Xiaozhe Yang

1H NMR-based metabolomics study on repeat dose toxicity of fine particulate matter in rats after intratracheal instillation

Systemic metabolic effects and toxicity mechanisms of ambient fine particulate matter (PM2.5) remain uncertain. In order to investigate the mechanisms in PM2.5 toxicity, we explored the endogenous metabolic changes and possible influenced metabolic pathways in rats after intratracheal instillation of PM2.5 by using a 1H nuclear magnetic resonance (NMR)-based metabolomics approach. Liver and kidney histopathology examinations were also performed. Chemical characterization demonstrated that PM2.5 was a complex mixture of elements. Histopathology showed cellular edema in liver and glomerulus atrophy of the PM2.5 treated rats. We systematically analyzed the metabolites changes of serum and urine in rats using 1H NMR techniques in combination with multivariate statistical analysis. Significantly reduced levels of lactate, alanine, dimethylglycine, creatine, glycine and histidine in serum, together with increased levels of citrate, arginine, hippurate, allantoin and decreased levels of allthreonine, lactate, alanine, acetate, succinate, trimethylamine, formate in urine were observed of PM2.5 treated rats. The mainly affected metabolic pathways by PM2.5 were glycine, serine and threonine metabolism, glyoxylate and dicarboxylate metabolism, citrate cycle (TCA cycle), nitrogen metabolism and methane metabolism. Our study provided important information on assessing the toxicity of PM2.5 and demonstrated that metabolomics approach can be employed as a tool to understand the toxicity mechanism of complicated environmental pollutants.

Genome-wide transcriptional analysis of cardiovascular-related genes and pathways induced by PM2.5 in human myocardial cells

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.

Combined effect of silica nanoparticles and benzo[a]pyrene on cell cycle arrest induction and apoptosis in human umbilical vein endothelial cells

Particulate matter (PM) such as ultrafine particulate matter (UFP) and the organic compound pollutants such as polycyclic aromatic hydrocarbon (PAH) are widespread in the environment. UFP and PAH are present in the air, and their presence may enhance their individual adverse effects on human health. However, the mechanism and effect of their combined interactions on human cells are not well understood. We investigated the combined toxicity of silica nanoparticles (SiNPs) (UFP) and Benzo[a]pyrene (B[a]P) (PAH) on human endothelial cells. Human umbilical vascular endothelial cells (HUVECs) were exposed to SiNPs or B[a]P, or a combination of SiNPs and B[a]P. The toxicity was investigated by assessing cellular oxidative stress, DNA damage, cell cycle arrest, and apoptosis. Our results show that SiNPs were able to induce reactive oxygen species generation (ROS). B[a]P, when acting alone, had no toxicity effect. However, a co-exposure of SiNPs and B[a]P synergistically induced DNA damage, oxidative stress, cell cycle arrest at the G2/M check point, and apoptosis. The co-exposure induced G2/M arrest through the upregulation of Chk1 and downregulation of Cdc25C, cyclin B1. The co-exposure also upregulated bax, caspase-3, and caspase-9, the proapoptic proteins, while down-regulating bcl-2, which is an antiapoptotic protein. These results show that interactions between SiNPs and B[a]P synergistically potentiated toxicological effects on HUVECs. This information should help further our understanding of the combined toxicity of PAH and UFP.

Cytotoxicity induced by fine particulate matter (PM 2.5 ) via mitochondria-mediated apoptosis pathway in human cardiomyocytes

Although the strongly causal associations were between fine particulate matter (PM2.5) and cardiovascular disease, the toxic effect and potential mechanism of PM2.5 on heart was poorly understood. Thus, the aim of this study was to evaluate the cardiac toxicity of PM2.5 exposure on human cardiomyocytes (AC16). The cell viability was decreased while the LDH release was increased in a dose-dependent way after AC16 exposed to PM2.5. The reactive oxygen species (ROS) generation and production of malondialdehyde (MDA) were increased followed by the decreasing in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The damage of mitochondria was observed by ultra-structural analysis and MMP measurement. The apoptotic rate of AC16 were markedly elevated which was triggered by PM2.5. In addition, the proteins involved in mitochondria- mediated apoptosis pathway were measured. The protein levels of Caspase-3, Caspase-9 and Bax were up-regulated while the anti-apoptotic protein, Bcl-2 was down-regulated after AC16 exposed to PM2.5. In summary, our results demonstrated that mitochondria-mediated apoptosis pathway played a critical role in PM2.5-induced myocardial cytotoxicity in AC16, which suggested that PM2.5 may contribute to cardiac dysfunction.

