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Differential effects of tannic acid on cisplatin induced nephrotoxicity in rats
FEBS Letters 581 (2007) 2027–2035
Differential effects of tannic acid on cisplatin induced nephrotoxicity in rats Kulbhushan Tikoo*, Deepak Kumar Bhatt, Anil Bhanudas Gaikwad1, Vikram Sharma1, Dhiraj G. Kabra1 Laboratory of Chromatin Biology, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India Received 7 February 2007; revised 31 March 2007; accepted 10 April 2007 Available online 24 April 2007 Edited by Varda Rotter
Abstract Cisplatin is a widely used antineoplastic drug. Major drawback of cisplatin therapy is its nephrotoxicity. The objective of this study was to check the effect of tannic acid on cisplatin induced nephrotoxicity. Post-treatment of tannic acid prevents cisplatin (5 mg/kg) induced nephrotoxicity and decreases poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 and hypoacetylation of histone H4. In contrast, co-treatment of tannic acid potentiates the nephrotoxicity. Comparative nephrotoxicity studies show that co-treatment of tannic acid with reduced dose of cisplatin (1.5 mg/kg) developed almost similar nephrotoxicity. MALDI protein profiling of plasma samples provides indirect evidence that tannic acid co-treatment increases bioavailability of cisplatin. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cisplatin; Nephrotoxicity; poly(ADP-ribose) polymerase; Histone H4; p-p38 and Tannic acid
1. Introduction Cisplatin (CP) remains the drug of choice in many platinum based chemotherapy regimens. It acts by damaging DNA owing to platination to form covalent platinum DNA adducts [1]. DNA damage elicits a series of signal transduction cascades, involving chromatin remodeling, which eventually lead to DNA repair, cell cycle arrest or apoptosis [2–5]. The therapeutic effects of cisplatin are dose dependent but the chief limit to its promising efficacy as an antineoplastic drug is its nephrotoxicity [6,7]. Use of other means to prevent nephrotoxicity e.g. hydration protocols [8], antioxidant supplementation [9] have proven to be partially successful due to the repeated dosing schedules of CP. Therapeutic interventions aimed at ameliorating renal damage require the use of combination * Corresponding author. Fax: +91 172 2214692. E-mail address: [email protected] (K. Tikoo). 1
Authors contributed equally.
Abbreviations: CP, cisplatin; PARP, poly(ADP-ribose) polymerase; PARG, poly(ADP ribose) glycohydrolase; BUN, blood urea nitrogen; MAPK, mitogen activated protein kinases; ROS, reactive oxygen species
therapies. This indicts the involvement of reactive oxygen species (ROS) generation as a mediator in inflicting nephrotoxic insult in CP therapy. Interaction of cisplatin with CYP2E1 results in the generation of reactive oxygen metabolites that causes renal injury and initiates apoptosis. Recent report shows that cytochrome P450 2E1 null mice provide novel protection against cisplatin induced nephrotoxicity and apoptosis [10]. Polyphenolics are known for their potent antioxidant and free radical scavenging properties. It significantly affects the pathways which are involved in the detoxification of chemical carcinogens particularly those in which CYP2E1 and NAD(P)H:quinone oxidoreductase (NQO1) are involved [11]. It is also reported that administration of tannic acid (gallotannin) resulted in a marked decrease in the expression of hepatic CYP2E1, but there are no reports which address the relevance of this polyphenol with respect to its role in mediating or preventing the toxic effects. In addition to tannic acid, other tannins have been shown to exert various biological effects ranging from anti-inflammatory to anticancer and antiviral effects [12–16]. The mechanism underlying the antiinflammatory effects of tannins include the scavenging of radicals; antioxidant effect [17,18] and inhibition of the expression of inflammatory mediators, such as some cytokines [12], including nitric oxide synthase, and cylooxgenase2[19]. Renal tubular apoptosis has been implicated as a final common pathway in response to a variety of cellular stresses at intensity below the threshold for necrosis [20,21]. Poly(ADPribose) polymerase (PARP), an enzyme involved in DNA damage and repair. It is also activated in conditions of oxidative stress caused by depletion of antioxidant enzymes by CP [22,23]. Poly(ADP-ribose) glycohydrolase (PARG) is another enzyme that keeps PARP in active state by hydrolytic cleavage of poly(ADP-ribose) (PAR) [24–26]. Inhibition of PARP can be obtained by the use of PARP/PARG inhibitors [27]; PARG inhibitors have shown cytoprotection against various tissue injuries in vivo as well as in vitro cell systems [28]. Tannic acid, a PARG inhibitor showed cytoprotection against ischemic reperfusion injury where oxidative stress is known to play a key role [29,30]. Novel strategies aimed at minimizing nephrotoxicity while enhancing its antineoplastic efficacy are incomplete until a more basic understanding of its toxicity is obtained. The objective of the present study was to study the effect of tannic acid in cisplatin induced nephrotoxicity.
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.04.036
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2. Materials and methods 2.1. Reagents and solutions cis-Diamminplatinumdichloride (cisplatin, CP) was obtained from Dabur India Pvt. Ltd. as a gift sample. Tannic acid was obtained from Merck, Germany. Trypsin was purchased from Promega Corporation (UK). All the other chemicals were purchased from Sigma (St. Louis, MO). Cisplatin solution was made freshly in saline (0.9% NaCl). Tannic acid was prepared freshly in phosphate buffer of pH 7.4. 2.2. Animal treatment and experimental protocol 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 Sprague–Dawley rats in the weight range of 170–190 g which were procured from the central animal facility of the institute, kept at controlled environmental conditions with room temperature 22 ± 2 C and 12 h light/dark cycles. Rats were divided at random into different groups, eight animals in each group. In co-treatment group single dose of tannic acid (10 mg/kg) was administered just after the CP (5 mg/kg) treatment. In post-treatment group three consecutive doses of tannic acid (10 mg/kg) were given from next day of CP treatment. Drugs were administered by intraperitoneal (i.p.) route. For comparative nephrotoxicity profile, rats were divided randomly into two groups, received either a single injection of CP (1.5 mg/kg,) or co-treatment of CP (1.5 mg/kg) with single dose of tannic acid (10 mg/ kg). Rats were weighed prior to injection and four day after CP treatment. Blood samples were collected on fifth day, from the retro orbital plexus of rats under light ether anesthesia in heparinized centrifuge tubes and immediately centrifuged at 2300 · g for the separation of plasma. Plasma was stored at 20 until assayed. 2.3. Estimation of plasma creatinine, blood urea nitrogen (BUN) and plasma albumin Plasma creatinine concentration was measured by the picric acid colorimetric method [31]. A picric acid solution was made by a tenfold dilution of saturated picric acid with 1% NaOH. Plasma (50 ll) was added to picric acid–NaOH solution (200 ll) in a micro titer plate. The samples were allowed to incubate for 15 min before being read at 490 nm in a micro plate reader. A standard curve was prepared on the same assay plate to derive the creatinine concentration. BUN and plasma albumin were determined colorimetrically using commercially available standard kits. 2.4. Histopathological procedures Rats 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. Kidneys were removed from the animal, decapsulated, sliced transversely, and paraffinembedded for light microscopic evaluation. Histopathological changes in kidney structure were assessed in at least 25 randomly selected tissue sections from each group studied. Sections were stained with Meyer’s hematoxylin and eosin to examine cell structure. Slides were observed under Olympus microscope (Model no. BX51).
2.5. Nuclei isolation and Western blotting Nuclei were isolated and acid soluble proteins were extracted according to method described previously [32]. For nuclear protein, nuclei were suspended in buffer (1% NP-40, 10 mM Tris, 10 mM NaCl, 10 mM EDTA, 10 lg/ml aprotinin, 10 lg/ml leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF)) and its protein concentration was determined by the method of Lowry et al. [33]. For Western blot analysis, nuclear proteins were transferred onto PVDF membrane (Bio-Rad) and immunoblot analysis was performed by using the anti-acetyl histone H4 (rabbit monoclonal 1:5000, Upstate), anti p38, anti p-p38 (rabbit 1:500, Santa Cruz Biotechnology Inc.), anti-PARP (rabbit 1:250, Upstate), anti actin (rabbit 1:2500, Sigma), and HRP-conjugated secondary antibodies (anti-rabbit, Calbiochem). Proteins were detected with the enhanced chemiluminescence (ECL) system and ECL Hyperfilm (Amersham Pharmacia Biotech). 2.6. MALDI plasma protein profiling For MALDI plasma protein profiling, 16 SD rats of weight 170– 190 g were divided into four groups, four animals in each group. In the first group vehicle for CP and tannic acid was administered, in second group CP (5 mg/kg) was injected, in the third group co-treatment of tannic acid (10 mg/kg) with cisplatin (5 mg/kg) was given and the fourth group was treated with tannic acid (10 mg/kg). Blood samples were collected and plasma was separated by centrifugation for 10 min at 2300 · g and stored at 20 C until analysis. After 20 times dilution of plasma, plasma protein was estimated [33]. Then 10 lg proteins from each sample was digested for over night at 37 C with 100 ng of modified trypsin (Promega Corporation, UK). For protein mass spectra 1 ll of the digested protein was mixed with 1 ll alpha-cyano hydroxy cinnamic acid solution matrix, dried and washed twice with 0.1% Trifluoroacetic acid. Protein profiles were accumulated from dried-droplet deposits of matrix/peptide mixture by MALDI-TOF mass spectrometer (Bruker Daltonics flexanalysis), externally calibrated using peptide standard mix (Bruker Daltonics) and 200 shots were accumulated to give an acceptable spectrum. For each digest, triplicate MALDI spots were deposited and analyzed. Ion signals above a signal-to-noise (S/N) ratios of 10 were considered for comparison. Peak intensities ratios of treated to control samples were calculated by averaging the peak intensities followed by t-tests of the intensity of each individual peaks. 2.7. Statistical analysis Data are expressed as means ± S.E.M. Statistical comparison between different groups were done using one-way analysis of variance (ANOVA) followed by tukey multiple comparison test, to detect the difference between various groups. P value less than 0.05 was considered to be significant.
