scholarly journals Microscale In Vitro Assays for the Investigation of Neutral Red Retention and Ethoxyresorufin-O-Deethylase of Biofuels and Fossil Fuels

PLoS ONE ◽  
2016 ◽  
Vol 11 (9) ◽  
pp. e0163862 ◽  
Author(s):  
Sebastian Heger ◽  
Kerstin Bluhm ◽  
Julia Brendt ◽  
Philipp Mayer ◽  
Nico Anders ◽  
...  
2018 ◽  
Author(s):  
Esther E. Omaiye ◽  
Kevin J. McWhirter ◽  
Wentai Luo ◽  
James F. Pankow ◽  
Prue Talbot

ABSTRACTWhile JUUL electronic cigarettes (ECs) have captured the majority of the EC market with a large fraction of their sales going to adolescents, little is known about their cytotoxicity and potential effects on health. The purpose of this study was to determine flavor chemical and nicotine concentrations in the eight currently marketed pre-filled JUUL EC cartridges (“pods”) and to evaluate the cytotoxicity of the different variants (e.g., “Cool Mint” and “Crème Brulee”) using in vitro assays. Nicotine and flavor chemicals were analyzed using gas chromatography/mass spectrometry in pod fluid before and after vaping and in the corresponding aerosols. 59 flavor chemicals were identified in JUUL pod fluids, and three were >1 mg/mL. Duplicate pods were similar in flavor chemical composition and concentration. Nicotine concentrations (average 60.9 mg/mL) were significantly higher than any EC products we have analyzed previously. Transfer efficiency of individual flavor chemicals that were >1mg/mL and nicotine from the pod fluid into aerosols was generally 35 - 80%. All pod fluids were cytotoxic at a 1:10 dilution (10%) in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. Most aerosols were cytotoxic in these assays at concentrations >1%. The cytotoxicity of aerosols was highly correlated with nicotine and ethyl maltol concentrations and moderately to weakly correlated with total flavor chemical concentration and menthol concentration. Our study demonstrates that: (1) some JUUL flavor pods have high concentrations of flavor chemicals that may make them attractive to youth, and (2) the concentrations of nicotine and some flavor chemicals (e.g. ethyl maltol) are high enough to be cytotoxic in acute in vitro assays, emphasizing the need to determine if JUUL products will lead to adverse health effects with chronic use.


2020 ◽  
Vol 10 (2) ◽  
pp. 213-220 ◽  
Author(s):  
Mahmoud Abudayyak ◽  
Elif Guzel ◽  
Gül Özhan

Purpose: The wide application of cupric oxide nanoparticles (copper (II) oxide, CuO-NPs) in various fields has increased exposure to the kind of active nanomaterials, which can cause negative effects on human and environment health. Although CuO-NPs were reported to be harmful to human, there is still a lack information related to their toxic potentials. In the present study, the toxic potentials of CuO-NPs were evaluated in the liver (HepG2 hepatocarcinoma) and intestine (Caco-2 colorectal adenocarcinoma) cells. Methods: After the characterization of particles, cellular uptake and morphological changes were determined. The potential of cytotoxic, genotoxic, oxidative and apoptotic damage was investigated with several in vitro assays. Results: The average size of the nanoparticles was 34.9 nm, about 2%-5% of the exposure dose was detected in the cells and mainly accumulated in different organelles, causing oxidative stress, cell damages, and death. The IC50 values were 10.90 and 10.04 µg/mL by MTT assay, and 12.19 and 12.06 µg/mL by neutral red uptake (NRU) assay, in HepG2 and Caco-2 cells respectively. Apoptosis assumes to the main cell death pathway; the apoptosis percentages were 52.9% in HepG2 and 45.5% in Caco-2 cells. Comet assay result shows that the highest exposure concentration (20 µg/mL) causes tail intensities about 9.6 and 41.8%, in HepG2 and Caco-2 cells, respectively. Conclusion: CuO-NPs were found to cause significant cytotoxicity, genotoxicity, and oxidative and apoptotic effects in both cell lines. Indeed, CuO-NPs could be dangerous to human health even if their toxic mechanisms should be elucidated with further studies.


