Mutagenesis Advance Access originally published online on April 24, 2007
Mutagenesis 2007 22(4):275-280; doi:10.1093/mutage/gem013
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Cytotoxicity in cultured mammalian cells is a function of the method used to estimate it
Genetic Toxicology, Safety Assessment, AstraZeneca, R&D Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Up to prescribed limits, the maximum test compound concentrations used in mammalian cell genotoxicity assays in vitro are determined by cytotoxicity, unless limited by solubility in solvents or culture medium. However, ‘cytotoxicity’ is different in the various test systems, both in the methods used to estimate it and the levels of toxicity that must be achieved. For example, in cytogenetic assays, the acceptable level of toxicity is defined as a ‘significant reduction (>50%)’ in cell number, culture confluency or mitotic index (MI) (OECD 473, ICH S2A), whereas mutation tests require relative total growth or cloning efficiency (CE) to be reduced by 80–90% (OECD 476, ICH S2A). In this study using mouse lymphoma cells, it was shown that, for a variety of agents with differing modes of action, cytotoxicity varies considerably depending on the method used to estimate it. Specifically, trypan blue exclusion, MI and binucleate incidence all grossly underestimate cytotoxicity in comparison with cell growth or CE. If the performance of different test systems is to be compared, or if data from different assays are to be used for the meaningful assessment of a novel chemical entity, it is essential that similar methods to determine cytotoxicity are used for them all. The purpose of this paper is not to recommend a specific method to determine cytotoxicity, although it can be argued that any such method must quantify the proportion of cells capable of division following treatment, but rather to draw attention to the fact that apparent toxicity depends upon the method used to estimate it.
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Introduction |
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In all in vitro genotoxicity tests used for regulatory submission, compounds must be tested up to prescribed maximum concentrations (5000 µg/ml or 10 mmol/l), the limit of solubility in solvent or culture medium, or the highest level allowed by cytotoxicity. Testing excessively toxic concentrations can lead to problems with data interpretation or may cause artefactual positive results (1
,2). Accordingly, accurate assessment of cytotoxicity is critical for assay validity. The methods to be used to assess toxicity and the recommended levels to be achieved are defined in OECD and ICH guidelines (3,4,5) but these differ between test systems. For example, cytogenetic assays, including in vitro micronucleus tests require cell number, confluency, mitotic index (MI) (3) or binucleate index (BI) (6) all to be reduced by 50% or more. In contrast, cell mutation assays require relative total growth (RTG) or cloning efficient to be reduced by 80–90% and 
30% reduction in viability estimated by trypan blue exclusion (TBE) has been suggested for the comet assay (7). During in-house development of a micronucleus test using mouse lymphoma L5178Y tk+/– cells, comparisons were made between several standard methods used to estimate cytotoxicity, i.e. RTG, cloning efficiency (CE), MI, BI (in cytochalasin B-blocked cultures) and TBE. The compounds selected had well-established, different mechanisms of toxicity, i.e. 2,4-dinitrophenol (DNP) (uncoupler of oxidative phosphorylation), mitomycin C (cross-linking agent), 4-nitroquinoline-N-oxide (NQO) (DNA damaging agent) and colchicine and carbendazim (aneugens). |
Materials and methods |
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All chemical and reagents were purchased from Sigma–Aldrich (Dorset, UK) unless otherwise stated.
Test chemicals
Colchicine, DNP, mitomycin C and NQO were used without further purification. A purified sample of carbendazim was generously supplied by Prof. J. M. Parry (University of Wales, Swansea, UK). All were dissolved in dimethyl sulphoxide (DMSO) except colchicine, which was dissolved in water.
Cell line and culture conditions
Mouse lymphoma L5178Y cells, clone 3.7.2c, were obtained from Dr J. Cole, (MRC Cell Mutation Unit, University of Sussex, Brighton, UK). They were confirmed to be free from mycoplasma infection and had the expected karyotype (8), including two copies of chromosome 11 detected by fluorescent in situ hybridization (A. Doherty, personal communication). Cells were cultured in RPMI 1640 medium (Invitrogen, Paisley, UK) supplemented with 10% heat inactivated donor horse serum (DHS), 2 mmol/l L-glutamine, 2 mmol/l sodium pyruvate, 1% Pluronic F68, 200 IU/ml penicillin, 200 µg/ml streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2 in air.
Treatment with test chemicals
All chemicals were tested in the absence of any exogenous metabolizing system. For 4-h exposure (DNP, mitomycin C, NQO), 107 cells were suspended in 20 ml RPMI containing 2.5% DHS; for 24-h exposure (carbendazim, colchicine), 4 x 106 cells were suspended in 20 ml RPMI with 10% DHS. In all cases, solvent or test chemical solution was added to the medium at 1% v/v. There were two cultures at each test concentration of test chemical, and four cultures in the solvent controls.
