Effects of methotrexate on hepatic and testicular tissues in male rabbits: Histological, hormonal and biochemical analysis

1. Study the main histological alterations of methotrexate on testicular and hepatic tissues.
2. Conduct the testosterone, FSH, LH hormone levels in serum after exposing to methotrexate.
3. Detects AST, ALT serum levels.

Introduction

Due to its effectiveness, methotrexate (MTX), a folic acid antagonist, has been utilized in malignant clinical practice for many years as an anticancer medicine (1,2). It has already been reported that methotrexate is toxic to various body organs, including the gastrointestinal, hematologic, and central neurological systems (3,4). The liver is one of the body's most essential organs since it serves as a hub for the metabolism of nutrients like proteins, carbs, and lipids, as well as the excretion of waste products from metabolism (5). Hepatotoxicity can be caused by various risk factors, including hereditary, non-genetic, and environmental ones (6). Anticancer medications, antiretroviral drugs, alcohol, and several medical conditions like age, sex, and diseases all have pharmaceuticals whose metabolites cause liver damage (7). Once administered at some very low doses, methotrexate (MTX) seems to affect the liver and alter its histology. Malaise, nausea, vomiting, diarrhea, headaches, minor alopecia, and fever are more often reported harmful side effects. A different kind of hepatotoxicity, including cirrhosis, is carried on by low-dose therapy (8). It has been observed that taking MTX causes an increase in aminotransferase levels in the blood and has been associated with the development of liver conditions such as cirrhosis and liver fibrosis (9). Methotrexate's toxic side effects have been seen in various animals, including rats, mice, rabbits, and dogs. Clinically, one of the significant limitations of using methotrexate at the required levels is the hepatotoxicity resulting from long-term use. MTX has been shown to lead to a reduction in the antioxidant enzymes. Due to its antioxidant properties, MTX-induced antioxidant enzyme depletion may contribute to an increase in reactive oxygen species (ROS). Part of the reason for MTX's effects is that it makes more ROS directly, which makes it hazardous. "Oxidative stress" is caused by an imbalance between the generation of ROS and antioxidant defenses, which may result in several clinical and pathological disorders (10), commonly hepatic and renal toxicity (11). The reproductive system has also been implicated in MTX effects. Both human and animal male gonads (testes) undergo cellular alterations (12). Animal studies have demonstrated abnormal spermatogenesis, cytotoxicity, spermatocyte, Sertoli cell, and Leydig cell degeneration (13,14). According to studies, oxidative stress is a key factor in the pathophysiology of MTX-induced testicular injury (15). An increase in reactive oxygen radicals (ROS) has been associated with atrophy in the testicular seminiferous tubules and death in spermatocytes (14,16). The shape of the testis was reportedly disrupted by oxidative stress, which also permanently destroyed the germ cells (17,18). Methotrexate testicular toxicity is a significant adverse effect that might cause infertility.

Materials and methods

Ethical approval

The study was conducted in accordance with the declaration of University of Kerbala, College of Veterinary Medicine, Animal Usage Protocol Committee No.9/30 on 15/6/2022.

Animals and experimental design

Six sexually mature domestic male rabbits aged 8-10 weeks, obtained from the veterinary faculty of Kerbala University animal house, were used for the study. The Animal Ethics Committee of Kerbala University's Veterinary Medicine College approved the set of criteria applied to this work. The body weight of the animals ranged from 1-1.5 kg. The rabbits were given time to acclimate under consistent temperature, humidity, and light/dark cycle conditions. Before the experiment, the animals were given free access to regular food and drinking water. Food was carefully selected and confined to vegetables and lettuce. They were divided into two groups, having three animals in each group. The control group (group 1) was administered normal saline. Methotrexate was administered to rabbits of group 2 at the dose rate of 50 mg per kg body weight intraperitoneally daily for two weeks (19). At the end of the experiment, all the animals were sacrificed, and blood was collected. After scarification, the liver and testis were swiftly extracted and fixed in 10% neutral buffered formaldehyde. Serums were prepared for liver function tests and evaluation of Testosterone and FSH.

Chemicals

Methotrexate 50 mg/ml was obtained from Neova Biogene Pvt Ltd (India).

