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An intrachromosomal recombination assay (“DEL”) based on a genetically
engineered HIS3 locus in S. cerevisiae [45] has been developed both as a colonycounting and a well-counting assay. Nine chem icals were used in a proof of principle
for higher throughput screening in 96- or 384-well format that had been modified into
a colorimetric assay [46]. The assay is reported to detect carcinogenic compounds
including those for which a genotoxicity mechanism has not previously been reported.
At present, the assay is not widely available.
7.5.4 Chromosome Damage and Aberration Assays
Chromosome damage and aberration assays may be classified into two classes: (1)
chromosome aberration tests in which all the chromosomes in a cell are examined and
(2) micronucleus tests in which chromosomes outside the nucleus in smaller
micronuclei are examined. In the first class, gross rearrangements of chromosomes
are examined including translocations, large insertions/deletions, the loss or gain of
whole chromosomes, and double- or single-stranded DNA breakage. Since chromosomes only condense and become visible during mitosis, most test protocols require
treatment with a mitotic poison that causes cells to accumulate at metaphase (such
cytochalasin B). In this procedure, all cells in the exposed population can be scored
rather than the subpopulation in mitosis at the analytical time point (OECD 473).
Some tools and systems are on the market for aiding mitotic indexing and chromosome aberration scoring (Pathfinder technology from IMSTAR, Paris, France).
Metasystems GmbH (Altlussheim, Germany) also provides a metaphase locating
software called Metafer MSearch. Such systems have the potential to speed up the
identification of metaphase cells, and use chromosome “painting” to help identify the
type of aberration. At present, automated high-throughput scoring of translocations,
insertions, and deletions has not replaced microscopic examination and it remains
unclear how widespread such systems will become. In a recent review of methods
focusing on human cells, the authors commented that “Even the best automated
system may never replace a skilled observer” [47]. These methods are not yet
sufficiently mature for profiling.
The micronucleus test (MNT) is effective in the automated detection of clastogens
and aneugens. Nuclear membrane forms around chromosomes and chromosome
fragments that fail to segregate into nuclei during anaphase. This might be a
consequence of failures in the mitotic machinery or chromosome breakage that
generates fragments without centromeres. These smaller “micronuclei” can be
identified and counted using two quite different approache s: high-resolution imaging
and flow cytometry. In both cases, DNA-specific dyes are crucial in resolving nuclei
and micronuclei. Imaging is reliant on careful optimization of the parameters used to
distinguish DNA-containing bodies from other membrane-bound compartments.
Diaz and coworkers [48] published an evaluation of an automated MNT assay
using fluorescent microscopy coupled with image analysis software from Cellomics
(Pittsburg, KS, USA). The results showed high concordance with data collected by
manual scoring. The speed of scoring limited throughput to 11 compounds per day (at
multiple dilutions, with and without S9 activation). This throughput approaches the
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minimum levels required for screening. Although the sample preparations remain
relatively complex, the ever-increasing processing power and data storage available to
developers will increase the assay throughput. Alternative imaging systems and
analysis software are also available. IMSTAR and Metasystems reduce analysis times
to 4–5 min per slide rather than the 25 min to score manually.
Flow cytometric assessment generates results that reproduce microscopic methods
for the in vivo assessment of micronucleus frequency in peripheral blood micronucleated reticulocytes [49] and the method is increasingly applied in in vitro
assessments. Litron Laboratories (Rochester, NY, USA) recently launched their
“MicroFlow
In Vitro” kit for the in vitro micronucleus test following a six
compound interlaboratory evaluation [50]. This method permits 50 samples to be
analyzed over the course of 3.5 h including incubation times, leading to a possible
throughput of 40 compounds per week or 2000 compounds per year.
It is important for the screener to recognize the inherent problems in specificity
observed in chromosome aberration assays. Only about 50% of compounds with
positive results are likely to be hazardous to rodents (or humans).
7.5.5 The “Comet” Assay
The electrophoresis of the nuclear content of individual cells followed by DNA
staining provides microscopic images reminiscent of comets hurtling across the night
sky—giving the name to the comet assay. It identifies strand-breaking agents.
