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Walmsley R.M.,Gentronix | Tate M.,University of Manchester
Methods in Molecular Biology | Year: 2012

Mutagens, clastogens, and aneugens cause increased expression of the human GADD45a gene. This has been exploited in the GreenScreen HC genotoxicity assay in which the gene's expression is linked to the expression of green fluorescent protein (GFP). The host for the reporter construct is the human lymphoblastoid cell line TK6. It was chosen for its growth as a cell suspension, which allows simple pipette transfers, and for its wild-type p53 competent status. P53 is required for proper GADD45a expression, and more generally for genome stability. TK6 is a karyotypically stable cell line. The GreenScreen assays were designed to facilitate screening, and this is reflected in its microplate format and low compound requirement. Protocols are available for testing with and without S9 as a source of exogenous metabolic activation. Data is collected either spectrophotometrically or by flow cytometry, and a simple spreadsheet converts raw data into dose-response curves, and provides a statistically significant positive or negative result. Extensive validation has demonstrated that in contrast to other in vitro mammalian genotoxicity assays, the GADD45a assays have both high sensitivity and specificity-they very rarely produce misleading positive results. © 2012 Springer Science+Business Media, LLC.


Topham C.H.,University of Manchester | Billinton N.,Gentronix | Walmsley R.M.,University of Manchester | Walmsley R.M.,Gentronix
Toxicological Sciences | Year: 2012

The in vitro mammalian genotoxicity tests identify some carcinogens not identified by the bacterial Ames test. However, historically they have produced rather more misleading predictions of carcinogenicity than the Ames test. This liability has been reduced in pharmaceutical testing by lowering the top-testing dose and rejecting data from excessively toxic doses. It also stimulated the development of new assays with inherently higher specificity. Among these, the GADD45a-GFP assay has been recognized as a maturing technology by the International Life Sciences Institute Health and Environmental Sciences Institute In Vitro Genetic Toxicity Emerging Technologies and New Strategies workgroup and has been concluded to be suitable for inclusion in a battery of high throughput screening by the U.K. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment. GADD45a is induced by compounds that cause damage to or missegregation of chromosomes, and is implicated in the stimulation of repair or apoptosis where damage is overwhelming. It is therefore important to understand whether this causes a liability in the assay to produce misleading positives for nongenotoxic inducers of apoptosis. Compounds hypothesized to stimulate apoptosis in the GADD45a-GFP assay or to induce GADD45a in the absence of genotoxic stress, such as p53 activators, NF-κB and Bcl-2 inhibitors were selected. Apoptosis induction was monitored using Annexin V binding and caspase 3/7 activation assays. The majority of compounds tested were negative in the GADD45a-GFP assay. The few that generated positive data were also found positive in concurrent comet assay and/or micronucleus tests. The data presented here demonstrate that the GADD45a-GFP assay is not vulnerable to the generation of misleading positive results by apoptosis inducers. © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.


Scott H.,Gentronix | Walmsley R.M.,Gentronix | Walmsley R.M.,University of Manchester
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2015

Boronic acids and their derivatives have been exploited for their pharmacological activity and their utility as intermediates in the synthesis of novel non-boron containing compounds. A recent study reported that boronic acids are bacterial mutagens. Here, results are reported from the testing of nine boronic acids using the pan-mechanistic eukaryotic GADD45a genotoxicity assays, BlueScreen HC and GreenScreen HC. Positive results were produced for one compound in GreenScreen and four compounds in BlueScreen. Only negative results were produced when tested with S9 metabolic activation. These data suggest that there is not a general genotoxic liability in eukaryotes, within this chemical domain. Furthermore, they are not potent eukaryotic genotoxins: positive results were produced only at concentrations between 1. mM and 10. mM. Their presence as low concentration contaminants or impurities would be unlikely to produce misleading positive results for a test material. © 2014 Elsevier B.V.


Patent
Gentronix | Date: 2013-04-10

The present invention relates to methods for detecting for the presence of an agent that putatively causes or potentiates DNA damage comprising subjecting a cell (containing a DNA sequence encoding a reporter protein operatively linked to a human GADD45 gene promoter and a human GADD45 gene regulatory element arranged to activate expression of the DNA sequence in response to DNA damage) to an agent; and monitoring the expression of the reporter protein from the cell. The invention also concerns expression cassettes, vectors and cells which may be used according to such a method and also modified media that may be employed in fluorscence assays and in preferred embodiments of the method of the invention.


