Improving Quality in Nanoparticle-Induced Cytotoxicity Testing by a Tiered Inter-Laboratory Comparison Study.

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

Physicochemical and toxicological evaluation of silica nanoparticles suitable for food and consumer products collected by following the EC recommendation.

Specific information about the particle size distribution, agglomeration state, morphology, and chemical composition of four silica samples, used as additives in food and in personal care products, were achieved with a combination of analytical techniques. The combined use of differential centrifugal sedimentation (DCS), sedimentation field flow fractionation (SdFFF), and scanning and transmission electron microscopy (SEM and TEM) allows to classify the water dispersed samples as "nanomaterials" according to the EC definition. The mechanical stirring and the ultrasound treatment were compared as dispersion methods. The particle surface chemical composition, determined by particle-induced X-ray emission (PIXE) and X-ray photoelectron spectroscopy (XPS), assessed the different levels of purity between the pyrogenic and the precipitated silica and highlighted particle surface chemical composition modifications in the outer shell when dispersed by mechanical stirring. The potential toxic effects of silica on intestinal Caco-2 cells were investigated using MTS assay and by measuring lactate dehydrogenase (LDH) release and caspases 3/7 activity after 24 h of incubation. No or limited decrease of cell viability was observed for all particles regardless of dispersion procedure, suggesting a relative innocuity of these silica samples.

Towards predicting the lung fibrogenic activity of MWCNT: Key role of endocytosis, kinase receptors and ERK 1/2 signaling.

Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-ß or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-ß in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-ß- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT.

Role of p38MAPK and oxidative stress in copper-induced senescence.

In the present work, we indicate that copper is involved in the senescence of human diploid fibroblasts and we describe mechanisms to explain it. Using different techniques, we show for the first time an accumulation of copper in cells during replicative senescence. This accumulation seems to be co-localized with lipofuscin. Second, we observed that an incubation of cells with copper sulfate induced oxidative stress, antioxidant response and premature senescence. Antioxidant molecules reduced the appearance of premature senescence. Third, we found that Nrf2 transcription factor was activated and regulated the expression of genes involved in antioxidant response while p38(MAPK) regulated the appearance of premature senescence.

Copper(II) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response.

The potential toxic effects of two types of copper(II) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major role in the activation of AP-1. In addition, cytotoxicity, inflammatory and antioxidative responses and activation of intracellular transduction pathways induced by rod-shaped CuO NPs were more important than spherical CuO NPs. Measurement of Cu(2+) released in cell culture medium suggested that Cu(2+) cations released from CuO NPs were involved only to a small extent in the toxicity induced by these NPs on HepG2 cells.

Differential toxicity of copper (II) oxide nanoparticles of similar hydrodynamic diameter on human differentiated intestinal Caco-2 cell monolayers is correlated in part to copper release and shape.

The potential toxic effects of copper oxide (CuO) nanoparticles (NPs) were studied on differentiated Caco-2 cell monolayers, a classical in vitro model of human small intestine epithelium. Two types of CuO NPs, with different specific surface area, different sizes as raw material but the same hydrodynamic diameter in suspension, differentially disturbed the monolayer integrity, were cytotoxic and triggered an increase of the abundance of several transcripts coding for pro-inflammatory cytokines and chemokines. Specific surface area was not a major variable explaining the increased toxicity when intestinal epithelium is exposed to rod-shaped CuO NPs, compared with spherical CuO NPs. The results suggest that release of Cu(II) cations and shape of these CuO NPs are likely to be implicated in the toxicity of these CuO NPs.

Pro-inflammatory effects of different MWCNTs dispersions in p16(INK4A)-deficient telomerase-expressing human keratinocytes but not in human SV-40 immortalized sebocytes.

