PFOS induces behavioral alterations, including spontaneous hyperactivity that is corrected by dexamfetamine in zebrafish larvae

Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae

Illicit drugs in Emergency Department patients injured in road traffic accidents

Urine and blood samples from 1730 drivers involved in road accidents (July 2012- December 2015) were analyzed for the evaluation of driving under influence of drug of abuse according to the Lombardia Region guideline. The 22.5 % of urine screenings tested positive for at least one class of drugs. 10.6% of the 1730 drivers were under the influence of drug, being blood concentration above the cut-off limit for at least one active substance; the prevalence of illicit drugs in blood was cocaine (5.7 %), cannabinoids (3.7 %), opiates (1.4 %), methadone (1.4 %), amphetamines (0.2 %). Trend in prevalence showed similar percentage (about 5 %) of cocaine and cannabinoids consumption in the last two years. Poly-drug of abuse consumption emerged in the 10.4 % of the positive blood and alcohol was above the legal limit in 47 % of the subjects driving under the influence of drugs

Determination of Glucocorticoids in UPLC-MS in Environmental Samples from an Occupational Setting

Occupational exposures to glucocorticoids are still a neglected issue in some work environments, including pharmaceutical plants. We developed an analytical method to quantify simultaneously 21 glucocorticoids using UPLC coupled with mass spectrometry to provide a basis to carry out environmental monitoring. Samples were taken from air, hand-washing tests, pad-tests and wipe-tests. This paper reports the contents of the analytical methodology, along with the results of this extensive environmental and personal monitoring of glucocorticoids. The method in UPLC-MS turned out to be suitable and effective for the aim of the study. Wipe-test and pad-test desorption was carried out using 50 mL syringes, a simple technique that saves time without adversely affecting analyte recovery. Results showed a widespread environmental pollution due to glucocorticoids. This is of particular concern. Evaluation of the dose absorbed by each worker and identification of a biomarker for occupational exposure will contribute to assessment and prevention of occupational exposure

The effects of developmental exposure to PFOS on startle response in 6 dpf zebrafish larvae.

<p>(A) Typical vibrational startle response in a 6 dpf zebrafish larvae. Control larvae display a single bout of swimming activity after triggering the stimulus (arrowhead), followed by a prolonged silent period. Note that in control larvae, the swimming bout induced by the vibrational stimulus is considerably more robust than the spontaneous bouts (analysis shown in B), and is followed by a period of inactivity. In contrast, in larvae exposed to 1 mg/L PFOS, the vibrational stimulus is followed by a prolonged sequence of swimming bouts that have the amplitude similar to spontaneous bouts. The time interval between triggering the stimulus and the initiation of the bout is defined as latency to startle (analysis shown in D). The delay between the end of the startle bout and the following bout (analysis shown in E) can be defined as the latency to resume spontaneous swimming activity in control, but not in larvae exposed to 1 mg/L PFOS. (B) Comparison of the distance moved within spontaneous vs. stimulation-induced swimming bouts. Control larvae and larvae exposed to 0.1 mg/L PFOS consistently display a robust increase in the amplitude of activity bout in response to vibrational stimulation. In contrast, zebrafish larvae exposed to 1 mg/L PFOS swam significantly longer distances within spontaneous bout, but do not increase the distance moved in response to the vibrational stimulation. (C) Average distance moved integrated over 1s timebins. The increase in distance moved is accounted for by a single, more robust bout in controls (see A). In contrast, the amplitude of the startle response in larvae exposed to 1 mg/L PFOS is significantly larger than in controls, and it is presumably accounted for by more than 1 bout. In addition, the larvae exposed to 1 mg/L PFOS remain hyperactive for about 4 s after stimulation. The higher variability before in spontaneous activity before and after the startle response can be explained by irregularity in occurrence of spontaneous bouts. Note also that the larvae exposed to 1 mg/L PFOS display an increase in activity before the stimulation. (D, E) Analysis of the latency to startle (D) and inactive period (E). Zebrafish larvae exposed to 1 mg/L PFOS have longer latency to startle, and have shorter inactive period than controls or larvae exposed to 0.1 mg/L PFOS. B - repeated measures ANOVA followed by unequal N HSD post-hoc test; * p<0.05 startle vs. spontaneous; § p<0.05 PFOS exposed vs. controls. C –repeated measures ANOVA followed by unequal N HSD, or Dunnett's post-hoc test, respectively; § p<0.05 PFOS exposed vs. control; * p<0.05 vs. baseline. D, E - ANOVA followed by Dunnett's post-hoc test; * p<0.05 PFOS exposed vs. controls. The number of independent observations is indicated at the bottom of each column in D and E. The graphs in B and C are based on the same number of observation as reported in D and E.</p

The structure of spontaneous locomotion in zebrafish larvae and mice during the active phase of the circadian cycle (light for zebrafish larvae, dark for mice).

<p>(A) Representative sequence of spontaneous activity (20 s) recorded at 6 dpf in one control and one larva exposed to 1 mg/L PFOS. In the control larva, the spontaneous activity consists of short, evenly spread bouts of swimming. In the larva exposed to 1 mg/L PFOS, the spontaneous activity consists of clusters of intense activity separated by extended periods of inactivity. Background noise (displacement below 0.2 mm/frame) shaded in gray; inset in top panel – magnification of a representative bout of spontaneous activity. (B) Quantification of frequency of spontaneous bouts of activity. Note the dramatic decrease in bout frequency induced by exposure to 1 mg/L PFOS. (C) Average distance moved during one spontaneous bout of activity. Zebrafish larvae exposed to 1 mg/L PFOS have a hyperactive phenotype characterized by a 2.5 fold higher distance swum during spontaneous bouts of activity. (D) Illustrative sequence of spontaneous locomotion during the active phase of the circadian cycle in one control and one mouse exposed to 3 mg/kg/dy PFOS during gestation. The spontaneous locomotor activity (visits) in the homecage is integrated over consecutive, non-overlapping 10 min bins and spline-interpolated for clarity. (E, F) Similar to the pattern found in zebrafish larvae, the mice exposed to 3 mg/kg PFOS display less frequent (E), but more intense (F) bouts of activity. B, C, E, F – one-way ANOVA followed by Dunnett's post-hoc test; * p<0.05 PFOS exposed vs. control. The number of independent observations is indicated at the bottom of each column in B, C, E and F.</p

The effects of acute administration of dexamfetamine on spontaneous swimming and on startle response.

<p>(A) Dexamfetamine displays a bell-shaped dose-dependence of spontaneous bout frequency, but does not alter the distance swam per bout in controls and in larvae exposed to 0.1 mg/L PFOS. In contrast, dexamfetamine monotonically increases the frequency of spontaneous bouts of activity, and reduces the distance moved per bout in larvae exposed to 1 mg/L PFOS. (B) Average rate of response to vibrational stimulation. At baseline, the rate of response is significantly lower in the larvae exposed to 1 mg/L PFOS than in controls. Upon administration of dexamfetamine, the rate of response is increased only at 1 µM in controls, and at both doses in the larvae exposed to 1 mg/L PFOS. (C) Acute dexamfetamine administration alters the spontaneous activity before and after stimulation (presumably by altering the frequency of spontaneous bouts; see also A), but does not influence the amplitude of the startle response (accounted for by a single bout; see also D) in controls and larvae exposed to 0.1 mg/L PFOS. In larvae exposed to 1 mg/L PFOS, the amplitude of the startle response is not altered (presumably accounted for by more than one bout; see also D and E), but the duration of hyperactivity following the vibrational stimulation is shortened by both 1 and 10 µM dexamfetamine (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094227#pone.0094227.s005" target="_blank">Figure S5</a>). (D) Dexamfetamine restores the amplitude-modulation of the activity bouts in larvae exposed to 1 mg/L PFOS by decreasing the distance moved for spontaneous bouts. (E) Dexamfetamine shortens the inactive period only in controls and in the larvae exposed to 0.1 mg/L PFOS. A, D - factorial ANOVA followed by unequal N HSD post-hoc test; § p<0.05 PFOS exposed vs. control; * p<0.05 vs. baseline. B- repeated measures ANOVA, followed by Dunnett's post hoc test with the first 3 samples as control (baseline); * p<0.05 vs. baseline. E - repeated measures ANOVA followed by unequal N HSD post-hoc test; * p<0.05 startle vs. spontaneous; § p<0.05 PFOS exposed vs. controls. The number of independent observations is indicated at the bottom of each column in A, B and E. Graphs in C and D are based on the same number of observation as reported in B and E.</p

