Life Sciences 69 (2001) 879–889

 

0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
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Inhibition of brain monoamine oxidase activity 

by the generation of hydroxyl radicals 

Potential implications in relation to oxidative stress

 

Ramón Soto-Otero

 

a,

 

*, Estefanía Méndez-Álvarez

 

a

 

Álvaro Hermida-Ameijeiras

 

a

 

, Inés Sánchez-Sellero

 

b

 

,

Angelines Cruz-Landeira

 

b

 

, Manuel López-Rivadulla Lamas

 

b

 

a

 

Grupo de Neuroquímica, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, 

Universidad de Santiago de Compostela, San Francisco 1, E-15782 Santiago de Compostela, Spain

 

b

 

Departamento de Toxicología Forense, Instituto de Medicina Legal, Facultad de Medicina, 

Universidad de Santiago de Compostela, San Francisco 1, E-15782 Santiago de Compostela, Spain

 

Received 20 October 2000; accepted 5 January 2001

 

Abstract

 

Monoamine oxidase (MAO) is an enzyme involved in brain catabolism of monoamine neurotrans-

mitters whose oxidative deamination results in the production of hydrogen peroxide. It has been docu-
mented that hydrogen peroxide derived from MAO activity represents a special source of oxidative
stress in the brain. In this study we investigated the potential effects of the production of hydroxyl rad-
icals (

 

 

OH) on MAO-A and MAO-B activities using mitochondrial preparations obtained from rat brain.

Ascorbic acid (100 

 

m

 

M

 

) and Fe

 

2

 

1

 

 (0.2, 0.4, 0.8, and 1.6 

 

m

 

M

 

) were used to induce the production of

 

 

OH. Results showed that the generation of 

 

 

OH significantly reduced both MAO-A (85–53%) and

MAO-B (77–39%) activities, exhibiting a linear correlation between both MAO-A and MAO-B activi-
ties and the amount of 

 

 

OH produced. The reported inhibition was found to be irreversible for both

MAO-A and MAO-B. Assuming the proven contribution of MAO activity to brain oxidative stress,
this inhibition appears to reduce this contribution when an overproduction of 

 

 

OH occurs.

© 2001

Elsevier Science Inc. All rights reserved.

 

Keywords:  

 

Monoamine oxidase; Hydroxyl radical; Enzyme inhibition; Oxidative stress; Brain mitochondria; Rat

 

Introduction

 

Monoamine oxidase (MAO) is an FAD-dependent enzyme localized in the outer mem-

brane of the mitochondria which plays an essential role in the turnover of monoamine neu-

 

* Corresponding author.  Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, San

Francisco1, E-15782 Santiago de Compostela, Spain. Tel.: 

 

1

 

34 981 563 100, ext 12210; fax: 

 

1

 

34 981 582 642.

 

E-mail address

 

: bnsoto@usc.es (R. Soto-Otero)

 

880

 

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

 

rotransmitters such as dopamine, serotonine and noradrenaline. It occurs in at least two
forms, MAO-A and MAO-B, with different specificities for substrates and inhibitors. The
cloning of cDNAs for MAO-A and MAO-B has demonstrated that both isoenzyme forms are
encoded by different genes, associating their different specificities for substrates and inhibitors
to their corresponding primary structures [1]. However, the potential contribution of the
membrane lipid environments on MAO-A and MAO-B specificities remain unsolved [2,3]. It
catalyzes the oxidative deamination of biogenic amines to their corresponding aldehydes, which
is accompained by the reduction of molecular oxygen to hydrogen peroxide (H

 

2

 

O

 

2

 

) [4,5].

As is well-known, H

 

2

 

O

 

2

 

 is a reactive oxygen species (ROS) which, through the Fenton re-

action, can generate a hydroxyl radical (

 

 

OH), this being considered the most damaging free

radical for living cells due to its high reactivity [6]. The involvement of 

 

 

OH in neuronal loss

has been postulated in cerebral ischemia [7], in aging [8], in Parkinson’s disease [9], and in
Alzheimer’s disease [10].

Although, H

 

2

 

O

 

2

 

 is also formed during mitochondria respiration, the amount of H

 

2

 

O

 

2

 

 gen-

erated by MAO activity greatly exceeds the amount produced during electron flow [5], which
identifies the activity of this enzyme as a process with a considerable toxic potential. This
suggestion is corroborated 

 

in vivo

 

 by the diminution in brain production of H

 

2

 

O

 

2

 

 observed in

rats by the inhibition of MAO activity with pargyline [11]. In spite of the existence of two en-
zymatic scavenging systems to protect cells from the presence of H

 

2

 

O

 

2

 

, catalase and glu-

tathione peroxidase, the brain levels of these two enzymes are very low compared to those
found in other tissues [12].

