Combinations of antioxidants protect oral fibroblasts from ROS inducing agents

Objective: Oxidative damage of soft oral tissues may result from exposure to chemicals or biochemicals found in teeth whitening products, dental restorations, tobacco, and alcohol. Also, metals in fillings, braces, bridges and other prosthodontic restorations such as, zinc (Zn), copper (Cu), and nickel (Ni), may have a cytotoxic activity. Our working hypothesis is that oral tissues are susceptible to the toxic effects of stressors such as metal ions, hydrogen peroxide (H₂O₂), ethanol (EtOH) and nicotine (Nic), decreasing cell viability/DNA synthesis and producing elevated reactive oxygen species (ROS). In this study, we investigated specific natural occruing polyphenols and turmeric derivative antioxidants (AO) in combinations that counteracted the effects of these stressors on cultured oral fibroblast proliferation and ROS production.

Method: Oral fibroblasts were pre-treated with stressors for 30- 60 minutes. The cells were then treated with 10^-5 M of the bioactive AO mixtures; resveratrol, ferulic acid and tetrahydrocurcuminoid (RFT), phloretin, ferulic acid and resveratrol (PFR), phloretin, ferulic acid and tetrahydrocurcuminoid (PFT) for 24 hours.  Cell viability and DNA synthesis were monitored by 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium ⁽MTS) and 5-bromo-2-deoxyuridine (BrdU) incorporation assays respectively. Total ROS was measured with dichlorodihydrofluorescein diacetate (H₂DCFDA).  

Result: Incubation of oral fibroblasts in the presence of the stressors (H₂O_2, EtOH or Nic) for 30 min or Zn, Cu and Ni for 30-60 min caused a dose-dependent decrease of DNA synthesis and number of viable cells, and increased total ROS activity.  AO treatment counteracted the insults by restoring DNA synthesis levels and cell viability, and decreasing total ROS activity.

Conclusion: The AO combinations of RFT, PFR and PFT protected oral fibroblasts from the detrimental effects of H₂O₂, EtOH, Nic and all metal salts tested by decreasing total ROS and increasing cell viability and DNA synthesis.