mutations in oxidative extract used in this study contained 21.87 g catechins /100g stress defense genes viz. superoxide dismutases (sodA, green tea) was measured by HPLC. 2.1.2.Lyophilized anthocyanins pigments from red grape
hydroperoxidereductase marc: The lyophilized anthocyanin pigment was prepared
susceptibility of MG1655 was studied. These genes encode according to the method reported by Revilla, et al., (37) and
enzymatic defense system against ROS, regulating their modified by El-Dakak (38). It was composed mainly of
intracellular steady-state level(32).
delphinidin 3-O-glucoside (Df-Gl), cyanidin 3-O-glucoside In another case, oral antimicrobial agents belonging (Cy-Gl), petunidin 3-O- glucoside (Pt-Gl), peonidin 3-O- to beta-lactams, quinolones, macrolides, tetracycline and the glucoside (Pn-Gl), malvidin 3-O-glucoside (Mv-Gl), trimethoprim-sulfamethaxazole combination are among the malvidin 3-O- acetylglucoside (Mv-Gl-Ac) and malvidin 3- most prescribed classes of drugs in medical practice(33).
O-p-coumarylglucoside (Mv-Gl-Cou) as measured by Again, knowledge of the potential side effects considered in HPLC. Total amount equivalent to 89.84g/ 100g lyophilized the light of various patient associated factors such as genetic anthocyanin pigment. make-up, renal and liver function, underlying disease, drug Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com
2.1.3.Lyophilized β-carotene pigment from Carrot pulp
Rats were observed daily for the appearance of any waste:HPLC analysis indicated that β-carotene was found to
symptoms of discomfort that might be related to studied be as 83.8% in the lyophilized β-carotene pigment; which treatments. Body weight (BW) of the rats was recorded daily was prepared according to the method reported by Chen
before administration of studied additives. At the end of &Tang (39) andmodified byEl-Dakak (38).
experiment period, the percentage of weight gain was 2.1.4. Drugs:Azithromycin (Zithromax® 250 mg and 500
expressed [(final weight – beginning weight) / beginning mg tablets) was a product of Pfizer, New York. 2.2.3. Weight of reproductive organs
2.1.5. Animals:MaleWister strain albino rats weighing45 g
± 2 (3 weeks age) obtained from Research Institute of At the end of the experimental period, rats were weighted Ophthalmology, Giza, Egypt, were used for the study. Rat and killed by diethyl ether. Testes, epididymis, seminal cubes were fed on laboratory chow and tap water ad vesicles and prostate were cut off, washed in ice-cooled libitum4 months.The animals were housed in separate saline solution0.15M KCl to remove blood and weighed. stainless steel cages raised in a well-ventilated room with Organ weights were recorded after autopsy and represented 12-h light/dark cycle. as weight per 100 g body weight (42).
Right hemiprostate and seminal vesicle with the adherent 2.2. Methods
coagulating gland were removed and stored at –20°C until 2.2.1.Experimental design:All animal rights have been
fructose content was determined (43).
