Co-culture of human alveolar epithelial (A549) and macrophage (THP-1) cells to study the potential toxicity of ambient PM2.5: a comparison of growth under ALI and submerged conditions.

Fine particulate matter (PM2.5) in the ambient atmosphere is strongly associated with detrimental health effects. However, these particles from various sources and regions are unlikely equally toxic. While animal studies are impractical for high-throughput toxicity testing, appropriate in vitro models are urgently needed. Co-culture of A549 and THP-1 macrophages grown at air-liquid interface (ALI) or under submerged conditions was exposed to same concentrations of ambient PM2.5 to provide accurate comparisons between culture methods. Following 24-h incubation with PM2.5 collected in Harbin in China, biological endpoints being investigated include cytotoxicity, reactive oxygen species (ROS) levels and pro-inflammatory mediators. The co-culture grown under submerged condition demonstrated a significant increase in ROS levels and all tested pro-inflammatory indicators [interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor-α] in mRNA expression and released protein levels. Similar but a declining response trend was observed using the same PM2.5 incubation after grown at ALI. We further observed a significant increase of PM2.5-induced phosphorylation of p38 MAPK and activation of NF-κB p65 in a dose-dependent trend for co-cultures grown under submerged condition. These results provide important implications that culture conditions (ALI versus submerged) can induce different extents of biological responses to ambient PM2.5; the co-culture grown at ALI is less likely to produce false-positive results than submerged culture. Hence, culture conditions should be discussed when comparing in vitro methods used for high-throughput PM2.5 toxicity assessment in future.

Toxicity assessment of electronic cigarettes.

Sale of electronic cigarette (e-cigarette) products has exponentially increased in the past decade, which raise concerns about its safety. This updated review provides the available toxicology profile of e-cigarettes, summarizing evidence from in vitro and in vivo studies. Data regarding which components in e-liquids exhibit potential toxicities are inconsistent. Some studies have reported that nicotine plays a significant role in inducing adverse outcomes and that solvents alone do not induce any adverse effects. However, other studies have suggested that nicotine is not associated with any adverse outcomes, whereas solvents and flavorings are the key components to elicit considerable deleterious effects on cells or animals. In addition, most of the studies that have compared the toxicity of e-cigarettes with tobacco cigarettes have suggested that e-cigarettes are less toxic than tobacco cigarettes. Nevertheless, scientific evidence regarding the toxicity profile of e-cigarette is insufficient owing to the lack of a standardized research approach. In the future, scientific toxicology data derived from standardized testing protocols including nicotine, ingredients analysis, the various e-cigarette devices made from different materials are urgently needed for thorough toxicology assessment. This review aims to update the toxicity profiles, identify knowledge gaps, and outline future directions for e-cigarettes research, which would greatly benefit public health professionals.

Ambient fine particulate matter induce toxicity in lung epithelial-endothelial co-culture models.

Epidemiological and toxicological studies have reported that ambient fine particulate matter (PM2.5) exposure are linked to adverse effects of cardiopulmonary system. An in vitro suitable model that assesses the interaction among various cell types should be developed to explain the toxic mechanisms occurred in cardiopulmonary system. The Transwell culture method was used to establish bi-culture consisting of A549 alveolar epithelial cells monoculture in apical chamber and EA.hy926 endothelial cells in the basolateral chamber, while tri-culture systems consisting of co-culture (A549 cells and THP-1 differentiated macrophages) in the apical chamber and also EA.hy926 endothelial cells in the basolateral chamber. Ambient PM2.5 collecting from Shanghai city in China was used for experiments. Our results showed that apical exposure of co-cultured cells to PM2.5 (20, 60, 180 µg/ml) for 24 h elicited stronger inflammatory responses than apical exposure of monocultured A549. Endothelial function was assessed via detecting gene expression in EA.hy926 cells, exposure of co-cultured cells induced more vigorous ICAM-1 and caveolin-1 mRNA expression in the tri-culture model than monocultured cells at the same dose of PM2.5 in the bi-culture model. Particles uptake were observed in both epithelial cells and endothelial cells according to TEM images. In conclusion, PM2.5 were able to pass through epithelial barrier and deposited in endothelium to further induce direct effect on endothelium function. The tri-culture system was more realistic and sensitive model to evaluate the impact of particles on the cardiopulmonary system than the bi-culture system. Therefore, the tri-culture system will contribute to explaining of the relationships between PM2.5 and cardiopulmonary diseases.

Effects of Fine Particulate Matter (PM2.5) on Systemic Oxidative Stress and Cardiac Function in ApoE(-/-) Mice.

AIM: In this study, we aimed to explore the toxic mechanisms of cardiovascular injuries induced by ambient fine particulate matter (PM2.5) in atherosclerotic-susceptible ApoE(-/-) mice. An acute toxicological animal experiment was designed with PM2.5 exposure once a day, every other day, for three days. METHODS: ApoE(-/-) and C57BL/6 mice were randomly categorized into four groups, respectively (n = 6): one control group, three groups exposed to PM2.5 alone at low-, mid-, and high-dose (3, 10, or 30 mg/kg b.w.). Heart rate (HR) and electrocardiogram (ECG) were monitored before instillation of PM2.5 and 24 h after the last instillation, respectively. Cardiac function was monitored by echocardiography (Echo) after the last instillation. Biomarkers of systemic oxidative injuries (MDA, SOD), heart oxidative stress (MDA, SOD), and NAD(P)H oxidase subunits (p22phox, p47phox) mRNA and protein expression were analyzed in mice. The results showed that PM2.5 exposure could trigger the significant increase of MDA, and induce the decrease of heart rate variability (HRV), a marker of cardiac autonomic nervous system (ANS) function with a dose-response manner. Meanwhile, abnormal ECG types were monitored in mice after exposure to PM2.5. The expression of cytokines related with oxidative injuries, and mRNA and protein expression of NADPH, increased significantly in ApoE(-/-) mice in the high-dose group when compared with the dose-matched C57BL6 mice, but no significant difference was observed at Echo. In conclusion, PM2.5 exposure could cause oxidative and ANS injuries, and ApoE(-/-) mice displayed more severe oxidative effects induced by PM2.5.

Rat lung response to ozone and fine particulate matter (PM2.5) exposures.

Exposure to different ambient pollutants maybe more toxic to lung than exposure to a single pollutant. In this study, we discussed the inflammation and oxidative stress responses of rat lung caused by ozone and PM2.5 versus that of rats exposed to saline, ozone, or single PM2.5 . Wistar rats inhaled 0.8 ppm ozone or air for 4 h and then placed in air for 3 h following intratracheal instillation with 0, 0.2 (low dose), 0.8 (medium dose), 3.2 (high dose) mg/rat PM2.5 dissolved in sterile saline (0.25 mL/rat), repeated twice per week for 3 weeks, the cumulative doses of PM2.5 in animals were 1.2, 4.8, and 19.2 mg. Rats were sacrificed 24 h after the last (sixth) exposure. The collected bronchoalveolar lavage fluid (BALF) was analyzed for inflammatory cells and cytokines. Lung tissues were processed for light microscopic and transmission electron microscopic (TEM) examinations. Results showed that total cell number in BALF of PM2.5 -exposed groups were higher than control (p < 0.05). PM2.5 instillation caused dose-trend increase in tumor necrosis factor alpha (TNF-α), interleukin-6, lactate dehydrogenase, and total protein of BALF. Exposure to ozone alone only caused TNF-α significant change in above-mentioned indicators of lung injury. On the other hand, ozone could enhance PM2.5-induced inflammatory changes and pathological characters in rat lungs. SOD and GSH-Px activities in lung were reduced in PM2.5-exposed rats with and without prior ozone exposure compared to control. To determine whether the PM2.5 and ozone affect endothelium system, iNOS, eNOS, and ICAM-1 mRNA levels in lung were analyzed by real-time PCR. These data demonstrated that inflammation and oxidative stress were involved in toxicology mechanisms of PM2.5 in rat lung and ozone potentiated these effects induced by PM2.5. These results have implications for understanding the pulmonary effects induced by ozone and PM2.5.

[Effects of ozone and fine particulate matter (PM2.5) on rat cardiac autonomic nervous system and systemic inflammation].

OBJECTIVE: To study the effects of ozone and fine particulate matter (PM2.5) on rat cardiac autonomic nervous system and systemic inflammation and the interconnected relationship of these two pathways. METHODS: 48 Wistar rats were randomly assigned to 8 groups (n = 6). Rats were intratracheal instillation of 0.2, 0.8 and 3.2 mg/rat PM2.5 as low-dose, medium-dose and high-dose PM2.5 alone exposure groups. Ozone combined PM2.5 groups were exposed to 0.8 ppm ozone 4 hours and then instilled 0.2, 0.8 and 3.2 mg/rat PM2.5 separately. Rats only inhaled 0.8 ppm ozone for 4 hours as ozone alone exposure group. Control rats were instilled with saline. Each exposure protocol applied twice a week for three continuous weeks. Systolic blood pressure and electrocardiograph (ECG) morphology of rats were recorded at 24 hours post-exposure of the last exposure. Serum TNF-alpha, IL-6 and CRP were analyzed. Right ventricles were used as pathological examination with HE staining. RESULTS: HRV indices in PM2.5 alone and combined exposure groups showed significant difference compared to control. However, heart rate only reduced significantly in ozone combined PM2.5 groups. Blood pressure rose significantly in combined groups and high-dose PM2.5 alone exposure. TNF-alpha and IL-6 increased significantly in PM2.5 alone/and ozone groups. CRP showed obvious dose-dependent relationship in PM2.5 alone and combined groups. Cardiac histopathology results revealed that PM2.5 deposition and myocardial inflammation were also observed in high-dose PM2.5 rats. CONCLUSION: Ozone can enhance the cardiac autonomic nervous system and systemic inflammation induced by PM2.5.

