- Email: [email protected]
Corrigendum to “Report from the in vitro micronucleus assay working group” [Mutat. Res. 540 (2003) 153–163]
Mutation Research 564 (2004) 97–100
Corrigendum
Corrigendum to “Report from the in vitro micronucleus assay working group” [Mutat. Res. 540 (2003) 153–163]夽 Micheline Kirsch-Voldersa,∗ , Toshio Sofunib , Marilyn Aardemac , Silvio Albertinid , David Eastmonde , Michael Fenechf , Motoi Ishidate Jr.g , Stephan Kirchnerd , Elisabeth Lorgeh , Takeshi Moritai , Hannu Norppaj , Jordi Surrall´esk , Annelies Vanhauwaerta , Akihiro Wakatal a
k
Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Peinlann 2, Brussels 1050, Belgium b NovusGene Inc., Tokyo, Japan c The Procter and Gamble Co., Miami Valley Laboratories, Cincinnati, OH, USA d F. Hoffmann-La Roche Ltd., Basel, Switzerland e Environmental Toxicology Graduate Program, University of California, Riverside, California f CSIRO Health Sciences and Nutrition, Adelaide, BC, Australia g Independent Consultant for Genotoxicity Testing for Chemicals, Tokyo, Japan h Biologie Servier, Toxicology Center, Gidy, France i Pre-Clinical Development Department, GlaxoSmithKline K.K.n. Tokyo, Japan j Finnish Institute of Occupational Health, Helsinki, Finland Group of Mutagenesis, Department of Genetics and Microbiology, Universitat Aut`onoma de Barcelona, Barcelona, Spain l Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan Available online 23 August 2004
Valuable comments were received regarding the difficulty in understanding our recommendations on how to assess cytotoxicity in cells blocked with cytochalasinB. We recommended in our paper that if toxicity is found, at least three √ analyzable test concentrations with no more than a 10-fold spacing between the concentrations should be tested, with the highest concentration exhibiting approximately 60% toxicity (if achievable). The limit of 60% toxicity is based on limited data which show that in some cases (i.e. some 夽 doi
of the original article:10.1016/j.mrgentox.2003.07.005. Corresponding author. Tel.: +32 2 629 34 23; fax: +32 2 629 27 59. E-mail address: [email protected] (M. Kirsch-Volders). ∗
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.07.002
aneugens), a very steep toxicity curve is observed and very closely spaced doses in the range of 50–60% toxicity need to be evaluated. Kirchner et al. (personal communication, 2002) (Table 2) indicated that in validation experiments the lowest observed effective dose (LOEDs) for the aneugens diethylstilbestrol and vincristine showed a relative cell count (RCC) of 42% and 43%, respectively; this corresponds to a toxicity of approximately 60%. These compounds might not have been found to be micronucleus inducers if they had not been tested up to the 60% toxicity level. In particular cases, toxicity levels within 50–60% or more could be accepted when keeping in mind appropriate dose spacing and the possibility of mitotic block (e.g. by measuring MI). Additional studies with different types of
98
M. Kirsch-Volders et al. / Mutation Research 564 (2004) 97–100
Table 1 Influence of cell death and cytostasis on cytotoxicity index Ideal control
CBPI Cytotoxicity (%)a Cytotoxicity (%)b Cell death (%) Cytostasis (%)a Cytostasis (%)b
Compound A
2 0 0 0 0 0
Compound B
Dose 1
Dose 2
Dose 1
Dose 2
1.5 50 50 0 50 50
1.2 80 80 0 80 80
1.6 50 37 20 30 37
1.4 80 60 50 30 60
CBPI: cytochalasin-B dependent proliferation index. Compound A is cytostatic agent that does not include cell death. Compound B is an agent that induces both cell death and cytostasis. a Expressed as proportion of all cells, alive and dead. b Expressed as proportion of viable cells only.
aneugens comparing toxicity between 50 and 60% and micronucleus induction would provide valuable information to assess the probability of missing an effect of these compounds at lower toxicity levels. The working group was aware that 60% toxicity as measured in an assay without cytochalasin-B may differ from 60% toxicity as measured in an assay with cytochalasin-B.
