Tannic acid prevents azidothymidine (AZT) induced hepatotoxicity and genotoxicity along with change in expression of PARG and histone H3 acetylation

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Toxicology Letters 177 (2008) 90–96

Tannic acid prevents azidothymidine (AZT) induced hepatotoxicity and genotoxicity along with change in expression of PARG and histone H3 acetylation Kulbhushan Tikoo ∗ , Anupama Tamta 1 , Idrish Yunus Ali 1 , Jeena Gupta 1 , Anil Bhanudas Gaikwad 1 Laboratory of Chromatin Biology, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160062, Punjab,India Received 26 August 2007; received in revised form 26 December 2007; accepted 26 December 2007 Available online 18 January 2008

Abstract Azidothymidine (AZT) is known to decrease HIV virus replication and is one of the most frequently prescribed antiretroviral drugs used for AIDS treatment. Dose-limiting toxicities are the major curse associated with AZT therapy. Recently, we have reported that tannic acid; a PARG inhibitor prevents cisplatin induced nephrotoxicity. The present work was conceived to study the effect of tannic acid on AZT induced hepatotoxicity and genotoxicity. AZT induces increase in plasma levels of ALT, AST and alkaline phosphatase along with increase in micronucleus (MN) count in peripheral blood. Suggesting, AZT is hepatotoxic and genotoxic to mice. Treatment of tannic acid protects AZT induced hepatotoxicity by decreasing the ALT, AST and alkaline phosphatase levels. It also significantly reduces the oxidative damage by preventing reduction in glutathione and decreasing the level of malondialdehyde in liver of AZT treated mice. In addition, tannic acid decreases the PARG expression, PARP cleavage and histone H3 acetylation in liver of AZT treated mice. Moreover, treatment of tannic acid also decreases MN count in peripheral blood, suggesting its anti-mutagenic effect. In light of these findings we suggest the potential role of tannic acid treatment in preventing AZT induced toxicity. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: AZT; Tannic acid; Oxidative stress; Hepatotoxicity; Micronucleus and genotoxicity

1. Background Azidothymidine (AZT) is a potent inhibitor of HIV replication and the first clinically approved drug for AIDS. Mechanism involved in therapeutic action of this nucleoside analogue includes the incorporation of the AZT triphosphate into newly synthesized DNA template (Wurtzer et al., 2005), which results in chain termination of DNA synthesis and inhibition of viral

Abbreviations: AZT, azidothymidine3 -azido-3 -deoxythymidine; ROS, reactive oxygen species; PAR, poly (ADP-ribose); PARG, poly (ADP-ribose) (PAR) glycohydrolase; PARP, poly (ADP-ribose) polymerase; AST, asparatate aminotransferase; ALT, alanine aminotransferase; MN, micronucleus; NCEs, normochromatic erythrocytes; NRTIs, nucleoside reverse transcriptase inhibitor; ERK, extracellular signal regulated kinase. ∗ Corresponding author. Tel.: +91 172 2214682–87; fax: +91 172 2214692. E-mail address: [email protected] (K. Tikoo). 1 These authors contributed equally to this work. 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2007.12.012

reverse transcriptase (Cossarizza and Moyle, 2004). The major limitations of AZT chemotherapy are clinical toxicities that include dose-related bone marrow suppression manifested as severe anaemia and leucopenia, hepatic abnormalities, myopathy, limited brain uptake and short half-life in plasma and the rapid development of resistance against the virus. Pharmacokinetic studies in humans have shown that the AZT plasma half-life is approximately 1 h. Repeated and higher doses of AZT are administered for maintaining therapeutic levels in plasma, thus leading to bone marrow toxicity (Skoblov et al., 2004). These drugs are also known to produce genotoxic manifestations that include mutagenesis, chromosomal aberrations and telomere shortening which eventually lead to micronucleus (MN) formation in erythrocyte (Sussman et al., 1999; Olivero et al., 2005). Metabolic pathways that result in the phosphorylation of AZT play a crucial role in AZT-DNA incorporation (Olivero et al., 1999) and may be altered after its prolonged treatment. Recently, Olivero et al. reported that thymidine kinase

