Mechanism of miR-21 targeting Smad7 in pulmonary fibrosis of A549 cells induced by beryllium sulfate / 环境与职业医学

Background The pathogenesis of beryllium-induced pulmonary fibrosis is unknown and there is no specific treatment for the disease as yet. MicroRNA (miRNA) may play a role in the process of beryllium-induced pulmonary fibrosis. Objective To construct a microRNA-21 (miR-21) interfering cell line, and to investigate the effect of miR-21 on beryllium sulfate (BeSO4)-induced fibrosis in human lung adenocarcinoma alveolar basal epithelial cells (A549 cells) and its potential mechanism. Methods The miR-21 target genes were predicted by the online database miRBase and verified by experiments using dual luciferase reporter gene. After transfecting A549 with miR-21interference lentivirus, puromycin was used to select a stable cell line. An in vitro model of pulmonary fibrosis was established using BeSO4 infecting A549 cells with a concentration of 10 μmol·L−1 and an exposure time of 48 h. Then the treated cells were divided into control group, model group, miR-21 interference group, and miR-21 interference control group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the relative expression level of miR-21 gene. Western blotting was used to detect the relative expression levels of TGF-β1/Smads pathway related proteins [Smad2, Smad3, p-Smad2, p-Smad3, Smad7, and transforming growth factor-β1 (TGF-β1)], myofibrosis cell marker α-smooth muscle actin (α-SMA), andextracellular matrix collagen-I (COL-I) and collagen-Ⅲ (COL-Ⅲ). Results The miRBase predicted that miR-21 had a binding site with Smad7, and the results of the dual luciferase reporter gene experiment showed that the target gene of miR-21 was Smad7. The construction of miR-21 interfered with A549 cell line was successful. Compared with the control group, the relative expression of miR-21 gene in the model group increased by 97.57%; the relative expression of Smad7 protein in the model group decreased by 15.48%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ increased by 13.55%, 35.72%, 18.35%, 35.75%, 25.52%, 31.58%, 24.61%, and 11.66% respectively (P<0.05). Compared with the interference control group, the miR-21 gene expression level in the interference group decreased by 28.96%; the relative expression of Smad7 protein increased by 19.07%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ decreased by 8.01%, 19.95%, 14.56%, 19.37%, 11.95%, 10.96%, 18.81%, and 31.36% repectively (P<0.05). There was no statistically significant difference in the gene abd protein expression levels of each gene between the model group and the interference control group (P>0.05). Conclusion In an in vitro model of pulmonary fibrosis induced by beryllium compounds, miR-21 may promote fibrosis by targeting Smad7 to regulate the TGF-β1/Smad signaling pathway.

Elastic scattering of 9Be+80Se and the breakup threshold anomaly / Dispersión elástica de 9Be+80Se y la anomalía de umbral de ruptura

Abstract We study the threshold anomaly displayed in the energy dependence of optical potentials describing elastic-scattering angular distributions of weakly bound nuclei with three different models: a) a semi-microscopic double folding potential with two free parameters, b) a phenomenological Wood-Saxon potential with six free parameters and c) an exponential potential with two free parameters. In this kind of nuclei, the coupling to the breakup channel is reflected as an increasing of the imaginary part of the potential as the bombarding energy decreases below the Coulomb barrier, while the real part of the potential (linked to the imaginary part by the dispersion relation) decreases in strength. This behavior is called breakup threshold anomaly (BTA). In this work, we apply this analysis to experimental data for the elastic scattering in the 9Be+80Se systemat eleven bombarding energies around the Coulomb barrier, obtained at the 20 MV tandem accelerator of the TANDAR Laboratory at Buenos Aires. Using the covariance-matrix method to estimate the uncertainties, the presence of the BTA in this system is unambiguously determined, independently of the model chosen for the nuclear potential.

