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Title
Modulation of oxidative events by multivalent manganese complexes in brain tissue

Permalink
https://escholarship.org/uc/item/90f4b1h2

Journal
Free Radical Biology and Medicine, 31(6)

ISSN
0891-5849

Authors
HaMai, D
Campbell, A
Bondy, SC

Publication Date
2001-09-15

DOI
10.1016/S0891-5849(01)00639-6

License
CC BY 4.0

Peer reviewed

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University of California
 Free Radical Biology & Medicine, Vol. 31, No. 6, pp. 763–768, 2001
Copyright © 2001 Elsevier Science Inc.
Printed in the USA. All rights reserved
0891-5849/01/$–see front matter

PII S0891-5849(01)00639-6

Original Contribution
MODULATION OF OXIDATIVE EVENTS BY MULTIVALENT MANGANESE
COMPLEXES IN BRAIN TISSUE

DIEM HAMAI, AREZOO CAMPBELL, and STEPHEN C. BONDY

Department of Community and Environmental Medicine, Center for Occupational and Environmental Health,
University of California, Irvine, Irvine, CA, USA

(Received 7 June 2001; Accepted 15 June 2001)

Abstract—Manganese toxicity can evoke neuropsychiatric and neuromotor symptoms, which have frequently been
attributed to profound oxidative stress in the dopaminergic system. However, the characterization of manganese as a
pro-oxidant remains controversial because antioxidant properties also have been associated with this metal. The current
study was designed to address these disparate findings concerning the oxidative properties of manganese. The apparent
ability of manganese in its divalent form to promote formation of reactive oxygen species (ROS) within a cortical
mitochondrial-synaptosomal (P2) fraction was completely abolished by the addition of one five hundredth of its molarity
of desferroxamine (DFO), a trivalent metal chelator. This large ratio and the high specificity of DFO for trivalent metal
ions discounted the possibility of inhibition of ROS generation by direct sequestration of divalent manganese, and
implied the trace presence of a trivalent metal. Further analysis suggested that this trace metal was manganic rather than
ferric ion. Ferric ion was able to dampen the reactive oxygen species-generating capacity of manganous chloride,
whereas manganic ion markedly promoted this property attributed to manganous ion. Such findings of the potent effects
of trace amounts of trivalent cations upon Mn2⫹-related free radical generation offer resolution of earlier disparate
findings concerning the oxidative character of manganese. © 2001 Elsevier Science Inc.

Keywords—Manganese, Reactive oxygen species, Oxidative stress, Pro-oxidant, Antioxidant, Neurotoxicity, Transi-
tion metals, Free radicals

INTRODUCTION transition metals to modulate and transport oxygen, the

presence of free transition metals that are not bound to
Chronic exposure to manganese devastates the central
proteins creates the potential danger for promotion of
nervous system with symptoms strikingly similar to
unwanted free radical reactions. Such an excess of tran-
schizophrenia and Parkinsonism [1,2]. Brain lesions,
sition metals may exert a pro-oxidant effect and initiate
marked by neuronal degeneration [3], are focal within
neuronal degeneration.
brain regions that are particularly active in oxidative
The pro-oxidant effects of transition metals have been
metabolism and have a significant dopamine content,
confirmed repeatedly in vitro and in vivo studies [5–7]. A
such as the rat hypothalamus or primate substantia nigra
small increase in levels of free iron within cells can
[4]. Due to their high consumption of oxygen, these
enhance lipid peroxidation and dopamine auto-oxidation
regions tend to generate large quantities of hydrogen
[8 –10]. Similarly, in its oxidation from the univalent to
peroxide (H2O2) and superoxide anion (O2•⫺). These
the divalent form, copper reacts with lipid hydroperox-
oxidative species readily participate in single-electron
ides to generate diene conjugates, and eventually cyto-
transfers with “catalytic” transition metals to generate
toxic alkoxyl, peroxyl, and lipid radicals [11]. The oxi-
oxygen-derived free radicals, or highly reactive oxygen
dation of iron, copper, and other transition metals is
species (ROS). Although the cell normally relies on
thought to catalyze the homolytic fission of hydrogen
peroxide to hydroxyl radical and hydroxyl ion in the
Address correspondence to: Dr. Diem HaMai, Department of Com- Fenton reaction [12].
munity and Environmental Medicine, University of California, Irvine,
Irvine, CA 92697-1825, USA; Tel: (949) 824-8077; Fax: (949) 824- The potential involvement of oxidative damage in
2793; E-Mail: [email protected]. manganese neurotoxicity suggests that manganese may

