Free Radical Biology and Medicine 209 (2023) 239–251

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

The comparison of antioxidant properties and nutrigenomic redox-related

activities of vitamin C, C-vitamers, and other common ascorbic
acid derivatives
Patrycja Jakubek a, b, *, Klaudia Suliborska a, Monika Kuczyńska a, Muhammad Asaduzzaman a,
Karol Parchem a, Izabela Koss-Mikołajczyk a, Barbara Kusznierewicz a, Wojciech Chrzanowski a,
Jacek Namieśnik a, Agnieszka Bartoszek a
a
Faculty of Chemistry, Gdańsk University of Technology, 80-233, Gdansk, Poland
b
Laboratory of Mitochondrial Biology and Metabolism, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093, Warsaw, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: The term ‘vitamin C’ describes a group of compounds with antiscorbutic activity of L-ascorbic acid (AA). Despite
Antioxidant activity AA’s omnipresence in plant-derived foods, its derivatives have also been successfully implemented in the food
Redox homeostasis industry as antioxidants, including the D-isomers, which lack vitamin C activity. This study aimed to determine
Oxidative damage
the relationship between redox-related activities for five derivatives of AA using electrochemical, chemical, and
Gene expression
Essential nutrients
biological approaches. Here we report that AA, C-vitamers, and other commonly consumed AA derivatives differ
in their redox-related activities. As long as the physiological range of concentrations was maintained, there was
no simple relationship between their redox properties and biological activity. Clear distinctions in antioxidant
activity were observed mostly at high concentrations, which were strongly correlated with electrochemical and
kinetic parameters describing redox-related properties of the studied compounds. Despite obvious similarities in
chemical structures and antioxidant activity, we showed that C-vitamers may exhibit different nutrigenomic
effects. Together, our findings provide a deeper insight into so far underinvestigated area combining chemical
properties with biological activities of commonly applied AA derivatives.

1. Introduction high as 3 g administered 6 times a day resulted in the peak plasma

The term essential nutrients refers to substances that are required for
the proper physiological function of human organism, but cannot be
produced endogenously – either at all or in sufficient amounts – and
thus, must be provided daily with food [1]. Among such dietary com­
ponents, some display antioxidant activity, e.g., vitamin C. The term
“vitamin C” describes a group of L-ascorbic acid (AA) derivatives, which
possess the biological activity of vitamin C. Such structurally related
compounds can be also named vitamers, or in this particular case –
C-vitamers [1]. Vitamin C is widely distributed in foods, mostly of plant
origin, and its high content was reported in black currants, peppers, kiwi
fruits, papayas, and oranges [2]. The dietary intake of vitamin C de­
termines its plasma concentration, which reaches 40–80 μM in healthy
individuals consuming several servings of fruits and vegetables daily. In
the case of oral supplementation of AA by healthy volunteers, doses as
concentrations not exceeding 220 μM [3]. In tissues, ascorbate accu­
mulates in millimolar concentrations that are not possible to be reached
in plasma unless pharmacological doses are provided intravenously.
Except for plant-derived sources of vitamin C, AA derivatives can be also
consumed as food additives. The group of ascorbates used in the food
industry includes, among others, L-ascorbic acid (E300, AA), sodium
L-ascorbate (E301, NaA), and calcium L-ascorbate (E302, CaA), which
exhibit the biological function of vitamin C, as well as D-isoascorbic acid
(E315, iAA, also known as erythorbic acid) and sodium D-isoascorbate
(E316, iNaA, also known as sodium erythorbate), which lack biological
activity of vitamin C (Fig. 1a). The enrichment of food products in AA
and its derivatives increases their nutritional and sensory value, inhibits
browning, stabilizes flavour and colour as well as protects against po­
tential spoilage and oxidation of other food components [4]. Besides,
different C-vitamers can be also consumed in the form of dietary

* Corresponding author. Nencki Institute of Experimental Biology of Polish Academy of Sciences, 3 Pasteur St., 02-093, Warsaw, Poland.
E-mail address: p.jakubek@nencki.edu.pl (P. Jakubek).

