Abstract
Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were exactly identical; (3) the power of the Comet assay to detect a low level of genotoxic potential can be elevated to a level higher than that of MN test by using DNA resynthesis inhibitors, such as araC and HU.
Highlights
Many methods have been used to identify genotoxic substances, including the detection of DNA damage, chromosome aberrations, and gene mutations both in vitro and in vivo
The Comet assay is essentially a method of detecting single-strand breaks (SSBs), we have shown that a low level of SSB as the initial damage cannot be detected by the Comet assay because these SSBs disappear following a repair event and that SSBs as initial DNA damage can be well detected in the acellular Comet assay [4]
The development of initial damage into alkali-labile sites following repair events is an important factor supporting the sensitivity of Comet assay, it is assumed that DNA resynthesis and rejoining events following SSB formation reduce the sensitivity of Comet assay in detecting DNA damage such as alkylated bases and bulky base adducts
Summary
Many methods have been used to identify genotoxic substances, including the detection of DNA damage, chromosome aberrations, and gene mutations both in vitro and in vivo. When the sensitivity of a genotoxicity testing method is regarded as high, it means that it can detect wide variety of compounds with unknown genotoxic potential and that the assay can detect a low level of genotoxic potential by known genotoxic compounds. The former is very important in order to avoid pseudonegative results. The genotoxic dose response curve for genotoxic compounds is thought to reach zero without having a no-response level at a low Journal of Nucleic Acids dose This statement forms the basis of the “nonthreshold concept” in the risk assessment, which describes the absence of a threshold in genotoxic potential.
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