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1, the enzyme responsible for AZT mono-phosphorylation, is down-regulated during long-term exposure and thus appears to be associated with AZT induced inhibition of replication and accumulation of cells in S-phase (Olivero, 2007). In addition, activation of poly-ADP-ribose polymerase and accelerated NAD+ catabolism have been observed in case of AZT treated animals. ROS mediated oxidative damages, activated ADP-ribosylation reactions and accelerated NAD+ catabolism play important role in the development of cardiomyopathy in animal model and liver toxicity in AZT treated AIDS patients (Szabados et al., 1999; Virag, 2005). The metabolism of poly (ADP-ribose) is mediated by PARG, key enzyme regulating PARP activation. Tannic acid, a PARG inhibitor has been shown to reduce cell death which is mediated by oxidative stress (Ying et al., 2001; Uchiumi et al., 2004). Tannic acid (Gallotannin) is reported to prevent many ROS mediated drug toxicities. It is one of the important as well as functionally active antioxidant among polyphenols, possessing antioxidant, anticancer and antimutagenic properties (Zhao et al., 2005). The mechanisms underlying the protective effect of tannins include the scavenging of radicals and inhibition of superoxide radicals. Tannic acid treatment resulted in significant recovery of hepatic glutathione levels and phase-II metabolizing enzymes. It also, significantly decreases lipid peroxidation, xanthine oxidase, hydrogen peroxide generation and liver damage (Sehrawat et al., 2006). Polyphenols have been reported to quench lipid peroxidation, prevent DNA oxidative damage, and scavenge hydroxyl radical (Lin et al., 2001). The ability of several polyphenols to chelate iron or copper ions has been ascribed to their antioxidant activity (Andrade et al., 2005). Recently, we have reported the differential effects of tannic acid on cisplatin induced nephrotoxicity (Tikoo et al., 2007a). Hence, present work was under taken to study the effect of tannic acid on AZT induced toxicity. 2. Materials and methods 2.1. Chemicals AZT was generously provided as gift from Ranbaxy, India. Tannic acid was purchased from Merck. All the chemicals were purchased from Sigma (St. Louis, MO, USA), unless otherwise mentioned.

2.2. Animal and drug treatment All the experiments were approved by the Institutional Animal Ethics Committee (IAEC) and complied with the NIH guidelines on handling of experimental animals. Experiments were performed on male Swiss albino mice (20 ± 2 g). Mice were randomly divided into four groups containing six animals in each, namely, control, tannic acid control, AZT treated and AZT along with tannic acid. Different doses of AZT (400, 800 and 1200 mg kg−1 , p.o.) was studied for optimization of toxicity. At 800 mg kg−1 dose of AZT moderate liver toxicity and genotoxicity was observed in mice. Hence, further study with tannic acid was carried out with this dose. AZT was administered (800 mg kg−1 , p.o.) for 28 days. Treatment of tannic acid (5 mg kg−1 , i.p.) was done after 30 min of AZT treatment for 28 days. Similarly, in tannic acid control group tannic acid (5 mg kg−1 , i.p.) was administered for 28 days.

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2.3. Measurement of biochemical parameters Blood samples were collected from the retro orbital plexus of mice under light ether anesthesia in heparinized centrifuge tubes and immediately centrifuged at 2300 × g for the separation of plasma. Plasma was stored at −80 ◦ C until assayed. The plasma was used for the estimation of ALT, AST and alkaline phosphatase. Estimations were carried out as per manufacturer’s instruction provided with commercially available kits (Accurex Ltd., Mumbai, India).

2.4. Assessment of oxidative stress markers Oxidative stress markers were determined as described previously (Tikoo et al., 2007b). Briefly, after sacrificing mice by cervical dislocation, the livers were excised and rinsed with normal saline and weighed. After weighing, liver tissue was minced properly and the homogenate was prepared in cold phosphatebuffered saline (pH 7.4) and centrifuged at 700 × g. Supernatant was collected and used for estimation of thiobarbituric acid reacting substances (TBARS). The lipid peroxide level in animal tissues was measured according to method described by Ohkawa et al. (1979). For reduced glutathione, liver tissues were homogenized in 10 ml ice-cold homogenizing buffer combined with sulphosalicylic acid with two 10 s burst of tissue disintegrator. This tissue homogenate was used for measuring GSH content. The GSH content was estimated according to Ellmans’ method (Ellman, 1959).

