Age- related Decrease in Rat Liver Catalase …abap.co.in/sites/default/files/CTBT_9_1_11.pdf ·...

Current Trends in Biotechnology and PharmacyVol. 9 (1) 88-96 January 2015, ISSN 0973-8916 (Print), 2230-7303 (Online)

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AbstractCatalase is an antioxidant enzyme that

catalyzes mainly the transformation of hydrogenperoxide, a reactive oxygen species, into waterand oxygen. Reactive oxygen species causeoxidative stress that plays an important role inaging process. Results of the present studysuggest that elevation of oxidative stress in liverduring aging process is due to down regulationof catalase as a consequence of increasemethylation of CpG island in its promoter regionwhich spans from -268 to +52 with respect totranslation initiation codon.

Keywords: Rat, catalase promoter, methylation,oxidative stress, aging.

IntroductionCatalase (EC 1.1.11.1) is an oxido-

reductase enzyme and an important member ofantioxidant defence system of aerobes. It playsa key role in protecting cells against reactiveoxygen species (ROS) by breaking downhydrogen peroxide molecules those aregenerated due to dismutation of superoxideradicals by superoxide dismutase (7). Ratcatalase gene is a single copy gene containing13 exons and spans 33 kb. Promoter region ofrat catalase is characterized by having eightCAAAT boxes, five GT boxes and lacks a TATAbox. The gene has multiple initiation sites (18).It has been suggested that multiple complex

formation of C/EBP-β, a transcriptional factor,which binds to catalase promoter, plays a crucialrole in its transcription (24).

Alteration in DNA methylation is consideredas a biomarker of aging (15) and has significantrole in the development of age related diseases(27). It is reported that levels of DNA methylationin the promoter regions of genes are graduallyaugmented during aging (25). However, the agerelated methylation is tissue specific (26) andindividual gene exhibits differential methylationpatterns in different tissues during progressionof age (12). Although, gene silencing byhypermethylation of promoter regions of genesin case of cancer is an established fact (10), itsrole in gene regulation of normal tissues underdifferent physiological conditions needs attention.

Liver catalase has the dominant role inintracellular detoxification of hydrogen peroxide,and several studies have confirmed its downregulation with age (21,17,4,14). However, thebiochemical basis of down regulation of catalaseduring aging is not well explained and warrantsfurther study. Since regulation of expression ofseveral genes are governed by methylation oftheir respective promoters (12), it is pertinent toexplore the role of age on catalase expressionby investigating the methylation status of itspromoter. Information on the role of methylationin regulating the expression of catalase is notavailable except for a study by Min et al. (2010)

Age- related Decrease in Rat Liver Catalase Expression isAssociated with Change in its Promoter Methylation

Pattern

Suresh Kumar Bunker, Jagneshwar Dandapat and G.B.N.Chainy*Department of Biotechnology, Utkal University, Bhubaneswar 751004, India

*For Correspondence - [email protected]

Methylation Status of Rat Catalase Promoter


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(16), where decreased expression of catalase isrecorded due to hypermethylation of CpG islandII in its promoter, as a consequence ofaugmentation of ROS in hepatocellularcarcinoma cells. To understand the role ofpromoter methylation in catalase expression wecompared methylation state of a specific regionof catalase promoter (4421 to 4740) which spansfrom -268 to +52 with respect to translationinitiation codon (+1 indicates A in the ATGtranslation start sites of catalase gene) along withits activity and translated products in the liver ofyoung (30 days), adult (90 days) and old (700days) male rats. In addition, lipid peroxidationlevel as an index of oxidative stress has beendetermined to establish a relationship betweencatalase expression and level of oxidative stress.

Materials and MethodsAnimals and experimental design: Male Wistarrats (Rattus norvegicus), obtained from theNational Institute of Nutrition (Hyderabad, India)were housed and breed in the animal room ofthe Department under standard conditions (12 hlight and 12 h darkness cycle) as per guidelinesof the Institutional Animal Ethics Committee(IAEC) regulated by the Committee for thepurpose of Control and Supervision ofexperiments on Animals (CPCSEA), Governmentof India. In the present study young (30 days),adult (90 days) and old (700 days) rats were used.Rats were sacrificed by decapitation under etheranesthesia after recording their respective bodyweights. Liver was dissected out immediately,cleaned in cold normal saline (0.9 %, w/v), patdried on filter paper and stored at -80 0C.

