Oxygenated Water could augment Doxorubicin to act against Diethylnitrosamine induced Hepatocellular...

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Oxygenated Water could augment Doxorubicin to act against Diethylnitrosamine induced Hepatocellular Carcinoma in Rats Mohamed AM. El-Boreay*, Ahmed M. Mansour, Mohamed F. ELShafie and Gouda K. Helal Pharmacology and Toxicology Department, Faculty of Pharmacy (boys), Al-Azhar University, Nasr-City, Cairo, Egypt.

* Corresponding author: Mohamed AM. El-Boreay, e-mail: [email protected]

ABSTRACT The present study was undertaken to evaluate the possible synergistic effect of oxygenated water with doxorubicin against diethylnitrosamine-induced hepatocellular carcinoma in rats. Hepatocellular carcinoma was induced by diethylnitrosamine added to the drinking water at a dose of (100 mg/L) for 8 consecutive weeks followed by 8 weeks of drinking tap water. Diethylnitrosamine administration caused elevation in serum liver enzymes, serum tumor markers, caspase-3 content and depletion of the antioxidant enzymes in the liver tissue. This was confirmed by histopathological investigations. Interestingly, treatment with oxygenated water (5 ml/kg/day, p.o) concurrently with intravenous doxorubicin administration via tail vein (2.5 mg/kg/week) for 4 weeks starting from the 13th week corrected serum liver enzymes, caused significant decrease in serum tumor markers, increased the hepatic tissue content of caspase-3, restored the depleted antioxidant enzymes and corrected the histopathological finding. In conclusion, oxygenated water enhanced the doxorubicin efficacy, most likely due to the anti-hypoxic effect.

Keywords: Hepatocellular Carcinoma; Diethylnitrosamine; Oxygenated water; Doxorubicin; Hypoxia.

1. INTRODUCTION Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer related death in the world [1]. The etiology of HCC can be diverse but most of the cases occur secondary to chronic infection either by hepatitis B virus or by hepatitis C virus or both [2]. Diethylnitrosamine (DENA) is a potent environmental carcinogen usually present in tobacco products, alcoholic beverages, cheese, soybean, processed meats and agricultural products [3]. DENA used to induce liver cancer in animal models as its metabolized by cytochrome P450 2E1 enzyme (CYP2E1) with subsequent generation of reactive oxygen species (ROS) that cause depletion of the antioxidant enzymes, oxidative stress, lipid peroxidation, alternation of deoxyribonucleic acid (DNA) structure and formation of alkyl DNA adducts [4- 7].

Doxorubicin (DOX) is one of the early anthracyclines, it is isolated from streptomyces peucetius and it has been considered one of the effective chemotherapeutic agents for HCC treatment but its use is hampered due to its cardiotoxicity [8]. Doxorubicin resistance is arising from the inability of the drug to accumulate inside cancer cells and cause apoptosis due to the overexpression of the multi drug resistance transporters and the overexpression of a hypoxia induced protein called N-myc downstream-regulated gene-1 (NDRG1) with subsequent decrease in doxorubicin mediated apoptosis [9, 10]. Hypoxia is a state in which the oxygen concentration is below the normal level and its response is mediated through the hypoxia-inducible factor-1 (HIF-1) [11]. Most of the solid tumors including HCC are characterized by a higher state of hypoxia inside the

Received: 20 May 2017 Accepted: 20 June 2017 Online: 01 July 2017

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tumor tissue due to rapid proliferation of cells, increased oxygen consumption and inadequate blood supply [12, 13]. In response to hypoxia and low oxygen level hypoxia inducible factor alpha (HIF-ɑ) which are oxygen sensitive are stabilized due to the inhibition of prolyl hydroxylation then rapidly accumulates and subsequently translocates to the nucleus to heterodimerizes with the β-subunit and recruits further co-activators [14, 15]. Hypoxia inducible factor-ɑ increases the expression of vascular endothelial growth factor (VEGF), pyruvate dehydrogenase kinase (PDK1), glucose transporter 1 (GLUT 1) and Multi drug resistant 1 (MDR1) genes which leads to the induction of the angiogenesis, inhibition of oxidative phosphorylation, increase rate of glycolysis, lactic acidosis and chemotherapy resistance [16- 26]. Tumor hypoxia can be used as a base for selective cancer therapy either by using pro-drugs activated by hypoxia or by using hyperbaric oxygen (HBO) to reduce the hypoxia [27- 29]. Another new method for targeting the tumor hypoxia is emerged in which the excessive production of hydrogen peroxide (H2O2) by the cancer cells and its reactivity toward manganese dioxide nanoparticles (MnO2 NPs) is used for simultaneous production of oxygen and the diminishing of hypoxia inside the tumor tissue [30, 31]. Oxygenated water is a new formula strategy used for delivering oxygen to cancer cells and diminishing the hypoxia inside the tumor depending on the reactivity of MnO2 toward H2O2 and the production of water enriched with oxygen because of this reaction. Therefore, the objective of the present study is to evaluate the possible effect of the oxygenated water alone or when combined with doxorubicin against DENA-induced HCC.