https://doi.org/10.1016/j.freeradbiomed.2023.10.400
Received 11 September 2023; Received in revised form 17 October 2023; Accepted 19 October 2023
Available online 20 October 2023
0891-5849/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
P. Jakubek et al.
supplements with recommended supplementation doses, which often
exceed the recommended dietary allowance of vitamin C by several-fold.
Based on the available evidence, the European Food Safety Authority
has conducted thorough evaluations and determined that the con­
sumption of AA and its derivatives does not pose any health risks, thus
there is no need to define an acceptable daily intake for these com­
pounds [5–7].
Vitamin C plays multiple essential roles in the human organism, most
of which stem from its ability to donate electrons, which makes it a
physiologically essential reducing agent [8]. In light of this, it is some­
what surprising that to date no complex evaluation of the (electro)
chemical properties as well as antioxidant and nutrigenomic activities of
C-vitamers and other AA derivatives has been performed. Therefore, in
this study, we aimed to determine the relationship between chemically
defined antioxidant properties of vitamin C, C-vitamers, and other
common AA derivatives and their impact on redox homeostasis in a
cellular setting mimicking the human alimentary tract. To the best of our
knowledge, such a comprehensive investigation combining chemistry
with the biological effects of five different AA derivatives, going beyond
food processing applications, has not been performed so far. The
chemical assessment of antioxidant activity was conducted by applying
electrochemical techniques such as potentiometric titration (PT) and
differential pulse voltammetry (DPV) as well as simple spectrophoto­
metric tests employing ABTS and DPPH radicals. With the use of the
ABTS and DPPH tests, we established stoichiometric n10 values that
reflect the number of radical molecules that can be scavenged by one
molecule of an antioxidant within a fixed predefined (10 min) reaction
time. Next, we determined the ability of AA derivatives to modulate
protein and lipid oxidation kinetics using another cell-free system
mimicking intestinal conditions.
In the biological part of this study, human colon adenocarcinoma
HT29 cell line was used as a cellular model representing human intes­
tinal epithelium that is directly exposed to ingested food. When cultured
under standard growth conditions (25 mM glucose, 10% fetal bovine
serum (FBS)), HT29 cells exhibit the phenotype of undifferentiated
colonic epithelial cells [9]. Moreover, the proteomic analysis of cell lines
and scrapings of the human intestinal epithelium showed that HT29
cells express proteins that are characteristic of human intestinal
epithelium in vivo [10]. As such, on one hand, they resemble intestinal
cells, and on the other hand, their metabolism is characteristic of the
metabolism of cancer cells, thus they are more sensitive to changes in
redox environment compared to normal cells [11]. We needed a model
resembling human alimentary tract but at the same time enabling the
detection of even very slight differences in response to antioxidant
treatments. The experimental design involved the assessment of cyto­
toxicity (MTT test), cellular antioxidant activity (CAA assay), genotox­
icity, and DNA protection against oxidative damage (comet assay) as
well as microarray analysis of changes in the expression of 84
redox-related genes. The results of this study shine some new light on
antioxidant activity regarding complex relationships between chemical
structures, electrochemistry as well as biological activity at both cellular
and nutrigenomic levels.
2. Materials and methods
2.1. Chemicals and reagents
AA and its derivatives investigated in this study included: L-
(+)-ascorbic acid (AA), D-(− )-isoascorbic acid (iAA), sodium L-
(+)-ascorbate (NaA), sodium D-(− )-isoascorbate (iNaA) and calcium L-
(+)-ascorbate (CaA), which were purchased from Sigma-Aldrich (USA).
Hexacyanoferrate(III) potassium (K3[Fe(CN)6]), iron(III) chloride
hexahydrate (FeCl3 × 6H2O), sodium thiosulfate pentahydrate
(Na2S2O3 × 5H2O) were purchased from Sigma Aldrich (USA) and used
in potentiometric titration experiments. All solutions were prepared using
purified water (QPLUS185 system, Millipore, USA), except for FeCl3 ×
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Free Radical Biology and Medicine 209 (2023) 239–251
6H2O and Na2S2O3 × 5H2O, which were dissolved in 1 M HCl. Spec­
trophotometric tests were conducted with DPPH (1-diphenyl-2-picrylhy­
drazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt) radicals from Sigma Aldrich (USA). Their stock
solutions were prepared using analytical grade methanol purchased
from Avantor Performance Materials (Poland). ABTS and DPPH solu­
tions were prepared in analytical grade methanol, as discussed in detail
in the response for Reviewers and indicated in Method description in
Section 2.3. Acetone and ethanol were used in other procedures (as
mentioned in the next sentence), but not for stock solutions of ABTS and
DPPH. Fresh free-range hen eggs obtained from a local Polish eco-farm
to avoid pesticide contamination were used for the preparation of li­
posomes. Reagents applied for the preparation of liposomes included
acetone, methanol, ethanol (Avantor Performance Materials, Poland),
and hexane (Merck, USA), all of analytical grade. Hemoglobin isolated
from bovine blood (in the form of lyophilized powder), 4-(2-hydrox­
yethyl)-1-piperazineethanesulfonic acid (HEPES) (≥99.5%) from Sigma
Aldrich (USA), and nitrogen (99.996%) from Oxygen S.c. (Poland) were
used for monitoring lipid peroxidation. The Protein Carbonyl content assay
kit, bovine serum albumin (BSA), and Bicinchoninic acid (BCA) kit were
purchased from Sigma Aldrich (USA) and used for the determination of
the inhibition of protein oxidation. All reagents for cell culture such as
phosphate-buffered saline (PBS), McCoy’s 5A medium, trypsin, FBS, and
antibiotics (penicillin and streptomycin) were purchased from Sigma
Aldrich (USA). For the MTT test, thiazolyl blue tetrazolium bromide
(MTT) and dimethyl sulfoxide (DMSO) from Sigma-Aldrich (USA) were
used. The CAA assay was carried out using the OxiSelect Cellular Anti­
oxidant Assay kit purchased from Cell Biolabs, Inc. (USA), and Hanks’
balanced salt solution (HBSS) from Sigma Aldrich (USA). A hydrogen
peroxide solution (30% v/v) as well as all ingredients used for the
preparation of buffers for comet assay were purchased from Sigma
Aldrich (USA) unless stated otherwise. For RNA isolation and assessment
of gene expression, the RNeasy Mini Kit, RNase-Free DNase set, QIAsh­
redders, RT2 First Strand Kit, RT2 SybrGreen qPCR Mastermix, and RT2
Profiler PCR Arrays for Oxidative Stress (PAHS 065Z) were provided by
Qiagen (Germany).
2.2. Electrochemical measurements
2.2.1. Standard reduction potential by potentiometric titration
The PT and mathematical analysis of experimental titration curves
were performed as described before [20]. Briefly, PT was performed
using JENCO 6230 N equipment (China) and platinum measuring
electrode (Micro Combination Redox Electrode, Ag|AgCl inner reference
electrode (RE), JENCO, China). The titration was carried out using a
solution of K3[Fe(CN)6]. The tested compounds were dissolved at a
concentration of 1 mg/mL using 0.01 M PBS (pH = 7.4 ± 0.1), except for
CaA. The latter compound was dissolved in ultra-pure water due to its
low solubility in phosphate buffers. The solution of CaA had a pH equal
to 3.83 ± 0.01, which was measured at the equivalence point of the
titration curve; this value was further used in calculations. The mea­
surements were carried out at 37 ◦ C (± 0.1 ◦ C). Finally, the titration
curves E = f(Vtitr.) were analyzed using the sigmoidal 5-parameter model
[20] by SigmaPlot 13.0 software (Systat Software Inc., UK). The po­
tential at the equivalence point was read directly from the mathematical
model and subsequently, the standard reduction potential (E0) of a given
redox couple was calculated. The accurate potential of RE (ϵ) was
measured by the titration of FeCl3 × 6H2O and Na2S2O3 × 5H2O, whose
E0 values are known [20,21].
2.2.2. Antioxidant power by differential pulse voltammetry
The DPV was performed as described previously [21] with minor
modifications. Briefly, 6 mM stock solutions of the studied compounds
were prepared using 0.1 M sodium phosphate buffer (pH = 7.4 ± 0.1) as
a solvent, except for CaA, whose solution was prepared in 0.1 M KCl (pH
= 7.0 ± 0.1). The DPV was carried out using Gamry Reference 600
P. Jakubek et al.
potentiostat (Gamry Instruments, Inc., USA). The three-electrode system
consisting of a glassy carbon electrode (BASi, USA) (working electrode
(WE)), platinum wire (counter electrode (CE)), and the Ag|AgCl elec­
trode (reference electrode, RE) from Hydromet S.c. (Poland) was
applied. The area of WE (AWE) was equal to 0.162 cm2, which was
determined by cyclic voltammetry of 1 mM K3[Fe(CN)]6 solution in 0.1
M KCl. The DPV measurements were recorded in the potential range of
− 0.3 to +0.9 V. The other settings included the potential scan rate (v) of
0.1 V s− 1, the pulse height of 0.05 V, the pulse time of 0.1 s, and the
sample period of 0.5 s. After every measurement, the surface of WE was
polished by alumina suspension (Buehler, USA) on the microcloth pads
(BASi, USA) and then rinsed with ultra-pure water and methanol. The
electrochemical cell was cleaned with a solution of 95% H2SO4 with
0.002% of KMnO4 (w/v).
The value of antioxidant power (AOP) was calculated using Equation
(1) and was expressed in W•cm− 2:
AOE
AOP = (1)
tf − ti
where: tf-ti – the difference between the time of the starting point of an
oxidation peak (ti) and its end (tf) [s], AOE – a specific parameter of
antioxidant energy [J × cm− 2] calculated according to Equation (2):
1 ∑
f
AOE = − (E − ε) • I • dt (2)
AWE i
where: E − an applied potential vs. RE [V], ε – a correction term (taking
into consideration the liquid junction between the WE and RE, identified
in the same way as described in Section 2.2.1), I – current measured in
relation to the background [A], t – potential sampling time [dt = 0.5 s].
2.3. Antioxidant activity by spectrophotometric tests
The colorimetric determination of the antioxidant activity of inves­
tigated compounds in a cell-free system and subsequent analysis of ob­
tained results were carried out as described previously [20]. Briefly, the
absorbance of the methanol solution of ABTS or DPPH radical was
adjusted to 0.8 ± 0.05 at 734 nm and 0.9 ± 0.05 at 515 nm, respectively.
Stock solutions of tested compounds were prepared in 96% ethanol and
diluted (using the same solvent) to concentrations falling within a linear
range of the assay. Measurements of absorbance were performed using a
TECAN Infinite M200 spectrophotometer (Tecan Group Ltd.,
Switzerland) after 10 min of incubation at 25 ◦ C, 37 ◦ C, or 41 ◦ C. The
results of antioxidant activity determinations were calculated into
stoichiometry value (n10) as described previously [20].
2.4. Kinetics of protein and lipid oxidation
2.4.1. Preparation of liposomes
The phospholipid (PL) fraction from hen egg yolk was isolated using
methanol followed by PL precipitation in acetone cooled to − 20 ◦ C, as
described previously [12]. PL fraction was dissolved in a mixture of
chloroform/methanol (2:1 v/v) to reach the concentration of 5 mg/mL.
Then, the solvent mixture was evaporated under reduced pressure using
a rotary evaporator at a temperature below 40 ◦ C. The solvent residue
was removed with a gentle stream of nitrogen for 30 min. The resulting
thin lipid film was hydrated at room temperature in HEPES buffer (10
mM, pH 7.2). For this purpose, the buffer was added to the round bottom
flask containing lipid film, and the content was stirred in the nitrogen
atmosphere using an orbital shaker at 37 ◦ C for 30 min. The obtained PL
vesicles (1 mg/mL) were then sonicated at the ambient temperature (8
min, 20 kHz) using Vibra-Cell VCX 750 ultrasound sources fitted with a
3 mm diameter titanium micro-tip (Sonics, USA). Power delivery was
controlled as an amplitude percentage that was set at 40%. A pulsed
duty cycle of 30 s on and 10 s off was used for all experiments. The
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Free Radical Biology and Medicine 209 (2023) 239–251
obtained liposomes were diluted with HEPES buffer (10 mM, pH 7.2) to
the final PL concentration of 0.2 mg/mL. Then, the liposomes were
sonicated again as described above to obtain small unilamellar vesicles
(SUVs). The liposome suspension was filtered on a 0.2 μm Acrodisc sy­
ringe filter with a nylon membrane to remove potential titanium parti­
cles [13]. The liposome size distribution and dispersity (polydispersity
index, PDI) were determined using a non-invasive black scatter light
method (NIBS) [14]. For this purpose, a Zetasizer Nano ZS (Malvern,
United Kingdom) equipped with helium-neon laser diffraction operating
at 633 nm was employed. All measurements were performed at 37 ◦ C.
The obtained PL fraction was used to observe the potential modulation
of lipid peroxidation and protein carbonylation processes by AA
derivatives.
2.4.2. Modulation of lipid peroxidation
The kinetics of lipid peroxidation was assessed by monitoring the
formation of conjugated dienes in the structure of unsaturated fatty
acids (UFAs) build-in PL molecules. For this purpose, liposome suspen­
sion (0.2 mg/mL) was placed in a quartz cell with a 1 cm path length and
incubated in the spectrophotometer thermostat at 37 ◦ C for 7 min. Then,
aqueous solutions of AA or its derivatives were added to obtain a con­
centration in the range of 10–100 μM and the mixture was incubated for
a further 2 min. The liposome oxidation was initiated by the addition of
an aqueous solution of hemoglobin in the final concentration of 2.5 μM
(10 μM based on haem iron), which was considered as t = 0 min. The
kinetics of oxidation was examined by monitoring the change of ab­
sorption at the wavelength of 234 nm at 37 ◦ C for 120 min at 1 min
intervals using a NanoDrop 2000c spectrophotometer (Thermo Scienti­
fic, USA). The oxidation rates were determined as the slope of the
regression line for the linear range of absorbance at 234 nm versus re­
action time. All experiments were carried out in three independent
repetitions.
2.4.3. Modulation of protein carbonylation
The effect of AA and its derivatives on protein oxidation was assessed
by quantification of the formed carbonyl groups (aldehyde and ketone)
in the structure of a model protein, i.e., BSA. For this purpose, the
Protein Carbonyl content assay kit (Sigma Aldrich) was used following
the protocol available on the manufacturer’s website (https://www.
sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents
/263/399/mak094bul-mk.pdf). BSA solution in HEPES buffer (10 mM,
pH 7.4) was mixed with liposomes (prepared as described in section
2.4.1) to obtain the final BSA and PL concentrations of 0.5 mg/mL each.
Then, the mixture was sonicated as already described to obtain SUVs.
Finally, to initiate the protein oxidation, 2 mL of BSA/PL mixture was
incubated with 0.34 mg/mL of hemoglobin and different concentrations
of tested compounds (10–1000 μM) or HEPES buffer (control) for 4 h at
37 ◦ C. After the incubation, proteins were precipitated from the mixture
using 50 μL of 100% trichloroacetic acid (TCA) solution, vortexed, left
on ice for 5 min, and then centrifuged at 13,000 × g for 5 min. Further
steps were performed as described in the manufacturer’s protocol.
Briefly, protein suspension was mixed with 2,4-dinitrophenylhydrazine
(DNPH), and the samples were incubated at room temperature for 10
min. Afterwards, 100% TCA was used to precipitate proteins, the sam­