It is understood that the baseline for calculation of cytotoxicity is given by the data obtained with cultures of untreated cells. Hence, the above-mentioned 60% refers to the ratio of the toxicity measured in treated cells to the toxicity measured in untreated cells, giving as an example data (Kirchner et al., personal communication, 2002) shown in Table 2 of our original paper.
Table 2 Nucleation data from an experiment using human lymphocytes (20 h treatment started 48 h after culture initiation) Dose (g/mL)
Replicate
Cell stage
Total cells scored
Mono
Bi
Multi
CBPIa Value
RIb Cytotoxicity (%)∗
Value
Cytotoxicity (%)
Solvent
A B C D
155 274 302 263
821 710 685 687
24 16 13 50
1000 1000 1000 1000
1.87 1.74 1.71 1.79
0.87 0.74 0.71 0.79
160.3
A B
404 399
591 596
5 5
1000 1000
1.60 1.61
22
0.60 0.61
22
221.9
A B
499 694
499 306
2 0
1000 1000
1.50 1.31
47
0.50 0.31
47
307.1
A B
811 755
189 245
0 0
1000 1000
1.19 1.25
72
0.19 0.25
72
425.0
A B
1000 1000
0 0
0 0
1000 1000
1 1
100
0 0
100
CBPI (note that all values are calculated using rounded values to two decimal places): Using the sample data for concentration 221.9 g/mL as presented in the table, CBPI can be calculated as follows: Replicate A: mononucleate cells = 499/binucleate cells = 499/multinucleate cells = 2/total cells analysed = 1000. Replicate B: mononucleate cells = 694/binucleate cells = 306/multinucleate cells = 0/total cells analysed = 1000. A replicate CBPI = 499 + 2 (499) + 3 (2)/1000 = 1.50. B replicate CBPI = 694 + 2 (306) + 3 (0)/1000 = 1.31. Treated group mean CBPIT = 1.41. The vehicle control group mean CBPIC = 1.78. Cytotoxicity = 100 − 100[(CBPIT − 1)/(CBPIC − 1)]. The cytotoxicity for the concentration of 221.9 g/mL is therefore: 100 − 100[(1.41 − 1)/(1.78 − 1)] = 47%. b RI (note that all values are calculated using rounded values to two decimal places): Using the sample data for concentration 221.9 g/mL as presented in the table, RI can be calculated as follows: Replicate A: binucleate cells = 499/multinucleate cells = 2/total cells analysed = 1000. Replicate B: binucleate cells = 306/multinucleate cells = 0/total cells analysed = 1000. A replicate RI = 499 + 2 (2)/1000 = 0.50. B replicate RI = 306 + 2 (0)/1000 = 0.31. Treated group mean RIT = 0.41. The vehicle control group mean RIC = 0.78. Relative replication index in treated cultures = (RIT /RIC ) × 100. The RI for the concentration of 221.9 g/mL is: 0.41/0.78 × 100 = 53%. This indicates a value relative to the control. Expressed as a percentage cytotoxicity the value is: 100 − 53 = 47%. a
M. Kirsch-Volders et al. / Mutation Research 564 (2004) 97–100
This was probably not clear enough and we propose with this short note a clarification of the ways to appropriately calculate cytotoxicity in the in vitro MN assay with cytochalasin-B. Assessment of toxicity is essential in determining the appropriate dose range for assessing the genotoxic potential of a test compound. Cytotoxicity is a general term, which may result at the cellular level from different events: necrosis, apoptosis, cell cycle delay, mitotic block, mitotic slippage, etc. Therefore, definition of these terms is important. In our view a cytotoxic event would include the following: 1. inhibition of cell division (i.e. a cytostatic event); 2. induction of necrosis (cell death); 3. induction of apoptosis (cell death). The level of cytotoxicity measured will depend on whether it includes all these measurements or only one or two of them. Kirsch-Volders et al. [1] and Fenech
99