Following treatment, the cells were centrifuged, washed once and re-suspended in 50 ml RPMI containing 10% DHS (4-h exposure) or counted and adjusted to 2 x 105 per ml (24-h exposure).
All chemicals were tested in at least two independent experiments.
Mutation assay
Mutation at the thymidine kinase (tk) locus was assessed for all test compounds by resistance to trifluorothymidine (TFT). The method used was the standard microtitre method employed in this laboratory essentially as described by Clements (9).
Micronucleus assays
The number of micronuclei per 1000 mononucleate cells was assessed for all test compounds in cultures without cytochalasin B. The micronucleus incidence per 1000 binucleated cells in cytochalasin B-arrested cultures was assessed for colchicine, carbendazim and NQO. Cytochalasin B was added at the end of the 24-h treatment period and removed 24 h later.
Methods used to determine cytoxicity
Relative cloning efficiency
CE was determined 1 or 2 days after the end of treatment. To estimate CE, cells were plated out at 1.6 cells per well in 96-well microtitre plates and it was calculated from the zero term of the Poisson distribution, P(0), the proportion of wells in which a colony had not grown. Relative cloning efficiency (RCE) for an individual culture was the CE expressed as a percentage of the mean control CE. RCE was calculated for all test chemicals. It should be noted that the CE values in this study were not corrected for the numbers of cells lost during the treatment period.
RTG, relative suspension growth and population doubling
Cultures were incubated for a further day after the initial CE estimates and counted. RTG was calculated as the product of suspension growth (SG) and RCE. SG was calculated for 4-h and 24-h exposure periods as follows:
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RTG for an individual culture was the product of SG and CE and was expressed as a percentage of the mean control RTG. RTG was calculated for all test chemicals.
Population doubling (PD) was calculated as follows:
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Relative PD for an individual culture was its PD expressed as a percentage of the control PD. PD was calculated using rounded cell numbers for clarity for the examples shown in Tables V and VI.
Mitotic index One day after the end of treatment with test chemical, slides were prepared by centrifuging 2 x 104 cells in a Cytospin 3 (Shandon) centrifuge (800 rpm for 8 min), and the cells were fixed with 3 : 1 methanol : acetic acid and stained with acridine orange. It should be noted that, because the mouse lymphoma cells were in exponential growth, colcemid or colchicine was not added to increase the number of metaphases. The number of cells in mitosis per 1000 cells was counted and expressed as a percentage of the control mean. MI was calculated for all test chemicals.
Binucleate index Immediately after test compound treatment, cytochalasin B in DMSO was added to the cultures to give a final concentration of 3 µg/ml. Cells were incubated for 1 day at 37°C and Cytospin slides were prepared, fixed and stained as for MI. The number of binucleate cells per 1000 cells was calculated and expressed as a percentage of the control mean. BI was calculated for all test chemicals except DNP and mitomycin C.
Trypan blue exclusion At the end of treatment with test compounds, a sample of cells were mixed with trypan blue (0.4% in water) at a ratio of 4 : 1 and the number of viable cells (not stained) counted using a haemocytometer. Viability was expressed as a percentage of control number of cells excluding trypan blue. Although numbers of trypan blue staining cells were not counted and it is recognized that these may be lost from the population relatively quickly, since TBE was estimated at the end of the treatment period, and because the cell numbers were the same as the control except at concentrations giving greatly increased toxicity estimated by some of the other parameters, this is considered acceptable for the purposes of these comparisons. TBE was measured for all test chemicals except DNP.
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Results |
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2,4-Dinitrophenol
Representative results for DNP are shown in Table I and Figure 1. A concentration-related increase in both TFT-resistant mutant frequency and micronucleus frequency in mononucleate cells was seen. Concentration-related decreases in survival assessed by RTG, RSG and RCE were seen with <10% survival at the highest concentrations. Markedly less toxicity was indicated by MI with 59% survival seen at the highest concentration analyzed in comparison with 3 and 1% with RCE and RTG, respectively.
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Colchicine
Representative results for colchicine are shown in Table II and Figure 2. A concentration-related increase in micronuclei in mononucleated cells was seen but the increase in binucleated cells was less convincing. However, it should be noted that these increases were seen only at concentrations reducing RTG or RSG to <10%. Further, at a concentration (0.063 µmol/l) reducing RTG, RSG and RCE to <5%, MI was increased and both BI and TBE were virtually unaffected. The increase in MI was obviously expected from the mode of action of colchicine.
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Carbendazim
Representative results for carbendazim are shown in Table III and Figure 3. Concentration-related increases were seen in TFT-resistant mutant frequency and micronuclei in both mononucleate and binucleate cells. Significant increases in all three parameters were seen with concentrations reducing RTG to 10%. However, at this concentration, which also gave 21% RCE and RSG, there was no reduction in MI, BI or TBE.
