Assessing oxidative stress induction ability and oxidative potential of PM2.5 in cities in eastern and western Japan.

Oxidative stress is an important cause of respiratory diseases associated with exposure to PM2.5. Accordingly, acellular methods for assessing the oxidative potential (OP) of PM2.5 have been evaluated extensively for use as indicators of oxidative stress in living organisms. However, OP-based assessments only reflect the physicochemical properties of particles and do not consider particle-cell interactions. Therefore, to determine the potency of OP under various PM2.5 scenarios, oxidative stress induction ability (OSIA) assessments were performed using a cell-based method, the heme oxygenase-1 (HO-1) assay, and the findings were compared with OP measurements obtained using an acellular method, the dithiothreitol assay. For these assays, PM2.5 filter samples were collected in two cities in Japan. To quantitatively determine the relative contribution of the quantity of metals and subtypes of organic aerosols (OA) in PM2.5 to the OSIA and the OP, online measurements and offline chemical analysis were also performed. The findings showed a positive relationship between the OSIA and OP for water-extracted samples, confirming that the OP is generally well suited for use as an indicator of the OSIA. However, the correspondence between the two assays differed for samples with a high water-soluble (WS)-Pb content, which had a higher OSIA than would be expected from the OP of other samples. The results of reagent-solution experiments showed that the WS-Pb induced the OSIA, but not the OP, in 15-min reactions, suggesting a reason for the inconsistent relationship between the two assays across samples. Multiple linear regression analyses and reagent-solution experiments showed that WS transition metals and biomass burning OA accounted for approximately 30-40% and 50% of the total OSIA or the total OP of water-extracted PM2.5 samples, respectively. This is the first study to evaluate the association between cellular oxidative stress assessed by the HO-1 assay and the different subtypes of OA.

Source contributions to multiple toxic potentials of atmospheric organic aerosols.

Fine particulate matter (PM2.5) in the atmosphere is of high priority for air quality management efforts to address adverse health effects in human. We believe that emission control policies, which are traditionally guided by source contributions to PM mass, should also consider source contributions to PM health effects or toxicity. In this study, we estimated source contributions to the toxic potentials of organic aerosols (OA) as measured by a series of chemical and in-vitro biological assays and chemical mass balance model. We selected secondary organic aerosols (SOA), vehicles, biomass open burning, and cooking as possible important OA sources. Fine particulate matter samples from these sources and parallel atmospheric samples from diverse locations and seasons in East Asia were collected for the study. The source and atmospheric samples were analyzed for chemical compositions and toxic potentials, i.e. oxidative potential, inflammatory potential, aryl hydrocarbon receptor (AhR) agonist activity, and DNA-damage, were measured. The toxic potentials per organic carbon (OC) differed greatly among source and ambient particulate samples. The source contributions to oxidative and inflammatory potentials were dominated by naphthalene-derived SOA (NapSOA), followed by open burning and vehicle exhaust. The AhR activity was dominated by open burning, followed by vehicle exhaust and NapSOA. The DNA damage was dominated by vehicle exhaust, followed by open burning. Cooking and biogenic SOA had smaller contributions to all the toxic potentials. Regarding atmospheric OA, urban and roadside samples showed stronger toxic potentials per OC. The toxic potentials of remote samples in summer were consistently very weak, suggesting that atmospheric aging over a long time decreased the toxicity. The toxic potentials of the samples from the forest and the experimentally generated biogenic SOA were low, suggesting that toxicity of biogenic primary and secondary particles is relatively low.

Scavenger receptor MARCO contributes to cellular internalization of exosomes by dynamin-dependent endocytosis and macropinocytosis.

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor class-A protein that is expressed on the cell surface of macrophages. MARCO mediates binding and ingestion of unopsonized environmental particles, including nano-sized materials. Exosomes are cell-derived, nano-sized vesicles (40-150 nm) that can contain lipids, RNA, DNA, and various proteins. Exosomes play an essential role in cell-to-cell communication via body fluids. However, mechanisms for the recognition and internalization of exosomes by recipient cells remain poorly characterized. In this study, cellular association of serum-derived fluorescent exosomes and 20-nm fluorescent nanoparticles (positive control) was compared between MARCO-expressing (CHO-MARCO) and control (CHO-CT) CHO-K1 cells to examine whether MARCO expression by recipient cells mediates the cellular uptake of exosomes and environmental nanoparticles. Fluorescence microscopic studies and quantitative analyses revealed that the cellular associations of both exosomes and 20-nm nanoparticles were greater in CHO-MARCO cells than in CHO-CT cells. Exosomes and nanoparticles colocalized with green fluorescent protein (GFP)-MARCO in cells transfected with GFP-MARCO-encoding constructs . Furthermore, inhibitory studies showed that actin reorganization and dynamin are involved in the MARCO-mediated cellular internalization of exosomes. These results indicated that MARCO plays a role in the uptake of exosomes.

Effects of diesel exhaust-derived secondary organic aerosol (SOA) on oocytes: Potential risks to meiotic maturation.

Particulate air pollution (PM 2.5) is a worldwide concern. Growing epidemiological evidence has shown pathophysiological effects of PM 2.5, not only on cardiovascular system but also on reproductive performance. The composition and physicochemical properties of PM 2.5 vary depending on the emission sources, climate conditions, and complex chemical reactions in the air. These factors make it difficult to understand the cause and mechanistic details of the adverse health effects of PM 2.5. Here, we show potential impacts of PM 2.5 on oocyte maturation in mice by utilizing diesel exhaust-derived secondary organic aerosol (SOA), a major component of urban PM 2.5. We found that the SOA destabilized microtubules of mouse oocytes and p-benzoquinone is one of the candidates for the microtubule-destabilizing compounds. We propose that some biologically reactive components of PM 2.5 should be prioritized for the regulation of atmospheric quality.

Aerosol Health Effects from Molecular to Global Scales.

Poor air quality is globally the largest environmental health risk. Epidemiological studies have uncovered clear relationships of gaseous pollutants and particulate matter (PM) with adverse health outcomes, including mortality by cardiovascular and respiratory diseases. Studies of health impacts by aerosols are highly multidisciplinary with a broad range of scales in space and time. We assess recent advances and future challenges regarding aerosol effects on health from molecular to global scales through epidemiological studies, field measurements, health-related properties of PM, and multiphase interactions of oxidants and PM upon respiratory deposition. Global modeling combined with epidemiological exposure-response functions indicates that ambient air pollution causes more than four million premature deaths per year. Epidemiological studies usually refer to PM mass concentrations, but some health effects may relate to specific constituents such as bioaerosols, polycyclic aromatic compounds, and transition metals. Various analytical techniques and cellular and molecular assays are applied to assess the redox activity of PM and the formation of reactive oxygen species. Multiphase chemical interactions of lung antioxidants with atmospheric pollutants are crucial to the mechanistic and molecular understanding of oxidative stress upon respiratory deposition. The role of distinct PM components in health impacts and mortality needs to be clarified by integrated research on various spatiotemporal scales for better evaluation and mitigation of aerosol effects on public health in the Anthropocene.

Differential Regulation of IL-1ß and IL-6 Release in Murine Macrophages.

Asbestos and silica (exogenous danger) and adenosine triphosphate (ATP, endogenous danger-signaling molecule) synergistically increase IL-1ß release from endotoxin-primed macrophage, which is mediated by NOD-like receptor protein 3 (NLRP3) inflammasome. However, the conversion of pro-IL-1ß to its active form seems to depend on the macrophage cell types. In the present study, bone marrow-derived macrophages (BMM) and three murine macrophage cell lines, J774.1, J774A.1, and RAW264.7 were exposed to ATP or fibrous titanium dioxide (FTiO2) in the presence or absence of lipopolysaccharide (LPS), and the concentrations of IL-1ß and IL-6 in both cell lysates and in the culture media were measured by immunoblotting to differentiate active form of IL-1ß from pro-IL-1ß. IL-1ß release was synergistically increased when the cells were exposed to both LPS and ATP or FTiO2, while IL-6 was readily released by LPS alone. IL-1ß released into the culture medium was pro-IL-1ß in J774.1 and RAW264.7, and most of the pro-IL-1ß remained inside the cells. In contrast, the active form of IL-1ß was released together with pro-IL-1ß from J774A.1 and BMM after the co-stimulation. J774A.1 and BMM express apoptosis-associated speck-like protein contains a carboxyl-terminal CARD (ASC) while J774.1 and RAW264.7 do not or only faintly express ASC, and accordingly, caspase-1, which converts pro-IL-1ß to its active form, is activated only in J774A.1 and BMM. Collectively, the canonical inflammasome pathway is not activated in J774.1 and RAW264.7, and the apparent synergistical increase of IL-1ß in the culture medium mostly reflects the leakage of pro-IL-1ß from these cells.

Nano-Sized Secondary Organic Aerosol of Diesel Engine Exhaust Origin Impairs Olfactory-Based Spatial Learning Performance in Preweaning Mice.

The aims of our present study were to establish a novel olfactory-based spatial learning test and to examine the effects of exposure to nano-sized diesel exhaust-origin secondary organic aerosol (SOA), a model environmental pollutant, on the learning performance in preweaning mice. Pregnant BALB/c mice were exposed to clean air, diesel exhaust (DE), or DE-origin SOA (DE-SOA) from gestational day 14 to postnatal day (PND) 10 in exposure chambers. On PND 11, the preweaning mice were examined by the olfactory-based spatial learning test. After completion of the spatial learning test, the hippocampus from each mouse was removed and examined for the expressions of neurological and immunological markers using real-time RT-PCR. In the test phase of the study, the mice exposed to DE or DE-SOA took a longer time to reach the target as compared to the control mice. The expression levels of neurological markers such as the N-methyl-d-aspartate (NMDA) receptor subunits NR1 and NR2B, and of immunological markers such as TNF-α, COX2, and Iba1 were significantly increased in the hippocampi of the DE-SOA-exposed preweaning mice as compared to the control mice. Our results indicate that DE-SOA exposure in utero and in the neonatal period may affect the olfactory-based spatial learning behavior in preweaning mice by modulating the expressions of memory function-related pathway genes and inflammatory markers in the hippocampus.

Effects of diesel engine exhaust origin secondary organic aerosols on novel object recognition ability and maternal behavior in BALB/c mice.

Epidemiological studies have reported an increased risk of cardiopulmonary and lung cancer mortality associated with increasing exposure to air pollution. Ambient particulate matter consists of primary particles emitted directly from diesel engine vehicles and secondary organic aerosols (SOAs) are formed by oxidative reaction of the ultrafine particle components of diesel exhaust (DE) in the atmosphere. However, little is known about the relationship between exposure to SOA and central nervous system functions. Recently, we have reported that an acute single intranasal instillation of SOA may induce inflammatory response in lung, but not in brain of adult mice. To clarify the whole body exposure effects of SOA on central nervous system functions, we first created inhalation chambers for diesel exhaust origin secondary organic aerosols (DE-SOAs) produced by oxidation of diesel exhaust particles caused by adding ozone. Male BALB/c mice were exposed to clean air (control), DE and DE-SOA in inhalation chambers for one or three months (5 h/day, 5 days/week) and were examined for memory function using a novel object recognition test and for memory function-related gene expressions in the hippocampus by real-time RT-PCR. Moreover, female mice exposed to DE-SOA for one month were mated and maternal behaviors and the related gene expressions in the hypothalamus examined. Novel object recognition ability and N-methyl-D-aspartate (NMDA) receptor expression in the hippocampus were affected in male mice exposed to DE-SOA. Furthermore, a tendency to decrease maternal performance and significantly decreased expression levels of estrogen receptor (ER)-α, and oxytocin receptor were found in DE-SOA exposed dams compared with the control. This is the first study of this type and our results suggest that the constituents of DE-SOA may be associated with memory function and maternal performance based on the impaired gene expressions in the hippocampus and hypothalamus, respectively.

Effects of acute single intranasal instillation of secondary organic aerosol on neurological and immunological biomarkers in the brain and lung of BALB/c mice.

Recently, we have reported that primary particles from diesel exhaust affect nervous system, immune system, and learning ability in mice. Currently, in vivo and in vitro studies have shown that secondary organic aerosol (SOA) generated from the coal-fired power plant induced adverse effects in lung and heart. However, the effect of SOA on central nervous system is still unknown. In the present study, using potential biomarkers recognized in previous studies of primary particles, we investigated the effect of acute single administration of SOA on the expression levels of various biomarkers in the brain and lung of mice. We generated the SOA by addition of ozone (O(3)) to the diesel exhaust particle (DEP). Eight-week-old male BALB/c mice were administered DEP or DEP+O(3) (SOA) (50 µg/50 µl/mouse) intranasally. Twenty-four hour after acute single exposure to SOA, olfactory bulb, hippocampus and lung from all mice were collected and mRNA expressions of neurological and immunological biomarkers were examined using real-time RT-PCR analysis and histological examination. Proinflammatory cytokines, their transcription factor and neurotrophin mRNA were remarkably increased in lung of mice exposed to SOA but not in the brain. Microarray data showed that changes of the inflammatory reaction and metabolizing enzyme gene cluster were observed in the brain and lung. Our findings suggested that an acute single exposure of SOA does not affect biomarkers in the brain of normal healthy individuals. Our present results also clearly indicate that SOA induces inflammatory responses in the lung by modulating proinflammatory cytokines, transcription factor and inflammatory responsive neurotrophins.

Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 cells.

The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20nm-1µm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC(50) value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1µg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure.