Mutagenesis vol.3 no.6 pp.455-458, 1988
DISCUSSION FORUM
The role of mammalian cell mutation assays in mutagenicity and
carcinogenicity testing
C.F.Arlett and J.Cole
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, Sussex
BN1 9RR, UK
Recent debates (Ashby, 1986a,b, 1988; Gatehouse and Tweats,
1986, Gamer and Kirkland, 1986, Ennever et al., 1987; Ishidate
and Harnois, 1987; Tweats and Gatehouse, 1988) in these
columns have been concerned with a rational design for testing
compounds for their genotoxic potential. Much of the debate is
concerned with the relevance and efficiency of in vitro shortterm tests, in particular the mammalian cell gene mutation assay.
This debate is particularly timely since the United Kingdom
Department of Health and Social Security are at present reviewing
the Committee on Mutagenicity (COM) guidelines, and their
recommended strategy of testing.
Both chromosomal and gene mutations may give rise to
heritable genetic disease. Current knowledge indicates that
carcinogenesis also involves both point mutation and chromosome
changes. A direct role of mutagenesis is seen in the modulation
of oncogenes (Sukumar et al., 1983; Marshall et al., 1984; Bos
et al., 1985). The role of chromosome changes can be seen at
two levels. First, in examples of reciprocal translocation whereby
deregulation of normal levels of transcriptional control lead to
malignancy, as with the myc proto-oncogene in Burkitt's
lymphoma (Croce, 1986), or where an altered gene product may
be involved as in chronic myelogenous leukaemia (Fainstein
et al., 1987). Second, via non-reciprocal exchanges which may
involve deletion or addition of chromosomal material. Deletions
in 13q 14.11 in retinoblastoma (Strong et al., 1981) or Ilpl3
in Wilms tumour (Koufos et al., 1985) are now well documented.
The involvement of both mutagenic and clastogenic events is
emphasized in examples such as retinoblastoma (Cavenee et al.,
1983) and perhaps bilateral acoustic neurofibromatosis (Rouleau
et al., 1987) where the tumour is thought to arise when, following
a primary mutation, the normal allele may be lost by deletion.
In the case of multiple endocrine neoplasia type 2A, the primary
defect maps to chromosome 10 (Simpson et al., 1987), and the
'tumour suppressing' sequences subject to deletion, to
chromosome 1 (Mathew et al., 1987).
We might then expect short-term tests which detect both
end points to be most valuable. The Ames test only detects point
mutations in DNA organized in a prokaryotic genome and thus
it is generally considered that at least one mammalian in vitro
test is also required. The question is, which one?
Clastogenicity assay
The clastogenicity assays have, in addition to their current legal
status considerable justification for this role since:
(i) they have been available for years (Evans, 1986) and have
been used to monitor changes in lymphocyte cultures from
individuals in hazardous occupations such as radiation
workers (Evans et al., 1979). They thus assume authority
through usage;
© IRL Press Limited, Oxford, England
(ii) the assays test chromosomal changes over the whole genome;
(iii) it is believed thatrelativelyfew compounds exist which cause
gene mutations and which are not also clastogenic.
Negative points with respect to the clastogenicity assays are:
(i) only chromosome changes are detected and the mechanisms
by which these changes occur are poorly understood. At least
50% of chromosome changes are not viable and thus not
relevant to carcinogenesis. In order to lead to a recognizable
tumour any change must occur in a viable cell;
(ii) they are expensive, the current cost in the UK being in the
region of ~ 5K£ for a single unreplicated test, in addition
they demand considerable skill in scoring and interpretation
at the microscope;
(iii) they are subject to deficiencies in experimental design. Any
suggestion that the test should be expanded and replicated
would make the assay even more expensive;
(iv) A list of compounds (Table I) has recently been assembled
which have been assessed as being positive in mammalian
cell gene mutation assays but negative in both Ames and
clastogenicity tests.
Mammalian cell gene mutation assays
The latter point suggests that mammalian cell gene mutation
assays are a necessary component of the primary in vitro screen
and we wish to make a number of comments about this and the
assays themselves.
Actual evidence for non-clastogenic mutagens is hard to come
by, and we are grateful to David Kirkland, Microtest, who has
compiled the information in the Table from published sources.