Lipid Peroxidation, catalase activity andcatalase western blotting: A 20% (w/v)homogenate of stored liver samples (-80 0C) wasprepared in 50 mM phosphate buffer, pH 7.4 witha Potter-Elvejhem type motor-driven glass teflonhomogenizer. Supernatant obtained aftercentrifugation of the homogenate at 1000 x g for15 min at 40C was used to determine the level ofLPx by monitoring the formation of thiobarbituricacid reactive substances (TBA-RS) in thepresence of 0.02% (w/v) butylated

hydroxytoluene to suppress the endogenousperoxidation product (19). The concentration ofTBA-RS was calculated from its extinction co-efficient, 1.56 × 105 M-1 cm-1 and was expressedas nmoles TBA-RS formed per mg protein. Apart of the homogenate was treated with tritonX-100 (final concentration 0.1 %, v/v) for 15 minand centrifuged at 10,000 x g for 15 min at 4 0C.The supernatant was used for catalase assay(4,1). One unit of CAT activity is defined as theamount of enzyme capable of catalyzing thedecomposition of 1 µmol of H

2O

2 per minute. The

activity was calculated by taking 43.6 M-1 cm-1asthe molar extinction co-efficient of H

2O

2 and finally

expressed in microkat /mg protein. Proteinconcentration of samples was estimatedaccording to the method of Bradford (2) by takingBSA as standard. Western blotting of catalasewas performed with 1000 x g supernatant of liverhomogenates prepared in 50 mM Tris-HCl buffer,pH 7.4, containing 150 mM NaCl, 104 mM PMSF,100 ìM E-64, 80 ìM aprotinin, 100 ìM leupeptin,1% triton X-100 and 0.1% SDS to avoid proteindegradation as described earlier. In brief, 25 mgof protein was resolved in 12% SDS-PAGE andtransferred to PVDF membrane at 23 mA currentfor 1h. The membrane was blocked in 5%blocking solution for 1h at room temperature. Theblot was then incubated with rabbit polyclonalanti-G3PDH (1:1000) and anti-Catalase (1:5000)for 1h at room temperature. The membrane waswashed 3 times (each 10 minutes) with washingsolution and subsequently incubated with HRP-conjugated anti-rabbit goat IgG (1:7500 forG3PDH and 1:10000 for catalase) for 1h at roomtemperature. Again the membrane was washed3 times (each 10 minutes) with washing solution.After washing, specific immunoreactive proteinswere detected in X-ray film with the help of ECLkit and band intensities were measured bydensitometry.

Methylation specific restriction enzymedigestion: 5 µg liver DNA isolated as perSambrook et al. (22) was digested in 100 µlreaction volume with 10 units of restrictionenzymes (MspI, HpaII; BamHI and HaeIII)

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separately in respective buffers as supplied bymanufacturer. Samples were incubated inshacking water bath at 37 °C for 16 h. Theenzymes were inactivated according tomanufacturer’s protocol and the digests werestored at 4 °C. Two sets of primers were used inthe present study. They have different bindingsites in the catalase promoter region. Primer S1(5'-ATCTCCCCAGCCTCTTCCCAT-3') bindscatalase promoter which contains five CCGGsites, whereas, primer S2 (5'-CTCCTTCCAATCCTGTCCCTTCTAGA-3') bindscatalase promoter which contains three CCGGsites. For both the forward primers, commonreverse primer As (5'-AGATGAAGCAGTGGAAGGAGC-3') was used. Size of PCRproducts for S1 and S2 are 320 bp and 254 bp,respectively. PCR was performed in a Thermalcycler (My Cycler, BIO RAD) using above genespecific primers in a final reaction volume of 25µl. Each PCR sample was prepared with positiveand negative controls. PCR reaction mixtureswere composed of 2 μl of restriction digests(containing 100 ng digested DNA), 2.5 μl of 10Xbuffer, 1.5 μl of 25 mM MgCl