Although it has several handling steps, it is a relatively simple assay. The comet
“head” contains giant (75 mm) supercoiled loops of DNA liberated from higher order
chromatin packaging by high salt treatment. The “tail” contains loops in which there
has been at least one single-stranded break leading to a more extended relaxed
supercoil. More single-strand breaks lead to the formation of more relaxed loops and
a greater proportion of staining migrates into the tail. Under alkaline conditions,
double-strand breaks, or two single-strand breaks in the same loop, lead to fragmentation of the loops and altered tail structure. Smaller fragments of DNA including
mitochondrial DNA (5.6 mm) and apoptotic fragments are beyond the resolution of the
gels, and do not contribute to the head or tail staining. Thus, the comet assay, in
alkaline or neutral conditions, should detect single- and double-stranded breaks, but
not “pure” aneugens. In alkaline conditions, breakage can occur at alkaline labile sites
including sites where the base has been lost from the sugar/phosphate backbone.
Modified assays in which lesion-specific nuclease treatment is included can be used to
reveal oxidative damage. Readers are referred to Collins and coworkers [51] for more
detail on data generation and interpretation.
In the routine comet assay, a user can operate at a maximum throughput of 8–12
compounds per week, limited by sample and cell preparation, imaging, and scoring.
There are a number of imaging and software analysis solutions currently available
from companies such as IMSTAR, Metasystems, and Perceptive Instruments Ltd
(Haverhill, UK). The assay has also been developed into a higher throughput
format that accommodates four compounds each tested at 10 dilutions in a 96-well
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microplate. In this method, cells are still transferred to slides for scoring [52], and with
robotic handling data from six compounds per day can be analyzed. A new protocol in
development utilizes a multichamber plate that can be used for both cell treatment and
electrophoresis [53]. The throughput is potentially 1500 compounds per year, which
approaches the minimum requirements for late profiling.
7.5.6 DNA Adduct Assessment
A general and sensitive approach for the measurement of DNA lesions formed with
nonradioactive carcinogens has been descr ibed. Normal and adducted nucleotides,
generated by nuclease digests of DNA modified in vivo or in vitro with a compound of
interest, are labeled with
32
P and detected and quantified after thin-layer chromatography (TLC). Such analysis of DNA adducts is used in mechanistic studies but
presently not suited to profiling because of the complexities in sample preparation and
analysis and subsequent low throughput. There are no formal guidelines for testing.
7.5.7 Gene Expression Assays
Microbes and metazoans are exposed to a variety of toxic stresses and have evolved
appropriate defenses and repair mechanisms. Some of these systems are regulated at
the protein level and others are regulated at the transcriptional level, allowing the
development of reporter assays. These transcriptional responses can be used to
provide an earlier marker for genotoxin exposure in a whole population of cel ls.
This is opposed to the detection of the endpoints discussed above in which genotoxic
stress leads to fixation of mutations or chromosomal aberrations/damage in a small
subpopulation.
Prokaryotic The first generation of reporters exploited the DNA damage inducible
genes of the SOS operon [54] in bacteria. These genes variously encode proteins
involved in excision repair, recombinational repair, and DNA polymerase. Reporters
for both the sfiA gene (SOS chromotest, [55]) and umuC [56] have been used to drive
b-galactosidase synthesis which can be assessed using a colorimetric assay. A review
of SOS chromotest data from 751 compounds [57] revealed that for the 452
compounds, which also had Ames data, there was agreement between the tests for
82% of compounds. A number of alternative SOS reporters have been developed to
drive expression of the lux operon, which allows luminometric data collection. These
include reporters for recA, uvrA, alkA [58], and umuC [59] as well as the commercially available Vitotox assay (Gentaur Molecular Products BVBA, Brussels, Belgium), which exploits the recN gene [60]. In a limited comparison of data from seven
genotoxins and six environmental samples generated using recA-lux, umuC, and sfi
reporter assays, the umuC test performed the best [61]. Although these systems are
less accurate in prediction of Ames data than the more cumbersome fluctuation tests,
their simplicity and low compound requirement are advantages for use.
