Search

The phosphorylation of histone H2AX in Serine 139 (gamma‐H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma‐H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma‐H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma‐H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma‐H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma‐H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma‐H2AX staining allows the detection of tissue‐specific effects in situ. Moreover, since gamma‐H2AX staining can be performed on formalin‐fixed and paraffin‐embedded tissue sections generated during repeated‐dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals. Environ. Mol. Mutagen. 60:4–16, 2019. © 2018 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

It is often assumed that genotoxic substances will be detected more easily by using in vitro rather than in vivo genotoxicity tests since higher concentrations, more cytotoxicity and static exposures can be achieved. However, there is a paucity of data demonstrating whether genotoxic substances are detected at lower concentrations in cell culture in vitro than can be reached in the blood of animals treated in vivo. To investigate this issue, we compared the lowest concentration required for induction of chromosomal damage in vitro (lowest observed effective concentration, or LOEC) with the concentration of the test substance in blood at the lowest dose required for biologically relevant induction of micronuclei in vivo (lowest observed effective dose, or LOED). In total, 83 substances were found for which the LOED could be identified or estimated, where concentrations in blood and micronucleus data were available via the same route of administration in the same species, and in vitro chromosomal damage data were available. 39.8 % of substances were positive in vivo at blood concentrations that were lower than the LOEC in vitro, 22.9 % were positive at similar concentrations, and 37.3 % of substances were positive in vivo at higher concentrations. Distribution analysis showed a very wide scatter of > 6 orders of magnitude across these 3 categories. When mode of action was evaluated, the distribution of clastogens and aneugens across the 3 categories was very similar. Thus, the ability to detect induction of micronuclei in bone marrow in vivo regardless of the mechanism for micronucleus induction, is clearly not solely determined by the concentration of test substance which induced chromosomal damage in vitro., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

In 2008 we published recommendations on chemicals that would be appropriate to evaluate the sensitivity and specificity of new/modified mammalian cell genotoxicity tests, in particular to avoid misleading positive results. In light of new data it is appropriate to update these lists of chemicals. An expert panel was convened and has revised the recommended chemicals to fit the following different sets of characteristics: • Group 1: chemicals that should be detected as positive in in vitro mammalian cell genotoxicity tests. Chemicals in this group are all in vivo genotoxins at one or more endpoints, either due to DNA-reactive or non DNA-reactive mechanisms. Many are known carcinogens with a mutagenic mode of action, but a sub-class of probable aneugens has been introduced. • Group 2: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests. Chemicals in this group are usually negative in vivo and non-DNA-reactive. They are either non-carcinogenic or rodent carcinogens with a non-mutagenic mode of action. • Group 3: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce gene mutations in mouse lymphoma cells, chromosomal aberrations or micronuclei, often at high concentrations or at high levels of cytotoxicity. Chemicals in this group are generally negative in vivo and negative in the Ames test. They are either non-carcinogenic or rodent carcinogens with an accepted non-mutagenic mode of action. This group contains comments as to any conditions that can be identified under which misleading positive results are likely to occur. This paper, therefore, updates these three recommended lists of chemicals and describes how these should be used for any test evaluation program., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)