Established in 1999 and based in Manchester, UK, Gentronix is an innovative biotechnology company helping to accelerate the pace of drug development in the pharmaceutical industry. Gentronix develops in vitro screening assays for the accurate identification of genotoxic compounds.

GreenScreen® products are cell-based assays for use in genetic toxicity screening using simple, rapid and high-throughput protocols.  The patented assay system is designed for use much earlier in drug discovery than the standard test battery for genotoxicity, and provides positive results that are more accurately predictive of in vivo risk than the current in vitro mammalian assays.

GreenScreen HC is a novel human cell-based screening assay for genotoxicity. The GreenScreen HC assay uses TK6 host cells which are p53 competent and familiar to most genetic toxicology laboratories. A patented GFP reporter system exploits the proper regulation of the DNA damage response gene GADD45a. The assay delivers both high specificity and high sensitivity and detects all common mechanistic classes of genotoxin. The 96-well microplate format takes about 20 minutes to prepare and delivers results after 48 hour incubation. A single microplate is sufficient for the simultaneous testing of 4 compounds over 9 serial dilutions. The protocol is also readily automated using standard laboratory equipment. For more information see GreenScreen HC.

Gentronix's latest release is GreenScreen HC S9, which incorporates exogenous metabolic activation using S9 fraction to allow the detection of pro-genotoxic compounds.  The 96 well microplate layout is similar to the GreenScreen HC assay with the additional pro-genotoxic control compounds.  Test articles are incubated with GreenScreen cells for 3 hours in the presence of S9 fraction, followed by a 45 hour recovery period in fresh medium.  Data is collected using flow cytometry.  For more information see GreenScreen HC S9.

GreenScreen GC is the name of our yeast cell-based screening assay for genotoxicity and cytotoxicity. A single microplate is sufficient for the simultaneous testing of 4 compounds over 9 serial dilutions. The protocol is also readily automated using standard laboratory equipment. A key advantage of using yeast is that aliquots of the culture can be stored in a laboratory freezer and are easily resuscitated ready for use after a 72 hour (i.e. over weekend) incubation. The stock culture can be used immediately for assays and remaining stocks are disposed of after 1 week. There is no requirement for on-going passage and maintenance of cell lines, and thus the assay can be handled in a basic laboratory setting. It has a lower specificity and sensitivity than GreenScreen HC.  See: GreenScreen GC

GreenScreen EM is a specially formulated version of GreenScreen GC used for environmental monitoring of genotoxic and cytotoxic contaminants. GreenScreen EM comprises a convenient cuvette based protocol and dedicated portable instrumentation. Principal applications are in the monitoring and characterisation of the genotoxicity and cytotoxicity of effluents and contaminated surface waters and soils. See: GreenScreen EM

Not for companies testing on their own behalf. Gentronix has negotiated a license with Amersham Biosciences (now GE Healthcare) to use the GFP gene in our assay, which allows us to distribute our products worldwide. Individual users do not need a license to use GreenScreen products.  However, Contract Research Organisations should contact Gentronix to discuss the requirements for using GreenScreen to provide a toxicology testing service to third parties.

We have completed trials of GreenScreen HC and GC in the laboratories of major European and US pharmaceutical companies and Contract Research Organisations. They were impressed with the technology, and Gentronix is now supplying product on a regular basis to these organisations.  Over 55 pharmaceutical and fine chemical companies in 11 countries are now using GreenScreen HC as part of their genotoxicity profiling strategy.  Additionally, we have generated a wealth of data from other evaluations. We are happy to share the results of both our published trials (See: Literature page) and unpublished collaborations with prospective customers and partners with a suitable confidentiality agreement in place.

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 the initiation of chemistry development 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 has 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.

Around 30 papers, articles and presentations describing our technology can be downloaded from this website. See: Literature page

We are happy to discuss your requirements, and make suitable arrangements for demonstration of the assay at a convenient location. Please contact us to discuss this. See: Contact page

We want your first experience of GreenScreen 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 assessment of transferability, 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. We would also assess the suitability of your microplate readers, 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 GreenScreen to interested members of your staff. Companies we have visited have found this approach to be valuable in minimising both their time and effort required to get started with GreenScreen and the amount of ongoing technical support subsequently required.

We do not provide reagents free of charge; however we adopt a flexible and low cost approach to enable customers to evaluate GreenScreen HC and GC. Please contact us to discuss your specific requirements. See: Contact page

Yes, we can test compounds for customers in our laboratories at Gentronix as a contract research service. Both catalogue and proprietary compounds can be tested, using 'blind' studies if necessary. We receive compounds and test them in the GreenScreen HC assay in accordance with our standard protocols. We usually request 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, usually limited by solubility. We will also run a single 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 would aim to complete the testing in approximately 2 weeks from receipt of compound.  For more details see: Outsourced service with GreenScreen HC.

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. For low numbers of compounds, the cost is 250 GBP per compound using kit reagents, 750 GBP for contract testing outsourced to Gentronix. 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 kit sizes are based on multiples of 20 compounds for GreenScreen HC, GreenScreen HC S9 and GreenScreen GC, however other bulk sizes for 100+ compounds can be produced on request.  The testing 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 in GreenScreen HC or GC by manual pipette in one day, using one microplate reader.  For the GreenScreen HC S9 assay the throughput is determined by the speed of the flow cytometer and how many 96 well microplates can be comfortably analysed per day.  Flow cytotmeters with a high-throughput microplate sampler will process plates faster than transferring the contents of microplates to sample tubes.

It depends on the liquid-handling method used. A multi-probe robotic platform may take approx. 10 minutes to prepare an assay plate for GreenScreen HC or GC once reagents and cells have been prepared, 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 HC S9 assay reads by flow cytometer at a single 48 hour time point.  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.  The new GreenScreen HC S9 assay was launched in 2007 and is available both in the form of reagent kits and as part of Gentronix's service testing offering. See: GreenScreen HC and Metabolic Activation.

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 (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.

The terms and conditions of sale expressly forbid such action. Furthermore, each GreenScreen kit comes with Gentronix's Quality Control assurance specific to the batch of cell culture and media supplied. Sub-culturing or diluting the cells invalidates that guarantee of reproducible performance. Finally, clever chemistry is employed in such a way that the assay strain attenuates upon subculture, and rapidly becomes unreliable.

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 results from a screen of the LOPAC collection of 1280 compounds are in press. Data can be shared under a confidentiality agreement with Gentronix so as not to prejudice future publications of original data. It is estimated that in excess of 5000 compounds have been tested so far by customers or collaborators of Gentronix.

GreenScreen GC has been 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.

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.