TY - JOUR
AU - Walmsley, R.M.
AB - Abstract The yeast (Saccharomyces cerevisiae) RAD54-GFP DNA repair reporter assay (GreenScreen® assay, GSA) can be used for early genotoxicity screening in drug discovery. During the initial validation of this preregulatory assay, a subset of known genotoxic compounds that did not give reproducibly clear positive GSA results was identified. Cell permeability, inherent drug resistance mechanisms, metabolic activation and compound solubility were identified as possible barriers to the detection of specific compounds. In this study three types of modification to the existing assay protocol were explored in order to address these possibilities: (i) modification of the reporter host strain by deletion of genes involved in cell wall integrity or with products functioning as efflux pumps (PDR5, ERG6, SNQ2, YOR1); (ii) expression in the host yeast of human phase I metabolic activation genes and (iii) variation in the test solvent system for compounds with poor aqueous solubility. The modifications described and the assay results presented show how the assay may be tailored to suit specific classes of test compound in a more analytical mode. Improvements in assay sensitivity were seen in the detection of some genotoxins using yeast cell wall mutants and those expressing human cytochrome P450 genes. Introduction The authors have previously reported the construction and assessment of a yeast-based genotoxicity assay system (GreenScreen Assay or GSA) utilizing a DNA damage repair reporter (1). In the reporter strain, there is a replicative plasmid containing the promoter of the DNA damage inducible yeast RAD54 gene fused to a gene encoding green fluorescent protein (GFP; 2–5). The principle behind the GSA is that induction of the RAD54 promoter, owing to DNA damage, results in the production of the extremely stable GFP by the yeast cells, which is readily detected by its fluorescence when illuminated with blue light. Following overnight exposure of the yeast culture to a test substance, both fluorescence and light absorbance measurements are recorded. The level of GFP fluorescence induction gives a measure of the genotoxicity of the substance. The extent of growth inhibition resulting from exposure to the test substance (relative total growth, RTG), is also determined by comparing the extent of proliferation of treated cells with that of untreated cells. This provides a measure of cytotoxicity. The assay is performed on a series of dilutions of the compound, allowing calculation of the lowest effective concentration (LEC) for each toxicity measurement. RAD54 encodes a structural element of the homologous recombinational repair pathway and the Rad54 protein exhibits a double-stranded DNA-dependent ATPase activity that facilitates recombinational repair, mediated by the homologous pairing and DNA strand exchange protein Rad51 (6–9). The entire genome is the target for DNA damage whereas the whole cell is the target for toxicity. This contrasts with reverse mutation assays which detect DNA damage at a genetic locus, such as the Salmonella HIS operon in the Ames test (10). The GSA has been developed for use in 96-well microplate format. The full regulatory battery of genotoxicity tests (11) comprising in vitro and in vivo tests for the evaluation of mutations and chromosomal damage are very low throughput, time consuming and compound hungry. As a consequence, the testing is not done early in discovery; it would be too expensive for large numbers of compounds. The speed, high throughput and low compound requirement of GSA made it an attractive screening tool earlier in drug discovery. To assess its value, a preliminary screening validation study of the assay was carried out using a panel of 102 compounds (1). This study demonstrated that the GSA detects a different but overlapping spectrum of compounds to bacterial genotoxicity assays. It was concluded that the combination of GSA with an in silico structure–activity relationship (SAR) screen and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests and thus make a valuable contribution to the early selection of compounds going forward in drug discovery. The original validation study (1) set out to discover the range of compounds detected as genotoxic with a single definitive protocol. This was an important criterion, as screening tests per se cease to be screening tests if they have to be run in different, compound dependent formats. All compounds were tested in a phosphate-buffered medium containing 1% DMSO. The assays were carried out in the absence of exogenous metabolic activation (i.e. without the addition of rat liver homogenate S9 fraction). Under these set conditions, the screening validation study demonstrated that the assay was sensitive to a broad spectrum of mutagens and notably the clastogens. As expected, a number of typical eukaryotic drug targets were identified including topoisomerases (etoposide), the mitotic spindle apparatus (colchicine) and DNA polymerases (aphidicolin). Furthermore, it showed that the metabolic capacity of yeast cells is sufficient to identify several compounds that require metabolic activation by rat liver S9 in bacterial genotoxicity assays (e.g. neutral red, 2-amino-4-nitrophenol, proflavin hemisulfate). The existence of more complex metabolic activation in yeast was suggested by the detection of cimetidine [that most likely acts through conversion to nitrosocimetidine (12,13) by the yeast cells], safrole, urethane and thiourea, (carcinogens not detected by the Ames test, even with the addition of S9 fraction). Within the published validation set (1) there was a subset of compounds, positive in one or more of the regulatory genotoxicity test battery, that either gave reproducible negative results or poorly reproducible positive GSA results. It is these compounds that are considered in the present paper. They fall broadly into two categories, compounds where transport in or out of the cell could be a limiting factor and compounds that require specific elements of mammalian metabolism (lacking in yeast cells) in order to be detected as genotoxic. For the first category of compounds, it is the cell wall, together with the outer membrane and associated proteins, which presents a potential barrier to influx and efflux of chemical compounds. Molecular size is often cited as an issue for yeast because of its robust cell wall. If we consider Lipinski's ‘rule of 5’, a drug-like compound typically has a molecular mass <500 (14). Amongst the positive genotoxins in the validation set, bleomycin (MW 1400), etoposide (MW 589), streptonigrin (MW 506) and crystal violet (MW 408) are all bulky molecules; so it is clearly simplistic to dismiss the yeast cell wall as too fine a physical sieve. However, it is possible that size was a contributory factor to problems with the detection of the genotoxicity of aphidicolin (MW 339) and camptothecin (MW 348). Nevertheless, the properties of the impinging molecule are not the only possible restricting factors in cellular access. It is also necessary to consider features of the yeast cell wall and membrane. Yeast cells have a typically eukaryotic inner cell membrane comprising a protein rich lipid bilayer. This membrane is protected by a complex outer cell wall that consists of almost equal amounts of β-glucans and mannoproteins (mannan). Mannoproteins are present on the outer surface of the cell wall, with glucans on the inner surface. A third component, chitin, represents <1% of the total mass of the wall (15). Ergosterol is an essential component of the yeast cell membrane. Just like cholesterol, found in mammalian cell membranes, ergosterol affects membrane fluidity and permeability (16). Yeast cells appear to be resistant to the toxicity of more drugs than higher eukaryotic cells and this reflects the contrast between the protected homeostasis of the metazoan and the free-living habitat of the protists. Aside from the additional physical barrier provided by the cell wall, yeasts otherwise share with higher eukaryotes a dynamic biochemical defence against xenobiotics, mediated by a network