What is immediately obvious is that all mutation data in Table
I are from the L5178Y T K + / - assay. However, this is not
always the case, and John Asquith, Toxicol Laboratories
(personal communication), has an example of a compound which
was negative for chromosome aberrations in L5178Y cells but
mutagenic in both L5178Y TK+/— and Chinese hamster ovary
cells. Since this compound is subject to commercial security full
details are not available to us. Note also that a number of rodent
non-carcinogens are mutagenic to these mammalian cells, a point
we will return to later. We regard the high incidence of positive
results for sister-chromatid exchanges as being evidence for the
interaction of the compounds listed in Table I with DNA.
A further example known to us (Lee et al., in preparation)
is l-methoxy-l,3,5-cycloheptatriene (MCHT) which was
regarded as Ames negative and negative for both in vitro human
lymphocyte chromosome aberrations and in vivo mouse bone
marrow tests, but positive for gene mutation in the V79 HPRT
assay (±S9) and for the L5178Y T K + / - assay (+S9 only).
However, upon further testing (Arlett et at., in press) we have
shown that in L5178Y cells, MCHT induced micronuclei and
trifluorothymidine (TFT) resistant mutants mainly of the small
colony class but did not induce a significant number of ouabain
resistant mutants. Thus, in these cells (in the presence of Aroclorinduced rat S9) MCHT acts as a clastogenic mutagen. Our
experience with MCHT reminds us of the possibility that what
455
C.F.Arlett and J.Cole
TaWe I. Chemicals giving positive mammalian cell gene mutation results,
but negative in Ames and in vitro chromosomal aberration tests
Chemical
Ref
Carc(Q or
non-Carc(N)
Benzoin
Benzyl acetate
Chloroacetic acid
Chlorobenzene
Chlorodibromomethane
CI acid yellow 73
Cinnamyl anthranilate
1,2-Dichlorobenzene
Dtcyclohexylthiourea
1,2-Epoxybutane
FD and C yellow no. 6
Geranyl acetate
Isophorone
Linthocholic acid
Malaoxon
Pentachloroethane
Sulfisoxazole
1,1,1,2-Tetrachloroethane
Trichloroethylene
3
3
2
3
3
3
3
3
2
2
3
3
3
1
3
3
3
3
3
N
C
SAL
L5178Y
(TK+/-)
CA
9
c
c
c
c
N
N
•7
N
N
C
N
N
C
N
C
c
• = positive in SCE.
** = equivocal result in SCE.
SAL = Salmonella.
CA = chromosome aberration.
? = not known.
1. Shelby and Stasiewicz, 1984.
2. McGregor el at., 1987a.
3. Tennant etal., 1987.
appears a + or — in Table I may, in fact, be based upon insecure,
perhaps poor, data or reflect species differences perhaps mediated
through differences in metabolic activation. Each compound in
Table I should be treated on its individual merits. For example,
one of the listed compounds benzyl acetate, was negative in the
Ames test (+ or — rat or Syrian hamster S9) and negative for
sister chromatid exchanges and chromosome aberrations in
Chinese hamster cells (+ or -S9). A dose related mutation
response was recorded for the L5178Y T K + / - assay (+S9 only,
NTP Technical Report Series no. 250, and positive in the absence
of S9, C.Riach and W.Caspary, personal communication).
However, to date we have been unable to detect either clastogenicity or mutagenicity in L5178Y TK+/— cells with this
compound (J.Cole et al., unpublished results), using micronuclei
and 6-thioguanine, trifluorothymidine and ouabain resistance as
end points. This leads us to believe that a detailed examination
of all the raw data for all end points which went into Table I
is needed and, in addition, further testing of the compounds may
also be required to clarify their mechanisms of action. For
example Colin Riach (Inveresk Research International, Ltd) has
already informed us that 1,2-epoxybutane is Ames positive when
tested in the vapour phase. Here we would point out that any
subsequent interpretation is bound to be biased by the assignment of + or — in any table made by the most authoritative
assessor. In the absence of access to the raw data there is no
way any fresh independent assessment may be made. Thus we
perceive a real problem whereby unsubstantiated + or — scores
may be repeatedly quoted in the literature. In this connection we
would remind readers that Mutagenesis provides a valuable
facility for computerized storage of data.
The present situation is that until a case to the contrary is made
we must accept the possibility that a number of non-clastogenic
mutagens exist.
456
What of the mammalian cell gene mutation assays themselves?
Experience with these assays to date has given them a reputation
for being (i) difficult to perform, (ii) expensive and (iii) over
sensitive ( T K + / - ) or insensitive (V79). In response, we would
like to make the following points:
(i) While skill and care is required in setting up mammalian
cell gene mutation assays scoring requires relatively little
skill and training and may readily be automated. In 1986
a Third Health and Safety Executive sponsored UKEMS trial
was set up in order to establish and test improved protocols
(Cole, 1988). It was fortunate that at the same time two
UKEMS guidelines groups had been charged with
developing statistical packages appropriate to these assays.
