Ebselen

Protective efficacy of thymoquinone or ebselen separately against arsenic-induced hepatotoxicity in rat

Abstract
Arsenic (As) exposure is associated with adverse health outcomes to the living organisms. In the present study, the hepato-protective ability of thymoquinone (TQ), the active principle of Nigella sativa seed, or ebselen (Eb), an organoselenium compound, against As intoxication in female rats was investigated. For this purpose, animals were allocated randomly into control, As (20 mg/kg), TQ (10 mg/kg), Eb (5 mg/kg), As+TQ, and As+Eb groups that were orally administered for 28 consecutive days. Arsenic exposure resulted in hepatic oxidative damage which was evi- denced by marked decreases in antioxidant parameters (superoxide dismutase (SOD), catalase (CAT), glutathione per- oxidase (GPx), glutathione reductase (GR), and glutathione (GSH)) concomitant with high malondialdehyde (MDA) level. Furthermore, As toxicity induced significant elevations in liver accumulation of As, serum hepatic indices (as- partate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin (TB)), and apoptotic marker (B cell lymphoma 2(Bcl2), Bcl-2-associated X protein (Bax), and caspase 3) levels. Additionally, notable increments in hepatic fibrotic markers (epidermal growth factor (EFG) and transforming growth factor beta 1 (TGF-β1)) associated with high nitric oxide, interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and myeloperoxidase (MPO) levels were noticed following As intoxication. Biochemical findings were well-supported by hepatic histopathological screening. The co-treatment of As-exposed rats with TQ or Eb considerably improved liver function and antioxidant status together with lessened hepatic As content, inflammation, apoptosis, and fibrosis. The overall outcomes demonstrated that TQ or Eb ameliorates As-induced liver injury through their favorable antioxidant, anti-inflammatory, anti-apoptotic, and fibrolytic properties.

Introduction
Arsenic (As) is an extremely hazardous metal that is abundant in the environment through natural and anthropogenic activi- ties (Mumtaz et al. 2020). Natural processes such as weathering and volcanic eruptions help in mobilizing As to the environment (Samelo et al. 2020). Additionally, As is involved in various industrial processes such as manufactur- ing of pigments, glass, pesticides, and metal alloys. Using of As-based pesticides also aggravated As pollution in the ground water (AL-Megrin et al. 2020). In this scenario, expo- sure to As may be environmental or occupational that occurs mainly via drinking water, inhalation, or skin contact. Following the As absorption through the intestine, it is metab- olized in the liver through oxidative methylation and glutathi- one conjugation (Metwally et al. 2020). Therefore, the liver is considered the major target organ of As toxicity and tumori- genesis. Plenty of former studies reported massive liver injury following As intoxication such as hepatomegaly, portal hyper- tension, fibrosis, cirrhosis, and hepatic cell carcinoma (Liu and Waalkes 2008; Hsu et al. 2016).

Several factors are contributing to the development of he- patic injury following As intoxication including oxidative stress (Thangapandiyan et al. 2019). As was found to generate reactive oxygen species (ROS) that triggers hepatotoxicity and liver cell death (Dkhil et al. 2020). ROS is mainly gener- ated in the mitochondria, inhibits the mitochondrial electron transport chain’s complex I, and reduces membrane integrity which consequently enhances a cascade of radical reactions with additional generation of other ROS (Al-Megrin et al. 2020; Nutt et al. 2005). Furthermore, As induces oxidative stress by reacting with thiol (─SH) group and subsequently suppresses antioxidant defense mechanism and glutathione system (Das et al. 2010). The management of As toxicity relies on administration of antidote such as British anti- lewisite (BAL; dimercaprol) which act as metal-chelating agents. Nevertheless, using these agents is associated with side effects such as disturbing essential metal elements, hepat- ic and renal injuries in addition to their non-specificity, and low therapeutic index (Bodaghi-Namileh et al. 2018); there- fore, new therapeutic approaches are needed to alleviate As toxic effects with better outcomes.Antioxidant agents have gained attention for their efficacy to alleviate the liver damage produced following As exposure (Xu et al. 2019). Nigella sativa plant belongs to the family Ranunculaceae and is widely growing in North Africa, Southwest Asia, and Southern Europe (Ali and Blunden 2003). This plant is extensively used for the treatment of dif- ferent disorders owing to its healing potentials (Ahmad et al. 2013). Thymoquinone (TQ), 2-isopropyl-5-methyl-1,4-ben- zoquinone, is the major active component extracted from the volatile oil of Nigella sativa seeds (Zeinvand-Lorestani et al. 2018). TQ had been used as an antihypertensive, antitumor, antidiabetic, anti-inflammatory, antioxidant, and pain- relieving agent (Hassan et al. 2019). The hepato-protective effect of TQ was previously illustrated against different chemicals as lead, paraquat, and methotrexate (El-Sheikh et al. 2015; Mabrouk et al. 2016; Zeinvand-Lorestani et al. 2018). TQ is involved in synthesis of essential antioxidant enzymes such as DT-diaphorase and heme oxygenase as well as activation of nuclear factor erythroid 2 like 2 which is responsible for the protection of cells against oxidative stress (Kundu et al. 2014). Besides its antioxidant role, TQ contrib- utes to alleviate tissue inflammation by inhibition of pro- inflammatory cytokines as interleukin 1 (IL-1β) and tumor necrosis factor alpha (TNF-α) as well as activation of anti- inflammatory ones such as interleukin 10 (IL-10) (Elsherbiny and El-Sherbiny2014).

