Biological impact of sequential exposures to allergens and ultrafine particle-rich combustion aerosol on human bronchial epithelial BEAS-2B cells at the air liquid interface.

The prevalence of allergic diseases is constantly increasing since few decades. Anthropogenic ultrafine particles (UFPs) and allergenic aerosols is highly involved in this increase; however, the underlying cellular mechanisms are not yet understood. Studies observing these effects focused mainly on singular in vivo or in vitro exposures of single particle sources, while there is only limited evidence on their subsequent or combined effects. Our study aimed at evaluating the effect of subsequent exposures to allergy-related anthropogenic and biogenic aerosols on cellular mechanism exposed at air-liquid interface (ALI) conditions. Bronchial epithelial BEAS-2B cells were exposed to UFP-rich combustion aerosols for 2 h with or without allergen pre-exposure to birch pollen extract (BPE) or house dust mite extract (HDME). The physicochemical properties of the generated particles were characterized by state-of-the-art analytical instrumentation. We evaluated the cellular response in terms of cytotoxicity, oxidative stress, genotoxicity, and in-depth gene expression profiling. We observed that single exposures with UFP, BPE, and HDME cause genotoxicity. Exposure to UFP induced pro-inflammatory canonical pathways, shifting to a more xenobiotic-related response with longer preincubation time. With additional allergen exposure, the modulation of pro-inflammatory and xenobiotic signaling was more pronounced and appeared faster. Moreover, aryl hydrocarbon receptor (AhR) signaling activation showed to be an important feature of UFP toxicity, which was especially pronounced upon pre-exposure. In summary, we were able to demonstrate the importance of subsequent exposure studies to understand realistic exposure situations and to identify possible adjuvant allergic effects and the underlying molecular mechanisms.

Construction of an In Vitro Air-Liquid Interface Exposure System to Assess the Toxicological Impact of Gas and Particle Phase of Semi-Volatile Organic Compounds.

Anthropogenic activities and industrialization render continuous human exposure to semi-volatile organic compounds (SVOCs) inevitable. Occupational monitoring and safety implementations consider the inhalation exposure of SVOCs as critically relevant. Due to the inherent properties of SVOCs as gas/particle mixtures, risk assessment strategies should consider particle size-segregated SVOC association and the relevance of released gas phase fractions. We constructed an in vitro air-liquid interface (ALI) exposure system to study the distinct toxic effects of the gas and particle phases of the model SVOC dibutyl phthalate (DBP) in A549 human lung epithelial cells. Cytotoxicity was evaluated and genotoxic effects were measured by the alkaline and enzyme versions of the comet assay. Deposited doses were assessed by model calculations and chemical analysis using liquid chromatography tandem mass spectrometry. The novel ALI exposure system was successfully implemented and revealed the distinct genotoxic effects of the gas and particle phases of DBP. The empirical measurements of cellular deposition and the model calculations of the DBP particle phase were concordant.The model SVOC DBP showed that inferred oxidative DNA damage may be attributed to particle-related effects. While pure gas phase exposure may follow a distinct mechanism of genotoxicity, the contribution of the gas phase to total aerosol was comparably low.

Exposure to naphthalene and ß-pinene-derived secondary organic aerosol induced divergent changes in transcript levels of BEAS-2B cells.

The health effects of exposure to secondary organic aerosols (SOAs) are still limited. Here, we investigated and compared the toxicities of soot particles (SP) coated with ß-pinene SOA (SOAßPin-SP) and SP coated with naphthalene SOA (SOANap-SP) in a human bronchial epithelial cell line (BEAS-2B) residing at the air-liquid interface. SOAßPin-SP mostly contained oxygenated aliphatic compounds from ß-pinene photooxidation, whereas SOANap-SP contained a significant fraction of oxygenated aromatic products under similar conditions. Following exposure, genome-wide transcriptome responses showed an Nrf2 oxidative stress response, particularly for SOANap-SP. Other signaling pathways, such as redox signaling, inflammatory signaling, and the involvement of matrix metalloproteinase, were identified to have a stronger impact following exposure to SOANap-SP. SOANap-SP also induced a stronger genotoxicity response than that of SOAßPin-SP. This study elucidated the mechanisms that govern SOA toxicity and showed that, compared to SOAs derived from a typical biogenic precursor, SOAs from a typical anthropogenic precursor have higher toxicological potency, which was accompanied with the activation of varied cellular mechanisms, such as aryl hydrocarbon receptor. This can be attributed to the difference in chemical composition; specifically, the aromatic compounds in the naphthalene-derived SOA had higher cytotoxic potential than that of the ß-pinene-derived SOA.

Effect of Atmospheric Aging on Soot Particle Toxicity in Lung Cell Models at the Air-Liquid Interface: Differential Toxicological Impacts of Biogenic and Anthropogenic Secondary Organic Aerosols (SOAs).