Resumen Estudiamos la anomalía de umbral presente en la dependencia con la energía de potenciales ópticos que describen la dispersión elástica de núcleos débilmente ligados, usando tres tipos de potenciales ópticos para la descripción de las distribuciones angulares: a) un potencial semimicroscópico doblemente plegado con dos parámetros libres, b) un potencial fenomenológico tipo Wood-Saxon con seis parámetros libres y c) un potencial exponencial con dos parámetros libres. En este tipo de núcleos, el acoplamiento al canal de ruptura se refleja como un aumento de la parte imaginaria del potencial al decrecer la energía de bombardeo por debajo de la barrera de Coulomb, mientras que la parte real (vinculada a la parte imaginaria por la relación de dispersión) disminuye en intensidad. Este comportamiento se denomina anomalía de umbral de ruptura (BTA). En el presente trabajo, se aplica este análisis a datos experimentales concernientes a la dispersión elástica en el sistema 9Be + 80Se a 11 energías de bombardeo alrededor de la barrera de Coulomb, obtenidos con el acelerador de 20MV del Laboratorio TANDAR en Buenos Aires. Se utilizó el método de matriz de covarianza para estimar las incertidumbres, se determina inequívocamente la presencia de la BTA en este sistema, independientemente del modelo elegido para el potencial nuclear.

Effect of titanium powder on the bond strength of metal heat treatment

PURPOSE: Ni-Cr alloy does not contain Beryllium, causing the metal compound to form oxides in the furnace but by using Titanium as a chemical catalyst the forming of the oxides can be controlled, and by controlling the impurities formed on the metal surface, the possibility of the Ni-Cr alloy bond strength being increased can be analysed. MATERIALS AND METHODS: Titanium was used as a chemical catalyst in the porcelain for the oxidation of beryllium-free metal (Ni-Cr) alloy. The T1 group, which does not use Titanium power as a chemical catalyst is a reference model for comparison. The T2 group and T3 group used 10 g and 20 g of Titanium power, respectively. They are fabricated to observe the shear bond strength and surface properties. There was no significance when One-way ANOVA analysis/Tukey Honestly Significant Difference Test was conducted for statistical analysis among groups (P > 0.05). RESULTS: Results of measuring the three-point flexural bond strength of the Ni-Cr alloy and thickness of the oxide film. Experiment T3 using 20 g Titanium chemical catalyst: 39.22 ± 3.41 MPa and 6.66 µm, having the highest bond strength and thinness of oxide film. Experiment T2 using 10 g Titanium chemical catalyst: 34.65 ± 1.39 MPa and 13.22 µm. Experiment T1 using no Titanium chemical catalyst: 32.37 ± 1.91 MPa and 22.22 µm. CONCLUSION: The T2 and T3 experiments using Titanium chemical catalyst showed higher bond strength for the Ni-Cr alloy and lower thickness of oxide film than experiment T1, and the titanium catalyst being able to increase bond strength was observed.

Mechanism of beryllium sulfate-induced apoptosis in human embryonic lung fibroblast / 中国职业医学