Ebselen (Eb), 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a lipid-soluble organoselenium compound, with promising antioxidant properties (Azad and Tomar 2014). Its lipid solu- bility facilitates its entrance to the cell. Eb possess strong anti- lipoperoxidative and antioxidant actions through a glutathione peroxidase (GPx)–like action (Mohammed et al. 2018). It acts as a scavenger for free radicals and protects the cells against the injurious effects of ROS, and reactive nitrogen species (Azad and Tomar 2014; Gabryel and Małecki 2006). Additionally, it was reported that Eb exhibited anti-inflamma- tory, anti-thrombotic, and anti-atherosclerotic activities (Chew et al. 2010; Lindenblatt et al. 2003). Hence, the goal of the current investigation was to explore the possible hepato- protective impacts of TQ or Eb against As-mediated hepatic damage through assessing the As deposition, liver function markers, oxidative stress indices, inflammatory mediators, ap- optotic proteins, and changes in liver tissue architecture.Sodium arsenite (As; CAS number 7784-46-5), ebselen (Eb; CAS number 60940-34-3), and thymoquinone (TQ; CAS number 490-91-5) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All other used chemicals were of high analytical grade.

Forty-two healthy female Sprague-Dawley rats of 8–10 weeks old and 150–180 g were obtained from the animal facility of the VACSERA (Cairo, Egypt). Tested rats were acclimatized for 10 days in plastic cages (595 × 380 × 200 mm3) under controlled conditions of 20 to 22 °C temperature, 65% relative humidity, and normal photoperiod (12 h light and dark). Each four female rats was housed in one cage. Standard rat pellet chow was used for feeding animals with free access to drink- ing water. All experimental protocols were undertaken in line with the Committee of Research Ethics for Laboratory Animal Care, Department of Zoology and Entomology, Faculty of Science, Helwan University (approval no., HU2019/Z/12), in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, 8th edition (NIH Publication no. 85–23, revised 1985).Group I (control group): animals were received orally normal saline (0.9% NaCl) containing 0.1 % DMSO. Group II (TQ group): animals were treated orally with TQ (10 mg/kg b.wt) based on a previous report (Kassab and El-Hennamy2017).Group III (Eb group): animals were treated orally with Eb (5 mg/kg b.wt) based on the dose experimented by Yang et al. (2000).Group IV (As group): animals were received orally As at a dose of 20 mg/kg b.wt according to Yadav et al. (2009). As was dissolved in normal saline (0.9% NaCl) contain- ing 0.1 % DMSO.Group V (As+TQ group): animals were treated orally with As and after 1 h, they received orally TQ using the same doses.Group VI (As+Eb group): animals were treated orally with As and after 1 h, they received orally Eb using the same doses.

