Reduction reactivity of catecholamines and their ability to promote a Fenton reaction

Article abstract of DOI:10.1016/j.ica.2016.07.044

Several studies have assigned catecholamines a pro-oxidant role and have therefore correlated catecholamines with the development of different pathophysiological processes. This pro-oxidant effect could be due to the Fenton reaction (i.e., Fe(II)?+?H₂O₂?→?Fe(III)?+? OH?+?OH^?), which is known to be utilized by catecholamines to reduce Fe(III) and O₂. In this work, the ability of a few catecholamines (i.e., dopamine, epinephrine and norepinephrine) to reduce Fe(III) to Fe(II) and O₂to H₂O₂and to produce OH radicals by the Fenton reaction was evaluated at different pH values. The catecholamines were observed to produce Fe(II) and H₂O₂at different pH values. Therefore, OH radical production was enhanced at pH values where only Fe(III) reduction was observed. At pH values near 7.0, the catecholamines did not enhance the production of OH radicals. Instead, the catecholamines acted as antioxidants forming bis-complexes, i.e., [Fe(LH)₂]⁺. The catecholamines sequestered iron from the reaction system and thereby prevented iron from reacting with other compounds such as H₂O₂.

Full text of DOI:10.1016/j.ica.2016.07.044

Accepted Manuscript
Research paper
Reduction reactivity of catecholamines and their ability to promote a Fenton
reaction
Victoria Melin, Adolfo Henríquez, Claudia Radojkovic, Brigitte Schwederski,
Wolfgang Kaim, Juanita Freer, David Contreras
PII:
S0020-1693(16)30422-4
http://dx.doi.org/10.1016/j.ica.2016.07.044
ICA 17179
DOI:
Reference:
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Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
10 May 2016
2 July 2016
26 July 2016
Please cite this article as: V. Melin, A. Henríquez, C. Radojkovic, B. Schwederski, W. Kaim, J. Freer, D. Contreras,
Reduction reactivity of catecholamines and their ability to promote a Fenton reaction, Inorganica Chimica Acta
(
2016), doi: http://dx.doi.org/10.1016/j.ica.2016.07.044
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Reduction reactivity of catecholamines and their ability to promote a Fenton
reaction
a,b
Victoria Melin* , Adolfo Henríquez , Claudia Radojkovic , Brigitte Schwederski , Wolfgang
a
c
d
d
Kaim , Juanita Freer and David Contreras* .
a,b
a,b
a
Faculty of Chemical Science, University of Concepción, Concepción, Chile.
b
Biotechnology Center, University of Concepción, Concepción, Chile.
c
Faculty of Pharmacy, University of Concepción, Concepción, Chile.
d
Institute of Inorganic Chemistry, Universität Stuttgart, Stuttgart, Germany.
*
Corresponding authors:
Biotechnology Center, University of Concepción, Casilla 160-C, Concepción, Chile.
Email address: victoriamelin@udec.cl (Victoria Melin) and dcontrer@udec.cl (David Contreras)
Abstract
Several studies have assigned catecholamines a pro-oxidant role and have therefore correlated
catecholamines with the development of different pathophysiological processes. This pro-
-
oxidant effect could be due to the Fenton reaction (i.e., Fe(II) + H
which is known to be utilized by catecholamines to reduce Fe(III) and O
of a few catecholamines (i.e., dopamine, epinephrine and norepinephrine) to reduce Fe(III) to
Fe(II) and O to H and to produce •OH radicals by the Fenton reaction was evaluated at
different pH values. The catecholamines were observed to produce Fe(II) and H at different
2 2
O
→ Fe(III) + •OH + OH ),
2
. In this work, the ability
2
2 2
O
2 2
O
pH values. Therefore, •OH radical production was enhanced at pH values where only Fe(III)
reduction was observed. At pH values near 7.0, the catecholamines did not enhance the
production of •OH radicals. Instead, the catecholamines acted as antioxidants forming bis-
+
] . The catecholamines sequestered iron from the reaction system and
complexes, i.e., [Fe(LH)
2
2 2
thereby prevented iron from reacting with other compounds such as H O .