ples were left on ice for another 5 min and subsequently centrifuged. The
supernatant was removed, and the remaining pellet was suspended in
ice-cold acetone, treated with ultrasounds, and centrifuged. The samples
were washed twice with acetone to remove unbound DNPH. After the
second wash, the pellet was dissolved in 6 M guanidine solution, and
100 μL of the obtained solution was transferred to the 96-well plate (in
three repetitions per each sample). The measurement of absorbance was
carried out at 375 nm using a TECAN Infinite M200 plate reader. The
protein oxidation was expressed as a content of carbonylated proteins.
The amount of carbonylated proteins in a sample per well was calculated
from Equation (3):
P. Jakubek et al.
A375
Ccp = •V (3)
εmM • l
where: Ccp – the content of carbonylated proteins in a well [nmol], A375
– the absorbance at 375 nm, ƐmM – the millimolar extinction coefficient
1
(22 mM M•cm ), l – an optical path in a well of a 96-well plate (0.2893 cm),
V – the total reaction volume in a well (100 μL).
To determine the amount of protein per sample, 5 μL of protein
suspension was transferred to another 96-well transparent plate and
BCA assay was performed. The amount of carbonylated proteins per mg
of protein was calculated using Equation (4):
Ccp
CP = • 1, 000 • D (4)
P
where: CP – the total content of carbonylated proteins [nmol/mg pro­
tein], P – the amount of protein from standard well x 20 [μg/well], 1000
– conversion factor (from μg to mg), D – sample dilution.
2.5. Cell culture
Human colon adenocarcinoma HT29 cells were provided and
authenticated by the American Type Culture Collection (ATCC, USA).
The cells were cultured in McCoy’s medium supplemented with 10%
FBS and penicillin/streptomycin and grown in a humidified atmosphere
(37 ◦ C, 5% CO2) in a Smart cell incubator (Heal Force, China). Cultured
cells were regularly checked for mycoplasma contamination using
Universal Mycoplasma Detection Kit from ATCC (USA). The solutions of
antioxidants used in cell-based tests were additionally sterilized by
passing through Millex sterile R33 mm (0.22 μm) syringe-driven filters
(Millipore, USA) to avoid contamination.
2.5.1. Cell growth by MTT test
MTT assay was performed to monitor the growth of HT29 cells
exposed to the investigated compounds as described previously [15].
Briefly, cells were seeded in 96-well tissue culture plates at the density of
5,000 cells per well and were allowed to settle for 24 h at 37 ◦ C. Then,
the cells were treated either with purified water (as a control) or with
solutions of tested antioxidants for 6, 24, and 72 h within the final range
of concentrations from 0.01 μM to 10 mM. All cells were allowed to grow
for the total incubation time of 72 h, then cell growth was estimated by
MTT assay. The measurement of absorption of formazan solutions was
performed at 540 nm using a TECAN Infinite M200 plate reader. The
effect of tested AA derivatives was calculated and expressed as a per­
centage of cell growth determined for control cells treated with an
appropriate solvent only (% control).
2.5.2. Cellular antioxidant activity by CAA assay
The procedure and analysis of CAA test results have been previously
described in detail [16]. In brief, HT29 cells were seeded in 96-well
plates (30,000 cells per well) and were allowed to settle for 24 h at
37 ◦ C. Then, the cells were treated with aqueous solutions of investi­
gated ascorbates for 1 h in the concentration range from 1 to 1000 μM.
All steps of the CAA assay (Cell Biolabs) were performed according to
the manufacturer’s protocol available online (https://www.cellbiolabs.
com/sites/default/files/STA-349-cellular-antioxidant-activity-assay-kit
.pdf). The measurements of fluorescence were carried out every 5 min
for 60 min at 480/530 nm (ex./em.) with TECAN Infinite M200 plate
reader. Calculations of CAA units were performed as described earlier
[20].
2.5.3. Genotoxicity and protection against DNA fragmentation by comet
assay
HT29 cells were seeded in 24-well tissue culture plates at the density
of 100,000 cells per well and were allowed to settle for 24 h at 37 ◦ C. For
genotoxicity testing, cells were treated for 24 h with tested ascorbates
dissolved in sterile ultra-pure water in the concentration range from 1 to
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Free Radical Biology and Medicine 209 (2023) 239–251
1000 μM. The cells treated with solvent only served as negative controls,
whereas cells treated with 200 μM hydrogen peroxide for 1 h served as a
positive control. For protection against DNA damage tests, the treatment
conditions were modified as follows: right after the 24 h treatment with
tested antioxidants at final concentrations of 50, 100, or 200 μM was
finished, the medium was removed and HT29 cells were subjected to
additional 1 h exposure under cell culture conditions to 150 μM
hydrogen peroxide solution prepared in fresh medium. Afterwards,
comet assay was performed as described previously [17]. The cell lysis
was carried out overnight, while DNA unwinding in electrophoretic
buffer lasted 20 min and was followed by 30 min of electrophoresis (26
V, 300 mA) in darkness. DNA was stained with cold SybrGreen solution
for 30 min and washed with cold ultra-pure water for another 5 min. The
stained nuclei were examined under a fluorescence microscope (Zeiss,
ImagerZ2, USA) and scanned using the Metafer4 scanning system
(Germany). For genotoxicity testing, the analysis was performed
employing the Metafer4 system and involved counting 200 consecutive
nuclei per gel. For testing protection against DNA damage, the analysis
was performed with Comet Score 2.0 software and involved counting
100 consecutive nuclei per gel. The results of comet assay were
expressed as %DNA in the comet tail.
2.6. Microarray analysis
HT29 cells were seeded in 24-well tissue culture plates at a density of
100,000 cells per well and were allowed to settle for 24 h at 37 ◦ C. Then,
the cells were treated with AA and CaA at 50 and 200 μM concentrations
for 24 h (37 ◦ C, 5% CO2). The tested compounds were dissolved in ultra-
pure water and sterilized with the aid of syringe filters (0.22 μm). Total
RNA isolation was performed with the use of the RNeasy Mini kit pro­
tocol with a homogenization step including the use of QIAshredders
(Qiagen, Germany). The additional on-column digestion of gDNA was
applied with the aid of the RNase-free DNase Set (Qiagen, Germany).
The quality and quantity of isolated RNA were estimated using Nano­
drop 2000c built-in software (Thermo Scientific, USA). The isolated
RNA was stored at − 80 ◦ C until use. The amount of mRNA used for
reverse transcription (RT) was always equal to 500 ng. RT was con­
ducted using the RT2 First Strand kit according to the manufacturer’s
protocol. The obtained cDNA was immediately diluted in qPCR master
mix using the RT2 SYBR Green kit according to the instructions supplied
by the manufacturer (Qiagen, Germany). All instructions are available
online at https://www.qiagen.com/us/resources/resourcedetail?
id=f4b13eaa-884f-4357-abe6-1a5f9469bc32&lang=en. The prepared
master mix was subsequently aliquoted over 96-well RT2 Profiler PCR
human oxidative stress array consisting of 84 human genes related to
oxidative stress response and 3 controls assessing RT efficiency, 3 pos­
itive PCR controls, a gDNA contamination control as well as reference
genes (Supplementary Table S1). The RT-PCR was performed according
to the protocol of the manufacturer using LightCycler 96 (Roche,
Switzerland). Data analysis was performed using software available on
the manufacturer’s website (https://dataanalysis.qiagen.com/pcr/arra
yanalysis.php). Data were normalized based on the geometric aver­
aging of 3 best-performing reference genes (B2M, HPRT1, and RPLP0).
The relative changes in gene expression were calculated with the aid of
the comparative threshold cycle method (ΔΔCt) [20].
2.7. Statistical analysis
Statistical analysis was carried out using Prism 8.0 software package
(GraphPad Software, Inc., USA); the level of statistical significance was
set at p-value <0.05. All results were expressed as means ± SD of three
independent experiments.
P. Jakubek et al.
3. Results
3.1. Standard reduction potential and antioxidant power
PT is a technique that allows monitoring of the oxidation of a com­
pound in the bulk solution and as such, measuring the thermodynamic
parameter E0 of a given redox couple. The parameter E0 describes the
compound’s ability to accept electrons and is strongly related to the
presence of redox-active moieties in its structure. The lower the E0, the
better electron donor a molecule is, which means that it exhibits
stronger antioxidant (i.e., reducing) properties. According to PT mea­
surements carried out for studied ascorbates, the antioxidant activity
among the studied group of compounds was shown to increase in the
following order: AA < NaA < iAA < iNaA < CaA (Table 1, Fig. 1b).
Among tested compounds, the strongest antioxidant turned out to be
CaA with E0 value almost 10-fold lower compared to other AA de­
rivatives. The sodium salts of both isomers of AA exhibited stronger
reducing properties than their parental forms, whereas the L-stereoiso­
mers turned out to be weaker electron donors than their corresponding
D-stereoisomers.
The AOP calculated based on DPV determinations combines three
significant factors that describe the electrooxidation at the surface of
WE: a value of the transferred charge Qa (area under the voltammetric
curve, the higher it is, the stronger the antioxidant properties are), the
rate of charge transfer expressed by the time of its transfer t (the shorter it
is, the faster the electrooxidation process) as well as the charge transfer
energy expressed by the Ep,a (the higher it is, the stronger the antioxi­
dant). Again, CaA exhibited the strongest antioxidant properties, here
taking into account both thermodynamic and kinetic aspects, since its
electrooxidation process was characterized by the highest values of Ip,a
and Ep,a (Table 1). The values of AOP were shown to be positively
correlated with the reducing properties of studied compounds (Pearson
r = − 0.9762, p-value 0.0008).
3.2. Antioxidant activity by ABTS and DPPH tests
Spectrophotometric tests used in this study were modified in a way to
reflect also the kinetics of redox reactions [18]. Therefore, the antioxi­
dant activity was expressed as the stoichiometric n10 value that de­
scribes how many molecules of a radical were scavenged by one
molecule of an antioxidant within 10 min reaction time. Stoichiometric
value n10 is calculated as a tangent of a linear relationship between
concentrations of reduced radical and studied compounds at three
different temperatures: room temperature (25 ◦ C), the average physio­
logical temperature of the human body (37 ◦ C), and elevated tempera­
ture (41 ◦ C), which in the case of humans may be accompanied by
excessive ROS formation and requires medical intervention. As shown in
Fig. 1c,e, C-vitamers and other studied AA derivatives were significantly
better scavengers of ABTS than of DPPH radicals, regardless of the
temperature (Fig. 1d,f, p-values are gathered in Supplementary
Table S2). In both tests, the strongest and the weakest radical scavengers
Table 1
The characterization of the oxidation (in the bulk solution by potentiometric titration, PT) and electrooxidation (at the surface of WE by differential pulse voltammetry,
DPV) of ascorbic acid and its derivatives. The values of standard reduction potential (E0) were calculated based on PT measurements carried out at 37 ◦ C. The values of
anodic peak potential (Ep,a), anodic peak current (Ip,a), charge passed under the anodic waves (Qa), antioxidant energy (AOE), time of reaction (t) and antioxidant
power (AOP) were determined by DPV at 25 ◦ C; potential scan rate v = 0.1 V s− 1, ε = 0.103 V. Results are presented as means ± SD. Three independent experiments
were performed for each compound and technique.
Compound E0 [V] Ep,a + ϵ [V] Ip,a [μA]
Method PT DPV DPV
AA 0.287 ± 0.002 0.233 ± 0.010 15.7 ± 1.60
NaA 0.272 ± 0.002 0.163 ± 0.005 15.6 ± 0.20
iAA 0.258 ± 0.002 0.218 ± 0.007 14.15 ± 0.01
iNaA 0.246 ± 0.002 0.256 ± 0.010 11.26 ± 1.03
CaA 0.021 ± 0.002 0.266 ± 0.012 25.1 ± 2.60
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Free Radical Biology and Medicine 209 (2023) 239–251
at each tested temperature were CaA and iNaA, respectively. In the ABTS
test, the antioxidant activity of AA and its isomer iAA was the same at
higher temperatures, while at room temperature, iAA turned out to be a
significantly better radical scavenger. In the DPPH test, AA exhibited
stronger antioxidant activity than iAA, regardless of the temperature.
NaA was a better radical scavenger than its isomer iNaA in both tests at
any tested temperature. All p-values were gathered in Supplementary
Table S3.
3.3. Modulation of lipid peroxidation
The impact of AA and its derivatives on the kinetics of lipid peroxi­
dation was used as another parameter describing their antiradical ac­
tivity. The method was based on monitoring the initiation of lipid
oxidation by the formation of primary lipid oxidation products. Specif­
ically in our study, we monitored the formation of conjugated dienes in
the structure of UFAs build-in the structure of liposome PLs induced by
the presence of haem iron. As shown in Fig. 2a, all tested compounds at
concentration range of 20–75 μM significantly increased the reaction
rate of liposome PL oxidation in the presence of haem iron compared to
the control (p-values are gathered in Supplementary Table S4). For all
tested compounds, there was a trend of accelerating the PL oxidation
along with the increasing concentration of tested antioxidants. Howev­
er, when the maximum reaction rate was reached, a further increase in
the concentration of AA or its derivatives led to a decrease in the reac­
tion rate. Based on the obtained results, we found very strong and sta­
tistically significant correlations between the concentration of the tested
compounds and the acceleration of lipid oxidation (Supplementary
Table S5). Among tested compounds, the strongest accelerator of lipid
peroxidation in the presence of haem iron turned out to be iAA in the
concentration range between 40 and 50 μM, while at the highest con­
centrations – its parent compound, AA (Fig. 2a, Supplementary
Table S6). CaA turned out to be a significantly stronger accelerator of
lipid peroxidation compared only to NaA and solely at the lowest tested
concentration (Fig. 2a, Supplementary Table S6).
3.4. Modulation of protein carbonylation
To determine the ability of AA and its derivatives to protect proteins
against oxidative damage, the system developed to study the liposome
PL peroxidation was used, with one modification, namely a model
protein (BSA) was added to the reaction mixture. After the oxidation
forced by the reaction mixture components, proteins were extracted,
and carbonyl derivative content was measured. The obtained results
showed that all tested AA derivatives, except CaA, slightly inhibited
protein carbonyl formation in the whole tested concentration range
(Fig. 2b). The strongest protective effect among tested compounds
showed AA, followed by NaA and iAA, especially at the highest tested
concentrations (Fig. 2b). L-stereoisomers tended to show a stronger
protective effect than D-stereoisomers and sodium salts of both isomers
of AA exhibited weaker protective properties than their parental forms,
Qa [μC⋅cm− 2] AOE [μJ⋅cm− 2] t [s] AOP [μW⋅cm− 2]
DPV DPV DPV DPV
16.6 ± 0.6 215.7 ± 18.7 5.5 ± 0.5 39.3 ± 0.8
18.3 ± 0.7 153.3 ± 0.7 5.0 ± 0.3 30.7 ± 1.9
16.1 ± 0.1 186.5 ± 1.4 5.8 32.9 ± 0.7
14.8 ± 0.3 217.6 ± 41.9 5.8 ± 0.6 37.7 ± 5.9
41.7 ± 2.2 659.3 ± 38.1 6.1 ± 0.3 107.6 ± 0.9
P. Jakubek et al. Free Radical Biology and Medicine 209 (2023) 239–251