BACKGROUND: Secondary organic aerosols (SOAs) formed from anthropogenic or biogenic gaseous precursors in the atmosphere substantially contribute to the ambient fine particulate matter [PM ≤2.5µm in aerodynamic diameter (PM2.5)] burden, which has been associated with adverse human health effects. However, there is only limited evidence on their differential toxicological impact. OBJECTIVES: We aimed to discriminate toxicological effects of aerosols generated by atmospheric aging on combustion soot particles (SPs) of gaseous biogenic (ß-pinene) or anthropogenic (naphthalene) precursors in two different lung cell models exposed at the air-liquid interface (ALI). METHODS: Mono- or cocultures of lung epithelial cells (A549) and endothelial cells (EA.hy926) were exposed at the ALI for 4 h to different aerosol concentrations of a photochemically aged mixture of primary combustion SP and ß-pinene (SOAßPIN-SP) or naphthalene (SOANAP-SP). The internally mixed soot/SOA particles were comprehensively characterized in terms of their physical and chemical properties. We conducted toxicity tests to determine cytotoxicity, intracellular oxidative stress, primary and secondary genotoxicity, as well as inflammatory and angiogenic effects. RESULTS: We observed considerable toxicity-related outcomes in cells treated with either SOA type. Greater adverse effects were measured for SOANAP-SP compared with SOAßPIN-SP in both cell models, whereas the nano-sized soot cores alone showed only minor effects. At the functional level, we found that SOANAP-SP augmented the secretion of malondialdehyde and interleukin-8 and may have induced the activation of endothelial cells in the coculture system. This activation was confirmed by comet assay, suggesting secondary genotoxicity and greater angiogenic potential. Chemical characterization of PM revealed distinct qualitative differences in the composition of the two secondary aerosol types. DISCUSSION: In this study using A549 and EA.hy926 cells exposed at ALI, SOA compounds had greater toxicity than primary SPs. Photochemical aging of naphthalene was associated with the formation of more oxidized, more aromatic SOAs with a higher oxidative potential and toxicity compared with ß-pinene. Thus, we conclude that the influence of atmospheric chemistry on the chemical PM composition plays a crucial role for the adverse health outcome of emissions. https://doi.org/10.1289/EHP9413.

A comparative study of persistent DNA oxidation and chromosomal instability induced in vitro by oxidizers and reference airborne particles.

Adverse health effects driven by airborne particulate matter (PM) are mainly associated with reactive oxygen species formation, pro-inflammatory effects, and genome instability. Therefore, a better understanding of the underlying mechanisms is needed to evaluate health risks caused by exposure to PM. The aim of this study was to compare the genotoxic effects of two oxidizing agents (menadione and 3-chloro-1,2-propanediol) with three different reference PM (fine dust ERM-CZ100, urban dust SRM1649, and diesel PM SRM2975) on monocytic THP-1 and alveolar epithelial A549 cells. We assessed DNA oxidation by measuring the oxidized derivative 8-hydroxy-2'-deoxyguanosine (8-OHdG) following short and long exposure times to evaluate the persistency of oxidative DNA damage. Cytokinesis-block micronucleus cytome assay was performed to assess chromosomal instability, cytostasis, and cytotoxicity. Particles were characterized by inductively coupled plasma mass spectrometry in terms of selected elemental content, the release of ions in cell medium and the cellular uptake of metals. PM deposition and cellular dose were investigated by a spectrophotometric method on adherent A549 cells. The level of lipid peroxidation was evaluated via malondialdehyde concentration measurement. Despite differences in the tested concentrations, deposition efficiency, and lipid peroxidation levels, all reference PM samples caused oxidative DNA damage to a similar extent as the two oxidizers in terms of magnitude but with different oxidative DNA damage persistence. Diesel SRM2975 were more effective in inducing chromosomal instability with respect to fine and urban dust highlighting the role of polycyclic aromatic hydrocarbons derivatives on chromosomal instability. The persistence of 8-OHdG lesions strongly correlated with different types of chromosomal damage and revealed distinguishing sensitivity of cell types as well as specific features of particles versus oxidizing agent effects. In conclusion, this study revealed that an interplay between DNA oxidation persistence and chromosomal damage is driving particulate matter-induced genome instability.

Adenine derivatization for LC-MS/MS epigenetic DNA modifications studies on monocytic THP-1 cells exposed to reference particulate matter.

The aim of this study was to explore the impact of three different standard reference particulate matter (ERM-CZ100, SRM-1649, and SRM-2975) on epigenetic DNA modifications including cytosine methylation, cytosine hydroxymethylation, and adenine methylation. For the determination of low levels of adenine methylation, we developed and applied a novel DNA nucleobase chemical derivatization and combined it with liquid chromatography tandem mass spectrometry. The developed method was applied for the analysis of epigenetic modifications in monocytic THP-1 cells exposed to the three different reference particulate matter for 24 h and 48 h. The mass fraction of epigenetic active elements As, Cd, and Cr was analyzed by inductively coupled plasma mass spectrometry. The exposure to fine dust ERM-CZ100 and urban dust SRM-1649 decreased cytosine methylation after 24 h exposure, whereas all 3 p.m. increased cytosine hydoxymethylation following 24 h exposure, and the epigenetic effects induced by SRM-1649 and diesel SRM-2975 were persistent up to 48 h exposure. The road tunnel dust ERM-CZ100 significantly increased adenine methylation following the shorter exposure time. Two-dimensional scatters analysis between different epigenetic DNA modifications were used to depict a significantly negative correlation between cytosine methylation and cytosine hydroxymethylation supporting their possible functional relationship. Metals and polycyclic aromatic hydrocarbons differently shapes epigenetic DNA modifications.

Metabolomic signatures of low birthweight: Pathways to insulin resistance and oxidative stress.

Several studies suggest that low birthweight resulting from restricted intrauterine growth can leave a metabolic footprint which may persist into adulthood. To investigate this, we performed metabolomic profiling on 5036 female twins, aged 18-80, with weight at birth information available from the TwinsUK cohort and performed independent replication in two additional cohorts. Out of 422 compounds tested, 25 metabolites associated with birthweight in these twins, replicated in 1951 men and women from the Hertfordshire Cohort Study (HCS, aged 66) and in 2391 men and women from the North Finland Birth 1986 cohort (NFBC, aged 16). We found distinct heterogeneity between sexes and, after adjusting for multiple tests and heterogeneity, two metabolites were reproducible overall (propionylcarnitine and 3-4-hydroxyphenyllactate). Testing women only, we found other metabolites associated with lower birthweight from the meta-analysis of the three cohorts (2-hydroxy-butyric acid and γ-glutamylleucine). Higher levels of all these metabolites can be linked to insulin resistance, oxidative stress or a dysfunction of energy metabolism, suggesting that low birthweight in both twins and singletons are having an impact on these pathways in adulthood.

Metabolomic identification of a novel pathway of blood pressure regulation involving hexadecanedioate.

High blood pressure is a major contributor to the global burden of disease and discovering novel causal pathways of blood pressure regulation has been challenging. We tested blood pressure associations with 280 fasting blood metabolites in 3980 TwinsUK females. Survival analysis for all-cause mortality was performed on significant independent metabolites (P<8.9×10(-5)). Replication was conducted in 2 independent cohorts KORA (n=1494) and Hertfordshire (n=1515). Three independent animal experiments were performed to establish causality: (1) blood pressure change after increasing circulating metabolite levels in Wistar-Kyoto rats; (2) circulating metabolite change after salt-induced blood pressure elevation in spontaneously hypertensive stroke-prone rats; and (3) mesenteric artery response to noradrenaline and carbachol in metabolite treated and control rats. Of the15 metabolites that showed an independent significant association with blood pressure, only hexadecanedioate, a dicarboxylic acid, showed concordant association with blood pressure (systolic BP: ß [95% confidence interval], 1.31 [0.83-1.78], P=6.81×10(-8); diastolic BP: 0.81 [0.5-1.11], P=2.96×10(-7)) and mortality (hazard ratio [95% confidence interval], 1.49 [1.08-2.05]; P=0.02) in TwinsUK. The blood pressure association was replicated in KORA and Hertfordshire. In the animal experiments, we showed that oral hexadecanedioate increased both circulating hexadecanedioate and blood pressure in Wistar-Kyoto rats, whereas blood pressure elevation with oral sodium chloride in hypertensive rats did not affect hexadecanedioate levels. Vascular reactivity to noradrenaline was significantly increased in mesenteric resistance arteries from hexadecanedioate-treated rats compared with controls, indicated by the shift to the left of the concentration-response curve (P=0.013). Relaxation to carbachol did not show any difference. Our findings indicate that hexadecanedioate is causally associated with blood pressure regulation through a novel pathway that merits further investigation.

The accuracy and responsiveness of continuous noninvasive arterial pressure during rapid ventricular pacing for transcatheter aortic valve replacement.

