Archives of Gerontology and Geriatrics 58 (2014) 427–433

A critical period in lifespan of male rats coincides with increasedoxidative stress

Dileep Kumar, Syed Ibrahim Rizvi *

Department of Biochemistry, University of Allahabad, Allahabad, U.P. 211002, India

A R T I C L E I N F O

Article history:

Received 8 August 2013

Received in revised form 26 October 2013

Accepted 14 November 2013

Available online 23 November 2013

Keywords:

Rat

Aging

Lifespan

Oxidative stress

Blood

A B S T R A C T

The oxidative stress theory of aging has provided the best possible explanation for the processes which

accompany aging and has received much support, however, in the last few years there have been

questions regarding the validity of this theory. We have conducted experiments to determine an array of

oxidative stress parameters in blood of male rats at various intervals (1, 4, 8, 12, 18 and 24 months)

during their entire lifespan. Established protocols were used to measure plasma antioxidant capacity,

erythrocyte plasma membrane redox system (PMRS), lipid and protein oxidation in erythrocytes and

plasma, and erythrocyte glutathione (GSH). Our results on the total plasma antioxidant potential, PMRS

in erythrocytes, protein and lipid peroxidation, and intracellular reduced GSH provide evidence that

oxidative stress is minimal till approximately one-third of the total lifespan (8 months) and there is a

spurt in oxidative stress between 8 and 12 months. The identification of a period (corresponding to 8–12

months) in the lifespan of rats coinciding with an spurt in oxidative stress is an interesting finding. No

such report is available in humans or in any other model systems during aging.

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1. Introduction

Despite considerable research effort, aging continues to be themost intriguing biological phenomenon. Although till date nogenes have been identified which cause aging, certain genes havebeen shown to control lifespan through modulation of signalingpathways (Bartke, 2008; Finch & Ruvkun, 2001). Denham Har-man’s free radical theory of aging in 1956 provided a convincingargument for the accumulation of oxidative damage beingresponsible for the progressive and functional deterioration withage (Harman, 1956). This theory which later became the ‘OxidativeStress theory of aging’ was the best possible explanation for theprocesses which accompany aging and received continued support(Barja, Cadenas, Rojas, Lopez-Torres, & Perez-Campo, 1994;Beckman & Ames, 1998; Droge & Schipper, 2007) however notall available data mainly concerning lifespan determinations andeffect of antioxidant supplementation could validate this theory(Buffenstein, Edrey, Yang, & Mele, 2008; Perez et al., 2009). Theoxidative stress theory now does not seem invincible and remainsembattled. It is thus imperative that further evidences aregenerated to verify the usefulness of this theory in the light ofcurrent knowledge.

* Corresponding author. Tel.: +91 9415305910.

E-mail address: sirizvi@gmail.com (S.I. Rizvi).

0167-4943/$ – see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.archger.2013.11.006

Reactive oxygen species (ROS) production is inseparable fromaerobic metabolism; however living systems have evolved to limitthe deleterious effect of ROS through several enzymatic and nonenzymatic antioxidant systems. There is considerable evidence forthe oxidative damage to macromolecules under normal physio-logical conditions (Gil et al., 2006; Inal, Kanbak, & Sunal, 2001;Sohal & Weindruch, 1996) suggesting that antioxidant repairmechanisms cannot completely avoid ROS mediated oxidativeinsult (Gil del Valle, 2011; Lenaj, 2001). The shift of redox balancetoward oxidative state during aging has been linked to thedevelopment of state of chronic inflammation (Hensley, Robinson,Gabbita, Salsmasn, & Floy, 2000; Stadtman, 2004) and predisposesthe development of various clinical conditions (Kuor-o, 2001).

Age related decline in plasma antioxidant capacity has beenreported in humans (Rizvi & Maurya, 2007), this alteration hasbeen shown to correlate with markers of lipid and proteinoxidative stress both in plasma and erythrocytes (Pandey & Rizvi,2010). In earlier reports we have shown the upregulation oferythrocyte PMRS, which is involved in transferring of reducingequivalents from inside the cell to extracellular acceptors, as afunction of human age (Rizvi, Jha, & Maurya, 2006), the increasedactivity of PMRS has been related to regeneration of ascorbate inthe plasma. It has been hypothesized that the increased activity oferythrocyte PMRS is a protective mechanism for mitigating theincreased oxidative stress (Rizvi, Pandey, Jha, & Maurya, 2009), alink between erythrocyte PMRS activity and lifespan has also beenhypothesized (Rizvi, Kumar, Chakravarti, & Singh, 2011).