Fig. 1. Antioxidant activity of vitamin C, C-vitamers, and other common ascorbic acid derivatives determined by electrochemical and spectrophotometric ap­
proaches. a, Chemical structures of studied compounds. b, The antioxidant activity of studied compounds expressed as standard reduction potential (E0) determined
by potentiometric titration (PT) at 37 ◦ C and antioxidant power (AOP) calculated based on the differential pulse voltammetry (DPV) results at 25 ◦ C with a potential
scan rate v = 0.1 V s− 1. Results are presented as means ± SD of three independent measurements. Different letters indicate significant differences in one-way ANOVA
with Tukey’s post hoc test for E0 or Kruskal-Wallis test for AOP with statistical significance set at p-value <0.05. c-f, The relationship between scavenged ABTS or
DPPH radicals and the concentration of ascorbic acid and its derivatives at 25, 37, and 41 ◦ C. Panels d and f show corresponding n10 value axes. The results are
expressed as means ± SD of three independent experiments carried out in triplicates. The abbreviations used refer to: AA, L-ascorbic acid; iAA, D-isoascorbic acid;
CaA, calcium L-ascorbate; NaA, sodium L-ascorbate; iNaA, sodium D-isoascorbate.

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P. Jakubek et al. Free Radical Biology and Medicine 209 (2023) 239–251

