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New psychoactive substances (NPS), often designed as (legal) substitutes to conventional illicit drugs, are constantly emerging in the drug market and being commercialized in different ways and forms. Their use continues to cause public health problems and is therefore of major concern in many countries. Monitoring NPS use, however, is arduous and different sources of information are required to get more insight of the prevalence and diffusion of NPS use. The determination of NPS in pooled urine and wastewater has shown great potential, adding a different and complementary light on this issue. However, it also presents analytical challenges and limitations that must be taken into account such as the complexity of the matrices, the high sensitivity and selectivity required in the analytical methods as a consequence of the low analyte concentrations as well as the rapid transience of NPS on the drug market creating a scenario with constantly moving analytical targets. Analytical investigation of NPS in pooled urine and wastewater is based on liquid chromatography hyphenated to mass spectrometry and can follow different strategies: target, suspect and non-target analysis. This work aims to discuss the advantages and disadvantages of the different data acquisition workflows and data exploration approaches in mass spectrometry, but also pays attention to new developments such as ion mobility and the use of in-silico prediction tools to improve the identification capabilities in high-complex samples. This tutorial gives an insight into this emerging topic of current concern, and describes the experience gathered within different collaborations and projects supported by key research articles and illustrative practical examples. Refereed/Peer-reviewed

The magnesium transporters TRPM7 and MagT1 are overexpressed in osteoblastogenesis. We have shown that silencing either TRPM7 or MagT1 accelerates the osteogenic differentiation of human bone mesenchymal stem cells. Here we demonstrate that the simultaneous downregulation of TRPM7 and MagT1 inhibits cell growth and activates autophagy, which is required in the early phases of osteoblastogenesis. In TRPM7/MagT1 downregulating cells the expression of two transcription factors required for activating osteogenesis, i.e. RUNX2 and OSTERIX, is induced more than in the controls both in the presence and in the absence of osteogenic stimuli, while COL1A1 is upregulated in co-silencing cells as much as in the controls. This explains why we found no differences in calcium deposition. We conclude that one of the two transporters should be expressed to accelerate osteogenic differentiation.

The presence of drugs of abuse in drinking water was described for the first time about 10 years ago, in 2008. The data today report their presence in several cities in Europe, America, and Asia, showing that the problem is global. These substances enter the water cycle through sewage systems, and cities where wastewater treatment systems are insufficient could have higher levels of illicit drugs in tap water. Illicit drugs are also ubiquitous pollutants in the aquifers and pose a potential risk to health when people drink contaminated water for their whole life. Studies have been carried out to assess their potential effects on the environment and also on the population, but the limited information on potential effects at trace levels should not be overlooked. Every day new illicit substances, some even active at low concentrations such as fentanyls, are synthesized and put on the market with a total lack of toxicological information and are now detectable in drinking water. This short review presents the occurrence and health risk of illicit drugs in drinking water. In our era of megacities, urban planners must consider these aspects in territorial planning.

Wastewater-based epidemiology is an innovative approach to estimate the consumption of chemicals and their exposure patterns in a population, on the basis of measurements of biomarkers in wastewater. This method can provide objective real-time information on xenobiotics directly or indirectly ingested by a population. This approach was used to examine the exposure of the Martinique population to the three classes of pesticides: triazines, organophosphates and pyrethroids. Martinique island (French West Indies) is a closed market and has been closely monitored since the early 2000's when contamination with chlordecone, an organochlorine insecticide widely applied between 1972 and 1993 in banana plantations, became a critical political issue. The aim of this study was to identify and quantify the patterns of human exposure and compare the results to those from other countries. Wastewater was collected as 24-h composite samples and analysed for selected urinary pesticide metabolites by liquid chromatography-tandem mass spectrometry. Organophosphate and pyrethroid metabolites were found in all the samples up to 330 ng/L, while triazines were found only at trace levels. Mass loads indicated higher exposure to pyrethroids than in some cities in Europe, but lower exposure to triazines and organophosphates. The estimated human intake for pyrethroids was close to the Acceptable Daily Intake, but importation of these pesticides to Martinique was low. This study illustrates the high human exposure with indoor pesticide use in comparison to its use in agriculture.

