Ambient air pollution is considered a major environmental health threat to pregnant women. Our previous work has shown an association between exposure to airborne particulate matter (PM) and an increased risk of developing pre-eclamspia. It is now recognized that many pregnancy complications are due to underlying placental dysfunction, and this tissue plays a pivotal role in pre-eclamspia. Recent studies have shown that PM can enter the circulation and reach the human placenta but the effects of PM on human placental function are still largely unknown. In this work we investigated the effects of airborne PM on trophoblast cells. Human, first trimester trophoblast cells (HTR-8/SV) were exposed to urban pollution particles (Malmö PM2.5; Prague PM10) for up to seven days in vitro and were analysed for uptake, levels of hCGβ and IL-6 secretion and proteomic analysis. HTR-8/SVneo cells rapidly endocytose PM within 30 min of exposure and particles accumulate in the cell in perinuclear vesicles. High doses of Prague and Malmö PM (500–5000 ng/ml) significantly decreased hCGβ secretion and increased IL-6 secretion after 48 h exposure. Exposure to PM (50 ng/ml) for 48h or seven days led to reduced cellular growth and altered protein expression. The differentially expressed proteins are involved in networks that regulate cellular processes such as inflammation, endoplasmic reticulum stress, cellular survival and molecular transport pathways. Our studies suggest that trophoblast cells exposed to low levels of urban PM respond with reduced growth, oxidative stress, inflammation and endoplasmic reticulum stress after taking up the particles by endocytosis. Many of the dysfunctional cellular processes ascribed to the differentially expressed proteins in this study, are similar to those described in PE, suggesting that low levels of urban PM may disrupt cellular processes in trophoblast cells. Many of the differentially expressed proteins identified in this study are involved in inflammation and may be potential biomarkers for PE.
Citation: Familari M, Nääv Å, Erlandsson L, de Iongh RU, Isaxon C, Strandberg B, et al. (2019) Exposure of trophoblast cells to fine particulate matter air pollution leads to growth inhibition, inflammation and ER stress. PLoS ONE 14(7): e0218799. https://doi.org/10.1371/journal.pone.0218799
Editor: Hai-Yan Lin, Chinese Academy of Sciences, CHINA
Received: November 20, 2018; Accepted: June 10, 2019; Published: July 18, 2019
Copyright: © 2019 Familari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the FP7 Ideas: European Research Council ReproUnion (http://reprounion.eu/) and Swedish Research Council Forte Funding (Stiftelsen för Miljöstrategisk Forskning) 2016-00572 (https://forte.se/en/) to MF, CI, SRH & EM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: SRH holds a patent related to diagnosis and treatment of pre-eclampsia and is co-founder of A1M Pharma and Preelumina Diagnostics (www.a1m.se). The patent (Diagnosis and treatment of preeclampsia - 201500335) held by SRH does not pertain to any material in this manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. MF, AN, LE, RdI, CI, BS, TL & EM declare no conflicts of interest.
Abbreviations: DE, differentially expressed; ER, endoplasmic reticulum; PAHs, polyaromatic hydrocarbons; PE, pre-eclampsia; PM, particulate matter
Ambient air pollution, considered a major environmental health threat to pregnant women and their offspring , is a mixture of gases and particles in the air. When present in sufficient concentration, or of sufficient hazardous types, it affects human health (1). One main source of airborne particulate matter (PM) in urban regions are motor vehicle emissions, which can also contain various metals and toxic compounds such as mutagenic polyaromatic hydrocarbons (PAH) .
Previous epidemiological studies by us [3, 4] and others [5–8] have reported the adverse effects of ambient air pollution on pregnancy-related maternal health as well as affecting the health of the offspring by complications such as low birth weight and pre-term births. In particular, we have shown that the severity of disease is linked to increased exposure to traffic-related air pollution [3, 4, 9]. Dadvand and colleagues have also found that air pollution from traffic-related sources has the most impact for adverse pregnancy outcomes . The risk estimates for air pollution are comparable to those for high maternal age and being overweight .
Air pollution levels in urban cities of Sweden generally meet the WHO guidelines for annual mean concentrations of different fractions of PM . However, we have reported an association between air pollution exposure during pregnancy and a higher risk of pre-eclampsia (PE) for some Swedish cities [3, 4], suggesting that even low levels of air pollution are hazardous.
PE is a clinical syndrome afflicting 3–7% of pregnant women, and is one of the leading causes of maternal mortality and morbidity, especially in developing countries . With its aetiology still largely unknown, the diagnosis of PE is based on maternal clinical manifestations; high blood pressure and proteinuria manifesting after 20 weeks of gestation. The predominant view suggests that the disease evolves in the utero-placental unit, most likely due to defective placentation leading to uneven blood perfusion, inflammation and oxidative stress , and as it progresses, gives rise to maternal endothelial damage causing the clinical manifestations. Placental oxidative stress also triggers immune and inflammatory responses in the mother . Although normal pregnancy is characterised by a mild systemic inflammatory response, in PE these responses are more extensive . However, the role of inflammation in PE alone, or with intra-uterine growth restriction, is not well understood. One suggested pathway between air pollution and pre-eclampsia (PE) is through the placenta .
It is now recognized that many pregnancy complications are due to underlying placental pathology or dysfunction . In addition, we [3, 4] and a number of others [5–8, 17–21] have confirmed a link between levels of PM pollution and pregnancy complications such as preterm births and pre-eclampsia, disorders in which placental dysfunction plays a pivotal role. It is well-established in rodents that inhaled PM during pregnancy not only reach the placenta but affect this tissue and the fetus [22, 23]. In particular, PM cause increased levels of cytokine release into the circulation, and inflammation within the placenta [24–26].
In humans, Nemmar and colleagues  demonstrated that inhaled labelled carbon particles (less than 100nm) could be detected in the circulation of healthy volunteers within 1 minute following exposure and could still be detected 60 minutes later. In addition, an increase in the release of proinflammatory cytokines (e.g. IL-6, IL-1b) into the human circulation following exposure to PM has been demonstrated . Changes in placental functions including altered gene expression and increases in markers of oxidative stress [28, 29], mitochondrial dysfunction and altered methylation profiles [30–32], and endothelial and villi pathology  have been reported following inhalation of PM during human pregnancy. More recently, Qin and colleagues found that exposure to high levels of urban PM2.5 (particles ≤ 2.5 μm) induced cell-cycle arrest, inhibited migration and invasion of extravillous trophoblast cells . A recent report by Liu et al.  indicated that carbonaceous PM are able to translocate from the lungs to the placenta and are engulfed by resident macrophages (Hofbauer cells).
In this study we have examined the effects of urban PM2.5 and PM10 (particles ≤10μm) on cultured first trimester trophoblast cell endocytosis, cell growth and gene expression.
Materials and methods
Urban particulate matter (PM)
Two sources of urban PM were used: Malmö PM2.5 (≤ 2.5 μm) and Prague PM10 (average size < 4 μm). Prague PM10, a standard reference material, was obtained from Sigma Aldrich (NIST2786). Originally collected in 2005 in Prague, Czech Republic, PM10 is representative of PM collected in an urban environment and not specific for any area of Prague. Prague PM10 has been chemically characterised and was used to compare the Malmö PM chemical composition analysis undertaken in this study, and to test their biological activity on human trophoblast cells. Apoptosis and increased levels of reactive oxygen species following exposure of human bronchial epithelial cells to Prague PM10 (up to 500 μg/ml) has been reported .
Malmö PM2.5 particles were collected over 26 days in April-May 2017 at 3 m height, and 4 m from a street crossing with an annual average daily traffic (2017) of 28000 vehicles in central Malmö, using a high-volume cascade impactor (BGI900, Mesa Labs, USA). The impactor samples air (0.9 m3/min) and collects all particles smaller than 2.5 μm on a polypropylene filter. The collected PM2.5 consists of particles generated by traffic and biomass burning, from both within the city and from east/south east (Poland) and west (Copenhagen). Time resolved mass concentration measurements of PM2.5 were conducted with a tapered element oscillating microbalance (TEOM 1400AB) and of black carbon (soot) and organic carbon content with an aethalometer (AE33). These analyses indicated mean PM2.5 was 6.1 ± 3.1 μg/m3 and 23.9 ± 10.3% of the particles were generated by biomass burning. Additional sources are mainly traffic-related, both locally generated and in-transported particles. The collected particles were extracted using pure methanol and dried in a vacuum evaporator. The dried collected particles were analysed by gas chromatography-mass spectrometry (GC-MS) for PAH  and by inductively coupled plasma mass spectrometry (ICP-MS) for metal composition according to standard procedures. While the overall composition of the Prague and Malmö PM sample were similar, the levels of PAH in the Malmö PM sample were 1.5–5-fold lower than in the Prague PM sample (NIST Certificate of Analysis Standard Reference Material 2786, 2016, Gaithersburg, MD, USA), depending on the particular PAH. Notably, greater concentrations (2–100-fold) of metals were found in the Prague PM samples than in the Malmö PM sample (See Supporting information S1 Table). Analysis of nitro-PAHs and oxy-PAHs were not included in this study.
Both PM samples were provided as powdered material that required sonication for resuspension, which is standard procedure in air pollution studies using cultured cells [35, 37, 38]. To disperse PM in solution, samples were subjected to a two-step sonication in 0.5 ml of RPMI in 1.5 ml tubes (Eppendorf). Sonication with the probe immersed into the solution, at 50 W, 0.05 cycle, 20% amplitude for 1 min was undertaken using UP50H Ultrasonic Processor, (Hielscher Ultrasound), followed by ultrasonication at 4°C, 120W, 15 min using Mettler Electronics Ultrasonic Bath. To avoid settling of PM, aliquots were sonicated in 500 μl RPMI immediately before use. Unexposed control cells were treated with RPMI without PM, which was subjected to the same sonication procedure. Cells were exposed to PM in RPMI, or RPMI alone (unexposed vehicle controls), or the supernatant of dispersed PM after centrifugation for 10 min at 14,000 rpm at room temperature.
Placental exposure considerations
While the actual exposure rate of the placenta to PM is not known, it is well established that PM ≤ 2.5 μm, can and do bypass lung epithelial phagocytic cells to enter directly into the circulation . The concentrations of PM used in this study are based on two key factors: 1. Our previous epidemiological studies showing that there is an association between women exposed to ambient air pollution in Malmö and low birth weights and pre-term births [3–4]; 2. The average daily mean ambient PM2.5 of 20–30 μg/m3 in Malmö Sweden, (compared to Swedish national average < 10 μg/m3). Therefore, we used the following parameters to estimate a realistic exposure dose: daily mean ambient PM 2.5 rate of 25 μg/m3 in Malmö Sweden, changes in cardio-respiratory physiology during pregnancy  and lung PM clearance capacity [39, 41]. We calculated that pregnant women were exposed to between 50–500 ng of PM2.5 per day, and thus the dose-response curve for our studies ranged from 0.5 to 5000 ng/ml. Details are provided in Supporting Information S1 Text.
Trophoblast cell culture
Primary placental trophoblast cells can be isolated from term placentae. However, the yield of cells is variable, and due to genetic heterogeneity, the levels of secreted factors among placentae are also highly variable. To ensure reproducibility, we used the HTR-8/SVneo cell line, a human first trimester transformed placental trophoblast cell line, purchased from American Type Culture Collection (ATCC Cell Lines, CRL-3271, Lot Number 64275781). Cells were expanded in RPMI culture medium (RPMI-1640 Medium Gibco, ThermoFisher Scientific) supplemented with 5% fetal bovine serum (Life Technologies) and 100 units/ml Penicillin-Streptomycin (Gibco)]. Six-well culture plates (ThermoFisher) were seeded with 3 x 105 (acute treatment, 48 hours) in 1 ml of RPMI, and exposure to urban particulate matter at varying doses from 5 ng to 5000 ng/ml began 24 hours later. Cells were exposed to a single dose of PM without media change for 48hrs. The initial seeding capacity throughout this study was chosen to prevent cells reaching confluence in the exposure period, which may have confounded our results.
Analysis of trophoblast cell function and gene expression
To investigate whether PM had effects on markers of placental biology, we harvested culture medium for determination of concentrations of secreted hCGβ and progesterone (Clinical Biochemistry Laboratory, Lund Hospital), as well as IL-6 (human IL-6 ELISA Kit, Invitrogen), 48 hours after exposure of PM. All treatments were assayed in triplicate in each experiment. Following removal of culture medium, cells were washed twice, trypsinized, harvested and counted. All experiments were repeated three times and data expressed as mean ± SD and analysed by one-way ANOVA followed by post-hoc tests, incorporating a Bonferroni correction for multiple comparisons, where p < 0.05 was considered significant.
Previous studies have shown altered expression of a range of genes in PE [42, 43]. To investigate whether these genes were altered after PM exposure, we used custom qPCR arrays (see Supporting Information S1 Text for gene expression methodology and selection rationale.) to examine expression of these genes after 48hours of exposure to PM.
Analysis of endocytosis
HTR-8/SVneo cells were plated on glass coverslips, coated with 1% gelatin (Sigma Aldrich) in phosphate buffered saline (PBS, Gibco) at a density of 1 x 103 cells per coverslip before placing in 12-well plates with 1 ml RPMI. Cells were plated directly onto coverslips (for histological staining), or after pre-staining in 2 ml of 2 x 10−6 M PKH67 (green fluorescent cell linker mini kit for general cell membrane labelling, MINI67, Sigma Aldrich) prepared according to manufacturer’s protocol, for 5 min at room temperature.
After 24 hours, cells were exposed to different doses of Malmö or Prague PM in RPMI; control cells were unexposed. Cells received a single dose of PM, and media was changed every 48 hours. The cells were harvested after a further 24–168 hours of PM exposure, washed four times in PBS, before fixing in 4% paraformaldehyde (in PBS, pH 7.0; Sigma Aldrich) for 20 min at room temperature. Following fixation, cells were washed three times in PBS before staining in hematoxylin and eosin (H&E, n = 2 experiments); or washed once in PBS, stained with DAPI nuclear stain for 10 min before a further two washes in PBS (fluorescence studies, n = 4 experiments).
Bright field images were taken using an Olympus BX60 microscope with cellSens Entry micro-imaging software. Fluorescent images were taken using a Zeiss LSM800 Airyscan Confocal with Zen Blue 2.1 Software at the Biological Optical Microscopy Platform at the University of Melbourne. In each of four experiments, fluorescent images (one per quadrant) of the PKH67-labelled vesicles and DAPI-stained nuclei were taken per sample.
Image J software  was used to quantify the number of PKH67-labelled vesicles and DAPI-stained nuclei in the same field. Data are expressed as mean ± SD number of PKH67-labelled vesicles/nuclei or number of nuclei per field and analysed by 2-way ANOVA with post-hoc tests (Holm-Sidak correction) using GraphPad Prism 7.04.
Quantitative proteomic analysis
For proteomic analysis, 3 x 105 HTR-8/SVneo cells were seeded in T-75 culture flasks (Nunc, ThermoFisher Scientific) and exposed to Prague PM (50 ng/ml) once (n = 3, Acute) or daily (n = 3, Chronic) and cells harvested after 48 hours (single dose without media change) or 7 days (both daily doses and media changes), respectively, and compared to unexposed controls (n = 4) receiving vehicle only with media changes daily for the same periods. Cells were washed 4 times in PBS, before removal with trypsin, pelleted and stored at -80°C for analysis.