2. MATERIALS AND METHODS 2.1 Drugs and Chemicals Doxorubicin hydrochloride (2 mg/ml) was obtained from EBWE Pharma, Unterach, Austria. DENA, Bovine serum albumin (BSA), 1,1,3,3-Tetraethoxypropane, Ellman’s reagent, Pyrogallol, Trichloroacetic acid (TCA), Reduced glutathione, Thiobarbituric acid (TBA), Oxidized glutathione (GSSG), Nicotinamide adenine dinucleotide phosphate (NADPH), 1-Chloro-2,4-dinitrobenzene, Cumene hydroperoxide and Glutathione reductase were obtained from Sigma-Aldrich (St. Louis, MO). Hydrogen peroxide (H2O2), Manganese dioxide (MnO2), Potassium dihydrogen phosphate (KH2PO4), Dipotassium hydrogen phosphate (K2HPO4), O-phosphoric acid and N-butanol were obtained from El-Gomhoreya Chemicals (Cairo, Egypt). All other chemicals, solvents and reagents were of analytical grade. 2.2 Preparation of Oxygenated Water Oxygenated water was prepared according to the following equation:

2H2O2+MnO2→2H2O+O2↑+MnO2↓ Two g of MnO2 were added to 20 mL of H2O2 and the reaction was left for 15 min to complete then the mixture was filtrated using filter paper. Two mL of the filtrate were added to 58 mL of distilled water and mixed well and every 200 g rat was given 1 mL of the mixture via the oral route. The dose and duration of the treatment were determined based on pilot studies carried out in our laboratory, as there were no previous studies performed on oxygenated water regarding its anticancer or its synergetic effect to the traditional chemotherapy against HCC before. The amount of dissolved oxygen in the water was determined before the start of the experiments in comparison to tap water at the National Research Center, Cairo, Egypt. The principle of oral oxygen therapy was described early by Eble MJ et al, who indicated that oral administration of oxygen-enriched water increased the dissolved amount of oxygen in blood in head and neck carcinomas patients [32]. . 2.3 Animals Male adult Swiss albino rats weighing 200–220 g were obtained from the breeding colony maintained at the animal house of the Nile Pharmaceutical Co., Cairo, Egypt. Animals were caged in six groups, given a rodent chew and water ad libitum and maintained at 25°C and 55±5% relative humidity with 12-h light–dark cycles. Animals were subjected to an adaptation period of two weeks in the animal house of Faculty of Pharmacy (boys), Al-Azhar University, Cairo, Egypt, before starting the experiment. The experiment was conducted in accordance with the ethical guidelines for investigations in laboratory animals and in compliance with the Guide for the Care and Use of Laboratory Animals [33]. 2.4 Experimental Design Seventy-two male adult Swiss albino rats were allocated into six groups (12 rats each) as follows: Group 1: Received saline for 16 weeks and served as negative control group. Group 2: Received a daily dose of (5 ml/kg/day, p.o) obligatory from the freshly prepared oxygenated water for 4 consecutive weeks starting from the 13th week and served as the oxygen water group. Group 3: Received DENA in their drinking water on a daily dose of (100 mg/L) for 8 consecutive weeks followed by 8 weeks of drinking tap water and served as tumor induced group [34]. Group 4: Tumor induced group and received a daily dose of (5 ml/kg/day, p.o) obligatory of the freshly prepared oxygenated water for 4 consecutive weeks starting from the 13th week. Group 5: Tumor induced group and received doxorubicin I.V at a dose of (2.5 mg/kg) via the tail vein once weekly for 4 consecutive weeks starting from the 13th week [35].



