Journal
ARCHIVES OF TOXICOLOGY
Volume 95, Issue 8, Pages 2825-2838Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00204-021-03102-3
Keywords
Comet assay; Cross-links; Oxidized bases; Alkylated bases; Mechanism of action; In vitro
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Funding
- BIOGENSA project of the Ministry of Science, Innovation and Universities of the Spanish Government [AGL70640-R]
- European Regional Development Fund (ERDF)
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The field of mechanistic toxicology is becoming increasingly important for human health risk assessment. Researchers have successfully increased the sensitivity and specificity of detecting DNA damage by modifying the comet assay.
Mechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.
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