D. Kumar, S.I. Rizvi / Archives of Gerontology and Geriatrics 58 (2014) 427–433428

The study on aging related biochemical parameters in humansis dependent on many variables such as genetic factors, tempera-ture, activity, and nutrition. Thus the conclusions drawn fromhuman based studies may not be very relevant when validation ofoxidative stress theory of aging is at stake. The present study wasundertaken to determine markers of oxidative stress of plasma anderythrocytes in male rats, kept in controlled laboratory conditions,at different ages ranging from 1 month to 24 months. We report theage dependent changes in plasma total antioxidant capacity, PMRS,protein carbonyl, advanced oxidation protein products (AOPP),reduced GSH, and lipid peroxidation product malondialdehyde(MDA).

2. Materials and methods

2.1. Chemicals

(2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ). 4,7-Diphenyl-1,10-phe-nanthroline disulfonic acid disodium salt (DPI), reduced GSH,2,4-dinitrophenylhydrazine (DNPH), and dithiobis nitro benzoicacid (DTNB) was procured from Sigma Aldrich, India. All otherchemicals were of analytical grade available from Merck, India andHIMEDIA Labs, India.

2.2. Animal model and study protocol

The experiment was carried out with 48 male Wistar rats of 1, 4,8, 12, 18 and 24 months old, containing eight animals in each agegroup (n = 8). They were housed in a temperature controlled room(25 � 5 8C) with 12-h light–dark cycles. All rats were fed with anormal laboratory diet of nutrient rich pellets containing total energyas fat, protein and carbohydrates, and had free access to drinking water.

2.3. Collection of blood, isolation of red blood cells and plasma

During experimental period, rats were sacrificed under lightanesthesia. Blood samples were collected by cardiac puncture into10 unit/ml heparin rinsed anticoagulant syringes, and then redblood cells were pelleted by centrifugation at 800 � g for 10 min at4 8C. After the removal of plasma (immediately frozen at �80 8Cuntil use for biochemical assays), buffy coat, and the upper 15% ofpacked red blood cells (PRBCs), the RBCs were washed twice withcold phosphate buffered saline (PBS) (0.9% NaCl and 10 mmol L�1

Na2HPO4; pH 7.4) and then used for experiment. All protocols forexperiments were approved by the Animal Care and EthicsCommittee of University of Allahabad.

2.4. Measurement of biochemical parameters in blood

Lipid profile, SGOT, SGPT, creatinine, and urea were measuredusing reagent kits from Erba Diagnostics, Mannheim, Germany.Blood glucose values were determined using an Accu-Chek ActiveGlucometer (Roche Diagnostics, Mannheim, Germany).

2.5. Erythrocyte membrane isolation

Erythrocyte ‘ghosts’ from leukocyte-free RBCs were prepared byfollowing the method of Dodge, Mitchell, and Hanahan (1963),with slight modifications. Briefly, washed and packed erythrocyteswere lysed by adding 10 volume of 5 mM phosphate buffer pH 7.4(at 4 8C). After leaving on ice for 30 min, the erythrocytemembranes were packed by centrifugation at 20,000 � g for10 min at 4 8C and the hemoglobin-containing supernatant wasremoved. The erythrocyte membranes were then washed threetimes by suspending in fresh buffer followed by centrifugationunder the same conditions. Finally, the membranes were

suspended in hypotonic 5 mM buffer followed by centrifugationunder the same conditions and then resuspended in 5 mMphosphate buffer pH 7.4. Protein was estimated in red cellmembrane preparation following the method of Lowry, Rosebrough,Farr, and Randall (1951), using bovine serum albumin as a standard.

