Nickel oxide nanoparticles exposure as a risk factor for male infertility: "In vitro" effects on porcine pre-pubertal Sertoli cells.

Lately, nickel oxide nanoparticles (NiO NPs) have been employed in different industrial and biomedical fields. Several studies have reported that NiO NPs may affect the development of reproductive organs inducing oxidative stress and, resulting in male infertility. We investigated the in vitro effects of NiO NPs on porcine pre-pubertal Sertoli cells (SCs) which undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposure at two subtoxic doses of NiO NPs of 1 µg/ml and 5 µg/ml. After NiO NPs exposure we performed the following analysis: (a) SCs morphological analysis (Light Microscopy); (b) ROS production and oxidative DNA damage, gene expression of antioxidant enzymes (c) SCs functionality (AMH, inhibin B Real-time PCR analysis and ELISA test); (d) apoptosis (WB analysis); (e) pro-inflammatory cytokines (Real-time PCR analysis), and (f) MAPK kinase signaling pathway (WB analysis). We found that the SCs exposed to both subtoxic doses of NiO NPs didn't sustain substantial morphological changes. NiO NPs exposure, at each concentration, reported a marked increase of intracellular ROS at the third week of treatment and DNA damage at all exposure times. We demonstrated, un up-regulation of SOD and HO-1 gene expression, at both concentrations tested. The both subtoxic doses of NiO NPs detected a down-regulation of AMH and inhibin B gene expression and secreted proteins. Only the 5 µg/ml dose induced the activation of caspase-3 at the third week. At the two subtoxic doses of NiO NPs a clear pro-inflammatory response was resulted in an up-regulation of TNF-α and IL-6 in terms of mRNA. Finally, an increased phosphorylation ratio of p-ERK1/2, p-38 and p-AKT was observed up to the third week, at both concentrations. Our results show the negative impact of subtoxic doses NiO NPs chronic exposure on porcine SCs functionality and viability.

Chronic use of Datura stramonium cigarettes and late diagnosis of bullous emphysema in a smoker of marijuana and tobacco.

Unconventional inhaled therapy as a treatment for respiratory diseases became very common during the 19th century. Here, we present the case of a 52-year-old patient who smoked Datura stramonium cigarettes, tobacco cigarettes, and cannabis, with only an early diagnosis of asthma. The patient was admitted to our hospital with acute respiratory syndrome, characterized by worsening dyspnea, cough, and an acute episode of dyspnea and chest tightness. The combined chronic use of both D. stramonium cigarettes and cannabis masks the progression of chronic obstructive lung damage due to tobacco cigarette smoking because of the lack of clinical signs and symptoms.

Night shift work and lymphoma: results from an Italian multicentre case-control study.

BACKGROUND: Night shift work can disrupt circadian rhythm and cause chronic sleep deprivation, which might increase the risk of lymphoma through immunosuppression and oxidative stress. MATERIAL AND METHODS: We investigated the association between night shift work and risk of lymphoma subtypes in 867 incident cases and 774 controls, who participated in a multicentre Italian study between 2011 and 2017. Based on questionnaire information, occupational experts assessed the lifetime probability of night shift work, the total number of night shifts and years of night shift work among study participants. OR and 95% CI for lymphoma and its major subtypes associated with night shift work was calculated with logistic regression, adjusting by age, gender, education, study area, marital status and family history of haemolymphatic cancer. RESULTS: Ever working night shifts was associated with an increase in the risk of chronic lymphocytic leukaemia (CLL) (OR 1.9, 95% CI 1.14 to 3.32), which was highest after a 15-34 years latency. However, there was not a linear increase in risk by probability of exposure, years of night shift work, nor lifetime number of night shifts whether under rotating or permanent work schedules. Risk of lymphoma overall, B cell lymphoma (BCL), its major subtypes other than CLL, and other less prevalent BCL subtypes combined did not show an association. CONCLUSIONS: We found conflicting evidence of an association between night shift work and the risk of CLL. We did not observe an association with other lymphoma subtypes.

