First-in-human controlled inhalation of thin graphene oxide nanosheets to study acute cardiorespiratory responses.

Graphene oxide nanomaterials are being developed for wide-ranging applications but are associated with potential safety concerns for human health. We conducted a double-blind randomized controlled study to determine how the inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at a concentration of 200 µg m-3 or filtered air were inhaled for 2 h by 14 young healthy volunteers in repeated visits. Overall, graphene oxide nanosheet exposure was well tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of highly purified and thin nanometre-sized graphene oxide nanosheets was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures at a clinical setting for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other two-dimensional nanomaterials in humans. Clinicaltrials.gov ref: NCT03659864.

Challenges and Recommendations in Assessing Potential Endocrine-Disrupting Properties of Metals in Aquatic Organisms.

New tools and refined frameworks for identifying and regulating endocrine-disrupting chemicals (EDCs) are being developed as our scientific understanding of how they work advances. Although focus has largely been on organic chemicals, the potential for metals to act as EDCs in aquatic systems is receiving increasing attention. Metal interactions with the endocrine system are complicated because some metals are essential to physiological systems, including the endocrine system, and nonessential metals can have similar physiochemical attributes that allow substitution into or interference with these systems. Consequently, elevated metal exposure could potentially cause endocrine disruption (ED) but can also cause indirect effects on the endocrine system via multiple pathways or elicit physiologically appropriate compensatory endocrine-mediated responses (endocrine modulation). These latter two effects can be confused with, but are clearly not, ED. In the present study, we provide several case studies that exemplify the challenges encountered in evaluating the endocrine-disrupting (ED) potential of metals, followed by recommendations on how to meet them. Given that metals have multiple modes of action (MOAs), we recommend that assessments use metal-specific adverse outcome pathway networks to ensure that accurate causal links are made between MOAs and effects on the endocrine system. We recommend more focus on establishing molecular initiating events for chronic metal toxicity because these are poorly understood and would reduce uncertainty regarding the potential for metals to be EDCs. Finally, more generalized MOAs such as oxidative stress could be involved in metal interactions with the endocrine system, and we suggest it may be experimentally efficient to evaluate these MOAs when ED is inferred. These experiments, however, must provide explicit linkage to the ED endpoints of interest. Environ Toxicol Chem 2023;42:2564-2579. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Is pulmonary inflammation a valid predictor of particle induced lung pathology? The case of amorphous and crystalline silicas.

Although inflammation is a normal and beneficial response, it is also a key event in the pathology of many chronic diseases, including pulmonary and systemic particle-induced disease. In addition, inflammation is now considered as the key response in standard settings for inhaled particles and a critical endpoint in OECD-based sub-acute/ chronic animal inhalation testing protocols. In this paper, we discuss that whilst the role of inflammation in lung disease is undeniable, it is when inflammation deviates from normal parameters that adversity occurs. We introduce the importance of the time course and in particular, the reversibility of inflammation in the progression towards tissue remodelling and neoplastic changes as commonly seen in rat inhalation studies. For this purpose, we used chronic inhalation studies with synthetic amorphous silicas (SAS) and reactive crystalline silica (RCS) as a source of data to describe the time-course of inflammation towards and beyond adversity. Whilst amorphous silicas induce an acute but reversible inflammatory response, only RCS induces a persistent, progressive response after cessation of exposure, resulting in fibrosis and carcinogenicity in rodents and humans. This suggests that the use of inflammation as a fixed endpoint at the cessation of exposure may not be a reliable predictor of particle-induced lung pathology. We therefore suggest extending the current OECD testing guidelines with a recovery period, that allows inflammation to resolve or progress into altered structure and function, such as fibrosis.

Application of quantitative mineralogy to determine sources of airborne particles at a European copper smelter.

