Carotenoid Uptake and Protection in Cultured RPE - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 327

In another study of protective effects of lutein, cultures of primary human RPE cells were
incubated for 2 h with 40 mM lutein, after which any remaining lutein in the culture medium was
washed away and the medium was replaced with phosphate buffered saline (Roberts et al., 2002).
Lutein did not cause any changes in cell morphology or any increase in DNA damage assessed by
the comet assay (Roberts et al., 2002). Cultures with and without lutein were irradiated either with
visible light (>400 nm; 2.5 J/cm 2 ) to mimic light reaching the adult human retina or with UVB light
above 300 nm (0.08 J/cm 2 ) to mimic the UV light reaching the retina in young children. Visible light
increased DNA damage only slightly and it has been prevented completely by lutein. UVB irradia-
tion caused extensive DNA damage which was also completely prevented in the presence of lutein
(Roberts et al., 2002).
Carotenoids are excellent singlet oxygen quenchers, so it may be expected that they will be
particularly efi cient at protecting cells against photosensitized damage involving singlet oxygen.
However, to be able to act as a singlet oxygen quencher, a carotenoid must be in the immediate
proximity of the photosensitizer (within 220 nm) (Kuimova et al., 2009; Redmond and Kochevar,
2006). Thus subcellular localization of the source of singlet oxygen and carotenoids are likely
to play a major role in the effectiveness of carotenoids in protection against photosensitized 1 O 2 .
Interestingly, we and others have demonstrated that despite accumulation of large concentrations of
lutein or zeaxanthin inside ARPE-19 cells, no signii cant protection against photosensitized damage
could be observed (Kanofsky and Sima, 2006; Rozanowska et al., 2004b; Wrona et al., 2004). The
photosensitizers tested included rose bengal (Rozanowska et al., 2004b), merocyanine 540 (Wrona
et al., 2004), acridine orange, and cis -di(4-sulfonatophenyl)diphenylporphine (Kanofsky and Sima,
2006). Rose bengal exists in an anionic form at neutral pH and, despite association with membrane
lipids, it is not permeable through the plasma membrane. Merocyanine 540 binds to cellular mem-
branes and the pattern of its distribution varies in different cell lines between the plasma membrane,
mitochondria, and lysosomes (Chen et al., 2000). Acridine orange and cis -di(4-sulfonatophenyl)
diphenylporphine are localized mainly in lysosomes (Kanofsky and Sima, 2006). It may be sug-
gested that lutein or zeaxanthin have not exerted any protective effects on photosensitized damage
induced by these photosensitizers because they were not colocalized in the same subcellular com-
partment and/or were bound to XBPs which limited their ability to act as singlet oxygen quenchers.
In contrast, synthetic carotenoid derivatives that colocalize with photosensitizers in lysosomes or
mitochondria of cultured ARPE-19 cells have been shown to offer a substantial protective effect
against photosensitized damage (Kanofsky and Sima, 2006). Clearly, the subcellular localization of
lutein and zeaxanthin and determination of whether they are present in free forms or are bound to
proteins requires elucidation.
Lutein and zeaxanthin have also been tested as potential protection against formation of lipo-
fuscin in cultured RPE (Sundelin and Nilsson, 2001). Lipofuscin is a complex aggregate of lipids
and proteins including several l uorophores and photosensitizers which accumulate in the RPE
mainly as a result of incomplete lysosomal digestion of POS (Ró . anowska and Ró . anowski,
2008; Rozanowska and Sarna, 2005). Zeaxanthin and lutein have been tested in primary cultures
of rabbit and bovine RPE cells fed POS under 40% oxygen, conditions leading to rapid accumula-
tion of lipofuscin-like inclusions (Sundelin and Nilsson, 2001). The cultured cells were supple-
mented with antioxidants 4 days before the i rst feeding with POS, and every 48 h thereafter.
Xanthophylls were injected into the culture medium directly from their 2 mM stock solutions
in tetrahydrofuran in the presence of butylated hydroxytoluene to give a i nal concentration of
10 mM. Administration of zeaxanthin or lutein led to a substantial, up to ~60% inhibition of accu-
mulation of l uorescent inclusions in rabbit RPE. However, a morphometric analysis of inclusion
bodies in bovine RPE showed that lipofuscin accumulation was diminished by 16% or less in
xanthophyll supplemented cells (Sundelin and Nilsson, 2001). This discrepancy may be explained
if xanthophylls interrupt the pathway leading to the formation of some lipofuscin l uorophores,
while not preventing from overall POS oxidation and formation of products nonsusceptile to
lysosomal digestion.