Role of zinc oxide nanoparticles in alleviating hepatic fibrosis and nephrotoxicity induced by thioacetamide in rats.

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Role of zinc oxide nanoparticles in alleviating hepatic fibrosis and nephrotoxicity induced by thioacetamide in rats. Samir AE Bashandy1*, Abdulaziz Alaamer2, Sherif A Abdelmottaleb Moussa 2,3, Enayat A. Omara4 1

Department of Pharmacology, Medical Division, National Research Centre, 33 EL Bohouth st. (former EL Tahir St.), Dokki, Giza, Egypt. P.O.12622.

2

Committee of Radiation and Environmental Pollution Protection (CREPP), Department of Physics, College of Science, Al- Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh, Saudi Arabia 3

Biophysics Group, Biochemistry Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Giza, Egypt. 4

Department of Pathology, Medical Division, National Research Centre

Corresponding Author: *

Samir AE Bashandy, Department of Pharmacology, Medical Division, National Research Centre, 33 EL Bohouth st., Dokki, Giza, Egypt. P.O.12622. Email: [email protected] Tel.: (002) 01121752112

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Abstract The present research studied the influence of zinc oxide nanoparticles (ZnO-NPs; 5mg, 7.5mg, 10 mg/Kg IP) on the liver and kidney injuries motivated by thioacetamide (TAA; 100mg/Kg IP).Each treatment was carried out three times weekly for eight weeks .ZnO-NPs relieved the decrease of hepatic or renal reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) induced by TAA. Moreover, ZnO-NPs lowered tissues malondialdehyde (MDA, an indicator for lipid peroxidation). TAA treatment led to a significant increase in plasma inflammatory markers (TNF-α, IL-6), liver enzymes (Gamma-glutamyltransferase (GGT), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and kidney function parameters (Creatinine, urea, uric acid). However, these parameters were reduced after treatment with ZnO-NPs. In addition, the hepatic fibrosis markers, Hydroxyproline level and α -smooth muscle actin immunopositive stain were lowered by ZnO-NPs. The protective effect of ZnO-NPs in respect to biochemical changes was also confirmed by histopatholgoical, and immunohistochemistry studies in the liver and kidney sections. Our results suggested that ZnONPs may attenuate TAA-toxicity via suppression of oxidative stress. Key words: zinc oxide nanoparticles, hepatic fibrosis, kidney injury, oxidative stress, inflammatory markers, histopathology, immunohistochemistry.

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Introduction Nanoparticles are famed as engineered structures with a diameter smaller than 100 nm. NPs have become an important tool in many industries including healthcare. Nanoparticles (NPs) are used in different applications, including drug carrier. NPs have significant medical value due to their probable applications in the treatment of many diseases and technology (Ali et al. 2016; Ober and Gupta 2011). Among various nanoparticles, zinc oxide nanoparticles (ZnO-NPs) have significant features and are used in different cases including sunscreens, biosensors, pigments, food additives, production of rubber, medicine and electronic materials (Smijs and Pavel 2011). Some studies have shown that ZnO-NPs can induce cytotoxic effects (Najafzadeh et al. 2013), while other reports indicated their antibacterial (Taccola et al. 2011) and antioxidant activities (Nagajyothi et al. 2015). Moreover, ZnO-NPs demonstrated anti-inflammatory activities as evidenced by suppressing both mRNA and protein expressions of iNOS, COX-2, IL-1b, IL-6 and TNF-α (Nagajyothi et al. 2015). ZnO-NPs showed a protective effect against doxorubicin induced male gonadotoxicity through their antioxidant potential (Badkoobeh et al. 2013). ZnONPs ameliorate the oxidative stress in diabetic rats as appeared by the increase in the activities of mRNA expression levels of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase and a decrease of malondialdehyde (Afifi and Abdelazim 2015). Zinc is essential trace element and it is a necessary part of superoxide dismutase (SOD) enzyme that plays antioxidant defense system (Perry et al. 2010). Zinc is included in the structure of metalloenzymes and hormonal complexes. It plays important role in biological processes as growth and division of cells (MacDonald 2000), osteogenesis (Popp et al. 2007), and immune response (Overbeck et al. 2008). Moreover, topical zinc may stimulate wound healing by enhancing re-epithelialization,and decreasing inflammation and bacterial growth (Agren

