Ameliorative Effect of Naringenin in 5-Fluorouracil-Induced Hepatotoxicity: An Experimental Study on Albino Wistar Rats

5-Fluorouracil (5-FU), a classified antineoplastic, pyrimidine antimetabolite second most frequently used, a powerful chemotherapeutic agent that has been used for decades to treat a variety of malignancies (Famurewa et al., 2019; Chelpuri et al., 2022). It has anticancer effects in a variety of ways, including suppression of the thymidylate synthase enzyme (Longley, 2003) and activation of the protein p53, as well as effects on cell cycle control and G1/S arrest and used in the treatment of various types of cancer, including colorectal, breast, skin, head, neck and liver cancer (Gelen et al., 2017). Apart from its benefits, it has many side effects in a variety of organs including myelosuppression, emesis, mucositis, nausea and toxicity to other organs, particularly cardiotoxicity, hematotoxicity, nephro and hepatotoxicity (Muhammad, Sallam and El-Abhar, 2020) Cytotoxic consequences of 5-FU is mainly by two modes of action-its metabolic product 5-fluoro-2-deoxyuridine5-monophosphate (FdUMP) blocks thymidylate synthase, results in a deficit in thymidine, inhibits DNA synthesis. In addition, it incorporates into RNA, inhibits RNA synthesis and causes cell toxicity (Chibber, 2011). Approximately 90% of 5-FU drug catabolized by dihydropyrimidine dehydrogenase (DPD) into dihydrouracil mainly in the liver, results in degradation products of alpha-fluoro-beta-alanine (FBAL) in inactive form, thus responsible for hepatotoxicity (Badawoud, 2017; Rashid et al., 2014).

Several studies have been postulated on naturally available compounds to mitigate various toxicities with anti-oxidant, anti-apoptotic and anti-inflammatory properties, thereby decreasing cell death and increasing natural remedies for anticancer drugs side effects (Renushe et al., 2022; Yousef et al., 2017). 

NG -4,5,7-trihydroxy flavanone is a phytochemical present in citrus fruits (grapefruit and oranges) and tomatoes and synthesized from an aromatic amino acid, phenylalanine and present as glycosylated form (Moghaddam et al., 2020). Biological properties of NG include anti-inflammatory (Leopoldini et al., 2011), cardioprotective, hematoprotective, hepatoprotective (Renugadevi and Prabu, 2009; Sravathi et al., 2022), nephroprotective and anti-carcinogenic properties (Ekambaram, Rajendran, Magesh and Sakthisekaran, 2008). In addition to a direct antioxidant property by free radical scavenging activity, NG can induce the endogenous anti-oxidant system. It has an anti-oxidant effect due to its OH substituent, which reacts highly against ROS. The OH can donate its H to free radicals to increase antioxidant capacity (Reddy et al., 2008).

All chemicals are procured from Qualigens Private Limited, India (Mumbai) and SRL Private Limited, India. 5-FU was procured from celon laboratories private limited, Hyderbad, India. Naringin (CAS No: 10236-47) was obtained from Sigma (SAC-St Louis, MO, USA).
 
Experimental animals
 
Healthy male albino Wistar rats (48), age-3 months, weighing 180-220 g, were bought from Jeeva Life Science (ISO 9001:2015 certified company), Hyderabad, India. They were acclimatized for 10 days and maintained at a temperature of 25±2°C, 12 hours (h) of a light-dark cycle (12:12) and humidity (45-55%) throughout the current experimental period. In addition, all the rats were given with standard pellet diet (low-fat and nutritionally balanced food) and ad libitum deionized water for 28 days.
 
Ethical statement
 
The Experiment was carried out according to the guidelines and prior approval of the Institutional Animal Ethics Committee (No.9/24/C.V.Sc., Hyd. IAEC-Rats/ 12.06.2021) and work is carried at Department of Pharmacology and Toxicology, P.V. Narsimha Rao Telangana Veterinary University, College of Veterinary Sciences, Rajendranagar, Hyderabad, India during 2021.
 
Experimental design
 
The Present experiment aimed to study the ameliorative effects of NG against 5-Fluorouracil-induced hepatotoxicity in rats on 14^th and 28^th day. A total of forty-eight (48) healthy adult male rats were separated into four groups, with twelve animals in one group (Fig 1).