The aim of the present study was to investigate the potential 

 

in vitro

 

 effects of the produc-

tion of 

 

 

OH on both MAO-A and MAO-B activities. MAO activity was determined in crude

mitochondrial fractions obtained from rat brain and 

 

 

OH was generated using a mixture of

ascorbic acid (AA) and ferrous iron (Fe

 

2

 

1

 

).

 

Methods

 

Chemicals

 

Kynuramine dihydrobromide, AA, and bovine serum albumin were purchased from Sigma

Chemical Co. (St. Louis, MO, USA). Clorgyline hydrochloride and R(-)-deprenyl hydrochlo-
ride was obtained from Research Biochemicals International (Natick, MA, USA). Ferrous
chloride tetrahydrate was purchased from Fluka Chemie AG (Buchs, Switzerland). Tereph-
thalic acid (THA), disodium salt was from Aldrich Chemical Co. (Milwaukee, WI, USA).
The water used for the preparations of solutions was of Milli-RiOs/Q-A10 grade (Millipore
Corp., Bedford, MA, USA). All remaining chemicals used were of analytical grade and were
purchased from Fluka Chemie AG (Buchs, Switzerland). Fresh stock solutions of AA and
Fe

 

2

 

1

 

 were prepared immediately before each experiment in water and a buffer solution

(Na

 

2

 

PO

 

4

 

/KH

 

2

 

PO

 

4

 

 isotonized with KCl, pH 7.4), respectively.

 

Preparation of brain mitochondria

 

Male Sprague-Dawley rats weighing 200–250 g were used. The rats were received from

the breeder at least four days before sacrifice, and were kept on a 12:12 light-dark schedule

 

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

 

881

 

with 

 

ad libitum

 

 access to food and water. Animals were stunned with carbon dioxide and

killed by decapitation. Brains were immediately removed and washed in ice-cold isolation
medium (pH 7.4, Na

 

2

 

PO

 

4

 

/KH

 

2

 

PO

 

4

 

 isotonized with sucrose). Brain mitochondrias were then

obtained by diferential centrifugation with minor modifications to a previously published
method [13]. Briefly, after removing blood vessels and pial membranes, the brains were man-
ually homogenized with four volumes (w/v) of the isolation medium. Then, the homogenate
was centrifuged at 900 

 

g

 

 for 5 min at 4 

 

8

 

C. The supernatant was centrifuged at 12,500 

 

g

 

 for

15 min. The mitochondria pellet was then washed once with isolation medium and recentri-
fuged under the same conditions. Finally, the mitochondrial pellet was reconstituted in a
buffer solution (Na

 

2

 

PO

 

4

 

/KH

 

2

 

PO

 

4

 

 isotonized with KCl, pH 7.4) and stored in aliquots under

liquid nitrogen.

The protein concentration of mitochondrial suspensions was determined according to the

method of Markwell et al. [14], using bovine serum albumin as the standard.

 

Determination of MAO activity

 

MAO activity was measured by a spectrophotometric assay based on the original proce-

dure of Weissbach et al. [15], as previously reported [13]. A Ultrospec III spectrophotometer
(Pharmacia Biotech, Uppsala, Sweden) with a cuvette holder thermostatized at 37 

 

8

 

C was

used. (-)-Deprenyl (250 nM) and clorgyline (250 nM) were used as irreversible and selective
inhibitor to assay MAO-A and MAO-B activity, respectively. Mitochondrial incubations
were performed in a buffer solution (Na

 

2

 

PO

 

4

 

/KH

 

2

 

PO

 

4

 

 isotonized with KCl, pH 7.4) at a final

protein concentration of 1 mg/ml. A 5 minute preincubation of the irreversible inhibitor and
the mitochondria preparation was made, followed by the concurrent addition of AA (100

 

m

 

M

 

) and Fe

 

2

 

1

 

 (0.2, 0.4, 0.8 or 1.6 

 

m

 

M

 

). After 5 min of incubation, kynuramine was added as

a non selective substrate at concentrations equal to the corresponding K

 

M

 

 value (90 

 

m

 

M

 

 for

MAO-A and 60 

 

m

 

M

 

 for MAO-B). All concentrations are final concentrations. The formation

of 4-hydroxyquinoline (4-OHQ) was then followed at 314 nm for 5 min.