VMR in zebrafish larvae and novelty-induced hyperactivity in mice.

<p>(A) The dark pulse induces a similar pattern of fluctuations in the frequency of spontaneous bouts in larvae exposed to 0.1 mg/L PFOS as in controls. Larvae exposed to 1 mg/L PFOS respond with an increase in frequency that does not vary over time. In addition, the frequency of spontaneous bouts is restored directly to baseline level after the dark pulse. (B) Hyperactivity and habituation in zebrafish larvae during the dark pulse; 30 s timebins. (C) Quantification of total distance and IOC in zebrafish larvae. (D) Novelty-induced hyperactivity in mice. (E) Quantification of total distance moved and habituation rate (estimated by IOC) in mice. Note the similarity between the dose-response curves of the effect of exposure to PFOS on habituation in zebrafish and mice. (F) Swimming activity in zebrafish larvae at the transition between light and dark (gray shaded areas). The larvae exposed to 1 mg/L PFOS display hyperactive episodes of higher magnitude that last considerably longer than in controls. * p<0.05 PFOS exposed vs. control; ANOVA followed by Dunnett's post-hoc test. The number of independent observations is indicated at the bottom of each column in C and E. Graphs in A and B are based on the same number of observations reported in C; graphs in D are based on the same number of observations as in E.</p

Abstracts from the 23rd Italian congress of Cystic Fibrosis and the 13th National congress of Cystic Fibrosis Italian Society

Cystic Fibrosis (CF) occurs most frequently in caucasian populations. Although less common, this disorder have been reported in all the ethnicities. Currently, there are more than 2000 described sequence variations in CFTR gene, uniformly distributed and including variants pathogenic and benign (CFTR1:www.genet.sickkids.on.ca/). To date,only a subset have been firmily established as variants annotated as disease-causing (CFTR2: www.cftr2.org). The spectrum and the frequency of individual CFTR variants, however, vary among specific ethnic groups and geographic areas. Genetic screening for CF with standard panels of CFTR mutations is widely used for the diagnosis of CF in newborns and symptomatic patients, and to diagnose CF carrier status. These screening panels have an high diagnostic sensitivity (around 85%) for CFTR mutations in caucasians populations but very low for non caucasians. Developed in the last decade, Next-Generation Sequencing (NGS) has been the last breakthrough technology in genetic studies with a substantial reduction in cost per sequenced base and a considerable enhancement of the sequence generation capabilities. Extended CFTR gene sequencing in NGS includes all the coding regions, the splicing sites and their flankig intronic regions, deep intronic regions where are localized known mutations,the promoter and the 5'-3' UTR regions. NGS allows the analysis of many samples concurrently in a shorter period of time compared to Sanger method . Moreover, NGS platforms are able to identify CFTR copy number variation (CNVs), not detected by Sanger sequencing. This technology has provided new and reliable approaches to molecular diagnosis of CF and CFTR-Related Disorders. It also allows to improve the diagnostic sensitivity of newborn and carrier screeningmolecular tests. In fact, bioinformatics tools suitable for all the NGS platforms can filter data generated from the gene sequencing, and analyze only mutations with well-established disease liability. This approach allows the development of targeted mutations panels with a higher number of frequent CF mutations for the target populationcompared to the standard panels and a consequent enhancement of the diagnostic sensitivity. Moreover, in the emerging challenge of diagnosing CF in non caucasians patients, the possibility of customize a NGS targeted mutations panel should increase the diagnostic sensitivity when the target population has different ethnicities