2018 ◽  
Author(s):  
Βασιλική Τσαρπαλή

Σκοπός της παρούσας μελέτης ήταν η εκτίμηση του περιβαλλοντικού κινδύνου από τα ευρέως χρησιμοποιούμενα ιμιδαζολικά ιοντικά υγρά (ILs), τετραφθοροβορικό 1-βούτυλο-3-μεθυλιμιδαζόλιο (1-butyl-3-methylimidazolium tetrafluoroborate /([bmim][BF4]) και τετραφθοροβορικό 1-μέθυλο-3-οκτυλιμιδαζόλιο (1-octyl-3-methylimidazolium tetrafluoroborate /[omim][BF4]), με την χρήση οργανισμών βιο-ενδεικτών και in vitro βιολογικών μοντέλων. Παράλληλα, καθώς η πιθανότητα χρήσης διαλυμάτων ILs-οργανικού διαλύτη σε ένα πλήθος εφαρμογών είναι αυξημένη, μελετήθηκε η επίδραση της ακετόνης στην ικανότητα των ILs να επάγουν φαινόμενα τοξικότητας. Αναλυτικότερα, πραγματοποιήθηκαν δοκιμές τοξικότητας με την χρήση των φυτοπλαγκτονικών ειδών Dunaliella tertiolecta και Scenedesmus rubescens, των καρκινοειδών Thamnocephalus platyurus και Artemia franciscana, των τροχοζώων Brachionus calyciflorus και Brachionus plicatilis, του δίθυρου μαλακίου Mytilus galloprovincialis, παρουσία ή απουσία ακετόνης (0.06% v/v). Πέραν της ικανότητάς τους να προκαλούν θνησιμότητα στους εκτιθέμενους οργανισμούς, διερευνήθηκε η πιθανότητα επαγωγής κυτταροτοξικών (τεχνική Neutral Red Retention Time), νευροτοξικών (ενζυμική δραστικότητα ακετυλ-χολινεστεράσης/AChE) και γενοτοξικών (συχνότητα εμφάνισης μικροπυρήνων/MN, τεχνική ανάλυσης κομητών/MS assay) επιπτώσεων, καθώς και φαινομένων οξειδωτικής καταπόνησης (προσδιορισμός επιπέδων σουπεροξειδικών ανιόντων, προσδιορισμός επιπέδων μηλονικής διαλδεϋδης/ MDA) σε ιστούς (αιμόλεμφος/αιμοκύτταρα και βράγχια) του είδους M. galloprovincialis. Επιπλέον, μελετήθηκε η κυτταροτοξική και μεταλλαξιγόνος δράση των ILs, εφαρμόζοντας την τεχνική των μικροπυρήνων με τη χρήση της κυτταροχαλασίνης-Β (CBMN), σε καλλιέργειες ανθρώπινων λεμφοκυττάρων. Παράλληλα, για να μελετηθεί καλύτερα ο μηχανισμός δράσης των ILs και η αλληλεπίδρασή τους με την ακετόνη πραγματοποιήθηκε ανάλυση LC-MS-TOF, και in vitro μελέτες σε απομονωμένα αιμοκύτταρα του μυδιού M. galloprovincialis, έπειτα από επώαση τους με τον χαοτροπικό παράγοντα υδροχλωρική γουανιδίνη (1 mM). Τέλος, προκειμένου να διερευνηθεί η επίδραση των φυσικοχημικών παραμέτρων, όπως η αλατότητα, στην φυσιολογική απόκριση των φυτοπλαγκτονικών οργανισμών (ρυθμός αύξησης, επίπεδα χλωροφύλλης α και ολικών καροτενοειδών), πραγματοποιήθηκε έκθεση καλλιεργειών D. tertiolecta στα ILs υπό διαφορετικές αλατότητες (30 και 35‰). Σύμφωνα με τα αποτελέσματα, τα ILs μειώνουν σημαντικά τη βιωσιμότητα των εκτιθέμενων οργανισμών και προκαλούν σημαντικές προ-παθολογικές καταστάσεις. Ειδικότερα, το [οmim][BF4] που φέρει μεγαλύτερη αλκυλική αλυσίδα, εμφανίζει μεγαλύτερη τοξικότητα, σε σχέση με το [bmim][BF4] και το μίγμα [bmim][BF4]-[οmim][BF4], προκαλώντας σημαντικές διαταραχές στη φυσιολογία των εκτιθέμενων ατόμων/κυττάρων, πιθανό λόγω της ικανότητας του IL να αλληλεπιδρά με τα φωσφολιπίδια και τις πρωτεΐνες των κυτταρικών μεμβρανών, ενεργοποιώντας μια σειρά αλυσιδωτών αντιδράσεων στο εσωτερικό των κυττάρων. Το είδος και η έκταση των επαγόμενων φαινομένων επηρεάζεται σημαντικά από την παρουσία της ακετόνης, η οποία αναστέλλει σε σημαντικό βαθμό τις τοξικές επιπτώσεις του [omim][BF4]. Σύμφωνα με την ανάλυση LC-MS-TOF, φαίνεται πως η ακετόνη μεταβάλλει το ιξώδες και την λιποφιλικότητα των ILs και κατ’ επέκταση την ικανότητά τους να αλληλεπιδρούν με τις βιολογικές μεμβράνες. Τέλος, η τοξικότητα των ILs φαίνεται πως εξαρτάται σε μεγάλο βαθμό από τις φυσικοχημικές παραμέτρους του περιβάλλοντος, καθώς επηρεάζουν την ικανότητα των κυττάρων να ανταπεξέρχονται σε διαφορετικές συνθήκες stress. Συγκεκριμένα, η αλατότητα φάνηκε να παίζει σημαντικό ρόλο στο ρυθμό αύξησης του μικροφύκους D. tertiolecta. Συμπερασματικά, τα αποτελέσματα της παρούσας μελέτης αμφισβητούν την ορθότητα του χαρακτηρισμού των ιμιδαζολικών ILs ως «Πράσινοι» διαλύτες. Παράλληλα, αναδεικνύει για πρώτη φορά το σημαντικό ρόλο της ακετόνης στη ρύθμιση του τοξικού χαρακτήρα των ILs, συμβάλλοντας με αυτό τον τρόπο στην προσπάθεια παραγωγής, ανάπτυξης και χρήσης περισσότερο περιβαλλοντικά φιλικών ILs.


