Sara Negri

Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2R4496C+/−). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2R4496C+/− myocytes (P=0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2R4496C+/− myocytes (P=0.001). DADs and triggered activity were abolished by ryanodine (10 &mgr;mol/L) but not by K201 (1 &mgr;mol/L or 10 &mgr;mol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2R4496C+/− mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2–FKBP12.6 interaction both in WT and RyR2R4496C+/− either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2R4496C+/− myocytes and ventricular arrhythmias in RyR2R4496C+/− mice; and (3) RyR2–FKBP12.6 interaction in RyR2R4496C+/− is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex.

Short Communication: Flecainide Exerts an Antiarrhythmic Effect in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia by Increasing the Threshold for Triggered Activity

Rationale: Flecainide prevents arrhythmias in catecholaminergic polymorphic ventricular tachycardia, but the antiarrhythmic mechanism remains unresolved. It is possible for flecainide to directly affect the cardiac ryanodine receptor (RyR2); however, an extracellular site of action is suggested because of the hydrophilic nature of flecainide. Objective: To investigate the mechanism for the antiarrhythmic action of flecainide in a RyR2R4496C+/− knock-in mouse model of catecholaminergic polymorphic ventricular tachycardia. Methods and Results: Flecainide prevented catecholamine-induced sustained ventricular tachycardia in RyR2R4496C+/− mice. Cellular studies were performed with isolated RyR2R4496C+/− myocytes. Isoproterenol caused the appearance of spontaneous Ca2+ transients, which were unaffected by flecainide (6 &mgr;mol/L). Flecainide did not affect Ca2+ transient amplitude, decay, or sarcoplasmic reticulum Ca2+ content. Moreover, it did not affect the frequency of spontaneous Ca2+ sparks in permeabilized myocytes. In contrast, flecainide effectively prevented triggered activity induced by isoproterenol. The threshold for action potential induction was increased significantly (P<0.01), which suggests a primary extracellular antiarrhythmic effect mediated by Na+ channel blockade. Conclusions: Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in RyR2R4496C+/− mice; however, at variance with previous reports, we observed minimal effects on intracellular Ca2+ homeostasis. Our data suggest that the antiarrhythmic activity of the drug is caused by reduction of Na+ channel availability and by an increase in the threshold for triggered activity.

Subclinical abnormalities in sarcoplasmic reticulum Ca2+ release promote eccentric myocardial remodeling and pump failure death in response to pressure overload

OBJECTIVES This study sought to explore whether subclinical alterations of sarcoplasmic reticulum (SR) Ca(2+) release through cardiac ryanodine receptors (RyR2) aggravate cardiac remodeling in mice carrying a human RyR2(R4496C+/-) gain-of-function mutation in response to pressure overload. BACKGROUND RyR2 dysfunction causes increased diastolic SR Ca(2+) release associated with arrhythmias and contractile dysfunction in inherited and acquired cardiac diseases, such as catecholaminergic polymorphic ventricular tachycardia and heart failure (HF). METHODS Functional and structural properties of wild-type and catecholaminergic polymorphic ventricular tachycardia-associated RyR2(R4496C+/-) hearts were characterized under conditions of pressure overload induced by transverse aortic constriction (TAC). RESULTS Wild-type and RyR2(R4496C+/-) hearts had comparable structural and functional properties at baseline. After TAC, RyR2(R4496C+/-) hearts responded with eccentric hypertrophy, substantial fibrosis, ventricular dilation, and reduced fractional shortening, ultimately resulting in overt HF. RyR2(R4496C+/-)-TAC cardiomyocytes showed increased incidence of spontaneous SR Ca(2+) release events, reduced Ca(2+) transient peak amplitude, and SR Ca(2+) content as well as reduced SR Ca(2+)-ATPase 2a and increased Na(+)/Ca(2+)-exchanger protein expression. HF phenotype in RyR2(R4496C+/-)-TAC mice was associated with increased mortality due to pump failure but not tachyarrhythmic events. RyR2-stabilizer K201 markedly reduced Ca(2+) spark frequency in RyR2(R4496C+/-)-TAC cardiomyocytes. Mini-osmotic pump infusion of K201 prevented deleterious remodeling and improved survival in RyR2(R4496C+/-)-TAC mice. CONCLUSIONS The combination of subclinical congenital alteration of SR Ca(2+) release and pressure overload promoted eccentric remodeling and HF death in RyR2(R4496C+/-) mice, and pharmacological RyR2 stabilization prevented this deleterious interaction. These findings suggest potential clinical relevance for patients with acquired or inherited gain-of-function of RyR2-mediated SR Ca(2+) release.