considered during all periods of the experiments according 2.2.4.Spermatozoal activity
to the Animal Care and Use Committee (IACUC) The Resazurin Reduction Test (RRT) was applied for guidelines. Fifty four male albino rats (9/group of six each) determining spermatozoal activity (44). RRT depends on the
wererandomlyallotted into experimental groups as follows. ability of metabolically active spermatozoa to reduce the Group1 (negative control) were administered with distilled resazurin dye (blue), with maximum absorption at 615 nm water (vehicle for the drug). The other rats treated with (A615), to resorufin (pink) with a maximum absorption of 20mg/ kgbw of azithromycinby oral gavage once daily at an 580 nm (A580). The ratio of the optical densities of reduced interval of 24 hr. between successive treatments,for 3 days to oxidized form, i.e., 580 to 615 nm can be used to evaluate every two weeks(which represents the therapeutic dose of the various grades of semen sample. Semen samples were the drug in humans),then divided into 8 groups; group II centrifuged at 2500 g for 20 min. The seminal plasma rats,which also served as the positive control.After supernatant was applied for test according to manufacturer's azithromycin administration, rats in groups III and IV were constructions using kits purchased from Bio-diagnostic, co-treated orally with Vitamin E (3IU /kg bw), β-carotene (50mg/kg bw) in corn oil, per day; respectively.Ingroups V, 2.2.5. Biochemical assay
VI and VIIrats were co-treated orally with anthocyanin At the end of experimental period, blood samples were (71mg /kg bw), green tea (120mg/kg body weight), with collected from the eye plexuses of animals by a fine selenium (0.1 mg/kg bw) as sodium selenite in deionized capillary glass tubes and placed immediately on ice. Blood water a day, respectively.Lastly, groups VIII and VIIIIrats serum samples were collected into dry clean centrifuge were co-treated orally with anthocyanins (24mg /kg bw)+ tubes; the serum was separated after centrifugation for 10 green tea extract (40mg/kg bw) + Se (0.03mg sodium sel/kg min at 3000 rpm (1500 xg) and kept at –20 ºC until analysis. bw)in deionized water; Vitamin E (1.5 IU /kg bw), β- Testosterone and interstitial cell-stimulating hormone carotene (25mg/kg bw) in corn oil, per day; respectively(40,
(ICSH) concentrations were measured in triplicate by 41). The Antioxidants application was given throughout the
ELISA according to the producer's instructions (Gama whole trial except time of drug administration. 2.2.2.Body weight gain (BWG)
2.2.6.Biochemical analysis of testis
Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com The right testis homogenate was centrifuged at 800 testis and epididymis as compared with the control animals rpm for 20 min at 4°C. The supernatant was used for (P<0.05) (Table 1). Azithromycin also caused decrease in
biochemical analysis. weights of seminal vesiclesand prostate gland when 2.2.6. 1.Protein
expressed as mg/ 100gm body weight(Table 1). Co-
The protein level in the testis homogenate was administration of azithromycin with different treatments assessed by the method described by Gornall&Bardawill
significantly prevented the decrease in the weights of the (45)to express other biochemical parameters as per mg
prostategland. However, the remained accessory glands proteinusing kits obtained from Randox Laboratories Ltd., weights significantly increased by different antioxidant. Co- Antrim, United Kingdom. treatment of selenium (0.1 mg/kg bw) recorded the highest 2.2.6. 2.Estimation of enzymatic antioxidants
value in the relative testicular weights and weight of seminal The superoxide dismutase (SOD),catalase (CAT), vesicles. However, co-treatment of anthocyanin (24mg /kg and glutathione peroxidase (GSH-PX) in the testis were bw)+ green tea(40mg/kg bw) + selenium (0.03mg/kg bw) estimated according to (46- 48)respectively, using kits
appeared to be the highest value in the relative cauda obtained from Biodiagnostic. epididymis weight.It is to theorizethat is the weight of 2.2.6. 3.Lipid peroxidation
sexual accessory gland decrease following azithromycin The Lipid peroxidation (Malondialdehyde,MDA) administration may be explained by reduction of plasma was estimated according to Metlzer et al (49), using kits
level of testicular androgens. obtained from Biodiagnostic. Table 1.Body weight gain and weight of reproductive
2.2.6.4. Fructose content
organs of male rats in four months of treatments. Fructose content of accessory sex glands (testis, Weight of organs (mg/ 100 g BW)
right hemiprostate and seminal vesicle with the adherent Daily intake
weight gain
coagulating gland) wasdetermined using the procedure of Mann (50) using kits purchased from Bio-diagnostic, Egypt.
2.6. Histopathological studies
Different sections of studied reproductive organs (left testis, control,
basal diet)
caudaepididymes, seminal vesicles and prostate) were prepared for histological examination (51).
