Introduction
Methotrexate (MTX), an analog of folic acid, is one of the frequently used chemotherapeutic agents, especially in treating hematological malignancies and solid tumors. It is a well-established and effective treatment for different types of rheumatic diseases, severe psoriasis, and bowel diseases (like Crohn’s disease) [1]. Its anticancer activity involves the prevention of RNA and DNA syntheses through the inhibition of dihydrofolate reductase enzyme responsible for the conversion of folate to tetrahydrofolate [2]. Clinically, although the drug has some comparative advantages over some chemotherapeutic agents, its use is associated with hepatotoxicity [3]. Studies have reported a rate of 7% hepatotoxicity in cancer patients on MTX chemotherapy. Its hepatotoxic effect ranges from elevations in serum aminotransferases to changes in kidney histology [4]. Also, the use of high-dose MTX or prolonged use may result in hepatic fibrosis and cirrhosis [5]. The precise mechanism of MTX hepatotoxicity is unclear. However, it has been associated with increased activities of mediators of inflammation such as interleukin 1β, tumor necrosis factor α, and interleukin 6. Also, oxidative and nitrosative stress characterized by the production of free radicals and impaired redox balance have been reported. Furthermore, Lipid Peroxidation (LPO) which is a form of oxidative stress has been associated with MTX-induced hepatotoxicity [6].
Cimetidine (CT) is used for the management of patients with gastric ulcer and as prophylaxis for gastric aspiration syndrome. It is used during surgery to prevent stress-induced ulceration [7]. CT works by blocking H2 receptors on the stomach lining thereby preventing the interaction of histamine with H2 receptors, thus preventing acid secretion [8]. Besides its antihistamine effect, it has scavenging activities on monochloramine and hypochlorous acid, known as cytotoxic oxidants produced by inflammatory cells such as neutrophils. Also, CT can scavenge hydroxyl radicals, singlet oxygen, and peroxy radicals at a high constant rate [9]. This property can be used for the treatment of diseases characterized by free radical-mediated oxidative stress. Furthermore, studies suggest that it can be repurposed for the treatment of hepatotoxicity because it acts as an antidote for acetaminophen poisoning in rats [10]. Also, it inhibits hepatotoxicity caused by carbon tetrachloride (CCl4) in rats [11] and was effective in a rabbit model of rifampicin and isoniazid-induced hepatotoxicity [12]. Similarly, CT abrogates cocaine-induced hepatotoxicity in mice [13] and radiation-induced hepatotoxicity in rats [14]. 
The time of drug administration is crucial because it can affect therapeutic outcomes [15]. It has been reported that the trough to peak ratio of some drugs is directly related to the dosing time interval and individual response [16]. The time of medication administration is an important factor in the treatment or management of ailments and drug toxicities. It can affect the kinetics of a drug due to several factors, including circadian rhythms [17]. Timing in terms of post-treatment, pre-treatment, and co-treatment with xenobiotics have been used to abrogate or ameliorate toxicities caused by drugs and chemical agents [18, 19]. The best prospect in terms of therapeutic outcome considering the aforementioned treatments will probably depend on the kinetics of the drug and patient’s response. The current study assessed the protective effect of time-modulated CT on a rat model of MTX-induced hepatotoxicity.  
Materials and Methods
Drugs 
MTX  was manufactured by Zuvius Lifesciences Ltd., (India), while CT was manufactured by Shandong Shenglu Pharmaceutical Company Ltd. (China). All other chemical substances used are of analytical grade. The aforementioned drugs were obtained from a pharmacy shop
Experimental animals
Adult male albino rats (200 g-250 g) were obtained from the experimental animal facility of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Madonna University, Elele, Rivers State, Nigeria. The rats were placed in cages (6 per cage) under natural light condition and temperature. The rats were fed with standard rodent chow and given water ad libitum and allowed for two weeks to acclimatize. This study was approved (Pharm/Res/No.33/ 2019) by the Research Ethics Committee of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Madonna University, Elele, Nigeria. The rats  were handled based on the regulation of the European Parliament and Council on the Protection of Experimental Animals, 2010 [20].
Experimental protocol
Thirty-six adult albino rats were divided into 6 groups of  6 rats in each group. 
The rats in group A (control) were injected intraperitoneally (IP) with one dose of 9% normal saline (0.2 mL) for 24 h. The rats in group B were treated IP with CT (20 mg/kg)  [21] diluted with normal saline for 24 h. The rats in group C were treated IP with one dose of MTX (20 mg/kg)  [22] dissolved in normal saline for 24 h. The rats in group D (pre-treatment group) were treated IP with CT (20 mg/kg)  one hour before treatment with one dose of MTX (20 mg/kg)  for 24 h.  The rats in group E (co-administration group) were co-treated IP with CT (20 mg/kg) and MTX (20 mg/kg) for 24 h. The rats in group F (post-treatment group) were treated IP with one dose of MTX (20 mg/kg) one hour before treatment with one dose of CT (20 mg/kg) for 24 h.
Sacrifice of animals
Rats were sacrificed after drug treatment with diethyl ether, and blood samples were collected from the heart. The blood samples were centrifuged (1200g for 20 min) and serum samples were obtained for the evaluation of liver function indices. Liver samples were collected, weighed, and preserved for histological assessment. Also, liver samples were collected, washed in ice-cold 1.15% potassium chloride, and homogenized in 0.1 M Tris-HCl buffer, pH 7.4. The homogenates were centrifuged (1500g for 20 min) and the supernatants were extracted and evaluated for biochemical parameters.
Evaluation of biochemical parameters
Serum and liver gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), conjugated bilirubin (CB), aspartate aminotransferase (AST), and total bilirubin (TB) levels were evaluated using commercial test kits (Randox Laboratories UK). Liver malondialdehyde (MDA) was assessed according to Buege and Aust study [23] and superoxide dismutase (SOD) as reported by Sun and Zigman [24]. Catalase (CAT) was evaluated according to Aebi [25] and glutathione peroxidase (GPx) according to Rotruck et al. [26]. Reduced glutathione (GSH) was evaluated as described by Sedlak and Lindsay [27] and total protein as described by Lowry et al. [28].
Histological examination of the liver
Liver samples were fixed in formal saline for 24 h, processed routinely, and embedded in paraffin blocks. Processed tissues were sectioned (3-5 μm thick), and slides were prepared and stained with hematoxylin and eosin. Stained sections were examined for histological changes under a light microscope.


