Mode‐of‐action analysis of the effects induced by nicotine in the in vitro micronucleus assay

Abstract

Nicotine's genotoxic potential has been extensively studied in vitro. While the results of mammalian cell‐based studies have inferred that it can potentially damage chromosomes, in general and with few exceptions, adverse DNA effects have been observed primarily at supraphysiological concentrations in nonregulatory assays that provide little information on its mode‐of‐action (MoA). In this study, a modern‐day regulatory genotoxicity assessment was conducted using a flow cytometry‐based in vitro micronucleus (MN) assay, Good Laboratory Practice study conditions, Chinese hamster ovary cells of known provenance, and acceptance/evaluation criteria from the current OECD Test Guideline 487. Nicotine concentrations up to 3.95 mM had no effect on background levels of DNA damage; however, concentrations above the point‐of‐departure range of 3.94–4.54 mM induced increases in MN and hypodiploid nuclei, indicating a possible aneugenicity hazard. Follow‐up experiments designed to elucidate nicotine's MoA revealed cellular vacuolization, accompanying distortions in microtubules, inhibition of tubulin polymerization, centromere‐positive DNA, and multinucleate cells at MN‐inducing concentrations. Vacuoles likely originated from acidic cellular compartments (e.g., lysosomes). Remarkably, genotoxicity was suppressed by chemicals that raised the luminal pH of these organelles. Other endpoints (e.g., changes in phosphorylated histones) measured in the study cast doubt on the biological relevance of this apparent genotoxicity. In addition, three major nicotine metabolites, including cotinine, had no MN effects but nornicotine induced a nicotine‐like profile. It is possible that nicotine's lysosomotropic properties drive the genotoxicity observed in vitro; however, the potency and mechanistic insights revealed here indicate that it is likely of minimal physiological relevance for nicotine consumers. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Baseline effects of non-flavored e-liquids in the in vitro micronucleus assay

“…However, we have shown that by complementing a conventional genotoxicity endpoint like micronucleus with MoA endpoints (such as DDR signaling proteins and cell cycle status), the data can be potentially better understood and rationalized, even in a limited cell line like CHO-WBL. Our recent findings (including the work of Smart et al 37 ) show that, in the absence of changes in cellular DNA content (e.g. G 2 M accumulation and endoreduplicated DNA), increases in the HDN endpoint alongside those in micronuclei might be indicative of a misleading (false positive) genotoxic response, at least in this cell model.…”

“…Interestingly, a recent comprehensive in vitro assessment of the genotoxic hazard potential of nicotine made similar observations on the same endpoints. 37 Although DDR biomarkers such as gH2AX and pH3 are being increasingly used in genetic toxicology assessments, the reliability of these endpoints is entirely dependent on the fidelity of the signaling pathways that underpin them. 28,36,[38][39][40][41][42] In the present study, owing to the contradictory nature of the In vitro MicroFlow™ and Multi-Flow™ findings, we investigated whether the pharmacologically active NFEL component nicotine could modulate DDR signaling pathways and mask cellular responses to genotoxic stress.…”

“…Interestingly, a recent comprehensive in vitro assessment of the genotoxic hazard potential of nicotine made similar observations on the same endpoints. 37…”

The in vitro ToxTracker and Aneugen Clastogen Evaluation extension assay as a tool in the assessment of relative genotoxic potential of e-liquids and their aerosols

“…Nicotine levels are often used as an indication of exposure in the testing of cigarette- and NGP-derived samples ( 28 , 76 , 77 ). However, nicotine has also been extensively characterised for its toxicological action both in vitro and in vivo and has been observed to induce genotoxic outcomes, including chromosome damage, in addition to oxidative stress ( 3 , 26 , 34 , 78–81 ). However, these effective concentrations are often at supraphysiological levels ( 3 , 28 , 82 ) (measured physiological nicotine levels range from 10 to 37 ng/ml ( 83) ) and, therefore, have limited physiological relevance.…”

“…This difference (in nicotine form) constitutes a (minor) change in EVP formulation. Further to this, when tested alone, nicotine has been demonstrated to have a number of toxic effects both in vitro and in vivo ( 3 , 26 , 34 ) and, in particular, has demonstrated genotoxic effects in vitro , albeit at supraphysiological concentrations ( 3 ).…”