scholarly journals Lessons learned in integrating traditional and NAM-based evidence to assess skin sensitization potential

2021 ◽  
Author(s):  
Allison Greminger ◽  
Katy Goyak ◽  
Joe Frasca ◽  
Colin North
Keyword(s):  
Biological Activity ◽  
Lessons Learned ◽  
Weight Of Evidence ◽  
In Vitro Assays ◽  
Adverse Outcome Pathway ◽  
Skin Sensitization ◽  
Reaction Products ◽  
Poorly Soluble

Classification of chemicals as skin sensitizers have traditionally relied on a small set of in vivo tests. Difficult to test substances, such as poorly soluble, mildly irritating, or those of Unknown or Variable Composition Complex reaction products or Biological Materials (UVCBs), producing weak or borderline results in Local Lymph Node Assay (LLNA), often benefit from additional data integration in a weight of evidence (WOE) approach. Advances in multiple testing and non-testing methodologies (in vivo, in vitro, and in silico) can now provide clarity and confidence in concluding on skin sensitization potential. Here we present several case studies using a WOE approach with difficult to test substances and highlight the utility of Toxicological Prioritization IndexTM (ToxPi™) as a comparative visualization and integration tool of toxicology studies. The three test chemicals chosen represent two poorly soluble substances, tetrakis (2-ethylbutyl) orthosilicate and decyl palmitate, and one UVCB substance, alkylated anisole. Data from in vivo and in vitro assays representing multiple key events within the skin sensitization adverse outcome pathway (e.g., direct peptide reactivity assay, human cell line activation test, GARD®Skin, LLNA) were either gathered from publicly available sources or specifically generated. Incorporating the data on our test chemicals as well as chemicals of a known sensitization class (sensitizer, irritating non-sensitizer, non-sensitizer) into ToxPi™ revealed biological activity profiles which were used to support class prediction for the three test chemicals. Using this method, the biological activity profiles for all three test chemicals were most consistent with a non-sensitizing class. This paper demonstrates that visualizing the WOE using mechanistic data maximizes the value of all data to the overall assessment of skin sensitization potential by reducing the uncertainty associated with any one individual assay.

scholarly journals Targeted Pathway-based In Vivo Testing Using Thyroperoxidase Inhibition to Evaluate Plasma Thyroxine as a Surrogate Metric of Metamorphic Success in Model Amphibian Xenopus laevis

2020 ◽  
Vol 175 (2) ◽  
pp. 236-250 ◽  
Author(s):  
Jonathan T Haselman ◽  
Jennifer H Olker ◽  
Patricia A Kosian ◽  
Joseph J Korte ◽  
Joseph A Swintek ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
In Vitro Assays ◽  
Adverse Outcome Pathway ◽  
Sodium Iodide Symporter ◽  
Chemical Safety ◽  
Compensatory Response ◽  
Amphibian Metamorphosis ◽  
In Vivo Testing

Abstract Chemical safety evaluation is in the midst of a transition from traditional whole-animal toxicity testing to molecular pathway-based in vitro assays and in silico modeling. However, to facilitate the shift in reliance on apical effects for risk assessment to predictive surrogate metrics having characterized linkages to chemical mechanisms of action, targeted in vivo testing is necessary to establish these predictive relationships. In this study, we demonstrate a means to predict thyroid-related metamorphic success in the model amphibian Xenopus laevis using relevant biochemical measurements during early prometamorphosis. The adverse outcome pathway for thyroperoxidase inhibition leading to altered amphibian metamorphosis was used to inform a pathway-based in vivo study design that generated response-response relationships. These causal relationships were used to develop Bayesian probabilistic network models that mathematically determine conditional dependencies between biochemical nodes and support the predictive capability of the biochemical profiles. Plasma thyroxine concentrations were the most predictive of metamorphic success with improved predictivity when thyroid gland sodium-iodide symporter gene expression levels (a compensatory response) were used in conjunction with plasma thyroxine as an additional regressor. Although thyroid-mediated amphibian metamorphosis has been studied for decades, this is the first time a predictive relationship has been characterized between plasma thyroxine and metamorphic success. Linking these types of biochemical surrogate metrics to apical outcomes is vital to facilitate the transition to the new paradigm of chemical safety assessments.

scholarly journals Skin sensitization

2015 ◽  
Vol 34 (12) ◽  
pp. 1222-1230 ◽  
Author(s):  
DA Basketter ◽  
IR White ◽  
JP McFadden ◽  
I Kimber
Keyword(s):  
Safety Evaluation ◽  
In Vitro Assays ◽  
Skin Sensitization ◽  
Human Data ◽  
Allergic Skin ◽  
Local Lymph Node ◽  
Allergic Contact ◽  
Risk Managers

Skin sensitization associated with allergic contact dermatitis is a common health problem and is an important consideration for toxicologists in safety assessment. Historically, in vivo predictive tests have been used with good success to identify substances that have the potential to induce skin sensitization, and these tests formed the basis of safety evaluation. These original tests are now being replaced gradually either by in vitro assays or by further refinements of in vivo methods such as the local lymph node assay. Human data have also been available to inform classification decisions for some substances and have been used by risk managers to introduce measures for exposure reduction. However, humans encounter hazards in the context of exposure rather than in the form of intrinsic hazards per se, and so in this article, we have examined critically the extent to which human data have been used to refine classification decisions and safety evaluations. We have also evaluated information on the burden of human allergic skin disease and used this to address the question of whether, and to what extent, the identification and evaluation of skin sensitization hazards has led to an improvement of public and/or occupational health.

scholarly journals Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment

2020 ◽  
pp. 153537022096696
Author(s):  
Leonardo Lima Fuscaldi ◽  
Joaquim Teixeira de Avelar Júnior ◽  
Daniel Moreira dos Santos ◽  
Daiane Boff ◽  
Vívian Louise Soares de Oliveira ◽  
...  
Keyword(s):  
Biological Activity ◽  
Antimicrobial Peptide ◽  
Mammalian Cells ◽  
Antimicrobial Agents ◽  
Sodium Dodecyl ◽  
Alpha Helix ◽  
In Vitro Assays ◽  
Control Group

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC50) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC50 [LyeTx I mnΔK] ⋙ EC50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

scholarly journals Machine Learning-based Biomarkers Identification and Validation from Toxicogenomics - Bridging to Regulatory Relevant Phenotypic Endpoints

2020 ◽  
Author(s):  
Sheikh Mokhlesur Rahman ◽  
Jiaqi Lan ◽  
David Kaeli ◽  
Jennifer Dy ◽  
Akram Alshawabkeh ◽  
...  
Keyword(s):  
Machine Learning ◽  
Dna Damage ◽  
Excision Repair ◽  
Dna Recombination ◽  
In Vitro Assays ◽  
Adverse Outcome Pathway ◽  
Dna Damage And Repair ◽  
Repair Pathways

ABSTRACTHigh-throughput in vitro assays and AOP-based approach is promising for the assessment of health and ecotoxicological risks from exposure to pollutants and their mixtures. However, one of the major challenges in realization and implementations of the Tox21 vision is the urgent need to establish quantitative link between in-vitro assay molecular endpoint and in-vivo phenotypic toxicity endpoint. Here, we demonstrated that, using time series toxicomics in-vitro assay along with machine learning-based feature selection (MRMR) and classification method (SVM), an “optimal” number of biomarkers with minimum redundancy can be identified for prediction of phenotypic endpoints with good accuracy. We included two case studies for in-vivo carcinogenicity and Ames genotoxicity prediction with 20 selected chemicals including model genotoxic chemicals and negative controls, respectively, using an in-vitro toxicogenomic assay that captures real-time proteomic response data of 38 GFP-fused proteins of S. cerevisiae strains covering biomarkers indicative of all known DNA damage and repair pathways in yeast. The results suggested that, employing the adverse outcome pathway (AOP) concept, molecular endpoints based on a relatively small number of properly selected biomarker-ensemble involved in the conserved DNA-damage and repair pathways among eukaryotes, were able to predict both in-vivo carcinogenicity in rats and Ames genotoxicity endpoints. The specific biomarkers identified are different for the two different phenotypic genotoxicity assays. The top-ranked five biomarkers for the in-vivo carcinogenicity prediction mainly focused on double strand break repair and DNA recombination, whereas the selected top-ranked biomarkers for Ames genotoxicity prediction are associated with base- and nucleotide-excision repair. Current toxicomics approach still mostly rely on large number of redundant markers without pre-selection or ranking, therefore, selection of relevant biomarkers with minimal redundancy would reduce the number of markers to be monitored and reduce the cost, time, and complexity of the toxicity screening and risk monitoring. The method developed in this study will help to fill in the knowledge gap in phenotypic anchoring and predictive toxicology, and contribute to the progress in the implementation of tox 21 vision for environmental and health applications.TOC Art

041. ENDOCRINE DISRUPTING IMPACTS IN RECEIVING WATERS OF THE SYDNEY BASIN

2010 ◽  
Vol 22 (9) ◽  
pp. 13
Author(s):  
R. Lim
Keyword(s):  
Water Reuse ◽  
Environmental Flows ◽  
Aquatic Systems ◽  
Weight Of Evidence ◽  
In Vitro Assays ◽  
Treatment Technology ◽  
Endocrine Disrupting ◽  
Treatment Technologies

