Introduction

Malignancy is the second dominant reason of mortalities all over the world. Although cancer commonly affects people after they have completed their families, a serious minority are diagnosed at a younger age (1). In spite of the fact that the incidence of cancer in children all over the world is low, the age-optimized frequency is seventy to 160 newly diagnosed cases for each millions of children per year at the age of zero to 14 (2). These patients have to complaint not only with the effect of having cancer and the immediate complications of treatment, but also with the possibility that surgery, radiotherapy or chemotherapy may lead to temporary or permanent squeal as gonadal damage, infertility, and organs toxicity (3,4). Imatinib (1^st generation Tyrosine kinase inhibitors (TKIs)) is a recent anticancer agent that is strongly inhibited human BCR-ABL1-positive tumor (5,6), that make it a particularly suitable well planned agent for the treatment CML and ALL. Imatinib also induced an inhibitory action on the expression of c-Kit -TK receptors in the alimentary tract which is contributed in the pathogenesis of gastrointestinal stromal tumor-GIST (7,8).

In addition, imatinib induces an inhibition to the PDGF-α and PDGF-β receptors (9) which may allow more therapeutic using. The major adverse effects enroll severe neutropenia and thrombocytopenia, oedema, fluid retention, nausea, mild diarrhoea, skin rashes, arthralgia, myalgia, bone pain, acute renal failure (10,11), and hepatotoxicity (12). The safety margin of imatinib, and liability to induce organ toxicity, has been studied in a conflicting manner in current years (13). There is a report of the GIST patient or CML patient (males) suffered from with gynaecomastia and hydrocele following exposure to imatinib mesylate (14). Little is known about the effect of early exposure to imatinib. This work aims to evaluate the repercussion of exposure (in vivo) of male albino rats to imatinib mesylate (at neonatal or infantile periods) using histological analysis of their testes in several end points and in adulthood using rat which is preferred as a suitable and an attractive alternate animal model for human.

Materials and methods

This experimental work was conducted on rats (male albino) purchased from Animal House of Veterinary College, university of Mosul, Mosul, Iraq.

Throughout the investigations the rats were breed under controlled normal environmental laboratory conditions and animal facility and were housed in an air-conditioned room with 12-hours light and dark cycles, where the temperature 23 ±2ºC and relative humidity 66-71% were kept stable. They were put in an individual manner in plastic cages (England) measuring 47×34 × 18 cm lined with wood chips (15) provided from Animal House. Animals were let to be comfort for a ten days before any procedure was performed (16), and supplied with free access of water ad libitum and pelleted standardized food (commercial rodent chow) (17). All rats received humane care, and procedures enrolling animals and their care were conducted in conformity with international roles and programs and the articles on animals accepted. The experiments were performed during the light section (18,19).

Animals were randomly assigned. Intact pups exposed to oral imatinib (Glivec^®, STI 571; Novartis- purchased from Ibn-Sena Teaching Hospital, Mosul) at neonatal and early infantile period on PND 1 to PND10 (n=32) using gavage with 24 gage needle and they were represented the treated group. They were received imatinib 200 mg/kg once daily for 10 days starting from PND 1 to PND10. While control group includes age and weight matched pup which received D. W following the same program applied to imatinib group (n=4 for each). Each treated pup was observed for clear signs of toxicity for the next four hours, and mortality throughout the next 24hours (20).

Imatinib doses choosen were justified to be in accordance with those used in clinical treatment regimens (21) (400-800 mg/d or 340-590 mg/m2 as the weight is 70 kg) dose surface area according to body-weight, f×mg/kg=mg/m2, f is a factor equaling to 6. 0 in rats (22).

Pups were categorized into 4 subgroups, according to the ages of euthanasia: 15 days postpartum (n=8); 40days postpartum (n=8); 70 days postpartum (n=8) and 140 days postpartum (n=8) (18).

Euthanization of rat with ether (19,23) was done 24 h after the final dose was given at laboratory of postgraduate studies of Department of Anatomy, Nineveh College of Medicine, University of Nineveh, Mosul, Northern Iraq.

After longitudinal incision gonads were excised and fixed in Bouin's solution (24). The histological analysis was conducted after processes of fixation, dehydration, clearing, impregnation and embedding in paraffin. Then tissues were submitted to sectioning and staining (Harris Hematoxylin and Eosin stain (H&E) Ten randomly selected testicular sections from each rat were analyzed blindly for any disorganization of cytoarchetecture of seminiferous tubules, evidence of vacuoles, gaps and abnormal cells in the seminiferous epithelium and sloughed cells, degeneration of seminiferous epithelium and interstitial spaces in order to achieve qualitative assessment (25).

On the other hand, sections were examined quantitavely (via visopan Reichert, Austria) where the diameter of seminiferous tubules (STD), epithelial height of seminiferous tubules (SEH), mean number of Sertoli cells and Leydig cell were obtained using eyepiece (16), and counted in fifty tubules of each sample at 400X magnification (26,27).

In addition, Johnsen' scores were used to analyze the effect of drug on spermatogenesis (28,29) The photomicrographs will be captured via Leica microscope with digital camera installed using planapochromatic objectives.

