Mutagenesis Advance Access originally published online on November 15, 2005
Mutagenesis 2005 20(6):449-454; doi:10.1093/mutage/gei062
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
An assessment of the utility of the yeast GreenScreen assay in pharmaceutical screening
1Faculty of Life Sciences, University of Manchester, Jackson's Mill, Manchester M60 1QD, UK, 2Johnson&Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, Department of ADME/TOX, B-2340 Beerse, Belgium and 3Genetics Department, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
In this paper we describe an initial reproducibility study of 12 proprietary compounds followed by the assessment of 51 marketed pharmaceuticals and, lastly, a summary of the data so far from 2698 proprietary compounds from the Johnson & Johnson (J&J) compound library, in the yeast GreenScreen® assay (GSA). In this assay, a reporter system in the yeast cells employs the DNA damage inducible promoter of the RAD54 gene, fused to the extremely stable green fluorescent protein (GFP). The assay proved to be very robust, the Excel templates provided by Gentronix with the assay interfaced well with in-house J&J systems with little adaptation, the assay was very rapid to perform and used very little compound. The results confirm previous work which suggests that the yeast GSA detects different classes of genotoxic compounds to the Ames assay and as a result can help screen out important genotoxic compounds at the pre-regulatory test phase that are missed by Ames-test-based screens alone. A combination of SAR evaluation of genotoxicity plus an Ames-test-based screen and the GSA provides a powerful pre-regulatory test battery to aid in the selection of successful drug candidates.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Pre-regulatory genotoxicity screening strategies are currently changing because of increased compound throughput and a general move to accumulate genotoxicity data in the early discovery phase of compound development to facilitate compound selection decisions. The ‘cut down’ versions of regulatory tests give a close (but not absolute) match of the results of the corresponding full tests, but compound requirement and total effort means these are not really screening solutions. Alternatively there are microplate-based bacterial screens: reversion assays related to the Ames test (e.g. Ames II), and SOS response reporters [UmuC, (1
,2); Vitotox (3)]. These can be automated and use less compound. The Ames II assay in particular is highly predictive of the full Ames assay (4). The principle drawback of the bacterial tests is their lack of eukaryotic targets and their inability to detect genotoxic events other than mutagenicity, which can be detected by mammalian cell assays. It is desirable therefore to identify new screening tests that reduce the number of genotoxic compounds that are not detected in bacterial tests but subsequently test positive in more costly mammalian cell tests.
Several publications have described the development and procedures for a yeast-based genotoxicity test system in which the activity of the repair system is assessed (5–8). A reporter system in the yeast cells combines the DNA damage inducible promoter of the RAD54 gene, fused to the extremely stable green fluorescent protein (GFP). Induction of the RAD54 promoter due to DNA damage results in increasingly fluorescent cells. The assay is called the GreenScreen GC assay (GSA). Recently, Cahill et al. (9) reported results from assays of 102 compounds in a screening validation study. The study demonstrated that GSA detects a different spectrum of genotoxins to bacterial genotoxicity assays and it was suggested that together with an in silico structure activity relationship (SAR) screen, and a high throughput bacterial screen, GSA would provide an effective preview of the regulatory battery of genotoxicity tests. Compounds were chosen for that validation study to cover different types of DNA damaging agents, and as such the majority of the compounds tested had positive data from at least one of the regulatory tests. Since the majority of the test compounds were not pharmaceuticals, data from the standard regulatory tests with the selected compounds were rarely complete.
To evaluate the decision making potential of the yeast-based assay in screening, we undertook four different studies. In an initial study, 12 coded proprietary compounds were tested in the Gentronix laboratories in Manchester. In the second study these compounds were retested by J&J's Beerse laboratories, along with five further coded proprietary compounds. Eleven non-proprietary compounds for which Gentronix screens had provided data were also tested in Beerse to ensure reproducibility of data between different laboratories using different robotic liquid handling systems. In the third study 51 coded compounds (all marketed pharmaceuticals from various companies) were tested in Beerse. In the final ongoing study at Beerse, 2698 proprietary pharmaceutical compounds have so far been tested in a long-term assessment of the potential of GSA in pre-regulatory screening. Whilst it is not generally acceptable to publish toxicology studies in which the identities of the compounds are withheld (10) the volume of this data makes a significant contribution to assessment of the utility of the GSA.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Strains and plasmids
The yeast strains, plasmids and growth media (F1) used in this study have been described previously (5
,6). The Saccharomyces cerevisiae strain FF18984 (MATa, leu2-3,112 ura3-52 lys2-1 his7-1) was obtained from Francis Fabre (French Atomic Energy Commission,CEA, Fontenay-aux-Roses, France). The strain has been neither modified to increase permeability nor sensitized to DNA damage by mutations.
The reporter strain (GenT01) is FF18984 containing a nuclear, episomally replicating, multiple copy plasmid bearing the entire upstream non-coding DNA sequence of the S.cerevisiae RAD54 gene, fused to a yeast codon-optimized derivative of the Aequorea victoria (jellyfish) GFP gene (11). The control strain (GenC01) is FF18984 containing an identical plasmid except that two base pairs have been removed at the start of the GFP gene, such that no GFP is made. The methods summarized below were precisely as described in Cahill et al. (9), except that compounds were tested at a final concentration of 2% DMSO (c.f. 1% DMSO in the Cahill study).
GreenScreen microplate preparation
Assays were carried out in 96-well, black, clear-bottomed microplates (Matrix ScreenMates, Cat. No. 4929, Apogent Discoveries, USA). The assays were performed using a liquid handling robot (MicroLabS single probe, Hamilton GB Ltd., Birmingham, UK or Genesis 8-probe robot, Benelux, Belgium) in a protocol designed to test up to four compounds on a single 96-well microplate. The 8-probe robot completes microplate filling in <5 min. Microplates can also be filled rapidly and effectively using a multi-channel pipette.
Compounds were first dissolved in 100% DMSO to produce 400 µl of stock standard at 25 mg/ml. This volume was sufficient for concurrent Ames II and Vitotox testing with some material left over for re-testing if necessary. Initially 40 µl of the stock standard were used for the GSA. 40 µl were added to 960 µl of water to produce a standard at 1 mg/ml in 4% DMSO. The standard 1 mg/ml stock was used to make two identical dilution series across the microplate and a compound ‘control’. To achieve this, 150 µl of the test chemical solution were put into two wells in the first column of a microplate. Each sample was serially diluted by transferring 75 µl into 75 µl of 4% DMSO, mixing, and then taking 75 µl out and into the next well. This produced two rows, each consisting of nine dilutions of 75 µl/well across the microplate (the extra 75 µl from Column 9 were discarded).
Compounds which proved to be excessively cytotoxic to yeast or particularly insoluble at this concentration were re-tested after further diluting the stock standard with DMSO, to produce a concentration in the sub-cytotoxic or soluble range respectively.
Controls were added as follows:
- Compound alone, to provide information on the compound's absorbance/fluorescence.
- Yeast cultures diluted with 4% DMSO alone, to give a measure of maximum proliferative potential.
- MMS as a genotoxicity control: ‘high’ = 0.00125% v/v; ‘low’ = 0.0001875% v/v.
- Methanol as a cytotoxicity control: ‘high’ = 3.5% v/v; ‘low’ = 1.5% v/v.
- Growth medium alone, to confirm sterility/lack of contamination
). 75 µl of the yeast suspension were added to each well of the diluted chemical: GenT01 to one series and GenC01 to the second series of each compound, and to appropriate standards and controls (i.e. GenT01 to MMS containing wells and GenC01 to methanol containing wells). After the plates were filled, they were sealed using a gas permeable membrane (Breath-easy, Diversified Biotech, USA) and then incubated without shaking, overnight (16–20 h) at 25°C. This protocol produced compound dilutions in a final concentration of 2% DMSO (top compound test concentration of 500 µg/ml) in contrast to the 1% used in the Cahill et al. (9) study.
Ames II
The manufacturer's protocol (Endotell AG, Switzerland) was followed in Beerse with the following modifications. Cells were exposed to sample compound for 90 min without shaking. Each compound was tested at eight concentrations and 96 wells of a 384-well plate were used for each compound concentration. The top concentration tested for each compound was 500 µg/ml.
DEREK
DEREK for Windows version 7.0 was used in Beerse to make predictions of mutagenicity in Salmonella.
Compounds chosen for the studies
In the preliminary study, 12 coded proprietary pharmaceutical compounds were tested in the Gentronix laboratories in Manchester, UK (Table I, compounds A–L). They were chosen by the Beerse laboratory because some had complementary data in bacterial and mammalian cell assays. An additional five proprietary compounds were tested with these original 12 in the Beerse laboratories (Table I, compounds M–Q). These compounds were also tested in Beerse using the Vitotox test, an SOS bioluminescence assay in Salmonella typhimurium from Thermo Labsystems. Eleven commercially available compounds previously tested by Gentronix in Manchester were also tested in Beerse, to compare results from the different robotic liquid handling systems, and to assess reproducibility at the different sites.
|
Snyder and Green (12
) reviewed the genotoxicity of marketed pharmaceuticals and for the present study 51 compounds were sourced from their review of 467 drugs (Table II). The principal criteria for selection were that the compounds should be readily available and have data (positive or negative) from the Ames test and at least one of the mammalian tests in the standard battery.
|
In the fourth part of this study, an additional 2698 proprietary compounds (to date) have been assessed at Beerse using the GSA alongside J&J's routine, pre-regulatory Ames II screen. Validation studies invariably concentrate on a relatively small group of diverse non-proprietary compounds, where regulatory test data is known. In contrast to this, proprietary collections do not reflect the ‘chemical universe’: there are groups of chemicals that are all almost identical and large areas of chemical space not represented at all. Since most genotoxicity testing is done with proprietary compounds it is clearly of interest to learn how screening tests perform in this environment. Pre-regulatory screening proceeds in parallel with other screening activities that greatly reduce the collection to a small number of leads for development. Thus, data for regulatory genotoxicity tests is only available for a relatively small number of compounds from the screen.
Data collection and handling
Following overnight incubation, GFP reporter fluorescence and yeast culture absorbance data were collected from the microplates using a Tecan Ultra-384 microplate reader (Tecan, Benelux, Belgium): excitation 485 nm/emission, 535 nm with an additional dichroic mirror (reflectance 320–500 nm, transmission 520–800 nm). Absorbance was measured through a 620 nm filter. The data were transported into a Microsoft Excel data analysis template, and converted to graphical format (see typical data from the GSA, annotated in Figure 1). Absorbance data give an indication of reduction in proliferative potential and these data were normalized to the untreated control (=100% growth). Fluorescence data were divided by absorbance data to give ‘brightness units’, the measure of average GFP induction per cell. These data were normalized to the untreated control (=1). In order to correct for induced cellular autofluorescence and intrinsic compound fluorescence, the brightness values for the GenC01 strain were subtracted from those of GenT01. This makes visual assessment of the data more reliable.
|
Positive genotoxicity results were assessed according to Cahill et al. (9
) and reflect a statistically significant increase in brightness compared to the untreated control. It is set at 1.3 (i.e. a 30% increase) and this is >3 times the standard deviation of the background. The LEC is the lowest test compound concentration that produces a relative GFP induction >1.3 threshold.
Comparisons
Published results from other genotoxicity tests and cancer studies with the marketed pharmaceuticals have been tabulated (Table II) and discussed in Appendix I (included in supplementary on-line data). They include the Ames test, mouse lymphoma assay (MLA), in vitro and in vivo cytogenetics (chromosome aberration assays), rodent micronucleus test (MNT), mammalian mutagenesis (CHO cell or lung V79 hypoxanthine-guanine phosphoribosyl transferase HGPRT) and rodent carcinogenicity. The data have been collected both from the Snyder and Green (12) paper as well as the peer-reviewed literature, the US 2004 Physicians Desk Reference (13) and freely available internet resources. These include CCRIS (Chemical Carcinogenesis Research Information System: http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CCRIS), NTP (National Toxicology Program Reports: http://ntp-server.niehs.nih.gov), IARC (International Agency for Research on Cancer: http://www.iarc.fr), NIOHS (National Institute for Occupational Health and Safety: http://www.cdc.gov/niosh/homepage.html), the USEPA (Environmental Protection Agency: http://www.epa.gov/iris/search.htm) and data sheets from individual pharmaceutical companies. Chromosome aberration test data (CHL) were largely drawn from Ishidate et al. (14). Specificity was calculated as the percentage of negative GSA results for compounds which were clearly negative carcinogens or non- genotoxic carcinogens (correct negative calls).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Preliminary study of 12 J&J proprietary compounds by Gentronix in Manchester
The 12 coded compounds were tested by Gentronix as described. Results are presented in Table I (Compounds A–L). In this collection of compounds with limited mammalian test data, GSA positives are more likely to be positive in mammalian cell tests than the bacterial tests. Six of the compounds tested positive in GSA, of which three were also positive (Compounds C, D and F) in the in vivo MNT but negative in Ames, Ames II and the Vitotox assay. For the three remaining GSA positives, two were also positive in bacterial tests (Compounds G and J), but had no mammalian cell data, and one was negative in bacterial tests and mammalian cell assays (Compound K). In contrast, three further compounds that were positive or equivocal in the bacterial assays were negative in GSA and mammalian assays (Compounds B, E and M).
Further testing of J&J proprietary compounds by J&J in Beerse, and comparison of robotic liquid handling systems
The 12 J&J proprietary compounds above (Table I: A–L) plus 5 additional J&J proprietary compounds (Table I: Compounds M–Q) were tested in Beerse. The GSA gave the same results in Beerse as it did in Manchester for the first 12 compounds (A–L). Of the five new compounds, one (Compound M) was positive in all three bacterial tests but negative in GSA and mammalian tests. The other four were negative in all tests. A further 11 commercially available compounds (for which GSA data had already been collected in Manchester), were also tested on different days, using either the Hamilton robot, the Tecan robot or both. The two robots produced the same results as each other for compounds tested on different days, and the compound results reproduced those collected in Manchester (data not shown). Together these results underlined the robustness of the assay in that the results with the same compounds at the same concentrations were comparable between the two laboratories and similar conclusions were reached.
For the bacterial assays, Ames II and full GLP Ames were in agreement for 13 out of 13 compounds, Vitotox was in agreement with Ames II for 14 out of 17 compounds. In this small collection, the GSA is useful in the identification of compounds which are negative in bacterial tests, but positive in mammalian cell tests. An interesting observation was that Compound D (positive) is identical to Compound Q (negative) except that they are optical isomers.
Study of 51 marketed pharmaceuticals
Synder and Green (12) have reviewed data on 467 marketed pharmaceuticals. Two hundred and six compounds had data from Ames tests and at least two mammalian cell tests, and of these 51 were available for study. The test results on these compounds from Snyder and Green (12) have been updated and are tabulated with the GreenScreen results in Table II. A discussion of each compound and an overall assessment of their genotoxicity and carcinogenicity (where data is available) are given in Appendix I (ibid). What follows is a summary.
These pharmaceuticals are all useful drugs in widespread use, and there is clear consensus on the positive benefit to risk ratio. Not surprisingly, and in contrast to the compound collection assessed in the screening validation paper where prevalence of genotoxins was very high, the prevalence of genotoxins, genotoxic carcinogens and positives in GSA is very low in the data for the pharmaceuticals presented in this work. As a consequence it is not meaningful to extract sensitivity data from the table. Low prevalence gives greater robustness to the specificity calculation. There are 35 compounds which are clearly negative carcinogens or non-genotoxic carcinogens. Of these 32 are negative in GSA, giving a specificity of 91% (32 out of 35) which rises to 94% (32 out of 34) if the antifungal (Itraconazole) is excluded.