It has been possible to integrate the two enterprises such that
we now believe that robust protocols are capable of
generating statistically sound data although there are
numerical problems with the V79 and CHO Chinese hamster
tests. We anticipate full reports in these pages in due course.
(ii) The cost of a repeated full mammalian cell gene mutation
assay is approximately the same at 5K£ as a single clastogenicity assay.
(iii) An oft-repeated complaint is that the L5178Y T K + / - test,
which is the most frequently used mammalian cell gene
mutation assay, is hyper-sensitive. As we pointed out in our
earlier comments on the results in Table I it is clearly giving
positive genotoxicity results for 'non-carcinogens'. Examples
of the use of this assay are provided by Clive et al. (1979)
and more recently by Wangenheim and Bolcsfoldi (1988).
In the latter study a number of non-carcinogens such as
dimethyl sulphoxide, glucose and urea were shown to be
positive on purely statistical grounds. This led the authors
to propose an arbitrary '4-fold rule' for judging the carcinogenic potential of chemicals tested in the assay. Inspection of the data indicate considerable variation in the control
(spontaneous) mutant frequencies and the experimental
design would not have satisfied the requirements of the proposed UKEMS recommendations for statistical analysis.
Meeting these requirements would, we believe, remove the
need for 'N-fold' arbitrary rules in the assessment of
genotoxic classification. We suggest that given a proper experimental design and application of appropriate statistics
many of the apparent 'positives' would have been excluded.
In a recent study conducted in accordance with the up-to-date
UKEMS protocol Leigh Henderson at Huntingdon Research
Centre (personal communication) found that of 50 compounds
examined in routine testing only two gave highly significant
positive responses (one of which was a reference compound, and
the other a related compound), two gave smaller positive
responses (both also related compounds) and one was equivocal.
Theremaining45 were all clear negatives. While this result might
be fortuitous, since we have no details of the compounds under
test, it does not seem to us to reflect 'super-sensitivity' of this
assay. Thus we suggest that a properly designed and executed
L5178Y TK+/— assay will generate sound results.
One aspect of assays based on hprt is that they may be
perceived as insensitive. There are several reasons why hprt-based
assays might be less sensitive for routine screening than the tkbased L5178Y assay.
(i) The spontaneous mutant frequency to 6-thioguanine
resistance is generally about an order of magnitude lower
than trifluorothymidine resistance. Thus, many more cells
Mammalian cell mutation assays
must be treated, maintained and plated to obtain a similar
level of statistical precision. This is a practical problem which
it is possible to overcome, although it is more difficult in
attached cells such as V79.
(ii) The long expression time required for the selection of
6-thioguanine-resistant mutants (6—9 days) may mean that
a class of slow-growing mutants (which are detected in the
tk-based assay, 2 - 3 day expression time) is lost. Potentially
this could be overcome by the use of 8-azaguanine as
selective agent for hprt mutants (expression time 2 - 3 days)
although it cannot be used for mouse cells,
(iii) the hprt gene may be less mutable than the tk gene, or the
mutations may not be detected by current methodology.
(iv) Some classes of chromosome events such as very large
deletions, loss of the whole chromosome, recombination and
gene conversion may be detected in systems based on the
autosomal tk gene but not on the X-linked hprt gene.
The relative contribution of these possibilities cannot be
resolved at present. However, the expanded protocols developed
by McGregor et al. (1987b) indicate that the insensitivity in V79
and CHO Chinese hamster cells is intrinsic rather than a
consequence of the limitations of size of experiment practicable
with cells growing attached to surfaces. The insensitivity may
arise as a consequence of the location of the gene of interest close
to a vital gene(s) on the X chromosome. Target size does not
seem an appropriate explanation since the hprt gene is 40 kb in
comparison with the tk gene at 10 kb. However, this dose not
take into account the size of the transcribing sequence.
We believe that the continued use of the Chinese hamster assays
must surely be called into question in those situations where we
seek to attempt an evaluation of the genotoxic potential of a
compound which is as complete as possible.