Group 6: Tumor induced group and received oxygenated water concurrently with doxorubicin at the same mentioned dose starting from the 13th week. Two rats from each group were used for histopathological examination. 2.5 Serum and Tissue Preparation One week after the last dose of doxorubicin, blood samples were collected from retro-orbital venous plexus under light ether anesthesia in non-heparinized tubes. Serum was separated by centrifugation at 4000 rpm for 20 min then stored at –20°C until analysis. After blood collection, all animals were sacrificed by cervical dislocation, livers were dissected out, rinsed with isotonic sterile saline (0.9 %), blotted dry on a filter paper and weighed. Then, they were stored at –80°C until homogenized in ice-cold 0.15 M KCl (w/v) using a Sonicator homogenizer (4710 Ultrasonic homogenizer; Cole-Parmer, Vernon Hills, Chicago, IL) to prepare 10 % (w/v) homogenate. The homogenate was then divided into aliquots and used for the determination of the liver contents of total protein, caspase-3, reduced glutathione (GSH) and malondialdehyde (MDA) as well as the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). 2.6 Biochemical Analysis Activities of serum enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in addition to levels of triglycerides (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), total bilirubin, direct bilirubin and total protein (TP) were determined colorimetrically using commercial assay kits from Biodiagnostics, (Cairo, Egypt). Lactate dehydrogenase (LDH) activity was determined kinetically according to standard methods using commercial assay kits from Spinreact (St. Esteve d’en Bas, Girona, Spain). Gamma-glutamyl transferase (GGT) activity was determined kinetically according to standard methods using commercial assay kits from Egyptian Company for Biotechnology (Spectrum) (Cairo, Egypt). Very low density lipoprotein-cholesterol (VLDL-C) concentration was calculated according to the following equation: VLDL-C = TC - (LDL-C+ HDL-C) Indirect bilirubin concentration was calculated according to the following equation: Indirect bilirubin = Total bilirubin – Direct bilirubin Serum alpha-fetoprotein (AFP) and alpha-L-fucosidase (AFU) levels as well as liver tissue caspase-3 content were assayed using rat specific ELISA kits supplied by My-Bio-Source Co. (San Diego, USA), ELAab Co. (Wuhan, China) and CUSABIO Co. (China) respectively,

according to the manufacturer instructions based on the ELISA quantitative sandwich immunoassay technique. Total protein content of hepatic tissue was determined according to the method of Lowry et al [36] using BSA as a standard protein. The activity of SOD enzyme was determined according to the method of Marklund and Marklund [37] while the CAT activity was evaluated by the method of Claiborne [38]. Reduced glutathione was measured according to Ellman’s method [39] and the concentrations were obtained from the standard curve constructed using serial dilutions of GSH while the GST activity was assessed by the method of Habig et al ([40]. In the meantime, the GR activity was assayed by the procedure adopted by [41] and the GPX activity was measured according to the method of Lawrence and Burk [42]. Lipid peroxidation was determined by estimating the level of thiobarbituric acid reactive substances (TBARSs) measured as MDA according to method of Uchiyama and Mihara [43] and the concentrations were obtained from the standard curve which was constructed using serial dilutions of 1,1,3,3-tetraethoxypropane. 2.7 Histopathological Examination of the Liver Tissue samples were taken from the livers of rats from different groups and fixed in 10 % neutral buffered formalin for 24 h and washing was done in tap water and then serial dilutions of alcohol (methyl, ethyl and absolute ethyl). Specimens were cleared in xylene and embedded in paraffin at 56°C in a hot air oven for 24 h. Paraffin wax tissue blocks were prepared for sectioning at 4 μm thicknesses by a sledge microtone. The obtained tissue sections were collected on glass slides, deparaffinized, stained by hematoxylin and eosin stain and then examination was done through a light electric microscope [44]. 2.8 Statistical Analysis of Data All values are presented as means ± standard error of the means (SEM). Statistical analysis was performed using Graph Pad Prism version 5 (Graph- Pad, San Diego, CA, USA). Comparison between different groups was carried out using One-way Analysis of Variance (ANOVA) followed by Tukey–Kramer multiple comparison tests. Difference was considered significant at p ≤ 0.05

