N-tert-butyl hydroxylamine, a mitochondrial antioxidant, protects human retinal pigment epithelial cells from iron overload: relevance to macular degeneration

Abstract

Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in the elderly in developed countries. AMD patients have elevated levels of iron within the retinal pigment epithelia (RPE), which may lead to oxidative damage to mitochondria, disruption of retinal metabolism, and vision impairment or loss. As a possible model for iron-induced AMD, we investigated the effects of excess iron in cultured human fetal RPE cells on oxidant levels and mitochondrial cytochrome c oxidase (complex IV) function and tested for protection by N-tert-butyl hydroxylamine (NtBHA), a known mitochondrial antioxidant. RPE exposure to ferric ammonium citrate resulted in a time- and dose-dependent increase in intracellular iron, which increased oxidant production and decreased glutathione (GSH) levels and mitochondrial complex IV activity. NtBHA addition to iron-overloaded RPE cells led to a reduction of intracellular iron content, oxidative stress, and partial restoration of complex IV activity and GSH content. NtBHA might be useful in AMD due to its potential to reduce oxidative stress, mitochondrial damage, and age-related iron accumulation, which may damage normal RPE function and lead to loss of vision.

Figure 1

Time-dependent increase in the levels of total iron in RPE cells treated with 250 µM FAC. Changes in total iron levels were measured using ICP. FAC exposure for 1–4 days promoted a time-dependent increase in the levels of total iron in RPE cells. The data represent mean ± sd of 3 independent experiments (***P<0.001).

Figure 2

Iron overload induced dose- and time-dependent increase in the levels of oxidative stress in RPE. A) Effect of different concentrations of FAC (4 days’ treatment) on the levels of intracellular oxidants. Changes in oxidative stress were measured with the oxidant-sensitive dye H₂DCF. The significant increase in oxidant levels was induced by the concentrations of FAC of 250 µM or greater. The data represent mean ± sd of 6 samples from 3 different experiments (***P<0.001 vs. control). B) Time-dependent increase in the levels of intracellular oxidants in RPE cells treated with 250 µM FAC. The data represent mean ± sd of 6 samples from 3 different experiments (**P<0.01, ***P<0.001 vs. control).

Figure 3

Effects of NtBHA posttreatment on iron homeostasis and oxidative stress levels in RPE cells. A) NtBHA posttreatment resulted in a decrease in levels of total iron in RPE cells treated with FAC (250 µM, 4 days) as determined by ICP. RPE cells were treated with NtBHA (250 µM, 16 h) after FAC exposure, and demonstrated a significant decrease in the levels of total iron compared with RPE cells treated with medium alone (0 µM NtBHA). The data represent mean ± sd of 3 independent experiments (**P<0.01, ***P<0.001). B) Effect of iron overload and NtBHA posttreatment on the levels of labile iron in RPE cells. Different treatments are indicated as follows: control = untreated RPE cells; NtBHA = RPE cells treated with 250 µM NtBHA; FAC, 4 days = RPE cells treated with 250 µM FAC for 4 days; FAC + 0 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days and posttreated with NtBHA-free cell medium; FAC + 250 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days and posttreated with 250 µM NtBHA. Changes in labile iron levels were measured with Fe-sensitive calcein dye. FAC treatment for 4 days promoted significant decrease in calcein fluorescence, indicating increased labile iron concentration. Treatment with 250 µM NtBHA of iron-loaded cells reduced the levels of intracellular labile iron compared with RPE cells treated with medium alone (0 µM NtBHA). The data represent mean ± sd of 7 samples from 4 independent experiments (***P<0.001). C) Effect of NtBHA posttreatment on the levels of intracellular oxidants in iron-treated RPE cells. RPE cells were treated overnight (16 h) with different concentrations of NtBHA after 4 days of FAC exposure. Changes in oxidant levels were measured with oxidant-sensitive dye H₂DCF. Data are expressed as mean ± sd of 3 independent experiments. Each experiment was performed in duplicate (**P<0.01, ***P<0.001). D) Fluorescein diacetate dye was used as a control to check for potential changes in RPE dye loading. Different treatments promoted no significant changes in fluorescein signal indicating no difference in loading differently treated RPE cells with dyes.

Figure 4

Effect of iron overload and NtBHA treatment on GSH levels in RPE cells. Control = untreated RPE cells; NtBHA = RPE cells treated with 250 µM NtBHA for 16 h; FAC, 4 days = RPE cells treated with 250 µM FAC for 4 days; FAC + 0 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days and posttreated with NtBHA-free cell medium for 16 h; FAC + 250 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days and posttreated with 250 µM NtBHA for 16 h. NtBHA treatment caused a significant increase in GSH levels in both control and FAC-treated RPE cells. The data represent mean ± sd of 5 samples from 3 different experiments (*P<0.05, ***P<0.001).

Figure 5

Effect of iron overload and NtBHA treatment on ferritin and transferrin receptor (TfR) expression in RPE cells. A) Different treatments are indicated: Contr = control untreated RPE cells; NtBHA = RPE cells treated with 250 µM NtBHA for 16 h; FAC, 4 days = RPE cells treated with 250 µM FAC for 4 days; FAC + 0 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days, then treated with NtBHA-free cell medium for 16 h; FAC = 250 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days, then treated with 250 µM NtBHA for 16 h. B) Effect of NtBHA (250 µM, 16 h) treatment on ferritin levels in RPE cells from 3 different donors.

Figure 6

Effect of iron overload and NtBHA treatment on mitochondrial complex IV activity. Control = untreated RPE cells; NtBHA = RPE cells treated with 250 µM NtBHA; FAC, 4 days = RPE cells treated with 250 µM FAC for 4 days; FAC + 0 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days, then treated with NtBHA-free cell medium; FAC + 250 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days, then treated with 250 µM NtBHA. Changes in the enzyme activity were measured using kinetic colorimetric assay. FAC treatment for 4 days caused a significant decrease in complex IV activity. Treatment with 250 µM NtBHA of iron overloaded cells resulted in a significant increase of complex IV activity compared with RPE cells treated with medium alone (0 µM NtBHA). Iron-loaded RPE cells treated with 250 µM NtBHA demonstrated significantly higher levels of complex IV activity compared with RPE cells treated with medium alone (0 µM NtBHA). The data represent mean ± sd of 3 independent experiments (*P<0.05 vs. control, **P<0.01).