Estrogenic activity in wastewater treatment plants through in vitro effect-based assays: Insights into extraction phase.

Effluents of wastewater treatment plants can abundantly spread endocrine disrupting chemicals in the environment. To improve water quality monitoring, the use of effect-based tools that measure estrogenic activity has been suggested, however their results could be influenced by different factors. This study compared the estrogenic activity of wastewater samples extracted with two stationary phases and tested with two in vitro effect-based assays to investigate whether and how stationary phases and assays could influence biomonitoring data. During four seasonal periods, the effluents of six WWTPs located in northern Italy were sampled. After the extraction using two different stationary phases (HLB, C18), the samples (n = 72) were tested using two effect-based assays: a gene reporter luciferase assay on mammalian cells (MELN) and yeast estrogen screen assay (YES). The results showed that estrogenic activity of HLB extracts was significantly different from the activity of C18 extracts, suggesting that extraction phase can influence biomonitoring data. Moreover, the estrogenic activity was overall higher using gene reporter MELN assay than using YES assay, suggesting that, due to difference in cell membrane permeability and metabolic activation, the applied cell model can affect the biomonitoring results. Finally, from the comparison between the activity of the final effluent and the environmentally safe estrogenic levels in surface waters, MELN data suggested that the activity of this effluent may pose an environmental risk, while YES data showed that it should not be considered a threat to the receiving surface waters. This study pointed out that a standardized approach is needed to assess the estrogenic activity of waters; it reported important data to select the most suitable stationary phase for samples extraction (samples extracted with C18 sorbent showed higher estradiol equivalent concentration values) and the most appropriate bioassay (gene reporter luciferase MELN assay was more sensitive than YES assay) to assess the environmental risk, thus protecting human health.

Atmospheric endocrine disruptors: A systematic review on oestrogenic and androgenic activity of particulate matter.

The alarming human health effects induced by endocrine disruptors (ED) have raised the attention of public opinion and policy makers leading worldwide to regulations that are continuously improved to reduce exposure to them. However, decreasing the exposure levels is challenging because EDs are ubiquitous and exposure occurs through multiple routes. The main exposure route is considered ingestion, but, recently, the inhalation has been hypothesized as an important additional route. To explore this scenario, some authors applied bioassays to assess the endocrine activity of air. This review summarizes for the first time the applied methods and the obtained evidences about the in vitro endocrine activity of airborne particulate matter (PM) collected outdoor. Among the bioassay endpoints, (anti)oestrogenic and (anti)androgenic activities were selected because are the most studied endocrine activities. A total of 24 articles were ultimately included in this review. Despite evidences are still scarce, the results showed that PM can induce oestrogenic, antioestrogenic, androgenic and antiandrogenic effects, suggesting that PM has an endocrine disrupting potential that should be considered because it could represent a further source of exposure to EDs. Although it is difficult to estimate how much inhalation can contribute to the total burden of EDs, endocrine activity of PM may increase the human health risk. Finally, the results pointed out that the overall endocrine activity is difficult to predict from the concentrations of individual pollutants, so the assessment using bioassays could be a valuable additional tool to quantify the health risk posed by EDs in air.

Determination of hexavalent chromium in textiles of daily use by ion chromatography and dermal risk assessment.

The determination of hexavalent chromium in textiles and clothes is challenging since during extraction, the original oxidation state should not be altered. Since, as a matter of fact, current analytical methods are focused only on total chromium determination, the purpose of this research is to develop a reliable analytical method for the determination of Cr(VI) in textiles and tissues of daily use for a reliable application of risk analysis models, which are usually based on total Cr data. After optimization, a 0.0025 M Na3PO4 extraction solution was selected for the extraction of Cr(VI) from textiles. This solution minimizes possible interconversion redox reactions and interference, and provides good extraction recoveries (88.4 ± 2.5% - 105.5 ± 0.6 %, according to Cr(VI) concentration) and quantitation limits (0.017 mg/kg), fully complying the current limits set for Cr(VI) in textiles in contact with skin, and for leachable Cr(VI). The developed method was validated investigating intra-day repeatability (n = 10) and inter-day repeatability (n = 30) which were below 12%, and matrix effect which was below 6% confirming the precision of the method and the negligibility of a matrix interference during the whole analysis. The method, which was proved to be suited also for bioaccessibility studies in saliva and sweat, was applied to the analysis of tank top, coloured paper napkin, polyamide tights, panties, highlighting Cr(VI) content in the panties only at very low concentration (0.028 mg/kg). As verified by ECHA and US EPA approaches, this amount does not pose a non-carcinogenic risk for human health. As regards carcinogenic risk, considering both adult and child exposure, the dermal contact with the panties poses an acceptable risk (R ≤ 10-6).

