Effects of chrysotile exposure in human bronchial epithelial cells: Insights into the pathogenic mechanisms of asbestos-related diseases

BACKGROUND: Chrysotile asbestos accounts for > 90% of the asbestos used worldwide, and exposure is associated with asbestosis (asbestos-related fibrosis) and other malignancies; however, the molecular mechanisms involved are not fully understood. A common pathogenic mechanism for these malignancies is represented by epithelial–mesenchymal transition (EMT), through which epithelial cells undergo a morphological transformation to assume a mesenchymal phenotype. In the present work, we propose that chrysotile asbestos induces EMT through a mechanism involving a signaling pathway mediated by tranforming growth factor beta (TGF-β). OBJECTIVES: We investigated the role of chrysotile asbestos in inducing EMT in order to elucidate the molecular mechanisms involved in this event. METHODS: Human bronchial epithelial cells (BEAS-2B) were incubated with 1 μg/cm2 chrysotile asbestos for ≤ 72 hr, and several markers of EMT were investigated. Experiments with specific inhibitors for TGF-β, glycogen synthase kinase–3β (GSK-3β), and Akt were performed to confirm their involvement in asbestos-induced EMT. Real-time polymerase chain reaction (PCR), Western blotting, and gelatin zymography were performed to detect mRNA and protein level changes for these markers. RESULTS: Chrysotile asbestos activated a TGF-β–mediated signaling pathway, implicating the contributions of Akt, GSK-3β, and SNAIL-1. The activation of this pathway in BEAS-2B cells was associated with a decrease in epithelial markers (E-cadherin and β-catenin) and an increase in mesenchymal markers (α-smooth muscle actin, vimentin, metalloproteinases, and fibronectin). CONCLUSIONS: Our findings suggest that chrysotile asbestos induces EMT, a common event in asbestos-related diseases, at least in part by eliciting the TGF-β–mediated Akt/GSK-3β/SNAIL-1 pathway. CITATION: Gulino GR, Polimeni M, Prato M, Gazzano E, Kopecka J, Colombatto S, Ghigo D, Aldieri E. 2016. Effects of chrysotile exposure in human bronchial epithelial cells: insights into the pathogenic mechanisms of asbestos-related diseases. Environ Health Perspect 124:776–784; http://dx.doi.org/10.1289/ehp.140962

cellular apoptosis mitochondrial function and confers resistance to The arginine metabolite agmatine protects

is dedicated to innovative approaches to the study of cell and molecular physiology. It is published 12 times AJP -Cell Physiolog

Plant extracts containing cinnamaldehyde, eugenol and capsicum oleoresin added to feedlot cattle diets: Ruminal environment, short term intake pattern and animal performance

The objective was to evaluate effects of adding a blend of essential oil compounds on ruminal fermentation and animal performance of feedlot cattle in comparison to sodium monensin. In Exp. 1, 24 angus steers (initial weight 141±6.6kg) were blocked by weight into 4 groups and randomly allocated to 8 pens of 3 steers. Treatments were monensin (46.7mg/kg dietary dry matter (DM)), or plant extracts (PE; 266mg/steer/d of cinnamaldehyde and eugenol+133mg/steer/d of capsicum oleoresin) added to a mineral mixture. The experiment lasted 84d and was divided in 2 periods of 0-44 and 45-84d. Diets were fed once daily and consisted of a corn grain based concentrate fed ad libitum, plus 200g alfalfa hay/steer/d as fed. The DM intake, average daily gain (ADG), feed conversion ratio (FCR) and rate of backfat deposition (BFD) were determined throughout the study. Short term intake patterns were evaluated by visual appraisal. In Exp. 2, two ruminally fistulated steers were used in a crossover design to determine how the ruminal fermentation variables pH, NH 3-N and volatile fatty acids (VFA) were affected by PE or monensin. Compared to monensin, PE did not alter overall DM intake (0.124kg/BW 0.75 versus 0.123kg/BW 0.75), FCR (0.21 versus 0.20), BFD (1.87mm/mo versus 1.76mm/mo), or longissimus dorsi muscle (LM) area (6.56cm 2/mo versus 6.69cm 2/mo) for PE and monensin, respectively. However, a treatment×period interaction occurred (P=0.02) for ADG, with steers fed PE having a higher (P=0.01) ADG in the second period (1.43kg/d versus 1.23kg/d for PE and monensin, respectively). Short term intake patterns were not altered by PE compared to monensin, as steers visited the feeders a similar number of times and the length of each visit was also similar (11.5min versus 10.6min and 8.28min versus 9.57min for PE and monensin, respectively). Although ruminal pH was not affected (5.55 versus 6.05 for PE and monensin, respectively), ruminal NH 3-N was lowered by PE (10.78mg/dl versus 20.05mg/dl, P=0.02). Ruminal total VFA concentrations did not differ between treatments (80.7mM versus 62.5mM), and feeding PE did not alter ruminal acetate (48.5mol/100mol versus 58.2mol/100mol), or propionate (32.8mol/100mol versus 25.2mol/100mol, P=0.65) proportions. Results show that steers fed PE performed equivalently to those fed monensin in a high concentrate diet, and that some productive variables were improved with PE feeding.Fil: Geraci, José I.. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Garciarena, Alberto D.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Gagliostro, Gerardo Antonio. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Beauchemin, Karen A.. Lethbridge Research Centre; CanadáFil: Colombatto, Dario. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Casein kinase 2 phosphorylates recombinant human spermidine/spermine N1-acetyltransferase on both serine and threonine residues