Effects of ozone and fine particulate matter (PM(2.5)) on rat system inflammation and cardiac function.

In order to understand the toxic mechanisms of cardiovascular system injuries induced by ambient PM(2.5) and/or ozone, a subacute toxicological animal experiment was designed with exposure twice a week for 3 continuous weeks. Wistar rats were randomly categorized into 8 groups (n=6): 1 control group, 3 groups exposed to fine particulate matters (PM(2.5)) alone at 3 doses (0.2, 0.8, or 3.2 mg/rat), 1 group to ozone (0.81 ppm) alone and 3 groups to ozone plus PM(2.5) at 3 doses (0.2, 0.8, or 3.2 mg/rat). Heart rate (HR) and electrocardiogram (ECG) was monitored at approximately 24-h both after the 3rd exposure and the last (6th) exposure, and systolic blood pressure (SBP) was monitored at approximately 24-h after the 6th exposure. Biomarkers of systemic inflammation and injuries (CRP, IL-6, LDH, CK), heart oxidative stress (MDA, SOD) and endothelial function (ET-1, VEGF) were analyzed after the 6th exposure. Additionally, myocardial ultrastructural alterations were observed under transmission electron microscopy (TEM) for histopathological analyses. Results showed that PM(2.5) alone exposure could trigger the significant increase of CRP, MDA, CK, ET-1 and SBP and decrease of heart rate variability (HRV), a marker of cardiac autonomic nervous system (ANS) function. Ozone alone exposure in rats did not show significant alterations in any indicators. Ozone plus PM(2.5) exposure, however, induced CRP, IL-6, CK, LDH and MDA increase, SOD and HRV decrease significantly in a dose-response way. Meanwhile, abnormal ECG types were monitored in rats exposed to PM(2.5) with and without ozone and obvious myocardial ultrastructural changes were observed by TEM. In conclusion, PM(2.5) alone exposure could cause inflammation, endothelial function and ANS injuries, and ozone potentiated these effects induced by PM(2.5).

[Time shift invariant properties of commonly used growth models and their application].

Growth analysis is of significance in estimating the age of organisms, but in reality, the initial time for this estimation adopted by different authors is not always the same. For example, in the growth analysis of mammals, the age could be birth age or conceptional age. Such a discrepancy in time, the so-called time shift, may generate different results in parameter-simulating process. In this paper, the time-shift invariant properties of commonly used three-parameter growth models, i.e., Spillman, Logistic, Gompertz and Bertalanffy models, were analyzed, and the results showed that these four models all had the invariant characters under any time shift, namely, no matter which initial time (age) of estimation was adopted, the same fitness was obtained. A case study was made with the growth data of Phodopus roborovskii and by adopting both birth age and conceptional age.

[Application of the nasal lining flap to correct the wide unilateral complete cleft lip].

OBJECTIVE: To study use of nasal lining flap in correction of the wide complete unilateral cleft lip. METHODS: 78 patients with wide complete cleft lip were. operated, firstly to be sure. That the lip length on both sides was equal. Then the nasal lining flap was used to enhance the height of the lip on cleft side for keeping symmetry with the non-cleft side. The rest of procedures was the same as "Millard" method. RESULTS: 84.6 percent of patients had good appearance at one and half years after surgery. CONCLUSIONS: It is an effective way to take advantage of the nasal lining flap to correct the wide complete unilateral cleft lip.

Anatomy of the nasal cartilages of the unilateral complete cleft lip nose.

The purpose of this study was to disclose the relationship between the anomaly of the cartilaginous framework and the nasal deformity of cleft lip. The noses of six stillborn infants with unilateral complete cleft lip were carefully dissected. The size and weight of the lower lateral cartilages were measured to determine whether there was a significant difference between the normal and involved sides. The position of the nasal cartilages was observed, and the distance between them was measured to determine whether they were normal. The surgical dissection revealed that the lower lateral cartilages from both sides were asymmetrical in three dimensions, indicating the displacement of the lower lateral cartilage on the involved side. There was displacement of the cartilaginous septum and the upper lateral cartilage. The statistical evaluation did not demonstrate a significant difference between weight and size of the two sides. One of the major causative factors of nasal deformity is displacement of the nasal cartilages. There is no hypoplasia of nasal cartilage in newborn infants with cleft lip.