Serum reproductive hormones and liver markers measurements

The FSH, LH, and testosterone levels were determined using ready-made kits (BT lab, Chinese) and ELISA (Biotech, USA) on serum samples taken from experimental animals. The manufacturer's recommendations examined each hormone. For AST and ALT assessments, using sanymed kits (China), liver activities were evaluated using a spectrophotometric method at a wavelength λ:340 nm and 37°C. The results were represented as U/L.

Histological analysis

Animals in all the groups were sacrificed, and liver and testis samples were collected and fixed in 10% formalin; after fixation, the liver tissues were processed routinely for embedding in paraffin (20). Tissue sections of 4 μm were stained with hematoxylin-Eosin (H&E) to evaluate the parenchymal and stromal structure of the liver tissues, Seminiferous tubules, and testicular interstitial spaces. After being stained with hematoxylin and eosin, the sections were examined under a light microscope for histopathological assessment (21).

Results

Results of liver histopathology

Based on the slides' microscopic descriptions, the normal control group was observed to have a radial arrangement and stroma (Figure 1), preserved hepatic cells of intact cytoplasm, prominent nucleus, visible portal tracts, and central veins (Figure 2). In MTX group, we noticed significant hepatocyte vacuolar degeneration, considerable sinusoidal dilatation (Figure 3), and intense congestion compared with the control-saline group (Figure 4). A significant infiltration of periportal inflammatory cells and early necrotic features indicates hepatitis. (Figures 5 and 6). In addition to severe bile duct epithelial hyperplasia (Figure 7).

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Figure 1: There is vacuolar degeneration of hepatocytes of a control rabbit liver, normal hepatic architecture (white arrow) cords arranged radially around the central vein (black arrow). H&E, 10x.

Figure 2: There is vacuolar degeneration of hepatocytes of a control rabbit that showed normal hepatocellular structure, hepatocyte (H) as a large cell with distinct borders and granular cytoplasm around the central vein (C.V) (black arrow). H&E, 40x.

Figure 3: Photomicrograph of the liver of a rabbit treated with 50 mg/kg Methotrexate, showing severe congestion and dilatation of central vein (black arrow), loss of normal architecture (yellow arrow). H&E, 4x.

Figure 4: Photomicrograph of a Methotrexate MTX-induced liver histopathological changes revealed significant hepatocellular injury represented by hepatocytes cytoplasmic vacuolation (black arrow). H&E, 10x.

Figure 5: Photomicrograph of a Methotrexate MTX-treated liver tissue revealed marked periportal inflammatory cells infiltration (black arrow), mild to moderate portal vein congestion (yellow arrow), and significant necrotic nuclear changes (red arrow). H&E, 10x.

Figure 6: Photomicrograph of a Methotrexate MTX -treated liver tissue, revealed significant, severe chronic periportal inflammatory cells infiltration, mainly macrophages (black arrow), vascular congestion (yellow arrow), and severe hepatocellular necrotic changes manifested by nuclear karryohexis (red arrow). H&E, 40x.

Figure 7: Photomicrograph of a Methotrexate MTX--induced liver tissue injury showed severe bile duct epithelial hyperplasia (black arrow), portal vein dilatation (yellow arrow), and significant inflammation (red arrow). H&E, 40x.

Testicular histopathology

MTX group testes showed histological alterations compared to control group testes, spermatocyte disintegration, and tubular cell necrosis (Figures 8-10). The main histological findings include reduced mature spermatozoa in the lumen edge of the seminiferous tubules and epithelial degeneration, irregular morphology, and deformation of germ cells (Figure 11).

Figure 8: The best Photomicrograph of the testis in the control group showed normal morphology of seminiferous tubules (black arrow); consider the germinal cells' orderly arrangement (yellow arrow). H&E, 10x.

Figure 9: Photomicrograph of testis in the control group, showed normal morphology of seminiferous tubules (black arrow), normal germinal epithelia (yellow arrow), and presence of mature spermatozoa towards tubular lumen (red arrow). H&E, 40x.

Figure 10: Photomicrograph of testis in MTX group showed disarrangement in seminiferous tubule morphology (black arrow) disruption of germinal epithelia (yellow arrow). H&E, 10x.

Figure 11: Photomicrograph of testis in MTX group, showing immature germinal epithelia of seminiferous tubules (black arrow), reduction in mature spermatozoa (yellow arrow), and degeneration of spermatocytes (red arrow). H&E, 40x.