[2,3] proposed inclusion of nuclearity, necrosis and apoptosis, in addition to cell counting, for assessment of cytotoxicity. Using a simple example involving an ideal control and two agents, one mainly inducing cytostasis without cell death and the other inducing cell death as well as cytostasis, it is relatively easy to see that the cytotoxicity index will vary depending on whether dead/dying cells are included or not (Table 1 and Fig. 1) (Fenech, personal communication, 2004). If cell death is not included, the estimation of cytotoxicity is an indirect, not perfect one. At the present time for genotoxicity screening purposes, cytotoxicity is measured practically as a change in cell numbers and/or cell proliferation, and not directly as cell death. At the Plymouth IWGT workshop on the in vitro micronucleus test the extent of inhibition that is required to adequately detect clastogens and aneugens was debated extensively and measures for the assessment of toxicity were recommended:
Fig. 1. Influence of cell death and cytostasis on cytochalasin-B dependent proliferation index (CBPI). Compound A is a cytostatic agent that does not induce cell death. Compound B is an agent that induces both cell death and cytostasis. DC: dead cell due to necrosis or apoptosis.
100
M. Kirsch-Volders et al. / Mutation Research 564 (2004) 97–100
i. determining cell proliferation in both treated and control cultures, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; ii. other markers, such as confluency, apoptosis or necrosis, were mentioned as additional markers providing valuable information. It appears that there is uncertainty regarding which formula suggested by the IWGT group should be applied in calculating cytotoxicity in tests that use cytochalasin-B. The formula selected should give relative values (treated versus untreated) depending on the endpoints (number of cell cycles, number of nuclei) assessed. For further clarification, two alternatives are given below, with worked examples: (1) Cytokinesis-block proliferation index (CBPI) CBPI indicates the number of cell cycles per cell during the period of exposure to cytochalasin-B: No. of mononucleate cells + 2 × no. of binucleate cells + 3 × no. of multinucleate cells CBPI = Total no. of cells Cytotoxicity should be calculated as follows: CBPIT − 1 Cytotoxicity = 100 − 100 CBPIC − 1 where T: test chemical treatment culture and C: vehicle control culture. A CBPI of 1 in a treated culture (all cells are mononucleate) is equivalent to 100% “cytotoxicity”, i.e. 60% would still reflect a cell division rate of ∼40% during treatment. This formula is the closest to relative cell count (RCC) which is one of the proposed measurements to assess “cytotoxicity” when cell lines are used in the absence of cytochalasin-B. (2) Replication index (RI) Replication index (RI) indicates the relative number of nuclei in treated cultures compared to control cultures:
(No. of binucleate cells + 2 × no. of multinucleate cells)T / total no. of cellsT × 100. RI = (No. of binucleate cells + 2 × no. of multinucleate cells)C / total no. of cellsC Therefore, to measure the reduction in treated cultures due to chemical treatment: Cytotoxicity = 100 − RI
1. Worked examples Table 2 gives data from an experiment using human lymphocytes exposed for 20 hours to a test chemical starting 48 h after culture initiation. Cytochalasin-B was present for the duration of treatment. 2. Conclusion In conclusion, the proposed calculations allow the estimation of toxicity levels, i.e. the relative inhibition of proliferation. It is obvious that both ways to calculate the index of cytotoxicity versus cell proliferation relative to the experimental control give the same value. It is important to emphasise that the 60% level was recommended based on experience that the activity of aneugenic compounds that display a very steep toxicity curve may not be detected at lower toxicity levels. References [1] M. Kirsch-Volders, A. Elhajouji, E. Cundari, P. Van Hummelen, The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction, Mutat. Res. 392 (1997) 19–30. [2] M. Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81–95. [3] M. Fenech, A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division, Mutagenesis 15 (4) (2000) 329– 336.