BACKGROUND: The accuracy of measurement of the continuous noninvasive arterial blood pressure (CNAP) technique is unknown during sudden cardiocirculatory arrest. METHODS: In 33 patients undergoing elective transfemoral aortic valve implantation procedures under analgesic sedation, invasive arterial blood pressure (IAP) was compared with a CNAP device during episodes of severe hypotension (functional cardiocirculatory arrests by rapid pacing) and the remaining time without severe hypotension. Systolic, diastolic, and mean pairs of blood pressure measurements were extracted for both groups and were analyzed by Bland-Altman plots. The responsiveness of the CNAP technique was assessed in the various phases of severe hypotension concerning time and amplitude of changes. RESULTS: Overall CNAP accuracy (bias), calculated by subtracting IAP from CNAP, was -6.3 ± 18.9, 7.4 ± 10.5, and 4.0 ± 11.3 mm Hg (mean ± SD, systolic, diastolic, and mean). Bias increased during episodes of severe hypotension to 11.8 ± 14.5, 13.8 ± 12.4, and 12.9 ± 12.4 mm Hg. The percentage of agreements (95% confidence interval) between the blood pressure pairs with a difference ≤15 mm Hg was 58.5% (57.9-58.6), 75.8% (75.5-76.0), 82.2% (81.9-82.4; systolic, diastolic, mean) for all data and 56.4% (54.2-58.9; P = 0.71), 53.2%* (51.1-56.0), and 57.4%* (56.3-59.1; *P < 0.001) during rapid pacing. The responsiveness of mean CNAP and mean IAP did not differ significantly during the various phases of rapid pacing. CONCLUSIONS: The stand-alone CNAP monitor (model 500at, software V3.5) accurately and rapidly measures the changes of blood pressure that occur during sudden development of cardiocirculatory arrest and recovery to baseline blood pressures. CNAP monitors the duration of the arrest.

A subtle interplay between three Pex11 proteins shapes de novo formation and fission of peroxisomes.

The organization of eukaryotic cells into membrane-bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These organelles are formed either through a growth and division process from existing peroxisomes or de novo from the endoplasmic reticulum (ER). Among the proteins involved in the biogenesis of peroxisomes, peroxins, members of the Pex11 protein family participate in peroxisomal membrane alterations. In the yeast Saccharomyces cerevisiae, the Pex11 family consists of three proteins, Pex11p, Pex25p and Pex27p. Here we demonstrate that yeast mutants lacking peroxisomes require the presence of Pex25p to regenerate this organelle de novo. We also provide evidence showing that Pex27p inhibits peroxisomal function and illustrate that Pex25p initiates elongation of the peroxisomal membrane. Our data establish that although structurally conserved each of the three Pex11 protein family members plays a distinct role. While ScPex11p promotes the proliferation of peroxisomes already present in the cell, ScPex25p initiates remodeling at the peroxisomal membrane and ScPex27p acts to counter this activity. In addition, we reveal that ScPex25p acts in concert with Pex3p in the initiation of de novo peroxisome biogenesis from the ER.

PEX11 family members are membrane elongation factors that coordinate peroxisome proliferation and maintenance.

Dynamic changes of membrane structure are intrinsic to organelle morphogenesis and homeostasis. Ectopic expression of proteins of the PEX11 family from yeast, plant or human lead to the formation of juxtaposed elongated peroxisomes (JEPs),which is evocative of an evolutionary conserved function of these proteins in membrane tubulation. Microscopic examinations reveal that JEPs are composed of independent elongated peroxisomes with heterogeneous distribution of matrix proteins. We established the homo- and heterodimerization properties of the human PEX11 proteins and their interaction with the fission factor hFis1, which is known to recruit the GTPase DRP1 to the peroxisomal membrane. We show that excess of hFis1 but not of DRP1 is sufficient to fragment JEPs into normal round-shaped organelles, and illustrate the requirement of microtubules for JEP formation. Our results demonstrate that PEX11-induced JEPs represent intermediates in the process of peroxisome membrane proliferation and that hFis1 is the limiting factor for progression. Hence, we propose a model for a conserved role of PEX11 proteins in peroxisome maintenance through peroxisome polarization, membrane elongation and segregation.

Gbx2 and Otx2 interact with the WD40 domain of Groucho/Tle corepressors.

One of the earliest organizational decisions in the development of the vertebrate brain is the division of the neural plate into Otx2-positive anterior and Gbx2-positive posterior territories. At the junction of these two expression domains, a local signaling center is formed, known as the midbrain-hindbrain boundary (MHB). This tissue coordinates or "organizes" the development of neighboring brain structures, such as the midbrain and cerebellum. Correct positioning of the MHB is thought to depend on mutual repression involving these two homeobox genes. Using a cell culture colocalization assay and coimmunoprecipitation experiments, we show that engrailed homology region 1 (eh1)-like motifs of both transcription factors physically interact with the WD40 domain of Groucho/Tle corepressor proteins. In addition, heat shock-induced expression of wild-type and mutant Otx2 and Gbx2 in medaka embryos demonstrates that Groucho is required for the repression of Otx2 by Gbx2. On the other hand, the repressive functions of Otx2 on Gbx2 do not appear to be dependent on corepressor interaction. Interestingly, the association of Groucho with Otx2 is also required for the repression of Fgf8 in the MHB. Therefore Groucho/Tle family members appear to regulate key aspects in the MHB development of the vertebrate brain.