www.elsevier.com/locate/brainres

Brain Research 1052

Research Report

Catalase inhibition by amino triazole induces oxidative stress

in goldfish brain

Tetyana V. Bagnyukovaa, Olena Yu. Vasylkiva, Kenneth B. Storeyb, Volodymyr I. Lushchaka,*

aDepartment of Biochemistry, Institute of Natural Sciences, Vassyl Stefanyk Precarpathian National University,

57 Shevchenko Street, 76025, Ivano-Frankivsk, UkrainebDepartment of Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6

Accepted 5 June 2005

Available online 14 July 2005

Abstract

The effects of in vivo inhibition of catalase by 3-amino 1,2,4-triazole (AMT) on the levels of damage products resulting from reactive

oxygen species attack on proteins and lipids as well as on the activities of five antioxidant and associated enzymes were studied in the brain

of goldfish, Carassius auratus. Intraperitoneal injection of AMT at a concentration of 0.1 mg/g wet weight caused a gradual decrease in

brain catalase activity over 72 h, whereas higher AMT concentrations (0.5 or 1.0 mg/g) reduced catalase activity by about two-thirds within

5–10 h. AMT effects on antioxidant enzyme activities and oxidative stress markers were studied in detail using fish treated with 0.5 mg/g

AMT for 24 or 168 h. The levels of thiobarbituric acid-reactive substances (a lipid damage product) increased 6.5-fold by 24 h after AMT

injection but fell again after 168 h. The content of carbonylproteins (CP) also rose within 24 h (by ¨2-fold) and remained 1.5-fold higher

compared with respective sham-injected fish after 168 h. CP levels correlated inversely with catalase activity (R2 = 0.83) suggesting that

catalase may protect proteins in vivo against oxidative modification. The activities of both glutathione peroxidase and glutathione-S-

transferase increased by ¨50% and 80%, respectively, in brain of AMT-treated fish and this might represent a compensatory response to

lowered catalase activity. Possible functions of catalase in the maintenance of prooxidant/antioxidant balance in goldfish brain are discussed.

D 2005 Published by Elsevier B.V.

Theme: Other systems of the CNS

Topic: Brain metabolism and blood flow

Keywords: Goldfish; Catalase; 3-Amino 1,2,4-triazole; Glutathione peroxidase; Carbonylprotein

1. Introduction

Most living organisms depend on ATP generation by

oxygen-based metabolism, but one consequence of oxygen

dependence is the production of reactive oxygen species

(ROS) such as superoxide (O2�), hydrogen peroxide (H2O2),

and hydroxyl radical (SOH), mainly as byproducts of

oxidative metabolism. The mitochondrial electron transport

chain and a variety of cellular oxidases are the main sources

of ROS generation [38]. ROS can attack multiple cellular

0006-8993/$ - see front matter D 2005 Published by Elsevier B.V.

doi:10.1016/j.brainres.2005.06.002

* Corresponding author. Fax: +1 38 03422 31574.

E-mail address: lushchak@pu.if.ua (V.I. Lushchak).

constituents, including proteins, nucleic acids, and lipids. To

cope with the damaging actions of ROS, organisms have

evolved multiple systems of antioxidant defense. So-called

low-molecular weight antioxidants include metabolites such

as glutathione, ascorbic acid, tocopherol, uric acid, etc.,

whereas high-molecular weight defenses include enzymes

such as superoxide dismutase (SOD), catalase, glutathione

peroxidase (GPx), and glutathione-S-transferase (GST)

[12,14,17,24]. These enzymes that deal directly with radical

species and the damage caused by them to macromolecules

constitute the first line of antioxidant enzymatic defense,

whereas other enzymes including glutathione reductase

(GR) and glucose-6-phosphate dehydrogenase (G6PDH)

contribute to the renewal of reducing power. GR catalyzes

(2005) 180 – 186

T.V. Bagnyukova et al. / Brain Research 1052 (2005) 180–186 181

the NADPH-dependent reconversion of oxidized gluta-

thione to reduced GSH, whereas G6PDH is a primary

source of NADPH synthesis.

Under normal physiological conditions, a relatively low

steady-state level of ROS in cells is the result of a balance

between ROS formation in prooxidant processes and ROS

elimination by enzymatic and non-enzymatic scavengers.

Constitutive activities of antioxidant enzymes are coordi-

nated to ensure the optimal defense against exo- and

endogenic ROS generation. Each enzyme carries out

specific functions, although an overlap between some

enzymes may exist; for example, both catalase and GPx

can degrade hydrogen peroxide [15,17,32]. The roles of

these two antioxidant enzymes in physiological defense

against peroxides are still not understood in full. Some

studies suggested that catalase was the main H2O2-

detoxifying enzyme [3,4,8,21], whereas other investigations

found that GPx was more efficient at H2O2 detoxification

compared to catalase [11,34]. One approach that can be used

to elucidate the physiological functions of antioxidant

enzymes in cells/organs is to modify their content. For

example, a controlled decrease in the amount of catalase

activity can be achieved by administering 3-amino 1,2,4-

triazole (AMT), an irreversible inhibitor of catalase [30].

Brain has intrinsically low to moderate activities of

catalase and GPx, whereas SOD is prevalent in normal brain

tissue [14,15,28,29]. Brain is an organ in which homeostasis

must be strictly maintained, based on a high dependence on

oxidative phosphorylation. Since a major source of ROS is

the leakage of electrons from the electron transport chain,

organs with a high dependence on ATP generation by

oxidative phosphorylation need effective ways to detoxify

O2� and H2O2. On the other hand, certain level of ROS is

needed as signaling molecules involved in normal function-

ing of brain, and they may modify the signal transduction

pathways [7]. This study aimed to investigate the effect of

catalase inhibition by AMT on the antioxidant enzyme

defenses and oxidative damage to proteins and lipids in

goldfish brain in order to clarify the role of catalase in the

protection of tissue proteins against ROS attack and

illuminate possible mechanisms that could substitute for

catalase function when the enzyme was inhibited.

2. Materials and methods

2.1. Chemicals

Phenylmethylsulfonyl fluoride (PMSF), butylated hydro-

xytoluene (BHT), yeast glutathione reductase (GR), 1-

chloro-2,4-dinitrobenzene (CDNB), reduced glutathione

(GSH), oxidized glutathione (GSSG), glucose-6-phosphate

(G6P), ethylenediamine-tetraacetic acid (EDTA), sodium

azide, and 3-amino 1,2,4-triazole (AMT) were purchased

from Sigma Chemical Co. (USA). N,N,NV,NV-tetramethyle-

thylenediamine (TEMED), NADP+, and NADPH were from

Reanal (Hungary), Tris–HCl was from Bio-Rad, and

guanidine–HCl was from Fluca. All other reagents were

of analytical grade.

2.2. Animals and experimental conditions

Goldfish (Carassius auratus L.) of both sexes weighing

20–70 g were purchased at a local fish market (Ivano-

Frankivsk, Ukraine) and were kept in dechlorinated tap water

and fed with standard fish food. Temperature was maintained

at 18 T 1 -C with a natural light–dark cycle with light from

about 8:00 am to 5:00 pm. Goldfish were acclimated to these

conditions for at least 1 month before experimentation.

For preliminary investigations of the effect of AMT on

catalase inhibition, fish were injected intraperitoneally with

AMT diluted in physiological saline (0.9% NaCl) at final

concentrations 0.1, 0.5, or 1.0 mg/g wet body weight (gww).

The volume of injected solution was 0.45% of body weight.

Controls were injected with 0.9% NaCl alone. Fish were

killed by transspinal dissection at 5, 10, 24, 48, 72, 120, or

168 h after the treatment. The brain was quickly removed

and used immediately to measure catalase activity.

For the next study, fish were injected with AMTsolution at

a final concentration of 0.5mg/gww (the volume injected was

0.3% of body weight). Another group of goldfish was treated

with the same volume of 0.9% NaCl. Control fish were not

treated. After 24 or 168 h, fish injected with AMT or NaCl

solutions were sampled, and brain tissue was processed

immediately to measure the parameters of interest.

2.3. Indices of oxidative stress

Tissue samples were homogenized (1:10 w/v) using a

Potter–Elvjeham glass homogenizer in 50 mM potassium

phosphate (KPi) buffer, pH 7.0, containing 0.5 mM EDTA

and a few crystals of PMSF, a protease inhibitor. A 250 Alaliquot of this homogenate was then mixed with 0.5 ml of

10% (final concentration) trichloroacetic acid (TCA) and

centrifuged for 5 min at 13,000 � g in an Eppendorf

centrifuge. Carbonylprotein (CP) levels were measured in

the resulting protein pellets, and thiobarbituric acid reactive

substances (TBARS) contents were assayed in the super-

natants using a spectrophotometer SF-46 (LOMO, USSR).

Carbonyl derivatives of proteins were detected by

reaction with 2,4-dinitrophenylhydrazine (DNPH) [20].

Resulting 2,4-dinitrophenylhydrazones were quantified

spectrophotometrically in guanidine chloride solution which

was used to solubilize protein pellets. The amount of CP in

the resulting supernatants was evaluated at 370 nm using a

molar extinction coefficient of 22 � 103 M�1 cm�1 [20].

The values were expressed as nanomoles of CP per protein

milligram in the guanidine chloride solution.

The decomposition of lipid hydroperoxides produces

low-molecular weight products, including malondialdehyde,

which can be measured by the TBARS assay [35].

Malondialdehyde and other aldehydes when boiled with

Fig. 1. The time course of catalase activity in brain of goldfish injected with

different concentrations of amino triazole (AMT): (A) 0.1 mg/gww, (B) 0.5

mg/gww, (C) 1 mg/gww. Each experimental point represents one fish,

whereas the control (zero time) value is a mean of three independent

determinations.

T.V. Bagnyukova et al. / Brain Research 1052 (2005) 180–186182

thiobarbituric acid at acid pH give a pink colored product

which can be assayed spectrophotometrically. Absorption

was measured at 535 nm, and a molar extinction coefficient

of 156 � 103 M�1 cm�1 was used for calculation of TBARS

concentration [35]. The values are expressed as nanomoles

of TBARS per gram wet weight of tissue (gww).

2.4. Assay of antioxidant enzyme activities

Tissue homogenates prepared as for TBARS/CP assay

were centrifuged at 4 -C for 15 min at 15,000 � g in a K-24

centrifuge (Germany). Supernatants were removed and used

for enzyme activity assays using a spectrophotometer SF-46

(LOMO, USSR). SOD activity was assayed as a function of

its inhibitory action on quercetin oxidation [2]. One unit of

SOD activity is defined as the amount of enzyme (per

milligram protein) that inhibits the quercetin oxidation

reaction by 50% of maximal inhibition. In our case, the

maximal inhibition was about 90%. Activities of catalase,

glutathione reductase (GR), and glucose-6-phosphate dehy-

drogenase (G6PDH) were measured as described previously

[2], and activities of selenium-dependent glutathione per-

oxidase (GPx) and glutathione-S-transferase (GST) were

measured as in [26]. One unit of catalase, GPx, GST, GR, or

G6PDH activity is defined as the amount of the enzyme that

consumes 1 Amol of substrate or generates 1 Amol of

product per minute; activities were expressed as interna-

tional units (or milliunits) per protein milligram.

2.5. Protein measurements and statistics

Protein concentration was measured by the Bradford

method with Coomassie Brilliant Blue G-250 [9] using

bovine serum albumin as a standard. Data are presented as

means T SEM. The analyzed parameters were virtually the

same for males and females, therefore, the data for both

sexes were combined. Statistical analysis was performed

using a Student’s t test. Inhibition values for SOD activity

were calculated using an enzyme kinetics computer program

[10]. Correlation analysis was performed using Excel

program software.

3. Results and discussion

3-Amino 1,2,4-triazole (AMT) is widely used to inhibit

catalase and thereby to investigate the physiological

functions of the enzyme [1,6,21,32]. In goldfish brain, the

inhibiting effect of AMT was dose- and time-dependent.

AMT at a concentration of 0.1 mg/gww gradually reduced

catalase activity over 72 h before reaching a minimum value

of ¨35% of control that was maintained over 168 h (Fig. 1,

curve A). At higher concentrations of AMT, 0.5 and 1 mg/

gww, a sharp reduction in activity to 20–40% of control

was seen within 5–10 h after the treatment, and then

catalase activity remained low over the remainder of the

experimental course (Fig. 1, curves B and C). Injection of

physiological saline in sham-treated fish did not influence

catalase activity. Analogous changes in catalase activity

were found in brain of two frog species, Rana ridibunda and

Rana esculenta, when treated with the same AMT doses

[27].

Catalase depletion by AMT treatment has been tested in

several different animal models; depletion typically leads to

oxidative stress and stimulates lipid peroxidation [11,14]. It

seems that this effect is tissue-specific. In frogs chronically

treated with AMT, the intensity of lipid peroxidation was

unchanged in liver and kidney [19,20], whereas brain, lung,

and heart showed much more marked changes with an

increase in TBARS levels of up to 3-fold [5,21]. In goldfish

brain, 24 h treatment with AMT (0.5 mg/gww) resulted in a

sharp, approximately 6.5-fold, increase in TBARS content

comparing to sham-injected group (Fig. 2A). However, this

accumulation was reversed after 168 h, suggesting that other

antioxidant defense mechanisms were switched on to

compensate for the reduced catalase activity and/or deal

with intensified lipid peroxidation. Another index of

oxidative stress, the content of carbonylproteins (CP),

showed a different pattern of response to catalase depletion.

The levels of CP in brain of both AMT-treated groups rose

by about 2.2- and 1.5-fold after 24 and 168 h, respectively,

when compared to respective sham-controls (Fig. 2B). It

should be noted that treatment with physiological saline

alone also resulted in increased brain CP levels at the 168 h

time point. However, even taking into account the last, one

can conclude that catalase inhibition led to progressive

protein oxidation over the experimental time course.

A disturbance to the balance of antioxidant defenses in

cells (such as by catalase depletion) may result in compen-

satory alterations by other components of antioxidant

defense including enzyme activities or the levels of low-

molecular weight antioxidants [24,32]. For example, long-

Fig. 3. The effect of AMT on the activities of antioxidant enzymes in

goldfish brain: (A) catalase, (B) glutathione peroxidase (GPx), (C)

glutathione-S-transferase (GST), (D) glutathione reductase (GR). Other

information as in Fig. 2.

Fig. 2. The effect of AMT on oxidative stress markers in goldfish brain: (A)

thiobarbituric acid-reactive substances, (B) carbonylproteins. Time (hours)

after injection is denoted as 24 and 168; ‘‘c’’ denotes injection with 0.9%

NaCl, ‘‘a’’ denotes injection with 0.5 mg/gww AMT in 0.9% NaCl. Data are

means T SEM, n = 5–7. Significant differences from the indicated groups

are P < 0.05.

T.V. Bagnyukova et al. / Brain Research 1052 (2005) 180–186 183

term inhibition of catalase resulted in elevated activities of

SOD and GR as well as increased levels of glutathione and

ascorbate in frogs, Rana perezi [22,23]. Treatment with

AMT increased GSH levels in rat brain [6], and both GSH

content and SOD activity were enhanced after depletion of

catalase with AMT in Musca domestica [1]. AMT injection

also caused some changes in antioxidant enzyme activities in

goldfish brain (Fig. 3). AMT injection (0.5 mg/gww) again

caused the expected decrease in catalase activity; levels fell

to 68 and 59% of respective sham-control values after 24 and

168 h, respectively. Oppositely, GPx and GST activities in

brain rose. GPx activity was not affected by 24 h treatment

with AMT, but activity rose by 46% after 168 h (Fig. 3B).

GST activity increased by 1.8-fold at 24 h post-injection

(Fig. 3C). GST activity also increased by a similar amount in

fish treated with 0.9% NaCl or AMTafter 168 h. The activity

of GR in brain of control fish was low (6.51 T 0.54 mU/mg

protein). Treatment with AMT caused a transient decrease in

GR activity to ¨67% of control after 24 h, although this

reduction was not significant compared to 24 h sham-

injected group. GR activity returned to near control values

after 168 h (Fig. 3D). The activity of SOD was 50.5 T10.8 U/mg protein in brain of control fish and did not

change significantly in the experimental groups (data not

shown). Similarly, G6PDH activity (7.85 T 0.27 mU/mg

protein in controls) was not affected by AMT treatment

(data not shown).

Correlation analysis suggested connections between CP

levels and the activities of some of antioxidant enzymes. A

strong inverse correlation between catalase activity and CP

content (R2 = 0.83) was seen; as catalase activity varied by

about 2-fold, CP levels were changed about 4-fold (Fig.

4A). This suggests that catalase depletion leads to a

dramatic rise in protein oxidation which is not apparently

compensated for by other H2O2-catabolyzing defenses.

Which specific proteins may be among the oxidized ones?

It is known that even antioxidant enzymes are sensitive to

Fig. 4. The relationships between catalase activity and carbonylprotein

levels (A) or glutathione-S-transferase (GST) activity (B). Each point

represents one experimental group (n = 5–7).

T.V. Bagnyukova et al. / Brain Research 1052 (2005) 180–186184

oxidative inactivation by different kinds of ROS [14,16,37].

For example, in budding yeast, Saccharomyces cerevisiae,

acatalasemic mutants had significantly lower G6PDH

activity than the wild type did. The activity of G6PDH

correlated positively with catalase activity and inversely

with CP levels. Hence, disruption of catalase genes resulted

in increased CP levels. G6PDH might be among the

oxidized proteins and, as a result, showed a loss of activity

[25]. In our study, catalase inhibition did not affect the

G6PDH activity in goldfish brain, but another enzyme, GR,

showed reduced activity after 24 h of AMT treatment when

levels of both oxidative damage products (TBARS, CP)

were highly increased. This could indicate that GR is among

cellular protein that is susceptible to oxidative damage. GR

has a cysteine residue in its active center which is easily

modified by some low-molecular weight products of lipid

oxidation (e.g. 4-hydroxy-2-nonenal or malonic dialde-

hyde), and this modification may lead to enzyme inactiva-

tion [36,37]. Reversed GR activity at 168 h post-injection

might be a consequence of enhancement of antioxidant

enzyme up-regulation and protein synthesis de novo. Hence,

the rebounded activity may reflect the exceeding of new

synthesis over enzyme inactivation. However, to clarify

which kinds of proteins might be oxidized, further inves-

tigations are needed.

Catalase inhibition in goldfish brain was accompanied by

enhancement of the activities of two glutathione metaboliz-

ing enzymes, GPx and GST. Activities of both enzymes

correlated inversely with catalase activity with high coef-

ficients of correlation, 0.60 and 0.85, respectively (Fig. 4B).

Both catalase and GPx can catabolyze H2O2, but GPx has

much higher affinity for H2O2 than catalase has [18],

suggesting that this enzyme may play a more important role

in vivo at low H2O2 concentrations whereas the role of

catalase increases under severe oxidative stress [17,32].

Clearly, the rise in GPx activity in goldfish brain could

compensate, at least partially, for the loss of catalase

activity. In addition, GPx catalyzes the decomposition of

some organic hydroperoxides including phospholipid per-

oxides in membranes, the activity which is relevant to

phospholipid hydroperoxide GPx [17,31]. Elevated GPx

activity after 168 h might be responsible for the reversal of

TBARS accumulation; TBARS were initially high after 24 h

of AMT treatment but fell again by 168 h possibly because

of an enhanced capacity for lipid peroxide decomposition.

The other enzyme of interest, GST, is involved in

detoxification of xenobiotics and aldehydic products of

lipid peroxidation, such as the fatty acid hydroperoxides, 4-

hydroxy-2-nonenal, and malondialdehyde [16]. GST con-

verts them to non-toxic species by conjugation with GSH

[14,17]. Catalase inhibition in goldfish brain might lead to

increased concentrations of H2O2 and result in increased

lipid peroxidation due to elevated production of hydroxyl

radicals via the Fenton reaction. Therefore, the elevation of

GST activity is possibly a response to elevated levels of

lipid peroxidation products and may be responsible for a

decrease in TBARS levels after 168 h of AMT treatment. A

tight connection of GST activity and TBARS levels has

been found earlier in hyperoxia-treated goldfish [28].

Compensatory activation of glutathione-metabolizing

enzymes as well as increased GSH levels have been

reported in several studies [1,6,23]. For example, in Chinese

hamster cells, elevated concentrations of hydrogen peroxide

led to increased activity of g-glutamylcysteine synthetase,

the enzyme that catalyzes the rate-limiting step of gluta-

thione synthesis [33]. The resulting rise in intracellular GSH

levels would be an adaptive response to oxidative stress.

GSH has a dual role in cells being a free radical scavenger

itself as well as a substrate for GPx and GST. In goldfish

brain, the level of total glutathione is rather high, ¨670

nmol/gww, only by about 20% lower than in kidney [26].

These relatively high concentrations suggest the importance

of this tripeptide in brain antioxidant defense.

It should be remembered that ROS including H2O2

accomplish a dual role in living systems. On one hand, they

can oxidize cell components and lead to, for example,

inactivation of certain enzymes. On the other hand, ROS are

known to be involved in oxygen sensing and signal

T.V. Bagnyukova et al. / Brain Research 1052 (2005) 180–186 185

transduction as second messengers. Thus, H2O2 is impli-

cated in up-regulation of antioxidant defenses via ROS-

sensitive transcriptional factors such as NF-nB, AP-1, etc.[16,19]. Moreover, H2O2 can serve a signaling molecule in

the oxygen response. It has been shown in HepG2 cells that

erythropoietin gene expression is under the control of O2-

dependent H2O2 production [13]. Clearly, data obtained by

us should be the result of two processes: possible

inactivation by elevated levels of hydrogen peroxide and

synthesis de novo of new molecules. Since posttranslational

modification of most antioxidant enzymes is unknown, the

registered rise of some enzyme activities might be due to

up-regulation of respective genes.

The changes in oxidative stress markers and antioxidant

defenses in goldfish brain after AMT treatment can be

divided into two phases, an ‘‘acute phase’’(24 h) and ‘‘phase

of adaptation’’(168 h). After 24 h of catalase inhibition,

oxidative stress had developed as confirmed by the

accumulation of protein and lipid oxidative damage

products. At the same time, GR activity decreased. After 7

days, TBARS levels had returned to control values,

indicating that compensatory protective mechanisms that

provide defense against lipid damage and/or repair damaged

lipids had been activated. On the other hand, CP continued

to accumulate over time. The activities of GPx and GST

rose in response to catalase depletion. These changes could

provide compensatory mechanisms for detoxifying H2O2 or

elevated amounts of hydroperoxides. However, these

enzyme changes were apparently not sufficient to protect

against protein oxidation and the accumulation of CP

occurred. In other words, this suggests that catalase is

needed under normal physiological conditions to protect

proteins from oxidation. It has its own specific functions in

the maintenance of tissue prooxidant/antioxidant balance

which cannot be completely compensated for by changes in

the activities of enzymes of the glutathione redox cycle.

Acknowledgments

We are grateful to J.M. Storey for critical reading of the

manuscript and to N. Borysevych, O. Chahrak, and L.

Luzhna for technical assistance. Special gratitude is given to

anonymous referees recommendations which helped to

improve the manuscript. Supported in part by a discovery

grant from the Natural Sciences and Engineering Research

Council of Canada to KBS.

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