The induction of vasculature formation is proposed to be a significant mechanism behind the non-genotoxic carcinogenicity of a chemical. The vasculature formation model used in this study is based on the coculture of hum...The induction of vasculature formation is proposed to be a significant mechanism behind the non-genotoxic carcinogenicity of a chemical. The vasculature formation model used in this study is based on the coculture of human primary HUVECs and hASCs. This model was used to develop an assay to assess the induction of vasculature formation. Three assay protocols, based on different conditions, were developed and compared in order to identify the optimal conditions required. Some serum supplements and growth factors were observed to be essential for initiating vasculature formation. Of the studied putative positive reference chemicals, aspartame, sodium nitrite, bisphenol A and nicotine treatment led to a clear induction of vasculature formation, but arsenic and cadmium treatment only led to a slight increase. This human cell-based assay has the potential to be used as one test within a next generation testing battery, to assess the non-genotoxic carcinogenicity of a chemical through the mechanism of vasculature formation induction.
Marine sponge extracts are known to contain potentially toxic compounds that have biological activities of possible pharmacological interest. Thus, it is vital that biological models are used for the preliminary toxicity...Marine sponge extracts are known to contain potentially toxic compounds that have biological activities of possible pharmacological interest. Thus, it is vital that biological models are used for the preliminary toxicity screening of such extracts. The present study reports the use of , a low-cost plant-based model, to assess the cytotoxicity and genotoxicity of marine sponge crude extract (SCE). Pre-germinated onion bulbs, exposed for 96 hours to different concentrations of SCE (ranging from 0.3125 to 20 μg/ml), were used to determine general cytotoxicity. Root length as well as morphological abnormalities were recorded. Genotoxicity was assessed by exposing the root tips to SCE (0.3125-20 μg/ml) and the appropriate controls for 48 hours, and then staining with acetocarmine. The Mitotic Index (MI), Mitotic Phase Indices (MPIs) and chromosomal aberrations were evaluated and recorded. SCE inhibited root growth (EC = 10.34 μg/ml) and elicited a mitodepressive effect (LC = 1.95 μg/ml) in a dose-dependent and significant manner. In addition, macroscopic alterations as well as chromosomal aberrations were detected. Overall, our findings indicate that crude extract exhibits cytotoxic and genotoxic activity, suggesting that it might contain substances with anti-proliferative/anticancer potential that could be subject to further characterisation.
The SARS-CoV-2 outbreak focused global attention on the shortcomings of the drug discovery process. It led to its acceleration in several areas, particularly in the processes associated with the development and approval...The SARS-CoV-2 outbreak focused global attention on the shortcomings of the drug discovery process. It led to its acceleration in several areas, particularly in the processes associated with the development and approval of COVID-19 vaccines. This situation contrasts with the low approval rates of new drugs for respiratory system diseases (e.g. asthma, chronic obstructive pulmonary disease, cancer, tuberculosis), which are leading causes of morbidity and mortality worldwide. In this context, innovation in respiratory system drug discovery is surely needed, and it is most likely to succeed through the use of preclinical models that are cost-effective, high-throughput and generate predictive human-relevant outcomes. Here, we highlight several non-animal new approach methodologies (NAMs) and their applications in respiratory research. We describe their potential uses for efficacy and toxicity assessments, to optimise the drug development process and reduce the high failure rates in clinical trials.
The failure rate for the translation of drugs from animal testing to human treatments remains at over 92%, where it has been for the past few decades. The majority of these failures are due to unexpected toxicity - that...The failure rate for the translation of drugs from animal testing to human treatments remains at over 92%, where it has been for the past few decades. The majority of these failures are due to unexpected toxicity - that is, safety issues revealed in human trials that were not apparent in animal tests - or lack of efficacy. However, the use of more innovative tools, such as organs-on-chips, in the preclinical pipeline for drug testing, has revealed that these tools are more able to predict unexpected safety events prior to clinical trials and so can be used for this, as well as for efficacy testing. Here, we review several disease areas, and consider how the use of animal models has failed to offer effective new treatments. We also make some suggestions as to how the more human-relevant new approach methodologies might be applied to address this.
