Figure 4. Comparison of reactive oxygen species generation in retinal pigment epithelium cells transfected with CYP1B1 variants of Leu432Val polymorphism. A: Cells were transfected with recombinant CYP1B1 clones with either the Leu432 or Val432 variant, trypsinized, washed with PBS, and treated with 400 nM 17β-estradiol. Approximately equal numbers of transfected, untransfected, and H₂O₂-treated cells (used as positive control) were incubated with CM-H2DCFDA and fluorescence was measured as described in Methods section. Basal fluorescence was subtracted from all measurements. B: To substantiate the generation of fluorescence due to ROS, cells subjected to similar treatment as that of panel A were further treated with or without 2 mM NAC (n-acetyl cysteine) before treatment with 17β-estradiol. As seen in the figure a significant decrease in fluorescence in NAC-treated cells were observed compared to the untreated ones.