Established in 1999 and based in Manchester, UK, Gentronix provides services and solutions that help companies optimize the development of drugs and other chemicals, by reducing rates of attrition due to toxicity and ensuring safer products across a wide range of chemistry driven industries.

Gentronix’ primary focus is on providing better hazard identification assays for genotoxicity through the use of our patented human cell GADD45a reporter assays; GreenScreen HC and BlueScreen HC.  Gentronix supplies kits of reagents and cell lines for these assays to allow researchers to conduct the assays within their own laboratories.  Additionally Gentronix provides comprehensive contract services for genotoxicity screening in GreenScreen HC, BlueScreen HC and related in vitro assays including Comet and Micronucleus tests, as well as a wide range of ADME and predictive toxicity endpoints.

GreenScreen HC is the accurate and rapid, human cell-based assay for determining potential genotoxic liability in new chemical entities in pharmaceutical development and for use in a range of other chemical industry sectors, including agrochemicals, cosmetics, personal care products and medical device/biomaterials.

With a very low compound requirement the assay can be used early in the discovery process to obtain valuable information on perceived genotoxic hazard.  The assay uniquely delivers both high specificity and high sensitivity and detects all mechanistic classes of genotoxin.

The GreenScreen HC assay uses p53-competent, human-derived, TK6 cells, familiar to most genetic toxicology laboratories.  The cells host a patented Green Fluorescent Protein (GFP) gene reporter system to detect the up-regulation of GADD45a, a growth arrest and DNA damage gene.  GADD45a plays a central role in mediating the adaptive cellular response to genotoxic stress and DNA damage. 

The detection of pro-genotoxins, i.e. chemicals which require metabolic activation to become genotoxic, is achieved by performing parallel assays in the absence and presence of post-mitochondrial rat liver fraction (S9), the latter with flow cytometric scoring.  The assays are conducted in 96-well microplate format and results are delivered after 48 hour incubation. A single microplate is sufficient for the simultaneous testing of 4 compounds over 9 serial dilutions. The protocols are also readily automated using standard laboratory equipment
 

BlueScreen HC is based on the GreenScreen HC genotoxicity assay using the same human-derived, p53-competent TK6 cells to host a patented reporter system that detects the up-regulation of the GADD45a gene. However, in place of Green Fluorescent Protein, BlueScreen HC utilizes a luciferase from the marine copepod Gaussia princeps to generate a flash luminescent output.  BlueScreen HC delivers both high specificity and high sensitivity and detects all mechanistic classes of genotoxin, just like GreenScreen HC.

The 96-well microplate format is rapid to set up and results are generated after 48 hours’ incubation. A single microplate is sufficient for the simultaneous testing of 4 compounds, each over 8 serial dilutions.  The luminescent output makes the assay well suited to analysing compounds with very high autofluorescence, such as those commonly found in personal care and cosmetic products, which otherwise might confound other assays with fluorescence outputs.  The superior signal-to-noise ratio also makes the assay more suitable for adaptation to even higher throughput screening in 384-well microplate format.

GreenScreen GC is a yeast cell based assay for genotoxicity and cytotoxicity screening.  The assay employs a genetically engineered brewer’s yeast strain, Saccharomyces cerevisiae, which hosts a Green Fluorescent Protein (GFP) gene reporter system to detect the up regulation of the DNA repair gene RAD54.   As a result, cells that sustain chromosome damage produce increasing quantities of GFP.  The assays are conducted in 96-well microplate format and results are delivered after 16 hour incubation.  A single microplate is sufficient for the simultaneous testing of 4 compounds over 9 serial dilutions.

The components of the assay are available from Gentronix and are currently used in locations with only basic laboratory equipment, for example where apparatus for mammalian cell passage is not available, or in environmental and industrial laboratories for environmental monitoring applications.   The assay has a lower sensitivity compared to the GreenScreen HC human-cell based assay and is not performed with the addition of S9 fraction, hence for applications in drug discovery the assay has largely been superseded by GreenScreen HC.
 

No.  Gentronix has negotiated licences with Amersham Biosciences (now GE Healthcare) and Prolume Limited to use the GFP and Gaussia Luciferase genes respectively in our assays.  These allow us to distribute our products worldwide.  Individual users do not need to obtain a separate licence to use GreenScreen HC and BlueScreen HC products within their own laboratories, and need only sign a straightforward end user label licence before using supplied kit materials.
 

