Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents

Abstract

The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, "misleading" or "irrelevant" positives. We set out to develop a rapid and effective follow-up testing strategy that would predict whether apparent in vitro micronucleus-inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous-type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp-positive chromatin, cleaved caspase 3-positive chromatin, ethidium monoazide bromide-positive chromatin, polyploid nuclei, phospho-histone H3-positive (metaphase) cells, tetramethylrhodamine ethyl ester-negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ-H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave-one-out approach indicated that a promising base model includes γ-H2AX shift and change in phospho-histone H3-positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26-30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput-compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results.

DNA damage foci: Meaning and significance

“…Rad51 analysis to test for the involvement of homologous recombination, see below) and analysis of foci induction in different genetic backgrounds such as mutants defective in different DNA repair pathways can provide important mechanistic insights in genotoxicity testing and are therefore prime candidates for inclusion in the battery of follow‐up tests for substances that tested positive in the Ames/ E. coli bacterial mutagenicity assay [Aardema, ]. Multiplexing with other established genotoxicity markers such as the micronucleus test has also been considered [Bryce et al, ]. Moreover, the applicability of foci assays not only to cell cultures but also to tissue sections following in vivo exposure [Qvarnstrom et al, ; Somaiah et al, ; Rothkamm et al, ] opens up exciting opportunities to produce ‘genotoxicity maps’ across the different organs and cell types of the body.…”

Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach

“…γH2AX is a biomarker of DNA double strand breaks that appears to be applicable to in vitro genotoxicity testing strategies [Audebert et al, ; Smart et al, ; Garcia‐Canton et al, ; Nikolova et al, ]. As several previous reports have suggested, when γH2AX readings are coupled with mitotic cell (phospho‐histone H3‐positive event) readings, additional information is gained that is useful for discriminating between clastogenic and aneugenic activities [Bryce et al, ; Cheung et al, Khoury et al, ].…”

Glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union assessment and its differences with IARC

“…The induction of DNA strand breaks observed in mice treated intraperitoneally with doses close to or in excess of the LD 50 has been associated to secondary effects of cytotoxicity (JMPR 2006; Kier 2015). Modes of action associated with secondary cytotoxicity should be excluded from the assessment of the intrinsic genotoxicity potential (Bryce et al 2014; Kitamoto et al 2015). …”

“…Secondary genotoxic effects produced by cytotoxicity should also be distinguished from true genotoxic potential (Bryce et al 2014; Kitamoto et al 2015). In fact, the UN and EU guidance recommends carcinogenicity and genotoxicity studies to be conducted on individual chemicals, limiting testing of mixtures/formulations to cases where synergistic effects are expected (United Nations 2015).…”