RESEARCH ARTICLE

Effect of dehydroepiandrosterone (DHEA) on monoamineoxidase activity, lipid peroxidation and lipofuscinaccumulation in aging rat brain regions

Pardeep Kumar Æ Asia Taha Æ Deepak Sharma ÆR. K. Kale Æ Najma Z. Baquer

Received: 6 November 2007 / Accepted: 11 February 2008 / Published online: 29 February 2008

� Springer Science+Business Media B.V. 2008

Abstract Dehydroepiandrosterone (DHEA), one of

the major steroid hormones, and its ester have

recently received attention with regard to aging and

age-related diseases like Alzheimer and others.

DHEA is synthesized de novo in the brain and its

substantial fall with age has been shown to be

associated with neuronal vulnerability to neurotox-

icity processes. Thus, DHEA is considered to be a

neuroactive pharmacological substance with potential

antiaging properties. A prominent feature that accom-

panies aging is an increase in monoamine oxidase

(MAO). Increased MAO activity with correlated

increase in lipid peroxidation in the aging rat brain

supports the hypothesis that catecholamine oxidation

is an important source of oxidative stress. The

progressive accumulation of lipofuscin in neuronal

cells is one of the most characteristic age related

changes, an increase in body weight was also

observed at 24 months. The objective of this study

was to observe the changes in monoamine oxidase

activity, lipid peroxidation levels and lipofuscin

accumulation occurring in aging rat brain regions,

and to see whether these changes are restored to

normal levels after exogenous administration of

DHEA (30 mg/kg/day for 1 month). The results

obtained in the present work revealed that normal

aging was associated with significant increases in the

activity of monoamine oxidase, lipid peroxidation

levels and lipofuscin accumulation in brain regions of

4, 14 and 24 months age group male rats. The present

study showed that DHEA treatment significantly

decreased monoamine oxidase activity, lipid perox-

idation and lipofuscin accumulation in brain regions

of aging rats, the increased body weight at 24 months

also decreased more than the age matched controls. It

can therefore be suggested that DHEA’s beneficial

effects seemed to arise from its antioxidant, antiobe-

sity, antilipofuscin, antilipidperoxidative and thereby

anti-aging actions. The results of this study will be

useful for pharmacological modification of the aging

process and development of new drugs for age related

disorders.

Keywords Aging � Brain �Dehydroepiandrosterone (DHEA) �Lipid peroxidation � Lipofuscin �Monoamine oxidase

Abbreviations

AD Alzheimer’s disease

CNS Central nervous system

DHEA Dehydroepiandrosterone

P. Kumar � A. Taha � D. Sharma � N. Z. Baquer (&)

Neurobiology Laboratory School of Life Sciences,

Jawaharlal Nehru University, New Delhi 110067, India

e-mail: nzbaquer@hotmail.com; nzbaquer@gmail.com

P. Kumar � R. K. Kale

Cancer and Radiation Biology Laboratory, School of Life

Sciences, Jawaharlal Nehru University, New Delhi

110067, India

123

Biogerontology (2008) 9:235–246

DOI 10.1007/s10522-008-9133-y

DHEAS Dehydroepiandrosterone-sulphate

DMSO Dimethylsulphoxide

GABA Gamma-aminobutyric acid

MAO Monoamine oxidase

MDA Malondialdehyde

NMDA N-methyl-D-aspartate

PD Parkinson’s disease

ROS Reactive oxygen species

TBARS Thiobarbituric acid reactive substance

Introduction

Aging is one of the biological processes shared by all

living organisms. The intricate causes of the aging

process are still a matter of extensive speculation

giving rise to many theories, in particular, the role of

reactive oxygen species (ROS) is nowadays prere-

quisite in understanding this process (Abrass 1990).

Although no single theory has been generally

accepted, the free radical theory of aging by Harman

(1993), predicts the popular hypothesis that the rate

of aging is dependent on the level of oxidative status

i.e. the balance between pro-oxidants and anti-

oxidants and the consequent oxidative damage.

Dehydroepiandrosterone (DHEA, 3\beta[-

hydroxy-5-androsten-17-one) and its sulphate ester

(DHEAS) are the most abundant steroid hormones in

human circulation, with 90–95% of the overall

production originating from synthesis by the steroi-

dogenic enzyme P450c17 within the adrenal zona

reticularis. DHEA secretion exhibits a characteristic

age-associated pattern (Reiter et al. 1977; Orentreich

et al. 1984). DHEA, DHEAS and pregenenolone are

known to be synthesized by the brain and are

considered as neurosteroids (Racchi et al. 2003).

Besides, DHEA is considered to be the youth

hormone (Celec and Starka 2003) (Fig. 1). DHEA

influences neuronal activity via interaction with

neurotransmitter receptors including N-methyl-D-

aspartate (NMDA), sigma and gamma-aminobutyric

acid (GABA) receptors (Bergeron et al. 1996),

thereby suggesting a putative anti-depressant action

for DHEA. DHEA is also capable of preventing many

age-dependent morphological, physiological and

behavioral alteration in the central nervous system

and has therefore been considered to be a neuroactive

pharmacological substance with potential antiaging

properties (Baulieu and Robel 1996; Wolf and

Kirschbaum 1999). DHEA has been shown to be

antiobese (Yen et al. 1977) and to have effect on

longevity (Lucas et al. 1985), improvement in lipid

profiles and protection against development of ath-

erosclerosis, osteoporosis (Labrie et al. 1997) and

modulation of immunological mechanisms (Khorram

et al. 1997) Moreover, DHEA has been shown to

protect hippocampal neurons against neurotoxin

induced cell death (Cardounel et al. 1999). DHEA

effects also include the modulation of NMDA

receptor functions (Baulieu and Robel 1996; Weaver

et al. 1997), the preservation of calcium homeostasis

and antioxidant activities, mainly by scavenging

reactive oxygen species (ROS) (Vedder et al. 1999;

Boccuzzi et al. 1997).

A prominent feature that accompanies aging is an

increase in monoamine oxidase, an enzyme respon-

sible for the metabolism of biologically important

active amines and oxidative deamination of these

amines produces ammonia and hydrogen peroxide

with potential toxicity. Regulation of this enzyme is

very important for normal neuronal activity. High

level of MAO has been linked to depression and to

Parkinson’s disease (Hauptmann et al. 1996). The

MAO enzyme has a crucial role in neurophysiology

since it inactivates neuro-transmitter monoamines

like dopamine, noradrenaline and serotonin. There

Fig. 1 Structure of Dehydroepiandrosterone and its sulfate

236 Biogerontology (2008) 9:235–246

123

are two enzymatic scavenging systems, catalase and

glutathione peroxidase to protect cells from the

presence of hydrogen peroxide, the levels of these

latter two enzymes however are low in brain com-

pared to other tissue (Marklund et al. 1982; Genet

et al. 2002). In addition, the hydrogen peroxide

generated by mitochondrial monoamine oxidase does

not easily reach the cytosolic catalase compartment.

These facts make catecholaminergic and serotonergic

neurons particularly vulnerable to the oxidative stress

caused by increased MAO activity (Sinet et al. 1980).

Several authors reported high brain MAO-B activity

in neurodegerative disease such as PD and AD

without any changes in MAO-A enzyme activity

(Benedetti and Dostert 1989; Saura et al. 1994).

Monoamine oxidase occurs at least in two forms,

MAO-A and MAO-B, with different specificities for

substrates and inhibitors. MAO A normally occurs in

adrenergic, noradrenergic and, in most cases, in

dopaminergic neurons, while MAO B is unexpectedly

predominant in serotoninergic neurons (Jahng et al.

1997). MAO A preferentially degrades serotonin

(5-HT), adrenaline and noradrenaline (NE), while

MAO B displays greater affinity for phenylethyl-

amine (PEA) and benzylamine (Fowler and Tipton

1984). Dopamine and tyramine are considered a

substrate for both MAO forms. The selective inhib-

itor of MAO-A is clorgyline. In contrast, the selective

inhibitor of MAO-B is deprenyl. There is also

evidence that MAO-B inhibitors improve the quality

of life in the elderly (Knoll 1993).

Malonaldialdehyde is one of the end products in

the lipid peroxidation process (Hagihara et al. 1984).

Earlier studies have revealed that there is an increase

in the lipid peroxides of aged liver and brain

homogenates (Miyazawa et al. 1993). Lipid peroxide

levels were found to be significantly higher in brains

of 18 months old as compared to 4 months old rats

(Noda et al. 1982; Moorthy et al. 2005).

Lipofuscin is a morphological structural entity and

is mainly accumulated in post-mitotic cells of brain.

The accumulation of lipofuscin in cells occurs

because it is undegradable and cannot be removed

from the cells via exocytosis. As age progresses,

the lipofuscin content per neuron as well as the

number of pigmented neurons has been shown to

increase in a linear fashion in many regions of the

brain (Sharma et al. 1993; Drach et al. 1994; Moorthy

et al. 2005). Intraneuronal accumulation of lipofuscin

is considered to be a marker of neuronal aging, and its

formation appears to be integrative and proportional

to the occurrence of lipid peroxidation. In addition,

an age-related increase in lipid peroxidation has been

shown to be directly correlated with the gross level of

lipofuscin accumulation (Sharma et al. 1993). Thus,

intraneuronal accumulation of lipofuscin is consid-

ered to be a marker of neuronal aging (Sohal and

Brunk 1989).

Though the literature available on DHEA may

have gathered a lot of knowledge on its beneficial

influence on immunological, cardiovascular, athero-

genic and kidney disorders, the reports suggesting

the anti-aging capabilities of DHEA in brain are

still preliminary and need extensive experimental

verification, especially in the context of normal

aging in animal models (Khorram et al. 1997).

Thus, the aim of this study was to investigate the

effects of exogenous administration of DHEA on

the following age-related parameters, namely body

weight, MAO activity, lipid peroxidation and

intraneuronal deposits of fluorescence contents, i.e.

lipofuscin (age pigment), in different brain regions

of 4, 14 and 24 months age groups in control and

DHEA treated rats. Since, brain regions may differ

in their vulnerability to aging (Cardozo-Pelaez et al.

2000), in the present work DHEA effect has been

observed in cerebral hemispheres, cerebellum and

brain stem, which are known to have different age

sensitivities.

Materials and methods

Animals

Male albino rats of the Wistar strain of different ages

namely 4, 14, and 24 months (n = 6 for each age

group) were used for all the experiments. Animals

were maintained in the animal house facility of

Jawaharlal Nehru University, New Delhi, India, at a

constant temperature of 25�C, humidity of 55% and

12 h dark and 12 h light cycle (light from 06:00 to

18:00 h). The animals were fed standard chow rat

feed (Hindustan lever Ltd., India) and water ad

libitum. All animal experiments were approved by

the JNU-Institutional Animal Ethics Committee; all

the institutional guidelines were adhered to in the

care and treatment of the animals.

Biogerontology (2008) 9:235–246 237

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Hormone administration

Animals of different age groups 4, 14 and 24 months

old were given intraperitoneal injection of DHEA

dissolved in dimethylsulphoxide (DMSO) at a dose of

30 mg/kg/day for 1 month (n = 6 for each group).

Control rats (n = 6 for each group) of age groups 4,

14 and 24 months were injected with the same

amount of the DMSO (vehicle). There was no

treatment on the day of the sacrifice. After 30 days

of hormone treatment experimental animals of all the

groups were sacrificed and brain regions were

isolated for further study. Dose of DHEA was based

on the studies of Garcia et al. 1995; Wen et al. 2001;

Flood et al. 1988 and as used by our group earlier

(Sinha et al. 2005). Only male rats were used because

we did not want to deal with the variations that occur

in females due to cycling estrogen levels and results

from one gender can be applicable to other.

Preparation of homogenate and separation

of subcellular fractions

Animals were sacrificed by cervical dislocation.

Whole brain was rapidly excised, and washed with

chilled normal saline. Tissue homogenates (1:10) of

the cerebral hemispheres were prepared as described

by Mayanil et al. (1982). The homogenizing medium

contained 0.25 M sucrose supplemented with

0.12 mM dithiothreitol buffered with 0.02 M trieth-

anolamine hydrochloride buffer, pH 7.4. All the

procedures were carried out at 4�C. Homogenates

were centrifuged at 1,000 rpm for 10 min to remove

nuclei and cell debris. The supernatant obtained was

further centrifuged at 12,000 rpm for 45 min on

SORVALL 5 CB refrigerated centrifuge. The super-

natant fraction was separated from the pellet and was

used as the soluble fraction. The pellet obtained,

containing crude synaptosomes (mitochondria and

synaptosomes) was washed twice and resuspended in

the same volume of the homogenizing medium. The

synaptosomal and the supernatant fractions were used

for determination of monoamine oxidase activity.

Synaptosomes, the isolated terminal portions of axons

that behave as metabolically autonomous mini-nerve

cells, provide a good experimental model to evaluate

neural degenerative processes and peroxidative events

in cerebral hemispheres. Synaptosomal preparation

preserves the functional activity of presynaptic

terminals thus they have proven very useful in the

study of various synaptic events, including uptake,

storage, synthesis and release of neurotransmitters.

Assay of monoamine oxidase (MAO)

The MAO activity was measured in synaptosomal

and supernatant fractions isolated from 4, 14 and

24 months old rat cerebral hemispheres in control and

DHEA treated rats. Monoamine Oxidase was deter-

mined according to the method of (Catravas et al. 1977)

as modified by Mayanil et al. (1982). Homogenates

were treated in cold with triton X-100 (0.5% final

concentration for 30–40 min). The assay mixture

(1 ml) contained the following in the final concentra-

tions, Tris/HCl: 0.05 mM (pH 7.4); Kynuramine

dihydrobromide: 0.22 mM; MgCl2: 0.08 mM and

150–200 lg of enzyme protein. The reaction was

started by adding the substrate, kynuramine and

incubated at 37�C for 90 min. The reaction was stopped

by the addition of 65 ll of 0.5 M-NaOH and 150 ll of

10% of ZnSO4. The mixture was heated in a boiling

water bath for 10 min and centrifuged at 10,000 rpm

for 10 min using a microfuge. The amount of reaction

product, 4-hydroxyquinoline formed was determined

spectrophotometrically in the supernatant against a

standard curve of the product formed by measuring the

increase in absorbance at 330 nm. The reagent blank

was prepared by replacing kynuramine with water. One

unit of enzyme is defined as one lmole of 4-hydroxy-

quinoline produced per mg protein per min at 37�C.

Measurement of lipid peroxidation

The formation of lipid peroxides was measured in the

whole homogenate of the cerebral hemispheres,

cerebellum and brain stem. The formation of mal-

ondialdehyde (MDA) an end product of fatty acid

peroxidation was measured spectrophotometrically at

532 nm by using a thiobarbituric acid reactive

substance (TABRS) essentially by the method of

Genet et al. 2002. Results are expressed as nmole of

MDA formed/mg protein.

Histological localization and distribution

of lipofuscin

Intraneuronal Lipofuscin accumulation in the cerebral

hemispheres, cerebellum and brain stem was

238 Biogerontology (2008) 9:235–246

123

observed in 5 micron thick paraffin embedded,

deparaffinised sections according to the method

described by Riga and Riga (1974) as used by Bala

et al. (2006) by fluorescence microscopy using a

Zeiss Orthomat microscope equipped with fluores-

cence attachments with Ploemipak Epi illuminator,

H2 cube (wide band), and exciter filter 390–490 nm

was used.

Protein estimation

Protein was estimated in the whole homogenate and

subcelluar fractions by the method of Bradford

(1976) using bovine serum albumin (BSA) as a

standard.

Statistical analysis

All data were calculated as means ± SEM of 4–6

separate values. The body weight, brain weight and

protein concentration were analyzed using one-way

ANOVA test followed by Turkey–Kramer multiple

comparison tests. The specific activity of enzyme and

MDA levels in brain regions were analyzed using

two-way ANOVA followed by post hoc bonferroni

test to determine the statistical comparison between

control and various experimental groups with differ-

ent ages. Levels of significance were evaluated with

P-values.

Chemicals

Kynuramine dihydrogen bromide, 4-hydroxyquino-

line and thiobarbituric acid were purchased from

Sigma Chemicals Company, USA. All other chem-

icals were of analytical grade and were brought from

SRL and Qualigen.

Results

General parameters

The changes in general parameters like body and

brain weight, and protein concentration in whole

homogenates, synaptosomal and supernatant fractions

of brain regions from different age groups namely 4,

14 and 24 months control and DHEA treated rats are

presented in Table 1.

The body weight increased in the control of

different groups with aging when compared to

4 months rats. DHEA treatment lead to a significant

decrease in body weight by nearly 30% (P \ 0.001)

at 24 months rats, compared to age matched control

rats. No significant changes were however, seen in

the 4 and 14 months DHEA treated rats. There were

no significant changes in brain weight when com-

pared to age matched control animals in all groups.

In different regions of brain, the protein content

was increased with age as compared to the 4 months

Table 1 Body weight, brain weight and protein concentration of 4, 14 and 24 months of control and DHEA treated aging male rats

General parameters Age in months/treatments

4 14 24

Control DHEA Control DHEA Control DHEA

Body wt (g) 338.6 ± 4.3 330.6 ± 6.2 491.2 ± 18.4 462.2 ± 10.1 618.5 ± 10.0 436.1 ± 10.4a

Brain wt (g) 1.59 ± 0.01 1.64 ± 0.02 1.71 ± 0.02 1.73 ± 0.01 1.79 ± 0.02 1.81 ± 0.03

Protein (mg/g)

Cerebral hemisphere WH 32.75 ± 1.6 35.12 ± 1.1 31.62 ± 1.4 38.87 ± 1.4c 42.06 ± 1.9 48.94 ± 1.8c

Synaptosomes 14.5 ± 1.5 15.1 ± 2.1 19.5 ± 2.5 22.2 ± 2.9 17.0 ± 2.7 18.2 ± 1.4

Supernatant 19.3 ± 1.9 20.7 ± 1.3 28.14 ± 2.2 29.1 ± 1.8 30.6 ± 2.7 33.2 ± 1.9

Cerebellum WH 30.56 ± 1.7 34.87 ± 1.8 33.89 ± 1.5 39.98 ± 1.9 41.91 ± 1.7 44.65 ± 1.5

Brain stem WH 30.43 ± 1.5 34.90 ± 1.7 34.77 ± 1.7 38.23 ± 1.3 41.47 ± 1.3 48.67 ± 1.7

Each value is a mean of ± SEM of five or more separate experiments. The comparison of each treated group is with the age-matched

control value. Statistical analysis is done by one-way ANOVA followed by Turkey–Kramer multiple comparison tests. P values areaP \ 0.001, bP \ 0.01, cP \ 0.05. DHEA—Dehydroepiandrosterone, WH—Whole homogenate

Biogerontology (2008) 9:235–246 239

123

old animals. In 14 months DHEA treated rats there

was a significant increase in protein concentration in

whole homogenate from cerebral hemisphere by 18%

(P \ 0.05) and 17% (P \ 0.05) in cerebellum as

compared to control groups. In 24 months DHEA

treated age group rats there were significant changes

in the protein content in cerebral hemispheres and

brain stem by 15% (P \ 0.05) and 16% (P \ 0.05)

respectively when compared with age matched con-

trol groups. There was no significant change in

protein content in all age groups of rats in synapto-

somal and supernatant fractions before and after

DHEA treatment.

Monoamine oxidase (MAO)

The changes in activity of MAO measured in

synaptosomal and supernatant fractions of cerebral

hemisphere in control and hormone treated rat brains

of different age groups i.e. 4, 14 and 24 months old

male rats showed significant increases in enzyme

activity with increasing age from 4 to 24 months.

Results are shown in Table 2.

In synaptosomal fraction, MAO activity was

increased significantly with age as compared to the

young (4 months old) in control and DHEA treated

animals. In 4 months old DHEA treated rat groups

there was no significant changes in MAO activity in

the synaptosomal fraction compared to 4 months

controls rats. In 14 months old DHEA treated groups

there was significant decrease in MAO activity by

18% (P \ 0.01) compared to 14 months controls rats.

In 24 months DHEA treated rat groups there was a

significant decrease in MAO activity by 44%

(P \ 0.001) compared to 24 months controls rats.

Results are shown in Table 2.

In the supernatant fraction, MAO activity signi-

ficantly increased with age as compared to the young

(4 months) in control and DHEA treated animals. In

4 months DHEA treated groups there were no

significant changes in MAO activity compared to

4 months control rats. In 14 months DHEA treated

groups there was a significant decrease in MAO

activity by 13% (P \ 0.05) compared to 14 months

control rats. At 24 months of DHEA treated rat

groups showed a significant decrease in MAO

activity, 48% (P \ 0.001) compared to 24 months

control rats. Results are shown in Table 2.

Age and regional variations in malondialdehyde

(MDA) levels and effect of DHEA treatment

The malondialdehyde (MDA) levels as a measure-

ment of lipid peroxidation in the whole homogenate

of the different age group male rats i.e. 4, 14 and

24 months control and DHEA treated animals in

different brain regions and results are given as nmole

MDA/mg protein. In different regions of the brain,

the MDA levels were increased significantly with age

as compared to the young (4 months) in control and

DHEA treated animals. The results are presented in

Table 3. As can be seen from Table 3 there was a

regional variation in the malondialdehyde levels in

brain regions with brain stem showing the lowest

levels and this pattern of decrease was also seen with

aging i.e. at both 14 and 24 months age groups.

In cerebral hemispheres, there was no significant

change in MDA levels in the 4 months DHEA treated

Table 2 Monoamine oxidase activity in synaptosomal and supernatant fractions in cerebral hemispheres of 4, 14 and 24 months of

control and DHEA treated aging male rats

Enzyme activity (U/mg protein/min)

Age in months/

treatments

Synaptosomal fraction Supernatant fraction

Control DHEA Control DHEA

4 0.317 ± 0.034 0.266 ± 0.033 0.268 ± 0.012 0.242 ± 0.012

14 0.927 ± 0.04 0.604 ± 0.02b 0.763 ± 0.012 0.606 ± 0.010c

24 1.29 ± 0.061 0.598 ± 0.015a 0.977 ± 0.082 0.698 ± 0.011a

Each value is a mean of ± SEM of five separate experiments. The comparisons of DHEA values are with the control values. Fisher’s

P values are aP \ 0.001,bP \ 0.01, cP \ 0.05. One unit of enzyme activity is defined as one lmole of 4-hydroxyquinoline produced

per mg protein per minute at 37�C

240 Biogerontology (2008) 9:235–246

123

rats when compared to age matched controls. In

14 months DHEA treated rat groups there was a

significant decrease in MDA levels by 35%

(P \ 0.01) when compared with respective control

rat groups. In 24 months DHEA treated rat groups

there was a significant decrease in MDA levels by

54% (P \ 0.001) when compared with the respective

controls. The results are presented in Table 3.

In cerebellum, there was no significant change in

MDA levels in the 4 months DHEA treated rats when

compared with age matched controls. In 14 months

DHEA treated rat groups there was a significant

decrease in MDA levels by 15% (P \0.01) when

compared with respective control rat groups. At

24 months, DHEA treated rat groups there was a

significant decrease in MDA levels by 52%

(P \ 0.001) when compared with respective control

age group. The results are presented in Table 3.

In brain stem, there was no significant change in

MDA levels in the 4 months DHEA treated rats when

compared to age matched controls. In 14 months

DHEA treated rat groups there was a significant

decrease in MDA levels by 21% (P \ 0.05) when

compared with the respective control age group. At

24 months, DHEA treated rat groups there was a

significant decreases in MDA levels by 29%

(P \ 0.001) when compared with the respective con-

trol age group. The results are presented in Table 3.

Effect of DHEA treatment on lipofuscin

accumulation

Deposition of lipofuscin in control and DHEA treated

male rats namely 4, 14 and 24 months in cerebral

hemisphere, cerebellum and brain stem are presented

in Fig. 2A and B, respectively. The lipofuscin content

in neurons was visualized in different regions of

aging rat brain. There was an age related increase in

the number of lipofuscin containing neurons and a

decrease in the non-pigmented neurons. In 4 months

age group control rats very small amount of lipofus-

cin deposition can be visualized in both DHEA

treated and control rats. In 14 months age groups rats

DHEA treatment decreased the lipofuscin deposition

when compared with respective control age groups.

The treatment with DHEA in 24 months old age

group rats was more effective in reducing the

lipofuscin deposition when compared to other age

groups.

Discussion

A vast number of evidences implicate that aging is

associated with a decrease in antioxidant status and

that age-dependent increase in lipid peroxidation is a

consequence of diminished antioxidant protection

(Schuessel et al. 2006). It has been shown that the

concentration of DHEA, a neurosteroid ‘‘antiaging’’

hormone, declines with aging in the brain (Kazihnit-

kova et al. 2004). In the present study, in vivo DHEA

administration was tested for its tentative neuropro-

tection and antioxidative role.

Lhullier et al. (2004) demonstrated that in vitro

effect of DHEA on synaptosomal glutamate release

depends on the age of rats, it decreases the basal

glutamate release from synaptosomes of old rats

(12 months), with no effect on young rats (17 days).

Table 3 Malondialdehyde (MDA) levels in whole homogenates of 4, 14 and 24 months of control and DHEA treated aging male rat

brain regions

Age in months/

treatment

Brain regions

Cerebral hemisphere Cerebellum Brain stem

nmoles of MDA/mg protein

Control DHEA Control DHEA Control DHEA

4 0.317 ± 0.034 0.266 ± 0.033 0.284 ± 0.015 0.274 ± 0.026 0.268 ± 0.012 0.242 ± 0.012

14 0.927 ± 0.04 0.604 ± 0.02b 0.919 ± 0.029 0.785 ± 0.017b 0.763 ± 0.012 0.606 ± 0.010c

24 1.29 ± 0.061 0.598 ± 0.015a 1.15 ± 0.057 0.554 ± 0.053a 0.977 ± 0.082 0.698 ± 0.011a

Each value is a mean of ± SEM of five separate experiments. The comparisons of DHEA values are with the control values.

Statisitical analysis is done by one-way ANOVA followed by Turkey–Kramer multiple comparison tests. P-values areaP \ 0.001,bP \ 0.01, cP \ 0.05

Biogerontology (2008) 9:235–246 241

123

DHEA and DHEAS (100 nm) can prevent/reduce the

neurotoxicity of NMDA, both in vitro and in vivo

models (Kimonides et al. 1998).

DHEA hormones act as signals on the reproductive

system and on the nutritional state of the animals.

The present results showed that the increased body

weight of the animals was decreased in DHEA treated

24 months age group male rats when compared to

their respective control groups. This could be due to

the decreased level of the hormone in the aging

animals, or due to the inhibition of de novo synthesis

of lipid and decreased functioning of glucose 6

phosphate dehydrogenase enzyme, which is essential

for the synthesis of fat from glucose (Berdanier et al.

1993). Cleary (1991) reported evidence concluding

that DHEA affects mitochondrial respiration leading

to the weight loss.

Monoamine oxidase activity in the brain is

involved in the catabolism of several neurotransmit-

ters such as dopamine, noradrenaline and serotonin,

Fig. 2 The lipofuscin

accumulation (yellow

autofluoresence shown by

white arrowheads) in

cerebral hemispheres,

cerebellum and brain stem

of 4, 14 and 24 months

aging male rats. (A)

Control, (B) DHEA treated

242 Biogerontology (2008) 9:235–246

123

accompanied by the reduction of molecular oxygen to

hydrogen peroxide, (Hauptmann et al. 1996) which in

the presence of iron generates �OH radicals through

the Fenton reaction. The involement of �OH in

neuronal loss has been postulated in cerebral ische-

mia, aging and Alzheimer’s disease (Richardson et al.

1992). Oreland and Gottries (1986) explained this

age-related increase in brain MAO activity by the

increase in extrasynaptosomal astroglia. Saura et al.

(1994) also demonstrated similar age dependent

increase in brain and heart MAO activities.

The results of the present study show that DHEA

treatment significantly decreases the increased MAO

activity in both synaptosomal and supernatant frac-

tions in aging male rats. Considering the important

role attributed to MAO activity in the generation of

ROS (Marklund et al. 1982), this decrease can be

regarded as a mechanism which reduces the contri-

bution of MAO activity to oxidative stress in DHEA

treated rats. Further, DHEA effect also correlated

with decreased lipid peroxidation levels and lipofus-

cin accumulation. DHEA administration in aging

male rats thereby results in decrease in MAO activity,

reduction in lipid peroxidation and lipofuscin

accumulation.

In the present experiments significant effects of

DHEA administration on synaptosomal MAO were

observed as compared to the supernatant fraction.

Study of Liu et al. (1996) reported that mitochondial

fraction showed a greater increase in lipid peroxida-

tion and protein oxidation than cytosol with

immobilization stress in brain of male rats. The

recovery of MAO activity by DHEA therapy to aging

rats is also compatible with the possibility that DHEA

plays a role in neurotransmission either as a neuro-

transmitter or as a neuromodulator, may be directly

interacting with the neurotransmitter receptors in

brain (Compagnone and Mellon 2000).

The present results demonstrated a significant

decrease in lipid peroxidation in DHEA treated male

rat brain in 14 and 24 months age groups as

compared to age matched control groups. These

findings agree with Rodriguez and Ruiz (1992) which

showed that peroxidative damage in plasma increased

with aging process in healthy human subjects.

Previous studies demonstrated that deprenyl, an

antidepressant and a MAO inhibitor administration,

decreased the lipid peroxidation level in the brain

(Alper et al. 1999; Kaur et al. 2003). It can therefore

be concluded that the long-term hormone (DHEA)

treatment at lower dose, given to aging male animals

in the present experiments may contribute towards

diminished oxidative stress.

Furthermore brain regions respond differently to

the exogenous DHEA treatment. However with

advancing age this inter regional difference becomes

narrow, which suggests that in old age, responsive-

ness of neurons to exogenous DHEA increases in all

the brain regions. An age-related significant increase

in lipid peroxidation was seen to be highest in the

cerebral hemispheres, which indicates that the most

sensitive area to be affected by free radicals is

cerebral hemispheres during the aging process.

Marx et al. (2000) reported that DHEA makes the

tissue more resistant to lipid peroxidation and

behaves almost parallel to a-tocopherol, a potent free

radical scavenger in terms of TBA production. In

young rats, high level of endogenous DHEA may not

allow exogenously administrated DHEA to accom-

modate its receptor binding sites. With an age related

decline in the endogenous DHEA levels, the respon-

siveness of neurons to exogenous DHEA

predominates as observed in the present investigation

and earlier also (Sinha et al. 2005). The present study,

elucidates that the exogenous DHEA treatment is

more effective at old age and is beneficial in

maintaining and normalizing MDA, a product of

lipid peroxidation levels in the brain regions of older

animals.

The results showed increased MAO activity with

correlated increase in lipid peroxidation in the aging

rat brain, which supports the hypothesis that cate-

cholamine oxidation is an important source of

oxidative stress, and also provide evidence that

lipofuscin deposition increased with age in all three

brain regions (Kaur et al. 2003). Regarding lipofuscin

accumulation, the cerebral hemispheres were found

to be more vulnerable with aging as compared to

cerebellum and brain stem. It was also observed that

the age related changes in the number of neurons

containing lipofuscin were higher in 24 months old

rat as compared to 4 and 14 months old male rats.

Progressive neuronal lipofuscin storage is also asso-

ciated with increase of oxidative stress, decrease of

antioxidative defense, accumulation of mtDNA muta-

tions, increased number of damaged defective and

impaired giant mitochondria with a low rate of

degradation, as well as decrease in the number and

Biogerontology (2008) 9:235–246 243

123

area of normal and functional healthy mitochondria

(Fosslien 2001; Terman and Brunk 2004). Pertaining

to the relationship between age and morphological

form of lipofuscin, it was observed in the present

work that the age related changes in the number of

neurons containing lipofuscin were higher in

24 months old rats as compared to the 14 months

old control animals.

DHEA treatment of aging animals, particularly in

24 months old rats, showed a decrease in lipofuscin

deposition in neurons and an increase in the number

of neurons without lipofuscin in the three different

brain regions when compared with the respective

control age group rats. The decrease of lipofuscin in

aging rats may also increase the neuronal activity in

the brain, which may also be due to the decreased

level of lipid peroxidation and decreased level of

oxidative stress in the aging brain with hormone

treatment. Increased oxidative stress, leading to

increased lipid peroxidation may contribute to the

aging of neural tissue considered an important factor

both in lipofuscinogenesis and aging (Kaur et al.

2001, 2003). In the present study, administration of

DHEA decreased the level of lipid peroxidation,

thereby reducing the deposition of lipofuscin in

different regions of older rat brain. Earlier work

from our laboratory, showed age-associated altera-

tions in the levels of antioxidative enzymes during

normal aging in the brain which could be restored to

almost 3 months old levels in brain regions with

exogenous DHEA administration (Sinha et al. 2008).

The present observations, suggest that DHEA

administration in male rats, provided better protection

at 24 months old rats as compared with the 4 and

14 months age groups, to the brain regions and

synaptosomal fraction from free radicals, by activa-

tion of antioxidant status in the brain region and

synaptosomes. The present study showed that DHEA

treatment significantly decreased monoamine oxidase

activity, lipid peroxidation and lipofuscin accumula-

tion in brain regions of aging rats, the increased body

weight at 24 months also decreased more than the age

matched controls. It may therefore be proposed that

DHEA administration may prevent the deleterious

effects of free radicals, changes in neurotransmitter

concentration and accumulation of lipofuscin in the

brain region, as a consequence, delaying the onset of

age-related disorders.

Acknowledgements The authors Pardeep Kumar, Dr. Asia

Taha and Prof. N.Z. Baquer are grateful to the financial support

from Council of Scientific and Industrial Research in the form

of junior and senior research fellowships from Indian Council

of Medical Research and emeritus fellowship from University

Grants Commission, New Delhi, India respectively.

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