Mechanisms of toxic action of Ag, ZnO and CuO nanoparticles to selected ecotoxicological test organisms and mammalian cells in vitro : a comparative review.

Silver, ZnO and CuO nanoparticles (NPs) are increasingly used as biocides. There is however increasing evidence of their threat to "non-target" organisms. In such a context, the understanding of the toxicity mechanisms is crucial for both the design of more efficient nano-antimicrobials, i.e. for "toxic by design" and at the same time for the design of nanomaterials that are biologically and/or environmentally benign throughout their life-cycle (safe by design). This review provides a comprehensive and critical literature overview on Ag, ZnO and CuO NPs' toxicity mechanisms on the basis of various environmentally relevant test species and mammalian cells in vitro. In addition, factors modifying the toxic effect of nanoparticles, e.g. impact of the test media, are discussed. Literature analysis revealed three major phenomena driving the toxicity of these nanoparticles: (i) dissolution of nanoparticles, (ii) organism-dependent cellular uptake of NPs and (iii) induction of oxidative stress and consequent cellular damages. The emerging information on quantitative structure-activity relationship modeling of nanomaterials' toxic effects and the challenges of extrapolation of laboratory results to the environment are also addressed.

. Schematic representation of the scope of the current review.

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Table SI Number of papers in ISI WoS (May 5 th and 6 th , 2013) concerning Ag, ZnO or CuO NPs and different test organisms or cells. The search terms for different nanoparticles were as follows: Topic= (nano* AND silver); Topic=(nano* AND ZnO OR Zinc oxide); Topic=(nano* AND CuO OR cupric oxide OR copper oxide). These search terms were refined with different terms for test organisms indicated. In bold are designated the organisms groups that were further addressed for retrieving information on toxicity mechanisms. The % values in the brackets are calculated for selected organisms (in bold    SIV Highlights for molecular toxicity mechanisms of silver nanoparticles (Ag NP) in mammalian cell cultures. Numbers (e.g., Ag NP5) indicate the primary size of nanoparticles in nm. The information is summarized in Figure 3 in the main text.
Highlights References Effect on cell viability (LDH, MTT, XTT assay) Ag + and Ag NP were toxic in the following order: Ag NP5 > Ag + > Ag NP20 > Ag NP50 in A549, HepG2, MCF-7, SGC-7901 cell lines. Ag + and Ag NP were toxic in L929 fibroblasts in the following order Ag NP20 > Ag + > Ag NP80 > Ag NP113. In RAW 264.7 macrophages, Ag NP20 and Ag + had similar toxicity. Hussain et al. 2005;Arora et al. 2008;Kim et al. 2009;Liu et al. 2010;Park et al. 2010;Nowrouzi et al. 2010;Foldbjerg et al. 2011;Park et al. 2011;Szmyd et al. 2013 Cellular uptake Relatively higher total concentrations of Ag were present in HepG2 cells exposed to Ag NP5 than to Ag NP20 and Ag NP50. Hussain et al. 2005;Kim et al. 2009;Liu et al. 2010;Nowrouzi et al. 2010;Park et al. 2010;Foldbjerg et  In RAW 264.7 macrophages Ag NP20 induced more ROS than Ag NP80 and Ag NP113. ROS was induced only near cytotoxic concentrations. Hussain et al. 2005;Kim et al. 2009;Foldbjerg et al. 2011;Park et al. 2011 Depletion of glutathione (GSH) In HepG2 cells only Ag NP5 and not Ag + , Ag NP20 or Ag NP50 depleted GSH level. Hussain et al. 2005;Arora et al. 2008;Liu et al. 2010;Park et al. 2010 Regulation of ROSresponsive genes In HepG2 cells the genes of metallothionein 1b and glutathione peroxidase (GPx) were induced by Ag + and not Ag NP10. Kim et al. 2009 Inhibition of ROS-quencing enzymes Superoxide dismutase (SOD) activity decreased after exposure of HepG2 cells to Ag + , Ag NP5 and Ag NP20 and not to Ag NP50. Arora et al. 2008;Liu et al. 2010;Nowrouzi et al. 2010 DNA damage DNA damage in HepG2 and A549 cells exposed to Ag + or Ag NP was prevented by pretreatment with antioxidant Nacetylcysteine. DNA damage in RAW 264.7 cells exposed to Ag NP5-113 was not significant and not the primary cause of the cell death. Arora et al. 2008;Kim et al. 2009;Foldbjerg et al. 2011;Park et al. 2011;Szmyd et al. 2013 Changes in cell morphology Ag NP20 and not Ag NP60 induced IL-8 in A549 cells at 0.35 µg/L.

