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Air pollution has been a major environment-related health threat. Most of the studies on PM2.5 toxicity have verified on the cardiovascular system and endothelial cells. However, researches on PM2.5-induced myocardial-related toxicity are limited. This study aims to fully understand the toxic effects of PM2.5 on human myocardial cell (AC16) and explore its molecular mechanism based on microarray analysis and bioinformatics analysis. Microarray data analysis manifested that PM2.5-induced toxicity affected expression of 472 genes compared with the control group, including 166 upregulated genes and 306 downregulated genes in human myocardial (AC16) cells. GO analysis showed that cellular processes such as immune response, cell maturation, embryonic heart tube morphogenesis, cellular response to electrical stimulus, skeletal muscle tissue regeneration, and negative regulation of signal transduction were upregulated, while regulation of transcription (DNA-dependent), rhythmic process, protein destabilization apoptotic process, and innate immune response were downregulated. The pathway analysis indicates that cell signaling pathways such as cytokine-cytokine receptor interaction, NF-κB signaling pathway, chemokine signaling pathway, endocrine and other factor-regulated calcium reabsorption, HTLV-I infection, and cell adhesion molecules (CAMs) were upregulated, while the TGF-β signaling pathway was downregulated. In addition, Signal-net showed that the TUBA4A, ADRBK2, BRIX1, SMC4, EIF5B, PRMT1, ATG4B, and NDC80 genes were significantly decreased, while the expression of the KRT6B gene was markedly increased compared with the control group. All the genes were verified by qRT-PCR. This study had provided new bioinformatics evidences in PM2.5-induced myocardial tissue toxicity which is necessary for further cardiovascular system toxicity studies.

This paper presents a hybrid information fusion approach that integrates the cloud model and the D–S evidence theory to perceiving safety risks using sensor data under uncertainty. The cloud model provides an uncertain transforming tool between qualitative concepts and their quantitative expressions and uses the measurement of correlation to construct Basic Probability Assignments. An improved evidence aggregation strategy that combines the Dempster’ rule and the weighted mean rule is developed to get rid of counter-intuitive dilemma existing in a combination of high-conflict evidence. A three-layer information fusion framework consisting of sensor fusion, factor fusion, and area fusion is proposed to synthesize multi-source information to get the final fusion results. The developed cloud D–S approach is applied to the assessment of the safety of a real tailings dam in operation in China as a case study. Data information acquired from 28 monitoring sensors is fused in a continuous manner in order to obtain the overall safety level of the tailings dam. Results indicate that the developed approach is capable of achieving multi-layer information fusion and identifying global sensitivities of input factors under uncertainty. The developed approach proves to perform a strong robustness and fault-tolerant capacity, and can be used by practitioners in the industry as a decision tool to perceive and anticipate the safety risks in tailings dams.

Our previous research showed that endosulfan triggers the extrinsic coagulation pathway by damaging endothelial cells and causes hypercoagulation of blood. To identify the mechanism of endosulfan-impaired endothelial cells, we treated human umbilical vein endothelial cells (HUVECs) with different concentrations of endosulfan, with and without an inhibitor for Notch, N-[N-(3, 5-difluorophenacetyl)-1-alanyl]S-Phenylglycinet-butylester (DAPT, 20 μM), or a reactive oxygen species (ROS) scavenger, N-Acetyl-l-cysteine (NAC, 3 mM), for 24 h. The results showed that endosulfan could inhibit cell viability/proliferation by increasing the release of lactate dehydrogenase (LDH), arresting the cell cycle in both S and G2/M phases, and inducing apoptosis in HUVECs. We also found that endosulfan can damage microfilaments, microtubules, and nuclei; arrest mitosis; remarkably increase the expressions of Dll4, Notch1, Cleaved-Notch1, Jagged1, Notch4, Hes1, and p21; and significantly induce ROS and malondialdehyde production in HUVECs. The presence of DAPT antagonized the above changes of cycle arrest, proliferation inhibition, and expressions of Dll4, Notch1, Cleaved-Notch1, Hes1, and p21 caused by endosulfan; however, NAC could attenuate LDH release; ROS and malondialdehyde production; apoptosis; and the expression levels of Dll4, Notch1, Cleaved-Notch1, Notch4, and Hes1 induced by endosulfan. These results demonstrated that endosulfan inhibited proliferation through the Notch signaling pathway as a result of oxidative stress. In addition, endosulfan can damage the cytoskeleton and block mitosis, which may add another layer of toxic effects on endothelial cells.

Supplemental material, Supplymentary_Materials-Baseline_characteristics for Sodium-glucose cotransporter 2 inhibitors and fracture risk in patients with type 2 diabetes mellitus: a meta-analysis of randomized controlled trials by Yake Lou, Ying Yu, Junchao Duan, Sining Bi, Khaing Nyein Chan Swe, Ziwei Xi, Yanan Gao, Yujie Zhou, Xiaomin Nie and Wei Liu in Therapeutic Advances in Chronic Disease

Exposure to fine particulate matter (PM2.5) is closely linked with cardiovascular diseases. However, the underlying mechanism of PM2.5 on cardiac function remains unknown. This study was aimed to investigate the role of microRNA-205 (miR-205) on PM2.5-induced myocardial inflammation and cardiac dysfunction. PM2.5 increased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), following by decreased cell viability and antioxidant enzymes, resulting in apoptosis of cardiomyocytes (AC16). The histopathological and ultrastructural analysis demonstrated that PM2.5 caused myocardial damage via interstitial edema, inflammatory cell infiltration, and myocardial fiber destruction. PM2.5 enhanced the release of inflammatory factors in AC16 cells and heart tissue. Microarray analysis and dual-luciferase reporter gene assays demonstrated that PM2.5-induced down-regulation of miR-205 regulated interleukin 1 receptor-associated kinase 2 (IRAK2), which further activated the TNF receptor-associated factor 6 (TRAF6)/nuclear transcription factor-κB (NF-κB) signaling pathway in vivo. Moreover, the chemical mimics of miR-205 markedly inhibited the IRAK2/TRAF6/NF-κB signaling pathway, whereas the chemical inhibitors of miR-205 amplified PM2.5-induced activation of the IRAK2 signaling pathway in vitro. In summary, our results found that PM2.5 could trigger myocardial toxicity via miR-205 negative regulating the IRAK2/TRAF6/NF-κB signaling pathway. Our study suggests that miR-205 could be a promising target molecule for mitigating the hazardous effects of PM2.5 on the cardiovascular system.