Data were recorded using excel program. Data were analyzed using Microsoft SPSS software version 17. Value is considered as significant when it P<0.05.

Results

All animals remain alive during the experimental work. At necropsy, no clear gross tissue abnormalities were observed of any animal. The histopathological analysis of the testes of pups that received repeated doses of imatinib at neonatal period or at neonatal /infantile periods showed that the by PND15, loss of differentiated germ cells was shown in testicular sections from treated animals (Figure 1).

Figure 1: A photomicrograph of a section obtained from rats at PND15 after treatment with 200mg/kg/day of imatinib on PND1-PND10, proliferation of spermatogonia is noticed (red arrow) (H&E×250).

At PND40, sections of control rats showed normal architecture of seminiferous tubule and interstitial tissue (Figure 2). While disturbance in the arrangement of the seminiferous tubules, germ cells depletion, epithelial cells sloughing, intraepithelial cells spaces, an appearance of some multinucleated cells and some apoptotic cells, and interstitial edema were the most evident features of testicular sections of treated animals. Sertoli cell also showed alterations in their morphology in these rats. Their nuclei showed abnormal shape and some of them were far from the basal membrane or even in the tubular lumen. Finally, Sertoli cell only tubular sections were seen (Figure 2) while an appearance of eosinophilic bodies was also noticed in some sections (Figures 2-5) (Table 1).

Figures 2: A photomicrograph of a testicular section obtained from rats at PND40 after treatment with 200mg/kg/day of imatinib on PND1-PND10 with disturbed seminiferous tubules, depleted germ cells, presence of vacuolated and detached Sertoli cells (green arrow), multinucleated cells (double blue arrow). Sertoli only cells feature is seen in one of the tubules (H&E×250).

Figures 3: A photomicrograph of testicular section obtained from rats at PND40 after treatment with imatinib on PND1-PND10 with disorganized cells and apoptosis (arrow) (H&E×250).

Figure 4: A photomicrograph of a testicular section obtained from rats at PND40 after treatment with 200mg/kg/day of imatinib on PND1-PND10 with germ cells depletion (H&E×250).

Figure 5: A photomicrograph of a testicular section obtained from rats at PND40 after treatment with 200mg/kg/day of imatinib on PND1-PND10 with vacuolation (arrow) of Sertoli cells (H&E×250).

In addition, The Seminiferous tubular diameter of this group was significantly reduced mean 117.2±2.5 µm. The epithelial height of the tubules was significantly reduced at this group mean 17.3±3.1 µm. These sections revealed that mean number of Sertoli cells/seminiferous tubule was 10.5±2.2, while that of Leydig cells was 3. 0± 1. 6., however, some sections exhibited Sertoli only cells feature (Figure 2) (Table 1 and 2).

Qualitatative Analysis of Testicular sections obtained from rats euthanized at PND70 reveals intense several alterations (Table 1). The most obvious features were germ cells loss and sloughing, disorganized appearance of the seminiferous tubules, detachment of some seminiferous tubules from their basement membrane, presence of vacuolated and detached Sertoli cells, some multinucleated cells were noticed, some apoptotic cells were seen, interstitial edema, and retention of elongated spermatid (Figures 6-9). Moreover, histological analysis demonstrated that these animals presented decreased Johnsen’s scores in relation to those in control animals (Table 1).

 Figure 6: A photomicrograph of a testicular section obtained from rats at PND70after treatment with 200mg/kg/day of imatinib on PND1-PND10 with disorganized appearance of the seminiferous tubules and some multinucleated cells (red arrow). (H&E×250).

Quantitative analysis

The seminiferous tubular diameter and their epithelial height of this subgroup at PND70 were diminished in comparison to those in the control rats, with mean of 125 ±4.1 µm and 7.0±1.1 µm respectively. Moreover, the number of Sertoli cells per seminiferous tubule was raised with mean of 18.2±0.2. Mean Leydig cells number was 7.0±0.1 (Table 2).

At PND140 the Qualitatative Analysis of testicular sections revealed presence of intraepithelial spaces, sloughing of epithelial cells, arrest at spermatid stage and disorganized appearance of the seminiferous tubules. Johnsen’s scores of 5.1±0.9 indicated incomplete recovery of spermatogenesis (Figure 10).

Figure 7: A photomicrograph of a testicular section of rat at PND70 after treatment with 200mg/kg/day of imatinib on PND1-PND10 with germ cells depletion, interstitial edema (arrow), and vacuolation (double arrow) (H&E×250).

Figure 8: A photomicrograph of a testicular section of rat at PND70 after treatment with 200mg/kg/day of imatinib on PND1-PND10 with germ cells depletion, interstitial edema. Detached tubule from their basement membrane (arrow) (H&E×250).

Figure 9: A photomicrograph of a section of rat at PND70 after treatment with of imatinib at PND1-PND10 with disorganization of the seminiferous tubules and germ cells depletion (arrow) (H&E×250).

Table 1: early and late term effects of imatinib on testicular histology

*P-value is considered as significant when P<0.05.

Table 2: Effects of imatinib on some testicular morphological parameters at various endpoints

*P-value is considered as significant when P<0.05.