Experimental Gerontology 34 (1999) 365–374

Effects of age and manganese (II) chloride on peroxidase
activity of brain and liver of the teleost,
Channa punctatus
S.B. Nayaka, B.S. Jenab, B.K. Patnaikb*
a

Government Science College, Chatrapur, Orissa, India
Department of Zoology, Berhampur University, Berhampur 760 007, Orissa, India

b

Received 2 October 1998; received in revised form 5 January 1999; accepted 7 January 1999

Abstract
Fish provide enormous spectrum of longevity and thus present the possibility of multiple
mechanisms of senescence. Oxidative stress as a causative agent of senescence and the protective
role of antioxidant enzymes were tested in the teleost, Channa punctatus taking peroxidase (POD)
(EC 1.11.1.7) as the representative enzyme. The activity of POD in brain and liver declined during
maturation phase (young vs middle-aged). During senescence phase (middle-aged to old) the
enzyme activity increased in liver but remained stabilized in brain. The degree of increase in
peroxidase activity following in vitro MnCl2 treatment was always higher in liver than in brain.
The rate of MnCl2 induced increase in POD activity of both tissues showed an increasing trend
with age. However statistical significance was observed only in brain during senescence phase. No
significant loss of enzyme activity in both the tissues and greater degree of increase by MnCl2 in
brain suggest that antioxidant capacity is not impaired in old murrels. © 1999 Elsevier Science
Inc. All rights reserved.
Keywords: Peroxidase; Liver; Brain; Age; Manganese (II) chloride; Murrel

* Corresponding author. Tel.: ϩ91-0680-282222; fax: ϩ91-0680-202322

0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 2 1 - 2

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1. Introduction
Aerobic organisms rely on oxygen for an efficient production of energy in the form of
ATP. But during normal physiological and metabolic processes that are essential to the cell,
oxygen is also the source of reactive oxygen species (ROS) which have deleterious effects
on vital structures and functions. ROS includes O2⅐ (Ϫ) and ⅐ OH free radicals, singlet oxygen,
and hydroperoxides. Aerobic organisms have antioxidant defenses to counteract the toxic
effects of ROS (Yu, 1994; Gille & Sigler, 1995). The free radical theory of aging envisages
that while the generation of ROS increases with advancing age, the protective defense
mechanisms weaken. As a result some ROS escape physiological scavenging, subsequently
inflicting molecular damage in lipids, proteins and nucleic acids. An accumulation of damage
with age cause functional derangement associated with aging (Harman, 1992).
The availability of oxygen differs between aquatic and terrestrial environments. The
requirement and consumption of oxygen also differ between aquatic and terrestrial species
of vertebrates. Similarly poikilothermic vertebrates have lower rates of oxygen consumption
(metabolic rate) than homeotherms. A strongly positive relationship exists between the rate
of oxygen consumption of aerobic animals and rate of generation of oxygen radicals in their
tissues (Davies et al., 1982; Sohal et al., 1989). Such circumstances demand that the free
radical theory of oxygen toxicity and the antioxidant defense mechanisms should be tested
in various phylogenetic groups to support the general theory and/or to indicate the deviations.
So far there are limited observations on the effect of age on antioxidant enzyme activities
of poikilothermic vertebrates (Patnaik, 1994). No significant change in activities of antioxidant enzymes (superoxide dismutase, SOD; catalase, CT; glutathione peroxidase, GSPx and
glutathione reductase, GR) was observed in the brain (Barja et al., 1990) and lungs (PerezCampo et al., 1993) of the aging frog, Rana perezi. Longitudinal studies in the same species
also indicated a similar trend in SOD and GR activities of liver (Lopez-Torres et al., 1993).
Catalase activity was found to increase with age in liver (Jena & Patnaik, 1992) and brain
(Jena et al., 1998) of the garden lizard. When mature rainbow trout and black bullheads were
compared with the immature counterparts, SOD and GR activities of liver and extrahepatic
tissues were found to be decreased in the former. On the other hand, whereas GSPx and CT
activities decreased in liver and kidney during maturation, the same parameters increased in
gill and muscle tissues (Otto & Moon, 1996). It appears that different organs have different
age-related patterns in antioxidant enzymes. The above cited examples suggest that agerelated trends in antioxidant enzymes of poikilothermic vertebrates in general and fishes in
particular are still unclear. Therefore, there is a need to conduct elaborate studies in different
species.
Fish provide enormous spectrum of longevity ranging from 2 to more than 150 years
(sturgeon, Acipenser fulvescens) and thus provide the possibility of multiple mechanisms of
senescence (Finch, 1990). The use of fish as experimental model for aging studies has many
advantages: the cost of rearing and maintenance is low, as they are poikilotherms the rate of
aging can be manipulated through temperature, in contrast to mammals where cessation of
growth is prerequisite for the appearance of senescence and many species show indeterminate growth, suggesting a different pattern of correlation between growth and aging (Woodhead, 1979).

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367

Channa punctatus, the Indian murrel has proved to be a suitable animal model for
gerontological studies (Mahapatro & Patnaik, 1993; Patnaik et al., 1994). The life span of
this species is short (5– 6 years). The age of the individuals can be determined easily. The
physiology of the species has been studied extensively. That the species undergoes senescence has been confirmed by other aging parameters, i.e. increase in mortality rate and
crosslinking of collagen (Gantayat & Patnaik, 1980), decrease in metabolism (Satapathy &
Patnaik, 1980), and increase in lipofuscin content (Nayak and Patnaik, unpublished data).
With an intention to make exhaustive studies on the status of oxidative stress and antioxidative enzymes in a single species the present study concerns estimation of peroxidase
activities in brain and liver and their regulation by manganese (II) chloride.

2. Materials and methods
2.1. Animals
Male murrels (Channa punctatus) have a short life span of 5– 6 years and their rate of
growth declines with age. The age of the individuals is accurately determined by counting
growth rings in scales and opercular bone (Qasim & Bhatt, 1966). While individuals without
a ring are considered as Ͻ1 year and with 1 ring as 1 year old (young), those with 2–3 rings
and 4 – 6 rings are 2–3 years (middle-aged) and 4 – 6 years old (old), respectively.
Live and healthy murrels were collected from the ponds of Berhampur localities during
May to July and maintained in aquaria (60 cm long ϫ 45 cm wide ϫ 45 cm high) at a water
temperature of 26 Ϯ 2°C. They were exposed to 12 h light/12 h dark cycle and fed with
adequate numbers of live earthworms on alternate days as described by Mahapatro and
Patnaik (1993). After at least two feedings the animals were used for the experiments.
2.2. Preparation of tissue homogenate and supernatant
Each fish was killed by stunning. The brain and liver were dissected out and immediately
transferred to separate beakers containing chilled potassium phosphate buffer (0.1 M, pH
7.0) to clear the adherent materials. The tissues were placed between the folds of Whatman
No. 1 filter paper to absorb the superficial fluid and then weighed. A 0.2% tissue homogenates were prepared in potassium phosphate buffer (0.1 M, pH 7.0) using a Potter-Elvehjem
type homogenizer with teflon pestle (Remi RQ 127A, Bombay). The tissue homogenates
were centrifuged at 3,500 rev./min for 10 min in a Hettich EBA III Centrifuge “Germany”
kept in a cold chamber maintained at 5– 8°C. The supernatants served as the crude enzyme
preparation for the assay of peroxidase activity.
2.3. Assay of peroxidase (EC 1.11.1.7) activity
The peroxidase activity was measured following the method of Wadhwa et al. (1988). In
the experimental set the reaction mixture included 2.0 ml of 0.1 M potassium phosphate
buffer (pH 7.0), 1.0 ml pyrogallol (0.005 M), 1.0 ml of a 0.05 M hydrogen peroxide “H2O2”

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Table 1
Morphometric parameters of male murrels (Channa punctatus) of three different age groupsa
Age group

Body weight
(g)

Y
(Ͻ1–1 yr)

18.6 Ϯ 8.8
(11)

MA
(2–3 yrs)

62.7 Ϯ 17.4
(11)

0
(4–6 yrs)

122.9 Ϯ 17.4
(11)

P

Ͻ0.001
Ͻ0.001

Length
(cm)
11.9 Ϯ 2.0
(11)
18.4 Ϯ 1.7
(11)
22.5 Ϯ 1.2
(11)

P

Ͻ0.001
Ͻ0.001

a
Here and in tables 2, 3 and 4 data represent mean Ϯ SD. Numbers in parentheses indicate number of animals
used. P refers to level of significance. Y, young; MA, Middle-aged; O, old.

solution and 1.0 ml supernatant (crude enzyme preparation) in a total volume of 5 ml. A
control was also run simultaneously without the pyrogallol solution and the volume was
adjusted with potassium phosphate buffer. The in vitro effect of MnCl2 was studied by
including 0.1 ml of a MnCl2 solution at a final concentration of 333 ␮M in another set of
assay mixture. To maintain the total volume as 5 ml in assay mixture containing metal salt
the volume of potassium phosphate buffer was adjusted accordingly. The reaction was
started by adding 1 ml of supernatant to each of experimental, metal salt-treated and control
sets. After 5 min at 25°C, the reaction was stopped by adding 0.5 ml H2SO4 (5% v/v). The
optical density was measured at 430 nm in a JASCO 7800 UV/visible spectrophotometer
(Japan). After suitable corrections with the control, the enzyme activity was expressed as
O.D. of purpurogallin formed/mg protein/5 min.
2.4. Protein content of supernatant
Protein precipitated from 0.1 ml of supernantant with an equal volume of 10% ice-cold
trichloroacetic acid was solubilized in a known volume of 0.1 N NaOH solution. The
quantity of protein in the sample was determined following the method of Lowry et al. (1951)
using a standard curve prepared with bovine serum albumin (Loba Chemie Indo-Austranal
Co., Bombay).
One-way analysis of variance (ANOVA) and Student’s t-tests were performed to evaluate
the statistical significance of the results.

3. Results
The morphometric parameters (body weight, g; length, tip of the snout-to-the end of the
longest caudal fin ray, cm) of the murrels showed positive correlations with advancing age
(Table 1). The basal enzyme activity was significantly higher in liver than in brain of all the
age-groups of fishes (Table 2). One way analysis of variance (ANOVA) indicated

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369

Table 2
Age and tissue differences in peroxidase activity (O.D. of purpurogallin formed/mg protein/5 min) in murrels
Age group
Y
(Ͻ1–1 yr)
MA
(2–3 yrs)
O
(4–6 yrs)

Enzyme activity
(Brain)

Enzyme activity
(Liver)

P between brain and liver of
corresponding age group

0.154 Ϯ 0.006
(8)
P Ͻ 0.001; Ϫ18.8%
0.125 Ϯ 0.016
(11)
P, NS; ϩ8.0%*
0.135 Ϯ 0.021
(9)

0.659 Ϯ 0.08
(6)
P Ͻ 0.02; Ϫ16.5%
0.550 Ϯ 0.072
(11)
P Ͻ 0.05; ϩ11.4%
0.613 Ϯ 0.044
(11)

Ͻ0.001
Ͻ0.001
Ͻ0.001

* NS, not significant at 0.05 level.

significant difference in POD activity among the three age groups of control [Brain, F (2,
24) ϭ 15.0, P Ͻ 0.001; Liver, F (2, 24) ϭ 8.7; P Ͻ 0.01] and MnCl2 treated samples
[Brain, F (2,24) ϭ 7.0; P Ͻ 0.01; Liver, F (2, 24) ϭ 11.0; P Ͻ 0.001]. The numbers in
brackets following F indicate degrees of freedom n1 and n2 and F represents variance
ratio between three age groups. The peroxidase (POD) activity decreased during maturation (young vs middle-aged) in both brain (18.8%, P Ͻ 0.001) and liver (16.5%, P Ͻ
0.02). While in liver there was a significant increase (11.4%, P Ͻ 0.05) in enzyme activity
during senescence phase (middle-aged to old); the parameter remained stabilized in brain
during that phase (Table 2).
3.1. In vitro effect of MnCl2 on POD activity
MnCl2 induced an increase in POD activity of both the tissues. But in a majority of cases
the degree of stimulation was significantly higher in liver than in brain of corresponding age
groups (Tables 3 and 4). The degree of increase of POD by MnCl2 showed an increasing
trend with advancing age. But statistical significance was observed only in brain during
senescence phase (middle-aged to old) (Table 4).

4. Discussion
H2O2 is decomposed by both catalase and peroxidases. But the types of peroxidase vary.
One type of selenium dependent glutathione peroxidase uses H2O2 as substrate and is known
as H2O2-GSHPx. There are also selenium-dependent and selenium-independent glutathione
peroxidases which use organic hydroperoxides like cumene-OOH as substrates (cumeneOOHGSHPx). The distribution of various types of peroxidases is both tissue and species
dependent (Igarashi et al., 1983). There has been much debate regarding the relative
importance of catalase and GSPx for detoxification of H2O2. Both are equally capable of
decomposing H2O2 (Flohe, 1982), GSPx is, effective at high H2O2 concentrations and
catalase at low H2O2 concentrations (Fridovich & Freeman, 1986). Experiments with

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Table 3
In vitro effect of manganese (II) chloride on the peroxidase activity (O.D. of purpurogallin formed/mg
protein/5 min) in brain and liver of male murrels of three different age groupsa
Age group

Enzyme activity
(Brain)

P between CB and
corresponding EB

CB1 0.154 Ϯ 0.006
(8)
Y
(Ͻ1–1 yr)

MA
(2–3 yrs)

O
(4–6 yrs)

Enzyme activity
(Liver)
CL1 0.659 Ϯ 0.08
(6)

Ͻ0.001

EB1 0.222 Ϯ 0.022
(8)
CB2 0.125 Ϯ 0.016
(11)

EL1 1.041 Ϯ 0.116
(6)
CL2 0.550 Ϯ 0.072
(11)

Ͻ0.001

EB2 0.177 Ϯ 0.026
(11)
CB3 0.135 Ϯ 0.021
(9)

EL2 0.914 Ϯ 0.134
(11)
CL3 0.613 Ϯ 0.044
(11)

Ͻ0.001

EB3 0.204 Ϯ 0.029
(9)

P between CL and
corresponding EL

Ͻ0.001

Ͻ0.001

Ͻ0.001

EL3 1.001 Ϯ 0.079
(11)

a

CB1, CB2, CB3 are controls for brain, CL1, CL2, CL3 are controls for liver, EB1, EB2, EB3 are MnCl2-treated
for brain and EL1, EL2, EL3 are MnCl2-treated for liver.

controlled decrease in enzyme activity by antibodies (Michiels et al., 1988) or chemicals
(Michiels & Remacle, 1988) and controlled increase in antioxidant activity through microinjection (Raes et al., 1987; Michiels et al., 1994) have consistently shown that GSPx is the
key enzyme of the antioxidant system under normal conditions and conditions of oxidative
stress. In the present investigation we have estimated the activity of peroxidase (POD;
Table 4
Age and tissue differences in the degree of increase (%) in POD activity by manganese (II) chloride

Age group

Brain

Y
(Ͻ1–1 yr)

44.9 Ϯ 15.6
(8)

MA
(2–3 yrs)

40.6 Ϯ 10.2
(11)

O
(4–6 yrs)

P, between age
groups

52.4 Ϯ 11.4
(9)

* NS, not significant at 0.05 level.

Liver

P, between age
groups

58.0 Ϯ 2.7
(6)
NS*
Ͻ0.05

66.2 Ϯ 11.2
(11)
63.0 Ϯ 5.7
(11)

P, between brain
vs. liver of
corresponding
age group
NS

NS

NS

Ͻ0.001
Ͻ0.02

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371

H2O2-oxidoreductase, EC 1.11.1.7), which includes peroxide-degrading enzymes both specific and nonspecific.
Even though brain was not included in their studies, the liver showed the highest activities
of catalase and glutathione peroxidase among the tissues studied in various species of fish
(Radi et al., 1985; Otto & Moon, 1996). In trout the activities of catalase and total glutathione
peroxidase were higher in liver than in brain (Perez-Campo et al., 1993). Our observations
also indicate higher POD activity in liver than in brain of murrels. Similar tissue differences
were reported in total GSPx activity of rat (Barja et al., 1990). The cause of such differences
could be due to high rate of free radical generation in liver than in brain. Moreover, even
though the brain is very susceptible to oxidative damage, it is not particularly enriched in
antioxidant enzymes (Giuffrida Stella & Lajtha, 1987; Rokyta et al., 1996).
The age changes in GSPx activity of different tissues of mammals showed an inconsistent
pattern. They did not show a significant change in brain of aging rat (Carrillo et al., 1992;
Nistico et al., 1992). Hepatic GSPx was shown either to increase (Sastre et al., 1992) or to
decrease (Matsuo et al., 1992) with age in rats. While in an earlier report we observed
insignificant age changes in brain catalase activity of murrel (Jena et al., 1998), the present
results indicate a decline in POD activity during maturation phase (young vs middle-aged)
only. This could be correlated with increased lipid peroxidation during that period (unpublished data) and enhanced H2O2 generation due to growth of brain tissue (Vertechy et al.,
1993). In liver a similar trend of decrease in POD activity and increase in lipid peroxidation
(unpublished data) existed for the maturation phase but whereas the enzyme activity increased during the senescence phase the lipid peroxidation remained unchanged. Our observation that POD activity declines during maturation phase is similar to the findings of Otto
and Moon (1996) regarding hepatic GSPx activity. It appears from the available literature
that, at least in brain, changes in catalase and peroxidase activities of fishes are mostly
maturation-related rather than senescence-related. Further studies on other species of fish are
needed to confirm senescent changes in antioxidant enzymes.
In addition to findings in specific activity the study of the regulatory changes in enzyme
is an important aspect. Our previous findings indicated that the degree of inhibition of brain
catalase by manganese chloride increased with age in murrels (Jena et al., 1998). In contrast,
we report here that the degree of increase of brain POD activity increased with age more
particularly during the senescent phase (middle-aged to old). The degree of increase in
hepatic POD activity was not age-dependent. The interesting finding is that while one
enzyme (catalase) required for the detoxification of H2O2 is inhibited by MnCl2 the other
enzyme (POD) is activated by the same metal salt and the degree of both the changes are
age-dependent. It seems that both the enzymes operate in a cooperative manner in that while
one is inhibited the other one is activated to eliminate H2O2. That antioxidant enzymes act
cooperatively and even in synergy has been suggested earlier (Michiels et al., 1994).
Inhibition of GSH cycle enzymes by BSO or BCNU but not the inhibition of catalase by
aminotriazole decreased the resistance of endothelial cells to free radicals (Suttorp et al.,
1986). Depending on the concentration, MnCl2 is known to have both protective and toxic
effect. Mn(II) is reported to be a scavenger of superoxide radical (Archibald & Fridovich,
1982). It reduces ⅐ OH to yield Mn(OH)2ϩ (Chang and Kosman, 1989) and activates a variety
of enzymes (West et al., 1974) including horse radish peroxidase (McEldoon & Dordick,

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1991). On the other hand, prolonged Mn(II) exposure induces the formation of reactive
oxygen species leading to impairment of antioxidant system (Desole et al., 1995). But it
appears that the antioxidant enzymes respond to the effects of Mn(II) differently comparable
to the results obtained with other metal ions. Although on one hand exposure to increase in
CuSO4 concentration (5, 10, 25, and 50 ppm) caused a decrease in catalase activity of liver,
on the other hand it stimulated GSPx activity in the same tissue of carp (Radi & Matkovics,
1988). Similarly, in response to trichlorobiphenyl (TCB), catalase activity declined and
GSPx activity increased in the liver of rainbow trout (Otto & Moon, 1995).
From our earlier and present studies it is observed that there is no decrease in the activities
of two enzymes, i.e. catalase and peroxidase involved in detoxification of H2O2 indicating no
weakening of antioxidant capacity in old murrels. Moreover, the MnCl2 inhibition of catalase
activity was compensated by simultaneous induction of POD activity thereby maintaining the
detoxification of H2O2.

Acknowledgments
The authors thank CSIR (New Delhi) for awarding a Research Associateship to B.S.J. and
authorities of Berhampur University for providing laboratory facilities.

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