Supplemental material, Supplymentary_Materials-Primary_outcomes_and_eligiblity_criteria for Sodium-glucose cotransporter 2 inhibitors and fracture risk in patients with type 2 diabetes mellitus: a meta-analysis of randomized controlled trials by Yake Lou, Ying Yu, Junchao Duan, Sining Bi, Khaing Nyein Chan Swe, Ziwei Xi, Yanan Gao, Yujie Zhou, Xiaomin Nie and Wei Liu in Therapeutic Advances in Chronic Disease

Supplemental material, Supplymentary_Materials-Search_strategtplusInfluence_analysisplusFunnel_plot for Sodium-glucose cotransporter 2 inhibitors and fracture risk in patients with type 2 diabetes mellitus: a meta-analysis of randomized controlled trials by Yake Lou, Ying Yu, Junchao Duan, Sining Bi, Khaing Nyein Chan Swe, Ziwei Xi, Yanan Gao, Yujie Zhou, Xiaomin Nie and Wei Liu in Therapeutic Advances in Chronic Disease

Fine particle matter (PM

Accumulating evidences support that exposure to fine particulate matter (PM

Amorphous silica nanoparticles (SiNPs) are not only abundant in nature, but also the second largest engineering nanomaterials in terms of annual output. Respiratory exposure is the main route for SiNPs to enter the human body. A large number of studies have focused on the respiratory toxicity of SiNPs and demonstrated that SiNPs could induce pulmonary tissue damage, inflammation, fibrosis, and even the malignant transformation of bronchial epithelial cells, while the size-dependent toxicity of SiNPs and their underlying biological mechanisms remain unclear. In this regard, a transcriptomics study would be conductive to gaining a better understanding of the toxic mechanism. In the present study, microarray analysis was performed to investigate the genome-wide transcriptional alteration induced by different sizes of SiNPs in human primary bronchial epithelial cells (BEAS-2B). To determine the effect of the particle size on the toxicity, nanoparticles of two sizes (41 nm and 61 nm) and submicron particles of one size (206 nm) were introduced. The bioinformatics analysis results indicated that: (1) the number of differentially expressed genes in the three SiNP-treated groups increased with the particle size decreasing; (2) the genes involved in the immune and inflammatory response, gene expression, signal transduction, endoplasmic reticulum stress, oxidative stress, cell metabolism, and cell proliferation were gradually upregulated with the particle size decreasing, while the genes related to the morphological development of the respiratory system were gradually downregulated with the particle size decreasing; (3) the modes of action of the two nanoparticles overlapped with each other to some degree, and there existed many different modes compared to those from the submicron particles; (4) both the silica nanoparticles affected the pathways associated with the cell entry of silica nanoparticles, autophagy and lysosomal dysfunction, endoplasmic reticulum stress, inflammatory response, DNA damage, and gene expression, as well as apoptotic resistance and cancer. To the best of our knowledge, this is the first study that has reported the alteration trend of gene expression profiles with the change in silica particle size. Our study provides a great deal of information on the toxic mechanisms underlying the respiratory toxicity induced by SiNPs, and can also serve as an experimental basis for the toxicity and safety evaluation of silica nanoparticles.

Cardiac fibrosis is associated with fine particulate matter (PM2.5) exposure. In addition, whether high-fat diet (HFD) could exacerbate the PM2.5-induced cardiac injury was unevaluated. Thus, this study was aimed to investigate the combined effects of PM2.5 and HFD on cardiac fibrosis. The echocardiography and histopathological analysis showed that co-exposure of PM2.5 and HFD had a significant deleterious effect on both cardiac systolic and diastolic function accompanied the myofibril disorder and myocardial fibrosis in C57BL/6 J mice than exposed to PM2.5 or HFD alone. The augmented oxidative damage and increased α-SMA area percentage were detected in heart tissue of mice exposed to PM2.5 and HFD together. PM2.5 upregulated the expressions of cardiac fibrosis-related special markers, including collagen-I, collagen-III, TGF-β1, p-Smad3 and total Smad3, which had more pronounced activations in co-exposure group. Meanwhile, the factorial analysis exhibited the synergistic interaction regarded to the combined exposure of PM2.5 and HFD. Simultaneously, PM2.5 and palmitic acid increased intracellular ROS generation and activated the TGF-β1/Smad3 signaling pathway in cardiomyocytes. While the ROS scavenger NAC had effectively attenuated the ROS level and suppressed the TGF-β1/Smad3 signaling pathway. Taken together, our results demonstrated combined exposure to PM2.5 and HFD could aggravate cardiac fibrosis via activating the ROS/TGF-β1/Smad3 signaling pathway.

Epidemiological studies have confirmed that PM

Up to now, while some toxicological studies have identified pulmonary fibrosis immediately induced by long-term PM

To find the correlation between exposure to PMA combination of computer and manual retrieval was used to search for keywords in PubMed (385 records), Cochrane Library (20 records), Web of Science (419 records) and Embase (325 records). Finally, ten epidemiological articles were considered in this meta-analysis. Stata 13.0 was used to examine the heterogeneity among the studies and to calculate the combined effect value (OR, odds ratio) by selecting the corresponding models. Sensitivity analysis and publication bias test were also performed.Meta-analysis indicated that there was an association between PMThere is a significant link between exposure to PM

Diesel exhaust particles (DEPs) are common airborne ultrafine particles (UFPs); however, few studies have examined their effects on the gastrointestinal tract. To investigate the interaction of gut microbiota and DEPs‐induced colonic injury, adult C57BL/6 mice are kept in whole‐body inhalation chambers and exposed to filtered room air (FRA) or DEPs (300 µg m−3) 1 h per day for 28 consecutive days. DEPs exposure results in colon epithelial injury with inflammatory cell infiltration and mucus depletion. Abundance of Lactobacillus in murine feces is transiently increased following 7‐day DEPs exposure and then decreased until the end of 28‐day exposure. A reduction of the colonic mucus layer thickness is observed in mice receiving gut microbiota from DEPs‐exposed mice. Mechanistically, RNA‐sequencing suggests disruption of the nitrogen metabolism pathway in DEPs‐exposed NCM460 cells. Upregulation of carbonic anhydrase 9 (CA9) expression levels is observed in epithelia following DEPs exposure both in vivo and in vitro. Oral administration of probiotics protects the mice against DEPS‐induced colon epithelial injury. The results strongly suggest the involvement of gut microbiota in response to DEPs exposure and subsequently epithelial injury in vivo. Supplementation with probiotic may be a potential way to protect against UFPs‐induced colon epithelial injury.