Casein kinase 2 purified from human erythrocyte cytosol has been found to phosphorylate human spermidine/spermine N1-acetyltransferase (SSAT) expressed as a fusion protein in E. coli and purified to homogeneity with a specific activity similar to that reported for pure human SSAT. The amino acid sequence of the protein revealed not less than four phosphorylable residues, optimal target for protein kinase 2 phosphorylation being flanked by acid residues in position +1 and +3. Our results indicate that most 32P-phosphate is taken up by Ser residues, as evidenced by HCl hydrolysis and electrophoresis and that the phosphorylation extent is modulated by the physiological polyamine concentration. Partial digestion with trypsin at a low concentration for less than one hour preferentially hydrolyzes Lys-Arg-Arg in position 141-143 of the SSAT suggesting that the Ser-phosphorylated residues are located in the C-terminus of the protein, probably Ser 146 and 149

Simple proxies of insulin resistance identify obese metabolic dysfunction-associated fatty liver disease subjects with advanced liver disease

Aims: Insulin resistance (IR) plays a pivotal role in the pathogenesis of Metabolic dysfunction-Associated Fatty Liver Disease (MAFLD), which can progress to liver fibrosis. We examined the relationship of different IR scores with markers of MAFLD severity in obese individuals. Materials and methods: In this retrospective observational study, 346 non-diabetic, overweight/obese individuals with newly diagnosed MAFLD (age 50.2 ± 13.3 years, 34% females, BMI 30.8 ± 4.4 kg/m2 ) underwent liver stiffness (LS) and controlled attenuation parameter (CAP) measurements by Fibroscan® to assess liver fibrosis and steatosis. Biochemical data were collected to calculate surrogate markers of IR (Homoeostasis model assessment - insulin resistance index [HOMA-IR], triglyceride-glucose index, triglyceride by HDL ratio), liver fibrosis (Nonalcoholic Fatty Liver Diseases fibrosis score, fibrosis-4 score, Aspartate aminotransferase to platelet ratio index) and steatosis (fatty liver index, hepatic steatosis index). Results: All three IR scores were associated with CAP, while only HOMA-IR positively correlated with LS (r = 0.275, p < 0.0001), independent of age and sex, BMI, transaminases, and fibrosis markers. Insulin-resistant individuals (HOMA-IR >2.5, n = 165) had higher liver enzymes, CAP and LS, with a 4-fold increased risk of severe liver disease (LS >9.7 kPa, OR 4.42[1.95-10.01], p = 0.0002). Among HOMA-IR components, fasting plasma insulin (FPI) was independently associated with LS (r = 0.270, p < 0.0001). ROC AUC for HOMA-IR and FPI to predict severe liver disease were virtually identical (0.748 and 0.758, respectively). Conclusions: HOMA-IR is independently associated with non-invasive markers of MAFLD severity in overweight/obese individuals. This relationship is largely mediated by hyperinsulinemia, regardless of BMI. Measuring insulin levels in MAFLD individuals might be useful to identify those at risk of liver fibrosis

Inhibition of agmatine transport in liver mitochondria by new charge-deficient agmatine analogues.

The charge of the agmatine analogues AO-Agm [N-(3-aminooxypropyl)guanidine], GAPA [N-(3-aminopropoxy)guanidine] and NGPG [N-(3-guanidinopropoxy)guanidine] is deficient as compared with that of agmatine and they are thus able to inhibit agmatine transport in liver mitochondria. The presence of the guanidine group is essential for an optimal effect, since AO-Agm and NGPG display competitive inhibition, whereas that of GAPA is non-competitive. NGPG is the most effective inhibitor (K(i)=0.86 mM). The sequence in the inhibitory efficacy is not directly dependent on the degree of protonation of the molecules; in fact NGPG has almost the same charge as GAPA. When the importance of the guanidine group for agmatine uptake is taken into account, this observation suggests that the agmatine transporter is a single-binding, centre-gated pore rather than a channel

Phosphorylation of recombinant human spermidine spermine N-1-Acetyltransferase by CK1 and modulation of its binding to mitochondria: A comparison with CK2

