Chronic inhalation of biomass smoke is associated with DNA damage in airway cells: involvement of particulate pollutants and benzene

This study examined whether indoor air pollution from biomass fuel burning induces DNA damage in airway cells. For this, sputum cells were collected from 56 premenopausal rural women who cooked with biomass (wood, dung, crop residues) and 49 age‐matched controls who cooked with cleaner liquefied petroleum gas. The levels of particulate matters with diameters of less than 10 and 2.5 µm (PM₁₀ and PM_2.5) in indoor air were measured using a real‐time aerosol monitor. Benzene exposure was monitored by measuring trans,trans‐muconic acid (t,t‐MA) in urine by HPLC‐UV. DNA damage was examined by alkaline comet assay in sputum cells. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in sputum cells were measured by flow cytometry and spectrophotometry, respectively. Compared with controls, biomass users had 4 times higher tail percentage DNA, 37% more comet tail length and 5 times more Olive tail moment (p < 0.001) in inflammatory and epithelial cells in sputum, suggesting extensive DNA damage. In addition, women who cooked with biomass had 6 times higher levels of urinary t,t‐MA and 2‐fold higher levels of ROS generation concomitant with 28% depletion of SOD. Indoor air of biomass‐using households had 2–4 times more PM₁₀ and PM_2.5 than that of controls_. After controlling potential confounders, positive association was found between DNA damage parameters, particulate pollution, urinary t,t‐MA and ROS. Thus, long‐term exposure to biomass smoke induces DNA damage in airway cells and the effect was probably mediated, at least in part, by oxidative stress generated by inhaled particulate matter and benzene. Copyright © 2011 John Wiley & Sons, Ltd.     

Changes in RANKL and osteoprotegerin expression after chronic exposure to indoor air pollution as a result of cooking with biomass fuel

The impact of indoor air pollution as a result of cooking with unprocessed biomass on membrane‐bound and serum receptor activator of nuclear factor‐kappa ligand 1 (RANKL), its soluble decoy receptor osteoprotegerin (OPG) and osteoclast precursor CD14⁺CD16⁺ monocytes was investigated. Seventy‐four pre‐menopausal women from eastern India using biomass and 65 control women who cooked with cleaner liquefied petroleum gas were enrolled. PM₁₀ and PM_2.5 levels in their indoor air were measured with real‐time aerosol monitors. The levels of membrane‐bound RANKL on leukocytes and percentage CD14⁺CD16⁺ monocytes in the subjects' blood were assayed by flow cytometry. Soluble RANKL and OPG in serum were measured by ELISA. The results showed that PM₁₀ and PM_2.5 levels were significantly higher in the indoor air of biomass‐using households. Compared with the control women, the levels of CD4⁺ and CD19⁺ lymphocytes and circulating granulocytes with elevated levels of membrane‐bound RANKL were higher in biomass users. The serum levels of RANKL were increased by 41% whereas serum OPG was reduced by 22% among biomass users. The absolute number of CD14⁺CD16⁺ monocytes was significantly increased in biomass users than the control women. After controlling for potential confounders, PM₁₀ and PM_2.5 levels were found to be positively associated with leukocyte and serum RANKL and CD14⁺CD16⁺ monocyte levels, but negatively with serum OPG. From these results, we can conclude that chronic exposure to biomass smoke increased membrane‐bound and soluble RANKL and circulating osteoclast precursors but decreased OPG, suggesting an increased risk of bone resorption and consequent osteoporosis in biomass‐exposed women of a child‐bearing age. Copyright © 2015 John Wiley & Sons, Ltd.     