3. Results 3.1. Cisplatin induced changes in body weight CP induced nephrotoxicity can easily be assessed by decrease in body weight, urine out put and water intake. CP treatment leads to a significant decrease in body weight as
Table 1 Changes in body weight and biochemical parameters by co and post treatment of tannic acid on CP induced nephrotoxicity in SD rats Treatment groups
Change in body wt (g)
BUN (mg/dl)
Creatinine (mg/dl)
Albumin (g/dl)
Vehicle control (0.9% saline) Tannic acid (10 mg/kg) control co-treatment Tannic acid (10 mg/kg) control post-treatment CP treated (5 mg/kg) CP (5 mg/kg) + tannic acid (10 mg/kg) co-treatment CP (5 mg/kg) + tannic acid (10 mg/kg) post-treatment
19 ± 2 17 ± 1 9.4 ± 1.3 (–)11 ± 3***a (–)14 ± 2***ab (–)8 ± 2.5***a
21 ± 0.79 20 ± 1.07 19 ± 0.23 85 ± 11.7***a 124 ± 12***ab 40 ± 4.18***ab
0.68 ± 0.04 0.84 ± 0.08 0.79 ± 0.02 2.18 ± 0.24***a 4.35 ± 0.21***a 1.25 ± 0.15**ab
3.67 ± 0.03 3.34 ± 0.08 2.9 ± 0.04*a 3.27 ± 0.09***a 3.08 ± 0.13***a 2.8 ± 0.09**ab
Biochemical parameters were estimated after 5 days of CP (5 mg/kg) and tannic acid (10 mg/kg) treatment, as described in Section 2. All values are expressed as means ± S.E.M. (n = 8), ***P < 0.001, **P < 0.01, *P < 0.05, a vs vehicle control group, b vs tannic acid group or CP treated group.
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served suggesting development of nephrotoxicity (data not shown).
Table 2 Comparative nephrotoxicity profiling Treatment groups
BUN (mg/dl)
Creatinine (mg/dl)
Vehicle control (0.9% saline) Tannic acid control CP treated (5 mg/kg) CP treated (1.5 mg/kg) CP (1.5 mg/kg) + tannic acid (10 mg/kg)
21 ± 0.79 20 ± 1.07 85 ± 11.7*** 28 ± 3.67*** 62 ± 11***
0.68 ± 0.04 0.84 ± 0.08 2.18 ± 0.24*** 1.17 ± 0.08*** 1.38 ± 0.05***
Changes in biochemical parameters by co-treatment of tannic acid on cisplatin induced nephrotoxicity in SD rats. Biochemical parameters were estimated after 5 days of cisplatin (5 mg/kg and 1.5 mg/kg) and tannic acid (10 mg/kg) treatment, as described in Section 2. All values are expressed as means ± S.E.M. (n = 5), ***P < 0.001 vs vehicle control group.
compared to control rats (Table 1). Significant increase in water intake and urine output of CP treated rats was also ob-
3.2. Changes in BUN, creatinine and albumin levels by co- and post-treatment of tannic acid with cisplatin CP treated rats developed polyuric acute renal failure as assessed by measuring different biochemical parameters like BUN, plasma creatinine and plasma albumin. The BUN level in CP treated rats increased significantly to reach 85 ± 11.7 mg/ dl (21 ± 0.79 mg/dl in control rats). Plasma creatinine level was also increased significantly to reach 2.18 ± 0.24 mg/dl (0.68 ± 0.04 mg/dl in control rats) after CP treatment. Similarly, plasma albumin level decreased significantly to reach 3.27 ± 0.09 g/dl (3.67 ± 0.03 g/dl in control rats). Co-treatment of tannic acid with CP further increases the nephrotoxicity as assessed by significant increase in BUN to reach 124 ± 12 mg/ dl (85 ± 11.7 mg/dl in CP treated rats), plasma creatinine to reach 4.35 ± 0.21 mg/dl (2.18 ± 0.24 mg/dl in CP treated rats)
Fig. 1. Histopathological changes in kidney after co- and post-treatment of tannic acid along with cisplatin. Transverse section of normal control rat kidney (A) and (B), CP treated kidney (C) and (D), co-treatment of tannic acid (E) and (F) and post-treatment of tannic acid (G) and (H). Sections were stained with Mayer’s hematoxylin counterstained with eosin and observed under magnification of 40· and 100· as described in Section 2. fi indicates cells undergoing apoptotic cell death.
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and decrease plasma albumin level 3.08 ± 0.13 g/dl (3.27 ± 0.09 g/dl in CP treated rats). In contrast to co-treatment of tannic acid, post-treatment of tannic acid significantly decreases the nephrotoxicity in the CP treated rats. The BUN and plasma creatinine level in tannic acid post treated rats decreased significantly from 85 ± 11.7 to 40 ± 4.18 mg/dl and 2.18 ± 0.24 to 1.25 ± 0.15 mg/dl, respectively (Table 1). 3.3. Effect of co-treatment of tannic acid with reduced dose of cisplatin (comparative nephrotoxicity) Co-treatment of tannic acid with CP increases the nephrotoxicity of cisplatin. Comparative nephrotoxicity study was planned to find out whether tannic acid co-treatment with reduced dose of CP (1.5 mg/kg) leads to a similar nephrotoxicity profile as with the routinely used dose of CP (5 mg/kg). Table 2 shows that co-treatment of tannic acid with CP (1.5 mg/kg) developed almost equivalent polyuric acute renal failure as in
A
116 kDa 85 kDa 66 kDa
PARP Actin 480
***b
PARP % of Control
400 320
*a
240 160
*b
80 0 a
B
b
c
CP (5 mg/kg) treated rats. There is significant increase in BUN levels 62 ± 11 mg/dl (21 ± 0.79 mg/dl in control rats) and plasma creatinine levels 1.38 ± 0.05 mg/dl (0.68 ± 0.04 mg/dl in control rats). 3.4. Cisplatin induces histopathological changes in rat kidney In the histopathology of kidney, while the normal control group displays normal kidney histology (Fig. 1A and B), extensive tubular epithelial cellular necrosis, desquamation, vacuolization and swelling were observed in the CP treated kidneys (Fig. 1C and D). Co-treatment of tannic acid with cisplatin increases the tubular cell damage (Fig. 1E and F). However, post-treatment of tannic acid resulted in remarkable improvement in the histological appearance and reduction in tubular cell damage (Fig. 1G and H). 3.5. Cisplatin induces PARP cleavage and phosphorylation of p38 Several reports suggest the role of PARP and phosphorylation of p38 in cisplatin induced nephrotoxicity [23,34]. We studied the PARP cleavage and phosphorylation of p38 in the kidney of the cisplatin treated rats. Fig. 2A, lane b shows that after cisplatin treatment there is significant increase in PARP cleavage as compared to control group (Fig. 2A, lane a) and it increases further by co-treatment of tannic acid (Fig. 2A, lane c). However, there is a decrease in PARP cleavage by the post-treatment of tannic acid (Fig. 2A, lane d). Suggesting that PARP activation is more pronounced in the co-treatment group as compared to the CP treated group. Compared to control animals phosphorylation of p38 is found to be increased by cisplatin treatment (Fig. 2B, lanes a and b) and it is further increased by co-treatment (Fig. 2B, lane c). But there is significant decrease in phosphorylation of p38 by the post-treatment of tannic acid (Fig. 2B, lane d) as compared to CP treated rats. Suggesting that kidney cells are undergoing apoptotic cell death and co-treatment of tannic acid potenti-
d
p-p38 Ac-H4 p38 Total H4 210
**a
120
150
100
***b
120 90 60 30 0
Ac-Histone H4 % of Control
p-p38 % of control
180
***b
80
***b
60
***a
40
*b
20
a
b
c
d 0
Fig. 2. Effect of tannic acid co-treatment and post-treatment on PARP cleavage and phosphorylation of p38 in cisplatin treated kidney. (A) Western blot of PARP and (B) phosphorylation of p38. Lane a, total cellular proteins of kidney of control animal; lane b, total cellular proteins of kidney of cisplatin treated animal; lane c, total cellular proteins of kidney of tannic acid co treated animal and lane d, total cellular proteins of kidney of tannic acid post treated animal. Anti actin and anti p38 antibody was used as loading control. Results were normalized with respect to actin and p38 band intensities in respective controls. Similar results were obtained in three independent set of experiments. ***P < 0.001, **P < 0.01 and *P < 0.05 a vs control, b vs cisplatin.
a
b
c
d
Fig. 3. Effect of tannic acid co-treatment and post-treatment in cisplatin treated rat kidney on histone H4 acetylation. Lane a, acid soluble nuclear proteins of control animal: lane b, acid soluble nuclear proteins of cisplatin treated animal: lane c, acid soluble nuclear proteins of tannic acid co treated animal and lane d, acid soluble nuclear proteins of tannic acid post treated animal. Anti H4 antibody was used as a loading controls. Results were normalized with respect to H4 band intensities in respective controls. Similar results were obtained in three independent set of experiments. ***P < 0.001 and *P < 0.05 a vs control, b vs cisplatin.