Can oestrogenic activity in air contribute to the overall body burden of endocrine disruptors?

Endocrine disruptors (EDCs) are emerging contaminants that are harmful to health. Human exposure occurs mainly through ingestion or dermal contact, but inhalation could be an additional exposure route; therefore, this study was conducted to evaluate the oestrogenic activity of airborne particulate matter (PM). Outdoor PM was collected for a year in five Italian sites and extracted with organic solvents (four seasonal extracts/site). The oestrogenic activity was assessed using a gene reporter assay (MELN), and the risk to human health through inhalation was quantified using the results. Moreover, extracts were analysed to assess cytotoxicity (WST-1 and LDH assays) on human bronchial cells (BEAS-2B). The extracts induced a significant cytotoxicity and oestrogenic activity. Oestrogenic activity showed a seasonal trend and was correlated with concentrations of benzo(a)pyrene and toxic equivalency factor. Although a low inhalation cancer risk was found, this study confirmed that oestrogenic activity in air could contribute to overall health risks due to EDC exposure.

Occurrence of human pathogenic viruses in drinking water and in its sources: A review.

Since many waterborne diseases are caused by human pathogenic viruses, virus monitoring of drinking water (DW) and DW sources is crucial for public health. Therefore, the aim of this review was to describe the occurrence of human pathogenic viruses in DW and DW sources; the occurrence of two viruses proposed as novel indicators of human faecal contamination (Pepper mild mottle virus and Tobacco mosaic virus) was also reported. This research was focused on articles that assessed viral occurrence using molecular methods in the surface water used for DW production (SW-D), groundwater used for DW production (GW-D), DW and bottled-DW (BW). A total of 1544 studies published in the last 10 years were analysed, and 79 were ultimately included. In considering the detection methods, filtration is the most common concentration technique, while quantitative polymerase chain reaction is the most common quantification technique. Regarding virus occurrence in SW-D, GW-D, and DW, high percentages of positive samples were reported for adenovirus, polyomavirus and Pepper mild mottle virus. Viral genomes were frequently detected in SW-D and rarely in GW-D, suggesting that GW-D may be a safe DW source. Viral genomes were also detected in DW, posing a possible threat to human health. The lowest percentages of positive samples were found in Europe, while the highest were found in Asia and South America. Only three articles assessed viral occurrence in BW. This review highlights the lack of method standardization and the need for legislation updates.

Assessment of Five Pesticides as Endocrine-Disrupting Chemicals: Effects on Estrogen Receptors and Aromatase.

Pesticides are widely applied all over the world, and pesticide exposure can induce different biological effects posing a possible threat to human health. Due to their effects on the endocrine system, some pesticides are classified as endocrine disruptors. The aim of the study is to assess the interference of five pesticides on estrogen biosynthesis and estrogen signaling. Three neonicotinoid insecticides (Acetamiprid, Clothianidin, and Thiamethoxam), a carbamate insecticide (Methiocarb) and a herbicide (Oxadiazon) were tested. The effect of pesticides on estrogen biosynthesis was studied through an ELISA assay using a recombinant form of human aromatase, the enzyme that catalyzes the transformation of androgens to estrogens. Moreover, the effect of pesticides on estrogen signaling was assessed using a gene reporter assay on MELN cells, which measures estrogen receptor-mediated estrogenic activity. The results of the ELISA assay showed that the pesticides did not alter aromatase activity (no interference with estrogen biosynthesis), while the results of the gene reporter assay showed that only Methiocarb was able to alter estrogen signaling at high doses. The estrogenic activity of Methiocarb, expressed as 17ß-estradiol equivalency factor (EEF), was equal to 8.0 × 10-8. In conclusion, this study suggested that Methiocarb should be considered a potential endocrine disruptor.

Oestrogenic Activity in Girls with Signs of Precocious Puberty as Exposure Biomarker to Endocrine Disrupting Chemicals: A Pilot Study.

The relationship between endocrine disrupting chemical (EDC) exposure and Precocious Puberty (PP) was investigated in this pilot study, involving girls with signs of PP (P) and pre-pubertal girls (C). Risk factors for PP were assessed through questionnaires, while 17ß-oestradiol (E2) levels and oestrogenic activity were quantified on sera. The oestrogenic activity, expressed as E2 equivalent concentration (EEQ), was applied as EDC exposure biomarker. Questionnaires showed a low EDC knowledge, a high EDC exposure, and a potential relationship between some habits at risk for EDC exposure and PP. EEQs were similar between C and P; however, they were significantly higher in girls living in an urban environment than in girls living in a rural environment, suggesting a potential higher EDC exposure in cities. The results of this pilot study highlighted the need to raise awareness on EDCs and can be considered a starting point to clarify the relationship between EDC exposure and PP.

