Heat-not-burn tobacco products (IQOS) have emerged as potentially reduced-risk alternatives to combustible cigarettes (CSs), yet their inhalation toxicity profile remains incompletely characterized. This study evaluated the comparative respiratory and systemic effects of IQOS aerosol vs. CS smoke through whole-body inhalation exposure in an experimental rat model. Twenty-one male Wistar albino rats (8-10 weeks, 250-300 g) were randomly assigned to three groups (n = 7): control (ambient air), CS exposure (10 mg nicotine/day, ∼6 cigarettes/session), and IQOS exposure (equivalent nicotine dose, ∼8 HeatSticks/session). A custom-designed whole-body inhalation exposure system, developed in collaboration with Istanbul Technical University, delivered standardized aerosols following Health Canada Intense puffing parameters (55 mL puff volume, 27.5 mL/s flow rate, 2-s puff duration, 30-s inter-puff interval) during daily 1.5-h sessions over 21 consecutive days. Peripheral blood samples were collected on Days 7, 14, and 21 for assessment of plasma inflammatory markers (IL-1β, IL-6, TNF-α, and hs-CRP), oxidative stress parameters (total oxidant status [TOS], total antioxidant status, oxidative stress index), and DNA damage in isolated mononuclear leukocytes using the alkaline comet assay. Plasma cotinine was quantified as the primary biomarker of systemic nicotine absorption. Terminal lung tissue analysis on Day 21 evaluated local respiratory oxidative stress, inflammatory cytokine concentrations, and tissue cotinine deposition. . Plasma TOS increased progressively in both exposure groups (CS: 9.2 ± 1.82 to 14.71 ± 1.65 µmol H2O2 Eq/L; IQOS: 6.81 ± 1.25 to 11.49 ± 1.30 µmol H2O2 Eq/L), with CS demonstrating significantly higher oxidative burden. DNA damage in circulating cells escalated markedly (CS: 21.74 ± 2.51% to 47.76 ± 3.67% tail DNA; IQOS: 16.22 ± 1.87% to 33.44 ± 2.41% tail DNA). Lung tissue analysis revealed significant 'elevation of inflammatory markers and oxidative stress parameters' in both exposure groups. While IQOS inhalation induced less severe toxicity than CS smoke, it produced significant respiratory and systemic adverse effects including oxidative stress, inflammatory responses, and DNA damage. These findings challenge claims of substantially reduced harm for heat-not-burn products and support continued regulatory oversight of inhaled tobacco delivery systems.
Carbon monoxide (CO) occupies an unusual position at the boundary between environmental toxicology and therapeutic pharmacology. The same molecule is regulated as an involuntary toxic exposure in public-health and occupational settings, yet it is also being explored as a controlled therapeutic agent in acute inflammation, organ preservation, wound repair and selected local-delivery indications. This critical narrative review was written to clarify three issues that have repeatedly distorted interpretation of the field: the quantitative endogenous baseline of CO (a haem-derived pool that can approach roughly 150 μmol/L in blood, far above a trace contaminant), the analytical validity of CO donor systems and the need to keep environmental exposure limits separate from drug-development risk assessment. Structured scoping searches of PubMed, Web of Science and the Cochrane Library were used to identify mechanistic, toxicological, donor chemistry, exposure metric and regulatory literature through November 2025. We place particular weight on post-2018 evidence showing that widely used ruthenium carbonyls, especially CORM-2 and CORM-3, do not behave as reliable CO donors under many physiological conditions and that several historical biological effects may reflect ruthenium reactivity rather than CO signalling. We argue that CO studies should be read through a donor-validation filter that favours gaseous CO, physiologically characterised organic donors, gas-entrapping materials and measured exposure metrics such as carboxyhaemoglobin (COHb). To make this operational, we propose a three-tier evidence scheme for grading CO biology and a minimum reporting set for CO intervention studies. The most defensible translational opportunities are short, monitored and reversible interventions in acute or localised settings; chronic systemic inhalation for diffuse disease remains less plausible because systemic COHb elevation cannot be confined to the diseased tissue. For applied toxicology, a coherent framework for CO therefore requires validated exposure metrics, explicit donor chemistry and strict separation of therapeutic benefit-risk assessment from population-protective environmental limits.
Background: Osteoporosis, a debilitating bone disease characterized by low bone mineral density, poses a large burden on the population. Current pharmacological treatment options are limited, with antiresorptive drugs being the current first-line option. Recent research has shown that the endocannabinoid system, modulated by endocannabinoids and phytocannabinoids, may influence bone remodeling. As the prevalence of cannabis use and research into its medicinal potential continues to increase, its therapeutic potential has emerged. Objective: To investigate the role of Δ9-tetrahydrocannabinol (THC) inhalation in treating osteoporosis in a rodent model. Methods: Adult female Sprague-Dawley rats underwent ovariectomy (OVX) to induce osteoporosis. Four weeks post-OVX, rats received either THC or Air treatment via inhalation for 8 weeks. A control group of age-matched rats received sham surgery. Rats were then euthanized at 38 weeks old, and hindlimb samples were collected for caliper, microCT, and biomechanical analyses. Results: OVX Air-treated rats showed significantly decreased trabecular bone volume, trabecular bone volume fraction, trabecular separation, trabecular tissue mineral density, trabecular number, and connective density compared to sham surgery controls. Compared with Air controls, OVX rats treated with THC showed increased endosteal volume (20%; p < 0.05). THC-treated rats also showed decreased trabecular bone mineral density (2%; p < 0.005) and trabecular thickness (14%; p < 0.05). Conclusions: Chronic inhaled THC worsened key aspects of trabecular bone microarchitecture. Results do not support use of THC vapor as a therapeutic agent for osteoporosis and suggest THC may adversely affect bone quality in estrogen-deficient states. Further research is needed to evaluate dose-dependent effects and to distinguish the skeletal impacts of different cannabinoid components.
Inhalation of ultrafine particles (UFP) mediates systemic vascular impairment which is, in part, driven by elevated rates of oxidant generation. One significant source of oxidant production in the vascular compartment is the purine catabolizing enzyme, xanthine oxidoreductase (XOR). However, mechanisms linking XOR and/or endothelial glycosaminoglycan (GAG)-sequestered XOR to vessel dysfunction allied to UFP inhalation remain underexplored. Based on known interactions between UFP and the liver, we hypothesized that exposure could lead to hepatic release of XOR to the circulation which subsequently contributes to vascular impairment. Utilizing our murine hepatocyte-specific XOR knockout (XORHep-/-) model (loss of function) in conjunction with reintroducing exogenous XOR (restoration of function) we demonstrate a specific role for liver-derived XOR in the pathogenesis of UFP-induced vascular impairment. Exposure of mice as well as in vitro exposure of hepatocytes to our model UFP, nano titanium dioxide (nTiO2) results in the upregulation and active release of XOR. Drinking water supplemented with the XOR inhibitor febuxostat or nitrite (NaNO2-) partially prevented nTiO2-induced impairment of vascular reactivity. Interestingly, nitrite appears to cause a down-regulation of hepatic XOR. XORHep-/- mice were partially protected against both impairment of endothelial dependent dilation and augmented angiotensin II constriction. To further demonstrate the role of circulating XOR in nTiO2-induced impairment of vessel reactivity, XORHep-/- mice had circulating XOR restored by i. v. Injection prior to exposure, which eliminated the protection of the hepatic knockout. It is important to note that acute restoration of intraluminal XOR in isolated vessels did not alter endothelial-dependent dilation or angiotensin II constriction. As such, we interrogated potential downstream mediators of XOR effects on endothelial function and found a decrease in the repressive trimethylation of lysine 9 on histone 3. Together these findings demonstrate that circulating XOR is a key contributor to endothelial dysfunction caused by UFP exposure. However, the impairment is not acute in nature and might involve epigenetic-mediated alterations in gene expression.
