Differential Micronuclei Induction in Human Lymphocyte Cultures by Imidacloprid in the Presence of Potassium Nitrate

Humans are exposed to pesticides as a consequence of their application in farming or their persistence in a variety of media, including food, water, air, soil, plants, animals, and smoke. The interaction of pesticides with environmental factors may result in the alteration of their physicochemical properties. Square wave cathodic stripping voltammetry (SW-CSV), a technique that simulates electrodynamically the cellular membrane, is used to investigate whether the presence of potassium nitrate (KNO3) in the culture medium interferes with the genotoxic behavior of imidacloprid. The cytokinesis block micronuclei (CBMN) method is used to evaluate imidacloprid's genotoxicity in the absence or presence of KNO3 in the culture medium and, as a consequence, its adsorption by lymphocytes. Comparing micronuclei (MN) frequencies in control and imidacloprid-treated blood cell cultures, statistically significant differences were not detected. KNO3 did not induce MN frequencies compared to control. Statistically significant differences in MN frequencies were observed when blood cell cultures were treated with imidacloprid in the presence of increasing concentrations of KNO3. SW-CSV revealed that by increasing KNO3 molarity, imidacloprid's concentration in the culture medium decreased in parallel. This finding indicates that imidacloprid is adsorbed by cellular membranes. The present study suggests a novel role of a harmless environmental factor, such as KNO3, on the genotoxic behavior of a pesticide, such as imidacloprid. KNO3 rendered imidacloprid permeable to lymphocytes, resulting in elevated MN frequencies

Respiratory and immune response to maximal physical exertion following exposure to secondhand smoke in healthy adults

© 2012 The Authors. Published by PLOS. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1371/journal.pone.0031880We assessed the cardiorespiratory and immune response to physical exertion following secondhand smoke (SHS) exposure through a randomized crossover experiment. Data were obtained from 16 (8 women) non-smoking adults during and following a maximal oxygen uptake cycling protocol administered at baseline and at 0-, 1-, and 3- hours following 1-hour of SHS set at bar/restaurant carbon monoxide levels. We found that SHS was associated with a 12% decrease in maximum power output, an 8.2% reduction in maximal oxygen consumption, a 6% increase in perceived exertion, and a 6.7% decrease in time to exhaustion (P<0.05). Moreover, at 0-hours almost all respiratory and immune variables measured were adversely affected (P<0.05). For instance, FEV 1 values at 0-hours dropped by 17.4%, while TNF-α increased by 90.1% (P<0.05). At 3-hours mean values of cotinine, perceived exertion and recovery systolic blood pressure in both sexes, IL4, TNF-α and IFN-γ in men, as well as FEV 1/FVC, percent predicted FEV 1, respiratory rate, and tidal volume in women remained different compared to baseline (P<0.05). It is concluded that a 1-hour of SHS at bar/restaurant levels adversely affects the cardiorespiratory and immune response to maximal physical exertion in healthy nonsmokers for at least three hours following SHS. © 2012 Flouris et al.Published versio

Study of the biological activity of various pesticides in relation to their physicochemical behaviour

Pesticides are extensively used in agriculture, all over the world, and in recent years their use has dramatically increased. Humans are exposed to them either through occupational exposure via inhalation or skin contact during the preparation of the solutions to be sprayed or during spraying as cultivators, or as consumers through the residues that remain in food, smoke, air, soil, water, plants and animals. This thesis aims in evaluating the biological activity of pesticides in combination to their physicochemical parameters. Specifically, it was attempted to investigate whether their genotoxicity is determined by their physicochemical characteristics and as a consequence by environmental parameters. Methidathion and Imidacloprid, two commonly used pesticides were chosen in this study. Methidathion is a non-systemic organophosphorus insecticide acting as an inhibitor of Cholinesterase. Imidacloprid is a systemic chforo-nicotinyl insecticide with soil, seed and foliar uses and acts as acetylcholine receptors antagonist. To evaluate the genotoxicity of the above pesticides, the Cytokinesis Block Micronuclei Assay (CBMN) in human lymphocytes in vitro, was selected. To investigate the physiochemical characteristics of those pesticides and their presence inside or outside the cell, the Cathodic Stripping Voltammetry Assay was chosen (CSV). In addition, an in vivo study of the cytogenetic damage in farmers occupationally exposed to different mixtures of pesticides was performed. Furthermore there was an in vivo evaluation of genotoxicity of Imidacloprid in rats. The experimental results of Methidathion showed that the environmental parameter pH, has a catalytic effect on its formula, rendering it more dangerous. In vitro experiments, aiming to evaluate the genotoxicity of Imidacloprid in human lymphocytes with the use of CBMN in correlation with the data from the experimental results of the CSV assay with and without the presence of ΚΝΟ₃, showed an alteration of its physiochemical behavior due to the presence of ΚΝΟ₃. The factor affecting the action of Imidacloprid is the charge of the molecule which forces the molecule to bind to the cell membrane and prevents it from penetrating into the cell. With the presence of ΚΝΟ₃ the charge is neutralized and the pesticide penetrates the cell. The presence of a harmless and abundant in nature substance, such as ΚΝΟ₃, can dramatically alter the action of a pesticide, such as Imidacloprid transforming it from a friendly to human organism into a harmful one. Furthermore, the results from the thermodynamic behavior of the adsorption of Imidacloprid with the use of CSV, showed that Imidacloprid's adsorption is higher on hydrophobic surfaces at low temperatures, and that explains its higher toxicity on cool-blooded insects versus its lower towards warm-blooded organisms. The present study contributes to the clarification of the mechanism of action of those two pesticides. The results obtained from biological as well as physicochemical experiments showed that inter-scientific approach can provide key information of immediate environmental interest.Τα φυτοφάρμακα αποτελούν σημαντικούς περιβαλλοντικούς ρυπαντές, στους οποίους εκτίθεται καθημερινά ο άνθρωπος με συνεχώς αυξανόμενο ρυθμό. Ο άνθρωπος εκτίθεται στα φυτοφάρμακα είτε μέσω της επαγγελματικής του ενασχόλησης ως καλλιεργητής, κατά τη διάρκεια ψεκασμών, είτε ως καταναλωτής, μέσω των υπολειμμάτων που παραμένουν σε τρόφιμα, καπνό, αέρα, έδαφος, νερό, φυτά και ζώα. Σκοπός της διατριβής είναι η μελέτη της βιολογικής δράσης φυτοφαρμάκων σε συνδυασμό με τη φυσικοχημική τους συμπεριφορά. Ειδικότερα επιδιώκεται να διερευνηθεί αν η γενοτοξική δράση τους καθορίζεται από τις φυσικοχημικές τους ιδιότητες και κατ’ επέκταση από τις περιβαλλοντικές συνθήκες. Επιλέχτηκαν δύο φυτοφάρμακα ευρείας χρήσης, το Methidathion και το Imidacloprid. Το Methidathion είναι οργανοφωσφορικό εντομοκτόνο, που δρα σαν ανταγωνιστής της χολινεστεράσης. To Imidacloprid είναι χλωρο-νικοτινικό εντομοκτόνο ευρέως φάσματος, το οποίο δρα ως ανταγωνιστής της ακετυλοχολίνης και ανήκει στα νικοτινοειδή, μια νέα κατηγορία φυτοφαρμάκων. Για την εκτίμηση της γενοτοξικής δράσης των παραπάνω φυτοφαρμάκων επιλέχθηκε η τεχνική των μικροπυρήνων (Micronucleus Assay) σε ανθρώπινα λεμφοκύτταρα in vitro με τη χρήση κυτταροχαλασίνης-Β. Για τη μελέτη της φυσικοχημικής συμπεριφοράς καθώς και της παρουσίας τους στον εσωκυττάριο ή εξωκυττάριο χώρο εφαρμόστηκε η τεχνική της καθοδικής βολταμετρίας με προσυγκέντρωση (Cathodic Stripping Voltammetry). Επίσης, πραγματοποιήθηκε in vivo μελέτη σε ομάδα αγροτών του νομού Ηρακλείου που χρησιμοποιούσαν μεταξύ άλλων φυτοφαρμάκων και το Methidathion. Συγχρόνως μελετήθηκε in vivo η δράση του Imidacloprid σε επίμυες. Τα αποτελέσματα των πειραμάτων για το Methidathion έδειξαν ότι ο περιβαλλοντικός παράγοντας pH, επηρεάζει καταλυτικά τη δομή του, ενισχύοντας την επικινδυνότητά του. Τα in vitro πειράματα προσδιορισμού της γενοτοξικής δράσης του Imidacloprid με την τεχνική των μικροπυρήνων σε καλλιέργειες ανθρώπινων λεμφοκυττάρων σε συνδυασμό με την τεχνική της καθοδικής βολταμετρίας με προσυγκέντρωση, παρουσία και απουσία ΚΝΟ₃, έδειξαν μεταβολή της φυσικοχημικής συμπεριφοράς του φυτοφαρμάκου εξαιτίας της παρουσίας του ΚΝΟ₃. Ο παράγοντας που επηρεάζει τη δράση του Imidacloprid είναι το φορτίο του μορίου το οποίο το κρατά προσκολλημένο στη μεμβράνη του λεμφοκυττάρου και δεν το αφήνει να εισχωρήσει στο εσωτερικό. Παρουσία ΚΝΟ₃ , το φορτίο του Imidacloprid εξουδετερώνεται με αποτέλεσμα να εισχωρεί ελεύθερα στο εσωτερικό του κυττάρου. Τα παραπάνω πειράματα έδειξαν ότι η παρουσία μιας ακίνδυνης και άφθονης στη φύση ένωσης, όπως το ΚΝΟ₃ μπορεί να αλλάξει δραματικά τον τρόπο δράσης ενός φυτοφαρμάκου όπως το Imidacloprid και να το μετατρέψει από φιλικό προς τον άνθρωπο σε αρκετά επικίνδυνο. Επίσης, από την μελέτη της θερμοδυναμικής συμπεριφοράς του Imidacloprid με την χρήση της καθοδικής βολταμετρίας με προσυγκέντρωση, προέκυψε ότι το συγκεκριμένο φυτοφάρμακο παρουσιάζει μεγαλύτερη δραστικότητα σε χαμηλές θερμοκρασίες γεγονός που το καθιστά πιο επικίνδυνο για τους ψυχρόαιμους οργανισμούς (έντομα) και λιγότερο για τους θερμόαιμους (θηλαστικά). Η εργασία αυτή συνεισφέρει στην αποσαφήνιση του μηχανισμού δράσης των δύο αυτών φυτοφαρμάκων. Τα αποτελέσματα αυτά που προέκυψαν, τόσο βιολογικά όσα και φυσικοχημικά, δείχνουν ότι η διεπιστημονική προσέγγιση μπορεί να δώσει χρήσιμες απαντήσεις σε ζητήματα περιβαλλοντικού ενδιαφέροντος

Differential Micronuclei Induction in Human Lymphocyte Cultures by Imidacloprid in the Presence of Potassium Nitrate

Humans are exposed to pesticides as a consequence of their application in farming or their persistence in a variety of media, including food, water, air, soil, plants, animals, and smoke. The interaction of pesticides with environmental factors may result in the alteration of their physicochemical properties. Square wave cathodic stripping voltammetry (SW-CSV), a technique that simulates electrodynamically the cellular membrane, is used to investigate whether the presence of potassium nitrate (KNO 3 ) in the culture medium interferes with the genotoxic behavior of imidacloprid. The cytokinesis block micronuclei (CBMN) method is used to evaluate imidacloprid&apos;s genotoxicity in the absence or presence of KNO 3 in the culture medium and, as a consequence, its adsorption by lymphocytes. Comparing micronuclei (MN) frequencies in control and imidacloprid-treated blood cell cultures, statistically significant differences were not detected. KNO 3 did not induce MN frequencies compared to control. Statistically significant differences in MN frequencies were observed when blood cell cultures were treated with imidacloprid in the presence of increasing concentrations of KNO 3 . SW-CSV revealed that by increasing KNO 3 molarity, imidacloprid&apos;s concentration in the culture medium decreased in parallel. This finding indicates that imidacloprid is adsorbed by cellular membranes. The present study suggests a novel role of a harmless environmental factor, such as KNO 3 , on the genotoxic behavior of a pesticide, such as imidacloprid. KNO 3 rendered imidacloprid permeable to lymphocytes, resulting in elevated MN frequencies. KEYWORDS: genotoxicity, micronuclei (MN), pesticides, imidacloprid, potassium nitrate (KNO 3 ), square wave cathodic stripping voltammetry (SW-CSV) INTRODUCTION The human diet is characterized by the presence of numerous naturally occurring chemicals in trace amounts. All these chemicals, constituents of processed and nonprocessed food, have been mainly considered as harmless to human health. Among the chemicals, potassium nitrate (KNO 3 ) is found as a preservative in processed food Humans are exposed to a large number of industrially made chemicals, designed for various purposes, among which are those to be used for crop protection. Several of these chemicals are responsible for serious negative side effects, such as biochemical malfunctions or genetic instability Pesticides constitute a heterogeneous class of chemicals representing an important group of environmental pollutants. In Europe, more than 3 million tons are released into the environment yearly Imidacloprid, a systemic chloronicotinyl insecticide with soil, seed, and foliar uses, blocks the nicotinergic neuronal pathway that is more abundant in insects than in warm-blooded animals. It is a General Use Pesticide (GUP) classified by the EPA as a class II and class III toxicity agent The genotoxic and mutagenic activities of certain pesticides have been studied both in in vitro and in vivo systems using cytogenetic end points, such as the cytokinesis block micronuclei (CBMN) method A rather important technique to simulate electrodynamically the adsorptive ability of cellular membranes is square wave cathodic stripping voltammetry (SW-CSV), using a hanging mercury drop electrode (HMDE). The HMDE is a capillary electrode with a mercury drop at its edge. The mercury drop acts as an electrode surface. It has been widely used in environmental and biological samplings. Its function is based on the application of a fixed voltage between 200 and -1200 mV, targeted at the redox of the electroactive species, such as imidacloprid, which has been adsorbed on its surface. Electrons flowing between the adsorbed electroactive species and the electrode surface are expressed in current (measured in nanoamperes) that corresponds with these species in solution. Electroactive species move towards the electrode surface by diffusion, while they are adsorbed via electrostatic interactions The present work focuses on the synergistic action between an environmental factor, such as KNO 3 , and a pesticide, such as imidacloprid. The possible side effects of KNO 3 on imidacloprid genotoxicity towards cultured human lymphocytes were investigated, using both the CBMN method and the SW-CSV. MATERIALS AND METHODS Reagents Ultrapure Milli-Q water, Millipore Academic system, was used throughout the experiments. Imidacloprid (RDH 37894; purity: 99.9% by HPLC) obtained from Sigma-Aldrich and KNO 3 (GA 18517; purity: 99.9% by HPLC) obtained from Fluka were used without further purification. Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid&apos;s Genotoxicity TheScientificWorldJOURNAL (2010) 10, 80-89 82 Imidacloprid stock solution (100 μM = 25.6 ppm) was prepared by dissolving 1.18 mg of insecticide in 46.07 ml of Milli-Q water and stored in the dark at 4 o C. This imidacloprid concentration is well below its solubility limit [26], while it corresponds to the mean value of previously reported concentrations The buffer solution used for voltammetric measurements was Britton-Robinson, prepared from a stock solution containing 10 mM phosphoric acid, 10 mM boric acid, and 10 mM acetic acid adjusted to the desired pH value, with NaOH. This buffer system is commonly used in analogous voltammetry experiments Apparatus and Software For the voltammetric measurements of imidacloprid, an electrochemical analyzer (Model TraceLab50, Radiometer Analytical) was used to control the voltage of a three-electrode system. As a working electrode, a Radiometer HMDE was used with a drop area of 3 mm 2 controlled by pneumatic connection with nitrogen (99.999%) at p = 1 bar. The reference electrode (Ag/AgCl-KCl = 3 M), type TR020, and auxiliary platinum electrode, type TM020, for POL150 were also from Radiometer. This system was connected with a Pentium II PC. All voltammetric measurements were carried out at 2 o C. As shown previously, low temperature facilitates the adsorption of imidacloprid on the mercury drop surface Instrumental Parameters The SW-CSV technique was used with: Cell parameters -Electrode HMDE, stirrer at 525 rpm, purge time 600 sec, accumulation time (t acc ) 60 sec, waiting time 10 sec, Hg-drop growth time 0.7 sec Signal parameters -Accumulation potential (E acc ) = -1060 mV[25], step duration 0.04 sec, step amplitude 1 mV, pulse amplitude +50 mV Current range -Minimum 10 nA, maximum 10 mA, under our experimental conditions the detection limit for imidacloprid was 0.25 μM. The pH measurements were carried out with a CRISON GLP21 pH-meter. Micronucleus Test in Human Lymphocytes In Vitro Blood samples were obtained from two healthy nonsmokers, without previous known contact with pesticides. Donors aged between 20 and 25 years. Whole blood (0.5 ml) was added to 6.5 ml Ham&apos;s F-10 medium (Invitrogen), 1.5 ml fetal calf serum (Invitrogen), and 0.3 ml phytohemagglutinin (Invitrogen) to stimulate cell division. The appropriate Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid&apos;s Genotoxicity TheScientificWorldJOURNAL (2010) 10, 80-89 83 chemicals were added 41 h postculture initiation to final imidacloprid concentration of 20 μM, and of KNO 3 to final concentrations of 2, 10, and 20 μM in separate treatments. In mixed treatments, the final concentrations were 20 μM for imidacloprid plus 5, 10, 15, and 20 μM for KNO 3 . Mitomycin-C (Sigma) at final concentration of 1.5 μM served as positive control. Cultures were incubated at 37 o C for 72 h. Three hours after the addition of the chemicals, 6 μg/ml cytochalasin-B (Sigma) was added at 44 h postculture initiation. Cells collected by centrifugation at 72 h postculture initiation, fixed with freshly made methanol/acetic acid (Riedel-de Haen/Merck) mixture (3:1 v/v) after mild hypotonic treatment, were stained with Giemsa (Fluka) The Cytokinesis Block Proliferation Index (CBPI), given by the equation: CBPI = M 1 + 2M 2 + 3(M 3 + M 4 )/N, where M 1 , M 2 , M 3 , and M 4 correspond to the numbers of cells with one, two, three, and four nuclei and N is the total number of cells Analytic Determination of Imidacloprid from Human Lymphocyte Cultures One milliliter of supernatant, derived from 72-h blood cultures in the presence of imidacloprid, was mixed with 9 ml of absolute ethanol Statistical Analysis Statistical analysis of MN data was made by the G-test for independence on 2x2 tables. This test is based on the general assumption of the χ 2 analysis, but offers theoretical and computational advantages RESULTS SW-CSV The effect of adsorbed imidacloprid on cultured cells was further investigated by the CBMN method. Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid&apos;s Genotoxicity TheScientificWorldJOURNAL Micronucleus Test in Human Lymphocytes In Vitro Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid&apos;s Genotoxicity TheScientificWorldJOURNAL (2010) 10, 80-89 85 Our data of 20 + 5 combined mixtures indicated marginally no significant statistical differences in both BNMN and MN frequencies (p = 0.057) compared to the control value. In the meantime, the same data appear to have a possible inducible effect, as their absolute values are almost twofold increased from their corresponding control values. A similar condition was observed at the concentration of 20 + 10 μΜ with regard to BNMN frequencies. The imidacloprid and KNO 3 cytotoxic effect was evaluated by the determination of CBPI. Regarding the cytotoxic index in all tested cytogenetic end points (see The reported control and positive control frequencies of MN were consistent to the literature, and authenticate our experimental procedure and treatment observations The presence of KNO 3 at concentrations higher than 10 μΜ in combination with 20 μΜ imidacloprid in culture media induces a statistically significant increase of MN frequencies (p &lt; 0.05 and 0.01, respectively) compared to control, while in separate treatments no statistical differences are observed, as seen in DISCUSSION Skepticism has gradually developed over studying the synergistic action of pesticides and their genotoxic side effects to humans and other organisms. Many variables intervene in the study system, rendering it too complex, not easily reproducible, and increasing the workload exponentially. In addition, scientists agree that the synergistic effects of agricultural chemical mixtures on human health are controversial and further studies on this issue are needed The present study poses an additional speculation on studying the synergistic effects between pesticides and environmental factors. For this purpose, the CBMN method was used in parallel to the SW-SCV electroanalytical technique. There is a relationship between polarographic and/or voltammetric behavior and genotoxic properties of organic compounds. By studying the mechanisms of their electrode reactions, useful clues can be provided to elucidate the mechanism of their interaction with living cells and their fate in the environment SW-CSV was used to study the effect of KNO 3 , a -harmless‖ chemical, on imidacloprid&apos;s genotoxicity to human lymphocyte cultures in vitro. This analytical voltammetric technique simulates electrodynamically the cellular membrane by applying a certain potential to a HMDE. By this system, the adsorptive ability of the cellular membrane towards imidacloprid, in the presence or absence of KNO 3 , could be evaluated. The CBMN method was used to evaluate imidacloprid genotoxicity in the absence or presence of KNO 3 . Neonicotinoids, including imidacloprid, are the most important new class of insecticides of the past 3 decades. They are increasingly replacing the organophosphates and methylcarbamates. Neonicotinoids have low activity on mammals relative to insect nicotinic acetylcholine receptors, providing a mechanistic basis for their safety 87 Controversial results regarding imidacloprid&apos;s genotoxic effect to cultured human lymphocytes were previously reported The negative in vitro genotoxic result in our findings from the separate treatment of 20 μM imidacloprid and the absence of imidacloprid&apos;s genotoxic effect in separate treatments on human lymphocytes in vitro at concentrations up to 400 μM, as well as the weak genotoxic effect on rat bone marrow in vivo A gradual reduction of imidacloprid&apos;s SW signal in culture medium with increasing KNO 3 concentration was observed by voltammetry. This is an indication that imidacloprid interacting with KNO 3 is adsorbed by lymphocytes. In the absence of KNO 3 , this reduction is not observed, while the SW signal is not altered by the presence of KNO 3 (see The calculated data from the electroanalytical method and genotoxicity experiments using the combination of imidacloprid and KNO 3 in cultured human lymphocytes denote the possibility of imidacloprid becoming genotoxic upon its interaction with a -harmless‖ chemical such as KNO 3 (see Our present data indicate that the combination of -harmless‖ environmental factors with pesticides that have not been classified as genotoxic to humans may interact, resulting in the alteration of the pesticides&apos; physicochemical properties. Such alteration may convert harmless-to-humans pesticides into harmful ones with unidentified side effects. The latter could be an indication of reconsidering our classification of natural or man-made chemicals as harmless or potentially harmful ones. Thus, further studies, including genotoxic, electroanalytical, physicochemical, and molecular assessment, are required before drawing final conclusions on the separate and/or combined with environmental factors action of pesticides. ACKNOWLEDGMENT