Impact of the COVID-19 pandemic and work-related stress in Umbrian healthcare workers during Phase 1 in Italy.

BACKGROUND: Depression, anxiety, psychological distress, and poor sleep quality increased in healthcare workers (HCWs) during the COVID-19 pandemic. The aim of the study was to assess levels of psychological distress in Umbrian HCWs during the COVID-19 Phase 1 lockdown along with exploring the relationship between sociodemographic/occupational factors. METHODS: Data on sociodemographic and occupational characteristics, change of job description, economic losses and emergency involvement and SARS-CoV2 infections in the workplace were collected using an anonymous online survey sent by healthcare professional associations. Data concerning psychological healthcare distress, were collected anonymously using BIAS 20 (stress balance) and Depression Anxiety Stress Scales (DASS-21). RESULTS: One thousand and one healthcare workers responded to the questionnaire. Biological risk at work was perceived by all HCWs, less so from psychologists and more so from those working in hospitals. Stress symptoms (DASS21 >14) were associated with a younger age group (OR 0.98; 95% CI 0.97-0.99) and less work experience (OR 0.98; 95% CI 0.96-0.99). Younger age was also associated with anxiety symptoms (DASS 21 >7) (OR 0.98; 95% CI 0.97-0.99), as well as graduate/post graduate education level (OR 2.04; 95% CI 1.14-3.63). Working as an independent contractor was a risk factor for high stress health impact (OR 2.00; CI 1.40-2.86) and stress (OR 1.87; CI 1.20-2.92), anxiety (OR 1.89; CI 1.22-2.92) and depression (OR 1.57; CI 1.10-2.22) symptoms. CONCLUSIONS: Our study showed a possible relationship between healthcare type of employment and distress symptoms during Covid19 pandemic phase 1. Results of our study should be confirmed in other Italian healthcare settings and could serve as a preliminarily baseline for multidisciplinary Italian collaboration.

Occupational exposure to glyphosate and risk of lymphoma:results of an Italian multicenter case-control study.

BACKGROUND: The International Agency for Research on Cancer (IARC) recently classified glyphosate, the most used herbicide worldwide, as a probable human carcinogen. We inquired into the association between occupational exposure to glyphosate and risk of lymphoma subtypes in a multicenter case-control study conducted in Italy. METHODS: The Italian Gene-Environment Interactions in Lymphoma Etiology (ItGxE) study took place in 2011-17 in six Italian centres. Overall, 867 incident lymphoma cases and 774 controls participated in the study. Based on detailed questionnaire information, occupational experts classified duration, confidence, frequency, and intensity of exposure to glyphosate for each study subject. Using unconditional regression analysis, we modelled risk of major lymphoma subtypes associated with exposure to glyphosate adjusted by age, gender, education, and study centre. RESULTS: Very few study subjects (2.2%) were classified as ever exposed to glyphosate. Risk of follicular lymphoma (FL) was elevated 7-fold in subjects classified as ever exposed to glyphosate with medium-high confidence, 4.5-fold in association with medium-high cumulative exposure level, 12-fold with medium-high exposure intensity, and 6-fold with exposure for 10 days or more per year. Significant upward trends were detected with all the exposure metrics, but duration. The overall p-value for an upward trend with four independent metrics was 1.88 × 10- 4. There was no association with risk of lymphoma (any subtype), Non Hodgkin Lymphoma, B-cell lymphoma, or the major lymphoma subtypes other than FL. CONCLUSIONS: Our findings provide limited support to the IARC decision to classify glyphosate as Group 2A human carcinogen.

Effects of Titanium Dioxide Nanoparticles on Porcine Prepubertal Sertoli Cells: An "In Vitro" Study.

