Figure 5
Combination of OH-PCBs inhibits hTERT expression without reduction of metabolic activity. (A) Left, Jurkat T cells were incubated with serial dilutions of a OH-PCB mix, containing equal amounts of 3-OHCB 28 and 3′-OHCB 28, as well as 3-OHCB 101 and 4-OHCB 101, for 48 hours. The following concentrations of the OH-PCB mix were used: 50 µM (Σ OH-PCB = 61.54 mg/L), 5 µM (Σ OH-PCB = 6.154 mg/L), 500 nM (Σ OH-PCB = 615.4 μg/L) and 50 nM (Σ OH-PCB = 61.54 µg/L). hTERT gene expression (left) and mitochondrial and cellular viability (right) were measured and evaluated as described in Fig. 3. Mean ± SD of three different experiments each with a total of three replicates are shown. Statistical analysis was performed by an unpaired, two-tailed Student’s t-test. Statistically significant differences are indicated (**P < 0.01; ****P < 0.0001). (B) Jurkat T cells were preincubated for 4 hrs. with 500 nM OH-metabolites mix. ATP production was measured as luminescent signal after adding ATP detection reagent from ToxGloTM-assay. Mean ± SD of three replicates are shown. Statistical analysis was performed by an unpaired, two-tailed Student’s t-test. Statistically significant differences are indicated (****P < 0.0001). (C) hTERT gene expression in the presence and absence of pyruvic acid. Jurkat T cells were treated with 500 nM OH-metabolites mix for 48 hrs. Where indicated, 24 hrs. before treatment start, pyruvic acid was added to the culture medium. hTERT expression was evaluated by RT-PCR as described. Mean ± SD of three different experiments each with a total of three replicates are shown. Statistical analysis was performed by an unpaired, two-tailed Student’s t-test. Statistically significant differences are indicated (**P < 0.01). (D) Production of reactive oxygen species (ROS) in Jurkat T cells in the presence of OH-PCB mix. Jurkat T cells preincubated with 4,5-diaminofluorescein-2-diacetate (DAF-2DA) were treated with 500 nM OH-PCB mix (Σ OH-PCB = 615.4 µg/L) for 4 hours and ROS production was measured as fluorescence signal. Mean ± SD of three replicates are shown. Statistical analysis was performed by an unpaired, two-tailed Student’s t-test. Statistically significant differences are indicated (*P < 0.05).