 

Group-1: Control-treated as sham received normal saline orally for 28 days.
Group-2: Toxic group-5-FU at a dose rate of 20 mg/kg b.wt for first 5 days-IP.
Group-3: Served as ameliorative group-NG (100 mg/kg b.wt) per orally for 28 days.
Group-4: Served as combination group-5-FU (@ 20 mg/kg b. wt/day) for 1^st 5 days, NG at a dose rate of 100 mg/kg b. wt/ day 28 days.
 
Blood sample collection
 
Blood was collected approximately 2 mL from each rat from retro-orbital plexus and allowed to clot for 3-4 h, later centrifuged at 8,000 revolutions per minute (rpm) for 10 min and collected serum stored at -20°C until further biochemical analysis by using semiautomatic ELISA reader (Thermo Scientific, USA) and Erba Mannheim biochemical kits (Transasia Biomedicals Ltd., Solan, Himachal Pradesh, India). After blood collection, rats were sacrificed on the 14th day and 28th day by using CO₂ chamber.
.
Liver enzymes estimation
 
ALT and AST were estimated as per the modified International Federation of Clinical Chemistry (IFCC) method (Shaw et al., 1983). Alkaline phosphatase (ALP) was procured from Erba Diagnostics and determined by a sandwich enzyme-linked immunosorbent assay (ELISA technique by using ELISA kit).
 
Analysis of oxidative stress indices
 
Tissue samples were collected in liquid nitrogen and stored at -80°C to study oxidative stress parameters (GSH, SOD and TBARS). One gram of tissue sample (liver), along with 10 mL of Tris HCl buffer (pH 7.2), was placed into homogenizer to get homogenate, followed by centrifugation at 4,000 rpm for 2 minutes (min).

The LPO markers concentration was estimated usinga a standard protocol (Balasubramanian et al., 1988). Briefly, 100 µL of homogenate, 1 mL of 10% trichloroacetic acid (TCA) and 1 mL of 0.67% of TBA were taken to a tightly stoppered tube and samples were heated at 96°C for 45 min. Following cooling, the contents were centrifuged for 5 min and the supernatant was read at 535 nm against blank.

Reduced GSH was estimated according to Ellman’s method (Ellman and biophysics, 1959) to know antioxidant status,  by mixing tissue homogenate supernatant with 5, 5' dithiobis-2- nitrobenzoic acid (DTNB) and incubating for 10 min and absorbing light at 412 nanometers (nm).

For SOD estimation, tissue homogenate reaction involves the generation of superoxide by mixing pyrogallol autoxidation by scavenging superoxide anion radicals and expressed as SOD units (one unit of SOD is the amount in mg of protein required to inhibit the MTT reduction by 50%) (Madesh et al., 1998).
 
Estimation of inflammatory cytokine storm
 
Inflammatory biomarkers were obtained using Enzyme-linked immune sorbent assay (ELISA) kit from Genelia, Krishgen Bio systems (Mumbai). Primary antibody-coated wells were washed five times with 200 µL of wash buffer. 100 µL of samples were added and incubated overnight. Add 100 µL of the secondary antibody and incubate, then add a biotin-labelled antibody and wash with wash buffer. Add Substrate solution to form (TMB) to microwells and absorbance is measured at 450 nm. Units are measured in pg/ mg protein.
 
Histopathology of heart tissue
 
Soon after sacrifice, liver tissues were collected and fixed in 10 per cent neutral buffer formalin (NBF) for 48 hours. Samples were washed under running water and subjected to ascending graded series of alcohol for dehydration, then cleared with xylene. Followed by buries with embedding medium (Paraffin), dyed with haematoxylin and eosin (H&E). The standard procedure described the sections observed under light microscopic examination (Luna, 1968).
 
Immunohistochemical analysis of heart tissues
 
For Immunoexpression, sections were deparaffinized in a hot air oven and cleared with xylene. Then, hydrated the sections in descending graded series of alcohol for each 5 min. For antigen retrieval, sections were placed in proteinase K (20 µg/mL) and blocked the sections with 3% BSA and washed with buffer solution. Sections were incubated with primary antibodies overnight. After 24 h, sections were washed with buffer and incubated for 30 min with diaminobenzidine (DAB) chromogen for 10 min. Stain with hematoxylin and observe immunoexpression intensity under Light microscopic examination (Chelpuri et al., 2022).
 
