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Ultrastructural Pathology

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Suppression of acetaminophen-inducedhepatocyte ultrastructural alterations in rats usinga combination of resveratrol and quercetin

Suliman Al Humayed, Bahjat Al-Ani, Abbas O. El Karib, Abdullah S. Shatoor,Refaat A. Eid, Shahid Aziz, Javed I. Wani & Mohamed A. Haidara

To cite this article: Suliman Al Humayed, Bahjat Al-Ani, Abbas O. El Karib, Abdullah S. Shatoor,Refaat A. Eid, Shahid Aziz, Javed I. Wani & Mohamed A. Haidara (2019): Suppression ofacetaminophen-induced hepatocyte ultrastructural alterations in rats using a combination ofresveratrol and quercetin, Ultrastructural Pathology, DOI: 10.1080/01913123.2019.1680585

To link to this article: https://doi.org/10.1080/01913123.2019.1680585

Published online: 19 Oct 2019.

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Suppression of acetaminophen-induced hepatocyte ultrastructural alterations inrats using a combination of resveratrol and quercetinSuliman Al Humayeda, Bahjat Al-Ani b, Abbas O. El Karibb, Abdullah S. Shatoora, Refaat A. Eidc, Shahid Aziza,Javed I. Wania, and Mohamed A. Haidarab,d

aDepartment of Internal Medicine, College of Medicine, King Khalid University, Abha, Saudi Arabia; bDepartment of Physiology, College ofMedicine, King Khalid University, Abha, Saudi Arabia; cDepartments of Pathology, College of Medicine, King Khalid University, Abha, SaudiArabia; dPhysiology Department, Kasr Al-Aini Faculty of Medicine, Cairo University, Giza, Egypt

ABSTRACTIngestion of a toxic dose of the analgesic drug, acetaminophen (also called paracetamol or APAP),is among the most common causes of acute liver injury in humans. We tested the hypothesis thatthe combined polyphenolic compounds, resveratrol (RES) and quercetin (QUR), can substantiallyprotect against hepatocyte ultrastructural damage induced by a toxic dose of APAP in a rat modelof APAP-induced acute liver injury. The model group of rats received a single dose of APAP (2 g/kg), whereas the protective group of rats was pretreated for 7 days with combined doses of RES(30 mg/kg) and QUR (50 mg/kg) before being given a single dose of APAP. All rats were thensacrificed 24 hours post APAP ingestion. Harvested liver tissues were prepared for transmissionelectron microscopy (TEM) staining, and liver homogenates were assayed for biomarkers ofinflammation, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and oxidativestress, such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase(GPx). In addition, blood samples were assayed for the liver injury enzyme alanine aminotransfer-ase (ALT) as an indicator of liver damage. TEM images showed that APAP overdose induced acuteliver injury as demonstrated by profound hepatocyte ultrastructural alterations, which weresubstantially protected by RES+QUR. In addition, APAP significantly (p < 0.05) modulated TNF-α,IL-6, MDA, SOD, GPx, and ALT biomarkers, which were completely protected by RES+QUR. Thus,RES+QUR effectively protects against APAP-induced acute liver injury in rats, possibly via theinhibition of inflammation and oxidative stress.

ARTICLE HISTORYReceived 12 January 2019Revised 8 October 2019Accepted 10 October 2019

KEYWORDSHepatocyte ultrastructure;acetaminophen; resveratrol;quercetin; animal model

Introduction

Acetaminophen (APAP)-induced hepatotoxicity iscommon in both humans and experimental animalmodels after an accidental or intentional ingestion ofan overdose of the drug.1 It is themost common agentof intentional self-harm andAPAP poisoning claimedthe life of 284 persons aged 12 years and over between1993 and 1996 in England and Wales, UK.2 In addi-tion, about 50% of acute liver failure admitted cases inthe United States of America are caused by APAPpoisoning.3,4 APAP is metabolized in the liver, andhepatotoxic metabolites that represent about 10% ofthe whole metabolites are rapidly inactivated by glu-tathione (GSH) to protect the hepatocytes.5 But, withthe drug overdose, for example, the elevated levels ofliver toxic metabolites, mainly N-acetyl-p-benzoqui-noimine (NAPQI), rapidly deplete GSH and

covalently modify cellular proteins, leading to thegeneration of high levels of reactive oxygen species(ROS) and depletion of the ATP, which results inmitochondrial damage and hepatocyte injury.6

However, it was postulated that depletion of 90% ofGSH in hepatocytes is critically necessary for thedevelopment of cell necrosis.7 In addition, hepaticinflammatory cytokines are also reported to beinvolved in APAP-induced liver injury.8

Quercetin and resveratrol are polyphenolic anti-oxidants found in fruits, vegetables, and grains.9,10

They have been widely known to have potent cardi-ovascular protective and therapeutic effects viascavenging ROS11 and have anti-inflammatoryeffects12,13, inhibit lipid peroxidation14, inhibit plate-let aggregation and thrombus formation15, preventapoptosis, and promote cell survival16,17 and liverprotection against hepatic sinusoidal obstruction

CONTACT Mohamed A. Haidara haidaram@hotmail.com Department of Physiology, College of Medicine, King Khalid University, Abha 61421, SaudiArabia

ULTRASTRUCTURAL PATHOLOGYhttps://doi.org/10.1080/01913123.2019.1680585

© 2019 Taylor & Francis Group, LLC

and hepatic steatosis.18,19 In addition, both quercetinand resveratrol were reported to inhibit APAP-induced ROS levels in liver tissue homogenates andthe blood of rats and mice.20,21 However, neitherquercetin nor a combination of quercetin and resver-atrol has been used before to study the protection ofhepatocyte ultrastructure upon acetaminophenintoxication in an animal model. Therefore, thisstudy was designed to investigate the degree of pro-tection by resveratrol and quercetin given in combi-nation against APAP intoxication to the hepatocyteultrastructure and compare it with the level of pro-tection provided by these agents to known liverinjury biomarkers.

Methods

Reagents and assay kits

Quercetin (C15H10O7, CAS Number 117-39-5) waspurchased from Sigma-Aldrich (St. Louis, MO,USA) and was prepared daily and freshly by dissol-ving in a normal saline solution (0.9% NaCl) to thefinal concentration of 50 mg/ml. Resveratrol(C14H12O3, Cat No. R5010) was also purchasedfrom Sigma-Aldrich (St. Louis, MO, USA) and wasprepared daily and freshly by dissolving in a salinesolution (0.9%NaCl) containing 20% hydroxypropylcyclodextrin (American Maize-Products Co.,Hammond, IN, USA) to a final concentration of30 mg/kg. Assay kits for the determination of mal-ondialdehyde (MDA, Cat No. NWK-MDA01) werepurchased from NWLSS (Vancouver, BC, Canada).Superoxide dismutase (SOD) assay kit was pur-chased from Cayman Chemical, Cat. No.706002.ELISA kits for the determination of the levels ofinterleukin (IL)-6 (Cat No. ELR-IL6-001) were pur-chased from RayBio, GA, USA. ELISA kits for thedetermination of tumor necrosis factor-alpha (TNF-α) (Cat No. ab46070) were purchased from Abcam,Cambridge, UK. Assay kits for the determination ofserum levels of alanine aminotransferase (ALT) werepurchased from Human Co., Germany.

Animals

All experimental procedures were approved by themedical research ethical committee at King KhalidUniversity and according to the Guide for the Care

and Use of Laboratory Animals published by theUS National Institutes of Health (NIH publicationNo.85-23, revised 1996). Sprague Dawley rats (n =18) weighing 170–200 g were used in this study.All rats were bred and housed in the researchcenter of King Khalid University, College ofMedicine (Abha, Saudi Arabia), at a temperatureof 23 ± 1°C and a 12 h light:12 h dark cycle. Ratshad free access to tap water and fed standardlaboratory chow during the acclimatization period.

Experimental design

After a one-week adaptation period, rats were ran-domly assigned into three groups (n= 6; each) andwere distributed in their corresponding cages and clas-sified as follows: (1) Control group: rats received nor-mal saline daily for 7 days; (2) APAP-intoxicated group(Model group): rats received normal saline for 7 con-secutive days and thengivena single dose ofAPAP (2 g/kg, orally)22; and (3) RES+QUR+APAP group: ratswere pretreated with RES (30 mg/kg)23 and 50 mg/kgQUR24 for 7 consecutive days and then administeredwith a single dose of APAP (2 g/kg, orally). APAP wasadministered to the desired groups one hour after thelast dose of treatment on day 7, and all treatment in allgroups was administered i.p. in a final volume of 1 ml.All animals were sacrificed on day 8.