Systemic inflammatory changes and increased oxidative stress in rural Indian women cooking with biomass fuels

The study was undertaken to investigate whether regular cooking with biomass aggravates systemic inflammation and oxidative stress that might result in increase in the risk of developing cardiovascular disease (CVD) in rural Indian women compared to cooking with a cleaner fuel like liquefied petroleum gas (LPG). A total of 635 women (median age 36 years) who cooked with biomass and 452 age-matched control women who cooked with LPG were enrolled. Serum interleukin-6 (IL-6), C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) were measured by ELISA. Generation of reactive oxygen species (ROS) by leukocytes was measured by flow cytometry, and erythrocytic superoxide dismutase (SOD) was measured by spectrophotometry. Hypertension was diagnosed following the Seventh Report of the Joint Committee. Tachycardia was determined as pulse rate > 100 beats per minute. Particulate matter of diameter less than 10 and 2.5 μm (PM₁₀ and PM_2.5, respectively) in cooking areas was measured using real-time aerosol monitor. Compared with control, biomass users had more particulate pollution in indoor air, their serum contained significantly elevated levels of IL-6, IL-8, TNF-α and CRP, and ROS generation was increased by 37% while SOD was depleted by 41.5%, greater prevalence of hypertension and tachycardia compared to their LPG-using neighbors. PM₁₀ and PM_2.5 levels were positively associated with markers of inflammation, oxidative stress and hypertension. Inflammatory markers correlated with raised blood pressure. Cooking with biomass exacerbates systemic inflammation, oxidative stress, hypertension and tachycardia in poor women cooking with biomass fuel and hence, predisposes them to increased risk of CVD development compared to the controls. Systemic inflammation and oxidative stress may be the mechanistic factors involved in the development of CVD.     

Respiratory symptoms,lung function decrement and chronic obstructive pulmonary disease in pre-menopausal Indian women exposed to biomass smoke

Abstract

Background: The impact of chronic exposure to smoke from biomass burning on respiratory health has been examined.

Methods: Six-hundred and eighty-one non-smoking women (median age 35 years) from eastern India who cook exclusively with biomass (wood, dung and crop residues) and 438 age-matched women from similar neighborhood who cook with liquefied petroleum gas (LPG) were examined. Pulmonary function test was done by spirometry. The concentrations of particulate matter having diameter of < 10?µm (PM₁₀) and < 2.5?µm (PM_2.5) in indoor air was measured by real-time aerosol monitor.

Results: Compared with LPG users, biomass users had greater prevalence of upper (50.9 versus 28.5%) and lower respiratory symptoms (71.8 versus 30.8%) and dyspnea (58.4 versus 19.9%). They showed reduction in all parameters measured by spirometer especially in mid-expiratory volume. PM₁₀ and PM_2.5 concentration in biomass using kitchen were 2–3-times more than LPG-using kitchen, and the decline in spirometry values was positively associated PM₁₀ and PM_2.5 levels in indoor air after controlling education, family income and kitchen location as potential confounders. Overall, 29.7% of biomass users and 16.4% of LPG users had deficient lung function, and restrictive type of deficiency was predominant. Chronic obstructive pulmonary disease (COPD) was diagnosed in 4.6% of biomass and 0.9% of LPG users. Women who predominantly used dung cake and did not possess separate kitchen had poorer lung function.

Conclusion: Cumulative exposure to biomass smoke causes lung function decrement and facilitates COPD development even in non-smoking and relatively young pre-menopausal women.     

Involvement of EGF receptor signaling and NLRP12 inflammasome in fine particulate matter‐induced lung inflammation in mice

