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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2014 Jul 2;171(17):3980–3987. doi: 10.1111/bph.12421

Toxicological effect of engineered nanomaterials on the liver

A Kermanizadeh 1,✉, B K Gaiser 1, H Johnston 1, D M Brown 1, V Stone 1
PMCID: PMC4243972  PMID: 24111818

Abstract

The liver has a crucial role in metabolic homeostasis, as it is responsible for the storage, synthesis, metabolism and redistribution of carbohydrates, fats and vitamins, and numerous essential proteins. It is also the principal detoxification centre of the body, removing xenobiotics and waste products by metabolism or biliary excretion. An increasing number of studies have shown that some nanomaterials (NMs) are capable of distributing from the site of exposure (e.g. lungs, gut) to a number of secondary organs, including the liver. As a secondary exposure site the liver has been shown to preferentially accumulate NMs (>90% of translocated NMs compared with other organs), and alongside the kidneys may be responsible for the clearance of NMs from the blood. Research into the toxicity posed by NMs to the liver is expanding due to the realization that NMs accumulate in this organ following exposure via a variety of routes (e.g. ingestion, injection and inhalation). Thus it is critical to consider what advances have been made in the investigation of NM hepatotoxicity, as well as appraising the quality of the information available and gaps in the knowledge that still exist. The overall aim of this review is to outline what data are available in the literature for the toxicity elicited by NMs to the liver in order to establish a weight of evidence approach (for risk assessors) to inform on the potential hazards posed by NMs to the liver.

Linked Articles

This article is part of a themed section on Nanomedicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-17

Keywords: liver, nanomaterials, toxicity, oxidative stress, inflammation

The rapid expansion of technological, scientific and commercial uses of atomic- or molecular-scale materials, their assembly and their unique properties, has led to an escalating interest in the fields of nanoscience, nanotechnology and nanomedicine (Maynard et al., 2006). In 2013, there were over 1300 consumer products on the market that claim to contain aspects of nanotechnology. These include a wide range of electronics, optics, and consumer products for soil and water remediation, or for medical uses such as therapeutics, diagnostics and drug delivery (Project on Emerging Nanotechnologies, 2013). However, due to their unique chemical and physical properties (size, shape, composition, charge, crystal structure, solubility, electrical conductance, etc.) there is concern that some nanomaterials (NMs) could be hazardous for people living and working with these materials (Hoet et al., 2004; Johnston et al., 2012). The small size of particulate NMs results in a high surface area to volume ratio, which potentially offers a greater biological activity per given mass compared with larger-size materials (Oberdorster et al., 2005). In addition to this, the surface reactivity per unit surface area can be greater at the nanoscale due to higher curvature of surface (Bhattacharya et al., 2012).

The prefix ‘nano’ has been specifically coined for materials containing tens to thousands of atoms, with dimensions in the scale of less than 100 nm (Buzea et al., 2007). It is this small size that is fundamental to the field of nanotechnology, although other particle parameters also determine their physical, biological and toxicological properties (Jin et al., 2008).

As the potential for public and occupational exposure is likely to rise with increasing production of NMs, there is an urgent need to consider the possibility of any detrimental health consequences of all the different types of NMs produced. Health risk is assessed based upon the level of exposure to the manufactured NM, toxicity of the material in question, route of exposure and the persistence in the organism of the particular material.

The lungs and the gastrointestinal tract are in constant contact with the external environment so it is not surprising to find that these systems are primary exposure sites for NMs (Chen et al., 1999; Sadauskas et al., 2009). It has been shown that different types of NMs can translocate from these primary exposure sites (Sadauskas et al., 2009). As a secondary exposure site, the liver is extremely important, as it has been shown to accumulate NMs at much higher quantities compared with other organs (Semmler-Behnke et al., 2008; Sadauskas et al., 2009).

Hepatocytes constitute the major cellular compartment of the liver (approximately 65% of total liver volume). These cells are polyhedral multifaceted parenchymal cells with eight or more faces, and range between 25 and 30 μm in diameter (Kmiec, 2001). Hepatocytes participate in almost all functions that are attributed to the liver. They play a substantial role in the metabolism of exogenous and endogenous lipids and catabolism of blood-derived cholesterol-enriched proteins (Kmiec, 2001). These cells are also responsible for the manufacture of important serum proteins such as complement components and acute-phase proteins crucial in the mammalian innate immune system (Kmiec, 2001). Moreover, hepatocytes are capable of synthesizing numerous hormones and cytokines (Stadnyk et al., 1990; Dong et al., 1998; Kermanizadeh et al., 2013b,c; Gaiser et al., 2013).