Copper processing operations, such as smelters and refineries, can produce airborne particles that may impact the health of workers. At these operations, worker exposure to chemicals are regularly monitored to ensure that regulatory compliance with occupational exposure limit values (OELVs) are maintained. Determining the type of airborne particles present is important for characterizing the composition of dust exposures and better understanding the relationship between worker exposure and health. Routine methods of analysis (e.g., chemical assay) are unable to differentiate between phases containing the same elements and may result in ambiguity. A novel approach of a combination of Quantitative Evaluation of Materials by Scanning Electron Microscope (QEMSCAN) and chemical characterization was used here to evaluate airborne and settled dust collected at key locations throughout a copper smelter in Europe. The copper (Cu) phases present in the airborne dust are indicative of the activities performed at specific locations. In the batch preparation area where Cu concentrate is received, significant amounts of Cu were carried in sulfidic minerals (chalcocite, chalcopyrite/bornite, >40%), whereas near the anode and electric furnace, the majority of Cu in dust was carried in metallic and oxidic phases (60-70%). Particle size analysis of the settled dust indicates that the sulfidic and oxidic Cu minerals are more likely to become airborne over metallic Cu. Furthermore, overall Cu concentrations decreased with particle size where metallic and oxidic Cu dominate, which suggests that differences in the proportion of Cu forms present in the dust will impact how much Cu ends up in the respirable fraction. These results highlight the need to understand the characterization of Cu in dust in order to set better OELVs.

Issues in the inhalation toxicity testing and hazard assessment for low density particulate materials such as synthetic amorphous silica (SAS).

Inhalation toxicity testing of particulate materials is mandated for classification. According to CLP, particulate materials should be tested as marketed and many particulate materials are marketed as non-respirable particles. However, OECD TG 413 requires exposure to particle sizes that are respirable and reach the alveoli. The requirement for exposure of rats to respirable particles is thus in contrast to CLP and requires the application of high shear forces. The exposure to artificially small particles causes a number of issues that hamper the interpretation of the results of the testing. These issues are aerosol altering in the exposure system, assessment of the adversity of the inflammatory lung responses, inclusion of recovery groups, and extrapolation of the results to humans exposed under occupational condition. In addition, effects of many particulate materials after testing according to OECD 413 are not intrinsic properties, but a general reaction of the lung to the deposited material, show very similar NOAECs for chemical diverse materials, and often are completely reversible.

Influence of systemic copper toxicity on early development and metamorphosis in Xenopus laevis.

The primary objective of the present study was to examine the influence of early systemic toxicity resulting from copper (Cu) exposure on metamorphic processes in Xenopus laevis. A 28-day exposure study with copper, initiated at developmental stage 10, was performed using test concentrations of 3.0, 9.0, 27.2, 82.5, and 250 µg Cu/L. The primary endpoints included mortality, developmental stage, embryo-larval malformation, behavioral effects, hindlimb length (HLL), growth (snout-vent length [SVL] and wet body weight), and histopathology. The 28-day LC50 value with 95% confidence intervals was 61.2 (51.4-72.9) µg Cu/L with 250 µg Cu/L resulting in complete lethality. Developmental arrest in the 82.5 and delay in the 27.2 µg Cu/L treatments was observed as early as study day 10 continuing throughout the remainder of exposure. SVL-normalized HLL, body weight, and SVL in the 27.2 and 82.5 µg Cu/L treatments were significantly decreased relative to control. At 82.5 µg Cu/L, and thyroid gland size was markedly reduced when compared with controls consistent with the stage of developmental and growth arrest. Concentration-dependent findings in the intestine, liver, gills, eyes, and pharyngeal mucosa were consistent with non-endocrine systemic toxicity. These were prevalent in the 9.0 and 27.2 µg Cu/L treatment groups but were minimally evident or absent in the 82.5 µg/L group, which was attributed to developmental arrest. In conclusion, developmental delay in larvae exposed to 27.2 and 82.5 µg Cu/L was the result of systemic toxicity occurring in early development prior hypothalomo-pituitary-thyroid axis (HPT)-driven metamorphosis and was not indicative of endocrine disruption.