Fig. 2. Redox-related activities of vitamin C, C-vitamers, and other common ascorbic acid derivatives. a, The impact of ascorbic acid and its derivatives on the
modulation of phospholipid oxidation in the presence of haem iron expressed as a maximum reaction rate (Vmax). The results are presented as means ± SD of three
independent experiments. Different letters indicate significant differences in one-way ANOVA with Tukey’s post hoc test with the level of significance set at p-value
<0.05. b, The ability of tested ascorbic acid derivatives to protect proteins (BSA) from oxidative damage. Results represent means ± SD of three independent
measurements. Statistically significant differences were determined by one-way ANOVA with Dunnett’s test vs. C+ and marked as (*) p-value <0.05, (***) p-value
<0.001, (****) p-value <0.0001; C+, carbonylated proteins not treated with antioxidants, C-, native BSA. c, The impact of ascorbic acid derivatives on cellular
antioxidant activity in the HT29 cell line. The results are expressed as means ± SD of three independent experiments carried out in triplicate. Statistical analysis was
carried out by one-way ANOVA with Tukey test with the level of significance set at p-value <0.05. Different letters represent statistically significant differences
between specific treatments. d,e, The genotoxicity of ascorbic acid derivatives towards HT29 cells, and their antigenotoxic impact in HT29 cells exposed to H2 O2 . The
cells were treated with studied ascorbate derivatives for 24 h (genotoxicity) or for 24 h prior to exposure to H2 O2 (antigenotoxic activity) and then submitted to the
comet assay procedure. The results are expressed as the mean percentage of DNA in comet tail ± SD from three independent experiments carried out in duplicate and
compared with C-, negative control (cells treated with solvent only) and C+, positive control (exposure to 150 μM H2 O2 ). Statistically significant differences were
determined by one-way ANOVA with Dunnett’s test and marked as (*) p-value <0.05. f, The impact of studied ascorbic acid derivatives on the growth of HT29 cells
determined by MTT assay after 6, 24, and 72 h treatment with tested compounds. Cell growth is expressed relative to control non-treated cells whose growth is
regarded as 100%. Results are means of three independent experiments carried out in quadruplicate. Statistically significant differences were determined by one-way
ANOVA with Dunnett’s test and marked as (*) p-value <0.05, (**) p-value <0.01, (***) p-value <0.001, (****) p-value <0.0001. The abbreviations used refer to: AA,
L-ascorbic acid; iAA, D-isoascorbic acid; CaA, calcium L-ascorbate; NaA, sodium L-ascorbate; iNaA, sodium D-isoascorbate.

