Exploring the role of oxidative stress and mitochondrial dysfunction in β-damascone-induced aneuploidy.

Background: The rose ketone β-damascone (β-Dam) elicits positive results in the in vitro micronucleus (MN) assay using human lymphocytes, but shows negative outcomes in the Ames test and combined in vivo MN and comet assays. This has led to the interpretation that the in vitro MN result is a misleading positive result. Oxidative stress has been suggested as an indirect mode of action (MoA) for in vitro MN formation, with the α, β-unsaturated carbonyl moiety of the β-Dam chemical structure expected to cause misleading positive results through this MoA.

Spontaneous histiocytic sarcoma originating from the epididymis in a CD-1 mouse.

We report a histiocytic sarcoma originating from the epididymis observed in a 110-week-old male CD-1 mouse in a carcinogenicity study. At necropsy, no lesions were observed in the epididymis. Histologically, a neoplastic lesion was observed in the cauda of the epididymis that was well demarcated from the surrounding tissues.

A solvent-free squeezing method for extraction of collected mass from aerosols of electronic cigarettes and heated tobacco products.

Previous in vitro toxicological assessments have demonstrated that almost no mutagenic and genotoxic activities in electronic cigarette (e-cigarette) and heated tobacco product (HTP) aerosols were detected even at the maximum recommended concentration. To accurately compare the toxicity levels between cigarette smoke and e-cigarette or HTP aerosols, higher exposure concentrations increasing the possibility to detect toxicity in in vitro tests are necessary, while avoiding solvent-induced toxicity. This study aimed to develop a solvent-free extraction method to obtain concentrated aerosol extracts for improved toxicological evaluation.

RNA sequencing analysis of early-stage atherosclerosis in vascular-on-a-chip and its application for comparing combustible cigarettes with heated tobacco products.

Our previous study showed promising results in replicating early-stage atherosclerosis when vascular endothelial cells (VECs) were exposed to cigarette smoke (CS) extract via M0 macrophages. We used an organ-on-a-chip system as an alternative to animal testing to model atherosclerosis, which is a complex disease involving endothelial and immune cell communications. By incorporating macrophages into the vascular-on-a-chip system, we aimed to mimic the indirect effects of inhalable substances, such as CS, on VECs.

Development of an in vitro human alveolar epithelial air-liquid interface model using a small molecule inhibitor cocktail.

Background: The alveolar epithelium is exposed to numerous stimuli, such as chemicals, viruses, and bacteria that cause a variety of pulmonary diseases through inhalation. Alveolar epithelial cells (AECs) cultured in vitro are a valuable tool for studying the impacts of these stimuli and developing therapies for associated diseases. However, maintaining the proliferative capacity of AECs in vitro is challenging.

Proof of concept for quantitative adverse outcome pathway modeling of chronic toxicity in repeated exposure.

Adverse Outcome Pathway (AOP) is a useful tool to glean mode of action (MOE) of a chemical. However, in order to use it for the purpose of risk assessment, an AOP needs to be quantified using in vitro or in vivo data. Majority of quantitative AOPs developed so far, were for single exposure to progressively higher doses.

A revision of the multiple-path particle dosimetry model focusing on tobacco product aerosol dynamics.

To assess the health impact of inhaled aerosols, it is necessary to understand aerosol dynamics and the associated dosimetry in the human respiratory tract. Although several studies have measured or simulated the dosimetry of aerosol constituents, the respiratory tract focus areas have been limited. In particular, the aerosols generated from tobacco products are complex composites and simulating their dynamics in the respiratory tract is challenging.

Development of an Sensitisation Test Using a Coculture System of Human Bronchial Epithelium and Immune Cells.

Chemical respiratory sensitisation is a serious health problem. However, to date, there are no validated test methods available for identifying respiratory sensitisers. The aim of this study was to develop an sensitisation test by modifying the human cell line activation test (h-CLAT) to detect respiratory sensitisers and distinguish them from skin sensitisers.

Effects of aerosols from heated tobacco products with flavors on the discoloration of bovine tooth enamel.

Objectives: This in vitro study assessed the potential of tooth discoloration by aerosols generated from three heated tobacco products (HTPs) with different specifications: in-direct heating tobacco system platform 1.0a (IT1.0a), in-direct heating tobacco system platform 2.

Development of a micronucleus test using the EpiAirway™ organotypic human airway model.

Background: The use of organotypic human tissue models in genotoxicity has increased as an alternative to animal testing. Genotoxicity is generally examined using a battery of in vitro assays such as Ames and micronucleus (MN) tests that cover gene mutations and structural and numerical chromosome aberrations. At the 7th International Workshop on Genotoxicity Testing, working group members agreed that the skin models have reached an advanced stage of maturity, while further efforts in liver and airway models are needed [Pfuhler et al.

Binary classification of users of electronic cigarettes and smokeless tobacco through biomarkers to assess similarity with current and former smokers: machine learning applied to the population assessment of tobacco and health study.

Background: Exposure to harmful and potentially harmful constituents in cigarette smoke is a risk factor for cardiovascular and respiratory diseases. Tobacco products that could reduce exposure to these constituents have been developed. However, the long-term effects of their use on health remain unclear.

Oxidative stress-mediated epidermal growth factor receptor activation by cigarette smoke or heated tobacco aerosol in human primary bronchial epithelial cells from multiple donors.

The epidermal growth factor receptor (EGFR) signaling pathway has essential roles in maintaining homeostasis of various tissues by regulating cell proliferation and differentiation. Deregulation of the EGFR signaling pathway is associated with various chronic diseases including chronic obstructive pulmonary disease. Cigarette smoke (CS) is known to activate EGFR, which is linked to chronic obstructive pulmonary disease.

