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.

Keywords: aneugenicity; genotoxic mechanism; lysosomotropism.

Figure 1

Nicotine‐induced effects in CHO‐WBL cells following 24‐hr exposure, as measured by the flow cytometry‐based in vitro MN assay (n = 2). (A) MN, HDN, and RPD endpoints. (B) BMD approach‐derived PoD for the MN endpoint.

Figure 2

Representative images of nicotine‐induced distortions in α‐tubulin‐related microtubules (green: α‐tubulin; blue: nuclear DNA; : vacuole; : aberrant metaphase; []: multinuclear cell). (A) Solvent‐treated control. (B) 3.95 mM. (C) 4.93 mM. (D) 5.92 mM. (E) 6.90 mM. (F) 8.88 mM.

Figure 3

Nicotine‐induced effects on the rate and extent of tubulin polymerization in a cell‐free assay alongside the impact of colchicine and paclitaxel. (A) V _max. (B) Maximum amount of tubulin produced.

Figure 4

Representative images of centromere and nuclei co‐localization in nicotine‐exposed CHO‐WBL cells (green: centromere; blue: nuclear DNA). (A) Solvent‐treated controls. (B) 5.92 mM. (C) 6.41 mM.

Figure 5

Nicotine‐induced NRU vis‐à‐vis cytotoxicity measured via RPD in the in vitro MN assay (n = 5 for the NRU endpoint; n = 2 for the RPD endpoint).

Figure 6

Nicotine‐induced effects in the in vitro MN assay in the absence and presence of lysosomal pH‐raising chemicals (n = 3). (A) MN endpoint. (B) RPD endpoint.

Figure 7

Impact of 24‐hr nicotine exposure on histone phosphorylation (n = 3) and cell cycle changes (n = 2) vis‐à‐vis the effects of the positive controls MMS, colchicine (COL), and paclitaxel. (A) γH2AX. (B) Phospho‐serine¹⁰‐H3. (C) Proportion of cells with G₁, S, G₂M, polyploid (supra‐tetraploid), and sub‐diploid (i.e., HDN and MN) DNA content.

Figure 8

Nicotine metabolite‐induced effects in the in vitro MN assay following 24‐hr exposure (n = 2). (A) MN endpoint (bars) and HDN endpoint (lines). (B) RPD endpoint.