Mutagenesis, Vol. 14, No. 3, 265-270,
May 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Discussion Forum |
The mouse lymphoma assay in the wake of ICH4—where are we now?
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, East Sussex BN1 9RR, UK and 1 Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA
Background
The ultimate goal of short-term tests should surely be to detect the complete spectrum of heritable effects or genetic events involved in the aetiology of cancer. Thus the types of mutational events that must be detected include point mutations, deletions, translocations, rearrangements, aneuploidy and mitotic recombination/gene conversion. The mouse lymphoma assay (MLA) has been widely used for many years for determining the potential for chemicals to cause the full broad spectrum of mutational damage (Applegate et al., 1990
; Mitchell et al., 1997). Substantial research has been conducted to understand the capabilities (and limitations) of the assay, its proper conduct and the appropriate interpretation of test data (Clive et al., 1979, 1990; Hozier et al., 1981, 1982, 1989; Moore-Brown, 1981; Cole et al., 1983, 1990, 1991; Turner et al., 1984; Moore et al., 1985a,b, 1989; Blazak et al., 1986; Doerr et al., 1989; Applegate et al., 1990; Moore and Doerr, 1990; Liechty et al., 1993, 1994, 1996; Zhang et al., 1996; Mitchell et al., 1997; Clark et al., 1998).
Historically the various international regulatory agencies have required that chemicals be evaluated for their ability to induce point mutations and chromosomal damage. While in recent years some regulatory agencies have chosen to recommend the MLA to meet both of these requirements for the in vitro mammalian assay (Dearfield et al., 1991; Department of Health and Human Services, 1997), others have required the conduct of an in vitro chromosome aberration assay. Thus, it has been necessary for pharmaceutical companies to conduct both an in vitro chromosome aberration analysis and a gene mutation assay (usually the MLA) in order to register their products in the world market.
To facilitate the international registration of pharmaceuticals the International Conference on Harmonisation (ICH) was formed to reach consensus concerning both testing requirements and protocol guidelines. The recent Fourth ICH (ICH4) document entitled Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals acknowledged that the thymidine kinase (tk)-based MLA detects both gene mutations and clastogenic effects and that, subject to the use of appropriate test protocols, `the various in vitro tests for chromosomal damage and the mouse lymphoma tk assay yield results with a high degree of congruence for compounds that are regarded as genotoxic but yield negative results in the bacterial reverse mutation assay' and are therefore `currently considered interchangeable when used together with other genotoxicity tests in a standard battery for genotoxicity testing of pharmaceuticals, if these test protocols are used'.
In addition to concluding that the MLA and chromosome aberration analysis are `interchangeable', the ICH4 Committee made two very specific (and very controversial) protocol recommendations for the MLA: (i) in the case of a negative result following 3–4 h treatment, a continuous treatment of 24 h without S9 was considered advisable; (ii) the Committee stated a preference for the microwell cloning protocol rather than the soft agar method which has traditionally been used by most laboratories conducting the MLA. These recommendations were made based on data generated in a trial initiated under the ICH auspices, the majority of the data from which were at the time largely unpublished in the peer reviewed literature.
In this paper, it is our aim to stimulate discussion, at a rather critical time, of the important issues surrounding the use of the MLA in the light of these ICH4 recommendations. Subjects to be tackled include: (i) a brief historical perspective of the development of the assay; (ii) a survey of the available mechanistic information that describes the capabilities of the assay and the possibility that the MLA is `equivalent' to in vitro chromosome aberration and aneuploidy tests; (iii) the importance of colony sizing; (iv) the justification for the newly recommended ICH4 `appropriate test protocols'; (v) issues that we feel are important to address before such protocol variations become `Tablets of Stone'.
