Lysosomotropic-related limitations of the BALB/c 3T3 cell-based neutral red uptake assay and an alternative testing approach for assessing e-liquid cytotoxicity

• Published in: Toxicology in vitro Vol. 61; p. 104647
• Main Authors: Cudazzo, Gianluca, Smart, Daniel J., McHugh, Damian, Vanscheeuwijck, Patrick
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2019; Elsevier Science Ltd
• Subjects: Adenosine triphosphate; Animals; Assaying; ATP; Biological Assay; Bone marrow; Cell Line; Cell Survival - drug effects; Chloroquine; Cytotoxicity; E-liquid; Electronic Nicotine Delivery Systems; Flow cytometry; Humans; Liquids; Lysosomes - metabolism; Lysosomotropism; Mammalian cells; Mice; Muscles; Neuroimaging; Neutral Red - metabolism; Nicotine; Nicotine - toxicity; Organic chemistry; Smoking; Smooth muscle; Tetrazolium salt; Tobacco; Toxicity; Cytotoxicity; E-liquid; Lysosomotropism; Nicotine
• ISSN: 0887-2333; 1879-3177; 1879-3177
• DOI: 10.1016/j.tiv.2019.104647

Cytotoxicity assays are used to quantify the cytotoxic potential of chemicals. The neutral red uptake (NRU) assay is one of these assays and is routinely used in the pharmaceutical, cosmetic, and tobacco industries. In the context of e-cigarette development, an NRU assay-based screen was implemented to evaluate the cytotoxic potential of e-liquids. E-liquids induced a biphasic response in the BALB/c 3T3-based assay. The NRU initially increased in a concentration-dependent manner before decreasing following treatment with higher concentrations until NRU was abolished. Experiments were performed to characterize the mechanism underlying this biphasic signal. Nicotine alone was found to induce the same biphasic effects, while inducing concentration-dependent decreases in relative cell counts (RCC). Imaging and flow cytometry data revealed that the increases in NRU likely resulted from nicotine-induced vacuolization via a lysosomotropic mechanism. In support of this, two lysosomotropic agents, chloroquine and lapatinib, induced similar profiles. Nicotine's effects were also translatable, as brain-, lung-, bone marrow-, and smooth muscle-derived mammalian cells responded with the biphasic NRU signal. However, like RCC, three other cytotoxicity endpoints, resazurin, adenosine triphosphate, and water soluble tetrazolium salt (WST)-8, were not subject to these effects. The WST-8 assay is proposed as an alternative to screen the cytotoxic potential of e-liquids. •Biphasic effects driven by nicotine were observed in the Neutral Red Uptake cytotoxicity assay.•The mechanism underlying these effects is linked to nicotine's lysosomotropic properties.•Effects were characterized at the organelle level using imaging and flow cytometry, and via other lysosomotropic agents.•Nicotine-induced changes to the acidic compartments of multiple mammalian cell lines were also observed.•The water soluble tetrazolium salt-8 assay was demonstrated to be a favorable alternative to screen the cytotoxic potential of e-liquids.