We tested whether multi-walled carbon nanotubes (MWCNTs) induce oxidative stress and a pro-inflammatory response in human N-hTERT telomerase-immortalized keratinocytes, in human SZ95 SV-40 immortalized sebocytes and in in vitro reconstructed epidermises. MWCNTS were tested in various dispersion states, from raw and agglomerated particles to isolated entities obtained by sonication in the presence of dispersive agents (hydroxypropylcellulose and Pluronic F108). It was observed that: (a) Contrary to individualized MWCNTs, agglomerated particles prepared by suspension into pure water increased the intracellular levels of reactive oxygen species as well as the expression and secretion of interleukin-8 in N-hTERT cells; (b) the inflammatory signature of MWCNTs in N-hTERT cells, drawn by transcriptomic analysis with low-density microfluidic cards, included various other cytokines such as interleukin-6 or C-C motif ligand 3; (c) the pro-inflammatory effects of MWCNTs, as assessed by interleukin-8 transcript level and protein release, were not observed in SZ95 cells; and (d) the secretion of interleukins-1α and -8 from in vitro reconstructed epidermal tissues, used as specific markers for skin irritation and sensitization, was unaffected in presence of MWCNTs, confirming that the cornified layer is an efficient barrier against MWCNTs.

Validation of the calibrated thrombin generation test (cTGT) as the reference assay to evaluate the procoagulant activity of nanomaterials.

We validated a preclinical toxicological screening assay and provided guidelines to evaluate the potential impact of nanoparticles (NPs) on blood coagulation. Five NPs with various physicochemical properties were studied using several existing methods of clotting times and thrombin generation assays in human normal pool plasma. In both recalcification clotting time (RCT) and calibrated thrombin generation test (cTGT), the NPs exhibited procoagulant activity (SiO2 ≥ SiC ≥ TiC > CuO > CB) but cTGT was more sensitive and relevant than RCT. Thus, the cTGT appears as a reference assay to investigate the nanoparticle (NP) procoagulant activity in human plasma. It should be used as the reference toxicity test for evaluating the effects of nanomaterials on coagulation cascade. In addition, we also showed that the use of the Pluronic F-108 dispersant and/or the sonication for the NP suspension preparation may mask their procoagulant activity and thus should be avoided.

Cytotoxicity of multi-walled carbon nanotubes in three skin cellular models: effects of sonication, dispersive agents and corneous layer of reconstructed epidermis.

The effects of multi-walled carbon nanotubes were investigated in SZ95 sebocytes, IHK keratinocytes and reconstructed human epidermises. Carbon nanotubes were subjected to dispersion protocols leading to different agglomeration states. Toxicological methods were chosen and adapted in order to ensure compatibility with nanotubes. Results show that: (i) Water-suspended nanotubes, as micrometric agglomerates, were not harmful to skin cells, except minor effects in keratinocytes, (ii) mild sonication slightly decreased nanotube agglomeration but increased cytotoxicity on keratinocytes, (iii) addition of hydroxypropylcellulose or Pluronic F108, which improved nanotube dispersion, masked the harmful effects of sonicated nanotubes. Altogether, these results indicate that carbon nanotubes induced cytotoxicity in human keratinocytes after a short exposure (24-48 h), particularly when they were sonicated before cell incubations. However, the cytotoxic effects of raw and sonicated nanotubes could be prevented in presence of dispersive agents. No cytotoxic effects were observed in SZ95 sebocytes or in stratified epidermises reconstructed in vitro.

Casein kinase 2 inhibition decreases hypoxia-inducible factor-1 activity under hypoxia through elevated p53 protein level.

HIF-1 (hypoxia-inducible factor-1) is the main transcription factor involved in the adaptation of cells to hypoxia. In addition to regulation of HIF-1alpha protein level, HIF-1 activity is also enhanced by several pathways involving asparagine hydroxylation and phosphorylation. Here, we investigated the relationship between casein kinase 2 (CK2), p53 and HIF-1. An increase in p53 protein level and transcriptional activity was observed when CK2 was inhibited by different inhibitors under normoxia and hypoxia. This increase was in parallel with a decrease in HIF-1 activity without changes in HIF-1alpha protein level, indicating a regulation of its transcriptional activity. Similar results were obtained using CK2alpha siRNA. Ectopic overexpression of p53 also led to an inhibition of HIF-1 activity. Conversely, CK2 inhibition had no effect in p53-null cells indicating that the inhibitory effect of CK2 inhibitors requires the presence of p53. p53 activity was not required because overexpression of a p53 mutated in its DNA-binding domain exerted the same effect as wild-type p53 and because the effect of CK2 inhibitors was still observed when p53 activity was inhibited by pifithrin-alpha. Since CK2 activity is increased in hypoxic conditions, this process provides one more mechanism to ensure enhanced HIF-1 activity under such conditions.