Human vasculature-on-a-chip with macrophage-mediated endothelial activation: The biological effect of aerosol from heated tobacco products on monocyte adhesion.

Heated tobacco products (HTPs) are expected to have the potential to reduce risks of smoking-associated cardiovascular disease (CVD). However, mechanism-based investigations of the effect of HTPs on atherosclerosis remain insufficient and further studies under human-relevant situations are desired for deeper understanding of the reduced risk potential of HTPs. In this study, we first developed an in vitro model of monocyte adhesion by considering macrophage-derived proinflammatory cytokine-mediated endothelial activation using an organ-on-a-chip (OoC), which provided great opportunities to mimic major aspects of human physiology.

Chemical and toxicological comparison of emissions from a heated tobacco product and the 1R6F reference cigarette.

It has previously been found that, compared with cigarette smoke, the aerosols generated by heated tobacco products contain fewer and lower harmful and potentially harmful constituents (HPHCs) and elicit lower biological activity in  models and lower smoking-related exposure biomarker levels in clinical studies. It is important to accumulate such scientific evidences for heated tobacco products with a novel heating system, because different heating system may affect the quantitative aspect of the amount of HPHCs and the qualitative aspect of the biological activity of the aerosol generated. Here, the chemical properties of, and toxicological responses to aerosols emitted by DT3.

Oral Absorption across Organotypic Culture Models of the Human Buccal Epithelium after E-cigarette Aerosol Exposure.

Inhaled aerosols are absorbed across the oral cavity, respiratory tract, and gastrointestinal tract. The absorption across the oral cavity, which is one of the exposure routes, plays an important role in understanding pharmacokinetics and physiological effects. After aerosol exposure from e-cigarettes, tissue viability studies, morphological observation, and chemical analyses at the inner and outer buccal tissues were performed using organotypic 3D in vitro culture models of the buccal epithelium to better understand the deposition and absorption on the inner and outer buccal tissues.

evaluation of mutagenic, cytotoxic, genotoxic and oral irritation potential of nicotine pouch products.

Non-clinical in vitro studies were conducted to investigate the characteristics of extracts from tobacco free nicotine pouches alongside a reference snus product and/or 1R6F reference cigarette. investigations were conducted in the Neutral Red Uptake (NRU) cytotoxicity assay, Bacterial Reverse Mutation (Ames) assay, and in vitro Mammalian Cell Micronucleus (MN) assay. These products were also investigated for their oral irritation potential in the EpiGingival™ 3D tissue model.

Donor-to-donor variability of a human three-dimensional bronchial epithelial model: A case study of cigarette smoke exposure.

Three-dimensional (3D) cultured primary cells are used to predict the toxicity of substances towards humans because these 3D cultures closely mimic the physiological architecture of tissues. Nonetheless, it is important to consider primary-cell-specific variability for endpoint selection and appropriate evaluation of toxicity because donor-dependent characteristics may be retained even in in vitro cell cultures. In this report, 3D differentiated bronchial epithelial cells from three donors were used to investigate donor-to-donor variability, with an aqueous extract of cigarette smoke (CS) used as the test substance.

Effects of antioxidant capacity on micronucleus induction by cigarette smoke in mammalian cells.

We have compared micronucleus (MN) induction by cigarette smoke in the L5178Y, TK6, and CHL/IU cell lines. The test sample was total particulate matter of 3R4F reference cigarette smoke, suspended in DMSO. After 3-h treatment, with or without a rat liver S9 metabolic activation system, followed by 24-h recovery, dose-dependent MN increases were seen in all cell lines.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

Background: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies).

LC/MS analysis of three-dimensional model cells exposed to cigarette smoke or aerosol from a novel tobacco vapor product.

A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotine-containing aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells.

In vitro long-term repeated exposure and exposure switching of a novel tobacco vapor product in a human organotypic culture of bronchial epithelial cells.

Next-generation tobacco products and nicotine delivery systems such as heat-not-burn tobacco products and electronic cigarettes, the usage of which is expected to have a beneficial impact on public health, have gained popularity over the past decade. However, the risks associated with the long-term use of such products are still incompletely understood. Although the risks of these products should be clarified through epidemiological studies, such studies are normally performed based on each product category, not product-by-product.

Novel biomarker genes which distinguish between smokers and chronic obstructive pulmonary disease patients with machine learning approach.

Background: Chronic obstructive pulmonary disease (COPD) is combination of progressive lung diseases. The diagnosis of COPD is generally based on the pulmonary function testing, however, difficulties underlie in prognosis of smokers or early stage of COPD patients due to the complexity and heterogeneity of the pathogenesis. Computational analyses of omics technologies are expected as one of the solutions to resolve such complexities.

Regulation of NRF2, AP-1 and NF-κB by cigarette smoke exposure in three-dimensional human bronchial epithelial cells.

Cigarette smoke (CS) is a complex mixture of chemicals and interacts with various physiological processes. We previously reported that nuclear factor erythroid 2-related factor 2 (NRF2) was the most sensitive transcription factor to aqueous CS extract (AqCSE) exposure in monolayer cultured human bronchial epithelial cell lines. Recently, in vitro three-dimensional (3D) culture models have been used to supplement pharmacological and toxicological assessments.

Multi-omics analysis: Repeated exposure of a 3D bronchial tissue culture to whole-cigarette smoke.

Cigarette smoke (CS) is a major risk factor in the development of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease. A comprehensive investigation of the biological impacts of chronic CS exposure on lung tissue is therefore important for understanding the pathogenesis of lung disease. We used three-dimensional (3D) organotypic human bronchial tissue cultures and metabolomics, transcriptomics, and proteomics to investigate changes in biological processes affected by repeated whole-CS exposure.