Cross-sectional Study of Workers Employed at a Copper Smelter-Effects of Long-term Exposures to Copper on Lung Function and Chronic Inflammation.

OBJECTIVE: The aim of the study was to assess the effect of exposure to copper-containing dust on lung function and inflammatory endpoints among workers of a German copper plant, effects rarely studied before. METHODS: One hundred four copper-exposed smelter workers and 70 referent workers from the precious metal and lead facilities were included, with different metal exposures in both groups due to the different process materials. Body plethysmography, exhaled nitric oxide (FeNO) measurements, and blood sampling were conducted in all workers. Smoking status and the use of respiratory protective equipment were considered. In a subgroup of 40 nonsmoking volunteers (28 copper-exposed and 12 referents), sputum biomarkers were assessed. RESULTS: Median lung function values of both copper-exposed and the referent groups were within reference ranges of "healthy" individuals, and statistical differences between the groups were mostly not evident. Similarly, differences in blood and sputum biomarkers were too small to be biologically relevant. CONCLUSION: The results suggest the absence of the detectable effects of copper-containing dust exposure on lung function or chronic inflammation within the investigated cohort.

Inhalation toxicity of copper compounds: Results of 14-day range finding study for copper sulphate pentahydrate and dicopper oxide and 28-day subacute inhalation exposure of dicopper oxide in rats.

Inhalation exposure to copper may occur during a range of occupational activities and the purpose of this study was to characterise the toxicological response to repeated inhalation of two copper compounds, representative of copper substances in large-scale production/use. Crl:CD(SD) rats were repeatedly exposed to aerosols of dicopper oxide (Cu2O) or copper sulphate pentahydrate (CuSO4.5 H2O) for 14-days as part of a range finding study at normalised copper doses of 0.18, 0.71, 1.78 and 8.9 mg/m3 Cu. Within a 28-days main study (Cu2O only), animals were repeatedly exposed to 0.2, 0.4, 0.8 and 2.0 mg/m3 Cu2O following OECD TG 412. The main study also consisted of satellite groups exposed for 1-, 2- or 3- weeks as well as a 13-week post-exposure recovery period group. Repeated exposure for 14-days to both copper compounds, normalised for copper content, led to an acute influx of polymorphonuclear leukocytes (neutrophils) and macrophages whilst only CuSO4.5 H2O exposure resulted in epithelial hyperplasia. This differential response may reflect the highly dissolvable nature of CuSO4.5 H2O in lung lining fluid leading to a release of copper ions at the epithelial surface whilst Cu2O is relatively indissolvable at neutral pH. In the 28-day study with Cu2O, an increase in cellularity was also evident in both histological and BALF samples and was dose-related with minimal to mild (neutrophilic) inflammation observed > 0.4 mg/m3 in the lung tissue sections and significant increases from 0.2 mg/m3 in BALF. There were no minimal haematological findings, no clinical findings and systemic organs were unaffected by inhalation exposure to dicopper oxide. The lung cellular response was limited to alveolar histiocytosis and neutrophil influx with no evidence of epithelial hyperplasia or fibrosis and all lung biomarkers returned to control levels within the post-exposure recovery period. Interestingly, the satellite groups showed that this acute cellular response followed a biphasic rather than monotonic pattern with a peak in lung biomarkers between weeks 1-3 and reduction thereafter. This reduction in lung biomarkers occurred during continued exposure and may indicate an adaptive response to copper exposure. Overall, these results show that repeated exposure to copper compounds results in an acute cellular response with no associated pathology and which fully resolved after the cessation of exposure. Therefore, the cellular response is evidence of a controlled and adaptive response associated with the removal of Cu2O from the alveolar surface.

Bioaccessibility as a determining factor in the bioavailability and toxicokinetics of cadmium compounds.