The increasing use of nanomaterials in a variety of industrial, commercial, medical products, and their environmental spreading has raised concerns regarding their potential toxicity on human health. Titanium dioxide nanoparticles (TiO2 NPs) represent one of the most commonly used nanoparticles. Emerging evidence suggested that exposure to TiO2 NPs induced reproductive toxicity in male animals. In this in vitro study, porcine prepubertal Sertoli cells (SCs) have undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposures at both subtoxic (5 µg/ml) and toxic (100 µg/ml) doses of TiO2 NPs. After performing synthesis and characterization of nanoparticles, we focused on SCs morphological/ultrastructural analysis, apoptosis, and functionality (AMH, inhibin B), ROS production and oxidative DNA damage, gene expression of antioxidant enzymes, proinflammatory/immunomodulatory cytokines, and MAPK kinase signaling pathway. We found that 5 µg/ml TiO2 NPs did not induce substantial morphological changes overtime, but ultrastructural alterations appeared at the third week. Conversely, SCs exposed to 100 µg/ml TiO2 NPs throughout the whole experiment showed morphological and ultrastructural modifications. TiO2 NPs exposure, at each concentration, induced the activation of caspase-3 at the first and second week. AMH and inhibin B gene expression significantly decreased up to the third week at both concentrations of nanoparticles. The toxic dose of TiO2 NPs induced a marked increase of intracellular ROS and DNA damage at all exposure times. At both concentrations, the increased gene expression of antioxidant enzymes such as SOD and HO-1 was observed whereas, at the toxic dose, a clear proinflammatory stress was evaluated along with the steady increase in the gene expression of IL-1α and IL-6. At both concentrations, an increased phosphorylation ratio of p-ERK1/2 was observed up to the second week followed by the increased phosphorylation ratio of p-NF-kB in the chronic exposure. Although in vitro, this pilot study highlights the adverse effects even of subtoxic dose of TiO2 NPs on porcine prepubertal SCs functionality and viability and, more importantly, set the basis for further in vivo studies, especially in chronic exposure at subtoxic dose of TiO2 NPs, a condition closer to the human exposure to this nanoagent.

Industrial Air Pollution and Respiratory Health Status among Residents in an Industrial Area in Central Italy.

The area of Civitavecchia (Lazio region, Central Italy) has been a reason of concern in the past because of environmental air contamination. The aim of this study was to evaluate the association between air pollution from different sources and respiratory symptoms and lung function in the population. A sample of 1177 residents underwent medical examination and lung function tests. Information on individual characteristics, histories of exposure and medical history were collected through a validated questionnaire. Long-term exposure to industrial, harbour, biomass combustion emissions (PM10) and urban traffic (NOx) at residential address was assessed using a Lagrangian dispersion model. The associations between exposure and wheezing and dyspnea were assessed using logistic regression models, while modified Poisson regression models were used to evaluate cough with phlegm. Relationships between exposure and lung function were analysed using linear mixed-effects models and cross-correlation. PM10 emissions from the harbour were associated with lower lung function parameters (FEV1: ß = -0.12, 95% CI -0.21 -0.03; p = 0.02; FEV1/FVC: ß = -1.67, (-3.10 -0.23); p = 0.02. This association was observed also in healthy subjects, but not in females. We found, even if at low exposure level, an effect of environmental PM10 exposure from harbour on lung function.

Effects of nicotine on porcine pre-pupertal sertoli cells: An in vitro study.

Smoke components, such as nicotine and its major metabolites, cross the blood-testis barrier and are detectable in the seminal plasma of both active smokers and individuals exposed to cigarette smoke. In vivo studies in a rat model have further demonstrated that nicotine exposure reduces the weight of the testis, as well as the number of spermatocytes and spermatids, and affects the ultrastructure of Sertoli cells (SC) - which serve as sentinels of spermatogenesis - causing intense germ cell sloughing in the tubular lumen that compromises offspring fertility. This study sought to determine the effects of nicotine on the viability and function of purified pig pre-pubertal SC. Nicotine exposure reduced the mRNA expression and protein levels of anti-Mullerian hormone (AMH) and inhibin B and impaired FSH-r sensitivity via the downregulation of FSH-r and aromatase gene expression compared to untreated SC. Overall, our study suggests that nicotine can significantly alter extracellular matrix and tight junction protein gene expression (e.g., laminin, integrin, and occludin), thus compromising cross-talk between the interstitial and tubular compartments and enhancing blood-testis barrier (BTB) permeability via downregulation of the mitogen-activated protein kinase (MAPK) pathway. These findings further elucidate a potential mechanism of action underlying nicotine exposure's detrimental effects on SC function in vivo.

