An in vitro comparison of the toxicological profiles of ground tire particles (TP) and actual tire and road wear particles (TRWP) emissions.

There is currently limited data on the potential effects of tire and road wear particles (TRWP) on human health. TRWP include tire fragments, but also road wear materials, dust, adsorbed gaseous pollutants and different types of inclusions that could affect their hazard profiles. Due to their availability and lower complexity, ground tire particles (TP) are often used in toxicological studies. However, this makes it difficult to draw firm conclusions about the potential hazard of actual TRWP. Here, we compared the in vitro toxicological profile of ground TP and actual TRWP emissions of similar size collected from road traffic. For this purpose, TP and TRWP were separately incubated with alveolar macrophages for 24 h, and the cellular response was evaluated in terms of cytotoxicity, proinflammatory response and oxidative stress. Both TP and TRWP induced neither significant cytotoxicity nor oxidative stress, but triggered a concentration-dependent proinflammatory response, as evidenced by increased TNF-α production. The level of TNF-α production was slightly higher with TRWP than with TP, independent of the particle dose. All in all, the pulmonary toxicity of TRWP could be due primarily to the tire tread inclusions and only marginally to other particle components (i.e. road wear materials, dust …). Although these preliminary results need to be confirmed by further analysis, they could be useful for tire manufacturers in the production of safer-by-design tires.

In vitro toxicological evaluation of aerosols generated by a 4th generation vaping device using nicotine salts in an air-liquid interface system.

BACKGROUND: Electronic cigarettes (EC) have gained popularity, especially among young people, with the introduction of fourth-generation devices based on e-liquids containing nicotine salts that promise a smoother vaping experience than freebase nicotine. However, the toxicological effects of nicotine salts are still largely unknown, and the chemical diversity of e-liquids limits the comparison between different studies to determine the contribution of each compound to the cytotoxicity of EC aerosols. Therefore, the aim of this study was to evaluate the toxicological profile of controlled composition e-liquid aerosols to accurately determine the effects of each ingredient based on exposure at the air-liquid interface. METHODS: Human lung epithelial cells (A549) were exposed to undiluted aerosols of controlled composition e-liquids containing various ratios of propylene glycol (PG)/vegetable glycerin (VG) solvents, freebase nicotine, organic acids, nicotine salts, and flavoured commercial e-liquids. Exposure of 20 puffs was performed at the air-liquid interface following a standard vaping regimen. Toxicological outcomes, including cytotoxicity, inflammation, and oxidative stress, were assessed 24 h after exposure. RESULTS: PG/VG aerosols elicited a strong cytotoxic response characterised by a 50% decrease in cell viability and a 200% increase in lactate dehydrogenase (LDH) production, but had no effects on inflammation and oxidative stress. These effects occurred only at a ratio of 70/30 PG/VG, suggesting that PG is the major contributor to aerosol cytotoxicity. Both freebase nicotine and organic acids had no greater effect on cell viability and LDH release than at a 70/30 PG/VG ratio, but significantly increased inflammation and oxidative stress. Interestingly, the protonated form of nicotine in salt showed a stronger proinflammatory effect than the freebase nicotine form, while benzoic acid-based nicotine salts also induced significant oxidative stress. Flavoured commercial e-liquids was found to be cytotoxic at a threshold dose of ≈ 330 µg/cm². CONCLUSION: Our results showed that aerosols of e-liquids consisting only of PG/VG solvents can cause severe cytotoxicity depending on the concentration of PG, while nicotine salts elicit a stronger pro-inflammatory response than freebase nicotine. Overall, aerosols from fourth-generation devices can cause different toxicological effects, the nature of which depends on the chemical composition of the e-liquid.

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.

Biological effects of brake wear particles in mammalian models: A systematic review.