Epidemiological studies have shown that exposure to ambient fine particulate matter (PM_2.5) is associated with respiratory diseases. Lung inflammation is a central feature of many pulmonary diseases, which can be induced by PM_2.5 exposure. However, the mechanisms underlying PM_2.5‐induced lung inflammation remain unclear. To characterize the role of epidermal growth factor receptor (EGFR) and inflammasome in PM_2.5‐induced lung inflammation in mice, 30 BALB/c mice were intrabroncheally instilled with saline and PM_2.5 suspension (4.0 mg/kg b.w.) for 5 consecutive days, respectively. Bronchoalveolar lavage (BAL) was conducted and BAL fluid (BALF) was collected. The levels of reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), epidermal growth factor (EGF), CXCL1, interleukin (IL)?1β, and IL‐18 in BALF were determined using ELISA. mRNA levels of IL‐6, IL‐1β, IL‐18, CXCL1, IL‐10, NLRP3, Caspase‐1, and NLRP12 in lung tissues were determined by RT‐PCR. Phospho‐EGFR (Tyr1068) and phospho‐Akt (Thr308) in lung tissues were examined using immunohistochemical staining and Western blotting, respectively. Protein levels of Caspase‐1, NLRP3, NF‐κB‐p52/p100, and NF‐κB‐p65 in bronchial epithelium were examined using immunohistochemical staining. It was shown that PM_2.5 exposure induced lung inflammation. Levels of total protein, ROS, iNOS, EGF, and CXCL1 and cell number in the BALF of mice exposed to PM_2.5 were markedly elevated relative to the control. mRNA levels of CXCL1, IL‐1β, and IL‐18 in lung tissues of PM_2.5‐exposed mice were increased in comparison with the control. However, level of NLRP12 mRNA in lung tissues of PM_2.5‐exposed mice was reduced. Phospho‐EGFR (Tyr1068) and phospho‐Akt (Thr308) levels in the lungs of PM_2.5‐instilled mice were higher than those in the lungs of the control. The protein levels of NF‐κB‐p52/p100 and NF‐κB‐p65 in bronchial epithelium of PM_2.5‐exposed mice were also increased compared with the control. This study suggests that EGF‐EGFR‐Akt‐NF‐κB signaling and NLRP12 inflammasome may be associated with PM_2.5‐induced lung inflammation in mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1121–1134, 2017.     

Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells

Epidemiological studies show a positive correlation between the air levels of fine particulate matter (PM_2.5) and cardiovascular disorders, but how PM_2.5 affects cardiomyocytes has not been studied in great deal. The aim of the present study was to obtain an insight into the links among intracellular levels of reactive oxygen species (ROS), apoptosis and mitogen‐activated protein kinases (MAPKs) in rat cardiac H9c2 cells exposed to PM_2.5. H9c2 cells were incubated with PM_2.5 at 100–800 µg ml^–1 to evaluate the effects of PM_2.5 on cell viability, cell apoptosis, intracellular levels of ROS and expression of apoptosis‐related proteins as well as activation of MAPKs. PM_2.5 decreased cell viability, increased the cell apoptosis rate and intracellular ROS production in a concentration‐dependent manner. PM_2.5 decreased the Bcl‐2/Bax ratio and increased cleaved caspase‐3 levels. A Western blots study showed up‐regulation of phosphorylated MAPKs including extracellular signal‐regulated protein kinases (ERKs), c‐Jun NH₂‐terminal kinases (JNKs) and p38 MAPK in the PM_2.5‐treated cells. The p38 MAPK inhibitor SB239063 attenuated whereas the ERKs inhibitor PD98059 augmented the effects of PM_2.5 on apoptosis and the expression of related proteins. In conclusion, PM_2.5 decreases cell viability and increases apoptosis by enhancing intracellular ROS production and activating the MAPKs signaling pathway in H9c2 cells. The MAPKs signaling pathway could be a new promising target for clinical therapeutic strategies against PM_2.5‐induced cardiac injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Inhibitory effects of protopanaxatriol type ginsenoside fraction (Rgx365) on particulate matter-induced pulmonary injury

Inhalation of fine particulate matter (PM_2.5) is associated with elevated pulmonary injury attributed to the loss of vascular barrier integrity. Black ginseng (BG), steamed 9 times and dried ginseng, and its major protopanaxatriol type ginsenosides (ginsenoside Rg4, Rg6, Rh4, Rh1, and Rg2) exhibited various biological activities including anti-septic, anti-diabetic, wound healing, immune-stimulatory, and anti-antioxidant activity. The aim of this study was to investigate the beneficial effects of Rgx365 (a protopanaxatriol type rare ginsenosides fraction) on PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Permeability, leukocyte migration, activation of proinflammatory proteins, generation of reactive oxygen species (ROS), and histology were examined in PM_2.5-treated EC and mice. Rgx365 significantly scavenged PM_2.5-induced ROS, inhibited ROS-induced activation of p38 mitogen-activated protein kinase (MAPK), activated Akt in purified pulmonary EC, which helped maintain endothelial integrity. Further, Rgx365 reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in PM-induced mouse lung tissues. Data suggested that Rgx365 might exhibit protective effects in PM-induced inflammatory lung injury and vascular hyperpermeability.     

