Development and validation of a minimally invasive protocol for assessing oxidative stress markers in exfoliated oral cells.

OBJECTIVES: This study aimed to develop and validate a minimally invasive protocol for characterizing oxidative stress markers in exfoliated oral cells. MATERIALS AND METHODS: Exfoliated oral cells were collected from healthy volunteers. The protocol included the utilization of specific fluorescent probes to measure intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and reduced glutathione (GSH). Cells from each volunteer were divided into the positive and negative control groups, which were, respectively, exposed or not to hydrogen peroxide (H2 O2 ) aiming to induce the oxidative stress. Measurements of cell fluorescence were performed using a microscope equipped with epifluorescence. RESULTS: The results showed that cells exposed to H2 O2 exhibited significantly higher intracellular expression of ROS compared to unexposed cells (positive control: 3851.25 ± 1227.0 vs, negative control: 1106.07 ± 249.6; p = 0.0338). On the contrary, cells exposed to H2 O2 displayed decreased expression of ΔΨm (p = 0.0226) and GSH (p = 0.0289) when compared to the negative control group (ΔΨm positive control: 14634.39 ± 1529.0 vs, negative control: 18897.60 ± 2338.0; and GSH positive control: 9011.08 ± 1900.0 vs, negative control: 15901.79 ± 2745.0). CONCLUSIONS: The developed protocol proved to be effective in detecting and quantifying oxidative stress biomarkers, such as ROS, ΔΨm and GSH, in exfoliated oral cells. This minimally invasive approach offers a promising method to assess oxidative stress expression and may be clinically relevant in the evaluation of oral diseases associated with oxidative stress.

Use of giant unilamellar lipid vesicles as antioxidant carriers in in vitro culture medium of bovine embryos.

Giant unilamellar vesicles (GUVs) are composed of lipophilic layers and are sensitive to the action of reactive oxygen species (ROS). The use of GUVs as microcarriers of biological macromolecules is particularly interesting since ROS produced by gametes or embryos during in vitro culture can induce the opening of pores in the membrane of these vesicles and cause the release of their content. This study investigated the behavior of GUVs [composed of 2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)] in co-culture with in vitro produced bovine embryos, as well as their embryotoxicity and effectiveness as cysteine carriers in culture medium. Embryonic developmental rates were unaffected, demonstrating the absence of toxicity of GUVs co-cultured with the embryos. No increase of intracellular ROS levels was observed in the embryos co-cultured with GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not increase oxidative stress. Variations in the diameter and number of GUVs demonstrated their sensitivity to ROS produced by embryos cultured under conditions that generate oxidative stress. Encapsulation of cysteine in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of this antioxidant in the medium. In conclusion, the use of GUVs in in vitro culture was found to be safe since these vesicles did not promote toxic effects nor did they increase intracellular ROS concentrations in the embryos. GUVs were sensitive to oxidative stress, which resulted in structural changes in response to the action of ROS. The possible slow release of cysteine into the culture medium by GUV rupture would therefore favor the gradual supply of cysteine, prolonging its presence in the medium. Thus, the main implication of the use of GUVs as cysteine microcarriers is the greater effectiveness in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos.