Monitoring psychoactive substance use at six European festivals through wastewater and pooled urine analysis.

The consumption of psychoactive substances is considered a growing problem in many 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 six music festivals across Europe by investigating wastewater 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 chromatography-mass 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 attendees may be monitored anonymously by analysing only a few samples which allows identifying the local profiles of use of different drugs within a wide 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.

Illicit drugs and pharmaceuticals in swimming pool waters.

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.

Recent advances in analytical methods for the determination of 4-alkylphenols and bisphenol A in solid environmental matrices: A critical review.

The problem of endocrine disrupting compounds (EDCs) in the environment has become a worldwide concern in recent decades. Besides their toxicological effects at low concentrations and their widespread use in industrial and household applications, these pollutants pose a risk for non-target organisms and also for public safety. Analytical methods to determine these compounds at trace levels in different matrices are urgently needed. This review critically discusses trends in analytical methods for well-known EDCs like alkylphenols and bisphenol A in solid environmental matrices, including sediment and aquatic biological samples (from 2006 to 2018). Information about extraction, clean-up and determination is covered in detail, including analytical quality parameters (QA/QC). Conventional and novel analytical techniques are compared, with their advantages and drawbacks. Ultrasound assisted extraction followed by solid phase extraction clean-up is the most widely used procedure for sediment and aquatic biological samples, although softer extraction conditions have been employed for the latter. The use of liquid chromatography followed by tandem mass spectrometry has greatly increased in the last five years. The majority of these methods have been employed for the analysis of river sediments and bivalve molluscs because of their usefulness in aquatic ecosystem (bio)monitoring programs. Green, simple, fast analytical methods are now needed to determine these compounds in complex matrices.

First interlaboratory exercise on non-steroidal anti-inflammatory drugs analysis in environmental samples.

Comparability of monitoring data are essential for any meaningful assessment and for the management of environmental risks of emerging pollutants. The reliability and comparability of data at European level is often limited, because analytical methods for emerging pollutants are often not fully validated, not harmonized or not suitable for all relevant matrices. This paper describes a collaborative interlaboratory exercise for the analysis of non-steroidal anti-inflammatory drugs (NSAIDs) residues in freshwater and wastewater, held in the framework of the EU project "Network of reference laboratories for monitoring of emerging environmental pollutants" (NORMAN). The NSAID compounds selected in this study were ketoprofen, naproxen, ibuprofen and diclofenac. Thirteen laboratories distributed along nine European Countries (Austria, France, Germany, Greece, Italy, Slovak Republic, Slovenia, Spain, and Switzerland) took part in this exercise, 126 samples were analyzed and a total number of 473 values in duplicate were collected. Samples selected in this study include environmental water (river water and waste water) and artificial water (fortified environmental and distilled water) with different ranges of complexity. Two analytical methods were proposed by the organiser; one is based on the use of solid phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the second one is based on SPE followed by gas-chromatography-mass spectrometry (GC-MS), however, in the first round some different approaches were also admitted. The main goals of this interlaboratory comparison were to evaluate the available analytical schemes for NSAID analysis in natural waters, to evaluate the repeatability (r) and reproducibility (R) between participating laboratories, and to evaluate the influence of the analytical method and sample matrices on the results.

PCB concentrations in some foods from four European countries.

Samples of salmon, butter and cabbage from Belgium, Italy, Spain and Portugal were analysed for their content in total, non-dioxin-like (as represented by the so-called seven indicator-PCBs: congeners 28, 52, 101, 118, 138, 153 and 180) and dioxin-like PCBs (mono-ortho and non-ortho PCBs). Salmon and cabbage from Belgium, and butter from Portugal and Belgium, contained less total and non-dioxin-like PCBs than those from other countries. Samples from Italy had the highest concentrations. Similar patterns were observed for dioxin-like PCBs (as represented by the TCDD-equivalents of toxicity, WHO-TEQs), with the lowest values in Belgium and Portugal for salmon, in Portugal for butter and in Belgium for cabbage. Differences up to five-fold in PCB concentrations and TEQ values were seen among commodities from the four countries. The implication is that it might be worthwhile monitoring, with selection of the least contaminated commodities, to reduce the PCB exposure of the general population. This could have health consequences, because daily intakes are higher than the tolerable levels for a considerable part of the European population.