1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.


1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.


2018 ◽  
Vol 21 (3) ◽  
pp. 215-221
Author(s):  
Haroon Khan ◽  
Muhammad Zafar ◽  
Helena Den-Haan ◽  
Horacio Perez-Sanchez ◽  
Mohammad Amjad Kamal

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.


Author(s):  
Nidhi Sharma ◽  
Arti Singh ◽  
Ruchika Sharma ◽  
Anoop Kumar

Aim: The aim of the study was to find out the role of auranofin as a promising broad spectrum antibacterial agent. Methods: In-vitro assays (Percentage growth retardation, Bacterial growth kinetics, Biofilm formation assay) and In-silico study (Molegro virtual docker (MVD) version 6.0 and Molecular operating environment (MOE) version 2008.10 software). Results: The in vitro assays have shown that auranofin has good antibacterial activity against Gram positive and Gram negative bacterial strains. Further, auranofin has shown synergistic activity in combination with ampicillin against S. aureus and B. subtilis whereas in combination with neomycin has just shown additive effect against E. coli, P. aeruginosa and B. pumilus. In vivo results have revealed that auranofin alone and in combination with standard drugs significantly decreased the bioburden in zebrafish infection model as compared to control. The molecular docking study have shown good interaction of auranofin with penicillin binding protein (2Y2M), topoisomerase (3TTZ), UDP-3-O-[3- hydroxymyristoyl] N-acetylglucosaminedeacetylase (3UHM), cell adhesion protein (4QRK), β-lactamase (5CTN) and arylsulphatase (1HDH) enzyme as that of reference ligand which indicate multimodal mechanism of action of auranofin. Finally, MTT assay has shown non-cytotoxic effect of auranofin. Conclusion: In conclusion, auranofin in combination with existing antibiotics could be developed as a broad spectrum antibacterial agent; however, further studies are required to confirm its safety and efficacy. This study provides possibility of use of auranofin apart from its established therapeutic indication in combination with existing antibiotics to tackle the problem of resistance.


2019 ◽  
Vol 16 (2) ◽  
pp. 116-127 ◽  
Author(s):  
Ashwani Kumar ◽  
Vineet Mehta ◽  
Utkarsh Raj ◽  
Pritish Kumar Varadwaj ◽  
Malairaman Udayabanu ◽  
...  

