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Huge reductions in incinerators' emissions occurred over time, and results of older studies cannot be directly generalized to modern plants. We conducted a systematic review of the epidemiologic evidence of the health effects of incinerators, classifying plants in three generations, according to emission limits. A systematic search identified 63 epidemiologic studies, published in English, investigating health effects of incinerators on humans. We focused on cancer, cardio-cerebrovascular diseases (CVD) and respiratory diseases, pregnancy outcomes and congenital anomalies. Only six studies in the general population were on third generation incinerators providing data on pregnancy outcomes and congenital anomalies. Given the heterogeneity of methods, the abundance of ecological/semi-ecological studies and the lack of reliable quantitative measures of exposure in several studies we did not perform any meta-analysis. No excesses emerged concerning all cancers and lung cancer. An excess of non-Hodgkin lymphoma was reported in some earlier studies, but not for second generation plants. Possible excesses of soft tissue sarcomas were confined to earlier incinerators and the areas closer to the plants. No clear association emerged for CVD and diseases of the respiratory system. Several different pregnancy outcomes were considered, and no consistent association emerged, in spite of a few positive results. Studies were negative for congenital anomalies as a whole. Sporadic excesses were reported in a few studies for specific types of anomalies, but no consistent pattern emerged. Evaluation of the evidence was hindered by heterogeneity in reporting and classification of outcomes across studies. Direct evidence from third generation plants is scarce. Methodological issues in study design (mainly related to exposure assessment, confounding and ecological design) and analysis make interpretation of results complex. In spite of this, the overall evidence suggests that, if there were any excesses at all for older incinerators, they were modest at most. Additional monitoring of third generation plants needs to overcome methodological weaknesses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