The reversibility of the inhibition was determined by dialysis using a Biodialyser® (Sigma

Chemical Co.) with an ultrafiltration membrane of a nominal molecular weight limit of
10,000 [16]. Mitochondrial preparations were preincubated at 37 

 

8

 

C for 15 min in the ab-

sence (control) or presence of both AA (100 

 

m

 

M

 

) and Fe

 

2

 

1

 

 (0.8 

 

m

 

M

 

). The resulting mixtures

were then dialysed at 4 

 

8

 

C using 250 ml of outer buffer (Na

 

2

 

PO

 

4

 

/KH

 

2

 

PO

 

4

 

 isotonized with

KCl, pH 7.4). The outer buffer was replaced with fresh buffer every 2 hours for a time period
of 10 h. Finally, the dialysed mixtures were then assayed for MAO-A and MAO-B activity.

 

Monitoring of 

 

 

OH formation

 

The generation of 

 

 

OH was fluorimetrically monitored using a modification to a previously

published method [17] in which THA is used as a chemical dosimeter of 

 

 

OH. A luminis-

cence spectrometer Model LS50B (Perkin-Elmer, Norwalk, CT, USA) was used. The cuvette
holder was thermostatically maintained at 37 

 

8

 

C and a magnetic stirrer was used for a conti-

nous mixing of the sample. For each assay, 2000 

 

m

 

l of a buffer solution (Na

 

2

 

PO

 

4

 

/KH

 

2

 

PO

4

isotonized with KCl, pH 7.4) containing 10 mM THA (final concentration) were incubated in
a quartz cuvette for 5 min to reach the temperature. An aliquot of phosphate buffer (pH 7.4)
was added to complete the final volumen of the incubation to 2.5 ml. Then, 100 

ml of AA

882

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

(100 

mM) and 20 ml of varying concentrations of Fe

2

1

 (0.2, 0.4, 0.8 or 1.6 

mM) were added.

All the concentrations are final concentrations. The monitoring of 

OH formation was imme-

diately initiated and maintained for the subsequent 5 min, using 312 nm and 426 nm as exci-
tation and emission wavelengths, respectively.

Statistical analysis

All results are expressed as means

6SEM. Data were tested for significant differences be-

tween means by a two-way Student’s t-test. Significance was indicated when p was equal to
or less than 0.05. Data analysis was aided by use of the computer program Origin® v. 6.0
(Microcal Software Inc., Northampton, MA, USA).

Results

In this study we investigated the in vitro effects of the generation of 

OH on MAO-A and

MAO-B activities using crude mitochondrial fractions obtained from rat brain. In order to op-
timize the analytical assay for MAO-A and MAO-B determination, we previously studied the
effects of the concentration of both clorgyline and (-)-deprenyl on MAO-A and MAO-B ac-
tivities, respectively. Fig. 1 shows the results obtained in this study. From the reported data,
we selected the concentration of 250 nM because it represents the lowest concentration of in-

Fig. 1. Effects of clorgyline on MAO-A activity and (-)-deprenyl on MAO-B activity after preincubation of mito-
chondrial preparations with 1

mM of (-)-deprenyl or clorgyline, respectively. Each point represents the mean6S.E.M.

from three determinations.

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

883

hibitor which guarantees a 100% inhibition of the corresponding MAO isoform. After prein-
cubation of mitochondrial preparations with clorgyline (250 nM) or (-)-deprenyl (250 nM)
for 5 min, K

M

 values for MAO-A and MAO-B were estimated by linear regression analysis

of the corresponding Lineweaver-Burk plots using the following concentrations of
kynuramine: 20, 40, 80, and 140 

mM for MAO-A and 20, 40, 60, 80, and 100 mM for MAO-B.

Under the here reported experimental conditions, MAO-A and MAO-B activities (con-
trols) were of 1.521

60.083 nmol 4-OHQ/mg protein?min and 4.56260.037 nmol 4-OHQ/

mg protein

?min, respectively.

As shown in Fig. 2, the production of 

OH was achieved by the use of the combined action

of AA (100 

mM) and Fe

2

1

 (0.2, 0.4, 0.8, and 1.6 

mM), with 

OH formation being dependent on

the Fe

2

1

 concentration used. To assess the production of 

OH, the area under the curve (AUC)

was used as an “impregnation factor”, and was calculated using the graph package Origin®.

The effect of the concurrent preincubation of AA and Fe

2

1

 with the mitochondrial prepara-

tions for 5 min was a significant reduction in both MAO-A and MAO-B activities, which was
dependent on Fe

2

1

 concentration used and consequently on the 

OH production achieved. Fig.