Corrigendum
Corrigendum to “Report from the in vitro micronucleus assay working group” [Mutat. Res. 540 (2003) 153–163]夽 Micheline Kirsch-Voldersa,∗ , Toshio Sofunib , Marilyn Aardemac , Silvio Albertinid , David Eastmonde , Michael Fenechf , Motoi Ishidate Jr.g , Stephan Kirchnerd , Elisabeth Lorgeh , Takeshi Moritai , Hannu Norppaj , Jordi Surrall´esk , Annelies Vanhauwaerta , Akihiro Wakatal a
k
Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Peinlann 2, Brussels 1050, Belgium b NovusGene Inc., Tokyo, Japan c The Procter and Gamble Co., Miami Valley Laboratories, Cincinnati, OH, USA d F. Hoffmann-La Roche Ltd., Basel, Switzerland e Environmental Toxicology Graduate Program, University of California, Riverside, California f CSIRO Health Sciences and Nutrition, Adelaide, BC, Australia g Independent Consultant for Genotoxicity Testing for Chemicals, Tokyo, Japan h Biologie Servier, Toxicology Center, Gidy, France i Pre-Clinical Development Department, GlaxoSmithKline K.K.n. Tokyo, Japan j Finnish Institute of Occupational Health, Helsinki, Finland Group of Mutagenesis, Department of Genetics and Microbiology, Universitat Aut`onoma de Barcelona, Barcelona, Spain l Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan Available online 23 August 2004
Valuable comments were received regarding the difficulty in understanding our recommendations on how to assess cytotoxicity in cells blocked with cytochalasinB. We recommended in our paper that if toxicity is found, at least three √ analyzable test concentrations with no more than a 10-fold spacing between the concentrations should be tested, with the highest concentration exhibiting approximately 60% toxicity (if achievable). The limit of 60% toxicity is based on limited data which show that in some cases (i.e. some 夽 doi
of the original article:10.1016/j.mrgentox.2003.07.005. Corresponding author. Tel.: +32 2 629 34 23; fax: +32 2 629 27 59. E-mail address: [email protected] (M. Kirsch-Volders). ∗
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.07.002
aneugens), a very steep toxicity curve is observed and very closely spaced doses in the range of 50–60% toxicity need to be evaluated. Kirchner et al. (personal communication, 2002) (Table 2) indicated that in validation experiments the lowest observed effective dose (LOEDs) for the aneugens diethylstilbestrol and vincristine showed a relative cell count (RCC) of 42% and 43%, respectively; this corresponds to a toxicity of approximately 60%. These compounds might not have been found to be micronucleus inducers if they had not been tested up to the 60% toxicity level. In particular cases, toxicity levels within 50–60% or more could be accepted when keeping in mind appropriate dose spacing and the possibility of mitotic block (e.g. by measuring MI). Additional studies with different types of
98
M. Kirsch-Volders et al. / Mutation Research 564 (2004) 97–100
Table 1 Influence of cell death and cytostasis on cytotoxicity index Ideal control
CBPI Cytotoxicity (%)a Cytotoxicity (%)b Cell death (%) Cytostasis (%)a Cytostasis (%)b
Compound A
2 0 0 0 0 0
Compound B
Dose 1
Dose 2
Dose 1
Dose 2
1.5 50 50 0 50 50
1.2 80 80 0 80 80
1.6 50 37 20 30 37
1.4 80 60 50 30 60
CBPI: cytochalasin-B dependent proliferation index. Compound A is cytostatic agent that does not include cell death. Compound B is an agent that induces both cell death and cytostasis. a Expressed as proportion of all cells, alive and dead. b Expressed as proportion of viable cells only.
aneugens comparing toxicity between 50 and 60% and micronucleus induction would provide valuable information to assess the probability of missing an effect of these compounds at lower toxicity levels. The working group was aware that 60% toxicity as measured in an assay without cytochalasin-B may differ from 60% toxicity as measured in an assay with cytochalasin-B.
