Allopurinol and Febuxostat Equally Attenuate Methotrexate-Induced Testicular Injury In Rats Via Activation Of EGF/ ERK 1/2 /HO ‐ 1 Signaling Pathway.

Methotrexate (MTX) is commonly used in the management of several malignancies and autoimmune disorders; however, testicular damage is one of the most detrimental effects of MTX administration. In the current study, we evaluated the possible protective effect of xanthine oxidase inhibitors either purine analogue; allopurinol (ALL) or non-purine analogue; febuxostat (FEB) in testicular injury induced by MTX in rats. Gonadotoxicity was induced by a single dose of MTX (20 mg/kg, i.p.). ALL and FEB were administered orally in the following daily doses (100, 10 mg/kg, respectively) for 15 days. Total and free testosterone were measured in serum. In addition, total antioxidant capacity (TAC), epidermal growth factor (EGF), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), extracellular signal-regulating kinase1/2 (ERK1/2), and nitric oxide (NO) end products were measured in testicular tissues. At the same time, immunoexpression of HO-1in testicular tissue was measured. Histopathological examination was done. ALL and FEB increased total and free serum testosterone. Both drugs showed a signicant reduction in testicular MDA, NO, TNF-α levels with an increase in TAC, EGF, and ERK1/2 levels in testicular tissue. Furthermore, both drugs enhanced HO-1 immunoexpression in testicular tissue. All these ndings were parallel to the preservation of normal testicular architecture in rats treated with ALL and FEB. In conclusion, All and FEB were protective against testicular damage induced by MTX through anti-inammatory, anti-apoptotic, and antioxidant actions which might be through activation of the EGF/ERK1/2/HO-1 pathway. At the same time, no signicant difference between ALL and FEB was noticed in this protective effect.


Introduction
Methotrexate, a folic acid antagonist, is a commonly prescribed medication with diverse indications including treatment of several types of cancer and autoimmune diseases. According to the most recent WHO's list of essential medicines, methotrexate (MTX) was included [1]. [2].
However, the unwanted effect of methotrexate on high proliferating organs represents a major concern. Testicular injury is among the limiting adverse effects of methotrexate ( [2]). Exposure to MTX causes testicular damage. Such effect is mediated through oxidative stress, apoptotic changes, in ammation and disturbance in blood supply [3].
Febuxostat (FEB) is a nonpurine XO inhibitor, is a more potent serum uric acid-lowering action compared with ALL [5]. FEB also antagonizes oxidative stress and in ammation in several models of cell injury.
Furthermore, It was reported that FEB reduces cardiotoxicity, nephrotoxicity, hepatotoxicity and pulmonary in ammation that are induced by certain cancer chemotherapeutic agents [6].
The present study evaluated and compared the expected protective effects of ALL and FEB on testicular damage induced by MTX in rats. The mechanisms of their effects were explored.

Ethics
Animal handling, medications, and scari cation were carried out following the guidelines for the care of experimental animals indicated by Institutional Ethical Committee (Faculty of Medicine, Minia University, Egypt) and according to the NIH Guide for taking care and use of laboratory animals (Approval number 325:11/2019).

Animals and experimental design
Adult male albino rats (200-250g, age 8-10 weeks) were purchased from the National Research Centre, Giza, Egypt. Rats housed in laboratory cages and feed a standard diet of rat chaw and tap water. Rats allowed 7 days to acclimate to the surroundings before beginning any experimentation. The temperature was maintained at 22 ± 2 °C. A 12/12 h light/dark cycle was maintained.

Experimental design
Rats were allocated to four groups (n=8) as follows: Group 4: Received oral febuxostat (10 mg/kg/d) for 14 day and at 11 th day received a single i.p. injection of MTX (20 mg/kg) [9].
After completing the experiment, blood gathered from the anesthetized rats and sera stored at −80 °C for determination of testosterone. Testis were dissected and washed with phosphate buffer solution and stored at −80°C for measuring TAC, MDA, NO, EGF and TNF-α. The other half of testis immersed in Bouin's uid for histological study.

