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Skin repair requires the activation of keratinocytes and is mediated by controlled inflammation and cell migration and proliferation, ending with the regeneration of well-differentiated cell layers. Whey derivatives contain galactooligosaccharides (GOS), which have potential beneficial effects on wound healing due to their activity as toll-like receptor ligands, although their direct nonprebiotic effects in the skin have not yet been described. In this study, we investigated the effects of different whey-derived products and purified GOS on a human keratinocyte cell line. We found that the inflammatory cytokine interleukin-8 (IL-8) was upregulated by nuclear factor kappa B (NF-kB) signaling triggered by whey derivatives and GOS and that wound healing was accelerated by promoting cell migration and the loss of E-cadherin in the absence of epithelial–mesenchymal transition. Interestingly, the treatments enhanced the mitochondrial function in association with the translocation of the Forkhead Box O1 (FOXO-1) transcription factor. Finally, we detected the increased expression of the differentiation markers induced by GOS and whey derivatives. All together, our results show that GOS-containing products can promote wound closure and skin health by direct activity on keratinocyte functions. Among the preparations tested, the fermented compound produced by autochthonous microorganisms was the most active in modulating keratinocyte activity, supporting the biological value of whey derivatives for health.

In our recent studies, we have developed a thermodynamic biochemical model able to select the resonant frequency of an extremely low frequency electromagnetic field (ELF-EMF) specifically affecting different types of cancer, and we have demonstrated its effects in vitro. In this work, we investigate the cellular response to the ELF electromagnetic wave in three-dimensional (3D) culture models, which mimic the features of tumors in vivo. Cell membrane was modelled as a resistor–capacitor circuit and the specific thermal resonant frequency was calculated and tested on two-dimensional (2D) and three-dimensional (3D) cell cultures of human pancreatic cancer, glioblastoma and breast cancer. Cell proliferation and the transcription of respiratory chain and adenosine triphosphate synthase subunits, as well as uncoupling proteins, were assessed. For the first time, we demonstrate that an ELF-EMF hampers growth and potentiates both the coupled and uncoupled respiration of all analyzed models. Interestingly, the metabolic shift was evident even in the 3D aggregates, making this approach particularly valuable and promising for future application in vivo, in aggressive cancer tissues characterized by resistance to treatments.

Vitamin D has been described as a differentiative hormone, but this definition is reductive for a molecule targeting every tissue, produced in its active form by many kinds of cells and effective on the whole life of cells by different mechanisms, which lead to nuclear, non-genomic and mitochondrial effects [...]

Vitamin D performs a differentiating, metabolic and anti-inflammatory function, through genomic, non-genomic and mitochondrial mechanisms of action [...]

Study question Can morphokinetic features included into Machine Learning (ML) algorithms identify cleavage-stage embryos with the best chance to reach the expanded blastocyst stage on day 5? Summary answer A ML algorithm based on early morphokinetic features can identify cleaving embryos that will reach the expanded blastocyst stage on day 5. What is known already To date, the conventional morphology assessment of cleaving human embryos has a limited predictive power on further embryo developmental potential. The morphokinetic analysis using Time-Lapse systems (TLS) was introduced in order to provide a new tool to identify dynamic biomarkers of embryo quality. More recently, ML approach has been applied for the analysis of specific embryo-related features, aiming at developing predictive algorithms to assess the embryo development potential. Study design, size, duration We retrospectively analysed 575 embryos obtained from 80 women aged 25–42 years, with normal BMI, AFC≥8, day 3 FSH2.5 ng/ml, no diagnosis of polycystic ovary syndrome or endometriosis. These patients underwent IVF at our IVF Unit between March 2018 and March 2020; their embryos were cultured using the Geri plus® TLS and a single blastocyst transfer was performed. Participants/materials, setting, methods A total number of 29 morphological and morphokinetic parameters were considered to build six different ML algorithms. The performance to assess which was the best-fitting algorithm was calculated using the ROC curve considering accuracy (% of embryos correctly classified by the algorithm), Cohen-kappa coefficient (measurement of the agreement among features), mean number of TP (embryos correctly classified as undergoing developmental arrest), mean number of TN (embryos uncorrectly classified as undergoing developmental arrest). Main results and the role of chance Overall, 210 embryos progressed to the expanded blastocyst stage on day 5 (BL group), whereas 365 displayed developmental delay or arrest at any stage (nBL group). Among the six different algorithms, the best-fitting algorithm was obtained using the Kbest features selection approach combined with a Random Forrest evaluation strategy. This algorithm was based on 7 variables: embryo morphological score on day 2, pronuclear fading time (tPNf), completion time of cleavage to two, four and eight cells (t2, t4, and t8 respectively), time intervals t4-t3 and t8-t4. The algorithm showed an AUC of 0.78, with an accuracy of 0.73, a Cohen-kappa of 0.41, a mean TP number of 302/365 embryos in the nBL group and a mean TN number of 120/210 embryos in the BL group. Mean false positive (FP) and false negative (FN) numbers were of 63 and 90.2, respectively. Limitations, reasons for caution The results obtained in this study may not be generalizable to patients with other clinical characteristics, to other time-lapse systems or different laboratory settings. The predictive power of the algorithm should be validated prospectively on a larger number of embryos. Wider implications of the findings: The current study represents a preliminary analysis for the development of hierarchical predictive models for embryo assessment based on their developmental potential, that embryologists will be able to apply as a support for decision-making. Trial registration number Not applicable

Purpose: The aim of this study was to assess whether the intraoperative use of the cold eye irrigation balanced salt solution (BSS) could have a protective effect in preventing the anterior chamber flare and conjunctival hyperemia and, thus, in reducing patients discomfort after phacoemulsification. Materials and methods: About 214 patients were enrolled and randomly divided into: patients whose eye were irrigated with BSS at ~ 20°C (Group 1) and patients whose eye were irrigated with BSS at 2.7°C (Group 2). Anterior chamber flare, visual analogue score and conjunctival hyperemia were evaluated at 1, 3, 5, and 30 days after surgery. Results: In patients of Group 2 the anterior chamber flare, the visual analogue score and the conjunctival hyperemia, used as parameters to evaluated clinical inflammation, at 1 day after surgery were significantly lower than those in Group 1 who received BSS solution at operating room temperature ( p Conclusion: Our study provided evidence supporting the efficacy of the treatment with cold irrigation solution on reduction of anterior chamber flare, pain and conjunctival hyperemia already at 1 day after phacoemulsification, suggesting that cooling procedure was fully effective at controlling early post-operative inflammation.

Simple Summary Malignant pleural mesothelioma is a lung tumor associated with asbestos exposure, with a poor prognosis, and a difficult pharmacological approach. Asbestos exposure is very toxic for the lungs, which counteract this toxic effect by activating some antioxidant defense proteins. When these proteins are more active that in normal conditions, as in several cancers, these tumors become able to survive and resist to stress or chemotherapy. In our laboratory, we collected cellular samples of mesothelioma and non-transformed mesothelium from Hospital’s Biobank and we evaluated these proteins. Our results demonstrated these proteins are upregulated in mesothelioma cells and not in non-transformed mesothelium. This event could be associated to toxic effects evoked by asbestos exposure, highlighting the need in the future to monitor asbestos-exposed people by measuring biomarkers identified, in the attempt to identify them as possible predictive markers and potential pharmacological targets addressed to improve mesothelioma prognosis. Abstract Although asbestos has been banned in most countries around the world, malignant pleural mesothelioma (MPM) is a current problem. MPM is an aggressive tumor with a poor prognosis, so it is crucial to identify new markers in the preventive field. Asbestos exposure induces oxidative stress and its carcinogenesis has been linked to a strong oxidative damage, event counteracted by antioxidant systems at the pulmonary level. The present study has been focused on some redox-sensitive transcription factors that regulate cellular antioxidant defense and are overexpressed in many tumors, such as Nrf2 (Nuclear factor erythroid 2-related factor 2), Ref-1 (Redox effector factor 1), and FOXM1 (Forkhead box protein M1). The research was performed in human mesothelial and MPM cells. Our results have clearly demonstrated an overexpression of Nrf2, Ref-1, and FOXM1 in mesothelioma towards mesothelium, and a consequent activation of downstream genes controlled by these factors, which in turn regulates antioxidant defense. This event is mediated by oxidative free radicals produced when mesothelial cells are exposed to asbestos fibers. We observed an increased expression of Nrf2, Ref-1, and FOXM1 towards untreated cells, confirming asbestos as the mediator of oxidative stress evoked at the mesothelium level. These factors can therefore be considered predictive biomarkers of MPM and potential pharmacological targets in the treatment of this aggressive cancer.

The segmentation of the in vitro fertilization (IVF) cycle, consisting of the freezing of all embryos and the postponement of embryo transfer (ET), has become popular in recent years, with the main purpose of preventing ovarian hyperstimulation syndrome (OHSS) in patients with high response to controlled ovarian stimulation (COS). Indeed cycle segmentation (CS), especially when coupled to a GnRH-agonist trigger, was shown to reduce the incidence of OHSS in high-risk patients. However, CS increases the economic costs and the work amount for IVF laboratories. An alternative strategy is to perform a fresh ET in association with intensive luteal phase pharmacological support, able to overcome the negative effects of the GnRH-agonist trigger on the luteal phase and on endometrial receptivity. In order to compare these two strategies, we performed a retrospective, real-life cohort study including 240 non-polycystic ovarian syndrome (PCO) women with expected high responsiveness to COS (AMH &gt, 2.5 ng/mL), who received either fresh ET plus 100 IU daily human chorionic gonadotropin (hCG) as luteal support (FRESH group, n = 133), or cycle segmentation with freezing of all embryos and postponed ET (CS group, n = 107). The primary outcomes were: implantation rate (IR), live birth rate (LBR) after the first ET, and incidence of OHSS. Overall, significantly higher IR and LBR were observed in the CS group than in the FRESH group (42.9% vs. 27.8%, p &lt, 0.05 and 32.7% vs. 19.5%, p &lt, 0.05, respectively), the superiority of CS strategy was particularly evident when 16–19 oocytes were retrieved (LBR 42.2% vs. 9.5%, p = 0.01). Mild OHSS appeared with the same incidence in the two groups, whereas moderate and severe OHSS forms were observed only in the FRESH group (1.5% and 0.8%, respectively). In conclusion, in non-PCO women, high responders submitted to COS with the GnRH-antagonist protocol and GnRH-agonist trigger, CS strategy was associated with higher IR and LBR than the strategy including fresh ET followed by luteal phase support with a low daily hCG dose. CS appears to be advisable, especially when &gt, 15 oocytes are retrieved.