3. RESULTS AND DISCUSSION 3.1 Effect on Hepatocellular Integrity, Function and Synthetic Capacity As shown in the Table 1 administration of DENA drastically increased the activities of ALT (250 %), AST (193 %), ALP (150 %), LDH (345 %) and GGT (400 %) in addition to levels of TG (257 %), TC (166 %), LDL-C (304 %), VLDL-C (340 %), total bilirubin (450 %),



direct bilirubin (987 %) and indirect bilirubin (121 %) while the level of HDL-C has been significantly reduced by about 46 % compared to the normal group. DENA treatment significantly impaired the synthetic function of the liver and this appeared as a reduction of the serum TP by 58 % when compared to the normal group (Table 1). Tumor induced group that treated with oxygenated water showed a significant reduction in the serum activities of ALT, AST, ALP, LDH and GGT by 13 %, 17 %, 12 %, 17 % and 20 % respectively in addition to significant reduction in the levels of TG, TC, LDL-C, VLDL-C, total bilirubin, direct bilirubin and indirect bilirubin by 9 %, 12 %, 11 %, 18 %, 24 %, 26 % and 17 % respectively compared to the DENA treated group (Table 1). This group also showed a non significant increase in the serum level of HDL-C by 2 % and a significant increase in the serum level of TP by 28 % compared to the DENA treated group (Table 1). The tumor induced group that treated with doxorubicin showed a significant reduction in the serum activities of ALT, AST, ALP, LDH and GGT by 20 %, 25 %, 26 %, 29 % and 33 % respectively in addition to significant reduction in the levels of TG, TC, LDL-C, VLDL-C, total bilirubin, direct bilirubin and indirect bilirubin by 21 %, 26 %, 26 %, 36 %, 37 %, 43 % and 20 % respectively compared to the DENA treated group (Table 1). This group also showed a non significant increase in the serum level of HDL-C by 8 % and a significant increase in the serum level of TP by 51 % compared to the DENA treated group (Table 1). On the other hand, the tumor induced group that treated with both oxygenated water and doxorubicin showed a significant reduction in the serum activities of

ALT, AST, ALP, LDH and GGT by 37 %, 45 %, 41 %, 50 % and 67 % respectively in addition to the levels of TG, TC, LDL-C, VLDL-C, total bilirubin, direct bilirubin and indirect bilirubin by 33 %, 45 %, 49 %, 58 %, 66 %, 76 % and 38 % respectively compared to the DENA treated group (Table 1). The combined treatment group also showed a significant increase in the serum levels of HDL-C and TP by 32 % and 86 % respectively compared to the DENA challenged group (Table 1). The group that administrated only oxygenated water showed mild significant elevation of the serum levels of ALT, AST and LDH by about 25 %, 22 % and 32 % respectively compared to the control group (Table 1). 3.2 Effect on Serum Levels of Tumor Markers Serum levels of tumor markers AFP and AFU have been significantly elevated in DENA treated group by 376 % and 262 % respectively compared to the normal group while the tumor induced group that treated with oxygenated water showed a significant reduction in the serum levels of AFP and AFU by 14 % and 13 % respectively compared to the DENA treated group (Table 1). Tumor induced group that was treated with doxorubicin showed a significant reduction in levels of those tumor markers by 30 % and 28 % respectively compared to the DENA treated group (Table 1). The levels of those tumor markers have been significantly reduced in the tumor induced group that was treated with both oxygenated water and doxorubicin by about 64 % and 54 % respectively when compared to the DENA treated group (Table 1).

Table 1: Effect of DENA with or without treatment by oxygenated water and doxorubicin alone or in combination on serum liver function tests and tumor markers.

Test Control Oxygenated water alone

DENA DENA+ oxygenated water

DENA+ doxorubicin

DENA+ oxygenated water + doxorubicin

ALT (U/ml) 26.00±1.14 32.50±1.61 91.00±1.05 79.13±1.11 ᵇ 72.98±1.27 ᵇ 57.75±1.56 ᵇ AST (U/ml) 43.78±1.51 53.50±1.61 128.00±2.84 106.40±2.30 ᵇ 95.76±2.68 ᵇ 70.00±1.64 ᵇ ALP (U/L) 81.00±3.65 90.53±3.08 202.50±5.31 178.10±2.12 ᵇ 149.40±2.86 ᵇ 119.00±4.00 ᵇ LDH (U/L) 205.40±5.82 270.60±9.27 913.20±24.82 760.50±16.64

