Testicular Morphological Changes Produced by Fluoroquinolones in Adult Male Albino Rats

Objectives: The objective of this research work was to observe the testicular morphological changes produced by fluoroquinolones in the reproductive organs of adult male albino rats, and to see whether these changes are reversible after discontinuation of the drugs. Study Design: Experimental randomized control trial. Duration and Place of Study: This Study was carried out at Department of Pharmacology, Post Graduate Medical Institute (PGMI), Lahore extending over a period of 12 weeks. Materials and Method: Eighty adult male albino rats weighing 200 – 300 gms were randomly selected and divided into four groups i.e. A, B, C & D, having 20 animals in each group. A, B & C, were the experimental groups & D served as control group. All the groups were further divided into sub groups 1 & 2. Three fluoroquinolones i.e. Ciprofloxacin (135 mg / kg / day), Ofloxacin (75 mg / kg / day) & Enoxacin (12.5 mg /kg/ day) were given to the groups A, B & C respectively for 42 days. Animals of group D received distilled water only. Animals of sub groups A1, B1, C1 & D1 were sacrificed on 42 nd day and testicular tissue was obtained for morphological study. Animals of sub groups A2, B2, C2 & D2 were sacrificed on 84 th day and testicular tissue for morphological changes was taken. No of leydig cells, height of epithelium and diameter of seminiferous tubules were taken as experimental parameters for morphological changes. Results: The study indicated statistically significant (P < 0.05) decrease in height of epithelium, diameter of seminiferous tubules and no. of leydig cells in experimental groups as compared to the control groups. Conclusion: The changes observed in morphology could lead to decrease in sperm count and testosterone levels. This study suggests gonadotoxic potentials of fluoroquinolones and adds concern to the indiscriminate and widespread use of fluoroquinolones and recommends more rational use of these drugs.


Introduction
A decline in male fertility has been observed over the past 50 years.The quality of semen regarding the sperm count has been deteriorated.The biological significance of this change is supported by a concomitant increase in the incidence of genitourinary abnormalities such as cryptorchidism, hypospadias and testicular cancer suggesting a growing impact of the factor with serious effects on male gonadal function. 1Environmental factors, drugs and chemicals are likely to cause changes in semen quality.The environmental factors also include the misuse of drugs and chemicals. 2The effects of diet on reproductive potentials of males are not that much elaborated.8][9] Antibiotic prescriptions are most commonly observed in most of the unrelated conditions which is also observed in the patients who attend the fertility clinics. 10Patients who acquire assisted conception show indication of reproductive tract infection. 11,12Ciprofloxacin is usually used by andrologist, urologists, and fertility specialists prior to the in vitro fertilization procedures for the bacterial infections and also to combat the increased concentration of leukocytes in the semen regardless the evidence of bacterial infection is found or not. 13iprofloxacin belongs to fluoroquinolones which is synthetic antibiotic.The fluoroquinolone antibiotics are an important, frequently prescribed group of medications.The recommended indications for fluoroquinolones include skin & soft tissue infections (SSTI), respiratory tract infections (RTI) sexually transmitted diseases (STD), and urinary tract infections (UTI).Since the introduction of fluoroquinolones in 1980's, one hundred million patients or even more have received the agents. 15UTIs are most commonly treated with fluoroquinolones in North America and Western Europe. 16Expanded-spectrum quinolones 17 which include norfloxacin and ciprofloxacin are highly active for eradication of bacteriuria in UTIs (> 90% cases) and gram negative bacteria. 18Fluoroquinolones are also effective against agents of atypical pneumonia i.e. chlamydia and mycoplasma and against intracellular pathogens such as legionella and some mycobacteria, including mycobacterium tuberculosis and mycobacterium avium complex.As a class, fluoroquinolones are generally well tolerated and safe.The most commonly detected adverse effects of fluoroquinolones include the CNS disturbance, GI reactions and skin irritations.The adverse effects of newer fluoroquinolones are dose dependent having steep dose response relationship. 19he present study was aimed at defining the testicular morphological changes produced by fluoroquinolones such as ciprofloxacin, ofloxacin and enoxacin.No. of leydig cells, height of epithelium and diameter of seminiferous tubules were taken as experimental parameters for morphological changes and whether these changes are reversible after discontinuing the drugs.