Statistical analysis
Values are presented as Mean±SEM. Variables were statistically analyzed by one-way Analysis of Variance (ANOVA), followed by Tukey’s post hoc test. The significance level was set at P<0.05, P<0.01, and P<0.001 for different comparisons.
Results
Effects on the body, and liver weights and liver function parameters
The body and liver weights, and serum TB, CB AST, GGT ALT, LDH, and ALP levels were normal (P>0.05) in CT-treated rats when compared to control. However, significant (P<0.001) elevations in serum TB, CB, AST, GGT, ALT, LDH, and ALP levels occurred in MTX-treated rats when compared to control (Table 1, Figures 1-8). The observed increases in the aforementioned parameters were 231.8%, 324.5%, 347.8%, 279.2%, 337.6%, 274.9%, and 315.1%, respectively.  In contrast, serum TB, CB, AST, GGT, ALT, LDH, and ALP levels were significantly decreased rats in post-treated (P<0.05), co-treated (P<0.01), and pre-treated (P<0.001) with CT when compared to MTX-treated rats (Figures 1-8).

Effects on liver tissue biochemical parameters
The liver levels of AST, GGT, ALT, LDH, and ALP were normal (P>0.05) in rats treated with CT when compared to control. However, the aforementioned parameters were significantly (P<0.001) elevated in rats treated with MTX when compared to control (Table 2). The observed increases in liver levels of AST, GGT, ALT, LDH and ALP were 295.3%, 300.8%, 275.4%, 355.4% and 297.0%, respectively.  On the other hand, liver levels of AST, GGT, ALT, LDH, and ALP were significantly reduced in rats post-treated (P<0.05), co-treated (P<0.01) and pre-treated (P<0.001) with CT when compared to MTX-treated rats (Table 2).

Effects on liver oxidative stress indices and histology
Liver SOD, CAT, GPX, GSH, and MDA levels were normal (P>0.05) in rats treated with CT. The liver levels of SOD, CAT, GPx, and GSH significantly (P<0.001) decreased whereas MDA significantly (P<0.001) increased in rats treated with MTX when compared to control (Table 3). In contrast, liver SOD, CAT, GPx, and GSH levels significantly increased whereas MDA levels significantly decreased in rats post-treated (P<0.05), co-treated (P<0.01), and pre-treated (P<0.001) with CT when compared with to MTX-treated rats (Table 3). Normal liver histology was observed in the control group (Figure 8A) whereas hepatocyte necrosis was observed in rats treated with MTX (Figure 8B). Also, the liver of rats post-treated with CT showed inflammatory cells (Figure 8C). The liver of rats pre-treated with CT showed ihepatocyte necrosis (Figure 8D) whereas the liver of rat co-treated with CT showed steatosis (Figure 8E).