Road traffic is a major contributor to air pollution through aerosols both from exhaust emissions (EE) and non-exhaust emissions (NEE). NEE result from mechanical abrasion of brakes and tires, erosion of road surfaces and resuspension of road dust into the atmosphere by passing traffic. EE have been thoroughly studied and have decreased over time due to a stricter control. On the other hand, NEE have not received such attention and there is currently no legislation to specifically reduce NEE particles. Consequently, NEE relative part has become prevalent, potentially making of these emissions a major human health concern. The aim of this systematic review was to provide an overview of the current state of knowledge on the biological effects of brake wear particles, a type of NEE. To this end, we conducted a bibliographic search of two databases (PubMed and Web of Science) on June 1, 2023, focusing on the toxicological effects of brake wear particles induced in vitro and in vivo. We excluded reviews (no original experimental data), papers not written in English, studies performed in non-mammalian models and papers where no toxicity data were reported. Of the 291 papers, 19 were found to be relevant and included in our analysis, confirming that the assessment of the brake wear particles toxicity in mammalian models is still limited. This review also reports that brake wear particles can induce oxidative stress, proinflammatory response and DNA damage. Finally, some perspectives for further research and measures to mitigate the risk of brake wear emissions are discussed.

Can the impact of micro- and nanoplastics on human health really be assessed using in vitro models? A review of methodological issues.

Because of the many advantages they offer (strength, low cost, durability, lightweight, resistance, etc.), plastics are integral part of our daily life with a production constantly rising. However, their waste management is still inadequate, resulting in their release and accumulation in the environment, representing a main source of pollution. Their degradation results in debris of variable size including microplastics (0.1 µm-5 mm) and even nanoplastics (<0.1 µm), whose potential impact on ecosystems and human health have raised concerns. The potential adverse effects they may cause have been evaluated using both in vitro and in vivo models. However, due to some specific characteristics of micro- and nanoplastics, there are challenging questions about whether conventional in vitro tests are appropriate for evaluating their toxicity. For example, low-density plastics float on the surface of the culture medium and cannot come into contact with cells adhering to the bottom of the culture plates, which prevents proper evaluation of potential adverse effects and leads to misinterpretation of toxicological assays. In this review, we discuss the main issues related to the evaluation of micro- and nanoplastics toxicity using conventional in vitro assays. A literature survey has allowed to propose some solutions to circumvent these issues including the use of mathematical models to accurately determine the dose of particles delivered to cells, advanced 3D models (organoids), inverted cell culture models, cell cultures at the air-liquid interface or under dynamic conditions. Finally, we propose some perspectives and recommendations for further research on the in vitro evaluation of micro- and nanoplastics toxicity, underlining the importance of using standardized protocols for comparison purposes and samples and experimental conditions more representative of real-life exposure.

Biological effects of Tire and Road Wear Particles (TRWP) assessed by in vitro and in vivo studies - A systematic review.

Air quality is a critical issue because even small amounts of air pollutants can cause significant adverse health effects. Road traffic is a major contributor to air pollution both through aerosols from exhaust emissions (EE) and non-exhaust emissions (NEE). The latter result from mechanical abrasion of brakes and tires, erosion of road surfaces and resuspension of road dust into the atmosphere by passing traffic. EE have been extensively characterized and have declined over time due to mitigation measures. By contrast, NEE have been less studied, are not tightly regulated and there are limited data on their toxicity. Thus, NEE relative part has become prevalent, potentially making of these emissions a major human health concern. The aim of this systematic review was to provide an overview of the current state of knowledge on the biological effects of NEE. We paid particular attention to the toxicological effects of Tire and Road Wear Particles (TRWP) induced in vitro and in vivo in mammalian models. To this end, we performed a bibliographic search in two databases (PubMed and Web of Science). Of the 400 papers, 22 were found to be relevant and included in our analysis, confirming that the assessment of the TRWP toxicity in mammalian models is still limited. This review also reports that oxidative stress and inflammation are the main mechanisms underlying the toxicity of TRWP.

Considerations on dosimetry for in vitro assessment of e-cigarette toxicity.

Electronic cigarettes (or e-cigarettes) can be used as smoking cessation aid. Some studies tend to show that they are less hazardous than tobacco cigarettes, even if it does not mean they are completely safe. The huge variation in study designs assessing in vitro toxicity of e-cigarettes aerosol makes it difficult to make comparisons and draw robust and irrefutable conclusions. In this paper, we review this heterogeneity (in terms of e-cigarette products, biological models, and exposure conditions) with a special focus on the wide disparity in the doses used as well as in the way they are expressed. Finally, we discuss the major issue of dosimetry and show how dosimetry tools enable to align data between different exposure systems or data from different laboratories and therefore allow comparisons to help further exploring the risk potential of e-cigarettes.