Patent
Gentronix | Date: 2013-10-14

The present invention relates to methods for detecting for the presence of an agent that putatively causes or potentiates DNA damage comprising subjecting a cell (containing a DNA sequence encoding Gaussia luciferase (GLuc) reporter protein operatively linked to a human GADD45 gene promoter and a human GADD45 gene regulatory element arranged to activate expression of the DNA sequence in response to DNA damage) to an agent; and monitoring the expression of the GLuc reporter protein from the cell. The invention also concerns expression cassettes, vectors and cells which may be used according to such a method and also modified media that may be employed in assays and in preferred embodiments of the method of the invention.


Grant
Agency: GTR | Branch: Innovate UK | Program: | Phase: Feasibility Study | Award Amount: 147.34K | Year: 2013

The human genome is complex and well protected by an array of systems that sense damage, and initiate repair. When damage is overwhelming, cells are usually forced into suicide to minimise the generation of viable mutant cells that might develop into tumours. Direct DNA damage is not the only threat to the genome. Tissues such as liver, kidney, muscle etc are different because they express different subsets of genes from the same human genome. Active genes are more loosely packaged than inactive genes; inactive genes are more likely to have modified DNA bases compared to active genes. This regulation, above the DNA sequence, defines the science of epigenetics. The distinctive and lifethreatening cells found in tumours also have distintive epigenetic signatures, and in this project it is aimed to determine whether it is possible to detect epicarcinogens in a simple lab test that can detect changes in these signatures of gene expression.


Patent
Gentronix | Date: 2010-03-26

The present invention relates to methods for detecting for the presence of an agent that putatively causes or potentiates DNA damage comprising subjecting a cell (containing a DNA sequence encoding Gaussia luciferase (GLuc) reporter protein operatively linked to a human GADD45 gene promoter and a human GADD45 gene regulatory element arranged to activate expression of the DNA sequence in response to DNA damage) to an agent; and monitoring the expression of the GLuc reporter protein from the cell. The invention also concerns expression cassettes, vectors and cells which may be used according to such a method and also modified media that may be employed in assays and in preferred embodiments of the method of the invention.


Gentronix Limited the specialist genetic toxicology provider is delighted to announce that it has become a full member of the UK GLP monitoring programme for the provision of genotoxicity testing. The company has built a GLP team with extensive industrial experience in the performance of GLP studies and full membership of the programme is a landmark step for Gentronix as it continues to provide an extended range of genotoxicity services to its customers. As part of the regulatory process for many sectors, new materials have to be tested to show that they are safe for use. An important part of this regulatory testing is to demonstrate that a chemical does not cause damage to DNA. The regulatory testing, which must be performed to GLP standards is required to show that a chemical or a metabolite of the chemical does not cause mutations or structural changes that could potentially lead to cancer formation or inheritable disease. No single regulatory assay can detect all types of DNA damage, therefore a battery of assays is used in order to fully characterise a compound. The first stage in this process is the in vitro assessment of genotoxicity. The Ames reverse mutation assay (OECD 471) uses genetically modified bacteria that can only grow in growth limiting media conditions if a mutation occurs that allows the bacteria to again synthesize key nutrients. In order to test for structural changes the in vitro micronucleus assay (OECD 487) has been widely adopted. This assay can be conducted in a number of different cell types with human lymphocytes and p53 competent TK6 lymphoblastoid cells being routinely used. Gentronix now offers both Ames and in vitro micronucleus testing under GLP using the most up to date OECD protocols to generate studies ready for incorporation into regulatory dossiers. John Nicholson, CEO of Gentronix commented "Gentronix is delighted to have achieved GLP compliance and to have further expanded its portfolio of genotoxicity assay offerings, so that with our accurate and speedy turnround standard, we can be recognised as the "go-to" genotoxicity company." Gentronix Limited is a UK company specialising in genotoxicity for a range of industries including; pharmaceuticals, chemicals, agrochemicals, personal care, consumer products, flavours, fragrances and taste enhancers, and medical devices. Gentronix provides services and solutions helping a wide range of chemistry driven companies develop safer products more quickly at lower cost from early screening to GLP studies. Gentronix is the inventor and owner of GreenScreen HC and BlueScreen HC human cell based assays for the pan-mechanistic detection of genotoxic hazard and offers these alongside an increasing range of non GLP and GLP assays for genetic toxicology. To find out more please visit our website: http://www.gentronix.co.uk or email us at info@gentronix.co.uk GLP is a set of principles that provide a framework within which the consistency and quality of non-clinical health or environmental safety tests on the properties of chemical compounds, extracts and mixtures can be ensured. It is a legal requirement for regulatory submission in the fields of pharmaceuticals, agrochemicals, veterinary medicines, industrial chemicals, cosmetics, food and feed additives, and biocides, that such studies should be conducted in compliance with the principles of GLP. Contact For more information please contact; Dr. Steve Beasley, Commercial Director Email: steve.beasley@gentronix.co.uk Tele: +44(0)1625-238-742