Cell cycle arrest
In HepG2 cells Ag NP and Ag + caused cell cycle arrest in S phase the following order: Ag NP5 > Ag + > Ag NP20 > Ag NP50. Liu et al. 2010;Park et al. 2010

Apoptosis/ Necrosis
In HT-1080 and A431 cells apoptosis prevailed at low and necrosis at high concentrations of Ag NP. Less than 10% of HepG2cells were positive for early apoptosis after exposure to cytotoxic concentrations of Ag NP. Less than 20% of A549cells were positive for early apoptosis and about 50% cells were positive for necrosis after exposure to AG NP. Arora et al. 2008;Liu et al. 2010;Nowrouzi et al. 2010;Foldbjerg et al. 2011;Szmyd et al. 2013 Table SV Highlights for molecular toxicity mechanisms of copper oxide nanoparticles (nCuO) in mammalian cell cultures. The information is summarized in Figure 3 in the main text.
Highlights References Effect on cell viability (LDH, MTT, XTT assay) nCuO were among the most toxic NPs to A549 cells.Cu 2+ was less toxic in A549 and HepG2cells than nCuO. Cu 2+ contributed to less than 50% of the overall cytotoxicity from nCuO exposure in A549 cells. Cu 2+ chelators D-penicillamine and desferoxamine failed to mitigate the cytotoxicity of nCuO in HEp-2 cells. Sonication of the nCuO increased the cytotoxicity in A549 cells. Karlsson et al. 2008;Fahmy and Cormier 2009;Lanone et al. 2009;Ahamed et al. 2010;Cronholm et al. 2011;Cho et al. 2012;Perreault et al. 2012;Piret et al. 2012;Zhang et al. 2012;Wang et al. 2012 Cellular uptake Preferential accumulation of Cu ions from nCuO was observed in sulphur-rich areas of HepG2 cells. CuO entered into the A549 cells through endocytosis. A fraction of nCuO was not excreted by A549 cells because of the deposition in the mitochondria and nucleus. Cronholm et al. 2011;Piret et al. 2012;Wang et al. 2012 Intracellu lar ROS: 2',7'-H 2 DCFDA, MitoSox Red Pretreatment with antioxidant N-acetylcysteine mitigated the cytotoxicity of nCuO in A549 cells (Cho et al., 2012). Karlsson et al. 2008;Fahmy and Cormier 2009;Piret et al. 2012;Zhang et al. 2012;Wang et al. 2012 Depletion of GSH and lipid peroxidati on Depletion of reduced glutathione and induction of lipid peroxidation Fahmy and Cormier 2009;Ahamed et al. 2010;Perreault et al. 2012 Regulatio n of ROSquencing enzymes CAT and GR activity decreased, GPx activity increased and SOD activity did not change after exposure of HEp-2 cells to 30 nm nCuO. SOD and CAT activity increased after exposure of A451 cells to 65 nm nCuO. Fahmy and Cormier 2009;Ahamed et al. 2010

DNA damage
Increased levels of p53, Rad51 and MSH2 in A549 cells exposed to CuO. DNA fragmentation and micronucleus formation in Neuro-2A cells at low sub-toxic concentrations of nCuO.Oxidative DNA damage after 4-h exposure, temporal activation of p38 and p53 after 4-h exposure and irreversible DNA damage after 8-h exposure of nCuO to A549 cells. Low levels of DNA damage after 4-h exposure of nCuO to A549 cells. Karlsson et al. 2008;Ahamed et al. 2010;Cronholm et al. 2011;Perreault et al. 2012;Wang et al. 2012

Changes in cell morpholo gy
The number of A549 cells with nCuO particles attached to the cell surfaces was higher when cells were exposed to nCuO in serum-deficient medium compared to medium with serum. Accumulation of nCuO particles in A549 cells decreased lysosomal activity and caused the appearance of secondary Cronholm et al. 2011;Wang et al. 2012 9/13 lysosomes.

Apoptosis/ Necrosis
Intracellular calcium release, mitochondrial membrane depolarization and release of pro-apoptotic factors in BEAS-2B cells. Collapse of mitochondrial membrane potential, mitochondrial swelling and damaged structural integrity in isolated rat liver mitochondria after exposure to ZnO. The mode of cell death in HepG2 cells exposured to ZnO was ROS-triggered mitochondria mediated apoptosis (Bax upregulation, Bcl2 down-regulation and JNKp38, p38, p53 and caspase-9 activation. Xia et al. 2008;Li et al. 2012;Sharma et al. 2012 GSH -reduced glutathione, MGST1 -microsomal glutathione transferase 1, SOD-superoxide dismutase