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Mitomycin C
At the single concentrations of mitomycin C tested using 4-h exposure (0.75 µmol/l), significant increases in micronuclei in mononucleated cells and TFT-resistant mutant frequency were seen in all experiments (Table IV). However, mean toxicity data (Figure 4) show survival of <10% estimated by RCE or RTG, but virtually no effect on MI or TBE.
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4-Nitroquinoline-N-oxide
At the single concentrations of NQO tested using 4-h (1 µmol/l) or 24-h exposure (0.33 µmol/l), significant increases in micronuclei, in both mononucleated and binucleated cells, and TFT resistance were seen in all experiments (Table IV). Representative cytotoxicity results for NQO following 4-h and 24-h exposure are shown in Figures 5 and 6, respectively. As with mitomycin C, marked toxicity was seen using RTG and, to a lesser extent, RCE but MI, BI and TBE all indicated lower levels of toxicity.
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Discussion |
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The mouse lymphoma tk assay is routinely used in this laboratory for regulatory testing purposes, but in order to provide mechanistic information and screens with higher throughput, both an in vitro micronucleus test and a comet assay using mouse lymphoma cells have been explored. In order to compare results between the three tests, it was thought necessary to use the same maximum concentrations in them all. Therefore, various methods that have been proposed to measure cytotoxicity in them were compared and their performance, with some possible reasons for the differences between them, are discussed below.
The international workshop on genotoxicity test procedures working group on the mouse lymphoma TK assay unanimously agreed that RTG should be the cytotoxicity measure to be used for concentration selection and data evaluation (10), so it was used as the reference point in this study. Extensive analysis of mouse lymphoma data from a large number of tests from different laboratories showed conclusively that it is essential to take into account the numbers of cells lost during treatment with test agents (10) and when this was done, cloning efficiency (RCE), cell growth (RSG) and RTG all gave very similar results for the majority of compounds. The results here are consistent with this conclusion, although it should be noted that RCE was not measured immediately after treatment and was not corrected for cells lost during the treatment period; therefore, RCE in this study slightly underestimated cytotoxicity in comparison with RSG and RTG.
It is now generally agreed that the best estimate of cytotoxicity for in vitro cytogenetic assays using Chinese hamster overy (CHO) cells measures cell growth from the start of treatment until sampling for metaphase analysis. Average generation time was initially suggested as a better alternative to MI, which was considered to be of limited value (11), but, more recently, PD has been recommended as a more accurate estimate of growth suppression (12). Although PD is essentially similar to RTG in that increase in cell number is the major component, the calculation differs significantly by comparing counts as log ratios, i.e. increases are compared exponentially rather than linearly. Also, the magnitudes of reduction indicating adequate cytotoxicity has been achieved differ, with 50% PD and 10–20% RTG recommended for CHO cytogenetics and mouse lymphoma TK assays, respectively. However, the apparent disparity may be rather smaller than it appears when the cell division rates and the times at which the cells are counted are taken into consideration. CHO cells have an average cell generation time of 12.9–14.8 h (11) resulting in typical control cultures undergoing 
1.2 PD by 20 h after the start of treatment (12). In contrast, mouse lymphoma cells in this laboratory have a PD time of 8–10 h and typical control cultures have undergone 1.5 PD 22 h after the start of a 3-h treatment period and a further 2.5 PD in the next 24 h. Therefore, the estimate of cell growth, RSG, contributing to RTG is made over a time where control cultures would have typically gone through at least 4 PD. Finally, it should be noted that RSG, by not taking into consideration the exponential increases in cell numbers once they have started to divide, overestimates toxicity in comparison with PD. After 4-h treatment with 3.5 mmol/l DNP, RSG is reduced to 15% but when calculated as PD this is 34% (Table V). Similarly, after treatment with concentrations of colchicine and carbendazim giving RSGs of 20 and 21%, the equivalent PDs are 63 and 66%, respectively (Table VI). Clearly, time of counting after the start of treatment in relation to the cell generation time is important and it seems likely that, after two to four cell divisions, 50% PD and 20% RSG will be similar for different cell types and treated with various agents.
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In comparison with RTG (or any of the parameters measuring increase in cell numbers even taking into account the differences between RSG and PD outlined above), both BI and MI measured 1 day after treatment grossly underestimated cytotoxicity for all the compounds tested. However, this is not surprising since neither parameter takes account of the actual cell numbers present in a population, merely the proportion of them that are attempting to progress through mitosis or cytokinesis. Consequently, any cells that have died and disintegrated before BI or MI is measured are ignored, and those that have sustained lethal damage and will subsequently die cannot be estimated. This point is illustrated by colchicine and carbendazim which have virtually no effect on BI or MI at concentrations that reduce RTG, RSG and Day 1 RCE to <5%. In agreement with this, BI and the related proliferative index measurement (13
) have also been reported to underestimate toxicity in comparison with cell growth for long-term human T-cell cultures (14). MI has also previously been shown to correlate poorly with rate of cell division (11). The only example here where MI differed significantly from BI was colchicine which, as expected, increased MI significantly through its mode of action.