Discussion
This study assessed the protective effect of time-modulated cimetidine against MTX-induced hepatotoxicity in a rat model. An important requirement in toxicological experiments is the assessments of the effects of xenobiotics on organ and body weights [29]. In this study, the induction of hepatotoxicity with MTX did not alter body and liver weights. Serum aminotransferases (AST and ALT) are used for differential diagnosis of liver disease  and higher levels show liver dysfunction [30]. Also, serum activities of CT, TB, LDH, and GGT correlate with liver function and elevated activities are observed in hepatic disorders [31]. The serum and liver activities of CT, CB, AST, GGT, ALT, LDH, and ALP were high in rats treated with MTX. This finding agrees with previous studies [32]. The elevations in the activities of the aforementioned indexes are clear signs of hepatic injury [33]. However, the activities of CB, TB, AST, GGT, ALT, LDH, and ALP decreased in rats’ pre-treated, co-treated, and post-treated with CT. The decreases in the activities of the aforementioned parameters were most seen in rats pre-treated with CT. 

Oxidative Stress (OS) is a condition that favors oxidant generation and depletion of antioxidants leading to tissue injury [34]. In the current study, we observed decreases in liver antioxidant (CAT, GPX SOD, and GSH) activities in MTX-treated rats. This observation is a sign of OS which is consistent with earlier studies [35]. However, marked up-regulation in the activities of liver antioxidants were noted in rats’ pre-treated, co-treated, and post-treated with CT. MDA is a metabolite of free radical-induced LPO cascade. It is an essential indicator of OS and the breakdown of lipid layers [36]. The current study observed elevated MDA level in MTX-treated rats which is an indication of LPO that is consistent with earlier reports [37]. However, decreased LPO characterized by low levels of MDA were noted in rats’ pre-treated, co-treated, and post-treated with CT. The lowest level of MDA was observed in rats pre-treated with CT. The current study showed hepatocyte necrosis in MTX-treated rats which is consistent with previous findings [38].  

Pre-treatment, offered, but co-treatment, and post-treatment with CT did not offer significant protection against MTX-induced hepatocyte necrosis. The exact mechanism of MTX-induced hepatotoxicity is not clear, but some mechanisms have been proposed. These mechanisms include direct toxic effect on the liver [39], inhibition of DNA synthesis, mitochondria damage, OS, and decreased hepatic folate levels by MTX polyglutamates [40]. The prevention of MTX-induced hepatotoxicity by CT might be due to its inhibitory effect on the above-mentioned mechanisms. CT has oxygen-radical scavenging activity. It scavenges hydroxyl radicals [41] and superoxide anions thereby preventing OS and LPO [42, 43]. Also, it can inhibit cytochrome P-450 monooxygenation reactions, which are associated with the production of toxic oxygen species [44]. Furthermore, the kinetics of CT in humans is similar to that of rats. It reaches peak plasma concentration in 45-75 minutes after administration resulting in blood levels averaging 2.8 μmol/L (0.7 mg/L). Its maximum activity is achieved when blood levels exceeded 0.5 mg/L [45]. Therefore, pre-treatment with CT produces the best hepatoprotective activity probably because its peak plasma concentration was attained before treatment with MTX. CT might have inhibited the activity of MTX before reaching its hepatic target. Furthermore, MTX undergoes hepatic and intracellular metabolism to polyglutamated forms, which are essential for its pharmacological and hepatotoxic activity [45]. Studies have shown that CT can inhibit the hepatic microsomal oxidation system [46]. Therefore, at peak plasma concentration, pre-treatment with CT might have inhibited the hepatic biotransformation of MTX by hepatic microsomal oxidation system leading to reduced production of its polyglutamated forms. 
Pre-treatment, co-treatment, and post-treatment with CT attenuate MTX-induced hepatotoxicity, but the maximum effect was seen in rats pre-treated with CT.
Ethical Considerations
Compliance with ethical guidelines
The directive (2010/63/EU) of the European :union: Parliament on the handling of laboratory animals for scientific purposes was used for this study.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Authors' contributions
Conceptualization, original draft preparation, data curation, manuscript review and editing, and data analysis: Elias Adikwu; Original draft preparation, data curation, manuscript review and editing, data analysis, animal handling: Emmanuel Nnaedozie.
Conflict of interest
The authors declare no conflict of interest.


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