# Berry e-liquid flavour toxicities are not equal on an alveolar-capillary barrier cell model according to two exposure methods

## TL;DR

This study shows that berry-flavored e-liquids without nicotine can harm lung cells differently depending on the flavor and exposure method.

## Contribution

The novelty is comparing two exposure methods to reveal unequal toxicity of berry-flavored e-liquids on an alveolar-capillary barrier model.

## Key findings

- Different berry flavors caused varying levels of toxicity in lung cell models.
- Exposure methods (liquid dilution vs. aerosol trapping) led to distinct toxicity outcomes.
- Chemical analysis identified flavor-specific compounds that may explain toxicity differences.

## Abstract

E-cigarettes are often presented as a smoking cessation tool, less harmful than tobacco and are now a regular feature of everyday life. However, the fruity and sweet flavours are attractive to teenagers who can use them recreationnaly. The main risk of this phenomenon is becoming trapped in nicotine addiction. Although e-liquids, which combine nicotine, humectants and flavouring agents, seem to affect the pulmonary system and in particular the alveolar-capillary barrier (ACB), data on nicotine-free e-liquids remain scarce. The aim of this work is to study the toxicity of nicotine-free e-liquids/aerosols flavoured with berries (Strawberry, Raspberry, Blackberry, Blueberry) using two exposure methods. First, we validated an in vitro model of the ACB using epithelial NCI-H441 and endothelial EA.hy926 cells. Secondly, barrier integrity, production of oxidative species and cytotoxicity were assessed by transepithelial resistance (TER) measurement, MitoSOX® and LDH test, respectively, after exposure to two different methods: dilution of the e-liquid and aerosol trapping in the culture medium. Finally, a non-exhaustive analysis of the chemical compounds found in berry e-liquids was performed to identify potentially toxic compounds specific to certain flavours. The model mimicked phenotypically and functionally the ACB. The two exposure methods tested revealed significant differences in terms of e-liquid/aerosol toxicity on the ACB, probably due to variations in aromatic and degradation products. Flavours are not all equal in terms of cell toxicities, making it essential to chemically identify the compounds responsible for these different degrees of toxicity.

Infographic illustrating a four-step research workflow: Step one shows validation of an in vitro alveolar-capillary barrier model using NCI-H441 and EA.hy926 human cell lines. Step two compares exposure methods to berries-flavored e-liquids and trapped aerosols with physicochemical alignment by osmolality. Step three investigates toxicity through LDH release, barrier integrity, and oxidative stress, highlighting concentration and flavor-dependent effects. Step four features chemical characterization of e-liquids using analytical equipment and a Venn diagram, suggesting cellular responses correspond to unique compositions of each flavor.