Overview of MLA development and assay characterization
Phase I. Initial protocol development
The L5178Y tk+/– mouse lymphoma assay was first described in 1972 by Clive and co-workers (Clive et al., 1972a,b). It was soon apparent that it had advantages as a mutation test, including: (i) rapid growth in suspension culture to high cell density, which provided for the very large numbers of cells necessary for a statistically valid test to be treated and subcultured for the required expression time before being subjected to mutant selection; (ii) the relatively short time (48 h) required for the expression of newly induced mutants made the assay more cost effective and also meant that fluctuations in the relative proportion of mutants due to clonal expansion and variable fitness of some newly induced mutants assumed less importance.
Following their original publication, Clive and his collaborators undertook a large-scale investigation of the potential and optimal conduct of the assay (Clive et al., 1979). The investigations included the use of trifluorothymidine (TFT) to select tk mutants (Moore-Brown, 1981), a comparison of the hprt and tk loci (Moore and Clive, 1982), an analysis of the best expression time for tk mutant selection and a description of distinct `large' and `small' colony tk mutants (Clive et al., 1979; Moore and Clive, 1982; Moore et al., 1985a,b). The large and small colony mutants figure in the conduct and information generated by the assay.
Early on during this phase, a protocol was devised for use in the National Toxicology Program (NTP) trials, the results of which were ultimately published in Tennant et al. (1987). However, it also became apparent during the 1970s that the MLA was acquiring a controversial reputation and a number of groups experienced a variety of problems with the assay (summarized in Clive et al., 1987).
Phase II. Resolution of protocol issues, evaluation of the ability of the MLA to detect chemical clastogens and aneugens and detailed mutant analysis
Protocol improvement and the use of a microwell cloning procedure.
During the 1980s, many of the protocol issues that had caused disillusionment with the reproducibility of the MLA were resolved (Moore-Brown et al., 1981; Moore and Howard, 1982; Brusick, 1986; Mitchell et al., 1997), crucially the role of agar quality in the growth and recovery of small colony mutants (Meyer et al., 1986).
In order to overcome the latter problem, a protocol variation that had initially been validated using L5178Y wild-type cells (Cole et al., 1983) involving cloning cells in liquid medium in 96-well microtitre trays instead of in soft agar in Petri dishes was successfully applied to tk+/– cells (Cole et al., 1986). A detailed comparison of cloning tk+/– cells in agar and microtitre trays showed that under optimal conditions the two methods gave essentially similar results when both sets of plates were scored by eye so that all small colonies were counted (J.Cole and D.Clive, unpublished observations, 1988). Oberly et al. (1997) confirmed the similar mutagenic response using the two procedures, while noting that cloning efficiency, as well as spontaneous and induced mutant frequencies, were somewhat higher using microwell cloning.
It is important to stress here that the microtitre (or microwell) protocol variation does not represent a major difference in approach to the MLA, since all other aspects of the assay remain unchanged. However, the use of the microwell variation does have implications when the results of assays from different laboratories using the two techniques are compared.
Comparison of chromosome aberration frequency with small-colony mutant induction.
Using a variety of chemicals, Doerr and Moore conducted an extensive evaluation of the correlation between gross chromosome aberration induction in mouse lymphoma cells and small colony tk mutant induction (Doerr et al., 1989; Moore and Doerr, 1990). From these studies it was clear that chemicals that induce small colony tk mutants also induce gross aberrations in the mouse lymphoma cells. As expected, they found no simple mathematical relationship between the two responses but it was concluded that small colony mutants result from event(s) that not only prevent the expression of the tk gene but also cause the cells to grow slowly. Such mutants could result from alterations that: (i) affect expression of the tk and linked loci (chromosome 11 events); (ii) affect the tk and other single essential loci in the genome, i.e. multiple point mutations; (iii) affect the tk locus and have a chromosome event (other than chromosome 11) that results in slow growth. Given that large colony tk mutants have normal growth rates they must result from events that affect the tk locus and possibly other loci, but not any loci that would make the cell grow slowly.
Chromosomal analysis of large and small mutant colonies.
Initially, detailed banded chromosome analysis of colonies selected from agar plates showed that a high percentage of small colony mutants have a wide variety of visible chromosome 11b aberrations, while large colonies do not, leading to the conclusion that large colonies are the result of single gene mutations, while small colonies represent chromosomal mutations (Hozier et al., 1981). However, more recently chromosome painting analysis of colonies selected using the microwell protocol (Zhang et al., 1996) demonstrated that ~25% of large colonies did show rearranged chromosome 11, compared with 75% of small colonies, and that the majority of the chromosomal abnormalities involved complex rearrangements often involving an increase in chromosome 11 material.
The NcoI restriction fragment length polymorphism (RFLP) analysis. Using the restriction enzyme NcoI and RFLP analysis of mutant colonies Applegate et al. (1990) demonstrated that mutation in both large and small colonies was often associated with loss of the entire tk+ allele. Since this was accompanied in some cases by duplication of the remaining tk– allele, it was conjectured that somatic recombination, including mitotic recombination or gene conversion, might account for loss of tk activity in at least some cases.
Subsequent analysis (Clive et al., 1990) confirmed that overall while the large majority (91%) of the small colonies demonstrated loss of the tk11b allele, it was also lost in a substantial proportion (~70%) of large colonies. For some point mutagens such as ethylmethane sulphonate, which induce primarily but not exclusively large colony mutants, <25% of the large colony mutants lost the entire tk+ allele. In other cases, such as the very potent point mutagen 2-amino-6-N-hydroxyadenine, induction of multiple point mutations may result in a more complex relationship between colony size and observed damage (Clive et al., 1991; Moore et al., 1991).
Microsatellite analysis (Liechty et al., 1994) has revealed that relatively large portions of chromosome 11b can be deleted in small colony mutants and also gave results that are consistent with aneuploidy induction, i.e. mutants that showed two copies of chromosome 11a (and no 11b) had only the chromosome 11a-linked microsatellite sequences.
Thus, to date, the combined NcoI, microsatellite and chromosome analyses suggest that quite complex large deletions, possibly extending outside the tk gene, particularly in small colony mutants, may be involved in the induction of tk mutants. However, while the spectrum of mutations detected by the MLA may include point mutation and intragenic deletion, mitotic non-disjunction (aneuploidy), translocation and allele loss, mitotic recombination and gene conversion, it should be noted that not all such events are always detected with equal efficiency following treatment with particular test substances.
p53 status of L5178y mouse lymphoma cells.
Very recently it has been demonstrated that L5178Y cells contain two mutant p53 alleles (Clark et al., 1998), one an exon 4 point mutation that gives a stop codon, thus producing a non-functional protein, the other a point mutation in exon 5. In L5178Y/TK+/– 3.7.2C cells, the exon 4 mutant allele is linked (on chromosome 11b) with the tk+ allele and the exon 5 mutant allele is linked (on chromosome 11a) with the tk– allele. It is possible that the dysfunctional p53 protein in L5178Y cells may account for the sensitivity of these cells to mutagens and for the assay's capability to detect the chromosomal rearrangements and mitotic recombination often seen in the later stages of cancer development.
The importance of optimal small colony mutant detection.
Finally, it has been acknowledged that the utility of the assay depends on an ability to detect the broad range of mutagenic events represented by optimal detection of both large and small colonies and the protocol for the conduct of the MLA has been updated to emphasize this point (Dearfield et al., 1991; Clive et al., 1995). This has been endorsed by the recent US EPA Phase III Workgroup which evaluated MLA literature published from 1976 to 1993 (Mitchell et al., 1997). The database included 602 chemicals of which a high proportion (~30%) could not be evaluated by the evaluation criteria established by the expert Workgroup. A major reason for this data deficiency, notably in the NTP MLA dataset, was failure to demonstrate adequate small colony detection in positive controls (Moore et al., 1999a).
ICH4 and the Japanese Trial: is a new protocol justified?