Of the seven drugs that tested positive in GSA, four are also positive in cytogenetic assays (see below). Only one is positive with Ames (Clomiphene). It also causes DNA breakage in various cell types (see appendix) and at least one report of MNT in vitro. Of the remaining six, Itraconazole and Ciclopirox are antifungals and whilst data from such compounds are more difficult to interpret, in the same way as antibiotics present problems to the Ames test, Ciclopirox has a plausible mechanism for genotoxicity in yeast cells (see appendix). It is also positive for in vitro chromosome aberration. Lansoprazole and Pyrimethamine (not antifungals) are positive with in vitro chromosome aberration studies; the latter is also positive with in vivo MNT and MLA. Sertraline, although carcinogenic is negative in other genotoxicity assays. It is likely that the tumours seen are due to non-genotoxic mechanisms. Azelastine is negative in all other studies.
Of the 51 pharmaceuticals tested, 44 (86%) were negative in GSA. This is a very much higher proportion than that in the 2004 screening validation paper of Cahill et al. (9) (47 negatives out of 102; 46%). Reassuringly for a collection of pharmaceuticals, 15 (marked superscript 1 in Table II) of the 51 compounds have only negative data available for genotoxicity, GSA and carcinogenicity, where available. There are a further 17 compounds (marked superscript 2 in Table II) that are negative in GSA and that have positive carcinogenicity, tumorigenicity or benign polyp formation data. For this group there is plausible data to support the view that the mechanism of carcinogenesis is non-genotoxic and they are not regarded as a risk to patients (see Appendix I in supplementary on-line data). These are useful drugs and it would be unhelpful if a screening assay had produced false positive results. There are five GSA negative compounds (marked superscript 3 in Table II) that have positive carcinogenicity data and the mechanism of action is probably genotoxic. The seven remaining compounds (marked superscript 4 in Table II) are negative in GSA with no carcinogenicity data, and have negative or sporadic positive genotoxicity data.
To determine how representative this subgroup of pharmaceuticals was, a simple comparison was made between the numbers of each regulatory test performed within the group of 51 and the Snyder collection as a whole (Table III). This subgroup has a slightly higher proportion of compounds with at least one positive result from the standard battery (43.1% compared with 28.7% in the larger set of 352 compounds), and this is mainly accounted for by a higher proportion of Ames positive and in vitro cytogenetics positive compounds. This is however a much lower proportion than that in the data from 102 compounds previously published in the validation study (9) in which 80% had positive data from at least one of the standard battery of tests. This is important as the prevalence of positive compounds in a test data set has a profound impact on the usefulness of the calculated ‘predictivity’ of an assay, especially where compound data sets contain a disproportionally high number of compounds with positive data (15). A discussion of each compound and an overall assessment of their genotoxicity and carcinogenicity (where data is available) is given in Appendix I.
|
Study of 2698 J&J compounds
J&J put 2698 potential drug candidates through the GSA during pre-regulatory screening. 2351 have also been tested with Ames II. Of the 2351, 164 (7%) were positive in Ames II, with and/or without S9 metabolic activation and 176 (7.5%) were positive in the GSA. Twelve (7%) of the 176 GSA positives were positive in Ames II. This underlines the fact that the Ames II and GSA assays each detect a different but overlapping spectrum of genotoxins, reflecting both the differences between prokaryotic and eukaryotic test organisms and their different endpoints (mutation and DNA damage-induced transcription respectively). As part of the company's policy in compound selection, compounds with Ames II positive data were rejected. The archived GreenScreen data was referenced when later regulatory tests on the Ames II negative compounds gave positive regulatory genotoxicity data. Ten such compounds emerged that gave positive results in the mouse lymphoma assay and/or the in vivo micronucleus assay. Of these, six were positive in GSA. No in vivo micronucleus assay negatives were positive in GSA. These results underline how the GSA can be used to help prevent important genotoxic drug candidates from persisting in development using valuable resources that could be used to develop compounds more likely to be successful drug candidates.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Pharmaceutical companies now produce a surfeit of research compounds for efficacy testing through techniques such as combinatorial chemistry. Once screening for activity has taken place a large number of ‘hits’, e.g. binding to a relevant pharmacological receptor, can be produced, which can be taken forward as ‘leads’ worthy for consideration as candidate drugs. It is then necessary to know if these hits possess properties other than potential efficacy that deem them worthy as potential development candidates. These properties include various pharmaceutical properties, metabolic stability, permeability, solubility, potential for drug–drug interactions, ease of manufacture and so on, i.e. drug-like properties. Screens are carried out to further filter out undesirable compounds based on such properties, with the intention that when a compound is selected for major regulatory development studies the chances of success are increased. Positive genetic toxicity findings in regulatory tests can often be enough to terminate development. If such results are found when major regulatory pre-clinical development studies are in progress, this can mean that a severe loss of time and money will have occurred by this point. Apart from these immediate losses, development of other promising and ultimately successful drugs will have been delayed. Thus there is a major need for accurate pre-regulatory screens that can help filter out genotoxins at an early stage when investment is relatively negligible for any one compound.
Taking these results in conjunction with the earlier Cahill et al. (9) study, it is interesting to note how profoundly chemical space can influence the results from different screening systems. Contrast, for example, the 12 Ames II positives amongst 176 GSA positives (7%) in this study of 2351 proprietary compounds with the 32 Ames positives amongst 50 GSA positives (64%) in the Cahill study of non-proprietary compounds. This serves as a salutary reminder that the evaluation of new tests by focussing on studies of well-known compounds does not necessarily help in the assessment of utility in collections of novel chemicals. This chemical space difference is also reflected in the data from a subset of 1487 of the 2698 compounds that were also tested with the (prokaryotic) Vitotox assay. In contrast to the high concordance with the Ames test in other studies (e.g. 16), in this study the Vitotox test was less successful in identifying Ames positives. Of 88 Ames II positive compounds, just six were Vitotox positive.
The results presented in this paper of known and candidate drugs, extend the previously published validation study of a wide selection of test compounds (mainly reference genotoxins and non-pharmaceuticals) to support the use of the GSA, in conjunction with in silico SAR and Ames-based bacterial screens, to help ensure the best chance of a particular compound giving negative results in regulatory genotoxicity tests. Of particular value is the demonstration that the GSA can be used in relatively high throughput screening of an actual pharmaceutical library of compounds, to detect compounds that are likely to be genotoxic in mammalian cells but missed by Ames II or other bacteria-based screens.
|
Supplementary material |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
Supplementary material is available at Mutagenesis online.