Hypoxia protects HepG2 cells against etoposide-induced apoptosis via a HIF-1-independent pathway.

Tumor hypoxia has been described to increase the resistance of cancer cells to radiation therapy and chemotherapy. It also supports the invasiveness and metastatic potential of the tumor. However, few data are available on the transduction pathway set up under hypoxia and leading to this resistance against anti-cancer therapies. HIF-1, the main transcription factor activated by hypoxia, has been recently shown to participate to this process although its role as an anti- or a pro-apoptotic protein is still controversy. In this study, we showed that hypoxia protected HepG2 cells against etoposide-induced apoptosis. The effect of hypoxia on cell death was assayed by measuring different parameters of the apoptotic pathway, like DNA fragmentation, caspase activity and PARP-1 cleavage. The possible implication of HIF-1 in the anti-apoptotic role of hypoxia was investigated using HIF-1alpha siRNA. Our results indicated that HIF-1 is not involved in the hypoxia-induced anti-apoptotic pathway. Another transcription factor, AP-1, was studied for its potential role in the hypoxia-induced protection against apoptosis. Specific inhibition of AP-1 decreased the protection effect of hypoxia against etoposide-induced apoptosis. Together, all these data underline that hypoxia could mediate its anti-apoptotic role via different transcription factors depending on the cellular context and pro-apoptotic stimuli.

Hypoxia-inducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic cells against tert-butyl hydroperoxide-induced apoptosis.

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1alpha short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.

Hypoxia and CoCl2 protect HepG2 cells against serum deprivation- and t-BHP-induced apoptosis: a possible anti-apoptotic role for HIF-1.

Hypoxia inducible factor-1 (HIF-1) is the main transcriptional factor activated by hypoxia. Besides the well-described role assigned to HIF-1 in the adaptation of cells to hypoxia, different recent data describe a possible role for HIF-1 in the modulation of apoptosis. However, this precise role is not yet clearly understood. In this study, chemical and physiological hypoxia, which were shown to induce HIF-1alpha stabilization and HIF-1 activation, were shown to inhibit apoptosis induced in HepG2 cells by two different pro-apoptotic conditions, serum deprivation- and t-BHP-induced oxidative stress. Indeed, hypoxia reduced DNA fragmentation, caspase activation, and PARP cleavage induced by these two pro-apoptotic conditions. These results are very interesting because it is a clear demonstration that hypoxia and chemical hypoxia have a direct protective effect on apoptotic cell death induced by two different stimuli. This observation is an important data in understanding how tumor growth can occur in challenging environmental conditions.

Mitochondria permeability transition-dependent tert-butyl hydroperoxide-induced apoptosis in hepatoma HepG2 cells.

Tert-butyl hydroperoxide (t-BHP) has been demonstrated to induce apoptosis in hepatoma cell line HepG2, but poor data were available on the signaling pathway initiated by t-BHP. In this work, we studied in details the apoptotic pathways induced in HepG2 cells by t-BHP. DNA fragmentation, activation of caspases and cytochrome c release were demonstrated. Permeability transition pore inhibitors prevented the DNA fragmentation and caspase activation induced by t-BHP. In addition, changes in the mitochondrial membrane potential were detected: hyperpolarization preceded loss of membrane potential. It also preceded caspase activation which occurred before the induction of DNA fragmentation. Taken together, these results emphasize the central role played by mitochondria in the initiation of apoptosis in HepG2 cells exposed to oxidant agents.

Caspase activation precedes PTP opening in TNF-alpha-induced apoptosis in L929 cells.