of ATP-binding cassette (ABC) transporter proteins. These promote active efflux of a wide range of drugs and other compounds. In the yeast genome, 22 genes have been identified with putative ABC transporter activity (17). The three major ABC transporter genes in yeast are PDR5 (pleiotropic drug resistance), YOR1 (yeast oligomycin resistance, a multispecific organic anion transporter) and SNQ2 (sensitivity to 4-nitroquinoline-N-oxide, resistance to singlet O2 generating photosensitizers). PDR5, YOR1 and SNQ2, have overlapping functions and substrate specificities; if each gene is deleted individually, the remaining two will compensate. If all three genes are deleted, drug resistance is impaired. Drug resistance in yeast has been reviewed previously (17,18). For the second category of compounds, the limiting factor to the detection of genotoxicity was drug metabolism. In the mammalian liver, this can be divided into phase I and phase II effects. During phase I, functional groups are added to the drug, or existing functional groups are transformed through oxidation, reduction, hydroxylation, hydrolysis and so on. Phase II metabolism or conjugation involves the masking of functional groups by modifications, such as acetylation, glycosylation and glutathione conjugation. Together, the two phases increase drug hydrophilicity to aid elimination by renal excretion. Both phases are enzymatic with phase II involving enzyme classes, such as N-acetyl transferases, glutathione S-transferases and sulphotransferases, and the major enzyme family responsible for phase I activation being the cytochrome P450s (CYPs) (19). Yeast has at least three functional CYP type enzymes (20–22), and the binding of various chemical carcinogens, including benzo[a]pyrene and cyclophosphamide to yeast cytochrome P450 has been demonstrated in vitro (23). However, the range of CYP enzymes expressed by yeast provides somewhat limited substrate specificity relative to the promiscuous collection expressed in humans. An obvious group of genotoxins with poor detection identified in the screening validation assay (1) was the primary aromatic amines and amides. Nine such compounds were tested. Three were positive genotoxins in GSA: 2-amino-4-nitrophenol and 1-naphthylamine which require S9 in the Ames assay and 9-aminoacridine which is direct acting. The remaining six were negative in GSA and positive in other in vitro genotoxicity tests: 2-acetamidofluorene, 2-aminoanthracene, 4,4-oxydianiline and o-anisidine which require S9 in the Ames test, and aniline and 4-aminophenol, which are negative with and without S9 in the Ames test. Most probably, cytochrome P450 1A2 is the missing activity for some of these, though there might be more than one explanation for those also negative with S9 in the Ames test. None of the six GSA negatives amongst the aromatic amides are particularly bulky compounds. It is worth noting that the aromatic amine motif is well known and as a consequence, it is recognized by in silico SAR programmes. Other compounds clearly missed as genotoxic in GSA but detected in tests with S9 addition were cyclophosphamide and aflatoxin B1, reflecting the incomplete phase I metabolism of yeast compared with that of mammalian cells. Yeast possesses effective Phase II antioxidants and there is experimental evidence demonstrating the reduction of cumene hydroperoxide by glutaredoxins in yeast (24) that most probably accounts for failure to detect genotoxicity of this and other oxidative compounds. A similar explanation might account for the failure to detect the (disputed) genotoxicity of hydroquinone. It would be possible to reduce the glutathione pools by genetic modification, but this would result in a more general sensitization to oxidative damage. Low glutathione pools, resulting from a naturally occurring mutant GSH1 allele, have been shown to have pleiotropic results on the analysis of DNA repair mutants and drug sensitivity (25). This acts as a reminder that it is expedient when considering drug metabolism in yeast, as well as in other systems, to remember that low phase I activity might be as similar as high phase II activity to provide an explanation for conflicts between results from different tests (e.g. whole animals carcinogenicity studies, animal cell tests, cells with or without S9 fraction, etc.). In the previously published validation study (1), the yeast strain used to perform the genotoxicity and cytotoxicity assessments did not carry any deliberately introduced mutations (other than those providing auxotrophic markers used for identification and laboratory manipulation purposes). This paper describes the application of a variety of genetic strategies to reduce the cell's capacity for drug resistance. A collection of yeast strains was constructed in which single or multiple genes required for cell wall integrity and/or multi-drug resistance were deleted. The genes targeted were ERG6, which encodes a sterol methyltransferase involved in the later stages of ergosterol biosynthesis, and the three multi-drug resistance ABC transporters PDR5, YOR1 and SNQ2. Data are also presented for experiments with compounds that only give positive genotoxicity data with the Ames test in the presence of metabolic activation. In this study, these compounds were tested in strains engineered to express the human cytochrome P450 enzymes 1A2 (narrow specificity) and 3A4 (broad specificity) in order to increase the metabolic capacity of the yeast cells [see (19) for relative proportions of drugs metabolized by CYP1A2 and CYP3A4]. CYP expression was used in preference to addition of exogenous rat liver S9 extract, since the DNA damage reporter system used relies on expression GFP, the detection of which is not compatible with presence of S9 (data not shown). In all these experiments, we have primarily sought to look for predictable improvements in sensitivity rather than change from negative to positive, although the finding that a change in solvent can make an unexpected change from negative to positive is a reminder that although complex metabolic solutions are compelling, simple matrix effects can be just as relevant. Materials and methods Yeast strains The wild-type Saccharomyces cerevisiae yeast strain and growth medium (F1) used have been described previously (1–3). The wild-type 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]. A full list of strains used in this study is given in Table I. Table I. S.cerevisiae strains used in this study Strain   Genotype   ‘Wild-type’ (FF18984)  (MATa leu2-3, 112, ura3-52, lys2-1, his7-1)  pdr5-Δ  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, pdr5-Δ::loxP  pdr5-Δ/erg6-Δ  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, pdr5-Δ::loxP, erg6-Δ::loxP  pdr5-Δ/yor1-Δ/snq2-Δ  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, pdr5-Δ::loxP, yor1-Δ::loxP, snq2-Δ::loxP-KanMX4-loxP  Int 022A  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, RAD54-GFP, d-leu2::rDNA  Strain   Genotype   ‘Wild-type’ (FF18984)  (MATa leu2-3, 112, ura3-52, lys2-1, his7-1)  pdr5-Δ  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, pdr5-Δ::loxP  pdr5-Δ/erg6-Δ  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, pdr5-Δ::loxP, erg6-Δ::loxP  pdr5-Δ/yor1-Δ/snq2-Δ  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, pdr5-Δ::loxP, yor1-Δ::loxP, snq2-Δ::loxP-KanMX4-loxP  Int 022A  MATa leu2-3, 112, ura3-52, lys2-1, his7-1, RAD54-GFP, d-leu2::rDNA  View Large Construction of cell wall mutant host strains The wild-type yeast (FF18984) was modified to increase permeability, by deletion of genes involved in drug efflux or cell membrane function. These deletion mutants (Table I) were generated by the PCR-mediated short flanking homology, Cre-loxP gene replacement method described by Güldener et al. (26). The plasmid pUG6 (26) was used as a template for synthesis of loxP-KanMX4- loxP disruption cassettes of ∼1600 bp in length. The ‘loxP’ system allows the removal of the KanMX marker after gene deletion, so that further deletions can be made using the KanMX selection system. For gene deletion, yeast cells were transformed with 5–10 µg of