Serum testosterone, FSH, LH, AST and ALT

The figure 12A and 12B demonstrates that MTX-treated rabbits had considerably lower serum FSH LH levels compared to control, which is a symptom of reproductive injury. MTX caused significant drops in serum testosterone levels in rats, a sign of reproductive impairment (Figure 12C). ALT activity in the MTX group was considerably higher than in the Control-saline group (Figure 12E). There was no statistically significant difference in the AST levels across the groups (Figure 12D).

Figure 12: Evaluation of MTX effects on rabbits' serum FSH(A), LH(B), Testosterone(C), AST(D), and ALT(E) levels for the two groups of rabbits: control group (CTRL), MTX-treated group (MTX).

Discussion

One of the drugs used to treat anti-metabolites is MTX, which is recommended for various neoplastic illnesses. However, MTX toxicity has been linked to several organ systems (22). Low dosages of MTX may cause alterations in the liver's histology and, over time, may result in a distinct kind of hepatotoxicity. However, high-dose administration, like in leukemia, can cause severe liver tissue destruction, an increase in hepatic enzymes, cirrhosis, and progressive fibrosis (23,24). Histopathological analysis of the current investigation revealed that MTX significantly caused histopathological modifications and hepatic tissue injury. This was consistent with earlier research (25,26), which discovered that MTX can cause hepatic lesions like focal fibrosis (27). These lesions could be recognized because the liver is the primary site of MTX metabolism into the toxic agent 7-hydroxy-MTX (28,29). Although the precise mechanism by which MTX causes hepatotoxicity is not entirely understood, it can be primarily related to the production of oxidative stress (30). As a result, other papers indicated that taking MTX causes the depletion of antioxidant enzymes and the production of free radicals (31,32), consistent with our findings. According to several studies, administering anti-cancer medications can cause qualitative and quantitative changes in gametes (33,34). Like other chemical products like pesticides, MTX also affected fertility (35-38). It has been documented that giving male rats anticancer medications like cisplatin and Adriamycin results in desquamation of germ cells, degeneration, and a decrease in seminiferous diameter and germinal cell thickness (39,40). The most prevalent long-term morbidities associated with chemotherapy are generally regarded as gonadal dysfunctions connected to chemotherapy and their impact on reproductive health (41). Yet, MTX damages testicular tissue by activating the body's endogenous inflammatory process in the cell (42).

 In addition, MTX injection caused testicular injury that was evident in the sloughing, atrophy, and degeneration of germ cells in the seminiferous tubules (43). The current study on rabbits' male reproductive system supports this. Particularly the degeneration of germ cells and reduced spermatogenesis. According to studies (44,45), the etiology of testicular injury caused by MTX is linked to high levels of oxidative stress, which activate the inflammatory response; our results support earlier studies on other tissues that demonstrated MTX to cause severe histological damage and modifications (46,47). Moreover, spermatogenetic abnormalities doubt the progression of spermatogenetic stages due to Sertoli cell destruction (48). In the same line, Nouri et al. (49) noted that spermatogenesis in MTX-treated rats had not achieved its full capability and had been altered. These modifications might result from MTX's oxidative activity. Hepatotoxicity is demonstrated by an increase in serum ALT activity in the MTX-treated rabbit group. However, AST in our experiment did not differ significantly across groups. For liver pathology, serum ALT is more specific.

 AST is a sign of long-term liver toxicity (50). This means that our study time might not be long enough to detect the AST alteration. MTX can potentially disrupt healthy spermatogenesis and ultimately result in infertility since it speeds up DNA damage, apoptosis, and uncontrolled apoptosis in germ cells. In addition to lowering the number and motility of sperm and the levels of serum testosterone, MTX may also shorten seminiferous tubules and slough the epithelium (51).

Conclusion

Our analysis demonstrates the significance of MTX-induced testicular and hepatic injury. Methotrexate administration caused severe damage-related degenerative changes, such as spermatocyte disintegration and a reduction in mature spermatozoa, hepatic lobular alterations with inflammatory cell infiltration, congestion, and hepatocyte degeneration. To better understand how methotrexate works, additional molecular and ultrastructural research is necessary.

Acknowledgment

I would like to express my special thanks to all the efforts provided throughout the time working on this study and for all the support during the project's completion.

Conflicts of interest

There are no conflicts of interest.