Mechanisms of Ageing and Development

ml dwelopmlt
ELSE.VIER 81 (1995) 15-25

Oxidative stress and aging in the Mongolian

gerbil (Meriones unguiculatus)

Rajindar S. Sohal *, Sanjiv Agarwal, Barbara H. Sohal

Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA

Received 3 February 1995; revision received 13 March 1995; accepted 3 April 1995

Abstract

The objective of this study was to determine if aging in the gerbil, tieriones unguiculatus,
is associated with elevation in the level of oxidative stress. Studies were conducted on the
brain, heart, kidney, liver and testis of young (3-6 months), adult (15 months), and old
(23-25 months) animals. Oxidative damage to proteins, measured as the concentration of
protein carbonyls and loss of activity of glucose-6-phosphate dehydrogenase, and to DNA,
measured as the concentration of 8-hydroxydeoxyguanosine, increased with age of the
animals. There was no appreciable age-related change in the activity of alkaline proteases,
which preferentially degrade oxidized protein. Rates of mitochondrial superoxide anion
radical and hydrogen peroxide generation also increased with age, most notably in the heart.
Antioxidative defenses, measured as activities of superoxide dismutase, catalase and glu-
tathione peroxidase and concentration of glutathione, did not exhibit a uniform pattern of
age-related changes. However, when the antioxidative potential of the tissue homogenates
was measured as their susceptibility to undergo protein oxidation, in response to experimen-
tally-induced oxidative stress, using X-irradiation, tissues of the old animals were signifi-
cantly more vulnerable than those of the young animals. Results of this study are interpreted
to indicate: (i) that the level of molecular oxidative damage to DNA and proteins increases
with age, and (ii) that the increased oxidative damage is due to both an elevation in the rates
of oxidant generation and an increase in the susceptibility of tissues to oxidative damage.

Keywords: Aging; Oxidative stress; Oxygen free radicals; Gerbil; Protein damage; DNA
damage

* Corresponding Author, Tel.: + 1 214 768 2732; Fax: + 1 214 768 3955.
Abbreviations: G-6-PDH, glucose-6-phosphate dehydrogenase; GPx, glutathione peroxidase; I-OHdG,
8-hydroxydeoxyguanosine; PHPA, p-hydroxyphenylacetate; ROM, reactive oxygen metabolites; SOD,
superoxide dismutase.

0047-6374/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0047-6374(94)01578-N
16 R.S. Sohal et al. 1Mech. Ageing Dev. 81 (1995) 15-25

1. Introduction

The hypothesis that oxidative stress is a major causal factor in the aging
process in aerobic organisms is presently under intense scrutiny. Ever since its
formal enunciation, in 1956 [l], this concept has been undergoing progressive
modifications prompted by the contemporaneous advances in the biochemistry of
reactive oxygen metabolites (ROM). In current terms, the hypothesis can be
stated as follows [2]:
‘ROM are constantly generated in aerobic organisms, primarily in mitochondria,
under normal physiological conditions. Despite the existence of overlapping mecha-
nisms of enzymatic and nonenzymatic antioxidative defenses, a small proportion of
ROM is detectable under steady-state conditions, inflicting ubiquitous oxidative
molecular damage. Some such damage is irreversible, exponentially accumulates
with age and is a major contributing factor in the age-related loss of functional
capacity.’
The validity of this or any other general hypothesis of aging can, of course,
only be established on the basis of its applicability to a wide array of species.
Because of their relatively lengthy life span, the study of the aging process in
mammals has, in the past, been primarily limited to two species, namely the
mouse and the rat. More recently, the Mongolian gerbil has also been used in a
few age-related studies [3,4], which thus provides an additional model for testing
various hypotheses of aging, especially for the purpose of determining whether or
not a particular phenomenon is shared by species other than the rat or mouse.
This is particularly desirable because of the variability in the patterns of the
aging process in different species.
In the context of the above rationale, one purpose of this study was to
determine whether or not the age-related changes in the gerbil, pertaining to
oxidative stress related parameters, follow a pattern similar to that observed in
other mammalian and non-mammalian species.
Another objective of this study was to obtain a comprehensive pattern
of various parameters associated with oxidative stress, e.g. rates of ROM
generation, antioxidative defenses and molecular oxidative damage. Previous
studies have tended to focus on individual components of the oxidative stress-re-
lated indicators rather than a broad spectrum of associated alterations. The
present approach is deemed to provide a better understanding of the relationship
between oxidative stress and the aging process.

