Introduction:
Electronic cigarettes (EC) represent a safer alternative to tobacco, however, there is limited information on adverse health effects, especially after long term use. Among potential harmful constituents of EC aerosols nicotine is the major addictive chemical and is a precursor for carcinogenic tobacco-specific nitrosamines (TSNAs). Multiple studies have highlighted the ability of nicotine to compromise blood brain barrier (BBB) integrity, altering transport and receptor systems. One knowledge gap is the effect of e-fluids on enzymes responsible for metabolism of toxicants including carcinogens, Therefore, the aim of this study was to examine the effect of nicotine and e-fluid on the expression of cytochrome P450 enzymes (CYPs) in an in vitro model of human BBB, the hCMEC/D3 cell line.

Methods:
The hCMEC/D3 cells were used to investigate time and dose-dependent responses of nicotine or e-fluid and EC aerosol condensate (normalised to nicotine concentration). High quality RNA was extracted and analysed by qRT-PCR for the expression of genes encoding enzymes involved in nicotine metabolism and bioactivation of procarcinogens (CYP1A1, CYP1A2, CYP2A6, CYP2E1, CYP2A13, CYP3A4) and recently described extrahepatic CYP isoforms (CYP2U1 and CYP2S1), which are highly expressed in BBB.

Results:
qRT-PCR results were normalised using UBC reference gene and expressed as a fold change between non treated and treated cells (n=3, p<0.05). Initial analyses revealed a time-dependent increase in mRNA expression levels of CYP2A6, CYP2U1, CYP2E1 and CYP2S1 following exposures with the highest fold change values after 24h. There was no expression of CYP3A4, CYP1A2 or CYP2A13 in hCMEC/D3 cells.

Conclusions:
Significant induction of mRNA expression for CYP2A6, CYP2E1, CYP1A1 and CYP2S1 following treatment with nicotine and e-fluid, suggests that BBB may play an active role in nicotine metabolism and possibly contribute to the bioactivation of procarcinogens.

Funding:
Funded by European Commission Horizon 2020 programme.