Development of a protocol of isolation of nanoparticles from patients' broncho-alveolar lavages for their in vitro toxicity assessment.

To investigate potential correlations between human exposure to inhaled particles and pathological effects, the biological monitoring of nanoparticles in broncho-alveolar lavages (BAL) from patients has been proposed. To better understand the underlying mechanisms of toxicity, we propose to couple this biomonitoring of nanoparticles to their in vitro toxicity assessment. However, BAL obtained from regular clinical practice are conditioned with sodium hypochlorite solution (in a 50% v/v ratio), which is toxic to cells. The aim of this study was to develop a protocol to neutralize sodium hypochlorite, allowing to properly investigate the toxicity of the nanoparticles BAL contain. We first tried to neutralize chemically the sodium hypochlorite using H2O2, ascorbic acid or sodium ascorbate but this approach was unsuccessful. In addition, standard toxicology assays (MTT, LDH) could not be used because of interference with neutralizing solutions. We thus changed strategy and used ultracentrifugation to isolate nanoparticles from the sodium hypochlorite solution, with satisfactory extraction yields (88 to 100%). We then incubated the extracted nanoparticles with macrophages from the RAW264.7 cell line and assessed the cell viability and pro-inflammatory response. This study can be used as a proof-of-concept for further study of the biological impact of nanoparticles. This approach paves the way for studies aiming at a better understanding of the aetiology of some idiopathic diseases and underlying mechanisms.

Data-Driven Quantitative Intrinsic Hazard Criteria for Nanoproduct Development in a Safe-by-Design Paradigm: A Case Study of Silver Nanoforms.

The current European (EU) policies, that is, the Green Deal, envisage safe and sustainable practices for chemicals, which include nanoforms (NFs), at the earliest stages of innovation. A theoretically safe and sustainable by design (SSbD) framework has been established from EU collaborative efforts toward the definition of quantitative criteria in each SSbD dimension, namely, the human and environmental safety dimension and the environmental, social, and economic sustainability dimensions. In this study, we target the safety dimension, and we demonstrate the journey toward quantitative intrinsic hazard criteria derived from findable, accessible, interoperable, and reusable data. Data were curated and merged for the development of new approach methodologies, that is, quantitative structure-activity relationship models based on regression and classification machine learning algorithms, with the intent to predict a hazard class. The models utilize system (i.e., hydrodynamic size and polydispersity index) and non-system (i.e., elemental composition and core size)-dependent nanoscale features in combination with biological in vitro attributes and experimental conditions for various silver NFs, functional antimicrobial textiles, and cosmetics applications. In a second step, interpretable rules (criteria) followed by a certainty factor were obtained by exploiting a Bayesian network structure crafted by expert reasoning. The probabilistic model shows a predictive capability of ≈78% (average accuracy across all hazard classes). In this work, we show how we shifted from the conceptualization of the SSbD framework toward the realistic implementation with pragmatic instances. This study reveals (i) quantitative intrinsic hazard criteria to be considered in the safety aspects during synthesis stage, (ii) the challenges within, and (iii) the future directions for the generation and distillation of such criteria that can feed SSbD paradigms. Specifically, the criteria can guide material engineers to synthesize NFs that are inherently safer from alternative nanoformulations, at the earliest stages of innovation, while the models enable a fast and cost-efficient in silico toxicological screening of previously synthesized and hypothetical scenarios of yet-to-be synthesized NFs.

Physicochemical Transformations of Silver Nanoparticles in the Oro-Gastrointestinal Tract Mildly Affect Their Toxicity to Intestinal Cells In Vitro: An AOP-Oriented Testing Approach.