2. Materials and methods

2.1. Animals
Breeding pairs, and retired breeders (15- 16 months old) of Mongolian gerbils
were obtained from Tumblebrook Farms, Westbrook, MA, and subsequently
maintained in our animal care facility. Animals were fed on Harlan Teklad
rodent diet ad libitum.
 R.S. Sohal et al. 1 Me& Age&g Dev. 81 (1995) 15-25 17

2.2. Experimental procedures

Males were used for experimental studies. The various procedures used in this
study have been described in detail in previous reports from this laboratory.
Therefore, only a brief description of the methods is provided here. The concentra-
tion of 8-hydroxydeoxyguanosine (8-OHdG) was estimated in DNA hydrolysates
by HF’LC using an ESA Coulochem Detector as reported previously [5,6]. Protein
carbonyl content was measured according to Levine et al. [7] using the 2,4-dinitro-
phenyllhydrazine procedure [S]. Protease activity was assayed by the fluorescamine
method [9], as described by us in detail previously [IO]. The rate of superoxide
anion radical (Or) generation was measured in the submitochondrial particles as
superoxide-dismutase (SOD)-inhibitable reduction of acetylated ferricytochrome c,
[ 11,121. The rate of H20, generation was measured in isolated mitochondria
fluorometrically, using the method of Hyslop and Sklar [13], by monitoring the
oxidation of p-hydroxyphenylacetate (PHPA) coupled to the enzymatic reduction
of H,O, by horseradish peroxidase, as described before [ll].
SOI> activity was measured in the homogenates of the tissues by the direct
method of Misra and Fridovich [14], as described previously [15]. Catalase activity
was determined according to Luck [16], as outlined elsewhere [ 151. Glutathione
peroxi’dase (GPx) activity was assayed by the procedure of Paglia and Valentine [ 171
using H,Oz as a substrate, as described previously [15], and the activity of
glucose-6-phosphate dehydrogenase (G-6-PDH) by following the rate of NADP +
reduction [18], using the procedure of Beutler [19]. Concentration of total glu-
tathione was measured by a modification of the method suggested by Tietze [20], as
described previously [21].

3. Results

3. I. L(fe span of gerbils

The strain of gerbils used in this study (from Tumblebrook Farms), has also been
employed by various other research groups working on this species. Although
mortality characteristics of this strain have not been very rigorously established,
retired breeders, usually about 15- 16 months of age, have been frequently used as
the aged group. A high rate of mortality around 2 years of age has been observed
by us and others [4], which is similar to that occurring in several strains of
laboratory mice and rats. We have previously reported that to obtain a representa-
tive cross-sectional sampling of aging populations, studies should be limited to the
period prior to the onset of high mortality, indicated by the sharp downward slope
of survivorship curves, because the survivors progressively represent subsets of
cohorts that are aging relatively more slowly [22]. Therefore, 3- to 6-, 15-, and 23-
to 25-month-old gerbils, representing, respectively, young, middle, and old age were
used in this study.

3.2. EjTect of age on molecular oxidative damage

A comparison of oxidative damage to DNA and proteins was made in the brain
and the heart of the gerbils at 5, 15, and 25 months of age. DNA damage, measured
18 R.S. Sohal et al. / Mech. Ageing Dev. 81 (1995) IS-25

as the concentration of 8-hydroxydeoxyguanosine (8-OHdG), increased progres-

sively with age in both organs (Fig. 1A). The increase between 5 and 25 months of
age was 69% (P < 0.0001) in the brain and 56% (P < 0.0001) in the heart.
Protein oxidative damage was measured as protein carbonyl content in the
homogenates of brain, heart, kidney, and testis. There was a steady age-associated
increase of carbonyls in all these organs (P < 0.0001) except kidney, where the
increase did not reach statistical significance (Fig. 1B). The magnitude of the
age-related increases between 5 and 25 months of age were quite comparable in the
brain (28%) and heart (33%) but much higher in the testis (72%).
Because age-associated changes in the activity of alkaline proteases can affect the
steady-state level of protein carbonyls, protease activity was compared in the brain
and heart of 5- and 25-month-old gerbils. No significant age-related differences in
the proteolysis of oxidized protein were observed in the brain (1.5 f 0.04 and 1.4
+ 0.05 pmol glycine equiv./h/mg protein in 5- and 25-month-old animals, respec-
tively), whereas a 13% decline was observed in the heart (1.5 I~I 0.01 and 1.3 +
0.04 pmol glycine equiv./h/mg protein in 5- and 25-month-old animals, respec-
tively).
Protein oxidative damage was also determined on the basis of loss of activity of
glucose-6-phosphate dehydrogenase, which is particularly susceptible to metal-cata-
lyzed oxidations in vitro [23]. A notable decline in the enzyme activity occurred in
the brain (42%; P < 0.0001) and the heart (37%; P < O.OOOl),whereas the kidney
showed only a minor decrease (Fig. 1C).

