Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

A novel PGPR strain homologous to Beijerinckia fluminensis induces biochemical and molecular changes involved in Arabidopsis thaliana salt tolerance.

The use of PGPR is widely accepted as a promising tool for a more sustainable agricultural production and improved plant abiotic stress resistance. This study tested the ability of PVr_9, a novel bacterial strain, homologous to Beijerinckia fluminensis, to increase salt stress tolerance in A. thaliana. In vitro plantlets inoculated with PVr_9 and treated with 150 mM NaCl showed a reduction in primary root growth inhibition compared to uninoculated ones, and a leaf area significantly less affected by salt. Furthermore, salt-stressed PVr_9-inoculated plants had low ROS and 8-oxo-dG, osmolytes, and ABA content along with a modulation in antioxidant enzymatic activities. A significant decrease in Na+ in the leaves and a corresponding increase in the roots were also observed in salt-stressed inoculated plants. SOS1, NHX1 genes involved in plant salt tolerance, were up-regulated in PVr_9-inoculated plants, while different MYB genes involved in salt stress signal response were down-regulated in both roots and shoots. Thus, PVr_9 was able to increase salt tolerance in A. thaliana, thereby suggesting a role in ion homeostasis by reducing salt stress rather than inhibiting total Na+ uptake. These results showed a possible molecular mechanism of crosstalk between PVr_9 and plant roots to enhance salt tolerance, and highlighted this bacterium as a promising PGPR for field applications on agronomical crops.

Nocturnal Heart Rate Variability Might Help in Predicting Severe Obstructive Sleep-Disordered Breathing.

Obstructive sleep apnea (OSA) can have long-term cardiovascular and metabolic effects. The identification of OSA-related impairments would provide diagnostic and prognostic value. Heart rate variability (HRV) as a measure of cardiac autonomic regulation is a promising candidate marker of OSA and OSA-related conditions. We took advantage of the Physionet Apnea-ECG database for two purposes. First, we performed time- and frequency-domain analysis of nocturnal HRV on each recording of this database to evaluate the cardiac autonomic regulation in patients with nighttime sleep breathing disorders. Second, we conducted a logistic regression analysis (backward stepwise) to identify the HRV indices able to predict the apnea-hypopnea index (AHI) categories (i.e., "Severe OSA", AHI ≥ 30; "Moderate-Mild OSA", 5 ≥ AHI < 30; and "Normal", AHI < 5). Compared to the "Normal", the "Severe OSA" group showed lower high-frequency power in normalized units (HFnu) and higher low-frequency power in normalized units (LFnu). The standard deviation of normal R-R intervals (SDNN) and the root mean square of successive R-R interval differences (RMSSD) were independently associated with sleep-disordered breathing. Our findings suggest altered cardiac autonomic regulation with a reduced parasympathetic component in OSA patients and suggest a role of nighttime HRV in the characterization and identification of sleep breathing disorders.

A follow-up study on workers involved in the graphene production process after the introduction of exposure mitigation measures: evaluation of genotoxic and oxidative effects.

During nanomaterial (NM) production, workers could be exposed, particularly by inhalation, to NMs and other chemicals used in the synthesis process, so it is important to have suitable biomarkers to monitor potential toxic effects. Aim of this study was to evaluate the effectiveness of the introduction of exposure mitigation measures on workers unintentionally exposed to graphene co-pollutants during production process monitoring the presumable reduction of workplace NM contamination and of early genotoxic and oxidative effects previously found on these workers. We used Buccal Micronucleus Cytome (BMCyt) assay and Fpg-comet test, resulted the most sensitive biomarkers on our first biomonitoring work, to measure the genotoxic effects. We also detected urinary oxidized nucleic acid bases 8-oxoGua, 8-oxoGuo and 8-oxodGuo to evaluate oxidative damage. The genotoxic and oxidative effects were assessed on the same graphene workers (N = 6) previously studied, comparing the results with those found in the first biomonitoring and with the control group (N = 11). This was achieved 6 months after the installation of a special filter hood (where to perform the phases at higher risk of NM emission) and the improvement of environmental and personal protective equipment. Particle number concentration decreased after the mitigation measures. We observed reduction of Micronucleus (MN) frequency and oxidative DNA damage and increase of 8-oxodGuo excretion compared to the first biomonitoring. These results, although limited by the small subject number, showed the efficacy of adopted exposure mitigation measures and the suitability of used sensitive and noninvasive biomarkers to bio-monitor over time workers involved in graphene production process.

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P < .05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during l-asparaginase treatment.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

Validation of a Bioanalytical Method for the Determination of Synthetic and Natural Cannabinoids (New Psychoactive Substances) in Oral Fluid Samples by Means of HPLC-MS/MS.

New psychoactive substances (NPS) represent an important focus nowadays and are continually produced with minimal structural modifications in order to circumvent the law and increase the difficulty of identifying them. Moreover, since there are a high number of different compounds, it is arduous to develop analytical screening and/or confirmation methods that allow the identification and quantification of these compounds. The aim of this work is to develop and validate a bioanalytical method for detecting new synthetic drugs in biological samples, specifically oral fluid, using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS) with minimal sample pretreatment. Oral fluid samples were simply centrifuged and denaturized with different rapid procedures before injection into the LC-MS/MS system. Calibration curves covered a linear concentration range from LOQ to 100 ng/mL. Validation parameters such as linearity, precision, accuracy, selectivity, matrix effect and thermal stability were evaluated and showed satisfactory results, in accordance with US Food & Drug Administration guidelines. The inter-day analytical bias and imprecision at two levels of quality control (QC) were within ±15% for most compounds. This method was able to identify and calculate the concentration of 10 NPS validated in this biological sample, even in the presence of matrix effect.

Urinary biomarkers of nucleic acid oxidation and methylation in workers exposed to low concentrations of benzene.

The study aims to investigate the influence of exposure to low concentrations of benzene on urinary biomarkers of nucleic acid oxidative damage and methylation. Benzene exposure was characterized for 93 coke production workers by measuring both airborne benzene and S-phenylmercapturic acid (SPMA) and unmodified benzene (U-B) in urine samples, collected at the end of the shift (ES) and at the next morning before shift (next BS). In the same urinary samples, biomarkers of nucleic acid oxidative damage and methylation were determined. Urinary concentrations of cotinine and creatinine were also determined to evaluate the smoking effect and to normalize urinary concentrations of analytes, respectively. The biomarkers of benzene internal dose, of oxidative damage (8-hydroxyy-7,8-dihydroguanine, 8-hydroxy-7,8-dihydroguanosine and 8-hydroxy-7,8-2'deoxyguanosine) and some of the biomarkers of nucleic acid methylation (5-Methyl-Cytosine, 1-Methyl-Guanine and 7-Methyl-Guanine) were higher in the ES than the next BS samples. Positive associations between ES 5-Methyl-Cytosine and both SPMA and U-B were found. In conclusion, occupational exposure to low levels of benzene seems to be related to urinary ES 5-Methyl-Cytosine that could be a possible biomarker to evaluate the changes of the nucleic acid methylation status.

The Relationship Between Widespread Pollution Exposure and Oxidized Products of Nucleic Acids in Seminal Plasma and Urine in Males Attending a Fertility Center.

BACKGROUND: In recent decades, there has been an increase in male infertility, and in many cases, the etiology remains unclear. Several studies relate male hypo-fertility to xenobiotic exposure, even if no data exist about multiple exposure at the environmental level. METHODS: The study involved 86 males with diagnosis of idiopathic male infertility (IMI), and 46 controls with no alteration in sperm characteristics. Seminal plasma (SP) and urine samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify biomarkers of exposure (the main metabolites of benzene, toluene, 1,3-butadiene, 3-monochloropropanediol, styrene, and naphthol) and effect (oxidized products of nucleic acids). RESULTS: Biomarker concentrations were similar in subjects with IMI and controls even if a stronger correlation between biomarkers of exposure and effects were observed in SP. Data show that, both in SP and urine, most metabolites were inter-correlated, indicating a simultaneous co-exposure to the selected substances at the environmental level. Principal component analysis showed in SP the clustering of mercapturic acids indicating a preferential metabolic pathway with Glutathione (GSH) depletion and, consequently, an increase of oxidative stress. This result was also confirmed by multivariable analysis through the development of explanatory models for oxidized products of nucleic acids. CONCLUSIONS: This study highlights how oxidative stress on the male reproductive tract can be associated with a different representation of metabolic pathways making the reproductive tract itself a target organ for different environmental pollutants. Our results demonstrate that SP is a suitable matrix to assess the exposure and evaluate the effects of reproductive toxicants in environmental/occupational medicine. The statistical approach proposed in this work represents a model appropriate to study the relationship between multiple exposure and effect, applicable even to a wider variety of chemicals.

Idiopathic pulmonary fibrosis and occupational risk factors.

Idiopathic pulmonary fibrosis (IPF) is a rare lung disease of unknown origin that rapidly leads to death. However, the rate of disease progression varies from one individual to another and is still difficult to predict. The prognosis of IPF is poor, with a median survival of three to five years after diagnosis, without curative therapies other than lung transplantation. The factors leading to disease onset and progression are not yet completely known. The current disease paradigm is that sustained alveolar epithelial micro-injury caused by environmental triggers (e.g., cigarette smoke, microaspiration of gastric content, particulate dust, viral infections or lung microbial composition) leads to alveolar damage resulting in fibrosis in genetically susceptible individuals. Numerous epidemiological studies and case reports have shown that occupational factors contribute to the risk of developing IPF. In this perspective, we briefly review the current understanding of the pathophysiology of IPF and the importance of occupational factors in the pathogenesis and prognosis of the disease. Prompt identification and elimination of occult exposure may represent a novel treatment approach in patients with IPF.

Novel sample-substrates for the determination of new psychoactive substances in oral fluid by desorption electrospray ionization-high resolution mass spectrometry.

A reliable screening and non invasive method based on the use of microextraction by packed sorbent coupled with desorption electrospray ionization-high resolution mass spectrometry was developed and validated for the detection of new psychoactive substances in oral fluid. The role of different sample substrates in enhancing signal intensity and stability was evaluated by testing the performances of two polylactide-based materials, i.e. non-functionalized and functionalized with carbon nanoparticles, and a silica-based material compared to commercially available polytetrafluorethylene supports. The best results were achieved by using the non-functionalized polylactide substrates to efficiently ionize compounds in positive ionization mode, whereas the silica coating proved to be the best choice for operating in negative ionization mode. LLOQs in the low µg/L, a good precision with CV% always lower than 16% and RR% in the 83(±4)-120(±2)% range, proved the suitability of the developed method for the determination of the analytes in oral fluid. Finally, the method was applied for screening oral fluid samples for the presence of psychoactive substances during private parties, revealing mephedrone in only one sample out of 40 submitted to analysis.

Sulphurous thermal water inhalation impacts respiratory metabolic parameters in heavy smokers.

