Melatonin Attenuates Methotrexate-Induced Reduction of Antioxidant Activity Related to Decreases of Neurogenesis in Adult Rat Hippocampus and Prefrontal Cortex

Previous studies have revealed that the side effects of anticancer drugs induce a decrease of neurogenesis. Methotrexate (MTX), one of anticancer drugs, can induce lipid peroxidation as an indicator of oxidative stress in the brain. Melatonin has been presented as an antioxidant that can prevent oxidative stress-induced neuronal damage via the activation of antioxidant enzymes associated with the increase of neurogenesis. The aims of the present study are to examine the neuroprotective effect of melatonin on the neurotoxicity of MTX on neurogenesis and the changes of protein expression and antioxidant enzyme levels in adult rat hippocampus and prefrontal cortex (PFC). Male Sprague-Dawley rats were assigned into four groups: vehicle, MTX, melatonin, and melatonin+MTX groups. The vehicle group received saline solution and 10% ethanol solution, whereas the experimental groups received MTX (75 mg/kg, i.v.) and melatonin (8 mg/kg, i.p.) treatments. After the animal examination, the brains were removed for p21 immunofluorescence staining. The hippocampus and PFC were harvested for Western blot analysis and biochemical assessments of malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD). The immunofluorescence result showed that coadministration with melatonin diminished p21-positive cells in the hippocampal dentate gyrus, indicating a decrease of cell cycle arrest. Melatonin reduced the levels of MDA and prevented the decline of antioxidant enzyme activities in rats receiving MTX. In the melatonin+MTX group, the protein expression results showed that melatonin treatment significantly upregulated synaptic plasticity and an immature neuron marker through enhancing brain derived neurotrophic factor (BDNF) and doublecortin (DCX), respectively. Moreover, melatonin ameliorated the antioxidant defense system by improving the nuclear factor erythroid 2-related factor 2 (Nrf2) in rats receiving MTX. These findings suggested that the effects of melatonin can ameliorate MTX toxicity by several mechanisms, including an increase of endogenous antioxidants and neurogenesis in adult rat hippocampus and PFC.


Introduction
The anticancer agents have been found to have various adverse effects in several organs [1][2][3][4]. Previous studies have found that "Chemobrain" described as the cognitive alterations in the brain can occur in cancer patients receiving anticancer drugs [5,6]. Methotrexate (MTX) is one of anticancer drugs classified in the antimetabolite group and readily crosses the blood-brain barrier (BBB). The mechanism and action of MTX function through inhibiting thymidylate synthase (TS) and blocking deoxythymidine monophosphate (dTMP) synthesis, resulting in an inhibition of DNA synthesis [7]. Interestingly, several studies have previously postulated the neurotoxicity of MTX in the brain [8][9][10][11][12]. In animal models, MTX-induced degeneration of neurons in the subgranular zone (SGZ) in the dentate gyrus of hippocampus linked to impairment of spatial and recognition memories. These memories were controlled by the hippocampus and PFC, respectively [10][11][12]. Accumulation of oxidative stress is an essential process that correlates with the degeneration of newly born neurons, whereas enzymatic antioxidants function as free radical scavengers that act as the major source of protection against oxidative stress [13]. Several studies have shown that hippocampal neurogenesis in the SGZ of the dentate gyrus has a clear correlation with oxidative stress and antioxidant enzyme levels [14][15][16][17]. A recent study has demonstrated that MTX markedly induces oxidative stress and reduces antioxidant enzyme levels in the hippocampus and PFC [18]. Moreover, MTX treatment causes cell cycle arrest in proliferating cells in the SGZ in the hippocampal dentate gyrus [18]. Therefore, it is believed that exposure to MTX can reduce neurogenesis, and this is closely related to memory deficits.