As the main components of fine particulate matter (PM2.5), silica nanoparticles (SiNPs) and benzo[a]pyrene (B[a]P) have attracted increasing attention recently. However, co-exposure to SiNPs and B[a]P causes pulmonary injury by aggravating toxicity via an unknown mechanism. This study aimed at investigating the toxicity caused due to long-term co-exposure to SiNPs and B[a]P on pulmonary systems at low dose using human bronchial epithelial (BEAS-2B) cells. The characterizations of SiNPs and B[a]P were done by transmission electron microscopy (TEM) and zeta potential granulometry. Cytotoxicity is evaluated using cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) activity; oxidative stress, cell cycle and apoptosis were assessed by flow cytometry, and inflammatory factors were detected using a Luminex xMAP system. Results show an obvious inhibition of cell proliferation and a marked increase in the LDH expression in the BEAS-2B cells after long-term co-exposure. Furthermore, long-term co-exposure is the most potent in generating intracellular ROS, thus causing inflammation. Cellular apoptotic rate is enhanced in the co-exposed group at low dose. Moreover, the long-term co-exposure induces significant cell cycle arrest, increasing the proportion of cells at the G2/M phase, while decreasing those at the G0/G1 phase. This study is the first attempt to reveal the severe synergistic and additive toxic effects induced by SiNPs and B[a]P co-exposure for long-term in BEAS-2B cells even at low dose.

Long-term exposure to fine particulate matter (PMHere, we investigated the PMIn vitro, PMPM

Nowadays, due to the wide use of amorphous silica nanoparticles (SiNPs), their adverse effects on human beings are attracting more attention. Understanding the interaction between SiNPs and cells is a fundamental step for toxicity assessment. Therefore, the current study is aimed at elaborating the internalization process, subcellular distribution, ultrastructure damage, and cytotoxicity of two different sizes of SiNPs (Nano-Si64 and Nano-Si46) in L-02 cells. The results indicate that the smaller-sized SiNPs, Nano-Si46, accumulate in cells more efficiently and produce a stronger cytotoxic effect than Nano-Si64. Both types of nanoparticles can accumulate in L-02 cells through the active endocytotic pathway and passive diffusion, and distribute within endocytotic vesicles or freely in cytoplasma and organelles. Microvillus fracture, membrane injury, mitochondria damage, degranulation of the rough endoplasmic reticulum, lamellar-like structure, lysosome destruction, autophagosomes, and autophagy-lysosomes are found in L-02 cells. Oxidative damage and direct interaction between SiNPs and subcellular structure are responsible for the toxicity.

This study was to investigate the combined toxicity of silica nanoparticles (SiNPs) and methylmercury (MeHg) on cardiovascular system in zebrafish (Danio rerio) embryos. Ultraviolet absorption analysis showed that the co-exposure system had high absorption and stability. The dosages used in this study were based on the NOAEL level. Zebrafish embryos exposed to the co-exposure of SiNPs and MeHg did not show any cardiovascular malformation or atrioventricular block, but had an inhibition effect on bradycardia. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased gradually in SiNPs, MeHg, co-exposure groups, respectively. Co-exposure of SiNPs and MeHg enhanced the vascular endothelial damage in Tg(fli-1:EGFP) transgenic zebrafish line. Moreover, the co-exposure significantly activated the oxidative stress and inflammatory response in neutrophils-specific Tg(mpo:GFP) transgenic zebrafish line. This study suggested that the combined toxic effects of SiNPs and MeHg on cardiovascular system had more severe toxicity than the single exposure alone.

Given the severe situation of world-wide particulate matter air pollution, it is urgent to explore the combined effects of particulate matter components on cardiovascular system. Using zebrafish model, this study was aimed to determine whether the low level co-exposure to silica nanoparticles (SiNPs) and benzo[a]pyrene (B[a]P) had a pronounced cardiovascular toxicity than the single exposure to either SiNPs or B[a]P alone. The FTIR and TGA analysis showed that the co-exposure system possessed of high absorption and thermal stability. Embryos exposed to SiNPs or B[a]P alone did not show cardiac toxicity phenotype at the NOAEL level. However, embryos co-exposed to SiNPs and B[a]P exhibited pericardial edema and bradycardia. While ROS generation remained unaffected, the co-exposure induced significant neutrophil-mediated inflammation and caused erythrocyte aggregation in caudal vein of embryos. Microarray analysis and STC analysis were performed to screen the cardiovascular-related differential expression genes and the expression trend of genes in each group. The co-exposure of SiNPs and B[a]P significantly enhanced the expression of proinflammatory and procoagulant genes. Moreover, the co-exposure markedly increased the phosphorylated AP-1/c-Jun and induced TF expression, but not NF-κB p65. This study for the first time demonstrated the inflammatory response and blood hypercoagulable state were triggered by the combination of SiNPs and B[a]P at low level exposure.

Objective Find the correlation between particulate matter (PM) and biomarkers related to blood coagulation, offer medical evidence to sensitive indicators and carry out early diagnosis of cardiovascular diseases. Method A combination of computer and manual retrieval was used to search for the keywords in PubMed (584 records), Cochrane Library (28 records), Web of Science (162 records) and Embase (163 records). Finally, a total of 25 articles were included in this meta-analysis. Stata 13.0 was applied to examine the heterogeneity among the studies and to calculate the combined effect estimates, percent variation (%) and 95% CI by selecting corresponding models. Additionally, sensitivity analysis and publication bias test were also conducted. Results Meta-analysis indicated that there was an association between PM2.5 exposure (per 10 µg/m3 increase) and fibrinogen. With the increase of PM2.5 exposure (per 10 µg/m3 increase), the content of fibrinogen revealed a high level (2.26%; 95% CI: 1.08–3.44%); and the increase of UFPs exposure (per 5000/cm3 increase) was correlated with some biomarkers such as cell surface antigen and protein ligand including ICAM-1, sCD40L, P-selectin, E-selectin and PAI-1 that indirectly related to blood coagulation, yielding a percent variation of 10.83% (95% CI: 3.49%−18.17%). Conclusion This meta-analysis expounded that PM-related biomarkers were associated with blood coagulation, and the relationship with fibrinogen was much stronger.