TQ or Eb was dissolved in DMSO and diluted with normal saline (0.9% NaCl) to the required concentration prior to the treatment. An equal volume of DMSO was given to the con- trol and As groups to balance the concentration of DMSO gavaged to all treated groups to reach a final DMSO concen- tration of 0.1%.At the end of the 28 days of exposure, rats were decapitated 24 h after the last treatment after sodium pentobarbital (Sigma-Aldrich, St. Louis, Missouri, USA) at a dose of 300 mg/kg. Blood was obtained from the abdominal aorta using a syringe, and the serum was separated after being centrifuged at 1300×g for 10 min. The liver tissue was quickly separated, weighed, and washed in neutral ice-cold 0.1-M Tris-HCl (Sigma-Aldrich, St. Louis, Missouri, USA) two times. Tissue homogenate was prepared by mixing liver tissue with 10 volumes of ice-cold 0.1-M Tris-HCl (pH 7.4), followed by centrifugation at 1300×g for 10 min at 4 °C. The resultant supernatants were used for conducting biochemical assays. Representative liver tissue slices were taken for histopatholog- ical examination and As quantification.

The As concentration in hepatic tissue was determined accord- ing to the method described by Datta et al. (2010). Firstly, the samples were digested by a mixture of three acids: nitric acid, perchloric acid, and sulfuric acid at 10:4:1 ratio, diluted with deionized Millipore water and passed through a Whatman filter paper No. 4 (Rankem, India) to obtain 10 ml of total volume. After that, 5 ml of concentrated hydrochloric acid (HCl) was added to it with shaking followed by adding 1 ml of potassium iodide (5% w/v) and ascorbic acid (5% w/v) mixture and incubation for 45 min for conversion of arsenate to arsenite. Millipore water was added up to 50 ml to make the final volume. An atomic absorption spectrometer (AAS) equipped with vapor-generation accessories (Varian AA240 model AAS) was employed to determine As concentrations. The instrumental conditions were lamp, arsenic hollow cath- ode lamp; wavelength, 193.7 nm; slit width, 0.5 nm; lamp current, 10.0 mA; vapor type, air/acetylene; air flow, 10.00 L/min; and inert gas for hydride generation, Argon. Both of the reducing agent (aqueous solution of 0.6% sodium borohy- dride was prepared in 0.5% w/v sodium hydroxide) and acid (HCl 40%) were prepared just before use. The used standards were 5, 10, 20, and 40 μg/l and prepared similarly as the test sample.Serum parameters (ALT, ALP, AST, and total bilirubin) as well as hepatic ones (ALT, ALP, and AST) of liver functions were calorimetrically measured using standard kits (Biodiagnostic, Giza, Egypt) following the manufacturer’s in- formation based on the methods described by Reitman and Frankel (1957), Walter and Gerade (1970), and Belfield and Goldberg (1971) respectively.

After fixation of liver specimens in 10% neutral-buffered formaldehyde for 24 h, they were dehydrated in ethyl alcohol, cleared in xylene, and embedded in molten paraffin wax. Afterwards, sections of4–5-μm thickness were prepared and stained with hematoxylin and eosin. Histopathological assess- ment of hepatic tissue was performed using a light microscope (Nikon; Eclipse E200-LED, Tokyo, Japan). For a semi- quantitative comparison of the structural changes, the abnor- malities in the liver sections were graded from 0 (normal structure) to 3 (severe pathological changes).Glutathione (GSH) level was evaluated in the deproteinized supernatant fraction by reaction of GSH with 5,5-dithiobisnitrobenzic acid to give a yellow coloration accord- ing to Ellman (1959).Lipid peroxidation was assessed in the liver tissue homoge- nates and expressed in terms of malondialdehyde (MDA) ac- cording to the method described by Janero (1990). Briefly, MDA in the sample reacted with thiobarbituric acid to form MDA-TBA adduct, red fluorescent compound, which can be easily measured spectrophotometrically.Hepatic nitrite/nitrate level was determined spectrophotomet- rically at 540 nm according to the Griess reaction and expressed as micromoles per milligram of protein as reported by Green et al. (1982).Activities of superoxide dismutase (SOD) and catalase (CAT) in liver homogenate were measured by the change in color intensity at 539 nm and 240 nm as described by Fisher et al. (2003) and Aebi (1984) respectively. Glutathione peroxidase (GPx) and glutathione reductase (GR) were estimated by means of a spectrophotometer following the methods stated by De Vega et al. (2002).

Pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α; catalog number: RTA00), and interleukin 6 (IL-6; catalog number: R600B) were measured as mentioned in the supplier instructions of R&D Systems ELISA kits (Minneapolis, Minnesota, USA) and expressed as picogram/ g tissue.MPO activity reflects the activity of neutrophil during inflam- mation. MPO activity in liver homogenate was measured spectrophotometrically at 460 nm using the O-dianisidine method and expressed in units/mg of protein (Bradley et al. 1982).For the illustration of the anti-apoptotic role of TQ or Eb against As-induced hepatotoxicity in female rats, levels of Bcl-2, caspase 3, and Bax were evaluated. Commercial ELISA kits were used to measure Bax (BioVision, Inc., Milpitas, CA, USA; catalog number: E4513) and Bcl-2 (Cusabio, Wuhan, China; catalog number: CSB-E08854r), following the manufacturers’ procedures. Furthermore, cas- pase 3 level was assessed by a colorimetric assay kit (Sigma-Aldrich, St. Louis, Missouri, USA; catalog number: CASP3C-1KT).The incidence of fibrosis in liver tissue was assessed by mea- suring levels of transforming growth factor beta 1 (TGF-β1, catalog number: MB100B) and epidermal growth factor (EGF, catalog number: DY3214) in the tissue homogenates using ELISA kits (R&D System, Minneapolis, MN, USA) following the manufacturers’ specifications.Data are presented as means ± SD and evaluated by one-way analysis of variance using the SPSS software (version 17). The tested groups were compared using Duncan’s test as a post hoc test. The acceptable level of significance was set at p ˂ 0.05.

Results
The As concentrations in hepatic tissues of female rats in response to different treatments are shown in Fig. 1. Hepatic As content was distinctly elevated (p < 0.05) in the As- exposed group compared with that in the control group. Furthermore, hepatic specimens from the As+TQ- and As+ Eb-exposed groups revealed significant declines (p < 0.05) in As concentrations in comparison with the sole treatment with arsenic.To estimate the liver dysfunction (Table 1), total bilirubin levels in serum samples together with the enzymatic activities of AST, ALT, and ALP in serum and liver samples were measured. Regarding the serum results, significant increases (p < 0.05) were observed in the level of total bilirubin as well as AST, ALT, and ALP enzymatic activities in As-treated rats in respect to the con- trol. However, the dual treatment with TQ and As resulted in notable reductions (p < 0.05) in the abovementioned parameters compared with that of the As-administered group. Furthermore, the group exposed to As+Eb showed significant declines (p < 0.05) in the level of total bilirubin and the enzyme activities of ALT and ALP only, while the activity of AST did not show any significant difference compared with the group exposed to sodi- um arsenite only. On the other hand, the hepatic AST, ALT, and ALP enzyme activities showed marked decreases (p < 0.05) in As-treated group compared with those of the control. Additionally, significant increments were noticed in the liver function indicators, AST, ALT, and ALP, in hepatic specimen of As+TQ and As+Eb groups as compared with that in As- exposed rats. The hepatic architecture of control and exposed rats is demonstrated in Fig. 2. Hepatocytes of control group (2A), TQ (2B), and Eb (2C) groups revealed normal his- tological structure of arranged hepatic cords. In contrast, As-exposed group (2D) showed noticeable pathological alterations such as fat deposition, inflammatory cell infil- tration, and hepatocytes necrosis. Furthermore, the ad- ministration of TQ or Eb with As (2E and 2F) had a rescue effect on As-induced hepatocytic alterations. However, some necrotic area was seen in TQ or Eb co- treated rats. Additionally, pathological scoring analysis showed that TQ or Eb treatment ameliorative the histo- pathological changes following As intoxication (p < 0.05) (Table 2).Levels of oxidative stress biomarkers in the liver of con- trol and exposed rats are shown in Fig. 3. The level of hepatic GSH was markedly lower (p < 0.05) in the livers of As-exposed rats than in control rats. Additionally, As intoxication induced significant lipid peroxidation in the liver which was evidenced by a significant elevation (p < 0.05) in MDA levels in As-exposed group in respect to the control group. Furthermore, NO production showed a significant rise (p < 0.05) in the As-administered group compared with that of the control group. On the other hand, co-administration of TQ or Eb to arsenic-treated rats resulted in a