Various studies have found that antioxidant exposure can stimulate adult neurogenesis in animal models [19][20][21]. Melatonin (N-acetyl-5-methoxytryptamine) has outstanding properties as an antioxidant and is widely used in experimental studies examining the hippocampus and PFC [22][23][24][25]. It is well known that melatonin functions directly as a free radical scavenger and indirectly as reducing oxidative stress through the stimulation of antioxidant enzymes [26,27]. This hypothesis was supported by an important role of melatonin in encouraging Nrf2 that plays an influential capacity in antioxidant defense systems [14,17,28,29]. In addition, the benefit of melatonin has been shown to promote DCX functions in the hippocampus, which has the potential for neuronal migration and is normally known as markers of premature neurons [11,[30][31][32][33][34]. The regulative function of melatonin in adult neurogenesis is modulated by neurotrophic factors, for example, BDNF, which plays a crucial role in synaptic plasticity and memories [17,25,35]. As shown in the previous studies, melatonin ameliorated memory deficits in both spatial and recognition memories and improved the BDNF levels in the adult rat hippocampus [17,36].
In addition to the interesting studies in the effects of melatonin on memory and neurogenesis, the present study focused on neuroprotective effects of melatonin on molecular mechanism related to neurogenesis in the MTX-induced rats. Therefore, the present study examined that melatonin administration mitigated MTX-induced cell cycle arrest, oxidative stress, and a decline of antioxidant enzymes and levels of DCX, Nrf2, and BDNF protein expression in hippocampus and PFC. However, these side effects of MTX were alleviated by cotreatment with melatonin.  [33,37,38]. MTX and leucovorin (LCV) were purchased from Pharmachemie B.V., Haarlem, Netherlands. LCV was used to reduce toxicity of MTX [8,39].

Experimental Procedures.
Rats were randomly arranged into four groups (n = 14 animals/group): vehicle, MTX, melatonin, and melatonin+MTX. In the vehicle group, 10% ethanol solution (10% ethanol diluted in saline solution) was given by intraperitoneal (i.p.) injection to rats at 7 : 00 pm. from day 1 to day 15, and a dose of saline solution (0.9% NaCl, Thai Nakorn Patana Co., Ltd., Nonthaburi, Thailand) was administered by intravenous (i.v.) injection at 10 : 00 am. on days 8 and 15 of the experiment. In the melatonin group, melatonin (8 mg/kg) was given by i.p. injection to rats at 7 : 00 pm. from day 1 to day 15. In the MTX group, a dose of MTX (75 mg/kg) was given by i.v. injection at 10 : 00 am. on days 8 and 15 of the experiment. After each MTX injection, LCV (6 mg/kg, at hour 18 h, and 3 mg/kg, at hour 26, 42, and 50) was administered to rats, whereas rats in the vehicle group received an identical volume of saline solution by i.p. injection. Rats in the melatonin+MTX group received equal volumes of melatonin and MTX at the corresponding time points (Figure 1).

Tissue Sample Preparation.
On day 3 after the end of the experimental duration, the rats in each group were euthanized by rapid stunning and decapitated. For p21 immunofluorescence study, a cerebral hemisphere was immediately harvested and immersed in 30% sucrose solution (Ajax Finechem, Auckland, New Zealand) for 3 h at 4°C [20]. Next, an optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) was used to embed the brains, and followed by immediate transferring, the embedded brains to liquid nitrogen cooled isopentane (Sigma-Aldrich, St. Louis, MO, USA) [40]. The other left and right cerebral hemispheres of the hippocampus and PFC were harvested and immediately transferred to liquid nitrogen for Western blotting and biochemical examinations. All tissue samples were transferred to a -80°C freezer until analysis.