2.5. Histopathology of liver Histopathology was performed as described previously (Tikoo et al., 2007a,b). Briefly, mice were anesthetized under light ether anesthesia, after surgery circulating blood was removed by cardiac perfusion with 0.1 M PBS (pH 7.4; 20–50 ml). After clearance of circulating blood, 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) was perfused for another 5 min (100–200 ml of fixative) to fix the tissues. Liver was removed from the animal, sliced transversely, and paraffin-embedded for light microscopic evaluation. Histopathological changes in liver structure were assessed in at least 25 randomly selected tissue sections from each group studied. Sections were stained with Mayer’s hematoxylin and eosin to examine cell structure.

2.6. Micronucleus count Peripheral blood slides for determining the frequency of micronucleated erythrocytes were prepared on the day of sacrifice. Slides were fixed in absolute methanol, and stained with Giemsa. For each, 2000 uniformly stained normochromatic erythrocytes (NCEs) were scored to determine the frequency of micronucleated cells, reflecting genetic damage and reflect events that occurred approximately 2–30 days previously. MN count is expressed as MN-NCEs per 1000 NCEs (% NCEs).

2.7. Protein isolation and Western blotting Nuclei isolation and western blotting were performed as described previously (Tikoo et al., 2007a,b). Briefly, total proteins were isolated from tissue homogenates by sonication and nuclei were isolated. Nuclei were suspended in low salt buffer [1% NP-40, 10 mM Tris, 10 mM NaCl, 10 mM EDTA, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin and 1 mM PMSF (phenyl methyl sulfonyl fluoride)] prior to sonication and its protein concentration was determined by Lowry method. Immunoblot analysis was performed by using Anti PARP (rabbit 1:1000, cell signaling) and anti-actin (rabbit 1:2500, Sigma, St. Louis, MO, USA) and HRP-conjugated secondary antibodies (anti-rabbit) from Santa Cruz, CA. Proteins were detected with the enhanced chemiluminescence (ECL) system and ECL Hyperfilm (Amersham Pharmacia Biotech, UK Ltd., Little Chalfont, Buckinghamshire, England).

2.8. Immunohistochemistry Liver was taken out as described earlier and processed on an automatic tissue processor (Leica). Sections (4 ␮m) were cut and mounted on slides coated

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with 3-aminopropyl triethoxy-silane. Slides were dried overnight at 20 ◦ C, then dewaxed with heat (50 ◦ C for 10 min), xylene and rehydrated in graded ethanol before washing in water. Sections were then introduced to high temperature (60 ◦ C) four times (5 min each) under 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval, after which they were allowed to stand for 20 min at room temperature. Sections were then washed twice (5 min each) in Tris-buffered saline (TBS; 0.05 M, pH 7.2) and TBST (0.05 M), respectively and blocked with normal 10% goat serum in TBS for 60 min. The primary antibody (anti Ac-histone H3, upstate and anti PARG, upstate) was diluted 1:100 in TBS and sections were incubated overnight at 4 ◦ C. Antibody solution was removed and the sections were washed in TBS twice (5 min each), then incubated for 60 min with alkaline phosphatase tagged secondary antibody (HRP-conjugated, anti-rabbit from Santa Cruz) diluted 1:1000 in TBS, followed by two washes of 5 min each in TBS. Sections were finally incubated in nitro-blue tetrazolium (337.5 ml), 5bromo-4-chloro-3-indolylphosphate and levamisole (0.001 M) in 10 ml TBS for 30–120 min. Counterstaining was performed with 0.5% aqueous light green or Harris’s hematoxylin. Sections were dehydrated in absolute ethanol and cleared in xylene before cover slipping with DPX (Meltzer et al., 1997). Immunohistochemical changes in liver structure were assessed in at least 10 randomly selected tissue sections from each group studied. The intensity of spot was graded from 1 to 4 (1, slight or no color; 2, very low color; 3, moderate blue color; and 4, very intense blue color) (Ilnytska et al., 2006). The immunohistochemistry score was expressed as mean ± S.E.M. for each experimental group.