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The SOS-umuC assay allows three compounds to be assessed over seven dilutions
on a 96-well microplate. A user can prepare up to 15 assay microplates per day
translating to 180 compounds profiled per week, or around 10,000 per year.
Eukaryotic (Yeast Cells) The first eukaryotic gene regulation assay (Gr eenSc reen
GC, Gentronix Ltd, UK), used Green Fluorescent Protein (GFP) as a reporter for
induction of the yeast RAD54 gene—a member of the recombinational repair family
of genes [62]. A screening validation study [63] demonstrated that the assay
detected a different spectrum of compounds to bacterial genotoxicity assays. It
was suggested that in conjunction with a high-throughput bacterial screen, the two
assays wo uld provide an e ffective preview of the regulatory battery of genotoxicity
tests. This proposal was subsequently justified in a study of 2698 proprietary
compounds from the Johnson & Johnson (J&J) compound library [40]. Four
compounds per 96-well microplate were tested with robust host cells and rapid
assay time. It was possible to assess up to 160 compounds per week even
without automation. Beyond this throughput, compound supply and data collection
become the rate-limiting factors. An alternative RAD54 reporter has also been
described [64].
7.5.7.1.3 Eukaryotic (Human Cells) Broader reservation about yeast tests led to
the development of a human cell assay in which GFP was used as a reporter for
induction of the GADD45a gene [65]. Significantly, the human TK6 cell line was
chosen not only because of its origins, but also due to its wild-type p53 status.
Other mammalian cell lines used commonly are insufficient or deficient in p53 and
this compromises the effectiveness of the DNA damage response. The resultant
GADD45a-GFP assay (GreenScreen HC, Gentronix Ltd) responds to all classes
of genotoxin including S9-generated metabolites [66]. In contrast to the regulatory
in vitro mammalian assays, the results demonstrate high specificity without
compromising sensitivity. This 96-well microplate assay is becoming widely
employed and its transfer to other laboratories has been systematically evaluated [67]. The format of the GADD45a-GFP assay is very similar to the yeast assay.
Four compounds are tested on a 96-well microplate although the assay incubation
time is longer (48 h instead of 16 h) due to longer doubling time of the host cells.
Manually, a compound throughput of 80–100 per operator per week is possible.
This can be significantly improved using robotics and/or a reduced number of
compound dilutions. In a recent study using this h igher throughput assay format, a
library of 1266 pharmacologically active compounds (LOPAC, Sigma-Aldrich
Co. Ltd) was screened [68]. The potential throughput from this assay format
could be as high as 36,000 compounds per year.
A second p53 responsive reporter has been described. This reporter exploits
elements of the p53R2 gene, which encodes a subunit of ribonucleotide reductase
linked to a luciferase gene [69, 70]. It has been validated against diverse mechanistic
classes of genotoxin. To perform this assay, the p53 wild-type cell line MCF-7 is
transiently transfected with two plasmids: one with the p53R2 reporter and the second
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with constitutively expressed control (driven by elements of CMV promoter).
Between 4 and 6 h after transfection, cells are exposed to test materials (five dilutions)
for 24 h, then washed three times, lysed, and assayed. It is not yet known how well the
assay transfers to other laboratories, and the protocol is quite complex for adaptation
to high throughput.
7.6 THE “OMICS”
The completion of the human genome project has led to new disciplines within
genetic toxicology—the “omics.” The study of global gene expression from transcription and translation, to a protein’s various posttranslational states and intracellular location(s), as well as tools that are beginning to give clues about the more
common toxi cological response patterns, increasingly provide techniques for novel
drug target identification. These technologies and the interpretation of their data are
not yet sufficiently developed for screening at the throughput required for profiling
and are not discussed in this chapter. However, in an integrated approach to safety
assessment, genetic toxicolo gists will increasingly have access to data from broader
toxicogenomic approaches. This data is already becoming useful in confirming
mechanism of drug action and adverse reaction. There have already been proposals
for a “new paradigm” in preclinical safety assessment in which in vitro and in silico
approaches are com bined [71].