Sulphurous thermal water inhalations have been traditionally used in the treatment of airway diseases. In vivo and in vitro studies reported that they ameliorate mucus rheology, mucociliary clearance and reduce inflammation. Cigarette smoking induces an inflammatory damage, with consequent remodeling of respiratory airways, which in turn affect pulmonary functions. Despite the anti-inflammatory effects of H2S are clinically documented in several airway inflammatory diseases, data on the effects of sulphurous thermal water treatment on pulmonary function and biomarkers of airways inflammation in smokers are still scant. Therefore, we investigated whether a conventional cycle of sulphurous thermal water inhalation produced changes in markers of respiratory inflammation and function. A cohort of 504 heavy current and former smokers underwent 10-day cycles of sulphurous thermal water inhalation. Pulmonary function and metabolic analyses on exhaled breath condensate were then performed at day 0 and after the 10-day treatment. Spirometric data did not change after spa therapy, while exhaled breath condensate analysis revealed that a single 10-day cycle of sulphurous water inhalation was sufficient to induce a statistically significant increase of citrulline levels along with a decrease in ornithine levels, thus shifting arginine metabolism towards a reduced nitric oxide production, i.e. an anti-inflammatory profile. Overall, sulphurous thermal water inhalation impacts on arginine catatabolic intermediates of airways cells, shifting their metabolic balance towards a reduction of the inflammatory activity, with potential benefits for smokers.

Higher Number of Night Shifts Associates with Good Perception of Work Capacity and Optimal Lung Function but Correlates with Increased Oxidative Damage and Telomere Attrition.

Sleep deprivation and the consequent circadian clock disruption has become an emergent health question being associated with premature aging and earlier chronic diseases onset. Night-shift work leads to circadian clock misalignment, which is linked to several age-related diseases. However, mechanisms of this association are not well understood. Aim of this study is to explore in night-shift workers early indicators of oxidative stress response and biological aging [oxidized/methylated DNA bases and leukocytes telomere length (LTL)] and late indicators of functional aging [lung function measurements (FEV1 and FVC)] in relation to personal evaluation of work capacity, measured by work ability index (WAI). One hundred fifty-five hospital workers were studied within the framework of a cross-sectional study. We collected physiological, pathological, and occupational history including pack-years, alcohol consumption, physical activity, and night shifts, together with blood and urine samples. Relationships were appraised by univariate and multivariate ordered-logistic regression models. We found that workers with good and excellent WAI present higher FEV1 (p< 0.01) and number of night-work shifts (p<0.05), but they reveal higher urinary levels of 8-oxoGua (p<0.01) and shorter LTL (p<0.05). We confirmed that higher work ability was prevalent among chronological younger workers (p<0.05), who have also a significant reduced number of diseases, particularly chronic (p<0.01) and musculoskeletal diseases (p<0.01). The new findings which stem from our work are that subjects with the highest work ability perception may have more demanding and burdensome tasks; they in fact present the highest number of night-shift work and produce unbalanced oxidative stress response that might induce premature aging.

Determination of synthetic and natural cannabinoids in oral fluid by solid-phase microextraction coupled to gas chromatography/mass spectrometry: A pilot study.

In 2008, several synthetic cannabinoids were detected in herbal smoking blends sold on websites and in the so-called "smart shops". These compounds, as well as new psychoactive substances, flooded the market of illicit drugs and are sold at street level. Development and validation of rapid analytical methods for the detection and quantification of synthetic cannabinoids in biological matrices are essential for the investigation of pharmacokinetic, pharmacodynamic and toxicological properties. In this pilot study, the potential of direct immersion solid-phase microextraction coupled to GC/MS for the determination of synthetic and natural cannabinoids in oral fluid was proved. Validation proved the suitability of the developed method for the determination of the analytes at trace levels in oral fluid: linearity was assessed in the LOQ - 1000 ng/mL range, whereas a good precision was observed with CV below 20%. The method was then applied to more than 100 real oral fluid samples collected from a population of young students, showing a positivity rate of 3.8%.

Non-invasive techniques to assess restrictive lung disease in workers exposed to free crystalline silica.