Statistical analysis
 
Data regarding the study were analysed statistically using a one-way Analysis of variance (ANOVA) using the statistical package for social sciences (SPSS) version 15.0. Duncan’s multiple comparison tests were done for comparison among groups and the significance level was set at P<0.05 (Snedecor and Cochran, 1994).

Serum biochemical parameters
 
Sustainable hepatotoxicity was observed by significantly elevated levels of ALT, AST, ALP and albumin (IU/L) recorded in group 2 by 5-FU in comparison with other groups. Treatment with NG considerably significantly reduced increase levels indicating a decrease in liver damage caused by 5-FU was recorded in group 4 rats on the 14^th and 28^th day of the experiment (Fig 2).


 
Oxidative stress parameters
 
Studies on tissue (liver) oxidative stress indices showed a significant (P<0.05) increase in TBARS levels in 5-FU group compared with other groups. There is no significant difference between group-1 and 3 rats. However there are significantly reduced TBARS levels with treatment with NG. The significantly (P<0.05) decreased concentrations of GSH and SOD in the 5-FU treated group on the 14^th and 28^th day of the experiment compared to the control group. However, the values were significantly increased in group-4 compared to group-2 rats, variation in the values of antioxidant levels helped suggest the reduction in 5-FU induced tissue damage by NG (Fig 3).


 
Cytokine profile
 
Pro-inflammatory cytokines levels (TNF-α, IL-1β, NF-κB and IL-10) and anti-inflammatory cytokines levels (IL-10) in the tissues of liver homogenate were estimated by using ELISA. Elevated levels of pro-inflammatory cytokine levels in 5-FU treated group were observed and while significantly (<0.0001) lowered in levels of anti-inflammatory cytokine concentrations-IL-10 levels were observed and it suggested that 5-FU has an inflammatory effect in the liver. Whereas, NG significantly decreased in group-4 compared with group-2 rats in levels of pro-inflammatory cytokine levels and significantly increased in levels in IL-10, indicating NG has boosted an anti-inflammatory response which helps to lesson decrease liver damage and reverse the negative response caused by 5-FU (Fig 4).


 
Histopathology
 
The liver sections of group 1 and 3 rats showed normal architecture of hepatic central vein (CV) and portal triad with the normal radiating appearance of hepatic cords with uniform sinusoids (Fig 5 A, D). The liver sections of group 2 on 14^th day showed vascular changes, including moderate congestion of CV, dilatation and congestion of portal vein (PV), parenchymatous changes including vacuolar degeneration and necrosis of hepatocytes, dilatation of sinusoids, marked Mononuclear cells (MNCs) infiltration; hepatocytes showed a swollen to pyknotic nuclei (Fig 5 B,C). On 28^th day of the experiment, the liver showed severe dilation of sinusoids, severe vascular degeneration in hepatic parenchyma, hepatocellular necrosis, a complete distortion of sinusoids, focal oedema, intense congestion of portal vein (PV) and mild hyperplasia of bile duct epithelium (Fig 5  F, G, H).

The liver sections of group 4 rats on 14^th and 28^th day of the experiment showed mild dilation and congestion of CV and PV, mild dilation of sinusoids with mild degenerated changes in the hepatic cord. The reconstructive appearance of hepatic lobules with radiating appearance of hepatic cords with uniform size of hepatic nuclei (Fig 5 E,I).


 
Immunoexpression of NF-êB and Caspaes-3
 
In the present study, strong immunopositive expression of inflammatory marker (NF-κB) and pro-apoptotic (Caspase-3) were observed in group-2, indicating the release of inflammatory cytokines and apoptotic protein. Whereas intensity in immunoexpression was reduced in group-4, indicating that NG has anti-inflammatory and apoptotic effects at the molecular level. There were no changes in immunoreactivity of NG group-3 gives information about the safety of the compound (Fig 6).