The purpose of this study of the Italian Society of Reference Values (SIVR) was to provide the reference values for metals in the urine of the Italian general population. Thirteen sampling centres situated in the north, centre and south of Italy took part in this project. Each sampling centre selected 20 adult subjects. The sample was made up of 120 male and 140 female non-smokers or smokers who had abandoned the habit at least 5 years previously, aged between 18 and 60 years. Urine samples were obtained from the same subjects in June and again in November of the same year. During collection of these two samples, we administered an ad hoc questionnaire designed to assess factors that might influence exposure and the results of the study such as personal characteristics, occupational or extra-occupational activities, dietary habits, the presence of dental fillings containing amalgam, the frequency and mode of exposure to passive smoking and exhaust gases of motor vehicles (traffic). Therefore, the urinary levels of thirteen trace elements (Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V) were determined by Inductively Coupled Plasma Mass Spectrometry. The concentrations obtained (95 th percentile) are in μg/L: 0.034, 0.900, 2.24, 0.600, 24.0, 0.013, 1.53, 4.44, 2.64, 0.022, 0.095, 0.759 and 0.855 for Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V, respectively. Further studies with a larger number of subjects are needed in order to continue the biomonitoring of the Italian general population and to monitor the modified levels over time., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Inhaled particles have been shown to produce systemic changes in DNA methylation. Global hypomethylation has been associated to viral sequence reactivation, possibly linked to the activation of pro-inflammatory pathways occurring after exposure. This observation provides a rationale to investigate viral sequence (both exogenous and endogenous) methylation in association to metal-rich particulate matter exposure. To verify this hypothesis, we chose the Wp promoter of the Epstein-Barr Virus (EBV-Wp) and the promoter of the human-endogenous-retrovirus w (HERV-w), respectively as a paradigm of an exogenous and an endogenous retroviral sequence, to be investigated by bisulfite PCR Pyrosequencing. We enrolled 63 male workers in an electric furnace steel plant, exposed to high level of metal-rich particulate matter., Results: Comparing samples obtained in the first day of a work week (time 0-baseline, after 2 days off work) and the samples obtained after 3 days of work (time 1-post exposure), the mean methylation of EBV-Wp was significantly higher at baseline compared to post-exposure (mean baseline = 56.7%5mC; mean post-exposure = 47.9%5mC; p-value = 0.009), whereas the mean methylation of HERV-w did not significantly differ. Individual exposure to inhalable particles and metals was estimated based on measures in all working areas and time spent by the study subjects in each area. In a regression model adjusted for age, body mass index and smoking, PM and metal components had a positive association with EBV-Wp methylation (i.e. PM10: β = 5.99, p-value < 0.038; nickel: β = 17.82, p-value = 0.02; arsenic: β = 13.59, p-value < 0.015)., Conclusions: The difference observed comparing baseline and post-exposure samples may be suggestive of a rapid change in EBV methylation induced by air particles, while correlation between EBV methylation and PM/metal exposure may represent a more stable adaptive mechanism. Future studies investigating a larger panel of viral sequences could better elucidate possible mechanisms and their role in pro-inflammatory pathways leading to systemic health effects., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Continuous exposure to particulate air pollution (PM) is a serious worldwide threat to public health as it coherently links with increased morbidity and mortality of cardiorespiratory diseases (CRD), and of type 2 diabetes (T2D). Extracellular vesicles (EVs) are circular plasma membrane fragments released from human cells that transfer microRNAs between tissues. In the present work it was explored the hypothesis that EVs with their encapsulated microRNAs (EVmiRNAs) contents might mediate PM effects by triggering key pathways in CRD and T2D., Methods: Expression of EVmiRNAs analyzed by real-time PCR was correlated with oxidative stress, coagulation and inflammation markers, from healthy steel plant workers (n=55) with a well-characterized exposure to PM and PM-associated metals. All p-values were adjusted for multiple comparisons. In-silico Ingenuity Pathway Analysis (IPA) was performed to identify biological pathways regulated by PM-associated EVmiRNAs., Results: Increased expression in 17 EVmiRNAs is associated with PM and metal exposure (p<0.01). Mir-196b that tops the list, being related to 9 different metals, is fundamental in insulin biosynthesis, however three (miR-302b, miR-200c, miR-30d) out of these 17 EVmiRNAs are in turn also related to disruptions (p<0.01) in inflammatory and coagulation markers., Conclusions: The study's findings support the hypothesis that adverse cardiovascular and metabolic effects stemming from inhalation exposures in particular to PM metallic component may be mediated by EVmiRNAs that target key factors in the inflammation, coagulation and glucose homeostasis pathways., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In the last few years, many reports have confirmed the presence of WU, KI and Merkel cell (MC) polyomaviruses (PyV) in respiratory samples wordwide, but their pathogenic role in patients with underlying conditions such as cystic fibrosis is still debated. To determine the prevalence of MCPyV, WUPyV and KIPyV, we conducted a 1-year-long microbiological testing of respiratory specimens from 93 patients with cystic fibrosis in Brescia, Italy. We detected PyV DNA in 94 out of 337 analysed specimens. KIPyV was the most common virus detected (12.1%), followed by WUPyV (8.9%) and MCPyV (6.8%). We found an intriguing association between the presence of MCPyV and the concurrent isolation of Pseudomonas aeruginosa, as well as with the patient status, classified as chronically colonized with P. aeruginosa. Our study adds perspective on the prevalence and the potential pathogenic role of PyV infections., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Epidemiological studies have shown associations of particulate matter (PM) exposure with hypercoagulability and thrombosis. Extracellular circulating histones have recently been identified as novel mediators of inflammatory and procoagulant responses. The potential roles of extracellular histones in PM-related hypercoagulability have yet not been investigated., Objectives: In 63 steel workers, we evaluated the effects of exposure to PM and PM metal components on two extracellular histone modifications (H3K4me3 and H3K9ac); and the association of H3K4me3 and H3K9ac with coagulation markers., Methods: Extracellular H3K4me3 and H3K9ac were determined in plasma through enzyme-linked immunosorbent assays. Coagulation markers included endogenous thrombin potentials (ETPs), tissue-type plasminogen activator antigen (t-PA) and D-dimer. Exposure to PM with aerodynamic diameters <1 μm (PM1) or <10 μm (PM10) and PM10 metal components were estimated for each participant., Results: The coagulation marker ETP, measured in the presence of soluble thrombomodulin (ETP TM+), showed significant positive associations with PM1 (β=107.84, p=0.03), PM10 (β=83.06, p=0.02), and zinc (β=75.14, p=0.03); and a marginal association with iron (β=122.58, p=0.07). Additional PM effects were observed on t-PA, D-dimer, and ETP TM+. PM1 exposure was associated with increased plasma H3K4me3 and H3K9ac (β=0.20, p=0.02; β=0.16, p=0.05, respectively). H3K4me3, but not H3K9ac, was associated with zinc (β=0.13, p=0.03) and iron (β=0.32, p=0.01) contained in PM. ETP TM+ was increased in association with higher plasma H3K4me3 (β=0.50, p=0.05) and H3K9ac (β=0.54, p=0.05)., Conclusions: This observational study suggests potential roles of extracellular histones in PM-induced hypercoagulability. Experimental studies are warranted to further characterize these findings., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The study has been performed on tears of apparently healthy subjects who live and work in urban and rural areas, respectively. After the collection the following elements were investigated: chromium (Cr); arsenic (As); copper (Cu); zinc (Zn); selenium (Se); rubidium (Rb); barium (Ba); lead (Pb) and cobalt (Co). Significantly higher values of As were found in subjects living and working in rural areas as compared to those found in urban area residents (0.290 vs. 0.025; p<0.001). Conversely, Ba and Pb were significantly lower in rural area residents (1.10 vs. 2.50, p=0.027 and 1.70 vs. 1.10, p=0.057, respectively). Our data show that trace elements analysis in tears is possible; further studies could define if it could be a reliable biomarker in persons exposed to high concentration of trace elements due to working or environmental reasons., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Some organochlorine compounds, such as polychlorinated biphenyls (PCBs), have a tendency to bioaccumulate in humans and predators at the top of the food chain. We have recently confirmed the transplacental transfer of these compounds and the present study has been designed on the same material with the aim of investigating their potential health effects on newborns from 70 pregnant women, resident in a Northern Italy industrial town. Organochlorine compounds [namely, p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), p,p'-dichlorodiphenyldichloroethene (p,p'-DDE), hexachlorobenzene (HCB), and PCBs] have been analyzed both in cord and maternal serum, placenta, and maternal subcutaneous adipose tissue by GC-MSD. p,p'-DDT levels in the adipose tissue resulted significantly (p<0.05) related to birth length. Mothers of neonates born by preterm programmed caesarean delivery showed significantly (p<0.005 for both) higher serum p,p'-DDE serum concentrations and p,p'-DDT levels in the adipose tissue, as compared to mothers delivering at term., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Particulate matter (PM) exposure has been linked to increased risk of cardiovascular disease, possibly resulting from hypercoagulability and thrombosis. Lung and systemic inflammation resulting from PM inhalation may activate blood coagulation, but mechanisms for PM-related hypercoagulability are still largely unknown., Objectives: To identify coagulation mechanisms activated by PM in a population with well-characterized exposure., Methods: We measured prothrombin time (PT), activated partial thromboplastin time, endogenous thrombin potentials (ETPs) with/without exogenous triggers and with/without soluble thrombomodulin, tissue-type plasminogen activator (t-PA) antigen, D-dimer and C-reactive protein (CRP) in 37 workers in a steel production plant with well-characterized exposure to PM with aerodynamic diameter of < 1 mum (PM(1)) and coarse PM (PM(10) - PM(1)). Blood samples were collected from each subject on the first (baseline) and last (postexposure) day of a 4-day work week. We analyzed differences between baseline and postexposure levels using a paired Student's t-test. We fitted multivariate mixed-regression models to estimate the associations of interquartile range PM(1) and coarse PM exposure with parameter levels., Results: None of the parameters showed any significant changes from baseline in postexposure samples. However, exposure levels were associated with shorter PT (beta[PM(1)] = -0.33 s, P = 0.08; beta[PM(coarse)] = - 0.33 s, P = 0.01), and higher ETP without exogenous triggers and with thrombomodulin (beta[PM(1)] = + 99 nm min, P = 0.02; beta[PM(coarse)] = + 66 nm min, P = 0.05), t-PA (beta[PM(1)] = + 0.72 ng mL(-1), P = 0.01; beta[PM(coarse)] = + 0.88 ng mL(-1), P = 0.04), and CRP (beta[PM(1)] = + 0.59 mg L(-1), P = 0.03; beta[PM(coarse)] = + 0.48 mg L(-1), P = 0.01)., Conclusions: PM exposure did not show any short-term effect within the week of the study. The association of PM exposure with PT, ETP and CRP provides some evidence of long-term effects on inflammation and coagulation.

