Methotrexate is used as a cancer treatment of vast types of oncological disorders and also used in other disorders as psoriasis, rheumatic diseases and inflammatory 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 first 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.
Inflammation has a role in MTX-induced testicular cytotoxicity. previous studies report an increase in circulating pro-inflammatory 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-inflammatory cytokines such as TNF-α [20].
In current study, after administration of MTX, cellular TNF- α activity significantly 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 significant decrease in MDA, NO and TNF-α levels and increased in TAC in testis. These findings 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 inflammatory status in different tissues liver [22], [23], [24] [8]. In consistence with our results, Amirshahrokhi reported the antioxidant and anti-inflammatory effects of FEB [9]. These effects result from FEB ability to anatgonize oxidative stress and inflammation induced by XO. FEB also antagonized cardiotoxicity induced by doxorubicin, cisplatin-induced nephrotoxicity, nephritis and lung inflammation [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 inflammation 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 significantly 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 inflammation aggravation in stimulating apoptosis emphasizes that the anti-apoptotic effect of ALL and FEB might be related to amelioration of oxidative stress and inflammation. Our results are supported by previous findings 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 inflammation, 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 findings run parallel to improvement of oxidative stress, inflammation, 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 ERK1/2, one of MAPKs, and has a pivotal role in the cellular death and survival [38]. Moreover, ERK1/2 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 [40–42]. Surprisingly, the current results are supported by reports exhibited the link between activation of EGF and induction of ERK1/2 [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 infiltration of inflammatory cell and exudates, whereas HO-1 downregulation enhanced inflammatory exudates, illustrating the role of HO-1 in modulating inflammatory 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 inflammation, 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 ERK1/2 pathway modulation [40].
These findings are shedding light on the contribution of EGF, ERK1/2 and HO-1 in oxidative stress, apoptosis and inflammatory cascade induced during testicular cytotoxicity. Surprisingly, EGF receptor-mediated signaling pathways affected ERK1/2 that inturn induced HO-1 [44]. This finding can support the assumption that inhibition EGF, ERK1/2 and HO-1 signaling pathway can provoke apoptosis, oxidative stress and inflammation 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, confirmed the protection afforded by ALL and FEB in testis damage. To the best of our knowledge this is the first 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-inflammatory, and anti-apoptotic actions. Furthermore, no significant difference was noticed between ALL and FEB in a such valuable effect.