763
764 D. HAMAI et al.

also catalyze the Fenton reaction. In spite of extensive Assay for reactive oxygen species formation
research efforts, the evidence for such speculations of
Reactive oxygen species (ROS) were assayed using
manganese pro-oxidant activity, based primarily on use
2⬘,7⬘-dichlorofluorescin diacetate (DCFH-DA) [22].
of the divalent salt manganese chloride (MnCl2), has
DCFH is capable of being oxidized to the fluorescent
been conflicting. Manganese has been implicated in en-
2⬘,7⬘-dichloro-fluorescein by reactive oxygen species.
hancing dopamine auto-oxidation and oxidative nigral
Although the identity of the ultimate species responsible
[10,13–15], and in inflicting damage similar to that of
for the oxidation of DCFH is not known [23], the utility
other mitochondrial toxins such as carbon monoxide and
of this probe as a sensitive measure of ROS in biological
cyanide [16]. In evaluations of the oxidative character of
systems, including isolated subcellular cerebral systems,
manganese, several groups have attributed accelerated
has been documented [24].
ROS formation to both the divalent and trivalent states of
P2 suspensions were diluted in 19 volumes of HEPES
the metal [17,18]. However, other reports contend that
buffer. Test compounds were pipetted into 96 well clus-
divalent manganese ion scavenges the ROS species su-
ter plates (Costar, Corning Inc., Corning, NY, USA) with
peroxide and hydroxyl radicals even when SOD activity
four replicates per sample, and suspended in 100 ␮l of
is inhibited [19], and attenuates oxygen toxicity in the
Tris-buffer per well. A volume of 100 ␮l, containing 5 ␮l
Lactobacillus planetarium and related bacteria deficient
of P2 fraction and 95 ␮l of 5 ␮M DCFH-DA, was added
in the superoxide dismutase enzyme [20]. Additional
to each well for a final volume of 200 ␮l.
anti-oxidant effects, including catalase-like dispropor-
Given the light-sensitive nature of the probe, assays
tionation of H2O2 [21], inhibition of iron-induced lipid
were carried out in a FL600 spectrofluorometer (Biotek
peroxidation and copper-dependent low-density lipopro-
Instruments, Inc., Winooski, VT, USA). The microplate
tein conjugation [7] have also been demonstrated using
reader allowed for fast light excitation and precise fluo-
the same divalent salt MnCl2. Conflicting findings such
rescence capturing, thus minimizing photo-oxidation of
as these have complicated the characterization of man-
the probe [25]. The incubation temperature was main-
ganese as either a pro-oxidant or an antioxidant.
tained at 37°C. Excitation and emission were set respec-
The current study was designed to address these in-
tively at 485 nm and 530 nm. The fluorescence from each
consistencies by focusing on the oxidative capacity of
well was monitored, digitized, and stored on a computer
Mn2⫹ and Mn3⫹, the two primary valence states of
using KC4 (Version 4.0) (Biotek) software. Parallel
manganese in biological tissues. The findings indicate
blanks with no DCFH-DA were included, and this value
that manganese indeed may promote unwanted free rad-
was subtracted from readings of samples to correct for
ical reactions, however, the redox dynamics between its
autofluorescence of fractions. Additionally, parallel
lower and higher valence states differ from that of other
blanks with no P2 loaded with DCFH-DA served as an
pro-oxidant metals. The key finding is that the trace
indicator of photo-oxidation of DCFH-DA. A DCF stan-
presence of a trivalent metal ion is required for the
dard curve (1–500 nM) was used for conversion of
pro-oxidant activity associated with excess Mn2⫹. The
fluorescence to pmol DCF formed/␮g protein/2 h.
catalytic effect of nanomolar concentrations of the triva-
lent ion on the oxidation of Mn2⫹ and redox cycling may
account for earlier conflicting reports pertaining to the Protein determination
oxidative properties of manganese salts. Protein content was assayed using the method of
Bradford [26].
MATERIALS AND METHODS
Statistical analysis
Tissue preparation Statistical analysis was performed using ANOVA and
Brains obtained from 2–3 month old male B6C351 Fisher’s Least Significant Difference Test to assess the
strain mice were excised on ice, and the cerebral cortex significance of differences between groups. The accep-
was weighed and homogenized in 10 vol. of 0.32 M tance level of significance was p ⬍ .01 using a two-tailed
sucrose and centrifuged at 1800 ⫻ g for 10 min. The distribution.
resulting supernatant fraction was centrifuged at
31,500 ⫻ g for 10 min to yield the crude mitochondrial/ RESULTS
synaptosomal pellet (P2). This was taken up in HEPES
Differential promotion of ROS formation by divalent
buffer to a concentration of 0.1 g-eq/ml. The composi-
and trivalent manganese
tion of the HEPES buffer was (mM): NaCl, 120; KCl,
2.5; NaH2PO4, 1.2; MgCl2, 0.1; NaHCO3, 5.0; glucose, The rate of generation of ROS in a cerebral cortical
6.0; CaCl2, 1.0; and HEPES, 10 at pH 7.4. mitochondrial-synaptosomal fraction was enhanced by
 Manganese and oxidative events 765

Fig. 1. Reactive oxygen species production as measured by DCF

formation in P2 cortical fractions following in vitro exposure to MnCl2 Fig. 3. Modulatory effects of trivalent iron and manganese salts on the
and MnAc3. The DCF formation is plotted against the log concentra- formation of reactive oxygen species by MnCl2. Values are mean ⫾ SE
tion. Values are mean ⫾ SE of three independent experiments realized of three independent experiments realized in quadruplicate. *Value of
in quadruplicate. *Value differs (p ⬍ .01) with that of the control. manganese-treated fractions differs (p ⬍ .01) with that of the control;
(Error bars are too small to visualize). ⫹value of fractions treated additionally with either MnAc3 or FeCl3
differs (p ⬍ .01) with the corresponding value of fractions treated with
MnCl2 alone.

100 to 500 ␮M of MnCl2 (Mn2⫹), whereas manganic

acetate (Mn3⫹) affected a similar increase in ROS for-
available ferric ions (Fe3⫹) from the preparations. ROS
mation at an order of magnitude lower in concentration
formation by Mn3⫹ was attenuated by DFO at a molar
(0.5 to 5 ␮M) (Fig.1).
ratio of ten to one. The rate of ROS formation was
reduced yet the metal ion retained significant pro-oxidant
Manganous-promoted ROS formation quenched by activity. This minor reduction was attributed to seques-
chelator DFO tration of a significant amount of manganic ion.
In comparison, the effect of DFO on attenuation of
Previous studies in this laboratory have found that ROS formation by Mn2⫹ was far greater than that of
interactions between different metal species, specifically Mn3⫹. Most remarkably, ROS levels were reduced be-
lead and aluminum, as well as the transition metals iron, low those of basal values in untreated subcellular frac-
copper, and chromium, accelerate ROS formation [27, tions, even at a molar ratio of 500 to 1 of Mn2⫹ to DFO
28]. To ascertain the contributions, if any, of iron to (Fig. 2). Because quenching occurred at such a large
manganese-promoted ROS formation, desferroxamine ratio between divalent manganese and DFO, the possi-
(DFO), a widely used chelator of trivalent iron and bility of inhibition of ROS generation by sequestration of
aluminum, was incubated with each of the manganese Mn2⫹ by DFO was discounted. The low concentrations
salts (Fig. 2). This was intended to deplete traces of of DFO required to quench ROS implied the trace pres-
ence of a trivalent metal.

