FIGURE 5.

APOE genotype modulates reactive oxygen species and antioxidant levels. (A) Total reactive oxygen species (ROS) levels were measured with 2′,7′-dichlorofluorescein diacetate (DCFDA; n = 4). DCFDA diffuses into the cell where it is deacetylated to a non-fluorescent compound by cellular esterases. Reactive oxygen species then oxidize the deacetylated DCFDA to form 2′, 7′-dichlorofluorescein, which fluoresces green. APOE4 brain endothelial cells exhibit lower total cellular ROS as evidenced by ∼32% lower DCFDA fluorescence. Of the different types of ROS, H₂O₂ is the most abundant and accounts for the majority of the DCFDA signal and (B) specific levels of H₂O₂ are lower in APOE4 brain endothelial cells. After the addition of exogenous H₂O₂, (C) levels of total reactive oxygen species (DCFDA) are still higher with APOE3 (n = 4), suggesting an upregulation of pathways to limit H₂O₂ levels in APOE4 brain endothelial cells, such as peroxisome or other enzymatic activity. For other ROS, while there are (D) no changes in peroxynitrite levels with APOE4, (E) there are ∼31% higher cellular O₂^– levels. These data are consistent with the idea that higher mitochondrial metabolism can result in the accumulation of O₂^–. Indeed, specifically for mitochondrial ROS, there are (F) ∼29% higher O₂^– (MitoSOX Red O₂^– Indicator which accumulates in mitochondria and is oxidized by O₂^–) and (G) ∼7% higher hydroxyl radical (OH580 probe which is oxidized by hydroxyl radicals) levels in APOE4 brain endothelial cells. The effects and levels of reactive oxygen species are in part regulated by antioxidant systems. Heme is an essential iron-containing compound with pleiotropic functions from respiration, oxygen transport and xenobiotic modification to modulation of reactive oxygen species levels. Heme is produced from glycine and succinyl CoA in a series of reactions that starts in the mitochondria, continues in the cytoplasm, and is then completed in the mitochondria. Heme can be degraded into bilirubin, which is an antioxidant and anti-inflammatory molecule. APOE4 brain endothelial cells have lower levels of (H) heme and (I) bilirubin, and (J) a lower ratio of GSH:GSSG (indicating more oxidative stress; APOE3 n = 5, APOE4 n = 6). Data is expressed as mean ± S.E.M. *p < 0.05 by Student’s t-test with n = 6 (unless otherwise specified above).