Biochemical measurements in testicular tissue
Levels of MDA, TAC and NOx measured calorimetrically using kit according to the manufacturer's instructions. TNF-α, ERK 1/2 , and EGF levels in testicular tissue homogenate was assessed by using ELISA kits according to manufacturer's instructions.

Determination of serum testosterone
Serum testosterone measured by ELISA kit guided by the manufacturer's instructions.
2.6. Histological study 2.6.1. Histopathological examination Hematoxylin-eosin staining was used and examined by optical microscope. Tissue damage in testis was graded and by multiplying each area grade by the percentage of totally occupied surface, the nal result for each testis was calculated. It was scored as the following: grade 1; normal testicular structure with intact germinal epithelium, grade 2; testicular Injury with germinal cells (less orderly and non-cohesive) together with closely packed seminiferous tubules, grade 3; testicular Injury with disorganized germinal cells with nuclear pyknotic and less distinct seminiferous tubule borders and grade 4; closely packed seminiferous tubules with coagulative necrosis of the germinal cells. The pathologist who examined and graded the histological changes in the testes was blinded to the type of the treatment of each animal. Grade 1 is de ned by normal tissue [10].

Immunohistochemical study of caspase-3 and Heme oxygenase-1 in testicular tissue
Immunohistochemical examination of caspase-3 and HO-1 were performed using monoclonal mouse antibodies. Guided by anti-caspase-positive cells percentage in the eld, immunoexpression was semiquantitatively scored as follows: 0, when absent; 1, positive cells were less than 10%; 2, when positive cells were from 10% to 50%; 3, when positive cells were from 51% to 90% and 4, when positive cells were more than 91%. HO-1 immunoreactivity was evaluated as positive cells number (based on positive staining percentage) and staining intensity. Extent was scored as: 0, less than 5%; 1, from 6% to 25%; 2, from 26% to 50% and 3, more than 50%. Intensity of staining was scored as: 0, weak; 1, moderate; 2, strong. The scoring was calculated by multiplying extent score by intensity [11,12].

Statistical analysis
Mean ± SEM is used to express results. One-way analysis of variance (ANOVA), followed by Tukey's HSD post hoc test for multiple comparisons were used. P value < 0.05 is considered signi cant. Graph Pad Prism7 program was used to analyze data.

Effect on serum testosterone.
Levels of free and total serum testosterone were decreased in MTX group when compared to control group. However, when compared to MTX group, the serum levels of free and total testosterone were increased in MTX+ALL and MTX+FEB groups [ Table 1]. No signi cant difference was noticed between MTX+ALL and MTX+FEB groups.  As compared to control group, testicular TNF-α was elevated in MTX group. In groups treated with ALL and FEB, testicular TNF-α was decreased, as compared to MTX group. The level of testicular TNF-α in MTX+FEB was higher than its level in MTX+ALL. Levels of EGF in testicular tissue were reduced in MTX group, as compared to control group. However, in MTX+ALL and MTX+FEB groups, levels of EGF in testicular tissue were increased, as compared to MTX group. Levels of ERK1/2 in testicular tissue were reduced in MTX group, as compared to control group. However, in MTX+ALL and MTX+FEB groups, levels of EGF in testicular tissue were increased, as compared to MTX group [ Table 3]. The changes in TNF-α, EGF and ERK1/2 were insigni cant in MTX+FEB group, as compared to MTX+ALL group.

Morphometric Results
The results of grading of testicular damage are shown in Table 4. An increase in total score of testicular tissue damage in MTX group when compared to control group. In contrast, reduction in total scoring of tissue damage in MTX+ALL and MTX+FEB groups, as compared to MTX group.

Discussion
Methotrexate is used as a cancer treatment of vast types of oncological disorders and also used in other disorders as psoriasis, rheumatic diseases and in ammatory bowel diseases [13].
Methotrexate Testicular toxicity has robust side effect causing subsequent infertility. Data from previous studies stated a disorganization in the seminiferous tubules of the testis, a reduction in sperm number, and sperm DNA destruction after MTX administration [14]. In current work, we evaluated the contribution of xanthine oxidase inhibitors in abrogation acute chemotherapy testicular cytotoxic effect. To the best of our knowledge, this is the rst in vivo study to observe a protective effect of purine analogue XO inhibitor; febuxostat versus non-purine analogue XO inhibitor; allopurinol, in testicular damage induced by MTX in rats.
MTX toxicity accumulates ROS and depletes antioxidants and hence damaging cellular macromolecules [15]. lipid peroxidation with consequent cell damage results from reaction of polyunsaturated fatty acids and membrane lipids with reactive oxygen species [16]. Also, NO is increased as a result of inducible nitric oxide synthase activation [17]. The reaction of ROS and NO produces the versatile oxidant peroxynitrite that can damage DNA and other cellular molecules [18]. Similarly, in the current work, MTX administration caused reduction in TAC and increase in MDA and NO in testes.
In ammation has a role in MTX-induced testicular cytotoxicity. previous studies report an increase in circulating pro-in ammatory cytokines with MTX administartion [19]. The former action caused by activation of the transcription factor NF-Kb through exaggerated production of ROS and the activated NF-kB has a direct relation to enhancement of the pro-in ammatory cytokines such as TNF-α [20].
In current study, after administration of MTX, cellular TNF-α activity signi cantly increased. Many studies illustrate that, TNF-α has a role in the pathogenesis of MTX-induced testicular injury [21].
In current study rats received ALL and FEB showed a signi cant decrease in MDA, NO and TNF-α levels and increased in TAC in testis. These ndings are in accordance with previous studies demonstrated that ALL possess antioxidant properties, which may be attributable to inhibition of xanthine oxidase which results in attenuation of oxidative stress, enhanced the antioxidant defenses and amelioration of in ammatory status in different tissues liver [22], [23], [24] [8]. In consistence with our results, Amirshahrokhi reported the antioxidant and anti-in ammatory effects of FEB [9]. These effects result from FEB ability to anatgonize oxidative stress and in ammation induced by XO. FEB also antagonized cardiotoxicity induced by doxorubicin, cisplatin-induced nephrotoxicity, nephritis and lung in ammation [25,26].
Caspase 3, is a crucial enzyme in programmed cell death pathway is implicated in germ cell apoptosis with MTX administration [27]. In current work, MTX-induced caspase-3 expression in testis, which agrees with [21] who reported that MTX-induced elevation in caspase 3 immunostaining. Induction of oxidative stress and in ammation are two supposed mechanisms induce apoptosis of testicular cells [28]. At the same time, activation of proapoptotic proteins may have a role in gonadotoxicity induced by MTX [14,29].
Interestingly, ALL or FEB signi cantly decreased testicular caspase-3 when supplemented with MTX. Other reports showed that XO-inhibitors are able to antagonize apoptosis [30].
The well-acknowledged participation of oxidative stress induction and in ammation aggravation in stimulating apoptosis emphasizes that the anti-apoptotic effect of ALL and FEB might be related to amelioration of oxidative stress and in ammation. Our results are supported by previous ndings about the antagonism of germ cell apoptosis and improvement of spermatogenesis with ALL treatment [31].
One of the key growth factors regulating cell survival is EGF that has been shown in Leydig cells, Sertoli and peritubular cells. By binding to EFG receptors, IT activates an extensive network of signal transduction pathways that regulate expression of both pro-and anti-apoptotic proteins that was effective in blocking the apoptosis [32]. Also, withdrawal of EGF from the extracellular environment increased the sensitivity to apoptotic stimuli [32]. Furthermore, EGF antagonized in ammation, ROS generation in many models of cell damage [33], [34], [35], [36], [37].