1. Introduction
Catecholamines, including dopamine, epinephrine and norepinephrine, are hormones and
neurotransmitters of the nervous system (Figure 1). The roles of these chemicals in the body is
widespread and includes several physiological processes[1]. In addition to their physiological
functions, catecholamines can participate in chemical reactions that generate harmful molecules
at the cellular level. In autoxidation reactions, catecholamine is oxidized by O , resulting in the
2
•
2
-
generation of superoxide radicals (O
) and semiquinone, which can be further oxidized to
•
-
quinone by reducing another O molecule[2, 3]. The radical O
2
2 2 2 2
is converted into H O and O
by a disproportionation reaction. Semiquinone and quinones produced during the oxidation
process are also harmful to the cellular environment[4-7]. Because of their ability to produce
reactive oxygen species (ROS), catecholamines have been attributed with the development of
several diseases associated with oxidative stress, among which include neurodegenerative
diseases such as Parkinson's disease[8, 9]. These diseases also affect iron homeostasis[10,
11]. When treated with catecholamines, the iron concentration in substantia nigra increased by
approximately 35% compared with normal physiological iron levels[12]. In the human body, iron
should remain bound to proteins due to the potential for iron to produce free radicals, especially
hydroxyl radicals (•OH)[13].
Figure 1: Catecholamines structures.
The main source of •OH radicals in biological systems comes from the Fenton reaction[14]. For
2 2
this reaction to occur, Fenton reagents, Fe(II) and H O , are necessary. The reaction
mechanism was proposed by Haber and Weiss in 1932. The •OH radicals act as the primary
oxidizing species in reaction (1)[15]. During the Fenton reaction, Fe(III) is formed; subsequently,
2 2
Fe(III) reacts with H O to produce Fe(II) by a “Fenton-like” reaction (2)[16]. The Fenton-like
reaction is three orders of magnitude slower than the Fenton reaction and thereby represents

the limiting step in the redox cycle of a Fenton system. The reaction mechanism of the Fenton-
like reaction involves the production of perhydroxyl radicals (i.e., •OOH)[17].
–
-1 -1
2 2
Fe(II) + H O → Fe(III) + •OH + OH k ≈70 L•mol •s [18] (1)
+ -1 -1
→ Fe(II) + •OOH + H k = 0.01 L•mol •s [19] (2)
Fe(III) + H
O
2 2
+
3
Like other catechols, catecholamines (LH
) can form complexes with iron[20-22]. Figure 2
shows the primary iron complexes that are typically present in aqueous solutions. The formation
2+
of the bidentate mono-complex ([Fe(LH)] ) is not dependent on any specific iron species in
3
+
2+
acidic aqueous solutions (i.e., [Fe] , [Fe(OH)] or [Fe(OH)
+
] )[20]. The formation of a
2
3+
monodentate complex ([Fe(LH )] ) was suggested by Xu and Jordan[23]. However, the
2
2+
formation of [Fe(LH)] is favorable due to a chelating effect. As the pH value increases, the
+
] ) is favored at pH close 7.0. At approximately pH 10 the
formation of the bis-complex ([Fe(LH)
formation of a tris-complex ([Fe(LH)
life due to the reduction of Fe(III)[24].
2
2
+
3
]) is predominant. Furthermore, [Fe(LH)] has a short half-
+
3
Figure 2: Primary Fe(III)-catecholamine complexes present in aqueous solutions. LH
: triprotic
catecholamine.
At physiological pH values, the formation of iron-catechol complexes is sufficiently favorable to
extract iron from stored proteins in the body[25-29]. Catecholamines behave as non-innocent
ligands, i.e., Fe(III) is reduced within the coordination sphere[25]. Therefore, catecholamines
could amplify oxidative pathological states by promoting the bioavailability of iron for the
production of free radicals by the Fenton reaction[23].
Paris et al. studied the effects of dopamine iron complexes in vitro under physiological
conditions[26]. The study highlighted the cellular toxicity of these complexes due to ROS
formation (the production of •OH radicals was observed).