The widespread use of silver nanoparticles (Ag NPs) in food and consumer products suggests the relevance of human oral exposure to these nanomaterials (NMs) and raises the possibility of adverse effects in the gastrointestinal tract. The aim of this study was to investigate the toxicity of Ag NPs in a human intestinal cell line, either uncoated or coated with polyvinylpyrrolidone (Ag PVP) or hydroxyethylcellulose (Ag HEC) and digested in simulated gastrointestinal fluids. Physicochemical transformations of Ag NPs during the different stages of in vitro digestion were identified prior to toxicity assessment. The strategy for evaluating toxicity was constructed on the basis of adverse outcome pathways (AOPs) showing Ag NPs as stressors. It consisted of assessing Ag NP cytotoxicity, oxidative stress, genotoxicity, perturbation of the cell cycle and apoptosis. Ag NPs caused a concentration-dependent loss of cell viability and increased the intracellular level of reactive oxygen species as well as DNA damage and perturbation of the cell cycle. In vitro digestion of Ag NPs did not significantly modulate their toxicological impact, except for their genotoxicity. Taken together, these results indicate the potential toxicity of ingested Ag NPs, which varied depending on their coating but did not differ from that of non-digested NPs.

Putative adverse outcome pathways for silver nanoparticle toxicity on mammalian male reproductive system: a literature review.

BACKGROUND: Adverse outcome pathways (AOPs) are conceptual frameworks that organize knowledge about biological interactions and toxicity mechanisms. They present a sequence of events commencing with initial interaction(s) of a stressor, which defines the perturbation in a biological system (molecular initiating event, MIE), and a dependent series of key events (KEs), ending with an adverse outcome (AO). AOPs have recently become the subject of intense studies in a view to better understand the mechanisms of nanomaterial (NM) toxicity. Silver nanoparticles (Ag NPs) are one of the most explored nanostructures and are extensively used in various application. This, in turn, has increased the potential for interactions of Ag NPs with environments, and toxicity to human health. The aim of this study was to construct a putative AOPs (pAOP) related to reproductive toxicity of Ag NPs, in order to lay the groundwork for a better comprehension of mechanisms affecting both undesired toxicity (against human cell) and expected toxicity (against microorganisms). METHODS: PubMed and Scopus were systematically searched for peer-reviewed studies examining reproductive toxicity potential of Ag NPs. The quality of selected studies was assessed through ToxRTool. Eventually, forty-eight studies published between 2005 and 2022 were selected to identify the mechanisms of Ag NPs impact on reproductive function in human male. The biological endpoints, measurements, and results were extracted from these studies. Where possible, endpoints were assigned to a potential KE and an AO using expert judgment. Then, KEs were classified at each major level of biological organization. RESULTS: We identified the impairment of intracellular SH-containing biomolecules, which are major cellular antioxidants, as a putative MIE, with subsequent KEs defined as ROS accumulation, mitochondrial damage, DNA damage and lipid peroxidation, apoptosis, reduced production of reproductive hormones and reduced quality of sperm. These successive KEs may result in impaired male fertility (AO). CONCLUSION: This research recapitulates and schematically represents complex literature data gathered from different biological levels and propose a pAOP related to the reproductive toxicity induced by AgNPs. The development of AOPs specific to NMs should be encouraged in order to provide new insights to gain a better understanding of NP toxicity.

Graphene oxide incorporating carbon fibre-reinforced composites submitted to simultaneous impact and fire: Physicochemical characterisation and toxicology of the by-products.

The toxicological profile of particulates released from carbon fibre-reinforced composites (CFC) incorporating nanoadditives, under impact and fire conditions (e.g. aircraft crash), is unknown to date. Our aim was to investigate the effects of simultaneous impact and fire on the physicochemical features of the particles released from CFCs produced from a graphene oxide (GO)-reinforced epoxy resin and the consequences on its toxicological profile. CFC samples with (CFC + GO) or without GO (CFC) were subjected to simultaneous impact and fire through a specific setup. Soot and residues were characterised and their toxicity was compared to that of virgin GO. Virgin GO was not cytotoxic but induced pro-inflammatory and oxidative stress responses. The toxicity profile of CFC was similar for soot and residue: globally not cytotoxic, inducing a pro-inflammatory response and no oxidative stress. However, an increased cytotoxicity at the highest concentration was potentially caused by fibres of reduced diameters or fibril bundles, which were observed only in this condition. While the presence of GO in CFC did not alter the cytotoxicity profile, it seemed to drive the pro-inflammatory and oxidative stress response in soot. On the contrary, in CFC + GO residue the biological activity was decreased due to the physicochemical alterations of the materials.