2.7. Statistical analysis
(Control
treated with
All values are means ± SD obtained from eight animal 20mg kg-1bw
groups (six of each). Data were analyzed with SAS software (SAS Institute, Cary, N.C.) using SAS analysis of Vitamin E(3
variance(PROC ANOVA). Significant differences between IU /kg bw)
means were determined by Duncan's multiple range test (P < (50mg/kg
3. Results
3.1.Weight gain and reproductive organs weight
The body weight gain and relative testicular weights and (120mg/kg
accessory glands weights are presented in Table 1. The body
weights of the treated rats did not show significant changes Selenium (0.1
mg/kg bw)
(Table 1). In the group treated with azithromycin alone,
there was significant decrease in relative weights of the Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com (mg/kg BW)
[Anth (24mg
/kg bw)+ ,
(40mg/kg
(0.03mg/kg
control, basal diet)
[Vitamin E(
(Control 1.23±0.14g
0.74±0.18f
1.5 IU /kg
treated with 20mg kg-1bw
carotene
of azithromycin)
(25mg/kg
Abbreviations; t = testes, p = prostate, c = cauda epididymis, sv = seminal Vitamin E(3 IU /kg bw)
4.89±0.09c
1.38±0.22e
vesicles. Values are means ± SD (n=6);within each classvalueswith different superscripted letters in the same column are significantly different (p < 0.05). β-carotene(50mg/kg bw)
2.48±0.30e
3.2.Serum the testosterone (T) and interstitial cell-
1.69±0.12f
stimulating hormone (ICSH) concentrations
The levels of T and ICSH in rats treated with azithromycin, Vitamin E, β-carotene, anthocyanin, green tea, 5.52±0.36b
2.43±0.2bc
and selenium are shown in Table 2. Azithromycin
extracted(120mg/kg bw)
administration significantly decreased serum T and ICSH concentration (P < 0.05) compared to the control group. Co- Selenium (0.1 mg/kg bw)
8.55±0.42a
3.68±0.30a
administration of Vit E, β-carotene, anthocyanin, green tea, selenium, mixture of the (anthocyanin+ green tea + Anthocyanin+ Green tea
3.43±0.36d
2.83±0.74b
selenium) and mixture of the (Vitamin E+ β-carotene) extract +Se
increased (P <0.05) the testosterone levels by (276, 32), (91, 147), (31, 146), (325, 230), (559, 399), (164, 283) and (310, [Anthocyanin (24mg /kg
373)respectively, relative to the azithromycin-treated bw)+ , green tea extract
(40mg/kg
(0.03mg/kg bw)]
Table 2. Testosterone (T) and interstitial cell-stimulating
hormone (ICSH) level of secretion in blood serum of male Vitamin E+ β-carotene
5.32±0.28b
3.49±0.20a
rats in four months of treatments. [Vitamin E( 1.5 IU /kg
(25mg/kg bw)]
Values are means ± SD (n=6);within each classvalueswith different superscripted letters in the same column are significantly different (p < 0.05). Daily intake
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ISSN 2349 – 4425 www.americanij.com
3.3.Fructose content and spermatozoal activity in the
reproductive organs
Fructose content of the four tested reproductive organs were determined and represented in Fig. 2.
Azithromycin administration significantly decreased the fructose content of prostate and seminal vesicle plus coagulating gland in relation to the negative control group. Clearly, all treatments applied in the present study led to significant increase in the fructose level in the sexual studied 3.3.Antioxidant and testicular markers enzymes
organs. However, the severest treatment was selenium In order to explore the possibility that azithromycin treatment (G7).Although, the use of mixture of vitamin E interferes with antioxidant defense system and thereby with β-carotene (G9), especially seminal vesicle(Fig. 2).
induces oxidative damage to rat testes, the antioxidant Therefore, thespermatozoal activity was selected using levels, testicular marker enzymes and marker of oxidative resazurin reduction test (RRT), as a ratio between A580 to stress were evaluated. The activity of SOD and CAT in the A615 (RRTR). All groups, insignificant (p>0.05) alterations post-mitochondrial fraction of rat testes decreased were recorded in spermatozoal activity after treatment with significantly by 31% and 53% in the rats treated with AZ different antioxidants(Fig. 2). However, animals were
compared with the corresponding group of control rats (Fig
subjected to 20 mg AZ/kgbw showed significant (p<0.05) 4, 5). This decrease was prevented by 31% and 80%,
reduction in their spermatozoal activity. All treatments led to respectively, on co-administration with Vit E. Treatment significant (p<0.05) elevation in spermatozoal activity of all with β-carotene increased the activity of SOD by 33% and tested reproductive organs, except the treatment with that of CAT by 92%. However co-administration with selenium (G7) that led to insignificant change in selenium (G7) or vitamin E + β-carotene (G9) recorded the spermatozoal activity of caudaepididymes and testes (Fig.2).