3.3. Rates of mitochondrial superoxide anion and Hz02 production

The rates of mitochondrial O,- (Fig. 2A) and H,O, (Fig. 2B) generation were
measured in the brain, heart, kidney and liver of 3-, 15-, and 24-month-old gerbils.
The rank order of the rate of O,- generation by submitochondrial particles in the
young (3-month-old) animals was: heart > kidney > liver > brain. The rates of

Fig. 1. (A) Age-related changes in 8-OHdG concentration in the DNA from the brain and heart of
gerbils. DNA from tissue homogenates was enzymatically hydrolyzed to nucleosides and the amount of
8-OHdG was determined by HPLC equipped with an electrochemical detector. Values are average +
S.E.M. of 4-5 determinations; (B) Comparison of protein carbonyl content in tissue homogenates of
gerbils of different ages. Carbonyl content was measured by the DNPH method of Levine et al. [7].
Results are average f S.E.M. of 334 determinations; (C) Glucose-6-phosphate dehydrogenase activity
in the brain, heart, and kidney of 6-, 15-, and 23-month-old gerbils. Enzyme activity in the cytosolic
fraction of tissue homogenates was measured spectrophotometrically by following the rate of reduction
of NADP+ at 340 nm. Results are average f S.E.M. of 3-8 determinations.
 R.S. Sohal et al. / Mech. Ageing Dev. 81 (1995) 15-25 19

0 0.0
Brain Heart Kidney Liver Brain Heart Kidney

Fig. 2. (A) Rates of mitochondrial OIT generation from brain, heart, kidney and liver of 3-, IS-, and
24-month-old gerbils. The rate of 02- production was measured in submitochondrial particles as
SOD-inhibitable reduction of acetylated cytochrome c. Both the system and the reference cuvette
contained 0.2- 1.0 mg submitochondrial protein, 0.6 pmol antimycin A, 7 mM succinate, and 7.8 pmol
acetylated cytochrome c; 200 units of SOD/ml were added to the reference cuvette. Results are average
_+ S.D. of 3-5 determinations; (B) Rates of H,O, release by mitochondria from brain, heart, and
kidney of 3-, 15-, and 24-month-old gerbils. Rate of H,O, release was measured in isolated mitochondria
as an increase in fluorescence due to the oxidation of PHPA during the coupled reduction of H,O,,
catalyzed by horseradish peroxidase. The reaction mixture included 20-200 fig protein, 500 fig PHPA,
4 units of horseradish peroxidase and 7 mM succinate. Results are average IfI S.D. of 4-6 determina-
tions.

OzT generation increased progressively between 3 and 24 months of age in the

heart, kidney, and liver, whereas the age-associated increase (57%) in the brain
occurred only between 3 and 15 months of age (all P-values < 0.0001). The
percent increase in 02- production between 3 and 25 months of age was: heart
(93%), kidney (510/o), liver (66%).
The rate of H,Oz generation was measured in intact isolated mitochondria from
brain, heart, and kidney without using any respiratory inhibitors, e.g. antimycin A
(Fig. 2B). The rank order of the rate of mitochondrial Hz02 release at all ages was:
heart > brain > kidney. Progressive increases in the rates of H20, release were
observed in all the three organs, between 3 and 24 months of age (38% in brain, P
< 0.0008; 73% in heart, P < 0.0001; and 272% in kidney, P < 0.0001).