Based on studies of pH influence on the oxidation of benzyl alcohol by
catecholamine/Fe(II)/H systems, the oxidation of benzyl alcohol was found to be dependent
2
O
2
on iron speciation in the catecholamine complexes[27]. The maximum level of oxidation of
benzyl alcohol was observed at pH values near 3.4, which was similar to the optimal pH value
observed for other catecholate-iron systems[28]. In these reports, the reactivity was determined
only by the oxidation of an aromatic substrate. However, the roles of Fe(III) reduction and •OH
radical production were not explored. Furthermore, the ability for catecholamines to induce
oxidative stress through the Fenton reaction in a biological system was not tested. In the
present study, the capacity for catecholamine-driven systems to produce ROS was tested on
human umbilical vein endothelial cells (HUVECs), a thin monolayer of cells that acts as the first
barrier between blood and tissue.
2
. Material and Methods
.1. Reagents
2
Catechol, dopamine hydrochloride, (±)-epinephrine hydrochloride, DL-norepinephrine
hydrochloride, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid (ferrozine), (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Life Technologies) and 5,5-
dimethyl-1-pyrroline N-oxide (DMPO) were all purchased from Sigma. Potassium fluoride,
[
bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane (BIS-tris, Calbiochem), dichloro-dihydro-
fluorescein diacetate (DCFH-DA, Calbiochem), H 30%, ferric nitrate, ferrous sulfate, sodium
2 2
O
acetate and acetic acid 100% were supplied by Merck.
All reagents were used without additional purification.
2.2. General Procedures
All reagent solutions were prepared in the dark under argon atmospheres. The ionic strengths of
-
1
all solutions were adjusted to 0.10 mol•L with KNO
3
. All experiments were performed at 20 ±
0.1°C in triplicate (n= 3).
For experiments where a pH adjustment was required, different buffers were used. A 0.050
-
mol•L BIS-tris buffer was used to maintain pH values from 6.0-7.0; a 0.050 mol•L acetate
1
-1

buffer was used for pH values of 4.0-5.5; and HNO
.0. The pH of each solution was adjusted prior to experimentation using a 3 Start Thermo
Orion pH meter.
3
was used to regulate pH values lower than
4
2.3. Reduction of Fe(III) by spectrophotometric measurements
The reduction of Fe(III) by each tested catecholamine was determined at pH values between
-
1
2
3 3
.0 and 7.0. The final concentrations in the systems were 0.15 mmol•L Fe(NO ) and 1.5
-
1
mmol•L catechol or catecholamine. The reaction was initiated by adding Fe(III).
Quantification of reduced Fe(III) was determined by spectrometry by measuring the levels of
Fe(II) formed at different reaction times (UV-vis diode array spectrophotometer, Agilent 8453).
Briefly, the reduction of Fe(III) resulted in the production of colored complexes between the
metal and the chelating 3-(pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine).
Absorbances were measured at 567 nm[29]. This technique was a variation of that disclosed by
Chen and Pignatello[30].
Based on the linear range of the constructed absorbance curve, the initial rate of reduction of
Fe(III) was determined. The maximum reduction of Fe(III) was observed 24 h after the start of
the reaction.
Furthermore, the relationship between the reduction of Fe(III) at different reaction times with the
2
absorbance of the respectively formed [Fe(LH)] was determined at pH 3.4. The absorption
+
2+
band corresponding to the [Fe(LH)] complex (λmáx= 700 nm[21]) was monitored using UV-
visible spectrometry. The absorbance was monitored in a "stopped flow" system for fast kinetics
(RX2000, Applied Photophysics) using the same reagent concentrations as those used in the
reduction of Fe(III).
2
.4. Reduction of O
2
The level of O
2
reduction was indirectly determined by monitoring the consumption of O
2
in air-
in
the solution was monitored every 3 seconds for a period of 24 h in a biological oxygen
-
1
saturated solutions at pH 3.4 and 7.0 ([O
2
]= 250 µmol•L at 20°C[11]).The percentage of O
2
-1
monitoring system YSI 5300A with a sensitivity of 3 µmol•L
2
O . The experiments were

