Differential Cytotoxicity of Flavored E-Liquids with and without Nicotine on Neonatal Human Melanocytes from Lightly and Darkly Pigmented Donors: A Preliminary Report

Abstract

The increasing use of e-cigarettes (ECs) has raised public health concerns due to the observed cytotoxic effects in both in vitro and in vivo studies. Infants and young children, being particularly vulnerable groups, exhibit heightened susceptibility to potential hazards arising from maternal use of ECs, as well as exposure to second-hand and third-hand aerosols emitted by ECs. Melanocytes are neural-crest-derived cells that regulate multiple biological functions. Melanocyte death, triggered by chemical exposure, is a known etiological cause of pigmentation abnormalities and neurodevelopmental disorders. Prior reports have demonstrated nicotine-induced differential cytotoxicity to neonatal human melanocytes derived from lightly pigmented (LP) and darkly pigmented (DP) donors. We recently reported that the vehicle base propylene glycol (PG) in e-liquid can alter the functions of LP melanocytes. However, to date, the effects of e-liquid flavors on LP and DP cells remain unexplored. Hence, in this preliminary study, a panel of twenty EC refill liquids comprising ten popular flavors (strawberry, grape, banana, vanilla, butterscotch, cinnamon, menthol, chocolate, cola, and tobacco), where each flavored e-liquid contained either 0 or 18 mg/mL nicotine, was examined for in vitro cytotoxicity to neonatal human melanocytes derived from LP and DP donors. Our results reveal that of the ten flavors, five (menthol, cinnamon, vanilla, tobacco, and banana) were highly cytotoxic, with their half-maximal inhibitory concentration (IC50) values within the tested concentration ranges. Moreover, the cytotoxicity of the specific flavors menthol, cinnamon, and vanilla was enhanced in the presence of nicotine, indicative of interactive effects, with nicotine and flavor contributing to greater melanocyte injury. The cytotoxicity of menthol (both with and without nicotine) and cinnamon (without nicotine) e-liquids was found to be higher in LP cells as compared to DP cells. In contrast, nicotine-containing vanilla e-liquid induced higher cytotoxicity in DP cells than LP cells. Only three flavors, cola (without nicotine), strawberry (without nicotine), and chocolate (without nicotine), were non-cytotoxic to both LP and DP cells. The findings that popular flavors in e-liquids induced moderate to high degree of melanocytotoxicity even in the absence of nicotine suggests that ECs are not harmless. This information may assist EC users identify particular flavors in refill liquids that may be detrimental to melanocytes. A first-screen identification of flavors in e-liquids that show a racial/ethnicity dependence can provide a baseline to identify cytotoxicity concentration ranges for popular flavors and help inform the regulatory guidelines for EC toxicity to young children and youth.