Abstract
The identification of genotoxic agents and their potential for genotoxic alterations in an organism is crucial for risk assessment and approval procedures of the chemical and pharmaceutical industry. Classically, testing strategies for DNA or chromosomal damage focus on in vitro and in vivo (mainly rodent) investigations. In cell culture systems, the alkaline unwinding (AU) assay is one of the well-established methods for detecting the percentage of double-stranded DNA (dsDNA). By establishing a reliable lysis protocol, and further optimization of the AU assay for the model organism Caenorhabditis elegans (C. elegans), we provided a new tool for genotoxicity testing in the niche between in vitro and rodent experiments. The method is intended to complement existing testing strategies by a multicellular organism, which allows higher predictability of genotoxic potential compared to in vitro cell line or bacterial investigations, before utilizing in vivo (rodent) investigations. This also allows working within the 3R concept (reduction, refinement, and replacement of animal experiments), by reducing and possibly replacing animal testing. Validation with known genotoxic agents (bleomycin (BLM) and tert-butyl hydroperoxide (tBOOH)) proved the method to be meaningful, reproducible, and feasible for high-throughput genotoxicity testing, and especially preliminary screening.
Highlights
Maintenance of genome integrity is an organism’s top priority to ensure a healthy life and successful reproduction of the species (Jackson and Bartek 2009)
Alkaline unwinding was initially developed for cell culture systems (Daniel et al 1985; Hartwig et al 1993) and is a frequently utilized genotoxicity test, even though it does not belong to the primal OECD genotoxicity tests
Similar to the alkaline COMET assay (which is not part of the primal OECD genotoxicity tests), using alkaline unwinding one can quantify DNA strand breaks caused by genotoxins via direct interactions with the DNA; alkali labile sites; or as a consequence of transient strand discontinuities resulting from nucleotide and base excision repair (Garberg et al 1988; OECD 2016)
Summary
Maintenance of genome integrity is an organism’s top priority to ensure a healthy life and successful reproduction of the species (Jackson and Bartek 2009). The genomic DNA is under constant attack of extrinsic and intrinsic genotoxic agents which may result in genetic alterations in somatic and/ or germ cells, which in turn might manifest target
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