Alessandro Meduri,1 Giovanni William Oliverio,1 Loredana Bergandi,2 Gabriella De Salvo,3 Rino Frisina,4 Cosimo Mazzotta,5,6 Pasquale Aragona1 1Biomedical, Dental and Morphological and Functional Images Sciences Department, University of Messina, Messina, Italy; 2Department of Oncology, University of Torino, Turin, Italy; 3Eye Unit, University Hospital Southampton Foundation Trust, Southampton, UK; 4Department of Neurosciences - Ophthalmology, University of Padova, Padua, Italy; 5Departmental Ophthalmology Unit, Alta Val d’Elsa Hospital, Siena, Italy; 6Siena Crosslinking Center, Siena, ItalyCorrespondence: Loredana BergandiDepartment of Oncology, University of Torino, Via Santena 5 Bis, Turin, 10126, ItalyTel +34 0116705843Fax +34 0116705840Email loredana.bergandi@unito.itPurpose: To evaluate the postoperative effect on central retinal macular thickness of a cooled irrigating eye solution used during cataract surgery.Patients and Methods: In this prospective, single-center study, 100 eyes of 50 patients (26 males and 24 females) were evaluated with spectral domain optical coherence tomography (SD-OCT) before and after phacoemulsification for senile cataract. Eyes were randomly divided into two groups based on the irrigating solution used during surgery: Group 1, 50 eyes received intraoperative irrigating solution at room temperature (∼ 20.0± 0.1°C); and Group 2, 50 fellow eyes received cold intraoperative irrigating solution (2.7± 0.1°C). Changes in central macular thickness (CMT) were evaluated in both groups by SD-OCT macular raster scan for the nine Early Treatment of Diabetic Retinopathy Study (ETDRS) subfields and total macular volume, performed pre-surgery, and 1 and 4 weeks post-surgery.Results: Despite there being no significant differences in variables between the two groups preoperatively, significant increases in CMT were observed at 1 week after surgery in both groups (p=0.02 and p=0.03, respectively), as well as in total macular volume (p< 0.0001 and p=0.02, respectively). Inter-subgroup analysis showed a significant reduction in CMT (p=0.03) and total macular volume (p=0.001) at 1 week post-surgery in Group 2 compared to Group 1, whereas no significant differences were observed at 4 weeks.Conclusion: The use of a cooled irrigating eye solution during phacoemulsification may be beneficial in preventing the possible development of postoperative macular thickening. Further clinical studies may support this finding.Keywords: cataract surgery, cystoid macular edema, central macular thickness, spectral domain optical coherence tomography, phacoemulsification, cold intraoperative irrigating solution

Vitamin D and beta-glucans are both immunostimulants. Vitamin D exerts its beneficial effects on many components of the immune system. In macrophages, the hormone modulates both phagocytic activity and cytokine production, therefore, it plays an important role in mediating the innate immune response to infection. The immunomodulatory properties of beta-glucans are attributed to the ability of these fungal cell wall polysaccharides to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes and macrophages. The intracellular signaling pathways activated by beta-glucans lead to enhanced phagocytosis and cytokine response. In this study we investigated the possible potentiation of immunomodulatory properties of the combined treatment with vitamin D and beta-glucans. The effects of 100 nM 1,25-dihydroxyvitamin D3 or 100 µg/mL beta-glucans were evaluated in human macrophages in terms of cytokine production, intracellular vesicle acidification and changes in energy metabolism, three hallmarks of macrophage antimicrobial activation. We found that all the analyzed parameters were enhanced by the co-treatment compared to the response to single molecules. The results of this study support the validity of a novel therapeutic approach that could boost the immune response, taking advantage of the synergy between two natural compounds.

Vitamin D and TGF-β exert opposite effects on epithelial-mesenchymal EMT transition. Here we report a novel mechanism of action of TGF-β that promotes the counteracting activity of vitamin D; in two models of human epithelial-mesenchymal EMT transition we demonstrated for the first time that TGF-β strongly induced the expression of vitamin D receptor (VDR) and that 1,25(OH)2D3 was able to contrast the TGF-β-driven EMT transition by transcriptional modulation. In human bronchial epithelial cells the effects of TGF-β on EMT transition markers (E-Cadherin expression and cell motility) were reversed by pre-treatment and co-treatment with 1,25(OH)2D3, but not when the hormone was given later. Silencing experiments demonstrated that the inhibition of TGF-β activity was VDR-dependent. 1,25(OH)2D3 abrogated the mitochondrial stimulation triggered by TGF-β. In fact we showed that 1,25(OH)2D3 repressed the transcriptional induction of respiratory complex, limited the enhanced mitochondrial membrane potential and restrained the increased levels of mitochondrial ATP; 1,25(OH)2D3 also decreased the production of reactive oxygen species promoted by TGF-β. Overall, our study suggests that the overexpression and activity of VDR may be a regulatory response to TGF-β signaling that could be exploited in clinical protocols, unraveling the therapeutic potentiality of 1,25(OH)2D3 in the prevention of cancer metastasis.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

The fight against a multifaceted incurable disease such as cancer requires a multidisciplinary approach to overcome the multitude of molecular defects at its origin. Here, a new thermophysical biochemical approach has been suggested and associated with the use of electromagnetic fields to control the growth of cancer cells. In particular, thermodynamic analysis of the heat transfer is developed in correlation with cellular parameters such as the volume/area ratio. We propose that the electromagnetic wave, at the specific frequency calculated as the characteristic response time of any cell type to the external thermal perturbation, can affect resonant intracellular molecular oscillations. The biochemical model hypothesizes that microtubules are stabilized, and the impact is predicted on cell growth, migration and mitochondrial activity. Experimental validation of the theoretical results shows that the thermodynamic analysis allows the application of the specific electromagnetic field able to decrease cancer cell invasion and proliferation.

The efficacy of the very low frequency electromagnetic field in cancer treatment remains elusive due to a lack of explanatory mechanisms for its effect. We developed a novel thermodynamic model that calculates for every cell type the frequency capable of inhibiting proliferation. When this frequency was applied to two human cancer cell lines, it reduced their growth while not affecting healthy cells. The effect was abolished by the inhibition of calcium fluxes. We found evidences of an enhanced respiratory activity due to the increased expression of the elements of the respiratory chain and oxidative phosphorylation, both at the mRNA and protein level. The respiratory burst potentiated the production of reactive oxygen species but was not associated to increased levels of ATP, leading to the conclusion that the energy was readily spent in the adaptive response to the electromagnetic field. Taken together, our data demonstrate that, regardless of individual molecular defects, it is possible to control cancer cells with a specific irradiation that imposes a mitochondrial metabolic switch, regulating calcium fluxes and deleterious to cancer growth. This approach lays the foundations for a personalized cancer medicine.

Breast cancer patients under 40 years of age who are candidate to chemotherapy with alkylating drugs may undergo controlled ovarian stimulation (COS) with recombinant human follicle-stimulating hormone (rhFSH) in order to get fertility preservation by mature oocyte cryostorage. The direct effect(s) of exogenous rhFSH on the chemosensitivity of breast cancer is currently unknown. To clarify this issue, we incubated four different breast cancer cell lines with rhFSH (10 IU/L, 24 h) and then we exposed them to doxorubicin (DOX) or cyclophosphamide (CPA). The effect(s) of rhFSH on human breast cancer cells treated with DOX or CPA was measured in terms of (1) cell viability, (2) cytotoxicity, (3) multidrug resistance (MDR) genes and proteins expression and activities, and (4) hypoxia-inducible factor 1-alpha (HIF-1α) activation. Pretreatment with rhFSH significantly increased the viability of breast cancer cells after treatment with DOX or CPA, and reduced the lactate dehydrogenase leakage and reactive oxygen species production. Moreover, after preincubation with rhFSH, the MDR proteins (Pgp, MPR1, and BCRP) expression and activity resulted upregulated and the HIF-1α pathway activated. In addition, the use of a widely used HIF-1α inhibitor, the 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), prevented the rhFSH effect on the onset of MDR. Taken together, these observations suggest that a short exposure to rhFSH induces chemoresistance to DOX and CPA in human breast cancer cells via HIF-1α activation.

Introduction Cardiovascular diseases are the leading cause of mortality worldwide. Apical periodontitis (AP) has been associated with an increased risk of cardiovascular diseases. A correlation has been shown between chronic AP and endothelial dysfunction (ED), but there is no evidence to indicate ED improves after endodontic treatment in patients with periapical lesions. The aim of this study was to investigate vascular and molecular markers of early ED before and after root canal treatment in young adults with chronic AP. Methods Twenty control subjects and 21 patients with AP were assessed at baseline. The AP patients were also evaluated 2 and 12 months post-treatment. Endothelial flow reserve was assessed via an endothelial function test, and enzyme-linked immunosorbent assays were used to evaluate plasma levels of proinflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor alpha; vasoconstrictor ED marker endothelin (ET)-1; circulating endothelial adhesion markers intercellular adhesion molecule 1 (ICAM-1)/CD54 and soluble vascular cellular adhesion molecule (sVCAM)-1/CD106; soluble CD14; and the endothelial leukocyte adhesion molecule (E-selectin). Results AP was associated with increased serum levels of ET-1, ICAM-1, E-selectin, IL-1, and sCD14, suggesting early vascular ED, with no macroscopic evidence of a reduction in endothelial flow reserve. Root canal treatment ameliorated inflammation and early ED, lowering plasma levels of IL-1, sCD14, ET-1, ICAM-1/CD54, and E-selectin to those of control subjects. Conclusions Our findings suggest that AP may drive early vascular ED and that the endodontic therapy of AP ameliorates early ED.

Diabetes and cancer are common, chronic, and potentially fatal diseases that frequently co-exist. Observational studies clearly indicate that the risk of several types of cancer is increased in diabetic patients and a number of cancer types have shown a higher mortality rate in patients with hyperglycemic associated pathologies. This scenario could be due, at least in part, to a lower efficacy of the cancer treatments which needs to be better investigated. Here, we evaluated the effects of a prolonged exposure to high glucose (HG) to the response to chemotherapy on human colon adenocarcinoma HT29 and LOVO cell lines. We observed that hyperglycemia protected against the decreased cell viability and cytotoxicity and preserved from the mitochondrial DNA lesions induced by doxorubicin (DOX) and 5-fluorouracil (5-FU) treatments by lowering ROS production. In HT29 cells the amount of intracellular DOX and its nuclear localization were not modified by HG incubation in terms of Pgp, BCRP, MRP1, 5 and 8 activity and gene expression. On the contrary, in LOVO cells, the amount of intracellular DOX was significantly decreased after a bolus of DOX in HG condition and the expression and activity of MPR1 was increased, suggesting that HG promotes drug chemoresistance in both HT29 and LOVO cells, but in a different way. In both cell types, HG condition prevented the susceptibility to apoptosis by decreasing the ratio Bax/Bcl-2 and Bax/Bcl-XL and diminished the level of cytosolic cytochrome c and the cleavage of full length of PARP induced by DOX and 5-FU. Finally, hyperglycemia reduced cell death by decreasing the cell percentage in sub-G1 peak induced by DOX (via a cell cycle arrest in the G2/M phase) and 5-FU (via a cell cycle arrest in the S phase) in HT29 and LOVO cells. Taken together, our data showed that a prolonged exposure to HG protects human colon adenocarcinoma cells from the cytotoxic effects of two widely used chemotherapeutic drugs, impairing the effectiveness of the chemotherapy itself.

AIM: To evaluate whether the Q-switched Nd:YAG laser treatment applied in routine capsulotomy elicits oxidative stress in aqueous and vitreous humors. METHODS: Thirty-six patients who had to undergo a 25 gauge pars plana vitrectomy due to vitreoretinal disorders were enrolled, 15 of them underwent a Q-switched Nd:YAG laser capsulotomy 7d before vitrectomy due to posterior capsule opacification (PCO) (Nd:YAG laser group) while the remaining 21 patients were not laser treated before vitrectomy (no Nd:YAG laser group). Samples of the aqueous and vitreous humors were collected during vitrectomy from all patients for the assessment of oxidative parameters which were compared between the Nd:YAG laser group and no Nd:YAG laser group. Thiobarbituric acid reactive substances (TBARS), a product of membrane lipid peroxidation, nitrite levels, the antioxidative activities of SOD and catalase, the 4-HNE-protein conjugate formation, indicating structural modifications in proteins due to lipoperoxidation, were assessed in aqueous and vitreous samples. RESULTS: In the human vitreous humor TBARS levels are significantly higher in the Nd:YAG laser group compared to the no Nd:YAG laser group and importantly, there is a significant correlation between the TBARS levels and the total energy of Nd:YAG laser used during capsulotomy. Moreover the anti-oxidative activities of SOD and catalase were significantly decreased by Nd:YAG laser treatment, both in aqueous and vitreous humors. In accordance with the TBARS data and anti-oxidative enzyme activities, significantly higher levels of proteins were conjugated with the lipoperoxidation product 4-HNE in the aqueous and vitreous humors in the Nd:YAG laser-treated group in comparison to no Nd:YAG laser group. CONCLUSION: These data, clearly suggest that any change that Q-switched Nd:YAG photo disruption may cause in the aqueous and vitreous compartments, resulting in a higher level of oxidative damage might be of considerable clinical significance particularly by accelerating the aging of the anterior and posterior segments of the eye and by worsening the intraocular pressure, the uveal, the retinal (especially macular) pathologies.

A glass belonging to the system SiO2-P2O5-CaO-MgO-Na2O-K2O was modified by introducing two different amounts of manganese oxide (MnO). Mn-doped glasses were prepared by melt and quenching technique and characterized by means of X-ray diffraction (XRD), scanning electron microscopy (SEM) observation and energy dispersion spectrometry (EDS) analysis. In vitro bioactivity test in simulated body fluid (SBF) showed a slight decrease in the reactivity kinetics of Mn-doped glasses compared to the glass used as control; however the glasses maintained a good degree of bioactivity. Mn-leaching test in SBF and minimum essential medium (MEM) revealed fluctuating trends probably due to a re-precipitation of Mn compounds during the bioactivity process. Cellular tests showed that all the Mn-doped glasses, up to a concentration of 50 μg/cm(2) (μg of glass powders/cm(2) of cell monolayer), did not produce cytotoxic effects on human MG-63 osteoblasts cultured for up to 5 days. Finally, biocompatibility tests demonstrated a good osteoblast proliferation and spreading on Mn-doped glasses and most of all that the Mn-doping can promote the expression of alkaline phosphatase (ALP) and some bone morphogenetic proteins (BMPs).

The purpose of this study was to evaluate the early inflammatory response following osseous resective surgery (ORS) with Piezosurgery compared to commonly used diamond burs. A sample was selected of 24 bilateral posterior sextants requiring ORS in 12 chronic periodontitis patients in a split-mouth design. In 12 sextants, bone recontouring was performed using a piezoelectric device. In the contralateral sextants, rotary instruments were used. Sextants treated with Piezosurgery obtained similar 12-month clinical results but lower postsurgical gene expression of interleukin-1β (IL-1β), a well-known proinflammatory cytokine, and lower patient morbidity compared with sextants treated with rotary instruments. In spite of the longer surgical time, the use of Piezosurgery for ORS seems to promote more favorable wound healing compared with rotary instruments, as the lower pain and the low levels of IL-1β mRNA at the surgical sites suggest a milder underlying inflammatory response.

The metabolic actions of the ghrelin gene-derived peptide obestatin are still unclear. We investigated obestatin effects in vitro, on adipocyte function, and in vivo, on insulin resistance and inflammation in mice fed a high-fat diet (HFD). Obestatin effects on apoptosis, differentiation, lipolysis, and glucose uptake were determined in vitro in mouse 3T3-L1 and in human subcutaneous (hSC) and omental (hOM) adipocytes. In vivo, the influence of obestatin on glucose metabolism was assessed in mice fed an HFD for 8 wk. 3T3-L1, hSC, and hOM preadipocytes and adipocytes secreted obestatin and showed specific binding for the hormone. Obestatin prevented apoptosis in 3T3-L1 preadipocytes by increasing phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 signaling. In both mice and human adipocytes, obestatin inhibited isoproterenol-induced lipolysis, promoted AMP-activated protein kinase phosphorylation, induced adiponectin, and reduced leptin secretion. Obestatin also enhanced glucose uptake in either the absence or presence of insulin, promoted GLUT4 translocation, and increased Akt phosphorylation and sirtuin 1 (SIRT1) protein expression. Inhibition of SIRT1 by small interfering RNA reduced obestatin-induced glucose uptake. In HFD-fed mice, obestatin reduced insulin resistance, increased insulin secretion from pancreatic islets, and reduced adipocyte apoptosis and inflammation in metabolic tissues. These results provide evidence of a novel role for obestatin in adipocyte function and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

Asbestos exposure increases the risk of asbestosis and malignant mesothelioma (MM). Both fibrosis and cancer have been correlated with the Epithelial to Mesenchymal Transition (EMT)&mdash, an event involved in fibrotic development and cancer progression. During EMT, epithelial cells acquire a mesenchymal phenotype by modulating some proteins. Different factors can induce EMT, but Transforming Growth Factor &beta, (TGF-&beta, ) plays a crucial role in promoting EMT. In this work, we verified if EMT could be associated with MM development. We explored EMT in human mesothelial cells (MeT-5A) exposed to chrysotile asbestos: we demonstrated that asbestos induces EMT in MeT-5A cells by downregulating epithelial markers E-cadherin, &beta, catenin, and occludin, and contemporarily, by upregulating mesenchymal markers fibronectin, &alpha, SMA, and vimentin, thus promoting EMT. In these cells, this mechanism is mediated by increased TGF-&beta, secretion, which in turn downregulates E-cadherin and increases fibronectin. These events are reverted in the presence of TGF-&beta, antibody, via a Small Mother Against Decapentaplegic (SMAD)-dependent pathway and its downstream effectors, such as Zinc finger protein SNAI1 (SNAIL-1), Twist-related protein (Twist), and Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), which downregulate the E-cadherin gene. Since SNAIL-1, Twist, and ZEB-1 have been shown to be overexpressed in MM, these genes could be considered possible predictive or diagnostic markers of MM development.