Cadmium toxicity occurs where there is absorption and accumulation of cadmium ions (Cd2+) in tissues beyond tolerable levels. Significant differences in the release of Cd2+ from cadmium compounds in biological fluids, like gastric fluid, may indicate differences in bioavailability and absorption. This means that direct read-across from high solubility cadmium compounds to lower solubility compounds may not accurately reflect potential hazards. Here, the relative bioaccessibility in gastric fluid of cadmium telluride and cadmium chloride was evaluated using in vitro bioelution tests whilst the toxicokinetic behavior of these two compounds were compared after dietary administration for 90 days in male and female Wistar Han rats following OECD TG 408. Cadmium chloride was highly bioaccessible, whilst cadmium telluride showed low solubility in simulated gastric fluid (90 % and 1.5 % bioaccessibility, respectively). This difference in bioaccessibility was also reflected by a difference in bioavailability as shown by the difference in the liver and kidney concentrations of cadmium after repeat oral exposure. Feeding at doses of 750 and 1500 ppm of cadmium telluride did not result in tissue cadmium levels above the lower limit of quantification (LLOQ). In contrast, feeding with a lower test substance concentration yet higher concentration of bioaccessible cadmium (30 ppm cadmium chloride) resulted in tissue accumulation of cadmium. Only slight, non-adverse changes in hematology and clinical chemistry parameters were seen at these doses, indicating an absence of significant cadmium mediated toxicity towards target organs (kidney and liver), reflected in minimal cadmium accumulation in these organs. This study demonstrates that bioelution tests can help determine the bioaccessibility of cadmium, which can be used to estimate the potential for target tissue toxicity based on known toxicokinetic profiles and threshold levels for cadmium toxicity, while reducing and refining animal testing.

Nanotoxicology: The Need for a Human Touch?

With the ever-expanding number of manufactured nanomaterials (MNMs) under development there is a vital need for nanotoxicology studies that test the potential for MNMs to cause harm to health. An extensive body of work in cell cultures and animal models is vital to understanding the physicochemical characteristics of MNMs and the biological mechanisms that underlie any detrimental actions to cells and organs. In human subjects, exposure monitoring is combined with measurement of selected health parameters in small panel studies, especially in occupational settings. However, the availability of further in vivo human data would greatly assist the risk assessment of MNMs. Here, the potential for controlled inhalation exposures of MNMs in human subjects is discussed. Controlled exposures to carbon, gold, aluminum, and zinc nanoparticles in humans have already set a precedence to demonstrate the feasibility of this approach. These studies have provided considerable insight into the potential (or not) of nanoparticles to induce inflammation, alter lung function, affect the vasculature, reach the systemic circulation, and accumulate in other organs. The need for further controlled exposures of MNMs in human volunteers - to establish no-effect limits, biological mechanisms, and provide vital data for the risk assessment of MNMs - is advocated.

Nanotoxicology data for in silico tools: a literature review.

The exercise of non-testing approaches in nanoparticles (NPs) hazard assessment is necessary for the risk assessment, considering cost and time efficiency, to identify, assess, and classify potential risks. One strategy for investigating the toxicological properties of a variety of NPs is by means of computational tools that decode how nano-specific features relate to toxicity and enable its prediction. This literature review records systematically the data used in published studies that predict nano (eco)-toxicological endpoints using machine learning models. Instead of seeking mechanistic interpretations this review maps the pathways followed, involving biological features in relation to NPs exposure, their physico-chemical characteristics and the most commonly predicted outcomes. The results, derived from published research of the last decade, are summarized visually, providing prior-based data mining paradigms to be readily used by the nanotoxicology community in computational studies.

Practices and Trends of Machine Learning Application in Nanotoxicology.