ᵇ 646.50±16.36 ᵇ

455.80±9.77 ᵇ

GGT (U/L) 3.98±0.10 4.58±0.12 19.90±0.55 15.92±0.49 ᵇ 13.34±0.55 ᵇ 6.59±0.16 ᵇ TG (mg/dL) 112.00±4.11 128.80±6.20 400.20±9.49 364.80±9.49 ᵇ 315.10±5.69 ᵇ 270.40±5.40 ᵇ TC (mg/dL) 114.80±2.28 129.90±2.02 305.30±3.22 268.30±4.66 ᵇ 224.50±4.75 ᵇ 167.50±4.17 ᵇ LDL-C (mg/dL) 45.47±1.08 60.14±1.89 183.80±6.10 163.40±5.80 ᵇ 135.10±4.02 ᵇ 93.20±4.91 ᵇ VLDL-C (mg/dL) 21.72±1.09 26.15±1.17 95.55±3.07 78.48±1.72 ᵇ 61.42±1.19 ᵇ 40.01±1.30 ᵇ HDL-C (mg/dL) 47.60±1.64 43.65±1.15 25.94±1.03 26.35±1.62 28.03±1.78 34.31±1.22 ᵇ Total bilirubin (mg/dL) 0.89±0.02 0.99±0.02 4.91±0.09 3.73±0.08 ᵇ 3.08±0.04 ᵇ 1.65±0.02 ᵇ Direct bilirubin (mg/dL) 0.34±0.02 0.41±0.02 3.69±0.10 2.71±0.09 ᵇ 2.10±0.05 ᵇ 0.90±0.03 ᵇ Indirect bilirubin (mg/dL)

0.55±0.01 0.57±0.03 1.22±0.06 1.01±0.04 ᵇ 0.97±0.04 ᵇ 0.75±0.03 ᵇ

TP (g/dL) 8.10±0.15 7.78±0.25 3.44±0.14 4.40±0.15 ᵇ 5.20±0.15 ᵇ 6.44±0.19 ᵇ AFP (pg/ml) 23.51±1.17 24.23±1.19 111.80±2.83 96.15±4.29 ᵇ 78.26±3.62 ᵇ 40.75±3.21 ᵇ AFU (pg/ml) 6.82±0.14 7.29±0.45 24.68±0.64 21.37±1.00 ᵇ 17.70±1.22 ᵇ 11.47±0.60 ᵇ

Data are expressed as means ± SEM of six rats per group. a Significantly different from the control group; b Significantly different from the DENA group; c Significantly different from the DENA+ oxygenated water group; d Significantly different from DENA+ doxorubicin group using one-way analysis of variance followed by Tukey’s test for multiple comparison between groups at p ≤ 0.05



3.3 Effect on Hepatic Tissue Levels of Caspase-3 as Apoptotic Marker DENA administration significantly increased the content of caspase-3 in the liver tissue by 89 % compared to the normal control while the tumor induced group that treated with oxygenated water showed a mild significant increase in the liver content of caspase-3 by 19 % compared to the DENA treated group (Table 2). Tumor induced group that was treated with doxorubicin showed a highly significant increase in this apoptosis marker content by 58 % compared to the DENA challenged group (Table 2). The tumor induced group that was treated with both oxygenated water and doxorubicin showed a highly significant increase in the caspase-3 content in the liver tissues by 96 % compared to the DENA administrated group probably due to the induction of apoptosis in cancer tissues (Table 2). 3.4 Effect on Oxidative Stress Markers Superoxide dismutase and CAT activities in the liver tissue have been significantly decreased in the DENA challenged group by 85 % and 77 % respectively compared to control group while their activities in the tumor induced group that treated with oxygenated water have been significantly increased by 77 % and 150 % respectively compared to DENA treated group (Table 2). Reduced glutathione content, GST, GR and GPx activities have been significantly depleted in the liver tissue of DENA treated group by 92 %, 81 %, 70 % and 75 % respectively compared to the control group while their

