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Abstract
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.
Highlights
During the last years, the Adverse Outcome Pathway (AOP) concept has arisen as a pragmatic tool in the toxicological evaluation of all kind of chemicals based on a more human relevant mechanistic toxicology
We measured relative suspension growth (RSG) using the proliferation assay with a broad range of concentrations of each chemical to perform the final assays with non-cytotoxic concentrations (RSG 48 h after the treatment > 80%) and including a cytotoxic concentration (Table 1)
When comparing hOGG1-sensitive sites in treated versus non-treated cells, significant differences were found from 0.625 mM of KBrO3 onwards. human alkyladenine DNA glycosylase (hAAG) and Endo III did not show any activity in KBrO3-treated cells, and no increase in hAAG- or Endo III-sensitive sites was detected compared to non-treated cells at any of the tested concentrations
Summary
The Adverse Outcome Pathway (AOP) concept has arisen as a pragmatic tool in the toxicological evaluation of all kind of chemicals based on a more human relevant mechanistic toxicology. Different assays are available for measuring KE, such as the alkaline comet assay (single cell gel electrophoresis) which is a method to measure DNA damage levels, strand breaks (SB) and alkalilabile sites (ALS) (apurinic/apyrimidinic-AP-sites or baseless sugars), at the level of individual cells. Its versatility makes it a widely used technique, as it can be applied to any eukaryotic cell type, including disaggregated tissues from which single cells or nuclear suspensions can be obtained (Vasquez 2012; Azqueta and Collins 2013; Jackson et al 2013; Asare et al 2016). If the DNA integrity of a nucleoid is disrupted by a SB, supercoiling is relaxed and part of the DNA will extend due to the electrophoretic force giving a comet-like image when stained with a nucleic acid specific dye and evaluated by fluorescence microscopy; whereas if DNA remains undamaged, supercoiling is preserved and, no comet tail is formed (Collins 2004)
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