marked recovery of the abovementioned parameters. Significant improvements (p < 0.05) in the oxidative stress markers were detected in the groups ex- posed to As+TQ and As+Eb in respect to the As-treated- only group. Results are expressed as mean ± SD (n = 10 fields). # or $ refers to the statistical significance at p < 0.05 against the control and As groups respectively.The disturbances in the antioxidant enzymes were assessed in liver tissue. Rats exposed to As exhibited marked low levels (p < 0.05) of hepatic SOD, CAT, GR, and GPx when compared with controls. On the other hand, the dual treatment of As with either TQ or Eb enhanced significantly (p < 0.05) the activities of the aforementioned enzymes in respect to the animals in- toxicated with As only as shown in Fig. 4. The adverse impacts of As intoxication and the potential ame- liorative role of TQ or Eb on hepatic inflammatory biomarkers were illustrated. In As-treated rats, levels of IL-6, TNF-α, and MPO were significantly augmented (p < 0.05) in liver tissue in comparison with that of the control group. However, TQ or Eb co-administration resulted in significant decline (p < 0.05) in levels of the abovementioned parameters in the groups exposed to As+TQ and As+Eb compared with the sole treat- ment with As (Fig. 5).To evaluate whether apoptotic parameters were triggered fol- lowing As intoxication, caspase 3, Bax, and Bcl2 were mea- sured in liver homogenate of As-administered animals. Compared with control, As-exposed group exhibited signifi- cant increments (p < 0.05) in the levels of caspase 3, Bax, and Bcl2 in liver tissue. Notably, the groups exposed to As+TQ and As+ Eb showed significant reductions (p < 0.05) in the levels of the tested apoptotic markers in the liver compared with those given As treatment (Fig. 6).In comparison with the control group, the fibrosis marker results in liver tissue indicated that TGF-β1 and EGF were significantly augmented (p < 0.05) in As-exposed animals. Animals exposed to As+TQ and As+Eb, on the other hand, presented a significant decrease (p < 0.05) in both fibrotic biomarkers as compared with As-treated rats as displayed in Fig 7. Discussion Antioxidants of natural origin are gradually gaining the re- searchers’ attention to be in place of conventional chelating agents in treatment of As intoxication. In this context, the refers to the statistical significance at p < 0.05 against the control and As groups respectively therapeutic potentials of TQ or Eb, phyto-antioxidants, were investigated in the current study against As-mediated hepatic injury in female rats with special reference to oxidative stress, inflammation, apoptosis, fibrosis, and histopathological changes. After absorption of As from GIT, it is reduced to arsenite in the liver, and the arsenic metabolites are excreted through the urine (Vahter 2002). Liver is considered the major site of As detoxification due to the abundance of GSH in hepatic tissue. GSH exerts its action as an antioxidant by As conjugation for cellular efflux followed by biliary excretion (Liu et al. 2001). Additionally, hepatic tissue is also the main site for As meth- ylat ion v ia methyl tran sfer as e enzyme a nd S - adenosylmethionine (SAM) as a substrate (Thomas 2007). In accordance with other studies, the As concentration in he- patic tissue was higher in As-treated group compared that in the control group (Li et al. 2017; Zhang et al. 2017). On the other side, the groups exposed to As+TQ and As+Eb displayed significant lower hepatic As concentrations than the group exposed to As only. The metal chelation efficacy of Eb was illustrated previously as it enhances the metal ex- cretion from the body and subsequently lowering its concen- tration (Ismail 2019). Furthermore, it was previously reported that TQ co-administration significantly diminished the metal ion accumulation. The TQ ability to lessen metal deposition may refer to its ability to prevent the depletion of intracellular GSH and the latter can bind heavy metals which have high affinity to thiol groups (Fouad and Jresat 2015). The enzymatic activities of AST, ALT, and ALP as well as total bilirubin level are sensitive indicators for diagnosis of hepatocellular injury. Significant hepatocellular alterations were reported in As toxicity as cellular necrosis and degener- ation, associated with loss of functional integrity of hepatic membrane with consequent leakage of these enzymes from the cytoplasm into the circulation (Jalaludeen et al. 2016; Kharroubi et al. 2014). Such enzymatic leakage across the damaged membrane to the blood