Angela Gambelunghe

Blood cadmium concentrations in the general population of Umbria, Central Italy

The aims of this study were (a) to assess blood cadmium (B-Cd) concentrations and to establish a tentative reference interval; (b) to identify significant determinants of B-Cd, in a population from Umbria, Central Italy, which was not occupationally exposed to cadmium (Cd). Four hundred and thirty-four healthy blood-donors volunteered to answer a questionnaire and provide a blood sample for B-Cd analysis, which was performed by graphite furnace atomic absorption spectrophotometry. Blood Cd concentrations ranged from non-detectable values, i.e. below 0.1 microgram/l up to 3.4 micrograms/l and were not normally distributed. The median values and the 95th percentiles were 0.7 and 2.0 micrograms/l, respectively. Concentrations of B-Cd were more than double in smokers than in non-smokers, median values being 1.1 micrograms/l and 0.5 microgram/l, respectively. In current smokers, B-Cd values correlated with the number of cigarettes smoked daily (rs = 0.40, P = 0.0001) and with the cumulative exposure to cigarette smoke (rs = 0.35, P = 0.0001). Concentrations of B-Cd correlated with age in the non-smokers, but not in the smokers and were significantly higher in women than in men only in the non-smokers. Both in smokers and non-smokers, B-Cd concentrations were similar in subjects living in urban or in rural areas. In the whole study population the lower and the upper tentative reference limit were < 0.1 and 2.2 micrograms/l, respectively, as computed by a non-parametric rank-based method. The upper limit was approximately double in smokers than in non-smokers (3.1 micrograms/l and 1.6 micrograms/l, respectively). Our results show that B-Cd concentrations in a general population from Umbria are in the range reported for general populations in Northern Italy and other European Countries. Smoking was the strongest determinant of B-Cd concentrations and age had a lesser effect.

Reactive oxygen species induce apoptosis in bronchial epithelial BEAS-2B cells by inhibiting the antiglycation glyoxalase I defence: involvement of superoxide anion, hydrogen peroxide and NF-κB

Reactive oxygen species (ROS) are implicated in the regulation of apoptosis through a number of distinct mechanisms depending on cell type and stimulation conditions. Glyoxalase I (GI) metabolizes methylglyoxal (MG) and MG-derived advanced glycation end products (AGEs) known to cause apoptosis. This study examined the possible role of GI among the mechanisms of ROS-driven apoptosis in human bronchial epithelial BEAS-2B cells exposed to wood dust and signaling pathways by which these reactive species regulate GI expression. Our results showed that wood dust generated distinct ROS (superoxide anion, and hydrogen peroxide) by selectively inhibiting the enzymatic activity of superoxide dismutase or glutathione peroxidase and catalase enzymes. These ROS caused a dramatic inhibition of the antiglycation GI enzyme, leading to the intracellular accumulation of the pro-apoptotic AGE, argpyrimidine (AP) and programmed cell death via a mitochondrial pathway. Pre-treatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, prevented these events. Hence, ROS-induced apoptosis in BEAS-2B cells occurred via a novel mechanism relying on GI inhibition and AP accumulation. We interestingly found that superoxide anion and hydrogen peroxide induced a diverse apoptosis level by differently inhibiting GI via NF-κB pathway. Since maintenance of an intact epithelium is a critically important determinant of normal respiratory function, the knowledge of the mechanisms underlying its disruption may provide insight into the genesis of a number of pathological conditions commonly occurring in wood dust occupational exposure. Our findings suggest that the antioxidant NAC may merit investigation as a potential preventive agent in wood dust exposure-induced respiratory diseases.

Low-level exposure to lead, blood pressure, and hypertension in a population-based cohort.

BACKGROUND Environmental lead exposure is a possible causative factor for increased blood pressure and hypertension, but large studies at low-level exposure are scarce, and results inconsistent. OBJECTIVE We aimed to examine the effects of environmental exposure to lead in a large population-based sample. METHODS We assessed associations between blood lead and systolic/diastolic blood pressure and hypertension in 4452 individuals (46-67 years) living in Malmö, Sweden, in 1991-1994. Blood pressure was measured using a mercury sphygmomanometer after 10min supine rest. Hypertension was defined as high systolic (≥140mmHg) or diastolic (≥90mmHg) blood pressure and/or current use of antihypertensive medication. Blood lead was calculated from lead in erythrocytes and haematocrit. Multivariable associations between blood lead and blood pressure or hypertension were assessed by linear and logistic regression. Two-thirds of the cohort was re-examined 16 years later. RESULTS At baseline, mean blood pressure was 141/87mmHg, 16% used antihypertensive medication, 63% had hypertension, and mean blood lead was 28µg/L. Blood lead in the fourth quartile was associated with significantly higher systolic and diastolic blood pressure (point estimates: 1-2mmHg) and increased prevalence of hypertension (odds ratio: 1.3, 95% confidence interval: 1.1-1.5) versus the other quartiles after adjustment for sex, age, smoking, alcohol, waist circumference, and education. Associations were also significant with blood lead as a continuous variable. Blood lead at baseline, having a half-life of about one month, was not associated with antihypertensive treatment at the 16-year follow-up. CONCLUSIONS Low-level lead exposure increases blood pressure and may increase the risk of hypertension.

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.

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.

Crystalline silica Min-U-Sil 5 induces oxidative stress in human bronchial epithelial cells BEAS-2B by reducing the efficiency of antiglycation and antioxidant enzymatic defenses

Reactive oxygen species (ROS) play an important role as mediators of pulmonary damage in mineral dust-induced diseases. Studies carried out to date have largely focused on silica-induced production of ROS by lung phagocytes. In this study we investigated the hypothesis that crystalline silica Min-U-Sil 5 can induce elevations in intracellular ROS in human bronchial epithelial cells BEAS-2B, via an indirect mechanism that involves ROS-inducing intracellular factors, through a reduction of antiglycation (glyoxalase enzymes) and antioxidant (paraoxonase 1 and glutathione-S-transferases) enzymatic defenses. The results show that crystalline silica Min-U-Sil 5 causes a significant reduction in the efficiency of antiglycation and antioxidant enzymatic defenses, paralleled by an early and extensive ROS generation, thus preventing the cells from an efficient scavenging action, and eliciting oxidative damage. These results confirm the importance of ROS in development of crystalline silica-induced oxidative stress and emphasize the pivotal role of antiglycation/antioxidant and detoxifying systems in determining the level of protection from free radicals-induced injury for cells exposed to crystalline silica Min-U-Sil 5.

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 TGFβ/TGFβ receptor (TGFβR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, β1 and β5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and β3 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 TGFβ 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 TGFβ/TGFβR signaling.

Chromium VI-induced apoptosis in a human bronchial epithelial cell line (BEAS-2B) and a lymphoblastic leukemia cell line (MOLT-4).

Hexavalent chromium compounds are well-documented human carcinogens. In vitro experiments show Cr (VI) induces cell death by apoptosis by activating p53 protein. The aim of this study was to evaluate Cr (VI)-induced apoptosis in a human bronchial epithelial cell line (BEAS-2B) and in a lymphoblastic leukemia cell line (MOLT-4). Cr (VI) caused a dose- and time-dependent increase in the apoptosis rate in both cell lines. Western blotting showed increased p53 protein expression in MOLT-4 cells, but not in BEAS-2B cells, after exposure to 0.5 and 3 &mgr;M hexavalent chromium for 12 hours and 4 hours, respectively. Apoptotic cell death induced by Cr (VI) was not decreased by pretreatment with caspase-3, -8, and -9 inhibitors. These preliminary results provide evidence of Cr (VI)-induced apoptosis, which deserves further investigation in occupationally exposed workers.

Phosphatidylserine metabolism modification precedes manganese- induced apoptosis and phosphatidylserine exposure in PC12 cells

Long-term exposure to high manganese (Mn) levels can lead to Parkinson-like neurological disorders. Molecular mechanisms underlying Mn cytotoxicity have been not defined. It is known that Mn induces apoptosis in PC12 cells and that this involves the activation of some signal transduction pathways. Although the role of phospholipids in apoptosis and signal transduction is well-known, the membrane phospholipid component in Mn-related damage has not yet been investigated. Phosphatidylserine (PS) facilitates protein translocation from cytosol to plasma membrane and PS exposure on the cell surface allows macrophage recognition of apoptotic cells. This study investigates the effects of MnCl2 on PS metabolism in PC12 cells, relating them to those on cell apoptosis. Apoptosis induction decreased PS radioactivity of PC12 cells incubated with radioactive serine. MnCl2 reduced PS radioactivity even under conditions that did not affect cell viability or PS exposure, suggesting that the effects on PS metabolism may represent an early event in cell apoptosis. Thus the latter conditions that also induced a greater PS decarboxylation were utilized for further investigating on the effects on PS synthesis, by measuring the activity and expression of PS-synthesizing enzymes, in cell lysates and in total cellular membranes (TM). Compared with corresponding controls, enzyme activity of MnCl2-treated cells was lower in cell lysates and greater in TM. Evaluating the expression of two isoforms of PS-synthesizing enzyme (PSS), PSSII was increased both in cell lysate and TM, while PSSI was unchanged. MnCl2 addition to control cell lysate reduced enzyme activity. These results suggest Mn plays a dual role on PS synthesis. Once inside the cell, Mn inhibits the enzyme/s, thus accounting for reduced PS synthesis in lysates and intact cells. On the other hand, it increases PSSII expression in cell membranes. The possibility that this occurs to counteract the direct effects of Mn ions on enzyme activity cannot be excluded. The effects on membrane enzyme activity and expression may also participate to PS exposure, observed at longer periods of treatment, by increasing membrane PS content.

Phosphatidylserine metabolism in human lymphoblastic cells exposed to chromium (VI).

Objective: Hexavalent chromium (Cr(VI)) compounds are widely found in different working environments. These compounds can cause apoptosis in human cells, but the mechanisms underlying chromium-induced apoptosis are not clear. A marker of apoptosis is the exposure of phosphatidylserine on cell membrane and the modification of phosphatidylserine metabolism. The aim of this study was to verify whether chromium could cause phosphatidylserine exposure and modification of its metabolism in human lymphoblastic leukemia cell line (MOLT-4). Methods: Phosphatidylserine exposure was evaluated by annexin V binding whereas phosphatidylserine metabolism was studied measuring the incorporation of [3H]serine. Results: Cell treatment with Cr(VI) increases phosphatidylserine exposure and cell apoptosis, but decreases the incorporation of [3H]serine into phosphatidylserine in a dose- and time-dependent manner. Conclusions: The Cr(VI)-induced apoptosis also through modification of phosphatidylserine exposure and metabolism.