disruption cassette DNA using a modified lithium acetate method (26). Verification of the correct disruption of the target gene was performed by analytical PCR on whole yeast colonies (27). Oligonucleotide primers were designed which annealed to the flanking regions of the target gene and to sites internal to the KanMX marker. Transformants selected for further study had PCR products corresponding in length to flanking region of the target gene plus the corresponding fragment of the KanMX4 marker (data not shown). The double deletion mutant pdr5-Δ/erg6-Δ was generated as follows: excision of the KanMX marker from the single pdr5-Δ::loxP-KanMX4-loxP mutant, by the action of Cre-recombinase expressed from plasmid pSH47 (26), followed by a second gene deletion step, targeting the ERG6 locus. The triple mutant, pdr5-Δ/yor1-Δ/snq2-Δ was generated by three successive Cre-loxP targeted gene deletions. At each stage of strain construction, confirmation of deletion of the correct target region was confirmed by analytical PCR, as described above. RAD54 reporter constructs Two RAD54-GFP reporter systems were used in this study, an episomal reporter plasmid (not integrated into the genome) and a chromosomal reporter (sequence integrated into the genome). Episomal RAD54-GFP reporter. The replicative GFP expressing plasmid pGENT01 and out-of-frame control plasmid pGENC01, have been described previously (1,3). They were used to transform the wild-type strain (FF18984), to give strains GenT01 and GenC01. The cell wall/ABC transporter mutants were also transformed with theses plasmids and the resulting strains were used to investigate the effects of cell wall/permeability mutations (PDR5, YOR1, SNQ2, ERG6). The pGEN001 plasmid contains the entire upstream non-coding DNA sequence of the Saccharomyces cerevisiaeRAD54 gene fused to a yeast codon-optimized derivative of the Aequorea victoria (jellyfish) GFP gene (28). The plasmids are maintained during cell growth and division by selection of uracil prototrophy, conferred by the presence on both plasmids of the yeast URA3 marker gene. Integrated RAD54-GFP cassette. In order to avoid competition between different plasmids within the yeast cell, it was decided to integrate the RAD54-GFP reporter construct for studies using the strains expressing human genes from replicative plasmids (see below). In a previous study, integration of the cassette at the HO locus (2) was reported, but signal output was considerably lower than that from the episomal plasmid. To improve expression from the integrated cassette it was targeted to the rDNA array of chromosome XII (P.W. Hastwell and R.M. Walmsley, manuscript in preparation). Studies from other laboratories have reported the successful integration of tens of copies of other genes at this locus (29). This reporter strain (Int022A) was then transformed with a centromeric plasmid bearing either one of the human CYPs 1A2 or 3A4, along with the human NADPH-cytochrome P450 oxidoreductase gene (hOR) (Figures 1 and 2). Fig. 1. View largeDownload slide Plasmid map for pMT3A4-Kan, coding for human CYP3A4 and cytochrome P450 NADPH oxidoreductase (hOR). Fig. 1. View largeDownload slide Plasmid map for pMT3A4-Kan, coding for human CYP3A4 and cytochrome P450 NADPH oxidoreductase (hOR). Fig. 2. View largeDownload slide Plasmid map for pPOP-A, coding for human CYP1A2 and cytochrome P450 NADPH oxidoreductase (hOR). Fig. 2. View largeDownload slide Plasmid map for pPOP-A, coding for human CYP1A2 and cytochrome P450 NADPH oxidoreductase (hOR). Cytochrome P450 yeast expression plasmids Plasmids and cloning vectors used and generated in this study are listed in Table II. Table II. Plasmids used and constructed in this study Plasmid   Description/main features   Size (bp)   Source/reference   pGENT01  Episomal RAD54-GFP reporter plasmid  10 861  Walmsley Lab  pGENC01  Negative control reporter plasmid, as pGENT01, but 2 bp removed (GFP out of frame)  10 859  Walmsley Lab  pRS416  Yeast Centromeric Shuttle Vector  4891  (31)  pUG6  Plasmid containing loxP-KanMX-loxP sequence  4009  (26)  pSH47  Cre-recombinase expression plasmid, GAL promoter control    (26)  pCS513  Plasmid bearing human CYP1A2 and human oxidoreductase, each with GAPDH promoter control  18 271  (30)  pAAH5  Plasmid bearing the human CYP3A4 gene sequence    Prof. Steven Kelly, University of Swansea  pGEM®-T Easy  Cloning vector which allows direct ligation of PCR products  3015  Promega  pMT001  pRS416 with GAPDH promoter/hOR PCR product inserted into Xho1/BamH1 sites  7723  This study  pMT005  pRS416 with GAPDH promoter/hOR PCR product inserted into Xho1/BamH1 sites and GAPDH promoter (440 bp) into Pac1/Asc1 sites  8870  This study  pMT004  pRS416 with GAPDH promoter/hOR PCR product inserted into Xho1/BamH1 sites, GAPDH promoter (440 bp) into Pac1/Asc1 sites and loxP-kanMX-loxP (1600 bp) at Not1 site  10 486  This study  pMT3A4-kan  pMT004 with hCYP3A4 (1829 bp) inserted as a Pac1/Asc1 fragment  11 674  This study  pPOP-A  pRS416 GAPDH-hCYP1A2 and GAPDH hOR (5396 bp) inserted at XbaI site  10 287  This study  Plasmid   Description/main features   Size (bp)   Source/reference   pGENT01  Episomal RAD54-GFP reporter plasmid  10 861  Walmsley Lab  pGENC01  Negative control reporter plasmid, as pGENT01, but 2 bp removed (GFP out of frame)  10 859  Walmsley Lab  pRS416  Yeast Centromeric Shuttle Vector  4891  (31)  pUG6  Plasmid containing loxP-KanMX-loxP sequence  4009  (26)  pSH47  Cre-recombinase expression plasmid, GAL promoter control    (26)  pCS513  Plasmid bearing human CYP1A2 and human oxidoreductase, each with GAPDH promoter control  18 271  (30)  pAAH5  Plasmid bearing the human CYP3A4 gene sequence    Prof. Steven Kelly, University of Swansea  pGEM®-T Easy  Cloning vector which allows direct ligation of PCR products  3015  Promega  pMT001  pRS416 with GAPDH promoter/hOR PCR product inserted into Xho1/BamH1 sites  7723  This study  pMT005  pRS416 with GAPDH promoter/hOR PCR product inserted into Xho1/BamH1 sites and GAPDH promoter (440 bp) into Pac1/Asc1 sites  8870  This study  pMT004  pRS416 with GAPDH promoter/hOR PCR product inserted into Xho1/BamH1 sites, GAPDH promoter (440 bp) into Pac1/Asc1 sites and loxP-kanMX-loxP (1600 bp) at Not1 site  10 486  This study  pMT3A4-kan  pMT004 with hCYP3A4 (1829 bp) inserted as a Pac1/Asc1 fragment  11 674  This study  pPOP-A  pRS416 GAPDH-hCYP1A2 and GAPDH hOR (5396 bp) inserted at XbaI site  10 287  This study  View Large Construction of the CYP3A4 expression plasmid, pMT3A4-Kan The hOR sequence was PCR amplified from plasmid pCS513 [obtained from Dr Christian Sengstag, ETH Zurich, Swiss Federal Institute of Technology, Zurich, (30)]. PCR primers were designed such that a XhoI restriction site was introduced at the 5′ end of the sequence and a BamH I restriction site at the 3′ end. The resulting 2895 bp fragment represented both the coding sequence of the gene and the upstream yeast GAPDH promoter. This PCR product was cloned into the vector pGEM®-T Easy (Promega) and sequenced, followed by ligation into the XhoI and BamH I cut sites of the yeast centromeric shuttle vector pRS416 (31), yielding plasmid pMT001. The GAPDH promoter required to control CYP expression was then introduced into the plasmid as a BamH I/Pac I fragment (after PCR amplification from pCS513). The fragment (440 bp) was cloned into pGEM®-Teasy followed by restriction with BamH I and Pac I, and ligation into pMT001, immediately downstream of the 2895 bp hOR fragment, to give plasmid pMT005. This construct was further modified to introduce the KanMX marker in order to aid future manipulation steps in yeast. To achieve this the loxP-KanMX4-loxP sequence was cut from plasmid pUG6 (26) as a Not I fragment (∼1600 bp) then ligated into the Not I site of pMT005 to give plasmid pMT004. Finally, the hCYP3A4 gene was introduced. CYP3A4 was PCR amplified from plasmid pAAH5 (obtained from Professor Steven Kelly, University of Swansea), incorporating a Pac I restriction site at the 5′ end and an Asc I site at the 3′ end. The resulting 1829 bp PCR product was cloned in to the vector pGEM®-T Easy (Promega) and