3.2. Effect of tannic acid on oxidative damage in AZT treated mice Tannic acid treatment showed significant change in TBARS and GSH levels in AZT treated mice. AZT treated mice show higher levels TBARS as compared to control mice (see Table 1). Treatment with tannic acid significantly reduces the level of TBARS in liver of AZT treated mice. TBARS level close to control animal levels suggests protection from oxidative stress. GSH plays a pivotal role in the defense against oxidative stress, as a cofactor of glutathione peroxidases (selenium dependent and independent) which participates in the elimination of hydrogen peroxide and lipid hydroperoxides. Glutathione content in the liver was significantly lower in AZT treated mice relative to the control group (see Table 1). However, treatment of tannic acid results in elevation of GSH level in liver. Suggesting, prevention against AZT induced oxidative damage by elevating GSH and decreasing TBARS levels. 3.3. Effect of tannic acid on micronucleus count in peripheral blood of AZT treated mice

2.9. Statistical analysis Experimental values are expressed as mean ± S.E.M. Comparison of mean values between various groups was performed by one way-analysis of variance (one way-ANOVA) followed by post hoc Tukey test. P value <0.05 was considered to be significant.

3. Results 3.1. Effect of tannic acid on plasma ALT, AST and alkaline phosphatase level in AZT treated mice ALT and AST is an enzyme marker of liver damage, as these are elevated in many liver diseases. Treatment of AZT causes a significant increase in plasma levels of ALT and AST, suggesting hepatotoxicity (see Table 1). However, treatment of tannic acid significantly reduced the increased plasma levels of ALT and AST levels to normal in AZT treated mice, indicating its hepatoprotective effect (see Table 1). Alkaline phosphatase is an enzyme, or more precisely a family of related enzymes that is produced in the bile ducts and sinusoidal membranes of the liver but it is also present in many other tissues. An increase in the level of serum alkaline phosphatase occurred on AZT treatment. However, treatment of tannic acid decreases it up to certain extent (see Table 1). Suggesting, tannic acid prevents AZT induced hepatotoxicity.

Micronuclei represent fragments of chromosomes or entire chromosomes that do not get incorporated into the daughter nuclei during cell division. Drugs that have clastogenic (induce chromosomal breakage) as well as aneugenic activities (damage to the spindle apparatus) cause an increase in the MN frequency. Treatment of AZT leads to a significant increase in MN count in peripheral blood as compared to control animal (see Table 1 and Fig. 1). However, treatment with tannic acid results in significant reduction in MN count in peripheral blood, indicating its anti-mutagenic effect. 3.4. Tannic acid treatment prevents histopathological changes in liver of AZT treated mice Tannic acid shows its protective effect by reduction in vacuolation, fine inflammatory infiltrations (around single necrotic hepatocytes, these included hyperplastic Kupffer cells and single lymphocytes) and fatty degeneration of hepatocytes most frequently around the hepatic triad or in the form of foci spread all over the parenchyma caused by AZT treatment in liver (see Fig. 2). Thus, our histopathological data supports the protection observed in biochemical and oxidative stress parameters by tannic acid treatment.

Table 1 Effect of tannic acid on ALT, AST, alkaline phosphatase, MDA, GSH and micronucleus count in AZT treated mice Alkaline phosphatase (U/I) Control Tannic acid control AZT AZT + tannic acid

178 180 368 208

± ± ± ±

6 14 40***a 60**b

AST (U/I) 27 24 68 37

± ± ± ±

4 4 11***a 3**b

ALT (U/I) 57 43 90 55

± ± ± ±

2 2 8***a 4**b

TBARS (␮M/mg protein) 0.07 0.07 0.17 0.09

± ± ± ±

0.01 0.01 0.02***a 0.01**b

GSH (␮M/mg protein) 43 42 16 36

± ± ± ±

Each value is represented as mean ± S.E.M. (n = 6), a vs Control, b vs AZT treated ***P < 0.001, **P < 0.01 and *P < 0.05.

