Tobacco as well as nicotine replacement therapy products contain relative high levels of nicotine. Research on the toxicity of nicotine has mainly focused on the interaction with nicotinic cholinergic receptors, but a lysosomotropic response of nicotine is also reported. The latter may explain recent research that indicates that nicotine interact with the autophagy response in cells. The monomer 2-hydroxyethylmethacrylate (HEMA) found in resin-based
dental materials is known to leak after curing, thereby causing patient exposure. HEMA has a cytotoxic potential in vitro, and it is suggested that cellular defence against HEMA-induced damage involves autophagy. For nicotine-addicted patients treated for dental decay with resin-based dental materials, combined exposure to nicotine and HEMA is likely to occur. The aim of this study was to investigate the individual and combined effect of nicotine and
HEMA exposure on cell viability and autophagy.

For this in vitro study, a human tongue squamous carcinoma cell line (PE/CA-PJ49) was used as a model. Cells were cultured and exposed to HEMA and nicotine individually and in combination. Cells were also treated with an inhibitor of lysosomal proteins. Cell viability was measured using MTT assay in addition to phase-contrast microscopy pictures to map
morphological changes. Specific antibody was used to detect changes in levels of the autophagy related protein SQSTM1/p62 after different exposures. Data was quantified using western blot and Odyssey CLx Infrared System.

Results showed that exposure to nicotine or HEMA alone had no effect on cell viability. However, combined exposure to nicotine and HEMA showed reduction of the viability in cells with increasing concentrations. Results showed that HEMA induced both synthesis and degradation of p62. This imply increased autophagy flux. Further, cells exposed to nicotine
showed significant increased level of p62. Results indicates that this increase was caused by decreased lysosomal degradation, implying inhibition of autophagy.

This study has shown that combined exposure to nicotine and HEMA affects the cell viability in a synergistic manner. The results support that autophagy protects cells against HEMAinduced toxicity. The impairment of autophagy pathway may explain the observed synergistic toxicity of combined exposure to nicotine and HEMA.



Solveig Uvsløkk1, Jan T. Samuelsen1, Tarja Tanner2
1Nordisk institutt for Odontologiske Materialer, Oslo, Norway.
2Department of Cariology, Endodontology and Pediatric Dentistry, Research Unit of Oral Health Sciences, University of Oulu, Finland.

Presented at: The Norwegian Society of Pharmacology and Toxicology (NSFT) 47th annual winter meeting at Beitostølen, Norway, 24-27 January 2019.