This study investigated nucleic acid oxidation associated with exposure to benzene at low levels in 239 workers recruited among traffic policemen, taxi drivers and gasoline pump attendants of the city of Parma (Italy). Biomarkers of exposure, namely urinary t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA), urinary cotinine, and urinary biomarkers of nucleic acid oxidation, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) were determined by liquid chromatography-tandem mass spectrometry. Relevant polymorphisms of NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferases M1-1 (GSTM1), T1-1 (GSTT1), and A1 (GSTA1) were characterized by polymerase chain reaction-based methods in a subgroup of subjects. Biomarkers of nucleic acid oxidation were correlated with each other (r> or =0.32, p<0.0001) and with exposure biomarkers (r> or =0.28, p<0.0001). Multiple linear regression models including age, sex and smoking habits as independent variables demonstrated that benzene exposure is associated with oxidation damage to nucleic acid, particularly to RNA (p<0.0001) and is modulated by the NQO1 polymorphism. The study confirmed a significant modulating effect of GSTM1 (p=0.010), GSTT1 (p=0.023) and GSTA1 (p=0.048) polymorphisms on S-PMA excretion, with a significant interaction between GSTM1 and both GSTT1 and GSTA1 (p=0.006 and p=0.037, respectively)., (2010 Elsevier Ireland Ltd. All rights reserved.)

A close link between stress protein up-regulation and oxidative damage may provide a novel therapeutic tool to counteract nephrotoxicity induced by toxic metals in the human population, mainly in children, of industrialized countries. Here we analysed the time course of the expression of several heat shock proteins, glucose-regulated proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of inorganic mercury and lead. Concomitantly, we used morphological and biochemical methods to evaluate metal-induced cytotoxicity and oxidative damage. In particular, as biochemical indicators of oxidative stress we detected reactive oxygen species (ROS) and nitrogen species (RNS), total glutathione (GSH) and glutathione-S-transferase (GST) activity. Our results clearly demonstrated that mercury increases ROS and RNS levels and the expressions of Hsp25 and inducible Hsp72. These findings are corroborated by evident mitochondrial damage, apoptosis or necrosis. By contrast, lead is unable to up-regulate Hsp72 but enhances Grp78 and activates nuclear Hsp25 translocation. Furthermore, lead causes endoplasmic reticulum (ER) stress, vacuolation and nucleolar segregation. Lastly, both metals stimulate the over-expression of MTs, but with a different time course. In conclusion, in NRK-52E cell line the stress response is an early and metal-induced event that correlates well with the direct oxidative damage induced by mercury. Indeed, different chaperones are involved in the specific nephrotoxic mechanism of these environmental pollutants and work together for cell survival.

Background: Polychlorinated biphenyls (PCBs) may interact with several thyroid functions. Severe environmental pollution by PCBs has been observed in Brescia, Italy, due to the presence of a factory that produced these compounds until 1984., Objectives: We aimed to investigate the correlation between serum levels of total PCBs and PCB 153 and thyroid hormone serum levels in adults in a cross-sectional population-based study., Methods: A total of 527 subjects were enrolled; they were face-to-face interviewed about their history of thyroid disease and provided a blood sample under fasting conditions. The serum levels of free and total triiodothyronine (FT3 and TT3), thyroxine (FT4 and TT4) and thyroid-stimulating hormone (TSH), anti-thyroperoxidase (anti-TPO) and anti-thyroglobulin (anti-TGA), and lipid-adjusted total PCBs and PCB 153 were determined., Results: There was no statistically significant difference in total PCB and PCB 153 concentrations in subjects with and without thyroid disease. Inverse, weak, statistically significant correlations were found between total PCBs and FT3 (Spearman's r=-0.09) and between total PCBs and PCB 153 and TSH (Spearman's r=-0.16 and -0.12, respectively). However, multiple regression analysis with FT3 and TSH serum levels as dependent variables and total PCBs and PCB 153 serum levels as predictors, including age, gender and BMI, showed a positive, rather than negative, association of PCBs and PCB 153 with FT3 and no association with TSH., Conclusions: This study does not support the hypothesis that relatively high PCB environmental exposure can determine substantial alterations in thyroid function among adult people.