Water reuse for a number of activities including potable water and replacement of environmental flows is becoming more significant due to the prolonged drought Australia has recently experienced. There is also much debate regarding potential impacts of compounds such as steroid endocrine disrupting chemicals (EDCs), pharmaceuticals and personal care products (PPCPs), and persistent organic pollutants (POPs) to environmental and human health. This paper presents an overview of findings on some EDCs in the Sydney Basin to assess the environmental risk they pose. A tiered approach, using a suite of endpoints spanning in vitro (e.g., estrogen receptor binding assay, the 2-hybrid yeast test) to in vivo (using the mosquitofish (Gambusia holbrooki) to assess vitellogenin induction, and morphological and behavioural changes) studies was conducted on aquatic systems receiving urban and treated sewage effluents. In vitro bioassays suggest low levels of estrogenicity in sewage contaminated waterways. Both estradiol (E2) and estrone (E1) were identified in all river water samples, suggesting that sewage contamination is widespread. The synthetic hormone, ethynylestradiol (EE2), was below detection limits in all samples tested. Results indicate that the STPs were not the only source of EDCs in aquatic systems within the Sydney area. Improvements in treatment technologies in STPs have substantially reduced EDC levels in final effluent as indicated by a reduction inendocrine disrupting effects on the mosquitofish over several years of study. In addition, advanced tertiary treatment technology removed EDCs to levels below that measurable by in vitro assays and in vivo fish testing. This tiered weight of evidence approach provided insights to the risks EDCs in sewage effluent produced from current treatment technologies have on the environment.

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

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.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

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.

Neurotoxicity of Pesticides: The Roadmap for the Cubic Mode of Action

2020 ◽  
Vol 27 (1) ◽  
pp. 54-77 ◽  
Author(s):  
Bogdan Bumbăcilă ◽  
Mihai V. Putz
Keyword(s):  
Biological Activity ◽  
Wiener Index ◽  
Laboratory Animals ◽  
Covalent Bonds ◽  
Organophosphate Compounds ◽  
Sar Studies ◽  
Structure Activity ◽  
Cubic Structure

Pesticides are used today on a planetary-wide scale. The rising need for substances with this biological activity due to an increasing consumption of agricultural and animal products and to the development of urban areas makes the chemical industry to constantly investigate new molecules or to improve the physicochemical characteristics, increase the biological activities and improve the toxicity profiles of the already known ones. Molecular databases are increasingly accessible for in vitro and in vivo bioavailability studies. In this context, structure-activity studies, by their in silico - in cerebro methods, are used to precede in vitro and in vivo studies in plants and experimental animals because they can indicate trends by statistical methods or biological activity models expressed as mathematical equations or graphical correlations, so a direction of study can be developed or another can be abandoned, saving financial resources, time and laboratory animals. Following this line of research the present paper reviews the Structure-Activity Relationship (SAR) studies and proposes a correlation between a topological connectivity index and the biological activity or toxicity made as a result of a study performed on 11 molecules of organophosphate compounds, randomly chosen, with a basic structure including a Phosphorus atom double bounded to an Oxygen atom or to a Sulfur one and having three other simple covalent bonds with two alkoxy (-methoxy or -ethoxy) groups and to another functional group different from the alkoxy groups. The molecules were packed on a cubic structure consisting of three adjacent cubes, respecting a principle of topological efficiency, that of occupying a minimal space in that cubic structure, a method that was called the Clef Method. The central topological index selected for correlation was the Wiener index, since it was possible this way to discuss different adjacencies between the nodes in the graphs corresponding to the organophosphate compounds molecules packed on the cubic structure; accordingly, "three dimensional" variants of these connectivity indices could be considered and further used for studying the qualitative-quantitative relationships for the specific molecule-enzyme interaction complexes, including correlation between the Wiener weights (nodal specific contributions to the total Wiener index of the molecular graph) and the biochemical reactivity of some of the atoms. Finally, when passing from SAR to Q(uantitative)-SAR studies, especially by the present advanced method of the cubic molecule (Clef Method) and its good assessment of the (neuro)toxicity of the studied molecules and of their inhibitory effect on the target enzyme - acetylcholinesterase, it can be seen that a predictability of the toxicity and activity of different analogue compounds can be ensured, facilitating the in vivo experiments or improving the usage of pesticides.

In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

2018 ◽  
Vol 21 (3) ◽  
pp. 215-221
Author(s):  
Haroon Khan ◽  
Muhammad Zafar ◽  
Helena Den-Haan ◽  
Horacio Perez-Sanchez ◽  
Mohammad Amjad Kamal
Keyword(s):  
In Silico ◽  
Docking Studies ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Chronic Obstructive ◽  
Lipoxygenase Inhibitors ◽  
Lipoxygenase Inhibition ◽  
In Silico Studies

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.

Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

2019 ◽  
Vol 16 (3) ◽  
pp. 175-180
Author(s):  
Fengjin Hao ◽  
Yueqin Feng ◽  
Yifu Guan
Keyword(s):  
Biological Activity ◽  
Botulinum Toxin ◽  
Cell Membrane ◽  
Western Blot ◽  
Biological Function ◽  
C6 Cells ◽  
Green Fluorescence ◽  
Bv2 Cells

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity. Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25. Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

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