Effect of Curcumin on Protein Damage Induced by Rotenone in Dopaminergic PC12 Cells.

Oxidative stress is considered to be a key factor of the pathogenesis of Parkinson's disease, a multifactorial neurodegenerative disorder characterized by reduced dopaminergic neurons in the substantia nigra pars compacta and accumulated protein aggregates. Rotenone is a worldwide-used pesticide that induces the most common features of Parkinson's by direct inhibition of the mitochondrial complex I. Rotenone-induced Parkinson's models, as well as brain tissues from Parkinson's patients, are characterized by the presence of both lipid peroxidation and protein oxidation markers resulting from the increased level of free radical species. Oxidation introduces several modifications in protein structure, including carbonylation and nitrotyrosine formation, which severely compromise cell function. Due to the link existing between oxidative stress and Parkinson's disease, antioxidant molecules could represent possible therapeutic tools for this disease. In this study, we evaluated the effect of curcumin, a natural compound known for its antioxidant properties, in dopaminergic PC12 cells treated with rotenone, a cell model of Parkinsonism. Our results demonstrate that the treatment of PC12 cells with rotenone causes severe protein damage, with formation of both carbonylated and nitrotyrosine-derived proteins, whereas curcumin (10 µM) co-exposure exerts protective effects by reducing the levels of oxidized proteins. Curcumin also promotes proteasome activation, abolishing the inhibitory effect exerted by rotenone on this degradative system.

Genetic overlap between autoimmune diseases and non-Hodgkin lymphoma subtypes.

Epidemiologic studies show an increased risk of non-Hodgkin lymphoma (NHL) in patients with autoimmune disease (AD), due to a combination of shared environmental factors and/or genetic factors, or a causative cascade: chronic inflammation/antigen-stimulation in one disease leads to another. Here we assess shared genetic risk in genome-wide-association-studies (GWAS). Secondary analysis of GWAS of NHL subtypes (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and ADs (rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis). Shared genetic risk was assessed by (a) description of regional genetic of overlap, (b) polygenic risk score (PRS), (c)"diseasome", (d)meta-analysis. Descriptive analysis revealed few shared genetic factors between each AD and each NHL subtype. The PRS of ADs were not increased in NHL patients (nor vice versa). In the diseasome, NHLs shared more genetic etiology with ADs than solid cancers (p = .0041). A meta-analysis (combing AD with NHL) implicated genes of apoptosis and telomere length. This GWAS-based analysis four NHL subtypes and three ADs revealed few weakly-associated shared loci, explaining little total risk. This suggests common genetic variation, as assessed by GWAS in these sample sizes, may not be the primary explanation for the link between these ADs and NHLs.

Protein carbonylation in dopaminergic cells exposed to rotenone.

Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic neurons and the most common features of Parkinson's disease in animal models. It acts as a mitochondrial complex I inhibitor that impairs cellular respiration, with consequent increase of reactive oxygen species and oxidative stress. This study evaluates the rotenone-induced oxidative damage in PC12 cells, focusing particularly on protein oxidation. The identification of specific carbonylated proteins highlighted putative alterations of important cellular processes possibly associated with Parkinson's disease. Carbonylation of ATP synthase and of enzymes acting in pyruvate and glucose metabolism suggested a failure of mechanisms ensuring cellular energy supply. Concomitant oxidation of cytoskeletal proteins and of enzymes involved in the synthesis of neuroactive molecules indicated alterations of the neurotransmission system. Carbonylation of chaperon proteins as well as of proteins acting in the autophagy-lysosome pathway and the ubiquitin-proteasome system suggested the possible formation of cytosolic unfolded protein inclusions as result of defective processes assisting recovery/degradation of damaged molecules. In conclusion, this study originally evidences specific protein targets of rotenone-induced oxidative damage, suggesting some possible molecular mechanisms involved in rotenone toxicity.