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however, statistically significant differences occurred only in the case of
the highest tested concentrations (p-values can be found in Supple­
mentary Table S7). In the case of CaA, the content of protein carbonyl
derivatives increased indicating pro-oxidative effects, which was not
observed for other tested compounds.
3.5. Antioxidant activity by CAA assay
CAA assay has been recognized as a more biologically relevant test to
measure antioxidant activity compared to chemical tests since it in­
volves the aspects of cellular absorption and metabolism of redox-active
compounds [19,20]. As shown in Fig. 2c, the CAA values of investigated
AA derivatives were low to moderate at a wide range of concentrations
(reaching up to 200 μM). Within this range, no significant differences
between AA derivates could be observed. Only at higher concentrations
(≥500 μM), the impact of studied compounds on the reducing capacity
of cells was noticeably enhanced. At both the highest concentrations
used (500 and 1000 μM), CaA prominently increased the CAA values of
HT29 cells compared to other ascorbate derivatives. At the highest
tested concentration (1000 μM), CaA caused almost complete inhibition
of probe oxidation (83% in the range of maximum 100%). The second
strongest ascorbate derivative was NaA. The CAA values for other
compounds did not, however, significantly differ from those determined
for NaA. Regarding the CAA values of compounds applied to HT29 cells
at the highest concentration (1000 μM), the order of antioxidant activity
rises as follows: iAA < AA < iNaA < NaA < CaA.
3.6. Genotoxicity and protection against oxidative DNA damage by comet
assay
To determine whether AA derivatives could exert genotoxic effects in
HT29 cells as well as to check if they prevent H2 O2 -induced DNA frag­
mentation, cells were first incubated with tested compounds for 24 h.
When the treatment was finished, the cells were subjected either to the
comet assay procedure (in the case of genotoxicity determination) or to
1 h treatment with 150 μM H2 O2 (to assess protection against oxidative
genotoxic insult), followed by the comet assay procedure.
The results obtained using comet assay showed that neither low
(achievable with normal diet) nor high concentrations (achievable only
by pharmacological interventions) of AA derivatives induced any DNA
fragmentation in HT29 cells (Fig. 2d). Further verification of the ability
of the studied compounds to protect cells against H2 O2 -induced oxida­
tive DNA damage was conducted using physiologically relevant con­
centrations, chosen based on the following criteria: average plasma
concentration of healthy individual (50 μM), moderate (100 μM), and
intense oral supplementation (200 μM) [3]. At neither of these con­
centrations, AA derivatives protected cells against H2 O2 -induced DNA
fragmentation (Fig. 2e). In both variants of comet assay, in the case of
non-treated cells (negative control, C-), the damage did not exceed 3 %
of DNA in a comet tail. In a second variant of the test, pre-treatment of
cells with higher concentrations of C-vitamers increased DNA frag­
mentation induced upon genotoxic treatment with H2 O2 , which in some
cases reached statistical significance.
3.7. Cell growth by MTT test
As shown in Fig. 2f, shorter treatments (6 and 24 h) of HT29 cells
with AA, iAA, CaA, and NaA did not significantly affect their growth in
the range of concentrations reaching up to 1 mM. Only after 72 h the cell
growth was slightly inhibited, however, by no more than 20%. In
contrast, a notable (however, statistically insignificant) cell growth
stimulation was observed after shorter treatments with iNaA, which
decreased to basal level after 72 h. All compounds applied to the cells at
the highest dose, that is 10 mM, occurred to drastically reduce cell
growth regardless of incubation time. The only exception was 10 mM
AA, which after the shortest treatment only slightly inhibited cell
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growth.
3.8. Nutrigenomic analysis
The impact of C-vitamers on the expression of redox-related genes
was studied with the use of microarrays consisting of 84 genes involved
in oxidative stress response and antioxidant defense systems. The com­
pounds selected for genomic analysis were: AA selected as a parent
compound and representative of the whole group as well as a human
exogenous antioxidant performing important endogenous functions, and
CaA chosen as the strongest antioxidant according to E0, AOP, n10 values
and CAA values at 500 and 1000 μM. The concentrations selected for cell
treatment reflect the physiologically relevant situation: 50 μM repre­
sents the average plasma concentration of AA in healthy individuals,
who consume fruits and vegetables daily, while 200 μM is the highest
plasma concentration of AA that could be reached after the prolonged
supplementation not exceeding maximum tolerated dose [3]. Besides,
the latter concentration could be easily achieved in the gastrointestinal
cells directly exposed to ingested food.
As shown in Fig. 3., the treatment with the lower concentration (50
μM) of AA significantly downregulated the expression of only two genes,
CYBB and RNF7 by 3.7-fold and 1.8-fold, respectively. These genes are
involved in the cellular response to oxidative stress as well as ROS-
generation mechanisms of immune response. Six other genes involved
in protection against oxidative stress (GPX1, GSTZ1, PDLIM1, PRDX5,
PRDX6, PRNP) showed a trend toward being downregulated. A statis­
tically significant, nonetheless slight (1.3-fold) upregulation by 50 μM
AA was observed only for OXSR1 encoding a protein called oxidative-
stress responsive kinase 1. A trend towards upregulation was observed
for four other genes (PRDX1, PRDX3, SOD1, TXN) encoding proteins that
indirectly or directly participate in H2 O2 metabolism. In turn, 50 μM
CaA did not significantly affect the expression of any of the investigated
genes. Only expression of two genes, MGST3 and PRDX4, involved in
xenobiotic metabolism and H2 O2 reduction, showed a slight trend to­
wards upregulation (1.4-fold). Thus, despite the presence of a common
AA structure in both investigated derivatives, their impact on the tran­
scription of redox-related genes in HT29 cells was very different at 50
μM treatment.
The treatment of HT29 cells with the higher concentration (200 μM)
of AA had a greater impact on the downregulation of RNF7 gene (2.3-
fold) compared to the exposure to a lower concentration described
above. Other downregulated genes were PTGS2 (2-fold decrease), as
well as PDLIM1 (1.7-fold decrease) and PRDX6 (1.4-fold decrease)
involved in protection against oxidative stress. The upregulation upon
cell treatment with 200 μM AA was observed for GCLC and GCLM (1.5-
fold and 1.75-fold, respectively), which encode two subunits of
glutamate-cysteine ligase, the rate-limiting enzyme in glutathione syn­
thesis as well as for PRDX4 (peroxiredoxin 4) and TXN (thioredoxin),
indirectly involved in recycling of GSH from GSSG (upregulated by 1.4-
fold and 1.9-fold, respectively). The expression of genes encoding
cytosolic SOD1 and mitochondrial PRDX3 was increased 2.1-fold each,
both of which support the removal of ROS. In the case of 200 μM CaA
treatment, CCL5, encoding a protein that mediates inflammatory pro­
cesses was the only significantly upregulated gene (2.2-fold). Other
genes that showed a trend towards upregulation (approximately 2-fold)
were GCLM, PRDX1, PRDX3, and SOD1, all involved in ROS
detoxification.
4. Discussion
The antioxidant properties of AA and its derivatives (CaA, NaA) or
isomers (iAA, iNaA) have been assumed to be alike and current research
on these compounds is focused mostly on their applicability in food
processing [21–24]. However, research aimed to explore their biological
activity, beyond the minimum required for allowance to use them as
food additives, as well as complex characterization of their redox-related
P. Jakubek et al. Free Radical Biology and Medicine 209 (2023) 239–251

Fig. 3. Nutrigenomic activity of vitamin C and one of the C-vitamers – calcium ascorbate. a, Heatmap presenting modulation of 84 oxidative stress response and
antioxidant defense genes in HT29 cells after 24 h treatment with ascorbic acid (AA) and calcium ascorbate (CaA) at 50 or 200 μM concentrations. b, Relevant
changes in the expression of genes observed following the indicated treatment. The results are calculated based on three independent experiments. Data and sta­
tistical analysis were performed at QIAGEN’S GeneGlobe Data Analysis Center following the manufacturer’s instructions. Statistically significant changes (p-values
<0.05) are highlighted in grey. Full names of investigated genes can be found in Supplementary Table S1.

properties, has been lacking. Therefore, in this study we examined the
hypothesis of whether the (electro)chemical properties of different AA
derivatives determine their antioxidant effects in a biological setting
using human colon adenocarcinoma HT29 cells as in vitro cellular model
of the gastrointestinal tract [25].
The antioxidant activity of studied AA derivatives was evaluated
using different methodological approaches. Two electrochemical pa­
rameters describing compounds’ reducing properties were determined
by electrochemical methods: (i) E0, the most commonly used parameter
characterizing the thermodynamic behaviour of redox-active com­
pounds [26], and (ii) AOP, combining both thermodynamics and ki­
netics of redox processes [27,28]. Both parameters were previously
successfully applied as predictors of bioactivity of redox-active com­
pounds [18,27,28]. According to E0 and AOP, the strongest antioxidant
activity among tested compounds was exhibited by CaA (Fig. 1b and
Table 1), which is not surprising as its chemical structure contains two
ascorbate moieties that are released as a result of dissociation in an
aqueous environment. In line with the electrochemical parameters, CaA
was also the strongest scavenger of both ABTS and DPPH radicals,
commonly used to assess the antioxidant activity of plant foods and
beverages [29]. Indeed, electrochemical parameters and n10 values were
strongly correlated as shown in Fig. 4. Such strong relationships between
electrochemical parameters (E0, AOP, which express thermodynamic
behaviour of a compound) and n10 values (involving the aspect of redox
reaction kinetics) were also shown in our previous studies on
redox-related properties of different polyphenols, i.e. quercetin, nar­
ingenin (aglycones), rutin, naringin (glycosides) or their mixtures, as
well as for different green tea catechins [18,27]. In turn, the CAA assay,
which is considered a more biologically relevant method compared to
ABTS or DPPH tests, revealed that as long as HT29 cells were exposed to
AA and its derivatives at concentrations achievable in plasma i.e. not
higher than 200 μM [3], the impact on the antioxidative cellular barrier
was mostly indistinguishable. However, vitamin C while orally ingested
can reach much higher concentrations in the intestinal lumen [30],
which further accumulate in high concentrations in tissues such as, e.g.,
liver, pancreas, or kidneys [31]. Therefore, such exposures were also
investigated. In line with (electro)chemical parameters, CaA again
appeared to exert the strongest antioxidant activity but only when
applied to cells at a concentration of 500 μM or higher (Fig. 2c). In
general, the majority of strong and statistically significant correlations
between CAA values and (electro)chemical parameters (E0, AOP, and n10
values) occurred for higher (200–500 μM) concentrations of AA and its
derivatives (Fig. 4, p-values are gathered in Supplementary Table S8). It
could be assumed that such high concentrations of physiologically
relevant antioxidants may override the redox homeostatic capacity of
cells, which we previously observed also in the case of catechins [18].
After the assessment of antioxidant activity using three different
approaches, in the next step, we examined how AA and its derivatives
affect the oxidation processes of biomolecules, as these are biologically
relevant targets of ROS. Initially, the kinetics of lipid peroxidation and
protein carbonylation was investigated in a cell-free system. For this
purpose, we used haem iron as a physiological and effective initiator of
lipid peroxidation [32]. Moreover, this form of iron, when consumed in
excess, seems to be an important factor in the induction of intestinal