Emerging evidence supports the idea that a dysfunction in cell metabolism could sustain a resistant phenotype in cancer cells. As the success of chemotherapeutic agents is often questioned by the occurrence of multidrug resistance (MDR), a multiple cross-resistance towards different anti-cancer drugs represent a major obstacle to cancer treatment. The present study has clarified the involvement of the carbon metabolites in a more aggressive tumor colon adenocarcinoma phenotype and in a chemoresistant mesothelioma, and the role of pyruvate treatment in the reversion of the potentially related resistance. For the first time, we have shown that human colon adenocarcinoma cells (HT29) and its chemoresistant counterpart (HT29-dx) displayed different carbon metabolism: HT29-dx cells had a higher glucose consumption compared to HT29 cells, whereas human malignant mesothelioma (HMM) cells showed a lower glucose consumption compared to HT29 cells, accompanied by a lower pyruvate production and, consequently, a higher production of lactate. When treated with pyruvate, both HT29-dx and HMM cells exhibited a re-established accumulation of doxorubicin and a lower survival ability, a decreased activity of multidrug resistance protein 1 (MRP1) and a restored mitochondrial respiratory chain function, improving the effectiveness of the chemotherapeutic agents in these resistant cancer cells.

The behavior of fluoride ions in the human organism is a classic example of double-edged sword. On the one hand the daily supplementation with fluoride is undoubtedly an important preventing factor in protecting teeth from caries, and, as an important mitogenic stimulus for osteoblasts, it may enhance mineral deposition in bone, but on the other hand fluoride, above a threshold concentration, has been demonstrated to be toxic. We present here a brief review of fluoride metabolism and exposure, its use in caries prevention and its effects on bone, followed by an updating about the main hypotheses concerning its mechanism of action and toxicity. The effects of fluoride have been related mainly to its ability to evoke the activation of G proteins and the inhibition of phosphotyrosine phosphatases, leading to an intracellular increase of tyrosine phosphorylation and activation of the mitogen-activated protein kinase pathway, and its capacity to cause generation of reactive oxygen species. We present also a unifying hypothesis accounting for these apparently different effects, although the available experimental models and conditions are highly variable in the literature. A lot of experiments still need to be performed to clarify the positive and negative effects of fluoride. Finding the mechanisms accounting for fluoride toxicity is an important point: indeed, the use of fluoride has been proposed in the preparation of new biomaterials to be inserted in the bone, in order to improve their stable and safe integration.

Vitamin D receptor (VDR) mediates many genomic and non-genomic effects of vitamin D. Recently, the mitochondrial effects of vitamin D have been characterized in many cell types. In this article, we investigated the importance of VDR not only in mitochondrial activity and integrity but also in cell health. The silencing of the receptor in different healthy, non-transformed, and cancer cells initially decreased cell growth and modulated the cell cycle. We demonstrated that, in silenced cells, the increased respiratory activity was associated with elevated reactive oxygen species (ROS) production. In the long run, the absence of the receptor caused impairment of mitochondrial integrity and, finally, cell death. Our data reveal that VDR plays a central role in protecting cells from excessive respiration and production of ROS that leads to cell damage. Because we confirmed our observations in different models of both normal and cancer cells, we conclude that VDR is essential for the health of human tissues.

The interplay between oocyte and surrounding cumulus cells (CCs) during follicular growth influences oocyte competence to undergo fertilization and sustain embryo development. The expression of many genes and proteins in CCs has been suggested as potential biomarker of oocyte competence in human in vitro fertilization (IVF). In the present study, we analyzed 90 human cumulus-oocyte complexes obtained during IVF procedure: 30 CCs were analyzed using quantitative real-time polymerase chain reaction and 60 CCs using Western blotting analysis to detect gene and protein expression of some enzymes related to oxidative stress, that is, the 3 nitric oxide synthase (NOS) isoforms and heme oxygenase 1 (HO-1). In the group of 60 CCs, we also investigated the expression and phosphorylation of IkBα, a known inhibitor of the nuclear factor κB (NF-κB) pathway, which controls several redox-sensitive genes. The expression of the messenger RNAs (mRNAs) was related to the oocyte morphological analysis performed by polarized light microscopy and to the occurrence of normal fertilization after intracytoplasmic sperm injection. We observed that the amount of iNOS and HO-1 mRNAs and proteins is significantly higher, and that in the meanwhile the NF-κB pathway is activated, in CCs corresponding to oocytes that were not fertilized in comparison to CCs whose corresponding oocyte showed normal fertilization. Instead, no correlation between the fertilization and the oocytes' morphological data was observed. These results suggest that the increase in iNOS and HO-1 mRNAs expression in CCs is a negative index of oocyte fertilizability and might be an useful tool for oocyte selection.

A series of Sr-substituted hydroxyapatites (HA), of general formula Ca(10-x)Srx(PO4)6(OH)2, where x=2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl2, in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in HA has a positive effect on MG-63 cells. In contrast, Sr ions alone, at the concentrations released by Sr-HA (1.21-3.24 ppm), influenced neither cell proliferation nor differentiation. Thus the positive effects of Sr in Sr-HA materials are probably due to the co-action of other ions such as Ca and P.