Short Preirradiation of TiO2 Nanoparticles Increases Cytotoxicity on Human Lung Coculture System.

Anatase titanium dioxide nanoparticles (TiO2 NPs) are used in a large range of industrial applications mainly due to their photocatalytic properties. Before entering the lung, virtually all TiO2 NPs are exposed to some UV light, and lung toxicity of TiO2 NPs might be influenced by photoexcitation that is known to alter TiO2 surface properties. Although the TiO2 NPs toxicity has been extensively investigated, limited data are available regarding the toxicity of TiO2 NPs that have been pre-exposed to UV light, and their impact on humans remains unknown. In this study, five types of TiO2NPs with tailored physicochemical features were characterized and irradiated by UV for 30 min. Following irradiation, cytotoxicity, pro-inflammatory response, and oxidative stress on a human lung coculture system (A549 epithelial cells and macrophages differentiated from THP-1 cells) were assessed. The surface charge of all samples was less negative after UV irradiation of TiO2 NPs, and the average aggregate size was slightly increased. A higher cytotoxic effect was observed for preirradiated TiO2 NPs compared to nonirradiated samples. Preirradiation of TiO2 NPs had no significant impact on the pro-inflammatory response and oxidative stress as shown by a similar production of IL-8, TNF-α, and reactive oxygen species.

Impact of the Physicochemical Features of TiO2 Nanoparticles on Their In Vitro Toxicity.

The concern about titanium dioxide nanoparticles (TiO2-NPs) toxicity and their possible harmful effects on human health has increased. Their biological impact is related to some key physicochemical properties, that is, particle size, charge, crystallinity, shape, and agglomeration state. However, the understanding of the influence of such features on TiO2-NP toxicity remains quite limited. In this study, cytotoxicity, proinflammatory response, and oxidative stress caused by five types of TiO2-NPs with different physicochemical properties were investigated on A549 cells used either as monoculture or in co-culture with macrophages differentiated from the human monocytic THP-1 cells. We tailored bulk and surface TiO2 physicochemical properties and differentiated NPs for size/specific surface area, shape, agglomeration state, and surface functionalization/charge (aminopropyltriethoxysilane). An impact on the cytotoxicity and to a lesser extent on the proinflammatory responses depending on cell type was observed, namely, smaller, large-agglomerated TiO2-NPs were shown to be less toxic than P25, whereas rod-shaped TiO2-NPs were found to be more toxic. Besides, the positively charged particle was slightly more toxic than the negatively charged one. Contrarily, TiO2-NPs, whatever their physicochemical properties, did not induce significant ROS production in both cell systems compared to nontreated control groups. These results may contribute to a better understanding of TiO2-NPs toxicity in relation with their physicochemical features.

Influence of the physicochemical features of TiO2 nanoparticles on the formation of a protein corona and impact on cytotoxicity.

Due to their unique properties TiO2 nanoparticles are widely used. The adverse effects they may elicit are usually studied in relation to their physicochemical features. However, a factor is often neglected: the influence of the protein corona formed around nanoparticles upon contact with biological media. Indeed, although it is acknowledged that it can strongly influence nanoparticle toxicity, it is not systematically considered. The aim of this study was to characterize the formation of the protein corona of TiO2 nanoparticles as a function of the main nanoparticle properties and investigate potential relationship with the cytotoxicity nanoparticles induce in vitro in human lung cells. To that purpose, five TiO2 nanoparticles differing in size, shape, agglomeration state and surface charge were incubated in cell culture media (DMEM or RPMI supplemented with 10% fetal bovine serum) and the amount and profile of adsorbed proteins on each type of nanoparticle were compared to their toxicological profile. While nanoparticle size and surface charge were found to be determinant factors for protein corona formation, no clear impact of the shape and agglomeration state was observed. Furthermore, no clear relationship was evidenced between the protein corona of the nanoparticles and the adverse effect they elicited.

The effects of different bisphenol derivatives on oxidative stress, DNA damage and DNA repair in RWPE-1 cells: A comparative study.

Bisphenol A (BPA) is a well-known endocrine disruptor and it is widely used mainly in the plastics industry. Due to recent reports on its possible impact on health (particularly on the male reproductive system), bisphenol F (BPF) and bisphenol S (BPS) are now being used as alternatives. In this study, RWPE-1 cells were used as a model to compare cytotoxicity, oxidative stress-causing potential and genotoxicity of these chemicals. In addition, the effects of the bisphenol derivatives were assessed on DNA repair proteins. RWPE-1 cells were incubated with BPA, BPF, and BPS at concentrations of 0-600 µM for 24 h. The inhibitory concentration 20 (IC20 , concentration that causes 20% of cell viability loss) values for BPA, BPF, and BPS were 45, 65, and 108 µM, respectively. These results indicated that cytotoxicity potentials were ranked as BPA > BPF > BPS. We also found alterations in superoxide dismutase, glutathione peroxidase and glutathione reductase activities, and glutathione and total antioxidant capacity in all bisphenol-exposed groups. In the standard and modified Comet assay, BPS produced significantly higher levels of DNA damage vs the control. DNA repair proteins (OGG1, Ape-1, and MyH) involved in the base excision repair pathway, as well as p53 protein levels were down-regulated in all of the bisphenol-exposed groups. We found that the BPA alternatives were also cytotoxic and genotoxic, and changed the expressions of DNA repair enzymes. Therefore, further studies are needed to assess whether they can be used safely as alternatives to BPA or not.

Antioxidants and selenocompounds inhibit 3,5-dimethylaminophenol toxicity to human urothelial cells.

Exposure to alkyl anilines may lead to bladder cancer, which is the second most frequent cancer of the urogenital tract. 3,5-dimethylaniline is highly used in industry. Studies on its primary metabolite 3,5-dimethylaminophenol (3,5-DMAP) showed that this compound causes oxidative stress, changes antioxidant enzyme activities, and leads to death of different mammalian cells. However, there is no in vitro study to show the direct effects of 3,5-DMAP on human bladder and urothelial cells. Selenocompounds are suggested to decrease oxidative stress caused by some chemicals, and selenium supplementation was shown to reduce the risk of bladder cancer. The main aim of this study was to investigate whether selenocompounds organic selenomethionine (SM, 10 µmol/L) or inorganic sodium selenite (SS, 30 nmol/L) could reduce oxidative stress, DNA damage, and apoptosis in UROtsa cells exposed to 3,5-DMAP. 3,5-DMAP caused a dose-dependent increase in intracellular generation of reactive oxygen species, and its dose of 50 µmol/L caused lipid peroxidation, protein oxidation, and changes in antioxidant enzyme activities in different cellular fractions. The comet assay also showed single-strand DNA breaks induced by the 3,5-DMAP dose of 50 µmol/L, but no changes in double-strand DNA breaks. Apoptosis was also triggered. Both selenocompounds provided partial protection against the cellular toxicity of 3,5-DMAP. Low selenium status along with exposure to alkyl anilines can be a major factor in the development of bladder cancer. More mechanistic studies are needed to specify the role of selenium in bladder cancer.

Evaluation of skin irritation potentials of different cosmetic products in Turkish market by reconstructed human epidermis model.

Human skin is a protective barrier against the toxic effects of cosmetics. Marketing of cosmetic products with ingredients tested on animals was prohibited in 2013. Since then, safety evaluation of cosmetic products is performed by using alternative in vitro toxicity tests. In vitro 3-D reconstructed human epidermis (RhE) tissue models are now used to define skin irritation/corrosion potentials of cosmetic ingredients and end-products. The main aim of this study was to evaluate skin irritation potentials of topically used cosmetic end-products which were marketed in Turkey during 2015-2017, by using the EpiDerm in vitro 3D-human skin model. Sixty widely used cosmetic products were collected from different markets/cosmetic shops. Among hair care products, only one shampoo was found to be strong/severe skin irritant/possible corrosive while 22 shampoos were moderate skin irritant and 11 shampoos were moderate to mild skin irritant. Among 6 skin care products, one was found to be moderate to mild skin irritant. We can suggest that alternative in vitro tests should continuously be used to test both the ingredients and the final cosmetic formulations.