stream resulted in significant decline in their activities in hepatic tissue along with high serum levels (Al-Olayan et al. 2014). Supporting former stud- ies, marked high activities of serum AST, ALT, ALP, and TB content paralleled with low hepatic enzymes were observed in our study in As-intoxicated rats in comparison with that in control rats ( Muthumani and Miltonprabu 2015). Histopathological screening demonstrated substantial hepatic lesions in As-treated group as fat deposition, portal inflamma- tion, inflammatory cell infiltration, and hepatocytes necrosis which supported the biochemical findings and ensured the previous studies (Bashir et al. 2006; Jalaludeen et al. 2016; Sankar et al. 2015; Zhang et al. 2013). However, co- supplementation of rats with TQ restored the enhanced levels of liver function markers induced by As owing to its strong antioxidant activity and this is consistent with previous studies (El-Sheikh et al. 2015; Hassanein and El-Amir2017; Zeinvand-Lorestani et al. 2018). Additionally, marked im- provements of these indices were noticed in As+Eb-exposed group in respect to As-exposed group that was formerly ob- served by Basarslan et al. (2013) in hepatotoxicity induced by radio-contrast media in rats. The hepato-protective effects of TQ or Eb have formerly been reported by histopathological studies (Hassanein and El-Amir2017; Ismail 2019). Hepatotoxicity due to As exposure is mediated by various mechanisms. Mitochondria are the main site of ROS genera- tion through the electron transport chain; therefore, they have the central role in the hepatocellular damage (Ott et al. 2007). As accumulation inside the mitochondria is greater than any other organelle because the membrane affinity for As is high. Arsenic is able to suppress complex I respiratory chain with subsequent ROS overproduction and oxidative stress (Ramanathan et al. 2003). Significant alterations in antioxi- dant parameters and lipid peroxidation under As intoxication have been reported previously in animal models (Al-Brakati et al. 2019; Jalaludeen et al. 2016; Mehrzadi et al. 2018; Messarah et al. 2012). Our results revealed notable exhaustion of the hepatic antioxidant system of As-intoxicated rats. GSH is a non-enzymatic tripeptide and multifunctional antioxidant which found in most body cells especially hepatocytes and exerts its antioxidant actions through multiple mechanisms (Nili-Ahmadabadi et al. 2011). The present results revealed that the hepatic GSH content was markedly decreased in As- treated group. This decline may refer to the reduction of As5+ into As3+ by GSH or the oxidation of GSH by free radicals resulted from As metabolism or the high affinity of As to sulfhydryl groups in GSH structure (Bodaghi-Namileh et al. 2018). Also, our findings revealed notable inhibition to hepat- ic antioxidant enzymes (SOD, CAT, GR, and GPx) in As- exposed group that may refer to consumption of these enzymes to neutralize the superoxide anion over-generation during As metabolism (Sankar et al. 2015). High levels of intracellular ROS generated by As exposure are involved in cell membrane damage via lipid peroxidation, which is a basic mechanism of As hepatic injury. In line with previous results, As treatment increased markedly the perox- idation of lipids as evidenced by high MDA content in respect to the control (Jalaludeen et al. 2016; Mehrzadi et al. 2018). Excessive production of NO (a pro-oxidative molecule) in liver tissue is involved in oxidative damage as it can interact with superoxide anion to generate peroxynitrite, ONOO−, (a stronger oxidizing agent) which induces further damage to the cellular components. Liver of As-exposed rats displayed prominent increments in NO levels compared with that in the control which is in accordance with other authors (Mehrzadi et al. 2018, Messarah et al. 2012). The co-treatment with TQ or Eb enhanced the antioxidant response in liver of As-exposed rats in comparison with the sole treatment with As. Noteworthy elevations were noticed in the antioxidant enzymatic activities and GSH content paralleled with reduction of NO and MDA levels in liver of TQ+As and Eb+As groups compared with that in As-exposed group. It was reported that TQ is a potent radical scavenger and has the ability to alleviate the cellular oxidative stress and lessening the associated lipid peroxidation (El-Sheikh et al. 2015; Hassan et al. 2019). Ebselen enhances the reduction of ROS in a manner resembling GPx. Briefly, the oxidation state of the selenium in Eb is changed, and the resting state selenol (EBS-SeH) is oxidized