Wastewater-based viral surveillance was proposed as a promising approach to monitor the circulation of SARS-CoV-2 in the general population. The aim of this study was to develop an analytical method to detect SARS-CoV-2 RNA in urban wastewater, and apply it to follow the trends of epidemic in the framework of a surveillance network in the Lombardy region (Northern Italy). This area was the first hotspot of COVID-19 in Europe and was severely affected. Composite 24 h samples were collected weekly in eight cities from end-March to mid-June 2020 (first peak of the pandemic). The method developed and optimized, involved virus concentration using PEG centrifugation, and one-step real-time RT-PCR for analysis. SARS-CoV-2 RNA was identified in 65 (61%) out of 107 samples, and the viral concentrations (up to 2.1 E + 05 copies/L) were highest in March-April. By mid-June, wastewater samples tested negative in all the cities corresponding to the very low number of cases recorded in the same period. Viral loads were calculated considering the wastewater daily flow rate and the population served by each wastewater treatment plant, and were used for inter- city comparison. The highest viral loads were found in Brembate, Ranica and Lodi corresponding to the hotspots of the first peak of pandemic. The pattern of decrease of SARS-CoV-2 in wastewater was closely comparable to the decline of active COVID-19 cases in the population, reflecting the effect of lock-down. This study tested wastewater surveillance of SARS-CoV-2 to follow the pandemic trends in one of most affected areas worldwide, demonstrating that it can integrate ongoing virological surveillance of COVID-19, providing information from both symptomatic and asymptomatic individuals, and monitoring the effect of health interventions., Graphical abstract Unlabelled Image

Objective(s) Endometriosis is a major cause of infertility and disability for women, caused by the presence of inflammatory endometrial implants in extrauterine locations. Among the constituents involved in the immune response during the development of endometriosis, several chemokines, including interferons (IFNs) may have a role in the pathogenesis of this disease. The aim of this preliminary study was to investigate the anti-proliferative and anti-migratory activities of type I IFNs (IFN-α2b and IFN-β1a) in primary endometrial stromal cells (ESCs) isolated from women with deeply infiltrating endometriosis (DIE). Study Design The study subjects included 7 women ranged in the age from 27 to 37 years with diagnosis of DIE (Stage III and IV). Collected primary ESC monolayers, isolated from endometriotic nodules, were incubated with various concentrations (from 1 to 1000 IU/ml) of IFN-α2b or IFN-β1a. Result(s) IFN-β1a had a significantly higher activity in hampering the proliferation of cells compared to IFN-α2b. This effect could be related to the induction of apoptosis and cell cycle arrest in S phase, observed in ESCs during incubation with IFN-β1a. Moreover, IFN-β1a was more potent than IFN-α2b in inhibiting migration and EGF-induced ERK activity of primary ESCs. Conclusion(s) The inhibitory in vitro effect on ESC proliferation and migration of IFN-β1a was much more potent than IFN-α2b. These preliminary data offer the rationale for future preclinical and clinical trials using IFN-β1a as a new tool for the therapy and tertiary prevention in patients with DIE.

The occurrence of illicit drugs (cocaine, opioids, amphetamines and cannabis derivatives), some of their metabolites and 48 pharmaceuticals, was investigated in pool and source waters in ten Italian indoor swimming pools. The samples were analyzed by highperformance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), after solid phase extraction (SPE). Cocaine and its metabolites were found in nine swimming pools, at concentrations from 0.3 to 4.2 ng/L for cocaine, 1.1 to 48.7 ng/L for norcocaine, 0.7 to 21.4 ng/L for benzoylecgonine and 0.1 to 7.3 ng/L for norbenzoylecgonine. Opioids, amphetamines and cannabis derivatives were never detected. The most frequent pharmaceuticals were anti-inflammatory drugs: ibuprofen was found in all the pool waters, with a maximum 197 ng/L and ketoprofen was detected in 9/10 samples (maximum 127 ng/L). Among anticonvulsants, carbamazepine and its metabolite, 10,11-dihydro-10,11dihydroxycarbamazepine, were frequent in swimming pool water (8/10 samples) at concentrations up to 62 ng/L. The cardiovascular drug valsartan was also found frequently (8/10 samples), but at lower concentrations (up to 9 ng/L). Other pharmaceuticals were detected occasionally and at lower concentrations (atenolol, enalapril, paracetamol, hydroclorothiazide, irbesartan and dehydro-erythromycin). Carbamazepine, irbesartan and dehydroerythromycin were detected at very low levels (up to 5 ng/L) in only one of the four source water samples. A quantitative risk assessment showed that the health risk for humans to these substance in swimming pool waters was generally negligible, even for vulnerable subpopulations such as children and adolescents.

Removal rates for pharmaceuticals and dyes have been modelled using so-called quasi-SMILES, which are representations of the above processes. Quasi-SMILES is an extend of the simplified molecular input-line entry system (SMILES) where, in addition to information on the molecular structure, the codes of physicochemical conditions are included. In addition, these codes can be a representation for various eclectic circumstances, such as presence or absence of light, impact of x-Rays beems, as well seasons (e.g. summer—winter). Analysis of quasi-SMILES of pharmaceuticals by Monte Carlo technique, applied via the CORAL software, shows it is possible to build predictive models using a one-variable correlation between optimal (flexible) descriptors and the removal rates. Removal rates used to build the model were obtained from recent publications including seasonal differences. The statistical characteristics of the best models for removal rates of pharmaceuticals and dyes are quite good for external validation set.