Biochemical measurements

Blood samplesAt the end of the experimental period, blood sam-ples were collected by cardiac puncture underanesthesia (sodium thiopentone at 40 mg/kgbody weight) after an overnight fast of 12 hours.These blood samples were collected without anyanticoagulant, left for 10 min, and then centri-fuged for 10 minutes at 4,000 r/min to obtainserum, which was stored at −20°C until furtherbiochemical analysis for determination of serumliver injury enzyme ALT.

Preparation of liver homogenatesAs we previously reported25, part of the liversobtained from the rats of all groups were freshlywashed with phosphate-buffered saline (PBS), pH7.4. Then, they were homogenized with an ultrasonichomogenizer in cold phosphate buffer, pH 7.4,

2 S. AL HUMAYED ET AL.

containing ethylene-diamine-tetra-acetic acid(EDTA). The supernatant obtained from each ratwas aliquot in separate tubes and stored at – 70°Cfor later determination of levels of TNF-α, IL-6, MA,SOD, and glutathione peroxidase (GPx) according tomanufacturer’s instructions.

Transmission electron microscopy (TEM)As we previously reported26, small pieces of 1 mm3

of the liver were fixed at 4°C in 4% bufferedglutaraldehyde (SERVA, Frankfurt, Germany)with 0.2 M cacodylate buffers (TAAB essentialfor microscopy, Aldermaston, Berks, UK) and pro-cessed for electron microscopic examination.Contrasted ultrathin sections with uranyl acetateand lead citrate (LobaChemie Pvt. Ltd., Colaba,Mumbai, India) were examined and photographedusing the Philips EM 208S transmission electronmicroscope (FEI Company, Eindhoven, TheNetherlands) at accelerating voltage of 80 kV.

Determination of serum levels of ALT and liverhomogenate levels of TNF-α¸ IL-6, MDA, SOD, andGPxOn day 8, animals were sacrificed, and liver func-tion was evaluated by assessing serum levels ofliver injury enzyme ALT using ELISA kits accord-ing to the manufacturer’s instructions. Tissuelevels of TNF-α, IL-6, MDA, SOD, and GPx weredetermined using ELISA kits according to themanufacturer’s instructions.

Statistical analysis

The data were expressed as mean ± standard devia-tion (SD). Data were processed and analyzed usingthe SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA).One-way ANOVA was done followed by Tukey’spost hoc test. Pearson correlation statistical analysiswas done for the detection of a probable significancebetween two different parameters. Results were con-sidered significant if p ≤ 0.05.

Results

Induction of acute liver injury in rats by APAP

We induced the disease in the model group of ratsby APAP overdose (2 g/kg body weight), which

was confirmed after 8 days as shown by high bloodand liver tissue levels in biomarkers of liver injuryand a profound damage to hepatocytes (Figure 1).APAP induced hepatotoxicity which caused abouta fivefold increase in ALT (Figure 1a) anda twofold decrease in the levels of the antioxidantSOD (Figure 1b), and TEM images of liver sec-tions (Figure 1c,d) confirmed liver injury andabnormal changes to hepatocytic ultrastructure.Representative TEM image (5,000×) of liver sec-tions obtained from the model group (Figure 1d)shows altered hepatocyte ultrastructure and con-densation of chromatin masses with no nucleolus,and cytoplasm displays many lipid droplets, swol-len mitochondria, dilation of endoplasmic reticu-lum, and damaged intercellular space. However,a TEM image of another hepatocyte at similarmagnification prepared from liver sections of thecontrol group (Figure 1c) shows an unremarkablehepatocyte, with centrally placed nuclei andcrowded cytoplasm with organelles, particularlyrough endoplasmic reticulum and mitochondria.