A point in favour of mammalian cell gene mutation assays,
whether they function as a primary screen, as seems appropriate
to us at this time, or in a confirmatory role, follows from the
fact that some are able to detect both gene mutations and
chromosomal events. For the hprt assays dependent upon
6-thioguanine or 8-azaguanine resistance, both translocations and
small deletions can be recognised (Thacker, 1985). With the tk
gene in mouse lymphoma cells large deletions and rearrangements
involving the gene result in the generation of 'small colony' TFT
resistant mutants (Hozier et al., 1985). 'Large colony' TFT
resistant mutants may represent point mutations or small deletions
within the tk gene itself (Moore et al., 1985). Good examples
of the use of large and small colonies with the TFT selective
system and a correlated response with 6-thioguanine selection
is given by the response of L5178Y cells to procarbazine (Clive
etal., 1988) or sodium fluoride (Cole etal, 1986).
For particular compounds for which more extensive information could be justified (for example to resolve apparent
mutagenicity positive/clastogenicity negative compounds) we
should like to recommend the use of an integrated L5178Y assay
system. This should be based upon UKEMS protocols and
statistical designs, and should include an assessment of large and
small TFT-resistant colonies, ouabain resistance and micronucleus
induction combined within a 48-h expression time. There would
also be the opportunity for evaluating 6-TG resistance (at a 7-day
expression time) in the same populations of treated and control
cells. The requirement for a clastogenicity assay would be met
by TFT resistant small colony mutants and micronuclei and the
assay would have the added bonus of detecting any nonclastogenic mutagens. Examples of where we have utilised this
system are seen with MCHT and BA. A similar integrated system
would be possible using the human lymphoblastoid cell line TK6
(Yandell et al., 1986). Recent studies suggests that it is possible
to select large and small colony tk mutants although the system
is as yet less well characterized than the L5178Y TK+/— assay
and the cells have been shown to be repair defective (Dean and
Fox, 1984). Chromosome aberrations, micronuclei, and hprt
mutations are also readily scored in these cells.
Finally, for us, the most compelling feature of mammalian cell
gene mutation assays which should commend their continued use
is the possibility of analysis of the nature of the genetic change
which gives rise to the mutant phenotype in viable cells. The
observation of viable mutants in vitro raises the possibility that
such mutants might also have an in vivo existence. Studies such
as those described by Meuth and Arrand (1982) on the nature
of mutations in an endogenous gene (aprt) and the potential for
mutant analysis in shuttle vector systems (Du Bridge and Calos,
1988) gives mammalian cell gene mutation assays a unique role
in understanding the mechanisms of mutagenesis in mammalian
and ultimately human cells.
Conclusions
Given that both chromosomal and gene mutational events are important in the generation of heritable disease and cancer, assays
for genotoxic potential which test for both end points would
appear most valuable. In this context assays for mammalian cell
gene mutation must be the most relevant of the battery of short
term tests which are available to us, and, in addition, changes
are detected in viable cells and the potential exists for analysis
of the nature of any genetic change. At present consideration is
being given to the possible exclusion of mammalian gene cell
mutation assays from the basic package of screening tests
recommended in the UK. Two arguments are put forward in
support of this proposal. First, that clastogenicity assays, which
are believed to be more reliable, serve to detect all mutagens.
This supposes that no non-clastogenic mutagens exist. At present
the evidence is that few such compounds do exist. Nevertheless,
until a concrete case is made we must adopt a position of caution
and accept the existence of genotoxic agents which will only be
detected by the mammalian cell gene mutation assays. Such assays
must, therefore, remain as a primary screen. The second
argument is based upon a belief that these assays deserve a poor
reputation as being difficult to perform and generate spurious
results. Within the United Kingdom a new generation of protocols
have been developed embodying experimental designs which
should provide data suitable for a statistical evaluation of results.
It is envisaged that the use of these new protocols will generate
sound data overcoming the objection of poor performance.
Whether or not the tests continue as a primary or supplementary
package we strongly recommend use of the new protocols.
We believe that until the problem of the non-clastogenic
mutagens is resolved (which may take up to 5 years), it is
premature to exclude the mammalian cell gene mutation assays
from the primary screen. We hope that such a reprieve will give
an opportunity to demonstrate the quality and relevance of data
which can be obtained from these assays. It will also provide
an opportunity to evaluate the strengths and weaknesses of the
individual assays available to us.
Acknowledgements
We are indebted to Professor B.A.Bridges for much helpful comment and to the
many 'interested parties' who have urged us to assemble these comments on the
relevance and practice of mammalian cell gene mutation assays. The views
expressed, however, are the responsibility of the authors alone.
457
C.F.Ariett and J.Cde
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