Emerging Contaminants (ECs) are ubiquitous in waters, arousing concern because of their potential risks for human health and the environment. This study investigated the presence of multiple classes of ECs in 21 wells over the drinking water network of Milan, in the most inhabited and industrialized area of Italy, and assessed the risks for consumers. Samples were analyzed using liquid chromatography coupled to mass spectrometry. Human risk assessment (HRA) was conducted by comparing the measured concentrations with drinking water thresholds from guidelines or calculated in this study; first considering the exposure to each single EC and then the entire mixture. Thirteen ECs were measured in the low ng/L range, and were generally detected in less than half of the wells. Pharmaceuticals, perfluorinated substances, personal care products, and anthropogenic markers were the most frequently detected. The results of the HRA excluded any risks for consumers in each scenario considered. This is one of the most comprehensive studies assessing the presence of a large number of ECs in the whole drinking water network of a city, and the risks for human health. Results improve the limited information on ECs sources and occurrence in drinking water and help establishing guidelines for regulatory purposes.

The occurrence of several classes of emerging contaminants (ECs) was assessed in the River Lambro basin, one of the most urbanized and industrialized areas of Italy. The study aims were to identify the main sources of ECs, quantify their amounts circulating in the water cycle, and study their fate in the aquatic environment. More than 80 ECs were selected among pharmaceuticals (PHARM), personal care products (PCPs), disinfectants (DIS), illicit drugs (IDs), perfluorinated compounds (PERF), alkylphenols and bisphenol A (Alk-BPA), and anthropogenic markers (AM). Specific analytical methods were developed for quantitative analysis based on solid phase extraction and liquid chromatography tandem mass spectrometry. ECs were measured in rivers upstream and downstream of the main city (Milan), and in untreated and treated wastewater from Milan to assess the contribution to river contamination, and in superficial and deep groundwater in the city area to study the relationship between river and groundwater contamination. Samples were collected in a two-year monitoring campaign. Almost all ECs were ubiquitous in untreated wastewater, at concentrations up to the μg/L range, and the most abundant classes were PHARM and AM. Removals during different wastewater treatment processes were studied and the most stable substances were PHARM, PCPs and PERF. The mass loads increased for all the classes of ECs along the River Lambro basin. A mass balance was done in the river basin and allowed to identify the main sources of contamination, which were domestic, from treated or untreated wastewater, for PHARM, PCPs and IDs, mainly industrial for PERF, and both industrial and domestic for Alk-BPA. The study of AM helped to identify direct discharges of untreated wastewater. A substantial contribution of surface water to groundwater contamination was observed. This study improves the knowledge on occurrence, sources and fate of multiple classes of ECs in a highly urbanized area providing useful information to help the establishment of EU regulations for ECs.

Alcohol use has increased in the last years and is a priority public health issue worldwide. Wastewater-based epidemiology (WBE) is a reliable approach for estimating substance use at the population level and can complement population surveys and sales statistics to obtain objective and updated information on alcohol use in a community. This work reports the first nationwide study of alcohol consumption conducted in Italy by WBE, covering 11.4% of the Italian population. A total of 264 raw wastewater samples (24-h composite) were collected from 17 Italian cities in 2013–2014 and one large city (Milan) over a 7-year period (2013–2019). Besides of normal days, special periods/events (i.e. Halloween, Milan Fashion Week and summer holidays) were studied in Milan. The urinary biomarker of ethanol (namely, ethyl sulphate) was measured by liquid chromatography-tandem mass spectrometry and was used to estimate alcohol consumption. Results were statistically evaluated to assess spatial and temporal differences and were compared with data from other epidemiological indicators. In 2013, alcohol consumption was significantly higher in small cities than in large ones but no significant differences were observed in relation to the geographical position. An increase in alcohol use was observed in seven large cities from 2013 to 2014. The 7-year monitoring in Milan showed no marked changes, except for remarkable increases in 2014 and 2019. Alcohol consumption significantly increased (ca. 25%) during weekends in Italy and in Milan, and during special periods/events, for instance summer holidays and Halloween. WBE estimates were in agreement with national prevalence data, demonstrating that WBE is suitable for objectively evaluating geographic, annual and weekly patterns of alcohol consumption at local and national scales. WBE information would be helpful to implement and evaluate measures for reducing alcohol consumption and preventing the related health risks.