Proteomics on cell pellets was performed at the Proteomics Core Facility at Sahlgrenska Academy, University of Gothenburg (http://proteomics.cf.gu.se/proteomics). A Tandem Mass Tag (TMT) set containing ten isobaric tags or labels (10-plex) allowed comparison of ten samples in one experiment for analysis by Mass Spectrometry (MS). In total, 100 μg protein per sample was trypsin digested into peptides and uniquely tagged at the N-terminals before analysis by nano-Liquid Chromatography-MS/MS. Proteins were identified in MS-raw data using Proteome Discoverer version 1.4 (ThermoFisher Scientific) against the Human Swissprot Database version March 2017 (Swiss Institute of Bioinformatics, Switzerland) and relative quantification was based on the ratio of reporter ion intensities/isobaric labelling). The low variability and the high sensitivity of the methodology used at the Proteomics Core Facility, University of Gothenburg, allows fold change of ≥1.2, if p-value is <0.05, to be considered significant. Student’s t-test was used to determine significant fold changes compared to unexposed controls, and together with Ingenuity Pathway Analysis (IPA, Qiagen) and Reactome Analysis (https://reactome.org/) to determine the biological relationships. Heatmaps were generated by Morpheus, https://software.broadinstitute.org/morpheus) for differentially expressed proteins (at p < 0.05 level of significance, FC > 1.2), and IPA analysis was used to generate diagrammatic representation of significant pathways and networks affected by PM exposure.
Reverse Phase Protein Arrays (RPPA) were performed at the Victorian Centre for Functional Genomics (VCFG), and the ACRF Translational RPPA platform at Peter MacCallum Cancer Centre Melbourne, for proteomic validation studies as previously described . Briefly, snap frozen protein lysates of PM exposed and control cells (n = 3 samples per group) were homogenised in CLB1 buffer (Zeptosens, Bayer), and quantified. Using a Caliper ALH3000 liquid handling robot (Perkin Elmer), samples were serially diluted before spotting onto ZeptoChips (Zeptosens) in duplicate using a Nano-Plotter-NP2.1 non-contact arrayer (GeSim). Chips were blocked, incubated with pre-validated primary antibodies diluted 1:500 for 20 hours before applying Alexa Fluor647 anti-rabbit secondary antibody (1:1000, 4 hours) (#Z-25308, ThermoFisher Scientific). Zeptosens instrument and software (version 3.1) were used to scan and calculate the relative fluorescence intensity. All samples were normalised to the background values reported in the secondary antibody-only negative controls. The antibodies used in the array, have been experimentally verified and highly-specific for signalling pathway components but include only a limited selection of pathway proteins in phosphorylated and non-phosphorylated form.
Pollution particles induce decreased cell growth and increased endocytosis
To examine the effects of the pollution particles on placental cells we exposed HTR-8/SVneo cells to Malmö and Prague particles over a culture period of 7 days (Fig 1) and examined the endocytotic responses, using the PKH67 fluorescent dye, and counted the number of cells at each time-point using DAPI nuclear dye.
(A-C): Fluorescence images showing PKH67 membrane stain (green), nuclei DAPI stain (blue) after 24 hr of PM exposure. Control cells show very few PKH67-stained endosomes (A, arrowhead). Cells exposed to Malmö PM show many small endosomes IB, arrowheads), whereas larger endosomes were detected in the Prague-treated cells (C, arrowheads), often in perinuclear region. Scale bars: 20 μm. (D-E): Quantification of numbers of endocytotic vesicles per cell (D) and cell number per field (E). Histograms display mean ± SEM. Significance of statistical analyses of particle-treated cultures with respective controls are indicated by: *, P<0.05; **, p<0.001; ***p<0.0001 (2-way ANOVA and t-test with Holm-Sidak correction for multiple comparison). See Supporting information S1 Fig and S1 Movie showing uptake of endocytic vesicles.
PKH7 is a dye that labels cell surface membranes as well as endosomes and phagosomes . There was very little labelling of internalised vesicles by PKH67 in unexposed control cells (Fig 1A). By contrast, in the Malmö PM-treated cells there were numerous small, fluorescently labelled vesicles in the cytoplasm (arrowheads, Fig 1B). However, in the Prague PM-exposed cells these fluorescently labelled vesicles were more numerous and larger (arrowheads, Fig 1C). Similarly, images of H&E stained cells showed fine particles (inset, Fig 1B) in the Malmö PM exposed cells and much larger particles in the Prague PM-exposed cells (inset, Fig 1C).
Quantification of PKH67 fluorescently labelled endocytotic vesicles in control and pollution particle-exposed HTR-8/SVneo cells showed statistically significant, time-dependent decreases in the numbers of endocytotic vesicles per cell from 30 min to 24 h and 7 days in all cultures (Fig 1D), consistent with this being a pulse labelling experiment and that the vesicle-bound fluorescent marker is likely processed during the culture period. However, both Malmö and Prague particle-exposed cells showed a significantly higher number of endocytotic vesicles per cell (p<0.001) when compared to the respective control cultures at each time-point (Fig 1D).
Observation of the cells during the culture period suggested that in the particle-exposed dishes there were fewer cells. Quantification of cell numbers at 30 min, 24h and 7 days after exposure showed that all cultures (vehicle-controls and particle-exposed) showed significant increases (p<0.0001) in cell number at each time-point over the 7-day culture period (Fig 1E), consistent with the normal expansion of these cells in culture. However, at 7 days, both the Malmö and Prague particle-exposed cultures showed significantly reduced numbers of cells (p<0.0001, Fig 1E) compared to controls, suggesting the growth of the cells in particle-exposed cultures was significantly inhibited. At 24 h in culture, only the Malmö cells showed a significant decrease (p = 0.03) in number, compared to respective controls (Fig 1E). There was no significant difference between the two particle-exposed cultures at any time-point (Fig 1E).
PM effects on protein secretion
To investigate whether PM had effects on markers of placental biology, we examined the levels of several factors : hCGβ (specific trophoblast pregnancy marker), IL-6 (marker of placental inflammation), progesterone (marker for trophoblast adhesion via MMP9) in the culture media. Exposure of HTR-8/SVneo cells for 48 h to varying concentrations of PM showed that only high doses of Prague (500 and 5000 ng/ml, Fig 2A) and Malmö PM (5000 ng/ml, Fig 2B) significantly decreased hCGβ secretion. Interestingly, exposure to Malmo PM supernatant at 500 or 5000 ng/ml also decreased hCGβ secretion (p < 0.01). Progesterone secretion was unaffected (see Supporting information S2 Fig).
The level of hCGβ (A, B) and IL-6 (C, D) secretion was measured in culture supernatant following exposure for 48 hours with varying doses of Prague (A, C) or Malmö (B, D) PM or PM-conditioned media. Results are expressed as ± S.D of triplicate wells. * p < 0.05. Abbreviations: Control (CTL), PM-conditioned media (S-).
By contrast, PM treatment for 48 h with high concentrations of Prague PM (500 and 5000 ng/ml) and Malmö PM (5000 ng/ml), caused significantly increased secretion of IL-6 (Fig 2C). Similarly, treatment of cells with the culture supernatants (500 ng/ml Prague PM and Malmö PM) also caused increased IL-6 secretion.
PM effects on gene expression
As exposure to the PM affected cellular secretion, we also examined whether there were changes in gene expression by quantitative RT-PCR. Exposure to Malmö or Prague PM at varying doses from 5 ng to 5000 ng/ml for 48 hours did not affect expression of any of the assayed genes (GAPDH, 18S, NFE2L2, MMP9, COX10, HMOX1, HIF1A, PECAM1 and ITGA5) compared to the expression in unexposed control cells (See Supporting information S2 Table for gene expression data).