2.6. Measurement of total antioxidant activity by FRAP

The total antioxidant potential of the plasma was determinedusing a modification of the ferric reducing ability of plasma (FRAP)assay reported by Benzie and Strain (1996). FRAP reagent wasprepared from 300 mmol L�1 acetate buffer, pH 3.6, 20 mmol L�1

ferric chloride and 10 mmol L�1 2,4,6-tripyridyl-s-triazine madeup in 40 mmol L�1 hydrochloric acid. All three solutions weremixed together in the ratio 10:1:1 (v/v/v) respectively, 3 ml ofFRAP reagent was mixed with 100 ml of plasma and the contentswere mixed thoroughly. The absorbance was read at 593 nm at 30 sintervals for 4 min. Aqueous solution of known Fe (II) concentra-tion in the range of 100–1000 mmol L�1 was used for calibration.Using the regression equation the FRAP values (mmol Fe (II)/L) ofthe plasma was calculated.

2.7. Measurement of erythrocyte PMRS activity

The activity of the erythrocyte PMRS was measured by thereduction of ferricyanide as described earlier (Rizvi et al., 2006).Briefly, packed RBC (0.2 ml) were suspended in PBS containing5 mM glucose and 1 mM freshly prepared potassium ferricyanideto a final volume of 2.0 ml. The suspensions were incubated for30 min at 37 8C and then centrifuged at 800 � g at 4 8C. Thesupernatant collected was assayed for ferrocyanide content using4,7-diphenyl-1,10-phenanthroline disulfonic acid disodium salt,absorption was recorded at 535 nm (0 = 20,500 M�1 cm�1). Theresults are expressed in mmol ferrocyanide/ml PRBC/30 min.

2.8. Determination of membrane and plasma protein carbonyls

Erythrocyte membrane protein carbonyls were measuredaccording to procedure of Levine et al. (1990). Erythrocytemembrane samples (0.2 mL) in PBS/0.4 mL plasma were takenin 2 tubes as test and control samples. A total of 4.0 mL of10 mmol L�1 2,4-DNPH, prepared in 2 mmol L�1 HCl, was added tothe test sample, and 4.0 mL of 2 mmol L�1 HCl alone was added tothe control sample. The contents were mixed thoroughly andincubated for 1 h in the dark at 37 8C. The tubes were shakenintermittently every 10 min to facilitate reactions with proteins.After shaking, 20% trichloroacetic acid (TCA) (w/v) was added toboth tubes, and the mixture was left on ice for 10 min. The tubeswere then centrifuged at 850 � g for 20 min to obtain proteinpellets. The supernatant was carefully aspirated and discarded. Theprotein pellets were washed 3 times with ethanol–ethyl acetate(1:1, v/v) solution to remove unreacted DNPH and lipid remnants.Finally, protein pellets were dissolved in 6 mmol L�1 guanidinehydrochloride and incubated for 10 min at 37 8C. The insolublematerials were removed by centrifugation. Carbonyl content wasdetermined by taking the spectra of the supernatant at 370 nm.Each sample was read against the blank. The carbonyl content wascalculated using an absorption coefficient of 22000 mol L�1 cm�1,and data were expressed in nmol mg�1 protein.

2.9. Assay of AOPP

Determination of AOPP levels was performed by modification ofthe method of Witko-Sarsat et al. (1996). 2 ml of plasma wasdiluted with 1:5 in PBS, 0.1 ml of 1.16 M potassium iodide was thenadded to each tube, followed by 0.2 ml acetic acid after 2 min. The

Fig. 1. Erythrocyte PMRS measured as marker of oxidative stress during rat aging.

Values expressed in micromole ferrocyanide/ml PRBC/30 min. Data are represented

as mean � SD (n = 8). Significant (P < 0.05) difference was obtained only between 8

and 12 month groups. Comparisons between any other two consecutive age groups

were non significant.

D. Kumar, S.I. Rizvi / Archives of Gerontology and Geriatrics 58 (2014) 427–433 429

absorbance of the reaction mixture was immediately read at340 nm against a blank containing 2 ml of PBS, 0.1 ml of KI, and0.2 ml of acetic acid. The chloramine-T absorbance at 340 nm beinglinear within the range of 10–100 mmol L�1, AOPP concentrationswere expressed as mmol L�1 chloramine-T equivalents.

2.10. Determination of erythrocyte reduced GSH

Erythrocyte GSH was measured following the method of Beutler(1984). The method is based on the ability of the –SH group toreduce 5,50-dithiobis,2-nitrobenzoic acid (DTNB) and form ayellow colored anionic product whose optical density is measuredat 412 nm. Concentration of GSH is expressed in mg mL�1 PRBCsand was determined from standard plot.

2.11. Determination of erythrocyte MDA content

Erythrocyte MDA was measured according to the method ofEsterbauer and Cheeseman (1990), with slight modification.Packed erythrocytes (0.2 ml) were suspended in 3 ml PBS contain-ing 0.5 mM glucose, pH 7.4. The suspension (0.2 ml) was added to1 ml of 10% TCA and 2 ml of 0.67% thiobarbituric acid (TBA), boiledfor 20 min at 90–100 8C and then cooled. Subsequently the mixturewas centrifuged at 1000 � g for 5 min and the absorbance ofsupernatant was read at 532 nm. The concentration of MDA inerythrocytes was calculated using extinction coefficient(e = 31,500) and is expressed as nmol mL�1 of packed erythrocytes.

2.12. Statistical analysis

Data are expressed as mean � SD for eight independentexperiments. Differences between the groups were assessed byone-way ANOVA using PRISM version 5.01 software package forWindows. Post hoc testing was performed for intergroup compar-isons using Bonferroni test. To detect differences between successivegroups (ages 1 and 4 month, 4 and 8 month, 8 and 12 month, 12 and18 month, and 18 and 24 month), the data was further analyzed bynon parametric Kruskal Wallis test. Post hoc analysis was performedusing Dunn test. A probability (P) value of less than 0.05 wasconsidered as statistically significant.

3. Results

Table 1 gives the values of plasma lipid profile, SGOT, SGPT,Creatinine, Urea and blood glucose in all ages of rats ranging from 1month to 24 months. The values representing age-dependentvariations are in agreement with reported findings (El-Wakf,Hassan, El-said, & El-Sai, 2008; Sampson, Hebert, Booe, &Champney, 1998). Except for a significant (P < 0.05) change inLDL between 8 and 12 months age groups, no significant variation

Table 1Biochemical indices of the blood in rats aged 1, 4, 8, 12, 18 and 24 months.

1 month (Values

are mean � SD)

4 month (Values

are mean � SD)

8 month

are mean

Blood glucose (mg/dl) 78 � 12 80 � 13 83 � 1

Total cholesterol (mg/dl) 87 � 05 90 � 07 96 � 0

HDL (mg/dl) 46 � 04 44 � 05 43 � 0

LDL (mg/dl) 37.50 � 06 42.36 � 8 49.89 � 0

Triglyceride (mg/dl) 84 � 11 85 � 07 88 � 0

SGOT (m/L) 101 � 07 105 �0 9 129 � 1

SGPT (m/L) 3.47 � 0.92 4.59 � 1.49 6.09 � 1

Urea (mg/dl) 39.0 � 4.03 42.90 � 4.38 43 � 7

Creatinine (mg/dl) 0.354 � 0.03 0.374 � 0.07 0.369 � 0

Data are represented as mean � SD (n = 8). One way ANOVA with post hoc Bonferroni was u

observed between any other age group when compared with the respective preceding age* Significant at 0.05 level.

is observed between any other parameter when comparison isdone between an age group and its respective preceding age group.

The activity of PMRS in erythrocytes plotted against rat age (inmonths) is shown in Fig. 1. Albeit a slow increase as a function ofage, there is no significant change in PMRS activity till 8 months ofage. There is a significant (p < 0.05) increase in PMRS activitybetween 8 and 12 months, thereafter there is gradual increase till24 months. The result of plasma antioxidant potential in terms ofFRAP is shown in Fig. 2. There is no significant change in FRAPvalues till 8 weeks and then a significant (p < 0.05) drop isobserved between 8 and 12 months.

The results of protein carbonyl formation in erythrocytemembrane as a function of rat age are given in Fig. 3. There isan increase in protein oxidation with age, however the there is amarked increase between 8 and 12 months which is significant(p < 0.05). The pattern of protein carbonyl in plasma as a functionof rat age is similar to results obtained for membrane carbonylformation (Fig. 4). The AOPP measured in plasma as a function ofrat age is shown in Fig. 5. The AOPP also shows an elevationbetween the ages of 8–12 months.