In this work, we studied the apoptotic pathway in murine fibrosarcoma cells L929 exposed to tumor necrosis factor alpha (TNF-alpha). DNA fragmentation, activation of caspases, cytochrome c release and poly (ADP-ribose) polymerase cleavage were demonstrated. We showed that the proapoptotic proteins Bid and Bax as well as caspase 8 are involved in the initiation of this apoptotic pathway triggered by TNF-alpha. Indeed, inhibition of caspase 8 could prevent TNF-alpha-induced DNA fragmentation. Furthermore, Bid and Bax translocation into mitochondria were already evidenced after 6 h. In contrast, permeability transition pore inhibitors did not prevent the DNA fragmentation induced by TNF-alpha. In addition, these events were not associated with changes in the mitochondrial membrane potential nor with the loss of ATP, which only occurred after 16 h. Taken together, these results underline the fact that TNF-alpha is able to induce caspase-dependent apoptosis in L929 in the absence of permeability transition pore opening.

Soluble factors released by virus specific activated cytotoxic T-lymphocytes induce apoptotic death of astroglioma cell lines.

Astrocytomas and astrogliomas represent the most common types of primary tumors in human central nervous system and are associated with high mortality due to the absence of efficient therapy. Here we demonstrate that, upon antigen-specific activation, cytotoxic T-lymphocytes (CTLs) secrete products that inhibit proliferation and induce apoptosis in a significant proportion of astroglioma cell lines. This effect is tumor specific in that normal cultured astrocytes do not develop apoptotic changes upon exposure to supernatant of activated CTLs. Experiments with purified lymphokines and lymphokine specific blocking antibodies indicate that synergistic activities of tumor necrosis factor (TNF)-alpha and interferon (INF)-gamma are required for the apoptosis inducing effect on some astroglioma cell lines. However, this effect appears to be dependent on additional factors produced by activated CTLs. Our results suggest that local application of factors released by activated CTLs or induction of CTL migration and activation in the tumor site may have a therapeutic effect in patients with astrogliomas.

CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2.

HIF-1 (hypoxia-inducible factor-1) is the major transcription factor that is specifically activated during hypoxia. This transcription factor is composed of two subunits: HIF-1alpha and ARNT (aryl hydrocarbon receptor nuclear translocator). ARNT is constitutively expressed, whereas HIF-1alpha is targeted to proteasome degradation by ubiquitination during normoxia. In hypoxia, HIF-1alpha is stabilized and translocates to the nucleus, where it binds to ARNT. The active HIF-1 induces expression of various genes whose products play an adaptive role to the new conditions induced by hypoxia. Besides the role played by HIF-1 in the adaptation to hypoxia, recent data describe a possible role for HIF-1 in the modulation of apoptosis. According to some authors, hypoxia induces apoptosis. However, it has also been reported that hypoxia could protect cells against apoptotic cell death induced by various agents such as serum deprivation and incubation in the presence of chemotherapy agents. These contradictory data suggest that HIF-1 could display either a proapoptotic or an antiapoptotic role according to the conditions. In order to study how HIF-1 can modulate apoptosis, we studied whether hypoxia or cobalt chloride, a chemical inducer of HIF-1, could influence apoptosis induced by tert-butyl hydroperoxide (t-BHP), serum deprivation, or both in hepatoma cell line HepG2. HepG2 cells were incubated 8 hours under normoxia or hypoxia in the presence of t-BHP with or without CoCl2. CoCl2 reduced the apoptotic death of HepG2 cells induced by t-BHP and serum deprivation, as measured by DNA fragmentation. This effect was confirmed by measurement of the caspase activity. Moreover, hypoxia also prevented t-BHP- or serum deprivation-induced DNA fragmentation and caspase activation-however, to a lower extent than CoCl2. These different data suggest a possible antiapoptotic role of HIF-1. More experiments are needed to define if HIF-1 actually plays an active role in cell death protection and to determine the exact mechanism underlying this effect.

Is HIF-1alpha a pro- or an anti-apoptotic protein?

Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor specifically activated by hypoxia. It induces the expression of different genes whose products play an adaptive role for hypoxic cells and tissues. Besides these protective responses, HIF-1 and/or hypoxia have also been shown to be either anti-apoptotic or pro-apoptotic, according to the cell type and experimental conditions. More severe or prolonged hypoxia rather induces apoptosis that is, at least in part, initiated by the direct association of HIF-1alpha and p53 and p53-induced gene expression. On the other hand, HIF-1alpha dimerized with ARNT, as an active transcription factor, can protect cells from apoptosis induced by several conditions. This review is aimed to describe the different mechanisms that account for these opposite effects of HIF-1alpha.