via ROS to selenenic acid (EBS- SeOH). Selenenic acid is then reduced by GSH to the active selenol through a selenenyl sulfide intermediate (EBS- SeSG)(Azad and Tomar 2014; Ismail 2019). The protective effect of Eb against heavy metal toxicity can be explained that by binding of the selenium atom in Eb with metal ions, Eb can trigger the gene expression of cytoprotective genes, as antiox- idant enzymes that can scavenge hydrogen peroxide radical formed by metal (Rusetskaya and Borodulin 2015).Concerning As-mediated hepatic inflammation, significant rises were found in the levels of IL-6, TNF-α, and MPO in As- treated rats compared with that of the control and this is in agreement with other authors (Prabu and Muthumani 2012; Souza et al. 2018). Due to the ability of As to generate excess ROS and induction of oxidative stress, it enhances overpro- duction and gene expression of pro-inflammatory cytokines and other inflammatory mediators including IFN-γ, IL-1β, TNF-α, and NF-κB. The activation of NF-κB further causes upregulations of gene expression of other pro-inflammatory genes including cytokines, chemokines, and adhesion mole- cules (Bodaghi-Namileh et al. 2018). MPO, heme peroxidase, stimulates the generation of ROS, including hypochlorous ac- id (HOCl) in presence of halide and hydrogen peroxide. The MPO/HOCl system has a pivotal role in the antimicrobial activity of neutrophils. Furthermore, MPO is a local mediator of tissue oxidative stress and inflammation (Ndrepepa 2019). On the other hand, co-treatment with TQ or Eb exhibited a notable anti-inflammatory role in As-intoxicated hepatocyte. TQ lessened the inflammation by inhibition of oxidative stress and release of the inflammatory mediators such as NO, TNF-α, and IL-1β (Al-Malki and Sayed 2014). The palliative action of Eb against tissue inflammation is achieved through downregulation of gene expressions of TNF-α and pro- inflammatory cytokines (IL-6, IL-8, monocyte chemoattractant protein-1, and cyclooxygenase 2) (Tewari et al. 2009).This study also revealed the apoptotic damage of hepato- cytes induced by As exposure that was evidenced by marked increases in caspase 3, Bax, and Bcl2 in the liver of As-treated group compared with that in control and this is concomitant with previous studies (Bashir et al. 2006; Bodaghi-Namileh et al. 2018). Arsenic has the ability to induce cellular apoptosis through translocation of cytochrome-c from mitochondria to the cytoplasm which initiate the caspase pathway and subse- quent formation of apoptosomes (Bodaghi-Namileh et al. 2018). Interestingly, livers of As+TQ- and As+Eb-treated rats showed noteworthy decreases in apoptotic markers compared with that of As-treated group revealing the marked anti- apoptotic effect against As-induced hepatic injury through their powerful ROS scavenging capacity. Also, TQ regulates the mRNA gene expression levels of p53, caspase enzymes, and the Bax/Bcl-2 ratio (Sheikhbahaei et al. 2016). Furthermore, Eb suppressed effectively the mitochondrial ap- optosis pathway by lessening the release of cytochrome-c and caspase 3 activation (Azad and Tomar 2014). Regarding the effect of As exposure on the fibrotic markers, notable rises in both TGF-β1 and EGF were ob- served in liver of As-intoxicated group compared with that of the control. TGFα and EGF are secreted by hepatic stellate cell (the major effector in liver fibrosis) and stimulate its pro- liferation (Lee and Friedman 2011). Arsenic-induced fibrosis was reported via activation of TGF-β/Smad pathway as a result of upregulation of NADPH oxidase and ROS genera- tion (Dai et al. 2019; Pan et al. 2011). Co-treatment with TQ or Eb attenuated As-induced hepatic fibrosis reflecting their po- tent hepato-protective efficacy to diminish As adverse effects which is concomitant with other reports (Abdelghany et al. 2016; Wasser et al. 2001). Although our findings showed obvious elevation in the levels of fibrotic markers (TGF-β1 and EGF) following As intoxication, no fibrotic hepatocytes were recorded in examined liver sections in As-exposed group. Conclusion From the current study, we concluded that oral As adminis- tration induced hepatic damage and dysfunction in female rats which were triggered by significant imbalance in oxidant/ antioxidant status. The oxidative damage was associated with induction of inflammation, apoptosis, fibrosis, and marked histological alterations. Co-treatment with TQ or Eb lessened the liver dysfunction induced by As exposure due to their potent antioxidant, anti-inflammatory, and Ebselen anti-apoptotic activities.