Mutation Research 653 (2008) 63 69
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and
Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres
Development of a highthroughput yeast-based assay for detection of
metabolically activated genotoxins
Xuemei Liu", Jeffrey A. Kramer, Jonathan C. Swaffield, Yi Hu, Guixuan Chai, Alan G.E. Wilson
Drug Metabolism, Pharmacokinetics, and Toxicology, Lexicon Pharmaceuticals, Inc., 8800 Technology Forest Place, The Woodlands, TX 77381, United States
a r t i c l e i n f o a b s t r a c t
Article history:
The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it
Received 24 January 2008
is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery
Received in revised form 14 March 2008
phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment
Accepted 19 March 2008
of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput
Available online 1 April 2008
screening and several strains of Salmonella must be employed to ensure that different types of DNA damage
can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant
Keywords:
value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires
Yeast
numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is
Dual luciferase
challenging. Another critical component of such screens is that they incorporate the capability of metabolic
Highthroughput
activation to ensure that no genotoxic metabolites are generated.
Metabolic activation
Genetic toxicology We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity,
Mutagens
and which incorporates metabolic activation. The assay has a low compound requirement as compared to
Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding
Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated
into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the
RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual
luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample.
The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are
interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due
to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation
using positive and negative control agents. This assay is efficient, requires little time and low amounts of
compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform,
and yields data that accurately and reproducibly detects DNA damage.
Whereas the normal yeast cell wall, plasma membrane composition and the presence of active trans-
porters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did
show concordance with regulatory mutagenicity assays, many of which require metabolic activation and
are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our
hands this assay performs as well as or better than other commercially available genetox assays. Fur-
thermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus
this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay
represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Rapid, highthroughput biological assays that measure chem-
ically induced mutations can provide  early warnings of
Abbreviations: GFP, green fluorescent protein; DSB, double strand break; EMS, mutagenicity and genotoxicity. Among these assays, bacterial
ethylmethanesulfonate; 2AAF, 2-acetylaminofluorene; 4AAF, 4-acetylamino-
screens are generally robust, economical, and well characterized.
fluorene; MMS, methylmethane sulfonate; 2AA, 2-aminoanthracene; 4NQO1,
The most widely used systems employ microplate-based bacterial
4-nitroquinoline-N-oxide; 2NF, 2-nitrofluorene; DMN, dimethylnitrosamine; YNB,
screens: the Ames test (Ames II) and the SOS response reporters
yeast nitrogen base; RLU, relative light units.
" [1 6]. The principle drawback of the bacteria-based tests is that
Corresponding author. Tel.: [phone]; fax: [phone].
E-mail address: [e-mail] (X. Liu). they lack eukaryotic chromosomes; thus they are unable to detect
1383-5718/$  see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2008.03.006
64 X. Liu et al. / Mutation Research 653 (2008) 63 69
clastogenic and aneugenic events [7]. Chromosome damage can 2.3. Luciferase reporter plasmids
lead to genomic rearrangements such as deletions, transloca-
The Renilla and firefly luciferase gene sequences were amplified by PCR
tions, and amplifications [8,9], and bacteria-based tests are not
from pRL-CMV and pGL-3-Basic plasmids (Promega Corp., Madison, WI), respec-
suitable for revealing such chromosomal aberrations. Typically,
tively. The Renilla primers used were forward: 5 -GGGCAAGCTTCCCTTATGAC-
chromosome breakage is scored microscopically [3]. However, this
TTCGAAAGTTTATGATCC-3 and reverse: 5 -GGGCCAGCTGTTATTGTTCATTTTTGAG-
technique remains labor intensive, time consuming, and its appli- AAC-3 . The firefly primers used were forward: 5 -GGGCAAGCTTCCCTTATGGAA-
GACGCCAAAAACATAAAG-3 and reverse: 5 -GGGCCAGCTGTTACACGGCGATCTTTCC-
cation for screening in drug discovery is severely limited. Therefore,
GCCC-3 . For both Renilla and firefly genes the forward primer incorporates a unique
it is desirable to have a screening test that reduces the false negative
HindIII site into the amplified product, whereas a unique PvuII site was introduced
results of bacterial tests, and which is amenable to highthroughput
to the end of reverse primers.
methods. The assay strain contains two nuclear, integrative replicating, single copy plas-
mids YIp358 (URA3) and YIp368 (LEU2) ordered from the ATCC (Manassas, VA) [25].