Cadmium exposure activates Akt/ERK Signaling and pro-inflammatory COX-2 expression in human gallbladder epithelial cells via a ROS dependent mechanism

Gallbladder cancer (GBC) is the commonest biliary tract cancer with an ill-defined etiology. We examined the role of Cd⁺² exposures in a primary human gallbladder (GB) cell line model in this study. Cd⁺² exposures induced decreased cell viability, reactive oxygen species (ROS) generation, altered Akt/ERK signaling pathway activation, PGE₂ and COX-2 expression in a human primary gallbladder epithelial cell model. Pharmacological inhibitors were used to determine the key drivers of elevated COX-2 expression due to Cd⁺² exposure. Our results show Cd⁺² causes a dose-dependent reduction in GB cell viability (EC50 value – 18.6 μM). Dose-dependent activation of phospho-Akt and phospho-ERK signaling pathways via increased phosphoprotein expression was observed due to Cd⁺². Signaling activation of Akt and ERK was prevented by 5 mM N-Acetyl Cysteine (NAC), establishing the role of ROS as a key driver in the activation process. Importantly, we observed Cd⁺² also caused a dose dependent change in the COX-2 and PGE₂ expression levels. PI3K-Akt and NF-kB signaling pathways play a key role in Cd⁺² exposure induced COX-2 activation in the gallbladder epithelial cells. In conclusion, our study measures the toxicological effects of Cd⁺² exposures on human GB epithelial cells for the first time and establishes the role of Cd⁺² as a possible driver of the Akt/ERK pathway overactivity and chronic inflammation in gallbladder carcinogenesis.     

The toxic effects of indoor atmospheric fine particulate matter collected from allergic and non‐allergic families in Wuhan on mouse peritoneal macrophages

Recent studies have shown that fine particulate matter (PM_2.5) is associated with multiple adverse health outcomes and PM_2.5‐induced oxidative stress is now commonly known as a proposed mechanism of PM_2.5‐mediated toxicity. However, the association between allergic symptoms in children and exposure to PM_2.5 has not been fully elucidated, particularly the role of PM_2.5 on the indoor environment involved in allergy or non‐allergy is unknown. The aim of the present study was to explore whether indoor PM_2.5 from the homes of children with allergic symptoms had more increased risks of allergy than that of healthy ones and then compare the toxicity and inflammatory response of them. In this study, indoor PM_2.5 was collected from the homes of schoolchildren with allergic symptoms and those of healthy ones respectively, and components of PM_2.5 were analyzed. PM_2.5‐mediated oxidative damage and inflammatory response were further evaluated in mouse peritoneal macrophages based on its effects on the levels of reactive oxygen species accumulation, lipid peroxidation, DNA damage or cytokine production. It seems that oxidative stress may contribute to PM_2.5‐induced toxicity, and PM_2.5 from the allergic indoor environment produced more serious toxic effects and an inflammatory response on mouse peritoneal macrophages than that from a non‐allergic indoor environment. Copyright © 2015 John Wiley & Sons, Ltd.     

Household coal combustion,indoor air pollutants,and circulating immunologic/inflammatory markers in rural China

The study aim was to investigate whether household bituminous (“smoky”) coal use and personal exposure to combustion emissions were associated with immunologic/inflammatory marker levels. A cross-sectional study of healthy never-smoking women from rural Xuanwei and Fuyuan, China was conducted, which included 80 smoky coal and 14 anthracite (“smokeless”) coal users. Personal exposure to fine particulate matter (PM_2.5) and benzo[a]pyrene (BaP) was assessed using portable devices, while 67 circulating plasma immunologic/inflammatory markers were measured using multiplex bead-based assays. Multivariable linear regression models were employed to estimate associations between smoky coal versus smokeless coal use, indoor air pollutants, and immunologic/inflammatory markers. Six markers were altered among smoky coal users compared to smokeless coal, including significantly decreased interferon-inducible T-cell alpha chemoattractant (CXCL11/I-TAC), and increased serum amyloid P component (SAP). CXCL11/I-TAC was previously found to be reduced in workers exposed to high levels of diesel engine exhaust, which exhibits similar constituents as coal combustion emissions. Further, there was evidence that elevated PM_2.5 and BaP exposure was associated with significantly diminished levels of the serum amyloid A (SAA); however, the false discovery rates (FDRs) were >0.2 after accounting for multiple comparisons. Inflammatory processes may thus mediate the carcinogenic effects attributed to smoky coal emissions.     

Urban particulate matter activates Akt in human lung cells

The normally picturesque Cache Valley in northern Utah is frequently reported to have the worst particulate (PM) air pollution in the United States. Numerous epidemiological studies conducted elsewhere have associated PM exposure to a variety of cardiovascular diseases and early mortality. We have previously shown that Cache Valley PM (CVPM) is pro-inflammatory, through a variety of mechanisms involving the release of inflammatory cytokines, unfolded protein response, ER stress, and C-reactive protein (CRP). This study was undertaken to determine whether Cache Valley PM (CVPM) would activate Akt, an upstream mechanism common to these events. Human lung (BEAS-2B) cells were treated with either fine (PM_2.5) or coarse (PM₁₀) particles (12.5 and 25 μg/ml) for periods up to 24 h. PM-exposed cells exhibited Akt activation as evidenced by phosphorylation at Thr³⁰⁸ and Ser⁴⁷³. Events downstream of Akt activation such as NF-κB activation were observed at 1 and 24 h, but IκB phosphorylation occurred only at 24 h, indicating that mechanisms of PM-mediated NF-κB activation are time dependent. Akt and NF-κB related inflammatory cytokine IL-1α, and IL-6 and the chemokine IL-8 were upregulated in treated cells at 6 and 24 h. The calpain inhibitor leupeptin limited Akt phosphorylation to Ser⁴⁷³ and reduced release of IL-1α, IL-6, and IL-8, indicating that calpain or similar protease(s) are involved in PM-induced activation of Akt and subsequent release of inflammatory cytokines. Our data indicate that PM activates Akt, which may play a role in the pro-inflammatory response to PM exposure.     

Outdoor wood furnaces create significant indoor particulate pollution in neighboring homes

Abstract

Context: The use of outdoor wood furnaces (OWFs) is common in many parts of the United States. Little published information exists on the concentrations of outdoor and indoor fine particulates found near OWFs.

Objective: To compare PM_2.5 (cts) and PM_0.5 (cts) particle concentrations inside four Connecticut homes located 30.5–259?m from OWFs, and inside six Connecticut control homes located more than 2?km from the nearest OWF.

Materials and methods: PM_2.5 (cts) and PM_0.5 (cts) measurements were made with a Dylos light-scattering particulate counter.

Results: Mean PM_2.5 (cts) concentrations were 4.21 times as great in the four OWF exposed homes than the six control homes (0.302?×?10⁶ counts/m³ versus 0.0718 counts?×?10⁶/m³ p?<?0.001). The mean PM_2.5 (cts) concentrations inside the four OWF exposed homes roughly corresponds to a mass PM_2.5 of 37?µg/m³, which is above the US EPA 24-h PM_2.5 limit of 35?µg/m³. Mean PM_0.5 (cts) concentrations were 3.44 times as great in the four OWF exposed homes than in the six control homes (0.657 versus 0.191?×?10⁶/m³ p?<?0.001). Mean PM_2.5 (cts) and PM_0.5 (cts) concentrations were significantly higher in the house 259?m from an OWF as compared with the mean of the six control homes.

Conclusion: Existing regulations, such as the present Connecticut law requiring a 61 meter distance between an OWF and neighboring homes, are not adequate to protect the health of neighboring residents.     

PM10 impairs the antioxidant defense system and exacerbates oxidative stress driven cell death

The aim of this study was to investigate the effect of airborne particulate matter with a mean aerodynamic diameter of ≤10 μm (PM₁₀) on oxidative stress markers and antioxidant enzymatic activity and its relevance in the face of acute oxidative challenge in a human lung epithelial cell line (A549). PM₁₀-induced reactive oxygen species (ROS) generation and oxidative damage with no changes in cellular viability. In addition, PM₁₀ decreased glutathione (GSH) levels (54.9%) and the activity of the antioxidant enzymes superoxide dismutase (65%), catalase (31.2%), glutathione reductase (61.5%) and glutathione-S-transferase (42.39%). Trolox, a scavenger of reactive species, prevented the increase of ROS generation and the decrease in GSH levels but partially prevented PM₁₀-induced oxidative damage. Interestingly, it was unable to avoid the decrease in the activity of antioxidant enzymes. Finally, the survival of the cells previously exposed to PM₁₀ and challenged with hydrogen peroxide was significantly lower. We conclude that the impairment in the antioxidant defense system induced by PM₁₀ weaken ROS detoxification which exacerbates cell death when these cells are exposed to an acute oxidative challenge.     

Seasonal variation in the toxicological properties of size-segregated indoor and outdoor air particulate matter

Ambient air particulate matter (PM) as well as microbial contaminants in the indoor air are known to cause severe adverse health effects. It has been shown that there is a clear seasonal variation in the potency of outdoor air particles to evoke inflammation and cytotoxicity. However, the role of outdoor sources in the indoor air quality, especially on its toxicological properties, remains largely unknown. In this study, we collected size segregated (PM_10–2.5, PM_2.5–0.2 and PM_0.2) particulate samples with a high volume cascade impactor (HVCI) on polyurethane foam and fluoropore membrane filters. The samples were collected during four different seasons simultaneously from indoor and outdoor air. Thereafter, the samples were weighed and extracted with methanol from the filters before undergoing toxicological analyses. Mouse macrophages (RAW264.7) were exposed to particulate sample doses of 50, 150 and 300 μg/ml for 24 h. Thereafter, the levels of the proinflammatory cytokine (TNF-α), NO-production, cytotoxicity (MTT-test) and changes in the cell cycle (SubG₁, G₁, S and G₂/M phases) were investigated. PM_10–2.5 particles evoked the highest inflammatory and cytotoxic responses. Instead, PM_2.5–0.2 samples exerted the greatest effect on apoptotic activity in the macrophages. With respect to the outdoor air samples, particles collected during warm seasons had a stronger potency to induce inflammatory and cytotoxic responses, whereas no such clear effect was seen with the corresponding indoor air samples. Outdoor air samples were associated with higher inflammatory potential, whereas indoor air samples had overall higher cytotoxic properties. This indicates that the outdoor air has a limited influence on the indoor air quality in a modern house. Thus, the indoor sources dominate the toxicological responses obtained from samples collected inside house.     

Oxidative stress and apoptosis are induced in human endothelial cells exposed to urban particulate matter

Correlations between exposure to particle matter (PM) with an aerodynamic diameter ⩽ 2.5 or 10 μm (PM_2.5 and PM₁₀, respectively) with cardiovascular effects have been demonstrated recently. Endothelial cells seem to play a relevant role in the responses to PM due to their participation in pro-inflammatory events. In this study we determined the effect of PM_2.5 and PM₁₀ from Mexico City on human endothelial cells by means of evaluating reactive oxygen species (ROS), nitric oxide (NO), NF-κB translocation and cell death. For this purpose we used human umbilical vein endothelial cells (HUVEC) as a model.The production of ROS was determined by the reduction of H₂DCFDA and NO by Griess reagent. The translocation of NF-κB was evaluated by Electrophoretic Mobility Shift Assay (EMSA) and the cellular death by the translocation of phosphatidylserine. TNF-α was used as a positive control for endothelial cell activation.PM_2.5 and PM₁₀ induced the production of ROS (77% and 126% increase, respectively, vs. control) and NO (up to 132% and 233% increase, respectively, vs. control). PM_2.5 and PM₁₀ also induced the nuclear translocation of NF-κB. All these events were associated with apoptosis. In conclusion, the activation of HUVEC induced by PM_2.5 and PM₁₀ is related with an oxidative stress, suggesting that these particles may participate in the development of cardiovascular and inflammatory diseases.     

Ginsenoside Rg1 reduces toxicity of PM2.5 on human umbilical vein endothelial cells by upregulating intracellular antioxidative state

Ambient airborne particulate matter (PM) is an important environmental pollutant responsible for many human diseases. Oxidative stress is suggested to be involved in PM-induced cell injury. The present study is designed to study unsalutary effects of the organic extracts of PM with an aerodynamic diameter of less than 2.5 μm (PM_2.5) and protective effect of Ginsenoside Rg1 (Rg1) against PM_2.5 on human umbilical vein endothelial cells (HUVECs) in vitro. Cytotoxic effects of the organic extract PM_2.5 on HUVECs were measured by means of HUVEC cell viability and the generation of intracellular reactive oxygen species (ROS). Expression of heme oxygenase-1(HO-1) and Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Nrf2 cytoplasm–nucleus location were assayed. The present results showed that PM_2.5 (50–800 μg/ml) decreased HUVEC viability and increased intracellular generation of ROS and malondialdehyde (MDA) in a concentration dependent manner, but increased HO-1 expression without concentration dependence. Rg1 (10 and 40 μg/ml) diminished PM_2.5-induced HUVEC viability, decrease ROS and MDA generation, increased HO-1 and Nrf2 expression and promoted Nrf2 translocation to nucleus in a concentration dependent manner. These results suggested that organic extracts of PM_2.5 increase oxidative stress and decrease cell viability; Rg1 antagonize PM_2.5-induced excess oxidative stress; HO-1 expression increase and Nrf2 translocation to nucleus may be involved in the effects of both PM_2.5 and Rg1 on HUVECs.     

NADPH oxidase isoform selective regulation of endothelial cell proliferation and survival

Proliferation and apoptosis of endothelial cells are crucial angiogenic processes that contribute to carcinogenesis and tumor progression. Emerging evidence implicates the regulation of proliferation and apoptosis by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H₂O₂). In the present study, we investigated the roles of the ROS-generating Nox4- and Nox2-containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in proliferation of human endothelial cells by examining the impact of these enzyme systems on (1) specific proliferative and tumorigenic kinases, extracellular regulated kinase1/2 (ERK1/2) and Akt, (2) cytoskeletal organization, and (3) the mechanisms that influence cellular apoptosis. ROS production and the expression of NADPH oxidase subunit Nox4, but not Nox2, were markedly higher in proliferating than in quiescent endothelial cells. Addition of the H₂O₂ scavenger catalase or downregulation of Nox4 protein with specific siRNA reduced ROS levels, cell proliferation, and ERK1/2 phosphorylation but had no effect on either cell morphology or caspase 3/7 activity. Although downregulation of Nox2 protein with siRNA also reduced ROS production and cell proliferation, it caused an increase in caspase 3/7 activity, reduced Akt phosphorylation, and caused cytoskeletal disorganization. Therefore, in endothelial cells, Nox4-derived H₂O₂ activates ERK1/2 to promote proliferation, whereas Nox2-containing NADPH oxidase maintains the cytoskeleton and prevents apoptosis to support cell survival. Our study provides a new understanding of the molecular mechanisms that underpin endothelial cell survival and a rationale for the combined suppression of Nox4- and Nox2-containing NADPH oxidases for unwanted angiogenesis in cancer.     