Risks and benefits of PVC in medical applications.

The safety of using PVC in the medical field has been recently challenged due to the toxic activity it allegedly exerts on exposed patients. The environmental repercussions of disposing of PVC, once its use has terminated, represent an additional point of debate, used to sustain the advisability of abolishing PVC. The reasons that have led some to request the abolition of PVC involve valid questions of principle, perhaps, but they lack a technical evaluation of the benefit-risk ratio and the possible consequences this action would have on patients and on healthcare personnel. The purpose of this paper is therefore to help bring the terms of the question back into the realm of evidence and proof, attempting to formulate a brief picture of what is known, in terms of PVC uses in the clinical field, evaluating the benefits and risks to human health and to the environment, also in relation to possible alternatives, and discussing the margins of uncertainty that emerge. Evidence supports the conclusion that PVC is an important weapon in the complex arsenal medicine has at its disposal to care for patients and cure diseases. Though its use can be considered safe, recent surveys have identified in some patients possibility of risks associated with DEHP, the principal plasticizer of PVC for medical applications. Studies are in progress to eliminate these margins of risk and increase the safety for patients.

Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping.

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.

Presence of therapeutic drugs in the environment.

Therapeutic drugs can contaminate the environment because of metabolic excretion, improper disposal, or industrial waste. To assess the extent of this contamination, we listed drugs thought to be putative priority pollutants according to selected criteria, and measured them in Lombardy, Italy. Most drugs were measurable in drinking or river waters and sediments, suggesting that pharmaceutical products are widespread contaminants, with possible implications for human health and the environment.

Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element.

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis.

Level, sources and toxicity of polychlorinated biphenyls in the Italian diet.

We used the duplicate portion method to measure the daily dietary intake of total and congener-specific polychlorinated biphenyls (PCB) and to assess their potential toxicity in a group of 20 subjects consuming a typical Italian diet. The mean +/- SD intake of total PCB, measured by GC-MS, was 3.72 +/- 1.51 micrograms/person/day, comparable to values reported in similar studies world-wide, with individual intakes varying within one order of magnitude, from 0.97 to 10.59 micrograms/person/day. The di-ortho congeners 153, 18 and 138 were the PCB found in the highest concentrations (respectively 13.8%, 11.4% and 10.9% of the total) while the non-ortho coplanar congeners (77, 126 and 169) amounted to 0.5% of the total. The corresponding levels of toxicity (TCDD-like TEQ values ascribable to PCB) ranged from 4.6 up to 119 pg/person/day of TCDD-equivalents in 18 subjects, i.e. presumed no-risk levels, but with peaks of 2109 and 4553 pg/person/day in two subjects with significant intakes of the congener 126. Principal components analysis and redundancy analysis showed dairy products, meat and fish were the principal sources of PCB, and vegetables those with the highest toxicity index in the Italian diet.

Identification and measurement of endogenous beta-oxidation metabolites of 8-epi-Prostaglandin F2alpha.

F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2alpha, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi-PGF2alpha, in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2alpha is the only identified metabolite of 8-epi-PGF2alpha in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another major endogenous metabolite, 2,3-dinor-8-epi-PGF2alpha, in human and rat urine. The identity of these compounds, present at the pg/ml level in urine, was proven by a number of complementary approaches, based on: (a) immunoaffinity chromatography for selective extraction; (b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated rat hepatocytes; and (d) chemical synthesis of the enantiomer of 2,3-dinor-5, 6-dihydro-8-epi-PGF2alpha as a reference standard. In humans, the urinary excretion rate of both dinor metabolites is comparable with that of 8-epi-PGF2alpha. Both metabolites increase in parallel with the parent compound in cigarette smokers, and they are not reduced during cyclooxygenase inhibition. Another beta-oxidation product, 2, 3,4,5-tetranor-8-epi-PGF2alpha, was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major beta-oxidation products of 8-epi-PGF2alpha are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF2alpha formation and degradation in vivo.

Reduction of urinary 8-epi-prostaglandin F2 alpha during cyclo-oxygenase inhibition in rats but not in man.

