Figure 6: PZ induces early glutathione depletion and accumulation of reactive oxygen species that correlates with CYP1A2 activity.
(a) Glutathione depletion in HLCs as measured by the ratio of glutathione (GSH) to oxidized glutathione (GSSG) after treatment with pazopanib (PZ) or acetaminophen (APAP) at indicated concentrations for 4 hours. A reduction in GSH/GSSG ratio with respect to control indicates a depletion of GSH or its conversion to GSSG in the treatment group. (b–c) Reactive oxygen species (ROS) accumulation in HLCs was measured by CellROX Green fluorescence. HLCs were treated with indicated concentrations of PZ or APAP for 4 hours and in the final hour of treatment 10 μM CellROX Green was supplemented to the medium. (b) Representative images of CellROX fluorescence in NHT1 and HT3 showing higher levels of intracellular ROS in HLCs treated with 10 μM PZ and 50 mM APAP compared to control, particularly increased nuclear staining of CellROX. Magnification 100×. (c) Quantification of CellROX intensity by flow cytometry where mean fluorescent intensity was measured relative to vehicle control-treated cells for each HLC. (d) After 4 hours, relative ROS accumulation (from (c)) is correlated with relative GSH/GSSG ratio (from (a)) in HLCs treated with PZ. Greater depletion of GSH, indicated by a lower GSH/GSSG is accompanied by lesser ROS accumulation in HLCs. (e,f) Correlation of PZ-induced GSH depletion and basal activity of PZ-metabolizing CYPs in HLCs, as previously determined in Fig. 2c and d. Relative GSH/GSSG ratio is inversely correlated with basal CYP1A2 activity levels (e) but not with CYP3A4 activity levels (f). For (a) data represents mean ± SE from three independent experiments in HLCs from differentiation initiated at 3 different times; **p < 0.001 comparing PZ vs. DMSO control; ***p < 0.0001 comparing PZ vs. DMSO control; ###p < 0.0001 comparing APAP vs. DMSO control; †p < 0.05 comparing HT2 vs. NHT1 and NHT2-HLCs treated with PZ. For c. data represents mean ± SE from two independent experiments. For (d–f) each data point represents an individual HLC, indicated by same color legend in (d) Correlation coefficient, R and p-values are determined by Pearson’s correlation.