In an attempt to explore the contribution of EGF pathway in testicular damage, we evaluate EGF in testes which showed that, MTX reduced testicular EGF level, as compared to control group. In contrast, ALL and FEB induced expression of EGF in testicular tissue. These ndings run parallel to improvement of oxidative stress, in ammation, and apoptosis status noticed with ALL and FEB.
In order to go deeper in understanding the mechanistic pathway underlying the protective effect of EGF which increased in treated groups we examined the status of downstream tyrosine kinase ERK 1/2 , one of MAPKs, and has a pivotal role in the cellular death and survival [38]. Moreover, ERK 1/2 signaling inhibition caused subsequent induction of apoptosis-associated proteins [39]. In current work, p-ERK 1/2 increased in the testicular tissue exposed to MTX + ALL and increased in MTX + FEB groups when compared with MTX intoxicated rats. Interestingly, recent studies demonstrated that several drugs provide protective effect through activating ERK signaling cascade [40][41][42]. Surprisingly, the current results are supported by reports exhibited the link between activation of EGF and induction of ERK 1/2 [43].
Heme oxygenase (HO)-1, member of the family of heat shock proteins (HSP-32), plays critical roles in physiological iron homeostasis, antioxidant defense and antiapoptotic effects [44]. Furthermore, induction of HO-1 results in reduction in in ltration of in ammatory cell and exudates, whereas HO-1 downregulation enhanced in ammatory exudates, illustrating the role of HO-1 in modulating in ammatory response [45].
Interestingly, MTX in the current study reduced HO-1 expression as evidenced by the undeniable elevation in semiquantitative scoring of immunoreactive cells for HO-1. Running in the same stream, Mahmoud and co-workers reported that MTX reduced expression of HO-1 [46]. In contrast, ALL and FEB upregulated HO-1 in testicular tissue which agrees with previous numerous studies stated the enhancing effect of xanthine oxidase inhibitors on HO-1 expression and linked that to the cytoprotective effects of ALL and FEB in different models of tissue injury. In these models of tissue injury, the amelioration of HO-1 expression was directly related in ammation, oxidative stress and apoptosis inductions. On the other hand, ALL and FEB reverted the reduction of HO-1 expression which was restorative to tissue integrity and functions [47], [48], [49]. The current induction of HO-1 is supported with data revealed that activation of HO-1 gene occurred via regulation of ERK1/2 and this enhances cell survival [50]. Furthermore, it was stated that protection against cell damage attained by HO-1 signaling induction involved ERK 1/2 pathway modulation [40].
These ndings are shedding light on the contribution of EGF, ERK 1/2 and HO-1 in oxidative stress, apoptosis and in ammatory cascade induced during testicular cytotoxicity. Surprisingly, EGF receptormediated signaling pathways affected ERK 1/2 that inturn induced HO-1 [44]. This nding can support the assumption that inhibition EGF, ERK 1/2 and HO-1 signaling pathway can provoke apoptosis, oxidative stress and in ammation and vice versa.
At the end of the day, the undeniable increase of serum testosterone and normalization of histopathological picture in ALL and FEB groups, as compared to MTX group, con rmed the protection afforded by ALL and FEB in testis damage. To the best of our knowledge this is the rst time to study the comparative effect of purine analogue versus non-purine analogue xanthine oxidase inhibitors. On the light of the previous data, we found that ALL and FEB are equipotent in their cytoprotective effect in testicular damage.
In conclusion, EGF through subcellular activation of ERK1/2/HO-1 pathway might have a crucial role in the protective effect of ALL and FEB in MTX induced testicular damage as shown through antioxidant, anti-in ammatory, and anti-apoptotic actions. Furthermore, no signi cant difference was noticed between ALL and FEB in a such valuable effect. that all data were generated in-house and that no paper mill was used.

Figure 1
Effect on testis histopathological picture. A representative photomicrograph of a section in testis tissue (20x). A) control group, B) MTX group, necrobiosis and nuclear pyknosis were detected in some of spermatogonial cells of the seminiferous tubules, C) MTX+ALL group; well-organized normal germinal epithelium, D) MTX+FEB group; a well-preserved tubular structures and normal germ cells.

Figure 2
Effect on caspase-3 immunoreactivity in testicular tissue (40x). A; control group, B; MTX group, C; MTX+ALL group and D; MTX+FEB group.