Ambient air pollution is recognized as a major threat to those with cardiovascular disease (CVD), especially among old adults within this high risk group. Heart rate variability (HRV) is a marker of cardiac autonomic system, which links air pollution and CVD. However, the relationship between PM and HRV has been inconsistently reported. To investigate the associations of PM2.5 and HRV in old adults whose average age was 55 years old or above, we conducted a meta-analysis of nineteen longitudinal studies including nine short-term and ten long-term studies. In the short-term exposure group, per 10 μg/m3 increase of PM2.5 was associated with decreases in the time-domain measurements, for SDNN −0.39% (95% CI: −0.72%, −0.06%) and for RMSSD −1.20% (95% CI: −2.17%, −0.23%) and in frequency-domain measurements, for LF −2.31% (95% CI: −3.85%, −0.77%) and for HF −1.87% (95% CI: −3.45%, −0.29%); In the long-term exposure group, per 10 μg/m3 increase of PM2.5 was associated with decreases in the time-domain measurements, for SDNN −0.92% (95% CI: −2.14%, 0.31%) and for RMSSD −1.96% (95% CI: −3.48%, −0.44%) and in frequency-domain measurements, for LF −2.78% (95% CI: −4.02%, −1.55%) and for HF −1.61% (95% CI: −4.02%, 0.80%). Exposure to PM2.5 is associated with decreased indicators of HRV in older adults suggesting an affected cardiac autonomic system upon exposure, which may explain the association between PM2.5 and risk of CVD in older adults. Long-term exposure to PM2.5 was more strongly associated with indicators of HRV than short-term exposure.

Background Ambient fine particulate matter (PM2.5) is a major threat to cardiovascular health. Endothelial dysfunction is the initiating event associated with the PM2.5-induced cardiovascular disease (CVD). A sensitive marker of endothelial function-circulating von Willebrand factor (vWF), is an independent predictor of adverse clinical outcome in CVD patients. PM2.5 exposure may cause CVD, but the reports of relationship between short-term PM2.5 exposure and circulating vWF are inconsistent. Objective To explore the influence of short-term PM2.5 exposure on circulating vWF. Methods By using a combination of computer and manual retrieval, a systematic literature retrieval was conducted on PubMed, Cochrane Library, Web of Science, Embase and Scopus databases up to October 2019. The heterogeneity among studies was tested by Stata 12.0, and the pooled %-change (percentage change per 10 μg/m3 increase in PM2.5) and its 95% confidence interval (95%CI) were calculated by using random effect model. Sensitivity analysis and publication bias detection were also carried out. Results 12 articles were included in this meta-analysis. Short-term PM2.5 exposure (per 10 μg/m3 increase) was associated with the increased vWF (%-change = 0.41, 95%CI: 0.11–0.71). The pooled effect estimates of subgroup with PM2.5 exposure level Conclusion Short-term PM2.5 exposure is associated with the increased circulating vWF. It suggests that short-term PM2.5 exposure causes endothelial dysfunction.

Nowadays, the great majority of toxicological studies have focused on immediate cardiovascular effects of simultaneous exposure to long-term or short-term PM2.5; yet, whether the persistent vascular fibrosis will be induced after short-term PM2.5 exposure and its related underlying mechanisms remain unclear. In this study, we adopted SD rats treated with PM2.5 for 1 month and followed by 12 months and 18 months recovery. Results from Doppler ultrasonography and histopathological analysis found that PM2.5-evoked vascular fibrosis was comprised of structural injury, including thickening of aortic media and carotid intima media thickness (CIMT), narrow left common carotid artery (LCCA), collagen deposition, impaired elasticity and functional alterations in aortal stiffness during long-term recovery. The protein expression levels of collagen I, collagen III, proliferating cell nuclear antigen (PNCA), TGF-β and osteopontin (OPN) remained elevated trends in PM2.5-treated groups for the related period than in control groups. Additionally, PM2.5 upregulated the protein expression levels of superoxide dismutase 2 (SOD2), mitochondrial fission related proteins (Drp1 and Fis1), while downregulated the protein expression levels of mitochondrial fusion related proteins (Mfn2 and OPA1). Moreover, PM2.5 significantly activated the mitophagy-related protein expression, including LC3, p62, PINK, Parkin. In summary, our results demonstrated that short-term PM2.5 exposure could trigger mitophagy, further lead to mitochondrial dysfunction which regulated persistent vascular fibrosis during long-term recovery.

Fine particle matter (PM2.5) has been extensively reported to contribute to the pathogenesis of pulmonary diseases. Recently, metformin has been reported to attenuate PM2.5 associated respiratory and cardiovascular injury, but the underling mechanism has not been discovered. Here, we performed comprehensively bioinformatics analysis and fully validation experiment to investigate the protection role of metformin and underling mechanism with RNAseq profile in GEO database. A combination of various bioinformatics tools including edgeR, principal component analysis (PCA), K-Means clustering, Gene Set Enrichment Analysis (GSEA), GO and KEGG enrichment were performed to identify the TLRs/MyD88/NF-κB axis functional as the key signaling transduction during PM2.5 associated toxicity. PM2.5 activated TLRs/MyD88/NF-κB pathway and resulted in significantly generation of IL-6, TNF-α, mitochondrial damage, decreasing of cell viability and increased LDH activity in RAW264.7 cells. Metformin significantly attenuated the production of IL-6, mitochondrial damage, cell viability and LDH activity by limiting TLRs/MyD88/NF-κB pathway. The siRNA against AMPKα2 or negative control were transfected to RAW264.7 cells to identify whether metformin protects PM2.5-induced cytotoxicity in an AMPKα2-dependent manner. Pretreatment with metformin significantly attenuated PM2.5 induced decreasing of cell viability and increased LDH activity, as well as inhibited the TLRs/MyD88/NF-κB pathway in both siControl or siAMPKα2 cells. Taken together, our results indicate that metformin protects against PM2.5-induced mitochondrial damage and cell cytotoxicity by inhibiting TLRs/MyD88/NF-κB signaling pathway in an AMPKα2 independent manner.

