The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Only MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.     

Gene expression profiles reveal distinct immunological responses of cobalt and cerium dioxide nanoparticles in two in vitro lung epithelial cell models

Fragmentary knowledge exists on cellular signaling responses underlying possible adverse health effects of CoO- and CeO₂-nanoparticles (NP)s after inhalation.     

Gene profiles of THP-1 macrophages after in vitro exposure to respiratory (non-)sensitizing chemicals: Identification of discriminating genetic markers and pathway analysis

It is recognized that respiratory sensitization is a hazard of high concern. Despite international regulatory requirements there is no established protocol for the identification of chemical respiratory sensitizers. New tests should be based on mechanistic understanding and should be preferentially restricted to in vitro assays. The major goal of this study was to investigate the genetic response of human THP-1 macrophages after contact with respiratory (non-)sensitizers, and to identify genes that are able to discriminate between both groups. THP-1 macrophages were exposed during different time points to 3 respiratory sensitizers, 2 irritants, and 1 skin sensitizer. Gene expression changes were evaluated using Agilent Whole Human Genome arrays. Fisher Linear Discriminant Analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory (non-)sensitizing chemicals. Among the 20 most discriminating genes which were categorized into molecular and biological Gene Ontology (GO) terms, EIF4E, PDGFRB, SEMA7A, and ZFP36L2 could be associated with respiratory sensitization. When categorizing the top-1000 genes into biological GO terms, 24 genes were associated with immune function. Using a pathway analysis tool, platelet-derived growth factor signaling was observed to be activated in THP-1 macrophages in the context of respiratory sensitization.     

煤焦沥青提取物对BEAS-2B细胞Keap1-Nrf2/ARE通路的作用机制

目的研究煤焦沥青烟提取物对BEAS-2B细胞Nrf2、Keap1、NQO1的基因及其蛋白表达的影响,以探讨煤焦沥青烟提取物在造成细胞氧化损伤过程中Nrf2-Keap1/ARE通路的作用机制。方法设立未处理对照组、0.1%DMSO溶剂对照组、5μg/ml B(a)P阳性对照组、6μmol/L全反式维甲酸组、6μmol/L全反式维甲酸预处理0.5 h后5μg/ml CTP染毒组和CTP染毒组(1、5、10和20μg/ml),染毒3、6、12和24 h后提取总RNA;RT-PCR检测Nrf2、Keap1、NQO1 mRNA的相对表达量;Western blot测定Nrf2、Keap1、NQO1蛋白的相对表达量。结果不同CTP染毒剂量作用后各个基因mRNA相对表达量差异无统计学意义(P0.05),而不同时间点相对表达量差异有统计学意义(P0.05)。Western blot结果显示,随着CTP烟提取物染毒浓度的增加,Keap1蛋白无明显变化,NQO1蛋白表达量逐渐上升;Nrf2蛋白在5μg/ml染毒浓度时表达最高,在5μg/ml CTP染毒浓度作用下,BEAS-2B细胞Nrf2蛋白表达量随时间逐渐升高,在染毒6 h后达到最高,之后表达量又呈下降趋势。结论煤焦沥青烟提取物作用于BEAS-2B细胞后,代谢酶NQO1表达上调,推测Keap1-Nrf2/ARE通路可能通过上调细胞保护性基因NQO1的表达而对抗煤焦沥青毒性作用,降低细胞氧化损伤。     

Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers

The aim of this study was to explore the usefulness of a human monocyte cell line in the development of in vitro models for predictive testing of contact sensitizers. Several studies have shown that contact sensitizers induce CD86 expression and enhanced internalization of MHC class II molecules in dendritic cells (DCs). We used THP-1, a human monocyte cell line, as a replacement for DCs for evaluation of these phenotypical alterations as predictive endpoints for contact sensitizers. Known sensitizers and irritants were evaluated. After 24-h exposure to samples, the expression of CD86 on THP-1 cells was measured by flow cytometry. Sensitizers such as dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), eugenol, p-phenylenediamine (PPDA) and ammonium tetrachloroplatinate (Pt) enhanced CD86 expression on THP-1 cells, while nickel sulfate, cobalt sulfate and irritants such as methylsalicylate (MS), sodium dodecyl sulfate (SDS) and dimethyl sulfoxide (DMSO) did not augment CD86 expression. A synergistic effect was observed when DNCB and IFN- were added simultaneously to a culture of THP-1 cells. Furthermore, internalization of MHC class II molecules was observed when the cells were treated with some of sensitizers for 2 h. The inducing effects of chemicals on the two phenotypical alterations were the same. These results suggest that these test systems can be used to predict contact-sensitizing ability of chemicals as an in vitro sensitization assay.     

