Tiziana Schilirò

Analysis of environmental endocrine disrupting chemicals using the E-screen method and stir bar sorptive extraction in wastewater treatment plant effluents

Endocrine disrupting chemicals (EDCs) have become a major issue in the field of environmental science due to their ability to interfere with the endocrine system. Recent studies show that surface water is contaminated with EDCs, many released from wastewater treatment plants (WWTP). This pilot study used biological (E-screen assay) and chemical (stir bar sorptive extraction-GC-MS) analyses to quantify estrogenic activity in effluent water samples from a municipal WWTP and in water samples of the recipient river, upstream and downstream of the plant. The E-screen assay was performed on samples after solid phase extraction (SPE) to determine total estrogenic activity; the presence of estrogenic substances can be evaluated by measuring the 17-beta-estradiol equivalency quantity (EEQ). Untreated samples were also assayed with an acute toxicity test (Vibrio fischeri) to study the correlation between toxicity and estrogenic disruption activity. Mean EEQs were 4.7 ng/L (+/-2.7 ng/L) upstream and 4.4 ng/L (+/-3.7 ng/L) downstream of the plant, and 11.1 ng/L (+/-11.7 ng/L) in the effluent. In general the WWTP effluent had little impact on estrogenicity nor on the concentration of EDCs in the river water. The samples upstream and downstream of the plant were non-toxic or weakly toxic (0<TU<0.9) while the effluent was weakly toxic or toxic (0.4<TU<7.6). Toxicity and estrogenic activity were not correlated. At most sites, industrial mimics, such as the alkylphenols and phthalates, were present in higher concentrations than natural hormones. Although the concentrations of the detected xenoestrogens were generally higher than those of the steroids, they accounted for only a small fraction of the EEQ because of their low estrogenic potency. The EEQs resulting from the E-screen assay and those calculated from the results of chemical analyses using estradiol equivalency factors were comparable for all samples and closely correlated.

Occupational exposure to formaldehyde and biological monitoring of Research Institute workers

AIM The aim of this study was to verify the presence of a relationship between formaldehyde exposure in the work environment with biological markers of exposure and of effect. METHODS Exposure to formaldehyde (FA) of 36 workers in different laboratories of a Cancer Research Institute and biomarkers of exposure, such as formaldehyde human serum albumin conjugate (FA-HSA) and biomarkers of effect, such as chromosome aberration (CA), micronuclei (MN) and sister chromatid exchanges (SCEs) were measured in peripheral blood lymphocytes of the same workers. RESULTS Individual FA levels of exposure ranged from 4.9 microg/m(3) to 268.7 microg/m(3). Subjects with high FA exposure showed a significant increase of the biomarker of exposure FA-HSA, but biomarkers of effect did not show any significant differences. CONCLUSIONS A significant relationship was observed between occupational exposure to FA and a biological marker of exposure (FA-HSA). The markers of effect used (CA, MN and SCE) failed to indicate the presence of genetic damage.

Ambient Air Levels and Occupational Exposure to Benzene, Toluene, and Xylenes in Northwestern Italy

The purpose of this study was to determine benzene, toluene, and xylenes air pollution in two cities in Italy (Biella and Torino) having different traffic intensities and to investigate whether new environmental conditions occurred consequent to the changes of gasoline composition in Europe during the last 20 yr. Furthermore, three types of urban occupational exposure (petrol pump attendants, traffic policemen, and municipal employees) to the same hydrocarbons were compared to verify three different expected levels of exposure. Results in Biella demonstrate a direct relationship between traffic density and level of human exposure to these pollutants. Air concentrations for benzene were 2.3 w g/m 3 in a suburban area having low traffic and 10.3 w g/m 3 in the central area having high traffic. The comparison to trend analysis recently carried out in Torino indicates it is possible to improve the situation in the central area of Biella by adopting the same traffic limitations imposed in Torino. Personal sampling devices demonstrated that only the petrol pump attendants show, by means of a multivariate analysis, statistically significant higher levels of benzene compared to the other two professional categories, in both winter and summer. Values found in the present study for petrol pump attendants were around 1 mg/m 3 . Environmental and occupational exposure to benzene, toluene, and xylenes could be largely lowered by adopting preventive measures including traffic restrictions, the reduction of aromatic chemical content in gasoline, and the recovery of gasoline vapors at petrol pump stations.