7.7 USING DATA FROM IN VITRO PROFILING: CONFIRMATORY
TESTS, FOLLOW-UP TESTS, AND THE LINK TO SAFETY ASSESSMENT
AND IN VIVO MODELS
The principal aim of a genotoxicity screening program is to reduce the proportion
of compounds that give positive results in IND-enabling GLP studies. The default
action for a compound with a positive genotoxicity result during profiling tests
would be removal from the collection. Profiling occurs when the properties of
compounds identified in other screening tests are already channeling into the
strategy of chemistry lead optimization. A positive result may not end a program,
but would immediately reduce the ranking of the compound and provide valuable
actionable information to the medicinal chemistry team. Only in very particular
cases would a potent genotoxin be carried forward. If the choice of compounds is
not broad due to the therapeutic indication, discovery, or patent strategy or the
nature of the target, then there will be two avenues for immediate follow-up of a
positive result. First, lead optimization chemists can be alerted to the need to
focus on the segregation of useful pharmacology from unwanted genotoxicity
facilitated by knowledge of the known structural alerting motifs (Section 7.4.2).
Second, the compound profile should be annotated or flagged to indicate the
possible need for follow-up by safety assessment teams to consider mechanism
of action.
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7.7.1 Annotations from Screening Data
All annotations/alerts should carry basic information including the assay type, the
result, the top dose tested, the solvent, and any additional information generated such
as control, lowest effective concentration, magnitude of effect, and associated toxicity
data. Annotations and appropriate alerts should be included even where the result is
negative and should be specific to the screening strategy.For a strategy based solely on
mutation endpoints, a negative result should generate an alert that genotoxicity
assessment is incomplete, and aneugenicity and clastogenicity have not been assessed. For a strategy based solely on aneuge nicity or clastogenicity, a negative result
should generate an alert that assessment is incomplete and an in vitro mutation has not
been assessed. For a strategy using a reporter assay that is not endpoint-specific, a
negative result should generate an alert for a complementary assay such as a bacterial
assay after a eukaryotic screen or a eukaryotic assay after a bacterial screen.
Most of the in vitro assays are described with an additional protocol for the
assessment of metabolites generated by the monooxygenases (CYPs) and various
conjugation systems. It is unclear whether genuine high-throughput screening assays
would be performed routinely with a source of exogenous metabolism such as the
rodent liver extract S9. This seems unlikely because of S9 handling difficulties in a
high-throughput situation: S9 is heat labile and frozen samples create problems
associated with rethawing, etc. If there are no data from assays incorporating S9, then
an alert should be generated to reflect that metabolites have not been investigated. This
alert might be strengthened by linkage to data from metabolic liability assays or in
silico approaches. In due course it is expected that there will be validated, metabolically competent cell lines for metabolite assessment.
7.7.2 Can a Genetic Toxicity Profile Assist with In Vivo Testing Strategies?
Current regulatory practice already requires that a positive in vitro result is followed up
with two in vivo tests.The first regulatory in vivo assay is routinely the micronucleustest
using either bone marrow or peripheral blood cells from only one sex in a rodent. With
appropriateplanningit is generallypossibleto use differenttissuesfromthe sameanimals
forthe second test. Thisis generallyselectedto assess the in vivo significanceof the result
of the in vitro test giving a positive result. Thus a positive result from a mutation assay
suggests the use of an in vivo test for DNA damage/mutagenesis such as unscheduled
DNAsynthesis(UDS OECD 486), or
32
P-postlabellingto detectDNA adduct formation
(noOECDguideline).If thepositiveresultis only in thepresenceofmetabolicactivation,
further metabolism studies are often instigated to determine if the metabolite formed in
vitro (by a rat liver extract) is actually formed or is inactivated in vivo.
A recent review identified 120 rodent carcinogens where the in vivo MNT gave
negative or equivocal results, and there were other in vivo data to consider. It was
apparent that UDS has very poor sensitivity [72] and adduct assessment is generally
reserved for compounds where reactive metabolites are suggested. The alkaline
comet assay is now considered an appropriate alternative. The other alterative is to use
one of the genetically engineered rodent mutation assays (MutaMouse, Big Blue),
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though these are expensive and time-consuming, so unlikely to be used routinely for
both tissues/endpoints. Advice regarding choice of tissue will be dependent on other
factors such as the target tissue of the drug. This information may not be available
when screening alerts are recorded and is beyond the scope of this chapter.