Resveratrol plus quercetin protects hepatocyteultrastructural alterations induced by APAP

To determine whether giving two antioxidantstogether can work to completely protect the ultra-structure of hepatocytes against damage induced byAPAP, one group of rats was pretreated for 7 dayswith RES+QUR and then administered with a singledose of APAP (Figure 2). Representative TEMimages (5,000×) of liver sections display hepatocyteswith normal architecture in the control animal group(Figure 2a,b) surrounded by an intact intercellularspace. It shows round centrally located nuclei with atleast one nucleolus and intact nuclear memebrane,and the cytoplasm displays many organelles, parti-cularly rough endoplasmic reticulum andmitochon-dria. Capillary sinusoid with Kupffer cell anderythrocytes were also seen. TEM images that repre-sent liver sections of APAP-treated rats (Figure 2c,d)display pyknotic nuclei with nuclear disaggregationof granular elements, mitochondrial swelling, lyso-somes, dilation of endoplasmic reticulum, and cyto-plasmic steatosis (lipid droplets). Also, damagedintercellular space and pleomorphic capillary sinu-soid with Kupffer cell and erythrocytes were alsoseen. Treatment of APAP rats with RES+QUR

ULTRASTRUCTURAL PATHOLOGY 3

(Figure 2e,f) significantly protected the hepatocellu-lar architecture of rats as demonstrated by normalnuclei and cytoplasmic compartments. However,very few cytoplasmic vacuoles can be seen.

Resveratrol plus quercetin protects themodulation of liver injury enzyme and oxidative,antioxidant, and inflammatory biomarkersinduced by APAP in the blood and liver tissues

Tissue inflammation, oxidative stress, and augmen-tation of ALT are known to be involved in thepathology of acute liver injury in animal modelsand humans.27 To investigate the level of inhibitionof modulation of these biomarkers by combinedinjections of resveratrol and quercetin for 7 daysprior to APAP hepatic intoxication, we measuredALT, MDA, SOD, GPx, TNF-α, and IL-6 in all ratgroups (Figures 3 and 4). As shown in Figure 3a–d,

acute liver injury induced by APAP significantly(p < 0.05) modulated MDA, SOD, GPx, and ALT,which were completely protected by RES+QUR.

In addition, APAP significantly (p < 0.05)increased liver tissue levels of TNF-α and IL-6compared to the control group, which were alsocompletely inhibited by RES+QUR (Figure 4a,b).

Discussion

In this report, we investigated the status of hepa-tocytic ultrastructure in APAP-induced acute liverinjury in a rat model of the disease in the presenceand absence of resveratrol plus quercetin. Also,our protective approach using both agents wasalso used to assess levels of oxidative, inflamma-tory, and liver injury biomarkers. Therefore, ratswere pretreated for 7 days with resveratrol plusquercetin prior to the induction of acute liver

Figure 1. Induction of acute liver injury in rats by APAP.Blood levels of ALT (a) and liver tissue levels of SOD (b) were measured one day post oral ingestion of rats (n = 6) with APAP (2 gm/kg; model group) compared to vehicle-ingested control group (n = 6). Results represent the mean (±SD), and experiments wereperformed in triplicate. *p < 0.0001 versus control. TEM images (5,000×) of harvested tissues obtained from the liver of the modelgroup (d) compared to the control group (c) rats are visualized using transmission electron microscopy. There are many fat dropletsand damaged mitochondria and rough endoplasmic reticulum in the hepatocyte obtained from the liver of the model group (APAP).Arrows point to the plasma membrane surrounding the hepatocyte. Abbreviations: N, nucleus; nu, nucleolus; L, lipid droplet; m,mitochondria; RER, rough endoplasmic reticulum; ne, nuclear envelop; chr, chromatin.

4 S. AL HUMAYED ET AL.

injury by APAP overdose. Here, we report theability of resveratrol plus quercetin to markedlyinhibit hepatocyte ultrastructural alterations andcompletely inhibit the induction of biomarkers ofinflammation, oxidative stress, and liver injury inAPAP-induced acute hepatotoxicity in rats.

Our conclusions are supported by the data indi-cating that APAP caused a profound damage tothe hepatocyte ultrastructure (Figures 1d and 2c,d)and induced biomarkers of liver injury, ALT

(Figure 1a); oxidative stress, MDA (Figure 3a);and inflammation, TNF-α and IL-6 (Figure 4a,b),which were substantially protected by RES+QUR(Figures 2–4). Also, our data demonstrate thatAPAP caused an inhibition of the antioxidants,SOD and GPx, which were significantly augmen-ted by RES+QUR to control levels (Figure 3b,c).Our results were thus consistent with our workinghypothesis that the combined polyphenolic com-pounds, resveratrol (RES) and quercetin (QUR),

Figure 2. Resveratrol and quercetin protect against APAP-induced hepatocyte ultrastructural damage in rats.TEM images (5,000×) of the liver obtained at the end of the experiment, one day post APAP ingestion in different groups of ratsused in this study; Control group (a and b), APAP group (c and d), and RES+QUR+APAP group (e and f). Most hepatocytes in animalsintoxicated with APAP had evidence of steatosis, shrinkage of nuclei with dense nuclear chromatin and swollen mitochondria. Bycomparison, rats pretreated with RES+QUR showed minimal cytoplasmic and nuclear changes. Note that arrows point to intercellularspaces between hepatocytes. Abbreviations: N, nucleus; nu, nucleolus; L, lipid droplet; V, vacuoles; m, mitochondria; RER, roughendoplasmic reticulum; ne, nuclear envelop; chr, chromatin; S, capillary sinusoid; K, Kupffer cell; R, erythrocytes.

ULTRASTRUCTURAL PATHOLOGY 5

Figure 3. Resveratrol and quercetin protect against APAP-induced modulation of MDA, SOD, GPx, and ALT biomarkers in rats.Liver homogenate levels of MDA (a), SOD (b), and GPx (c) and blood levels of ALT (d) were measured at the end of the experiment,one day post APAP ingestion in different groups of rats used in this study; Control group, APAP group, and RES+QUR+APAP group.Results represent the mean (±SD); n = 6 for each group. Experiments were performed in triplicate. *p < 0.05 versus control, **p <0.05 versus APAP.

Figure 4. Resveratrol and quercetin inhibit APAP-induced TNF-α and IL-6 biomarkers in rats.Liver homogenate levels of TNF-α (a) and IL-6 (b) were measured at the end of the experiment, one day post APAP ingestion indifferent groups of rats used in this study; Control group, APAP group, and RES+QUR+APAP group. Results represent the mean(±SD); n = 6 for each group. Experiments were performed in triplicate. *p < 0.05 versus control, **p < 0.05 versus APAP.

6 S. AL HUMAYED ET AL.

can substantially protect against hepatocyte ultra-structural damage caused by a toxic dose of APAPin a rat model of APAP-induced acute liver injury.

Promising therapeutic effects of quercetin andresveratrol on hepatotoxicity-induced acute andchronic liver injuries via the inhibition of oxidativeand nitrosative stress, inflammation, apoptosis, andNF-kB pathways were reported28-30, which are inagreement with our findings shown in Figures 3and 4 and our recent report.31 However, conflictingdata on the protective/treatment effects of resvera-trol alone against APAP-induced acute liver injury inrats have been reported.32,33 A recently publishedwork32 reported significant inhibition of liver iNOSimmunostaining and hepatocyte ultrastructuredamage by RES (10 mg/kg) given 20 minutes postAPAP (1 g/kg) injection in rats. However, adminis-tering triple-dose RES (30 mg/kg) at the same timewith APAP (1 g/kg) to rats caused a weak inhibitionof blood ALT and no effect on the ALT levels incultured hepatocytes prepared from these animals.33

In addition, the same group33 found no significantpathological changes in the liver tissue of the modelgroup (APAP) stained with H&E. However, ourH&E images demonstrated substantial liver damageinduced by APAP (data not shown). Our treatmentprotocol (2 g/kg APAP) versus their treatment pro-tocol (1 g/kg APAP) could be the reason for suchdifferences.

Finally, our biochemical data that showed RES+QUR completely inhibited APAP-induced biomar-kers of liver injury, inflammation, and oxidativestress are almost in agreement with our TEM imagesthat showed a substantial protection of hepatocyteultrastructure, which points to the importance ofelectron microscopy as a tool besides the biochem-ical investigations to monitor the progress of acuteliver injury. Therefore, we conclude that pretreat-ment with resveratrol plus quercetin can effectivelyprotect against hepatocyte ultrastructural alterationsand inhibits inflammatory and oxidative stress bio-markers in a rat model of APAP overdose-inducedhepatotoxicity.

Acknowledgments

The authors extend their appreciation to the Deanship ofScientific Research at King Khalid University for funding thiswork through the Research Group Project under grant number

(R.G.P1./100/40). Also, the authors would like to thankDr Mariam Al-Ani from Dental Care Partnership, SuttonColdfield, Birmingham, UK, for proofreading the manuscript.

Disclosures

The authors declare no conflict of interest.

Funding

This work was supported by the King Khalid University[Grant number KKU-Project No. R.G.P1./100/40.].

ORCID

Bahjat Al-Ani http://orcid.org/0000-0001-5773-1160

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