sequenced. Finally, it was excised from pGEM®T Easy and ligated as a Pac I/Asc I fragment into pMT004, to give plasmid pMT3A4-Kan. Figure 1 shows a diagram of the final plasmid. Construction of the CYP1A2 expression plasmid, pPOP-A Plasmid pCS513 (30) was digested with restriction enzyme Xba I, to yield a 5396 bp fragment, which comprised both the hOR and CYP1A2 genes (each with an upstream yeast GAPDH promoter region). This fragment was ligated into the XbaI cut site of pRS416 (31), yielding plasmid pPOP-A. Figure 2 shows a diagram of the final plasmid. Microplate preparation The standard assay protocol of Cahill et al. (1) was used as described, except where stated. In brief, four compounds were tested simultaneously in 96-well, black, clear-bottomed microplates. Compounds under test were dissolved in 4% DMSO v/v, then serially diluted in microplates to give nine concentrations in volumes of 75 µl. The compounds tested are listed in Table III. Table III. Results of cytotoxicity and genotoxicity assessments for cell wall mutants Compound  Molecular weight (g/mol)  Highest conc. tested (µg/ml)  Assay results                                       Growth inhibition                 Genotoxicity-GFP induction                       WT    pdr5    pdr5/erg6    pdr5/snq2/yor1    WT GSA    pdr5    pdr5erg6    pdr5/snq2/yor1            LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)   Test chemicals                                          Aphidicolin  338.5  20  −  ND  −  20  ++  10  ++  2.5  +  20  ++  5  ++  5  −  ND      Camptothecin  348.4  15  −  ND  −  ND  ++  1.88  ++  3.75  −  ND  +  15  ++  3.75  ++  3.75      Catechol  110.1  900  +  450  ++  225  ++  7.03  ++  225  +  900  +  900  −  ND  ++  225      Crystal violet  408.0  2  ++  0.5  ++  0.5  ++  0.25  ++  0.5  ++  0.25  ++  0.15  ++  0.01  ++  0.25      Mitomycin C  334.3  250  ++  31.3  ++  15.6  ++  3.91  ++  31.3  +/−*  ND  −  ND  −  ND  −  ND      4-Nitroquinoline-N-oxide  190.2  0.5  ++  0.25  ++  0.25  ++  0.13  ++  0.13  +  0.25  +  0.25  +  0.25  −  ND  Standard genotoxins                                          Bleomycin sulphate  ∼1400  40  ++  5  ++  10  ++  1.25  +  20  ++  10  ++  10  ++  10  −  ND      MMS  110.1  32.5  ++  8.13  ++  8.13  ++  2.03  ++  4.06  ++  1.02  ++  1.02  ++  1.02  ++  1.02      MNNG  147.1  2.5  ++  0.31  ++  0.63  ++  0.63  ++  0.63  ++  0.31  ++  0.31  ++  0.08  −  ND      N-Nitroso-N-methyl urea  103.1  250  ++  31.3  ++  62.5  ++  62.5  ++  62.5  ++  31.3  ++  31.3  ++  7.81  −  ND      Urethane  89.1  10  −  ND  +  10  ++  2.5  +  10  +  10  +  10  −  ND  +  10  Compound  Molecular weight (g/mol)  Highest conc. tested (µg/ml)  Assay results                                       Growth inhibition                 Genotoxicity-GFP induction                       WT    pdr5    pdr5/erg6    pdr5/snq2/yor1    WT GSA    pdr5    pdr5erg6    pdr5/snq2/yor1            LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)   Test chemicals                                          Aphidicolin  338.5  20  −  ND  −  20  ++  10  ++  2.5  +  20  ++  5  ++  5  −  ND      Camptothecin  348.4  15  −  ND  −  ND  ++  1.88  ++  3.75  −  ND  +  15  ++  3.75  ++  3.75      Catechol  110.1  900  +  450  ++  225  ++  7.03  ++  225  +  900  +  900  −  ND  ++  225      Crystal violet  408.0  2  ++  0.5  ++  0.5  ++  0.25  ++  0.5  ++  0.25  ++  0.15  ++  0.01  ++  0.25      Mitomycin C  334.3  250  ++  31.3  ++  15.6  ++  3.91  ++  31.3  +/−*  ND  −  ND  −  ND  −  ND      4-Nitroquinoline-N-oxide  190.2  0.5  ++  0.25  ++  0.25  ++  0.13  ++  0.13  +  0.25  +  0.25  +  0.25  −  ND  Standard genotoxins                                          Bleomycin sulphate  ∼1400  40  ++  5  ++  10  ++  1.25  +  20  ++  10  ++  10  ++  10  −  ND      MMS  110.1  32.5  ++  8.13  ++  8.13  ++  2.03  ++  4.06  ++  1.02  ++  1.02  ++  1.02  ++  1.02      MNNG  147.1  2.5  ++  0.31  ++  0.63  ++  0.63  ++  0.63  ++  0.31  ++  0.31  ++  0.08  −  ND      N-Nitroso-N-methyl urea  103.1  250  ++  31.3  ++  62.5  ++  62.5  ++  62.5  ++  31.3  ++  31.3  ++  7.81  −  ND      Urethane  89.1  10  −  ND  +  10  ++  2.5  +  10  +  10  +  10  −  ND  +  10  LECs underlined indicate an increase in sensitivity of >2 dilutions, compared with the corresponding WT result, or positive result in mutant strain, compared with a negative result in the WT (standard GenTO1 RAD54-GFP GSA reporter strain). All compounds tested using 2% DMSO as solvent. * Positive using 1% DMSO as solvent, as previously published (1). View Large Stationary phase cultures for wild-type, pdr5-Δ, pdr5-Δ/yor1-Δ/snq2-Δ and CYP bearing strains were diluted to an optical density (OD600nm) = 0.2 in double strength F1 growth medium. An aliquot of 75 µl of the yeast suspension was added to each well of the diluted chemical: test strain (RAD54 reporter) to one series and the control strain to the second series of each compound and controls. Addition of the cells dilutes the final DMSO concentration to 2%. For assays using the pdr5-Δ/erg6-Δ strain, the starting inoculum was doubled to (OD600nm) = 0.4 to compensate for the impaired growth phenotype previously observed with this strain. Microplates were sealed using gas permeable membranes (Breath-easy, Diversified Biotech, USA) and incubated without shaking (unless stated) overnight (16–20 h) at 25°C. For specified assays using CYP expressing yeast strains, a low glucose growth medium (0.5% cf. 2% in standard protocol) was used and the microplates were shaken during overnight incubation. This modification to the standard protocol was used such that (i) cultures would be well oxygenated as CYP enzymes are monooxygenases and (ii) growth would be aerobic rather than anaerobic; respiration is repressed and yeast grow fermentatively in the presence of high levels of glucose, i.e. 2%. Routine assay controls, summarized here but explained in detail in (1), were included for all compounds tested, i.e. (i) test compound alone, to provide information on the compound's inherent absorbance and fluorescence; (ii) yeast cultures diluted with 2% DMSO alone, to give a measure of maximum proliferative potential; (iii) MMS as a genotoxicity control: ‘high’ = 0.00125% v/v, ‘low’ = 0.0001875% v/v; (iv) methanol as a cytotoxicity control: ‘high’ = 3.5% v/v, ‘low’ = 1.5% v/v; (v) growth medium alone, to confirm sterility. Data collection and handling Following overnight incubation with test compound, GFP reporter fluorescence and yeast culture absorbance data were collected from the microplates using a Tecan Ultra-384 microplate reader (Tecan UK). Fluorescence, excitation 485 nm/emission 535 nm with an additional dichroic mirror (reflectance 320–500 nm, transmission 520–800 nm); absorbance, 620 nm filter. The raw data collected were used to calculate growth inhibition and fluorescence induction values using software based on the following method (1). Absorbance data were normalized to the untreated control (=100% growth). During the assay, the cells settle and grow on the base of the microplate. Flocculation has rarely been observed using this assay, hence measurement of absorbance was judged to be a suitable method for estimating cell number which lends itself easily to a high throughput screening assay. 