Most recently attention has been drawn to the assessment of PigA mutations
[73,74]. This X-linked gene provides a readily scored mutant phenotype as a
consequence of its essential role in the anchoring of GPI proteins in the membrane.
Mutant cells are distinguished by their inability to bind GPI-linked proteins such as
CD59, CD55, and CD24. Furthermore, wild-type cells are killed by Aerolysin, a
bacterial toxin that uses the GPI anchor to mediate lysis. Thus, aerolysin resistance is a
direct selection for PigA mutants that can be cloned and sequenced to determine the
nature of the mutation.
A positive result from a chromosome aberratio n assay (MNT,cytogenetic analysis,
or small colonies in MLA), suggests a second in vivo assay for clastogenesis or
aneugenesis. Centromere and DNA staining of mitotic cells allows the distinction of
aneugens and clastogens.
If positive result s in reporter assa ys specifically developed for profiling screening
are obtained, in vivo MNT is conducted as a follow-up. Additional information may
also be generated during the regulatory in vitro testing. Kirkland and Speit [72]
recommend the comet assay as a second in vivo test, because the limited data available
suggests that this assay identifies more of the carcinogens missed by MNT.
7.8 WHAT TO TEST, WHEN, AND HOW
Genotoxicity data should become an important part of a compound’s profile. It can
provide value in decision making at all stages in discovery from libraries containing
millions of compounds to leads progressing to candidate selection. For entire libraries,
screening could allow segregation of genotoxins into a sublibrary. If a new therapeutic
campaign is initiated where genotoxicity is allowable or expected, such as antineoplastics or antivirals, then those compounds are included; if genotoxicity is unacceptable, then these compoundsare excluded. As previouslydiscussed,no screen detects all
genotoxins. Conducting a library screen does not eliminate genotoxicity concerns and
appropriate alerts remain for unassessed hazards. Furthermore, the chemical differences that evolve from hit to lead to drug-like candidate introduce newstructuralmotifs.
None of the genotoxicity assays described above has yet been used for large
libraries. Although most assa y developers have access to the instrumentation
required for developing HTS or ultr a-HTS methods, a progressive longer term
strategy to collect the data over a period of at least a year is a feasible approach for
screening large sets.
Hit profiling is more feasible as it is generally anticipated that the initial target screen
willgenerate <1%hits (10,000–100,000 compoundsper year). All compoundswould
benefitfromthe early inclusionof genotoxicitydata in the profile.Only four of the assays
discussed have the capacity for thisscreeningscale:the bacterialAmesII,SOS reporters,
the yeast RAD54-GFP reporter and the human GADD45a-GFP reporter. The bacterial
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Ames II and SOS reporters provide an early warning for Ames positives. The yeast
RAD54-GFPreporterandthehumanGADD45a-GFPreporterpreview humananeugens
and clastogens, missed by Ames. The most effective approach to screening would
combine the bacterial, yeast, and now the human tests. As early as 2004, Kitching
et al. [75] compiled validation data for 71 compoundsfrom the GreenScreen GC (yeast)
test with published SOS/umu data. The results were as follows:
.
54 compounds (76%) had positive data in cancer studies.
.
32 compounds (45%) were positive in GreenScreen GC.
.
32 compounds (45%) compounds were positive with SOS/umu.
.
22 compounds (31%) were positive for both tests.
.
Each test had 10 unique positives.
