Development Of Alternative Methodologies Research Articles

Radon alpha track counting on solid state nuclear track detector by an ImageJ-based software macro

Radon (222Rn) is a radioactive gas emanating from geological materials. Inhalation of this gas is closely related to an increase in the probability of lung cancer if the levels are high. The usual methodology for the quantification of radon by passive methods is the use of etched solid-state nuclear track detectors, frequently in combination with optical microscopes or image scanning for image acquisition and software-based image processing for track counting. Currently available commercial instrumentation, as the Radosys microscopy system, is quite expensive, so the development of alternative methodologies is desirable.In this work, a simple, fast and low-cost image acquisition system for the determination of tracks in chemically etched CR-39 solid-state nuclear track detectors to quantify 222Rn alpha tracks has been proposed. The image of the detector surface is obtained by a conventional light stereoscopic microscope, transmitted by a CCD camera into the computer, and analyzed by the ImageJ open-source software. This methodology was employed to analyze 45 samples collected in dwellings and caves located in the region of Extremadura (Southwest Spain). Results show a good correlation coefficient of r2 = 0.98 between the reference and purposed methodology and excellent repeatability, demonstrating that the system enables routine counting tracks for radon measurement as an alternative to the Radosys microscopy instrument.

An easy and low-cost biomagnetic methodology to study regional gastrointestinal transit in rats.

The identification of gastrointestinal (GI) motility disorders requires the evaluation of regional GI transit, and the development of alternative methodologies in animals has a significant impact on translational approaches. Therefore, the purpose of this study was to validate an easy and low-cost methodology (alternate current biosusceptometry- ACB) for the assessment of regional GI transit in rats through images. Rats were fed a test meal containing magnetic tracer and phenol red, and GI segments (stomach, proximal, medial and distal small intestine, and cecum) were collected to assess tracer's retention at distinct times after ingestion (0, 60, 120, 240, and 360min). Images were obtained by scanning the segments, and phenol red concentration was determined by the sample's absorbance. The temporal retention profile, geometric center, gastric emptying, and cecum arrival were evaluated. The correlation coefficient between methods was 0.802, and the temporal retention of each segment was successfully assessed. GI parameters yielded comparable results between methods, and ACB images presented advantages as the possibility to visualize intrasegmental tracer distribution and the automated scan of the segments. The imaging approach provided a reliable assessment of several parameters simultaneously and may serve as an accurate and sensitive approach for regional GI research in rats.

Predicting the safety of medicines in pregnancy: A workshop report

The framework for developmental toxicity testing has remained largely unchanged for over 50 years and although it remains invaluable in assessing potential risks in pregnancy, knowledge gaps exist, and some outcomes do not necessarily correlate with clinical experience. Advances in omics, in silico approaches and alternative assays are providing opportunities to enhance our understanding of embryo-fetal development and the prediction of potential risks associated with the use of medicines in pregnancy. A workshop organised by the Medicines and Healthcare products Regulatory Agency (MHRA), “Predicting the Safety of Medicines in Pregnancy – a New Era?”, was attended by delegates representing regulatory authorities, academia, industry, patients, funding bodies and software developers to consider how to improve the quality of and access to nonclinical developmental toxicity data and how to use this data to better predict the safety of medicines in human pregnancy. The workshop delegates concluded that based on comparative data to date alternative methodologies are currently no more predictive than conventional methods and not qualified for use in regulatory submissions. To advance the development and qualification of alternative methodologies, there is a requirement for better coordinated multidisciplinary cross-sector interactions coupled with data sharing. Furthermore, a better understanding of human developmental biology and the incorporation of this knowledge into the development of alternative methodologies is essential to enhance the prediction of adverse outcomes for human development. The output of the workshop was a series of recommendations aimed at supporting multidisciplinary efforts to develop and validate these alternative methodologies.

A unified in silico model based on perturbation theory for assessing the genotoxicity of metal oxide nanoparticles.

Nanomaterials (NMs) are an ever-increasing field of interest, due to their wide range of applications in science and technology. However, despite providing solutions to many societal problems and challenges, NMs are associated with adverse effects with potential severe damages towards biological species and their ecosystems. Particularly, it has been confirmed that NMs may induce serious genotoxic effects on various biological targets. Given the difficulties of experimental assays for estimating the genotoxicity of many NMs on diverse biological targets, development of alternative methodologies is crucial to establish their level of safety. In silico modelling approaches, such as Quantitative Structure-Toxicity Relationships (QSTR), are now considered a promising solution for such purpose. In this work, a perturbation theory machine learning (PTML) based QSTR approach is proposed for predicting the genotoxicity of metal oxide NMs under various experimental assay conditions. The application of such perturbation approach to 6084 NM-NM pair cases, set up from 78 unique NMs, afforded a final PTML-QSTR model with an accuracy better than 96% for both training and test sets. This model was then used to predict the genotoxicity of some NMs not included in the modelling dataset. The results for this independent data set were in excellent agreement with the experimental ones. Overall, that thus suggests that the derived PTML-QSTR model is a reliable in silico tool to rapidly and cost-efficiently assess the genotoxicity of metal oxide NMs. Finally, and most importantly, the model provides important insights regarding the mechanism of the genotoxicity triggered by these NMs.

DNA Fingerprint: a Lab Protocol for High School Students

INTRODUCTION. The amount of new discoveries in molecular biology as well as the speed with which technological advances have been occurring and presented to students increases the complexity, the dynamics and the fluidity of the school environment. However, the approaching methods and the teaching resources used in classroom remains the most traditional ever. Therefore, the development of alternative methodologies which can overcome the deficiencies in schooling-learning process and motivate students for science field is needed. OBJECTIVE. Thus, we aimed to standardize a laboratorial protocol for simulate DNA fingerprint technique directed for high school students. MATHERIAL AND METHODS. Once the original protocol with DNA samples requires expensive reagents and equipment, practical activities were performed with protein samples. Protein extraction protocols used for seven distinct samples were tested: food supplements; milk serum; blood serum; powdered milk; animal; plant; rice bran. Each protocol efficacy was tested in SDS-PAGE electrophoresis. RESULTS AND DISCUSSION. Best samples were chosen for simulate the band patterns in an electrophoresis gel ordinarily found in DNA fingerprint methods. COMCLUSIONS. Therefore, such results may be used for forensic and paternity tests simulations in an alternative teaching activity for high school students, which could arise the opportunity to approach the basics principles of science technology and research. However, more tests are required in order to reproduce the results with cheaper protocols and make its viability in the class. Nonetheless, results accomplished in this work may be reproduced by school teachers in a partnership with academy labs in order to bring biotechnology resources to students life.

Ensaio do linfonodo local murino (LLNA) na diferenciação entre a dermatite de contato alérgica e de contato irritante: um estudo da expressão de marcadores de superfície de linfócitos T

Introduction: The Local Lymph Node Assay (LLNA) was developed as an alternative to Buhler and Maximization assays. It is applied to discriminate substances that are able to induce contact dermatitis and the endpoint is cell quantification in mice auricular lymph nodes. Although recommended by international agencies involved in the development of alternative methodologies, LLNA still needs to be improved. Objective: In this context, the goal of this study was to investigate possible differences in lymphocyte subpopulation patterns among mice treated with irritants and dermosensitizers. Method: Animals were treated with sensitizers dinitrochlorobenzene (DNCB) and paraphenylenediamine (PPD) and irritants sodium lauryl sulfate (SLS) and tritonX-100 (TX-100) for 3 days, using dorsum area of both ears. The percentage of different lymphocyte subpopulations were analyzed by flow cytometry. Ears of animals were also evaluated for possible pathological alterations. Results: Differences were observed in CD4+ CD25+ and CD4+ CD69+ cells, as well as in the proliferation of these subpopulations. The histopathological analysis of the ears showed no difference between the treatment with either dermosensitizers or irritants. Conclusions: T lymphocyte phenotyping might still be a useful tool in the development of an assay to differentiate between dermosensitizers and irritants. Moreover, these results may contribute to improving knowledge on this field and helping in the search of a correlate in vitro assay.

Quantitative Metabolite Profiling of an Amino Group Containing Pharmaceutical in Human Plasma via Precolumn Derivatization and High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.

Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable "heteroelement" (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups. The presence of a known number of I atoms in both the drug molecule and its metabolites enabled a cross-validation of the newly developed derivatization procedure and quantification based on monitoring of the introduced Br. The formation of the derivatives was quantitative, providing a 4:1 stoichiometric Br/NH2 ratio. The derivatives were separated via reversed-phase HPLC with gradient elution. Bromine was determined via ICPMS at a mass-to-charge ratio of 79 using H2 as a reaction gas to ensure interference-free detection, and iodine was determined at a mass-to-charge ratio of 127 for cross-validation purposes. The method developed shows a fit-for-purpose accuracy (recovery between 85% and 115%) and precision (repeatability <15% RSD). The limit of quantification (LoQ) for Br was approximately 100 μg/L.

Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models

As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.

Priorities in the development of alternative methodologies in the pharmaceutical industry.

Promotion of animal welfare is an underlying and laudable goal for toxicologists and there is good reason to adopt practical, focused, investigative approaches towards this aim. Pharmaceutical regulatory toxicology can be subdivided into the areas of systemic (target organ), reproductive, genetic and topical toxicology, as well as immunotoxicology and oncology. These areas can be assessed for prioritisation as to where adoption of measures to promote any or all of the 3 Rs (reduction, replacement, refinement) would lead to the most tangible benefit for animals. These measures can range, for example, from replacement of animal experimentation with alternative in vitro techniques, to adoption of regulatory protocols that reduce the number of animals required. This paper is confined to consideration of in vitro technology in terms of reducing/replacing laboratory animal use, and a suggested list of criteria for prioritisation is potential for:- Regulatory acceptability Reducing development cost Reducing animal numbers Promoting welfare aspects Elucidating toxic mechanisms Usefulness in compound selection Advancing the science of toxicology Clear messages emerge from such an analysis which could influence prioritisation of the application of in vitro toxicology from the standpoints of animal welfare, feasibility and resources.