1. 8-epi-prostaglandin (PG) F2 alpha, a major F2 isoprostane, is produced in vivo by free radical-dependent peroxidation of lipid-esterified arachidonic acid. Both cyclo-oxygenase isoforms (COX-1 and COX-2) may also form free 8-epi-PGF2 alpha as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8-epi-PGF2 alpha in vivo is mostly COX-independent and urinary 8-epi-PGF2 alpha is therefore an accurate marker of 'basal' oxidative stress in vivo. 2. To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8-epi-PGF2 alpha vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg-1 day-1; indomethacin: 4 mg kg-1 day-1) were given i.p. to rats kept in metabolic cages. In vivo formation of 8-epi-PGF2 alpha was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha). All compounds were selectively measured by immunopurification/gas chromatography-mass spectrometry. 3. Naproxen reduced urinary excretion of 2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha but, unexpectedly, also that of 8-epi-PGF2 alpha (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8-epi-PGF2 alpha in liver and plasma remained unchanged after indomethacin. 4. These findings prompted us to re-assess the contribution of COX activity to the systemic production of 8-epi-PGF2 alpha in man. We gave naproxen (1 g day-1) to healthy subjects (four nonsmokers and four smokers). Urinary 8-epi-PGF2 alpha remained unchanged in the two groups (9.63 +/- 0.99 before vs 10.24 +/- 1.01 after and 20.14 +/- 3.00 vs 19.03 +/- 2.45 ng h-1 1.73 m-2), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11-dehydro-TXB2 and 2,3-dinor-TXB2; > 50% for 2,3-dinor-6-keto-PGF1 alpha). 5. To investigate whether rat COX-1 produces 8-epi-PGF2 alpha more efficiently than human COX-1, we measured the ex vivo formation of 8-epi-PGF2 alpha and TXB2 simultaneously in whole clotting blood. Serum levels of 8-epi-PGF2 alpha and TXB2 were similar in rats and man. 6. We conclude that a significant amount of COX-dependent 8-epi-PGF2 alpha is present in rat but not in human urine under normal conditions. This implies that urinary 8-epi-PGF2 alpha cannot be used as an index of near-basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.

Measurement of urinary 8-Epi-prostaglandin F2alpha, a novel index of lipid peroxidation in vivo, by immunoaffinity extraction/gas chromatography-mass spectrometry. Basal levels in smokers and nonsmokers.

8-Epi-prostaglandin F2alpha (8-epi-PGF2alpha) is an F2-isoprostane recently identified as a marker of free radical-catalyzed lipid peroxidation in vivo and potential mediator of oxidative damage. Currently, endogenous 8-epi-PGF2alpha is measured by gas chromatography-mass spectrometry after lengthy sample preparation. We extracted and purified 8-epi-PGF2alpha in one step from biological samples on immunoaffinity columns prepared with an anti-8-epi-PGF2alpha antiserum, raised in our laboratory. Quantitation was done by stable-isotope dilution gas chromatography/negative-ion chemical ionization mass spectrometry, with selected ion recording. Carboxylate anions of the pentafluorobenzyl ester trimethylsilyl ether derivative of 8-epi-PGF2alpha and [2H4]8-epi-PGF2alpha were monitored (m/z 569 and 573). Basal urinary excretion of 8-epi-PGF2alpha can be accurately and rapidly measured by this method. Under normal conditions rats (n = 30) excreted 2.18 +/- 0.68 ng/24 h. In healthy nonsmoking young volunteers, urinary excretion of 8-epi-PGF2alpha, measured three times on alternate days, was fairly constant (CV 2-10%). Nonsmokers excreted significantly less 8-epi-PGF2alpha than age-matched smokers (8.08 +/- 2.3 vs. 18.40 +/- 4.77 ng/h/1.73 m2; n = 6; p < 0.005), as reported by others using different methods.

Utility of Hydrogen and Methane Breath Tests in Combination with X-Ray Examination after a Barium Meal in the Diagnosis of Small Bowel Bacterial Overgrowth after Jejuno-Ileal Bypass for Morbid Obesity.

