Induction of DNA double-strand breaks by monochlorophenol isomers and ChKM in human gingival fibroblasts

Abstract

Phenol has been traditionally used in dental treatment as a sedative for the pulp or as disinfectant for carious cavity and root canal. However, phenol is regarded as a mutagenic and carcinogenic agent and its use in dental practice is now therefore restricted. Monochlorophenols are derivatives of phenol, which are still used clinically as root canal disinfectants, they are even more active antiseptics/disinfectants than phenol, and the so-called Walkhoff (ChKM) solution makes use of monochlorophenol for root canal disinfection. Ingredients in the ChKM solution are the monochlorophenol compound 4-chlorophenol (4-CP), camphor, and menthol. In literature, the use of the ChKM solution is controversial because of a possible DNA toxicity of the ingredient 4-CP. However, it is unknown whether ChKM can really induce DNA damage in human oral cells. In this study, the induction of DNA double-strand breaks (DSBs) by ChKM and monochlorophenol compounds (2-chlorophenol, 2-CP; 3-chlorophenol, 3-CP; and 4-chlorophenol, 4-CP) was tested in human gingival fibroblasts (HGFs). DNA DSBs (foci) induced in HGFs unexposed and exposed to monochlorophenols or ChKM solution were investigated using the γ-H2AX DNA focus assay, which is a direct marker for DSBs. DSBs result in the ATM-dependent phosphorylation of the histone H2AX. When cells were exposed to medium or medium + DMSO (1 %) (negative controls), an average of 3 foci per cell were found. In positive control cells (H₂O₂ + medium, or H₂O₂ + medium + DMSO (1 %), an average of 35 foci each were found. About 20 DSB foci per cell were found, when HGFs were exposed to 2-CP (4 mM), 3-CP (2.3 mM), 4-CP (2.1 mM), or ChKM (corresponding to 1.5 mM 4-CP). Our results show increasing DNA toxicities in the order of 2-CP < 3-CP < 4-CP < ChKM solution. An additive DNA toxicity was found for 4-CP in combination with camphor in the ChKM solution, compared to the 4-CP alone. No significant differences regarding multi-foci cells (cells that contain more than 40 foci) were found when HGFs were exposed to the EC₅₀ concentrations (given in parenthesis) of ChKM (1.5 mM), 4-CP (2.1 mM), or 2-CP (4 mM). Significantly fewer multi-foci cells were found when HGFs were exposed to the EC₅₀ concentration (given in parenthesis) of 3-CP (2.3 mM), compared to the EC₅₀ concentrations of ChKM, 4-CP, or 2-CP. Monochlorophenol compounds and/or ChKM solution can induce DSBs in primary human oral (cavity) cells, which underscores their genotoxic capacity.