2, 0.2 ìl of 10 mM

dNTPs, 0.2 μl of 25 µM forward and reverseprimers and 1.5 Units of TrueTM Start Taq DNApolymerase (Fermentas Life Science), andvolume was adjusted with autoclaved milli Qwater. PCR conditions were 5 min at 95 ºC, 1step cycling, 30 s at 94 ºC, 30 s at 59 ºC (S1)and 52 ºC (S2), 60 s at 72 ºC for 32-35 cyclesfollowed by 5 min final extension at 72 ºC.

Methylation specific primer PCR (MSP-PCR):To determine methylation status of catalasepromoter by MSP-PCR, we have chosen a partof catalase promoter that spans from -184 to -34as described in bioinformatic analyses (Materialsand Methods section). One µg of liver DNA wastreated with sodium bisulfite using the EpitectBisulfite conversion kit. To analyze themethylation level of the catalase promoter,primers for MSP were designed using the methprimer software-The Li Lab (www.urogene.org/methprimer). In brief, 50 ng bisulfite-treatedgenomic DNA was amplified using both

methylation-specific and unmethylation-specificprimers. Methylation specific primer sequenceswere 5'-GAGTTTTAGTGGTTAATTAGGAGGC-3'(Forward) and 5'-GTAAAACAAA AAAACCGAACGAA-3' (reverse). Unmethylation specificprimer sequences were 5'-GTTTTAGTGGTTAAT TAGGAGGTGG-3'(forward) and 5'-ACATAAAACAAAAAAACCAAACAAA-3 '(reverse). PCR conditions were 95oC for 5minutes; 35 cycles of 95oC for 30s, 54oC for 30s,72oC for 60s and 7 minutes final extension at72oC.

PCR products were analysed on 1.5%agarose gels using ethidium bromide staining andanalyzed under gel documentation system (BIORAD, Universal Hood II). Methylation andunmethylation status were determined by theband intensities of the PCR products.

Bioinformatics : Part of the rat catalase gene(Gene Bank AH 004967.1) spanning 320 bp (from4421 to 4740) was analyzed for CpG island usingthe CpG Island Searcher programme http://cpgislands.usc.edu/cpg.aspx). The CpG islandwas defined as a DNA sequences of 200 bp withGC content more than 50%. CpG island ofcatalase gene has 65.7% GC content, andobserved CpG /expected CpG ratio is 0.65.Moreover, the aforesaid region containstranscriptional as well as translational start pointswith several CCAAT sites. To recognize bindingsites for transcription factor we used theTFSEARCH programme http://www.cbrc.jp/research/db/TFSEARCH .html).

Statistical analysis: Data was presented asmean ± standard deviation (SD) and wereanalyzed by one-way analysis of variance(ANOVA) followed by Duncan’s new multiplerange tests to find out the level of significanceamong the mean values. Band intensities ofwestern blots and PCR products were quantifiedusing computer assisted densitometry Image-Quant TL, Image Analysis Software v2003.Relative densities were determined as the ratioof catalase band/G3PDH (internal control) band.



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Statistical significance between groups wasconsidered at p <” 0.001 levels.

ResultsLiver lipid peroxide level was found to be

35% higher in old rats in comparison to youngrats (Table 1). Liver catalase activity showed a35% decline in old rats in comparison to youngones. Relative level of its translated productsdecreased by 26% in liver of old rats than youngones (Table 1; Fig. 2). Ratios of PCR products ofdigested DNA samples by isoschizomers (MspI/HpaII) of different age groups of rats arepresented in Table 1. Presence and absence ofPCR products in BamHI and HaeIII digestedsamples using S1 and S2 primers served aspositive and negative controls for isoschizomer-digestion experiments, respectively (Fig. 3a,b).Ratios of band intensities of the PCR productsof MspI to HpaII for primers S1 and S2 were 49%and 65% lower in old age in comparison to youngrats, respectively (Table 1; Fig. 3c ,d). On theother hand, band intensity of MSP products ofcatalase promoter was 132% higher in the liverof 700 days rats than 30 days old ones (Table 1and Fig. 3e).

DiscussionOxidative stress is considered as a

hallmark in the process of aging (8). The elevatedlevel of LPx in liver tissues of 700 days old rats

suggests the induction of oxidative stress in theold age. The decrease in catalase expression in700 days old rats and hence a reduction inantioxidant activity could explain the elevation inlipid peroxides in aged rats. Our data are also inaccordance with earlier reports whichdemonstrated reduce catalase activity along withelevated lipid peroxidtion in the liver of aging rats(6, 17, 21). The results of the present studyadvocate that decreased expression of catalasein aged rats may be associated with impairmentof liver functions due to oxidative predominanceas advocated by Schmukar (23).

Considering the important role ofcatalase in protecting liver from oxidative stress,we thought to explore the potential mechanism

Fig. 1. Selected CpG island of promoter region of rat catalase gene from 4421 to 4740 (-268 to + 52) wasconsidered for study of methylation pattern by restriction enzyme digestion and MSP PCR. Meth Primerprogram was used to select CpG island and primers for methylation specific PCR (MSP). All parameterswere used as a default values.

Fig. 2. Representative western blots of rat livercatalase of young (30 days), adult (90 days) and old(700 days) rats and G3PDH, used as internal control.

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Fig. 3. Representative PCR products of promoter region of rat liver catalase gene of different age groupsafter digestion with MspI and HpaII restriction enzymes. DNA samples were taken from young (30 days),adult (90 days) and old (700 days) rats. (a) PCR product of MspI (lane 2 to 4) and HpaII (lane 5 to 7) with S1and As primers (b) PCR products of MspI (lane 2 to 4) and HpaII (lane 5 to 7) with S2 and As primers. (c)PCR product of BamHI (lane 2 to 4) and HaeIII (Lane 5 to 7) with S1 and As (d) with S2 and As primers andloaded against 100 bp DNA ladder (lane 1). (e) Representative MSP PCR products in the liver of young(lane 2 to 3), adult (lane 4 to 5) and old rats (lane 6 to 7) and loaded against 100 bp DNA ladder (lane 1). U-represent unmethylated specific PCR product amplified with unmethylated specific primers, M- representmethylated specific PCR product amplified with methylated specific primers.



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behind the down regulation of catalase with aging.Results of the present investigation suggest thatdifferential methylation status of CpG islands ofcatalase promoter is possibly associated with theage dependent reduction in catalase expression.The region of catalase promoter as depicted infig.1 (4421 to 4740) selected for the presentstudy, is characterized by having eighteen CpGdinucleotides and several CCAAT sites. Theregion is also characterized by the presence ofbinding sites for several transcriptional factors

such as AP1, SP1, C/EBPα, C/EBPβ andGATA1and GATA2 (18, 24). Bioinformaticsanalysis of the same region also supports thepresence of the binding sites of the abovetranscription factors. To understand the role ofmethylation in the expression of catalase withrespect to aging, we assessed methylation statusof the same region of catalase promoter byanalysing its sensitivity to methylation specificisoschizomers and methylation specific PCR.Conventionally it is believed that MspI cleaves

Table1. Age related changes in lipid peroxidation, catalase activity, translated products, ratio of band intensitiesof PCR products of digested DNA samples of MspI to HpaII and band intensities of the MSP products of thepromoter region of catalase gene in the liver of young (30 days), adult (90days) and old (700 days) old malerats. For western blotting relative band intensities were determined as the ratio of catalase band/G3PDH(internal control) band. PCR products were quantified by densitometric analyses and expressed as arbitraryunits (a.u.). Each data represent mean ± S.D. of five animals. Data having different superscripts in same rowdiffers significantly (p<” 0.05) from each other.The ratio of band intensities of PCR products of digested DNAsamples and MSP products of young rats was taken as 100%.