The decisions we make on chemical safety, for consumers, workers and the environment, must be based on the best scientific data and knowledge available. Rapid advances in biology, in cell-based technologies and assays, a...The decisions we make on chemical safety, for consumers, workers and the environment, must be based on the best scientific data and knowledge available. Rapid advances in biology, in cell-based technologies and assays, and in analytical and computational approaches, have led to new types of highly relevant scientific data being generated. Such data enable us to improve the safety decisions we make, whilst also enabling us to avoid animal testing. Stimulated by the UK and EU bans on animal testing for cosmetics, Next Generation Risk Assessment (NGRA) approaches, which integrate various types of non-animal scientific data, have been established for assessing the safety of chemical ingredients used in cosmetics and other consumer products. In stark contrast, the chemicals regulations in Europe and other parts of the world have not kept pace with modern safety science and regulators are now mandating even more animal testing. Urgently closing this science-regulation gap is essential to upholding the EU's legislative requirement that any animal testing is a last resort. The ongoing revisions of UK and EU chemicals strategy and regulations provide an opportunity to fundamentally change the design and assessment paradigm needed to underpin safe and more sustainable innovation, through applying the best science and tools available rather than continuing to be anchored in animal tests dating back many decades. A range of initiatives have recently been launched in response to this urgent need, in the UK as well as in the EU.
Moore MM, Abraham I, Ballantyne M
… +25 more, Behrsing H, Cao X, Clements J, Gaca M, Gillman G, Hashizume T, Heflich RH, Hurtado S, Jordan KG, Leverette R, McHugh D, Miller-Holt J, Phillips G, Recio L, Roy S, Scian M, Simms L, Smart DJ, Stankowski LF, Tarran R, Thorne D, Weber E, Wieczorek R, Yoshino K, Curren R
The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting assays, to assess potential...The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.
Dengue is an arboviral (insect-transmitted) infection of global concern. Currently, there are still no specific dengue antiviral agents to treat the disease. Plant extracts have been used in traditional medicine for trea...Dengue is an arboviral (insect-transmitted) infection of global concern. Currently, there are still no specific dengue antiviral agents to treat the disease. Plant extracts have been used in traditional medicine for treating various viral infections - thus, in the present study, aqueous extracts of dried flowers of (AM), whole plant of (MP) and leaves of (PG) were investigated for their potential capacity to inhibit dengue virus infection of Vero cells. The maximum non-toxic dose (MNTD) and the 50% cytotoxic concentration (CC) were determined by using the MTT assay. A plaque reduction antiviral assay was carried out with dengue virus types 1 (DV1), 2 (DV2), 3 (DV3) and 4 (DV4), in order to calculate the half-maximum inhibitory concentration (IC). AM extract inhibited all four virus serotypes tested; MP extract inhibited DV1, DV2 and DV4, but not DV3; PG extract inhibited DV1, DV2 and DV4, but not DV3. Thus, the results suggest that AM is a promising candidate for the pan-serotype inhibition of dengue viral activity.
In recent decades, it has become clear that in many fields - such as drug development, particularly with regard to drug dosage and specific disease treatment - the sex of a patient must be taken into consideration, in vi...In recent decades, it has become clear that in many fields - such as drug development, particularly with regard to drug dosage and specific disease treatment - the sex of a patient must be taken into consideration, in view of the fact that male and female physiology and pathophysiology show many differences of practical concern. While, in the last decade or so, considerable efforts have been undertaken to consider the sex of the animals during the planning of experiments, this topic has just started to be acknowledged in studies. Cells in such studies seem mainly to be used according to their availability, without considering the sex of the original donor. Even when such information is available, experimental data are reported without overtly detailing this information. In recent years, the increasing complexity of models (e.g. stem cell-based, 3-D cultures, organoids, or organ-on-a-chip technologies) has contributed to systems that better resemble the human situation. However, the issue of the sex of the experimental organisms being used has not yet been properly taken up by the cell culture community. Thus, alongside the increasing complexity of multicell-type models, we now see models that incorporate cells from both male and female origin - this representing, in fact, a genetic chimaera. Here, we aim to discuss where we are currently, with respect to considering the sex of any animals or humans used in experiments, and we try to identify what is lacking in the cell culture field, in order to help facilitate change.
There is an ongoing aim to replace animal and laboratory models with methods. Such replacement requires the successful validation and comparably good performance of the alternative methods. We have developed an predic...There is an ongoing aim to replace animal and laboratory models with methods. Such replacement requires the successful validation and comparably good performance of the alternative methods. We have developed an prediction system for human clinical pharmacokinetics, based on machine learning, conformal prediction and a new physiologically-based pharmacokinetic model, i.e. ANDROMEDA. The objectives of this study were: a) to evaluate how well ANDROMEDA predicts the human clinical pharmacokinetics of a previously proposed benchmarking data set comprising 24 physicochemically diverse drugs and 28 small drug molecules new to the market in 2021; b) to compare its predictive performance with that of laboratory methods; and c) to investigate and describe the pharmacokinetic characteristics of the modern drugs. Median and maximum prediction errors for the selected major parameters were 1.2 to 2.5-fold and 16-fold for both data sets, respectively. Prediction accuracy was on par with, or better than, the best laboratory-based prediction methods (superior performance for a vast majority of the comparisons), and the prediction range was considerably broader. The modern drugs have higher average molecular weight than those in the benchmarking set from 15 years earlier ( 200 g/mol higher), and were predicted to (generally) have relatively complex pharmacokinetics, including permeability and dissolution limitations and significant renal, biliary and/or gut-wall elimination. In conclusion, the results were overall better than those obtained with laboratory methods, and thus serve to further validate the ANDROMEDA system for the prediction of human clinical pharmacokinetics of modern and physicochemically diverse drugs.
The pathological accumulation of quinolinic acid (QA) is often associated with neuritis and neuronal cell death in several neurodegenerative diseases, through the overproduction of free radicals. Urolithin B and aurapten...The pathological accumulation of quinolinic acid (QA) is often associated with neuritis and neuronal cell death in several neurodegenerative diseases, through the overproduction of free radicals. Urolithin B and auraptene have been reported to exert potent antioxidant effects - however, little is known about the protective effects of these compounds against QA-induced neurotoxicity. Therefore, this study aimed to explore the protective effects of urolithin B and auraptene against QA-induced neurotoxicity in the SH-SY5Y neuroblastoma cell line. The MTT assay was used to evaluate cell viability, and flow cytometry was carried out to evaluate effects on the cell cycle and apoptosis. The intracellular levels of reactive oxygen species (ROS) were also determined. Our findings showed that auraptene at non-toxic concentrations had no protective effect on QA-induced toxicity. However, urolithin B at concentrations of 0.6 μM and 2.5 μM enhanced the viability of cells treated with QA. Moreover, while the percentage of apoptotic cells (i.e. in the sub-G1 phase) was shown to significantly increase after QA treatment, pre-treatment with urolithin B reduced the number of these apoptotic cells. Furthermore, urolithin B, as an antioxidant, also significantly reduced QA-induced ROS production. Our findings suggest that urolithin B may possess potent antioxidant and neuroprotective effects against QA-induced neurotoxicity that merit further investigation.
Stem cell-based therapy presents an attractive alternative to conventional therapies for degenerative diseases. Numerous studies have investigated the capability of human umbilical cord-derived mesenchymal stem cells (hU...Stem cell-based therapy presents an attractive alternative to conventional therapies for degenerative diseases. Numerous studies have investigated the capability of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) to contribute to the regeneration of cardiomyocytes, and the results have encouraged further basic and clinical studies on the MSC-based treatment of cardiomyopathies. This study aimed to determine the potential of cardiomyogenic transcription factors in differentiating hUC-MSCs into cardiac-like cells MSCs were isolated from umbilical cord tissue and were transduced with the transcription factor genes, and , via infection with lentiviruses, to promote differentiation into the cardiomyogenic lineage. Gene and protein expression were analysed with qPCR and immunocytochemical staining. After transduction, differentiated cardiac-like cells showed significant expression of cardiac genes and proteins, namely GATA-4, Nkx-2.5, cardiac troponin I (cTnI) and myosin heavy chain (MHC). The cardiomyogenic-induced group significantly overexpressed cardiac-specific genes (). Expression of the calcium channel gene was also significantly increased, while the sodium channel gene was downregulated in the transduced hUC-MSCs, as compared to non-transduced cells. The results suggest that GATA-4 and Nkx-2.5 interact synergistically in the activation of downstream cardiac transcription factors, demonstrating the functional convergence of hUC-MSC differentiation into cardiac-like cells. These findings could potentially be utilised in the efficient production of cardiac-like cells from stem cells; these cardiac-like cells could then be used in various applications, such as for implantation in infarcted myocardium, and for drug screening in toxicity testing.
Neuhaus W, Reininger-Gutmann B, Rinner B
… +67 more, Plasenzotti R, Wilflingseder D, De Kock J, Vanhaecke T, Rogiers V, Jírová D, Kejlová K, Knudsen LE, Nielsen RN, Kleuser B, Kral V, Thöne-Reineke C, Hartung T, Pallocca G, Rovida C, Leist M, Hippenstiel S, Lang A, Retter I, Krämer S, Jedlicka P, Ameli K, Fritsche E, Tigges J, Kuchovská E, Buettner M, Bleich A, Baumgart N, Baumgart J, Meinhardt MW, Spanagel R, Chourbaji S, Kränzlin B, Seeger B, von Köckritz-Blickwede M, Sánchez-Morgado JM, Galligioni V, Ruiz-Pérez D, Movia D, Prina-Mello A, Ahluwalia A, Chiono V, Gutleb AC, Schmit M, van Golen B, van Weereld L, Kienhuis A, van Oort E, van der Valk J, Smith A, Roszak J, Stępnik M, Sobańska Z, Reszka E, Olsson IAS, Franco NH, Sevastre B, Kandarova H, Capdevila S, Johansson J, Svensk E, Cederroth CR, Sandström J, Ragan I, Bubalo N, Kurreck J, Spielmann H
The adoption of on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated t...The adoption of on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. 's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.
The use of 3-D cell culture models in cancer research has yielded substantial gains in knowledge on various aspects of tumour biology. Such cell culture models could be useful in the study of head and neck squamous cell...The use of 3-D cell culture models in cancer research has yielded substantial gains in knowledge on various aspects of tumour biology. Such cell culture models could be useful in the study of head and neck squamous cell carcinoma (HNSCC), where mimicking intratumoral and intertumoral heterogeneity is especially challenging. Our research aims to establish 3-D spheroid models for HNSCC that reproduce the connections between tumour cells and the surrounding microenvironment. The aims of this study were to determine the optimal conditions for the culture and use of spheroids from HNSCC cell lines and optimal timepoint for using the spheroids obtained, to evaluate the effects of coculture with tumour-specific fibroblasts on spheroid formation, and to investigate spheroid responses to cisplatin treatment. Four HNSCC cell lines (UMSCC-11A, UMSCC-11B, UMSCC-22B and UD-SCC-01) were seeded in flat or round bottom well ultra-low attachment spheroid plates, and spheroid formation was evaluated. The HNSCC cell lines were then cocultured with stromal cells of the tumour microenvironment, producing an accelerated formation of dense spheroids. The viability of cells within the spheroids was assessed during cell culture by using a fluorescent dye. Our results suggest that: three out of the four cell lines tested could form usable spheroids with acceptable viability; the addition of stromal cells did not improve the number of viable cells; and the use of round bottom well plates supported the formation of a single spheroid, whereas flat bottom well plates led to the formation of multiple spheroids of different sizes.
Substances of abuse have the potential to cause addiction, habituation or altered consciousness. Most of the research on these substances focuses on addiction, and is carried out through observational and clinical studie...Substances of abuse have the potential to cause addiction, habituation or altered consciousness. Most of the research on these substances focuses on addiction, and is carried out through observational and clinical studies on humans, or experimental studies on animals. The transposition of the EU into Italian law in 2014 (IT ) includes a ban on the use of animals for research on substances of abuse. Since then, in Italy, public debate has continued on the topic, while the application of the Article prohibiting animal research in this area has been postponed every couple of years. In the light of this debate, we briefly review a range of methodologies - including animal and non-animal, as well as patient or population-based studies - that have been employed to address the biochemical, neurobiological, toxicological, clinical and behavioural effects of substances of abuse and their dependency. We then discuss the implications of the Italian ban on the use of animals for such research, proposing concrete and evidence-based solutions to allow scientists to pursue high-quality basic and translational studies within the boundaries of the regulatory and legislative framework.
Culture of care in Laboratory Animal Science (LAS) refers to a commitment toward improving animal welfare, scientific quality, staff wellbeing, and transparency for all stakeholders, ensuring that the animals and personn...Culture of care in Laboratory Animal Science (LAS) refers to a commitment toward improving animal welfare, scientific quality, staff wellbeing, and transparency for all stakeholders, ensuring that the animals and personnel involved are treated with compassion and respect. A strong culture of care can be established by the proactive implementation of the Three Rs, sharing best practices, caring for and respecting animals and colleagues, empowering staff, taking responsibility for our actions, and having a caring leadership. Culture of care, when established, should be evaluated continuously, in order to foster its progress and persistence. Even though several tools for assessing the culture of care within an institution have been proposed, an ultimate standard for measuring the concept is lacking. Here, we review the culture of care concept and propose the 'Capability Maturity Model' as a means of quantifying culture of care in the laboratory animal setting.