UK based Gentronix Limited, the specialist genetic toxicology company, today announced it has signed a three year technology access deal with one of China's most prestigious research intensive institutes, Shanghai Institute Materia Medica (SIMM), Chinese Academy of Sciences (CAS). Under the terms of the multi-year agreement, Gentronix will provide training & installation support and on-going reagent supply to SIMM scientists to enable use of its proprietary technology, GreenScreen HC, for the detection of chemicals that may cause DNA damage. The human cell based technology will be used to further expand the ongoing SIMM - Laboratoires Servier joint research initiative set up in 2012 as the collaborators look to expand the range of predictive assays used at SIMM to better understand the properties and potential hazards of new drug candidates. GreenScreen HC makes use of the human derived TK6 cell line, genetically modified to carry a reporter construct that allows for the generation and detection of Green Fluorescent Protein (GFP) in response to up-regulation of a key gene, GADD45a following DNA stress inducing events. GreenScreen HC is a pan-mechanistic detection assay capable of detecting all classes of genetic toxin, as well as being able to detect genotoxins formed through metabolic activation using S9 mix. Prof Yang Ye, Deputy Director of SIMM, commented 'As we expand our research initiative with Laboratoires Servier in novel drug discovery, it is important for SIMM to have access to the best technology as it looks to both advance its collaborative research activities whilst at the same time train the scientists of tomorrow. GreenScreen HC provides us with an excellent capability for early genetic toxicology hazard assessment' For Nancy Claude, Director of Non Clinical Safety at Servier, the implementation of Greenscreen HC at the SIMM-Servier joint laboratory is an important step for the early stage toxicity screening conducted in both parties as it will contribute to the selection of the best drug candidates quickly and using a minimal amount of compound. John Nicholson, Chairman and CEO of Gentronix further commented 'This represents our first GreenScreen installation in China and an important development as we increase usage of our technology globally. SIMM has a notable history in research and we look forward to working with their scientists as they expand their drug profiling activities'. Gentronix Limited is a UK company specialising in genotoxicity for a range of industries including; pharmaceuticals, chemicals, agrochemicals, personal care, consumer products, flavours, fragrances and taste enhancers, and medical devices. Gentronix provides services and solutions helping a wide range of chemistry driven companies develop safer products more quickly at lower cost from early screening to GLP studies. Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS) evolved from the Institute of Materia Medica of Peking Academy of Sciences founded in 1932. Through several generations of effort, SIMM has become one of the leading interdisciplinary research centers in China. It is recognized worldwide by its outstanding achievements in both basic and applied research fields. SIMM's mission is to provide a comprehensive solution in drug discovery and development. By combining basic and applied research efforts and through cross-fostering between chemistry and biology, scientists at SIMM carry out studies toward the elucidation of the structural basis of bioactive substances, discovery of new targets or mechanisms of action, comprehensive pre-clinical evaluation of drug candidates, and promotion of commercialization, thereby playing an indispensable role in building China's drug innovation capabilities. The major research directions of SIMM include drugs against diseases seriously endangering the health of Chinese people like tumors, cardiovascular diseases, neurological diseases, metabolic diseases, autoimmune diseases as well as infectious diseases. Another task for SIMM is to strengthen research and development of Traditional Chinese Medicine (TCM). SIMM ranks at the top in China for its drug discovery and development activities. More than 70 drugs have been developed since its establishment. Of these, 10 were listed in the Chinese Pharmacopoeia. A number of drug candidates are currently under pre-clinical evaluations. More information is available at: english.simm.cas.cn To find out more please visit our website: http://www.gentronix.co.uk or email us at info@gentronix.co.uk   For more information contact: Dr Steve Beasley, Commercial Director, Gentronix Limited; Email: steve.beasley@gentronix.co.uk Tele: +44(0)1625-238-742


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