Archives of Toxicology
Antibiotic–Antimycotic (penicillin, streptomycin and
Amphotericin B, 100×), GlutaMAX™-l (l-alanine-l-glu-
tamine, 100×) and 4′,6-diamidino-2-phenylindole, dihydro-
chloride (DAPI), fuorescein isothiocyanate-dextran (FITC)
10.000 MW were from Gibco (Thermo Fisher Scientifc,
Inc., MA, USA). Punctually, Gibco® Antibiotic–Antimycotic
is used to prevent bacterial and fungal contamination. This
solution contains 10,000 units/mL of penicillin, 10,000 µg/
mL of streptomycin, and 25 µg/mL of Gibco Amphotericin
B. The antibiotics penicillin and streptomycin prevent bac-
terial contamination of cell cultures due to their efective
combined action against Gram-positive and Gram-negative
bacteria. Amphotericin B prevents fungal contamination of
cell cultures due to its inhibition of multi-cellular fungus
and yeast. Particularly, Penicillin was originally purifed
from the fungus Penicillium and acts by interfering directly
with the turnover of the bacterial cell wall and indirectly
by triggering the release of enzymes that further alter the
cell wall. Streptomycin was originally purifed from Strep-
tomyces griseus. It acts by binding to the 30S subunit of the
bacterial ribosome leading to inhibition of protein synthesis
and death in susceptible bacteria. Amphotericin B is an anti-
fungal agent that prevents the growth of fungi and yeast by
causing an increase in fungal plasma membrane permeabil-
ity. Fetal bovine serum (FBS), 3-(4,5-dimethyl-thiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT), 2′,7′-dichlorodi-
hydrofuorescein diacetate (DCDHF-DA) were purchased
from Sigma-Aldrich Co. (St. Louis, MO, USA). Crystal
violet was from Merck Millipore Corp. (Darmstadt, Ger-
many). Trans-Resveratrol (trans-RSV) (>99% HPLC Grade)
(5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-1,3-benzenediol)
from Zhejiang Chempharm (Lian Hua Feng Rd., Hang-
zhou, China) was kindly given by Temis Lostaló S.A Phar-
maceutical Laboratories. Rhodamine-conjugated phalloidin,
Mitotracker Red CMXRos and MitoSOX Red were from
Molecular Probes (Eugene, OR, USA). All-trans-retinal
(CAS 116-31-4) was purchased from Santa Cruz Biotech-
nology Inc. (Santa Cruz, CA, USA).
δ=3.31 ppm. Coupling constants are reported in Hertz (Hz).
13C NMR spectrum was proton decoupled and is referenced
to the middle peak of the solvent CD3OD at δ=49.0 ppm.
Splitting patterns are designated as: s, singlet; d, doublet; t,
triplet; q, quadruplet; dd, double doublet, etc.
A2E synthesis
A2E was synthesized according to Parish et al. (1998)
with slight modifcations applied in Alaimo et al. (2019).
In brief, all-trans-retinal (50 mg, 175 μmol) was mixing
with ethanolamine (70 μL, 75 μmol) in dichloromethane
(3 mL) containing acetic acid (5 μL) and stirred in the dark
at RT for 3 days. The solvent was evaporated to dryness.
The residue was purifed by silica gel column chromatogra-
phy using CH3OH:CH2Cl2 (5:95) and CH3OH:CH2Cl2:TFA
(8:92:0.001), obtaining A2E (23.8 mg, 51%) with 9% of iso-
A2E (estimated by 1H-NMR). 1H NMR (CDCl3) δ1.02 (s,
12H), 1.48 (m, 4H), 1.64 (m, 4H), 1.70 (s, 6H), 2.00 (s,
3H), 2.03 (m, 4H), 2.14 s, (3H), 2.15 (s, 3H), 3.74 (t, 2H, t,
J=5.2 Hz), 4.61 (t, 2H, J=5.1 Hz), 6.13 (d, 1H, J=16.0 Hz),
6.16 (d, 1H, J=11.0 Hz), 6.17 (d, 1H, J=16.0 Hz), 6.27 (d,
1H, J = 16.0 Hz), 6.41 (d, 1H, J = 11.0 Hz), 6.55 (d, 1H,
J=16.2 Hz), 6.60 (d, 1H, J=16.1 Hz), 6.70 (s, 1H), 6.75 (d,
1H, J=15.9 Hz), 7.07 (dd, 1H, J=11.2, 15.9 Hz), 7.82 (d,
1H, J=1.0 Hz), 7.92 (dd, 1H, dd, J=1.1, 7.0 Hz), 8.40 (dd,
1H, J=11.2, 15.7 Hz), 8.54 (d, 1H, J=7.0 Hz).
Cell culture
ARPE-19 (ATCC®CRL-2302™) cell line which is devoid
of endogenous A2E (Sparrow and Cai 2001) was kindly
provided by Dr.Jeremias Gastón Galletti and Dr. Mauricio
Guzmán (Instituto de Medicina Experimental IMEX-CONI-
CET, Buenos Aires, Argentina). ARPE-19 is a spontane-
ously arising human RPE cell line that maintains normal
RPE cells in vivo (Dunn et al. 1996). Cells were cultured
in DMEM supplemented with 10% heat-inactivated FBS,
2.0 mM glutamine, 100 units/ml penicillin, 100 µg/mL
streptomycin and 0.25 µg/mL amphotericin in a humidifed
atmosphere of 5% CO2–95% air and 37 °C. Medium was
renewed three times per week. ARPE-19 cells were cultured
and used up to 15 passages maximum. For all experiments,
ARPE-19 cells were detached with 0.05% trypsin-EDTA,
diluted with DMEM 10% FBS and re-plated into multi-well
plates to yield 70–80% confuent cultures after 24 h. Experi-
mental conditions: A2E or RSV was delivered in culture
media for 24 h. In the experiments that involved the pres-
(Albuquerque et al. 2015; Lee et al. 2015; Moine et al. 2018)
Final concentration of dimethyl sulfoxide (DMSO) and
ethanol did not exceed 0.25%. DMSO and ethanol added to
the samples did afect neither cell viability, morphology nor
other parameters tested in this study. Water was distilled and
deionized using a MILLI-Q water system. Others chemicals
used were of analytical grade.
Spectral data of trans‑RSV by 1H‑nuclear magnetic
resonance (NMR)
1H NMR and 13C NMR spectra were recorded at room tem-
perature (RT) in CD3OD as solvent using a Bruker AM-500
NMR instrument operating at 500.14 MHz and 125.76 MHz
1
1
for H and 13C, respectively. The H NMR spectrum is
referenced to the middle peak of the solvent CD3OD at
1 3