highest elevation percent in the activity of SOD by 48%, In general, the obtained data indicated that, the treatments 45% and that of CAT by 134% and 121%, but treatment with selenium (G7) or vitamin E + β-carotene (G9)recorded with anthocyanin (G5) and green tea (G6) recorded the the highest value except only the treatment with selenium lowest elevation percent in the activity of SOD by 25%for (G7) that led to insignificant change in spermatozoal activity each and that of CAT by 42% and 29%. of testes, however treatment with selenium (G7), β-carotene Testicular GSH-Px decreased significantly by 47% (4) and it's mixture with vitamin E (G9) recorded the highest in the AZ-treated rats relative to the controls (Fig. 6).All
value in caudaepididymes and (Fig.2). Moreover, the co-
treatment with antioxidants increased the level of the GSH- admiration with different antioxidants led to attitude the Px to normal except co-administration with selenium (G7) hazard effect of Azithromycin on the spermatozoal activity. that recorded the highest value in GSH-Px. In Contrary, anthocyanin and green tea extracttreatments gave the lowest elevation percent in the activity of GSH-Px. Volume 2 2015 Issue 1 DEC-JAN AIJCSR

ISSN 2349 – 4425 www.americanij.com revealed atrophy and degeneration of seminiferous tubules associated with interstitial edema (Fig.8).
3.3.Markers of oxidative damage
The level of MDA, a marker of lipid peroxidation, testicular MDA increased significantly (p<0.05) in rats treated with AZ. These increment was markedly (p<0.05) decreased by administration of all treatmentscomparing to AZ-treated rats (Fig.7). Rationally, co-administration with
selenium (G7) recorded the lowest value in MDA. 3.4. Histological changes
The photomicrographs in Fig. 8 illustrated the different
histopathologic changes that were observed in the testis of animals given various treatments used in this work. Administration of AZ caused severehistopathologic lesion such as vacuolations and degeneration of spermatogoneal cells lining seminiferous tubules. Normal seminiferous tubules were noticed in testes of control negative rat (G1). No histopathological changes were noticed in testes of rat from group 3 (Fig.8). Increase diameter of seminiferous
tubule was noticed in testes of rat from groups 4 and 7 (Fig.8). However, examined sections from groups 5 and 6
revealed atrophy of seminiferous tubules (Fig.8). Apparent
normal seminiferous tubules were observed in testes of rats from group 8 (Fig.8). Meanwhile, tests of rat from group 9
Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com Fig (8) Histopathological image of Testes tissue from control rats, AZ and AZ plus different antioxidants-treated animals after in four months of treatments G1:Control (-) (Normal control, basal diet), G2:Control
(+) (Control treated with 20mg kg-1bw of azithromycin),
G3:Vitamin E(3 IU /kg bw), G4:β-carotene (50mg/kg bw),
G5:Anthocyanin (71mg /kg bw),G6:Green tea extracted
(120mg/kg
bw),G8:[Anthocyanin (24mg /kg bw)+ , green tea extract
(40mg/kg bw) + Se (0.03mg/kg bw)], andG9: [Vitamin E(
1.5 IU /kg bw), +β-carotene (25mg/kg bw)].
Normal histopathological image of epididymis were observed in all groups, except epididymis of control negative rat (AZ administration) in group 2 showed vacuolation of epithelial lining tubules (Fig.9).
Fig.9Histopathological image of epididymis tissue from
control rats , AZ and AZ plus different antioxidants-treated animal after in four months of treatments G1:Control (-) (Normal control, basal diet), G2:Control
(+) (Control treated with 20mg kg-1bw of azithromycin),
G3:Vitamin E(3 IU /kg bw), G4:β-carotene (50mg/kg bw),
G5:Anthocyanin (71mg /kg bw),G6:Green tea extracted
(120mg/kg
bw),G8:[Anthocyanin (24mg /kg bw)+ , green tea extract
(40mg/kg bw) + Se (0.03mg/kg bw)], andG9: [Vitamin E(
1.5 IU /kg bw), +β-carotene (25mg/kg bw)].
Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com Microscopically, no histopatholgical change were noticed in seminal vesicle of rat from group 1(control-ve) and group 4 (Fig.10). However, seminal vesicle of group 2 (control+ ve
rat) revealed atrophy and shortening of epithelial lining (Fig.10). Meanwhile, seminal vesicle of rat from groups 3,
5, 7, and 9 showed hyperplasia of epithelial lining (Fig.10).