Abstract: Cytosolic spermidine/spermine acetyltransferase (SSAT) catalyzes the acetylation of the N-l-propylamino groups of spermine and spermidine. The enzyme has a very short half-life and is rapidly induced by various stimuli. Once acetylated, these polyamines are subjected to the action of polyamine oxidase, which, besides initiating polyamine catabolism, may produce reactive oxygen species that in turn trigger modifications in subcellular compartments such as mitochondria. The present work evaluates the ability of the cAMP-independent Ser/ Thr-protein kinase CK1 to phosphorylate SSAT. Results demonstrate that SSAT is phosphorylated by CK1, in sites distinct from those phosphorylated by CK2. Moreover, both phosphorylation processes are involved in the uptake of SSAT into rat liver mitochondria. Although CK2 is less effective than CK1 in phosphorylating SSAT, CK2 phosphorylation is much more powerful in preventing binding of SSAT to mitochondrial structures. These results suggest the involvement of CK1- and CK2-mediated SSAT phosphorylation in regulating the contents of polyamines and SSAT itself within subcellular compartments and implicate SSAT and polyamines as indirect modulators of progression through the cell Cycle

Agmatine is transported into liver mitochondria by a specific electrophoretic mechanism

Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism the driving force of which is ΔΨ (electrical membrane potential). Although this process showed strict electrophoretic behaviour, qualitatively similar to that of polyamines, agmatine is most probably transported by a specific uniporter. Shared transport with polyamines by means of their transporter is excluded, as divalent putrescine and cadaverine are ineffective in inhibiting agmatine uptake. Indeed, the use of the electroneutral transporter of basic amino acids can also be discarded as ornithine, arginine and lysine are completely ineffective at inducing the inhibition of agmatine uptake. The involvement of the monoamine transporter or the existence of a leak pathway are also unlikely. Flux-voltage analysis and the determination of activation enthalpy, which is dependent upon the valence of agmatine, are consistent with the hypothesis that the mitochondrial agmatine transporter is a channel or a single-binding centre-gated pore. The transport of agmatine was non-competitively inhibited by propargylamines, in particular clorgilyne, that are known to be inhibitors of MAO (monoamine oxidase). However, agmatine is normally transported in mitoplasts, thus excluding the involvement of MAO in this process. The I(2) imidazoline receptor, which binds agmatine to the mitochondrial membrane, can also be excluded as a possible transporter since its inhibitor, idazoxan, was ineffective at inducing the inhibition of agmatine uptake. Scatchard analysis of membrane binding revealed two types of binding site, S(1) and S(2), both with mono-co-ordination, and exhibiting high-capacity and low-affinity binding for agmatine compared with polyamines. Agmatine transport in liver mitochondria may be of physiological importance as an indirect regulatory system of cytochrome c oxidase activity and as an inducer mechanism of mitochondrial-mediated apoptosis

Steatosis/steatohepatitis: how sustainable is the non-invasive instrumental differential diagnosis in clinical practice?

Aim: Simple, rapid, and non-invasive methods for the early diagnosis of non-alcoholic steatohepatitis (NASH) in patients with fatty liver are an unmet need in clinical practice. Transient elastography (TE), commonly used for measuring liver stiffness (LS), which is significantly influenced by both liver fibrosis and inflammation is a promising tool.Methods: We studied retrospectively the impact of TE in a cohort of 98 consecutive asymptomatic patients with fatty liver who underwent a liver biopsy [21 non-alcoholic fatty liver (NAFL) and 77 NASH] and TE on the same day at the Hepatology Unit of University Hospital of Pisa. Patients positive for HBsAg, anti-HCV, HIV, autoantibodies, drug-induced liver disease, Wilson’s disease, hemochromatosis, alpha-1 antitrypsin deficiency, type 2 diabetes, or neoplasia were excluded.Results: NAFL patients were younger (42.5 years vs. 47.7 years, P = 0.02) and with lower BMI (25.5 kg/m2vs. 28.8 kg/m2, P < 0.001) than NASH patients. TE was higher in NASH than NAFL patients (8.1 kPa vs. 5.4 kPa, P = 0.01). Age, BMI, TE, and total/LDL cholesterol were statistically significantly different between NAFL and NASH patients, but with multivariate analysis only BMI (P = 0.009) and TE (P = 0.031) were independent predictors of NAFL/NASH with AUROCs of 0.771 and 0.754, respectively. A score combining TE and BMI (TE*BMI) showed the best AUROC (0.817, by De Long test, P = 0.01) to differentiate NAFL/NASH (P = 0.005).Conclusion: Ultrasound based LS measure qualifies as a candidate tool for the early screening of NASH in fatty liver patients provided that its measure is properly standardized and tested in large prospective studies enrolling patients with different clinical and histological features