Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration.

BACKGROUND: Nanoparticles (NPs) can potentially cause adverse effects on organ, tissue, cellular, subcellular and protein levels due to their unusual physicochemical properties. Advances in nanotechnology have identified promising candidates for many biological and biomedical applications. Since the properties of NPs differ from that of their bulk materials, they are being increasingly exploited for medical uses and other industrial applications. The aim of the present study was to investigate the particle-size effect of gold nanoparticles (GNPs) on the hepatic tissue in an attempt to cover and understand the toxicity and the potential threat of their therapeutic and diagnostic use.
METHODS: To investigate particle-size effect of GNPs on the hepatic tissue, a total of 70 healthy male Wistar-Kyoto rats 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 the hepatocytes, portal triads and the sinusoids. The alterations in the hepatocytes were mainly summarized as hydropic degeneration, cloudy swelling, fatty degeneration, portal and lobular infiltrate by chronic inflammatory cells and congestive dilated central veins.
CONCLUSIONS: The induced histological alterations might be an indication of injured hepatocytes due to GNPs toxicity that became unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs. These alterations were size-dependent with smaller ones induced the most effects and related with time exposure of GNPs. The appearance of hepatocytes cytoplasmic degeneration and nuclear destruction may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the hepatocytes to undergo atrophy and necrosis. More histomorphologcal, histochemical and ultrastrucural investigations are needed in relation of the application of GNPs with their potential threat as a therapeutic and diagnostic tool.

Introduction

In vivo studies in rats exposed to aerosols of GNPs revealed that the NPs were rapidly taken into the system with the highest accumulation in the lungs, aorta, esophagus and olfactory bulb [1]. Moreover, particles of nano-dimension are believed to be more biologically reactive than their bulk counter parts due to their small size 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 medicinal value hence GNPs are thought also to be relatively non-cytotoxic [3]. Yet there are differing reports of the extent of the toxic nature of these particles owing to the different modifications of the GNPs, surface functional attachments and shape and diameter size of the nanospheres [4,5]. Moreover, the metallic nature of the metal derived NPs and the presence of transition metals encourages 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 and other normal cellular functions may be disrupted and are currently undergoing further investigation [11,12]. The toxicity of NPs is being addressed by a number of standardized approaches with in vitro, in vivo as well as detailed genomic or biodistribution studies [12].

It has been shown that NPs may produce in vitro toxicity in some cell-based assays, but not in others. This may be a result of interference with the chemical probes, differences in the innate response of particular cell types, or other factors [13]. In addition, GNPs are used as carriers for the delivery of drugs and genes [14].

The histological and the histochemical characterization in the hepatic tissues due to GNPs have not yet been identified and documented. In the present study, an attempt has been made to characterize the possible histological alterations in the hepatic tissues following intraperitoneal administration of GNPs and, if so, whether are related to the size of these NPs and the time of exposure.

The present study was carried out to investigate the particle-size effect of GNPs on the hepatic tissue in an attempt to cover and understand the toxicity and potential threat of their therapeutic and diagnostic use in relation with the time of exposure.

Materials and methods

A total of 70 healthy male Wistar-Kyoto rats obtained from the Laboratory Animal Center (College of Pharmacy, King Saud University, Saudi Arabia). The rats nearly of the same age (12 weeks old) and weighing 220-240 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 intraperitonealy in rats for periods of 3 or 7 days as shown in Table 1.

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)

The rats were maintained on standard laboratory rodent diet pellets and were housed in humidity and temperature-controlled ventilated cages on a 12 h day/night cycle. Two animals from each group were killed by dislocation of the neck at intervals of 3 and 7 days of treatment with GNPs. All experiments were conducted in accordance with the guidelines approved by King Saud 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 formalin (10%), then dehydrated, with grades of ethanol (70, 80, 90, 95 and 100%). Dehydration was then followed by clearing the samples in 2 changes of xylene. Samples were then impregnated with 2 changes of molten paraffin wax, then embedded and blocked out. Paraffin sections (4-5 um) were stained with hematoxylin and eosin the conventional histological and stain according to Pearse [15]. Stained sections of control and treated rats were examined for alterations in the architecture, portal triads, hepatocytes, sinusoids and for the presence of degeneration, necrosis, fatty change and portal fibrosis.

Results and discussions

Size and morphology of different gold nanoparticles

The 10 and 20 nm GNPs show spherical shape while the 50 nm GNPs show hexagonal shape. The mean size for GNPs was calculated from the images taken by the transmission electron microscope (TEM). The 10 nm GNPs was of mean size of 9.45 ± 1.33 nm, 20 nm GNPs was of mean size of 20.18 ± 1.80 and the 50 nm GNPs was of mean size of 50.73 ± 3.58. The high electron densities of GNPs as well as the homogeneity of the particles shape and size make them highly conspicuous under the TEM. In addition, relatively simple methods can be applied to obtain the populations of GNPs of different average sizes which allow simultaneous detection of several targets.

Histological Alterations

In comparison with the control group, the following histological alterations were detected in the liver tissue of GNPs-treated rats. These histological alterations were observed in Figures 1,2,3,4,5,6,7,8,9,10,11 and 12 and can be summarized as shown in Table 2.

