Protective effects of hesperidin on ionizing radiation-induced liver damage

Amac Karaciger cogunlukla ust batin, sag alt akciger, distal ozafagus tumorleri icin veya tum vucut radyoterapi (RT) uygulamasinda radyasyona maruz kalmaktadir. Bu calismada iyonizan radyasyonun karaciger dokusunda neden oldugu oksidatif stresi uzerine hesperidinin koruyucu etkisinin arastirilmasi amaclanmistir. Gerec ve Yontemler 24 yetiskin erkek rat rastgele 4 gruba ayrildi. Kontrol grubuna sadece fizyolojik salin, Grup HES’e 15 gun 50 mg/kg hesperidin, Grup RAD’a sadece irradyasyon yapildi ve Grup HES + RAD’a 15 gun boyunca 50 mg/kg hesperidin verildi, 15. gun sonunda abdominopelvik bolgeye 10 Gy dozunda radyasyon uygulandi. Radyasyon uygulandiktan 24 saat sonra Total antioksidan kapasite (TAK) ve malondialdehid (MDA) tayini icin karaciger ve kan ornekleri alindi ve ayrica histopatolojik inceleme yapildi. Bulgular Grup RAD ile karsilastirildiginda, plazma ve doku MAD duzeyi Grup HES + RAD'da anlamli olarak azaldi (p=0.002). Hem plazma hem de dokudaki TAK, HES + RAD grubunda anlamli olarak daha yuksek bulundu  (sirasiyla, p = 0.002, p = 0.004). Grup RAD’da , histolojik olarak portal alanda odem, sinuzoitlerde dilatasyon, hepatositlerde belirgin olarak sime, intrasitoplazmik vakuolizasyon ,arada nekroz, belirgin sinuzoidal dilatasyon, santral ven dilatasyonu ve konjesyon izlendi. Nukleer hipertrofi belirgindi. Grup HES+RAD, Grup RAD ile karsilastirildiginda periportal odem, santral ven dilatasyonu ve konjesyon histolojik olarak belirgin degildi Sonuc Radyoterapinin lipit peroksidasyonunda artisa ve antioksidan kapasitede azalmaya neden oldugu; Ratlarda 15 gun boyunca 50 mg/kg/gun hesperidin uygulamasinin, radyasyonun neden oldugu karaciger hasarinda gorulen histolojik degisiklikleri ve oksidatif stresi azalttigi gorulmustur.


Introduction
Excessive reactive oxygen species (ROS) formation can induce oxidative stress, leading to cell damage that can culminate in cell death. ROS are produced by living organisms as a result of normal cellular metabolism and environmental factors, such as air pollutants or ionizing radiation [1]. Cellular exposure to ionizing radiation leads to oxidizing events that alter atomic structure through direct interactions of radiation with target macromolecules . Further, the oxidative damage may spread from the targeted to neighboring [2]. The destructive effect of ionizing radiation results from reactive oxygen species including hydrogen peroxide (H2O2), superoxide anion (O-2) hydroxyl radicals that develop from dissolution of water [3]. Another mechanism of action of RT is alteration of the cell homeostasis, modifiying the signal conduction, increasing DNA damage and consequently making the cell proper for apoptosis [4].
Radiotherapy (RT) is among the most common and most important techniques used for cancer treatment [5].
Liver is mostly exposed to radiation during RT to the upper abdomen, the right lobe of the lung, distal esophagus tumors or total body irradiation. Radiation may lead to cellular damage, and clinical and laboratory findings of liver dysfunction [6,7].
The use of antioxidants either in the diet or as therapeutic agents might offer protection against radiation induced damage [8].
Phenolic compounds are mainly divided into phenolic acids and flavonoids. The structure of phenolic compounds plays an important role in the radical scavenger effect and metal chelating property. Flavonoids constitute a significant group of phenolic compounds, and more than 4000 flavonoids have been detected and they have been classified according to molecular structures [9]. Flavonoids have drawn attention of the researchers due to their properties of being radical scavengers, regulators of enzymatic activity, acting as antibiotic, anti-allergenic, anti-diarrhetic, anti-ulcer and antiinflammatory drugs [10].The best defined characteristic of flavonoids is their acting as anti-oxidants, which removes free radicals and reactive oxygen species [11,12].
The present study was aimed to investigate the protective effect of hesperidin administered via the peroral route in rats with radiation-induced liver damage.

Material and Methods
The study was approved by the Institutional Animal

Irradiation of animals
The experimental model of anaesthetized rats for irradiation was used, as described by Parihar et al. (13). The animals in Groups RAD and HES+RAD were anaesthetized with an intraperitoneal injection of 100 mg/kg ketamine, then four rats in the prone position were administered a single nonlethal dose of 10 Gy using a 6-MV linear accelerator at a dose rate of ~1 Gy/min with the source axis distance (SAD) technique and a 1.0-cm bolus material on the surface. A computed tomography simulation of a rat was performed with 1-mm slices, and a dose calculation was performed with the Eclipse treatment planning system (ver. 8.9; Varian Medical Systems, Palo Alto, CA, USA). The animals were returned to their home cages following irradiation. Control animals were anaesthetized but not exposed to radiation. All irradiations were performed between 08:00 and 09:30.

Chemical analysis
Tissue samples were cut into small pieces and then homogenized in phosphate-buffered saline (pH 7.4) using a glass-Teflon homogenizer (Ultra Turrax IKA T18 Basic) for 2 min at 5,000 rpm. The homogenate was then centrifuged (5,000 g, 15 min). The supernatant was used for the analysis.

Statistical analysis
All analyses were performed with the 'R' software (ver. 3

Results
The results of the biochemical assessments of peroxidation and antioxidant capacity parameters are shown in Tables 1 and 2.