Examined sections of rat from group 6 showed focal necrosis of epithelial lining (Fig.10). Marked atrophy and
necrosis of epithelial lining were observed in seminal vesicle of rat group 8 (Fig.10).
Fig 10 Histopathological image of seminal vesicle tissue from control rats, AZ and AZ plus different antioxidants-treated animals after in four months of G1:Control (-) (Normal control, basal diet), G2:Control (+) (Control
treated with 20mg kg-1bw of azithromycin), G3:Vitamin E(3 IU /kg bw),
G4:β-carotene (50mg/kg bw), G5:Anthocyanin (71mg /kg
bw),G6:Green tea extracted (120mg/kg bw),G7:Selenium (0.1 mg/kg
bw),G8:[Anthocyanin (24mg /kg bw)+ , green tea extract (40mg/kg bw) + Se
(0.03mg/kg bw)], andG9: [Vitamin E( 1.5 IU /kg bw), +β-carotene (25mg/kg
Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com The microscopic investigation of prostate glandof group 2 (control+ ve rat) revealed necrosis associated with massive leucocyte cells infiltration (Fig.11). Meanwhile prostate of
rat from group 1 (control-ve) and group 3 showed no histopathological changes (Fig.11). However, prostate gland
of ratsfrom groups 4, 5 and 8 revealed hyperplasia and vaculation of epithelial lining prostatic acini(Fig.11).
Meanwhile, examined sections from groups 6 and 7 showed interstitial edema (Fig.11). On the other hand, prostate gland
of rat from group 9 revealed distension of the acini with eosinophilic protein secretion(Fig.11) and hyperplasia of
epithelial lining some acini in another examined sections from the same group 9(Fig.11).
Fig. 11 Histopathological image of Prostate tissue
from control rats, AZ and AZ plus different
antioxidants-treated animals after in four months of
treatments
G1:Control (-) (Normal control, basal diet), G2:Control (+) (Control
treated with 20mg kg-1bw of azithromycin), G3:Vitamin E(3 IU /kg bw),
G4:β-carotene (50mg/kg bw), G5:Anthocyanin (71mg /kg
bw),G6:Green tea extracted (120mg/kg bw),G7:Selenium (0.1 mg/kg
bw),G8:[Anthocyanin (24mg /kg bw)+ , green tea extract (40mg/kg bw) + Se
(0.03mg/kg bw)], andG9: *Vitamin E( 1.5 IU /kg bw), +β-carotene (25mg/kg
4. Discussion
A review of literature suggest that antimicrobial agents can affect male reproductive functions including sperm counts, motility, morphology, spermatogenesis and germ cell integrity (35,29,52-55).Azithromycin is an azalide,
a subclass of macrolide antibiotics, structurally modified from erythromycin. Its efficacy and potential to inhibit microorganisms had led to its increased use. It is well- known that Azithromycin is widely used to treat or prevent certain bacterial infections, most often those causing middle ear infections, strep throat, pneumonia, typhoid, and Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com sinusitis. AZ has been shown to induce a number of serum testosterone (Table 2)may be AZ was able to reduce
biochemical disorders and increasing number of evidence the rate limiting enzymes of testosterone synthesis, indicates that it has risk of hepatotoxicity and cardio-toxicity indicating a hindered production of the latter this isin as adverse effect (34).This study appears to be the first
agreement with that of Ghosh&Dasupta (60) who reported
reporting the ability of the drug to cause oxidative damage that gentamycin is aminoglycoside antibiotic make similar to rats testicular function in rats. For our Knowledge, this is the first time to reveal the AZ- Regarding low fructose level in reproductive organs induced oxidative stress, decreased antioxidant levels, lipid (Fig. 2)seems to beconnected with the decreases in
peroxidation and histopathological changes in the testis, testosterone levels. This explanation was also supposed by epididymis and seminal vesicle. In the present study, Melis (43),as fructose is the nutrient substance present in
administration of AZ did not affect the body weight of the seminal plasma, and is initiated and controlled by testicular animals but specifically caused a significant reduction in the androgens (50). In this respect, hormonal deficiency causes
weights of testis, epididymis and seminal vesicle. The a decrease or even disappearance of seminal fructose.In fact, weight of the testis is largely dependent on the mass of the treatment with androgens restores the ability of the differentiated spermatogenic cells; the reduction in the accessory glands to produce this sugar (61). Moreover,