OBJECTIVES: To compare the reliability of spirometry and body plethysmography in detecting restrictive lung disease in clay excavation workers exposed to free crystalline silica (FCS). The exhaled breath condensate (EBC) biomarkers of oxidative stress were also assessed in order to evaluate early lung damage. METHODS: The study involved 62 workers (58 males and 4 females) at a company that extracts and processes clay. RESULTS: Body plethysmography (total lung capacity below the lower normal limit) and spirometry respectively indicated restrictive pattern prevalence rates of 22.6% and 1.6%. EBC 4-hydroxynonenale levels were not sufficiently sensitive to highlight a restrictive deficit, but did distinguish low and high rates of occupational exposure. There was no correlation between plethysmography values and the intensity or duration of exposure. CONCLUSIONS: Only one out of 14 cases of restrictive deficit diagnosed on the basis of body plethysmography values was also identified by means of spirometry. This finding supports the need to use body plethysmography in the health surveillance of clay workers exposed to FCS.

[Laboratory animal allergy.] / Allergia da animali da laboratorio.

SUMMARY: Laboratory animal allergy (LAA) is caused by an immunological hypersensitivity reaction to highmolecular- weight antigens that are present in laboratory animals' urine, dander and saliva. All laboratory animal facility personnel who regularly come in contact with laboratory animals, such as technicians, researchers, cleaning staff, veterinarians and even administrative staff, are at risk of developing LAA. Generally, most epidemiological studies indicate a LAA prevalence ranging from 6% to 44% and an incidence ranging from 9% to 30%. Prevalence and incidence data vary widely because the diagnosis is not uniformly defined: some diagnoses are made solely on the basis of symptoms, whereas others also require a positive skin test or confirmation of the presence of laboratory animal allergen-specific IgE antibodies.

7-Hydroxymatairesinol improves body weight, fat and sugar metabolism in C57BJ/6 mice on a high-fat diet.

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro.

Biomarkers of exposure to stainless steel tungsten inert gas welding fumes and the effect of exposure on exhaled breath condensate.

The respiratory tract is the main target organ of the inhaled hexavalent chromium (Cr-VI) and nickel (Ni) contained in stainless steel (SS) welding fumes (WFs). The aim of this study was to investigate the Cr and Ni content of the exhaled breath condensate (EBC) of SS tungsten inert gas (TIG) welders, and relate their concentrations with oxidative stress and inflammatory biomarkers. EBC and urine from 100 SS TIG welders were collected pre-(T0) and post-shift (T1) on a Friday, and pre-shift (T2) on the following Monday morning. Both EBC and urinary Cr concentrations were higher at T1 (0.08 µg/L and 0.71 µg/g creatinine) and T0 (0.06 µg/L and 0.74 µg/g creatinine) than at T2 (below the limit of detection [LOD] and 0.59 µg/g creatinine), and EBC Ni concentrations generally remained

Reference Intervals for Urinary Cotinine Levels and the Influence of Sampling Time and Other Predictors on Its Excretion Among Italian Schoolchildren.

(1) Background: Environmental Tobacco Smoke (ETS) exposure remains a public health problem worldwide. The aims are to establish urinary (u-) cotinine reference values for healthy Italian children, to evaluate the role of the sampling time and of other factors on children's u-cotinine excretion. (2) Methods: A cross-sectional study was performed on 330 children. Information on participants was gathered by a questionnaire and u-cotinine was determined in two samples for each child, collected during the evening and the next morning. (3) Results: Reference intervals (as the 2.5th and 97.5th percentiles of the distribution) in evening and morning samples were respectively equal to 0.98⁻4.29 and 0.91⁻4.50 µg L-1 (ETS unexposed) and 1.39⁻16.34 and 1.49⁻20.95 µg L-1 (ETS exposed). No statistical differences were recovered between median values found in evening and morning samples, both in ETS unexposed and exposed. Significant predictors of u-cotinine excretions were ponderal status according to body mass index of children (ß = 0.202; p-value = 0.041 for evening samples; ß = 0.169; p-value = 0.039 for morning samples) and paternal educational level (ß = -0.258; p-value = 0.010; for evening samples; ß = -0.013; p-value = 0.003 for morning samples). (4) Conclusions: The results evidenced the need of further studies for assessing the role of confounding factors on ETS exposure, and the necessity of educational interventions on smokers for rising their awareness about ETS.