Parameters Age (days)

30 90 700

3.67±0.19a 4.21±0.07b 4.98±0.10c

Lipid peroxidation (nmol

TBARS/mg protein)

(%) (100 ± 5 %) (114 ± 1.9 %) (135 ± 2.7 %)

Catalase activity

(µkat/mg protein) 13.74±0.89a 10.85±0.51b 8.93 ±0.46c

(%) (100 ± 6.74) (87 ± 4.09) (65 ± 3.34)

Catalase translated product 1.85±0.06a 1.61±0.05b 1.38±0.06c

(Integrated density value)

(%) (100 ± 3.24) (87 ± 2.71) (74 ± 3.21)

Ratio MspI/HpaII

With S1 primer 1.45±0.90a 1.05±0.01b 0.74±0.12c

(%) (100±6.0a) (72±0.9b) (51±16.0c)

With S2 primer 1.59a±0.15 1.01±0.12b 0.56±0.07c

(%) (100±9.0a) (64±1.02b) (35±12.0c)

Catalase MSP product 52.30 ±4.97a 123.34±10.64b 172.10±13.75c

(Integrated density value)(%) (100±9.50a) (136±11.74b) (232±18.54c)

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CCGG site when its cytosines are unmethylated(CCGG) or the internal cytosine is methylated(Cm5CGG) whereas, HpaII cleaves CCGG whenits cytosines are unmethylated (28). Presenceof PCR bands of DNA digested samples (MspI/HpaII) of three age groups suggest that all fiveCCGG sites present in the specific region ofcatalase promoter are resistant to enzymaticcleavage. Decrease in intensities of PCRproducts of digested samples of old rats are 49%less in comparison to young animals. Thisindicates a change in methylation pattern ofcatalase promoter, which may be due to changingof sensitivity of CCGG sites to MspI and HpaII.It is possible that, in young age cytosines ofCCGG are methylated in such a manner that theyconfer resistance to MspI. It is to mention herethat MspI fails to cleave the sequence if cytosinesare methylated at m5CCGG or m5Cm5CGG (13) orat 5'-Cm5CGGCC-3' (3, 11). On the other hand,HpaII fails to cleave the sequence if its cytosinesare methylated (Cm5CGG or Cm4CGG or m5CCGGor m4CCGG) or hydroxymethylated (hm5Cm5CGG).Therefore, it is possible that changes inmethylation pattern of cytosines of CCGG in oldage make it more vulnerable to MspI and moreresistant to HpaII, resulting in the decrease ofthe ratio. The same region of catalase promoter(-184 to -34) containing 11 CpG dinucleiotides isabout 200% more methylated in liver of old ratsin comparison to young ones as evident frommethylation specific PCR (MSP) investigations.Observed inverse relationship betweenexpression of catalase and methylation of itspromoter in relation to age is in good confirmationwith a earlier report, where a negative relationbetween the expression of liver glucokinsae andmethylation of its promoter in old age wasobserved (9). It has been demonstrated thatdecrease catalase expression in hepatocellularcarcinoma cell lines is attributed to diminishedexpression of Oct-I, a transcription factor, thatregulates catalase expression (20). It is not outof context to mention that expression of catalasein hepatocellular carcinoma cells is reported toreduce as a result of hypermethylation of CpG

island II in its promoter, as a consequence ofaugmentation of ROS (16).

In summary, we have demonstrated that,hypermethylation of hepatic catalase promoteris associated with the down regulation of catalaseexpression in aged rats and contributes toelevated oxidative stress in liver. However, thedetailed mechanisms that link oxidative stress,down regulation of catalase expression,hypermethylation of catalase promoter and agingstill merit further investigation

AcknowledgementsSKB is thankful to the CSIR, Govt. of

India, for providing the fellowship and GBNCthanks the UGC, New Delhi for the award ofEmeritus Fellowship. Authors also thank to theHead, Department of Biotechnology, UtkalUniversity for providing necessary facilities tocarry out the work. Authors express their gratitudeto DST (DST-PURSE) and the DBT, Govt. ofIndia, New Delhi for financial support.

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