Cytotoxicity, genotoxicity, and enzyme activity effects of UV filters and their chlorinated by-products in human lung epithelial cells (A549).

Transformation and toxicity studies of UV filter diethylamino hydroxybenzoyl hexyl benzoate in the swimming pools

Human biomonitoring (HBM) has become a crucial tool for assessing exposure to emerging chemicals. We analyzed 250 24-h urine samples from the German Environmental Specimen Bank (ESB), collected between 2000 and 2021, for exposure to diethylamino hydroxybenzoyl hexyl benzoate (DHHB), a UV filter increasingly used in sunscreens. Three major metabolites were examined: 2-(4-diethylamino)-, 2-(4-ethylamino)-, and 2-(4-amino)-2-hydroxybenzoyl)benzoic acid (DHB, EHB, AHB), with detection rates of 18°%, 13°%, and 87°%, respectively. While EHB and DHB were specific to DHHB, AHB suggested other exposure sources, making it unreliable for assessing DHHB exposure. DHB and EHB were first detected in 2012, with increased detection rates thereafter. The median daily intake of 37ng/kg bw/d was much lower than the derived no-effect level of 2900mg/kg bw/d, indicating low risk from DHHB exposure. However, since the analyzed ESB samples were collected in winter, they likely reflect exposure from other products and the environment rather than sunscreen-related exposure. Recently, concerns have emerged regarding the DHHB impurity di-n-hexylphthalate (DnHexP), a reproductive toxicant not authorized in the EU. Retrospective analysis of oral DHHB dosing experiments indeed revealed impurity related dose-dependent excretion of DnHexP metabolites (MnHexP, oxidized 5-OH-MnHexP, and 5-oxo-MnHexP). Due to uncertainties in dose allocation, only a rough excretion fraction of 45°% for MnHexP was derived. Our findings suggest that the DHHB impurity DnHexP may contribute to DnHexP exposure in sunscreen users applying products with contaminated DHHB. Given DnHexP's toxicity, this warrants re-assessment of DHHB's safety in cosmetics and enhanced surveillance of both DHHB and DnHexP in HBM studies.

Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of delta-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the delta-subunit may form multimeric channels with alpha beta gamma-ENaC. Reverse transcription-PCR revealed that delta-ENaC is co-expressed with alpha beta gamma-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that delta-ENaC is co-expressed with alpha-, beta-, and gamma-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for delta alpha beta gamma-ENaC differed from those of both alpha beta gamma- and delta beta gamma-ENaC (n = 6). The contribution of the delta subunit to P(Li)/P(Na) ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of delta alpha beta gamma-ENaC differed from those of alpha beta gamma- and delta beta gamma-ENaC. Co-immunoprecipitation of delta-ENaC with alpha- and gamma-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of delta-ENaC with other subunits could account for heterogeneity of native epithelial channels.

Occurrence and formation of disinfection by-products in the swimming pool environment: A critical review

Silver nanoparticles are increasingly used in various products, due to their antibacterial properties. Despite its wide spread use, only little information on possible adverse health effects exists. Therefore, the aim of this study was to assess the toxic potential of silver nanoparticles (<100 nm) in human lung epithelial (A549) cells and the underlying mechanism of its cellular toxicity. Silver nanoparticles induced dose and time-dependent cytotoxicity in A549 cells demonstrated by MTT and LDH assays. Silver nanoparticles were also found to induce oxidative stress in dose and time-dependent manner indicated by depletion of GSH and induction of ROS, LPO, SOD, and catalase. Further, the activities of caspases and the level of proinflammatory cytokines, namely interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly higher in treated cells. DNA damage, as measured by single cell gel electrophoresis, was also dose and time-dependent signicants in A549 cells. This study investigating the effects of silver nanoparticles in human lung epithelial cells has provided valuable insights into the mechanism of potential toxicity induced by silver nanoparticles and warrants more careful assessment of silver nanoparticles before their industrial applications.

