Long-Term Effect of Ciprofloxacin on Testicular Tissue: Evidence for Biochemical and Histochemical Changes

Due to the enhanced antibiotic resistance observed in various farm animal species, administration of antibiotics to control and/or manage microbial diseases may impose certain hazards (1). According to previous findings, a number of antimicrobial agents have been associated with damaged spermatogenesis (2). The fluoroquinolones are known as the most important group of antibiotics against different bacterial diseases in humans, poultry and animals (1, 3). Fluoroquinolones exert good bactericidal activity against a number of bacterial agents, including E. coli, Hibiscus, Pseudomonas, Staphylococcus and Chlamydia species (4).

Ciprofloxacin (CPFX) is a second-generation fluoroquinolone broad-spectrum antibiotic used to treat a number of gram-positive and -negative bacteria that cause infections of the bones and joints, and respiratory and urinary tracts. It mainly acts through inhibition of a type II topoisomerase, DNA gyrase, which is necessary to unwind replicated prokaryotic DNA. CPFX is routinely administered by urologists and fertility specialists in order to control male reproductive infections. Its side effects occur most frequently in the gastrointestinal tract and central nervous system. Allergic and cardiovascular reactions are additional adverse effects observed during treatment with CPFX (5, 6). It has been reported that CPFX significantly impairs both testicular function and structure in rats (7, 8). Following administration of CPFX, high levels of this drug were detected in prostatic tissue and seminal fluid (9). Abd-Allah et al. (10) have reported that administration of CPFX significantly reduced sperm count, motility and daily sperm production in rats, all which might adversely affect male fertility.

Leydig and Sertoli cells play key roles in spermatogenesis and cell lineage metabolism. These cells are considered to be important cells for intratesticular endocrine function (11, 12). Any disruption in their physiologic correlation with the germinal epithelium (GE) would enhance CPFX-induced damages in testicular tissue. However, the cytoplasmic biochemical alterations in GE and the role of inflammation in spermatogenesis and spermiogenesis processes are enigmatic (13, 14). Therefore the primary aim of the present study is to illustrate the histochemical alterations of cytoplasmic carbohydrate supplement, unsaturated fatty acids (lipid foci) and cytoplasmic lipase enzyme modifications. Additionally, we have evaluated tissue alkaline phosphatase (ALP) in seminiferous tubules (STs) as a biomarker enzyme for inflammation. The final aim of the present study was to determine the serum levels of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH ) and their association with histopathological changes in the testes.

Although the therapeutic and prophylactic effects of CPFX on different gram-positive and -negative bacteria has been well documented, various studies reported that even short term administration of CPFX promoted male reproductive toxicity (8, 10). Several reports indicated that the therapeutic dose of CPFX in prolonged and short time consumption remarkably decreased sperm hyper activation and motility (16, 17). Although it has been reported that CPFX in therapeutic dose resulted in considerable apoptosis of spermatogonia cells (17), the exact mechanism and/or pathophysiology for CPFX-induced damages in testicular tissue has not been determined.

Our findings showed that long term administration of CPFX resulted in remarkable alterations in intracellular biochemical supplements. The intracytoplasmic carbohydrate ratio decreased in spermatogenesis cell lineage and simultaneously the lipid foci supplement and lipase enzyme increased in the cytoplasm of these cells. An increase in ALP level was mainly manifested in degenerated GE. Significant changes also occurred in serum levels of testosterone, LH and FSH.

Previous studies have shown that glucose transporters are the main ways to transfer glucose to the STs; carbohydrates are the major sources of energy for hyper-mitotic cells (18). Thus any degeneration could result in interruption of glucose passage to the STs and ultimately to GE (12).

In the present study, both spermatogonia cells and spermatocytes had PAS-negative reactions in CPFX-treated animals. Thus, following CPFX administration, there was decreased glucose transport and/or metabolism in spermatocytogenesis and the spermatogenesis cell lineage which resulted in a switch in their energy source from glucose to lipids. As a result, cytoplasmic lipid foci increased in the cytoplasms of these cells, particularly in the first three layers. Simultaneously, due to insufficient energy, these cells were unable to synthesize essential proteins and therefore they underwent apoptosis and disruption (19, 20). Since there were no significant differences in studied parameters between low and high dose CPFX-treated animals, we concluded that CPFX had the capability to impact the cellular biochemical supplements, even at in low doses.

The increase of lipase enzyme synthesis in CPFX-treated groups revealed that the metabolic pathway of the cell lineage in the first three layers of the GE was altered by using the lipid sources from different biological activities. The number of Sertoli cells with SB-positive cytoplasm increased in CPFX-treated groups which showed the effects of CPFX in lipid accumulation in Sertoli cells. Lipid supplement in Sertoli cells varies with different conditions. Forinstance during phagocytosis of residual bodies or damaged cells, the intracytoplasmic ratio of lipids increases in their cytoplasm (12, 21). Our ALP staining was in good accordance with obtained results as the testes of the CPFX-treated animals exhibited strong ALP reactions, particularly in the disrupted cells. According to these findings it would be more logical to say that the number of ALP-positive disrupted GE cells increased in STs with an eventual elevation in phagocytosis.

According to previous reports, constant levels of FSH and LH are essential for initiating and supporting the spermatogenesis process (22, 23). Hence degeneration of Sertoli and Leydig cells in the CPFX-treated groups may be associated with severe alterations in serum LH and FSH levels. On the other hand, Leydig cells control Sertoli cell physiological bioactivities by the synthesis of testosterone (22, 23). FSH increases the synthesis of androgen binding protein by Sertoli cells which is needed to maintain high concentrations of testosterone (22, 24). Our histochemical findings confirmed the above reports. The increase in the number of Leydig cells with positive ALPstained cytoplasms and the decreased number of cells with lipidophilic cytoplasm in CPFX groups suggested that the biosteroid activity of the Leydig cells reduced in CPFX-dosed animals. Thus, we concluded that remarkable degeneration of Leydig cells after administration of different doses of CPFX resulted in significant reduction in serum testosterone levels; this reduction in testosterone was responsible for the Sertoli cell degeneration and consequent GE disintegration.

Following CPFX administration the spermatogonia, spermatocyte and Sertoli cells in STs switch their energy source from glucose to lipids. Thus, inadequate energy supplement leads to cellular degeneration. Impairment of Leydig cells in testosterone synthesis negatively impacts this pathological process by affecting Sertoli cells.