Metabolic impact induced by total, water soluble and insoluble components of PM 2.5 acute exposure in mice

Fine particulate matter (PM2.5) has been listed as an important environmental risk factor for human health. However, the systemic biological effects on metabolic responses induced by PM2.5 and its components were poorly understood. This study was aimed to evaluate the toxicity of different components of PM2.5 at molecular level via metabolomics approach. In the present study, we adopted a 1H NMR-based metabolomics approach to evaluate metabolic profiles in mice after acute exposure to Total-PM2.5, water soluble components of PM2.5 (WS-PM2.5) and water insoluble components of PM2.5 (WIS-PM2.5). First, we characterized the morphological features and chemical composition of PM2.5. Then, the metabolites changes of serum and urine in mice were systematically analyzed using 800 MHz 1H NMR techniques in combination with multivariate statistical analysis. Total-PM2.5 exposure affected metabolites mainly involved in amino acid metabolism, protein biosynthesis, energy metabolism and metabolism of cofactors and vitamins. WS-PM2.5 exposure influenced lipid metabolism and carbohydrate metabolism. WIS-PM2.5 exposure mainly perturbed amino acid metabolism and energy metabolism. The results suggested that acute exposure to the Total-PM2.5, WS-PM2.5 and WIS-PM2.5 in mice exhibited marked systemic metabolic changes. In addition, the insoluble fraction of PM2.5 contributed greatly to the toxicity of PM2.5.

Cellular pathways involved in silica nanoparticles induced apoptosis: A systematic review of in vitro studies

Silica nanoparticles (SiNPs) have been found to pass through biological barriers and get distributed in the human body. They induce cell apoptosis via various mechanisms in body organs. To understand these mechanisms, we carried out systematic review of in vitro studies on SiNPs-induced cell apoptosis. Office of Health Assessment and Translation approach for Systematic Review and Evidence Integration was used to identify 14 studies dating from the year 2000 to current. Four studies showed an increase in DNA damage, cell cycle arrest, proapoptotic factors and decrease in antiapoptotic factors resulting to apoptosis. Eight studies showed induction of mitochondrial dysfunction, Bax upregulation, Bcl-2 downregulation, and caspase-3, -7, -9 activities increase. Increase in FADD, TNFR1 and Bid proteins was observed in one study, while the other NO production and caspase-3 activity was increased. These studies found the potency of SiNPs to induce cell apoptosis through DNA damage, mitochondrial, tumor necrosis factor, and nitric oxide related pathways.

Low-dose combined exposure of nanoparticles and heavy metal compared with PM2.5 in human myocardial AC16 cells

The co-exposure toxicity mechanism of ultrafine particles and pollutants on human cardiovascular system are still unclear. In this study, the combined effects of silica nanoparticles (SiNPs) and/or carbon black nanoparticles (CBNPs) with Pb(AC)2 compared with particulate matter (PM)2.5 were investigated in human myocardial cells (AC16). Our study detected three different combinations of SiNPs and Pb(AC)2, CBNPs and Pb(AC)2, and SiNPs and CBNPs compared with PM2.5 at low-dose exposure. Using PM2.5 as positive control, our results suggested that the combination of SiNPs and Pb(AC)2/CBNPs could increase the production of reactive oxygen species (ROS), lactate dehydrogenase leakage (LDH), and malondialdehyde (MDA) and decrease the activities of superoxide dismutase (SOD) and glutathione (GSH); induce inflammation by the upregulation of protein CRP and TNF-α, and apoptosis by the upregulation of protein caspase-3, caspase-9, and Bax while the downregulation of protein Bcl-2; and trigger G2/M phase arrest by the upregulation of protein Chk2 and downregulation of protein Cdc2 and cyclin B1. In addition, the combination of CBNPs and Pb(AC)2 induced a significant increase in MDA and reduced the activities of ROS, LDH, SOD, and GSH, with G1/S phase arrest via upregulation of Chk1 and downregulation of CDK6 and cyclin D1. Our data suggested that the additive interaction and synergistic interaction are the major interaction in co-exposure system, and PM2.5 could trigger more severe oxidative stress, G2/M arrest, and apoptosis than either co-exposure or single exposure.