performed in the presence or absence of Fe(III). In these systems, the final concentrations were
-
.5 mmol•L for catecholamine or catechol and 0.15 mmol•L for Fe(III).
1
-1
1
For all compounds, the initial rate of reduction of O
2
(or O
2
consumption) was determined from
consumption after 24
the linear range of the O
2
concentration curve early in the reaction. The O
2
h was also determined.
2 2 2
The concentration of H O produced by the reduction of O was determined after 24 h using a
-1
reflectometric method (Merck, Reflectoquant) at a detection limit of 5.8 µmol•L H
O .
2 2
2.5. Hydroxyl radical production
To assess the effect of catecholamines on the •OH radical production by Fenton-like systems,
comparative studies were performed in the absence and presence of each catecholamine. Due
to the reported ability of catechol to amplify Fenton-like reactions, comparisons between the
catecholamine-driven Fenton-like systems and catechol-driven Fenton-like systems were
performed for all determinations[28, 31-33]. The studied Fenton-like systems are listed in Table
1.
Table 1. Studied Fenton-like systems.
Systems
Name
Fe(NO
Fe(NO
Fe(NO
3
)
3
)
3
)
3
+ H
+ H
+ H
2
2
2
O
2
+ DMPO
Unmodified system
3
O
2
+ DMPO + Catechol
Catechol-driven Fenton-like system
Catecholamine-driven Fenton-like system
3
O + DMPO + Catecholamine*
2
Hydroxyl radicals (•OH) were detected using a DMPO spin-trapping method by EPR
spectroscopy[34]. The •OH production was determined at pH values between 2.0 and 7.0. The
-
1
-1
final concentrations in the systems were 0.15 mmol•L Fe(NO
)
3 3
, 1.5 mmol•L catechol or
and 40 mmol•L DMPO. Reactions were initiated by adding
Fe(III). Samples were subsequently transferred via syringe to a capillary Aqua X in a Bruker
108 TMH/9701 instrument. The EPR spectra were recorded on the X band of a Bruker
-
1
-1
2 2
catecholamines, 1.5 mmol•L H O
4

ESP300 spectrometer equipped with a Bruker ER035M gaussmeter and an HP 5350B
microwave counter. The magnetic fields were set at the highest intensity peaks; variations in the
peak heights were followed. The amounts of DMPO/•OH adduct produced were considered
proportional to the heights of these peaks[35]. The EPR experiments were performed at room
temperature (approximately 20°C).
2.6. Determining comparative parameters
The obtained data were normalized according to a method reported by Contreras et al.[31]. The
maximum amount of •OH radicals produced in each system and the time required to reach this
value were determined and compared.
2.7. Cell cultures
Cell culture studies were performed using HUVECs (Lonza). Cells were grown in gelatin-coated
dishes, using M-199 medium supplemented with 10% newborn calf serum and 10% fetal bovine
serum, until approximately 90% confluence. Cell passages were carried out with 0.1%
trypsin/EDTA.
All experiments were performed in 96-well plates at 20,000 cells per well cultured at 37°C and
-
1
5
% CO
2
for 2 days. The Fe(III)-catecholamine complexes were formed by adding 25 nmol•L of
catecholamine to M-199 medium without phenol red (pH 7.2–7.4) containing
-
1
2
50 nmol•L Fe(NO
catecholamine complex formation was verified using a UV-vis diode array spectrometer (Agilent
453). Prior to testing, the cells were incubated with M-199 1% fetal bovine serum for 4 h[36].
3 3
) and allowing 30 min for complex formation at room temperature. Fe(III)-
8
-
Then,1 nmol•L of the Fe(III)-catecholamine complexes was added to the M-199 medium (pH
1
-
1
7
2 2
.2–7.4) in the presence or absence of 10 nmol•L of H O .
To assess cell viability, the cultures were incubated with MTT reagent following manufacturer
recommendations. MTT was added to cultures 4 h after the treatments with the Fe(III)-
catecholamine complexes. The MTT reagent is reduced to a purple-derived formazan
precipitate in living cells and can be measured at 540 nm after solubilization with DMSO[37].
Before analyzing the results, the correlation between the number of living cells and formazan
formation was confirmed (R= 0.985).

The quantification of the produced ROS in HUVECs treated with the Fe(III)-catecholamine
complexes was performed using a DCFH-DA probe. In the presence of ROS, fluorescent
compounds were formed (λex= 485 nm; λem= 522 nm)[38]. These compounds were detected
using a fluorescent spectrometer (Synergy 2 Alpha Biotek Instruments) after 4 h of incubation
with the Fe(III)-catecholamine complexes. Fluorescence emission was considered proportional
to the amount of intracellularly produced ROS[39].
2.8. Statistical Analyses
To determine the significance of the effect of catecholamines on the parameters analyzed in this
study, ANOVA and post-test (Tukey) calculations were carried out to compare differences
between means. All statistical analyses were performed using GraphPad InStat 3 (GraphPad
Software, Inc.) software.
3
. Results and Discussion
.1. Reduction of Fe(III)
3
Research studies investigating pollutant removal and wood biodegradation via the Fenton
reaction driven by catechols have correlated Fe(III) reduction with the amount of oxidized
substrates (mainly by •OH radicals)[33, 40, 41]. To determine the relationship between •OH
radical production and the ability for catecholamines to reduce Fe(III), the initial rate of reduction
R
of Fe(III) (I ) at an optimum pH of 3.4 was measured (Figure 3).

Figure 3. Initial rate of reduction of Fe(III) (I
R
) for catechol and catecholamines at pH 3.4
(n=3).
At pH 3.4, catechol reduces Fe(III) more rapidly than catecholamines. Previous studies had
reported that the limiting step in the reduction of Fe(III) was an electron transfer between one of
the deprotonated oxygen atoms to Fe(III)[20]. Therefore, catecholamines may have a slower
electron transfer rate than catechol. This could be due to an electron withdrawing substituent in
catecholamines (Hammett constant for dopamine > 0)[42] that may decrease the electron
density on the hydroxyl groups in the ring and thereby decrease the electron transfer rate to
Fe(III).
2+
The relationship between the kinetics for Fe(III) reduction and changes in [Fe(LH)] absorbance
was investigated (Figure 4). For all studied compounds, the amounts of reduced Fe(III)
increased exponentially until quasi-steady states were reached. For all assayed catechols, the
2+
amounts of reduced Fe(III) decreased linearly with [Fe(LH)] concentration (R≥-0.995).These
results agreed with those of previous works wherein the increases in oxidative capacities of
2+
Fenton and Fenton-like systems were related to the presence of [Fe(LH)] [27].

2+
Figure 4. Absorbance of Fe(III)-catecholamine mono-complex ([Fe(LH)] ) (black line)
and reduced Fe(III) concentration (white circles) at pH 3.4. a) Catechol; b) dopamine; c)
epinephrine; d) norepinephrine (n=3).
To correlate the relationship between catecholamines with oxidative stress under physiological
conditions, the ability to reduce Fe(III) at pH 7.0 was determined. At pH 7.0, Fe(III) reduction
2
was not observed for any catecholamine, even in presence of O . Thus, it was not possible to
determine whether the reduction of Fe(III) occurred. Furthermore, under these conditions, the
formation of an intermediate Fe(II)-ferrozine complex was unknown because Fe(II) chelation
+
] complex.
occurred inside the [Fe(LH)
2
To determine the pH range for the reduction of Fe(III) by catecholamines, I
was determined (Figure 5).
R
as a function of pH

Figure 5. Initial rate of reduction of Fe(III) (I
R
) at different pH values. Red square: catechol;
green circle: dopamine; blue triangle: epinephrine; magenta rhombus: norepinephrine (n=3).
At different pH values, catechol and catecholamines showed similar I
value was obtained at pH 3.4. Above pH 3.4, I values sharply declined and eventually dropped
to undetectable levels at pH values greater than 5.5. The I
R R
trends. A maximum I
R
R
trends agreed with the pH range for
2
+
+
] is the main species, and
which [Fe(LH)] is the main species[24]. Above pH 5.5, [Fe(LH)
2
Fe(III) reduction was not observed. Fe(III) reduction was also studied for all systems in the
presence of O . Under these conditions, no significant changes in I were observed.
2
R
2 2
3.2. Autoxidation of catecholamines and H O production
The oxidation of catechols by O
2
or autoxidation, may lead to the formation of H
O
2 2
(a Fenton
reagent). Thus, the autoxidation of catecholamines was monitored by O
solution in the presence or absence of Fe(III).
2
consumption in the
2 2
At pH 3.4, no significant O consumption was observed. Fe(III) did not catalyze significant O
2
consumption. The formation and subsequent decomposition of [Fe(HL)] was previously
+
verified by spectrometry (Figure 4, black line).
At pH 7.0, catecholamines and catechol were oxidized in air-saturated solutions at different
rates (Figure 6a). Epinephrine had the highest initial rate of O
2
consumption and had consumed
-
1
all O
2
in the solution (250 µmol•L ). This was consistent with the results reported by Chen et

al.[43], wherein a comparative study by cyclic voltammetry showed that epinephrine had the
lowest oxidant potential at pH 7.
Figure 6. a. Initial rate of consumption of O
2
for catechol and catecholamines at pH 7.0 in
consumption after 24 h of reaction
absence (white bar) or presence (grey bar) of Fe(III). b. O
2
for catechol and catecholamines at pH 7.0 in absence (white bar) or presence (grey bar) of
Fe(III) (n=3).
When the autoxidation of catecholamines was performed in the presence of Fe(III), increases in
2
the initial rate of O consumption were observed (Figure 6a, gray bar). These increases were
26% for catechol and exceeded 250% for dopamine and epinephrine (Figure 6). These results
were consistent with Halliwell’s generalization[44]. The mechanism by which metals, such as
Fe(III), promoted catecholamine oxidation has been related to the formation of Fe(III)-
catecholamine complexes and interactions with available d-orbitals to overcome spin
restrictions[45].
Other researchers have shown that the autoxidation of catecholamines produces semiquinones
and other reaction products with the ability to reduce Fe(III) to Fe(II)[46]. Because of this, we
expected our reaction system to produce these products by catecholamine autoxidation.
However, Fe(III) reduction at pH 7.0 was not observed (i.e., free Fe(II) was not found in solution
after 24 h of autoxidation). This could be due to 4 possibilities: a) Fe(III) was not reduced in the
+
+
[
Fe(LH)
2 2
] complex; b) Fe(III) was reduced, but Fe(II) was not released from the [Fe(LH) ]
complex; c) Fe(III) was reduced and released into the aqueous solution, but was reoxidized to

Fe(III) by dissolved oxygen or d) a few of the oxidation products precipitated and iron was
trapped or remained bound in a heterogeneous phase.
As the main product of O
quantifiable concentrations of
norepinephrine were oxidized (14±2 and 19±1 µmol•L , respectively). The absence or low
concentration of H in solutions where catechol or catecholamine were oxidized could be due
to the consumption of this reagent by reacting with the semiquinone through an organic Fenton
reagent[47]. This could also cause a decrease in the H concentration after 24 h.
2
reduction, the concentration of H
2 2
O after 24 h was determined. Only
2 2
H O
were found in solutions where dopamine and
-1
2 2
O
2 2
O
3.3. •OH radical production by Fenton-like systems
Catecholamines can reduce Fe(III) and O
2
and thereby produce reagents for a Fenton reaction.
reduction with
was observed only at pH values near 7.0 or higher. In this way, •OH radical production
was evaluated at an optimal pH= 3.4 and at pH 7.0.
However, Fe(III) reduction was only observed at pH values below 5.5, and O
2
2 2
H O
The maximum signal of the DMPO/•OH adduct observed, Smax, and the time required for each
system in reaching that signal, Tmax, were used to compare the •OH radical production in the
Fenton-like systems (Table 2).
Table 2. Comparative parameters of •OH radical production by Fenton-like systems at pH 3.4.
System
t
min)
max
S
max
(AU)
(
1.02 ± 0.01
1.35 ± 0.03
7.84 ± 0.17
23.6 ± 0.10
13.6 ± 0.65
2.15 ± 0.07
Unmodified system
31.93 ± 3.55
13.16 ± 0.16
8.08 ± 0.21
10.58 ± 0.11
Catechol-driven Fenton-like system
Dopamine-driven Fenton-like system
Epinephrine-driven Fenton-like system
Norepinephrine-driven Fenton-like system
The driven systems reached higher values of Smax than that for the unmodified system.
According to the literature for similar systems[27, 31], these results indicate that catecholamines
increased the reactivity of Fenton-like systems. Catechol-driven systems produced the highest