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Fig. 4. (Electro)chemical parameters describing antioxidant activity are
strongly correlated with cellular antioxidant activity of high concentrations of
ascorbic acid and its derivatives. Correlations between values of standard
reduction potential (E0) as well as other electrochemical parameters (Ep,a, Ip,a,
Qa, and AOP) and values expressing antioxidant activity of ascorbic acid and its
derivatives determined by chemical (n10 values for ABTS and DPPH tests) and
biological (CAA) approaches examined using Pearsons’s coefficients. The larger
the dot, the stronger the correlation; the dark blue colour indicates a strong
positive correlation, dark red colour shows a strong negative correlation.
oxidative stress and potentially contribute to the development of some
diseases including colorectal cancer [33]. The obtained data indicated
that in the presence of haem iron, lower concentrations of AA and its
derivatives accelerated PL oxidation. Such results are not surprising
since ascorbate is known to act in a concentration-dependent manner as
not only an antioxidant but also a propagator of prooxidant effects on
lipids in the presence of transition metal ions such as iron or copper as
shown by reactions 1–3:
Fe2+ + H2 O2 (or LOOH) → Fe3+ + HO• (or LO• ) + OH− (R1)
Fe3+ + H2 O2 (or LOOH) → Fe2+ + HOO• (or LOO• ) + H+ (R2)
Fe3+ + AA → Fe2+ + AA•− + 2H+ (R3)
3+
Reaction (R1) leading to the formation of Fe is markedly faster
than reaction (R2), in which Fe2+ becomes restored (reaction rate co­
efficients k equal 5.7 × 102 M− 1s− 1 and 2.6 × 10− 3 M− 1s− 1, respec­
tively) [34,35]. The presence of AA and its derivatives contributes to the
acceleration of Fe2+ regeneration, as shown by the reaction (R3). Ulti­
mately, this propagates reaction (R1), leads to increased ROS produc­
tion, and results in the acceleration of lipid oxidation. Furthermore, our
data showed that AA and iAA had a greater contribution to increased PL
oxidation when compared to their sodium salts (NaA and iNaA). Indeed,
such an effect could be attributed to their acidic forms since it has been
shown that a slight decrease in pH value could increase lipid oxidation
mediated by hemoglobin in cod microsomal membranes [36]. This
observation can be related to the pH-induced changes in hemoglobin
structure leading to the acceleration of lipid oxidation [37]. In turn,
when the maximum lipid oxidation rate was achieved, a further increase
in the concentration of AA derivatives led to a decrease in the oxidation
rate. This effect may be related to the fact that at high concentrations
reducing compounds are present in sufficient amounts to also scavenge
ROS generated in a reaction mixture [38].
In the case of protein carbonylation, AA and its two derivatives (NaA,
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Free Radical Biology and Medicine 209 (2023) 239–251
iAA) showed the most potent protective effects when applied at a high
physiologically relevant concentration (100 μM) (Fig. 2b), with AA
turning out to be significantly stronger than both NaA and iAA (Sup­
plementary Table S7). At lower concentrations, again, mostly AA and
then NaA showed statistically significant albeit slight protection against
protein carbonylation. Interestingly, it has been shown that 5-week
supplementation with vitamin C (400 mg/day, a form of ascorbate
was not specified) can indeed reduce the level of protein carbonylation
but only in healthy volunteers with low baseline ascorbate level (base­
line ascorbate ~30 μM). Individuals with normal baseline vitamin C
status (~50 μM) did not benefit from supplementation [39]. In contrast,
CaA, which occurred to be the strongest antioxidant in (electro)chemical
and cellular tests (Fig. 1b,d,f and 2c), showed an unexpected proox­
idative effect at most tested concentrations, with 30 μM concentration
exhibiting the most potent effects (Fig. 2b). Altogether, these results
show that the impact of AA and its derivatives on protection against
protein carbonylation is concentration-dependent, as well as that AA
derivatives are less effective than the parent compound. In line with our
findings, some reports have previously shown that AA can either
decrease (at 1 mM concentration) or increase (at 10 mM concentration)
levels of protein carbonylation [40,41]. In the latter case, it has been
suggested that ascorbic acid could serve as a potential source of protein
carbonylation due to its autoxidation to dehydroascorbic acid, which
can further react with amino groups in proteins and generate products of
Maillard reaction and finally, advanced glycation end products [40,42].
Other biomolecules liable to oxidative damage are nucleic acids.
Therefore, the ability of physiologically-relevant concentrations of AA
and its derivatives to provide protection against H2 O2 -induced oxidation
of DNA in HT29 cells was examined by comet assay. So far numerous
observational as well as interventional studies showed that vitamin C at
plasma concentrations exceeding 50 μM protects the integrity of DNA
under oxidative stress conditions in vivo [43]. However, somewhat
surprisingly, in this study preincubation of HT29 cells with high albeit
physiologically-relevant concentrations of C-vitamers, i.e. AA, NaA, and
CaA, followed by short genotoxic H2 O2 treatment resulted in increased
levels of DNA fragmentation (Fig. 2e). Such an effect can be explained by
the already discussed prooxidative behaviour of ascorbates resulting
from their involvement in Fenton reactions (reactions R1-R3). The
previously reported DNA damage induced in cellular systems has been
usually observed for millimolar concentrations of ascorbate [44,45].
The ability of ascorbate to prevent oxidative DNA damage in HT29 cells
has been also earlier reported [46]. In the latter case, experimental
conditions and concentrations used markedly differed from those
applied in our study, i.e., vitamin C-treated HT29 cells were kept in
suspension during the experiment, and part of the cells was treated with
only 10 μM H2 O2 for the last 10 min of the total 60 min vitamin C
treatment with this antioxidant [46]. In such circumstances, the direct
chemical neutralization of H2 O2 by AA could occur not related to the
redox status of the cells as investigated in our system. Although there is
much evidence that vitamin C may protect DNA against oxidative
damage, there is still a lot of inconclusive and contradictory data. The
protective effect of vitamin C against DNA damage may depend on the
dose and incubation time and, as suggested by some researchers, it re­
quires further insight [35].
In this study, the HT29 cell line was used as in vitro model of the
intestinal epithelium, however, some data interpretations may also refer
to the malignant origin of these cells [9,25]. It is widely recognized that
quickly proliferating cancer cells are characterized by enhanced ROS
generation and upregulated endogenous antioxidant capacity, which
creates a microenvironment favourable for the further proliferation of
cancer cells [47,48]. There is also evidence from in vivo studies that a
surplus of exogenously administered antioxidants can support mela­
noma and lung cancer progression [49,50]. In our study, iNaA turned
out to be the only compound that showed a trend of notable enhance­
ment of HT29 cell growth, even though its redox-related activity did not
stand out among other AA derivatives. Seeing that no evidence of the
P. Jakubek et al. Free Radical Biology and Medicine 209 (2023) 239–251