Accumulating evidences support that exposure to fine particulate matter (PM2.5) could cause inflammation of the airway, but its underlying mechanisms are less known. Our study aimed to explore the potential effect of non-canonical NF-κB signaling pathway in airway inflammation, which caused by PM2.5, and the possible regulatory relationship between miR-6747–5p and NF-κB2. The histological analysis from in vivo study manifested that PM2.5 could induce the exudation and infiltration of polymorphonuclear leukocytes (PMNs). Immunohistochemistry results of lung tissues showed that PM2.5 increased ICAM-1, 6Ckine, SDF-1 and BAFF positive staining with a dose-dependent manner. In addition, PM2.5 could induce the p52 nuclear translocation to trigger non-canonical NF-κB signaling pathway in lung tissues and BEAS-2B cells. Targetscan reporter gene assay showed that there was a target regulatory relationship between miR-6747–5p and NF-κB2. Besides, the chemical mimics of miR-6747–5p weakened the activation of non-canonical NF-κB signaling pathway induced by PM2.5. In summary, exposure to PM2.5 could trigger airway inflammation by activating the non-canonical NF-κB signaling pathway, which may be related to the negative feedback regulation mechanism of miR-6747–5p. Our findings will give new ideas into the toxic effects of airway inflammation triggered by PM2.5.

Increasing environmental exposure to silica nanoparticles (SiNPs) and limited cardiotoxicity studies posed a challenge for the safety evaluation and management of these materials. This study aimed to explore the adverse effects and underlying mechanisms of subacute exposure to SiNPs on cardiac function in rats. Results from echocardiographic, ultrastructural and histopathological analysis found that SiNPs induced cardiac contractile dysfunction, accompanied by incomplete myocardial structures, disordered sarcomere segments, interstitial edema and myocyte apoptosis in heart. Levels of myocardial enzymes and inflammatory factors were markedly increased in both serum and heart tissue, accompanied by elevated levels of oxidative damage occurred in the hearts of SiNPs-treated rats. SiNPs significantly upregulated the expressions of inflammation and contraction-related proteins, including JNK, p-JNK, c-Jun, TF and PAR1. Lentivirus transfection of JNK shRNA showed the low-expression of JNK-facilitated F-actin and inhibited TF in the SiNPs-treated cardiomyocytes. Moreover, SiNPs activated the mRNA and protein levels of JNK/TF/PAR1 pathway, and these effects were significantly dampened after JNK knock down. Our results demonstrate that SiNPs trigger myocardial contractile dysfunction via JNK/TF/PAR1 signaling pathway.

Epidemiological evidence showed that the particulate matter exposure is associated with atherosclerotic plaque progression, which may be related to foam cell formation, but the mechanism is still unknown. The study was aimed to investigate the toxic effects and possible mechanism of PM

Figure S1. Effects of SiNPs on cellular adhesion molecule expression in SD rats’aortic arch sections were detected by immunohistochemical analysis. The expression of PECAM-1was increasing in a dose-dependent manner compared to the control group. (A) Control (B) 1.8 mg/kg·bw (C) 5.4 mg/kg·bw (D) 16.2 mg/kg·bw. Table S1. Primers used for qRT-PCR. Table S2. Hydrodynamic size and zeta potential of SiNPs in distilled water, DMEM, DMEM (10% serum) and normal saline as dispersion medium at different time points.Table S3. The top microRNA-gene ranked by degree over 5 in Signal-Net analysis. Table S4 The summary of 16 significant pathways involved in 11 microRNA. (PDF 1631 kb)

Silica nanoparticles (SiNPs) have been widely used in human health related products, such as food additives, cosmetics and even drug delivery, gene therapy or bioimaging. Recently, a first-in-human clinical trial based on polyethylene glycol (PEG)-modified SiNPs had been approved by US FDA to trace melanoma. However, as a nano-based drug delivery system, its biocompatibility and vascular toxicity are still largely unknown. Thus, we synthesized the fluorescent SiNPs to explore the biocompatibility and vascular endothelial function, and compare different biological effects caused by PEG-modified and unmodified SiNPs in cells and zebrafish model. The characterizations of SiNPs and PEG-modified SiNPs were analyzed by TEM, SEM, AFM and DLS, which exhibited relatively good stable and dispersive. Compared with SiNPs, PEG-modified SiNPs had markedly reduced the inflammatory response and vascular damage in Tg (fli-1: EGFP) and Tg (mpo: GFP) transgenic zebrafish lines, respectively. Consistent with the in vivo results, the PEG-modified SiNPs had been found to significantly decline the levels of ROS, inflammatory cytokines and mitochondrial-mediated apoptosis in vascular endothelial cells compared to SiNPs, and the ROS scavenger NAC could effectively alleviate the above adverse effects induced by nanoparticles. Our results suggested that the PEG-modified SiNPs could become more safety via increasing the biocompatibility and decreasing cellular toxicities in living organisms.

Up to now, while some toxicological studies have identified pulmonary fibrosis immediately induced by long-term PM2.5 exposure, there has been no evidence indicating, whether short-term exposure can lead to post-exposure development of pulmonary fibrosis. Here, we treated rats with PM2.5 for 1 month (10 times), followed by normal feeding for 18 months. 18F-FDG intake, which is linked with the initiation and development of pulmonary fibrosis in living bodies, was found to gradually increase in lung following exposure through micro PET/CT imaging. Histolopathological examination revealed continuous deterioration of pulmonary injury post-exposure. Collagen deposition and hydroxyproline content continued to increase all along in the post-exposure duration, indicating pulmonary fibrosis development. Chronic and persistent induction of pulmonary inflammatory gene expression (Tnf, Il1b, Il6, Ccl2, and Icam1), epithelial mesenchymal transition (EMT, reduction of E-cadherin and elevation of fibronectin) and RelA/p65 upregulation, as well as serum inflammatory cytokine production, were also found in PM2.5-treated rats. Pulmonary oxidative stress, manifested by increase of MDA and decrease of GSH and SOD, was induced during exposure but disappeared in later post-exposure duration. These results suggested that short-term PM2.5 exposure could lead to sustained post-exposure pulmonary fibrosis development, which was mediated by oxidative-stress-initiated NF-κB/inflammation/EMT pathway.

To demonstrate the combined adverse effect and the mechanism of silica nanoparticles (SiNPs) with 57.66 ± 7.30 nm average diameter and high-fat diet (HFD) on Wistar rats, 60 male Wistar rats were randomly divided into six groups (n = 10): Control group, SiNPs group, HFD group, 2 mg kg−1 SiNPs + HFD group, 5 mg kg−1 SiNPs + HFD group and 10 mg kg−1 SiNPs + HFD group. HFD was administrated for 2 weeks for the rats in advance and SiNPs were supplied every 3 d for 48 d subsequently. The present study illustrated that both HFD and SiNPs could decrease sperm concentration, mobility rates, increase abnormality rates, damage testicular structure, reduce spermatogonium numbers and spermatoblast numbers, reduce ATP levels, and affect expression of regulatory factors for meiosis in testis. HFD and SiNPs further damaged the sperm and lowered the ATP level and expression of factors associated with meiotic signaling pathway compared with the HFD without SiNPs in testicular tissue of Wistar rats. These results suggested that SiNPs significantly promoted reproductive toxicity induced by HFD in Wistar rats, which provides novel experimental evidence and an explanation for magnified reproductive toxicity triggered by SiNPs in HFD rats.

Lin Feng,1,2 Xiaozhe Yang,1,2 Yanfeng Shi,1,2 Shuang Liang,1,2 Tong Zhao,1,2 Junchao Duan,1,2 Zhiwei Sun1,2 1Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing 100069, PR China; 2Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, PR China Background: The harmful effects following the release of nanomaterials into environment are of great concern today. Purpose: In this study, subacute effect due to co-exposure to low-dose silica nanoparticles (SiNPs) and lead acetate (Pb) on cardiovascular system was detected in Sprague Dawley male rats. Materials and Methods: Histopathological and ultrastructural changes of heart, aortic arch and abdominal aorta were detected. Blood routine and blood biochemistry examinations were used to show the changes of blood components. The fibrinolytic and plasmin factors, inflammation-related factors and myocardial-related enzyme in serum were analysised by ELISA and Western blot assay. Results: Histopathological and ultrastructural examination of heart, aortic arch, and abdominal aorta showed that serious damage occurred in co-exposure group (n=6/group). Blood routine examination showed that leukocytosis and thrombocytopenia increased markedly, while changes in the erythrocyte count were not obvious in the co-exposure group. The expression of alanine transaminase (ALT) decreased obviously in co-exposure group, while no significant changes were noted in the expression of aspartate aminotransferase (AST), cholesterol (CHO), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) in the co-exposure group on blood biochemistry analysis. In addition, data from ELISA analysis showed that the levels of fibrinolytic and plasmin factors, including thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), tissue-type plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI), and antithrombin III (AT III), were decreased, while those of human fibrinogen (FIB) and D-dimer (D2D) increased significantly in the co-exposure group. Moreover, the myocardial-related enzyme in serum, tested by ELISA, and cardiovascular-related protein expression of atrial natriuretic peptide and brain natriuretic peptide, tested by Western blot assay, was increased in the heart. Furthermore, the expression of inflammation factors such as C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) was increased in heart tissue subjected to combined exposure, which was manifested by Western blot assay, while the protein levels of angiotensin II (ANG II) and endothelin 1 were (ET-1) elevated in blood vessels in the co-exposure group. Conclusion: In conclusion, the major interactions involved in subacute toxicity due to co-exposure to low doses of SiNPs and Pb on cardiovascular system were expected to be additive and synergistic in nature. Co-exposure to SiNPs and Pb could aggravate the cardiovascular toxicity via endothelial damage, hypercoagulation, and cardiac injury in vivo. Keywords: SiNPs, Pb, combined exposure, cardiovascular toxicity, in vivo

Junchao Duan,1,2 Shuang Liang,1,2 Lin Feng,1,2 Yang Yu,1,2 Zhiwei Sun1,2 1Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing, PR China; 2Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing, PR China Background: As a promising nanocarrier in biomedical fields, silica nanoparticles (SiNPs) could transfer from the circulatory system to multiple organs. Among these, blood–liver molecular exchange is a critical factor in biological response to NPs. However, the potential effect of SiNPs on hepatic lipid metabolism is unclear. In this study, we employed three models to attempt discover whether and how SiNPs disturb hepatic lipid metabolism in vivo and in vitro. Methods: Firstly we used ICR mice models to evaulated the effects of SiNPs on the serum and hepatic lipid levels through repeated intravenous administration, meanwhile, the protein expressions of protein markers of lipogenesis (ACC1 and FAS ), the key enzyme of fatty acid β-oxidation, CPT1A,and leptin levels in liver were detected by western blot. For verification studies, the model organism zebrafish and cultured hepatic L02 cells were further performed. The TLR5 and adipocytokine-signaling pathway were verified. Results: Inflammatory cell infiltration and mild steatosis induced by SiNPs were observed in the liver. Cholesterol, triglyceride, and low-density lipoprotein cholesterol levels were elevated significantly in both blood serum and liver tissue, whereas the ratio of high-density:low-density lipoprotein cholesterol was markedly decreased. Protein markers of lipogenesis (ACC1 and FAS) were elevated significantly in liver tissue, whereas the key enzyme of fatty acid β-oxidation, CPT1A, was decreased significantly. Interestingly, leptin levels in the SiNP-treated group were also elevated markedly. In addition, SiNPs caused hepatic damage and steatosis in zebrafish and enhanced hyperlipemia in high-cholesterol diet zebrafish. Similarly, SiNPs increased the release of inflammatory cytokines (IL1β, IL6, IL8, and TNFα) and activated the TLR5-signaling pathway in hepatic L02 cells. Conclusion: In summary, our study found that SiNPs triggered hyperlipemia and hepatic steatosis via the TLR5-signaling pathway. This suggests that regulation of TLR5 could be a novel therapeutic target to reduce side effects of NPs in living organisms. Keywords: silica nanoparticles, hyperlipemia, hepatic steatosis, hepatic lipid metabolism, TLR5-signaling pathway

