The 3D reconstructed skin micronucleus assay: considerations for optimal protocol design

Mutagenesis ◽  
2019 ◽  
Author(s):  
Darren Kidd ◽  
Sarah Phillips ◽  
Teresa Chirom ◽  
Nicky Mason ◽  
Robert Smith ◽  
...  
Keyword(s):  
Statistical Power ◽  
Treatment Protocol ◽  
Micronucleus Assay ◽  
Metabolic Activation ◽  
Reconstructed Skin ◽  
Replication Index ◽  
Optimal Protocol ◽  
Further Development ◽  
The Eu

Abstract Implementation of the seventh amendment to the EU Cosmetics Directive has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006, further development occurred and a standard protocol was published in 2011. To evaluate and optimise the assay at Covance Laboratories, we tested nine chemicals [4-nitrophenol (4-NP), cyclohexanone (CH), 2-ethyl-1,3-hexanediol (2-EHD), methyl methansulfonate (MMS), mitomycin C (MMC), ethyl nitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and vinblastine (VIN)] using the EpiDerm™ 3D skin model (MatTek Corporation®, IVLSL, Bratislava, Slovakia) and compared the data using the standard 48-h treatment regimen and also an emerging 72-h treatment protocol. The EpiDerm™ tissue has reportedly some metabolic capacity but data using 48-h treatments has provided mixed results. Our investigations demonstrate that the two chemicals requiring metabolic activation (BaP and CPA) were negative following the 48-h protocol but were clearly positive following 72-h treatment. Furthermore, Replication Index (RI) data showed higher RI values in vehicle control treatments (indicating increased cell division) across the treatment set following 72-h treatments. A general greater magnitude of micronucleus (MN) induction was also observed following test chemical treatment. These data suggest that the 72-h treatment protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. For further assay optimisation, we compare the statistical power of scoring cells from duplicate or triplicate cultures per treatment concentration and provide recommendations.

Natural products from the traditional Moroccan pharmacopoeia: A potential lead for the prevention and treatment of COVID-19

2020 ◽  
Vol 11 (SPL1) ◽  
pp. 1278-1285
Author(s):  
Mohamed Yafout ◽  
Amine Ousaid ◽  
Ibrahim Sbai El Otmani ◽  
Youssef Khayati ◽  
Amal Ait Haj Said
Keyword(s):  
Medicinal Plants ◽  
Plant Extracts ◽  
Molecular Mechanisms ◽  
Scientific Literature ◽  
Treatment Protocol ◽  
World Health ◽  
The World ◽  
Health Organization ◽  
Promising Source

The new SARS-CoV-2 belonging to the coronaviruses family has caused a pandemic affecting millions of people around the world. This pandemic has been declared by the World Health Organization as an international public health emergency. Although several clinical trials involving a large number of drugs are currently underway, no treatment protocol for COVID-19 has been officially approved so far. Here we demonstrate through a search in the scientific literature that the traditional Moroccan pharmacopoeia, which includes more than 500 medicinal plants, is a fascinating and promising source for the research of natural molecules active against SARS-CoV-2. Multiple in-silico and in-vitro studies showed that some of the medicinal plants used by Moroccans for centuries possess inhibitory activity against SARS-CoV or SARS-CoV-2. These inhibitory activities are achieved through the different molecular mechanisms of virus penetration and replication, or indirectly through stimulation of immunity. Thus, the potential of plants, plant extracts and molecules derived from plants that are traditionally used in Morocco and have activity against SARS-CoV-2, could be explored in the search for a preventive or curative treatment against COVID-19. Furthermore, safe plants or plant extracts that are proven to stimulate immunity could be officially recommended by governments as nutritional supplements.

Thematic Priorities For Sustainable Development of Ukraine-Romania Cross-Border Cooperation (The Case Study of Chernivtsi Region)

2020 ◽  
pp. 205-219
Author(s):  
Valentyna Bohatyrets ◽  
Liubov Melnychuk ◽  
Yaroslav Zoriy
Keyword(s):  
Foreign Policy ◽  
Quantitative Research ◽  
Legal Framework ◽  
Cultural Development ◽  
Cross Border ◽  
Strategic Orientations ◽  
The Impact ◽  
Further Development ◽  
Cross Border Cooperation ◽  
The Eu

This paper seeks to investigate sustainable cross-border cooperation (CBC) as a distinctive model of interstate collaboration, embedded in the neighboring borderland regions of two or more countries. The focus of the research revolves around the establishment and further development of geostrategic, economic, cultural and scientific capacity of the Ukrainian-Romanian partnership as a fundamental construct in ensuring and strengthening the stability, security and cooperation in Europe. This research highlights Ukraine’s aspirations to establish, develop and diversify bilateral good-neighborly relations with Romania both regionally and internationally. The main objective is to elucidate Ukraine-Romania cross-border cooperation initiatives, inasmuch Ukraine-Romania CBC has been stirring up considerable interest in terms of its inexhaustible historical, cultural and spiritual ties. Furthermore, the similarity of the neighboring states’ strategic orientations grounds the basis for development and enhancement of Ukraine-Romania cooperation. The authors used desk research and quantitative research to conclude that Ukraine-Romania CBC has the impact not only on the EU and on Ukraine multi-vector foreign policy, but it also has the longer-term global consequences. In the light of the current reality, the idea of introducing and reinforcing the importance of Cross-Border Cooperation (CBC) sounds quite topical and relevant. This research considers a number of explanations for Ukraine-Romania Cross-Border Cooperation as a key element of the EU policy towards its neighbors. Besides, the subject of the research is considered from different perspectives in order to show the diversity and complexity of the Ukraine-Romania relations in view of the fact that sharing common borders we are presumed to find common solutions. As the research has demonstrated, the Ukraine-Romania cross border cooperation is a pivotal factor of boosting geostrategic, economic, political and cultural development for each participant country, largely depending on the neighboring countries’ cohesion and convergence. Significantly, there is an even stronger emphasis on the fact that while sharing the same borders, the countries share common interests and aspirations for economic thriving, cultural exchange, diplomatic ties and security, guaranteed by a legal framework. The findings of this study have a number of important implications for further development and enhancement of Ukraine-Romania cooperation. Accordingly, the research shows how imperative are the benefits of Romania as a strategic partner for outlining top priorities of Ukraine’s foreign policy.

scholarly journals Novel Hybrid Compounds Containing Benzofuroxan and Aminothiazole Scaffolds: Synthesis and Evaluation of Their Anticancer Activity

2021 ◽  
Vol 22 (14) ◽  
pp. 7497
Author(s):  
Elena Chugunova ◽  
Gabriele Micheletti ◽  
Dario Telese ◽  
Carla Boga ◽  
Daut Islamov ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Normal Liver ◽  
Mitochondrial Pathway ◽  
Reaction Pathways ◽  
Aromatic Nucleophilic Substitution ◽  
X Ray ◽  
Hybrid Compounds ◽  
Aromatic Nucleophilic Substitution Reaction ◽  
Further Development

A series of novel hybrid compounds containing benzofuroxan and 2-aminothiazole moieties are synthesized via aromatic nucleophilic substitution reaction. Possible reaction pathways have been considered quantum-chemically, which allowed us to suggest the most probable products. The quantum chemical results have been proved by X-ray data on one compound belonging to the synthesized series. It was shown that the introduction of substituents to both the thiazole and amine moieties of the compounds under study strongly influences their UV/Vis spectra. Initial substances and obtained hybrid compounds have been tested in vitro as anticancer agents. Target compounds showed selectivity towards M-HeLa tumor cell lines and were found to be more active than starting benzofuroxan and aminothiazoles. Furthermore, they are considerably less toxic to normal liver cells compared to Тamoxifen. The mechanism of action of the studied compounds can be associated with the induction of apoptosis, which proceeds along the mitochondrial pathway. Thus, new hybrids of benzofuroxan are promising candidates for further development as anticancer agents.

scholarly journals Role of Surface Chemistry in the In Vitro Lung Response to Nanofibrillated Cellulose

Nanomaterials ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 389
Author(s):  
Kukka Aimonen ◽  
Satu Suhonen ◽  
Mira Hartikainen ◽  
Viviana R. Lopes ◽  
Hannu Norppa ◽  
...  
Keyword(s):  
Micronucleus Assay ◽  
Nanofibrillated Cellulose ◽  
In Vitro Toxicity ◽  
Surface Functional Group ◽  
Fiber Morphology ◽  
Surface Charges ◽  
Cell Viability Assay ◽  
Wide Range ◽  
Group Density

Wood-derived nanofibrillated cellulose (NFC) has emerged as a sustainable material with a wide range of applications and increasing presence in the market. Surface charges are introduced during the preparation of NFC to facilitate the defibrillation process, which may also alter the toxicological properties of NFC. In the present study, we examined the in vitro toxicity of NFCs with five surface chemistries: nonfunctionalized, carboxymethylated, phosphorylated, sulfoethylated, and hydroxypropyltrimethylammonium-substituted. The NFC samples were characterized for surface functional group density, surface charge, and fiber morphology. Fibril aggregates predominated in the nonfunctionalized NFC, while individual nanofibrils were observed in the functionalized NFCs. Differences in surface group density among the functionalized NFCs were reflected in the fiber thickness of these samples. In human bronchial epithelial (BEAS-2B) cells, all NFCs showed low cytotoxicity (CellTiter-GloVR luminescent cell viability assay) which never exceeded 10% at any exposure time. None of the NFCs induced genotoxic effects, as evaluated by the alkaline comet assay and the cytokinesis-block micronucleus assay. The nonfunctionalized and carboxymethylated NFCs were able to increase intracellular reactive oxygen species (ROS) formation (chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate assay). However, ROS induction did not result in increased DNA or chromosome damage.

scholarly journals Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1929
Author(s):  
Tereza Cervena ◽  
Andrea Rossnerova ◽  
Tana Zavodna ◽  
Jitka Sikorova ◽  
Kristyna Vrbova ◽  
...  
Keyword(s):  
Cytochalasin B ◽  
Culture Media ◽  
Methodological Approach ◽  
Micronucleus Assay ◽  
Toxicity Testing ◽  
In Vitro Toxicity ◽  
Testing Strategies ◽  
Transmission Electron ◽  
The Impact

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

scholarly journals Phytotoxicity and Other Adverse Effects on the In Vitro Shoot Cultures Caused by Virus Elimination Treatments: Reasons and Solutions

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 670
Author(s):  
Katalin Magyar-Tábori ◽  
Nóra Mendler-Drienyovszki ◽  
Alexandra Hanász ◽  
László Zsombik ◽  
Judit Dobránszki
Keyword(s):  
Adverse Effects ◽  
Physiological Condition ◽  
Harmful Effect ◽  
Shoot Cultures ◽  
Virus Elimination ◽  
In Vitro Shoots ◽  
Further Development ◽  
In Vitro Shoot Cultures ◽  
Harmful Effects

In general, in vitro virus elimination is based on the culture of isolated meristem, and in addition thermotherapy, chemotherapy, electrotherapy, and cryotherapy can also be applied. During these processes, plantlets suffer several stresses, which can result in low rate of survival, inhibited growth, incomplete development, or abnormal morphology. Even though the in vitro cultures survive the treatment, further development can be inhibited; thus, regeneration capacity of treated in vitro shoots or explants play also an important role in successful virus elimination. Sensitivity of genotypes to treatments is very different, and the rate of destruction largely depends on the physiological condition of plants as well. Exposure time of treatments affects the rate of damage in almost every therapy. Other factors such as temperature, illumination (thermotherapy), type and concentration of applied chemicals (chemo- and cryotherapy), and electric current intensity (electrotherapy) also may have a great impact on the rate of damage. However, there are several ways to decrease the harmful effect of treatments. This review summarizes the harmful effects of virus elimination treatments applied on tissue cultures reported in the literature. The aim of this review is to expound the solutions that can be used to mitigate phytotoxic and other adverse effects in practice.

scholarly journals The Implications of Regulatory Framework for Topical Semisolid Drug Products: From Critical Quality and Performance Attributes towards Establishing Bioequivalence

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 710
Author(s):  
Tanja Ilić ◽  
Ivana Pantelić ◽  
Snežana Savić
Keyword(s):  
Statistical Power ◽  
Failure Modes ◽  
In Vitro Release ◽  
Optimal Number ◽  
Drug Products ◽  
Pharmaceutical Equivalence ◽  
And Performance ◽  
Low Sensitivity

Due to complex interdependent relationships affecting their microstructure, topical semisolid drug formulations face unique obstacles to the development of generics compared to other drug products. Traditionally, establishing bioequivalence is based on comparative clinical trials, which are expensive and often associated with high degrees of variability and low sensitivity in detecting formulation differences. To address this issue, leading regulatory agencies have aimed to advance guidelines relevant to topical generics, ultimately accepting different non-clinical, in vitro/in vivo surrogate methods for topical bioequivalence assessment. Unfortunately, according to both industry and academia stakeholders, these efforts are far from flawless, and often upsurge the potential for result variability and a number of other failure modes. This paper offers a comprehensive review of the literature focused on amending regulatory positions concerning the demonstration of (i) extended pharmaceutical equivalence and (ii) equivalence with respect to the efficacy of topical semisolids. The proposed corrective measures are disclosed and critically discussed, as they span from mere demands to widen the acceptance range (e.g., from ±10% to ±20%/±25% for rheology and in vitro release parameters highly prone to batch-to-batch variability) or reassess the optimal number of samples required to reach the desired statistical power, but also rely on specific data modeling or novel statistical approaches.

scholarly journals Satureja subspicata and S. horvatii Extracts Induce Overexpression of the BCl-2 Family of Anti-apoptotic Genes and Reduce Micronuclei Frequency in Mice

2018 ◽  
Vol 13 (6) ◽  
pp. 1934578X1801300
Author(s):  
Jasmina Čakar ◽  
Naida Kadrić Lojo ◽  
Anja Haverić ◽  
Maida Hadžić ◽  
Lejla Lasić ◽  
...  
Keyword(s):  
Human Lymphocyte ◽  
Micronucleus Assay ◽  
Balkan Peninsula ◽  
Lymphocyte Culture ◽  
Cytotoxic Activities ◽  
Cytotoxic Effects ◽  
Apoptotic Genes ◽  
Apoptotic Activity

Satureja subspicata and S. horvatii are endemic species of the Balkan Peninsula and often used in traditional medicine in Bosnia and Herzegovina to treat different health conditions. We aimed to analyze the unevaluated apoptotic, genotoxic and cytotoxic effects of two Satureja species, as well as their content of phenolics that are mainly responsible for the plant's biological activity. Apoptotic and geno/cytotoxic activities of S. subspicata and S. horvatii were investigated in vitro in human lymphocyte culture and in vivo in mice. The content of the main phenolics in plant extracts was determined by ultra-high pressure liquid chromatography-MS-MS (UHPLC–MS/MS). Genotoxic and cytotoxic activities of Satureja extracts were evaluated in vitro by applying a cytokinesis-block micronucleus cytome assay in human lymphocyte culture and in vivo applying a mice reticulocytes micronucleus assay. SALSA RT-MLPA R011-C1 apoptosis assay was used for measuring the relative expression of 44 genes associated with the regulation of the apoptotic pathways in human lymphocyte cultures treated with different concentrations of two Satureja extracts. The first analysis of phenolic compounds in S. horvatii and S. subspicata determined by an UHPLC-MS/MS method revealed high levels of rosmarinic and caffeic acids. Minor genotoxic potential was determined in relation to the tested concentrations while no cytostatic and cytotoxic effects were revealed in vitro. However, when applied in concentrations of 200 mg/kg per os, aqueous extracts of two Satureja species significantly decreased frequency of reticulocytes micronuclei in treated mice against controls. Extracts of S. subspicata and S. horvatii in concentrations of 0.2 mg/mL, regardless of solvent used, downregulated pro-apoptotic and upregulated anti-apoptotic genes, showing anti-apoptotic activity. Our results indicate that the registered anti-genotoxic and anti-apoptotic activity is most likely related to the high level of phenolic acids (particularly rosmarinic and caffeic) in the tested extracts.

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