Machine Learning (ML) techniques have been applied in the field of nanotoxicology with very encouraging results. Adverse effects of nanoforms are affected by multiple features described by theoretical descriptors, nano-specific measured properties, and experimental conditions. ML has been proven very helpful in this field in order to gain an insight into features effecting toxicity, predicting possible adverse effects as part of proactive risk analysis, and informing safe design. At this juncture, it is important to document and categorize the work that has been carried out. This study investigates and bookmarks ML methodologies used to predict nano (eco)-toxicological outcomes in nanotoxicology during the last decade. It provides a review of the sequenced steps involved in implementing an ML model, from data pre-processing, to model implementation, model validation, and applicability domain. The review gathers and presents the step-wise information on techniques and procedures of existing models that can be used readily to assemble new nanotoxicological in silico studies and accelerates the regulation of in silico tools in nanotoxicology. ML applications in nanotoxicology comprise an active and diverse collection of ongoing efforts, although it is still in their early steps toward a scientific accord, subsequent guidelines, and regulation adoption. This study is an important bookend to a decade of ML applications to nanotoxicology and serves as a useful guide to further in silico applications.

Length-dependent toxicity of TiO2 nanofibers: mitigation via shortening.

Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO2 nanofibers (TiO2 NF) cause a dose-dependent decrease of cell viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO2 NF of industrial origin. Long TiO2 NF (L-TiO2 NF) were more cytotoxic than their shortened counterparts (S-TiO2 NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO2 NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO2 NF, S-TiO2 NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO2 NF but not to S-TiO2 NF, suggesting that short-term macrophage activation in vitro may not be always a reliable indicator of persistent inflammation in vivo. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence in vitro as well as inflammation development in vivo. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO2 NF toxic effects.

Assessment of the physicochemical properties of chrysotile-containing brake debris pertaining to toxicity.

Grinding and drilling of chrysotile asbestos-containing brake pads during the 20th century led to release of chrysotile, resulting in varying levels of workplace exposures of mechanics. Despite exposures, excess risk of mesothelioma remains in doubt. Objectives: The toxicity of particulates is primarily derived through a combination of physicochemical properties and dose and as such this study aimed to determine properties of asbestos-containing brake debris (BD) which may influence pathogenicity and potential of mesothelioma. Materials and Methods: Chrysotile-containing brake pads were ground - to reflect occupational activities, aerosolized, and size-fractionated to isolate respirable fractions. Analysis of morphology, biodurability, surface charge, and interactions with macrophages were undertaken. Results: The respirable fraction of BD contained ∼15-17% free chrysotile fibers thereby constituting a small but relevant potential long fiber dose. Acellular biodurability studies showed rapid dissolution and fragmentation of chrysotile fibers that was consistent for pure chrysotile control and BD samples. Conclusions: The long, free, respirable chrysotile fibers were present in BD, yet were of low bio-durability; incubation in artificial lysosomal fluid led to destruction of free fibers.

Application of Bayesian networks in determining nanoparticle-induced cellular outcomes using transcriptomics.

Inroads have been made in our understanding of the risks posed to human health and the environment by nanoparticles (NPs) but this area requires continuous research and monitoring. Machine learning techniques have been applied to nanotoxicology with very encouraging results. This study deals with bridging physicochemical properties of NPs, experimental exposure conditions and in vitro characteristics with biological effects of NPs on a molecular cellular level from transcriptomics studies. The bridging is done by developing and implementing Bayesian Networks (BNs) with or without data preprocessing. The BN structures are derived either automatically or methodologically and compared. Early stage nanotoxicity measurements represent a challenge, not least when attempting to predict adverse outcomes and modeling is critical to understanding the biological effects of exposure to NPs. The preprocessed data-driven BN showed improved performance over automatically structured BN and the BN with unprocessed datasets. The prestructured BN captures inter relationships between NP properties, exposure condition and in vitro characteristics and links those with cellular effects based on statistic correlation findings. Information gain analysis showed that exposure dose, NP and cell line variables were the most influential attributes in predicting the biological effects. The BN methodology proposed in this study successfully predicts a number of toxicologically relevant cellular disrupted biological processes such as cell cycle and proliferation pathways, cell adhesion and extracellular matrix responses, DNA damage and repair mechanisms etc., with a success rate >80%. The model validation from independent data shows a robust and promising methodology for incorporating transcriptomics outcomes in a hazard and, by extension, risk assessment modeling framework by predicting affected cellular functions from experimental conditions.