Epidemiological evidence suggests that fine particulate matter (PM2.5) in air pollution promotes the formation of deep venous thrombosis. However, no evidence is available on the effects of PM2.5 lead to disseminated intravascular coagulation (DIC). For the first time, this study explored the effects of PM2.5 on DIC via coagulation disorders in vivo. SD rats received intratracheal instillation of PM2.5 once every three days for one month. Doppler ultrasound showed that the pulmonary valve (PV) and aortic valve (AV) peak flow were decreased after exposure to PM2.5. Fibrin deposition and bleeding were observed in lung tissue and vascular endothelial injury was found after exposure to PM2.5. Expression of thrombomodulin (TM) in vessel was downregulated after PM2.5-treated, whereas the levels of proinflammatory factors and adhesion molecules (IL-6, IL-1β, CRP, ICAM-1 and VCAM-1) were markedly elevated after exposure to PM2.5. Tissue factor (TF) and the coagulation factor of FXa were increased, while vWF was significantly lowered induced by PM2.5. Thrombin-antithrombin complex (TAT) and fibrinolytic factor (t-PA) were elevated, while there was no significantly change in the expression of anticoagulant factors (TFPI and AT-III). To clarify the relationship between PM2.5 and DIC, we examined the general diagnostic indices of DIC: PM2.5 prolonged PT and increased the expression of D-dimer but decreased platelet count and fibrinogen. In addition, the gene levels of JAK1 and STAT3 showed an upward trend, whereas there was little effect on JAK2 expression. And inflammatory factors (IL-6, IL-1β and TNF) in blood vessels of were up-reglated in PM2.5-treated rats. In summary, our results found that PM2.5 could induce inflammatory response, vascular endothelial injury and prothrombotic state, eventually resulted in DIC. It will provide new evidence for a link between PM2.5 and cardiovascular disease., Graphical abstract Unlabelled Image, Highlights • PM2.5 triggered vascular endothelial injury via inflammatory response. • PM2.5 activated TF-dependent coagulation and caused coagulation dysfunction. • PM2.5 induced fibrin deposition and bleeding in pulmonary and resulted in DIC.

Autophagy dysfunction is a potential toxic effect of nanoparticles. Previous studies have indicated that silica nanoparticles (SiNPs) induce macroautophagy/autophagy dysfunction, while the precise mechanisms remain uncertain. Hence, the present study investigated the molecular mechanisms by which SiNPs enhanced autophagosome synthesis, which then contributed to autophagy dysfunction. First, the effects of SiNPs on autophagy and autophagic flux were verified using transmission electron microscopy, laser scanning confocal microscopy, and western blot assays. Then, the activation of endoplasmic reticular (ER) stress was validated to be through the EIF2AK3 and ATF6 UPR pathways but not the ERN1-XBP1 pathway, along with the upregulation of downstream ATF4 and DDIT3. Thereafter, the ER stress inhibitor 4-phenylbutyrate (4-PBA) was used to verify that SiNP-induced autophagy could be influenced by ER stress. Furthermore, specialized lentiviral shRNA were employed to determine that autophagy was induced via specific activation of the EIF2AK3 and ATF6 UPR pathways. Finally, the 2 autophagic genes LC3B and ATG12 were found to be transcriptionally upregulated by downstream ATF4 and DDIT3 in ER stress, which contributed to the SiNP-enhanced autophagosome synthesis. Taken together, these data suggest that SiNPs induced autophagosome accumulation via the activation of the EIF2AK3 and ATF6 UPR pathways in hepatocytes, which offers a new insight into detailed molecular mechanisms underlying SiNP-induced autophagy dysfunction, and specifically how UPR pathways regulate key autophagic genes. This work provides novel evidence for the study of toxic effects and risk assessment of SiNPs.

The mechanisms of systemic pulmonary inflammation and toxicity of fine particulate matter (PM

PM

Although the strongly causal associations were between fine particulate matter (PM

Exposure to PM2.5 has been strongly linked to endothelial dysfunction. However, the underlying mechanism of PM2.5 on the vascular endothelial function is poorly understood. This study examined the toxic effect and underlying mechanism of PM2.5 on human umbilical vein endothelial cells (HUVECs). Decreased cell viability and increased LDH activity were observed in the PM2.5-treated HUVECs in a dose-dependent manner. The production of ROS, MDA, and the inhibition of SOD activity were also triggered by PM2.5 in HUVECs. In addition, PM2.5 increased the intracellular levels of proinflammatory cytokines (IL-6, TNF-a, IL-1β, IL-8 and CRP), cell adhesion molecules (ICAM-1, VCAM-1) and tissue factor (TF), resulted in endothelial activation. For an in-depth study, the protein levels of IL-6, JAK1 and STAT3 were up-regulated significantly, while the expression of JAK2 and SOCS1 were down-regulated gradually in PM2.5-treated HUVECs in a dose-dependent manner. These results show that PM2.5 triggered endothelial activation via upregulation of the IL-6 dependent JAK1/STAT3 signaling pathway. This will provide new insights into the toxic effects and mechanisms of cardiovascular diseases triggered by ambient air pollution.

The toxicity mechanism of nanoparticles on vertebrate cardiovascular system is still unclear, especially on the low-level exposure. This study was to explore the toxic effect and mechanisms of low-dose exposure of silica nanoparticles (SiNPs) on cardiac function in zebrafish embryos via the intravenous microinjection. The dosage of SiNPs was based on the no observed adverse effect level (NOAEL) of malformation assessment in zebrafish embryos. The mainly cardiac toxicity phenotypes induced by SiNPs were pericardial edema and bradycardia but had no effect on atrioventricular block. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased in a dose-dependent manner. Microarray analysis and bioinformatics analysis were performed to screen the differential expression genes and possible pathway involved in cardiac function. SiNPs induced whole-embryo oxidative stress and neutrophil-mediated cardiac inflammation in Tg(mpo:GFP) zebrafish. Inflammatory cells were observed in atrium of SiNPs-treated zebrafish heart by histopathological examination. In addition, the expression of TNNT2 protein, a cardiac contraction marker in heart tissue had been down-regulated compared to control group using immunohistochemistry. Confirmed by qRT-PCR and western blot assays, results showed that SiNPs inhibited the calcium signaling pathway and cardiac muscle contraction via the down-regulated of related genes, such as ATPase-related genes (atp2a1l, atp1b2b, atp1a3b), calcium channel-related genes (cacna1ab, cacna1da) and the regulatory gene tnnc1a for cardiac troponin C. Moreover, the protein level of TNNT2 was decreased in a dose-dependent manner. For the first time, our results demonstrated that SiNPs induced cardiac dysfunction via the neutrophil-mediated cardiac inflammation and cardiac contraction in zebrafish embryos.