Airborne micro- and nanoplastics are emerging environmental contaminants of increasing concern, yet their inhalation toxicity and pulmonary fate remain insufficiently understood. In this study, we investigated how particle morphology and surface oxidation influence pulmonary inflammation and clearance using environmentally relevant, size-controlled polystyrene (PS) nanoplastics. Spherical and fragmented PS were generated via bottom-up and top-down approaches, respectively, and subjected to ultraviolet irradiation to simulate environmental aging. Fragmented and ultraviolet (UV)-aged nanoplastics exhibited enhanced surface oxidation and a higher intrinsic oxidative potential than pristine spherical particles. Following a single pulmonary exposure in mice via pharyngeal aspiration at doses of 25-100 μg per mouse, these environmentally transformed nanoplastics induced more severe acute pulmonary inflammation. In particular, UV-aged fragmented PS (100 μg per mouse) increased neutrophil counts by 3.9-fold compared with pristine spherical PS, along with increased pro-inflammatory cytokine production, which was closely associated with particle-derived oxidative reactivity. Although acute inflammatory responses were largely reversible, time-course analysis of lung burden after a single non-overload exposure (25 μg per mouse) revealed delayed pulmonary clearance of fragmented nanoplastics relative to spherical particles, with estimated clearance half-lives of 13.5 days for pristine spherical PS and 27.4 days for UV-aged fragmented PS. Overall, this study demonstrates that nanoplastics most relevant to real-world environmental exposure may pose a greater risk of respiratory health effects, along with prolonged lung residence.
Tris(1-chloro-iso-propyl) phosphate (TCIPP) emerged as the predominant organophosphorus flame retardants (OPFRs) in both dust and air environments. This study investigated its respiratory toxicity using mouse inhalation model. Molecular docking and dynamics simulations revealed that TCIPP could binds to glutathione peroxidase 4 (GPX4) with an affinity of -5.3098 kcal/mol, stabilizing the protein conformation. Inhalation TCIPP exposure (34 μg/m3 for 1 h) significantly reduced GPX4 expression and GSH levels in lung tissue. Histopathological examination indicated severe pulmonary injury, including inflammatory cell infiltration, alveolar wall thickening and apoptosis after TCIPP inhalation. Besides, DHE staining revealed that the ROS level increased by 94.7% in the lung after TCIPP exposure. Meanwhile, the results revealed a 50.7% increase in lung iron content (Fe2+), along with elevated ferroptosis markers (p53, Transferrin Receptor 1 (TFR1)) and reduced FTH1 levels following TCIPP inhalation. These effects were counteracted by the ferroptosis inhibitor Fer‑1 (0.8 mg/kg), which also attenuated pathological damage, ROS accumulation, and NLRP3‑mediated pyroptosis. Concurrently, electron microscopy analysis demonstrated mitochondrial cristae membrane disruption and shrinkage, accompanied by dysregulated expression of mitochondrial dynamics regulators (down‑regulated Mfn1/2 and up‑regulated Drp1), all of which were reversed by Fer‑1. Collectively, the results indicate that TCIPP inhalation induces GPX4‑dependent ferroptosis, promoting inflammatory responses and mitochondrial imbalance, ultimately driving respiratory toxicity. Ferroptosis thus represents a potential therapeutic target for organophosphorus flame retardant‑associated lung injury, which may aid in biomarker discovery for related pulmonary diseases.
Organophosphate flame retardants (OPFRs) such as tributyl phosphate (TBP) are widely used industrial additives increasingly detected in the environment and human tissues, yet their inhalation toxicity remains poorly characterized. In this study, we employed the ex vivo porcine precision-cut lung slice (pPCLS) model to investigate the cytotoxic, oxidative stress, and structural effects of aerosolized TBP under both acute and repeated exposure conditions. Using the Vitrocell®, pPCLS were exposed to TBP aerosols at deposited surface concentrations (µg/cm²) within ranges commonly applied in ALI-based inhalation toxicology models and selected to simulate cumulative low-dose exposure scenarios reported for organophosphate flame retardants in indoor environments. Acute exposure (24 h) caused a reduction in tissue viability, increased reactive oxygen species (ROS) production, and enhanced caspase-3 activation, indicating mitochondrial stress and apoptosis induction. There is a potential tendency for markers (K-RAS, p53, and MMP-15) to increase. Histological analyses revealed disruption of extracellular matrix architecture. Repeated low-dose exposure (5 × 24 h) led to progressive tissue disorganization and early signs of fibrogenic remodeling, evidenced by increased collagen deposition, p53 expression, reduced tissue viability, and sustained oxidative stress. These findings suggest that cumulative subcytotoxic exposure to aerosolized TBP can initiate pathways associated with chronic pulmonary injury. The pPCLS model demonstrated high reproducibility, preserved lung architecture, and responsiveness to chemical insult, reinforcing its translational relevance for respiratory toxicology. Overall, our results provide mechanistic insight into TBP-induced lung toxicity and highlight precision-cut lung slices as a useful model for evaluating inhalation hazards of industrial chemicals.