It is understood that the baseline for calculation of cytotoxicity is given by the data obtained with cultures of untreated cells. Hence, the above-mentioned 60% refers to the ratio of the toxicity measured in treated cells to the toxicity measured in untreated cells, giving as an example data (Kirchner et al., personal communication, 2002) shown in Table 2 of our original paper.
Table 2 Nucleation data from an experiment using human lymphocytes (20 h treatment started 48 h after culture initiation) Dose (g/mL)
Replicate
Cell stage
Total cells scored
Mono
Bi
Multi
CBPIa Value
RIb Cytotoxicity (%)∗
Value
Cytotoxicity (%)
Solvent
A B C D
155 274 302 263
821 710 685 687
24 16 13 50
1000 1000 1000 1000
1.87 1.74 1.71 1.79
0.87 0.74 0.71 0.79
160.3
A B
404 399
591 596
5 5
1000 1000
1.60 1.61
22
0.60 0.61
22
221.9
A B
499 694
499 306
2 0
1000 1000
1.50 1.31
47
0.50 0.31
47
307.1
A B
811 755
189 245
0 0
1000 1000
1.19 1.25
72
0.19 0.25
72
425.0
A B
1000 1000
0 0
0 0
1000 1000
1 1
100
0 0
100
CBPI (note that all values are calculated using rounded values to two decimal places): Using the sample data for concentration 221.9 g/mL as presented in the table, CBPI can be calculated as follows: Replicate A: mononucleate cells = 499/binucleate cells = 499/multinucleate cells = 2/total cells analysed = 1000. Replicate B: mononucleate cells = 694/binucleate cells = 306/multinucleate cells = 0/total cells analysed = 1000. A replicate CBPI = 499 + 2 (499) + 3 (2)/1000 = 1.50. B replicate CBPI = 694 + 2 (306) + 3 (0)/1000 = 1.31. Treated group mean CBPIT = 1.41. The vehicle control group mean CBPIC = 1.78. Cytotoxicity = 100 − 100[(CBPIT − 1)/(CBPIC − 1)]. The cytotoxicity for the concentration of 221.9 g/mL is therefore: 100 − 100[(1.41 − 1)/(1.78 − 1)] = 47%. b RI (note that all values are calculated using rounded values to two decimal places): Using the sample data for concentration 221.9 g/mL as presented in the table, RI can be calculated as follows: Replicate A: binucleate cells = 499/multinucleate cells = 2/total cells analysed = 1000. Replicate B: binucleate cells = 306/multinucleate cells = 0/total cells analysed = 1000. A replicate RI = 499 + 2 (2)/1000 = 0.50. B replicate RI = 306 + 2 (0)/1000 = 0.31. Treated group mean RIT = 0.41. The vehicle control group mean RIC = 0.78. Relative replication index in treated cultures = (RIT /RIC ) × 100. The RI for the concentration of 221.9 g/mL is: 0.41/0.78 × 100 = 53%. This indicates a value relative to the control. Expressed as a percentage cytotoxicity the value is: 100 − 53 = 47%. a
M. Kirsch-Volders et al. / Mutation Research 564 (2004) 97–100
This was probably not clear enough and we propose with this short note a clarification of the ways to appropriately calculate cytotoxicity in the in vitro MN assay with cytochalasin-B. Assessment of toxicity is essential in determining the appropriate dose range for assessing the genotoxic potential of a test compound. Cytotoxicity is a general term, which may result at the cellular level from different events: necrosis, apoptosis, cell cycle delay, mitotic block, mitotic slippage, etc. Therefore, definition of these terms is important. In our view a cytotoxic event would include the following: 1. inhibition of cell division (i.e. a cytostatic event); 2. induction of necrosis (cell death); 3. induction of apoptosis (cell death). The level of cytotoxicity measured will depend on whether it includes all these measurements or only one or two of them. Kirsch-Volders et al. [1] and Fenech
99
