- Email: [email protected]
Fourth international workgroup on genotoxicity testing: Results of the Comet assay workgroup
Abstracts / Toxicology 226 (2006) 12–77
importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have integrated the databases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not doselimiting. Reference
2.
3.
4.
Hayashi, M., MacGregor, J.T., Gatehouse, D.G., Adler, I.-D., Blakey, D.H., Dertinger, S.D., Krishna, G., Morita, T., Russo, A., Sutou, S., 2000. Environ. Mol. Mutagen. 35 (3), 234–252.
doi:10.1016/j.tox.2006.05.053 Fourth international workgroup on genotoxicity testing: Results of the Comet assay workgroup Brian Burlinson
5.
Huntingdon Life Sciences, UK E-mail address: [email protected] As part of the ongoing IWGT initiative, a series of workshops were held as part of the ICEM/EMS joint conference in San Francisco, September 2005. The main purpose of this workshop was to enhance the ability of the Comet assay to be used for regulatory decision making with the primary focus being the rodent alkaline (pH >13) Comet assay as a replacement/alternative for the in vivo unscheduled DNA synthesis (UDS) assay. Topics and conclusions were: 1. Number of dose levels In situations where evident toxicity is not present, i.e. maximum dose level of 2 g/kg is reached, can the
6.
35
limit dose only be tested? The group decision was no, because downturns in response exist (bell-shaped dose-response curve). Also, positive responses at multiple dose levels reinforce the biological relevance of the result. Does the method used to process tissues, i.e. isolation of nuclei or whole cells, make a difference? The consensus was that there were not enough data to decide although data that were presented suggested that the issue was unlikely to be a problem. However, any international validation study should consider both processing methods for different tissues using reference chemicals with diverse mechanisms of action and covering a range of potencies. Do we need to include measures of cytotoxicity? The consensus was “yes” and suggested methods included dye exclusion tests for membrane integrity and/or metabolic competency. Determining the frequency of “dead” cells (hedgehogs or ghost cells) by the neutral diffusion assay was also suggested as a useful measure of cytotoxicity. The final method was using histopathology to evaluate levels of necrosis and apoptosis when results were positive. It was pointed out that there was a need to standardise ways to present histopathological findings. Image analysis (IA) or manual scoring The consensus was that IA is preferred but not required. Hedgehogs (ghost or dead cells) should be excluded from IA data collection although determining their frequency may be useful for data interpretation. If IA is used then percentage of tail DNA appeared to be the most linearly related to dose and the easiest to compare across studies and therefore should always be presented. Data on the distribution of migration among cells should also be presented. Need to develop and include historical negative/ positive control data The minimal number of studies needed was not defined but should be enough studies to demonstrate the stability of the negative/positive controls. Criteria for determining the acceptability of new studies, based on historical control data, should be developed for each tissue by each lab. There was some discussion on the background responses for negative controls and there was a consensus that negative controls should exhibit measurable DNA migration. A suggestion was made that to detect crosslinking agents a mean value of around 10–20% tail DNA is needed. Minimal reporting standards It was agreed that to ensure that all studies can be independently evaluated a minimum reporting
36
Abstracts / Toxicology 226 (2006) 12–77
Mouse lymphoma L5178Y tk+/− cells were treated with dimethylsulphoxide (DMSO) as a standard solvent; agents known to induce oxidative damage: gamma irradiation and potassium bromate (KBrO3 ) and wellestablished alkylating agents: methyl methansulphonate (MMS) and ethylnitrosourea (ENU). No increase in DNA break sites was seen with FPG, ENDOIII or hOGG1 following treatment with 1–6% DMSO (Table 1). With gamma irradiation (1–10 Gy), there was a significant increase in breaks with all three enzymes (Table 1). Treatment with KBrO3 (0.25–2.5 mmol/L), showed similar increases in breaks with both FPG and hOGG1 but increases in breaks with ENDOIII were only seen at the highest concentration 2.5 mmol/L (Table 1). Following treatment with MMS and ENU, there were significant increases in breaks with FPG and ENDOIII but, in contrast, there was no additional damage with hOGG1 (Table 1). The data indicate that FPG, ENDOIII and hOGG1 are all able to detect oxidative DNA damage. However, FPG and ENDOIII also detect high levels of damage to DNA following alkylation and, therefore, are not specific for oxidative DNA damage alone. hOGG1 does not detect alkylation damage and, therefore, appears to be more specific for oxidative DNA damage, in particular 8-oxoguanine.
standard for regulatory submissions and publications will be developed. This standard will be consistent with OECD in vivo genetic toxicology test method guidelines. Previous publications have covered some aspects of protocol design and reporting (Hartmann et al., 2003; Wiklund and Agurell, 2003). References Hartmann, A.A., Agurell, E., Beevers, C., Brendler-Schwaab, S., Burlinson, B., Clay, P., Collins, A., Smith, A., Speit, G., Thybaud, V., Tice, R.R., 2003. Mutagenesis 18, 45–51. Wiklund, S.J., Agurell, E., 2003. Mutagenesis 18, 167–175.
doi:10.1016/j.tox.2006.05.054 Oral Abstracts hOGG1 is specific for detecting 8-oxoguanine in the modified comet assay Catherine C. Smith, Mike O’Donovan, Elizabeth Martin Genetic Toxicology, Safety Assessment, AstraZeneca, Mereside, Alderley Park, Cheshire SK10 4TG, UK E-mail address: (C.C. Smith)
[email protected]
The specificity and sensitivity of the comet assay can be enhanced by incubating lysed cells with lesionspecific endonucleases that recognise certain damaged bases. The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the use of formamidopyrimidine DNA-glycosylase (FPG) in the comet assay to measure levels of oxidative damage, in particular 8-oxoguanine (Gedik and Collins, 2005). In the present study, the use of FPG was compared with endonuclease III (ENDOIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1) for detecting different types of DNA lesions.
Acknowledgements We would like to thank Andrew Collins for his kind donation of the FPG and ENDOIII enzymes and Debbie Godwin and Karen Oldman (AstraZeneca) for performing statistical analyses. References Gedik, C.M., Collins, A., ESCODD (European Standards Committee on Oxidative DNA Damage), 2005. FASEB J. 19, 82–84.
doi:10.1016/j.tox.2006.05.055 Table 1 The effect of FPG, ENDOIII and hOGG1 on TI following treatment with DMSO (6%), gamma irradiation (10 Gy), KBrO3 (2.5 mmol/L), MMS (23 mol/L) and EMS (2 mmol/L) Treatment
DMSO (6%) Gamma (10 Gy) KBrO3 (2.5 mmol/L) MMS (23 mol/L) ENU (2 mmol/L)
% Tail intensity No enzyme
+FPG
± ± ± ± ±
6.6 28.0 91.3 94.0 62.6
3.7 15.0 2.1 4.5 14.6
1.3 4.3 0.5 2.5 3.2
± ± ± ± ±
+ENDOIII 1.7 7.1* 5.0* 2.3* 4.9*
Mean ± S.D. from at least three independent experiments; * p < 0.001, one-sided ANOVA.
5.0 34.3 42.1 47.9 61.8
± ± ± ± ±
1.2 7.2* 3.7* 11.7* 3.6*
+hOGG1 3.9 19.6 89.8 4.9 19.2
± ± ± ± ±
4.8 4.3* 6.0* 1.2 5.8
importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have integrated the databases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not doselimiting. Reference
2.
3.
4.
Hayashi, M., MacGregor, J.T., Gatehouse, D.G., Adler, I.-D., Blakey, D.H., Dertinger, S.D., Krishna, G., Morita, T., Russo, A., Sutou, S., 2000. Environ. Mol. Mutagen. 35 (3), 234–252.
doi:10.1016/j.tox.2006.05.053 Fourth international workgroup on genotoxicity testing: Results of the Comet assay workgroup Brian Burlinson
5.
Huntingdon Life Sciences, UK E-mail address: [email protected] As part of the ongoing IWGT initiative, a series of workshops were held as part of the ICEM/EMS joint conference in San Francisco, September 2005. The main purpose of this workshop was to enhance the ability of the Comet assay to be used for regulatory decision making with the primary focus being the rodent alkaline (pH >13) Comet assay as a replacement/alternative for the in vivo unscheduled DNA synthesis (UDS) assay. Topics and conclusions were: 1. Number of dose levels In situations where evident toxicity is not present, i.e. maximum dose level of 2 g/kg is reached, can the
6.
