As a member of HESI’s Genetic Toxicology Testing Committee (GTTC), we co-published an article in Environmental and Molecular Mutagenesis entitled “Critical Evaluation of Methods for the Identification of Aneugens”. This article presents the strengths, weaknesses, opportunities, and threats for a variety of technologies currently being used to discriminate aneugens from potentially DNA reactive substances (i.e., mutagens/clastogens). This working group concluded that the “newer, high information content, semi-automated approaches were considered preferable to traditional microscopy-based FISH and CREST techniques”.

Current gold-standard for aneugen detection

Visual methods to identify micronuclei containing either centromeric DNA or kinetochores has been used for decades via fluorescent in situ hybridization (FISH) or immunolabeling with anti-kinetochore antibodies (isolated from CREST patients), respectively. These “FISH/CREST” techniques became popular with regulatory toxicologists as they were examples, mentioned in OECD test guidelines, of how to identify the etiology of induced micronuclei (see OECD TGs 474 and 487). Since aneugen exposure leads to non-disjunction, the resulting micronucleus (MN) will contain a whole chromosome and light up in both FISH/CREST endpoints. Conversely, clastogenic agents would be negative in either FISH/CREST assays, and predominantly induce MN that have no fluorescent signal.

Enter New Approach Methods (NAMs)

In concert with other GTTC working group members, we aimed to shed light on present day aneugen detection using a variety of NAMs. Specifically, multiplexed high content imaging and flow cytometry. Both platforms have numerous advantages but the 2 that stand out are less analysis time and the ability to easily evaluate a wider area of the dose response curve. This latter point is critical as aneugens often have steep cytotoxicity curves, which may hinder efficient detection if employing FISH/CREST on slides from a MN assay with typical 2-3 fold dose spacing.

ToxTracker ACE (Aneugen & Clastogen Evaluation) Technology

Temporal cell cycle analysis via flow cytometry can detect aneugens by measuring mitotic delay (G2/M blocks) and induction of hyper/polyploid cells (>4N DNA content). By combining ToxTracker with cell cycle analysis, we can easily discriminate between the 2 mechanisms of MN formation; aneugenicity and clastogenicity. This is important during drug development and risk assessment as if an aneugenic (or non-DNA reactive) mode of action is empirically determined, safety margins can be calculated as there are low exposures which elicit no adverse biological effects (ie., a threshold). ToxTracker ACE can be used for this as a screening platform or when building weight of evidence once a positive MN test result is encountered.

To learn more, please contact us using the link below or check out the full publication.