content and activities in the tumor induced group that treated with oxygenated water have been significantly increased by 151 %, 105 %, 46 % and 75 % respectively when compared to the DENA treated group (Table 2). In addition, to the antioxidant depletion in the DENA treated group the MDA content in the liver tissue has been significantly increased by 539 % compared to the control group while its content in the tumor induced group that was treated with oxygenated water has been significantly decreased by 22 % when compared to the DENA challenged group (Table 2). The tumor induced group that was treated with doxorubicin as expected showed no improvement in the antioxidant enzymes but it even showed a deterioration in the activities of SOD, GR and GPx compared to the DENA administrated group while its liver content of MDA showed a significant decrease by 17 % compared to the DENA intoxicated group (Table 2). The liver tissue activities of SOD and CAT have been significantly increased in the tumor induced group treated with oxygenated water and doxorubicin together by 271 % and 239 % respectively compared to the DENA challenged group (Table 2). The GSH content and the activities of GST, GR and GPx in this group have been significantly increased by 589 %, 220 %, 142 % and 190 % respectively compared to the DENA treated group (Table 2). In addition, the combined treatment significantly reduced the MDA content in the liver tissues by 58 % compared to the DENA treated group (Table 2).

Table 2: Effect of DENA with or without treatment by oxygenated water and doxorubicin alone or in combination on liver content of caspase-3, MDA and antioxidant enzymes.

Test Control Oxygenated water alone


DENA+ doxorubicin

DENA+ oxygenated water+ doxorubicin

Caspase-3(ng/g tissue) 3.65±0.17 4.51±0.12 6.90±0.24 8.56±0.48 ᵇ 10.88±0.52 ᵇ 13.53±0.53 ᵇ

SOD (U/mg protein) 4.50±0.13 4.30±0.10 0.68±0.02 1.21±0.03 ᵇ 0.57±0.03 2.54±0.05 ᵇ

CAT (U/mg protein) 1.21±0.03 1.142±0.02 0.27±0.01 0.69±0.03 ᵇ 0.29±0.01 0.94±0.02 ᵇ

GSH (µmole/g tissue) 14.20±0.32 13.12±0.30 1.10±0.03 2.76±0.10 ᵇ 1.25±0.05 7.59±0.43 ᵇ

GST (U/mg protein) 76.17±2.25 71.67±1.74 14.33±1.08 29.33±1.22 ᵇ 14.69±1.05 45.83±1.30 ᵇ

GR (U/mg protein) 4.10±0.08 3.88±0.07 1.21±0.04 1.76±0.09 ᵇ 1.06±0.04 2.93±0.06 ᵇ

GPx (U/mg protein) 11.52±0.27 10.72±0.33 2.89±0.08 5.05±0.26 ᵇ 2.71±0.08 8.38±0.35 ᵇ

MDA(nmol/g tissue) 32.78±1.29 45.86±1.84 209.40±9.15 158.40±3.77 ᵇ 174±6.68 ᵇ 88.43±3.01 ᵇ

Data are expressed as means ± SEM of six rats per group. a Significantly different from the control group; b Significantly different from the DENA group; c Significantly different from the DENA+ oxygenated water group; d Significantly different from DENA+ doxorubicin group using one-way analysis of variance followed by Tukey’s test for multiple comparison between groups at p ≤ 0.05

3.5 Finding of the Histopathological Examination Histopathological examination of the liver sections of the control group showed no histopathological alteration and normal histological structure of the central vein and the surrounding hepatocytes in the parenchyma (Figure 1A, Table 3). On the other hand,

the group that administrated only oxygenated water showed neither anaplasia nor dysplasia but congestion in the central and portal vein with degeneration in the surrounding hepatocytes (Figure 1B, Table 3). The DENA administrated group showed focal areas of anaplasic hepatocytes forming HCC with trabecular



pattern in the hepatic parenchyma with the compression of the adjacent surrounding cells (Figure 1C, Table 3). The group of rats administrated DENA followed by oxygenated water starting from the 13th week showed focal areas of anaplastic hepatocytes as well as hepatocytomegalic cells in the hepatic parenchyma (Figure 1D, Table 3). The DENA and

doxorubicin administrated group showed multiple focal areas of dysplasia and degeneration in the hepatic parenchyma (Figure 1E, Table 3). The hepatic parenchyma of the rats administrated DENA followed by oxygenated water and doxorubicin together showed focal areas of ballooning degeneration and dysplasia with dilatation in the central vein (Figure 1F, Table 3).