3 illustrates the MAO activity found under the different experimental conditions assayed, and
shows that MAO activities ranged from 85% to 53% (of control) for MAO-A and from 77%
to 39% (of control) for MAO-B. As can be seen, the reduction observed in both MAO-A and

Fig. 2. Representative fluorimetric recording of the production of 

OH from the oxygen disolved in the incubation

medium (pH 7.4, Na

2

PO

4

/KH

2

PO

4

 isotonized with KCl) in the presence of AA (100 

mM) and different concentra-

tions of Fe

2

1

. THA (10 mM) was used to detect the production of 

OH and the wavelenghts of excitation and

emission were of 312 nm and 426 nm, respectively.

884

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

MAO-B activities were in all cases statistically significative. In addition, we verified that the
reported inhibition is dependent on the preincubation time (data not shown), which is clearly
related to the amount of 

OH generated.

In this study we attempted to correlate the MAO inhibition found with the amount of 

OH

generated by the system AA

1Fe

2

1

. For this reason we submited the data of MAO activities

versus 

OH accumulation expressed as AUC to a lineal regression analysis. As shown in Fig. 4,

the correlation coefficients obtained were r 

5 0.894 for MAO-A and r 5 0.953 for MAO-B,

which confirms the existence of a direct correlation between MAO inhibition and the produc-
tion of 

OH.

For reversibility studies, mitochondrial preparations were preincubated in the absence

(control) or presence of the both AA (100 

mM) and Fe

2

1

 (0.8 

mM). As shown in Table 1, after

dialysis of the mitochondrial preparations, neither MAO-A nor MAO-B activity were recov-
ered, compared to the corresponding controls obtained in the absence of AA

1Fe

2

1

. Further-

more, as shown in Table 1, the inhibition found was very similar to that found with non-dialysed
samples preincubated with AA

1Fe

2

1

.

Although, we performed some mitochondria incubations in the presence of Fe

2

1

 (0.8 

mM)

alone, we did not found statistically significative differences in both MAO-A and MAO-B ac-
tivities when compared with their corresponding controls (in the absence of Fe

2

1

).

Fig. 3. Effects of the production of 

OH on MAO-A and MAO-B activities. MAO activity was assayed after 15

min of incubation of the mitochondrial preparation with AA (100 

mM) and the corresponding concentration of

Fe

2

1

. Controls for MAO-A and MAO-B activities were obtained in the absence of AA and Fe

2

1

. Data are mean 

6

SEM derived from 4 separate experiments. Statistical significance: * p

, 0.05; ** p, 0.01; *** p, 0.001 as com-

pared to the corresponding control (Student’s t-test).

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

885

Discussion

MAO activity in the brain is involved in the catabolism of several neurotransmitters such

as dopamine, noradrenaline, and serotonine. Obviously, the catabolism of these neurotrans-
mitters by MAO involves the formation of H

2

O

2

 which in the presence of iron generates 

OH

through the Fenton reaction. It has been reported that the main mechanism of the cytotoxicity
produced by H

2

O

2

 is precisely via the generation of 

OH [18]. Despite the existence of two

enzymatic scavenging systems, catalase and glutathione peroxidase, to protect cells from the
presence of H

2

O

2

, it is well-known that the brain levels of these two enzymes are low com-

pared to other tissue [12]. In addition, the H

2

O

2

 generated by mitochondrial monoamine oxi-

dase does not easily reach the catalase compartment [4]. Evidently, these facts make cate-
cholaminergic and serotonergic neurons particularly vulnerable to the oxidative stress caused
by MAO activity. In addition, it has been suggested that there are two other factors that may
contribute to enhance the damaging potential of this metabolic pathway: a) the reported in-
crease of MAO activity with age in human brain [19,20], which facilitates increased produc-
tion of H

2

O

2

 and b) the capacity shown by neuromelanine to bind iron [21], which promotes

the generation of specific-sites for the formation of 

OH. The involvement of MAO activity in

the pathogenesis and progresion of Parkinson’s disease has been previously postulated [22].

Fig. 4. Variations of MAO-A and MAO-B activities with the generation of 

OH. The production of 

OH is repre-

sented by the area under the curve (AUC) from the corresponding fluorimetric recording obtained in the condi-
tions used for the determination of MAO activity. Values are means 

6 SEM of four experiments. The linear regre-

sion lines for each set of data are shown.

