Nanofibers, particularly multi-walled carbon nanotubes, have attracted attention for their exceptional properties, but concerns remain about their potential health hazards due to their fiber-like morphology. Although bio-durable nanofibers may cause cancer upon inhalation, only rigid nanofibers may exhibit morphology-driven pathogenicity. Since no validated methods exist for assessing their rigidity, alternative approaches are needed that comply with the 3R principles (Replacement, Reduction, Refinement) and the European Commission efforts to foster alternatives to animal testing. This study aims to advance the development of a harmonized test method for nanofibers toxicity by comparing effects of selected carbon-based nanomaterials (NMs) with different morphologies: a nanofiber (Mitsui-7-JRCNM40011a), an elongated material (NM-400) and a particle (Printex-90). Therefore, in vitro toxicological screening and proteomic investigations were employed using differentiated THP-1 (dTHP-1) macrophage-like cells. First, we evaluated cytotoxicity and pro-inflammatory responses of the different dTHP-1 phenotypes (M0, M1 and M2) to evaluate their sensitivity, and thus selected the M0 phenotype for further oxidative and lysosomal investigations: Mitsui-7-JRCNM40011a caused, besides increased cytotoxicity and pro-inflammatory effects, oxidative stress and lysosomal dysfunction. Moreover, decreased levels of 25 lysosomal proteins, including five cathepsins, were detected. These findings deepen the understanding of nanofiber-related toxicity, supporting the development of a reliable in vitro testing strategy.
Nanofibers (NFs) are characterized by having at least two external dimensions in the nanoscale and have emerged as a focal point for research and development. Multi-walled carbon nanotubes (MWCNTs), which are among the most extensively studied NFs, exhibit a variety of valuable properties, including thermal and electrical conductivity, making them advantageous for many industrial applications, such as in high performance batteries and medical biosensors. However, their potential health hazards remain a subject of concern, particularly due to their morphological similarities to asbestos fibers, known to cause fibrosis, cancer and mesothelioma. Understanding the potential of NFs to cause asbestos-like pathogenicity upon inhalation is crucial to enable the responsible usage of such materials.
The fiber pathogenicity paradigm (FPP) outlines that the morphology and bio-durability of fibers can potentially lead to asbestos-like lung diseases like fibrosis, cancer and pleural mesothelioma, an aggressive form of cancer almost uniquely associated with asbestos exposure. The World Health Organization (WHO) outlined for occupational settings counting criteria for fibers with potentially carcinogenic properties. These criteria stipulate that fibers exceeding lengths > 5 μm, with a respirable diameter < 3 μm, and an aspect ratio > 3:1, pose significant risks to human health upon inhalation exposure if they also meet the additional criterion of being sufficiently bio-durable. However, NFs represent a challenge to the FPP. As the diameter of a fiber decreases, the fiber gains flexibility and becomes more prone to tangling. Studies with thin MWCNTs indicate that those with diameters below 30 nm (the proposed bending threshold for MWCNTs tend to form spherical agglomerates, losing their fibrous nature. The toxicity of entangled MWCNTs would align more closely with bio-persistent granular particles.
Risk assessment of the pathogenicity of fibers currently relies on data obtained from animal testing, which is time-consuming and raises both ethical and scientific concerns. In addition, the European Commission is making efforts to phase out animal testing in the area of chemical safety assessment. The challenges when using rodents for the assessment of fiber toxicity relate to the long latency periods for tumor development, which occur toward the end of their lifespan when they are susceptible to spontaneous tumor development. This makes it difficult to determine the true extent of the carcinogenic properties of NFs. Therefore, to ensure the safe and sustainable use of rapidly emerging novel NFs, there is an urgent need for alternative assessment strategies, e.g., in form of new approach methodologies (NAMs), in particularly high throughput in vitro assays. However, so far only a few NAMs are validated, established as OECD test guidelines (TGs), and accepted for regulatory risk assessments. Existing NAM-based OECD TGs cover for example, skin corrosion/irritation and serious eye damage/irritation, which are acute effects associated with substance toxicity. However, when considering NF toxicity, long-term effects addressing potential outcomes like fibrosis and cancer become essential.