Wastewater-based epidemiology (WBE) can be a useful tool to face some of the existing challenges in monitoring the use of new psychoactive substances (NPS), as it can provide objective and updated information. This Europe-wide study aimed to verify the suitability of WBE for investigating the use of NPS. Selected NPS were monitored in urban wastewater by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The main classical illicit drugs were monitored in the same samples to compare their levels with those of NPS. Raw composite wastewater samples were collected in 2016 and 2017 in 14 European countries (22 cities) following best practice sampling protocols. Methcathinone was most frequent (>65% of the cities), followed by mephedrone (>25% of the cities), and only mephedrone, methcathinone and methylone were found in both years. This study depicts the use of NPS in Europe, confirming that it is much lower than the use of classical drugs. WBE proved able to assess the qualitative and quantitative spatial and temporal profiles of NPS use. The results show the changeable nature of the NPS market and the importance of large WBE monitoring campaigns for selected priority NPS. WBE is valuable for complementing epidemiological studies to follow rapidly changing profiles of use of drugs. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4396]

Smoking cigarettes and drinking coffee are common habits in today's society. However, it is not easy to get up-to-date information on smoking prevalence and caffeine consumption as it is usually obtained from population surveys. To overcome this limitation and complement epidemiological information, we employed wastewater-based epidemiology (WBE) to gain a picture of the consumption of cigarettes and caffeine per day per person in Italy. A nationwide study was conducted by measuring two urinary metabolites of nicotine (cotinine and trans-3'-hydroxycotinine) and a caffeine metabolite (1,7-dimethyluric acid) in untreated wastewater from 16 cities. The spatial profiles of use depended mostly on the specific population habits in each city, not on the geographical area or the population size. The patterns of consumption were stable over the week, except in Milan where the use of both substances decreased on Sundays, probably because there were no commuters. In Milan, the use of nicotine decreased from 2013 to 2015. There was a significant relationship between smoking and consumption of products containing caffeine (mainly coffee), thus in cities where more cigarettes were smoked, more caffeine was drunk. These results are generally in accordance with findings from epidemiological studies, but provide some additional local profiles of use and closely follow changes over the years. This information could be useful for healthcare professionals and policy-makers to monitor progress towards the reduction of prevalence in tobacco use, and set up new health campaigns.

Bone loss is a well known early event in astronauts and represents one of the major obstacle to space exploration. While an imbalance between osteoblast and osteoclast activity has been described, less is known about the behavior of bone mesenchymal stem cells in microgravity. We simulated microgravity using the Random Positioning Machine and found that mesenchymal stem cells respond to gravitational unloading by upregulating HSP60, HSP70, cyclooxygenase 2 and superoxyde dismutase 2. Such an adaptive response might be involved in inducing the overexpression of some osteogenic transcripts, even though the threshold to induce the formation of bone crystal is not achieved. Indeed, only the addition of an osteogenic cocktail activates the full differentiation process both in simulated microgravity and under static 1G-conditions. We conclude that simulated microgravity alone reprograms bone mesenchymal stem cells towards an osteogenic phenotype which results in complete differentiation only after exposure to a specific stimulus.

Illicit drugs have been recognized as emerging aquatic pollutants due to their presence in aquatic ecosystems up to µg/L level. Among these, the synthetic psycho-stimulant drug amphetamine (AMPH) is commonly found in both surface and wastewaters worldwide. Even though the environmental occurrence of AMPH is well-known, the information on its toxicity towards non-target freshwater organisms is completely lacking. This study investigated the imbalance of the oxidative status and both oxidative and genetic damage induced by a 14-day exposure to two concentrations (500 ng/L and 5000 ng/L) of AMPH on the freshwater bivalve Dreissena polymorpha by the application of a biomarker suite. We investigated the activity of antioxidant enzymes (SOD, CAT and GPx), the phase II detoxifying enzyme GST, the lipid peroxidation level (LPO) and protein carbonyl content (PCC), as well as primary (Single Cell Gel Electrophoresis assay) and fixed (DNA diffusion assay and Micronucleus test) genetic damage. Our results showed that a current realistic AMPH concentration (500 ng/L) did neither cause notable imbalances in enzymatic activities, nor oxidative and genetic damage to cellular macromolecules. In contrast, the bell-shaped trend of antioxidants showed at the highest tested concentration (5000 ng/L) suggested an overproduction of reactive oxygen species, leading to oxidative damage, as confirmed by the significant increase of protein carbonylation and DNA fragmentation.