[2,3] proposed inclusion of nuclearity, necrosis and apoptosis, in addition to cell counting, for assessment of cytotoxicity. Using a simple example involving an ideal control and two agents, one mainly inducing cytostasis without cell death and the other inducing cell death as well as cytostasis, it is relatively easy to see that the cytotoxicity index will vary depending on whether dead/dying cells are included or not (Table 1 and Fig. 1) (Fenech, personal communication, 2004). If cell death is not included, the estimation of cytotoxicity is an indirect, not perfect one. At the present time for genotoxicity screening purposes, cytotoxicity is measured practically as a change in cell numbers and/or cell proliferation, and not directly as cell death. At the Plymouth IWGT workshop on the in vitro micronucleus test the extent of inhibition that is required to adequately detect clastogens and aneugens was debated extensively and measures for the assessment of toxicity were recommended:
Fig. 1. Influence of cell death and cytostasis on cytochalasin-B dependent proliferation index (CBPI). Compound A is a cytostatic agent that does not induce cell death. Compound B is an agent that induces both cell death and cytostasis. DC: dead cell due to necrosis or apoptosis.
100
M. Kirsch-Volders et al. / Mutation Research 564 (2004) 97–100
i. determining cell proliferation in both treated and control cultures, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; ii. other markers, such as confluency, apoptosis or necrosis, were mentioned as additional markers providing valuable information. It appears that there is uncertainty regarding which formula suggested by the IWGT group should be applied in calculating cytotoxicity in tests that use cytochalasin-B. The formula selected should give relative values (treated versus untreated) depending on the endpoints (number of cell cycles, number of nuclei) assessed. For further clarification, two alternatives are given below, with worked examples: (1) Cytokinesis-block proliferation index (CBPI) CBPI indicates the number of cell cycles per cell during the period of exposure to cytochalasin-B: No. of mononucleate cells + 2 × no. of binucleate cells + 3 × no. of multinucleate cells CBPI = Total no. of cells Cytotoxicity should be calculated as follows: CBPIT − 1 Cytotoxicity = 100 − 100 CBPIC − 1 where T: test chemical treatment culture and C: vehicle control culture. A CBPI of 1 in a treated culture (all cells are mononucleate) is equivalent to 100% “cytotoxicity”, i.e. 60% would still reflect a cell division rate of ∼40% during treatment. This formula is the closest to relative cell count (RCC) which is one of the proposed measurements to assess “cytotoxicity” when cell lines are used in the absence of cytochalasin-B. (2) Replication index (RI) Replication index (RI) indicates the relative number of nuclei in treated cultures compared to control cultures:
(No. of binucleate cells + 2 × no. of multinucleate cells)T / total no. of cellsT × 100. RI = (No. of binucleate cells + 2 × no. of multinucleate cells)C / total no. of cellsC Therefore, to measure the reduction in treated cultures due to chemical treatment: Cytotoxicity = 100 − RI
1. Worked examples Table 2 gives data from an experiment using human lymphocytes exposed for 20 hours to a test chemical starting 48 h after culture initiation. Cytochalasin-B was present for the duration of treatment. 2. Conclusion In conclusion, the proposed calculations allow the estimation of toxicity levels, i.e. the relative inhibition of proliferation. It is obvious that both ways to calculate the index of cytotoxicity versus cell proliferation relative to the experimental control give the same value. It is important to emphasise that the 60% level was recommended based on experience that the activity of aneugenic compounds that display a very steep toxicity curve may not be detected at lower toxicity levels. References [1] M. Kirsch-Volders, A. Elhajouji, E. Cundari, P. Van Hummelen, The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction, Mutat. Res. 392 (1997) 19–30. [2] M. Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81–95. [3] M. Fenech, A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division, Mutagenesis 15 (4) (2000) 329– 336.