The endocrine disrupting activity of surface waters and of wastewater treatment plant effluents in relation to chlorination

The present study investigated the biological quantification of estrogenic activity in the effluent of a wastewater treatment plant (WWTP) and in the recipient river in north-western Italy. Samples of the WWTP effluent and those of river water upstream and downstream the WWTP were taken from September 2006 to May 2007. The effluent was evaluated in the presence and absence of chlorination. The E-screen assay, with human estrogens receptor-positive MCF-7 BUS breast cancer cells, was performed on samples after solid-phase extraction to determine the total estrogenic activity by measuring the 17beta-estradiol equivalent quantity (EEQ). In order to study the correlation between estrogenicity and toxicity, untreated samples were also assayed with the acute toxicity test Microtox. Furthermore, to determine the efficiency of the chlorination process, all the samples were analysed for disinfection by-products (trihalomethanes) and Escherichia coli. The mean EEQs were 5.0 ng/L (+/-6.1 ng/L) upstream of the plant, 6.7 ng/L (+/-7.4 ng/L) downstream from the plant and 23.3 ng/L (+/-20.4 ng/L) in the WWTP effluent. The difference between upstream and downstream of the treatment plant was not significant. Chlorinated water samples had lower estrogenic activity and E. coli concentrations, but had greater toxicity and higher trihalomethane concentrations. Estrogenic activity was not correlated with toxicity. These results suggest that the WWTP effluent had little impact on the estrogenic activity of the recipient river.

Endocrine disrupting activity in fruits and vegetables evaluated with the E-screen assay in relation to pesticide residues.

Food is likely to be one of the most important routes of human exposure to endocrine disrupting compounds (EDCs). In the present study, we evaluated the total estrogenic activity of fruits and vegetables, which was calculated using the human breast cancer cell line (MCF-7 BUS) proliferation assay (E-screen), in relation to pesticide residues. We analysed 44 food samples, 30 fruits and 14 vegetables. Of these samples, 10 did not contain any pesticide residues. The other 34 samples contained from 1 to 7 pesticide residues in concentrations ranging from 0.03 to 1.91 ppm. Estrogenic activity was detected in the 59% of samples tested. The positive controls used were 17-β-estradiol (E2), the phytoestrogen genistein and the pesticide endosulfan. The average value of estradiol equivalency quantity (EEQ) for all positive samples was 0.15±0.32 μg/100g. A low correlation was found between the concentration of pesticide residues and the EEQ values (Spearman correlation r=0.376 and p=0.012). Using values obtained from the literature, we compared the estrogenic activity of food samples with the intrinsic content of phytoestrogens, but we found no correlations. Our results also suggested that the calculated intake of dietary EDCs might represent a concentration comparable to the normal endogenous estrogen concentration in human blood.

Combined Analysis of Chromosomal Aberrations and Glutathione S-transferase M1 and T1 Polymorphisms in Pathologists Occupationally Exposed to Formaldehyde

The formaldehyde (FA) genotoxic potential in occupationally exposed individuals is conflicting. A relevant indoor-air FA pollution was found in hospitals and scientific institutions where FA is used as a bactericide and tissue preservative. In the present study, we evaluated the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes from workers in pathology wards who have been exposed to FA, compared with a group of unexposed subjects. The subjects were also analyzed for the GSTM1 and GSTT1 metabolic gene polymorphisms. The exposed subjects showed a significant increase in the frequency of CA per cell and in the percentage of cells with aberrations compared to control subjects. The different GST genotypes did not affect the level of cytogenetic damage since CA frequencies were not statistically different between the GST “null” genotypes and the GST “positives”. The generalized linear models showed that the number of CAs and cells with CAs increased with age, but, independent of age, it was significantly higher in the experimental rather than in the control group. Cubic-spline regression confirmed the linear relationship between CAs and age, but it provided evidence for a non-linear relationship between CAs and the number of years of FA exposure. Similar results were observed when the model included the number of cells with CAs as dependent variables. Our results demonstrate that air FA induces CAs even consequently to low levels of daily exposure, indicating an increased risk of genetic damage for workers exposed to this air pollutant.

PM10 size distribution of metals and environmental-sanitary risk analysis in the city of Torino.