This study reports an investigation of omeprazole and venlafaxine parent substances and metabolites in Italian municipal influent wastewaters (IWWs). These pharmaceuticals were selected because they are widely consumed in Italy, but are poorly detected in waste and surface water. The aim of the study was to identify the most relevant pharmaceuticals metabolites in wastewater in order to improve the prioritization step and choose priority pollutants for environmental monitoring campaigns. This was done by investigating omeprazole, venlafaxine and their main metabolites in 30 IWWs from ten Italian cities and by comparing results with information from pharmacokinetic studies. Analysis was performed by solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS). We searched for 23 omeprazole and four venlafaxine metabolites using data-dependent and MS/MS methods. Parent omeprazole was never present in the samples. Six omeprazole metabolites were found in IWWs. Venlafaxine and two metabolites were present in all the samples. The metabolic profiles in Italian IWW agreed with results in IWW from Spain and with urinary excretion profiles from pharmacokinetic studies. Comparing results from different sources was useful to improve the identification of pharmaceuticals metabolites in environmental samples and to focus the attention of future studies on the most relevant compounds.

The quantitative determination of urinary biomarkers in raw wastewater has emerged in recent years as a promising tool for estimating the consumption of illicit drugs, tobacco and alcohol in a population and for comparing local and temporal trends. In this study, a three-year monitoring campaign (2012-2014) was conducted to compare alcohol and cocaine use in two European cities (Santiago de Compostela, Spain, and Milan, Italy) by wastewater analysis. Ethyl sulphate and benzoylecgonine were used, respectively, as biomarkers of ethanol and cocaine consumption and cocaethylene as an indicator of co-consumption of both substances. Biomarkers were measured using liquid chromatography-tandem mass spectrometry and concentrations were converted to rates of consumption using specific correction factors. Results were statistically compared in terms of geographic and temporal tendencies. Alcohol intake was significantly higher in Santiago than in Milan (13.6L versus 5.1L ethanol/1000 people day, averages). Cocaine use was higher in Milan than in Santiago de Compostela (800 versus 632 mg/1000 people day, averages). A significant higher consumption of both alcohol and cocaine was observed during the weekends (~23-75% more than on weekdays) in both cities. In terms of years, slight changes were observed, but no clear trends as representative of the whole year could be identified because of the limited number of days sampled. Co-consumption was evaluated using the cocaethylene/benzoylecgonine ratio, which was higher during the weekend in both cities (58% in Santiago and 47% in Milan over the non-weekend day means), indicating a greater co-consumption when cocaine is used as a recreational drug. Wastewater-based epidemiology gave estimates of alcohol and cocaine use in agreement with previous wastewater studies and with recent European surveillance and prevalence data, and weekly profiles of use and preferential patterns of consumption could be plot.

The authors regret for inconvenience caused but several affiliations should be corrected as follows: Erika Castrignanὁ , Zhugen Yang , Edward J. Feil , Richard Bade , Sara Castiglioni , Ana Causanilles , Emma Gracia-Lor , Felix Hernandez , Benedek G. Plosz , Pedram Ramin , Nikolaos I. Rousis , Yeonsuk Ryu , Kevin V. Thomas , Pim de Voogt , Ettore Zuccato , Barbara Kasprzyk-Hordern , ∗ Department of Chemistry, Faculty of Science, University of Bath, Bath, BA2 7AY, United Kingdom Department of Biology and Biochemistry, University of Bath, Bath, BA27AY, United Kingdom Research Institute for Pesticides and Water, University Jaume I, Avda. Sos Baynat s/n, E-12071, Castellὁn, Spain School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5000, Australia Norwegian Institute for Water Research (NIVA), Gaustadalleen 21, 0349, Oslo, Norway Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Department of Environmental Health Sciences, Via Mario Negri 2, 20156, Milan, Italy KWR Watercycle Research Institute, Chemical Water Quality and Health, P.O. Box 1072, 3430 BB, Nieuwegein, the Netherlands Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 94248, 1090 GE, Amsterdam, the Netherlands Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet, Building 115, 2800, Kgs. Lyngby, Denmark Process and Systems Engineering Center (PROSYS), Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229,2800, Kgs. Lyngby, Denmark Department of Analytical, Environmental & Forensic Sciences, School of Population Health & Environmental Sciences, King's College London, London, SE19NH, United Kingdom School of Water, Energy and Environment, Cranfield University, Cranfield, MK43 0AL, United Kingdom Department of Chemical Engineering, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom Queensland Alliance for Environmental Health Science (QAEHS), University of Queensland, 20 Cornwall Street, Woolloongabba, QLD, 4102, Australia Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Avenida Complutense s/n, Madrid, Spain The authors would like to apologise for any inconvenience caused.

The consumption of psychoactive substances is considered a growing probleminmany communities. Moreover, new psychoactive substances (NPS) designed as (legal) substitutes to traditional illicit drugs are relatively easily available to the public through e-commerce and retail shops, but there is little knowledge regarding the extent and actual use of these substances. This study aims to gain new and complementary information on NPS and traditional illicit drug use at sixmusic festivals across Europe by investigatingwastewater and pooled urine. Samples were collected, between 2015 and 2018, at six music festivals across Europe with approximately 465.000 attendees. Wastewater samples were also collected during a period not coinciding with festivals. A wide-scope screening for 197 NPS, six illicit drugs and known metabolites was applied using different chromatographymass spectrometric strategies. Several illicit drugs and in total 21 different NPS, mainly synthetic cathinones, phenethylamines and tryptamines, were identified in the samples. Ketamine and the traditional illicit drugs, such as amphetamine-type stimulants, cannabis and cocaine were most abundant and/or frequently detected in the samples collected, suggesting a higher use compared to NPS. The analyses of urine and wastewater is quick and a high number of attendeesmay be monitored anonymously by analysing only a fewsampleswhich allows identifying the local profiles of use of different drugswithin awide panel of psychoactive substances. This approach contributes to the development of an efficient surveillance system which can provide timely insight in the trends of NPS and illicit drugs use. (C) 2020 Elsevier B.V. All rights reserved.

Illicit drugs are considered to be emerging aquatic pollutants since they are commonly found in freshwater ecosystems in the high ng L(-1) to low μg L(-1) range concentrations. Although the environmental occurrence of the most common psychoactive compounds is well known, recently some investigations showed their potential toxicity toward non-target aquatic organisms. However, to date, these studies completely neglected that organisms in the real environment are exposed to a complex mixture, which could lead to dissimilar adverse effects. The present study investigated the oxidative alterations of the freshwater bivalve Dreissena polymorpha induced by a 14-d exposure to an environmentally relevant mixture of the most common illicit drugs found in the aquatic environment, namely cocaine (50 ng L(-1)), benzoylecgonine (300 ng L(-1)), amphetamine (300 ng L(-1)), morphine (100 ng L(-1)) and 3,4-methylenedioxymethamphetamine (50 ng L(-1)). The total oxidant status (TOS) was measured to investigate the increase in the reactive oxygen species' levels, while the activity of antioxidant enzymes and glutathione S-transferase were measured to note the eventual imbalances between pro-oxidant and antioxidant molecules. In addition, oxidative damage was assessed by measuring the levels of lipid peroxidation and protein carbonylation. Significant time-dependent increases of all the antioxidant activities were induced by the mixture. Moreover, the illicit drug mixture significantly increased the levels of carbonylated proteins and caused a slight variation in lipid peroxidation. Our results showed that a mixture of illicit drugs at realistic environmental concentrations can impair the oxidative status of the zebra mussel, posing a serious hazard to the health status of this bivalve species.

Perfluorinated substances are listed among emerging contaminants because they are globally distributed, environmentally persistent, bioaccumulative and potentially harmful. In a three-year monitoring campaign (2010-2013) we investigated the occurrence, sources and fate of nine perfluoroalkylcarboxylic acids and three perfluoroalkylsulfonic acids, in the most industrialized region of Italy. Composite samples were collected in influents and effluents of wastewater treatment plants (WWTPs), in the main rivers flowing through the basin, and in raw groundwater and finished drinking water. Samples were analyzed by liquid chromatography tandem mass spectrometry. Perfluorinated substances were not removed in WWTPs and those receiving industrial wastes discharged up to 50 times the loads of WWTPs receiving municipal wastes. The mass balance of the emissions in the River Lambro basin showed continuously increasing contamination from north to south and differences in the composition of homologues in the west and east sides of the basin. Ground and drinking water were contaminated in industrial areas, but these substances were removed well in Milan. Contamination from industrial sources was prevalent over urban sources, contributing to 90% of the loads measured at the closure of the basin. The River Lambro was confirmed as one of the main sources of contamination in the Po River. © 2014 Elsevier B.V. All rights reserved.

a b s t r a c t This study shows the evaluation of the possible use of the freshwater bivalve Dreissena polymorpha for the removal of some recalcitrant contaminants, namely many pharmaceuticals and drugs of abuse that are not sufficiently removed from civil wastewaters. This mollusk has an enormous filtering capability and is highly resistant to natural and anthropogenic stresses and to a significant bioaccumulation of lipophilic contaminants. All these characteristics may be particularly useful for the removal of compounds not easily eliminated by conventional wastewater treatment processes. To verify this hypothesis an experimental study was conducted at the pilot scale using a pilot plant installed in the largest wastewater treatment plant of Milan (Milano-Nosedo, Italy). First, we presented results obtained in several preliminary tests in order to evaluate the capability of zebra mussel specimens to survive in different wastewater mixtures, its filtering capacity and the possible influence of bio- and photo-degradation in the abatement of the molecules of interest. Finally, data obtained in the final tests demonstrated a capacity of this filter-feeder to reduce the concentrations of several pharmaceuticals and drugs of abuse higher than that obtained by the simple natural sedimentation, suggesting a possible implementation of the bio-filtration process in wastewater management. © 2014 Elsevier B.V. All rights reserved.