1 5 2***a 3**b

MN Count (MN/1000 cells) 2 2 13 5

± ± ± ±

0.33 0.41 0.50***a 0.35*b

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Fig. 1. Representative Giemsa stained peripheral blood slides of AZT treated mice showing micronucleus. (A) Control, (B) tannic acid, (C) AZT and (D) AZT + TA. Arrow indicates micronucleus.

3.5. Effect of tannic acid on expression of PARG and PARP in liver of AZT treated mice It has been reported that PolyADP-ribose polymerases (PARPs) catalyzes post-translational modification of nuclear

proteins by polyADP-ribosylation (Cohen-Armon et al., 2007). The catalytic activity of the PARP-1 is stimulated by DNA strand breaks and PARP-1 activation is required for initiation of DNA repair. PARG is another enzyme involved in regulating activation of PARP (Ying et al., 2001; Uchiumi et al., 2004). Therefore,

Fig. 2. Representative Mayer’s hematoxylin- and eosin-stained sections from control (A), control tannic acid treated (B), AZT treated (C) and tannic acid treated AZT rat liver (D). Magnification is at 100×.

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we studied the expression of PARG in the liver of AZT treated mice. Fig. 3C shows that after AZT treatment there is significant increase in PARG expression as compared to control group. However, tannic acid, a PARG inhibitor prevents AZT induced increase in expression of PARG (see Fig. 3D) as well as the cleavage of PARP in mice liver (see Fig. 4, lane d).

acetylation suggested us to check the level of histone acetylation in AZT treated mice. Fig. 3C shows that there is increase in histone H3 acetylation in liver after AZT treatment. Whereas, treatment of tannic acid prevents the increase in acetylation of histone H3 (see Fig. 3D). 4. Discussion

3.6. Effect of tannic acid on acetylation of histone H3 in liver of AZT treated mice Recent report suggests that PARP-1 activation enhanced ERK-induced Elk1-phosphorylation and core histone acetylation in cortical neurons treated with nerve growth factors and in stimulated cardiomyocytes (Cohen-Armon et al., 2007). These observations and a link between PARP-1 activation and histone

Our study demonstrates treatment of tannic acid (5 mg kg−1 ) along with AZT (800 mg kg−1 ) for 28 days shows a protective effect on the cellular damage in mice liver. Treatment of AZT is reported to cause severe side effects; one of them is dilated severe liver toxicity. Probable mechanism for AZT induced toxicity is alterations in liver mitochondrial DNA, alteration in oxidative phosphorylation coupling and changes in fine ultra structure of

Fig. 3. Immunostaining of PARG in liver sections of control (A), control tannic acid treated (B), AZT treated (C) and tannic acid treated AZT rat liver (D). Magnification is at 100×. Similar results were obtained in three independent set of experiments. Each value is represented as mean ± S.E.M. (n = 3), a: vs Control and b: vs AZT treated ***P < 0.001 and **P < 0.01.

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Fig. 4. Western blot of PARP in kidney after tannic acid treatment in AZT treated mice. Where, lane a: control, lane b: AZT, lane c: tannic acid and lane d: tannic acid + AZT. Results were normalized with respect to actin in respective controls. Similar results were obtained in three independent set of experiments. All the values were represented as mean ± S.E.M. (n = 3), ***P < 0.001; a: vs Control and b: vs AZT.

liver mitochondria. However, the precise mechanism of toxicity is still unknown. Generation of reactive oxygen species (ROS) in mitochondria plays an important role in development of AZT induced toxicity in rat. AZT is reported to cause lipid peroxidation and oxidative damage to DNA in mouse cardiac mitochondria (De la Asuncion et al., 1999, 2004). Role of peroxide production in oxidative damage caused by AZT has been documented in hepatocytes (Majid et al., 1991). Antioxidants like vitamin C and E have been reported to prevent the AZT induced glutathione depletion (De la Asuncion et al., 2004). Our results indicate that tannic acid treatment prevents AZT induced hepatotoxicity by decreasing the lipid peroxidation and increasing glutathione in AZT treated mice. The antioxidant activity of tannic acid has been previously attributed to its capacity to form a complex with iron ions, interfering with the Fenton reaction. At low doses in in vitro system tannic acid possesses an *OH trapping activity (Andrade et al., 2005), which may be another possible mechanism for protection against hepatotoxicity. Oxidative stress also leads to an increase in liver enzymes which are specific marker of liver toxicity. Treatment of AZT leads to a significant increase in the levels of liver enzymes like alkaline phosphatase, ALT and AST. However, treatment of tannic acid significantly reduced the elevated levels of these enzymes, suggesting its role in preventing hepatotoxicity. Our histopathological data also supports the protection observed in biochemical parameters, such as reduction in vacuolation, fine inflammatory infiltrations (around single necrotic hepatocytes, these included hyperplastic Kupffer cells and single lymphocytes) and fatty degeneration of hepatocytes, most frequently around the hepatic triad or in the form of foci spread all over the parenchyma caused by AZT treatment. Treatment of tannic acid decreases central vessel congestion and degenerative changes in hepatocytes of AZT treated mice liver. AZT treatment leads to

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enlargement of central vein and cell lining suggestive of centrilobular necrosis which is partially improved by treatment of tannic acid. Nucleoside reverse transcriptase inhibitor (NRTIs), inhibits viral replication because they lack a hydroxyl group at the 3rd position of the ribose ring and when incorporated into viral DNA, act as chain terminators (Meng et al., 2000). For NRTIs to be effective against HIV, they must be taken up by the host cell, phosphorylated by a series of cellular enzymes to the triphosphate form, bind at the polymerase active site and can mimic as a substrate that can be incorporated into viral DNA by HIV-1 RT. Because NRTIs are analogs of normal nucleotides, they can also be incorporated into the DNA of the host and that results in toxicity. The other possible mechanism leading to hepatotoxicity is generation of ROS, which causes DNA fragmentation. DNA damage results into MN formation which circulates in peripheral blood and can be counted in blood smear. Our results clearly show the genotoxic potential of AZT at a minimum dose of 800 mg kg−1 , as there is presence of MN in the peripheral blood of AZT treated animals. Treatment of tannic acid (5 mg kg−1 ) decreases the frequency of micronuclei in AZT treated animals. Tannic acid has been reported to have anticlastogenic and antimutagenic properties in vivo. Oral administration of tannic acid reduced the frequencies of micronuclei induced by mitomycin C, ethyl nitrosourea (ENU) or 4-nitroquinoline 1-oxide in mouse bone marrow cells (Sasaki et al., 1990). Poly(ADP-ribose)-polymerase-1 (PARP-1) and poly(ADPribose) (PAR) are emerging key regulators of chromatin superstructure and transcriptional activation (Rapizzi et al., 2004). Activated PARP synthesizes branching (ADP-ribose)n polymers from NAD and attaches the polymer to glutamate or aspartate residues of suitable acceptor proteins, including PARP1 itself (automodification), histones, DNA repair enzymes and transcription factors. Tannic acid has been reported to inhibit PARG, the catabolic enzyme of PAR metabolism (Bakondi et al., 2002; Erdelyi et al., 2005). Moreover, recently we have reported the protective effect of tannic acid on cisplatin induced nephrotoxicity. In cisplatin treated rats, treatment of tannic acid inhibits PARG thereby instigating a negative feedback inhibition loop leading to inactivation along with a decrease in expression of PARP (Tikoo et al., 2007a). Decrease in AZT induced hepatotoxicity by tannic acid treatment may well be explained if we assume that tannic acid inhibits PARG activation (see Fig. 3D). Chromatin is a compact structure, and decondensation or hyperacetylation facilitates the “opening” of higher order chromatin structure to facilitate access of transcriptional activators and proteins, and simultaneously becomes more susceptible for ROS mediated damage, this may lead to severe toxicity. This chromatin remodeling is necessary to allow the access of the DNA repair machinery to the damaged areas of chromosomes. DNA damage induced by UV light irradiation increases acetylation of histones, indicating that relaxation of chromatin is an initial step in DNA repair. Histones are hyperacetylated after DNA damage for allowing interaction of DNA repair machinery to DNA (Ramanathan and Smerdon, 1986). Similarly, recent report shows that PARP activation also enhances core histone acetylation (Cohen-Armon et al., 2007). Our results also show

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