Chemical Research in Toxicology

The increasing e-cigarette use worldwide presents an urgent need to characterize their nicotine delivery property, brain stimulation and potential long-term health effects. We constructed an end-to-end system enabling combustible-cigarette (c-cigarette) and e-cigarette aerosol generation, animal exposure, and effect assessment. The system consists of (1) a 10-channel aerosol generator resembling human smoking/vaping scenarios, (2) nose-only and whole-body exposure chambers suitable for long- or short-duration studies, (3) a lab protocol for animal exposure and collecting arterial and venous blood <1 minute after the exposure, and (4) chromatograph and mass spectrometry to quantify nicotine concentrations in aerosol and biospecimens. We applied the system in a proof-of-principle study characterizing in vivo nicotine delivery after e-cigarette aerosol inhalation. Groups of Sprague-Dawley rats were exposed to e-cigarette aerosols for 1, 2 and 4 minutes, respectively. Arterial and venous blood samples were collected immediately after the exposure. We also directly compared nose-only and whole-body exposure approaches. After nose-only e-cigarette aerosol exposure, the nicotine concentration in arterial blood was substantially higher (11.32 ng/mL in average) than in veins. Similar arterio-venous concentration difference was observed in whole-body exposure experiments. In summary, we described a complete system ideal for e- and c-cigarette in vivo nicotine kinetics and long-term health research. Our findings highlight arterial blood as the suitable bio-specimen for e-cigarette nicotine delivery studies. HighlightO_LIWe constructed a combustible- and e-cigarette aerosol generation - exposure - effect assessment system resembling real world human smoking/vaping scenarios. C_LIO_LIProof-of-principle study characterized in vivo nicotine delivery from e-cigarette aerosol to arterial and venous blood at high temporal resolution. C_LIO_LIAfter exposure, the nicotine concentration was substantially higher (11.32 ng/mL) in arterial blood than in veins. C_LIO_LIOur results suggest arterial blood as the suitable bio-specimen to study nicotine delivery and brain stimulation. C_LI

BackgroundUS sales of oral nicotine pouches (ONPs) have rapidly increased, with cool/mint-flavored ONPs the most popular. Restrictions on sales of flavored tobacco products have either been implemented or proposed by several US states and localities. Zyn, the most popular ONP brand, is marketing Zyn-"Chill" and Zyn-"Smooth" as "Flavor-Ban Approved", probably to evade flavor bans. At present it is unclear whether these ONPs are indeed free of flavor additives that can impart pleasant sensations such as cooling. MethodsSensory cooling and irritant activities of "Flavor-Ban Approved" ONPs, Zyn-"Chill" and "Smooth", along with "minty" varieties (Cool Mint, Peppermint, Spearmint, Menthol), were analyzed by Ca2+ microfluorimetry in HEK293 cells expressing the cold/menthol (TRPM8) or menthol/irritant receptor (TRPA1). Flavor chemical content of these ONPs was analyzed by GC/MS. ResultsZyn-"Chill" ONP extracts robustly activated TRPM8, with much higher efficacy (39-53%) than the mint-flavored ONPs. In contrast, mint-flavored ONP extracts elicited stronger TRPA1 irritant receptor responses than Zyn-"Chill" extracts. Chemical analysis demonstrated the presence of WS-3, an odorless synthetic cooling agent, in Zyn-"Chill" and several other mint-flavored Zyn-ONPs ConclusionsSynthetic cooling agents such as WS-3 found in Flavor-Ban Approved Zyn-"Chill" can provide a robust cooling sensation with reduced sensory irritancy, thereby increasing product appeal and use. The label "Flavor-Ban Approved" is misleading and may implicate health benefits. Regulators need to develop effective strategies for the control of odorless sensory additives used by the industry to bypass flavor bans.