Occupational risk factors for idiopathic pulmonary fibrosis in Southern Europe: a case-control study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia of unknown cause associated with the histopathologic and/or radiologic pattern of usual interstitial pneumonia (UIP). Occupational risk factors have been proposed to be associated with UIP. The aim of this case-control study is to evaluate the relationship between UIP pattern and occupational exposure in Southern Europe. METHODS: Sixty nine cases with a UIP radiological pattern at CT-scan were selected from a clinical database of the University Hospital of Perugia, Umbria, between January 2010 and December 2013. Controls (n = 277) not reporting doctor diagnosed pulmonary fibrosis, were ascertained casually among general population from the same catching area of cases. Data were collected by a questionnaire used previously in a similar study. Logistic regression models, adjusted for gender, age and smoking, were performed to evaluate the association between UIP and occupational exposure. RESULTS: Farmers, veterinarians and gardeners (OR = 2.73, 95%CI = 1.47-5.10), metallurgical and steel industry workers (OR = 4.80, 95%CI = 1.50-15.33) were occupations associated with UIP. Metal dust and fumes and organic dust were risk factors for UIP. Increasing the length of occupational exposure in jobs at risk of pulmonary fibrosis, increased the risk of having UIP. CONCLUSIONS: This case control study confirm partially the results from previous similar studies. Some discrepancies could be explained by the different geographical origins of the population under study, reflecting also different occupational exposures.

Levodopa-responsive breathing discomfort in Parkinson's disease patients.

In Parkinson's disease (PD), respiratory disturbances have been reported and the effect of levodopa on respiratory function remains controversial. The objective of this study was to evaluate pulmonary function utilizing spirometric and subjective evaluations in mild to moderated PD. Thirty-four consecutive sporadic PD patients (Hoehn and Yahr scale: 1-3) were prospectively evaluated using clinimetric scales, spirometry and modified Borg scale, all in off- and on-conditions. To check the respiratory function, a follow-up was performed at 4 years in a subgroup of these patients. Spirometric results were normal for all patients in both the on- and off-conditions at baseline. After levodopa administration, in addition to a significant improvement in subjective state of breathing discomfort, the mean forced expiratory volume in 1 s (FEV1), vital capacity (VC), forced vital capacity (FVC) values and their mean percentages predicted values (FEV1%, VC%, FVC%) were significantly increased (p < 0.05). Moreover, residual volume, total lung capacity, and the FEV1/FVC ratio were not significantly different for the ON and OFF conditions. At 4-year follow-up no resulting variations in the baseline values for FEV1%, FVC% or VC% were revealed. The results from this prospective study suggest that PD patients report frequently pulmonary discomfort. Levodopa improves respiratory symptoms. Pulmonary restrictive and obstructive dysfunctions, when not present at baseline, might not be present at 4-year follow-up.

Activation of the aryl hydrocarbon receptor and risk of lymphoma subtypes.