A comparative evaluation of urinary biomarkers was carried out to characterize benzene exposure in a group of 100 traffic policemen of the city of Parma (Italy). All subjects were monitored once, in two consecutive days characterized by similar climatic conditions but preceded by two windy days. Benzene ambient concentration measured by municipal air monitoring stations was 1 microg/m(3) (Day 1) and 2 microg/m3 (Day 2). Personal exposure to ambient concentrations of benzene, toluene, ethylbenzene and xylene (BTEX) was assessed by using Radiello((R)) passive-diffusive samplers in a subgroup of 24 workers. Benzene metabolites, t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA) were determined by isotopic dilution liquid chromatography-tandem mass spectrometry on spot urine samples collected at the end of the shift. Urinary benzene (U-B) was determined by solid-phase microextraction gas chromatography-mass spectrometry. Airborne benzene concentration expressed as median [and interquartile range] was 6.07 [0.28-9.53] microg/m(3), as assessed by personal sampling. Urinary concentrations of biomarkers in the whole group were 41.8 [34.1-89.8] microg/g creatinine for t,t-MA, 0.67 [0.23-1.32] microg/g creatinine for S-PMA, and 0.16 [0.13-0.26] microg/l for U-B. Smokers eliminated significantly higher concentrations of unchanged BTEX and benzene metabolites than non-smokers (p < 0.05). When traffic policemen were distinguished into indoor (n=31) and outdoor workers, no significant differences were observed for either airborne benzene or urinary biomarkers. Significantly lower concentrations of S-PMA and U-B were determined in samples collected at Day 1 as compared to Day 2 (p < 0.0001 and p = 0.003, respectively) suggesting that these biomarkers are enough sensitive and specific to detect changes in airborne benzene concentration even at few microg/m(3).

An integrated approach based on ambient and biological monitoring, the latter including both biomarkers of exposure and susceptibility, was applied to characterize benzene exposure in a group of 37 taxi drivers of the city of Parma (Italy). Airborne benzene concentrations were assessed by 24 h personal sampling and work-shift sampling inside the taxicab using passive samplers (Radiello). Benzene metabolites, trans,trans-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA), and urinary cotinine as biomarker of smoking habits were measured by isotopic dilution liquid chromatography tandem mass spectrometry in both pre-shift (PS) and end-of-shift (EOS) samples. Urinary benzene (U-B) levels were determined by solid-phase microextraction gas chromatography-mass spectrometry in EOS samples. Relevant polymorphisms of microsomal epoxide hydrolase, NAD(P)H:quinone oxidoreductase, glutathione S-transferases M1-1 (GSTM1), T1-1, and A1 were characterized by PCR-based methods. Mean airborne benzene concentration was 5.85 +/- 1.65 microg/m3, as assessed by 24 h personal sampling integrating for work-shift, indoor or general environment activities. Significantly, higher benzene concentrations were detected in the taxicab during the work-shift (7.71 +/- 1.95 microg/m3, p < 0.005). Smokers eliminated significantly higher concentrations of U-B and S-PMA than non-smokers in EOS samples [geometric mean (geometric S.D.): 2.58 (4.23) versus 0.44 (1.79) microg/l for U-B; 3.79 (1.50) versus 2.14 (1.87) microg/gcreat. for S-PMA, p < 0.002]. Within smokers, S-PMA concentrations significantly increased at the end of the work-shift compared to pre-shift values (p < 0.05). t,t-MA showed a similar behaviour, although differences were not significant. In the narrow range examined, no correlation was observed between air benzene concentration and urinary biomarkers. All benzene biomarkers but EOS t,t-MA were correlated with U-cotinine (p < 0.05). GSTM1 polymorphism significantly modulated S-PMA excretion, as subjects bearing the GSTM1pos genotype [3.61 (1.15) microg/gcreat.] excreted significantly higher S-PMA concentrations than GSTM1null subjects [2.19 (1.18) microg/gcreat., p < 0.05].

Study Objectives: The aim of this study was to characterize the elemental composition of exhaled breath condensate (EBC) in order to identify new biomarkers of exposure and susceptibility in COPD patients. Serum pneumoproteins were used as lung-specific biomarkers of effect., Design: EBC was obtained from 50 healthy subjects, 30 healthy smokers, 30 asthmatics, and 50 patients with stable COPD, and was collected by cooling exhaled air. Trace elements and toxic metals in the samples were measured by means of inductively coupled plasma-mass spectrometry and electrothermal atomic absorption spectroscopy. The serum pneumoproteins were immunoassayed., Results: The EBC of COPD subjects had higher levels of such toxic elements as lead, cadmium, and aluminum, and lower levels of iron and copper, than that of the nonsmoking control subjects. There were no between-group differences in surfactant protein (SP)-A and SP-B levels. Clara-cell protein and SP-D levels were negatively and positively influenced, respectively, by tobacco smoke., Conclusions: Our results show that toxic metals and transition elements are detectable in the EBC of studied subjects. We propose new biomarkers of exposure as a means of assessing the target tissue dose of carcinogenic and pneumotoxic substances from tobacco smoke or polluted workplaces, and the use of the transition elements involved in redox systems of oxidative stress as disease biomarkers associated with effect or susceptibility. Together with biomarkers of effect, such as serum pneumoproteins, the elemental composition of EBC may be clinically useful in distinguishing similar diseases.

Chromium speciation has attracted attention because of the different toxicity of Cr(III), which is considered relatively non-toxic, and Cr(VI), which can cross cell membranes mainly as a chromate anion and has been classified as a class I human carcinogen. The aims of the present study were to measure soluble Cr(VI) levels in environmental samples, to develop a simple method of quantifying Cr(VI) in exhaled breath condensate (EBC), and to follow the kinetics of EBC Cr(VI) in chrome plating workers.Personal air samples were collected from 10 chrome platers; EBC was collected from the same workers immediately after the work shift on Tuesday and before the work shift on the following Wednesday. Environmental and EBC Cr(VI) levels were determined by means of colorimetry and electrothermal absorption atomic spectrometry, respectively.The method of detecting Cr(VI) in environmental air was based on the extraction of the Cr(VI)-diphenylcarbazide (Cr(VI)-DPC) complex in 1-butanol, whereas EBC Cr(VI) was determined using a solvent extraction of Cr(VI) as an ion pair with tetrabutylammonium ion, and subsequent direct determination of the complex (Cr(VI)-DPC) in EBC.Kinetic data showed that airborne Cr(VI) was reduced by 50% in airway lining fluid sampled at the end of exposure and that there was a further 50% reduction after about 15 h. The persistence of Cr(VI) in EBC supports the use of EBC in assessing target tissue levels of Cr(VI).