BackgroundSince late 2022, the sale of most flavoured tobacco products has been prohibited in California, including menthol cigarettes. Tobacco companies responded by introducing "non-menthol" cigarettes in which menthol was replaced with WS-3, an odorless synthetic cooling agent to elicit cooling sensations similar to menthol. Legislation enacted in 2024 banned the addition of cooling characterizing flavours in tobacco products in California. However, the industry continues to market "non-menthol" cigarettes in the state, with very similar package designs. It is unknown whether these cigarettes contain a cooling agent. MethodsAvailable Newport-branded "non-menthol" cigarettes were purchased in California in 2025, extracted and tested for sensory cooling activity by Ca2+ microfluorimetry of HEK293T cells expressing the human TRPM8 cold/menthol receptor. Chemical analysis was performed by gas chromatography - mass spectrometry (GCMS). "Non-menthol" and menthol cigarettes marketed in 2023-24 served as controls. ResultsExtracts from Newport-branded "non-menthol" cigarettes marketed in California in 2025 did not elicit sensory cooling activity. Chemical analysis confirmed the absence of menthol and any of the major commercial synthetic cooling agents. ConclusionsThe tobacco industry removed sensory cooling agents from "non-menthol" cigarettes marketed in California. However, this did not result in the market withdrawal of "non-menthol" cigarettes in the state. "Non-menthol" cigarettes in California continue to be marketed with package designs resembling those of former menthol cigarettes, signaling the potential presence of a characterising flavour.

Evaluation of unintended immunotoxicity represents an important component of nonclinical safety assessment, as perturbation of immune function may increase susceptibility to infection, impair vaccine responses, and disrupt immune homeostasis. Regulatory guidance, including the ICH S8 Immunotoxicity Guideline, recommends a weight-of-evidence approach in which observations from conventional toxicological endpoints are integrated with functional immune assays to support interpretation of immune system effects. The present study applied an integrated immunotoxicity evaluation framework to examine concordance among structural, functional, and cellular immune endpoints in male Sprague-Dawley rats using a well-characterized immunosuppressive reference compound. Hematological evaluation revealed leukopenia characterized primarily by lymphocyte depletion. Reductions in spleen and thymus weights were accompanied by histopathological evidence of lymphoid depletion in multiple immune tissues, including spleen, thymus, lymph nodes, Peyers patches, and bone marrow. Functional immune competence was assessed through hemagglutination antibody response to sheep red blood cells and delayed-type hypersensitivity assays, both of which demonstrated marked suppression of adaptive immune responses. Flow cytometric immunophenotyping further demonstrated substantial reductions in B-cell populations and decreases in CD4 and CD8 T-cell counts, whereas NK cell populations were comparatively less affected. The concordance of hematological alterations, lymphoid tissue changes, impaired functional immune responses, and lymphocyte subset depletion provides integrated evidence of immune system perturbation. These findings demonstrate that complementary immunotoxicity endpoints collectively support hazard characterization of immune system effects under GLP conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/713556v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@beaf9dorg.highwire.dtl.DTLVardef@fb9f10org.highwire.dtl.DTLVardef@187ff06org.highwire.dtl.DTLVardef@1780dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Determining environmental chemical carcinogenicity is an urgent need as humans are increasingly exposed to these chemicals. In this study, we determined the carcinogenicity of wide variety real-life exposure chemicals in large scale. To determine chemical carcinogenicity, we have developed carcinogenicity prediction models based on the hybrid neural network (HNN) architecture. In the HNN model, we included new SMILES feature representation method, by modifying our previous 3D array representation of 1D SMILES simulated by the convolutional neural network (CNN). We used 653 molecular descriptors modeled by feed forward neural network (FFNN), and SMILES as chemical features to train the models. We have developed three types of machine learning models: binary classification models to predict chemical is a carcinogenic or non-carcinogenic, multiclass classification models to predict severity of the chemical carcinogenicity, and regression models to predict median toxic dose of the chemicals. Along with the hybrid neural network (HNN) model that we developed, Random Forest (RF), Bootstrap Aggregating (Bagging) and Adaptive Boosting (AdaBoost) methods were also used for binary and multiclass classification. Regression models were developed using HNN, RF, Support Vector Regressor (SVR), Gradient Boosting (GB), Kernel Ridge (KR), Decision Tree with AdaBoost (DT), KNeighbors (KN), and a consensus method. For binary classification, our HNN model predicted with an average accuracy of 74.33% and an average AUC of 0.806, for multiclass classification, the HNN model predicted with an average accuracy of 50.58% and an average micro-AUC of 0.68, and for regression model, the consensus method achieved R2 of 0.40. The predictive performance of our models based on a highly diverse chemicals is comparable to the literature reported models that included the similar and less diverse molecules. Our models can be used in identifying the potentially carcinogenic chemicals for a wide variety of chemical classes.

There has been a substantial rise in e-cigarette (e-cig) use or vaping in the past decade, prompting growing concerns about their adverse health effects. Recently, e-cig manufacturers have been using synthetic cooling agents, like WS-23 and WS-3, to provide a cooling sensation without the "menthol taste". Studies have shown that aerosols/vapes generated by e-cigs can contain significant levels of reactive oxygen species (ROS). However, studies investigating the role of synthetic coolants in modulating ROS levels generated by e-cigs are lacking. This study seeks to understand the potential of synthetic coolants, e-cigarette additives that have become increasingly prevalent in e-liquids sold in the United States (US), on acellular ROS production. Aerosols were generated from e-liquids with and without synthetic coolants through a single-puff aerosol generator; subsequently, acellular ROS was semi-quantified in H2O2 equivalents via fluorescence spectroscopy. Our data suggest that adding WS-3 to e-liquid base (PG:VG), regardless of nicotine content, has a minimal impact on modifying e-cigarette-generated acellular ROS levels. Additionally, our data also suggest that the addition of WS-23 to nicotine-containing e-liquid base significantly modifies e-cigarette-generated acellular ROS levels. Together, our data provide insight into whether adding synthetic coolants to e-liquids significantly impacts vaping-induced oxidative stress in the lungs.

E-cigarettes have attracted significant attention as a safer substitute for conventional tobacco smoking. However, they have introduced new inhalable toxicants, including benzaldehyde-propylene glycol acetal (BPGA)--a chemical adduct produced by cherry-flavoured e-cigarettes. The health risks associated with such flavour-derived acetals remain insufficiently elucidated at the cellular level. This study investigated the role of BPGA in the progression of epithelial-to-mesenchymal transition (EMT)-like changes in alveolar epithelial cells (A549 cells). A549 cells exposed to various concentrations of BPGA were analysed for cell viability, morphology, mitochondrial function, lysosomal health, and cytoskeletal integrity using viability assays and fluorescence imaging. Intracellular reactive oxygen species (ROS) production was quantified using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Antioxidant enzyme expression, inflammatory responses, and EMT-associated phenotypic alterations were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence (IF) assays. Exposure of alveolar epithelial cells to BPGA caused a concentration-dependent decrease in cell viability. BPGA exposure resulted in mitochondrial membrane depolarisation, lysosomal damage, cytoskeletal changes, and stress fibre formation, which altered cell morphology. It significantly increased intracellular ROS production. As a result, antioxidant enzyme levels were upregulated as a protective response. However, during severe oxidative stress, this response was overwhelmed. Excess ROS disrupted cellular homeostasis and initiated apoptosis, though not completely. ROS also acted as a signalling molecule, promoting the upregulation of inflammatory mediators. These changes were associated with altered EMT marker expression, suggesting that BPGA might drive EMT-like remodelling. In conclusion, BPGA, a chemical adduct from e-cigarette vapour, induces alveolar injury by promoting oxidative stress, inflammation, and EMT-related changes, which may explain a mechanism by which e-cigarette exposure could lead to lung injury and pulmonary fibrosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/724520v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@f7739dorg.highwire.dtl.DTLVardef@1c74f11org.highwire.dtl.DTLVardef@180aeeorg.highwire.dtl.DTLVardef@75ae14_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

RATIONALEThe ban of menthol cigarettes is one of the key strategies to promote smoking cessation in the United States. Menthol cigarettes are preferred by young beginning smokers for smoking initiation. Almost 90% of African American smokers use menthol cigarettes, a result of decades-long targeted industry marketing. Several states and municipalities already banned menthol cigarettes, most recently California, effective on December 21, 2022. In the weeks before Californias ban took effect, the tobacco industry introduced several "non-menthol" cigarette products in California, replacing previously mentholated brands. Here, we hypothesize that tobacco companies replaced menthol with synthetic cooling agents to create a cooling effect without using menthol. Similar to menthol, these agents activate the TRPM8 cold-menthol receptor in sensory neurons innervating the upper and lower airways. METHODSCalcium microfluorimetry in HEK293t cells expressing the TRPM8 cold/menthol receptors was used to determine sensory cooling activity of extracts prepared from these "non-menthol" cigarette brands, and compared to standard menthol cigarette extracts of the same brands. Specificity of receptor activity was validated using TRPM8-selective inhibitor, AMTB. Gas chromatography mass spectrometry (GCMS) was used to determine presence and amounts of any flavoring chemicals, including synthetic cooling agents, in the tobacco rods, wrapping paper, filters and crushable capsule (if present) of these "non-menthol" cigarettes. RESULTSCompared to equivalent menthol cigarette extracts, several California-marketed "non-menthol" cigarette extracts activated cold/menthol receptor TRPM8 at higher dilutions and with stronger efficacies, indicating substantial pharmacological activity to elicit robust cooling sensations. Synthetic cooling agent, WS-3, was detected in tobacco rods of several of these "non-menthol" cigarette brands. Crushable capsules added to certain "non-menthol" crush varieties contained neither WS-3 nor menthol but included several "sweet" flavorant chemicals, including vanillin, ethyl vanillin and anethole. CONCLUSIONTobacco companies have replaced menthol with the synthetic cooling agent, WS-3, in California-marketed "non-menthol" cigarettes. WS-3 creates a cooling sensation similar to menthol, but lacks menthols characteristic "minty" odor. The measured WS-3 content is sufficient to elicit cooling sensations in smokers, similar to menthol, that facilitate smoking initiation and act as a reinforcing cue. Regulators need to act quickly to prevent the tobacco industry from bypassing menthol bans by substituting menthol with synthetic cooling agents, and thereby thwarting smoking cessation efforts.

Electronic cigarettes (e-cigs) have a strong foothold in the marketplace as a product to replace tobacco cigarette usage. Despite many researchers investigating the use of e-cigs and possible health issues, there is still controversy concerning how to evaluate and use e-cig condensates. Therefore, to identify factors that influence in vitro e-cig studies, we examined parameters that can impact experimental outcomes. We generated high wattage e-cig aerosol condensate (ECAC) to determine reproducible conditions to evaluate ECAC with respect to cellular survival. Cytotoxicity of ECAC was independent of serum conditions. However, cytotoxicity of ECAC is altered by treatment duration and by physical factors, including cell seeding density and volume of ECAC used. In addition, interactions between ECAC components and cells, as well as the culture vessel surface, diminish the bioavailability of ECAC components in vitro and altered the results obtained. Moreover, the cell seeding density changes reactive oxygen species production in response to ECAC exposure. Our data indicated that normalized ECAC doses (ECAC weight per cell) better reflect the toxicity of ECAC than nominal doses (ECAC percentage). These results provide factors for researchers to consider in the design of in vitro experiments using ECAC.

The transition toward animal-free safety assessment of chemicals has accelerated the development of New Approach Methodologies (NAMs) for predicting skin sensitization. However, individual in silico models and experimental NAM assays frequently produce inconsistent or contradictory results, limiting their reliability when used in isolation. To address this challenge, we present a tiered integrated assessment framework implemented through the open source SaferSkin application, which enables systematic comparison and integration of multiple predictive models and experimental data within a transparent weight-of-evidence workflow. In this case study, a diverse set of 21 reference compounds was evaluated using a battery of in silico approaches, including the OECD QSAR Toolbox, VEGA, CASE Ultra and additional machine-learning models implemented within SaferSkin. The platform enables side-by-side comparison of predictions and integration of experimental data through Bayesian network models, allowing probabilistic updating of predictions as new evidence becomes available. Our results demonstrate that reliance on any single predictive model is insufficient for robust hazard identification due to frequent disagreement between models. In contrast, consensus interpretation across multiple modelling approaches combined with targeted experimental evidence substantially improves predictive confidence. The integrated weight-of-evidence framework showed strong concordance with reference classifications and was further supported by independent validation using the Pred-Skin Bayesian model. Importantly, the tiered workflow enables resolution of ambiguous cases. For example, lower-tier predictions for ethyl (2E,4Z)-deca-dienoate were inconsistent across models, whereas targeted third-tier testing using the SENS-IS assay identified the compound as a strong sensitiser (GHS Category 1A). Overall, this study demonstrates how integrated modelling, Bayesian evidence updating and targeted NAM testing can reduce uncertainty in skin sensitization assessment. The SaferSkin framework provides a transparent and reproducible approach for implementing Next Generation Risk Assessment (NGRA) strategies and supports the development of animal-free regulatory toxicology and Safe-and-Sustainable-by-Design chemical innovation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/711911v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@b59ca0org.highwire.dtl.DTLVardef@13de455org.highwire.dtl.DTLVardef@599358org.highwire.dtl.DTLVardef@d87fd1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

Ethylene oxide (EtO) is a highly reactive industrial chemical and classified as a known human carcinogen with a putative mutagenic mode of action (MOA). Its genotoxic potential is primarily mediated through alkylation of DNA, resulting in the formation of the mutagenic adduct O6-(2-hydroxyethyl)-2-deoxyguanosine (O6-HE-dG). The N7-(2-hydroxyethyl)guanine (N7-HE-G) adduct is formed in greater abundance and is generally considered to be non-mutagenic. However, dose-response relationships of these DNA adducts, particularly at low inhalation exposure levels (i. e., below 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 the present study, male and female B6C3F1 mice were exposed to EtO (0, 0.05, 0.1, 0.5, 1, 50, 100, and 200 ppm) 6 hours/day for 28 consecutive days. Immediately following the last exposure, DNA was extracted from lung, liver, bone marrow, and mammary gland, and further utilized to measure DNA adduct levels 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 ([&le;]1 ppm) and increased sharply and exposure-disproportionately in the high-dose range ([&ge;]50 ppm). Despite a very low limit of detection, O6-HE-dG, in contrast, was not detected at exposures <50 ppm in any tissue consistent with at most a shallow linear exposure response. At higher exposures ([&ge;]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 non-target (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 inform both quantitative cancer risk assessment and genotoxic hazard characterization.

Acute respiratory distress syndrome (ARDS) involves death of lung epithelial cells. ARDS is a leading reason behind mortality in respiratory infections. Here we show a proof-of-concept that a Fullerene nanoformulation can be used for the regeneration of cells treated with apoptosis-inducing molecules, suggeting its potential for ARDS therapy.

Tattoos and permanent make-ups, involving dermal injection of chemicals, are increasingly popular, with 30-40% of young adults in North America and Europe having at least one tattoo. Recent studies highlighted a link between tattooing and health risks, including skin cancers and lymphomas. This study comprehensively catalogs tattoo ink chemicals and investigates their potential adverse effects, addressing the urgent need for greater understanding of tattoo-related health concerns. First, 364 unique tattoo chemicals were identified from various scientific and regulatory sources, with nearly half functioning as pigments. Hazardous chemicals were identified, revealing carcinogens, endocrine disruptors, neurotoxicants, and dermal toxicants. A regulatory analysis based on the European Unions cosmetic regulations, revealed gaps in regulations of such hazardous chemicals. Curated chemical-disease associations highlighted that some tattoo chemicals are known to cause dermatitis. Further, diverse toxicological information, including experimental results from REACH dossiers, were integrated to construct stressor-AOP network linking 151 chemicals to 362 AOPs, revealing potential carcinogenic mechanisms associated with tattoo ink chemicals. A systems biology approach revealed potential immunomodulatory effects associated with these chemicals. Finally, all findings have been made available through the online database Tattoo Ink Chemicals and associated Toxicities Knowledgebase (TICToK; https://cb.imsc.res.in/tictok), which can support risk assessment and sustainable tattoo practices. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/668261v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@11c06f1org.highwire.dtl.DTLVardef@10bc82borg.highwire.dtl.DTLVardef@82b4eeorg.highwire.dtl.DTLVardef@1347792_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tox21 assays compile extensive chemical bioactivity data across diverse biological targets, making them widely utilized resources for in silico model development. Nuclear receptor-specific assays within this dataset are particularly valuable for screening potential endocrine disrupting chemicals. This study presents a comprehensive benchmarking of diverse machine learning (ML), deep learning (DL), and transformer-based architectures with varied chemical feature representations across nuclear receptor assays. First, 43 datasets associated with 18 nuclear receptors within Tox21 assays were systematically curated from ToxCast invitrodb v4.3. Upon testing across these datasets, model performance was found to be dependent on the degree of class imbalance. Tree-based ML models such as random forest (RF) and extreme gradient boosting (XGBoost) trained on descriptors, or combination of descriptors and fingerprints, consistently outperformed in datasets with higher proportions of active chemicals (>10%), while DL models showed greater robustness for those with moderate proportions (5-10%). Further analysis revealed that approximately 40% of misclassified active chemicals occupied structurally isolated regions of the chemical space, suggesting absence of close structural analogues in the training set potentially contributed to their misclassification. External validation using in vitro and in vivo androgen and estrogen receptor bioactivity data showed generally good concordance. Finally, a systematic literature review revealed that the models in this study span wider range of architectures, feature representations, and assay endpoints, and are broadly comparable to or better than existing work. Overall, insights from this study can inform the development of more reliable in silico tools supporting new approach methodologies for nuclear receptor bioactivity predictions.

