Carotenoid Uptake and Protection in Cultured RPE - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 334

It is noteworthy that some epidemiological studies have found that lycopene, but not lutein nor
zeaxanthin, is substantially decreased in serum of AMD patients compared with age-matched
control subjects (Cardinault et al., 2005).
In the case of photosensitized oxidation where both singlet oxygen and free radicals are involved,
an even simpler explanation of synergistic action is credible—inhibition of the free radical chain
reaction by a-tocopherol or ascorbate can protect the carotenoid from free radical-mediated degra-
dation so it can function longer as a singlet oxygen quencher (Wrona et al., 2003, 2004). Interactions
such as these may explain synergistic protection offered by a combination of antioxidants observed
in many systems (Bohm et al., 1998a,b, 2001; Wrona et al., 2003, 2004).
These possible cooperative effects of antioxidant mixtures have been tested in ARPE-19 cells
supplemented with zeaxanthin and a-tocopherol or ascorbate and exposed to photosensitized action
of merocyanine 540 for up to 60 min (Wrona et al., 2004). To assess cell viability mitochondrial
activity was determined and endogenous cholesterol was employed as a reporter of the damage
pathway (Girotti and Korytowski, 2000). Interaction of cholesterol with singlet oxygen leads to
formation of a specii c product, 5a-cholesterol hydroperoxide, which can be detected by high per-
formance liquid chromatography using electrochemical detection. Decomposition of 5a-cholesterol
hydroperoxides or interaction of cholesterol with free radicals leads to the formation of other cho-
lesterol hydroperoxides, such as 7a,b-cholesterol hydroperoxides. Supplementation of cells solely
with zeaxanthin or a-tocopherol has provided no signii cant protection of ARPE-19 cells from cell
death even though a-tocopherol alone exerted a signii cant inhibitory effect on photoformation of
7a,b-cholesterol hydroperoxides and zeaxanthin alone inhibited photoformation of 5a-cholesterol
hydroperoxide (Wrona et al., 2004). The supplementation of cells with 0.5 mM ascorbate alone offered
a small protection against cell death but was detectable only after 10 min of irradiation with visible
light. Interestingly, ascorbate signii cantly inhibited photoformation of 5a-cholesterol hydroper-
oxide, and this effect was signii cant even after 60 min of irradiation. Combinations of zeaxanthin
with either a-tocopherol or ascorbate provided a signii cant synergistic protection of cell viability
for up to 30 min of irradiation and an inhibitory effect against photoformation of both, 5a- and 7a,b
-cholesterol hydroperoxides for up to 60 min of irradiation.
As mentioned previously, in the AMD retina iron metabolism is compromised (He et al., 2007;
Wong et al., 2007). Thus, it is of interest to determine the effects of potential antioxidants in the
presence of iron. In an in vitro study of ARPE-19 cells, addition of a lipophilic iron complex led to
about a ninefold increase in the photosensitized yield of 7a,b-cholesterol hydroperoxides (Wrona
et al., 2004). In the presence of the iron, ascorbate exerted pro-oxidant effects, while the effects of
a-tocopherol, zeaxanthin, or their combination were still protective (Wrona et al., 2004). Thus, it
appears that the effects of potential antioxidants are strongly dependent on the sources of oxidative
damage. The same antioxidant may be protective under certain conditions and exert deleterious
effects when the conditions are changed. Therefore a detailed understanding of the sources of the
oxidative damage is required in order to design an adequate antioxidant mixture.
Another study looking at the effect of a combination of antioxidants on protection of ARPE-19
cells against oxidative damage used a lipophilic extract of tomatoes containing carotenoids at
concentrations of 1.2mg of b-carotene, 0.75mg of lycopene, 0.05mg of lutein, and 0.17mg of
a-tocopherol/g of dry weight of tomato powder (Chichili et al., 2006). The mixture offered a sub-
stantial protection against oxidative damage induced by hydrogen peroxide in the absence and pres-
ence of sodium nitrate. Hydrogen peroxide induced extensive carbonylation of cellular proteins and
formation of thiobarbituric acid reactive substances. Exposure of cells to both, H 2 O 2 and NaNO 2 ,
led to tyrosine nitration. All these effects were substantially diminished upon supplementation of
cells with the tomato extract. Further studies are needed to determine whether the same outcome
can be achieved upon supplementation of cells with a mixture of those carotenoids without possible
additional components from tomatoes, which even at trace concentrations might upregulate cellular
antioxidant defense mechanisms (Baur and Sinclair, 2006; Dinkova-Kostova and Talalay, 2008).