886

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

The results of the present study show that both MAO-A and MAO-B are inhibited by the

generation of 

OH. As previously described, the generation of 

OH was achieved with the

combined action of AA

1Fe

2

1

. However, no changes in either MAO-A or MAO-B activities

were found when MAO activity was determined in the presence of Fe

2

1

 alone. Evidently,

under these conditions, the H

2

O

2

 produced by MAO activity gives 

OH (Fenton reaction). For

this reason, the reported data shows that very probably the amount of 

OH produced during

MAO activity is insufficient to cause MAO inhibition. Nevertheless, additional studies will
be required to further evaluate the potential contribution of long term MAO activity in the
presence of Fe

2

1

 to MAO inhibition. It is important to point out that the concentrations used

of both AA and Fe

2

1

 are close to those considered physiological [23,24] and lower than those

used by other authors to induce lipid peroxidation [25]. In addition, the concentrations of AA
and Fe

2

1

 used in this study were found necessary to guarantee different rates of 

OH produc-

tion, which enable us to prove the existence of a direct correlation between 

OH production

and MAO activity. It has been reported that copper, zinc superoxide dismutase (Cu,Zn-SOD)
is able to catalyze the formation of 

OH from H

2

O

2

 by a mechanism which involves the par-

ticipation of free copper released from the oxidatively damaged enzyme [26,27]. However, as
only Mn-SOD is present in the mitochondria, and given that this form does not catalyze this
reaction [26], then its potential involvement on this present study may be discarded.

The reversibility studies showed that the reported inhibition was irreversible for both

MAO-A and MAO-B. However, it was not possible to know if this inhibition is due to the ox-
idation of the enzyme by 

OH or to a modification of the lipid environment of MAO in the

mitochondria membrane caused by a potential peroxidation process [25]. Evidently, further
studies of the molecular mechanism of this inhibition will be of great interest.

Taking into consideration the important role attributed to MAO activity in the generation

of ROS [5,11], the here reported inhibition might be regarded as a mechanism which reduces
the contribution of MAO activity to oxidative stress when an overproduction of 

OH was

reached. Thus, the results of this study seem to show that MAO activity does not contribute
greatly to sustained 

OH production , which thus limits its suggested involvement in neuro-

degenerative processes to the initiation of lipid peroxidation on biological membranes. Evi-
dently, the start of lipid peroxidation is sufficient to trigger a cascade of reactions leading to
cell damage.

Table 1
Reversibility of MAO-A and MAO-B inhibition by 

OH production

Activity

b

 

(% Inhibition)

Preparation

a

MAO-A

MAO-B

Control

1.521

60.0083 (0%)

4.562

60.037 (0%)

Non-dialysed

1.016

60.010 (33%)

2.354

60.072 (48%)

Dialysed

1.009

60.013 (33%)

2.311

60.095 (49%)

a

Biological preparations were incubated in the absence (control) or in the presence of AA (100 

mM) 1 Fe

2

1

(0.8 

mM) at 37 8C for 15 min and then dialysed or not at 4 8C for 10 hours.

b

Values represent means 

6 SEM (n5 4) and are expresed as nmol 4-OHQ/mg protein·min.

R. Soto-Otero et al. / Life Sciences 69 (2001) 879–889

887

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a well-known dopaminergic

proneurotoxin widely used to investigate the pathogenesis and progression of Parkinson’s
disease [28]. This drug is bioactivated in the astrocytes by the action of MAO-B to give the
1-methyl-4-phenylpyridinium ion (MPP

1

) [29,30], which is actively taken up by the presyn-

aptic dopamine uptake system and accumulated within these nerve terminals [31]. Then,
MPP

1

 acts causing inhibition on the mitochondrial oxidation of NAD

1

-linked substrates in

dopaminergic neurons, and thus leads to a depletion of ATP and consequently causes cell
death [32]. However, the molecular mechanism of MPTP neurotoxicity has been also associ-
ated with the capacity shown by this drug to produce oxidative stress through the generation
of 

OH [33,34]. Although, MPTP affects the nigrostriatal dopaminergic system in a wide var-

ity of animal species [35], there are notorious differences in reaction among the different an-
imal species to this compound. Thus, primates [36] and mice [37] are sensitive to MPTP to
different degrees while rats are practically insensitive [38]. Assuming that MPTP is activated
by MAO and MPTP induced the generation of 

OH, the inhibition of MAO activity by 

OH

production might be involved in the different sensitivity of the different species to this pro-
neurotoxin. Evidently, to corroborate this hypothesis it might be particularly useful to investi-
gate the inhibitory properties of 

OH production against MAO activity from different animal

species.

Acknowledgments

This research has been supported by Grant MP97-0033 from the Dirección General de

Enseñanza Superior e Investigación Científica, Madrid, Spain.

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