Download Docosahexaenoic acid improves endogen antioxidant

Docosahexaenoic acid improves endogen antioxidant defense in ARPE-19 cells
P. Bogdanov1, F. Gasso1 and J.C. Domingo2
1Brudy
Technology, Barcelona, Spain; 2Deparment of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain.
Fax: +34 93 4021219. e-mail: [email protected]/[email protected]
RESULTS
RESULTS
Generation of intracellular reactive oxygen species (ROS) in retinal cells was produced by 2,2 -azobis-2propionimidinedihydrochloride (AAPH), rotenone (ROT), antymicin A (ANT A), xanthine/xanthine oxidase
(X/XO) and buthionine sulfoximine (BSO) in order to induce the cellular damage associated with lipid
peroxidation and DNA damage. Intracellular ROS was measured by the oxidation of fluorescent probes
(H2DCFDA and DHR123 from Molecular Probes). Lipid peroxidation of retinal cells was estimated as
thiobarbituric acid-reactive substances (TBARS) according to the established methods corrected for protein
content of the lysates (Micro BCA protein assay reagent kit from Pierce Biotechnology). Oxidative DNA
damage of retinal cells was estimated by detection of 8-oxodeoxyguanine (8-OHdG) using the OxyDNA
Assay Kit (Biotrin). Cellular reduced glutathione (GSH) level was estimated by glutathione Assay Kit
(Sigma-Aldrich). The values of GSH concentration obtained were normalized against the protein
concentration of the lysate and expressed as µM/mg protein. DHA in triglyceride structure obtained by
enzymatic synthesis (DHA TG 70e, Algatrium plus®) was obtained from Brudy Technology. Other DHA
was obtained from commercial suppliers.
Figure 2.- Perceptual protection of DHA TG 70e on ARPE-19 cells expressed as reduction in intracellular
oxidant species production. (A) Effect of DHA TG 70e dose on 40 mM AAPH oxidation. (B) Effect of different
stressor inducers on DHA TG 70e protection in ARPE-19 cells. Each bar represents Mean ± SEM (n>4). ∗
p<0.05 by Mann-Whitney test was considered statistically significant, (A) DHA Low-dose vs. DHA Middle or
High-dose treatment.
A
B
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A
Figure 1.- Time course of fluorescence intensities of H2DCF-DA (A) or DHR 123 (B) probes in ARPE-19 after
exposure to 40 mM of AAPH. Control cells (red line) and 0,5 µM DHA treated cells (green line). Each data point
represents the mean of data from six wells (n=6) expressed as fluorescence intensity in arbitrary units.
A
∗
% [GSH] (µM/mg prot)
-56%
B ∗
#
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Figure 4.- Effect of BSO treatment in perceptual protection (A) and on GSH intracellular levels (B) of DHA TG 70e
treated ARPE-19 cells. Each bar represents Mean ± SEM (n>4). ∗ p<0.05 by Mann-Whitney test was considered
statistically significant, (A) BSO treated vs. BSO untreated cells and (B) DHA and/or BSO treated vs. Control cells.
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Figure 5.- Perceptual protection of DHA on ARPE-19 cells expressed as reduction in intracellular oxidant
species production. (A) Effect of DHA content and synthetic method on 40 mM AAPH oxidation. (B) Effect of
chemical structure on DHA 70 protection in ARPE-19 cells. Each bar represents Mean ± SEM (n>4). ∗
p<0.05 by Mann-Whitney test was considered statistically significant, (A) DHA low or middle content vs.
DHA TG 70e treatment at same dose and (B) DHA FFA or EE vs. DHA TG 70e treatment.
CONCLUSIONS
n 
n 
B ∗
B
∗
∗
n 
Figure 3.- Generation of TBARS (A) and 8-OHdG (B) in DHA TG 70e treated ARPE-19 cells before and following
oxidative stress. Each bar represents Mean ± SEM (n>4) of the perceptual variation of each parameter vs.
stressed control. ∗ p<0.05 by Mann-Whitney test was considered statistically significant, DHA TG 70e treated vs.
Control. # p<0.05 by Mann-Whitney test was considered statistically significant, Pre- vs. Post-oxidative stress.
% PROTECCTION
-49%
% PROTECTION
% PROTECTION
∗
B
RESULTS
A
B
%[8-OHdG] (UA/mg prot)
METHODS
A
%[MDA] (µM/mg prot)
INTRODUCTION
Docosahexaenoic acid (DHA) is a major dietary Omega-3 fatty acid being to a major structural lipid of
retinal photoreceptor outer segment membranes. DHA may affect photoreceptor membranes function by
altering permeability and lipid phase properties concerning retinal cell signalling mechanisms involved in
phototransduction. The DHA in the retinal cells (ARPE-19) plays an integral role in the visual cycle, and any
malfunction of the ARPE-19 could generate retinal degenerative diseases such as retinitis pigmentosa and
age-related macular degeneration (ARMD). Under diseased conditions, disturbance in the prooxidantantioxidant balance leads to oxidative stress. Oxidative stress may be a major pathogenic factor in ARMD, as
the potency of retinal defense and repair systems necessary to operate in response to chronic environmental
and metabolic energy–oxidant load is reduced in the aging eye. The retina is particularly susceptible to
oxidative damage because of its high consumption of O2 and its constant exposure to light. The use of
antioxidants and mineral supplements can preserve vision en patients with ARMD and reduce the rate of
disease progression. Lipid peroxidation is one of the consequences of oxidative damage and has been
suggested as a general measurement cell injury. The aim of this study, therefore, was to analyze the effect of
DHA supplementation on the modulation of the redox cellular balance in retinal cells. Reactive oxygen
species (ROS) are intimately involved in the oxidative damage of retina and are used by mammalians cells as
signalling molecules for redox regulation. Extensive evidence suggests that DHA may have a protective role
against retinal tissue although it is easily conceivable that DHA is very sensitive to oxidative stress due to its
high unsaturation. Moreover, structural features of DHA was studied: synthetic origin (enzymatic or
chemical), content (fish oil with 20%, 50% or 70%) and chemical structure (free fatty acid, FFA; ethyl ester,
EE or triglyceride, TG)
n 
The application of the Omega-3 fatty acids in ocular field requires a manufactured products
according to pharmaceutical standards that include quality control steps to insure purity and
structural quality that it can condition it effectiveness.
The incorporation of the DHA TG 70e (Algatrium plus®) in the cellular membrane not only
does not promote the lipid peroxidation but that acts in a low concentration as an activator of
the cellular antioxidant network probably as a result of an adaptive response of the cell
(International Patent PCT/EP2006/070016).
The DHA TG 70e (Algatrium plus®) is an essential cellular antioxidant that protects the
oxidation of cell membranes and DNA damage by free radicals. It works by increasing the
concentration of intracellular glutathione which neutralizes a large number of free radicals.
Unlike a conventional antioxidant products that exerts its activity by scavenging free radicals,
DHA TG 70e enhances the natural mechanism of cell protection against free radicals
(International Patent PCT/EP2006/070016).
Thus, the increase in the intracellular concentration of glutathione provides a unique and
complete protection to maintain the antioxidant network in optimal conditions against free
radicals of different origins, including endogenous mitochondrial ROS.