Intracellular GSH levels in rats as a function of age is shown inFig. 6. The GSH increases significantly (p < 0.05) from 1 month to 8months and then there is a drop in level which is prominentbetween 8 and 12 months and again between 18 and 24 months.Lipid peroxidation measured in terms of MDA is plotted against ratage in Fig. 7. There is an age dependent increase in erythrocyteMDA with a prominent spurt between 8 and 12 months. Thedifferences between groups for all the parameters (p-value)reported in the results were determined by Dunn post hoc test.

(Values

� SD)

12 month (Values

are mean � SD)

18 month (Values

are mean � SD)

24 month (Values

are mean � SD)

0 79 � 11 75 � 12 83 � 09

9 119 � 08 128 � 09 140 � 16

6 35 � 07 33 � 05 30 � 07

8 87.55 � 09* 104.29 � 10 125.53 � 16

8 109 � 09 123 � 10 139 � 15

0 137 � 12 154 � 15 169 � 14

.05 11.45 � 2.21 13.8 � 2.98 17.03 � 3.07

.09 57 � 5.89 55 � 7.69 67 � 7.34

.08 0.437 � 0.02 0.457 � 0.03 0.468 � 0.03

sed for comparison between any two consecutive age groups. No significant variation is

group.

Fig. 2. Total antioxidant capacity of plasma measured in terms of FRAP value as a

function of rat age. FRAP value is expressed as mmolFe (II) per l of plasma. Data are

represented as mean � SD (n = 8). Significant (P < 0.05) difference was obtained only

between 8 and 12 months. Comparisons between any other two consecutive age

groups were non significant.

Fig. 4. Protein carbonyl content in plasma measured as a function of oxidative stress

marker during rat aging. The concentration of the protein carbonyl content is

expressed as nmoles L�1 of plasma. Data are represented as mean � SD (n = 8).

Significant (P < 0.05) difference was obtained only between 8 and 12 months.

Comparisons between any other two consecutive age groups were non significant.

Fig. 3. Erythrocyte membrane protein carbonyls (protein oxidation index)

measured as a function of oxidative stress marker during rat aging. Protein

carbonyl level expressed as nmoles/mg protein. Data are represented as mean � SD

(n = 8). Significant (P < 0.05) difference was obtained only between 8 and 12 months.

Comparisons between any other two consecutive age groups were non significant.

Fig. 5. Plasma AOPPs level measured as free radical mediated protein oxidation

during rat aging. Concentration of AOPP is expressed as mmol L�1 of chloramine-T

equivalents. Significant (P < 0.05) difference was obtained only between 8 and 12

months (One way ANOVA post hoc Bonferroni test). Comparisons between any

other two consecutive age groups were non significant.

Fig. 6. Erythrocyte reduced GSH content measured as antioxidant level.

Concentration of GSH is expressed in milligram per milliliter packed

erythrocytes. Data are represented as mean � SD (n = 8). Significant (P < 0.05)

difference was obtained between the age groups 8 and 12 months. Comparisons

between any other two consecutive age groups were non significant.

Fig. 7. Erythrocyte lipid peroxidation (MDA) measured as a free radical mediated

oxidative stress marker during rat aging. *P < 0.05 compared with 8 month old rat

values. Concentration of MDA is expressed as nmol mL�1 of packed erythrocytes.

Data are represented as mean � SD (n = 8). Significant (P < 0.05) difference was

obtained only between 8 and 12 months. Comparisons between any other two

consecutive age groups were non significant.

D. Kumar, S.I. Rizvi / Archives of Gerontology and Geriatrics 58 (2014) 427–433430

D. Kumar, S.I. Rizvi / Archives of Gerontology and Geriatrics 58 (2014) 427–433 431

4. Discussion

The importance of measurement of blood oxidative stressbiomarkers is highlighted by a recent report showing that proteinand lipid oxidation markers measured in blood provide a reliableindication about the redox status in skeletal muscle, heart and liver(Veskoukis, Nikolaidis, Kyparos, & Kouretas, 2009). Althoughoxidative stress may damage the red cell itself, the mass effectof large quantities of ROS leaving the red cell have a tremendouspotential to damage other components of the circulation (Johnson,Goyette, Ravindranath, & Ho, 2005).