BackgroundAirway fibrosis is one of the pathological characteristics of severe asthma. Transforming growth factor (TGF)-β has been known to promote epithelial-mesenchymal transition formation and to play a role in the progression of tissue fibrosis. Cellular communication network factor 2 (CCN2) and fibronectin (FN) are well-known markers of EMT and fibrosis. However, whether AREG is involved in TGF-β-induced CCN2 and FN expression in human lung epithelial cells is unknown.MethodsAREG and FN were analyzed by immunofluorescence staining on ovalbumin-challenged mice. CCN2 and FN expression were evaluated in human lung epithelial (A459) cells following TGF or AREG treatment for the indicated times. Secreted AREG from A549 cells was detected by ELISA. Cell migration was observed by a wound healing assay. Chromatin immunoprecipitation was used to detect the c-Jun binding to the CCN2 promoter.ResultsAREG and FN expression colocalized in lung tissues from mice with ovalbumin-induced asthma by immunofluorescence staining. Moreover, TGF-β caused the release of AREG from A549 cells into the medium. Smad3 siRNA down-regulated AREG expression. AREG also stimulated CCN2 and FN expression, JNK and c-Jun phosphorylation, and cell migration in A549 cells. AREG small interfering (si) RNA inhibited TGF-β-induced expression of CCN2, FN, and cell migration. Furthermore, AREG-induced CCN2 and FN expression were inhibited by EGFR siRNA, a JNK inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). EGFR siRNA attenuated AREG-induced JNK and c-Jun phosphorylation. Moreover, SP600125 downregulated AREG-induced c-Jun phosphorylation.ConclusionThese results suggested that AREG mediates the TGF-β-induced EMT in human lung epithelial cells through EGFR/JNK/AP-1 activation. Understanding the role of AREG in the EMT could foster the development of therapeutic strategies for airway remodeling in severe asthma.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Inhaled polymyxins are associated with toxicity in human lung epithelial cells that involves multiple apoptotic pathways. However, the mechanism of polymyxin-induced pulmonary toxicity remains unclear. This study aims to investigate polymyxin-induced metabolomic perturbations in human lung epithelial A549 cells. A549 cells were treated with 0.5 or 1.0 mM polymyxin B or colistin for 1, 4, and 24 h. Cellular metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and significantly perturbed metabolites (log2 fold change [log2FC] ≥ 1; false-discovery rate [FDR] ≤ 0.2) and key pathways were identified relative to untreated control samples. At 1 and 4 h, very few significant changes in metabolites were observed relative to the untreated control cells. At 24 h, taurine (log2FC = -1.34 ± 0.64) and hypotaurine (log2FC = -1.20 ± 0.27) were significantly decreased by 1.0 mM polymyxin B. The reduced form of glutathione (GSH) was significantly depleted by 1.0 mM polymyxin B at 24 h (log2FC = -1.80 ± 0.42). Conversely, oxidized glutathione (GSSG) was significantly increased by 1.0 mM both polymyxin B (log2FC = 1.38 ± 0.13 at 4 h and 2.09 ± 0.20 at 24 h) and colistin (log2FC = 1.33 ± 0.24 at 24 h). l-Carnitine was significantly decreased by 1.0 mM of both polymyxins at 24 h, as were several key metabolites involved in biosynthesis and degradation of choline and ethanolamine (log2FC ≤ -1); several phosphatidylserines were also increased (log2FC ≥ 1). Polymyxins perturbed key metabolic pathways that maintain cellular redox balance, mitochondrial β-oxidation, and membrane lipid biogenesis. These mechanistic findings may assist in developing new pharmacokinetic/pharmacodynamic strategies to attenuate the pulmonary toxicities of inhaled polymyxins and in the discovery of new-generation polymyxins.

Rationale: The ongoing COVID-19 pandemic highlights the need to develop novel anti-pneumonia interventions against emerging and established pathogens. We previously reported a therapeutic dyad of immunostimulatory small molecules that induces innate immunity, termed inducible epithelial resistance, against a wide range of pneumonia-causing pathogens, including coronaviruses and influenza viruses. This combination (“Pam2-ODN”) is comprised of a Toll-like receptor (TLR)-2/6 agonist, Pam2CSK4, and a TLR-9 agonist, ODN M362, that stimulate protective responses from lung epithelial cells and promotes synergistic survival benefits and microbicidal effects that exceeds the additive effects of treatment with individual ligands. Here, we investigate the immunomodulatory signaling mechanism of Pam2-ODN that reduces susceptibility to viral infection in lung epithelial cells. Methods: Transcriptional responses of human and mouse lung epithelial cells to influenza A H1N1 or SARS-CoV-2 (GSE147507) or Pam2-ODN (GSE289984) were analyzed using R and IPA software to build host-based antiviral innate immune pathways to infections and identify relevant transcriptional factors (TFs) involved. Isolated human or mouse lung epithelial cells were stimulated with PBS or Pam2-ODN and challenged with influenza A H3N2 or coronavirus OC43 to study transcriptional control of relevant TFs by high-throughput methods of immunofluorescence (IF) staining, in-cell western blotting and imaging flow cytometry (IFC). Results: Network-based enrichment analysis reduced infection of epithelial cells with Pam2-ODN against both coronavirus and influenza through inhibition of viral budding and viral RNA replication. In silico prediction of pathogen-specific host-based responses with Pam2-ODN included disruption of SARS-CoV-2's IL-1, 6 and 8 signaling, and inhibition of influenza A's anti-interferon mechanisms. Functional enrichment analysis revealed activation of these host innate immune responses by Pam2-ODN prior to viral exposure through activity of NF-kB/RelA and AP-1/cJun. IFC and IF confirmed an NF-kB-dependent transcriptional cooperation of RelA and cJun with Pam2-ODN in both mouse and human lung epithelial cells. Phospho-kinetic studies revealed an early transient activity of RelA-(pS536), followed by a sustained cJun-(pS73) signal in lung epithelial cells after Pam2-ODN. Upon viral infection, Pam2-ODN treated cells activated cJun-(pS73) signaling more rapidly in response to both influenza and coronavirus infection. Coronavirus-induced activation of RelA-(pS536) after infection was suppressed in Pam2-ODN treated cells. Conclusion: Inducible epithelial resistance by Pam2- ODN enhances broad host-based antiviral innate immunity responses through signaling pathways of RelA and cJun, which also abrogate virus-specific pathogenic mechanisms. Furthermore, phospho-signaling control of these transcriptional responses in lung epithelium suggest a novel pathogen-dependent antiviral response immune mechanism to infection.

Effects of the novel poly(methyl methacrylate) (PMMA)-encapsulated organic ultraviolet (UV) filters on the UV absorbance and in vitro sun protection factor (SPF)

Particulate matter causes telomere shortening and increase in cellular senescence markers in human lung epithelial cells

Photokinetics of oil soluble 1,3,5-Triazine UV filters in combination with Butyl Methoxydibenzoylmethane or with Diethylamino Hydroxybenzoyl Hexyl Benzoate

Ethyl pyruvate modulates adhesive and secretory reactions in human lung epithelial cells

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.

Current understanding of mechanisms of ischemia-reperfusion-induced lung injury during lung preservation and transplantation is mainly based on clinical observations and animal studies. Herein, we used cell and systems biology approaches to explore these mechanisms at transcriptomics levels, especially by focusing on the differences between human lung endothelial and epithelial cells, which are crucial for maintaining essential lung structure and function. Human pulmonary microvascular endothelial cells and human lung epithelial cells were cultured to confluent, subjected to different cold ischemic times (CIT) to mimic static cold storage with preservation solution, and then subjected to warm reperfusion with a serum containing culture medium to simulate lung transplantation. Cell morphology, viability, and transcriptomic profiles were studied. Ischemia-reperfusion injury induced a CIT time-dependent cell death, which was associated with dramatic changes in gene expression. Under normal control conditions, endothelial cells showed gene clusters enriched in the vascular process and inflammation, while epithelial cells showed gene clusters enriched in protein biosynthesis and metabolism. CIT 6 h alone or after reperfusion had little effect on these phenotypic characteristics. After CIT 18 h, protein-biosynthesis-related gene clusters disappeared in epithelial cells; after reperfusion, metabolism-related gene clusters in epithelial cells and multiple gene clusters in the endothelial cells also disappeared. Human pulmonary endothelial and epithelial cells have distinct phenotypic transcriptomic signatures. Severe cellular injury reduces these gene expression signatures in a cell-type-dependent manner. Therapeutics that preserve these transcriptomic signatures may represent new treatment to prevent acute lung injury during lung transplantation.