In 1994, in response to the ICH Committee discussions suggesting that the MLA could be used to detect the clastogenicity of chemicals, a group of Japanese Laboratories instigated a collaborative trial(s) designed to investigate the ability of the MLA to detect clastogens and in particular aneugens. Initially only the microwell protocol was chosen although later some laboratories using agar cloning were included; the study was ultimately extended to include 45 Japanese Laboratories in addition to seven other laboratories with several years experience using the assay (Sofuni et al., 1996). The 40 chemicals studied in the trial included 34 clastogens and/or spindle poisons as defined by chromosome aberration (CA) tests. Each chemical was tested blind by two or three laboratories. The results were compared with the historical database of in vitro CA tests, including the Chinese hamster lung fibroblast (CHL/IU) tests undertaken over a period of years by the Japanese (Matsuoka et al., 1996; Honma et al., 1999). Initial reports suggested that using the `standard' MLA protocol of 3–4 h treatment time, while many clastogens were deemed `positive', nine of 34 tested were negative. However, it has been reported that when treatment time was extended to 24 h (comparable with the long continuous exposure employed in the CHL assays, where treatment covering 1.5 cell cycles is recommended to detect `all' clastogens) a further seven were deemed positive (including the spindle-damaging agents colchicine, vincristine and diethylstilbestrol). Overall, of the combined results for short and long treatment times, it has been claimed that, using the microwell assay, only three of 34 (8.8%) chemicals were MLA-negative/CA-positive, two of which had not been tested in the MLA using the 24 h protocol. However, it has also been reported that following 24 h treatment both specificity and sensitivity were reduced when the cells were plated in agar.
As a result of this work, a number of recommendations and comments were made on the conduct of the MLA (Sofuni et al., 1997), including heterogeneity and the use of single or duplicate cultures, cytotoxicity parameters, relative survival and relative total growth, strategies for dose range finding and the statistical analysis of MLA data. These issues are obviously not specific to the microwell version of the MLA and indeed apply in one form or another to all mammalian cell in vitro assays. Moreover, since they have been the subject of considerable discussion in the past, and will no doubt be the subject of workshops in the future (e.g. the proposed UKEMS revision of Statistical Guidelines), they will not be addressed further here.
The issue we do wish to address here is that while the largely unpublished data from the Japanese Trial led the ICH4 committee to conclude that the MLA is indeed capable of detecting most substances that induce chromosome aberrations (including aneugens) and thus that the CA assays and the MLA are therefore currently considered interchangeable, it resulted in two controversial recommendations being noted in the ICH4 document: (i) in the case of a negative result following 3–4 h (treatment with and without S9), a continuous treatment of 24 h without activation was considered advisable; (ii) a requirement for the use of the microwell cloning protocol rather than agar cloning.
While we recognize the considerable feat involved in organizing and executing a large trial involving so many laboratories, including a substantial proportion previously inexperienced in the MLA, and endorse the general conclusion that the MLA is capable of detecting a wide spectrum of clastogens, these recommendations raise immediate questions that cannot be ignored.
Agar versus microwell cloning
Two issues need airing before the question `Is there loss of specificity and/or sensitivity when cells are cloned in agar after 24 h treatment?' is addressed.
Firstly, as we noted above, there is no sound theoretical reason why the two cloning procedures should differ when assessing the mutagenic potential of test substances and, indeed, under optimal conditions both give essentially similar responses (J.Cole and D.Clive, unpublished results). However, in practice, mutant frequencies (both spontaneous and induced) are frequently considerably higher when microwell cloning is employed. Two explanations have been suggested.
- (i) Agar plates are generally scored by automated colony counting, where some colonies may be obscured while others are too small to be `seen' by the colony counter. In contrast, colonies of all sizes are easily detected on the microtitre plates, which are routinely scored by eye (with or without use of the vital dye MTT to aid viable colony detection).
(ii) The use of liquid medium in the wells provides less stringent growth requirements for the small colonies, which appear to be at a selective disadvantage in agar and may fail to survive in sub-optimal conditions.