OBJECTIVE: To investigate the effect of beryllium sulfate( BeSO_4) on apoptosis of human embryonic lung fibroblast( MRC-5 cell). METHODS: MRC-5 cells were cultured in vitro and randomly divided into 6 groups: a control group,a low-,medium- and high-dose BeSO_4 group,an antagonist group,and an activator group. The former 4 groups were given final concentrations of 0,1,10 and 100 μmol / L of BeSO_4,respectively. The combined treatment of BeSO_4and2-aminoethoxydiphenyl borate( 10 μmol / L final concentration) was used in the antagonist group. The combined treatment of BeSO_4 and inositol triphosphate( IP3)( 10 μmol / L final concentration) was used in the activator group. After 24 and48 hours of culture,the cells were harvested. The apoptosis of MRC-5 cells was detected by flow cytometry. The intracellular calcium ion( Ca~(2+)) was detected using laser scanning confocal microscope. Quantitative real-time polymerase chain reaction was used to detect the relative expression of IP_3RⅢ and B-cell lymphoma-2( BCL-2) mRNA and the protein expression of IP_3RⅢ and IP3 were detected by enzyme-linked immunosorbent assay. RESULTS: The apoptosis rates of cells in the 3 BeSO_4 dose groups at the time points of 24 and 48 hours were lower than those in the control group at the same time points( P < 0. 05). The apoptosis rate of the antagonist group was lower than those in medium-dose BeSO_4 group and control group at the same time points( P < 0. 05). At the time point of 48 hours,the apoptosis rate of the activator group was lower than that of control group( P < 0. 05) and higher than that of the medium-dose BeSO_4group( P < 0. 05). As for the Ca~(2+)concentration at time point of 24 hours,the low-dose BeSO_4 group was lower than the control group( P < 0. 05),and the high-dose BeSO_4 group was higher than the control group( P < 0. 05). The Ca~(2+)concentrations at time point of 48 hours in the medium- and high-dose BeSO_4 groups were lower than that in the control group( P < 0. 05). Compared with the medium-dose BeSO_4 group and control group at time points of 24 and 48 hours,the Ca~(2+)concentrations in the antagonist group decreased( P < 0. 05),while thoes of the activator group increased( P < 0. 05). The expression of BCL-2and IP_3RⅢmRNA in the 3 BeSO_4 groups,the activator and antagonist group were higher than those of the control group( P <0. 05). The expression of IP3 R Ⅲ protein at the time point of 24 hours in the medium-dose BeSO_4 group,the activator group and the antagonist group were lower than that of control group( P < 0. 05). The expression of IP_3RⅢ protein at the time point of 48 hours,the high-dose BeSO_4 group was lower than the control group( P < 0. 05); the activator and antagonist groups were higher than the medium-dose BeSO_4group( P < 0. 05). The expression of IP3 protein in the lowand medium-dose BeSO_4 groups and the activator group were higher than that in the control group( P < 0. 05). The expression of IP3 protein in activator group was higher than the medium-dose group( P < 0. 05). CONCLUSION: BeSO_4 might change the Ca~(2+)concentration and inhibite the apoptosis of MRC-5 cell through regulating the IP3 R / Ca~(2+)pathway,IP3 can improve the decrease of Ca~(2+)concentration in MRC-5 cells induced by BeSO_4.

Effects of DNA methylation of thymocyte differentiation antigen-1 in beryllium sulfate induced human fetal lung fibroblast fibrosis / 中国职业医学

OBJECTIVE: The change of DNA methylation of thymocyte differentiation antigen-1( Thy-1) was observed in beryllium sulfate( Be SO4) stimulated human fetal lung fibroblast( MRC-5 cell) to explore the effects of Thy-1 in Be SO4 induced lung fibrosis. METHODS: MRC-5 cell culture in vitro model was used. The final concentrations of Be SO4were1. 0,10. 0 and 100. 0 μmol / L( low-,medium- and high-dose groups). The control was untreated. Other 2 intervention groups were the 5-azacytidine( AZC) intervention group( 10. 0 μmol / L of AZC and 10. 0 μmol / L Be SO4) and the trichostatin A( TSA) intervention group( 0. 5 μmol / L of TSA and 10. 0 μmol / L Be SO4). The cells were collected 24,48 and 72 hours after exposure. Real-time quantitative polymerase chain reaction( PCR) was used to determine the relative expression of collagen typeⅠ( Col Ⅰ),collagen type Ⅲ( Col Ⅲ),α-smooth muscle actin( α-SMA) and Thy-1 mRNA.The nested landed methylation specific PCR was used to detect the Thy-1 DNA methylation level. RESULTS: At 24 hours,the relative expression level of Col Ⅲ mRNA in MRC-5 cells showed an increasing trend with increasing dose( P < 0. 05);at 48 and 72 hours,the relative expression levels of Col Ⅰ,Col Ⅲ and α-SMA mRNA in MRC-5 cells increased with the increasing dose( P < 0. 05). All these 3 indicators in MRC-5 cells of 3 dose groups increased with the increase of expose time( P < 0. 05). The relative expression level of Thy-1 mRNA in MRC-5 cells of all 3 dose groups were lower than that in control( P < 0. 05). The relative expression level of Thy-1 mRNA of the high-dose group was lower than that of the lowdose group( P < 0. 05). The Thy-1 DNA methylation levels in the medium- and high-dose groups were both higher than that of the control( P < 0. 05). The Thy-1 DNA methylation levels of the 3 dose groups increased with the increasing dose( P < 0. 05). The Thy-1 DNA methylation levels of MRC-5 cells in the 2 intervention groups were higher than that of the control( P < 0. 05),but there was no significant difference when compared with the medium-dose group( P > 0. 05).CONCLUSION: Be SO4 stimulation can induce the fibrosis of MRC-5 cells. In this process,the Thy-1 DNA methylation level increases,while the Thy-1 mRNA expression level decrease. Thy-1 DNA methylation might be one of the important mechanisms of lung fibrosis induced by Be SO4.