Contract Research Organisations wishing to offer the GreenScreen HC assay as a service to customers should contact Gentronix for more details since certain licence conditions exist for use of GreenScreen HC that CROs need to be aware of .

There are no restrictions on the use of BlueScreen HC for CROs wishing to provide this assay as a service to customers
 

Currently over 100 pharmaceutical, fine chemical and biotechnology companies, located in 17 countries have used the GreenScreen HC assay to screen for genotoxicity liability in their development compounds.  Gentronix is now regularly supplying GreenScreen HC or BlueScreen HC assay materials, or performing contract services in our laboratory, for many of these companies.

Gentronix has conducted international ring trials of the GreenScreen HC and GreenScreen HC S9 assays in the laboratories of major European and US pharmaceutical companies and Clinical Research Organisations, the results of which have been published in the peer reviewed literature.  Additionally, we have  generated a wealth of data from other evaluations of large and diverse compound collections, the majority of which have also been published.  (See our Literature pages).

A major need exists within the pharmaceutical industry to accurately identify potentially toxic compounds as early as possible in the drug discovery process. This enables elimination of these compounds from the programme or further medicinal chemistry to reduce the toxicity of promising lead candidates. The current regulatory battery of in vitro and in vivo assays is effective in identifying DNA damaging agents (positive genotoxicity results), however recent studies of in vitro regulatory data have shown the battery to be less effective in the identification of non-genotoxins (negative results). The result of this poor accuracy is that additional effort including expensive animal testing is required to distinguish real genotoxins from false positive results.

The GreenScreen HC and BlueScreen HC assays are ideally suited to fulfil this early screening role.  Their accuracy, speed and low compound requirement means they can be deployed early in the discovery process, and the valuable information on genotoxic liability obtained can be acted upon before significant investment is made in progressing lead molecules that are ultimately unlikely to reach the clinic.
 

Gentronix has published around 40 papers and articles describing our assay technology, its various applications and a wealth of validation data.  See our Literature pages to link to and download our published papers, or to request reprints.

When kits are purchased by a user for the first time, Gentronix will usually supply our scientists to run training and installation of the assay in their laboratory.  This includes hands on demonstration of the assay, optimization of laboratory equipment especially microplate readers and flow cytometers, and providing training in data processing and interpretation.  If you are simply interested in seeing the assay run in situ we also welcome visitors to our laboratory in Manchester, UK.  Please contact us to discuss your requirements.

We want your first experience of GreenScreen HC or BlueScreen HC to be a good one and to make it work first time in your hands; hence we prefer to visit you at your laboratories to guide you through the first set of assays. We appreciate that you employ very competent scientists, but we have learned during our international assessments of transferability and years of experience, that like any new assay, a period of familiarisation is needed, and this is speeded by having an expert on hand. During the visit we would explain and demonstrate the assay protocol, run a series of standard compounds and collect and interpret the data. Importantly we would also set up and optimise protocols for your microplate reader and flow cytometer if appropriate, advise on the automation of the assay if liquid handling robotics are to be used, address any compound specific issues and, if requested, give a presentation about our assay technology to interested members of your staff.

All the companies we have visited have found this approach to be valuable in minimising both their time and effort required to get started with GreenScreen HC or BlueScreen HC and the amount of ongoing technical support subsequently required.

We do not provide reagents free of charge, however we do adopt a flexible and cost effective approach to enable customers to access our genotoxicity assays via reagent kits or contract service.  Please contact us to discuss your specific requirements.

Yes, we can test compounds for customers in our laboratories at Gentronix as a contracted service. Both catalogue and proprietary compounds can be tested, using 'blind' studies if necessary. We receive compounds and test them in the GreenScreen HC and GreenScreen HC S9 assay in accordance with our standard protocols. We usually request just a 10 mg pre-weighed sample of each compound, from which we will test the compound over 9 serial dilutions from a maximum concentration of typically 1 mg/ml, unless limited by solubility. We will also run a repeat test if the concentration range initially chosen shows excessive cytotoxicity, compound precipitation or autofluorescence, in order to obtain an unambiguous result. Upon completion of the testing we will provide a report that will give you a summary of the assay features, details of the protocol performed and discussion of the results. We aim to complete the testing within 2 weeks from receipt of compound.

The true cost of any assay involves consideration of a number of elements (labour, consumables, reagents, instrument depreciation and overheads). The cost of GreenScreen reagents is governed by a price-volume matrix taking account of the volume and frequency of use. This matrix accommodates for the various stages of the drug discovery process where GreenScreen can be used e.g. 'hits to lead' screening, lead optimisation screening or pre-regulatory lead candidate screening.  We are happy to provide a quotation based on your estimated annual usage, please contact us.