Secondly, while for the reasons discussed above the microwell procedure is considered by many workers to be preferable, when agar and microwell cloning are compared it should be recognized that both methods have advantages and disadvantages. An advantage of the agar method is that the plating procedure and automated colony scoring are certainly less time consuming and tedious than using microtitre plates. Conversely, a disadvantage of the microtitre method is that although it is not apparently susceptible to loss of small colonies, at present the plates are scored by eye and the colonies are simply classified as `large' or `small' by the scorer. This is somewhat subjective and does not generate the histograms of colony size produced by automated scoring of agar plates, which are the basis for designating colonies as large or small using the agar method. Lack of experience in scoring microtitre plates may be one reason why the results in the different laboratories taking part in the Japanese trial had such widely variable results for positive and negative controls (Moore et al., 1999b).
It is, however, highly probable that in the future automated scoring of microtitre plates using image analysis will be more widely available which may well overcome this disadvantage. There are additional issues which must be considered, particularly when using the microtitre method. These are discussed in more detail in a further publication (Moore et al., 1999b).
We recognize that many laboratories worldwide have had considerable experience over many years with the MLA using agar cloning and are naturally reluctant to forgo a large database unless agar is clearly proven to be inferior to the microwell protocol. It is clear to us that direct comparisons of results from different laboratories using the two procedures is certain to be problematic if small colonies are substantially under-represented in agar for some reason. Thus, any claim that 24 h exposure to a test substance results in loss of both specificity and sensitivity when cells are cloned in agar requires considerably more systematic experimentation, with the relevant data being made available in the literature for discussion before final conclusions can be drawn.
- We recommend that experiments should be undertaken by a number of laboratories experienced in both cloning procedures. A large population of cells should be treated with appropriate chemicals (e.g. MMS and colchicine) for 4 and 24 h under carefully defined conditions (see below), subcultured for the expression time (again see below) and then split and plated in either soft agar in Petri dishes or liquid medium in microtitre plates. Both automated and manual scoring should be used for scoring the agar plates to ensure that any discrepancy in mutant frequency was not due to underscoring of small colonies by the colony counter.
The requirement for 24 h treatment to detect some aneugens
The suggestion in ICH4 that in the case of a negative result following 3–4 h treatment (with and without S9) a continuous treatment of 24 h without activation was considered advisable is also open to debate. No data have been published to support these recommendations and none has been made available for us to evaluate. In the `standard' MLA protocol, considerable care is taken to ensure that the cells are growing optimally during the 4 h treatment period. Large numbers of cells (6–10x106) are treated and since L5178Y cells have a very short cell cycle time (~9–10 h), cells in all phases of the cell cycle are exposed to the test substance. It is certainly possible that in relatively rare cases, treatment during more than one cell cycle is necessary to induce mutations (see Cole et al., 1982). Furthermore, since the genotoxicity of non-DNA targeted chemicals such as spindle poisons or nucleotide analogues may well be cell cycle dependent, treated and untreated cultures should be carefully monitored during (and after) the 24 h treatment period, to enable an accurate assessment of both cell division time and cytotoxicity. In addition, to obtain reproducible results and avoid artefacts, paying considerable thought to culture conditions during long exposure may be necessary; simply prolonging exposure from 3 to 24 h under standard short treatment time conditions may not be advisable. At present it is unclear whether treatment conditions such as cell density, serum concentration, pH, shaken versus static cultures, treatment vessel and the optimal expression time for newly induced mutations after 24 h treatment, all of which may be relevant and affect the result, were taken into consideration in the Japanese trial.
- We strongly recommend that further experimental work on 24 h treatment should be undertaken as a matter of urgency. This should include: (i) optimizing culture conditions during treatment; (ii) a detailed examination of cell cycle effects and toxicity during and after long treatment (including a comparison of RTG and %survival); (iii) treatment with suitable toxic/non-clastogenic control substances (such as DMSO); (iv) an examination of expression time for newly induced mutants, since the optimal time may well be affected by long treatment with substances that are affecting the cell cycle.