2.5. Biochemical Assessments. The cold deionized water was used to homogenize the hippocampal and PFC tissues, and the tissue homogenates were centrifuged at 13,000 rpm at 4°C for 10 min. The supernatant was gently collected, and the protein concentrations were analyzed using the Nano-Drop spectrophotometer (NanoDrop™ 2000, Thermo Fisher Scientific, USA). All biochemical assessments were detected  3 Oxidative Medicine and Cellular Longevity coronal plane to get the whole dentate gyrus (bregma point -2.3 to -6.3 mm) [42] using a cryostat (Cryostat Series HM550 Microm international, A.S. Science Co., Ltd., Walldorf, Germany) and collected into 24-well plates containing the cryoprotective buffer. Sections were incubated in blocking buffer (20% normal goat serum (Abcam, Cambridge, UK) in PBS containing 0.5% Triton X-100) for 1 h and incubated with an anti-p21 (1 : 100, Santa Cruz Biotechnology, TX, USA) at 4°C overnight. The next day, sections were washed with buffer (PBS containing 0.3% Triton X-100), followed by incubating with an Alexa Fluor 488 rabbit anti-mouse IgG (1 : 300, Invitrogen, San Diego, CA, USA) for 1 h, and were labeled with propidium iodide (PI) (1 : 6000, Sigma-Aldrich, St. Louis, MO, USA). Nine sections from every 8th brain section of the entire length of the dentate gyrus were selected from each rat for staining [43]. The p21-positive cells within the three cells of the internal rim of the dentate gyrus were scored, and a total positive cell count of nine sections from each rat were multiplied by 8 for quantification. Fluorescence images were taken at 10× and 40× magnifications using a fluorescence microscope (Nikon ECLIPSE 80i, Melville, NY, USA).

SOD Levels.
MTX treatment induced the decline of SOD levels in the hippocampus. SOD levels in the MTX group were significantly lower than the vehicle group. Data represents mean ± SEM of the hippocampal SOD levels, vehicle: 24:55 ± 2:69 unit/mg protein; MTX: 14:49 ± 1:57 unit/mg protein; MEL: 27:66 ± 1:41 unit/mg protein; and MEL+MTX: 23:64 ± 1:23 unit/mg protein (p < 0:05, p < 0:01, p < 0:001, Figure 3(d)). Moreover, treatment with melatonin in MTX-treated rats improved SOD levels in the PFC. The SOD levels in the PFC in the MEL+MTX group were significantly greater than the MTX group, but this was not detected in comparison with the vehicle group.  Figure 4(d)).  Figure 5).

Discussion
Chemotherapy-induced deterioration of memory and hippocampal neurogenesis has been recently reported [44]. MTX is a chemotherapeutic medication used to treat cancer, neoplastic disorders, and rheumatoid arthritis. However, the use of this drug is restricted because of its side effects on multiple organs such as hepatotoxicity, renal toxicity, and neurotoxicity [3,4,8]. The present work aimed to study the adverse effects of MTX on hippocampus and PFC, which are the brain areas that are related to neurogenesis and cognitive functions [45,46].