Subsequently J&J assessed 2698 potential drug candidates through the GreenScreen yeast assay during preregulatory screening [40]. Two thousand three hundred
fifty one compounds were also tested with Ames II. One hundred sixty four (7%) of the
2351 compounds were positive in Ames II, with and/or without S9 metabolic
activation and 176 (7.5%) were positive in the GreenScreen. Twelve (7%) of the
176 GreenScreen-positive compounds were positive in Ames II. These results
emphasize that the Ames II and GreenScreen assays each detect a different but
overlapping spectrum of genotoxins, reflecting both the differences between prokaryotic and eukaryotic test organisms and their different endpoints (mutation and
DNA damage-induced transcription, respectively). Recently, the same group tested
1684 compounds using the GreenScreen HC (human cells) and Ames II assays (data
not published). There was a 31 compound overlap between the two assays. This again
confirms the different endpoints covered by bacterial and mammalian cell tests and
reveals how genotoxicity results can vary for different compound collections.
A combination of two genotoxicity tests is a better screening strategy than
conducting one assay. At earlier stages of discovery when chemical development
is still in progress, one test is presumably sufficient. Given that in silico methods are
quite effective in the identification of Ames-positive compounds based on structural
alerts, a single eukaryotic screening test is preferred.
Profiling during lead optimization might generate 2,000–10,000 compounds per
year. Genetic toxicity screening using eukaryotic models such as GreenScreen HC is
already occurring at this level in some of the larger pharmaceutical companies.
7.9 CHANGES TO REGULATORY GUIDELINES CAN INFLUENCE
SCREENING STRATEGY
The current ICH S2B guidelines require both Ames and in vitro mammalian test data.
Since Ames data causes the greatest concern, most screening methods use one of the
bacterial screens as well as one of the MNT screening tests using flow cytometry or
imaging readouts. Earl y mammalian genotoxicity screens provide a useful preview of
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the regulatory MNT. However, MNT is known to have high prevalence of positive
results (35% or more) in which many are false positives, not confirmed in in vivo
testing. Consequently, many potentially valuable nonhazardous leads might be
discarded based on MNT results before the selection of candidat es is complete or
require a large follow-up effort. To reduce this risk, a secondary screen of MNT
positives with the higher specificity GADD45a-GFP (GreenScreen HC) test would
identify the subset of compounds liable to give positive results in later in vivo tests.
Many of the remaining GADD45a-GFP negatives could become useful drugs once
mechanistic studies establish the nonrelevance of the positive MNT data.
The problems generated by the poor specificity of the current regulatory in vitro
mammalian tests has led to the proposal of new testing strategies published in the US
Federal Register as/or ICH S2(R1). These proposals contain two significant changes
(Table 7.1). First, the maximum testing dose in the in vitro mammalian tests should be
reduced from 10 to 1 mM to reduce the generation of misleading positive results due to
high toxicity. The second change is that two different options for data submission are
proposed. Option one is essentially the same as the current requirement except for the
lowerdosing.Option two is more radical: The submissionrequiresAmes test for in vitro
dataand two differentin vivo endpoints.It is proposedthatthese endpointscan be obtained
fromthe same animalexposure study. For compounddiscovery and development, option
2 may be preferredsincethereis valuein generatingprofilingdata in in vitromammalian
assays during screening. The bacterial screens are valuable in as a preview of the
regulatory Ames test results. Hence, there is now a broad selection of assays available
that help avoid in vivo failures by compounds inevitably undetected by Ames. In the
absenceofinformative in silico data,a high-specificityeukaryotic testwould be preferred.
7.10 SUMMARY
There are now highly specific genotoxicity screening assays that can provide reliable
hazard warnings, data early enough for medicinal chemists. Individual prokaryotic
and eukaryotic tests detect different but overlapping classes of genotoxins. It has been
demonstrated that the overall level of genotoxicity in a library that can be detected by
these approaches is similar to the level of candidate attrition due to genotoxicity. This
strategy can be facilitated using in silico approaches to identify embedded genotoxic
structural moieties that can be potentially avoided. This suggests that early profiling
will lead to development of more predictive in silico tools, which in turn will cause a
significant reduction in late stage failure due to genotoxicity. It also offers an overall
increase in efficiency in later regulatory genotoxicity safety assessment by providing
an early alert for projects likely to require mechanistic investigation.
ACKNOWLEDGMENT
Andy Scott (Unilever) is thanked for the preparation of Figure 7.1. Nick Billinton is
thanked for his contributions to the screening sections.
ACKNOWLEDGMENT 347
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