RESEARCH Open Access

Histological alterations in the liver of rats inducedby different gold nanoparticle sizes, doses andexposure durationMohamed Anwar K Abdelhalim1* and Bashir M Jarrar2

Abstract

Background: Nanoparticles (NPs) can potentially cause adverse effects on organ, tissue, cellular, subcellular andprotein levels due to their unusual physicochemical properties. Advances in nanotechnology have identifiedpromising candidates for many biological and biomedical applications. Since the properties of NPs differ from thatof their bulk materials, they are being increasingly exploited for medical uses and other industrial applications. Theaim of the present study was to investigate the particle-size effect of gold nanoparticles (GNPs) on the hepatictissue in an attempt to cover and understand the toxicity and the potential threat of their therapeutic anddiagnostic use.

Methods: To investigate particle-size effect of GNPs on the hepatic tissue, a total of 70 healthy male Wistar-Kyotorats were exposed to GNPs received 50 or 100 ul of GNPs infusion of size (10, 20 and 50 nm for 3 or 7 days).

Results: In comparison with respective control rats, exposure to GNPs doses has produced alterations in thehepatocytes, portal triads and the sinusoids. The alterations in the hepatocytes were mainly summarized ashydropic degeneration, cloudy swelling, fatty degeneration, portal and lobular infiltrate by chronic inflammatorycells and congestive dilated central veins.

Conclusions: The induced histological alterations might be an indication of injured hepatocytes due to GNPstoxicity that became unable to deal with the accumulated residues resulting from metabolic and structuraldisturbances caused by these NPs. These alterations were size-dependent with smaller ones induced the mosteffects and related with time exposure of GNPs. The appearance of hepatocytes cytoplasmic degeneration andnuclear destruction may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interferingwith the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turnmay induce stress in the hepatocytes to undergo atrophy and necrosis. More histomorphologcal, histochemicaland ultrastrucural investigations are needed in relation of the application of GNPs with their potential threat as atherapeutic and diagnostic tool.

Keywords: gold nanoparticles, size, hepatic tissue, histology, hydropic degeneration, nanotoxicity, rats

IntroductionIn vivo studies in rats exposed to aerosols of GNPsrevealed that the NPs were rapidly taken into the systemwith the highest accumulation in the lungs, aorta, eso-phagus and olfactory bulb [1]. Moreover, particles ofnano-dimension are believed to be more biologically

reactive than their bulk counter parts due to their smallsize and larger surface area to volume ratio [1,2].Gold in its bulk form has long been considered an

inert, noble metal with some therapeutic and even med-icinal value hence GNPs are thought also to be relativelynon-cytotoxic [3]. Yet there are differing reports of theextent of the toxic nature of these particles owing to thedifferent modifications of the GNPs, surface functionalattachments and shape and diameter size of the nano-spheres [4,5]. Moreover, the metallic nature of the metal

* Correspondence: abdelhalimmak@yahoo.com1Department of Physics and Astronomy, College of Science, King SaudUniversity, Saudi ArabiaFull list of author information is available at the end of the article

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© 2012 Abdelhalim and Jarrar; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

derived NPs and the presence of transition metalsencourages the production of reactive oxygen species(ROS) leading to oxidative stress [6,7].Although some scientists consider NPs as nontoxic,

other studies reporting the toxic effects induced by NPs[8-10]. Although some NPs may appear to be nontoxic,other cellular mechanisms such as cell signaling andother normal cellular functions may be disrupted andare currently undergoing further investigation [11,12].The toxicity of NPs is being addressed by a number ofstandardized approaches with in vitro, in vivo as well asdetailed genomic or biodistribution studies [12].It has been shown that NPs may produce in vitro toxi-

city in some cell-based assays, but not in others. Thismay be a result of interference with the chemicalprobes, differences in the innate response of particularcell types, or other factors [13]. In addition, GNPs areused as carriers for the delivery of drugs and genes [14].The histological and the histochemical characteriza-

tion in the hepatic tissues due to GNPs have not yetbeen identified and documented. In the present study,an attempt has been made to characterize the possiblehistological alterations in the hepatic tissues following

intraperitoneal administration of GNPs and, if so,whether are related to the size of these NPs and thetime of exposure.The present study was carried out to investigate the

particle-size effect of GNPs on the hepatic tissue in anattempt to cover and understand the toxicity and poten-tial threat of their therapeutic and diagnostic use in rela-tion with the time of exposure.