Tubular epithelium represents the primary target of mercuric ions (Hg(2+)) nephrotoxicity. Although widely investigated, the mechanisms of Hg(2+) cell uptake, accumulation and excretion all along the nephron remain largely unknown. In the present study, native distal tubular-derived Madin-Darby canine kidney (MDCK) cells exposed to subcytotoxic (micromolar) HgCl(2) concentrations were used for investigating specific mechanisms involved in the tubular response to toxic metals. Inductively coupled plasma-mass spectrometry (ICP-MS) was firstly used for assessing HgCl(2) solubility and then for quantifying Hg(2+) cell uptake. Exposed to HgCl(2), MDCK cells showed a rapid, but transient, Hg(2+) accumulation. The metallic cation was found to affect cell density and morphology, being these effects related to the dose and the time of exposure. In parallel, an Hg(2+)-induced up-regulation of endogenous MRP1 and MRP2 export pumps, a significant HgCl(2)-dependent induction of protective cellular thiols and an increase in the glutathione conjugates metabolism were also observed. The functional suppression of MRPs activity, obtained by MK-571 treatment, increased the Hg(2+) cell content and the sensitivity of MDCK cells to HgCl(2). Our results demonstrate that, in MDCK cells, inorganic Hg(2+) promotes the activation of specific detoxifying pathways that may, at least partly, depend on the activity of MRP transporters.

The present work tested the estrogenic activity of three weak environmental estrogens p,p'DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane], p,p'DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] and betaBHC [beta-benzene-hexachloride] in the transgenic estrogen-reporter mouse model (ERE-tK-LUC). By a time dependent analysis of the transgenic reporter expression (luciferase), we showed that all these chemicals modulated the estrogen receptors (ERs) in the whole body, although with a different efficacy and depending upon the tissue analyzed. Peak activity was registered at 16 h of treatment with 5000 microg/kg of each compound. Organochlorines are lipophylic molecules that accumulate in fat. During weight loss they are mobilized and their concentration increases in blood. We tested whether after experimental accumulation in fat tissue, followed by a 48 h period of fasting, these compounds could be modulated to reach sufficient levels to activate the ERs in target tissues. This experimental setting produced results that were different from those obtained following acute treatments. In loaded mice, fasting induced betaBHC mobilization resulted in strong ER activation in the liver, lung, eye, cerebellum, hypothalamus and cortex. p,p'DDT mobilization had no effect in these tissues, but efficiently acted in the testis, where, on the contrary, betaBHC inhibited reporter expression. During fasting, betaBHC, p,p'DDT and the metabolite p,p'DDE increased in blood concentration, from 2.7 +/- 0.36, 0.65 +/- 0.01 and 0.48 +/- 0.06 microg/ml to 9.51 +/- 1.1, 4.98 +/- 0.77 and 6.0 +/- 0.71 microg/ml, respectively. We conclude that these organochlorines modulate differently the expression of estrogen regulated genes in a tissue- and compound-specific manner and that their action is dependent on the energy balance. Moreover, we show that this mouse model is suitable to detect the estrogenic activity of chemicals with variable structures such as alkyl phenols and polychlorobiphenyls.

The polychlorinated biphenyls (PCBs) have been demonstrated to be inducers of hepatic microsomal enzymes and some of their effects such as hormonal imbalance, and alteration of lipid and porphyrin metabolism could be ascribed to this mechanism. For this reason, the urinary excretion of D-glucaric acid (DGA), an indirect indicator of enzymatic induction, was suggested as a biological marker of effect following exposure to PCBs. The aim of the present study was to investigate whether any inductive effects resulting from exposure to these compounds through ingestion of contaminated food could be detected early by measuring urinary DGA (U-DGA). U-DGA was measured in 73 subjects exposed to PCBs due to ingestion of PCB-contaminated food and levels ranged from 1.7 to 12.4 mmol/mol creatinine, with a mean value of 5.96 mmol/mol creatinine. These values were higher than those usually found in the general population. Sex and smoking habits did not affect U-DGA excretion, while age and alcohol intake were significantly correlated with U-DGA excretion, a finding in agreement with the results of other investigations. Neither total PCB blood concentration nor PCB chlorine content was significantly correlated with U-DGA excretion, and only the PCB 138 congener was weakly correlated with U-DGA levels. The results indicate that, for exposure to PCB resulting in blood concentrations up to 394 microg/l, no statistically significant effect of these persistent organochlorine compounds on human enzyme induction could be demonstrated, as measured by DGA urinary excretion.

The aim of the present study is to evaluate the influence of the genetic polymorphism of two enzymes involved in the biotransformation of xenobiotics, cytochrome P450 1A1 (CYP1A1) and glutathione-S-transferase M1 (GSTM1), on the urinary levels of 1-hydroxypyrene (1-OH-P) in workers exposed to polycyclic aromatic hydrocarbons (PAHs) and in unexposed workers (controls). The study group consisted of 30 controls recruited among employees of a service company and 171 PAHs-exposed workers from two electric steel plants and an iron foundry (all males, ranging between 18 and 60 years of age). Determination of airborne PAHs and urinary 1-OH-P was performed by high-performance liquid chromatography (HPLC) with fluorimetric detection. Polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was used to determine the genetic polymorphisms of CYP1A1 (CYP1A1*2A and CYP1A1*2B) and GSTM1. No influence of the genetic polymorphism of CYP1A1 and GSTM1 on the urinary levels of 1-OH-P was observed in this study.