Introduction There is evidence to suggest that an association exists between oral infections and coronary heart disease (CHD). Subjects presenting lesions of endodontic origin (LEOs) or pulpal inflammation had an increased risk of developing CHD. However, findings concerning systemic manifestations of apical periodontitis (AP) remain controversial. An association between CD14 gene polymorphisms and atherosclerosis-associated diseases has been shown, but there are no data regarding an association between CD14 polymorphism and AP. This study evaluated associations between clinical oral health status, CD14 polymorphisms, and CHD. Methods A case-controlled clinical trial was designed to compare middle-aged adults with acute myocardial infarction or unstable angina (n = 51) within 12 months of the acute event defined as first manifestation with healthy controls (n = 49). Participants were matched for age, sex, and socioeconomic status. Indicators of oral disease and compliance were evaluated. CD14 polymorphisms were analyzed by restriction fragment length polymorphism–polymerase chain reaction. Results CHD subjects had a higher prevalence of oral diseases and lower compliance to oral preventive strategies than healthy controls. Multivariate analysis showed a positive association between missing teeth (odds ratio [OR] = 1.37; 95% confidence interval [CI], 1.02–1.85), the number of LEOs (OR = 4.37; 95% CI, 1.69–11.28), chronic periodontitis (OR = 5.87; 95% CI, 1.17–29.4), and CHD. No statistically significant association emerged between the CD14 C(−260)T and the CD14 C(−159)T polymorphism, endodontic or periodontal disease, and CHD. Conclusions Chronic oral diseases may increase the risk of CHD and may be an unconventional risk factor for CHD.

Some soda-lime-phospho-silicate glasses, such as Hench's Bioglass(®) 45S5, form bone-like apatite on their surface when bound to living bone. To improve their osteointegration for clinical purposes, the fluoride insertion in their structure has been proposed, but we recently showed that fluoride causes oxidative damage in human MG-63 osteoblasts, via inhibition of pentose phosphate oxidative pathway (PPP) and its key enzyme glucose 6-phosphate dehydrogenase (G6PD). In the same cells we have now investigated the role of nitric oxide (NO) in these effects. Fluoride-containing bioactive glasses and NaF caused, as expected, release of lactate dehydrogenase in the extracellular medium, accumulation of intracellular malonyldialdehyde, inhibition of PPP and G6PD: we have now observed that these effects were significantly reverted not only by superoxide dismutase (SOD) plus catalase (scavengers of reactive oxygen species), but also by N-monomethyl l-arginine (l-NMMA, a NOS inhibitor) and 2-phenyl-4,4,5,5,-tetramethylimidazoline-1oxyl 3-oxide (PTIO, a NO scavenger). Moreover the two highest concentrations of both fluoride-containing bioglasses and NaF caused increase of nitrite (a stable derivative of NO) levels in the culture supernatant, which was inhibited by l-NMMA, erythrocytes, PTIO and SOD/catalase, and increase of intracellular NO synthase (NOS) activity. The incubation with bioglasses or NaF increased also the phosphorylation of Ser(1177) in the endothelial NOS isoform. Furthermore, the NO donor spermine NONOate was able to inhibit G6PD activity in vitro, and this effect was partly reverted by PTIO. Therefore our results suggest that most cytotoxic effects of fluoride are mediated by the production of NO: reactive oxygen species are important, causing NOS phosphorylation. We also observed, for the first time, that Tempol, but not SOD/catalase, besides inhibiting the oxidative stress induced by fluoride, also scavenges fluoride ions. For this reason it is not a selective inhibitor of the oxidative effects of fluoride.

Phosphate-based glasses with different amounts of titanium dioxide (TiO2), having the following molar composition 50P(2)O(5)-30CaO-9Na(2)O-3SiO(2)-3MgO-(5-x) K2O-xTiO(2), (where x = 0, 2.5, 5 mol %), were synthesised and characterized in terms of solubility (according to ISO 10993-14), and in vitro biocompatibility using human MG-63 osteoblast cells. Dissolution tests were carried out in Tris-HCl (pH 7.4) to simulate the physiological pH and in citric acid (pH 3.0) to simulate an acidic environment. The weight loss decreased with increasing TiO2 content, a process further enhanced in acidic medium. TiO2 reduced the pH changes usually caused by the dissolution products released. Cellular tests showed that all the glasses studied (0-5 mol % TiO2) and TiCl4, used to investigate the biocompatibility of titanium ions, did not produce cytotoxic effects on human MG-63 osteoblasts for up to 5 days in culture. On the basis of these results, we suggest that TiO2-containing phosphate glasses could be promising substrates for bone tissue engineering applications. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 99A: 295-306, 2011.

This study was aimed at evaluating whether the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is altered in platelets from patients with an acute coronary syndrome (unstable angina and acute myocardial infarction). We investigated 10 patients with unstable angina (UA), 14 with acute myocardial infarction (AMI) and 14 age and sex-matched healthy subjects. The serum markers of platelet activation (sP-selectin), inflammation (TNF-alpha and erythrocyte sedimentation rate), thrombotic state (fibrinogen) and plaque disruption were significantly higher in both UA and AMI patients compared to the healthy controls. In their platelets we assessed the cGMP levels in basal conditions and after stimulation with sodium nitroprusside (SNP), and performed Western blot analysis of homogenates to measure the expression of soluble guanylate cyclase isoforms. Basal levels of cGMP (pmol/10(10) platelets) were significantly higher in platelets from UA patients (1,097 +/- 111; p0.0001) and AMI (1,122 +/- 77; p0.0001) compared to those collected from healthy controls (497 +/- 80). The platelets of AMI patients exhibited a lack of cGMP increase after SNP stimulation in comparison with UA patients. The phosphorylation of upstream (Akt1 protein kinase alpha and endothelial NO synthase) and downstream (vasodilator-stimulated phosphoprotein, VASP) signaling proteins of the NO/cGMP pathway was investigated: serine phosphorylation in Akt1, eNOS and VASP was enhanced in platelets from UA and AMI patients when compared to controls. Furthermore, in AMI patients the inhibitors of guanylate cyclase and cGMP-dependent protein kinase did not revert the VASP phosphorylation. These data suggest that platelets from AMI patients are more resistant to SNP stimulation, not only as cGMP production, but also in terms of VASP activation. From these ex vivo results we hypothesize that the increased inflammatory state which often accompanies patients with cardiovascular diseases might promote a platelet preactivation resulting in their reduced sensitivity to NO.

