Assessment of estrogenic and androgenic activity in PM10 air samples from an urban, industrial and rural area in Flanders (Belgium) using the CALUX bioassay.

BACKGROUND: Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. OBJECTIVES: The objective of this study was to investigate and compare the overall estrogen and androgen activating potential of PM10 air samples at an urban, rural and industrial location in Flanders, using a human in vitro cell bioassay. METHODS: PM10 samples were collected on glass fiber filters every six days between April 2013 and January 2014 using a high-volume sampler. Extraction was executed with a hexane/acetone mixture before analysis using a recombinant estrogen- or androgen responsive human carcinoma cell line. Results were expressed as bioanalytical equivalents (BEQs) per cubic meter of air. RESULTS: High fluctuations in estrogenic activity were observed during the entire sampling period, with median BEQs of 32.1, 35.9 and 31.1 fg E2-Eq m(-)³ in the industrial, urban and rural background area, respectively. Estrogenic activity was measured in 70% of the samples, while no androgenic activity was observed in any of the samples. The estrogenic activity in the industrial area was positively correlated with the airborne concentration of the sum of the non-carcinogenic PAHs pyrene and fluoranthene (rho=0.48; p<0.01) and the sum of the carcinogenic PAHs (rho=0.36; p=0.05). CONCLUSIONS: This study showed that no androgenic activity was present in PM10 and that although the median estrogenic activity was rather low and comparable in the three locations, high fluctuations in estrogenic response exist over time. While atmospheric PAHs contributed to the observed estrogenic response, especially in the industrial area, the chemicals responsible for the majority of estrogenic activity remain to be identified.

Identification of PM10 characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B).

Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10 in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013-2014 PM10 was sampled (24h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The reduction in cell viability was significantly correlated with BC, Cd and Pb. The induction of IL-8 in Beas-2B cells was significantly associated with Cu, Ni and Zn and endotoxin. Endotoxin levels explained 33% of the variance in IL-8 induction. A significant interaction between ambient temperature and endotoxin on the pro-inflammatory activity was seen. No association was found between OP and the cellular responses. This study supports the hypothesis that, on an equal mass basis, PM10 induced biological effects differ due to differences in PM10 characteristics. Metals (Cd, Cu, Ni and Zn), BC, and endotoxin were among the main determinants for the observed biological responses.

Gene expressions changes in bronchial epithelial cells: markers for respiratory sensitizers and exploration of the NRF2 pathway.

For the classification of respiratory sensitizing chemicals, no validated in vivo nor in vitro tests are currently available. In this study, we evaluated whether respiratory sensitizers trigger specific signals in human bronchial epithelial (BEAS-2B) cells at the level of the transcriptome. The cells were exposed during 6, 10, and 24h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4×44K oligonucleotide arrays. A limited number of 11 transcripts could be identified as potential biomarkers to identify respiratory sensitizers. Three of these transcripts are associated to immune system processes (HSPA5, UPP1, and SEPRINE1). In addition, the transcriptome was screened for transcripts that are differentially expressed compared to vehicle control for each chemical. The results show that the NRF2-mediated oxidative stress response is activated in the cell line after stimulation with all of the chemicals that were selected in our study, and that - at the level of gene expression - this pathway shows no potential to discriminate between any of the three compound groups: respiratory sensitizers, skin sensitizers, or electrophilic respiratory irritants.

Gene markers in dendritic cells unravel pieces of the skin sensitization puzzle.

The underlying events of how dendritic cells (DC) are capable of evoking an antigen-specific skin sensitization response are not yet understood. Recently, we revealed a set of genes in human cord blood CD34(+) DC (CD34-DC) that show a discriminating behaviour after skin sensitizing exposure. Based on their differential expression, an in vitro assay was developed to identify chemicals as sensitizing or not. This study was designed to investigate the genes' involvement in the DC response to skin sensitizers and as such gain insights in the sensitization cascade. Functional connection of the marker genes was inquired by constructing a molecular network using Ingenuity software. By real-time RT-qPCR, we established the effective expression of 3 additional gene transcripts in the generated network in CD34-DC, of which CREB1 and TNF-alpha were significantly altered in expression by sensitizing versus non-sensitizing exposure. Next, it was tested whether the discriminating response of CCR2 and COX2 marker genes was translated at the protein level in CD34-DC exposed to 3 sensitizers versus 3 non-sensitizers. Significantly differential protein expression of CCR2 and COX2 was confirmed using flow cytometry. Our results indicate that the marker genes may be functionally relevant in DC mediated skin sensitization.

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.

THP-1 monocytes but not macrophages as a potential alternative for CD34+ dendritic cells to identify chemical skin sensitizers.

Early detection of the sensitizing potential of chemicals is an emerging issue for chemical, pharmaceutical and cosmetic industries. In our institute, an in vitro classification model for prediction of chemical-induced skin sensitization based on gene expression signatures in human CD34+ progenitor-derived dendritic cells (DC) has been developed. This primary cell model is able to closely mimic the induction phase of sensitization by Langerhans cells in the skin, but it has drawbacks, such as the availability of cord blood. The aim of this study was to investigate whether human in vitro cultured THP-1 monocytes or macrophages display a similar expression profile for 13 predictive gene markers previously identified in DC and whether they also possess a discriminating capacity towards skin sensitizers and non-sensitizers based on these marker genes. To this end, the cell models were exposed to 5 skin sensitizers (ammonium hexachloroplatinate IV, 1-chloro-2,4-dinitrobenzene, eugenol, para-phenylenediamine, and tetramethylthiuram disulfide) and 5 non-sensitizers (l-glutamic acid, methyl salicylate, sodium dodecyl sulfate, tributyltin chloride, and zinc sulfate) for 6, 10, and 24 h, and mRNA expression of the 13 genes was analyzed using real-time RT-PCR. The transcriptional response of 7 out of 13 genes in THP-1 monocytes was significantly correlated with DC, whereas only 2 out of 13 genes in THP-1 macrophages. After a cross-validation of a discriminant analysis of the gene expression profiles in the THP-1 monocytes, this cell model demonstrated to also have a capacity to distinguish skin sensitizers from non-sensitizers. However, the DC model was superior to the monocyte model for discrimination of (non-)sensitizing chemicals.

Gene profiles of a human bronchial epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis.

Respiratory sensitization is a concern for occupational and environmental health in consumer product development. 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 alterations in gene expression of human bronchial epithelial (BEAS-2B) cells after exposure to respiratory sensitizers and respiratory non-sensitizing chemicals, and to identify genes that are able to discriminate between both groups of chemicals. BEAS-2B cells were exposed during 6, 10, and 24h to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x 44K oligonucleotide arrays. Fisher Linear Discriminant Analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory sensitizing and respiratory non-sensitizing chemicals. The 10 most discriminative genes were BC042064, A_24_P229834, DOCK11, THC2544911, DLGAP4, NINJ1, PFKM, FLJ10986, IL28RA, and CASP9. Based on the differentially expressed genes, pathway analysis was used to identify possible underlying mechanisms of respiratory sensitization. We demonstrated that in bronchial epithelial cells the canonical PTEN signaling pathway is probably the most specific pathway in the context of respiratory sensitization. Results are indicative that the BEAS-2B cell line can be used as an alternative cell model to screen chemical compounds for their respiratory sensitizing potential.

Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis.

There are currently no accepted biological prediction models for assessing the potential of a substance to cause respiratory sensitization. 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 alterations in gene expression of human alveolar epithelial (A549) cells after exposure to respiratory sensitizing and non-respiratory sensitizing chemicals, and to identify genes that are able to discriminate between both groups of chemicals. A549 cells were exposed during 6, 10, and 24 h to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x44K oligonucleotide arrays. A Fisher linear discriminant analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory sensitizing and respiratory non-sensitizing chemicals. Among the 20 most discriminating genes, which were categorized into molecular and biological gene ontology (GO) terms, CTLA4 could be associated with asthma and/or respiratory sensitization. When categorizing the top-1000 genes into biological GO terms, 22 genes were associated with immune function. Using a pathway analysis tool to identify possible underlying mechanisms of respiratory sensitization, no known canonical signaling pathway was observed to be activated in the A549 cell line.

Gene expression signatures in CD34+-progenitor-derived dendritic cells exposed to the chemical contact allergen nickel sulfate.

The detection of the sensitizing potential of chemicals is of great importance to industry. A promising in vitro alternative to the currently applied animal assays for sensitization testing makes use of dendritic cells (DCs) that have the capability to process and present antigens to naive T cells and induce their proliferation. Here, we studied changes in gene expression profiles after exposing DCs to the contact allergen nickel sulfate. CD34+-progenitor-derived DCs, initiated from 3 different donors, were exposed to 60 microM nickel sulfate, during 0.5, 1, 3, 6, 12 and 24 h. cDNA microarrays were used to assess the transcriptional activity of about 11,000 genes. Significant changes in the expression of 283 genes were observed; 178 genes were up-regulated and 93 down-regulated. These genes were involved in metabolism, cell structure, immune response, transcription, signal transduction, transport, and apoptosis. No functional information was found for 74 genes. Real-time RT-PCR was used to confirm the microarray results of 12 genes. In addition, 3 DC maturation markers not present on the microarrays (DEC205, DC LAMP and CCR7) were analyzed using real-time RT-PCR and found to be up-regulated at several time points. Our data indicate that a broad range of biological processes is influenced by nickel. Some processes are clearly linked to the immune response and DC maturation, others may indicate a toxic effect of nickel.

Phenotypic alterations and IL-1beta production in CD34(+) progenitor- and monocyte-derived dendritic cells after exposure to allergens: a comparative analysis.

Dendritic cells (DC) have been shown to capture and process antigens and play an initiating role in contact sensitization. Cells with dendritic morphology can be generated in vitro either from CD34(+) cord blood cells or from CD14(+) peripheral monocytes. The aim of this study was to determine the state of maturation/activation of both populations after exposure to several concentrations of four well-established model allergens (nickel sulfate, eugenol, alpha-hexylcinnamaldehyde and 2,4,6-trinitrobenzene sulfonic acid) or the irritant sodium dodecyl sulfate. We analyzed the surface expression of CD86, CD83 and HLA-DR and the production of IL-1beta. DC from the two sources were generated separately in two laboratories, but challenged using identical test protocols. Using both DC populations it was possible to detect the allergens under investigation, though minor differences regarding effective concentrations were noted. The non-responsiveness of CD34-DC to CIN was probably due to non-optimal concentrations. Ni(2+), known as a moderate allergen in vivo, showed the most prominent effect in both cell systems. CD86 expression was the most reliable phenotypic marker for the in vitro identification of allergens. Due to substantial individual variations it was difficult to draw any definite conclusions as to the relevance of IL-1beta production as an activation endpoint. We conclude that both test systems are able to respond to allergens, but CD34-DC must be exposed to higher concentrations to demonstrate significant phenotypic changes. On the other hand, Mo-DC from only some of the donors reacted to allergens, in contrast to CD34-DC, which responded to allergens irrespective of the donor, thus necessitating the use of Mo-DC cultures from several blood donors.

In vitro tests for haematotoxicity: prediction of drug-induced myelosuppression by the CFU-GM assay.

In a prevalidation study, a standard operating procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggest promising applications for developing and validating other in vitro methods for use in haematotoxicology.