To study why the symptoms of abdominal bloating occurring in a number of patients after jejuno-ileal bypass for morbid obesity become resistant to antibiotics, we used a method which combined a hydrogen breath test after lactulose with an X-ray examination of the abdomen after barium. Ten operated patients with bloating symptoms resistant to antibiotics, ten operated patients without symptoms or with pre-existing symptoms, that had remitted after antibiotic treatment and ten nonoperated obese controls were investigated. There was a significant correlation between post-surgical symptoms persisting after antibiotics and the exhalation of large amounts of hydrogen of colonic origin (> 100 parts per million) after lactulose. Furthermore, symptomatic patients had high prevalence of colonic motility disorders (slow transit). In these patients, treatment with a prokinetic (cisapride 40 mg/kg/day for 10 days) reduced colonic transit time, colonic hydrogen production and bloating symptoms. Abdominal symptoms in these patients may therefore have other causes than small bowel bacterial overgrowth alone. All operated patients with persistent abdominal bloating should therefore be investigated before starting empirical treatment with antibiotics.

Role of bile acids and metabolic activity of colonic bacteria in increased risk of colon cancer after cholecystectomy.

Since the metabolic activity of the colonic flora plays a definite role in colon cancer and an increased incidence of this disease is reported after cholecystectomy, we studied the metabolic activity of the colonic flora in a group of postcholecystectomy patients and matched controls by measuring, as representative end products of the bacterial metabolism, their fecal bile acids (BA), fecal 3-methylindole (SK) and indole (IN), and respiratory methane and hydrogen. Patients had significantly higher SK and lower IN, and, among BA, higher lithocholic (LCA) and chenodeoxycholic acid concentrations and LCA/deoxycholic acid ratio in the stools than controls. Similar differences from controls were reported for colon cancer. Comparable bacterial metabolic activities are thus operative in the large bowel of postcholecystectomized and colon cancer patients. This supports the biological plausibility of the association of cholecystectomy and colon cancer.

The effects of S(-) and R(+) sulpiride, metoclopramide, cisapride and domperidone on the small intestine suggest DA2-receptors are involved in the control of small intestinal transit time in rats.

To study the effect of intraperitoneal S(-)sulpiride (1-15 mg/kg), R(+)sulpiride (5-10 mg/kg), metoclopramide (1-15 mg/kg), cisapride (10 mg/kg) and domperidone (5-10 mg/kg) on intestinal progression, rats were given the test drug followed by oral lactulose. Their hydrogen excretion was used to calculate the small bowel transit time (SBTT) and maximum peak time (MPT). Metoclopramide (7.5 mg/kg) had the greatest effect on SBTT (-25%), followed by S(-)sulpiride and domperidone. S(-)sulpiride (10 mg/kg) had the greatest activity on the MPT (-35.2%) followed by metoclopramide. R(+)sulpiride and cisapride did not modify SBTT and MPT. In conclusion S(-)sulpiride is the isomer active on intestinal transit and DA2-receptors seem important targets in the modulation of intestinal progression, since S(-)sulpiride, metoclopramide and domperidone are DA2-receptor antagonists, and R(+)sulpiride and cisapride are not. The H2 breath test proved a valid method for measuring the effect of drugs on the small intestine in animals.

Indomethacin-induced enteropathy: effect of the drug regimen on intestinal permeability in rats.

To set up and characterize reproducible, long-standing small intestinal inflammation in rats, animals were given three different oral regimens of indomethacin (Ind): a bolus of 10 mg/kg in water and three daily doses of 2, 4, and 8 mg/kg Ind in (a) the drinking water or (b) the standard diet. The effect of Ind on the small intestine was monitored by measuring intestinal permeability (IP). The three-day regimen seemed more suitable than a bolus dose to induce long-standing inflammatory modifications in the rat small intestine and Ind administered in the drinking water gave more consistent modifications of IP and more reproducible results than Ind in food. IP seemed a suitable tool for detecting these inflammatory changes in the small-intestinal physiology. This model could be used to assess the effect of new drugs on inflammation.

High-performance liquid chromatographic determination of desmosine and isodesmosine after phenylisothiocyanate derivatization.

A new sensitive and selective high-performance liquid chromatographic method for the analysis of desmosine and isodesmosine in human and rat tissues is described. This method requires a purification step with column chromatography, followed by precolumn derivatization phenylisothiocyanate. The reaction products are then separated by isocratic chromatography on a C18 column and quantitated by ultraviolet detection at 254 nm. The recovery of standards of both compounds added to tissue samples and analysed by this method is usually greater than 90%, and the absolute detection limit is 0.5 ng for both compounds. The method is sensitive enough to measure both substances in tissue fragments of 30 mg of wet mass, which means that it can be used to study elastin in small human biopsies.