Gene expression profiles and bioinformatics analysis of human umbilical vein endothelial cells exposed to PM2.5

Cardiovascular system is demonstrated the main target of PM2.5 and the objective of this study was to explore the toxic effect and molecular mechanisms caused by PM2.5 in primary human umbilical vein endothelial cells (HUVECs) using microarray and bioinformatics analysis. The results showed that 591 genes were differentially expressed triggered by PM2.5, of which 174 genes were down-regulated, while 417 genes were up-regulated. Gene ontology analysis revealed that PM2.5 caused significant changes in gene expression patterns, including response to stimuli, immune response, and cellular processes. Pathway analysis and Signal-net analysis suggested that endocytosis, chemokine signaling pathway, RNA transport, protein processing in endoplasmic reticulum (ER) and autophagy regulation were the most critical pathways in PM2.5-induced toxicity in HUVECs. Moreover, gene expression confirmation of LIF, BCL2L1, CSF3, HMOX1, RPS6, PFKFB, CAPN1, HSPBP1, MOGS, PREB, TUBB2A, GABARAP by qRT-PCR indicated that endocytosis might be involved in the cellular uptake of PM2.5 by forming phagosomes, and subsequently inflammation, hypoxia and ER stress was occurred, which finally activated autophagy after PM2.5 exposure in HUVECs. In summary, our data can serve as fundamental research clues for further studies of PM2.5-induced toxicity in HUVECs.

PM2.5-induced alteration of DNA methylation and RNA-transcription are associated with inflammatory response and lung injury.

The mechanisms of systemic pulmonary inflammation and toxicity of fine particulate matter (PM2.5) exposure remains unclear. The current study investigated the inflammatory response and lung toxicity of PM2.5 in rats following intratracheal instillation of PM2.5. After repeated (treated every 3 days for 30 days) PM2.5 exposure, total protein (TP), lactate dehydrogenase (LDH) activity and inflammatory cytokines including interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) levels in bronchoalveolar lavage fluid (BALF) were markedly elevated. The expression levels of IL-6, IL-1β, TNF-α and NF-κB in rat lung tissue and BEAS-2B cells were significantly upregulated after PM2.5 exposure. Histopathological evaluation suggested that the major pathological changes were alveolar wall thickening and inflammatory cell infiltration of the lungs. Genome wide DNA methylation and RNA-transcription analysis was performed on human bronchial epithelial cells (BEAS-2B) to explore the potential mechanisms in vitro. PM2.5 induced genome wide DNA methylation and transcription changes. Differentially methylated CpGs were located in gene promoter region linked with CpG islands. Integrated analysis with DNA methylation and transcription data indicated a clear bias toward transcriptional alteration by differential methylation. Disease ontology of differentially methylated and expressed genes addressed their prominent role in respiratory disease. Functional enrichment revealed their involvement in inflammation or immune response, cellular community, cellular motility, cell growth, development and differentiation, signal transduction and responses to exogenous stimuli. Gene expression validation of ACTN4, CXCL1, MARK2, ABR, PSEN1, PSMA3, PSMD1 verified their functional participation in critical biological processes and supported the microarray bioinformatics analysis. Collectively, our data shows that PM2.5 induced genome wide methylome and transcriptome alterations that could be involved in pulmonary toxicity and pathological process of respiratory disease, providing new insight into the toxicity mechanisms of PM2.5.

Co-exposure of silica nanoparticles and methylmercury induced cardiac toxicity in vitro and in vivo

The released nanoparticles into environment can potentially interact with pre-existing pollution, maybe causing higher toxicity. As such, assessment of their joint toxic effects is necessary. This study was to investigate the co-exposure cardiac toxicity of silica nanoparticles (SiNPs) and methylmercury (MeHg). Factorial design was used to determine the potential joint action type. In vitro study, human cardiomyocytes (AC16) were exposed to SiNPs and MeHg alone or the combination. Higher toxicity was observed on cell viability, cell membrane damage in co-exposure compared with single exposure and control. The co-exposure enhanced the ROS, MDA generation and reduced the activity of SOD and GSH-Px. In addition, the co-exposure induced much higher cellular apoptotic rate in AC16. In vivo study, after SD rats exposed to SiNPs and MeHg and their mixture by intratracheal instillation for 30days, pathological changes (myocardial interstitial edema) of heart were occurred in co-exposure compared with single exposure and control. Moreover obvious ultra-structural changes, including myofibril disorder, myocardial gap expansion, and mitochondrial damage were observed in co-exposure group. The activity of myocardial enzymes, including CK-MB, ANP, BNP and cTnT, were significantly elevated in co-exposure group of rat serum. Meanwhile, the cardiac injury-linked proteins expression showed an increase in SERCA2 and decreased levels of cTnT, ANP and BNP in co-exposure group. Factorial design analysis demonstrated that additive and synergistic interactions were responsible for the co-exposure cardiac toxicity in vitro and vivo. In summary, our results showed severe cardiac toxicity induced by co-exposure of SiNPs and MeHg in both cardiomycytes and heart. It will help to clarify the potential cardiovascular toxicity in regards to combined exposure pollutions.