weight of the testis may be due to decreased number of germ fructose, when converted by spermatozoa to lactic acid cells, inhibition of spermatogenesis and steroidogenic provides an important source of energy for sperm cells. Our enzyme activity (56).The reduction in testis and epididymis
studydemonstrated the relationship between the reduction in weights is due to the marked parenchymal atrophy in spermatozoal activity and the low level of fructose (Fig. 2
germinal cell layer thickness and the other deteriorated and Fig. 3)as statedby earlier studies (62)as well as other
histopathologic findings in testis, along with decreased RRT researchers Melis (43). The only parameter that appears to
ratio, which were observed in AZ-treated rats in the present be possibly improved with oral antioxidant therapy is sperm study. Similar findings were reported earlier by Turk et al.,
motility. Many studies have shown small but significant (57).The observed weight loss of the accessory sex organs
improvements in sperm motility with supplementation of may be due to reduced bioavailability of sex hormones such selenium (63), Vitamin E (64), Vitamin E and selenium
as testosterone, luteinizing hormone, follicle stimulating (65), glutathione (66) and Astaxanthin(67), as well
hormone and inhibit B, the levels of which in the circulation Many years ago,MacLeod (68) noted that
indicate the reproductive endocrine status of the male as has incubation of sperm under conditions of high oxygen tension been stated beforewith Schrade, (58). All antioxidants
lead to a rapid loss of their motility. The addition of the administrated significantly mitigated the effect of AZ on the antioxidant catalase to the medium preserved sperm motility testicular weight and other accessory reproductive organs prompting MacLeod (68)to suggest that sperm must
affected by the drug.More recently Elias & Nelson, (55)
produce hydrogen peroxide during normal oxidative have emphasized that testosterone is very essential in metabolism. Since this publication, it has evolved that three spermatogenesis. It was reported that testosterone is inter-related mechanisms account for oxidative stress- essential in growth and association with follicle stimulating mediated male infertility—impaired motility, impaired hormone acts on the somniferous tubules to initiate and fertilization and oxidative DNA damage. maintain spermatogenesis. Earlier, Louvetet al., (59)point
In normal situation the seminal plasma contains antioxidant out that interstitial cell-stimulatinghormone (ICSH), also mechanisms which are likely to quench these ROS and known as Lutenizing hormone (LH) stimulates Leydig cell protect against the damage to spermatozoa(29). It well
production of testosterone. In accordingly, the level of these known that bactericidal antibiotics can induce cellular death two sexual hormones; testosterone (T) and ICSH were through a common oxidative damage mechanism that relies determined in blood serum of the investigated rats (Table
on the production of ROS. Through their various primary 2).Azithromycin administration significantly decreased
targets, antibiotics can activate cellular respiration, which Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com leads to the formation of superoxide and the release of iron mg of Vitamin E and 225 mg of selenium (65) or 300 mg of
from iron-sulfur clusters. Free iron then activates a chemical Vitamin E alone (64) have been shown in placebo controlled
reaction (the Fenton reaction) to produce ROS in the form of studies to reduce sperm MDA levels. Finally, a well- hydroxyl radicals (OH•) (30).The administration of AZ
designed RCT of 2 months treatment with 1 g of Vitamin C induced marked oxidative stress and altered the antioxidant and Vitamin E reported a very significant reduction in sperm status in rats and its mechanism of toxicity appears to DNA damage (75). This finding is supported by non-
precede through the generation of free radicals or depletion controlled studies also reported a reduction in sperm DNA of the antioxidant systems(34).