You will find that GreenScreen is indeed very cost-effective and here are a few comments on the other costs involved in running the assay: Labour - Approximately one third of a full time employee's time is required to manually set up, read, and report 20 compounds per day, although this throughput could be significantly increased by the use of liquid handling robots. Consumables: are inexpensive - one 96 well plate and two breathable membranes are required for testing 4 compounds. Reagents: very small quantities of the genotoxic standard compound methyl methanesulfonate are required. For GreenScreen HC additional consumables (cell culture flasks) and reagents (RPMI media and additives) are required for the continuous passaging of cell lines, however, these are common to many cell culture laboratories. Instrument depreciation - GreenScreen can run on one of several standard fluorescent/absorbance readers, and indeed you may already have a compatible reader in house. See: GreenScreen HC and GC Instrumentation

Presently there are two kit sizes available for GreenScreen HC and GC: for 20 or 100 compounds. The throughput is principally determined by the liquid-handling used for preparation, i.e. manual pipettes or robotic. The minimum run size is 4 compounds (i.e. one microplate) yet up to 40 compounds can be tested by manual pipette in one day, using one microplate reader.

It depends on the liquid-handling method used. A multi-probe robotic platform may take 5 - 10 minutes to prepare an assay plate for GreenScreen HC or GC, i.e. serially dilute test compounds, add standard compounds and then add the two cell strains. Whilst by hand using programmable multi-channel pipettes it takes around 20 minutes per plate. The GreenScreen HC assay requires two reading time points at 24 and 48 hours. The GreenScreen GC assay requires only an overnight incubation with a read time point at 16 - 20 hours.

This depends on the maximum concentration you wish to test. Usually this is either a fixed amount, or the limit of cytotoxicity or solubility. The assay requires only approx. 0.5 ml of test solution per test. To test from a concentration of 1 mg/ml, if the compound is soluble in the aqueous diluent (2% v/v DMSO) then 1 mg or less of test sample is sufficient. If the compound is to be pre-solubilised in DMSO then typically 10 mg of sample is required.

The assay has been optimised to run 4 compounds per microplate, each over 9 concentrations, usually using serial 50/50 dilutions. The assay software recognises and interprets data from the defined microplate layout. This wide dilution series also allows the user to capture the toxicologically relevant concentration range in a single run, without the need for a range finding experiment. The magnitude of dilution across the microplate can be altered, however the number of dilutions can not.

The usual solvent is aqueous DMSO (2% v/v for GreenScreen HC and 4% v/v for GreenScreen GC), however, others such as pure water or ethanol (4% v/v) can be used. Thus the assay is suited to test compounds that are stored or pre-solubilised in DMSO.

As eukaryotic cells, both the TK6 cells (GreenScreen HC) and yeast cells (GreenScreen GC) are equipped with some components of human metabolism such that some pro-mutagenic compounds known to require metabolic activation can be detected using GreenScreen assays. However, like the other in vitro genotoxicity test systems, these cell lines lack the inherent metabolic competency of the liver. The standard GreenScreen GC assay is not compatible with the use of S9 due to spectroscopic interference. GreenScreen HC can be used with S9 using a flow cytometer to collect the data and protocols for this application are currently being devised by Gentronix. See: GreenScreen HC and Metabolic Acivation.

It is likely that most of the equipment required to perform the GreenScreen assay is already available in your laboratory.

After extensive in-house evaluation, Gentronix recommends several manufacturer's microplate readers, including models from Bio-Tek (Synergy2), BMG LABTECH (PHERAstar and POLARstar Optima), Perkin Elmer (Victor 3 and Envision), Tecan (ULTRA, Evolution, Infinite F500 and Safire2), Thermo Scientific (Varioskan Flash) and Molecular Devices (SpectraMax M2e, M5 and M5e). Some other plate readers can be used for the GreenScreen assay, but may give reduced performance compared to the recommended instruments. The data output should exportable into Microsoft Excel. The fluorescence readings are commonly made at 485 nm excitation and 535 nm emission wavelengths, however a standard fluorescein filter set, commonly supplied with most plate readers, will normally suffice. Absorbance measurements are made through the clear-bottomed plate at a wavelength in the region of 600 nm. Although less convenient, separate readers can be used to measure fluorescence and absorbance if necessary. For more details see GreenScreen HC and GC Instrumentation page. The availability of an option to make fluorescence polarisation measurements allows the use of a GreenScreen protocol to test compounds that exhibit very high autofluorescence at the same wavelengths as GFP.

Yes, GreenScreen HC and GC are designed for use with both handheld multi-channel pipettes and liquid handling robots. If a moderately high throughput is required, i.e. greater than 20 compounds / day, the use of robotic liquid handling to set up the assay microplates may be a more convenient option. We have used a variety of commercially available single and multi-probe liquid handling robots to produce assay plates, including those supplied by BioTek, Hamilton and Tecan.

The cell lines are fully validated and guaranteed for up to 3 months of continuous culture after which fresh reagents can be obtained from Gentronix. A second set of aliquots of frozen cell cultures are supplied with each kit in case a replacement is required due to a loss of cell lines during the three month period, due for example to mishandling or breakdown of incubation equipment. Each GreenScreen kit comes with Gentronix's Quality Control assurance specific to the batch of cell culture and media supplied.

By making use of a second, non-fluorescent yeast strain the GreenScreen assay protocol is able to accurately correct for the autofluorescence properties of the majority of test compounds. For highly autofluorescent compounds a patented protocol using fluorescence polarisation can also be used to discriminate between the GFP-derived assay signal and compound autofluorescence, exploiting the high fluorescence anisotropy of GFP.

The GreenScreen software alerts the user if the compound is significantly absorbing at the wavelengths used. For most coloured compounds tested to date, the concentration at which the toxic range of the compound is detected by the assay is sufficiently dilute as to present few problems. For highly coloured compounds, such as chemical dyes, a separate experiment diluting the dye in the absence of cells is able to correct up to an optical density of ~5.0 OD units.

To aid the analysis of compounds with low aqueous solubility, the assay can be run with a final concentration of DMSO of 1% v/v in GreenScreen HC and 2% v/v in GreenScreen GC, allowing the testing of samples stored or pre-solubilised in DMSO.

Yes, the data analysis software which produces a detailed report on the genotoxicity and cytotoxicity of each set of 4 compounds per microplate is supplied with the assay kit at no extra cost. As periodic software updates become available these will be sent to all current GreenScreen users.

An initial validation of GreenScreen HC with 75 well-characterised compounds was published in 2006 (See: Literature page). Pre-publication data on 66 marketed pharmaceuticals is available and a screen of the LOPAC collection of 1280 compounds is underway. Data can be shared under a confidentiality agreement with Gentronix so as not to prejudice future publications of original data. In excess of 3500 compounds have been tested so far by customers or collaborators of Gentronix.

GreenScreen GC has been extensively validated by a number of in house and external trials.  The latest of a study of 305 compounds published in 2007 can be obtained from the Literature Page.

Results from the published validation studies show that in comparison with other test data GreenScreen demonstrates high specificity in the detection of genotoxic carcinogens without compromising sensitivity. Hence, a positive result in GreenScreen is highly predictive of a positive result in the regulatory test battery. Results are highly reproducible using a standardised, validated protocol. GreenScreen detects genotoxic chemicals with a wide variety of mechanisms of action, including those with specific eukaryotic targets, and a different but overlapping spectrum of compounds to bacterial assays.

GreenScreen HC scores the same positives as GreenScreen GC, however being a human cell-based assay GreenScreen HC detects a wider spectrum of compounds. Specifically: Aneugens, since these don't damage DNA, but mis-segregate whole chromosomes. The yeast RAD54 assay only sees damaged DNA; Nucleoside analogues - as yeast are often more selective as to which substances they incorporate; and mammalian-specific topoisomerase inhibitors.

See: Results Output and Interpretation page for a typical result output from the GreenScreen assay.

The following are examples of genotoxic compounds detected with GreenScreen HC.
Direct acting genotoxins: Busulfan, Cisplatin, Ethyl nitrosourea, MNNG, 4-NQO, ICR-191
Topoisomerase inhibitors: Camptothecin, Doxorubicin, Etoposide.
Aneugens: Benomyl, Colchicine, Griseofulvin, Paclitxel, Vincristine sulphate.
Nucleotide analogues: 5-Azacytidine, 5-Fluorouracil, Azidothymidine (AZT).
DNA synthesis inhibitors: Aphidicolin, Hydroxyl urea, Pyrimethamine.
Reactive oxygen species generators: Bleomycin sulphate, Hydrogen peroxide.