Occupational and environmental medicine traditionally dealt with elements, particularly with heavy metals. The interest was justified by the wide exposure in the workplace and in the general environment and by the evidence of their specific biological and toxicological effects. During the last 2 decades of 20th century the availability of indicators of exposure or of internal dose has substantially increased thanks to improvement in AAS-ETAAS techniques and to the entrance of ICP-MS into the field of biological monitoring. There are now more and more demands for controlling pre-analytical and analytical factors, for analysing biological matrices in addition to blood and urine and for setting up methods for elements not yet extensively studied in respect to their possible biological or toxicological role. Finally, deeper knowledge has to be reached in order to evaluate the significance of elements and, possibly, of their species in biological fluids at current doses and in order to face their effects, especially those in the first portion of the dose-response curve, which is going to be the main field of interest of occupational and environmental toxicology for the next few years., (Copyright 2002 Elsevier Science B.V.)

The results of a polycentric study to assess the reference values of urinary mercury (U-Hg) in four Italian cities are presented. A total of 383 subjects were selected on the basis of standardised criteria by a questionnaire on personal habits, lifestyle, occupational or non-occupational exposure to Hg, medical history, number and area of dental amalgams. U-Hg was determined by hydride generation atomic absorption method (HG-AAS), with a detection limit of 0.5 microg/l and by flow injection (FI) inductively coupled plasma mass spectrometry (ICP-MS), with a detection limit of 0.03 microg/l. The median value of U-Hg, determined by HG-AAS, was 0.78 microg/g creatinine (0.75 for males and 0.83 for females), with 5 degrees and 95 degrees percentiles, respectively, of 0.17 and 3.66 microg/g creatinine. When determined by FI ICP-MS, the median value was 0.79 microg/g creatinine (0.77 for males and 0.79 for females) with 5 degrees and 95 degrees percentiles of, respectively, 0.12 and 5.02 microg/g creatinine. Among the independent variables, city of origin, area of dental amalgams, fish intake and tobacco smoking significantly influenced the U-Hg levels. The U-Hg reference values from this survey are lower than those from other recent investigations, probably due to characteristics and selection of the examined individuals and to the strict control of pre-analytical and analytical factors of variability.

This paper presents the results of a polycentric study carried out in seven different areas, organized by the Italian Society of Reference Values (SIVR) for assessing reference values of lead in blood (B-Pb) at the current doses of the metal to general population. The estimated arithmetic mean for B-Pb in males was of 45.1 microg/l and 30.6 microg/l in females; the 95th centile was 100 and 60 for males and females, respectively. The main variables influencing B-Pb levels were gender, age, BMI, outside sport practice, alcohol consumption and smoking habits, while the geographic area and the urban residence did not affect the metal concentration in blood.

Although it is generally accepted that reference values can be included among the instruments of modern occupational medicine, problems arise when applying them from clinical chemistry to the needs of occupational medicine. Here some general aspects regarding reference values beginning from their theoretical basis, their significance and importance and their possible use in occupational medicine are reviewed. Furthermore, their relationship with other more familiar 'guideline values', such as action levels and limit values, is demonstrated.

Urinary levels of 1-hydroxypyrene in a general adult population group are studied. Experimental data are not normally distributed; statistical analysis required a base 10 logarithmic transformation of data. The concentrations of urinary 1-hydroxypyrene measured were expressed as microgram g-1 urinary creatinine and are comparable with those reported by other authors, both for smoker and non-smoker subgroups. Multiple regression analysis shows that, for smokers, the number of cigarettes smoked per day and the body mass index (BMI) significantly influence the levels of urinary 1-hydroxypyrene expressed as microgram g-1 urinary creatinine, whereas no personal or behavioural variable (age, sex, alcohol consumption, dietary intake of pyrene, BMI) modified the 1-hydroxypyrene levels for non-smokers.

In this study we evaluated the effects of two heavy metals, lead and manganese, on the release of some glycohydrolases of lysosomal origin. N-acetyl-beta-D-glucosaminidase and its major isoenzymes, beta-D-glucuronidase and alpha-D-galactosidase. We have studied release of these enzymes in vitro from peripheral mitogen-activated lymphocytes from healthy subjects after addition of Pb or Mn to the medium and their plasma levels in individuals exposed at work to Pb (31 subjects) or to manganese (36 subjects), versus matched controls. We also determined the plasma levels in a general population (417 subjects). The enzymatic activities were assayed fluorimetrically with 4-methylumbelliferyl-glycosides as substrates. Particular attention was given to some technical aspects: enzymatic activity was preserved by addition of ethylene glycol and stable liquid material was employed for calibration purposes. N-acetyl-beta-D-glucosaminidase isoenzymes were separated by a routine chromatofocusing procedure on PBE 94. The addition of both metals to lymphocytes inhibits lysosomal enzyme release. These data were supported by the plasma levels for the exposed subjects, in which enzyme levels were significantly decreased after either type of exposure. In the general population of subjects not professionally exposed, the effect of lead appears to be masked by concomitant effects of alcohol consumption. Undoubtedly, some heavy metals can alter distribution of glycohydrolases of lysosomal origin between the intra- and extracellular environment, probably interfering with membrane mechanisms. Lysosomal enzymes seem to behave as sensitive biomarkers for early subclinical changes that might later lead to clinical disease.