Inhalation is a major route of chemical exposure for both consumers and workers. Physiologically-based kinetic (PBK) modeling is a promising tool to understand the absorption, distribution, metabolism, and excretion (ADME) of inhaled chemicals and to predict systemic concentrations of chemicals in humans. New Approach Methodologies (NAMs) can help generate essential input parameters for PBK models. However, validated NAM-based test methods to assess uptake of inhaled chemicals are currently lacking. Reliable information on respiratory uptake is required to determine relevant exposure concentrations for evaluation of systemic effects using NAMs. This manuscript describes a project that aims to apply robust and reliable in vitro models to study cellular uptake, intracellular accumulation, absorption and systemic exposure of chemicals following inhalation. To evaluate the robustness and predictivity of different NAM-based barrier models, to examine appropriate in vitro to in vivo scaling strategies, and to assess sensitivity and uncertainty in the resulting PBK models, the project will focus on relatively data-rich chemicals, specifically per- and polyfluoroalkyl substances (PFAS). While some have been widely explored and others remain data-poor, the entire chemical family is of interest due to its health hazards. Therefore, the work combines experimental and modeling approaches by generating in vitro data on the respiratory uptake and benchmark this to existing human in vivo data, developing biokinetic models to better understand chemical fate within the test systems, and refining inhalation PBK models to improve estimates of systemic uptake. Read-Across (RAx) will be employed as data gap filling technique to infer on the apparent permeability of non-tested PFAS. Together, the in vitro and in silico results will inform and parameterize PBK models, ultimately enabling more reliable predictions of systemic availability. The project will deliver a workflow to combine in vitro and in silico methods to assess the uptake of inhaled substances, that could be modified and applied to other inhaled substances. Standardized in vitro models for respiratory uptake will improve the evaluation of inhalation as a route of exposure contributing to systemic effects, which is a key requirement for quantitative in vitro to in vivo extrapolation (qIVIVE) and supports the implementation of next-generation risk assessment (NGRA).
Formaldehyde is highly reactive, causing irritation and acute and chronic toxicity in target tissues (the eyes, respiratory tract, and skin) following inhalation and/or skin contact. Moreover, formaldehyde is a local carcinogen after inhalation exposure. It induces nasal squamous cell carcinoma in laboratory animals and nasopharyngeal carcinoma in humans, albeit less convincingly. While formaldehyde has been evaluated and classified by different organizations and committees in recent years, the question has arisen of how to deal with substances that release formaldehyde. These releasers decompose in aqueous media and are frequently used as biocides in water-miscible metalworking fluids, adhesives, paints, disinfectants, and cosmetics. In this context, the MAK Commission has developed a concept for differential evaluation regarding carcinogen classification and MAK value derivation. This approach is based on hydrolysis rates as a function of pH, temperature, and concentration under physiological conditions. Furthermore, vapor pressure is considered to determine whether the formaldehyde releaser will be present as a vapor or aerosol upon inhalation. This concept is demonstrated using four examples that lead to different carcinogen classifications and MAK value derivations. Additionally, the releasers were evaluated for percutaneous absorption and sensitization.
A triple-blinded, independent panel of five experts was engaged to review information on the cancer mode of action (MOA) of vinyl acetate monomer (VAM) to provide input on key decisions for the calculation of No-Significant-Risk-Level (NSRL) values using best available science. Based upon the weight of evidence, the panel expressed a strong preference for using effects observed at the point of contact for the NSRL rather than in using those observed in systemic tissues. None of the systemic endpoints were identified as useful for quantitative risk assessment. A cancer MOA involving cytotoxicity and regenerative cell proliferation was best supported by the weight of evidence (mean score of +3.8 ± 1.1; scale of -5 to +5). In contrast, a cancer MOA involving direct genotoxicity was not supported for VAM (mean score of -3.6 ± 2.1). Separate NSRL values were derived for inhalation and oral routes. Based upon the decisions recommended by the panel at each step of the process, inhalation and oral NSRL values of 20,000 and 110,000 μg/day were calculated, respectively. Confidence in the inhalation and oral NSRL values was considered high and medium, respectively by the panel. Human exposures at or below these NSRL levels are not expected to pose a significant cancer risk.
Plastic fragmentation leads to widespread microplastic (MP) pollution. Inhalation of airborne MPs is a key human exposure route. Evidence links MPs to respiratory diseases, but causal mechanisms and real-world risks remain unclear. This review synthesizes evidence on human exposure to airborne MPs and their role in major respiratory diseases. It aims to identify critical knowledge gaps and improve the framework for assessing inhalation health risks. This is a systematic review study. PubMed, Scopus, and Web of Science were searched (up to 15 June 2025) using keywords for MPs and respiratory diseases. Two researchers independently screened articles, extracted data, and assessed the quality of the studies. A total of 17 studies were included. MPs are found in human lungs and can induce inflammation. Evidence associates chronic exposure with increased risks of COPD, asthma, and pulmonary fibrosis. A major limitation is that experimental animal studies use MP concentrations vastly exceeding environmental levels, limiting real-world risk extrapolation. Research is also disproportionately focused on polystyrene (PS), while occupational data implicate other polymers. Airborne MPs are a respiratory toxicant, but definitive causal evidence is constrained by a critical gap between experimental and environmentally relevant concentrations and a polymer-specific research bias. Future studies must prioritize environmentally relevant exposures, investigate high-risk polymers, and integrate exposomics with advanced toxicology.
Formic acid is widely used in rubber latex coagulation, especially in semiurban and rural industrial settings. Its unregulated handling poses significant occupational health hazards. The objectives of the case series are to document the clinical, toxicological, and autopsy findings in six fatal cases of formic acid poisoning among rubber industry workers in Tripura, India. To document the clinical, toxicological, and autopsy findings in six fatal cases of formic acid poisoning among rubber industry workers in Tripura, India. This retrospective case series analyzed clinical records, autopsy findings, histopathology, and toxicology reports of six deceased individuals exposed to formic acid through ingestion or inhalation. All victims were rubber factory or plantation workers; five deaths were suicidal and one accidental. Common features included metabolic acidosis, gastrointestinal corrosion, pulmonary edema, and multi-organ failure. Histopathology revealed acute tubular necrosis, gastric mucosal necrosis, and alveolar edema. Toxicology confirmed lethal levels of formic acid. These cases highlight critical gaps in occupational safety, chemical handling practices, and mental health support in industrial environments handling corrosive agents like formic acid.
BZ (3-quinuclidinyl benzilate) is a potent antimuscarinic incapacitating agent for which acute toxicity remains insufficiently defined because available evidence is limited, heterogeneous, and largely historical. In this study, an oral-first in silico workflow was applied to characterize the predicted acute toxicity of BZ, with quantitative emphasis on oral LD50 estimation and exploratory qualitative contextualization of dermal and inhalation hazards. The workflow combined qualitative hazard screening, quantitative prediction of acute oral toxicity, validation against structurally and toxicologically relevant reference compounds, and heuristic interspecies scaling sensitivity analysis of rat-derived oral theoretical LD50 (t-LD50) values. Qualitative screening consistently indicated elevated toxicological concern. BZ was assigned to Cramer Class III in Toxtree, and binary classifiers concordantly predicted toxicity for the oral route, whereas dermal and inhalation classifications were less consistent across tools. Quantitative modelling further refined this profile. Most estimators converged within a relatively narrow oral t-LD50 range, while a smaller subset produced higher values, indicating a stable central toxicity band with model-dependent upper-range extension. Validation against reference compounds showed heterogeneous model performance, supporting interpretation within a multi-model weight-of-evidence framework rather than reliance on a single estimator. Heuristic interspecies scaling using three simplified approaches yielded different absolute scaled values but preserved the relative ranking of the source rat-derived oral predictions, indicating internal consistency of this sensitivity analysis. These findings indicate that BZ exhibits a reproducible predicted acute oral toxicity signal that can be captured by an uncertainty-aware multi-model computational strategy. Although such an approach does not replace compound-specific experimental toxicology or higher-tier toxicokinetic modelling, it provides a structured first-tier evidence base for BZ and illustrates the value of integrated in silico methods for the assessment of rare and poorly characterized hazardous compounds.
Combustion of composite solid propellants generates complex aerosols containing metallic nanoparticles (NPs) and acidic gases, raising concerns about inhalation toxicity. Here, we investigated the effects of alumina (Al₂O₃) NPs, alone or combined with hydrochloric acid (HCl), in a human alveolar-capillary barrier (ACB) model exposed at the air-liquid interface. The model consisted of hAELVi alveolar epithelial cells co-cultured with HPMEC-ST1.6 R pulmonary microvascular endothelial cells and exposed using a Vitrocell Cloud system under acute or repeated scenarios. Deposited Al₂O₃ NPs doses corresponding to low-to-high alveolar exposure scenarios were 0.007, 1.35 ± 0.1 and 2.3 ± 0.2 µg/cm², with or without HCl at 1.37 mM. Barrier integrity, viability, inflammatory and remodeling mediators, oxidative stress and DNA damage were assessed 24 h after the final exposure. The co-culture formed a tight functional barrier with high transepithelial electrical resistance, low permeability and organized junctional protein expression. Acute exposure to the highest Al₂O₃ NPs dose induced a compartment-specific inflammation-related response, based on the biomarkers investigated, with increased IL-1α, IL-8, IL-1β, CCL2, DKK1 and angiogenin, without cytotoxicity or barrier disruption. Repeated exposure shifted the response toward moderate cytotoxicity at the highest dose, with viability decreasing to 77% for Al₂O₃ NPs alone and 66% for Al₂O₃ NPs with HCl, while permeability remained unchanged. No significant oxidative stress or γ-H2AX-associated DNA damage was detected. HCl co-exposure did not enhance Al₂O₃ NPs toxicity. However, because HCl was administered in dissolved form prior to nebulization, the contribution of potential gas-particle interactions occurring in combustion plumes could not be evaluated and the results should not be interpreted as evidence for or against synergistic effects arising from gas-particle interactions occurring in combustion plumes. These findings show that exposure pattern critically shapes alveolar-capillary responses, supporting human air-liquid interface co-culture models for mechanistic inhalation toxicology.
Understanding intrapulmonary pharmacokinetics (PK) following inhalation remains a significant challenge in drug development and repurposing. Current lung sampling methods include bronchoalveolar lavage (BAL), biopsies, and the more recent bronchosorption technique, which enhances regional specificity while reducing potential quantification errors. This study aimed to develop a pulmonary population physiologically based pharmacokinetic (PBPK) model for inhaled salbutamol by integrating data from all three sampling techniques to improve PK predictions and to compare different sampling strategies to optimize future study designs. A population-based minimal PBPK model was developed using data from a previously published study (NCT03524066) investigating salbutamol's pulmonary and plasma PK in 13 healthy volunteers after inhalation. Simulations assessed the impact of permeability on pulmonary PK profiles and BAL-derived epithelial lining fluid (ELF)-to-plasma ratios using salbutamol as a reference compound. Stochastic simulation-estimation (SSE) methods were employed to assess the feasibility of different sampling strategies for estimating key parameters of the PBPK model. First, we evaluated using one or two sampling techniques within a single bronchoscopy session. Second, we compared uniform and staggered bronchosorption-based sampling strategies for drugs from different permeability categories. The minimal PBPK model described pulmonary PK of salbutamol across the lung and estimated the unbound tissue-plasma partition coefficient for the lung ( K p , u , lung ) and the effective permeability ( P eff ) of salbutamol as 11.0 and 0.543 m/h, respectively. Inter-individual variabilities (IIV) were found on plasma clearance and lung deposition fraction. No significant IIV was detected on K p , u , lung or P eff . Simulations indicated that low-permeability drugs exhibited higher concentrations in the ELF, while high-permeability drugs accumulated more in lung tissues, after inhalation. Results from SSE showed that bronchosorption plus biopsy were the most informative two-technique combination and bronchosorption alone was the best single-technique option. Additionally, the optimal sampling strategy for both uniform and staggered sampling depended on drug permeability, with early time points favoured for high-permeability drugs and later or broader windows needed for low-permeability drugs. A pulmonary population PBPK model for inhaled salbutamol was developed by integrating detailed intrapulmonary data from bronchoalveolar lavage, biopsy, and bronchosorption. The study revealed that parameter estimates of K p , u , lung and P eff were sensitive to the sampling technique. Staggered sampling strategies mitigated the risk of biased estimates, though the ideal sampling windows varied by drug's permeability. These findings support model-informed, permeability-driven study design in inhaled drug development.
The primary purpose of this study was to examine patterns in the reliance on respiratory tract irritation as a toxicological basis for occupational exposure limits (OELs), focused on U.S.-based OELs. This study pursued three aims: (1) to evaluate the degree to which OELs are based on respiratory irritation, or more broadly, on respiratory effects; (2) to assess the variability in the OEL values across organizations; and (3) to determine how well surrogate endpoints align with respiratory irritation for OELs having respiratory tract effects as their critical effect. To address these aims, a database was developed that listed the chemical OELs from the German MAK (Maximale Arbeitsplatz-Konzentration) Commission, U.S. National Institute for Occupational Safety and Health (NIOSH), U.S. Occupational Safety and Health Administration (OSHA), California OSHA, American Conference of Governmental Industrial Hygienists (ACGIH®), and the Workplace Environmental Exposure Levels Committee (WEEL; initially derived under the American Industrial Hygiene Association (AIHA®), and after 2012 via Toxicology Excellence for Risk Assessment (TERA)). Information on inhalation toxicity, specifically respiratory irritation, was also included. Analyses of the collated data found the following. Most chemicals in the database with at least one OEL had three or four OELs from different organizations; there was variability among the OEL numeric values, but most chemicals differed by a factor of less than 10. The respiratory tract was the site of the critical or co-critical effect (i.e., the one occurring at the lowest concentration) for ∼50% of the chemicals in the database, illustrating the importance of consideration of this target organ in OEL development. Inconsistencies between GESTIS-curated Globally Harmonized System (GHS) Hazard (H) codes and OEL documentation regarding respiratory effects were identified, suggesting that the potential for respiratory effects may not be adequately addressed in assigning H codes. Neither skin nor eye irritation-related H codes are sufficiently sensitive to predict potential respiratory irritation, so other surrogate measures are needed to evaluate the potential to cause respiratory irritation of chemicals for which inhalation toxicity data are not available.
An albumin-humanized mouse model was generated via crossbreeding between albumin-transgenic and albumin-null mice and characterized. Immunoblot analyses confirmed that only human, but not mouse, albumin was expressed, and they revealed that the level of serum human albumin in the albumin-humanized mouse was 5-6 times the level in the albumin-transgenic mouse. Serum albumin levels (measured in plasma) were 3-4 times lower, whereas globulin levels were 2 times higher, in the albumin-humanized than in wild-type mice. The transgene integration site was localized via optical genome mapping to mouse chromosome 14, between 2 marker positions that are 143-kilobase pairs apart. A neighboring gene (alpha fetal protein) to albumin on the human chromosome was confirmed absent in the transgenic mouse. A transgene-mouse genome junction sequence was further identified, enabling a new protocol to distinguish between hemizygous and homozygous albumin-transgenic mice. Transgenic human albumin formed covalent adducts with naphthalene 1,2-oxide (NAO) ex vivo at the Cys34 residue, which was recovered in the peptide 31LQQCPF36 following tryptic/chymotryptic digestion. LQQC∗[NAO]PF, structurally confirmed by mass spectral comparisons with synthetic adduct standards, was also detected in protease-digested plasma from homozygous albumin-humanized mice that were exposed to naphthalene through intraperitoneal injection (200 mg/kg) or inhalation (20 ppm). Pharmacokinetic analysis of plasma NAO-albumin, in comparison with NAO-glutathione, revealed a later time at maximal concentration (9.6 versus 2 hours), lower maximal concentration (0.21 versus 12 μM), and longer elimination half-life (79.7 versus 2.3 hours) for NAO-albumin following intraperitoneal naphthalene injection; the persistence of NAO-albumin adduct was also demonstrated following inhalation naphthalene exposures. SIGNIFICANCE STATEMENT: An albumin-humanized mouse was generated and characterized to demonstrate its utility for detecting in vivo formation of reactive metabolite adducts with human albumin. Key findings, including detection of the albumin adduct with naphthalene 1,2-oxide (NAO) at Cys34 following exposure of mice to naphthalene, a ubiquitous pollutant and possible human carcinogen, and persistence of the NAO-albumin adduct in plasma relative to NAO-glutathione adducts, indicate that the model will be valuable for discovering biomarkers of exposure and potential toxicity for numerous drugs and other xenobiotics.
Respiratory diseases, encompassing chronic inflammatory conditions, interstitial fibrotic disorders, acute infectious diseases, and pulmonary malignancies, represent a profound global health burden with unacceptably high morbidity and mortality rates. Historically, the pharmaceutical pipeline for respiratory therapeutics has suffered staggering attrition rates during clinical development. This is primarily due to the fundamental inability of conventional two-dimensional cell cultures and in vivo animal models to faithfully recapitulate the complex three-dimensional architecture, multicellular heterogeneity, and human-specific physiological dynamics of the pulmonary system. To bridge this critical translational gap, lung organoids-self-organizing, three-dimensional microphysiological constructs derived from pluripotent or adult stem cells-have emerged as a useful human-cell-based platform. This comprehensive review critically evaluates current lung organoid technologies, elucidating their derivation pathways and capacity for high-fidelity disease modeling. We analyze their application in dissecting the pathogenesis of chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, viral infections including SARS-CoV-2, and non-small cell lung cancer. Furthermore, we highlight their important role in predictive toxicology for assessing environmental inhalation hazards, cosmetic safety, and drug-induced lung injury, aligning with evolving regulations prioritizing alternatives to animal testing. Despite their immense potential, widespread clinical and industrial translation is currently impeded by biological bottlenecks: the absence of functional vascularization, incomplete immune integration, and reliance on undefined xenogeneic matrices. We systematically examine bioengineering strategies addressing these limitations-including synthetic hydrogels, microfluidic organ-on-a-chip platforms, and 3D bioprinting-to overcome translational hurdles, accelerate precision medicine, and improve respiratory pharmacology.
Smoking-related disease is related to inhalational toxicant exposure. Here, we assessed levels of 17 analytes, which have a potential link to smoking-related disease, in aerosol from four variants of the ElfBar Elfa vaping product compared with cigarette smoke levels or safety limits. Carbon monoxide, NNK and NNN, acrolein, benzo[a]pyrene, 1,3-butadiene, benzene, cadmium and arsenic were below detectable or quantifiable levels in all vaping product aerosols. For formaldehyde and acetaldehyde, these were between 99.6% and 99.9% lower than levels in 3R4F reference cigarette smoke depending on the variant assessed. For metals quantifiable in the aerosols, assumed potential exposure levels among users were all significantly lower than USP permissible daily exposure levels for inhalational medicines or below NIOSH-recommended exposure limits. Overall, toxicant exposure among smokers completely switching to Elfa vaping product use would be significantly reduced, or eliminated, compared with continued smoking. Further, metal exposure is likely not of toxicological concern.
Ethylene oxide (EtO) is a highly reactive industrial chemical and known human carcinogen with a mutagenic mode of action (MOA). Its genotoxicity is primarily mediated through alkylation of DNA, forming the mutagenic adduct O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6-HE-dG), albeit in small quantities, and the more abundant but less- or nonmutagenic N7-(2-hydroxyethyl)guanine (N7-HE-G) adduct. However, dose-response relationships of these DNA adducts, particularly at low inhalation exposure levels (< 3 ppm), remain unknown. These data are necessary to inform the biological plausibility of different statistical dose-response models that have been applied to human or animal data used for cancer risk assessment. In this study, B6C3F1 mice were exposed to EtO (0 to 200 ppm) for 6 h/d over 28 consecutive days. DNA adducts in lung, liver, bone marrow, and mammary gland were quantified using highly sensitive mass spectrometry platforms. N7-HE-G was detected in all tissues and exposure groups, showing linear dose-response relationships in the low-dose range (≤ 1 ppm) and increased sharply and exposure-disproportionately in the high-dose range (≥ 50 ppm). Despite high sensitivity, O6-HE-dG was undetectable in any tissue at exposure < 50 ppm, reflecting adduct levels that are below the current quantifiable limit. At higher exposures (≥ 50 ppm), O6-HE-dG exhibited a dose-response pattern of N7-HE-G. Notably, the mammary gland, despite being anatomically distant from the site of inhalation, exhibited the second-highest levels of both adducts at higher doses. This study provides the first reliable quantitative dose-response evidence of DNA adducts in tumor target and nontarget (liver) tissues across a wide range of EtO exposures. The two DNA adducts differ markedly in their abundance, repairability and mutagenic potential and together provide a molecular MOA dose-response framework to provide the biological foundation for informing quantitative cancer risk assessment and genotoxic hazard characterization.