Figure 5.

Carotenoids induce ROS production in HepG2 cells. A–F) HepG2 cells were treated with vehicle only (A), 2 μM zeaxanthin (B), 0.5 μM 3,3′-didehydrozeaxanthin (C), 2 μM 3,3′-didehydrozeaxanthin (D), 2 μM lutein (E), or 2 μM dihydrolutein derivatives (F). After 2 h of incubation, the nonfluorescent carboxy-H₂DCFDA dye was added to treated cells. In the presence of ROS, the reduced fluorescein compound is oxidized and emits bright green fluorescence. Representative images were taken under a fluorescent microscope at ×20. G) HepG2 cells were treated with increasing amounts of 3,3′-didehydrocarotenoids (0.1, 0.3, 0.6, 1.0, 1.5, and 2 μM), respectively. Treated cells were harvested by centrifugation and resuspended in 1× PBS. Fluorescence was determined by emission of light at 525 nm and measured in absorbance units per milligram of protein. Assay was performed in duplicate and repeated 3 times. H, I) HepG2 cells were treated with vehicle only (H) or 2 μM of β,β-carotene (I). J) HepG2 cells were transfected with a vector for the expression of murine BCDO2. After 2 d, cells were treated with 2 μM of β,β-carotene. After 2 h, nonfluorescent carboxy-H₂DCFDA dye was added to treated cells (H–J). Representative images were taken under a fluorescent microscope at ×40. K) Nontransfected HepG2 cells and HepG2 cells transfected with a plasmid for recombinant murine BCDO2 expression were treated with different amounts of β,β-carotene. After 2 h, nonfluorescent carboxy-H₂DCFDA dye was added, and cells were harvested by centrifugation and redissolved in 1× PBS. Fluorescence was determined by emission of light at 525 nm. Data represent values from 3 experiments. *P < 0.05. L) Immunoblot analysis of HepG cells from K confirms the expression of recombinant murine V5-tagged BCDO2. In addition, levels of the mitochondrial marker protein COXIV indicate that amounts of mitochondria are not grossly altered on overexpression of recombinant BCDO2.