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035
ates apoptosis while post-treatment of tannic acid prevents it by decreasing PARP cleavage and phosphorylation of p38. 3.6. Hypoacetylation of histone H4 in cisplatin induced nephrotoxicity CP is known to cause histone H3 phosphorylation and hyperacetylation of histone H4 in cancer cells in vitro [35].
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We studied the changes in level of acetylation of histone H4 in the kidney of the cisplatin treated rats. As compared to control rat kidney, CP treatment decreases acetylation of histone H4 (Fig. 3, lanes a and b). In case of co-treatment of tannic acid there is further decrease in acetylation of H4 as compared to CP treated rats (Fig. 3, lane c). However, in contrast to cotreatment, post-treatment of tannic acid prevents decrease in
Fig. 4. Mass spectra of plasma proteins showing changes in plasma protein binding by co-treatment of tannic acid on cisplatin treated rats. MALDI mass spectra of plasma protein (10 lg) digested with trypsin. Plasma samples of control (A) cisplatin treated (B) tannic acid (C) and tannic acid co administered along with cisplatin (D). Blood was taken out after 15 min of treatment; plasma was separated from blood, digested by modified trypsin (100 ng) and subjected to MALDI TOF analyses as described in Section 2. Similar results were obtained in three independent set of experiments.
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the acetylation of histone H4 in CP treated rats (Fig. 3, lane d). Our data and other reports suggest that CP induces different histone modifications in different cell types. 3.7. MALDI plasma protein profiling Potentiation of nephrotoxicity by co-treatment of tannic acid and comparative nephrotoxicity studies suggested us to check the bioavailability of CP after tannic acid co-treatment. We applied mass spectroscopic approach which provides indirect evidence regarding CP/tannic acid and plasma protein
binding. Plasma protein spectra were taken after different treatments. In each spectrum, 200–300 peaks were observed, ion signals having sufficient S/N ratios were considered useful in further analyses (Fig. 4). In comparison to tryptic digested plasma of control animals major peptide peaks (e.g. peptides having m/z 1266, 1299, 1440, 1455 and 1479) were suppressed after cisplatin treatment in plasma taken after 15 min of drug injection (Fig. 4B). Tannic acid treatment also suppresses same peaks which are suppressed by CP and were present in control samples (Fig. 4C).
Fig. 4 (continued)
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035
Peptide mass (m/z) - 1479
A
Control
200000
Peak intensity
Cisplatin Co-treatment
150000
Tannic acid 100000
a
***
a
***
50000
a
***
0
B
Peptide mass peak (m/z)-1479 0.5
Cisplatin
Peak intensity ratio (treatment/control)
Co-treatment 0.4
Tannic acid
0.3 0.2 0.1 0
Fig. 5. Peak intensity obtained from mass spectra of plasma protein (10 lg) digested with modified trypsin (100 ng). Peak intensities ratios of treated to control samples were calculated by averaging the peak intensities followed by t-tests of the intensity of the peak. (A) Comparative change in peak intensity of albumin peptide identified by MSMS spectra and having mass (m/z) 1479 after 15 min of drug treatment. (B) Bar diagram of peak intensity ratio of peptide mass (m/ z) 1479 after 15 min of drug treatment. Results are expressed as means ± S.E.M. of three independent experiments performed in triplicates, ***P < 0.001 a vs control.
Furthermore, co-treatment of tannic acid with CP resulted in further suppression of these peaks in the mass spectra of tryptic digested plasma proteins (Fig. 4D). These results indicate the involvement of competitive binding between CP and tannic acid to form cisplatin–protein or tannic acid–protein adducts. The possibility of same binding site for CP and tannic acid is indicated by the suppression of same peaks e.g. major peptide peak of mass 1479 by CP as well as tannic acid (Fig 5A and B).
4. Discussion Present study was conceived to study the effect of tannic acid on CP induced nephrotoxicity. Here, we provide evidence that co-treatment of tannic acid with CP increases the nephrotoxicity while the post-treatment prevents it up to certain extent. Nephrotoxic damage by cisplatin shows increase in BUN and creatinine levels. Comparative nephrotoxicity profiling suggests that tannic acid increases the CP induced nephrotoxicity. The elevated levels of BUN and creatinine suggest an increase in the toxicity of cisplatin in the presence of tannic acid and this potentiating effect of tannic acid can be observed at both therapeutic as well as sub therapeutic dose levels. Moreover, histopathology examination of kidney sections also supports our biochemical data. These results indicate the central role of tannic acid in potentiating the nephrotoxic effect of CP and point towards its profound clinical implications. It can be argued that tannic acid also shows a protective effect
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owing to its antioxidant action but the fact that this polyphenol exerts a modulatory effect depending upon the time of administration. CP induced apoptosis involves PARP cleavage, caspase-3 and bcl-2 in renal epithelial cells [36]. We observed a significant increase in PARP cleavage in CP treated rat kidneys, indicating cells are undergoing apoptotic cell death. Co-treatment of tannic acid resulted in further increase in PARP cleavage while post-treatment prevents its cleavage. Decrease in cleavage of PARP in post-treatment group might be due to the inhibition of PARG by tannic acid, which is a known PARG inhibitor [29,30]. Tannic acid inhibits PARG thereby instigating a negative feedback inhibition loop leading to inactivation along with a decrease in expression of PARP. Oxidants are potent activators of mitogen activated protein kinases (MAPKs), including p38 MAPK [37–39]. CP stimulates the production of ROS, including hydroxyl radicals. The formation of hydroxyl radicals in CP treated kidney cells, both in vitro and in vivo, involves the release of iron from the heme groups of cytochrome P-450 2E1 [10]. As far as downstream signaling is concerned, CP reportedly causes activation of all three MAPKs in kidney both in vitro and in vivo [34]. However the role of p38 MAPK inhibition in CP induced nephrotoxicity has recently been demonstrated [40]. We also observed similar results, as phosphorylation of p38 is increased in cisplatin treated rats. Increase in the MAPK activation is thought to play a central role in mediation of CP induced nephrotoxic damage as it has been shown that pharmacological inhibition of p38 MAPK results in decreased nephrotoxicity in vivo [40]. Co-treatment of tannic acid along with CP resulted in further increase in phosphorylation of p38, where as post-treatment of tannic acid resulted in a decrease in phosphorylation of p38. Suggesting that it is the time of tannic acid treatment (co- or post-treatment) which decides whether to potentiate or to prevent CP induced nephrotoxicity. Downstream target of PARP or MAPK signaling is chromatin so we studied the role of chromatin dynamics in transducing the upstream signals. We therefore investigated change in acetylation of histone H4 in CP induced nephrotoxicity. CP induces phosphorylation of histone H3 and hyperacetylation of Histone H4 in cancer cells [35]. However, CP induces hypoacetylation of histone H4 in rat kidney. Co-treatment with tannic acid further reduces the acetylation of histone H4, where as post-treatment results in reverting acetylation to control level. It is widely believed that acetylation status of histones correlates well with the transcriptional activation of genes [41]; hyperacetylation signifies a transcriptionaly active state while hypoacetylation leads to a transcriptionally repressed state. These findings substantiate the pivotal role played by chromatin dynamics in mediating toxic responses. Although, this process could occur by MAPK dependent or by independent recruitment of deacetylating enzymes which leads to chromatin condensation and transcriptional repression but our data definitely suggests that global change in histone acetylation may serve as predictive markers for drug toxicity and efficacy which is of fundamental importance in determining the effectiveness of anticancer drugs. Tannic acid per se does not cause any nephrotoxicity at the doses used for this study and cisplatin is not reported to interact with this polyphenol. Tannins are known to bind covalently with proteins [42]. The strongest interactions among tannins are found between BSA and tannic acid [42]. CP is also
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reported to bind with proteins in an irreversible manner [43,44]. Our data suggest that the both tannic acid as well as cisplatin bind to albumin in plasma and share similar binding sites. CP is also known to form DNA adduct but out of 10 molecules only one enters the nucleus and rest nine bind to proteins [45]. It is already reported that tannic acid lends a conspicuous change in the conformation of BSA in vitro [42]. Moreover, by translating the MS–MS spectra to amino acid sequence by MASCOT search, we found that the most predominant peak 1479 corresponds to that of albumin (data not shown). Similar in vitro studies with BSA, tannic acid and cisplatin also showed results identical to what we found in tryptic digest of plasma samples (data not shown). Suppression of same peptide peaks by cisplatin and tannic acid treatment provides indirect evidence that tannic acid increases the bioavailability of cisplatin which may be responsible for increased nephrotoxicity. These findings substantiate the need of further studies to delineate the roles of polyphenols in general and tannic acid in particular in different cancer models. These studies will aid in the development of a novel therapeutic dose design which is a fulcral aspect of cancer research. Acknowledgments: This work was supported by grant from Department of Biotechnology, Govt. of India (Grant BT/PR4005/BRB/10/ 331/2003) and National Institute of Pharmaceutical Education and Research (NIPER).