Background: Cholinesterase inhibitors are the first line of therapy for the management of Alzheimer’s disease (AD), however, it is now established that they provide only temporary and symptomatic relief, besides, having several inherited side-effects. Therefore, an alternative drug discovery method is used to identify new and safer ‘disease-modifying drugs’. Methods: Herein, we screened 646 small molecules of natural origin having reported pharmacological and functional values through in-silico docking studies to predict safer neuromodulatory molecules with potential to modulate acetylcholine metabolism. Further, the potential of the predicted molecules to inhibit acetylcholinesterase (AChE) activity and their ability to protect neurons from degeneration was determined through in-vitro assays. Results: Based on in-silico AChE interaction studies, we predicted quercetin, caffeine, ascorbic acid and gallic acid to be potential AChE inhibitors. We confirmed the AChE inhibitory potential of these molecules through in-vitro AChE inhibition assay and compared results with donepezil and begacestat. Herbal molecules significantly inhibited enzyme activity and inhibition for quercetin and caffeine did not show any significant difference from donepezil. Further, the tested molecules did not show any neurotoxicity against primary (E18) hippocampal neurons. We observed that quercetin and caffeine significantly improved neuronal survival and efficiently protected hippocampal neurons from HgCl2 induced neurodegeneration, which other molecules, including donepezil and begacestat, failed to do. Conclusion: Quercetin and caffeine have the potential as “disease-modifying drugs” and may find application in the management of neurological disorders such as AD.


2020 ◽  
Vol 16 ◽  
Author(s):  
Haicheng Liu ◽  
Yushi Futamura ◽  
Honghai Wu ◽  
Aki Ishiyama ◽  
Taotao Zhang ◽  
...  

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.


2020 ◽  
Vol 16 ◽  
Author(s):  
Benedetta Bocchini ◽  
Bruna Goldani ◽  
Fernanda S.S. Sousa ◽  
Paloma T. Birmann ◽  
Cesar A. Brüning ◽  
...  

Background: Quinoline derivatives have been attracted much attention in drug discovery and synthetic derivatives of these scaffolds present a range of pharmacological activities. Therefore, organoselenium compounds are valuable scaffolds in organic synthesis because their pharmacological activities and their use as versatile building blocks for regio-, chemio-and stereoselective reactions. Thus, the synthesis of selenium-containing quinolines has great significance, and their applicability range from simple antioxidant agents, to selective DNA-binding and photocleaving agents. Objective: In the present study we describe the synthesis and antioxidant activity in vitro of new 7-chloroN(arylselanyl)quinolin-4-amines 5 by the reaction of 4,7-dichloroquinoline 4 with (arylselanyl)-amines 3. Methods: For the synthesis of 7-chloro-N(arylselanyl)quinolin-4-amines 5, we performed the reaction of (arylselanyl)- amines 3 with 4,7-dichloroquinoline 4 in the presence of Et3N at 120 °C in a sealed tube. The antioxidant activities of the compounds 5 were evaluated by the following in vitro assays: 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric ion reducing antioxidant power (FRAP), nitric oxide (NO) scavenging and superoxide dismutase-like activity (SOD-Like). Results: 7-Chloro-N(arylselanyl)quinolin-4-amines 5a-d has been synthesized in yields ranging from 68% to 82% by the reaction of 4,7-dichloroquinoline 4 with arylselanyl-amines 3a-d using Et3N as base, at 120 °C, in a sealed tube for 24 hours and tolerates different substituents, such as -OMe and -Cl, in the arylselanyl moiety. The obtained compounds 5a-d presented significant results with respect to the antioxidant potential, which had effect in the tests of inhibition of radical’s DPPH, ABTS+ and NO, as well as in the test that evaluates the capacity (FRAP) and in the superoxide dismutase-like activity assay (SOD-Like). It is worth mentioning that 7-chloro-N(arylselanyl)quinolin-4-amine 5b presented excellent results, demonstrating a better antioxidant capacity when compared to the others. Conclusion: According to the obtained results 7-chloro-N(arylselanyl)quinolin-4-amines 5 were synthesized in good yields by the reaction of 4,7-dichloroquinoline with arylselanyl-amines and tolerates different substituents in the arylselanyl moiety. The tested compounds presented significant antioxidant potential in the tests of inhibition of DPPH, ABTS+ and NO radicals, as well as in the FRAP and superoxide dismutase-like activity assays (SOD-Like).


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