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 negative control strain were subtracted from those of the reporter strain. The genotoxicity and cytotoxicity positive decision thresholds are explained in ref. (1). The absorbance (cytotoxicity) threshold is set at 80% of the maximum extent of yeast cell proliferation on each microplate (i.e. the cell density reached by the untreated control cells). In Table IV, (+) is recorded if one or two compound dilutions produce a final cell density <80% threshold, (++) is recorded if either (i) three or more compound dilutions produce a final cell density <80% threshold or (ii) at least one compound dilution produces a final cell density <50%; (−) is recorded when no compound dilutions produce a final cell density <80% threshold. The lowest effective concentration (LEC) is the lowest test compound concentration that produces a final cell density <80% threshold. Table IV. Results of cytotoxicity and genotoxicity assessments for compounds tested in the presence of expressed CYP 3A4 or 1A2 Test chemical  Molecular weight (g/mol)  Highest conc. tested (µg/ml)  Assay results                               Growth inhibition             Genotoxicity-GFP induction                   RAD54-GFP integrant    RAD54-GFP integrant + CYP1A2    RAD54-GFP integrant + CYP3A4    RAD54-GFP integrant    RAD54-GFP integrant + CYP1A2    RAD54-GFP integrant CYP3A4            LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)   Aflatoxin B1  312.3  40  −    +  40  NT    −    +  20  NT    Cyclophosphamide                                  Standard assay protocol  279.1  50 000  −    NT    +  50  −    NT    +  25      Low glucose/shaking protocol      +  50      +  50  −        +  25  Colchicine                                  Standard assay protocol  399.4  1.25  −        −                    Low glucose/shaking protocol      −    NT    −    −        ++  0.156      N-Nitrosodimethylamine                                  Standard assay protocol  74.1  6.25  +  6.25  NT    +  6.25  −    NT    ++  1.56      Low glucose/shaking protocol      +  6.25      +  6.25  −        −    Test chemical  Molecular weight (g/mol)  Highest conc. tested (µg/ml)  Assay results                               Growth inhibition             Genotoxicity-GFP induction                   RAD54-GFP integrant    RAD54-GFP integrant + CYP1A2    RAD54-GFP integrant + CYP3A4    RAD54-GFP integrant    RAD54-GFP integrant + CYP1A2    RAD54-GFP integrant CYP3A4            LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)     LEC (µg/ml)   Aflatoxin B1  312.3  40  −    +  40  NT    −    +  20  NT    Cyclophosphamide                                  Standard assay protocol  279.1  50 000  −    NT    +  50  −    NT    +  25      Low glucose/shaking protocol      +  50      +  50  −        +  25  Colchicine                                  Standard assay protocol  399.4  1.25  −        −                    Low glucose/shaking protocol      −    NT    −    −        ++  0.156      N-Nitrosodimethylamine                                  Standard assay protocol  74.1  6.25  +  6.25  NT    +  6.25  −    NT    ++  1.56      Low glucose/shaking protocol      +  6.25      +  6.25  −        −    LECs underlined indicate an increase in sensitivity of >2 dilutions for a CYP expressing strain, compared with the corresponding RAD54-GFP reporter result. The majority of results represent average data obtained for at least two independent assays. NT, not tested. View Large The genotoxicity threshold is set at 1.3 (i.e. a 30% increase) and this is greater than three times the SD of the background. A detailed explanation for the assay threshold levels applied has been reported previously (1). A positive genotoxicity result (+) is concluded if one or two compound dilutions produce a relative GFP induction >1.3 threshold. A strong positive genotoxicity result (++) is concluded if either (i) three or more compound dilutions produce a relative GFP induction >1.3 threshold or (ii) at least one compound dilution produces a relative GFP induction >1.6 threshold. A negative genotoxicity result (−) is concluded where no compound dilutions produce a relative GFP induction >1.3 threshold. In the case of genotoxicity, the LEC is the lowest test compound concentration that produces a relative GFP induction >1.3 threshold. Assay repetition For studies of cell wall mutants (Table III), the number of independent assays for each strain was as follows: Test compounds. (a) Aphidicolin: WT, pdr5, 4, pdr5/erg6, 3, pdr5/snq2/yor1, 2; (b) camptothecin: WT, pdr5, 4, pdr5/erg6, 2, pdr5/snq2/yor1, 3; (c) catechol: WT, 4, pdr5, 3, pdr5/erg6, 1, pdr5/snq2/yor1, 2; (d) 4-nitroquinoline-N-oxide: WT, 4, pdr5, 3, pdr5/erg6, 2, pdr5/snq2/yor1, 3; (e) crystal violet and mitomycin C, single assay per strain. Crystal violet was tested as it has been used to demonstrate increased permeability of mutated Salmonella strains used for the Ames test, but since the data did not show any remarkable change for any of the strains tested, the line of enquiry was not pursued further. Mitomycin C was included for only one set of assays owing to the fact that the compound was prohibitively expensive. Standard genotoxins. MMS, MNNG, N-nitroso-N-methyl urea: WT, pdr5: two assays per strain; pdr5/erg6, pdr5/snq2/yor1: one assay per strain; urethane and bleomycin: one assay per strain. Less assay repetition was included for these standard compounds as we already had demonstrated unequivocal positive genotoxicity data with the standard WT reporter strain (GenT01). For studies of CYP expressing strains (Table IV and Figure 4), assay repetition was as follows: aflatoxin B1, three independent assays per strain; cyclophosphamide and colchicine, two independent assays per strain/growth condition; N-nitrosodimethylamine: standard growth protocol, four independent assays per strain; one assay per strain was performed with the low glucose/shaking protocol (as a positive genotoxicity result had already been demonstrated with the standard growth conditions in the CYP expressing strain). For comparison of ethanol versus DMSO solvent systems (Figure 5), there were three independent assays for each compound tested. For clarity, the data shown in the tables and figures represent the average result for each compound or strain combination tested. All assay results were within one serial dilution of the average sensitivity values (LEC) shown. Choice of compounds Table III lists compounds used in this study. Each compound was chosen for specific reasons. Mitomycin C, aphidicolin and catechol were chosen because they were only just above the threshold of genotoxicity; camptothecin was a positive genotoxin in the original study of Afanassiev et al. (5), but was subsequently not found to be reproducibly positive in the screening protocol; crystal violet was chosen because of its role in the assessment of permeability in the Ames Salmonella test strains; 4NQO was chosen both as a known substrate of the ABC transporter SNQ2 (32) and also for the narrow concentration range of its detection at the limits of tolerable toxicity; aflatoxin B1 (33), cyclophosphamide (34), N-nitrosodimethylamine and colchicine were chosen as substrates of the common human cytochrome P450 enzymes used in this study; o-anisidine and aniline were chosen as aromatic amines, negative in the screening study and difficult to dissolve using the standard protocol; the last five compounds shown in the table were all positive genotoxins in GSA (1). Results For each compound assayed, growth inhibition and genotoxicity controls (methanol and MMS) were used to demonstrate that the cells were responding in a dose-dependent manner (data not shown). Each of the mutant host strains was transformed with both pGenC01 and pGenT01 plasmids. The control plasmid provided a check for non-specific increases in fluorescence, owing to factors other than RAD54-GFP transcription (data not shown; however, all data were in the expected range for the GenC01 control strain). All compounds were tested in aqueous 2% DMSO v/v. Fluorescence inductions are plotted for test compound concentrations giving RTGs of ≥30%. Growth inhibition data were plotted for the GenC01 strain, which invariably mirror the data of the test strain. Ampicillin was tested as a standard negative compound (not cytotoxic or genotoxic) and as expected was negative for all the mutant strains (data not shown). Deletion of genes involved in cell membrane integrity and drug efflux affects assay sensitivity Cytotoxicity (growth inhibition) and genotoxicity (fluorescence induction) assay data for 11 test compounds were generated. Results from the wild-type yeast strain were compared with those obtained using the three different mutant host strains, i.e. two strains carrying deletion(s) in ABC transporter genes (pdr5-Δ and pdr5-Δ/yor1-Δ/snq2-Δ), and one strain carrying deletions in both an ABC transporter gene and a gene involved in cell membrane biosynthesis (pdr5-Δ/erg6-Δ). The strains carried either the GFP reporter plasmid (pGenT01), or the negative control plasmid (pGenC01). Table IV summarizes the data for all strains and compounds tested. An example of the assay dose response data obtained for one of the compounds where cellular accumulation of the drug was believed to influence assay sensitivity is illustrated using camptothecin in Figure 3. The single (pdr5-Δ) mutant gave similar cytotoxicity and genotoxicity results to the wild-type strain. Both the double (pdr5-Δ/erg6-Δ) and triple (pdr5-Δ/yor1-Δ/snq2-Δ) mutants were more sensitive to the cytotoxic and genotoxic effects of the drug, indicating that these mutations affected drug accumulation in the yeast cells. Fig. 3. View largeDownload slide Relative total growth (cytotoxicity) and RAD54-GFP reporter (genotoxicity) data for camptothecin (average data for at least two independent assays per strain) and bleomycin (single experiment for each strain). Closed circle, WT; open circle, pdr5-Δ strain; inverted closed triangle, pdr5-Δ/erg6-Δstrain; inverted open triangle, pdr5-Δ/yor1-Δ/snq2-Δ strain. Fig. 3. View largeDownload slide Relative total growth (cytotoxicity) and RAD54-GFP reporter (genotoxicity) data for camptothecin (average data for at least two independent assays per strain) and bleomycin (single experiment for each strain). Closed circle, WT; open circle, pdr5-Δ strain; inverted closed triangle, pdr5-Δ/erg6-Δstrain; inverted open triangle, pdr5-Δ/yor1-Δ/snq2-Δ strain. The data generated for the control genotoxins with the wild-type strain were similar to those reported in the screening validation paper (1). For example, bleomycin (Figure 3) had a marked effect on RTG (++), apparent over a wide concentration range. The GFP induction threshold is passed at low toxicity and there are three data points above (++). In the mutant strains, the cytotoxicity effects were similar to wild-type, except in the pdr5-Δ/yor1-Δ/snq2-Δ mutant which is less sensitive (Figure 3). The GFP induction was qualitatively similar in the mutants (++) though, again, the exception is pdr5-Δ/yor1-Δ/snq2-Δ where induction did not pass the threshold (−). Expression of human cytochrome P450 3A4 and 1A2 genes by an integrated host reporter yeast strain allows the detection of compounds predicted to be activated by phase I human liver metabolism In these experiments, the integrated RAD54-GFP reporter strain, Int022A, was transformed with replicative plasmids carrying either expressed human CYP3A4 and human NADPH oxidoreductase (pMT3A4-Kan, Figure 1) or human CYP1A2 and human NADPH oxidoreductase (pPOPA, Figure 2). The genotoxicity and cytotoxicity results obtained were compared with those from the Int022A RAD54-GFP reporter strain. The results for the four compounds tested, colchicine, cyclophosphamide, N-nitrosodimethylamine (tested with CYP 3A4 expression) and aflatoxin B1 (CYP1A2 expression) are summarized in Table IV, whereas Figure 4 illustrates growth inhibition and genotoxicity data for cyclophosphamide (tested with CYP3A4 expression) and aflatoxin B1 (CYP1A2 expression). Fig. 4. View largeDownload slide Relative total growth (cytotoxicity) and RAD54-GFP reporter (genotoxicity) data for strains expressing human cytochrome P450 enzymes. Left panel, cyclophosphamide (average data for two independent assays per strain). Open circle, RAD54-GFP integrant strain; closed circle, RAD54-GFP integrant strain, low glucose/shaking; inverted closed triangle, CYP3A4 strain; inverted open triangle, CYP3A4 strain, low glucose/shaking. Right panel, aflatoxin B1 (average data for three independent assays per strain). Closed circle, RAD54-GFP integrant strain; open circle, CYP1A2 strain. Fig. 4. View largeDownload slide Relative total growth (cytotoxicity) and RAD54-GFP reporter (genotoxicity) data for strains expressing human cytochrome P450 enzymes. Left panel, cyclophosphamide (average data for two independent assays per strain). Open circle, RAD54-GFP integrant strain; closed circle, RAD54-GFP integrant strain, low glucose/shaking; inverted closed triangle, CYP3A4 strain; inverted open triangle, CYP3A4 strain, low glucose/shaking. Right panel, aflatoxin B1 (average data for three independent assays per strain). Closed circle, RAD54-GFP integrant strain; open circle, CYP1A2 strain. For colchicine the largest and only clear genotoxicity responses were obtained with the strain containing CYP3A4 culture shaken in a low glucose medium. For N-nitrosodimethylamine, a genotoxicity response was observed only with the strain containing CYP3A4 culture using the standard glucose concentration, without shaking. With cyclophosphamide, cytotoxicity was negligible in the unshaken RAD54-GFP reporter culture but became apparent in shaken cultures with or without the expressed CYP3A4. For genotoxicity there was a positive result only in the CYP3A4 expressing strain, with the greatest expression at high concentration from the shaken culture (Figure 4). Growth inhibition and genotoxicity results for aflatoxin B1 (Figure 4) tested with CYP1A2 expression show that the compound only gave a positive genotoxicity result (+) in the presence of CYP1A2 expression, although a small subthreshold increase in response was seen in the RAD54-GFP reporter strain. Use of ethanol as a solvent improves the detection of the genotoxicity of aromatic amines Figure 5 shows growth inhibition and genotoxicity assay data obtained when assays were performed in two different solvent systems, 2% DMSO (standard protocol) and 0.5% ethanol v/v. The standard genotoxic compound MMS gave very similar clear positive cytotoxicity results with both solvent systems. MMS was also clearly genotoxic when diluted in 2% DMSO (++) although dilution in 0.5% ethanol gave higher induction ratios. Both aniline and o-anisidine were cytotoxic in both solvent systems (++), as seen with MMS. In 0.5% ethanol, aniline gave a clear trend in induction with increasing concentration towards the GFP induction threshold (1.3), whereas this effect was less pronounced in 2% DMSO. o-Anisidine gave a positive genotoxicity signal (+) when dissolved in 0.5% ethanol, but this was not seen using 2% DMSO as solvent. The fact that neither compound was positive in 2% DMSO confirmed data from the screening validation study. Control experiments were carried out with solvent alone as the test compound to show that the effect seen was not because of the genotoxic effects of ethanol (data not shown). Fig. 5. View largeDownload slide Relative total growth (cytotoxicity) and RAD54-GFP reporter (genotoxicity) data for aniline, o-anisidine and MMS using 2% DMSO (closed circle) or 0.5% ethanol (open circle) as the assay solvent; average data for three independent assays. Fig. 5. View largeDownload slide Relative total growth (cytotoxicity) and RAD54-GFP reporter (genotoxicity) data for aniline, o-anisidine and MMS using 2% DMSO (closed circle) or 0.5% ethanol (open circle) as the assay solvent; average data for three independent assays. Discussion The broad rationale for the generation of strains ablated for genes required for normal permeability was conservative and based on published studies of gene functions. The specific aim was to discover how the whole mutant phenotype would affect the performance of the RAD54-GFP genotoxicity assay in practice. Looking at the results as a whole it is clear that no single strain mutation strategy was effective in improving the detection of genotoxins compared with the wild-type strain. Each mutant showed improved detection of particular genotoxic compounds but the secondary effects of the mutation(s), either on growth rate or increased sensitivity to other toxic properties of the compound, reduced or removed sensitivity to other genotoxins. Different laboratory strains of S.cerevisiae have distinctive genotypes and phenotypes. This even extends to quite unexpected differences between near isogenic strains. Early in the history of yeast genetic engineering, it was noted that plasmid transformed yeast cells can have slower growth rates than their untransformed progenitors (35). During the systematic deletion of yeast genes following the generation of the genome sequence (36), 1.5% of gene knockout strains had unlinked mutations in essential genes. The strains described in the present study were all constructed in the same genetic background and at least five independent GFP reporter transformants were analysed to minimize confounding effects. The first group of mutants studied had genes involved in cell membrane integrity and drug efflux deleted (cell wall mutants), PDR5 mutations are reported to confer reduced drug resistance in ERG gene mutants (37). ERG6 is not an essential gene, but mutant strains exhibit diminished growth rates, reduced mating frequencies and low transformation rates [reviewed by Jensen-Pergakes et al. (38)]. ERG6 mutants have been shown to have increased permeability to dyes and cations, such as Li2+ and Na2+ (39). The pdr5 mutation had marginal effects on the growth inhibitory effects of the test chemicals and the improved detection of the topoisomerase inhibitor camptothecin was balanced by lack of detection of the DNA cross-linking agent mitomycin C. The pdr5-Δ/erg6-Δ double mutant strain was generally more sensitive to growth inhibiting properties of the test compounds, with increased growth inhibition at concentrations where the wild-type strain was effective in the detection of genotoxicity. The strain did not detect the genotoxicity of catechol or urethane. However, all other test compounds found to be genotoxic using the wild-type strain were genotoxic with this strain, and in the case of aphidicolin and camptothecin, there was a noticeable increase in assay sensitivity (aphidicolin, LEC decreased from 20 µg/ml in the wild-type to 5 µg/ml in the mutant; camptothecin, LEC 3.75 µg/ml in the mutant, negative in the wild-type). This strain was also more sensitive to the genotoxic effects of MMS and N-nitroso-N-methyl urea (LECs 0.08 versus 0.31 µg/ml and 7.81 versus 31.3 µg/ml, respectively). Overall this was the most useful of the ‘cell wall’ mutants, giving the broadest improvement in assay sensitivity. The cell wall mutant (pdr5-Δ/snq2-Δ/yor1-Δ) was more sensitive to the genotoxic activity of catechol, reflected by a drop in LEC from 900 to 225 µg/ml, compared with the wild-type. Sensitivity was also increased for the genotoxicity of camptothecin. Although this was a useful mutant with respect to these two compounds, it was less useful for other compounds; five compounds which tested positive for genotoxicity with the wild-type strain appeared negative for genotoxicity with the pdr5-Δ/snq2-Δ/yor1-Δ strain: aphidicolin, 4-NQO, bleomycin, MNNG, N-nitroso-N-methyl urea. These results demonstrate the need to distinguish between screening and analytical tests. The addition of this strain to a battery of tester strains would provide a broader coverage of chemical space, but used as a single test strain it would make a less effective assay. Previously Lichtenberg-Frate et al. (40) looked at the same three combinations of mutations in a different yeast genetic strain background and a smaller group of compounds that would not have revealed this limitation. The expression of human cytochrome P450 genes was performed using a host strain with the RAD54-GFP reporter cassette integrated into the rDNA array. CYP1A2 has quite a narrow substrate specificity and was chosen because it had previously been shown effective in a different yeast strain using a mutational endpoint (30). In this study, using a different strain, expression also led to the predicted increased sensitivity to the toxicity and genotoxicity of aflatoxin B1 compared with wild-type. CYP3A4 exhibits a broader substrate specificity in mammalian cells and this was also the case in this yeast study, producing positive results for three compounds that were negative in the integrated strain without CYP expression. Although these two CYP expression studies gave promising results, Table IV also reveals the surprises that come from apparently simple modifications; colchicine is genotoxicity positive in the strain with an episomal plasmid borne reporter but was negative in the reporter integrant strain. The detection of colchicine was limited to the low glucose/shaking protocol, whereas detection of N-nitrosodimethylamine was limited to the standard protocol. As with the ‘cell wall’ mutants, these strains together increase the capacity to identify compounds requiring metabolic activation, but they would need to be used in combination and with varying protocols. The positive genotoxicity result seen with the mitotic spindle poison colchicine was surprising, since in the evaluation study published by Parry in 1993 (41), colchicine was negative when tested using fungal end point assays (S.cerevisiae strains D6 and D61 and Aspergillus nidulans). However, since a positive result with the current yeast assay was only observed in the presence of CYP3A4 expression, the result may relate to the accumulation of the metabolites of colchicine, i.e. 3-demethylcolchicine and 2-demethylcolchicine. Many candidate pharmaceutical compounds precipitate from solution as soon as they are diluted out of DMSO into aqueous solvents and such insolubility can prejudice their development. In our initial validation study (1), solubility was a problem with some of the compounds tested, e.g. the aromatic amines. Ethanol is extremely well tolerated by yeasts and the data for aniline and o-anisidine clearly show that it is a good solvent for testing of aromatic amines, although the improved assay sensitivity observed might be owing to an increase in cell wall and/or membrane permeability caused by ethanol. This might also be the case for other solvent systems and deserves further investigation. Satisfactory results for the regulatory genetic toxicology tests are required for all marketed pharmaceuticals, and the same rigour may be required eventually for novel foods, cosmetics, pesticides and industrial effluents. Screening is an important tool where large numbers of samples are examined, but screening tests are limited in the range of compounds detectable by being, of necessity, restricted to a single protocol. There is a battery of genotoxicity tests because no single test is sufficient to sufficiently reduce the risk carried forward into animal and human tests. An unfortunate but inevitable consequence of this is that there are not infrequently contradicting results. In such cases, additional data can be helpful in choosing compounds for development, e.g. in situations where there is a need to resolve conflicting genotoxicology data obtained from mammalian and bacterial cell tests; in particular, in the case of test compounds which are negative in bacterial mutagenicity tests, but give positive genotoxicity results in mammalian cell systems only at toxic exposure levels. In these cases an opportunity to use a panel of yeast tester strains with the RAD54-GFP test system may allow genotoxicity to be assessed at non-toxic concentrations. This paper demonstrates that the RAD54-GFP DNA repair reporter assay can be used in a variety of formats that broaden its sensitivity. Conflict of interest statement R.J.Walmsley is a senior lecturer at the University of Manchester, and the Founder and Scientific Director of Gentronix Ltd, the company that markets the commercial version of the RAD54-GFP genotoxicity assay. A.W.Knight and N.Billinton are employees to Gentronix Ltd. The other authors have no conflicts to declare. 3 Present address: Department of Pharmacy and Pharmaceutical Sciences, Coupland 3 Building, University of Manchester, Oxford Road, Manchester M13 9PL, UK We also thank Francis Fabre for providing S.cerevisiae strain FF18984, Stefan Wölfl and Sandra Reichardt at the Hans Knoll Institute, Jena for providing pdr5 and erg6 mutant strains and Paul Cahill at Gentronix for assistance with assays. This work was supported by a UK Department of Trade and Industry Knowledge Transfer Partnership Award to Gentronix Ltd and the University of Manchester. We thank Stephen Oliver, Andy Hayes, Daniela Delneri and David Gardner for their advice and useful discussions. References 1. 