In Vitro Biological Effects of E-Cigarette on the Cardiovascular System-Pro-Inflammatory Response Enhanced by the Presence of the Cinnamon Flavor.

The potential cardiovascular effects of e-cigarettes remain largely unidentified and poorly understood. E-liquids contain numerous chemical compounds and can induce exposure to potentially toxic ingredients (e.g., nicotine, flavorings, etc.). Moreover, the heating process can also lead to the formation of new thermal decomposition compounds that may be also hazardous. Clinical as well as in vitro and in vivo studies on e-cigarette toxicity have reported potential cardiovascular damages; however, results remain conflicting. The aim of this study was to assess, in vitro, the toxicity of e-liquids and e-cigarette aerosols on human aortic smooth muscle cells. To that purpose, cells were exposed either to e-liquids or to aerosol condensates obtained using an e-cigarette device at different power levels (8 W or 25 W) to assess the impact of the presence of: (i) nicotine, (ii) cinnamon flavor, and (iii) thermal degradation products. We observed that while no cytotoxicity and no ROS production was induced, a pro-inflammatory response was reported. In particular, the production of IL-8 was significantly enhanced at a high power level of the e-cigarette device and in the presence of the cinnamon flavor (confirming the suspected toxic effect of this additive). Further investigations are required, but this study contributes to shedding light on the biological effects of vaping on the cardiovascular system.

Characterization of the elemental and particle load of patient exhaled breath condensate and comparison with pulmonary lavages.

In the field of biomonitoring, exhaled breath condensate (EBC) is described as a potentially useful matrix for assessing inhalation exposure biomarkers in a non-invasive way. However, it is still unclear to what extent EBC is representative of the deep lung. To address this knowledge gap, EBC, bronchial washes (BWs), and bronchoalveolar lavages (BALs) were collected from 82 patients suffering from interstitial lung diseases (ILDs). The particulate contents and elemental composition of EBC, BW, and BAL were then compared in the same patients. The size distribution of particles in EBC was assessed with dynamic light scattering while inductively coupled plasma mass spectrometry was used to quantify its elemental composition. In addition, transmission electron microscopy coupled with energy dispersive x-ray spectrometry were used to further characterize samples of interest. EBC was found to be representative of both the sub-micron and nano-sized particle fractions of BAL and BW, with lower overall levels of elements in EBC than in BW and BAL. Silicon (Si) was the main component for all respiratory matrices with median levels of 2525µg l-1, 5643µg l-1and 5169µg l-1in the nano/ion fractions of EBC, BAL and BW, respectively. Moreover, Si levels in EBC from patients in this study were elevated compared to the levels reported in the literature for healthy subjects. Interestingly, Si levels in the EBC of ILD patients were inversely related to those in BAL and BW. In conclusion, the particulate content of EBC is associated with the lung particle burden and potentially correlates with pathologies, rendering it a relevant biomonitoring technique for the occupational and clinical fields.

Experimental and Computational Nanotoxicology-Complementary Approaches for Nanomaterial Hazard Assessment.

The growing development and applications of nanomaterials lead to an increasing release of these materials in the environment. The adverse effects they may elicit on ecosystems or human health are not always fully characterized. Such potential toxicity must be carefully assessed with the underlying mechanisms elucidated. To that purpose, different approaches can be used. First, experimental toxicology consisting of conducting in vitro or in vivo experiments (including clinical studies) can be used to evaluate the nanomaterial hazard. It can rely on variable models (more or less complex), allowing the investigation of different biological endpoints. The respective advantages and limitations of in vitro and in vivo models are discussed as well as some issues associated with experimental nanotoxicology. Perspectives of future developments in the field are also proposed. Second, computational nanotoxicology, i.e., in silico approaches, can be used to predict nanomaterial toxicity. In this context, we describe the general principles, advantages, and limitations especially of quantitative structure-activity relationship (QSAR) models and grouping/read-across approaches. The aim of this review is to provide an overview of these different approaches based on examples and highlight their complementarity.

Nano-delivery to the lung - by inhalation or other routes and why nano when micro is largely sufficient?

Respiratory diseases gather a wide range of disorders which are generally difficult to treat, partly due to a poor delivery of drugs to the lung with adequate dose and minimum side effects. With the recent developments of nanotechnology, nano-delivery systems have raised interest. In this review, we detail the main types of nanocarriers that have been developed presenting their respective advantages and limitations. We also discuss the route of administration (systemic versus by inhalation), also considering technical aspects (different types of aerosol devices) with concrete examples of applications. Finally, we propose some perspectives of development in the field such as the nano-in-micro approaches, the emergence of drug vaping to generate airborne carriers in the submicron size range, the development of innovative respiratory models to assess regional aerosol deposition of nanoparticles or the application of nano-delivery to the lung in the treatment of other diseases.

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.

Structure-Activity Relationship of Graphene-Based Materials: Impact of the Surface Chemistry, Surface Specific Area and Lateral Size on Their In Vitro Toxicity.

Predictive toxicity and structure-activity relationships (SARs) are raising interest since the number of nanomaterials has become unmanageable to assess their toxicity with a classical case-by-case approach. Graphene-based materials (GBMs) are among the most promising nanomaterials of this decade and their application might lead to several innovations. However, their toxicity impact needs to be thoroughly assessed. In this regard, we conducted a study on 22 GBMs to investigate their potential SARs by performing a complete physicochemical characterization and in vitro toxicity assessment (on RAW264.7 cells). We used GBMs of variable lateral size (0.5-38 µm), specific surface area (SSA, 30-880 m²/g), and surface oxidation (2-17%). We observed that reduced graphene oxides (RGOs) were more reactive than graphene nanoplatelets (GNPs), potentially highlighting the role of GBM's surface chemistry and surface defects density in their biological impact. We also observed that for GNPs, a smaller lateral size caused higher cytotoxicity. Lastly, GBMs showing a SSA higher than 200 m²/g were found to induce a higher ROS production. Mechanistic explanations are proposed in the discussion. In conclusion, pairing a full physicochemical characterization with a standardized toxicity assessment of a large set of samples allowed us to clarify SARs and provide an additional step toward safe-by-design GBMs.

Assessing biological oxidative damage induced by graphene-based materials: An asset for grouping approaches using the FRAS assay.

Graphene-based materials (GBMs) are extremely promising and their increasing number urges scientists to conduct more and more toxicity studies. However, case-by-case approaches are rarely the best options in the earliest phases of industrial processes. Grouping can show great assets in this context: it is defined as the process of gathering substances into a common group. Oxidative stress being a major mechanism of nanotoxicity, an important grouping criterion is the surface reactivity, for which a relevant assessment is the FRAS (ferric reducing ability of the serum) assay. However, the application of the FRAS to GBMs is questioned due to their hydrophobicity. In this study, we explored the relevance and feasibility of the FRAS for grouping, working on 22 GBMs and 2 carbon blacks. We concluded that with few adjustments, the FRAS method appeared perfectly adapted to these materials and allowed a classification as "reactive" or "non-reactive" in agreement with results of ROS production for 84% of our GBMs. While not self-sufficient for toxicity assessment, the FRAS presents interesting qualities: it is fast, cheap, and simple. Therefore, we recommend studying GBMs using the FRAS as a step of a grouping process, a complement to other assays or as an early screening tool.

Relationship between Occupational Exposure to Airborne Nanoparticles, Nanoparticle Lung Burden and Lung Diseases.

The biomonitoring of nanoparticles in patients' broncho-alveolar lavages (BAL) could allow getting insights into the role of inhaled biopersistent nanoparticles in the etiology/development of some respiratory diseases. Our objective was to investigate the relationship between the biomonitoring of nanoparticles in BAL, interstitial lung diseases and occupational exposure to these particles released unintentionally. We analyzed data from a cohort of 100 patients suffering from lung diseases (NanoPI clinical trial, ClinicalTrials.gov Identifier: NCT02549248) and observed that most of the patients showed a high probability of exposure to airborne unintentionally released nanoparticles (>50%), suggesting a potential role of inhaled nanoparticles in lung physiopathology. Depending on the respiratory disease, the amount of patients likely exposed to unintentionally released nanoparticles was variable (e.g., from 88% for idiopathic pulmonary fibrosis to 54% for sarcoidosis). These findings are consistent with the previously performed mineralogical analyses of BAL samples that suggested (i) a role of titanium nanoparticles in idiopathic pulmonary fibrosis and (ii) a contribution of silica submicron particles to sarcoidosis. Further investigations are necessary to draw firm conclusions but these first results strengthen the array of presumptions on the contribution of some inhaled particles (from nano to submicron size) to some idiopathic lung diseases.

Graphene-Based Materials In Vitro Toxicity and Their Structure-Activity Relationships: A Systematic Literature Review.

The unique properties of graphene-based materials (GBMs) placed them among the most exciting nanomaterials of the past decade. Scientists and industry are looking forward to working with not only efficient but also safe, sustainable GBMs. Designing a safer-by-design GBM implies to acquire the knowledge of which physicochemical characteristics (PCCs) can increase toxicity. In this systematic review, we extracted data from the literature to provide the available information about the structure-activity relationship of GBMs. 93 papers studying a total of 185 GBMs are included. Graphene oxides (GOs) and few-layer graphenes (FLGs) are the most studied GBMs. While reduced graphene oxides were often classified as poorly oxidant and weakly cytotoxic, graphene quantum dots were mostly moderately or highly cytotoxic. FLGs demonstrated relationships between median size and oxidative stress, between lateral size and both cytotoxicity and oxidative stress, and between thickness and cytotoxicity. We also underline relationships between median size, lateral size, and thickness of GOs and oxidative stress. However, it appears difficult to highlight clear structure-activity relationships for most PCCs and biological end points because despite a large amount of available data, the GBMs are often too poorly characterized in terms of PCCs descriptors and the biological end points investigation is not standardized enough. There is an urgent need for a better standardization of the experimental investigation of both PCCs and biological end points to allow research teams to play a part in the collaborative work toward the construction of a safer-by-design GBM through a better understanding of their key toxicity drivers.

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.

Combined effects of nanoparticles and other environmental contaminants on human health - an issue often overlooked.

Air pollution is considered as a major public health issue worldwide. It consists of a complex mixture of pollutants including nanoparticles to which we are increasingly exposed to due to the dramatic development of the nanotechnologies and their incidental or intentional release in the environment. Consequently, some concerns have raised about the combined toxicity of air particulates and other air pollutants on human health. However, the interactions between the contaminants and their resulting combined toxicity are often overlooked. Indeed, the biological effects triggered by nanoparticles are usually assessed focusing on individual nanoparticles, while their interaction with co-contaminants can deeply impact, either positively or negatively, their biodistribution, fate in the organism and toxicological profile (additive, synergistic or antagonistic responses). This paper presents a bibliographic review on the combined toxicity of nanoparticles and co-pollutants and discusses the underlying mechanisms. It also highlights the scarcity of data in the current literature, arguing for an urgent need to take into account the mixture effects to be more representative of real-life conditions for a better and accurate human health risk assessment and management.

Exploring graphene-based materials' genotoxicity: inputs of a screening method.

Graphene-based materials (GBMs) are promising nanomaterials, and several innovations depend on their use. However, the assessment of their potential hazard must be carefully explored before entering any market. GBMs are indeed well-known to induce various biological impacts, including oxidative stress, which can potentially lead to DNA damage. Genotoxicity is a major endpoint for hazard assessment and has been explored for GBMs, but the available literature shows conflicting results. In this study, we assessed the genotoxicity of 13 various GBMs, one carbon black and one amorphous silica through a DNA damage response assay (using a human respiratory cell model, BEAS-2B). Concurrently, oxidative stress was assessed through a ROS production quantification (DCFH-DA assay using a murine macrophage model, RAW 264.7). We also performed a full physicochemical characterization of our samples to explore potential structure-activity relationships involving genotoxicity. We observed that surface oxidation appears linked to genotoxicity response and were able to distinguish several groups within our studied GBMs showing different genotoxicity results. Our findings highlight the necessity to individually consider each nanoform of GBMs since the tested samples showed various results and modes of action. We propose this study as a genotoxicity assessment using a high-throughput screening method and suggest few hypotheses concerning the genotoxicity mode of action of GBMs.