Cytotoxicity Evaluation of a Primer by an ARE Reporter Assay

Objectives: Monomers that contain a methacryloyloxy group are routinely used in dental practice. Nowadays, minimal intervention approaches become mainstream in the dental field. After removing all infected areas, we usually apply a dental primer directly without dilution to the dentin even if the primer is applied in close proximity to the pulp. We previously showed that an anti-oxidant responsive element (ARE)-firefly luciferase reporter assay system could be used for evaluating cytotoxicity of methyl methacrylate and its derivatives. Our previous study suggested that introduction of a hydroxyl group into methacrylate-based resin monomers increases their intracellular electrophilic reactivity and cytotoxicity. The purpose of this work was to study the cytotoxicity of a resin primer (PANAVIA V5 Tooth Primer, Kuraray Noritake Dental Inc., Japan) using the ARE-luciferase reporter assay system.
Methods: HepG2 cells stably expressing an ARE-regulated luciferase reporter gene (HepG2-AD13 cells) were seeded in 24-well culture plate at a density of 6.0 × 10⁵ cells/mL and pre-cultured for 24 hours. The cells were then cultured for 6 hours with various concentrations of the primer. Thereafter, cells in each well were lysed and assayed for firefly luciferase activity by adding the luminescence substrates. Luminescence was measured in relative light units using a luminometer. The ARE activity based on the luminescence intensity was expressed as fold activation to the control value obtained without additives. (N=4)
Results: PANAVIA V5 showed a bell-shaped dose-response curve for ARE activation, and stimulated the reporter expression strongly in a low concentration range (1000-fold dilution). At 0.3 and 1.0 µL/mL, the primer increased the reporter luciferase expression by approximately 50-fold and 40-fold, respectively.
Conclusions: This study suggested that not only monomers but also products used in dental clinics could be evaluated by using the ARE reporter assay system.