Manganic but not ferric enhanced

manganous-promoted ROS formation

Because DFO at nanomolar concentrations abolished

Mn2⫹-promoted ROS formation, the possible presence
of very low concentrations of a trivalent metal ion was
investigated to try to account for the pro-oxidant activity
associated with Mn2⫹. To simulate the effect of trivalent
metal ions, which are intrinsically available in trace
amounts in brain tissue, Fe3⫹ and Mn3⫹ salts were added
at very low concentrations (500 nM–5 ␮M) to different
Fig. 2. Effect of DFO on manganese-generated ROS formation. P2
cortical fractions were incubated with or without one of the manganese samples incubated with MnCl2 (Fig. 3). While additions
salts. Values are mean ⫾ SE of three independent experiments realized of nanomolar concentrations of the ferric salt reduced
in quadruplicate. *Value of manganese-treated fractions differs (p ⬍ Mn2⫹-promoted ROS formation, additions at the same
.01) with that of the control; ⫹value of fractions treated additionally
with DFO differs (p ⬍ .01) with the corresponding value of fractions concentrations of the Mn3⫹ salt potentiated Mn2⫹-pro-
treated with one of the manganese salts alone. moted ROS formation. Additions of the Mn3⫹ salt alone
766 D. HAMAI et al.

claims of Mn as a pro- or antioxidant. Rather such

disparate characterizations of manganese can be recon-
ciled by our finding that the pro-oxidant activity associ-
ated with divalent manganese depends on the trace pres-
ence of a trivalent metal ion, most probably manganic,
Mn3⫹. Although both valences of Mn have been associ-
ated with accelerated generation of ROS, the findings of
this current study reveal a resistance to oxidation by
Mn2⫹ that contrasts with the readiness of the lower
valence states of iron and copper to become oxidized to
higher valence levels.
Fig. 4. Inhibition by DFO on manganese-based ROS generation. P2 An apparent ability of manganese in the divalent state
cortical fractions were incubated with or without MnAc3, and DFO.
Values are mean ⫾ SE of three independent experiments realized in to promote the formation of ROS within a cortical mi-
quadruplicate. *Value of manganese-treated fractions differs (p ⬍ .01) tochondrial-synaptosomal fraction was found. However,
with that of the control; ⫹value of fractions treated additionally with the addition of one five hundredth of its molarity of
MnAc3 and/or DFO differs (p ⬍ .01) with the corresponding value of
fractions treated with MnCl2 alone. desferroxamine (DFO), a trivalent metal chelator, com-
pletely abolished the pro-oxidant activity of Mn2⫹. The
large molar ratio between additions of Mn2⫹ and DFO,
at nanomolar concentrations did not promote ROS for- and the high specificity of DFO for trivalent metal ions,
mation above basal levels (Fig. 1). negated the possibility of inhibition of ROS generation
by sequestration of the divalent cation by the chelator.
Moreover, the trace presence of a trivalent metal was
Chelation of manganic ion by DFO neutralized implicated as a promoting factor in Mn2⫹-related gener-