Kupffer cells (KCs; 20% of total cell numbers in a healthy liver) are the resident macrophages of the liver. These cells represent the largest number of macrophages in the mammalian body. KCs eliminate both soluble and particulate antigens from the portal circulation and are responsible for the clearance of gut-derived bacteria and potential bacterial toxins such as endotoxins and peptidoglycans (Kmiec, 2001). These cells resemble other macrophages in the body – characterized by numerous microvillus projections, blebs and lamellipodia (Tiegs and Lohse, 2009). KCs are generally concentrated in the periportal region of the liver, which allows them to monitor the blood entering the organ (Kmiec, 2001). It is hypothesized that due to the constant exposure to low levels of gut-derived bacterial products, KCs are in a permanent semi-activated state (Tiegs and Lohse, 2009). Under pathological conditions, bacteria that bypass the intestinal barrier are the most important activators of KCs. These macrophages have an extremely large array of surface receptors designed for identification of most gut-derived antigens. In addition, similar to other macrophages, once activated they are capable of producing an array of mediators involved in a wide range of functions including: protein degradation, modulation of cell function and defence mechanisms and cytotoxicity (Baffy, 2009). Although KCs have the ability to initiate and sustain an immune response to eliminate pathogenic antigens, under normal circumstances they are extremely important in the maintenance of liver tolerance (in which the organ does not mount an immune response to the antigen; Tiegs and Lohse, 2009). It is understood that following the initial activation and production of a pro-inflammatory response, KCs release IL-10, which down-regulates the production of TNF-α and IL-6 and other potentially damaging pro-inflammatory cytokines (Tiegs and Lohse, 2009).

Although hepatocytes and KCs make up the large majority of the cell population of the liver, the organ also contains other cell types that are crucial to its normal function. These cells could also potentially be involved in the overall response of the liver to NMs and include sinusoidal endothelial cells (Fainboim et al., 2007), hepatic stellate cells, CD1d – restricted T-cells, natural killer T-cells, αβ T-cells, γδ T-cells, pit cells (natural killer cells – CD3-CD56+) and small numbers of B lymphocytes (Tiegs and Lohse, 2009). In addition, there is a subset of professional antigen presenting cells resident in the liver, the dendritic cells (DC) responsible for processing and presenting antigens to lymphocytes. These consist of both myeloid and plasmacytoid DCs (Baffy, 2009).

Effects of engineered NMs on the liver – a summary of in vitro studies

Numerous studies have investigated the nanotoxicological effects of a wide range of engineered NMs in the liver utilizing in vitro models. This section will summarize some of these studies while attempting to form conclusions from the data available in the literature. This can be used to establish future testing strategies aimed at assessing the hepatic toxicity of NMs.

In a set of recent studies [FP7-funded risk assessment of engineered nanoparticles (ENPRA) project] the acute toxicological effects of a large panel of engineered NMs to C3A (a human hepatocellular carcinoma cell line) and primary human hepatocytes were investigated. The panel of NMs included two zinc oxide materials (ZnO; uncoated 100 nm and triethoxycaprylylsilane coated 130 nm), two different multi-walled carbon nanotubes (MWCNTs; D: 5–35 L: 700–3000; D: 6–20 L: 700–4000), Ag (<20 nm), a 7 nm TiO2 anatase, two rutile TiO2 NMs (10 and 94 nm) and two derivatives of the 10 nm rutile with positive and negative covalent functionalization (Kermanizadeh et al., 2013a,b,c). Doses were expressed as μg·cm−2 because many of the NMs in this study settled relatively rapidly (80–0.16 μg·cm−2 equating to 256–0.5 μg·mL−1). The authors noted that the Ag NMs elicited the greatest level of cytotoxicity [24 h lethal concentration 50 (LC50) – 2 μg·cm−2], followed by the uncoated ZnO (24 h LC50 – 7.5 μg·cm−2) and coated ZnO (24 h LC50 – 15 μg·cm−2) materials. The ZnO NMs were found to be about 40–50% soluble, which could account for their toxicity. In contrast the Ag NM was <1% soluble suggesting that solubility was less important for driving the Ag NM-induced effects. The LC50 was not attained in the presence of any of the other engineered NMs (up to 80 μg·cm−2).

