Fig 1.
Methodological framework of cell culture and analyses.
1) U87 and IMR-32 cells were maintained in DMEM+10%FBS. Cells were exposed for 3, 5 or 10 days to a solution containing different F concentrations. After the exposure period, the following analyses were performed: 2) Viability assay; 3) General cell metabolism by MTT reduction; 4) Apoptosis/Necrosis; 5) ATP levels; 6) Cell morphology; 7) Oxidative Biochemistry assays by measuring Reactive oxygen species, 8) Oxidative Biochemistry assays by measuring GSH/GSSG ratio, 9) Oxidative Biochemistry assays by measuring Lipid Peroxidation; and 10) Comet assay.
Fig 2.
Fluoride toxicity on central nervous system human cells.
Cell viability was performed on A) IMR-32 and B) U87 in different time and F concentrations. Live and dead cells were counted after 3, 5 and 10 days of F exposure. U87 cell death apoptosis or necrosis were evaluated after 10 days of 0.22μg/mL F by C) 3/7 caspase assay and D) membrane integrity analysis respectively. Data were expressed as mean ± SD. Statistical analysis were performed by one-way ANOVA followed by Tukey’s test. Statistical significance compared to control group were showed by * when p<0.05.
Fig 3.
General cell metabolism (GCM) status of A) IMR-32 and B) U87 cells were performed by MTT assay in different times and F concentrations.
GCM was indicated as MTT absorbance divided by percentage of viable cells. ATP synthesis was evaluated on C) IMR-32 and D) U87 after 10 days of 0.095 and 0.22μg/mL F. Data were expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. Statistical significance compared to control group was showed by * when p<0.05.
Fig 4.
IMR-32 cells morphometric analyses after 10 days of F exposure to 0.095 and 0.22μg/mL.
Cells were stained by 10% Giemsa and cellular morphology was observed under inverted phase contrast microscope and photomicrography were analyzed by ImageJ. The following parameters were evaluated: A) Nuclei area B) nuclear perimeter and C) rounded index were evaluated. Photomicrography of control (D and G), 0.095μg/mL (F and H) and 0.22μg/mL (F and I). Data were expressed as mean ± SD.
Fig 5.
U87 cells morphometric analyses after 10 days of F exposure to 0.095 and 0.22μg/mL.
Cells were stained by 10% Giemsa and cellular morphology was observed under inverted phase contrast microscope and photomicrography were analyzed by ImageJ. The following parameters were evaluated: A) Nuclei area B) nuclear perimeter and C) rounded index were evaluated. Photomicrography of control (D and G), 0.095μg/mL (F and H) and 0.22μg/mL (F and I). Data were expressed as mean ± SD. Arrows shows decreased cellular density and narrowing of cytoplasmic projections.
Fig 6.
Oxidative stress parameters of fluoride exposure on U87 cells.
A) GSH/GSSG ratio, B) Reactive oxygen species (ROS) and B) Lipid peroxidation (TBARS) were evaluated after 10 days of 0.095 and 0.22μg/mL F expo-sure. Data were expressed as mean ± SD. Statistical analysis were performed by one-way ANOVA followed by Tukey. Statistical significance compared to control group was showed by * whenever p<0.05.
Fig 7.
DNA fragmentation of U87 glial cells after 10 days of 0.095 and 0.22μg/mL F exposure was analyzed by comet assay.
Data were showed as % of DNA on head and tail and expressed as mean ± SD. Statistical analysis were performed by one-way ANOVA followed by Tukey. Statistical significance compared to control group was showed by * whenever p<0.05.