Materials and methodsA total of 70 healthy male Wistar-Kyoto rats obtainedfrom the Laboratory Animal Center (College of Phar-macy, King Saud University, Saudi Arabia). The ratsnearly of the same age (12 weeks old) and weighing220-240 gm of King Saud University colony were used.Animals were randomly divided into groups, 12 GNPs-treated rats groups and one control group (NG).The 10, 20 and 50 nm GNPs were administered intra-

peritonealy in rats for periods of 3 or 7 days as shownin Table 1.The rats were maintained on standard laboratory

rodent diet pellets and were housed in humidity andtemperature-controlled ventilated cages on a 12 h day/

Table 1 The intraperitoneal administration of 10, 20 and 50 nm GNPs in rats for periods of 3 or 7 days

Groups Infusion dose, size and exposure duration of GNPs

Control received no gold nanoparticles (GNPs; n = 10)

G1A received infusion of 50 μl GNPs of size 10 nm for 3 days (n = 5)

G1B received infusion of 50 μl GNPs of size 10 nm for 7 days (n = 5)

G2A received infusion of 50 μl GNPs of size 20 nm for 3 days (n = 5)

G2B received infusion of 50 μl GNPs of size 20 nm for 7 days (n = 5)

G3A received infusion of 50 μl GNPs of size 50 nm for 3 days (n = 5)

G3B received infusion of 50 μl GNPs of size 50 nm for 7 days (n = 5)

G4A received infusion of 100 μl GNPs of size 10 nm for 3 days (n = 5)

G4B received infusion of 100 μl GNPs of size 10 nm for 7 days (n = 5)

G5A received infusion of 100 μl GNPs of size 20 nm for 3 days (n = 5)

G5B received infusion of 100 μl GNPs of size 20 nm for 7 days (n = 5)

G6A received infusion of 100 μl GNPs of size 50 nm for 3 days (n = 5)

G6B received infusion of 100 μl GNPs of size 50 nm for 7 days (n = 5)

40 x 1 40 x 3

Figure 1

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- 40 x 3 40 x 7

100 x 3

Figure 2

40 x 3 40 x 5

Figure 3

40 x 3 40 x 4

Figure 4

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night cycle. Two animals from each group were killedby dislocation of the neck at intervals of 3 and 7 days oftreatment with GNPs. All experiments were conductedin accordance with the guidelines approved by KingSaud University Local Animal Care and Use Committee.Fresh portions of the lateral lobes of the liver from

each rat were cut rapidly, fixed in neutral buffered for-malin (10%), then dehydrated, with grades of ethanol(70, 80, 90, 95 and 100%). Dehydration was then fol-lowed by clearing the samples in 2 changes of xylene.Samples were then impregnated with 2 changes of mol-ten paraffin wax, then embedded and blocked out. Par-affin sections (4-5 um) were stained with hematoxylin

40 x 2 Figure 5

(40 x 1)

40 x 10 40 x 8

(100 x 2)

Figure 6

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and eosin the conventional histological and stainaccording to Pearse [15]. Stained sections of control andtreated rats were examined for alterations in the archi-tecture, portal triads, hepatocytes, sinusoids and for thepresence of degeneration, necrosis, fatty change andportal fibrosis.

Results and discussionsSize and morphology of different gold nanoparticlesThe 10 and 20 nm GNPs show spherical shape whilethe 50 nm GNPs show hexagonal shape. The mean sizefor GNPs was calculated from the images taken by thetransmission electron microscope (TEM). The 10 nm

(40 x 1)

40 x 3 100 x 3

(40 x 9)

Figure 7

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GNPs was of mean size of 9.45 ± 1.33 nm, 20 nm GNPswas of mean size of 20.18 ± 1.80 and the 50 nm GNPswas of mean size of 50.73 ± 3.58. The high electrondensities of GNPs as well as the homogeneity of the par-ticles shape and size make them highly conspicuousunder the TEM. In addition, relatively simple methodscan be applied to obtain the populations of GNPs of dif-ferent average sizes which allow simultaneous detectionof several targets.

Histological AlterationsIn comparison with the control group, the following his-tological alterations were detected in the liver tissue ofGNPs-treated rats. These histological alterations were

observed in Figures 1,2,3,4,5,6,7,8,9,10,11 and 12 andcan be summarized as shown in Table 2.The cloudy swelling might be exhibited as a result of

disturbances of membranes function that lead to mas-sive influx of water and Na+ due to GNPS effects. Cellu-lar swelling might be accompanied by leakage oflysosomal hydrolytic enzymes that lead to cytoplasmicdegeneration and macromolecular crowding [16].Hydropic degeneration is a result of ion and fluid

homestasis that lead to an increase of intracellular water[17]. The vacuolated swelling of the cytoplasm of thehepatocytes of the GNPs treated rats might indicateacute and subacute liver injury induced by these NPs.Some studies indicate that nuclear polymorphism isseen in hepatic dysplasia and carcinomatous lesion [18].Binucleation represents a consequence of cell injury andis a sort of chromosomes hyperplasia which is usuallyseen in regenerating cells [19].Occasional inflammatory cells infiltration was seen in

the portal triads and the perioral zones of GNPs treatedrats. The infiltrate cells were mainly lymphocytes andplasma cells. This infiltration was more prominent after7 days of administration and in rats received 100 μlthan those received 50 μl. The appearance of inflamma-tory cells in the hepatic tissue may suggest that GNPscould interact with proteins and enzymes of the hepaticinterstitial tissue interfering with the antioxidant defensemechanism and leading to reactive oxygen species

100 x 1 100 x 4

100 x 2 100 x 3

Figure 8

40 x 4 Figure 9

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(ROS) generation which in turn may imitate an inflam-matory response [20].The sinusoidal Kupffer cells became prominent and

increased in number due to GNPs exposure. Thischange was more prominent at 10 nm GNPs with doseof 100 μl than 20 nm and 50 nm GNPS and more after7 days of administration than rats exposed to GNPs for3 days. Kupffer cells activation might indicate thatGNPs activate the phagocytic activity of the sinusoidalcells by increasing the number of Kupffte cells to helpin removing the accumulated GNPs where lysosomesare involved in the intracellular breakdown into smallmetabolic products. The produced Kupffer cells hyper-plasia might be correlated with the amount of injuriousto the hepatic tissue induced by GNPs intoxication and

represents a defense mechanism of detoxification. Kupf-fer cell hyperplasia is contributed to hepatic oxidativestress [21].Fatty change was observed in some swelling hepato-

cytes of rats exposed to 100 μl of 10 nm GNPs and to les-ser extent in the ones exposed to larger particles. Thishepatic liposis was more frequent in rat exposed to theGNPs for 7 days than those received the treatment for 3days. Hepatocytes fatty change might be due to lipid per-oxidation that leads to rough endoplasmic damage anddetachment of the cytoplasmic lipoprotein and indicateabnormal fat metabolism [22]. The seen hepatocytesabnormal retention of lipids in the present investigationinduced by GNPs might indicate toxic injury to liver inthe form of hepatocytes liposis by these particles.

100 x 1 40 x 2

Figure 10

40 x 7 100 x 8

40 x 8

Figure 11

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Sporadic spotty well-defined necrosis was noticed insome hepatocytes of GNPs treated rats. This alteration wasdetected in the liver of rats exposed to 10 nm size particlesand to lesser extent with 20 nm particles but was not seenwith those exposed to 50 nm size particles. Apoptic altera-tion might be followed organelles swelling specially mito-chondria, endoplasmic reticulum and rupture of lysosomeswhich might lead to amorphous eosinophilic cytoplasm asan initial sign in the sequence of hepatocytes necrosisbefore shrinking and dissolution of nulei [23]. The seenhepatocytes necrosis due to GNPs exposure might indicateoxidative stress on these cells by glutathione depletion.

ConclusionsHistological alterations by GNPs exposure as shown inthe results of the present work could be an indication of

injured hepatocytes due to GNPs toxicity that becomeunable to deal with the accumulated residues resultingfrom metabolic and structural disturbances caused bythese particles. One might conclude that these alterationsare size-dependent with smaller ones induced moredamage with relation with the time exposure of GNPs.The appearance of hepatocytes cytoplasmic degenera-

tion and nuclear destruction may suggest that GNPsinteract with proteins and enzymes of the hepatic tissueinterfering with the antioxidant defense mechanism andleading to reactive oxygen species (ROS) generationwhich in turn may induce stress in the hepatocytes toundergo atrophy and necrosis.More histomorphologcal, histochemical and ultrastru-

cural investigations are needed to correlate the biomedi-cal application of GNPs with the potential threat oftheir therapeutic and diagnostic use.