To examine changes in protein expression we conducted proteomic analyses on the cells following Acute (48 hour) and Chronic (7 day) exposure with Prague PM (50 ng/ml) and used bioinformatic analyses to examine the most significantly affected pathways and processes. In total, 29 proteins were differentially expressed after acute exposure. These are listed in Fig 3 with their fold change compared to unexposed control cells, and replicate values represented in a heat map, where red and blue indicate up-and down-regulation, respectively. Of the 29 differentially expressed proteins, three were down-regulated and 26 were up-regulated (Fig 3).
Heatmap showing replicate expression intensities for control (CTL) and acute exposed samples with matching table showing levels of differential protein (UniProt code and description) expression (FC, fold change) following acute PM exposure (48 hr, 50 ng/ml, single dose). Shades of red and blue indicate relative levels of up- or down-regulation respectively. The Heatmap was generated with Morpheus (https://software.broadinstitute.org/morpheus).
The IPA analyses of acute PM exposure indicated that the most significant pathways affected were phagosome maturation, retinoic acid receptor (RAR) activation (p<0.02). However, of the proteins associated with these canonical pathways only 2/148 and 2/190 proteins, respectively showed expression changes. Clustering of cellular functions into networks revealed 2 major networks (amino acid metabolism/molecular transports/small molecule biochemistry, and cell death and survival/cellular compromise/cancer). Merging these two networks revealed major nodes involving TNF and VEGF signalling in the extracellular space, amyloid beta precursor protein, prion protein in the membrane and TP53, Fos a zinc finger E-box binding homeobox factor (ZEB2) and the estrogen receptor in the nucleus (Fig 4).
IPA network diagram showing combination of the top two networks (‘Amino acid metabolism, molecular transport, small molecule biochemistry’ and ‘Cell death and survival, cellular compromise, cancer’) affected in trophoblast cells exposed to Prague PM (50 ng/ml) for 48 h. Actual up- and down-regulated proteins are shown in red and green respectively, predicted activated and inhibited proteins are shown in orange and blue respectively, orange lines indicate activation, blue lines inhibition, yellow lines indicate inconsistent findings and grey lines no effect predicted. The Networks & Functional analyses were generated through the use of Ingenuity Pathways Analysis, Qiagen.
Following chronic exposure to Prague PM (7 days, 50 ng/ml daily), 47 proteins were differentially expressed compared to unexposed controls (Fig 5). Of the 47 proteins, 7 were down regulated and 40 were upregulated.
Heatmap showing replicate expression intensities for control (CTL) and chronic exposed samples with matching table showing levels of differential protein (UniProt code and description) expression (FC, fold change) following chronic PM (7 day, 50 ng/ml daily) exposure. Shades of red and blue indicate relative levels of up- or down-regulation respectively. The Heatmap was generated with Morpheus (https://software.broadinstitute.org/morpheus).
The top five canonical pathways affected were antigen presentation (p<0.0027; 2/38 proteins), autoimmune thyroid disease signalling (p<0.004; 2/47), graft-versus-host disease signalling (p<0.0042; 2/48), acute phase response signalling (p<0.0048; 3/170) and NRF2-mediated oxidative stress response (p<0.0069; 3/193). Clustering of cellular functions into networks revealed 2 major networks (cellular movement/cell cycle/cellular development and cell death and survival/cancer/organismal injury and abnormalities). Merging of these networks implicated 36 focus molecules involved in extracellular signalling (VEGF, Midkine, IGFBP2, immunoglobulins, interferon alpha) acting through membrane proteins (amyloid precursor protein, TNFRSF10B, NTRK1 EGFR), converging on the Ras-MAPK cascade and AKT in the cytoplasm and Fos/Jun, NFkB, RB1 and HMGA1 in the nucleus (Fig 6).
IPA network diagram showing combination of the top two networks (‘Cellular development, cellular growth and proliferation, connective tissue development and function’ and ‘Cell-to-cell signalling and interaction, drug metabolism, molecular transport’) affected in trophoblast cells following chronic Prague PM (7 days, 50 ng/ml daily) exposure. Actual up- and down-regulated proteins are shown in red and green respectively, predicted activated and inhibited proteins are shown in orange and blue respectively, orange lines indicate activation, blue lines inhibition, yellow lines indicate inconsistent findings, and grey lines no effect predicted. The Networks & Functional analyses were generated through the use of Ingenuity Pathways Analysis, Qiagen.
We initially used western blotting analysis to validate proteomic findings but given the low percentage changes in protein abundance, this method was not sensitive enough to show the differences between control and PM exposed cells (see S3A Fig). Instead we used RPPA analysis, which confirmed the pathway activation predicted by IPA (see S3B Fig). Notably, TP53 following acute PM exposure, as well as AKT and ERK, following chronic PM exposure, were activated. These proteins are highlighted in orange in Figs 4 and 6, respectively.
There is a strong association between levels of PM pollution and pregnancy complications such as preterm births and pre-eclampsia ([3–7], disorders in which placental dysfunction plays a pivotal role . Inhaled particles can be detected in the circulation within 1 minute  and a recent study has shown that particulate matter can be found within cells in the placenta , suggesting this tissue is also a target for air pollution.
Similarly, rodents exposed to PM via inhalation during gestation developed morphological changes in the placenta . Moreover, inhaled particles, such as nanoparticles, can reach the mouse placenta , and exposure to diesel particles can cause DNA damage  and increased cytokine production in the fetus  and in the placenta , suggesting transplacental transfer of pollution particles from the maternal to the fetal circulation. Consistent with this, analysis of samples of full-term placenta exposed to air pollution PM showed high levels of heavy metals in the syncytiotrophoblast layers and it was proposed that trophoblast cells actively phagocytosed and concentrated metal-containing particles .
We attempted in this study to calculate a realistic exposure rate based on our previous epidemiological data studies showing that there is an association between women exposed to ambient air pollution in Malmö and low birth weights and pre-term births [3–4]. As the average daily mean ambient PM2.5 is between 20–30 μg/m3 in Malmö, Sweden, (compared to Swedish national average < 10 μg/m3), we began our calculations assuming pregnant women were exposed to PM2.5 of 25 μg/m3 daily. We calculated that pregnant women were exposed to between 50–500 ng of PM2.5 per day. However, in other cities, such Taiyuan City, China  that have higher levels of average daily ambient levels of PM2.5, testing higher concentrations in the microgram to low milligram range on trophoblast cells would be highly appropriate.
In this study we have shown, using an in vitro approach that PM particles are internalised by endocytosis in human trophoblast cells for at least 7 days, with many of the internalised vesicles accumulating in the perinuclear region. Moreover, the initiation of particle endocytosis is very rapid, occurring within 30 minutes of exposure with both Malmö and Prague PM. While at this time-point there was no clear effect on growth of the cells, the number of cells in cultures exposed to PM for 7 days was significantly decreased, suggesting PM exposure inhibits cell growth in vitro. In addition to changes in growth, there are alterations in a trophoblast pregnancy marker (hCGβ) and inflammation (IL-6) but not in placental differentiation (progesterone) within 48 hours. By contrast, Wang and colleagues  reported a significant decrease in progesterone levels following exposure of trophoblast cells to industry-derived PM2.5. While we do not know the reason for the differences in results between studies, we have used HTR-8/SVneo cells (a first trimester transformed cell line from human chorionic villi explants), and Wang et al used JEG-3 cells, which are derived from a human choriocarcinoma. Thus, it is possible the difference in results relate to the different origin of the cell lines used.
While by RT-PCR there were no significant changes in several genes that have been associated with hypoxia (NFE2L2, HIF1A, COX10, HMOX1) and cell adhesion (MMP9, PECAM1 and ITGA5) after 48 hours exposure, the proteomic analyses of cells exposed to Prague PM, acutely (48 h) or chronically (7 days), showed significant changes in 29 and 47 proteins respectively. These findings confirm that PM enter trophoblast cells by endocytosis and can alter cellular pathways.
The acute inflammatory (IL-6) and dysfunctional (hCGβ) responses observed in the HTR-8/SVneo cells are consistent with earlier studies, which showed an increase in the release of proinflammatory cytokines (e.g. IL-6, IL-1b) into the human circulation following exposure to PM . Further support for an inflammatory response is provided by increases in interferon-induced protein (IFIT3, ), and a phosphodiesterase (PDE7A, ), which have been associated with innate immune system activation and inflammation, respectively, and are upregulated following acute PM exposure. Both Acute and Chronic PM exposure led to increases in PDE7A, which has been suggested as a target to alleviate inflammation associated with various inflammatory diseases [53, 54] and underscores the activation of inflammation in trophoblast cells following exposure to air pollution.
The acutely increased expression of prion protein (PRNP, ), chaperone proteins, DNAJC14  and FAM107B , the endo-lysosomal trafficking protein (MGRN1)  and aldehyde dehydrogenase (ALDH1A3) , which can be upregulated following acute cell stressors, suggests that the PM-treated trophoblast cells exhibit a stress response. The up-regulation of TP53, suggests that these cells may be more prone to cell death. Consistent with this, there was an observed loss of cells from these cultures with time. Changes in human placental functions including altered gene expression and increases in markers of oxidative stress [28, 29], mitochondrial dysfunction and altered methylation profiles [30–32], and endothelial and villi pathology  have been reported following inhalation of PM during pregnancy.
While the Acute response to PM appeared to affect amino acid metabolism, molecular transport and cell death and survival, the predominant chronic response to PM appeared to involve changes in immune signaling affecting antigen presentation, autoimmune responses and oxidative stress responses. While neither the Prague nor Malmö PM have been analysed for bioaerosols, other traffic-pollution particulate matter, including PM2.5 and PM10, have been shown to contain endotoxins, which cause inflammatory and innate immune responses in the respiratory system [60–62]. Consistent with this, the clustering of the dysregulated proteins into the ontologies of antigen presentation, acute phase response signalling, NRF2-mediated oxidative stress response and innate immune system activation, following chronic PM exposure suggest that the Malmö and Prague particles may contain microorganism fragments or endotoxins that activate inflammatory and innate immune responses in placental cells. Bacterial endotoxins can trigger inflammation via interactions with Toll-like receptors (TLR/CD14)  and the changes in IFIT3 and IL-6 at 48 h as well as the implication of NFkB involvement in the chronically exposed cells at 7 days are consistent with an endotoxin response .
The upregulated expression of endocytosis and intracellular transport proteins (REPS2, IFT43, TBC1D10A, PXDC1, RAB35,) are consistent with the uptake of PM into endosomes or phagosomes, which normally would eventually fuse with lysosomes for degradation. The accumulation of vesicular PM in the perinuclear region for up to 7 days suggests that the process of degradation is dysfunctional. Consistent with this, there is dysregulated expression of KLH29, RAB35 and TCPR1 proteins, which are involved in fusing endosomes to lysosomes. Moreover, the expression of ER stress related proteins TMX4 , REEP1 , TNFAIP8 ) and cell death associated proteins (TNFRSF10B , TRAIL , suggest that the PM has disrupted ER-phagosome interactions  and induced an ER stress-like response.
Various stimuli can lead to ER stress, including endotoxins, drugs and inflammation followed by overloaded or misfolded proteins . Therefore, alternative mechanisms by which PM exposure may lead to ER stress in trophoblast cells is by increased internalisation of membrane receptors, and innate immune system activation, via the upregulation of AMPN (also known as CD13) and GBP1, which are known to regulate Toll-Like receptor internalisation (63) or antigen internalisation respectively (64). This may lead to increased activation of signalling cascades, increased inflammation as evidenced by LPCAT2 and MDK, which are known to be upregulated during inflammation (65, 66) and dysregulation of chaperone proteins (increases in DNAJC1, FAM107B and decrease in HSPBAP1) as shown in this study. Importantly, trophoblast cells are known to induce platelet-activating factor (PAF) upon inflammatory stimulation, via the activity of acetyl-transferase, LPCAT2, which was upregulated (+1.24 fold) in the chronically exposed cells. The fact that acetylated-PAF was found to be much higher in PE than in normal placentas (67), reinforces the parallels between PM-exposed trophoblasts and dysfunctional placentas in PE.
An intriguing finding in the acute response was the dysregulated expression of several metal ion transport proteins (SLC3A2, SLC7A5, TRIM59, KCNMA1). As both Malmö PM2.5 (See Supplementary Information S1 Table) and Prague PM10 contain metals including mercury, copper, iron, zinc and lead, it is plausible that these expression changes are adaptive responses by the cell to cope with these heavy metals and thus modulate oxidative stress that can lead to superoxide and hydroxyl radicals . In this context the expression of PRNP protein may provide a link between metal exposure and cell signalling as it has been implicated in diverse cellular functions including placenta development, metal ion transporter, signalling and cytoskeletal associated protein . In the IPA network analyses for Acute exposure, PRNP upregulation was linked to activation of several signalling proteins including ERK1, P38 MAPK, FOSL1 and TP53, as well as chaperone proteins (HSPB1, DNAJC14) and APP. Similar to the compensatory expression of ion transport genes, the dramatic increases (44–66%) in ALDH1A3 following chronic or acute PM exposure may be a response to the high levels of various aldehydes that are present in urban traffic-derived PM. AKT and ERK pathways were predicted to be activated by IPA analysis and these were confirmed to show increased phosphorylation by the RPPA analyses.
The differentially expressed proteins detected in the PM-exposed HTR-8/SVneo cells are similar to proteins and genes that are differentially expressed in PE and IUGR. For instance, PRNP is highly upregulated in PE compared to normal pregnancy levels  and transcriptome profiling of 200 placentas, revealing that DNAJC14 was one of five hub proteins that were significantly associated with fetal growth restriction . Other differentially expressed proteins found in our study, following Acute (FAM107B) and Chronic PM exposure (PRICKLE2, TNFAIP8, DAG1, PSAP, IFT43 and ZNF652) were also present in placental expression networks associated with birth weight restriction . This leads to the suggestion that PM exposed trophoblast cells may activate similar cell stress pathways as occurs in PE and foetal growth restriction.
It is now well-established that different sized inhaled PM can cross into the human circulation directly, or indirectly via phagocytosis by immune cells that migrate to the lymphatic system where most are cleared via the kidney and gastrointestinal tract [39, 74, 75], and can reach the placenta . Our studies suggest that even low levels of urban PM can have damaging effects on trophoblast cells, even over short exposure periods. Trophoblast cells actively take up urban PM by endocytosis and may respond to the particles themselves, or their components such as endotoxins, reactive metals, or PAHs. The accumulation of the vesicular-bound particles in the perinuclear region, appears to lead to changes in trafficking proteins with subsequent activation of ER stress, growth inhibition, oxidative stress and inflammation. Many of the dysfunctional cellular processes ascribed to the differentially expressed proteins in this study are similar to those found in PE and IGUR, suggesting that air pollution particulate matter may contribute to these conditions.
S1 Fig. Effect of PM exposure on trophoblast progesterone secretion.
The level of Progesterone secretion was measured in culture supernatant following exposure for 48 hours with varying doses of Prague (A) or Malmö (B) PM or PM-conditioned media. Results are expressed as ± S.D of triplicate wells. * p < 0.05. Abbreviations: Control (CTRL), PM-conditioned media (S-).
This fig shows corresponding stained cells (Nucleus-DAPI Blue staining, and vesicles-PKH67 green staining) used to prepare z-stack and its animation.
S3 Fig. Validation of proteomic studies.