References [1] Jamieson, E.R. and Lippard, S.J. (1999) Structure, Recognition, and Processing of Cisplatin-DNA Adducts. Chem. Rev. 99, 2467– 2498. [2] Green, C.M. and Almouzni, G. (2002) When repair meets chromatin. First in series on chromatin dynamics. EMBO Rep. 3, 28–33. [3] Kornberg, R.D. and Lorch, Y. (1999) Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98, 285–294. [4] Thoma, F. (1999) Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair. EMBO J. 18, 6585–6598. [5] Tyler, J.K. and Kadonaga, J.T. (1999) The ‘‘dark side’’ of chromatin remodeling: repressive effects on transcription. Cell 99, 443–446. [6] Gonzales-Vitale, J.C., Hayes, D.M., Cvitkovic, E. and Sternberg, S.S. (1977) The renal pathology in clinical trials of cis-platinum (II) diamminedichloride. Cancer 39, 1362–1371. [7] Meyer, K.B. and Madias, N.E. (1994) Cisplatin nephrotoxicity. Miner. Electrolyte Metab. 20, 201–213. [8] Cornelison, T.L. and Reed, E. (1993) Nephrotoxicity and hydration management for cisplatin, carboplatin, and ormaplatin. Gynecol. Oncol. 50, 147–158. [9] Gemba, M. and Fukuishi, N. (1991) Amelioration by ascorbic acid of cisplatin-induced injury in cultured renal epithelial cells. Contrib. Nephrol. 95, 138–142. [10] Liu, H. and Baliga, R. (2003) Cytochrome P450 2E1 null mice provide novel protection against cisplatin-induced nephrotoxicity and apoptosis. Kidney Int. 63, 1687–1696. [11] Krajka-Kuzniak, V. and Baer-Dubowska, W. (2003) The effects of tannic acid on cytochrome P450 and phase II enzymes in mouse liver and kidney. Toxicol Lett. 143, 209–216. [12] Feldman, K.S., Sahasrabudhe, K., Lawlor, M.D., Wilson, S.L., Lang, C.H. and Scheuchenzuber, W.J. (2001) In vitro and In vivo inhibition of LPS-stimulated tumor necrosis factor-alpha secretion by the gallotannin beta-D -pentagalloylglucose. Bioorg. Med. Chem. Lett. 11, 1813–1815.
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035 [13] Fong, H.H., Bhatti, W. and Farnsworth, N.R. (1972) Antitumor activity of certain plants due to tannins. J. Pharm. Sci. 61, 1818. [14] Mota, M.L., Thomas, G. and Barbosa Filho, J.M. (1985) Antiinflammatory actions of tannins isolated from the bark of Anacardium occidentale L. J. Ethnopharmacol. 13, 289–300. [15] Uchiumi, F., Maruta, H., Inoue, J., Yamamoto, T. and Tanuma, S. (1996) Inhibitory effect of tannic acid on human immunodeficiency virus promoter activity induced by 12-O-tetra decanoylphorbol-13-acetate in Jurkat T-cells. Biochem. Biophys. Res. Commun. 220, 411–417. [16] Van Molle, W., Vanden Berghe, J., Brouckaert, P. and Libert, C. (2000) Tumor necrosis factor-induced lethal hepatitis: pharmacological intervention with verapamil, tannic acid, picotamide and K76COOH. FEBS Lett. 467, 201–205. [17] Hagerman, A.E., Riedl, K.M. and Rice, R.E. (1999) Tannins as biological antioxidants. Basic Life Sci. 66, 495–505. [18] Erdelyi, K., Kiss, A., Bakondi, E., Bai, P., Szabo, C., Gergely, P., Erdodi, F. and Virag, L. (2005) Gallotannin inhibits the expression of chemokines and inflammatory cytokines in A549 cells. Mol. Pharmacol. 68, 895–904. [19] Lee, S.J., Lee, I.S. and Mar, W. (2003) Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 activity by 1,2,3,4,6penta-O-galloyl-beta-D -glucose in murine macrophage cells. Arch. Pharm. Res. 26, 832–839. [20] Lieberthal, W., Fuhro, R., Andry, C.C., Rennke, H., Abernathy, V.E., Koh, J.S., Valeri, R. and Levine, J.S. (2001) Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells. Am. J. Physiol. Renal Physiol. 281, F693–F706. [21] Lieberthal, W., Triaca, V. and Levine, J. (1996) Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis. Am. J. Physiol. 270, F700–F708. [22] Burkle, A., Chen, G., Kupper, J.H., Grube, K. and Zeller, W.J. (1993) Increased poly(ADP-ribosyl)ation in intact cells by cisplatin treatment. Carcinogenesis 14, 559–561. [23] Shiomi, M., Wakabayashi, Y., Sano, T., Shinoda, Y., Nimura, Y., Ishimura, Y. and Suematsu, M. (1998) Nitric oxide suppression reversibly attenuates mitochondrial dysfunction and cholestasis in endotoxemic rat liver. Hepatology 27, 108–115. [24] Masutani, M., Nakagama, H. and Sugimura, T. (2003) Poly(ADP-ribose) and carcinogenesis. Genes Chromosomes Cancer 38, 339–348. [25] Miwa, M. and Sugimura, T. (1971) Splitting of the ribose–ribose linkage of poly(adenosine diphosphate-robose) by a calf thymus extract. J. Biol. Chem. 246, 6362–6364. [26] Sevigny, M.B., Silva, J.M., Lan, W.C., Alano, C.C. and Swanson, R.A. (2003) Expression and activity of poly(ADP-ribose) glycohydrolase in cultured astrocytes, neurons, and C6 glioma cells. Brain Res. Mol. Brain Res. 117, 213–220. [27] Falsig, J., Christiansen, S.H., Feuerhahn, S., Burkle, A., Oei, S.L., Keil, C. and Leist, M. (2004) Poly(ADP-ribose) glycohydrolase as a target for neuroprotective intervention: assessment of currently available pharmacological tools. Eur. J. Pharmacol. 497, 7–16. [28] Genovese, T. et al. (2004) Treatment with a novel poly(ADPribose) glycohydrolase inhibitor reduces development of septic shock-like syndrome induced by zymosan in mice. Crit. Care Med. 32, 1365–1374. [29] Ying, W., Sevigny, M.B., Chen, Y. and Swanson, R.A. (2001) Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death. Proc. Natl. Acad. Sci. USA 98, 12227–12232. [30] Ying, W. and Swanson, R.A. (2000) The poly(ADP-ribose) glycohydrolase inhibitor gallotannin blocks oxidative astrocyte death. Neuroreport 11, 1385–1388. [31] Allcock, G.H., Hukkanen, M., Polak, J.M., Pollock, J.S. and Pollock, D.M. (1999) Increased nitric oxide synthase-3 expression in kidneys of deoxycorticosterone acetate-salt hypertensive rats. J. Am. Soc. Nephrol. 10, 2283–2289. [32] Tikoo, K., Gupta, S., Hamid, Q.A., Shah, V., Chatterjee, B. and Ali, Z. (1997) Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver. Biochem. J. 322, 273–279. [33] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035 [34] Arany, I., Megyesi, J.K., Kaneto, H., Price, P.M. and Safirstein, R.L. (2004) Cisplatin- induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells. Am. J. Physiol. Renal Physiol. 287, F543–F549. [35] Wang, D. and Lippard, S.J. (2004) Cisplatin-induced posttranslational modification of histones H3 and H4. J. Biol. Chem. 279, 20622–20625. [36] Zhan, Y., van de Water, B., Wang, Y. and Stevens, J.L. (1999) The roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of renal epithelial cells. Oncogene 18, 6505–6512. [37] Guyton, K.Z., Liu, Y., Gorospe, M., Xu, Q. and Holbrook, NJ. (1996) Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J. Biol. Chem. 271, 4138–4142. [38] Khadaroo, R.G., Parodo, J., Powers, K.A., Papia, G., Marshall, J.C., Kapus, A. and Rotstein, O.D. (2003) Oxidant-induced priming of the macrophage involves activation of p38 mitogenactivated protein kinase through an Src-dependent pathway. Surgery 134, 242–246. [39] Peus, D., Vasa, R.A., Beyerle, A., Meves, A., Krautmacher, C. and Pittelkow, M.R. (1999) UVB activates ERK1/2 and p38
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[40] [41] [42]
[43] [44]
[45]
signaling pathways via reactive oxygen species in cultured keratinocytes. J. Invest. Dermatol. 112, 751–756. Ramesh, G. and Reeves, W.B. (2005) p38 MAP kinase inhibition ameliorates cisplatin nephrotoxicity in mice. Am. J. Physiol. Renal Physiol. 289, F166–F174. Struhl, K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12, 599–606. Labieniec, M. and Gabryelak, T. (2006) Interactions of tannic acid and its derivatives (ellagic and gallic acid) with calf thymus DNA and bovine serum albumin using spectroscopic method. J. Photochem. Photobiol. B 82, 72–78. Cole, W.C. and Wolf, W. (1980) Preparation and metabolism of a cisplatin/serum protein complex. Chem. Biol. Interact. 30, 223– 235. Perera, A., Jackson, H., Sharma, H.L., McAuliffe, C.A. and Fox, B.W. (1992) A comparative binding of platinum anti-tumour compounds to plasma proteins in the rat (in vivo) and mouse (in vitro). Chem. Biol. Interact. 85, 199–213. Goodsell, D.S. (2006) The molecular perspective: cisplatin. Oncologist 11, 316–317.