Figure 1

GNPs-normal rat

Figure 2

GNPs-treated rat received 50 μl of 10 nm particles for 3 days

Figure 3

GNPs-treated rat received 100 μl of 10 nm particles for 7 days

Figure 4

NPs-treated rat received 50 μl of 20 nm particles for 3 days

Figure 5

GNPs-treated rat received 50 μl of 10 nm particles for 7 days

Figure 6

GNPs-treated rat received 50 μl of 50 nm particles for 3 days

Figure 7

GNPs-treated rat received 100 μl of 10 nm particles for 3 days

Figure 8

GNPs-treated rat received 100 μl of 10 nm particles for 7 days

Figure 9

GNPs-treated rat received 100 μl of 20 nm particles for 3 days

Figure 10

GNPs-treated rat received 100 μl of 20 nm particles for 7 days

Figure 11

GNPs-treated rat received 100 μl of 50 nm particles for 3 days

Figure 12

GNPs-treated rat received 100 μl of 50 nm particles for 7 days

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 by chronic 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 central vein (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 dilated congested 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 lymphocytic infiltrate (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 of liver 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 and dilated 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 liver hepatocytes with binucleation or even trinuclei noticed; no other effects (100 × 2)

The cloudy swelling might be exhibited as a result of disturbances of membranes function that lead to massive influx of water and Na+ due to GNPS effects. Cellular swelling might be accompanied by leakage of lysosomal hydrolytic enzymes that lead to cytoplasmic degeneration 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 the hepatocytes of the GNPs treated rats might indicate acute and subacute liver injury induced by these NPs. Some studies indicate that nuclear polymorphism is seen in hepatic dysplasia and carcinomatous lesion [18]. Binucleation represents a consequence of cell injury and is a sort of chromosomes hyperplasia which is usually seen in regenerating cells [19].

Occasional inflammatory cells infiltration was seen in the portal triads and the perioral zones of GNPs treated rats. The infiltrate cells were mainly lymphocytes and plasma cells. This infiltration was more prominent after 7 days of administration and in rats received 100 μl than those received 50 μl. The appearance of inflammatory cells in the hepatic tissue may suggest that GNPs could interact with proteins and enzymes of the hepatic interstitial tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may imitate an inflammatory response [20].

The sinusoidal Kupffer cells became prominent and increased in number due to GNPs exposure. This change was more prominent at 10 nm GNPs with dose of 100 μl than 20 nm and 50 nm GNPS and more after 7 days of administration than rats exposed to GNPs for 3 days. Kupffer cells activation might indicate that GNPs activate the phagocytic activity of the sinusoidal cells by increasing the number of Kupffte cells to help in removing the accumulated GNPs where lysosomes are involved in the intracellular breakdown into small metabolic products. The produced Kupffer cells hyperplasia might be correlated with the amount of injurious to the hepatic tissue induced by GNPs intoxication and represents a defense mechanism of detoxification. Kupffer cell hyperplasia is contributed to hepatic oxidative stress [21].

Fatty change was observed in some swelling hepatocytes of rats exposed to 100 μl of 10 nm GNPs and to lesser extent in the ones exposed to larger particles. This hepatic liposis was more frequent in rat exposed to the GNPs for 7 days than those received the treatment for 3 days. Hepatocytes fatty change might be due to lipid peroxidation that leads to rough endoplasmic damage and detachment of the cytoplasmic lipoprotein and indicate abnormal fat metabolism [22]. The seen hepatocytes abnormal retention of lipids in the present investigation induced by GNPs might indicate toxic injury to liver in the form of hepatocytes liposis by these particles.

Sporadic spotty well-defined necrosis was noticed in some hepatocytes of GNPs treated rats. This alteration was detected in the liver of rats exposed to 10 nm size particles and to lesser extent with 20 nm particles but was not seen with those exposed to 50 nm size particles. Apoptic alteration might be followed organelles swelling specially mitochondria, endoplasmic reticulum and rupture of lysosomes which might lead to amorphous eosinophilic cytoplasm as an initial sign in the sequence of hepatocytes necrosis before shrinking and dissolution of nulei [23]. The seen hepatocytes necrosis due to GNPs exposure might indicate oxidative stress on these cells by glutathione depletion.

Conclusions

Histological alterations by GNPs exposure as shown in the results of the present work could be an indication of injured hepatocytes due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these particles. One might conclude that these alterations are size-dependent with smaller ones induced more damage with relation with the time exposure of GNPs.

The appearance of hepatocytes cytoplasmic degeneration and nuclear destruction may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the hepatocytes to undergo atrophy and necrosis.

More histomorphologcal, histochemical and ultrastrucural investigations are needed to correlate the biomedical application of GNPs with the potential threat of their therapeutic and diagnostic use.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

AMAK and JBM have analyzed data, interpreted and written the final draft of this manuscript. The animal model used in this study was obtained from the Laboratory Animal Center (College of Pharmacy, King Saud University, Saudi Arabia). AMAK has conceived the study and its design and obtained research grants for this study. Moreover, both authors have read and approved the final manuscript.