Toxicology in Vitro最新文献

Air pollution represents a growing threat to human health, particularly affecting the respiratory system through the inhalation of gaseous pollutants and fine particles that reach the alveolar region composed mainly of alveolar epithelial type I (AT1) and type II (AT2) cells at the air-liquid interface (ALI). To reduce reliance on animal models, physiologically relevant and experimentally accessible in vitro models are increasingly needed for inhalation toxicology. In this study, we developed and characterized a simple and robust human alveolar coculture model at ALI using commercially available cell lines, A549 (AT2 phenotype-like) and Ci-hAELVi (AT1 phenotype-like). Two ALI acclimatization periods (24 h and six days) were evaluated under incubator control and clean air exposure conditions. The coculture exhibited stable metabolic activity, controlled proliferation, and reduced variability compared to monoculture. The model expressed complementary AT1- (Podoplanin, Caveolin-1, Aquaporin-5, HTI56) and AT2 pneumocyte-associated markers (surfactant proteins and HTII280) at the gene and protein levels, resulting in a mix of both the alveolar epithelial phenotypes. Importantly, the coculture model maintained epithelial integrity and functional stability during prolonged ALI exposure for up to 72 h, exceeding the typical exposure window of monoculture system. In addition, responsiveness of coculture with ZnO aerosol and lipopolysaccharides exposure, was tested demonstrating its functional relevance. Overall, this relevant coculture model provides a valuable in vitro tool for preliminary toxicological screening.

Organ-specific multicellular in vitro models are used to mimic the lung-blood-brain axis, and to assess the nanomaterials (NMs) safety in humans. We employed a triculture lung model, a whole-blood model, an astrocytes-neurons coculture to examine health outcomes by three cerium dioxide (CeO₂) NMs and silver (Ag) nanowires. Endpoints included cytotoxicity, gene expression, genotoxicity, inflammatory markers at the air-liquid interface (ALI), complement activation, and secondary toxicity in astrocytes-neurons coculture. Post-exposure, CeO₂-3.5 nm high-dose decreased cell viability, no DNA damage was detected. At epithelial-macrophages interface, CeO₂-50 nm upregulated surfactant protein A (SPA), cell surface death receptor (FAS), and heme oxygenase-1 (HMOX1), whereas CeO₂-3.5 nm downregulated SPA. Ag-nanowires upregulated HMOX1, macrophage inflammatory protein-1β (MIP-1β), granulocyte colony-stimulator factor (G-CSF), chemokine C-X-C-motif ligand 1 (CXCL1). At endothelial side, CeO₂-50 nm and - 3.5 nm, and Ag-nanowires upregulated HMOX1. In whole-blood model, CeO₂-3.5 nm high-dose reduced terminal complement complex (TCC) proteins, while CeO₂-50 nm and Ag-nanowires increased them. Nanomaterials activated CD11b⁺ on granulocytes and monocytes. Ag-nanowires conditioned-medium (CM) on astrocytes-neurons coculture, decreased cell viability. CeO₂-50 nm CM upregulated IL1β, NFκB, and HMOX1. Overall, CeO₂-3.5 nm exhibits lung toxicity; CeO₂-50 nm CM triggers inflammatory response and Ag-nanowires CM may induce cytotoxicity in brain cells.

While suitable as a universal supplement in cell culture, fetal calf serum (FCS) has several downsides, including its bovine origin and batch-to-batch variability. In this study, we investigated human platelet lysate (HPL) as a potential replacement for FCS when culturing an extravillous trophoblast cell line. HTR8/SVneo cells were cultured in conventional medium supplemented with 10% FCS or serum-free medium supplemented with varying concentrations of HPL for 24 h or 3 weeks, whereafter LDH release, metabolic activity and cytokine release were assessed. Cellular viability and cell counts were measured for each passage during the 3 weeks of culturing. Cells were successfully cultured in all media. Media with 1% and 10% HPL provided the best yields, but culturing in 10% HPL also increased LDH release. Medium composition had a direct effect on IL-6 release and affected IL-8 release after three weeks of culturing. Furthermore, HPL interfered with the detection of RANTES at higher HPL concentrations. Based on our findings, we recommend culturing HTR8/SVneo cells in serum-free medium with 1% HPL. However, we suggest that researchers assess how medium composition affects relevant outcomes before implementing a serum free culture protocol.

A fragrance ingredient's potential to induce skin sensitization is an important factor in risk assessment. The key events in the skin sensitization adverse outcome pathway (AOP) can be evaluated with new approach methodologies (NAMs) for hazard identification, potency, and point of departure (PoD) derivation, distinguishing between sensitizers and non-sensitizers. The first key event is the covalent binding of an electrophilic material to skin proteins, which can be assessed using peptide reactivity assays (e.g., the Direct Peptide Reactivity Assay (DPRA)). Some sensitizers are not directly electrophilic and require biotic (pro-hapten) or abiotic (pre-hapten) activation before reacting with proteins. The Peroxidase Peptide Reactivity Assay (PPRA) was developed to characterize the reactivity of certain pre-/pro-haptens. The PPRA incorporates a horseradish peroxidase-hydrogen peroxide (+HRP/P) oxidation system to mimic the activation required for some pre-/pro-haptens to become protein-reactive and therefore potentially sensitizing. An evaluation of chemical structures for their suitability as substrates for +HRP/P resulted in the development of 5 structural activity groups that were defined based on the ability of a chemical to act as an HRP/P substrate. The goal of this study was to apply these sub-groups to understand the applicability domain of the PPRA in analyzing the sensitization potential of pre-/pro-hapten fragrance ingredients. The PPRA expanded hazard identification for 20 of the 88 known sensitizers of the 99 pre-/pro-hapten fragrance ingredients analyzed.

Multiple drug resistance (MDR) among antibiotic-resistant bacteria poses a major challenge in modern medicine, prompting interest in alternative antimicrobials like mouthwashes and toothpastes. Plasma-activated liquids (PALs), produced by exposing liquids to cold atmospheric plasma (CAP), are enriched with reactive oxygen and nitrogen species (RONS) that may have antimicrobial properties. However, their biocompatibility with mammalian cells remains underexplored. In this study, three formulations were subjected to CAP: (L1) zinc sulfate and calcium chloride, (L2) xylitol and sodium fluoride, and (L3) deionized water. Using a dielectric barrier discharge (DBD) system, we assessed the physicochemical, cytotoxic, and whitening effects of the resulting PALs. In vitro assays showed that PALs were non-cytotoxic to L929 fibroblast cells at concentrations up to 1.5% for PAL1, 10% for PAL2, and 15% for PAL3. PAL2 and PAL3 preserved over 90% cell viability. Scratch assays indicated that while nontreated (NT) NT1 and NT3 reduced fibroblast migration, CAP treatment enhanced cell movement over time. PAL1 did not significantly affect proliferation but increased metabolic activity by 25%, suggesting a selective pro-survival effect. Hydrogen peroxide was the predominant RONS: 6.9 mg·L^-1 in PAL3, 5.8 mg·L^-1 in PAL1, and 2.7 mg·L^-1 in PAL2. All PALs demonstrated measurable whitening of human enamel, with PAL3 showing the most significant colour change (ΔE). These results support the potential of PALs as safe, multifunctional agents for use as modern mouthwashes.