Differential effects of tannic acid on cisplatin induced nephrotoxicity in rats Kulbhushan Tikoo*, Deepak Kumar Bhatt, Anil Bhanudas Gaikwad1, Vikram Sharma1, Dhiraj G. Kabra1 Laboratory of Chromatin Biology, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India Received 7 February 2007; revised 31 March 2007; accepted 10 April 2007 Available online 24 April 2007 Edited by Varda Rotter
Abstract Cisplatin is a widely used antineoplastic drug. Major drawback of cisplatin therapy is its nephrotoxicity. The objective of this study was to check the effect of tannic acid on cisplatin induced nephrotoxicity. Post-treatment of tannic acid prevents cisplatin (5 mg/kg) induced nephrotoxicity and decreases poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 and hypoacetylation of histone H4. In contrast, co-treatment of tannic acid potentiates the nephrotoxicity. Comparative nephrotoxicity studies show that co-treatment of tannic acid with reduced dose of cisplatin (1.5 mg/kg) developed almost similar nephrotoxicity. MALDI protein profiling of plasma samples provides indirect evidence that tannic acid co-treatment increases bioavailability of cisplatin. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cisplatin; Nephrotoxicity; poly(ADP-ribose) polymerase; Histone H4; p-p38 and Tannic acid
1. Introduction Cisplatin (CP) remains the drug of choice in many platinum based chemotherapy regimens. It acts by damaging DNA owing to platination to form covalent platinum DNA adducts [1]. DNA damage elicits a series of signal transduction cascades, involving chromatin remodeling, which eventually lead to DNA repair, cell cycle arrest or apoptosis [2–5]. The therapeutic effects of cisplatin are dose dependent but the chief limit to its promising efficacy as an antineoplastic drug is its nephrotoxicity [6,7]. Use of other means to prevent nephrotoxicity e.g. hydration protocols [8], antioxidant supplementation [9] have proven to be partially successful due to the repeated dosing schedules of CP. Therapeutic interventions aimed at ameliorating renal damage require the use of combination * Corresponding author. Fax: +91 172 2214692. E-mail address: [email protected] (K. Tikoo). 1
Authors contributed equally.
Abbreviations: CP, cisplatin; PARP, poly(ADP-ribose) polymerase; PARG, poly(ADP ribose) glycohydrolase; BUN, blood urea nitrogen; MAPK, mitogen activated protein kinases; ROS, reactive oxygen species
therapies. This indicts the involvement of reactive oxygen species (ROS) generation as a mediator in inflicting nephrotoxic insult in CP therapy. Interaction of cisplatin with CYP2E1 results in the generation of reactive oxygen metabolites that causes renal injury and initiates apoptosis. Recent report shows that cytochrome P450 2E1 null mice provide novel protection against cisplatin induced nephrotoxicity and apoptosis [10]. Polyphenolics are known for their potent antioxidant and free radical scavenging properties. It significantly affects the pathways which are involved in the detoxification of chemical carcinogens particularly those in which CYP2E1 and NAD(P)H:quinone oxidoreductase (NQO1) are involved [11]. It is also reported that administration of tannic acid (gallotannin) resulted in a marked decrease in the expression of hepatic CYP2E1, but there are no reports which address the relevance of this polyphenol with respect to its role in mediating or preventing the toxic effects. In addition to tannic acid, other tannins have been shown to exert various biological effects ranging from anti-inflammatory to anticancer and antiviral effects [12–16]. The mechanism underlying the antiinflammatory effects of tannins include the scavenging of radicals; antioxidant effect [17,18] and inhibition of the expression of inflammatory mediators, such as some cytokines [12], including nitric oxide synthase, and cylooxgenase2[19]. Renal tubular apoptosis has been implicated as a final common pathway in response to a variety of cellular stresses at intensity below the threshold for necrosis [20,21]. Poly(ADPribose) polymerase (PARP), an enzyme involved in DNA damage and repair. It is also activated in conditions of oxidative stress caused by depletion of antioxidant enzymes by CP [22,23]. Poly(ADP-ribose) glycohydrolase (PARG) is another enzyme that keeps PARP in active state by hydrolytic cleavage of poly(ADP-ribose) (PAR) [24–26]. Inhibition of PARP can be obtained by the use of PARP/PARG inhibitors [27]; PARG inhibitors have shown cytoprotection against various tissue injuries in vivo as well as in vitro cell systems [28]. Tannic acid, a PARG inhibitor showed cytoprotection against ischemic reperfusion injury where oxidative stress is known to play a key role [29,30]. Novel strategies aimed at minimizing nephrotoxicity while enhancing its antineoplastic efficacy are incomplete until a more basic understanding of its toxicity is obtained. The objective of the present study was to study the effect of tannic acid in cisplatin induced nephrotoxicity.
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.04.036
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2. Materials and methods 2.1. Reagents and solutions cis-Diamminplatinumdichloride (cisplatin, CP) was obtained from Dabur India Pvt. Ltd. as a gift sample. Tannic acid was obtained from Merck, Germany. Trypsin was purchased from Promega Corporation (UK). All the other chemicals were purchased from Sigma (St. Louis, MO). Cisplatin solution was made freshly in saline (0.9% NaCl). Tannic acid was prepared freshly in phosphate buffer of pH 7.4. 2.2. Animal treatment and experimental protocol 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 Sprague–Dawley rats in the weight range of 170–190 g which were procured from the central animal facility of the institute, kept at controlled environmental conditions with room temperature 22 ± 2 C and 12 h light/dark cycles. Rats were divided at random into different groups, eight animals in each group. In co-treatment group single dose of tannic acid (10 mg/kg) was administered just after the CP (5 mg/kg) treatment. In post-treatment group three consecutive doses of tannic acid (10 mg/kg) were given from next day of CP treatment. Drugs were administered by intraperitoneal (i.p.) route. For comparative nephrotoxicity profile, rats were divided randomly into two groups, received either a single injection of CP (1.5 mg/kg,) or co-treatment of CP (1.5 mg/kg) with single dose of tannic acid (10 mg/ kg). Rats were weighed prior to injection and four day after CP treatment. Blood samples were collected on fifth day, from the retro orbital plexus of rats under light ether anesthesia in heparinized centrifuge tubes and immediately centrifuged at 2300 · g for the separation of plasma. Plasma was stored at 20 until assayed. 2.3. Estimation of plasma creatinine, blood urea nitrogen (BUN) and plasma albumin Plasma creatinine concentration was measured by the picric acid colorimetric method [31]. A picric acid solution was made by a tenfold dilution of saturated picric acid with 1% NaOH. Plasma (50 ll) was added to picric acid–NaOH solution (200 ll) in a micro titer plate. The samples were allowed to incubate for 15 min before being read at 490 nm in a micro plate reader. A standard curve was prepared on the same assay plate to derive the creatinine concentration. BUN and plasma albumin were determined colorimetrically using commercially available standard kits. 2.4. Histopathological procedures Rats 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. Kidneys were removed from the animal, decapsulated, sliced transversely, and paraffinembedded for light microscopic evaluation. Histopathological changes in kidney structure were assessed in at least 25 randomly selected tissue sections from each group studied. Sections were stained with Meyer’s hematoxylin and eosin to examine cell structure. Slides were observed under Olympus microscope (Model no. BX51).
2.5. Nuclei isolation and Western blotting Nuclei were isolated and acid soluble proteins were extracted according to method described previously [32]. For nuclear protein, nuclei were suspended in buffer (1% NP-40, 10 mM Tris, 10 mM NaCl, 10 mM EDTA, 10 lg/ml aprotinin, 10 lg/ml leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF)) and its protein concentration was determined by the method of Lowry et al. [33]. For Western blot analysis, nuclear proteins were transferred onto PVDF membrane (Bio-Rad) and immunoblot analysis was performed by using the anti-acetyl histone H4 (rabbit monoclonal 1:5000, Upstate), anti p38, anti p-p38 (rabbit 1:500, Santa Cruz Biotechnology Inc.), anti-PARP (rabbit 1:250, Upstate), anti actin (rabbit 1:2500, Sigma), and HRP-conjugated secondary antibodies (anti-rabbit, Calbiochem). Proteins were detected with the enhanced chemiluminescence (ECL) system and ECL Hyperfilm (Amersham Pharmacia Biotech). 2.6. MALDI plasma protein profiling For MALDI plasma protein profiling, 16 SD rats of weight 170– 190 g were divided into four groups, four animals in each group. In the first group vehicle for CP and tannic acid was administered, in second group CP (5 mg/kg) was injected, in the third group co-treatment of tannic acid (10 mg/kg) with cisplatin (5 mg/kg) was given and the fourth group was treated with tannic acid (10 mg/kg). Blood samples were collected and plasma was separated by centrifugation for 10 min at 2300 · g and stored at 20 C until analysis. After 20 times dilution of plasma, plasma protein was estimated [33]. Then 10 lg proteins from each sample was digested for over night at 37 C with 100 ng of modified trypsin (Promega Corporation, UK). For protein mass spectra 1 ll of the digested protein was mixed with 1 ll alpha-cyano hydroxy cinnamic acid solution matrix, dried and washed twice with 0.1% Trifluoroacetic acid. Protein profiles were accumulated from dried-droplet deposits of matrix/peptide mixture by MALDI-TOF mass spectrometer (Bruker Daltonics flexanalysis), externally calibrated using peptide standard mix (Bruker Daltonics) and 200 shots were accumulated to give an acceptable spectrum. For each digest, triplicate MALDI spots were deposited and analyzed. Ion signals above a signal-to-noise (S/N) ratios of 10 were considered for comparison. Peak intensities ratios of treated to control samples were calculated by averaging the peak intensities followed by t-tests of the intensity of each individual peaks. 2.7. Statistical analysis Data are expressed as means ± S.E.M. Statistical comparison between different groups were done using one-way analysis of variance (ANOVA) followed by tukey multiple comparison test, to detect the difference between various groups. P value less than 0.05 was considered to be significant.
3. Results 3.1. Cisplatin induced changes in body weight CP induced nephrotoxicity can easily be assessed by decrease in body weight, urine out put and water intake. CP treatment leads to a significant decrease in body weight as
Table 1 Changes in body weight and biochemical parameters by co and post treatment of tannic acid on CP induced nephrotoxicity in SD rats Treatment groups
Change in body wt (g)
BUN (mg/dl)
Creatinine (mg/dl)
Albumin (g/dl)
Vehicle control (0.9% saline) Tannic acid (10 mg/kg) control co-treatment Tannic acid (10 mg/kg) control post-treatment CP treated (5 mg/kg) CP (5 mg/kg) + tannic acid (10 mg/kg) co-treatment CP (5 mg/kg) + tannic acid (10 mg/kg) post-treatment
19 ± 2 17 ± 1 9.4 ± 1.3 (–)11 ± 3***a (–)14 ± 2***ab (–)8 ± 2.5***a
21 ± 0.79 20 ± 1.07 19 ± 0.23 85 ± 11.7***a 124 ± 12***ab 40 ± 4.18***ab
0.68 ± 0.04 0.84 ± 0.08 0.79 ± 0.02 2.18 ± 0.24***a 4.35 ± 0.21***a 1.25 ± 0.15**ab
3.67 ± 0.03 3.34 ± 0.08 2.9 ± 0.04*a 3.27 ± 0.09***a 3.08 ± 0.13***a 2.8 ± 0.09**ab
Biochemical parameters were estimated after 5 days of CP (5 mg/kg) and tannic acid (10 mg/kg) treatment, as described in Section 2. All values are expressed as means ± S.E.M. (n = 8), ***P < 0.001, **P < 0.01, *P < 0.05, a vs vehicle control group, b vs tannic acid group or CP treated group.
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served suggesting development of nephrotoxicity (data not shown).
Table 2 Comparative nephrotoxicity profiling Treatment groups
BUN (mg/dl)
Creatinine (mg/dl)
Vehicle control (0.9% saline) Tannic acid control CP treated (5 mg/kg) CP treated (1.5 mg/kg) CP (1.5 mg/kg) + tannic acid (10 mg/kg)
21 ± 0.79 20 ± 1.07 85 ± 11.7*** 28 ± 3.67*** 62 ± 11***
0.68 ± 0.04 0.84 ± 0.08 2.18 ± 0.24*** 1.17 ± 0.08*** 1.38 ± 0.05***
Changes in biochemical parameters by co-treatment of tannic acid on cisplatin induced nephrotoxicity in SD rats. Biochemical parameters were estimated after 5 days of cisplatin (5 mg/kg and 1.5 mg/kg) and tannic acid (10 mg/kg) treatment, as described in Section 2. All values are expressed as means ± S.E.M. (n = 5), ***P < 0.001 vs vehicle control group.
compared to control rats (Table 1). Significant increase in water intake and urine output of CP treated rats was also ob-
3.2. Changes in BUN, creatinine and albumin levels by co- and post-treatment of tannic acid with cisplatin CP treated rats developed polyuric acute renal failure as assessed by measuring different biochemical parameters like BUN, plasma creatinine and plasma albumin. The BUN level in CP treated rats increased significantly to reach 85 ± 11.7 mg/ dl (21 ± 0.79 mg/dl in control rats). Plasma creatinine level was also increased significantly to reach 2.18 ± 0.24 mg/dl (0.68 ± 0.04 mg/dl in control rats) after CP treatment. Similarly, plasma albumin level decreased significantly to reach 3.27 ± 0.09 g/dl (3.67 ± 0.03 g/dl in control rats). Co-treatment of tannic acid with CP further increases the nephrotoxicity as assessed by significant increase in BUN to reach 124 ± 12 mg/ dl (85 ± 11.7 mg/dl in CP treated rats), plasma creatinine to reach 4.35 ± 0.21 mg/dl (2.18 ± 0.24 mg/dl in CP treated rats)
Fig. 1. Histopathological changes in kidney after co- and post-treatment of tannic acid along with cisplatin. Transverse section of normal control rat kidney (A) and (B), CP treated kidney (C) and (D), co-treatment of tannic acid (E) and (F) and post-treatment of tannic acid (G) and (H). Sections were stained with Mayer’s hematoxylin counterstained with eosin and observed under magnification of 40· and 100· as described in Section 2. fi indicates cells undergoing apoptotic cell death.
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and decrease plasma albumin level 3.08 ± 0.13 g/dl (3.27 ± 0.09 g/dl in CP treated rats). In contrast to co-treatment of tannic acid, post-treatment of tannic acid significantly decreases the nephrotoxicity in the CP treated rats. The BUN and plasma creatinine level in tannic acid post treated rats decreased significantly from 85 ± 11.7 to 40 ± 4.18 mg/dl and 2.18 ± 0.24 to 1.25 ± 0.15 mg/dl, respectively (Table 1). 3.3. Effect of co-treatment of tannic acid with reduced dose of cisplatin (comparative nephrotoxicity) Co-treatment of tannic acid with CP increases the nephrotoxicity of cisplatin. Comparative nephrotoxicity study was planned to find out whether tannic acid co-treatment with reduced dose of CP (1.5 mg/kg) leads to a similar nephrotoxicity profile as with the routinely used dose of CP (5 mg/kg). Table 2 shows that co-treatment of tannic acid with CP (1.5 mg/kg) developed almost equivalent polyuric acute renal failure as in
A
116 kDa 85 kDa 66 kDa
PARP Actin 480
***b
PARP % of Control
400 320
*a
240 160
*b
80 0 a
B
b
c
CP (5 mg/kg) treated rats. There is significant increase in BUN levels 62 ± 11 mg/dl (21 ± 0.79 mg/dl in control rats) and plasma creatinine levels 1.38 ± 0.05 mg/dl (0.68 ± 0.04 mg/dl in control rats). 3.4. Cisplatin induces histopathological changes in rat kidney In the histopathology of kidney, while the normal control group displays normal kidney histology (Fig. 1A and B), extensive tubular epithelial cellular necrosis, desquamation, vacuolization and swelling were observed in the CP treated kidneys (Fig. 1C and D). Co-treatment of tannic acid with cisplatin increases the tubular cell damage (Fig. 1E and F). However, post-treatment of tannic acid resulted in remarkable improvement in the histological appearance and reduction in tubular cell damage (Fig. 1G and H). 3.5. Cisplatin induces PARP cleavage and phosphorylation of p38 Several reports suggest the role of PARP and phosphorylation of p38 in cisplatin induced nephrotoxicity [23,34]. We studied the PARP cleavage and phosphorylation of p38 in the kidney of the cisplatin treated rats. Fig. 2A, lane b shows that after cisplatin treatment there is significant increase in PARP cleavage as compared to control group (Fig. 2A, lane a) and it increases further by co-treatment of tannic acid (Fig. 2A, lane c). However, there is a decrease in PARP cleavage by the post-treatment of tannic acid (Fig. 2A, lane d). Suggesting that PARP activation is more pronounced in the co-treatment group as compared to the CP treated group. Compared to control animals phosphorylation of p38 is found to be increased by cisplatin treatment (Fig. 2B, lanes a and b) and it is further increased by co-treatment (Fig. 2B, lane c). But there is significant decrease in phosphorylation of p38 by the post-treatment of tannic acid (Fig. 2B, lane d) as compared to CP treated rats. Suggesting that kidney cells are undergoing apoptotic cell death and co-treatment of tannic acid potenti-
d
p-p38 Ac-H4 p38 Total H4 210
**a
120
150
100
***b
120 90 60 30 0
Ac-Histone H4 % of Control
p-p38 % of control
180
***b
80
***b
60
***a
40
*b
20
a
b
c
d 0
Fig. 2. Effect of tannic acid co-treatment and post-treatment on PARP cleavage and phosphorylation of p38 in cisplatin treated kidney. (A) Western blot of PARP and (B) phosphorylation of p38. Lane a, total cellular proteins of kidney of control animal; lane b, total cellular proteins of kidney of cisplatin treated animal; lane c, total cellular proteins of kidney of tannic acid co treated animal and lane d, total cellular proteins of kidney of tannic acid post treated animal. Anti actin and anti p38 antibody was used as loading control. Results were normalized with respect to actin and p38 band intensities in respective controls. Similar results were obtained in three independent set of experiments. ***P < 0.001, **P < 0.01 and *P < 0.05 a vs control, b vs cisplatin.
a
b
c
d
Fig. 3. Effect of tannic acid co-treatment and post-treatment in cisplatin treated rat kidney on histone H4 acetylation. Lane a, acid soluble nuclear proteins of control animal: lane b, acid soluble nuclear proteins of cisplatin treated animal: lane c, acid soluble nuclear proteins of tannic acid co treated animal and lane d, acid soluble nuclear proteins of tannic acid post treated animal. Anti H4 antibody was used as a loading controls. Results were normalized with respect to H4 band intensities in respective controls. Similar results were obtained in three independent set of experiments. ***P < 0.001 and *P < 0.05 a vs control, b vs cisplatin.
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035
ates apoptosis while post-treatment of tannic acid prevents it by decreasing PARP cleavage and phosphorylation of p38. 3.6. Hypoacetylation of histone H4 in cisplatin induced nephrotoxicity CP is known to cause histone H3 phosphorylation and hyperacetylation of histone H4 in cancer cells in vitro [35].
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We studied the changes in level of acetylation of histone H4 in the kidney of the cisplatin treated rats. As compared to control rat kidney, CP treatment decreases acetylation of histone H4 (Fig. 3, lanes a and b). In case of co-treatment of tannic acid there is further decrease in acetylation of H4 as compared to CP treated rats (Fig. 3, lane c). However, in contrast to cotreatment, post-treatment of tannic acid prevents decrease in
Fig. 4. Mass spectra of plasma proteins showing changes in plasma protein binding by co-treatment of tannic acid on cisplatin treated rats. MALDI mass spectra of plasma protein (10 lg) digested with trypsin. Plasma samples of control (A) cisplatin treated (B) tannic acid (C) and tannic acid co administered along with cisplatin (D). Blood was taken out after 15 min of treatment; plasma was separated from blood, digested by modified trypsin (100 ng) and subjected to MALDI TOF analyses as described in Section 2. Similar results were obtained in three independent set of experiments.
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K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035
the acetylation of histone H4 in CP treated rats (Fig. 3, lane d). Our data and other reports suggest that CP induces different histone modifications in different cell types. 3.7. MALDI plasma protein profiling Potentiation of nephrotoxicity by co-treatment of tannic acid and comparative nephrotoxicity studies suggested us to check the bioavailability of CP after tannic acid co-treatment. We applied mass spectroscopic approach which provides indirect evidence regarding CP/tannic acid and plasma protein
binding. Plasma protein spectra were taken after different treatments. In each spectrum, 200–300 peaks were observed, ion signals having sufficient S/N ratios were considered useful in further analyses (Fig. 4). In comparison to tryptic digested plasma of control animals major peptide peaks (e.g. peptides having m/z 1266, 1299, 1440, 1455 and 1479) were suppressed after cisplatin treatment in plasma taken after 15 min of drug injection (Fig. 4B). Tannic acid treatment also suppresses same peaks which are suppressed by CP and were present in control samples (Fig. 4C).
Fig. 4 (continued)
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035
Peptide mass (m/z) - 1479
A
Control
200000
Peak intensity
Cisplatin Co-treatment
150000
Tannic acid 100000
a
***
a
***
50000
a
***
0
B
Peptide mass peak (m/z)-1479 0.5
Cisplatin
Peak intensity ratio (treatment/control)
Co-treatment 0.4
Tannic acid
0.3 0.2 0.1 0
Fig. 5. Peak intensity obtained from mass spectra of plasma protein (10 lg) digested with modified trypsin (100 ng). Peak intensities ratios of treated to control samples were calculated by averaging the peak intensities followed by t-tests of the intensity of the peak. (A) Comparative change in peak intensity of albumin peptide identified by MSMS spectra and having mass (m/z) 1479 after 15 min of drug treatment. (B) Bar diagram of peak intensity ratio of peptide mass (m/ z) 1479 after 15 min of drug treatment. Results are expressed as means ± S.E.M. of three independent experiments performed in triplicates, ***P < 0.001 a vs control.
Furthermore, co-treatment of tannic acid with CP resulted in further suppression of these peaks in the mass spectra of tryptic digested plasma proteins (Fig. 4D). These results indicate the involvement of competitive binding between CP and tannic acid to form cisplatin–protein or tannic acid–protein adducts. The possibility of same binding site for CP and tannic acid is indicated by the suppression of same peaks e.g. major peptide peak of mass 1479 by CP as well as tannic acid (Fig 5A and B).
4. Discussion Present study was conceived to study the effect of tannic acid on CP induced nephrotoxicity. Here, we provide evidence that co-treatment of tannic acid with CP increases the nephrotoxicity while the post-treatment prevents it up to certain extent. Nephrotoxic damage by cisplatin shows increase in BUN and creatinine levels. Comparative nephrotoxicity profiling suggests that tannic acid increases the CP induced nephrotoxicity. The elevated levels of BUN and creatinine suggest an increase in the toxicity of cisplatin in the presence of tannic acid and this potentiating effect of tannic acid can be observed at both therapeutic as well as sub therapeutic dose levels. Moreover, histopathology examination of kidney sections also supports our biochemical data. These results indicate the central role of tannic acid in potentiating the nephrotoxic effect of CP and point towards its profound clinical implications. It can be argued that tannic acid also shows a protective effect
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owing to its antioxidant action but the fact that this polyphenol exerts a modulatory effect depending upon the time of administration. CP induced apoptosis involves PARP cleavage, caspase-3 and bcl-2 in renal epithelial cells [36]. We observed a significant increase in PARP cleavage in CP treated rat kidneys, indicating cells are undergoing apoptotic cell death. Co-treatment of tannic acid resulted in further increase in PARP cleavage while post-treatment prevents its cleavage. Decrease in cleavage of PARP in post-treatment group might be due to the inhibition of PARG by tannic acid, which is a known PARG inhibitor [29,30]. Tannic acid inhibits PARG thereby instigating a negative feedback inhibition loop leading to inactivation along with a decrease in expression of PARP. Oxidants are potent activators of mitogen activated protein kinases (MAPKs), including p38 MAPK [37–39]. CP stimulates the production of ROS, including hydroxyl radicals. The formation of hydroxyl radicals in CP treated kidney cells, both in vitro and in vivo, involves the release of iron from the heme groups of cytochrome P-450 2E1 [10]. As far as downstream signaling is concerned, CP reportedly causes activation of all three MAPKs in kidney both in vitro and in vivo [34]. However the role of p38 MAPK inhibition in CP induced nephrotoxicity has recently been demonstrated [40]. We also observed similar results, as phosphorylation of p38 is increased in cisplatin treated rats. Increase in the MAPK activation is thought to play a central role in mediation of CP induced nephrotoxic damage as it has been shown that pharmacological inhibition of p38 MAPK results in decreased nephrotoxicity in vivo [40]. Co-treatment of tannic acid along with CP resulted in further increase in phosphorylation of p38, where as post-treatment of tannic acid resulted in a decrease in phosphorylation of p38. Suggesting that it is the time of tannic acid treatment (co- or post-treatment) which decides whether to potentiate or to prevent CP induced nephrotoxicity. Downstream target of PARP or MAPK signaling is chromatin so we studied the role of chromatin dynamics in transducing the upstream signals. We therefore investigated change in acetylation of histone H4 in CP induced nephrotoxicity. CP induces phosphorylation of histone H3 and hyperacetylation of Histone H4 in cancer cells [35]. However, CP induces hypoacetylation of histone H4 in rat kidney. Co-treatment with tannic acid further reduces the acetylation of histone H4, where as post-treatment results in reverting acetylation to control level. It is widely believed that acetylation status of histones correlates well with the transcriptional activation of genes [41]; hyperacetylation signifies a transcriptionaly active state while hypoacetylation leads to a transcriptionally repressed state. These findings substantiate the pivotal role played by chromatin dynamics in mediating toxic responses. Although, this process could occur by MAPK dependent or by independent recruitment of deacetylating enzymes which leads to chromatin condensation and transcriptional repression but our data definitely suggests that global change in histone acetylation may serve as predictive markers for drug toxicity and efficacy which is of fundamental importance in determining the effectiveness of anticancer drugs. Tannic acid per se does not cause any nephrotoxicity at the doses used for this study and cisplatin is not reported to interact with this polyphenol. Tannins are known to bind covalently with proteins [42]. The strongest interactions among tannins are found between BSA and tannic acid [42]. CP is also
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reported to bind with proteins in an irreversible manner [43,44]. Our data suggest that the both tannic acid as well as cisplatin bind to albumin in plasma and share similar binding sites. CP is also known to form DNA adduct but out of 10 molecules only one enters the nucleus and rest nine bind to proteins [45]. It is already reported that tannic acid lends a conspicuous change in the conformation of BSA in vitro [42]. Moreover, by translating the MS–MS spectra to amino acid sequence by MASCOT search, we found that the most predominant peak 1479 corresponds to that of albumin (data not shown). Similar in vitro studies with BSA, tannic acid and cisplatin also showed results identical to what we found in tryptic digest of plasma samples (data not shown). Suppression of same peptide peaks by cisplatin and tannic acid treatment provides indirect evidence that tannic acid increases the bioavailability of cisplatin which may be responsible for increased nephrotoxicity. These findings substantiate the need of further studies to delineate the roles of polyphenols in general and tannic acid in particular in different cancer models. These studies will aid in the development of a novel therapeutic dose design which is a fulcral aspect of cancer research. Acknowledgments: This work was supported by grant from Department of Biotechnology, Govt. of India (Grant BT/PR4005/BRB/10/ 331/2003) and National Institute of Pharmaceutical Education and Research (NIPER).
References [1] Jamieson, E.R. and Lippard, S.J. (1999) Structure, Recognition, and Processing of Cisplatin-DNA Adducts. Chem. Rev. 99, 2467– 2498. [2] Green, C.M. and Almouzni, G. (2002) When repair meets chromatin. First in series on chromatin dynamics. EMBO Rep. 3, 28–33. [3] Kornberg, R.D. and Lorch, Y. (1999) Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98, 285–294. [4] Thoma, F. (1999) Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair. EMBO J. 18, 6585–6598. [5] Tyler, J.K. and Kadonaga, J.T. (1999) The ‘‘dark side’’ of chromatin remodeling: repressive effects on transcription. Cell 99, 443–446. [6] Gonzales-Vitale, J.C., Hayes, D.M., Cvitkovic, E. and Sternberg, S.S. (1977) The renal pathology in clinical trials of cis-platinum (II) diamminedichloride. Cancer 39, 1362–1371. [7] Meyer, K.B. and Madias, N.E. (1994) Cisplatin nephrotoxicity. Miner. Electrolyte Metab. 20, 201–213. [8] Cornelison, T.L. and Reed, E. (1993) Nephrotoxicity and hydration management for cisplatin, carboplatin, and ormaplatin. Gynecol. Oncol. 50, 147–158. [9] Gemba, M. and Fukuishi, N. (1991) Amelioration by ascorbic acid of cisplatin-induced injury in cultured renal epithelial cells. Contrib. Nephrol. 95, 138–142. [10] Liu, H. and Baliga, R. (2003) Cytochrome P450 2E1 null mice provide novel protection against cisplatin-induced nephrotoxicity and apoptosis. Kidney Int. 63, 1687–1696. [11] Krajka-Kuzniak, V. and Baer-Dubowska, W. (2003) The effects of tannic acid on cytochrome P450 and phase II enzymes in mouse liver and kidney. Toxicol Lett. 143, 209–216. [12] Feldman, K.S., Sahasrabudhe, K., Lawlor, M.D., Wilson, S.L., Lang, C.H. and Scheuchenzuber, W.J. (2001) In vitro and In vivo inhibition of LPS-stimulated tumor necrosis factor-alpha secretion by the gallotannin beta-D -pentagalloylglucose. Bioorg. Med. Chem. Lett. 11, 1813–1815.
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035 [13] Fong, H.H., Bhatti, W. and Farnsworth, N.R. (1972) Antitumor activity of certain plants due to tannins. J. Pharm. Sci. 61, 1818. [14] Mota, M.L., Thomas, G. and Barbosa Filho, J.M. (1985) Antiinflammatory actions of tannins isolated from the bark of Anacardium occidentale L. J. Ethnopharmacol. 13, 289–300. [15] Uchiumi, F., Maruta, H., Inoue, J., Yamamoto, T. and Tanuma, S. (1996) Inhibitory effect of tannic acid on human immunodeficiency virus promoter activity induced by 12-O-tetra decanoylphorbol-13-acetate in Jurkat T-cells. Biochem. Biophys. Res. Commun. 220, 411–417. [16] Van Molle, W., Vanden Berghe, J., Brouckaert, P. and Libert, C. (2000) Tumor necrosis factor-induced lethal hepatitis: pharmacological intervention with verapamil, tannic acid, picotamide and K76COOH. FEBS Lett. 467, 201–205. [17] Hagerman, A.E., Riedl, K.M. and Rice, R.E. (1999) Tannins as biological antioxidants. Basic Life Sci. 66, 495–505. [18] Erdelyi, K., Kiss, A., Bakondi, E., Bai, P., Szabo, C., Gergely, P., Erdodi, F. and Virag, L. (2005) Gallotannin inhibits the expression of chemokines and inflammatory cytokines in A549 cells. Mol. Pharmacol. 68, 895–904. [19] Lee, S.J., Lee, I.S. and Mar, W. (2003) Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 activity by 1,2,3,4,6penta-O-galloyl-beta-D -glucose in murine macrophage cells. Arch. Pharm. Res. 26, 832–839. [20] Lieberthal, W., Fuhro, R., Andry, C.C., Rennke, H., Abernathy, V.E., Koh, J.S., Valeri, R. and Levine, J.S. (2001) Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells. Am. J. Physiol. Renal Physiol. 281, F693–F706. [21] Lieberthal, W., Triaca, V. and Levine, J. (1996) Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis. Am. J. Physiol. 270, F700–F708. [22] Burkle, A., Chen, G., Kupper, J.H., Grube, K. and Zeller, W.J. (1993) Increased poly(ADP-ribosyl)ation in intact cells by cisplatin treatment. Carcinogenesis 14, 559–561. [23] Shiomi, M., Wakabayashi, Y., Sano, T., Shinoda, Y., Nimura, Y., Ishimura, Y. and Suematsu, M. (1998) Nitric oxide suppression reversibly attenuates mitochondrial dysfunction and cholestasis in endotoxemic rat liver. Hepatology 27, 108–115. [24] Masutani, M., Nakagama, H. and Sugimura, T. (2003) Poly(ADP-ribose) and carcinogenesis. Genes Chromosomes Cancer 38, 339–348. [25] Miwa, M. and Sugimura, T. (1971) Splitting of the ribose–ribose linkage of poly(adenosine diphosphate-robose) by a calf thymus extract. J. Biol. Chem. 246, 6362–6364. [26] Sevigny, M.B., Silva, J.M., Lan, W.C., Alano, C.C. and Swanson, R.A. (2003) Expression and activity of poly(ADP-ribose) glycohydrolase in cultured astrocytes, neurons, and C6 glioma cells. Brain Res. Mol. Brain Res. 117, 213–220. [27] Falsig, J., Christiansen, S.H., Feuerhahn, S., Burkle, A., Oei, S.L., Keil, C. and Leist, M. (2004) Poly(ADP-ribose) glycohydrolase as a target for neuroprotective intervention: assessment of currently available pharmacological tools. Eur. J. Pharmacol. 497, 7–16. [28] Genovese, T. et al. (2004) Treatment with a novel poly(ADPribose) glycohydrolase inhibitor reduces development of septic shock-like syndrome induced by zymosan in mice. Crit. Care Med. 32, 1365–1374. [29] Ying, W., Sevigny, M.B., Chen, Y. and Swanson, R.A. (2001) Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death. Proc. Natl. Acad. Sci. USA 98, 12227–12232. [30] Ying, W. and Swanson, R.A. (2000) The poly(ADP-ribose) glycohydrolase inhibitor gallotannin blocks oxidative astrocyte death. Neuroreport 11, 1385–1388. [31] Allcock, G.H., Hukkanen, M., Polak, J.M., Pollock, J.S. and Pollock, D.M. (1999) Increased nitric oxide synthase-3 expression in kidneys of deoxycorticosterone acetate-salt hypertensive rats. J. Am. Soc. Nephrol. 10, 2283–2289. [32] Tikoo, K., Gupta, S., Hamid, Q.A., Shah, V., Chatterjee, B. and Ali, Z. (1997) Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver. Biochem. J. 322, 273–279. [33] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
K. Tikoo et al. / FEBS Letters 581 (2007) 2027–2035 [34] Arany, I., Megyesi, J.K., Kaneto, H., Price, P.M. and Safirstein, R.L. (2004) Cisplatin- induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells. Am. J. Physiol. Renal Physiol. 287, F543–F549. [35] Wang, D. and Lippard, S.J. (2004) Cisplatin-induced posttranslational modification of histones H3 and H4. J. Biol. Chem. 279, 20622–20625. [36] Zhan, Y., van de Water, B., Wang, Y. and Stevens, J.L. (1999) The roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of renal epithelial cells. Oncogene 18, 6505–6512. [37] Guyton, K.Z., Liu, Y., Gorospe, M., Xu, Q. and Holbrook, NJ. (1996) Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J. Biol. Chem. 271, 4138–4142. [38] Khadaroo, R.G., Parodo, J., Powers, K.A., Papia, G., Marshall, J.C., Kapus, A. and Rotstein, O.D. (2003) Oxidant-induced priming of the macrophage involves activation of p38 mitogenactivated protein kinase through an Src-dependent pathway. Surgery 134, 242–246. [39] Peus, D., Vasa, R.A., Beyerle, A., Meves, A., Krautmacher, C. and Pittelkow, M.R. (1999) UVB activates ERK1/2 and p38
2035
[40] [41] [42]
[43] [44]
[45]
signaling pathways via reactive oxygen species in cultured keratinocytes. J. Invest. Dermatol. 112, 751–756. Ramesh, G. and Reeves, W.B. (2005) p38 MAP kinase inhibition ameliorates cisplatin nephrotoxicity in mice. Am. J. Physiol. Renal Physiol. 289, F166–F174. Struhl, K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12, 599–606. Labieniec, M. and Gabryelak, T. (2006) Interactions of tannic acid and its derivatives (ellagic and gallic acid) with calf thymus DNA and bovine serum albumin using spectroscopic method. J. Photochem. Photobiol. B 82, 72–78. Cole, W.C. and Wolf, W. (1980) Preparation and metabolism of a cisplatin/serum protein complex. Chem. Biol. Interact. 30, 223– 235. Perera, A., Jackson, H., Sharma, H.L., McAuliffe, C.A. and Fox, B.W. (1992) A comparative binding of platinum anti-tumour compounds to plasma proteins in the rat (in vivo) and mouse (in vitro). Chem. Biol. Interact. 85, 199–213. Goodsell, D.S. (2006) The molecular perspective: cisplatin. Oncologist 11, 316–317.