Titanium dioxide nanoparticles cause apoptosis in BEAS-2B cells through the caspase 8/t-Bid-independent mitochondrial pathway

To understand the underlying mechanism for apoptosis induced by titanium dioxide nanoparticles (TNP), human airway epithelial cell line was cultured to investigate the relevant apoptosis pathways. Our results showed that the levels of reactive oxygen species and morphological apoptosis increased in a dose-dependent manner whereas cell viability decreased in a similar manner in response to TNP exposure in the BEAS-2B cells. The activities of caspase 3 and PARP were also increased in parallel to the morphological apoptosis. Levels of caspase 9 increased significantly whereas there were no detectable changes in caspase 8 and t-Bid in the TNP treated cells. Caspase 9 inhibition blocked the TNP-induced activation of caspase 3 significantly. The levels of bax, cytochrome C, p53 and bcl-2 also changed reflecting the activation of intrinsic apoptosis pathway. Our results provide solid evidence that apoptosis in BEAS-2B cells exposed to TNP occurred via a mitochondrial apoptosis pathway independent of caspase 8/t-Bid pathway.     

Analysis of nicotine-induced DNA damage in cells of the human respiratory tract

Epithelium of the upper and lower airways is a common origin of tobacco-related cancer. The main tobacco alkaloid nicotine may be associated with tumor progression. The potential of nicotine in inducing DNA mutations as a step towards cancer initiation is still controversially discussed. Different subtypes of nicotinic acetylcholine receptors (nAChR) are expressed in human nasal mucosa and a human bronchial cell line representing respiratory mucosa as a possible target for receptor-mediated pathways. In the present study, both cell systems were investigated with respect to DNA damage induced by nicotine and its mechanisms.Specimens of human nasal mucosa were harvested during surgery of the nasal air passage. After enzymatic digestion over night, single cells were exposed to an increasing nicotine concentration between 0.001 mM and 4.0 mM. In a second step co-incubation was performed using the antioxidant N-acetylcysteine (NAC) and the nAChR antagonist mecamylamine. DNA damage was assessed using the alkali version of the comet assay. Dose finding experiments for mecamylamine to evaluate the maximal inhibitory effect were performed in the human bronchial cell line BEAS-2B with an increasing mecamylamine concentration and a constant nicotine concentration. The influence of nicotine in the apoptotic pathway was evaluated in BEAS-2B cells with the TUNEL assay combined with flow cytometry.After 1 h of nicotine exposure with 0.001, 0.01, 0.1, 1.0 and 4.0 mM, significant DNA damage was determined at 1.0 mM. Further co-incubation experiments with mecamylamine and NAC were performed using 1.0 mM of nicotine. The strongest inhibitory effect was measured at 1.0 mM mecamylamine and this concentration was used for co-incubation. Both, the antioxidant NAC at a concentration of 1.0 mM, based on the literature, as well as the receptor antagonist were capable of complete inhibition of the nicotine-induced DNA migration in the comet assay. A nicotine-induced increase or decrease in apoptosis as assessed by the TUNEL assay in BEAS-2B could not be detected.These results support the hypothesis that oxidative stress is responsible for nicotine-induced DNA damage. Similar results exist for other antioxidants in different cell systems. The decrease in DNA damage after co-incubation with a nAChR antagonist indicates a receptor-dependent pathway of induction for oxidative stress. Further investigations concerning pathways of receptor-mediated DNA damage via nAChR, the role of reactive oxygen species and apoptosis in this cell system will elucidate underlying mechanisms.     

Differential modulation of CXCR4 and CD40 protein levels by skin sensitizers and irritants in the FSDC cell line

The development of non-animal methods for skin sensitization testing is an urgent challenge. Some of the most promising in vitro approaches are based on the analysis of phenotypical and functional modifications induced by sensitizers in dendritic cell models. In this work, we evaluated, for the first time, a fetal skin-derived dendritic cell line (FSDC) as a model to discriminate between sensitizers and irritants, through analysis of their effects on CD40 and CXCR4 protein expression. The chemicals concentrations were chosen based on a slight cytotoxicity effect (up to 15%). Protein levels were evaluated by Western blot and immunocytochemistry, after stimulation with the skin sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulphate (NiSO(4)), the non-sensitizer 2,4-dichloronitrobenzene (DCNB), and the irritants sodium dodecyl sulphate (SDS) and benzalkonium chloride (BC). All sensitizers tested increased CD40 and CXCR4 levels. In contrast, irritants decreased both proteins levels, with a more pronounced effect on CXCR4. In agreement with these results, dendritic cells derived from human peripheral blood monocytes-derived dendritic cells (MoDC) showed a similar response pattern to the skin sensitizer and irritant tested, PPD and SDS, respectively. In conclusion, evaluation of CD40 and CXCR4 proteins in chemical-treated FSDC may represent a useful tool in a future in vitro test for sensitizing assessment.     

Silica nanoparticles induce cytokine responses in lung epithelial cells through activation of a p38/TACE/TGF-α/EGFR-pathway and NF-κΒ signalling

Amorphous silica nanoparticles (SiNPs) have previously been shown to induce marked cytokine (interleukin-6; IL-6 and interleukin-8; CXCL8/IL-8) responses independently of particle uptake in human bronchial epithelial BEAS-2B cells. In this study the involvement of the mitogen-activated protein kinases (MAP-kinases), nuclear factor-kappa Β (NF-κΒ) and in particular tumour necrosis factor-α converting enzyme (TACE) and—epidermal growth factor receptor (EGFR) signalling pathways were examined in triggering of IL-6 and CXCL8 release after exposure to a 50 nm silica nanoparticle (Si50). Exposure to Si50 increased phosphorylation of NF-κΒ p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK), but not extracellular signal regulated kinases (ERK). Inhibition of NF-κΒ and p38 reduced the cytokine responses to Si50, whereas neither JNK- nor ERK-inhibition exerted any significant effect on the responses to Si50. Increases in membrane-bound transforming growth factor-α (TGF-α) release and EGFR phosphorylation were also observed after Si50 exposure, and pre-treatment with inhibitors of these pathways reduced the release of IL-6 and CXCL8, but did not affect the Si50-induced phosphorylation of p38 and p65. In contrast, p38-inhibition partially reduced Si50-induced TGF-α release, while the p65-inhibition was without effect. Overall, our results indicate that Si50-induced IL-6 and CXCL8 responses in BEAS-2B cells were regulated through combined activation of several pathways, including NF-κΒ and p38/TACE/TGF-α/EGFR signalling. The study identifies critical, initial events in the triggering of pro-inflammatory responses by nanoparticles.     

Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells

Cytochrome P450 (CYP) 2A13 is mainly expressed in the respiratory system and has the ability to metabolize aflatoxin B(1) (AFB(1)). However, the role of CYP2A13-mediated AFB(1) metabolism and its consequences in human lung epithelial cell is not clear. Therefore, the objectives of this study were to investigate the significance of CYP2A13 in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis. To achieve these objectives, CYP2A13 was stably over-expressed in immortalized human bronchial epithelial BEAS-2B cells (B-2A13) and its significance in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis was compared to cells with stably expression of CYP1A2 (B-1A2), the predominant AFB(1) metabolizing enzyme in liver, as well as CYP2A6 (B-2A6) as controls. AFB(1) induced B-2A13 cytotoxicity and apoptosis in a dose- and time-dependent manner. The cytotoxic and apoptotic effects of AFB(1) were significantly remarkable in B-2A13 cells than those of B-1A2 and B-2A6 cells. The increased expression of pro-apoptotic proteins, such as C-PARP, C-caspase-3, and Bax, and decreased expression of anti-apoptotic proteins, such as caspase-3, Bcl-2, and p-Bad further confirmed the data of AFB(1)-induced cytotoxicity and apoptosis. Furthermore, increased DNA adduct was observed in B-2A13 after AFB(1) treatment as compared to B-1A2 cells and B-2A6 cells. Finally, treatment with nicotine, a competitor of AFB(1), and 8-methoxypsoralen (8-MOP), an inhibitor of CYP enzyme, further confirm the critical role of CYP2A13 in AFB(1)-induced cytotoxicity and apoptosis. Collectively, these findings suggest adverse effects of AFB(1) in respiratory diseases mediated by CYP2A13.     

PXJ-41/M2-1转染人肺腺癌PAa细胞及其稳定表达

目的探讨外源性呼吸道合胞病毒(RSV)M2—1基因在人肺腺癌贴PAa细胞中获得稳定表达的可行性。方法将携有RSVM2—1基因的真核表达载体PxJ41／M2—1，在脂质体介导下，导入人肺腺癌PAa细胞株。通过以18筛选获得阳性克隆，并继续培养，用RT—PCR及免疫组织化学方法检测M2—1在人肺腺癌PAa细胞内的稳定表达情况。结果经RT—PCR方法扩增到M2—1620bP特异性条带。免疫组织化学证实，转染PxJ41／M2—1后的PAa细胞内有M2—1蛋白的表达。     

Evaluation of in vitro cytotoxicity and genotoxicity of copper–zinc alloy nanoparticles in human lung epithelial cells

In the present study, in vitro cytotoxic and genotoxic effect of copper–zinc alloy nanoparticles (Cu–Zn ANPs) on human lung epithelial cells (BEAS-2B) were investigated. XTT test and clonogenic assay were used to determine cytotoxic effects. Cell death mode and intracellular reactive oxygen species formations were analyzed using M30, M65 and ROS Elisa assays. Genotoxic effects were evaluated using micronucleus, comet and γ-H2AX foci assays. Cu–Zn ANPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements. Characterization of Cu–Zn ANPs showed an average size of 200 nm and zeta potential of −22 mV. TEM analyses further revealed the intracellular localization of Cu–Zn ANPs in cytoplasm within 24 h. Analysis of micronucleus, comet and γ-H2AX foci counts showed that exposure to Cu–Zn ANPs significantly induced chromosomal damage as well as single and double stranded DNA damage in BEAS-2B cells. Our results further indicated that exposure to Cu–Zn ANPs significantly induced intracellular ROS formation. Evaluation of M30:M65 ratios suggested that cell death was predominantly due to necrosis.