Characterising known and new chemical compounds for skin sensitisation provides a basis for the development of safer products where ingredients are exposed to skin. By including new approaches, such as tiered testing strategies and integrated data analysis, it is possible to develop next generation products adhering to emerging regulations, scientific evidence and animal welfare principles. To ensure data integrity during such assessment the OECD provides characterisation guidelines and Defined Approaches (DAs) to uniform work-flows. In this study we developed and applied the integrated characterisation tool called <<SaferSkin>> to compare the results of different DAs for eight compounds and included results obtained from current OECD guidance and emerging methods. We tested two compounds with unclear or indeterminate results with the SENS-IS assay to explore the value of the experiment in strengthening the weight of evidence and arriving at a clearer conclusion.

Flavoring chemicals utilized in electronic nicotine delivery systems (ENDS) have been shown to result in an increase in cellular inflammation, meanwhile, the effects of fruit and tobacco flavors on lung inflammation by nose-only exposures to mice are relatively unknown. We hypothesized that C57BL/6J mice exposed to flavored e-cigarettes would result in an increase in lung inflammation. C57BL/6J mice were exposed to air, propylene glycol/vegetable glycerin (PG/VG), and e-liquids "Apple", "Cherry", "Strawberry", "Wintergreen", and "Smooth & Mild Tobacco", for one hour per day for a three day exposure. Quantification of flavoring chemicals was measured by proton nuclear magnetic resonance spectroscopy (1H NMR), differential cell counts by flow cytometry, pro-inflammatory cytokines/chemokines by ELISA, and matrix metalloproteinase levels by western blot. Exposure to PG/VG, Apple, and Smooth & Mild Tobacco resulted in an increase in neutrophil cell count in lung bronchoalveolar lavage fluid (BALF). Strawberry exposure increased KC levels in BALF while in lung homogenate KC levels were increased in PG/VG, Cherry, and Smooth & Mild Tobacco exposure. Exposure to PG/VG and Cherry increased IL-6 levels and in all exposed mice there was a male-specific decrease in MCP-1 levels in lung homogenate. Mice exposed to PG/VG, Apple, Cherry, and Wintergreen resulted in an increase in MMP2 levels. Our results indicate that female mice exposed to cherry flavored e-liquids and male mice exposed to tobacco flavored e-liquids resulted in an increase in inflammation, while exposure to mint flavored e-liquids resulted in a decrease in inflammatory cytokine and an increase in tissue repair proteins. This study revealed that flavored-based e-cigarette exposure elicited sex-specific alterations in lung inflammation, with cherry flavors/benzaldehyde eliciting female-specific increases in inflammation. This highlights the toxicity of flavored chemicals and the further need for regulation of flavoring chemicals.

Silver has been used as a biocide for centuries, mostly in health-oriented applications. However, as a biocide, silver is toxic not only to its intended targets, mainly bacteria and fungi, but also to all living cells. Because of this toxicity, it is desirable to use forms of silver that maximize the required biocidal activity while minimizing the amount of silver that will be released in the environment at the end of life of the product. Silver nano objects are a good compromise for such requirements. The high surface to volume ratio allows for good reactivity and thus good biocidal activity, while the small amount of silver present in nano objects allows for a limited environmental release at the product end of life. In this work, we tested three types of silver nano objects. The first type, polyvinylpyrrolidone-coated silver nanoparticles (nAg-PVP) were used as a control nanoparticle, as this type of nanoparticle is now widespread. We also manufactured and tested maltodextrin-coated silver nanoparticles (nAg-MD) and micrometric (20 {micro}m in two dimensions and a few nanometers in the third one) silver flakes ({micro}AgSF). For these three silver nano objects, we investigated the biocidal activity by stringent tests using both Staphylococcus aureus and Escherichia coli as target bacteria. In addition, we investigated toxicity on mammalian macrophages or keratinocytes cell lines, as well as on an insect hemocyte cell line. Our results showed that the two innovative silver nano objects (nAg-MD and even more {micro}AgSF), showed both a better bactericidal activity and a lesser toxicity than the reference nAg-PVP nanoparticles. In addition, we also checked that beyond toxicity, the silver nano objects did not induce an inflammatory reaction, making them safer to use.

The environmental fragmentation of plastic waste leads to the formation of micro- and nanoplastics (MNPs), which pose serious ecological and human health concerns. Despite increasing interest in their biological effects, many studies rely on artificial, uniform particles that fail to mimic the diverse physical and chemical characteristics of real-world MNPs. To address this limitation, we developed the Accelerated Plastic Aging in Suspension (APAS) system--a scalable, reproducible method that mimics natural aging processes by combining ultraviolet (UV) radiation, thermal stress, and mechanical shear to generate environmentally relevant MNPs from commonly used polymers. We used APAS to fragment polyethylene terephthalate (PET), polyamide 6 (Nylon), and polyacrylonitrile (PAN), and observed time-dependent degradation, including the spontaneous formation of nanoplastics (<100 nm). Flow cytometry revealed substantial increases in particle number and reductions in average particle size over 12 weeks. Imaging flow cytometry confirmed consistent generation of heterogeneous, irregular particles across replicate batches. High-resolution imaging via AFM, TEM, and SEM confirmed the presence of nanoplastics with textured and irregular morphologies. Chemical characterization showed APAS aging altered particle surface charge and induced polymer-specific changes in autofluorescence and Raman spectral profiles, consistent with oxidative surface modifications. Laser Direct Infrared (LDIR) imaging further confirmed structural and chemical changes in polymer spectra post-aging. Functionally, under physiologically relevant shear flow conditions, endothelial cells internalized APAS-generated PET MNPs at significantly higher levels than polystyrene (PS) beads of similar size. Uptake was enhanced particularly under oscillatory flow, highlighting the influence of particle physicochemical properties on cellular interactions. Together, these findings demonstrate the ability of the APAS system to produce complex and realistic MNPs for use in environmental and toxicological studies. The system enables generation of nanoplastics and supports more accurate modelling of biological exposure scenarios compared to conventional synthetic particles.

Ethylene oxide (EtO) is primarily used as an intermediate in the manufacture of chemicals, with a minor use as a sterilant for medical equipment and food products. It is a direct-acting alkylating agent that reacts with cellular macromolecules, including proteins and DNA. EtO has been shown to induce tumors in rodents and humans. DNA reactivity has been the postulated mode of action (MOA) for its carcinogenicity. The current study has investigated the dose response for EtO-induced genetic damage to inform the biological plausibility of a dose-response model for cancer risk assessment. Male and female B6C3F1 mice were exposed to 0, 0.05, 0.1, 0.5, 1, 50, 100, or 200 ppm EtO by whole-body inhalation (6 hours/day for 28 days, 7 days/week). Mutagenicity was assessed by determining the frequency of mutant Pig-a phenotype in reticulocytes (RET) and mature red blood cells (RBC) on Day 28. Cytogenetic damage was evaluated by the erythrocyte micronucleus (MN) test in blood samples collected on Days 5 and 28. EtO is a relatively weak genotoxicant with treatment-related increases in Pig-a and MN frequencies being seen primarily at 200 ppm. The hockey-stick shaped dose response for genetic damage may be conservatively interpreted as being no more than a linear response with a single slope. Thus, a cancer risk assessment dose-response model consisting of a single linear slope throughout the exposure range is biologically plausible and consistent if EtO were acting through a mutagenic MoA for its carcinogenicity.

Glioblastoma multiforme (GBM) is the most common and biologically aggressive brain tumour. The current standard therapy for GBM consists in surgical resection, followed by radiotherapy and chemotherapy. Yet, the treatment is limited due to the area for the surgical resection and for the inability of some drugs to cross the brain blood barrier, leading to a general prognostic of no more than a year. Cold atmospheric plasma (CAP) is a new approach in the treatment of this challenging disease. CAP interaction with cells is dependent on physical and chemical factors, with different plasma discharges, cell type, and culture conditions leading to different CAP activity. Considering the plasma self-adaptation that different plasma discharge modes can undergo, which leads to different interaction plasma/cells, the characterization of a new device is essential. In this study we analysed the effect of a novel large pin-to-plate non-thermal atmospheric plasma on U-251 MG cells under different conditions. The analysis of reactive oxygen and nitrogen species (RONS) on plasma, media and cells were also assessed. We were able to demonstrate that the pin-to-plate device is cytotoxic to GBM cells in a dose, time and ROS dependent manner. The measurements of RONS on plasma/media also give us an insight on the chemical effect of this novelty device, and the possibility to better understand the use of this device as a promising GBM therapy.