3.4. Age-related changes in antioxidative defenses

Activities of total SOD, catalase, and glutathione peroxidase and the concentra-
tion of glutathione were compared in the homogenates of brain, heart, kidney, and
liver at 3, 15, and 24 months of age (Fig, 3A). The age-related pattern of SOD
activity was similar in all three organs, i.e. a significant increase in activity occurred
between 3 and 15 months of age (P < O.OOOl),whereas no significant changes were
noted subsequently.
The age-related pattern of catalase activity varied in different organs (Fig. 3B).
Activity was extremely low in the brain and showed relatively little age-related
variat:ion, whereas the activity increased with age in the heart (P < 0.0001) and
declined in the kidney (P -C 0.0001).
20 R.S. Sohai et al. 1 Mech. Ageing Dev. 81 (1995) 15-25

Glutathione peroxidase activity followed a relatively uniform pattern in each of

the three organs, but there was no clear age-related trend (Fig. 3C). For example,
the activity decreased between 3 and 15 months of age, but increased between 15
and 24 months of age in all the four organs. Glutathione concentration exhibited an
uneven age-related pattern, i.e. it declined progressively in the brain (Fig. 3D);
however, in the kidney a comparable decline occurred between 3 and 15 months of
age with no further loss thereafter. There was relatively little age-related change in
glutathione concentration in the heart, whereas a U-shaped pattern was observed in
the liver.

3.5. Eflect of age on susceptibility to oxidative damage

On the basis of the variability in the age-related pattern of individual antioxida-
tive defenses, described above, it is hazardous to infer whether or not the antioxida-
tive potential of the tissues is altered by the aging process. Comparison of the
ability to withstand experimentally-induced oxidative stress in vivo was previously
suggested to provide a possible overall measure of the antioxidative efficiency of the
tissues [24]. A comparison of protein carbonyl content was made between the
homogenates of the brain and heart of 5- and 25-month-old animals following
exposure to 6 krad (60 Gy) X-irradiation (Fig. 4). Irradiation-induced increase was

Brain Heart Kidney Liver Brain Heart Kidney Liver

0 0
Brain Heart Kidney Liver Brain Heart Kidney Liver

Fig. 3. Activities of superoxide dismutase (A), catalase (B), glutathione peroxidase (C), and concentra-
tion of glutathione (D) in the tissue homogenates of 3-, 15-, and 24-month-old gerbils. Values are
average f S.D. of 3-6 determinations
 R.S. Sohal et al. / Mech. Ageing Den 81 (1995) 15-Z 21

5 25
Age (month) Age (month)
Fig. 4. Effect of X-irradiation on protein carbonyl content in homogenates of the brain and the heart of
S- and 25-month-old gerbils. Tissues were homogenized in 5 mM phosphate buffer (10% w/v) pH 7.4,
0.1% Triton-X and protease inhibitors aprotenin, leupeptin and pepstatin, and exposed to 6 krad of
irradiation delivered at 200 rad/min. Values are average k S.E.M. of 3-6 determinations.

38% in the brain and 20% in the heart from young animals whereas corresponding
increases in the old animals were 211% and 152%.

4. Discussion

The results of this study indicate that DNA and protein oxidative damage
increases during life in tissues of the gerbil. Rates of mitochondrial Ozl and H,O,
production increase, whereas the antioxidative defenses, as reflected by the suscep-
tibility to withstand induced oxidative damage, decline during aging. The individual
components of the antioxidative system however do not exhibit a coherent age-re-
lated pattern.
The most fundamental assumption of the oxidative stress hypothesis of aging is
that molecular oxidative damage is ubiquitous and increases with age in causal
assoc:iation with the loss of cellular function. Previous studies by Ames and
colleagues [25,26] have shown that oxidative damage to DNA also increases with
age in the tissues of the rat. An age-related increase in protein carbonyl content has
been reported in the gerbil brain [3] and the rat liver [27]. Protein carbonyl content
as well as the concentration of 8-OHdG were previously reported by us not only to
increase with age in the mouse, but, quite importantly, caloric restriction of the
mice, that results in an extension of their life span, was found to retard the
age-a,ssociated increase in protein carbonyls [l l] and 8-OHdG content [28]. Experi-
mental studies on the housefly also indicated that oxidative damage to proteins [8]
and DNA [6] not only increased with age, but was also inversely correlated with the
life expectancy of the flies. Extension of the life span of the flies by lowering the
level of physical activity was found to correspondingly lessen the accumulation of
prote:in carbonyls and 8-OHdG content [6,8]. Similarly, extension of the life span in
transgenic Drosophila melanogaster, overexpressing Cu-Zn SOD and catalase, was
22 R.S. Sohal et al. / Mech. Aping Dev. 81 (1995) 15-25

also accompanied by a relatively lower amount of age-associated increase in the

concentration of protein carbonyls in the flies [29]. Therefore, on the basis of the
available information, two generalizations may be made: (i) oxidative damage to
tissues increases with age, and (ii) the steady state level of damage is decreased by
the experimental regimes that extend life span. What remains obscure, however, is
whether the age-related loss of cellular functions is due to generalized oxidative
molecular damage or whether it is a consequence of the oxidative damage to certain
key molecules or intracellular loci. Identification of the key targets of oxidative
damage would facilitate the elucidation of the mechanisms by which oxidative stress
may cause functional attrition.
Three different hypotheses concerning the causes of the age-associated increase in
protein oxidative damage were tested in this study, namely (i) increased rates of
ROM generation, (ii) decline in antioxidative defenses, and (iii) decline in protease
activity. The rate of mitochondrial OZT and H,O, increased with age. However, a
point worth noting is that the rates of mitochondrial oxidant generation not only
differ in various tissues, but the relative age-related increase in the rates of
production also varies [l 1,30,3 13. Age-related increase in mitochondrial oxidant
generation has also been noted in the rat [32], mouse [l 11,and the housefly [33], and
would thus appear to be a general phenomenon associated with aging. The
mechanism causing such an elevation is not fully known; however, experimentally-
induced oxidative damage to the inner mitochondrial membrane has been shown to
increase the rate of mitochondrial H,O, generation [33,34]. It is therefore plausible
that age-related increase in mitochondrial oxidant generation is due to self-inflicted
oxidative damage to the inner mitochondrial membrane.
Numerous studies have been conducted to determine whether or not the antiox-
idative defenses decline during aging (reviewed in [2,35]). Such studies have usually
involved measurements of some of the main components of the antioxidative
defense network, e.g. activities of SOD, catalase, glutathione peroxidase and
glutathione reductase, and concentration of glutathione. However, due to differ-
ences in the age-dependent changes among different antioxidants, no overall
age-related pattern has yet emerged on the basis of such studies. This point is also
demonstrable in the present study. For example, the SOD activity increased in the
brain, heart, kidney, and liver; catalase activity decreased in the kidney, but tended
to increase in the heart, whereas glutathione activity was unchanged in the heart,
but declined in the brain. Although nothing concerning the overall antioxidative
efficiency of the gerbil tissues can be confidently concluded on the basis of such
conflicting changes, the susceptibility of the tissue homogenates to undergo protein
oxidation, induced by X-irradiation, increases with age. Two possibilities can be
offered to explain this phenomenon. The first is that there is indeed a decline in
overall antioxidative defenses during aging. The postulated decline would probably
be occurring in some component(s) of the antioxidative defenses that remains
unidentified on the basis of the present study. The second possibility is that
loosely-bound transition metals, catalyzing the scission of H,O, into hydroxyl free
radical and hydroxyl ion, may be more abundant in the tissues of the old as
compared to the young animals. An age-related increase in vulnerability to oxida-
 R.S. Sohal et al. 1 Mech. Ageing Dev. 81 (1995) 15-25 23

tive damage was previously reported in the housefly in vivo as well as in vitro [24].
On the basis of the results of these studies, we suggest that determinations of
antioxidative defenses should include the resistance of the tissues to induced
oxidative stress.
The third possibility that age-associated increase in the concentration of oxidative
molecular damage, specifically to proteins, is due to a decline in the cellular ability
to degrade such products is not clearly supported by the present results because of
the lack of any significant difference in alkaline protease activity in the brain
between the young and the old animals and only a relatively small age-related
decline in the heart. A similar situation was previously observed by us in the rat
[lOI.
In conclusion, studies on the Mongolian gerbil tissues confirm previous findings
in other mammals that oxidative molecular damage increases during aging. This
appears to be due to both an increased generation of oxidants and an increase in
susceptibility to oxidative damage.

Acknowledgements

This study was supported by a grant ROl AG7657-06 from National Institutes of
Health-National Institute on Aging. The technical assistance of Miss Kim Dawson
is gratefully acknowledged.

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