Genetic polymorphisms in the interleukin-1 gene cluster have been associated with the severity of periodontal diseases featured by a variable degree of destruction of connective tissue and bone, such as periodontitis and periimplantitis. This study was aimed to investigate if a link exists between such interleukin-1 gene polymorphisms and the development of gingival recessions during orthodontic treatment in Italian children.We evaluated, in 74 young Italian patients of both sexes, the -889 C/T polymorphism of the interleukin-1alpha gene and the -511 C/T and +3954 C/T polymorphisms of interleukin-1alpha gene by polymerase chain reactions-restriction fragment length polymorphism method using NcoI, AvaI and TaqI as restriction enzymes.No association of interleukin-1 genotypes investigated and gingival recession occurring during orthodontic treatment were identified.In the population studied specific interleukin-1 genotypes (linked to a higher susceptibility to bone resorption in periodontal disease) there does not appear to be any association with the development of gingival recessions during orthodontic treatment.

It is generally accepted that salivary components are important for dental health, but to date no clear correlation has been found between one or more of these components and the outcome of dental caries. The identification of salivary factors preventing, favoring or signaling dental caries might help to control the disease. In the present study, western blotting analysis of whole saliva from 20 healthy caries-free children showed the presence of the soluble form of CD14, a bacterial pattern-recognition receptor for many bacterial components that is involved in the innate immune response. The identity of the protein was confirmed through N-terminal sequencing by Edman degradation, and by partial sequencing with mass spectrometry of tryptic peptides. Conversely, CD14 was completely absent in the saliva of 20 age-matched patients affected by two to eight carious lesions, but appeared in their saliva a few weeks after dental restoration. These results suggest that the absence of salivary soluble CD14 could represent an useful index of caries activity, and might be used to detect early carious lesions not visible by oral inspection.

Bioactive glasses such as Hench's 45S5 have applications to tissue engineering and bone repair: the insertion of zinc has been proposed to improve their bone-bonding ability and to slacken their dissolution in extracellular body fluids. In view of a potential clinical application, we have investigated whether zinc-containing 45S5 (HZ) glasses might be cytotoxic for human MG-63 osteoblasts. In our experimental conditions, after 24 h of incubation HZ glasses released significant amounts of Zn2+ and induced in MG-63 cells release of lactate dehydrogenase (index of cytotoxicity) and the following indexes of oxidative stress: (i) accumulation of intracellular malonyldialdehyde, (ii) increased activity of pentose phosphate pathway, (iii) increased expression of heme oxygenase-1, (iv) increased activity of Cu,Zn-superoxide dismutase, (v) decreased level of intracellular thiols. These effects were inversely related to the zinc content of glass powders, were mimicked by ZnCl2 solutions and were prevented by either metal chelators (EDTA, NTA) or the antioxidant ascorbate, suggesting that Zn2+ released fastly from HZ glasses can cause MG-63 cell damage via an oxidative stress. This work highlights the importance of designing Zn-containing bioactive glasses without cytotoxic effects and gives supplementary information about the prooxidant role of zinc in living systems.

The cytotoxic effects of asbestos are partly mediated by the production of free radicals, including nitric oxide (NO). SV40 has been suggested to synergize with asbestos in the pathogenesis of malignant mesothelioma. Crocidolite asbestos fibers induced in human mesothelial and malignant mesothelioma cells a significant increase of NO synthase activity and expression, which was absent in SV40-infected cells. Furthermore, SV40 infection prevented the NFκB activation elicited by crocidolite in both mesothelial and mesothelioma cells. These data suggest that SV40, by inhibiting the synthesis of NO, could favor the survival of transformed, potentially neoplastic cells.

Artemisinin is a natural product used as an alternative drug in the treatment of severe and multidrug-resistant malaria. In the present work we show that artemisinin shares with other sesquiterpene lactones the ability to inhibit the activation of the nuclear factor NF-kB: by this mechanism, artemisinin, as well as parthenolide, inhibits nitric oxide synthesis in cytokine-stimulated human astrocytoma T67 cells. These results suggest that artemisinin, in addition to its antiparasitic properties, could also exert a therapeutic effect on neurological complications of malaria.

Objective— In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. Methods and Results— Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[ 3 H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP–dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. Conclusions— Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP–dependent protein kinase.

The cytotoxicity of asbestos has been related to its ability to increase the production of reactive oxygen species (ROS), via the iron-catalyzed reduction of oxygen and/or the activation of NADPH oxidase. The pentose phosphate pathway (PPP) is generally activated by the cell exposure to oxidant molecules. Contrary to our expectations, asbestos (crocidolite) fibers caused a dose- and time-dependent inhibition of PPP and decreased its activation by an oxidative stress in human lung epithelial cells A549. In parallel, the intracellular activity of the PPP rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), was significantly diminished by crocidolite exposure. This inhibition was selective, as the activity of other PPP and glycolysis enzymes was not modified, and was not attributable to a decreased expression of G6PD. On the opposite, the incubation with glass fibers MMVF10 did not modify PPP and G6PD activity. PPP and G6PD inhibition did not correlate with the increased nitric oxide (NO) production elicited by crocidolite in A549 cells. Experiments with the purified enzyme suggest that crocidolite inhibits G6PD by directly interacting with the protein. We propose here a new mechanism of asbestos-evoked oxidative stress, wherein fibers increase the intracellular ROS levels also by inhibiting the main antioxidant pathway of the cell.