Numerous studies suggest that the SGZ of dentate gyrus in the hippocampus is where neurogenesis occurs through-out life [47,48]. Earlier reports have demonstrated that the toxicity of MTX impaired hippocampal neurogenesis. MTX induces decreases in neurogenesis in the SGZ of the hippocampal dentate gyrus including proliferation and survival of cells and immature neurons using immunofluorescence studies [10][11][12]. Many previous studies have shown that immature neurons are most commonly characterized by doublecortin or DCX [30,31]. DCX is a microtubuleassociated protein that usually expresses in immature neurons develop into new neurons [49]. In migrating cells, DCX binds to microtubules and results in promoting the movement of neuronal cells [50]. Therefore, DCX is important for neuronal migration by regulating microtubule stabilization [51]. The present study was confirmed more precisely with the protein expression levels. The result shows that the toxicity of MTX significantly reduced the DCX levels in the hippocampus. This is comparable to previous reports that depict that the DCX levels in the hippocampus are dramatically reduced in MTX-treated rats [18,52]. A previous report demonstrates that melatonin is a pharmacological intervention protecting against chemotherapyinduced neurogenic impairment [44]. The present study clearly shows that melatonin can ameliorate DCX expression levels in the melatonin+MTX group. In line with previous researches, melatonin obviously upregulates the level of immature neurons in the SGZ of the dentate gyrus in adult rats treated with chemotherapy such as MTX and 5-  Oxidative Medicine and Cellular Longevity fluorouracil (5-FU) and antiepileptic drug such as valproic acid (VPA) [11,33,34]. Moreover, DCX stabilizes microtubules to promote microtubule polymerization, which is necessary for neurite and dendrite formation [49]. Previous studies have reported that melatonin administration in C57BL/6 mice increases DCX-labeled cells especially imma-ture neurons with more mature dendrites in the hippocampal dentate gyrus [30,31]. Extending more dendritic complexity of new generated neurons into the granule cell layer in the dentate gyrus and then the molecular layer to organize synaptic connection is essential for the integration of new neurons into the hippocampal circuitry [53,54]. Further study in the present work confirms one of the negative consequences of MTX on neurogenesis in the SGZ of the dentate gyrus is cell cycle arrest using p21 immunofluorescence examination [55]. The p21 is well known as a cyclin-dependent kinase (CDK) inhibitor, which promotes both CDKs inhibition and cycle arrest during the G1/S phase activated by various stimuli [56]. Focus on the present study, an improvement of p21-positive cells in the MTX group indicates upregulation of cell cycle arrest. Corresponding to our previous research, rats receiving MTX showed an increase in p21-positive cells in the SGZ of the hippocampal dentate gyrus [18,52]. It is concluded that the toxicity of MTX stimulates cell cycle arrest, which has a negative effect on neurogenesis. However, the present research postulates the protective effect of melatonin against MTX-induced cell cycle arrest in the hippocampus. The results demonstrate the decrease of p21-positive cells in the SGZ of hippocampal dentate of rats in the melatonin+MTX group. Our previous study suggested that cotreatment with melatonin in 5-FUtreated rats could relieve the increase of cell cycle arrest [17]. Altogether, melatonin could inhibit oxidative stress  Oxidative Medicine and Cellular Longevity caused by side effects of MTX, resulting in the decrease of cell cycle arrest in the hippocampus. Therefore, the present study shows that melatonin can improve neurogenesis and inhibit cell cycle arrest in MTX-treated rats.
Associations between neurogenesis and BDNF have been found in several studies [57,58]. BDNF is a neurotrophin family of growth factors and plays important activities in several fields of brain functions, for example, cell survival, learning, and memory [58][59][60]. Signaling pathway of BDNF enhances the cAMP response element binding protein (CREB) transcription factor, which regulates the nerve cell survival and increases synaptic plasticity and neurogenesis [57,61]. Changes in synaptic plasticity caused by various factors such as age and depression have been investigated [62,63]. For example, depression reduces synaptic plasticity in the hippocampus via downregulation of synaptic proteins and growth factors. Depression disturbs expression of Nmethyl-D-aspartate (NMDA) receptor gene in the PFC, which is very important for controlling synaptic plasticity [63]. In addition, a previous study has reported that fluoxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant, mitigates memory impairments and depletion of hippocampal neurogenesis in rats caused by VPA associated with decreasing of BDNF levels in the hippocampus [64]. MTX also causes memory impairment in adult