A cross-sectional study was conducted in 35 male subjects randomly selected from workers of a ferroalloy production plant and exposed to manganese (Mn) oxides; the objective was to detect early signs of neurologic impairment. The subjects' mean age was 39.4 years (SD, 8. 4); the average exposure duration was 14.5 years (range, 5-29 years). A control group of industrial workers not exposed to neurotoxic chemicals and comparable in age and confounding factors was recruited. The intensity of Mn exposure was moderate, as reflected by airborne Mn concentrations in total dust averaging 193 [corrected] micro g/m3. Mn levels in blood (MnB) and urine (MnU) were significantly higher in the Mn-exposed workers than in control workers. A relationship (not found with MnU) was found between MnB and a cumulative exposure index calculated on the basis of air concentration and exposure history for each subject (r = 0.52; r2 = 0.27; P = 0.002). Psychomotor function scores were lower among Mn-exposed subjects. The Aiming score was negatively correlated with MnB in the exposed group. The olfactory threshold did not differ between the two groups, although it was negatively associated with MnU in the exposed group. The white blood cell count results were significantly higher in Mn-exposed subjects than in controls. These findings show that an increase in Mn body burden is associated with an impairment of motor functions, whereas the increased excretion of Mn is related to an increased olfactory perception. Changes in numbers of leukocytes could indicate possible interferences of Mn with the immunological system., (Copyright 1997 Academic Press.)

Controlling occupational exposure to chemical substances in biomedical laboratories is very complex because of the high number of compounds involved, and the rapid changes in analytical techniques and methods. On the other hand, evidence of increased mortality for malignant lymphoma and leukemia has been observed in laboratory technicians. This calls for more precise procedures of risk assessment to identify the specific substances that may be responsible for these effects. A model of hazard identification is presented, which includes the following steps: identification, classification and periodical updating of the chemical substances used in each analytical technique; evaluation of the variables that can influence the exposure; quantification of exposure using environmental measurements. The availability of such a procedure can be useful for the organization of health and epidemiological surveillance programs for the laboratory workers.

Quality assurance (QA) concerns the validity of all the analytical processes (from collection of the samples to interpretation of the results). It is not an abstract concept but must be adapted to the different situations such as the different exposure levels, the different analytical methods, and the context of use (risk assessment procedures, research, routine determinations). The main requirements in QA programmes regard the control of all the known sources of preanalytical and analytical variations, while the instruments with which adequate QA can be implemented are the certified materials and the quality control programmes (quality manual, internal and external quality controls). Another important concept in QA is that measurements must be placed a different metrological levels: at the highest there are the methods (definitive, reference) to be used for assessing accuracy of routine methods. QA programmes should enable a grading of biomarkers (from experimental only to full evaluated) and of the laboratories in order to identify the significance of the test and to assess the level at which a laboratory could operate.

In a preliminary study of 11 men randomly selected in a ferro-alloy plant and of 15 control subjects, platelet monoamine oxidase (MAO) and serum dopamine beta-hydroxylase (DBH) activities were measured. A tendency towards lower MAO-B activity in the exposed workers as compared to control subjects (t = 1.95; P = 0.06) was found whereas DBH activity was similar. In the exposed group, a dose-effect relationship was noted between a manganese (Mn) cumulative exposure index (CEI) and DBH activity (R2 = 0.40, P < 0.05). Since DBH is an expression of catecholamine release, the relative increase in such activity could be envisaged as a compensatory mechanism to a reduced turnover rate as reflected by MAO-B activity. Owing to the limited sample size, these findings should be confirmed by further epidemiological and experimental studies.

The progressive improvement of hygienic conditions in the workplace has increased the importance of obtaining detailed information on extra-occupational factors that might influence the levels of the biological indicators. This information is indispensable both when subjects belonging to the general population are selected for establishing 'reference values' and when subjects occupationally exposed to specific chemical substances are studied. In non-occupationally exposed subjects the biological indicator levels may be influenced by circumstances which enhance absorption of the substance in question. Examples of interference factors considered for biological indicators of main metals are: gender, age, smoking habits, alcohol consumption and dietary habits. In occupationally exposed subjects the levels of the biological indicators can be influenced by factors that interfere with the metabolism of the substances absorbed in the workplace. In particular, factors such as alcohol, drugs and tobacco appear to play an important role in modifying the biological indicator levels in the occupationally exposed. Ethanol can inhibit as well as induce the metabolism of solvents. Inhibition occurs after excessive ingestion of ethanol, whereas induction occurs in subjects who regularly consume alcohol. There are several examples of inhibition of the metabolism of solvents by alcohol in man, occurring at levels of exposure frequently encountered in the workplace, also within the 'occupational exposure limits', (OEL). Conversely, there are very few studies on the effects of induction, which presumably occur only when the exposure levels greatly exceed OEL. Among drugs, analgesics seem to play a particular role in interfering with the metabolism of solvents. Since cigarette smoking is frequently associated with alcohol ingestion at present it is difficult to extrapolate the isolated effect of smoking on the metabolism of solvents. In order to facilitate interpretation of the results of biological monitoring, we propose to prepare informative sheets for the main substances which will contain information on factors that can influence the levels of the indicators.

Reference values for blood-cadmium levels (B-Cd) are available for only a limited number of geographical areas and for particular population strata (sex, age, smoking habits). This paper, in agreement with the TRACY guidelines, describes and discusses the criteria used to rank published papers on reference values for cadmium retrieved by Medline and Toxline between 1976 and 1991. The TRACY criteria deal with the grading of published papers in terms of their suitability for calculating provisional reference values. Only four out of 18 papers were considered suitable for the TRACY project. The four articles were finally used via meta-analysis to provide provisional reference values for smokers and non-smokers. The comparison of results obtained using published statistics and individual data is used to discuss the appropriateness of meta-analysis in the case of cadmium. Due to the availability of large enough studies and to the clear differences across countries, the suitability of a compound upper reference limit to B-Cd levels seems limited.