Classification, labelling and packaging of the most popular e-cigarette refill liquids across 9 European countries

Background Classification, Labelling and Packaging (CLP) of the e-cigarette refill liquids adopts the UN Globally Harmonised System of Classification and Labelling of Chemicals (GHS) into the EU. Regarding article 17 where a substance and mixture classified as hazardous must bear a label and article 35, where substances and mixtures must acquire packaging requirements, our study aimed to evaluate the level of compliance of these products within the European market. Methods Through cross referencing of two data sources, we identified examples of the most popular websites and their marketed brands, as also the most popular point of sale e-cigarettes, in nine European Member States (Greece, France, Poland, Germany, Holland, the United Kingdom, Spain, Romania and Hungary). An optical evaluation from two independent researchers followed for all the e-liquids (n=122). Evaluation was conducted on the vial, on the external package and leaflet if included. Results Results showed that almost all products evaluated were in near compliance with the two articles based on CLP regulations. The 89.3% (109) of the samples contained hazard pictograms while 80.3% (98) contained hazard statements. Additionally, the use of child-resistant fastening (CRF) systems and tactile warnings of danger (TWD) were frequently found on the vials and external packages (90.2% vs. 80.3%, respectively). Conclusions This is the first study to evaluate the labelling/packaging practices and the technical design/safety features of e-cigarettes in 9 EU MS before the implementation of Tobacco Products Directive. Funding: This work was supported by a grant from the European Commission (Horizon2020 HCO-6-2015; EUREST-PLUS: 681109)

Determination of DNA damage and telomerase activity in stanozolol-treated rats

Anabolic androgenic steroids (AAS) are performance -enhancing drugs commonly abused by atheletes. Stanozolol is a synthetic testosterone-derived anabolic steroid. Although it is well known that AAS have several side-effects, there are only few toxicological studies available on the toxic effects and mechanisms of action of stanozolol. The aim of this study was to investigate the genotoxic effects of stanozolol and to determine its effects on telomerase activity in Sprague-Dawley male rats. For this purpose, 34 male rats were divided into 5 groups as follows: i) the control group (n=5); ii) the propylene glycol (PG)-treated group (n=5); iii) the stanozolol-treated group (n=8); iv) the PG-treated group subjected to exercise (n=8); and v) the stanozolol-treated group subjected to exercise (n=8). PG is used as a solvent control in our study. Stanozolol (5 mg/kg) and PG (1 ml/kg) were injected subcutaneously 5 days/week for 28 days. After 28 days, the animals were sacrificed, and DNA damage evaluation (comet assay) and telomerase activity assays were then performed using peripheral blood mononuclear cells (PBMCs). Telomerase activity was measured by using the TeloTAGGG Telomerase PCR ELISA PLUS kit. The results of this study revealed that stanozolol treatment induced DNA damage, while exercise exerted a protective effect. Stanozolol treatment without exercise stimulation was associated with a significant increase in telomerase activity in the PBMCs

Porphyridium purpureum microalga physiological and ultrastructural changes under copper intoxication

The present work assessed the effect of copper (Cu) on cell dynamics and structure of the microalga Porphyridium purpureum (Rhodophyta, Bangiophycidae). Ultrastructure of the microalga was investigated and fluorescence of chlorophyll a and phycoerythrin, and content of reactive oxygen species (ROS) were estimated by flow cytometry. The number of cells did not show statistically significant differences at concentrations of 50 and 100 mu g/L of Cu compared to the control, whereas 150 mu g/L of Cu inhibited population growth. The fluorescence of chlorophyll a increased following exposure to Cu 100 mu g/L and fluorescence of phycoerythrin enhanced by Cu 150 mu g/ L. There was no alteration in the above indicators at other concentrations. The content of ROS increased with increasing Cu concentration in a dose-dependent manner. The population size structure was also changed by Cu as the number of cells sized 4-6 mu m was increased in the presence of Cu, especially with Cu 150 mu g/L. Changes in the topography of thylakoids grew larger with Cu concentration

In Vitro Assessment of Poly-N-Vinylpyrrolidone/Acrylic Acid Nanoparticles Biocompatibility in a Microvascular Endothelium Model

An amphiphilic copolymer of N-vinyl-2-pyrrolidone and acrylic acid&mdash;namely, p(VP-AA)-OD6000 (p(VP-AA))&mdash;was synthesized to prepare p(VP-AA) nanoparticles (NPs). Furthermore, the copolymer was linked with CFSE, and the so-prepared nanoparticles were loaded with the DiI dye to form D nanoparticles (DNPs). In this study, as demonstrated by immunofluorescence microscopy, immunofluorescence, and confocal microscopy, DNPs were readily taken up by human microvascular endothelial cells (HMEC-1) cells in a concentration-dependent manner. Upon uptake, both the CFSE dye (green stain) and the DiI dye (red stain) were localized to the cytoplasm of treated cells. Treatment with p(VP-AA) did not affect the viability of normal and challenged with LPS, HMEC-1 cells at 0.010 mg/mL and induced a dose-dependent decrease of these cells&rsquo; viability at the higher concentrations of 0.033 and 0.066 mg/mL (p &le; 0.01; p &le; 0.001, respectively). Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon p(VP-AA) NPs treatment by assessing the expression of adhesion molecules (E-Selectin, ICAM-1, and V-CAM). NPs treatments at concentrations utilized (p = NS) did not affect individual adhesion molecules&rsquo; expression. p(VP-AA) NPs do not activate the endothelium and do not affect its viability at pharmacologically relevant concentrations

What is the content of the electronic cigarette liquids according to their flavor? An evaluation in nine European Union countries before TPD implementation

Introduction Safety data on the chemical content of the electronic cigarette liquids depending on their flavors are scarce. Aims and objectives: This study aimed to identify and evaluate the content of e-liquids according to their flavor in the most common e-liquid products in the EU. Methods Within the Horizon2020, EUREST-PLUS study, 122 of the most commonly sold e-liquids in 9 European Countries (France, Poland, Germany, the Netherlands, United Kingdom, Spain, Romania, Hungary and Greece) were randomly selected and purchased. E-liquids were then divided into 7 groups according to their flavor. These groups are: Tobacco (35), Fruits (44), Menthol-mint (9), Non-alcoholic drinks (10), Alcohol (5), Desserts-sweets (11) and other flavors (8). A qualitative chemical analysis was performed using gas chromatography-mass spectrometry technique companied with solid phase microextraction (GC-SPME-MS), while the identification of the compound was done by NIST or Wiley library. Results After analyzing the total number of the samples, 232 compounds were detected in total. The 101 of these compounds were detected in Fruit flavor, 52 in the Tobacco flavor, 30 in the Non-alcoholic drinks flavor, 26 in the Desserts-sweets flavor, 24 in the alcohol flavor, 12 in the other flavors category and 15 in menthol-mint flavor. Of the detected compounds, Nonanal and Oxime-, methoxy-phenyl were the most frequently detected compound (15 and 14 times in tobacco flavor respectively) and also present in all flavors. These compounds are strong irritants to skin and eye. Additionally, banana oil, which was detected in 12 Fruit flavor samples and in most flavors, can cause skin, eye and respiratory irritation, narcotic effects and damage to organs through prolonged or repeated exposure Conclusions To our knowledge, this is the first study to examine the chemical content of the most common e-liquids in the EU depending on the flavor, indicating that several flavors could be more hazardous. Stricter strategies for these products are necessary. Funding This work was supported by a grant from the European Commission (Horizon2020 HCO-6-2015; EUREST-PLUS: 681109; Vardavas)