To this end, the Adverse Outcome Pathways (AOPs) serves as a framework that allows to compile existing knowledge to understand long-term toxicological effects. Therefore, the AOPs are a crucial foundation for the design of testing strategies. AOPs link the molecular initiating event (MIE) to the adverse outcome through a well-defined sequence of key events (KEs) across molecular, cellular, organ, and organism levels, based on the current knowledge. Each KE represents a measurable biological change. Specifically for fiber toxicity, multiple AOPs, such as AOP171, AOP303, and AOP409, have been developed. Common to these AOPs is the induction of inflammation, with the macrophage response to fiber exposure playing a critical role in initiating the inflammatory cascade. In particular, when alveolar macrophages encounter rigid fibers of critical lengths, the fundamental mechanism for particle clearance, the phagocytosis by the macrophage, is compromised, leading to a state known as frustrated phagocytosis. The inability to successfully phagocytose large fibers triggers a cascade of detrimental effects, ultimately leading to cell death. These effects are accompanied by lysosomal disruption the release and generation of reactive oxygen species (ROS) and pro-inflammatory mediators, which recruit additional immune cells, such as circulating monocytes. Following extravasation, monocytes are maturated in the tissue to non-activated (M0) macrophages, which are further polarized by local stimuli to type 1 (M1) or type 2 (M2) macrophages. M1 macrophages are found promptly at the affected site to release pro-inflammatory cytokines (inflammatory macrophages), while M2 macrophages appear later to counteract the pro-inflammatory activity of the M1 phenotype by secreting anti-inflammatory mediators (anti-inflammatory macrophages).
Differentiated human monocytic THP-1 macrophage-like cells (dTHP-1) are a prominent cell model frequently used for studying macrophage responses to fibers owing to a very limited number of better alternatives. Compared to primary human monocyte-derived macrophages, which are short-lived, finite, and have high inter-doner variability, dTHP-1 cells are more robust cell model that might be easier to standardize. Moreover, they are widely used for studying immunology, infectious, and inflammatory diseases, among others. Also, they are utilized in the Human Cell Line Activation Test (h-CLAT), a component of one of the limited number of NAM-based OECD test guidelines. THP-1 cells can be readily differentiated into the M0 phenotype, and further polarized into the M1 and M2 macrophage phenotypes, offering versatile possibilities for toxicity studies and appearing well suited for use in a harmonized fiber testing strategy. While previous research mainly focused on M0 macrophages, recent endeavors have incorporated M1 and M2 phenotypes. However, there is considerable variation in the experimental conditions used for differentiation and polarization. Therefore, a crucial starting point for establishing the testing procedure is to select the most appropriate macrophage phenotype. The selected dTHP-1 phenotype must provide the most sensitive readouts for NF exposure, particularly to the varying rigidity of the NFs, as it is imperative to extend the FPP to encompass this parameter. The lack of validated methods for assessing NF rigidity presents a significant challenge. Therefore, different approaches are required, such as investigating the effects of NFs on cellular responses.
In a previous study, we conducted a meta-analysis of over one hundred existing omics datasets from both human and rodent in vitro and in vivo data, demonstrating the potential of this approach of identifying relevant signatures for different morphologies of carbon-based NMs, including entangled and rigid NFs. Although this information contributes to the development of a harmonized fiber testing strategy, distinct responses to the various morphologies of the evaluated carbon-based materials were observed at the level of cellular pathways. No specific genes or proteins were identified as potential biomarkers. To deepen the mechanistic understanding of NF toxicity, and aiming at advancing the development of a harmonized testing strategies for NFs, in this work we investigated the suitability of different dTHP-1 phenotypes. To this end, we assessed the cytotoxicity and cellular responses towards Printex-90, NM-400, and Mitsui-7-JRCNM40011a -- representing a spherical particle, an entangled fiber, and a carcinogenic rigid fiber, respectively. The assays address cytotoxicity, pro-inflammatory cytokine release, and oxidative stress and lysosomal integrity. To further unravel the underlying cellular toxicity mechanisms caused by the morphologically different carbon NMs, we additionally employed proteomic profiling using an advanced tandem mass tag (TMT) labelling protocol. Our findings highlighting the inherent limitations of certain dTHP-1 cell models for comprehensively evaluating fiber toxicity, and provide a molecular description of the morphological-driven toxicity for Mitsui-7-JRCNM40011a.