S3 Fig A shows the results of western blotting probing for the upregulated proteins following chronic exposure to PM: ALPP, TSCD22D4, PDE7A and downregulated HSPBAP1. The very small changes in protein abundance identified via proteomics and SRM, were difficult to detect using this methodology. S3 Fig B shows the results of RPPA analysis following chronic and acute exposure to PM. These results confirm the predictions of activated pathways following IPA analysis, namely AKT and ERK following chronic PM exposure, and TP53 following acute PM exposure.
S1 Movie. Animation showing internalised Prague PM in trophoblast cells.
Prague PM10 24 hr Exposure.avi; Z-stack animation, showing the presence of internalised vesicles with PKH7 staining in cells exposed to PM after 24 hr exposure.
S1 Table. Concentration of different PAHs and metals from urban PM2.5 samples collected in Malmö, Sweden.
Table showing the concentration (ng/mg) of 32 individual PAHs and 14 metals determined in the urban PM2.5 samples collected in Malmö, Sweden. The PAH levels found in the Malmö PM sample were somewhat lower, a factor of 1.5–5 dependent on compound, compared to published results of the PM Prague sample (NIST Certificate of Analysis Standard Reference Material 2786, 2016, Gaithersburg, MD, USA). Notably, even greater differences were found for the metals, a factor 2 to 100 times higher for the individual metals, in the Prague PM samples than in the Malmö PM sample. Analysis of nitro-PAHs and oxy-PAHs were not included in this study but will be the focus of future work.
S2 Table. Effect of PM exposure on trophoblast gene expression.
The expression of selected genes (GAPDH, 18S, NFE2L2, MMP9, COX10, HMOX1, HIF1A, PECAM1 and ITGA5) in trophoblast cells following exposure of varying doses (ng/ml) of Prague (S4 Table A) or Malmö (S4 Table B) PM for 48 hours. Values are based on relative expression (ΔΔCt values, using methodology developed by Pfaffl, 20011) compared to unexposed control cells. Values in the Table are Average (av) ± Standard Deviation (std), n = 6 per sample per gene per PM concentration, compared to unexposed control cells equal to 1. We found no significant changes in expression for any gene at any exposure concentration. 1Pfaffl, M. W (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research. 29:2002–2007.
S1 Text. Physiological dose considerations, methodology and rationale for gene expression analysis.
a. Physiological dose considerations based on Malmo average daily ambient concentration of PM2.5. b. Methodology and rationale for gene expression analyses of PM exposed trophoblast.
We thank Drs Gabriella Segal and Ellie Cho for their invaluable assistance with Confocal Microscopy at the Biological Optical Microscopy Platform, University of Melbourne. We thank A/Prof Kaylene J Simpson and Arthi M Macpherson from the Victorian Centre for Functional Genomics and the ACRF Translational RPPA platform at Peter MacCallum Cancer Centre for generating the RPPA data.
- 1. WHO. Review of evidence on health aspects of air pollution–REVIHAAP Project. 2013.
- 2. Atkinson R, Arey J. Mechanisms of the gas-phase reactions of aromatic hydrocarbons and PAHs with OH and NO3 radicals. Polycyclic Aromatic Compounds. 2007;27(1):15–40.
- 3. Malmqvist E, Jakobsson K, Tinnerberg H, Rignell-Hydbom A, Rylander L. Gestational diabetes and preeclampsia in association with air pollution at levels below current air quality guidelines. Environ Health Perspect. 2013;121(4):488–93. pmid:23563048
- 4. Malmqvist E, Liew Z, Kallen K, Rignell-Hydbom A, Rittner R, Rylander L, et al. Fetal growth and air pollution—A study on ultrasound and birth measures. Environ Res. 2017;152:73–80. Epub 2016/10/16. pmid:27741452.
- 5. Lee PC, Roberts JM, Catov JM, Talbott EO, Ritz B. First trimester exposure to ambient air pollution, pregnancy complications and adverse birth outcomes in Allegheny County, PA. Maternal and child health journal. 2013;17(3):545–55. Epub 2012/05/01. pmid:22544506
- 6. Pedersen M, Stayner L, Slama R, Sorensen M, Figueras F, Nieuwenhuijsen MJ, et al. Ambient air pollution and pregnancy-induced hypertensive disorders: a systematic review and meta-analysis. Hypertension. 2014;64(3):494–500. pmid:24935943.
- 7. Pedersen M, Halldorsson TI, Olsen SF, Hjortebjerg D, Ketzel M, Grandstrom C, et al. Impact of Road Traffic Pollution on Pre-eclampsia and Pregnancy-induced Hypertensive Disorders. Epidemiology (Cambridge, Mass). 2017;28(1):99–106. Epub 2016/09/21. pmid:27648591
- 8. Wang YY, Li Q, Guo Y, Zhou H, Wang X, Wang Q, et al. Association of Long-term Exposure to Airborne Particulate Matter of 1 mum or Less With Preterm Birth in China. JAMA Pediatr. 2018;172(3):e174872. pmid:29297052
- 9. Backes CH, Nelin T, Gorr MW, Wold LE. Early life exposure to air pollution: how bad is it? Toxicology letters. 2013;216(1):47–53. Epub 2012/11/21. pmid:23164674
- 10. Dadvand P, Ostro B, Amato F, Figueras F, Minguillon MC, Martinez D, et al. Particulate air pollution and preeclampsia: a source-based analysis. Occupational and environmental medicine. 2014;71(8):570–7. Epub 2014/04/01. pmid:24683010.
- 11. WHO. WHO Air quality guidelines for particulate matter, ozone, nitrogen dioxide and sulfur dioxide. Global update 2005. Summary of risk assessment. 2006.
- 12. Gupte S, Wagh G. Preeclampsia-eclampsia. Journal of obstetrics and gynaecology of India. 2014;64(1):4–13. Epub 2014/03/04. pmid:24587599
- 13. Hansson SR, Naav A, Erlandsson L. Oxidative stress in preeclampsia and the role of free fetal hemoglobin. Frontiers in physiology. 2014;5:516. pmid:25628568
- 14. Redman CW, Sargent IL. Placental stress and pre-eclampsia: a revised view. Placenta. 2009;30 Suppl A:S38–42. pmid:19138798.
- 15. Borzychowski AM, Sargent IL, Redman CW. Inflammation and pre-eclampsia. Semin Fetal Neonatal Med. 2006;11(5):309–16. pmid:16828580.
- 16. Ilekis JV, Tsilou E, Fisher S, Abrahams VM, Soares MJ, Cross JC, et al. Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Am J Obstet Gynecol. 2016;215(1 Suppl):S1–s46. Epub 2016/03/15. pmid:26972897
- 17. van Eeden SF, Tan WC, Suwa T, Mukae H, Terashima T, Fujii T, et al. Cytokines involved in the systemic inflammatory response induced by exposure to particulate matter air pollutants (PM(10)). American journal of respiratory and critical care medicine. 2001;164(5):826–30. Epub 2001/09/11. pmid:11549540.
- 18. Ruckerl R, Schneider A, Breitner S, Cyrys J, Peters A. Health effects of particulate air pollution: A review of epidemiological evidence. Inhal Toxicol. 2011;23(10):555–92. Epub 2011/08/26. pmid:21864219.
- 19. van den Hooven EH, de Kluizenaar Y, Pierik FH, Hofman A, van Ratingen SW, Zandveld PY, et al. Chronic air pollution exposure during pregnancy and maternal and fetal C-reactive protein levels: the Generation R Study. Environ Health Perspect. 2012;120(5):746–51. pmid:22306530
- 20. Giovannini N, Schwartz L, Cipriani S, Parazzini F, Baini I, Signorelli V, et al. Particulate matter (PM10) exposure, birth and fetal-placental weight and umbilical arterial pH: results from a prospective study. J Matern Fetal Neonatal Med. 2018;31(5):651–5. Epub 2017/03/10. pmid:28277922.
- 21. Wylie BJ, Matechi E, Kishashu Y, Fawzi W, Premji Z, Coull BA, et al. Placental Pathology Associated with Household Air Pollution in a Cohort of Pregnant Women from Dar es Salaam, Tanzania. Environ Health Perspect. 2017;125(1):134–40. Epub 2016/06/11. pmid:27286442
- 22. Blum JL, Chen LC, Zelikoff JT. Exposure to Ambient Particulate Matter during Specific Gestational Periods Produces Adverse Obstetric Consequences in Mice. Environ Health Perspect. 2017;125(7):077020. Epub 2017/09/13. pmid:28893721
- 23. Liu Y, Wang L, Wang F, Li C. Effect of Fine Particulate Matter (PM2.5) on Rat Placenta Pathology and Perinatal Outcomes. Med Sci Monit. 2016;22:3274–80. Epub 2016/09/16. pmid:27629830
- 24. Gurgueira SA, Lawrence J, Coull B, Murthy GG, Gonzalez-Flecha B. Rapid increases in the steady-state concentration of reactive oxygen species in the lungs and heart after particulate air pollution inhalation. Environ Health Perspect. 2002;110(8):749–55. Epub 2002/08/03. pmid:12153754
- 25. Hougaard KS, Jensen KA, Nordly P, Taxvig C, Vogel U, Saber AT, et al. Effects of prenatal exposure to diesel exhaust particles on postnatal development, behavior, genotoxicity and inflammation in mice. Part Fibre Toxicol. 2008;5:3. Epub 2008/03/12. pmid:18331653
- 26. Campagnolo L, Massimiani M, Vecchione L, Piccirilli D, Toschi N, Magrini A, et al. Silver nanoparticles inhaled during pregnancy reach and affect the placenta and the foetus. Nanotoxicology. 2017;11(5):687–98. Epub 2017/06/18. pmid:28618895.
- 27. Nemmar A, Hoet PH, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF, et al. Passage of inhaled particles into the blood circulation in humans. Circulation. 2002;105(4):411–4. pmid:11815420.
- 28. Saenen ND, Plusquin M, Bijnens E, Janssen BG, Gyselaers W, Cox B, et al. In Utero Fine Particle Air Pollution and Placental Expression of Genes in the Brain-Derived Neurotrophic Factor Signaling Pathway: An ENVIRONAGE Birth Cohort Study. Environ Health Perspect. 2015;123(8):834–40. Epub 2015/03/31. pmid:25816123
- 29. Dutta A, Khramtsova G, Brito K, Alexander D, Mueller A, Chinthala S, et al. Household air pollution and chronic hypoxia in the placenta of pregnant Nigerian women: A randomized controlled ethanol Cookstove intervention. Sci Total Environ. 2018;619–620:212–20. Epub 2017/11/18. pmid:29149745.
- 30. Janssen BG, Munters E, Pieters N, Smeets K, Cox B, Cuypers A, et al. Placental mitochondrial DNA content and particulate air pollution during in utero life. Environ Health Perspect. 2012;120(9):1346–52. Epub 2012/05/26. pmid:22626541
- 31. Clemente DBP, Casas M, Janssen BG, Lertxundi A, Santa-Marina L, Iniguez C, et al. Prenatal ambient air pollution exposure, infant growth and placental mitochondrial DNA content in the INMA birth cohort. Environ Res. 2017;157:96–102. Epub 2017/05/24. pmid:28535425.
- 32. Maghbooli Z, Hossein-Nezhad A, Adabi E, Asadollah-Pour E, Sadeghi M, Mohammad-Nabi S, et al. Air pollution during pregnancy and placental adaptation in the levels of global DNA methylation. PLoS One. 2018;13(7):e0199772. Epub 2018/07/07. pmid:29979694
- 33. Qin Z, Hou H, Fu F, Wu J, Han B, Yang W, et al. Fine particulate matter exposure induces cell cycle arrest and inhibits migration and invasion of human extravillous trophoblast, as determined by an iTRAQ-based quantitative proteomics strategy. Reprod Toxicol. 2017;74:10–22. pmid:28843701.
- 34. Liu N, Miyashita L, McPhail G, Thangaratinam S, Grigg J. PA360 Abstract: Do inhaled carbonaceous particles translocate from the lung to the placenta?. European Respiratory Society International Congress; Paris2018.
- 35. Ru Q, Xiong Q, Chen L, Tian X, Yue K, Ma B, et al. Lipopolysaccharide accelerates fine particulate matter-induced cell apoptosis in human lung bronchial epithelial cells. Int J Occup Med Environ Health. 2018;31(2):173–83. pmid:29021634.
- 36. Kliucininkas L, Martuzevicius D, Krugly E, Prasauskas T, Kauneliene V, Molnar P, et al. Indoor and outdoor concentrations of fine particles, particle-bound PAHs and volatile organic compounds in Kaunas, Lithuania. J Environ Monit. 2011;13(1):182–91. Epub 2010/11/18. pmid:21082095.
- 37. Bekki K, Ito T, Yoshida Y, He C, Arashidani K, He M, et al. PM2.5 collected in China causes inflammatory and oxidative stress responses in macrophages through the multiple pathways. Environ Toxicol Pharmacol. 2016;45:362–9. pmid:27393915.
- 38. Lasagni Vitar RM, Tau J, Janezic NS, Tesone AI, Hvozda Arana AG, Reides CG, et al. Diesel exhaust particles (DEP) induce an early redox imbalance followed by an IL-6 mediated inflammatory response on human conjunctival epithelial cells. Exp Eye Res. 2018;171:37–47. Epub 2018/03/11. pmid:29524384.
- 39. Kastury F, Smith E, Juhasz AL. A critical review of approaches and limitations of inhalation bioavailability and bioaccessibility of metal(loid)s from ambient particulate matter or dust. Sci Total Environ. 2017;574:1054–74. Epub 2016/09/28. pmid:27672736.
- 40. Soma-Pillay P, Nelson-Piercy C, Tolppanen H, Mebazaa A. Physiological changes in pregnancy. Cardiovascular journal of Africa. 2016;27(2):89–94. Epub 2016/05/24. pmid:27213856
- 41. Tschernig T, Pabst R. What is the clinical relevance of different lung compartments? BMC pulmonary medicine. 2009;9:39. Epub 2009/08/13. pmid:19671154
- 42. Mary S, Kulkarni MJ, Malakar D, Joshi SR, Mehendale SS, Giri AP. Placental Proteomics Provides Insights into Pathophysiology of Pre-Eclampsia and Predicts Possible Markers in Plasma. Journal of proteome research. 2017;16(2):1050–60. Epub 2016/12/29. pmid:28030762.
- 43. Yang JI, Kong TW, Kim HS, Kim HY. The Proteomic Analysis of Human Placenta with Pre-eclampsia and Normal Pregnancy. Journal of Korean medical science. 2015;30(6):770–8. Epub 2015/06/02. pmid:26028931
- 44. Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 2017;18(1):529. Epub 2017/12/01. pmid:29187165
- 45. Pearson HB, Li J, Meniel VS, Fennell CM, Waring P, Montgomery KG, et al. Identification of Pik3ca Mutation as a Genetic Driver of Prostate Cancer That Cooperates with Pten Loss to Accelerate Progression and Castration-Resistant Growth. Cancer Discov. 2018;8(6):764–79. pmid:29581176.
- 46. Costa MA. The endocrine function of human placenta: an overview. Reprod Biomed Online. 2016;32(1):14–43. Epub 2015/12/01. pmid:26615903.
- 47. Veras MM, Damaceno-Rodrigues NR, Caldini EG, Maciel Ribeiro AA, Mayhew TM, Saldiva PH, et al. Particulate urban air pollution affects the functional morphology of mouse placenta. Biol Reprod. 2008;79(3):578–84. pmid:18509159.
- 48. Reliene R, Hlavacova A, Mahadevan B, Baird WM, Schiestl RH. Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice. Mutat Res. 2005;570(2):245–52. Epub 2005/02/15. pmid:15708583.
- 49. de Melo JO, Soto SF, Katayama IA, Wenceslau CF, Pires AG, Veras MM, et al. Inhalation of fine particulate matter during pregnancy increased IL-4 cytokine levels in the fetal portion of the placenta. Toxicology letters. 2015;232(2):475–80. Epub 2014/12/08. pmid:25481569.
- 50. Kaiglova A, Reichrtova E, Adamcakova A, Wsolova L. Lactate dehydrogenase activity in human placenta following exposure to environmental pollutants. Physiological research. 2001;50(5):525–8. Epub 2001/11/13. pmid:11702858.
- 51. Wang C, Yang J, Hao Z, Gong C, Tang L, Xu Y, et al. Suppression of progesterone synthesis in human trophoblast cells by fine particulate matter primarily derived from industry. Environ Pollut. 2017;231(Pt 1):1172–80. Epub 2017/09/25. pmid:28935403.
- 52. Bulgari O, Dong X, Roca AL, Caroli AM, Loor JJ. Innate immune responses induced by lipopolysaccharide and lipoteichoic acid in primary goat mammary epithelial cells. Journal of animal science and biotechnology. 2017;8:29. Epub 2017/04/12. pmid:28396748
- 53. Giembycz MA, Smith SJ. Phosphodiesterase 7A: a new therapeutic target for alleviating chronic inflammation? Current pharmaceutical design. 2006;12(25):3207–20. Epub 2006/10/06. pmid:17020529.
- 54. Lelubre C, Medfai H, Akl I, Leentjens J, Kox M, Pickkers P, et al. Leukocyte phosphodiesterase expression after lipopolysaccharide and during sepsis and its relationship with HLA-DR expression. Journal of leukocyte biology. 2017;101(6):1419–26. Epub 2017/03/31. pmid:28356347.
- 55. Lo RY, Shyu WC, Lin SZ, Wang HJ, Chen SS, Li H. New molecular insights into cellular survival and stress responses: neuroprotective role of cellular prion protein (PrPC). Molecular neurobiology. 2007;35(3):236–44. Epub 2007/10/06. pmid:17917112.
- 56. Joshi S, Wang T, Araujo TLS, Sharma S, Brodsky JL, Chiosis G. Adapting to stress—chaperome networks in cancer. Nature reviews Cancer. 2018. Epub 2018/05/26. pmid:29795326.
- 57. Nakajima H, Ishigaki Y, Xia QS, Ikeda T, Yoshitake Y, Yonekura H, et al. Induction of HITS, a newly identified family with sequence similarity 107 protein (FAM107B), in cancer cells by heat shock stimulation. International journal of oncology. 2010;37(3):583–93. Epub 2010/07/29. pmid:20664927.
- 58. Chhangani D, Mishra A. Mahogunin ring finger-1 (MGRN1) suppresses chaperone-associated misfolded protein aggregation and toxicity. Sci Rep. 2013;3:1972. Epub 2013/06/13. pmid:23756845
- 59. Singh S, Brocker C, Koppaka V, Chen Y, Jackson BC, Matsumoto A, et al. Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress. Free Radic Biol Med. 2013;56:89–101. Epub 2012/12/01. pmid:23195683
- 60. Alexis NE, Lay JC, Zeman K, Bennett WE, Peden DB, Soukup JM, et al. Biological material on inhaled coarse fraction particulate matter activates airway phagocytes in vivo in healthy volunteers. J Allergy Clin Immunol. 2006;117(6):1396–403. pmid:16751003.
- 61. Liu H, Zhang X, Zhang H, Yao X, Zhou M, Wang J, et al. Effect of air pollution on the total bacteria and pathogenic bacteria in different sizes of particulate matter. Environ Pollut. 2018;233:483–93. pmid:29101891.
- 62. Morakinyo OM, Mokgobu MI, Mukhola MS, Hunter RP. Health Outcomes of Exposure to Biological and Chemical Components of Inhalable and Respirable Particulate Matter. Int J Environ Res Public Health. 2016;13(6). pmid:27314370
- 63. Kelly FJ, Fussell JC. Size, source and chemical composition as determinants of toxicity attributable to ambient particulate matter. Atmospheric Environment. 2012;60:504–26. http://dx.doi.org/10.1016/j.atmosenv.2012.06.039.
- 64. Silva Z, Verissimo T, Videira PA, Novo C. Protein disulfide isomerases: Impact of thapsigargin treatment on their expression in melanoma cell lines. International journal of biological macromolecules. 2015;79:44–8. Epub 2015/04/29. pmid:25912252.
- 65. Allison R, Edgar JR, Pearson G, Rizo T, Newton T, Gunther S, et al. Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia. The Journal of cell biology. 2017;216(5):1337–55. Epub 2017/04/09. pmid:28389476
- 66. Kim JS, Park J, Kim MS, Ha JY, Jang YW, Shin DH, et al. The Tnfaip8-PE complex is a novel upstream effector in the anti-autophagic action of insulin. Sci Rep. 2017;7(1):6248. Epub 2017/07/26. pmid:28740220
- 67. Li T, Su L, Lei Y, Liu X, Zhang Y, Liu X. DDIT3 and KAT2A Proteins Regulate TNFRSF10A and TNFRSF10B Expression in Endoplasmic Reticulum Stress-mediated Apoptosis in Human Lung Cancer Cells. The Journal of biological chemistry. 2015;290(17):11108–18. Epub 2015/03/15. pmid:25770212
- 68. Edagawa M, Kawauchi J, Hirata M, Goshima H, Inoue M, Okamoto T, et al. Role of activating transcription factor 3 (ATF3) in endoplasmic reticulum (ER) stress-induced sensitization of p53-deficient human colon cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through up-regulation of death receptor 5 (DR5) by zerumbone and celecoxib. The Journal of biological chemistry. 2014;289(31):21544–61. Epub 2014/06/19. pmid:24939851
- 69. Nunes-Hasler P, Demaurex N. The ER phagosome connection in the era of membrane contact sites. Biochimica et biophysica acta. 2017;1864(9):1513–24. Epub 2017/04/23. pmid:28432021.
- 70. Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell. 2010;140(6):900–17. Epub 2010/03/23. pmid:20303879
- 71. Makzhami S, Passet B, Halliez S, Castille J, Moazami-Goudarzi K, Duchesne A, et al. The prion protein family: a view from the placenta. Frontiers in cell and developmental biology. 2014;2:35. Epub 2014/11/05. pmid:25364742
- 72. Donadio S, Alfaidy N, De Keukeleire B, Micoud J, Feige JJ, Challis JR, et al. Expression and localization of cellular prion and COMMD1 proteins in human placenta throughout pregnancy. Placenta. 2007;28(8–9):907–11. Epub 2007/01/27. pmid:17254632.
- 73. Deyssenroth MA, Peng S, Hao K, Lambertini L, Marsit CJ, Chen J. Whole-transcriptome analysis delineates the human placenta gene network and its associations with fetal growth. BMC genomics. 2017;18(1):520. Epub 2017/07/12. pmid:28693416
- 74. Grunig G, Marsh LM, Esmaeil N, Jackson K, Gordon T, Reibman J, et al. Perspective: ambient air pollution: inflammatory response and effects on the lung’s vasculature. Pulm Circ. 2014;4(1):25–35. pmid:25006418
- 75. Elder A, Oberdorster G. Translocation and effects of ultrafine particles outside of the lung. Clin Occup Environ Med. 2006;5(4):785–96. Epub 2006/11/18. pmid:17110292.