Effects of Beryllium on Human Serum Immunoglobulin and Lymphocyte Subpopulation

To investigate the effects of short-term exposure of beryllium on the human immune system, the proportion of T-lymphocytes such as CD3+, CD4+, CD8+, CD95, and NK cells, andthe proportion of B cells and TNFalpha level in peripheral blood and immunoglobulins in the serum of 43 exposed workers and 34 healthy control subjects were studied. External exposure to beryllium was measured by atomic absorption spectrometer as recommended by the NIOSH analytical method 7300. T lymphocyte subpopulation analysis was carried out with flow cytometer. The working duration of exposed workers was less than 3 months and the mean ambient beryllium level was 3.4 microg/m3, 112.3 microg/m3, and 2.3 microg/m3 in molding (furnace), deforming (grinding), and sorting processes, respectively (cited from Kim et al., 2008). However, ambient beryllium level after process change was non-detectable (< 0.1 microg/m3). The number of T lymphocytes and the amount of immunoglobulins in the beryllium-exposed workers and control subjects were not significantly different, except for the total number of lymphocytes and CD95 (APO1/FAS). The total number of lymphocytes was higher in the beryllium-exposed individuals than in the healthy control subjects. Multiple logistic regression analysis showed lymphocytes to be affected by beryllium exposure (odd ratio = 7.293; p < 0.001). These results show that short-term exposure to beryllium does not induce immune dysfunction but is probably associated with lymphocytes proliferation.

Measurements of the fallout flux of beryllium-7 and its variability in the soil

The aim of this study was to examine the beryllium-7 behavior in the soil. Natural variability of beryllium-7 concentration was calculated to be about 23 percent (relative standard deviation), and the depth distribution could be approximated by an exponential decay in bare soil, with an average penetration depth in the soil about 1 cm. The nuclide was not found below 2 cm depth, which confirmed its utilization to infer the erosion processes as a tracer of soil surface. The maximum beryllium-7 concentration in the analyzed period was about 40 Bq.kg-1.

Berílio-7 é um radionuclídeo cosmogênico, com meia-vida de 53 dias, produzido pelo processo de espalação de átomos de oxigênio e nitrogênio dentro da troposfera e estratosfera. Após sua produção, este é transportado até a superfície terrestre pela deposição úmida e seca. A precipitação seca contribui somente com 3-8 por cento do inventário total. Medidas de berílio-7 no solo podem serem usadas para indicar movimento de solo da camada superficial e este estudo objetiva examinar o comportamento de berílio-7 no solo. Variabilidade natural do inventário de berílio-7 é em torno de 23 por cento (desvio padrão relativo). A distribuição em profundidade de berílio-7 pode ser aproximada por uma função exponencial no solo nu, com uma profundidade média de distribuição no solo em torno de 1 cm. O berílio-7 não foi encontrado abaixo da profundidade de 2 cm para o tipo de solo estudado, o que confirma sua utilização para avaliar processo de erosão superficial como um traçador de solo superficial. A concentração máxima de berílio-7 no período analisado é em torno de 40 Bq.kg-1.

An Outbreak of Acute Beryllium Disease in a Compound Metal Alloy Factory / 대한산업의학회지