Yeast (Saccharomyces cerevisiae) provide a valuable system for
The vectors YIp358 and YIp368 were digested with HindIII and PvuII to remove
the analysis of recombination in eukaryotes [10,11]. Several publica-
lacZ, purified by agarose gel electrophoresis, and ligated with firefly and Renilla
tions have described the development of a yeast-based genotoxicity
genes generated via PCR to produce the YIp358Firefly and YIp368Renilla vectors,
test in which the DNA damage-inducible promoter of the RAD54
respectively.
To generate a PGK1-YIp368Renilla promoter fusion construct, the PGK1 pro-
gene is fused to the green fluorescent protein (GFP)1 [12 15]. In
moter was amplified by PCR from yeast (HMS-1) genomic DNA using primers
yeast, the RAD54 protein participates in the recombinational repair
forward: 5 -GGGCGGATCCGAAGTACCTTCAAAGAATGGGGTCTCATC-3 and reverse:
of double-strand DNA breaks together with the RAD51, RAD52,
5 -GGGCAAGCTTTTGTTTTATATTTGTTGTAAAAAGTAG-3 , which incorporated unique
RAD55, and RAD57 proteins. In this process, RAD54 interacts with
BamHI and HindIII sites, respectively [19]. The RAD51-YIp358Firefly reporter
RAD51 and stimulates DNA strand exchange, promoted by RAD51 construct was initiated by introducing a KpnI site at -1.3kb of the RAD51 5
NTS, and the HindIII sites at the third codon. The primers used were for-
protein [16,17]. For several reasons, GFP is an unsuitable reporter for
ward: 5 -AGATTAATGGTACCTATTTTGTGTTGGGGTTGTTTTTGGGACC-3 and reverse:
this study because the GFP signal is invariably contaminated with
5 -AGACACATAAGCTTACATATGACGATAACAAATTAGTAGGCC-3 . The final vectors
endogenous (or media-based) autofluorescence [13 15] although
bear the up-stream noncoding DNA sequence of the S. cerevisiae PGK1 promoter
there are methods to mitigate this [18]. Moreover, the limited
in front of Renilla luciferase reporter and RAD51 gene fused with firefly luciferase
reporter. All the sequences were verified using the dye terminator cycle sequencing
metabolic capability of yeast requires the use of the autofluorescent
method on an ABI PRISM.
nicotinamide nucleotides (NADPH) and S-9 mix for the activation
of most promutagens.
2.4. Construction of luciferase reporter yeast strain
To overcome autofluorescence limitations, we employed two
luciferase genes as reporters, and this dual luciferase assay pro- Yeast transformation was as described elsewhere; a lithium acetate protocol was
employed [26]. Double transformants were selected on Ura DO, Leu DO SC-media
vides advantages for measuring gene expression in yeast. First,
plates [27]. Individual colonies were picked and streaked twice for clonal purification
the sensitivity of the luciferase assay allows the reporter to be
ć%
before storage at -80 C. Nonpromoter containing YIp358Firefly and YIp368Renilla
integrated into the genome and still provide measurable signal,
vectors were also transfected and used for background evaluation.
alleviating the issue of copy number variation observed with epi-
2.5. RAD51 dual luciferase assay
somal reporters [19]. Second, the use of a constitutive promoter to
drive expression of one of the reporters provides an internal con-
Assays were carried out in 96-well, white microplates (Dynex Technologies,
trol for cell numbers. In the current study, we have used the RAD51
Chantilly, VA). Before treatment with possible genotoxic chemicals, yeast cells were
ć%
promoter as an inducible reporter of DNA damage, and the PGK1
grown in YNB-URE-LEU medium at 30 C and 200 rpm orbital shaking for 24 h
promoter as the constitutive internal control. The RAD51 gene is a to achieve logarithmic growth. Cells were collected and reseeded (75 L, 3 × 105
cells/mL) into a 96-well, clear plate (Becton Dickinson Labware, Franklin Lakes, NJ).