pro-oxidant activity of manganous ion ation of ROS, given that DFO binds more tightly to
trivalent ions than to divalent ones by at least 11 orders
To inquire further whether Mn3⫹ may be the metal of magnitude [29]. Further analysis demonstrated that
being chelated by DFO, promotion of ROS production in very low concentrations of Fe3⫹ ion were able to dampen
samples incubated with MnCl2 was tested in the presence the oxidative potential of Mn2⫹, whereas small amounts
and absence of nanomolar and low micromolar concen- of Mn3⫹ could markedly promote the ROS-generating
trations of MnAc3 (500 nM–2 ␮M) and DFO (500 nM–5 capacity of Mn2⫹. This suggested that the trace metal in
␮M) (Fig. 4). A concentration of 500 nM DFO com- manganous salts, which was complexed by DFO, was
pletely neutralized the effect of the added 500 nM Mn3⫹, primarily Mn3⫹ rather than Fe3⫹. Although DFO forms
and reduced ROS production to levels similar to those the most stable complex with Fe3⫹ [29], the ability of
found when Mn2⫹ was added alone. Furthermore, an DFO to sequester trivalent metals such as Al3⫹ and
excess of DFO (1 ␮M), intended to complex with the Mn3⫹ has been utilized to study chelation therapy and
added 500 nM Mn3⫹ and any trace amounts of Mn3⫹ metal coordination chemistry [30 –33].
already pre-existing in solution, completely abolished the Mn3⫹ added alone at nanomolar concentrations was
excess generation of ROS observed with addition of not sufficient to elevate basal levels of ROS formation,
Mn2⫹ to basal levels. yet the same concentration range added to samples incu-
bated with Mn2⫹ led to significant pro-oxidant activity.
DISCUSSION Moreover, nanomolar concentrations of DFO treatment
of samples incubated with manganous and manganic
The valence state of transition metals, such as iron salts returned the rate of ROS generation to basal levels.
and copper, has been established as a critical factor in the This implied an interaction between the divalent and
ability of the metal to catalyze ROS formation in the trivalent states from which the capacity of Mn2⫹ to
Fenton reaction, yet experimental use of Mn has been catalyze ROS generating reactions may be determined.
largely limited to that of the divalent salt. This may have These data demonstrate the reluctance by manganese
been a critical oversight given that the redox cycling, in the divalent form to undergo oxidation. Despite the
which is necessary for the generation of ROS described feasibility of manganese catalyzing Fenton-like reactions
by the Fenton reaction, involves at least two different [34], the low reduction potential of the aqueous divalent
valences. Indeed, studies of other valence states of man- ion may not permit its interaction with H2O2 to produce
ganese and their potential to promote oxidative stress OH• at a measurable rate [29] under normal cellular
have been sparse and conflicting [7,18,19,28]. concentrations and redox balance. However, the oxida-
The findings of this current study do not dispute either tion of Mn2⫹ may be facilitated by its interaction with
 Manganese and oxidative events 767

