Optimization of the in vitro comet assay as a tool for mechanistic risk assessment

Abstract

Genotoxicity evaluation is of key importance in the health risk assessment of substances to which humans are exposed, as it has been long established that certain genotoxic compounds are able to damage DNA entailing severe consequences for human health, such as cancer. Current strategies of genotoxicity testing consider mainly final effects in DNA: mutations and chromosomal aberrations. However, a mechanistic approach more relevant to humans, in which not only classical endpoints but also mechanistic events (e.g., DNA oxidation or alkylation) are integrated and considered for risk assessment, is becoming more necessary. In this context, modifications to the in vitro comet assay protocol, which measures strand breaks and alkali labile sites in its standard version, arise as a promising alternative method in the detection of premutagenic lesions. The aim of this thesis was to develop and validate a new tool for in vitro genotoxicity testing, based on the comet assay, that can be used in the elucidation of different mechanisms of action. This approach may represent a good candidate for complementing current in vitro genotoxicity testing batteries. The combination of the comet assay with lesion-specific enzymes is used to detect altered bases. A review about this version of the assay revealed that formamidopyrimidine DNA glycosylase (Fpg) is the most used enzyme, used for the detection of oxidized bases. In total, 12 different enzymes have been combined with the comet assay to detect other lesions such as alkylated bases, presence of uracil, pyrimidine dimers or AP-sites. The areas of application in which the enzyme-modified comet assay has been more extensively used are in vitro genotoxicity testing and human biomonitoring. For the detection of alkylated bases, two non-commercially available bacterial enzymes, 3-methyladenine DNA glycosylase II (AlkA) and 3-methyladenine DNA glycosylase (AlkD), have been sporadically employed. In this thesis, a commercial human alkyladenine DNA glycosilase (hAAG) was successfully applied for the first time. Moreover, the use of hAAG together with other different enzymes (non-commercial Fpg and commercial Fpg, Endonuclease III -Endo III- and human 8-oxoguanine DNA glycosylase -hOGG1-), with various specificities towards oxidized lesions, was optimized to be used on a single assay using a medium throughput format (i.e., 12 minigels/slide). To this aim, the incubation conditions when using the widely used 2 gels/slide format and the medium-throughput 12 minigels/slide format was assessed. This comparison highlighted that is crucial to perform enzyme titration experiments using the same protocol, equipment and the format that are going to be used in the final experiments. Moreover, in order to detect DNA cross-links, an extra DNA lesion which may be difficult to detect by mean of enzymes, an already known modification of the comet assay was set up using the same throughput format. Finally, both comet assay modifications were validated using TK-6 cells treated with non-cytotoxic concentrations of nine compounds with several mechanisms of action: oxidizing and alkylating agents, cross-linkers, a bulky-adducts inducer and non-genotoxic compounds. The combination of the results of both modifications allowed to clearly differentiate the induced lesions, with the exception of the bulky adducts, which was expected. Moreover, no DNA lesions was detected in cells treated with the non-genotoxic compounds. Both Fpg enzymes (non-commercial and commercial one) gave same results. The in vitro comet assay modified with several enzymes together with the cross-links modification increases significantly the comet assay ability to detect different premutagenic lesions, providing genotoxic mechanistic information about the type of damage. Its application might be very relevant in the current regulatory context.