OBJECTIVES: We conducted an epidemiological survey to inquire into an outbreak of acute pneumonitis after two reported cases of interstitial lung disease. METHODS: The study subjects were 45 workers from a compound metal alloy factory. We reviewed the factory's industrial hygiene data along with the results of a special health examination, including pulmonary function tests, simple chest X-rays, and high resolution computed tomography. RESULTS: The air concentrations of beryllium ranged from 0.42 microgram/m3 to 112.3 microgram/m3, and the mean concentration of urinary beryllium were 1.53+/-0.79 microgram/g of creatinine in the molding workers, 1.41+/- 0.50 microgram/g of creatinine in the casting workers, and 1.16+/-0.53 microgram/g of creatinine in the sorting workers. The rates for cough (p=0.054), dyspnea (p=0.030), and the use of medical services (p=0.018) were higher in the molding workers than in the non-molding workers. The incidence rate of acute interstitial lung disease was higher for the molding process (32.0%) than for the non-molding process (5.0%) (p=0.012). The time of employment for all patients was prior to December 1st, 2002. CONCLUSIONS: Since most of the patients were molding workers, and all of the patients had worked without a ventilation system, this outbreak of acute interstitial lung disease was regarded as acute beryllium disease. Although the direct cause of the epidemic was the beryllium fumes, the fundamental cause was improper control of the work environment. Therefore, the means for preventing avoidable epidemics of occupational diseases are discussed.

An Outbreak of Acute Beryllium Disease in a Compound Metal Alloy Factory / 대한산업의학회지

OBJECTIVES: We conducted an epidemiological survey to inquire into an outbreak of acute pneumonitis after two reported cases of interstitial lung disease. METHODS: The study subjects were 45 workers from a compound metal alloy factory. We reviewed the factory's industrial hygiene data along with the results of a special health examination, including pulmonary function tests, simple chest X-rays, and high resolution computed tomography. RESULTS: The air concentrations of beryllium ranged from 0.42 microgram/m3 to 112.3 microgram/m3, and the mean concentration of urinary beryllium were 1.53+/-0.79 microgram/g of creatinine in the molding workers, 1.41+/- 0.50 microgram/g of creatinine in the casting workers, and 1.16+/-0.53 microgram/g of creatinine in the sorting workers. The rates for cough (p=0.054), dyspnea (p=0.030), and the use of medical services (p=0.018) were higher in the molding workers than in the non-molding workers. The incidence rate of acute interstitial lung disease was higher for the molding process (32.0%) than for the non-molding process (5.0%) (p=0.012). The time of employment for all patients was prior to December 1st, 2002. CONCLUSIONS: Since most of the patients were molding workers, and all of the patients had worked without a ventilation system, this outbreak of acute interstitial lung disease was regarded as acute beryllium disease. Although the direct cause of the epidemic was the beryllium fumes, the fundamental cause was improper control of the work environment. Therefore, the means for preventing avoidable epidemics of occupational diseases are discussed.

Immunotoxicology of beryllium lung disease

Beryllium induces non-caseating granulomatous inflammation in humans exposed to the metal dust or fumes in both occupational and non-occupational settings. The resulting condition, chronic beryllium disease (CBD), affects principally the lungs, lymphatics, and skin and continues to plague modern industry. Beryllium exerts several important immunotoxic effects, including induction of a beryllium-antigen specific adaptive immune response and the triggering of inflammatory and innate immune responses. Genetic susceptibility plays a role in CBD adaptive immune responses, mainly mediated through single nucleotide polymorphisms in HLA-DP and, to a lesser extent, HLA-DR. The adaptive response is characterized by influx and proliferation of CD4+ central and effector memory T cells expressing Th1 cytokines. Insights into the immunopathogenesis of CBD have implications for the understanding of other immune-mediated granulomatous disorders and for metal antigen behavior.

Immunotoxicology of Beryllium Lung Disease

Beryllium induces non-caseating granulomatous inflammation in humans exposed to the metal dust or fumes in both occupational and non-occupational settings. The resulting condition, chronic beryllium disease (CBD), affects principally the lungs, lymphatics, and skin and continues to plague modern industry. Beryllium exerts several important immunotoxic effects, including induction of a beryllium-antigen specific adaptive immune response and the triggering of inflammatory and innate immune responses. Genetic susceptibility plays a role in CBD adaptive immune responses, mainly mediated through single nucleotide polymorphisms in HLA-DP and, to a lesser extent, HLA-DR. The adaptive response is characterized by influx and proliferation of CD4+ central and effector memory T cells expressing Th1 cytokines. Insights into the immunopathogenesis of CBD have implications for the understanding of other immune-mediated granulomatous disorders and for metal antigen behavior.