PM2.5‐induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/NF‐κB‐dependent pathway

The aim of this study was to explore the intracellular mechanisms underlying the cardiovascular toxicity of air particulate matter (PM) with an aerodynamic diameter of less than 2.5 µm (PM_2.5) in a human umbilical vein cell line, EA.hy926. We found that PM_2.5 exposure triggered reactive oxygen species (ROS) generation, resulting in a significant decrease in cell viability. Data from Western blots showed that PM_2.5 induced phosphorylation of Jun N‐terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen‐activated protein kinase (MAPK) and protein kinase B (AKT), and activation of nuclear factor kappa B (NF‐κB). We further observed a significant increase in expressions of intercellular adhesion molecule‐1 (ICAM‐1) and vascular adhesion molecule‐1 (VCAM‐1) in a time‐ and dose‐dependent manner. Moreover, the adhesion of monocytic THP‐1 cells to EA.hy926 cells was greatly enhanced in the presence of PM_2.5. However, N‐acetylcysteine (NAC), a scavenger of ROS, prevented the increase of ROS generation, attenuated the phosphorylation of the above kinases, and decreased the NF‐κB activation as well as the expression of ICAM‐1 and VCAM‐1. Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and NF‐κB inhibitor (BAY11‐7082) significantly down‐regulated PM_2.5‐induced ICAM‐1 and VCAM‐1 expression as well as adhesion of THP‐1 cells, but not JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580), indicating that ERK/AKT/NF‐κB is involved in the signaling pathway that leads to PM_2.5‐induced ICAM‐1 and VCAM‐1 expression. These findings suggest PM_2.5‐induced ROS may function as signaling molecules triggering ICAM‐1 and VCAM‐1 expressions through activating the ERK/AKT/NF‐κB‐dependent pathway, and further promoting monocyte adhesion to endothelial cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Titanium dioxide nanoparticles increase plasma glucose via reactive oxygen species‐induced insulin resistance in mice

There have been few reports about the possible toxic effects of titanium dioxide (TiO₂) nanoparticles on the endocrine system. We explored the endocrine effects of oral administration to mice of anatase TiO₂ nanoparticles (0, 64 and 320 mg kg^–1 body weight per day to control, low‐dose and high‐dose groups, respectively, 7 days per week for 14 weeks). TiO₂ nanoparticles were characterized by scanning and transmission electron microscopy (TEM) and dynamic light scattering (DLS), and their physiological distribution was investigated by inductively coupled plasma. Biochemical analyzes included plasma glucose, insulin, heart blood triglycerides (TG), free fatty acid (FFA), low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), total cholesterol (TC), tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐6 and reactive oxygen species (ROS)‐related markers (total SOD, GSH and MDA). Phosphorylation of IRS1, Akt, JNK1, and p38 MAPK were analyzed by western blotting. Increased titanium levels were found in the liver, spleen, small intestine, kidney and pancreas. Biochemical analyzes showed that plasma glucose significantly increased whereas there was no difference in plasma insulin secretion. Increased ROS levels were found in serum and the liver, as evidenced by reduced total SOD activity and GSH level and increased MDA content. Western blotting showed that oral administration of TiO₂ nanoparticles induced insulin resistance (IR) in mouse liver, shown by increased phosphorylation of IRS1 (Ser307) and reduced phosphorylation of Akt (Ser473). The pathway by which TiO₂ nanoparticles increase ROS‐induced IR were included in the inflammatory response and phosphokinase, as shown by increased serum levels of TNF‐α and IL‐6 and increased phosphorylation of JNK1 and p38 MAPK in liver. These results show that oral administration of TiO₂ nanoparticles increases ROS, resulting in IR and increasing plasma glucose in mice. Copyright © 2015 John Wiley & Sons, Ltd.