AcknowledgementsThe authors are very grateful to National Plan of Science and Technology (NPST).This research was financially supported by the National Science and TechnologyInnovation Plan (NSTIP), Research No. 08-ADV206-02 and Research No. 09-NAN670-02, College of Science, King Saud University, Saudi Arabia.

Author details1Department of Physics and Astronomy, College of Science, King SaudUniversity, Saudi Arabia. 2College of Applied Medical Sciences, Al-JoufUniversity, P.O. Box 2014, Skaka-Al-Jouf, Saudi Arabia.

Authors’ contributionsAMAK and JBM have analyzed data, interpreted and written the final draft ofthis manuscript. The animal model used in this study was obtained from the

100 x 2 Figure 12

Table 2 Histological alterations induced in GNPs-treated rats

Figure 1 GNPs-normal rat demonstrating benign looking hepatic lobule and hepatocytes surrounding a central vein

Figure 2 GNPs-treated rat received 50 μl of 10 nm particles for 3 days demonstrating: Congested central vein with portal infiltrated bychronic inflammatory cells (40 × 3); Liver section showed widely dilated congested central vein (40 × 7); necrotic hepatocytes (100 × 3)

Figure 3 GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating: Liver section widely dilated congested centralvein (40 × 3) with hepatocytes disarray and cloudy swelling of liver hepatocytes (40 × 5)

Figure 4 GNPs-treated rat received 50 μl of 20 nm particles for 3 days demonstrating: lobular infiltrate by chronic inflammatory cells (40× 3) congested hepatic sinusoids containing red blood cells (40 x4)

Figure 5 GNPs-treated rat received 50 μl of 10 nm particles for 7 days demonstrating dilated congested blood sinusoids (40 × 2)

Figure 6 GNPs-treated rat received 50 μl of 50 nm particles for 3 days demonstrating: Widely dilated congested central vein with dilatedcongested liver sinusoids with hepatocytes cloudy swelling (40 × 1); Fatty degeneration focally affecting the liver lobules (40 × 10) and(40 × 8); Fatty degeneration focally affecting the liver lobules (100 × 2)

Figure 7 GNPs-treated rat received 100 μl of 10 nm particles for 3 days demonstrating: Liver hepatocytes with lobular lymphocyticinfiltrate (40 × 1); prominent diffuse fatty degeneration (40 × 3) & (100 × 3); prominent diffuse fatty degeneration (40 × 3) & (100 × 3);congested dilated blood sinusoids (40 × 9); scattered fatty degeneration of liver hepatocytes with focus of inflammatory cells infiltrate ofliver lobules; diffuse fatty degeneration

Figure 8 GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating: Dilated congested blood vessels (100 × 1);congested blood sinusoids (40 × 4); fatty degeneration of liver hepatocytes (100 × 2); increased sinusoidal Kupffer cells (100 × 4)

Figure 9 GNPs-treated rat received 100 μl of 20 nm particles for 3 days demonstrating: Inflammatory cells infiltrate of liver lobules anddilated congested central vein (40 × 4)

Figure 10 GNPs-treated rat received 100 μl of 20 nm particles for 7 days demonstrating: Liver hepatocytes with binucleated cells (100 ×1); dilated congested central vein (40 × 2)

Figure 11 GNPs-treated rat received 100 μl of 50 nm particles for 3 days demonstrating: Scattered lobular inflammatory cells infiltrate (40× 7) and (100 × 8); scattered few cloudy swellings on (40 × 8)

Figure 12 GNPs-treated rat received 100 μl of 50 nm particles for 7 days demonstrating: No major pathological effect seen. Just few liverhepatocytes with binucleation or even trinuclei noticed; no other effects (100 × 2)

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Laboratory Animal Center (College of Pharmacy, King Saud University, SaudiArabia). AMAK has conceived the study and its design and obtainedresearch grants for this study. Moreover, both authors have read andapproved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 20 May 2011 Accepted: 25 January 2012Published: 25 January 2012

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doi:10.1186/1477-3155-10-5Cite this article as: Abdelhalim and Jarrar: Histological alterations in theliver of rats induced by different gold nanoparticle sizes, doses andexposure duration. Journal of Nanobiotechnology 2012 10:5.

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