The Toxicity of AgNPs

Since previous studies have demonstrated the biodistribution and bioavailability of AgNPs in a variety of animals, the toxicity of AgNPs is of interest.

1. In vivo studies

The in vivo studies on the toxicity of AgNPs have been conducted by using laboratory animals, including rats, mice, invertebrates, and fishes, and demonstrated that AgNPs are toxic to all tested animals in a dose-dependent manner (Buffet et al., 2014;

Kim and Ryu, 2013; Myrzakhanova et al., 2013). The exposure methods in rats and mice include oral ingestion, inhalation, intraperitoneal injection, and intravenous injection;

those in invertebrates (such as mussels and oyster), fishes, and fish embryos are mainly immersing (Buffet et al., 2014; Gagne et al., 2013; Kim and Ryu, 2013; Myrzakhanova et al., 2013).

For laboratory rats and mice, the concentrations and treatment time periods of AgNPs were ranged, respectively, from 1 to 1000 mg/kg and 3 to 90 days in the oral

exposure experiments; the concentrations and treatment time periods of AgNPs were ranged from 1.32 to 1.91 x 10⁷ particles/cm³ and 14 ~ 19 days in the inhalation exposure experiments, respectively (Hadrup and Lam, 2014; Kim and Ryu, 2013; Sung et al., 2009).

Previous studies of inhalation toxicity of AgNPs in laboratory rats found several negative health effects, including 1) significantly increased numbers of goblet cells and the amount of mucin of respiratory tract (1.32 x 10⁶ particles/cm³, 1.98 to 64.9 nm AgNPs; 6 hours/day, 5 times a week for 4 weeks)(Hyun et al., 2008), 2) pulmonary inflammation, decreased pulmonary function, and bile duct hyperplasia (2.9 x 10⁶ particles/cm³, 2 to 65 nm AgNPs; 6 hours/day, 5 times a week for 13 weeks)(Sung et al., 2009), 3) alteration of gene expression in the brain with motor neuron disorders, neurodegenerative disease, and altered immune cell function (1.91 x 10⁷ particles/cm³, 22.18 ± 1.72 nm AgNPs; 6 hours/day, 5 times a week for 2 weeks)(Lee et al., 2010). On the other hand, early studies of oral toxicity of AgNPs in laboratory rats found several negative health effects, including 1) weight loss (5 mg/kg) and damage to intestinal epithelial cells (20 mg/kg) (3 to 20 nm AgNPs for 21 days) (Shahare and Yashpal, 2013), 2) weight loss, pigmentation in ileum, increases in alkaline phosphatase (ALKP) activity and serum cholesterol level, and bile duct hyperplasia (56 ± 1.46 nm AgNPs for 90 days) (Kim et al., 2008; Kim et al., 2010). Apart from previous studies with high concentrations of AgNPs exposure, there were some studies conducted of relatively low concentrations of AgNPs. Sardari et al.

(2012) had found that rats orally exposed to 1 to 2 mg/kg/days of 70 nm AgNPs for 30 days could induce hepatitis, necrosis of glomerular cells/proximal tubular epithelial cells, changes in normal splenic architecture. In addition, significantly increased ALKP and aspartate transaminase (AST) activities and changes in the levels of cytokines, and mild inflammation of renal cortex in the mice exposed to 1 mg/kg/days of 42 nm AgNPs for 28 days by oral administration (Park et al., 2010a). As above, the toxicity caused by

AgNPs in laboratory animals included weight loss, damage to the alimentary/hepatobiliary system, abnormalities in hematology/biochemistry, genotoxicity, neurotoxicity, and immunotoxicity.

In previous studies of invertebrates (such as mussels and oyster), fishes, and fish embryos, the exposure concentrations and exposure time periods of AgNPs were ranged, respectively, from 0.0008 to 50 mg/L and 1 to 14 days. These studies demonstrated that AgNPs could cause disruption of the ionic regulation/steroidogenesis, histological alterations of gills (telangiectasia, circulatory disturbances, epithelial lifting, epithelial desquamation, deformed lamellae, and epithelial hyperplasia), neurotoxicity, immunotoxicity, cytotoxicity, and genotoxicity (Degger et al., 2015; Gagne et al., 2013;

Hawkins et al., 2015; Kim and Ryu, 2013; Kwok et al., 2016; Thummabancha et al., 2016;

Wu and Zhou, 2013).

2. In vitro studies

Numerous studies of in vitro toxicity of AgNPs have been published, which include human cancer cell lines (such as skin carcinoma cells [A431], human lung adenocarcinoma cells [A549], human hepatoma cells [HepG2]), rainbow trout hepatocyte, rainbow trout gill cells, and Japanese medaka fibroblast cells (OLHNI2) (Kim and Ryu, 2013; Zhang et al., 2014; Zhang et al., 2016). Although the possible mechanisms of toxic effects caused by AgNPs are not fully understood, previous studies have suggested that the AgNPs can cause cytotoxicity (i.e. mitochondrial dysfunction and apoptosis) and genotoxicity (i.e. DNA damage, formation of micronuclei, cell cycle arrest) via reactive oxygen species (ROS)-dependent pathway and ROS-independent pathway (Kim and Ryu, 2013; Zhang et al., 2014; Zhang et al., 2016). An early study has found that AgNPs can penetrate through the cell membrane, be ionized in the cytoplasm, and then induce negative effects, which is considered a Trojan-horse type mechanism (Park et al., 2010b).

Furthermore, recent studies have demonstrated that AgNPs cause cytotoxicity through the disruption of normal autophagic flux (Mao et al., 2016; Mishra et al., 2016).

2.1 ROS-dependent pathway

Reactive oxygen species (ROS) are by-products of cellular oxygen metabolism and mainly produced during mitochondrial respiration in eukaryotic cells. When there are increased amounts of ROS accumulating in cells, this condition is known as oxidative stress (Kim and Ryu, 2013). It is reported that the AgNPs can induce oxidative stress by increasing the amount of intracellular ROS with glutathione depletion, lipid peroxidation enhancement, DNA damage, cell cycle alteration, and cell proliferation inhibition, and these changes ultimately lead to cell death (Kim and Ryu, 2013; Zhang et al., 2014).

2.2 ROS-independent pathway

A previous study using HepG2 and Caco2 cell lines showed that AgNPs could cause damages to the DNA and mitochondria without increasing oxidative stress (Sahu et al., 2014). Farkas et al. (2010) demonstrated that the integrity of cell membrane and the metabolic activity of rainbow trout hepatocytes were significantly decreased without increased oxidative stress. These findings suggest that the cytotoxicity caused by AgNPs may not be associated with a ROS-dependent pathway.

3. AgNPs and Ag ion

Because Ag ions are consistently dissolved from AgNPs, the toxicity of AgNPs is actually due to the dissolved Ag ions and/or AgNPs is still controversial. Some studies suggested that the dissolved Ag ions are the cause of toxicity induced by AgNPs (Lubick, 2008) or AgNPs may act as the Trojan-horse to bring Ag ions into the cells and induce toxic effects (Farkas et al., 2010). However, there were several studies indicating that 1) the toxicity of AgNPs was higher than that of pure Ag ions, 2) only AgNPs could cause formation of micronuclei, and 3) the alteration on gene expression induced by AgNPs was

different from that by pure Ag ions (Kim et al., 2009a; Piao et al., 2011; Sahu et al., 2014).

Therefore, the toxic mechanisms of AgNPs and Ag ions may be different.

4. Factors Affecting the Toxicity of AgNPs

The toxicity of AgNPs is influenced by not only cell type and exposure time/concentration, but also the different coatings and sizes of AgNPs (Kim and Ryu, 2013; Riaz Ahmed et al., 2017; Zhang et al., 2014; Zhang et al., 2016)

5. The effect of AgNPs on immune system

According to the previous in vivo studies, AgNPs would enter the blood circulation through alimentary/respiratory administrations, and thus the negative effects caused by AgNPs to the leukocytes should be of concern. Several studies on the negative effects of AgNPs on human leukocytes have demonstrated that AgNPs could cause several effects in human neutrophils, including morphological alterations, cytotoxicity, atypical cell death, inhibition of de novo protein synthesis, increased production of the CXCL8 chemokine (IL-8), and impaired lysosomal activity (Liz et al., 2015; Poirier et al., 2014;

Poirier et al., 2016; Soares et al., 2016). Cytotoxicity and inhibition of lymphocyte proliferation in lymphocytes and macrophages were also revealed (Huang et al., 2016a;

Shin et al., 2007). However, the effects of AgNPs on the functional activities of human neutrophils and lymphocytes are still poorly understood.