homolog of bacterial recA which promotes the pairing of homol-
All the test compounds were dissolved in 15% DMSO to make 30,000, 15,000, 3000,
ogous DNA molecules and strand exchange reactions [16,20 23];
1500, 300 and 150 g/mL. The test compounds were then added to yeast cells (10 L
therefore, we surmised that our assay would assist in the detection
to make final concentration 2000, 1000, 200, 100, 20, and 10 g/mL in the incubation
of DNA-damaging agents.
systems) combined with 65 L of 11% S-9 mix to make 5% final concentration of S-9;
or a phosphate buffer according to bacterial Ames assay [28]. Since previous results
We evaluated RAD51-based induction of luminescence using 28
have shown that DMSO increases yeast DEL recombination [10], the final concen-
model compounds. The testing compounds included DNA dam-
tration for DMSO in the medium is 1%. After 18 h treatment as previously described
aging agents, many of which are promutagens, and well-known
[29], 40 L cells were harvested for assessment of dual luciferase activity in 200 L
reagents employed as controls in the regulatory test. Our data show
passive lysis buffer (Promega luciferase kits). Luciferase activity was determined for
that the assay has a high level of concordance with regulatory muta- 25 L aliquots of cell lysates in 100 L lysis buffer mixed with 25 L of the luciferase
assay solution, and read on a Tecan Ultra-384 (Tecan UK Ltd., Theale, UK) in a 96-
genicity assays, and suggests that yeast dual luciferase reporter
well plate format. Changes in induction ratio (fold changes) were calculated using
system affords a new tool to identify metabolically activated muta-
the equation (F = firefly, R = Renilla):
gens and genotoxins.
Ftreated/Rtreated
Fold changes = .
FDMSO/RDMSO
2. Materials and methods
The results are expressed as the mean Ä… S.D. where n equals six.
2.1. Caution
2.6. Ames assay
All the positive controls were handled in accordance with NIH Guidelines for
the Laboratory Use of Chemical Carcinogens [24].
Ames assays were performed as described by Mortelmans and Zeiger [28].
All solvents and chemicals were purchased from Aldrich Chemicals (Milwaukee,
Briefly, the tester strains TA100 and TA98 were combined with S-9 mix or buffer,
WI) or Sigma (St. Louis, MO) and the purities are greater than 99%. Aroclor1254-
test or control article, as well as a trace of histidine and molten agar (Moltox, Inc.,
induced rat liver S-9 homogenate was obtained from Molecular Toxicology Inc.
Boone, NC). After 48 h, the background lawn density was scored, followed by count-
(Boone, NC). The dual luciferase assay kit (No. E1960) was purchased from Promega
ing the number of revertant colonies. Mutation results are reported as numbers
Corp. (Madison, WI).
of revertants per plate. The results are expressed as the mean Ä… S.D. based on six
dilutions scored in triplicate.
2.2. Yeast strains and growth media
2.7. Data analysis
The haploid strain S. cerevisiae YPH499 was obtained from Stratagene (La Jolla,
CA). The strain has not been modified to increase permeability or sensitivity to DNA
Data analysis was similar to that described by others [29]. For each com-
damage. Transfected yeast strains were grown on yeast nitrogen base (YNB) with
pound, data from three independent experiments were collected and the mean
URA and LEU dropout medium as previously described [19].
and standard deviation was calculated. A compound was considered genotoxic in
X. Liu et al. / Mutation Research 653 (2008) 63 69 65
the luciferase assay if it caused at least a 1.6-fold induction as compared to the
DMSO and the observed increase was concentration dependent. If the test com-
pounds showed cytotoxicity (both firefly and Renilla signals are lower than twofold
of background) or precipitation in more than three concentrations (totally six con-
centrations were screened), further dilutions need to be tested. A compound was
considered negative, if it did not satisfy the above mentioned criteria but led to a
cytotoxic effect or precipitation was found in the medium within the treated con-
centration range. Methylmethane sulfonate (MMS), at concentrations of 250 and
750 g/mL served as a positive control mutagen for experiments performed in the
absence of metabolic activation. 2-Aminoanthracene (2AA) at concentrations of 13.3
and 66.7 g/mL was used as positive control for studies in the presence of metabolic
activation.
3. Results
3.1. Background
An important criterion for many reporter assays is the back-
ground signal. To evaluate background luminescence resulting from
the spontaneous oxidation of luciferin with components in the
yeast lysate and S-9, we performed both firefly and Renilla luciferase
assays using the double transfected nonpromoter YIp358Firefly
and YIp368Renilla vector strains. The background signals for the
firefly luciferase assay (mean Ä… S.D.) with and without S-9 were
85 Ä… 49 and 96 Ä… 47 relative light units (RLU), respectively. Renilla
luciferase assay yielded 61 Ä… 30 RLU without S-9 and 94 Ä… 31 RLU
with S-9. The background signals for lysis buffer without adding
luciferase substrate were 105 Ä… 42 RLU without S-9 and 96 Ä… 47 RLU
with S-9. Thus, in both assays the background signal was negligi-
ble.
3.2. Sensitivity, reproducibility, and stability
Other important criteria for reporter assays are sensitivity, lin-
earity, reproducibility, and stability. The luciferase yeast strain was
ć%
grown to exponential phase in YNB-URE-LEU medium at 30 C.
The cell numbers were quantified by hemocytometer, serial dilu-
tions were prepared, and between 600 and 50,000 cells were
used per assay for both firefly and Renilla luciferase activities.
Our data showed that both the firefly and Renilla luciferase activ-
ities increased linearly with increasing numbers of cells (Fig. 1A
and B), and the correlation of firefly to Renilla luciferase activi-
ties was very strong (Fig. 1C). The firefly/Renilla activity ratio was
calculated at each cell concentration (Fig. 1D), and the average
mean value was 0.086. The error between replicates was slightly
higher at lower cell concentrations. Therefore, 16,000 25,000 cells
per assay were employed for validation studies. The yeast dual
luciferase assay was also tested with different concentrations of
S-9 (1, 2, 5, and 10%) co-incubation with eight replicates. The fire-
fly luciferase assay signals were 1648 Ä… 234 (0% S9), 1765 Ä… 210
(1% S9), 1626 Ä… 251 (2% S9), 1480 Ä… 247 (5% S9), and 1386 Ä… 125
(10% S9) RLU. Renilla luciferase assay yielded 23,634 Ä… 2034 (0%
S9), 22,374 Ä… 1757 (1% S9), 22,853 Ä… 1907 (2% S9), 22,199 Ä… 2410
(5% S9), and 19,586 Ä… 1982 (10% S9) RLU. The concentration range
for S9 is based on those used in the bacterial Ames assay
[28]. Our data showed that as the S9 concentration increased,
mean of three replicate assays performed at each dilution. (C) Firefly vs. Renilla
luciferase activity is linear in 600 51,200 range of cell concentrations analyzed.
These data are from graphs (A) and (B) and the correlation coefficient is indicated (R).
(D) Firefly and Renilla activity ratio is independent of the number of cells assayed.
The data represent those shown in (A) and (B). The bars indicate standard deviation.
(E) Stability of firefly and Renilla luciferase activities in cell lysates with or without
S-9. Yeast cells were lysed in passive lysis buffer, and the ratios firefly and Renilla
Fig. 1. Sensitivity, reproducibility, and stability of yeast luciferase assay. Yeast strain
luciferase activity were determined immediately as day 0. The lysates were then
containing the RAD51firefly and PGK1Renilla reporters was grown in YNB-URA-LEU
ć%
stored in -20 C and luciferase assays were performed on the same aliquots of the
medium to exponential phase. The cell numbers were quantified by hemocytometer,
lysate on days 1 and 7. The data shown represent the means from three independent
and dilutions were prepared such that the indicated number of cells was used to
experiments, with the bars showing standard deviation.
assay both firefly (A) and Renilla (B) luciferase activities. The data represent the
66 X. Liu et al. / Mutation Research 653 (2008) 63 69
Table 1
Twenty-eight compounds tested in yeast dual luciferase assay
Compounds CAS no. Salmonella Carcinogenicitya
Rat Mouse
Urethane 51-79-6 + + +
Ethylmethane sulfonate 62-50-0 + + +
Dimethylnitrosamine 62-75-9 + + +
b
Vinblastin 865-21-4 --
4-Acetylaminofluorene 28322-02-3 + -
2-Acetylaminofluorene 53-96-3 + + +
Etoposide 33419-42-0 -
Bleomycin 9041-93-4 -
2-Aminoanthracene 613-13-8 +
Cyclophosphamide 50-18-0 + + +
Mitomycin C 50-07-7 + +
Sodium azide 26628-22-8 + -
4-Nitroquinoline-N-oxide 56-57-5 + + +
Methylmethane sulfonate 66-27-3 + +
Cisplatin 15663-27-1 + +
Arsenic III oxide 1327-53-3 --
Methyl viologen 1910-42-5 +
Nalidixic acid 389-08-2 - + -
2-Nitrofluorene 607-57-8 + +
Taxol 33069-62-4 -
Ara C 147-94-4 -
Chlorambucil 305-03-3 + + +
Actinomycin D 50-76-0 - +
Caffeine 58-08-2 -- -
HCl 7647-01-0 --
NaOH 1310-73-2 -- -
MeOH 67-56-1 -
DMSO 67-68-5 -- -
a
Data are collected from Carcinogenic Potency Project (University of California,
Berkeley).
b
Data are not available.
Fig. 2. Effect of MMS and 2AA on induction of firefly luciferase. Yeast cells were
treated with different concentrations of MMS, 2AA, and vehicle for 18 h. (A) MMS.
(B) 2AA + Aroclor1254-induced rat liver S-9. The data represent the means from three
cytotoxic to yeast cells. Thus, 5% S-9 was chosen as the working
independent experiments, with the bars showing standard deviation.
concentration.
the cytotoxicity is slightly increased. Although the S-9 concen-
3.4. Induction by other DNA-damaging agents
trations might raise concerns about the reproducibility of the
firefly/Renilla ratios, our data demonstrate that those theoreti-
In order to evaluate the ability of the dual luciferase assay to
cal concerns do not raise any practical problems with this assay
detect mutagens with and without metabolic activation, we exam-
(Fig. 1E).
ined the levels of RAD51-based luciferase induction using a variety
To determine the stability of yeast lysate for the purposes of
of DNA-damaging agents. Tables 1 and 2 list chemicals that were
the genotoxicity assay, lysates were examined over time. The dual
tested in the yeast dual luciferase assay, including both cytotoxic
luciferase strain was assayed immediately after lysis; and the fire-
genotoxins and noncytotoxic nongenotoxins. The dual luciferase
fly/Renilla activity ratio at this point was defined as day 0. The
assay was performed in parallel, with and without metabolic acti-
ć%
lysates were then stored at -20 C and luciferase assays were per-
vation (Aroclor1254-induced rat liver S-9), using the same reagents
formed on the same lysate aliquots on days 1 and 7. The data (Fig. 1E)
and single cell purified colonies as a source of the tester cells. MMS
ć%
demonstrate that the yeast lysates are stable up to 7 days at -20 C
was employed as a positive control for the induction of RAD51.
but variation between replicates increased slightly as a function of
2AA was used as a positive control for the S-9 metabolic activation
time. Therefore the dual luciferase reporter assay is useful for geno-
luciferase studies. The background luminescence was measured
toxicity studies with respect to sensitivity, reproducibility, linearity,
using 1% DMSO treatment. All of the direct-acting mutagens
and stability.
including MMS, EMS, sodium azide, 2-nitrofluoren (2-NF), and 4-
nitroquinoline-N-oxide (4NQO1) were shown to be positive in our
3.3. Effect of MMS and 2AA on induction of firefly luciferase system. The cross-linking agents (mitomycin C and cisplatin) and
a reactive oxygen species generator (methyl viologen dichloride)
The system was optimized by using the alkylating agent MMS, were also evaluated and showed greater than 1.6-fold RAD51 induc-
which mutates DNA and promotes activation of the intra-S-phase tion. The negative controls, caffeine, HCl, NaOH, MeOH, and DMSO,
checkpoint response [30,31]. We selected 2AA as a model com- did not show any RAD51 induction in the dual luciferase assay.
pound for optimizing the metabolic activation conditions using We also tested clastogens that are negative in the Ames assay,
the Aroclor 1254-induced liver S-9. The mutagenic potencies of including: etoposide, vinblastin, arsenic III oxide, bleomycin, taxol,
MMS and 2AA were measured by serial dilutions of MMS or 2AA araC, chlorambucil, and actinomycin D. No induction was observed
in the exponentially growing cells, with or without S-9 activa- for taxol, chlorambucil, araC, and actinomycin D but induction was
tion. Fig. 2A and B demonstrate that both MMS and 2AA cause a seen with etoposide, vinblastin, arsenic III oxide, and bleomycin.
dose-dependent stimulation of RAD51 gene expression. Five and Because the dual luciferase assay could detect promutagens, we
ten percent S-9 caused similar inductions of luminescence with selected 2AAF, 4AAF, dimethylnitrosamine, and cyclophosphamide
test concentrations of 2AA, but 10% S-9 treated alone was slightly as model compounds for metabolic activation evaluation. All of
X. Liu et al. / Mutation Research 653 (2008) 63 69 67
Table 2
Comparison between yeast dual reporter assay and published dataa
Compounds Luciferase Assay Genotoxicity Referenceb
Concentrations Lowest Detectable Maximum With S-9 Without S-9
( g/mL) Concentration ( g/mL) Fold Changes
Urethane 25,000-50 12,500 (cytotoxic) 1.8 + - [45]
Ethylmethane sulfonate 2500-5 500 1.9 + + Alkylation of DNA [46]
Dimethylnitrosamine 100-0.2 50 2.5 + - Require metabolic activation [47]
Vinblastin 500-1 250 (cytotoxic) 1.9 + + [48]
4-Acetylaminofluorene 1250-2.5 313 3.7 + - Require metabolic activation [49]
2-Acetylaminofluorene 1250-2.5 313 3.4 + - Require metabolic activation [50]
Etoposide 1200-2.4 300 (cytotoxic) 1.9 + + [51]
Bleomycin 250-0.5 50 (cytotoxic) 2.4 + + Cleavage of DNA. [52]
2-Aminoanthracene 500-1 50 2.9 + + Require metabolic activation [53]
Cyclophosphamide 1000-2 250 2.2 + - Require metabolic activation [54]
Mitomycin C 200-0.4 100 (cytotoxic) 1.9 + + Alkylation of DNA [55]
Sodium azide 5-0.01 2.5 (cytotoxic) 2.2 + + [56]
4-Nitroquinoline-N-oxide 30-0.06 0.5 (cytotoxic) 2.6 + + [50]
Methylmethane sulfonate 2000-4 196 (cytotoxic) 11 + + Alkylation of DNA [57]
Cisplatin 30-0.06 15 (cytotoxic) 2.2 + + [58]
Arsenic III oxide 1250-2.5 125 (cytotoxic) 1.8 + + [59]
Methyl viologen 1000-2 100 (cytotoxic) 1.8 + + [60]
Nalidixic acid 500-1 25 (cytotoxic) 2.1 + + [61]
2-Nitrofluorene 100-0.2 5 (cytotoxic) 2.2 + + [62]
Taxol 1000-2 -- -
- [63]
Ara C 1000-2 -- -
- [64]
Chlorambucil 1000-2 -- -
- Alkylation of DNA [65]
Actinomycin D 250-0.5 -- -
- Inhibition of DNA and RNA synthesis [66]
Caffeine 500-1 -- -
- Negative
HCl 10%-0.1% -- -
- Negative
NaOH 400-0.8 -- -
- Negative
MeOH 10%-0.1% -- -
- Negative
DMSO 1% -- -
- Negative
a
Cells were treated with compounds for 18 h. Values are expressed by three determinations.
b
Experimental details are described in Section 2.
c
Data are collected from published literature.
these four compounds exhibited a dose-dependent induction of 4. Discussion
RAD51 (Fig. 3).