HD-PTP (PTPN23) is a non-transmembrane protein tyrosine phosphatase which contributes to the signal transduction pathways involved in the regulation of cell migration and invasion. We here demonstrate in T24 bladder carcinoma cells that HD-PTP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. In addition, treatment of the cells with the calpain inhibitor calpeptin results in the redistribution of endogenous HD-PTP to the periphery of the cells. Since (i) calpains are overexpressed in some tumors and (ii) the downregulation of HD-PTP enhances cell migration and invasion, we propose that HD-PTP degradation by calpains might result in the acquisition of a more aggressive phenotype in neoplastic cells.

We have previously shown that cancer cells can protect themselves from apoptosis induced by type 1 interferons (IFNs) through a ras-->MAPK-mediated pathway. In addition, since IFN-mediated signalling components STATs are controlled by PPAR gamma we studied the pharmacological interaction between recombinant IFN-beta and the PPAR-gamma agonist troglitazone (TGZ). This combination induced a synergistic effect on the growth inhibition of BxPC-3, a pancreatic cancer cell line, through the counteraction of the IFN-beta-induced activation of STAT-3, MAPK and AKT and the increase in the binding of both STAT-1 related complexes and PPAR-gamma with specific DNA responsive elements. The synergism on cell growth inhibition correlated with a cell cycle arrest in G0/G1 phase, secondary to a long-lasting increase of both p21 and p27 expressions. Blockade of MAPK activation and the effect on p21 and p27 expressions, induced by IFN-beta and TGZ combination, were due to the decreased activation of STAT-3 secondary to TGZ. alone also increased p21 and p27 expression through STAT-1 phosphorylation and this effect was attenuated by the concomitant activation of IFNbeta-induced STAT-3-activation. The combination induced also an increase in autophagy and a decrease in anti-autophagic bcl-2/beclin-1 complex formation. This effect was mediated by the inactivation of the AKT-->mTOR-dependent pathway. To the best of our knowledge this is the first evidence that PPAR-gamma activation can counteract STAT-3-dependent escape pathways to IFN-beta-induced growth inhibition through cell cycle perturbation and increased autophagic death in pancreatic cancer cells. (C) 2011 Elsevier Inc. All rights reserved.

Wastewater analysis can provide estimates of illicit drug (ID) consumption in local communities.We used repeated raw wastewater analysis in urban wastewater treatment plants to estimate loads of cocaine, heroin, methamphetamine, and cannabis consumed daily by the inhabitants of two cities in Northern Italy, Milan and Como, from 2005 to 2009.Daily cocaine loads did not change in Milan from 2005 to 2008 but fell 45% in 2009 (ANOVA, p0.001, followed by Tukey-Kramer HSD test [2009 vs. others], p0.05), and there was a similar drop in Como (41%, p0.0001, t-test). Heroin also fell from 2008 to 2009 in Milan (66%, ANOVA, p0.001, followed by Tukey-Kramer [2009 vs. others], p0.05) and Como (26%, p=0.017, t-test). However, methamphetamine, which had risen in Milan from 2005 to 2008, rose further in 2009 (Kruskal-Wallis test, p0.001, followed by Steel-Dwass [2009 or 2008 vs. previous], p0.05), and cannabis, which was falling from 2005 to March 2009, rose again in September 2009 (40%, p=0.027, t-test).Results suggest a trend toward a decrease in consumption of costly ID, such as cocaine and heroin. This might be due to a reduction in the number of consumers and/or to a change in their behaviour since there was also an increase in the consumption of less expensive ID. This itself might reflect a drop in consumers' money supply, caused by the economic crisis. Wastewater analysis was useful to estimate ID consumption levels in local communities in real time and promptly identify changes in trends.