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. Google Scholar 2. Walmsley,R.M., Billinton,N. and Heyer,W-D. ( 1997) Green fluorescent protein as a reporter for the DNA damage-induced Gene RAD54 in Saccharomyces cerevisiae. Yeast , 13, 1535–1545. Google Scholar 3. 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. Biosens. Bioelectron. , 13, 831–838. Google Scholar 4. 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 polarisation for the enhanced detection of green fluorescent protein (GFP) in the presence of cellular auto-fluorescence and other green fluorescent compounds. Analyst , 125, 499–506. Google Scholar 5. Afanassiev,V., Sefton,M., Anantachaiyong,T., Barker,G., Walmsley,R. 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. , 464, 297–308. Google Scholar 6. Petukhova,G., Stratton,S. and Sung,P. ( 1998) Catalysis of homologous pairing by yeast Rad51 and Rad54 proteins. Nature , 393, 91–94. Google Scholar 7. Mazin,A.V., Bornarth,C.J., Solinger,J.A., Heyer,W.D. and Kowalczykowski,S.C. ( 2000) Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament. Mol. Cell , 6, 583–592. Google Scholar 8. Van Komen,S., Petukhova,G., Sigurdsson,S., Stratton,S. and Sung,P. ( 2000) Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol. Cell , 6, 563–572. Google Scholar 9. Solinger,J.A, Lutz,G., Sugiyama,T., Kowalczykowski,S.C. and Heyer,W-D. ( 2001) Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament. J. Mol. Biol. , 307, 1207–1221. Google Scholar 10. Ames,B.N. ( 1974) A combined bacterial and liver test system for detection and classification of carcinogens as mutagens. Genetics , 78, 91–95. Google Scholar 11. ICH Harmonised Tripartite Guideline, S2B, Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. 1997.7.16 (http://www.fda.gov/cder/guidance/1856fnl.pdf—accessed May 2005). Google Scholar 12. De Flora,S. and Picciotto,A. ( 1980) Mutagenicity of cimetidine in nitrite-enriched human gastric juice. Carcinogenesis , 1, 925–930. Google Scholar 13. Ichinotsubo,D., Mower,H. and Mandel,M. ( 1981) Mutagen testing of a series of paired compounds with the Ames Salmonella testing system. Progr. Mutat. Res. , 1, 298–301. Google Scholar 14. Lipinski,C.A., Lombardo,F., Dominy,B.W. and Feeney,P.J. ( 2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. , 46, 3–26. Google Scholar 15. Tomlin,G.C., Hamilton,G.E., Gardner,D.C., Walmsley,R.M., Stateva,L.I. and Oliver,S.G. ( 2000) Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis. Microbiology , 146, 2133–2146. Google Scholar 16. Bammert,G.F. and Fostel,J.M. ( 2000) Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol. Antimicrobial Agents Chemother. , 44, 1255–1265. Google Scholar 17. Rogers,B., Decottignies,A., Kolaczkowski,M., Carvajal,E., Balzi,E. and Goffeau,A. ( 2001) The pleiotropic drug ABC transporters from Saccharomyces cerevisiae. J. Mol. Microbiol. Biotechnol. , 3(2), 207–214. Google Scholar 18. Kolaczkowski,M., Kolaczkowska,A., Luczynski,J., Witek,S., Goffeau,A. ( 1998) In vivo characterization of the drug resistance profile of the major ABC transporters and other components of the yeast pleiotropic drug resistance network. Microb. Drug Resist. , 4, 143–158. Google Scholar 19. Evans,W.E. and Relling,M.V. ( 1999) Pharmacogenomics: Translating functional genomics into rational therapeutics. Science , 286, 487–491. Google Scholar 20. Kelly,L., Lamb,D.C., Corran,A.J., Baldwin,B.C., Parks,L.W. and Kelly,D.E. ( 1995) Purification and reconstitution of activity of Saccharomyces cerevisiae P450 61, a sterol D22-desaturase. FEBS Lett. , 377, 217–220. Google Scholar 21. Briza,P., Breitenbach,M., Ellinger,A. and Segall,J. ( 1990) Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae. Genes Dev. , 4, 1775–1789. Google Scholar 22. Aoyama,Y., Yoshida,Y. and Sato,R. ( 1984) Yeast cytochrome P-450 catalyzing lanosterol 14a-demethylation. II. Lanosterol metabolism by purified P-45014DM and by intact microsomes. J. Biol. Chem. , 259, 1661–1666. Google Scholar 23. Kelly,S.L., Kelly,D.E., King,D.J. and Wiseman,A. ( 1985) Interaction between yeast cytochrome P-450 and chemical carcinogens. Carcinogenesis , 6, 1321–1325. Google Scholar 24. Collinson,E.J., Wheeler,G.L., Garrido,E.O., Avery,A.M., Avery,S.V. and Grant,C.M. ( 2002) The yeast glutaredoxins are active as glutathione peroxidases. J. Biol. Chem. , 277, 16712–16717. Google Scholar 25. Brendel,M., Grey,M., Maris,A.F., Hietkamp,J., Fesus,F., Pich,C.T., Dafre,L., Schmidt,M., Eckardt-Schupp,F. and Henriques,JAP ( 1998) Low glutathione pools in the original pso3 mutant of Saccharomyces cerevisiae are responsible for its pleiotropic sensitivity phenotype. Curr. Genet. , 33, 4–9. Google Scholar 26. Güldener,U., Heck,S., Fielder,T., Beinhauer,J. and Hegemann,J.H. ( 1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. , 24, 2519–2524. Google Scholar 27. Huxley,C., Green,E.D. and Dunham,I. ( 1990) Rapid assessment of S.cerevisiae mating type by PCR. Trends Genet. , 2, 236. Google Scholar 28. Cormack,B.P., Bertram,G., Egerton,M., Gow,NAR, Falkow,S. and Brown AJP. ( 1997) Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology , 143, 303–311. Google Scholar 29. Lopes,T.S., Klootwijk,J., Veenstra,A.E., Van der Aar,P.C., Van Heerikhuisen,H., Raue,H.A. and Planta,R.J. ( 1989) High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene , 79, 199–206. Google Scholar 30. Sengstag,C., Eugster,H.P. and Wurgler,F.E. ( 1994) High promutagen activating capacity of yeast microsomes containing human cytochrome P450 1A and human NADPH-cytochrome P450 reductase. Carcinogenesis , 15, 837–843. Google Scholar 31. Sikorski,R.S. and Hieter,P. ( 1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics , 122, 19–27. Google Scholar 32. Haase,E., Servos,J. and Brendel,M. ( 1992) Isolation and characterisation of additional genes influencing resistance to various mutagens in the yeast Saccharomyces cerevisiae. Curr. Genet. , 21, 319–324. Google Scholar 33. Langouet,S., Johnson,W.W., Guillouzo,A. and Guengerich,F.P. ( 1998) Detoxication of aflatoxin B1 as a model for carcinogen metabolism. In Vitro Mol. Toxicol. , 11, 95–101. Google Scholar 34. Ren,S., Yang,J., Kalhorn,T.F. and Slattery,J.T. ( 1997) Oxidation of cyclophosphamide to 4-hydroxycyclophosphamide and deschloroethylcyclophosphamide in human liver microsomes. Cancer Res. , 57, 4229–4235. Google Scholar 35. Danhash,N., Gardner,D.C.J. and Oliver,S.G. ( 1991) Heritable damage to yeast caused by transformation. Biotechnology  9 179–182. Google Scholar 36. Niedenthal,R., Riles,L., Guldener,U., Klein,S., Johnston,M. and Hegemann,J.H. ( 1999) Systematic analysis of S.cerevisiae chromosome VIII genes. Yeast , 15, 1775–1796. Google Scholar 37. Kaur,R. and Bachhawat,A.K. ( 1999) The yeast multidrug resistance pump, Pdr5p, confers reduced drug resistance in erg mutants of Saccharomyces cerevisiae. Microbiology , 145, 809–818. Google Scholar 38. Jensen-Pergakes,K.L., Kennedy,M.A., Lees,N.D., Barbuch,R., Koegel,C. and Bard,M. ( 1998) Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants. Antimicrobial Agents Chemother. , 42, 1160–1167. Google Scholar 39. Bard,M., Lees,N.D., Burrows,L.S. and Kleinhans,F.W. ( 1978) Differences in crystal violet uptake and cation-induced death among yeast sterol mutants. J. Bacteriol. , 135, 1146–1148. Google Scholar 40. Lichtenberg-Frate,H., Schmitt,M., Gellert,G. and Ludwig,J. ( 2003) A yeast-based method for the detection of cyto and genotoxicity. Toxicol. In Vitro , 17, 709–716. Google Scholar 41. Parry,J.M. ( 1993) An evaluation of the use of in vitro tubulin polymerisation, fungal and wheat assays to detect the activity of potential chemical aneugens. Mutat. Res. , 287, 23–28. Google Scholar Author notes 1Faculty of Life Sciences, The Mill, University of Manchester, Sackville Street, Manchester M60 1QD, UK and 2Gentronix Limited, The Fairbairn Building, 72 Sackville Street, Manchester M60 1QD, UK © The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org
TI - Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP genotoxicity assay
JO - Mutagenesis
DO - 10.1093/mutage/gei044
DA - 2005-06-28
UR - https://www.deepdyve.com/lp/oxford-university-press/genetic-modification-and-variations-in-solvent-increase-the-mAXx0nQXLM
SP - 317
EP - 327
VL - 20
IS - 5
DP - DeepDyve
ER -