Endosulfan, a persistent organic pollutant, is widely used in agriculture as a pesticide. The aim of the present study was to evaluate the blood toxicity of different doses of endosulfan in Wistar rats. The experimental sample was composed of four groups, a control group that did not receive endosulfan and three endosulfan-exposed groups that respectively received 1, 5, or 10 mg/kg/day (doses below LD50), of endosulfan for 21 days. The results showed that endosulfan significantly decreased the prothrombin time (PT) and upregulated the activated coagulation factors VIIa, Xa, and XIIIa; thrombin-antithrombin complex (TAT); and P-selectin. Plasma levels of tissue factor (TF) and malondialdehyde (MDA) were increased in the endosulfan groups. The activated partial thromboplastin time (APTT) and the level of activated coagulation factor IXa showed no obvious changes. Immunohistochemical results showed increased expression of von Willebrand factor (vWF) and the inflammatory cytokine interleukin (IL)-1β in the groups exposed to endosulfan. The pathology and electron microscopy results showed impaired vascular tissue accompanied by the exfoliation of endothelial cells and mitochondrial damage in the endosulfan-exposed groups. In summary, our results suggest that endosulfan damages endothelial cells via oxidative stress and the inflammatory response, leading to the release of TF and vWF into the blood. The TF and vWF in the blood may activate extrinsic coagulation factors and platelets, thus triggering the extrinsic coagulation pathway. There were no obvious effects on the intrinsic coagulation pathway.

Recent years, air pollution has been a serious problem, and PM

The multinucleation effect of silica nanoparticles (SiNPs) had been determined in our previous studies, but the relative mechanisms of multinucleation and how the multinucleated cells are generated were still not clear. This extensional study was conducted to investigate the mechanisms underlying the formation of multinucleated cells after SiNPs exposure. We first investigated cellular multinucleation, then performed time-lapse confocal imaging to certify whether the multinucleated cells resulted from cell fusion or abnormal cell division. Our results confirmed for the first time that there are three patterns contributing to the SiNPs-induced multinucleation in HepG2 cells: cell fusion, karyokinesis without cytokinesis, and cytokinesis followed by fusion. The chromosomal passenger complex (CPC) deficiency and cell cycle arrest in G1/S and G2/M checkpoints may be responsible for the cell aberrant cytokinesis. The activated MAPK/ERK1/2 signaling and decreased mitosis related proteins might be the underlying mechanism of cell cycle arrest and thus multinucleation. In summary, we confirmed the hypothesis that aberrant cytokinesis and cell fusion resulted in multinucleation in HepG2 cells after SiNPs exposure. Since cell fusion and multinucleation were involved in genetic instability and tumor development, this study suggests the potential ability of SiNPs to induce cellular genetic instability. These findings raise concerns with regard to human health hazards and environmental risks with SiNPs exposure.

Silica nanoparticles (SiNPs) play a vital role in medical applications such as drug delivery and cancer therapy. SiNPs can translocate into the bloodstream through all the possible routes of entry. However, there have been scarce studies on the pre-thrombotic effect of SiNPs and the mechanism of the pre-thrombotic state in vivo. We specifically focused on the changes of platelet function and blood coagulation in Wistar rats after 7 consecutive days of intravenous injection of SiNPs (52 nm). The platelet aggregation assay, structural changes of platelet membrane glucoproteins, coagulation test, coagulant/anti-coagulant and fibrinolytic factors and the possible molecular mechanism of pre-thrombotic state formation were performed. Our results demonstrated a significant increase in platelet aggregation rate and platelet activation after SiNP exposure. The clotting time was significantly shortened while fibrinogen (FIB) contents were increased. There were sustained increases in coagulation factors and thrombin–antithrombin complex (TAT) expression induced by SiNPs. Antithrombin III (AT-III) of the SiNP-treated groups was significantly decreased while the concentrations of the tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (t-PA) and D-dimer were elevated. The phosphorylation of nuclear factor-κB/p65 (NF-κB/p65) and activator protein-1/c-Jun (AP-1/c-Jun) and the protein levels of JNK were increased after SiNP exposure. In summary, our results revealed that SiNPs induced the hypercoagulable and pre-thrombotic state in rats through the interaction between platelet activation, coagulation system hyperfunction, anti-coagulation and fibrinolytic resistance. Direct interactions between SiNPs and coagulation factor XII (F XII) and the JNK-NF-κB/AP-1 pathway might be involved in the regulation of pre-thrombotic state formation.

Fine particle matter (PM) is correlated with male reproductive dysfunction in animals and humans, but the underlying mechanisms remain unknown. To investigate the toxic mechanism of PM, 32 male Sprague-Dawley (SD) rats were exposed to saline or PM2.5 with the doses of 1.8, 5.4, and 16.2 mg/kg.b.w. via intratracheal instillation, respectively, one time every 3 days, in total times for 30 days. Sperm concentration, hormone level, the expressions of BTB-associated protein and the mitogen-activated protein kinase (MAPK) pathway, tumor necrosis factor α and transforming growth factor β3 levels were detected. The results showed a decrease in sperm number, testosterone and luteinizing hormone levels and altered ultrastructure of BTB in testis of rat after exposure to PM2.5 . The protein levels of N-Cadherin, Occludin, Claudin-11, and Connexin-43 were significantly decreased in the testes. TGF-β3 content in testes showed increase, with the p-p38/p38 MAPK ratio also increasing after PM2.5 exposure. These results demonstrate that PM2.5 restrained the expressions of BTB-associated proteins through activating TGF-β3/p38 MAPK pathway and decreasing testosterone secretion, and therefore lead to the damage of BTB resulting in the decrease of sperm quality, which might be the potential reasons for its negative effects on spermatogenesis and male reproduction.

Fine particulate matter (PM

Given that the effects of ultrafine fractions (