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1990).There are several mechanisms through which zinc lessens oxidative stress in cells. In particular, it has been found that zinc suppresses synthesis of anti-inflammatory cytokines that bring active forms of oxygen (Valko et al. 2005). Thioacetamide is used to motivate hepatic failure and hepatocyte damage in experimental animal models (Sarkar and Sil 2007), hence, it is a potent hepatotoxic and hepato-carcinogenic compound. Thioacetamide (TAA) induces hepatotoxicity through an unstable reactive metabolite, thioacetamide-S-dioxide which promotes generation of reactive oxygen species (ROS) via binding covalently to macromolecules (Yogalakshmi et al. 2010). Liver fibrosis can originate from Lipid peroxidation induced by TAA (Müller et al. 1991), where the generated free radicals activate the hepatic stellate cells and increase the deposition of extracellular matrix components. Also, cytokines, such as tumor necrosis factor, interleukins and transforming growth factor seem to involve in fibrogenesis (Friedman 1993). Since ZnO-NPs have various pharmacological effects like antidiabetic, anti-inflammatory and antioxidant, the present study was conducted to estimate the protective effect of zinc oxide nanoparticles against thioacetamide induced kidney and liver injuries, fibrosis and oxidative stress.

Materials and methods Chemicals Thioacetamide (TAA) was purchased from Sigma (St. Louis, MO, USA). Other chemicals and reagents were of high analytical grade and were purchased from standard commercial suppliers. TAA was prepared freshly by dissolving in sterile distilled water and stirred well until all crystals were dissolved

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Preparation of zinc oxide nanoparticles Zinc oxide nanoparticles were prepared by the sol gel method using 2 g of zinc acetate dehydrate as precursor dissolved in 14 ml of methanol, under magnetic stirring for 2 h. Then, the resulting solution undergos rapid drying to obtain a white powder. The obtained powder was calcined at 500 °C for 2 h to obtained ZnO nanoparticles. The crystalline phases of the obtained nanopowder were identified by X-ray diffraction (XRD) using a Bruker D5005 powder X-ray diffractometer. Crystallite size was estimated from the Debye-Sherrer Equation and found to be 38–54 nm. Furthermore, the obtained nanopowder was characterized in terms of morphology, structure, and optics characteristics using a number of analytical techniques reported by Omri et al 2016. Preparation of zinc oxide nanoparticles suspension The nanoparticles of zinc oxide were suspended in 1% sodium carboxy methyl cellulose as stabilizer or surfactant, stirred with a magnetic stirrer for 5 minutes and then dispersed by ultrasonic vibration for 15 min (Wang et al. 2008). In order to avoid the aggregation of the particles, fresh suspension was prepared before every use. Animals and experimental groups Forty male albino (Sprague-Dawley) rats weighing 150 ± 10 g. were used in the study. Each 4 rats stayed with each other in polyethylene cage with standard conditions of the light cycle, temperature, and humidity. The rats were obtained from animal house, National Research Centre, Egypt. The rats were adapted in laboratory condition for ten days and then they were grouped into five groups (ܰ =8) as follows: Group I: Normal control receiving the equivalent amount of vehicle for eight Weeks.

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Group II: Rats treated with thioacetamide (TAA) at dose level of 100mg/Kg (Bahcecioglu et al. 2015) (I.P) three times weekly for 8 weeks. Group III: Rats given a combination of low dose (5mg/kg I.P) of ZnO-NPs (Torabi et al. 2013) and TAA (100mg/Kg I.P) three times weekly for 8 weeks. GroupIV: Rats given a combination of middle dose of ZnO-NPs (7.5mg/kg I.P) and TAA (100mg/Kg I.P) three times weekly for 8 weeks. GroupV: Rats given a combination of high dose of ZnO-NPs (10mg/kg I.P) (Torabi et al. 2013) and TAA (100mg/Kg I.P) three times weekly for 8 weeks. Ethics Statement This experiment was carried out accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publication No. 85–23, revised 1996) and under regulations of Animal Care and Use of National Research Centre in Egypt. All sampling was performed under light anesthesia with ether. Preparation of samples After eight weeks of the experiment, blood samples were collected from a retero orbital vein and separated plasma was stored in Eppendorf tubes at -30˚ for biochemical analysis. Immediately after blood sampling, animals were victimized by cervical dislocation under ether anesthesia and livers and kidneys were collected for biochemical and histopathological examinations. Liver and kidney were rapidly removed, washed in ice-cooled saline, plotted dry and weighed. A weighed part of each tissue was homogenized with ice-cooled saline (0.9% NaCl) to prepare homogenate. The homogenate was then centrifuged at 3000 rpm for 10 min. at 5°C using a cooling centrifuge (Laborzentrifugen, Sigma, Germany). The supernatant was used for various analysis. The remaining portion of liver or kidney was fixed immediately in 10%

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neutral buffered formalin, processed for light microscopy to get (5µm) paraffin sections and stained with Hematoxylin & Eosin (H &E) to verify histological details, Masson’s trichrome staining (MTs) to illustrate the collagen fibers. Moreover, some liver sections were subjected to immunochemistry. Immunohistochemistry Examination for α -smooth muscle actin Liver sections were deparaffinized in xylene and rehydrated in graded alcohol. The tissues were pretreated with 10 mM citrate buffer. The slides were washed with phosphate buffered saline (PBS), and blocked. Sections were incubated overnight at 4 °C in a humidified chamber with one of the following primary antibodies: mouse monoclonal antibody to α-SMA diluted 1:100. The sections were rinsed again with PBS then incubated with a biotinylated goat anti -rabbit and mouse antibody for 10 min. The sections were rinsed again with PBS. Finally, sections were incubated with Streptavidin peroxidase. To visualize the reaction, slides were incubated for 10 min with 3, 3′-diaminobenzidine tetrahydrochloride. The slides were counterstained with haematoxylin then dehydrated and mounted. Primary antibodies were omitted and replaced by PBS for negative controls. In each field, the immunopositive (dark brown) area was recorded. Biochemical investigation: Blood plasma was used for colorimetric determination of AST, ALT, GGT, bilirubin, glucose, creatinine, urea, uric acid and protein by using kits from Salucea Company, Netherlands. Moreover, plasma inflammatory markers, IL-6 and TNF-α (R and D Systems, USA) or hepatic hydroxyproline (Koma, Biotechnology, Seoul, Korea) were determined by enzyme-linked Immunosorbent assay, ELISA. On the other hand tissue (Hepatic and renal) MDA, SOD, CAT, and GSH were evaluated using colorimetric assay kits according to the instructions of the manufacturer (Bio Diagnostic, Egypt).

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Statistical analysis The degree in variability of results was expressed as means ±standard error of means (SEM). Data were evaluated by one-way analysis of variance (ANOVA) followed by LSD comparison test. The level of significance was accepted at P < 0.01. Results Oxidative stress markers Malondialdehyde is an indicator of lipid peroxidation that causes oxidative damage to tissues. The data presented in table1 showed a significant increase of hepatic MDA of treated groups (TAA, TAA+ZnONPs-L, TAA+ ZnONPs-M, TAA+ ZnONPs-H) by 116%, 68%, 20% and 33% respectively compared with control group. On the other hand, MDA of rats treated with TAA and ZnO-NPs decreased significantly as compared to TAA group. Similar results were recorded in renal tissues .Moreover, the treatment of rats with TAA only led to a significant decrease in hepatic and renal GSH, CAT, and SOD. The injection of ZnONPs to rats given TAA alleviated the decrease of antioxidant enzymes or GSH. Inflammatory markers Both plasma IL6 and TNF-α elevated significantly in TAA-given group as compared with control (Figs 1, 2). ZnONPs reduced the effect of TAA on previous two parameters. In addition, ZnO-NPs mitigated the increase of hepatic hydroxyproline evoked by TAA (Fig.3). Liver and kidney function tests Table 2 demonstrated that GGT, ALT, AST and bilirubin values of TAA-treated rats are significantly higher than those of control rats. Injection of TAA and ZnONPs in combination 8


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caused a significant decrease in liver enzymes tested when compared with TAA group. Moreover, ZnO-NPs prevented the decrease of plasma protein produced by TAA. Also, urea. Uric acid and creatinine levels elevated significantly in TAA group and ZnONPs reduced this elevation. Histopathological result Liver The control group showed a normal histological structure of the central vein and surrounding hepatocytes, blood sinusoids and round nuclei were noticed (Fig.4 A). Light microscopic examination of the TAA group showed disruption of the architecture of hepatic tissues and collagen fibers deposition around central vein and the portal tract, and pseudolobulation of hepatocytes with fibroblasts, widely of the hepatocytes associated with necrosis, as well as inflammatory cells infiltration in the portal area and central vein, dilatation in the blood sinusoids, pyknotic nuclei were also, observed (Fig. 4B). Treatment with TAA and ZnONPs-L (5 mg/kg) showed moderated improvement in liver tissues, dilatation in the central vein associated with hydropic degeneration in most of the hepatocytes. Also, strands of collagen fibers with inflammatory cells infiltration in portal tract and central vein area (Fig. 4C). In the group treated with TAA and nano zinc medium dose (7.5mg/kg) showed the liver nearly normal hepatic parenchyma with only with minimal necrosis, a few tiny of collagen fibers and inflammatory cells infiltration (Fig. 4D). On the other hand, TAA intoxicated rats administered with nano zinc high dose (10mg/kg) revealed marked regeneration of the hepatocytes with preservation of the normal hepatic

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architecture, minimal of necrosis, inflammatory cells infiltration and thin fibrotic septa (Fig.4 E). Kidney Kidney sections of the control group showed normal architecture of renal tissue with number of glomeruli and renal tubules (Fig. 5A). Kidney sections of the TAA-treated group showed remarkable changes in the renal tissue. These changes include shrinkage of the glomeruli; wide urinary space, necrosis, degeneration vacuolation in the renal epithelia tubular, pyknotic and apoptotic nuclei were observed. Furthermore, presence of hyaline cast in renal tubular lumens, interstitial lymphocytic infiltration and congestion were also observed (Fig.5 B). The histological examination of the kidney of treated with TAA and ZnONPs-L (5 mg/kg) showed moderated degeneration in the renal epithelia tubular and glomeruli, mildly wide urinary space, and hemorrhage between tubules were observed (Fig.5 C). After treatment TAA group with ZnONPs-M (7.5mg/kg) showed mild degeneration in the renal epithelia tubular and glomeruli, and hemorrhage between tubules were observed (Fig.5 D). High dose of ZnONPs (10mg/kg) administration showed improvement effects in the TAA treated group with mild degeneration the renal epithelia tubular and glomeruli (Fig.5 E). Masson trichrome staining In the normal group, the liver tissues were usual minimal of collagen fibers (Fig. 6A). Intoxicated rat TAA revealed thick collagen fibers were detected in the liver tissues were extended from portal to central area with formation pseudolobulation of hepatocytes (Fig. 6B). ZnONPs low dose (5 mg/kg) treatment group showed decreased collagen fibers and were exist around portal and central vein area (Fig. 6C). Nano zinc of medium and high dose treatment

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groups(7.5,10mg/kg) showed remarkable decrease in collagen fibers as compared with TAA group (Figs. 6D and 6E). Immunohistochemical staining of α -SMA Immunohistochemical staining for α -SMA expression was performed to visualize activated hepatic stellate cells (HSCs) and progression of liver fibrosis. In the control group, was positively expressed weakly in the portal and central vein area (Fig. 7A). Immunohistochemical staining for α-SMA revealed a marked increase in the number of activated HSCs in the liver of CCl4-treated rat; the brown granules that indicate positive expression were distributed in the portal and central vein area owing to the proliferated fibrous tissues and fibrous septa (Fig. 7B). Treatment with TAA and nano zinc medium and high dose (Figs.7 D and 7 E) was more effective in suppressing α-SMA than treatment with low dose ZnONPs (Fig.7 C). However, α-SMA immunopositivity was substantially reduced by the treatment with nano zinc in a dose-dependent manner.

Discussion Oxidative stress is a major mechanism that initiates and progress liver or kidney injuries. Our organs have been provided with an antioxidant system to combat oxidative stress (Li et al. 2015). Thioacetamide is familiar hepatotoxic producing free radicals and oxidative stress through its Soxide metabolite (thioacetamide-S dioxide), an unstable and reactive metabolite (Yogalakshmi et al. 2010) .The present study clarified the decrease of oxidative stress results from TAA via ZnONPs as evidenced by a decrease of hepatic MDA and increase of GSH, SOD, and CAT. Antioxidant and anti-inflammatory properties of ZnO-NPs were reported by some investigators (Nagajyothi et al.2015; Li et al. 2017). Zinc exerts antioxidant activity by various mechanisms as induction of metallothioneins, a powerful scavenger of free radicals (Suntres and Lui 1990), 11


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protection sulphhydryl groups against oxidation, keeping the intracellular levels of reduced glutathione (Bray and Bettger 1990) and reduction the generation of lipid peroxidation products (Bao et al. 2013) .The antioxidant enzymes SOD and catalase showed a defense against oxidative cell injury. SOD is a metal-containing enzyme that stimulates the break-down of superoxide into oxygen and hydrogen peroxide. It conserves the concentration of superoxide radicals at low levels and therefore plays an important role in the defense against oxidative stress (Fridovich 1997). Catalase, an enzyme found mainly in peroxisomes, decomposes hydrogen peroxide to water and oxygen. Zinc has a vital role in the regulation of cellular glutathione, which is vital to cellular antioxidant defense (Parat et al. 1997). Zinc is a vital part of superoxide dismutase (SOD) enzyme that control free radicals and oxidative stress ( Peixoto et al. 2009) . Thioacetamide is a vigorous hepatorenal toxic agent which manifested by histopathological changes and biochemical analysis of the present work. Our results demonstrated a significant increase in plasma liver enzymes (GGT, ALT, and AST) or bilirubin and a decrease of protein due to TAA treatment. The increase in plasma enzymatic activities is linked to liver damage and change of membrane characteristics result in the leakage of enzymes and therefore its level elevated in the plasma. The increase in liver enzymes may indicate hepatocytes necrosis (Gressner et al. 2007). Also, plasma creatinine, urea, and uric acid elevated significantly in TAA group which may indicate renal insufficiency and tubular injury (Özen et al. 2004) .The pathological changes observed in liver and kidney of TAA group can be attributed to increase of tissue MDA, an indicator of lipid peroxidation which alters the physiological functions of cell membranes and plays an important role in cellular membrane damage through free radical chain reaction mechanism(Wong-Ekkabut et al. 2007) .It was reported that ZnO-NPs are able to protect cell membrane integrity against oxidative stress damage, increase antioxidant enzyme

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levels ,decrease MDA and free radicals levels (Dawei et al. 2009) .The decrease of pathological changes here in ZnO-NPs + TAA group is likely due to the decrease of lipid peroxidation, enhancement of antioxidant enzymes(catalase and SOD) and preserve the tissue GSH. Oxidative stress is closely related to the pathological damage of hepatic fibrosis that represents a common response to chronic liver injury (Poli 2000) .Liver fibrosis is a wound healing response to hepatocellular injury. It includes deposition of extracellular matrix by activated hepatic stellate cells (HSCs) (Reeves and Friedman 2002). Activated HSCs show an increased expression of matrix genes and a-smooth muscle actin (aSMA) as well as enhancement proliferation. Activated HSCs are responsible for the overproduction of collagens during hepatic fibrosis (Poonkhum et al. 2011). Inflammation, induced by oxidative stress, is a key event in HSC activation (Greenwel et al. 2000). Several pro-inflammatory cytokines, chemokines and adhesion molecules initiate the activation of HSC (Pinzani and Macias-Barragan 2010). The present results demonstrate a significant increase in hepatic hydroxyproline concentration and α -SMA expression of rats treated with TAA. Moreover, thick collagen fibers were detected in the liver sections of TAA group stained with Masson trichrome. Hydroxyproline is one of the most amino acids present in collagen following hydroxylation of proline moiety. The presence of hydroxyproline in the extracellular matrix (ECM) produced by activated hepatic stellate cells (HSCs) preserves the integrity and function of liver cells. Its level in liver tissues comprises a superior limiting factor which could signify correctly the rates and progression of liver fibrogenesis (Wynn 2008). The development of fibrosis depends mainly on the incorporation of proline into procollagen via hydroxylation to hydroxyproline that correlated with the rates of collagen synthesis and can be used in the assessment of collagen content. So the level of this amino acid is considered a marker of collagen synthesis and degradation during the pathogenesis of fibrosis (McAnulty et al 1991).

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Our work has shown that ZnO-NPs could inhibit rat liver fibrosis development as appeared from a significant decrease of hepatic hydroxyproline level and decrease of α-SMA-positive cells and collagen bundles. It is reported that zinc supplementation inhibited ethanol and acetaldehydeinduced activation of HSCs on different levels, acting as an antioxidant and inhibition of several markers of HCS activation (Szuster-Ciesielska et al. 2009). It appears that zinc has different ways to protect liver against fibrosis. Zinc can reduce transforming growth factor beta induced epithelial differentiation and fibroblast activation, which are common features of tissue fibrosis (Sörensen-Zender et al. 2015). In addition, ZnO-NPs can control fibrosis through lowering of hepatic lipid peroxidation which participates in fibrosis development (Situnayake et al. 1990). Moreover, ZnO-NPs here mitigate the increase of IL-6 and TNF-α stimulated by TAA. TNF is involved in the pathogenesis of various inflammatory liver diseases (Dong et al. 2016). Interlukin-6 was strongly associated with fatty liver disease and it was highly specific in a diagnose of non-alcoholic steatohepatitis (Tarantino et al. 2009). It has been affirmed that serum TNF and IL-6 levels are increased in patients with acute or chronic hepatitis infection (Knobler and Schattner 2005; Xia et al. 2015). Interleukin (IL)-1β and tumor necrosis factoralpha production were increased in HL-60 cells under zinc deficiency assuming an important role of zinc in proinflammatory cytokine regulation, this should encourage research in the use of zinc supplementation for treatment of inflammatory diseases (Wessels et al. 2013). Since interlukin -6 dysregulates enzymatic antioxidant defenses (Mathy-Hartert et al. 2008), we can suggest that ZnO-NPs may protect hepatic or renal damage induced by TAA via reducing levels of IL-6 and lipid peroxidation.

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In conclusion, it is probable that ZnO-NPs can inhibit hepatic fibrosis or renal toxicity induced by TAA through lowering oxidative stress and inflammatory markers and improved the antioxidant status of the hepatic and renal tissues.

Conflict of interest: The authors have declared that no competing interests exist.

Acknowledgment The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at Al Imam Mohammad Ibn Saud Islamic University (IMSIU), for its funding of this research through the research Project No. 371212.

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Situnayake, R. D., Crump, B. J., Thurnham, D. I., Davies, J. A., Gearty, J.,and Davis, M. 1990. Lipid peroxidation and hepatic antioxidants in alcoholic liver disease. Gut, 31(11), 1311- 1317. PMID: 2253918. Smijs, T. G., and Pavel, S. 2011. Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness. Nanotechnol. Sci. Appl. 4(1): 95-112. PMID: 24198489. Sörensen-Zender, I., Bhayana, S., Susnik, N., Rolli, V., Batkai, S., Arpita, B., and Schiffer, M. 2015. Zinc-α2-glycoprotein exerts antifibrotic effects in kidney and heart. J. Am. Soc. Nephrol. 26(11): 2659–2668. PMID: 25788525. Suntres, Z. E., and Lui, E. M. 1990. Biochemical mechanism of metallothionein-carbon tetrachloride interaction in vitro. Biochem. Pharmacol. 39(5): 833-840. PMID: 2310409. Szuster-Ciesielska, A., Plewka, K., Daniluk, J., and Kandefer-Szerszeń, M. 2009. Zinc supplementation attenuates ethanol-and acetaldehyde-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS) production and by influencing intracellular signaling. Biochem. Pharmacol. 78(3): 301-314. PMID: 19376089. Taccola, L., Raffa, V., Riggio, C., Vittorio, O., Iorio, M. C., Vanacore, R. and Cuschieri, A. 2011. Zinc oxide nanoparticles as selective killers of proliferating cells. Int. J. Nanomed. 6: 1129-1140. PMID: 21698081. Tarantino, G., Conca, P., Pasanisi ,F., Ariello, M., Mastrolia, M., Arena, A., Tarantino, M., Scopacasa. F., Vecchione, R.2009.Could inflammatory markers help diagnose nonalcoholic Steatohepatities? .Eur. J. Gastroenterol. Hepatol. 21(5):504-11.PMID: 19318968

Torabi, M., Kesmati, M., Harooni, H. E., and Varzi, H. N. 2013. Effects of nano and 20


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Conventional zinc oxide on anxiety-like behavior in male rats. Indian J. pharmacol. 45(5): 508-512. PMID: 24130388. Valko, M., Morris, H., and Cronin, M. T. 2005. Metals, toxicity and oxidative stress. Curr. Med. Chem. 12(10): 1161-1208. PMID: 15892631. Wang, B., Feng, W., Wang, M., Wang, T., Gu, Y., Zhu, M., and Chai, Z. 2008. Acute toxicological impact of nano-and submicro-scaled zinc oxide powder on healthy adult mice. J. Nanopart. Res. 10(2): 263-276. springer.com/article/10.1007/s11051-007-9245-3. Wessels, I., Haase, H., Engelhardt, G., Rink, L., Uciechowski, P. 2013. Zinc deficiency Induces production of the proinflammatory cytokines IL-1β and TNFα in promyeloid cells via epigenetic and redox-dependent mechanisms. J. Nutr. Biochem. 24 (1) 289–297. PMID : 22902331. Wong-Ekkabut, J., Xu, Z., Triampo, W., Tang, I. M., Tieleman, D. P., and Monticelli, L. 2007. Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study. Biophys. J . 93(12): 4225-4236. PMID: 17766354. Wynn, T. A. 2008. Cellular and molecular mechanisms of fibrosis. J pathol. 214(2): 199-210. PMID: 18161745. Xia, C., Liu, Y., Chen, Z. and Zheng`, M. 2015. Involvement of interleukin 6 in hepatitis B viral infection. Cell Physiol. Biochem. 37(2): 677-686. PMID: 26343270. Yogalakshmi, B., Viswanathan, P., and Anuradha, C. V. 2010. Investigation of antioxidant, anti- inflammatory and DNA-protective properties of eugenol in thioacetamide-induced liver injury in rats. Toxicology, 268(3): 204-212. PMID: 20036707.

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Legends Fig.1.Effect of zinc oxide nano particles on plasma interleukin 6 of rats treated with TAA L: Low dose of ZnONPs,M : Medium dose of ZnONPs,H : High dose of ZnONPs *: Significant difference compared to control at P≤ 0.01 @: Significant difference compared to TAA at P≤ 0.01

Fig.2.Effect of zinc oxide nano particles on plasma tumor necrosis factor-alpha of rats treated .

with TAA L: Low dose of ZnONPs,M : Medium dose of ZnONPs,H : High dose of ZnONPs * : Significant difference compared to control at P≤ 0.01. @: Significant difference compared to TAA at P≤ 0.01.

Fig.3. Effect of zinc oxide nano particles on hepatic hydroxyproline of rats treated with TAA L: Low dose of ZnONPs,M : Medium dose of ZnONPs,H : High dose of ZnONPs * : Significant difference compared to control at P≤ 0.01. @: Significant difference compared to TAA at P≤ 0.01.

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Fig.4. A. Photomicrograph of a section of a liver from the control group showing normal histological structure of the central vein (CV) and surrounding hepatocytes (H) and round nuclei (N). B. Photomicrograph of a section of a liver from TAA showing disruption of the architecture of hepatic tissues and collagen fibers deposition around central vein and portal tract, and pseudolobulation of hepatocytes with fibroblasts (arrow), widely of the hepatocytes associated with degeneration and necrosis (star), with pyknotic nuclei (arrowhead) C. Photomicrograph of a section of a liver from TAA and nano zinc low dose showing moderated improvement in liver tissues, with necrosis (star), with pyknotic nuclei (arrowhead) in the hepatocytes. Also, strands of collagen fibers with inflammatory cells infiltration in portal tract and central vein area (thin arrow). D. Photomicrograph of a section of a liver from TAA and nano zinc medium dose group showing the liver sections of the liver exhibited apparent nearly normal hepatic parenchyma with only a few tiny of collagen fibers and inflammatory cells infiltration (thin arrow). Necrosis (star), with pyknotic nuclei (arrowhead) in the hepatocytes was also noticed. E. Photomicrograph of a section of a liver from TAA and nano zinc high dose showing marked regeneration of the hepatocytes with preservation of the normal hepatic architecture, minimal of necrosis (star) with pyknotic nuclei (arrowhead), inflammatory cells infiltration and thin fibrotic septa (thin arrow). (H and E, X 400).

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Fig. 5.A. Photomicrograph of the kidney of control rat showing normal architecture of renal tubular epithelial cells (T) and glomeruli (G) and urinary spaces (Us). B. Photomicrograph of the kidney of rat treated with TAA showing glomerular shrinkage (G), wide urinary space, (US), degeneration vacuolation (V), hyaline cast (H) in the renal epithelia tubular, and interstitial hemorrhage, panoptic (star) and apoptotic nuclei (arrowhead) were observed C. Photomicrograph of the kidney of rat treated with TAA and low dose of nano zinc showing mild degeneration in the renal epithelia tubular (T) and glomerui (G), mildly wide urinary space (US), and hemorrhage between tubules (H). D. Photomicrograph of the kidney of rat treated with TAA and median dose of nano zinc showing mild degeneration the renal epithelia tubular (T) and with pyknotic nuclei (arrowhead) E. Photomicrograph of the kidney of rat treated with TAA and high dose of nano zinc showing, generally intact tubular epithelial cells and glomeruli appear normal with mild degeneration some renal epithelia tubular (T) and glomeruli (G). (H and E, X 400)

Masson trichrome staining Fig.6. A. Photomicrograph of a section of a liver from the control group showing minimal of collagen fibers B. Photomicrograph of a section of a liver from TAA showing thick collagen fibers were detected in the liver tissues were extended from portal to central area with formation pseudolobulation of hepatocytes C. Photomicrograph of a section of a liver from TAA and nano zinc low dose showing decreased collagen fibers and were exist around portal and central vein area

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D. Photomicrograph of a section of a liver from TAA and nano zinc medium dose group showing remarkably decreased of collagen fibers E. Photomicrograph of a section of a liver from TAA and nano zinc high dose showing remarkably decreased of collagen fibers as compared with TAA group (Masson trichrome staining X 400)

Immunohistochemical staining of α-SMA Fig.7. A. Photomicrograph of a section of a liver from the control group showing weakly satin in the portal and central vein area B. Photomicrograph of a section of a liver from TAA showing marked increase in the number of the brown granules in portal area and central area owing to the proliferated fibrous tissues and fibrous septa C. Photomicrograph of a section of a liver from TAA and nano zinc low dose showing moderately brown granules in portal area and central area D. Photomicrograph of a section of a liver from TAA and nano zinc medium dose group showing mild brown granules in portal area and central area E. Photomicrograph of a section of a liver from TAA and nano zinc high dose showing mild brown granules in portal area and central area. (α-SMA stain, X 400)

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Table1.Effect of nano zinc oxide on Hepatic and renal oxidative stress markers of rats treated with thioacetamide Parameter

Organ

MDA(nmol /mg)

Liver

Control 4.70±0.14

TAA 10.19±0.28*

TAA+ZnONP- L 7.94±0.14*@

TAA+ZnONP- M 5.64±0.15*@

TAA+ZnONP- H 6.26±0.13*@

Kidney

7.63±0.58

18.36±1.19*

14.12±0.37*@

9.75 ±0.45*@

11.13±0.39*@

Liver

6.73±0.19

3.56±0.14*

4.80±0.11*@

5.46±0.26*@

5.24±0.12*@

Kidney

3.53±0.15

1.74±0.11*

2.58±0.08*@

3.01±0.11*@

3.00±0.19*@

Liver

12.37±0.71

5.87±0.21*

7.25±0.23*@

10.00 ±0.31*@

8.62±0.25*@

Kidney

6.38±0.25

3.06±0.22*

3.94 ±0.21*@

5.00±0.25*@

4.62±0.30*@

Liver

222.13±4.96

155.38±1.82* 175.50±2.52*@

216.00±4.57@

207.95±6.24@

Kidney

48.75±1.40

19.50±0.53*

48.00±0.81@

43.13±2.11@

GSH(mg/g tissue)