amounts of •OH radicals. This was reflected in the Smax values, which were at least 240% higher
than those obtained in other systems. Catecholamine-driven systems required longer time to
reach Smax with epinephrine-driven systems requiring the most time to reach Smax. A logarithmic
R
relationship between Smax and I was observed (R=0.9984; Figure 7). This confirmed Fe(III)
reduction as the primary mechanism that promotes •OH radical production in the Fenton
reaction by catechol compounds.
Figure 7. Relationship between maximum signal value of DMPO/•OH adduct (Smax)and I
.4.
R
at pH
3
At pH 7.0, all Fenton-like systems showed slower •OH radical production than at pH 3.4.
Although the signal of the DMPO/•OH adduct was detectable, it was below the limit of
quantification. This was consistent with results obtained in studies on Fe(III) reduction, which
showed that the amount of reduced Fe(III) was significant only at acidic pH below 5.5.
2 2
Although catecholamine oxidation can produce H O , the pH at which this happens was not
2+
consistent with the pH at which the [Fe(LH)] complex, which was responsible for increased
reactivity, was formed. Together, these results indicate that at approximately pH 5.5,
+
catecholamines coordinated with iron in aqueous solutions to form [Fe(LH)
this complex prevented the entry of H
prevented the formation of •OH radicals through the Fenton reaction. This antioxidant
2
] . The formation of
2 2
O into the metal coordination sphere and therefore,

mechanism of catecholamines confirmed the reports of other studies that have also shown the
ability for catecholamines to scavenge several other radical species[48-50].
3.4. ROS production in cell cultures
To verify the inability to increase ROS production in Fenton-like systems under physiological
conditions, an in vitro study was performed. The concentration of reagents used in cultures was
the same order of magnitude as the concentration of catecholamines found in blood[51, 52].At
this concentration, the Fenton-like reagents did not produce significant decreases in the number
of cells in each culture (p˃0.05, Figure 8a).
ROS production in HUVECs incubated in the presence of Fe(III)-catecholamine complexes
showed no significant differences between the controls and the systems with Fenton-like
reagents (p˃0.05, Figure 8b). The exposure to Fenton-like reagents did not result in increases
in ROS production beyond normal cell metabolism. These results were consistent with those
observed in •OH radical production at pH 7.0, where no quantifiable radical signals were
observed.
Figure 8. a. Cell viability of HUVECs and b. production of reactive oxygen species (ROS) in
HUVECs incubated for 4 h in the presence of Fe(III)-catecholamine complex in M-199 medium
(pH7.2-7.4; n = 3).

4. Conclusion
Catecholamines have been associated with the development of oxidative stress by Fenton
reactions. These compounds reduce Fe(III) to Fe(II) and O to H and thereby produce
2
2 2
O
Fenton reagents. However, the presence of catecholamines in the studied systems did not
increase the production of ROS at physiological conditions. Accordingly, catecholamines could
2 2
not promote the Fenton reaction at pH values near 7.0, even in presence of excess of H O . At
+
]
this pH, the [Fe(LH)
2
complex did not have the ability to reduce Fe(III). However at acidic pH
of 3.4 the ability to reduce Fe (III) was closely related to the change in the amount of •OH
+
radicals that can be produced by a driven Fenton reaction. The [Fe(LH)
2
]
complex also did not
allow the participation of iron in Fenton-like reactions. According to these results, oxidative
stress mediated by catecholamine-driven Fenton reactions requires microenvironments with pH
2 2
values below 5.5 and external sources of H O .
Acknowledgments
The financial support for this work was provided by FONDECYT (grant nos. 1131101 and
160100), FONDEQUIP (grant no. EQM140075), FONDAP Solar Energy Research Center,
1
SERC-Chile (grant no. 15110019), MECESUP (grant no. UCH0601, Ph.D. Fellowship to Victoria
Melin) and CONICYT (Ph.D. grant nos. 21100880 and 24121439).

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Graphical abstract

Graphical abstract
The reduction of Fe(III) and O
2
by catecholamines occurrs at different pH range. Although
by their autoxidation, these compounds only promote •OH
radical production by Fenton reaction at pH value wherein Fe(III) is reduced. This is because
OH radical production is related to initial rate of Fe(III) reduction.
2 2
catecholamines can produces H O
•

Highlights
•
Catecholamines can reduce Fe(III) at acidic pH values up to pH 5.5
•
Fe(III) reduction by catecholamines is correlated with •OH production by Fenton
reaction
•
•
Catecholamines promote O
2 2 2
reduction to H O at pH near 7.0
Catecholamines cannot promote Fenton reactions at pH near 7.0