Validation of the ability of the microwell/MLA to detect aneugens
If the detection of aneugens is considered an essential aspect of an in vitro mammalian cell assay and MLA is to be considered equivalent to in vitro cytogenetics assays in this respect there is an urgent need for a comprehensive validation of the assay's postulated ability to detect aneugens. This aspect of the MLA has yet to be addressed adequately. In the past, the large majority of mutant analyses have been undertaken following selection in agar and examples of aneugenicity have been detected. Further characterization of mutants picked from both soft agar and microwells is necessary, to confirm the spectrum of mutants induced by specific treatments, particularly following treatment with aneugens. Picking and expanding mutants from microwell plates is in practice considerably easier than from agar plates, provided that elementary precautions are taken to ensure the single cell origin of each colony analysed. This could best be achieved by plating the cells in microtitre trays at suitable cell density such that each `positive' well would have a high probability of containing a colony of single cell origin. It is also important that colonies should be randomly selected for analysis and should cover the full size range, so that the spectrum of mutations induced by the chosen test substance would be established.
Two analytical methods are available, banded chromosome analysis and/or treatment of the chromosome preparations with mouse chromosome-specific paints including chromosome 11. Chromosome preparations can be analysed by fluorescence in situ hybridization to determine any chromosome rearrangements present in the tk– mutants compared with the parental tk+/– strain. Ideally it would be best to use both methods to maximize the information gained. In addition, it would be useful to analyse the mutants using microsatellite analysis. Much of the published molecular analysis, to date, includes only southern analysis that detects the presence or absence of the tk allele. There is no theoretical reason why loss of the tk+ allele by itself would make the cell slow growing. In order to understand what is happening, one must look beyond the tk locus. Microsatellite analysis can identify the approximate size of the genetic alteration. Molecular and chromosome analysis should be performed upon sufficient colonies selected from the whole dose range and from the untreated culture.
Summary and recommendatons
We feel strongly that the two new recommendations for assay performance are premature. Before new MLA protocols promoting the microwell cloning and requiring 24 h treatment become `Tablets of Stone', we recommend the following.
- Before discarding the well-understood soft agar protocol, it is essential that further comparisons of the two protocols be made. There is currently insufficient scientific evidence that one protocol is superior to the other. A comparison of agar and microwell cloning in the same experiment by laboratories with demonstrable expertise in small colony detection is required, and is being undertaken (B.Myhr and M.Moore, personal communication).
- A comparison of 4 and 24 h treatment, in the latter case paying particular attention to treatment conditions, cellular growth and mutant expression time, is essential. Both the agar and the microwell cloning methods should be used and compared in side by side experiments.
- We feel that the MLA is fully capable of detecting the clastogenic potential of chemicals. Its efficiency in detecting aneuploidy is less well documented and requires further investigation.
With regard to the `interchangeability' or `equivalence' of the the MLA and CA analysis, we offer the following conclusions. Clearly, it has been amply demonstrated by both tk mutant analysis and detailed experiments in which both gene mutation and chromosomal damage (as detected by chromosome aberration or the micronucleus test) have been assessed (Doerr et al., 1989; Cole et al., 1990; Moore and Doerr, 1990) that the MLA is capable of detecting chromosomal damage induced by a wide range of compounds. However, while chromosome aberration tests and the MLA both detect chromosome damage they are not exact equivalents and different compounds result in different relative potencies when assessed by the two tests. This must be so, since the MLA detects damage in viable cells capable of forming colonies, in addition to detecting a wider range of genetic damage than can be visualized by gross chromosome aberration analysis. We consider this to be an advantage of using a well-conducted MLA as part of a test battery. While both cytogenetic analysis and the MLA can provide important information in assessing the ability of a chemical to cause genetic damage, it is clear that if one assay is to be selected for general use, we believe that the MLA provides information that is more complete and more relevant for the hazard characterization phase of risk assessment.
Notes
This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names of commercial products constitute endorsement or recommendation for use.
2 To whom correspondence should be addressed. Email: moore.martha{at}epamail.epa.gov 
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Received on September 21, 1998; accepted on January 20, 1999.
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