Although a number of studies have been conducted on the protective effect of natural agents against chemotherapeutic drugs, we believe that this is the first study to look into the potential protective effect of NG against 5-fluorouracil-induced chronic liver damage against oxidative stress, inflammation, apoptosis and DNA damage at two intervals that is on 14^th and 28^th day. Although 5-FU is a powerful anti-neoplastic medication, its hepatotoxic side effects may reduce its effectiveness in treating cancer. On the other hand, NG, a phytochemical used as an ameliorative agent against hepatoxicity through lowered oxidative stress, inflammation and apoptotic damage in various studies (Shirani et al., 2020). We select NG among many products because it has more absorption into the body with an increase in bioavailability than naringin powder (Massaro et al., 2023). This study, therefore, evaluates the chronic effects of NG at different intervals.

ALT, AST and ALP are the most important biological markers of cellular damage and toxicity (Gelen et al., 2017; Vanithasree et al., 2011). 5-FU is primarily metabolized in the liver to fluoro-beta-alanine, which is an inactive form that may be responsible for hepatic damage (Saif et al., 2009). The liver injury further causes membrane damage, which allows enzymes to circulate and be detected in the serum. It might be caused by an excess production of free radicals and oxidation products in the liver, which cause damage to the membranes and endothelial lining of the hepatic vessels, resulting in liver dysfunction and a change in the permeability of the hepatocytes cell membrane, allowing them to enter into the bloodstream (Dimitriu et al., 2015). A significant elevation of these enzyme activities has been used as an indicator of acute liver injury in agreement with the observations of Afolabi and Harish with an increase in the concentration of serum enzymes by administering 5-FU (Harish et al., 2021; Tavakoli Pirzaman et al., 2023). In the present study, 5-FU might have suppressed the growth of cellular proteins and RNA synthesis, resulting in a low level of serum proteins due to oxidative stress. ROS may induce inflammation, causing protein damage and lowering the ability of the liver to synthesize new proteins, leading to decrease in these values as report by  Badawoud and Harish, who stated that 5-FU caused a decrease a in TP due to oxidative stress in liver (Badawoud et al., 2017; Harish et al., 2021).

In group 4, the mean values of serum ALT, AST and ALP were significantly decreased and a significant increase in the mean values of TP and albumin was observed when compared to group 2 rats on the 14^th and 28^th day of the experiment, which could be due to ameliorative effect of NG by protecting sub-cellular damage and macromolecules (especially lipids) from oxidative injury (Caglayan et al., 2018).

ROS directly affects various biological components, leading to cellular damage and necrosis in the liver (Sehitoglu et al., 2015). Lipid peroxidation is one of the mechanisms involved in tissue damage through ROS formation. TBARS is one of the most widely used assay for measuring lipid peroxidation as MDA is a better predictor of oxidative damage (Al-Asmari et al., 2016). In our current study, a significant increase in TBARS levels indicates that 5-FU causes oxidative stress and our results are consistent with previous findings (Du et al., 2023).

The removal of ROS in normal healthy cells is achieved by a radical scavenging mechanism that includes catalase (CAT), superoxide dismutase (SOD) and reduced GSH. Oxidative stress can arise due to an increase in ROS production or a decrease in antioxidant defence (Ebrahimi et al., 2023).

GSH is an antioxidant that protects against toxic damage by combating ROS via free radical production. The absence of GSH in tissues reduces the cell’s defenses against oxidative stress. Our findings suggest that 5-FU precipitated GSH reservoirs, consistent with earlier findings (Manvitha et al., 2019; Afolabi et al., 2016).

SODs are a ubiquitous family of enzymes, efficiently catalysing the dismutation of superoxide anions to molecular oxygen which is transformed by catalase and GSH-Px into water, thus reducing free radical-mediated cell damage (Aikemu et al., 2016). It is regarded as the first defence against superoxide anions, which are abundant during 5-FU redox cycling (Sengul  et al., 2021). The reduction in the lung SOD values in the present study could be due to damage by disrupting the oxidant-antioxidant balance and increased inflammation (Arab et al., 2018; Rashid et al., 2014; Sengul et al., 2021).

Cytokine secretion is an inflammatory mediator contributing pathogenesis of tissue injury (Laverty et al., 2010). Oxidant stress directly triggers a cascade of inflammatory pathways. The transcription factor, NF-κB is thought to be pivotal in this process. It up-regulates genes of pro-inflammatory cytokines TNF-α, IL-1β, NF-κB (Prisciandaro et al., 2011) involved in liver toxicity (Refaie et al., 2022).

In the present study, a significant (P<0.05) elevation was noticed in the concentration of inflammatory cytokines in homogenized tissue of group 2 rats when compared with group 1 and 4 rats on 14^th and 28^th day of the experiment. This might be due to 5-FU ability to cause excessive ROS generation, which stimulates multiple signaling pathways, including the redox-sensitive NF-κB transcription and MAPK pathway, resulting in various gene expressions for TNF-α (Elghareeb et al., 2021; Famurewa et al., 2019; Gelen et al., 2017).

In group 4, the concentration of inflammatory cytokines was significantly (P<0.05) decreased and IL-10 significantly increased on the 14th and 28th day of experiment when compared with group 2, which might be due to the antioxidant and anti-inflammatory defense mechanism of NG against the production of ROS. NG decrease TNF-α and its associated abnormalities by interactions with intracellular signaling cascade via stimulation of Nrf2 pathway (Zhang et al., 2015),  thus reducing phosphorylation of Caspase-3 to suppress apoptosis and NF-κB Toll like receptor 4 (TLR4) stimulation, thus inhibiting pro-inflammatory cytokines (Chen et al., 2012). These findings are in accordance with the observations of earlier observations of Mahmoud (Mahmoud, 2013).

The microscopic changes of liver sections in group 2 rats revealed mild congestion of CV, vacuolar degeneration in the hepatic parenchyma, sinusoidal dilation and mild dilation and congestion of hepatic PV, infiltration of inflammatory cells in the portal triad were also observed on 14^th day of the experiment. On 28^th day liver section of group 2 showed severe dilatation of sinusoids, severe vacuolar degeneration in the hepatic parenchyma, mild bile duct hyperplasia, severe congestion of PV and complete distortion of sinusoids. These changes might be due to toxic accumulation of intermediate metabolites and ROS production. The microscopic findings correlate positively with the elevated mean values of liver biochemical profiles viz., ALT, AST and ALP. Similar results were observed in 5-FU and CCL2 induced hepatotoxicity  (Gelen et al., 2017; Ayaz et al., 2017; Jyothi et al., 2009).

In group 4 rats, the liver sections showed a mild degree of microscopic changes like mild congestion of CV and PV, mild dilation of sinusoids, mild infiltration of inflammatory cells and reconstructive appearance of hepatic cords on 14^th and 28^th day of the experiment. These ameliorative actions may be due to the antioxidant action of NG on hepatic cords (Mahmoud, 2013).

NF-κB is a crucial redox-sensitive transcription factor implicated in the formation of liver abnormalities (Luedde and Schwabe, 2011). In our study, the intensity of immunoexpression of NF-κB increased, which indicates that 5-FU will cause inflammation and lead to liver damage, coinciding with previous works. In addition, the level of intensity increases in caspase-3 helps to know that 5-FU will cause damage through the apoptotic pathway (Gelen et al., 2017). These results also suggest that the intensity of NF-κB and caspase-3 decrease gives an idea of anti-apoptotic and inflammatory effect of NG, which helps to regulate liver function (Muhammad et al., 2020).

In conclusion, NG treatment could lessen liver damage caused by 5-FU in rats. Taken together, we conclude that 5-FU exposure on the 14^th and 28^th day causes severe liver damage through increased liver enzymes, oxidative enzymes and inflammatory cytokines, histopathology and immunohistochemistry. However, the intensity of lesions increases with an increase in time. At the same time, NG powder could improve the restoration of biochemical oxidative enzymes due to anti-apoptotic, anti-oxidant anti-inflammatory properties. The intensity of liver injury decreases on the 28^th day, suggesting that chronic intake of natural flavonoid products will restore damage caused by various chemotherapeutic agents. Furthermore, future studies were required to understand the detailed mechanisms of ameliorative effect using molecular techniques

We would like to thank P.V. Narsimha Rao Telangana Veterinary University.

All the authors were hereby declared no conflict of interest.