All NMs significantly increased IL-8 protein production at 24 h post exposure. Meanwhile no significant change in TNF-α, IL-6 or C-reactive protein was detected. Urea and albumin production were measured as indicators of hepatic function. These markers were only altered by the coated and uncoated ZnO, which significantly decreased albumin production (Kermanizadeh et al., 2013c). Furthermore, a dose-dependent decrease in the cellular glutathione content following exposure of the C3A cells to Ag, the ZnO and the MWCNTs was observed suggesting oxidative stress (Kermanizadeh et al., 2012). Intracellular reactive oxygen species (ROS) levels were also measured and shown to increase significantly following exposure of the C3A to the NMs with relatively low toxicity (MWCNT and TiO2). The antioxidant Trolox in part prevented the detrimental effect of NMs on cell viability, and suppressed the NM-induced IL-8 production after exposure to all materials with the exception of the Ag NM (Kermanizadeh et al., 2012). In contrast, following a 4 h exposure of C3A cells to sub-lethal doses of the NMs, the largest amount of DNA damage was induced by two of the TiO2 samples with relatively low toxicity (7 nm and the positively charged 10 nm materials; Kermanizadeh et al., 2012). These findings indicate that cytotoxicity alone is not sufficient to rank the hazard of NMs in vitro and that they vary in their mechanism of toxicity. Therefore a battery of tests may be required for a comprehensive in vitro toxicity analysis.

The use of hepatocyte cell lines as a replacement for animal models has been heavily criticized mainly due to low expression of metabolic enzymes. The same authors compared the response of primary human hepatocytes to the C3A cell line with respect to their toxicological response to six of the NMs mentioned earlier (two ZnO, two MWCNTs, one Ag and one positively functionalized TiO2; Kermanizadeh et al., 2013b). The cell line was comparable to the primary hepatocytes with regards to the cytotoxic effects of the NMs with the same rating of toxicity being generated in both primary hepatocytes and the C3A cell line (Ag > uncoated ZnO > coated ZnO). The LC50 was not attained in the presence of the MWCNTs and the TiO2 NMs (Kermanizadeh et al., 2013b). All NMs significantly increased IL-8 production, with no change in levels of TNF-α and IL-6 (Kermanizadeh et al., 2013b). Furthermore NM uptake was similar for both the primary hepatocytes and C3A cells as investigated by transmission electron microscopy (TEM; Kermanizadeh et al., 2013b). This study demonstrated that the C3A cell line is a good model for investigating NM-induced hepatocyte responses with respect to uptake, cytotoxicity, pro-inflammatory effects and cytokine secretion.

The majority of in vitro nanotoxicological experiments only utilize a single-cell type. However, it is difficult to gain a realistic understanding of how NMs affect an organ when investigating one single-cell type alone. In a recent set of trials, primary rat KCs were incorporated into a primary rat hepatocyte culture (C. Filippi, A. Kermanizadeh and V. Stone, in preparation). The cells were then exposed to Ag (<20 nm) and positively charged anatase TiO2 NMs (10 nm) for a 24 h period. The data suggest that KCs are important in the overall liver response to NMs and play an important role in the orchestration of the response. This was highlighted by the up-regulation of TNF-α and IL-6 observed when KCs were present when compared with a hepatocyte only in vitro system.

Numerous studies have identified Ag NMs as being relatively toxic to hepatocytes. For instance, in a recent study investigating the toxicological effects of a 24 h exposure to Ag (35 nm) and cerium dioxide (CeO2 < 25 nm) NMs on the human hepatocyte cell line (C3A) and primary trout hepatocytes, the Ag NMs were found to be more toxic than the CeO2 NMs to the hepatocytes with an LC50 of 50 μg·mL−1 for the C3A cells and 1000 μg·mL−1 for the trout hepatocytes (Gaiser et al., 2011). Additionally confocal microscopy confirmed that agglomerates of both Ag and CeO2 NM were internalized by the hepatocytes (Gaiser et al., 2011).

In a similar study in the human hepatoma cell line HepG2, Ag NMs (5–10 nm) were again shown to be highly toxic to the cells as demonstrated by the MTT, Alamarblue and lactate dehydrogenase (LDH) assays, with an LC50 of 1.95–3.38 μg·mL−1 depending on the assay utilized (24 h exposure; Kim et al., 2009). In this study, the toxicity observed was not associated with the release of Ag+ ions. Furthermore it was suggested that the mechanism of toxicity was oxidative as the Ag NM toxicity was reduced following pretreatment with the hydrophilic antioxidant N-acetylcysteine (Kim et al., 2009).

Similarly, exposure of human chang liver cells to Ag NMs (28–35 nm) resulted in a reduction in cell viability (with an LC50 of 4 μg·mL−1), ROS production and reduced glutathione depletion (Piao et al., 2011). The authors suggest that Ag induced a decrease in cell viability via a mechanism involving apoptosis and DNA fragmentation.

In another study, C3A cells were exposed to Ag (<20 nm) NMs for 24 h. The Ag NMs were again shown to be highly cytotoxic (LC50 – 5 μg·cm−2; Gaiser et al., 2013). These NMs were detected within the cytoplasm and the nucleus of hepatocytes. There was also an increased secretion of the neutrophil chemo-attractant IL-8 from the hepatocytes following exposure to sub-lethal concentrations of the Ag NMs (Gaiser et al., 2013).

Treatment of Wistar primary liver cells with 40 and 80 nm Ag NMs for 24 h resulted in a significant decrease in mitochondrial membrane potential and an ADP-induced depolarization of the mitochondria. Hence, the authors suggested that the Ag NMs have detrimental effects on liver mitochondrial function (Teodoro et al., 2011).

Looking at the literature available the data seem to indicate that Ag NMs (with seemingly different physiochemical properties) are relatively, highly cytotoxic to hepatocytes in vitro. Another important point worth mentioning is that in most studies oxidative stress has been suggested as the mechanism for the toxicity observed in these liver models.

Exposure of the HepG2 hepatocyte cell line to single-walled carbon nanotubes (SWCNTs; 1000 nm with diameter rage of 1–6 nm) induced oxidative stress and increased the proportion of apoptotic cells (Yuan et al., 2012). In the same study, the hepatocytes were exposed to graphene oxide (GO) nanosheets (lateral dimension of 100 nm and a height of 1 nm). The authors observed that the GO NMs were less cytotoxic and suggested that these materials are more biocompatible with the hepatocytes in vitro (Yuan et al., 2012).

It is extremely difficult to come to a definitive conclusion about the adverse effects of carbon nanotubes on the liver due to the sparse number of studies that have been carried out. However the data available seem to suggest that the nanotubes only induce low level toxic effects to hepatocytes in vitro.

In order to establish the adverse effect of a 4 nm TiO2 NM a number of human and rat hepatocyte cell lines including the human hepatocellular carcinoma cell line (SMMC-7721), human liver cell line (HL-7702), rat hepatocarcinoma cell line (CBRH-7919) and rat liver cell line (BRL-3A) were utilized. Despite the varying degrees of cytotoxicity between the different cell types, an LC50 was not obtained at concentrations up to 100 μg·mL−1 suggesting the relatively low cytotoxicity of these NMs (Sha et al., 2011). However, exposure of these cells of TiO2 NM was associated with increased cellular ROS and decreased intracellular glutathione (GSH) levels (Sha et al., 2011).

Exposure of primary trout hepatocytes to 5 nm anatase TiO2 for 96 h resulted in an LC40 at 30 μg·mL−1. Furthermore, the authors did not observe any increased ROS formation above the control levels (Thomas et al., 2011). In a similar study exposure of HepG2 cells to 30–70 nm anatase TiO2 for 24 h resulted in significant oxidative DNA damage and apoptosis, as demonstrated by an up-regulation in the expression of p53, Bax, Apaf-1 and cyto-1 within the cells (Shukla et al., 2013).

Currently there are conflicting views on the adverse effects of TiO2 NMs in vitro. However, the majority of data suggest that these NMs are of relatively low toxicity. This being said many authors have reported adverse effects at sub-lethal doses of these NMs particularly with regards to genotoxicity.

Exposure of rat liver slices to 5 nm Au NMs for 24 h did not result in any cytotoxic effects as assessed utilizing the LDH and MTT assays, despite the uptake of NMs by the hepatocyte (this seems to indicate that uptake is not necessarily equated with cytotoxicity; Dragoni et al., 2012). In addition there was no reduction in intracellular glutathione levels within the cells up to concentrations of 500 μM (Dragoni et al., 2012).

It is difficult to summarize and compare all the nanotoxicological in vitro data available in the liver as all the experiments have a number of different variables. Differences exist between NMs (even if they are the same type), in the concentrations utilized, preparation of NMs, exposure times, the use of cell lines or primary cells, species and the media and the serum protein used in each experiment. However, there are some recognizable patterns among most if not all of the available literature. Firstly, different NMs can be roughly classified according to their cytotoxicity to liver cells in vitro in the order of Ag (Table 1 demonstrates the toxic nature of different Ag NMs in different studies) > ZnO > SWCNT > Al2O3 > TiO2 > MWCNT > Ce2O3 and Au. It appears that in most instances the highly soluble NMs are more toxic than their insoluble counterparts (however, exceptions to this do exist i.e. Kim et al., 2009; Kermanizadeh et al., 2013c). A note of caution is therefore advised, as some or most of the toxicity mentioned earlier might be due to release of metal ions. Despite this some studies have shown that there is a clear ‘nano-effect’, which exceeds the toxicity of the equivalent amount of soluble metals. One suggested mechanism of soluble NM toxicity is the transport of materials into the cells and dissolution in the acidic environment of the lysosomes, with large amounts of ions being released (Stern et al., 2012). In addition NMs, such as carbon nanotubes, are known to have metal impurities, which may cause oxidative stress when released inside the cell. In addition enzymatic biodegradation of carbon nanotubes could contribute to their overall toxicity.

Table 1.

The toxicity of Ag NMs in a selected number of studies

Publication Ag NM utilized Model(s) 24 h – LC50
Gaiser et al., 2011 Ag 35 nm C3A cells 50 μg·mL−1
Primary trout hepatocytes 1000 μg·mL−1
Kim et al., 2009 Ag 5–10 nm HepG2 cells 1.95–3.38 μg·mL−1
Piao et al., 2011 Ag 28–35 nm Chang liver cells 4 μg·mL−1
Kermanizadeh et al., 2012; 2013b,c Ag <20 nm C3A cells 1.25 μg·cm−2
Primary human hepatocytes 2.5 μg·cm−2
Primary rat Kupffer cells and hepatocytes 1.25 μg·cm−2
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In the majority of experiments oxidative stress has been suggested as the main mechanism of toxicity and sub-lethal changes to the liver cells in vitro. The recent abundant evidence suggesting the involvement of oxidative stress in the pathogenesis of various disorders and diseases has attracted much attention both in the scientific community and general public. ROS and other free radicals are critical intermediates in the normal physiology and pathophysiology of the liver in particular with regards to the hepatocytes (Diesen and Kuo, 2009). ROS are essential for many normal physiological functions. They are implicated in cell signalling and are considered to be the second messengers that can trigger cytokine, hormone and growth factor release from certain cells (Diesen and Kuo, 2009). ROS can also affect gene expression, as well as playing a role in the normal induction of apoptosis, although the exact mechanisms are currently unclear (Diesen and Kuo, 2009). Since ROS are ubiquitous in the normal physiology of so many processes, it is not surprising that when excess ROS are produced they subsequently affect many normal functions of a healthy cell. ROS are important in the creation of oxidative stimuli required for normal physiological homeostasis of hepatocytes. However, the equilibrium between ROS generation and the antioxidant defence within a cell can be disrupted resulting in an overall net oxidative stress (Kang, 2002). In the liver, free radicals triggered by ROS are created by the neutrophils, KCs, mitochondria and cytochrome P450 (Kang, 2002). The damage created by oxidative stress can affect all cells within the liver by inducing inflammation, ischaemia, apoptosis and necrosis (Lieber, 1997). It is believed that ROS also affect signal transduction pathways that when unbalanced may lead to hepatic inflammation, necrosis, fibrosis and/or apoptosis (Diesen and Kuo, 2009).

The data from the literature indicate that the use of sub-lethal NM concentrations is critical with regards to mechanistic studies, otherwise the formation of any meaningful conclusions about the cause and effect and the mechanism of action is almost impossible. Finally, it seems that NMs with relatively low toxicity may still possess sub-lethal effects with toxicological consequences.

Effects of engineered NMs on the liver – a summary of in vivo studies

Exposure via the i.v. route

A bio-distribution study in which adult Wister rats were exposed to 20 nm Au NMs via an i.v. route, and in which NM localization was examined after 1 day, 1 week and 2 months showed that the NMs accumulated very rapidly in the liver (1 day – 49.4 ± 50.4 ng·g−1; 1 week 64.8 ± 39.7 ng·g−1; 2 months 72.2 ± 40.5 ng·g−1; Balasubramanian et al., 2010). The authors also showed changes in the expression of genes in the organ related to detoxification, lipid metabolism and the cell cycle.

In another set of experiments i.v. treatment of male Wistar rats with 15 and 55 nm amorphous silica NMs (50 mg·kg−1) and 2, 20 and 200 nm Au NMs (6 μg of Au NMs per animal) resulted in DNA damage in the liver by the silica NMs only (no genotoxicity was observed following exposure to any of the Au NMs; Downs et al., 2012). An influx of leukocytes as well as increased necrosis and apoptosis was observed following exposure to the silica NMs in the liver (Downs et al., 2012).

A recent study investigated the effects of i.v. administration of a panel of NMs, consisting of two ZnO materials (coated 100 nm and uncoated 130 nm), two MWCNTs, one Ag (<20 nm) and one 10 nm positively charged rutile TiO2, on the liver of C57/BL6 mice (Kermanizadeh et al., 2013a). The animals were injected with either a single dose of NM (12.8 μg per animal) or three doses (6.4 μg per animal) every 24 h. Animals were killed 6, 24, 48 and 72 h after the single i.v. injection or 72 h after the triple injection regime. A wide array of NMs induced a neutrophil influx into the liver as early as 6 h post i.v. injection. However, the neutrophils were only involved in the initial phases of the immune response against the NMs as the leukocyte numbers had returned to control levels after 48 h. Furthermore the authors investigated whether Ag and the TiO2 NMs depleted glutathione in the liver and found no significant effect on total GSH following exposure to the chosen NMs after a 24 h exposure (Kermanizadeh et al., 2013a). Finally, the authors noted an up-regulation of IL-10, CXCL2 and ICAM-1 mRNA as well as a decrease in C3 and IL-6 in the livers of animals treated with the NMs (Kermanizadeh et al., 2013a).

In female Wistar rats injected with 20 nm Ag NMs i.v., an up-regulation of a number of pro-inflammatory genes was observed in the liver after 24 h, including MIP-2, IL1R-1 and TNF-α (Gaiser et al., 2013). Particles were detected by TEM in hepatocytes and KCs in both the cytoplasm and nuclei. Reduced glutathione levels in the liver were unaltered (Gaiser et al., 2013).

Changes in the expression of genes have also been observed in the livers of BALB/c mice 30 min following i.v. injections of to 4 and 100 nm PEG Au-coated NMs; microarray analysis showed the changes occurred in genes associated with apoptosis, cell cycle, inflammation and metabolic processes (Cho et al., 2009). The authors did not notice any significant differences between the effects of 4 and 100 nm Au NMs (Cho et al., 2009).

KCs were identified as being very important for the removal of 2 and 40 nm Au NMs following their i.v. administration to C57/BL6 mice (Sadauskas et al., 2007). Also i.v. injections of these NMs resulted in their rapid accumulation in the resident liver macrophages (Sadauskas et al., 2007).

Intratracheal instillation (i.t.) route

In a study in which female Wistar rats were exposed to 1.4 and 18 nm Au NMs i.t., the labelled NMs translocated (up to 8% of total dose) from the lungs to secondary organs, one of which was the liver (1% of total administered Au NMs; Semmler-Behnke et al., 2008). It is not surprising to note that the authors suggest that the route of the exposure is crucial in the proportion of NMs ending up in secondary organs (Semmler-Behnke et al., 2008). In another study in which C57/BL6 mice were treated with 2, 40 and 100 nm Au NMs administered i.t., the 2 nm Au materials translocated to the liver (Sadauskas et al., 2009).

A recent study in which male Sprague-Dawley rats were exposed to 20 nm CeO2 NMs via the i.t. route resulted in the accumulation of NMs in the liver, elevations in serum alanine transaminase levels and a reduction in albumin levels (Nalabotu et al., 2012). The authors also showed that the animals exposed to the NMs had a reduced overall liver weight, enlarged hepatocytes, sinusoidal dilatations and an accumulation of granular materials. The authors suggest that exposure to CeO2 via the lungs (i.t.) can result in detrimental effects in the liver (Nalabotu et al., 2012).

In another study the oxidative effect (glutathione depletion) and gene expression response of C57/BL6 mice liver tissue 24 h following the i.t. administration of NMs (via the lungs) was assessed. The mice were exposed to two ZnO materials (uncoated 100 nm and triethoxycaprylylsilane coated 130 nm), two MWCNTs (D: 5–35 L: 700–3000; D: 6–20 L: 700–4000), Ag (<20 nm), 7 nm TiO2 anatase, two rutile TiO2 NMs (10 and 94 nm) and two derivatives of the 10 nm rutile with positive and negative covalent functionalization (1, 4, 8, 16, 32, 64 and 128 μg of different NMs per animal; I. Gosens et al., submitted). The study showed that the i.t. instilled Ag, ZnO and positively charged TiO2 resulted in acute distal effects on the liver that involved glutathione depletion, while exposure to all NMs, with the exception of the MWCNTs, resulted in changes in gene expression in the liver most of which were anti-inflammatory genes. The authors did not associate these changes with toxicity.

In a recent study, exposure of C57/BL6 BomTac pregnant mice to carbon black Printex 90 via the i.t. route resulted in detrimental effects on the newborn (Jackson et al., 2012). The newborn mice showed changes in the levels of expression of mRNA of hepatic genes associated with inflammation, cell cycle and lipid metabolism. Furthermore the authors concluded that the effects were more pronounced in the female offspring (Jackson et al., 2012).

Oral route

The oral route exposure of male Swiss albino mice to 30 nm ZnO NMs resulted in the accumulation of materials within the liver. This accumulation was associated with an elevation in the serum levels of alanine aminotransferase and alkaline phosphatase, as well as pathological lesions in the liver (Sharma et al., 2012). ZnO NM exposure resulted in oxidative stress, significant DNA damage and the induction of apoptosis in the liver compared with control animals (Sharma et al., 2012). It is important to state that there are very few ingestion studies in which the effects of NMs on the liver have been investigated.

In summarizing the liver-related in vivo experiments, a few comparable outcomes are notable. Firstly, the route of exposure is extremely important in determining the proportion of the NM dose that reaches the liver. The largest proportion of NM dose reaching the liver occurred following an i.v. exposure. The size of the NM itself also seems to be important as smaller NMs reach the liver in higher quantities (Sadauskas et al., 2009) especially following translocation from the lungs (in reality, this is due to the levels of NM penetrating through the lung rather than a specific liver effect). It has also been suggested that the route of exposure is extremely important in determining which proteins are acquired on the surface of NMs (Johnston et al., 2012). This protein corona can and does influence the toxicity of the NM in the liver (Elbakry et al., 2012; Johnston et al., 2012). Therefore the overall toxicity of NM is not only dependent on its physico-chemical characteristics, but also on surface proteins recognized by the cells (i.e. how a NM is coated; Fadeel, 2012).

In vitro versus in vivo systems comparisons and limitations

As it has been shown that the potential of NMs for translocation to the liver is a realistic prospect and this organ accumulates a large proportion of the total translocated dose. Therefore, it is essential that the dangerous effects of NM exposure on normal liver function are thoroughly investigated. Although huge advances have been made in identifying the potential nanotoxicological effects on the liver, there are still gaps in the literature.

Very few studies have attempted to make a direct comparison between in vitro and in vivo liver models. However, two recent sets of trials conducted as part of European funded projects – ENPRA and InLiveTox have attempted to make this comparison. In these studies the adverse effects of a panel NMs on the liver were assessed using a hepatocyte cell line, primary human hepatocytes and liver tissues from exposed animals to determine if the response observed in the in vitro systems was indeed mirrored and representative of cells in vivo. The results show that there are some promising comparisons between certain NM-induced end points using the cell line (C3A) and primary liver cells (primary human hepatocytes), primary mice and rat liver tissue (Table 2). The data from these two recent studies suggest that simple in vitro test models can be extremely valuable in predicting the potential liver response in vivo. However, in vitro studies have some major limitations that need to be discussed.

Table 2.

General similarities for certain investigated end points between the ENPRA and InLiveTox projects (table offers a simplified summary)

End point C3A cells Primary human hepatocytes Rat liver Mice liver
Cytotoxicity YES YES NA NA
Antioxidant depletion YES NA YES NO (i.v. exposure) / YES (i.t. exposure)
Changes in gene expression YES NA YES YES
DNA damage YES NA NA NO
Cytokine production YES YES NA YES
NM Uptake YES YES YES YES
Functional markers YES YES NO NA
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NA, not tested or not relevant.

It is often very difficult to make a direct comparison between cells in vitro and tissue responses in vivo. At best, in vitro findings can act as an indicator of possible in vivo responses. One principle reason for this is that the comparisons between the systems are rarely like for like, that is cytotoxicity in an in vitro system is not inflammation in vivo, and the utilization of doses that would cause liver cells to die in vivo would be unrealistic and unethical. In addition, these high doses would mask any sub-lethal effects. The limitations continue as an organ is never comprised of only a single cell type, and crosstalk between different cell types and different organs is essential in the overall response to a toxic challenge. This being said increasing numbers of new sophisticated in vitro models such as three-dimensional culture, tissue slices and fluidic models, as used in the InLiveTox project, are being developed in order to improve in vitro risk assessment, with a view to reducing, refining and replacing animal studies.

In an in vitro system, soluble NMs remain trapped in the well, whereas soluble material constituents can disperse in an in vivo model. Likewise, many particle types will be removed from the site of deposition by phagocytes and eventually excreted. As already mentioned it is very unlikely that any NM will reach the liver without a protein coating, which may influence its overall toxicity to the organ. It is often very difficult to reproduce the exact protein corona in an in vitro study; however, the route of exposure and translocation can be used to improve the preparation and dispersion of NMs.

Although some attempts have been made to improve in vitro testing systems, the use of animal models is still in all probability the most reliable representation of a whole organ/body response to foreign materials such as NMs. However, the high cost and ethical implications of any in vivo study must be fully appraised.

The future

With the advances in the fields of nanotechnology and nanomedicine, the potential for public and occupational exposure is likely to increase, so there is an urgent need to consider the possibility of any detrimental health effects associated with this increased exposure to NMs. Hence it is crucial to identify the dangers associated with NM exposure both in vitro and in vivo, consequently assembling a knowledge base of the human health effects associated with NM exposure (Lin et al., 2012). Engineered NMs are manufactured from a diverse group of substances and can have very diverse physico-chemical characteristics such as size, shape, surface charge, surface reactivity, crystalline phase, polarity, solubility or impurities. Hence, a range of materials with different characteristics needs to be evaluated for a comprehensive toxicity profile, which would allow structure activity relationships to be generated. Likewise, standardization of methods such as particle preparation and exposure conditions are essential to be able to compare studies carried out in different laboratories – ensuring that any differences in toxicological responses are due to the materials and not the methodology.

Therefore one of the most important reasons for conducting nanotoxicology studies is to provide a knowledge base towards assessing the risks associated with realistic NM exposures. The findings obtained in such studies should provide hazard data for the NMs that will be used for risk assessment purposes to determine any health implications associated with these NMs. For this purpose there are certain areas in liver nanotoxicology in which knowledge is severely lacking.

To our knowledge there have been very few studies if any that have investigated the effects of NMs on the liver following inhalation exposure (in all reality the most prominent route of NM exposure). It is important to note that inhalation and instillation are not always comparable – for example the deposition patterns of the particles in the lung can vary between the two methods meaning that potential adverse effects observed following intratracheal administration might not necessarily be seen following inhalation.

One of the most important paradigms of risk assessment is exposure. From the literature it is evident that there is a clear lack of studies in which low realistic relevant exposure scenarios have been employed that can be used for risk assessment purposes concentrating on the liver. Hence, there is a real need for long-term studies in which animals are exposed to low doses of NMs via different routes (i.e. inhalation, ingestion and i.v. routes). This will be the only means by which a realistic and reliable liver risk assessment model can be formulated.

Conflict of interest

The authors declare that they are no competing interests.

Glossary

DC

dendritic cell

ENPRA

risk assessment of engineered nanoparticles

KC

Kupffer cells

LC50

concentration of the chemical that killed 50% of the cells

MIP-2

macrophage inflammatory protein 2-alpha

MWCNT

multi-walled carbon nanotube; NM, nanomaterial

ROS

reactive oxygen species

SWCNT

single-walled carbon nanotube

TEM

transmission electron microscopy

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