|
Acknowledgments |
|---|
Conflict of Interest Statement: R. M. Walmsley is Founder and Chief Scientific Officer, D. J. Tweats is a Consultant and P. A. Cahill, N. Billinton and A. Knight are all employed by Gentronix Ltd, the remaining authors have no conflicts to declare.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +44 161 306 4174; Fax: +44 161 236 0409; Email: richard.walmsley{at}manchester.ac.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
|---|
-
1. Reifferscheid,G., Heil,J., Oda,Y. and Zahn,R.K. (1991) A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples. Mutat. Res., 253, 215–222.[CrossRef][Web of Science][Medline]
2. Yasunaga,K., Kiyonari,A., Oikawa,T., Naoki Abe,N. and Yoshikawa,K. (2004) Evaluation of the Salmonella umu test With 83 NTP Chemicals. Environ. Mol. Mutagen., 44, 329–345.[CrossRef][Web of Science][Medline]
3. van der Lelie,D., Regniers,L., Borremans,B., Provoost,A. and Verschaeve,L. (1997) The VITOTOX® test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics. Mutat. Res., 389, 279–290.[Web of Science][Medline]
4. Flückiger-Isler,S., Baumeister,M., Braun,K., Gervais,V., Hasler-Nguyen,N., Reimann,R., Van Gompel,J., Wunderlich,H.-G. and Engelhardt,G. (2004) Assessment of the performance of the Ames IITM assay: a collaborative study with 19 coded compounds. Mutat. Res., 558, 181–197.[Web of Science][Medline]
5. Walmsley,R.M., Billinton,N. and Heyer,W.-D. (1997) Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 from Saccharomyces cerevisiae. Yeast, 13, 1535–1545.[CrossRef][Web of Science][Medline]
6. Billinton,N., Barker,M.G., Michel,C.E., Knight,A.W., Heyer,W.-D., Goddard,N.J., Fielden,P.R. and Walmsley,R.M. (1998) Development of a green fluorescent protein reporter for a yeast genotoxicity biosensor. Biosensors and Bioelectronics, 13, 831–838.[CrossRef][Web of Science][Medline]
7. Knight,A.W., Goddard,N.J., Fielden,P.R., Gregson,A.L., Billinton,N., Barker,M.G. and Walmsley,R.M. (2000) The application of fluorescence polarization for the enhanced detection of green fluorescent protein (GFP) in the presence of cellular auto-fluorescence and other green fluorescent compounds. The Analyst, 125, 499–506.[CrossRef]
8. Afanassiev,V., Sefton,M., Anantachaiyong,T., Barker,M.G., Walmsley,R.M. and Wölfl,S. (2000) Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances. Mutat. Res. Genet. Toxicol. Environ. Mutagenesis, 464, 297–308.[CrossRef]
9. Cahill,P.A., Knight,A.W., Billinton,N., Barker,M.G., Walsh,L., Keenan,P.O., Williams,C.V., Tweats,D.J. and Walmsley,R.M. (2004) The GreenScreen® genotoxicity assay: a screening validation programme. Mutagenesis, 19, 105–119.
10. Phillips,D.H., (2004) The pros and cons of publishing genetic toxicology studies in which the identities of the compounds are withheld. Mutagenesis, 19, 1.
11. Cormack,B.P., Bertram,G., Egerton,M., Gow,N.A.R., Falkow,S. and Brown,A.J.P. (1997) Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology, 143, 303–311.
12. Snyder,R.D. and Green,J.W.(2001) A review of the genotoxicity of marketed pharmaceuticals. Mutat. Res., 488,151–169.[CrossRef][Web of Science][Medline]
13. Physicians' Desk Reference 2004, Thomson PDR, Montvale, NJ, USA.
14. Ishidate,M. Jr, Harnois,M.C. and Sofuni,T. (1988) A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures. Mutat. Res. 195, 151–213.[Web of Science][Medline]
15. Cooper,J.A., Saracci,R. and Cole,P. (1979) Describing the validity of carcinogen screening tests. Br. J. Cancer, 39, 87–89.[Web of Science][Medline]
Received on July 18, 2005; revised on October 10, 2005; accepted on October 13, 2005.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. W. Knight, L. Birrell, and R. M. Walmsley Development and Validation of a Higher Throughput Screening Approach to Genotoxicity Testing Using the GADD45a-GFP GreenScreen HC Assay J Biomol Screen, January 1, 2009; 14(1): 16 - 30. [Abstract] [PDF]
|
|
|
|
 |
|
 A. W. Knight, N. Billinton, P. A. Cahill, A. Scott, J. S. Harvey, K. J. Roberts, D. J. Tweats, P. O. Keenan, and R. M. Walmsley An analysis of results from 305 compounds tested with the yeast RAD54-GFP genotoxicity assay (GreenScreen GC) including relative predictivity of regulatory tests and rodent carcinogenesis and performance with autofluorescent and coloured compounds Mutagenesis, November 1, 2007; 22(6): 409 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Jacobson-Kram and J. F. Contrera Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part I: Early Screening for Potential Human Mutagens Toxicol. Sci., March 1, 2007; 96(1): 16 - 20. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||