Table 3: Effect of DENA with or without treatment by oxygenated water and doxorubicin alone or in combination on histopathological findings of liver tissues

Histopathological Findings

Control Oxygenated water alone


DENA+ doxorubicin

DENA+ oxygenated water+ doxorubicin

Anaplasia 0 0 +++ ++ 0 0 Dysplasia 0 0 0 0 +++ + None = 0; mild=+; moderate=++; severe = +++

Figure 1: Histology of the liver samples of the control, oxygenated water alone, DENA, DENA+ oxygenated water group, DENA+ doxorubicin group and DENA+ oxygenated water+ doxorubicin group. (A) Control group: Showing normal histological structure of the central vein and surrounding hepatocyte in the hepatic parenchyma; (B) Oxygenated water alone treated group: Showing

congestion in central and portal veins with degeneration of the surrounding hepatocytes; (C) DENA treated group: Showing anaplastic hepatocytes forming HCC with trabecular pattern with compression of adjacent hepatocyte; (D) DENA+ oxygenated water treated group: Showing focal area of anaplastic hepatocytes with hepatocytomegaly and compression of the surrounding

ones; (E) DENA+ doxorubicin treated group: Showing multiple focal area of dysplasia and degeneration in hepatocytes; (F) DENA+ oxygenated water+ doxorubicin treated group: Showing focal area of dysplasia and ballooning degeneration in

hepatocytes of the parenchyma with dilatation of the central vein. Hematoxylin–eosin staining, magnification 40×.



There is a constant need to find new treatment for HCC because even with the recent research there is no effective treatment that can cause complete recovery leading to tumor recurrence or toxicity [45]. The present work aimed to find out if administration of oxygenated water either alone or combined with doxorubicin can improve the therapeutic outcome during HCC treatment in rat model. Diethylnitrosamine intoxication at a dose of (100 mg/L) for 8 consecutive weeks in drinking water resulted in significant elevation of the serum ALT, AST, ALP, LDH, GGT, lipid profiles, bilirubins and decreased the serum levels of TP and HDL-C. These data are in agreement with the reported literature [46- 51]. DENA also caused significant elevation in HCC related tumor markers including AFP and AFU besides increasing liver tissue content of caspase-3. These results are in line with the reported literature [52- 55]. DENA administration caused depletion of the antioxidant enzymes and increased lipid peroxidation in the liver tissue of the rats along with formation of anaplastic hepatocytes with trabecular pattern in the histopathology examination of the liver tissue and those results were supported by the reported literatures [56- 61]. In the current study, oxygenated water improved the liver functions, tumor markers and histopathological findings. This could be due to its anti-hypoxic effect, which could be responsible for the increase in caspase-3 content, restoration of the liver antioxidant enzymes and the reduction in lipid peroxidation. This can be explained by the reported literature which indicated that hypoxia causes overexpression of the activator protein 1 (AP-1) gene and survivin that have antiapoptotic activities besides causing excessive production of ROS, which cause depletion of the antioxidant enzymes and lipid peroxidation [62- 66]. On the other hand, the combined treatment showed a better improvement in the previously mentioned parameters. This effect could be considered as synergistic effect between oxygenated water and doxorubicin as previously reported by He C et al, who indicated that down regulation of HIF-2ɑ, which overexpressed because of hypoxia improves the efficacy of doxorubicin in treatment of HCC [67]. Therefore, it could be suggested that correction of hypoxia by oxygenated water could augment the doxorubicin effect. In the present study, oxygenated water administrated alone to normal rats caused congestion in the portal and central vein with degeneration in the surrounding hepatocytes accompanied with significant elevation in the serum ALT, AST and LDH. This toxicity may have happened due to the presence of few traces of H2O2 in the administrated oxygenated water due to incomplete conversion of H2O2 by the catalyst (MnO2). Those traces could lead to activation of the kupper cells and the

release of thromboxane A2 (TXA2) that increase the portal pressure leading to the portal vein congestion and the degeneration in the surrounding hepatocytes [68]. In addition, it may have contributed to the therapeutic efficacy of the oxygenated water as it has been reported that H2O2 augments doxorubicin cytotoxicity due to enhancing its anti-proliferative effect [69]

4. CONCLUSION Oxygenated water has shown potential synergistic effect with doxorubicin in the treatment of HCC most likely due to its anti-hypoxic effect. Further studies are needed to elucidate the exact mechanisms under the synergistic effect of oxygenated water with doxorubicin or other traditional cytotoxic drugs.

5. ACKNOWLEDGEMENTS The authors are grateful to Dr. Adel B. Kholoussy, Professor of Histology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt, for examining and interpreting the histopathological data of this study.

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