In addition to the end-points used in mutation and cytogenetics assays, a limited amount of data were generated in this study using TBE since this has been proposed as the toxicity measure in a protocol for regulatory submissions for an in vitro micronucleus test using CHO cells (15,16). It has also been proposed for use in the in vitro comet assay (7) and was recommended as one of the measures to be used in the in vitro alkaline elution assay using rat hepatocytes (17). As with BI and MI, results showed TBE grossly underestimated cytotoxicity in comparison with RTG. Again, this is not surprising and the reasons for it have been appreciated for some time. To quote from Freshney's standard text on basic tissue culture technique (18): ‘Most viability tests rely on a breakdown in membrane integrity that is determined by the uptake of a dye to which the cell is normally impermeable, e.g. trypan blue etc .... However, this effect is immediate and does not always predict ultimate survival. Furthermore, dye exclusion tends to overestimate viability .... While short-term tests are convenient and are usually quick and easy to perform, they reveal only cells that are dead (i.e. permeable) at the time of the assay. Frequently, however, cells that have been subjected to toxic influences (e.g. irradiation, antineoplastic drugs) show an effect several hours, or even days, later. The nature of the tests required to measure viability in these cases is necessarily different, since by the time the measurement is made, the dead cells may have disappeared. Therefore, long-term tests are used to determine survival rather than short-term toxicity. Survival implies the retention of regenerative capacity and is usually measured by plating efficiency.’
The issue of ‘dead or dying’ cells has been addressed by Rossman's group (19) using arsenite in a human osteosarcoma line, comparing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and clonal survival (essentially RCE). This showed that, because arsenite induces delayed toxicity, killing cells after its removal from the medium, NR and MTT gave results comparable with CE only if an interval of at least 48 h was allowed after the end of treatment. Similarly, it has been suggested that with rat hepatocytes in vitro, TBE is more useful if performed after a short recovery period (17). Any method, such as TBE, MTT or NR, which essentially determines cell function will underestimate cytotoxicity if performed immediately after the end of treatment with toxic agents.
The purpose of this paper is not to recommend any particular method to measure cytotoxicity for any of the mammalian cell genotoxicity assays but rather to draw attention to the considerable differences between them. The over sensitivity of both the mouse lymphoma TK assay and in vitro clastogenicity tests has recently been highlighted (20,21) and possible areas for improvement are being considered by the International Life Sciences Institute/Health and Environmental Sciences Institute Subcommittee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity Testing. One of the generally agreed points is the need to re-examine the maximum level of cytotoxicity to be tested and the most appropriate measures to estimate it. Further, the ICH guidelines are currently being reviewed and possible changes include the addition of the in vitro micronucleus test to those currently accepted or, in contrast, the omission of any in vitro mammalian cell assay from the standard test battery. If any meaningful comparison of the performance of difference assays is to be made, responses must be measured at concentrations resulting in death/survival of a similar percentage of cells in the various test systems and excessive cytotoxicity is likely to increase the number of irrelevant positive results. It has been suggested that the cytotoxicity measurement should be related to the genotoxic end-point in a particular assay, but this can be challenged by the argument that any response that occurs only at concentrations killing >90% of the target population is of limited value. Reduction in survival to 10% RTG is generally agreed to be the limit for the mouse lymphoma TK assay but some apparently more conservative estimates, e.g. 50–60% reduction in MI or BI or TBE, may actually result in cell mortality much >90%.
In conclusion, the data presented here do not allow a firm recommendation to be made on a single or best estimate of cytotoxicity to be used for all in vitro mammalian cell genotoxicity assays. It can be argued that, for cell lines which are able to grow at clonal density such as CHO and L5178Y, CE is the best method to measure survival but most genetic toxicologists would probably agree that some method of enumerating cells capable of going through a few cell divisions after treatment is required, and PD which takes exponential cell growth into account would appear to be better than linear comparisons.
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Acknowledgments |
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The authors wish to thank the following AstraZeneca staff: Ann Doherty for cytogenetic analyses, Catherine Smith for TBE data and Katie Clare for excellent technical assistance.
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* To whom correspondence should be addressed. Tel: +44 1625 513749; Fax: +44 1625 231281; Email: mike.odonovan{at}astrazeneca.com
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Received on August 2, 2006; revised on February 8, 2007; accepted on March 2, 2007.
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