Estrogenic activity of biological samples as a biomarker.

Biological assays can evaluate the cumulative effect of a mixture, considering synergistic/antagonistic interactions and effects of unknown/unconsidered compounds. Therefore, their application could increase in the next years also to analyse biological samples. The aim of this review is to discuss the methodological approach and the application of estrogenic activity assays in human biological samples. 75 research articles were analysed and divided according to whether they used these assays: i) to quantify the level of estrogens and/or as a biomarker of estrogenic status ii) as a biomarker of exposure to endocrine disrupting chemicals (EDCs). For the first purpose, some authors extracted biological samples while others tested them directly without any treatment. The study of these methodologies outlined that the methodology applied influenced the specificity of analysis. The estrogenic activity biomarker was used to analyse physiological variations of estrogens, pediatric diseases, hormone-dependent diseases and estrogen suppression/enhancement after pharmaceutical treatments. For the second purpose, some authors extracted samples while others tested them directly, some authors divided endogenous estrogens from xenoestrogens while others tested samples without separation. The analysis of these methodologies outlined some limitations related to the efficiency of extraction and the incorrect separation of some compounds. The studies which applied this EDC biomarker showed that it was correlated with some EDCs, it varied according to the exposure of the population and it allowed the identification of some relationships between EDC exposure and breast cancer, type 1 diabetes and adverse health effects on children. In conclusion, the estrogenic activity of biological samples can be a useful tool: to quantify low levels of 17ß-estradiol, to assess the combined effect of endogenous estrogens and xenoestrogens, to estimate the estrogenic status providing considerable insight into physiological or pathological conditions, to evaluate EDC presence implementing the existing knowledge about EDC exposure and adverse health effects.

Molecular Basis for Endocrine Disruption by Pesticides Targeting Aromatase and Estrogen Receptor.

The intensive use of pesticides has led to their increasing presence in water, soil, and agricultural products. Mounting evidence indicates that some pesticides may be endocrine disrupting chemicals (EDCs), being therefore harmful for the human health and the environment. In this study, three pesticides, glyphosate, thiacloprid, and imidacloprid, were tested for their ability to interfere with estrogen biosynthesis and/or signaling, to evaluate their potential action as EDCs. Among the tested compounds, only glyphosate inhibited aromatase activity (up to 30%) via a non-competitive inhibition or a mixed inhibition mechanism depending on the concentration applied. Then, the ability of the three pesticides to induce an estrogenic activity was tested in MELN cells. When compared to 17ß-estradiol, thiacloprid and imidacloprid induced an estrogenic activity at the highest concentrations tested with a relative potency of 5.4 × 10-10 and 3.7 × 10-9, respectively. Molecular dynamics and docking simulations predicted the potential binding sites and the binding mode of the three pesticides on the structure of the two key targets, providing a rational for their mechanism as EDCs. The results demonstrate that the three pesticides are potential EDCs as glyphosate acts as an aromatase inhibitor, whereas imidacloprid and thiacloprid can interfere with estrogen induced signaling.

Shape-engineered titanium dioxide nanoparticles (TiO2-NPs): cytotoxicity and genotoxicity in bronchial epithelial cells.

The aim of this study was to evaluate cytotoxicity (WST-1 assay), LDH release (LDH assay) and genotoxicity (Comet assay) of three engineered TiO2-NPs with different shapes (bipyramids, rods, platelets) in comparison with two commercial TiO2-NPs (P25, food grade). After NPs characterization (SEM/T-SEM and DLS), biological effects of NPs were assessed on BEAS-2B cells in presence/absence of light. The cellular uptake of NPs was analyzed using Raman spectroscopy. The cytotoxic effects were mostly slight. After light exposure, the largest cytotoxicity (WST-1 assay) was observed for rods; P25, bipyramids and platelets showed a similar effect; no effect was induced by food grade. No LDH release was detected, confirming the low effect on plasma membrane. Food grade and platelets induced direct genotoxicity while P25, food grade and platelets caused oxidative DNA damage. No genotoxic or oxidative damage was induced by bipyramids and rods. Biological effects were overall lower in darkness than after light exposure. Considering that only food grade, P25 and platelets (more agglomerated) were internalized by cells, the uptake resulted correlated with genotoxicity. In conclusion, cytotoxicity of NPs was low and affected by shape and light exposure, while genotoxicity was influenced by cellular-uptake and aggregation tendency.