In order to evaluate the urinary excretion of cobalt, a study was carried out in two plants producing diamond segments and sintered wires for stone cutting. In both plants, the highest Co exposures were found during mixing and granulation of Co powders, generally performed 1-2 times per week for 1-2 h in the first plant and 3-4 h in the other. Environmental hygiene conditions were good in the first plant and poor in the other with environmental Co concentrations around the ACGIH TLV (50 micrograms/m3) in the first and many times higher (up to 8000 micrograms/m3) in the second. In two workers employed in the above mentioned jobs. Cobalt in urine (CoU) was determined in samples collected during the first day, before shift and about 2, 4, 7 and 10 h after the beginning of exposure; samples were also collected on the 2nd, 3rd and 4th day, before and at the end of the shift. The study showed that Co in urine rapidly increased in the hours following the cessation of exposure, with a peak of elimination about 2-4 h after exposure, and a subsequent decrease (more rapid for the first 24 h) in the following days. Such a pattern was apparently independent of the degree of exposure, since it was detected in both plants and confirmed also in an industrial hygienist with lower Co exposure. The data indicate that for a correct evaluation of Co exposure through CoU determination, variables such as type and characteristics of the job and most importantly, the times at which biological samples are collected should be carefully considered.

Two types of selection are used to identify subjects who make up reference groups: 'a posteriori' selection starting from a large number of subjects who are then subsequently selected and 'a priori' selection wherein the group to be examined is defined on the basis of preestablished criteria. The main variability factors are biological and those depending on the modalities of selection of groups and determination of the analysis. The nature of the element is also particularly important, i.e. whether an essential element must be considered only a micropollutant or whether it should be considered essential and at the same time a micropollutant. In addition two other factors which play an important role are the temporo-spatial modifications induced by natural or anthropogenic phenomena which influence the modality and degree of exposure in man. Two criteria are generally used in the production of reference groups: exclusion/inclusion and partitioning criteria. While in clinical chemistry the health status represents an important exclusion factor, in the case of metals, the environmental conditions and the function of organs and apparatuses involved in the toxicokinetics of metals can be considered as main criteria. All the other variables such as sex, age, life-style are partitioning criteria and enable us to obtain reference values for more and more homogeneous subgroups.

In order to evaluate whether hypertension can be considered as a confounding factor in the setting up of reference values for blood lead, we examined the results of a cross sectional study which evaluated the relationship between lead in blood and hypertension in a sample of 254 males and 271 females of a general population not occupationally exposed to lead. The statistical analysis and in particular the multiple logistic regression showed that, even if some well-known confounding factors such as age, sex, overweight, smoking and alcohol are taken into account, blood lead levels are well correlated with hypertension. The results suggest that even modest lead absorption is able to influence the probability of being hypertensive. The relationship between blood lead and hypertension and their relationship with the main confounding factors involved in the determination of reference values of metals in blood are discussed.

Neutron activation analysis-electrothermal atomic absorption spectroscopy (ETA-AAS) and inductively coupled plasma atomic emission spectrometry (ICP-AES) have been used for the determination of 46 elements in urine, 35 in blood and 26 in serum of unexposed Italian subjects living in the same region (Lombardy). The results allowed the proposal of reference values for various elements determined in more than 350 healthy subjects, these being Ag, Al, As, Be, Bi, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Tl, V, Zn, in urine; Ag, As, Bi, Cd, Cr, Co, Cu, Hg, Pb, Se, Tl, Zn in blood; and Ag, Al, Be, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Se, Tl, V, Zn in serum (or plasma). For all other elements indicative values are suggested. In addition to the mean value and the "reference range", a "range of uncertainty" and an upper limit above which metabolic abnormalities could be expected have also been defined on the basis of simple statistical considerations.

Urinary mutagenic activity detected by the bacterial fluctuation assay, using Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA with and without metabolic activation (S9 mix), was studied in a group of 21 workers exposed to inorganic lead and a control group of 22 non-occupationally exposed subjects. Occupational exposure to inorganic lead had no effect on urinary mutagenicity in the strains considered, with or without metabolic activation. In smokers (exposed and non-exposed), urinary mutagenic activity appeared to increase compared to non-smokers (exposed and non-exposed), only with Salmonella typhimurium TA98 in the presence of S9 mix.

Using a sensitive ETA ASS method, urinary thallium (Tl-U) has been measured in a group of non-exposed individuals and in two groups of workers with suspected industrial exposure. In non-exposed subjects (72 healthy males aged 41 +/- 11 years) the Tl-U mean value was 0.22 mu/l (range 0.05-0.61). Significantly higher values were found in two groups of workers (30 and 21 subjects) employed in two cement factories and two cast iron foundries (mean Tl-U value: 0.38 and 0.33 mu/l, respectively; range 0.06-1.2). Thallium has been determined in coal, raw materials and, in one factory, also in environmental samples, but no detectable levels were found. The analytical problems connected to urinary thallium determination and the preliminary results of biological monitoring in non-exposed individuals and in workers are discussed.

The concentration of total urinary chromium (Cr-U) in two different groups of healthy occupationally non-exposed subjects living in the Italian regions of Lombardia and Veneto has been determined using an ETA-AAS direct method. The age, sex, residence and smoking habits were correlated with CrU excretion. The mean CrU value found in subjects living in Lombardia (n = 310, males 170 and females 140) was 0.59 + 0.26 microgram/l (range 0.08 - 2.10); in Veneto subjects (n = 238, males 187 and females 51) the mean value was 0.52 +/- 0.31 microgram/l (range 0.05 - 1.90). A significative difference between male and female values and a decrease of CrU levels with age increasing have been evidenced in both groups. Smoking habits seem to influence metal urinary levels while, on the other hand, no influence of living sites was observed. Some analytical problems of ETA-AAS Cr determination systems are discussed and "normal" CrU values measured in this study are compared with data reported in the literature.