Currently, screening of DNA damaging agents is achieved with
3.5. Comparisons of dual luciferase assay with regulatory tests a battery of standardized genetic toxicity assays that have the abil-
ity to monitor both cytotoxic and genotoxic effects [32,33]. These
We compared the relative concordance of our assay to the reg- assays provide high precision and predictivity of potential mech-
ulatory assays using the published data (Tables 1 and 2). anisms of genetic toxicity. Negative findings in these assays are
a strong indication that carcinogenicity is unlikely. In contrast,
indications with multiple assays that a sample can produce DNA
damage or subcellular damage is a strong indication that additional
testing may be required. Whilst this is true in relation to the detec-
tion of human carcinogens as positive in these regulatory assays,
it has been shown that the established in vitro mammalian assays
in particular have very poor specificity and thus there is a need
for developing new assays to improve specificity. These regulatory
approaches provide reliable prediction of human carcinogenic haz-
ard, but time and cost factors limit the number of chemicals that
can be evaluated in these systems and most screens are still applied
late in preclinical drug development.
Measurement of altered gene expression to detect DNA dam-
age response in living cells has shown value in preclinical safety
assessment [34]. While damage-inducible genes have been iden-
tified in numerous organisms, regulation of the damage response
has been most extensively characterized in Escherichia coli and S.
cerevisiae [12,34 37]. A rapid turn-around yeast-based genotoxicity
test has been reported in which RAD54 gene induction is moni-
Fig. 3. Effect of 2AAF (closed bar 625 g/mL; dotted bar 313 g/mL; hashed
tored as induction of green fluorescence protein in yeast [12,13,18].
bar 62.5 g/mL; open bar 31.3 g/mL), 4AAF (closed bar 625 g/mL; dotted bar
The RAD54 protein performs crucial functions in the repair of DNA
313 g/mL; hashed bar 62.5 g/mL; open bar 31.3 g/mL), DMN (closed bar
50 g/mL; dotted bar 25 g/mL; hashed bar 12.5 g/mL; open bar 2.5 g/mL),
double-strand breaks (DSBs), which are the major genotoxic lesions
and cyclophosphamide (closed bar 500 g/mL; dotted bar 250 g/mL; hashed bar
induced by radiation and clastogens. Unrepaired DSBs can cause
50 g/mL open bar 25 g/mL) on induction of firefly luciferase with 5% S9. The data
loss of chromosomes or cell death. If misrepaired, DSBs can give rise
shown represent the means of three replicates, with the bars showing standard
to mutations and chromosomal rearrangements; therefore, DSBs, if
deviation.
68 X. Liu et al. / Mutation Research 653 (2008) 63 69
unrepaired, can lead on to cellular changes that may contribute applied in this assay making this assay suitable for the detection of
to stages in the carcinogenic process in multicellular organisms mutagenicity in the early drug discovery screening.
[38 41]. InS. cerevisiae RAD51, RAD52, RAD54, and RAD55 have been In summary, previous studies have shown that RAD51 expres-
assigned to one epistasis group, the recombinational repair group. sion is controlled at the level of transcription, with levels varying
In response to DSBs, the RAD51 protein binds to single-stranded in response to DNA damage [17,20,35,43,44]. The stable transfor-
DNA and helps to scan double-stranded DNA until a homologous mation of YPH499 with dual reporter constructs is, therefore, a
sequence is found, where it forms a nucleofilament on the single- useful addition to the predictive tests for genotoxicity and muta-
stranded DNA and catalyzes homologous stand exchange together genicity, especially because the assay detects a DNA DSB response.
with RAD54 [17,20,42]. Therefore, it maybe that the RAD51 gene This simple, rapid assay is likely to be a valuable addition as an
used in this study is more sensitive than the RAD54 gene because early screen for the mutagenicity and genotoxicity of mutagens and
of its apparently early induction in response to DNA damage. promutagens.
Reporter genes have been used extensively to monitor
DNA damage-induced responses, including -galactosidase, CAT, Acknowledgements
luciferase, and GFP. Among them, GFP may be the most commonly
used. The major drawback of GFP assays is that many cellular We thank Melinda M. Albright, Suma Gopinathan, Joe J. Shaw,
metabolites and crucial cellular extracts in culture media exhibit and Lance Ishimoto for useful suggestions during the preparation
intense autofluorescent. Such autofluorescence background can of this manuscript.
cause experimental inaccuracy in GFP assays. Additionally, the nor-
malization of GFP assays has routinely been achieved by quantifying
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