Fig. 3.

Detection of ROS and oxidative DNA damage. The accumulation of intracellular ROS was evaluated by two independent techniques: Synergy 2 fluorescent microplate reader (Part A) and flow cytometry (Part B) in cells pre-loaded with 2′,7′-dichlorofluorescin-diacetate (H2DCFDA). R508 and R508/T cells were treated with the oil-PAHs at dilutions 1:100 and 1:500 in the presence or absence of ROS scavengers: N-acetylcysteine (NAC; 1 mM) and Trolox (100 μM) for 72 h. The cells treated with DMSO (vehicle) were used as control. Data represent average values ± SD calculated from three independent experiments in duplicates (n = 6). ^*Indicates ROS accumulation values which are statistically different from the values obtained from DMSO treated samples. ^**Indicates statistically significant difference from PAHs 1:500 (paired Student’s t-test P ≤ 0.05). Part C: Immunocytofluorescent detection of 8-oxo-dG (green fluorescence) in exponentially growing R508 and R508/T cells treated with oil-PAHs (1:500 dilution) for 14 days. Data represent average values ± SD calculated from three independent experiments in duplicate in which at least 50 cells per analysis were evaluated (n = 6). ^*Indicates values which are statistically different from the value obtained from DMSO (vehicle) treated samples (paired Student’s t-test P ≤0.05). Part D: Evaluation of lipids peroxidation in cells exposed to oil-PAHs (1:500 and 1:100) for 72 h. Data represent average values ± SD calculated from three independent experiments (n = 3). Part E: Detection of 8-oxo-dG in urine sample from mice fed with the oil-PAHs. The SV129 mice were fed once a day with the vehicle (DMSO diluted with H₂O 1:100) or with the oil-PAH fraction. Data represent average values ± SD calculated from three independent experiments in which three mice per condition were evaluated (n = 9). ^*Indicates value which is statistically different from the value obtained from DMSO (vehicle) fed mice (paired Student’s t-test P ≤ 0.05).