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.

Oxidative activity of ammonium persulfate salt on mast cells and basophils: implication in hairdressers' asthma.

Background: Persulfate salts are components of bleaching powders widely used by hairdressers during hair-bleaching procedures. Hairdressers are at high risk for occupational asthma and rhinitis, and ammonium persulfate is the main etiologic agent. Objective: To explore the effects of ammonium persulfate on human albumin, mast cells, and basophils in order to evaluate a possible effect of ammonium persulfate oxidizing activity in the mechanism of ammonium persulfate-induced occupational asthma. Methods: High-performance liquid chromatography/mass spectrometry was performed on ammonium persulfate-incubated human albumin. The activation of LAD2 human mast cell and KU812 human basophil cell lines incubated with ammonium persulfate was evaluated. CD63 expression on persulfate-in-vitro-incubated blood basophils from nonexposed healthy controls (n = 31) and hairdressers with work-related respiratory symptoms (n = 29) was assessed by flow cytometry. Results: No persulfate-albumin conjugate was found. An oxidative process on tryptophan and methionine was detected. Ammonium persulfate induced reactive oxygen species (ROS) generation and the degranulation of LAD2 and KU812 cells. Human basophils from healthy controls, incubated in vitro with ammonium persulfate, showed increased CD63 expression and ROS production. In hairdressers with ammonium persulfate-caused occupational asthma (positive persulfate challenge), basophil-CD63 expression was higher than in those with a negative challenge and in healthy controls. Conclusions: Ammonium persulfate incubated with human albumin did not generate any adduct but oxidized some amino acids. This oxidizing activity induced human mast cell and basophil activation which might be crucial in the mechanism of persulfate-induced occupational asthma and rhinitis.

Dimethylformamide metabolism following self-harm using a veterinary euthanasia product.

Background. A veterinary euthanasia drug containing embutramide, mebezonium, tetracaine, and dimethylformamide (DMF; T-61® or Tanax®) may cause serious manifestations or even fatalities after self-poisoning. Immediate toxicity is mainly due to a general anesthetic and due to a neuromuscular blocking agent, while delayed hepatotoxicity seems related to the solvent DMF. The protective role of N-acetylcysteine (NAC) administration remains debatable. Material and methods. Two male veterinarians (50- and 44-year-old) attempted suicide by injecting T-61 in the precordial area for the first one, and by ingesting 50 mL for the second. Both received NAC (for 14 days in the first case and only for 20 h in the second). Urine was collected for the serial determination of DMF, N-methylformamide (NMF), and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC). Results. Both patients developed only mild signs of liver injury. The metabolite of DMF, NMF, appeared rapidly in the urine, while a further delay was necessary for AMCC excretion. The kinetics of elimination of DMF and DMF metabolites were slightly slower than those reported in exposed workers. Conclusions. While both patients had a favorable outcome, there is no clear evidence that NAC could directly influence NMF and AMCC excretion. Further investigations of NMF and AMCC excretion, with and without NAC, would be indicated.

Measurement of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine by high-performance liquid chromatography with direct ultraviolet detection.

A new high-performance liquid chromatographic (HPLC) method is described for the determination of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), the final product of the conjugation reaction between a metabolic intermediate of N,N-dimethylformamide (DMF) and glutathione. Urine samples were purified by C(18) solid-phase extraction and then directly analysed by HPLC with an Aminex Ion Exclusion HPX-87H column maintained at 25 degrees C and a UV detector set at 196 nm. Under isocratic conditions (2.4 mM sulphuric acid, flow-rate=0.6 ml/min) AMCC eluted at 20.2 min. The reproducibility (C.V.%) was 1.3-2.7% (intra- and inter-assay, N = 5); the accuracy was 98.0+/-1.7% at 10 mg/l and 101.9+/-1.5% at 800 mg/l (mean+/-SD, N = 3). AMCC was measured in urine from 22 exposed subjects. A strong correlation was found between AMCC and environmental DMF [AMCC (mg/g creatinine)=3.40xDMF (mg/m(3)) + 3.07; r=0.95], while in the urine of 20 unexposed subjects the concentration of AMCC was constantly below the detection limit of the method (0.9 mg/l in urine). The method described appears to be useful for the biological monitoring of DMF exposure.

Monitoring Surface Contamination by Antineoplastic Drugs in Italian Hospitals: Performance-Based Hygienic Guidance Values (HGVs) Project

Antineoplastic drugs (ADs) will continue to represent a potential risk for personnel involved in the handling of these compounds and great concerns have been raised by the presence of ADs in many surveyed workplaces. Eight hospitals were investigated by means of wipe sampling for surface residue determination. Each wipe sample was tested for five ADs considered suitable exposure markers. Cyclophosphamide (CP), gemcitabine (GEM), 5-fluorouracil (5-FU), platinum-containing drugs (Pt), and epi-doxorubicin (EPI) contamination levels were measured in 85 per cent of the studied pharmacies and 93 per cent of outpatient care units (OpCUs). This study showed that 83 out of 349 samples were positive in Pharmacies, this proportion being statistically significant (χ2 = 42.9, p < 0.001). The positive samples provided evidence of at least one substance with levels greater than the limit of detection (LOD). The two most frequently detected substances were Pt (42%) and CP (30%). These accounted for 72 per cent of the whole dataset, followed by 5-FU and GEM. Based on the 90th percentile of wipe sampling data distribution, we suggest hygienic guidance values (HGVs) of 3.6, 1.0, 0.9, and 0.5 ng cm-2 for CP, 5-FU, GEM and Pt, respectively, as the best target levels of the surface contamination load in Italian pharmacies. The approach of proposing guidance values at the 90th percentile of results obtained from workplaces with good hygiene practice was found to be a simple and practical way of controlling occupational exposure. HGVs were challenged in this study as technical threshold limits to benchmark AD residual surface contamination at workplaces.

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.

Urinary levels of metal elements in the non-smoking general population in Italy: SIVR study 2012-2015

The purpose of this study of the Italian Society of Reference Values (SIVR) was to provide the reference values for metals in the urine of the Italian general population. Thirteen sampling centres situated in the north, centre and south of Italy took part in this project. Each sampling centre selected 20 adult subjects. The sample was made up of 120 male and 140 female non-smokers or smokers who had abandoned the habit at least 5 years previously, aged between 18 and 60 years. Urine samples were obtained from the same subjects in June and again in November of the same year. During collection of these two samples, we administered an ad hoc questionnaire designed to assess factors that might influence exposure and the results of the study such as personal characteristics, occupational or extra-occupational activities, dietary habits, the presence of dental fillings containing amalgam, the frequency and mode of exposure to passive smoking and exhaust gases of motor vehicles (traffic). Therefore, the urinary levels of thirteen trace elements (Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V) were determined by Inductively Coupled Plasma Mass Spectrometry. The concentrations obtained (95th percentile) are in μg/L: 0.034, 0.900, 2.24, 0.600, 24.0, 0.013, 1.53, 4.44, 2.64, 0.022, 0.095, 0.759 and 0.855 for Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V, respectively. Further studies with a larger number of subjects are needed in order to continue the biomonitoring of the Italian general population and to monitor the modified levels over time.