Evidence is now clear for the presence of a PMRS in allorganisms including bacteria, yeast, animals and plants (Crane,Sun, Clark, Grebing, & Low, 1985; Rubinstein & Luster, 1993). It isaccepted that PMRS is involved in transferring reducing equiva-lents from intracellular donors to extracellular acceptors mainlyoxidized ascorbate. In this way the PMRS helps the cells to respondto changes in redox potential thereby regulating a variety ofphysiological functions including cell metabolism, ion channels,growth and death (Lane & Lawen, 2009; Principe, Avigliano, Savini,& Catani, 2010).

The PMRS has been extensively studied in erythrocytesbasically due to the fact that erythrocytes lack mitochondriaand PMRS is the only mechanism for transplasma membraneelectron transport. Importantly erythrocytes encounter a variety ofoxidants in the blood during their lifespan. Recent reports showthat erythrocyte PMRS plays an important role in providingprotection against oxidative stress during human aging and in type2 diabetes mellitus (Rizvi & Srivastava, 2010).

The male Wistar rats have been reported to have an averagelifespan of 24 months (Masoro, 1980; Schlettwein-Gsell, 1970).Our observation of an age-dependent increase in erythrocyte PMRSactivity in rats until 24 months of age corroborates our earlierresults on humans wherein we have reported an age-dependentincrease in erythrocyte PMRS activity till 80 years. This observationalso substantiates our earlier hypothesis for the dependence ofanimal lifespan on PMRS activity (Rizvi et al., 2011). To the best ofour knowledge this is the first report of an age dependentdocumentation of PMRS activity in rats.

Our observation of a sudden increase (52.28%) in PMRS activitybetween 8 and 12 months of age is interesting. Given the role ofPMRS to mitigate oxidative stress, our observation prompts us tohypothesize that there is sudden increase in plasma oxidative statebetween 8 and 12 months which triggers a compensatory responsereflected by increased PMRS activity. Significantly no such spurt inPMRS activity has been observed during aging in humans (Rizviet al., 2006), although humans also show an increasing erythrocytePMRS activity as a function of age. The FRAP results corroborate ourview that plasma anti oxidation potential sharply decreases(123.80%) between 8 and 12 months of age in rats.

Protein oxidative damage mediated by ROS is particularlyimportant in aging (Voss & Siems, 2006). Oxidation of protein canlead to protein carbonyls which are formed on amino acids lysine,proline, arginine and threonine. Peptide bond oxidative cleavagecan also release fragments which can form carbonyl derivatives(Stadtman & Levine, 2003). Protein carbonyl formation is oftenused as a measure of severe oxidative damage, it accompaniesprotein dysfunction, associated with higher thermosensitivity andhydrophobicity (Berlett & Stadtman, 1997). Cakatay et al. (2003)working on rat skeletal muscle reported a significant increase inprotein carbonyl levels and nitrotyrosine in ‘‘young’’/old vs ‘‘old’’/young rats, however they observed no correlation betweenmarkers of oxidative protein damage and lipid peroxidation. Inearlier reports we have reported an increased protein carbonylformation during aging in humans (Pandey, Mehdi, Maurya, &Rizvi, 2010), similar results are observed in rats however, the spike

in carbonyl formation between 8 and 12 months reflects anincreased plasma/cellular oxidation state between this period oflifespan. Oxidation of amino acid side chains often result instructural change and/loss of functions making the proteinsusceptible to degradation.

AOPPs are defined as dityrosine containing cross linked proteinproducts and are considered as reliable marker for estimating thedegree of protein oxidation (Witko-Sarsat et al., 1996). AOPPs canbe formed in vitro by exposure of serum albumin to hypochlorousacid (HOCl). Thus, oxidative stress induces AOPP formation byreaction of plasma proteins with chlorinated oxidants. AOPP hasbeen considered as novel markers of oxidant mediated proteindamage. The age-dependent increase in AOPP in humans hasalready been reported (Pandey et al., 2010). Age related increase inplasma AOPPs level signifies increased oxidative. Albiet a smallincrement, there is no significant increase in AOPP formation until8 months of age in rats, however a spike is observed between 8 and12 months. We did not observe such an effect in humans (Pandeyet al., 2010).