35
limit dose only be tested? The group decision was no, because downturns in response exist (bell-shaped dose-response curve). Also, positive responses at multiple dose levels reinforce the biological relevance of the result. Does the method used to process tissues, i.e. isolation of nuclei or whole cells, make a difference? The consensus was that there were not enough data to decide although data that were presented suggested that the issue was unlikely to be a problem. However, any international validation study should consider both processing methods for different tissues using reference chemicals with diverse mechanisms of action and covering a range of potencies. Do we need to include measures of cytotoxicity? The consensus was “yes” and suggested methods included dye exclusion tests for membrane integrity and/or metabolic competency. Determining the frequency of “dead” cells (hedgehogs or ghost cells) by the neutral diffusion assay was also suggested as a useful measure of cytotoxicity. The final method was using histopathology to evaluate levels of necrosis and apoptosis when results were positive. It was pointed out that there was a need to standardise ways to present histopathological findings. Image analysis (IA) or manual scoring The consensus was that IA is preferred but not required. Hedgehogs (ghost or dead cells) should be excluded from IA data collection although determining their frequency may be useful for data interpretation. If IA is used then percentage of tail DNA appeared to be the most linearly related to dose and the easiest to compare across studies and therefore should always be presented. Data on the distribution of migration among cells should also be presented. Need to develop and include historical negative/ positive control data The minimal number of studies needed was not defined but should be enough studies to demonstrate the stability of the negative/positive controls. Criteria for determining the acceptability of new studies, based on historical control data, should be developed for each tissue by each lab. There was some discussion on the background responses for negative controls and there was a consensus that negative controls should exhibit measurable DNA migration. A suggestion was made that to detect crosslinking agents a mean value of around 10–20% tail DNA is needed. Minimal reporting standards It was agreed that to ensure that all studies can be independently evaluated a minimum reporting
36
Abstracts / Toxicology 226 (2006) 12–77
Mouse lymphoma L5178Y tk+/− cells were treated with dimethylsulphoxide (DMSO) as a standard solvent; agents known to induce oxidative damage: gamma irradiation and potassium bromate (KBrO3 ) and wellestablished alkylating agents: methyl methansulphonate (MMS) and ethylnitrosourea (ENU). No increase in DNA break sites was seen with FPG, ENDOIII or hOGG1 following treatment with 1–6% DMSO (Table 1). With gamma irradiation (1–10 Gy), there was a significant increase in breaks with all three enzymes (Table 1). Treatment with KBrO3 (0.25–2.5 mmol/L), showed similar increases in breaks with both FPG and hOGG1 but increases in breaks with ENDOIII were only seen at the highest concentration 2.5 mmol/L (Table 1). Following treatment with MMS and ENU, there were significant increases in breaks with FPG and ENDOIII but, in contrast, there was no additional damage with hOGG1 (Table 1). The data indicate that FPG, ENDOIII and hOGG1 are all able to detect oxidative DNA damage. However, FPG and ENDOIII also detect high levels of damage to DNA following alkylation and, therefore, are not specific for oxidative DNA damage alone. hOGG1 does not detect alkylation damage and, therefore, appears to be more specific for oxidative DNA damage, in particular 8-oxoguanine.
standard for regulatory submissions and publications will be developed. This standard will be consistent with OECD in vivo genetic toxicology test method guidelines. Previous publications have covered some aspects of protocol design and reporting (Hartmann et al., 2003; Wiklund and Agurell, 2003). References Hartmann, A.A., Agurell, E., Beevers, C., Brendler-Schwaab, S., Burlinson, B., Clay, P., Collins, A., Smith, A., Speit, G., Thybaud, V., Tice, R.R., 2003. Mutagenesis 18, 45–51. Wiklund, S.J., Agurell, E., 2003. Mutagenesis 18, 167–175.
doi:10.1016/j.tox.2006.05.054 Oral Abstracts hOGG1 is specific for detecting 8-oxoguanine in the modified comet assay Catherine C. Smith, Mike O’Donovan, Elizabeth Martin Genetic Toxicology, Safety Assessment, AstraZeneca, Mereside, Alderley Park, Cheshire SK10 4TG, UK E-mail address: (C.C. Smith)
[email protected]
The specificity and sensitivity of the comet assay can be enhanced by incubating lysed cells with lesionspecific endonucleases that recognise certain damaged bases. The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the use of formamidopyrimidine DNA-glycosylase (FPG) in the comet assay to measure levels of oxidative damage, in particular 8-oxoguanine (Gedik and Collins, 2005). In the present study, the use of FPG was compared with endonuclease III (ENDOIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1) for detecting different types of DNA lesions.
Acknowledgements We would like to thank Andrew Collins for his kind donation of the FPG and ENDOIII enzymes and Debbie Godwin and Karen Oldman (AstraZeneca) for performing statistical analyses. References Gedik, C.M., Collins, A., ESCODD (European Standards Committee on Oxidative DNA Damage), 2005. FASEB J. 19, 82–84.
doi:10.1016/j.tox.2006.05.055 Table 1 The effect of FPG, ENDOIII and hOGG1 on TI following treatment with DMSO (6%), gamma irradiation (10 Gy), KBrO3 (2.5 mmol/L), MMS (23 mol/L) and EMS (2 mmol/L) Treatment
DMSO (6%) Gamma (10 Gy) KBrO3 (2.5 mmol/L) MMS (23 mol/L) ENU (2 mmol/L)
% Tail intensity No enzyme
+FPG
± ± ± ± ±
6.6 28.0 91.3 94.0 62.6
3.7 15.0 2.1 4.5 14.6
1.3 4.3 0.5 2.5 3.2
± ± ± ± ±
+ENDOIII 1.7 7.1* 5.0* 2.3* 4.9*
Mean ± S.D. from at least three independent experiments; * p < 0.001, one-sided ANOVA.
5.0 34.3 42.1 47.9 61.8
± ± ± ± ±
1.2 7.2* 3.7* 11.7* 3.6*
+hOGG1 3.9 19.6 89.8 4.9 19.2
± ± ± ± ±
4.8 4.3* 6.0* 1.2 5.8