The aryl hydrocarbon receptor (AhR) is a transcription factor implicated in several pathways known to be relevant in lymphomagenesis. Aim of our study was to explore the link between AhR activation and risk of lymphoma subtypes. We used a Dual-Luciferase Assay® and a luminometer to detect the activation of the luciferase gene, in HepG2 cells transfected with a specific reporter systems, by a 50 ml serum aliquot of cases of diffuse large B cell lymphoma (N = 108), follicular lymphoma (N = 85), chronic lymphocytic leukemia (N = 72), multiple myeloma (N = 80), and Hodgkin lymphoma (N = 94) and 357 controls who participated in the multicentre Italian study on gene-environment interactions in lymphoma etiology (ItGxE). Risk of each lymphoma subtype associated with AhR activation was calculated with polytomous logistic regression adjusting by age, gender, and study centre. The overall prevalence of AhR activation ranged 13.9-23.6% by subtype, and it varied by study area (8-39%). Risk associated with AhR activation was moderately elevated for follicular lymphoma (OR = 1.56, 95% CI 0.86, 2.80) and chronic lymphocytic leukemia (OR = 1.56, 95% CI 0.83, 2.96). Despite our inconclusive findings about the association with risk of lymphoma subtypes, we showed that the Dual-Luciferase Assay can be reliably and easily applied in population-based studies to detect AhR activation.

No evidence of DNA damage by co-exposure to extremely low frequency magnetic fields and aluminum on neuroblastoma cell lines.

Whether exposure to 50-60Hz extremely low frequency magnetic fields (ELF-MF) exerts neurotoxic effects is a debated issue. Analogously, the potential role of Aluminum (Al) in neurodegeneration is a matter of controversial debate. As all living organisms are exposed to ELF-MF and/or Al daily, we found investigating the early effects of co-exposure to ELF-MF and Al in SH-SY5Y and SK-N-BE-2 human neuroblastoma (NB) cells intriguing. SH-SY5Y5 and SK-N-BE-2 cells underwent exposure to 50Hz ELF-MF (0.01, 0.1 or 1mT) or AlCl3 (4 or 40µM) or co-exposure to 50Hz ELF-MF and AlCl3 for 1h continuously or 5h intermittently. The effects of the treatment were evaluated in terms of DNA damage, redox status changes and Hsp70 expression. The DNA damage was assessed by Comet assay; the cellular redox status was investigated by measuring the amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) while the inducible Hsp70 expression was evaluated by western blot analysis and real-time RT-PCR. Neither exposure to ELF-MF or AlCl3 alone induced DNA damage, changes in GSH/GSSG ratio or variations in Hsp70 expression with respect to the controls in both NB cell lines. Similarly, co-exposure to ELF-MF and AlCl3 did not have any synergic toxic effects. The results of this in vitro study, which deals with the effects of co-exposure to 50Hz MF and Aluminum, seem to exclude that short-term exposure to ELF-MF in combination with Al can have harmful effects on human SH-SY5Y and SK-N-BE-2 cells.

Glyoxalase I drives epithelial-to-mesenchymal transition via argpyrimidine-modified Hsp70, miR-21 and SMAD signalling in human bronchial cells BEAS-2B chronically exposed to crystalline silica Min-U-Sil 5: Transformation into a neoplastic-like phenotype.

Glyoxalase I (Glo1) is the main scavenging enzyme of methylglyoxal (MG), a potent precursor of advanced glycation end products (AGEs). AGEs are known to control multiple biological processes, including epithelial to mesenchymal transition (EMT), a multistep phenomenon associated with cell transformation, playing a major role in a variety of diseases, including cancer. Crystalline silica is a well-known occupational health hazard, responsible for a great number of human pulmonary diseases, such as silicosis. There is still much debate concerning the carcinogenic role of crystalline silica, mainly due to the lack of a causal demonstration between silica exposure and carcinogenesis. It has been suggested that EMT might play a role in crystalline silica-induced lung neoplastic transformation. The aim of this study was to investigate whether, and by means of which mechanism, the antiglycation defence Glo1 is involved in Min-U-Sil 5 (MS5) crystalline silica-induced EMT in BEAS-2B human bronchial epithelial cells chronically exposed, and whether this is associated with the beginning of a neoplastic-like transformation process. By using gene silencing/overexpression and scavenging/inhibitory agents, we demonstrated that MS5 induced hydrogen peroxide-mediated c-Jun-dependent Glo1 up-regulation which resulted in a decrease in the Argpyrimidine-modified Hsp70 protein level which triggered EMT in a novel mechanism involving miR-21 and SMAD signalling. The observed EMT was associated with a neoplastic-like phenotype. The results obtained provide a causal in vitro demonstration of the MS5 pro-carcinogenic transforming role and more importantly they provide new insights into the mechanisms involved in this process, thus opening new paths in research concerning the in vivo study of the carcinogenic potential of crystalline silica.