metal colloids and ligands, such as oxy-hydroxides, Fe3⫹, and those of closed shell ions, such as those in the
available in the biological environ [35]. Such catalysts alkaline earth family (e.g., Mg, Ca) [38]. The stability of
can alter the electronegativity of the metal ion [36] and this electron configuration and the reluctance to lose a
increase exponentially the rate constant of the oxidation d-electron accounts for the poor reducing ability of
reaction [37], thus overcoming the difficulty of the in- Mn2⫹ [36]. Contrastly, Mn3⫹ has much higher transition
trinsically low redox potential of the Mn2⫹ ion. probabilities compared to Mn2⫹ [38]. Its four unpaired
Mn3⫹ or intermediate complexes that are significantly electrons in the usual high-spin configuration are similar
stronger oxidizing agents may serve a similar catalytic to those of the lower oxidation state of iron, Fe2⫹, and
role in the oxidation of Mn2⫹. Given the redox potential render the ion with marked instability and high reduction
between the two ions, Mn3⫹ or ionic intermediates could and oxidation potential. Its high spin d4, Mn3⫹ is inclined
induce a shift in the electron density of the divalent ion either to lose one electron in the antibonding eg set of
to enhance its ability to oxidize or reduce. Effectively, an
orbitals to maximize ligand field stabilization or to gain
interaction between Mn2⫹ and ion catalysts could desta-
an electron to maximize electron exchange energy [36].
bilize the divalent ion and change the overall redox status
The hybrid properties of both closed shell and transition
to one that is more favorable to the oxidation of the
metal ions in Mn may manifest themselves as redox
divalent ion and the acceleration of ROS generation.
bioenergetics atypical of other transition metals.
Mn 2⫹ ⫹ H 2 O 2 7 关Mn 2⫹ ⫺ OH 7 Mn 3⫹ ⫺ • OH兴 7 Mn 3⫹ ⫹ HO • ⫹ OH ⫺
The different properties of Mn2⫹ and Mn3⫹ and their
intermediate complexes
interaction during oxidative events may be particularly
relevant in the context of biological systems. Although
Similarly, the dampening effect of the pro-oxidant activ- the divalent state is the most stable form of manganese in
ity observed with trace additions of Fe3⫹ suggest that aqueous solutions, the trivalent state is the dominant state
this trivalent form of iron may also alter the redox status of manganese in many biological tissues and in natural
of Mn2⫹. The symmetry of the d-electron configuration water sources [38]. In blood, free, and ␣2-macroglob-
of manganous and ferric ions may confer stability to both ulin-bound Mn2⫹ is oxidized to Mn3⫹, possibly by fer-
the redox couples of manganese (Mn2⫹ 4 3 Mn3⫹) and roxidase I or ceruloplasmin, and is then bound to trans-
iron (Fe2⫹ 4 3 Fe3⫹) by reinforcing the low redox ferrin [39]. Within cells, manganese is found
potential of one another, thereby mitigating some of the predominantly in mitochondria in the divalent form,
pro-oxidant activity potentiated by Mn3⫹. where its uptake and export may be via a calcium carrier
The resistance of Mn2⫹ to oxidation differs with the [38]. The high oxidative activity of some brain regions
readiness of the lower valence states of iron and copper may facilitate oxidation of small amounts of divalent
to change to higher valence levels. For these latter tran- manganese to the trivalent state, thereby initiating Fenton
sition metals, the lower valence state exhibits greater cycling.
redox potential, higher reactivity, and greater readiness In summary, manganous ion has no intrinsic oxidant
to catalyze free radical-generating reactions. The higher potential. However, Mn2⫹may act in a pro- or antioxi-
valence state Mn3⫹ showed significant redox potential, dant manner, depending on the trace presence of either
while the lower valence state Mn2⫹ was found to be a Mn3⫹ or Fe3⫹ ion, respectively. Catalytic amounts of
modest pro-oxidant only with the trace presence of an-
trivalent manganese may promote Fenton-type redox cy-
other trivalent metal. Such a marked difference between
cling, whereas ferric iron can attenuate this process. The
the redox potential of the two valences contrast with
potent effects of trace amounts of trivalent cations upon
other transition metals, suggesting that manganese may
manganese-related free radical events may account for
possess redox dynamics that rely heavily on other medi-
the discordant literature on this subject. Our findings can
ating factors before it undergoes free radical-generating
reactions. reconcile reports of inhibition of iron-based ROS pro-
The known unusual ionic properties of its divalent and duction by manganese [40,7], and descriptions of the
trivalent states of manganese validate the potential exis- apparently intrinsic ROS-generating capacity of manga-
tence of atypical redox differentials. In contrast to other nese in both divalent and trivalent forms [18]. The co-
transition metals, manganese is most stable in aqueous, existence of both the divalent and trivalent forms of
neutral pH solutions with the oxidation number of ⫹2. manganese in biological tissues underscores the potential
Mn2⫹ is an S-state ion in the usual high-spin configura- metabolic significance of interactions of manganese in
tion with an approximately spherical distribution of the different valence states. This should serve as an impetus
five unpaired 3d-electrons about the nucleus. The half- to reframe the design of studies on manganese intoxica-
filled 3d shell of Mn2⫹ imparts the ion with a stability tion—in terms of not only elements involved, but also of
analogous to that of the higher oxidation states of iron, valence states.
768 D. HAMAI et al.

Acknowledgement — This work was supported in part by NIH Grant radical by manganous complexes: in vitro. Arch. Biochem. Bio-
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