tumorigenic impact of iNaA has been noted in the past in vivo studies
[51,52], the observed trend should be replicated using other (colon)
cancer cell lines before drawing any firm conclusions. In contrast,
millimolar concentrations of AA derivatives caused significant inhibi­
tion of cell growth and such an effect has been thoroughly documented
in scientific literature [45,53,54]. The ascorbate-induced inhibition of
cell growth may be a result of disrupted redox homeostasis towards
oxidative stress since ascorbate at millimolar concentrations (i) un­
dergoes auto-oxidation and triggers the generation of H2 O2 that can be
further transformed into much more reactive hydroxyl radical (HO• )
and (ii) depletes intracellular GSH and NADPH pools [55]. Such high
concentrations of ascorbates can be achieved solely through intravenous
injection, at least in the bloodstream [56].
Finally, we looked at the impact of AA on cellular redox-related
function at the genomic level for two compounds that behaved differ­
ently in previous experiments – AA and CaA – despite obvious chemical
structural similarity (Fig. 3a). Since these experiments were intended to
spot even subtle changes in the patterns of gene expression, alterations
in the expression of genes, which failed slightly to reach statistical sig­
nificance (p-values between 0.05 and 0.1) were also included in Fig. 3b
to indicate a trend in gene expression patterns. The reason was that in
cells treated with physiologically relevant concentrations of non-toxic
dietary antioxidants, minimal influence on transcriptome can be ex­
pected. In contrast, such major specific alterations in gene expression
may be seen, for example, in drug research. As depicted in Figs. 3 and 5,
the treatment of cells with a low concentration of AA (considered
optimal in the context of plasma concentrations) caused significant
downregulation of CYBB, which makes one of the subunits of one of the
major ROS-generating enzymes – NADPH oxidase [57]. Such results
could suggest a decrease in the generation of O•−2 , and thus alleviation of
oxidative stress. On the other hand, a trend towards decreased expres­
sion of SOD3, which acts in the extracellular milieu, might suggest either
lowered antioxidant protection or no need to even induce it. At the same
time, the expression of SOD1, which converts O•− 2 to less reactive H2 O2 ,
was significantly upregulated, but only after treatment with a higher
concentration of AA. The possible enhanced production of H2 O2 did not
seem to be counterbalanced by the changed expression of other genes.
This would suggest the increased preservation of H2 O2 , which besides
being one of ROS, is known to mediate intracellular signalling [58].
Furthermore, it is commonly known that there is a strong interplay
between AA and GSH in plants and animals, including humans, in terms
of the maintenance of redox homeostasis [59,60]. Increased expression
of GCL, one of the subunits of enzyme participating in the synthesis of
GSH, may also suggest that AA induced enhanced preservation of the
GSH pool. Upregulation of TXN may also indirectly affect the regener­
ation of GSH, since TXN is utilized by PRDXs, which are other enzymes
engaged in the neutralization of H2 O2 [61]. Here, mitochondrial PRDX3
as well as cytosolic PRDX1 and PRDX4 were upregulated, while other
cytosolic isoforms – PRDX5 and PRDX6, were downregulated. Based on
the obtained results, it is hard to notice any clear pattern of changes in
gene expression, which would suggest definite more antioxidant or
pro-oxidant effects.
Interestingly, AA and CaA did not occur to support cellular redox
homeostasis unidirectionally. In contrast to AA, the majority of changes
in the expression of redox-related genes observed after the treatment
with CaA, missed to reach statistical significance. Such an observation at
first may seem rather counterintuitive, seeing that CaA occurred to be a
significantly stronger antioxidant in terms of its (electro)chemical
properties, which stood out among the rest of the tested derivatives.
However, previously we made similar observations for glutathione,
whose activity was weaker compared to the group of apparently stron­
ger reducing agents – catechins [18]. In that case, the treatment of HT29
cells with glutathione affected different sets of redox-related genes than
catechins, whose low, physiologically-relevant concentrations had an
impact on the expression of solely 3 genes, which was diminished after
treatments with higher concentration [18]. It could be hypothesized that
the lack of notable effects on the expression of the antioxidant defense
system after treatment with strong reducing agents such as CaA could be
derived from the proper redox balance maintained in cells after treat­
ment, so the expression of antioxidant genes was no longer needed. The
observed diversified cellular response to the treatment with AA and its
calcium salt rather points to the fine regulation of endogenous systems
of antioxidant protection preventing both oxidative and reductive stress
occurrence [62]. The explanation of a similar phenomenon has been
previously proposed by our group in the case of sulfiredoxin-mediated
maintenance of redox homeostasis in response to the treatment with
catechins [63].

Fig. 5. Nutrigenomic effects of vitamin C. a, Cellular response to oxidative stress upon treatment with vitamin C represented by ascorbic acid (AA) and calcium
ascorbate (CaA) can be modulated at the level of gene expression. b, Genes affected by the treatment of HT29 cells with AA and CaA are involved in numerous
pathways related to ROS neutralization, antioxidant protection, and cellular metabolism. Genes whose expression was significantly changed in response to vitamin C
treatments are written in bold (p-value <0.05), other affected genes refer to p-values between 0.05 and 0.1. The abbreviations refer to: CAT, catalase; CCL5, che­
mokine (C–C motif) ligand 5; CYBA, cytochrome b-245, alpha polypeptide; CYBB, cytochrome b-245, beta polypeptide; Cys, L-cysteine; DHA, dehydroascorbic acid;
FTH1, ferritin, heavy polypeptide 1; GCL, glutamate cysteine ligase; GCLC, glutamate-cysteine ligase, catalytic subunit; GCLM, glutamate-cysteine ligase, modifier
subunit; Glu, glutamic acid; Gly, glycine; GPX, glutathione peroxidase; GSH, glutathione; GSS, glutathione synthetase; GSSG, glutathione disulphide; GSTZ1,
glutathione transferase zeta 1; MGST3, microsomal glutathione S-transferase 3; NADP(H), nicotinamide adenine dinucleotide phosphate; OXSR1, oxidative-stress
responsive 1; PDLIM1, PDZ and LIM domain 1; PRDX, peroxiredoxin; PRNP, prion protein; PTGS2, prostaglandin-endoperoxide synthase 2 (prostaglandin G/H
synthase and cyclooxygenase); RNF7, ring finger protein 7; SOD, superoxide dismutase; SVCT, sodium-dependent vitamin C transporter; TXN, thioredoxin; TXNR,
thioredoxin reductase; VIMP, selenoprotein S.

249
P. Jakubek et al.
5. Conclusions
In conclusion, our study shows that integrating both electrochemical
and biological approaches is essential to comprehensively understand
and potentially predict the behavior of redox-active compounds within
biological systems. This integration is crucial because electrochemistry
underpins the chemical foundation of redox-related biological activities
of antioxidants. We found that the distinctive behaviour of the studied
ascorbate derivatives in cellular antioxidant activity occurred solely at
higher concentrations achievable either in the intestinal lumen (being in
contract with ingested food) or in various tissues (e.g., liver) as a result
of ascorbate accumulation. The CAA values, obtained for higher con­
centrations of ascorbates, were shown to be strongly correlated with
electrochemical and kinetic parameters describing redox processes. In
the case of modulation of oxidative damage, the differences between the
studied ascorbate derivatives were observed already at low concentra­
tions that could be considered physiologically relevant. At the nutrige­
nomic level, the distinct cellular response observed in the treatment
with AA and its calcium salt, which happens to exhibit the highest
antioxidant potency among AA derivatives, may suggest fine regulation
of redox homeostasis. This result warrants deeper investigation. Future
research should intend to confirm these findings in more in vitro and
then in vivo models to verify whether different redox-related properties
of AA derivatives can have, so far omitted, health implications. If
confirmed, these findings may be relevant to the designing of nutra­
ceuticals, dietary supplements, or even trials investigating the effec­
tiveness of vitamin C in cancer therapies.
Author contributions
Conceptualization: A.B.; Formal analysis: P.J.; Investigation: P.J., K.
S., M.K., M.A., K.P., I.K.-M.; Methodology: M.K., B.K., W.C.; Visualiza­
tion: P.J.; Roles/Writing - original draft: P.J., K.P., I.K.-M., Writing -
review & editing: P.J., K.P., I.K.-M., A.B., Funding acquisition J.N., Re­
sources: A.B.; Supervision: A.B.
Declaration of competing interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was funded by the National Science Centre (Poland)
through the MAESTRO 6 grant programme (application number 2014/
14/A/ST4/00640). Part of the findings included in this manuscript
(results of spectrophotometic tests, MTT, CAA, comet assays, and
microarrays analysis) has been included in the doctoral thesis of P.
Jakubek entitled “Chemical and biological evaluation of antioxidant
activity of endogenous redox-active compounds compared to plant-
derived exogenous antioxidants” [2022, Gdańsk University of Technol­
ogy], the electronic version of which was published in the open, insti­
tutional, digital repository of Gdańsk University of Technology,
Pomeranian Digital Library.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.freeradbiomed.2023.10.400.
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