damage with the use of a combination of Vitamin C and E In connection, animals treated with AZ in this study (400 mg each), β-carotene (18 mg), zinc and selenium (76)
showed decreased activities of antioxidant enzymes otherwise a combination of acetylcysteine, 180 mg Vitamin SOD,CAT and GSH-PX in the testis. This decrease in the E, 30 mg β-carotene and essential fatty acids (73).
antioxidant defense molecules led to a concomitant increase Various agents have been attempted to protect from in the level of MDA. The inactivation of the antioxidant and/or prevent the side effects of many chemotherapeutics. enzymes may be caused by excess ROS generated in the Flavonoids are among these agents and are found in almost system (69). Our data concluded that co-administration of
all food categories such as fruits and vegetables being the Vit E, β-carotene, anthocyanin, green tea extract, mixture of main source. Flavonoids have many functions, such as the (anthocyanins+ green tea extract+ Se) or mixture of the phenolic antioxidants, scavengers of free radicals, chelating (Vitamin E+ β-carotene) with AZ significantly attenuated agents, and modifiers of various enzymatic and biologic the effects of AZ drug on the antioxidant enzymes and reactions (77). Anthocyanins and green teais a naturally
further suggests that impairment of male fertility through occurring plant polyphenol that exhibits antioxidative induction of oxidative stress. Olayinka&Ore (34)observed
properties both in vivo and in vitro. In fact, they have been a significant reduction in the activities of SOD, CAT and shown to exert a potent scavenging action on both O - GST in the liver of azithromycin- treated animals. The ˙OH, as well as lipid peroxidationbeside increase the significantly decreased activity of the ROS scavenging activities of SOD and Catalase(78- 80).
enzyme, SOD and CAT, by exposure to azithromycin, The declining of lipid peroxidation in all samples conforms to previous report on macrolide antibiotics (70).
studied apparently indicates that all treatments potently This may be due to the damaging effects of free radicals scavenged the free radicals (O2 - and ˙OH), and suppressed possibly generated by the action of the drug a clear oxidative oxidative DNA damage. The antioxidant activity of Vit E, β- stress has been achieved in the testis. Lipid peroxidation is a carotene, anthocyanin, green tea, process of oxidative degredation of polyunsaturated fatty (anthocyanin+ green tea extract + selenium) or mixture of acids that result in impaired membrane structure and the (Vitamin E+ β-carotene) and mitigation of ROS- function (71).
induced depletion of GSH-Px and CAT activities also show In the main line this prospectus, several studies that all used antioxidants in the present study especially Se, have reported that levels of ROS within semen can be Vitamin E and β-carotenehas strong antioxidant activity. reduced by augmenting the scavenging capacity of seminal The proposed mechanism of action within which plasma using oral antioxidant supplements. The oral may profoundly includethe non-enzymatic antioxidants antioxidant Astaxanthin(67), carnitine(72) or a combination
present within semen include -tocopherol (Vitamin E), of antioxidants such as acetylcysteine, β-carotene, Vitamin glutathione, carotenoids, and flavonoids. These agents E and essential fatty acids (73) directlybinding cure with
principally act by directly neutralizing free radical activity reduce seminal ROS levels. likewise,a randomized control chemically. However, they also provide protection against study comparing 3 months of Vitamin E (600 mg/day) free radical attack by two other mechanisms. Albumin in treatment with placebo has confirmed this reduction in another nutrient system can intercept free radicals by seminal ROS levels (74). Furthermore, a combination of 400
Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com becoming oxidized itself, thereby sparing sperm from Free radicals, otherwise damage cellular lipids by attack(81).