Machine learning prediction of nanoparticle in vitro toxicity: A comparative study of classifiers and ensemble-classifiers using the Copeland Index.

Nano-Particles (NPs) are well established as important components across a broad range of products from cosmetics to electronics. Their utilization is increasing with their significant economic and societal potential yet to be fully realized. Inroads have been made in our understanding of the risks posed to human health and the environment by NPs but this area will require continuous research and monitoring. In recent years Machine Learning (ML) techniques have exploited large datasets and computation power to create breakthroughs in diverse fields from facial recognition to genomics. More recently, ML techniques have been applied to nanotoxicology with very encouraging results. In this study, categories of ML classifiers (rules, trees, lazy, functions and bayes) were compared using datasets from the Safe and Sustainable Nanotechnology (S2NANO) database to investigate their performance in predicting NPs in vitro toxicity. Physicochemical properties, toxicological and quantum-mechanical attributes and in vitro experimental conditions were used as input variables to predict the toxicity of NPs based on cell viability. Voting, an ensemble meta-classifier, was used to combine base models to optimize the classification prediction of toxicity. To facilitate inter-comparison, a Copeland Index was applied that ranks the classifiers according to their performance and suggested the optimal classifier. Neural Network (NN) and Random forest (RF) showed the best performance in the majority of the datasets used in this study. However, the combination of classifiers demonstrated an improved prediction resulting meta-classifier to have higher indices. This proposed Copeland Index can now be used by researchers to identify and clearly prioritize classifiers in order to achieve more accurate classification predictions for NP toxicity for a given dataset.

The toxicology of chrysotile-containing brake debris: implications for mesothelioma.

The global use of "asbestos" in various commercial products has led to a wide range and pervasive legacy of disease. One such use of chrysotile asbestos was brake pads and was utilized commonly in automobiles and heavy vehicles. The result of incorporation of chrysotile into brake pads is associated with the exposure of mechanics fitting and servicing vehicles to liberated chrysotile fibers. Despite the proven exposure, the relative risk of malignant mesothelioma (MM) in this occupational population is broadly seen as low. The toxicity of particulates, including fibers such as chrysotile, is driven by a combination of dose and physicochemical properties. As such, it is plausible that chrysotile released from brake pads may have undergone modification, thereby altering the pathogenicity profile. The impact of high sheer stress causing shortening of long fibers, heat modification, binding of resin matrix to the fiber surface on the relative toxicity of brake debris with regards to MM is considered. It is apparent that released chrysotile can undergo significant modification, reducing the long fiber dose although not all modifications may lead to reduced toxicity.

Silica modification of titania nanoparticles enhances photocatalytic production of reactive oxygen species without increasing toxicity potential in vitro.

Titania (TiO2) nanoparticles were surface modified using silica and citrate to implement a 'safe-by-design' approach for managing potential toxicity of titania nanoparticles by controlling surface redox reactivity. DLS and zeta-potential analyses confirmed the surface modification, and electron microscopy and surface area measurements demonstrated nanoscale dimensions of the particles. Electron paramagnetic resonance (EPR) was used to determine the exogenous generation of reactive oxygen species (ROS). All the produced spray dried nanotitania lowered levels of ROS when compared to the corresponding dispersed nanotitania, suggesting that the spray drying process is an appropriate design strategy for the control of nano TiO2 ROS reactivity. The modification of nanotitania with silica and with citrate resulted in increased levels of ROS generation in exogenous measurements, including photoexcitation for 60 minutes. The dichlorodihydrofluorescein (DCFH) assay of dose-dependent production of oxidative stress, generated by pristine and modified nanotitania in macrophages and alveolar epithelial cells, found no significant change in toxicity originating from the generation of reactive oxygen species. Our findings show that there is no direct correlation between the photocatalytic activity of nanotitania and its oxidative stress-mediated potential toxicity, and it is possible to improve the former, for example adding silica as a modifying agent, without altering the cell redox equilibrium.

Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a (Ink4a/Arf).

Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard.