Hydroxyethyl methacrylate is genotoxic but not mutagenic in human gingival fibroblasts as assessed by Duplex Sequencing.

Abstract

Objectives: Hydroxyethyl methacrylate (HEMA) induces adverse effects, including the generation of reactive oxygen species (ROS) and genotoxicity. The study aimed to utilize Duplex Sequencing (DS), a highly accurate next generation sequencing technology, to analyze mutagenicity in human gingival fibroblasts (HGF) exposed to a wide concentration range of HEMA.

Methods: We determined HEMA concentrations that are not excessively cytotoxic using Hoechst33342 assays to avoid secondary effects. ROS generation was quantified using 2,7-dichlorofluorescin diacetate, while genotoxicity was assessed by comet assay. To analyze the mutagenicity of HEMA, HGF were treated with 1 µM to 3.25 mM HEMA for 24 h, followed by a recovery phase. DS was used to detect mutations in isolated DNA.

Results: HEMA induced ROS at 3.25 mM (191 % ± 57 %) and 1.5 mM (170 % ± 18 %) (p < 0.05; one-way ANOVA with Dunnett's post-hoc test). We observed increased genotoxicity at 3.25 mM (olive tail moment: 1.56 ± 0.46) and 1 mM HEMA (0.96 ± 0.16) in comparison to the control (0.26 ± 0.08, p < 0.05; linear mixed model corrected by Scheffé's method). Nonetheless, no mutagenicity was detected, as the mutation rate in HEMA-treated HGF cells (9.3 × 10⁻⁸-1.2 × 10⁻⁷) was comparable to that of the control (1.3 × 10⁻⁷).

Significance: Although HEMA has potentially mutagenic properties such as the formation of ROS and genotoxicity, no HEMA-induced mutations could be detected in HGF using DS under the specific experimental conditions and concentrations investigated. We posit that, during the recovery phase, either damaged DNA is repaired or apoptosis is initiated.