ROS mediated lipid peroxidation may lead to loss of membraneintegrity and cell death (Niki, 2013). MDA has been shown to crosslink erythrocyte phospholipids and proteins, leading to loss ofmembrane integrity, functions and ultimately diminished celllifespan. MDA accumulation can also affect anion transport andband 3 associated enzymes, phosphofructokinase and glyceralde-hydes 3 phosphate dehydrogenase (Dumaswala, Zhuo, Jacobsen,Jain, & Sukalski, 1999). Several reports confirm increase in MDAduring aging in erythrocytes in humans (Kumar, 2011; Mehdi,Singh, & Rizvi, 2012). An age-dependent increase in MDA level hasbeen reported in heart of rats (Van der Loo et al., 2003). Our resultson rats also show an increased MDA as a function of age, theobservation of increase between 8 and 12 months reflectsincreased oxidative stress during this period of life and corrobo-rates other markers.

GSH plays an important role in detoxification of H2O2. Inerythrocytes it is a major antioxidant protecting importantproteins such as spectrin, the oxidation of which may lead tochange in membrane fluidity (Carroll et al., 2006). Besidesaugmentation of antioxidant defence, GSH also plays an importantrole in maintenance of SH groups in Hb and other enzymes inreduced state. The importance of GSH during condition of oxidativestress is thus critical for the cell (Dumaswala et al., 2001).

Studies show that red cells are important as biological carriersof GSH by biosynthesis and therefore provide an importantdetoxifying system within the circulation (Dumaswala et al., 2001;Sharma, Awasthi, Zimniak, & Awasthi, 2000). GSH synthesis inerythrocytes is dependent upon the availability of L-cysteine. Thedecrease in erythrocyte GSH level during human aging has beenreported by us and other workers (Rizvi & Maurya, 2007), howeverwe provided the first evidence of the age-dependent decline in L-cysteine influx in erythrocytes (Rizvi & Maurya, 2008). A depletionof erythrocyte GSH may cause release of iron accompanied by lipidperoxidation and hemolysis (Comporti, Signorini, Buonocore, &Ciccoli, 2002). Our observation of an increase in GSH upto 8 monthsof age is an interesting finding, although we cannot explain thisfinding on the basis of our present results however this could be anadaptative response which some authors have termed ‘prosurvivalsignaling’ (Liochev, 2013; Van Raamsdonk & Hekimi, 2012). Thedecrease in GSH after 8 months is expected since this period isaccompanied with continuous increase in oxidative stress.

Our results on redox parameters in rats over a full lifespan of 24months reinforce the view that there is an increase in oxidativestress as a function of age, however an interesting fact emergesthat the level of oxidative stress is minimal till 8 months of life. Wehave no explanation regarding the observation of a suddenincrease in oxidative stress between 8 and 12 months of age in

D. Kumar, S.I. Rizvi / Archives of Gerontology and Geriatrics 58 (2014) 427–433432

rats. Several leads such as activation of vitagenes (Calabrese et al.,2011), the activation of nuclear factor 2 (Nrf2) which is beingreferred as a ‘master regulator’ for the expression of hundreds ofgenes involved in antioxidant response including immune andinflammatory responses, tissue remodeling and fibrosis, carcino-gemesis and metastasis, and cognitive dysfunction (Hybertson,Gao, Bose, & McCord, 2011), or any possible life history ‘trade offs’(Monaghan, Metcalfe, & Torres, 2009) may possibly explain thisobservation.

In conclusion, we provide evidence for the increase in oxidativestress during aging in rats for their full lifespan. The observation ofa spurt in oxidative stress between 8 and 12 months, correspond-ing to approximately one-third – midway of the total lifespan, is aninteresting finding. No such report is available in humans or in anyother model systems. We are however prompted to hypothesizethat given the heterogeneity of living conditions for humans,earlier reports may have overlooked subtle ‘critical’ periods.Further research is needed to understand the full implications ofthese observations.

Declaration of interest

The authors declare no conflicts of interest.

Acknowledgement

The authors are grateful to University Grants Commission, NewDelhi for financial support in the form of grant F 37-392/2009 toSIR.

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