rats, and toxicity of MTX causes an impairment of spatial and recognition memories linked to the degeneration of neurons in the SGZ of the dentate gyrus. Spatial and recognition memories are regulated by the hippocampus and PFC, respectively [10][11][12]. Moreover, our other studies have confirmed that MTX induces a decrease of BDNF level in the adult rat hippocampus and PFC [18,52]. In line with the present work,   [17]. Consistent with the present study, the result demonstrates that melatonin can upregulate BDNF expression in the hippocampus and PFC of rats receiving MTX. These findings demonstrate that the cotreatment with melatonin in MTX-treated rats stimulated the levels of BDNF in both the hippocampus and PFC, which has an important function in neurogenesis and synaptic plasticity related to learning and memory [57,65]. Several researches have reported the function of oxidative stress in neurodegenerative disorders [66][67][68]. MTX could upregulate ROS and induce oxidative stress in multiple organs [3,4,9,69]. The present work demonstrated that rats in the MTX group had an accumulation of MDA levels in both hippocampus and PFC. It is well known that MDA is the final product of lipid peroxidation and commonly used as the most popular and dependable markers to determine oxidative stress [70]. Therefore, lipid peroxidation caused by an increment of ROS accumulation leads to cell membrane leakage, resulting in changes of protein structure and function, and DNA damage [71,72]. Our prior researches have shown that MTX enhances accumulation of MDA levels and decreases in the levels of antioxidant enzymes in the hippocampus and PFC in adult rats [18,52]. Here, the present experiment found that co-administration with melatonin can decrease the levels of MDA in MTX-treated rats. In the present study, moreover, MTX enhanced an accumulation of oxidative stress that is verified by the reduction of the SOD, CAT, and GPX levels in the hippocampus and PFC. These results reveal that receiving MTX is a high-risk factor to generate oxidative stress and induces the reduction of antioxidant enzymes required to maintain ROS balance in the hippocampus and PFC.
Melatonin has antioxidant capability and acts as a free radical scavenger [26], which directly inhibits reactive oxygen species (ROS) and reactive nitrogen species (RNS) [73]. To restrict overexpression of ROS, both enzymatic and nonenzymatic systems of endogenous antioxidants control ROS balance [74]. Melatonin also diminishes oxidative stress via activating the enzymatic antioxidant defenses including SOD, CAT, and GPX [14,24,28]. The present study found that melatonin conceivably improved antioxidant activity in both the hippocampus and PFC in rats receiving MTX. Additionally, increases in antioxidant enzyme levels are related to an increment of Nrf2 expression in both the hippocampus and PFC. The most important biological activity under Nrf2 control is the redox homeostasis in the cells [75]. When cells are under oxidative stress, the mechanism of Nrf2-ARE (antioxidant response element) signaling pathway is responsible for the expression of many antioxidant enzymes such as CAT, SOD, and GPX [76]. These important enzymes actively act via ameliorating oxidative stress and free radicals in the cells [77]. Therefore, Nrf2 is one of the important proteins that regulates antioxidant defense systems and strongly acts as a therapeutic target to treat neurodegenerative disorders related to oxidative stress [78]. A recent work revealed that rats in the melatonin+MTX group showed a significant increase in Nrf2 level in the hippocampus and PFC in comparison with the MTX group. Various studies have demonstrated that the outstanding properties of melatonin strongly enhance Nrf2 functions in several mechanisms to rescue traumatic brain injury and neuroinflammation [28,75,79,80]. Altogether, the discovery from the present work indicates that melatonin markedly exhibits neuroprotective effects, which are mediated by activation of Nrf2 in MTX-induced rats. Therefore, the present study reveals an effective role of Nrf2 correlated with an increase in ROS scavenging enzymes and neurogenesis.

Conclusion
In conclusion, these findings highlighted the side effects of MTX that are strongly related to oxidative stress-suppressed neurogenesis and induced an increase of cell cycle arrest in the hippocampal dentate gyrus. Here, toxicity of MTX also promoted lipid peroxidation and restrained antioxidant enzyme levels and protein expression of DCX, Nrf2, and BDNF. From further investigations, we found that melatonin had a potential to relieve the side effects of MTX by enhancing the antioxidant enzymes and Nrf2 expression to reduce the cell oxidative stress for maintaining the ROS balance and redox homeostasis in the hippocampus and PFC. Moreover, an impairment of neurogenesis and memories was restored by increasing BDNF expression that regulated cell survival and synaptic plasticity via the neuroprotective effect of melatonin.