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CORESTA Meeting, Smoke Science/Product Technology, 2023, Cancun, STPOST 05

Identification of a novel mode of action of vanillin derivative compound veratraldehyde

WATANABE T.; MUNAKATA S.; TAKAHASHI T.; HASHIZUME T.
Japan Tobacco Inc., Scientific Product Assessment Center, Yokohama, Kanagawa, Japan

Compounds that test positive in in vitro micronucleus tests (MNTs) need to be followed up with in vivo MNTs. However, it is common for these compounds to test negative in in vivo MNTs, known as “misleading positives.” To reduce in vivo follow-up tests, we developed an in vitro mode of action (MoA) analysing platform which consists of ToxTracker assay and high-content imaging analysis (HCA) to classify MN-inducing compounds according to their MoA: clastogenic, aneugenic, or reactive oxygen species–generating. To demonstrate the utility of our platform, we analysed the MoA of a flavouring compound veratraldehyde (VER), a derivative of vanillin in which the hydroxyl group of vanillin is replaced with a methoxy group. Both VER and vanillin are known to be misleading positives in in vitro MNTs. To reveal the MoA of these compounds, we employed the ToxTracker assay which uses flow cytometer to analyse six reporters. The results showed that VER activated one of the two reporters associated with DNA damage, characteristic of aneuploidy, whereas vanillin did not activate any of these six reporters. A subsequent ToxTracker ACE (Aneugen Clastogen Evaluation) assay showed that VER induced cell cycle defects, indicative of aneugenicity. HCA combined with nucleus staining and γ-H2AX immunostaining revealed that VER also induced an abnormal nuclear morphology but minimal DNA damage. Additionally, staining microtubules with fluorescent dye resulted in a decrease in the fluorescence signals, indicating modulation of microtubule dynamics. From a perspective of chemical structure, we considered that VER interacts with the colchicine binding pocket of tubulin to modify its polymerization because VER has adjacent methoxy groups, similar to colchicine. A novel MoA by which VER induces micronuclei in vitro was revealed through the collective results of this study, thus demonstrating the utility of our platform that combines ToxTracker assays with HCA.