The consumption of illicit drugs causes indisputable societal and economic damage. Therefore it is necessary to know their usage levels and trends for undertaking targeted actions to reduce their use. Recently, a new approach (namely sewage epidemiology) was developed for the estimation of illicit drug use based on measurements of urinary excreted illicit drugs and their metabolites in untreated wastewater. This review aims at critically evaluating the published literature and identifying research gaps of sewage epidemiology. Firstly, the existing analytical procedures for the determination of the four most used classes of illicit drugs worldwide (cannabis, cocaine, opiates and amphetamine-like stimulants) and their metabolites in wastewater are summarized and discussed. The focus lies on the sample preparation and on the analysis with chromatographic techniques coupled to mass spectrometry. Secondly, back-calculations used to transform measured concentrations in wastewater (in ng/L) into an amount of used illicit drug (in g/day per 1000 inhabitants or doses/day per 1000 inhabitants) are discussed in detail for the four groups of illicit drugs. Sewage epidemiology data from Spain, Belgium, UK, Italy, Switzerland and USA are summarized and compared with data from international organisations, such as the European Monitoring Centre for Drug and Drug Addiction (EMCDDA) and the United Nations Office on Drugs and Crime (UNODC). The results derived from wastewater analysis show in general good agreement with existing prevalence data (percentage of a population that uses illicit drugs at a given time) and demonstrate the potential of sewage epidemiology. However, this review confirms that future work should focus on further optimisation and standardisation of various important parameters (e.g. sample collection and back-calculations). In the future, sewage epidemiology could be used in routine drug monitoring campaigns as a valuable tool in addition to the classical socio-epidemiological studies for the determination of local, national and international illicit drug use.

Pharmaceutically-active compounds are regularly and widely released into the aquatic environment in an unaltered form or as metabolites. So far, little is known about their potential detrimental effects on algae populations which can ultimately impact nutrient cycling and oxygen balance. For our analysis, the common microalga Pseudokirchneriella subcapitata (P. subcapitata) was exposed to a mixture of 13 drugs found in Italian wastewaters and rivers. Traces of pharmaceuticals investigated were detected in treated algal cells, except for cyclophosphamide and ranitidine, indicating that these algae are able to absorb pharmaceutical pollutants from the environment. The effects of the treatment were investigated by Amplified Fragment Length Polymorphism (AFLP) assessment of DNA damage and 2-DE proteomic analysis. While no genotoxic effect was detected, proteomic analysis showed that algae are sensitive to the presence of drugs and that, in particular, the chloroplast is affected. © 2010 Elsevier B.V.

It is now well established that residues from therapeutic drugs consumed by humans can end up, through the sewage system, in the surface water of populated areas. Given that the global production of major illicit drugs is comparable to that of widely used pharmaceuticals, we tested for the presence of drugs of abuse (cocaine, opioids, amphetamines and cannabis derivatives), some related opioid pharmaceuticals (codeine and methadone) and/or their metabolites in Italian and British surface waters. Having identified residues of all major drugs of abuse in raw and treated urban wastewater, we now measured their levels in several rivers and lakes by a selective multi-residue assay based on liquid chromatography-tandem mass spectrometry. Recoveries in surface water were generally higher than 80%, with overall variability of the method lower than 10%. LODs were generally lower than 0.2 ng/L, and LOQs were lower than 0.6 ng/L, with few exceptions. Many of the tested substances were found in both rivers and lakes, at concentrations ranging from high pg/L to high ng/L, with loads in rivers in the range of tenths to hundreds of grams per day. Our data indicate that residues of drugs of abuse have become widespread surface water contaminants in populated areas. Since most of these residues still have potent pharmacological activities, their presence in the aquatic environment may have potential implications for human health and wildlife.

A preliminary investigation has been carried out on the occurrence and effects of antibiotics used in Italian aqua-culture with the objective of identifying priorities for monitoring programmes. According to the information available on the most pertinent and diffuse fish diseases and their related therapies, the presence of flumequine and oxytetracycline in sediments sampled from two trout farms and three sea-bass farms and in their surrounding environments was selected for an analytical investigation. The concentrations of oxytetracycline and flumequine varied up to a maximum of 246.3 and 578.8 mug/kg d.w., respectively. Flumequine was seen to have the highest toxicity in a bioluminescence assay with EC50 values varying within the range of 12-15 mg/l, while the EC50 values for oxytetracycline were within the range of 121-139 mg/l. The results of the present study indicate flumequine and oxytetracycline as priority chemicals to be monitored for possible environmental side effects of aquaculture in Italy. Apart from peak concentrations the chronic presence of flumequine and oxytetracycline in sediments both inside and outside farms should also be considered. In spite of the potential risks related to the use of antibiotics, the concentrations found in the sediments of the studied fish farms are significantly lower than those found in other areas. (C) 2003 Elsevier Ltd. All rights reserved.

orrigendum to “Assessing geographical differences in illicit drug onsumption—A comparison of results from epidemiological and astewater data in Germany and Switzerland” Drug Alcohol Depend. 161 (2016) 189–199] rederic Beena,∗, Lubertus Bijlsmab, Lisa Benagliaa, Jean-Daniel Bersetc, na M. Botero-Coyb, Sara Castiglionid, Ludwig Krause,f, Frank Zobelg, Michael P. Schaubh Alexander Bucheli i, Felix Hernandezb, Olivier Delemonta, Pierre Esseivaa, hristoph Ort j,∗