CAT(U/g tissue)

SOD(U/g tissue)

Treatment

34.12±1.22*@

Each value is the mean ±SE ,N=8 *Significant difference compared to control P≤0.01 @Significant difference compared to TAA P≤0.01 TAA : Thioactamide , ZnONP- L:Nano zinc oxide low dose , ZnONP- M: Nano zinc oxide medium dose ZnONP- H: Nano zinc oxide high dose MDA : Malondialdehyde , GSH : Reduced glutathione , CAT : Catalase , SOD: Superoxide dismutase


Page 28 of 36


Page 29 of 36

" (( +,- .

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7.41± 0.33*

) !"# $ 6.58 ±0.08*@

) !"#% 5.98±0.22@

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29.63±1.58

66.13±1.61*

48.50±0.71*@

35.13±1.17*@

44.75±1.66*@

53.13±1.59

144.25±2.39*

89.38±1.42*@

69.36±0.97*@

95.38±1.93*@

2 72±0.01

06±2 003

0 56±2 2134

0 0±2 2134

0 10±2 2134

0 28±2 293

2 70±2 2 34

2 57±2 2 34

2 11±2 2034

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67 90±0 :834

0 75±2 29

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5 75±2 05

6 92±2 0:3

9 :6±2 0:34

5 10±2 014

9 9:±2 034

2 90±2 2 8 9±0 05

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Fig.1.

J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS LIBRARY on 08/2 ly. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official Page 31 of 36

Fig.2.

J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS LIBRARY on 08/2 ly. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official

Page 32 of 36

Fig.3.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS LIBRARY on 08/20/17 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 33 of 36

Fig.4

146x165mm (600 x 600 DPI)


Page 34 of 36

Fig.5

130x147mm (600 x 600 DPI)

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MASSACHUSETTS LIBRARY on 08/20/17 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 35 of 36

Fig.6

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Page 36 of 36

Fig.7

146x165mm (600 x 600 DPI)

Erratum to: Role of quercetin and arginine in ameliorating nano zinc oxide-induced nephrotoxicity in rats.

Curcumin Attenuates Hepatotoxicity Induced by Zinc Oxide Nanoparticles in Rats.

Retinopathy Induced by Zinc Oxide Nanoparticles in Rats Assessed by Micro-computed Tomography and Histopathology.

Picroliv affords protection against thioacetamide-induced hepatic damage in rats.

Modulation of thioacetamide-induced hepatic inflammations, angiogenesis and fibrosis by andrographolide in mice.

Protection of rats from thioacetamide-induced hepatic fibrosis by the extracts of a traditional Uighur medicine Cichorium glandulosum.

Effect of oral administration of ethanolic extract of Vitex negundo on thioacetamide-induced nephrotoxicity in rats.

Prenatal development toxicity study of zinc oxide nanoparticles in rats.

The role of hypoxia-inducible factor-1α in zinc oxide nanoparticle-induced nephrotoxicity in vitro and in vivo.

Protective effect of olive and juniper leaves extracts on nephrotoxicity induced by thioacetamide in male mice.

Lactoferrin Enhanced Apoptosis and Protected Against Thioacetamide-Induced Liver Fibrosis in Rats.

Taurine release from brain slices in thioacetamide-induced hepatic encephalopathy in rats.

Antidiabetic activity of zinc oxide and silver nanoparticles on streptozotocin-induced diabetic rats.

Enhanced GABA release in cerebral cortical slices derived from rats with thioacetamide-induced hepatic encephalopathy.

Nitric oxide ameliorates zinc oxide nanoparticles-induced phytotoxicity in rice seedlings.

Anti-fibrotic Role of miR-214 in Thioacetamide-induced Liver Cirrhosis in Rats.

Effect of zinc oxide nanoparticles on dams and embryo-fetal development in rats.

Protective effect of zinc-induced metallothionein synthesis on gentamicin nephrotoxicity in rats.

Rapid degradation of zinc oxide nanoparticles by phosphate ions.

Neurotoxicity induced by zinc oxide nanoparticles: age-related differences and interaction.

Evaluation of 2-week repeated oral dose toxicity of 100 nm zinc oxide nanoparticles in rats.

Citrulline uptake in rat cerebral cortex slices: modulation by Thioacetamide -Induced hepatic failure.

Anticoccidial and antioxidant activities of zinc oxide nanoparticles on Eimeria papillata-induced infection in the jejunum.

Genotoxic effects of zinc oxide nanoparticles.