Estimating the Smoking Ban Effects on Smoking Prevalence, Quitting and Cigarette Consumption in a Population Study of Apprentices in Italy.

OBJECTIVES: We evaluated the effects of the Italian 2005 smoking ban in public places on the prevalence of smoking, quitting and cigarette consumption of young workers. DATA AND METHODS: The dataset was obtained from non-computerized registers of medical examinations for a population of workers with apprenticeship contracts residing in the province of Viterbo, Italy, in the period 1996-2007. To estimate the effects of the ban, a segmented regression approach was used, exploiting the discontinuity introduced by the application of the law on apprentices' smoking behavior. RESULTS: It is estimated that the Italian smoking ban generally had no effect on smoking prevalence, quitting ratio, or cigarette consumption of apprentices. However, when the estimates were applied to subpopulations, significant effects were found: -1% in smoking prevalence, +2% in quitting, and -3% in smoking intensity of apprentices with at least a diploma.

Peroxynitrite-mediated glyoxalase I epigenetic inhibition drives apoptosis in airway epithelial cells exposed to crystalline silica via a novel mechanism involving argpyrimidine-modified Hsp70, JNK, and NF-κB.

Glyoxalase I (Glo1) is a cellular defense enzyme involved in the detoxification of methylglyoxal (MG), a cytotoxic by-product of glycolysis, and MG-derived advanced glycation end products (AGEs). Argpyrimidine (AP), one of the major AGEs coming from MG modification of protein arginines, is a proapoptotic agent. Crystalline silica is a well-known occupational health hazard, responsible for a relevant number of pulmonary diseases. Exposure of cells to crystalline silica results in a number of complex biological responses, including apoptosis. The present study was aimed at investigating whether, and through which mechanism, Glo1 was involved in Min-U-Sil 5 crystalline silica-induced apoptosis. Apoptosis, by TdT-mediated dUTP nick-end labeling assay, and transcript and protein levels or enzymatic activity, by quantitative real-time PCR, Western blot, and spectrophotometric methods, respectively, were evaluated in human bronchial BEAS-2B cells exposed or not (control) to crystalline silica and also in experiments with appropriate inhibitors. Reactive oxygen species were evaluated by coumarin-7-boronic acid or Amplex red hydrogen peroxide/peroxidase methods for peroxynitrite (ONOO(-)) or hydrogen peroxide (H2O2) measurements, respectively. Our results showed that Min-U-Sil 5 crystalline silica induced a dramatic ONOO(-)-mediated inhibition of Glo1, leading to AP-modified Hsp70 protein accumulation that, in a mechanism involving JNK and NF-κB, triggered an apoptotic mitochondrial pathway. Inhibition of Glo1 occurred at both functional and transcriptional levels, the latter occurring via ERK1/2 MAPK and miRNA 101 involvement. Taken together, our data demonstrate that Glo1 is involved in the Min-U-Sil 5 crystalline silica-induced BEAS-2B cell mitochondrial apoptotic pathway via a novel mechanism involving Hsp70, JNK, and NF-κB. Because maintenance of an intact respiratory epithelium is a critically important determinant of normal respiratory function, the knowledge of the mechanisms underlying its disruption may provide insight into the genesis, and possibly the prevention, of a number of pathological conditions commonly occurring in silica dust occupational exposure.

In vitro cadmium effects on ECM gene expression in human bronchial epithelial cells.

Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFß/TGFß receptor (TGFßR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, ß1 and ß5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and ß3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFß and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFß/TGFßR signaling.