Biochemical parameters
MDA is associated with lipid peroxidation. Compared with the control group, the serum MDA level was significantly higher in Group RAD (p = 0.002) and was significantly decreased in Group HES+RAD (p = 0.002), ( Table 1). The MDA levels in the liver tissue was significantly higher in Group RAD (p = 0.002), (Table 1), while treatment with hesperidin significantly reduced lipid peroxidation in liver tissue in Group HES+RAD (p = 0.002), (Table 1). in Group RAD (p = 0.002) and was significantly increased in Group HES+RAD (p = 0.004), (Table 2). TAS levels in the liver tissue was significantly lower in Group RAD (p = 0.002), (Table 2), and was significantly higher in liver tissue in Group HES+RAD (p = 0.002), (Table 2).

Histopathological analysis
All figures demonstrate the histopathological changes in the liver tissues of rats in each group. Cord alignment was regular in the liver parenchyma. The portal area and central vein structures were normal (Fig 1A, 1B). In Group RAD, portal edema, sinusoidal dilation, mild mononuclear cell reaction, significant intra-cytoplasmic vacuolization, swelling in the hepatocytes, necrosis, significant sinusoidal dilation, central vein dilation and congestion were observed. Nuclear hypertrophy was evident (Fig 2A, 2B).   RT is an important therapeutic agent for cancer treatment. The main purpose of RT is applying a maximum dose of ionizing radiation to tumor tissue while causing minimum damage [5].
Radiation changes the cell structure, causing ionization and activation of atoms, damages the basic compounds and consequently leads to a visible biological lesion when absorbed by a viable cell and interacts with biological systems to produce excess ROS. ROS also negatively affect intracellular concentration of antioxidants [15].
As radiation is known to induce lipid peroxidation, supplementation of antioxidants either in the diet or as therapeutic agents are believed to play major role in reducing toxicity.
Srinivasan et al. [16] showed that pretreatment with ferulic acid, a dietary phenolic acid, significantly decreased the levels of thiobarbituric acid reactive substances (TBARS) and protected the hepatocytes against with increasing dose (1, 2 and 4 Gy) of γ-radiation induced cellular damage.
Chitra et al. [17] indicated that tocopherol used as an antioxidant relieved radiotherapy-induced oxidative damage.
In rats with pretreated hesperidin (100 mg/kg/d, b.w, orally for 7 days) compared to the control group, superoxide dismutase (SOD) activity, glutathione (GSH) and MDA concentration in the lungs significantly decreased 24 hours after exposed to γ-radiation 18 Gy [21].
Shaban et al. [22] showed that 8 Gy and 10 Gy caused significant increases in DNA-fragmentation and protection is more effective when 200 mg/kg hesperidin is given before rather than after exposure of rats testis to radiation.
It was also reported that SOD, catalase and glutathione peroxide enzyme activity decreased in rats which were exposed to 1,3,5 Gy of whole body irradiation, and these anti-oxidant enzyme activites significantly increased in the group in which hesperidin was administered (50mg/kg and 100 mg/kg) for 7 days after irradiation, and the decrease in AST, ALT, ALP, LDH and gamma-GT levels was maximum in rats exposed to 5 Gy radiation [23].
In our study, MDA, which indicates lipid peroxidation, was seen to significantly decrease and TAS, which reflects an anti-oxidant status, was seen to significantly increase in rats pretreated with hesperidin for 15 days (50 mg/kg) exposed On pathology examination in radiation-induced liver disease (RILD), obliteration and occlusion, retrograde congestion and secondary hepatic necrosis are observed in the central veins of the hepatic lobules [24].
Kalpana et al. [25] results revealed that in 4 Gy irradiated group there was anisocytosis of hepatocytes and some of the hepatocytes showed blast transformation, nuclear disintegration and some had pyknotic nuclei, in the 25mg/kg hesperidin pre-administered group most of the hepatocytes was within normal limits with occasional occurrence of macrovesicular type of fatty change In our study, portal edema, sinusoidal dilation, mild monomononuclear cell infiltration in some areas, significant intracytoplasmic vacuolization, necrosis, significant sinusoidal dilation, central vein dilation and congestion were observed in the single 10 Gy irradiated group. İn HES (50mg/kg) +RAD group, cordon arrangement is regular in portal area and surrounding parenchyma. Focal necrosis and hypertrophy in hepatocytes, mild dilation in sinusoids. And also liver damage was scored from a maximum of 24 points on eight criteria and the severity of the damage was determined using DSS. In RAD group had a significantly higher score than HES+RAD group (p < 0.001).This result supported that hesperidin alleviated the liver damage .
Radiation was found to be associated with severe chronic side effects such as late fibrosis, which could limit the prognosis besides leading to oxidative stress. Hesperidin (100 mg/kg or 200 mg/kg) was shown to have an anti-fibrotic property through preventing the increase in serum and hepatic parameters, caspase 3 gene expression, inducible nitric oxide synthase and alpha smooth muscle actin [26,27]. Since our study included the acute period after radiation, there was no fibrosis in the radiation group.
The limitation of this study was that liver function tests were not examined and the protective effect of hesperidin was not observed at different doses.
In conclusion, radiotherapy was found to lead to an increase in lipid peroxidation and a reduction in antioxidant capacity; 50 mg/kg/day hesperidin administration for 15 consecutive days was seen to reduce the histological changes of liver damage and oxidative stress in rats The treatment options are limited in RILD, and the disease may result in hepatic failure and death. Further human studies are required for investigating whether hesperidin administration before RT is useful or not insusceptible to RT-induced liver damage.