binding to membrane anionic phospholipids (34), proteins
Dietary deficiencies have been linked with sperm and DNA, finally the entire cell (88). Antioxidants, being
oxidative damage as stated by several research works. The free radical scavengers hamper the destructive effects of the Age and Genetic Effects in Sperm (AGES) study examined latter on cells and tissues. AZ-induced renal and hepatic the self-reported dietary intake of various antioxidants and toxicity in rats as explained by the photomicrograph of rats nutrients (vitamins C and E, β-carotene, folate and zinc) in a kidney and liver sections. The histological alterations group of 97 healthy nonsmokers and correlated this with observed in the liver of azithromycin - treated rats sperm quality (82). The pre study reported a significant
characterized by sinusoidal and portal congestion, and mild correlation between vitamin C intake and sperm periportal cellular infiltration by mononuclear cells coupled concentration and between vitamin E intake and total with the presence of protein casts in the kidney tubular progressively motile sperm. This is also consistent with lumen as well as severe cortical congestion and hemorrhage earlier reports of a significant link between seminal plasma is an indication of disruption of cellular architecture. All vitamin E levels and an increase in percentage of motile these abnormalities might have resulted due to the formation sperm (83).Alternatively, differences in the populations
of highly reactive radicals caused by the drug which studied may explain the discrepant results. Song et al., (84)
disrupted normal cellular functioning of the liver and the correlated sperm DNA damage with dietary antioxidant kidney(34). This phenomenon would have been responsible
intake in infertile men, while Silver et al., (85)examined this
for structural changes such as vacuoles, gaps, cytotoxicity relationship in healthy presumed fertile patients. Fertile men and atrophic changes in the testis and the other parts of the with low levels of oxidative attack may not be as dependent reproductive system (accessory sex organs). The vacuoles on seminal antioxidants for protection of their sperm DNA and gaps in the seminiferous epithelium appear due to integrity. Therefore, a dietary deficiency in antioxidants may dilatation of endoplasmic reticulum in Sertoli cells (89), and
not lead to sperm oxidative DNA damage in this fertile missing of germ cells from the epithelium (90), respectively.
The oxidatative stress in the testis should have affected the While many studies have shown improvements in sperm structure and functions of cell organelles that manifested as quality with antioxidant treatment, the ability of these histopathological changes. Since the oxidative stress-status changes to translate into improved chances of pregnancy is induces cellular and DNA damage (91), it is possible that
less clear. The Menevitnutraceutical is postulated to improve the cell death and consequent tubular atrophy, at least in a sperm quality by three complimentary mechanisms. First, it few tubules, would have been the response of seminiferous contains traditional antioxidants such as Vitamins C and E, epithelium to altered biochemical milieu in the testis. These selenium and lycopene to protect sperm from ROS already results therefore indicate that the oxidative stress induced produced. Second, it contains garlic which is known to have owing to an imbalance between the free radicals and an anti-inflammatory effect, thereby potentially reducing oxidants, has a key role in initiating tubular damage in the seminal leukocyte ROS production (86). Finally, Menevit
testis, just as it does in the liver and kidneys (34).
contains zinc, selenium and folate that are believed to play a Taken together, the present study reveals role in augmenting protamine packaging of sperm DNA thatadministration of therapeutic dose of AZ tomale rats (87), helping to protect sperm from ROS attack. While it is
induced oxidative stress by decreasing the antioxidant yet to be proven that combinational therapy such as system, increased lipid peroxidation and concomitantly Menevitimproves sperm DNA integrity, it appears logical impairs testicular function, spermatogenesis and epididymal that using several antioxidants with different modes of sperm physiology and morphology. All antioxidants action, together with an agent to reduce leukocyte ROS administrated in this study exhibited similarities in their production (23) is most likely to result in a beneficial effect.
capability to mitigate the toxic responses of AZ, which suggests that the adverse effects of AZ on the testes are at Volume 2 2015 Issue 1 DEC-JAN AIJCSR ISSN 2349 – 4425 www.americanij.com least in part due to impairment of the antioxidant defense [ 3 ] Martini M.C. Infertility. In: PernollM,l. system and further enhancement of lipid peroxidation. The ed. Current obstetric and gynecologic diagnosis inability of these antioxidants to fully protect the testes and treatment. Reproductive endocrinology and against AZ-induced toxicity suggests that the antibiotic infertility.7th ed. Norwalk (CT): Appleton & could mediate testicular damage through other mechanisms apart from oxidative stress. In view of the importance of this Lange.1991; 1025-1036. drug in clinical practice, the relevance of our study to [ 4 ] Sigman M, Lipshultz L.I., Howards humans merits further investigation on the mechanisms by which AZ causes testicular toxicity. 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