Materials and Methods
An experimental study was conducted at Pharmacology Department of PGMI, Lahore.The total duration of study was 12 weeks.80 male albino rats aging 7 weeks and weighing 200 to 300 grams were selected.Random division of rats was done into 4 groups named A, B, C & D with 20 animals in each group.Groups A, B & C were experimental and group D served as control.A further division of all the groups was done into sub groups 1 and 2. Three drugs, Ciprofloxacin (135 mg/kg/day), Ofloxacin (72 mg/kg/day) and Enoxacin (12.5 mg/kg/day) were given to the groups A,B and C respectively for 42 days.All the drugs were dissolved in distilled water before administration.The control group D was given distilled water only.Animals of sub groups A1, B1, C1 and D1 were sacrificed on 42 nd day and testicular tissue was obtained for morphological studies.While animals of sub groups A2, B2, C2 and D2 were sacrificed on 84 th days and testicular tissue was obtained for morphological studies.Morphological changes were evaluated by considering no. of leydig cells, height of epithelium and diameter of seminiferous tubules.Statistical analysis was done by using SPSS V. 16.ANOVA was applied for comparison among the various groups while P value less than 0.05 was considered significant.

Results
The mean height of epithelium was 180.73  In majority of control group animals (D1& D2) the lumen of the seminiferous tubules were filed by mature spermatozoa.All the cells of spermatogenic series and sertoli cell were healthy looking.The interstitial tissue was normal, richly vascularised and occupied by acitive healthy looking leydig cell (Fig 1,  2).
Most of the tubules were devoid of spermatids and the number of cell layers was reduced.The scattered early stage of spermatogenic cells were seen in the seminiferous tubules.The shed spermatogenic cells debris was seen in the lumen of a large number of seminiferous tubules (Fig. 4, 6, 8).

Discussion
The fluoroquinolones are anti-microbial agents with a broad spectrum of antibacterial activity.These are synthetic and effective after oral administration for a variety of infectious diseases.The fluoroquinolones have well documented therapeutic and adverse effects.However, the present study proposed that prolonged exposure to fluoroquinolones (ciprofloxacin, ofloxacin and enoxacin) even at therapeutic doses produced toxicity in reproductive activity of male albino rats.Morphological changes produced by fluoroquinolones in experimental groups are the evidence of such toxicity.
Degenerative changes in the seminiferous tubules are the evidence for genotoxicity.1][22] In this study mean diameter of seminiferous tubules was higher in group D (Control) in contrast to A, B and C (experimental groups) P < 0.001 which is significant, also shown in photomicrographs Fig. 1 to 8. Studies using rats treated with therapeutic doses of fluoroquinolones confirmed the observation in humans regarding the adverse effect of fluoroquinolones on spermatogenesis. 23hanges observed on light microscope include expansion of interstitial and intertubular space with vacuolization, degeneration and necrosis of interstitial (leydig) cells following which an exudation into the interstitium was developed and congestion in veins increased in all experimental groups as compared to control groups showing P value < 0.05 that is significant.Multiple comparison among study groups showed that Ciprofloxacin produced less morphological changes (Fig. 3 & 4) in testicular tissue as compared to Ofloxacin (Fig. 5 & 6) and Enoxacin (Fig. 7 & 8).These data were confirmed when compared with previous study conducted by khaki et al in 2008. 20Anderson et al (2012) showed that Ofloxacin at a dose of 72 mg/kg per day had almost the highest potential in terms of impairment of the rat testicular function. 24ther studies also added that fluoroquinolones have toxic effects on testicular functions of different animals .21,25-28

Conclusion
Evidence of morphological changes produced by fluoroquinolones in this study showed that these changes could lead to decrease in sperm count and serum testosterone levels.This study suggests that fluoroquinolones are potentially gonadotoxic therefore widespread and indiscriminate use of quinolones shall be discouraged.