The mechanisms responsible for negative biological effects due to airborne particulate matter (PM) exposure are still being studied, however the interactions between metals and biologic systems seem to be of primary importance. The aim of the study was to estimate a healthy risk linked to exposure to airborne PM10 metals by means of an environmental-sanitary risk assessment. Metals PM10 size distribution analysis was carried out in a central site of Torino city - Italy, then the Target Hazard Quotient (THQ) and the Cancer Risk (CR) were applied, according to standard EPA methods. All sampled metals were present on the different PM10 fractions, however some metals were distributed in some specific fractions: ANOVA test shows Cr, Cu, Mo and Pb as differently distributed among the eight fractions, rising the hypothesis of potential effects in specific tracts of respiratory system. Regarding the risk assessment, in general the CR was higher for an adult than for a child, conversely the THQ resulted higher for a child. While the concentrations of all the sampled metals appeared to be under control, their presence in the different PM10 fractions and their THQ and CR provided indications related to the body districts potentially in contact with these substances.

Cotinine and N-(2-hydroxyethyl)valine as markers of passive exposure to tobacco smoke in children

Large segments of populations, including children, are exposed to environmental tobacco smoke (ETS), a risk factor for lung cancer and heart, circulatory and respiratory diseases. Recently, ETS was classified as a class A carcinogen by USEPA, as carcinogenic to humans by IARC (group 1) and by the National Toxicology Program of the US National Institutes of Health. Cotinine, a product of the metabolism of nicotine, is measurable in urine and, correlates strictly and directly to ETS exposure, therefore representing a well-known internal dose marker. Another marker of active tobacco smoking is the N-(2-hydroxyethyl) valine (HOEtVal) which results from the reaction between ethylene oxide (EtO) and the N-terminal valine of hemoglobin. The aim of this study was the evaluations of ETS markers, namely urinary cotinine and HOEtVal measured in blood in 100 children with ages ranging between 3 and 13 years. Experimental findings show that cotinine, as a specific internal dose marker, and HOEtVal, as a nonspecific biological effective dose marker, both depend on the passive exposure to ETS as well as on the active habit of smoking.

Chemical characterisation and cytotoxic effects in A549 cells of urban-air PM10 collected in Torino, Italy.

Human type II alveolar cells (A549) were exposed to aqueous- and organic-solvent PM10 extracts to evaluate their effects on cell proliferation, proinflammatory cytokine release and cytotoxicity (assayed by lactate dehydrogenase, LDH, activity). PM10 samples collected in Torino (northwest Italy) were analysed for inorganic chemical species (bioavailable iron and secondary particulates) and endotoxins, which are potentially inflammatory promoters in human airways. During the sampling period, PM10 concentration was 55.4±39.1μg/m(3), secondary particles constituted 42±9% of the PM10 total mass, and bioavailable iron concentration was 0.078±0.095μg/m(3). PM10 inhibits cell proliferation and induces both IL-6 and LDH release in a dose- and time-dependent manner, with a seasonal trend. The different effects of aqueous and organic PM10 extracts demonstrate the importance of particle composition for the induction of cytotoxic effects on A549 cells. A first comparison between the biological effects induced by PM10 extracts and PM10 components was performed.

Monitoring air pollution effects on children for supporting public health policy: the protocol of the prospective cohort MAPEC study

Introduction Genotoxic biomarkers have been studied largely in adult population, but few studies so far have investigated them in children exposed to air pollution. Children are a high-risk group as regards the health effects of air pollution and some studies suggest that early exposure during childhood can play an important role in the development of chronic diseases in adulthood. The objective of the project is to evaluate the associations between the concentration of urban air pollutants and biomarkers of early biological effect in children, and to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in children. Methods and analysis Two biomarkers of early biological effects, DNA damage by the comet assay and the micronuclei (MN) test, will be investigated in oral mucosa cells of 6–8-year-old children. Concurrently, some toxic airborne pollutants (polycyclic aromatic hydrocarbon (PAH) and nitro-PAH) and in vitro air mutagenicity and toxicity in ultra-fine air particulates (PM0.5) will be evaluated. Furthermore, demographic and socioeconomic variables, other sources of exposures to air pollutants and lifestyle variables will be assessed by a structured questionnaire. The associations between sociodemographic, environmental and other exposure variables and biomarkers of early biological effect using univariate and multivariate models will be analysed. A tentative model for calculating the global absolute risk of having early biological effects caused by air pollution and other variables will be proposed. Ethics and dissemination The project has been approved by the Ethics Committees of the local Health Authorities. The results will be communicated to local Public Health Agencies, for supporting educational programmes and health policy strategies. LIFE+2012 Environment Policy and Governance. LIFE12 ENV/IT/000614.